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+{"text": "CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic \u03b1 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ~240 kb microdeletion at the 7q35 inherited from her father, a \u223c538 kb microduplication at the 15q13.3 region and a \u223c178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 ( Copy number variations (CNVs) are deletions or duplications of genomic DNA larger than 1 kilobase . RecurreCNVs may be inherited from a parent or arise from de novo in an individual . CNVs caCNTNAP2  7q35(146236230_146475758)\u00d71 pat), involving partially 69) gene A, 15q13.69) gene B and mic69) gene C, includSybr Green qPCR assay confirmed CMA data in our patient and demonstrated the paternal segregation of 7q35 microdeletion, whereas 15q13.3 and PAR1 microduplications were inherited from her mother with hypothyroidism. The healthy brother of the proband was a carrier of 15q13.3 and Xp22.33 microduplications; both inherited from his mother . Here, we report on a patient presenting with severe myoclonic epilepsy, microcephaly, hypertelorism, dysmorphisms as well as severe psychomotor delay and intellectual disability. CMA of the proband identified three different CNVs, a paternal 7q35 microdeletion in combination with two maternal microduplications at 15q13.3 and Xp22.33. CNTNAP2. This gene encodes for a protein of the neurexin superfamily, known as contactin-associated protein 2 (Caspr2), which is a transmembrane protein involved in cell\u2013cell interactions between neurons and glial cells and plays a crucial role in the axonal differentiation and guidance [The 7q35 microdeletion encompasses partially the intron 1 and the entire exon 2 of guidance . CNTNAP2 contains two important regulatory regions for the transcription factors Storkhead box 1A (STOX1A) and Forkhead box P1 (FOXP1) [CNTNAP2 are essential for the correct control of CNTNAP2 expression; a deletion within intron 1 covering the FOXP2 binding sites has been observed in patients suffering from language delay and autism [CNTNAP2 in combination with other chromosomal rearrangements has been observed in a patient with speech delay and ASD [It has been shown that intron 1 of  (FOXP1) . These td autism  and bipod autism . A simil and ASD .CNTNAP2 dysfunction and neurodevelopmental disorders. It has been described a patient with non-specific dysmorphic features, speech problems and learning disability having an intragenic deletion which disrupts the reading frame of the CNTNAP2 [CNTNAP2 gene in a subject with Alzheimer\u2019s disease (AD) [CNTNAP2 were observed in certain subjects with epilepsy, autism, bipolar disorder, schizophrenia and ADHD, indicating that CNTNAP2 was involved in several neurologic or psychiatric diseases [Other genetic studies support the link between  CNTNAP2 ,30. Impoase (AD) . In addidiseases .CNTNAP2 in the development of neurological diseases. Toma et al., in their large comprehensive study, showed that multiple classes of DNA variations such as SNPs, de novo variants and CNVs of CNTNAP2 are unlikely to play a role in the development of psychiatric or neurological disorders [Conflicting results have been described in the literature regarding the role played by isorders . CNTNAP2 (chr7:146203548-146334635) is not only present in affected individuals but also in an unaffected relative. Therefore, it was observed that this CNV deletion is unlikely to be highly penetrant and did not segregate with bipolar disorder in the above-described family [CNTNAP2 deletions may have incomplete penetrance, as a consequence of the variable expression of the CNTNAP2 allele [In addition, by studying a large family with bipolar disorder, they showed that a CNV deletion that removes the FOXP2 transcription binding site in intron 1 of d family . The lim2 allele . CNTNAP2 does not segregate with the disease, indicating that heterozygous CNTNAP2 deletions are not always fully penetrant [In addition, by studying an extended family, Toma C. et al. observed that a deletion which removes the FOXP2 binding site in intron 1 of enetrant .The chromosome 15q13 region has been observed to be a hotspot for CNVs because it contains low copy repeat elements (LCRs), which promote non-allelic homologous recombination (NAHR) resulting in chromosomal microduplications and microdeletions ,17, TourCHRNA7 gene, which codes for \u03b17 nicotinic acetylcholine receptor (\u03b17nAChRs) in the brain [The 15q13.3 microduplication encompasses the CHRNA7 dosage sensitivity was suggested as the cause of several of the neuropsychiatric and neurodevelopmental phenotypes detected in patients harbouring 15q13.3 CNVs [Although it has been shown that deletions at 15q13.3 in patients with neuropsychiatric phenotypes manifest incomplete penetrance (80%)  and much3.3 CNVs . 15q13.3 duplication have been shown to have incomplete penetrance in two patients with childhood-onset schizophrenia . FurtherCRLF2) gene mapped at the PAR1 on the short arm of the X and Y chromosomes (Xp22.33/Yp11.32) [Cytokine receptor-like factor 2 (mes Xp22./Yp11.32 Yp11.32) . The actYp11.32) . It has Yp11.32) . CurrentYp11.32) .CRFL2 were not yet described. CRLF2 copy number gain and therefore dysregulation of CRLF2 expression has been described only in patients with acute lymphoblastic leukaemia (ALL) [CRLF2 and other genes such as SHOX, CSF2RA and IL3RA associated with abnormality of the nervous system were annotated in the database of chromosomal imbalance and phenotype in humans (DECIPHER n. 289637 and n. 294106). Considering these findings, CRLF2 dysregulation could affect neuronal signalling and have an additive role in the development of the brain disorders observed in the proband, but further studies are needed to confirm this hypothesis.Until now, patients with neurodevelopmental phenotypes having microduplications at Xp22.33 involving only ia (ALL) . Two dupTo our knowledge, the concomitant presence of these three private CNVs in a patient has not previously been reported.The girl\u2019s father is a carrier of 7q35 microdeletion, and her mother was found to have microduplication of 15q13.3 and Xp22.33, the same as the proband\u2019s brother, but without neurodevelopmental phenotypes. This suggests that each of the CNVs alone described above are not pathogenic enough to induce clinical manifestations, whereas the simultaneous occurrence of these three CNVs  may induce the brain disorders observed in our patient.The two-hit model could be an explanation for phenotypic differences between probands and their carrier parents ,40. ThisCNTNAP2  [We do not exclude that microcephaly, severe psychomotor delay and dysmorphic signs observed in the proband could be due to the fact that proband\u2019s parents are third cousins. In support of this hypothesis, Rodenas-Cuadrado P. et al. observed facial dysmorphisms and psychomotor problems in a case report in which the proband\u2019s parents were consanguineous (first degree cousins) carrying a 203 kb deletion encompassing exons 2\u20133 of the 914.175) . It is essential to underline that the interpretation of the clinical significance of small CNVs (\u223c500 kb), such as those observed in this patient, is very challenging because their role in the etiopathogenesis of several neurological seems to be associated to different factors, among which multifactorial effects or epigenetic alteration modifying gene expression . ConsequThe main limitations of this study are the absence of direct sequencing and whole exome/genome sequencing for the proband and her parents. Sanger sequencing could give us important information regarding the identified variants, whereas whole exome/genome sequencing can be used to exclude other genetic factors that can contribute to the observed phenotypes. In addition, since family history showed that proband\u2019s parents are third cousins, it is possible that other genetic factors than those described by us could be involved in the development of the observed phenotype. CNTNAP2, CHRNA7 and CRLF2 genes in neurodevelopmental disorders.Importantly, future functional studies are needed to better understand the potential role of In conclusion, we hypothesise that the simultaneous presence of these three CNVs has an additive effect on phenotype, and together with other environmental or genetic factors, could be responsible for the clinical manifestations of the affected female described here."}
+{"text": "It is a known fact that road traffic accidents are responsible for nearly 1.3 million deaths and another 50 million injuries in a year around the globe. Depending on the economic development of the country, victim category may change. In countries like Sri Lanka Vulnerable Road Users (VRU) are more susceptible for road traffic accidents. According to police records pedestrian involved accidents were top the list among other victim categories up to 2015. At present motor cycle riders are recorded as the highest number of victims due to road traffic accidents and pedestrians are at the second place. This paper is about pedestrians involved road traffic accidents reported to Kandy Police station in Sri Lanka to investigate and report a comparison of accidents recorded. Data Collection By law it is mandatory to report all the road traffic accidents to the police station concerned. For this study, road traffic accidents reported to Kandy police station during the period 29.08.2017 to 31.12.2018 were considered. Kandy is the second largest city in Sri Lanka and Kandy has nearly 10% of total traffic related incidents reporting compared to rest of the Country. As explained by Ming Sun et al it is very important pay detailed information about the pedestrian involved accidents to find an acceptable solution to ensure their safety. As a start, summary of accident categories are summarized in the table 1. Table 1: Summary of victim categories reported to Kandy Police Station Severity Gender of the Victim Female Male Grand Total Pedestrian Others Pedestrian Others Fatal 13 2 10 10 35 Grievous 30 19 36 63 148 Grand Total 43 21 46 73 183 According to the table 1, it is evident that pedestrians are highly vulnerable to road traffic accidents in the study area concerned. Further, Table 2 is representation of the same data set in percentages. Table 2 : Percentages of road traffic accidents for Pedestrians and others Pedestrian Others Pedestrian Others Fatal 37.14% 5.71% 28.57% 28.57% 100.00% Grievous 20.27% 12.84% 24.32% 42.57% 100.00% It is important to check whether the accidents are prominent in weekdays or weekends. Table 3 is a summary of accidents summarized according to the day of the week; Table 3 Accident variation according to the day of the week Pedestrian Involved accident Other Accidents Weekdays 70 70 Weekends 19 23 Accident / day (Weekday) 14 14 Accident/ Day (Weekend) 9.5 11.5 According to the dataset considered, there is no difference of accidents per day for weekdays for pedestrian involved and others. But for Weekends, there is a difference of accidents per day on weekends. This may be due to more pedestrians travel during weekdays due to official commitments, schooling etc. Table4. Vehicles involved in Accidents Vehicle Category Victim Category Pedestrian others Motor cycle 31 17 Three wheelers 9 37 Other Vehicles 49 39 Grand Total 89 93 According to the table 4, Motor Cycles and Three wheelers are responsible for most of the Pedestrian involved accidents. Since Motor cycles and three wheelers also categorized as vulnerable road users similar to pedestrians, they should be educated about the importance of sharing facilities with their fellow road users. Table 5 Accident locations Female Male Pedestrian Others Pedestrian Others Fatal Pedestrian crossing 6 0 2 0 others 7 2 8 10 Grievous Pedestrian crossing 9 0 11 1 Others 21 19 25 62 Grand Total 43 21 46 73 as per the information collected and analyzed, pedestrian involved accidents are high at pedestrians crossings compared to other locations. It is a known fact that pedestrian movements, especially across roads are higher at pedestrian crossings, but making such places safer also an important responsibility of the authorities.Pedestrian involved accidents are at a very high percentage. This may be because of low property damage and minor injury relate accidents also considered. Due to various uncertainties and possible underreporting, only fatal accidents and accidents categories as grievous are considered. According to the dataset considered, pedestrians are at high risk on roads compared to other road users. Main culprits in making pedestrian related accidents are other groups of vulnerable road users namely Motorcycle riders and Three-wheeler drivers. Pedestrian related accidents are more prominent during weekends compared to other victim categories.Traffic Accidents, Pedestrian accidents, Vulnerable road users"}
+{"text": "In the original article, there was an error. We have identified four sentences where the words \u201cincrease\u201d and \u201cdecrease\u201d were mistakenly transposed. These four transpositions do not affect the integrity of the data and were the result of an error in translating the sign of the effect sizes from analyses to text. As a result of correcting these four transpositions, two sentences in the discussion contextualizing direction of change required modification.the Abstract sentences #9 and 11, the Results subsectionFKBP5 Change from Before to after MBSR Treatment sentence #1, the Discussionsubsection FKBP5 sentence paragraph 1sentence #1, paragraph 2 sentence #5, andparagraph 3 sentence #2.A correction has been made to Abstract sentence #9: There was a significant time x responder group interaction for methylation in FKBP5 intron 7 bin 2  whereby responders had an increase in methylation and non-responders had a decrease in methylation from before to after treatment in this region.Abstract sentence #11: Increases in FKBP5 methylation after treatment in responders as compared to decreases in non-responders suggest that effective meditation intervention may be associated with stress-related pathways at the molecular level.Results>Methylation of FKBP5>Change from Before to after MBSR Treatment>sentence #1: There was a significant time x responder group interaction for methylation in FKBP5 intron 7 bin 2  whereby responders had an increase in methylation and non-responders had a decrease in methylation from before to after treatment in this region. Discussion>FKBP5>sentence #1: Analyses of FKBP5 revealed increases in intron 7 methylation after treatment in responders while non-responders had decreases in methylation within a specific FKBP5 CpG site of bin 2.Discussion>FKBP5>Paragraph #2>sentence #4: Additionally, consistent with our findings is that despite examining a different regulator region of FKBP5 than our study (exon 1 promoter vs. intron 7 GRE in our study) both of these investigations showed that changes in methylation were related to better symptom response with corresponding measures of increased gene expression identified in the study of PTSD response to exposure therapy (24).Discussion>FKBP5>Paragraph #3>sentence #2: Collectively our findings as well as those of others are consistent with the hypothesis that non-pharmacologic interventions may facilitate stress reduction through the regulation of FKBP5 to feedback on glucocorticoid hyperactivity to reduce stress.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "At the molecular level, the circadian clock is regulated by a time delayed transcriptional-translational feedback loop in which the core proteins interact with each other rhythmically to drive daily biological rhythms. The C-terminal domain of a key clock protein PER2 (PER2c) plays a critically important role in the loop, not only for its interaction with the binding partner CRY proteins but also for the CRY/PER complex\u2019s translocation from the cytosol to the nucleus. Previous circular dichroism (CD) spectroscopic studies have shown that mouse PER2c (mPER2c) is less structured in solution by itself but folded into stable secondary structures upon interaction with mouse CRYs. To understand the stability and folding of human PER2c (hPER2c), we expressed and purified hPER2c. Three oligomerization forms of recombinant hPER2c were identified and thoroughly characterized through a combination of biochemical and biophysical techniques. Different to mPER2c, both thermal unfolding DLS and CD analyses suggested that all forms of hPER2c have very stable secondary structures in solution by themselves with melting temperatures higher than the physiological body temperature, indicating that hPER2c does not require CRY to fold. Furthermore, we examined the effects of EDTA, salt concentration, and a reducing agent on hPER2c folding and oligomerization. The ability of hPER2c forming oligomers reflects the potential role of hPER2c in the assembly of circadian rhythm core protein complexes. Per and Cry genes. After transcription and translation with time delays, PER and CRY proteins accumulate in the cytosol and form complexes that translocate into the nucleus to inhibit the function of the CLOCK/BMAL1 complex. Thus, PERs and CRYs regulate their own transcriptions to generate a negative feedback loop  = \u03b8obs/cnl where, c stands for the concentration of protein in moles, n for the number of residues and l for the path length of the cuvette. Furthermore, thermal denaturation studies were carried out by heating each sample from 5\u00b0C to 90\u00b0C with 5\u00b0C intervals, and the ellipticities were measured using the same parameter settings described above. At each temperature, the sample was equilibrated for 3 minutes prior to the CD measurements. The thermal unfolding profile of each sample was characterized using the mean residue ellipticity minima at 208nm ([\u03b8]208) to determine Tm values by fitting the Boltzmann sigmoid equation to [\u03b8]208 using Origin . The spectrum was further analyzed using BeStSel software to determine secondary structure content [http://pcddb.cryst.bbk.ac.uk/) having accession ID from CD0006240000 to CD0006242000.Far-UV CD measurement of the recombinant hPER2c was conducted using a J-1500 CD spectrophotometer (JASCO) connected to a Peltier temperature controller. Elution fractions of recombinant hPER2c from SEC were concentrated to approximately 15\u03bcM in the same SEC buffer . For each measurement, 400\u03bcl of sample was added in a 0.1cm path length rectangular quartz cuvette . The CD ellipticities  with sequence coverage of 68%. I-TASSER server provided the five best homology models of hPER2c with C-scores (confidence score)  of -2.33Recombinant hPER2c was purified to high purity by affinity chromatography and SEC  to excluThe size of all four forms of hPER2c was further confirmed by TEM . The hPEThe CD spectra of all four hPER2c forms were analyzed by BeStSel in order to determine their secondary structure compositions . The norPrevious studies speculate that the mPER2c domain gains secondary structure upon interacting with mCRY1 . The CD The thermal stability of hPER2c dimer, 20mer, and 40mer were analyzed by DLS . The hyd208) for all three forms of hPER2c were plotted against temperature and fitted with the Boltzmann sigmoid equation using Origin , suggesting that the 20mer is likely to convert into the metastable transition state under physiological temperature. Fitting of ellipticity values of hPER2c from this transition state to a completely unfolded state resulted a second Tm of 87.3\u00b0C (standard error 2.20\u00b0C), indicating that the high stability of the transition state far beyond physiological temperature 2+ ion waresidues , suggestrization .Using multiple analytical techniques including SEC, DLS, and TEM, three different and stable forms of recombinant hPER2c were observed with molecular weights close to 40mer, 20mer, and dimer. These different forms of hPER2c were then further characterized in terms of thermal stability and secondary structure composition using DLS and CD. Thermal stability studies from both CD and DLS show that the hPER2c 40mer has a higher stability than 20mer. Our study here reveals that, under physiological temperature, hPER2c 20mer is likely to adapt to a metastable state . ImportaMouse PER2c was suggested to be largely unstructured and becomes highly ordered once it interacts with CRY1 . In thisIt has been speculated that mammalian PER2 could form a homodimer with the rich coil-coil structure near the C-terminal domain . In our E.coli, where a reducing environment prevents the formation of the disulfide bonds. We speculated that the dimeric hPER2c could be an artificial product introduced during harvesting/purification process in vitro, where the reducing potential wasn\u2019t sufficient to prevent the formation of the disulfide bonds. The dimer of hPER2c with disulfide bonds should not exist in an eukaryotic cytosol which also has a reducing environment. Based on these observations, without the disulfide bonds, the hPER2c will form large oligomers. This is consistent to the observation that the hPER2c 40mer and 20mer are produced in the high concentration in our expression system.To investigate whether disulfide bonds involve in stabilities of all three forms of hPER2c, they were treated with 20mM DTT for up to 36hrs. No effects on 40mer and 20mer were observed. However, both DLS, SEC and TEM showed the appearance of oligomers and aggregated forms after DTT treatment of the hPER2c dimer . The levAs mentioned above, both CD and DLS thermal stability studies show that both 40mer and 20mer have high stability with melting temperatures. Furthermore, our secondary structural analyses show that both hPER2c 40mer and 20mer contained more helices and less strands when compared to the dimeric hPER2c. Overall, hPER2c 40mer has higher percentage of helices and better stability than 20mer. We speculate these two oligomer forms were stably formed with helical coil-coiled structures. Wheel plot analysis of the longest helix in hPER2c modeled structure (residue 1158 to 1175) showed a unique amphipathic property  with oneIt is noteworthy that the oligomerization of hPER2c only results in two different oligomeric forms with distinct molecular weights close to 40mer and 20mer instead of a continuous size distribution. In a recent study on the assembly products of the core circadian clock proteins in mouse liver cell, large cIn conclusion, we identified and characterized three different forms of recombinant hPER2c, 40mer, 20mer, and dimer. Our study provides experimental evidences to clarify the long-standing speculation about whether or not hPER2c indeed requires hCRYs to facilitate folding into a stable structure. Unlike the structure of mPER2c published before, our data clearly shows that hPER2c has very stable secondary structure and does not need to interact with other protein to fold. Despite the high homology between hPER2c and mPER2c, our results indicate significant differences in their thermal stabilities. The ability of hPER2c to form oligomers reflects its potentials in assembly of circadian protein complexes. The molecular mechanism of forming distinct oligomers with discrete sizes is unknown. High resolution structural determination of these forms of hPER2c is needed to fully understand the mechanisms of hPER2c oligomerization and its roles in the circadian rhythm.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(A) DLS profile of hPER2c 20mer measured as the protein was being concentrated. (B) DLS profile of hPER2c dimer measured as the protein was being concentrated.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe effect of the salt on the stability and polymerization of hPER2c 20mer (A) and dimer (B).(TIF)Click here for additional data file.S4 FigThe effect of the DTT and EDTA on the polymerization of hPER2c 40mer (A and B), 20mer(C and D) and dimer (E and F).(TIF)Click here for additional data file.S5 FigAliphatic residues are marked with blue squares; Hydrophilic residues are marked with red diamonds; Positively charged residues with black octagons. This wheel map is generated by EMBOSS pepwheel.(TIF)Click here for additional data file."}
+{"text": "After this article  and its Specifically:In Fig 1B, the pCEFL-GST and pCEFL-GST+MG132 panels appear to report the same image.\u25cbFig 2D VEGF in  appears \u25cbFig 2E VEGF in  appears \u25cbFig 2E \u03b2-actin in  appears Similarities were noted between the following western blot results reported in Fig 2 of  and Fig In response to the concerns about similarities between results reported in  and 3],,3], the PLOS ONE Editors retract this article.In light of the unresolved concerns and in line with the corresponding author\u2019s request, the YX agreed with retraction. The other authors either could not be reached or did not respond directly."}
+{"text": "Focal Cortical Dysplasias (FCD) are localized malformative brain lesions in epilepsy. FCD3a associated with hippocampal sclerosis, affects the superficial cortex and is presumed to have an \u2018acquired\u2019 rather than developmental origin. Precursor cells may arise outside neurogenic zones including cortical layer I. Our aim was to characterise subsets of glial progenitor cells in the superficial cortical layers, known to be involved in gliosis and gliogenesis and that could distinguish FCD3a from other subtypes.Using immunohistochemistry we quantified the density of glial progenitor subsets in superficial cortex layers using markers against PAX6, GFAP, Olig2 and PDGFR\u03b2 and proliferation marker MCM2 in ten FCD3a cases compared to 18 other FCD types and 11 non-FCD controls.Glial progenitor cells types were present in the cortical layer I and II in all FCD groups. GFAP cells frequently expressed PAX6 and significantly higher GFAP/PAX6 than GFAP/MCM2 cell densities were identified in the FCD3a group (p < 0.05). Olig2 cell densities were significantly higher in FCD3b than FCD3a (p = 0.002) and significantly higher GFAP/MCM2 compared to PDGFR\u03b2/MCM2 cell densities were identified in both FCD3b and FCD2 groups. There was no correlation between cell densities and the age of patients at surgery and between cortical regions.Immature and proliferative glial populations across FCD variants reflect reactive cell types and differences may provide insight into underlying pathomechanisms. Higher PAX6 expression in astroglial cells in FCD3a may indicate a switch to astrocytic maturation and enhanced superficial gliosis. Higher Olig2 and GFAP/MCM2 densities in FCD3b may reflect margins of the tumour infiltration zone rather than true cortical dysplasia. FCD3a principally involves the superficial cortical layers with abnormal clustering of neurones in layer II, reduction of neurones in the lower part of layer II/III and accompanying laminar gliosis . This FCThe superficial cortical layers are the last to form during development with complete maturation extending into post-natal period. Experimental studies show deficient Robo1-mediated signalling, that regulates normal neuronal migration to the superficial layers, results in abnormal distribution of neurones in layer II and III during the post-natal stages , reminisWe hypothesised that glial progenitor cell types are integral to both the development and phenotype of FCD3a. Our aim was to evaluate this using paired-box transcription factor (PAX6), which has developmental regulatory roles in differentiation and proliferation of astrocytes , Olig2 t239 surgical cases were selected from the archives of the Epilepsy Society Brain and Tissue Bank which included ten representative cases of FCD3a, eighteen cases with other FCD subtypes , seven cases with ILAE type I HS and no dysplasia and four non-epilepsy controls ; FCD subtypes were determined using current ILAE criteria  Table 1Table 1. Immunohistochemistry was carried out on 5 \u03bcm formalin-fixed paraffin-embedded brain sections using anti-NeuN , anti-MCM2 , anti-PAX6 , anti-GFAP  and anti\u2013PDGFR\u03b2  as described in previous studies . Chromog3Qualitative assessment of immunolabelling with NeuN labelling confirmed the subtype of FCD including the typical clustering of neurones in layer II that defines FCD3a as distinct from other FCDs and normal cortex B-C. PAX6Quantitative evaluation showed variability in labelling between cases . For sin4We observed a range of glial progenitor cell types in cortical layer I and II common to FCD types but with differences that could reflect their underlying aetiology. Our aim was to compare FCD3a which primarily involves the superficial cortex to other \u2018pan-cortical\u2019 FCD and one finding was higher PAX6/GFAP than GFAP/MCM2 co-labelled cells in FCD3a. PAX6 is a transcription factor and stem cell marker, primarily expressed in radial glia during development with roles in cortical patterning . In the PDGFR\u03b2 is known to be expressed in brain parenchymal cells other than pericytes, including NG-2 cells, and show dynamic changes in number, proliferation and distribution following seizures and cortical injury . In the 5Our findings highlight differences in glial regenerative populations between dysplasia types of relevance to their divergent underlying aetiologies and potentially to their pathophysiology , warrantThe authors report no declarations of interest."}
+{"text": "Aldehyde dehydrogenase 1A3 (ALDH1A3) has been implicated in the survival and proliferation of prostate cancer cells.We retrospectively reviewed our patients with advanced disease on adjuvant hormonal therapy after prostatectomy. Time to castration resistance stage was documented. And Immunohistochemistry analysis for ALDH1A3 was performed for those patient samples on tissue microarray. Bioinformatics anslysis was used for RNA sequencing data of both primary prostate cancer and metastatic castration resistance prostate cancer (mCRPC) from online datasets. Crispr-Cas9 was used to knock out ALDH1A3 in prostate cancer luminal cells, and morphologic analysis as well as the Gene Set Enrichment Analysis (GSEA) were facilitated to discover the mechanisms of the resistance phenotype.We found that the patients with ALDH1A3 low expression had shorter time to progression to castration resistance compared with those of higher expression group on adjuvant hormonal therapy after radical prostatectomy. The ALDH1A3 knockout cells gradually acquired resistance to androgen deprivation therapy, a few cells have been found in knockout group showing as that the spindle-like luminal cells in charcoal stripped medium. Furthermore, PI3K pathway activation has been confirmed by Western blot. The PI3K pathway inhibitor BEZ235 has been demonstrated that the acquired ADT resistance by ALDH1A3 down regulation could be rescued by PI3K pathway inhibitor.These results suggested a novel function for ALDH1A3 in development of mCRPC, and indicated PI3K pathway inhibitor has the potential in the treatment of a subgroup of mCRPC patients. Androgen deprivation therapy (ADT) is the standard of care for advanced prostate cancer or progression after localized definitive treatment. However, most patients eventually progress to a condition known as castration-resistant prostate cancer (CRPC), characterized by lack of response to ADT. Despite the several treatment options for this stage of disease, the impact on overall survival is less than optimal and, most importantly, there is no reliable biomarker to predict the response of the treatment or resistance. As a result, no standard guidance is available to optimally sequence approved treatments for individual patients. Since 2005, the next-generation sequencing (NGS) technologies  have madThe aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using nicotinamide adenine dinucleotide (NAD+). The level of ALDHs enzymatic activity has been regarded as a cancer stem cell (CSC) marker and seems to correlate with tumor aggressiveness  which haWe found, surprisingly, that ALDH1A3 was down regulated in metastatic castration resistant prostate cancer from previous sequencing data . CombingThe protocol to generate the tissue microarrays (TMAs) in this cohort has been described in our previous report . We retrThree datasets .The RNA sequencing data were downloaded from SU2C database. The median value of RPKM for ALDH1A3 was used as cut-off value, any sample which is higher than the median value was determined as ALDH1A3escribed . The GenThe human prostate cancer cell line  were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences  and maintained in RPMI medium with 10% fetal bovine serum within a humidified atmosphere containing 5% CO2 at 37\u2009\u00b0C.http://crispr.mit.edu/), targeting the first exon. The sequence of the guide is as follows\u2014ALDH1A3: 1- AGTTATGGCTACCACCAACG; 2-TAGTCTGCGGCGCACCGGCT; green fluorescent protein (GFP): GGCGAGGAGCTGTTCACCG. Then, we ligated the guide to the LentiCrispr-V2 system followed by Sanger sequencing validation [We designed the guide RNA for ALDH1A3 from , Phospho-Akt  and glyceraldehyde 3-phosphate dehydrogenase  were used in Western Blot assay in accordance with the manufacturer\u2019s instructions.p\u2009<\u20090.05 was considered to be statistically significant. All the statistical calculations were performed using GraphPad Prism v6.0 software .Differences in vitro experiment like cell numbers between groups were subjected to Student\u2019s t test. Our earlier work has demonstrated that ALDH1A3 highly expressed in human prostate, which had a strong correlation with primary prostate cancer luminal signature and could be a potential biomarker of AR signaling pathway. Then we moved on to investigate its expression in the castration resistant prostate cancer. We retrospectively reviewed 79 patients with advanced disease who underwent radical prostatectomy followed by adjuvant hormonal therapy in our center. It was indicated that negative expression of ALDH1A3 predicted shorter time progression to castration resistance on adjuvant hormonal therapy . The ALDH1A3 ranking the most significant changes in the whole list confirmed the results. The Gene Set Enrichment Analysis (GSEA) finally correlated ALDH1A3 signature with several biologic event. We found the ALDH1A3 signature had significant positive correlation with ERG signature and prostate cancer luminal signature, respectively   Fig.\u00a0a, b, andion Fig.\u00a0c, d.FigBased on the above results, we aimed to investigate the mechanisms of negative expression of ALDH1A3 in mCRPC samples. We designed small guide RNA targeting the functional exon of ALDH1A3 to knock out this gene on cell level to see the phenotype changes. After validation of the knock out efficiency Fig.\u00a0, we pickBased on the above GSEA results, the ALDH1A3 signature negatively correlated with PI3K-AKT-mTOR signaling pathway. We speculate that the ALDH1A3 loss could activate the PI3K pathway. In order to validate this hypothesis, we did Western blot assay to test the PI3K pathway activation to compare the ALDH1A3 wild type cells and knockout cells. The blotting showed that both in LnCaP and VCaP cells, phospho-AKT had been elevated in ALDH1A3 knockout group Fig.\u00a0, 5. NextOur previous study has demonstrated that ALDH1A3 is specifically expressed in luminal compartment in human prostate epitheliums. From the TCGA data of 333 primary prostate cancer, ALDH1A3 correlated with AR signaling pathway and corresponding luminal signature. It is also suggested that ALDH1A3 has a potential to be a predictor of survival in primary prostate cancer patients. In present study, with our single center follow up database, we performed IHC analysis on tissue microarray for patients with advanced disease upon adjuvant hormonal therapy after radical prostatectomy. The results showed that ALDH1A3 low-expression patients indicated shorter time to progression to castration resistance. The phenotype that we showed at the beginning has implications to understand the mechanisms of this prostate specific gene in the development of cancer progression. From the metastatic prostate cancer samples database, the RNA sequencing data demonstrated that ALDH1A3 was down regulated in mCRPC group compared with the primary prostate cancer. Next-generation sequencing (NGS) analysis has made it possible to reclassify different subtypes in a specific cancer by molecular changes. It indeed could provide benefits for clinical practice in oncology, such as diagnosis, prognosis, and treatment decisions. For example, patients with cancers of unknown primary (CUP), traditionally, are generally assumed to have a poor prognosis with a treatment in cytotoxic chemotherapy guided by histologic features and the pattern of metastatic spread. A new study showed that NGS may provide an opportunity for CUP patients to benefit from individualized therapies according to the targetable genomic alterations identified by tumor molecular profiling . In thatThanks to the RNA sequencing data, we found that the PI3K pathway signature had been highly correlated with ALDH1A3 signature. Then we speculated the PI3K signaling pathway activation might be due to ADT resistance. And we also confirmed this hypothesis by showing the up-regulation of phospho-AKT upon ALDH1A3 knockout. Furthermore, PI3K signaling pathway inhibitor BEZ235 could rescue the ADT resistance following ALDH1A3 knockout. The PI3K/AKT/mTOR pathway is altered in almost 50% of mCRPC through either PTEN inactivation or/and aberrant activation in PIK3CA/B . It\u2019s beIn conclusion, the NGS provides multiple new opportunities and tools to accelerate and facilitate the entire process of drug testing toward accelerated drug positioning and approval for precise and personalized medicine. It also allows researchers to discover some hidden pattern of the complex cancer. Such strategies include the development of inhibitors with a higher potency against their intended target, like ADT and Abiraterone, and the use of combination therapies incorporating inhibitors of parallel or alternative signaling pathways mediating acquired resistance, like the mTOR inhibitor BEZ235 in the treatment of PI3K-AKT-mTOR pathway activation. In this paper, we\u2019ve investigated alterations in the targeted gene leading to ADT resistance to mCRPC. Based on the RNA sequencing and experimental results, we found that PI3K pathway alteration or activation might be the cause of the resistance. We, then, rescued the ADT resistance by PI3K pathway inhibitor-BEZ235. The acquired resistance to ADT therapy by some patients with low level of ALDH1A3 could be overcome by combination therapy with PI3K pathway inhibitor, which will provide a new potential approach to the treatment of mCRPC.Additional file 1 : Table S1: A differential expression gene list as ALDH1A3 signature.Additional file 2 : Figure S1: Original data of western blot (ALDH1A3) in Fig.\u00a0ot ALDH1A in Fig.\u00a0Additional file 3 : Figure S2: Original data of western blot (GAPDH) in Fig.\u00a0Additional file 4 : Figure S3: Original data of western blot (ALDH1A3) in Fig.\u00a0Additional file 5 : Figure S4: Original data of western blot (p-AKT) in Fig.\u00a0Additional file 6 : Figure S5: Original data of western blot (GAPDH) in Fig."}
+{"text": "CHK2), in which gene mutations convey up to twofold higher risk for breast cancer but do not increase lung cancer risk. We have investigated the role of CHK2 and the related kinase checkpoint protein 1 (CHK1) in cell cycle regulation in primary breast and lung primary epithelial cells. At the molecular level, CHK1 activity was higher in lung cells, whereas CHK2 was more active in breast cells. Inhibition of CHK1 profoundly disrupted the cell cycle profile in both lung and breast cells, whereas breast cells were more sensitive toward inhibition of CHK2. Finally, we provide evidence that breast cells require CHK2 to induce a G2\u2013M cell cycle arrest in response of DNA damage, whereas lung cells can partially compensate for the loss of CHK2. Our results provide an explanation as to why CHK2 germline mutations predispose for breast cancer but not for lung cancer.Cancer is a life-threatening disease that affects one in three people. Although most cases are sporadic, cancer risk can be increased by genetic factors. It remains unknown why certain genes predispose for specific forms of cancer only, such as checkpoint protein 2 ( BRCA1) and 2 (BRCA2) strongly predispose for breast and ovarian cancer, whereas germline mutations in the DNA repair genes MSH2, MSH6, and MLH1 are associated with hereditary nonpolyposis colon cancer.Cancer is a disease that can arise in virtually any tissue. Most cases are the result of mutations that occur by chance. However, germline variants can affect cancer risk too. Many cancer predisposition genes (CPGs) have key roles in DNA repair, cell cycle control, and cell survival pathways, which are necessary to maintain genomic integrity. Surprisingly, despite their role in basic cellular programs, CPGs appear to affect cancer development across tissues differently. For instance, mutations of the DNA repair genes breast cancer protein 1 -checkpoint protein 2 (CHK2) pathway, which is activated in response to DNA DSBs, is more active in primary breast than in lung cells4. Interestingly, both ATM and CHK2 germline mutations predispose for breast cancer6.Why mutations in DNA repair genes predispose for specific cancer types is an outstanding mystery. Since many breast CPGs are involved in the repair of DNA double-strand breaks (DSBs)BRCA1, which regulates many processes, confer a 10\u201320% higher risk than BRCA2 mutations, which functions exclusively in DSB repair8. In addition, BRCA1 mutation carriers develop breast cancer at a younger age than BRCA2 mutation carriers9. Both BRCA1 and CHK2 play roles in cell cycle control11. Since a dysregulated cell cycle can lead to genetic errors and genomic instability, uncontrolled cell division is one of the hallmarks of cancer12. It is therefore possible that mutations in BRCA1 and CHK2 contribute to cancer development by deregulation of the cell cycle.However, DNA repair proteins may contribute to breast cancer risk in additional ways. For instance, mutations in 13. Second, several breast CPGs are known, whereas the genetic component of lung tumorigenesis appears to be very small14. We observed that breast and lung cells have a different cell cycle distribution, which is reflected in differential CHK1 and CHK2 activity. We provide evidence that breast cells depend on CHK2 to induce a G2 cell cycle arrest in response to DSBs, whereas lung cells appear to have compensatory mechanisms. These findings may help to explain why CHK2 germline mutations predispose for breast cancer but not for lung cancer.To understand differences in tissue-specific cancer risk, we focused on primary breast and lung cells for two reasons. First, breast and lung cancer are among the most common kinds of cancer, suggesting that they have a high cancer risk4. Interestingly, CHK1 and CHK2 also play roles in cell cycle regulation: CHK1 is required for checkpoints throughout the cell cycle, whereas CHK2 is mostly active during the G1 phase. We therefore set out to compare the cell cycle profile of breast and lung primary cells.We previously observed that the functionally related CHK1 and CHK2 play tissue-specific roles in the DNA damage response in primary breast and lung cells4. Consistent with the slow population doubling times, the majority of these cells were in G0\u2013G1 phase P21 Fig. 16. In aging Fig. .These results suggest that the differential activity of CHK1 and CHK2 in primary breast and lung cells affects cell cycle regulation.17.CHK1 and CHK2 are known to play key roles in linking DNA damage signaling to cell cycle control. We wondered whether the role of CHK1 and CHK2 in inducing a DNA damage cell cycle checkpoint was different in both cell types. To test this, we made use of the DNA DSB-inducing agent doxorubicin, which is known to induce a G2\u2013M arrest4 and activate CHK1 and CHK2. In both cell types, 16-h exposure to doxorubicin increased the G2\u2013M fraction . Since the CDC2/Cyclin B complex is a better substrate for tyrosine kinase Wee129 than CDC2 alone, we hypothesize that the drop in Cyclin B levels may be responsible for this decrease. Thus the initial inactivation of CDC2 triggers an early G2\u2013M arrest, whereas the drop in Cyclin B1 levels at a later stage may maintain G2\u2013M phase arrest. The mechanism responsible for the loss of Cyclin B1 may be the upregulation of P5318.Interestingly, we previously observed that CHK1 is preferentially upregulated after DNA damage in lung cells, whereas CHK2 activation was stronger in breast cells after DNA DSBs30. Concurringly, sporadic mutations of CHK1 and CHK2 have been found in most types of cancer. In addition, germline mutations of CHK2 appear to predispose for certain types of cancer. People who harbor truncating CHK2 mutations  have an approximately twofold increased risk of developing breast cancer32. Carriers also have an increased likelihood to develop prostate34 and colon cancer37, but, intriguingly, no increased risk of lung cancer39.Cell cycle checkpoints are important to prevent transmission of damaged DNA to the daughter cells, and hence CHK1 and CHK2 can protect cells against cancerCHK2 predisposes for breast cancer but not for lung cancer. Since mice harboring a CHK2*1100delC mutation did not show the tissue-specific bias observed in humans40, a better disease model is needed to understand CHK2-mediated cancer predisposition. The comparative analysis of healthy primary epithelial cells may provide further insights into the relation between loss of CHK2 and tissue-specific cancer development.Considering the pivotal importance of cell cycle arrests to prevent genomic instability, our data may provide an explanation for why loss of Supplementary Information"}
+{"text": "Hair follicles cycle through periods of growth (anagen), regression (catagen), rest (telogen), and release (exogen). Telogen is further divided into refractory and competent telogen based on expression of bone morphogenetic protein 4 (BMP4) and wingless-related MMTV integration site 7A (WNT7A). During refractory telogen hair follicle stem cells (HFSC) are inhibited. Retinoic acid synthesis proteins localized to the hair follicle and this localization pattern changed throughout the hair cycle. In addition, excess retinyl esters arrested hair follicles in telogen. The purpose of this study was to further define these hair cycle changes. BMP4 and WNT7A expression was also used to distinguish refractory from competent telogen in C57BL/6J mice fed different levels of retinyl esters from two previous studies. These two studies produced opposite results; and differed in the amount of retinyl esters the dams consumed and the age of the mice when the different diet began. There were a greater percentage of hair follicles in refractory telogen both when mice were bred on an unpurified diet containing copious levels of retinyl esters (study 1) and consumed excess levels of retinyl esters starting at 12 weeks of age, as well as when mice were bred on a purified diet containing adequate levels of retinyl esters (study 2) and remained on this diet at 6 weeks of age. WNT7A expression was consistent with these results. Next, the localization of vitamin A metabolism proteins in the two stages of telogen was examined. Keratin 6 (KRT6) and cellular retinoic acid binding protein 2 (CRABP2) localized almost exclusively to refractory telogen hair follicles in study 1. However, KRT6 and CRABP2 localized to both competent and refractory telogen hair follicles in mice fed adequate and high levels of retinyl esters in study 2. In mice bred and fed an unpurified diet retinol dehydrogenase SDR16C5, retinal dehydrogenase 2 (ALDH1A2), and cytochrome p450 26B1 (CYP26B1), enzymes and proteins involved in RA metabolism, localized to BMP4 positive refractory telogen hair follicles. This suggests that vitamin A may contribute to the inhibition of HFSC during refractory telogen in a dose dependent manner. The hair follicle has a self-cycling ability. It goes through a regeneration phase (anagen), a degenerative phase (catagen) involving the apoptosis and loss of the lower two-thirds of the hair follicle, a resting phase (telogen), and a release phase (exogen) . The traRetinoids is a general term for both synthetic and natural forms of vitamin A. The dietary form of vitamin A is retinyl esters. Retinyl esters are important for healthy hair ; since bThe active form of vitamin A is retinoic acid (RA). RA is synthesized at or near the site of action , by a feDgat1tm1Far null mice also altered the hair cycle by both lengthening the first anagen and shortening the second telogen  reduced zig-zag hairs, but did not block hair development. Recently, reduced retinal synthesis in Del(4Sdr16c5-Sdr16c6)1Nyk double null mice altered the hair cycle (In a previous study in C57BL/6J (B6) mice, retinyl esters dose dependently altered the hair cycle . Feeding telogen . These eethality . Conditiir cycle . These mir cycle . In contThe Jackson Laboratory  Institutional Animal Care and Use Committee approved all procedures. C57BL/6J  mice  were used.B6 Dams and female pups were fed the NIH 31 diet  throughout this study. Only females were used because they fight less, which reduces wounds. Wounds by themselves will initiate anagen, which would interfere with this study . Mice wen = 8), 28 (n = 9), or 56 (n = 10) IU retinyl acetate/g diet  for 16 weeks  until 12 weeks of age, and then switched to an American Institute of Nutrition (AIN)93 maintenance diet containing 4 (16 weeks  (Everts 16 weeks . All mic16 weeks . In addi16 weeks . The fir16 weeks  and reduull mice .n = 7), 28 (n = 10), or 56 (n = 6) IU retinyl acetate/g diet  for 12 weeks . Second generation female pups were used in this study . Six-wee12 weeks . At 6 weImmunohistochemistry was performed in serial sections as described previously . PrimaryBone morphogenetic protein 4 positive and negative telogen hair germs and dermal cells were counted from mice fed various levels of vitamin A in studies 1 and 2. The percent of hair follicles in refractory telogen was calculated in two steps. First, the number of BMP4 positive telogen hair germs was divided by the total number of telogen hair follicles to get the fraction of telogen hair germs in refractory telogen. Second, this fraction was multiplied by the percentage of telogen follicles calculated in the original diet studies  because Co-immunofluorescence was performed using a two-antibody co-localization system in frozen dorsal skin sections as previously described . PrimaryImmunohistochemistry was first performed with antibodies against KRT6 and CRABP2 using AEC+ (red) as described above with additional blocking steps using the streptavidin, biotin blocking kit . IHC was then performed with the Vector mouse anti-BMP4 antibody using the Mouse on Mouse ImmPRESS Peroxidase kit with some modifications . First, we doubled the concentration of blocking reagent and incubated the slides for 2 h. Second, we diluted the ImmPRESS reagent 1:1 in the provided 2.5% normal horse serum. Slides were then treated with DAB Eq (brown) for 10 min, counterstained and mounted as described above. The number of hair follicles with positive KRT6 or CRABP2 plus or minus dermal BMP4 was counted. The percent of positive KRT6 or CRABP2 hair follicles with dermal BMP4 was then calculated separately from the percent of KRT6 or CRABP2 hair follicles without dermal BMP4. An average of 26 hair follicles (range of 2\u201354) were scored per mouse. Four to seven mice per diet were analyzed. Images were taken with the Nikon Eclipse i80 microscope with the DS-Ri2 camera . Images were sized, cropped, composite figures were created, and whole images were adjusted for brightness, contrast, and color balance in Adobe Photoshop CS3. Unaltered images are in post-hoc test when significant treatments effects were seen. When there was a significant interaction between study and diet a one-way ANOVA was performed on the interaction variable followed by Tukey post-hoc test. When equal variances were not seen, each study was analyzed by Kruskal\u2013Wallis followed by Mann\u2013Whitney U test. P value < 0.05 is defined as statistical significance.Equal variance was first confirmed using Levene\u2019s test of equality of error variances using SPSS version 24 . Comparison among experimental groups was analyzed by univariate analysis of variance using a 2 \u00d7 3 full factorial model followed by Tukey Mice from study 1  on the 5Next, we attempted to determine the localization pattern of vitamin A metabolism proteins during telogen using BMP4 to mark refractory telogen. Dorsal skin was collected from B6 mice fed an unpurified diet at 70\u2013101 days of age and IHC performed in serial sections with antibodies against BMP4 (Abcam), KRT6, SDR16C5, RDH10, ALDH1A2, CRABP2, and CYP26B1. Using the Abcam antibody, all proteins localized to BMP4 positive telogen hair follicles , 3. SDR1This study was done to better define the impact retinyl esters have on the hair cycle. In mice bred on an unpurified diet, excess  levels of retinyl esters lead to a greater percentage of hair follicles in refractory telogen. Regardless of retinyl ester level fed, KRT6 and CRABP2 localized to the inner bulge cells almost exclusively in refractory telogen in these mice. SDR16C5, ALDH1A2, and CYP26B1 also localized to the hair follicle at greater intensities during refractory telogen in mice bred and fed an unpurified diet. In contrast, when mice were bred on a purified diet containing recommended  levels of retinyl esters the percent of hair follicles in refractory telogen decreased as retinyl ester levels increased. In addition, KRT6 and CRABP2 localized to the inner bulge cells during both competent and refractory telogen in adult mice fed recommended (4IU) and high (28IU) levels of retinyl esters. These data suggest that vitamin A may regulate HFSC in a dose and time dependent manner.tm1BlhKrt1-15crePGR)22Cot/J/Foxc1 null mice identified increased Aldh1a2 expression. Assay for transposase accessible chromatin then sequencing (ATAC-seq) analysis of the promoters of differentially expressed genes revealed a SMAD binding motif in the promoter of Aldh1a2, but not NFATC1 or FOXC1 binding sites. This suggests that BMP signaling may increase ALDH1A2 and RA synthesis during refractory telogen to contribute to HFSC quiescence.Bone morphogenetic protein 2, BMP4, and BMP6 inhibit HFSCs during refractory telogen . BMP2 anCyp26b1tm1Hh null mice decreased Lhx2 at embryonic day E16.5 and E18.5 along with altering several other HFSC regulatory genes and blocked hair follicle development (tm2(cre)Wrst;Cyp26b1tm1HhEn1 null mice. The finding that CYP26B1 localized to the HFSC and inner bulge in addition to the dermis in this study suggests that reduced RA may be critical within the HFSC and/or inner bulge cells but not the dermal papilla. CRABP2 shuttles RA to both RARs and CYP26B1 (Del(4Sdr16c5-Sdr16c6)1Nyk double null mice and excess RA in Dgat1tm1Far null mice; and an increase of hair follicles in refractory telogen following excess retinyl esters in study 1, reduced retinyl esters in study 2, and pharmacological doses of synthetic retinoids in the clinic.Retinoic acid may also regulate HFSC quiescence. Excess RA in the elopment . However CYP26B1 . This suRetinoic acid synthesis proteins localizing to the KRT6 positive inner bulge cells during telogen is similar to what was seen in the inner cell layer and companion layer during anagen . ALDH1A3Cyp26b1 expression decreased in these inner bulge cells in late exogen verses early exogen 22Cot/J, Foxc1 null mice and FGF18 injections revealed opposite effects based on the stage of the hair cycle the follicle was in at the time , arresting hair follicles in refractory telogen is a good thing. This is because CCCA in mice only occurs when hair follicles are in anagen . No miceThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Jackson Laboratory  Institutional Animal Care and Use Committee.HE and JS conceptualized, acquired funding, provided resources, provided supervision, and reviewed and edited the manuscript. HE administered the project and curated the data. LS, CV, EH, and HE performed the analyses. LS wrote the original draft. CV wrote the first draft of the revision. NK provided resources. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "In the article , there w\"The cohort was validated against a set of nearly 60,000 laboratory records matched manually by a team of research assistants who found that the approach had 6.3% overmatching (laboratories that were matched should not have been) and 1.4% undermatching (laboratories that should have been matched were not) [22].\"The last sentence reads:\"The cohort was validated against a set of nearly 60,000 laboratory records matched manually by a team of research assistants who found that the approach had 1.4% overmatching (laboratory records that were matched should not have been) and 6.3% undermatching (laboratory records that should have been matched were not) [22].\"The last sentence should have read:"}
+{"text": "Transcription factor 4 (TCF4) intron 3 is the main cause of Fuchs\u2019 endothelial corneal dystrophy (FECD) and may confer an increased risk of developing bipolar disorder (BD). Usage of alternative 5\u2032 exons for transcribing the human TCF4 gene results in numerous TCF4 transcripts which encode for at least 18 N-terminally different protein isoforms that vary in their function and transactivation capability. Here we studied the TCF4 region containing the CTG TNR and characterized the transcription initiation sites of the nearby downstream 5\u2032 exons 4a, 4b and 4c. We demonstrate that these exons are linked to alternative promoters and show that the CTG TNR expansion decreases the activity of the nearby downstream TCF4 promoters in primary cultured neurons. We confirm this finding using two RNA sequencing (RNA-seq) datasets of corneal endothelium from FECD patients with expanded CTG TNR in the TCF4 gene. Furthermore, we report an increase in the expression of various other TCF4 transcripts in FECD, possibly indicating a compensatory mechanism. We conclude that the CTG TNR affects TCF4 expression in a transcript-specific manner both in neurons and in the cornea.The CTG trinucleotide repeat (TNR) expansion in  TCF4 is expressed in almost every tissue type in human5. In the brain TCF4 expression peaks during late embryonic development and continues at a relatively high level during postnatal development7. Notably, transcription from TCF4 gene can begin at multiple mutually exclusive 5\u2032 exons leading to transcripts with varying composition of functional protein domains which modulate the ability of TCF4 to regulate transcription8.Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor that plays a vital role in the development of the nervous and immune system11. In addition, mutations in the gene regions included only in the longer isoforms of TCF4 have been associated with intellectual disability12. Alterations in TCF4 expression levels have been described in patients with depression13. An expansion of a CTG TNR, located in an alternative promoter region between TCF4 exons 3 and 4 (known as CTG 18.1), upstream of TCF4 5\u2032 exons 4a, 4b and 4c causes Fuchs' endothelial corneal dystrophy (FECD) and has been linked to Bipolar disorder (BD)15.TCF4 gene is implicated in susceptibility to schizophrenia, and mutations in TCF4 cause Pitt-Hopkins syndrome, a rare developmental disorder characterized by severe motor and mental retardation16. Out of the many genetic mutations associated with FECD the CTG TNR expansion in TCF4 is thought to be one of the major factors in the development of the disease, as the presence of a TCF4 allele with 50 or more CTG TNR-s confers an increased risk of developing the disease17. BD is a psychiatric disorder that affects up to 1% of the global population, causing severe mood alterations in affected patients18. It has been shown that a TCF4 CTG TNR expansion of over 40 CTG TNR-s is frequent in a subset of patients with a severe type of BD and that the repeat expansion in TCF4 may increase vulnerability to BD15.FECD is an ocular disorder associated with corneal edema and vision disruption having a varying prevalence between different populations affecting about 4% of people over 40 in the United StatesTCF4 CTG TNR expansion and the mRNA levels of TCF4 transcripts spanning the TCF4 CTG TNR region have produced contradictory results. A study by Foja et al.19 reported that TCF4 CTG TNR expansion is connected with a reduction in the levels of TCF4 transcripts beginning in the proximity of the CTG TNR, whereas a study by Okumura et al.20 has reported that TCF4 CTG TNR expansion is connected with an increase in overall TCF4 levels and in the levels of TCF4 transcripts beginning in proximity of the CTG TNR. Two other studies22 have reported no effect of the CTG TNR expansion on total TCF4 mRNA levels.Studies on the connection between the TCF4 CTG TNR expansion affects the levels of TCF4 transcripts and total TCF4 levels. Here, we hypothesized that the TCF4 CTG TNR region contains functional promoters that regulate the transcription of nearby 5\u2032 exons and that the activity of these promoters is altered by the length of TCF4 CTG TNR expansion. For this, we first characterized the TCF4 alternative promoter region containing the CTG TNR by identifying transcription start sites (TSS) and describing the splicing of nearby 5\u2032 exons 4a, 4b, and 4c. We then used luciferase reporter assay to investigate whether the CTG TNR expansion could influence TCF4 expression in primary neurons by affecting the ability of surrounding regulatory regions to promote transcription. Furthermore, RNA-seq data from corneal tissue of FECD patients with an expanded TCF4 CTG TNR was analyzed to determine the expression levels of TCF4 transcripts beginning both proximal and distal to the CTG TNR. Collectively, our results demonstrate that the CTG TNR expansion differentially modulates the activity of TCF4 promoters.It is currently unknown whether the TCF4 5\u2032 exons 4a, 4b and 4c, which are located between internal exons 3 and 4  spanning the TCF4 CTG TNR region from just downstream of exon 3 into 5\u2032 exon 4a and a longer sequence (TCF4 p4abc) spanning the entire TCF4 CTG TNR from just downstream of exon 3 to inside exon 4 . The luciferase reporter assay revealed that an extended CTG TNR with a length of 54, 67/70 or 144 repeats significantly reduced the activity of the promoter for both TCF4 p4a and p4abc . Our analysis revealed that FECD patients with an expanded CTG TNR displayed reduced levels of transcripts containing 8. The major TCF4 transcripts comprising of 5\u2032 exons 3b, 3c and 3d encode for isoform TCF4-B when spliced to internal exon 3; transcripts with exons 3b and 3d encode for isoform TCF4-C when spliced to exon 4; transcripts with exons 8a, 8bII and 8cII encode for isoform TCF4-D when spliced to exon 8; transcripts with exons 10a, 10b and 10c spliced to exon 10 encode for isoforms TCF4-A, TCF4-I and TCF4-H, respectively  and Fuchs\u2019 dystrophy (>\u200950)15. Soliman et al. has shown that the severity of FECD correlates with the length of the CTG TNR in TCF4 as patients with a CTG TNR expansion exhibited a more severe form of FECD, but the mechanism underlying this phenotype remains unknown30. It is important to note that our determination of TSS-s by 5\u2032 RACE and reporter experiments were done using human cerebellar RNA and rat cultured cortical neurons, respectively. Therefore, it would be interesting to conduct similar experiments in human corneal endothelial cells. This would help to translate our findings between different cell types and further validate the effect of the CTG TNR expansion on transcription also in FECD patients.Previous studies have indicated that the CTG TNR expansion in intron 3 of 28 revealed that the levels of TCF4 transcripts containing alternative 5\u2032 exons 4aI and 4aIII were reduced in the corneal endothelial cells of FECD patients with an expanded CTG TNR. These results support our findings that the TCF4 CTG TNR expansion reduces the activity of proximal downstream promoters linked to these 5\u2032 exons also in human corneal endothelium. Interestingly, an increase in the levels of TCF4 transcripts encoded by downstream alternative 5\u2032 exons distal to the CTG TNR was also noted, which may indicate a compensatory mechanism to rescue the levels of TCF4 protein arising from the deficit of transcripts encoding TCF4-C. This compensation phenomenon needs to be considered when studying TCF4 expression levels in FECD and other diseases connected with the TCF4 intronic CTG TNR and could explain why different research groups have published contradictory results concerning changes in TCF4 levels when studying FECD22. Our results indicate that the levels of TCF4 transcripts change bidirectionally in response to an expanded CTG TNR\u2014transcripts beginning near the repeat region decline just as Foja et al. reported19 while certain transcripts beginning downstream of the repeat region increase as reported here, and mask the decrease of long TCF4 transcripts. As the expression of different TCF4 transcripts decline and rise simultaneously the overall TCF4 levels may not change significantly in FECD as has been reported by Mootha et al.21 and O\u0142dak et al.22. In contrast, Okumura et al.20 found an increase in total TCF4 expression levels which is also evident in our RNA-seq analysis as we saw a slight rise in TCF4 expression when measuring the expression of internal exons present in all TCF4 transcripts (exons 10\u201321). Our results illustrate the importance of the exact transcript measured when studying TCF4 expression levels. The original RNA-seq study by Chu and others concluded that the TCF4 CTG TNR expansion increases the stability and thus the amount of expanded CTG repeat-including intronic RNAs in the corneal endothelium and causes comprehensive changes in splicing. No alterations in the overall expression of mature TCF4 mRNA was noted28. The study by Nikitina et al. was a data article and no conclusions were made27.Detailed analysis of previously published FECD RNA-seq datasets31. This can cause an accumulation of RNA polymerases in the CTG TNR and dissociation of the polymerase, leading to a decrease of full-length TCF4 transcripts beginning from upstream of the CTG repeat. An increase in the expression of 5\u00b4 exons spliced to exon 3 may be due to preferential splicing of transcripts to the exon before the CTG TNR (exon 3), as changes in splicing have been described before in diseases associated with repeat expansion31.Interestingly, we also detected an increase in the expression of 5\u2032 exons spliced to exon 3 encoding for TCF4-B in patients with FECD, showing that almost all the TCF4 promoters far upstream from the CTG TNR had increased activity due to the repeat expansion. However, the increase in upstream promoter activity did not reflect in the levels of transcripts containing exons 3 and 4. It is plausible that the CTG repeat expansion could regulate transcriptional elongation of RNA polymerase by slowing down the polymerase in the CTG TNR region8. Currently FECD research focuses mainly on the CTG TNR and missplicing of longer TCF4 transcripts in FECD17, and little research has been done to analyze the expression of all the TCF4 transcripts in FECD. Our detailed analysis provides new insight into FECD as we show that the CTG TNR directly modulates the expression of TCF4 which may be among the underlying causes for the development of the disease. Since TCF4 mRNAs detected in the present study are expressed virtually in all tissues, with high levels in the fetal and postnatal brain4, there may also be a similar correlation between the CTG TNR length and the expression levels of TCF4 transcripts in vivo in the brain which could predispose development of BD. However, it should be noted that the link between the CTG TNR expansion in TCF4 and BD has only been shown once and has not been reported by newer studies.We have previously shown that TCF4 protein isoforms can be divided into longer and shorter isoforms which vary in their function and transactivation capabilityn TNR containing TCF4 mRNAs are the cause of Fuchs' corneal dystrophy33. According to this mechanism, the TNR-carrying RNAs cluster RNA binding proteins, interfering with the splicing of various mRNAs. Of note, antisense therapy using Fuchs' dystrophy ex vivo cell models leads to inhibition of RNA foci and mis-splicing in\u00a0Fuchs' dystrophy35. Since the CTG TNR is located in the intron between exons 3 and 4, this repeat\u00a0is not included in the fully mature TCF4 mRNA4, but the CTG TNR is still included in the pre-mRNA of transcripts initiated at the upstream promoters .Strong evidence has also been provided in support of a mechanism in which the toxic (CUG)29 and unstable TNR-s may occur in both coding and noncoding regions, including promoters, introns and untranslated regions (UTR) of genes36. Among noncoding TNR-s, one of the most studied is the TNR repeat (CGG) located in the 5\u2032 UTR of Fragile X Mental Retardation (FMR1) gene. This TNR causes hypermethylation and silencing or increases in the expression level of the gene, depending on TNR length37. TNR diseases with TNR in the promoter region of the affected gene have been less studied. Recently, an intronic polymorphic CGG repeat in a conserved alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene, was shown to lead to hypermethylation of the promoter and transcriptional silencing of AFF3 expression in the brain38. However, the effect of TNR on promoter activity using transient expression analysis of promoters linked to TNR was not studied. Research on Friedreich ataxia, which is caused by an expansion of the intronic TNR (GAA) in the FXN gene, has revealed reduced expression of the gene in patient derived cell lines39. A hexamer repeat expansion (GGGGCC) located in the 5\u2032 regulatory region of the C9ORF72 gene, causing hereditary amyotrophic lateral sclerosis, has been shown to reduce the ability of the surrounding region to promote the expression of a reporter protein in human kidney and neuroblastoma cell lines40. Overall, these results indicate that expansion of TNR can alter the expression of the nearby genes. This is in agreement with our results showing that the expression levels of different TCF4 transcripts are altered in FECD due to the CTG TNR expansion.Repeat expansions have been associated with more than 40 diseasesTCF4 transcripts in FECD has displayed varying results. Analyzing only total TCF4 levels or levels of certain TCF4 transcripts can produce misleading results due to the complexity of the TCF4 gene and its regulation. The current study shows that the TCF4 CTG trinucleotide repeat expansion modulates the activity of nearby TCF4 promoters in a length dependent manner\u2014an expanded CTG TNR causes reduction in promoter activity. Analysis of RNA-seq datasets revealed that the expression levels of the many TCF4 transcripts are increased or decreased simultaneously in the cornea of FECD patients. Further work is needed to elucidate the exact mechanism how this repeat region affects TCF4 transcription and whether the changed TCF4 levels contribute to the development of FECD and BD.Taken together, our results help to explain why previous research on the levels of Human postmortem tissues were used to obtain DNA and RNA samples. All protocols using human tissue samples were approved by\u00a0Tallinn Committee for Medical Studies, National Institute for Health Development (Permit Number 402). All experiments were performed in accordance with relevant guidelines and regulations.TCF4 gene fragments were screened from human DNA samples for the TCF4 CTG TNR length and fragments with the desired CTG TNR length were amplified by PCR from 20\u00a0ng of genomic DNA in a 20\u00a0\u03bcl mixture using 0.4 units of Phusion Hot Start II (Thermo Scientific) and primer p4a_p4abc_F paired with the p4abc_R or p4a_R primer (Supplementary Table TCF4 p4abc) and the shorter (TCF4 p4a) sequence of the TCF4 gene  were acquired from human genomic DNA by PCR. A sixth synthetic DNA segment with 144 CTG repeats was ordered from GenScript. All the generated constructs were verified by sequencing as the length of TNR tended to be unstable in bacteria when producing plasmids . All experiments were performed in accordance with the relevant guidelines and regulations.41. Neurons grown 6\u00a0days in vitro were transfected with 180\u00a0ng firefly reporter construct and 20\u00a0ng pGL4.83[hRlucP/PGK1/Puro] as described previously4 for 4\u00a0h on a plate shaker using Lipofectamine 2000  with a reagent to DNA ratio 3:1. Two days after transfection neurons were lysed in 50\u00a0\u03bcl Passive Lysis Buffer (Promega) and luciferase reporter assay was performed using the Dual-Glo Luciferase Assay System (Promega) according to manufacturer\u2019s protocol. Luciferase signals were measured using the GENios Pro microtiter plate reader (Tecan). For analysis, the signals were first normalized to the signal of the Renilla luciferase and then normalized to the respective ratio in cells transfected with the 11 repeat CTG construct. One-way repeated-measures analysis of variance (ANOVA) with Greenhouse\u2013Geisser correction followed by Dunnett\u2019s post hoc test was used to determine the statistical significance compared to the luciferase signals from the 11 repeat CTG construct group.Prenatal rat cortical neurons were cultured as described previouslyTotal RNA from post-mortem adult human cerebellum was treated with Turbo DNase (Thermo Fisher Scientific) according to the supplier\u2019s protocol. 5\u2032 RACE analysis was carried out on human cerebellar RNA using the GeneRacer Kit (Thermo Fisher Scientific) according to manufacturer\u2019s protocol with primers outlined in Supplementary Tables 20 and a random hexamer primer mixture  according to the manufacturer\u2019s protocol. A negative control (\u2212\u2009RT) was also included where Superscript III reverse transcriptase was not added. After cDNA synthesis, PCR was performed in 20\u00a0\u03bcl using 3 units of Hot FirePol (Solis Biodyne) and primers listed in Supplementary Table For RT-PCR, cDNA was synthesized from human cerebellar RNA using 100 units of SuperScript III reverse transcriptase (Thermo Fisher Scientific) with oligo(dT)42 was used to locate potential TCF4 TSS-s. Both predicted TSS-s  and total counts of CAGE reads for the reverse strand (encoding for TCF4) were visualized in UCSC Genome Browser together with the EST-s from GenBank (accessed at 10.07.2020) in the area surrounding the TCF4 CTG TNR region . The FANTOM5 data can be accessed at https://fantom.gsc.riken.jp/5/datahub/hg19/reads/ctssTotalCounts.rev.bw.Cap Analysis of Gene Expression (CAGE) data from the Functional Annotation of the Mammalian Genome project phase 5 (FANTOM5)27 and SRP23860928) using prefetch tool (version 2.10.0) from the SRA toolkit. Reads in fastq format were extracted using fasterq-dump. Adapter and quality trimming was done using BBDuk (part of bbmap version 38.79) using the following parameters: ktrim\u2009=\u2009r k\u2009=\u200923 mink\u2009=\u200911 hdist\u2009=\u20091 tbo qtrim\u2009=\u2009lr trimq\u2009=\u200910 minlen\u2009=\u2009100 (minlen\u2009=\u200985 for data from PRJNA524323). Reads were mapped to hg19 genome  using STAR aligner (version 2.7.3a) with default parameters. To increase sensitivity for unannotated splice junctions, splice junctions obtained from the 1st pass were combined (per dataset) and filtered as follows: junctions on mitochondrial DNA and non-canonical intron motifs were removed; only junctions supported by at least 6 reads in the whole dataset were kept. The filtered junctions were added to the 2nd pass mapping using STAR. Intron-spanning reads were quantified using FeatureCounts (version 2.0.0) with the following parameters: -p -B -C -s 2 -J. To count reads from TCF4 extended exons (exons 4c and 7bII), reads crossing a region 2\u00a0bp 5\u2032 from the internal exon  were quantified using FeatureCounts and a custom-made saf file. Splice junctions in the TCF4 region showing less than 4 reads for the whole dataset were discarded, the rest of the splice junctions associated with TCF4 were manually curated and annotated according to Sepp et al.4. A custom R script was used to quantify the expression of different TCF4 splice variants. Reads crossing the indicated splice junctions were normalized using the number of all splice-junction crossing reads in the respective samples. Then, data summed by the Exon column (see Supplementary Table TCF4 internal exons and transcripts encoding different TCF4 protein isoforms. The annotated splice junction table for quantifying different TCF4 splice sites and transcripts encoding different isoforms is shown in Supplementary Table Raw RNA-seq data from corneal endothelium of FECD patients and controls (see Supplementary Table Supplementary Information 1.Supplementary Table S4."}
+{"text": "The durum wheat line DT696 is a source of moderate Fusarium head blight (FHB) resistance. Previous analysis using a bi-parental population identified two FHB resistance quantitative trait loci (QTL) on chromosome 5A: 5A1 was co-located with a plant height QTL, and 5A2 with a major maturity QTL. A Genome-Wide Association Study (GWAS) of DT696 derivative lines from 72 crosses based on multi-environment FHB resistance, plant height, and maturity phenotypic data was conducted to improve the mapping resolution and further elucidate the genetic relationship of height and maturity with FHB resistance. The Global Tetraploid Wheat Collection (GTWC) was exploited to identify durum wheat lines with DT696 allele and additional recombination events. The 5A2 QTL was confirmed in the derivatives, suggesting the expression stability of the 5A2 QTL in various genetic backgrounds. The GWAS led to an improved mapping resolution rendering the 5A2 interval 10 Mbp shorter than the bi-parental QTL mapping interval. Haplotype analysis using SNPs within the 5A2 QTL applied to the GTWC identified novel haplotypes and recombination breakpoints, which could be exploited for further improvement of the mapping resolution. This study suggested that GWAS of derivative breeding lines is a credible strategy for improving mapping resolution. Fusarium graminearum Schwabe produces mycotoxins that are harmful to human and animal health1. The contaminated grains are downgraded because they are unhealthy for human and animal consumption. Integrated FHB management provides the most effective control strategy and utilizes fungicide application, residue management, crop rotation, and growing cultivars with the highest available level of resistance. Growing cultivars with resistance is an efficient and cost-effective component of the strategy, consequently developing FHB resistant cultivars is one of the major objectives of wheat breeding programs where the disease is a problem. Only partial resistance has thus far been reported in bread wheat germplasm and durum wheat cultivars lack adequate resistance to FHB. The durum wheat line DT696 is a source of moderate FHB resistance adapted to the western Canadian Prairies2. Durum wheat cultivars derived from DT696 as a source of FHB resistance, such as Brigade, Transcend, and CDC Credence show improved FHB resistance relative to other elite durum wheat cultivars in production4. Using resistance available in adapted sources such as DT696 has the advantage of minimizing detrimental effects of linkage drag since DT696 is an advanced elite breeding line adapted to Canadian growing conditions.Fusarium head blight (FHB) is a devastating disease of wheat causing severe reduction in grain yield and quality in warm and moist growing regions of the world. The causal pathogen 2. The QTL on chromosome 5A (5A1 and 5A2) were consistently expressed over different field locations and years. The 5A1 QTL explained up to 20.8% and 5A2 up to 25.7% of FHB phenotypic variance, suggesting a moderate effect of these QTL. The same QTL were also reported in a backcross recombinant inbred line population from T. turgidum ssp. dicoccum line BGRC3487 and a derivative of DT696 (DT735)5. The QTL interaction analysis of the DT707 \u00d7 DT696 population indicated the presence of an additive effect between the 5A1 and 5A2 QTL2. The 5A1 QTL co-located with a moderate plant height QTL and 5A2 with a major maturity QTL, providing supporting evidence for associations between these developmental traits and FHB resistance2. Three hypotheses are postulated for the co-localization of 5A1 with plant height and 5A2 with maturity QTL. FHB resistance may be the result of disease escape in taller and later-maturing plants as a consequence of height and maturity genes per se. A second possibility is that height or maturity loci have both disease escape and physiological FHB resistance effects, in other words there is a pleiotropic effect of height and maturity genes on FHB resistance. Alternatively, linkage between FHB resistance genes and genes for the developmental traits exist within the QTL intervals. Effective utilization of these QTL in breeding programs and the identification of predictive markers for marker-assisted selection (MAS) will depend on deciphering the type of association with plant height and maturity.High-density mapping of FHB resistance quantitative trait loci (QTL) using a large bi-parental population (number of lines 427) from a cross between lines DT707 and DT696 identified five resistance QTL on chromosomes 1B, 2B, 5A (two loci), and 7A7. The association of an FHB resistance QTL with the major effect plant height gene Rht-B18 suggested the contribution of the dwarfing allele of Rht-B1 to FHB susceptibility10. Buestmayr et al.11 found an FHB resistance QTL on chromosome 5A from T. macha co-located with the Q-gene that controls plant height, and spike traits including anthesis date, and spike density and length. The FHB resistance has also been considered pleiotropic to anther extrusion as the effect on resistance because of the co-localization of Qfhs.ifa-5AS, a FHB resistance QTL from hexaploid wheat line Sumai 3 on chromosome 5A, with an anther extrusion QTL13. These studies further emphasize the importance of greater understanding the relationship of FHB resistance and developmental traits through high-resolution mapping of FHB resistance QTL, designing markers for MAS, and the effective utilization of these QTL for developing resistant cultivars. A recent fine mapping effort that projected the interval of Qfhs.ifa-5AS on the International Wheat Genome Sequencing Consortia (IWGSC) Refseq v1.013 along with the high-resolution mapping of the DT707 \u00d7 DT696 population2 proposed the co-localization of Qfhs.ifa-5AS with 5A1 QTL.The association of FHB resistance QTL with plant height and maturity is not new. Previous studies highlighted a negative correlation between plant height and FHB severity14. GWAS has been successfully used for the identification and mapping of FHB resistance QTL in a durum wheat diversity15 and a durum wheat breeding panel16. Thus, GWAS is valuable for validating and high-resolution mapping of the FHB resistance QTL previously identified through bi-parental mapping such as the 5A1 and 5A2 QTL.The validation of QTL in breeding lines with relatively uniform plant height and maturity may enable teasing apart the contribution of disease escape mediated by tall alleles of plant height QTL from the potential physiological resistance conferred by the 5A1 and late alleles of maturity from the 5A2 QTL. Since its introduction as a source of moderate FHB resistance in the durum wheat breeding program at the Swift Current Research and Development Centre of the Agriculture and Agri-Food Canada (AAFC SCRDC), DT696 and its derivatives have been used as parents in several hundred crosses. This has provided an opportunity for the application of a genome wide association study (GWAS) for improving mapping resolution of FHB resistance QTL through the identification of novel recombination events at the 5A1 and 5A2 loci. It has also provided the opportunity for validating the 5A1 and 5A2 QTL and deciphering the type of the association of FHB resistance with plant height and maturity reported repeatedly in previous studiesFhb1, an FHB resistance QTL on chromosome 3BS present in the hexaploid wheat line Sumai 3 have been identified19. Candidate resistance genes have been proposed for Sumai 3 FHB resistance loci Qfhs.ifa-5A, on chromosome 5A20 and Fhb2 on chromosome 6B21, and a Wuhan-1 resistance QTL on chromosome 2D22. High-resolution mapping of FHB resistance QTL in DT696 will contribute to the design of predictive markers for MAS, which will allow their rapid and effective utilization in breeding programs.High-resolution mapping of FHB resistance QTL is difficult because of the small effect of resistance loci with quantitative expression, variable expression across environments, their epistasis interaction with other loci, and their interactions with genetic background and developmental traits. To date, only resistance genes associated with 23. A subset of 223 lines representing the recombinants at the 5A1 and 5A2 QTL were phenotyped in multiple environments and subjected to GWAS using the 90\u2009K SNPs. This reduced the size of 5A2 QTL interval to 15.8 Mbp. An FHB resistance QTL in a low recombination region of chromosome 6B was also identified. A panel of 523 contemporary breeding lines from the AAFC SCRDC breeding program with relatively uniform plant height and maturity was genotyped with SNP markers associated with the 5A2 interval, assessed for FHB resistance and subjected to haplotype-FHB trait association analysis. Significant association between markers with the DT696 allele and FHB traits validated the markers for MAS and demonstrated that the contribution of 5A2 QTL to FHB resistance is partially independent of plant height and maturity. DT696 haplotype and novel recombination events were present in 1022 lines of the Global Tetraploid Wheat Collection (GTWC). The novel recombination events constitute valuable resources for further refining the genetic interval and identifying candidate gene. The presence of the DT696 haplotype in the diversity panel will provide opportunity for the utilization of the resistance in other regions of the world from which the diversity panel originated.The objectives of this study were to evaluate lines derived from DT696 from the AAFC SCRDC breeding program for FHB resistance, to sample recombination in the chromosome 5A region for high-resolution mapping of the FHB resistance QTL, to further elucidate the genetic relationship of height and maturity to FHB resistance, and to identify additional loci contributing to FHB resistance in the breeding lines. We genotyped 401 DT696 derivatives available from the AAFC SCRDC durum wheat breeding program with the 90\u2009K iSelect SNP assay2, covariance analysis conducted with either plant height or maturity assigned as a covariate to test the dependency of FHB resistance expression on these traits. The genotypic variation for FHB severity and index remained significant when either plant height or maturity was assigned as covariate.Variance analysis was conducted for the FHB traits  as well as plant height and maturity for 223 DT696 derivatives phenotyped in Morden and Brandon FHB nurseries in Manitoba, Canada in 2016 and 2017. The line variation for FHB severity and index, and plant height was significant in all environments except at Morden in 2016  plotted against the pair-wise genetic distance on average was 1.6\u2009cM in the DT696 derivative lines  decay calculated from pair-wise SNP LD squared allele frequency correlation . A two tailed t-test of plant height between lines carrying alternate alleles at Tdurum_contig27834_260 was significant (P\u2009=\u20092.78E-5) and lines carrying the dwarfing allele (17 out of 223 lines) were on average 10.6\u2009cm shorter than those with the alternate allele .Single nucleotide polymorphisms associated with plant height were revealed on chromosomes 1A (two SNPs), 2A (four SNPs) and two physically distant loci on chromosome 4B (23 and six SNPs) in the Morden 2016 environment, and on chromosome 3A (one SNP) at the Brandon location of the same year  adjusted P-value varied between the SNPs within this interval and was often lower in the 2017 than the 2016 environment  and a 3.7 Mbp interval between SNPs wsnp_Ex-c621_1231298 and Tdurum_contig_69612_781 (5A2\u20132) was lower (more significant) than their flanking markers in the 2016 environment. A large drop in LD r2 at wsnp_Ex-c621_1231298 suggested the presence of a frequent crossover in the DT696 derivatives at this locus. The physical position of the vernalisation gene VRN1 (TraesCS5A01G391700) inferred from the IWGSC Refseq. v1.0 annotation was between the 5A2\u20131 and 5A2\u20132 intervals  at Morden in 2017  was detected in DT696 derivatives between SNPs wsnp_Ex-c621_1231298 and wsnp_Ex-c621_1231444, however, rare recombination events were detected between the other SNPs spanning the 5A2\u20132 interval  in FHB index at Brandon in 2016 and 2017, and at Morden in 2017 but not in 2016  and Brandon  nurseries, while the difference among other haplotypes remained insignificant.A few SNPs spanning the 5A2 FHB resistance QTL interval inferred from the QTL mapping of the DT707\u00a0\u00d7\u00a0DT696 population were used to genotype 523 contemporary breeding lines developed at AAFC SCRDC. According to the physical positions, the interval formed by these SNPs covered the entire 5A2\u20131 and a portion of 5A2\u20132 interval. All lines carried the DT707 allele at the interval between SNPs RN1 Fig.\u00a0. The phedex Fig.\u00a0. This haKukri_c33022_198 and wsnp_Ex_c621_1231298; this was also a common breakpoint in the lines of GTWC  was lowered by high disease pressure, which could be similar to what we observed for 5A-2. Moreover, a genotype by environment interaction was expected based on the moderate heritability of FHB resistance in DT696 which was previously inferred from phenotyping the DT707 \u00d7 DT696 population at multiple field nurseries including Morden where the DT696 derivatives were phenotyped in the present study2. Such a genotype \u00d7 location interaction had lower impact on the expression of 5A2 QTL in the DT707 \u00d7 DT696 population than the derivatives as the 5A2 QTL was consistently expressed over environments in the previous mapping study on the DT707 \u00d7 DT696 population2, but was expressed only at Brandon location for the derivatives  was approximately 50% higher at Morden than Brandon. The effect of disease pressure on the expression of FHB QTL was observed by Clark et al.2, associated with both FHB resistance and plant maturity2. Such linkage decay helps to explain the rare occurrence of lines moderately resistant yet earlier maturing than DT696 within the DT696 derivative set. The association of developmental and FHB resistance genes at a single locus is not new. The co-localization of the plant height gene Rht8 and an FHB resistance QTL on chromosome 2D was previously reported26. Subsequently, Handa et al.14 identified genes associated with DON degradation as the basis for FHB resistance in this interval, demonstrating that Rht8 was not the sole contributor to FHB resistance. The availability of additional recombination events within the 5A2 QTL interval in the GTWC panel is an opportunity to validate further the possibility of a similar disassociation in the 5A2 QTL.The presence of DT696 derivative lines with equal and occasionally higher FHB resistance, but shorter and earlier maturity than DT696 suggests that the resistance may not be solely due to disease escape of the taller and later-maturing plants. Moreover, when plant height and maturity were assigned as covariates in the variance analysis of FHB trait means, the variance of lines remained significant, further supporting the minor effect of plant height and maturity on expression of FHB resistance in the derivatives. Nevertheless, moderate to low correlations of FHB traits with plant height and maturity indicated that FHB resistance is partially associated with the developmental traits in the DT696 derivatives. However, there is no evidence to suggest that the FHB resistance genes segregating in the population are inextricably associated with plant height and maturity. In fact, higher numbers of recombinants in the derivatives could allow the decay of linkage between genes for the developmental traits and those for FHB resistance within the 5A2 region, that was according to Sari Rht-B1 (Tdurum_contig27834_260) was associated with plant height only at Morden in 2016. The effect of the locus partially explains the shorter plant height of the DT696 derivatives at this location compared to Brandon. Despite a relatively large association of Tdurum_contig27834_260 (GWAS R2\u2009=\u20090.29) with plant height, it was not detected as an SNP significantly associated with FHB susceptibility in the GWAS. This is different from the results of He et al.10 that suggested that the Rht-B1 tall allele is associated with Type I FHB resistance , reflecting lower FHB incidence in taller plants. Even though Rht-B1 was not detected as an FHB resistance QTL in the GWAS, a two-tailed test found significant association between the SNP within this gene and FHB index at the Morden nursery in 2016  between lines with alternative alleles of Tdurum_contig27834_260. It could also be a consequence of the low frequency (8%) of the dwarfing allele of Rht-B1 in the derivative population. Nevertheless, the Type II resistance which is independent of Rht-B1 seems to be the major determinant of FHB resistance in the derivatives. This is supported by the non-significant variance of lines for FHB incidence across all four environments and the identification of the 5A2 and 6B FHB resistance loci only for FHB severity and index ratings, but not for FHB incidence.The SNP within Fhb1, which in a previous study was expressed in a wider range of winter wheat genetic backgrounds than the FHB resistance QTL QFhs.nau-2DL25. The consistency of expression in a diversity of genetic backgrounds improves the success of recovering the 5A2 FHB resistance QTL during breeding of elite durum germplasm and explains the success achieved with the improved FHB resistance in durum varieties, derived from DT696, such as Brigade, Transcend and CDC Credence4.The presence of four sub-populations in the DT696 derivatives suggested the presence of substantial genetic variation in the population. This variation would have originated from genetic diversity of the founders used for crossing with DT696  impeding further mapping resolution. In the present study, we genotyped 716 DT696 derivatives in order to select recombinant representatives at the 5A1 and 5A2 QTL interval for phenotyping. This likely allowed us to capture all existing recombination events in the DT696 derivatives. Additional recombinants at the 5A2\u20132 interval could be identified in future studies by stimulating recombination using biotic and abiotic treatmentsVRN1 from other genes conferring FHB resistance in the interval due to homogeneity at the VRN1 locus. The breeding populations were subjected to phenotypic selection for lines with reduced levels of FHB by the breeders. Two thirds of selected lines had either Hap 1  or Hap 4 , indicating that genotypic selection agreed with phenotypic selection in 75% of cases. Combining genotypic with phenotypic selection could enhance selection gain in the breeding programs31 over the 75% obtained using phenotypic selection alone in the present study. We were able to rule out an association of inoculation method with the expression of FHB resistance, by comparing the FHB index of haplotypes at Carman nursery with spray inoculation at 50% anthesis and Brandon with corn spawn inoculation. Because Hap 4 was significantly different from Hap 5 at both nurseries despite different inoculation methods, the inoculation method was not the source of variation. The resistance mediated by Hap 4 is not solely due to developmental traits as spray inoculations at 50% anthesis minimizes the possibility of disease escape associated with plant height and maturity33.SNPs associated with the 5A2 FHB resistance QTL were validated for marker-assisted selection on breeding lines of 13 AAFC SCRDC crosses. Not only did the application to contemporary breeding material allow the validation of SNPs for marker-assisted selection, it also allowed discriminating the effect of Forty-six GTWC lines with the DT696 haplotype provide opportunity at the international level for breeding resistance to FHB. Additional research is required to validate that these lines express resistance to FHB in their target environment. As expected, population size raised the recombination frequency at 5A2\u20132 interval. Assessing the phenotype of germplasm with the recombination events at 5A2\u20132 interval will be valuable to refine the interval towards identifying candidate genes.T. turgidum ssp. carthlicum line Blackbird34. A pedigree search of DT696 derivatives lines with resistance alleles in the 6B QTL indicated that all were derived from a single breeding population named A0580 (DT696/A0200H-056/3/Langdon-Dic3A (a resistant recombinant substitution line derived from crosses of T. turgidum spp. durum cv. Langdon with wild emmer wheat T. turgidum spp. dicoccoides)//SC9475-CX1*2/ND2710 (a hexaploid source of FHB resistance presumably derived from line Sumai 335) and most of them also carried the DT696 haplotype at 5A2 QTL. Considering the field location-dependent expression of both the 5A2 and 6B QTL in the DT696 derivatives, lines of A0580 with resistance alleles of both QTL are valuable source for developing cultivars with consistent expression of resistance over multiple environments.The FHB resistance locus on chromosome 6B in the DT696 derivatives was not detected from the previous QTL mapping study of the DT707 \u00d7 DT696 population. This suggests that resistance alleles are contributed from founder lines crossed with DT696 and its derivatives. Comparative mapping indicated that the interval defined by SNPs on chromosome 6B in our current study correspond to the Type II FHB resistance QTL detected from 2 minimized the possibility of environment affecting the expression of this QTL. Unlike the 5A2 QTL, the expression of 5A1 seems to be dependent on other loci or certain genetic backgrounds that were rarely present in DT696 derivatives. Weak expression of 5A1 QTL below a level detectable by the GWAS algorithm could be another reason the 5A1 locus was not detected in the derivatives.GWAS analysis of DT696 derivatives could not associate any SNPs within the 5A1 QTL interval with FHB resistance. Consistent detection of the 5A1 QTL in the QTL mapping study using the DT707 \u00d7 DT696 population in the same field locations used for GWAS of the derivatives (Morden nursery in 2016 and 2017)More work is needed to further resolve the 5A2 FHB resistance QTL. Efforts are underway to develop Near Isogenic Lines (NILs) carrying recombination break points in the 15.8\u2009Mb interval of the 5A2 FHB resistance QTL. Recombination break points will be mapped with the SNP markers identified in the present study and selected recombinants will be subjected to intense phenotyping to improve the mapping resolution. The NILs will also be subjected to further reverse genetic analysis to identify reliable markers required for high-precision MAS.Fhb1 and Qfhs.ifa-5AS, a complex genetic architecture is proposed for the 5A2 FHB resistance QTL, with the presence of at least two distinct loci (5A2\u20131-and 5A2\u20132) in the interval. GWAS along with haplotype analysis in the contemporary breeding lines enabled the validation of the 5A2 FHB resistance QTL in multiple genetic backgrounds, lending support to the transferability of resistance conferred by this locus to elite germplasm. This study led to the identification of the SNP markers desired for marker-assisted selection that will contribute to the speed and precision of gene pyramiding and should pave the way for improving FHB resistance in durum wheat.In conclusion, phenotyping of DT696 derivatives that carried historical recombination from 72 breeding populations, enabled selecting for early-maturing semi-dwarf lines with FHB resistance equal to or better than DT696 as candidates for developing resistant cultivar. GWAS resolved the 25.8 Mbp 5A2 interval obtained from the bi-parental QTL mapping into a 15.8 Mbp interval. Like 2. Thirteen contemporary breeding populations active in the breeding program were used for marker validation. These populations totalling 523 lines were developed from crosses between advanced breeding lines and the cv. Transcend which is a moderately susceptible durum variety derived from DT6964.A search was conducted within pedigrees of AAFC SCRDC durum wheat lines to identify DT696 derivatives among advanced breeding lines using an in-house script of Statistical Analysis System (SAS) software version 9.3  that enabled retrieval of pedigrees from crosses made with DT696 and its secondary and tertiary derivatives. This identified 72 populations with a total number of 716 lines derived from DT696  and 15-acetyl-deoxynivalenol (15ADON) producing strains of F. graminearum. The Morden nursery was inoculated approximately 2\u20133 weeks prior to heading, while at the Brandon nursery the first application of corn inoculum was done at six weeks after planting followed by another application two weeks after the first application. Colonized corn grains were broadcasted between the rows at the rate of 40\u2009g\u2009m\u22122 at Brandon, and at 8\u2009g per row performed twice, one week apart at Morden. The nursery at Brandon was irrigated three times a week with an overhead low-pressure mist irrigation system immediately upon completion of inoculation to provide moisture for inoculum development in the corn and to provide moisture on the spikes for disease development. The nursery at Morden was irrigated three times a week using Cadman Irrigation Travellers with Briggs booms.Phenotyping 223 DT696 derivatives for FHB resistance, plant height and relative maturity was conducted following the method described by Sari 2 pressurized backpack sprayer calibrated at 2 kPa at 50% anthesis with 50\u2009ml per row conidia suspension composed of a mixture of aggressive 3ADON and 15ADON producing strains of F. graminearum. The concentration of the conidial suspensions was adjusted to 5 \u00d7 104 conidia mL\u22121 using a hemocytometer and Tween 20 (1 drop per 100\u2009ml) was added to the suspension. Inoculation was repeated 4 d after the first inoculation. The nursery was mist irrigated in the evening, and the morning after each inoculation.Phenotyping of the 523 contemporary breeding lines was conducted in FHB nurseries located at Brandon and Carman in 2017. Lines at the Brandon nursery were subjected to the same inoculation protocol as DT696 derivatives lines with the experiment conducted as an unreplicated alpha-lattice field designs with 12 entries per incomplete block. At the Carman nursery, the spikes of the plots were spray-inoculated using a COFHB incidence (percentage of spikes showing symptoms) and severity (percentage of spike area infected) were recorded for each plot. FHB index was calculated from the incidence and severity rating data using the formula (incidence \u00d7 severity)/100. Plant height was measured on a representative plant from the soil surface to the tip of spikes excluding the awns. Relative maturity was rated using a 1\u20134 scale (1\u2009=\u2009latest and 4 earliest maturity) concurrent with the FHB rating at three weeks post anthesis, by pinching the seeds and comparing their moisture levels with the parents.et al.23. The SNP clustering was performed in Genomestudio software v. 2011.1  using the default clustering algorithm and following the workflow described by Cavanagh et al.36. Approximately 10% of total SNPs  were scorable. The order of markers on the physical map of the IWGSC Refseq v1.037 and durum wheat cv. Svevo38 and wild emmer wheat accession Zavitam reference assemblies39 was determined by aligning the 90\u2009K SNP source sequences using the Nucmer program available in Mummer v.3 software40. Only SNPs with missing data less than 10% and minor allele frequency of higher than 5% were retained. The retained SNPs were then used to calculate an identify-by-state genetic similarity matrix for all possible pairs of lines using Plink v1.0741. For lines showing \u2265 0.99 genotypic similarities, only one representative line with the lowest number of missing SNPs was maintained.A subset of DT696 derivatives (401 lines) were genotyped using the wheat iSelect 90\u2009K SNP genotyping assay following the method described by Wang 42. Lines were then genotyped with a Fluidigm  high-throughput genotyping system using nine KASP markers within the 5A1 and seven KASP markers within the 5A2 QTL interval. The genotypic data of these lines were then combined with those of the 401 lines obtained from the iSelect 90\u2009K SNP genotyping assay and a similarity matrix was generated using Flapjack v. 1.16.10.31 software43 to select 359 representative recombinant lines for phenotyping. Lines from the contemporary breeding populations used for validation of SNPs for MAS were genotyped using the same KASP markers with the Fluidigm high-throughput genotyping system.To select recombinant representatives at the 5A1 and 5A2 FHB resistance QTL on chromosome 5A, SNPs within the QTL interval were used for genotyping 374 additional DT696 derivatives. SNPs within the 5A1 and 5A2 interval were converted to Kompetitive Allele-Specific PCR (KASP) markers using the Polymarker pipeline44. The population structure was inferred from the model-based quantitative assessment of sub-population available in Faststructure v1.0 software45. Linkage disequilibrium (LD) squared allele frequency correlation (r2) was calculated for each pair of SNPs within each chromosome using Haploview software46. The overall pattern of LD decay in the population was estimated following the method described by Maccaferri et al.27 from pairwise LD r2 estimates of all chromosomes plotted against the genetic distance between SNPs inferred from the tetraploid wheat consensus genetic map24. A non-linear regression model was then fitted using the nonlinear least squares method in R v. 3.5.3 software (https://www.r-project.org/). The genetic distance (cM) at which the r2 was lower than the empirical threshold of 3.0 was considered as the average LD decay of the population.Of 359 derivatives phenotyped at multiple locations, 223 genotyped using iSelect 90\u2009K SNP genotyping assay, were subjected to GWAS. When markers were filtered for missing data and minor allele frequency (using the criteria described above), 6231 SNPs met the criteria for GWAS. Centered identity-by-state kinship matrix was calculated for these SNPs using Tassel v. 547 and Settlement of MLM Under Progressively Exclusive Relationship (SUPER)48 models of R package GAPIT v. 2 software49 with the kinship matrix generated as described above. The following models were tested with each of cMLM and SUPER methods: 1) no correction for population structure; 2) model corrected for population structure using the % membership coefficient generated by Faststructure for number of subpopulations (Q) of 3\u20137. The model with correction for the population structure of Q4 was superior to other models according to the quantile-quantile (QQ) plot generated by GAPIT, hence the probability (P) values of this model were used for determining the significant SNPs. The experiment-wise P value threshold was either 0.05 when the FDR adjusted P values were reported or based on Bonferroni adjusted P value which was equal to a marker-wise P value of 8.25 \u00d7 10\u22126. Assigning significant SNPs to QTL was implemented through anchoring the SNPs to the tetraploid wheat consensus genetic map24 and durum wheat cv. Svevo genome assembly38, and comparing the genetic and physical position of QTL reported in the previous QTL mapping study2. The FDR adjusted P values were plotted against LD r2 values extracted from Haploview to associate recombination break points with changes in significance of P values.GWAS was conducted with the 6,231 SNPs, FHB traits , plant height and relative maturity data using compressed mixed linear (cMLM)et al.38. The haplotype and recombination break points were compared between the GTWC and DT696 derivatives at 22 SNPs within the 5A2 QTL interval. Since DT696 and cv. Strongfield were common to both datasets and cv. Strongfield had an identical haplotype to DT707 for the 22 SNPs spanning the 5A2 interval, we could reclassify the genotypic data of the GTWC to either a DT696 or DT707 haplotype. Lines with missing records or heterozygous at these 22 SNPs were excluded from analysis. One-dimensional hierarchical cluster analysis and haplotype visualization was performed using the dendextend and ComplexHeatmap packages of R v.3.5.3.Haplotypes and recombination breakpoints in the GTWC were based on the genotypic data generated using the 90\u2009K iSelect SNP genotyping assay described by Maccaferri t-test with correction for heteroscedastic distribution  of data in Microsoft Excel.Variance and covariance analyses were conducted using SAS v.9.3. To estimate the variance due to line, the mixed model was used with genotype assigned as a fixed effect and block assigned as random effect. To estimate the variance due to genotype when plant height and maturity were assigned as covariates, the mixed model was used with line and either plant height or relative maturity assigned as fixed effects, while block was assigned as a random effect. Spearman\u2019s rank correlation analysis was conducted between each of the FHB traits and plant height and maturity using PROC CORR of SAS v.9.3. The significant difference between lines of different haplotypes was inferred using a two-tailed Supplementary Information\u00a01.Supplementary Information\u00a02.Supplementary Information\u00a03.Supplementary Information\u00a04."}
+{"text": "Endometrial carcinoma (EC) has a worldwideincidence of 8.4 per 100,000 persons per year and is aleading cause of cancer mortality in women in developedcountries after ovarian and cervical cancer . Theass"}
+{"text": "The penultimate sentence of the first paragraph should have referred to a 0.8% weight loss among patients receiving placebo. The final sentence should have read that weight loss with bimagrumab was greater than the 1-year FDA threshold of 5% for registration of new medicines for treating obesity and overweight. This article has been corrected.1In the Original Investigation titled \u201cEffect of Bimagrumab vs Placebo on Body Fat Mass Among Adults With Type 2 Diabetes and Obesity: A Phase 2 Randomized Clinical Trial,\u201d"}
+{"text": "The percentage of patients in the healthy control group who reported worsened quality of life should have read 9.5. This article has been corrected.1In the Research Letter titled \u201cSequelae in Adults at 6 Months After COVID-19 Infection,\u201d"}
+{"text": "Scoliosis is an abnormal bending of the body axis. Truncated vertebrae or a debilitated ability to control the musculature in the back can cause this condition, but in most cases the causative reason for scoliosis is unknown (idiopathic). Using mutants for somite clock genes with mild defects in the vertebral column, we here show that early defects in somitogenesis are not overcome during development and have long lasting and profound consequences for muscle fiber organization, structure and whole muscle volume. These mutants present only mild alterations in the vertebral column, and muscle shortcomings are uncoupled from skeletal defects. None of the mutants presents an overt musculoskeletal phenotype at larval or early adult stages, presumably due to compensatory growth mechanisms. Scoliosis becomes only apparent during aging. We conclude that adult degenerative scoliosis is due to disturbed crosstalk between vertebrae and muscles during early development, resulting in subsequent adult muscle weakness and bending of the body axis. Diseases that result in bending of the spine are more common in elderly populations. Camptocormia (a 45 degree anterior bent of the lower joints of the spine) has an average age of onset of 66 years  and frommyoD [The segmentation clock controls an essential process in the vertebrate embryo and leads to the formation of the somites, transient structures which will later contribute to the formation of one of the most well-known and characteristic structure of all vertebrates, the vertebral column. This structure together with the axial muscles act to control axial posture and function. During early stages of development, the pre-somitic mesoderm is segmented from anterior to posterior by the periodic expression of genes from the Notch-Delta family . This exmyoD , 9. In cmyoD , 11. MormyoD , 13. MyomyoD   single, -/-; her7-/- double, and her1-/-; her7-/-; tbx6her1-/- triple mutants). These mutants show varying levels of somite segmentation defects and subsequently mild axial skeletal phenotypes [-/-; her7-/-, tbx6-/- and her1-/-; her7-/-; tbx6-/-her1 at adult and embryonic stages. We found that scoliosis is not coupled to vertebral defects and may correlate with early perturbations of muscle formation during development. Strikingly, -/-; her7-/-her1 double mutants develop scoliosis with the same penetrance as wild type animals, despite showing aberrant vertebral skeletal phenotypes. In contrast, loss of Tbx6 function greatly increased the chance of an animal developing scoliosis with age. Finally, we present evidence that this correlates with severe muscle patterning defects during embryogenesis and propose that vertebrae and muscle segmentation are not directly coupled during development.It has been suggested that human patients with congenital scoliosis may have early abnormalities during somitogenesis . The numenotypes . The str-/-; her7 -/-her1 and -/-; her7-/-; tbx6-/-her1 present mild defects in the segmentation of the axial skeleton (fusion and hemivertebrae) with a 100% incidence. This is also true for 80% of fss (tbx6-/-) mutants [As we have previously shown  zebrafish mutants for  mutants . Later o-/-tbx6 third generation animals developed scoliosis, similar to first generation animals 3 out of 8 (38%) evaluated at a similar age . By the end of the experiment (12 months post fertilization) all -/-; her7-/-; tbx6-/-her1 first generation animals (8/8) had developed scoliosis. Similarly, in the third generation, all animals (7/7) -/-; her7-/-; tbx6-/-her1 animals developed scoliosis at 14 months post fertilization  of the third generation and 38% (3/8) of the time lapse individuals developed visible scoliosis at 12 months post fertilization. All of the -/-; her7-/-her1 mutants had vertebral fusions and hemivertebrae in more than one position within the axial skeleton. This value was the same as the wild type scoliosis recurrence. In wild type individuals, scoliosis was first detected at 5 months post fertilization in 1 out of 22 fish (4.5%) and at 6 months post fertilization in 1 out of 8 (12%). At the end of the analyzed period (1 year) 38% (3/8) of wild type animals had developed scoliosis. These individuals had no vertebral defects. Presentation of scoliosis therefore did not correlate with axial skeletal phenotypes in wild type animals. An additional observation, showed that 64% of the females (9/14 that developed scoliosis during the whole experiment) had the first measurable sign of scoliosis at 3 months post fertilization compared to 0 % of males  ilar age  and 2D. lization . By 12 mcoliosis  and 2D;  mutants . Despiteof males .-/-tbx6 mutants, the muscle volume at the pelvic fin and the anal fin was also statistically different from wild type  beginning of the pectoral fin, (2) end of pectoral fin, (3) pelvic fin and (4) anal fin. The first and second positions were selected as the majority of the scolioses were observed in the fish at these levels. The musculature of the fish changes in volume significantly at different anterio-posterior positions, such that anterior myomeres are larger than those in posterior region. By standardizing the locations of all measurements of muscle volume for each animal, we could compare absolute muscle volumes between animals. This allowed us to ensure that any differences in muscle volume were not due to inherent differences in muscle volume along the trunk or due to differences in animal size \u20133E. A twild type  and 3I. Three-way correlation analysis was performed in order to establish if the length of the fish had an effect on the muscle volume or the angle of deviation from the body axis. It was found that the length of the fish has a negligible correlation with the angle of deviation of the body axis . There is a low positive correlation between muscle volume and fish length  and a low negative correlation between muscle volume and angle of deviation . An interpretation of these correlation scores is that severity of scoliosis correlates with a lower muscle mass but is independent of the length of the fish.-/-; her7-/-; tbx6-/-her1 or -/-tbx6 mutants    to deterFM1954A) . Therefo-/-tbx6 and the triple -/-; her7-/-; tbx6her1-/- mutants cavities can be seen in the fast muscle  could be measured with an average size of 1.105\u03bcm \u00b1 0.66. In -/-; her7-/-; tbx6-/-her1 cavities were found in 18 out of 20 optical sections, 40 cavities (between 1 and 5 per section) were measured with an average size of 1.407\u03bcm \u00b1 0.93. In order to determine whether the cavities contained cells that do not express muscle fiber markers, whole mount DAPI staining was performed. In all -/-; her7-/-; tbx6-/-her1 and -/-tbx6 embryos imaged at 32hpf (n=8 for each genotype), the cavities did not contain any cells  embryos were analyzed. In all mutants the fast  and slow (F59 positive) muscle fibers lose their metameric organization \u20134L. In at muscle \u00b4, 4D\u00b4. Tt muscle \u00b4. In tbxny cells . In addi regions \u00b4. The sl present \u00b4 or the  present \u00b4 as prev mutants .-/-; her7-/-her1 mutants have a significantly smaller region of fast muscle compared to the wild type at 32 hpf , but only start to develop scoliosis as they age (after six weeks of age). In the -/-; her7 -/-her1 mutants there is no correlation between the position or amount of vertebral defects and the risk of developing scoliosis. If vertebral defects were correlated with scoliosis, we would predict that all animals with significant vertebral aberrations would present scoliosis. There is a high incidence of vertebral defects in -/-; her7-/-her1 mutants ranging from 1-12 defects per animal at 25 dpf [-/-;her7-/-her1 mutants should show a high incidence of scoliosis. However, the incidence of scoliosis was the same as in the wild type animals examined at a similar age. When evaluating whether muscle morphology in -/-; her7-/-her1 mutants was related to where scoliosis occurred along the body axis we did not observe any difference in muscle volume at regions where scoliosis occurred relative to other axial positions. Furthermore, there were no lesions in fast or slow muscle during embryogenesis in -/-; her7-/-her1 mutants. In contrast, -/-; her7-/-; tbx6-/-her1 mutant animals showed a lower muscle volume in regions in which scoliosis was observed and also had perturbed slow and fast muscle development.In order to understand more about the link between the somite clock and adult degenerative scoliosis, we analysed zebrafish mutants defective for three clock segmentation genes: t 25 dpf  and betw-/-; her7-/-her1 and -/-; her7-/-; tbx6-/-her1 mutants this was unrelated to the skeletal phenotype. The higher scoliosis incidence in the -/-; her7-/-; tbx6-/-her1 mutants is probably due to the muscle defects caused by the loss of Tbx6 activity. It is possible, that -/-; her7-/-her1 mutants recover from early muscle patterning defects by compensatory growth, but in an absence of Tbx6 function, this cannot occur. In -/-tbx6 and -/-; her7-/-; tbx6-/-her1 mutants the muscle contains many cavities that originated from embryonic stages. There is no recovery of the early muscle defects in these mutants, potentially due to a deficit in the myogenic precursor population at embryonic stages, in turn due to the role of Tbx6 in regulating differentiation of dermamyotome progenitor cells [her1 and her7, results in long lasting muscle phenotypes that appear to be correlated with scoliosis. The occurrence of muscle phenotypes in compound -/-; her7-/-; tbx6-/-her1 mutants occurs independently of skeletal abnormalities as there is no correlation of skeletal abnormalities relative to the presence of scoliosis between genotypes.Despite differences in adult muscle phenotype and embryonic muscle development between or cells . In the or cells . Thus, p-/-tbx6 mutant individual in this study that did not develop scoliosis had higher muscle mass than its scoliotic siblings  implying that muscle mass and scoliosis are directly correlated. In human patients, differences between the left and right-side muscles at the position of axial curvature have been documented. In the bent side the muscle becomes overstretched, while in the opposite side the muscles are shorter and tighter [In humans, spinal muscles are essential for stabilising the spine, and changes in muscle mass have been linked to axis curvatures. It is known that upon spine curvature, muscles adjacent to the curvature are generally weaker and progressively deform , 19. Our tighter . We did  tighter , 37.-/-; her7-/-; tbx6-/-her1 and -/-tbx6 individuals can be explained by differences in total number of individuals quantified. Regardless, in both generations, there is a trend towards scoliosis, which is much higher than in wild type.In elderly patients there is no difference in scoliosis prevalence between males and females . In our tbx6 function leads to the strongest scoliotic phenotype. The -/-tbx6  mutant zebrafish and human patients with mutations in TBX6 have similar skeletal phenotypes including hemivertebrae, butterfly vertebrae and rib abnormalities [TBX6 is available for comparison. Our findings from zebrafish intriguingly suggest that scoliosis-associated genes may be needed for normal muscle patterning at early embryonic stages and it is the disruption of early muscle organisation that may underlie scoliosis onset later in life.Our data show that loss of malities . In humamalities . In zebr-/-; her7 -/-her1 mutants but not in -/-tbx6 or -/-; her7-/-; tbx6-/-her1 mutants. Nonetheless, in -/-tbx6 and the triple -/-; her7-/-; tbx6-/-her1 mutant embryos, the slow and fast muscle fibers show cavities depleted of nucleated cells , is a similar phenotype described [Him is regulated by Notch signalling [-/-tbx6 mutants have a more than 50% reduction in the number of Pax3+ Pax7+ muscle progenitor cells at embryonic stages and unusually large fast muscle fibres that persists until later larval stages [tbx6 interacts with Mesp-b and Ripply1 to regulate myogenesis in zebrafish [The fast muscle domain and fast fiber cross sectional area were decreased in ed cells , which ced cells . Adult hreported . This phescribed . Him inhescribed . It is kgnalling . Interesgnalling , 46. Thil stages . In addiebrafish . Hence, -/-; her7-/-, tbx6-/-her1 and triple -/-; her7-/-; tbx6-/-her1 can serve as a model for adult scoliosis, because they reproduce the muscle and bone indicators present in clinical patients. Previously, it has been proposed that human patients with congenital scoliosis, may have early abnormalities during somitogenesis [In summary, we have shown that the zebrafish mutants for 2, 0.33 mM MgSO4) at 28\u00b0C. For anesthesia, a 0.2 % solution of 3-aminobenzoic acid ethyl ester (Sigma), containing Tris buffer, pH 7, was used.The work was carried out at the Hubrecht Institute (the NL), according to local laws. Ethical approval was obtained through the relevant DEC committee (HI 10.1801). Standard husbandry conditions applied, according to FELASA guidelines . Embryossa38869fss and hu2526her7 mutants were provided by Jeroen den Hertog (Hubrecht Institute). The gSAIzGFFM1954A line was obtained by a gene trap method [her1 in the her7hu2526 background, were generated as described in Lleras-Forero et al. [The p method . Double o et al. . DNA waso et al. .Olympus SZX16 stereoscope with a Leica DFC450C camera.Hematoxylin/eosin (H&E) was performed on sagittal and transverse cryo-sections (15\u03bcm) of adult zebrafish according to standard procedures. H&E sections were photographed on a Nikon eclipse NI with a DS-Ri2 camera and 4x Plan Fluor objective. Alizarin Red staining of bone was performed as described previously . Whole mTM plus Cyanine 3 system . For detection of nuclei DAPI was used in which embryos were fix at 32 hpf as described above and then washed twice for 10 minutes in PBS. Staining was performed with DAPI solution  in Eppendorf tubes, at 4 degrees in the dark, over two nights.32 hpf embryos were dechorionated and fixed in 4% PFA overnight. Phalloidin (Invitrogen Alexa Fluor 546) staining was performed according to Goody, 2013 . StaininImage acquisition was performed by embedding embryos in 0.8% agarose and visualising on a Leica SP8 confocal microscope using a 20X objective  for the whole mounts and a 40x objective  for the cloacal area acquisitions.Olympus SXZ16 stereomicroscope (1.5X PlanApo objective) connected to a DFC450C Leica camera. Immediately afterwards, the individual was returned to warm E3 media without anesthesia. Test subjects were returned to their specific tank in the animal facility only when they were completely awake and moving. This procedure was repeated at 3, 6, 9 and 12 months after fertilization. From 3 months onwards, a picture was taken using a Sony Xperia mobile. Sedation and photography did not take more than two minutes per animal and did not compromise survival.Embryos from mutant crosses were kept in E3 at 28\u00b0C until 7 dpf. They were then housed at a density of 50 embryos in a 3 Liter tank. At six weeks post fertilization, eight juvenile animals, with fully developed swim bladder, from either mutant or wildtype embryos were housed individually in the animal facility. Individuals were fed tetrahymena in combination with Gemma 75 for the first two weeks, followed by Artemia and Gemma 150 for the following two weeks. The same day each individual was anesthetized (as described above) and photographed using an \u00b0 rotation, with 400 ms exposure time. Reconstructions were performed using NRecon (Version 1.7.1.0). Each individual scan overlapped its neighbouring scans in its respective batch by 200 mm, to ensure robust concatenation. For One-year-old fish were fixed in 4% paraformaldehyde overnight at 4 degrees; afterwards an incision was made in the abdomen and the organs were removed. Staining for soft tissue visualization with Micro CT was performed according to Descamps et al., 2014  for 9 daReconstructions were processed using ImageJ/Fiji . ConcateUsing a Leica SP8 confocal with a 40x plan Apo objective  and a Z-step size of 0.27\u03bcm, an image from the cloacal region from six 32 hpf embryos , was taken. Five slides within each image were randomly chosen and the area was measured manually using Fiji. The data point plotted in the graph is the mean of the 5 slides within one embryo.Cross-sectional area quantification was done in the images of the cloacal region described above. Using Fiji, 5 randomly selected fibers were manually measured in the slides described above. Each point in the graph represents one embryo (25 different fibers). The statistical significance of the data was measured using a two-tailed Student\u2019s t-test with unknown variance in Excel 2016 (Microsoft).-/-tbx6 and -/-; her7-/-; tbx6-/-her1 were measured in the optical sections stained by Phalloidin as described above. Using Fiji, the longest vertical distance of each cavity was measured in \u03bcm. The average and standard deviation were calculated for each mutant.Cavity size in In order to measure accurately if individual zebrafish had developed scoliosis and if the scoliosis progressed over time, the individual pictures from the time lapse were used. A straight line was drawn from the tip of the premaxilla, through the middle of the eye to the caudal fin. In fish lacking scoliosis or with a perturbation of the body axis the line ends exactly at the apex of the caudal fin. In a fish with a bent body axis, the line deviates from this position. The angle between the position of the line coming from the premaxilla to the new position of the apex was manually measured. In humans, the spine is considered deformed when the Cobb angle is \u2265 10 degrees . The samSupplementary FiguresSupplementary Custom Macros"}
+{"text": "Our aim was to determine whether carbon dioxide (CO2) levels in the first 3 days after birth reflected abnormal EtCO2 levels in the delivery suite, and hence, a prolonged rather than an early insult resulted in IVH. In addition, we determined if greater EtCO2level fluctuations during resuscitation occurred in infants who developed IVH. EtCO2 levels during delivery suite resuscitation and CO2 levels on the neonatal unit were evaluated in 58 infants . Delta EtCO2 was the difference between the highest and lowest level of EtCO2. Thirteen infants developed a grade 3\u20134 IVH (severe group). There were no significant differences in CO2 levels between those who did and did not develop an IVH (or severe IVH) on the NICU. The delta EtCO2 during resuscitation differed between infants with any IVH (6.2 (5.4\u20137.5) kPa) or no IVH (3.8 (2.7\u20134.3) kPA) (p < 0.001) after adjusting for differences in gestational age. Delta EtCO2 levels gave an area under the ROC curve of 0.940 for prediction of IVH.Abnormal levels of end-tidal carbon dioxide (EtCOConclusion: The results emphasize the importance of monitoring EtCO2 levels in the delivery suite. Our aim then was to study EtCO2 levels in the delivery suite during resuscitation of preterm infants and their relationship to the subsequent CO2 levels within the first 3 days after birth and the development of IVH.Improvements in neonatal intensive care have resulted in decreased mortality rates of preterm infants. The development of intraventricular haemorrhage (IVH), however, can result in long-term adverse outcomes. Several studies of preterm infants have demonstrated that abnormal levels of carbon dioxide  were associated with severe IVH development, as was a greater degree of fluctuation in pCO2 levels within the first 4 days after birth [2 variability [2 can adversely affect cerebral oxygenation and perfusion and the electrical activity of the brain [2 levels during resuscitation in the delivery suite were associated with IVH development but also inflations resulting in larger tidal volumes and lower EtCO2 levels [2 levels during resuscitation in the delivery suite would be at greatest risk of developing an IVH.In one study of very low birth weight infants, not only high but also low partial pressures of carbon dioxide pCO were assiability . Furtherhe brain ; such flhe brain . We have2 levels . Those f2 levels . A furthWe reviewed the records of infants born at less than or equal to 33 weeks of gestational age who required resuscitation in the delivery suite between March 2010 and March 2014. A certain part of their data was reported in a previous paper [2 levels.The resuscitation protocol, together with the monitoring methodology, has previously been described . The cliTE) of each inflation and the breath-by-breath minute ventilation. The highest EtCO2 was recorded as were the number of inflations with tidal volumes more than 6 mls/kg  or end-tidal CO2 levels of less than 4.5 kPa (low EtCO2 levels) and the number of inflations with both high tidal volumes and low EtCO2 levels as previously described [2 (delta EtCO2) during resuscitation was determined by calculating the difference between the highest and lowest EtCO2 value during resuscitation in delivery suite.The resuscitation recordings were analysed to determine the duration of resuscitation, the number of inflations, the inflation pressure, the positive end expiratory pressure (PEEP), inflation time (Ti), mean airway pressure (MAP), expiratory tidal volume  during each of the first 3 postnatal days was determined by calculating the difference between the highest and lowest CO2 value on each day. Volume targeted ventilation was not used on the NICU during the study period.The unit\u2019s routine policy was to insert arterial lines for blood gas sampling, and in the absence of such, access capillary sampling was undertaken. Blood gases were recorded on the intensive care observation charts every 4 to 6 h depending on the clinical status of the infant, and the hourly ventilator settings were collected at the time as each arterial gas result. Venous blood gases were not analysed in this study. Data on the maximum, minimum and average pCOThe medical records were examined to determine whether the infants developed an IVH which was diagnosed from cranial ultrasounds. As per unit policy, cranial ultrasounds were performed on admission to the unit for all infants born below 34 weeks of gestation. Cranial ultrasounds were repeated on day 3, between days 7 and 10, days 14 and 21 and days 28 and 30. All cranial ultrasounds were reported by a neonatal consultant. The IVHs were reported as grades I to IV using the Papile classification . Severe U test or the chi-square test as appropriate. As infants who develop IVH tend to be more immature, regression analyses were performed to determine if the results remained statistically significant after correcting for differences in gestational age. The strength of any relationships of EtCO2 values during resuscitation with CO2 levels during the first 3 days after birth were assessed by calculating Spearman correlation coefficients. The performance of factors identified as significantly related to IVH development from the regression analysis were assessed by receiver operator characteristic curve analysis and estimation of the corresponding area under the curve (AUC). Statistical analyses were performed using JMP statistical software  and SPSS software 25.0 .The data were tested for normality using the Kolmogorov-Smirnov test and found to be non-normally distributed; hence, differences were assessed for statistical significance using the Mann-Whitney 2 traces which were unsuitable for analysis, a further 24 were excluded due to a large leak throughout and 12 excluded due to missing neonatal unit observation charts. The remaining 58 infants were included in this study and had a median gestational age of 27.3 (interquartile range (IQR) 24.9\u201329.0) weeks and a birth weight of 0.90 (0.72\u20131.21) kg. The infants who were and were not included in this study did not differ significantly with regard to birth weight (p = 0.893) or gestational age (p = 0.785). Fifty-three of the 58 infants were ventilated on day 1 after birth. Thirty infants developed no IVH with 28 infants developing an IVH. Seven infants developed a grade 1, eight infants grade 2, five infants grade 3 and the remaining eight infants a grade 4 IVH, that is 13 infants developed a severe IVH. All infants who developed an IVH did so in the first 3 days after birth. Eleven infants developed an IVH on day 1 (9 grade 1\u20132), nine on day 2 (5 grade 1\u20132) and eight on day 3 (1 grade 1\u20132). There were no significant differences in CO2 levels on the NICU and IVH development.Three hundred and seventy infants were born at less than or equal to 33 weeks of gestation within the study period. There were 150 infant recordings available for analysis, but 56 had recordings of tidal volume or EtCO2 during resuscitation in the no-IVH group was 8.0 (7.1\u20138.4) kPa and 10.0 (8.9\u201311.2) kPa in the IVH group; the difference remained statistically significant after adjusting for differences in gestational age between the two groups (p = 0.0002). The highest EtCO2 during resuscitation in the no/non-severe group IVH was 8.2 (7.4\u20139.9) kPa and 10.2 (8.6\u201311.1) kPa in the severe IVH group; the difference remained statistically significant after adjusting for differences in gestational age between the two groups (p = 0.037). There was no significant difference in the mean tidal volume during resuscitation between infants in the no/non severe IVH group and the severe IVH group (p = 0.621).The highest EtCO2 during resuscitation in the no-IVH group was 4.2 (3.2\u20135.2) kPa and 3.5 (3.0\u20134.5) kPa in the IVH group, but this was not statistically significant after adjusting for the difference in gestational age between the two groups (p = 0.11). The lowest EtCO2 during resuscitation in the no/non-severe IVH group was 4.0 (3.2\u20135.1) kPa and 3.1 (2.7\u20133.5) kPa in the severe IVH group; the difference remained significant after adjusting for the difference in gestational age between the two groups (p = 0.041).The lowest EtCO2 on delivery suite and the highest pCO2 on the neonatal unit on days 2  and 3 .There were weak correlations between the highest end-tidal CO2 during resuscitation was significantly different between the no/non-severe IVH (4.3 (3.1\u20135.6) kPa) and severe IVH groups (7.2 (5.9\u20137.6) kPa) (p = 0.003) and between infants with any IVH (6.2 (5.4\u20137.5) kPa) or no IVH (3.8 (2.7\u20134.3) kPa) (p < 0.001) after adjusting for differences in gestational age. The delta CO2 level during resuscitation weakly correlated with the delta CO2 level on the neonatal unit on days 2  and 3 .The delta EtCO2 on the delivery suite had an AUC of 0.856 and the delta CO2 during resuscitation had an AUC of 0.940. A delta EtCO2 equal or greater than 4.9 kPa predicted any IVH development with 96.4% sensitivity and 83.3% specificity.The receiver operator characteristic curve analysis demonstrated that in predicting any IVH, the maximum level of EtCO2 and greater degree of fluctuation of EtCO2 levels during resuscitation. In addition, we highlight that the delta EtCO2 level in the delivery suite had both a high sensitivity and specificity in predicting IVH development. Importantly, we have shown that abnormal EtCO2 levels during resuscitation did not necessarily translate into abnormal levels on the neonatal unit. Our findings, then suggest that it is early abnormal carbon dioxide levels in preterm infants that are important in IVH development. Previously, initial post-delivery levels of pCO2 were found to be significantly higher in infants with cerebral haemorrhage than those without, but by 8 h after birth, those differences were no longer significant [2 levels in the delivery suite, but not the neonatal unit, with IVH development. Some studies have suggested that sicker infants undergo more blood gas analysis than more stable infants, hence, allowing detection of a greater degree of fluctuation in levels of carbon dioxide [2 during resuscitation in all infants included in our cohort, and therefore, the outcomes relating to fluctuation of carbon dioxide are not influenced by the infant\u2019s degree of sickness. We speculate that by the time infants are admitted to the neonatal unit, clinicians have greater access to routine monitoring and would readily re-check abnormal levels of pCO2 by blood gas analysis, hence, limiting the degree of fluctuation between extremes of values and thereby achieving tighter control. This would explain our findings that it is the delivery suite levels of CO2 that are more influential regarding IVH development and why the extreme levels of carbon dioxide seen during resuscitation do not translate to neonatal unit levels of CO2 during the subsequent 72 h. We did not use volume targeted ventilation during the study period and this would have likely reduced fluctuations in CO2 levels on the NICU.We have demonstrated that preterm infants who developed an IVH had a higher maximum level of EtCOnificant , which a2 levels that were associated with IVH development, but this was no longer significant after adjustment for gestational age. Nevertheless, we highlight fluctuations in EtCO2 that are highly predictive of IVH development. Hence, it is important to avoid both high and low levels of EtCO2 during resuscitation. Our results emphasize that respiratory function monitoring should be helpful in the labour suite. It has been previously shown that resuscitators were unable to accurately assess chest wall movement [2 levels during resuscitation.As previously mentioned , we did movement , 12, andmovement . We havemovement . Eye tramovement  suggesti2 levels and fluctuation of EtCO2 levels during resuscitation and how they correlated with both carbon dioxide levels in the first 3 days after birth and IVH development. We monitored EtCO2 levels during resuscitation, yet on the neonatal unit, we have analysed levels of CO2 from blood gas measurements. Previous studies, however, have found EtCO2 to be as accurate as arterial monitoring when trending carbon dioxide levels [Our study has strengths and some limitations. This is the first study to evaluate EtCOe levels . We did 2 levels in the delivery suite was associated with a significantly increased risk in the development of IVH. Those results emphasise the need for EtCO2 monitoring during resuscitation in the delivery suite.In conclusion, a greater degree of fluctuation of EtCO"}
+{"text": "Mycobacterium tuberculosis. Under the hypoxic condition, phosphorylated DevR interacts with multiple binding sites at the promoter region of the target genes. In the present work, we carried out a detailed computational analysis to figure out the sensitivity of the kinetic parameters. The set of kinetic parameters takes care of the interaction among phosphorylated DevR and the binding sites, transcription and translation processes. We employ the method of stochastic optimization to quantitate the relevant kinetic parameter set necessary for DevR regulated gene expression. Measures of different correlation coefficients provide the relative ordering of kinetic parameters involved in gene regulation. Results obtained from correlation coefficients are further corroborated by sensitivity amplification.The DevRS two-component system plays a pivotal role in signal transmission and downstream gene regulation in  Mycobacterium tuberculosis . Co. CoRv313hspX. The ordering of the parameters related to expression of hspX in terms of correlation values (absolute)  compared to the other synthesis rates associated with hspX. On the other hand, the activation and inactivation rates (kb3 and ku3) related to the formation of active P1 (P1*) show a lesser correlation. These two opposing factors work together to determine the effective contribution of distal binding site P1 in the expression of hspX as reported by Chauhan et al. [KD (= ku/kb) value associated with the distal binding site P1 acts as a bottleneck for its proper functionality. Due to this reason, the mutant pmutP1 (mutation at P1) showed \u223c 30% of the wild type GFP expression, as reported by Park et al. [Next, we check sensitivity of the parameters associated with ute) see  areksm4k et al.  Fig 3C)hspX. Theksm7 which takes care of synthesis of GFP due to the cooperative effect of all the three binding sites  of hspX. The synthesis rates ksm5 and ksm6 due to P2 and S, respectively, come next. As P2 lies close to the transcription start point (TSP) of hspX, the GFP synthesis rate due to P2 is higher than that of S. It is important to note that, mutation at P2 (pmutP2) shows \u223c 53% of the wild type expression. Thus mutation at both primary binding sites P1 and P2 (pmutP1P2) shows only \u223c 12% of wild type expression and agrees with Park et al. [kb4 & kb5) and unbinding (ku4 & ku5) rates with GFP for the activation of P2 and S show that preference of RP is almost equal for P2 and S due to their close proximity.The next parameter that shows high correlation with GFP is k et al. . The cornarK2-Rv1738 system. For these pair of genes we quantify the correlation coefficients  starts producing the transcripts with maximum efficiency. Thus, activation of P1 serves as a bottleneck for narK2 transcripts. On a similar note, the parameter sets  and  controls the activation of the sites P2 and S2, respectively.Both the parameters Rv1738, the ordering of the parameters in terms of correlation values (absolute) are  reduces further and has a weak role in the overall production of transcripts. The weak contribution of P1 is observed in the expression profile of the mutant pBmutP1 [narK2). Transcription rate ksm13 from distal secondary site S2 is least and appears at the end of ordering. In between ksm9 and ksm13, the sensitivity of different binding rates appear.In  are see ksm19>ksRv3134c, hspX and narK2-Rv1738. The parameters with a high correlation coefficient with the output (GFP) indeed show a high level of sensitivity amplification Click here for additional data file.S1 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S2 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S3 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S4 Figksrp and kdrp) and GFP (ksg and kdg). Each panel consists of 103 independent simulation data.The output (GFP in nM) as a function of synthesis and degradation rates of phosphorylated DevR ((PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 TableList of all kinetic parameters used in the model.(PDF)Click here for additional data file.S2 Table5 indipendent run for synthesis and degradation rate parameters of four genes.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S3 Table5 indipendent run for all the input parameter with output of Rv3134c.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S4 Table5 indipendent run for all the input parameter with output of hspX.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S5 Table5 indipendent run for all the input parameter with output of narK2.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S6 Table5 indipendent run for all the input parameter with output of Rv1738.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file."}
+{"text": "H3K27me3) as a new immunohistochemistry (IHC) marker for MPNST have recently available to assist pathologists in differentiating MPNST from other histologic mimics. We aim to study the expression pattern of H3K27me3 in MPNST and its histologic mimickers and their association with the clinicopathological data. Diagnosis of malignant peripheral nerve sheath tumor (MPNST) is rather challenging due to its divergent morphologic heterogeneity and lack of specific ancillary test. The emergence of H3K27 trimethylation  for 3 tiers system . The clinicopathological data were retrieved from the patients\u2019 record. A total of 59 benign and malignant spindle cell tumours , 4 leiomyosarcoma, 1 spindle cell liposarcoma, 1 solitary fibrous tumour, 2 low grade fibromyxoid sarcoma and 1 unclassified spindle cell sarcoma), diagnosed from January 1998 to April 2018 in Hospital Universiti Sains Malaysia (HUSM) were tested for H3K27me3 expression which is statistically significant as compared to its histologic mimics (p<0.001). Similar findings (p=0.026) were also observed in high grade MPNST (81.8%), intermediate grade MPNST (100%) and 0% in low grade MPNST. A total of 61.1% (11/18 MPNST) showed loss of H3K27me3, combined with other panel of markers, is useful in MPNST diagnosis to differentiate it from the histological mimickers.  Malignant peripheral nerve sheath tumour (MPNST) is particularly rare with an incidence of 0.001%  in the general population and representing approximately 4% of all soft tissue sarcomas . This soft tissue neoplasm has gained an interest due to its diversity of histologic appearances, overlapping pattern, the rarity of the tumour, and the need of supportive and confirmatory tests which often renders MPNST as diagnostically challenging neoplasm. All these may justify to the delay in diagnosis. MPNST is rare, aggressive soft tissue sarcomas associated with poor prognosis. H3K27me3) as a new immunohistochemistry marker for MPNST have recently available to assist pathologists in differentiating MPNST from other histologic mimics. However, it also remains conflicting results for H3K27me3. Prieto-Granada et al., (2016) found that H3K27me3 immunohistochemistry has good sensitivity and robust specificity for the diagnosis of MPNST, while others noted loss of expression in other type of sarcomas too, which included radiation-associated angiosarcomas  and dedifferentiated chondrosarcoma . In this study we investigated the expression of H3K27me3 in differentiating MPNST from other types of spindle cell tumour and its association with its histological grading and clinicopathological data.The diagnosis of MPNST relies on morphology and IHC interpretation. Differentiating MPNST from other types of spindle cell neoplasm is important for prognosis and management. Several markers have been suggested to distinguish MPNST from other soft tissue tumours. These include S-100 protein, SOX10, epithelial membrane antigen (EMA), cytokeratins, CD34, HMB45 and desmin . None of these markers are specific for MPNSTs and mostly used to narrow down the diagnosis of spindle cell tumour. The emergence of H3K27 trimethylation . The clinicopathological data for all cases which included age, clinical presentation, site and depth of tumour involvement, tumour size, type of sample, patients\u2019progress such as presence of local recurrence and metastasis after diagnosis, were also obtained from patients\u2019 record notes. The study obtained its ethical clearance from the Ethical Committee, Universiti Sains Malaysia (JEPEM Code: USM/JEPeM/17020148).Histological assessment The tissue slides were deparaffinized and hydrated using standard procedures and stained with hematoxylin and eosin (H&E). The slides were viewed using Olympus CX31 light microscopy to assess the morphology of tumour and grading of MPNST. The grading system is based on tumour differentiation, mitotic count and tumour necrosis in FNCLCC grading system. A 3-tiered grading system with low-grade (LG), intermediate grade (IG) and high-grade (HG) MPNSTs is used, with the LG end being characterized by neurofibroma (NF)-like tumors composed of a cellular proliferation of bland spindle cells that appear to be in a continuum with the so-called atypical/cellular neurofibroma. The typical morphologic picture defining a conventional HG MPNST is represented by a fascicular, monomorphic spindle cell proliferation with a \u201cmarbled\u201d low-power appearance and areas of geographic necrosis. Finally, examples exhibiting histomorphologic features that deviated from these classic LG and HG patterns were classified under intermediate MPNST .Immunohistochemical (IHC) staining for H3K27 trimethylationH3K27me3 at 1:200 was applied to the sections. Sections of mimickers without primary antibodies served as negative controls, and sections of mimickers stained with primary antibodies for each antibody served as positive controls. All these were run together with each batch of staining. Tissue from colon was used as positive control for H3K27me3 staining.IHC staining was performed to the paraffin-embedded tissue blocks according to standard procedures (Abcam Code Ab192985) using pressure cooker antigen retrieval (Tris-EDTA buffer solution pH 9.0) method. The monoclonal primary antibody Its expression was quantified in the various samples examined using a scoring method adopted from Cleven et al., (2016). A mean percentage of nuclear staining was determined at a magnification of 400x, and scored 0 (0 cell positive), 1+ (1-24% of cell positive), 2+ (25-49% of cell positive) and 3+ (>50% of cell positive). The 0 score is considered loss of expression. Microscopy for immunoreactivity was evaluated by a single independent pathologist who is blinded to the case. Data were statistically analyzed by IBM SPSS version 24, using chi-square test or fisher-exact test. Epidemiological and clinicopathological data A total of 59 participants involved in the present study. A total of 18 MPNST cases and 41 of its histologic mimickers , 4 synovial sarcoma, 3 fibrosarcoma, 2 gastrointestinal stromal tumour (GIST), 4 leiomyosarcoma, 1 spindle cell liposarcoma, 1 solitary fibrous tumour, 2 low grade fibromyxoid sarcoma and 1 unclassified spindle cell sarcoma) were included in this study. The result of the data showed that the mean age of the participants is 40.76 years old (SD=17.21). Our study shows a bimodal distribution of MPNST patients which were most prevalence in the 12-year-old and younger age group and at 40-59 year old age group with mean age of diagnosis was 32.83 (SD=20.54). Ten out of 18 (55.6%) of MPNST cases were sporadic and 44.4% (8 out of 18) were NF-1 associated MPNST.Morphologically 14 cases (77.8%) were classical spindled type, 3 cases (16.7%) were malignant triton tumour and 1 case (5.6%) was epithelioid MPNST. For histologic grades, 77.8% (11/18) of MPNST cases were high grade, 11.1% (2/18) were intermediate grade and low grade cases respectively. In term of patients\u2019 outcomes, 7 out of 18 MPNST cases (38.9%) appeared with no local recurrence or distant metastases. Six out of 18 (33.3%) cases noted to have distant tumour metastasis at diagnosis and less than 5 years after diagnosis. Lungs, intraspinal and bone were the predominant sites of metastases. Three out of 18 cases (16.7%) show disease recurrence and 2 out of 18 cases (11.1%) resulted in death less than 5 years after diagnosis.Pattern of expression of H3K27me3 in MPNSTH3K27me3 in MPNST. Of the 18 tumours evaluated for H3K27me3 expression, 14 are high grade MPNST and remaining 2 each are of intermediate grade and low grade MPNST. Majority of high grade MPNST (9/14) and all of the intermediate grade MPNST showed loss of H3K27me3 expression. None of the low-grade cases show loss of H3K27me3 expression. However, 1 case of high grade MPNST (5.6%) show strong immunoreactivity towards H3K27me3 expression .We evaluated the expression of pression . In our Pattern of expression of H3K27me3 in MimickersH3K27me3   .In general, the malignant peripheral nerve sheath tumour is typically seen in adult life because most tumour occur in patients 20 to 50 years of age with a median age of about 35 . In our study, the median age was 28.5 years old ranged from the youngest age of 3 year old and the eldest was 81 year old. Although MPNST is most prevalent in adolescents and young adults, our study noted 4 cases (22.2%) of the MPNST cases were found in the 12 and younger age group. MPNST in childhood are recognized but rare and it may affect patient in a wide age range from birth to eighth decades. This was supported by Kudesia et al., (2014), in which the incidence of MPNST among paediatric patient, whom age less than 5 months old was only about 1.7%. In this study, we noted that among MPNST cases, 8 out of 18 patients has associated background history of neurofibromatosis 1 (NF1) with skin manifestation. Among patient with NF1, majority are diagnosed with MPNST at slightly earlier age with equal male to female distribution as compared to those with sporadic MPNST .In our study, we found that the commonest site of tumour were upper extremities, followed by the trunk and upper extremities. This findings showed similarity with previous studies . They found this tumour occur predominantly in the extremities, followed by the trunk/retroperitoneum and head and neck region. This findings explain the fact that most MPNSTs arise in association with major nerve trunks, including the sciatic nerve, brachial plexus and sacral plexus. Besides, it has been described at various anatomical sites including intraspinal and various visceral .A tumour can behave like benign, such as in the form of well circumscribed mass, superficial mass with indolent presentation and this may lead to delay in the patients\u2019 management. This type of tumour is either asymptomatic or may cause minimal discomfort. Many patients reported pain and neurological deficits at time of presentation. In our study, this commonly slow enlarging mass may exhibits rapid growth and we found that all of MPNST cases in our study presented with lesions more than 5cm as compared to benign peripheral nerve sheath tumour which in majority of cases presented with tumour less than 5cm at time of diagnosis. In majority of cases the duration of the tumour are less than 1 year upon diagnosis. MPNSTs have a relatively poor prognosis in comparison with other soft-tissue malignancies. The five-year survival for adults and children varies from 35% to 50% . Most MPNSTs are aggressive, high grade sarcomas with a high likelihood of local recurrence (40% to 65%), and distant metastases (40% to 68%.) . From previous case series, they found that MPNST frequently metastasize to the lungs followed by bone whereas lymph node metastases are uncommon . Other sites of metastasis include liver, brain, soft tissue, skin, and retroperitoneum. In our study, nine cases showed distant metastases and local recurrence with majority metastasize to the lung followed by other sites including intraspinal, liver and testes. However, the data from our study were limited because some of the patient had missing data or defaulted follow up. We had difficulty in tracing record of the patient and getting adequate information, especially those who were previously referred case and treated outside of our center. MPNSTs have poor outcome if untreated. Unfortunately, two of our patient developed lung metastases and succumbed to death after diagnosis with advanced MPNST in spite of a wide excision and additional chemotherapy. Although MPNST is rare in the paediatric age group, based on our study, diagnosis should be considered in children without NF1 with a rapidly evolving and painful mass in the distribution of a peripheral nerve. Early diagnosis and referral to multidisciplinary team are important in ensuring the best diagnosis and optimal therapy in this young age. In this study, we would like to emphasize on the important of this marker to help identify MPNST early in order for patient to get appropriate treatment earlier.From our study, the predominant subtype was spindle cell type (77.8%). It is very crucial to differentiate it from other type of spindle cell sarcoma. The diagnosis of spindle MPNST is challenging especially if the mass arising from unusual site. In one of our paediatric cases, it was arising from a pelvic region and the morphological pattern was purely spindle. In this case, synovial sarcoma was one of the differentials and interestingly it showed moderate to strong nuclear immunoreactivity to TLE1 in which rendered more challenges to the pathologist. However, TLE-1 positivity can also be seen in about 15% of MPNST and about 8% in Solitary Fibrous Tumour . Here, we could see the role of H3k27me3 in which it showed loss of its expression thus, it confirmed the diagnosis of MPNST especially when the molecular test is not available in the centre to demonstrate the presence of translocation t in synovial sarcoma.There are several reported cases of a rare spindle MPNST arising in a peripheral nerve, in which the clinical and radiological findings were indistinguishable from benign peripheral nerve sheath tumour, lead to insufficient initial treatment. Based on morphological criteria including high cellularity, nuclear atypia and mitotic activity, pathologist try to differentiate MPNST from atypical neurofibroma. Microscopically the tumours were composed of highly cellular spindle-shaped cells arranged in hypercellular and hypocellular areas with perivascular hypercellularity, characteristic of MPNST .H3K27me3 IHC. MPNST is the malignant counterpart of peripheral nerve sheath tumour and the main differential diagnosis. Logically a greater degree of cellularity, pleomorphism and higher mitotic activity are criteria used to classify the lesion as malignant. A distinctive, rare subtype of MPNST that raises separate differential is characterized by a predominance of large epithelioid cells, such as epithelioid MPNST. These tumours are relatively more common in superficial sites and strongly express S100 protein. In our study only one epithelioid MPNST case encountered and similar to previous study, it expressed diffuse and strong S100 protein and showed weak immunoreactivity towards Although S100 protein is a marker of nerve sheath differentiation, it has limited diagnostic utility as MPNSTs are often negative or very focally positive, (50-90%). Small percentage of MPNST cases (2%-15%) were found to express TLE-1. However the positive expression were either weak (1+) or moderate (2+) staining . Therefore, in practice usually strong staining (3+) for TLE-1 argues strongly against a diagnosis of MPNST. Accurate diagnosis is crucial in choosing the correct treatment given. In this study we found one of the cases was diagnosed and treated as Ewing Sarcoma as initial biopsy of the case was reported as possible Ewing sarcoma. However, the final diagnosis from the excised tumour, it was malignant peripheral nerve sheath tumour. The discrepancy of the diagnoses showed the heterogeneity of the tumour itself which may resembled different morphological features in a small biopsy. Over the past two decades, a few studies have shown that diagnostic agreement in soft tissue neoplasm between primary institutional diagnosis and reviewer diagnosis ranges from 53% to 73% . Overall diagnostic discrepancies range from 28% to 35%, of which minor discrepancies constitute 7% to 16% and major discrepancies 11% to 25% . The rarity of soft tissue tumours and its constantly evolving histopathological criteria, poses a challenge for general pathologists resulting in referral to centres with a soft tissue pathologist. The implication of this is that an inappropriate panel of IHC stains would be performed, with or without IHC misinterpretation . Thus, additional diagnostic markers are needed to aid in this often challenging differential diagnosis .H3K27me3 and altered DNA methylation .In a study on cancer development by epigenetic modifications, they have found that proteins of the polycomb group which are transcriptional repressors that modify chromatin, regulate cell fate, and promote cancer growth actually play an important role in pathogenesis of MPNST . In MPNST, inactivation of polycomb repressive complex II (PRC2) promotes cell proliferation and tumor growth. Inactivation of PRC2 is therefore expected to result in both loss of H3K27me3 could be of diagnostic aid . Other study suggest that PRC2 inactivation in MPNST may occur during progression to higher grades . In our study, immunohistochemistry of H3K27me3 proved to show loss of expression in 61.1% of MPNST, comparable to the findings of a previous study which showed 51% negative for H3K27me3 , and 69% negative for H3K27me3 of all MPNSTs . It showed that MPNST group have the highest number of H3K27me3 negative expression, while mimickers group mostly displayed strong immunoreactivity towards H3K27me3 (H3K27 expression and type of cases (p<0.001). These findings suggest that H3K27me3 loss is definitely a more specific marker for MPNST and useful in differential diagnosis, particularly for tumors that lack expression of supportive Schwann cell markers  and among high grade spindle cell sarcomas. Many studies had shown that inactivation of the polycomb repressive complex 2 (PRC2), has recently been identified in 70\u201390% of malignant peripheral nerve sheath tumour . The recent discovery of frequent inactivation of PRC2 in MPNSTs suggests that immunohistochemical detection of H3K27me3 . Our finH3K27me3 (61.1%) whereas only 7.3% were loss in non-MPNSTs (histologic mimickers). The findings were also observed by Cleven et al., (2016), who found that 24 out of 341 (7%) cases of other spindle cell tumours were negative for H3K27me3. From this study, we found that the marker also played a role in differentiating benign peripheral nerve sheath tumour and malignant peripheral nerve sheath tumour. In our study, both neurofibroma and schwannoma show moderate to strong expression of H3K27me3 with a p value of <0.001. This indicate that there is a significance difference in expression of H3K27me3 between benign peripheral nerve sheath tumour and malignant peripheral nerve sheath tumour . In our study, 25% of synovial sarcoma (SS) and 33.3% of fibrosarcomatous dermatofibrosarcoma protuberans (DFSP) showed loss of H3K27 trimethylation expression. The 25% of SS cases warrant molecular test for diagnostic confirmation. On the other hand in another study by Cleven et al. (2016) on MPNST and other spindle cell tumours, loss of H3K27me3 was reported in 60% of synovial sarcomas and 38% of DFSP. Our results are almost in agreement with the above study in which loss of expression was also observed in non-MPNST. This highlight the possibility that this antibody is not specific to MPNST. Majority of MPNST showed loss of h tumour . This isHowever, the marker was significantly showed to be useful in differential diagnosis of MPNST when combine with other ancillary test .H3K27me3 expression and MPNST histologic grades is another important finding. In our study, loss of H3K27me3 was observed in 64.3% of high grade, all of the intermediate grade and none of the low grade MPNST. Our study showed there was significant association between the histologic grades and H3K27me3 expression. Most of the H3K27me3-negative MPNST (9/18) were high grade with 3 cases showed local recurrence, 4 cases showed distant metastases and 2 cases end up with death while none of the low grade or intermediate grade having any local recurrence or distant metastases. This is in keeping with previous findings where loss of positivity for H3K27me3 is associated with poor prognosis in MPNST . In this study, there is no significant association between morphologic subtypes of MPNST and the expression of H3K27me3 immunoreactivity. This was supported by Prieto-Granada et al., (2016) where they found no significant differences in staining between different MPNST subtypes. However, all malignant triton tumour showed loss of expression and associated with poor prognosis and more aggressive behavior . We also found that there was no significant difference in the expression of H3K27me3 immunoreactivity between NF1 associated MPNST and sporadic MPNST.The association between H3K27me3 in majority of sporadic MPNST cases as compared to NF1-associated MPNST and conclude that H3K27me3 analysis has good sensitivity and robust specificity for the diagnosis of MPNST, particularly without clinical history of NF-1. Prieto-Granada et al., (2016) found that the sensitivity of H3K27me3 for sporadic MPNST and radiation-induced MPNST are 94 % and 93%, respectively. However, further study needs to be carried out to determine the sensitivity and specificity of this marker for diagnosis of MPNST. For the clinical management of these patients, it may be of value to be able to identify MPNST among these sarcomas to select patients who might be candidates for novel targeted therapeutic approaches .Recent study by Prieto-Granada et al., (2016) showed loss of expression of H3K27me3 is a potentially good immunohistochemical diagnostic marker. Though we found it is not to be a specific marker, but it definitely will give an added value when use in combination with other IHC panels especially in differentiating MPNST from its histologic mimickers. Since most of the panel of IHC in diagnosis MPNST are of limited value, our results had shown that"}
+{"text": "In the Surveillance Summary \u201cPrevalence of Autism Spectrum Disorder Among Children Aged 8 Years\u2014Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2016,\u201d errors occurred in the Abstract and Table 2 and Table 3.39%. On page 11, the second footnote of Table 2 should have referenced Supplementary Table 7. On page 11, the fourth column header in Table 3 should have been IQ \u226470. On page 1 in Results, the percentage of girls with intellectual disability should have been"}
+{"text": "Porcine circovirus-associated diseases (PCVADs) cause considerable economic losses in industrial pork production in the field. To minimize the economic losses due to PCVAD, porcine circovirus type 2 (PCV2) vaccines have been developed, and there is widespread vaccination worldwide today. However, limited information is available concerning the current status of PCV2 infection in the field on the Asian continent. The present study aimed to assess sero- and viral dynamics of PCV2 from 12 PCV2-contaminated pig herds with vaccination against PCV2 in Southern and Central Taiwan. In particular, the level of PCV2 load during the window period for seroconversion using real-time polymerase chain reaction and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Our results revealed that pig herds showed slight or no seroconversion after three to four weeks post-PCV2 immunization. The presence of PCV2 was observed during the window period for seroconversion in all herds. In conclusion, natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field. Circoviridae together with the genus Cyclovirus  was observed in the sow vaccination against PCV2 at 2-4 weeks pre-farrowing group, followed by the mass vaccination group , and the difference was not statistically significant between those two groups . The levThe serodynamics of the PCV2 antibody in pigs during the suckling to slaughter stages from different sow vaccination programs were measured by ELISA and are shown in 3 PCV2 genome copies/\u00b5l) or low PCV2 loads (<103 PCV2 genome copies/\u00b5L) indicated PCVAD or subclinical PCV2 infection, respectively [3 PCV2 genome copies/\u00b5L) was detected at 7 weeks old in 2 of 10 pigs in Farm A. Subsequently, ten of ten pigs (100%) and 9 of 10 pigs (90%) had PCV2 viremia at 11 and 15 weeks old, respectively  was detected at 11 weeks old in 4 of 10 pigs  in both Farm E and F, respectively . High PC 10 pigs . Subsequ 10 pigs .1.52 PCV2 genome copies/\u00b5L in Farm J (3 PCV2 genome copies/\u00b5L)  in Farm K . High PCn Farm J . This man Farm J . Subsequpies/\u00b5L) .Natural exposure to PCV2 should be considered starting at 11\u201312 weeks of age in the most analyzed pig farms. Regarding the production system , naturalPCV2 is one of the major primary infectious agents causing mortality in PRDC in the field. PCVADs have caused enormous economic losses in industrial pork production worldwide . FortunaTo compare the level of PCV2 MDA, it is not surprising that MDA was highly significantly lower in the sow without vaccination against PCV2 group than in the sow with mass or pre-farrowing vaccination groups. However, the highest CV of PCV2 MDA was also observed in the sow without PCV2 vaccination group. These results indicated that gilts and sows in pig herds have been naturally exposed to field viruses. Thus, sow vaccination against PCV2 should be a critical concern in the field. Our results indicated that a high level of PCV2 MDA was observed in the sow vaccination against PCV2 at 2\u20134 weeks in the pre-farrowing group, followed by the mass vaccination group. Fraile et al.  indicateTo compare the sero- and viral dynamics of PCV2, despite the pig production system, sow and piglet vaccination programs, all pig herds, except the Farm G herd, showed a decrease of PCV2 total antibody levels during 3\u20134 to 6\u20137 weeks of age, reaching the lowest levels of 0.25 to 1.24 S/P values. The natural exposure of PCV2 as measured by real-time PCR occurred in all herds, and seroconversion of PCV2 antibodies was subsequently observed. Regarding natural exposure of PCV2 during the growing to fattening period, better protection against natural exposure of PCV2 under different types of vaccine, vaccination timing or two shots of PCV2 immunization worth to further investigated. This result is in accordance with a previous study; natural PCV2 infection can occur late in growing to fattening pigs, and viremia can last very long until slaughter ,31. BaseConsistent with previous field observations ,11,12, nOur study revealed the cross-sectional nature of the sero- and viral dynamics of PCV2 in twelve PCV2-contaminated pig herds in the field. The presence of PCV2 was observed during the window period for seroconversion in all herds. Natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field."}
+{"text": "In the original article, there was a typographic error in the introduction regarding the human population and number of pigs in Denmark.The text should read:\u201cIn 2017 (3rd quarter), Denmark had 3,226 pig farms with 12.69 million animals (5) compared to a human population of 5.76 million individuals (6).\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "Entropy [1,*, Chi-Fang Chen 1,*, Wei-Chun Hu 1 and Pieretti Nadia 2Shashidhar Siddagangaiah that should be corrected to the following order in this paper [1,*, Chi-Fang Chen 1,*, Wei-Chun Hu 1 and Nadia Pieretti 2Shashidhar Siddagangaiah We have found an error in the order of one author\u2019s name and surname in the article recently published in Entropy :ShashidThe authors would like to apologize for any inconvenience caused to the readers by these changes."}
+{"text": "The current investigation provides information on supportive services caregivers said they would be interested in if they were made available. Participants were recruited from the Birmingham, AL metro area and received a $25 gift card for completing a telephone interview. Of the 38 caregivers enrolled, 18 (47.37%) reported being currently employed and working an average of 33.92 hours per week (range = 5 \u2013 60). Participants were caring for individuals with average scores on the AD8 Dementia Screening Scale of 7.3 and 10.4 on the Clinical Dementia Scale Sum of Boxes (above the cutoffs for probable dementia). Thirty-three caregivers (87%) reported being interested in at least one of the services listed with differences observed for services that would be preferred by working vs. non-working caregivers. These data will be utilized to provide initial support for a multicomponent intervention that may be effective in reducing negative outcomes within Black caregivers."}
+{"text": "FGF4 retrogene on CFA 12 have opened up new areas of investigation to further define the pathophysiology of premature IVDD. While preliminary data from studies investigating FGF4 retrogenes in IVDD implicate FGF4 overexpression as a major disease factor, they have also highlighted knowledge gaps in our understanding of intervertebral disc herniation which is a complex and multifactorial disease process.Premature degeneration of the intervertebral disc and its association with specific chondrodystrophic dog breeds has been recognized for over a century. Several lines of evidence including disease breed predisposition, studies suggesting heritability of premature intervertebral disc degeneration (IVDD) and association of a dog chromosome 12 (CFA 12) locus with intervertebral disc calcification have strongly supported a genetic component in IVDD in dogs. Recent studies documenting association of IVDD with an overexpressing  The list of inherited neurological disorders in companion and production animals is ever expanding. There are over 120 known genetic variants for neurological disorders in dogs alone . Beyond FGF4 associated with insertion of FGF4 retrogenes on CFA12 and CFA18 appear to have broader influences on limb length across many breeds and are the only genes to have been implicated in body size in across-breed association studies  and has been used to describe extreme differences in limb length in several dog breeds, such as the Basset Hound, Dachshund, and Pekingese . Historiral disc .Fibroblast Growth Factor 4 is most highly expressed in the embryonic ectoderm, axial, paraxial, and lateral plate mesoderm, and tail bud   for Hansen Type I intervertebral disc disease  and polyA tracts (class 1 templated sequence insertion polymorphism) , 3534, 3orphism) .FGF4 retrogenes have been associated with clinical phenotypes in dogs.Retrocopies have historically been considered to be inactive in most cases due to lack of appropriate regulatory elements as well as genetic alterations that remove open reading frames, and documented association with disease states is relatively uncommon. However, retrotransposition is believed to play an important role in genome evolution  and as tFGF4 retrogene is 3,209 bp long (Gen Bank accession no. MF040221) and consists of the parental chromosome 18 FGF4 exons  with common 5\u2032 UTR and exons but a shorter 3\u2032 UTR and is inserted within a LINE element on the parental chromosome 18]. The CFA12 FGF4 retrogene is transcriptionally active resulting in a 20-fold increase in FGF expression in neonatal intervertebral disc from dogs homozygous for the CFA12 FGF4 retrogene insertion compared to disc from neonatal dogs with just the parental FGF4 genes  appeared and produced an obvious phenotype, strong selection was likely applied to retain them.Although we have little ancient historical data documenting IVDD prior to the late nineteenth century , we do kFGF4 retrogenes generally reflects the morphological phenotype and susceptibility to IVDD we recognize in clinical practice (FGF4 retrogene copy (CFA12 or CFA18) tend to have skeletal dysplasia with moderately short limbs, while those carrying both copies, such as Dachshunds, Corgis and Bassett Hounds, have the most severe form of disproportionate dwarfism. The breeds with a higher frequency of the CFA12 FGF4 insertion are the same breeds identified in the last 50 years as being predisposed to IVDD , it does not appear to be directly associated with development of IVDD since several of the highly susceptible short legged breeds carrying the CFA12 FGF4 retrogene such as Beagles, French Bulldogs and Cocker Spaniels do not carry the CFA18 FGF4 retrogene and interestingly contrast with the short legged breeds such as Cairn Terriers and West Highland White Terriers that are rarely reported to have IVDD and carry only the CFA18 FGF4 retrogene . Sunsertion , 42.FGF4 retrogene was present in 40 breeds, the CFA18 FGF4 retrogene in 32 breeds and both retrogenes in 23 breeds  in the general population of breeds such as Beagles, Dachshunds, French Bulldogs, and most spaniel breeds, and not surprisingly, this frequency increases when looking at the population of dogs that present with clinical IVDD . Consistent with the skeletal dysplasia phenotype common to both retrogenes, presence of 1 or 2 copies of either the CFA12 or CFA18 FGF4 retrogenes was also associated with \u201csmaller\u201d dogs (based on body weight).Genotyping of over 3,000 dogs from 75 breeds showed that the CFA12 3 breeds  42). Th. ThFGF4 FGF4 retrogene is associated with a younger age of presentation for decompressive surgery (mean 6.1 vs. 8.5 years) (FGF4 retrogene had a significantly younger age at surgery (median 3.7 years) compared to other breeds homozygous for the CFA12 FGF4 retrogene such as Dachshunds (median age 6.5 years)   42). In. InFGF4 5 years) , 52 thatFGF4 retrogene frequency also described dogs with calcified (Type I) IVD to be significantly smaller (median 8.1 vs. 25 kg) and to have a significantly younger age at presentation for surgery (5.5 vs. 9 years) compared to non-calcified IVD (Type II) dogs  compared to surgically treated animals with fibrous/non-calcified disc herniation/protrusion   dogs . Supporty 0.149) .FGF4 retrogene (84.8%) compared to 1 copy of the CFA12 FGF4 retrogene (63.8%) compared to zero copies (18.5%)  . Multivao 1 copy . Presencfication .FGF4 retrogene location on chromosome 12 was done using high vs. low radiographic calcification scores in Dachshunds  Dachshunds. This population would be necessary for a successful genome mapping study and is consistent with the CFA12 FGF4 retrogene dosage effects on radiographically apparent calcification subsequently demonstrated following identification of the CFA12 retrogene , relative risk for IVDD  associated with the CFA12 FGF4 retrogene ranged from 5.5 in Chihuahuas to 15.1 in mixed breed dogs.Prospective data determining the risk for IVDD associated with presence of the CFA12 erformed . LookingFGF4 retrogene. Clarification of these pathophysiological and clinical variables in the presentation of IVDD is important if genotyping is to be used as a tool for reduction in disease incidence. A more comprehensive picture will also increase breeder and owner confidence for eradication strategies that may potentially require major alterations in breed standards for those breeds with high allele frequencies.Several aspects of the clinical presentation of IVDD in dogs are not easily explained by a simple presence or absence of the CFA12 FGF4 retrogene allele frequency  as the major determinant of ratio changes. However, several genetic factors are likely to be contributing to conformation including CFA12 and 18 FGF4 retrogenes. Clinically affected Dachshunds were reported to have longer epaxial muscle fascicle lengths in one study, which could be a secondary effect of clinical IVDD; however, it is interesting to note that FGF4 is also a key signaling molecule in the development of myofibers and tendon of epaxial muscles arising from somitic mesoderm  length were associated with increased risk in one study  while a mesoderm , 68.FGF4 retrogene status of dogs within the Standard Wire Haired population which can be inferred from the successful GWAS mapping using this Dachshund subtype (FGF4 retrogene (FGF5 gene (likely loss of function based on recessive inheritance)  , 70. Altng limbs , 71. Redng limbs  and it iPO2 gene . RSPO2 sPO2 gene , an impoPO2 gene  and downPO2 gene .FGF4 retrogene presence and other factors) based on breed has been reported  gene associated with screw tail and brachycephaly  would provide further insight into the relative effects of the two retrogenes.Co-expression of the CFA12 and CFA18 e modest , potentie modest . The CFA the IVD  althoughFGF4 retrogenes documented to date may facilitate its expression in other chromosomal locations as well. This provokes the question of how many other times the FGF4 gene has been retrotransposed and then eliminated by selective breeding, or if additional retrocopies remain in some breeds and are responsible for other morphological phenotypes or contribute to IVDD.The location of a large CpG island in the two FGF4 retrogene were found in 12% (46/378) of dogs presenting for IVDD related surgery and with documented presence of calcified intervertebral discs  are defined, may have specific molecular mechanistic similarities to FGF4 overexpression and IVDD  in dogs. However, less definitive data are available regarding genetics of IVDD and associated low back pain and sciatica in the general human population. Heritability of risk factors for IVDD in humans was initially established using twin studies  may sometimes conflict with dog breeding phenotypic goals.The most critical unanswered question relating to the CFA12 FGF4 retrogene has been seen in almost all IVDD affected breeds, even those with high allele frequencies. In breeds with a high degree of segregation it should be possible to reduce or eliminate the retrogene from the population.Positively, some degree of segregation of the CFA12 FGF4 retrogenes. In high IVDD risk breeds such as Dachshunds, Bassets, Corgis and Pekingese that have both the CFA12 and CFA18 FGF4 retrogene, breeding away from the CFA12 FGF4 retrogene, while still maintaining the aesthetically desirable shortness in stature contributed by the CFA18 FGF4 retrogene is possible.Even in high allele frequency breeds, such as the Dachshund, there are frequency differences between populations [0.98 in USA/UK samples and 0.94 in Swiss samples ], indicaFGF4 retrogene, so that the allele frequency can be reduced.The dominant nature of the CFA12 FGF4 retrogene on IVDD and the very high allele frequency in some breeds means that eradication may be challenging. The Wire Haired Dachshund was developed through crossbreeding with Schnauzer and Terrier breeds which may explain the lower frequency of the CFA12 susceptibility locus and lower incidence of IVDD in some wire haired populations. Long term strategies may require a combination of testing and selection of heterozygous dogs, outbreeding, cross breeding, and alteration in breed standards, maybe with inclusion of additional CFA12 FGF4 retrogene-negative phenotypes within the breed standards. Whatever the future holds, from a veterinary perspective, current data suggest that breeding priorities should be for dogs with fewer copies of the CFA12 All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The results showed that excess Na addition allowed us to compensate evaporation of sodium during the synthesis and sintering. Er2O3 was added as sintering aid to limit abnormal grain growth caused by h\u2013BN addition. The modification of amount of Na and Er2O3 addition resulted in high purity KNN samples with tetragonal structure and apparent density higher than 97%. Finally, piezoelectric features of prepared dense samples were measured and presented.Analysis of dense Potassium Sodium Niobate (KNN) ceramic obtained by hot pressing (HP) method at 1100 \u00b0C are presented in this paper. The synthesis of KNN-based piezoelectrics meets the following challenges\u2014low density of material, uncontrolled K/Na ratio, multiphase composition and formation of different KNN structures. The classical hot pressing approach results in contamination by carbon originating from graphite molds. The proposed hexagonal Boron Carbide (h-BN) layer between green sample and graphite mold could protect samples from carbon contamination. Additionally, the presence of h-BN may decrease the formation of oxygen vacancies, which allows us to maintain the semiconductor features of the KNN structure. Remaining issues were addressed with the addition of excess Na and Er T3. Its specific positioning combined with proper compositions stands for many functional properties, including ferromagnetism or thermoelectricity. KNN materials consist of two phases: ferroelectric potassium niobate (K1\u2212xNbO3) and antiferroelectric sodium niobate (NaxNbO3) which differ in transition but crystallize in the almost the same perovskite structure. The basic transition dependency of K/Na ratio results in possibility of obtaining functional perovskite structure with outstanding piezoelectric properties, given that mentioned stoichiometric proportions are preserved, because of morphotropic phase boundary (MPB) that it creates in the tetragonal-rhombohedral transition [Ferroelectric materials, such as described above (except for Polyvinylidene Fluoride-PVDF), used as piezoelectrics have the crystalline structure of perovskite, which is commonly known as a structure similar to BaTiOansition .The main challenge of implementing KNN lead-free piezoelectric materials is the proper synthesis. While material can be relatively simply obtained by high temperature processing of uniaxially pressed samples, its expected properties could be decreased due to the small density of samples. Amongst possible reasons of this situation, most possible are: evaporation of sodium, alkali vacancies created during sintering and lack of liquid phase in the microstructure of pure KNN ,8,9.2 and Ar atmospheres respectively showed, that similar improvements of properties can be obtained by changing the environment [In face of those problems, a proper solutions were pursued by many researching teams experimenting with both environment and composition. Shimizu et al. showed that pure alkali niobate ferroelectric and dielectric properties can be significantly improved using only low oxygen partial pressure processes . Those sironment . Moreoveironment .0.5Nb0.5)O3 (CSN), it was shown, that sealed ceramic samples were denser that their unsealed counterparts. The overall improvement of electrical properties was also noted [Yang et al., while obtaining transparent lead-free ferroelectric ceramics, went one step further, employing sealed sintering process and comparing it to sintering in air. Although used KNN materials were differing due to the doping of Ca(Scso noted . Additioso noted .0.44Na0.52Li0.04)(Nb0.86Ta0.10Sb0.04)O3 in which lithium subtracts either potassium or sodium atoms, while alkaline earth metals are in niobium positions [33) with relatively high conversion ratios. Due to those findings, later iterations of solution were to follow [In addition to process changes, various dopants were reported to improve KNN synthesis processes and were considered as a possible solution to this problem. The simplest idea of doping with alkaline earth metals was proposed by Mali\u010d et al. but was generally lacking significant, if any, positive effect on densification of samples . Previouositions  and resuo follow ,18, showo follow .3+ substitution in perovskite structure could reduce coupling factor of materials but greatly improve its other piezoelectric properties [2O3 which was investigated by Zhao et al. and shown that it could result in creation of highly desired liquid phase during sintering [Another chance of improvement came with the involvement of rare earth metals oxides. Those materials were expected to improve samples, based on previous research, performed to improve PZT material systems. Those studies showed that Yboperties . In caseoperties  alongsidintering .2O3, were employed. In comparison to previous research [The aim of this study was to obtain a dense KNN-based material, using industrially applicable process, using the listed findings and preliminary studies performed prior to this paper, which would be tested in stress sensor. To achieve this task, dopants of rare-earth elements, namely Erresearch , processresearch . The tex0.5Na0.5NbO3), the following commercial reactants were used\u2014Nb2O5 powder of CHANGSHA EASCHEM CO. Ltd.  and K2CO3, Na2CO3 carbonate powders produced by Lach-Ner . In the first step of KNN synthesis, the carbonate reactants were annealed at 150 \u00b0C for 24 h in order to remove residual water. Then K2CO3, Na2CO3 and Nb2O5 powders with 5 wt.% and 10 wt.% excess of sodium carbonate were mixed in ceramic mortar. The extra amount of Na2CO3 was added to compensate sodium evaporation process during synthesis. Thus prepared powders were dry homogenized in ball mill using 10.0 mm diameter silicon nitride milling media for 24 h. The reactants mixture were placed into alumina crucible with lid and heated up in air atmosphere to temperature of 950 \u00b0C, in which they were annealed for 2 h. The crushed and grinded KNN reaction bed was analyzed by X-ray diffraction method. The qualitative and quantitative X-ray Diffraction (XRD)/Rietveld phase composition analysis was made using PANalytical (model) diffractometer , equipped with Cu anode supported by X-pert HighScore software  for quantitative analysis of the samples.To synthesize KNN . having 0.65 m2/g specific surface area, 99.5% purity and 8.69 g/cm3 helium density. Due to company information average grain size of powder was estimated to d50 = 1.4 \u00b5m. The powder batches were dry homogenized in rotary mill using 5.0 mm Si3N4 grinding media for 24 h. Then mixtures were screened by 0.5 mm mesh sieve and placed in 25.0 mm diameter hot pressing graphite molds. To prevent excessive contamination of green samples by carbon, the graphite parts of the mold were covered by graphite foil and next it was sprayed by hexagonal boron nitride (h-BN) powder, as schematically showed in The obtained KNN powders were then mixed with 1 wt.% and 2 wt.% of Er2CO3 extra addition was manufactured in order to check: density, microstructure formation and phase composition. Cooling rate of HP process for those materials was very low of 3 \u00b0C/min. to reduce internal stresses caused by phase transformation and polycrystal formation during heat treatment.Prepared powders were hot pressed (HP) under 25 MPa of pressure for 2 h at 1100 \u00b0C in argon flow with heating/cooling rate of 10 \u00b0C/min and 8 \u00b0C/min. respectively. The applied pressure accelerated sintering, polycrystal densification and reduced evaporation of sodium. The obtained dense KNN sinters had diameter of 25.0 mm and thickness of 5.0 mm. Additionally, polycrystalline KNN reference sample without sintering activator and Na2O3 for O K\u03b1 line, Er2O3 for Er M\u03b1 and Er L\u03b1 lines. During calculations pressure variation method based on gas compensated technique was used. In order to confirm influence of HP applied pressure on material texturing, the reference sample was taken for ultrasonic measurement of longitudinal wave propagation in parallel and perpendicular directions to pressing axis. The ultrasonic examination was made by UZP-1  apparatus equipped with 1 MHz heads. In order to measure piezoelectric signal generated by samples in simulated real conditions, selected materials were subjected to poling process in custom built apparatus for 1 h in 2 kV/mm in silicon oil environment. It was followed by measurement of electric signal generated by piezoelectric properties of previously poled samples subjected to repetitive cyclic compression by custom built apparatus with force 150 N and 200 N for 2000 cycles. This equipment allows us to apply repetitive cyclic compression to rectangular or circular samples with set load between 100 to 1000 N. The analysis of these signals was performed by DasyLab 2016 software  in order to minimize distortions during measurements.The water contact with the sample is harmful due to the hygroscopic feature of KNN, for this reason, all samples handling was carefully proceeded in dry and no moisture conditions. The density measurements of KNN polycrystals was conducted in helium pycnometer AccuPyc II 1340 produced by Micrometrics Company . An average density value was calculated based on 30 measurement points. For microscopic observation purposes the sintered samples were included in resin and then polished by Struers polisher TegraPol-21 equipped with TegraForce-5 grinding discs under set load of 35 N and rotation speed of 300 rpm. The process was carried out in isopropanol environment. Surface preparation was done by diamond discs followed by final polishing with 0.25 um colloidal silica, which enabled mechanical etching of polycrystal to reveal microstructure details. Scanning electron microscope (SEM) FEI NOVA NANO SEM 200  was used for preliminary observations. Fracture morphology was recorded by use of secondary electrons (SE), while polished samples were observed in backscatter electron (BSE) mode. Detailed microstructural examinations were performed by use of scanning electron microscope FEI QUANTA 3D FEG SEM  equipped with energy dispersive spectrometer (EDS) . The applied energy of the electron beam was 15 keV (sufficient enough to excite the maximum intensity of Er L\u03b1 line) and beam current of 11 nA. Microstructure observations and microbeam analysis was performed in low vacuum conditions. Carbon dioxide gas was used to eliminate any reaction of the KNN with water vapor, which is the default gas in Variable Pressure SEM measurements. ZAF correction procedure was used for calculations. The following standards have been used for quantitative analysis: NaCl crystal for Na K\u03b1 line, KBr for K K\u03b1 line, pure niobium for Nb L\u03b1 line, Al4Nb10O30 in quantity of 83.5 wt.% and NaNbO3 in quantity of 11.5 wt.%, while planned stoichiometric KxNa1\u2212xNbO3 existed as minor phase in quantity of approximately 5.0 wt.%. That may be caused by deficiency of sodium in the system, due to evaporation occurred at both synthesis and hot-pressing steps. The X-ray diffraction patterns obtained for reference sample revealed presence of two major phases: K3 measured by helium method on the polycrystalline grinded sample. Then, the material was examined by SEM. Morphological features of KNN fracture are presented in Relative density of manufactured reference material was estimated as 98.4% based on the theoretical one of 4.55 g/cmThe ultrasonic analysis of longitudinal wave propagation examined in perpendicular directions of the reference sinter confirms texturing influence of HP process, what is visible in the wave propagation anisotropy reaching 25%. The measurement directions and wave velocity are collected in 0.5Na0.5NbO3, Er2O3 sintering activator was added because of its positive effect in formation of both orthorhombic and tetragonal structures which enhance piezoelectric properties of obtained sinters [2CO3 and addition of 1 wt.% or 2 wt.% of Er2O3 were subjected to density measurements with results collected in 3, which confirmed that all of samples have more than 97% of relative density which is enough for piezoelectric generator manufacturing. This suggests that material may be strengthened because of low concentration of microstructure defects. Complete density data is presented in In order to obtain materials containing K sinters . The hot2CO3 and Er2O3 sintering aid allowed us to obtain almost pure KNN material in the tetragonal crystallographic structure. The increase of Er2O3 from 1 wt.% to 2 wt.% content and excess of Na2CO3 addition from 5 wt.% to 10 wt.% led to orthorhombic structure of K0.5Na0.5NbO3 and small quantities of KNN doped by erbium. However, the same mixture with both additions of 10 wt.% of Na2CO3 and 2% Er2O3 resulted in shift to orthorhombic structure. Lattice parameters of detected samples are gathered in The results of quantitative and qualitative XRD analysis presented in 2O3 is presented in The obtained KNN sinters were subjected to microstructural and chemical analysis by SEM-EDS method. The material fracture with addition of 1 wt.% of Er2CO3 resulted in non-stoichiometric composition of KNN in some parts of the material. The nucleation rate of fine grains was previously confirmed to be enhanced by the excess amount of Na2CO3 introduced to the system [Results of average elemental composition measured by EDS technique for KNN taken from two randomly chosen flat areas of the polished N5E1 sample are collected in e system .2CO3 excess from 5 wt.% to 10 wt.% resulted in a much more uniform fractures morphology as shown in 2O3 from 1 wt.% to 2 wt.%. Based on the results of our previous work [xNa1\u2212xNbO3 were formed due to higher sodium content, which was enhanced by the excess amount of Na2CO3 introduced to the system.The N5E1 polycrystal is characterized by some elongated and geometrically oriented grains as marked in ous work , it can 2CO3 exhibited much lower values. This proves that small overabundance of Na in materials can be beneficial for piezoelectric properties but must be carefully optimized.Microstructure of sample N5E1 should have the best piezoelectric properties from all prepared samples thanks to its tetragonal crystallographic structure as shown in 2CO3 added to powder mixtures have a positive influence on maintaining the desired tetragonal KxNa1\u2212xNbO3 stoichiometry. Material with 5 wt.% excess of Na2CO3 and 1 wt.% of Er2O3 (N5E1 sample) which possess domain microstructure, generated highest electric signal of all samples for both 150 N and 200 N load cyclic compression. Combination of 10 wt.% Na2CO3 and 2 wt.% Er2O3 (N10E2 sample) additions led to extensive formation of monoclinic KNN with low piezoelectric properties. The hot pressing process applied in KNN manufacturing stimulated texturing of microstructure, which was confirmed by ultrasonic measurements indicating anisotropy of properties. In case of reference sample, an addition of hexagonal boron nitride leads to sintering intensification and extensive grain growth up to 50 \u00b5m. The experiment proved that it is possible to obtain KNN polycrystalline samples characterized by large dimensions. The diameter of obtained sinters was of 25 mm with thickness of 5 mm. The main phases detected were either orthorhombic or tetragonal KNN. Obtained sinters generated relatively high piezoelectric signal under cyclic compression.The layer of h-BN on green samples was initially introduced as protective layer to prevent the excessive carbon contamination during hot pressing. Morphology of the samples in the areas close to sample surface showed a formation of much larger grains comparing to the grains in the central area of samples, which could result from limited diffusion of boron inside the samples during hot pressing. The excess amount of Na"}
+{"text": "Scientific Reports 10.1038/srep23220, published online 21 March 2016Correction to: This Article contains an error in Figure 2. During the final preparation of the figures prior to publication, the wrong version of Figure 2 containing an incorrect graph legend was used. The correct Figure 2 appears below as Figure"}
+{"text": "Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9\u201310 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9\u201310 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9\u201310 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9\u201310 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9\u201310 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype\u2013phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases. The disease is characterized by a peculiar distribution of muscle weakness affecting facial and shoulder girdle muscles. Abdominal muscles also become affected, leading to a characteristic hyperlordotic posture. The selective and precocious weakness of tibialis anterior muscle is typical at lower limb, eventually followed by proximal leg muscles involvement4.Facioscapulohumeral muscular dystrophy  has prevalence of one in 8\u201320,0005. Two genetically distinct disease subtypes, FSHD1 and FSHD2, have been described up to now6. FSHD1 is associated with contractions of a polymorphic macrosatellite repeat on chromosome 4q35.27. This region consists of tandemly arrayed 3.3\u00a0kb D4Z4 repeat elements ranging from 11 to\u2009>\u2009100 repeat units in healthy subjects. Individuals displaying FSHD symptoms and carrying D4Z4 alleles with 10 or fewer repeat units are genetically defined as FSHD18. They represent 95% of people with FSHD.Presently, FSHD diagnosis is based on molecular findingshttp://www.musclegenetable.fr). FSHD1 and FSHD2, considered clinically undistinguishable, are characterized by DNA hypomethylation of the 4q35 D4Z4 array9.FSHD2 defines 5\u201310% of affected individuals carrying two D4Z4 arrays in the healthy range (>\u200910 repeat units). FSHD2 is defined as the cause of SMCHD1 mutation in OMIM and Gene Table  have been recently added to the list of possible contributors to disease onset and modifiers of disease severity, and also to explain a digenic inheritance for FSHD25; even though more extended analysis suggest the limited contribution of these factors27. This clinical and molecular complexity has complicated FSHD diagnosis, clinical practice and genetic counseling. Based on the above considerations, how can we interpret and use the result of the FSHD molecular test?FSHD is characterized by reduced penetrance and wide variability in the clinical expression among patients and within families28, making the definition of a clear cut-off point problematic.Here, we investigate the occurrence of the FSHD classical phenotype in 134 index cases and 110 relatives carrying alleles with 9\u201310 D4Z4 repeat units from the Italian National Registry for FSHD (INRF). Alleles of this size, named borderline D4Z4 reduced alleles (bDRA), are considered the upper size of the diagnostic range and pose the major diagnostic challenges. In fact, a wide phenotypic spectrum including atypical or incomplete phenotypes with no affected relatives is frequently observed in subjects carrying a bDRAwww.fshd.it)29. Out of 1340 index cases bearing a DRA with 1\u201310 repeats, we identified 166 subjects (14.6%) carrying a bDRA . Clinical and molecular analysis was extended to all available and willing to participate relatives. Phenotypic characterization was performed on 134 index cases and 110 relatives carrying a bDRA from 58 unrelated families. Seventy-six index cases did not have available relatives carrying a bDRA .In this study, we enrolled subjects carrying a bDRA. Index cases were identified through the INRF considering a 9-year time-window (2008\u20132016). The INRF database contains clinical and molecular data of subjects examined by the Italian Clinical Network for FSHD 31. Subjects were further classified on the basis of the clinical signs considering typical and atypical features, as listed in the CCEF : (1) subjects presenting facial and scapular girdle muscle weakness typical of FSHD , (2) subjects with muscle weakness limited to scapular girdle or facial muscles , (3) asymptomatic without any functional motor impairment (FSHD score 0) or healthy subjects , (4) subjects with myopathic phenotype presenting clinical features not consistent with FSHD canonical phenotype . Age at onset was estimated as self-reported anamnestic record32.We used the Comprehensive Clinical Evaluation Form (CCEF) for the clinical characterization of each subjectEcoRI, EcoRI/BlnI. Digested DNA was separated by pulsed field gel electrophoresis (PFGE) in 1% agarose gels, as previously described18. Allele sizes were estimated by Southern hybridization with probe p13E-11 of 7\u00a0\u03bcg of EcoRI-digested, EcoRI/BlnI-digested genomic DNA extracted from peripheral blood lymphocytes, electrophoresed in a 0.4% agarose gel, for 62\u201364\u00a0h at 35\u00a0V, alongside an 8\u201348\u00a0kb marker (Bio-Rad). Participants carrying alleles of 36\u201341\u00a0kb (9\u201310 D4Z4 units) in size were included in the study. Within the European population, pathological D4Z4 contractions usually are associated with the permissive 4qA haplotype in the subtelomeric region of chromosome 4q. The qA polymorphism was assessed by HindIII digestion and hybridization with qA probe. Restriction fragments were detected by autoradiography or by using the Typhoon Trio system . To verify that the obtained shortened D4Z4 fragment on chromosome 4 has a causative 4qA haplotype, an additional HindIII Southern blot was performed as suggested for the molecular diagnosis of FSHD133.DNA was prepared from isolated lymphocytes according to standard procedures. In brief, restriction endonuclease digestion of DNA was performed in agarose plugs with the appropriate restriction enzyme: t test or ANOVA.We used descriptive statistics for quantitative variables (mean and standard deviation) and qualitative variables (relative frequencies). Associations between qualitative variables were assessed using chi-square test or Fisher exact test. Associations between clinical parameters were described and tested using Pearson r correlation coefficient. Associations between quantitative variables and qualitative variables were evaluated using The INRF database was approved by the Provincial Ethics Committee of Modena (2712/CE). Informed written consent was obtained from all study participants, in accordance with the ethical standards of the 1964 Declaration of Helsinki.This manuscript does not contain any individual person\u2019s data in any form. Each patient was identified by a unique alphanumeric identification code and all data were made anonymous.The clinical characterization of 134 index cases carrying a bDRA  in all patients falling in categories A, B, or D. We therefore evaluated whether clinical categories may represent different stages of disease progression that is pre-symptomatic carriers are included in category C, mildly affected individuals are in category B and fully manifesting subjects are in category A. If this is the case one would expect category C subjects to be the youngest and category A the oldest. Figure\u00a0Among the 73 index cases classified as category A, 2 presented severe facial weakness and were classified as subcategory A1, 25 showed the classical pattern of facial weakness (subcategory A2), 46 presented mild facial involvement (subcategory A3) . Thus, subcategory B1 patients have a milder clinical phenotype.Facial-sparing scapular myopathy is often detected in clinical practice and it must be distinguished from other forms of myopathy including scapular peroneal syndromeThree index cases, aged 31, 52 and 70\u00a0years, were classified as subcategory B2 showing facial weakness without scapular girdle involvement. They presented a mild motor impairment, FSHD score 1, 1, 4 respectively. Two of them had isolated weakness of facial muscles.Out of 134 index cases, 25 index cases (18.7%) were classified as category D. Sixteen patients were identified as subcategory D1 for the presence of additional atypical features, more frequently including prevalent pelvic girdle weakness and axial involvement with bent spine and dropped head. Nine index cases (6.7%) presented phenotypes inconsistent with FSHD, such as isolated axial weakness (i.e. bent spine syndrome), or other clinical conditions described in Table\u00a0There was also the case of a woman  from 58 unrelated families  as reported in Supplementary Table\u00a01.We then analyzed whether the clinical category of the index case can predict the phenotype observed in the relatives carrying the same bDRA. We subdivided the probands in two groups on the basis of the CCEF phenotype observed in their relatives. One group included 19 probands whose relatives had classical FSHD or incomplete phenotype (categories A and B); the other group included 39 probands whose relatives were healthy or had a complex phenotype (categories C and D). This analysis shows that the distribution of clinical categories among the 58 probands subdivided on the basis of the phenotypic categories of their relatives does not significantly differ . Cases are distributed on the basis of the size of D4Z4 alleles reported as number of Repeat Units and the correspondent molecular weight as kilobases(kb).Supplementary Figure 2. Selection of index cases and their relatives carrying a bDRA for genotype\u2013phenotype correlation analysis.Supplementary Figure 3. Clinical categories of CCEF (from reference ).Supplementary Table 1. Distribution of probands on the basis of the clinical phenotype of relatives."}
+{"text": "An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.Cytoplasmic calcium ([Ca Because Ca2+ acts as a stress signal, it is one of the first responses plants have to environmental stimuli. This fast response is achieved through an influx of extracellular Ca2+ and an efflux of stored Ca2+ from the vacuole, endoplasmic reticulum (ER), and mitochondria that is achieved via a concentration gradient between the intracellular Ca2+ stores and the cytoplasm ..2+-bindiM domain .2+ sensors in plants are driven by constitutive promoters such as Cauliflower Mosaic Virus 35S (35S) and Ubiquitin 10 (UBQ10) [2+ sensors enabled the functional study of [Ca2+]cyt changes across entire plant organs [2+]cyt signature. While [Ca2+]cyt changes typically observed in the entire plant organ likely originate from more than one cell type, we lack tools to monitor these changes within specific tissues or cells cyt changes in roots treated with chemicals previously shown to induce [Ca2+]cyt increases in plants.To date, expression of CGaMP and other genetically-encoded Ca (UBQ10) ,13. Altht organs , it is lor cells . To addrromoters . We demo2+]cyt sensors that are available to the scientific community are driven by constitutive promoters. A. thaliana lines expressing the UBQ10:GCaMP3 construct, which we generated and used previously to study [Ca2+]cyt oscillations in root hairs   solution without ATP to roots. We found that applying MS solution triggered a rise in [Ca2+]cyt that was delayed and lower in amplitude compared to the [Ca2+]cyt change caused by ATP  . In mamm2+]cyt . While p [2+]cyt ,26,27. C decline A 5,25].,25.2+]cyd by ATP A. This oresponse ,20,27,282+]cyt signatures. For example, the PIN2:GCaMP3 lines, which expressed GCaMP3 in the epidermis and cortex , NaCl treatment resulted in cell-specific [Ca2+]cyt transients in the early elongation zone [UBQ10:GCaMP3 exhibited late-onset [Ca2+]cyt transients  in cells of the elongation zone  using freshly prepared MS plates for planting seeds and (2) pretreating roots two times with MS solution prior to application of the actual chemical solution. These modifications in our treatment protocols were tested using ATP which typically generated peaks with the highest amplitude.As shown in the preceding sections, the 0.5X MS control solution induced [Cad cortex . Althoug2+]cyt transients triggered by the MS solution were significantly dampened when fresh plates were used for the experiments . Under these modified growth and pretreatment conditions, the ATP-induced [Ca2+]cyt increases were lower in amplitude when compared to the [Ca2+]cyt increases shown in 2+]cyt transients, these [Ca2+]cyt responses were better separated from the [Ca2+]cyt responses induced by MS solution, particularly in lines expressing UBQ10:GCaMP3 and those in which GCaMP3 was expressed in root surface cell types and the cortex , and analyzed the relationship between chemicals using Analysis of Variance (ANOVA) with Tukey Post-Hoc test (3+ showed similar responses (except ATHB8:GCaMP3 to Al3+) while adapted ATP:MS showed significantly slower reaction times to both ATP and glutamate  [2+]cyt transients when compared to UBQ10:GCaMP3 lines , is required for root growth recovery after encountering high salinity conditions cyt transients in the cortex revealed by the PEP:GCaMP3-expressing lines could be a mechanism to maintain root growth integrity. The cell type GCaMP3 lines described here should be useful in testing this hypothesis.The line that was most similar to . (2010)  . The [Ca2+]cyt transients observed after MS treatment were reminiscent of touch-induced [Ca2+]cyt responses [2+]cyt signaling [2+]cyt imaging involved planting seed of the reporter lines on a thin layer of agarose to secure the root while at the same time allowing for the root cells to be accessible to the various chemical treatments  (http://neb.com) and cloned into a modified pCAMBIA1390 vector cyt-dependent GCaMP3 fluorescence, images of growing roots were acquired every 1 s for 10 to 15 min using the Leica SP8-X confocal microscope. Images were captured at a scanning speed of 600 MHz and pixel resolution of 512 \u00d7 300. Baseline GCaMP3 fluorescence of the roots was first acquired for 1 min after placing seedlings on the stage of the microscope. Twenty \u00b5L of the treatment solution was then added on top of the agarose medium where the root was growing using an adjustable volume pipette while imaging continued. From the collected images, the average fluorescence intensity was acquired by marking a rectangular region of interest corresponding to the specific cell type using the rectangular selection tool of the SPX-8 LAS software. GCaMP3 fluorescence (F) values were normalized using the formula To measure [Caonstruct . Another"}
+{"text": "S1 TableMeasurements were done on 3, 4 and 5 days-old seedlings. The presented values are the mean of twelve replicates per accession and treatment.(XLS)Click here for additional data file.S2 TableValues were obtained by dividing the RGR presented in (XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableThe threshold used to declare these SNPs significant is 5% Bonferroni. If the gene was found in the respective analysis, it is denoted with a 1 in the table, where a value of 0 indicates no significant association.(XLSX)Click here for additional data file."}
+{"text": "Figure 3 as published. Parts A and B were labeled in the incorrect order, the order of these panels within the figure was altered during reviewer responses and the figure legend was not updated correctly in the process. The correct legend appears below.In the original article, there was a mistake in the legend for Figure 3. (A) MCAv was determined continuously by transcranial doppler ultrasound (TCD) and presented as change in MCAv from rest in each protocol for each participant. (B) PETCO2 was determined by measurement of breath-by-breath respiratory gas exchange and ventilation. Data were averaged for 1 min during seated rest (Rest); over 30 s during the final minute of four 4 min high intensity (85% HRmax) interval bouts ; across the duration of four 30 s supramaximal (200% Wmax) sprint intervals , and over a 30 s period, 15 s into the recovery following each bout in both interval protocols (Reco 1 \u2013 4). Data are presented as mean \u00b1 SD (n = 24). Significance  between time points is denoted by * for differences from resting values;\u20ac for differences between interval (ex) and recovery (reco). Significant differences between protocols are denoted by \u03a9.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "The authors issue this Correction to address the following issues that were noted after this article  was publIn Questions were raised about the lack of loading control data reported in  for the As demonstrated in Fig 2A and For Figs 2B-2D, different amounts of purified iNOSfl proteins were loaded in the plus versus minus CaM lanes so as to obtain approximately equivalent iNOSfl levels (130 KD bands) across lanes within each gel or blot. Loading volumes needed to achieve equivalent iNOSfl levels and were determined through standardization experiments for which data were not reported in the article. The iNOS blots in Fig 2B and 2D served as the controls for the CaM blot in Fig 2B and the heme gel in Fig 2C, respectively. For each of these experiments, duplicate gels were prepared for the control and experimental assays by loading the same purified iNOSfl proteins on parallel gels.In The article\u2019s Data Availability statement says, \u201cAll relevant data are within the paper.\u201d The underlying data were not included with  but are PLOS ONE\u2019s Editorial Board reviewed the updated figures, supporting data files, and information provided above regarding western blot controls and advised that they address the previous concerns and errors in the original manuscript. The Academic Editor commented that the corrections do not change the article\u2019s conclusions.A member of The authors apologize for the errors in the published article.S1 File(PPT)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 FileThe blot image provided for Fig 2D is a digital image of the original blot reported in the published figure; levels were adjusted in the image file so that band intensities would align approximately with bands observed by the Ponceau S staining. The original film from the Fig 2D blot experiment is no longer available.(ZIP)Click here for additional data file.S4 File(TIF)Click here for additional data file.S5 File(JPG)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(ZIP)Click here for additional data file.S9 File(ZIP)Click here for additional data file."}
+{"text": "Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity. Published 12, October 2018We recently discovered an error in the original Figure 3A, in which the RIP quantification figure in Suz12 was accidentally duplicated from Ezh2. This occurred inadvertently during the conversion of histogram plot to dot format with mislabeled file name of raw data. The figure has been corrected so that the corrected Figure 3A now reflects the correct quantification. The correction does not affect or change any of the conclusions of the manuscript. We apologize for the mistake and any inconvenience or confusion this error may have caused:Corrected Figure 3Original Figure 3The article has been corrected accordingly."}
+{"text": "KCNE1B blurs the genetic basis of KCNE1-LQTS patients\u201d [KCNE1B, absent from the GRCh37 version of the human genome, is an artifact introduced in GRCh38:KCNE1 (T) is different from the reference nucleotide for the paralogue position of the KCNE1B pseudogene (C), according to GRCh38  capture panel [KCNE1 SNV, while the mother is wild type. PCR was performed using primers that, according to GRCh38, should not be able to distinguish between KCNE1 and KCNE1B , the mother is wild type and, therefore, the daughter could not possibly inherit two variant alleles from her parents  and homozygous KCNE1 reference (C/C) genotypes for rs1805128, with no sign of the reference KCNE1B (T) allele (Supplementary Table\u00a0We have PCR amplified the regions containing the positions for rs1805127 and rs1805128 SNPs on DNA from lymphoblastoid cell lines from ten individuals representing ten different ethnic origins from four continents in the same conditions unable to distinguish In regard to the Viewpoint by Pantou et al. \u201cThe potential presence of the highly similar paralogue gene KCNE1B is an artifact that should be reviewed and curated. This would reduce the probabilities of false negative results caused by misalignment of variant reads originating from the true and clinically relevant KCNE1 locus to the KCNE1B region of the GRCh38 reference genome.In our opinion, these data, together with those presented by Pantou et al., strongly suggest that Supplementary Materials and MethodsSupplementary Table 1Supplementary Table 2Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3"}
+{"text": "Scanning tunnelling microscopy is used to compare the properties of PTCDA molecules adsorbed on a partly CaF1-covered Si(111) surface with deposition on thicker CaF2/CaF1/Si(111) films. The identification of mostly single molecules on the CaF1/Si(111) interface layer is explained by the presence of atomic-size defects within this layer. Geometry-optimisation calculations using density functional theory reveal a geometry on CaF2(111) of nearly flat-lying PTCDA molecules with two oxygen atoms displaced towards calcium surface ions. This geometry is in agreement with the experimental observations.Thin insulating films are commonly employed for the electronic decoupling of molecules as they enable a preservation of the intrinsic molecular electronic functionality. Here, the molecular properties of 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) adsorbed on insulating CaF The study of molecular adsorption on thin insulating films is motivated by the possibility to investigate and utilise molecular properties in their largely undisturbed state . MoleculA particularly well-studied case of surface-specific molecular properties is the adsorption of 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) on metal \u201312, semi2 thin films grown on Si(111) surfaces, with PTCDA as the probe molecule. In particular, the adsorption properties of PTCDA on CaF2/CaF1/Si(111), CaF1/Si(111), as well as Si(111)-(7 \u00d7 7) surfaces are experimentally investigated by high-resolution scanning tunnelling microscopy (STM) and the adsorption geometry on a CaF2(111) slab is theoretically modelled using density functional theory (DFT). A prominent difference of the molecular properties on the different surface areas is the presence of mostly single molecules in CaF1/Si(111) regions, while ultrasmall molecular assemblies are experimentally observed on thicker CaF2 films. A rather flat-lying geometry is found from geometry-optimisation calculations of a single PTCDA molecule on a CaF2 slab using DFT, whereby an interaction between two carbonyl oxygen atoms and two surface calcium ions leads to a slight deformation of the PTCDA molecule.Here, the understanding of molecular adsorption on insulating thin films is extended by studying an insulator-on-semiconductor system, namely CaFSample preparation and STM experiments were performed under ultrahigh-vacuum conditions. Highly B-doped p-type Si(111) samples  were used as substrates. The (7 \u00d7 7) reconstruction was formed by flash cycles and the (7 \u00d7 7) surface quality was checked by STM imaging.2 material  was deposited from an e-beam evaporator  on silicon samples held at about 600 \u00b0C by direct-current heating. Further details on the sample preparation and CaF2/Si(111) surface properties can be found in [Ub. Gwyddion [CaFfound in \u201329. PTCDfound in . Image dGwyddion  was used2 triple layers. The lowest triple layer was held fixed. A tolerance of 10\u22124 Ha/Bohr was used.Density functional theory calculations were performed using cp2k (http://www.cp2k.org)  and para1-covered Si(111)-(7 \u00d7 7) and CaF2/CaF1/Si(111) surfaces. Representative overview images acquired at 77 K on the three different surfaces areas are reproduced in PTCDA adsorption was studied by STM after deposition on partly CaF2 films on Si(111) requires the formation of a CaF1 interface layer as the first step. This interface layer is generated by an interface reaction between CaF2 and the silicon surface [2 to CaF1 and F. The dissociated fluorine atoms mostly desorb from the surface, likely in the form of SixF molecules [2 layers can then be grown stoichiometrically on the interface layer by successive CaF2 deposition. The CaF1/Si(111) surface has a (1 \u00d7 1) termination after etching the Si(111)-(7 \u00d7 7) reconstruction.The growth of ordered CaF surface \u201329, wherolecules \u201329. Thic1/Si(111) areas  thick film is shown in On the CaFsee also  is the psee also  was only1 and CaF2 areas. A detailed statistical analysis is shown in 2 direction from more than 1000 molecules in a total of 10 images. The CaF2 directions were determined from filled-state imaging of the (7 \u00d7 7) reconstruction [2 on Si(111) at the chosen growth parameters, the CaF2 direction is identical to the Si  direction that was determined from STM images. Three equivalent CaF2 directions are depicted at the right of the exemplary image in 2 direction, especially after deposition on samples held at room temperature. The width of the peaks is attributed to uncertainties of the angle measurement and the asymmetry to a statistical artefact. However, it is also noteworthy that the intermediate minimum in the distribution is less pronounced in measurements performed after PTCDA deposition on cooled samples, which suggests an increased barrier at the estimated sample temperature of 140 K to arrive in the optimum adsorption geometry.The STM data in truction . Due to 1/Si(111) and CaF2/CaF1/Si(111) areas from measuring the pairwise molecular separations of a total of about 400 (1600) molecules on the CaF1 (CaF2) area in the STM image in 2/CaF1/Si(111) areas, while no clear preference is apparent from the distribution on the CaF1/Si(111) interface layer areas. As the surface lattice periodicities are identical for the interface and subsequent layers due to the extremely small mismatch between the silicon and CaF2 lattice constants [1 areas can rather be explained by nucleation at defects present in the interface layer. This is substantiated by data in 2 surfaces are mostly defect-free.A second statistical analysis was performed for the nearest-neighbour distances between nearby PTCDA molecules on CaFonstants , the abs2(111) slab (only the top layer is shown in 2(111) surface. In this geometry, the PTCDA molecule is aligned with the long axis along the  direction, which corresponds to an alignment with an angle of 30\u00b0 off a CaF2 direction  CaF2(111) [2(111) is likely the result of an interplay between the carbonyl oxygen\u2013calcium attraction and the repulsion between the other oxygen atoms and the surface fluorine atoms, as well as the resulting deformation of the PTCDA molecule.The PTCDA molecule is in a nearly flat-lying geometry, with the exception that two of the four carbonyl oxygen atoms are displaced out of the PTCDA plane towards a surface calcium ion. An attraction between the molecular oxygen and surface calcium atoms is suggested from an oxygen\u2013calcium distance of about 2.8 \u00c5. Note that this distance is larger than 2.4 \u00c5 recently found for the oxygen\u2013calcium distance of a carboxylic acid moiety of a ferrocene derivative that binds vertically to CaF2(111) . The oxy3 of the electron density difference \u0394\u03c1 = \u03c1full \u2212 \u03c1slab \u2212 \u03c1PTCDA. This difference was calculated as a difference between the electron densities of the full (\u03c1full), CaF2 slab (\u03c1slab), and PTCDA gas phase (\u03c1PTCDA) systems. The main finding is electron accumulation below the carbonyl oxygen atoms, in agreement with the attractive interaction with the surface calcium atom already identified before from the oxygen displacement.2(111) system are depicted in 1 thin film is of strong LUMO character, whereas the filled-state image  as well as the three lowest unoccupied molecular orbitals  as calculated with cp2k for the PTCDA/CaFte image  on the Chin film  reveals A slightly deformed adsorption geometry including a small tilt of the molecule with respect to the surface plane is furthermore in agreement with the experimental observation of a slight asymmetry in the imaged lobe height, see for example 1/Si(111), and CaF2/CaF1/Si(111) surface areas were studied by STM at low temperatures. Single molecules were identified on the CaF1/Si(111) interface layer while ultrasmall molecular assemblies were found on the CaF2/CaF1/Si(111) areas. The presence of mostly single PTCDA molecules in CaF1 regions is rationalised by nucleation at defects present within the CaF1 interface layer. In contrast, the CaF2/CaF1 layer is mostly defect-free. A statistical analysis revealed a preferred molecular orientation of the long molecular axis along a CaF2 direction, in full agreement with the DFT-calculated optimum adsorption geometry. The DFT-based analysis furthermore revealed a nearly flat-lying molecular adsorption geometry with a downward displacement of two carbonyl oxygen atoms. Based on an analysis of the electron density difference, these atoms are attracted towards the surface calcium ions. A comparison of calculated molecular orbital shapes with the experimental STM data suggests a strong influence of the LUMO in filled-state STM imaging on the CaF1 interface layer. Instead, the absence of long-range order on the CaF2 films is explained by a mismatch of the common PTCDA motifs with the CaF2 surface structure.Adsorption properties of PTCDA molecules on Si(111)-(7 \u00d7 7), CaF"}
+{"text": "In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the  jnk gene family is known to act within this pathway but its specific role in heart development has been difficult to establish using mouse mutants because genes within the family overlap in function and embryos mutant for more than one jnk gene die before the heart is fully formed. In these studies, we used zebrafish to determine the role of jnk1 in heart development and found that a specific gene transcript from zebrafish jnk1a is needed to produce the full complement of cardiomyocytes that form the first part of the ventricular heart chamber. We show that this happens because jnk1a is required for the normal expression of hand2, a gene required to make ventricular heart cells. Furthermore, we can fully reverse the abnormalities in jnk1a embryos by increasing hand2 gene activity. This part of the zebrafish ventricular chamber is equivalent to the left ventricle in humans and understanding how jnk genes work may help understand why some human heart malformations are associated with an underdeveloped left ventricle.The planar cell polarity pathway is a genetic cascade important for normal development of the heart. The  Vangl2, Dvl2 and Celsr1 have been shown to cause cardiac malformations  \u20135, but fiewed in ), specifiewed in . Inactiviewed in ,9 and Rhiewed in  also proSOS1 found in patients with Noonan syndrome, has been shown to directly activate Jnk  x35 cycles, 72\u00b0C (5min). These conditions ensured products obtained were within the linear phase of PCR (jnk1a) and NheI-HF  and gel densitometry performed using Fiji [ef1\u03b1 value and with regard to the efficiency of the PCR reaction as established with plasmids containing full length transcripts  using a high-speed digital video camera (640x480 pixels at 127 frames/sec) . Data was analysed using Fiji  and simuEmbryos were incubated in 10mM BrdU  in 15% DMSO for 30 min on ice at the 10-somite stage (ss). Then, embryos were washed in E3 medium and allowed to develop until 12ss. After fixing in 4% PFA overnight at 4\u00b0C, embryos were stored in methanol at -20\u00b0C. Embryos were rehydrated in PBS, then permeabilized using Proteinase K and post-fixed in PFA 4%. After incubation in 2N HCl for 1 hour, standard immunohistochemistry using anti-BrdU and anti-GFP antibodies was carried out. The proportion of BrdU-positive cardiomyocytes was assessed as above.TUNEL staining  was performed as previously described  in wholemyl7:gfp embryos at the 14 ss were hand dechorionated and oriented in 1.5% low melting agarose. A 20-image stack , using a 20x objective at 3\u03bcm intervals, was acquired every 6 minutes between 15\u201316 ss to 19\u201320 ss. Manual tracking was performed using Fiji [An established protocol  with mining Fiji  and XY ci, yi) and the final  coordinatesMean speed calculated from each measurable displacement per unit time between the initial  and the final  coordinatesMean velocity calculated from the absolute displacement per unit time between initial  and, depending on the number of groups to compare, either Student t test or ANOVA with Tukey's post-hoc analysis was performed. Data is presented as mean and SEM.S1 Figjnk1a and jnk1b genes are capable of producing exon 7 and short C-terminus containing transcripts that fully match human JNK1 transcripts. However, whilst Ex8 derived from jnk1b matches the human peptide the Ex8 from jnk1a is divergent and contains a serine rather than threonine residue (*). In contrast whilst the long C-terminal extension provided by jnk1a matches the human, the jnk1b long terminal extension is highly divergent and differs by 9/39 amino acids including insertion of an additional threonine residue.Black text indicates identical amino acids, green text indicates favourable amino acid substitutions and red text divergent amino acid residues. Exons 2\u20136 and 9\u201312 are common to all transcripts and highly conserved. Both (TIF)Click here for additional data file.S2 FigA) specificity of primers used in RT-PCR splicing assay. Primers sets to identify Ex7 and Ex8 containing variants of both jnk1a and jnk1b transcripts were assessed against all 8 transcripts contained within plasmids, using primers for the plasmid backbone (M13) as loading control. Restriction enzyme digests showing original PCR product and complete digestion by enzyme to identify C-terminal extension in jnk1a and jnk1b transcripts. (B) Graphical representation of gel densitometry indicating the splicing assay was carried out during the linear phase of amplification for jnk1a and jnk1b Ex7 and Ex8 PCR (Arbitrary logarithmic Units). (C) PCR with plasmid containing transcripts were used as positive controls for PCR reactions and indicated efficiency of each reaction.((TIF)Click here for additional data file.S3 FigA) Prior to heart formation all transcripts are present throughout the embryo at the start of gastrulation (6hpf) and the end of gastrulation (10hpf). The most abundant transcripts by RT-PCR splicing assay are indicated by dashed lines. (B) WISH for all jnk1a and jnk1b alternatively spliced transcripts at 24hpf and 48hpf. Transcripts with obvious expression within heart\u2014jnk1a Ex7Lg and jnk1b Ex8Sh\u2014are indicated by arrows. (C) Close up of heart in all jnk1a transcripts at 24 and 48hpf. Outline of heart indicated by dashed lines. Absent expression indicated by arrow heads and expression in heart indicated by arrows in jnk1a Ex7Lg, jnk1a Ex8Lg and jnk1b Ex8Sh. (D) WISH demonstrated expression of jnk2 and jnk3 in the developing brain (arrows) but not heart (arrowheads). (E) RT-PCR of extracted hearts indicates that jnk2 is expressed at low level at 24 and 48 hpf, whilst there is late onset of jnk3 gene expression by 48hpf. ef1alpha is used as a loading control.Chromogenic WISH is not quantitative but indicates the extent of expression. ((TIF)Click here for additional data file.S4 Fign = 3 clutches in all cases.(TIF)Click here for additional data file.S5 FigA) Cardiac morphology at 28 hpf. All ventricular cardiomyocytes are labelled by MF20 antibody (red) and atrial cells by both MF20 and S46 antibody (appear yellow). Normal appearances seen in wildtype (wt) embryos. Ventricular hypoplasia (indicated by size of white bar) is seen in MZjnk1a null mutants but is not seen in MZjnk1b null mutants. There is no additional ventricular hypoplasia in MZjnk1a/MZjnk1b double mutants. (B) MF20 antibody staining to identify somites/skeletal muscle blocks in wild type (wt) and MZjnk1a/MZjnk1b mutants (C) Somite counting excludes global somatic developmental delay in MZjnk1a/MZjnk1b null mutants.  TUNEL labelling at 12ss stage in wild type (wt) and MZjnk1a/MZjnk1b mutants. Myocytes in the ALPM are identified by Mef2 antibody. The percentage TUNEL positive cells in Mef2 population is unchanged in MZjnk1a/MZjnk1b null mutants.  BrdU incorporation in cardiomyocytes from 10ss to 12ss. Quantification shows that there is no difference in BrdU incorporation index in MZjnk1a/MZjnk1b null mutants compared to wild type (wt) controls. ns = nonsignificant.((TIF)Click here for additional data file.S1 TableGuide RNA sequences of CRISPR Cas9 mutant production and Genotyping primers used for selection of mutants. Transcript cloning indicated are transcript sequences; add additional sequence for Gateway or restriction enzyme cloning. RT-PCR splicing assay primers, see RT-PCR primers for jnk2 and jnk3 and in-situ hybridization primer sequences used to produce sense and non-sense probes.(DOCX)Click here for additional data file.S2 TableNucleotide sequences for all jnk1a and jnk1b transcripts based on results of bacterial subcloning and translation into peptide sequences.(DOCX)Click here for additional data file.S1 Movie(MOV)Click here for additional data file.S2 Movie(MOV)Click here for additional data file."}
+{"text": "Dear Editor,IDH1-mutated relapsed or refractory acute myeloid leukemia (AML) showed an overall response rate of 41.6% and a complete remission rate of 21.6% with a median duration of response of 8.2\u00a0months [IDH1 mutations do not show a clear prognostic effect in AML patients who are treated with standard induction and consolidation therapy [The first clinical IDH1 inhibitor ivosidenib as a single agent in  therapy . It is uWe evaluated the mIDH1 inhibitor BAY1436032 in sequential or simultaneous combination with cytarabine plus doxorubicin in a previously reported IDH1 mutant PDX mouse model , the per2The findings from our preclinical study show that simultaneously combining an IDH1mutant inhibitor with cytarabine plus doxorubicin substantially inhibits leukemia in vivo. These findings are in accordance with our previous study in which we showed a synergistic effect of simultaneous administration of an IDH1 inhibitor with the hypomethylating agent azacitidine compared with sequential administration .\u00a0A phaseESM 1(DOCX 22 kb)"}
+{"text": "Understanding the optical and electronic mechanisms that govern charge separation, transport and recombination in these devices will enhance their current conversion efficiencies under illumination to sunlight. In this work, density functional theory with Perdew-Burke Ernzerhof (PBE) functional approach was used to explore the optical and electronic properties of three modeled TiO2 brookite clusters, (TiO2)n=5,8,68. The simulated optical absorption spectra for (TiO2)5 and (TiO2)8 clusters show excitation around 200\u2013400 nm, with (TiO2)8 cluster showing higher absorbance than the corresponding (TiO2)5 cluster. The density of states and the projected density of states of the clusters were computed using Grid-base Projector Augmented Wave (GPAW) and PBE exchange correlation functional in a bid to further understand their electronic structure. The density of states spectra reveal surface valence and conduction bands separated by a band gap of 1.10, 2.31, and 1.37 eV for (TiO2)5, (TiO2)8, and (TiO2)68 clusters, respectively. Adsorption of croconate dyes onto the cluster shifted the absorption peaks to higher wavelengths.A range of solution-processed organic and hybrid organic\u2212inorganic solar cells, such as dye-sensitized and bulk heterojunction organic solar cells have been intensely developed recently. TiO In recent years, there has been alarming increase in energy demand across the globe ,2,3; fos2 has attracted a wide range of interests in the research community owing to its proven ability for many applications in the medical field .The absorption spectra of (TiOT method  in which2)5 and (TiO2)8 complex presented were computed using GPAW and TD-DFT [2)5 also shows a slight absorption peak at around 400 nm, suggesting the absorption peak at higher wavelength than (TiO2)8. Maximum peak height indicates that the higher absorbance is notable in (TiO2)8 brookite absorption spectra than in the (TiO2)5 absorption spectra. The results of the absorption spectra of (TiO2)5 and (TiO2)8 generally agrees with findings from literature in the sense that TiO2 is majorly sensitive in the UV region of the solar spectrum owing to its wide band gap (3.0\u20133.2 eV) [The absorption spectra of  ,65.2)5, (TiO2)8 and (TiO2)68 nanoclusters were computed using GPAW [2)5, (TiO2)8 and (TiO2)68 are presented in The density of states and the projected density of states of  state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals as illustrated in d and p orbitals, while contributions from oxygen atoms are minimal.The density of states (DOS) is composed of the surface valence and conduction bands separated by a wide band gap. The PDOS results for the clusters show that both the oxygen and titanium atomic orbitals contributes to the valence states, with the oxygen 22 investigated in this work is shown in the 2 brookite semiconductor material employed in this study was used to generate models of the three TiO2 clusters. Brookite TiO2 has an orthorhombic crystalline structure with eight formula units in the orthorhombic cell.The structural model for brookite polymorph of TiOThe optimized molecular geometries of the croconate dyes CR1 and CR2 considered for this study are presented in the 3), which is an alkyl derived from methane, with one carbon atom bonded to three hydrogen atoms while CR2 contains an electron withdrawing carboxyl group (\u2013COOH), which is an organic compound situated in the carboxylic acid, one carbon atom bonded to two oxygen atoms and one hydrogen atom.CR1 contains electron donating methyl group (CH2)5, (TiO2)8 and (TiO2)68 brookite clusters through the diketo group as presented in 2)5, (TiO2)8 and (TiO2)68 brookite clusters are investigated in this section. A fascinating feature about croconate dyes is their good solvatochromic shift, they can absorb photons in the near infrared region [2 complexes were relaxed upon adsorption.The croconate dyes CR1 and CR2 are adsorbed onto the surface of 5 is 3.93 eV, CR2-(TiO2)5 is 5.53 eV, CR1-(TiO2)8 is 0.75 eV, CR2-(TiO2)8 is 0.68 eV, CR-(TiO2)68 is 4.74 eV, and CR2-(TiO2)68 is 4.95 eV.The computed adsorption energies are presented in 2 nanocluster [2)8 brookite cluster than the one with the electron withdrawing moiety (CR2). However, the dye with the electron withdrawing moiety (CR2) binds more strongly to (TiO2)5 and (TiO2)68 brookite cluster than the dye with the electron donating methyl (CR1). The results suggest that the dye molecules react differently to different surfaces and sizes of TiO2 brookite clusters.The positive adsorption energies denote the binding ability of the dye molecules to the surface of TiOocluster . The res2)5 are presented in 2)5 cluster. The absorption maxima of (TiO2)5 brookite cluster, which was located around 200 nm has now shifted to higher wavelength around 700\u2013900 nm as shown in 2)8 are presented in 2)8 cluster and it is evident that the absorption maxima with the dyes have shifted to higher wavelength. The absorption maxima of (TiO2)8 brookite cluster that was located around 200 nm has now shifted to higher wavelength around 600\u2013900 nm. In both cases, the CR1 and CR2 dye molecules show a bathochromic shift upon adsorption of the dyes on TiO2 cluster. The bathochromic shift observed after adsorption of CR1 and CR2 dye molecules on TiO2 brookite cluster suggests good optical properties of the dye molecules and corroborates with reports from literature that the adsorption of dye molecules on TiO2 improves its optical response and helps overcome its limited spectral sensitivity in the UV region, thereby improving its photocatalytic properties and overall DSSCs device efficiencies [The optical spectra of CR1 and CR2 dyes adsorbed on the (TiOciencies .2)5, (TiO2)8 and (TiO2)68 brookite cluster are presented in 2 clusters where the unoccupied electronic states are situated. This suggests good electronic coupling between the occupied excited state of the dye and the unoccupied acceptor levels of the semiconductor conduction band. The localization of the HOMO electronic level on the dye molecules and the LUMO electronic level on the TiO2 clusters implies efficient separation of charge upon adsorption and electron injection from the dye excited state into the TiO2 semiconductor conduction band.The isodensity surfaces of the molecular orbital involved in photoexcitation of CR1 and CR2 dye molecules adsorbed on 5, (TiO2)8 and (TiO2)68 were computed using GPAW and PBE exchange correction functional. The TDOS and PDOS spectra are presented in 2)5, dyes-(TiO2)8 and dyes-(TiO2)68, respectively. The DOS is composed of the surface valence and conduction bands separated by a wide band gap. The density of states for the clean (TiO2)5, (TiO2)8 and (TiO2)68 clusters before the dyes were adsorbed were presented previously in 2 cluster alone and the DOS spectrum of croconate dyes adsorbed on TiO2 clusters, it is observed that the adsorption of the CR1 and CR2 dye molecules results in new occupied molecular orbitals introduced to the band gap of the TiO2 clusters upon the absorption of the dye molecules as seen in 2. Comparing between the DOS spectra of all the clean (TiO2)5, (TiO2)8 and (TiO2)68 clusters and the DOS spectra of the dyes adsorbed on the clusters, it is clear that the adsorption of the dyes on TiO2 clusters results in a shift of the conduction band edge of TiO2 to higher energy levels, and consequently narrowing of the band gap between the occupied valence states and the unoccupied conduction band. Additionally, upon adsorption of the dye molecules on the TiO2 clusters, the DOS results reveal that the process introduces new occupied electronic orbitals between the two states where there was a broad band gap initially and in the conduction band of the TiO2 clusters.In order to understand further the electronic structure of CR1 and CR2 dye molecules adsorbed on TiO2)5, dyes-(TiO2)8 and dyes-(TiO2)68 show the contribution of the atomic orbitals in the occupied states and unoccupied states. The PDOS results for the clean clusters showed that both the oxygen and titanium atomic orbitals contributes to the valence states, the oxygen 2p atomic orbitals contributes mostly to the highest occupied valence band state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals. The valence band is dominated by the p atomic orbitals of oxygen with a little contribution from the p atomic orbitals of titanium. The major contribution in the conduction band comes from the titanium orbitals, especially the d and p orbitals. The PDOS spectra of all the dyes-(TiO2)5, dyes-(TiO2)8 and dyes-(TiO2)68 complexes show major contributions from the 3d orbitals of titanium, 2p orbitals of carbon, 2p orbitals oxygen, s orbitals of hydrogen to the valence states. Carbon p orbitals contribute majorly to the conduction band.The PDOS spectra of the dyes-n where n = 5, 8 and 68 were modeled from the optimized ground state bulk structure of TiO2 brookite. (TiO2)5 brookite cluster is a nanostructured form of brookite TiO2 modeled from the bulk structure with dimension repeated in x, 2)8 brookite cluster with the stoichiometry of (TiO2)8 is modeled from the bulk structure of brookite, while (TiO2)68 is a supercell which was created from the repetition of the optimized TiO2 brookite bulk structure unit cell in repeated by 2 \u00d7 2 \u00d7 2 \u00c5 in three dimensions. The trend for TiO2 is (TiO2)n with Ti and O in the ratio 1:2, respectively. The clusters were then exported to (GPAW) [The bulk structure of brookite TiOorted in . The thro (GPAW)  softwareo (GPAW)  for furt2 brookite clusters that were generated are presented in 2)5 brookite nanocluster with five titanium and 10 oxygen atoms cleaved from brookite bulk structure as described in the previous section. 2)8 comprising eight titanium and 16 oxygen atoms whereas 2)68 supercell 2 \u00d7 2 \u00d7 2 \u00c5 comprising 68 titanium atoms and 136 oxygen atoms.All DFT calculations were performed within an atomic simulation environment (ASE) with GPAW software while the structures were visualized with Avogadro. GPAW is a Python based program-package formulated with density-functional theory (DFT) combined with the grid space projector-augmented wave (GPAW). The three TiO2 as well as the main features of oxygen vacancies in rutile and anatase polymorphs. GPAW implements the projector-augmented wave method with the smooth wave-functions and electron density represented on real space grids. The structures were considered to have converged when the maximum forces that were acting on all the atoms were about 0.05 N. The periodic boundary conditions were implemented on the supercell during the relaxation and was set to none for the nonperiodic brookite cluster models. The atoms of the cluster were reoriented during the relaxation until the ground state structure was reached, where they became stable and the forces converged to a maximum value of 0.05 N.All the structures were relaxed in vacuum using GPAW with the PBE. The exchange correlation energy was approximated within the generalized gradient approximation PBE  whereby The UV/Vis, total density of states (TDOS) and partial density of states (PDOS) of the nanocluster structures were computed using the trajectory files obtained from the relaxed structures. The UV/Vis was calculated in vacuum and the TDOS and PDOS were computed using the PBE functional.2)5, (TiO2)8 and (TiO2)68 clusters. We also investigated the adsorption of visible and near infra-red light harvesting croconate dyes onto the three TiO2 brookite clusters, namely (TiO2)5, (TiO2)8 and (TiO2)68. Our findings reveal that (TiO2)5 cluster shows higher wavelength absorption than the corresponding (TiO2)8 and (TiO2)68 clusters owing to its miniaturized size. The band gap of (TiO2)5 cluster also shows a smaller band gap than the corresponding (TiO2)8 and (TiO2)68 clusters, which generally suggests that the nanostructure of TiO2 will enhance more charge transport for solar cell applications. The simulated optical absorption spectrum for (TiO2)5 and (TiO2)8 cluster shows excitation around 200\u2013400 nm, with (TiO2)8 cluster showing higher absorbance than the corresponding (TiO2)5 cluster. The computed density of states and the projected density of states spectra for (TiO2)5, (TiO2)8 and (TiO2)68 clusters reveal surface valence and conduction bands separated by energy band gaps of 1.10, 2.31, and 1.37 eV for (TiO2)5, (TiO2)8 and (TiO2)68 clusters, respectively. The projected density of states spectrum reveals that 2p atomic orbitals contribute mostly to the highest occupied valence band state, whereas the lowest unoccupied state of the conduction band is mainly dominated by the contributions of titanium 3d atomic orbitals. Our findings showed that the adsorption of croconate dyes on the cluster shifted the absorption spectrum to higher wavelengths. The absorption maxima of the clusters alone located in the UV region was shifted to the visible and near infra-red region of the solar spectrum, making it possible to absorb in the whole spectral region. The isodensity surfaces show that HOMO were more localized on the dye while the LUMO were delocalized on the TiO2 semiconductor, depicting that electrons were transferred from the dye excited states to the semiconductor acceptor states. Our findings generally show that the optical and electronic properties of TiO2 cluster vary with the size of the cluster.First principle calculations based on density functional theory (DFT) and time dependent (TD-DFT) were used to investigate the optical and electronic properties of  less susceptible to UV degradation. The research also provides significant findings for the fabrication of brookite semiconductor based DSSCs.Findings from this research are of good significance as the theoretical knowledge and the results could be a guide to experiments on dye molecules adsorbed onto brookite polymorph for application as electron transporting material in nanostructured TiO"}
+{"text": "Immunohistochemistry (IHQ) is commonly used for the detection of mismatch repair proteins deficiency (MMRD). One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2.PMS2 mutations (R134* germline mutation in one tumour and M1R and c.1239delA somatic mutation in the other) as the primary event and somatic MSH6 mutation (c.3261dupC) as the secondary event in both tumours.We review the frequency of this MMRD IHC pattern among 108 colorectal (CRCs) and 35 endometrial cancers in our files with loss of expression of at least one protein, and present two CRCs showing loss of PMS2 and MSH6 protein expression (1.9% of CRCs). NGS analysis of these tumours identified This study suggests that Next Generation Sequencing (NGS) tumour analysis should be considered in the algorithm of Lynch syndrome screening to detect MMR gen somatic mutation in inconclusive cases. MLH1 promoter hypermetylation, the most common immunohistochemical pattern observed in MMRD tumours is MLH1/PMS2 loss [MLH1 germline mutation, whereas MSH2/MSH6 loss occurs in LS due to MSH2 germline mutations [PMS2 [MSH6 [Mismatch repair proteins deficiency (MMRD) can be studied by different methods in tumour tissue, but the most commonly used in the routine practice are immunohistochemistry (IHC) to analyze the expression of MMR proteins  and/or microsatellite instability (MSI) analysis . Since tMS2 loss . This pautations . In addiS2 [MSH6 , 6, respPMS2 mutations (germline in one tumour and somatic in the other) as the primary event and somatic MSH6 mutation as the secondary event in both tumours. These cases illustrated the utility of NGS on tumour tissue for LS screening in inconclusive cases.One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2 . In thisCRCs and ECs with loss of any MMR protein diagnosed between 2010 and 2019 were identified in our Laboratory Information System. Indications for the study of MMR protein expression have changed along this period of time from screening for LS in patients with CRC and Bethesda criteria to universal screening, not only for the identification of patients with LS, but also to select the more appropriate chemotherapy or immunotherapy treatment. In the same way, universal screening for LS by IHC has been implemented in our centre for all ECs. In two cases, IHQ of MMR protein and NGS were realized \u20139 [see AWe identified 108 CRCs and 35 ECs with loss of at least one MMR protein. The different patterns of expression and their relative frequencies are presented in Table\u00a0Patient 1 was a 41-year old female without a personal history of cancer, including father and grandfather with bladder cancer and a brother with melanoma. She presented in 2007 with a pT3N0 tumour of the right colon  and received adjuvant Capecitabine. In 2016 a liver mass of 12\u2009cm was identified and the patient received 3\u2009cycles of neoadjuvant chemotherapy (FOLFOX). The pathologic examination showed the metastatic nature of the lesion and poor response to chemotherapy (approximately 10% of tumour regression). There was no evidence of disease in the last follow-up in 2020. Liver metastases showed loss of PMS2 and MSH6, but intact expression of MLH1 and MSH2 . The tumour showed loss of PMS2 and MSH6 staining with intact staining for MLH1 and MSH2 [see Additional\u00a0file\u00a0NGS was performed in the liver metastasis in patient 1 and in the primary tumour in patient 2. Poor DNA integrity precluded NGS analysis of the primary tumour in patient 1.PMS2, accompanied by a somatic missense mutation (G271S). Patient 2 portrayed two putative truncating somatic mutations (M1R and c.1239delA) in PMS2. Interestingly, tumour samples from both patients portrayed the same somatic frameshift mutation in MSH6 (c.3261dupC). In addition, a second missense somatic mutation located in a close genomic position to the previously one, was detected in both patients   located in a mononucleotide tract, typical of MMRD.Our data tend to support the hypothesis that in both tumours genetic alterations in PMS2 mutation, no PMS2 alterations were reported in the other patient. The authors also observed a somatic mutation in the same mononucleotide tract in MSH6 in three additional CRCs with MLH1/PMS2 loss, although the subjacent genetic or epigenetic alteration of these three cases was not reported.Although a complete review of all published series about MMRD is out of the scope of this study, we have only identified two similar cases included in the series reported by McCarthy et al. , who fouPMS2 (47%) or MLH1 (24%) germline mutation. These germline MLH1 mutations produced MLH1 proteins that retain antigenicity but are non-functional compromising stability of MLH1-PMS2 complexes and inducing PMS2 loss. Kato et al. [MLH1 promoter hypermetylation. Only 1 out of 5 patients tested for germline mutations carried a PMS2 germline mutation and any of them carried a MLH1 germline mutations. Finally, Pearlman et al. [PMS2.Previous studies have reported that isolated loss of PMS2 represents approximately 0.4% of CRCs and 2.2\u20137% of ECs with MMRD, respectively , 12. Then et al.  reportedPMS2 mutations than among LS patients carrying germline mutations in other MMR genes  [Tumours with isolated PMS2 loss seem to have specific clinicopathological features. The risk of developing CRC is lower among LS patients carrying germline In patient 1, MSH6 loss of expression occurred only in liver metastases and after the use of chemotherapy. MSH6 loss expression has been reported in some CRCs after the use of neoadjuvant therapy . AlthougPOLE. Whereas one of the mutations was reported as probably benign in previous reports, the somatic mutation E277G detected in the tumour of patient 1 has been reported as germline and pathogenic in one family with familial CRC [These two cases showed a somatic mutation in the exonuclease domain of lial CRC . The rolPMS2 germline and/or somatic mutations as the possible primary event and somatic mutations of MSH6 as a possible secondary event of these cases. NGS tumour analysis should be considered in the algorism of LS screening for inconclusive cases.Summarizing, loss of PMS2 and MSH6 expression with intact expression of MLH1 and MSH2 is very infrequent and was observed in 1.9% of CRC with IHC-MMRD, but not in ECs. NGS analysis of two cases demonstrated Additional file 1. Immunohistochemistry: Clones and criteria used for the interpretation. Massive parallel sequencing: Description of the NGS panel. Sanger sequencing: Primers used and variants obtained from Sanger sequencing.Additional file 2. Figure case 2.Additional file 3. 2013 CARE Checklist."}
+{"text": "Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment. Osteoblast lineage commitment is regulated by a coordinated set of extra cellular stimuli and developmentally-regulated signaling pathways, including those mediated by bone morphogenic proteins (BMPs), Wnt-ligands, steroid hormones, and growth factors, among others . FollowiWe discuss research demonstrating the role of epigenetic mechanisms in controlling gene transcription during mesenchymal cell commitment to the osteogenic lineage. We first overview key components of epigenetic mechanisms that control gene expression in mammals and then describe how these epigenetic processes contribute to regulate transcription of the Runx2 and Sp7 genes, two critical osteogenic master regulators.Nuclear DNA is organized as chromatin. Nucleosomes are the fundamental units of this chromatin and are composed of an approximately 147 bp DNA segment wrapped around an octamer of histone proteins  . A portiMolecular components that regulate chromatin organization and transcription are critical players during cell differentiation. Among them the large group of \u201chistone post-translational modifications\u201d (HPTMs), which are considered a principal \u201cepigenetic\u201d mechanism . HPTMs mGenetic studies allowed the identification of the Polycomb Group (PcG) and the Trithorax Group (TrxG) complexes, which mediate inhibition and activation of transcription, respectively, during embryogenesis . One sigSeveral mammalian TrxG complexes, including COMPASS (Complex of Proteins Associated with Set1a/b) and the Mixed Lineage Leukemia (MLL1\u20135)-containing COMPASS-like complexes have been reported . TrxG-meSet1-COMPASS mediates global genomic deposition of H3K4me3 in most mammalian cells and therefore its function is tightly associated with transcriptional activation of a large number of genes. MLL2-COMPASS-like has been shown responsible for H3K4me3 deposition at promoters in embryonic stem cells (ESCs), contributing to transcriptional upregulation of homeobox genes . InteresChromatin domains with decreased enrichment of H3K4me3 or H3K27me3 can also be maintained through the activity of lysine demethylases . In partMethylation at H3K9  is strongly associated with the formation of highly compacted and transcriptionally repressed chromatin (heterochromatin) . The metcis or by transferring histone octamers in an ATP-dependent manner. This changes the exposure of regulatory motifs thereby facilitating or preventing recognition by cognate factors  activities (Genomic DNA can be methylated at cytosines that are followed by guanosines (CpG dinucleotides). The DNA methyl-transferases that mediate this modification belong to a well-conserved family of proteins that include both maintenance (Dnmt1) and tivities . Most retivities . DNA demtivities . Importativities .Enrichment of H3K9me3/2 at transcriptionally-inactive chromatin can be associated with the presence of 5mCpG. This is due to the ability of the proteins that \u201cwrite\u201d and \u201cread\u201d these two epigenetic marks to form complexes  thereby Differentiation of mesenchymal stem cells (MSCs) to the osteogenic lineage requires the expression and function of two master transcription factors: Runx2 (Runt Related Transcription Factor 2) and Sp7 . These factors bind to and control the expression of numerous downstream target genes that code for proteins that are necessary for establishing the osteoblast phenotype . Runx2 aActive transcription of the Runx2 and Sp7 genes is accompanied by an epigenetic landscape that includes increased H3K4me3 enrichment at both promoter regions . Hence, It has been determined that an enrichment of H3K4me1 accompanies transcriptional silence at the Runx2 and Sp7 genes in non-osteoblastic and mesenchymal uncommitted cells ,b. As MLMaintenance of chromatin with low H3K4me3 enrichment (and hence elevated H3K4me1) at the Runx2 promoter can be also achieved by binding and activity of the H3K4 demethylase Jarid1b/Kdm5b , which cFindings from several teams revealed that regulating the repressive mark H3K27me3 represents an important step during the differentiation of MSCs toward osteoblasts. It was demonstrated that the activity of the H3K27 demethylases Utx/Kdm6a and Jmjd3/Kdm6b is critical for erasing this mark from the promoter of the Runx2 and Sp7 genes  and hencThe repressive mark H3K9me3 plays an important role during differentiation of MSCs to the osteoblastic lineage ,b. H3K9mAlthough most studies support the role of the H3K27me3 and H3K9me3 marks during repression of both master osteogenic genes, recent analyses indicate that for certain cells, this role may have to be re-evaluated. Thus, analysis of uncommitted human umbilical cord-derived Wharton Jelly MSCs (WJ-MSCs) showed that the Runx2-P1 promoter region does not exhibit significant enrichment of H3K9me3 and H3K27me3 . InteresHistone lysine acetylation also plays a relevant role during bone formation as it supports increased expression of critical genes for osteoblast differentiation . ChromatDNA methylation (5mCpG) can regulate osteogenic differentiation of MSCs. Addition of the DNA methyl-transferase inhibitor 5-Azacytidine (5-Aza) to human and murine mesenchymal cells enhances their ability to engage osteogenesis by reducing 5mCpG enrichment at regulatory regions of osseous genes . In partex vivo. Recent results demonstrate that the substrate stiffness at which hMSCs are grown in culture can induce rapid and stable changes in chromatin organization and the associated epigenetic landscapes that permanently up- or down-regulate genes (Controlling lineage commitment in MSCs represents a critical challenge to overcome skeletal deficiencies in patients. Extensive research during the last 20 years has shed light into potential targeting of specific epigenetic mechanisms that control bone-related gene expression. Future studies will need to consider the influence of the mechanical environments at which MSCs are maintained and differentiated te genes , hence mMM analyzed the data and wrote most parts of the manuscript. MC wrote parts of the text and prepared the figures. GN analyzed the data, wrote parts of the text and helped preparing the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "FOXA2 haploinsufficiency (FOXA2+/\u2212 iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2\u2212/\u2212 iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2\u2212/\u2212 iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with  Monogenic diabetes (MD) is caused by a mutation or defect in a single gene-regulating beta-cell development and/or function3. Several heterozygous mutations in the TFs expressed during pancreatic development are associated with a specific form of MD, known as MODY. However, homozygous mutations of the same TFs lead to neonatal diabetes, which can be associated with pancreatic hypoplasia/agenesis in some mutations4. This indicates that the onset and severity of the diabetes phenotype are correlated with the dosage of the TF expression during pancreatic development.During human development, early endodermal tissue becomes specified toward a pancreatic fate before evagination of pancreatic buds, populated with pancreatic progenitors. All adult pancreatic cells are originated from the same progenitors expressing a group of transcription factors (TFs), including PDX1, SOX9, FOXA2, NKX6.1, HNF6, and PTF1A5. Foxa2 knockout mice die at an early embryonic stage and show developmental defects in the foregut (FG) and neural tube8. During pancreatic development, FOXA2 is expressed at early stages starting from the endoderm stage and its protein level is increased during the endocrine specification stage9, while the exocrine and ductal cells express a low level of FOXA29. Mouse studies showed that Foxa2 is important for islet development and beta-cell functionality12 and its specific deletion in beta-cells leads to hyperinsulinemic hypoglycemic phenotype14. In human, previous reports demonstrated that patients with heterozygous FOXA2 mutations develop hyperinsulinemia, hypoglycemia, hypopituitarism, endodermal organ defects, and craniofacial abnormalities16. Recent genomic studies found that type 2 diabetes (T2D) risk alleles are associated with FOXA2-bound enhancers in human17. Another recent study reported a patient with diabetes due to a heterozygous missense variant in FOXA2, indicating that FOXA2 defect can lead to MD18. FOXA2 is known to regulate the expression of multiple TFs controlling pancreatic endocrine cell fate and insulin secretion20.FOXA2 is expressed in several tissues and performs distinct functions as evident in the phenotypes of mouse models21, which do not fully reflect human phenotypes. Recent progress in the induced pluripotent stem cell (iPSC) technology allowed us to generate in vitro models to study human genetic diseases22. To our knowledge, there is only one recent study that used FOXA2 knockout hESCs (FOXA2\u2212/\u2212 hESCs) to study FOXA2 during pancreatic progenitor differentiation23. To better understand the role of FOXA2 in beta cell development and diabetes progression, we established iPSCs from a patient with a heterozygous deletion in FOXA2 (FOXA2+/\u2212 iPSCs)24. We showed a number of key genes essential for pancreatic development were dysregulated in FOXA2+/\u2212 iPSC-derived pancreatic cells and the results obtained were further validated by generating CRISPR/Cas9-mediated FOXA2 knockout (FOXA2\u2212/\u2212 iPSCs). Our results suggest that FOXA2 have a crucial role in pancreatic endocrine development in human.Most of the information available on FOXA2 functions have been obtained from animal studiesFOXA2 gene 24 and from a healthy control 25. In this study, we generated additional iPSC lines from another healthy control (Ctr2-iPSCs). All the established iPSC lines showed the expression of the pluripotency markers and showed the ability to differentiate into the three germ layers  ] and a deletion  as expected24 and as reported in the patient sample26  and endocrine progenitors (EPs). FOXA2 protein and mRNA levels were markedly reduced in PP2 derived from FOXA2+/\u2212 iPSCs , HNF1A, and PAX4 as well as the exocrine precursor markers, PTF1A and AMYLASE. Interestingly, all these pancreatic markers were significantly downregulated in PP2 derived from FOXA2+/\u2212 iPSC lines compared to those derived from Ctr-iPSCs  and 427 upregulated  differentially expressed genes (DEGs) in PP2 derived from FOXA2+/\u2212 iPSCs compared to those generated from Ctr-iPSCs  and 564 upregulated  DEGs in EPs derived from FOXA2+/\u2212 iPSCs compared to those generated from Ctr-iPSCs + cells. However, the expression levels of INS, NKX6.1, C-PEP, and GLUCAGON (GCG) were downregulated as examined by immunostaining and flow cytometry in stage 7 derived from FOXA2+/\u2212 iPSCs compared to those derived from Ctr-iPSCs  and KCl stimulation compared to low glucose levels  and high (20\u2009mM) glucose and KCl stimulation was measured. Beta cells derived from FOXA2els Fig. . We did SCs Fig. . HoweverSCs Fig. . These rFOXA2+/\u2212 iPSCs and to exclude the possibility of cell line variations, we used CRISPR/Cas9 to KO the FOXA2 gene in the iPSCs (FOXA2\u2212/\u2212 iPSCs)  controls. Characterization of both FOXA2\u2212/\u2212 iPSC lines showed that they were pluripotent and displayed normal karyotype  derived from FOXA2+/\u2212 iPSCs and FOXA2\u2212/\u2212 iPSCs  at the PP2 stage on reversing the phenotype associated with FOXA2 deficiency  with hypoglycemic hyperinsulinemic phenotype due to two different FOXA2 haploinsufficiency carrying the genetic information of the patient. Our results indicate that FOXA2 haploinsufficiency negatively impacted endocrine islet development by downregulating key TFs associated with islet cell development and upregulating genes associated with neuronal development and WNT and BMP pathways. Taken together with other recent reports23, our findings suggest that FOXA2 defects may lead to monogenic diabetes based on the gene dosage. However, it will be of interest to perform further studies to investigate how FOXA2 works with other TFs to main normal pancreatic endocrine development in human.In conclusion, our current study provides the first human iPSC model for The protocols to obtain blood samples were approved by the Institutional Review Board (IRB) of Sidra Medicine (no. 1702007608) and QBRI/HBKU (no. 2018-002). Informed consent forms were signed by human subjects.FOXA2 heterozygous deletion  and healthy controls (Ctr1-iPSCs and Ctr2-iPSCs) as we previously reported25. All lines were extensively characterized as previously reported25. The first four stages of the pancreatic differentiation were performed using our protocol27 and for further differentiation to beta cells, Rezania et al.28 protocol was used  for 4 days during stage 4 and the control cells were treated with DMSO. For BMP inhibition, the cells were treated every day during stage 4 with 100\u2009ng/mL NOGGIN, while the control cells were treated with 50\u2009ng/mL of NOGGIN. The cells were collected at end of stage 4 to examine the effect of WNT and BMP inhibition on the dysregulated genes associated with 29. RT-PCR and qPCR were performed as previously reported25.  experiments and data analysis were performed as we previously reportedFOXA2 iPSC lines from two control iPSC lines (Ctr1 and Ctr2). The cells were transfected with a plasmid expressing spCas9 and gRNA  using Lipofectamine Stem Transfection Reagent following the manufacturer\u2019s instructions (ThermoFisher Scientific)  and high (20\u2009mM) glucose, which was repeated two times. In the end, the clusters were incubated in low glucose with\u200930\u2009mM KCL to depolarize the cells and release their C-PEP (C-PEPTIDE) contents. The C-PEP concentration in the collected supernatants was measured using Human C-Peptide ELISA Kit . Each value of C-PEP level of low or high glucose condition was normalized to its own total content of C-PEP in each sample. The stimulation indices were determined through the ratio between the C-PEP level at high glucose concentration to its own basal level secretion at the two challenges.At the end of day 2 of stage 4, the cells were dissociated with TrypLE and resuspended in the differentiation medium with 10\u2009\u03bcM Y-27632. The resuspended cells were either transfected with FOXA2 plasmid , PDX1 plasmid , or the empty vectors. Transfection was carried out using the Lipofectamine\u2122 3000 Transfection Reagent following the manufacturer\u2019s instruction . At 48\u2009h post-transfection (end of stage 4), the cells were harvested for RNA and protein extraction. Some experiments were continued until the end of stage 7 (beta-cell stage) for further analyses.t-test by Prism 8.At least three biological replicates were used in most of the experiments and statistical analysis was carried out using unpaired two-tailed Student\u2019s Supplementary Table 1: Differentiation MediaSupplementary Table 2:Antibody detailsSupplementary Table 3: Primer listSupplementary Table 4: Top downregulated genes in PP2 derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 5: Top Upregulated genes in PP2 derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 6: Top downregulated genes in the EPs derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 7: Top upregulated genes in the EPs derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 8: gRNA sequences for FOXA2Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6"}
+{"text": "Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (<\u20091%) subclonal cancer specific RE insertions is complicated.Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of\u00a0Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions.This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.The online version contains supplementary material available at 10.1186/s13100-020-00228-6. Retroelements (REs) in the human genome comprise more than 1.5 million copies that arise as a result of a process called retrotransposition. Three major classes of REs are LTR retroposons, LINEs, and SINEs that include the primate-specific Alu family. Only a tiny portion of REs is still capable of retrotransposition including more than one hundred of autonomous LINE-1 (L1) copies , 3 and aThe method is based on consecutive selective amplification of both 5\u2032 and 3\u2032 flanking regions adjacent to the copies of retroelements belonging to young RE subfamily of interest Fig.\u00a0. For detAt the next step fragmented DNA is ligated to stem-loop DNA adapters containing Unique Molecular Identifiers (UMI) . These rNext, the biotinylated primer specific to AluYa5 (or AluYb8) fragment containing 3 diagnostic mutations for Ya5 (or specific 7\u2009nt fragment for Yb8) is used for linear amplification of retroelement 5\u2032 flanks. The biotinylated primer along with the biotinylated dCTP is used to introduce biotin into generated ssDNA molecules. After linear amplification the obtained product is denatured, the biotinylated 5\u2032 flank copies are captured and removed from the reaction with the use of streptavidin-coated magnetic beads. Importantly, the restricted and adaptor-ligated genomic DNA template remains intact. This DNA is used for the second linear amplification with the primer complementary to the same region characteristic for AluYa5 (or AluYb8) but in the opposite direction (towards the 3\u2032 flank of the insertion). This product is also captured by streptavidin-coated magnetic beads. The remaining initial template DNA is used to confirm insertions by locus-specific PCR. Captured linear DNA is amplified in the subsequent PCR to generate libraries of AluYa5 (AluYb8) flanks (separately for 5\u2032 and 3\u2032 flank). The second PCR is used for sample barcoding and generates libraries ready for sequencing on Illumina. The resulting fragments consist of a short part of the Alu repeat, adjacent genomic flank (5\u2032 or 3\u2032), a part of the adapter with UMI, and standard parts necessary for Illumina sequencing including sample barcodes. 3\u2032 flanking library also includes the polyA tail of the Alu.After sequencing, we use a custom computational pipeline  to map ahttp://dbrip.brocku.ca/) or 1000 genomes project databases [To evaluate method sensitivity, we used polymorphic Alu insertions. Using our method we first identified coordinates of AluYa5 insertions in the genomes of four healthy individuals . Then we compared these coordinates and identified in total 137 AluYa5 insertions that are absent in the genome of individual D1 and present in the genome of one of the individuals D2, D3, or D4 , 37 out of 45 Alu for individual D3 (0.3%) and 36 out of 48 Alu for individual D4 (0.1%) have nearest 3\u2032 restriction site in the range of 30\u2013529\u2009bp (same as for the 5\u2019flank). Next, we searched the 3\u2019flank library for the insertions that are present in both 5\u2032 and 3\u2032 flank libraries. As a result, we were able to find 32\u201334 out of 36 insertions present in 1% (D2) of cells, 26\u201329 out of 37 insertions present in 0.3% (D3) of cells and 16\u201318 out of 36 insertions present in 0.1% (D4) cells  of insertions present in 1% of the cells in a sample can still be found with sequencing depth of 2 million reads. As expected, for the cells with 10 times lower concentration (0.1%) even 15 million of reads is not enough to catch 100% of the insertions, however, 28 out of 48 (approximately 60%) are repeatedly found in both replicates. Around 20% of insertions are still repeatedly detectable with sequencing depth of 2 million in both replicates . We plotted normalized UMI count in replicate 1 versus UMI count in replicate 2 for each of the identified Alu insertion from individuals D1-D4  that left after two rounds (5\u2032 and 3\u2032 flank) of linear amplification and capture was used as a template for locus-specific PCR. We selected 10 non-reference Alu insertions which have a distance between 5\u2032 and 3\u2032 flanks restriction sites in the range 400\u2013600\u2009bp. Next, we removed short (100\u2013300\u2009bp) template DNA molecules from the mixture by magnetic beads. This procedure depletes DNA fragments that do not contain Alu insertions  originating from the genome of individual D1. After that, we performed locus-specific PCR with the primers corresponding to unique flanking regions of each insertion. We observed PCR product of expected length for 4 out of 4 insertions present in the genome of D2 (1% of cells), for 3 of 4 insertions present in the genome of D3 (0.3% of cells) and for 1 of 2 insertions present in the genome of D4 (0.1% of cells). Obtained PCR products were sequenced by Sanger method which in all cases allowed to confirm the insertions by the alternative method and were able to identify TSD indicating true Alu insertions.Using our method, we performed a search for cancer-specific Alu Ya5 and AluYb8 insertions in 6 paired  colorectal cancer samples. Libraries of 5\u2032 and 3\u2032 flanks were prepared using SeqURE method and sequenced. For each 5\u2032 flank library we obtained a minimum of 2,173,258 reads and the difference between tumor and normal samples in each pair was less than 2-fold. After data processing and artifact filtration, we compared lists of Alu insertion coordinates in paired normal and tumor samples. Only the insertions with 2 or more different UMI present in the tumor sample and absent in corresponding paired normal sample were considered tumor-specific candidates. We identified 10 potential tumor-specific Alu insertions in the 5\u2032 flank libraries of 5 out of 6 tumor samples (see Table\u00a0n\u2009=\u20093) we failed to get PCR product. The obtained PCR products for 4 tumor-specific insertions were sequenced by Sanger method. In all cases we confirmed tumor-specific L1HS insertions.To further proof the developed copy-capture approach we identified cancer-specific insertions of another family \u2013 L1HS in the same set of paired tumor/normal samples. As amplification and sequencing of 5\u2019flanking regions of L1 insertions is yet challenging we generated only the library of 3\u2032 flanks, using the same experimental approach as for Alu 3\u2032 flank libraries. Genomic DNA was digested with another pair of restriction enzymes and ligated to adapted with the same pan-handle like structure (see methods for details). After linear amplification with L1HS specific biotinylated primer 3\u2032 flank copies were extracted with streptavidin-coated magnetic beads and amplified in the subsequent 2 round PCR to generate ready-for-sequencing sample-barcoded libraries. Same as for Alu libraries, template DNA stays intact and is used for validation by locus-specific PCR. For each library we obtained a minimum of 219,970 reads and the difference between tumor and normal samples in each pair was less than 2-fold. In six paired samples combined we found 1119 L1HS insertions including 854 reference insertions (hg38), 188 previously known non-reference insertions  and 77 In the recent years it is becoming widely accepted that RE transcriptional and transpositional activity can play a significant role in cancer. New insertions can disrupt cellular genes  either by direct integration into a gene , 29 or vWe use a modified selective amplification approach , 31 to gThe significant drawback of this approach is the inability to identify TSD \u2013 the hallmark of the retrotransposition event. As a result, even with very high sensitivity and vigorous artifact filtration pipelines, many potential insertions are not confirmed by the locus-specific PCR. This limitation can be overcome by the amplification of both flanking regions (5\u2032 and 3\u2032). However, this approach is not reliable when detecting insertions present in a minor fraction of cells if different aliquots of DNA from the same sample are used for library preparation and validation. Here we consecutively amplify both adjacent genomic regions of each insertion from the same template which significantly increases the chance to retrieve both flanks and identify TSD. In addition, sequence of one flank is used to validate the insertion found in the library of another flank: when we know the insertion coordinate from one library we can predict the length of another flank as we use restrictase for DNA fragmentation. If the corresponding sequence has suitable length for amplification and sequencing but not found in another flank library \u2013 this indicates false insertion. Moreover, template genomic DNA used for libraries preparation stays intact and can be used for locus-specific PCR to confirm insertions. Using spike-in controls we show that this approach can indeed produce reliable results. Most of the insertions that have corresponding sequences in 5\u2032 and 3\u2032 flank libraries were confirmed by locus-specific PCR from the same DNA template. We also directly characterized the sensitivity of the method. We show that the majority of insertions present in as little as in 1% of cells can be reliably (in two replicates) identified with the sequencing depth of only 2 million of 150\u2009+\u2009150 paired reads (of 0.6 Gb) per sample. This corresponds to 0.2x human genome coverage. It should be mentioned that uneven amplification efficiency of different flanking sequences results in different sequencing coverage for each particular RE insertion. As it was shown previously  amplificUnlike L1 insertions, tumor-specific Alu insertions are rare in cancers including colorectal cancer . As a prTo further validate the method, we searched for tumor specific L1HS insertions in the same set of samples using the same methodological approach. We identified 10 candidate insertions with UMI counts ranging from 2 to 16 and fully confirmed 4 of them by orthogonal approach. These results indicate successful application of our method on real samples, and at the same time, demonstrate the necessity for sequencing of both flanks for reliable tumor-specific RE identification. Additionally, the identified tumor specific L1HS insertions indicate that at least 3 out of 6 analyzed colorectal tumor samples were subjected to retropositional activity. At the same time, we accurately showed absence of tumor specific Alu insertions in all 6 samples. Taken together, these facts indicate that Alu insertions in cancer occur much rarer than L1 and active L1 machinery alone is insufficient for Alu retropositional activity.Most of the previously described techniques for targeted RE capture like RC-seq  use origSix colorectal cancer samples were obtained during R0 partial colectomy from patients with stage II or III colorectal adenocarcinoma (5 patients) or carcinoma in situ (1 patient) treated at the A.N. Ryzhikh National Medical Research Centre for Coloproctology. The study was approved by the local ethical committee and all the patients gave standard informed consent. Tumor and normal tissue fragments were taken under the supervision of a pathomorphologist in the shortest possible time after intestinal resection (not more than 30\u2009min). DNA was extracted with QIAamp DNA Mini Kit (Qiagen) according to manufacturer\u2019s protocol.PBMC of four healthy donors were isolated from peripheral blood by standard Ficoll-Paque  centrifugation protocol. T-cells from different individuals were stained by the following antibodies: individual D2 CD3-eFluor450 , individual D3 - CD3-FITC , individual D4 CD3-PC5 . Cells of individual 1 were not stained. FACS sorting was performed in 5 replicates by BD FACS Aria III. Cells were gated based on side and forward scatter. Each cells mixture contained 50,000 cells of individual D1, 500 cells of individual D2, 150 cells of individual D3 and 50 cells of individual D4. After sorting three out of five aliquots were resorted to evaluate the resulting number of cells in the mixture. DNA from sorted cells was isolated using QIAGEN DNeasy Blood and Tissue Kit (Qiagen).To avoid possible contamination, we first prepared libraries from cell mixtures and sequenced them. After that we made libraries from DNA of individuals D1-D4 separately to identify individual-specific Alu insertions present in the genome of individuals D2, D3 or D4 and absent in the genome of individual D1.Thirty\u2009ng of genomic DNA was digested by incubation in 10 \u03bcl of 1x FD buffer with 5\u2009U of FspBI and 5\u2009U of Csp6I  for 30\u2009min at 37\u2009\u00b0C. For adapters ligation fragmented DNA was diluted by 20 \u03bcl of 1x FD Buffer with 20\u2009\u03bcmol of ATP (Thermo Fisher Scientific), 50\u2009pmol of SL-adapter  for 30\u2009min at 37\u2009\u00b0C and 30\u2009min at 65\u2009\u00b0C. For adapters ligation fragmented DNA was diluted by 20 \u03bcl of 1x FD Buffer with 20\u2009\u03bcmol of ATP (Thermo Fisher Scientific), 50\u2009pmol of SL-adapter-2  and used for the 2nd amplification to obtain DNA fragments of opposite (5\u2032) L1HS flanks. The amplification profile, capture and purification condition were the same as for 3\u2032- flank libraries. The captured amplicons (copies) were indexed at the same condition as 3\u2032 flanks amplicons, purified with AmPure XP beads, mixed equimolarly and sequenced on Illumina MiSeq paired end 150\u2009+\u2009150.Raw sequencing reads were processed as described previously . As a rePrimers for the specific genomic regions were designed with primer-blast and GeneRunner programs. Twenty-five\u2009\u03bcl PCR reactions containing 200\u2009\u03bcM of each of dNTP, 0.2\u2009\u03bcM of each of forward and reverse locus-specific primers (Additional file Additional file 1: Supplementary Table 1. A detailed description of individual-specific Alu insertions.Additional file 2: Supplementary Table 2. TSD identification.Additional file 3: Supplementary Table 3. Oligonucleotide sequences used for library preparation and locus-specific PCR."}
+{"text": "The sirtuin (SIRT) proteins are a highly conserved family of NAD+-dependent deacetylases that regulate histones as well as non-histone proteins. Seven sirtuin genes have been identified (SIRT1 to SIRT7) in mammals. SIRT1, SIRT6, and SIRT7 are primarily localized in the nucleus. SIRT2 is localized mainly in the cytoplasm. SIRT3, SIRT4, and SIRT5 are often located in the mitochondria. Therefore, the sirtuin family proteins exert their diverse functions at various cellular locations and regulate metabolism, stress responses, growth and aging processes. The sirtuin proteins are often considered as nutrient sensors. This study assessed the expression of sirtuin genes in C2C12 muscle cells under glucose stress conditions at different time points. Expression of all seven sirtuins was confirmed by Real-Time PCR analysis. SIRT1 (24 h) and SIRT3 (6 h and 15 h) are highly expressed under low glucose (2.7 mM) and high glucose (25 mM) conditions, whereas SIRT2 and SIRT4, SIRT5, SIRT6 and SIRT7 expressions were either relatively lower or there was no significant change under glucose stress conditions. Our results indicate that SIRT1 and SIRT3 demonstrated the greatest fluctuation in response to glucose stress, whether high or low glucose. These findings will help elucidate the role of sirtuins in the regulation of cellular processes, including metabolism. It will also help to enhance our understanding of the roles of sirtuin genes in regulation of blood sugar fluctuation in normal persons and diabetic patients, as well as in elderly individuals, many of whom are insulin resistant and \u201cprediabetic\u201d or diabetic."}
+{"text": "C-C motif ligand 2 (CCL2) was originally reported as a chemical mediator attracting mononuclear cells to inflammatory tissue. Many studies have reported that CCL2 can directly activate cancer cells through a variety of mechanisms. CCL2 can also promote cancer progression indirectly through increasing the recruitment of tumor-associated macrophages into the tumor microenvironment. The role of CCL2 in cancer progression has gradually been understood, and various preclinical cancer models elucidate that CCL2 and its receptor C-C chemokine receptor 2 (CCR2) are attractive targets for intervention in cancer development. However, clinically available drugs that regulate the CCL2\u2013CCR2 axis as anticancer agents are not available at this time. The complete elucidation of not only the oncological but also the physiological functions of the CCL2\u2013CCR2 axis is required for achieving a satisfactory effect of the CCL2\u2013CCR2 axis-targeted therapy. C-C motif ligand 2 (CCL2) was discovered first among CC chemokines in 1989 and helped to propel subsequent discoveries of other CC chemokines . The bioThere is a number of studies suggesting that CCL2 can activate cancer cells through a variety of mechanisms. Initially, CCL2 was focused on and studied as a key molecule in the migration and proliferation of prostate cancer cells . CCL2 diAlthough a great number of the studies focusing on the function of CCL2 in cancer progression used prostate and breast cancer cells, more recent CCL2 studies have incorporated colorectal, lung, gynecological, gastric, and other types of cancer cells. Akt activation by phosphatidylinositol phosphate kinase gamma (PIPKI\u03b3)-mediated STAT3 phosphorylation induces CCL2 expression in colorectal cancer cells and silencing PIPKI\u03b3 greatly decreases CCL2 expression at both the mRNA and protein levels, leading to reduced chemotaxis of cancer cells to macrophages . In metaSince CCL2 is also known by the alias monocyte chemoattractant protein-1 and activates macrophages, inactivation of cancer cells by the inhibition of the CCL2\u2013CCR2 axis are partly presumed to be caused by the reduction of tumor-associated macrophages (TAMs) at the primary and metastatic site, inducing the destruction of the immunosuppressive tumor microenvironment and the susceptibility to the antitumor T lymphocyte response. TAMs play a major role in cancer progression by affecting diverse processes such as immunosuppression, angiogenesis, and tumor cell proliferation and metastasis during cancer progression ,41,42. IMyeloid-derived suppressor cells (MDSCs) also foster cancer cell activity by suppression of T lymphocytes and natural killer cells, while simultaneously they can recruit regulatory T lymphocytes to further promote immunosuppression . DeletioCAFs are stromal components and play an important role in tumor progression by regulating the tumor microenvironment and primarily releasing proteolytic enzymes, growth factors, and cytokines ,59. PancConsistent with the role of CCL2 in cancer progression, serum CCL2 levels and CCL2 expression levels in tumor tissue have been reported as potential biomarkers in a variety of cancer patients .The usefulness of serum CCL2 in prostate cancer patients was supported by a study using the serum samples of 255 and 124 men with and without prostate cancer, respectively, which reported that serum CCL2 levels in men with prostate cancer were significantly higher than those without. In addition, among the 255 patients with prostate cancer, those with serum CCL2 level \u2265 320 pg/mL had significantly poorer overall survival and prostate cancer-specific survival, higher TNM stages, and worse histological grade than those with serum CCL2 level < 320 pg/mL, indicating the potential of serum CCL2 level as a prognostic biomarker as well . The outWhen the preoperative serum CCL2 concentration of 45 colorectal cancer patients was measured, the 5-year survival rate was significantly better in the group with low preoperative CCL2 level than in the group with high preoperative CCL2 level . In a stSerum CCL2 was significantly higher in HCC patients in a study that measured CCL2 levels from serum samples from 98 resectable HCCs, 101 patients with chronic hepatitis B, and 100 asymptomatic hepatitis B and C virus carriers. In addition, the serum CCL2 also correlated with the disease stage of HCC and the combination of alpha-fetoprotein and CCL2 has significantly superior discriminative ability than alpha-fetoprotein alone . In examSerum CCL2 levels in 135 breast cancer patients were measured and elevated and serum CCL2 levels were significantly correlated with advanced cancer stage and lymph node metastasis . ImmunohA study of 212 pancreatic cancer patients found that CCL2 serum levels in pancreatic cancer patients were significantly higher than in normal healthy subjects . In a stCCL2 and CCR2 expression was analyzed by immunohistochemical staining and its correlations with clinicopathologic features and prognosis were evaluated in 221 patients with diffuse large B-cell lymphoma, and high expression of CCL2 or CCR2 was correlated with clinicopathological characteristics and indicated significantly poorer overall survival and progression-free survival . In a stAs a result of examining the peripheral blood of 60 patients with gastric cancer and 20 healthy subjects, preoperative serum levels of CCL2 in patients with gastric cancer were significantly higher than those in healthy controls. In addition, preoperative serum CCL2 was significantly correlated with lymph node metastasis . A studyIn a retrospective study including 86 ovarian cancer patients, 67 benign ovarian cysts, and 42 healthy women, ovarian cancer patients had significantly higher serum CCL2 levels, and serum CCL2 levels were statistically significantly correlated with histological malignancy . In a stComparing 142 lung cancer patients with 141 non-lung cancer subjects, CCL2 was significantly elevated in lung cancer patients . WesternExamining CCL2 levels in urine samples from 60 bladder cancer patients and 20 control healthy subjects revealed CCL2 levels in the urine of patients with bladder cancer correlated significantly with TNM stage and cancer grade . ImmunohExamination of serum CCL2 and TNF-\u03b1 in 297 patients with pretreatment laryngeal cancer showed that CCL2 and TNF-\u03b1 levels were independent predictors of overall survival and distant metastasis-free survival . ImmunohThe effects of the orally administered CCR2 inhibitor PF-04136309 in combination with leucovorin (folinic acid), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) in patients with treatment-naive locally advanced pancreatic ductal adenocarcinoma were assessed in a phase Ib study; ultimately, 16 (49%) of 33 patients receiving FOLFIRINOX plus PF-04136309 achieved an objective tumor response, with local tumor control achieved in 32 (97%) patients . HoweverLosartan, a type I angiotensin II receptor antagonist, is reported to have the potential to act as direct CCR2 antagonists. Losartan and its primary metabolite, EXP-3174, inhibit CCL2-mediated monocyte recruitment to the metastatic site through the inhibition of CCL2-induced Erk1/2 activation and significantly reduce the metastatic burden in breast and colon cancer models . SimilarPreclinical studies using the mouse model were also reported. Inhibition of CCL2 improved radiation resistance using mice transplanted with pancreatic duct adenocarcinoma cell lines . In a prAlthough many studies have proven the key roles of the CCL2\u2013CCR2 axis in tumor progression in a variety of cancers, there are no clinically available drugs that can modulate the CCL2\u2013CCR2 axis as anticancer agents so far. As described earlier, a high serum CCL2 level may reflect the activity of cancer cells because CCL2 is produced by them; however, some studies have reported conflicting results that a low serum CCL2 level contributed to a worse prognosis in cancer patients . This diThe performance of the drug itself in exerting its potential effect to suppress cancer cell activity is also important to consider. Measurement of free CCL2 concentration following carlumab administration revealed a rapid fall in free CCL2 as expected; however, post-treatment free CCL2 concentrations rapidly rebounded and quickly exceeded pretreatment serum concentrations. These unexpected results suggest that carlumab was ineffective at suppressing free CCL2 concentration for a clinically meaningful period of time. As no apparent increase in CCL2 production rate was observed when the dose was increased from 1 to 50 mg/kg in cynomolgus monkeys, dose-escalation might be needed to adequately and constitutively suppress free CCL2 concentration . PF-0413The CCL2\u2013CCR2 axis functions physiologically in various organs such as the lungs and digestive tract where a variety of immune cells accumulate. Blockade of the CCL2\u2013CCR2 axis potentially causes unexpected effects in such organs. Although it is unclear whether each adverse event attributes to the blockade of the CCL2\u2013CCR2 axis because PF-04136309 was used in combination with cytotoxic agents in a single-arm study, pulmonary toxicity and diarrhea as well as myelosuppression were reported as severe adverse events in clinical trials . AlthougIn summary, to achieve sufficient efficacy of CCL2\u2013CCR2-targeted therapy for cancer patients, it is not sufficient to target cancer cells or immune cells alone and it is necessary to fully elucidate the physiological functions of the CCL2\u2013CCR2 axis against a variety of normal cells expressing CCR2."}
+{"text": "This study aims to identify the role of synovial fluid levels of a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) for the prediction of intraarticular steroid injection success in knee osteoarthritis (OA).A total of eighty-four advanced stage knee OA patients (42 with stage 3 OA and 42 with stage 4 OA) were enrolled in the study. Baseline and posttreatment outcomes were determined using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Pretreatment synovial fluid ADAMTS9 and ADAMTS4 levels were measured by enzyme linked immunosorbent assay (ELISA). \u2018\u2019Total WOMAC score regression of 18% and above\u2019\u2019 was taken as a minimal clinically important difference (MCID) to indicate improvement. Determining the best predictors of intraarticular steroid injection success in both groups was evaluated by multiple logistic regression analyses.= 0.026). Decreasing synovial fluid ADAMTS9 levels (odds ratio (OR): 0.625, 95% confidence interval (CI): 0.437\u20130.893) were found to be predictive for the WOMAC score percent change \u226518 in all OA patients (P= 0.010). Decreasing ADAMTS9 levels in synovial fluid  were predictive for MCID in stage 3 OA patients (P\u00a0= 0.039). Synovial fluid ADAMTS9 levels were significantly lower in the stage 4 OA group when compared with the stage 3 group. The level of synovial fluid ADAMTS9 was statistically significantly lower in the WOMAC score percent change \u226518% than the WOMAC score percent change <18% group in Stage 3 OA group (PThe lower levels of ADAMTS9 in synovial fluid may be used in conjunction with high WOMAC scores in the prediction of intraarticular steroid injection success and advanced stage knee OA patients. Knee osteoarthritis (OA) is a destructive joint disease in which joint manifestations include progressive loss of cartilage, cartilage calcification, osteophyte formation, and subchondral bone remodelling. Osteoarthritis is graded according to radiological findings of changes in the joints. The Kellgren and Lawrence (K&L) scoring system is used to denote the radiological stage in OA. In advanced stages of OA, progressive changes occur due to cartilage degradation [1]. The extracellular matrix (ECM) of cartilage consists of type 2 collagen and aggrecan, which is a significant proteoglycan [1]. Progressive changes in cartilage in OA are dependent on active cell processes mediated by cytokines and proteases [1]. The role of synovial inflammation in the progression of OA is well known [2]. Several studies demonstrated that inflammation of the synovial membrane played a role in the progress of joint degeneration by increasing the release of proteases, such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), in the fibroblasts of cartilage . Members of the ADAMTS family, especially ADAMTS4 and ADAMTS9 are already known to bind and degrade ECM components and contribute to joint destruction processes in arthritis [5]. Zhang et al. [6] showed that ADAMTS4 levels in synovial fluid were significantly lower in late-stage OA than in early-stage OA. Peng et al. [7] reported that ADAMTS4 in synovial fluid might be a potential biomarker for cartilage degeneration. Previously, the aggrecanases ADAMTS4 and ADAMTS5 were shown to contribute to cartilage destruction and to be responsible for aggrecan degradation, especially in patients with early-stage OA [8]. Gok et al. [9] suggested that the cytosine adenine (CA) repeat polymorphism of the ADAMTS9 gene could be used to determine the radiological progression of OA.Although recent studies have already shown that ADAMTS4 and ADAMTS9 are involved in cartilage destruction in OA , the possible role of these matrix metalloproteinases in OA progression and their utility in the evaluation of treatment success remains unclear. This study aimed to determine the significance of synovial fluid ADAMTS4 and ADAMTS9 levels in OA progression, particularly in the assessment of the response to intraarticular steroid injection in advanced stage OA patients.Eighty-four advanced stage knee OA female patients (42 with stage 3 OA and 42 with stage 4 OA) with matching body mass index (BMI) were recruited consecutively from the outpatient clinic of Konya Bey\u015fehir State Hospital in 2018. The diagnosis of knee OA was determined according to the radiographic features. The radiological stage was based on the K&L scale, which is accepted as a reference standard of the World Health Organization. It requires the presence of all five radiological criteria: osteophytes on the joint side, periarticular ossicles, joint area narrowing (JSN), small pseudocyst regions within the subchondral bone and a change in the formation of the bone ends. Inclusion criteria of OA patients: patients with radiological features  and stage 4  [12], women who have not received any intraarticular and/or systemic OA therapy before and a BMI between 30 kg/m2 and 18 kg/m2. Exclusion criteria: patients with other knee joint diseases , infectious diseases, septic arthritis, obesity, neurological or neuromuscular diseases, bone tumours, osteoporosis or trauma related fractures, diabetes mellitus, Addison\u2019s disease, immune system disorder, rheumatological diseases and previous use of systemic steroids and intraarticular hyaluronic acid injections. Also, OA women who did not comply with stage 3 and stage 4 criteria, that is, no changes in X-ray or changes in only osteophyte or JSN, were not included in the study.All participants provided written informed consent, and the study ethics committee approval was obtained from the local Ethical Committee of the University . Clinical examination was performed, and anthropometric measurements, concomitant diseases  were recorded for all participants included in the study. Knee function was assessed before and 1 month after the treatment with intraarticular steroid injection, using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). The WOMAC score is composed of 24 parameters that include pain (score range: 0\u201320), stiffness (score range: 0\u20138), and functional impairment (score range: 0\u201368) [13]. Improvement in the WOMAC score after intraarticular injection was assessed within an average of one to 3 months. \u2018\u2019Total WOMAC score regression of 18% and above\u2019\u2019 was taken as a minimal clinically important difference (MCID) to indicate improvement [14].Only advanced stage OA patients who received an intraarticular injection of the steroid methylprednisolone acetate (40 mg to 1 mL) were included in the study. Patients managed with surgical treatment were excluded. Knee joint synovial fluid samples were obtained using sterile needles. The samples were stored at \u201380 \u00b0C until the day of analysis. Synovial fluid levels of ADAMTS4 and ADAMTS9 were analyzed using Eastbiopharm branded human ADAMTS4 and ADAMTS9 enzyme linked immunosorbent assay (ELISA) kit with an immunoassay device (Immulite 2000) and presented in pg/mL and ng/mL, respectively. The blood reference range of the ADAMTS9 assay kit is 5\u2013400 ng/mL, and the blood reference range of the ADAMTS4 assay kit is 5\u20131000 pg/mL. The intraassay and interassay coefficients of variation of the kit were lower than 10% and 12%, respectively.Data analysis was performed using SPSS for Windows, version 22 . Whether the distributions of continuous variables were normal or not was determined using the Kolmogorov\u2013Smirnov test, homogeneity of variances was evaluated using the Levene test. Continuous variables were shown as mean \u00b1 standard deviation (SD) or median (min\u2013max), where applicable. While Student\u2019s t-test compared the mean differences between groups, the Mann\u2013Whitney U test was applied for comparisons of the median values. Patients with advanced OA, according to the K&L scale, were grouped into stage 3 and stage 4 OA. The optimal cutoff points of synovial fluid ADAMTS9 levels in advanced stage OA cases were evaluated by receiver operating characteristic (ROC) analyses, calculating the area under the curve (AUC) as giving the maximum sum of sensitivity and specificity for the variables test. Stage 3 and stage 4 groups were subgrouped into those with a WOMAC score percent change \u226518% and a WOMAC score percent change <18%. Synovial fluid ADAMTS9, synovial fluid ADAMTS4 levels and other variables were compared with Student\u2019s t-test, responding and not responding to the improvement in WOMAC score in stage 3 and stage 4 groups. Also, categorical comparisons were performed using the \u03c72-test. Finally, multiple logistic regression analysis was performed separately in stage 3, stage 4, and all advanced stage knee OA patients to determine the effect of independent variables associated with response to intraarticular therapy. Adjusted odds ratios (ORs) and 95% confidence intervals (CI) were calculated for each variable. A P-value of less than 0.05 was considered statistically significant.n= 42; stage 4,n= 42) were enrolled in the study. Their baseline anthropometric and biochemical characteristics are given in Table 1. The mean age of the patients with stage 3 OA was 62.38 \u00b1 6.78 years, and patients with stage 4 OA was 67.64 \u00b1 5.63 years. The mean age of the patients with stage 4 OA was significantly higher than the stage 3 OA group (P< 0.001). There were no statistically significant differences among smokers in the groups (P= 0.766).A total of 84 BMI matched OA patients with advanced stage disease . WOMAC scores before intraarticular injection were 82.12 \u00b1 6.18 and 65.21 \u00b1 16.26. WOMAC scores after intraarticular injection were 67.26 \u00b1 7.21 and 53.19 \u00b1 12.72 respectively for stage 3 and stage 4 OA patients. WOMAC scores before and after intraarticular injection were significantly higher in the stage 3 OA group compared to the stage 4 OA group (P< 0.001and P < 0.001). Synovial fluid ADAMTS4 levels were 128.51 \u00b1 27.79 pg/mL and 131,84 \u00b1 35.47 pg/mL respectively in stage 3 and 4 OA groups. There were no statistically significant differences among synovial fluid ADAMTS4, WOMAC score change and concomitant diseases  between groups (Table 1).Synovial fluid ADAMTS9 levels were 5.92 \u00b1 1.94 ng/mL in stage 3 OA and 5.02 \u00b1 1.35 ng/mL in stage 4 OA cases . The patients with stage 4 OA had significantly lower levels of ADAMTS9 when compared with stage 3 OA group ; cutoff levels were determined, and AUC values were calculated. The AUC, best cutoff values, sensitivity, and specificity for distinguishing the groups for each parameter are given in Table 2. Synovial fluid ADAMTS9 levels were found to be statistically significant. When the groups that responded and did not respond to the treatment were compared within stage 3 and stage 4, the synovial fluid ADAMTS9 levels were 5.04 \u00b1 1.44 ng/mL in the WOMAC score percent change \u226518% group, and 6.43 \u00b1 2.01 ng/mL in the WOMAC score percent change <18% group.= 0.026). There were no statistically significant differences among age, smoking, synovial fluid ADAMTS4, and concomitant diseases between the groups in stage 3 (Table 3). Age, smoking, synovial fluid ADAMTS9, synovial fluid ADAMTS4, and concomitant diseases were not statistically different between WOMAC score percent change \u226518% and WOMAC score percent change <18% in stage 4 (Table 4).The level of synovial fluid ADAMTS9 was statistically significantly lower in the WOMAC score percent change \u226518% than the WOMAC score percent change <18% group in stage 3 OA (P= 0.010) (Table 5).At first, logistic regression analysis was performed to determine the variables associated with an 18% improvement in the WOMAC (MCID) of intraarticular steroid injection in all OA patients. Decreasing synovial fluid ADAMTS9 levels  were found to be predictive for the MCID in all OA patients  of intraarticular steroid injection treatment in stage 3 and stage 4 OA patients, respectively. Only decreasing ADAMTS9 levels in synovial fluid  were predictive for MCID in stage 3 OA patients (P\u00a0= 0.039) (Table 6). Age, smoking, synovial fluid ADAMTS9, synovial fluid ADAMTS4, and concomitant diseases were not statistically associated with MCID in stage 4 (Table 6). Advanced stage OA is a chronic disease associated with progressive cartilage degeneration. Known triggers of OA include mechanical factors, age, and inflammation [15]. In recent years, neither systemic nor intraarticular medical treatment is effective in stopping the progression of OA [15]. Surgical treatment is the last resort in patients with OA. Several studies have focused on factors in the progression of OA in the elderly population. Kevorkian et al. [16] showed that the expression of ADAMTS1, ADAMTS5, ADAMTS9, and ADAMTS15 genes was significantly decreased and that the expression of ADAMTS16, ADAMTS2, ADAMTS14, and ADAMTS12 genes was increased in OA cartilage, compared to normal cartilage. Naito et al. [17] demonstrated that ADAMTS4 might play an important role in the degradation of aggrecan in human osteoarthritic cartilage. In our study, we found no significant differences in synovial fluid ADAMTS4 levels of stage 3 and stage 4 OA patients. However, the ADAMTS9 levels in the synovial fluid of stage 4 OA patients were significantly lower than those of stage 3 OA patients. Also, the WOMAC score in stage 4 OA group was significantly lower than in the stage 3 OA group. Pretreatment WOMAC scores were higher in the stage 3 OA group when compared with the stage 4 OA group, possibly pointing out the ongoing cartilage degeneration.In human chondrocyte cell lines, Coughlan et al. [10] showed that both ADAMTS5 and ADAMTS9 appeared to be important in cartilage ECM formation and turnover, and that depletion of ADAMTS5 and ADAMTS9 will lead to improved ECM formation and neocorticalization. Yaykasli et al. [11] demonstrated that leptin increased the expression of ADAMTS4, ADAMTS5, and ADAMTS9 genes and cartilage degeneration in human chondrocytes. Yang et al. [18] revealed abnormal increases in Cysteine-rich protein\u00a061(Cyr61) and ADAMTS4 protein levels in OA tissues and chondrocytes. In the same study, Cyr61 reduced ADAMTS4 protein expression in chondrocytes, and Cyr61 interacted with ADAMTS4 to affect OA progression. Stanton et al. [19] found no significant phenotypic abnormalities in ADAMTS4 and ADAMTS5 knockout mice. Still, they found a significant reduction in the severity of surgically induced OA in ADAMTS5 knockout groups compared with ADAMTS4 knockout groups [19]. The present study demonstrates the prediction of intraarticular treatment success in stage 3 OA patients with synovial fluid ADAMTS9 levels. Intraarticular steroid injection was ineffective in stage 4 OA patients due to the severity of cartilage degeneration.In conclusion, we think that ADAMTS9\u2019s low level of synovial fluid is significant in the stage 3 group where cartilage degeneration is continuing, and is meaningless in the stage 4 group where cartilage has wholly degenerated, there is no longer any extracellular matrix structure and there will be no regeneration. With this result, the significantly low level of ADAMTS9 may be related to the fact that intraarticular or systemic treatments will still be effective due to the ECM protease in the synovial fluid of stage 3 OA patients. The limitations of our study are mainly the lack of evaluation of other proteases and proteoglycans, which are risk factors of OA, a small number of patients and the controls had a history of joint injury and had been operated upon the knee for different reasons. The lack of a control group and early-stage OA is the limitation of the study. WOMAC 20,50, and 70 indexes could not be obtained due to the limited number of patients. The fact that no questionnaire related to the psychosocial status of patients has been conducted is another limitation of our study. Further prospective studies in larger cohorts are needed to validate the results of the present study. The authors would like to thank the staff at Bey\u015fehir State Hospital and to all patients who participated in the study."}
+{"text": "This study examined the prevalence of loneliness and relationship with cooking and eating habits among older adults using data collected in December 2019 from a nationally representative sample age 50-80 through the National Poll on Healthy Aging. Older adults who live alone were more likely those who lived with others (19% vs. 8%) and those with a high PHQ-2 score were more like than those with a low PHQ-2 score (20% vs. 11%) to report that they do not cook dinner most days (0-2 times/week). Those who rated their diet as fair/poor were more likely to indicate they cook few meals a week (0-2) compared to those who reported a good or very good/excellent diet (18% vs. 11 % vs 8%). Those with a high PHQ-2 score were less likely than those with a low PHQ-2 score to say they do major food shopping less than once a week (31% vs 47%). Those with a high PHQ-2 score were more likely to say that that always ate alone in the last week compared to those with a low PHQ-2 score (17% vs 7%). These findings demonstrate that older adults who lived alone, had a higher PHQ-2 score, and had a poorer diet were more likely to cook and do grocery shopping less often. Strategies and policies to support older adults to address depressive symptoms and to increase cooking and improve diet may have many health and social benefits and will be explored through this session."}
+{"text": "IntroductionThis article is a retrospective analysis of the neurosurgical census at our institution to determine an optimal threshold for brain tissue oxygenation (PbtO2). The use of brain tissue oxygen monitoring has been in place for approximately\u00a0three decades but data suggesting optimal thresholds to improve outcomes have been lacking. Though there are multiple modalities to monitor cerebral oxygenation, the monitoring of brain tissue oxygen tension has been deemed the gold standard. Still, it is not clear exactly how reductions in PbtO2 should be treated\u00a0or what appropriate thresholds to treat might be. The aim of our study was to determine if our threshold of 28 mmHg for a good functional outcome could be correlated to the Glasgow Coma Scale (GCS) and Glasgow Outcome Scale (GOS).MethodsA retrospective analysis of the Arrowhead Regional Medical Center (ARMC) Neurosurgery Census was performed. Patients from 2017-2019 who had placement of Licox\u00ae cerebral oxygen monitoring sensors \u00a0were included in the analysis. Fifteen patients were consecutively identified, all of which presented with traumatic brain injury (TBI). Data on age, gender, days in the intensive care unit (ICU), days before discharge or end of medical care, admission GCS, hospital length of stay, GOS, maximum and minimum PbtO2 values for five days following insertion, minimum and maximum intracranial pressures (ICPs), and brain temperature were included for analysis.\u00a0Patient data were separated into two groups; those with consistently higher PbtO2 scores \u00a0and those with inconsistent/lower PbtO2 scores . Standard student t-tests were used to find potential statistical differences between the groups (\u03b1 = 0.05).ResultsThere were seven patients in the consistently high PbtO2 category (\u2265 28 mmHg)\u00a0and eight patients in the inconsistent/low PbtO2 category (<28 mmHg). The average maximum and minimum PbtO2 for the group displaying worse outcomes (as defined by GCS/GOS) was 23.0 mmHg and 14 mmHg, respectively. Those with consistent Day 2 PbtO2 scores of \u2265 28 mmHg had significantly higher GCS scores at discharge/end of medical care (p < 0.05). Average GCS for the patient group with\u00a0>28 mmHg PbtO2 averaged over Days 2-5 group was 11.4 (n=7). Average GCS for the <28 group was 7.0 (n=8). The GCS for the >28 group was 63% higher than found in the <28 group (p = 0.03). GOS scores were significantly higher in those with consistently higher PbtO2 (\u2265 28) than those with lower PbtO2 scores (< 28). The averages were 3.5 in the higher PbtO2 group as compared to 2 in the lower PbtO2 group.ConclusionAlong with ICP monitors\u00a0and monitoring in the assessment of CPP, brain tissue oxygenation allows yet another metric by which to optimize treatment in TBI patients. At our institution, a PbtO2 level of \u2265 28 mmHg is targeted in order to facilitate a good functional outcome in TBI patients. Keeping patients at this level improves GCS and GOS at discharge/end of medical treatment. Given the observation that episodes of hypoxemia and ischemia following severe head injury increased mortality, the advent of brain tissue oxygenation monitoring in the 1990s\u00a0had become an important tool in the intensive care unit (ICU) to avoid these insults . Along wThe Licox\u00ae brain tissue oxygenation system  was introduced into clinical practice in 1993, and initially saw most of its use in the setting of traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) [Though the use of brain tissue oxygen monitoring has been in place for close to three decades with improvement in outcomes, data suggesting optimal thresholds to change outcomes have been lacking -5. ThougPreviously,\u00a0correlations between reduced brain tissue oxygenation and poor outcome in TBI patients have been observed . StudiesA retrospective analysis of the Arrowhead Regional Medical Center (ARMC) Neurosurgery Census was performed. Patients from 2017-2019 who had placement of Licox\u00a0cerebral oxygen monitoring sensors were included in this analysis. Fifteen patients were consecutively identified, all of which presented with traumatic brain injury (TBI). Data on age, gender, days in ICU, days before discharge/end of medical care, admission GCS, GCS at discharge/end of medical care, GOS, maximum and minimum PbtO2 values for five days following insertion, minimum and maximum ICPs, and brain temperature were included. Patients were separated by those with consistently high post Day 2 PbtO2 (> 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg). Day 2 PbtO2 values were chosen in order to allow the Licox\u00a0brain tissue monitoring device a period of calibration.We set the minimum requirement to be binned into the consistently high PbtO2 group as having at least \u2265 28 mmHg PbtO2 maximum for Days 3, 4, and 5 . This was the defined threshold below which to treat at our institution to attain a good functional outcome. Seven patients were identified with consistently higher PbtO2 scores (\u2265 28 mmHg), and eight patients were identified with inconsistent/lower PbtO2 scores (< 28 mmHg). Standard t-tests were used to find potential statistical differences between the consistently high and inconsistently high PbtO2 groups. All statistical analysis, plot, and graph construction was done using Microsoft Excel (2016)\u00a0with the Analysis ToolPak .The most common cause of TBI in our patient analysis occurred via motor vehicle accidents. Out of 15 patients, there were three deaths (20%). Our analysis included more men  than women , and most patients were within their first three decades of life. All patients were intubated, either in the pre-hospital setting or shortly after arrival, and were admitted to the ICU. Licox\u00a0monitors were placed following initial stabilization typically within the first 24-48 hours of admission. PbtO2 levels were recorded hourly by nursing staff. Daily maximum and minimum values were obtained for the first five days of cerebral tissue oxygenation monitoring  were separated by those with consistently high post Day 2 PbtO2 (\u2265 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg)  than those with lower PbtO2 scores (< 28 mmHg). The averages were 3.5 in the consistently high PbtO2 group compared to 2 in the inconsistently high/low PbtO2 group. The GOS for the >28 group was 62% higher than found in the <28 group (p = 0.01).External ventricular drain (EVD) length actually increases for those with consistently higher PbtO2. EVD length average is 20 days in the consistently high PbtO2 group as compared to 12 days in the inconsistent/low PbtO2 group. Those patients with consistently \u2265 28 mmHg PbtO2 scores had significantly longer hospital stays (average for \u2265 28 mmHg was 40 days compared with 12 days for those in the inconsistent/low PbtO2 group). Lastly, the consistently higher PbtO2 group had fewer deaths. Out of the 15 patients, three died, of which two had consistently low PbtO2 scores based on our threshold of 28 mmHg.Our threshold of 28 mmHg was also compared to 25 mmHg of PbtO2. We found that the length of EVD was not significantly longer . GOS is significantly higher in the higher PbtO2 (\u2265 28 mmHg) group . ICU stay (days) was not significantly different .\u00a0GCS was also found to be significantly higher in the 28 mmHg PbtO2 group .\u00a0Lastly, we compared our threshold of 28 mmHg to 20 mmHg of PbtO2. We found that GOS was significantly higher in the higher PbtO2 (\u2265 28 mmHg) group . GCS on discharge was significantly higher in the good group . ICU stay (days) was not significantly different . The length of time for EVD placement was significantly higher in the good group .Brain tissue oxygenation has become an important tool in the ICU to direct TBI patient interventions and to optimize treatment options. The management of TBI patients involves continued assessment and monitoring to prevent secondary injuries, such as hypotension and hypoxia\u00a0after the initial irreversible primary brain injury has occurred. Prompt assessment of cerebral oxygenation is important because studies have shown that patients are most at risk for cerebral ischemia during the first few days following injury .At our institution, we consider a partial oxygen pressure above 20 mmHg to be indicative of a functional outcome\u00a0and aim for readings above 28 mmHg for a good outcome . In thisIn our study, patients were separated by those with consistently high post Day 2 PbtO2 (> 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg) Table . We chosGOS\u00a0was significantly higher in those with consistently higher PbtO2 (> 28 mmHg) than those with lower PbtO2 scores (< 28 mmHg). The average\u00a0was 3.5 in the consistently high PbtO2 group as compared to 2 in the inconsistently high/low PbtO2 group. GOS for the >28 group was 62% higher than found in the < 28 group (p = 0.01).Controversy still exists as to the best placement of brain tissue oxygen monitor probes. Uninjured brain, injured brain, and penumbra surrounding the injured brain have all been advocated for, in regard to the placement of the probe. Some authors have waited two hours to record the first PbtO2 levels to allow the calibration of probes. Placement in all 15 patients was within the white matter of the frontal lobe\u00a0in an uninjured brain.\u00a0The Licox\u00a0system is able to measure both oxygen and temperature in the intracranial cavity, with the intention of providing additional metrics to inform clinical decision-making. Potential interventions include the augmentation of the ventilator settings, augmentation in CPP, and the addition of sedation . The potThe consistently higher PbtO2 group had fewer deaths. Out of the 15 patients, three died, of which two had consistently low PbtO2 scores, based on our threshold of 28 mmHg. This was in line with previous studies with a threshold similar to ours for PbtO2 levels. Erikkson et al.\u00a0found that PbtO2 levels below 29 mmHg in the first 72 hours predicted increased mortality in TBI patients . Though We did find that EVD length\u00a0and hospital stay\u00a0actually increases for those with consistently higher PbtO2. EVD length average was 20 days in the consistently high PbtO2 group compared to 12 days in the inconsistent/low PbtO2 group. Those patients with consistently high PbtO2 scores had significantly longer stays with an average of 40 days as compared with 12 days for those in the inconsistent/low PbtO2 group. We hypothesize that this increased length is a result of both increased mortality in the inconsistent or low PbtO2 groups, as well as more aggressive care in those patients who were deemed to have a better prognosis.The limitations of this study include the limited data points for PbtO2 as compared to similar studies in which PbtO2 is recorded by the minute. Additionally, our study would benefit from an increased patient population. Future studies may take into account these features to further delineate thresholds for PbtO2 treatment.Along with ICP monitors\u00a0and monitoring in the assessment of CPP, brain tissue oxygenation allows yet another metric by which to optimize treatment in TBI patients.\u00a0Previous methods of assessment of cerebral hypoxia relied on jugular bulb oximetry. Here, we describe the protocol used at our institution after the placement of brain tissue oxygen monitoring probes. We sought to keep PbtO2 levels above 28 mmHg for a good functional outcome. We demonstrate improvements in both GCS and GOS for patients who consistently had maximum PbtO2 levels above 28 mmHg on Days 3 to 5 while the monitoring system was in place. In addition, patients in this group had fewer deaths, in line with previous studies. However, this population did have a longer EVD duration as well as hospital stay duration. Future studies to better delineate thresholds should be performed."}
+{"text": "The correct author name order is Xinzhi Wang1, Qianqian Jiang1,2, Yichi Zhang1, Nannan Yuan1 and Jianguo Tang1.The author name order was incorrectly displayed as Qianqian JiangThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "Correction to: BMC Public Health (2020) 20:614https://doi.org/10.1186/s12889-020-08742-1It was highlighted that the original article  containeEthics approval and consent to participateThe QE team received initial approval for the AHME evaluation with \u201cExempt\u201d status from UCSF\u2019s Institutional Review on 13 June 2013. Ethical approval also was obtained from the Ghana Health Services Ethical Review Committee (ERC) and the Kenya Medical Research Institute (KEMRI). Prior to each round of data collection, the QE team received approval from all three ethical review boards for any changes made to the research protocol. Approvals for Round 2 (2017) of data collection were received: on 12 December 2017 from UCSF, 16 November 2017 from the ERC, and 17 January 2017 from KEMRI.Ethical approval for Round 1 of AHME\u2019s Client Exit Interviews was obtained from Marie Stopes International\u2019s Ethics Review Committee (MSI ERC) on 25 August 2017, the Ghana Health Service Ethics Review Committee (GHS ERC) on 20 November 2017, and Amref Health Africa Ethics and Scientific Review Committee (Amref ESRC) on 30 April 2018. Approvals for Round 2 were received from MSI ERC on 10 September 2018 and GHS ERC on 12 November 2018. The approval received from Amref ESRC on 30 April 2018 covered both Round 1 and Round 2 of data collection in Kenya."}
+{"text": "GPR41 and GPR43 appear to be derived from the same image.After publication of this article , concernGPR41 adipose tissue. The original images for GPR41 and GPR43 adipose tissue are provided in The authors apologize that an error was made in generating An Academic Editor reviewed the updated figure and underlying data and confirmed that they support the results and conclusions reported in the published article.The authors apologize for the error in the published article.With this Correction, the authors also provide underlying data supporting Fig 2 and Fig 3 of , in S3 aS1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(PZF)Click here for additional data file.S4 File(PZF)Click here for additional data file."}
+{"text": "Sirtuin1 (Sirt1) has a NAD (+) binding domain and modulates the acetylation status of peroxisome proliferator-activated receptor-\u03b3 coactivator-1\u03b1 (PGC1\u03b1) and Fork Head Box O1 transcription factor (Foxo1) according to the nutritional status. Sirt1 is decreased in obese patients and increased in weight loss. Its decreased expression explains part of the pathomechanisms of the metabolic syndrome, diabetes mellitus type 2 (DT2), cardiovascular diseases and nonalcoholic liver disease. Sirt1 plays an important role in the differentiation of adipocytes and in insulin signaling regulated by Foxo1 and phosphatidylinositol 3\u2032-kinase (PI3K) signaling. Its overexpression attenuates inflammation and macrophage infiltration induced by a high fat diet. Its decreased expression plays a prominent role in the heart, liver and brain of rat as manifestations of fetal programming produced by deficit in vitamin B12 and folate during pregnancy and lactation through imbalanced methylation/acetylation of PGC1\u03b1 and altered expression and methylation of nuclear receptors. The decreased expression of Sirt1 produced by impaired cellular availability of vitamin B12 results from endoplasmic reticulum stress through subcellular mislocalization of ELAVL1/HuR protein that shuttles Sirt1 mRNA between the nucleus and cytoplasm. Preclinical and clinical studies of Sirt1 agonists have produced contrasted results in the treatment of the metabolic syndrome. A preclinical study has produced promising results in the treatment of inherited disorders of vitamin B12 metabolism. Sirtuin1 (Sirt1) is one of the seven mammalian proteins belonging to the silent information regulator 2 (Sir2) proteins/Sirtuin family with highly conserved catalytic and nicotinamide adenine dinucleotide (NAD+) binding domain . Sirt1 hCalorie restriction regulates the expression levels of Sirt1 in a tissue-dependent manner . Sirt1 rThe metabolic syndrome is a global public health problem related to overnutrition. It is defined as a cluster of cardio-metabolic abnormalities that includes obesity, insulin resistance or diabetes mellitus type 2 (DT2), hypertension and dyslipidemia ,22. The Secretion of insulin by pancreatic beta cells is enhanced by Sirt1 in response to glucose stimulation ,35,36. CClinical studies evidenced decreased Sirt1 expression in adipose tissues of obese patients ,42 and iSirt1 plays an important role in the differentiation of skeletal muscle . InsulinVisceral low grade inflammation triggered by adipocytes contributes to the pathogenesis of obesity, insulin resistance and other outcomes of the metabolic syndrome ,59,60. BConverging evidences support an antagonistic crosstalk between Sirt1 and nuclear transcription factor Kappa B (NF-\u03baB) . DamaginMice with hepatocyte specific knock out of Sirt1 (Sirt1LKO) exposed to a high fat diet develop hepatic inflammation and ER stress . Liver iOxidative stress is associated with pathogenesis of complex metabolic diseases. Sirt1 activates Foxo transcription factors via the feedback loop ,76. ActiThe fetal programming hypothesis , also naThe decreased expression of Sirt1 plays a prominent role in the pathomechanisms of fetal programming produced by MDD in rats (vitamin B12 and folate) ,81,93,94MDD during pregnancy and lactation induces impaired fatty acid oxidation, reduced activity of complexes I and II, cardiac hypertrophy with enlargement of cardiomyocyte and liver steatosis in weaning rat pups ,96. ThesThe impaired cellular availability of vitamin B12 leads to ER stress related to decreased expression of Sirt1. ER stress is evidenced by increased expression and activation of elf2-\u03b1 and activating transcription factor 6 (ATF6) and may be favored by decreased expression of heat shock proteins (HSP). Decreased Sirt1 impairs the transcription of HSP by increasing the acetylation of heat shock protein factor 1 HSF1;  93]. Co. Co93]. Preclinical and clinical studies in the treatment of complex and inherited metabolic diseases have produced contrasted results in the evaluation of health benefits of activation of Sirt1 by natural and pharmaceutical small activating molecules.Resveratrol is a natural polyphenolic compound found in red wine and grape and in plants and fruits like berries and peanuts. Resveratrol activates Sirt1 and mimics the caloric restriction status known to be protective against the metabolic syndrome. For decades, the therapeutic use of resveratrol has been considered in regard to its anti-inflammatory, antioxidant, antiaging and anticancer properties ,102,103.Given the health benefits of resveratrol in experimental animal studies, clinical trials have been carried out to evaluate its effects in the prevention and treatment of metabolic syndrome. A preliminary exploratory trial showed that administration of 500 mg resveratrol twice per day for 60 days in type 1 diabetic patients decreased fasting plasma sugar (FPS) and hemoglobin A1C . A prospfa/fa rats [ob/ob mice by upregulating hepatic Sirt1 and GCN5 [Activators of Sirt1 such as SRT1720, SRT2183 and SRT1460 are 1000-fold more active than resveratrol. SRT1720 and resveratrol improve insulin sensitivity in nutritionally and genetically induced obesity and DT2 in mice . SRT1720/fa rats . SRT1720/fa rats . Further/fa rats . SRT1720/fa rats . SRT1720and GCN5 . A compuand GCN5 . At low and GCN5 . Recentland GCN5 .Phase 1 clinical trials showed that SRT2104, another small molecule activator of Sirt1 is safe and tolerable in healthy volunteers, including the elderly ,133. HowMTR-KO mice with brain specific invalidation of Mtr gene encoding MS enzyme [Some severe forms of inherited disorders of intracellular metabolism of vitamin B12 are resistant to conventional treatments. Decreased Sirt1 activity plays a central role in some of the pathomechanisms of these disorders. We therefore evaluated the effect of Sirt1 agonists in a preclinical study in fibroblasts from patients with cblG and cblC inherited defects of vitamin B12 metabolism and an original transgenic mouse model of methionine synthase deficiency specific to neuronal cells. Patient fibroblasts with cblC and cblG defects of vitamin B12 metabolism presented with endoplasmic reticulum stress, altered subcellular localization of HuR, HnRNPA1 and RBM10, global mRNA mislocalization and increased HnRNPA1-dependent skipping of interferon regulatory factor 3 (IRF3) exons. SRT1720 inhibited ER stress and rescued RBP and mRNA mislocalization and IRF3 splicing. Furthermore, Sirt1 activation by SRT1720 partially restored the methylation and phosphorylation of these RBPs in patients\u2019 fibroblasts. Interestingly, SRT1720, vitamin B12 and SAM treatment improved cognitive functions in conditional S enzyme . In partIn summary, preclinical and clinical studies of Sirt1 agonists have produced contrasted results in the treatment of the metabolic syndrome. A preclinical study has produced promising results in the treatment of inherited disorders of vitamin B12 metabolism.Numerous experimental evidence show a strong link between decreased Sirt1 and the pathological manifestations of metabolic syndrome by synergistic cellular and molecular mechanisms. It is noteworthy that over nutrition and MDD both produce a decrease of Sirt1. There are additive and synergistic effects of the MDD fetal programming and subsequent exposure to a high fat diet in adult life. The therapeutic prospects for using activators of Sirt1 in the treatment of disease outcomes of metabolic syndrome have not been conclusive to date. However, pharmacological activation of Sirt1 opens promising perspectives for the treatment of rare diseases of vitamin B12 metabolism with particular effects on reticulum stress and mislocalization of RBPs."}
+{"text": "The nanoparticles are synthesized by solvent-induced nanoprecipitation of [Tb2(TCAn)2] complexes  with the use of polystyrenesulfonate (PSS) as stabilizer. The temperature responsive luminescence behavior of PSS-[Tb2(TCAn)2] within 293\u2013333\u00a0K range in water is modulated by reversible changes derived from the back energy transfer from metal to ligand (M*\u2009\u2192\u2009T1) correlating with the energy gap between the triplet levels of ligands and resonant 5D4 level of Tb3+ ion. The lowering of the triplet level (T1) energies going from TCA1 and TCA2 to their brominated counterparts TCA3 and TCA4 facilitates the back energy transfer. The highest ever reported temperature sensitivity for intracellular temperature nanosensors is obtained for PSS-[Tb2(TCA4)2] (SI\u2009=\u20095.25%\u00a0K\u22121), while PSS-[Tb2(TCA3)2] is characterized by a moderate one (SI\u2009=\u20092.96%\u00a0K\u22121). The insignificant release of toxic Tb3+ ions from PSS-[Tb2(TCAn)2] within heating/cooling cycle and the low cytotoxicity of the colloids point to their applicability in intracellular temperature monitoring. The cell internalization of PSS-[Tb2(TCAn)2]  marks the cell cytoplasm by green Tb3+-luminescence, which exhibits detectable quenching when the cell samples are heated from 303 to 313\u00a0K. The colloids hold unprecedented potential for in vivo intracellular monitoring of temperature changes induced by hyperthermia or pathological processes in narrow range of physiological temperatures.The work introduces hydrophilic PSS-[Tb Narrow intensive emission bands and long lifetimes of excited states provide a higher signal-to-noise ratio than that for the organic NPs and quantum dots. Versatility of lanthanide emitters derives from the variation of lanthanide ions, while their ligand environment enables to modify both intensity and response ability of lanthanide-centered luminescence. The temperature dependence of lanthanide-centered luminescence is the well documented basis for development of noninvasive submicrometer luminescent thermometric mapping19. Real-time in vivo and in vitro monitoring of temperature in living cells is of great impact to record temperature fluctuations during magnetic hyperthermia (MHT) or photothermal therapy (PTT), two emergent modalities for thermal treatment of cancer. The inorganic NPs doped by lanthanide ions are widely documented as nanothermometers based on up-conversion luminescence21. Mostly, they utilize the luminescence intensity ratio (LIR) approach19. The presence of the thermally coupled f-levels is the prerequisite for the temperature sensitivity of the inorganic NPs doped by Er22, Tm18, Nd23 and Ho24 ions, where the population distribution of thermally coupled energy levels can be re-distributed with temperature change due to Boltzmann statistics. Temperature responsive behavior of the lanthanide-based NPs is greatly affected by the energy gap between the thermally coupled f-levels of lanthanide ions. Thus, both the nature of the doped or co-doped lanthanide ions and the architecture of the lanthanide-doped NPs are the tools for tuning their temperature sensitivity19. However, the temperature sensitivities (S) defined as the rate of change of the thermo-sensitive parameter with temperature are typically low, usually being below 1%\u00a0K\u22121 at the temperatures about 300\u00a0K for the major part of the reported lanthanide-doped inorganic NPs28.Lanthanide-based nanoparticles (NPs) have gained growing attention during recent decades due to their divergent application in cellular mapping and sensing. Specific photophysical characteristics of lanthanide compounds constituting the NPs significantly differentiate the latter from polymeric organic nanoparticles and quantum dots29. Greater temperature sensitivities of the molecular complexes, reported as up to 4.9%\u00a0K\u22121, make them very promising basis for development of the lanthanide-based thermometers34. It is worth noting that both encapsulation of lanthanide complexes and their deposition onto polymeric nanomaterial  induce some changes in the inner-sphere environment of lanthanide ions, which, in turn, affect the thermal behavior of the complexes. However, the effects of the nanoparticulate hosts on the temperature responsive behavior of the lanthanide complexes are insufficiently studied. Moreover, the ligand environment of the ions commonly applied for these purposes (Tb3+ and Eu3+) is restricted to 1,3-diketonates. This mainly derives from both good luminescence and convenient thermal behavior of the lanthanide diketonates. However, the low solubility of the complexes in specific solvents  affects the efficiency of the doping procedure restricting their use as molecular blocks of silica nanoparticles36.Lanthanide complexes where lanthanide ions are coordinated by organic ligands of different structure represent promising basis for thermo-responsive nanomaterial because of the presence of different mechanisms, with respect to those regulating the thermo-responsive behavior of lanthanide ions in the inorganic matrices6. The main advantage of this morphology is the facile and easy way of conversion of the molecular lanthanide complexes into the aqueous colloids. Moreover, any complexes, which are soluble in any water-miscible organic solvent, but insoluble in water, provide a basis for the transition from molecular to nanoparticulate form. However, this molecule-to-nano transformation can affect both inner- and outer-sphere environment of the terbium ions, which, in turn, changes the Tb3+-centered luminescence and its thermo-sensitivity.The luminescent nanoparticles with core\u2013shell morphology, where the core results from the solvent-induced nanoprecipitation of lanthanide complexes, while the hydrophilic shell derives from the deposition of the polyelectrolytes, represent very promising alternative to the polymeric nanoparticles doped by the lanthanide complexes3+ ions for both efficient Tb3+-centered luminescence and facile conversion into the aqueous luminescent colloids with high colloid stability37. The documented colloids were revealed as efficient fluorescent contrast agents with good cellular uptake behavior4. Moreover, some thiacalix[4]arene derivatives has been recently reported39 as the appropriate ligand environment of Tb3+ ions to generate the Tb3+-centered luminescence exhibiting the temperature responsive behavior in physiological temperature range. In particular, the dimeric 2:2 terbium complexes with the bromo-substituted thiacalix[4]arenes provide a promising basis for design of a temperature-sensitive nanomaterial39. This is the reason for focusing on the optimal conditions of an efficient conversion of the complexes from molecular blocks in the organic solutions to the water dispersed hydrophilic nanoparticles. The identification of main factors, including the structure of the ligands, influencing the temperature responsivity of the Tb3+-centered luminescence of the aqueous colloids based on the terbium complexes with four thiacalix[4]arene derivatives, will be discussed. Thus, the four thiacalix[4]arene derivatives bearing at the upper rims different substituents, including bromines 3\u00b76H2O, Alfa Aesar, 99.9%), gadolinium(III) nitrate hexahydrate (Gd(NO3)3\u00b76H2O, Sigma-Aldrich, 99.9%), triethylamine , D2O  (PSS) , DAPI (Sigma) and sodium chloride (Sigma-Aldrich) were used as received. N,N-Dimethylformamide (DMF) (Acros Organics) was twice distilled over P2O5. Thiacalixarenes TCA140, TCA241, TCA339 and TCA442 were synthesized according to published procedures. Both 1H NMR spectra of the thiacalix[4]arenes and their elemental analysis data  nitrate hexahydrate (Tb(NO3)3\u00b76H2O with TCAn in the 1:1 ratio, and the solution was basified by the excess amounts of triethylamine (TEA) taken in 1:8 (TCAn:TEA) ratio43. The resulted colloidal solution was sonicated for 30\u00a0min (sonication bath temperature was controlled to be 20\u2009\u00b1\u20092\u00a0\u00b0C). Excess amounts of PSS were separated from colloids via centrifugation  and supernatant drain. One layered PSS-coated aqueous colloids were obtained via their redispersion (using sonication) in bidistilled water .The synthetic procedure is based on the formation of nanosized cores derived from precipitation of water insoluble Tb(III) complexes with ligands TCAn (n\u2009=\u20091\u20134) with the following adsorption of polystyrenesulfonate (PSS) molecules on their surfaces. The precipitation is induced by the pouring of 0.5\u00a0ml of the complex DMF solution under intensive stirring (2200\u00a0rpm) 2.5\u00a0ml of PSS-containing aqueous solution . The complex formation in the DMF solution was achieved by mixing of the Tb(NO2(TCAn)2] (n\u2009=\u20091\u20134) was examined by measuring of Tb3+-centered luminescence in the aerated aqueous colloids at different temperatures. The temperature acquisition performed by the water circulation thermostat required 5\u00a0min.The thermo-behavior of PSS-[Tb2(TCAn)2]  at different temperatures was performed in the manner shown in Scheme The monitoring of the luminescence intensity and fluorescent microscopy imaging of M-Hela cells stained by PSS-arene derivative (TCA2) has been also introduced in the present work to vary hydrophobicity of calixarene derivatives upon addition of tert-butyl groups to facilitate the solvent-induced aggregation of their complexes and to compare with TCA1.Figure\u00a0TCAn n\u2009=\u2009\u20134 form t3+ ions in the DMF solutions evidences great similarity with TCA2. The steady state and time-resolved Tb3+-centered luminescence of the terbium complex with TCA1 in DMF reveals the insignificant deviation from the 2:2 complex with TCA2  of TCA2 was evaluated by the analysis of the fluorescence and phosphorescence spectra of the isostructural Gd3+ complexes in DMF  are very close to the values recently reported for TCA1 39. This allowed to assume that the sandwich-like structure of [Tb2(TCAn)2] complexes shown in Fig.\u00a02(TCAn)2] colloids.UV\u2013Vis study of the complex formation of TCA1 with TbCA2 Fig.\u00a0. The eneDMF Fig.\u00a0. It is w3+ ions and the excess of TEA tend to stay in the aqueous DMF solution due to their better solubility in water. The aqueous colloids resulting from the aggregation can be easily separated from the supernatant by centrifugation. The facile phase separation of the produced colloids provides the way to wash and analyze the Tb3+ in the supernatants. This enables to dispose of any water-soluble admixtures and to evaluate the extent of the Tb3+ complexes conversion from the molecular to the nanoparticulate state. The conversion extent was determined by spectrophotometric analysis of the Tb3+ ions in the supernatants, produced by the first phase separation after the synthesis, and the second one performed within the washing procedure. The percentages of the Tb3+ losses during the synthesis and the washing step are collected in Table 3+ ions during the synthetic procedure are about 3% relative to the initial concentration of the Tb3+ ions in the synthetic mixture, which indicates that the major extent of the complexes is converted from the molecular to the nanoparticulate state. Moreover, the EDS spectrum of the dried PSS-[Tb2(TCA3)2] colloids . The copper constituting of the grid is manifested by the Cu band in the spectrum 2], being about 1.7% for TCA1, while less than 1% for TCA2-4.The Tb2(TCAn)2] (n\u2009=\u20091\u20133)43, the electrokinetic potential values measured in the aqueous colloids range from \u2212\u200946 to \u2212\u200968\u00a0mV (Table 2(TCAn)2]-based cores, which are smaller than those measured by the DLS technique according to corresponding size distribution diagrams 2] colloids and their aggregation behavior are the well-known reasons for the deviation in the size-values measured by the different techniques44.Similar to PSS-[Gdn)2] n\u2009=\u2009\u2013343, then)2] n\u2009=\u2009\u2013343, then)2] n\u2009=\u2009\u2013343, the2(TCAn)2] colloids is manifested by four narrow bands peculiar for Tb3+-centered luminescence 2] complexes in the DMF solutions39, the luminescence intensity is the highest for the thiacalix[4]arenes TCA1 and TCA2 compared to the bromo-substituted ligands TCA3 and TCA4. The integral intensities of the four emission bands measured in PSS-[Tb2(TCAn)2] colloids in the same concentration and instrumental conditions presented in Table 5D4\u2009\u2192\u20097F6) and 543\u00a0nm (5D4\u2009\u2192\u20097F5) are about 0.7 for the PSS-[Tb2(TCAn)2] colloids, which is greater than the ratios 0.56\u20130.59 previously reported for [Tb2(TCAn)2] in the DMF solutions39. Since the ratio is commonly considered as the factor affected by changes in a ligand environment of terbium ions45, the observed difference can be explained by the hydration effect arisen from the solvent exchange on going from the DMF to the aqueous solutions.The steady state luminescence of PSS-[Tb3+ ions in [Tb2(TCAn)2] shown in Fig.\u00a038. Thus, the measurements of hydration numbers (q) of the Tb3+ ions in PSS-[Tb2(TCAn)2] were performed for confirming the complex stoichiometry. For this reason, in accordance with the well-known procedure46 the excited state lifetimes were measured for PSS-[Tb2(TCAn)2] in both H2O and D2O and reported in Table 46 and colloidal particles44 in H2O and D2O solutions allows calculating the number of coordinated water molecules with the uncertainty in q (number of water molecules) of\u2009\u00b1\u20090.5 by the use of the Horrocks equation  were calculated according to the equation presented in SI along with the hydration numbers (q) presented in Table avg values among the ligands TCAn (n\u2009=\u20091\u20133). For comparison, the average values of the complexes in DMF are reported in brackets along with those measured in the colloids. The comparison of the lifetime values measured in PSS-[Tb2(TCAn)2] when n\u2009=\u20092, 3 indicates that they are smaller than those measured for TCA1 or previously reported for TCAn (n\u2009=\u20092\u20134) in the DMF solutions of [Tb2(TCAn)2] 2] (n\u2009=\u20091\u20133). Thus, the sandwich-like coordination of Tb3+ ions leading to the 2:2 stoichiometry is predominant in these colloids, while the small extent of the monomeric complex forms with the greater hydration number of Tb3+ ions (q\u2009=\u20095)39 cannot be excluded because of the shorter \u03c4 values contributing to \u03c4avg 2] for TCA4 correlates with the hydration number equal to 3. The greater hydration number revealed in PSS-[Tb2(TCA4)2] versus PSS-[Tb2(TCAn)2] (n\u2009=\u20091\u20133) can be explained by the increased contribution of the more hydrated monomeric form.The lower average \u03c4-value of PSS-[Tb3+-centered luminescence for PSS-[Tb2(TCAn)2] is worth preceding by discussion of the main factors responsible for the temperature dependent luminescence of [Tb2(TCAn)2] in the solutions. As it has been previously documented39 the triplet energy level of the ligands is of the greatest impact on the thermo-behavior of [Tb2(TCAn)2] in solution. The recently reported invariance of the Tb3+-centered luminescence of complex [Tb2(TCA2)2] in solution upon heating within the range 293\u2013323 K39 results from the higher energy of the triplet levels relative to the resonant 5D4 level of Tb3+ ion . The difference between the energies of these levels in accordance with the Latva rule is enough to provide efficient ligand-to-metal energy transfer and insignificant energy back transfer48. The similarity in the triplet levels for TCA1 and TCA2 in the terbium complexes is the reason for the insignificant change in the luminescence of [Tb2(TCAn)2]  in the solutions within the temperature range 293\u2013323\u00a0K. However, the relative intensity of the main band at 545\u00a0nm of PSS-[Tb2(TCAn)2]  has a thermal behavior, manifested by the quenching under the heating to 333\u00a0K, which is most pronounced for TCA2 2] .The degradation of the colloids can be monitored by measuring the Tbds Table . This va50 can facilitate the degradation of the complexes during the luminescence measurements at different temperatures, where the samples are exposed to the irradiation for a long time (about twenty-thirty minutes). However, PSS-[Tb2(TCAn)2]  should be irradiated within two hours at least for the degradation of the luminescence on 20\u201330% relative to the initial value, which is much longer than the irradiation time for the heating\u2013cooling cycle. The DLS measurements of the colloids after the heating\u2013cooling cycle confirm the insignificant degradation of the colloids 2] when n\u2009=\u20093, 4 results in more pronounced quenching under the heating from 293 to 333\u00a0K 2]  being quenched by the heating to 333\u00a0K tends to recover after the cooling to 293\u00a0K, although the recovery is incomplete. It is worth noting that the previously reported energies of the triplet levels for ligands TCA3 (440\u00a0nm) and TCA4 (458\u00a0nm) reveal them as much worse antenna of Tb3+-centered luminescence than TCA1 and TCA2 due to the greater back energy transfer from the excited level of Tb3+ to the triplet level of the ligand39. The lifetime values of PSS-[Tb2(TCAn)2]  tend to decrease by 20% relative to the initial value under the same heating. The thermo-induced decrease in the lifetime values 2]  colloids than for the complexes in DMF solutions. However, the data in Fig.\u00a02(TCAn)2] between n\u2009=\u20093 and 4.The luminescence of PSS-[Tbn)2] n\u2009=\u2009, 4 beingn)2] n\u2009=\u2009, 4 beingn)2] n\u2009=\u2009, 4 being3+-losses after the heating\u2013cooling cycle, being no more than 3.5% relative to the initial concentration of PSS-[Tb2(TCAn)2] , indicates rather poor degradation of the complexes under their treatment within the cycle (Table 2(TCAn)2] becomes greater going from ligands TCA1 and TCA2 to the bromo-substituted ligands TCA3 and TCA4 2] tends to be larger with the increase of the number of the bromo-substituents of the ligand. The photobleaching explains the degradation of Tb3+-centered luminescence after the heating\u2013cooling cycle 2] and PSS-[Tb2(TCA4)2], respectively. Indeed, the aforesaid percentages of the degradation agree with the real time duration (about 20\u201325\u00a0min for more details see the Exp. Section) required for the luminescence measurements during the heating\u2013cooling cycle within 293\u2013333\u00a0K. It is worth noting that both nanoparticulate state and aqueous environment are the factors enhancing the photobleaching, since the degradation of [Tb2(TCA4)2] in the DMF solutions after continuous irradiation for a half of an hour is no more than 10% of the initial luminescence intensity39. The literature data on the photo-oxidation of the phenols enhanced by their chloro-substitution50 can be assumed as an explanation of the bromo-substitution effect on the photobleaching. Thus, the upper-rim bromination of the thiacalix[4]arene ligands is the reason for the partially-reversible temperature-induced quenching of PSS-[Tb2(TCA3)2] and PSS-[Tb2(TCA4)2] through the back energy transfer mechanism, although the increased photobleaching contributes to the quenching of the colloids reducing the reversibility of their thermal behavior.The Tble Table . HoweverCA4 Fig.\u00a0. MoreoveSI, defined by Eq.\u00a02]  colloids and plotted versus temperature in Fig.\u00a02(TCA3)2], and between 4.8\u20136.2 (heating) and 2.2\u20134.6 (cooling) for PSS-[Tb2(TCA4)2]. These numbers for SI are higher than the ones reported for inorganic lanthanide-based nanoparticles, being similar to the best ever reported examples and the highest ever reported temperature sensitivity for intracellular temperature nanosensors31. It is worth noting that the SI-values presented in Fig.\u00a02(TCA4)2] results from the contribution of the photo-induced complex transformations. It is worth assuming that high activity of the complexes at the nanoparticle/water interface is the reason for transformations of the complexes under their heating and prolonged irradiation. Nevertheless, these transformations result in the insignificant release of toxic for living cells Tb3+ ions, which can be regarded as a prerequisite of the use of PSS-[Tb2(TCAn)2] as intracellular detectors of temperature changes. Moreover, the extent of the luminescence reversibility after the heating\u2013cooling cycle for PSS-[Tb2(TCA3)2] seems to be promising for the reproducibility of the thermally-driven luminescence changes. The latter is important for monitoring of the intracellular temperature changes within several heating/cooling cycles under a cell treatment by hyperthermia. Taking into account that the temperature range can be reduced to 293\u2013318\u00a0K to fit into the physiological temperature interval, the reusability of PSS-[Tb2(TCA3)2] was evaluated in the narrower temperature range 2] n\u2009=\u2009, 4 collon)2] n\u2009=\u2009, 4 collo3+-luminescence under the heating and cooling of the aqueous dispersions performed within five heating/cooling cycles. The graph in Fig. n/I0 and In/I(n\u22121) values versus the number of cycles  designate the luminescence intensities of the initial colloids, and those at the beginning of n- and (n\u22121)-cycles, respectively) illustrates smaller degradation of the luminescence after the first cycle. This reveals the possibility to run five heating\u2013cooling cycles at least. Moreover, the SI-values calculated for the same temperature conditions (308\u00a0K) remain practically unchanged under the running of the cycles 2] colloids can be considered as cellular nanosensors convenient for recurrent monitoring of the temperature changes, while PSS-[Tb2(TCA4)2] nanoparticles with the greater thermo-sensitivity than PSS-[Tb2(TCA3)2] can be applied for single intracellular measurement. Both cytotoxicity and cell internalization of the colloids should be measured before their intracellular application.The data plotted in Fig.\u00a0les Fig. c. It is 2(TCAn)2] is worth preceding by the discussion of the previously reported data on the PSS-coated colloids based on different lanthanide complexes37. It has been, in particular, already estimated that the PSS-based exterior layer of the lanthanide complexes deposited onto the hard cores is a reason for greater cell internalization versus the same hard cores deposited by PSS-polyethyleneimine (PEI) bilayer, where the PEI-layer is the exterior37. Moreover, the previously published colloids exhibit rather low cytotoxicity, which makes them efficient cellular contrast agents in confocal microscopy imaging of cell samples37. Thus, cell viability measurements of the three cell lines incubated by the colloids has been performed. The results are presented in Fig. The presentation of the cellular uptake and cytotoxicity of PSS-[Tb2(TCA3)2] and PSS-[Tb2(TCA4)2] is observed. In particular, the IC50 values of PSS-[Tb2(TCA3)2] evaluated for M-Hela, Chang liver and HSF cell lines are 380, 300 and 340\u00a0\u00b5M respectively, while the same values for PSS-[Tb2(TCA4)2] are above 380\u00a0\u00b5M.It is worth noting that both colloids exhibit rather low cytotoxicity, although some difference in the cytotoxic effect of PSS-[Tb2(TCAn)2]  colloids under various incubation time indicate the cell internalization of the colloids. Moreover, the data reveal the gradual growth of the cell internalization (derivative of measured luminescence intensity) under the increased incubation time from one to eight hours. The fluorescence microscopy images 2] ) into M-Hela cells.The flow cytometry measurements Fig. d,e perfoes Figs.\u00a0,S6 illus2(TCA3)2] and PSS-[Tb2(TCA4)2] into M-Hela cells was visualized by both fluorescence microscopy and confocal images of the cell samples incubated by the colloids. Figure 2(TCA4)2] and confocal microscopy for the cell samples incubated for 24\u00a0h. No detectable difference between the colloids is revealed in their cell internalization. It is worth noting that the confocal image with the staining of the cell nuclei by the dye  details the location of PSS-[Tb2(TCA4)2] within cell cytoplasm. Moreover, Fig. 5c indicates that the nanoparticles are in close proximity to the cell nuclei, but do not enter them. The efficient cell internalization of the PSS-coated colloids seems to disagree with their high negative surface charge manifested by the \u03b6-values in Table 51. The protein-corona, in turn, is the well-known factor modifying the cell internalization of different types of nanoparticles52, which has been previously exemplified for the silica nanoparticles bearing sulfonate-groups at their surface53. Thus, efficient cell internalization of the PSS-coated colloids manifested by the marking of the cell cytoplasm provides a basis for their use as intracellular transducers of the temperature changes.The internalization of PSS-arenes with four tert-buthyl-, two or four bromo-substituents. The synthesis was based on solvent-induced nanoprecipitation of the Tb3+ complexes, while a hydrophilic coating of the cores by polystyrenesulfonate (PSS) molecules provides their colloid stability. The hydration number equal to 2 was evaluated by the time-resolved luminescence measurements for the nanoprecipitated complexes with the unsubstituted, tert-buthyl-substituted and dibromo-substituted ligands. This indicates that specific dimeric complexes with sandwich-like coordination mode of Tb3+ ions, which are predominant in the DMF solutions, remain unchanged after the nanoprecipitation. The greater (close to 3) hydration number obtained for the complexes with tetra-brominated thiacalix[4]arene can be explained by a contribution of the monomeric complexes with the greater hydration number of Tb3+ ions. The bromo-substitution of thiacalix[4]arene causes the quenching of the Tb3+-centered luminescence due to the energy back transfer, which increases with the heating in the temperature range 293\u2013333\u00a0K. The deviation of the luminescence intensities measured under the heating and cooling of the colloids indicates that the temperature-dependent luminescence of the colloids has a contribution coming from the irreversible transformation of the complexes. Thus, the greatest irreversible contribution to the heating-induced quenching of the luminescence makes the nanoparticles based on the complexes with tetra-brominated thiacalix[4]arene the most sensitive to the heating, but restricts their applicability to the single use only. However, the di-brominated ligands provide the optimal ligand environment of the Tb3+ ions in the nanoparticles for the recurrent temperature measurements for five heating/cooling cycles at least. The heating/cooling cycle results in the insignificant release of toxic Tb3+ ions from the nanoprecipitated complexes with the di-brominated thiacalix[4]arenes. This along with the low cytotoxicity of the PSS-coated colloids estimated for the three cell lines points to their applicability as intracellular sensors of temperature changes. The quick cell internalization of the colloids results in the efficient marking of the cell cytoplasm by green luminescence. The latter exhibit detectable quenching under the heating of the cell samples in the temperature range within 303\u2013313\u00a0K. This study demonstrates the successful application of complex colloidal structures for monitoring the intracellular temperature changes with unprecedented sensitivity, and may represent a significant step forward for accurate 2D temperature mapping in processes like hyperthermia or pathological processes.The work introduces synthesis of hydrophilic core\u2013shell nanoparticles exhibiting thermo-responsive TbSupplementary Information."}
+{"text": "Cell proliferation was significantly accelerated by knockdown of CLIC3 in MKN7 cells. On the other hand, the proliferation was attenuated by exogenous CLIC3 expression in human gastric cancer cells (KATOIII and NUGC-4) in which endogenous CLIC3 expression is negligible. Our results suggest that CLIC3 functions as a Cl\u2212 channel in the plasma membrane of gastric cancer cells and that decreased expression of CLIC3 results in unfavorable prognosis of gastric cancer patients.Pathophysiological functions of chloride intracellular channel protein 3 (CLIC3) in human gastric cancer have been unclear. In the tissue microarray analysis using 107 gastric cancer specimens, CLIC3 expression was negatively correlated with pathological tumor depth, and the patients with lower expression of CLIC3 exhibited poorer prognosis. CLIC3 was expressed in the plasma membrane of cancer cells in the tissue. CLIC3 expression was also found in a human gastric cancer cell line (MKN7). In whole-cell patch-clamp recordings of the cells expressing CLIC3, NPPB-sensitive outwardly rectifying Cl Gastric cancer is one of the most common malignant tumors in the abdominal region , 2. Vari2+-activated Cl\u2212 channel, also contributes to tumor invasion and poor prognosis of human gastric cancer i and reduced cell proliferation with G0/G1 arrest [\u2212]i affects the anti-cancer activity of paclitaxel, a microtubule-targeted chemotherapeutic drug, in MKN28 cells [\u2212]i also attenuates cell proliferation of prostate cancer PC3 cells [\u2212 homeostasis in gastric cancer cells, resulting in the enhancement of cancer cell growth. Thus, the plasma membrane expression of gastric CLIC3 may explain why CLIC3 function in gastric cancers is different from other cancers. Future study is necessary to clarify the mechanism of regulation of CLIC3 expression in gastric cancer cells.The activities of Cl1 arrest . Interes28 cells . A decreC3 cells . ChangesOur study clarifies that CLIC3 has a channel activity in the plasma membrane of gastric cancer cells, and that decreased expression of CLIC3 results in unfavorable prognosis of gastric cancer patients via stimulation of the cancer cell proliferation. These findings suggest that pathophysiological role of CLIC3 in plasma membrane is different from that in cytosol and organellar membrane of the cancer cells.Additional file 1: Table S1. Expression of CLIC3 and clinicopathological characteristics patients with gastric cancer.Additional file 2: Fig. S1. Enlarged image of tissue microarray analysis  from \u2212\u00a0100 mV to +\u00a0100 mV in the CLIC3-expressing HEK293T cells exposed to standard bathing solution (control: red) and the low Cl\u2212 bathing solution (blue)."}
+{"text": "Plasmodium falciparum parasite population diversity from two sites with low (pre-elimination) and high transmission in Senegal and Nigeria, respectively.Characterizing the genetic diversity of malaria parasite populations in different endemic settings (from low to high) could be helpful in determining the effectiveness of malaria interventions. This study compared P. falciparum malaria infected patients in Senegal (94) and Nigeria (93). Allelic polymorphism at merozoite surface protein 1 (msp1) and merozoite surface protein- 2 (msp2) genes were assessed by nested PCR.Parasite genomic DNA was extracted from 187 dried blood spot collected from confirmed uncomplicated msp1 and msp2 allelic families are the K1 and IC3D7 allelotypes in both Senegal and Nigeria. Multiplicity of infection (MOI) of greater that 1 and thus complex infections was common in both study sites in Senegal  than in Nigeria . The heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal  than isolates from Nigeria . In Senegal, K1 alleles was associated with heavy than with moderate parasite density. Meanwhile, equal proportions of K1 were observed in both heavy and moderate infection types in Nigeria. The IC3D7 subtype allele of the msp2 family was the most frequent in heavily parasitaemic individuals from both countries than in the moderately infected participants.The most frequent The unexpectedly low genetic diversity of infections high endemic Nigerian setting compared to the low endemic settings in Senegal is suggestive of possible epidemic outbreak in Nigeria. Plasmodium falciparum continues to be a significant public health havoc in many endemic countries in tropical and sub-tropical parts of the world  [(1\u2009\u2212\u2009\u03a3Pi2)], where n\u2009=\u2009sample size, Pi\u2009=\u2009allele frequency as described by . A p valA total of 187 malaria infected participants were recruited for the study with almost equal proportion from the two countries . The mean age was not different from each country and locality within the countries. Although, participants were randomly enrolled into the study, however, more males (108) partook in the study than females (79)  infections, K1/RO33 combination was the most frequent in both countries but higher in Senegal (17%), and Nigeria (7%). However, the K1/MAD20/RO33 trimorphic allelic infections were least frequent; Senegal (3%) and Nigeria (2%).For P. falciparum isolates from Nigeria (190-300\u00a0bp). Similar pattern was also observed with the MAD20 alleles, where sizes in Senegal ranged from 100-300\u00a0bp while those from Nigeria ranged from 200 to 300\u00a0bp. In addition, a different pattern was observed in RO33 where only one clone type was seen in Nigeria (160\u00a0bp) as against multiple clones in Senegal (160\u2013240\u00a0bp)  was different from that seen in msp2 gene, there was no difference in the frequency of isolates with IC3D7 allele in Senegal (57%) and Nigeria (58%), while the FC27 frequency was more prevalent in Senegal (26%) than in Nigeria (16%). Similar pattern was noticed with the IC3D7/FC27 dimorphic allelic family in both countries. Allele sizes of the IC3D7 variant of msp2 ranged from 300 to 700\u00a0bp in Senegal and 300\u2013800\u00a0bp in Nigerian P. falciparum isolates, while FC27 alleles varied in size from 200 to 600\u00a0bp in Senegal and 290\u2013550\u00a0bp in Nigeria  than the sites in Nigeria . Consequently, the heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal  than isolates from Nigeria . However, the heterozygosity of msp2 gene was not different for the two countries  from both countries. The K1 allele was more prevalent in individuals with higher parasitaemia infections (28) than those with moderate infections (18) from Senegal. However, in Nigeria, equal proportions of K1 were present in both moderate and high parasitaemic infections. Individuals with heavy infections from Senegal showed the highest proportion of K1/MAD20 (14) allele combination and same pattern was observed for the K1/RO33 mixture (13).The distribution of various allele types in both msp2 family showed high occurrence in high parasitaemic individuals from both countries  than in the moderately infected participants. Similar pattern was noticed with the FC27 allele  index which is related to the number of clones per infection and usually associated with the level of malaria transmission [msp1 and 2 genes were observed for both study locations in Senegal, but moderate MOI values were noted in Nigeria. Similar results for Senegal were reported by Niang et al. in 2017 [Control interventions targeting malaria parasite continues to face multiples hurdles from development of drug resistance in the parasite, insecticide resistance by the vectors, and unavailability of a reliable vaccine to confer the necessary protection against the parasite. Hence, there is need for continuous monitoring and evaluation of the effectiveness of control measures. Here, the diversity of smission \u201329 was amsp1 was more diverse in infections from Senegal than those from Nigeria. However, similar levels of diversity for msp2 was observed in both countries. This is in line with the differences in MOI probably due to differences in urbanization levels and recombination between genetically different clones. Indeed, there was a difference in the clonal structures of all allelic families  from both countries with Senegal showing the more sub-structured msp1 and msp2 populations. Nevertheless, similar trends has been observed in the Kingdom of Eswatini where a high genetic diversity was obtained in a low transmission area [Based on heterozygosity, which measures the level of genetic diversity at polymorphic loci, ion area . Circulamsp1 K1 allele in high and low parasite infections from both countries has been previously observed by various studies [The high occurrence of  studies , with th studies , 32, wes studies  and Iran studies .msp2 gene was also observed with both grades of parasite density, while no particular pattern was noticed with the FC27 allelotype. This finding is in agreement with the study of Hamid et al. in 2013 [Similarly, high frequency of IC3D7 allele type of  in 2013  where ICP. falciparum individuals should be evaluated in a bidirectional manner and a more holistic approach should be employed in determining the true epidemiological situation of any malaria endemic country as this will help in a more targeted control measure.Taken together, it can be concluded that evidence driving selection of these observed allelotypes in moderate and heavy Additional file 1: Table S1. Sequences of the primers used to amplify the msp 1 and msp 2 genes of P. falciparum isolates."}
+{"text": "Research found that post-selenization treatments are essential to produce stoichiometric thin films with desired crystallinity and orientation for the sputtered Sb2Se3. However, the influence of the sputtering process on Sb2Se3 device performance has rarely been explored. In this work, the working pressure effect was thoroughly studied for the sputtered Sb2Se3 thin film solar cells. High-quality Sb2Se3 thin film was obtained when a bilayer structure was applied by sputtering the film at a high (1.5 Pa) and a low working pressure (1.0 Pa) subsequently. Such bilayer structure was found to be beneficial for both crystallization and preferred orientation of the Sb2Se3 thin film. Lastly, an interesting power conversion efficiency (PCE) of 5.5% was obtained for the champion device.Magnetron sputtering has become an effective method in Sb It can be seen that although other devices presented decent OCV values, the final PCEs of these devices were restricted by the low FF and especially the poor SCJ, which were far inferior to that of the champion mixed-Sb2Se3 device. We attribute the unsatisfied SCJ of the devices sputtered at low working pressures (below 1 Pa) to the shunt paths caused by the pinholes within the films, whilst the low SCJ of the devices sputtered at elevated working pressures (above 1.5 Pa) could be attributed to the insufficiently thick absorber layer at such high working pressures, as shown in SEM images. In addition, the undesired grain orientations of other films  prepared under improper working pressures would increase the series resistance of the devices and thus lead to poor FF and SCJ [2Se3 device was much higher than that of the other samples in the wavelength region from 500 nm to 850 nm, suggesting less recombination losses of photogenerated carriers not only within the bulk film but also at the Sb2Se3/CdS heterojunction [The current density\u2013voltage  and a low working pressure (1.0 Pa) subsequently. Such bilayer structure was found to be beneficial for both crystallization and preferred orientation of the Sb2Se3 thin film. Further, shunt leakages induced by pinholes within the bulk film could be effectively hindered by using the bilayer structure. Finally, an interesting PCE of 5.5% was obtained for the champion device. In summary, the influence of working pressure on the device performance of sputtered Sb"}
+{"text": "After this article  was publSpecifically:\u25cb37 and 39 degree panels for HepG2\u25cb37 and 39 degree panels for Huh7The following panels in Fig 1A appear to report overlapping data:Similarities were noted between Tubulin data reported in Fig 1B  and Fig 6C (lanes 1\u20134). The similar lanes represent samples exposed to different experimental conditions.The corresponding authors commented that the invasion results for HepG2 and Huh7 cells were highly similar in the 37 and 39 degree groups. The original data underlying the results reported in Fig 1A are no longer available.The authors provided available image data to support the results reported in Figs 1B and 6C, but these data did not resolve the concerns. The MMP-2, MMP-9, and Tubulin data reported in Fig 6C appear to match image data provided in support of Fig 1B, albeit with different blot and lane labels, suggesting that all three panels of Fig 6 may have reported the wrong data. None of the image data provided appear to match the results reported in Fig 1B. The authors commented that no additional western blot data are available currently.PLOS ONE Editors retract this article.In light of the unresolved concerns, the LY, JZ and WL agreed with retraction and apologize for the issues with the published article. The other authors either could not be reached or did not respond directly."}
+{"text": "KCNQ1 gene. In rare cases, LOM of IC2 has been reported in families with KCNQ1 germline variants which additionally cause long-QT syndrome (LQTS). Thus, a functional link between disrupted KCNQ1 transcripts and altered IC2 methylation has been suggested, resulting in the co-occurrence of LQTS and BWS in case of maternal inheritance. Whereas these cases were identified by chance or in patients with abnormal electrocardiograms, a systematic screen for KCNQ1 variants in IC2 LOM carriers has not yet been performed.Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by disturbances of the chromosomal region 11p15.5. The most frequent molecular finding in BWS is loss of methylation (LOM) of the Imprinting Centre 2 (IC2) region on the maternal allele, which is localised in intron 10 of the KCNQ1 by MLPA and an amplicon-based next generation sequencing approach. We identified one patient with a splice site variant causing premature transcription termination of KCNQ1.We analysed 52 BWS patients with IC2 LOM to determine the frequency of germline variants in KCNQ1 transcription is required for the establishment of IC2 methylation, but that KCNQ1 variants cause IC2 LOM only in a small number of BWS patients.Our study strengthens the hypothesis that proper  Beckwith-Wiedemann syndrome  is a congenital imprinting disorder which was first described by Beckwith and Wiedemann in 1963 . Clinicainsulin-like growth factor 2 (IGF2) and H19 genes. The Imprinting Centre 2  controls the expression of the genes KCNQ1, KCNQ1OT1 and CDKN1C. In contrast to the IC1, the IC2 is maternally methylated leading to an expression of KCNQ1 and CDKN1C and a repression of KCNQ1OT1 on the maternal allele, while it is unmethylated on the paternal allele resulting in silencing of KCNQ1 and CDKN1C and expression of KCNQ1OT1  but more common in familial cases of BWS (20\u201340%) [The most frequent molecular cause of BWS is loss of methylation (LOM) of the IC2 (50\u201360% of patients) causing a biallelic expression of ed genes . Furthered genes ). Point (20\u201340%) . An epig(20\u201340%) .CDKN1C [KCNQ1 gene [KCNQ1, either by CNVs or single nucleotide variants (SNVs), causes a LOM of the IC2 [KCNQ1 and its regulatory elements, but not the IC2 itself. In mice, a maternally inherited poly(A) cassette, inserted into Kcnq1, terminates the transcription of Kcnq1 upstream of IC2 and results in LOM of the IC2, biallelic expression of Kcnq1ot1 and silencing of Cdkn1c and Kcnq1 [BWS mostly occurs sporadically, but in addition to inherited variants in CDKN1C , familiaCDKN1C , among tNQ1 gene \u201313. Some the IC2 \u201316. Remand Kcnq1 .KCNQ1 are the most frequent causes for congenital long-QT syndrome (LQTS) [KCNQ1 variant have a prolonged QT interval. Vice versa, 10% of the genetically affected patients who do not have a prolonged QT interval can be affected by a cardiac event until the age of 40\u2009years [KCNQ1 variants but remained undetected so far due to the reduced penetrance of LQTS variants. Up to now, six LQTS families have been described in which altered KCNQ1 transcripts lead to BWS in combination with LQTS when inherited from the mother or to isolated LQTS when transmitted from the father [KCNQ1 variant and can therefore be explained by the biallelic expression of KCNQ1 in the heart but monoallelic expression in other tissues [KCNQ1 variants in BWS patients associated with IC2 LOM is missing.Loss-of-function mutations in e (LQTS) , a herede (LQTS) ). The pe40\u2009years . It is ce father , 21\u201323. KCNQ1 among BWS patients with IC2 LOM.In our study, we therefore aimed to determine the frequency of pathogenic germline variants in KCNQ1 by multiplex ligation-dependent probe amplification (MLPA) did not reveal any deletion or duplication in the cohort of 52 BWS patients with IC2 LOM.Copy number analysis of the coding sequence of KCNQ1 was detected in one patient. The variant was located in the donor splice site of intron 1 of KCNQ1 . The change was also detected in the mother but not in the father. The variant affects the canonical donor splice site of intron 1   approach, heterozygosity for a splice variant in The boy was born at 26\u2009weeks of gestation by caesarean section due to premature placental separation and preeclampsia with a birthweight of 950\u2009g (50th percentile), a length of 33\u2009cm (30th percentile) and a head circumference of 23\u2009cm (15th percentile). He showed marked macroglossia, cleft palate, umbilical and inguinal hernias, hypospadias, ear lobe creases and a nevus flammeus in his face. He suffered from chronic respiratory insufficiency due to bronchopulmonary dysplasia as a consequence of his prematurity. He underwent multiple surgeries during the first months of life, including reduction of macroglossia. He showed a severe feeding disorder and required tube feeding for several years. His psychomotor development was significantly delayed putatively due to his premature birth. At the age of 3 years, he was able to walk without support, and he started speaking a few words at the age of 7 years. On the last examination at the age of eight and a half years, he was attending a school for special needs. His vision was restricted because of retinopathy of prematurity, and he needed regular controls for a nephrocalcinosis \u00b0I\u2013II.Clinical diagnosis of BWS was confirmed by molecular detection of IC2 LOM at the age of 2 months. Molecular karyotyping did not reveal any CNVs (resolution of 50\u2009kb). He had one healthy brother. His mother was healthy as well and did not show any BWS features. MLPA of the mother showed a normal methylation pattern.KCNQ1 and revealed a borderline prolonged QTc interval of 440\u2013450\u2009ms. The long-term ECG showed normal findings but sporadic extrasystoles. The mother\u2019s ECG revealed no pathologic findings.An electrocardiography (ECG) of the patient was performed after the results of the molecular analysis of KCNQ1 germline variants, we identified one case with a maternally inherited splice site variant (c.386+1G>T) affecting the splice donor site of intron 1 of KCNQ1. A variant at the same position with another base pair substitution (c.386 +1G>C) has been described by Valente et al. [KCNQ1 transcript. The resulting monoallelic expression of KCNQ1 could explain the LQTS phenotype as KCNQ1 is expressed biallelically in the heart [By analysing 52 BWS patients with IC2 LOM for e et al.  in a BWSe et al. . The authe heart . In tissKCNQ1 upstream of IC2 results in LOM. A deletion in KCNQ1 has been reported that does not include the IC2 but causes LOM, suggesting that transcription of the entire KCNQ1 gene is required for the establishment of the imprinting mark at the IC2 locus [KCNQ1 upstream of IC2 which leads to an IC2 LOM and BWS symptoms when inherited from the mother but to a normal phenotype in case of paternal inheritance [KCNQ1 results in a LOM of the IC2 and BWS when maternally inherited [KCNQ1 and one with a 160-kb KCNQ1 duplication. Both patients were affected by LQTS and BWS due to an IC2 LOM [Five further cases corroborate the hypothesis that interrupted transcription of C2 locus . Demars eritance . In a thnherited . Valente IC2 LOM . In our  IC2 LOM . The borIn fact, functional analyses have not been conducted but the effect of the splice site variant can be delineated from findings published recently by Valente et al. .KCNQ1 variants in BWS patients with IC2 LOM have not yet been performed. Demars et al. screened 78 BWS patients with IC2 LOM for CNVs and discovered one patient with a duplication including exon 2 and parts of intron 1 and 2 of KCNQ1. However, they did not perform ECG examinations [KCNQ1 mutation carriers without a QT prolongation [KCNQ1 germline variants and multiple miscarriages [KCNQ1 [KCNQ1 gene. However, our screening report has some technical limitations. The used MLPA kit does not cover exon 5 of KCNQ1 . In case of therapy refractoriness, left cardiac sympathetic denervation or an implantable cardioverter defibrillator (ICD) is necessary. Also, the patients should avoid competitive sports (e.g. swimming) and the intake of QT interval prolonging drugs (for review: ). The prKCNQ1 variants. The study was approved by the ethical committee of the Medical Faculty of the RWTH Aachen University (EK303-18).We investigated 52 BWS patients carrying an IC2 LOM. The molecular diagnosis had been confirmed by methylation-specific multiplex ligation-dependent probe amplification  according to the manufacturer\u2019s instructions. Multilocus imprinting disturbances (MLID) were excluded by screening with a MS-MLPA-based multilocus assay . Patients with MLID were excluded as they are probably caused by other (genomic) alterations than by KCNQ1 gene by MLPA  following the manufacturer\u2019s instructions.The patient\u2019s DNA samples were analysed for deletions or duplications of the KCNQ1 by an amplicon-based next generation sequencing approach. First, the exons of the whole coding region of KCNQ1 were amplified by polymerase chain reaction (PCR); primers and PCR conditions are listed in All patients were screened for SNVs in Additional file 1: Supplementary tables. Primer sequences and PCR conditions for the amplicon-based NGS analysis of KCNQ1."}
+{"text": "PSD95, DLGA, ZO-1) domains, to an extraordinarily large number of other proteins besides NUMB. Many of these interactions suggest additional roles for LNX1/2 proteins in the nervous system in areas such as synapse formation, neurotransmission and regulating neuroglial function. Twenty years on from their initial discovery, I discuss here the putative neuronal functions of LNX1/2 proteins in light of the anxiety-related phenotype of double knockout mice lacking LNX1 and LNX2 in the central nervous system (CNS). I also review what is known about non-neuronal roles of LNX1/2 proteins, including their roles in embryonic patterning and pancreas development in zebrafish and their possible involvement in colorectal cancer (CRC), osteoclast differentiation and immune function in mammals. The emerging picture places LNX1/2 proteins as potential regulators of multiple cellular signalling processes, but in many cases the physiological significance of such roles remains only partly validated and needs to be considered in the context of the tight control of LNX1/2 protein levels in vivo.Ligand of NUMB Protein X1 and X2 (LNX1 and LNX2) are E3 ubiquitin ligases, named for their ability to interact with and promote the degradation of the cell fate determinant protein NUMB. On this basis they are thought to play a role in modulating NUMB/NOTCH signalling during processes such as cortical neurogenesis. However, LNX1/2 proteins can bind, via their four PDZ ( Ligand of NUMB Protein-X) protein family evolved from a common ancestral protein that contained a RING  domain and four PDZ  domains [PDZ and RING). Mammalian LNX1 and LNX2 were named for their ability to interact with NUMB and have retained the ancestral RING plus four PDZ domains structure [Lnx1 gene by McGlade et al., this focus allows me to comprehensively summarize the published literature on these intriguing but incompletely understood proteins [The LNX , the NUMB-binding NPAY/F motif, four PDZ domains and a carboxyl terminal PDZ binding motif  Figure . A multiLNX1/2-like gene is present in some, but not all, invertebrate lineages [LNX1/2 orthologues, whereas those in Nematoda and Arthropoda do not. This indicates that LNX1/2 proteins are not essential in all invertebrate lineages. In the vertebrate lineage, duplication of an ancestral LNX1/2-like gene gave rise to LNX1, LNX2 and LNX2b. These three genes seem to be conserved in all vertebrate lineages with the exception of the loss of LNX2b in eutherian mammals. This loss of LNX2b is quite interesting in that the LNX2b pseudogene contributed several exons to the non-coding Xist RNA that control X-chromosome inactivation in eutherian mammals [A  mammals .LNX1/2-like genes in all vertebrate lineages suggests the acquisition of essential vertebrate-specific functions. Intriguingly, vertebrate LNX1/2 proteins have either an NPAY (LNX1) or NPAF  NUMB-binding motif, whereas the invertebrate proteins lack this motif [LNX1/2 orthologues in all vertebrates. Conversely however, this same observation tells us that the ancestral LNX1/2 protein probably evolved to perform cellular function(s) not related to an interaction with NUMB. It seems likely that vertebrate LNX1/2 proteins would also have retained some of these functions. Thus an important lesson from examining the evolutionary history of LNX1/2 proteins is that, despite their name, they should not be considered a \u2018one-trick pony\u2019 and are likely to have significant functions independent of their regulation of NOTCH signalling via NUMB.The maintenance of at least two The X-ray crystallographic structure of the LNX2 RING domain was determined by Nayak and Sivaraman in 2015  and provin vitro ubiquitination assays. They also showed for the first time that LNX2 could indeed ubiquitinate NUMB, something that had previously only been shown for LNX1. LNX2 autoubiquitination diminished but did not abolish this activity. Another finding from their study was that the ZnF-RING-ZnF LNX2 construct was present as a dimer in both the crystal structure and in solution. However, dimerization was not required for LNX2 autoubiquitination activity.Using LNX2 autoubiquitination as a readout of E3 ubiquitin ligase activity Nayak and Sivaraman  saw thatHaving determined the structure of the LNX2 RING domain with its flanking ZnF motifs, Nayak and Sivaraman  have rechttp://www.rcsb.org). Specifically, structures of mouse LNX1 PDZ2 , human LNX1 PDZ3 in complex with a peptide ligand from the coxsackievirus and adenovirus receptor (CAR) (PDB ID: 3B76) and human LNX2 PDZ2  have been submitted to the PDB. Most of these structures are not associated with any journal publication, though interestingly LNX2 PDZ2 was used in a recent, highly innovative study in which the application of strong electric field pulses to protein crystals was combined with time-resolved X-ray crystallography in order to probe the internal protein mechanics of a PDZ domain [Apart from the RING domain, structures of a number of individual PDZ domains are available in the Protein Data Bank  . This brThe fact that yeast two-hybrid screens failed to identify any peptides that bind to PDZ4 from either LNX1 or LNX2  is notewWhile we have learned a lot from the study of individual LNX1/2 domains, a structure of a full-length LNX1/2 protein would now be extremely informative. Such a structure would resolve the nature of PDZ1-mediated intra- and/or intermolecular interactions of LNX1/2 proteins. It would hopefully also reveal how the 3D arrangement of PDZ domains in a full-length LNX1/2 protein might orient ligands of specific PDZ domains as potentials substrates for ubiquitination by the ZnF-RING-ZnF domain.LNX1 and LNX2, the expression of lnx2b in zebrafish is discussed separately below. Northern blot analysis revealed expression of murine Lnx1 mRNA in adult heart, brain, lung, skeletal muscle and kidney with little or no message detected in spleen, liver and testis [LNX1 mRNA was likewise detected in adult heart, brain and kidney as well as the placenta and pancreas, whereas much lower levels were detected in lung, liver and skeletal muscle [Lnx1 mRNA seems to be expressed in many adult tissues. Notably, both these and subsequent studies [Lnx1 transcripts are expressed in the brain and spinal cord compared with other tissues. These transcripts are predicted to give rise to 70 and 62 kDa proteins that lack the ZnF-RING-ZnF domain present in the 80 kDa protein that is expressed in other tissues  that lack the catalytic ZnF-RING-ZnF region is intriguing. The p70 isoform is found across diverse vertebrate species and the short amino terminal sequence unique to this isoform is highly conserved . In addiLNX1/2 mRNA and protein levels in vivo have significant implications for interpreting reports on the role of LNX1/2 proteins in disease, and the physiological relevance of the many interactions of LNX1/2 proteins that have been identified to date. The seemingly tight control of LNX1/2 protein levels suggest that LNX1/2 proteins (i) may have general functions in the many cell types, but that they are only required at extremely low levels or (ii) have very specific roles in certain cell types in which they are expressed at higher levels but in a temporally or spatially restricted manner. The fact that LNX1/2 proteins are normally present in vivo at relatively low levels must be kept in mind when interpreting experiments that rely heavily on LNX1/2 overexpression.Overall the low expression levels of LNX1 and LNX2 proteins, and the possible lack of correlation between in vivo is not known, so the physiological relevance of a LNX1\u2013SHC interaction is unclear.LNX1 was first identified in a yeast two-hybrid screen to identify proteins that bind to the PTB domain of murine NUMB . The intThe ubiquitin ligase activity of LNX1 was confirmed and NUMB was shown to be a substrate for LNX1-mediated ubiquitination . UbiquitNUMB interacts with the NOTCH receptor and can negatively regulate NOTCH signalling in several contexts. NUMB is thought to achieve this inhibition of NOTCH signalling by modulating either the endocytosis of NOTCH or the trafficking of NOTCH post-endocytosis, however the exact mechanism is a matter of debate . Given NHes1 [Hes5 is unaffected and rather point to other functions of NUMB to explain the phenotypes observed in these mice [Importantly, some evidence that a loss of LNX1/2 function can lead to altered NOTCH signalling has emerged more recently. In zebrafish embryos expressing a dominant negative form of LNX2 it was found that NUMB levels were increased and the number of cells exhibiting NOTCH signalling was decreased, as assessed using a transgenic fluorescent protein-based reporter . In a diHes1 . Howeverese mice . In anotese mice .These observations make it clear that relating increased or decreased LNX1/2 protein expression to alterations in NOTCH signalling in a given context is not straightforward. Firstly, LNX1/2 may or may not affect NUMB levels depending on the particular isoform(s) of NUMB present; secondly, redundancy of function between LNX1/2 isoforms and between NUMB and NUMB-like need to be considered and thirdly, even if NUMB levels are altered this could affect pathways other than NOTCH signalling. Finally, it is possible that LNX1/2 may modulate NUMB and/or NUMB-like function independently of triggering their degradation, for example by altering NUMB or NUMB-like subcellular localization. Nevertheless, LNX1/2 proteins certainly can act as activators of NOTCH signalling through the ubiquitination of NUMB, but this mechanism must be carefully examined in each cellular context for the reasons outlined above.NUMB functions as a cell fate determinant in many types of neural progenitor cells, as well as in non-neural cells. During neurogenesis asymmetric localization of NUMB in dividing neural progenitors causes it to partition predominantly into one of the daughter cells\u2014specifying a distinct fate for this cell by antagonizing NOTCH signalling . For exa\u2212/\u2212 mice defects in the specification of ependymal cells were observed. These mice exhibit dramatically decreased levels of NUMB protein, which may at least in part explain the phenotype observed. Intriguingly, Gli3\u2212/\u2212 mice have increased LNX2 levels, particularly in the SVZ, leading the authors to speculate that LNX2 overexpression in Gli3\u2212/\u2212 mice may target NUMB for proteolytic degradation. While this mechanism seems plausible, a causal relationship between LNX2 and NUMB levels was not definitively demonstrated. Nevertheless, this is an interesting finding that begs the question of whether LNX2 plays a role in normal SVZ development beyond this proposed role in Gli3\u2212/\u2212 animals.While examining knockout mice lacking the Sonic hedgehog signalling component GLI3, Wang et al.  uncovereThe interaction of contactin-associated protein-4 (CASPR4) with LNX2 has also been proposed to play a role in neuronal differentiation . CASPR4 A distinct role for LNX1/2 proteins in cortical neurogenesis has been proposed based on their interaction with SHOOTIN1 . SHOOTINin vivo during normal brain development. Notably, gross brain morphology was found to be normal and no changes in NUMB levels were observed in mice lacking both LNX1 and LNX2 in the nervous system [The examples above indicate several potential roles for LNX1/2 proteins in regulating neurogenesis and neuronal differentiation and fit with the well-established role of NUMB in such processes. However, none could be considered definitive proof of such a role s system . FurtherLnx1 and Lnx2 mRNAs are significantly up-regulated during osteoclast differentiation in vitro. To probe the requirement for LNX2 the authors employed lentiviral transduction of shRNAs to knockdown LNX2 expression in bone marrow derived macrophages. This drastically decreased osteoclast formation in vitro and increased apoptosis of pre-osteoclasts. LNX2 knockdown also dampened the activation of the signalling pathways that promote osteoclast formation, namely M-CSF induced ARK and AKT activation, as well as RANKL induced NF-\u03baB and JNK activity. Notably LNX2 knockdown caused an increase in NUMB levels and decreased levels of the NOTCH2 intracellular domain, as well as decreased expression of the NOTCH target gene Hes1. NOTCH signalling is known to play multiple roles in maintaining bone homoeostasis and controlling osteoclast differentiation [in vitro differentiation system can be confirmed in vivo, perhaps by examining osteoclast differentiation and homoeostasis in detail in Lnx2 knockout mice.A possible role for mammalian LNX1/2 proteins in the differentiation of osteoclasts that seems to involve the modulation of NOTCH signalling via NUMB has recently been described . This inntiation ,48. ThesSeveral hundred proteins have been described as potentially interacting with LNX1/2 proteins and the overwhelming majority of these interactions are mediated by the LNX1/2 PDZ domains ,13,18,20The cytoplasmic domains of several components of epithelial tight junctions including CLAUDINS ,51, juncCLAUDINs comprise a large family of transmembrane proteins involved in the formation of tight junctions that maintain epithelial and endothelial barrier functions. CLAUDIN-1 cytoplasmic domain was found to interact with LNX1 via its carboxyl terminal PDZ binding motif . LNX1 ovJAM4 is a member of the immunoglobulin superfamily of adhesion molecules that localizes to tight junctions. JAM4 was shown to interact via its carboxyl terminal PDZ binding motif with PDZ2 of LNX1 and LNX1 was seen to partly co-localize with JAM4 and other tight junction components in epithelial cells . LNX1p70In vivo both CAR and LNX2 proteins are present in several tissues in embryonic day 16.5 mice and in particular co-expression of both protein was noted in a subset of blood vessels [in vivo is a major strength of these studies but the functional consequence of the interaction of LNX1/2 proteins with CAR either in epithelial or sperm cells remains to be established. Notably mice lacking LNX2 appear to have normal fertility, suggesting that LNX2 is not absolutely required for sperm development and fertilization in any case [CAR like JAM4 is a member of the immunoglobulin superfamily that can interact with LNX1 via its cytoplasmic tail . CAR is  vessels . CAR and vessels . The demany case . Similarany case .These studies suggest multiple ways in which LNX1/2 proteins might be involved in the formation or remodelling of tight junctions in epithelial cells. This may be relevant in the context of EMTs that occur developmentally and that also contribute to tumour development and metastasis . InteresPotential functions of LNX1/2 proteins in presynaptic nerve terminals are suggested by the characterization of interactions of LNX1/2 proteins with several presynaptic proteins. The finding that both LNX1 and LNX2 are predominantly expressed in neurons  suggestsProteomic analyses of the LNX1 interactome revealed an interaction of LNX1 with LIPRIN-\u03b1 proteins ,14. ThisA number of other proteins that play important roles in synapse formation and function have also been reported to interact with LNX1/2 proteins. These include MYCBP2 (PAM/HIGHWIRE/RPM-1), a well-known regulator of synapse formation  and CASPERBB2 is a member of the epidermal growth factor receptor family of receptor tyrosine kinases with important signalling functions in many contexts during embryonic development, including the differentiation of myelinating Schwann cells in the peripheral nervous system. ERBB2 was found to interact with the PDZ domain region of LNX1 and endogenous ERBB2 could be co-immunoprecipitated with LNX1 from mouse brain lysate . ImmunosLnx1 and Lnx2 mRNA was detected in purified T cells, the levels of LNX1 or LNX2 proteins were not assessed and are likely to be significantly lower than are achieved by heterologous expression. It will be important therefore to show that the endogenous levels of LNX1/2 proteins in T cells can affect CD8-\u03b1 levels and /or subcellular localization, perhaps by examining T cells under conditions of LNX1/2 knockdown or loss of function.CD8 is a T-cell co-receptor that can stabilize the interaction of the T-cell receptor with the class I MHC on antigen presenting cells and recruit effector proteins to the T-cell receptor. The cytoplasmic tail of CD8-\u03b1 was found to bind to both LNX1 and LNX2 . The intThe best characterized interactions of LNX1/2 proteins with transcriptional regulators are those of zebrafish LNX2b (described separately). However, a few interactions of mammalian LNX1 and LNX2 with nuclear proteins have been described. Several reports describe LNX1 as being partly localized in the nucleus ,58,59, aSki interacting protein (SKIP/SNW1) was identified in a yeast two-hybrid screen using PDZ1 of LNX1 as a bait . EmployiArmbruester et al.  describePDZ-binding kinase (PBK/TOPK) is another nuclear protein that has been shown to interact with LNX1 ,15,18. PThe non-receptor tyrosine kinase c-SRC can interact via its carboxyl terminus with both PDZ1 and PDZ3 of LNX1 . The twoZheng et al. . found ain vivo cellular contexts these interactions and potential modulatory influences of LNX1/2 proteins on cell signalling are relevant.It clear from the examples above that LNX1 has the ability to interact with, and in some cases ubiquitinate, key components of intracellular signalling pathways. Going forward, it will be interesting to determine in which LNX1 exon3 eliminates expression of the neuronally expressed LNX1p70 and p62 proteins, but is not expected to affect LNX1p80 expression in other tissues [https://www.mmrrc.org/catalog/sds.php?mmrrc_id=32436). However, only basic neurological and behavioural tests were performed, so subtle neural defects cannot be ruled out. The sole immunological abnormality reported is difficult to reconcile with the fact that only the neuronal LNX1 isoforms are eliminated in these mice.The first LNX1/2 loss-of-function mouse model was an isoform-specific LNX1 knockout generated by Lexicon Pharmaceuticals as part of a large-scale gene knockout programme. In this mouse deletion of  tissues . This moLNX2 knockout mouse was subsequently generated in my laboratory [LNX1exon3\u2212/\u2212 line mentioned above to generate LNX1/LNX2 double knockout (DKO) mice in which LNX2 is eliminated globally in all tissues, while the loss of LNX1 is expected to be restricted to the exon 3-containing LNX1 p70 and p62 isoforms in the CNS. These mice thus represent a loss-of-function model to study neuronal functions of mammalian LNX1 and LNX2. LNX1/LNX2 DKO mice are viable, healthy and fertile and do not exhibit gross neuroanatomical defects [\u2212/\u2212 mice was associated with decreased NUMB levels and abnormalities in SVZ formation [Lnx1 and Lnx2 mRNA expression.A boratory ,20. Sinc defects . Corticaormation   in zebrafish development have been extensively examined by Ro and Dawid  , making  markers . Lnx2b wectoderm . Taken tcdx4 during zebrafish embryogenesis has also been shown [Cdx4 expression is repressed by Tcf3 in the absence of Wnt signalling, whereas the transcriptional regulator E4f1 was shown to de-repress cdx4. Lnx2b can interact with E4f1, but promotes repression of cdx4 \u2014counteracting the action of E4f1. While Lnx2b can ubiquitinate E4f1 in cell-based ubiquitination assays, this did not lead to E4f1 degradation and its E3 ubiquitin ligase activity was not required to antagonize the de-repression of cdx4 by E4f1. Thus the interaction of Lnx2b with E4f1 appears to be sufficient to modulate the Tcf3 repression complex on the cdx4 promoter, although interactions of Lnx2b with other components of the complex could play a role.A distinct role for Lnx2b in modulating the expression of the caudal homeobox gene en shown . Cdx/cauPtf1a transcription factor that is required for differentiation of these cells. Other exocrine markers such as trypsin were also drastically diminished, whereas endocrine cell differentiation seemed unaffected. The specificity of this phenotype tallied with the expression of lnx2 mRNA in the ventral pancreatic bud from which exocrine cells are derived, but not in the dorsal bud from which endocrine cells arise. The mechanism underlying this phenotype appears to involve LNX2/2b-mediated regulation of Notch signalling via Numb, based on the observations that Numb knockdown substantially rescued the phenotype and that increased levels of Numb and decreased Notch signalling were seen in pancreatic cells of embryos expressing dominant negative Lnx2. Destabilization of Numb by Lnx2 and Lnx2b is proposed to promote Notch signalling in ventral bud-derived cells which appears to be important in the specification and/or proliferation of the exocrine precursor cell population [A third function for Lnx2 and -2b proteins during zebrafish development has also been revealed by Dawid et al. . In thispulation .in vivo. A key question is whether these functions are shared in other lineages, particularly in eutherian mammals that lack LNX2b. The absence of a clear mammalian Bozozok orthologue suggests that this function of Lnx proteins in very early embryonic patterning may be specific to fish. By contrast, Ro and Dawid [Together these studies in the zebrafish model represent the best mechanistic descriptions of LNX1/2 protein function nd Dawid  demonstrLNX1 mRNA was noted in gliomas compared with healthy brain tissue [LNX1 and LNX2 mRNA are expressed predominantly in neurons [LNX1 gene in brain tumours and osteosarcoma xenografts [KIT, PDGFR-\u03b1 and VEGFR2) and so amplification of LNX1 may be incidental. A few cases with specific amplification of LNX1 were reported [LNX1 missense mutations were found [LNX1 mRNA expression in HEK cells using siRNA led to cell cycle arrest and caused alterations in the expression levels of several cancer associated genes. Nie et al. [LNX1 mRNA and particularly LNX1 protein in tumours, the question of whether LNX1 actually promotes a cancer phenotype in vivo remains unclear and further evidence is required to demonstrate a compelling causative role for LNX1 in brain tumours.A number of reports have highlighted altered LNX1 expression in the context of brain tumours, although in most cases a causative role for LNX1 in driving tumorigenesis has not been demonstrated. Down-regulation of n tissue , though  neurons  and as snografts . It is nreported  and somere found , howeverre found  highlighe et al.  reportedLNX2 gene maps to chromosome 13q12.2\u2014a region that is frequently amplified in CRC. Examining genes that are amplified in this chromosomal region, Camps et al. [LNX2 mRNA to be overexpressed in CRC compared with normal colon mucosa as well as in CRC cell lines. Furthermore, it was shown that LNX2 knockdown using siRNA reduced the viability of CRC cell lines and induced apoptosis. Using genome-wide expression profiling they observed dysregulation of 680 genes upon silencing of LNX2 in a CRC cell line (SW480) that exhibits chromosome 13 amplification. The expression of multiple target genes of both the NOTCH and WNT/\u03b2-catenin signalling pathways were prominently decreased and alterations of upstream components of these pathways were noted at both the mRNA and protein levels. In particular, TCF7L2, a transcription factor that is a major effector of WNT signalling, and many of its target genes were down-regulated in LNX2 knockdown cells. Similarly, levels of NOTCH and the NOTCH ligand JAG1 as well as several NOTCH target genes were significantly decreased. Thus, LNX2 has the ability to simultaneously regulate two signalling pathways known to play critical roles in CRC tumorigenesis. Surprisingly, decreased NUMB protein levels were observed upon LNX2 knockdown\u2014the opposite of what one might expect for NUMB as a substrate for LNX2-mediated ubiquitination and an inhibitor of NOTCH signalling. Thus, the observed changes in NOTCH signalling cannot be explained simply by LNX2 modulating NUMB levels. Nevertheless, the findings suggest a credible model whereby overexpression of LNX2 as a consequence of LNX2 gene amplification promotes NOTCH and WNT/\u03b2-catenin signalling, which drives cell proliferation and prevents apoptosis in CRC cells [The most comprehensive studies linking LNX1/2 proteins to tumorigenesis are in relation to CRC ,35. The s et al.  found LNRC cells .LNX1 mRNA and protein were down-regulated in a population of putative cancer stem cells (side population cells) isolated from the CRC cell line HT29. Cancer stem cells in tumours are proposed to give rise to cells with highly proliferative and invasive phenotypes. LNX1 knockdown increased the number of cancer stem cells and led to the formation of more colonospheres in extreme limited dilution assays, and more tumours in mice injected with LNX1 knockdown HT29 cells. This suggested that LNX1 is a negative regulator of cancer stemness in CRC. Seeking a mechanism for this effect the authors examined LNX1 interacting proteins and found that knockdown of CAR, a known LNX1 ligand [LNX1 knockdown leads to increased CAR protein levels, suggesting that CAR may be involved in mediating the effect of LNX1 on cancer stemness, although the mechanism by which LNX1 regulated CAR levels and whether it involved LNX1-mediated ubiquitination of CAR was not examined. Finally, this study showed that tamoxifen increased LNX1 gene expression and could inhibit colonosphere formation in HT29 cells. Overall these findings suggest that LNX1 may act as a tumour suppressor and that up-regulating LNX1 expression may represent a novel therapeutic strategy to negatively regulate cancer stemness in CRC.A more recent study has now also implicated LNX1 in CRC . Here th1 ligand , caused LNX1 mRNA to be up-regulated in CD133\u2212 compared with CD133+ stem cells, suggesting that an involvement of LNX1 in cancer stemness may not be specific to CRC [LNX2 promoter region that was linked to increased susceptibility to diffuse large B-cell lymphoma [LNX2 gene to differential responses to the chemotherapeutic drug cytosine arabinoside in lymphoblastoid cells [Interestingly, a previous study of cancer stem cells in glioblastoma reported c to CRC . Furtherlymphoma  and anotid cells . These sLNX1 that are associated with susceptibility to Kawasaki disease\u2014an inflammatory paediatric condition that causes damage to the corony arteries and is thought to be triggered by an unidentified infection [LNX1 transcript levels in acute compared with convalescent Kawasaki disease. A different report found that blood levels of both LNX1 and LNX2 mRNAs were significantly elevated in chronic compared with acute Q fever [Coxiella burnetti infection and the acute form usually resolves by itself, whereas chronic Q fever develops as an endocarditis and is associated with an impaired immune response. The authors suggest that LNX1/2 expression could be used as a prognostic biomarker in Q fever patients. These reports, along with the previously discussed interaction of LNX1/2 proteins with CD8 [A couple of studies have found associations of LNX1 and LNX2 with infectious diseases. A GWAS found intronic SNPs in nfection . This st Q fever . Q feverwith CD8  suggest LNX2 mRNA expression in placental tissue was found to be associated with preeclampsia in pregnancy [LNX2 gene to be hypomethylated in smokers [Differential expression of regnancy , while a smokers . The funin vivo (with the notable exception of studies of zebrafish Lnx1/2 proteins). While our understanding of some basic aspects of LNX1/2 function is still incomplete, it is improving. It is now appreciated that LNX1/2 protein levels are tightly regulated. We are beginning to understand the differential functions of LNX1, LNX2 and LNX2b, as well as the neuronal and non-neuronal LNX1 isoforms. Fantastic structural insights into the mechanisms of LNX1/2 ubiquitin ligase activity have emerged in the past few years. The LNX1/2 interactome has been thoroughly explored using a variety of complementary approaches. Loss-of-function analyses of mammalian LNX1/2 proteins in both cellular and mouse models have been published. Many pieces of the puzzle are falling into place.Two decades of research have highlighted a multitude of potential functions for LNX1/2 proteins, based largely on their ability to interact with many binding partners . Howeverin vivo. The molecular basis for the subtle phenotypes already observed in mice lacking LNX1 and LNX2 proteins in the CNS can be dissected more precisely. The generation of mice lacking both LNX1 and LNX2 in all tissues will clarify proposed roles for LNX1/2 proteins in immune function and bone homeostasis and allow the role for LNX1/2 proteins in pancreas differentiation to be explored in mammals. Further structural studies will hopefully reveal structures of full-length LNX1/2 proteins and/or structures of LNX: substrate complexes. Translational studies will be also be needed to tease out the disease associations of LNX1/2 proteins, particularly in CRC, where both LNX1 and LNX2 have been implicated. These studies will determine whether LNX1/2 proteins are worth considering as potential drug targets, particularly as methods to inhibit both E3 ubiquitin ligases and PDZ domain interactions with small molecules develop [Going forward armed with this knowledge and the new research tools that are available, researchers in this field can be optimistic about putting more pieces of this puzzle together. The currently available and newly generated LNX1 and LNX2 knockout mice can be used to explore the significance of putative functions of LNX1/2 proteins  develop .Click here for additional data file."}
+{"text": "Identifying immunogens that induce HIV-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict T-cell epitopes for various HLA types.We tested the performance of the NetMHCpan4.0 computational neural network in re-identifying 93\u2009T-cell epitopes that had been previously independently mapped using the whole proteome IFN-\u03b3 ELISPOT assays in 6 HLA class I typed Ugandan individuals infected with HIV-1 subtypes A1 and D. To provide a benchmark we compared the predictions for NetMHCpan4.0 to MHCflurry1.2.0 and NetCTL1.2.p\u2009=\u20090.0000005). MHCflurry1.2.0 similarly predicted all but 2 of the peptides that NetMHCpan4.0 predicted and NetCTL1.2 predicted only 14 of the 93 experimental peptides.NetMHCpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched HLA class I alleles. Receiver Operator Characteristic (ROC) analysis gave an area under the curve (AUC) of 0.928. Setting NetMHCpan4.0 to predict 11-14mer length did not improve the prediction (37\u201379 of 93 peptides) with an inverse correlation between the number of predictions and length set. Late time point peptides were significantly stronger binders than early peptides (Wilcoxon signed rank test: NetMHCpan4.0 class I epitope predictions covered 95% of the epitope responses identified in six HIV-1 infected individuals, and would have reduced the number of experimental confirmatory tests by >\u200980%. Algorithmic epitope prediction in conjunction with HLA allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort. Computational algorithms were initially demonstrated as useful tools for predicting potential epitopes that might elicit quality T-cell responses , 2. Compag genes  in cynomag genes  necessarag genes . Computaag genes , therefoag genes \u201310.For HIV-1 vaccine design purposes an important consideration for the suitability of a computational algorithm is the breadth of discrete number of T-cell epitopes it generates that could reach particular levels of coverage  of circuin-silico approaches in epitope prediction and its application for vaccine design reported a meagre 22, 44%, and relatively higher 78% match for three computational tools namely YFPEITHI, CTLPRED and IEDB respectively [A reliable pan-HLA-specific algorithm NetMHCpan4.0 \u201320 that ectively . Using eet.al [The data used was from an independent study that did not include this analysis in its objectives. Experimental data of peptides previously mapped for HIV-1 epitope recognition of 6 individuals for a separate study . The web version of NetMHCpan4.0 [http://www.cbs.dtu.dk/services/NetMHCpan/) was configured to predict 9mer through 14mer epitopes for 22 HLA class I alleles  plot. The hit rate (sensitivity) and false hit rate (specificity) of binder predictions as determined by the NetMHCpan4.0 threshold of peptides within the top 2% (with a score of 2 or less) were calculated and the strength of the model was determined by calculating the area under the curve, AUC of the ROC plot \u201343. PearTo experimentally determine epitopes for 757 peptides spanning the whole HIV-1 proteome for clades A and D as well as both time points of the 6 individuals required a total of 4230 test assay wells. For each test subject these included 9 antigen proliferation wells, 384 culture ELISPOT wells and an average of 164 epitope mapping ELISPOT wells . Using the 22 HLA alleles represented in the study subjects we were able to computationally predict 95% of the experimentally mapped epitopes. This approach could have reduced the test assays by eliminating all the T-cell antigen proliferation and culture ELISPOT steps totalling to 3258 assay wells (77%) and leaving only 972 (23%) epitope mapping assays required. Applying a pooling strategy to the computational predictions similar to that used in the experimental pooling where each pool contained approximately 20 peptides with a coverage of 3 per peptide pool, the 923 potential peptides  eligible epitope mapping peptides) would make at most 46 pools. Consequently the computational prediction approach could have reduced the experimental assays by at least 80%.rs\u2009=\u20090.88  epitopes were experimentally mapped of which 12 were recognized at both baseline and later time points, 34 only at baseline and 54 only at the later time point. Comparison of the ranked computational score for Netmhcpan4.0 binders of early (n\u2009=\u200934) versus later peptides showed that the later time point predictions were stronger binders reaching statistical significance  (Fig.\u00a0The experimental peptide mapping data was derived from a baseline time point corresponding to HIV-1 Fiebig stages IV, V and VI (Table\u00a0In this analysis we showed that the computational method NetMHCpan4.0 predicted 95% of previously experimentally mapped HIV-1 epitopes in 6 HIV-1 infected individuals expressing a total of 22 different HLA class I alleles. In our IFN-\u03b3 ELISPOT assays we evaluated 757 17mer peptides overlapping by 11 amino acids and covering the whole HIV-1 subtype A1 and D consensus proteomes. Out of the 5 experimentally determined epitopes missed by the algorithm Table\u00a0, 4 were As the participants had been enrolled in the acute/early phase of HIV-1 infection and we had observed intra-participant changes in epitope recognition between early and late time points after infection, we compared the binding scores of confirmed epitopes at these time points and found a statistically significant change towards recognition of higher binding peptides as the infection entered the chronic phase. This might represent better support of the T-cell response directed at more stable HLA/peptide complexes as the infection progresses into chronicity.The NetMHCpan4.0 algorithm, which is based on binding affinity and integrates data on eluted naturally processed ligands, reflected optimal HLA class I binding for 9-mers, producing a decreasing number of predictions when the peptide size was increased from 9 to 11 amino acids. With a single exception, predicted binders between 11 and 14 amino acids included at least one 9mer predicted to bind on its own, suggesting a destabilizing effect of the extra amino acids beyond the canonical HLA class I binding pockets at positions 2 and 9 could account for fewer predictions.Important limitations are the lack of predictions of HLA class II restricted epitopes, which might have contributed to a fraction of IFN-\u03b3 ELISPOT responses. Approximately 5% of the computational predictions may be false positives that only increase the size of planned wet experiments and approximately 1% of true positives may also be missed.In this analysis, using NetMHCpan4.0, MHCflurry and NetCTL to predict previously experimentally mapped epitopes, we demonstrate that the computational methods reliably predict an acceptable portion of binder epitopes. We recommend the use of such computational methods to reduce the size of experiments required cost associated."}
+{"text": "Scientific Reports 10.1038/srep34626, published online 11 October 2016Correction to: This Article contains errors in the Supplementary Information file, where the size distributions of the nanoparticles given in Figure S12b and Figure S13c are incorrect. The correct Figures S12 and S13 are given below as Figs."}
+{"text": "ObjectiveTo compare 5 French (Fr) and 6 Fr guiding catheters regarding the volume of contrast administered, fluoroscopy time, and total procedure time during transradial percutaneous coronary intervention (PCI).BackgroundPrevious studies had compared 5 Fr and 6 Fr catheters and deemed 5 Fr catheters safe and effective.\u00a0In this study, we retrospectively compared the 5 Fr catheter to 6 Fr catheter with an attempt to eliminate the effect of inter-operator skill level variability.MethodsIn a single-center, retrospective cohort study, we randomly selected patients who had received PCI through transradial access using 5 Fr or 6 Fr catheters. The study involved two groups of 100 patients each. These groups were comprised of an equal number of cases from each operator. The primary endpoint was contrast medium volume. Secondary endpoints were fluoroscopy time and procedure time.ResultsLess contrast was used in the 5 Fr group vs. 6 Fr catheter group . PCI using 5 Fr catheters was associated with shorter fluoroscopy time  and shorter procedure time , but this was not statistically significant.ConclusionTransradial PCI using 5 Fr guiding catheters was associated with less contrast medium usage, but there was no advantage regarding procedure time or fluoroscopy time when compared to 6 Fr catheters. Similar to 6 Fr catheters, 5 Fr catheters achieved high PCI success rates through radial access when compared in the treatment of coronary lesions with the same level of complexity. Since its inception in 1978, the field of percutaneous coronary intervention (PCI) has been evolving to constantly better itself [Among the numerous medical devices manufactured,\u00a0only a few make the cut and are deemed to be the standard of care after being studied and tested in the field. Before the 6 Fr catheter was determined to be the standard of care, it had also been compared and tested against 7 Fr and 8 Fr catheters; and\u00a0it was crowned as the standard of care only after it was deemed to be effective -4. With Study design and patient populationOur study was a single-center, retrospective\u00a0cohort study designed to compare procedural variables of 5 Fr versus 6 Fr catheters in patients who underwent PCI using radial access. A total of 200 patients were involved in the retrospective analysis in this study. The study population was randomly selected from a pool of 463 patients that included all the consecutive PCI cases performed at St. Mary\u2019s Medical Center - Marshall University by four different interventional cardiologists with expertise in transradial procedures during May 2016-May 2017. All of the cases used radial access as initial access, and at least one stent was deployed using 5 Fr or 6 Fr as an initial catheter. Indications for the procedure were either elective for stable angina or emergent for non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI). The total study population was then divided into two groups: 5 Fr group and 6 Fr group. Of the total 463 cases that we considered, 265\u00a0were performed using 6 Fr catheter and 198 using 5 Fr catheter. Each of these two groups was then divided into four subgroups based on the operator  grade 3 anterograde flow without the occurrence of complications such as death from any cause, urgent coronary bypass surgery, or acute myocardial infarction.Data analysisBaseline clinical characteristics were analyzed for all patients included in the study. Results were expressed as proportions or mean with standard deviation (SD). The primary endpoint was the amount of contrast used in the procedure. Secondary endpoints included procedure time and fluoroscopy time.\u00a0All were continuous variables and they were assessed by t-test using SPSS Statistics version 24.0 . A p-value of <0.05 was considered statistically significant.A total of 200 patients were included in the analysis, with 100 each from the two study groups. Both groups consisted of unselected randomized patients in routine clinical practice with a diverse clinical profile. The two groups were well balanced regarding baseline characteristics  in the 5 Fr group, while crossover from 6 Fr to 5 Fr occurred in two cases (2%) in the 6 Fr group. As defined in the methods section, a 99% success rate was achieved in the 5 Fr group while 98% success rate was achieved in the 6 Fr group.\u00a0Compared with transradial PCI using 6 Fr catheters, the 5 Fr group had lower contrast use . PCI using 5 Fr catheters was associated with shorter fluoroscopy time\u00a0. Again,\u00a0procedure time was less with the 5 Fr catheters . The latter two findings were statistically insignificant.Initial prototypes of the 5 Fr and\u00a06 Fr catheters did not have a lumen large enough for an adequate stent insertion as the lumen was less than 0.058 inches; hence, these catheters could only be employed for diagnostic purposes . It was The appeal of 5 Fr catheter came to light when several studies reported favorable prospects of utilization.\u00a0In 2001, Sch\u00f6bel et al. reported a success rate of up to 95% in 5 Fr catheters used in transfemoral access site depending on the type of the lesion. However, it was also reported that 5 Fr catheters had limitations in the application\u00a0such as poor back-up support in long lesions as well as severe stenosis . Gobeil The 5 French catheter use has strongly been associated with decreased usage of contrast volume. Schussler et al. studied 5 Fr catheters with transradial PCI in preselected cases where they were shown to be effective. They concluded that the usage of 5 Fr catheters reduces\u00a0morbidity and the cost of the procedure since it required less amount of contrast medium and resulted in minimal complications . Rakhit Gobeil et al. compared the 5 Fr and 6 Fr in transradial PCIs . Their sThe success of the procedure was defined as\u00a0less than 50% residual stenosis with TIMI grade 3 anterograde flow without the occurrence of complications such as death from any cause, urgent coronary bypass surgery, or acute myocardial infarction. 5 Fr catheters were associated with a 99% success rate, which is higher than the 6 Fr group (98%). Previous studies have shown the superiority of the use of 5 Fr catheters in terms of success and complications rates; however, the success rate of 99% is significantly higher than the rate reported in older studies like Gobeil et al., which showed a success rate of 90% . In receDahm et al. conducted a randomized study of 5 Fr vs. 6 Fr transradial PCI aiming to ascertain whether the 5 Fr guiding catheter has equivalent procedural success and complication rates as the 6 Fr. Their study showed that transradial PCI of non-complex lesions can be successfully performed with either 5 Fr or 6 Fr guiding catheters, with 5 Fr showing higher procedural success rates and lower vascular access complications in comparison to 6 Fr guiding catheter . In thisOur findings revealed that transradial PCI using 5 Fr guiding catheters was associated with less contrast medium usage when compared to 6 Fr catheters. 5 Fr catheters would thus be the preferred choice for patients with reduced renal function or when minimizing the use of contrast medium is of concern. 5 Fr catheters did not have an advantage over 6 Fr catheters in terms of procedure time or fluoroscopy time. Similar to 6 Fr catheters, 5 Fr catheters can achieve a high PCI success rate through radial access when compared in the treatment of coronary lesions with the same level of complexity."}
+{"text": "Small ubiquitin\u2010related modifier (SUMO)\u2010specific protease 2 (SENP2) is essential for the development of healthy placenta. The loss of SENP2 causes severe placental deficiencies and leads to embryonic death that is associated with heart and brain deformities. However, tissue\u2010specific disruption of SENP2 demonstrates its dispensable role in embryogenesis and the embryonic defects are secondary to placental insufficiency. SENP2 regulates SUMO1 modification of Mdm2, which controls p53 activities critical for trophoblast cell proliferation and differentiation. Here we use genetic analyses to examine the involvement of SUMO2 and SUMO3 for SENP2\u2010mediated placentation. The results indicate that hyper\u2010SUMOylation caused by SENP2 deficiency can be compensated by reducing the level of SUMO modifiers. The placental deficiencies caused by the loss of SENP2 can be alleviated by the inactivation of gene encoding SUMO2 or SUMO3. Our findings demonstrate that SENP2 genetically interacts with SUMO2 and SUMO3 pivotal for the development of three major trophoblast layers. The alleviation of placental defects in the SENP2 knockouts further leads to the proper formation of the heart structures, including atrioventricular cushion and myocardium. SUMO2 and SUMO3 modifications regulate placentation and organogenesis mediated by SENP2. Genetic analyses reveal that hyper sumoylation caused by SENP2 deficiency can be compensated by reducing the level of SUMO modifiers. The placental deficiencies caused by the loss of SENP2 can be alleviated by the inactivation of gene encoding SUMO2 or SUMO3. SENP2 genetically interacts with SUMO2 and SUMO3 pivotal for the development of three major trophoblast layers. The alleviation of placental defects in the SENP2 knockouts further leads to the proper formation of the heart structures, including atrioventricular cushion and myocardium. Protein modification by SUMO2 and SUMO3 is essential for SENP2\u2010mediated placentation and organogenesis. Therefore, we used a genetic approach to test if SUMO2 and SUMO3 also modulate placental development mediated by SENP2. To perform genetic tests, mice carrying the deletion of SENP2 were crossed with those carrying SUMO2 and SUMO3 mutation to obtain the double mutants. At E10.5, three major trophoblast layers \u2014 the labyrinth, spongiotrophoblast, and TGC (trophoblast giant cell) in SUMO2+/\u2212 and SUMO3\u2212/\u2212 placentas are comparable to the wild type (Figure 2.2The alleviation of placental defects seemed to allow the embryo to survive after E12.5. We were able to recover SENP2\u2212/\u2212; SUMO2+/\u2212 embryos at E12.5 Figure A\u2010C. TherTo test if SUMO3 also modulates SENP2\u2010dependent embryonic and extraembryonic development, SENP2 nulls were crossed into the SUMO3 homozygous background. We successfully obtained the double knockout of E12.5 and E13.5 embryos Figure A\u2010C, M\u2010O,3Genetic analyses described in this study clearly demonstrate that SUMO2 and SUMO3 modulate SENP2\u2010dependent extraembryonic and embryonic development. Placental defects caused by hyper\u2010SUMOylation in the SENP2 knockouts can be alleviated by the reduction of SUMO modifiers. We succeed to prove the concept that hyper SUMOylation caused by the loss of SUMO proteases can be compensated by decreasing the level of SUMO modifiers. Although SUMO1 regulates the Mdm2/p53 pathway essential for cell cycle progression,SENP2 are not primary but secondary defects due to placental insufficiency. This is further supported by the current study showing that rescue of placental insufficiencies promotes the proper formation of the AV cushion and myocardium.It remains possible that the alleviation of extraembryonic and embryonic defects is independent of each other in the double mutants. However, our data show a tight link of heart deformities to placental deficiencies, suggesting these are dependent events. Furthermore, our demonstration of SENP2 dispensable for embryonic development strongly argues against a specific requirement of SENP2 in cardiac tissue during heart development.While removing one allele of SUMO3 has no effect on the SENP2\u2010null defects, placental and embryonic defects are alleviated by SUMO2 heterozygosity. Therefore, SUMO2 may play a more important role than SUMO3 in SENP2\u2010mediated placentation. The genetic analyses also suggest nonredundant functions of SUMO2 and SUMO3 in SENP2\u2010dependent regulation. Although SUMO2 and SUMO3 are ~95% identical, we do not find synergistic effects when both of them are reduced in the SENP2 nulls. It is possible that SUMO2 and SUMO3 modify distinct substrates critical for placentation. In addition to the SUMO1\u2010mediated modulation, SUMO2/3 may be critical for the regulation of S phase during cell cycle progression.44.1The SENP2, SUMO2 and SUMO3 knockout mouse strains and their genotyping methods were reported previously.4.2Samples were fixed, paraffin\u2010embedded, sectioned and stained with hematoxylin/eosin for histological evaluation.The authors declare no potential conflict of interest."}
+{"text": "FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2high MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2high MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2high MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2low MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong.The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits.  FOXP2 mutations in humans affect both the coordination of fine orofacial movements and language perception3. Because songbirds \u2013 like humans \u2013 need to learn most of their communicative vocalizations, they offer a unique model to study the role of FoxP2 (for nomenclature FOXP2/FoxP2 see Methods) for vocal learning and for the maintenance of learned vocalizations as adults4. Studying the relationship between FoxP2 and vocal learning in songbirds may inform the neurogenetic mechanism underlying the speech deficits in patients carrying FOXP2 mutations for the following reasons. The FoxP2 protein coding sequence is highly conserved between humans and songbirds as are the brain expression patterns, notably in the cerebellum and striatum7. Moreover, genetic manipulations of FoxP2 expression levels in the striatal song nucleus Area X during the critical phase of song learning lead to inaccurate and incomplete imitation of the tutor\u2019s song and more variable vocal production10. This phenotype bears similarities to the specific speech deficits called developmental verbal dyspraxia, DVD (or childhood apraxia of speech), that patients carrying FOXP2 mutations suffer from. The core-phenotype of DVD consists of altered precision, consistency and sequencing of movements underlying speech in the absence of neuromuscular deficits11. In addition, altered FoxP2 levels in adult Area X affect the dopaminergic modulation of corticostriatal signaling important to song variability and affect song maintenance13, stressing the fact that tight regulation of FoxP2 expression is a prerequisite for correct neural transmission in differentiated neural circuits. Additional effects of Foxp2 and its disruption on the embryonic development and the function of neural circuits have been described in mice23. Further evidence for the biological relevance of tight regulation of FoxP2 expression levels comes from the following studies. FoxP2 expression levels in Area X transiently increase during song learning but are lower in adults24. Singing decreases overall FoxP2 levels in Area X but not in the surrounding striatum and the degree of FoxP2 down regulation correlates with the amount of produced song27. This relationship is missing in deafened birds, pointing to an important role of auditory feedback for singing-driven FoxP2 down regulation27. How does singing affect FoxP2 expression at the cellular level? Medium spiny neurons (MSNs), the most abundant cell type in the avian striatum, predominantly express FoxP2 at low levels (FoxP2low) while a subset expresses FoxP2 at very high levels (FoxP2high). Both subtypes are not equally affected by singing; the density of FoxP2high MSNs is not measurably different after singing, contrary to the decreasing density of FoxP2low MSNs24. The authors hypothesized that the difference might be due to the neuronal age. Adult Area X constantly receives new MSNs that originate at the ventricular zone33. FoxP2high MSNs colocalize more frequently with a marker for new neurons than FoxP2low MSNs24. Recently we showed that new MSNs mature and participate in singing activity \u2013 as measured by immediate early gene activation \u2013 within a timeframe of six weeks34. Whether FoxP2 influences not only the function but also the integration of new neurons into existing circuits is still an open question. Based on the results of Thompson et al. (ref. 21) we hypothesized that FoxP2high MSNs are newly recruited into Area X and need to become FoxP2low MSNs before they can participate in singing. To test this, we labelled neuronal progenitors in adult zebra finches. At different time points after these cells had migrated into Area X, we quantified their expression levels of FoxP2 and whether they also expressed the immediate early gene expression EGR-1 after singing.The forkhead box P2 transcription factor (FOXP2) is linked to speech and language disorders. Heterozygous 37. Foxp2 expression levels vary in striatal MNSs and these differences may be relevant for the morphology of striatal MSNs. Dopamine receptor 1 (D1) expressing MSNs38 express Foxp2 at higher levels than dopamine receptor 2 (D2) expressing MSNs35. These differences in Foxp2 levels may be linked to anatomical differences between D1 and D2 MSNs39. Furthermore, in mice carrying humanized Foxp2 alleles (Foxp2hum/hum mice), Foxp2high MSNs are more numerous in the dorsal striatum and their MSNs have longer dendrites than wildtype mice37. Based on the latter results we hypothesized that FoxP2 levels of new MSNs in adult songbirds correlate with their neural morphology. To test this, we virally labelled neural progenitors and analyzed their FoxP2 expression, dendrite complexity and spine density after migration into Area X.In rodents, Foxp2 expression is associated with neurite outgrowth, neuronal morphology and synapse formation in cortico-striatal circuitshigh neurons. Neurons within the bottom 30% of the measured pixel intensity distribution were considered as FoxP2low. Because we were interested in the two extremes of the expression levels in this study, we did not analyze the new neurons with intermediate FoxP2 expression levels further . At 21 dpi and at 31 dpi 36.17% \u00b1 6.02 (SEM) and 34.91% \u00b1 2.53 (SEM) percent of all BrdU+ cells were FoxP2high neurons, respectively. At 42 dpi the percentage of FoxP2high cells had significantly decreased to 12.95% \u00b1 2.87 (SEM) on average  and 31 dpi  to 42 dpi . Interestingly there was a significant positive relationship with a low effect size between nucleus size and FoxP2 expression levels in all three experimental groups  and detected BrdU+/FoxP2+ cells after 21, 31 and 42 days post BrdU injection (dpi) in Area X of adult male zebra finches Fig.\u00a0. We foun26. Undirected singing is also associated with expression of the immediate early gene EGR-1 in Area X, which is therefore often used as a molecular readout of the neuronal activity associated with undirected song44. We hypothesized that FoxP2 levels in new FoxP2high neurons needed to be downregulated before activation by singing could occur, resulting in EGR-1 expression. Consequently, we did not expect to find BrdU+/FoxP2high/EGR-1+ MSNs in Area X. To test this, we analyzed BrdU+/EGR-1\u2009\u00b1\u2009/FoxP2+ cells in birds that had sung before sacrifice after 21 dpi, 31 dpi and 42 dpi.In adult zebra finches FoxP2 expression levels in Area X are behaviourally regulated. Undirected singing leads to downregulation of FoxP2 mRNA and proteinhigh/EGR-1+ cells in Area X  and 40.28% \u00b1 13.34 (SEM) of the new neurons that expressed FoxP2high were not activated by singing (BrdU+/FoxP2high/EGR-1-)  nor at 31 dpi . Contrary to our hypothesis we found BrdU+/FoxP2a X Fig.\u00a0 in all g1-) Fig.\u00a0. At 42 dhigh and FoxP2low new neurons at 31 dpi  and not mushroom spines  or stubby spines .Because of the relationship of FoxP2 levels and nucleus size we wanted to further characterize the morphology of new neurons expressing different FoxP2 levels. We used a lentiviral approach to express Green Fluorescent Protein (GFP) in progenitor cells at their place of birth in the lateral wall of the lateral ventricle Fig.\u00a0. After 3dpi Fig.\u00a0 and founons Fig.\u00a0. Becausesis Fig.\u00a0. At 42 d34.In the present study we investigated the dynamics of FoxP2 expression in adult-born MSNs in the striatal song nucleus Area X of adult male zebra finches. We show that the new MSN strongly expressed FoxP2 at their arrival in Area X from the ventricular zone (VZ) where they were born 21 days prior. During this stage and at intermediate maturation stages (31 days) one third of new MSNs expressed FoxP2 at high levels. At the late maturation stage (42 days) most new MSNs expressed FoxP2 at low levels Fig.\u00a0. Togethe34 here we asked if this was linked to FoxP2 levels. After singing FoxP2 mRNA and protein levels are lower in Area X tissue27 than when birds were silent whereas the expression of the immediate early gene EGR-1 increases linearly the more birds sing undirected song41. Analyzing expression levels in individual neurons revealed that only FoxP2low MSNs, but not Foxp2high MSNs seemed to be subject to singing induced FoxP2 downregulation24. We therefore hypothesized that FoxP2high MSNs were not yet connected into the circuit and therefore not regulated by singing. Our data contradict this hypothesis. We show that the induction of the immediate early gene EGR-1 in MSNs after singing was equally likely in FoxP2low and FoxP2high MSNs, suggesting that both were functionally incorporated into the song circuit.Since our previous work demonstrated that new MSNs participate in singing-associated neural activity in Area X45. In our study, the relationship between FoxP2 downregulation and song quantity was present in new MSNs at 42 dpi but not earlier and we therefore suggest that new MSNs start to receive auditory feedback signals between 31 and 42 dpi.Previous work showed that the degree of FoxP2 downregulation correlates with the quantity of produced song and depends on auditory feedback, which is relayed to Area X via the cortical song nucleus HVCFoxP2 promoter contains EGR-1 binding sites46 and that EGR-1 expression is crucial for functional integration of new neurons in the adult rodent hippocampus47.What might cause the age-dependent FoxP2 downregulation in new MSNs? One possibility is that intrinsic mechanisms, depending more on cell age than on extracellular inputs, downregulate FoxP2 during maturation. Another possibility is that EGR-1 gradually decreases FoxP2 levels during every singing event. This latter scenario is consistent with the findings that the low and FoxP2high MSNs. We now show differences between the morphology of adult generated MSNs that express FoxP2 at high or low levels; high FoxP2 levels were associated with greater dendrite complexity and higher dendritic spine density in comparison to neurons with low FoxP2 levels , a negative regulator of synaptogenesis19. Foxp2 specifically promotes corticostriatal synaptogenesis via the repression of Mef2c. Whether a similar mechanism shapes the integration of new MSNs into the avian corticostriatal network remains to be elucidated by future studies. In zebra finch Area X, two direct FoxP2 targets are associated with neuronal outgrowth; the very-low-density-lipoprotein receptor (VLDLR) and the contactin-associated protein-like 2 (CNTNAP2). Their expression correlates positively with FoxP2 in juveniles and in singing adults54. Thus, in new Area X neurons, VLDLR and CNTNAP2 would be expected to be highly activated during singing in FoxP2high MSN but not in FoxP2low MSNs and may thus generate the diverging MSNs morphology we found.What mechanisms might account for morphological differences between FoxP2high MSNs from former FoxP2low MSNs in later maturation phases. If these two subpopulations persist long-term, they might resemble striato-nigral and striato-pallidal MSNs of the direct and indirect pathway in mammals. These MSNs subtypes are morphologically and neurochemically different. Direct pathway MSNs express the dopamine receptor D1 and their dendrites are more complex than indirect pathway MSNs that express the dopamine receptor D255. High Foxp2 levels in D1 MSNs and low Foxp2 in D2 MSNs have been proposed to be linked to this anatomical dichotomy35. The avian direct and indirect pathway through the basal ganglia however is not characterized by different MSN projections but rather by direct and indirect pallidal-like output neurons that project from Area X to the thalamus56. To date, it is not known if MSN subtypes exclusively synapse on either direct or indirect pallidal-like neurons57. Contrary to mammalian MSNs, more than half of the Area X MSNs express multiple dopamine receptors58 so that they cannot be used as markers for indirect versus direct pathway neurons. Investigating potential avian MSNs subtypes and the developmental role FoxP2 plays in those will be of interest for future studies.We would like to propose some speculations regarding possible functions of two MSNs subpopulations that differ in FoxP2 expression levels. We found that these populations differed in nucleus size, dendritic complexity and spine density during an early time period of their integration into Area X. We do not know if the observed morphological differences persist long-term because of a lack of markers that could distinguish former FoxP234. General MSNs function is feed forward inhibition within a cortico-striatal-thalamo-cortical loop during singing59. We hypothesize that new MSNs in adult zebra finches are entrained to produce a correct firing pattern in a plastic phase during their maturation and thus may counteract song drift, as has been suggested before60. This process might be in influenced by varying FoxP2 expression levels and the resulting morphological differences between putative subpopulations of new MSNs. FoxP2high new neurons with a higher dendrite complexity and more dendritic spines might be more receptive to tuning than FoxP2low new neurons. For further interpretations of our findings it will be crucial to gain additional knowledge on the microcircuitry of Area X and on the role neurons play for its function.What might be the function of new MSNs in Area X and how is it affected by FoxP2 expression levels? Our previous study showed that once matured, new MSNs have similar characteristics as older, resident MSNs and are active during singingIn summary, FoxP2 expression levels vary in adult-born MSN at different maturation times after they have been recruited to Area X. We show that the different FoxP2 expression levels correlate with neuronal morphology and spine density. Varying FoxP2 expression levels during a specific time window might permit different target gene activation important for correct incorporation and function of new MSNs in Area X.61, FOXP refers to the human gene, Foxp refers to the mouse gene and FoxP refers to all other species. FOXP, Foxp2 and FoxP2 correspond to the protein product.We follow the nomenclature proposed byTaeniopygia guttata, age >120 days) were used in the present study, bred and housed at the Department of Animal Behaviour at Freie Universit\u00e4t Berlin. The colony was kept under a 12:12\u2009h light:dark-cycle with food and water ad libitum. All experiments were reviewed and approved by the veterinary department of the Freie Universit\u00e4t Berlin and by the ethics committee of the Regional Office for Health and Social Affairs Berlin and were performed in accordance with relevant guidelines and regulations. The permit numbers are G0116/13 and G0296/15.42 adult male zebra finches  in Area X at three time points, e.g. at 21 days , 31 days  or 42 days  after BrdU injections (dpi). In the second experiment we analyzed FoxP2 expression levels and the expression of the early growth response protein 1 (EGR-1) at 21 dpi , 31 dpi  and at 42 dpi (6 birds 156 neurons). In the third experiment we analyzed FoxP2 expression levels, dendrite morphology and spine density of new neurons that were labelled via lentiviral infection. In total we analyzed 52 neurons of 4 zebra finches  at 31 dpi and 23 neurons of 3 zebra finches at 42 dpi .For the analysis of FoxP2 levels and EGR-1 expression in newborn neurons 35 birds received BrdU (50\u2009\u00b5g/g) via intramuscular injections in the mornings for 5 consecutive days. Birds were assigned to three groups with different survival times .62.For FoxP2 expression level analysis after BrdU treatment or lentiviral injections, 17 birds were isolated in sound attenuated chambers for one night before sacrifice. In the following morning, birds were kept from singing by the experimenter sitting nearby for 1.5\u2009h after lights went on. For EGR-1 expression analysis in new neurons after singing, 18 birds were kept in sound attenuated chambers for three nights and were perfused in the morning of the 4th day 1.5\u2009h after the lights went on. During those 1.5\u2009h birds had to sing at least 150 motifs to be included in the subsequent analysis of EGR-1 expression. Vocalizations were continuously monitored via Sound Analysis Pro63 containing a GFP reporter gene was generated as described in Lois et al.63 and stereotactically injected into the ventricular zone of 7 birds under isoflurane anesthesia. Titers ranged from 2\u2009\u00d7\u2009106 and 3\u2009\u00d7\u2009107 viral particles/\u00b5l. Birds were fixed in a stereotaxic head holder, with the beak in a 45\u00b0 angle from the vertical axis. In each hemisphere, approximately 200\u2009\u00b5l of virus containing solution were injected into four sites. Following coordinates relative to the bifurcation of the midsagittal sinus were used: anterior-posterior 3.8\u20134.1, medial-latera \u22121.3/+1.3, dorsal-ventral \u22125.0, injection angle: 10\u00b0 lateral.To label progenitor cells at the lateral wall of the ventricle, the lentiviral expression vector pFUGWFor immunohistochemical staining birds were overdosed with isoflurane and immediately perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were dissected, post-fixed in 4% PFA for one night and washed for another night in PBS. Brains were cut sagitally or frontally into 50\u2009\u00b5m sections using a vibrating microtome . BrdU antigen retrieval required incubation in 2N HCl for 30\u2009min at 37\u2009\u00b0C and neutralization with borate buffer. GFP signal was enhanced via antibody staining. All immunostainings were performed according to standard protocols. The following primary antibodies were used: anti FoxP2 , anti EGR-1 , anti BrdU , anti GFP . Fluorescent secondary antibodies were the following: anti-rat-Alexa-Fluor-488 , anti-rabbit-Alexa-Fluor-568 , anti-goat-Alaxa-Fluor-647 . To visualize nuclei, all sections were counterstained with 4\u2032, 6-Diamidin-2-phenylindol .64. Only neurons with spiny long dendrites were included in the analysis. The Rolling Ball Background Subtraction plugin was used to subtract background. We measured the mean pixel intensity of nuclear FoxP2 expression, by positioning a circle of 4\u2009\u00b5m in diameter (12.56\u2009\u00b5m2) in\u00a0the center of the BrdU+ nucleus. In total we analyzed the intensity of the FoxP2 expression dependent fluorescence of 166 BrdU+ cells at 21 dpi (n\u2009=\u20095), 295 BrdU+ cells at 31 dpi (n\u2009=\u20096) and 272 BrdU+ cells at 42 dpi (n\u2009=\u20096). FoxP2high were defined as cells that fell into the top 30% of measured mean pixel intensities in one animal . We decided on the 30% value because it covered the FoxP2high expressing cells in the bimodal distribution of all FoxP2 intensities. We defined the neurons that fell into the bottom 30% of measured mean pixel intensities as FoxP2low. As for the FoxP2high cutoff, the bottom 30% contained the low-intensity peak of the bimodal density distribution. Because we particularly wanted to address the effect of high and low FoxP2 expression levels on neuronal properties, neurons with intermediate FoxP2 expression levels were not considered for further analysis. The Simple Neurite Tracer plugin (Fiji) was used to trace individual neurons and we analyzed their total branch length and number of primary dendrites. The traces were then used by the Sholl analysis plugin in Fiji65. We measured intersections of dendrites with concentric circles that were placed every 10\u2009\u00b5m starting from the center of the soma. The maximal number of intersections per neuron was extracted from the Sholl analysis dataset. For dendritic spine analysis images were deconvolved using the Tikhonov-Miller algorithm in the DeconvolutionLab plugin in Fiji66. Prior to deconvolution an individual point spread function was generated for each image using the Born and Wolf 3D optical model in the PSF Generator plugin in Fiji67. Semiautomated dendritic spine counts were performed using the software NeuronStudio68 that uses a spine classification algorithm. For spine classification, the default settings were used to classify spines as mushroom, stubby or thin spines: a head-to-neck ratio threshold of 1.1\u2009\u00b5m, a height-to-width ratio threshold of 2.5\u2009\u00b5m and a minimum mushroom head size of 0.35\u2009\u00b5m. A spine is considered mushroom if the head-to-neck ratio is above the threshold and its head is larger than 0.35\u2009\u00b5m. A spine is considered stubby if its head-to-neck ratio and its heights-to-width ratio are below threshold. In all other cases spines were classified as thin. On average, we analyzed spines densities on secondary dendrites along the length of 118\u2009\u00b5m \u00b1 1.92  per neuron. In total, we analyzed spines of 52 individual neurons of 4 animals in experimental group 31 dpi, and 23 neurons of 3 animals at 42 dpi. Additionally, we measured the dendrite diameter of 44 new neurons in 4 animals  using the line measuring tool in Fiji64. We took 5 measures on each of 3 secondary dendrites per neuron . The experimenter was blind to FoxP2 levels of individual neurons during the whole quantification process, because cells were selected for quantification based on their BrdU+ fluorescence or their EGR-1 fluorescence and FoxP2 fluorescence in a different channel was quantified last. The datasets generated and analysed during the current study are available from the corresponding author on request.Z-Stacks of BrdU+ or GFP+ cells in Area X were obtained with a SP8 confocal microscope (Leica). For FoxP2 scanning, all microscope settings were kept constant. Scans of BrdU+ nuclei were performed using a 63x lens , an image size of 1024\u2009\u00d7\u20091024 pixels and a z-stack size of 1\u2009\u00b5m. Whole neurons (GFP+) were imaged using a 63x lens, an image size of 2042\u2009\u00d7\u20092042 pixel and a z-stack size of 1\u2009\u00b5m. Acquired images were processed using the Fiji software package69. Significance level was p\u2009<\u20090.05 for all tests. Plots were generated using the ggplot package in R70. For the analysis of FoxP2high neurons we used a Kruskal-Wallis test followed by a Dunn\u2019s test for pairwise comparison. For the analysis of FoxP2low neurons we used ANOVA followed by a Bonferroni\u2019s multiple comparison test. The relationship of (a) FoxP2 levels and nuclear diameter as well as (b) FoxP2+ neurons and singing were determined using a linear regression analysis. Dendritic spine data  and Sholl data were analyzed using the paired Student\u2019s t-test. Data from the spine type analysis, mushroom head size, total dendrite length, number of primary dendrites and dendrite thickness were analyzed using the Mann Whitney test. Number of maximal intersections was analyzed using the Student\u2019s t-test. Choice of test was based on previous analysis for normality using the Shapiro-Wilk-test and variance analysis using the F-test or Levene\u2019s test.The software R was used to analyze data"}
+{"text": "To the editor,The separation distance recommended\u03bcm in diameterWe modeled expiratory flow during normal conversational speech as a jet of constant velocity of 1 m/s and 10 s in durationWhile the quiescent case Fig.\u00a0a support3It is also important to differentiate between outside and indoor gathering. Although it is likely that any virus-ladenSupplementary material 1 (GIF 2722 kb)Supplementary material 2 (GIF 2497 kb)Below is the link to the electronic supplementary material."}
+{"text": "SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1\u2009+\u2009SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90\u00a0min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring  SMA has an autosomal recessive inheritance pattern with an incidence of 1 in 6000\u201310,000 births4. SMA results from the loss or mutation of survival motor neuron 1 (SMN1) on the q arm (q13) of chromosome 5 and retention of the paralogous survival motor neuron 2 (SMN2)5. SMN1 and SMN2 are nearly identical save 5 key nucleotide differences at the 3\u2032 ends of the genes. SMN2 is functionally distinguishable from SMN1 by a single nucleotide difference (c.840C>T) in exon 7 that disrupts an exonic splice enhancer7. Even though close to 95% of SMA cases result from the loss of SMN1 and retention of SMN2, there is a wide spectrum of clinical disease severity based on the motor development milestones that are achieved8. Numerous studies using different assays and patient cohorts (reviewed in9) have demonstrated a strong inverse correlation between SMN2 copy number and disease severity. There are currently three USA Food and Drug Administration (FDA) approved therapeutic options; nusinersen , onasemnogene abeparvovec  and risdiplam 14. These agents target SMN2 expression. SMN2 copy number is used to design the therapeutic regimens for SMA patients receiving these approved therapies, with immediate treatment for patients with 2 or 3 copies of SMN216. A simple yet highly accurate and sensitive solution for SMN1 and SMN2 copy number detection is needed to meet the rapidly growing demand for informed SMA treatment.Proximal spinal muscular atrophy (SMA) is an early-onset neurodegenerative disease characterized by the loss of \u03b1-motor neurons in the anterior horn of the spinal cord which leads to muscle weakness and atrophySMN1 deletions20. MLPA is a multi-step method requiring post-PCR capillary electrophoresis and a long time-to-result (~\u200920\u00a0h)19. Alternatively, qPCR requires normalization with standard curves. More importantly, neither MLPA nor qPCR can consistently distinguish unit differences in SMN1 or SMN2 when the copy number is greater than 322. Genomic sequencing (GS) technology has made progress on copy number detection. A recent publication reported SMA diagnosis and carrier identification using whole genome sequencing and advanced copy number analysis software23. However, there are still limitations to GS for diagnosis including the cost, sample processing time, complex data analysis and the need for orthogonal validation.Current genotyping and copy number determination methods for SMA are complex, time consuming or lack resolution with higher copy numbers. Multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect 24. With dPCR, the prepared PCR sample is distributed across a large number of physically isolated micro-reaction partitions so that each partition will have single digit counts of template DNA or none at all26. The absolute concentration (and confidence level) of the target gene(s) can be calculated by counting the number of positive partitions (containing at least one target molecule) and the number of negative partitions (containing no target molecules) and using a Poisson-based statistical correction. dPCR can reliably and accurately measure SMN1 and SMN2 copy numbers over a wide range, between 0 and 6 copies27. Due to the optical configuration of first-generation dPCR platforms, most of the currently available multiplex assays are duplex or variations of a duplex assay. A recently published report on multiplex dPCR describes simultaneous identification of SMN1 and SMN2 copy numbers by using different signal amplitudes within the same optical channel28. The Combinati  Absolute Q Digital PCR System  alleviates the limitations previously noted to provide absolute quantification of a target gene within a sample for molecular diagnostics and prognosticsSMN1, SMN2, total SMN (SMN1\u2009+\u20092), and RPPH1, respectively  hybridizes to nucleotide c.840C while the SMN2 probe (VIC) hybridizes to nucleotide c.840T. The total SMN  targets intron 1\u2014a region that is identical between SMN1 and SMN2. The internal reference control is designed to target the highly conserved RPPH1 gene (TAMRA), which is consistently present as 2 copies30.The hydrolysis probe-based multiplex assay is designed to use FAM, VIC, TYE665, and TAMRA to identify SMN1 and SMN2 copy number assays previously developed on the QuantStudio 3D dPCR System , SMN2 only (NA23255), or both SMN1 and SMN2 (NA03815). Representative images of the dPCR results from the analysis software  were normalized against the reference gene (RPPH1) that contains 2 copies per genome. The scatter plots for SMN1 exon 7 , 3:0 (NA17117), and 1:1 (NA03815). For NA03815, the Absolute Q dPCR data showed copy numbers for SMN1 exon 7, SMN2 exon 7, and total SMN intron 1 as 1.1, 1.1 and 2.2, respectively.The optimized assay was first tested on control DNA samples containing are Fig.\u00a0A shows sn 7 Fig.\u00a0B, SMN2 en 7 Fig.\u00a0C, and ton 1 Fig.\u00a0D shows rEcoRI-digested genomic DNA. Genomic DNA up to 25\u00a0ng was also tested on the Absolute Q. There are no apparent differences in SMN1 and SMN2 copy number quantification completed with 2.5\u00a0ng template or with 25\u00a0ng genomic DNA (Supplementary Table The Absolute Q dPCR assays require less template genomic DNA (2.5\u00a0ng) than the QuantStudio 3D dPCR assays which uses 30\u201360\u00a0ng of SMN1, SMN2 and total SMN in 10 genomic DNA test samples from Coriell Cell Repositories were quantified using the SMA multiplex 4-color assay. All 10 Coriell DNA samples were run in triplicate on the Absolute Q dPCR System for assay verification and repeatability. The intra-assay variability\u2014measured by %CV\u2014between the SMN1  for the SMN2 assay and 0.987 (95% CI 0.963\u20130.996) for the total SMN assay. The SMN1 and SMN2 copy numbers measured with this multiplex assay are concordant with those provided by Coriell Cell Repositories, including one sample with 5 copies of SMN2. Additionally, the 6 copies of total SMN for NA03814 correctly matched the corresponding sums of SMN1 and SMN2 copy numbers. The total SMN copy numbers in all of the samples except for NA11254 were equal to their corresponding sums of SMN1 and SMN2 copy numbers.Copy numbers of MN1 Fig.\u00a0A, SMN2  within the cohort. The Absolute Q dPCR identified one of the patient-derived sample (MND10) as containing 1 copy of SMN1 and one copy of SMN2. This sample was from an individual harboring a SMN1(p.A2G) missense mutation27. The Absolute Q dPCR SMN1  with 5 copies of total SMN. Bland Altman analysis of the results from SMN1 , the total SMN copy number at exon 7 (4 copies) was greater than the sum of the SMN1 exon 7 (0 copies) and SMN2 exon 7 (2 copies) copy numbers. Based on this result, this sample was hypothesized to have 2 copies of either SMN1 or SMN2 containing a partial deletion of exon 7. The partial deletion of exon 7 in this sample was confirmed orthogonally by junctional PCR (data not shown) and by GS23.Absolute Q and QuantStudio 3D array dPCR SMA assays were compared on a set of blinded samples derived from patient lines n\u2009=\u200915). This set of samples contained gDNAs from 13 SMA patients and 2 non-SMA controls. Using the MN1 Fig.\u00a0A, SMN2  and the low DNA input amount (~\u20092 to 3\u00a0ng per reaction) facilitate single molecule partitioning. Finally, SMN1 and SMN2 genes, each approximately 28\u00a0kb in length, are both located in the same region of chromosome 5 but they are separated from each other by about 875\u00a0kb of genomic sequence. Mechanical sheering of the genomic DNA during DNA sample isolation may further fragment this region thereby reducing the likelihood of both genes being in the same partition. Eliminating the restriction digestion step in the copy number assay, therefore, simplifies the workflow of the assay. Future studies would assess the effects of specimen source and DNA isolation procedure on the need for either a shearing or digestion step prior to dPCR analysis.Some dPCR copy number variation assays rely on digestion of the template genomic DNA with a restriction endonuclease31. The shortened run-time along with minimal hands-on operation could facilitate platform adoption into diagnostic and prognostic settings.In addition to eliminating the need for a pre-PCR digestion step, the dPCR workflow is simplified by integration of multiple systems into a single instrument. The first-generation of dPCR systems have separate instruments for microfluidic partitioning of the PCR reactions and template DNA, PCR amplification and raw data acquisition. This required significant hands-on-time and training for each unique workflow, which, in turn, increased the probability of operator error or process variability. The Absolute Q dPCR System combines sample partitioning, thermocycling, and raw data acquisition into a single benchtop instrument. As a result, the Absolute Q dPCR System has an average run-time that is 73% faster than first-generation dPCR systemsSMN1 and SMN2 copy numbers, dPCR can also identify partial deletions in SMN1/SMN2 and gene conversion events that generate hybrid SMN genes. By comparing total SMN copy number to the sum of SMN1 exon 7 and SMN2 exon 7 copy numbers, partial deletions of SMN1 or SMN2 can be identified. One of the patient-derived cell lines tested in this study (MND003) contained a partial deletion of exon 7, which was confirmed orthogonally (data not shown). Additionally, one of the control samples (NA11254) possibly contains a partial deletion as the total SMN copy number was found to be 3 at intron 1 while the sum of SMN1 and SMN2 copy numbers at exon 7 was 2. The discrepant results with this sample are not a consequence of a lack of specificity in the intron 1 primers or probe since the in silico specificity scores for these sequences were high, i.e. all had a BLAT score of 22, which exceeds the minimal threshold of 2032. These results need to be confirmed orthogonally by junctional PCR or GS in order to verify that the partial deletion is indeed the reason for the discrepancy. Partial SMN deletions, particularly resulting from the loss of exons 7 and/or 8 (SMN1/2\u039478), have been previously reported in the literature using several approaches including MLPA and GS33. Gene conversion events can create hybrid SMN genes where part of the gene is SMN1 and another part is SMN234. The most commonly identified hybrid gene is SMN1 with c.840T (i.e. SMN2), however there are other SMN1/SMN2 hybrid gene variations. Other SMN1/SMN2 hybrid genes have different exon 7 inclusion efficiencies compared to SMN1 or SMN236. It is important to correctly identify hybrid SMN genes as they can potentially lead to unexpected responses to current therapeutic options. Future work involving the development of a panel of assays targeting the single nucleotide difference between SMN1 and SMN2 at exon 8, as well as the intronic single nucleotide differences, would be useful for the identification of partial deletions in SMN genes as well as hybrid SMN genes.In addition to determining SMN1 are present in cis on the same chromosome. A single nucleotide polymorphism within SMN1  is associated with the cis configuration of SMN137. In addition, this dPCR assay cannot detect intragenic point mutations in SMN1, which account for 3\u20135% of SMA cases8. Future studies are needed to develop dPCR assays that are able to detect these single nucleotide changes. These assays could potentially be integrated into an expanded multiplexing assay panel alongside the current SMN1/SMN2 differentiating dPCR assays described here.A limitation of the copy number assay described in this study is its inability to detect silent SMA carriers (2\u2009+\u20090) where both copies of 39. Dried blood spots (DBS) are a common source material for SMA newborn screening. A 3-mm diameter DBS typically yields\u2009~\u2009140\u00a0ng gDNA (in a volume of 50 \u03bcL) using column-based DNA extraction40. This study demonstrates that SMN1 and SMN2 copy numbers can be accurately measured with as little as 2\u20133\u00a0ng gDNA, so the Absolute Q and MAP plates could reliably be used for orthogonal validation of SMA carrier status or SMN2 copy number. As the technology is further developed, quadraplex dPCR could potentially become a primary SMA screening tool.Newborn screening of SMA is becoming more available and routine in the United States as well as many other countries41) and other technologies such as mass spectrometry ($3.00 per sample42). These per-sample costs are exclusive of the cost associated with labor, which is reduced with the lower hands-on time of the Absolute Q dPCR System.The acquisition cost for the Absolute Q dPCR System is comparable to high-end qPCR instrumentation, and somewhat lower than other currently available dPCR platforms. In this study, the consumable (SMA reagents and MAP plates) costs were $2.36 per test. This is less than typical per-sample costs for available droplet dPCR systems ($3.80 per sample24). We have demonstrated that this system can identify a rare single nucleotide variant (EGFR(T790M)) associated with non-small cell lung carcinoma as well as fusion genes (BCR-ABL1 and CCDC88C-FLT3) that are associated with different types of cancer29.The Absolute Q dPCR quadruplex assay approach could be extended to identification of copy number variations associated with other diseases, in addition to SMA. dPCR has been used to identify rare gene variants as well as differences in copy number of multiple genes associated with pediatric-onset disorders . NA03814 and NA03815 samples were from known SMA carriers, i.e. 1 copy of SMN1.Control samples of human genomic DNA (n\u2009=\u200910) were obtained from Coriell Cell Repositories  as described previously29. Approval for cell line generation was provided by the Nemours Institutional Review Board and this study is registered on ClinicalTrials.gov (NCT01754441 and NCT02532244). This study was approved by the Nemours Institutional Review Board (#764456). Written informed consent or assent was obtained for each patient-derived cell line. Each cell line was de-identified. For all samples tested, the tester was blinded to the previously measured SMN1 and SMN2 copy numbers. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or research committees and with the 1964 Helsinki declaration and its later amendments of comparable ethical standards.Patient-derived samples (n\u2009=\u200915) were procured from the Motor Neuron Diseases Research Laboratory . Genomic DNA was isolated from patient-derived fibroblast and lymphoblastoid cell lines and the 29. Each unit is loaded with 10 \u00b5L of sample mixture and more than 90% of the loaded sample is partitioned and analyzed in\u2009~\u200920,000 pico-scale partitions. The partition volume in MAP plates is defined by the physical dimensions of the microarray chambers and not by a stochastic process such as fluid emulsions, which is important because consistent partition volume is a critical component of the dPCR statistical correction model. The physical array ensures that all samples across all plates yield\u2009~\u200920,000 analyzed partitions and minimal sample is lost to dead volume or compromised partitions.The Combinati Absolute Q Digital PCR System consists of Microfluidic Array Partitioning (MAP) consumable plates and a fully integrated instrument that automates partitioning of reagents in the plate, PCR thermocycling and 5-color fluorescence image acquisition. The MAP plate has a standard microplate footprint capable of running up to 16 samples per dPCR runThe Absolute Q digital PCR System has a walk-away workflow identical to traditional qPCR. The MAP plate is loaded via pipette with 10 \u03bcL of PCR mix and an overlay of 10 \u03bcL Isolation Buffer in each well. The wells are then capped with specialized gaskets. The plate is then placed into the Absolute Q tray and retracted into the system. The Absolute Q uses positive pressure from an on-board compressor to partition the sample within the MAP plate without the need for microfluidic valves, sealing films or other moving parts. The MAP slides are constructed of a cyclo olefin polymer  film that seals the microfluidic features that are molded into a separate piece of thicker material. Four identical slides are bonded to a rigid, microtiter format plate frame that includes the loading wells to complete the plate assembly. The thin film becomes gas permeable when positive pressure is applied to a well containing reagents. As the reagent enters the microfluidic features, air is passed out of the partition through the film. This allows reagent to completely fill dead-ended partitions and prevents any bubbles from forming inside of the microfluidic features. The Isolation Buffer overlay follows the reagent and physically separates the reagent reaction volumes to complete the partitioning. Positive pressure applied to the consumable during the PCR thermocycling prevents any evaporation and ensures that bubbles will not form and disrupt the isolated micro-reactions.Before and after PCR thermocycling, entire arrays are imaged with up to five optical channels configured for the most commonly used dyes, including a ubiquitous quality control dye (ROX) used to verify proper partition filling and finding. The images taken before PCR are subtracted from the after-PCR images to remove background noise. Combinati Analysis software automatically applies optical crosstalk compensation and classifies the partitions using a convoluted neural network algorithm to eliminate false positives/ negatives and ensure robust quantification results. The full dPCR process occurs within the single benchtop instrument without operator\u2019s interaction after setting up the protocol parameters Fig.\u00a0. The str44 and UCSC In-Silico PCR  using default settings. For Primer-BLAST, each primer was required to have at least 2 total mismatches to unintended targets, including at least 2 mismatches with the last 5 base pairs at the 3\u2032 end. For In-Silico PCR, the minimum match size was 15 and a minimum BLAT32 score of 20, using the GRCh38/hg38 human genome assembly. Primers and probes were also evaluated for primer dimers and cross primer interactions using Multiple Primer Analyzer  with the optimal sensitivity value (3) for dimer detection. All of the oligos were synthesized by Integrated DNA Technologies and Thermo Fisher Scientific. The assay includes a total of 3 pairs of primers and 4 probes with the SMN1 and SMN2 targets sharing a common pair of primers. The FAM-labeled SMN1 and VIC-labeled SMN2 probes were designed utilizing the signature c.840 single nucleotide difference between exon 7 of SMN1 (c.840C) and SMN2 (c.840T). The oligos for total SMN copy number were designed upstream in intron 1 targeting both SMN1 and SMN2 genes, and the probe was labeled with TYE665. The TAMRA-labeled probe for RNase P  targeted the single exon region of RPPH1 gene . All primers were checked for target sequence specificity using NCBI Primer-BLASTSMN1/SMN2 primers, 900\u00a0nmol/L RPPH1 primers, 900\u00a0nmol/L total SMN primers, and 250\u00a0nmol/L of each probe. Each MAP plate well was loaded by hand via pipette with 10 \u00b5L of PCR reaction mix then overlaid with 10 \u00b5L Isolation Buffer. Gasket caps were placed and the plate was put into the Absolute Q and run with the following conditions: 3\u00a0min activation at 95\u00a0\u00b0C, 40 cycles of 5\u00a0s at 95\u00a0\u00b0C and 30\u00a0s at 62\u00a0\u00b0C. The fully automated Absolute Q Control Software  controls the sample digitization into the MAP partitions, thermal cycling, and imaging.The control DNA samples were diluted to 10\u00a0ng/\u00b5L working stock. The blinded DNA samples were measured using Qubit 4 fluorometer (Invitrogen) and diluted to 10\u00a0ng/\u00b5L. The PCR reaction mix contained 1X Combinati MasterMix, 2.5\u201325\u00a0ng human gDNA, 1800\u00a0nmol/L SMN1, SMN2 and total SMN are normalized by the reference control RPPH1 using the following equations:1\u2009=\u2009number of SMN1 positive partitions, N2\u2009=\u2009number of SMN2 positive partitions, N3\u2009=\u2009number of total SMN positive partitions and N4\u2009=\u2009number of RPPH1 positive partitions.The copy numbers were determined using the Absolute Q Analysis Software (v10.5.4). The software automatically calculates the optimal positive/ negative threshold and in this case the positive signals were five times higher than the negative signals. For each unit, the software displays the total viable partition count and the positive partition count. The software has a setting to identify the sample types as a copy number variation assay and applies the appropriate calculations to automatically display the copy numbers for the samples. The copy numbers of 45 using SigmaPlot v.12.0  was used to measure the agreement between dPCR methods.The coefficient of variability (%CV), defined as the standard deviation divided by the mean value for each set of replicates, was used to assess repeatability, or intra-assay precision. Reliability was also measured by the intraclass correlation coefficient (ICC) using SPSS v.25 . A Bland Altman agreement analysisSupplementary Table S1."}
+{"text": "Diabetes is an increasingly important risk factor for ischemic stroke and worsens stroke prognosis. Yet a large proportion of stroke patients who are eventually diabetic are undiagnosed. Therefore, it is important to have sensitive assessment of unrecognized hyperglycaemia in stroke patients.Secondary outcome analysis of a randomized controlled trial focussing on parameters of glucose metabolism and detection of diabetes and prediabetes in patients with acute ischemic stroke (AIS).A total of 130 consecutively admitted patients with AIS without previously known type 2 diabetes mellitus (T2DM) were screened for diabetes or prediabetes as part of secondary outcome analysis of a randomized controlled trial that tested lifestyle intervention to prevent post-stroke cognitive decline. Patients had the oral glucose tolerance test (OGTT) and glycated hemoglobin (HbA1c) measurements in the second week after stroke onset and after 1 year. The detection rates of diabetes and prediabetes based on the OGTT or HbA1c values were compared.By any of the applied tests at the second week after stroke onset 62 of 130 patients (48%) had prediabetes or T2DM. Seventy-five patients had results from both tests available, the OGTT and HbA1c; according to the OGTT 40 (53.3%) patients had normal glucose metabolism, 33 (44%) had prediabetes, two (2.7%) T2DM. In 50 (66.7%) patients the HbA1c results were normal, 24 (32%) in the prediabetic and one (1.3%) in the diabetic range. The detection rate for disorders of glucose metabolism was 10% higher  with the OGTT compared with HbA1c. After 1 year the detection rate for prediabetes or T2DM was 7% higher with the OGTT (26% relative difference).The study intervention led to a more favourable evolution of glycemic status after 1\u00a0year.The OGTT is a more sensitive screening tool than HbA1c for the detection of previously unrecognized glycemic disorders in patients with acute stroke with an at least a 25% relative difference in detection rate. Therefore, an OGTT should be performed in all patients with stroke with no history of diabetes. Trial registrationhttp://clinicaltrials.gov. Unique identifier: NCT01109836. Glucose abnormalities, either diabetes type 2 (T2DM) or impaired glucose tolerance (IGT) are common in patients with stroke and impair their prognosis \u20135. T2DM It is a longstanding paradox that the OGTT is used for diabetes diagnosis whereas HbA1c serves as monitoring parameter for antidiabetic treatment. As the recommended 2-h oral glucose tolerance test (OGTT) may be a time-consuming procedure, an International Expert Committee and the American Diabetes Association (ADA) have recommend the use of HbA1c as a diagnostic test  with\u2009\u2265\u20096The aim of the present study was to find out how well HbA1c results will compare with the determination of FPG and 2-h plasma glucose (2hPG) using an OGTT in screening for T2DM and prediabetes in patients with acute non-disabling ischemic stroke during the early post-acute period and after the 1-year follow-up.In this study we performed a retrospective analysis of the data regarding glucose metabolism that were collected as part of the protocol in the Austrian Polyintervention Study to Prevent Cognitive Decline after Ischemic Stroke (ASPIS) trial. The study details of ASPIS have been described earlier . In shorPatients were tested for diabetes or prediabetes with HbA1c or an OGTT at baseline in the first or second week after the stroke and at 1-year follow-up. Blood samples for FPG and 2hPG were taken in sodium fluoride tubes and analysed immediately with the hexokinase method in a Hitachi Laboratory systems machine. The World Health Organization (WHO) 1999 criteria for diabetes   had already T2DM according to patients\u2019 history, eight patients without previous diagnosis had no results for HbA1c or the OGTT available to allow classification at baseline, leaving 130 patients for further analysis  patients had normal glucose metabolism, 33 (44%) had prediabetes, two (2.7%) T2DM. According to the HbA1c results 50 (66.7%) were normal, 24 (32%) prediabetic and one T2DM (1.3%), see Table After 1 year, 28 patients were either study dropouts or had no further tests for glucose metabolism available, leaving 102 subjects for the follow-up analysis: the results of either tests revealed 56 patients as normal (54.9%), 36 as prediabetes (35.3%) and 10 (9.8%) with T2DM .67 (66%) patients had both tests, OGTT and HBA1c, available for direct comparison: according to OGTT 45 (67.2%) patients had normal glucose metabolism, 19 (28.4%) had prediabetes, and three (4.5%) T2DM. According to HbA1c results 51 (76.1%) were normal and 16 (23.9%) prediabetic. The observed agreement in classification of normal glucose metabolism was 62.7%, of prediabetes only 20.7%, the detection rate for prediabetes or T2DM was 9.9%  higher with OGTT, (see Table Next we looked how glycemic status changed over time in patients who had tests both at baseline and 1-year follow-up: Out of 102 patients 67 (65.7%) remained stable, 16 (15.7%) improved and 19/102 (18.6%) progressed to prediabetes or T2DM. Baseline variables of these three groups are shown in Additional file The main finding of this study was that in patients which subacute minor stroke the OGTT detects up to 25 to 29% more patients with prediabetes or T2DM than HbA1c regardless if performed early after the event or at 1-year follow-up. The odds that prediabetes was detected by the OGTT was 2.6 higher compared to HbA1c measurement alone. As in several previous studies , 12 disoDuring the 1-year observation the classification of glucose disorders in stroke patients in the trial was not invariable, although about two thirds of the patients did not change their original category. A multifactorial lifestyle-oriented intervention resulted in a non-significant statistical trend for a higher percentage of patients who improved in their glucose metabolism in the intervention group than the control group receiving a standard care. With improvements in lifestyle  or treatAmerican Diabetes Association has recommended HbA1c as the tool for diagnosing T2DM and defined a cut-off level 48\u00a0mmol/mol to indicate diabetes . Since lThere is also evidence that the parameters of the OGTT correlate better with outcomes of vascular disease than HbA1c. A German study among 1015 patients admitted for coronary angiography found T2DM in 149 patients by an OGTT, but only 42 by HbA1c  and the The strength of our study is that we ascertained consecutively patients admitted that were assessed and reassessed within the settings of a randomized controlled trial. Secondly, we performed a follow-up up investigation after 12\u00a0months and therefore our results do not rely only on measurements during the acute stroke period.Weak points of our study are the low number of patients investigated as this was a regionally conducted multi-centre trial and the considerable number of dropouts. Despites this weakness in quantity our results are robust over time from baseline to follow up and confirmatory with other studies. Furthermore, it can be stated that part of the hyperglycaemic states that were classified as diabetes and prediabetes at baseline can be interpreted as acute hyperglycaemic stress responses  especialIn addition, we found a trend for a lower rate of progression of disorders of glucose metabolism among those who underwent multimodal interventions for risk factor control compared with the control group. An improved detection of prediabetes and early T2DM using an OGTT could help to focus and tailor preventive interventions to improve or maintain cardiovascular and metabolic health.In the clinical evaluation of patients with acute stroke the assessment of the glycemic status is important in order to assign adequate preventive management to correct metabolic abnormalities that may influence the prognosis. The OGTT is a more sensitive method than HbA1c to detect and monitor disorders of glucose metabolism in patients with an ischemic stroke. The OGTT should be carried out in all stroke patients who have not been found to be diabetic before the stroke event.Additional file 1: Table S1. Baseline characteristics of patients in different groups of evolution of glucose metabolism (GM) between baseline and 12 months follow up. Both, OGTT and HbA1c were used for diagnosis, patients were classified to PD or T2DM if either the OGTT or HbA1c criteria were fulfilled."}
+{"text": "Actinoplanes sp. SE50/110 chromosome via the bacteriophage \u03c6C31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.The pSETT4 vector integrates into the  Actinoplanes sp. SE50/110 chromosome via the bacteriophage \u03c6C31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.The pSETT4 vector integrates into the  Actinoplanes sp. SE50/110 (strain ATCC 31044) is known as a natural producer of acarbose , assembled by gene splicing by overlap extension (gene SOEing) (For construction of pSETT4 SOEing) , and clo SOEing)  accordingap was digested with NdeI and KpnI and treated with shrimp alkaline phosphatase following the supplier\u2019s instructions . The tipA promoter was amplified from pSETGUS .The complete sequences of pSETT4"}
+{"text": "The pathogenic BRCA1/2 germline mutations contributed to Hereditary Breast and Ovarian Cancer (HBOC) susceptibility. The features of BRCA1/2 germline mutations in non-small cell lung cancer (NSCLC) have not been systematically studied. Here we performed the first study investigating the characteristics of pathogenic BRCA1/2 germline mutations in Chinese NSCLC patients and compared them with those from Chinese HBOC.Information on BRCA1/2 germline mutations from 9010 Chinese NSCLC patients were collected from available studies and analyzed, and compared with the BRCA1/2 germline mutations from Chinese HBOC BRCA1/2 database .19  pathogenic BRCA1 and 60  pathogenic BRCA2 germline mutations from NSCLC were identified. The carrier frequency of BRCA1/2\u00a0in Chinese NSCLC patients (86/9010\u2009=\u20090.95\u2009%) was significantly lower than that in Chinese breast and ovary cancer patients  (P\u2009<\u20090.001). We found that frameshift and nonsense mutations were the predominant types of BRCA1/2 mutation in NSCLC, with no obvious hot spot mutations. No significant difference in the ratio of frameshift and nonsense mutations was found between BRCA1 and BRCA2 in NSCLC. 5 out of 19 mutations in BRCA1 and 23 out of 60 mutations in BRCA2 were novel mutations found in NSCLC that have never been reported in Chinese HBOC. A trend of higher percentage of BRCA1 nonsense mutations in the carriers was revealed in NSCLC compared with HBOC, while no such difference was found in BRCA2 in all types of mutations.BRCA1/2 germline mutations from NSCLC exhibited distinct characteristics compared with those from HBOC in Chinese population, including lower carrier frequency than HBOC, higher ratio of nonsense mutations and carriers than HBOC, and novel BRCA1/2 germline mutations never found in HBOC. The germline mutations of BRCA1/2 have been comprehensively investigated in Hereditary Breast and Ovarian Cancer (HBOC). The interpretation of pathogenicity of BRCA1/2 germline mutations is based on the American College of Medical Genetics (ACMG) guidelines and provides strong tools for new mutation identification and interpretation [Apart from HBOC, the germline BRCA1/2 mutations have also been found in a variety of other cancers, including pancreatic cancer , lung caThe information on mutations and specific mutation sites were collected from three publications reporting the BRCA1/2 germline mutations in Chinese population \u201310. MutaThe information on mutations and specific mutation sites was downloaded from the LOVD database following the link below:https://databases.lovd.nl/shared/variants?search_owned_by_=%3D%22Xianqi%20Gao%22.The set of data was reported in a recent publication comprehensively reviewed and summarized the BRCA1/2 germline mutations from HBOC in Chinese population from 1999 to 2017 . Therefohttps://www.r-project.org/). Chi-square test and Fisher test were performed when rate or percentage was compared for significance. P\u2009<\u20090.05 was regarded as significant difference.Statistical analysis was performed, and figures were plotted with GraphPad Prism 5.0 software  or the R software  of lung cancer patients with pathogenic germline mutation, and in 32.35\u2009% (11/34) of patients with likely pathogenic germline mutations . TherefoIn this study, we also identified several novel pathogenic germline mutations in NSCLC that have never been found in Chinese HBOC patients. New germline mutations of BRCA1/2 have been emerging all the time in the past 20\u00a0years in HBOC . It appePARP inhibitors (PARPi) have been widely used in the treatment of HBOC, and their effectiveness have been proved by many studies at first-line and multiline levels , 18. MaiAdvances have also been made in the application of PARPi in cancers other than HBOC with BRCA1/2 germline or somatic mutations. For example, for metastatic pancreatic cancer patients with germline BRCA mutations who had not progressed during first-line platinum-based chemotherapy, progression-free survival was longer with maintenance Olaparib than with placebo . There aIn conclusion, our study systematically investigated the characteristics of BRCA1/2 germline mutations in Chinese NSCLC patients, and compared the features of the mutations with those from Chinese HBOC. We found that BRCA1/2 germline mutations in NSCLC had lower carrier frequency than HBOC, potential higher ratio of BRCA1 nonsense mutations and carriers than HBOC, and revealed several novel BRCA1/2 germline mutations that have never been reported in Chinese HBOC."}
+{"text": "Based on experiments conducted since this article\u2019s  publicatIn experiments done after the article was published, the authors observed signals for the negative control indicating that there was unspecific binding to the sepharose beads . FurtherGiven these findings, the result and conclusion statements referring to a physical interaction between BAG3 and HSPB7 are not supported. Specifically:The penultimate sentence of the ZBTB17-HSPB7 locus subsection of the Results: \u201cUsing GST pull-down experiment and co-immunoprecipitation we also observed that recombinant BAG3 interacts with HSPB7 .\u201din vitro experiments demonstrate a physical interaction of BAG3 with HSPB7  suggesting functional relationships between the 2 proteins that may be relevant for their genetic implication in DCM pathophysiology.\u201dThe fifth sentence of the second paragraph of the Discussion section: \u201cIn addition, our The seventh sentence of the second paragraph of the Discussion section: \u201cThe interaction signal of BAG3 Arg151 and Cys151 isoforms with HSPB7 was similar (data not shown), suggesting no direct effect of the polymorphism on HSPB7 binding.\u201dPLOS ONE\u2019s Editorial Board confirmed that the main conclusions of the article stand are not substantively affected by this issue.A member of The authors apologize for the errors in the published article.S1 File(PPTX)Click here for additional data file.S2 File(PPTX)Click here for additional data file."}
+{"text": "A role for phagocytosis in inducing cell death during thymocyte negative selection. Published 23, December 2019Timd4-/- RIPmOVA thymic slices. We had intended to show CD69 induction on RIPmOVA thymic slices treated with Annexin V in Figure 3b, which are the data described in the results as well as the figure legend, and these data are shown in the corrected version.In the original published version of the article, Figure 3b mistakenly showed the same graph as represented in Figure 4b, which shows CD69 induction on The corrected Figure 3 is shown here:The originally published Figure 3 is also shown for reference:Timd4-/-. The left column should be labelled Timd4+/-. The article has been corrected accordingly.In addition, in Figure 5-figure supplement 1b of the original published version, both columns of the graph are labelled The corrected Figure 5-figure supplement 1 is shown here:The originally published Figure 5-figure supplement 1 is also shown for reference:"}
+{"text": "Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana.False-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping.This was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20\u00b0C. P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1\u20132 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1\u20132 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1\u20132 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1\u20132 of the Pfhrp 2 gene.Of the 2,860 microscopically Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.The low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1\u20132 of the  Plasmodium falciparum parasites [The diagnosis of symptomatic malaria is typically based on smear microscopy, the gold standard . Howeverarasites , the cauarasites .Pfhrp 2 gene, which codes for the PfHRP 2 antigen, which both cause false negative RDT test results [Pfhrp 2 gene have been found to react positively with PfHRP 2-based malaria RDTs due to cross-reactivity of PfHRP 2 with PfHRP 3 [Pfhrp 2 [Despite the numerous advantages of malaria RDTs over smear microscopy, a few disadvantages exist. Some disadvantages include prolonged PfHRP2 antigen persistence after parasite clearance, which results in false positive test results as well as the prozone effect  and the  results , 10. Par PfHRP 3 . The PfH[Pfhrp 2 .Pfhrp 2 gene that result in false negative malaria RDT results are detrimental and have the potential of missing malaria positive patients in countries like Ghana where testing is largely by PfHRP2 RDTs. The symptomatic malaria patients who test negative may likely not receive appropriate antimalarial treatment [Deletions in the reatment , 14. To reatment .Pfhrp 2 gene can adversely affect the diagnostic accuracy of PfHRP 2 based malaria RDT kits, the WHO has recommended that countries with a Pfhrp 2 gene deletion parasite population at or above 5% should resort to the use of non-PfHRP 2-based RDT kits [Pfhrp 2 gene deletions in symptomatic malaria infections from high endemic countries such as Ghana is complicated by the fact that the infections are predominantly multiclonal [Pfhrp 2 gene deletions can be masked by the presence of co-infecting parasites with intact Pfhrp 2 genes.Since deletions in the RDT kits . Identifticlonal  and the Pfhrp 2 gene was in the Amazon region of Peru [Pfhrp 2 in Ghana, most of which have been conducted on asymptomatic malaria carriers and also without implementing the WHO recommendations [Pfhrp 2 and or Pfhrp 3 gene that result in false negative PfHRP 2-based malaria RDT results.The first reports of the existence of parasites with deletions in the  of Peru  and has  of Peru , includi of Peru . To datendations , 19. DueEthical approval for this study was obtained from the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana (study # 068/17-18). Written informed consent was obtained from each adult participant (\u226518years old) and caregivers of participants less than 18 years old provided parental consent. Written informed assent was also obtained from all participants aged between 12 and 17 years old. Each consenting or assenting participant was informed of the objectives, methods, anticipated benefits and risks of the study as well as their liberty to withdraw without any penalty.This was a cross-sectional survey among all symptomatic individuals seeking care in selected health care facilities in the ten regions of Ghana between May and August 2018.Pfhrp 2/3 gene deletions [As per the WHO protocol on determining the prevalence of eletions , a total\u00ae tube. The venous blood was tested on the CareStart\u00ae malaria PfHRP 2 RDT kit . Samples were classified as RDT positive if both the control and test lines were positive and RDT negative if only the control line but not the test yielded a positive result.After obtaining written informed consent (for adult participants), parental consent (for children under 18 years old), and assent (for children 12 to 17 years old) the study participants were enrolled into the study. Demographic data was obtained from each participant and entered in KoBoCollect and about 2 ml of venous blood collected into an EDTA vacutainer\u00ae No. 3 filter paper. The blood spots were air dried and stored individually in a Ziplock\u00ae bag with a desiccant. Packed cells were isolated from the remaining blood samples and stored in a 1.5 ml eppendorf\u00ae tube at -20\u00b0C until used.Thick and thin blood smears were also prepared using 6 \u03bcl and 2 \u03bcl of whole blood respectively. Filter paper blood spots were prepared by spotting four 50 \u03bcl drops of whole blood on to WhatmanThe blood smears were processed and then stained with 3% Giemsa for 45 min. The stained slides were subsequently viewed under 100X oil immersion microscope . Two ind\u00ae and positive by microscopy  were sorted out from the total samples collected. Genomic DNA was extracted from the dry blood spots (DBS) using the Chelex extraction method [Samples that were negative by the routinely used CareStart PfHRP 2 RDTn method  and from18S rRNA gene was adapted from Singh et al. [2, 133 nM each of forward (rPLU6) and reverse (rPLU5) primers (18S rRNA gene from 2 \u03bcl of DNA obtained using the Zymo extraction kit or 5 \u03bcl of Chelex extracted DNA (~40 ng) in the primary reaction. The nested PCR was performed using similar concentrations of reagents as in the primary reaction mix; however, rFal1 (forward) and rFal2 (reverse) primers were used to amplify 0.5 \u03bcl of the primary product in P. falciparum identification. The cycling parameters for the primary and nested reactions are listed in P. falciparum (MRA 102G) was used as a positive control and the no template control served as negative control.The nested PCR amplification of the h et al.  with slih et al. . Briefly primers  and 1 U et al. [msp 1 locus was amplified using nested PCR. The primary reaction contained 200 nM each of M1-0F and M1-0R primers in a reaction containing 2 \u03bcl of DNA obtained using the Zymo extraction kit or 5 \u03bcl of Chelex extracted DNA (~40 ng) and 0.1 \u03bcl One Taq DNA polymerase . In the secondary reaction, 1 \u03bcl of the primary PCR product was amplified using 200 nM each of M1-KF+M1-KR , M1- R033R+M1-R033F  and M1-MF+M1-MR  primer sets  of gDNA. Primers G-F4 and GNF (500 nM each) were used in the secondary reaction of amplify 2 \u03bcl of the primary PCR product. The cycling conditions for the primary and secondary PCR reactions included an initial denaturation at 95\u00b0C for 5 min followed by 35 cycles of a denaturation at 94\u00b0C for 30 seconds, primer annealing at 55\u00b0C (for primary) or 58\u00b0C (for secondary) for 1 minute and an extension at 68\u00b0C for 1 minute and a final extension at 68\u00b0C for 5 minutes.The Pfhrp 2 and Pfhrp 3 genes were amplified using the protocol described by Abdallah et al.,[Pfhrp2F1 (forward) and Pfhrp2R1 (reverse) primers for Pfhrp2 exon 2 and Pfhrp3F1 (forward) and Pfhrp3R1 (reverse) primers for Pfhrp 3 exon 2 were used to amplify 2 \u03bcl of DNA obtained using the Zymo extraction kit or 5 \u03bcl of Chelex extracted DNA (~40 ng) using 0.1 \u03bcl OneTaq DNA polymerase  in a 15 \u03bcl reaction containing 1.8 mM MgCl2 and 0.2 \u03bcM dNTP mix. The DNA was initially denatured at 95#x00B0;C for 3 min followed by 35 cycles of denaturation at 94\u00b0C for 30 seconds, annealing at 58\u00b0C (Pfhrp 2 gene) or 55\u00b0C (Pfhrp 3 gene) for 30 seconds and an extension at 68\u00b0C for 1 min. The final extension was performed at 68\u00b0C for 5 min and finally held at 4\u00b0C. Genomic DNA from Dd2 , HB3  and 3D7 , representing Pfhrp 2 deleted, Pfhrp 3 deleted and wild type parasites respectively were used as controls for the PCR amplifications.Exons 2 and 1\u20132 of the h et al., with minPfhrp 2 and Pfhrp 3 genes. The primary PCR reactions were carried out in a 15 \u03bcl reaction volume containing 2 \u03bcl of DNA obtained using the Zymo extraction kit or 5 \u03bcl of Chelex extracted DNA (~40 ng), 0.2 \u03bcM each of forward and reverse primers 2E12F1 and 2E12R1 for Pfhrp 2 and 3E12F1 and 3E12R1 for Pfhrp 3 respectively), 0.133 \u03bcM dNTPmix and 2.5 mM MgCl2 supplemented with 0.1 \u03bcl of OneTaq DNA polymerase . The secondary reactions were amplified using similar reaction conditions as the primary reaction, however, the primer sets were replaced with 2E12F2 and 2E12R2 for Pfhrp 2 or 3E12F2 and 3E12R2 for Pfhrp 3. The templates for the secondary reactions were 2 \u03bcl of a 1:100 dilution of the respective primary PCR products.A nested PCR protocol was used to amplify exon 1\u20132 of the PCR products (10 \u03bcl) were separated on 2.0% agarose gels containing 0.2 \u03bcg/ml ethidium bromide and subsequently viewed under UV illumination using a Bio-Print TX4 gel documentation system . All samples that yielded visible fragments after agarose gel electrophoresis were classified as positive for the particular PCR reaction.3), 50 ml liquid casein, 5 g polyvinyl alcohol, 5 g polyvinylpryrrolidine). Subsequently, 50 \u03bcl of PfHRP 2 and pan-Aldolase coated beads (1:500 dilution of each in elution buffer) was aliquoted into each well of the plate. The samples tested included a 50 \u03bcl aliquot of the test samples and negative control samples from 86 malaria na\u00efve people. The plates were washed three times with wash buffer. Anti-PfHRP 2 and anti-Aldolase (1:500) detection antibodies (50 \u03bcl) were then added to each well of the plate and Incubated for 45 mins with shaking. The plate was subsequently washed three times and then incubated for 30 mins with 50 \u03bcl of streptavidin-phycoerythrin (1:200) with shaking. After the final wash, the plates were read on the Luminex 200 (Luminex Corp. USA). The cutoff value for PfHRP 2 and pan-Aldolase antigen positivity was the lognormal mean fluorescence intensity (MFI) of the 86 malaria na\u00efve samples minus the background (bg) MFI signal \u00b1 3 SD.The Multiplex Bead Assay (MBA) was used to determine the PfHRP 2 and pan-Aldolase levels of the samples . BrieflyThe datasets were captured using KoBoCollect, extracted and cleaned in IBM SPSS version 22. IBM SPSS version 22 was used to generate the descriptive statistics including mean, standard error of the mean and the frequency. Kruskal-Wallis test (GraphPad Prism v5) was used to compare the median Regional parasite densities and the age of the study participants. Pearson\u2019s Chi squared test was used to compare the medians of the Regional distribution of male and female participants at a 5% level of significance.The MFI cut off value for positivity for the PfHRP2 and aldolase luminex data was set at 50 and 20 respectively. Spearman\u2019s correlation (GraphPad Prism v5) was used to determine the correlation between parasite density and parasite antigen  concentrations.Plasmodium parasites were identified in 1% (141/13136) of the samples that tested negative by PfHRP 2 RDT kits , leaving 134 false negative (FN) samples. Unfortunately, neither DBS nor whole blood was found for seven of these samples. The age of the patients with FN results ranged between 1 and 94 years and the age distribution were similar across the 10 Regions . Children aged \u226410 years constituted 22.6% and adults 50 years and above constituted 15.8% of the study population. The distribution of males and females across the Regions were similar , with males representing about 30% of the study population.Microscopy identified the presence of malaria parasites in 2890 of the 18618 samples with valid microscopy results. Microscopic densities of RDT kits . Out of The distribution of people carrying parasites that were identified as FN was nationwide, with the highest prevalence found in the Ashanti Region and the lowest prevalence in the Volta Region . The parWhole blood or filter paper samples were available for 127 out of the 134 samples, unfortunately neither whole blood nor DBS could be found for 7 samples.Plasmodium falciparum species PCR identified 116 out of the 127 FN samples as positive  of the 79 false negative samples contained infections with parasite densities of less than 200 p/\u03bcl (between 76 and 192 p/\u03bcl). All these 7 samples tested negative after 2 exon 2 . The samPfhrp 2 exon 2 gene deletions, only 4 (12.9%) tested negative for Pfhrp 2 exon1-2. Samples with gene deletions in both Pfhrp 2 exon 2 and Pfhrp 2 exon 1\u20132 were found in only three Regions, the Ashanti, Brong Ahafo and the Central, with the Central Region having the highest prevalence  of samples with deletions in both Pfhrp 2 exon 2 and exon 1\u20132  of the samples with The PfHRP 2 antigen was absent  in 32.9% (26/79) of the FN samples, whilst the aldolase antigen was absent  in 38% (30/79) of the FN samples. There were 27.8% (22/79) of the FN samples that were negative for both PfHRP 2 antigen and aldolase.About 68% (48/71) of the samples that had parasite densities >200 p/\u03bcl tested positive by aldolase luminex. Parasite density exhibited a significant strong positive correlation  with aldolase concentrations and a significant but weak correlation  with PfHRP 2 antigen concentration.Pfhrp 2/3 gene deletions amongst samples that tested positive for aldolase by luminex were predominant in the Ashanti Region and Northern Region  and the South Eastern (Volta and Greater Accra) parts of the country. The low number of false negative RDT results identified in this study is lower than 8.4% and 54% that have been reported in Uganda and Benin respectively . Low parPfhrp 2/3 gene deletions in samples from symptomatic malaria patients. Parasites with Pfhrp 2 exon 2 gene deletions accounted for about a third of the false negative samples. Parasites with Pfhrp 2/3 gene deletions were identified in all but one of the Regions in Ghana, the Eastern Region where no sample was identified to contain parasites with deletions in exon 2 of the Pfhrp 2 gene. All parasites with deletions in Pfhrp 2 exon1-2 also had deletions in Pfhrp 2 exon 2 and were mainly identified in 3 neighboring Regions in the lower mid-section of western Ghana. The pattern of distribution of parasites with deletions in both exon 2 and exon 1\u20132 of the Pfhrp 3 gene were like that of the Pfhrp 2 gene. Deletions at exon 2 were three times more prevalent than at exon 1\u20132 for both the Pfhrp 2 and Pfhrp 3 genes. The observed higher prevalence of deletions in exon 2 than exon 1\u20132 could be a true reflection of parasites with exon 2 gene deletions being more abundant than parasites with exon 1\u20132 gene deletions as has been reported in other studies such as an earlier report from India where parasites with Pfhrp 2 exon 2 gene deletions were at higher frequencies than parasites with Pfhrp 2 exon 1\u20132 gene deletions [Pfhrp 2 exon 2 PCR results, some studies have identified samples with higher frequencies of deletions in Pfhrp 2 exon 1\u20132 than Pfhrp 2 exon 2 [This study identified parasites with eletions . The vareletions , 38. Des2 exon 2 .Pfhrp 3 genes have been found to compensate for the absence of Pfhrp 2 exon 2 in a parasite, especially at high parasite densities and result in a positive PfHRP 2 based malaria RDT test due to the cross-reactivity of PfHRP 3 antigen with the PfHRP 2 antibodies on the RDT kits [Pfhrp 3 exon 2 gene and most likely the corresponding PfHRP 3 antigen in circulation in the false negative samples identified in this study did not result in a positive PfHRP 2 RDT result, even though some of the samples had parasite densities of greater than 1000 parasites per microlitre. The absence of PfHRP 3 antigen compensation has been observed in other studies including one from Kenya [Parasites with intact RDT kits , 40. Howom Kenya . PfHRP 3Pfhrp 2 exon 2 or due to the presence of some parasite isolates producing very low concentrations of aldolase. The prozone effect could have been one of the reasons why samples containing high concentration of PfHRP 2 antigen tested negative by the PfHRP 2 based RDT kit as has been suggested in an earlier report [Despite the high positive correlation between parasite density and aldolase activity, about 38% of the false negative samples had aldolase levels below the cut off for positivity, which suggests that some parasites isolates produced very low aldolase activity. The presence of PfHRP 2 antigen in the false negative samples that had no aldolase activity could have resulted from persisting PfHRP 2 antigen from a recently cleared infection containing parasites with intact r report .Pfhrp 2 exon 2 gene deletions. The confirmation of parasites with Pfhrp 2 gene deletions amongst symptomatic malaria patients with false negative RDT results is detrimental to malaria control in Ghana, especially in communities and facilities where malaria diagnosis is solely based on RDT results. The prevalence of parasites with deletions in the Pfhrp 2 gene circulating in symptomatic malaria patients could be more than identified in this study as only parasites contained in false negative samples were evaluated as opposed to all microscopy positive samples. Overall, the low prevalence of samples classified as false negative resulted in only a small fraction of the samples collected analyzed for Pfhrp 2 gene deletions.There were about a third of the aldolase positive false negative RDT results that were caused by parasites with Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance and monitoring of parasites with Pfhrp 2 gene deletions.The low prevalence but nationwide distribution of samples with false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in both exon 2 and exon 1\u20132 of the S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file."}
+{"text": "Plasmodium falciparum infections. Numerous genotypes of haptoglobin have been reported in malaria endemic populations. In this study, the relationship between haptoglobin genotypes and incidence of uncomplicated malaria in a cohort of children living in a malaria-endemic area of Uganda was determined.Haptoglobin (Hp) is an acute phase protein that takes part in systemic regulation of haem during This is an extension of a longitudinal study comprising of 423 children aged between six months and nine years, who were actively followed up for one year. Malaria episodes occurring in the cohort children were detected and the affected children treated with national policy drug regimen. Haptoglobin genotypes were determined by an allele-specific PCR method and their frequencies were calculated. A multivariate negative binomial regression model was used to estimate the impact of haptoglobin genotypes on incidence of uncomplicated malaria in the children\u2019s cohort. In all statistical tests, a P\u2013value of\u2009<\u20090.05 was considered as significant.The prevalence of the Hp 1\u20131, Hp 2\u20131 and Hp 2\u20132 genotypes in the children\u2019s cohort was 41%, 36.2% and 22.9%, respectively. The overall frequency for the Hp 1 allele was 59%, while Hp 2 allele occurred at a frequency of 41%. After adjustment of incidence rates for age, insecticide treated bed net (ITN) use and malaria history, the incidence of uncomplicated malaria for children carrying the Hp 2\u20132 genotype and those with the Hp 2\u20131 genotype was statistically similar (P\u2009=\u20090.41). Also, no difference in the incidence of uncomplicated malaria was observed between children carrying the Hp 1\u20131 genotype and those having the Hp 2\u20131 genotype (P\u2009=\u20090.84) or between Hp 2\u20132 Vs Hp 1\u20131 genotypes (P\u2009=\u20090.50).P. falciparum malaria severity are needed to understand the role of these genotypes in malarial protection.This study showed that the Hp 1\u20131 and Hp 2\u20131 genotypes each occur in nearly 4 in 10 children and the Hp 2\u20132 genotype occurs in 2 of every 10 children. No association with incidence of uncomplicated malaria was found. Additional studies of influence of haptoglobin genotypes on  Plasmodium falciparum malaria infections involves interplay of both parasite and host factors such as host genetic variability . About 88.2% of the participants\u2019 guardians (373 / 423) reported owning and using an insecticide treated bed net (ITN) within their households. Four hundred and three participant\u2019s guardians (95.3%) reported having ever administered an anti-malarial drug to the enrolled child. Throughout the 1-year of longitudinal follow up in this study, malaria episodes were not registered among 217 out of 423 children (51.3%). Among those who experienced malaria episodes  during the 1\u00a0year of follow up, the range of annual episodes per child was 1 to 9.Haptoglobin genotyping was performed successfully for 398 samples by determining the presence or absence of 935\u00a0bp, 1.2\u00a0kb and 1.4\u00a0kb DNA fragments corresponding to the Hp2, Hp1S and Hp1F genotypes, respectively. The Hp1-1, Hp2-1 and Hp2-2 genotypes were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency for the Hp1 allele was 59%, while Hp2 allele occurred at an allele frequency of 41%. The distribution of the Hp genotypes in the study cohort is presented in Table P. falciparum infection, were included in the final multivariate negative binomial regression model to determine the extent to which they were affected by the haptoglobin genotype after adjusting for other independent determinants of malaria incidence identified in an earlier study in the same children\u2019s cohort [This study is an extension of a longitudinal study carried out previously on the same cohort of children . In thiss cohort ; namely There was no statistically significant difference in the distribution of the Hp genotypes among children who did and did not experience uncomplicated malaria throughout the year as shown in Table P. falciparum infection in Dogon, but not in Fulani tribe [Haptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. However, no association with incidence of uncomplicated malaria was found. The present findings differ from results of prospective cohort studies carried out among Kenyan children, in which the Hp 2\u20132 genotype was associated with lower incidence of clinical malaria , 14. In ni tribe . The finP. falciparum infections [P. falciparum malaria severity are needed to further understand the role of these genotypes in malarial protection.In addition, Hp genotypes have been shown to influence plasma Hp levels , 10 needfections , 25. Thifections , 17. AddThe Hp1 allele was present at an allele frequency of 59%, yet no influence on the incidence of uncomplicated malaria by the Hp1 allele was observed. This is also in line with some earlier studies that found no clear associations between the Hp 1 allele and malaria susceptibility , 16. InsHaptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. Association of Hp genotypes with incidence of uncomplicated malaria was not observed. This lack of detectible association with uncomplicated malaria suggests that these alleles may have been maintained by protection from other infections. Similar studies in different settings are needed to confirm these findings."}
+{"text": "After this article  was publpCDNA3.1 and NSP4 panels in Fig 3AMID and C157 panels in Fig 5CThe authors noted that due to figure preparation errors the pCDNA3.1 and MID dot plots were mistakenly duplicated in the NSP4 and C157 panels, respectively. These errors are addressed in the revised Figs PLOS ONE\u2019s Editorial Board advised that the updated figures support the results as reported in the original article.A member of The original data are available upon request from the corresponding author for Figs 1B, The authors apologize for the errors in the published figures."}
+{"text": "Genetically engineered murine models involving chromosomal translocation genes have been generated using transgenic approaches or gene knockin.4 However, the closest model is the creation of bona fide chromosomal translocations, which recapitulate the physiologic levels of the fusion genes, the reduced dosage of the wild-type alleles and the contribution of the reciprocal products of the translocations. This has been achieved in vivo using the Cre-loxP system in embryonal stem cells,5 which is labor intensive and time consuming. The CRISPR/Cas9 system is a versatile tool for genomic engineering. Transduction of target cells with vectors encoding Cas9 and sgRNAs targeting two genomic loci causes two DNA double strand breaks (DSBs), which could induce chromosomal rearrangements as a result from non-homologous end joining-based DNA repair.6 This strategy has been used to model oncogenic chromosomal rearrangements in murine10 or human11 cells in vivo. However, given the challenges of delivering CRISPR/Cas9 vectors into hematopoietic cells, the penetrance of leukemogenesis has been shown with low frequency in vivo.11 Here we establish an efficient methodology to generate chromosomal translocations in mice hematopoietic cells based on a vector design enabling convenient lentiviral delivery of multiple sgRNAs to hematopoietic cells from a Cas9 expressing mouse strain.A wide range of chromosomal rearrangements have been identified in human cancers especially in leukemia.KMT2A (MLL) gene constitutes a hotspot for chromosomal translocations in human leukemia, resulting in over 135 different leukemia-associated KMT2A gene fusions.2KMT2A-MLLT1 defines a leukemia subtype with poor outcome. This translocation frequently constitutes the sole genomic lesion to drive the leukemia,12 providing a good model to establish this approach. We designed a strategy to induce Kmt2a-Mllt1 translocations in murine hematopoietic cells using the CRISPR/Cas9 system followed by transplantation into lethally irradiated recipient mice  with special designs: (1) The small size of the backbone with minimum interval sequences between each modules improves the virus titer; (2) The isocaudomer sites flanking the sgRNA cassettes facilitates the combination of multiple single sgRNAs from individual vector into one vector . We firstly cloned several individual sgRNAs targeting the equivalent introns of Kmt2a and Mllt1 that are orthologue regions in the human leukemia translocation . Transduction of this vector coding two sgRNAs in NIH 3T3 cells generated the Kmt2a-Mllt1 translocation efficiently .The 13 we crossbred the Cas9 mice with the inbred C57BL/6 mice and used the F1 progenies (Cas9GFP+/\u2212) as the bone marrow donors. Given the C57BL/6 haploidentical setting, these cells can reconstitute the hematopoietic system in lethally irradiated C57BL/6 recipient mice without inducing graft-versus-host disease. Transduction of these bone marrow cells with the sgRNA vector (sg_ K-M) lead to a detectable genomic rearrangement between the Kmt2a and Mllt1 locus at 4 days after transduction in ex vivo cultures  but not for lymphoid markers  or progenitor markers   in leukemia cells was confirmed by FISH using pan-chromosome probes Fig. E. The KmTCF3-HLF translocation defining a highly aggressive leukemia subtype for which so far no representative mouse model could be engineered.15 Given the frequent PAX5 heterozygous deletions and reduced gene expression identified in this leukemia,16 we tested the hypothesis if the Tcf3-Hlf translocation could induce a mouse leukemia when established in a Pax5 haploinsufficient context . We crossbred the Cas9 mice with the Pax5 heterozygous mice 17 and used the F1 progenies (Cas9GFP+/\u2212 Pax5+/+ or Cas9GFP+/\u2212 Pax5+/\u2212) as the bone marrow donors. Transduction of the bone marrow cells with validated sgRNAs targeting the equivalent introns of mice Tcf3 and Hlf loci (sg_T-H) lead to detectable Tcf3-Hlf translocation and the expression of the fusion product . The generation of the Tcf3-Hlf translocation was less effective than the Kmt2a-Mllt1 translocation, with \u223c0.16% of the cells detected by ddPCR to harbor the translocation .We next attempted to model the Tcf3-Hlf translocation was detected in genomic DNA from peripheral blood and bone marrow at 4 weeks after transplantation . However, no progression to leukemia could be detected even after a follow-up for up to 11 months. Post-mortem examination did not reveal any abnormality of spleen, kidney, liver and bone marrow cells and the Tcf3-Hlf translocation signal was undetectable in bone marrow cells . This suggests a selective disadvantage of cells carrying Tcf3-Hlf in vivo, confirming similar observations made with a knockin model of Tcf3-Hlf.18 Thus, despite the frequency of PAX5 haploinsufficiency in TCF3-HLF positive patients, deletion of Pax5 does not counteract the negative selection pressure by Tcf3-Hlf. Notably, the Kmt2a-Mllt1 myeloid leukemia can be established in the Pax5\u200a+/\u2212 background with similar dynamics as in the Pax5\u200a+/+ background confirming that a reduced Pax5 gene dosage does not influence the leukemogenic activity of Kmt2a-Mllt1 .After transplantation of the engineered cells into lethally irradiated C57BL/6 recipients, the Tcf3-Hlf was not sufficient to induce leukemia in this study the model can now be used to evaluate different cells of origin and to screen for required cooperative genetic events. Our approach can be employed to model any pair of genes as long as the orientation to the centromere does not result in dicentric chromosomes. Using the inbred Cas9 mouse strain will further simplify this approach. Besides, our vector design facilitates the combination of multiple sgRNAs to introduce additional oncogenic lesions . Thus, our strategy provides an efficient way to generate precise translocations, enabling functional modeling in mice and constitutes a framework to study the role of cooperative events and the cells of origins in leukemogenesis.Activation of oncogenes by fusion or juxtaposition to ectopic regulatory elements constitutes important cancer initiating events. Here, we established an efficient strategy using the CRISPR/Cas9 system to generate leukemogenic chromosomal translocations in vivo. We generated two types of translocations in bone marrow cells with frequencies of 0.6% and 0.16%. Although the generation of"}
+{"text": "Journal of Oncology and to the readership for any inconvenience caused. The corrected figure and legend are presented here.In the article titled \u201cUBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells\u201d ["}
+{"text": "FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids.Two transcribed retrocopies of the fibroblast growth factor 4 ( Gene retrocopies, often previously referred to as processed pseudogenes, are formed through the mRNA-mediated gene duplication of cellular gene transcripts . In mammRetrocopies are more likely to come from highly expressed genes , with soFGF4) retrocopies have been described previously in dogs on chr18 [FGF4L1 (CFA18) and FGF4L2 (CFA12) in this study. Both FGF4L1 and FGF4L2 are associated with forms of disproportionate dwarfism that are common across many popular dog breeds, and there is evidence that these genes have been under selection owing their strong phenotypic effects [FGF4L2 has also been associated with canine chondrodystrophy, a disorder characterized by premature degeneration of the intervertebral discs, which predisposes affected dogs to intervertebral disc herniation [FGF4L2, indicating the possibility of alternative risk loci for the disorder [Two expressed, polymorphic fibroblast growth factor 4 gene  to identify the presence of an intron-less retrocopy, followed by inverse PCR to identify the site of insertion. Five additional FGF4 retrocopies were then identified, sequenced, and characterized.Because two recent, functional FGF4 retrocopies, and thus all reads coming from FGF4 retrocopies are aligned to the parental FGF4 gene locus. To identify any such novel FGF4 retrocopies, aligned paired end Illumina sequence data in the region surrounding the FGF4 gene  were downloaded from the Sequence Read Archive and analyzed. Sequencing files were viewed in Integrative Genomics Viewer [FGF4 gene retrocopy somewhere in the genome as retrocopies lack introns, while discordant paired end reads, wherein one mate maps to the FGF4 gene locus and the other mate maps to another region of the genome, are indicative of the putative insertion site for an FGF4 retrocopy  were utilized for this approach ,23,24,25s Viewer . DiscordFGF4 retrocopies in DNA samples. A total of 164 surgical cases that were previously shown to have 0 copies of FGF4L1 and FGF4L2 were used for novel FGF4 retrocopy discovery [FGF4 retrocopies was tested by amplifying the region between exon 1 and exon 3 of FGF4 , and fragments were then circularized by ligation at final concentrations varying between 1 and 10 ng/\u03bcL using T4 DNA ligase according to the manufacturer\u2019s instructions for sticky end ligation . A set of inverted primers were designed that amplified circular DNA fragments containing the 5\u2032 end of the FGF4 retrocopy insertion (http://bioinfo.ut.ee/primer3/) [Whole-genome sequence data were not available for any of the individuals treated by surgical decompression for presumed IVDD. Therefore, a molecular approach was developed to test for novel iscovery . The pre of FGF4 . The ideetrocopy . When anerse PCR  was thennsertion . PCR wasrimer3/) .FGF4 retrocopies came from the Bannasch Canine Repository and were obtained under UC Davis Animal Care and Use Committee protocol 18,561 [FGF4 retrocopies identified via discordant paired end reads or inverse PCR (FGF4 gene were observed in a dataset of 722 canids to determine which single-nucleotide variants (SNVs) were unique to FGF4 retrocopies [All canine DNA samples used for retrocopy sequencing and subsequent population genotyping of the l 18,561  (Supplemerse PCR . Entire erse PCR . Variantrocopies .FGF4 retrocopy insertion sites were defined using the 4-Way Multiz Alignment & Conservation track for CanFam2 on the UCSC genome browser [Evolutionarily conserved elements (ECR) near the  browser , which s browser .FGF4 retrocopy for population genotyping, as previously described [FGF4 retrocopy produces a different size amplicon when the retrocopy is present . Multivariable linear regression was performed in R studio using the generalized linear model function with sex and FGF4 genotype included to identify any association with height.Height was measured in selected cases to determine if FGF4 retrocopies, FGF4L1 and FGF4L2, evidence for four additional FGF4 retrocopies in canids was observed in the whole-genome sequence dataset (FGF4 retrocopies include a copy on CFA27 (FGF4L3) seen in three Nova Scotia Duck Tolling Retrievers (NSDTR); a copy on CFA22 (FGF4L4) seen in two Norwich Terriers; a copy on CFA13 (FGF4L5) seen in a Belgian Malinois and a Dutch Shepherd; and a copy on CFA36 (FGF4L6) seen in two red wolves. Sequence read archive (SRA) accession numbers for these individuals are available in In addition to the two known  dataset . The novFGF4 gene locus aligning to a partial FGF4 retrocopy insertion in the CanFam3 reference genome at chr7:68,372,263\u201368,373,442. To confirm whether this was a real FGF4 retrocopy fragment or a mistake in the reference assembly, primers were designed flanking the insertion and the region was amplified in six Boxers. Five were heterozygous for the CFA7 partial FGF4 retrocopy insertion, and Sanger sequencing confirmed the sequence matched the reference genome. Because this retrocopy only contains the 3\u2032 UTR of the gene and has no ORF, this retrocopy fragment was not considered for further analysis.Discordant reads were also observed at the 3\u2032 end of the FGF4L1 nor FGF4L2 was then tested for the presence of any FGF4 retrocopy using an exon\u2013exon PCR assay. Four of these individuals tested positive for the presence of an FGF4 retrocopy. These samples were first tested for the other newly discovered FGF4 retrocopies. One sample, a Shetland Sheepdog, was heterozygous for FGF4L5. The medical history of this individual indicates that it received a hemilaminectomy to treat a mass that was not disc-related.A surgically treated population of 164 individuals that had neither FGF4 retrocopies were present in these individuals, indicating they contained a novel FGF4 retrocopy. Inverse PCR was then performed to discover the insertion site of the novel FGF4 retrocopies in these individuals, which was on CFA13 (FGF4L7) at approximately CFA13:25,020,600. The three dogs were all heterozygous for FGF4L7, and Sanger sequencing revealed that FGF4L7 is a full length FGF4 retrocopy.The three remaining dogs were all Pit Bull Terrier mixes that had received hemilaminectomies for IVDD at relatively young ages , and none of the newly discovered or previously defined FGF4 retrocopies were confirmed through PCR amplification and sequencing. The genomic location for the FGF4 retrocopies, their TSD, and genomic sequence surrounding the TSD are shown in FGF4L5 insertion site. Insertion sites for 6/7 of the FGF4 retrocopies had a low G/C content compared with the Canfam3 average of 41.3% . The number of evolutionarily conserved regions within 2.5 kb of the insertion sites is also reported in Novel of 41.3% . All FGFFGF4 retrocopy to the parental FGF4 sequence showed that each novel copy has a fully conserved ORF  were identified in either the ORF or the 5\u2032 UTR of any of the retrocopies. However, six SNVs were identified in the 3\u2032 UTR that differed from the reference genome FGF4 gene sequence. Analysis of a whole-genome sequencing variant calling dataset from 722 canids indicated that these SNVs are also present at the parental FGF4 gene  and an insertion . These indels were not identified in any canids other than the two red wolves in a whole-genome sequencing variant calling dataset, which included 46 gray wolves. The parental FGF4 locus was sequenced in seven red wolves to determine whether these variants also exist in the parental gene in red wolves or if they are unique to the retrocopy. While three individuals were heterozygous for the CFA18:48,416,575T>TA insertion at the parental FGF4 gene, CFA18:48,415,685CA C was not identified in any of the parental FGF4 sequences, indicating this variant may have occurred after retrotransposition and may be unique to FGF4L6.The red wolf FGF4 retrocopies were identified was utilized to determine allele frequencies of the FGF4 retrocopies. A complete list of FGF4 retrocopy genotyping results is available in FGF4L3 was only observed in the NSDTR breed in the whole-genome sequencing dataset, and was thus tested in 100 randomly selected NSDTR. The allele frequency of FGF4L3 was 8.5% in the NSDTR.A targeted population genotyping approach based on the breeds in which FGF4L4 had an allele frequency of 16.7% in Norwich Terriers (n = 30). Further testing for FGF4L4 in related terrier breeds identified this retrocopy in Norfolk Terriers , Border Terriers , and Skye Terriers . Given the previous association of FGF4 retrogenes with skeletal dysplasia, FGF4L4 genotype was also compared to height at the withers in 24 Border Terriers using multiple linear regression. The regression analysis identified no significant association between FGF4L4 and height in Border Terriers , although only one homozygous wild type individual was included (included .FGF4L5 was not identified in any other Shetland Sheepdogs (n = 58) or Belgian Malinois (n = 14). Australian Shepherds (n = 19) and Anatolian Shepherd dogs (n = 5) also tested negative for FGF4L5. No additional Dutch Shepherd samples were available for population genotyping of FGF4L5 in the breed. FGF4L6 was tested in 14 red wolf samples, 5 of which were heterozygous .FGF4L7 (n = 201), and all tested negative for the retrocopy. Because FGF4L7 was identified in dogs treated for IVDD and could be contributing to the disorder, all mixed breed dogs from the Bannasch Canine Repository that had been treated surgically for IVDD were also tested for FGF4L7 (n = 55), all of which tested negative. However, two discordant reads mapping to the FGF4L7 were subsequently identified in the whole-genome sequence data of a single Chinese village dog (SRR7107669). Several breeds developed in Asia were then tested for FGF4L7, including Chow Chow (n = 22), Pugs (n = 9), Pekingese (n = 8), and Tibetan terriers (n = 6), none of which tested positive. However, FGF4L7 was identified in Chinese Shar-Pei .Pit Bull Terriers and Pit Terrier Mixes were then tested for FGF4 retrocopies exist in canids in addition to the previously identified FGF4L1 and FGF4L2. Novel retrocopies appear to be breed or breed group specific, contain intact ORFs, and have not accrued mutations that differentiate them from parental FGF4 gene haplotypes. The FGF4 retrocopies were retrotransposed from FGF4 genes with distinct haplotypes, indicating that the same copy has not been retrotransposed multiple times. It is unclear whether any of these novel copies are expressed retrogenes, or in what tissue types they could be expressed. FGF4L7 was identified in three dogs treated surgically for IVDD, however, the significance relative to IVDD is unknown. The majority of IVDD surgical cases examined in this study that were not explained by FGF4L2 were found to have no FGF4 retrocopies, indicating that there are risk factors other than FGF4 retrocopies that predispose dogs to IVDD.Multiple recently transposed FGF4L1 and FGF4L2 has indicated that the FGF4 retrocopies are capable of expression [FGF4 gene is GC-rich and contains many evolutionarily conserved transcription factor binding sites that were previously hypothesized to be conducive towards expression of the retrocopies [FGF4L3 retrocopy likely affects expression. It has also been reported that the expression of retrocopies is highly dependent on the genomic environment of the insertion sites [FGF4L1 and FGF4L2 have inserted into regions containing nearby evolutionarily conserved elements (ECRs). Similarly, ECRs at all but one of the FGF4 retrocopy insertion sites may be conducive towards expression. The different genomic context at the insertion sites for FGF4L1 and FGF4L2 could also explain the different phenotypes between the copies. If expressed, the novel FGF4 retrocopies may show unique expression profiles, resulting in phenotypic associations other than height and IVDD. FGF4 is involved in several cellular processes including cell growth, tissue repair, tumor growth and invasion, and is also a well-known proto-oncogene [Evidence for the expression of both pression ,15. The on sites . Both FGoncogene ,33.FGF4L2 has been shown to have a major association with IVDD [FGF4L2 retrogene, implicating alternate causative factors [FGF4 retrogenes are logical candidates for these FGF4L2 negative IVDD cases, however the additional FGF4 retrocopies identified in this study do not appear to provide a compelling explanation for this group of dogs owing to the limited frequency of the retrogenes in affected animals. Although FGF4L7 was identified in three dogs treated surgically for IVDD, it was not seen in any other breeds in the surgically treated data set, and the breed with the highest identified allele frequency  is not known to be among the breeds highly predisposed to IVDD [FGF4L4 retrogene. Interestingly, FGF4L7 inserted 5 Mb downstream from the HAS2 gene, a gene that has been implicated in the Shar-Pei wrinkled skin phenotype as well as Familial Shar-Pei Fever [FGF4L7 in the breed.Although ith IVDD ,19,34, c to IVDD . Similarei Fever . Strong FGF4L4 was not found to be associated with height in Border Terriers, the majority of Border Terriers tested had either one or two copies of FGF4L4, and only one individual with 0 copies was identified. If the retrocopy has a dominant effect on height in the breed, more homozygous wild type individuals will need to be measured to determine any effect. FGF4L4 was also found at low allele frequencies in other related terrier breeds, including the Skye, Norwich, and Norfolk Terriers, and may have originated in a common progenitor to the terrier breed group. As dog breeds are known to be highly inbred [FGF4L1 is also very common in Norwich and Norfolk Terriers [FGF4L1 and FGF4L2, making them the first breed to be identified with three FGF4 retrocopies.While y inbred ,38, a hiTerriers , and theFGF4 retrocopies in canids appear to have been very recently retrotransposed with no new mutations differentiating them from the parental FGF4 gene. Even the red wolf FGF4 retrocopy, FGF4L6, is nearly identical to the red wolf specific FGF4 haplotype. Dating the FGF4 retrocopy insertions is difficult owing to their short length (3.2 kbp) and sequence identity to the parental gene sequence; however, the FGF4 retrocopies are identical to canine-specific FGF4 gene haplotypes, which are distinct from modern wolf FGF4 haplotypes  and a hedgehog (Echinops telfairi) also have FGF4 retrocopies, although they are only 61.2% and 90.6% identical to the parental genes, indicating they are not recent [FGF4 retrocopies not found in their reference genomes. Another possibility is that L1 mediated gene retrotransposition in general is occurring more frequently in canids. If this was the case, recent, polymorphic retrocopies may be more common in canids in a greater number of genes than just FGF4.All the plotypes . This cotal gene ,39. HoweFGF4L1 and FGF4L2, retrocopies of other genes may have phenotypic consequences. As such, the possibility of retrocopy insertions should be considered when scanning critical intervals for disease trait associations. Recently inserted gene retrocopies can result in overexpression of the parental gene product, resulting in gain of function, which could be deleterious [FGF4 gene; a similar approach could be generalized to all genes to identify other polymorphic gene retrocopies in canids. Similar to the FGF4 retrocopies, other polymorphic retrocopies may play an important role in both breed health and phenotypic variation across dogs.While next generation sequencing allows for the detection of polymorphic gene retrocopies, they often go unidentified or misidentified by common variant calling methods . Howevereterious . In this"}
+{"text": "The voltammogram of [(Cp2Ti)3HATN(Ph)6] shows six oxidation and three reduction waves. Solution spectra of [(Cp2Ti)3HATN(Ph)6] and of the electrochemically formed oxidation products show electronic transitions in the UV, visible and the NIR ranges. Density functional theory (DFT) and linear response time\u2010dependent DFT show that the three formally titanium(II) centers transfer an electron to the HATN ligand in the ground state. The optically excited transitions occur exclusively between ligand\u2010centered orbitals. The charged titanium centers only provide an electrostatic frame to the extended \u03c0\u2010electronic system. Complete active self\u2010consistent field (CASSCF) calculation on a structurally simplified model compound, which considers the multi\u2010reference character imposed by the three titanium centers, can provide an interpretation of the experimentally observed temperature\u2010dependent magnetic behavior of the different redox states of the title compound in full consistency with the interpretation of the electronic spectra.Multinuclear transition metal complexes bridged by ligands with extended \u03c0\u2010electronic systems show a variety of complex electronic transitions and electron transfer reactions. While a systematic understanding of the photochemistry and electrochemistry has been attained for binuclear complexes, much less is known about trinuclear complexes such as hexaphenyl\u20105,6,11,12,17,18\u2010hexaazatrinaphthylene\u2010tristitanocene [(Cp More complex?! The ground state of the three\u2010nuclear [(Cp2Ti)3HATN(Ph)6] complex is formed by spin states of different multiplicity and explains the rich electronic transition in the UV\u2010vis\u2010NIR ranges in different charge states. Contrary to common notion, IVCT transitions do not play a role in the NIR spectra. All steps are one electron processes (Table\u20052Ti)3HATN(Ph)6] allows the observation of even one more redox transition in the accessible potential range than for the previously reported related compoundThe voltammogram of [3HATN(Ph)6] and its oxidation and reduction products [(Cp2Ti)3HATN(Ph)6]3\u2212, [(Cp2Ti)3HATN(Ph)6]2\u2212, [(Cp2Ti)3HATN(Ph)6]1\u2212, [(Cp2Ti)3HATN(Ph)6]1+ and [(Cp2Ti)3HATN(Ph)6]2+ show electronic transitions in the UV, vis and NIR spectral ranges , transitions from metal centered d\u2010type orbitals to ligand orbitals (MLCT) and of course transitions between purely ligand centered orbitals (in the visible region mainly \u03c0\u2010\u03c0*). In multinuclear complexes transitions between d\u2010type orbitals of different metal centers in different valence states can be observed as a fifth type of electronic transition, commonly referred to as IVCT. However, an IVCT is only possible in systems with extensive state mixing involving two d orbitals from the metal centers and one ligand orbital which provides strong electronic coupling between the metal centers.Different types of electronic transitions can be observed in transition metal complexes. In common literature they are usually visualized by molecular orbital (MO) diagrams, where MOs are combined from ligand orbitals Figure\u2005a and met6 ligand at least one electron is supposed to be transferred from the three Cp2Ti centers to the ligand. The cyclic voltammetric data of HATN(Ph)6 in Figure\u20056]2\u2212 was unstable in the electrolyte solution. The combined magnetic and electrochemical data made the ground state plausible with a negatively charged ligand, although the exact number of transferred electrons cannot be ascertained with the available methods.Experimental magnetic moments of the solid compound at elevated temperature suggest the existence of more than two unpaired electrons in the ground state of the neutral complex.The measured spectra displayed in Figure\u2005not provide a full explanation for the spectra of the oxidized species, i.\u2009e. there is disappearance of the expected CT bands in experimental NIR spectra. For the HATN complexes, this is not surprising because the applicability of the single electron picture and the appropriate definition of the ground state require more detailed quantum chemical calculation as provided in Sections 2.3. to 2.5. of this paper. For [(Cp2Ti)3HATN(Ph)6] studied here, one must suspect appearance of multiple spin states. Those scenarios prevent a meaningful applicability of a single electron picture in addition to the anyway very crude assumptions required to condense electrochemical and spectroscopic data in one picture. Therefore, we strongly discourage to derive theoretical quantities such as orbital energies from experimental data in case high quality quantum chemical calculations are available.While this assignment seemed plausible at first sight, it can 2.33h symmetry two degenerate HOMOs can be found 3HATN(Ph)6]. Generally, the bent titanocene(II) fragment is often used in the preparative chemistry in order to introduce electron transfer processes as starting point for a broad range of subsequent reactions.2Ti leaves this transfer as a valid option.Each Cpe Figure\u2005b. The el2Ti)3HATN(Ph)6] in the NIR range  give first insights into the excitations observed in the experimental NIR spectra. When the HATN molecule is charged, the calculation reproduces similar electronic excitations as found for the . In this compound three electrons were transferred to the ligand and caused absorptions at 800\u2005nm and 920\u2005nm. Calculation on the TD\u2212B3LYP level confirmed that the observed spectra resulted from transitions from the highest occupied molecular orbital (HOMO) and a single occupied molecular orbital (SOMO) to the lowest unoccupied molecular orbital (LUMO). In this model compound the assignment was clear because no transition metals and d\u2010orbitals were involved in the coordination. However, up to six electrons of three TiCp2 complexes are available for a possible charge transfer in [(Cp2Ti)3HATN(Ph)6]. This significantly increases the complexity of the system comared to the system studied by Moilanen et\u2005al.This interpretation has precedence in literature. Moilanen et\u2005al.2Ti)3HATN(Ph)6] more precisely, four spin multiplicities  were calculated for the neutral species with PBE0. All multiplicities except S=3 show a strong spin contamination . The spin states S={0, 1, 2} are also energetically degenerate. The mixing of higher spin states leads to assumption of a strong multireference character produced by the titanium centers, which is reasonable for such a highly symmetrical system. As reported in the literature, high spin contamination of the ground state wavefunction can lead to even higher spin contamination in the excited states and therefore lead to prediction of unphysical excitations.In order to investigate the ground state of [(Cp2Ti)3HATN(Ph)6] can still be calculated with sufficient accuracy using TD\u2010DFT in the spin states S=1 and S=2. Also, the spin states S=0 and S=3 are energetically very close to each other but show no electronic excitations in the NIR range. Table\u2005However, the experimental spectrum of [3HATN(Ph)6], i.\u2009e. before any electronic excitation. Consequently, the titanium atoms are not involved in the electronic excitation process, but provide a charged frame for the excitation processes on the HATN(PH)6 ligand. Despite the good agreement of the theoretical excitations with experiment, the results are affected by spin degeneracy and spin contamination and therefore should be interpreted with caution. Both attributes indicate an intrinsic multi\u2010reference character. This character is caused by the titanium atoms and is not required to reproduce the observed NIR spectrum (cf. Section\u20052.5).Looking at the structure of the orbitals involved in the excitation process , it becomes clear that only ligand\u2010ligand excitations are observed. As hypothesized above, a charge transfer from the titanium atoms to the ligand takes place in the ground state of . All electron transfers are one electron processes clearly separated on the potential scale. The solution spectra of [(Cp2Ti)3HATN(Ph)6] and its oxidation and reduction products [(Cp2Ti)3HATN(Ph)6]3\u2212, [(Cp2Ti)3HATN(Ph)6]2\u2212, [(Cp2Ti)3HATN(Ph)6]1\u2212, [(Cp2Ti)3HATN(Ph)6]1+ and [(Cp2Ti)3HATN(Ph)6]2+ show electronic transitions in the UV, vis and NIR spectral ranges. DFT calculations on TD\u2010DFT and CASSCF/NEVPT2 levels show that three electrons are transferred from the formally titanium(II) centers to the bridging ligand in the ground state. However, the complete spectra can only be explained when assuming that spin states of different multiplicity form the ground state of [(Cp2Ti)3HATN(Ph)6]. The observed electronic excitations in the UV, Vis and NIR ranges are all transitions between ligand levels. Contrary to common notion, LMCT or IVCT transitions do not play a role. The same assignment rules can also be reproduced by calculating the isolated and appropriately charged HATN ligand. The temperature\u2010dependent magnetic properties can be explained the hybridization of different electronic configurations of different total spin with one valence electron located at each of the three Ti centers and one electron in a SOMO of the ligand.Three reduction waves and six oxidation waves are observed in voltammograms of [(Cp2Ti)3HATN(Ph)6] was synthesized as reported.6), Sigma Aldrich, Steinheim, Germany), silver perchlorate . Acetonitrile (MeCN) and tetrahydrofurane (THF) were dried according to standard procedures2Ti)3HATN(Ph)6] was solved in dry THF to a concentration of 0.3\u2005mM. complex and the pure HATN ligand.All calculations were performed using the program package ORCA in version 4.2.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file."}
+{"text": "It was highlighted that the original article  containeIncorrectThe returned similarity score is based on Jaccard similarity [18] of Morgan-style connectivity fingerprints [19] with a radius of up to 5.CorrectThe returned similarity score is based on Jaccard similarity [18] of Morgan-style connectivity fingerprints [19] with a radius of up to 3."}
+{"text": "Electrocatalysts exposed to ~3\u201330 ALD cycles of TiO2 exhibited overpotentials at 10 mA cm\u20132 of geometric current density that were several hundred millivolts lower than uncoated catalysts, with correspondingly higher specific activities. For example, the deposition of TiO2 onto IrO2 yielded a 9-fold increase in the OER-specific activity in 1.0 M H2SO4 . The oxidation state of titanium and the potential of zero charge were also a function of the number of ALD cycles, indicating a correlation between oxidation state, potential of zero charge, and activity of the tuned electrocatalysts.We report that TiO Broader context2 emissions future depends on the electrochemical production of fuels and commodity chemicals. In the absence of a substantial carbon tax, electrochemical production of these materials must be cost competitive with conventional production. The levelized cost of electrochemically produced chemicals depends heavily on operational expenses  and the balance of systems costs, and depends relatively less on the price of the catalyst.1 Therefore, one pathway to low cost electrochemical fuel and commodity chemical production is to reduce the OpEx by fabricating highly active catalysts. Current methods to enhance catalytic activity are limited or rely on computationally-expensive calculations. Simple tools that can be used to enhance the catalytic activity for a variety of chemical reactions, such as tuning catalysts through atomic layer deposition as presented here, are essential to developing low-cost electrochemical systems that can meet global energy and chemical demands.Realizing a low anthropogenic CO3 Despite potential advantages , the industrial use of many heterogeneous electrocatalysts is currently limited in part by suboptimal catalytic activity and/or selectivity. In addition, there are limited methods to tune the selectivity and activity of heterogeneous electrocatalysts.2 Methods and design tools such as doping, inducing strain, and mixing metal oxides have been used to improve the catalytic activity of heterogeneous electrocatalysts.7 The activity of heterogeneous electrocatalysts can also be tuned by applying thin layers of another material, leading to an altered surface charge density on the resulting composite material relative to the bulk charge density of either individual material.13 This approach has been widely used to alter the catalytic and electronic properties of core/shell nanoparticles, although additional tuning of the particle support structure is necessary to create an efficient heterogeneous electrocatalyst.15 Density functional theory calculations have shown that a single atomic layer of TiO2 on RuO2 should lead to enhanced selectivity for the chlorine-evolution reaction (CER) relative to the oxygen-evolution reaction (OER).9 Enhanced catalytic activity for the OER has been reported for WO3 photocatalysts coated with 5 nm of alumina, with the activity increase ascribed to an alteration in the electronic surface-state density.16 Enhanced catalytic activity has also been observed at the interface between TiO2 and RuO2, with charge transfer between RuO2 and TiO2 resulting in a mixed phasewithanintermediatechargedensity.5Highly active electrocatalysts are required for the cost-effective generation of fuels and commodity chemicals from renewable sources of electricity.2, RuO2, and F-doped SnO2 (FTO) were tuned and evaluated for the chlorine-evolution reaction (CER) and the oxygen-evolution reaction (OER). The CER provides a promising approach to infrastructure-free wastewater treatment as well as for the production of chlorine, an important industrial chemical whose global annual demand exceeds seventy million metric tons.18 The OER is the limiting half-reaction for water splitting that could provide hydrogen for transportation and could also provide a precursor to energy storage via thermochemical reaction with CO2 to produce an energy-dense, carbon-neutral fuel.19Herein, atomic layer deposition  has been used to tune the surface charge density, and consequently tune the catalytic activity, of electrocatalytic systems in a fashion consistent with estimates based on group electronegativity concepts . To test these predictions, the activities of the known electrocatalysts, IrO\u03c7) relative to the group electronegativity of RuO2 (\u03c7 \u2248 2.72), the most active catalyst for the OER in the benchmarking literature  as well as the most active catalyst for the CER.20 IrO2 (\u03c7 \u2248 2.78) and FTO (\u03c7 \u2248 2.88) were also investigated because they have higher electronegativities than RuO2, and therefore using ALD to overcoat these catalysts with TiO2 (\u03c7 \u2248 2.62) is expected to shift their surface electronic properties and catalytic activities towards that of RuO2, the optimal single metal oxide catalyst. These materials were also chosen because TiO2, IrO2, RuO2, and other materials are commonly used to form mixed metal oxide electrodes, most notably the dimensionally stable anode (DSA), in which TiO2 increases the anode\u2019s stability, but does not confer enhanced activity to the aggregated material.21Each material tested was selected based on its theoretical group electronegativity  were determined for IrO2, RuO2, and FTO as a function of the successive number of TiO2 ALD cycles  for the OER at 10 mA (cmgeo)\u20132 in 1.0 M H2SO4 and for the CER at 1 mA (cmgeo)\u20132 in 5.0 M NaCl adjusted to pH 2.0 with HCl. Current densities were chosen to produce >95% measured Faradaic efficiency for each catalyst , and current\u2013potential data were corrected for the solution resistance (<2.0 mV correction) as measured by electrochemical impedance spectroscopy (see ESI\u2020 for details). The three catalysts were prepared on substrates that had very low roughness to minimize effects in geometric overpotential measurements due to surface area differences. Specifically, electrocatalyst samples consisted of a ~300 nm metal\u2013oxide film sputter deposited on a (100)-oriented Si substrate, in the case of IrO2 and RuO2, or commercially available TEC 15 FTO glass substrates, in the case of FTO-based electrocatalysts. TiO2 overlayers were then deposited on top of the electrocatalysts. The microstructure of a typical IrO2-based electrocatalyst is shown in the cross-sectional scanning electron microscopy (SEM) image in Overpotentials (geo)\u20132 for bare RuO2 and IrO2 agreed well with values reported for catalysts prepared on similarly flat surfaces. We are unaware of comparable OER data for FTO or for CER catalysts.22 The overpotentials for IrO2 and FTO, for both the OER and CER, initially showed an improvement  with increasing ALD cycle number, before exhibiting an inflection point due to an increase in overpotential at higher ALD cycle numbers ) is a standard quantity for comparing the OER activity of heterogeneous electrocatalysts . For IrO2 and RuO2 catalysts, the OER specific activities of the uncoated catalysts were in good agreement with previously reported values.20 We are unaware of reported specific activities for FTO for the OER or for any catalyst for the CER. The specific activities for the OER and CER were characterized by volcano-type relationships as a function of the TiO2 ALD cycle number  in 0.5 M H2SO4. To compare these catalysts, we measured the roughness of our catalysts using AFM . For our catalysts, bare IrO2 exhibited a Tafel slope of ~60 mV dec\u20131 in good agreement with previously reported OER catalysts.24 As the activity of our IrO2 based catalyst increased from bare IrO2 to 10 TiO2 ALD cycles, the Tafel slope remained constant at ~60 mV dec\u20131 while the exchange current density (i0)increased from ~1 \u00d7 10\u20137 to ~2 \u00d7 10\u20135 mA (cmAFMSA)\u20132. Initially the IrOx/SrIrO3 catalyst also had an OER Tafel slope of ~60 mV dec\u20131 and an i0 of ~7 \u00d7 10\u20136 mA (cmAFMSA)\u20132. For the IrOx/SrIrO3, however, after a period of activation the Tafel slope improved dramatically to ~40 mV dec\u20131, which indicates a previously unknown OER mechanism, while the i0 deteriorated to ~3 \u00d7 10\u20137 mA (cmAFMSA)\u20132 . In our case, IrO2 coated with 10 ALD cycles of TiO2 exhibited lower overpotentials than the freshly prepared IrOx/SrIrO3 catalyst at current densities <1 mA (cmAFMSA)\u20132 and lower overpotentials than the activated IrOx/SrIrO3 catalyst at <0.02 mA (cmAFMSA)\u20132, but substantially higher overpotentials at the more industrially relevant current densities of >10 mA (cmAFMSA)\u20132.25 Further discussion on surface roughness, including AFM, and SEM sample characterization is presented in the ESI\u2020 .The specific activity \u20132. For thinly coated catalysts (3\u201310 cycles) the OER stability improved from about 1 h to about 4 h, while for thicker TiO2 coatings (>30 cycles) the OER stability increased to >9 h . The loss in activity for the OER for TiO2 coated samples was associated with a loss in the TiO2 coating as illustrated in X-ray photoelectron spectroscopy (XPS) measurements of the Ti 2p core level before and after electrochemical stability testing . For the CER, all catalysts were relatively stable over the 24 h testing period except for the FTO-based catalysts which followed the same trend as the OER, with thicker TiO2 coatings stabilizing the electrodes. XPS measurements of the stable CER catalysts indicated that the TiO2 overcoating was still present even after 24 h of continuous operation . These results indicate that, as prepared here, these catalysts are not long-term stable, and substantial work is needed to obtain an industrially relevant catalyst. Similarly prepared catalysts exhibit enhanced stability by making the catalyst material thicker, annealing the catalyst, or mixing SbxOy, TiO2, TaxOy, or SnO2 into the catalyst.28 It is possible that similar techniques could be used to enhance the stability of the catalysts presented in this work.To test the longevity of the enhanced catalytic performance with TiO2 is not readily explained by surface morphological changes of the electrocatalyst. Deposition of TiO2 does not substantially affect the electrochemically active surface area, a metric believed to be related to active site density, and changes in the surface area alone do not account for the magnitude of the enhancement in the specific activity . Furthermore, while high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and STEM electron dispersive X-ray spectroscopy (EDS) maps of IrO2 samples with 10 cycles of TiO2 ,32 consistent with expectations that addition of TiO2 does not fundamentally change the mechanism or the potential determining step for either reaction. Hypothesized mechanisms generally involve coordination of either OOH or OCl groups to unsaturated sites on the metal oxide in the potential determining reaction steps.35 FTO based catalysts exhibited very large overpotentials for both the CER and OER and had correspondingly high Tafel slopes in excess of 190 mV dec\u20131, potentially indicating a different, much less efficient mechanism than the process that controls the reactivity of the more active catalysts.The enhancement in catalytic performance observed with deposition of TiO of TiO2  indicateous film  and cataEZC) of the electrocatalysts were measured as a function of TiO2 thickness  and (S3) and Fig. S12\u2013S15 for details and discussion on handling thin TiO2 layers in EZC measurements). EZC thus yields insight into the strength of the bonds on the catalyst surface.37EZC is also qualitatively very similar to group electronegativity which describes how difficult it is for molecules to gain electrons and is correlated to OER activity . Additionally, EZC of metal electrodes has been correlated with metal\u2013oxygen single bond strengths which is also qualitatively similar to computationally derived oxygen binding energies which have long been correlated with electrocatalytic activity.38EZC, group electronegativity, and oxygen binding energies each have their strengths and weaknesses. EZC is measurable, but it is not easy to predict. Electronegativity is completely theoretical and very simple to calculate, but does not take into account more complex qualities of materials like edge sites. Oxygen binding energies are strongly theoretically grounded and can take into account complexities of materials like edge sites, but they are also relatively difficult to calculate. These strengths and weaknesses show that all these descriptors may be used complimentarily to predict and understand catalytic activity . Measured EZC values for bare RuO2 and IrO2  were consistent with previously reported values for Ru and Ir.39 We are unaware of reported EZC values for FTO. As the RuO2 and IrO2 samples were coated with increasing ALD cycles of TiO2 the EZC shifted from lower to higher potentials in both cases and eventually reached the value for bulk TiO2. This behavior is consistent with the expected trends for equilibrated group electronegativities. The EZC for bare FTO (450 mV vs. SCE) was less than that for bulk TiO2 and greater than bare IrO2 or RuO2. The FTO EZC decreased with increasing TiO2 cycles up to 10 cycles and as the TiO2 cycles increased beyond 10 the EZC increased until it reached the bulk value of TiO2 at large cycle numbers. The overall trend of the FTO EZC increasing to higher values with increasing TiO2 cycle number is consistent with group electronegativity arguments. However, the intermediate behavior where the EZC decreases and then increases is not well explained by group electronegativity and could, in part, arise from the complicated behavior of the F dopant atoms (further discussion on the limits of group electronegativity are found in the ESI\u2020). For all catalysts, the EZC continued to shift even beyond the point where TEM data indicated that the film is continuous (40 ALD cycles). This suggests that the exposed metal oxide is not fully responsible for the shift in EZC and that the surface TiO2 is likely responsible in part for the EZC shift. Shifts in EZC with incremental TiO2 deposition suggest that ALD can be used to tune the catalytic performance. These data reveal that the catalysts with the highest activity for the CER have EZC values between 50 and 75 mV vs. SCE  for the CER. Additionally, active OER and CER catalysts for all systems investigated have EZC values between 25 and 200 mV vs. SCE with the best OER catalysts having a somewhat higher EZC (~110 mV vs. SCE) than the best CER catalysts (~60 mV vs. SCE).To investigate the electrocatalysts\u2019 surface electronic properties the potentials of zero charge , stacked from bottom to top, for increasing ALD TiO2 thickness, with 0 cycles indicating the bare catalyst substrate. Deposition of low cycle numbers of ALD TiO2 on IrO2 and RuO2 produced Ti core-level peaks that were at ~456.6 eV and ~457.6 eV, which is consistent with previously reported binding energies for Ti3+ states.41 As the ALD cycle number increased, the Ti oxidation state for these samples gradually increased to its bulk oxidation state (~+4), and signals indicative of bulk TiO2 were eventually observed  and the thin TiO2 film, in which the chemical nature of the phase produces a more oxidized metal, with the mixed phase most likely dominated by Ti4+ sites.To further understand the surface states of the catalysts, X-ray photoelectron spectroscopy was used to measure the Ti oxidation state. observed . In the 2 cycles was accompanied by a peak shift of the Ti 2p3/2 peak relative to the bulk TiO2 peak position . The Ti 2p3/2 peak of the IrO2-and RuO2-based catalysts shifted from reduced, lower binding energies to the more oxidized, higher binding energies typical of bulk TiO2. The FTO-based Ti 2p3/2 peak shifts from more oxidized, high binding energies at low TiO2 cycles to lower binding energies for intermediate TiO2 cycles (10\u201340 cycles) before increasing again to higher binding energies at large TiO2 thicknesses (>60 cycles). The Ti 2p3/2 peak shift is qualitatively consistent with the variation in EZC with TiO2 cycle number suggesting that the change in the surface charge density is correlated with a change in the Ti oxidation state.The variation in the Ti oxidation state with ALD TiO2 thickness can be explained by charge transfer from the underlying metal oxide substrate. In this scenario, a more reduced Ti species present at low deposited cycles of TiO2 on IrO2 and RuO2 would be accompanied by a more oxidized metal oxide substrate. To confirm this hypothesis, we measured the Ir 4f, Ru 3d, and Sn 3d core-level photoemission . Unlike in the case of the Ti 2p spectra, the Ir 4f, Ru 3d, and Sn 3d core-level photoemission exhibited very small changes between the bare metal oxide substrate and those with varying thicknesses of TiO2. This was reflected in the peak shifts of the main peak for the Ir 4f, Ru 3d, and Sn 3d spectra with TiO2 thickness relative to that of the bare substrate , which were an order of magnitude lower than those for the Ti 2p core-level photoemission and mostly within the error of the measurement (\u00b10.1 eV). While peak fitting (see the ESI\u2020 for details) of these spectra indicates that initial deposition of TiO2 leads to a slightly more oxidized Ir and Ru state, and a slightly more reduced Sn state for FTO, no trend with thickness was observed for any of the substrates, and changes in the oxidation state of the underlying catalyst are likely below the detection limit for the techniques used in this study .The variation in the Ti oxidation state with TiO2 can tune surface electron densities of the catalyst in a direction consistent with predictions from group electronegativity concepts . Given that concomitant changes in electrochemical activity were observed with deposition of TiO2, these data indicate that ALD may be useful to tune the activity of other catalysts for diverse reactions, including those critical for renewable energy storage and wastewater treatment.In summation, surface characterization suggests that atomic layer deposition of low cycle numbers of TiO"}
+{"text": "This paper describes the impact of ligandacidity on the formation of VO2 nanocrystals from the solvothermalreaction of vanadyl acetylacetonate (VO(acac)2) with stoichiometricamounts of water. Carboxylic acids examined herein favor the formationof the monoclinic VO2(B) phase over the tetragonal VO2(A) phase as the concentration of water in the reaction increases.However, the threshold concentration of water required to obtain phase-pureVO2(B) nanocrystals increases as the pKa of the carboxylic acid decreases. We also observe thatincreasing the concentration of VO(acac)2 or the concentrationof acid while keeping the concentration of water constant favors theformation of VO2(A). Single-crystal electron diffractionmeasurements enable the identification of vanadyl carboxylate speciesformed in reactions that do not contain enough water to promote theformation of VO2. Increasing the length of the carbon chainon aliphatic carboxylic acids did not impact the phase of VO2 nanocrystals obtained but did result in a change from nanorod tonanoplatelet morphology. These results suggest that inhibiting therate of hydrolysis of the VO(acac)2 precursor either bydecreasing the ratio of water to VO(acac)2 or by increasingthe fraction of water molecules that are protonated favors the formationof VO2(A) over VO2(B).Vanadium dioxide (VO The most stable bulk phase, VO2(R), with a rutilestructure, is well known for its low-temperature metal-to-insulatortransition to VO2(M) and is used for smart window applications.2 Like VO2(M), the metastable tetragonal phase known asVO2(A) has also been investigated as a material for opticalswitches,4 while the metastable monoclinic phase knownas VO2(B) has been used as a cathode material in lithium-and sodium-ion batteries.11 All of these applications benefit from the solution processabilityafforded by nanocrystalline morphologies. Given the variation in theapplication of multiple crystal polymorphs of VO2, thereis considerable motivation to understand how to control which polymorphsform under various reaction conditions.Vanadium dioxide (VO2(A) andVO2(B) are typically synthesized using solvothermal methodsat elevated pressure.18 These methods often utilize V2O5 and a reductantin water, and control over the crystal phase is usually achieved byvarying the reaction temperature. VO2(A) is the major productobtained at reaction temperatures between 220 and 270 \u00b0C, andVO2(B) is the major product obtained at temperatures between180 and 200 \u00b0C.15 Varying the pressure by varying the volume of thereaction mixture within the pressure-sealed reaction vessel19 also impacts the phase of the resulting product.One study showed that a mixture containing V2O5 and oxalic acid reacting at 180 \u00b0C in a pressure-sealed autoclavecould result in phase-pure VO2(B) after 24 h and phase-pureVO2(A) after 7 days.20 However,this result could only be obtained with a filling ratio of 80/100mL in the pressure-sealed vessel: a filling ratio of 60/100 mL onlyyielded VO2(B) with no phase change present after 7 days.Nanocrystals of themetastable polymorphs VO2 nanocrystals. Specifically, we showed that decreasingthe concentration of water present in the solvothermal reaction mixtureof vanadyl acetylacetonate (VO(acac)2) and lauric acidin toluene to 4 equiv or less per vanadium center produces phase-pureVO2(A) nanocrystals, whereas using 20 equiv or more ofwater produces phase-pure VO2(B) nanocrystals.21 This approach has the added advantage of enablingaccess to smaller nanocrystals with dimensions less than 500 nm. Incontrast, reactions that use water as the solvent produce very largeparticles, usually nanorods that are several microns long.10 We hypothesize that the mechanismby which the concentration of water impacts the crystal phase of VO2 nanocrystals is through controlling the relative rates ofprecursor hydrolysis and condensation. Decreasing the concentrationof water slows hydrolysis and may allow nanocrystal nucleation viacondensation of partially hydrolyzed species. We suspect that condensationof these partially hydrolyzed species favors the formation of VO2(A) over VO2(B).Recently, we demonstrated that tuning the chemical rather thanphysical reaction conditions enables control over the crystal phaseof VOKa and steric bulk of organic carboxylic acid ligandsto vary the rate of precursor hydrolysis. We find that replacing lauricacid with a stronger acid, namely trifluoroacetic acid, expands therange of water concentrations that lead to the formation of VO2(A) nanocrystals by a factor of almost four, up to 15 equivof water per vanadium center. Increasing the concentration of trifluoroaceticacid and/or the VO(acac)2 precursor relative to water alsofavors the formation of VO2(A) over VO2(B).These experiments indicate that reaction conditions that suppressthe rate of precursor hydrolysis  favor the formationof VO2(A).21 In contrast tovarying the acidity of carboxylic acid, changing the chain lengthof aliphatic carboxylic acids does not alter the crystal phase ofthe product. This observation indicates that the steric bulk of theseligands does not alter the relative rates of precursor hydrolysisand condensation significantly enough to impact which crystal phaseof VO2 nucleates.Here, we test this hypothesisby tuning the p2 nanocrystals from VO(acac)2. We varied two properties of these ligands\u2014their acidityand their steric bulk\u2014to determine which property has the largestinfluence on the resulting nanocrystals. Our previous work demonstratesthat the amount of water present in a solvothermal reaction with VO(acac)2 and lauric acid in toluene determines both the crystal phaseof VO2 synthesized, as well as the length of the resultingnanorods.21 Using this synthesis methodology,we varied the amount of water present in the reaction from 0.5 to20 mmol (2\u201380 equiv per vanadium) in the presence of 4 equiv(1 mmol) of a selection of acids with varying pKa\u2019s shown in 2(A) products (or product mixtures containingVO2(A)) to phase-pure VO2(B) products as theamount of water present in the reaction increases past a particularthreshold concentration. This observation is consistent with our previouslyreported study in which the threshold concentration of water requiredfor the formation of phase-pure VO2(B) in the presenceof lauric acid (pKa = 5.3) was determinedto be 5 mmol (20 equiv per vanadium).21 Here we observe that decreasing the pKa of the carboxylic acid increases the threshold concentration ofwater required to obtain VO2(B) instead of VO2(A). For example, trifluoroacetic acid (pKa = 0.23) has a threshold concentration of 15 mmol (60 equiv per vanadium).Notably, the threshold water concentration observed in the absenceof acidic ligands is 3 mmol (12 equiv per vanadium). This observationsuggests that the minimum concentration of water required to formphase-pure VO2(B) is 12 equiv per vanadium center. Additionof carboxylic acids increases this threshold.We set out to investigate therole of carboxylic acid ligands inthe synthesis of VO2(A) or VO2(B)  of water by electron diffraction suggests that these productsare the corresponding vanadyl carboxylate compounds\u2014VO(benzoate)2 and VO(4-nitrobenzoate)2\u2014and that thesecompounds also crystallize as one-dimensional chains of octahedrallycoordinated vanadium ions with a structure that is entirely analogousto VO(acetate)2 (Supporting Information). The powder X-ray diffractionpattern of the product obtained from the reaction of VO(acac)2 with 4 equiv of trifluoroacetic acid and 4 equiv of waterresembles that of the other confirmed vanadyl carboxylate species.We therefore strongly suspect that this pattern corresponds to VO(trifluoroacetate)2. These data indicate that carboxylic acids can displace acetylacetonate,and 1 mmol (4 equiv) of water is insufficient to hydrolyze these speciesto form VO2. In a solvothermal reaction utilizing VO(benzoate)2 as a precursor in the absence of water and benzoic acid,the VO(benzoate)2 precursor is recovered. VO(benzoate)2 also forms upon reaction of 4 equiv of benzoic acid withVO(acac)2 in the absence of water under the same solvothermalconditions . This observation indicates that ligand exchangeto form the VO(carboxylate)2 species does not require thepresence of water.We also observe that in the presence of very low concentrationsof water (1 mmol or less), the addition of acetic, benzoic, 4-nitrobenzoic,and trifluoroacetic acid results in products whose powder X-ray diffractionpatterns do not match that of either VOr VO2(B) 2. Inthe2, VO(benzoate)2, and VO(4-nitrobenzoate)2 state that these species are insoluble in most solvents,which isconsistent with our observations.29 Although VO(trifluoroacetate)2 has been reported previously as a soluble monomeric complex,30 the species we isolated from the reaction ofVO(acac)2 in the presence of 4 equiv each of trifluoroaceticacid and water is not soluble in polar or nonpolar solvents. We thereforesuspect that this species is also a coordination polymer. VO(benzoate)2 and VO(4-nitrobenzoate)2 are reported to crashout immediately upon addition of vanadyl sulfate to an aqueous solutionof the corresponding carboxylate,29 indicatingthat monomeric carboxylate complexes cannot be isolated even in thepresence of excess acid. Titration of acetic acid into aqueous solutionsof the vanadyl ion VO2+ achieves a maximum of 1.5 boundacetates per solvated vanadium center,32 indicatingthat stable solutions in which all monomeric species are bound totwo acetate ligands cannot be achieved. Based on these previous reports,we conclude that monomeric vanadyl acetate, benzoate, or 4-nitrobenzoatespecies are highly unlikely to exist in nonpolar toluene solutionscontaining 4 equiv of carboxylic acid per vanadium, such as thoseused here.The earliest reports of coordination polymers ofVO(acetate)2 in the absence of water produces no reaction\u2014therecoveredreaction mixture still contains lauric acid and VO(acac)2 (see Supporting Information). Furthermore,we note that formation of VO2(A) occurs in the presenceof lauric acid and 0.5 or 1 mmol water, whereas at least 2 mmol water(8 equiv) is required to observe the formation of any VO2 species in the presence of acetic, benzoic, 4-nitrobenzoic, or trifluoroaceticacid 2 and lauric acid are intactin the mixture recovered from a solvothermal reaction run in the absenceof water, we cannot completely rule out the formation of some monomericvanadyl laurate species in solution or the presence of a small fractionof such species in the recovered reaction mixture.In contrast to benzoic acid, reaction of lauric acidwith VO(acac)ticacid 1. Wesus2 from the solvothermal reaction of VO(acac)2 involves hydrolysis of VO(acac)2 to form [VO(H2O)5]2+ followed by condensation of this speciesto generate VO2.21 This vanadyl aquo complex is stable under neutral aqueous conditions34 and has a pKa of 5.3\u20136.0.35 We proposed in our previous work that the relativerate of hydrolysis versus condensation controls whether VO2(A) or VO2(B) nuclei form. Slow hydrolysis enables condensationof partially hydrolyzed species, whereas faster hydrolysis promotesthe formation of the fully hydrolyzed species before significant condensationoccurs. We hypothesized that condensation of the fully hydrolyzedspecies favors the formation of VO2(B) nuclei, while condensationof partially hydrolyzed species favors the formation of VO2(A) nuclei. Here, we observe that addition of stronger carboxylicacids increases the threshold concentration of water required to obtainphase-pure VO2(B) nanocrystals instead of VO2(A). This observation is consistent with our hypothesis that therate of hydrolysis controls the crystal phase of VO2 becauseincreasing the strength of the organic acid should inhibit hydrolysisof VO(acac)2. More acidic reaction conditions result ina higher ratio of positively charged hydronium ions to water molecules,which lowers the concentration of neutrally charged water availableto hydrolyze the VO(acac)2. Hydronium ions are much lessnucleophilic than neutral water and therefore less reactive towardhydrolysis.Previousreports have posited that the mechanism for the formationof VO2(A) over VO2(B) by conductinga series of reactions in which we varied the relative concentrationsof VO(acac)2, water, and trifluoroacetic acid (2(A) becomes the favored product as the concentration of either VO(acac)2 or trifluoroacetic acid increases relative to the concentrationof water. 2 from 0.25 to 1mmol while keeping the concentration of water constant at 15 mmolresults in a transition from VO2(B) products to VO2(A) products. Obtaining VO2(B) from a reactioncontaining 1 mmol of VO(acac)2 and 1 mmol trifluoroaceticacid requires increasing the amount of water present to at least 30mmol, whereas VO2(B) can be obtained using only 15 mmolof water in the presence of 0.25 mmol of VO(acac)2 and1 mmol trifluoroacetic acid. 2 is kept constant at 0.25 mmol and theratio of trifluoroacetic acid to water is increased by either decreasingthe concentration of water or increasing the concentration of acid.In both cases, higher ratios of trifluoroacetic acid to water favorthe formation of VO2(A).We further tested our hypothesis that slow hydrolysispromotesthe formation of VOtic acid 3. VO2(A)2(A) and VO2(A) is a more thermodynamicallystable phase than VO2(B), we investigated whether strongcarboxylic acids could mediate the conversion of VO2(B)nanocrystals to VO2(A). Previous reports show that conversionfrom VO2(B) to VO2(A) is possible under solvothermalconditions at high temperatures.9 For example, Zhang,et al. obtained VO2(A) nanocrystals upon reacting a solutioncontaining VO2(B) nanocrystals and water at 260 or 280\u00b0C for 48 h.3 Additionally, anotherreport also heated VO2(B) nanocrystals to 280 \u00b0C underhydrothermal conditions for 48 h to convert them to VO2(A).9 We investigated the possibilitythat lowering the temperature and using an acidic reaction environmentcould also instigate this transformation. 2(B) nanocrystalscan indeed be converted to VO2(A) when heated in toluenein the presence of 4 equiv of trifluoroacetic acid and 20 equiv ofwater at 230 \u00b0C over the course of 120 h. This conversion isindicated by the coalescence of two pairs of diffraction peaks at2\u03b8 = 14.4 and 15.3\u00b0 and 2\u03b8 = 29 and 30.2\u00b0 intosingle peaks at 2\u03b8 = 15 and 30\u00b0, respectively. The resultingpeaks correspond to the (110) and (220) planes of the VO2(A) structure. The overall decrease in the number of diffractionpeaks signals an increase in symmetry as the monoclinic VO2(B) structure converts to the tetragonal VO2(A) structure.The VO2(A) nanocrystals obtained from this reaction aresmaller than the initial VO2(B) nanocrystals (2(B) nanocrystals and re-nucleation as VO2(A), possibly using VO(trifluoroacetate)2 as areaction intermediate. We note that in reactions containing VO(acac)2, lauric acid, and 2 or 20 equiv of water, we do not observeany indications of phase interconversion over the course of a 24 hreaction period. Examining products present in these reaction mixturesafter various reaction times reveals that only one type of crystallineproduct is observed per reaction condition: VO2(A) forthe reaction containing 2 equiv of water and VO2(B) forthe reaction containing 20 equiv of water (see Supporting Information). This observation implies that forreactions starting from VO(acac)2, the final VO2 product phase nucleates directly from condensation of hydroxylatedspecies formed upon precursor hydrolysis.Since the presence of strong carboxylic acids favorsthe formationof VOcrystals 4. This c2 nanocrystals, we next investigatedthe impact of tuning the steric bulk of the carboxylic acid ligandon the solvothermal synthesis of VO2 nanocrystals. A selectionof aliphatic carboxylic acids with varying carbon-chain lengths waschosen, specifically butyric (4C), hexanoic (6C), heptanoic (7C),decanoic (10C), lauric (12C), and stearic (18C) acid. All of thesecarboxylates have relatively similar pKa\u2019s of \u223c4.8\u20135.3.36 Figure S14in the Supporting Information shows powderX-ray diffraction spectra obtained from reactions of VO(acac)2 in the presence of 4 equiv of each of these acids and 20equiv of water. These data demonstrate that VO2(B) is obtainedin every case. Likewise, the morphology of the nanocrystals remainsconstant with variation of the ligand chain length, except for stearicacid, as shown in Figure S14. Reactionscontaining aliphatic carboxylic acids with chain lengths less than18 carbons produce VO2(B) nanorods of average length 110\u2013150nm, while stearic acid yields nanoplatelets in addition to nanorods.Overall, since only one of the aliphatic carboxylic acids resultedin a difference in morphology, we conclude that varying the lengthof aliphatic carboxylic acids does not strongly influence the hydrolysisand growth of VO2(B) nanocrystals.After establishing that the acidity of the carboxylicacid ligandsimpacts the phase of VO2(B) nanocrystalssynthesized with carboxylic acids of various pKa\u2019s. Since all of the acids produce phase-pure VO2(B) with 60 equiv of water, these products were analyzed byscanning electron microscopy  nanocrystals from VO(acac)2 in thepresence of polyvinylpyrrolidone (PVP)18 and from V2O5 in the presence of oxalic acid.7 The first study discovered that changing theconcentration of PVP in the reaction impacted the morphology of theresulting nanocrystals. Nanorods formed in the absence of PVP, whileaddition of 3 mg/L PVP formed nanoflowers, and 54 mg/L PVP formednanocarambolas.18 Thus, the change in morphologyfrom nanoflowers to nanocarambolas occurred after an increase in PVPconcentration by a factor of 18. The second study found that using0.06 mol/L oxalic acid produced nanorods, while 0.12 mol/L oxalicacid produced a mixture of nanobundles and nanocarambolas.7 Both of these studies concluded that the interactionof ligands with the surface of the nanocrystal during growth impactsthe final morphology.38Finally, the reaction containing trifluoroaceticacid producednanocarambolas, which are elongated aggregates of stacked nanosheetswhose cross section resembles a six-pointed star.Supporting Information). At early reaction times (3 h), we observe nanorods with shortrod-like protrusions growing perpendicular to the long axis. After6 h, we observe fully formed nanocarambolas. We hypothesize that theshort protrusions observed at early times expand along the long axisof the original nanorod, forming sheets that result in the 6-armednanocarambola structure. Unlike other carboxylic acids used here,trifluoroacetic acid has two moieties that can engage in strong interactionswith the nanocrystal surface: the carboxylate group and the fluorineatoms. Carboxylic acid groups are well known to bind to the surfacesof metal oxide nanoparticles.40 In the case of TiO2, trifluoroacetic acid ligands were found to have a similarstructure-directing effect as the addition of HF\u2014both additivesresulted in the stabilization of {001} facets and the formation ofnanosheets in the case of HF and a mixture of nanosheets and truncatedoctahedrons in the case of trifluoroacetic acid.41 The authors also observed that aliphatic carboxylic acids,such as oleic acid, form truncated octahedrons, indicating that thecarboxylate group also stabilizes {001} facets but not to the sameextent as fluoride ions. The authors attributed the intermediate structure-directingeffect of trifluoroacetic acid to its partial degradation under solvothermalreaction conditions to form fluoride ions. We suspect that similardegradation of a fraction of the trifluoroacetic acid molecules maybe responsible for the formation of VO2(B) nanocarambolas.To explore the mechanism by which the unusual nanocarambolamorphologyforms, we varied the duration of the reactions run in the presenceof trifluoroacetic acid (see Ka and increasing the concentration of carboxylicacid ligands present in the reaction mixture while keeping the concentrationof water constant favors the formation of VO2(A), whereasincreased pKa and decreased acid concentrationfavor the formation of VO2(B). Furthermore, increasingthe concentration of the precursor relative to the concentration ofwater leads to VO2(A), whereas VO2(B) formsfrom reaction mixtures containing lower precursor-to-water ratios.These observations are consistent with our hypothesis that the rateof precursor hydrolysis relative to condensation controls which crystalphase nucleates. We identified VO(carboxylate)2 speciesas likely reaction intermediates formed prior to nucleation of theVO2 species. Finally, our results suggest that acidityalone likely does not have a strong impact on nanocrystal morphology.Instead, interactions involving substituents on the carboxylic acidligands, such as intermolecular \u03c0\u2013\u03c0 interactionsor interactions between fluoride ions formed upon degradation of \u2212CF3 groups and the nanocrystal surface, may play a significantrole in determining the morphology of VO2(B) nanocrystals.Overall, this work provides important insight into how carboxylicacids impact the crystal phase and morphology of VO2 nanocrystals.Our work demonstrates the effects of theacidity of the reactionmixture on the crystal phase and morphology of vanadium dioxide nanocrystalsobtained under solvothermal reaction conditions. Decreasing the p2, stearic acid, decanoic acid, heptanoicacid, hexanoic acid, and butyric acid purchased from Sigma-Aldrichand lauric acid purchased from Millipore were pumped into the gloveboxovernight in an evacuated antechamber before use. Benzoic acid and4-nitrobenzoic acid purchased from Oakwood, acetic acid purchasedfrom Fisher Scientific, and trifluoroacetic acid purchased from Sigma-Aldrichwere used without further purification.Prior to the addition of waterto the reaction mixtures, all manipulations were carried out in anMBraun inert atmosphere glovebox under a nitrogen atmosphere unlessotherwise stated. Autoclave reactors and Teflon liners were pumpeddown in an evacuated antechamber overnight prior to use in the glovebox.Molecular sieves  were activated by heatingat 200 \u00b0C under vacuum overnight. Toluene was dried over activatedsieves (20% by weight) for at least 24 h, then purged with nitrogenfor at least 1 h before being transferred via a cannula to a Schlenkflask. Toluene was then pumped into the glove box overnight in anevacuated antechamber and stored over activated 3 \u00c5 molecularsieves. VO(acac)Trifluoroacetic acid is bothvolatile and corrosive and should therefore be handled exclusivelyin a fume hood. Autoclave reactors become pressurized at elevatedtemperatures and should be handled with care. No attempts should bemade to open autoclave reactors unless they are at room temperature.2 and10 mL of toluene inside a nitrogen-filled glovebox. The autoclavereactor was sealed and removed from the glovebox. In ambient air,the autoclave reactor was opened, and the desired amount of waterwas added (3\u201320 mmol). A carboxylic acid (1 mmol) was addedto the reaction mixture either before  or after  removal from the glovebox.Tables S1 and S4 in the Supporting Information contain detailed lists of the masses and volumes of the variousreagents used in these reactions. After the addition of water, theautoclave reactor was sealed again and heated in an oven at 200 \u00b0Cfor 24 h before it was removed and allowed to cool to room temperature.The black solid product was collected by centrifugation under ambientconditions and washed with ethanol. The product was rotovapped todryness and stored in the glovebox under nitrogen.A 25 mL Teflon-lined autoclavereactor was charged with 0.25 mmol (0.068 g) VO(acac)2 (0.075\u20133 mmol) and 10 mL of toluene. The autoclave reactorwas sealed and removed from the glovebox. In ambient air, the autoclavereactor was opened and the desired amounts of water (0.5\u201320mmol) and trifluoroacetic acid (0.5\u20134 mmol) were added . The autoclavereactor was sealed again and heated in an oven at 200 \u00b0C for24 h before it was removed from the oven and allowed to cool to roomtemperature. The black solid product was collected by centrifugationunder ambient conditions and washed with ethanol. The product wasrotovapped to dryness and stored in the glovebox under nitrogen. Vanadylcarboxylate products were collected and purified using proceduresidentical to those used for the vanadium dioxide nanocrystals.A 25 mL Teflon-lined autoclavereactor was charged with the desired amount of VO(acac)2 nanocrystals. Single nanocrystals of VO(benzoate)2 and VO(4-nitrobenzoate)2 were structurally analyzedusing a Rigaku XtaLAB Synergy-ED electron diffractometer equippedwith a HyPix-ED detector (see Supporting Information for details).Powder X-ray diffractionmeasurements were performed using a Rigaku XtaLAB Synergy-S Dualflexsingle-crystal diffractometer operating in the powder collection modeusing Cu K\u03b1 radiation. Nanocrystalline powders were affixedto a Nylon loop (0.1 mm inner diameter) with a light coating of viscousoil and mounted on a goniometer for data collection. An FEI TecnaiF20 G2 scanning transmission electron microscope operated at 200 kVand a Zeiss Auriga scanning electron microscope with an InLens detectoroperated at 5\u201320 kV were used to analyze the morphology ofthe VO"}
+{"text": "This protocol was applied to the release of a phosphorylatedserine derivative and the nucleotide analogue AZT monophosphate. Nucleotiderelease in the presence of ATP and a kinase provides a diphosphate,demonstrating that this method can be applied to biological processes.Phosphate mono- anddiesters can be liberated efficiently fromboryl allyloxy (BAO) and related phosphotriesters by H Other phosphate prodrugs5 employ the esterase labile pivaloylmethyl and S-acyl-2-thioethyl groups, while cyclic phosphotriestergroups are activated by acidic hydrolysis and cytochrome P450 oxidation.Phosphate prodrugs addressthis problem 2, as see6 Elevated levels of H2O2 and its abilityto initiate chemical reactions create the potential for its use indrug release, illustrated by the release of fluorophores7 and cytotoxins8 fromboronates in cells and animals. Our prior studies in which boronateoxidation initiates fragmentation reactions that release polar groupsthrough the generation of boron enolates from boryl allyloxy (BAO)groups9 or hemiacetal intermediates from\u03b1-boryl ethers10 led us to explorethe potential of peroxide-mediated phosphate release. Herein we describethe development of a new phosphate group-release strategy based onthe peroxide-mediated oxidation of borylated phosphoesters  followed bythe addition of excess H2O2\u00b7urea (30 equiv)in D2O at 37 \u00b0C. The final concentration of the substratewas 2 mM with a CD3CN:D2O ratio of 7:3 (v/v).The resulting H2O2 concentration is higher thancellular levels,11 though prior relatedstudies10 show that this concentrationpredicts cellular responses. Product concentrations were quantifiedagainst an internal standard. The products of these reactions werevalidated by comparison to independently prepared and characterizedphosphates.127 and 8 areshown in 7 proceededrapidly and efficiently via the intermediate boron enolate, with 94%of the starting material being consumed within 18 min and phosphate 9 being produced in an equal amount. This experiment showeda pseudo-first-order rate constant of 2.3 \u00d7 10\u20133 L mol\u20131\u202fsec\u20131 and a half-lifeof 301 s.12 The reaction with 8 was more complex since two cleavage events are required. Spectraloverlap prohibited monitoring the formation of the phosphomonoester,though acrolein production is easily detected. The reaction showeda rapid consumption of 8, with the concentration of themonocleavage product 10 increasing and then ultimatelybecoming negligible at 24 min. Subsequent NMR analysis confirmed thatthe product was phosphate 11. Acrolein was formed inan 88% yield based on the two equiv that are expected from the cleavageof two BAO groups. The second BAO cleavage is significant, since itshowed that phosphate dianion release is faster than boron enolateprotonation. Additionally, the absence of a significant buildup ofthe monocleavage product indicates that the second cleavage is notsubstantially slower than the first. The pinacol esters partiallyhydrolyze to boronic acid intermediates prior to oxidation, althoughthis does not impact cleavage efficiency. Phosphate release was notobserved in the absence of peroxide or solely in the presence of urea.12Time courses for theoxidative cleavage reactions of Serveral substrates were synthesized and servedto highlight thescope of the process 1. The re12 releases phenylphosphate 13 somewhat more slowly than the breakdownof 8 (entry 1). While 90% of the starting material wasconsumed after18 min, dicleavage product was formed in 63% yield, and 27% of thecorresponding monocleavage product remained. This proceeded to 70%and 23% of the respective products after 25 min.12 We speculate that the rate arises from solubility issues,as turbidity was observed during the course of the reaction. The phosphorothioate 14 releases thiophosphate 15 in 79% yield after18 min and 94% yield after 49 min, with turbidity again being observedas a potential source of the slowed release. Delivering thiophosphates,which are substantially more stable toward enzymatic cleavage thanphosphates,13 is significant, regardlessof the release rate. Phosphodiesters that contain an aryl and an alkylgroup cleave selectively as shown in the conversion of 16 to 17. Aryl phosphates can be cleaved enzymaticallyto release monoalkyl phosphates.14 Compound 18, in which the BPin group is replaced by the recently reportedand more easily handled EPin group,15 releases 9 extremely efficiently, albeit somewhat more slowly. Consumptionwith quantitative product release did not occur until 32 min. Boronate 18 is significantly more hydrolytically stable than 7, which could be useful for applications in which the boronicacid analogue does not readily traverse a cell membrane. The diminishedrate of release will be valuable when a slower phosphate productionis therapeutically beneficial.The phosphotriester 16 leadingto concerns about its formation duringprodrug cleavage, though the release of a glutathione scavenger couldaugment the potency of these agents in numerous applications.17 Although our studies with deliberate acroleinrelease through the cleavage of BAO-containing molecules showed notoxicity,12 the potential for a competitivebiological response inspired us to prepare oxidatively cleavable boronateanalogues that mitigate byproduct electrophilicity. The \u03b1-borylphosphate 19 reacts with peroxide to form 9 within 12 min, indicating that this group reacts even faster thanthe BAO group. Boronates 20 and 22 (secBAO)are homologues of the original vinyl boronate and release methyl vinylketone and crotonaldehyde, respectively, in addition to 21. These byproducts are significantly less electrophilic than acrolein.18 The phosphates were prepared by the additionof the alcohols to dimethylphosphoryl chloride, since the phosphoramiditeprotocol was unsuccessful. Both of these substrates release dimethylphosphate in the presence of H2O2, though 20 showed an alternate, slower mechanism for release in theabsence of peroxide, making structurally similar phosphates unsuitablefor biological applications.12 This studyshows that a range of options exist should byproduct release leadto a competitive biological response.Acrolein is an inhalation toxinthat can sequester glutathione,23 served as a model for the conditionalrelease of phosphoserine. The process was conducted with the initialconcentration of 23 being 0.2 mM in biologically relevantPBS buffer, which is an order of magnitude more dilute than the previousstudies to observe the capacity for oxidative cleavage at low substrateand oxidant concentrations. Serine phosphate 24 was formedin 89% yield after 60 min and quantitatively after 140 min as determinedby HPLC, showing that phosphorylated amino acid units can be releasedunder conditional control in peptides and proteins.19 Insterestingly, replacing the BAO groups of 8 with p-borylbenzyl groups8 resultedin oxidation but only monocleavage.12 Thus,the BAO group is substantially more effective for releasing dianionicphosphates.20Serine derivative 25 in which the boronic acid(HO-BAO group) proved desirable for isolation purposes (211H NMR studies showed that 25 reacts withH2O2 as expected to release AZT monophosphate(AZTMP) 26 in 91% yield after 25 min. Moreover, the negativecontrol phosphate 27 proved to be completely inert underthese conditions.12We explored the potentialfor nucleotide release through the preparationof AZT-derived phosphate purposes 3.211H NM26 in the presence of ATP to form AZT diphosphate 28 and ADP , ATP (10 \u03bcM),and TMPK. Hydrogen peroxide (30 equiv) was added for the oxidativerelease studies of 25. After 1 h CellTiter-Glo reagentwas added, and luminescence was measured after 5 min. The resultsare shown in 28. The negative controls 27 and H2O2-free 25 failed to initiateATP consumption. The combination of 25 and H2O2 showed a level of ATP consumption equivalent to thatof the positive control 26, confirming that oxidativecleavage provides a nucleotide analogue that can engage in enzymaticreactions.Prior to conducting phosphorylationstudies, we showed that ATPis stable to H2O2. The reactions are rapid and efficient, evenat low substrate and peroxide concentrations. Applying the protocolto AZT monophosphate release demonstrated that the released nucleotideis a suitable substrate for enzymatic phosphorylation, validatingthe compatibility of the process with biomolecular transformations.The elevated levels of H2O2 in numerous diseasestates indicate that this approach will be applicable to the developmentof site-selective phosphate-containing prodrugs.We have shown that a range of organoboronates canbe incorporatedinto phosphotriesters to release phosphoesters upon exposure to H"}
+{"text": "APOL1 gene variants prevalent in 13% of this population contributes to the genetic component of the excess risk for nondiabetic kidney failure , Writing \u2013 Review & Editing)."}
+{"text": "Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson\u2019s disease (PD) and Crohn\u2019s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome. Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson\u2019s disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2.Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson\u2019s disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear.To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson\u2019s disease-linked mutations , suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease.Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson\u2019s disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson\u2019s disease. LRRK2 can cause monogenic Parkinson\u2019s disease (PD), and coding and non-coding variants in LRRK2 are associated with increased risk for developing sporadic PD and Crohn\u2019s disease  or VPS35 (D620N). Rab10 phosphorylation was significantly reduced with RAB12 knockdown in LRRK2 R1441G KI and VPS35 D620N KI A549 cells  knock-in, homozygous LRRK2 KO, homozygous RAB12 KO, and homozygous VPS35 D620N (GAT/ATT) knock-in was performed using CRISPR/Cas9. Sequence information for generating targeting gRNA, ssODN donor, and PCR primers are as follows:ice.synthego.com). To isolate monoclonal cell populations, edited cell pools were seeded at 1 cell/well using a single cell printer into 96- or 384-well plates. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and identified using the PCR-Sanger-ICE genotyping strategy described above.CRISPR/Cas9-mediated knockout of LRRK2 or RAB12 and CRISPR/Cas9-mediated knock-in of LRRK2 R1441G or VPS35 D620N in A549 cells was performed by Synthego Corporation . To generate these cells, ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA produced at Synthego were electroporated into the cells using Synthego\u2019s optimized protocol. Editing efficiency was assessed upon recovery, 48 hr post electroporation. Specifically, genomic DNA was extracted from a portion of the cells, PCR amplified, and sequenced using Sanger sequencing. The resulting chromatograms are processed using Synthego Inference of CRISPR edits software  were transduced with lentivirus carrying the transgene cassette for expression of TMEM192-3x-HA. A synthetic cDNA encoding TMEM192-3x-HA was cloned into pLVX-IRES-hygromycin lentiviral vector containing the CMV promoter. Stably expressing cells were selected using resistance to Hygromycin B  supplied in growth medium at 200 \u03bcg/mL for 21 days. Following selection, cells were screened for the stable expression of TMEM192-3x-HA in lysosomes by quantifying the percentage of cells with colocalization of anti-HA and anti-LAMP1 by immunofluorescence, and by monitoring cell lysates for expression TMEM192-3x-HA (~30 kDa) by western blot.For ICC, the following secondary antibodies (Thermo Fisher) were used at 1:1000 dilution: goat anti-mouse Alexa-Fluor 488 (A32723), goat anti-rabbit Alexa-Fluor 568 (A11036).For western blot analysis, the following secondary antibodies  were used at 1:20,000 dilution: IRDyes 800CW donkey anti-rabbit IgG (#926-32213), 680RD donkey anti-mouse IgG (#926-68072).A549 cells were transfected with Dharmacon SMARTpool siRNA targeting 14 Rab GTPases, LRRK2 and non-targeting scramble control , using DharmaFECT 1 . Cells were collected 3 days after transfection for protein or mRNA analysis.The total RNA was extracted from cells using RNeasy Plus Micro Kit . cDNA was synthesized from 1 to 2 \u03bcg of RNA using Superscript IV VILO master mix (Thermo Fisher #11756050). The cDNA was diluted threefold and 1 \u03bcL of diluted cDNA was used as template. To measure the relative expression levels of mRNAs by RT-qPCR, Taqman Fast Advanced Master Mix (Thermo Fisher #4444557) was used, together with gene-specific primers using TaqMan Assays (Thermo Fisher). GAPDH was used as the housekeeping gene. The PCR was run using QuantStudio 6 Flex Real-Time PCR System, 384-well (Thermo Fisher). Gene expression was analyzed using 2^(delta-delta Ct) method with GAPDH as internal controls.LRRK2, pS935 LRRK2, and pT73-Rab10 MSD assays were previously established . BrieflyCells were lysed in lysis buffer  supplemented with cOmplete tablet , phosSTOP (Roche #04906837001), and Benzonase nuclease . Cell lysates were prepared by incubating with NuPage LDS Sample Buffer  and NuPAGE Sample Reducing Agent  for 5 min at 95\u00b0C to denature samples. Lysates were loaded onto NuPAGE 4\u201312% Bis-Tris gels (Thermo Fisher). Proteins were transferred to nitrocellulose membranes  using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with Rockland blocking buffer at room temperature for 1 hr , incubated with primary antibody (diluted in Blocking Buffer) overnight at 4\u00b0C, and then with secondary antibodies  for 1 hr at room temperature. Odyssey CLx Infrared Imaging System (LI-COR) was used for western blot detection and quantitation.HEK293T cells and A549 cells were cultured in DMEM media (Thermo Fisher #11965-092) containing 1% Pen/Strep and 10% FBS . For LLOMe treatment, LLOMe  was added at 1 mM for 2 hr or 4 hr prior to fixation or lysing cells for downstream analysis. Nocodazole  was added at 25 \u03bc\u039c for 2 hr prior to fixation or live-cell imaging. Cells were routinely screened to confirm the absence of mycoplasma contamination.Doxycycline-inducible cell lines were generated to stably express WT Rab12 or a phospho-deficient mutant of Rab12 (S106A) in RAB12 KO A549 cells. Briefly, lentiviral constructs were generated by cloning 3XFLAG-RAB12 (or RAB12 S106A) into the pLVX-TetOne-Puro vector. Lentivirus was produced by transfecting the plasmids in HEK293T cells using Lenti-X Packaging Single Shots . The media containing lentivirus were collected from transfected cells and were further concentrated by 50-fold using Lenti-X Concentrator . RAB12 KO A549 cells were infected with lentivirus expressing WT 3XFLAG-RAB12 or 3XFLAG-RAB12 S106A mutant. Cells carrying the lentiviral vectors were selected with puromycin (1 \u03bcg/mL). To enable the expression of WT Rab12 or the Rab12 S106A mutant, doxycycline  was added in the cell culture for 3 days.x3 transgene as described previously , harvested by scraping into fresh KPBS and pelleted via centrifugation. Cell pellets were resuspended in KPBS + buffer , and cells were fractionated by passing the suspension through a 21 G needle five times followed by centrifugation at 800 \u00d7 g for 10 min. Post-nuclear supernatant (PNS) was harvested and incubated with anti-HA magnetic beads  for 15 min with end-over-end rotation. Lysosome-bound beads were washed three times with KPBS + buffer, and samples used for immunoblotting were eluted from beads by heating to 95\u00b0C for 10 min in 1\u00d7 NuPAGE LDS Sample Buffer (Thermo Fisher).Lysosomes were isolated by immunoprecipitation from cells expressing the TMEM192-HAeviously  with theFor analysis of pRab10 levels on isolated lysosomes, one confluent 15 cm plate of cells was used per experimental condition. Cells were treated with 1 mM LLOMe (or vehicle) for 2 hr at 37\u00b0C prior to isolation of lysosomes via anti-HA immunoprecipitation as described above. Lyso-IP were performed with 60 \u00b5L of anti-HA magnetic bead slurry per condition. Immunoblotting for pRab10 and pRab12 levels was performed in parallel with analysis of total Rab10 and Rab12 levels, as detailed above, using 20% of total immunoprecipitated material per condition. pT73 Rab10, pS106 Rab12, total Rab10, total Rab12, and HA band intensities were quantified from immunoblots using ImageStudio Lite software (LI-COR), and the phospho- and total Rab band intensities were normalized to HA band intensity within each experimental condition. Data were normalized to the median value within each replicate and was then normalized to the mean value of vehicle-treated WT samples across replicates. Calculations for the total fraction of Rab12 present on immunoprecipitated lysosomes were performed by extrapolating the quantitated western blot signal of both the IP and PNS fractions out to 100%, and then calculating the percent of total estimated Rab12 signal captured in the IP divided by the total estimated Rab12 signal present in the PNS sample.For analysis of LRRK2 levels on isolated lysosomes, three confluent 15 cm plates of cells (seeded 24 hr prior to the assay start) were used per experimental condition. Cells were treated with 1 mM LLOMe (or vehicle) for 4 hr at 37\u00b0C and then lysosomes were isolated via anti-HA immunoprecipitation as described above. Lyso-IP were performed with 150 \u00b5L of anti-HA magnetic bead slurry per condition. For immunoblot detection of endogenous LRRK2, 25% of the total immunoprecipitated material (per condition) was loaded onto a 3\u20138% Tris-Acetate gel (Thermo Fisher), fully resolved gels were transferred to nitrocellulose membranes, probed overnight at 4\u00b0C with a 1/500 dilution of mouse anti-LRRK2 , and imaged using standard immunoblotting protocol detailed above. LRRK2 and HA band intensity was quantified from immunoblots using ImageStudio Lite software (LI-COR), LRRK2 intensity was normalized to HA band intensity within each experimental condition, data was normalized to the median value within each replicate, and then was normalized to the mean value of vehicle-treated WT samples across replicates. Calculations for the total fraction of LRRK2 present on immunoprecipitated lysosomes were performed as for Rab12 (see above).WT, RAB12 KO, and LRRK2 KO A549 cells were seeded in 96-well plates , and then treated with vehicle or LLOMe (1 mM). After 2 hr, cells were fixed with 4% PFA for 15 min, permeabilized and blocked with blocking buffer  for 1 hr at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4\u00b0C. pT73 Rab10 antibody , Rab10 antibody , LAMP1 antibody , and HA antibody  were used in the study. After three washes with PBS/0.05% Triton X-100, secondary fluorescently labeled antibodies were diluted in blocking buffer and incubated for 1 hr at room temperature. DAPI (1:1000) and cell mask deep red  were diluted in PBS/0.05% Triton X-100 and incubated for 10 min. After three washes with PBS/0.05% Triton X-100, the cell plates were imaged on an automated confocal high-content imaging system  using a 63\u00d7 water immersion objective lens with excitation lasers  and preset emission filters. Channels were separated to avoid fluorescence crosstalk. A custom analysis was developed in the Harmony 4.9 image analysis software (Revvity) to enable image analysis. For analysis of puncta intensity, pT73 Rab10 or total Rab10 spots were defined using \u2018Finding Spots\u2019 building blocks, and the sum of corrected spot intensity per cell was used to measure puncta signals. The colocalization between pT73 Rab10 or TMEM192-HA puncta and LAMP1-positive lysosomes were measured with object-based analysis. Briefly, pT73 Rab10 or TMEM192-HA and LAMP1 spots were independently defined using separate \u2018Find Spots\u2019 building blocks. Colocalized pT73 Rab10 and LAMP1 spots or TMEM192-HA and LAMP1 spots were identified using the geometric center overlap method within the \u2018Select Population\u2019 tool. The average number of colocalized spots were calculated per cell from 16 fields per well  or 20 fields per well  and averaged across the well.For the colocalization analysis of Rab12 and organelle markers, HEK293T cells were transfected with mCherry-Rab12 plasmid (pcDNA3.1 vector) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were treated with vehicle or LLOMe (1 mM), with or without nocodazole (25 \u03bc\u039c). After 2 hr, cells were fixed and immunostained with a LAMP1 antibody  or GM130 antibody (Abcam ab52649). Cell plates were imaged on an automated confocal high-content imaging system  using a 40\u00d7 water immersion objective lens. For the colocalization analysis of LRRK2 and organelle markers, HEK293T cells stably expressing eGFP-LRRK2 were used. For the colocalization analysis of LRRK2 and Rab12, HEK293T cells stably expressing eGFP-LRRK2 were transfected with mCherry-Rab12 plasmid. After LLOMe and nocodazole treatment, cell plates were imaged using a 63\u00d7 water immersion objective lens.For image analysis, we used PCC to assess the colocalization between various pairs of fluorophores: (1) mCherry-Rab12 and 488 LAMP1-positive lysosomes, (2) mCherry-Rab12 and 647 GM130-positive Golgi, (3) eGFP-LRRK2 and 568 LAMP1-positive lysosomes, (4) eGFP-LRRK2 and 647 GM130-positive Golgi, and (5) mCherry-Rab12 and eGFP-LRRK2, as the PCC is a commonly used intensity-based measurement to quantify colocalization between two fluorophores. A custom analysis algorithm built in the Harmony image analysis software  was used to calculate PCC. For Rab12 or LRRK2 singly expressing cells, individual nuclei were identified with DAPI and cell boundaries were defined using either the mCherry or GFP fluorescent channel, respectively. Rab12-positive and LRRK2-positive cells were selected after thresholding for extremely low or high intensities. Lysosomes and Golgi were independently segmented using the \u2018Find Spots\u2019 building block in Harmony, and PCC for either Rab12 or LRRK2 was calculated on a per-cell basis within these two compartments. The average PCC score was calculated from ~30 fields per well across three independent biological replicates.For Rab12-LRRK2 colocalization, the number of Rab12-LRRK2 co-expressing cells was much lower than their single-expressing counterparts. Therefore, additional steps were taken to manually identify cells that expressed both mCherry-Rab12 and eGFP-LRRK2 at correct levels. mCherry-Rab12 and eGFP-LRRK2 PCC were calculated on a per-cell basis from ~3 wells across three independent biological replicates.Calculation of the percentage of Rab12 and LRRK2 in lysosomes was performed using the Harmony image analysis software  to determine the percentage of mCherry-Rab12 intensity in lysosomes compared to the whole cell. From Rab12-positive cells, lysosomes were segmented using the LAMP1 channel. mCherry-Rab12 sum intensity from lysosomes was calculated and divided by the total sum mCherry-Rab12 intensity within the entire cell. Values were calculated on a per-cell basis, averaged across ~30 fields per well, and across three independent biological replicates. A similar image analysis process was adapted for calculating the percentage of LRRK2 within lysosomes.WT and LRRK2 KO A549 cells were transfected with mCherry Rab12 plasmid (pcDNA3.1 vector) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were treated with vehicle or LLOMe (1 mM). After 2 hr, cells were fixed and immunostained with LAMP1 antibody . Cell plates were imaged on an automated confocal high-content imaging system  using a 63\u00d7 water immersion objective lens.For the image analysis, cells were identified through a nuclear stain (DAPI) and lysosomes were segmented with LAMP1 staining using the \u2018Find Image Region\u2019 building block in the Harmony 5.1 analysis software (Revvity). The total mCherry-Rab12 levels present in lysosomes were determined by calculating the mean fluorescence intensity of the 568 nm channel within the LAMP1 area. Values were measured from mCherry-Rab12 expressing cells  and averaged across wells (~4\u20136 wells per condition).2 and 37\u00b0C condition. The confocal images were taken every 10 min for 90 min in total.HEK293T cells were transfected with eGFP-LRRK2 and mCherry-Rab12 plasmids (pcDNA3.1 vectors) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were incubated with Hoechst 33342  and CellMask Deep Red Plasma membrane Stain  for 10 min. After replacing the cell culture media containing 1 mM LLOMe, the cell plates were immediately started for live-cell imaging on an automated spinning-disk confocal high-content imaging system  using a 40\u00d7 water immersion objective lens under 5% COFields of view containing cells co-transfected for eGFP-LRRK2 and mCherry-Rab12 were manually selected from the time lapse dataset across three independent experiments. For each field, channel, and timepoint, the z-stack was converted to a 2D image by maximum intensity projection, then the background intensity was estimated by smoothing the image with a Gaussian filter with a kernel standard deviation of 50 pixels (~14.8 \u03bcm) using the ndimage module in scipy v1.9.3 . The bacTo better visualize cells co-expressing low levels of LRRK2 and Rab12, the contrast limits were set to between 0 and 125 AU for LRRK2 and between 0 and 600 AU for Rab12. Cells were included in the segmentation if they (1) appeared morphologically healthy, (2) were visible throughout the time series, and (3) co-expressed LRRK2 and Rab12 at levels above background, but below the maximum contrast limit. Cells were segmented using the brush tool in napari using a brush size of 10 pixels (~3.0 \u03bcm). Cells were painted to the edge of the cell border including the nucleus but excluding signal from cell debris and other extracellular sources. Tracking cells across frames was typically possible through a combination of proximity and morphology, but where the assignment was ambiguous, those cells were excluded from further analysis (n=2). The resulting dataset contained 55 segmented cells.For each cell, the non-background subtracted LRRK2 and Rab12 signals were extracted and normalized to between 0.0 and 1.0 by subtracting 200 AU and then dividing by 800 AU. Values above 1.0 or below 0.0 were set to 1.0 or 0.0, respectively. For all pixels under the cell mask, the PCC I was calculated between the normalized LRRK2 and Rab12 signals using the pearsonr function in the scipy.stats module. Cell properties such as area, perimeter, and mean intensity in each channel were extracted for each timepoint using the regionprops function in scikit-image v0.19.3 . Cells wData are shown as mean \u00b1 SEM, and all statistical analysis was performed in GraphPad Prism 9. Unpaired (or paired) t-tests were used for statistical analyses of experiments with two treatment groups. For more than two groups, analysis was performed using one-way analysis of variance (ANOVA) with Tukey\u2019s multiple comparison, one-way ANOVA with Sidak\u2019s multiple comparison test, one-way ANOVA with Dunnett\u2019s multiple comparison test, repeated measures one-way ANOVA with Dunnett\u2019s multiple comparison or two-way ANOVA with Sidak\u2019s test, as indicated in figure legends. Comparisons were considered significant where *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.The pT73 Rab10 antibody used in these studies is available from the corresponding author upon reasonable request. This valuable study shows that Rab12 regulates LRRK2 activation via damaged lysosomes. The strength of evidence supporting the claim is compelling. Although key questions about the mechanism remain unanswered, these findings provide a useful template for further research. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article \"Rab12 regulates LRRK2 activity by promoting its localization to lysosomes\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Tone down conclusions throughout as indicated in the reviews.2. Verify the LRRK2 dependence of activation of rab10 phosphorylation.3. Demonstrate that Rab12 does in fact have a more important role than Rab29.4. Clarify the discrepancy in Figure 4 (see comments).5. The data do not show lysosomal accumulation of rab12 precedes LRRK2 (point #1 above) and clarify Fig. 3 as described by Reviewer #3.Reviewer #1 (Recommendations for the authors):1. Fig. 2G. The A549 R1441G cells show lower kinase activity than wild-type cells. Does this clone have less LRRK2? These cells should also be listed under Key Reagents.2. Figure 3E would benefit from nocodazole addition to most clearly demonstrate the true co-localization of Rab12 and LRRK2 on lysosomes (rather than concentration in a very crowded perinuclear area).3. Line 326. \"Rab12 and LRRK2 showed a primarily cytosolic localization [at baseline].\" Most Rabs are 50% on membranes or bound to GDI in cytosol. The images shown highlight massive concentration in one part of the cell upon LLOME addition and the faint, small-vesicle bound Rab12 would appear very diffuse without actually being non-membrane associated or \"cytosolic\". Perhaps the word \"diffuse\" would be more precise for the Rab?4. In the Lyso-IPs, there seems to be a lot of Rab10 on lysosomes yet by microscopy that is never seen. Perhaps the tag is pulling down some Golgi membranes for biosynthetic trafficking of the HA-tag? It might be worth mentioning that this represents a very small fraction of total Rab10 if that is the case. Also, the LAMP is lost in LLOME samples and this should also be mentioned.eLife paper but this is a short report in a competitive area where other complementary stories will bolster this work (Biorxiv doi: https://doi.org/10.1101/2023.02.17.529028).5. Normally mechanism is required for an Reviewer #3 (Recommendations for the authors):Although the overall role of Rab12 in activating LRRK2 is well supported by the data, there are several areas where the study could be improved.1. Although it is likely that the reported relationship between Rab12 and LRRK2 is generalizable to other cell types, this remains to be shown outside of A549 cells. Unapanta et al, bioRxiv, 2022 showed that Rab38 can also play an important role in activating LRRK2 in melanocytes and other cell types might employ other LRRK2 activation mechanisms. Although it is beyond the scope of this study, it will eventually be important to see how Rab12 KO in a mouse model affects LRRK2 activity across a range of tissues (similar to what the Alessi lab did for Rab29).2. It is puzzling that even though LLoMe-induced lysosome damage does not increase LRRK2-R1441G abundance at lysosomes , it still promotes LRRK2-dependent phosphorylation of Rab10 by LRRK2-R1441G . This result suggests that something more than recruitment to lysosomes is required for Rab12-dependent LRRK2 activation. At a minimum, some discussion of these results and the potential impacts of LRRK2 activation mechanisms would be helpful. The claim in lines 395-397 that these pathogenic variants are unable to further respond to additional lysosomal stress is undermined by the discrepancy between 4B vs 4D.3. Based on data in Figure 4A, it is claimed that lysosome damage causes LRRK2 to accumulate at lysosomes. However, in Figure 4D the same treatment fails to yield a similar increase in LRRK2 levels at lysosomes. These inconsistencies undermine confidence in these results and should be explained.4. In multiple figures, treatment with LLoMe results in a decrease in the amount of LAMP1 that is recovered in the TMEM192-HA \"lysosome\" immunoisolations. This effect was not explained. However, it raises a concern about whether LLoMe is truly triggering LRRK2 recruitment to lysosomes versus the possibility that there is also an effect on TMEM192-HA localization. Is LRRK2 getting recruited to lysosomes? Or is TMEM192-HA moving to a different compartment in the endolysosomal pathway? Some simple experiments that test the colocalization between TMEM192-HA and lysosome proteins +/- LLoMe treatment would help to address this.5. Fig. 3A shows phosphorylated Rab12 at lysosomes but does not show total Rab12 at lysosomes. Showing total Rab12 is required to support claims of regulated recruitment.6. The methods section lacks details for cell line generation and validation. How was Cas9 delivered? How were clones selected? How were mutations in Rab12 assessed?7. Details are lacking on the source and identity of the TMEM192-3xHA plasmid.eLife. Your revised article has been evaluated by Vivek Malhotra (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled \"Rab12 regulates LRRK2 activity by facilitating its localization to lysosomes\" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The main concerns that remained were:1) Title is not appropriate. See below comments from Reviewer #1.2) Some of the data interpretations were not appropriate. All reviewers commented on it.3) Please discuss alternate models, see point 3 from Reviewer #2.Reviewer #1 (Recommendations for the authors):I was really hoping to move this forward; unfortunately, the response to the reviewer comments is not sufficient as follows.1. The authors were asked to modify the title. As it stands, the title implies that Rab12 is on lysosomes which it is normally not, and that LRRK2 is only active on lysosomes which is also not true. The title is really important and either needs to indicate that Rab12 promotes localization to DAMAGED lysosomes or be otherwise modified.2. LRRK2 still produces pRab10 in Rab12 knockout cells. The authors claim on Page 13 that Rab12 is necessary and sufficient for Rab10 phosphorylation but this is not correct. It is clear that LRRK2 still produces pRab10 in cells lacking Rab12 from blots in 2A and 1D. In Figure 2C, the authors show significant variability in Rab10 levels. This highlights the importance of normalizing phosphoRab10 levels to total Rab10 levels. Please normalize data to total Rab10 and not GAPDH or other protein. This is standard in this field.3. The only data on Rab12 being on lysosomes comes from lyso-IPs. In order to understand the magnitude of the process being studied here the authors need to state the fraction of the total Rab12 and total LRRK2 in the LysoIPs of Fig. 3 and 4. This is essential. For example, it is possible that Rab12 increases LRRK2 on lysosomes from 2% of total to 4% of total. The reader needs this information.4. The authors were asked to carry out nocodazole experiments to document true lysosome co-localization. 293T cells are poorly adherent and give poor microscopy images with or without LLOME or nocodazole. Why didn't the authors use their A549 cells as in Fig. 3E? Note that a Golgi marker in these cells would have shown complete co-localization with lysosomes in this scenario-this is why nocodazole is so important to separate membrane compartments. The authors need a clear and well controlled experiment showing that the structures they visualize are lysosomes and not Golgi derived vesicles. This circles back to the title of the paper.Reviewer #2 (Recommendations for the authors):The authors have responded to all of the concerns raised. It is nice to see that LRRK2 is not required for the localization of rab12 to the lysosome. What is very clear is that LLOMe recruits rab12 to the lysosome, induces phosphorylation of rab10 and rab12 is required for phosphorylation of rab10. However, a discrepancy remains between the recruitment of LRRK2 to the lysosome by LLOMe, which is not as apparent for wt LRRK2 in Fig. 4D as in 4A, and the phosphorylation of rab10 induced by LLOMe . So it seems likely, as the authors have now included in the results, that mechanisms other than lysosomal recruitment of LRRK2 contribute to the phosphorylation of rab10. Importantly, they have also shown that LRRK2 is responsible for the induction by LLOMe , so I think this is fine, but they should probably include this important consideration in the discussion as well as the results.Reviewer #3 (Recommendations for the authors):The revised manuscript provides strong evidence that Rab12 can regulate the ability of LRRK2 to phosphorylate Rab10 and that this occurs at least in part by regulating the abundance of LRRK2 at lysosomes. Although key questions about the mechanism remain unanswered, these are still important findings. New data was added to the manuscript that helped to address concerns that were raised during the previous review. Below I have provided some suggestions for clarification to the text that should be addressable without the need for new experiments. However, I also remain concerned about some important results and their interpretation (points 1-).1. There is a discrepancy between the strong decrease in LAMP1 levels in the lysosome IP samples in Figure 3A and that is not seen in the LAMP1 immunofluorescence signal in 3C. This should be acknowledged and/or explained in the text.2. Lines 272-275 imply that previous studies (references 32 and 33) also saw reductions in LAMP1 levels on purified lysosomes following LLoMe treatment. However, I could not find evidence of this in the papers that were cited.3. There is still a discrepancy between Figure 4A-C where LLoMe activates LRRK2 (as measured by phosphor-Rab10) even in the context of VPS35-D620N and LRRK2-R1441G and Figure 4D where LLoMe does not change LRRK2 lysosome abundance in the same cells. The authors touched on this in the response to reviewers but the answer was not definitive. The description and interpretation of these results in the manuscript text is confusing as it is unclear to what extent LRRK2 kinase activity is regulated in response to these stimuli versus LRRK2 localization and proximity to substrates. The data suggests that there might be independent changes to LRRK2 localization and activity. It is not necessary for the authors to solve these puzzles but they should be more clearly presented.4. Line 168: The statement that Rab12 is necessary and sufficient is too strong. Some commentary is required to at minimum acknowledge that this study only examined A549 cells and that the extent that the findings are generalizable to other cell types remains unknown.5. Figure 3A and D and Figure 4A-D should also show the total cell lysates rather than just the IP results.6. Figure 4E and Line 505 over-reach in stating that Rab12 mediates LRRK2 recruitment to lysosomes. Proving this would require evidence that a direct interaction between LRRK2 and Rab12 is necessary for LRRK2 recruitment and activation. Essential revisions:Reviewer #1 (Recommendations for the authors):1. Fig. 2G. The A549 R1441G cells show lower kinase activity than wild-type cells. Does this clone have less LRRK2? These cells should also be listed under Key Reagents.The LRRK2 R1441G KI A549 cells show reduced Rab10 phosphorylation when assessed at the whole cell level. This variant is also associated with reduced LRRK2 levels as assessed by MSD-based analysis in cell lysates as shown in However, analysis of both LRRK2 levels and Rab10 phosphorylation on lysosomes isolated from LRRK2 R1441G KI cells shows an increase in both the lysosomal levels of LRRK2 and pT73 Rab10 with this PD-linked variant. These data demonstrate that the LRRK2 R1441G variant leads to a relative increase in LRRK2 activity at the lysosome.We have updated the Key Reagents list to also include all knock-in and knockout A549 cell models that were employed in this study. We thank the reviewer for pointing this omission out in our initial submission.2. Figure 3E would benefit from nocodazole addition to most clearly demonstrate the true co-localization of Rab12 and LRRK2 on lysosomes (rather than concentration in a very crowded perinuclear area).As suggested by the reviewer, we employed nocodazole treatment to assess whether Rab12 and LRRK2 colocalization is maintained when the clustering of late endosomes/lysosomes in the perinuclear region is disrupted by blocking microtubule assembly. Briefly, HEK293 cells overexpressing mCherry-Rab12 and eGFP-LRRK2 were pre-treated with nocodazole (50 uM) for 2.5 hours and then treated with either vehicle or LLOMe (1mM) for 1.5 hours in the continued presence of nocodazole. The distribution of mCherry-Rab12 and eGFP-LRRK2 was then assessed using high-content confocal imaging. Unfortunately, treatment with both LLOMe and nocodazole caused significant cytotoxicity, rendering us unable to quantify the extent of colocalization of Rab12 and LRRK2 with and without nocodazole treatment following lysosomal membrane permeabilization. We were able to observe a few examples of surviving cells in our studies. In these cells, we found that the apparent colocalization of Rab12 and LRRK2 (overlay in white) following LLOMe treatment was maintained with nocodazole treatment . Using this orthogonal approach, we also observed significant colocalization of pT73 Rab10 with the lysosomal marker LAMP1 following LLOMe treatment that is abolished by RAB12 deletion or LRRK2 deletion. These results provide additional confirmation that LRRK2 promotes Rab10 phosphorylation on lysosomes in a Rab12-dependent manner. As suggested by the reviewer, we have added new text (lines 326-328 of the updated manuscript) to address this point: \u201cWhile Rab10 has been reported to primarily localize to the Golgi and endosomes, our data show that a proportion of Rab10 is localized to lysosomes basally and in response to lysosomal damage.Dev Cell 2020 PMID: 32916093 and Eriksson et al\u00a0Cell Death Dis\u00a02020 PMID: 32409651). Our hypothesis is that the loss of LAMP1 following lysosomal membrane permeabilization may arise from the redistribution of LAMPs to the cytosol in small vesicles and/or targeted degradation via lysophagy, as suggested by the studies highlighted. Whereas LAMP1 levels are robustly reduced following LLOMe treatment, we observed a minimal impact on the levels of exogenously-expressed TMEM192-3xHA in isolated lysosomes . As suggested by the reviewer, we have added new text to our updated manuscript that discusses the reduction in LAMP1 observed following LLOMe treatment and potential explanations for this effect (lines 299-304).The reviewer also astutely noted the loss of LAMP1 detected in our isolated lysosomes following LLOMe treatment. This is an effect we have observed across our Lyso-IP experiments. Our data is consistent with previous studies that have also observed a loss of lysosomal transmembrane proteins following LLOMe treatment .We agree with the reviewer that the observations from this study open up many interesting questions regarding the mechanism by which Rab12 senses lysosomal damage and promotes enhanced LRRK2-dependent phosphorylation of Rab10 on lysosomes. We have submitted this as a short report to communicate our findings in a timely fashion given the competitive nature of this field, and we hope to provide additional mechanistic insight regarding this process in future work.Reviewer #3 (Recommendations for the authors):Although the overall role of Rab12 in activating LRRK2 is well supported by the data, there are several areas where the study could be improved.1. Although it is likely that the reported relationship between Rab12 and LRRK2 is generalizable to other cell types, this remains to be shown outside of A549 cells. Unapanta et al, bioRxiv, 2022 showed that Rab38 can also play an important role in activating LRRK2 in melanocytes and other cell types might employ other LRRK2 activation mechanisms. Although it is beyond the scope of this study, it will eventually be important to see how Rab12 KO in a mouse model affects LRRK2 activity across a range of tissues (similar to what the Alessi lab did for Rab29).in vivo by analyzing LRRK2-dependent Rab10 phosphorylation across different tissues in Rab12 KO mice . The authors observed reductions in phospho-Rab10 levels in Rab12 KO mouse lung with trends toward reduction in the large intestine and kidney. This study supports the relevance of Rab12 as a regulator of LRRK2 activity beyond the A549 cell model used in the present work. The authors could not assess the role for Rab12 in regulating LRRK2 activity in the brain given challenges in detecting pT73 Rab10 in brain tissue, and additional studies in human iPSC-derived CNS cells are warranted to better assess the consequences of Rab12 deletion in the brain. These additional studies will undoubtedly be a focus of future work by us (and others) but are beyond scope for the present manuscript.The reviewer raises a great question regarding whether Rab12 regulates LRRK2 activity in additional cell types beyond the A549 cell model used in the present work. In a recent preprint that also identified Rab12 as a key regulator of LRRK2 activation, the authors characterize the impact of loss of Rab12 2. It is puzzling that even though LLoMe-induced lysosome damage does not increase LRRK2-R1441G abundance at lysosomes , it still promotes LRRK2-dependent phosphorylation of Rab10 by LRRK2-R1441G . This result suggests that something more than recruitment to lysosomes is required for Rab12-dependent LRRK2 activation. At a minimum, some discussion of these results and the potential impacts of LRRK2 activation mechanisms would be helpful. The claim in lines 395-397 that these pathogenic variants are unable to further respond to additional lysosomal stress is undermined by the discrepancy between 4B vs 4D.The reviewer raises an important point regarding the potential mechanisms by which the LRRK2 R1441G variant leads to increased Rab10 phosphorylation at the lysosome and whether additional factors beyond LRRK2 localization may contribute to this effect. We focused on detecting effects on the localization of endogenously-expressed LRRK2 to avoid any potential artifacts caused by LRRK2 overexpression. Because of this, the amount of LRRK2 detected on isolated lysosomes is low, and technical variability in lysosomal isolations across independent experiments can complicate our ability to quantify smaller effects on LRRK2 levels on lysosomes. To add confidence in the effect of lysosomal damage on LRRK2 levels in LRRK2 R1441G and VPS35 D620N cells, we performed two additional, independent replicates. The updated compiled data confirm that both variants have elevated levels of LRRK2 on isolated lysosomes at baseline compared to WT cells and show a non-significant trend of a further increase in LRRK2 levels following LLOMe treatment .These data suggest that increased LRRK2 recruitment to lysosomes upon damage may at least, in part, contribute to elevated Rab10 phosphorylation observed in LRRK2 R1441G and VPS35 D620N KI cells with LLOMe treatment. The extent of lysosomal Rab10 phosphorylation is far greater than the extent of increase in LRRK2 levels following LLOMe treatment, suggesting that additional mechanisms beyond increased recruitment of LRRK2 to lysosomes may contribute to this effect on LRRK2 activation as suggested by the reviewer.In addition to updating Figure 4 with this new data, we have also modified the text to remove our conclusion that LLOMe treatment does not further impact LRRK2 levels in PD-linked variant cells and added new text to the results section (as described above) to highlight that additional mechanisms beyond increased LRRK2 recruitment to lysosomes may also contribute to LRRK2 activation following lysosomal damage in these cells.3. Based on data in Figure 4A, it is claimed that lysosome damage causes LRRK2 to accumulate at lysosomes. However, in Figure 4D the same treatment fails to yield a similar increase in LRRK2 levels at lysosomes. These inconsistencies undermine confidence in these results and should be explained.The reviewer astutely points out that Figure 4A shows a significant increase in LRRK2 levels on lysosomes in WT cells following LLOMe treatment, while Figure 4D shows a non-significant trend toward an increase in lysosomal LRRK2 levels under similar conditions. As described above, we think this variability is due to the low levels of LRRK2 detected on lysosomes upon endogenous expression and technical variability in recovery of lysosomes across experiments using the Lyso-IP method.PNAS 2018 PMID: 30209220, Herbst et al EMBO J 2020 PMID: 32643832, and Bonet-Ponce et al Science Adv 2020 PMID: 33177079), and further supported by orthogonal imaging-based studies , increasing confidence that lysosomal damage causes LRRK2 to accumulate at lysosomes.Our observations are consistent with previous work .The reviewer raises a valid concern regarding whether LLOMe treatment might confound our ability to successfully isolate lysosomes following lysosomal membrane permeabilization. As described in more detail in our response to point #4 from Reviewer #1, we consistently observe a depletion in LAMP1 levels on isolated lysosomes following LLOMe treatment while the lysosomal levels of TMEM192-3xHA were minimally impacted. Our hypothesis is that the loss of LAMP1 following lysosomal membrane permeabilization may arise from the redistribution of LAMPs to the cytosol in small vesicles and/or targeted degradation via lysophagy in response to lysosomal damage, as suggested by Lee et al We thank the reviewer for their suggestion to examine the lysosomal localization of TMEM192-3xHA with and without LLOMe treatment to increase confidence in the use of Lyso-IP to isolated damaged lysosomes. We assessed the colocalization of TMEM192-HA and the lysosomal marker LAMP1 treated with vehicle or LLOMe using high-content confocal imaging. We quantified the extent of colocalization between HA-tagged TMEM192 and LAMP1 and confirmed that LLOMe treatment has no impact on the localization of TMEM192 to lysosomes. This new data add confidence in the use of the Lyso-IP method to isolate damaged lysosomes and has been added to Figure 3- figure supplement 1.5. Fig. 3A shows phosphorylated Rab12 at lysosomes but does not show total Rab12 at lysosomes. Showing total Rab12 is required to support claims of regulated recruitment.We agree with the reviewer that it is important to show total Rab12 levels on isolated lysosomes at baseline and following LLOMe treatment to support our claims of regulated recruitment. This data can be found in Figure 3D of our updated manuscript, showing that Rab12 levels are significantly increased on lysosomes following LLOMe treatment and that this signal is lost in RAB12 KO cells to confirm the specificity of our antibody.6. The methods section lacks details for cell line generation and validation. How was Cas9 delivered? How were clones selected? How were mutations in Rab12 assessed?We thank the reviewer for suggesting that we expand on our methods section with f urther details on how our cell lines were generated and validated. Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA produced at Synthego were electroporated into the cells using Synthego's optimized protocol. Editing efficiency was assessed upon recovery, 48 hours post electroporation. Specifically, genomic DNA was extracted from a portion of the cells, PCR amplified and sequenced using Sanger sequencing. The resulting chromatograms are processed using Synthego Inference of CRISPR edits software (ice.synthego.com). To isolate monoclonal cell populations, edited cell pools were seeded at 1 cell/well using a single cell printer into 96 or 384 well plates. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and validated using the PCR-Sanger-ICE genotyping strategy to confirm successful gene deletion or editing.We have updated our Materials and Methods section to describe how these cell lines were generated and validated.7. Details are lacking on the source and identity of the TMEM192-3xHA plasmid.We have added additional details on the generation of the TMEM192-3xHA plasmid and the construct itself to the Materials and Methods section.Reviewer #1 (Recommendations for the authors):I was really hoping to move this forward; unfortunately, the response to the reviewer comments is not sufficient as follows.1. The authors were asked to modify the title. As it stands, the title implies that Rab12 is on lysosomes which it is normally not, and that LRRK2 is only active on lysosomes which is also not true. The title is really important and either needs to indicate that Rab12 promotes localization to DAMAGED lysosomes or be otherwise modified.We agree with the reviewer that the title for our manuscript is very important and something we want to make sure best describes the findings from our study. Based on the reviewer\u2019s comment, we had modified our title in our last resubmission to \u201cRab12 regulates LRRK2 activity by facilitating its localization to lysosomes\u201d to soften our language around the requirement of Rab12 in mediating LRRK2\u2019s localization to the lysosomes. We now appreciate that this did not fully address the reviewer\u2019s concern around the role that lysosomal damage plays in this recruitment. Based on the reviewer\u2019s feedback, we have further modified our title to \u201cRab12 is a regulator of LRRK2 activity and mediates its localization to damaged lysosomes.\u201d We thank the reviewer for their additional feedback on the title and are happy to further adjust the title as needed.2. LRRK2 still produces pRab10 in Rab12 knockout cells. The authors claim on Page 13 that Rab12 is necessary and sufficient for Rab10 phosphorylation but this is not correct. It is clear that LRRK2 still produces pRab10 in cells lacking Rab12 from blots in 2A and 1D. In Figure 2C, the authors show significant variability in Rab10 levels. This highlights the importance of normalizing phosphoRab10 levels to total Rab10 levels. Please normalize data to total Rab10 and not GAPDH or other protein. This is standard in this field.We appreciate the reviewer\u2019s comments around the requirement of Rab12 for LRRK2-dependent phosphorylation of Rab10, and we have provided new data and further revised the text to address these points. Using our sensitive MSD-based assay to quantify pT73 Rab10 levels under basal conditions, we observed a similar reduction in Rab10 phosphorylation with Rab12 knockdown or deletion as we also observed with LRRK2 knockdown or deletion  which led us to make this comment. As the reviewer noted, we do observe some residual Rab10 phosphorylation upon Rab12 knockdown/deletion when assessed by western blot analysis . This signal is also observed in LRRK2 KO A549 cells, which may be a result of background signal from western blot analysis or may also suggest that a minor proportion of Rab10 phosphorylation may be LRRK2- and Rab12- independent in this cell model as proposed by the reviewer. We appreciate this reviewer\u2019s point and have therefore modified the text to remove any reference to Rab12 being required or sufficient for LRRK2-dependent Rab10 phosphorylation. Specifically, we have changed the subsection heading of the results section on page 13 from \u201cRab12 is necessary and sufficient for LRRK2-dependent phosphorylation of Rab10\" to \u201cRab12 deletion attenuates LRRK2-dependent phosphorylation of Rab10.\u201dThe reviewer also commented on variability in total Rab10 levels across clones examined in Figure 2C and suggested we normalize pRab10 levels to total Rab10 levels. Phospho-Rab10 levels in Figure 2B were measured using a quantitative MSD-based assay and normalized for protein input. Issues with specificity and sensitivity precluded us from using an MSD-based assay against total Rab10, so western blot analysis was used to measure total Rab10 levels. To address the reviewer\u2019s concern, we have now normalized pRab10 levels (quantified using an MSD-based assay) to total Rab10 levels measured using western blot analysis. These new data have been added as a new supplementary figure . These new data confirm our original findings in Figure 2B, strongly suggesting that alterations in Rab10 levels do not explain the significant reduction in pRab10 levels observed in Rab12 KO cells. They are presented on page 13 of the revised manuscript.3. The only data on Rab12 being on lysosomes comes from lyso-IPs. In order to understand the magnitude of the process being studied here the authors need to state the fraction of the total Rab12 and total LRRK2 in the LysoIPs of Fig. 3 and 4. This is essential. For example, it is possible that Rab12 increases LRRK2 on lysosomes from 2% of total to 4% of total. The reader needs this information.We thank the reviewer for these comments and have now added several additional data panels related to them that we feel have strengthened the paper. To address the reviewer\u2019s question regarding the percentage of total LRRK2 and Rab12 present on isolated lysosomes, we quantified LRRK2, Rab12, and HA band intensity in WT cells from both the IP and PNS  samples used for Figure 3D and 4A. We then calculated the total amount of each species present in both sample types by extrapolation based on the amount of sample loaded for western blot analysis and determined the total percentage of each species recovered in the IP. This analysis indicates that ~2% of LRRK2 is on lysosomes at baseline, and LLOMe treatment roughly doubles this to ~4% present on lysosomes. Similarly, ~1% of total Rab12 is present on lysosomes at baseline, and LLOMe treatment increased this to ~1.5% present on lysosomes. This analysis has been added as new data to our updated manuscript in Figure 3- figure supplement 2G and is presented on page 22 of the revised manuscript. Our analysis of total HA recovery in the IP samples confirms that less than 100% of the total HA-labeled lysosomes present in the input were captured in all cases \u2013 by experimental design to ensure equal capture across conditions\u2013 which ultimately suggests that the calculated total LRRK2 and Rab12 levels present on lysosomes are likely underestimates of the true values.We had previously included imaging data that showed Rab12 localization to lysosomes basally that was significantly increased upon LLOMe treatment . As an orthogonal approach to address the reviewer\u2019s question, we quantified the percentage of LRRK2 and Rab12 on lysosomes at baseline and following LLOMe treatment using imaging-based analysis. Specifically, we overexpressed either fluorescently-tagged LRRK2 or Rab12 and quantified the % of LRRK2 or Rab12 on lysosomes by measuring the amount of LRRK2 or Rab12 signal that colocalized with LAMP1 normalized to the total LRRK2 or Rab12 signal per cell. Our imaging-based analysis suggested that ~5% of LRRK2 localized to lysosomes at baseline and that LLOMe significantly increased this to ~6%, and ~12% of Rab12 was observed at lysosomes basally and ~14% following LLOMe treatment. These data have been added as a new figure panel in Figure 3- figure supplement 2H and is presented on page 22 of the revised manuscript. This analysis required overexpression of both LRRK2 and Rab12 which also has its caveats with respect to recapitulating endogenous localization but nonetheless provides a useful complimentary approach.We employed two distinct methods to estimate the percentage of LRRK2 and Rab12 on lysosomes basally and following LLOMe treatment. Both methods support our conclusions that a portion of total LRRK2 and Rab12 localize to lysosomes and that lysosomal damage further increases their lysosomal localization. The potential caveats associated with both approaches make it difficult in the end for us to be confident about the precise quantification of both proteins on lysosomes. Nevertheless, we do agree with the reviewer that this data addresses an essential question and have therefore decided to include it with the caveat as stated on lines 733-738:\u201cThese results also show that a small percentage of Rab12 and LRRK2 are present on lysosomes at baseline and that lysosomal damage leads to a significant increase in the localization of both proteins to the lysosome , but precise quantification of the amount of Rab12 and LRRK2 on lysosomes under these conditions is difficult and warrants further study.\u201d4. The authors were asked to carry out nocodazole experiments to document true lysosome co-localization. 293T cells are poorly adherent and give poor microscopy images with or without LLOME or nocodazole. Why didn't the authors use their A549 cells as in Fig. 3E? Note that a Golgi marker in these cells would have shown complete co-localization with lysosomes in this scenario-this is why nocodazole is so important to separate membrane compartments. The authors need a clear and well controlled experiment showing that the structures they visualize are lysosomes and not Golgi derived vesicles. This circles back to the title of the paper.We thank the reviewer for their suggestion to use nocodazole to confirm the colocalization of LRRK2 and Rab12 and to more clearly understand if LRRK2 and Rab12 are recruited to lysosomes rather than the Golgi upon LLOMe treatment. In line with their suggestions, we have conducted new studies and added new figures to the manuscript that we feel have further strengthened the manuscript.eLife, 2019) and found that reducing the nocodazole treatment to 25 uM for 2 hours reduced toxicity and enabled subsequent analysis. We saw that the Golgi marker GM130 showed fragmented and dispersed punctate structures, confirming the effects of nocodazole treatment in this paradigm.To examine this, we conducted new studies using overexpression of eGFP-tagged LRRK2 and mCherry-tagged Rab12 to follow the localization of each protein at baseline and following lysosomal damage. We had initially tried to use A549 cells, but it turned out to be very challenging to identify a sufficient number of cells expressing both eGFP-LRRK2 and mCherry-Rab12 due to poor transfection efficiency in A549 cells . We therefore used HEK293T cells to address the reviewer\u2019s comment. We further optimized the concentration and duration of nocodazole treatment based on previously-published work  LLOMe treatment significantly increased the colocalization of Rab12 with the lysosomal marker LAMP1 but not with the Golgi marker GM130 (as quantified by Pearson\u2019s correlation coefficient). This co-localization was retained upon nocodazole treatment . These data support our hypothesis that lysosomal damage increases the localization of Rab12 to lysosomes and not Golgi-derived vesicles.2) LLOMe treatment also significantly increased the colocalization of LRRK2 with LAMP1 but not with the Golgi marker GM130 (quantified by Pearson\u2019s correlation coefficient). As was observed with Rab12, this co-localization was retained upon nocodazole treatment . These data add confidence to our conclusions that lysosomal damage increases the localization of LRRK2 to lysosomes.3) To add confidence that Rab12 and LRRK2 colocalize following LLOMe treatment, we quantified their colocalization using Pearson\u2019s correlation coefficient. We observed colocalization between Rab12 and LRRK2 following lysosomal damage and saw that this co-localization was preserved upon nocodazole treatment (new figure 3F). These data further support our conclusion that Rab12 and LRRK2 colocalize with one another following LLOMe treatment.Overall, these data add confidence that a portion of LRRK2 and Rab12 localize to lysosomes a baseline, LLOMe treatment induces increased localization of both LRRK2 and Rab12 to lysosomes but not to the Golgi, and LRRK2 and Rab12 colocalize following lysosomal damage. We thank the reviewer for their suggestion that has further strengthened our conclusions.Reviewer #2 (Recommendations for the authors):The authors have responded to all of the concerns raised. It is nice to see that LRRK2 is not required for the localization of rab12 to the lysosome. What is very clear is that LLOMe recruits rab12 to the lysosome, induces phosphorylation of rab10 and rab12 is required for phosphorylation of rab10. However, a discrepancy remains between the recruitment of LRRK2 to the lysosome by LLOMe, which is not as apparent for wt LRRK2 in Fig. 4D as in 4A, and the phosphorylation of rab10 induced by LLOMe . So it seems likely, as the authors have now included in the results, that mechanisms other than lysosomal recruitment of LRRK2 contribute to the phosphorylation of rab10. Importantly, they have also shown that LRRK2 is responsible for the induction by LLOMe , so I think this is fine, but they should probably include this important consideration in the discussion as well as the results.We are pleased that our previous submission addressed all of the concerns raised by this reviewer and thank them for their suggestions that have substantially improved our manuscript. We agree that additional mechanisms beyond lysosomal recruitment of LRRK2 may contribute to the phosphorylation of Rab10. In addition to the text that was added in the results section around this in our last submission, we have updated the discussion to also include this important consideration as suggested by the reviewer. Specifically, we have added the following text to the Discussion (lines 908-915):\u201cWe cannot rule out that additional mechanisms beyond increased proximity between LRRK2 and its Rab substrates may contribute to Rab10 phosphorylation following lysosomal damage, as the magnitude of Rab10 phosphorylation induced by LLOMe treatment was greater than the extent of LRRK2 recruitment to damaged lysosomes. Additional studies are warranted to determine how lysosomal membrane rupture triggers Rab12 recruitment, to identify other regulatory processes that may contribute to Rab phosphorylation upon lysosomal damage, and to better define how broadly such mechanisms are employed to drive LRRK2 activation in PD.\u201dReviewer #3 (Recommendations for the authors):The revised manuscript provides strong evidence that Rab12 can regulate the ability of LRRK2 to phosphorylate Rab10 and that this occurs at least in part by regulating the abundance of LRRK2 at lysosomes. Although key questions about the mechanism remain unanswered, these are still important findings. New data was added to the manuscript that helped to address concerns that were raised during the previous review. Below I have provided some suggestions for clarification to the text that should be addressable without the need for new experiments. However, I also remain concerned about some important results and their interpretation (points 1-).1. There is a discrepancy between the strong decrease in LAMP1 levels in the lysosome IP samples in Figure 3A and that is not seen in the LAMP1 immunofluorescence signal in 3C. This should be acknowledged and/or explained in the text.The reviewer correctly notes that we did not observe a significant decrease in LAMP1 signal assessed via imaging following LLOMe treatment, in contrast to the decrease observed in isolated lysosomes. LLOMe treatment leads to permeabilization of the lysosomal membrane, and these data may suggest that we are immunopurifying fragments of lysosomal membrane from which LAMP1 has been dissociated or perhaps degraded. We observed strong recruitment of Gal3 to isolated lysosomes following lysosomal damage, supporting some membrane integrity and glycocalyx remain after LLOMe treatment. Our imaging-based analysis did not show obvious differences in LAMP1 signal with LLOMe treatment, likely because our fixation and imaging conditions would capture lysosomal membrane fragments and dissociated LAMP1.To acknowledge this point, we have added the following text to the results section (lines 287-295):\u201cWhile LLOMe treatment reduced the levels of lysosomal-associated membrane protein 1 (LAMP1) in isolated lysosomes, the levels and localization of TMEM192-3xHA, the lysosomal membrane protein used to isolate lysosomes, were not significantly impacted by LLOMe treatment . We did not observe a loss of LAMP1 signal by immunofluorescence analysis, suggesting that LAMP1 may dissociate or be degraded from ruptured lysosomal membranes upon immunopurification. These data suggest that while LLOMe treatment results in lysosomal membrane damage, sufficient lysosomal integrity remains to enable purification of this subcellular compartment using TMEM192.\u201d2. Lines 272-275 imply that previous studies (references 32 and 33) also saw reductions in LAMP1 levels on purified lysosomes following LLoMe treatment. However, I could not find evidence of this in the papers that were cited.We appreciate that the inclusion of references 32 and 33 in our previous draft following our comment around potential explanations for the reduction in LAMP1 levels observed on isolated damaged lysosomes was not clearly elaborated. References 32 and 33 had been previously included to support our hypothesis that LAMP1 levels may be reduced due to enhanced lysophagy or redistribution to small vesicles upon damage. These papers showed that LLOMe treatment led to the redistribution of LAMP2 to the cytoplasm or increased the turnover of LAMP1, respectively. However, based on the great point raised by this reviewer that LAMP1 signal loss was not observed in our imaging-based studies, our speculation is that we are immunopurifying lysosomal membrane fragments from which LAMP1 has dissociated or been degraded. We have revised the text as described above in Point 1 to better reflect our thinking on this and removed reference to these papers entirely in the updated manuscrpt.3. There is still a discrepancy between Figure 4A-C where LLoMe activates LRRK2 (as measured by phosphor-Rab10) even in the context of VPS35-D620N and LRRK2-R1441G and Figure 4D where LLoMe does not change LRRK2 lysosome abundance in the same cells. The authors touched on this in the response to reviewers but the answer was not definitive. The description and interpretation of these results in the manuscript text is confusing as it is unclear to what extent LRRK2 kinase activity is regulated in response to these stimuli versus LRRK2 localization and proximity to substrates. The data suggests that there might be independent changes to LRRK2 localization and activity. It is not necessary for the authors to solve these puzzles but they should be more clearly presented.We sincerely appreciate the reviewer\u2019s feedback that the description and interpretation of our results around the magnitude of increase in Rab10 phosphorylation being greater than the increase in LRRK2 localization to lysosomes observed following LLOMe treatment should be edited to more clearly represent our ideas on the balance between activity versus localization. To further clarify our results and interpretation of our data, we have modified our results section to the following (lines 849-857):\u201cThe levels of LRRK2 on lysosomes were significantly increased in untreated LRRK2 R1441G KI cells and VPS35 D620N KI cells, suggesting that enhanced localization of LRRK2 to lysosomes and proximity to its Rab substrates may contribute to the elevated Rab10 phosphorylation observed on lysosomes at baseline in these cells . Rab10 phosphorylation was increased on lysosomes in response to LLOMe treatment while the levels of LRRK2 were not significantly impacted on lysosomes isolated from LRRK2 R1441G and VPS35 D620N KI cells, suggesting additional mechanisms beyond LRRK2 localization may also contribute to LRRK2 activation in response to lysosomal damage in these cells.\u201dFurther, we have added the following text to the Discussion (lines 908-915):\u201cWe cannot rule out that additional mechanisms beyond increased proximity between LRRK2 and its Rab substrates may contribute to Rab10 phosphorylation following lysosomal damage, as the magnitude of Rab10 phosphorylation induced by LLOMe treatment was greater than the extent of LRRK2 recruitment to damaged lysosomes. Additional studies are warranted to determine how lysosomal membrane rupture triggers Rab12 recruitment, to identify other regulatory processes that may contribute to Rab phosphorylation upon lysosomal damage, and to better define how broadly such mechanisms are employed to drive LRRK2 activation in PD.\u201d4. Line 168: The statement that Rab12 is necessary and sufficient is too strong. Some commentary is required to at minimum acknowledge that this study only examined A549 cells and that the extent that the findings are generalizable to other cell types remains unknown.Based on the reviewer\u2019s suggestions, we have removed our statement regarding Rab12 being necessary and sufficient. Specifically, we have revised the text in the results section (line 165 on page 10) from \u201cRab12 is necessary and sufficient for LRRK2-dependent phosphorylation of Rab10\u201d to \u201cRab12 deletion attenuates LRRK2-dependent phosphorylation of Rab10.\u201d Further, we have added the following text to the Discussion (lines 915-918 on page 31):\u201cAs our work focused on the role of Rab12 in A549 cells, it will also be important to understand whether Rab12 similarly regulates LRRK2 activation and localization to damaged lysosomes in other cell types.\u201d5. Figure 3A and D and Figure 4A-D should also show the total cell lysates rather than just the IP results.We thank the reviewer for their suggestion to include the results from total cell lysates in addition to our IP results. We have included side by side blots showing the western blot data from the immunoprecipitated samples as well as the PNS . These have been added to Figure 3- figure supplement 1B and F and Figure 4-figure supplement 1A-D.6. Figure 4E and Line 505 over-reach in stating that Rab12 mediates LRRK2 recruitment to lysosomes. Proving this would require evidence that a direct interaction between LRRK2 and Rab12 is necessary for LRRK2 recruitment and activation.We appreciate the reviewer\u2019s point regarding softening our language around Rab12 mediating LRRK2 recruitment to lysosomes without direct evidence that an interaction between these proteins is required for LRRK2 recruitment to damaged lysosomes. We have revised Figure 4E from \u201cRab12 mediates LRRK2 recruitment and Rab phosphorylation on lysosomes\u201d to \u201cRab12 regulates LRRK2 recruitment and Rab phosphorylation on lysosomes.\u201d Further, we have revised the sentence in the discussion to remove \u201cmediates\u201d and instead use the word \u201cregulates\u201d:\u201cOur findings provide key insight into the mechanism by which LRRK2 activity is increased in response to lysosomal damage by demonstrating that Rab12 regulates LRRK2 localization to ruptured lysosomes.\u201d"}
+{"text": "Legumes manage both symbiotic (indirect) and non-symbiotic (direct) nitrogen acquisition pathways. Understanding and optimising the direct pathway for nitrate uptake will support greater legume growth and seed yields.2-fixation pathway involving soil-borne rhizobia bacteria, the acquisition of nitrate and ammonia from the soil can also be an important secondary nitrogen source to meet plant N demand. The balance in N delivery between symbiotic N (indirect) and inorganic N uptake (direct) remains less clear over the growing cycle and with the type of legume under cultivation. In fertile, pH balanced agricultural soils, NO3\u2212 is often the predominant form of reduced N available to crop plants and will be a major contributor to whole plant N supply if provided at sufficient levels. The transport processes for NO3\u2212 uptake into legume root cells and its transport between root and shoot tissues involves both high and low-affinity transport systems called HATS and LATS, respectively. These proteins are regulated by external NO3\u2212 availability and by the N status of the cell. Other proteins also play a role in NO3\u2212 transport, including the voltage dependent chloride/nitrate channel family (CLC) and the S-type anion channels of the SLAC/SLAH family. CLC\u2019s are linked to NO3\u2212 transport across the tonoplast of vacuoles and the SLAC/SLAH\u2019s with NO3\u2212 efflux across the plasma membrane and out of the cell. An important step in managing the N requirements of a plant are the mechanisms involved in root N uptake and the subsequent cellular distribution within the plant. In this review, we will present the current knowledge of these proteins and what is understood on how they function in key model legumes . The review will examine their regulation and role in N signalling, discuss how post-translational modification affects NO3\u2212 transport in roots and aerial tissues and its translocation to vegetative tissues and storage/remobilization in reproductive tissues. Lastly, we will present how NO3\u2212influences the autoregulation of nodulation and nitrogen fixation and its role in mitigating salt and other abiotic stresses.Legumes have multiple pathways to acquire reduced nitrogen to grow and set seed. Apart from the symbiotic N The majority of HATS genes and encoded proteins are activated when soil NO3\u2212 concentrations are low    to be utilised. The transport process is mostly an energy dependent (active) process  has been linked to a NO3\u2212 efflux activity in response to ammonium (NH4+) toxicities and the subsequent acidification of the apoplast, while the NO3\u2212/peptide transporter NAXT allows for passive NO3\u2212 efflux across the PM in response to increased acidities around the roots  where homeostatic balance in N supply (direct supply) is offset against the N-dependent regulatory controls of the biological N2-fixing or acquisition symbiosis.The diversity of physiological responses to NON depleted soils via symbiosis with compatible soil rhizobia. The symbiosis results in the development of root nodules that house N2-fixing bacteroids within a cellular environment conducive to the fixation of atmospheric N2 to NH3  to support their N needs independent of the existing symbiosis. Furthermore, high levels of NO3\u2212 and or ammonium (NH4+) in the soil actively inhibits symbiotic N2-fixation fixation (SNF), which could be due to the activity of NO3\u2212 and NH4+ (AMT) transporters in legume roots and nodules . This dichotomy in N acquisition strategy provides flexibility to legumes to secure N to meet plant demand and to adjust to fluctuations in the availability of N in the soil. However, this also creates difficulties in managing the symbiotic partnership, which is negatively impacted by the concentration of reduced N in the soil  inhibition. There were differences in inorganic N uptake among the different legume species in field experiments, which was positively correlated to rapid lateral root expansion and soil colonisation  and their involvement in NO3\u2212 transport in plants and will unravel those activities previously characterised in the model legumes, Medicago truncatula (Medicago), Lotus japonicus (Lotus) and Glycine max (soybean). The review will then explore signalling activities of these transporters for root development and nodulation, their post-translational regulation and role of these transporters in legume nodules and their influence on N2-fixation, and nodule N homeostasis. Tissue NO3\u2212 transport, storage and redistribution will be investigated and their role in the mitigation of different abiotic stresses. We hope this review will highlight where the research gaps exist in this field and where future research is required to better understand this alternative N uptake pathway operating in all legumes.The rate of NO3\u2212 transporters are divided into four general classifications. The first is the large Nitrate Transporter 1/Peptide Transporter (NRT1/PTR/NPF) family , maize  and tomato (LeNrtl-l and LeNrtl-2)  NO3\u2212 transporter genes  was expressed during seed development. In Glycine soja, expression of GsNRT1.57, GsNRT1.96 (NPF 7), GsNRT1.84 (NPF1) are induced by N supply  enhances NO3\u2212 flux into Xenopus laevis oocytes. In plants, MtNPF6.8 expression in roots is enhanced when NO3\u2212 is absent in the growth media and repressed when present. MtNPF6.8 is also considered a transceptor by its ability to also mediate ABA transport and regulate primary root growth , which shows higher nodulation sensitivity to NO3\u2212 than WT plants. Mtnip-1 contains a lesion in the gene MtNIP/LATD (MtNPF1.7), which is an NPF ortholog  show defects in nodule vasculature development and a reduction in bacteroid nitrogenase activity. MtNPF7.6 is suggested to mediate NO3\u2212 uptake from the soil solution to deliver low concentrations to developing nodules through xylem to phloem transfer in vascular tissues. The rate and quantity of NO3\u2212 transport into nodules influences nodule development, possibly through mechanisms involving NO influence of LB activity and oxygen delivery to the bacteroids  showed defective nodule development  show a reduction in N2-fixation but no change to nodulation or nodule number. LjNPF3.1 is expressed in roots and in the nodule outer cortex . Shoot growth in the mutant was compromised but could be recovered at elevated NO3\u2212 supply (5\u00a0mM).The low-affinity NO3\u2212 transport (HATS) in plants. NRT2 transporters share structure similarity with NPFs having 12 transmembrane regions with a large hydrophilic loop between TM 6 and TM 7, although both families do not share sequence homology  was higher in plants grown with NO3\u2212 compared to N-deprived conditions. The expression of GmNRT2.1 and GmNRT2.2 has since been shown to be in the exodermis and epidermis of soybean roots, a similar expression pattern to AtNRT2.1 and AtNRT2.4 . Two NRT2 genes (LjNRT2.1 and CM0161.180 (LjNRT2.7)) showed strong induction while the other two genes (LjNRT2.2 and CM0001.20) showed no response to NO3\u2212  under both symbiotic and non-symbiotic conditions, Ljnrt2.4 mutants displayed significant reduction in shoot biomass, NO3\u2212 content and nitrogenase activity in nodules compared to the inoculated wild type. In Medicago, three NRT2 genes  have been identified with varied expression in roots, shoots and nodules  under minus N conditions or when supplied NO3\u2212   except for AtNRT2.7  . CLC genes have been found in various plants with seven CLC homologues identified in Arabidopsis  has shown similar affinities for NO3\u2212 and Cl\u2212 anions. GcCLC-c2\u2019s affinity for the Cl\u2212 anion is pH independent as compared to GmCLC1, where Cl\u2212 affinity was pH dependent  was downregulated alongside other NO3\u2212 transporter related genes (LjNRT2.1 and LjNRT2.1) in nodulated roots compared to roots without rhizobia inoculation  or suggests a nodulation enhanced repression of gene activity. It will be important to further explore this family to examine their potential involvement in salt tolerance and/or drought and their relationship to root symbiotic N status and whether resupply of NO3\u2212 to nodulated roots would activate their expression and activity.In legumes, GmCLC1 is located on the tonoplast and is induced by water and NaCl stress in soybean leaves and roots  have been shown to be nitrate transporters and located on the symbiosome membrane at late stage of nodule development. Both proteins are not related to NPF or NRT2 and are able to transport both NO3\u2212 and Cl\u22121 . NO3\u2212 sN concentrations. NO3\u2212 can also deregulate the rhizobia symbiosis and impact both root and nodule development in positive (low concentrations) or negative (high concentrations). The Medicago AtNPF6.3 ortholog, MtNPF6.8 has been reported as master nitrate signal sensor in primary root tips  of primary root growth in a Mtnpf6.8 mutant  has also been reported to have a role in ROS homeostasis  might be involved in ABA signalling. In addition to slow root growth and development, the latd mutant also showed abnormal nodule development, with infection thread arrest in root hairs with a rhizobial deprived primordium. ABA application fails to rescue the latd phenotype, suggesting that the nitrate induced nodule regulation by MtNFP1.7 is ABA independent  transcription factors are involved in regulating gene expression of NO3\u2212 transporters and assimilation genes  belonging to RWP-RK family of plant transcritiopn factors have emergered as major players of NO3\u2212 signalling pathways by activating the expression of root derived CLE peptides. Nod factor induces NIN expression which transcriptionally regulates up to three CLE genes, CLE-RS1/2/3 in roots. The encoded peptides are mobile signals that interact with respective receptors in shoots of different legume species including, the Lotus HYPERNODULATION ABERRANT ROOT1 (HAR1)  and Medicago   called NO3\u2212 responsive elements (NREs) located in the promoters of genes, including NRT2.1 and NIR1 , accumulated NO3- promotes the binding of NLP1 to the promoter of NRT2.1, which further increases NRT2.1 expression and consequently NO3- influx into the cell. The increased NO3\u2212 concentration in the cytosol triggers a nuclear enrichment of NLP4 which can then activate the expression of NRE regulated CLE-RS2 genes and the onset of the AON regulatory pathway   receptor. In Medicago, the high-affinity nitrate transporter, MtNRT2.1 has been reported recently in regulation of nodulation  and yield related traits  thus negatively regulating the primary NO3\u2212 response under low NO3\u2212 concentrations, while in contrast CIPK8 kinase acts a positive regulator for the low-affinity NO3\u2212 response  have conserved Ser residues at or near the same position in all NRT members  and ureides exported from determinate nodules .In most legumes, N to different sections of the nodule is important for maintaining N homeostasis and for the delivery of N out of the nodule to support growth for the rest of the plant  and nodule development. One mechanism was proposed where NO3\u2212 derived NO competitively influences the ability of leghemoglobin to bind oxygen and disrupt the delivery of oxygen to actively respiring bacteroids  is also expressed in vascular  cells  show defects in nodule vasculature development and a reduction in bacteroid nitrogenase activity, possibly through an accumulation of NO and a reduction in leghemoglobin expression. MtNPF7.6 is suggested to mediate NO3\u2212 uptake from the soil to deliver low concentrations into developing nodules through a xylem to phloem vascular transfer. A similar mechanism was reported in rice which showed OsNPF2.2\u2019s role in vasculature development of different organs in rice , has a role in nodulation where mutant plants (Mtlatd) produce defective nodules , shoot NO3\u2212 content increased as did leaf area and shoot growth. The disruption of basipetal NO3\u2212 transport did not impact the negative influence of NO3\u2212 on legume nodulation but also didn\u2019t disrupt N2-fixation capacities of the nodules  is expressed in the leaf petiole. AtNRT1.4 is a low-affinity NO3\u2212 transporter which when inactivated reduces the NO3\u2212 content in the petiole while increasing in the leaf lamina. Petiole NO3\u2212 content has been used to monitor N fertiliser demand in some plants  in Glycine soja leaves raises the possiblity that these transporters are involved in leaf NO3\u2212 homeostatis. Further investigations are required to explore their role in the recycling of N metabolites in plants.AtNPF4.6 (AtNRT1.2) transports both NON supply also motivates plants to transfer nutrients from older to younger leaves to support their growth. Both low and high-affinity transporters are involved in this remobilization. In this context, AtNPF2.13 (NRT1.7) mobilises nitrate from old to young leaves via phloem loading  also appear to influence the transfer of NO3\u2212 from old to young leaves (Hsu and Tsay AtNRT2.4 and AtNRT2.5) may also participate in the phloem loading and translocation of NO3\u2212 to aerial tissues. The N inducible, AtNRT2.4 is expressed in the phloem of leaves and its loss of activity reduces leaf NO3\u2212 content  is expressed in vascular tissues of siliques and in the funiculus suggesting a role in delivering NO3\u2212 to developing seeds. Early NO3\u2212 delivery was found to be important at the one to four cell stage of early embryogenesis where loss of supply resulted in abnormal embryo development  has also reported to be involved in NO3\u2013 transport into the embryo at the bent cotyledon stage of developing seeds . The transgenics grew better with significantly higher relative leaf water content and less relative electrolyte leakage than observed in WT plants. Moreover, the concentration of Cl\u2212 ions in the roots of transgenic plants was lower than the controls , a process which can influence external pH  to make it NO3\u2212 selective  was less prominent. This indicated Cl\u2212 uptake activity for both MtNPF\u2019s, with MtNPF6.5 like ZmNPF6.4 as could behave as a Cl\u2212 selective transporter. Under salt stress, mtnpf6.5\u20133 mutants showed reduced Cl\u2212 contents in roots and shoots (48\u201355% and 22\u201326%) than WT while mtnpf6.7 mutants showed Cl\u2212 levels to the WT. Longer primary roots with more lateral roots were developed in the Mtnpf5.6 and Mtnpf6.7 mutants under salt stress. However, in the presence of NO3\u2212 these phenotypic changes were abolished. Recently, the expression of GsNRT2.3, GsNRT2.4, GsNRT1.12, GsNRT1.43, GsNRT1.62 and GsNRT1.57 were found to be upregulated when Glycine soja plants were treated with alkaline salts (NaHCO3)  have been observed in wheat roots (TaNRT2.1)  and MtNRT3.1L2 (MTR_4g104730) help decrease arsenic (As) contamination in plants  declined when the expression of MtNRT3.1 was downregulated. Furthermore, complementation of MtNRT3.1L1 in nrt3.1 mutants showed that NRT3.1 alone or via NRT2.1/NRT3.1 confers As (V) tolerance.Heavy metal contamination poses a great threat to human health due to the potential absorption and incorporation into the food chain  have greatly expanded our knowledge of the processes managing NO3\u2212 transport to support both growth and seed development. The relationship between alternative N acquisition systems (N2-fixation and direct root uptake) are slowly starting to take shape identifying shared signalling pathways managing root and nodule development and the interdependency on alternative reduced N reserves to support early development of legumes subject to a rhizobial inoculation. The important next steps will be to define the regulatory controls limiting both nodulation and soil N to allow these systems to be used effectively together without penalties linked to carbon availabilities. Further work is required to understand the different layers of feedback mechanisms controlling N assimilation and the spatial (cell types) and temporal time periods they operate under. Genetic resources are improving in legumes, which will result in a rapid expansion of knowledge in this space to support the further growth and utilisation of legumes for sustainable protein production and the indirect benefits, through N deposition in the soil, weed and disease management afforded to other crops grown in rotation with legumes.Studies across several model plant systems (ZR. and BNK conceptualised the idea, ZR, MNS and BNK performed the literature survey and wrote the draft manuscript, FP-W provided necessary suggestions, ZR, FP-W and BNK edited and critically evaluated the draft manuscript prior to submission."}
+{"text": "Trem2 expression levels, there is more data available on how the human Trem2 gene functions in the pathophysiology of AD.Recent genetic studies have highlighted the central role of the innate immune system in Alzheimer\u2019s disease (AD) by identifying several risk variants in genes that are exclusively expressed within microglia in the brain. Most notably, rare variants, such as the R47H, R62H and H157Y mutations, in the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) gene increase risk for late-onset AD [Molecular Neurodegeneration, Tran et al. and Qiao et al. push the field of TREM2 biology forward through two uniquely and newly generated mouse models. Tran et al. explore the response of a new Trem2 R47H knock-in mouse model without cryptic splicing to acute demyelination following administration of cuprizone and within the context of early and late amyloidosis [Trem2 H157Y knock-in mouse model to study the role of the H157Y variant within the context of amyloidosis [In two research articles recently published in loidosis . Qiao etTrem2R47H mouse models, including the use of CRISPR/Cas9 and through bacterial artificial chromosome (BAC) technology. It has been reported that Trem2R47H knock-in models created through CRISPR exhibit aberrant splicing and reduced expression levels of Trem2 mRNA [Trem2 mRNA expression, but similar levels of human TREM2 protein in myeloid cells, compared to CV mice [Trem2R47H NSS  model compared to the Trem2R47H CSS (Cryptic Splice Site) model display expression of Trem2 at similar levels to wild type (WT) mice without evidence of cryptic splicing products  plaques in 5XFAD mice and showed that A\u03b2 levels and compaction are brain region-, age- and sex- specific in Trem2R47H NSS mice. Largely, they showed that the Trem2R47H NSS mice exhibited suppressed microglial activation early in disease that conversely shifted to a unique interferon signature with age. The earlier stage also showed impaired microglia-plaque interactions and less compacted plaques with increased dystrophic neurites. This impairment is lost at 12\u00a0months of age  Trem2H157Y/H157Y mice exhibit increased shedding of TREM2 as they displayed higher levels of soluble TREM2 (sTREM2) in serum and brain compared to the heterozygous (het) and WT groups. The hom Trem2H157Y/H157Y mice displayed enhanced A\u03b2 clearance and reduced total A\u03b2 burden at the late stage of A\u03b2 development, but minimal changes in the early stage of A\u03b2 deposition. Interestingly, there were no changes in synaptic markers like synaptophysin but an enhancement in paired-pulse facilitation and strengthened long-term potentiation recordings in the hom Trem2H157Y/H157Y mice compared to WT mice. This data supports a beneficial effect of the H157Y mutation on synaptic plasticity, presynaptic function, and on amyloid burden during the later stage of amyloidosis (Fig.\u00a0Trem2H157Y/H157Y mice have reduced gliosis and decreased Trem2 signaling compared to WT mice.In vivo understanding of a more rare Trem2 mutation the patient carries as not all have the identical effects on AD pathology. As Qiao et al. demonstrate, the Trem2 H157Y mutation did not affect A\u03b2 pathology at the early stage of A\u03b2 development but reduced A\u03b2 burden and increased A\u03b2 clearance at the later stage of amyloidosis. The in vivo effects of the H157Y mutation on A\u03b2 burden differ from what has been observed in vivo utilizing amyloidosis models with the R47H mutation [Trem2R47H NSS mice exhibit suppressed microglial reactivity early in amyloidosis that conversely shifts to a unique interferon signature with age and amyloid accumulation. These effects at the early and later times assessed by Tran et al. also differed based on brain region with noticeable sex differences.Both studies indicate that identifying the optimal time window to intervene in a disease like AD will be key to reach treatment effectiveness when targeting TREM2 function. Additionally, moving forward we may need to consider the specific type of mutation , 9. FurtThese two studies and others also highlight the importance of understanding the type of pathology and pathology severity in the brains of patients with AD when therapeutically targeting TREM2 function. Neuroanatomically, fibrillar A\u03b2 deposition occurs first and most severely in regions such as the precuneus and frontal lobes  during tTrem2H157Y/H157Y mice displayed increased production of sTREM2. It is certainly possible that the increase in sTREM2 is leading to some of these effects. What are the roles of TREM2 and sTREM2 on neuronal activity and function. How will the H157Y mutation and Trem2R47H NSS model affect tau pathology and neurodegeneration? While these and other critical questions remain, the findings from these two exciting papers solidify the growing idea that the effects TREM2 and microglia are context dependent and differ based on disease severity, brain region, age, sex, mutation type and the types of pathologies present. All these factors need to be better studied and considered when designing TREM2 targeting therapies with the goal of mitigating AD and other CNS disease associated pathologies.Overall, the authors\u2019 discoveries provide important new considerations to our approaches for therapeutically targeting TREM2, open new avenues to consider in advancing AD therapeutics and raise several interesting questions for further study. What is it about the H157Y mutation and relationship between microglia, TREM2, sTREM2 and A\u03b2 that results in decreased A\u03b2 when assessed at a late stage of disease? Qiao et al. show that the hom"}
+{"text": "Poststreptococcal glomerulonephritis (PSGN) is caused by prior infection with specific strains of group A streptococcus (GAS). PSGN is commonly characterised by significant complement C3 depression and maintained C4 levels, especially in children. The 2022-23 GAS outbreak has been associated with childhood morbidity and mortality in the UK due to invasive GAS infections but no PSGN outbreak has been described.st, 2019 and March 31st, 2023 and for patients under the age of 18 at the time of the request. We collected demographic information as well as concurrent anti antistreptolysin and anti-DNAase B antibody levels. We excluded 4 individuals who had only marginally decreased C3 levels .We reviewed the laboratory requests for complement C3 and C4 measurements received by the main East of England immunology laboratory for the 48 months between April 1We assigned cases as possible PSGN if C3 was low, C4 was normal and antistreptolysin serologies were not obtained. We assigned cases as probable PSGN if C3 was low, C4 was normal and antistreptolysin and anti-DNAase B antibody levels were elevated.24 unique patients aged 0-18 were identified (18 boys and 6 girls), 8 possible and 16 probable PSGN cases. All but two of the probable cases were boys. There were no cases of possible or probable PSGN between 9.3.20 and 20.3.22. Half of the observed cases have been observed within the last 12 months, mirroring the reported invasive GAS incidence in England.Table of possible and probable Post Streptococcal Glomerulonephritis cases, East of England Immunology LaboratoryPSGN is unusual in industrialized countries. The monitoring of C3 and C4 complement level requests suggests a possible undiagnosed concurrent to the GAS PSGN outbreak in the UK and the circulation of possible nephritogenic strains.All Authors: No reported disclosures"}
+{"text": "Figure 4A since both figures were derived from the same experiment. The original There was an error in Results section, subsection PPI inhibits the growth of gastric cancer cells in vitro and induces their ferroptosis: \u201cIn addition, using a specific fluorescent probe, FerroOrange, we revealed that PPI increased the levels of ferrous ions in both AGS and MKN-45 cells (A correction has been made to the 45 cells \u201d is corr45 cells .\u201dResults section, subsection PPI downregulates NRF2 and FTH1 in the gastric cancer cells: \u201cUsing Western blot assays to detect ferroptosis-related proteins in the cancer cells after PPI treatment in vitro, we found that PPI downregulated the expression of both NRF2 (in vitro, we found that PPI downregulated the expression of both NRF2 and FTH1 (A correction has been made to the oth NRF2  and FTH1oth NRF2  in both and FTH1  in both and FTH1 , indicatThe corrected"}
+{"text": "In this review, we highlight some of the key findings to date in the discovery of PROTACs targeting HDACs by HDAC class and HDAC isoenzyme, current gaps in PROTACs to target HDACs and future outlooks.Over the past three decades, we have witnessed the progression of small molecule chemical probes designed to inhibit the catalytic active site of histone deacetylase (HDAC) enzymes into FDA approved drugs. However, it is only in the past five years we have witnessed the emergence of proteolysis targeting chimeras (PROTACs) capable of promoting the proteasome mediated degradation of HDACs. This is a field still in its infancy, however given the current progress of PROTACs in clinical trials and the fact that FDA approved HDAC drugs are already in the clinic, there is significant potential in developing PROTACs to target HDACs as therapeutics. Beyond therapeutics, PROTACs also serve important applications as chemical probes to interrogate fundamental biology related to HDACs  A review on current proteolysis targeting chimeras (PROTACs) as chemical probes for histone deacetylase (HDAC) enzymes. N-\u03b5-acetyl-l-lysine amino acids in histone proteins. The reverse reaction, histone acetylation, can also be carried out by Histone Acetyltransferases (HATs). Histone acetylation and histone deacetylation play important roles in influencing chromatin structure and gene transcription.1 For example, histones with non-acetylated lysine residues encompass positive charges and are tightly associated with negatively charged DNA, termed heterochromatin.2 Alternatively, histones with acetylated lysine residues, with no charge on the lysine residue, are less tightly associated with DNA and are less compact in histone DNA packing, termed euchromatin.2 Additionally, the acetylated lysine residues on histones also serve as important recruitment sites for bromodomain-containing proteins that can further recruit HATs or chromatin remodelers capable of altering DNA accessibility to transcription factors.3 Hence, the fine balance between HDAC and HAT activities in the cell plays an important role in the regulation of gene expression, and such post-translational modifications are considered important epigenetic markers.4Eighteen histone deacetylase (HDAC) enzymes exist in humans, primarily characterised for their catalysis in the hydrolysis of acetyl groups from 5\u20138 p53, STAT3 and AR are important transcription factors whereby their overexpression or mutation can be associated with many cancers.9\u201311 While \u03b1-tubulin, another important cancer drug target, is a central component of the cytoskeleton and important for cell division.12Despite their role in histone deacetylation, the name Histone Deacetylase can perhaps be deceptive as the substrate specificity of HDACs can expand beyond histone proteins to non-histones such as p53, signal transducer and activator of transcription 3 (STAT3), the androgen receptor (AR) and \u03b1-tubulin as a few select examples.13 Of the eighteen HDAC enzymes in humans, eleven perform catalysis utilising a divalent Zn2+ ion in the HDAC active site, while the remaining seven are NAD+-dependent and termed Sirtuins (SIRTs).14 HDACs can be further subdivided into classes based on their sequence similarity to yeast HDACs Hda1 and Rpd3. The eleven-zinc dependent HDACs encompass class I , class II  and class IV (HDAC11), and the seven SIRTs encompass class III (SIRT1\u2013SIRT7). Class II can be further subdivided into class IIa  and class IIb (HDAC6 and HDAC10). These isoforms differ in their sub-cellular locations, catalytic activities and substrate specificities. For example, HDAC1, HDAC2 and HDAC3 are predominantly localised in the nucleus while the majority of other zinc-dependent HDACs can be found in either the nucleus or cytoplasm.15Higher eukaryotic organisms typically contain more HDAC isoforms, the common fruit fly only encodes 5 HDAC genes in comparison to the 18 HDACs in humans.16 In comparison, since the approval of the first rationally designed kinase inhibitor imatinib in 2001, over 74 small molecule inhibitors targeting kinases gained FDA approved by the end of 2021.17 One hurdle to the potential progression of new HDAC inhibitors is perhaps the toxicities that have been associated with current FDA-approved HDAC drugs. Side effects experienced by patients taking vorinostat include fatigue, gastrointestinal upset, thrombocytopenia and QT interval prolongation,18 more akin to a non-selective cytotoxic agent than a targeted cancer therapeutic. Many have hypothesised that these side effects are linked to the pan-HDAC inhibition of all eleven zinc-dependent HDAC enzymes by inhibitors such as vorinostat.16,19\u201322 It could be envisaged the global change in the acetylome caused by pan-HDAC inhibition could be leading to these undesired side effects. However, it is also important to note that the hydroxamic acid functional group, incorporated in three of four FDA approved HDAC targeting drugs, have also been associated with mutagenicity.23Given the importance of HDACs in gene transcription it is perhaps no surprise that a number of HDAC inhibitors have gained FDA approval . Vorinos16,19\u201322 The literature on small molecule HDAC inhibitors is vast and beyond the scope of this review. We recommend the review by Ho et al. for a comprehensive account of HDAC inhibitor design and development over the past thirty years.24 At the time of writing this review 135 clinical trials have been reported as active or recruiting in clinicaltrials.gov related to the search term \u2018Histone Deacetylase\u2019.25 Many of these trials are combination therapies with FDA approved HDAC inhibitors, but also contain new HDAC inhibitors, and hybrid dual functionalised HDAC inhibitors designed to target HDACs and other protein drug targets including kinases.26 Intriguingly, current HDAC inhibitors targeting a singular HDAC isoform in clinical trials are mainly represented by HDAC6.Despite this there is still hope and enthusiasm that the discovery of isoform selective HDAC inhibitors will lead to enhanced therapeutic efficacies with reduced side effects for specific diseases related to certain HDAC isoforms.in vivo in seven differing corepressor complexes adding an additional layer of complexity.27 The differing corepressor complexes each have distinct physiological roles in the cell, and hence targeting a HDAC containing corepressor complex with selectivity may also be essential to discovering new HDAC therapeutics with greater therapeutic efficacies and reduced side effects.28 It will be truly interesting to see how the future of Proteolysis Targeting Chimeras (PROTACs) can contribute to this area.Beyond isoform selectivity, HDAC1, HDAC2 and HDAC3 exist 29\u201331 Chemical probes have also proved important for validating the role of specific proteins in disease and their potential as drug targets.32,33 Most chemical probes are rationally designed to inhibit protein function. However, the discovery of cell permeable molecules, not only capable of protein inhibition, but also degradation of the target protein has created a new area of research in chemical probes.34\u201336 Perhaps the most well-known examples of this being proteolysis targeting chimeras (PROTACs). However, given the success of this strategy other protein degradation strategies such as molecular glues, lysosome-targeting chimeras (LyTACs), autophagosome-tethering compounds (ATTECs) and antibody-based PROTACs (AbTACs) are also being actively explored.37\u201339 To date, the majority of protein degradation strategies targeting HDACs has been focused on the design of PROTACs which will also be the focus of this review, however Toriki et al. recently reported molecular glues targeting HDAC1 and HDAC3 for degradation.40Small molecule chemical probes that exhibit high affinity and selectivity for their intended target protein serve important applications in addressing fundamental biological questions related to the target protein.41 the formation of a stabilised and cooperative ternary complex is often highlighted as important for degradation.42 This unique mechanism of action can lead to significantly enhanced protein selectivity. For example, the bromodomain inhibitor JQ1 is a pan-BET inhibitor, however when JQ1 is incorporated into a PROTAC, this results in the selective degradation of BRD4 over BRD2 and BRD3.43 Foretinib, a pan-kinase inhibitor, was found to bind 133 kinases of the 428 kinases screened by Bondeson et al.,44 however when functionalised into PROTAC 1 (utilising the Von Hippel-Lindau E3-ligand) it retained binding to just 52 of the 133 kinases  of 210 nM for p38\u03b1. The stability of the ternary complex formed between 1, p38\u03b1 and the VHL ligase complex was highlighted as more important for degradation than the binding affinity between p38\u03b1 and 1. Further studies revealed that modifying the connectivity of the VHL ligand to the linker in PROTAC 2, while maintaining the same foretinib POI binding ligand, can modify the selectivity of degradation for the p38\u03b4 isoform over previously degraded p38\u03b1.45 These are just a few examples of how PROTAC-mediated degradation can alter protein selectivity, and there are many recent and comprehensive reviews available on PROTACs.46\u201348PROTACs designed to degrade their target protein typically contain three components: a ligand to engage the protein of interest (POI), a ligand to initiate protein degradation (most commonly an E3-ligand), and a linker covalently bonding these two ligands. PROTACs result in poly-ubiquitination of the lysine amino acids on the POI resulting in eventual degradation by the proteasome. This transfer of ubiquitin to the POI requires the formation of a protein\u2013protein interaction between the POI and E3-ligase mediated by the PROTAC, kinases . In prot+-dependent SIRT2 published online in 2017.49 The first zinc-dependent HDAC targeting PROTAC was reported for HDAC6 in 2018.50 It is important to note PROTACs also have promise beyond chemical probes with approximately 20 PROTACs progressing to or already in clinical trials.46 The most prevalent protein targets for these PROTACs being AR, estrogen receptor (ER), Bruton's tyrosine kinase (BTK) and interleukin-1 receptor-associated kinase 4 (IRAK4). To date, as of writing, no PROTACs targeting HDACs are in clinical trials.Given such remarkable findings in modifying protein selectivity by utilising PROTAC mediated degradation over enzyme/receptor inhibition it is no surprise researchers wishing to modulate HDAC activity have been drawn to the PROTAC field. The first PROTAC targeting a Histone Deacetylase enzyme was NAD28 HDAC1 and HDAC2 share 83% amino acid sequence identity and exist in vivo interchangeably as components of six corepressor complexes , while HDAC3 exists in the SMRT/NCoR complex.28 HDAC8, unlike HDAC1-3, is not known to exist in corepressor complexes.Class I HDACs encompass HDAC1, HDAC2, HDAC3 and HDAC8. HDAC1, HDAC2 and HDAC3 are predominantly localised in the nucleus of the cell, while HDAC8 can be found in both the nucleus and cytoplasm.51 while HDAC3 has recently been shown to have a critical role in tumour development in Kras-mutant non-small cell lung cancer.52 HDAC8 has been found to be overexpressed in a number of cancers.53 To date, a number of PROTACs have been reported capable of degrading class I HDACs.54\u201368Of the HDAC family, HDAC1\u2013HDAC3 are attractive drug targets given that they are nucleus-localised and hence likely the most prominent HDACs in regulating histone deacetylation and gene transcription. The selective targeting of HDAC1 and HDAC2 has been highlighted as a potential strategy for treating B-cell acute lymphoblastic leukaemia (B-ALL),et al. carried out a proteomics study investigating HDAC degradation in the presence of 48 PROTACs that varied in HDAC ligand, linker length and E3 ligand.56 In this study HDAC1, HDAC2 and HDAC9 were the least frequently identified HDAC isoforms to undergo proteasome mediated degradation of 9 of the 11 zinc-dependent HDACs on PROTAC treatment (HDAC10 and HDAC11 are in low abundance in the KELLY cell line used in the study). On the other side of the spectrum, HDAC6, HDAC8 and HDAC3 were the most frequently identified HDAC isoforms to undergo proteasome mediated degradation with the 48 PROTACs used in the study. This seems to be the challenge in generating HDAC1 and HDAC2 selective PROTACs, separating HDAC1 and HDAC2 degradation from other isoform degradation such as HDAC3 and HDAC6 degradation, the latter of which seem more susceptible to PROTAC mediated degradation, and there are other examples of this beyond the study by Xiong et al.Although PROTACs have been reported capable of degrading HDAC1 and HDAC2 achieving the selective degradation of these isoforms has proved challenging. Xiong et al. were able to synthesise PROTAC 3, which was capable of degrading HDAC1 at a concentration of 10 \u03bcM in the acute myeloid leukaemia HL-60 cell line  approach Sinatra ell line .57 Howev50 value of 0.55 \u03bcM for HDAC1 and still degraded HDAC2 at low micromolar concentrations, while 5 exhibited DC50 values of 0.91 \u03bcM and 4.19 \u03bcM respectively for HDAC1 and HDAC2.58 These PROTACs are also effective degraders of HDAC3 , however we noticed a significant observation in that these PROTACs exhibit a hook effect for HDAC3. At concentrations greater than 1 \u03bcM HDAC3 abundance starts to increase rather than decrease, while reduced HDAC1 and HDAC2 abundance was still maintained at concentrations greater than 1 \u03bcM. This effect was also time dependent and at 36 hours HDAC3 abundance started to reduce again with 4 meaning caution must be taken not only with the PROTAC concentration used but also with time-dependent degradation. PROTACs 4 and 5 were capable of compromising cell viability in HCT116 cells with single-digit micromolar IC50 values, however they were only marginally enhanced in potency compared to the benzamide class I HDAC inhibitor CI-994. In a separate study, we investigated utilising the class I HDAC inhibitor entinsotat as inspiration for PROTAC 6.59 PROTAC 6 encompassing the triazole and carbamate moiety was also a degrader of HDAC1, HDAC2 and HDAC3 in HCT116 cells, but was reduced in degradation potency (HDAC1 DC50 = 2.8 \u03bcM) compared to 4 and 5. PROTAC 6 also exhibited a similar hook effect for HDAC3 although at higher concentrations, above 2.5 \u03bcM.PROTAC 4 exhibited a DCvice versa given the high amino acid sequence similarity between HDAC1 and HDAC2. With PROTACs 4 and 5 the dose response curves for HDAC1 and HDAC2 degradation mirror one another, however we have consistently observed greater HDAC1 degradation levels over HDAC2 degradation,54,58,59 at least suggesting this may be viable. Regards targeting HDAC1 and HDAC2 for degradation, there are PROTACs reported that degrade HDAC1 and HDAC2, however not with complete selectivity and work still needs to be done to fine tune HDAC1 and HDAC2 degradation over the degradation of other HDAC isoforms.A real feat of the PROTAC technology would to be selectively degrade HDAC1 over HDAC2 or 56,58,60,61 Using a hydrazide pan HDAC1-3 inhibitor, an uncommon HDAC ligand, Xiao et al. were able to synthesise and identify a HDAC3 selective degrader, 7, incorporating an alkyl linker and a VHL ligand in the PROTAC  compared to 4 and 5 that also degrade HDAC1 and HDAC2. 11 also did not compromise cell viability in HCT116 cells to the same levels as PROTACs 4 and 5 and the pan-HDAC1-3 inhibitor CI-994.In optimisation studies of PROTACs targeting HDAC1-3 for degradation we modified the attachment of the VHL E3-ligand to the linker in PROTAC 11.The selective degradation of HDAC3 by PROTACs seems to be achievable. PROTACs that recruit the VHL E3 ligase are more likely to promote the degradation of HDAC3. However, it is also important to note that the choice of cell line can have a crucial effect on the HDAC isoform degradation outcome.in vivo.56,62 One of few studies was carried out by Xiong et al., investigating the effects of 48 PROTACs on HDAC1-3 containing corepressor complexes by proteomics.55 Reduced abundance of GPS2, NCoR1 and NCoR2, components of the HDAC3-SMRT/NCoR complex, coincided with PROTACs capable of degrading HDAC3, including PROTACs 9 and 10. The reduced abundance of these HDAC3-SMRT/NCoR corepressor complex components was much more prominent with PROTACs recruiting the VHL E3 ligase compared to PROTACs recruiting the cereblon E3 ligase (HDAC ligand dacinostat). Intriguingly, MIER1\u20133, components of HDAC1/2 containing corepressor complexes, were also reduced in abundance with certain PROTACs, including 9 and 10, despite no observed significant reduction in HDAC1 and HDAC2 abundance. Three PROTACs recruiting the IAP E3 ligase were capable of reducing the abundance of GSE1 and HMG20B components of the CoREST complex. The authors hypothesised that such PROTACs can engage HDAC1 and HDAC2 in these corepressor complexes but only result in the degradation of these specific complex components and not the degradation of HDAC1 and HDAC2 themselves.Very few studies have investigated the effects of HDAC targeting PROTACs on the corepressor complexes that HDAC1, HDAC2 and HDAC3 exist 62 We compared the gene expression profile of multiple components of all seven HDAC1\u20133 containing corepressor complexes. Although not considered differentially expressed genes, genes of the HDAC3-SMRT/NCoR complex TBL1RX1, TBL1X and NCoR1 were exclusively only upregulated in the presence of the HDAC3 selective PROTAC 11, highlighting again, similar to the results by Xiong et al., that this corepressor complex does seem to be affected as whole by PROTAC-mediated degradation.We performed RNA-seq experiments with the HDAC1\u20133 inhibitor CI-994 and seven PROTACs capable of degrading HDAC1\u20133 with different degradation profiles side-by-side .More efforts are needed to investigate the effects of PROTAC-mediated degradation on corepressor complexes. This is a real advantage that the PROTAC-mediated degradation of HDACs could offer over HDAC inhibitors. However, discovering such PROTACs for HDAC1/2 complexes is non-trivial as HDAC1 and HDAC2 exist in six different corepressor complexes interchangeably, with each individual corepressor complex encompassing multiple protein partners. Additionally, the selective degradation of one of the six HDAC1/2 corepressor complexes may have minimal effects on HDAC1 or HDAC2 abundance overall, and such PROTACs could easily be overlooked.63\u201368 Again, this perhaps reflects the observation in the proteomic study by Xiong et al. that HDAC8 is one of the HDAC isoforms more prone to PROTAC mediated degradation along with HDAC3 and HDAC6. Utilising a previously developed selective HDAC8 inhibitor Chotitumnavee et al. were able to synthesise a selective PROTAC degrader of HDAC8  in comparison to HDAC8 inhibition and degradation by 12 . The authors noted PROTAC 12 also reduced levels of IKZF1 a known neo-substrate for the cereblon E3 ligase, however the E3-ligand pomalidomide alone did not show comparable potency in compromising cell viability. This perhaps suggests that the degradation of HDAC8 by PROTACs may be more effective than the selective inhibition of HDAC8 in compromising cell viability.A number of studies have now been reported on the PROTAC mediated degradation of HDAC8.of HDAC8 .64 PROTAet al. utilised a dual HDAC6-HDAC8 inhibitor for PROTAC design.65 Intriguingly, although the inhibitor from which PROTAC 13 was designed, is a more potent HDAC6 inhibitor than HDAC8 inhibitor, 13 was capable of approx. 30-fold more potent HDAC8 degradation over HDAC6 degradation in HCT-116 cells. 13 exhibited DC50 values of 147 nM and 4.95 \u03bcM respectively for HDAC6 and HDAC8. The authors suggest that 13 is more effective at forming a ternary complex with HDAC8 over HDAC6. No degradation of HDAC1 or HDAC3 was observed in the presence of 13. Acetylation of SMC-3 a known substrate for HDAC8 was also observed. Degradation with 13 was time-dependent with degradation initiated after 2 hours, reaching maximum HDAC8 degradation levels at 10 hours, and HDAC8 levels started to rise again at 24 hours. No cell viability studies were reported with 13.Sun et al. synthesised a series of PROTACs designed to selectively target HDAC8 for degradation in neuroblastoma cells, utilising a selective HDAC8 inhibitor as the basis for PROTAC design.66 PROTAC 14 was capable of dose-dependent HDAC8 degradation with a Dmax value (maximum percentage degradation) of 70% for HDAC8 at 10 \u03bcM and no degradation of HDAC1 or HDAC6 was observed. Degradation was again also time-dependent, with the maximum degradation of HDAC8 exhibited at 6 hours and recovery of HDAC8 to basal levels noted after 48 hours. Comparative VHL recruiting analogues of 14 exhibited no significant HDAC8 degradation. PROTAC 14 was also capable of increasing the acetylation of SMC-3 and exhibited anti-neuroblastoma activity.Darwish et al. utilised an indole scaffold, again a selective inhibitor of HDAC8, for the development of PROTACs to target HDAC8.67 Unique to this study VHL based PROTACs were capable of effectively degrading HDAC8, however they were again outperformed by cereblon recruiting PROTACs. PROTAC 15 exhibited a DC50 of 0.58 \u03bcM for HDAC8 in A549 cells with a Dmax value greater than 95%, and maximum HDAC8 degradation was observed at 18 hours. Again, similar to the results observed by Chotitumnavee et al. PROTACs outperformed the selective HDAC8 inhibitors from which they were originally derived in compromising cell viability in A549 cells. PROTAC 15 was also capable of long duration tumour regression in an A549 tumour mouse model.Huang et al. reported a highly potent HDAC8 targeting PROTAC with a single digit nanomolar DC50 value of 1.8 nM and Dmax value of 97% in MDA-MB-231 breast cancer cell line.6816 was also a degrader of HDAC6 but with a higher DC50 value of 38 nM. Rapid HDAC8 degradation was observed at 4 hours with 16, and HDAC8 abundance was reduced up to 48 hours after treatment. When 16 was investigated in jurkat cells the DC50 value modestly increased to 4.7 nM for HDAC8 and 78.5 nM for HDAC6 highlighting a modest drop in potency and HDAC8 selectivity in a different cell line. Although 16 only exhibited weak anti-proliferative activity in MDA-MB-231 cells it still outperformed the selective HDAC8 inhibitor from which it was derived and also inhibited migration of MDA-MB-231 cells whereby the selective HDAC8 inhibitor did not. In cell viability assays, and assays investigating apoptosis, 16 was more effective than its parent selective HDAC8 inhibitor. However, at the effective micromolar concentrations needed in these assays concurrent HDAC6 degradation with HDAC8 degradation may not necessarily be ruled out.Zhao 69 Li et al. reported that the treatment of HEK293 cells with the proteasome inhibitor MG132 results in an increased abundance of HDAC4, HDAC5, and HDAC7, with HDAC7 levels even further elevated again comparatively to HDAC4 and HDAC5.70 This suggests these HDAC enzymes are prone to proteasome mediated degradation in cells in their own right. Again, this finding correlates well with the proteomic study by Xiong et al.,56 after HDAC6, HDAC3 and HDAC8, the isoforms HDAC7, HDAC5 and HDAC4 respectively were the most frequently identified to be in reduced abundance on PROTAC treatment, with no PROTACs reported to reduce the abundance of HDAC9. In terms of gaining isoform selective degradation by PROTAC treatment within the class IIa enzymes only HDAC4 selective PROTACs have been reported to date.Class IIa HDACs encompass HDAC4, HDAC5, HDAC7 and HDAC9. These HDACs can be found in both the nucleus and cytoplasm and they exhibit significantly reduced deacetylase enzymatic activity on histone proteins compared to class I HDACs.et al. reported the first HDAC4 selective PROTACs to investigate the role of HDAC4 in Huntington's Disease  HDAC inhibitor, reported previously by Scott et al.73Macabuag  Disease .71 They 50 values of the hydroxamic acid-based PROTACs ranged from 28\u201348 nM with corresponding Dmax values of 77-83%. The TFMO-based PROTACs produced DC50 values ranging from 3\u20134 nM with associated Dmax values ranging from 72\u201385%. Interestingly, HDAC4 degradation efficiency was not impacted by varying the PEG linker length. PROTACs 17 and 18, which are representative degraders from each series, were capable of producing a dose-dependent degradation of HDAC4 in Jurkat E6-1 cells. Additionally, PROTAC 17 exhibited the hook effect at 10 \u03bcM. By employing multiplexed western blotting, Macabuag et al. evaluated isoform-selectivity of PROTACs 17 and 18 for HDAC4 over other class IIa HDAC isoforms, whereby Jurkat E6-1 cells were treated with the degrader compounds over 24 h at increasing concentrations. PROTACs 17 and 18 exhibited a dose-dependent degradation of HDAC4, while no degradation of HDAC1, HDAC5, HDAC7 or HDAC9 was observed; however, minor degradation of HDAC3 was noted for PROTAC 18 at 10 \u03bcM. The study proved that PROTACs 17 and 18 induced HDAC4-selective degradation with no degradation of other class IIa isoforms. Macabuag et al. also demonstrated concentration-dependent degradation of HDAC4 in mouse neuroblastoma cells (Neuro-2a) over 24 h with PROTAC 18.71 However, a 20-fold decrease in degradation efficiency was noted in Neuro-2a cells in comparison to the Jurkat cell line. Yet, again, demonstrating how the cell type can influence the degradation efficiency of the PROTAC.The DCet al. it was discovered that the selective degradation of HDAC6 was observed over the other eleven zinc-dependent HDACs (50 Given this result, and the number of potent HDAC6 PROTACs reported to date in the literature, it does seem that HDAC6 is the most amenable zinc dependent HDAC isoform to proteasome mediated degradation by PROTACs. Of the HDAC isoforms HDAC6 does contain a zinc-finger ubiquitin-binding domain,74 and it is tempting to speculate that this ubiquitin binding domain is a potential reason why HDAC6 is so amenable to proteasome mediated degradation by PROTACs over other HDAC isoforms. In direct comparison there are single digit nanomolar inhibitors reported for HDAC10,75 however to date, as of writing, no PROTACs targeting HDAC10 for degradation have been reported.Class IIb HDACs incorporate HDAC6 and HDAC10. Of the eleven-zinc dependent HDAC enzymes PROTACs targeting HDAC6 for degradation are probably the most frequently reported in the literature to date. When the first hydroxamic acid pan-zinc dependent HDAC inhibitor was incorporated into PROTAC 19 by K. Yang nt HDACs .50 Givenet al. reported the selective degradation of HDAC6 with PROTAC 19 incorporating a pan-HDAC inhibitor as the HDAC ligand,50 An et al. utilised the selective HDAC6 inhibitor Nexturastat A for inspiration in designing PROTACs to target HDAC6.76 PROTAC 20 incorporating Nexturastat A as the HDAC ligand and pomalidomide as the E3-ligand exhibited an impressive DC50 value of 3.8 nM in the B lymphoblast MM.1S. cell line (50 value of 1.21 \u03bcM compared to a value of 2.25 \u03bcM for Nexturastat A.After K. Yang ell line . No degret al. reported a potent and selective HDAC6 degrader 21 also utilising Nexturastat A as the HDAC ligand,77 with a different linker attachment position to the ligand to PROTAC 20. Again, PROTAC 21 exhibited a single digit nanomolar DC50 value of 1.6 nM in MM.1S cells and could initiate HDAC6 degradation within 1 hour of treatment. PROTAC 21 also degraded IKZF1/3 in a dose dependent manner, known neo-substrates of PROTACs incorporating thalidomide analogues to recruit the cereblon E3 ligase. Above concentrations of 100 nM this dual HDAC6 and IKZF1/3 degradation by 21 was found to be responsible for enhanced antiproliferation effects in MM.1S cells. To abolish IKZF1/3 degradation, K. Yang et al. were able to optimise cereblon recruiting E3-ligands and incorporate them into PROTACs such as 22 to promote the selective degradation of HDAC6 without the co-degradation of IKZFs.78 Highlighting that IKZF degradation does not always have to coincide with PROTACs incorporating cereblon recruiting E3-ligases. In a different study by H. Yang et al., the authors were able to demonstrate that alteration of the linker connectivity to Nexturastat A in PROTAC 23 still maintained potent DC50 values directly comparable to other HDAC6 targeting PROTACs.79Wu et al. reported PROTAC 24 recruiting the VHL E3 ligase to selectively degrade HDAC6 with a DC50 value of 7.1 nM and Dmax value of 95% in MM.1S cells.80 Utilising the VHL ligand for HDAC6 degradation required a longer linker length to target HDAC6 with similar Dmax values to cereblon recruiting HDAC6 PROTACs. One of the potential advantages of 24, similar to 22, is that IKZF1/3 neo-substrates were not degraded when recruiting the VHL E3 ligase and this could be considered a more selective chemical probe for studying HDAC6 related biology. No cell viability assays were reported with 24 in this study.K. Yang et al. designed and synthesised a structurally unique HDAC6 targeting PROTAC 25,81 based on the natural product indirubin as the ligand to engage HDAC6. PEG linkers were explored in this study and intriguingly the shortest linker containing one PEG unit was much more effective at HDAC6 degradation than the longer PEG linkers. 25 exhibited a HDAC6 DC50 value of 108.9 nM in K562 lymphoblast cells with no degradation of HDAC1 observed. The authors were interested in investigating NLRP3 inflammasome activation with 25, and 25 reduced the expression of NLRP3 in THP-1 cells. The selective HDAC6 inhibitor from which PROTAC 25 was derived was less effective in this response, and the inhibitor was also more cytotoxic to cells.Cao et al. utilised their novel solid phase synthesis approach to hydroxamic acid-based PROTACs in an attempt to discover PROTACs to selectively degrade HDAC6.82 They identified 26 and 27 with DC50 values of 3.5 nM and 19.4 nM respectively for HDAC6 in the leukaemia HL-60 cell line. No degradation of HDAC1 or HDAC4 was observed with 26 and 27. 26 and 27 were screened against a small panel of acute myeloid leukaemia cell lines and B-cell acute lymphoblastic leukaemia cell lines for their effects on cell viability. Despite its potent DC50 value for HDAC6, 27 did not compromise cell viability in the cell lines tested at concentrations ranging from 0.5\u201350 \u03bcM, the authors noted that this is an observation similar to others studying the effects of selective HDAC6 inhibitors on cell proliferation.83 PROTAC 26, on the other hand, did compromise cell viability in three of the AML cell lines tested and was capable of inducing apoptosis, but with IC50 values in the double digit micromolar ranges. This was a surprising result given that 26 is not only a selective HDAC6 degrader but also exhibits a similar inhibition profile to class I HDACs as the pan-HDAC inhibitor vorinostat in vitro. The authors hypothesised that the reduced permeability of 26 to the nucleus compared to vorinostat is perhaps responsible for the loss of potency in cell viability assays.Sinatra 84 Offering an alternative to the hydroxamic acid functional group which has previously been reported for mutagenic effects.23 One of the most intriguing findings of this study was that 28 and 29 were compromised in terms of their inhibition of HDAC6 compared to hydroxamic acid HDAC inhibitors, with IC50 values of 0.64 \u03bcM and 0.69 \u03bcM respectively. Yet, 28 and 29 were more potent degraders of HDAC6 in cells, with DC50 values of 131 nM and 171 nM respectively, both PROTACs exhibited a hook effect at 10 \u03bcM. Although extensive studies were not carried out their effects on cell viability, these PROTACs did not compromise cell viability in the MM.1S cells in which they were tested.The same group reported non-hydroxamic acid based PROTACs 28 and 29 that selectively degrade HDAC6 in MM.1S cells.+ to catalyse the hydrolysis of acetylated lysine residues and consist of the sirtuin family SIRT1-7. The first HDAC targeting PROTAC reported in the literature was targeting SIRT2 for degradation.49 PROTAC 30 was synthesised utilising click chemistry and the POI ligand incorporated a selective inhibitor of SIRT2 and thalidomide as the E3-ligand (49 Dose dependent degradation of SIRT2 was observed in HeLa cells with no degradation of SIRT1. SIRT2 degradation by 30 could be further visualised in living HeLa cells with GFP-tagged SIRT2.Class III HDACs utilise NAD3-ligand .49 Dose et al. reported a new SIRT2 inhibitor and incorporated it into PROTACs 31 and 32.8531 and 32 were capable of degrading SIRT2 at concentration range of 0.5\u201310 \u03bcM in MCF7 and BT-549 cell lines, with a hook effect observed at greater concentrations. Via degradation 32 was capable of reducing SIRT2 deacetylase and defatty acylation activity in cells, whereby the SIRT2 inhibitor from which PROTAC 32 was derived was not capable of reducing SIRT2 defatty acylation activity. This could be a major advantage to using PROTACs to degrade SIRTs over inhibition of their catalytic active site alone. At lower concentrations 32 was more effective at compromising cell viability in MCF7 and BT-549 cells than the inhibitor from which it was derived, however at higher concentrations the differences between 32 and the selective SIRT2 inhibitor were much less. The authors hypothesised this is due to the hook effect exhibited on SIRT2 in the presence of 32.J.Y. Hong 86 It is the smallest of the HDAC enzymes and is a much more effective fatty-acid deacylase than a histone deacetylase.87 HDAC11 does not exhibit significant effects on cell proliferation but has been noted as a potential target for metabolic disorders.88 Selective inhibitors of HDAC11 have been reported,89 however as of yet, at the time of writing, no PROTACs targeting HDAC11 for degradation have been reported. HDAC11 could potentially be a very amenable target for proteasome-mediated degradation as Long et al. noted its poly-ubiquitination and short half-life of 4 hours in BEAS-2B cells, which could be reversed in the presence of the proteasome inhibitor MG132.90HDAC11 is the singular HDAC isoenzyme in class IV and was the most recently discovered HDAC in 2002.via the proteasome, rather than solely inhibiting the catalytic active site, will allow researchers to probe the biology of HDACs in cells and in vivo beyond their enzymatic function alone. Five years ago, before the first HDAC targeting PROTAC was reported, this would have been non-trivial, at least by small molecule approaches in chemical biology. It is also clear that the selectivity of degradation is not only PROTAC dependent but also dependent on the cell line, an observation also made by others.91 Before PROTACs are used as chemical probes it would be recommended to first validate the selectivity of HDAC isoform degradation in the cell line of investigation. This seems to be even more imperative when a pan-HDAC inhibitor is incorporated as the HDAC ligand in the PROTAC.It is clear that PROTACs have the capacity to offer potent and selective degradation of individual HDAC isoforms in the cell. PROTACs are already being used as chemical probes to study biology related to individual HDAC isoforms. Degrading the HDAC enzyme et al.56 investigating HDAC isoform degradation in the presence of 48 PROTACs correlates well with the number of isoform-selective HDAC targeting PROTACs reported to date in the literature. PROTACs capable of the selective degradation of HDAC6 are the most frequently reported, followed by selective degraders of HDAC8 and selective degraders of HDAC3. Future challenges will likely involve dialling out the degradation of these isoforms while attempting to degrade another select HDAC isoform. It will also be interesting to witness the discovery of new E3-ligands and how these new ligands can influence or modify HDAC degradation selectivity. In regards to SIRTs much fewer studies have been carried out in comparison to Zn2+ dependent HDACs but the selective degradation of SIRT2 over SIRT1 certainly seems to have been achieved.Some HDAC isoforms certainly seem more prone to PROTAC-mediated degradation than others. The proteomics study carried out by Xiong et al. reported on the noncanonical and non-cytotoxic effects of selective HDAC6 inhibitors in reducing tumour growth in vivo by enhancing antitumor immune responses with immune checkpoint inhibitors in breast cancer.92 Additionally, the class I HDAC inhibitor tucidinostat was found to act synergistically with the aPD-L1 antibody to reduce solid tumours.93 There have been a number of studies recently reported demonstrating how HDAC inhibitors can prime and enhance the anti-tumour immune response of immune check point inhibitors and help overcome associated drug resistance to cancer immunotherapies in solid tumours.94 There are a significant number of active and recruiting clinical trials that are investigating combination therapies of HDAC inhibitors with immune checkpoint inhibitors.94 This could be another novel therapeutic application of PROTACs targeting HDACs. However, the fate of PROTACs targeting HDACs in a clinical context perhaps lies with the fate of exemplary PROTACs targeting AR and ER such as ARV-110 and ARV-471 etc.46 currently in clinical trials and their potential FDA approval status. If and when that time comes, it seems researchers will be primed and ready with a significant number of HDAC targeting PROTACs already available for optimisation to progress to clinical trials.In a therapeutic context, there are examples whereby HDAC targeting PROTACs are more effective at compromising cell viability in cancer cells than their counterpart HDAC inhibitor. There are also examples whereby the reverse is true, particularly when compared to a pan-HDAC inhibitor. However, in future studies, the therapeutic potential of HDAC targeting PROTACs should be investigated beyond cytotoxicity alone. For example, Banik J. T. H. and J. P. S. are inventors on the PCT patent application WO2021148811A1 and the following patents: JP2023-510947, US2023-0120211A1."}
+{"text": "As the world population ages, health and social care professionals are increasingly confronted with patients with chronic long-term conditions and multimorbidity, requiring an extensive assessment and integrated care management strategy. The aim of this paper was to systematically collect and assess evidence of interprofessional education and training strategies for Comprehensive Geriatric Assessment (CGA) to build a competent health workforce.A systematic review was conducted according to PRISMA guidelines and the databases Medline, CINAHL, Cochrane and Embase were searched for studies illustrating effectiveness of educational interventions for teaching and training CGA in an interprofessional context.Based on 21 identified studies, a great variability and heterogeneity in duration, setting and design of the interventions was identified. Promising results were found in the domains analysed, ranging from knowledge and skills; practices and behaviour; patient health outcomes; attitudes and perceptions to collaboration and quality of care.Education and training of transversal skills within a continuous learning approach is key to equip the health care workforce for successful CGA performance in an interprofessional environment.Further research in this field is recommended to strengthen the evidence-base towards development of a resilient and integrated health care workforce for an ageing population. Currently, European citizens reach an average life expectancy of 81 years at birth . Actuall3In this regard, the Comprehensive Geriatric Assessment (CGA) constitutes a multi-dimensional diagnostic tool to evaluate the clinical, functional and psychosocial status of geriatric patients and subsequently coordinate and monitor clinical and social care needs and interventions targeting individual resilience . Studies789Due to the multidimensional nature of the CGA, an inter-professional and team-based approach is inherent for its effective and efficient provision in daily practice. The concept of integrated care bundles coherent methods and models to produce collaboration, connectivity and coordination across professional and organizational boundaries . Even ifThe World Health Organization (WHO) advocates for policy strategies strengthening health and social workforce development, with a special focus on collaborative practice 14. In th\u201cstudents from two or more professions learn about, from and with each other to enable effective collaboration and improved health outcomes\u201d ,has proven to facilitate team-based approaches, preparing students for person-centered care delivery for an ageing population. Despite a growing body of evidence of research in this area, conceptualization, implementation and evaluation of inter-professional educational interventions remain topics in need for further investigation . AgainstBased on the current situation it was the aim of this review to pull together the most recent evidence on inter-professional education and training interventions to teach workforce from different disciplines, thereby facilitating coordinated delivery of CGA in daily practice.This systematic review was conducted in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist 19. The sThe search was limited to publications between January 2000 and September 2022 since contemporary studies rather respond to current evidence-based didactic and pedagogic approaches, thereby also taking into account possible e-learning and digitalization methods.Bibliographic management and deduplication was carried out with the bibliographic software Endnote and manually.In order to be included, studies had to meet the following inclusion criteria: 1) Experimental or quasi-experimental educational intervention studies or cohort studies; 2) applying a multi-professional, undergraduate and/or continuous professional development (CPD) education and training approach in the field of CGA in any setting; 3) and carrying out a self-assessed or objective evaluation of the intervention. Studies were excluded, if the intervention addressed professional silos only and focused on geriatric care in general without referring to geriatric assessments.Outcomes of interest were attitudes and perceptions of learners; knowledge, confidence, competence, abilities or skills; learner\u2019s behaviour and practices; as well as patients\u2019 health outcomes.Each included article was assessed for study quality by two independent reviewers  using the Medical Education Research Study Quality Instrument (MERSQI), a tool developed particularly to assess educational studies consisting of six different domains with an overall possible score range from 0 to 18 . For assNarrative synthesis of the data was performed, as no meta-analysis was carried out due to expected heterogeneity of outcome data.The applied search strategy yielded a total of 379 results, 138 additional results were identified on Google Scholar and via reference tracking. After deduplication (n = 23 results removed), title-/abstract screening (n = 310 results removed) and full-text screening (n = 163 results removed), 21 results were included for synthesis. The PRISMA 2020 flow diagram illustrates the screening process and demonstrates reasons for exclusion .Table 1  summarizes the 21 studies. As illustrated, these studies had been published between 2009 and 2021 with the majority being published between 2013 and 2018. Nearly all of the studies  were conducted in the USA, the rest was undertaken in Greece  and GermIncluded studies represented almost consistently quasi-experimental studies, either with a pre-/post-design [2124252627282930313222343536373840The majority of the studies  considered for this review addressed undergraduate health and social care professions\u2019 students whereas two studies evaluated educational interventions on a CPD level 27. Two p37Disciplines represented in each study were as follows: medicine  [212223242526272829313233343536373821222326272829303134353637392224252627283132343536373824272831323334353637383924252628323439402224262832362426283426322832212728Applied educational and training interventions showed a high degree of heterogeneity, ranging from training courses ; a Geria26233322293032343638Primary outcomes of interest varied as well, with studies analysing overall inter-professional learning experience 243739; a243728362526The majority of the studies used a self-assessment approach of learners [2125262728303132333436373840232924The mean MERSQI score for the included studies was 10.52 (SD 2.07) with a score range from 6.5 to 15.0. Results for each domain indicate a mean score of 1.43 (SD 0.44) for study design, 1.83 (SD 0.51) for sampling, 1.57 (SD 0.85) for type of data, 1.45 (SD 1.13) for validity of evaluation instrument, 2.69 (SD 0.52) for data analysis and 1.50 (SD 0.51) for outcome domain.Primary outcomes investigated in studies included in this review ranged from evaluation of general attitudes and perceptions of the learning experience; knowledge, skills, skill confidence and competencies; practices and behaviour; patient health outcomes as well as other particular measures such as collaborative environment, communication style and quality of care objectives. Attitudes and perceptions in the context of providing geriatric care in an inter-professional environment were analysed in nine studies 2122283036373839.36373821222833363738Positive effects were shown on the development of attitudes towards frailty syndrome and its management in daily clinic towards inter-professional collaboration  and prac3638Mixed results are reported by Renschler et al.  who founEleven studies investigated the domains knowledge, skills, (skills) confidence and competencies [21252627293031343540212527293134352630Turrentine et al.  describe2527Mixed results are reported in terms of (skills) confidence , self-asEveryday practices and team behaviour were analysed by two studies included in this review 24. The ePatient health outcomes were analysed by one study and are briefly presented here . ConsideIn terms of collaboration and quality of care, a randomized controlled trial conducted with medical and nursing students reported a post-intervention increase of evaluated care objective scores in the intervention and control group in all but one category: care of other symptoms. The intervention group achieved significant post-intervention scores in three categories: pain therapy (p = 0.006); guarding of patient\u2019s autonomy (p = 0.001) and integration of psychological aspects (p = 0.003). Moreover, a significant change in interaction initiation was observed in the intervention group (p = 0.0007) and there was a significant post-intervention increase in the number of uni-professional information items exchanged in both groups (p < 0.0001) and in subgroups (p = 0.0002-p = 0.004) .Byerly et al.  additionDefinition and development of transversal skills of tomorrow\u2019s health workforce will be key to enable different professions in health and social care to deliver high quality and patient-centred integrated care in health care systems of the future 44. SkillBased on the concept of ICP and \u201cadaptive expertise\u201d, the current paper describes the evidence-base for trainings offered for different health professional groups to perform CGA in inter-professional collaborative practice.The overall care rendered by CGA teams providing longitudinal assessment and care can be divided into six steps: data-gathering, discussion among the team, increasingly including the patient and/or caregiver as a member of the team, development of a treatment plan \u2013 together with the patient and/or caregiver, implementation of the treatment plan, monitoring response to the treatment plan and finally revising the treatment plan . Given t32The need for targeted inter-professional education and training initiatives in geriatric care has been highlighted by literature for some time 5152. Ind5153Studies included were of varying quality in study design, only one study using a randomized controlled trial approach design . DuratioGiven the heterogeneity of IPE interventions published in the papers included and this major weakness in the evaluation frameworks applied to evaluate the educational impact of training interventions, the data provided in this review should be reflected thoroughly. Two of the studies included 39 set upResults of this review need to be considered in light of its limitations. As already mentioned, the studies included present with a strong heterogeneity in terms of study design, outcomes and evaluation methodologies. This hampers the possibility to present a clear evidence-base and only allows drawing a cautiously positive picture of effectiveness of educational interventions to train CGA across health and social care professions, which is aggravated by the fact that not all studies provided full disclosure of educational content delivered and intervention execution. Most of the studies included presented self-reported data based on learners\u2019 perceptions instead of objective assessment results or patient health outcomes that may lead to questioning reliability and generalizability of effects.Despite the mentioned heterogeneity, the majority of studies included encompasses didactic approaches with a strong practical orientation, either in form of real patient encounters [222425303233363738262829313923The data on training formats, however, contrast with assessment methods applied as only two studies assessed practices and behaviour 24 and onNone of the studies used the Objective Structured Clinical Examination (OSCE) as assessment methodology. The OSCE is a widespread method and gold standard to assess clinical skills and competencies transmitted during educational and training initiatives as objectively as possible 57. Despi131560Although authors of this review adhered to guidelines for writing systematic reviews 19, some This systematic review highlights effects and possibilities to educate and train professionals in health and social care in conducting a CGA and shows promising results for inter-professional collaborative practice in geriatric care, when measured with students\u2019 self-assessment scales. There seems to be potential for introducing case-based and/or work-placed teaching methodologies reflecting the complexity of interacting physical, psychological/emotional, contextual and social factors evaluated during CGA for different professions, enabling a shared decision process during CGA of older people.However, further educational research, preferably reflecting effects, needs and progress of interprofessional collaborative practice in the European education/training landscape and focussing on effectiveness of complex care based training situations based upon standardized educational assessment frameworks is needed to strengthen the evidence-base, foster education and training standards as well as curricular development in an inter-professional context and thus adequately equip the health workforce to acquire adaptive expertise for delivery of CGA and, therefore, meet the needs of an ageing population.The additional file for this article can be found as follows:10.5334/ijic.7549.s1Supplemental material.Research protocol."}
+{"text": "In hereditary spastic paraplegias (HSPs), magnetic resonance imaging (MRI) scans of the brain typically show the involvement of the anterior part of the corpus callosum with abnormalities in the white matter fibers of the forceps minor(\u201cears of the lynx sign\u201d). However, these imaging findings are particularly associated with spastic paraplegia type 11 (SPG11) or spastic paraplegia type 15 (SPG15).NT5C2(p.[Leu468Pro] and p.[Gln542Argfs*71]), consistent with SPG45, of the SPG subtypes with thin corpus callosum.A child with spastic gait achieved independent walking at the age of 2 years, and 3 years later was referred to our service for investigation. A brain MRI demonstrated corpus callosum dysgenesis and peritrigonal white matter abnormality . Whole"}
+{"text": "Following the publication of this article , the corThe 2D gel electrophoresis image in Figure 1A in , and FigThe shVector panels in Figure 6A of  are simiThe shVector panel in Figure 7A of  is similIn the Actin panels in In \u25cb The xenograft tumor study is described as having 3 male mice per group, but the corresponding authors clarified that 2 male and 2 female mice were used per group.\u25cb 8% and 10% SDS-PAGE gels were used for western blot experiments, not 12% as stated.\u25cb Sequences of shRNAs against ALDOA and the non-targeting control are absent from the article. The corresponding authors provided the relevant sequences in The following inaccuracies/omissions were identified in the Methods section:The corresponding authors stated that data underlying all the results reported in the article are available, except for the raw image data underlying the western blots in Figure 5.The corresponding authors confirmed that the same 2D gel image and cohort of 7 patients are reported in four articles \u20134, and eThe underlying image data for the actin panels in For PLOS ONE Editors issue this Expression of Concern to notify readers of the above issues and relay the supporting data and updated figures provided by the corresponding authors.The S1 File(DOCX)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file."}
+{"text": "Poly(pyrazolyl)borate ligands have been obtained throughthe reactionof highly reactive haloboranes with in situ formed pyrazolides undervery mild conditions. This versatile synthetic method allows the selectivesynthesis of bis-, tris-, or tetrakis(pyrazolyl)borates. Furthermore,the method is compatible with the use of functional groups on theheterocyclic moieties of the poly(pyrazolyl)borates that were notaccessible to date. Strongly encumbered sodium and thallium(I) poly(pyrazolyl)borateswith a reduced donating ability have been obtained for the first time. Since then, poly(pyrazolyl)boratesof almost any transition metal have been prepared4 and used as enzymatic models,5 for the development of new materials,6 and as power catalysts in reactions as carbene or nitrene C\u2013Hinsertion, polymerization or carbonyl derivatizations.7 The success of this family of ligands resides in the possibilityof fine-tuning the electronic and steric properties of the metal complexesthrough the introduction of appropriate groups on the heterocyclicrings. However, despite the more than 4200 crystal structures of thistype of complexes that have been described,8 the simultaneous pyrazole decoration with bulky and electron-withdrawingsubstituents has not been possible to date.Poly(pyrazolyl)borate ligands were describedfor the first timeby Trofimenko during the 1960s.9 This transformationpresents drawbacks: (i) difficult control of the reaction stoichiometrywith possible formation of mixtures of dihydrobis(pyrazolyl)borates(Bpx), hydrotris(pyrazolyl)borates (Tpx) andtetrakis(pyrazolyl)borates (Tkpx), (ii) hazardous evolutionof hydrogen gas under high temperature conditions,10 (iii) limited functional group scope due to their sensibilityunder reductant conditions and (iv) pyrazoles containing simultaneouslyelectron-withdrawing and bulky substituents do not react under theseconditions due to reduced nucleophilicity and higher steric hindrance.The access to Tpx ligands presenting these characteristicscould widen the catalytic applications of their metal complexes dueto the increased electrophilicity,11 easierreduction,12 and higher stability of lowoxidation states13 of the correspondingmetal centers.The most commonroute to prepare poly(pyrazolyl)borates is thereaction of a high excess of the desired pyrazole derivative witha metal borohydride in the absence of solvent 1a.9 This14 very flammable reagents,in a reaction with the same disadvantages related before boratesBCl2 with nonsubstituted sodium pyrazolide.17The third generation of tris(pyrazolyl)boratesappeared with thesubstitution of the hydrogen atom with an alkyl or an aryl moiety.Alkyltris(pyrazolyl)borates were prepared from lithium alkylborohydridederivatives,d before 1b. In th)borates1c.16 AltHere we present a new practical and directmethodology for thepreparation of poly(pyrazolyl)borate ligands under very mild conditions,with wide applicability that yields exclusively the desired poly(pyrazolyl)boratederivative in good to excellent yields 2.3Tl), the usual entry for metalexchange.4a A dichloroborane dimethylsulfidecomplex (BHCl2\u00b7SMe2) was chosen as thehighly reactive boron source and 3-tert-butylpyrazole(1a) as the standard azaheterocycle borate(3aNa) just using the ca. stoichiometric amounts of all reagents on toluene atroom temperature for 24 h. Conditions optimization for tris(pyrazolyl)borateligands synthesis has been performed and is specified in the 3aTl was prepared in situ through standard proceduresto facilitate product purification.14aFirst, we focused on more extended thalium(I) tris(pyrazolyl)borates(erocycle 3. A baseerocycle . A smootxTl (3a\u2013jTl) using the optimized reaction conditions. An excellent 90% yieldwas obtained for complex 3bTl bearing the encumbering tert-butyl group at position3 and a bromine atom at position 4 of the heterocycle. This methodallows for the first time the preparation of boron scorpionate ligandswith a strongly electron-withdrawing nitro group in the presence ofa bulky substituent such as tert-butyl. Ligand 3cTl was prepared in a satisfactory93% yield. Introduction of the bulkier adamantyl group at position3 (3dTl) did not negatively affectthe product yield. All attempts to prepare 3bTl, 3cTl and 3dTl presenting electron-withdrawinggroups and highly hindering tert-butyl or adamantylsubstituents through literature methods were unsuccessful.9b The preparation of TpxTl bearing a mesityl group on position3 also proceeded smoothly in good yields with a hydrogen (3eTl) or a bromine (3fTl) atom on position 4 of the pyrazole ring.These optimized conditions were used in thepreparation of thalliumhydrotris(pyrazolyl)borate complexes using the selection of pyrazolederivatives, as shown in 1g bearing asensitive aldehyde substituent at position R4. As expected,the reaction proceeded smoothly, and the corresponding sodium complex 3gNa was obtained in 79% yield. Finally,we challenged the scope of the method using as the starting materialthe trisubstituted pyrazole 1h containing two methylgroups and one electron-withdrawing nitro group. The expected product 3hNa was obtained in a satisfactory86% yield. For pyrazoles 1g and 1h sodiumtris(pyrazolyl)borates were obtained directly, and sodium to thalliumexchange was not performed. The introduction of two encumbering isopropylgroups hinders the formation of 3iTl, and only traces of the expected product could be detectedeven at higher temperatures.We have also explored the compatibility of this newprocedure forthe synthesis of pyrazolylborates supporting functional groups ofspecial sensibility to reductant environments. We used our standardconditions in the reaction with pyrazole 1a\u2013g ,18 difficult to isolate as pure materials.19 The results obtained using acommercially available chloroborane dimethylsulfide complex (BH2Cl\u00b7SMe2) for a selection of thallium(I) Bpx complexes are shown in Tl3 were used, and pyrazole and base equivalents were adjustedto ca. ideal stoichiometric amounts with good results. Pyrazoles 1a\u2013c bearing a highly encumbering tert-butyl group cleanly produced the expected 2a\u2013cTl in good to excellentisolated yields. In these cases, the isolation of compounds 2a\u2013cTl is greatlyfacilitated by our selection of the boron source that allows fullcontrol of the 2:1 stoichiometry, thus avoiding contamination withother pyrazolylborates. However, the use of the standard conditionsfor 2e\u2013f yielded a complex reactionmixture due to the formation of the desired 2e\u2013fTl besides the two correspondingpyrazaboles with two bridgehead boron atoms borates borate.The scarce examples described of these ligands are almost restrictedto those presenting methyl or hydrogen groups on positions 3 and/or5 of the pyrazole rings due to the difficulty associated with thethermal introduction of the fourth heterocycle.22xNa (4a\u2013fNa) with highlyencumbering substituents. Alkaline salts of these ligands are a commonentrance to other metal complexes through metal exchange.22Further, we extended our procedure for the preparationof sterically challenging and rare tetrakis(pyrazolyl)borates.2cTl, 3bNa(OH2), and 4fTl were determined by single crystalX-ray diffraction borate ligands with excellent yields andcomplete regioselectivity.In conclusion, we have developed a usefuland versatile new syntheticprocedure for the selective preparation of Bp"}
+{"text": "Ab initio molecular dynamics (AIMD) simulations show the significance of asymmetric motion on a multidimensional potential energy landscape around the metal center for activated crossover from triplet ligand centered (3LC) to triplet metal centered (3MC) states on picosecond timescales. Significant entropic differences arising from the structural distributions of the 3LC and 3MC states revealed by the simulations are found to favor the forward crossover process. Simulations at different temperatures provide further insight into the interplay between structural and electronic factors governing the 3LC \u2192 3MC dynamics as a concerted two-electron energy transfer process, and the broader implications for photoinduced generation of high-energy 3MC states of interest for emerging photocatalytic applications are outlined.Excited state evolution of the rhodium( iii) complex [Rh(iii)(phen)2(NH3)2]2+  has been investigated theoretically to gain a better understanding of light-driven activation of high-energy metal centered states.Excited state evolution of the rhodium( One strategy to overcome this limitation is to activate high-energy 3MC states indirectly through energy transfer from another chromophoric unit with better light-harvesting capabilities.8 This can be achieved as an intramolecular activation from an initially excited CT or ligand centered (LC) state. Another challenge with the involvement of MC states in d6 complexes is the population of anti-bonding eg orbitals that frequently causes substantial excited state energy losses, associated with weakened metal\u2013ligand (M\u2013L) bonds.15,16 Activated crossover into high-energy 3MC states following initial LC excitation is, however, known from studies of prototype Rh(iii) complexes, as evidenced by experimental observations of dual photoluminescence (PL) from both the 3LC and 3MC states, as well as photochemical quenching experiments involving the 3MC state.17\u201326 The effect on the relative energy between the 3LC and 3MC and its impact on the state-to-state crossover efficiency was studied experimentally for a series of tris-phenanthroline derivatives, with the general formula [Rh(iii)(phen)2XY]n+ , by tuning the ligand field strength.19Transition metal complexes that combine efficient light-harvesting with long-lived, high-energy excited states are widely used in molecular photocatalysis and related photochemical applications.27\u201335 Some simulations have also covered the explicit interplay between excited state deactivation processes and solvent relaxation and reorganization processes.36Quantum chemical simulations offer significant opportunities to gain detailed insight into several different aspects of the excited state evolution of transition metal complexes. A variety of many-state quantum dynamical simulation methods have provided important insights into the ultrafast dynamics associated with initial relaxation processes within a dense manifold of excited states.3LC \u2192 3MC state crossover activation in the prototype Rh(iii) phenanthroline complex, [Rh(phen)2(NH3)2]3+, using ab initio molecular dynamics (AIMD) simulations conducted at the density functional theory (DFT) level. The [Rh(phen)2(NH3)2]3+ complex provides a good prototype system to investigate such 3LC \u2192 3MC crossover dynamics computationally as experimental observations of dual emission in the 170\u2013200 K temperature range point to a favorable 3LC\u20133MC balance, similar to what has also been reported for the [Rh(iii)(phen)3]3+ complex.19,24,37,38 We focus on intramolecular aspects of activated 3LC \u2192 3MC crossover dynamics on picosecond timescales, i.e. excited state dynamics taking place on the lowest excited triplet potential energy surface following any initial ultrafast ISC and IC processes as well as initial local relaxation of the initially populated 3LC state. This provides a largely complementary set of computational challenges compared to many recent studies focused on the earliest ultrafast multi-state relaxation processes, and places a greater focus on accounting for large-scale structural distortions on a complex multidimensional molecular energy landscape on timescales that can extend to tens of picoseconds and longer.In this work, we investigate the 23LC and 3MC states in the [Rh(phen)2(NH3)2]3+ complex were performed at the unrestricted DFT (uDFT) level of theory using the B3LYP* hybrid functional with reduced 15% Hartree\u2013Fock exchange (HFex) and the 6-311G basis set with the Stuttgart\u2013Dresden SDD effective core potential for the central Rh atom.39\u201342 An implicit Polarizable Continuum Model (PCM) was applied to account for a water solvent environment.43 The nature of the various states was confirmed by frequency analysis. All calculations were conducted using the Gaussian09 software.44Full geometry optimizations of structurally distinct relaxed ex)/6-311G/(water) in the 3MC and 3LC excited states were chosen as starting geometries for subsequent AIMD calculations using the atom centered density matrix propagation (ADMP) model.45 The size of the basis set was reduced to 3-21G in order to reduce the computational costs, and motivated by test calculations indicating only minor differences for the calculated energy differences between the relaxed 3LC and 3MC states according to a comparison with the larger 6-311G basis set. Explicit solvent structure and dynamics were neglected in this study focusing mainly on intramolecular rearrangement aspects, and an overall solvent response was approximated with the PCM model. The temperature dependence of the dynamics was investigated at constant selected temperatures  imposed by a standard thermostat. A minimum energy path was calculated as a complementary way to track the 3MLCT \u2192 3MC conversion (details in ESIOptimized structures at the B3LYP*(15% HF33.1iii)(phen)2(NH3)2]3+ complex from a first exploration of the triplet PES by means of unconstrained geometry optimizations and single point quantum chemical calculations. A single 3LC state minimum, identified as a local excitation on one of the phenanthroline ligands (designated as phen\u2032), as well as a set of four non-degenerate local minima of 3MC nature were identified by geometry optimizations. Different orientations of the eg orbitals relative to the M\u2013L bonds allow for up to six different 3MC states associated with Jahn\u2013Teller distortions along different M\u2013L axes. The C2 rotation axis, characteristic of the C2 point group to which [Rh(iii)(phen)2(NH3)2]3+ complex belongs more specifically, reduces the number of unique 3MC states to four. A graphical representation of the eg orbital orientations along different M\u2013L bonds, together with the names for the corresponding states adopted in this report , are described in g orbital set more strongly compared to the homoleptic parent complex [Rh(iii)(phen)3]3+. This potentially increases the energy offset between the distinct 3MC states. Indeed, different emission band broadening characteristics for the two Rh complexes of \u223c40 cm\u22121 in acetonitrile at 297 K for [Rh(iii)(phen)3]3+ and \u223c60 cm\u22121 in a water/ethylene mixture at 170 K for [Rh(iii)(phen)2(NH3)2]3+ provides a likely experimental indication of such differences in the MC state PES landscapes.19,38Several local triplet state minima were identified for the 3+ complex, with the aim to reveal the crossover dynamics as well as further properties associated with the population dynamics of thermally accessible states.The M\u2013L bonds of the relaxed 3.2iii)(phen)2(NH3)2]3+ after initial optical excitation and intersystem crossing into a 3LC excited state, we initially performed AIMD calculations at 170 K, 200 K, and 298 K.To better understand the triplet state dynamics in 2+ complex has, for example, also been reported at the uDFT level.48 All the bond elongations and contractions at 200 K suggest the population of a single dx2\u2212y2 d-type-orbital, and the M\u2013L bond values also correlate well with the calculated distances in the fully relaxed 3MC1 excited state.M\u2013L bond distance distribution plots are analysed in 3MC excited state is populated in this simulation. Two bond distributions with average distances of \u223c2.1 \u00c5 (blue and orange peaks) are shorter than the remaining four coordination sites in agreement with an eg-dx2\u2212y2 orbital assignment characteristic of the 3MC2 state  to 10.26 D (red peak) corresponding to the 3MC2 state (170 K simulation). Both 3MC1 and 3MC2 calculated dipole moments by uDFT minimization calculations (8.08 D and 10.74 D) reinforce the general state assignments, and the electronic identity of each 3MC state. Larger peak broadening additionally suggest that the 3MC1 is able to move over a larger accessible volume in the multidimensional PES compared to the 3MC2 state.A clearly different set of M\u2013L bond distance distribution profiles top  at the tC2 state . Other pFig. SI.7. The geo3MC dynamics at 298 K. At this higher temperature, the M\u2013L bond distributions access a wide range of distances during the simulation, and with more than one distribution peak. Furthermore, the dipole moment histogram at 298 K shows a bimodal distribution characteristic of alternating population of two relatively distinct local electronic states  are registered, mainly in the Rh \u2013 N1 bond reaching a distance of 2.23 \u00c5 right before the transition occurs. \u03b1SOMO orbital , after temporarily reaching a maximum Rh \u2013 N bond stretching of 2.92 \u00c5. This particular initial distortion could potentially represent a sweet spot for initiating photochemical reactions.First, an AIMD simulation at 330 K shown in the top panel of al 3a in  at the d3LC lifetime was extended significantly to \u223c17 ps in order to successfully capture a crossover event.Triplet crossover dynamics was also simulated at a lower temperature of 298 K as shown in 1 bond stretching to 2.6 \u00c5. Although the \u03b1SOMO orbital 2b shows population of the 3MC3 state rather than the 3MC4 observed in the previous simulated temperature, the 3LC excited state is still deactivated through a higher energy MC state. This initial population of the 3MC3 state is rapidly converted into the 3MC4 excited state in \u223c900 fs . An observed second populated 3MC4 state achieves even longer lifetimes up to \u223c2300 fs (tIb mark), before finally deactivating to the calculated lower energy 3MC2 excited state (tIIb mark).While the limited simulation data precludes a reliable Arrhenius analysis that can be correlated quantitatively with the experimentally observed temperature-dependence, it is already notable that the simulations over a range of moderate temperatures shows a clear temperature-dependence of the crossover rate. Furthermore, the transition times in the 1\u201320 ps time range are consistent with the crossover being an activated process with a small effective transition barrier in basic agreement with a relaxed minimum energy path . The metal center activation can in principle be achieved in a concerted or step-wise fashion via electron hopping followed by hole hopping or the equivalent reverse path as illustrated in 3LC \u2192 3MC mechanism in detail from the simulations, the weights of the molecular orbital coefficients from the metal d-atomic-orbitals in the \u03b1SOMO and beta semi-unoccupied molecular orbital (\u03b2SUMO) were tracked for a series of snapshots with an interval of 4 fs during a 400 fs time window around the transition region  than at 298 K so that the transition state (TS) is essentially reached without any initial geometry stretching. In contrast, a TS at 298 K is only reached by bond stretching after significantly longer state sampling. The two highest energy 3MC excited states behave as important intermediate states, reflecting the complexity of multi-step deactivation paths towards the ultimate population of the lowest 3MC2 state.Finally, the AIMD simulations also highlight the importance of statistical factors influencing the crossover dynamics beyond what can be extracted from static calculations of transition barriers and scans of reaction paths. In particular, the simulations indicate a clear significance of entropy factors related to the accessible PES volume for the different states. This effective area, as an intrinsic characteristic of the distinct excited states dynamics for both sampled temperatures, are already revealed in the trajectories within the phen M\u2013L bonds during the simulation time as represented in 3MC product state corresponds an increase of the reaction entropy. Since the 3LC \u2192 3MC transition is mainly ruled by M\u2013L Jahn\u2013Teller distortions, we assess the change in accessible coordinate space in the simulations by the six M\u2013L bond distributions. In this approximation, the accessible volume of each state can be expressed as the product of the standard deviations of each M\u2013L bond (\u03b4li) byThe larger accessible volume in the Sr) then relates with the Boltzmann constant kB and the partition function in terms of volume changes between the 3MC state (VMC) and the 3LC state (VLC) asThe entropy change of the crossover reaction (\u0394iii)(phen)2(NH3)2]3+ complex together with the final active volume V (phen)2(NH3)2]3+ on picosecond timescales. Initial results from quantum chemical calculations revealed a clear difference in the triplet potential energy landscape between well-defined 3LC states on the phen ligands on one hand, and a more complex 3MC potential energy landscape comprising several local minima with distinct structural and electronic characteristics on the other hand.AIMD simulations were used to investigate 3LC internal state dynamics reveal a narrow and well-defined range of accessible geometries closely constrained near the ground state geometry. In contrast, 3MC dynamics is revealed to involve significant structural mobility on a flat 3MC PES that includes facile transitions between several Jahn\u2013Teller distorted minima. The MC dynamics notably includes fast transitions through higher-energy MC states connecting different low-energy parts of the full 3MC PES. Notably, the large difference in thermally accessible coordinate space between the LC and MC parts of the triplet PES indicates that entropic effects will have a significant influence on the free energy balance and effective driving force governing the 3LC \u2192 3MC crossover dynamics.The AIMD simulations provide further insight into the multidimensional dynamics taking place on the complex triplet PES. First, 3LC \u2192 3MC process at different temperatures showed a significant influence of the temperature on the crossover rate, consistent with an activated process characterized by a small energy barrier. The simulations also reveal further details of the triplet dynamics proceeding via a complex cascade involving several local 3MC states following the initial crossover from the 3LC state. Analysis of the AIMD simulations also enabled an in-depth assessment of the electric rearrangements taking place during the 3LC \u2192 3MC crossover. In particular, the 3LC \u2192 3MC transition is seen to involve a rapid redistribution of both the excited electron and the accompanying hole from one of the phen ligands to the metal center that takes place in a fully concerted fashion on a sub-picosecond timescale. This corresponds to a spin-allowed intramolecular two-electron excitation energy transfer process that takes place dynamically as a structure-driven weakening of the ligand field splitting of the metal center. It is interesting to consider as a topic for further investigation that the two-electron nature of this process appears to provide a fundamentally different set of opportunities and limitations for tuning of the excited state landscape compared to the corresponding one-electron case encountered in more widely studied 3MLCT \u2192 3MC deactivation processes.Simulations of the 3LC \u2192 3MC transition provides useful insights for further developments of transition metal complexes that aim to utilize 3MC states for photochemical applications more broadly. In particular, 3LC \u2192 3MC crossover is confirmed to provide a promising approach to overcome problems associated with weak absorption and ultrafast initial energy losses from direct excitations into 3MC states more generally, as long as the full complexity of the triplet potential energy landscape and associated dynamics revealed by simulations is taken into consideration.Finally, the in-depth understanding of this \u03b1SOMO 3LC \u2192 3MC transition dynamics MP4 movie at 298 K is also available in the ESI.\u2020DFT energies and geometry details, trajectories and electron structure analysis of the MD simulations, NEB calculation analysis, and cartesian coordinates of the optimized structures are available in the ESI.PP supervised the project and IBL performed the calculations. PP and IBL contributed to the manuscript preparation.There are no conflicts to declare.SC-014-D3SC04381A-s001SC-014-D3SC04381A-s002"}
+{"text": "Flt4 knockout mice to investigate VEGFR3 signaling in lymphatic junctions. We show that loss of Flt4 prevents specialized button junction formation in multiple tissues and impairs interstitial absorption. Knockdown of FLT4 in human lymphatic endothelial cells results in impaired NOTCH1 expression and activation, and overexpression of the NOTCH1 intracellular domain in Flt4 knockout vessels rescues the formation of button junctions and absorption of interstitial molecules. Together, our data reveal a requirement for VEGFR3 and NOTCH1 signaling in the development of button junctions during postnatal development and may hold clinical relevance to lymphatic diseases with impaired VEGFR3 signaling.Lymphatic capillaries develop discontinuous cell-cell junctions that permit the absorption of large macromolecules, chylomicrons, and fluid from the interstitium. While excessive vascular endothelial growth factor 2 (VEGFR2) signaling can remodel and seal these junctions, whether and how VEGFR3 can alter lymphatic junctions remains incompletely understood. Here, we use lymphatic-specific   Lymphatic capillaries form discontinuous junctions called buttons to enable absorption of interstitial contents. Jannaway et al. show that vascular endothelial growth factor receptor 3 (VEGFR3) expression is required for the formation of button junctions and that NOTCH1 signaling can fully rescue button formation in the absence of VEGFR3. However, inherited genetic mutations can impair lymphatic vessel function and lymph flow to cause a collection of diseases known as congenital lymphedema. While several gene mutations have been identified in the past decade in congenital lymphedema patients,3 Button junctions first begin to form at E17.5 in the skin lymphatic vasculature and mature by 3 weeks after birth.4 While angiopoietin-2 and DLL4 signaling in lymphatic endothelial cells (LECs) regulate button junctions in lymphatic capillaries in the skin and intestine, respectively,6 the molecular mechanisms required for button formation during development are still poorly understood. Recently, genetic deletion of two VEGFA receptors, Flt1 and Nrp1, from vascular endothelium resulted in the zippering of existing intestinal lacteal buttons and prevented high-fat-diet-induced obesity in mice.7 The proposed cause of this zippering was increased VEGFA ligand availability and thus excessive activation of VEGFR2. This role for VEGFR2 was confirmed by a new model of viral infection that caused button junctions to revert to continuous zipper-like junctions.8 In contrast, a clear mechanism for VEGFR3 in button formation has not been elucidated.To prevent or clear edema, lymphatic vessels absorb the majority of interstitial fluid. Interstitial fluid absorption by lymphatic capillaries is permitted via the remodeling of continuous junction proteins, reminiscent of zippers, into punctate discontinuous junctions that have been referred to as buttons.Flt4 from all LECs postnatally (Flt4iLECKO). Our data reveal that VEGFR3 is required for button formation during a critical period of postnatal development but is later dispensable for button junction maintenance. We also show that overexpression of the NOTCH1 intracellular domain (NICD) is sufficient to rescue button formation in the absence of VEGFR3 and restores the function of lymphatic capillaries to absorb interstitial fluid. Our findings reveal that a VEGFR3/NOTCH1 signaling axis is required for button formation and interstitial absorption. Furthermore, the most common form of congenital lymphedema, Milroy disease, is caused by dominant-negative point mutations in the VEGFR3 gene, and these patients exhibit dysfunctional interstitial absorption. Thus, our findings here hold potential clinical relevance to this disease.Here, we identify VEGFR3 as a major regulator of button junction development during postnatal life. To better understand the role of VEGFR3 in lymphatic function, we induced the deletion of Flt4 was deleted from LECs by crossing Flt4fl/fl9 with a lymphatic-specific Prox1CreERT2 strain.10 While this Cre strain can delete genes highly efficiently with two injections of tamoxifen (TM),12 we and others have found that inactivation of Flt4 led to the reappearance of VEGFR3-competent LECs.13 Therefore, we administered TM every other day from P1 until analysis at P21 . To assess button junction development, we located tissues rich in lymphatic capillaries that remodeled their junctions into buttons. The lymphatic vessels of the diaphragm were previously used as a model for postnatal button formation.5 All lymphatic vessels in the diaphragm of control and Flt4iLECKO pups expressed the lymphatic capillary marker LYVE1  and fewer buttons (<1% vs. 60%) compared to littermate controls, indicating that VEGFR3 is required for button formation in the ear skin  expression in both the blood17 and lymphatic13 vasculature. In blood and lymphatic endothelium, DLL4 is the ligand for NOTCH receptor 1 and 4.19 We found that the expression of DLL4, NOTCH1, and NOTCH4  is cleaved and translocates to the nucleus to initiate gene transcription.20 Western blot for cleaved NICD1 and NICD4 in shFLT4-treated cells revealed that NICD1 activation was significantly decreased, whereas NICD4 had very low basal expression that did not change . Kdr was deleted from P1 with the same dose and frequency of TM as before , simultaneously deleting both receptors from P1   , signifi to ~3%) ; 36%\u201317% to ~3%) ; 11%\u201379%nockouts  and S5I Flt4iLECKO mice. Thus, we bred Flt4iLECKO mice with Rosa26-NICD1 mice  that express the NOTCH1 NICD, which induces constitutive NOTCH1 signaling after Cre recombination21  in the Flt4iLECKO mice. The NICD1 fragment also rescued the ability of lymphatic capillaries to absorb interstitial tracer injected into the ear. Finally, our data show that NOTCH1 signaling can accelerate button formation, so NOTCH1 should be investigated in future studies as a potential inducer of button formation.This study demonstrates that VEGFR3 is required for button junction formation and interstitial fluid absorption in multiple tissues. Because genetic deletion of VEGFR3 at a developmental time point when buttons are fully formed does not affect the number or morphology of buttons, we conclude that VEGFR3 signaling does not maintain button junctions and, further, that there is a critical postnatal developmental window wherein VEGFR3 must be activated to successfully form buttons. To investigate the mechanisms downstream of VEGFR3 activation, we cultured hdLECs with a 22 although these findings were challenged by studies suggesting that VEGFR2 only weakly activates DLL4 and instead proposed a role for VEGFR3 activating DLL4 and NOTCH signaling.24 In lymphatics, loss of VEGFR3 was shown to decrease DLL4 expression, which in turn decreased NOTCH1 signaling.13 Surprisingly, we found that DLL4 levels did not change at the protein level, although NOTCH1 activation was significantly decreased in FLT4 knockdown hdLECs. Importantly, we show that forced activation of NOTCH1 signaling in the absence of VEGFR3 restores the ability of lymphatic capillaries to form buttons and absorb interstitial fluid, identifying NOTCH1 as a potential druggable target even in the absence of VEGFR3. Future studies will be needed to probe which NOTCH ligands are inducing NOTCH1 cleavage and whether NOTCH ligands are tissue specific in the context of button formation.While previous studies have demonstrated that VEGFR3 and NOTCH receptor signaling are closely linked, a direct role for these specific receptors in button formation has not previously been demonstrated. VEGF-A/VEGFR2 signaling was shown to induce DLL4-NOTCH signaling during angiogenic sprouting,FLT4 gene cause the most common type of congenital lymphedema, called Milroy disease.26 Milroy patients completely fail to absorb interstitial tracer during a lymphoscintigraphy exam, similar to our Flt4iLECKO mice here.27 Given the known role of VEGFR3 as the main receptor required for lymphangiogenesis,28 it was first suspected that dermal lymphatic vessel aplasia caused Milroy disease.29 However, this idea was challenged when skin biopsies from the feet of Milroy patients were found to contain lymphatic vessels.27 Most mutations in FLT4 occur in the kinase domain, thereby abolishing the kinase activity of VEGFR3.30 Because VEGFR3 makes homodimers or heterodimers with VEGFR2 to signal, these point mutations act in a dominant-negative manner.32 The lack of a phenotype in the Kdr knockouts here argues against heterodimers being involved in button formation. Mice with a similar point mutation in Flt4, known as Chy mice, are a mouse model of congenital lymphedema.29 Thus, the Flt4iLECKO mice reported here that lack VEGFR3 protein likely mimic the dominant-negative mutations in the Milroy patients or Chy mice that have very low residual VEGFR3 signaling. Based on our present data, we hypothesize that a failure to form buttons may underlie the pathogenesis of the Milroy patients and that future work is needed to investigate our signaling mechanism in the Chy mice.Heterozygous point mutations in the 5 and mutations in ANGPT2 have since been identified in congenital lymphedema patients.33 Interestingly, a recent study found that inhibition of angiopoietin 2 or deletion of the angiopoietin receptors, Tie1 and Tie2, decreased the expression of VEGFR3 in LECs.34 However, to our knowledge, lymphoscintigraphy was not performed on these patients, so it is unknown whether they fail to absorb interstitial tracer and thus if defective button formation contributes to this form of lymphedema. Together with the findings here, it argues that a single signaling pathway, namely ANGPT2/VEGFR3/NOTCH1, is responsible for regulating button junction formation.Angiopoietin 2 is also required for button formation,35 A recent study found that acquired lymphedema patients have elevated levels of the proinflammatory mediator leukotriene B4 (LTB4),36 which inhibited VEGFR3 expression and phosphorylation and inhibited NOTCH signaling in LECs. Thus, it is tantalizing to speculate that the lymphatic capillaries of acquired lymphedema patients may have aberrantly zippered junctions as part of the disease progression. Further supporting our data, Tian et al. also showed that lymphatic-specific deletion of Notch1 prevented the absorption of an interstitial tracer and prevented a pharmacological rescue of tail lymphedema, indicating that NOTCH1 signaling must remain intact for LTB4 inhibition to be effective at preventing lymphedema. Given that we show here that overactivation of NOTCH1 signaling both rescues the button defect in Flt4iLECKO mice and accelerates button formation in healthy mice, it is likely that the deletion of Notch1 phenocopies the Flt4iLECKO mice and prevents button formation, but this remains to be tested in future studies.While congenital lymphedema is caused by genetic mutations, acquired lymphedema develops after injury to the lymphatic vasculature.Notch1fl/fl conditional knockout mice to answer this question. Another limitation extends to extrapolating our findings here to the Milroy patients. Here, we are deleting both alleles of the Flt4 gene, leading to a complete loss of protein and signaling, whereas the Milroy patients commonly harbor a point mutation in the kinase domain of Flt4 that acts in a dominant-negative manner.30 Although the patients have a severe loss of VEGFR3 signaling, there is likely some residual signaling remaining. Future studies using the Chy mouse strain will be needed to confirm our findings here regarding defective button junction formation.While we show here that VEGFR3 is needed for postnatal button formation and is dispensable for maintaining buttons, our study has a few limitations. One limitation is that we show that NOTCH1 NICD is capable of rescuing button formation in the absence of VEGFR3, but we have not determined whether NOTCH1 is required for normal button junction formation. We will need to conduct future studies using In summary, our data show that VEGFR3 is required for button junction formation in lymphatic capillaries and interstitial fluid absorption. We were able to rescue this defect by overexpressing NICD1 to enhance NOTCH1 signaling, restoring lymphatic function in these mice. Finally, we show that overexpression of NICD1 can accelerate button junction formation, suggesting that NOTCH1 may be an important inducer of button junction formation developmentally.jscallan@usf.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Josh Scallan  and the Kdrfl/fl strain (Strain#: 018977) were purchased from The Jackson Laboratory.37 Both male and female mice were used on a C57BL/6J background and displayed no sex differences in phenotypes, but individual mice were not sexed. Mice had ad libitum access to food and water. To breed the final mice used in this study, male mice expressing Prox1CreERT2 along with a homozygous floxed gene were crossed with females expressing only the homozygous floxed gene without Prox1CreERT2. One parent expressed a heterozygous Prox1GFP allele or R26-NICD allele. In this manner, littermate controls and knockouts were stained and quantified together but not every pup harbored the Prox1GFP allele. To delete genes at birth, pups were injected subcutaneously with TM (100 \u03bcg in5 \u03bcL of sunflower oil) from postnatal day P1 and then every other day after until analysis at P21. To delete genes in weanlings, mice were injected intraperitoneally (1 mg in 50 \u03bcL of sunflower oil) starting at P21 and then every other day after until analysis at P35. In experiments with the Prox1CreERT2;R26-NICD1 mice, tamoxifen was injected at P1 and P3, as previously published.11 All experiments were performed in accordance with the University of South Florida guidelines and were approved by the institutional IACUC committee.Mouse strains used in this study include FLT4 (shFLT4) (sequence: GAGAGACTTTGAGCAGCCATT)38 for 48 h (VectorBuilder). The virus was removed, and the cells grown for a further 48 h before collection of either protein or RNA.Primary human dermal lymphatic endothelial cells  were cultured on fibronectin-coated plates using EBM-2 media (PromoCell). All hdLECs were used at passage 6\u20137 for shRNA knockdown. Cells were infected with either a scrambled control (Scr) or an shRNA targeting Mice were anesthetized with ketamine and placed prone on an acrylic board. A 2% solution of Evans blue dye and 2% BSA diluted in sterile saline was freshly prepared prior to the experiment. Approximately 2 \u03bcL of the Evans Blue dye was injected into the center edge of the ear using a glass needle attached to a syringe. The glass needle was fabricated by pulling capillary glass on a vertical puller  before grinding the tip to a diameter of approximately 100 \u03bcm. Images of the dye-filled lymphatics in the ear were captured using a color camera (Zeiss Axiocam 208 Color) mounted on a dissection microscope (Zeiss Stemi 508).39 Mice were anesthetized with ketamine and placed prone on an acrylic board. A 25 \u03bcL glass syringe with a 30-gauge needle was used to inject 2 \u03bcL of the dye conjugate  into the mouse ear of P24 mice. Ears were immediately imaged on an Olympus MVX10 fluorescence stereomicroscope outfitted with a Photometrics Evolve 512 Delta EMCCD camera controlled by \u03bcManager open source software.Bovine serum albumin (BSA) was conjugated to Alexa 790 and purified through centrifugal molecular weight cutoff filters as previously described.11 Unless otherwise stated, all procedures were performed at 4\u00b0C on an orbital shaker . Briefly, tissues were fixed overnight with 2% PFA in PBS and then washed with PBS before permeabilizing for 1 h with PBS +0.3% Triton X-100 (PBST). Tissues were blocked for 2 h with 3% donkey serum in PBST and incubated overnight with primary antibodies diluted in PBST. After five 15-min washes with PBST, tissues were incubated with secondary antibodies diluted in PBST for 2 h on an orbital shaker at room temperature. Following five 15-min washes in PBST, the tissues were incubated with DAPI (Sigma) at room temperature for 5 min and then washed in PBS. Tissues were then mounted onto glass slides  using ProLong Diamond Antifade Mountant (Invitrogen) and stored at 4\u00b0C. Details of primary and secondary antibodies are outlined in the resource table. Images were acquired using a 40x water immersion objective on a Leica SP8 confocal microscope and analyzed with FIJI software (NIH). Figures were created using Adobe Photoshop.Whole-mount immunostaining was performed as described previously.7 Buttons were considered short, discrete, unconnected sections of VE-cadherin protein that were perpendicular to the cell membrane. To objectively distinguish between button, intermediate and zipper junctions, we measured and averaged the length of each junction type from 3 samples, allowing us to define a button junction as <4.72 \u03bcm in length, intermediate junction as >4.72 \u03bcm but <7.85 \u03bcm in length, and a zipper junction as >7.85 \u03bcm in length. Using these criteria, the total length for each junction type was calculated as a percentage of total junction length.The junction types were quantified using FIJI software from images of lymphatic capillaries. Three types of junctions have been defined \u2013 buttons, intermediate, and zipper junctions. We quantified each junction type by measuring junction lengths and then normalizing as a percentage of total length as previously published.Total protein was collected from hdLECs lysed with RIPA buffer  and the protein concentration was measured with a BCA protein assay kit . Gel electrophoresis was performed using the mini gel tank (Invitrogen), the iBlot 2 dry blotting system (Invitrogen), and the iBind automated western system (Invitrogen). Protein was visualized using SuperSignal west pico plus chemiluminescent substrate (ThermoFisher Scientific).Total RNA was collected from hdLECs according to manufacturer\u2019s instructions in the RNeasy plus mini kit (Qiagen). cDNA was synthesized according to manufacturer\u2019s instructions in the Advantage RT-for-PCR kit . The QuantStudio 6 Pro Realtime System (Applied Biosystems) was used to carry out quantitative RT-PCR using Taqman probes (ThermoFisher). The cycle threshold (Ct) value for GAPDH was used to normalize the Ct value for each gene of interest.All junction quantification was analyzed by two-way ANOVA with Sidak\u2019s post hoc test. All qRT-PCR data were analyzed by unpaired Student\u2019s t-test. All graphical data are presented mean \u00b1 standard deviation, except for 1"}
+{"text": "During April 17\u2013May 5, 2023, 13 monkeypox (mpox) cases were reported to the Chicago Department of Public Health (CDPH) after 2 months during which only a single case had been reported. The cluster was remarkable because it comprised more than 10 cases at a time when sporadic cases or small clusters  were being reported in the United States, and >69% of the persons in this cluster had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine.On May 9, 2023, CDPH issued a health alert,**3 and viral load <200 viral copies/mL). Three (8%) patients experienced concurrent sexually transmitted infections at the time of mpox diagnosis.During March 18\u2013June 12, 2023, 40 laboratory-confirmed mpox cases were identified in Chicago, including 22 (55%), five (13%), and 13 (33%), respectively, among patients who had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine, those who had received 1 vaccine dose of JYNNEOS vaccine, and those who had not received any vaccines for mpox . All casMost vaccinated patients in this cluster received 1 or 2 JYNNEOS vaccine doses during July\u2013August 2022; the timing of vaccination is similar to an mpox cluster in France involving vaccinated persons who acquired mpox >6 months after vaccination3). Preliminary sequencing results from one unvaccinated patient and three patients who had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine indicate that Monkeypox virus (MPXV) among these Chicago patients is consistent with the B.1 variant of clade IIb MPXV, the predominant variant during the 2022\u20132023 outbreak. Genomic sequences revealed very few point mutations relative to published MPXV genomes, with no changes predicted to cause amino acid changes or increased pathogenicity. In terms of risk factors, patients who received 2 doses of JYNNEOS or 1 dose of ACAM2000 had a median of three sex partners (range\u00a0=\u00a0one to 20) during the 3 weeks before symptom onset, compared with 1.5 (range\u00a0=\u00a00\u20136) among patients who received 1 dose of JYNNEOS vaccine and unvaccinated patients. Preliminary medical record review indicates that vaccinated patients experienced self-limited illness, managed in outpatient settings. Compared with patients who received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine, patients who received 1 dose of JYNNEOS or no vaccines experienced a higher prevalence of lesions affecting the genital (43% versus 6%) and ocular (29% versus none) mucosa. The two hospitalized patients in this cluster had not received any mpox vaccine and had advanced HIV (<200 CD4 cells/mmAlthough the cause of this cluster has not yet been determined, leading hypotheses include a potential high number of sexual exposures in a network with many vaccinated persons, decreased vaccine effectiveness due to waning of humoral immunity, or vaccine mishandling or administration errors. Health departments are encouraged to report vaccination status of mpox patients to CDC for rapid detection of similar clusters among persons who were previously vaccinated. Persons with known or suspected mpox exposures should isolate and seek testing if they develop mpox symptoms, even if they have been vaccinated. Temporary sexual behavior changes, such as limiting the number of new or multiple sex partners and limiting sex in settings where anonymous sexual contact with multiple partners occurs can also help prevent mpox. Persons eligible for vaccination, particularly those with advanced HIV and other immunocompromising conditions, should receive 2 doses of JYNNEOS vaccine. Additional research on the durability of JYNNEOS vaccine\u2013induced immunity is needed."}
+{"text": "Medication nonadherence is a major concern for many health care stakeholders. Improving medication adherence in health plan members who have both hypertension and diabetes is essential for the successful management of these chronic diseases, with anticipated outcomes in decreased health care utilization, all-cause mortality and cost.To (a) identify patients who are potentially nonadherent to antidiabetic or antihypertensive agents within 1 managed care organization and (b) determine the relationship of rates of medication nonadherence with 2 mail intervention programs that involved quarterly medication-specific profiles of patients with potential nonadherence sent to primary care physicians (PCPs) and general medication adherence letters sent to patients with potential nonadherence.The study sample consisted of commercial members, Medicare Advantage-Prescription Drug Plan (MA-PD) members and Medicare Prescription Drug Plan (PDP) members who filled prescriptions for antihypertensive and antidiabetic medications and utilized their managed care pharmacy benefit during each measurement quarter (3 months) in the 2-year study period. Nonadherence was defined as a medication possession ratio (MPR) less than 77.0% for 1 or more antihypertensives and/or antidiabetic medications for each standalone calendar quarter. The first intervention, letters to PCPs with patient-specific medication profiles for 2008 Q2, began 6-8 weeks after 2008 Q2 and continued for each standalone calendar quarter through the end of the study period in 2010 Q1 . We assumed that patient care was managed by PCPs for hypertension and diabetes treatment. The medication profile also included antihyperlipidemic medication claims information, but there was no adherence analysis performed for antihyperlipidemic medications. The second intervention, letters sent to potentially nonadherent patients, began 6-8 weeks after 2009 Q1 for patients with MPR less than 77% for 1 or more antidiabetic or antihypertensive medications in 2009 Q1 and continued for each standalone calendar quarter through the end of the study period in 2010 Q1.Because there were 2 different interventions, 2 baseline adherence rates were calculated, for 2008 Q2 for the PCP mailing and for 2009 Q1 for the patient mailing. Compared with the baseline nonadherence rate in 2008 Q2 (35.6%), a small increase in nonadherence was observed in 2008 Q3 (36.4%), following by 6 calendar quarters of lower rates of nonadherence with a 27.7% nonadherence rate in the last measurement period in 2010 Q1. Compared with the nonadherence rate of 30.8% in baseline 2 (2009 Q1), the patient mailings were associated with small increases in nonadherence to 31.4% in 2009 Q2 and 31.1% in 2009 Q3, respectively, followed by lower nonadherence rates in 2009 Q4 (29.2%) and 2010 Q1 (27.7%).A 2-part intervention that involved mailings to PCPs for patients with both diabetic and antihypertensive medications who were potentially nonadherent to at least 1 medication, followed 9 months later by a general mailing sent to these potentially nonadherent patients regarding medication adherence, was associated with apparent improvement. However, the effect of the 2-part intervention on medication nonadherence could not be isolated because of coincident disease management interventions in diabetes and hypertension during the 2-year study period."}
+{"text": "Pak1 deficiency in the cardiac atria have not been well explored. In this study, Pak1 cardiac-conditional knock-out (cKO) mice were studied under baseline and adrenergic challenge conditions. Pak1 cKO mice show atrial arrhythmias including atrial fibrillation (AF) in vivo, detected during anaesthetized electrocardiography without evidence of interstitial fibrosis upon Masson's trichrome staining. Optical mapping of left atrial preparations from Pak1 cKO mice revealed a higher incidence of Ca2+ and action potential alternans under isoprenaline challenge and differences in baseline action potential and calcium transient characteristics. Type-2 ryanodine receptor (RyR2) channels from Pak1 cKO hearts had a higher open probability than those from wild-type. Reverse transcription-quantitative polymerase chain reaction and Western blotting indicated that pCamkII\u03b4 and RyR2 are highly phosphorylated at baseline in the atria of Pak1 cKO mice, while the expression of Slc8a2 and Slc8a3 as a Na+\u2013Ca2+ exchanger, controlling the influx of Ca2+ from outside of the cell and efflux of Na+ from the cytoplasm, are augmented. Chromatin immunoprecipitation study showed that pCreb1 interacts with Slc8a2 and Slc8a3. Our study thus demonstrates that deficiency of Pak1 promotes atrial arrhythmogenesis under adrenergic stress, probably through post-translational and transcriptional modifications of key molecules that are critical to Ca2+ homeostasis.P21-activated kinase 1 (Pak1) signalling plays a vital and overall protective role in the heart. However, the phenotypes of This article is part of the theme issue \u2018The heartbeat: its molecular basis and physiological mechanisms\u2019. Abnormal \u2018phosphorylation states' of Ca2+-handling proteins occurs in several disease conditions including AF  approximately 50 mM) on the trans  side of the bilayer at 21\u00b0C. The luminal chamber was voltage-clamped at ground. The free [Ca2+] and pH of the solutions were measured using a Ca2+ electrode  and a Ross-type pH electrode  as previously described i under Ang II stress at single cell level.Our electrophysiological studies in intact animals and isolated atrial independently implicated out mice  that sho2+ alternans recorded in LA in particular when they are acutely exposed to isoprenaline stress in our study suggests the alteration of Ca2+ homeostasis that could be linked to either abnormal SR Ca2+ release or decrease in kinetics of SERCA2A and NCX currents and an increase in diastolic Ca2+ levels in as we found in Pak1 cKO mouse ventricular cardiomyocytes under baseline conditions [2+ reuptake into the SR via SERCA2A inhibition using the antagonist thapsigargin was demonstrated to increase the susceptibility to cellular Ca2+ alternans [et al. [Canditions . Upon islternans . Additio [et al.  have sho [et al. .2+ refilling time in Pak1 cKO ventricular myocytes when compared to WT littermates. This observation along with slow NCX and SERCA2A kinetics, may imply longer cytosolic Ca2+ transients. In fact, ventricular myocyte experimentation using total Pak1 KO mice have reported longer Ca2+ transient recovery time in KO than in WT mice [Pak1 cKO mice under baseline conditions in the present study.We previously  have als WT mice . The two2+ sensitivity of RyR2 channels from PAK1 cKO mice. This increase in RyR2 activity might have resulted from the higher phosphorylation levels at S2808 and S2814 detected in the PAK1 cKO heart preparations since enhanced phosphorylation at these sites has been linked to RyR2 dysregulation and increased RyR2-mediated cytosolic Ca2+ leak [2+ would probably impact on the consistency of local SR Ca2+-release events, providing an explanation of the cellular basis for an increase in arrhythmogenesis and abnormal Ca2+ transient observed in Pak1 cKO mice.Our data on RyR2 single-channel recordings revealed that channels derived from PAK1 cKO hearts exhibit a markedly higher Po than channels from control hearts, suggesting an increase in cytosolic Caa2+ leak \u201339. The Pak1 cKO mice with alterations of the activity of RyR2 and CamKII and expression of Serca2a and Ncx genes that are essential to Ca2+ homeostasis and electrophysiological stability in the atria. These changes may reflect multiple regulatory mechanisms that PAK1 is involved in Ca2+ handling proteins activity and expression at both post-translational and transcriptional levels. Firstly, in our previous study, we have established the action of PAK1 on the phosphatase PP2A and the resulting balance between kinase and phosphatase activity controlling LTCC and IK activity in sinoatrial node pacemaker cells [Pak1 cKO mouse atrial tissue. Admittedly, such mechanisms require more investigations in the future, while the downregulated SERCA2a and upregulated Slc8a2 and Slc8a3 transcripts in Pak1 cKO atrial tissue may reflect a complicated transcriptional regulation but important role of Pak1 as a regulator of cardiac SERCA2a and Slc8a2 and Slc8a3 through transcriptional mechanisms, which is consistent with our previous finding in the ventricular tissue [Our molecular studies then associated PAK deficiency in er cells ; An impoer cells . As a coer cells . Thus Par tissue .2+ homestasis in the atria through regulation of calcium handling proteins RyR2, Serca2a and NCX via either post-translational and transcriptional mechanisms, thus providing new insights into atrial Ca2+ signalling regulatory mechanism that has an implication in developing new therapeutic strategies for atrial tachyarrhythmias.In conclusion, our study demonstrates for the first time, to our knowledge, a key regulatory role of Pak1 for maintaining electrophysiological stability and Ca"}
+{"text": "Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol\u2014a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug. Candida albicans is an opportunistic pathogen causing severe morbidity and mortality in immunocompromised patients. Candida spp are the second most common cause of nosocomial bloodstream infection in the US, and cause about 70% of all fungal infections  / pellet weight. Equation 2: % DHE = [(A230/518) \u00d7 dilution factor]/pellet weight. % ergosterol = Equation 1 \u2013 Equation 2. The dilution factor is the dilution factor used for diluting the sterol-containing heptane solution with ethanol, 290 = E for ergosterol at 282 nm, and 518 = E for DHE at 230 nm -2H-tetrazolium hydroxide (XTT) (Alfa-Aesar) in PBS with 4 uL of menadione per/100 uL was added to each well. The plates were incubated for one hour at room temperature and then 50 uL of solution from each well was removed and read at 492 nm to determine metabolic activity of each well. The no-drug well averaged between the two duplicates was used to calculate the OD492 that indicates a 50% reduction. The concentration of drug that yielded a reduction in metabolic activity immediately less than 50% of the no-drug well was considered the MIC50. The heat map of the metabolic activity of these wells is shown as the medians of 6 biological replicates for each strain in For the broth-microdilution assay, cells were grown as stated above and 10Supplement 1Supplemental Figure S1: Quantification of small colony variants throughout passaging of four separate populations of cells that were exposed repeatedly to increasing concentrations of ethanol in medium A or repeatedly exposed to water in medium A. Quantification of small colony variants throughout passage numbers of four separate populations of cells that were exposed repeatedly to increasing concentrations of ethanol in media A or repeatedly exposed to water in media A. Green squares were repeatedly exposed to ethanol and black dots were repeatedly exposed to water. Y-axis shows % small colonies relative to total number of colonies. Bars represent the mean values of the populations at that passage number. Small colonies were classified as less than 50% of the size of the normal sized colonies on a plate by eye.Supplement 2Supplemental Figure S2: Enc and Wnc survival in 10% ethanol. Enc and Wnc survival in 10% ethanol. Colonies exposed for 4 hours in 10% ethanol or 10% water and plated on YPD-agar to count final colonies. The percentage of final colonies in 10% ethanol compared to 10% water were calculated and used to calculate a relative survival compared to SC5314 within each experiment (y-axis). Normal-sized colonies from ethanol populations (Enc) or water population (Wnc) were used. One-way ANOVA with Welch\u2019s correction was used for statistical testing with multiple comparisons. Median values with 95% confidence interval are plotted for 8\u201316 replicate cultures from 2\u20134 separate experiments for each strain.Supplement 3Supplemental Figure S3: Growth curves and doubling times of Esc strains and normal-sized colony strains in YPD. Isolated colonies were picked and grown for 20 hours to post-exponential phase and seeded into YPD in 96 well plates at an approximate OD600 of 0.05. Cells were grown for 48 hours at 30\u00b0C with constant shaking in a Biotek Epoch2 plate reader with OD600 read every 30 minutes. Three biological replicate cultures from each strain were used to graph the growth curves. Growth curves show the mean and standard deviation of each timepoint. The wildtype, background strain: SC5314, a culture from the repeat media-A (with no ethanol exposure) is shown as RepDMEM-2, and a normal sized colony from a repeat ethanol exposure population is shown as RepEtOH-8. Esc6, Esc7, and Esc8 are all shown as well. Timepoints 0\u201324 hours were used to calculate doubling times by performing a nonlinear regression in GraphPad Prism with the exponential plateau fit to find the rate constant: k. All R2 values were above 0.9331. k values were then used to calculate doubling times with the formula LN(2)/k. These were standardized to the SC5314 doubling times and then plotted in this figure as standardized doubling times.Supplement 4Supplemental Figure S4: Principal component analyses for SC5314 vs Esc6, Esc7, or Esc8 in 10% ethanol from RNA sequencing. Principal component analyses for SC5314 vs Esc6, Esc7, or Esc8 in 10% ethanol each from RNA sequencing. PC1 variance ranges from 81%\u201393% and PC2 variance ranges from 4%\u20139%. SC5314 is shown in red dots in each and Esc strains are shown in blue. Each dot represents a separate biological replicate culture that was sequenced.Supplement 5Supplemental Figure S5: Gene Ontology Terms of the Top 20 Processes of Significantly Enriched Transcripts in Esc strains exposed to ethanol compared to SC5314 exposed to ethanol. Gene Ontology Terms of the Top 20 Processes of Significantly Enriched Transcripts in Esc strains exposed to ethanol compared to SC5314 exposed to ethanol. Transcripts that were significantly enriched in Esc strains were used to determine GO term process clustering on Candida Genome Database GO term finder. The top 20 hits from each strain were added to the graph and shown on y-axis. Esc6 bars are shown in blue, Esc7 bars are shown in black, and Esc8 bars are shown in orange. X-axis shows the cluster frequency of each of these processes relative to the total number of genes in the respective sets of enriched transcripts used for the analysis.Supplement 6Supplemental Figure S6: Histogram Fit Curve of Esc6 Allele Depth Ratio. Histogram fit curve of Esc6 allele depth ratio is shown. Esc6 is used as an example of what was done for all of the strains that were sequenced. Binning was performed by calculating the number of genes that had allele depth ratios within bins of 0.05. The bin of 0\u20130.05 was set to an arbitrary number as this represents homozygous for the reference strain and should not be called in the sequence variant report. Then a trimodal fit was performed in excel and minima values were calculated and used for further determination of heterozygous and homozygous polymorphisms.Supplement 7Supplemental Figure S7: MIC50 values from broth-microdilution experiments of SC5314 and Esc6, Esc7, and Esc8. MIC50 values from broth-microdilution experiments of SC5314 and Esc6, Esc7, and Esc8. Six separate biological replicate experiments were conducted and plotted. MIC50 values were determined for each by measuring metabolic activity (XTT) and determining the well that represented a 50% reduction in metabolic activity. Geometric means with geometric SD are graphed and ordinary One-way ANOVA was performed for statistical significance.Supplement 8Supplemental Figure S8: Voriconazole Disk-Diffusion Images of SC5314 and Esc Strains. SC5314 and Esc strains  were grown to post exponential phase and then seeded onto YPD agar at a concentration of 105 cells per plate. A 1 ug disk of voriconazole was placed in the center of the plate. These plates were incubated at 30\u00b0C for 48 hours and then imaged with the same settings and converted to black and white images.Supplement 9Supplemental Figure S9: UPC2 to HSP90 Ratio of SC5314 vs Esc7 in different conditions. UPC2 to HSP90 Ratio of SC5314 vs Esc7 in different conditions. Arbitrary transcript values from RNA-seq data were used to calculate transcript ratios of UPC2 to HSP90 and were plotted (y-axis). Ordinary one-way ANOVA used for statistical significance. NS = not significant. * = p-value of 0.0377.Supplement 10Supplemental Table S1 Transcripts showing significant changes in abundance between SC5314 with ethanol vs water medium by RNASeqSupplement 11Supplemental Table S2 Transcripts showing significant changes in abundance between Esc7 with ethanol vs water medium by RNASeqSupplement 12Supplemental Table S3 Transcripts showing significant changes in abundance between Esc7 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 13Supplemental Table S4 Transcripts showing significant changes in abundance between Esc7 with water vs SC5314 with water medium by RNASeqSupplement 14Supplemental Table S5 Transcripts showing significant changes in abundance between Esc6 with ethanol vs water medium by RNASeqSupplement 15Supplemental Table S6 Transcripts showing significant changes in abundance between Esc8 with ethanol vs water medium by RNASeqSupplement 16Supplemental Table S7 Transcripts showing significant changes in abundance between Esc6 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 17Supplemental Table S8 Transcripts showing significant changes in abundance between Esc8 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 18Supplemental Table S9 Transcripts showing significant changes in abundance between Esc6 with water vs SC5314 with water medium by RNASeqSupplement 19Supplemental Table S10 Transcripts showing significant changes in abundance between Esc8 with water vs SC5314 with water medium by RNASeqSupplement 20Supplemental Table S11 Esc6 Genomic PolymorphismsSupplement 21Supplemental Table S12 Esc7 Genomic PolymorphismsSupplement 22Supplemental Table S13 Esc8 Genomic PolymorphismsSupplement 23Supplemental Table S14: Primers used in this study."}
+{"text": "BRCA1 and BRCA2 in metaplastic breast cancer. Their headline rate of 56% for BRCA1 is extremely high, but caution is necessary in translating this to all metaplastic cancers as there is likely a strong testing bias.In this issue Corso et al.  have carBRCA1 or BRCA2. A previous report of exome sequencing of 30 metaplastic breast cancers with paired normal tissue did not report any mutations in BRCA1 or BRCA2 [Metaplastic breast cancers are a heterogenous group of invasive breast cancers which share differentiation toward squamous or mesenchymal-appearing elements . The repor BRCA2 , althougor BRCA2 , 7.BRCA1 and BRCA2 genes, of which 23 (0.4%) were diagnosed with metaplastic breast cancers. They identified 13/23 (56.5%) with a BRCA1 pathogenic variant compared to 11.5%  with other breast cancer types (p\u2009<\u20090.0001). Interestingly one of the BRCA1 variants was found in one of the two cases with HER2 overexpression which is not usually a feature of BRCA1 cancers [BRCA1 driven than those that are oestrogen overexpressed [BRCA1 pathogenic variants in metaplastic breast cancer is about 10-fold less based on the detection rates in all breast cancers tested. This is because population testing studies outside clear founder populations only find around a 1% rate for BRCA1 [BRCA1 variant carriers were <53 years of age at diagnosis and 7/13 had a family history of breast or ovarian cancer with 2 being tested for a known variant in the family. Although the detection rate in this study is extremely high this bias in testing should mean a cautious approach to assessing the likelihood of a BRCA1 pathogenic variant in those with a sporadic metaplastic breast cancer aged >60 years as the detection rates are likely to be very much lower. For instance, the report of exome sequencing came from a cohort of 347 with over 2/3rds of tumours >51years at diagnosis and no BRCA1/2 variants were reported in 30 metaplastic tumours. Although the age range in the 30 cases undergoing exome sequencing was not recorded [BRCA1 variants if they had been seen, but large rearrangements that account for around 20% of BRCA1 pathogenic changes [In this edition of the journal Corso et al.  assessed cancers , 9. Althxpressed . The pred 13/23 5.5% with  changes  would noBRCA1 and BRCA2 Pathogenic Variant carriers and the diagnosis of metaplastic breast cancer should certainly not dissuade clinicians from ordering germline testing if other local criteria are met [BRCA2 in this study should not deter testing in this gene as they have also been reported and indeed shown to respond to PARPi [The Corso et al. report  clearly to PARPi .MLH1 and TP53. The TP53 associated tumour was a spindle cell sarcoma, but interestingly this was also the pathology in two of the BRCA1 carriers and indeed two BRCA1 carriers were noted to have squamous type. It is of note that other genes specifically associated with TNBC were not tested in all the samples and in particular more testing of PALB2, RAD51D, RAD51D, and BARD1 [Two further pathogenic germline variants were identified in nd BARD1 , 11 woul"}
+{"text": "This article has been corrected: The authors found an error in Figure 3B. During assembly of the figure, incorrect images of colonies formed by ACC cell lines in which ASXL1 was silenced by shRNA#1 (KD1) were used. The authors prepared a new Figure 3 using representative images from the original experiments. This correction has no impact on the main conclusion. The authors would like to apologize for any inconvenience caused.Figure 3 is presented below.New"}
+{"text": "The INO80 complex stood out in a large family of ATP-dependent chromatin remodelers because of its ATPase domain binding and translocating on DNA at the edge of nucleosomes, rather than at two helical turns from the center of DNA that is wrapped around nucleosomes. This unique property of INO80 was thought to account for its singular role in nucleosome placement at gene promoters in a DNA-sequence dependent manner that is crucial for transcription regulation. Now, we uncover INO80 functions differently than previously thought with its ATPase domain translocating on DNA close to the center of nucleosomes, like other remodelers. Our discovery also reveals the physical properties of the first ~36 bp of DNA on the entry side of nucleosomes is the main determinant for the DNA specificity of INO80 rather than the properties of the extranucleosomal DNA. The DNA sequence sensitive step of INO80 is after DNA is displaced from the histone octamer on the entry side of nucleosomes and 20 bp of DNA are moved out the exit side. We find the ATPase domain and Arp5 subunit of INO80 are likely involved in INO80\u2019s DNA specificity and the mechanism of INO80 remodeling is substantially different than originally proposed. Building on this observation, a scan was performed to find where single nucleotide gaps and nicks in nucleosomal DNA interfere with nucleosome movement, given DNA gaps/nicks are known to block DNA translocation of chromatin remodelers5. Gaps from Superhelical Locations (SHL)-2 to -6, where DNA enters nucleosomes, were found to efficiently block nucleosome mobilization by INO80 and helped confirm the idea that the ATPase domain of INO80 translocates at SHL-61. The following year cryo-EM revealed a mixed population of nucleosome bound Chaetomium thermophilum and human INO80 with the ATPase domain bound on nucleosomal DNA at SHL-6 or SHL + 2 positions7. Given the earlier biochemical data, it was assumed that the structure with the ATPase domain at SHL-6 represented the active form of INO80. Interestingly, the structure of INO80\u2019s ATPase domain bound at SHL-6 differs from that observed with Chd1 and Isw1a ATPase domains bound to SHL + 2 9. In the same year, the cryo-EM structure of yeast SWR1 complex-bound nucleosomes revealed its ATPase domain bound to DNA at SHL + 2, quite different than INO80 despite sharing numerous subunits and both belonging to the same sub-family of ATP-dependent chromatin remodelers10. Since then, INO80 is the only ATP-dependent chromatin thought to translocate on DNA near the edge of nucleosomes and this assumption has influenced many subsequent studies on the properties of this complex including its DNA sequence specificity.In 2017, chemical crosslinking and DNA footprinting techniques revealed that the ATPase domain of the INO80 chromatin remodeling complex associates with nucleosomal DNA at the SHL-6 position11. These studies found INO80 alone can correctly position more than 90% of nucleosomes at the + 1-position, next to the transcription start site. INO80 positioning of the + 1 nucleosome is not due to nucleosome spacing, a property that INO80 had been previously shown to exhibit, because the proper positioning of nucleosomes was not dependent on the density of nucleosomes assembled on DNA in these experiments13. These data show INO80 has intrinsic DNA sequence specificity and the DNA near promoters can arrest INO80 from further mobilizing nucleosomes. Examination of the physical properties of INO80 positioned + 1 nucleosomes compared to salt gradient dialysis (SGD) positioned nucleosome revealed linker DNA had more pronounced DNA propeller twist and the H2A-H2B bound DNA had less twist, corresponding to respectively more and less rigid DNA in the INO80 positioned than SGD nucleosomes14. From these experiments, the physical properties of extranucleosomal DNA was thought to restrict INO80 remodeling and is supported by the idea of the ATPase domain translocating on DNA close to where extranucleosomal DNA enters and the Arp8/Arp4/actin module bound to extranucleosomal DNA known to control INO80 nucleosome mobilization activity16. This premise however was not well experimentally validated when the remodeling activity of nucleosomes with rigid and flexible linker DNA showed only minor differences17.The DNA sequence specificity of INO80 was found using yeast genomic DNA reconstituted into chromatin and scanning for ATP-dependent chromatin remodeler(s) that could restore the same nucleosome positioning as observed in vivo 1. The physical properties of DNA displaced from H2A-H2B on the entry side of nucleosomes are critical for the efficient passage of DNA from the entry to the exit sides of nucleosomes. Although previously overlooked, we find that the DNA sequence bound to H2A-H2B is the primary driver for INO80\u2019s DNA sequence specificity and Arp5\u2019s grappler and DNA \u201cgatekeeping\u201d activity are likely involved.Currently, we find that our view of how INO80\u2019s ATPase domain engages nucleosomes is incomplete and we need to revise the models of INO80 remodeling nucleosomes. Our current data support INO80\u2019s ATPase domain translocating on DNA at SHL + 2 not SHL-6 and the ATPase contacts at SHL-6 being important in regulating INO80\u2019s remodeling activity. The DNA gaps shown earlier now appear to not block translocation but instead likely impact the physical properties of DNA being displaced at this region akin to that seen with DNA sequence specificity18. The side of nucleosomes with the 70 bp extranucleosomal DNA is the (\u2212) side in reference to the dyad axis and the (+) side is on the opposite side of the nucleosomes. In 70N5 nucleosomes the (\u2212) and (+) sides are where DNA respectively enter or exit nucleosomes when remodeled by INO80. Ino80, the catalytic subunit of the INO80 complex, is crosslinked to DNA at a new position from nt + 4 to + 18 from the dyad axis that was not previously observed and at nt \u221258 and \u2212 110/\u2212111, positions that had been previously reported16  ported16 . Peptideported16 . The DNA21. When comparing these models, the site crosslinked at SHL-6, spanning from amino acid 683 to 799, is consistent with both models, making it inconclusive as to which model is more likely correct . However, the region of Ino80 from amino acid 915 to 1080 is only proximal to the correct site in DNA when the ATPase domain is actively engaged at SHL + 2 and not at SHL-6 .We model key segments of yeast Ino80 that were absent in the available structures using Alpha Fold2 and align this structure to those of INO80 and SWR1 bound to nucleosomes to find if the region of Ino80 mapped to SHL + 2 and \u2212 6 are consistent with the ATPase domain actively translocating at SHL + 2 DNA footprinting shows INO80 extensively interacting with DNA similar to that observed by DNA crosslinking and highlights where the ATPase domain of Ino80 translocates on nucleosomal DNA. INO80 protects the upper strand of DNA at the SHL-1 to + 3 and SHL - 5/-6 consistent with where the ATPase domain of Ino80 binds . There i22. DNA initially moves 20 base pairs (bp) on the exit side of nucleosomes then rapidly advances an additional 12 bp for a total movement of 32 bp from the starting point, as evident by the 20 bp step being marginally detected and not increasing over time while DNA moving 32 bp continuously increases over a period of 5 minutes  that binds to the H3-H4 tetramer and then separately switching the flanking 36 bp of DNA (referred to as M2) that bind to the H2A-H2B dimer . There aThe outcomes of these experiments are quite revealing. Altering the flanking DNA (M2) has a more pronounced effect on INO80 nucleosome mobilization compared to changing the orientation of the core DNA (M1). Depending on the orientation of INO80 switching the flanking 36 bp DNA either stimulated or repressed nucleosome movement with 70N5 M2 nucleosomes being remodeled 16-fold less efficiently and the rate of remodeling of 5N70 M2 nucleosomes increasing at least 5-fold as estimated after remodeling for 30 minutes . The difIncorporation of the histone variant H2A.Z in place of H2A does not cause any differences in INO80\u2019s DNA sequence specificity and shows that DNA sequence specificity does not depend on the type of H2A present . Next, we determine if the DNA sequence specificity of INO80 is shared by other ATP-dependent remodelers that sense linker DNA length and space nucleosomes by comparing the properties of ISW2 to INO80. We noticed that while ISW2 did show some DNA sequence-dependent effects, they were generally less pronounced and opposite to what we observed with INO80 and demonstrates the uniqueness of INO80\u2019s DNA specificity .7. The rate of ATP hydrolysis with all three mutant nucleosomes are equivalent to the original 601 nucleosomes and indicates that the reduction of remodeling observed with replacing the 10 bp DNA (M2.2) is uncoupled from ATPase activity, consistent with earlier observations . Arp5 interactions near the acidic pocket appear not to be changed; however, its interaction with H3, close to the SHL2 position on DNA, is reduced when INO80 is bound to M2 nucleosomes . Furthermore, we find a loss of Ies6 crosslinking at residue 80 of H3 but not at residue 109 of H2B. These differences in Arp5 photocrosslinking were not observed at residues 109 of H2B or 56 of H4, confirming the site specificity of Arp5 loss. DNA footprinting and histone crosslinking indicate two changes in INO80 interactions: altered binding of both the ATPase domain and the Arp5/Ies6 module with the histone octamer and nucleosomal DNA that is tied to DNA sequence changes at the H2A-H2B interface.We map DNA movement during remodeling as described earlier using DNA photocrosslinking and cleavage to discern at which stage in remodeling is DNA sequence important. The first obvious difference when remodeling M2 nucleosomes is that DNA movement on the entry and exit sides of nucleosomes are uncoupled from each other with new DNA positions only detected on the exit side . A shortThe question arises as to where does the 20 bp come from to translocate out the exit side if no DNA movement is observed at the entry side of nucleosomes. We find that DNA displacement occurs on the entry side of M2 nucleosome, equivalent to that observed with 601 nucleosomes, as indicated by reduced crosslinking to DNA not coupled to DNA movement . DNA disChaetomium thermophilum of the ATPase domain engaging either SHL-6 or SHL + 2. Based on our findings, SWR1 and INO80 complexes likely engage nucleosomes more similarly than thought and the observed binding of the ATPase domain at SHL + 2 for INO80 is not an artifact of cryo-EM.The model of INO80 engaging and remodeling nucleosomes needs to be substantially revised starting with the ATPase domain\u2019s interaction and translocation on nucleosomal DNA. DNA photocrosslinking and peptide mapping reveals the ATPase domain of Ino80 contacts both SHL-6 and SHL + 2, bridging these two superhelical locations. It becomes evident from DNA footprinting and crosslinking that Ino80 binds simultaneously to both positions as indicated by the extent of the footprint at these positions and the distinct regions of the ATPase domain that are crosslinked. Several approaches are used to decipher at which of these two sites is the cleft region of the ATPase domain bound and therefore translocating on when ATP is added. Both structural modeling of where the ATPase domain is crosslinked to DNA and mapping where DNA translocation occurs when an ATP is analog is added indicates the active cleft region of the ATPase domain is bound and translocates on DNA at SHL + 2 rather than the previously stated SHL-6 position. These findings resolve two previous conundrums as to why the ATPase domains of SWR1 and INO80 complexes engage nucleosomes so differently when they are paralogs and share many of the same subunits and the observation in the original cryo-EM structures of human and 27More evidence for the ATPase domain of Ino80 actively translocating on DNA at SHL + 2 comes from the DNA sequence specificity of INO80 providing an effective way to capture early stages in INO80 remodeling. When INO80 nucleosome remodeling is dramatically slowed due to changes in the DNA sequence, we see DNA only be moved to new positions on the exit side of 70N5 M2 nucleosomes. Although no new DNA positions are observed on the entry side of nucleosomes, DNA is displaced where H2A-H2B binds at the same rate and extent as DNA translocation on the exit side. These data suggest DNA displacement near where the ATPase domain binds SHL-6 is likely coupled to DNA translocation at SHL + 2 and not due to DNA translocation at SHL-6 as originally proposed Next, DNA translocation is arrested after moving 20 bp on the exit side and uncouples the normally fast transition from moving 20 to 32 bp observed with the preferred DNA substrate (70N5 601 nucleosomes). These results indicate that the movement of 32 bp of DNA on the exit side occurs after DNA is displaced from the histone octamer on the entry side and this transition is the stage at which DNA sequence influences the rate of nucleosomes being moved by INO80 .33. In contrast to these other remodelers, we find that the contacts of the ATPase domain at SHL-6 is crucial for INO80\u2019s DNA sequence specificity and likely vital for INO80\u2019s positioning of + 1 nucleosomes near transcription start sites in vivo, while with these other remodelers only modest effects have been conferred on remodeling33.There are several clues as to how DNA sequence might alter INO80\u2019s binding to nucleosomes and thereby effect its ability to mobilize nucleosomes based on DNA footprinting and site-directed histone crosslinking. Binding of the ATPase domain at SHL-6 is altered by DNA sequence as seen with a loss of INO80 protection at SHL-5 on one strand of DNA and on the other strand at SHL-6 along with DNA lifting off from nt \u221265 to \u221267. The ATPase domain still binds to part of DNA at this region and is not completely lost consistent with some type of conformation change. The importance of the ATPase domain binding at SHL-6 for regulating INO80\u2019s DNA sequence specificity is validated by replacement of a 10 bp A/T rich DNA centered at SHL-6 with a G/C rich DNA decreasing the rate of nucleosome movement by a factor of 4\u20135. These data show the interactions of the ATPase domain at SHL-6 is an important part of the DNA sequence sensing ability of INO80. The ATPase domain of other remodelers including the SWR1 complex are known to have a secondary binding site at SHL6 while also primarily binding to SHL2, as observed here with INO807. The observation with M2 nucleosomes of DNA translocation at SHL + 2 aberrantly moving pass the dyad axis till the SHL-2 position is consistent with the loss of Arp5 contacts at SHL-2 and Arp5 restraining DNA movement between the two sides of nucleosomes.There appears to be more factors involved in the DNA sequence dependence of INO80 because replacement of the 10 bp of DNA at SHL-6 does not show the same extent of inhibition as seen by swapping the 36 bps of DNA bound to the H2A-H2B dimer. It appears likely the Arp5 subunit of INO80 is involved and we observe like the ATPase domain of Ino80 that Arp5 contacts are also DNA sequence dependent. The DNA binding domain of Arp5 binds differently to nucleosomal DNA when the sequence is changed as seen with a loss of protection at SHL-2 on both DNA strands. Second, we observe by site-directed histone crosslinking a loss of Arp5 crosslinking to residue 80 of histone H3 in M2 nucleosomes compared to 601. These changes indicate Arp5 interactions with nucleosomal DNA are perturbed in a DNA-sequence dependent manner rather than through a complete loss of their binding. The involvement of Arp5 is consistent with its previously suggested role as a \u201cgatekeeper\u201d that regulates DNA movement from one side to the other of nucleosomes1. We had previously interpreted DNA gaps in the SHL-2 to \u22126 region that interfere with INO80 mobilizing nucleosomes to be due to blocking DNA translocation1. Based on the current data, we realize this is incorrect because INO80 is not translocating on DNA at this region and the gaps instead are likely tied to the strong DNA dependence observed at this same location. Together these data suggest the physical properties of the DNA being displaced from the histone octamer, including flexibility/rigidity, are crucial for regulating the rate of nucleosome mobilization by INO80 which are impacted by DNA sequence or breaks in the DNA strand. Studies from Phillip Korber, Karl-Peter Hopfner and Sebastian Eustermann have shown that INO80 with yeast chromatin reconstituted in vitro preferentially stops mobilizing nucleosomes when encountering DNA that is highly enriched with a less negative propeller twist from SHL-3 to SHL-6, indicative of DNA flexibility at this region being important for the specificity of INO80 to position nucleosomes14. These data are consistent with our observations and further highlight the importance of DNA physical properties in this region. More work is needed to better understand how DNA shape effects this important step in INO80 remodeling.In summary, our data indicates the DNA sequence specificity of INO80 resides principally at the H2A-H2B region proximal to where linker DNA enters nucleosomes, the ATPase domain of Ino80 and the Arp5 subunit are involved, and the sequence impacts the ability of DNA to pass from the linker proximal to distal sides of the nucleosome. It seems that not only is DNA sequence important, but also the integrity of the DNA strand in this region of the nucleosomes based on earlier studies34.This revised understanding of INO80\u2019s mechanism raises questions about whether hexasomes are the correct substrate for INO80 and if hexasomes remodeled by INO80 would exhibit the same DNA sequence specificity as nucleosomes, given the importance of the DNA bound to the proximal H2A-H2B dimer that is typically absent in the hexasome experimentsSaccharomyces cerevisiae INO80 and ISW2 complexes were purified by immunoaffinity purification with 2 - FLAG (DYKDDDDK) epitopes attached at the C-terminus of the catalytic subunit. Yeast was grown in 15L YPD cultures , until the OD600 reached 5\u20136. Cells were harvested, washed with ice-cold water followed by ice-cold H-0.3 buffer  containing protease inhibitors . The cell pellet was collected and passed through a syringe to make yeast spaghetti, which were frozen in liquid nitrogen. The spaghetti was ground into fine powder by cryogenic grinding using (Spex freezer mill 6870). The yeast powder was suspended with ice-cold H-0.3 buffer with protease inhibitors, and nuclear proteins were extracted (S-100 extract) by ultracentrifugation at 100,000 g for 1 h using a Ti-55.2 rotor in an ultra-centrifuge (Thermo Fisher) at 4\u00b0C. The supernatant containing the soluble protein fraction from ultracentrifugation was incubated overnight at 4\u00b0C with the Anti-FLAG M2 agarose beads (Sigma Aldrich) (10 \u03bcl beads per ml S-100 extract) equilibrated with buffer H-0.3 with protease inhibitors. The resin was washed several times with buffer H-0.5 followed by H-0.1  with protease inhibitors. FLAG-tagged protein complexes were eluted from the resin with 1 mg/mL solution of 3X-FLAG peptide in buffer H-0.1 containing PMSF only (no other protease inhibitor). Complex purity and integrity was determined by analyzing samples on 4\u201320% gradient SDS\u2013polyacrylamide gels and staining with coomassie brilliant blue R-250 and SYPRO Ruby protein stain.Native wild-type The M1 and M2 601 DNA constructs were synthesized by IDT-DNA. The M1 construct was generated by swapping and taking the reverse complement of the middle 36 bp DNA fragments, whereas the M2 construct was generated by swapping and taking the reverse complement of the outer 36 bp DNA fragments. The 601 DNA sequence mutant M2.1, M2.2, and M2.3 constructs were generated by site directed mutagenesis using the NEB SDM Kit by swapping the nucleotide regions with the opposite M2 corresponding nucleotide regions.32P]-labeled DNA at 37\u00b0C by rapid salt-dilution with 3\u20135 \u03bcg of recombinant Xenopus laevis histone octamers . The DNA and histone octamer was serially diluted from 2 M to 280 mM NaCl in steps at 37\u00b0C. Reconstituted nucleosomes were examined on a native 4% polyacrylamide gel (35.36 acrylamide: 1 bisacrylamide) (PAGE) and the 32P signal was captured on phosphorimaging .Mononucleosomes were reconstituted with 1.7 \u03bcg of PCR-generated 70N5 and 5N70 DNA (70 and 5 bp of flanking DNA on either side of 145 bp of 601 nucleosome position sequence DNA) and 100 fmol of 5 \u00b4-labeled Ino80-FLAG markers of known molecular weights prepared by in vitro coupled transcription and translation using TnT\u00ae T7 Quick Coupled Transcription/Translation System (Promega) as described before 1. Data images in figures are representative of \u2265 3 experiments.Photoaffinity-labeled INO80 complex (after digestion of DNA and label transfer) was denatured with 0.4% SDS and heating at 90\u00b0C for 3 minutes, followed by buffer exchange using Amicon Ultra filters to remove SDS and FLAG peptides. C-terminal FLAG-tagged INO80 was purified by immobilization on ANTI-FLAG1. Briefly, Protease (ArgC) digested Ino80 fragments were resolved in 4\u201320% Tris-glycine SDS-polyacrylamide gels and transferred onto PVDF membranes using Bio-Rad Trans-Blot\u00ae electrophoretic transfer cell. The membranes were blocked with 5% fat-free milk in TBST , overnight at 4\u00b0C, washed with TBST, and incubated with mouse monoclonal ANTI-FLAG\u00ae M2-Peroxidase (HRP) antibody  diluted 1:1000 for 1 hour at room temperature. The blots were washed with TBST, and developed with SuperSignal\u2122 West Femto Maximum Sensitivity Substrate (Thermo Fisher) visualization using an Image Quant LAS 4000 . .Western Blots were performed as described before 36. dUMP and dCMP analogs coupled with p-azidophenacyl bromide were incorporated along with [\u03b1-32P] dGTP/dATP, in tandem, at specific positions. Photo-reactive DNA was reconstituted into nucleosomes and bound with saturating amounts of INO80 at 30\u00b0C for 30 min. The extent of enzyme binding was assessed on 4% native polyacrylamide gels. In all experiments > 90% of nucleosomes were bound by the enzyme. After binding, INO80 was crosslinked to DNA by UV irradiation , and DNA was digested with DNaseI and S1 nuclease for transfer of the radioactive label to the crosslinked protein(s)36. Protein subunits were separated on 4\u201320% SDS polyacrylamide gels and radiolabeled subunit(s) were visualized by phosphorimaging. Data images in figures are representative of \u2265 3 experiments.Site-specific photo-reactive DNA probes with 601-nucleosome positioning sequence for 70N5 nucleosomes were synthesized by enzymatic incorporation of modified nucleotides into double stranded DNAXenopus laevis histones with amino acids replaced by cysteine at specific positions  were expressed, purified, and reconstituted into octamers with other histones as discussed above. Homogeneous 70N5 601/M2 nucleosomes were reconstituted using the respective 601-nucleosome positioning sequence containing Cy5 label at one end. The PEAS [N-((2-pyridyldithio)ethyl)-4-azidosalicylamide] (Thermo Fisher) -conjugated nucleosomes were iodinated with [125I] (Perkin Elmer). Detailed protocol had been discussed and standardized earlier 16. Nucleosomes with photoreactive octamers were bound to saturating amounts of INO80 at 30\u00b0 C and were irradiated directly under UV  for crosslinking. After crosslinking, samples were treated with 100 mM DTT and incubated at 37\u00b0 C for 30 minutes to transfer [125I]-radiolabel to the crosslinked proteins. Samples were resolved on 4\u201320% SDS-polyacrylamide gels, dried and viewed by phosphor-imaging following 14\u201316 hours of exposure to identify the radiolabeled subunits. Data images in figures are representative of 3 individual experiments..Recombinant"}
+{"text": "GRAIN WIDTH AND WEIGHT 2  exhibit abnormally wide grains (UBIQUITIN SPECIFIC PROTEASE 15 (OsUBP15) promotes grain size and weight  proteasome pathway. The Ub-proteasome pathway participates in a wide range of physiological processes, including plant reproduction  was identified in an EMS-generated mutant population; smg3-1 displayed shorter panicles and reduced grain number per panicle; the reduced grain size was found to be due to defects in cell expansion  displayed similar defects, confirming the role of SMG3 as a positive regulator of grain size.A small grain mutant  revealed that SMG3 is localized to the endoplasmic reticulum where it colocalized with the ER-marker HDEL. At the whole plant level, the staining patterns of the pSMG3::SMG3-GUS showed that the SMG3 gene is expressed in the developing panicles and the roots. pathway . SMG3 inDGS1 gene, Li and colleagues generated four alleles in the ZH11 and KNK rice varieties , which displayed defects in grain size. Interestingly, the double mutant smg3-2 dgs1-1 showed a greater reduction in grain size than either single mutant alone. On the other hand, overexpression of DGS1 in a ZH11 background resulted in longer rice grains. The defects in the dgs1 mutant alleles resembled those of the smg3 mutant. Interestingly, both DGS1 and SMG3 genes were expressed in developing panicles. At the subcellular level, analysis of the GFP-DGS1 line indicates localization to the ER. To test if DGS1 is a functional E3 ligase, the authors expressed the protein in E. coli to conduct a biochemical analysis. Since a full DGS1 could not be expressed in E. coli they used a version lacking the transmembrane domain (DGS1\u0394TM) to perform a ubiquitination assay. This analysis indicated that DGS1 can autoubiquitinate; and that mutations in the RING domain in DGS1 abolished the activity.To identify other components in the ERAD pathway, the authors tested protein interactions between SMG3 and other known regulators of grain development in rice using yeast two-hybrid assays. The analysis indicated that SMG3 physically interacts with DECREASED GRAIN SIZE1 (DGS1), which has been previously characterized in the control of grain size regulation . The SMGsmg3-1 and dgs1 mutants were less sensitive to brassinosteroids. Additionally, the smg3 and dgs1 mutants had changes in gene expression consistent with changes in BR function . In contrast, the expression of BR-signaling genes BZR1 and GSK2 was decreased. Furthermore, in vitro experiments demonstrated the interaction between BRI1 and DGS1, also found in planta with split luciferase assays. This interaction is critical since DGS1 was identified as a functional E3 ubiquitin ligase and can ubiquitinate BRI1.A previous study of the SMG3 homolog in Arabidopsis (UBC32) indicated that this protein participates in the degradation of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1), so authors tested the relationship between SMG3, DGS1, and BR signaling in rice. They found that the dgs and smg3 mutants, suggesting that SMG3 and DGS1 affect the abundance and hence the function of the BRI1 receptor.To explore the effect of DGS1 on BRI1 function, the authors used an anti-BRI1 antibody to measure the accumulation of the BR receptor in different genetic backgrounds. The BRI1 protein levels were increased in the In summary, this work provides evidence for the participation of ER-associated degradation in plant hormone signaling in the control of reproductive development. Some interesting phenomena are still to be discovered regarding the involvement of SMG3 in the responses to abiotic stresses and its possible participation in root development."}
+{"text": "This article has been corrected: The authors found that they incorrectly submitted two images: Figure 2I (tumors from the NC and FBXO28 groups), showing upregulation of FBXO28 in BxPC-3 cells in a mouse tumorigenesis experiment, and Figure 5G (FBXO28+SMARCC2 48h panel), showing results obtained with transfected BxPC-3 cells in a wound healing experiment. The authors stated that these corrections were prepared using images from the original experiments and do not change the results or conclusions of this paper. The authors apologize for this error.Figure 2 and 5 are presented below.Corrected"}
+{"text": "Recent quality initiatives require that the routine annual therapeutic drug-monitoring (TDM) parameters for the high-risk medication digoxin include a measure of renal function and a serum potassium level but not a serum digoxin concentration (SDC) measurement. Several studies have shown that the majority of the SDCs obtained in hospital settings provide little clinically actionable information.To evaluate the appropriateness and utility of SDCs ordered in a medical group practice setting by categorizing the reason the SDC was ordered and identifying action taken in response to the result.The descriptive study was conducted as a retrospective, electronic medical record (EMR) review of 90 primary care patients with continuous prescriptions for digoxin current on their medication profile with no gaps in therapy for at least 2 years prior to an SDC result entered into the EMR between January 1, 2009, and September 30, 2009. The reason the SDC was ordered was abstracted independently by 2 reviewers, who then assigned it to 1 of 8 predefined indication categories based on previously published criteria and practice guidelines. A third reviewer resolved inter-reviewer discrepancy (n?=?1).A total of 90 patients with at least 1 SDC met inclusion criteria. Routine monitoring was the most frequent SDC order indication category with 35 patients (38.9%), 17 (48.6%) of whom did not have the recommended monitoring measures of potassium or renal function drawn concurrently. Patients were included in other categories as follows: confirmation of signs/symptoms of toxicity 30 (33.3%); assessment of factors altering pharmacokinetics 5 (5.6%); assessment of dosage change 5 (5.6%); assessment of drug interaction 3 (3.3%); assessment of clinical response 3 (3.3%); assessment of adherence 1 (1.1%); and other 2 (2.2%). Across all categories, a total of 19 (21.1%) of SDC results were outside the therapeutic range of 0.5 nanograms (ng) per mL and 2.0 ng per mL, 18 of which were below 0.5 ng per mL, with none of the subtherapeutic levels leading to a change in digoxin therapy. Only 1 patient (1.1%) had therapy changed in response to an elevated abnormal SDC result of 2.1 ng per mL and was in the routine monitoring category.The majority of SDC results obtained in our medical group setting did not lead to clinical action, such as dose adjustment or drug discontinuation. SDCs were commonly measured as part of routine monitoring, which is considered an inappropriate indication, and often without being accompanied by better markers for digoxin toxicity such as serum potassium levels and measures of renal function as recommended by drug-monitoring quality initiatives. Provider education is needed regarding the most indicative digoxin TDM parameters to obtain in order to satisfy quality initiatives."}
+{"text": "Following the publication of this article , concernThe P21 western blot panel in Fig 4A appears to have no detectable signal or background, nor does the panel present a positive control to confirm a successful blot.The Ad5/F35-siAPE1 panel in Fig 8A appears to partially overlap with the Ad5/F35-siAPE1+IR panel in Fig 8a and the Ad5/F35-siAPE1+IR panel in Fig 9a despite representing different experimental conditions.The Ad5/F35-siAPE1 panel in Fig 9A appears to partially overlap with the Ad5/F35-EGFP+IR panel when rotated despite representing different experimental conditions.The authors did not provide a response to the concern regarding the P21 western blot in Fig 4A nor was any underlying data provided to resolve this issue.The authors stated that the concerns related to Figs 8 and 9 were unintentional errors made during preparation of the figures but stated they were unable to source the original underlying data for both figures. The PLOS Editors remain concerned about the reliability of these figures.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the reliability and validity of these data, the All authors either did not respond directly to the final editorial decision or could not be reached."}
+{"text": "To measure the impact of clinical pharmacists in primary care practices who closely monitor patients older than 80 years after initiation of new medications. \u00a0The study was an uncontrolled pilot trial performed at a group-model health maintenance organization in the Pacific Northwest between August and December 1999. Forty-eight patients who were older than 80 years and were prescribed at least one new medication in their primary care clinic were called at home 3 to 6 days after starting a new medication and asked questions focusing on compliance and potential adverse drug events. \u00a0More than 20% of patients (10 of 48) had a clinically important change made as a result of the pharmacist telephone monitoring; 42% of patients (20 of 48) either experienced an undesired medication effect (14 of 48) or an inadequate effect (6 of 48). Pharmacists spent an average 11.3 minutes at an estimated cost of $6.40 per patient. \u00a0A simple, inexpensive pharmacist-based program to screen for medication problems after initiation of new medicines may improve the care to a population older than 80 years."}
+{"text": "After this article  was publSpecifically:The LRRK2 KO \u03b2-actin panel in Fig 4A appears similar to the LRRK2 WT p38 panel in Fig 9A.The authors stated that the above concern was due to errors made during preparation of the figures and stated that the panel in Fig 9A was correct. The authors provided the underlying panel and accompanying quantification data for Fig 4A  along wiIn reviewing this matter, PLOS noted that the primary data were not provided with the published article  contraryPLOS ONE Editors issue this Expression of Concern.In light of the above concern pertaining to data availability, the S1 File(PPTX)Click here for additional data file.S2 File(PZFX)Click here for additional data file.S3 File(PPTX)Click here for additional data file.S4 File(PZFX)Click here for additional data file."}
+{"text": "Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone\u2010modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3\u2010recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED\u2010associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2. Polycomb repressive complex 2 (PRC2) is composed of four core subunits \u2013 embryonic ectoderm development (EED), enhancer of zeste homologue 2, suppressor of Zeste 12, and RB\u2010binding protein 4/7. PRC2 catalyses methylation of H3K27, an enzymatic activity necessary for PRC2\u2010mediated epigenetic gene silencing during stem cell differentiation and transformation. PRC2 absent EED is incapable of recognizing binding sites to promote deposition of H3K27me3, results in a significant upregulation of genes that are normally associated with H3K27me marks. The nucleosome represents the smallest repeated structural unit of eukaryotic chromatin, consisting of DNA wrapping around histones,Drosophila as repressors of Hox genes and are gradually found in other species, such as human, mouse, Xenopus, and so on.Polycomb group (PcG) proteins are key epigenetic regulators that mediate heritable transcriptional silencing by modifying chromatin states and participating in the establishment and maintenance of cell fates during multicellular development.A vast number of studies report that the core subunits of the PRC2 complex show spatiotemporal specific expression patterns, indicating that they may have distinct functions.This review provides a comprehensive overview of EED in regulating stem cell fate and the underlying regulatory mechanisms during various organ morphogenesis, refining the framework for understanding the functions and mechanisms of PRC2\u2010mediated regulation of gene expression.2As a WD\u201040 repeat family protein, EED contains seven WD40\u2010repeat motifs at its C terminus preceded by an extended N\u2010terminal segment.3EZH2 adopts an autoinhibitory conformation through crystal structures of the inactive isolated catalytic domain, suggesting that structural rearrangement of EZH2 is likely required for PRC2 activation.The PRC2 crystal structures provide fundamental insights into the interaction with each subunit, substrate recognition, and allosteric activation of its enzymatic activity. Moreover, PRC2 is more active on di\u2010nucleosomes and higher\u2010order chromatin structures than on mononucleosomes or histone tails,PRC2 recruitment is modulated by many different factors, including the interaction of PRC2 subunits with DNA and histones, PRC1\u2010mediated H2AK119ub1, RNA and other histone modifications.4Mammalian heterochromatin contains great repressive chromatin domains, including H3K9me2/3\u2010modified constitutive heterochromatin and H3K27me2/3\u2010decorated facultative heterochromatin. The two categories of histone modifications are catalysed by Suv39h1/2 and PRC2, respectively.5For pluripotent stem cells to expand and differentiate into one or more specialized and committed cell types in the body, critical cell fate decisions and lineage commitment must be made during growth and development. As differentiating stem cells undergo the cascade of lineage decisions on branching points through epigenetic\u2010threshold modulation, specific genes must be switched on and genes associated with alternative lineages need to be repressed in a dynamic and timely manner. Next, we will dissect the dynamic roles of EED during ontogenesis to further insight into the PRC2 and its role in the specificity and diversity of lineage specification Figure\u00a0.5.1\u2212/\u2212 embryos result in a genome\u2010wide decrease in H3K27me1, H3K27me2 and H3K27me3.ESCs possess the ability to differentiate into all the cell types of tissues and organs.5.2The primordial germ cells (PGCs) are precursors of the oocyte and sperm, which transmit significant epigenetic information to the offspring.5.3During the development of the nervous system, the PRC2 complex plays a vital role in maintaining the self\u2010renewal and proliferation capacities of neural stem/progenitor cells (NSPCs).5.4The development and function of the cardiovascular system are vulnerable to epigenetic insults, one of which is epigenetic repressors PRC2.5.5Intestinal stem cells (ISCs), located at the bottom of the crypts, are a cell population self\u2010renew extensively and differentiates into all cell types within crypts and villi.5.6The skin is the first barrier that protects mammals against external insults and dehydration. Epidermal lineages derive from a single layer of multipotent skin progenitors named basal cells, which attach to underlying basement membranes separating the epidermis from the dermis.5.7The bone marrow (BM) contains haematopoietic and mesenchymal stem cells.5.8Bone formation involves two major distinct mechanisms: intramembranous and endochondral ossification.6In this study, we comprehensively and systematically reviewed the research advances on EED/PRC2 function regulating ontogenesis Table\u00a0. PRC2 coLiuyan Huang conceived, wrote, revised the manuscript and made the figure and table. Fanyuan Yu and Feifei Li revised the manuscript. Ling Ye, Chenglin Wang and Fanyuan Yu reviewed, revised and edited the manuscript. All authors read and approved the final manuscript.This work was supported by National Natural Science Foundation of China 81873708 (Chenglin Wang), 82201045 (Fanyuan Yu), Sichuan Province Science and Technology Program 2022JDRC0130 (Fanyuan Yu) and 2022ZYD0055 (Fanyuan Yu) and Young Elite Scientist Sponsorship Program by CAST 2022QNRC001 (Fanyuan Yu).The authors declare no potential conflicts of interest."}
+{"text": "Point 5 of the section \u2018Available diagnostic tools and their limitations\u2019 incorrectly states that the BIOLINE Onchocerciasis IgG4 ELISA (Abbott) is discontinued. As of July 2023, the ELISA kit is still available, catalog number 61EK11."}
+{"text": "A new study by Longo, Roy et\u00a0al. has solved the structure of the RAD51C\u2010XRCC3 (CX3) heterodimer with a bound ATP analog, identifying two main structural interfaces and defining separable replication fork stability roles. One function relates to the ability of RAD51C to bind and assemble CX3 on nascent DNA, with an impact on the ability of forks to restart upon replication stress. The other relates to effective CX3 heterodimer formation, required for 5\u2032 RAD51 filament capping, with effects on RAD51 filament disassembly, fork protection and influencing the persistence of reversed forks. A new study by Longo, Roy et\u00a0al. has solved the structure of the RAD51C\u2010XRCC3 (CX3) heterodimer with bound ATP analog, identifying two main structural interfaces. Studies of cancer mutations of previously unknown significance in the two interfaces define separable replication fork stability roles required for fork protection and restart. Comprehensive analyses of breast and ovarian cancer pedigrees and patients with Fanconi anemia, a rare disorder characterized by hematological and developmental defects, pinpointed RAD51C as a cancer predisposition gene . This fiFunctional inquiries on RAD51C have been going on for decades. This is due to its role in interacting and assisting RAD51's recombinase function , centralTwo main complexes of RAD51 paralogs are known, with RAD51C being common to both . In one Alvinella pompejana (ap), an extreme metazoan living in deep\u2010sea waters. Leaving out a small, resolution limiting N\u2010terminal domain (NTD) of RAD51C and using a non\u2010hydrolyzable ATP mimic bound in the active sites of both RAD51C and XRCC3, the authors solved high\u2010resolution crystal structures of the CX3 heterodimer. This revealed extensive contacts between the RAD51C C\u2010terminal domain (CTD) and XRCC3 NTD and CTD, with these subdomains connected by a linker polymerization motif (PM) of XRCC3. Although similar to RAD51 structures in terms of ATP binding [Important insights on these topics came recently through the teamwork of several groups, combining structure elucidation of the CX3 complex with functional studies of specific cancer variants . Ingenui binding , the CX3Relying on the 3D representation of the CX3 heterodimer, the authors noticed that the cancer and patient mutations can be largely segregated in the two distinct interaction regions, CTDi and NTDi , 2, 14. in\u2009situ protein interaction with nascent DNA replication forks (SIRF). Besides this separation of function, the authors attempted to predict hCX3\u2010RAD51 complexes using AlphaFold2. In this way, they realized that XRCC3, but not RAD51C, interacts with RAD51 [What is the nature of the persisting replication stress? A126 is located on the P\u2010loop required to bind the ATP phosphate and is capable of disrupting the CX3 interface. Indeed, while the G125V mutation greatly impacts the structure and causes loss of interaction with XRCC3 as observed by two\u2010hybrid, the A126T mutation reduces the interaction with XRCC3 upon replication stress, as measured by proximity ligation assays or by co\u2010immunoprecipitation. While the G264S mutation does not destabilize the interaction with XRCC3, it greatly reduced the interaction of RAD51C with nascent DNA at stalled forks as revealed by Thus, CX3 plays important hitherto unappreciated roles during DNA replication stress, using different interfaces to facilitate fork restart or to protect forks and resolve emerging reversed forks. This work creates the foundation to study the multiple CX3 functions relevant to cancer biology at the nexus with replication stress response.The authors declare no conflict of interest."}
+{"text": "Among the genes located in this region, PRRT2 codes for a member of the synaptic SNARE complex that allows the release of synaptic vesicles. PRRT2 is a candidate gene for ASD since homozygote mutations are associated with intellectual disability and heterozygote mutations cause benign infantile seizures, paroxysmal dyskinesia, or hemiplegic migraine. Here, we explored the contribution of PRRT2 mutations in ASD by screening its coding part in a large sample of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the human genome Diversity Panel. We detected 24 nonsynonymous variants, 1 frameshift (A217PfsX8) and 1 in-frame deletion of 6 bp (p.A361_P362del). The frameshift mutation was observed in a control with no history of neurological or psychiatric disorders. The p.A361_P362del was observed in two individuals with autism from sub-Saharan African origin. Overall, the frequency of PRRT2 deleterious variants was not different between individuals with ASD and controls. Remarkably, PRRT2 displays a highly significant excess of nonsynonymous (pN) vs synonymous (pS) mutations in Asia (pN/pS\u200a=\u200a4.85) and Europe (pN/pS\u200a=\u200a1.62) compared with Africa . We also showed that whole genome amplification performed through rolling cycle amplification could artificially introduce the A217PfsX8 mutation indicating that this technology should not be performed prior to PRRT2 mutation screening. In summary, our results do not support a role for PRRT2 coding sequence variants in ASD, but provide an ascertainment of its genetic variability in worldwide populations that should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.Inherited and  NLGN3/4X, SHANK2/3, NRXN1 and CNTNAP2 were shown to alter synaptic function and increase the risk for ASD de novo or inherited submicroscopic microdeletion/duplication at 16p11.2 have been associated with a variety of developmental/neuropsychiatric disorders including ASD, intellectual disability (ID), schizophrenia, or bipolar disorders, but also with body mass index \u22127; deletion OR:38.7 and 2.3\u00d710\u221213) et al. (2009) screened for rare variations in 8 candidate genes selected for their function and expression in the brain . None of them showed a significant association with ASD at the exception of SEZ6L2 for which a suggestive association was detected PRRT2 gene PRRT2 codes for a membrane protein that interacts with SNAP25, a protein from the synaptic vesicles release machinery (the SNARE complex). PRRT2 is a compelling candidate gene for ASD and other psychaitric diseases since a homozygote frameshift mutation (A217PfsX8) was shown to segregate with non-syndromic ID in one consanguineous family with 9 affected individuals PRRT2 mutations (including the recurrent frameshift A217PfsX8) were identified in individuals and families with benign infantile seizures (BIS), paroxysmal kinesigenic dyskinesia (PKD), hemiplegic migraine (HM), or episodic ataxia, variably associated (e.g. the ICCA syndrome that associates BIS with PKD) PRRT2 in a sample of 961 individuals from worldwide populations.Autism Spectrum Disorders (ASD) are characterized by impairments in reciprocal social communication, and repetitive, stereotyped and ritualistic behaviors PRRT2 was performed in individuals with ASD recruited by the PARIS  study at specialized clinical neuropsychiatric centers disposed in France and Sweden  from blood leukocytes or B-lymphoblastoid cell lines, and was extracted with phenol-chloroform , S2. Allproducts . The 3 cPRRT2 variants in individuals and controls was determined by a two-sided Fisher's exact test on a two-by-two contingency table. PolyPhen-2 (http://psort.hgc.jp) and PSORT (http://genetics.bwh.harvard.edu/pph2) were used to predict the functional impact of amino acid substitutions and trans-membrane segments, respectively. To estimate the selective pressure on PRRT2 in different populations (pN/pS), the number of synonymous SNPs or nonsynonymous SNPs was divided by the total number of synonymous or nonsynonymous positions in PRRT2. The number of synonymous and nonsynonymous sites of PRRT2 were calculated by DnaSP v5 The significance of differences in PRRT2 in a large cohort of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the HGDP We sequenced all coding exons of PRRT2 mutation screening or in the 1000 genomes (>2000 controls). The genomes of these two individuals were investigated using the Illumina 1M Duo SNP array. Using Identity by state (IBS) analysis, we showed that these two individuals were not relatives . In two individuals with ASD from sub-Saharan African origin, a 6 bp in-frame deletion (p.A361_P362del) was identified. The variant was transmitted by unaffected mothers and never observed in any individuals from our study and from other elatives , but werelatives . This PRof PRRT2 . In siliIn the control sample, we identified one individual carrying the frameshift mutation A217PfsX8 already described as causing autosomal dominant ICCA, BIS, PKD, HM or epilepsy) PRRT2 variants and trace the possible origin of them. For example, the predicted damaging P140A is a relatively frequent polymorphism present in Asia (7%), Oceania (5%), in Native American (7%), but more rare in Europe . The A214P variant was observed in Asian  and Native American populations. The P216L was observed in North Africa , Europe (<1%) and Asia (<1%). Interestingly, we observed a higher ratio of nonsynonymous vs synonymous variants in Asia compared with Africa. In Asia, we found only 1 synonymous variant (E127E) carried by a single individual  and 15 nonsynonymous variants  carried by 78 individuals . In contrast, in Africa, we found 3 synonymous variants  carried by 7 individuals  and only 1 nonsynonymous variant (P45S) carried by 2 individuals .We used a panel of DNA from individuals originated from worldwide populations  and S5. vs Africa EVS P\u200a=\u200a0.67; Europe HGDP vs Europe EVS P\u200a=\u200a1). In order to test whether this difference in selective pressure was specific to PRRT2, we used the data from EVS and extended our pN/pS calculation to all genes included in the 16p11.2 deletion  to synonymous polymorphism (pS) in each population sample . Usuallydeletion . We obsePRRT2 mutation screening should not be performed on DNA previously amplified through RCA.During the screening of the recurrent frameshift mutation \u2018A217PfsX8\u2019, we used whole genome amplified DNA for three individuals with ASD. Strikingly, we observed the A217PfsX8 mutation in these three samples for which the DNA was amplified using rolling circle amplification protocol  . We obtaKCTD13 gene are located within the deletion and can be considered possible driver(s) as well PRRT2 was indeed a very compelling candidate. Homozygous PRRT2 mutations were associated with clinical features also observed in a subset of individuals with ASD such as intellectual disability and epilepsy. PRRT2 encodes a putative membrane protein, localized at the synaptic membrane and interacting with SNAP25, a member of the SNARE protein complex involved in the release of synaptic vesicles. Our study was designed to identify new coding variants of PRRT2 in a relatively large sample of individuals with ASD and to ascertain the genetic variability of the gene in worldwide populations.Both deletions and duplications at 16p11.2 increase the risk for ASD, but the genes and the mechanisms involved remain largely unknown. Whereas several other strong candidate genes including the PRRT2 mutation enrichment in individuals with ASD compared with controls. None of the patients tested were deleted or duplicated for the 16p11.2 locus. In addition, we did not find causative PRRT2 mutations previously associated with neurological diseases such as PDK, ICCA, epilepsy or migraine in our cohort of patients. We observed a significant enrichment of maternally transmitted mutations in the individuals with ASD (9/10 transmitted by the mother). However, this apparent disequilibrium might have occurred by chance since to our knowledge, no transmission disequilibrium was reported for PRRT2 mutations in other neurological diseases. Taken together, these results indicate that mutations in PRRT2 are not a frequent cause of autism. Our study cannot however exclude that, in individuals with 16p11.2 deletions, haploinsufficiency of PRRT2 could increase the risk of ASD.We could not find any deleterious Secondly, we detected the presence of the deleterious truncating mutation A217PfsX8 in a control . This man had no psychiatric diseases and, to his knowledge, no history of any psychiatric disorders in his first and second degree relatives (parents and grand parents). This individual had no diagnostic for migraine and epilepsy, but was unavailable for further clinical explorations in order to detect mild signs of neurological problems. Similar incomplete penetrance of the A217PfsX8 mutation was observed in carriers from families with ICCA syndrome PRRT2 in worldwide populations. We identified 21 novel nonsynonymous variants including 17 never reported before and 4 already listed in the nucleotide variant database , whereas the pN/pS ratio in Asia and Europe is higher than expected and consistent with positive Darwinian selection PRRT2 seems to be the gene with the higher difference of selective pressure between Europe and Africa, but addition samples should be screened to ascertain if this difference is specific to PRRT2 or is also true for other genes within the deletion. Considering this heterogeneous pattern of selective pressure, it would be interesting to see if there is a higher prevalence of PRRT2 related disease in Asia and Europe compared to Africa. It would also be interesting to explore the genetic diversity of the binding partners of PRRT2 in order to decipher whether this lower selective pressure is restricted to PRRT2 or to other members of the same pathway.Thirdly, we ascertained the genetic variability of database . Despitein vivo and in vitro. For this reason, we strongly suggest that PRRT2 mutation screening should be restricted to native DNA and validate using Sanger sequencing. Interestingly, in the last release of the Exome Variant Server database, at the same location of the A217PfsX8 mutation , 10% of the PRRT2 allele contains an additional C  and 10% have a deletion of a C , but with very low average sample read depth (N\u200a=\u200a10). Given the very high penetrance of the A217PfsX8 mutation in PRRT2-related diseases, it is impossible that 20% of the PRRT2 alleles carry a frame shift mutation at this position. Especially, since we found the A217PfsX8 mutation in only 1 individual out of 1990 (Allelic frequency\u200a=\u200a0.025%). This high frequency of A217PfsX8 mutation in EVS could be related to the DNA amplification step required for whole exome sequencing. Studying the mechanism leading to the A217PfsX8 mutation was beyond the scope of our study, but our observation that a DNA amplification step through RCA could introduce this additional C/G might help in understanding the mechanism leading to A217PfsX8 mutation.Finally, we showed that whole genome amplification using multiple displacement amplification technology such as GenomiPhi and Repli-G could lead to the appearance of the A217PfsX8 mutation. The genomic region surrounding the mutation is made of 4 guanines and 9 cytosines and seems therefore to be at risk for mutation both PRRT2 mutations do not play a major role in the susceptibility to ASD and confirm that truncating mutations of PRRT2 are not fully penetrant. We also provide an ascertainment of the genetic diversity of PRRT2 in worldwide populations as well as important indication on the pitfalls for the mutation screening. All together, these results should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.In summary, our study indicates that Figure S1PRRT2 in Exome Variant server. The nucleotide positions are according to PRRT2 from NCBI37/hg19. The average coverage of 10\u00d7, 20\u00d7 and 40\u00d7 are indicated in red, yellow and green respectively.Sequence coverage of PRRT2 in Exome Variants Serveur. Total number of sample sequenced (in blue) and average read depth (purple) for (TIF)Click here for additional data file.Figure S2Multiple dimension scale (MDS) of the genetic distances between individuals from the Human Genome Diversity Pannel. Insert. MDS restricted to the African populations including the two individuals with ASD carrying the in frame deletion p.A361_P362del (triangles).(TIF)Click here for additional data file.Table S1Cohorts used in this study.(DOCX)Click here for additional data file.Table S2Ethnicity of the cohorts.(DOCX)Click here for additional data file.Table S3PRRT2 primers.(DOCX)Click here for additional data file.Table S4PRRT2 nonsynonymous variants identified in the HGDP.(DOCX)Click here for additional data file.Table S5PRRT2 synonymous variants identified in the HGDP.(DOCX)Click here for additional data file.Table S6PRRT2 coding variants present in dbSNP, Exome variant Server (EVS) and the 1000 genome project (1KG).(DOCX)Click here for additional data file.Table S7Identity by state (IBS) values for the 2 individuals with ASD carrying the mutation p.A361_P362del compared with the IBS from different African populations.(DOCX)Click here for additional data file.Table S8Evolutionary analysis of all genes located within the 16p11.2 deletion.(DOCX)Click here for additional data file."}
+{"text": "The NLSs of GRK5 and 6 bind DNA in vitro, whilst the NLS of GRK4 does not. Using mutants of GRK5 we identify the regions of GRK5 required for DNA-binding in vitro and nuclear localization in cells. The DNA-binding ability of GRK5 requires both the NLS and an N-terminal calmodulin (CaM)-binding site. A functional nuclear export sequence (NES), required for CaM-dependent nuclear export of the kinase, is also identified. Based on our observations we propose a model to explain how nuclear localization of GRK5 may be regulated. Notably, the nuclear localization of GRK5 and 6 is differentially regulated. These results suggest subfamily specific nuclear functions for the GRK4 subfamily members. Identification of GRK specific small molecule inhibitors of nuclear localization and/or function for the GRK4 subfamily may thus be an achievable goal.G protein-coupled receptor kinases (GRKs) act to desensitize G protein-coupled receptors (GPCRs). In addition to this role at the plasma membrane, a nuclear function for GRK5, a member of the GRK4 subfamily of GRKs, has been reported. GRK5 phosphorylates and promotes the nuclear export of the histone deacetylase, HDAC5. Here we demonstrate that the possession of a nuclear localization sequence (NLS) is a common feature of GRK4 subfamily members . However, the location of the NLS and the ability of these GRKs to bind DNA  The G protein-coupled receptor kinase (GRK) family of serine/threonine kinases were originally identified based on their ability to phosphorylate agonist occupied G protein-coupled receptors (GPCRs). GRK-mediated GPCR phosphorylation leads to recruitment of an arrestin family member, Arrestins 1\u20134, to the phosphorylated receptor. Arrestin binding sterically inhibits signaling via heterotrimeric G proteins, targets the receptor for internalization and initiates G protein-independent, arrestin-dependent, signal transduction The seven members of the GRK family are grouped into three subfamilies; the GRK1 (GRK1 and 7), GRK2 (GRK2 and 3) and GRK4  subfamilies Notably, expressing the N-terminus of GRK5 (GRK5-NT) has been shown to lead to accumulation and stabilization of the I\u03baB\u03b1/NF-kB complex in the nucleus and inhibition of NF-\u03baB activity in vivo and whether they represent the only means by which GRK5 promotes hypertrophy is not known. Similarly, we have very little information regarding how TAC promotes nuclear localization of GRK5 and whether the ability of GRK5 to bind DNA is required for GRK5-dependent hypertrophy. In this study we report the identification of an NES sequence in GRK5 and describe a variety of GRK5 mutants that differ in their ability to localize to the nucleus and/or their ability to bind DNA. These mutant constructs provide insights into the mechanisms regulating nuclear localization of GRK5 and provide useful tools for future dissection of the role of GRK5 in vivo.GRK5-mediated HDAC5 phosphorylation and the ability of GRK5 to regulate I\u03baB\u03b1 function represent two mechanisms whereby cardiac overexpression of GRK5 could cause pathological cardiac hypertrophy in response to pressure overload in mice. However, the extent to which either of these mechanisms are actually responsible for GRK5-dependent cardiac hypertrophy GRK5 is upregulated in the left ventricle in dilated cardiomyopathy and left ventricular overload disease in vitro suggests subfamily specific nuclear functions for the GRK4 subfamily of GRKs.In this study we also demonstrate that possession of an NLS is a common feature of the GRK4 subfamily of GRKs. However, the location and DNA-binding properties of the NLS differs between subfamily members. Furthermore although binding to calmodulin (CaM) promotes nuclear export of GRK5, nuclear localization of GRK6A is unaffected by this treatment. Thus even though both kinases bind CaM, and have NLSs with similar locations and DNA-binding properties their nuclear export is differentially regulated. Although a nuclear function for GRK5 has been identified, a role for GRK4 and GRK6 in the nucleus remains to be elucidated. The observation that the nuclear export of GRK5 and GRK6A are differentially regulated and that GRK4 fails to bind DNA in vivo. The identification of NLSs in other GRK4 subfamily members further indicates that investigating potential nuclear functions for these enzymes may also be a fruitful exercise.To summarize, the aim of the experiments detailed in this study were, through analysis of mutant GRK5 constructs, to gain more information on the regulatory mechanisms controlling its nuclear localization. Nuclear functions for GRK5 are only just beginning to be characterized. Our study identifies a mutant of GRK5 that reveals that nuclear localization and the ability to bind DNA are dissociable properties of the kinase. This mutant will be a useful tool for probing the functional relevance of the DNA-binding ability of GRK5 All cells were maintained in Dulbecco's modified Eagle medium (GIBCO) containing 10% fetal calf serum (Sigma) and penicillin and streptomycin  at 37\u00b0C, 5% CO2.GCG TGT GCA GCT GCT ACC GCC ATG AAC GGC GGG GAC CC TAC GAG ACC AAG GAT GCC  \u2013 3\u2032; GRK5 anti-sense primer, 5\u2032 \u2013 G GTC CCC GCC GTT CAT GGC GGT AGC AG\u2032TGCC  ACA CGC GGC ATC CTT GGT CTC GTA GGC \u2013 3\u2032. GRK2 sense primer, 5\u2032 \u2013 CAGCC AGC GCA GCT GCT GAT GCC ATG AAC GGC GGG GAC CC ACA CCG GAC AAG  \u2013 3\u2032; GRK2 anti-sense primer, 5\u2032 \u2013 G GTC CCC GCC GTT CAT GGC ATC AGC AG\u2032TGCC GGC CTT GTC CGG TGT GTG GCT  \u2013 3\u2032. Nucleotides in bold encode the mutated amino acids.The plasmids for pRK5-GRK5 GCA GCT GCT ATA AAG CGC GAA GAG GTG GAG CGG CC TTC CAG CAG AGG  \u2013 3\u2032; anti-sense primer, 5\u2032 \u2013 G CCGAGC AGC TGC CCT CTG CTG GAA GGG CG CTC CAC CTC TTC GCG CTT TAT  \u2013 3\u2032. Nucleotides in bold encode the mutated amino acids.pCMV5-GRK6A/B/C (human) were generous gifts from Dr Mario Tiberi  GCT AGA ATA GCT GCT AGG AAA GGT GAA GCT ATG GC\u2013 3\u2032; anti-sense primer, 5\u2032 - GC AGC AGC TAT TCT AGC TTT TTG TAG CTT TTT GCA GGCCAT AGC TTC ACC TTT CCT  \u2013 3\u2032. The location of the NLS in the splice variants of GRK4 is as follows; \u03b1 and \u03b3, residues 219\u2013227, lysines 221, 224 and 225 mutated to alanine; \u03b2 and \u03b4, 187\u2013195, lysine residues 189, 192 and 193 mutated to alanine. Integrity of mutant constructs was confirmed by DNA sequencing.pRK5-GRK4\u03b1/\u03b2/\u03b3/\u03b4 (human) were generous gifts from Richard Premont,  2 at 37\u00b0C, 5% CO2. Cells were subsequently immunolabelled with a mouse anti-GRK4-6 antibody  for 1 hr at room temperature. Confocal images were taken at room temperature using a Bio-Rad MRC 1024 laser scanning confocal system with Nikon Plan Apo 60x oil immersion lens and Optiphot 2 microscope using Bio-Rad Lasersharp 2000 software to acquire the images. The confocal plane shown represents a plane through the center of the nucleus as assessed by Hoechst staining. Images shown in a single figure were taken using the same microscope settings to ensure that cells with similar levels of GRK expression were compared within an experiment. Images were optimized for contrast in Adobe Photoshop, but no further manipulations were made. To quantify ionophore-dependent nuclear export at least 50 appropriately transfected cells were counted and GRK distribution scored as nuclear/cytosolic. Cells were deemed to show a cytosolic distribution if no transfected protein was detected in the nucleus. At least three separate transfections were scored per experiment.HEp2 cells (ATTC) were transfected by electroporation as described previously 2, 0.1% Triton X-100, 3 mM dithiothreitol, 0.1 M NaCl, 0.05 mM EDTA) for 1 hr at 4\u00b0C. Following incubation, the resin was washed and the amount of GRK retained on the resin determined by Western blot analysis. Films were quantified using a densitometer (Bio-Rad). In each experiment a control and experimental value were compared and significance determined using a t test. For competition assays, purified GRK6A (150 ng) was preincubated with the stated amounts of DNA  or RNA  for 30 min on ice before the DNA-binding assays were performed.As described previously GRK6A was expressed in, and purified from, baculovirus-infected SF9 cells as previously described 388RKEKVKRE395) The GRK4 subfamily contains three kinases, GRK4, GRK5 and GRK6. Four splice variants of human GRK4  and three of GRK6  have been identified. GRK5 contains an NLS located between amino acids 388 and 395  (in vitro suggests distinct nuclear functions for these enzymes.GRK5 binds DNA  its NLS . To deted bound) . GRK6A, d bound) , suggestd bound) . The amoin vitro DNA binding assay using purified GRK6A. Approximately 23 \u00b1 1.7% of the purified GRK6A (150 ng) incubated with native DNA-cellulose was retained by the resin  promotes nuclear export of GRK5  but not the cellulose support  for GRK4 distinct from that of GRKs 5 and 6. The NLS of GRK4 is similar in sequence and location to predicted putative NLSs in GRK1 and GRK7 All members of the GRK4 subfamily contain functional NLSs, however, the NLS sequence of GRKs 5 and 6 differ from that of GRK4 in sequence, location and DNA-binding properties. Thus the NLS of GRKs 5 and 6, but not 4, bind DNA 2+/CaM appears to be responsible for exposing the NES in the cytosolic pool of transfected wild type GRK5 since GRK5NTPB, which lacks the N-terminal CaM binding site, is almost entirely nuclear. The observation that both GRK5\u0394NES and GRK5NTPB are not exported from the nucleus in a Ca2+/CaM-dependent fashion further supports this contention since it demonstrates that CaM-dependent nuclear export is both NES and CaM-dependent. A role for CaM in mediating nuclear export has also been proposed for Ca2+/CaM dependent protein kinase I-\u03b1 where CaM-binding promotes binding of the kinase to the CRM1 nuclear export complex Analysis of the subcellular localization and DNA-binding properties of the seven GRK5 mutants used in this study suggests a potential model whereby GRK5 nuclear localization may be regulated . The wilin vitro  distribution of the kinase and that when the palmitoylation sites on GRK6A are mutated nuclear GRK6A can be detected in vitro. Given the complexity of regulatory mechanisms controlling the nuclear localization of GRK5 it is not beyond the realms of possibility that both sequences may participate in regulating the nuclear localization of GRK6, potentially, in a cell type specific fashion. That the nuclear export of GRK5 and 6 is differentially regulated may suggest a common nuclear function that is differentially modulated following activation of specific receptor subsets, or alternatively, that GRK5 and 6 perform distinct nuclear functions. Additionally, it suggests that it may be possible to design subfamily specific small molecule regulators of the nuclear functions of GRK5 and 6A by selectively blocking their nuclear import/export.Despite having NLSs with similar locations and DNA-binding properties and both being CaM-binding proteins, the nuclear export of GRK5 is Cain vitroThe crystal structure of GRK6A has been solved and suggests that this kinase may exist as a dimer in vivo model of pressure overload disease. Specifically the role GRK5 kinase activity, nuclear localization and DNA-binding ability plays in promoting pathological hypertrophy in response to TAC may reveal additional nuclear functions for this kinase. A more detailed understanding of the molecular features of GRK5 required to promote pathological hypertrophy may lead to the identification of regions on GRK5 or target molecules amenable to regulation via small molecule inhibitors.This study highlights both a structural diversity between the GRK2 and GRK4 subfamilies of GRKs and a potential functional diversity within the GRK4 subfamily. A nuclear function for GRK5, phosphorylation and inhibition of the MEF2 repressor HDAC5, has been identified. Presumably GRK6 and GRK4 also have nuclear functions. What these are and whether they vary in a subtype, or splice variant, specific fashion remains to be determined. In this study we identify a functional NES in GRK5 and describe a number of mutants of this kinase that vary in their ability to localize to the nucleus or to bind DNA. Analysis of the hypertrophic response following TAC in transgenic mice with cardiac specific overexpression of these mutants is anticipated to reveal more information on the role of GRK5 in this"}
+{"text": "SULF2 methylation in gastric cancer.Biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents could improve the clinical efficiency. The sulfatases(SULFs) play a critical role in the pathogenesis of a variety of human cancers. Here, we focused our investigation on the prognostic and predictive impact of SULF2 was analyzed in 100 gastric cancer samples. The in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed were determined by histoculture drug response assay(HDRA). Additionally, 56 gastric cancer patients treated with a modified FOLFOX regimen were retrospectively analyzed to further evaluate the prognostic and predictive impact of SULF2 methylation in gastric cancer.Promoter CpG island methylation of SULF2(SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2. Samples with SULF2U were more sensitive to cisplatin than those with SULF2M, while samples with SULF2M were more sensitive to irinotecan than SULF2U. There were no association between SULF2 methylation and in vitro sensitivity to docetaxel, gemcitabine and pemetrexed. SULF2 methylation was found to have a significant association with cisplatin efficacy and irinotecan efficacy. Among the 56 patients receiving the modified FOLFOX regimen, a significant association was observed between survival and SULF2 methylation status. Multivariate analysis revealed that SULF2 methylation was an independent prognostic factor of overall survival in gastric cancer patients treated with platinum-based chemotherapy.Methylated SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation may be a novel prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy. Gastric cancer is one of the most frequent causes of cancer-related death worldwide SULFs in gastric cancer, however, are non-conclusive, and the prognostic or predictive value of SULFs for chemosensitivity prediction remains unknown.Heparan sulfate proteoglycans (HSPGs) are coreceptors for heparin-binding growth factors and cytokines distributed on the cell surface and in the extracellular matrix. Two isoforms of the extracellular heparan sulfate 6-O-endosulfatases (SULFs), SULF1 and SULF2, have been discovered in mammals. Both proteins are secreted to the cell surface to modulate the sulfation of HSPGs SULF2, the gene encoding the SULF2 endosulfatase, and its association with in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan, and pemetrexed in 100 human gastric cancer samples. To this end, we performed a series of in vitro sensitivity tests on freshly-removed gastric tumor tissues and evaluated the possible use of SULF2 methylation status for predicting the chemotherapeutic efficacy of there agents. Then, we retrospectively analyzed the SULF2 methylation in a cohort of 56 gastric cancer patients treated with a modified FOLFOX regimen and concluded that SULF2U serves as an independent prognostic biomarker in gastric cancer patients treated with a modified FOLFOX regimen.In this study, we have analyzed the promoter CpG island methylation of All research involving human participants have been approved by the Human Research Protective Committee of Drum Tower Hospital Affiliated to Medical School of Nanjing University and written informed consent was obtained from all patients.in vitro evaluation of chemosensitivity by histoculture drug response assay (HDRA) Enrolled patients were those undergoing the gastrectomy in the Department of General Surgery of the Drum Tower Hospital, Nanjing, China during the period from August 2010 to October 2011. Eligibility criteria for enrollment into the study included the following parameters: (1) age >18 years; (2) histologically confirmed gastric adenocarcinoma, mucinous or signet ring cell adenocarcinoma; (3) no previous or concomitant malignancies other than gastric cancer; (4) no previous history of chemotherapy or radiotherapy, either adjuvant or palliative; and (5) adequate function of all major organs. Tissue samples were extracted from 100 freshly-removed gastric tumors. Each tumor tissue was divided into two parts: (1) one part was kept in 4\u00b0C Hanks\u2019 balanced salt solution with 1% penicillin/streptomycin after collection, and then sent to the laboratory within 15 min in 4\u00b0C, for in vitro sensitivity and resistance were 20 ug/ml for cisplatin 2. After histoculture, 100 \u00b5l of type I collagenase  and 100 \u00b5l of 3--2,5-diphenyl-2H- tetrazolium bromide (MTT) solution  were added to each culture well and incubated for another 16 hours. After extraction with dimethyl sulfoxide, the absorbance of the solution in each well was read at 540 nm.HDRA procedures were performed as previously reported by Furukawa and colleagues in vitro efficacy rate was also estimated based on the cut-off inhibition rate as follows: efficacy rate (%)\u200a=\u200anumber of sensitive cases/total number of cases.The absorbance per gram of cultured tumor tissue was calculated from the mean absorbance of tissue from 8 parallel culture wells, and the tumor tissue weight was determined before culture. The inhibition rate of each anti-cancer agent was calculated by using the following formula: Inhibition rate (%)\u200a=\u200a(1\u2013T/C) \u00d7100, where T is the mean absorbance of treated tumor / Weight and C is the mean absorbance of control tumor / Weight. The cut-off inhibition rates used to distinguish the sensitive and the resistant cases were adopted at 30%, 40%, 50%, and 60% for each drug tested. The \u22122 plus folinic acid 200 mgm\u22122 as a 2 h infusion on day 1, followed by a 22 h infusion of 5-FU 2000 mgm\u22122 on days 1 and 2, every two weeks. These 56 patients were further followed up and their survival time was calculated from the date of diagnosis to the date of the last follow-up or death from any cause.Of all patients, 56 with Eastern Cooperative Oncology Group (ECOG) performance status (PS) \u22642 received a modified FOLFOX (a combination of 5-FU and platinum) chemotherapy regimen after resection of primary tumors as follows: oxaliplatin 85 mgmThree 7-\u00b5m sections were prepared from primary tumor blocks that contained at least 80% tumor cells. After hematoxylin-eosin staining, the cancerous parts were microdissected and transferred into a microcentrifuge tube. DNA was isolated routinely and then was chemically modified by sodium bisulphite to convert all unmethylated cytosines to uracils while leaving methylcytosines unaltered SULF2 using the ABI Prism 7300HT Sequence Detection System (Applied Biosystems). Each PCR reaction contained genomic DNA 2 \u00b5l, SYBR Green PCR Mix  10 \u00b5l, water 7.7 \u00b5l, and primers 0.15 \u00b5l (10 \u00b5mol/l). The PCR conditions were 95\u00b0C for 10 min, followed by 45 cycles at 59\u00b0C for 30 s, 72\u00b0C for 30 s and 95\u00b0C for 30 s. Primers for SULF2 methylated PCR  were as follows: forward 5\u2032 TAAGTGTTTTTTTTATAGCGGC 3\u2032, reverse 5\u2032TACCGTAATTTCCGCTATC 3\u2032. Primers for SULF2 unmethylated PCR  were as follows: forward 5\u2032 GTTTATAAGTGTTTTTTTATAGTGGT3\u2019, reverse 5\u2032TACCATAATTTCCACTATCCCT 3\u2032. Each batch of reaction included a positive control from Methyltransferase (M.SssI)-treated human genomic DNA (fully methylated), a negative control from DNA samples which had been confirmed as unmethylated and another negative control without DNA. All tests were performed in duplicate. Results were validated for selected samples through combined bisulfite modification and bisulfite sequencing.MSP was performed to determine the methylation status of in vitro chemosensitivity efficacy were analyzed using the Fisher\u2019s exact probability test. All statistical tests were two-sided, and a P value of less than 0.05 was considered as statistical significance. Statistical analysis was performed using the SPSS, version 16.0.Values were expressed as means \u00b1 standard deviation. Differences in inhibition rates between groups were evaluated using the t-test. The possible associations of SULF2 methylation with clinical characteristics and SULF2 (SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2 . The RT-PCR amplification curves of SULF2M and SULF2U were shown in SULF2 methylation and any of the patients\u2019 characteristics, including sex, histology, tumor site, stage, histological grade and lymph node metastasis.The characteristics of all patients are shown in In vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed was successfully tested in all the samples. The mean inhibition rates for each tested drug are listed in SULF2U were more sensitive to cisplatin than those with SULF2M , while samples with SULF2M were more sensitive to irinotecan than SULF2U  and irinotecan efficacy . No other association between clinical characteristics and survival was found . A significant association was observed between survival and SULF2 methylation status. The median overall survival was 309 days (95% CI\u200a=\u200a236 to 382 days) for patients with SULF2M, and 481 days (95% CI\u200a=\u200a418 to 490 days) for those with SULF2U . A significant association was observed between survival and tumor site  the rate of SULF2M in human gastric cancer is around 30%; (ii) the first evidence for the SULF2U is associated with cisplatin sensitivity in cancer; (iii) evidence for the SULF2M is associated with irinotecan sensitivity in gastric cancer; (iv) a retrospective study and validation for SULF2 methylation in a cohort of 56 patients with gastric cancer, which allowed us to discover that SULF2U is an independent prognostic biomarker in gastric cancer patients treated with a modified FOLFOX regimen.Although previous studies have demonstrated the involvement of SULF2 in cancer pathogenesis, its value for chemosensitivity prediction remains unclear. This study focuses on the promoter CpG island methylation of SULFs were inconclusive. In one study, only 13 early-stage breast and gastric cancers, most of which were stage I, were analyzed by semi-quantitative RT-PCR for SULF1 expression SULF1 was prevalent in these two types of cancer. In another study, the expression of both SULF1 and SULF2 mRNA was determined by real-time RT-PCR for a large cohort of gastric cancer tissues, finding that SULFs were expressed at higher levels in gastric cancer as compared with normal tissues. In the current study we found by examining the SULF2 methylation in 100 gastric cancer samples, that the rate of SULF2M was approximately 30%, which indicated that predominate gastric cancer were SULF2U. Recent studies by integrated genomic analyses revealed that SULF2 acts as a downstream effector of p53, and that activation of p53 could lead to the SULF2U and up-regulation of SULF2. The possible link between p53 and SULF2 in growth factor signaling pathway suggested a possible role for SULF2 in cancer development and cancer patients\u2019 outcome SULF2 unmethylation and SULF2 up-regulation needs to be further tested in these samples. The different expression levels and methylation status of SULF2 between tumor and normal tissue also need to be further verified.In previous studies of gastric cancer, the findings on methylation status and expression levels of SULF2 methylation as chemosensitivity predictor are scarce. Methylation of the SULF2 promoter has been associated with better survival of resected lung adenocarcinoma patients, and also with a marginal improvement in survival of advanced NSCLC patients receiving standard chemotherapy  SULF2M were 134-fold more sensitive to topoisomerase I inhibitor than those with SULF2U. In the current study, we have demonstrated that gastric tumors with SULF2M are more sensitive to irinotecan than those with SULF2U. Furthermore, we demonstrated for the first time that SULF2 methylation is also a potential predictive biomarker for cisplatin efficacy. Gastric tumors with SULF2U were more sensitive to cisplatin than those with SULF2M, and gastric cancer patients with SULF2U showed lower mortality when receiving platinum-based chemotherapy. Although several predictive biomarkers have been identified for cisplatin, such as ERCC1 SULF2 unmethylation increases tumor sensitivity to cisplatin may lie on ubiquitin conjugating enzymes (UBE). It has been reported that SULF2 methylation results in increased expression of UBE SULF2M induced cisplatin resistance.Studies on SULF2 methylation and cisplatin sensitivity may partially explain this phenomenon. Treatment of gastric cancer with DNMT inhibitors could result in demethylation of SULF2 and consequently increase the sensitivity to cisplatin. Thus, in addition to the predictive impact, our data also supports the inclusion of a DNMT inhibitor in current treatment protocols for at least a subset of gastric cancer patients.A significant synergistic effect of cisplatin and DNA methyltransferase (DNMT) inhibitors 5-aza-dC (DAC) on cell viability was observed in the cisplatin-resistant AGS cell line but not in the cisplatin-sensitive MKN28 cell line SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation is a potential prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy.In conclusion, our study provides novel evidences that Figure S1The RT-PCR amplification curves of SULF2M and SULF2U. The red curve stands for amplification of SULF2M, and the blue curve stands for amplification of SULF2U. (TIF)Click here for additional data file."}
+{"text": "For step-and-shoot type delivery of intensity-modulated radiation therapy (IMRT), beam stability characteristics during the first few monitor units need to be investigated to ensure the planned dose delivery. This paper presents the study done for Siemens ONCOR impression plus linear accelerator before commissioning it for IMRT treatment. The beam stability for 6 and 15 MV in terms of dose monitor linearity, monitor unit stability and beam uniformity is investigated in this work. Monitor unit linearity is studied using FC65G chamber for the range 1-100 MU. The dose per MU is found to be linear for small monitor units down to 1 MU for both 6 and 15 MV beams. The monitor unit linearity is also studied with portal imaging device for the range 1-20 MU for 6 MV beam. The pixel values are within \u00b11\u03c3 confidence level up to 2 MU; for 1 MU, the values are within \u00b12\u03c3 confidence level. The flatness and symmetry analysis is done for both energies in the range of 1-10 MU with Kodak diagnostic films. The flatness and symmetry are found to be within \u00b13% up to 2 MU for 6 MV and up to 3 MU for 15 MV. Intensity-modulated radiation therapy (IMRT) is becoming the commonly chosen treatment modality because of its dose conformity to the concave tumor volumes, sparing surrounding normal organs. Siemens ONCOR Impression Plus linear accelerator with photon beam energies of 6 and 15 MV equipped with double-focused multileaf collimator (MLC) has been installed in our clinic. These accelerators are designed to deliver intensity-modulated fields using step-and-shoot technique. In this type of IMRT delivery, multiple segments are the result of the optimization process to conform the dose to the target volume. These segments are delivered in a stack arrangement. Each beam segment is delivered separately with the radiation beam turned off (paused)[The operation of the linear accelerator delivering low monitor unit segments needs to be investigated for every linear accelerator prior to commissioning the linear accelerator for IMRT. Many aut1max in the SP34 slab phantom.[1Dose monitor linearity and monitor unit stability at small monitor units for 6 and 15 MV were examined using a FC65G ionization chamber connected to DOSE1 electrometer. The measurements were done at 100 cm SSD with the chamber at dMonitor unit stability was studied with the same setup as described above. For the collimator setting of 10 cm \u00d7 10 cm, the ionization was recorded for 10 MU. It was compared with the cumulative ionization of 5 MU measured two times; 2 MU measured five times, 1 MU measured ten times. SimilarlFilm dosimetry was used to study dose uniformity for low monitor units. Beam flaFor low-MU linearity, ionization readings were recorded in the range of 1-100 MU. Similar study done for 15 MV is shown in Figures Figures For 15 MV , the varFor 15 MU, as shown in Flatness symmetry study was done with Kodak diagnostic films. Flatness was calculated using \u2018variation over mean\u2019 (80%) method. The symmetry value was obtained with \u2018point difference quotient\u2019 method. For 6 MVFor 15 MV beam, inplane and crossplane flatness for 1 MU is 4.11 and 4.88% respectively. For 2, 3 5 and 10 MU, the crossplane flatness values are within 2%. The inplane flatness is within 3% for 2, 5 and 10 MU; while it is 3.97% for 3 MU .The symmetry analysis for 15 MV  shows thFor 6 MV beam, the stability of the beam at low MU in terms of cGy/MU is within \u00b12%, assuming all other factors are proportional while converting the charge to cGy. The stability in terms of flatness and symmetry is also acceptable and is within \u00b13% up to 2 MU. The stability of 15 MV beam in terms of flatness and symmetry is within \u00b13% above 3 MU.However, it is suggested that segments with monitor units less than 5 MU should be avoided. The inverse planning system used at our center takes care of prevention of small MU segments such as 5 MU from the treatment plan. So for the range of monitor units used for patient treatment, the Siemens ONCOR Impression Plus linear accelerator has been delivering stable beams for step-and-shoot IMRT treatments."}
+{"text": "Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the  In vertebrates, major parts of the craniofacial skeleton derive from cranial neural crest cells (cNCCs) that previously migrated into the pharyngeal arches tfap2\u03b1 starting at 12 hours post fertilization (hpf). tfap2\u03b1 mutants show defects both in pre-migratory cNCC specification and in cartilage development Col2a1 in mouse , while in zebrafish both sox9a and sox9b are required for col2a1 expression. However, they play both distinct and overlapping functions in zebrafish chondrogenesis dlx2-expressing cNCC. After migration of the cNCCs into the pharyngeal arches, sox9a is expressed in cNCCs under control of dlx2aox9b is only expressed in the pharyngeal endoderm sox9a in the jef mutant leads to a complete absence of cartilage sox9b mutation causes an absence of ceratobranchials and a reduction of the two most anterior pairs of arches. Sox9a is essential for endochondral ossification, while sox9b has an additional role in formation of some dermal bones Runx2b expression Differentiation of cNCCs into mature chondrocytes is controlled by a number of transcription factors. In zebrafish, pre-migratory cNCCs express casanova (cas) and bonny and clyde (bon) mutants lack endodermal markers and present severe cartilage defects van gogh (vgo) mutants make pharyngeal endoderm but fail to form pouches, which leads to an absence of cNCC segmentation into distinct elements vgo and cas mutants restores the cartilaginous phenotype In addition to these endogenous patterning cues, cNCCs also receive external signals from surrounding cells and extracellular matrix, such as Fgf, BMP and Shh signals e.g. mitosis, differentiation and apoptosis Egr1 is expressed during embryogenesis in cartilage, bones, teeth, salivary glands, nasal glands, developing vibrissa, tendons and skeletal muscles egr1 gene is composed of two exons and one intron in situ hybridization before gastrulation gr1 expression was observed in the pharyngeal region until at least 48 hpf.The early growth response (EGR) family contains four highly conserved zinc finger (C2H2 type) transcription factors that mediate the cellular response to many stimuli inducing Egr1 knockout mice display sterility for both sexes and are smaller due to defects in adenohypophysis development and particularly LH\u03b2 expression Egr1 KO mice present increased bone resorption, a reduced bone mass and an increased production of the osteoclastogenic cytokine M-CSF (macrophage Colony-Stimulating Factor) in stromal cells of the bone matrix egr1 leads to a reduction of the eye size, while the retina and lens lack appropriate differentiation Egr1 is known to play various roles such as tumor promotion in prostate fsta), a known BMP antagonist. We also show that BMP signaling is required around 30 hpf in the neural crest cells for runx2b expression in chondrocytes and proper pharyngeal cartilage formation, while BMP signaling is absent in the pharyngeal region upon egr1 knock-down.In this study, we report that the transcription factor Egr1 plays a specific role in pharyngeal cartilage formation during zebrafish embryogenesis, while it is expressed in pharyngeal endoderm. We show that Egr1 is part of a regulatory cascade, together with the endodermal factors Runx3 and Sox9b, that suppresses expression of follistatin A (Danio rerio) were reared in a recirculating system from Techniplast, Italy at a maximal density of 7 fish/l. The water characteristics were as follows: pH\u200a=\u200a7.4, conductivity\u200a=\u200a500 \u00b5Scm-1, temperature\u200a=\u200a28\u00b0C. The light cycle was controlled . Fish were fed twice daily with dry powder (ZM fish food\u00ae) with size adapted to their age, and once daily with fresh Artemia salina nauplii (ZM fish food\u00ae). Larvae aged less than 14 days were also fed twice daily with a live paramecia culture. Wild type embryos from the AB strain were used and staged according to Kimmel Tg(hsp70l:dnBmpr-GFP)Zebrafish .Breeding: the day before breeding, 2 males and 2 females were placed in breeding tanks out of the recirculating system, with an internal divider to prevent unwanted mating. On the day of breeding, fish were placed in fresh aquarium water and the divider was removed to allow mating. Eggs were collected every 30 minutes and raised in E3  were synthesized by Gene Tools  and are complementary to the 5\u2032 sequence near the translation start site or to the splice junctions. MO stock solutions were prepared as suggested by Gene Tools. Tetramethylrhodamine dextran  was added at a concentration of 0.5% to sort correctly injected embryos a few hours after injection. The morpholino sequences are as follow:egr1: GGATTTAGTGCTTACCTCCAGCAAG.MO splicing egr1: TGCAGCCATCTCTCTGGAGTGTGCT.MO translation runx3: TGCTCGGGTCTACGGGAATATGCAMO translation fst1a: CTGACGTTTAGCATCCTTAGCATGMO translation CCTCTTACCTCAGTTACAATTTATA (Gene-Tools\u00ae).MO standard control: gr1 cDNA fragment starting at the ATG start codon and spanning the entire coding region was obtained by PCR-amplification from the cDNA clone egr1 mRNA was synthesized using mMessage mMachine Sp6 (Ambion\u00ae) and injected alone or co-injected with a morpholino at the one-cell stage with a microinjector (Narishige\u00ae).An eegr1 spl was examined by RT-PCR using SuperScript\u00ae from Invitrogen . mRNA of 50 injected embryos was extracted for each experiment. The primers used were: Zf-Egr1 forward: 5\u2032-CAGTTTGATCACCTTGCTGG-3\u2032; Zf-Egr1 reverse: 5\u2032-GGAAGACGTGGAAGAGGAAG-3\u2032.Efficiency of the splicing morpholino MOin situ hybridization were raised in presence of 0.003% of 1-phenyl-2-thiourea until the desired stage, fixed overnight in 4% of PFA at 4\u00b0C and stored in 100% methanol at \u221220\u00b0C until use. Visible in situ hybridizations (ISH) were performed as described Injected embryos for Whole-mount fluorescent immunohistochemistry was described in in situ hybridization were taken by Nikon\u00ae Eclipse 90i microscope and NIS-Elements microscope imaging software. Fluorescent images were acquired with a Leica\u00ae SP2 confocal microscope.Pictures of visible Cartilage was stained with Alcian Blue 8 GX (Sigma\u00ae). Four days old embryos were fixed in PFA 4% for 2h at room temperature, rinsed with PBST and finally stained overnight with 10 mM Mg Cl2/80% ethanol/0.04% Alcian Blue solution. Embryos were rinsed with 80% ethanol/10 mM MgCl2. Pigments were bleached in H2O2 3%/KOH 0,5% for 1 h. 25% glyc\u00e9rol/0,1% KOH, 50%Glycerol/0,1% KOH.hsp70l:dnBmpr-GFPTransgenic embryos 10 mM stock solution of dorsomorphin  was diluted in DMSO . Embryos at desired stage were placed into multiwell plates with dorsomorphin diluted in E3 rearing medium at the desired concentration during a specific time period. DMSO alone was used as control. Embryos were then rinsed several times with E3, raised in E3 and finally fixed at the desired stage.egr1 transcripts or against a control sequence (MOcon). Two different morpholinos against egr1 were used, one inhibiting egr1 splicing (MOegr1 spl) and the other preventing translation of egr1 mRNA (MO egr1 tr). Injection of MO egr1 spl efficiently inhibited egr1 splicing in embryos, as judged by RT-PCR of 2 days post fertilization (dpf) embryos  directed against  embryos , whereasPharyngeal and cranial cartilages were stained with Alcian Blue in 4 dpf embryos. Remarkably, in embryos injected with 8 ng of MOegr1 tr, branchial cartilages were completely absent, while Meckel\u2019s cartilage and the palatoquadrate were significantly reduced and the ceratohyal and hyosymplectic misshapen  . A similegr1 mRNA together with MO egr1 or MOcon. Co-injection of 75 pg of egr1 mRNA together with 8 ng MOegr1 tr rescued the development of pharyngeal and cranial skeleton in 87% of co-injected embryos   , while bpression . In contpression . Thus, fegr1 morphant embryos by in situ hybridization. Expression of early neural crest markers ap2a3 was not affected in egr1 morphants at 24 hpf  and 48 hpf  egr1 morphants .Runx2b is a transcription factor known to play a key role in chondrocyte maturation and is expressed in mesenchymal cartilage condensations in the viscerocranium starting at 40 hpf orphants . At 48 hegr1 spl , revealiegr1 expression is dispensable for early specification and migration of cNCCs, but is required for proper late chondrogenesis in pharyngeal arches.We conclude that egr1 expression pattern in the developing pharyngeal region. Whole-mount in situ hybridization against egr1 confirmed its expression in the pharyngeal region starting at 30 hpf and persisting until at least 48 hpf  and fli1 , which is expressed in pre-cartilage condensations and endothelium fli1 in the cartilage condensations while egr1 expression is located in stripes separating cartilages  and sox9a  mutant embryos, devoid of all molecular endodermal markers egr1 transcripts were observed at 48 hpf in the pharyngeal region as well as in different brain regions and in the heart. In contrast, in 48 hpf homozygous cas embryos , while expression is maintained in the brain, the duplicated hearts and increased in the fin buds.To determine precisely in which tissue  , reminiscent of the situation observed in egr1 morphants.At stages beyond 26 hpf, expression of the Sox9b transcription factor is localized in pharyngeal epithelium and endoderm; this factor indirectly regulates gr1 is expressed in pharyngeal endoderm, we investigated potential regulatory connections between the sox9b and egr1 genes. At 42 hpf, egr1 morphants do not express sox9b in the pharyngeal pouches and its expression in the brain is altered  (not shown). At 48 hpf, sox9b mRNA is still absent in the branchial arches of egr1 morphants, while a decreased expression relative to controls is observed in the two first pharyngeal arches   (sox9b mutant (b971sox9b) embryos, the egr1 expression pattern remains intact in the pharyngeal endoderm and epithelium as compared to wild type control siblings  . The obs11, 84%) . Converssiblings . Our resrunx3 is another gene expressed in pharyngeal endoderm and required for runx2b expression in the ventral pharyngeal chondrocytes egr1 and sox9b genes. Therefore, we decided to investigate the contribution of the runx3 gene to the regulatory cascade in pharyngeal endoderm by performing epistasis experiments.In zebrafish, in situ hybridization, we showed that runx3 expression at 48 hpf was similar in endodermal pouches of egr1 morphants  to that in control embryos      . We also73, 91%) . Importa47, 89%) . Consist98, 86%) . Finallyintained  indicatirunx3 morphants by alcian blue staining at 4 dpf, we observed that co-injection ofrunx3 mRNA caused a partial rescue in 89% of the larvae , however its co-injection with MO runx3 caused a partial rescue of the anterior part of the viscerocranium in 62% of the larvae (egr1 expression.), indicating that Runx3 is indeed acting through activation of When we tested the specificity of the effects observed in e larvae , while re larvae , while re larvae , while regr1 expression, which in turn is required for sox9b expression and, finally Sox9b presumably triggers an extracellular signal leading to runx2b expression in post-migratory cNCC and chondrogenesis.In conclusion, our results show that in pharyngeal endoderm Runx3 is required for egr1 morphants. Shh is expressed in pharyngeal endoderm gr1 morphants compared to control embryos at 48 hpf. Among the BMP factor family, bmp2a, bmp2b, bmp4, bmp5 and bmp7 are all expressed in pharyngeal endoderm. By in situ hybridization in Egr1 depleted embryos, no significant alteration of expression was observed in the pharyngeal region for any of these genes (data not shown).To determine which extracellular signal is controlled by the endodermal regulatory cascade, we decided to analyze different candidate ligands of the HH and BMP signaling pathways in casanova mutants, devoid of endoderm, sox9b mRNA was undetectable   (The gene coding for the secreted TGF\u00df/BMP antagonist follistatin A (Fsta) is expressed in presumptive cephalic mesendoderm at 8 hpf 96, 95%) ,B and fs61, 93%) ,D in theegr1 morphants, a clear over-expression of fsta  relative to controls was observed at two days of development    , or of S79, 97%)  displayefstaTo further characterize a putative function of Follistatin A in cartilage development, we decreased its expression by injecting a splicing morpholino against Taken together, these results indicate that the endodermal cascade of transcription factors Runx3, Egr1 and Sox9b is required to reduce the level of fsta expression in the pharyngeal region.hand2 and edn1 at 24 hpf in egr1 and fsta morphants. Expression of both ventral markers was maintained in the microinjected embryos  of type 1 larvae, 23% (47/197) of type 2 larvae and 6% (12/197) of larvae with a complete loss of viscerocanium (type 3), while after treatment between 27 hpf and 37 hpf, we observed only 6% (14/231) of type 1 larvae, 77% (177/231) of type 2 larvae and finally 17% (40/231) of type 3 larvae . Treatmehsp70l:dnBmpr-GFP)w30. We performed a heat shock at 37\u00b0C during 30 minutes at 29 hpf and compared the head cartilage at 4 dpf of the transgenic larvae to that of their non-transgenic siblings ((hsp70l:dnBmpr-GFP)w30 was performed after 48 hpf, almost all larvae presented normal cartilage  (type 1). These results clearly confirm that BMP signaling is required for pharyngeal cartilage formation between 27 hpf and 37 hpf.To confirm this requirement for BMP signaling at late stages, we also used an inducible dominant negative BMP receptor-GFP transgenic line , while it remained expressed in the ethmoid plate and cleithrum. Upon heat shock of (hsp70l:dnBmpr-GF)w30 transgenic animals at 29 hpf, we observed the same percentage  of embryos expressing runx2b only in the cleithrum at 48 hpf.The cartilage defects observed upon BMP inhibition are quite similar to those obtained in ed 37 hpf . Half ofrunx2b expression at 2 dpf, similar to the defects observed upon blocking of the Runx3-Egr1-Sox9b cascade in the endoderm.Taken together, these observations indicate that inhibition of BMP signaling between 29 and 37 hpf causes cartilage defects at 4 dpf and prevents fli1-GFP embryos. We used fli-GFP expressing embryos in order to visualize the cNCC through the presence of GFP. The embryos were injected with a control morpholino or the egr1 splicing morpholino and fixed at 32 hpf to perform immunohistochemistry  in pharyngeal endoderm and sox9b mutants or morphants display a dramatic reduction of pharyngeal cartilage at 4 dpf and a lack of runx2b expression at 48 hpf, while exogenously expressed Sox9b partially rescued the mutant phenotype unx2b expression in cNCC cells.We have shown that all three factors are coexpressed in pharyngeal endoderm starting at about 30 hpf and that each of them is absolutely required for pharyngeal cartilage formation. These results are in agreement with previous studies concerning the function of Sox9b and Runx3 in zebrafish. Expression of ox9b in endoderm and runx2b in cNCCs. Expression of egr1 in endodermal pouches was deduced from single and double fluorescent in situ hybridization experiments, co-expression of egr1 with cNCC markers such as dlx2a (not shown) or sox9a was never observed. The complete absence of egr1 mRNA in the pharyngeal region of cas mutants, lacking endoderm, further supports this conclusion. The defects were observed following gene knock-down using translation or splicing morpholinos and the specificity of these defects for Egr1 depletion was shown by the rescue experiments.Here, we introduce a new player by showing that the endodermal transcription factor Egr1 is required for cartilage formation and expression of segr1 and sox9b, while runx3 expression was not affected in egr1 morphants or sox9b mutants. Egr1 depletion decreases sox9b expression only, while conversely egr1 expression is not affected in sox9b mutants. Finally, sox9b mutants display normal expression of egr1 and runx3. We further show that the defects observed in runx3 morphants can be partially rescued by expression of exogenous runx3 or egr1 mRNA, clearly indicating that Runx3 is located upstream of Egr1. Taken together, these results establish a regulatory cascade where Runx3 activates expression of Egr1, which itself then activates sox9b transcription. This cascade is not required for pharyngeal endoderm formation or the survival of pharyngeal endodermal cells, as was previously shown for Runx3 morphants egr1 or runx3 morphants or sox9b mutants, expression of the endodermal markers nkx2.3 and sox17 is not altered compared to controls. In conclusion, we describe a regulatory cascade that operates mainly in the pharyngeal endoderm and controls expression of runx2b in cartilage mesenchyme as well as cartilage differentiation and morphogenesis. This control exerted by endodermal transcription factors is obviously mediated by an extracellular signaling pathway that remains to be described.Additional experiments revealed that Runx3 depletion led to decreased expression of both runx2b expression increases after 24 hpf and is most crucial during the period between 27\u201337 hpf. This result was confirmed by inducing the expression of a dominant-negative Bmp receptor runx3egr1 and sox9b gene expression in pharyngeal endoderm.One of the signaling pathways involved in cartilage and bone formation is the TGF\u00df/BMP pathway egr1 morphants, we did not detect any decrease of expression for any of these genes. During development, many processes require inhibition of BMP signaling by secreted BMP antagonists follistatin a (fsta) in the pharyngeal region at 48 hpf in egr1 morphants. Such a strong over-expression of Follistatin A would obviously lead to a perturbation of BMP signaling in the entire pharyngeal region and cause defects similar to the BMP inhibition experiments discussed above. Indeed, when we tested activation of the BMP pathway using antibodies against phospho-Smad1/5/8, we observed that egr1 knock-down dramatically reduces BMP signaling in the pharyngeal region. Pathway activation was abolished not only in the post-migratory cNCC clusters, but also in the neighboring tissues, potentially causing additional perturbations in skeletal morphogenesis.When we tested the expression of various extracellular signaling molecules  in Sox9 knock out mice Clearly, the function of BMP signaling in craniofacial development is conserved in vertebrates, including mouse and human, and the importance of antagonists such as Follistatin is also well documented. Different threshold levels of Bmp4 were shown in mouse to regulate various genes involved in craniofacial skeletal morphogenesis fsta, which needs to be tightly controlled so that the Bmp ligands can bind to their respective receptors located on cartilage precursors cells  in dorso-ventral patterning by specifying expression of dorso-ventral markers hand2 and edn1 at 24 hpf. This is consistent with the onset of expression after 24 hpf of the regulators Runx3, Egr1 and Sox9b in pharyngeal endoderm and with the observed lack of fsta overexpression in egr1 morphants at 24 hpf. An earlier function for control of BMP signaling by Follistatin A was previously shown in dorso-ventral patterning of the retina fsta expression was shown to be increased at 13 hpf upon knock-down of the TALE-class homeodomain transcription factor Meis1, leading to a decrease of BMP signaling in the optic vesicle. At 19 hpf, fsta expression was highly increased in meis1 morphants in all its expression domains fsta expression specifically in the pharyngeal region at later stages is taken over by the endodermal regulatory cascade described here.Interestingly, manipulation of the endodermal regulatory cascade leaves intact the early function of BMP signaling in dorso-ventral patterning, as indicated by the maintained expression of the ventral markers sox9a, whose expression is maintained in pharyngeal cartilage precursor cells and required for their differentiation sox9a expression. In contrast, the key regulatory gene for chondrogenesis and osteogenesis, runx2b is dramatically down-regulated in egr1 morphants. Its expression in pharyngeal cartilage precursor cells normally starts at 34 hpf fsta down-regulation for efficient activation by BMP signaling as described here. runx2b expression is down-regulated despite the normal expression of sox9a, conversely it is not affected in sox9a mutants Our results indicate that BMP signaling is required in cNCC cells after their migration to their ventral position in the pharyngeal arches to induce their differentiation into hypertrophic chondrocytes, but also to allow their subsequent migration leading to morphogenesis of the different head cartilages. Additional extracellular signals will also play a role, such as Fgfs or Hhs. At 24 hpf, cNCC cells express the early differentiation marker runx2b in presumptive head cartilage, but left nearly intact its expression in the developing cleithrum, a dermal bone. Similar observations were made in the sox9a mutants sox9a expression and fsta inhibition are mainly required for endochondral (replacement) bone formation.Interestingly, inhibition of BMP signaling by increased Follistatin A expression in our experiments led to a decreased expression of fsta gene, thereby allowing the correct activation of BMP signaling in cNCC cells required for their differentiation and morphogenesis of pharyngeal and basicranial cartilage. This cascade starts by increased expression of runx3, followed by activation of egr1 expression and finally sox9b expression in the pharyngeal endoderm.In conclusion, we show here that a regulatory cascade is active in pharyngeal endoderm that represses expression of the Figure S1casanova mutants, lacking endoderm, do not express egr1 at 48 hpf. Single in situ hybridization for egr1 in casanova mutants. Lateral  and dorsal  views, anterior to the left. Scale bars 100 \u00b5m.  Wild-type or heterozygous +/\u2212cas express egr1 in the pharyngeal endoderm (pe).  Homozygous \u2212/\u2212cas do not express egr1 in the pharyngeal region, but in the pectoral fins and in the two hearts of \u2212/\u2212cas embryos. Pectoral fin (pf), heart (h).(TIF)Click here for additional data file.Figure S2casanova mutants, lacking endoderm, do not express sox9b or fsta at 48 hpf. Lateral views of in situ hybridizations (A\u2013D) with indicated markers, anterior to the left. Scale bars 100 \u00b5m. Homozygous \u2212/\u2212cas do not express fsta  nor sox9b  compared to wild-type or heterozygous +/\u2212cas. Pharyngeal endoderm (pe), otic vesicle (ov).(TIF)Click here for additional data file.Movie S1Double fluorescent in situ hybridization for fli1  and egr1  in 47 hpf zebrafish embryos. Lateral view, anterior to the left, the movie proceeds from the left side going to the center of the embryo. fli1 is expressed in cranial neural crest cells  and in endothelium. fli1 displays the characteristic expression pattern in \u201cstripes\u201d of the pharygeal cartilage precursor cells. egr1 is expressed more inside of the embryo in the pharyngeal endoderm. fli1 and egr1 mRNAs never colocalize.(MP4)Click here for additional data file.Movie S2Double fluorescent in situ hybridization for sox9a  and egr1  in 72 hpf larvae. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. sox9a is expressed in pharyngeal cartilage cells and egr1 is expressed in pharyngeal endoderm. sox9a and egr1 mRNAs never colocalize and the egr1 expression domains surrounding the sox9a expression domains can be clearly observed.(MP4)Click here for additional data file.Movie S3Double fluorescent in situ hybridization for sox9b  and egr1  at 72 hpf. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. sox9b and egr1 are expressed in pharyngeal endoderm cells and colocalize in the same expression domains.(MP4)Click here for additional data file.Movie S4Double fluorescent in situ hybridization for fli1  and egr1  at 4 dpf. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. fli1 is expressed in pharyngeal cartilages and in endothelium. Egr1 is expressed in the phrayngeal endoderm and oral epithelium. fli1 and egr1 mRNAs never colocalize.(MP4)Click here for additional data file."}
+{"text": "Hepatitis C is prevalent among thalassemia patients in Iran. It is mainly transfusion mediated, in particular among patients treated before 1996 when blood screening was introduced.The current study aimed to investigate why patients still seroconvert to anti-HCV in Iranian thalassemia centers.During 2006-2007 sera were sampled from 217 anti-HCV positive thalassemia patients at nine thalassemia centers in Tehran and Amol city, where 34 (16%) patients had been infected after 1996. The HCV subtype could be determined by sequencing and phylogenetic analysis of partial NS5B and/or 5\u05f3NCR-core region in 130 strains.1a (53%) was predominant followed by 3a (30%), 1b (15%), and one strain each of 2k, 3k and 4a. Phylogenetic analysis revealed 19 clades with up to five strains diverging with less than six nucleotides from each other within subtypes 1a and 3a. Strains in seven clades were from nine patients infected between 1999 and 2005 and similar to strains from eight patients infected before 1996, indicating ongoing transmission at the centers. Further epidemiological investigation revealed that 28 patients infected with strains within the same clade had frequently been transfused at the same shift sitting on the same bed. An additional eight patients with related strains had frequently been transfused simultaneously in the same room.The results suggest nosocomial transmission at these thalassemia centers both before and after the introduction of blood screening. Further training of staff and strict adherence to preventive measures are thus essential to reduce the incidence of new HCV infections. Thalassemia major and thalassemia intermedia are both common transfusion dependent anemias in Iran. There are around 18000 known thalassemia patients in the country , which iThe current study aimed to characterize HCV strains from patients at thalassemia centers in Tehran in Tehran province and in Amol City in Mazandaran province to investigate possible nosocomial transmission at these centers.Two hundred seventeen thalassemia patients positive for anti-HCV were included in the study. Out of these, 112 patients were from seven thalassemia centers in Tehran, Zafar adult thalassemia clinic, Childrens\u05f3 medical center , Sodeh clinic, Aliasghar Hospital, Mofid Hospital, Special Medical center, Boali Hospital and from Shahid Bahonar hospital in Karaj City. The other 105 patients were from Amol thalassemia center at Imam Reza hospital in northern Iran. All staff members in Amol and Zafar adult thalassemia centers were anti-HCV negative. 100 \u00b5l plasma was added to 500 \u00b5l lysis buffers (0.5% SDS and 10mM EDTA) and 500 \u00b5l water saturated phenol. RNA was precipitated with isopropanol. The pellet was washed with 70% ethanol and dissolved in 20 \u00b5l distilled water. cDNA synthesis and semi-nested PCR were performed with primers hep101, hep102 and hep105 as previously described  to give The products obtained from the NS5B and 5'UTR-core regions were purified using GFX PCR DNA and Gel band purification kit . The sequencing reaction was carried out with the ABI PRISM TM Big Dye TM Terminator Cycle Sequencing Reaction Kit  using purified PCR products as templates. PCR products amplified within the NS5B region were sequenced with hep105, and those amplified within the 5'UTR-core region were sequenced with primers univ-1 and 186. The obtained sequences were aligned with 347 sequences of the corresponding region retrieved from Gen Bank  patients had been infected after 1996, most of them, 26 (76 %), were infected after 2000 . Fourtee Strain 752 in clade 11 was from a patient infected in 1999 and identical to strain 794 from a patient infected before 1996. Seven clades were formed by strains only from Amol (clades 1, 5, 6, 7, 8, 10 and 12; The frequencies of subtype 1b and 3a in hepatitis C infected thalassemia patients from Tehran were 25% and 22%, respectively, which were in contrast with the findings from the thalassemia centers in Amol, where 1a and 3a were the most prevalent subtypes. In addition, data from earlier Iranian studies showed subtypes 1a and 3a were the most common ones in the country with geographical differences in their distribution, and 1a was frequent among patients infected through blood transfusion -11. Seve"}
+{"text": "Increased expression of EZH2 correlates with aggressive clinical behavior in various malignancies. In this study, we aim to investigate the clinical and prognostic values of EZH2 expression and activity in tumor tissues and improve the risk stratification in patients with renal cell carcinoma after surgery. We analyzed EZH2 expression and its activity as indicated by H3K27me3 levels comprising 373 patients with renal cell carcinoma in our institute. Outcome was assessed as overall survival and disease free survival using Kaplan-Meier analysis. Prognostic values of EZH2 and H3K27me3 expression for clinical outcomes were evaluated by Cox regression analysis. We used receiver operating characteristic to calculate diagnostic accuracy. High EZH2 expression correlates with poor overall survival in all patients, especially in advanced RCC, which is an independent prognostic factor in disease free survival and overall survival. Compared with EZH2, H3K27me3 expression is not an independent prognostic factor. The expressions of H3K27me3 and EZH2 are not completely consistent, which might be due to complicated interaction of Polycomb Repressor Complex 2. A combination of EZH2 expression and TNM stage could have better prognostic value than do TNM stage or EZH2 expression alone in both sets for disease free survival and overall survival. These results imply that evaluating intratumoral EZH2 density might improve prognostic value to the TNM staging system and inform treatment decisions for patients with late-stage renal cell carcinoma.  Kidney and renal pelvis cancer were estimated to account for 65,150 new cases and 13,680 cancer-related deaths in the USA in 2013 .Histone modification is a crucial epigenetic modification. Recent findings suggested that SETD2, EZH2, and MLL2 methyltransferases, and UTX and JARID1C demethylases played crucial roles in the development of RCC ,6. HowevIn recent studies, overexpression of EZH2 is related to poor prognostic in cancers of the prostate, breast, melanoma, endometrium, and pancreas -13. HoweEthical approval was granted by the Clinical Research Ethics Committee of Zhongshan Hospital of Fudan University . Signed informed consent was obtained from all patients for the acquisition and use of patient tissue samples and anonymized clinical data.We enrolled patients from the Department of Urology, Zhongshan Hospital, Fudan University, and whose tumors were pathologically proven RCC. This database, started in 2005, is designed to prospectively study RCC patients undergoing nephrectomy. It includes patients\u2019 baseline clinical and outcome data. Fuhrman grading system and TNM stage were according to 2004 WHO criteria and the American Joint Committee on Cancer 2010 TNM classification respectively -16. The We analyzed the EZH2 and H3K27me3 expression by immunohistochemistry. The immunohistochemical procedure (IHC protocol F) was performed on an automated immunostainer . Primary antibodies against EZH2  and H3K27me3  were used in the procedure. Two observers (Shuai. J and Lei. Z), unaware of the clinical features and outcomes of patients, evaluated slides. As previously described, scores were calculated by multiplying the staining intensity and extension ranging from 0 to 12 for each tumor or non-tumor tissue score . BrieflyWestern blot was carried out as previously described . PrimaryDFS is defined as the interval between curative surgery and recurrence or metastasis. OS is defined as the interval from date of curative surgery until death from any cause. The patients were cencerd if they were still alive until the last follow-up. We assessed disease-free survival and overall survival using Kaplan-Meier method and log-rank test.2 test, Fisher\u2019s exact method, or CMH (Cochran-Mantel-Haenszel) \u03c72. Cox univariate analysis was performed. Those parameters with a p-value< 0.1 in the univariate analyses were included in a Cox multivariate proportional hazards regression model. We used ROC curves to assess sensitivity, specificity and respective areas under the curves (AUCs) with 95% (CI) for the prediction of survival. We used Logistic stepwise regression to estimate the EZH2, H3K27me3 and TNM diagnostic accuracy. The variable was removed if the p-value was greater than 0.05. A new variable predicted probability (p) for RCC was obtained by Logistic stepwise regression on the basis of an equation. All equations were provided in the We analyzed associations between EZH2 or H3K27me3 expression and clinical characteristics with \u03c7A total of 373 patients with RCC from two sets were included in this study. Clinical characteristics were much the same in each cohort. Most of the patients were men, and had clear-cell histology, Fuhrman grade II, and TNM stage I or II diseases. The median follow-up was 77 months (IQR 72-83) in the training set and 75 months (IQR 72- 82) in the validation set . RepreseDFS was our first outcome of interest. Ten patients in the training set and 11 patients in the validation set were excluded for distant metastasis before surgery or death from other causes . In bothTNM stage is the most common used clinical prognosticator for RCC patients. When compared with TNM stage, the predictive value of the EZH2 expression is much the same using ROC analysis in both sets   table 3table 3 . To deveEZH2, as a histone methyltransferase, catalyzes the trimethylation of the lysine 27 of histone H3 (H3K27me3). Our findings demonstrate that EZH2 is an independent poor prognostic factor for RCC patients undergoing nephrectomy. To our knowledge, it is the first study analysis the prognostic significance of EZH2 and H3K27me3 expression in RCC. Pathologic stages have proved to be the single most important prognostic factor for RCC. However, same cancer stage might still have diverse clinical outcomes. In this study, EZH2 expression is found to be predictive of survival of RCC patients independently. The prognostic value of TNM stage is further improved when EZH2 levels and TNM stage are combined as a predictive model. Moreover, EZH2 expression has a similar outcome predictive ability to TNM stage in ROC analysis and has good discriminatory power as a supplementary risk factor in patients with late-stage RCC. Evaluating EZH2 and H3K27me3 expression can be a clinically applicable procedure to discriminate patients with different prognosis. It could also lead to a more personalized treatment for RCC patients after curative surgery. Traditionally, cancer has been acknowledged as a genetic disease that is driven by the sequence of oncogenes or tumor suppressor genes alteration. However, epigenetic changes including histone modification, DNA methylation, and miroRNA regulation, are increasingly evident as a tumor developmental factor -21. OnceOur data showed that EZH2 has a better predictive outcome than H3K27me3 not only in late stage patients but also in all patients. Although several integrated staging systems (ISSs) have evolved and improved for prediction of prognosis and outcome for RCC, TNM staging system is still the most used clinical prognosticator and the essential guide to the evaluation of therapeutic options but is done on the basis of anatomical information ,29. MetaOur results prospectively suggest that high intratumoral EZH2 expression independently predicts poor postoperative outcome of RCC patients. Integration of intratumoral EZH2 expression levels into current TNM staging systems might improve prognostic value to patient survival and contribute to personalized therapy.Table S1New variable predicted probability (p) for RCC survival.(DOCX)Click here for additional data file.Table S2Clinical characteristics of patients according to the EZH2 and H3K27me3 expression in the training set (n=187).(DOCX)Click here for additional data file.Table S3Clinical characteristics of patients according to the EZH2 and H3K27me3 expression in the validation set (n=186).(DOCX)Click here for additional data file.Table S4Patients were excluded form DFS analysis.(DOCX)Click here for additional data file.Table S5Training set univariate analyses of factors associated with overall survival and disease free survival.(DOCX)Click here for additional data file.Table S6Validation set univariate analyses of factors associated with overall survival and disease free survival.(DOCX)Click here for additional data file.Table S7EZH2 expression positively correlates with H3K27me3 in both sets.(DOCX)Click here for additional data file."}
+{"text": "Thus the relation between ADA1 and asthma could be mediated by events occurring during the early extrauterine life. Moreover the increased prevalence of allergic diseases in western populations parallels the widespread practice of phototherapy during the neonatal period. These observations prompted us to reevaluate our previous data and show new observations.Several studies suggest a protective role of bilirubin against oxidative damage during the neonatal period. ADAData from 2729 previously studied subjects, from 53 subjects studied at birth and after 30 years and from a survey of phototherapy frequency in four Italian Hospital including 7392 newborns are reported.1*2 allele carriers are less represented among asthmatic subjects than in controls (p=0.0004). ADA1*2 allele carriers among newborns undergoing phototherapy for hyperbilirubinemia is higher when compared to newborns not undergoing this treatment (p=0.006). In infants treated by phototherapy, the maximum bilirubin level attained during the first few days of life positively correlated with the ADA1*2 allele dose (p=0.001). Among subjects studied at birth, allergic rhinitis and/or conjunctivitis are more frequent among those treated with phototherapy than among those not treated (p=0.046).ADA1*2 allele through an increase of bilirubin level in the neonatal period protects infants from oxidative stress and favours Th2\u2192Th1 switching thus preventing allergic manifestations in later periods of life.These observations support our hypothesis that ADA We also show (iii) a survey of the frequency of phototherapy in several hospitals.1 site controls ADA activity. Two codominant alleles, ADA1*1 and ADA1*2 are present and are associated with different enzymatic activity. The corresponding three common ADA1 phenotypes (11 is 15% more active than ADA1 2/1 and 30% more active than ADA1 2 (Adenosine deaminase (ADA) is a polymorphic enzyme present in all ammalian tissues . ADA is enotypes  have difn ADA1 2 .The ADA tissue enzymes consist of one or more molecules of RBC ADA and one molecule of adenosine deaminase complexing protein (ADPC) .ADPC is identical with CD26 (a T-cell activator molecule). ADA expression may be connected with T-cell activation , 16.ADA catalyses the irreversible deamination of adenosine to inosine. In purine metabolism, the classical function of the ADA enzyme is considered to be the regulation of intra-and extracellular levels of adenosine. ADA is present on the surface of many cell types, including lymphocytes where it acts as an ectoenzyme . A co-st1 polymorphism could modulate adenosine receptor activity in the respiratory tract via the signal transduction pathway of adenosine and other mediators.Current interest is focused on the effects produced by adenosine activation via surface adenosine receptors. Four adenosine receptors  exist on the surface of different cell types . In the Mice studies have shown a relationship between ADA and an inflammatory phenotype in the lungs, similar to human asthma. This includes extensive mast cell degranulation, eosinophilia, activation of alveolar macrophages, increase in baseline airway resistance and AHR \u201323.Some observations suggest that ADA activity may influence the expression of Th cytokine genes .We have reconsidered the following group samples:291 asthmatic children (sample 1) and 1417 control-children (sample 2) from the Italian population (120 asthmatic adults (sample 3) and 116 control adults (sample 4) from the Chinese Han population    (sample 7), 388 from Penne  (sample 8), 381 from the University of Rome La Sapienza (sample 9) and 1083 from the University of Rome Tor Vergata (sample 10) were considered . We have shown no difference between starch gel electrophoresis and DNA analysis.ADAphoresis  in samplChi square test of independence was performed by SPSS programs (25). Three way contingency table analysis was carried out according to Sokal and Rohlf .1 phenotypes in asthmatic patients and in controls. In both Italian and Chinese populations a significant associations between asthma and ADA1 phenotype (genotype) is observed. Carriers of ADA1*2 allele are less represented among asthmatics versus controls suggesting a protective role of this allele against asthmatic phenotypes.Table 1*2 allele among newborns treated with phototherapy for hyperbilirubinemia and among newborns not treated. The proportion of subjects carrying the ADA1*2 allele is significantly higher in newborn treated with phototherapy versus untreated newborns. This indicates that ADA1*2 allele is positively associated with higher bilirubin levels in the neonatal period.Table 1*2 allele dose.Table Table Table 1*2 allele and asthma and a positive correlation between ADA1*2 allele and bilirubin levels in the neonatal period. Parallel to these correlations are two epidemiological data: the widespread practice of phototherapy in the neonatal period and the rise of asthma and other allergic manifestations in Western populations. Preliminary data reported in Table The data shown in Tables 1*2 allele and asthma is mediated in part by the relationship between ADA1*2 and bilirubin level in the newborn. Our data support the hypothesis that ADA1*2 favouring high level of bilirubin has a beneficial effect on oxidative damage and in turn protects from allergic manifestations later in life.Because of the protective role of bilirubin from oxidative damage during the neonatal period, we speculate that the relationship between ADASubstances that contribute to antioxidant defence protect from asthma : it is p2\u2192Th1 subpopulations during the neonatal period has not been fully studied. Increasing bilirubin level ADA1*2 allele could decrease oxidative stress favouring Th2\u2192Th1 switch thus explaining the negative correlation between ADA1*2 and asthma.The role of oxidative stress on the switch Th2\u2192Th1 switch cannot be excluded.The positive relationship between phototherapy and allergy observed in the small sample of subjects studied at birth and after 30 years supports our hypothesis. The decrease of bilirubin level induced by phototherapy favours the oxidative stress resulting in an increased susceptibility to allergy. However, the possibility of a direct negative effect of light on oxidative stress and on ThThe long term iatrogenic consequences of phototherapy in the population are not yet known. An epidemiological study on a larger sample of subjects examined at birth and after few decades is needed."}
+{"text": "An ideal intracanal medicament should be able to eliminate any remaining intracanal microorganism. The aim of this study was to compare the antimicrobial effects of Bioglass 45S5 with calcium hydroxide on Enterococcus (E) faecalis in-vitro.Direct exposure test (DET) was used to evaluate the antimicrobial effect of Bioglass 45S5, calcium hydroxide and normal saline (control group) on 80 paper cones contaminated with E. faecalis suspension. All samples were aseptically transferred into BHI culture medium to quantify microbial concentration in periods of 1, 24, 48 and 72 hours. Turbidity of the culture medium was measured via optical density (OPD) method with a spectrophotometer (wavelength=540nm). Results were then analysed statistically using student t-test.Mean difference of optical density between Bioglass 45S5 and calcium hydroxide appeared insignificant within 1 hour of the test period (P>0.05); however calcium hydroxide showed significantly greater antimicrobial properties after 24 hours (P<0.05). Antimicrobial effect of both materials displayed significant increases with time.Although both Bioglass 45S5 and calcium hydroxide exhibited antimicrobial effects against E. faecalis, neither attained complete eradication of bacteria. However, calcium hydroxide seemed to have superior disinfecting effect. Bacteria and their by-products play a major role in periradicular diseases and its progress; in fact, they are the main interfering factors in the periapical repair process 2]. The . The 2].Recent microbiologic studies demonstrate wide-spread presence of resistant microorganisms such as E. faecalis and Candida Albicans in persistent canal infections and secondary infections in endodontic failures 8]9][10[9][108][[10[9]105]6][10[6][105][[10[6]. Thi. Thi13].Bioglass S53P4 is thought to be the most effective glass on pathogenic microorganisms . WaltimoThe aim of the current in vitro study was to compare the antimicrobial effect of bioactive glass 45S5 (Nova Bone) and calcium hydroxide on E. faecalis using a direct exposure test (DET).This experimental study comprised of eighty paper cones contaminated with E. faecalis (ATCC 19818). Sterile absorbent points #50 were floated in a 10(8) CFU/mL concentration of E. faecalis suspension for 5 minutes. The paper cones were then regarded as the study samples. All experimental procedures were carried out under strict aseptic conditions. The contaminated paper cones were randomly divided into four test and control groups as outlined below.Test group 1 (n=20): 5.1 g bioactive glass 45S5 powder , CaO (24.5%), Na (24.5%), P2O5 (20.6%)) mixed with 4mL sterile distilled water to form a toothpaste consistency paste.2  mixed with 12mL sterile distilled water to form a paste with the same consistency as the Bioglass mixture.Test group 2 (n=20): 6.2 g calcium hydroxide Ca(OH)The control negative group (n=20) was exposed to normal saline and autoclaved, whereas control positive samples were only exposed to normal saline. Direct exposure test (DET) was used for the evaluation of antimicrobial effects of study samples. Each paper cone was separately placed in a sterile petri plate completely covered with experimental materials or saline (control groups). Plates were placed in a 37\u00b0C, 100% humidity incubator. A total of 5 samples were aseptically removed from each group at 1, 24, 48, 72 hours intervals and irrigated with normal saline to completely eliminate materials. Each paper cone was then inserted into sterile lab tubes containing 5mL BHI (brain heart infusion broth) and placed in a 37\u00b0C incubator for 48 hours. To evaluate the microorganisms in BHI culture medium, turbidity was assessed with a spectrophotometer that measured optical density (OD/100) (at a wavelength of 540nm).The optical density of culture medium is directly related to the total of existing bacterial in the medium. Subculture of blood agar solid medium was used to identify the target microorganism and prove the existence of E. faecalis; hence, 0.01mL was pulled out of the liquid culture medium with a sterile loop and subcultured. The morphology of the colonies of E. faecalis was evaluated both macro and microscopically (gram staining) after 24 hours. All data were statistically analysed to verify normal distribution; results were then analysed using Student t-test. The level of significance was set at P=0.05.According to kolmogrov-smirnov test, all data exhibited normal distribution. The absence of microorganism growth in the negative control group proved the aseptic and sterile working condition. Growth of microorganisms in the positive control group indicated bacterial existence within the incubation periods. Therefore, uncertainty regarding any unwanted disturbing factors during the experiment could be eliminated.There was no significant difference between Bioglass and calcium hydroxide after 1 hour; however, after 24, 48 and 72 hours intervals calcium hydroxide showed superior antimicrobial properties (P<0.05). Antimicrobial effect of both materials increased with longer time intervals, both groups displayed significant decrease of optical density (OD/100) after 72 hours .Many in vitro studies have been carried out on the antimicrobial efficacy of different materials 11]18][[18][11][[[18][11]It has been shown that the efficacy of Bioglass 45S5 increases after the first 24 hours ; this isPrevious studies revealed more efficient results for antimicrobial effect of Bioglass S53P4 compared to Bioglass 45S5 19]. Zeh. Zeh19].A recent study reported that antiseptic effect of bioactive glass (BAG) S53P4 powder was stronger than that of calcium hydroxide. The formulation of Bioglass 45S5 is: Sio2 (45%), Na2o (24.5%), CaO (24.5%), P2O6 (6%), whereas, Bioglass S53P4 is Sio2 (53%), Na2o (23%), CaO (20%), P2O4 (4%).This effect was apparently not exclusively pH-related. The presence of dentin powder in suspension increased the efficacy of S53P4 against a multiplicity of oral microorganisms . The sizAntimicrobial characteristics of Ca(OH)2 is due to the release of OH ions into the environment, the increase of pH and therefore destruction of the microorganism cell wall 17]28] [28] 17][ [28] 17]A recent study, has reported that the combined effect of dentin powder and BAG is neither a matter of osmolarity or even cellular agglutination, nor is it linked to binding of bacteria to dentin powder in the presence of BAG. It was proved that only combinations of solid BAG and dentin powder and not their supernatants in suspension have an additive effect. Dentin powder with its complex surface acted as a recipient for ions in solution, and hence acted as a catalyst for the dissolution of the glass in aqueous suspension. It was this ionic flow between the glass and dentin powder that appeared to interfere with bacterial viability . It shouThe current study demonstrated that the antimicrobial effect of Bioglass 45S5 against E. faecalis was lower than Ca(OH)2, although it tends to increase with time. Considering all desirable properties of Bioglass, the material could well be incorporated into endodontics practise as an intracanal medicament in the future. Further research is recommended to find more effective formulations of Bioglass."}
+{"text": "The whole sequence is atom economic and the application of a multicomponent reaction assures diversity.An Ugi four-component reaction of propargylamine with 3-formylindole and various acids and isonitriles produces adducts which are subjected to a cationic gold-catalyzed diastereoselective domino cyclization to furnish diversely substituted spiroindolines. All the reactions run via an  The importance of nitrogen containing heterocyclic molecules in chemical biology is undisputed. The synthesis of such biologically interesting heterocycles is generally target-oriented, inspired by nature or randomly directed. In all these cases the design of a synthetic sequence to produce a library of diversely substituted molecules is the first and most important step. The basic concept of diversity-oriented synthesis (DOS) involves short reaction sequences, a strong focus on bond construction, and functional group compatibility \u20133. ReactAs an efficient activator of alkynes, gold has recently attracted a lot of attention \u201336. Many1a), propargylamine (2a), phenylacetic acid (3a) and tert-butylisonitrile (4a) in methanol at 50 \u00b0C gave the Ugi-adduct 5a with an excellent yield of 94%. With compound 5a in hand we were keen to apply the previously developed conditions for intramolecular hydroarylation [5a with 5 mol % of Au(PPh3)SbF6 in chloroform at room temperature produced the desired spiroindoline 6a in a moderate yield of 55% along with some unidentified byproducts  instead of TFA did not improve the outcome Cl (5 mol %) and AgSbF6 (5 mol %) were loaded along with chloroform (2 mL). Ugi product 5 (0.2 mmol) was added followed by TFA (1 equiv), and the reaction mixture was stirred at rt. After completion, the reaction mixture was partitioned between EtOAc (100 mL) and 2 N K2CO3 solution (2 \u00d7 50 mL). The organic layer was washed with brine (50 mL), dried over magnesium sulfate, and evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (10% diethyl ether in dichloromethane) to afford compound 6a\u2013q.To a screw capped vial Au(PPhFile 1Experimental section."}
+{"text": "Among the inherited disorders of blood, thalassaemia and Sickle Cell Diseases contributes to a major bulk of genetic diseases in India. Our aim was to verify the role of the Xmn1 polymorphism as a modulating factor in sickle cell patients and frequency of the polymorphism in Indian sickle cell patients. 60 sickle homozygous and 75 sickle beta thalassemia patients were included and 5 ml blood sample was collected from them. Screening of sickle patients was done by HPLC. An automated cell analyzer SYSMEX (K-4500 Model) was used to analyze the Complete Blood Count of patients. Xmn1 polymorphism analysis was done by PCR-RFLP and one-way ANOVA test was applied to analysis of variance between groups. Among the sickle patients 27 were heterozygous (+/\u2212) and 19 were homozygous (+/+) while 30 were heterozygous (+/\u2212) and 24 were homozygous (+/+) in sickle \u03b2-thalassemia patients. Extremely significant differences  of hematological parameters seen among patients with Xmn1 carrier and without the Xmn1 carrier. In our cases the clinical symptoms were barely visible and higher HbF level with Xmn1 carriers were found. Presence of Xmn1 polymorphism in sickle cell patients with higher HbF were phenotypically distinguished in the sickle cell patients. We conclude that the phenotypes of Indian sickle cell patients were greatly influenced by Xmn1polymorphism.X The sequence variation has been shown to increase Hb F levels in \u03b2-thalassaemia anemia.mn1 polymorphism on the phenotype of Indian sickle cell patient; thus our aim was to evaluate the role of the Xmn1 polymorphism as a modulating factor in sickle cell patients and its frequency.The C-T substitution at position \u2212158 of the Gy globin gene, referred to as the Xmn1-\u03b3 polymorphism, is a common sequence variant in all population groups, present at a frequency of 0.32 to 0.35.+ while 46 patients were HbS\u03b20). About 5 ml blood sample was collected from hematology outpatient department AIIMS; after taking their consent. Study was approved from institutional ethical committee. Clinical evaluation was done during physical examination  as well as laboratory evaluation. Age of onset of splenomegaly and jaundice was <10 year and the presence of anemia was evaluated through hemogram analysis. Complete blood count and red cell indices were measured by automated cell analyzer . Quantitative assessment of hemoglobin Hb F, Hb A, Hb A2 and Hb S and diagnosis of HbSS and HbS\u03b2-thalassemia was performed by high performance liquid chromatography . DNA extraction done by phenol chloroform method and DNA quantification done by nano drop spectrophotometer. Xmn1 polymorphism analysis was done by PCR-RFLP method as per Suttonmn1polymorphism was used to for comparison of clinical features.Subjects were 60 sickles homozygous and 75 sickle beta thalassemia patients (29 patients were HbS\u03b2mn1 polymorphism. Fourteen (23.33%) patient in sickle homozygous and 21(28%) patients in sickle \u03b2-thalassemia were normal for Xmn1 polymorphism. The frequency of the Xmn1 polymorphism was higher among sickle homozygous than sickle \u03b2-thalassemia patients. Clinical severity were improved with homozygous (+/+) Xmn1 polymorphism in sickle cell anemia as well as sickle \u03b2-thalassemia patients. Reticulocytes, haemoglobin and red cell indices were higher in Xmn1carriers than non carriers and found extremely statistically significant . The explanation to the improvement of RBCs in Xmn1 carriers is that the anemia was less and overall red cell indices and Hb value was improved. Hyperpigmentation was found in a few cases. Splenomegaly, gall stone, painful crisis, jaundice and frequency of blood transfusion in relation with HbF and Xmn1 polymorphism were lesser in Xmn1 carriers in comparison to non-carriers and found statistically significant . Details of haematological parameters and clinical parameters of HbSS and HbS\u03b2-thalassemia are given in Sixty sickle homozygous  and 75 sickle \u03b2-thalassemia  patients were characterized. Out of 60 sickle homozygous patients; 27 45%) were heterozygous (+/\u2212) and 19 (31.67%) were homozygous (+/+) while 30(40%) were heterozygous and 24(32%) were homozygous in sickle \u03b2-thalassemia for X5% were hmn1 site with elevated HbF expression has been previously published. The role of increased HbF response as an ameliorating factor has become evident in patients who were mildly affected despite being homozygotes or compound heterozygotes for \u03b20 or \u03b2+ thalassaemia.mn1 and frequency of blood transfusion was reported in thalassemic patients.21Fetal hemoglobin (HbF) genes are genetically regulated and the level of HbF and its distribution among sickle erythrocytes is highly variable. Hb F is the major genetic modulator of the hematological and clinical features of sickle cell disease with the Senegal and Saudi-Indian haplotype which gives its beneficial effects to the pateints.mn1 site with the Arab Indian haplotype is thought to be associated with high fetal hemoglobin concentration and confer a benign course of the disease. However, the clinical presentation of sickle cell disease in different regions in our country is highly variable.mn1 carriers while non carriers of Xmn1polymorphism were found worsened. Our cases of sickle cell patient showed the presence of C-T variation at position \u2212158 in the G\u03b3 gene, affect hematologically as well as clinically and increased production of HbF. Xmn1 carriers of HbS\u03b2-thalassemia patients had higher HbF than HbSS patients. The clinical features of HbS-\u03b2 thalassemia are extremely variable, ranging from a completely asymptomatic state to a severe disorder similar to homozygous sickle cell disease. This heterogeneity is likely to be due to the presence of different \u03b2-thalassemia alleles or interaction with modulating genetic factors like associated \u03b1-thalassemia and/or a gene for raised HbF production (Xmn1 polymorphism).mn1 site polymorphism is also likely to influence phenotypemn1 polymorphism absence reduction is associated with acquired HbF elevation.mn1polymorphism and phenotypic effect on Indian sickilers. However a study on HbE\u03b2-thalassemia report the phenotypic effect of Xmn1 polymorphismmn1 polymorphism in thalassemia intermedia patients.mn1 polymorphism and IVS 1-1 mutation leads to a milder phenotypic presentation causing a delay in onset of blood transfusions but dose not effect the amount of blood received /kg/year. However \u03b1-thalassemia also influence on the level of HbF in patients with sickle cell disease.mn1 polymorphism found higher amongst sickle cell anemia patient in comparison to sickle \u03b2 thalassemia. Sickle homozygous and sickle \u03b2-thalassemia patients showed clinical variation and this could be due to the association of Xmn1polymorphism, either in homozygous or heterozygous state. Presence of Xmn1polymorphism in sickle patients with higher HbF that improve phenotypic presentation in the sickle cell patients. A study from Western Iran in \u03b2-thalassemia patients report the presence of Xmn1 polymorphic site on both chromosomes (+/+) the level of Hb F tended to be increased compared to the absence of Xmn1 ( / ) and the presence of this polymorphic site caused a positive influence on Hb F production and the G\u03b3 percent which could improve the clinical symptoms of \u03b2-thalassemia patients.mn1polymorphism.The strong association of X"}
+{"text": "POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5\u2032 upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5\u2032 upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function.POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human  Dlx5-Dlx6 genes was found by the study of five affected members displaying hearing loss and craniofacial defects. The affected individuals shared a deletion of 5,115 bp. Bioinformatic analysis of this sequence indicated the presence of several HCNR, which in a transgenic mouse reporter assay, drove expression in the inner ear and developing bones The inner ear of vertebrates is one of the most complex sensory organs of the head and host two senses, the sense of hearing and the sense of balance. From an ectodermal layer adjacent to the hindbrain, the otic placode, a spheroid organ is generated by invagination/cavitation followed by a series of developmental processes such as patterning, cell diversification and morphogenesis. In the ventral portion of the inner ear, the cochlea or auditory organ emerges as an outpocketing of the otic vesicle, while in the dorsal portion of the otic vesicle the vestibular organs, semicircular canals and endolymphatic duct are developed. In each sensory organ, the main functional unit is composed by the hair-cells, the supporting cells and the sensory neurons that connect the hair-cells to the central nervous system POU4F3 (Brn3c) is specifically expressed in hair-cells and mutations in POU4F3 causes DFNA15, an autosomal dominant form of progressive hearing loss POU3F4 (Brn4) causes deafness type 3 (DFN3), characterized by a conductive hearing loss that results from stapes fixation and progressive sensorineural deafness POU3F4 is located in the X chromosome (Xql3-q22) being one of the most frequent causes of X-linked deafness. In rats, the Pou3f4 gene is expressed during embryonic development in the brain, the neural tube, and the otic capsule at E15.5 and E17.5 days Pou3f4 might be required for epithelial-mesenchymal interactions taking place during development POU3F4 gene was identified where several microdeletions also caused DFN3 phenotype, suggesting that regulatory regions were present in that region POU3F4 enhancer within a HCNR (HCNR 81728) was described to induce reporter expression in the otic mesenchyme. This enhancer lay within the smallest microdeletions shown to cause DFN3 Xenopus coding region, at position 970 Kb (HCNR 81675) and 170 Kb (HCNR 82478) of the pou3f4 coding unit that present otic vesicle enhancer activity in a zebrafish transgenesis assay. Here, we have analyzed the spatio-temporal activity of these enhancers, as well as the signaling pathways that initiate their activity. We found that the HCNRs display distinct temporal patterns of activation and; while HCNR 81675 is regulated by the retinoic acid (RA) signaling, the HCNR 82478 is regulated by the Fgf pathway and the HCNR 81728 enhancer is under the control of Hh. Finally we present direct evidence that the distal enhancer is bound in vivo by Sox2 and Pax2 transcription factors. Altogether, these data suggest that the regulatory apparatus of the pou3f4 is multiple, complex and integrate distinct developmental inputs.The POU proteins are transcription factors that bind to DNA through their POU-specific and POU-homeodomain regions and play essential roles during development. Several members of the POU family are expressed in the inner ear. The gene Zebrafish transgenic fishes have been maintained at the PRBB Animal Facility. Our Animal Facility in accordance with national and European regulations is registered as animal research center with the number B9900073. Veterinary welfare supervision and daily water check-ups are conducted  to ensure the animals good health status. Temperature, humidity, light intensity and noise control in the room are strictly monitorized to guarantee animal welfare. Zebrafish embryos have been sacrificed after being anesthetized with 0.016% tricaine when necessary. The experimental zebrafish procedures have been performed following the protocols (CEEA-PRBB ref JMC-07-1001-CPC and MM-08-1108BAE) approved by the Ethical Committee for Animal Research (CEEA) from Barcelona Biomedical Research Park (PRBB) according to the European Union regulations.pou3f4 HCNR 81675 and HCNR 82478 GFP zebrafish lines were obtained as described in http://pipeline.lbl.gov/cgi-bin/gateway2). Subsequently genome fragments were amplified by PCR from Xenopus tropicalis, cloned into the PCR8/GW/TOPO vector and stable zebrafish lines generated by the Tol2 transgenesis system.pou3f4 HCNR 81675 and HCNR 82478 GFP embryos were staged according to morphology and somite pair number. For life GFP imaging, tricaine-anesthesized embryos were mounted in slides with glycerol and images were taken under the microscope.Whole-mount in-situ hybridization (WISH) was performed according to standard protocols HCNR 81675 and HCNR 82478 GFP transgenic embryos were chorion punctured and incubated with different pharmacological inhibitors in system water, from 5.5, 7.5 or 9.5 hours post-fertilization (hpf) stage until 18\u201320 hpf or 36\u201340 hpf respectively. The battery of inhibitors included: 30 and 50 \u00b5M SU5402  to inhibit Fgf signaling, 100 \u00b5M DAPT  to inhibit Notch signaling, 20 \u00b5M DEAB (Sigma) to block RA synthesis, 30 \u00b5M Dorsomorphin (Biomol) to inhibit Bmp signaling and 45 \u00b5M cyclopamine A (CyA) to inhibit Hh signaling. DMSO diluted at 1/200 in system water or EtOH 95% was used as the carrier control treatment.pou3f4 HCNR 81675 developed embryos were frozen in tissue-tek OCT and sectioned (12 \u00b5m). Slides were fixed in \u221220\u00b0C methanol for 10 minutes and blocked-permeabilized in 10% fetal bovine serum (FBS), 3% bovine seric albumin (BSA) (Sigma) in 0.1% PBT (0.1% tween-20) for 90 minutes at room temperature. Slides were stained with rabbit anti-GFP (Takara) at 1\u2236400 overnight at 4\u00b0C and donkey anti-rabbit Alexa 488 (Molecular Probes) at 1\u2236400 for 90 minutes at room temperature, both in 0.1% PBT, 10% FBS, 3% BSA. Sections were mounted in mowiol for imaging. For double immunostaining, transgenic GFP was able to be detected after all the processing, thus pou3f4 HCNR 81675 embryos were sectioned, blocked-permeabilized and stained with mouse anti acetylated-tubulin (Sigma T6793) or rabbit anti-Pax2 (Zymed laboratories ref.71-6000) at 1\u2236400 overnight at 4\u00b0C and goat anti-mouse Alexa 546 or goat anti-rabbit Alexa 594 (Molecular Probes) at 1\u2236400 for 90 minutes at room temperature. Nuclei were stained with 4\u20326-diamidino-2-phenylindole  for 5 minutes and mounted in mowiol.For immunostaining after WISH, pou3f4 HCNR 81675 embryos were injected into the otic vesicle with a micromanipulator with the FM\u00ae 4-64FX reagent (Invitrogen) at 1\u22365 dilution in water, and then left in system water for 15 minutes, fixed in 4% paraformaldehyde (Sigma) and frozen in tissue-tek OCT (Sakura) for sectioning and imaging.Alive 3 day-old pou3f4 HCNR 81675 sequence, human and Xenopus sequences were analysed in rVISTA 2.0 (http://rvista.dcode.org/) that predicts evolutionarily conserved transcription factor binding sites. Moreover, the sequence of Xenopus was also analysed by Transfac 7.0 in the Gene Regulation portal (http://www.gene-regulation.com/index.html). Pax2 and Sox2 binding sites found in the pou3f4 HCNR 81675 were subjected to site-directed mutagenesis. Based on the Transfac motif and literature research, we mutated the core nucleotides of the Pax2 and Sox2 binding sites by designing primers that contained the Pax2 binding core GTGAATAG mutated into TCAAACAT or the Sox2 binding core ACAAAA mutated into GTGCTC. Mutant primers were used to introduce these point mutations into the pCR8/GW/TOPO TA\u00ae - HCNR 81675 construct using the Quik Change\u00ae XL Site-Directed Mutagenesis Kit (Stratagene). Transgenic zebrafish containing either Pax2 or Sox2 mutant binding sites as well as the double mutant for Pax2 and Sox2 motifs were generated using the ZED vector and Tol2 transposon/transposase transgenesis method In order to predict conserved TFB sites in the Xenopus embryos were dissected and used for Chromatin immunoprecipitation assay. ChIP analysis was performed with minor modifications as described previously Xenopus pou3f4 HCNR 81675 were designed , whereas primers for a CNR in the Xenopus haemoglobin-2 locus with no predicted Pax2 and Sox2 binding sites according to rVISTA and Transfac databases were used as negative control .ACA, reverse Pictures were acquired in a Leica DRM microscope using a Leica DFC300 FX camera and the Leica IM50 software. Adobe Photoshop CS2 software was used for photograph editing.Xenopus and zebrafish to identify several highly conserved non-coding regions (HCNR) in the 1 Mb 5\u2032 upstream region of the pou3f4 transcription start site with enhancer activity in the developing inner ear pou3f4 transcriptional start site  to detect GFP mRNA was performed. WISH in the same stage embryos revealed that, in both ectively . Thus, apou3f4 early expressed enhancers activate reporter GFP expression at different developmental stages in zebrafish, we next assayed whether the spatial pattern of expression in the inner ear also presented particularities. From 13 hpf to 24 hpf, GFP driven by HCNR 81675 is observed in almost the entire otic placode , the RA (DEAB) and Bmp (Dorsomorphin) pathways, smaller otic vesicles were observed since all three pathways play essential roles in otic placode formation pea3, neuroD, krox20, msx1 and ptc1 was done in inhibitor-treated embryos to confirm that each signaling pathway was abrogated at our working concentrations . These results were further confirmed by double labelling experiments. Double immunostaining with anti-GFP antibody and anti-Pax2a showed co-localization of both proteins in the same domain  observed in embryos transgenic for the HCNR 81675 enhancer, correlated with abrogation of  embryos . All togpax2a and sox2 are co-expressed in a similar domain than that promoted by the HCNR 81675 enhancer and retinoic acid inhibition results in loss of both, GFP and pax2a and sox2 expression, we then checked for Pax2 and Sox2 binding sites in the HCNR 81675 genomic sequence. TRANSFAC analysis of human and Xenopus HCNR 81675 sequences revealed a high number of conserved putative Transcription Factor Binding Sites (TFBS). Interestingly for the work presented here, a putative Sox motif and two Pax2/5/8 motifs where found in the sequence . PCR primers were designed to include the region of Pax2 and Sox2 binding sites. Otic vesicles from stage 30 to 34 Xenopus embryos were dissected and the chromatin was immunoprecipitated with either rabbit anti-Pax2 and rabbit anti-Sox2 antibodies or rabbit IgG as control. pou3f4 HCNR 81675 DNA was precipitated by the anti-Pax2 and anti-Sox2 antibodies but not by the isotopic antibody. Moreover, no binding of these two proteins in the Xenopus haemoglobin promoter which lacks Pax2 and Sox2 binding sites was detected, confirming the specificity of Pax2 and Sox2 binding to the endogenous Xenopus HCNR 81675  syndrome not only affect the POU3F4 coding sequence POU3F4 gene. Accordingly, we and others have recently identified several pou3f4 inner ear enhancers within this genomic region Hearing impairment is one of the most prevalent sensorineural defects in humans and in the last years many human ear disorders have been linked to mutations in over a hundred different genes POU3F4 expression mainly to the periotic mesenchyme in transgenic lacZ mouse embryos e POU3F4 coding unit several other evolutionary conserved sequences (from human to Xenopus) were identified Xenopus HCNR 81675 and HCNR 82478 specifically drive reporter GFP expression to the otic epithelium and at later stages to the inner ear sensory patches. HCNR 81675 directs reporter gene expression only to the otic vesicle, while HCNR 82478 acts as a general enhancer driving pou3f4 expression to all the tissues where it is expressed such as the otic vesicle, midbrain-hindbrain boundary, mesonephros and spinal cord. Both new otic enhancers are at each side of the previously identified POU3F4 enhancer pou3f4 expression to the otic territory indicating that Pou3f4 is a crucial transcription factor for inner ear development and that its expression needs very fine-tuned regulation. This is further exemplified by its distinct temporal activity. In stable transgenic fishes, GFP in the otic vesicle is switched on at different developmental stages, being the enhancer at HCNR 81675 active before the one at HCNR 82478, and this one before the regulatory element located in HCNR 81728. Moreover, transcriptional activation directed by the HCNR 82478 enhancer starts in the mesonephros, followed by activity in the midbrain-hindbrain boundary and finally to the otic vesicle. Later on, at 24\u201332 hpf GFP is no longer detected in the mesonephros and in contrast it is activated in the spinal cord.Previous work published by Ahn and colleagues described a human HCNR that specifically directs Hox and Irx gene clusters or the Sox2 gene Sox2, Kondoh and colleagues have nicely dissected out the genomic regulatory apparatus of the chicken Sox2 locus. Eleven enhancers were identified and from those, three and two enhancers control the expression of Sox2 in the spinal cord and otic placode, respectively.It has been well reported that developmental genes are very tightly regulated and thus in their loci several scattered enhancers are contained around or in the gene that regulate. Examples are found in the locus of the fgf3 and fgf8 results in complete ablation of the otic placode, while in chick and mice Fgfs from the mesoendoderm primarily control the process fgf expression POU3F4 mesenchymal enhancer; second, previous reports in mice indicated that Pou3f4 cooperates with Tbx1 during cochlear development Pou3f4 expression is reduced in conditional Tbx1 mutants pou3f4 expression, acting separately over distinct enhancers located at different HCNRs.Inner ear development is highly complex and many distinct signaling pathways regulate it. Fgf signaling for example is used throughout ear development to control distinct processes, initially is essential for the induction of the otic primordium from the ectoderm. In zebrafish embryos, loss of pou3f4 HCNR 81675 co-localized with the pax2a and sox2 expression domains in the otic vesicle. Since Sox and Pax but not Tbx binding sites were found in the HCNR 81675 sequence, we hypothesized that both Pax2 and Sox2 might be activating the enhancer in the zebrafish transgenic line. This was confirmed by chromatin IP and site-directed mutagenesis of the Pax2 and Sox2 binding sites that showed that Pax2 and Sox2 TF are bound directly to the enhancer and regulate GFP expression in vivo. Altogether, our results suggest that early pou3f4 expression directed by the HCNR 81675 enhancer may be regulated by retinoic acid and Sox2 and Pax2 transcription factors. Our mutagenesis analysis of the HCNR 81675 enhancer plus the fact that GFP is only found in domains of pax2a and sox2 co-expression, indicate that Pax2 and Sox2 transcription factors alone are not sufficient to activate this enhancer and act in a cooperative manner over the genomic locus. This would be similar to what has been reported by the cooperative interaction of Pax6 and Sox2 in the \u03b4-crystallin and N3 Sox2 enhancers GFP driven by pou3f4/Pou3f4 gene is expressed in the midbrain-hindbrain boundary, mesonephros and spinal cord, as well as the periotic mesenchyme in Xenopus and mouse embryos pou3f4 expression in the periotic mesenchyme and the activation of the pou3f4 enhancers in the otic epithelium in zebrafish. First, endogenous pou3f4 transcripts might be present in the otic epithelium at lower and undetectable levels by in situ hybridization; secondly developmental differences of expression might appear when enhancers are extracted from their genomic context. The latter hypothesis of an improper regulation of foreign enhancers is favoured by the fact the human enhancer described by Ahn et al. 2009 when inserted in mice Endogenous POU3F4 gene and their regulation may contribute to the further understanding of the function of POU3F4 in inner ear and its implications in DNF3.In conclusion, the description of the spatiotemporal activity of novel enhancers of Figure S1HCNR 81675 activity is not dependent on RA, Fgf, Notch, Bmp and Hh at 7.5 and 9.5 hpf. (A\u2013N) GFP is observed after treatment of HCRN 81675 transgenic embryos with pharmacological inhibitors of signaling pathways at 7.5 hpf (A\u2013G) and 9.5 hpf (H\u2013N). Orientation is anterior to the left and dorsal up.(TIF)Click here for additional data file.Figure S2Abrogation of different signaling target genes after treatment with specific signaling inhibitors. (A\u2013J) In situ hybridization for the Fgf, Notch, Retinoic Acid, BMP and Sonic Hedgehog target genes pea3 (A\u2013B), neuroD (C\u2013D), krox20 (E\u2013F), msxC (G\u2013H) and ptc1 (I\u2013J) to confirm inhibitor activity at our working concentrations. Dorsal view, orientation is anterior to the left.(TIF)Click here for additional data file.Figure S3HCNR 82478 activity is dependent on Fgf signaling when treated at 5.5 and 7.5 hpf. (A\u2013N) GFP is inhibited after treatment of HCRN 82478 transgenic embryos with 30 \u00b5M SU5402 at 5.5 hpf (A\u2013G) and 50 \u00b5M SU5402 at 7.5 hpf (H\u2013N). Orientation is anterior to the left and dorsal up in all images.(TIF)Click here for additional data file.Figure S4Endogenous expression pattern of pou3f4/Pou3f4 in Xenopus and mouse. (A\u2013B) In situ hybridization for pou3f4 mRNA in Xenopus embryos of stage 35 (A) and stage 42 (B). Note that the endogenous expression is detected at the periotic mesenchyme at stage 42, whereas at stage 35 pou3f4 is still not expressed. (C\u2013D\u2033) In situ hybridization for Pou3f4 mouse mRNA in mice embryos of stage E8.5 (C) and E16.5 (D). In mice, also Pou3f4 is expressed at the otic mesenchyme at later stages, shown in insets . Transverse sections shown in all panels.(TIF)Click here for additional data file."}
+{"text": "IFIH1, one common and four rare SNPs have been associated with lower risk for type 1 diabetes. Our aim was to test whether these type 1 diabetes-associated IFIH1 polymorphisms are associated with the occurrence of enterovirus infection in the gut of healthy children, or influence the lack of association between gut enterovirus infection and islet autoimmunity.Interferon induced with helicase C domain 1 (IFIH1) senses and initiates antiviral activity against enteroviruses. Genetic variants of HLA-DR4-DQ8/DR3-DQ2) as well as 375 children without this genotype were included for monthly fecal collections from 3 to 35 months of age, and genotyped for the IFIH1 polymorphisms. A total of 7,793 fecal samples were tested for presence of enterovirus RNA using real time reverse transcriptase PCR.After testing of 46,939 Norwegian newborns, 421 children carrying the high risk genotype for type 1 diabetes  as compared to wild-type homozygotes ; odds ratio 2.5, p\u200a=\u200a0.06. The association was stronger when infections were restricted to those with high viral loads . The lack of association between enterovirus frequency and islet autoimmunity reported in our previous study was not materially influenced by the IFIH1 SNPs.We found no association with frequency of enterovirus in the gut for the common IFIH1 polymorphisms have no, or only minor influence on the occurrence, quantity or duration of enterovirus infection in the gut. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection.We conclude that the type 1 diabetes-associated  Picornaviridae) cause mostly inapparent or subclinical infections; acute disease may range from minor illness to paralytic disease Enteroviruses (family IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation associated gene 5), encodes a cytoplasmic sensor that recognizes certain types of double stranded RNA (dsRNA) molecules, which are commonly produced during the replication of some RNA viruses. Signaling via IFIH1 triggers activation of NF-\u03baB and interferon regulatory pathways, and induces antiviral interferon responses in vivo effects of IFIH1 in mda5 knockout mice infected with coxsackievirus IFIH1Enteroviruses have been considered as possible environmental triggers or accelerators of islet autoimmunity leading to type 1 diabetes IFIH1 gene revealed that lower risk of type 1 diabetes is also associated with two rare variants with a presumed loss of function (nonsynonymous SNPs rs35744605 (E627X) and rs35667974 (I923V)), and with two noncoding variants affecting conserved splice sites (rs35337543 (1641+1G>C)) and rs35732034 (2807+1G>A)), odds ratios of about 0.5\u20130.7 IFIH1 polymorphisms to type 1 diabetes, and if enterovirus is involved here, remains unclear and paradoxical. Presumably, variants associated with reduced IFIH1 function would confer the host with a mild antiviral response to enterovirus infection. If so, this would influence the observed association between enterovirus frequency and type 1 diabetes in the population, and failure to account for IFIH1 SNPs in population studies could be hypothesized to \u201cconceal\u201d relations between enterovirus and islet autoimmunity or type 1 diabetes.Recently, deep sequencing of exons and splice sites in the IFIH1 polymorphisms associated with type 1 diabetes can predict frequency, viral load, or duration of gut infections with enterovirus. We further aimed to test whether our previously reported lack of association between enterovirus and islet autoimmunity was modified by taking these IFIH1 polymorphisms into account.The primary aim of the present study was to assess in healthy children whether the DRB1*04:01-DQA1*03-DQB1*03:02/DRB1*03-DQA1*05-DQB1*02 (DR4-DQ8/DR3-DQ2) which confers high risk for type 1 diabetes HLA DR4-DQ8/DR3-DQ2 genotype and 375 children without this genotype, as described in detail in The participants were identified and recruited in the MIDIA-study that during 2001\u20132007 screened 46,939 newborns from the Norwegian general population for the HLA genotype IFIH1 polymorphisms modified the risk of type 1 diabetes-related islet autoimmunity among children with the HLA-DR4-DQ8/DR3-DQ2 genotype  and the Norwegian Data Inspectorate.Fecal samples were tested for enterovirus as described earlier IFIH1 SNP , and four rare SNPs rs35744605 (E627X), rs35667974 (I923V), rs35732034  and rs35337543 . SNP genotyping of rare variant SNPs was performed with the MassARRAY system using iPLEX chemistry . The common SNP rs1990760 was genotyped using TaqMan technology with predesigned primer-probe mixes . Among children with at least one fecal sample tested for enterovirus (n\u200a=\u200a808), genotyping with the MassARRAY system failed in 5\u20138 DNA samples (<0.1%), and with TaqMan only two samples failed. Duplicate genotyping was performed in 5% randomly chosen samples, with 100% concordance.Subjects were genotyped for one common 2 test) in either of the polymorphisms. Haploview 4.2 was used to assess linkage disequilibria between the alleles of the typed SNPs. Association analyses were performed using logistic regression models with enterovirus infection (yes or no) as the response, individual SNP genotypes as predictors, and a random intercept to account for potential intra-individual correlation (clustering) of infections .The final sample for analysis included 796 children and their 7,793 fecal samples . There wApart from taking enterovirus positivity as an outcome, in further sensitivity analysis we restricted infections to those with above median quantity of enterovirus RNA among positive samples , or to the first enterovirus RNA positive samples among series of two or more consecutively positive samples. Prolonged infection episodes with at least two consecutively positive samples were assessed by excluding other positive samples.IFIH1 SNPs evaluated. This was modeled similarly as for the IFIH1 SNP-enterovirus relation, but with autoimmunity status as a covariate and an additional random intercept to specify matched set .The frequencies of enterovirus in fecal samples by genotypes of the five SNPs are shown in  the gut , we obse the gut . Here thP\u200a=\u200a0.01, P\u200a=\u200a0.09) (2<0.04), the above association is unlikely to be explained by linkage disequilibrium to other known diabetes-associated variants. The association of each IFIH1 SNP with enterovirus was similar among children with and without the type 1 diabetes-associated HLA DR4-DQ8/DR3-DQ2 high-risk genotype, respectively (data not shown).This suggestive association was strengthened when the analysis was restricted to infections with high viral loads  . As therIFIH1 polymorphisms into account would modify our previously published observation of no association between enterovirus frequency and islet autoimmunity IFIH1 rs1990760  IFIH1 SNP our data suggest that the per allele odds ratio is highly unlikely to be greater than about 1.2 . We cannot exclude weak to moderate effects of the rare variants because of limited power, but the possible existence of such weak effects of rare IFIH1 variants on enterovirus infections would have limited population impact on the epidemiology of enterovirus infections, if any at all.We are aware of only two previous studies relating mda5 knockout mice mda5 knockout mice with coxsackievirus have produced partially conflicting results, but one group showed significantly decreased production of type I IFN, but this was not correlated with increased virus titers If the suggestive association between the rare A variant of rs35732034 and frequency of high quantity enteroviral infections is true, this indicates a dominant or dose-dependent effect of the predicted interference with splicing of the intron enterovirus genus in type 1 diabetes pathogenesis seem likely, yet the limited numbers of available longitudinal studies of islet autoimmunity where enteroviral RNA have been tested as a risk factor have been inconsistent between populations and sources of the sample The evidence for the involvement of the IFIH1 alleles that had been previously associated with reduced type 1 diabetes risk were predicted to be loss of function variants, and may thus confer increased risk for enterovirus infection. We therefore reasoned that our previously published lack of association between enterovirus RNA and islet autoimmunity IFIH1 SNPs. However, adjusting for IFIH1 (rs1990760) and the exclusion of children with rare IFIH1 alleles did not influence the lack of association. A prospective cohort study recently reported lack of association with islet autoantibody development for genotypes of the IFIH1 SNP rs2111485, a SNP in close proximity and in strong linkage disequilibrium with rs1990760. While this may partially be explained by limited statistical power, their study showed an association with faster progression from autoimmunity to type 1 diabetes The IFIH1 variants previously associated with type 1 diabetes are loss of function variants, and experimental data showing an important role of IFIH1 in antiviral immunity to enteroviruses, out data suggests that these variants have no or just minor impact on the frequency and duration of gut infection with enterovirus in healthy infants. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection frequency.In conclusion, despite the predictions that rare Table S1The effect of IFIH1 genotypes on different measures of enterovirus infection in longitudinal fecal samples.(DOC)Click here for additional data file."}
+{"text": "Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. It has been proposed that UCP3 reduces production of reactive oxygen species (ROS) and oxidative damage. However, the mechanisms by which UCP3 attenuates ROS production are not well understood. Here we report that UCP3 interacts with the non-processed form of thioredoxin 2 (Trx2), a redox protein that is localized in mitochondria, but not processed Trx2, which is involved in cellular responses to ROS. The hydrophilic sequences within the N-terminal tail of UCP3, which faces the intermembrane space, are necessary for binding to Trx2. In addition, Trx2 directly associated with UCP3 through a mitochondrial targeting signaling sequence, was processed in the intermembrane space, and thereby allowing redox reactions. A bimolecular fluorescence complementation analysis demonstrated that the interaction of these proteins occurs in the mitochondrial intermembrane space. Furthermore, increased UCP3 expression significantly attenuated ROS production in isolated mitochondrial without effects on membrane potential, however this effect is lost by Trx2 knock down. These results suggest that UCP3 binds to Trx2 in the mitochondrial intermembrane space and attenuates ROS production."}
+{"text": "A transcription factor called Promyelocytic Leukemia Zinc Finger (PLZF) calibrates the balance between spinal cord progenitor maintenance and differentiation by enhancing their sensitivity to mitogens that are present in developing embryos. Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment. The embryonic spinal cord is organized into an array of discrete neural progenitor domains along the dorsoventral axis. Most of these domains undergo two periods of differentiation, first producing specific classes of neurons and then generating distinct populations of glial cells at later times. In addition, each of these progenitors pools exhibit marked differences in their proliferative capacities and propensity to differentiate to produce the appropriate numbers and diversity of neurons and glia needed to form functional neural circuits. The mechanisms behind this regional control of neural progenitor behavior, however, remain unclear. In this study, we identify the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) as a critical regulator of this process in the chick spinal cord. We show that PLZF is initially expressed by all spinal cord progenitors and then becomes restricted to a central domain, where it helps to limit the rate of neuronal differentiation and to preserve the progenitor pool for subsequent glial production. We also demonstrate that PLZF acts by promoting the expression of Fibroblast Growth Factor (FGF) Receptor 3, thereby enhancing the proliferative response of neural progenitors to FGFs present in developing embryos. Together, these findings reveal a novel developmental strategy for spatially controlling neural progenitor behavior by tuning their responsiveness to broadly distributed growth-promoting signals in the embryonic environment. The formation of neural circuits within the developing central nervous system (CNS) depends upon the spatially and temporally ordered generation of distinct classes of neurons and glia from multipotent neural stem and progenitor cells (NPCs). An essential feature of this progression is the ability of NPCs to self-renew in a manner that permits early-born cells such as neurons to form while maintaining a sufficient progenitor pool to generate later-born cell types such as glia. At the heart of this process is the interplay between mitogenic signals from the extracellular environment and cell intrinsic factors, which integrate this information to permit either progression through the cell cycle or the onset of terminal differentiation These features of NPCs are exemplified in the developing spinal cord, where many extrinsic and intrinsic factors regulating progenitor maintenance and differentiation have been characterized. In the early neural plate and tube, NPCs are organized in a proliferative neuroepithelial sheet and sustained by the mitogenic actions of several growth factors, particularly Fibroblast Growth Factors (FGFs). FGFs are broadly present in neural tissues and the surrounding mesoderm and act through receptor tyrosine kinases (FGFRs) expressed by NPCs throughout the course of neural development As development proceeds, NPCs become increasingly poised to undergo terminal differentiation through the actions of retinoids, which activate the expression of homeodomain and bHLH transcription factors such as PAX6 and OLIG2. These factors participate in the dorsoventral patterning of NPCs and promote the accumulation of proneural bHLH proteins needed to trigger cell cycle exit and neuronal differentiation The period of neurogenesis in the spinal cord is relatively brief, lasting for only a few days in chick and mouse development, after which time undifferentiated NPCs up-regulate expression of the pro-glial transcription factors SOX9 and NF1A, and begin to give rise to astrocyte and oligodendrocyte precursors Olig2 mutant mice. Olig2+ NPCs exhibit limited capacity for self-renewal, suggesting that Olig2 represses genes that promote NPC proliferation Zbtb16, which encodes the Promyelocytic Leukemia Zinc Finger (PLZF) transcription factor, as one of the most prominently elevated genes in the absence of Olig2 function  and e3 (HH stage 17)  stage 10] in a subset of SOX2tage 17) \u2013C, G\u2013H. + progenitors and is down-regulated as cells express the proneural transcription factor NEUROG2 and begin to form differentiated interneurons marked by LHX1/5 and NEUN staining , progenitors in the intermediate spinal cord up-regulate the expression of the early glial fate determinants SOX9 and NF1A, and begin to transform into astrocyte progenitors s emerge . Togethes emerge .Since PLZF is associated with stem and progenitor cell maintenance in other tissues, we sought to examine whether its function plays a comparable role in the developing spinal cord. We first investigated the consequences of elevating PLZF activity on NPC maintenance and neuronal differentiation. Expression vectors encoding PLZF and an IRES-nuclear Enhanced Green Fluorescent Protein (nEGFP) reporter cassette under the control of the cytomegalovirus enhancer-chick beta actin promoter were electroporated into the chicken spinal cord, and embryos collected 2 d later to assess changes in neuronal differentiation. In spinal cords electroporated with the empty vector, \u223c60% of transfected cells expressed NPC markers such as SOX2 while the remaining \u223c40% expressed neuronal markers such as NEUN . When PLASCL1 and NEUROG1 mRNA and a \u223c28% decrease in the number of NPCs expressing NEUROG2 protein relative to spinal cords transfected with the control vector  along with an IRES-nEGFP reporter cassette to identify the transfected cells. Electroporation of this construct into the spinal cord reduced PLZF levels to nearly background staining levels (HES5-2 and ID2 (+ or TUJ1+ neurons formed after PLZF knockdown (unpublished data). Perhaps accounting for this result, we found that PLZF loss was associated with a \u223c2-fold increase in the frequency of cells undergoing apoptotic cell death measured by activated CASPASE3 staining , which possess a nonsense mutation in the PLZF coding sequence that ablates its DNA binding function Lu/LuZbtb16 mutant mice (unpublished data). However, using a panel of lineage-restricted makers on the spinal cords from Lu/LuZbtb16 mutant and control littermates, we found that differentiation was increased among some interneurons whose progenitors normally express high levels of PLZF  and p2  central progenitor domains were reduced by \u223c12% in the Lu/LuZbtb16 mutants, and this decrease was mirrored by a >14% rise in the number of dI6, V1, V2a, and V2b interneurons distinguished by their expression of specific transcription factors including Bhlhe22 (Bhlhb5), Foxp2, Vsx2 (Chx10), and Gata3  or PLZF-transfected (\u223c5%) embryos  form of FGFR1 that forms nonfunctional heterodimers with FGFR3 and other FGFRs and blocks their downstream signaling activity ed cells . In conted cells .+ cells seen after electroporation with shPLZF alone  and ETV4 (PEA3), as well as feedback inhibitors of the pathway such as STAT3 is a noncanonical effector of FGF signaling +highFGFR3 intermediate region of the spinal cord compared to the PLZF\u2212lowFGFR3 dorsal spinal cord. For this analysis, PAX6 was used to monitor NPCs in place of SOX2. The extent of PAX6 expression completely overlaps with SOX2, and the high versus low levels of PAX6 in the intermediate and dorsal spinal cord served as a convenient proxy for assessing the presence or absence of PLZF  that can override the endogenous transcriptional repressor functions of PLZF and potentially other BTB/POZ proteins than with shRNA constructs selectively targeting PLZF. To date, we have identified five additional family members with expression in the developing spinal cord, suggesting there may be complementary functions with PLZF (Z.B.G. and B.G.N. unpublished observations). A comparable situation exists within the hematopoietic lineage where the lack of a prominent phenotype in either PLZF-null mice or in cell lines transfected with PLZF targeting shRNA is attributed to the presence of other BTB/POZ proteins such as the closely related FAZF The FGF pathway also receives inputs from other signaling networks and has extensive feedback regulatory mechanisms The growth and morphogenesis of the nervous system depends upon the ability of the FGFs to promote the rapid proliferation of NPCs and block neuronal differentiation highFGFR3 NPCs in the intermediate spinal cord display a heightened response to ectopically expressed FGF8 compared to their lowFGFR3 dorsal counterparts  In summary, PLZF and FGFR3 work in parallel with other FGFR, mitogen signaling pathways, and, most likely, other members of the BTB/POZ family, to modulate proliferation in the spinal cord and thereby permit NPCs to differentiate at characteristic rates and times in development. PLZF facilitates the mitogenic activity of the FGFs, which act in a STAT3-dependent manner to maintain a specific population of NPCs in a proliferative state and ensure that the necessary number of progenitors is available for the transition from neurogenesis to gliogenesis. Aberrant activation of FGFR3 and STAT3 has been observed in a multitude of human cancers Drosophila Engrailed repressor domain 5\u2032-cgcagctgagatcctagaaata-3\u2032 and 5\u2032-ttcagcctgaagcaccagctgg-3\u2032) into the plasmid pCIG-shRNA Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG CreOlig2GFPOlig2Fertilized chicken eggs were acquired from AA Lab Eggs, Inc. and McIntyre Poultry and Fertile Eggs. Eggs were incubated at 37\u00b0C and 60% humidity, staged, and electroporated with plasmid vectors as previously described 2, equilibrated overnight in 30% sucrose, frozen on crushed dry ice in OCT mounting media (Sakura Tissue-Tek), and cryosectioned. Prior to staining, slides were fixed in 4% paraformaldehyde for an additional 10 min at 4\u00b0C and then rinsed twice in PBS containing 2 mM MgCl2, for 10 min per wash. Slides were overlaid with 1 mL of X-Gal Staining Buffer  and placed in a humidified chamber at 37\u00b0C for several hours to overnight. Once signal had developed, slides were repeatedly rinsed in PBS with 2 mM MgCl2, coverslipped, and imaged using brightfield microscopy.Dissected tissue was briefly fixed in 4% paraformaldehyde at 4\u00b0C, rinsed repeatedly in PBS containing 2 mM MgClt test using GraphPad Prism 5.0 software.All images were collected using either a Zeiss Observer D1 microscope equipped with an Apotome optical imaging system or a Zeiss LSM5 Exciter confocal imaging system. Images were processed using Zeiss Axiovision and LSM Exciter software suites and Adobe Photoshop. Pixel intensity analysis of mRNA and protein expression was performed using NIH ImageJ software. Cell counts were performed manually and in most cases represented as the mean value of multiple images of tissue sections collected from several independent specimens as indicated in the figure legends. For in ovo electroporation experiments, all transfected cells were counted for each image analyzed, with, on average, 100 cells per image in experiments focusing on the ventral region of the spinal cord at e4 (HH 21), more than 350 transfected cells per image in experiments with an e5 (HH 25) endpoint, and over 900 cells per image in experiments with an e15 (HH 41) endpoint. Unless stated otherwise, results are expressed as fractional changes normalized to electroporation with empty vector controls set to a value of 1.0. The statistical significance of differences observed between experimental and control groups were assessed with the Student's Figure S1Olig2 mutant mice and demarcates neural progenitors in the developing mouse spinal cord.  Expression of Zbtb16 mRNA and PLZF protein in wild-type e10.5 mice. PLZF is broadly expressed by Sox2+ progenitors, including Olig2+ motor neuron progenitors, but absent from differentiated Isl1/2+ motor neurons. (C) PLZF expression is elevated in the ventral spinal cord of e10.5 Cre/CreOlig2 mice. Microarray expression profiling revealed that Zbtb16 mRNA levels were 2.86-fold elevated in Olig2 mutants, p\u200a=\u200a0.00126 (unpublished data), and comparable changes in PLZF protein staining are seen using immunohistochemistry. (F\u2013O) Analysis of wild-type mouse embryos at e9.5 and e11.5 shows that the pattern of PLZF expression is similar to that observed in chicken embryos. PLZF is initially expressed by all Sox2+ progenitors and then becomes restricted to a central domain bordered by Msx1 and Olig2 expression. PLZF is subsequently down-regulated as cells differentiate into TuJ1+ neurons. (P\u2013U) PLZF and Fgfr3 mRNA expression are highly overlapping at multiple stages of mouse development. Serial sections of e9.5, e11.5, and e12.5 spinal cords are shown.PLZF is increased in (TIF)Click here for additional data file.Figure S2GFAP expression.  Stable electroporation of chick embryos at e3 with CMV::PLZF expression vectors using the Tol2 transposon system does not lead to any change in the expression of either SOX9, NF1A, or GFAP when analyzed at e7.PLZF expression precedes the appearance of early glial progenitor markers, but its misexpression does not alter the normal course of their onset. (A\u2013J) Antibody costaining analysis of PLZF and two early markers of glial progenitor fate, SOX9 and NF1A. SOX9 expression in the chick commences on e4, whereas NF1A appears later at e5\u2013e6 (TIF)Click here for additional data file.Figure S3HES gene expression or dorsoventral pattern. (A\u2013B) Spinal cords transfected with PLZF did not exhibit any significant alteration in the mRNA expression of two of the principal Notch effector genes, HAIRY1 and HES5-2. Insets show the extent of transfection in the corresponding sections marked by the presence of nEGFP protein. (C\u2013D) PLZF+ cells in the intermediate spinal cord of e5 (HH 25) chick embryos express high levels of PAX6 protein (blue brackets). However, PLZF is largely absent from dorsal progenitors that express low levels of PAX6 protein (yellow brackets). (E\u2013H) PLZF misexpression does not alter the expression of the homeodomain proteins IRX3, PAX6, PAX3, or PAX7 that demarcate the boundaries of progenitor domains in the developing spinal cord. All electroporations were carried out at e3 (HH 17) and collected for analysis on e5 (HH 25).PLZF misexpression does not lead to changes in (TIF)Click here for additional data file.Figure S4Zbtb16) gene, which lacks the sites targeted by the shPLZF construct. (M) Chart displays the mean ventricular zone area \u00b1 SEM for embryos electroporated with the indicated plasmids relative to the untransfected contralateral sides of the spinal cord. Blue dotted lines demarcate the border of the contralateral VZ in each image. (N) Chart displays the mean number of transfected NPCs expressing NEUROG2 \u00b1 SEM, relative to empty vector controls. All electroporations were performed at e2 (HH 10) and collected at e4 (HH 21). In all panels, ***p<0.001 and ****p<0.0001. Counts were based on at least 12 images taken from \u22658 electroporated embryos.PLZF knockdown can be rescued by the coexpression of human PLZF. (A\u2013C) Electroporation of e3 (HH 17) chick spinal cords with a vector encoding PLZF shRNAs and an IRES-nEGFP transfection marker reduced endogenous PLZF protein expression at e5 (HH 25) by 93.7\u00b11.29%. Chart displays the mean pixel intensity of PLZF antibody staining \u00b1 SEM for spinal cords electroporated with the control or PLZF shRNA constructs, relative to PLZF expression on the nontransfected contralateral control sides. (D\u2013F) Electroporation with a vector producing a nontargeting control shRNA does not alter PLZF, SOX2, or NEUROG2 expression. (G\u2013L) The effects of PLZF knockdown on SOX2 and NEUROG2 expression are rescued by coelectroporation with an expression construct encoding the human PLZF ((TIF)Click here for additional data file.Figure S5Lu/LuZbtb16 (PLZF) mutant mice. The number of cells found in the PLZF-expressing p1 and p2 domains, though not the p0 domain, were significantly decreased in Lu/LuZbtb16 mutants, while progenitors in the adjacent pMN domain that does not normally express high levels of PLZF were unaffected. Each of these progenitor pools was identified by both the absence of the early neuronal differentiation marker Sox11  and the presence of specific patterning markers (AA). The pMN was distinguished by the expression of Olig2, the p2 by the expression of Nkx6.1 dorsal to Olig2+ cells, the p1 domain as being situated between zones of Nkx6.1 (p2) and Dbx1 (p0) expression, and p0 by the expression of Dbx1. (M\u2013X) The number of dI6, V1, V2a, and V2b neurons, which are normally derived from PLZF+ progenitors (Lu/LuZbtb16) mice. However, neurons that are not associated with PLZF+ progenitors, such as dI3 interneurons and motor neurons, are not changed.  Charts displaying the mean number of cells expressing the indicated progenitor or neuronal markers \u00b1 SEM relative to WT and Lu/+Zbtb16 littermate controls. Results are representative of >10 images collected from at least five embryos of each genotype. In all panels, *p<0.05, **p<0.01, and ***p<0.001. (AA) Summary of the transcription factors that define specific progenitor (p) pools and their neuronal progeny in the developing spinal cord.Reduced progenitor pools and excessive neuronal production in PLZF deficient mice. (A\u2013L) Several progenitor pools that express PLZF are reduced in genitors , are inc(TIF)Click here for additional data file.Figure S6FGFR and SPROUTY genes in the wild-type and PLZF-electroporated spinal cord. (A\u2013F) Analysis of FGFR1, FGFR2, and FGFR3 mRNA expression in e4 (HH 21) and e5 (HH 25) chick spinal cords. FGFR4 was not present in the spinal cord at any stage examined (unpublished data). (G\u2013I) PLZF misexpression at e3 (HH 17) increases FGFR3 expression in the e5 (HH 25) dorsal spinal cord, but does not alter the expression of either FGFR1 or FGFR2.  At e5 (HH 25), neither SPRY1 nor SPRY2 are expressed in the intermediate spinal cord where FGFR3 levels are normally high (C), nor were they elevated following PLZF misexpression at e3 (HH 17). (L) FGF2 mRNA is expressed by scattered cells throughout the e5 (HH 25) chick spinal cord.Expression of (TIF)Click here for additional data file.Figure S7STAT3 is expressed throughout the VZ of the e5 (HH 25) chick spinal cord. (F\u2013I) Both PLZF and FGFR3 misexpression at e3 (HH 17) increase the activity of a cotransfected STAT3 responsive-LacZ reporter construct when assessed at e4 (HH 21), suggesting that elevated FGF signaling can stimulate the activity of the STAT3 pathway. Results in (I) are represented as the mean activity of the STAT3-LacZ reporter \u00b1 SEM seen following PLZF or FGFR3 misexpression, relative to the activity of the reporter transfected with control plasmids. Counts were based on at least 10 images taken from 8\u201310 electroporated embryos. ****p<0.0001.PLZF and FGFR3 promote NPC maintenance through the STAT3 pathway. (A\u2013D) ERK1/2 phosphorylation is not observed in the central spinal cord of wild-type embryos or those electroporated with expression constructs producing constitutively active (ca) FGFR1, caFGFR3, or PLZF. (E) (TIF)Click here for additional data file.Figure S8p<0.0001.Coexpression of PLZF and FGF8 disrupts neuronal differentiation in a manner that recapitulates the expression of a constitutively activated form of FGFR3. (A\u2013O) The coexpression of PLZF with FGF8 leads to a significant expansion in the VZ marked by PAX6 expression. Effects were seen in both the dorsal spinal cord (yellow brackets and associated panels) and intermediate spinal cord (blue brackets and associated panels). This phenotype was fully penetrant and ranged from moderate (A\u2013E) to extremely severe (K\u2013O).  Misexpression of caFGFR3 increases the proportion of transfected cells expressing SOX2, similar to the effects seen with the concomitant misexpression of PLZF and FGF8 (A). (R) Chart displays the mean number of caFGFR3-transfected cells expressing SOX2 \u00b1 SEM, relative to transfection with an empty control vector. All electroporations were performed at e3 (HH 17) and collected at e5 (HH 25). Counts were based on at least 10 images taken from \u22658 embryos. ****(TIF)Click here for additional data file.Table S1Antibodies used for immunohistochemistry.(DOCX)Click here for additional data file.Table S2PCR primers used to create in situ probes.(DOCX)Click here for additional data file."}
+{"text": "Histone modification plays an important role in cell differentiation and tissue development. A recent study has shown that the dimethylation of lysine 4 residue on histone 3 (H3K4me2) marks the gene body area of tissue specific genes in the human CD4+ T cells and neural cells. However, little is known of the H3k4me2 distribution dynamics through the cell differentiation and tissue development.We applied several clustering methods including K-means, hierarchical and principle component analysis on H3K4me2 ChIP-seq data from embryonic stem cell, neural progenitor cell and whole brain of mouse, trying to identify genes with the H3K4me2 binding on the gene body region in different cell development stage and study their redistribution in different tissue development stages.A cluster of 356 genes with heavy H3K4me2 labeling in the gene body region was identified in the mouse whole brain tissue using K-means clustering. They are highly enriched with neural system related functions and pathways, and are involved in several central neural system diseases. The distribution of H3K4me2 on neural function related genes follows three distinctive patterns: a group of genes contain constant heavy H3K4me2 marks in the gene body from embryonic stem cell stage through neural progenitor stage to matured brain tissue stage; another group of gene have little H3K4me2 marks until cells mature into brain cells; the majority of the genes acquired H3K4me2 marks in the neural progenitor cell stage, and gain heavy labeling in the matured brain cell stage. Gene ontology enrichment analysis also revealed corresponding gene ontology terms that fit in the scenario of each cell developmental stages.We investigated the process of the H3K4me2 mark redistribution during tissue specificity development for mouse brain tissue. Our analysis confirmed the previous report that heavy labeling of H3K4me2 in the downstream of TSS marks tissue specific genes. These genes show remarkable enrichment in central neural system related diseases. Furthermore, we have shown that H3K4me2 labeling can be acquired as early as the embryonic stem cell stage, and its distribution is dynamic and progressive throughout cell differentiation and tissue development. Post-translational modifications of histones play important roles in regulating DNA activities and gene expressions in eukaryotic cells -4. In thth position on the N-terminal tail of histone 3) is an important histone mark and has been shown to bind to both gene promoter regions and enhancer regions and its binding is often associated with gene activation. A recent study demonstrated that in human CD4+ T cell and neural tissue, H3K4me2 shows specific binding patterns on the tissue-specific genes in their transcribed regions [H3K4me2  or group 3 (with heavy H3K4me2 acquired in NP stage). In addition, GO function analysis also suggests that groups 1 and 3 genes are more involved in essential neural functions such as transmission of nerve impulse and synaptic transmission while the group 2 (with heavy H3K4me2 only acquired in the latest stage) are more involved in developing and maintain synapse structures as a mature brain function. These observations imply that these disease-related genes are more involved in the essential neural tissue functions than in the advanced brain activities.Besides H3K4me2, many other histone marks as well as epigenetic events such as DNA-methylation are also involved and more insight about the differentiation process can be gained with more data available. In addition, the brain tissue data in this study is obtained from whole brain sample even though it is well known that different anatomical and functional regions of the brain have distinctive gene expression patterns and more refined analysis of region and function specific genes could be identified using similar approach as in this paper.In summary, we investigated the process of the H3K4me2 mark redistribution during tissue specificity development for mouse brain tissue. Our analysis confirmed the previous report that heavy labeling of H3K4me2 in the downstream of TSS marks tissue specific genes. These genes are not only highly enriched with neural system functions and pathways, but also highly involved in CNS diseases. Furthermore, we have shown that such labeling can be acquired as early as the embryonic stem cell stage, and its distribution is dynamic throughout cell differentiation and tissue development. The distribution of H3K4me2 on neural function related genes follows three distinctive patterns: a group of genes contain constant heavy H3K4me2 marks in the gene body from ES through NP to matured brain tissue stage; another group of gene have little H3K4me2 marks until cells mature into brain cells; the majority of the genes acquired H3K4me2 marks in the NP stage, and gain heavy labeling in the matured brain cell stage. GO enrichment analysis also revealed corresponding GO terms that fit in the scenario of each cell developmental stages.Three H3K4me2 ChIP-seq datasets are downloaded from NCBI Gene Expression Omnibus with accession number GSE11172 including murine embryonic stem (ES) cells, ES-derived neural progenitor (NP) cells, and whole brain (WB) tissues . The 36-In WB tissue, the cluster with an elevated H3K4me2 marking downstream of TSS was further divided into four clusters using K-means  to obtain a cluster of 356 genes with highly elevated H3K4me2 labeling downstream of TSS. This set of genes was compared with clusters obtained from ES and NP samples, and largely overlapped genes and clusters were further subject to GO enrichment analysis using the MetaCore software as well as the NIH DAVID tool.http://www.genego.com/metacore.php), as well as Database for Annotation, Visualization and Integrated Discovery . Principal Component Analysis (PCA) was performed on the same combined data using Matlab code.At the same time, the combined TSS profiles of the 356 genes from all three origins were clustered using hierarchical clustering in Matlab and three clusters were selected based on visual inspection: the cluster with constant H3K4me2 labeling from ES through NP to matured brain; the cluster with minimal H3K4me2 labeling in ES and NP stage, but gets heavy H3k4me2 labeling in brain tissue; and the cluster with minimal H3K4me2 labeling in ES stage, but gradually gains H3K4me2 labeling as the cells differentiate into NP cells and mature as brain tissue. Each cluster of genes was subject to gene set enrichment and network analysis using bioinformatics software MetaCore\u2122 by GeneGo (H3K4me2: dimethylation of lysine 4 residue on histone 3; ES: embryonic stem; NP: neural progenitor; WB: whole brain; GO: gene ontology; TSS: transcription start site; GnRH: Gonadotropin-releasing hormone; ChIP-seq: chromatin immunoprecipitation sequencing; CNS: central nerve system; PCA: principal component analysisThe authors declare that they have no competitng interests.JZ performed all the analysis and wrote the manuscript. JDP participated in the discussion and manuscript editing. KH designed the study and participated in the writing of the manuscript."}
+{"text": "There was a formatting error in Table 5 in the PDF. On page 8, an incorrect heading was given for the second page of Table 5. \"(A) Comparison of sequencing based point variants and variants between references\" should be \"(B) Coding SNP summary\"."}
+{"text": "Nosocomial infections in patients admitted in coronary intensive care unit (CCU) are frequently caused by potentially pathogen microorganism (PPM). The aim of the present study is 1) to determine the incidence of PPM in patients admitted in our CCU the last year 2) to identify the risk factors for colonization with PPM.Electronic medical records of all patients without previous infection who hospitalized in CCU unit from January since December 2010 were reviewed. During hospitalization, specimens were taken from the nasopharynx, blood and urine cultures and if applicable from the central or peripherals lines.49 patients were included in the study with mean age 63,73yrs (SD=15.45). 64% of the participants were colonized with PPM. The most common isolated pathogens were Staph.Epidermidis (36.7%), Klebsiella Pneumoniae (32.6%), Pseudomonas Aeruginosa (10.2%), Candida Albicans (8.2%) \u03ba\u03b1\u03b9 MRSA (4.1%). Risk factors for colonization with PPM were found the duration of stay in CCU  and the high levels of urea and creatinine .The rates of PPM were significant high. Proper attention should be given in the risk factors that were found to be correlated.None declared."}
+{"text": "Cigarette smoking has been shown as major environmental risk factor for rheumatoid arthritis (RA). Epidemiological studies indicate an association of cigarette smoking with development of RA ,2, althoRASF obtained from patients undergoing joint replacement surgery were stimulated with freshly prepared cigarette smoke extract (CSE) for 24 hours. Expression of HDACs was measured at the mRNA level by Real-time TaqMan and SYBR green PCR and at the protein level by immunoblot analysis. Global histone 3 (H3) acetylation was analyzed by immunoblot.Stimulation of RASF (n = 8-10) with CSE significantly enhanced the expression of HDAC1 , HDAC2  and HDAC3  at the mRNA level while the expression of HDAC 4-11 remained unchanged. On the protein level, expression of HDAC1 and HDAC3 were not altered, whereas the expression of HDAC2 protein was decreased in CSE stimulated RASF. No measurable changes in global acetylation of H3 were induced by CSE in RASF (n = 6).CSE specifically downregulates the expression of HDAC2 in RASF. Differential regulation of HDAC2 at the mRNA and protein level points to post-transcriptional degradation mechanisms induced by smoking. Even though global H3 acetylation was not changed by CSE, decreased HDAC2 levels might be associated with hyper-acetylation and thus increased expression of specific HDAC2 regulated genes."}
+{"text": "Oligomannose sugars on gp120 are of significance to HIV vaccine design because of the description of the broadly neutralizing mAb 2G12 and, more recently, of the potent and broadly neutralizing \u2018PGT\u2019 series of mAbs, which bind with high affinity to clusters of oligomannose moieties on gp120. Attempts to elicit oligomannose-specific antibodies have focused mainly on immunizing with antigenic clusters of synthetic oligomannose or natural oligomannose. These strategies have had limited success, suggesting that alternative approaches are needed. Here, we present the surface lipo-oligosaccharide (LOS) of Rhizobium radiobacter Rv3 that antigenically mimics the 2G12 epitope, as one potential new avenue of exploration.The chemical structure of the Rv3 OS was determined by NMR and its antigenic similarity to the 2G12 epitope determined by ELISA and X-ray crystallography. Immunogenicity of the Rv3 OS and its ability to elicit antibodies of 2G12-like specificity was assessed by immunizing mice with heat-killed Rv3 bacteria.The detailed chemical analysis of the Rv3 OS revealed that its carbohydrate backbone consists of a unique tetramannose backbone that is analogous to the D1 arm of mammalian oligomannose. 2G12 bound with at least similar affinity to purified Rv3 OS as reported for oligomannose. The 2.4 \u00c5-structure of the 2G12:Rv3 OS complex shows that 2G12 contacts all four mannosyl moieties in the Rv3 backbone that mimic the D1 arm, with the majority of contacts occurring with the terminal mannose disaccharide. Antibodies elicited by immunizing with heat-killed bacteria bound a synthetic tetramannose epitope and monomeric gp120.Although the elicited antibodies failed to exhibit the desired neutralizing activity, our data suggest that presentation of an antigenic analog of mammalian oligomannose in a bacterial context presents a novel avenue for pursuing immunogens to elicit oligomannose-specific HIV neutralizing antibodies. The development of Rv3-based bioconjugates using human carbohydrate vaccine carriers is ongoing."}
+{"text": "In postnatal skin the transcription factor Sox2 is expressed in the dermal papilla (DP) of guard/awl/auchene hair follicles and by mechanosensory Merkel cells in the touch domes of guard hairs. To investigate the consequences of Sox2 ablation in skin we deleted Sox2 in DP cells via Blimp1Cre and in Merkel cells via K14Cre. Loss of Sox2 from the DP did not inhibit hair follicle morphogenesis or establishment of the dermis and hypodermis. However, Sox2 expression in the DP was necessary for postnatal maintenance of awl/auchene hair follicles. Deletion of Sox2 via K14Cre resulted in a decreased number of Merkel cells but had no effect on other epithelial compartments or on the dermis. The reduced number of Merkel cells did not affect the number or patterning of guard hairs, nerve density or the interaction of nerve cells with the touch domes. We conclude that Sox2 is a marker of two distinct lineages in the skin and regulates the number of differentiated cells in the case of the Merkel cell lineage and hair follicle type in the case of the DP. \u2022Sox2 is a marker of two distinct lineages in the skin.\u2022Sox2 is required for postnatal maintenance of awl/auchene hair follicles.\u2022Loss of Sox2 results in a reduction in Merkel cells. The transcription factor Sox2 is involved in maintenance of the early, pluripotent stem cells of the eipiblast  and in rSox2 is expressed in the dermal papilla cells of guard/awl/auchene hair follicles  and in tWithin the epidermis Sox2 is expressed in a small population of mechanosensory cells known as Merkel cells . These nIn view of the key contributions of DP cells and Merkel cells to skin function and the observation that Sox2 is a marker of SKPs, we have investigated the consequences of deleting Sox2 in the DP and Merkel cell compartments.All experiments were approved by King's College London, Cambridge University and Cancer Research UK local ethics committees and performed under the terms of a UK government Home Office licence. Sox2fl/fl mice, in which flox sequences flank the Sox2 locus , were kiFlow cytometry was performed on dermal preparations as described previously  using a Gating criteria were as follows. Debris was gated out using forward and side scatter plots. Doublets and dead cells were also gated out and analysis was performed on live cells using GFP and APC channels. Gating for positively labelled cells was performed against negative control samples to less than 0.5% background.Preparation and immunostaining of conventional cryosections (5\u201330\u00a0\u03bcm thick) and whole mounts of tail epidermis, back skin and whisker pad were performed as described previously . Back skThe following primary antibodies were used at the dilutions indicated: Sox2 1:100 (R&D Systems), CD133 1:50 (eBioscience), Itga8 1:200 (R&D Systems), Dcc 1:100 (R&D Systems), K14 1:1000 (Covance), Blimp1 1:50 (eBioscience), Corin 1:100 (R&D Systems), K8 1:100 , NF-H 1:100 (Millipore), PGP9.5 1:100 (Dako), V-GLUT2 1:500 (Invitrogen), Rab3c 1:3000 (Abcam), synapsin II 1:200 (Abcam), Cav2.1 1:100 (Millipore), piccolo 1:2000 (SYSY) and GFP 1:500 (Invitrogen).Images of back skin whole mounts immunostained for PGP9.5 were acquired from the epidermal side using a confocal microscope. The Metamorph software tube formation application was used to quantitate the percentage of total skin area covered by nerves.Back skin was collected from E18.5 mice. Sample processing and image capture were carried out by Dr Jeremy Skepper, Anatomy Department, Multi-Imaging Centre, University of Cambridge. Tissues were fixed in 4% glutaraldehyde in 0.1\u00a0M HEPES buffer at pH 7.4 for 12\u00a0h at 4\u00a0\u00b0C and rinsed 5 times in 0.1\u00a0M HEPES buffer. Tissues were then treated with 1% osmium ferricyanide at room temperature for 2\u00a0h, rinsed 5 times in distilled water and treated with 2% uranyl acetate in 0.05\u00a0M maleate buffer at pH 5.5 for 2\u00a0h at room temperature. Tissues were rinsed in distilled water and dehydrated in an ascending series of ethanol solutions from 70% to 100%. This was followed by treatment with two changes of dry acetonitrile and infiltration with Quetol epoxy resin. Images were taken with a FEI Tecnai G2 operated at 120\u00a0Kv using an AMT XR60B digital camera running Deben software.E18.5 embryos were harvested from Blimp1Cre+/Sox2fl/wt\u00d7Sox2fl/fl intercrosses. Skin was dissected from the embryos and surgically sutured onto the backs of adult NSG mice. Sutures were removed after 1 week and grafts were harvested after 10 weeks.The number of Merkel cells per touch dome was quantitated in 7 mice per genotype. For each biological replicate 3 areas were analysed, corresponding to a total of 30 touch domes per mouse. Nerve density was analysed in 8 biological replicates, with 3 areas per mouse. Innervation of Merkel cells , touch domes per unit area and guard hairs per unit area were analysed in 4 biological replicates per genotype. In all cases data from the biological replicates were pooled. The D\u2019Agostino and Pearson omnibus test was used to examine normality of data distribution. Since none of the data followed a Gaussian distribution, a non-parametric Mann-Whitney test was applied to examine the significance of differences between datasets.In order to delete Sox2 in the DP we required a promoter that would drive Cre expression in the dermis at the appropriate stages of development. One candidate was the promoter of the transcription factor B lymphocyte induced maturation protein (Blimp1). Blimp1 has previously been shown to be required for maintenance but not initial specification of DP progenitors .To examine Blimp1 expression in developing skin we performed immunostaining with Blimp1 antibodies and also examined expression of GFP under the control of the Blimp1 transcriptional regulatory regions . Blimp1 Analysis of Blimp1 mRNA expression levels in previously published microarray datasets  revealedBlimp1+ cells have previously been shown to give rise to mature DP, the dermal sheath, and the upper (papillary) dermis . We therFlow cytometry showed that 67\u201375% of CD133+ cells in E18.5 and P2 dermis were also GFP+ H and I, The lineage relationship between dermal papilla, dermal sheath, and cells in the papillary region has not been clearly defined. Our results suggest that these cell types share a common embryonic origin and may be distinct from other fibroblast populations in the dermis.Our data on Blimp1 expression indicated that expression of Cre under the control of Blimp1 transcriptional regulatory elements could be used to ablate gene expression in all DP at the earliest stages of dermal papilla morphogenesis. Blimp1Cre mice were therefore crossed with Sox2fl/fl mice. Blimp1Cre+/Sox2fl/fl mice were born at the expected Mendelian ratio but died shortly after birth, probably because of Sox2 deletion in the brain. Analysis of Sox2 expression in Blimp1Cre/Sox2fl/fl wholemounts and thick cryosections of skin confirmed efficient ablation of Sox2 in the DP of E16.5 follicles, in contrast to control littermates A\u2013D.Expression of the DP markers CD133, Itga8, Corin and two specific markers of guard/awl/auchene DP, Dcc and Sox10 , was unaTo investigate whether Sox2 plays a role in the postnatal maintenance of hair follicles we grafted E18.5 Blimp1Cre/Sox2fl/fl and control littermate skin onto NSG immunodeficient mice. At the macroscopic level, 10 week old wild type and Sox2 null grafts were indistinguishable A and B. Merkel cells reside in the epidermal basal layer, directly linked to nerve cells containing NF-H positive neurofilaments A and B. Immunostaining for Sox2 and the Merkel cell marker Keratin 8 (K8)  revealed\u2212, littermates had Sox2+ Merkel cells  in K14CreSox2fl/fl skin  did not reveal a significant difference (We next prepared horizontal whole mounts  of E18.5fference F\u2013H. TherMerkel cells are excitatory cells that carry out synaptic processes . TransmiSox2 has long been recognised as a marker of embryonic pluripotency , and recCurrent approaches to modulating gene expression in the dermal papilla include driving Cre expression via dermal papilla specific or pan-fibroblast promoters . We founAblation of Sox2 in the DP did not affect hair follicle morphogenesis but did modulate hair follicle type. Since the decrease in awl/auchene follicles was correlated with an increase in zigzag hairs, rather than a decrease in overall follicle number, it is likely that Sox2 specifies follicle subtype, rather than being required for maintenance of awl/auchene follicles. We have previously shown that Sox2-expressing cells are required for formation of awl/auchene follicles in skin reconstitution assays  and our Our results highlight a distinction between those genes that are required for core DP functions and those that specify the identity of the dermal papillae of different types of follicle . ExampleWhen we ablated Sox2 in the epidermis via K14Cre, the number of Merkel cells per touch dome was reduced, confirming the recent studies of It has been suggested previously that Merkel cells play a role in attracting nerve growth into the skin during embryonic development . In suppSox2 was not required for expression of synaptic and voltage-gated signalling markers or formation of dense core synaptic vesicles in Merkel cells. Given that Sox2 is a well-established neural transcription factor, we speculate that other neural transcription factors are capable of compensating for the loss of Sox2 and maintaining expression of sufficient levels of synaptic proteins for synaptic vesicles to form. Indeed Sox2, which is repressed by the Polycomb complex , acts asIn conclusion, we have shown that Sox2 plays a role in Merkel cell differentiation and in specifying dermal papilla subtypes. Sox2 thus plays a role in lineage commitment of both epithelial and mesenchymal cells within the skin."}
+{"text": "PLoS Genetics sheds light onto how Cdk9 function is interconnected with the monoubiquitination of histone H2B in the fission yeast Schizosaccharomyces pombeTranscriptional regulation in eukaryotes requires the transcriptional machinery to negotiate the complexities of DNA packaged into chromatin. Specific modifications of the core histone proteins serve to regulate transcription and ensure that genes are expressed at the right place and the right time CDK9 controls transcriptional elongation, transcription-coupled mRNA processing, and histone modification One post-translational histone modification emerging as a key player in numerous processes is histone H2B monoubiquitination (H2Bub1). In mammals this modification appears to serve as an important tumor suppressor The new study by Sans\u00f3 et al. has unraveled additional details about the complex dance between the fission yeast ortholog of CDK9  and H2Bub1 S. pombe H2B ubiquitin ligase Brl2 both lead to septation defects, these can be rescued by blocking spCdk9. Furthermore, compound mutants of Spt5 and Set1 suggest that the phenotypic effects of H2Bub1 loss may be due to multifaceted downstream effects of H2Bub1 on both Spt5 (through spCdk9 recruitment) and H3K4me3 (through Set1). Further support for a homeostatic feedback mechanism is provided by studies of RNAPII occupancy in fission yeast with reduced spCdk9 activity. Consistent with opposing roles of H2Bub1 and spCdk9, the authors observed increased RNAPII occupancy in transcribed regions and decreased occupancy at gene 3\u2032 ends. Importantly, the combined mutation of spCdk9 and H2Bub1 rescued the H2B K119R mutation and displayed RNAPII occupancy similar to mutant spCdk9 alone.Does this mean that spCdk9 and H2Bub1 play opposing roles in fission yeast? Phenotypic studies suggest that this may be at least partially true. For example, while H2B K119R mutation or deletion of the These results demonstrate the intricacy and complexity of the choreographed tango between CDK9 orthologs and chromatin, and illustrate significant differences between yeast and metazoans. This study shows that although H2Bub1 requires Cdk9 activity in fission yeast, Bur1 in budding yeast"}
+{"text": "The paternal 5mC is converted in fertilized oocytes to 5hmC allowing for epigenetic reprogramming of sperm DNA. Human tumors contain much lower levels of 5hmC compared to normal tissue. Proliferating cells in a tumor have drastically reduced levels of 5hmC, suggesting that this stable base modification is lost during DNA replication. 5hmC is most abundant in cells of the nervous system. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation and this modification associates preferentially with gene bodies of activated neuronal function-related genes, in which gain of 5hmC is often accompanied by loss of H3K27me3. Importantly, gain of 5hmC is rarely associated with DNA demethylation suggesting that 5hmC is a rather stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet proteins leads to defects in neuronal differentiation suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development. We propose that the function of 5hmC in promoter regions is to \u201crepair\u201d inappropriate de novo DNA methylation but its exact mechanistic role in gene bodies is still unknown."}
+{"text": "IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1\u2013P3 in the human and mouse IGF2 locus. The expression of tammar IGF2 was predominantly from the P2 promoter, similar to humans. Expression of IGF2 was higher in pouch young than in the adult and imprinting was highly tissue and developmental-stage specific. Interestingly, while IGF2 was expressed throughout the placenta, imprinting seemed to be restricted to the vascular, trilaminar region. In addition, IGF2 was monoallelically expressed in the adult mammary gland while in the liver it switched from monoalleleic expression in the pouch young to biallelic in the adult. These data suggest a complex mode of IGF2 regulation in marsupials as seen in eutherian mammals. The conservation of the IGF2 promoters suggests they originated before the divergence of marsupials and eutherians, and have been selectively maintained for at least 160 million years.In mice and humans,  Human IGF2 has five promoters (huP1 and P0\u2013P3) and transcription start sites (TSS) each adjoining distinct 5\u2032 non-coding exons, while rodent IGF2 has only 4 (P0\u2013P3) Monodelphis domestica, detected a single product from total neonatal RNA using 5\u2032 RACE and identified only one 5\u2032 non-coding exon IGF2 isotype from a single promoter.Insulin-like growth factor 2 IGF2 is imprinted in the fetal liver and is paternally expressed from P1, P2 and P3 (orthologous to mouse P1\u2013P3 respectively) with dominant expression from the P2 promoter in the majority of tissues tested IGF2 has continued expression from P1 and P3 in addition to biallelic expression from huP1 (previously reported as P1) Human Igf2 is paternally expressed in the embryo, but biallelically expressed in the choroid plexus and leptomeniges, which are the only major sites of Igf2 gene expression in the adult rodents IGF2-P0 transcript is paternally expressed at high levels in fetal skeletal muscle, in the term placenta and at low levels in all adult tissues except the brain In the mouse P0-derived transcripts are only expressed in the labyrinthine layer of the placenta, whereas transcripts from P1\u2013P3 are found throughout the developing embryo and placenta with dominant expression from the P3 promoter Igf2 domain has 3 differentially methylated regions (DMRs), DMR0 surrounding the area that codes for the placental specific promoter P0, DMR1 located 5\u2032 of P1 and DMR2 located at the second coding exon IGF2 in mesoderm-derived tissues  without disrupting H19 imprinting, whilst maintaining IGF2 imprinting in the endoderm-derived tissues (liver and placenta) IGF2, therefore DMR2 is an activation site The mouse IGF2 control mechanisms amongst the eutherian mammals. Marsupials diverged from the eutherian mammals around 160 million years ago IGF2 is highly conserved and imprinted. However, very little is known about the regulation of this gene in marsupials. Most marsupials have a short pregnancy supported by a much less invasive placenta than typically seen in eutherian mammals, and all give birth to a highly altricial neonate IGF2 gene.The presence of a potential functional equivalent of mouse DMR2 IGF2 is imprinted in the whole bodies of opossum neonates IGF2 shows complete imprinting in the fetal liver, but has paternally biased expression in the placenta and brain and is biallelically expressed adult liver IGF2 in the neonatal opossum IGF2 gene that may account for the biased expression observed in the head and the placenta IGF2. This study investigates the evolution of the IGF2 regulatory regions in a marsupial, the tammar wallaby Macropus eugenii, and identified 3 promoters in the tammar IGF2 region.All experiments and wild animal collection were approved by the University of Melbourne Animal Experimentation Ethics Committee and the animal handling and husbandry procedures were in accordance with the National Health and Medical Research Council of Australia (2004) guidelines. Animals were collected under approval from the South Australian Department of Environment and Natural Resources.Tammar wallabies of Kangaroo Island, South Australia origin were held in our breeding colony in Melbourne. Pregnancy was initiated in females carrying an embryo in diapause by the removal of their pouch young (RPY) 2O  in a dilution of 1/24. Total RNA was DNase treated  to remove contaminating DNA and tested by performing a housekeeping gene PCR. Total RNA quality was assessed by running samples on a gel  and quantified with a nano-spectrometer . cDNA was synthesised using Roche Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Typically 4000 ng of total RNA was used in each cDNA synthesis reaction, with 1.1 \u00b5l of Oligo (dT)18 (50 \u00b5M), trilaminar yolk sac samples for qPCR analysis were amplified using random hexamer primers. All cDNA samples were diluted to 40 uL for a final concentration of 100 ug/ul. cDNA integrity was immediately assessed with GAPDH PCR.Genomic DNA (gDNA) was extracted from approximately 20 mg of snap frozen tissue using a Wizard Genomic DNA Purification Kit (Promega). Total RNA was extracted from liver, placenta and brain tissues using Tri-Reagent (Ambion) as described by the manufacturer, and from mammary glands using RNeasy Lipid Tissue Mini Kit (QIAGEN), with a final elution of RNA in 50\u201380 \u00b5l of RNAsecure HAll primers were designed using Primer3 (v. 0.4.0) IGF2 transcription start sites (TSS) we performed 5\u2032RACE, using both the 5\u2032 RACE System for Rapid amplification of cDNA ends, Version 2.0, (Invitrogen) and SMARTer RACE cDNA Amplification Kit (Clontech). Liver and placental RNA was used. Gene specific primers were designed in the first translated exon (To identify tammar ted exon  and werePCR products were subcloned in to the pGEM-T Easy vector and transformed in to JM109 competent cells (Promega). Plasmids were purified using the Wizard Plus SV Minipreps DNA Purification System (Promega) and sequences were obtained according to standard methods using ABI3130xl capillary genetic analysers, with BDV3.1 terminators.IGF2 transcripts in the brain, liver, mammary gland and placenta. Each sample was extracted and prepared as described above. In the brain expression profile, 1 female and 3 male samples were used in the day 10 pouch young (PY) group and all 3 of the day 50 PY group were male. In the liver, 1 female and 3 male samples were used in the day 10 PY group and 2 female and 2 male samples were used in the day 75 PY group. All of the adult samples were female. As there were no obvious outliers in the data, we decided it was acceptable to use both sexes.Quantitative real-time polymerase chain reaction (qPCR) was used to quantify the relative expression from each of the IGF2 qPCR primers were designed so the reverse primer encompassed the intron exon boundary between exon 2 and 3 and was used with the transcript specific primers  using the following conditions: 95\u00b0C for 10 min, followed by 45 cycles at 95\u00b0C for 15 sec and 61\u00b0C for 30 min and 72\u00b0C for 30 sec. A liver sample triplicate and a negative template triplicate were included on each plate as a calibrator and negative control respectively for each primer combination.PCRs were carried out in triplicates in 20-\u00b5l volumes consisting of FastStart Universal SYBR Green Master (Rox) (Roche), forward and reverse In addition to using the dissociation curve, PCR products from the first plate were run on a gel to confirm amplification of a single band and that the NTC wells were blank. The data was analysed in Microsoft Excel and R statistical package IGF2 allelic expression sample specific genotypes were identified using PCR and direct sequencing of gDNA. Two single nucleotide polymorphisms IGF2 primers for gDNA and transcript specific primers for cDNA  was used to optimise reaction efficiency and only 30\u201335 cycles of PCR were used to prevent saturation. For expression analysis GoTaq Green Master Mix (Promega) was used. PCR cycles consisted of 94 or 96\u00b0C for 2 min, followed by a maximum of 35 cycles of 30 sec at 94 or 96\u00b0C, 30 sec-1 min at 58\u201363\u00b0C, and 30 sec-1 min at 72\u00b0C, and a final extension at 72\u00b0C for 5 min.IGF2 snps. Of these samples 2 adult livers, 3 mammary glands, 4 placentas and 2 pouch young brains and livers were analysed for IGF2 imprinting. PCR products from cDNA and gDNA were resolved by gel electrophoreses and bands extracted . The purified product was then sequenced. Sequences were assessed by eye using the FinchTV (v.1.3.1) DNA sequence chromatogram trace viewer software. The relative peak height for each allele indicates biallelic  or imprinted  expression.Approximately 9 pouch young, 13 adults and 17 placental samples were screened for Using MethPrimer IGF2 gene is highly complex in eutherian mammals. This study confirmed that marsupials also have complex regulation of IGF2, and identified three evolutionarily conserved promoters, each with three distinct non-coding exons that showed tissue specific imprinting and expression in the tammar wallaby.The regulation of the imprinted IGF2, 5\u2032 RACE was performed on mRNA derived from two tissues, liver and placenta. Three distinct bands were amplified from the liver and placental samples, representing three distinct TSS (IGF2 may not have a liver specific promoter orthologous to human HuP1 or a placenta specific promoter orthologous to mouse P0.To determine how many different transcription start sites (TSS) there are in the tammar inct TSS . As addiinct TSS . Each bainct TSS . Three a+1 nucleotide in the Inr motif (where A+1 is the transcription start site) . A TATA-like sequence at -27 relative to the P3 A+1 nucleotide in the Inr motif, similar to the TATA-box observed at the mouse P3 At the mouse and rat P1 there are no TATA, CAT or GC boxes, but there are direct and inverted repeats of various length in the region upstream of the P1 non-coding exon IGF2, pairwise alignments using a 100 bp sliding window were performed in VISTA Using 20 kb genomic sequences of IGF2The opossum non-coding exon was highly conserved in the tammar genome and aligned to the tammar with 92% sequence identity approximately 300 bp upstream of the first tammar exon 1B TSS . This suIGF2 transcription in the liver peaks shortly after birth IGF2 in the tammar is similarly developmental stage-specific; expression is higher in the pouch young, during the time of rapid growth and nutrient transfer from the mother, compared to both fetal and adult stages IGF2 is synthesized in most tissues and its production declines rapidly in rodents soon after birth IGF2 mRNA peaks approximately 10 days after birth IGF2 expression peaks in the male pouch young between 70 and 100 days after birth and in females around 150 days IGF2 in liver was predominantly monoallelic from each of the 3 promoters in pouch young aged 12 and 72 days, and biallelic in both adults tested  identified by 5\u2032RACE are indicated by the arrows. ggaggag repeats are in boxes, CTCCCAAG repeats are highlighted in yellow and TGTCCC repeats are highlighted in green. TATA-boxs (TATAWAAR) are emboldened and red, CCATT and GC-box/Sp1-sites are underlined, Inr elements (YYANWYY) are emboldened and blue and DPE (RGWYV) sequences are emboldened and green. Exon sequences are in capitals. P1 is characterised by multiple repeat sequences. P2 has multiple TSSs and a high number of CCATT and GC-box/Sp1-sites. A TATA-Inr core promoter was identified around the fourth TSS. P3 is an Inr-DPE core promoter. Degenerate nucleotides represented using IUPAC codes.(PDF)Click here for additional data file.Figure S2Alignment of tammar exon 1B region with opossum exon 1 region. Opossum non-coding exon\u200a=\u200a1\u2013420 Tammar exon 1B\u200a=\u200a725\u20131104. Alignments were performed using ClustalW and were highlighted using BOXshade 3.31.(PDF)Click here for additional data file.Table S1Primers used in this study.(PDF)Click here for additional data file."}
+{"text": "Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of  The developing vertebrate limb is widely adopted as a model to study cell-cell signaling, pattern formation and morphogenesis, and has provided a wealth of knowledge of the function and regulation of specific transcription factors and signaling molecules Dlx and Msx homeobox transcription factors, expressed in the AER and the mesoderm of the limb buds, play an important morphogenetic role, although their functions and regulations are yet to be defined at the molecular level. Dlx5;Dlx6 double knock-out (DKO) mice are characterized by loss of the central digit(s) and/or fusion with the lateral ones DLX5;DLX6 region, and for which point mutations in the DNA-binding domain of DLX5 have recently been found Msx1 and Msx2 genes results in moderate-penetrance polydactyly of the forelimbs (FL) and oligodactyly of the hindlimbs (HL) (loss of the anterior digits and part of the tibia in the zeugopod) Proximo-distal limb development and digit extension is directly controlled by the signaling activity of the AER via expression of morphogens of the FGF family Dlx5, Dlx6, Msx1 or Msx2 shows evident limb defects in vitro the Dlx and Msx proteins compete for the same DNA binding sites, form heterodimers via their homeodomain and reciprocally inhibit their transcriptional activities Dlx genes are implicated in the differentiation of specific cell lineages, such as forebrain interneurons Dlx and Msx genes have been shown to cooperate only in specific cases Msx2 transcription in the AER, via homeodomain binding elements present in the gene\u2019s promoter Studies on the cellular and molecular functions of Dlx and Msx proteins during limb development have met with difficulties owing to several reasons. First, none of the single knockout for Dlx5;Dlx6 and Msx1 or Msx2 in limb development, by generating double and triple Dlx;Msx compound mutant mice and analyzing their limb phenotypes. The limb phenotype of Msx1;Dlx5;Dlx6 triple knock-out (TKO) mice shows features of the Msx1;Msx2 DKO phenotype Dlx5;Dlx6 control the expression of Msx2, but not of Msx1. In contrast, the limb phenotype of Msx2;Dlx5;Dlx6 TKO mice is consistent with a severe aggravation of the ectrodactyly defect. We also re-examine the spatio-temporal expression of Dlx and Msx genes in FLs and HLs and observe that in the AER these genes are co-expressed whereas in the anterior mesenchyme Msx expression precedes that of Dlx, ruling out a direct regulation of Msx by Dlx in this territory. Combining these findings with qPCR expression analyses on the limb buds of different compound embryos, and with ChIP data, we propose that two modes of regulation coexist during limb development: 1) a direct transcriptional regulation of Msx2 by Dlx proteins in the AER and, later on, in the limb mesoderm, and 2) a Bmp2- and Bmp4-mediated non-cell autonomous regulatory loop between the AER and the anterior limb mesoderm.We investigate possible interactions between lacZ/+Dlx5 (hereafter named Dlx5+/\u2212); neo/+Dlx5;Dlx6 lacZ/+; Msx1 and lacZ/+Msx2  have been previously reported +/\u2212Msx1 or the +/\u2212Msx2 single heterozygous parents with the +/\u2212Dlx5;Dlx6 double heterozygous ones, and then crossing the triple heterozygotes. Following mating, the day of the vaginal plug was considered as embryonic age 0.5 (E0.5).All animal procedures were reviewed and approved by the Ethical Committee of the University of Torino and University of Genova, the Italian Ministry of Health and the French Minist\u00e8re de l\u2019Enseignement sup\u00e9rieur et de la Recherche. No surgery or other manipulation on adult animals was used in this study. All efforts were made to minimize suffering. Generation and genotyping of the lacZ expression analysis E10.5 embryos were fixed for 15\u201330 min in 2% paraformaldehyde in PBS, while E14.5 embryos were fixed for 15\u201330 min in 4% PFA. X-gal staining was performed as described Cartilage staining (with Alcian Blue) of E14.5 embryos as well as bone and cartilage staining (with Alcian Blue and Alizarine Red) of E18.5 embryos were carried out as previously described in situ hybridization (WMISH), embryos at the desired age were dissected in cold RNAse-free PBS, fixed in cold 4% PFA for 12\u201316 hrs, rinsed with PBS, permeabilized by treatment with proteinase-K, prehybridized and hybridized as previously published Dlx5 probe comprised 780 bp and was linearized with EcoRI and transcribed with T7 RNA polymerase Dlx6 probe is a 350 bp fragment spanning exons 3\u20134 Msx1 probe was a 550 bp 3\u2032 spanning the homeodomain, containing exon 1; the Msx2 probe corresponded to 378 bp in the first exon of Msx2 cDNA, the Fgf8 probe corresponded to most of the mouse coding sequence, the Bmp4 probe (a kind gift from B. Hogan) contains the 3\u2032 UTR and most of the coding sequence from a mouse cDNA Gremlin corresponded to the entire murine coding sequence (a kind gift from R. Zeller). After hybridization, the signal was revealed with the NBT-BCIP chromogenic reaction.For RNA:RNA whole-mount +/\u2212;Dlx5\u2212/\u2212;Dlx6Msx1\u2212/\u2212) biological triplicates could be analyzed. Primer sequences were designed with the Primer Express online tool. RNA quality, primer efficiency and correct size were tested by RT-PCR and agarose gel electrophoresis. Standard curve were performed using WT cDNA with four calibration points: 1\u223610; 1\u223640; 1\u2236160; 1\u2236640. Specificity and absence of primer dimers was controlled by denaturation curves. Rps9 mRNA abundance was used for normalization . Pools of two half limbs from the same embryos were used to extract total RNA, using the Tissue Lyser II reagent followed by elution through RNA micro-kit plus (Qiagen). cDNA synthesis was done using standard conditions, 3 ng of each cDNA sample were used to carry out qPCRs on a CFX96 equipment (Biorad) using the SybrSafe system (Invitrogen). Samples were analyzed in technical triplicates, and for each genotypes (except for the We used the Position-Weight matrix (PWM) provided by JASPAR under accession PH0024.1. The score of a site was computed with standard log-likelihood ratios, using as null model the nucleotide frequencies computed over the whole intergenic fraction of the mouse genome. We considered for further analysis putative sites scoring 50% of the maximum possible score or better. We selected among the sites identified above the ones that are conserved in at least two of 8 vertebrate species (genome sequence version in parenthesis): mouse (mm9), human (hg19), cow (bosTau4), opossum (monDom5), platypus (ornAna1), chicken , frog (xenTro2), zebrafish (danRer6) and lamprey (petMar1). A site is defined as conserved with species S if it lies in a region of the mouse genome which is aligned with a region of the S genome and the aligned sequence in/S/is a site according to the same definition used for mouse sites. All genomic sequences and pre-computed \u201cNet\u201d alignments were obtained from UCSC.A ranked list of putative Dlx target genes was obtained from the sites determined above by associating each site conserved in at least one species to its closest Refseq mRNA, and then selecting the sites located either within 10 kb upstream of the TSS, or within the non-coding portion of the first exon, or in the first intron. We then associated to each putative target a score equal to the sum of the conservation scores (number of species) of its associated sites.DLX5-myc expression vectors  containing the full-length human DLX5 cDNA with an in-frame insertion of the myc-TAG at the C-terminus, was used as described DLX5-myc expression vector indicated above, by site directed mutagenesis and sequence verified .A DLX5 mRNA endogenously, but have been shown to respond to Dlx expression with activation of the p63 promoter DLX5-myc expression vectors were used for transfections, which yielded an efficiency of 35% . Chromatin was crosslinked, sonicated, immunoprecipitated with either the anti-myc TAG  or the anti-acetyl-Histone H4  antibodies and de-crosslinked according to instructions . Fragments of the human BMP2 and BMP4 loci spanning the identified conserved regions were PCR-amplified and analysed by gel electrophoresis .Previous evidences indicate that Dlx5 binds to homeodomain-responsive elements in a proximal region of the Dlx5;Dlx6 intergenic region Msx2 promoter Next, we screened conserved regions of the vertebrate genome for the presence of consensus Dlx DNA-binding sites, as defined by the Dlx5 PWM Msx1 and Msx2 genes were found in the top 10% of the list of putative Dlx targets, strengthening the possibility that Dlx proteins might directly regulate Msx expression.We then generated a ranked list of putative Dlx targets, based on the position of predicted conserved Dlx sites in the genome, as indicated in the Methods sections. The ranked list contains 3,051 Refseq mRNAs associated to 2,412 unique Entrez gene IDs (available upon request). The Dlx5, Dlx6, Msx1 and Msx2 in the FL and HL of normal embryos, at E10.5 and E11.5, by X-gal staining of heterozygous embryos carrying an allele with inserted lacZ reporter , and by WMISH (Dlx6)  of both the FL and the HL  and in the anterior HL mesoderm .We examined expression of H (Dlx6) . WMISH fd the HL , whereas compare  with O\u2013Pis stage . In the r margin . At this the AER -AB. HistMsx expression precedes that of Dlx in the HL anterior mesoderm, consequently in this location Dlx genes are unlikely to regulate Msx gene transcription cell-autonomously and Dlx and Msx proteins are unlikely to interact in the anterior limb mesoderm, at early stages.In summary, Dlx and the Msx genes during limb development, we used animals DKO for Dlx5;Dlx6 and analyzed the expression of the Msx-lacZ reporter in this genetic background. To do this, we compared the expression patterns of the lacZ reporter between +/\u2212Msx1 and +/\u2212Msx1;\u2212/\u2212;Dlx6\u2212/\u2212Dlx5 mice  mutant embryos, as compared to WT. In the \u2212/\u2212Msx2 mutant the expression level of Dlx5, Dlx6 and Msx1 was unchanged, whereas in the \u2212/\u2212Msx1 mutant the expression levels of Dlx5, Dlx6 and Msx2 were reduced by 65%, 40% and 15%, respectively. Moreover, in the posterior half of the HLs from the \u2212/\u2212Msx1 mutant we observed a reduction of 40% in the abundance of Dlx5 and Msx2 mRNA, whereas Dlx6 did not change  to quantify the reduction of  changed . We thent change .\u2212/\u2212;Dlx5\u2212/\u2212;Dlx6\u2212/\u2212Msx1 or \u2212/\u2212;Dlx5\u2212/\u2212;Dlx6\u2212/\u2212.Msx2 TKO newborns were obtained at a frequency lower than expected, and all died shortly after birth, due to severe craniofacial malformations and consequent breathing impairment. Quadruple knock-out embryos were never obtained in spite of several attempts.To reveal possible functional interactions between the Dlx and the Msx gene products during limb development, we generated TKO mice with genotype Msx2;Dlx5;Dlx6 TKO animals, the HLs were severely affected  and E18.5/birth . In affected  whereas affected . The lim2 allele .Msx1;Dlx5;Dlx6 TKO mice the FL were usually normal, although in one case (1/6) anterior polydactyly was observed (data not shown). Of note, polydactyly has been reported for the FLs in Msx1;Msx2 DKO mice with a moderate penetrance Msx1;Msx2 DKO usually exhibit loss of 1\u20132 anterior digits and of the tibia Dlx5;Dlx6Msx1;Msx2In Msx1;Msx2 DKO phenotype in the Msx1;Dlx5;Dlx6 TKO animals, which retain two functional Msx2 alleles, argues in favor of a severe reduction of Msx2 expression in the TKO limbs, as compared to the Dlx5;Dlx6 DKO.The appearance of features of the Msx1 allele in the context of Dlx5;Dlx6 DKO background  results in a 50% reduction of Msx2 mRNA in both the anterior and the posterior half of the embryonic HLs, as compared to WT. Moreover, expression of Msx2 in the anterior and posterior halves of Msx1 homozygus mutant HLs was reduced by 30% compared to Msx1 heterozygous HLs (data not shown). This indicates that reduced Msx2 expression may result from the combination of (1) loss of Dlx5 and Dlx6  the loss of Msx1, with the consequent decrease of Msx2 expression  but not significantly in the anterior mesoderm .We carried out WMISH for mesoderm . Bmp4 ex embryos , as expes region . We also embryos . Thus, wBmp4 expression is unchanged in the anterior mesoderm of the limbs; however this technique is poorly quantitative and cannot reveal minor changes in gene expression level. To further investigate the mechanism that might induce and/or sustain Msx2 expression in the anterior half of the HLs, we determined the relative abundance of Bmp2 and Bmp4 mRNAs by qRT-PCR in the anterior and posterior halves of the embryonic HLs (E11) with different genotypes. Expression of Bmp2 is mainly decreased in the anterior (about 50%), and minimally in the posterior half (20%) of the HLs from Dlx5;Dlx6 DKO embryos  than the posterior half (50%) (data not shown). Strikingly we observed a strong synergy between Msx1 and Dlx5;Dlx6 in controlling BMP expression. When combined with the Dlx5;Dlx6 DKO mutation, the loss of a single Msx1 allele was sufficient to reduce Bmp2 and Bmp4 expression to levels lower than 10% of that of control samples, both in the anterior and in the posterior halves of the HLs. Considering that Msx2 is a known target of Bmp2 and Bmp4 in several tissues Msx2 expression is reduced in the Msx1;Dlx5;Dlx6 TKO, and how this mutant shows a limb phenotype with similarities to the Msx1;Msx2 DKO.The WMISH results reported above suggest that  embryos . Bmp2 exr halves . Of noteFgf8 and Shh mRNAs in HL samples from the relevant genotypes (Fgf8 mRNA was slightly reduced in the absence of Dlx5 and Dlx6 (DKO samples), and was further drastically reduced when one Msx1 allele was eliminated in the absence of Dlx5;Dlx6. Similarly, the abundance Shh mRNA  was slightly reduced in the absence of Dlx5 and Dlx6, and was further reduced when one Msx1 allele was simultaneously eliminated. While reduced Fgf8 expression is not surprising Shh expression is novel and may contribute to the altered digit number and morphogenesis seen in the Msx1,Dlx5;Dlx6 TKO embryos.Finally, we determined the abundance of enotypes . The AbuBmp2 and Bmp4 loci by Dlx5, we first exploited the genome-wide predictions of conserved Dlx sites, described above. In the proximity of the Bmp2 locus we identified three Dlx consensus binding sequences, named B2-RE1, located 30 kb upstream of the transcription start site (TSS), B2-RE2 and B2-RE3, located respectively 3.5 kb and 18 kb downstream of the end of the Bmp2 transcript. All these Dlx binding elements are conserved, although not identical, in at least two vertebrate species, and fall within stretches of conserved genomic sequence , as well as an irrelevant sequence (IL10) did not show any enrichment (not shown). These results reinforce the notion that DLX proteins physically interact with four (out of five) conserved elements close to the BMP2 and the BMP4 loci, and might exert a direct transcription regulatory activity, relevant for normal limb development.To demonstrate that the Dlx5 protein physically binds to these putative responsive elements, we transfected a human Dlx5, Dlx6, Msx1 and Msx2 genes encode homeodomain transcription factors involved in limb development. The targeted disruption of each of these genes, individually, does not lead to limb defects, however double Msx1/Msx2Dlx5/Dlx6in vitroMsx2;Dlx5;Dlx6 TKO mice show an aggravation of the ectrodactyly phenotype, as compared to Dlx5;Dlx6 DKO, they do not present any obvious additional defect. Furthermore, inactivation of either Dlx5;Dlx6 or Msx2 does not modify the level of Msx1 expression, suggesting that the Msx2;Dlx5;Dlx6 TKO phenotype represents the exclusive contribution of the absence of Msx2 in the Dlx5;Dlx6 mutant context. This leads us to conclude that either Msx2 has a rather minor function in limb development, or that its function is largely compensated by Msx1, in agreement with the limited defects observed in +/\u2212;Msx2\u2212/\u2212Msx1 compound mutant animals Msx1;Dlx5;Dlx6 TKO mice display a limb defect which can be interpreted as the sum of the limb anomalies found in the Msx1;Msx2 DKO and in the Dlx5;Dlx6 DKO. These results strongly suggest that the expression of Msx2 is suppressed in this genetic context, and that therefore Dlx5;Dlx6 are genetically upstream of Msx2.Although Dlx5;Dlx6 DKO limbs Msx2 expression is diminished in the central sector of the AER. As starting at E10.5 Dlx and Msx genes are co-expressed in the AER, it is possible to hypothesize that in this territory Dlx proteins bind directly on the Msx2 promoter, as previously reported Msx1 and Msx2 precedes that of Dlx5 and Dlx6. This precludes the possibility of a direct regulation of Msx genes by Dlx proteins. Nevertheless, our qRT-PCR analyses show that Msx2 expression is reduced in the anterior limb mesoderm of Dlx5;Dlx6 DKO limbs, implying the existence of a non cell-autonomous mode of regulation between the AER and the anterior limb mesoderm. It is possible, therefore, that a diffusible protein, expressed by AER cells in a Dlx-dependent fashion, is required to initiate and/or sustain Msx2 expression in the anterior limb mesoderm.We show that in Msx genes are well documented downstream effectors of BMP signaling in several developing structures Gremlin, results in upregulation of both Msx1 and Msx2 expression Msx2 expression Msx1, in the dorsal ectoderm, induces the formation of ectopic AERs Msx1 and Msx2 leads to a phenotype that mimics, in some aspects, the loss of BMP signaling Msx genes are also upstream of Bmp4 in several developmental systems. During tooth germ development, mesodermal Msx1 is needed for efficient Bmp4 expression Msx1\u2212/\u2212 mice, can be rescued by a Bmp4-expressing transgene Msx genes are required in the mesoderm for maintenance of Bmp4 expression Msx genes, depending on the context and the developing structure. The possibility that Bmp2 and Bmp4 participate in a Dlx-Msx signaling loop between the limb bud AER and mesoderm is clearly in line with previously identified roles of these molecules.Noticeably, Dlx5;Dlx6 and mesodermal Msx2. Indeed, we find that Bmp2 and Bmp4 expression is significantly reduced in Dlx5;Dlx6 DKO hindlimbs, at E11 when no evident defect is yet visible. Reduction for Bmp2 and Bmp4 in the anterior part of the HL is too high  to be accounted for by the ectoderm alone, and rather indicates a downregulation of these Bmps in the mesoderm, too. This could mean that Bmps produced in the ectoderm activate Bmps also in the mesoderm, either directly or via Msx1. Indeed, further inactivation of one Msx1 allele in the context of the loss of Dlx5;Dlx6 nearly abolishes Bmp2 and Bmp4 expression, necessarily implying both the ectoderm and the mesoderm  accompanied, however, by less severe zeugopod defect. Thus, it appears that both AER- and mesenchyme-derived Bmps are involved in the Msx;Dlx defects. The possibility that misexpression of AER-Bmps alone directly cause the TKO defects is unlikely. Rather, in light of the well known self-regulatory system of signalling loops comprising FGFs, SHH and BMPs Fgf8 and Shh upon loss of Msx1, Dlx5 and Dlx6 genes, most likely the overall nature of the limb defects in TKO mutant embryos is a quantitative misregulation of the \u201cslow module\u201d of this loop. Dlx and Msx genes can be regarded as new players in this complex regulation. Since Shh participates in the \u201cslow module\u201d, a more critical role for it could be envisioned, as suggested by resemblance of the phenotype of Msx1;Dlx5;Dlx6 TKO limbs with that exhibited by Shh KO embryos The loss of mesenchymal In conclusion, we propose a model that involves a complex epithelial-mesodermal dialogue between Dlx and Msx , entailiDlx5;Dlx6 DKO vs. +/\u2212;Dlx5\u2212/\u2212;Dlx6\u2212/\u2212Msx2 vs. Msx2;Dlx5;Dlx6 TKO  the genetic inactivation of one allele, and b) the lack of Dlx5;Dlx6 genes.The importance of allelic dosage, hence quantitative gene expression, is increasingly being recognized, even when phenotypes are not evident see . As a fuMsx1;Dlx5;Dlx6 TKO animals, Msx2 expression is further reduced. A residual level is nonetheless observed; however, this is not sufficient to compensate for the loss of Msx1. In conclusion, allelic dosage and quantitative gene expression are crucial factors to be considered in the interpretation of a series of phenotypes, especially when related genes are involved.Likewise, in DLX5 and DLX6 genes cause the SHFM-type-1 congenital malformation when lost or mutated, while the MSX1 and MSX2 genes cause cleft palate and tooth agenesis. By crossbreeding mutant mouse strains, we show that the Dlx5;Dlx6 and Msx1;Msx2 genes cooperate for normal limb development and morphogenesis. At least two modes of regulation have emerged, one in which Dlx5;Dlx6 control expression of Msx2 cell-autonomously, the other in which the AER and the anterior mesenchyme interact non cell-autonomously, entailing Bmps as signaling molecules. We further show that the BMP2 and BMP4 loci comprise Dlx5-binding elements, occupied by Dlx5. Thus, the highly related homeodomain genes Dlx and Msx are two key players of a novel set of molecular and histological interactions during limb development.In human, the Figure S1Top. Location of predicted conserved Dlx binding sites in the Dlx5-Dlx6 intergenic genomic region. Sites are indicated with colour vertical bars (asterisk) and annotated with the species conservation. The chromosomal position and coordinates are also reported. The mammalian genomic conservation is reported on the bottom. The known i56i element is correctly predicted by the PWM bioinformatic approach we have adopted. Bottom. Same as above, relative to the Msx2 proximal promoter. Two known conserved Dlx binding sites are correctly predicted.(PDF)Click here for additional data file.Figure S2Quantification of the Msx2 mRNAs by qRT-PCR in the anterior and posterior halves of HLs from Msx1+/\u2212;Dlx5\u2212/\u2212;Dlx6\u2212/\u2212 embryos, relative to the corresponding WT samples (set\u200a=\u200a1).(PDF)Click here for additional data file.Figure S3Quantification of the Fgf8 and Shh mRNAs by qRT-PCR in the HLs from Msx1\u2212/\u2212 (top left), Dlx5\u2212/\u2212;Dlx6\u2212/\u2212 (top right), Msx1+/\u2212;Dlx5+/\u2212;Dlx6+/\u2212 (bottom left) and Msx1+/\u2212;Dlx5\u2212/\u2212;Dlx6\u2212/\u2212 (bottom right) embryos, relative to the corresponding WT samples (set\u200a=\u200a1).(PDF)Click here for additional data file.Table S1Sequences of the oligonucleotides used for real-time qPCR on mouse embryonic tissues.(PDF)Click here for additional data file.Table S2Sequences of the oligonucleotides used for ChIP analysis on the predicted Dlx elements near the human BMP2 and BMP4 loci.(PDF)Click here for additional data file.Table S3The Dlx5 Position-Weight matrix and results of the prediction of Dlx5 binding sites based on genomic conservation.(PDF)Click here for additional data file.Table S4Sequences of the mouse and human conserved genomic regions containing predicted Dlx5 binding sites near the BMP2 locus.(PDF)Click here for additional data file.Table S5Sequences of the mouse and human conserved genomic regions containing predicted Dlx5 binding sites near the BMP4 locus.(PDF)Click here for additional data file."}
+{"text": "Chrnb2\u2212/\u2212 mutants and from wildtype mice were obtained at postnatal day 4 (P4), during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1), a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1) and chemokine (C-C motif) ligand 21 (Ccl21) mRNAs in Chrnb2\u2212/\u2212 mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2\u2212/\u2212 mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2\u2212/\u2212 mutant strains reveals the effects of genetic background upon gene expression.Mice lacking expression of the \u00df2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2) display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC) axons to their dorsal lateral geniculate nuclei (dLGNs). Transcriptomes of LGN tissue from two independently generated  Chrnb2\u2212/\u2212 mutants) have served as a popular model for studying visual system development Chrnb2\u2212/\u2212 mutants the projections of the two eyes remain intermingled in the LGN at P8 but form an altered eye specific segregated pattern by P14 Chrnb2\u2212/\u2212 mutants compared to wild type (WT) animals Mutant mice provide an invaluable tool for studying the development and organization of the mammalian visual system Chrn2b\u2212/\u2212 mutant mice were intitally reported to lack retinal waves Chrnb2\u2212/\u2212 mutants do manifest retinal waves, though the waves are not normal in their spatial or temporal characteristics Coordinated firing of action potentials that sweep across the retina in a wavelike manner  occur in the WT retina from the late embryonic stage to eye opening in the mouse Chrnb2\u2212/\u2212 mutant animals are thought to reflect the abnormal retinal activity that occurs during the developmental period when key features of the visual system are being established. Whether or not the structural and functional abnormalities that have been documented in the Chrnb2\u2212/\u2212 mutants are driven by abnormal patterns of retinal activity during development, the aberrations displayed by the visual system of the Chrnb2\u2212/\u2212 mutants presumably also reflect the abnormal expression of molecules that play a role in forming the patterns of connections in the developing visual system. As a first step in probing this matter, in the present study we have used microarray technology to compare the expression of molecules in the retina and the LGN of the Chrnb2\u2212/\u2212mutants with those of WT animals during the period of eye specific segregation in the LGN.The aberrations in the structural and functional organization of the Chrnb2\u2212/\u2212 mutant mice were a kind gift from Dr. M Picciotto All experiments were performed in accordance with NIH and institutional guidelines regarding animal use and were approved by the campus animal use and care committee of the University of California, Davis. WT (C57BL/6J) mice were obtained from Jackson Laboratory , Picciotto (\u201cPic\u201d) Chrnb2\u2212/\u2212, Xu Chrnb2\u2212/\u2212, or C57BL/6J WT animals. Retinas from each animal were separated from the sclera and were combined. RNA was isolated with the RNAeasy Mini kit (Qiagen). A total of 500 ng of RNA was amplified with Ambion MessageAmp II-Biotin Enhanced reagents (AM1791) and aRNA yields were 57\u2013122 \u00b5g. Twenty micrograms of each aRNA target  were fragmented and hybridized to Affymetrix GeneChip Mouse 430 2.0 expression arrays using the Affymetrix GeneChip Fluidics Station and Affymetrix reagents (Affymetrix). Adult retinal tissue was prepared as above but only two male littermates of each type were sampled.Total retinas from three male P4 littermates from timed pregnancies were harvested and immediately placed on dry ice. Tissue was maintained at \u221280\u00b0C until RNA was prepared. Mice were homozygous Picciotto Total LGN  was isolated from a second set of male P4 littermates under the dissecting microscope. The cortex was removed and the LGNs were visualized and excised using an 18G needle. The LGN was isolated as a discrete entity without any surrounding thalamic tissue attached. Left and right LGN were combined for each animal and frozen on dry ice. RNA was prepared and amplified as described for retinas, but the aRNA yield from 500 ng of LGN RNA was much lower, 10\u201331 \u00b5g. Ten micrograms of each aRNA target  were fragmented and hybridized as described for retinal samples.Both the retinal and LGN tissue contain a mixture of cell types.Chip data were analyzed with dChip software Following euthanasia, eyes from WT and mutant animals were enucleated then fixed in 4% PFA in PBS for 30\u201345 minutes. After cryoprotection in a 25% sucrose solution each eye was embedded in OCT , sectioned at 10 \u00b5m on a Leica  cryostat, and mounted on glass slides. Brains from WT and mutant animals were harvested and the unfixed brain was embedded in OCT, frozen on dry ice, sectioned at 15 \u00b5m on a cryostat (Leica), mounted on glass slides and stored at \u221280\u00b0C until used. Before processing for immunohistochemistry, brain section slides were warmed to room temperature, fixed in 4% PFA for 2\u20134 minutes and washed in PBS.For immunostaining, sections were blocked in 10% normal donkey serum, 2% bovine serum albumin, and 0.3% Triton X in PBS for 2 hours, then incubated in blocking solution overnight at 4\u00b0C with primary antibodies used as follows: Anti-CHRNB2 M270 , anti-CHRNB2 , anti-CCL21 , anti-CDH1  and anti-SPP1 , followed by incubation with Alexa Fluor 568 or 594 fluorescent secondaries , or CY3  for 1 hour. Nuclei were visualized with DAPI  and sections were coverslipped using Vectashield . For control slides primary antibodies were omitted. Images were acquired on an Olympus FV500 confocal microscope . Brightness and contrast were adjusted using Adobe Photoshop.Retinal projections were traced by making intraocular injections of cholera toxin-\u00df (CTB) conjugated to fluorescent dyes (Molecular Probes). Twenty four hours prior to the required time points,  mice were anesthetized by immersion in ice water and intraocular injections of 1 \u00b5l of CTB conjugated to Alexa 488 (left eye) and Alexa 594 (right eye) in 0.5% saline were made into the far temporal region of the eye with a micro pipette. The eyes were treated with ophthalmic antibiotic and the animals placed on a warm surface until full movement was regained. After euthanasia, the brains were removed and immersion fixed with 4% paraformaldehyde for 48 hours. The brains were then sectioned at 50 \u00b5m on a vibratome (Leica), mounted on glass slides, cover slipped, then imaged on a Nikon Eclipse E600 upright microscope equipped with an ORCA-ER C4742-90 CCD camera (Hamamatsu Photonics) using a 10\u00d7 objective. Images were pseudo colored using Wasabi software (Hamamatsu).Qualitative RT-PCR was performed to validate the microarray assay for selected genes of interest. Qiagen OneStep RT-PCR reagents were used with the protocol recommended by the manufacturer. Ten to 20 ng of each template RNA was used per 25 \u00b5l reaction volume. Negative controls were run without added template. At 20\u201335 cycles aliquots of each reaction were withdrawn and run on agarose gels. Gel images have been adjusted for brightness and contrast with Adobe Photoshop. Actin control RT-PCRs are shown in Chrnb2 expression vs. those resulting from inter-strain differences. The importance of the contribution of the mouse background strain to the transcriptional profile has been noted by others Erdr1 and Traf4) differ between the two mutants in the P4 retina  reveals the genes truly affected by lack of 4 retina , while h4 retina . HoweverXlr3a gene, which is overexpressed in P4 mutant LGN (see below), is located on the X chromosome. The two Chrnb2\u2212/\u2212mutant strains potentially express combinations of X alleles from the 129/SvEv, HM1, DBA/2 and/or C57BL/6J lines Pisd-ps3 pseudogene family is reduced in mutants for both tissues and ages examined. However, two probe sets (1453144_at and 1453145_at) are also detected significantly less in Picciotto than in Xu Chrnb2\u2212/\u2212mutant mice, indicating a potential background strain effect for at least some of these loci.Expression of at least two genes which show significantly different expression between mutant and WT animals may also be affected by background strain. The Chrnb2\u2212/\u2212mutants are created by two distinct procedures. In the Picciotto mutant a small portion of the gene around the start codon and the signal peptide has been replaced with a lacZ-neomycin resistance construct Chrnb2 mRNA was dramatically lower in microarray analysis of tissues from both mutants  and six have known cell adhesion functions . Cdh1  expression is remarkably suppressed:  with significantly altered expression in P4 retina show an increased expression in the Chrnb2\u2212/\u2212mutants  and outer and inner plexiform layers at P4 compared to WT  shows reduced expression in both mutants in P4 LGN and in P4 and adult retinas by the selection criteria chosen , or the reduced expression in P4 LGN is localized. Expression of the immune response indicator CCL21 (chemokine (C-C motif) ligand 21; 6CKINE) is increased in P4 Chrnb2\u2212/\u2212mutant retina, but not in adult mutant retina , we assayed RNA populations in both mutant and WT (C57BL/6J) mouse LGN and retinal tissue at P4 by microarray hybridizations. Adult retinas were also assayed and gene expression compared to age matched WT animals.Recently, this laboratory has reported that two strains of Chrnb2\u2212/\u2212 mutants is heterozygous for at least one chromosomal locus Chrnb4\u2212/\u2212 mutants with C57BL/6J in a microarray analysis. The Chrnb4\u2212/\u2212 mutants used were generated by a process similar to the Xu Chrnb2\u2212/\u2212 mutants presented in this work Chrnb4 gene on chromosome 9 While the use of single-gene mutants to elucidate gene function is a powerful tool, many authors have cautioned that the methods by which these knockout animals are prepared may not reveal the true contribution of the knocked out gene to the transcriptional profile compared with \u201ccontrol\u201d animals Chrnb2 is located on Chr3 and examination of our data reveals that four genes we score as having significantly different expression are also located on chromosome 3: S100a10 and Cldn11 in P4 LGN, Cp in P4 retina, and Gstm1 in adult retina. Of these, S100a10 and Gstm1 lie very close to Chrnb2 at Chr3F and differences in their expression may be a result of the mutation process rather than the lack of CHRNB2.Chrnb4 gene, but our tissue, ages, and selection criteria all vary from that report Chrnb4 does lead to changes in expression of calcium ion binding proteins similar to our results for Chrnb2, indicating that this may be a pathway generally sensitive to alterations in nAChR subunit composition. While comparing both independently produced Chrnb2\u2212/\u2212 animals to the selected WT strain does not completely correct for allelic effects, our analysis of two independently generated mutants allows a more certain determination of transcripts affected by the lack of CHRNB2. At least two genes with consistently different expression between both mutants and WT . CCL21 is a ligand expressed by injured neurons that activates microglia as part of the inflammatory response. Retinas from mice lacking CHRNB2 show 8-fold overexpression of Ccl21 by microarray analysis. CCL21 is overexpressed in the OS, OPL and IPL of P4 Chrnb2\u2212/\u2212 retinas compared with WT. Photic injury to the adult retina has been reported to increase expression of one member of the CCL21a/b/c chemokine family, Ccl21a (aka Scya21a) by more than four-fold in the ONL In P4 retinas, mutation of Chrnb2\u2212/\u2212 mice, which have not been exposed to abnormally high light levels, synthesize large amounts of CCL21 in their retinas is not known. Unlike the pyknotic nuclei and other cellular abnormalities reported in photic injured retinas Chrnb2\u2212/\u2212 mutant mice. Adult brains of Picciotto Chrnb2\u2212/\u2212 mutants have been reported to show increased neuronal atrophy and microgliosis Why the P4 Cp) mRNA has also been reported to be upregulated in response to photic damage Cp mRNA in P4 Chrnb2\u2212/\u2212 mutant retina relative to WT  may be eliminated by lack of CHRNB2 in the retina, and that this transient defect in the adherens junction leads to subsequent compensatory overexpression of CRB1.Mutation of CRB1 (crumbs1) is associated with Leber's congenital retinopathy and retinitis pigmentosa in humans 2+ ion flow at the synapse. CRB1 is required to prevent light-induced retinal degradation in Drosophila Chrnb2\u2212/\u2212 LGN.CRB1 shares calcium binding properties with CHRNB2 and activation of nicotinic AChRs increases calcium trafficking at the synapse Chrnb2 is associated with a decrease in transcript expression in the P4 LGN as 32 of 36 transcripts with altered expression show a reduction of expression in the mutants , LYPD2  and CDH1, proteins with known axon guidance properties, are significantly lower in P4 mutant LGN compared to the age matched WT. Various effects of SPP1 on axon growth have been reported. Ries et al in vitro assay using purified rat E20 RGCs while in an in vitro growth assay of effects on dorsal root ganglia showed SPP1 in the substrate inhibited outgrowth and caused fasciculation Spp1\u2212/\u2212 mutant mice are grossly normal Spp1\u2212/\u2212 mutant mice appears equivalent to WT. Reduced SPP1 does not cause the segregation defect.Given the displacement of RGC axonal projections in the Ccl21 and Crb1, in Chrnb2\u2212/\u2212 mutant retinas at P4 and decreased expression of Spp1 mRNA in mutant LGNs at P4. Zoli et al Chrnb2\u2212/\u2212 mutant mice. Spp1 expression appears to be tissue-dependent in Chrnb2\u2212/\u2212 mutant mice, but whether it plays a role in neuronal injury response in these animals is not known.The association of SPP1 expression with neuronal degeneration in the rodent brain Chrnb2\u2212/\u2212 LGNs. LYPD2 is an Ly6 superfamily member closely related to LYNX1, a known modulator of \u03b14\u00df2 nAChRs Expression of Lypd2 is markedly downregulated in Chrnb2\u2212/\u2212 mutant LGN. While more detailed analysis is required to address whether genes isolated in the microarray experiments are associated with the reported visual system defects, the finding that CDH1 is specifically down-regulated in the Chrnb2\u2212/\u2212 mutant LGN is novel and intriguing. CDH1 and CDH2 (N-cadherin) are prototypes of a large family of adhesion molecules that play important roles in the development of the nervous system Cdh1 transcript was not found in embryonic or postnatal retinas tested until P14 CDH1 expression is strongly downregulated and is not detectable in P4 dh2 mRNA in retina and LGN is high and unaltered in P4 Chrnb2\u2212/\u2212 mutant mice. It has been established that cadherins, including CDH1 and CDH2, regulate cell-cell interactions through homophilic binding Chrnb2\u2212/\u2212 mutant visual system.Our microarray data show expression of CChrnb2\u2212/\u2212 mutants. Matter et al 2+ activation of transcription is abnormal and expression of CDH1 or LYPD2 in LGN neurons is weakened. The accuracy of neuronal connections in the LGN therefore may be compromised by the altered expression of axon guidance and adhesion molecules at the thalamic level.We recognize the limitations of extrapolating from microarray and RT-PCR analysis to protein expression. Nontheless, based on these RNA analyses, it is tempting to hypothesize that deficits of the axon growth regulators CDH1 and LYPD2 are responsible for the diffuse localization of ipsilateral RGC projections in the LGNs of Figure S1Actin controls for RT-PCRs. Actin amplification controls for RT-PCRs shown in (TIF)Click here for additional data file.Table S1Excel spreadsheets of present calls for all the microarrays.(XLS)Click here for additional data file.Table S2Excel spreadsheets of all mutant vs WT comparisons.(XLS)Click here for additional data file.Table S3Excel spreadsheets of all Pic vs Xu comparisons.(XLS)Click here for additional data file.Table S4Gene Abbreviations and Names from ,  and .(XLS)Click here for additional data file."}
+{"text": "SNF3 gene in the yeast Saccharomyces cerevisiae encodes a low glucose sensor that regulates expression of an important subset of the hexose transporter (HXT) superfamily. Null mutations of snf3 result in a defect in growth on low glucose concentrations due to the inability to relieve repression of a subset of the HXT genes. The snf3 null mutation phenotype is suppressed by the loss of either one of the downstream co-repressor proteins Rgt1p or Mth1p. The relief of repression allows expression of HXT transporter proteins, the resumption of glucose uptake and therefore of growth in the absence of a functional Snf3 sensor.The RGT1 and MTH1 genes (RGT1/rgt1\u0394 MTH1/mth1\u0394 snf3\u0394/snf3\u0394) but homozygous for the snf3\u2206 were found to grow on low glucose. Since null alleles in the heterozygous state lead to suppression, MTH1 and RGT1 display the phenomenon of combined haploinsufficiency. This observed haploinsufficiency is consistent with the finding of repressor titration as a mechanism of suppression of snf3. Mutants of the STD1 homolog of MTH1 did not display haploinsufficiency singly or in combination with mutations in RGT1. HXT gene reporter fusion assays indicated that the presence of heterozygosity at the MTH1 and RGT1 alleles leads to increased expression of the HXT2 gene. Deletion of the HXT2 gene in a heterozygous diploid, RGT1/rgt1\u0394 MTH1/mth1\u0394 snf3\u0394/snf3\u0394 hxt2\u0394/hxt2\u0394, prevented the suppression of snf3\u0394.Strains heterozygous for both the HXT2 is the gene responsible for restoration of growth under these conditions suggests that the numbers of repressor binding domains found in the regulatory regions of members of the HXT family may have biological relevance and enable differential regulation.These findings support the model of relief of repression as the mechanism of restoration of growth on low glucose concentrations in the absence of functional Snf3p. Further, the observation that  Saccharomyces cerevisiae. Glucose transport occurs via facilitated diffusion mediated by a group of homologous transmembrane hexose transporters, encoded by the HXT genes  prion allows growth on alternate substrates in the presence of glucose due to a specific interaction of the Pma1p and Std1p proteins fostering the creation of a stable Std1p-Rgt1p complex [HXT gene expression under specific physiological conditions.Thus unctions ,24. In can Std1p ,16,17 bu complex  stronglysnf3 function results in the inability to grow on low glucose solid media, it results in a dramatic increase in the time it takes to adapt to low glucose liquid media and a diminished capacity to achieve the same final OD of wild type strains [While the loss of  strains . The sporgt1 and mth1 in the snf3 null strains were examined to determine if these mutations can completely suppress the adaptation defect. The adaptation time of the snf3/snf3 mth1/mth1 (UCD2883), snf3/snf3 rgt1/rgt1 (UCD2884) strains and wild type (YPH501) are equivalent , , snf3/snHXT2 promoter was the most sensitive reporter to changes in regulatory machinery and promoter copy numbers, it was used to compare the strains that demonstrated some amount of single haploinsufficiency as well as the homozygous nulls of mth1 and rgt1 . Null of HXT2 were also constructed in a series of haploid mutations to assess the impact of loss of this transporter on suppression of the snf3 growth defect in a variety of backgrounds. The hxt2 null strains were transformed with the HXT2 gene and used as a control. Loss of the HXT2 gene did not impact growth of the wild type strain on low glucose, YPH500 . The growth of both haploid strains, snf3\u2206 hxt2\u2206 mth1\u2206  (Sector B) and snf3\u2206 hxt2\u2206 rgt1\u2206  (Sector C) was improved by transformation with the HXT2 gene (Panel B). Growth of the heterozygous diploid, snf3\u2206 / snf3\u2206 hxt2\u2206 / hxt2\u2206 mth1\u2206 /MTH1 rgt1\u2206 /RGT1 (Sector F) was greatly reduced by deletion of the HXT2 gene and became similar to the s\u2206 nf3 mutation (compare sector E to sector F). Thus the expression of combined haploinsufficiency requires the presence of a wild type copy of the HXT2 gene. The fact that growth of strains carrying the snf3 suppressors mth1\u2206  and rgt1\u2206  in a haploid state also requires the presence of the HXT2 gene suggests that expression of this transporter is important for growth under these low glucose conditions.The analysis of the impact of loss of repressor gene dosage on HXT gene promoter regions revealed a direct connection between the number of binding sites and the level of repression. Addition of binding sites to a reporter gene construct increased repression 10 to 50 fold [HXT2 promoter was shown to carry only three Rgt1 binding sites while the promoters of the HXT1 and HXT3 genes carried 13 and 15 putative binding sites for Rgt1p-mediated repression respectively [HXT2 repression. When considered in combination with the previous reports that 2 micron plasmids containing only the promoters of genes regulated by this complex can suppress the snf3 growth defect [Analysis of the distribution of Rgt1 binding sites in  50 fold . These aectively . Our finh defect , the cleHXT2 protein may be an important transporter for the transition between substrate concentrations and that the affinity of the transporter may be impacted by other factors within the cell. It is also possible that under very high substrate conditions the Hxt2 transporter plays an important regulatory role and that putative regulatory role rather than level of transporter protein expression is the key factor enabling growth of the heterozygous strain. A rapid induction of HXT2 may therefore confer a distinct growth advantage to the cells during adaptation to differing substrate concentrations. Differences in numbers of repressor binding sites could play a role in allowing expression of some transporters to occur under otherwise limiting conditions.These observations also suggest that repressor binding site number may represent yet another mechanism to differentially regulate the different hexose transporters. The Hxt2 transporter has been shown to exist in forms with differing affinity for substrate  althoughThis information adds unique insight to the model that has formed over the past decade since the roles of the corepressor proteins were first elucidated and this intriguing mechanism of transcriptional regulation began to be understood ,16,17. OSaccharomyces cerevisiae used in this study are listed in Table Saccharomyces Genome Deletion Project was purchased from Open Biosystems and used to make all complete null alleles that contain the KanMX resistance marker that provides resistance to G418. Genomic DNA was purified by using the MasterPure\u2122 Yeast DNA purification kit (Epicentre). Genomic DNA of the respective null strain from the deletion set was used as the template for PCR mediated gene disruption by amplifying approximately 200 base pairs of homology flanking each side of the deleted ORF. For all deletions that contain the HphMX resistance marker specifying resistance to Hygromycin B, the pYC140 plasmid [The strains of  plasmid  was used plasmid . All del plasmid . DiploidYeast strains were grown on Yeast Extract Peptone Media (YP) consisting of 1% yeast extract, 2% Bacto peptone and either 2% glucose, 2% galactose, or 0.05% glucose. Synthetic complete (SC) or synthetic dropout (SD) media were made according to established protocols  with theSTD1 was cloned into the p416TEF vector which contains the URA3 selectable marker [STD1 preceded by the strong constitutive TEF promoter and flanked by the CYC1 terminator sequence.The complete open reading frame of e marker  by usingHXT2, HXT3, and HXT4 were made in the CEN/ARS vector pRS416 which contains the URA3 selectable marker [HXT2, HXT3 and HXT4 were PCR amplified with 5\u2019 SacI and 3\u2019NotI overhangs and cloned into pRS416 to create pKD-HXT2, pKD-HXT3 and pKD-HXT4. The resulting plasmids were then sequenced to ensure the fidelity of the cloning. The E. coli LacZ gene from the one hybrid reporter vector described in [E. coli LacZ gene. All plasmids were transformed into the respective yeast strains as described in [The LacZ reporter fusions to the promoters of e marker . The proribed in  was amplribed in .HXT2 from YPH500 was constructed in the CEN/ARS vector pRS316, which contains the URA3 yeast selectable marker [HXT2 gene, along with 562bp of the upstream region, was PCR amplified using HiFi Taq (Invitrogen #11304-011) with primers containing 5\u2019 XhoI (5\u2019-CTCGAGTTTCCGTGAAATAGATTC-3\u2019) and 3\u2019 XbaI (5\u2019-TCTAGATATTAGTAGCCATTAGCC-3\u2019) overhangs. The resulting PCR product was cloned into the MCS of pRS316. The plasmid, now carrying a functional copy of HXT2 under its native promoter, was transformed into previously hxt2 null strains [A plasmid carrying a copy of e marker . The HXT strains .Strains were streaked out from glycerol stocks to YPD media and a separate single colony was used to inoculate three replicate starter cultures in Synthetic Complete (SC) 2% glucose media. Starter cultures were grown for 48 hours and used to inoculate 200 mL of SC 0.05% glucose plus Antimycin A to an OD580 of 0.02. Cultures were grown in 500 mL non baffled Erlenmeyer flasks at 30\u00b0C with shaking on an orbital shaker at 200 RPM and the OD monitored at 580 nm. All strains were examined in triplicate and each figure represents one experiment where all strains shown were grown at the same time. Error bars indicate the standard deviation of the average of the three triplicates.Overnight cultures of strains were grown in YP 2% glucose, washed once with sterile water and then re-suspended in sterile water to an OD580 of 1.0. The strains were then subjected to a 10 fold serial dilution and 10 \u03bcL of each was spotted on YP or SC plus 0.05% glucose and Antimycin A. Growth was assessed after 3 days of incubation at 30\u00b0C.LacZ data were performed using SAS .Strains with reporter plasmids were grown to mid log phase in SD minus uracil with 2% galactose, harvested by centrifugation and then resuspended in either SD minus uracil with 0.05% and Antimycin A or SD minus uracil with 2% galactose and allowed to grow for 3 hours in the respective media. Approximately 15 OD units were harvested by centrifugation and resuspended in assay buffer and assayed as described in . \u03b2-galacThere are no financial or non-financial competing interests for any of the authors.KLD and LFB planned all experiments. KLD did the work with the exception of the data provided in Figure KLD current address: Amyris Biotechnologies, 5885 Hollis Street, Suite 100, Emeryville, CA"}
+{"text": "Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.++/high Mg++. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon . RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low CaWe showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions. The discovery that the distension induced motor behaviour of the isolated intestine is regulated by the enteric nervous system (ENS) Recent concepts suggested that mechanosensitivity in the ENS is a property of many different classes of neurons Previous work mainly focused on mechanosensitive enteric neurones in the guinea pig. There are only a few studies which addressed the question of whether AH/Dogiel type II neurons behave as mechanosensors in regions outside the ileum or in mice. AH/Dogiel type II neurons were described in the mouse small and large intestine We argue that the functional relevance of RAMEN and the validity of the concept of multifunctional enteric neurons require that their existence is confirmed in other species and gut regions. The aim of this study was therefore to characterise properties of mechanosensitive myenteric neurons in the mouse ileum and colon. Mice were chosen for the obvious advantage to perform in the future studies in knockout models.All animal procedures are approved by the ethical committee of the government of Oberbayern (Germany) (55.2-1-54-2531.8-210-09) and according to the German guidelines for animal protection and animal welfare.2, 5% CO2; pH\u200a=\u200a7.40) Krebs solution containing (in mM): 117 NaCl, 4.7 KCl, 1.2 MgCl2 6 H2O, 1.2 NaH2 PO4, 25 NaHCO3, 2.5 CaCl2 2 H2O, 11 glucose. The mucosa and submucosa were gently removed in order to obtain myenteric plexus preparations still embedded between the two muscle layers. The preparations (5\u00d710 mm) were pinned onto a silicone ring that was placed in a recording chamber continuously perfused with 37\u00b0C carbogen bubbled Krebs solution with a rate of perfusion of 11 ml/min. To block synaptic transmission 20 min perfusion of the entire tissue with a low Ca++/high Mg++ Krebs solution, containing (in mM) 98 NaCl, 4.7 KCl, 16 MgCl2 6 H2O, 1.2 NaH2 PO4, 25 NaHCO3, 0.25 CaCl2 H2O, 11 glucose, was used.Male mice C57BI/6  16 weeks old were killed by cervical dislocation followed by exsanguination. The ileum or the distal colon was quickly removed and further dissected in Carbogen aerated (95% On-octylamino)-6-naphthyl]vinyl] pyridinium betaine) by local application through a microejection pipette loaded with 20 \u00b5M Di-8-ANEPPS dissolved in DMSO and pluronic F-127 containing Krebs solution. The dye staining did not change the electrophysiological properties of the nerve cells 2 or \u223c4 \u00b5m2 per pixel, respectively. The fluorescent images were acquired and processed by the Neuroplex 8.3 software (RedShirt Imaging). Individual cells can be identified since the dye incorporates into the membrane revealing the outline of individual cell bodies  which was connected to a motorized micromanipulator  to control advancement of the hair. The hair was placed 1 to 2 \u00b5m above the ganglion and then advanced to create ganglion deformation with a step size of 10 \u00b5m, then left in place for 1 min and finally retracted with a 10 \u00b5m step. In preliminary experiments mechanical stimulations were repeated at least three times at 5\u201310 min intervals to ensure that there was a genuine response rather than cell damage induced spike discharge. In addition, after the experiments, any cell damage could be excluded by immunohistochemical staining that allowed us to verify intact cell morphology.3 (0.1%)/horse serum  for 1 h at room temperature followed by 24 h and 3 h incubation with the primary and secondary antibody, respectively. As primary antibodies we used goat anti-choline acetyltransferase , rabbit anti-Neurofilament 145 kDa , rabbit anti-nitric oxide synthase  and goat anti-calretinin . As secondary antibodies we used donkey anti-rabbit conjugated to Carbocyanin , donkey anti-goat conjugated to Carbocyanin  or donkey anti-rabbit conjugated with biotin  and then streptavidin AMCA . Finally, specimens were washed in PBS, mounted on poly-l-lysine-coated slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 (0.1) containing 65% glycerol. The preparations were examined with an epifluorescence microscope (Olympus), equipped with appropriate filter blocks. Pictures were acquired with a video camera connected to a computer and controlled by Scion image software . Frame integration and contrast enhancement were employed for image processing.In order to study the neurochemical code of the mechanosensitive neurons immunohistochemistry was performed. Tissue specimens were fixed 4 h at room temperature in a solution containing 4% paraformaldehyde and 0.2% picric acid in 0.1 mol/l phosphate buffer and then washed (3\u00d710 min) in phosphate buffer. Whole mount preparations were first incubated in phosphate buffered saline (PBS)/NaNWe counted the number of dye-labeled neurons in the ganglia and analysed the number of mechanosensitive neurons per ganglion and the frequency of action potentials discharge. In addition, spike discharge frequency during the dynamic deformation was separately analysed. These values were then correlated with the degree of ganglion deformation. The duration of spike discharge and the occurrence of the last action potential were calculated. We additionally analysed the instantaneous frequency  and averaged the values for dynamic and sustained deformation as well as for the entire period.For signal and image analysis we used Neuroplex 8.3.2 (RedShirt Imaging), Igor Pro 6.03  and Image J 1.43u  software. For the movie editing the program Adobe Premiere Pro  was used. The statistical analyses and graphics were performed with Sigmaplot 12.0 . All data are presented as mean \u00b1 standard deviation or, when not normally distributed, as median values together with the 25% and 75% quartiles given in brackets. Differences in the spike frequency between several control stimulations were performed with a one way repeated measures analysis of variance (ANOVA) test. Action potential frequency fired from the same neuron after 2 subsequent stimuli were tested with a paired t-test. Correlation analyses were performed with Pearson Product Moment Correlation. Comparisons between data from the small and large intestine were made with Mann-Whitney Rank Sum Test. To test the differences in action potential frequency before and after blockade of synaptic transmission we used repeated measures ANOVA on ranks. Differences and correlations were considered significant when P was <0.05.In the ileum mechanical stimulation by intraganglionic volume injection was performed in 51 ganglia from 12 mice. The intraganglionic injection resulted in an initial dynamic deformation of the ganglia which lasted 306 [153/401] ms, followed by a sustained deformation which lasted for 150\u00b140 s; during this time the volume was redistributed in the ganglionic network and the ganglion regained its original shape see .The mean number of neuronal cell bodies per ganglion was 19\u00b16. Ganglion deformation evoked a spike discharge in 22\u00b111% neurons per ganglion (range 5\u201355%). Of these mechanosensitive neurons 20% (47 neurons) fired one action potential only. All other 188 mechanosensitive neurons fired at least 2 and up to 59 spikes  which fired during the dynamic deformation with at comparable frequencies (5 [5/5] Hz versus 3 [1.7/8.7] Hz) .The degree of deformation expressed as percentage change in the ganglionic area after intraganglionic volume injection was 5.1 [3.8/8.9] % (23 ganglia from 8 mice). Of note, the percentage of mechanosensitive neurons as well as the spike frequency during the dynamic deformation positively correlated (p<0.05) with the degree of ganglion deformation .Once started, the spike discharge lasted on average 239 [100/402] ms. The time of occurrence of the last action potential during burst firing was 389 [243/686] ms after the beginning of the dynamic deformation. To check for possible late onset responses we used longer recording periods in 4 ganglia and found that none of the responding neurons fired action potentials beyond 2 s after the stimulus.The spike discharge pattern was rapidly adapting see  and veryIn order to study the reproducibility of the responses we performed experiments (5 ganglia from 4 mice) with 3 mechanical stimulations 10\u201315 min apart. These stimulations provoked each time a similar change in ganglionic area which varied between the stimuli less than 0.21%. In these experiments we found a high reproducibility of the responses: the number of mechanosensitive neurons per ganglion  and the overall spike discharge  remained stable.In the distal colon mechanical stimulation was performed in 91 myenteric ganglia from 14 mice. Like for the ganglia in the ileum the intraganglionic volume injection resulted in a two phase\u2019s deformation. An initial dynamic deformation of the ganglia which lasted for 320 [248/433] ms was followed by a sustained deformation which lasted for 175\u00b135 s.The mean number of neuronal cell bodies per ganglion was 22\u00b110. Of these, 14\u00b19% neurons per ganglion responded to the mechanical stimulation (range 2\u201326%) . About 2Like RAMEN in the ileum, two subsequent stimuli within an interval of 2 s activated the same mechanosensitive neurons in the colon  without significant changes in the firing rate during the dynamic deformation (5 [2.5/10] Hz versus 7.5 [5/12] Hz).The degree of deformation calculated as percentage change in the ganglionic area was 3.4 [1.6/5.0] % (29 ganglia from 6 mice) and significantly smaller than in the ileum. Like in the ileum the percentage of mechanosensitive neurons as well as the firing frequency during the dynamic deformation was positively correlated  with the degree of ganglion deformation . In two Once started, the spike discharge lasted on average 540  ms. The last spike occurred 1,067  ms after the beginning of the dynamic deformation. The majority of mechanosensitive neurons (127 out of 159) showed a rapidly adapting spike discharge typical for RAMEN . The remLike their counterparts in the guinea pig myenteric plexus, RAMEN in the mouse myenteric plexus respond to von Frey hair probing with spike discharge during dynamic deformation. As already described in our study in guinea pig myenteric plexus, intraganglionic volume injection was much superior to von Frey hair stimulation regarding reliability and reproducibility of the response. This was mainly due to the inability to repeatedly position the hair exactly on the same spot. The disadvantage of the deformation evoked by volume injection was the slow recovery. Therefore, onset and rapid offset of the stimulus can only be controlled with von Frey hair probing. We used von Frey hair probing to specifically address the question whether RAMEN respond to loading (onset of deformation by advancing the hair) as well as to unloading (offset of the stimulus by retracting the hair). Such experiments were performed in 20 mechanosensitive neurons (17 ganglia from 3 mice). The spike frequency of RAMEN was identical between loading and unloading (1.4 [0.7/2.8] versus 1.4 [0.5/1.8] Hz) .We performed experiments  where we used in the same ganglia the von Frey hair probing and the intraganglionic volume injection. With the von Frey hair one deforms a relatively small area of the ganglion while distant areas of the ganglion showed no noticeable deformation see also .With the++/high Mg++ did not change in the ileum the number of mechanosensitive neurons responding to ganglion deformation . There was also no change in the overall spike discharge rate (3.5 [1.5/6.2] Hz versus 2.9 [1.5/4.9] Hz) or in action potential frequency during the dynamic deformation (7.7 Hz versus 6.7 Hz) (The blockade of synaptic transmission with low Ca 6.7 Hz) .++/high Mg++ (9.4 and 5.9 Hz).Likewise, in the colon the percentage of mechanosensitive neurons per ganglion remained unchanged . Moreover, the action potential frequency during the dynamic deformation 10.0 Hz versus 8.1 Hz  and the ++/high Mg++.Of note, the percentage of mechanosensitive neurons as well as the firing frequency during the dynamic deformation was still positively correlated (p<0.05) with the degree of ganglion deformation during synaptic blockade by low Ca+ concentrations are in equilibrium during continuous Krebs perfusion. We nevertheless performed experiments with intraganglionic injection of low K+ (1 mM) Krebs solution in order to account for the possible buffering capacity of glia cells. Intraganglionic injection of low K+ solution (12 colonic ganglia in preparations from 4 mice) activated 16\u00b19% neurons per ganglion which fired at 7.5 [3.2/9.8] Hz during the dynamic and at 2.8 [2.0/5.8] Hz during the sustained deformation. Both the percentages of mechanosensitive neurons as well as their firing frequencies were comparable to injection of Krebs solution containing 4.7 mM K+ (see data above).We assumed that the intra- and extraganglionic KBased on previous studies, the putative mechanosensitive myenteric neurons in the mouse (AH/Dogiel type II neurons) can be identified by NF145kDa-IR in the ileum Additional markers where used to identify the cholinergic or nitrergic phenotype of RAMEN. In the ileum (16 ganglia from 2 mice) double staining revealed that 79% of mechanosensitive neurons (62 out of 78 ) were ChAT-IR, 12% (9 out of 78) were NOS-IR and the remaining 9% (7 out of 78) were negative for both markers . ChAT-IRSmall neurons may be easier to activate because of their lower input resistance; but different cell sizes did not affect the identification of RAMEN. There was no difference in cells size between mechanosensitive neurons  and those that did not respond to ganglionic deformation .2 versus 61,232  \u00b5m2). This may explain why the degree of deformation was on average significantly higher in the ileal myenteric plexus (p\u200a=\u200a0.005). Nevertheless, the duration of the dynamic deformation was similar in both regions. Although the overall spike frequency as well as the firing frequency during the dynamic deformation did not significantly differ between the two regions, colonic RAMEN fired for a longer period than RAMEN in the ileum (p\u200a=\u200a0.003).The behaviour of most mechanosensitive neurons in the ileum and distal colon was comparable and characterised by rapidly adapting spike discharge. Therefore, the use of the term RAMEN seems appropriate. The number of neurons per ganglion was comparable in the ileum (19\u00b16) and distal colon (22\u00b110), despite the finding that myenteric ganglia in the ileum were significantly smaller than those in the colon  than in the in the colon (14\u00b19%). This is likely an experimental bias as the degree of ganglion deformation was in general smaller in the colon. Analysis of only those ganglia that were deformed to a similar degree (variation of \u22640.3% in ganglion area) revealed that neither the percentage of responding neurons nor the firing frequency during dynamic deformation was significantly different between the two regions: 19\u00b18% responding neurons in the ileum fired at 5.8 [4.2/9.9] Hz and 14\u00b16% responding neurons fired at 8.8 [6.9/9.6] Hz in the colon.This study identified in the mouse small and large intestine mechanosensitive neurons with characteristics and properties comparable to previously identified RAMEN in the guinea pig ileum By analogy to the guinea pig, mouse RAMEN fired action potentials during the dynamic deformation. The response declined with the decay in deformation (rapid adaptation) and ceased when the ganglion entered the phase of sustained deformation. This behaviour is not due to desensitization because spike discharge occurred in response to two consecutive mechanical stimuli applied within 2 seconds. This pattern of response is likely to reflect the type of muscle activity present in the small intestine. Phasic contractions are in fact dominant in the ileum Several results extended our previous findings in the guinea pig ileum. Firstly, RAMEN fired during the onset of ganglionic deformation by advancing the von Frey hair but also when releasing the force by retracing the hair. This suggested that they are able to encode dynamic deformations independent of whether we apply or release the force. Secondly, the proportion of RAMEN responding to deformation and their firing frequency strongly correlated with the degree of ganglionic deformation. Based on the result that there is still a positive correlation between firing rate of RAMEN and ganglion deformation after synaptic blockade, we conclude that sensitivity to deformation is not influenced by neural tone but rather an intrinsic property of RAMEN. Thirdly, we demonstrated that firing rate of mechanosensitive enteric neurons not only relates to the stimulus strength but, even more important, to the degree of ganglion deformation as a result of varying stimulus strengths. Our results are in line with previous suggestion that guinea pig enteric neurons respond to ganglion deformation that occurs during contraction and relaxation of the gut Markers for putative sensory Dogiel Type II neurons in the mouse ileum NK145KDa  and coloIn summary, our findings support the existence and functional relevance of RAMEN across species and regions. Basic properties of RAMEN, such as firing pattern in response to ganglion deformation, their proportion and cholinergic phenotype are almost identical in mouse and guinea pig. This suggests that RAMEN are fundamental for mechanosensitivity in the ENS.Movie S1Activation of enteric mechanosensitive neurons by ganglionic deformation. Slow motion movie of the ganglionic deformation during intraganglionic volume injection . The outlines of the neurons are visible because the voltage sensitive dye Di-8-ANEPPS incorporates into the outer membrane. The movie shows the deformation during the volume injection and at the same time the colour coded action potential discharge (red flashes). Locations of the 2 neurons that fire action potentials in response to intraganglionic volume injection are indicated by red arrows. Note that with this magnification activity in one neuron is detected by several pixels. The two traces to the left show the responses of the two mechanosensitive neurons. Each action potential corresponds to the colour coded activity (red flashes). In the upper part of the movie onsets of the dynamic and sustained deformations are indicated.(AVI)Click here for additional data file."}
+{"text": "Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4\u2013Msh5 complex has hindered an understanding of the structural aspects of Msh4\u2013Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4\u2013Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4\u2013Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4\u2013Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4\u2013Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.The Msh4\u2013Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4\u2013Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs .  The MutS homodimer in bacteria is involved in the repair of mismatches that occur during DNA replication The MutS homodimer has the shape of an oval disk with two channels of dimensions \u223c30\u00d720 and \u223c40\u00d720 \u00c5 with DNA passing through the larger channel Saccharomyces cerevisiae meiotic crossovers are initiated by the programmed introduction of \u223c140\u2013170 DNA double strand breaks (DSBs) by the Spo11 protein in combination with accessory factors S. cerevisiae have provided molecular details into the sequence of events during repair of DSBs into crossover products In S. cerevisiae msh4\u0394, msh5\u0394 mutants have strong defects in meiotic crossing over (2.5 fold decrease), spore viability (30\u201340%) and disjunction of homologous chromosomes Consistent with the physical studies, genetic and biochemical data support the role of the Msh4\u2013Msh5 complex in meiotic crossover formation. S. cerevisiae and mammals. A smaller subset of crossovers in these organisms is made through the Mus81-Mms4 pathway msh5 mutants showed more severe phenotypes compared to msh4 mutants. msh4/5 mutations were classified into three types based on crossover frequency and spore viability. These include a) mutations with spore viability and crossover frequency similar to that of msh4/5 \u201cnull\u201d mutations, b) mutations with intermediate defects in spore viability and crossing over and c) mutations with only crossover defects. Interestingly, mutations in equivalent positions in Msh4 and Msh5 ATPase and DNA binding domains were observed that had asymmetric effects on crossover frequency and spore viability.The Msh4\u2013Msh5, Mlh1\u2013Mlh3 complexes are part of the major crossover pathway in msh4/5 mutations. As no crystal structure is available for the Msh4\u2013Msh5 complex, homology modeling was used to generate a structural model for this complex using the hMSH2\u2013hMSH6 crystal structure as the template. Homology modeling has proved to be useful in a number of cases where crystal structures are not available for a protein msh4/5 mutations result in meiotic defects by two mechanisms: by affecting stability of the Msh4/5 proteins or interaction of the Msh4\u2013Msh5 complex with the DNA. The model has not only been used to explain the structural basis of the meiotic defects observed in the mutations but also to propose further mutations that may be analyzed. These include residues at the putative interface of the Msh4\u2013Msh5 complex, residues that may be involved in DNA binding and double mutations that may serve as compensatory mutations. Such information is useful to predict incompatibilities between segregating polymorphisms in MSH4 and MSH5 genes in populations. More generally the availability of a model for Msh4\u2013Msh5 structure will facilitate the design of new mutational studies of the complex, interpretation of MSH4/5 polymorphism data in populations and a mechanistic understanding of Msh4\u2013Msh5 function in meiotic crossing over.The aim of this study is to provide a structural basis for understanding Msh4\u2013Msh5 function as well as molecular explanation for each of these S. cerevisiae Msh4\u2013Msh5 based on alignment with the hMSH2\u2013hMSH6 complex (PDB code 2o8b) as the template. Alignments between the templates and targets obtained from automatic programs are considered unsatisfactory. This is because, automatic programs tend to introduce breaks in the middle of regular secondary structural elements, or align hydrophobic residues in the target with solvent exposed residues in the template among other reasons. Extensive manual intervention was therefore required to arrive at a high quality alignment suitable for the comparative modeling. Reasons for choosing hMSH2\u2013hMSH6 complex as the template are discussed below.Crystal structures of bacterial MutS, and eukaryotic MutS\u03b1 and MutS\u03b2 complexes are available The choice of using hMSH2\u2013hMSH6 as the template was intentional based on the rigorous analysis of the quality of alignment between Msh4 & hMSH2, Msh4 & hMSH6, Msh5 & hMSH2, Msh5 & hMSH6 and Msh4 & hMSH3 pairs. Poor sequence identity of the order of 20% amongst all pairs meant that templates could not be decided purely based on sequence identity. Therefore all possible alignments were assessed to decide the template. The curated alignment obtained has been provided in It was observed that there existed more cases of in/dels within regular secondary structures in case of hMSH2 as template for Msh4 and hMSH6 as template for Msh5. The assessment of the alignment involved the use of structural environments around the sequence. Also, features such as solvent accessibility and secondary structure of residues were considered. For example, buried apolar residues replaced by buried polar residues and exposed polar residues being replaced by exposed apolar residues were commonly seen when Msh4 was aligned with hMSH2 and Msh5 with hMSH6 as the template. So, if the templates are swapped in modeling Msh4 and Msh5, not only was the quality of alignment poor, the modeled structure had large number of short contacts and collapsed during the energy-minimization steps. Two non-bonded atoms are said to be in short contact if their inter-atomic distance is too short in comparison to the classic contact criteria proposed by Ramachandran and coworkers The Msh4\u2013Msh5 model was used to address asymmetry of the Msh4 and Msh5 subunits within the complex, to map interface residues between Msh4 and Msh5 and to analyze the interaction of the complex with Holliday junction DNA. These are discussed in further detail below.msh4 G639A and msh5 G648A have spore viability similar to msh4/5\u0394 mutants , Y480A (67%), V488A (40%), and L548A (50%) respectively. Reasons for their asymmetric phenotypes are outlined below. The ATP binding mutations msh4 G639A and msh5 G648A are poorly tolerated in both Msh4 and Msh5 because of structural constraint. The Glycine residues have positive \u03c6 values which is not comfortably adopted by non-Glycine residues. This is also indicated by the high conservation of these residues. The ATP hydrolysis residue, Msh5 R685 is involved in main chain hydrogen bonding to stabilize the \u03b2 sheet as shown in msh5 R685W mutation is therefore poorly tolerated compared to the equivalent mutation msh4 R676W. In the DNA binding domain, Msh5 D527 is a solvent exposed residue and hence leads to instability when mutated to a hydrophobic residue such as Alanine. The Msh5 Y480 in the DNA binding domain is involved in aromatic interactions as shown in Subunits of the MutS and MutS\u03b1 complexes show asymmetry for mismatch binding and ATP hydrolysis  mutants  [6]. But3. The images of the cavity are shown in 3. The Holliday junction is known to take up stacked conformations in the presence of metal ions Volume of the cavity in the Msh4\u2013Msh5 modeled structure was calculated to be 16676 \u00c5Most probable Msh4\u2013Msh5 interface residues were identified as discussed in The Msh4\u2013Msh5 model structure can be used to predict mutational changes that are compensatory. For example, in the putative interface region, Msh4 K819, D283 and K284 are involved in ionic interactions with Msh5 D732, H264 and D269 respectively. In principle if these residues are mutated such that the overall interaction is retained, for example K819D and D732K, the phenotype is expected to be close to the wild type. The ongoing efforts are directed towards design and generation of such mutants which will further our understanding of sequence-structure-function relationship of Msh4\u2013Msh5 complex.msh4/5 mutations are likely to cause meiotic defects by three main modes. The mutation may disrupt the structural integrity of local regions and hence affect the overall stability of the Msh4\u2013Msh5 complex. The mutation may disrupt the interaction between the Msh4 and Msh5 proteins and prevent complex formation. Finally, the mutation may affect DNA binding by the Msh4/5 proteins.The msh4/5 mutations cause significant meiotic defects  could not be explained with the Msh4\u2013Msh5 modeled structure. Yeast-two-hybrid analysis suggests sixteen mutations disrupt the Msh4\u2013Msh5 complex  have meiotic defects similar to msh4/5\u0394. In our model these involve residues stabilizing \u03b1-helical regions, residues part of the left handed \u03b1 helical region of the Ramachandran map, residues involved in aromatic-aromatic interactions, cation-pi interactions, ionic interactions, hydrogen bonding and buried or solvent exposed residues. A significant proportion of interactions involve hydrogen bonding of main chain and side chain atoms or side chain and side chain atoms. A detailed information on the residues that constitute this network has been indicated in Nine mutations, two in Residues stabilizing \u03b1-helical regionsMsh4 D139 and Msh5 D433 are involved in stabilizing \u03b1-helical regions. The Msh4 D139 serves as an N-cap residue to stabilize a helix four residues downstream. In the case of Msh5, N430 is not a good initiator of the \u03b1-helix and hence the D433 stabilizes the structure by means of a hydrogen bond between side chain of D433 and main chain amide of N430 as shown in Residues with conformations in left handed \u03b1-helical region of the Ramachandran mapMsh5 G648 and Msh4 G639 are two residues with a positive \u00f8 dihedral angle. These angles are accommodated only in the case of Glycine due to the lack of side chain. When these residues are mutated to Alanine with this combination of \u00f8 and \u03a8 angles the residues experience short contacts involving their side chains and hence destabilize the structure.Residue involved in aromatic-aromatic and cation-pi interactionsmsh5 W298A, Alanine has an aliphatic side chain which cannot participate in such an interaction and hence affects stability. The Msh5 W298 is also involved in a cation pi interaction with Msh5 R312 . These residues are involved in aromatic-aromatic interactions, cation-pi interactions and ionic interactions. A few residues are involved in stabilizing the DNA binding region. However, in two cases msh4 L493A and msh4 D532A no explanation could be provided on the basis of the modeled structure. This is mainly due to the high sequence variation between the hMSH2\u2013hMSH6 template and the Msh4\u2013Msh5 model in this region.In terms of severity of phenotypes, these set of mutations second the null mutations. There are nine such mutations, four in msh5 D680A mutant.Msh4 Y143, F194 and Msh5 Y480 are involved in aromatic-aromatic interactions with surrounding aromatic residues within a distance of 6 \u00c5 as shown for Y480 in msh4/5-t mutations. There are 9 such mutations distributed in both MSH4 and MSH5 . These residues are involved in aromatic-aromatic interactions (Msh5 Y486), cation-pi interactions (Msh4 F491) and ionic interactions  with surrounding residues in 6 \u00c5 radius. Msh4 E276 and N532 are involved in a tight packing which may be disturbed upon mutation to Alanine which has a smaller side chain. The Msh5 S416 is a residue proximal to DNA and hence may affect the stability of binding. Structural explanation could not be provided for the meiotic defects observed in mutations msh5 D76A and D539A.These mutations are deviant from the wild type only with respect to recombination frequency and have been described previously as S. cerevisiae Msh4\u2013Msh5 complex. The modeling studies suggest that Msh4 is most likely functionally similar to hMSH6 of the hMSH2\u2013hMSH6 complex and likewise Msh5 is similar to hMSH2. The model also explains the functional asymmetry between Msh4 and Msh5 with respect to ATP and DNA binding mutations S. cerevisiae Msh4 and Msh5 proteins have been analyzed by mutational studies msh4/5 mutations affecting crossover frequency and spore viability. The model can also facilitate the design of new mutational studies, design of structure based inhibitors of the Msh4\u2013Msh5 complex as well as predict the functional impact of polymorphisms in the MSH4, MSH5 genes. Such studies will be useful for understanding the mechanism of crossover formation by the Msh4\u2013Msh5, Mlh1\u2013Mlh3 pathway The Msh4\u2013Msh5 complex plays an important role in different stages of the meiotic recombination pathway. In the absence of a crystal structure for this complex, we built a homology model of the \u221210 suggesting high confidence and that both complexes are likely to adopt the same fold. In addition, structural alignments of the S. cerevisiae Msh4, Msh5 protein sequences were also obtained from Bioinfo metaserver or 3D-Jury which uses the alignment information that is predicted consistently by other reliable servers S. cerevisiae Msh2\u2013Msh6 complex is also known to bind Holliday junction structures which further justifies its use as a template Two possible templates (hMSH2\u2013hMSH6 and hMSH2\u2013hMSH3) are available for the modeling of Msh4\u2013Msh5 complex The model of Msh4\u2013Msh5 complex was built using MODELLER (version 9.10) auto-model program with added energy optimization steps Intra and inter protein interactions were identified using the PIC  server Homologues of Msh4 and Msh5 of not necessarily known 3-D structure were obtained by use of PSI-BLAST queried against the UNIPROT-SPROT database Volume measurements were made using the 3V server, which is known to use a robust algorithm to determine the size of cavities. We used 3V to calculate the size of the cavity formed by the Msh4\u2013Msh5 heterodimer and also to calculate the volume of Holliday junction DNA Figure S1Alignment of hMSH6, hMSH3 and Msh4 sequences. 2o8bB corresponds to the hMSH6 from the hMSH2\u2013hMSH6 complex and 3THY corresponds to hMSH3 from the hMSH2\u2013hMSH3 complex. Highlighted in yellow are the deletions that are unique to hMSH3.(PDF)Click here for additional data file.Figure S2Alignment of Msh4 and Msh5 amino acid sequences with the hMSH2\u2013hMSH6 complex (PDB code 2o8b). The alignment was used to model the Msh4\u2013Msh5 complex structure. Msh4/5 residues whose mutations cause null phenotype, intermediate defects in crossing over and viability or only crossover defects are shown in blue, green and red boxes respectively.(PDF)Click here for additional data file.Figure S3Structure based sequence alignment of Msh1\u20136 and hMSH2, hMSH3, hMSH6. Highly conserved positions are highlighted in red, positions with conservative substitutions are highlighted in a blue box with residues marked in red. DNA binding residues identified from literature survey are highlighted in a yellow box. Residues predicted by Multi-VORFFIP (MV) server to have high probability of DNA binding are highlighted in a black box.(PDF)Click here for additional data file.Figure S4Protocol used to model Msh4\u2013Msh5 structure. The sequences were submitted to three structure prediction servers and the alignment obtained from all these were compiled and manually curated to get final alignment which was provided to MODELER to build the structure. The structure was then energy minimized and checked for stereo-chemical quality .(PDF)Click here for additional data file.Table S1Sequence to structure mapping for Msh4.(XLSX)Click here for additional data file.Table S2Sequence to structure mapping for Msh5.(XLSX)Click here for additional data file.Table S3Explanation for meiotic defects observed in msh4/5 mutants based on the homology model.(DOCX)Click here for additional data file.Table S4Probability of each residue to bind/stabilize DNA.(XLSX)Click here for additional data file."}
+{"text": "Much of Figure 1 was removed due to a cropping error. Additionally, the color of each figure was incorrectly modified which may affect the readability. Please see the corrected figures here:Figure 1: Figure 2: Figure 3: Figure 4:"}
+{"text": "In studying the epidemiology of parvovirus 4 (PARV4) in Taiwan, we detected DNA in plasma of 3 mothers and their newborns with hydrops. In 1 additional case, only the mother had PARV4 DNA. Our findings demonstrate that PARV4 can be transmitted through the placenta. Parvoviridae family  LPGYNYVGPGNPL  and YKYPYVLGQGQDTL  YDYPYVLGHNQDTL .Transmission routes of human parvovirus 4 (PARV4), a recently discovered member of the To exclude the possibility of antibody cross-reaction between PARV4 and B19V, we tested plasma samples sent to our laboratory for confirmation of B19V infection with PARV4 immunoblot. Unexpectedly, we detected PARV4 DNA in plasma from a mother and her newborn with hydrops. Therefore, we examined samples from 5 additional infants with hydrops.During 2000\u20132009, our laboratory received blood samples from 6 infants with nonimmune idiopathic hydrops . Paired Antibodies to PARV4 and B19V were tested by immunoblots. DNA of PARV4 and B19V was detected by seminested and nested PCR, respectively. PARV4 immunoblot and PCR were performed according to the methods in our previous report (Four of the 5 mothers had immunoglobulin (Ig) M against PARV4 . Two of Only the mother and newborn of case A had detectable B19V DNA (genotype 1). By contrast, PARV4 DNA (genotype 2) was found in plasma of all but 1 of the 6 case-patients. The newborn negative for PARV4 DNA received a whole-blood exchange before sampling.,,,The first serologic study (Contrary to the epidemiology of PARV4 in Europe, studies in Africa found different transmission routes and a higher seropositive rate in blood donors and the general population. In Ghana, 8.6% of infants had PARV4 viremia (,PARV4 can be transmitted through nonparenteral routes (Maternal PARV4 infections were diagnosed by detection of PARV4 DNA in all 5 mothers; 4 of whom had IgM against PARV4. Using IgM against PARV4 as evidence of recent infection must be done cautiously because of persistent IgM against PARV4 (Persons with past B19V infection are expected to have IgG against B19V VP1 but not VP2 in immunoblot (In conclusion, PARV4 can be transmitted parenterally and placentally. Other transmission routes might exist and remain to be discovered. Prospective studies of PARV4 infection during pregnancy are needed to clarify the effect of PARV4 infection on fetal outcome.Infants with nonimmune idiopathic hydrops fetalis are not routinely checked for parvovirus B19 infection."}
+{"text": "Heat shock transcription factor 1 (HSF1) plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE) are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications. S. cerevisiae HSF directly activates the expression of genes whose protein products are involved in protein folding and degradation, ion transport, signal transduction, energy generation, carbohydrate metabolism, vesicular transport, cytoskeleton formation and other cellular functions All organisms are exposed to proteotoxic stresses that result in the accumulation of misfolded proteins. In response to these stresses cells have evolved adaptive responses to protect and stabilize cellular proteins until more favorable conditions for cell proliferation are encountered While mammalian cells express four distinct HSF proteins encoded by separate genes, HSF1 is the primary factor responsible for stress responsive gene transcription in vitro phosphorylation experiments and in vivo studies using either lexA or Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain. As such, many of the earlier studies exploring S303 and S307-dependet regulation of HSF1 activity have resulted in conflicting results. For example, previous phosphorylation experiments suggested that S307 was phosphorylated by ERK which, in turn, acted as an essential priming step for GSK3-dependent phosphorylation of S303 in vitro studies suggested that S303 could also be phosphorylated by a variety of mitogen activated protein kinases (MAPK) including the stress responsive MAPK p38 in vivo data suggested S303 phosphorylation could occur independently of S307 phosphorylation The activity of HSF1 is also thought to be negatively regulated through a number of post-translational modifications including phosphorylation, sumoylation and acetylation While the specific mechanism by which S303 and S307 phosphorylation repress HSF1 activity remains unclear, evidence has suggested that S303 and S307 phosphorylation represses the transactivation potential of HSF1 S. cerevisiae is unable to complement for the loss of the essential yeast HSF protein While HSF1 and the cognate HSEs are quite well conserved from yeast to humans, our previous results demonstrated that human HSF1 expressed in Here we report the use of the yeast assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. Our results suggest that S303 phosphorylation blocks human HSF1 homotrimerization thereby preventing human HSF1 activation and complementation of the loss of yeast HSF. Furthermore, we demonstrate that S303 phosphorylation also blocks HSF1 homotrimerization in mammalian cells. We show that phosphorylation of HSF1 S303 in yeast occurs via the action of the MAPK Slt2 and not via the action of GSK3 and we extend these findings to show that S303 phosphorylation also occurs independent of GSK3 in mammalian cells.When human HSF1 is expressed in yeast it is unable to homotrimerize, promote gene expression and complement for the loss of the essential yeast HSF protein To ascertain whether HSF1 is being phosphorylated in yeast, we employed a commercially available antibody specific for phospho-S303 (pS303). Because this antibody has not previously been characterized in the literature, we tested its specificity in human HeLa cells where S303 is known to be constitutively phosphorylated Consistent with a contribution to HSF1 repression  S303 is Human HSF1 S303 phosphorylation is known to promote sumoylation of lysine 298, which also contributes to the repression of HSF1 activity \u2212/\u2212 mouse embryonic fibroblasts (MEF) Previous reports have suggested that phosphorylation of S303 represses the ability of HSF1 to transactivate gene expression In addition to post-translational modifications, HSF1 activity is also thought to be repressed through intramolecular interactions between carboxyl- and amino-terminal coiled-coil domains and mutations in these domains render HSF1 constitutively trimerized, nuclear localized and bound to DNA in mammalian cells in vitro phosphorylation experiments have suggested that HSF1 is phosphorylated at S303 by glycogen synthase kinase 3 (GSK3) in vivo. To test if GSK3 contributes to HSF1 repression, we assayed human HSF1-dependent yeast growth in a strain also lacking the yeast GSK3 homolog Rim11. Supporting the notion that yeast GSK3 can repress human HSF1 activity in yeast we observed human HSF1-dependent yeast growth as well as HSF1 multimerization in the rim11\u0394 strain  in yeast phosphorylate human HSF1 at S303 to promote HSF1 repression, we assayed S303 phosphorylation in several previously generated protein kinase deletion strains obtained from the yeast gene deletion collection e strain . Homotriin yeast . In mamm\u0394 strain  also supin vitro phosphorylation experiments in vivo using lexA/Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain \u2212/\u2212 MEFs which lack endogenous HSF1. When we expressed S303A, S307A or S303/307A HSF1 mutants in hsf1\u2212/\u2212 MEFs we observed a modest elevation of Hsp70 expression under normal growth conditions  supplemented with protease inhibitors (Roche) and Halt phosphate inhibitor cocktail (Thermo Scientific Pierce). Proteins extracts were generated from mammalian cell culture using cell lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA assay and 80\u2013100 \u00b5g of total protein was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. HSF1 oligomerization was assessed using the amine-specific cross-linker ethylene glycol bis-succinimidyl succinate (EGS) (Pierce). Crosslinking analysis were carried out as described previously"}
+{"text": "Chronic hepatitis C virus infection and its sequela are major health problems facing the Egyptian community. The high prevalence and spread rates of the disease require serious actions to stop or decrease these rates. Determination of HCV genotypes and subgenotypes adds significant knowledge about the epidemiology of the disease, and provides an added value in the decision making process of what strategy to follow and what therapy response to expect. The molecular epidemiology and genetic variability of HCV variants circulating in Egypt still need further analysis.The study was held to evaluate the genotype and subgenotype of the hepatitis c virus circulating in Sharkia as one of the large governorates of Egypt, which was not included in any study for genotyping of the virus before.The HCV molecular epidemiology in Sharkia governorate was studied using direct sequencing and further phylogenetic analysis of a partial NS5B region of the HCV genome from 63 patients. HCV genotype and subtype were successfully determined in 62 out of 63 patients.The highest prevalent genotype was genotype 4a, which was found in 57 patients (92%) followed by 2 isolates (3%) with genotype 4o, 2 strains (3%) with genotype 1g and one isolate (2%) with genotype 4n.This molecular epidemiology study revealed high prevalence of HCV genotype 4, subtype 4a among Egyptian patients residing in Sharkia governorate, Egypt. Hepatitis C virus (HCV) infectionis estimated to affect 130\u2013170 million people worldwide (1). Although the clinical manifestation of HCV acute infection is generally mild or asymptomatic, it can lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The main indication for liver transplantation in the industrialized world is related to HCV infection .HCV is a positive-sensed single-stranded RNA virus belonging to the Flaviviridae family. The HCV genome is an RNA molecule of approximately 9600 nucleotides, structured in a coding region that contains one large open reading frame (ORF), flanked by non-translated regions (NTR) at the 5\u2019 and 3\u2019 ends encoding a polyprotein precursor of about 3,000 amino acids. The precursor is cleaved into at least 10 different proteins comprising the structural proteins, core, E1, E2, and p7 as well as the non-structural proteins, NS2, NS3, NS4A, NS4B, NS5A, and NS5B , following the manufacturer\u2019s instructions.HCV genotypes were identified via direct sequencing of non-structural (NS5B) viral genes with universal primers described by Murphy et al. 2007 , and sequenced using the dideoxynucleotide chain termination method with the ABI PRISM\u00aeBigDye Terminator Cycle Sequencing Reaction kit  Sequence analysis was performed with the ABITM3130 Genetic Analyzer . The sense and antisense primers described above were used as sequencing primers. The chromatogram sequencing files were inspected using Chromas 2.3 . The genotype of each sample was determined by comparing its sequence with those of HCV prototypes obtained from GenBank, followed by further genetic analysis. The DNA alignments were generated with ClustalX version2.0 software .HCV genoThe NS5B region corresponds to a 389 nt fragment covering the middle section of the NS5B gene of the HCV genome. The direct sequencing and phylogenetic analysis of the HCV NS5B gene was successful in 62out of 63 positive samples. Samples number Eg 21 and Eg 22 had been sequenced two times and both of them have exactly the same sequence in each run. They were expressed in the tree as one sample (Eg 21). The result of the study revealed that subtype 4a was the most prevalent in this population with a total number of 57 (92%).Subtype 1g and 4o were each identified in 2 samples (3%), and 4n was identified in 1 sample only (2%). Preliminary phylogenetic analysis containing 56 sequences (data not shown) was performed and allowed a selection of 37 additional GenBank sequences corresponding to the most related ones to the study isolates. These reference sequences were named after their accession numbers, subgenotype and country of origin. Partial NS5B coding sequences obtained from Egyptian samples were aligned with the reference sequences. The phylogenetic tree obtained by performing neighbor-joining (NJ) analysis of the alignment of sequences is shown in Three samples clustered in a different branch with reference sequences from genotype 4. HCV isolates Eg-10 and Eg-48 clustered with reference sequences of HCV genotype 4o from Egypt and from Canada with a high bootstrap value of 98%. The Eg-16 isolate clustered with three reference sequences of genotype 4n from Egypt, Canada and an Egyptian immigrant from the Netherlands, with a bootstrap value of 95%.Another 2 samples (Eg-40 and Eg-62) branched with reference sequences of HCV genotype 1g from Canada and from Egypt with a bootstrap value of 94%.Sequences were submitted to GenBank (accession numbers: JN203143 to JN203205).According to different reports, the prevalence of HCV in Egypt ranges from 11% to higher than 14% of the general population, 5\u20137 million people with active infection having detectable HCV RNA and over 500,000 new infections each year -17. In cIt is known that genotype and even subtypes differences of HCV havea high impact on interferon responsiveness and give an idea about the natural history of the disease . In EgypIn a study on 206 samples collected from the Kasr El-Aini School of Medicine and the National Cancer Institute, Cairo, Egypt, HCV genotype 4 was detected in 186 samples (90.3%). Five other subtypes could be also identified: subtypes 4a, 4d, 4m, 4n, and 4o. They also found that 9.7% of the patient\u2019swereinfected with HCV genotype 1g and 1a . In the Another study, from the Alexandria district, showed that 78% of the isolates were HCV genotype 4a variants, whereas the remaining identified variants were 4m (11%), 4o (5.5%), 4n (2.7%) and 4p (2.7%) . In IsmaIn conclusion, this study shows that HCV genotype 4ais predominant in the governorate of Sharkia, and it confirms that the Egyptian HCV epidemic is composed of multiple lineages of genotypes 4 and 1."}
+{"text": "FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER\u2013resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the  SCS3 and YFT2, the yeast homologs of mammalian FIT2, are part of a large genetic interaction network connecting lipid metabolism, vesicle trafficking, transcription, and protein synthesis. From these interactions we determined that yeast strains lacking SCS3 and YFT2 are defective in their response to chronic ER stress and cannot induce the unfolded protein response pathway or transcription of phospholipid biosynthetic genes in low inositol. Our findings suggest that the mammalian FIT genes may play an important role in ER stress pathways, which are linked to obesity and type 2 diabetes.The ability to form lipid droplets is a conserved property of eukaryotic cells that allows the storage of excess metabolic energy in a form that can be readily accessed. In adipose tissue, the storage of excess calories in lipid droplets normally protects other tissues from lipotoxicity and insulin resistance, but this protection is lost with chronic over-nutrition. The FAT storage-inducing transmembrane (FIT) proteins were recently identified as a conserved family of proteins that reside in the lipid bilayer of the endoplasmic reticulum and are implicated in lipid droplet formation. In this work we show that specific functions of the FIT proteins are conserved between yeast and humans and that  Eukaryotic cells store neutral lipids  in cytoplasmic lipid droplets (LDs) surrounded by a monolayer of phospholipids and associated proteins FIT gene expression decreases LD production in an adipocyte differentiation cell culture model and in zebrafish Fat storage-Inducing Transmembrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were identified as unique, evolutionarily conserved ER-resident proteins that affect the partitioning of TG into LDs FIT2 gene homologs, SCS3 and YFT2, that are readily identified as the only homologous genes in S. cerevisiae in BLAST searches using the full-length human protein as a query  Budding yeast contains two The metabolic pathways leading to the formation of neutral lipids and the hydrolytic reactions catalyzing their mobilization are conserved between yeast and mammalian cells FIT gene homologs, SCS3 and YFT2. Despite the distant evolutionary relationship between these genes in fungi, the data show that SCS3 and YFT2 have shared as well as unique functions. Common functions are also demonstrated between the proteins in yeast and human systems. However, in contrast to FIT gene knockdown experiments in higher eukaryotes, deletion of SCS3 and/or YFT2 does not noticeably impact the number or size of LDs. A genetic network centered on SCS3 and YFT2 is presented that identifies a multitude of aggravating and alleviating interactions that connect major biosynthetic processes critical for cell growth. We find that the functions of SCS3 and YFT2 relate lipid metabolism and signaling with transcription of phospholipid biosynthetic genes and protein synthesis. More specifically, the data show that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and suggest that the function of Scs3 and Yft2 impacts a regulatory response that serves to coordinate these processes.We have initiated studies in yeast to identify chemical and synthetic genetic phenotypes associated with deletions of the mammalian FIT genes in mammals YFT2 was an uncharacterized open reading frame (YDR319C) whose relationship  to SCS3 was not appreciated. Consequently, none of the studies conducted to date on SCS3 have taken into account its potential functional redundancy with YFT2. Phylogenetic evidence from 42 sequenced fungal genomes YFT2 arose by a segmental duplication of SCS3  elements and drives the expression of phospholipid biosynthetic enzymes and other lipid metabolic proteins SCS3 or YFT2 or both genes were hypersensitive to cerulenin as indicated by the reduced cell density at saturation  and sterol-14 reductase (Erg24) to inhibit ergosterol biosynthesis  but relates SCS3 function to increased translational fidelity based on the properties of a single base substitution within yeast 18S rRNA that also confers this phenotype SCS3 increased the toxicity of cadmium ions. Although the mechanism of this toxicity is not thoroughly understood, cadmium is known to cause lipid peroxidation and like many metals, affects membrane fluidity scs3\u0394 strains results from an accumulation of metal ion, potentially due to altered membrane composition and/or transporter function (see below). Inositol auxotrophy has been associated with deletion of SCS3 since the gene was first cloned scs3\u0394 strain was attributed to decreased levels of inositol-3-phosphate synthase (Ino1), the rate-limiting enzyme in the synthesis of phosphatidylinositol (PI) YFT2  YFT2 . Howeverof scs3\u0394  as well FIT2 in yeast, we used the fluorescent LD-specific dye BODIPY 493/503 to determine whether either SCS3 or YFT2 could drive the production of LDs in human embryonic kidney (HEK293) cells. Remarkably, transient expression of either SCS3 or YFT2 under the control of the CMV promoter induced the appearance of LDs in HEK293 cells similar to overexpression of the human and mouse FIT genes , suggest that more complex interpretations should be considered: Yeast may possess alternative mechanisms for storing neutral lipids in droplets. Thus, the absence of one mechanism due to deletion of SCS3 and YFT2 may be compensated by the presence of another. Differences in the size of LDs between S. cerevisiae and organisms where FIT gene-dependent changes have been observed could also be important: The average diameter of LDs in yeast ranges between 0.35\u20130.45 \u00b5m compared to 1.1 \u00b5m in C. parapsilosis and up to 100 \u00b5m in higher eukaryotes Given the complementation of mutant phenotypes by human IT genes  [10]. NoIT genes . Recentlhenotype . Taken aan cells  and the SCS3 and YFT2 and to uncover functional relationships with other genes and processes, we conducted synthetic genetic array (SGA) analysis using query strains deleted for either or both genes , we found that the total number of interactions increased significantly using the double mutant query  in at least one of the three screens and then compared their \u03b5 values for all three queries with the corresponding values for the overlapping verified genes. The scores were highly correlated  among the 221 \u201ctrue positives\u201d revealed a sharp cutoff for aggravating and alleviating interactions at \u22120.1 and 0.2, respectively, in the single mutant query screens . The samSCS3 and YFT2. Indeed, we identified many interactions with one or the other gene as well as interactions that required deletion of both genes  phosphatase that is localized to the ER and Golgi and functions in protein trafficking, secretion and cell wall maintenance. Deletion of SAC1 dramatically and selectively increases the level of PtdIns(4)P (8\u201312 fold) and causes missorting of Chs3 to the vacuole SAC1 mutant compromises cell wall maintenance SAC1 substantially reduces strain fitness (to 0.48 relative to wild-type) and this is further aggravated by deletion of either SCS3 or YFT2. However, the fitness of the triple mutant strain is significantly improved (to 0.78 relative to wild-type) indicating the strong alleviating effect of deleting both SCS3 and YFT2. On the other hand, deletion of CHS3 does not have a significant impact on strain fitness by itself but exhibits reduced fitness in combination with either SCS3 or YFT2. These effects are suppressed in the triple mutant strain. The converse of this pattern of genetic interactions was also seen  where each query gene-deletion had alleviating interactions with a particular array gene deletion that was reversed when both SCS3 and YFT2 were deleted. These types of genetic interactions indicate that the functions of SCS3 and YFT2 (or the processes that they impact) sometimes antagonize one another.Synthetic genetic interactions reveal functional relationships between genes th genes . These ge mutant . In otheSCS3 and/or YFT2. Five GO bioprocess terms were significantly over-represented in our data (SCS3 and YFT2 in the cell: They linked together processes (e.g. chromatin-transcription and secretion) that individually are among the most highly connected in the global genetic landscape SCS3 and/or YFT2 is very similar to the genome as a whole  followed by the Golgi and the mitochondrial envelope  that convert PE to PC in the terminal steps of the CDP-DAG pathway had the opposite phenotype  in combination with deletions of SCS3 and/or YFT2  since deletion of either one or both genes rescues the poor growth of a strain lacking Arg82, the inositol polyphosphate multikinase responsible for synthesizing IP4 and IP5  which function as controlling ligands for different biochemical activities P (noted above), suppresses defects associated with a temperature-sensitive mutant of the opposing PI 4-kinase, Stt4, localized at the plasma membrane sac1\u0394 strain by deletion of both SCS3 and YFT2 could involve or impact functions at the plasma membrane.A role for  and IP5 . DeletioSCS3 and YFT2 to the synthesis of phospholipids, sphingolipids and inositol phosphates are reflected in the cross-talk between these pathways involving lipid-derived second messengers such as phosphatidic acid (PA) and diacylglycerol (DAG) PAH1 and DGK1 which encode PA phosphatase (lipin) and DAG kinase, respectively. Deletions of PAH1 and DGK1, which catalyze the interconversion of PA and DAG SCS3 and YFT2  elevates the concentration of PA which up-regulates the transcription of phospholipid biosynthetic genes and drives a dramatic expansion of the nuclear/ER membrane  and restores normal levels of Ino1 protein SCS3, can be suppressed by deleting the Opi1 repressor INO-regulated genes, increased inositol synthesis and altered lipid composition scs3\u0394 strains  which is recruited to the INO1 promoter via its interaction with the DNA binding factor Ume6 SCS3 and YFT2 and this presumably coincides with reduced transcription of INO1 and other target genes in the triple mutants relative to the Rpd3(L) single mutants  and in Sgf73 which tethers this module to the rest of the SAGA complex INO-regulated genes SET2 and specific subunits of the Rpd3(S) complex (RCO1 and EAF3) exhibit aggravating interactions with SCS3 and YFT2 , deletions of multiple INO80 subunits led to alleviating rather than aggravating phenotypes in the scs3\u0394 yft2\u0394 strain (INO1 gene activation SCS3 and YFT2 rescuing the poor fitness of INO80 complex mutants that are deficient in a repressive function in transcription. This interpretation is supported by strong negative genetic interactions between the INO80 complex and the Rpd3(L) HDAC complex indicating that they function in redundant parallel pathways  pathway, namely the Ire1 sensor kinase/endoribonuclease and the Hac1 transcription factor INO1 gene transcription requires Ire1 and Hac1 and starvation for inositol  and ribosome assembly (e.g. Rei1)  . These rand YFT2 . These iSCS3 and YFT2 resulted in strong aggravating phenotypes with deletions such as SEY1 and ICE2 (proteins linked to ER morphology) and components of the secretory pathway  that causes secretory stress and activates the UPR as a result of defects in COPII-mediated vesicle formation YFT2, SCS3 or both genes in the sec13-1 strain caused an increasingly strong synthetic sick growth phenotype at the permissive temperature of 22\u00b0C  induce transcription of phospholipid biosynthetic genes (e.g. INO1) and activate the UPR (increase HAC1 pre-mRNA splicing) SCS3 and YFT2 deletion strains were significantly compromised for both of these responses  and 0.2% Tween 80 SCS3, YFT2 and OPI1 were generated de novo in BY4742 using standard one step gene replacement and dominant drug selectable markers Synthetic complete media contained 400 \u00b5M myo-inositol unless otherwise stated. Low or no inositol media was prepared from inositol-free YNB (Difco) and amino acid dropout mixes. YPO media contained 0.3% yeast extract, 0.5% peptone, 0.1% glucose, 0.5% KHSCS3 (scs3\u0394::natMX), YFT2 (yft2\u0394::natMX) or both genes (scs3\u0394::natMX yft2\u0394::URA3) and screened by standard SGA methods against an array of 4292 strains representing a healthy subset  of the viable gene-deletion collection hda robot. Digital images of the double or triple mutant colonies were captured and analyzed using ColonyImager software ura3\u0394::natMX), against the same deletion array.Query strains were derived from Y7092 his3\u0394::kanMX strain his3\u0394::kanMX colonies on the inner perimeter of each plate (the plate median) and of all 98 plates  was determined and used to normalize the colony sizes on each plate as follows: Normalized size\u200a=\u200aunnormalized size\u00d7343/plate median. Following normalization, the mean colony size (\u00b1 standard deviation) was calculated for each mutant (represented by 10 colonies in the control screens and six colonies in each query screen) and the data were filtered to remove outliers (>2 standard deviations from each mean). After recalculation of the mean and standard deviation for each colony, the difference in the pixel size of each mutant on the array was computed between the control and query screens. The significance of this difference was assessed by performing a two-tailed t-test  of the null hypothesis that the sizes of the colonies in the control and query screens are statistical the same. The fitness of the query strains relative to wild-type was determined by comparing the median his3\u0394::kanMX colony sizes on the inner perimeter for all plates in each query screen versus the control screen (i.e. 3108 colonies for each query and 5180 colonies for the control). The resulting relative fitness values  were used to calculate genetic interaction scores (\u03b5) as described below. The fitness of each array deletion strain as a single mutant or as a double or triple mutant (combining deletions of SCS3 or YFT2 or both genes) relative to wild-type was determined from the ratio of the mean colony size of the mutant to the median his3\u0394::kanMX colony size from the 5180 control colonies. Genetic interaction scores for digenic pairs were calculated as the difference between the observed and expected fitness values of the double mutants where expected fitness was calculated as the product of the fitness of the corresponding single mutants . For trigenic interactions, a similar multiplicative model was employed, \u03b5\u200a=\u200aWxyz \u2013 (Wx * Wyz), where Wxyz is the observed fitness of the triple mutant, Wx is the fitness of the array deletion strain and Wyz is Wscs3\u0394 yft2\u0394 (see above). The general applicability of this solution in the current study is satisfied by the modest 11% fitness defect (Wscs3\u0394 yft2\u0394\u200a=\u200a0.89) of the scs3\u0394 yft2\u0394 query strain relative to wild type. Unless otherwise indicated, subsequent analyses employed stringent criteria to select interactions with a high probability of representing true positives. These criteria included (i) \u226540 pixel size difference between single mutant and double or triple mutant colonies; (ii) a p value \u22640.01 representing the probability that the colony sizes of the strains being compared are significantly different from one another and (iii) \u03b5 scores \u2264\u22120.12 or \u22650.2. Similar criteria for high stringency cutoffs were applied in the large scale study of Costanzo et al SCS3. In an E-MAP of the early secretory pathway SCS3 were reported that had an S score <\u22121.5. We scored 22 of these and found 14 (64%) that met our stringent criteria . In addition, many genetic interactions with SCS3 were identified by Constanzo et al., p value (I\u03b5I>0.08 and p<0.05). From this group we determined that 140 unique interactions were scored in our dataset. Forty-eight of these met our stringent criteria and a total of 64 interactions (46%) were found when interactions were compared at the same p value limit (p<0.05).Each plate of the deletion array contained a two-colony perimeter of the same SCS3 and/or YFT2 and then calculated enrichment by hypergeometric distribution.Biological process annotations for genes on the viable gene-deletion array were obtained from supplementary data file S6 of Costanzo et al 600 readings from 100-well plates every 15 minutes. A single colony was inoculated at a starting OD600 of 0.1 into SD/MSG media that contained 0.1% Nonidet-P40. Doubling time and growth curve data were derived from three independent colonies per strain or condition as per established protocols Growth at 30\u00b0C with shaking was measured at using a Bioscreen C Microbiology Reader (Growth Curves USA), which recorded ODSI Materials and Methods: \u201cMicroscopy\u201d section. For yeast imaging an upright Olympus BX61 microscope with a sensicam QE cooled CCD camera (Black/White) and IP Lab 4.0.8 software was used at the Analytical Imaging Facility at the Albert Einstein College of Medicine. Cells were viewed with a 100X NA\u200a=\u200a1.4 oil objective and FITC filter. Sections (0.2 \u00b5m) were collected for each image and combined into a single projection using Image J (http://imagej.nih.gov/ij/). Cells, either early log phase (between OD600 nm 0.2 and 0.8), freshly saturated overnight or 5 day stationary phase cultures, were stained with 0.5 \u00b5g/\u00b5l BODIPY 493/503 (Invitrogen), for 5 to 10 min. prior to fluorescence microscopy. Cells were prepared as indicated from either SD/MSG, YPD, YPO or SC-Ino media and either imaged immediately or fixed in paraformaldehyde and permeablized in 0.1% Triton as described in HEK293 cells were imaged as described in Methods for RNA preparation and Northern analysis have been reported elsewhere SCS3 and YFT2 deletion strains was achieved by growing strains for 2 hours in 1 \u00b5Ci/ml of [1-14C]acetate (57 mCi/mmol) or for 6\u20138 generations in 10 \u00b5Ci/ml 32P-orthophosphate. Lipids were extracted by the two-step 4\u00b0C method 2 and dissolved in chloroform/methanol  for thin layer chromatography. Labeled neutral and phospholipids were separated by one-dimensional TLC in hexanes\u2236ethyl ether\u2236acetic acid (80\u223620\u22361) 20 (30\u223635\u223635\u22367) 600 of log phase wild-type and SCS3 and YFT2 gene-deletion cells were prepared in triplicate and analyzed by mass spectrometry. Total lipid species are reported as nmol/108 cells. Mass spectrometry was performed at the Kansas Lipidomics Research Center Analytical Laboratory.Metabolic labeling of lipids in log phase wild type and Figure S1SCS3 and YFT2 in the genomes of sequenced fungi. Evolutionary relationships were constructed using the sequences of 153 genes that are universally present in the 42 genomes shown Saccharomyces complex, the group of species that share the whole-genome duplication (WGD) and those with the variant genetic code (CTG). YFT2 and SCS3 orthologs were identified by Blast searches of yeast genomes at NCBI or the Broad Institute databases using the complete amino acid sequences for SCS3 or YFT2 or the corresponding signature sequences from the fourth transmembrane domain of each protein. FIT gene orthologs were identified in all organisms except P. chrysosporium, A. terreus and C. globosum. Figure was adapted from ref. Phylogenetic distribution of (TIF)Click here for additional data file.Figure S2SCS3 YFT2 gene-deletion strains. A HsFit expression complements synthetic growth defects in scs3\u0394 yft2\u0394 xxx\u0394 strains. The HsFit2 gene, controlled by a TDH3 promoter and a CYC1 terminator, was substituted for YFT2 by one-step gene replacement in the Y7092 scs3\u0394::URA3 query strain. SGA techniques were then used to recover wild-type, scs3\u0394 yft2\u0394 and HsFit2-expressing scs3\u0394 yft2\u0394 strains containing the indicated deletions from the viable gene-deletion array. Final haploid colonies generated in quadruplicate were printed onto SGA selection media and photographed at 48 hours. Each row of panels is deleted for a different array gene (indicated on the right). Wild-type , scs3\u0394 yft2\u0394 (\u0394\u0394) and HsFit2-expressing scs3\u0394 yft2\u0394 (\u0394\u0394 HsFit2) strains are annotated across the top. Note that only the ice2\u0394 strain is an inositol auxotroph and the growth phenotypes were assayed on SGA medium which contains excess inositol. B HsFit expression in the scs3\u0394 yft2\u0394 strain does not induce the UPR. Northern analysis of the distribution of HAC1 mRNA into unspliced Hac1u and spliced Hac1i forms and stable U3 snRNA was performed on RNA from early log phase cultures of wild-type, scs3\u0394 yft2\u0394 (\u0394\u0394) and HsFit2-expressing scs3\u0394 yft2\u0394 (\u0394\u0394 HsFit2) strains grown in the presence of excess inositol. The extent of Hac1 splicing is expressed as % of total Hac1 mRNA and is indicated under each lane. ER stress in untreated and DTT-treated cells is reported by the accumulation of spliced HAC1 mRNA.Expression of HsFit2 in (TIF)Click here for additional data file.Figure S3SCS3 YFT2 gene-deletion strains exhibit comparable lipid droplet phenotypes under various stress conditions. A. The indicated strains were grown in inositol-free media supplemented with 10 \u00b5M or 100 \u00b5M inositol . Early log phase and saturated overnight cultures were analyzed for lipid droplets by direct staining of live cells with BODIPY 493/503 as described in sec13-1 mutation on lipid droplet production in wild-type and SCS3 YFT2 gene-deletion strains. SGA methodology was used to introduce the conditionally-viable sec13-1 mutation as in Wild-type and (TIF)Click here for additional data file.Figure S4SCS3 YFT2 double gene-deletion strain and the SCS3 or YFT2 single gene-deletion strains, respectively. The data are represented in p value<0.01.Comparisons of genetic interaction scores between genes identified in two or more screens. Epsilon scores are compared for 167 (A) and 72 (B) strains that yielded genetic interactions with the (TIF)Click here for additional data file.Figure S5SCS3 and/or YFT2 genetic interactions  were manually examined in GBrowse for overlap with the coding and likely promoter regions of verified genes. From the resulting 40 dubious ORFs and their neighboring/overlapping verified gene-deletions we performed pairwise comparisons of the \u03b5 values obtained in scs3\u0394, yft2\u0394 and scs3\u0394 yft2\u0394 screens  and determined a Pearson correlation coefficient of 0.75.Correlation of genetic interaction scores between dubious ORFs and their genomic neighbors. Deletion strains corresponding to dubious ORFs in SGD that were contained in the set of 636 (TIF)Click here for additional data file.Figure S6SCS3/YFT2 gene-deletion strains. A. Neutral lipid content is comparable between a wild-type strain and strains deleted for SCS3 and/or YFT2. Log phase cells were metabolically labeled for 2 hours with 14C-acetate and lipids were extracted by the two-step 4\u00b0C method SCS3 and YFT2 does not affect total cellular lipid profiles. Lipids from log phase wild-type and scs3\u0394 yft2\u0394 cells were prepared from unlabeled log phase cells as described above and analyzed by quantitative mass spectrometry at the Kansas Lipidomics Research Center Analytical Laboratory. The abundance of lipid species is graphed as nmol/108 cells. Left panel: total polar lipids, right panel: total neutral lipid species. Error bars indicate the standard deviation from three biological replicate analyses. Lysophosphatidic acid, LPA; lysophosphatidylethanolamine, LPE; phosphatidic acid, PA; phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylglycerol, PG; phosphatidylinositol, PI; phosphatidylserine, PS; diacylglycerol, DAG; triaclyglycerol, TG.Lipid profiling of wild-type and (TIF)Click here for additional data file.Figure S7SCS3/YFT2 gene deletion strains. A CPY processing in wild-type and scs3\u0394 yft2\u0394 strains. Strains were transformed with a MET15 gene-containing plasmid prior to growth in methionine-minus SC media. Cells were pulse-labeled with Tran 35S-labeling reagent for 10 mins and chased with excess cold methionine and cysteine as depicted. Extract preparation and immuoprecipitation were as described p1) and Golgi-modified (p2) precursors and vacuolar mature form (mCPY) are indicated. B Tunicamycin hypersensitivity of SCS3 and YFT2 deletion strains. Two-fold serial dilutions of the indicated strains were spotted onto SC media containing 100 \u00b5M inositol with or without 1.0 \u00b5g/ml tunicamycin. Plates were photographed at 2 and 3 days respectively.CPY processing and tunicamycin sensitivity of (TIF)Click here for additional data file.Figure S8SCS3 YFT2 gene-deletion strains. Northern analysis of UPR induction after DTT treatment. Cells were grown to early log phase at 30\u00b0C in synthetic complete media, treated with 6 mM DTT for 1 hour and either left in DTT-containing media (panel A) or washed into fresh 30\u00b0C media lacking DTT (panel B). Cells were harvested over the time courses indicated. HAC1 mRNA (reporting both unspliced Hac1u and spliced Hac1i forms) was detected as described in Attenuation of the UPR in (TIF)Click here for additional data file.Table S1SCS3 and YFT2 Genetic Interactions.Stringent Set of (XLSX)Click here for additional data file.Table S2SCS3 and YFT2 Gene Interactions.GOSlim Component Analysis of (XLS)Click here for additional data file."}
+{"text": "The dorsal cochlear nucleus (DCN) is a first relay of the central auditory system as well as a site for integration of multimodal information. Vesicular glutamate transporters VGLUT-1 and VGLUT-2 selectively package glutamate into synaptic vesicles and are found to have different patterns of organization in the DCN. Whereas auditory nerve fibers predominantly co-label with VGLUT-1, somatosensory inputs predominantly co-label with VGLUT-2. Here, we used retrograde and anterograde transport of fluorescent conjugated dextran amine (DA) to demonstrate that the lateral vestibular nucleus (LVN) exhibits ipsilateral projections to both fusiform and deep layers of the rat DCN. Stimulating the LVN induced glutamatergic synaptic currents in fusiform cells and granule cell interneurones. We combined the dextran amine neuronal tracing method with immunohistochemistry and showed that labeled projections from the LVN are co-labeled with VGLUT-2 by contrast to VGLUT-1. Wistar rats were exposed to a loud single tone  for 6 hours. Five days after acoustic overexposure, the level of expression of VGLUT-1 in the DCN was decreased whereas the level of expression of VGLUT-2 in the DCN was increased including terminals originating from the LVN. VGLUT-2 mediated projections from the LVN to the DCN are likely to play a role in the head position in response to sound. Amplification of VGLUT-2 expression after acoustic overexposure could be a compensatory mechanism from vestibular inputs in response to hearing loss and to a decrease of VGLUT-1 expression from auditory nerve fibers. The dorsal cochlear nucleus (DCN) in the auditory brainstem is a major termination point of the auditory nerve and auditory nuclei Synaptic projections can be specifically associated with vesicular glutamate transporters that package glutamate into synaptic vesicles The vestibular nuclear complex lies in the floor of the fourth ventricle and consists of four major nuclei, namely the medial, lateral, superior and inferior vestibular nuclei. The vestibular nuclear complex controls eye movements, reflex postural head and neck, movements and balance during stance and gait as well as modulation and modification of autonomic function to maintain homeostasis during changes in body posture Previous studies have shown that cochlear kanamycin injections which produce deafness, resulted in a significant reduction of VGLUT-1 in the regions of the ventral and dorsal cochlear nucleus receiving auditory nerve inputs as well as an increase of VGLUT-2 in regions receiving non auditory inputs Here we characterised VGLUT-2 mediated projections from the lateral vestibular nucleus (LVN) to the DCN by combining dextran amine neuronal tracing method with immunohistochemistry. We also showed that exposure to loud sound resulted in an increase in the number of VGLUT-2 positive terminals originating from the LVN and discuss its role within the context of cross modal compensation of hearing deficit.Experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the Home Office and Leicester University's Ethical Committee .2PO4, 26 NaHCO3, 0.5 ascorbic acid, 0.1 CaCl2 and 4 MgCl2 . The brainstem was dissected out of the brain, oriented in its coronal or its sagittal plane and glued onto a mounting plate that was placed in a slicing chamber of a VT1000S vibroslicer  containing the same ice cold solution as described above. Axonal tracing was performed on the isolated brainstem as described below.Forty six Wistar and Lister Hooded rats P18 to P27 day-old were culled by decapitation and their brains were immediately transferred to ice-cold low sodium artificial solution containing (in mM): 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 2 CaCl2, and 1 MgCl2 (pH 7.4 when gassed with 95% O2 \u2236 5% CO2). Dextran amine dye (see below for composition) was delivered under binocular control, using a custom made combined pressure and electroporation system coupled to a double-barrelled glass pipette  as described in Barker et al.2 and 2.5 CaCl2 at pH 7.3. Axonal tracing was performed on brainstems extracted from control animals and 5 days after initial exposure to loud sound using age matched rats from the same litters.One millimetre thick slices were cut under binocular control using a MZ75 stereomicroscope (Leica Microsystems Ltd). Slices were cut from the top of the brainstem until the DCN was reached. Injections were subsequently performed below the tissue surface to a depth of 500 \u00b5m ensuring that subsequent sections contain both the LVN and the DCN  2\u2236 5% CO2 at 37\u00b0C for a period of 3 to 4 hours. After the incubation period, brainstems were either frozen down for immunohistochemistry (see below) or sliced before final observation. Slicing was performed by transferring the mounting plate with the glued brainstem to the vibroslicer slicing chamber and 120 \u00b5m thick slices were cut in the ice-cold low sodium artificial solution described above. Slices were performed up to a depth of 600 \u00b5m which contain both the DCN and the LVN. Slices were fixed in 4% paraformaldehyde for 10 minutes before being washed in 1\u00d7 PBS for 10 minutes. Slices were finally mounted on slides with 1% agarose and viewed with an Olympus Fluoview FV300 confocal microscope  for final observation.Brainstems attached to their mounting plates were then transferred to an incubation chamber containing the artificial cerebrospinal solution detailed above and bubbled with 95% OAxonal tracing was performed on the isolated brainstem as described above. Serial sections were taken in slices containing the DCN and LVN . Transmitted light images were taken using a Nikon eclipse TE2000 microscope and fluorescently labelled cells were imaged using an Olympus Fluoview FV300 confocal microscope. Transmitted light images were then overlaid and the outlines of the sections including the DCN were traced over using Powerpoint. Labelled cell bodies within the LVN were also overlaid on the reconstruction and then represented by red dots with each dot representing a cell body.Immunohistochemistry was performed on tissue extracted from control animals (see below) and 5 days after initial exposure to loud sound using age matched rats from the same litters. Brainstems were rapidly frozen in Tissue Tek (Sakura) after their incubation with dextran amine dye, using hexane and dry ice. Frozen tissue was processed within a month and sectioned at 20 \u00b5m using a cryostat. Slices were mounted on Polysine coated slides and fixed in 4% paraformaldehyde for 10 minutes. Slides were washed for 3\u00d75 min in 1\u00d7 Dullbeco's PBS (Invitrogen) with 0.1% Triton X-100 (PBS-T solution). After washing, sections were incubated with 1% bovine serum albumin and 1% goat serum in 1\u00d7 PBS with 0.1% Triton X-100 (blocking buffer) for 1 hour at 20\u00b0C. Sections were then incubated with anti-VGLUT antibodies  in blocking buffer overnight at 4\u00b0C. Slides were then washed with PBS-T for 6\u00d710 min. The secondary antibody  was applied for 2 h at 20\u00b0C. Sections were washed in PBS-T solution for 6\u00d710 min and then mounted with Prolong Gold antifade reagent (Molecular Probes).th, 50th and 75th percentile for each animal). The images were converted to 8 bit TIFF files in ImageJ (NIH software). Analysis was conducted as described in 2 using a 60\u00d7 objective) were counted for alternate coronal and sagittal sections. The labelled terminals were classified into two different subtypes, mossy fiber like terminals which have large irregular endings (\u22652 \u00b5m) and small boutons (<2 \u00b5m) Images were obtained using an Olympus Fluoview FV300 confocal microscope (Olympus Ltd). When imaging VGLUT immunoreactivity, confocal settings were determined using samples from animals aged P25 and the settings remained constant for all other imaging. To quantify VGLUT immunoreactivity sections were selected from an even distribution throughout the cochlear nucleus . Excitatory postsynaptic currents were elicited by stimulating the lateral vestibular nucleus with a concentric bipolar electrode  consisting of a platinum iridium inner pole of 50 \u00b5m and a stainless style outer diameter of 200 \u00b5m. Electrical pulses of 30\u201380 V (0.5\u20132 mA) and 0.1 ms duration were provided by a Digitimer DS2 A isolated stimulator  triggered by the pCLAMP software. Pharmacological blockers were bath applied using a peristaltic pump (Gilson Minipuls 3). Excitatory post synaptic currents were recorded in the presence of strychnine (10 \u00b5m) and gabazine (20 \u00b5m) and were blocked by NBQX disodium salt -quinoxalin) and D-AP5 . NBQX and D-AP5 were from Ascent Scientific , gabazine was from Tocris Bioscience . All the other drugs used in this study came from Sigma Aldrich . Once the whole cell recording was terminated, the pipette was gently removed from the cell by positive pressure and slices were fixed in 4% paraformaldehyde for 4 hours before being washed in 1\u00d7 PBS for 15 minutes. Slices were finally mounted onto slides with 1% agarose with the cover-slip sealed with nail polish to prevent dehydration. Slices were kept overnight at 4\u00b0C before final observation with the Olympus Fluoview FV300 confocal microscope.Experiments were conducted in P18\u201325 day old rats as the use of juvenile rats allowed reliable and stable whole cell recordings. Patch clamp recordings were performed in control animals (see below) and 5 days after initial exposure to loud sound using age matched rats from the same litters. The dissection of the brainstem from 14 Wistar rats was performed as described above. Sagittal slices (160\u2013180 \u00b5m thick) containing the LVN and the DCN were cut in low sodium artificial cerebrospinal solution before being transferred to the experimental chamber containing the artificial cerebrospinal solution (both solutions are described above). DCN cells were visualised with differential interference contrast (DIC) optics on an Axioskop microscope  with a 40\u00d7 N.A. 0.75 water-immersion lens. Whole-cell patch-clamp recordings were made from fusiform and granule cells using thick-walled glass pipettes  with a Multiclamp 700A amplifier , connected to an analogue to digital converter  filtered at 6 kHz (8-pole Bessel filter) and sampled at 20 kHz. Currents were recorded with pCLAMP 9.2 software (Axon Instruments). Cells were maintained at a holding potential of \u221270 mV and whole-cell access resistances of less than 10 M\u03a9 were not compensated. The intracellular solution contained 0.1% lucifer yellow and (in mM) 97.5 K gluconate; 32.5 KCl; 5 EGTA; 10 HEPES; 1 MgClWistar rats aged between 19 and 22 days were used (15 control and 15 acoustic over exposure) as this represents an age where the onset of hearing has already occurred Auditory brainstem response recordings were performed before AOE and 5 days after the first day of exposure to loud sound. Recordings were performed by inserting positive, negative and ground electrodes subcutaneously at the vertex, the mastoid and the back, respectively Means \u00b1 SEM were calculated using n samples which represented either the number of fields, terminals or grids as indicated in the text. N represents the number of animals for each condition. Statistical comparisons were performed by Mann-Whitney tests when comparing VGLUT-1 and VGLUT-2 intensities within regions of the cochlear nucleus and when comparing the effect of AOE on both VGLUT-1 and VGLUT-2 intensities in those regions. Although the test is non-parametric, it does assume that the two distributions are similar in shape and shape distributions were previously assessed as follows. Measurements of florescent intensity were taken along each layer of the DCN , in the MCD and in the shell region. Data were then compared using a one way ANOVA with Tukeys post hoc test. For all regions there was no significant difference in fluorescent intensity between all points. Unpaired T tests were used when comparing the size of labelled cell bodies in the LVN and when comparing terminal sizes under control conditions and after AOE; the percentage of VGLUT-2 labelled terminals originating from the LVN, the EPSC amplitude in control and exposed conditions. Significance was assessed for P<0.05. Data are represented as Mean \u00b1 SEM.The cochlear nucleus is divided into the ventral and the dorsal cochlear nucleus (DCN) When dextran amine was injected into the LVN , 2B synaTo assess the presence of functional synaptic projections between the LVN and the DCN, whole cell recordings were performed in sagittal slices containing both nuclei. Seven fusiform cells (N\u200a=\u200a6) were characterised by their location in the fusiform layer, their morphology (based on Lucifer yellow filling) and their passive properties , the shell region and the DCN molecular layer . Labelled terminals originating from the LVN failed to co-localize with VGLUT-1  with onl2 area,  to 4.7\u00b13.2 per 5000 \u00b5m2 area . The percentage of VGLUT-2 labelled terminals originating from the LVN increased from 1.6\u00b10.1%  to 5.8\u00b10.7%  P<0.001, unpaired t test). Examples of dextran amine labelled small boutons and mossy fiber terminals can be seen in Wistar rats were subjected to a loud 15 kHz tone for 2 times 3 hours . After 5 days, this procedure affected the auditory brainstem responses  and incrSo far, reported documentation of vestibular projections to the DCN remains scarce Our study shows that VGLUT-1 is strongly expressed in the MCD, the DCN molecular layer and the shell region. The MCD is primarily innervated by the axons of the type I spiral ganglion neurons The co-localization of VGLUT2 with LVN terminals in the DCN shown in this study confirms that the LVN pathway to the DCN is glutamatergic and demonstrates that projections from the LVN, like those from the spinal trigeminal nucleus and the cuneate nucleus Acoustic over exposure (AOE) can induce cochlear damage with resultant damage to the hair cells, spiral ganglion neurones and the myelin sheath of the auditory nerve An increase in the expression of VGLUT-2 could indicate a compensatory mechanism whereby specific synaptic inputs to the DCN are increased in response to the loss of synaptic activity from the auditory nerve. The inputs from the LVN would therefore consist of a compensatory mechanism originating from the vestibular system. Cross modal compensation has previously been demonstrated after deafferentation or sensory deprivation in other modalities It is of interest that vestibular conditions such as vertigo or vestibular schwannoma can result in tinnitus Figure S1Sagittal (top) and coronal (bottom) brainstem slice showing the site of injection of dextran amine in the dorsal cochlear nucleus DCN. Left, overlays of the brightfield and fluorescence photomicrographs at 3 hours post injection of dextran amine. Images on the right show the fluorescent micrographs. Scale bar 100 \u00b5m.(TIF)Click here for additional data file.Figure S2High magnification photomicrographs showing VGLUT-1 and VGLUT-2 positive puncta in the MCD (A), the shell region (B) and the layers of the DCN (C\u2013E). VGLUT-1 positive puncta are most densely located in the MCD and molecular layer of the DCN (A and C) whereas VGLUT-2 puncta show a greater density than VGLUT-1 in the shell region, the fusiform and deep layers . Scale bar 2 \u00b5m.(TIF)Click here for additional data file.Figure S3Examples of mossy fiber terminals originating from the LVN labelled with VGLUT-2 in control condition (left) and after AOE (middle). Note the presence of multiple VGLUT-2 labelled mossy fiber terminals after AOE. Scale bar 5 \u00b5m.(TIF)Click here for additional data file."}
+{"text": "Helicobacter pylori colonizes the human gastric mucosa, leading to a spectrum of gastric diseases in susceptible populations. Here we announce the draft genome sequences of strains HPARG8G and HPARG63. The data for both genome sequences provide insights regarding the diversity in gene content and rearrangement of the genomic islands commonly harbored by H.\u00a0pylori. Helicobacter pylori strain HPARG8G was isolated from a patient with gastric ulcer disease and strain HPARG63 was recovered from a patient with chronic gastritis. Here we report the draft genome sequences of both strains. A total of 785,600 reads with an average length of 435.02 nucleotides, corresponding to 33-fold coverage for strain HPARG8G and 100-fold coverage for HPARG63, were obtained with a 454\u00a0GS titanium pyrosequencer. De novo assembly was done using 454 Newbler version 2.6, yielding 47 contigs for HPARG8G and 34 contigs for HPARG63. The genomes were annotated using the RAST server  as well as 36 tRNAs and 2 rRNAs. The average G+C content was 38.98%. The HPARG63 strain genome sequence contains approximately 1,668,716\u00a0bp and 1,623 coding sequences and also has 36 tRNAs and 2 rRNAs, with an average G+C content of 38.79%. The H.\u00a0pylori P12 strain was predicted by RAST as one of the closest overall neighbors of HPARG8G and HPARG63. Both genomes displayed a conserved repertoire of housekeeping genes corresponding to various metabolic pathways. The two genomes each showed a complete cag pathogenicity island (cag PAI), with the complex rearrangement in the region delimited by dapB and murI genes described in the Mongolian gerbil-colonizing strain H.\u00a0pylori B8  under accession numbers CBKZ010000001 through CBKZ010000047 and CBKY010000001 through CBKY010000034.The first versions of the HPARG8G and HPARG63 sequences are accessible at the European Nucleotide Archive ("}
+{"text": "Hox genes to direct homeotic transformations or thebreadth of their patterning potential. To begin delineating Hoxgene function in neural development we used a mouse ES cell based system thatcombines efficient neural differentiation with inducible Hoxb1 expression. Geneexpression profiling suggested that Hoxb1 acted as bothactivator and repressor in the short term but predominantly as a repressor inthe long run. Activated and repressed genes segregated in distinct processessuggesting that, in the context examined, Hoxb1 blockeddifferentiation while activating genes related to early developmental processes,wnt and cell surface receptor linked signal transduction and cell-to-cellcommunication. To further elucidate aspects of Hoxb1 functionwe used loss and gain of function approaches in the mouse and chick embryos. Weshow that Hoxb1 acts as an activator to establish the full expression domain ofCRABPI and II in rhombomere 4 and as arepressor to restrict expression of Lhx5 andLhx9. Thus the Hoxb1 patterning activityincludes the regulation of the cellular response to retinoic acid and the delayof the expression of genes that commit cells to neural differentiation. Theresults of this study show that ES neural differentiation and inducibleHox gene expression can be used as a sensitive model systemto systematically identify Hox novel target genes, delineatetheir interactions with signaling pathways in dictating cell fate and define theextent of functional overlap among different Hox genes.The evolutionarily conserved Hox family of homeodomain transcription factorsplays fundamental roles in regulating cell specification along the anteriorposterior axis during development of all bilaterian animals by controlling cellfate choices in a highly localized, extracellular signal and cell contextdependent manner. Some studies have established downstream target genes inspecific systems but their identification is insufficient to explain either theability of  Hox target genes have beenidentified. Some studies have established direct and downstream target genes inspecific systems but their identification is insufficient to explain either theability of Hox genes to direct homeotic transformations or thediversity of their patterning potential.The evolutionarily conserved Hox family of homeodomain transcription factors playsfundamental roles in conferring regional identity and regulating cell specificationalong the anterior \u2013 posterior (AP) axis during development of all bilateriananimals Hox gene expression has been genetically manipulatedHox genes and accumulation of secondary effects in gain or lossof function genetic models present serious limitations. The elucidation of theprecise roles that Hox genes play in cell fate specification aswell as the identification of target genes and processes are key goals todeciphering the regulatory network underlying morphogenesis of the body plan.Furthermore, this may allow harnessing their patterning potential in the directeddifferentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS)cells to specific cell types.Two main general approaches have been used, a candidate target gene approach Hox gene expression and specific dorsoventral (DV) patterningcues define specific subclasses of neuronal progenitors in the developing hindbrainand spinal cord Hoxgenes act as integrators of AP and DV patterning mechanisms to generate specificclasses of neuronal progenitors and neurons for the appropriate AP levels of thehindbrain and the spinal cord. For example, Hoxb1 is specificallyexpressed in rhombomere 4 of the developing hindbrain. The specification of thisterritory and subsequent generation of r4 specific neuronal progenitors and neuronsdepend largely on Hoxb1 function. Disruption of the Hoxb1 gene inmice leads to transformation of the r4 territory into an r2-like state Hoxb1 in r2 causes homeotictransformation of r2 to a r4-like identity in chick Hoxb1 expression is responsible for the generation of facialbranchiomotor neurons and the suppression of serotonergic fate specification Hox genesdirect the generation of distinct motor neuron (MN) subtypes at hindbrain, brachial,thoracic and lumbar regions During development of vertebrate neural tube the combinatorial use ofHox gene function in neuraldevelopment we modeled the role of Hox genes in neural cell fate specification usinga mouse ES cell based system that affords the possibility of inducible Hoxb1expression. Using a differentiation protocol that generates a highly homogeneouspopulation of neural stem (NS) cells and inducible expression ofHoxb1 we showed that timely long term induction (8 days) of theHoxb1 transgene in ES cell derived NS cells resulted in thespecification of NS cells toward a hindbrain specific identity through theactivation of a rhombomere 4-specific genetic program and the repression of anteriorneural identity Hoxb1 mediates neural crest cell fate induction. This wassubsequently verified in vivoHoxb1 target genes,Hoxb2, Hoxa2, EphA2 andPhox2bHoxb1 targetgenes.To bypass limitations in delineating in vivo function we used loss and gainof function approaches using the chick and mouse developing embryos as model systemsand investigated the in vivo response of two up  and two down  regulated genes in ESderived NS cells. Hoxb1 is itself regulated by retinoic acid CRABPI and II. On the otherhand, Lhx5 and 9 mediate neuronal differentiationin vivo repression would correlate well with the finding thatHoxb1 blocks ES derived NS cell differentiation after mitogenwithdrawal Hoxb1 downstreamtarget genes in other approaches Hox target genes and processes, define binding sites andelucidate the interactions of Hox genes and extracellular signalsin dictating neural cell fate.Here we use this approach and microarray gene expression profiling to identifypotential novel Hoxb1 target genes and processes. To compare the long and short termeffects of Hoxb1 function and limit the number of potential target genes we used ashort term and a long term induction protocol. To validate the approach andelucidate aspects of Hoxb1 Animal studies were conducted in accordance with international guidelines andafter ethical approval of the competent Veterinary Service of Athens. The Hoxb1mouse mutants were described and genotyped as reported Tet-On/Hoxb1cells were as described previously +) and uninduced (Hoxb1\u2212) cells asdescribed earlier The generation and neural differentiation of the mouse ESTotal RNA was isolated from ES derived NS cells using the RNeasy kit (Qiagen)according to the manufacturer's instructions and digested by RQ1 DNase(Promega) to remove genomic DNA. First strand cDNA synthesis was performed withSuperscript II reverse transcriptase (Invitrogen) using random primers. Realtime PCR analysis was carried out in a Chromo4 DNA engine (Biorad), running thefollowing program: 95\u00b0C for 10 min, then 40 cycles of 95\u00b0C for 15 s,60\u00b0C for 40 s, followed by plate read. PCR reactions included 1x SYBRgreener PCR master mix (Invitrogen), 200 nM primer and 2 ul of template in a 25ul reaction volume. Primers were as follows (5\u2032 to 3\u2032):CRABPI F:GGAGATCAACTTCAAGGTCGGAG,CRABPI R: ATACTCCTCAGGGGAACTCGCATC,CRABPII F: ACATCAAAACCTCCACCACTGTGCGAAC,CRABPII R: CGTCATCTGCTGTCATTGTCAGGATCAGC,Lhx5 F: GACAAGGAAACCGCTAACAACG,Lhx5 R:GTGGACCCCAACATCTCAGACTCG,Lhx9 F: TACTTCAATGGCACTGGCACCG,Lhx9 R: TCCTTGGCATCTGGGTTATGG.in situ hybridization embryos were fixed overnight at4\u00b0C in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer saline(PBS). In situ hybridization was performed in whole embryos using probes formouse CRABPI and CRABPIILhx9Lhx9Lhx5in situhybridization, embryos were fixed again overnight at 4\u00b0C and then processedfor immunofluorescence. For immunofluorescence embryos were fixed in 4%PFA in PBS for 1\u20132 h at 4\u00b0. Embryos were cryoprotected with 30%sucrose in PBS and cryosectioned. Blocking was carried out in 10% normalgoat serum (NGS) with 0.1% triton for 1 h at RT. The cryosections wereincubated overnight at 4\u00b0C with the primary antibody diluted in 1%NGS, 0.1% triton in PBS. Primary antibodies used were as follows: rabbitanti-Hoxb1, 1\u2236400 (Covance), mouse Lhx5, 1\u2236100. Secondary antibodieswere anti-mouse and anti-rabbit Alexa 488 or Alexa 568 (Molecular Probes) usedat 1\u2236500. Images were acquired using a Leica TCS SP5 confocalmicroscope.For Hoxb1 cDNA was inserted into thepCAGGS-IRES-NLS-GFP expression vector in situhybridization or immunofluorescence.Chick embryos were staged according to Hamburger and Hamilton (HH)  and electroporated at HH stage 10\u201311. Chick embryos wereelectroporated with plasmid DNA at a concentration of 1.5 \u00b5g/\u00b5l. Thecoding regions of mouse Hoxb1 target genes and processes we usedthe stable line ESTet-On/Hoxb1 that allows for tight dox mediatedinducible expression of the Hoxb1 transgene at both the ES celland NS cell stages. However, inducible expression of the transgene couldmobilize the endogenous Hoxb1 autoregulatory loop only at theNS cell stage demonstrating the importance of cellular context forHoxb1 function and its analysis. Hoxb1induction using an 8-day long dox exposure resulted in the generation of r4specific neuronal progenitors Hoxb1 downstream effectors andcompare the short term and long term effects of Hoxb1expression we performed microarray gene expression analysis after a two day longexposure to dox  (HHstage 20) by whole mount in situ hybridization with the chickLhx5 in situ hybridization probe cLxh5 at HH is expressed in twodorsomedial stripes in r2 and r3  analysis showed that up regulatedand down regulated genes related to strictly distinct processes. The Hoxb1repressing activity was directed primarily towards differentiation related processeswhereas its activating functions were directed primarily towards early development,wnt and cell surface receptor linked signal transduction and cell-to-cellcommunication  upon cell cycle exit. To in vivo validate some of ourfindings we corroborated the effects of Hoxb1 on the expression patterns ofCRABPI, CRABPII, Lhx5 andLhx9 using in vivo loss and gain of functionmodels. CRABPI, CRABPII and Lhx5had a Hoxb1 dependent r4 specific expression pattern. It is worth noting that noneof them was identified as such in the aforementioned screens underlining thesensitivity of the approach presented here.Three other screens have been conducted to identify CRABP expression was initially associated with structures thatwere more sensitive to excess of RA CRABPI and II was activated byHoxb1 in ES derived NS and these findings were validated in themouse embryo since expression of both was down regulated in the r4 ofHoxb1\u2212/\u2212 reverting to expression patterns identical tothose of r2. Intriguingly, CRABPI is up regulated whereasCRABPII is down regulated in the resident territory of r4 motorneurons suggesting that maturation and/or specification of this subpopulation needsparticular shielding from RA exposure. Ectopic Hoxb1 expression inr2 through timely supply of extraneous RA converts the r2 trigeminal motor neuronsinto r4 facial motor neurons CRABPIand II expression. However, further studies are needed to provethis hypothesis and establish whether CRABPI/II are direct Hoxb1target genes. The localized expression of RARa in r4 and thelocalized expression of Cyp1B1, an atypical RA generatingcytochrome, in the ventral r4 RARa as a Hoxb1 downstream target in r4. The ES derivedNS cells are a mixture of different DV characters and this limits the detectioncapacity for markers that are exclusively expressed in distinct and narrow DVlevels. This can be bypassed by dorsalising or ventralising these cells withappropriate DV morphogenetic signals Cyp1b1 is induced in shh treated ES derivedHoxb1+ NS cells as well.Within the developing neural tube the diverse cellular distribution patterns ofretinoid receptors and retinoid binding proteins indicates that it is necessary tofine-tune levels of RA signaling for the specification of diverse of neuralsubpopulations. CRABPI and II are located in the cytoplasm and bind RA, a key playerin CNS pattern formation, neural specification and differentiation.Lhx genes) was regulated byHoxb1 in ES derived NS cells. Click here for additional data file.Table S2List of genes regulated in both short (s) and long (l) induction schemes.Classification is according to primary GO process assignment. If not anassignment has been made genes are labelled as non-classified.(XLS)Click here for additional data file.Table S3List of of common genes induced in Hoxb1-/- r4 and also regulated by Hoxb1 inES derived neural progenitors after long induction. At the top part of thelist the observed regulation is in the same direction and after the space itis in the opposite direction.(XLS)Click here for additional data file.Table S4List of common genes repressed in Hoxb1-/- r4 and also regulated in ESderived neural progenitors after long induction. At the top part of the listthe observed regulation is at the same direction and after the space it isin the opposite direction.(XLS)Click here for additional data file."}
+{"text": "Sox1, Sox2 and Sox3 are key regulators of NP differentiation and that their roles in CNS development are largely redundant. Nevertheless, mutation of each SoxB1 individually results in a different array of CNS defects, raising the possibility that SoxB1 proteins have subtly different functions in NP cells. To explore the mechanism of SOXB1 functional redundancy, and to identify genes that are most sensitive to loss of the Sox3 gene, we performed genome wide expression profiling of Sox3 null NP cells. Nineteen genes with abnormal expression were identified, including the homeobox gene Dbx1. Analysis of Sox3 null embryos revealed that Dbx1 was significantly reduced in the neural tube and developing brain and that SOX3 bound directly to conserved elements associated with this gene in cultured NP cells and in vivo. These data define Dbx1 as a direct SOX3 target gene whose expression, intriguingly, is not fully rescued by other SOXB1 transcription factors, suggesting that there are inherent differences in SOXB1 protein activity.SoxB1 sub-family of transcriptional regulators are expressed in progenitor (NP) cells throughout the neuroaxis and are generally downregulated during neuronal differentiation. Gain- and loss-of-function studies indicate that  Sry-related HMG box) family of transcription factors, of which 20 members have been identified in mammals. SOX genes generally have developmentally-regulated expression and play important roles in cell specification, self-renewal and differentiation in a broad range of embryonic contexts Sox3, and the closely related genes Sox1 and Sox2 (which together make up the SoxB1 subgroup), are expressed in neuroprogenitor cells throughout the neuroaxis and are down regulated upon differentiation Sox3 null mice exhibit specific CNS defects within the hippocampus, corpus callosum and hypothalamus despite the widespread Sox3 expression in NP cells throughout the developing brain SOX3 have a relatively mild phenotype that includes infundibular hypoplasia, hypothamalic-pituitary axis dysfunction and incompletely penetrance of intellectual disability Sox2 or Sox1 in mice also results in regionally-restricted defects as opposed to a general NP phenotype SOX3 is a member of the SOX  subpopulation of NP cells. This phenomenon can be explained by at least three possibilities. Firstly, given that subtle differences in Sox1, Sox2 and Sox3 gene expression have been identified in the developing CNS Sox3 null embryos despite expression of Sox2 in this structure While these studies provide strong support for SOXB1 functional redundancy, the existence of congenital CNS defects in Sox3 gene, we performed genome wide expression profiling of Sox3 null NP cells. Nineteen genes with abnormal/delayed expression were identified that included the homeobox gene Dbx1. In vivo analysis of Sox3 null embryos revealed that Dbx1 was significantly reduced in the neural tube and developing brain and that SOX3 bound directly to conserved elements associated with this gene in cultured NP cells and in vivo. These data define Dbx1 as a direct SOX3 target gene whose expression, intriguingly, is not fully rescued by other SOXB1 transcription factors, suggesting that there are inherent differences in SOXB1 protein activity.To explore the mechanism of SOXB1 functional redundancy, and to identify genes that are most sensitive to loss of the Sox3 null embryonic stem cells were previously targeted as described in . Sox3 null mice have been published previously R1 ES cells were maintained on irradiated MEFs in standard conditions and neurodifferentiated as monolayers using N2B27 as previously described Sox3 null Day 4 samples using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays (full dataset available at Gene Expression Omnibus: GSE53760). 2 way-ANOVA, using batch as a factor, was used to identify the significantly regulated genes. qRT-PCR validation was performed previously described RNA from differentiated cells was extracted using a RNeasy mini kit (Qiagen) and cDNA generated using a High Capacity RNA-to-cDNA kit (ABI). Expression profiling was performed on four wild type and four IHC was performed as described previously sample (non-enriched region) - Ctsample (peak region)] was >2 following normalisation to CtIgG and CtInput of the corresponding qPCR run.In vitro ChIP was performed as per Animal experiments were approved by the University of Adelaide Animal Ethics Committee (project number: S-2012-242). All studies were conducted in accordance with the principles of animal replacement and reduction and experimental refinement.Sox3 expression during N2B27 neuroinduction, we performed qRT-PCR . Sox3 expression was upregulated by Day 2, and increased further at Day 4 and Day 6. Consistent with these data, weak expression of SOX3 protein was evident in Day 2 cultures. By Day 4, robust expression of SOX3 was detected in most cells. At Day 6, robust expression of SOX3 could still be detected in most cells, although some cells (particularly those at the periphery of colonies) had begun to downregulate SOX3.To begin to identify SOX3 target genes in NP cells, we utilised the N2B27 culture system, which has previously been shown to generate NP cells from ESC with high efficiency  qRT-PCR  and immu qRT-PCR . At the Sox3 deletion on NP differentiation, we performed N2B27 neuroinduction using our previously-generated Sox3 null ES cells that contain a GFP reporter knock-in allele Sox3 has a major impact on induction of neural progenitors, expression of neural progenitor markers (including other SoxB1 genes) was analysed by qRT-PCR. No significant difference in Sox1, Sox2 or Pax6 expression was detected in samples utilized for microarray analysis, at Day 4  and Fezf2 (p\u200a=\u200a0.0228) showed significant down regulation in mutant cells (Efnb3 (p\u200a=\u200a0.0229), Cspg5 (p\u200a=\u200a0.0251), Fam174b (p\u200a=\u200a0.0065), Ddr2 (p\u200a=\u200a0.0386) and Slit1 (p\u200a=\u200a0.0456) was also confirmed in the independent samples. These data identifies a number of potential SOX3 targets in cultured NP cells.To identify putative SOX3 target genes, we compared the global gene expression profile of Day 4 NP cell cultures derived from nt cells . SignifiSox3 null NPs potentially represent genes that are direct SOX3 targets. To investigate this possibility, we examined the differentially expressed genes for SOX3 binding sites using a previously published SOX3 ChIP-seq data set generated using a comparable NP differentiation system Sox3 null NP cells. Over 83% of the differentially expressed genes  as embryos at this stage of development have an abundance of NP cells and a paucity of differentiating neurons. In addition, like Day 4 NP cell cultures, Sox1 and Sox2 expression in not significantly altered in Sox3 null embryos at this stage  using chromatin extracted from 10.5 dpc embryonic heads. Significant enrichment (p\u22640.001) of this SOX3 binding this site was observed in comparison to the IgG negative control  can function redundantly with SOX3 to \u201crescue\u201d the SOX3 deficiency. However, the lack of a complete phenotypic rescue suggests that, at least in some NP cells, the level of SOXB1 protein may be important for neurodevelopment. Alternatively, each of the SOXB1 factors could bind preferentially to a unique subset of SOXB1 targets. As a result, target gene expression resulting from the loss of a single SOXB1 gene would only be partially rescued by the other two SOXB1 factors. To explore this issue in more detail, we attempted to identify genes that were sensitive to the loss of SOX3 in NP cells. To do this we used a previously targeted Sox3 null ES cell line in which the single SOX3 exon was replaced with GFP Sox3 null NP cells resulted in rapid upregulation of Sox3/GFP at the mRNA and protein level. The NP markers Sox1 and Pax6 were also rapidly induced and showed comparable kinetics in the control and mutant cultures. In addition, expression of Sox2 was comparable between the two populations. Notably, no overt defects in the morphology or global gene expression (see below) were identified in Sox3 null NP cells. This is likely due to the expression of Sox1 and Sox2 in N2B27-induced NP cells and is consistent with the mild CNS phenotype of Sox3 null embryos.Despite the widespread expression of Sox3 were expressed in the majority of NP cells. Of over 24,000 genes assayed by microarray, only 19 had significantly different expression levels >1.4-fold at Day 4. Expression differences (when tested by qRT-PCR) were validated on independent samples for a majority of the targets, thereby confirming the authenticity of the microarray data. However, not all differentially expressed genes validated on independent samples . The relatively small number of differentially expressed genes is interesting to consider in the context of a recently-published ChIP-seq analysis of a similar NP cell type, which showed that SOX3 binds at thousands of genomic sites, many of which are likely to have regulatory functions To maximise the likelihood of identifying genes that are directly regulated by SOX3, we performed gene expression profiling at Day 4, the earliest time point at which high levels of Cspg5 has been associated with intellectual disability disorders Slit1 expression has been published in mice with abnormal corpus callosum development SOX3 mutations Sox3 null mice have abnormal corpus callosum development Interestingly, many of the genes identified by the microarray analysis have established roles in neurodevelopment. For example, Fezf2. Fezf2 has been shown to be important for the early development of the posterior hypothalamus/thalamus in zebrafish and mouse Fezf2 expression has been previously published to be downstream of the SOXC group genes Sox4 and Sox11, double deletion of which leads to a loss of Fezf2 expression. Regulation of Fezf2 by SOX4/11 gene is mediated by a cis acting enhancer binding site which, interestingly, was also identified as a SOX3 binding site in NP cells by ChIP-seq and independently within our lab  had significantly different expression that matched the genome-wide expression profiling analysis. The lack of in vivo validation for other targets may reflect a phenotypic or stage-specific difference in the NP cells induced by N2B27 differentiation compared with their 9.5 dpc embryonic counterparts. Consistent with this idea, expression analysis of Sox3 targets in Day 6 NP cells revealed that many genes no longer had significantly different expression levels or had greatly reduced expression fold changes  could be used to generate NP cells that have an equivalent SOXB1 dosage but which lack SOX3. In addition to the spinal cord, we identified a reduction in Dbx1 expression in the head of 10.5 dpc Sox3 null embryos. However, we were unable to determine whether this reflected a lack of Dbx1 expression in a specific region or general reduction in the Dbx1 expression level (data not shown).Dbx1 is a direct target of Sox3 Dbx1 expression in Sox3 null NPs. The data also suggest that the regulation of Dbx1 by SOX3 is complex and subtle differences between early or later expression or spinal cord/brain expression might be regulated any number of these sites. Further studies are needed to address this issue. Given the increasing ease with which the genome can be manipulated Dbx1 locus and assess their impact on Dbx1 expression in vitro and in vivo.A recent report by Oosterveen et al also indicated that Figure S1Expression of putative Sox3 targets at day 6 of neural progenitor differentiation. A: Putative targets Dbx1 and Slit1 relative gene expression levels by qRT-PCR at day 6 of N2B27 differentiation, . B: Putative targets Cp, Fezf2 and Efnb3 showing reduced and/or insignificant fold change at day 6 of N2B27 differentiation . RQ: relative quantification normalised to \u03b2-actin.(TIF)Click here for additional data file."}
+{"text": "We report that the high level of 5hmC present in neurons is enriched in active transcription units, and that surprisingly strong depletion of 5mC over gene bodies is observed for these genes. However, the relative contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndrome causing mutation of residue R133C in the MeCP2 methyl-CpG binding domain (MBD) preferentially inhibit 5hmC binding. Loss of MeCP2 does not alter the genomic distribution of 5hmC. These findings demonstrate that 5hmC and MeCP2 constitute a novel, cell specific epigenetic mechanism for regulation of chromatin structure and gene expression in the mammalian nervous system, and they provide new mechanistic insights into the pathophysiology of Rett Syndrome (RTT).The very high levels of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes and its accumulation within gene bodies suggests that the epigenetic mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem (ES) cells. Here we present the first quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC) and gene expression in identified, terminally differentiated CNS cell types"}
+{"text": "Avian influenza H5N1 virus is highly pathogenic partially because its H5 hemagglutinin contains a polybasic cleavage site that can be processed by proteases in multiple organs.Monoclonal antibodies (mAb) specific to the synthetic peptide of hemagglutinin polybasic cleavage site of H5N1 virus were raised and tested for their neutralizing potential.Purified mAb showed suppression of H5N1 pseudovirus infection on Madin-Darby Canine Kidney (MDCK) cells but the efficacy was less than 50%. Since those mAb are specific to the intact uncut polybasic cleavage site of hemagglutinin, their efficacy depends on the extent of hemagglutinin cleavage on the viral surface.Proteolytic analysis suggests the low efficacy associated with those mAb may be due to proteolytic cleavage already present on the majority of hemagglutinin prior to the infection of virus. Avian H5N1 highly pathogenic influenza virus was first isolated from sick geese in China during 1996 and later transmitted to human in Hon Kong during 1997 . The rapThere are many antiinfluenza measures available. For example, vaccination is a good defense against highly pathogenic influenza like the avian H5N1 virus ,16, but An antibody targeting the conserved epitopes on viral surface may be able to circumvent the antigenic drift and thus avoid the hit-and-miss situation associated with influenza vaccines. For example, the ectodomain of M2 channel protein is highly conserved among most strains of influenza A viruses and has been targeted as a broad spectrum epitope, but the antibody will only work on influenza A viruses and as the mutations accumulate at the ectodomain of M2 protein, they count against the efficacy of such antibody . The HA2The polybasic cleavage site on hemagglutinin is highly conserved among those highly pathogenic H5N1 viruses and its polybasic residue constituent should make this peptide fairly antigenic but discernable from other hydrophobic peptides and, therefore, this polybasic peptide is an interesting candidate as a broad spectrum epitope. Because the proteolytic cleavage of HA is a necessary step for an influenza virus to become infectious, we hypothesize that monoclonal antibody (mAb) specific to the polybasic cleavage site on hemagglutinin may be able to suppress virus infection by preventing HA cleavage by host proteases.The polybasic cleavage site on hemagglutinin is highly conserved among H5N1 avian influenza viruses ,30,31, bThose 7 hybridomas were cultured to raise ascitic fluid in nude mice and the mAb was purified by protein A resin . Briefly, monoclonal antibodies in the ascitic fluid was precipitated by 50% saturation of ammonium sulfate and redissolved in a minimal volume of phosphate buffered saline (PBS) before an overnight dialysis. Dialysed ascitic fluid was loaded onto a protein A column that had been equilibrated with 20 mM sodium phosphate pH7 buffer. The monoclonal antibody was eluted with 100 mM acetic acid pH2.8 and neutralized by 1 M Tris/HCl pH9. The immunoglobulin in elution was concentrated with Centricon  and the contents were determined by Bradford protein assay .One mg of pepsin (Sigma P6887) was predissolved in 200 \u03bcL of 0.1 M glycine acetate (pH 2.6). Twenty \u03bcL of mAb was added with an equal volume of pepsin and digested at 37\u00b0C for 40 min before 1 \u03bcL of 1 M Tris (pH 9) was added to alleviate the protein precipitation due to acidity. The mixture was further incubated for 30 min before 4 \u03bcL of 1 M Tris (pH9) was added to stop the reaction. An aliquot of 225 \u03bcL D-MEM was added to the antibody/pepsin mixture and 50 \u03bcL of the mixture was used for pseudovirus neutralization assay.nd antibody was used to probe the membrane at 1:5000 dilution for 1 hr. The immunoreactive spots were developed with a chemiluminescent substrate  and scanned with a typhoon scanner (Amersham).Three synthetic peptides were used to evaluate the epitope binding property of those 7 purified monoclonal antibodies. The full length 18 meric peptide, RERRRKKRGLFGAIAGFI, the N-half 8 meric peptide, RERRRKKR, and the C-half 10 meric peptide, GLFGAIAGFI, were synthesized at the Genomic Research Center of Academia Sinica. All 3 peptides were dissolved in DMSO at 2 mg/mL concentration. An aliquot of 5 \u03bcL (10 \u03bcg) was spotted on a 1 cm square of PVDF membrane . Four ten-fold serial dilutions were also spotted on the membrane similarly. The PVDF membranes were allowed for air dry and subject to immunoblotting after the membrane being rewetted by methanol/PBS. Blocking of the PVDF membrane was carried out with 2% non-fat milk dissolved in PBS. The purified monoclonal antibodies were diluted 500 fold in 2% non-fat milk and allowed for 1 hr incubation with the membrane. Horseradish peroxidase (HRP) conjugated anti-mouse 2GERRRKKRGLFGAIAGFIEGG, mutation underlined; 3) Anhui 2005 H5N1 pseudovirus, RERRR_KRGLFGAIAGFIEGG, which has a deletion of K344 or K345 at the polybasic site. The peptide sequence of antigens and viral cleavage sites were listed in Table To avoid using those highly pathogenic H5N1 viruses, pseudoviruses comprised of HA5 and NA1 antigens, Pol and Gag of HIV and a firefly luciferase reporter gene  were kinPseudovirus infection assay was performed on Madin-Darby Canine Kidney (MDCK) cells which were cultured in D-MEM supplemented with 10% FBS. Overnight cultured MDCK cells were incubated with pseudovirus in the presence or absence of mAb for 1 or 4 hours at 37\u00b0C before the pseudovirus mixture was removed by aspiration. Pseudovirus treated MDCK cells were cultured 48 hours further before the infectivity was determined. The infectivity of pseudovirus was determined by assaying the transfected and expressed luciferase activity in those MDCK cells. The MDCK cells were rinsed with PBS before being solublized with 60 \u03bcL Glo Lysis Buffer of Promega  for 2 hours. Fifty \u03bcL of cell lysate was mixed with an equal volume of Bright-Glo Luciferase Substrate in an opaque well/plate. The chemiluminescence was detected with a TopCount NXT of Perkin Elmer .The immunostain blot was carried out with a 4-20% gradient SDS-polyacrylamide gel and transferred to a PVDF membrane in a semidry blot transfer apparatus (BioRad). Both the primary and the secondary antibodies were diluted 1:5000 fold in PBS containing 2% non-fat milk. The stains were developed with chemiluminescent substrates of Invitrogen  and the band intensities were measured by a Typhoon scanner and analyzed by ImageQuant TL of GE Healthcare .All 7 mAb were purified to near homogeneousity by protein A resin Figure , but the2 can result in a better suppression during the pseudovirus infection assay. After pepsin treatment (Figure 2 remained low and none of the clones provided more than 50% suppression on pseudovirus infection (Figure Since H5N1 avian influenza virus is highly pathogenic and is not available for general laboratory use, we opted for a pseudovirus that expresses H5 and N1 surface proteins and contains a firefly luciferase reporter gene so that its infectivity can be assessed by luciferase activity. A preliminary neutralization scan was summarized in Figure t Figure , the effAll 7 monoclonal antibodies were subjected to epitope peptide analysis by dot blot. The result showed that clone A, C, 3C4 and 6B8 recognize the full length 18 meric peptide only and are reactive to as little as 0.1 \u03bcg of full length peptide (Figure To verify if these purified mAb can bind to hemagglutinin, mAb C, 3C4, and 6B8 were subjected to immunoblotting against recombinant hemagglutinin or virus lysate, as shown in Figure Because our mAb cannot recognize those cleaved HA1 or HA2 Figures  and 6, tThe authors declare that they have no competing interests.HJT is responsible for the overall research; LAC carried out the epitope peptide analysis and 293 cell expressed HA immunostain study; ALY supervised the overall progress. All authors have read and approved the final manuscript."}
+{"text": "Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 \u03bcg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 \u00d7 96 well plates) can easily be fully processed  in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20\u00b0C (but not 4\u00b0C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20\u00b0C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from  Systems biology involves the study of molecules in context as part of a larger system or network rather than in isolation and the development of mathematical models of the particular system being studied. Both the wet (laboratory-based) and dry (computer-based) experiments are used to inform each other and together generate a greater understanding of the biological system ,2. Over Arabidopsis thaliana seedlings. Depending on the available equipment, the method can be performed manually using multichannel pipettes or automated with a liquid handling robot. The developed protocols enable the study of gene expression by qPCR  in large numbers of samples .Historically, RNA extraction has been performed using organic solvents and phenol-chloroform . Similarlater solution .\u2022 RNA\u2022 1.5 ml eppendorf tubes.http://www.mbpinc.com/, Cat.# 3453). NOTE: If using alternative tubes, ensure that they are sufficiently strong to withstand bead beating in a mixer mill.\u2022 2 ml eppendorf tubes .\u2022 5 mm stainless steel cone balls  which in addition to the binding, wash and elution plates, contains buffers RA1, RA2, RA3 & RA4 and DNaseI. The kit requires the addition of 1% \u03b2-mercaptoethanol to buffer RA1 and ethanol to buffers RA3 and RA4 prior to use.\u2022 illustra RNAspin 96 RNA isolation kit .http://www.starlab.co.uk/, Cat.# E2896-0110).\u2022 1.1 ml 96 deepwell plates . To process 1\u00d7 96 well plate requires 8\u00d7 200 \u03bcl tips  and 144\u00d7 1000 \u03bcl tips .\u2022 Disposable tips for robot .http://www.binder-world.com).\u2022 Oven for preparing beads .\u2022 Bead-mill .\u2022 Plate centrifuge at room temperature capable of speed of 3,220\u00d7 g (e.g. Eppendorf 5810R centrifuge fitted with an A-4-81 swing-bucket rotor).http://www.nanodrop.com).\u2022 NanoDrop spectrophotometer Agilent 2100 Bioanalyser  with 8-channel liquid handling arm fitted with disposable tips (200 \u03bcl and 1 ml as detailed above) and a TeVacS vacuum unit. Although we used the vacuum unit with a custom built spacer to ensure the wash plate  was positioned at the correct height directly below the binding plate, a standard size spacer (number 6) can be used. Details of the robot deck layout are depicted in Figure Although our method was developed and performed on a high specification liquid handling robot, it can be easily transferred to other platforms.\u2022 Freedom Evo-2 150 liquid handling robot  seedlings carrying the ELF3:LUC transgene (EARLY FLOWERING 3) [CIRCADIAN CLOCK ASSOCIATED 1) seedlings [CAB2:LUC transgene (CHLOROPHYLL A/B-BINDING PROTEIN 2) [-2\u00b7s-1) at 22\u00b0C for 9 days.Wildtype ERING 3)  or CCA1-eedlings  carryingOTEIN 2)  were groThe concentration of purified RNA was measured using a NanoDrop spectrophotometer (ND1000). Typically 2 \u03bcl of each sample was used.http://www.agilent.com/) according to the manufacturer's instructions. The ladder used was RNA 6000 ladder  and 150-200 ng of each sample was loaded. All samples and the ladder were denatured at 70\u00b0C for 2 mins prior to analysis. The results were analysed using Agilent 2100 Expert software.The integrity and quality of purified RNA were analysed using an Agilent 2100 Bioanalyzer and a RNA 6000 Nano Assay kit  according to the manufacturer's instructions. cDNA was diluted 1/10 and 1 \u03bcl used for subsequent qPCR. qPCR analysis for isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) in a final volume of 10 \u03bcl was then performed using LightCycler 480 SYBR Green I Master mix on a LightCycler480 . The following primers each at 300 nM were used: IPP2 forward primer GTATGAGTTGCTTCTCCAGCAAAG and IPP2 reverse primer GAGGATGGCTGCAACAAGTGT [Purified total RNA (1 \u03bcg) was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis kit with oligo dT primers . Only half of the lysed sample was processed further to complete the RNA extraction procedure while the other half was stored in the freezer (-20\u00b0C) after lysis as backup in the case of some experimental catastrophe.later.1) Using clean tweezers, harvest 45-50 mg Arabidopsis seedlings per sample directly into 1.5 ml eppendorf tubes containing 500 \u03bcl RNAlater solution.2) Store samples overnight at 4\u00b0C to allow full penetration of the RNA: At this point, samples can safely be stored at -20\u00b0C for up to 10 months with no detrimental effect - see comments section below.3) Store samples at -20\u00b0C until sample lysis and completion of the RNA extraction procedure. NOTEIf the balls are not rinsed properly the acid not only causes rust and corrosion damage to the balls but would also degrade the RNA sample.4) Before use, 5 mm stainless steel cone balls (one per sample) are prepared by washing in 0.4 M HCl for 1 h, thoroughly rinsed using RNase-free water and then baked at 240\u00b0C for at least 4 h. NOTE: 5) Add prepared cone balls to 350 \u03bcl RA1 (containing 1% \u03b2-mercaptoethanol) in a 2 ml eppendorf tube (one per sample per tube).later solution and dab dry on some tissue paper.6) Carefully remove seedlings from RNA7) Transfer seedlings to prepared eppendorf tube containing stainless steel cone ball and RA1 lysis buffer (containing 1% \u03b2-mercaptoethanol).-1 for 2 \u00d7 2 mins. The tube positions are rotated between shaking steps as described in the manufacturer's operating manual. NOTE: Although the TissueLyser equipment we used is capable of processing 192 samples (2 \u00d7 96) in 1.5 ml tubes simultaneously, the size of the bead  required for efficient sample disruption and homogenisation necessitated the use of 2 ml eppendorf tubes. Consequently, samples have to be processed in two batches of 48. Although it is possible to use smaller stainless steel balls (e.g. 3-4 mm) and thus process samples in larger batches of 96 \u00d7 1.5 ml eppendorf tubes, we have found that this only works well with wild type samples or similar. In our hands, smaller bead sizes do not work well with mutant plant samples which often grow poorly and the resulting yield is very low. As our experiments usually include a variety of genetic manipulations, we routinely use 5 mm cone balls for efficient disruption and homogenisation of all samples.8) Homogenise the samples in two batches of 2\u00d7 24 tubes using a TissueLyser bead mill at a frequency of 25 s9) Centrifuge lysed samples at 11,000 \u00d7 g for 5 mins.10) Transfer supernatant to a filter plate.: Missing out this step subsequently results in clogged wells in the binding plate and drastically reduced yields.11) Centrifuge filter plate (over a 1.1 ml deepwell plate) at 3,200 \u00d7 g for 10 mins to remove debris. NOTEAt this point, lysed samples can be safely stored at -20\u00b0C for up to 6 months with no detrimental effects on purified RNA - see comments section below.12) 150 \u03bcl of the cleared lysed solution is transferred to another 1.1 ml deepwell plate to be processed for RNA extraction while the remainder is stored at -20\u00b0C as backup in case of experimental disaster. NOTE: either a centrifuge or vacuum manifold to pass the solutions through the plate. NOTE: We have modified the kit manufacturer's protocol to be used on plate centrifuges commonly found in labs  and all centrifugation steps were carried out in an Eppendorf 5810R centrifuge with an A-4-81 swing-bucket rotor. The plate sandwich formed by the binding plate combined with the plate included in the kit was too deep to be used in our centrifuge set up. Therefore the plate sandwich was composed of a binding plate over a 1.1 ml 96 deepwell plate not the plate provided with the kit. All vacuum steps were performed at -200 mbar for 2 \u00d7 60 secs. In our hands, greater pressure resulted in splashing of the sample and possible cross well contamination.The manual RNA extraction procedure is performed using multichannel pipettes and 13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare cleared lysed samples  for binding to the silica membrane by adding an equal volume of RA4 buffer (150 \u03bcl) to each well of 1.1 ml deepwell plate.15) Mix thoroughly by pipetting up and down 12 times.16) Transfer samples (300 \u03bcl) to the wells of the binding plate.17) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.18) Add 460 \u03bcl buffer RA3 to each well of the binding plates .19) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.20) Add 29 \u03bcl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.21) Add 460 \u03bcl RA2 buffer to each well of the binding plate.22) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.23) Add 800 \u03bcl RA3 buffer to each well of the binding plate.24) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.25) Add 460 \u03bcl RA4 buffer to each well of the binding plate.26) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.27) Proceed to elution (step 30 below).The automated RNA extraction procedure is performed using a liquid handling robot with an integrated vacuum unit on the robot deck to pass the solutions through the binding plate.13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare the robotic workstation and check that the actual deck layout matches the virtual one shown on the robot script with all labware  containing appropriate solutions  in the correct locations on the robot deck - see Figure 15) Place 1.1 ml deepwell plate containing 96 lysed samples  onto the appropriate position of the robot deck.16) Select appropriate robot script and run which will perform steps 17-28 as detailed below.17) Prepare lysed samples for binding to the silica membrane by the addition of an equal volume of RA4 buffer (150 \u03bcl) and mix thoroughly by pipetting (150 \u03bcl) up and down 12 times.18) Transfer samples (300 \u03bcl) to the wells of the binding plate.19) Apply vacuum  to pull the solution through the binding plate.20) Add 460 \u03bcl buffer RA3 to each well of the binding plates .21) Apply vacuum  to pull the solution through the binding plate.22) Add 29 \u03bcl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.23) Add 460 \u03bcl RA2 buffer to each well of the binding plate.24) Apply vacuum  to pull the solution through the binding plate.25) Add 800 \u03bcl RA3 buffer to each well of the binding plate.26) Apply vacuum  to pull the solution through the binding plate.27) Add 460 \u03bcl RA4 buffer to each well of the binding plate.28) Apply vacuum  to pull the solution through the binding plate.29) Remove binding plate from robot and proceed to elution (step 30 below).Regardless of whether the manual or automated method is used, the same elution procedure is followed.30) Centrifuge the plate at 3,220 \u00d7 g for 10 mins to ensure all ethanol is removed prior to elution.31) Add 50 \u03bcl RNase-free water to each well of the binding plate and incubate for 3 mins.32) Place binding plate over the 96 well elution plate (contained in the illustra kit) and elute purified RNA by centrifugation at 3,220 \u00d7 g for 3 mins.CAB2:LUC transgene which are elongated [ELF3:LUC transgene.The yields of extracted RNA purified using the manual methods (using a centrifuge or vacuum) and the automated (using vacuum) procedure were similar Figure . Althouglongated . As the longated , these 9260/280 ratio of between 1.8 and 2.2 (average 2.09) indicative of highly pure RNA [260/230 ratios, which are a measure of polysaccharide contamination, were much more variable. This often occurs with plant samples which typically have relatively high polysaccharide contents [260/230 ratio seemed to have no detrimental effect on subsequent use of the purified RNA for reverse transcription and real time-qPCR (see comments below). This may not be the case for other applications which would have to be individually assessed.The quality of the HTP purified RNA was assessed by UV spectrometry using a Nanodrop spectrophotometer Figure . All purpure RNA . The A26contents -18 howevThe integrity and quality of purified RNA can be analysed using denaturing gel electrophoresis and visualising the ribosomal RNA bands. However, this technique requires a relatively large amount of RNA to detect clearly visible bands and the RNA is not recoverable for subsequent use. Recently, this analysis has been largely replaced by the use of Bioanalyzers which use microfluidics and capilliary electrophoresis on chips. Bioanalyzers require much smaller amounts of RNA and are ideal when limited amounts of starting sample are available. We therefore assessed the purified RNA using Bioanalyzer microfluidic chips which demonstrated its consistently high quality producing distinct, sharp ribosomal peaks Figure .isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) gene expression was performed on eight scattered samples  from one of the 96 well plates  is sufficient for subsequent cDNA synthesis and analysis of expression by qPCR of over 500 genes in triplicate (based on single-plex qPCR) using our usual procedures [[Plant tissue contains large amounts of polysaccharides and a number of endogenous PCR inhibitors such as polyphenolic compounds which can co-purify with the RNA and inhibit downstream applications such as qPCR -18. The bidopsis . All tescedures [ and unpuTraditional methods of RNA extraction such as phenol-chloroform are cheaper to perform and produce a greater yield than kit based silica methods . However, modern health and safety practice encourages the use of safer alternatives (where available) to replace harmful or dangerous chemicals so, even on low throughput, many labs now routinely use silica-based methods (such as spin columns). In addition to simplifying sample handling procedures, the use of a 96 well plate format is cost effective as the cost per sample in the 96 well format is less than half that incurred when using individual mini spin columns. This is before other considerations such as staff time are factored in and represents a considerable saving for larger scale studies involving hundreds of samples. The 96 well plate format also lends itself to automation. Liquid handling robots used to be the preserve of industry however they are becoming more commonplace in academia particularly with the recent move to a more systems-based approach to studying biology. Liquid handling robots are also decreasing in price and becoming more affordable both for core facilities and individual laboratories. Small systems cost around the same as a plate format qPCR machine and can be used for multiple applications as many individual labs will not have sufficient sample throughput for a dedicated robot specifically for RNA extraction. Our optimised protocol can easily be implemented on other robot platforms providing flexibility to the available system.The automated RNA purification procedure is based on the adsorption of nucleic acids to silica membranes and can be performed on the open bench in contrast to a recently published HTP method using phenol chloroform  which relater used in our protocol) can be used. These have recently become commercially available and increasingly popular  or 10 months . Storage at 4\u00b0C also affects the quality of the RNA.Many of the issues encountered with plant RNA extraction protocols are thought to originate from the initial sampling procedure . In orde example ). These C Figure . AlthougWe also investigated the effects of freezing the lysed samples in RA1, the lysis buffer supplied with the illustra kit. Samples were processed immediately or after storage at -20\u00b0C for 2 days or 6 months prior to completion of the extraction procedure Figure . No signlater) and the ability to freeze lysed samples with no detrimental affects on the subsequently isolated RNA, provide a flexible sampling and storage scheme. When this is used in conjunction with our automated HTP RNA extraction procedure, it enables complex time series experiments critical for systems-based plant research.We describe a HTP silica membrane-based method for total RNA extraction from Arabidopsis seedlings in a 96 plate format. This procedure can be performed manually by using appropriate multichannel pipettes or automated with the use of a specialised liquid handling robot. The described protocols isolate RNA in sufficient quantity and of high quality suitable for sensitive downstream processes such as reverse transcription and qPCR. The procedure is time efficient and cost effective for the processing of large numbers of samples. We have used our automated method to investigate and verify sample storage options during the procedure. The combined use of an aqueous RNA stabilising solution (RNAThe authors declare that they have no competing interests.ESC and SK performed the experimental work and performed the data analysis. LEK conceived and coordinated the project and wrote the manuscript. All authors have read and approved the final manuscript."}
+{"text": "To the Editor: As someone with an interest in case reports (1-3), I read your editorial regarding the comeback of the case report with great interest (4). The three case reports that you published (5-7) are a great read. The case report by Nosan et al (6) mentions an \u201cantimongoloid palpebral slant,\u201d which is a term with pejorative connotations . A more informative and appropriate term would be downward-slanting palpebral fissures (10). One can only hope that with time inappropriate terms will disappear from medical discourse save for historical mention and medical journals should play a very important part in discouraging their use (11)."}
+{"text": "Diffusion-weighted magnetic resonance imaging (DW-MRI) is a quantitative MRI technique that provides physiological information by measuring the degree of water molecule diffusion within the extracellular space. It gives a quantitative measurement known as the apparent diffusion coefficient (ADC) value. The aim of the study is to show the influence of the menstrual cycle on breast ADC values and the relationship of the ADC to transverse relaxation (T2) value.b values . The T2 relaxation time was measured using T2w turbo spin echo (TSE) with four echo times . ADC and T2 maps were generated automatically by standard Philips software.Female volunteers had one MRI scan per week over 4 weeks using a 3 T MRI scanner. The ADC of the fibroglandular tissue was measured using a single-shot SE-EPI with four The study was performed on 11 healthy volunteers (23 to 41 years old) with a regular menstrual cycle. There is no significant difference between ADC and T2 values for the 4 weeks. Pearson's correlation coefficient indicated a negative correlation between ADC and T2 values. See Table The study shows that ADC values are not affected by the normal hormonal fluctuations during the menstrual cycle."}
+{"text": "Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions. Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Manipulation of these cell types could allow us to replace dead or diseased cells in our bodies and hence potentially provide a solution to a wide range of medical problems. However, before we can perform such manipulations, we need to understand how the stem cells are wired so that we are able to re-wire them in a logical way to produce the desired cell types. Here we have attempted to understand this wiring by using an RNAi screen in which each individual component of the cell is systematically removed and the consequences on cellular fate determined. We have identified hundreds of genes that are required for efficient loss of stem cell characteristics and hence conversion into other cell types. By studying a subset of these genes, we have been able to show that many converge on two related negative regulators of one of the key pathways that act to promote loss of stem cell identity. These negative regulators, Dusps, normally limit the ability of stem cells to change their function and hence be converted to different cell types. Embryonic stem cells and induced pluripotent stem cells (iPS cells) are currently generating intense interest due to their potential therapeutic roles in regenerative medicine  locus. This reporter gene is regulated in an analogous manner to endogenous rex1Rex1GFPd2 ES cells were maintained in media containing MEK and GSK inhibitors (2i) to maintain their ES cell status and treated with siRNAs pools targeting \u223c17,000 individual genes. After 24 hrs, cells were exchanged into fresh media lacking these inhibitors and the levels of GFP in each cell were assessed over time  (Rex1GFPd2) or ES cells containing an alternative reporter gene, where GFP is instead driven by the oct4  promoter, thereby providing an independent readout for the loss of pluripotency (tcf7l1(tcf3), jarid2, and dpy30jun and mbd3 which were both identified in two of these screens in addition to our own  . As our ipotency . These sipotency . These 3 our own . However our own . Thus cenras, dmbx1, jun, gli3, Having established the core network of genes working in concert with the GSK3 and ERK pathways we wanted to discover the relative contributions of these genes to the actions of the individual pathways. First we performed a counter screen in the presence of both pathway inhibitors (\u201c+2i\u201d) to eliminate siRNAs which promoted accumulation of GFP in the cells irrespective of the activity of the ERK and GSK3 pathways . This elWe then created a network out of the genes from the high confidence dataset identified in the \u201c2i\u201d screen based on previous knowledge of physical and functional interactions. Functionally related subnetworks could be identified, two of the most prominent of which are composed of genes encoding proteins associated with regulating chromatin modifications and sequence-specific DNA binding transcription factors . These gIn summary, by comparing single inhibitor assays, we have been able to subcategorise the genes required for embryonic stem cell differentiation and tentatively assign them to mediating or regulating the effects of either the ERK pathway, or the GSK3 pathway or both. Each pathway appears to require genes associated with overlapping and yet distinct biological processes.rex1 and nanog and the appearance of the early differentiation marker fgf5. The majority of the siRNAs tested showed increased rex1 and nanog expression relative to control siRNAs upon \u201c2i\u201d withdrawal (rex1 and nanog expression (2\u200a=\u200a0.81). Conversely, more than half of the siRNAs tested reduced the accumulation of fgf5 mRNA , and reduced levels of nestin after 5 days  or alternatively the genes might encode proteins which are directly phosphorylated by ERK. For example, it is known that transcriptional regulators such as Ets1, Jun and FoxO1 can all be phosphorylated by ERK in other situations Another key issue to address is whether the ERK and GSK3 pathways work together or in parallel manner, to target different substrates and ultimately control different gene expression programmes and biological functions in ES cells. The two pathway inhibitors have both distinct and overlapping affects on ES cells . The ES cells cultured under \u201c+2i\u201d conditions were adapted in serum/LIF culture conditions for at least 8 passages before the experiments were performed. Cells were stained for alkaline phosphatase expression using an alkaline phosphatase detection kit as described by the manufacturer's (Millipore).ES cells were generally maintained in NDiff N2B27 media  in the presence of the GSK3 inhibitor CHIR99021  and MEK inhibitor PD0325901  (\u201c+2i\u201d media) and were routinely passaged using Accutase  every other day. For differentiation, the media containing inhibitor was removed and replaced with NDiff N2B27 media. Where indicated, ES cells were maintained in serum/LIF conditions in media containing knockout DMEM (Invitrogen 10829-018), 15% heat inactivated FBS (Invitrogen 10082-147), 2 mM of Glutamax-1 supplement (Invitrogen 35050-038), 1% non-essential amino acids (Invitrogen 11140-035), 50 \u00b5M 2-mercaptoethanol (Invitrogen 31350-010) and 5\u00d7104/cm2 cells (ie 1.28\u00d7104 cells/well of 96 well plate) were plated out into a mixture of 0.3 \u00b5l of RNAi Max (Invitrogen) and 100 nM siRNA in 100 \u00b5l of \u201c+2i\u201d media for 24 hrs. All validation experiments used ON-TARGETplus siRNA SMART pools from Dharmacon.For RNAi, 4\u00d710gapdh, hmbs and tbp. The primer-pairs used for RT-PCR experiments are listed in Real time RT-qPCR was carried out as described previously Western blotting was carried out with the primary antibodies; Erk2 , phospho-ERK , Dusp1 , Dusp6  and Pou5f1 . All experiments were carried out in 96-well plates. The lysates were directly harvested in the 2\u00d7SDS sample buffer followed by sonication . The proteins were detected using infrared dye-conjugated secondary antibodies , and the signal was collected with a LI-COR Odyssey Infrared Imager and quantified using Odyssey software .The Ras activities were examined using Ras activation ELISA assay kit (Millipore) as described in the manufacturers' instructions. The total lysates used in the ELISA assay was normalised with the quantity of the proteins assayed by the BCA protein assay kit (ThermoScientific).Flow cytometric analysis was carried out using a LSRII flow cytometer and samples were loaded using HTS loader (BD Biosciences). For sampling, media was removed from each well. Single cell suspensions were generated by treating cells with accutase at 37\u00b0C for 7 mins followed by resuspendion in 0.03% BSA/PBS. Dead cells were stained by Sytox Red dead cell stain . The cells were analysed immediately after sampling. Each sample was analysed with 10,000 event counts with the flow rate at 1 \u00b5l/s. The resulting GFP profile (green channel) was created by gating with the right ranges of cell sizes based on forward and reverse scatter plot  and dead cells were gated away based on the Sytox Red stain profile (red [APC] channel).Rex1GFPd2 ES were grown in 96 well plates in the presence of \u201c2i\u201d and reverse transfection was performed using siGENOME siRNA pools . 24 hrs later, the \u201c2i\u201d media was removed and replaced with fresh NDiff N2 B27 media. After 28 hrs, the levels of GFP in the cells were determined by flow cytometry as described above. Each plate contained 8 control non-targeting siRNAs, and the positive control siRNAs against gsk3\u03b2 and fgf4. To take into account slight variations in the timing of pluripotency loss, the ratio of high GFP to low GFP expressing cells was established on each plate based on the non-targeting controls, allowing a threshold to be set as 1 (ie 50% high GFP and 50% low GFP). This threshold was used to determine the ratio of high to low GFP expressing cells in the other wells. The mean plus/minus standard deviation (SD) was calculated for each plate, and individual wells were scored positive if they exceeded 2\u00d7SD above or below this mean. The screen was performed in duplicate, with duplicate plates being analysed on different days. A final list of positive hits was determined by taking siRNAs which scored an average of 2\u00d7SD across both plates (or on a single plate where the duplicate well was defective in the case of 30 siRNAs), generally with both plates scoring >1.5\u00d7SD above the mean. However, an additional small number of siRNAs were scored as positive where the average score was >2.5\u00d7SD above the mean where only one plate had to score >1.5\u00d7SD above the mean, and also for seven siRNAs where the average score was >1.9\u00d7SD above the mean and both plates scored >1.9\u00d7SD above the mean.All liquid handling processes were performed using Biomek robotic system (Beckman Coulter). For the primary screen, Rex1GFPd2 or Oct4GFP ES cells were used and screens were performed as above except that ON-TARGETplus siRNA duplexes were used and GFP levels in Oct4GFP ES cells were determined 72 hrs after release from \u201c2i\u201d. Individual wells in each screen were scored as positive if the average GFP(+)/GFP(\u2212) ratio exceeded 1.25\u00d7SD above the non-targeting controls across both duplicate plates. Additional hits were considered as positive if they scored >0.8\u00d7SD above the mean in one validation screen and also scored >1.0\u00d7SD above the mean in the other.For the validation screens, either Rex1GFPd2 were used as for the validation screens but only one inhibitor (ie either CHIR99021 or PD0325901) was withdrawn. Wells were scored as positive if the average GFP(+)/GFP(\u2212) ratio exceeded 1.5\u00d7SD above the mean of the non-targeting controls.For the \u201c1i\u201d screens, For constructing networks, lists of gene names were uploaded into STRING GO term analysis was carried out using DAVID Bioinformatics Resources 6.7 (NIH) Figure S1Rex1GFPd2 ES cells grown for the indicated times in the presence of the inhibitors CHIR99021 and PD0325901 (\u201c2i\u201d) or upon removal of the inhibitors (\u201c\u22122i\u201d). The profiles corresponding to high GFP expressing cells are shown in dark green in (A). The effect of pre-treating cells with control non-targeting siRNAs  or siRNAs against fgf4 (blue) or gsk3 (red) on the GFP expression profile is shown in (B). The dashed line shows the position of the centre of the peak of the starting population of cells. (C) FACS profiles of GFP expression in Oct4GFP ES cells grown for the indicated times following removal of the inhibitors (\u201c\u22122i\u201d) in the presence of the indicated siRNA constructs (as labelled in B). Clear shifts in the distributions of high versus low GFP expressing cells can be observed between 27 and 30 hr for the Rex1GFPd2 ES cells and at 72 hrs for the Oct4GFP cells after removal of \u201c2i\u201d in the presence of fgf4 or gsk3 siRNAs.Kinetics of GFP expression level changes in the primary and validation library screens. (A and B) FACS profiles of GFP expression in (PDF)Click here for additional data file.Figure S2Rex1GFPd2 ES cells; column 2, validation \u201c2i\u201d screen using Rex1GFPd2 ES cells; column 3, validation \u201c2i\u201d screen using Oct4GFP ES cells; column 4, secondary \u201c1i\u201d screen using Rex1GFPd2 ES cells upon removal of CHIR99021; column 5 secondary \u201c1i\u201d screen using Rex1GFPd2 ES cells upon removal of PD0325901. Examples are shown for siRNAs which affect the ratio of high/low GFP expression (A) specifically when only the ERK pathway inhibitor is removed (ERK only), (B) only the GSK3 inhibitor is removed (GSK only), (C) either the ERK pathway or GSK inhibitors are removed (ERK/GSK) or (D) only when both the ERK pathway and GSK inhibitors are removed (ERK & GSK). (E) Example FACS profiles of GFP expression in the presence of control non-targeting siRNAs or siRNAs against the indicated genes from the primary \u201c2i\u201d screen, for hits which demonstrate enhanced loss of GFP expression. .Representative FACS profiles in each of the library screens. (A\u2013D) FACS profiles of GFP expression in the presence of control non-targeting siRNAs or siRNAs against the indicated genes. The numbers next to each gene name indicate plate and well numbers and the numbers above each graph are the corresponding GFP high/GFP low ratios. Example profiles from the following screens are provided; column 1, primary \u201c2i\u201d screen using (PDF)Click here for additional data file.Figure S3Enriched functional categories of genes in the high confidence dataset. Enriched classes of genes amongst the 292 high confidence hits resulting from the validation screens [depicted in (A)] were determined by searching for Gene Ontology (GO) terms using DAVID.  Data are shown graphically according to their relative P-values or  data are visualized using REVIGO. GO terms are grouped according to the level of complexity of the terms; (B and C) molecular function, (D and E) biological process and (G and H) cellular component.  data are plotted according to the size of the GO term category  and the significance of the association with the category (x-axis). The identities of the most significant categories are indicated. (F and I) Analysis of interacting networks of enriched GO terms from the molecular function (F) and the cellular component (I) categories, depicted using the \u201cinteractive graph\u201d view of REVIGO.(PDF)Click here for additional data file.Figure S4Rex1GFPd2 ES cells grown for the indicated times in the presence of the inhibitors PD0325901 and CHIR99021 (\u201c2i\u201d), upon removal of both of the inhibitors (\u201c\u22122i\u201d), or removal of either the PD0325901 or the CHIR99021 inhibitors (\u201c\u22121i\u201d). The data are shown graphically (A) and as FACS profiles of GFP expression 60 hrs after inhibitor removal (B).Kinetics of GFP expression level changes in the \u201c1i\u201d screens. GFP expression profiles of (PDF)Click here for additional data file.Figure S5Heatmap summary of the hits from the \u201c1i\u201d screens. The heatmap summary depicts the stratification of the hits according to their effects on the GFP(+)/GFP(\u2212) ratio upon withdrawal of the GSK3 inhibitor (GSK3) or MEK inhibitor (ERK). Red indicates an increased ratio and blue represents a decreased ratio, and intermediate colours given according to the scale bar. Hits are grouped as specific to the ERK or GSK3 pathways (left side map), related to either the ERK or the GSK3 pathways  or only functional when both pathways are active (PDF)Click here for additional data file.Figure S610, and coloured according to the bar shown below the figure. Terms corresponding to distinct functional groups are manually clustered (indicated on the left), and are then ranked according to their significance scores in the \u201cERK only\u201d dataset.Heatmaps of the enriched GO terms identified for genes corresponding to high confidence hits from the validation screens. The heatmap distribution of the full list of significant GO terms identified specifically associated with the ERK (133), or GSK3 (168) pathways and the total 274 validated hits. Each GO term is scored by \u2212log(PDF)Click here for additional data file.Figure S7Heatmaps of the specific subsets of enriched GO terms identified for genes corresponding to high confidence hits from the validation screens. Heatmap distributions of specific subsets of significant GO terms identified specifically associated with the categories of hits described in (PDF)Click here for additional data file.Figure S8Interaction networks of genes associated with high confidence hits from the validation and secondary screens. STRING network analysis of the core network formed by the 274 genes associated with signal-dependent loss of pluripotency and promoting early differentiation processes in the mouse embryonic stem cells. Genes are grouped according to common biological processes. (A) In addition to a highly connected central network Click here for additional data file.Figure S9nanog and rex-1 (B), and the early differentiation marker, fgf5 (C). The expression levels were normalised by the average of three reference genes  or gapdh (C). Data are shown for each siRNA duplex relative to the maximal expression exhibited in the presence of an siRNA pool (taken as 100). The blue dashed lines indicate the threshold level (>1.5 SD above (B) or below (C) the mean of the control [ctr] siRNAs). Genes which show changes exceeding (B) or below (C) this level, are indicated by red bars. Data are presented as means \u00b1 SEM and are the average of three biological replicates (n\u200a=\u200a3). Heatmap summaries (see rex1 (y-axis) and the reciprocal of fgf5 (x-axis) mRNA expression levels upon 2i withdrawal for 36 hrs and 48 hrs, respectively, are plotted following knockdown of individual genes (see B and C for details). Data are shown for each siRNA duplex relative to the maximal expression exhibited in the presence of an siRNA pool (taken as 100). Dotted lines represent the expression values >2 standard deviations above the mean of the negative control siRNAs. Red dots represent siRNA duplexes which promote elevated expression of nanog and lower levels of fgf5 (quadrant 1), whereas green (quadrant 3) and black (quadrant 4) dots represent siRNAs that cause changes at or below this threshold cut-off value for only one gene. The brown dot represents the negative control siRNAs. Blue dots (quadrant 2) represent siRNAs that cause elevated nanog expression but fail to show reductions in fgf5 expression.The effects of depletion of genes identified in the validation screen on the expression of markers of pluripotency and early differentiation. (A) Venn diagram (top) illustrating the number and a list of selected screen hits from each category used in the subsequent studies. (B and C) RT-qPCR analysis of the effects of depletion of the indicated genes on the mRNA expression levels of the ES-specific genes, (PDF)Click here for additional data file.Figure S10Western blot analysis of phospho-ERK levels upon depletion of genes identified in the secondary siRNA screens. ERK activation levels following 2i withdrawal (\u22122i) for 20 mins were determined by western blotting using a phospho-ERK-specific antibody  and normalized against total ERK levels (bottom panels). siRNAs against the indicated genes from the \u201c1i\u201d screens  or control non-targeting siRNAs (ctrl) are indicated. The presence or absence of inhibitors in the control samples is indicated (+/\u22122i). Dotted lines indicate where gels have been cut to remove irrelevant lanes and rejoined. The data shown are from one of three biological replicate experiments.(PDF)Click here for additional data file.Figure S11Summary of the effects of each screen hit on the activity of Ras and ERK. A heatmap summary see . The col(PDF)Click here for additional data file.Figure S12Rex1GFPd2 ES cells grown in the presence of LIF and released for 28 hrs. The profiles corresponding to high GFP expressing cells are shown in dark green. The effect of pre-treating cells with control non-targeting siRNAs or siRNAs against the indicated genes on the GFP expression profile under these conditions is shown. The ratio of high to low GFP expressing cells is shown quantitatively on the right as means \u00b1 SEM; n\u200a=\u200a2\u20133). The vertical lines represent the mean of the non-targeting control (ctrl) (solid line) and two standard deviations above this mean (dotted line).GFP expression level changes upon gene depletion under LIF conditions. FACS profiles of GFP expression in (PDF)Click here for additional data file.Figure S13dusp1, dusp5, dusp6 and dusp14 mRNA expression in mouse ES cells. Data are normalised by the average values of three reference genes (ref) and are presented as means \u00b1 SEM and are the average of three biological replicates (n\u200a=\u200a3). (A) The kinetics of dusp5 and dusp14 expression at the indicated times following 2i withdrawal. (B) The effects of the indicated inhibitors, either alone or in combination, on the expression of dusp5 and dusp14 at the indicated times following inhibitor withdrawal. (D and E) The effects of depletion of the indicated genes on dusp5 mRNA expression in the presence, (D) or dusp1, dusp5, or dusp5 mRNA expression in the absence, (E) of 2i. The blue dashed lines indicate the threshold level (2\u00d7 SD above/below the mean of the negative controls) and levels below and above this are indicated by red and pink bars, respectively. The average activity in the presence of control siRNA (ctrl) is shown by the solid grey line. (C and F) Dusp1 and Dusp6 protein expression measured by western blot analysis (bottom panels) was quantified and normalized by Pou5F1 levels . (C) The kinetics of Dusp1 and Dusp6 expression at the indicated times following 2i withdrawal. (F) The effects of depletion of the indicated genes on Dusp1 and Dusp6 expression in the presence of 2i. Data are the average of two experiments. (G) Active ERK levels were determined by the ratio of phospho-ERK (pERK)/total ERK (ERK) levels at the indicated times following 2i release for the indicated times in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to maximal levels with the control siRNA (taken as 100) and are presented as means \u00b1 SEM from the average of two biological replicates (n\u200a=\u200a2).The regulation of Dusp activity and ES cell differentiation (A\u2013B and D\u2013E) RT-qPCR analysis of (PDF)Click here for additional data file.Figure S14The role of Dusps in MAP kinase activation in ES cells. (A) Active ERK levels were determined by the ratio of phospho-ERK (pERK)/total ERK (ERK) levels at the indicated times following 2i release in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to maximal levels with the control siRNA (taken as 100) and are presented as means \u00b1 SEM from the average of two biological replicates (n\u200a=\u200a2). (B) Active levels of the indicated MAPKs were determined by the ratio of phosphorylated (p) to non-phosphorylated forms at the indicated times following 2i release in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to basal levels with the control siRNA in the presence of 2i (taken as 1) and are presented as means \u00b1 SEM from the average of two biological replicates (n\u200a=\u200a2).(PDF)Click here for additional data file.Table S1Rex1GFPd2 cells. (B) Positive hits from the validation screen in Rex1GFPd2 cells. (C) Positive hits from the validation screen in Oct4GFP cells. See Positive hits identified in the primary and validation siRNA screens for genes involved in escape from pluripotency. (A) Positive hits from the primary siRNA screen in (XLSX)Click here for additional data file.Table S2Rex1GFPd2 and Oct4GFP cells compared to negative controls. Hits were scored as positive according to the same criteria as those which scored positive for retention of pluripotency.Hits identified in the primary and validation siRNA screens for genes involved in maintaining pluripotency. Genes are listed when their depletion results in more loss of GFP-positive cells with both (XLSX)Click here for additional data file.Table S3Overlaps with other siRNA screens. Overlaps between genes identified in this paper as important for promoting loss of pluripotency and onset of differentiation  or the m(PDF)Click here for additional data file.Table S4Rex1GFPd2 and Oct4GFP cells and were considered as a high confidence group for further analysis.Positive hits identified in both of the validation siRNA screens for genes involved in escape from pluripotency. These hits scored positive in validation screens with both (XLS)Click here for additional data file.Table S5Rex1GFPd2 cells in the presence of the MEK and GSK3 inhibitors (\u201c+2i\u201d). These were not taken forward for further analysis due to the lack of a potential link to active ERK or GSK3 signalling.Hits eliminated in the counterscreen. Validated hits from the primary screens which also scored as positive in a screen with (XLS)Click here for additional data file.Table S6Summary of the point of action of siRNAs with respect to the ERK signaling pathway. Genes targeted by the siRNA constructs are grouped according to whether they act to potentiate from Ras activity, act downstream of Ras to activate ERK or play a role downstream from, or in parallel to, ERK signalling. Each column indicates the association of the genes with the ERK, GSK or both pathways, as determined by the \u201c1i\u201d screens see .(PDF)Click here for additional data file.Table S7PCR primers used in RT-qPCR. The sequences of both forward (F) and reverse (R) primers are provided.(PDF)Click here for additional data file."}
+{"text": "Typical flow velocities in the extraparenchymal pulmonary veins demonstrate two major antegrade flow waves: a biphasic systolic wave S1 and S2) and a monophasic early diastolic wave (D)  with RV-independent pulmonary circulation . Phase-contrast imaging of the pulmonary veins was performed on a 1.5 T MR scanner with velocity encoding set at 120 cm/s. We compared these flow patterns with the flow patterns of a control group of 10 patients (15 pulmonary veins) who had RV-dependant pulmonary circulation and underwent CMR due to other reasons .In all patients with RV-independent pulmonary circulation pulmonary vein flow curve showed a single systolic peak in early systole with the S2 peak consistently absent Figure .This study further supports the concept that the late systolic pulse (S2) of the pulmonary vein flow curve is caused by forward propagation of the right ventricular systolic pressure pulse. It also demonstrates that the early systolic pulse (S1) is independent of the right ventricle but dependent on the left atrial and left ventricular events."}
+{"text": "In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA notch3 expression is mostly in Bergmann glia and at low levels in few PCNA The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90  Teleost fish, like many non-mammalian vertebrates, display widespread neurogenesis in adulthood (see review(s) In the zebrafish dorsal telencephalon, the cellular composition of progenitors is mixed, with a fraction of cells that do not display glia characteristics intermingled with others that show markers and morphology typical of radial glia The Notch pathway is a conserved pathway throughout the animal kingdom and has been intensely studied for its crucial role in cell fate decision, proliferation and cell death during embryonic neural development  and notch3 (previously notch5) notch2 expression is excluded from the nervous system during embryonic development Multiple Notch receptors are present in the zebrafish genome, namely in situ hybridization and co-stained them with glial, neuronal, oligodendrocytic, endothelial and proliferation markers. In the adult zebrafish brain, we find all known Notch receptors to be expressed, except for notch2. Our analysis reveals that notch1a, notch1b and notch3 are expressed in all five neurogenic regions of the adult zebrafish brain and that they localize with the majority of the proliferating cells. Similar to embryonic stages, we also find overlapping and complementary expression patterns of Notch receptors in different adult neurogenic niches. The expression of Notch receptors in proliferating cells suggests that Notch signaling might be required at a basal level for maintaining proliferation of neural progenitors.Because the cellular composition of progenitor cells in the adult zebrafish brain shows great diversity and different responses of progenitors to Notch signaling has been reported in mammals, we analysed in detail the expression of the four Notch receptors in five adult zebrafish neurogenic niches. To this end, we characterized the expression pattern of Notch receptors by notch1a, notch1b and notch3 but not notch2 was detected in the adult brain. Thus notch2 is not further mentioned.We focused our analysis on five neurogenic zones: the dorsal and ventral telencephalon, the hypothalamus, the optic tectum, and the cerebellum. It has been shown that proliferating cells in these areas are periventricularly located notch1a, notch1b and notch3 are mostly expressed in cells lining the ventricular surface  for the receptors and olig2, a marker for oligodendrocytes. This revealed that all three Notch receptors are expressed in a subpopulation of olig2Tg(fli1:gfp) notch1a and notch3 localize in a subset of these GFP In the adult zebrafish telencephalon,  surface . In the  surface  \u2013C . Inrea (Dm) , B and ea (Dll) , B . Inobserved , B . In and Dll . These T and vT . To asse2 cells . Using tP  cells .In the dorsal telencephalon different cell populations have been previously characterized based on their proliferative status and presence of glial markers Fig , 3, .in situ hybridization analysis, cells positive for notch1a and notch1b are scattered along the ventricular surface of the dT  notch1a and notch1b are often co-expressed in proliferating cells, iv) notch3 is expressed in very few S100notch1a and notch1b which are present in roughly more than half of this population.All together, these results show that i) all three Notch receptors are expressed in approximately the same number of proliferating glial cells, ii) notch1a and notch1b are expressed in some, but not all of the S100notch3 is found throughout the ventricular zone of Vd  and the ventral nucleus of the ventral telencephalon (Vv) al cells , while nne of Vd .notch3 than for notch1a/1b  in groups of cells with different expression intensities , we detected single scattered notch1a , A  andnotch1b , B  celentricle . Moreovecaudally .notch1a and notch1b are arranged in a pattern that resembles the distribution of proliferating cells in this zone as previously shown notch3 is expressed in a very similar fashion to notch1b , at the level of the posterior recess, positive cells for  notch1b .notch3 covers most of the S100notch3 is also expressed in most of the S100notch3notch1a/3 shows that notch1a is expressed in part of the notch3 domain  of adult teleost fish, active proliferating cells are located in the dorsomedial area of the periventricular gray zone (PGZ) notch1a, notch1b and notch3 in dPGZ, mPGZ and PML  . notch3  as well . We thengenitors . A few cL and GL . Co-stais notch3 . Also nonotch1a is present predominantly in the progenitor niche, whereas notch1b is also strongly expressed in a few Bergman glia cells. notch3 is mostly excluded from the niche but expressed in a high proportion of the Bergman glia , a notch3 homologue has been identified, and the expression of this receptor was also found in ventricularly located cells of the adult brain et al.notch3 is expressed in most of the radial glia population of the dorsal telencephalon and hypothalamus neurogenic niches, but also that it is associated, together with notch1a and notch1b, with proliferating cells throughout the adult brain. A possible explanation for these differences might be that we have preformed the in situ hybridization on 12 notch2 expression in the adult brain, opposed to what has been previously reported Our expression pattern analysis is consistent with what had been observed in zebrafish larval brains, where In the mammalian brain several studies support the idea that NSCs present high Notch activity, essential for their maintenance and proliferation In the dorsal telencephalon we observed that approximately 20 notch1a, notch1b and notch3 are expressed in proliferating glial cells, which form the majority of the constitutively proliferating neural progenitor population . Considering that the cellular composition of the ventricular zone cells is very diverse, different cell types could be more or less responsive to variations in Notch signaling activity, and the outcome of such variations may be very different depending on the cell type/state and neurogenic niche. It could also well be that combinatorial signaling mediated by the activation of different Notch receptors triggers distinct responses in different cells or at different stages.Here we show that pulation . However is rare . As the notch3 is strongly expressed in the majority of S100notch1a and notch1b expressed in a subset of notch3notch3 might have a more prominent role in the glial lineage, whereas notch1a/1b expression might be related to their progenitor character  of the adult zebrafish brain show a pseudostratified neuroepithelial-like morphology and interkinetic nuclear migratio In chick spinal cord, a study reports that Notch activity increases at a higher frequency prior to mitosis notch3, and partially notch1b, are expressed in Bergmann glia in the adult zebrafish. Additionally, Notch1 receptor is expressed in mature neurons in the rodent cortex notch1a is expressed both in neural precursors and in differentiating neurons whereas notch1b and notch3 are found only in neural precursors notch1b in a HuC/D notch1b expression that we observed in neurons could indicate that also in the adult zebrafish brain Notch signaling might serve later stages of neuronal differentiation and/or neuronal activity.Notch signaling is not only active in germinal zones but also in non-proliferative areas. Similarly to the adult mouse cerebellum olig2 is a marker for both oligodendrocyte precursor cells (OPCs) and, in combination with other markers, for more mature oligodendrocytes olig2olig2olig2olig2, promotes the specification of OPCs olig2 subpopulation could represent immature oligodendrocytes.Oligodendrocytes are another cell type produced in neurogenic regions of the adult zebrafish brain notch3 expression is associated with the arterial fate during arterial-venous differentiation notch1a and notch3. This suggests that also in the adult zebrafish brain, Notch signaling might be important for vessel homeostasis or for the maintenance of arterial phenotype in some cells.Notch signaling is also involved in the development of the vascular system (see review(s) notch3 might be important for the glial properties of progenitors as well as other glial cells; ii) notch1a/1b might modulate progenitors proliferative status in a level-dependent manner Our results showed that Notch receptor expression domains overlap to a great extent in the neurogenic areas, but also that regional and cellular heterogeneity in the expression of different receptors exists in the adult zebrafish brain. The differences observed between receptor expression in certain niches could indicate that they are involved in distinct cellular processes. Therefore, we hypothesise that: i) predominantly All procedures were in accordance with the live animal handling and research regulations of the University and State of Saxony, Germany, review boards, the Regierungspr\u00e4sidium Dresden (permit AZ 24D-9168.11-1/2008\u20131 and \u22124). This institutional review board specifically approved this study.gol-b1 line in the AB genetic background fli1:GFP) Fish were kept under standard conditions as previously described in situ hybridization experiments, brains were cryosectioned in 14 in situ hybridization (FISH) stainings, brains were cryosectioned in 12 Whole heads with the brain exposed were fixed in 4notch1anotch1bnotch2notch3olig2in situ hybridization was done as previously described in situ reaction, with better sensitivity for detecting cells expressing lower levels of notch transcripts. All in situ hybridizations were done on at least five individuals. All probes were tested on embryos to confirm probe specificity. We have also perfomed negative controls: 1) with probe and without anti-DIG antibody, and 2) without probe and with anti-DIG antibody to assess for unspecific signal (antibody or tyramid trapping). In these tests no signal was detected under the fluorescence microscope.Riboprobe preparation was done as previously described Immunostainings for S100http://fiji.sc), CombineZP v7.0 (http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/News.htm) and Adobe Photoshop CS4. Figures were assembled using Adobe Illustrator CS4. Confocal images shown are single planes or maximum projections of up to 3 planes . Confocal images were taken on a Leica TCS-SP5, using the Leica HCX PL APO 40\u00d7/0.75 Dry and Leica HCX PL APO 63\u00d7/1.2 Water objectives. Image acquisition for the quantifications was done using the Leica HCX PL APO 40\u00d7/0.75 Dry objective. To minimize cross talk between the channels, sequential image acquisition was performed. Images were processed using Fiji (http://www.r-project.org/) using ANOVA followed by a post-hoc Tukey's HSD test.The quantifications of co-localization between the FISH, S100notch1a (n\u200a=\u200a4), 24 for notch1b (n\u200a=\u200a4) and 26 for notch3 (n\u200a=\u200a5). For data visualization and median comparison we used notched boxplots in R and modified them with Adobe Illustrator CS4. To confirm statistically significant differences in expression of each gene between apical and basal or between genes in the same nuclei position we applied the Wilcoxon rank-sum test or ANOVA followed by a post-hoc Tukey's HSD test, respectively. Fluorescence profile analysis was done using Fiji Plot Profile. Fluorescence values for each gene expression were normalized for background signal in the tissue and their corresponding maximum. Plots were done in Microsoft Ofice Excel 2007 and modified in Adobe Illustrator CS4.To analyse Notch receptor expression levels in proliferating cells of the ventral telencephalon we measured the mean grey value (MGV) of the FISH signal for each receptor. We selected the regions of interest based on the position of the PCNA Figure S1Notch receptor expression in telencephalic oligodendrocytes. Confocal images of double FISH showing the localization of Notch receptor (white) and olig2 (green) in the dorsal telencephalon parenchyme. A\u2013C, notch1a, notch1b and notch3 are expressed in a subpopulation of parenchymal olig2 olig2notch1bolig2(TIF)Click here for additional data file.Figure S2Notch receptor expression in fli1:gfp endothelial cells in the telencephalic parenchyme. Confocal images showing localization of notch1a and notch3 and gfp A\u2013Bnotch1a and notch3 expression in a few endothelial cells (white arrows); unfilled arrow in B indicates a notch3(TIF)Click here for additional data file.Figure S3Apical to basal gradient of Notch receptor expression in the ventral telencephalic niche. Fluorescence profile measurements of Notch receptor expression in the two indicated areas (dark grey rectangle in schematics) of the Vv proliferation zone. Stronger fluorescence signals are detected in proliferating cells Click here for additional data file.Figure S4notch3 expression in glia and proliferating cells of the adult zebrafish hypothalamus. Confocal images showing localization of notch3 mRNA by FISH (white), radial glia labelled with S100A, B, notch3 is expressed in most PCNA notch3A\u2013F, notch3 localizes with most S100notch3notch3 expression in the S100notch3. Abbreviations: Hc, caudal zone of the periventricular hypothalamus; Hd, dorsal zone of the periventricular hypothalamus; Hv, ventral zone of the periventricular hypothalamus: PR, posterior recess of the diencephalic ventricle. Scale bar \u200a=\u200a100 (TIF)Click here for additional data file.Figure S5Overlapping and complementary notch1a/3 expression in the adult zebrafish hypothalamus. Confocal images of double FISH showing the localization of notch1a (white), notch3 (red), and PCNA (green). Cross-sections at the indicated level through the diencephalon; hypothalamic area shown in the micrographs is indicated in the cross-section schematics. A\u2013C, notch1a is expressed in a subpopulation of notch3notch3notch1a expression partially overlaps with the notch3notch1anotch3(TIF)Click here for additional data file.Figure S6notch1a and notch1b expression in olig2  Confocal images of double FISH showing the localization of notch1a/1b (white), olig2 (green) and HuC/D (red) in the superficial layer of the optic tectum. Cross-sections at the indicated level through the mesencephalon; tectal area shown in the micrographs is indicated in the cross section schematic in A. A\u2013B, notch1a and notch1b are expressed in a subpopulation of olig2olig2B, notch1b is also expressed in olig2(TIF)Click here for additional data file.Table S1Localization of Notch receptor positive cells with glial and proliferation markers. Number of cells counted in adult zebrafish Dm and Dl telencephalic areas according to their notch expression and co-label with radial glial (S100(PDF)Click here for additional data file.Video S13D animation showing notch1a expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file.Video S23D animation showing notch1b expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file.Video S33D animation showing notch3 expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file."}
+{"text": "Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology.Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures.  Tbx5. Activation of Tbx5 in the forelimb-forming region of the developing embryos is essential for forelimbs to form and disruption of human TBX5 causes limb abnormalities. We show that activation of Tbx5 in a restricted territory is achieved through a combination of activation inputs that are present broadly throughout the embryo flank and dominant, repressive inputs present only in more caudal regions of the flank. The sum of these inputs yields restricted activation in the rostral, forelimb-forming flank. Our results explain how the regulatory switches that were harnessed for the acquisition of limbs during evolution operate and how they can be turned off during the evolution of limblessness in species such as the snake.The acquisition of limbs during vertebrate evolution was a very successful innovation that enabled this group of species to diversify and colonise land. It has become clear recently that the primary driver behind the evolution of new structures, such as limbs, is the acquisition of novel regulatory elements that control when and where genes are activated rather than the proteins encoded by the genes themselves acquiring novel functions. We have identified the regulatory element from a gene,  Tbx5 in nascent forelimbs and Tbx4 in hindlimbs Fgf10 in the limb mesenchyme Fgf8 expression in the apical ectodermal ridge (AER) and Fgf8 produced from the AER, in turn, maintains Fgf10 expression in mesenchyme to establish a positive feedback loop of Fgf signalling that maintains limb growth. Mutations in human TBX5 cause Holt-Oram Syndrome (HOS OMIM142900), a disorder characterised by upper limb and heart abnormalities TBX4 cause Small Patella Syndrome (SPS OMIM 147891), a disorder characterised by knee, pelvis and toe defects Tbx5 is the earliest marker of presumptive forelimb mesenchyme and because activation of this factor within a defined region of the LPM ultimately dictates the position at which the forelimbs will arise, identifying the factors that control activation of this Tbx5 expression domain will reveal the mechanisms employed that allowed the acquisition of limbs in vertebrates and that dictate forelimb position in the embryo.Forelimbs and hindlimbs are derivatives of the lateral plate mesoderm (LPM) that arise at fixed positions along the vertebrate body axis. Limb formation is initiated by limb induction signals from axial tissues Tbx5 is initially expressed in the forelimb-forming region of LPM prior to the emergence of a bud and it is subsequently restricted to the forelimb region as development proceeds. Tbx5 is essential for forelimb formation and this exclusive requirement is limited to a short time window when limb bud initiation occurs Tbx4, the paralog of Tbx5, is able to rescue forelimb formation following conditional deletion of Tbx5Tbx4/5 gene represented by AmphiTbx4/5 of the limbless cephalochordate, amphioxus, can fully compensate for the loss of Tbx5 in the mouse Tbx5 expression in the LPM was a critical step in the acquisition of limbs during vertebrate evolution.Hox genes are conserved homeodomain-containing transcription factors that are arranged in clusters in the genome. The chromosomal organization of the genes in the complex reflects their expression pattern along the rostro-caudal body axis to determine positional identity Tbx5 regulatory element sufficient for early forelimb expression Tbx5. However, since the ability to activate Tbx5 is not strictly restricted to Hox genes expressed only at forelimb level, the mechanism by which a rostro-caudal Hox code establishes forelimb-restriction of Tbx5 remained unknown. Here, we demonstrate how Hox paralogous group members act cooperatively to restrict expression of Tbx5 in the LPM, which ultimately determines the positions the forelimbs will emerge from the flank of the embryo. We show that mutations of a single Hox binding site in the Tbx5 forelimb regulatory element cause expanded reporter gene expression in caudal LPM. Rostral restriction in Tbx5 expression through repression in the caudal LPM is mediated by Hoxc8/9/10 genes and this repressive function is limited to Hox genes that are expressed in Tbx5-negative caudal LPM. We further map the Hoxc9 protein domains required to confer transcriptional repression that distinguishes these paralogs from other Hox proteins expressed throughout the flank of the embryo. Our results demonstrate how a nested, combinatorial code of Hox protein transcriptional activation and repression along the rostro-caudal embryo axis restricts Tbx5 expression to the forelimb and ultimately determines forelimb position.We have previously identified a Tbx5 gene that recapitulates the dramatic forelimb-restricted expression of this gene Tbx5 expression, we generated a series of constructs in which each individual Hbs site1-6 is mutated and tested their ability to activate a LacZ reporter gene in transgenic mice. While the Tbx5 int2(361) reporter construct drove forelimb-restricted expression of LacZ (Tbx5 int2(565)) . To distinguish the requirement for Hbs2 and 6bp3\u2032, we next mutated either of these sites )  that cont2(565)) . We haveess Tbx5 . These rse sites . Mutatioreporter , while mreporter  equivalereporter . These rThese results demonstrate that the binding sites within this regulatory element can be divided into 2 distinct functional groups. Hbs1 and 3-6 act as \u2018on\u2019 switches important for the amplitude of activation, whereas Hbs2 determines spatial resolution by hosting repressive complexes that restrict the domain of activation. This element can therefore have a binary function, serving as a site for the formation of transcriptional activation or repression complexes.Hox genes can activate this regulatory element Hox genes in chick and mouse embryos at stages when Tbx5 is first expressed in the forelimb-forming region. Tbx5 is first expressed at the level of somites 13\u201320 in chick and somites 4\u201311 in mouse embryos. As previously reported Hox4 and Hox5 paralogs overlap with that of Tbx5 in both mouse and chick embryos   construct  reporter, LacZ expression is repressed in the forelimb region   LacZ repficiency ), \u03b2-gal ficiency  indicatireporter  demonstrTbx5 expression, we co-electroporated Hoxc9 with a Tbx5 int2(361) reporter in which Hbs2 is mutated  showed clear repression of the reporter demonstrating that within the limits of this assay the entire N-terminus or the domain overlapping the junction between N1 and N2 is required for repressive activity  can reduce LacZ expression , suggestTbx5 as an assay, we have been able to distinguish the opposing transcriptional activities of different Hox paralogous group proteins. Hoxc9, as well as Hoxc8 and Hoxc10, that are normally expressed in the LPM caudal to the forelimb, can repress Tbx5 to restrict its expression to the forelimb level region of the LPM  is required for its repression in caudal LPM (Our results demonstrate that five of the six Hox binding sites (namely Hbs1 and Hbs3-6) within the udal LPM . One posudal LPM , suggesthttp://www.genomatix.de) is the presence of a 6-bp sequence named Pbx1-Meis1 complexes site located 3\u2032 of Hbs2 (6bp3\u2032). Pbx is a Hox co-factor that can attenuate Hox-mediated gene transcription by recruiting histone deacetylases (HDACs) Tbx5 forelimb regulatory element.An alternative model is that the transcriptional activity of the Hox complex bound at Hbs2 is determined by a co-factor(s). The sequence of Hbs2 is identical to the sequences of Hbs1 and Hbs3, therefore we compared the sequences surrounding these Hox binding sites. One distinguishing feature of Hbs2 identified using Mat Inspector  in the protein. The hexapeptide of AbdA represses dpp expression by inhibiting the function of a glutamine (Q)-rich C-terminal activation domain Tbx5 transcription, we suggest the model that Hoxc9 supresses Tbx5 expression by interaction with co-repressor(s) (in vitro translated proteins or nuclear extract from LPM (data not shown). Other potential collaborators are Smad proteins. In the Drosophila haltere, a Mad/Med/Shn complex works in combination with Ubx to repress Sal expression Tbx5 expression . Other candidate repressors are engrailed (En) and sloppy paired (Slp) since, in Drosophila, they form a complex with Hox, Exd and Hth to repress transcription Engrailed1 and Engrailed2, are expressed in LPM at pre-limb bud stages As it is unlikely that Hox protein itself directly represses essor(s) . One canTbx5 expression or that they share similar 3D structure domains in spite of their distinct amino acid sequences.We have shown that Hoxc8 and Hoxc10 have transcriptional repression ability similar to Hoxc9 . To gainTbx5 forelimb regulatory element reveals a direct link between patterning of the rostro-caudal axis of the embryo by Hox genes and the programme that controls positioning of the forelimb forming territory. A clear correlation between Hox expression and establishment of the forelimb territory of the LPM has previously been suggested Tbx5Hoxc9 is reduced Tbx5 (and the subsequent forelimb programme) being essential for emergence of an ectopic wing bud from this region. In the limbless python, Hoxc8 expression is rostrally expanded to the anterior limit of the trunk Tbx5-negative caudal LPM at pre-limb bud stages in chick and mouse , however, we did not detect expression of Hoxc9 in the forelimb-forming region, in contrast to the strong staining in caudal tissues. We therefore conclude that Hoxc9 is not present in the forelimb-forming region at stages when Tbx5 expression is first initiated. Later expression of Hoxc9 is not sufficient to cause detectable repression of the domain of Tbx5 already activated by Hox4/5 paralogous genes.A previous study has demonstrated the presence of and a function for Tbx5 expression, our study does not exclude the possibility that other caudally-expressed Hox genes have a similar repressive ability. We favour a model in which other caudally-expressed Hox paralogs have redundant functions in repression of Tbx5. Hoxc cluster null mice have no defects in the limb skeleton Tbx5 in these mutants have not been reported and we predict that the ectopic expansion of Tbx5 in caudal LPM would not cause any skeletal defects. Further analysis will be required to uncover the requirement of caudally\u2013restricted Hox paralogs, such as Hox8, Hox9 and Hox10 for Tbx5 repression in caudal LPM.While we have shown that Hoxc8, Hoxc9 and Hoxc10 can repress Tbx5 in the LPM, a similar Hox protein code is present in axial tissues  that do not express Tbx5. The activity of the forelimb regulatory element of Tbx5 is restricted to LPM and this LPM restriction is maintained following mutation of Hbs2 that leads to caudal expansion in expression. One possible explanation for LPM restriction is the presence of unknown repressors in axial tissues or alternatively additional factors, which are active exclusively in LPM, are required for Tbx5 expression. Odd-skipped related (Osr) genes are candidates as they are expressed in LPM, but excluded from axial tissues such as neural tube and somites Tbx5 forelimb regulatory element to test if reporter activity was lost. The activity of the element was unaffected, however, suggesting Osr genes are not required for Tbx5 LPM expression  complex that ultimately determines the caudal boundary of Tbx5 expression. Thus, the regulation of Tbx5 expression in the LPM represents an excellent system to understand the interactions between neighbouring Hox binding sites and how the consequent output is integrated.Our analysis of the http://www.genomatix.de). Transgenic embryos were generated by the Procedural Service section, NIMR by standard pronuclear microinjection techniques. Mouse embryos were staged according to LacZ transgene were identified by PCR using specific primers . Sequences surrounding putative Hox binding sites and the mutations induced are as followings, binding sites are shown in bold; Hbs1, ATTATTGGAAC; mut Hbs1, ATGCTTGGAAC; Hbs2, ATTATCGACTCTCA; mut Hbs2, ACGATCGACTCTCA; mut 6bp3\u2032, ATTATCGACTGCAA; mut Hbs2+6bp3\u2032, ACGATCGACGCTTA; Hbs3, TAATTCAGA; mut Hbs3, TCGTTCAGA; Hbs4, TTATTAAGGCC; mut Hbs4, TTGGCAAGGCC; Hbs5, TTATCTTGCCAT; mut Hbs5, TCGTCTTGCCAT; Hbs6, TTATTTTG; mut Hbs6, TCGTTTTG.For reporter analysis in chick and mouse, we used the BGZA reporter vector in situ hybridizations were carried out essentially as previously described Pitx1, Tbx5 and mouse Hox genes have been described previously Whole mount Fertilized chick embryos  were incubated at 38\u00b0C and staged according to Hamburger Hamilton (HH) In vitro translated proteins were produced using a TnT Coupled Reticulocyte Lysate System (Promega). Proteins were labelled with 35S-Methionine (PerkinElmer) to verify and quantify translation. LPM strips adjacent to somites 5\u201310  and lateral to somite 14 to its caudal extreme  were dissected from E9 mouse embryos. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce) following manufacturers instructions.32P by incubating with T4 polynucleotide kinase (NEB) for 30 minutes. 2 \u00b5l of in vitro translated protein or nuclear extract were blocked with 200 ng poly-dIdC, 2 \u00b5g of poly-dGdC or 2 \u00b5g of poly-dAdT in binding buffer  in a total volume of 22 \u00b5l for 15 minutes on ice. For super-shift, 2 \u00b5l of the antibody recognising flag epitope  was added to the binding reaction and incubated for a further 15 minutes. Then, 1 \u00b5l of 32P -labelled double-stranded oligonucleotides were mixed and incubated for 30 minutes. The protein\u2236DNA hybrids were resolved on 6% PAGE in 0.5xTBE.Double-strand oligonucleotides were labelled with Figure S1Tbx5 forelimb regulatory element. A\u2013C. The Tbx5 int2(361) LacZ reporter construct was electroporated with pCA\u03b2-dsRed-Express in the presumptive forelimb region of HH14 chick embryos. 24 hours later, the efficiency of electroporation was assessed by dsRed expression (A\u2013C) and embryos were stained for LacZ to analyze the enhancer activity (A\u2032\u2013C\u2032). Co-electroporation of pcDNA-mHoxc8 (B and B\u2032) and pcDNA-mHoxc10 (C and C\u2032) reduced LacZ expression.Hoxc8 and Hoxc10 can repress activity of the (TIF)Click here for additional data file.Figure S2LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative smad binding site and Hox binding sites are shown as green and blue boxes, respectively. B\u2013C, E9.5 embryos were stained for \u03b2-galactosidase for a mutated construct of Tbx5 int2(565) reporter.Disruption of a putative smad binding site does not expand (TIF)Click here for additional data file.Figure S3LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative Osr binding site (purple box) locates proximal to Hbs5 (blue box). B\u2013C, E9.5 embryos were stained for \u03b2-galactosidase for mutated constructs of Tbx5 int2(565) reporter.Disruption of a putative Osr binding site does not affect (TIF)Click here for additional data file."}
+{"text": "Activation of neutrophils sequestered in the alveolar milieu can cause the release of reactive oxygen species (ROS), increasingly regarded as key substances modulating epithelium dysfunction and disruption. These oxidants are generated by the neutrophil respiratory burst oxidase system that reduces molecular oxygen (O2) to superoxide (O2-). Alpha-1 antitrypsin (AAT) deficiency (AATD) provides us with the most definitive evidence for the physiological and clinical importance of AAT and in this study we examined the immunomodulatory activity of AAT and investigated whether neutrophil ROS production was regulated by AAT.Neutrophil O2- production in response to fMLP (10-6M) and IL-8 (10ng) was measured by a cytochrome C reduction assays respectively. Analysis of ERK phosphorylation in response to fMLP \u00b1 AAT was carried via Western blot analysis of neutrophil whole cell lysates. To verify fMLPs interaction with its cognate receptors fMLP R1 (FPR1) and fMLP R2 (FPR2), flow cytometry was used to measure levels of FITC labeled fMLP binding to the membranes of neutrophil.In this study we demonstrate using in vitro models that AAT modulates neutrophil O2- production elicited by fMLP and IL-8 in a dose dependant manner (P<0.05). Mechanisms of inhibition were investigated and in vitro studies revealed that AAT functions to inhibit fMLP signaling through inhibiting its interaction with its receptors FPR1 and FPR2 on the neutrophil plasma membrane.The potential of AAT as a regulator of neutrophil ROS production adds a new understanding to the role of AAT in health and disease."}
+{"text": "These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (\u2212/\u2212Trpm1) or a missense mutation in the pore domain of TRPM1 (tvrm27/tvrm27Trpm1). Hearing thresholds were evaluated in adult (4\u20135 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in \u2212/\u2212Trpm1 or tvrm27/tvrm27Trpm1 mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca The mechanotransduction function of hair cells occurs within the set of stereocilia on each hair cell. The hair cell stereocilia are coupled to one another by intercilliary links including tip links, which are protein structures that connect the tip of each stereocilium to the side of its tallest neighboring stereocilium TRP channels are tetramers comprised of six transmembrane polypeptide subunits that form permeable ion channels. TRP channels play prominent roles in calcium signaling and are ubiquitously expressed and participate in the perception of a wide variety of sensory stimuli including taste , temperature and light Another potential candidate from the TRP family is the transient receptor potential channel melastatin 1 (TRPM1). The TRPM family contains eight channels expressed in the stria vascularis, organ of Corti, outer and spiral ganglion cells TRPM1 causes the complete form of congenital stationary night blindness (cCSNB), an autosomal recessive disease that is characterized by depolarizing bipolar cell (DBC) dysfunction Trpm1 (\u2212/\u2212Trpm1) lacked the b-wave of the electroretinogram (ERG) tvrm27Trpm1, was recently identified in a chemical mutagenesis screen based on a similar ERG b-wave reduction tvrm27/tvrm27Trpm1 mutants showed an eye phenotype consistent with that reported for \u2212/\u2212Trpm1 mice with one important distinction: tvrm27/tvrm27Trpm1 mice retain expression of the TRPM1 protein on the dendritic tips of DBCs Trpm1 mouse mutants.In humans, a mutation in tm1Lex/tm1LexTrpm1, hereafter referred to as Trpm1\u2212/\u2212 mice, were generated by Lexicon Genetics and acquired from the European Mouse Mutant Archive (www.emmanet.org). tvrm27/tvrm27Trpm1 mice were derived from a mutagenesis program tvrm27Trpm1 changes a highly conserved alanine at position 1068 to threonine (p.A1068T) Trpm1\u2212/\u2212 and tvrm27/tvrm27Trpm1 mice were tested. The genotype of each mouse used in this study was confirmed by PCR (for the knockout and sibling controls) or DNA sequence analysis (for the point mutation tvrm27).All mice were cared for and treated in accordance to the Institutional Animal Care and Use Committee of Case Western Reserve University (CWRU). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and animal welfare guidelines at CWRU, USA. The protocol was approved by the Institutional Animal Care and Use Committee at CWRU (Protocol Number: 2012-0068).To evaluate hearing, auditory-evoked brain stem response (ABRs) was recorded following presentation of pure tone stimulus to the mouse ear at frequencies of 8, 16 and 32 kHz in accordance with previous descriptions Trpm1\u2212/\u2212 or tvrm27/tvrm27Trpm1 mice to controls (Trpm1+/\u2212 or Trpm1+/+ mice).The data were collected in Excel\u00ae ; statistical analysis was performed using GraphPad Prism\u00ae . A Mann-Whitney test was used to compare thresholds from Trpm1\u2212/\u2212 allele: Details of the targeted deletion of Trpm1 was reported previously GGCATGTGTAGCTACCACAG and KA1216: GCATAGTCCATGGACCTAGC or KA1215 and KA1217: GCAGCGCATCGCCTTCTATC), platinum Taq polymerase and PCR buffer as suggested by the manufacturer . The reaction mix was cycled 35 times at 94\u00b0C, 20 seconds, 55\u00b0C, 20 seconds and 72\u00b0C, 60 seconds. The PCR products were resolved on 2% agarose gel. An 840 base pair fragment for the wild-type and 280 base pair fragment for the knockout allele are expected.tvrm27Trpm1 allele: The genotype of the tvrm27Trpm1mice was confirmed by amplification of the target sequence by PCR (primers used KA1220: ATTCAGGGAGTGCTTGGTTG and KA1221: GGTATCTGCCACCCTCTCAG) followed by DNA sequence analysis. Genomic DNA (\u223c500 ng/reaction) obtained from tail biopsies were amplified using Platinum Taq DNA polymerase and reaction buffer supplied by the manufacturer . PCR was carried out for 35 cycles . The 639 base pair PCR product was resolved by agarose gel electrophoresis (2% agarose gel). Primer KA1220: ATTCAGGGAGTGCTTGGTTG was used to sequence the PCR product.Trpm1 mRNA is expressed in the cochlea of \u2212/\u2212Trpm1 mice, RT-PCR analysis was carried out as described previously Trpm1 message was detected by amplification of the target cDNA by PCR (primers used KA1223: ACCTCATGGTGAAGGACTGG and KA1224: TTCCTTTGAGCAAGGCAGTT). PCR amplification of \u03b2-actin cDNA  from each RT reaction served as a control for RNA  and RT-PCR protocol. RNA isolated from C57BL/6J (B6) mice cochlea served as a reference for the expected PCR product size for Trpm1 and \u03b2-actin from a wild-type tissue. PCR was carried out for 35 cycles . The PCR products (847 base pairs for Trpm1 and 932 base pairs for beta-actin) were resolved using agarose gel (2%) electrophoresis.To determine if TRPM1 and the fact that TRPM1 is expressed in hair cells Trpm1.Trpm1\u2212/\u2212 or tvrm27/tvrm27Trpm1 mice and age-matched controls. The ABR waveforms recorded from the Trpm1 mutants were comparable to those obtained from control mice. Trpm1 mutants, and ABR thresholds did not differ from control at any frequency examined .Physiological findings associated with mutations in examined . The gen\u2212 allele  or DNA smutation . We conf/\u2212 mouse . The dat\u2212/\u2212Trpm1 mice indicates that TRPM1 is not essential for the development of inner ear (vestibular and cochlear) structure or function. More specifically, adult mice display normal inner ear function indicating that hair cell MET function in the mouse inner ear is not dependent on TRPM1. Mice carrying a missense mutation in the pore domain of TRPM1, where the highly conserved alanine at position 1068 is converted to threonine (p.A1068T), also displayed no inner ear phenotype: The tvrm27/tvrm27Trpm1 mice (n\u200a=\u200a4) showed hearing thresholds in the normal range  consistent with that reported for \u2212/\u2212Trpm1 mice with an important difference: tvrm27/tvrm27Trpm1 mice retain expression of the TRPM1 protein on the dendritic tips of DBCs tvrm27/tvrm27Trpm1 mice to retain expression of the mutant TRPM1 protein in hair cells. Since comparable ABR thresholds were obtained in the \u2212/\u2212Trpm1 mutant and the loss of function mutant tvrm27/tvrm27Trpm1, we believe the knockout mutation in Trpm1 is less likely to trigger a compensatory response. Further, if TRPM1 mediated its function as a heteromer with a related channel protein, then we would expect to observe loss of hair cell function to some degree in the tvrm27/tvrm27Trpm1 mutants due to a dominant negative effect. Our data suggests that TRPM1 is less likely to function as heteromers in the ear.The second possibility for the lack of an auditory phenotype in TRPM1Trpm1 mouse mutants with different effects on gene expression and trafficking provides important tools with which to understand TRPM1 function in the body.The data presented in this report demonstrates that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is the mechanotransduction channel in mouse auditory hair cells. This conclusion is consistent with the lack of auditory involvement in patients with cCSNB due to mutations in TRPM1 is not essential for the development of hearing or balance in the mouse model and it is unlikely that TRPM1 is the hair cell mechanotransduction channel."}
+{"text": "The physical state of the patient before surgery is defined by the American Society Anesthesiology (ASA) physical status classification system. The Simplified Acute Physiology Score (SAPS II) provides an estimate for the risk of intrahospital death for ICU patients. The Sequential Organ Failure Assessment (SOFA) score is used to monitor the patient's condition during his/her stay in the ICU, assessing the extent of organ dysfunction or failure. Is the ASA physical status classification system a good prognostic index for determining postsurgical patient's admittance to the ICU? What is the evolution of these patients? Could we predict the outcome of these patients?A retrospective analysis of the ASA, SAPS II and SOFA of all postsurgical patients admitted to an ICU, between 1 May and 31 October 2010.Total ICU admissions: 323 patients, 118 being postsurgical patients . Maximum patient SOFA: between 0 and 19. Patient SAPS II: between 8 and 99. Of the 118 patients, five had ASA 5, a mortality of 100% being expected but only three died. The expected mortality rate of the three deceased  was 5.2%, 92.5%, 98.4%, respectively. The two patients who got better had a SAPS II of 21 and 56 with a maximum SOFA of 4 and 16, which means that they improved significantly, against all odds. Most ICU admitted patients were ASA 3 and ASA 4. Fifty per cent of ASA 3 patients presented a maximum SOFA between 0 and 5; maximum SOFA was higher in 34% of ASA 3 patients (5 to 10) with predicted ICU mortalities of up to 7% and 46%, respectively. Four patients of the ASA 3 group died. Of the ASA 4 patients, 43% had a maximum SOFA between 5 and 10, and 34% presented a lower maximum SOFA (0 to 5). In 10 (26%) ASA 4 patients, maximum SOFA exceeded 11 with a mortality ICU predicted rate of 56%. In fact, five died. The reason for admission to the ICU of the 20 patients with lower ASA  was a need for tighter monitoring or stabilization of postsurgical complications. Indeed, all deaths in the ASA 2 (1/17) and ASA 3 (4/38) groups were related to complications from co-morbidities.ASA 3 and ASA 4 patients are those who benefit the most from a stay in an ICU, enabling one to reduce mortality predicted by SAPS II and SOFA scores. The ASA physical status classification system is not a good indicator of mortality, but its association with SAPS II and maximum SOFA scores define more effectively the severity and prognosis of the postsurgical patient."}
+{"text": "Vitamin B12 is an essential micronutrient required for optimal hemopoetic, neuro-cognitive and cardiovascular function. Biochemical and clinical vitamin B12 deficiency has been demonstrated to be highly prevalent among patients with type 1 and type 2 diabetes mellitus. It presents with diverse clinical manifestations ranging from impaired memory, dementia, delirium, peripheral neuropathy, sub acute combined degeneration of the spinal cord, megaloblastic anemia and pancytopenia. This review article offers a current perspective on the physiological roles of vitamin B12, proposed pathophysiological mechanisms of vitamin B12 deficiency, screening for vitamin B12 deficiency and vitamin B12 supplementation among patients with diabetes mellitus. Vitamin B12 or cobalamin is a water soluble vitamin that plays a very fundamental role in DNA synthesis, optimal haemopoesis and neurological function. The clinical picture of vitamin B12 deficiency hence, is predominantly of features of haematological and neuro-cognitive dysfunction > 100 fl) with/without anaemia, ovalocytes, hyper segmented white blood cells (i.e. >5% of neutrophils with \u22655 lobes) and pancytopenia . Due to Several cross sectional studies -12 and c<150 pmol/l or total serum vitamin B12of 150\u2013250 pmol/l and holotranscobalamin \u226437 pmol/l and homocysteine \u226515 \u03bcmol/l.Due to the diverse definitions of vitamin B12 deficiency used in most studies and the cultural and religious beliefs in different regions of the world, comparison of the prevalence of vitamin B12 deficiency among T2DM patients and healthy general populations is difficult. In one population based study among 1048 elderly Finnish subjects aged 65\u2013100 years, the total prevalence of definite vitamin B12 deficiency was 12.1% . PreviouIn India, a country with a large proportion of vegetarians due to cultural and religious beliefs, very high prevalence of vitamin B12 deficiency among the general population has been reported. In one study by Yajnik et al. to determine the frequency of vitamin B12 deficiency and hyperhomocysteinemia among 441 healthy middle aged Indian men, vitamin B12 deficiency as defined by vitamin B12 concentrations <150 pmol/L was reported among 67% of the study participants . VegetarIn another cross sectional study among 175 healthy elderly Indian subjects aged >60 years, vitamin B12 deficiency also defined as vitamin B12 concentrations <150 pmol/L was reported among 16% of the study participants . ElevateIn the absence of contraindications like renal and hepatic dysfunction, recent guidelines advocate for the use of metformin as the first line glucose lowering agent concurrently with life style modification approaches ,25. DespThe risk of developing metformin associated vitamin B12 deficiency is greatly influenced by increasing age, metformin dose and duration of use ,18. In ath month [Decrease in vitamin B12 absorption and levels following metformin use typically starts as early as the 4th month . Clinicath month .The proposed mechanisms to explain metformin induced vitamin B12 deficiency among patients with T2DM include: alterations in small bowel motility which stimulates bacterial overgrowth and consequential vitamin B12 deficiency, competitive inhibition or inactivation of vitamin B12 absorption, alterations in intrinsic factor (IF) levels and interaction with the cubulin endocytic receptor . MetformType 1 DM (T1DM) is an auto immune condition that results from auto immune destruction of insulin secreting beta cells of the pancreas. It is invariably associated with other organ and non organ specific auto immune and endocrine conditions leading to development of autoimmune polyglandular syndromes .Pernicious anemia resulting from chronic autoimmune gastritis is highly frequent among patients with T1DM. Chronic autoimmune gastritis and pernicious anemia occurs in about 2% and up to 1% of the general population respectively. Among patients with T1DM, the prevalence is increased by 3 to 5 folds .Vitamin B12 deficiency due to pernicious anemia occurs frequently among patients with T1DM. In one cross sectional study done in South India among 90 patients with T1DM, low vitamin B12 levels were noted among 45.5% of the study subjects as defined by the manufactures\u2019 cut off point of <180 pg/ml and among 54% using the published cut off point of <200 pg/ml . No posiPatients with T1DM actively exhibit auto antibodies to intrinsic factor (AIF) type 1 and 2  and pariThe PCA inhibit secretion of intrinsic factor resulting into pernicious anemia, a condition which is 10 times more prevalent among type 1 diabetic patients than non diabetic patients. Type 1 AIF result into vitamin B12 deficiency by blocking the binding of vitamin B12 to IF. This prevents its transportation to its absorption site, the terminal ileum. These auto antibodies are found in 70% of patients with pernicious anemia .Primary autoimmune hypothyroidism and celiac disease are frequent co morbidities among patients with T1DM -38 and dVitamin B12 deficiency among patients with autoimmune hypothyroidism could be explained by the presence of antibodies to the gastric parietal cells and intrinsic factor, reduced oral intake, dyserythropoesis due to thyroid hormone deficiency and defective absorption due to reduced bowel motility, bowel wall oedema and bacterial overgrowth .Celiac disease which is a highly prevalent autoimmune mediated gastrointestinal condition occurs in 1-16% of T1DM patients compared to 0.3-1% in the general population . IngestiCurrently, there are no published guidelines advocating for routine screening for vitamin B12 deficiency among patients with T2DM. However among type 2 diabetic patients, it is clinically plausible to screen for vitamin B12 deficiency prior to initiation of metformin and later annually among elderly patients with history of long term use of metformin (\u22653-4 years), use of high doses of metformin (\u22652 g/day), clinically worsening diabetic distal polyneuropathy in the presence or absence of the discussed haematological abnormalities .The screening approach for vitamin B12 deficiency among diabetic patients and the general population is similar. Measurement of the serum vitamin B12 concentrations should be the preliminary screening step for vitamin B12 deficiency among patients with T2DM. Concentrations <200 pg/ml are usually diagnostic of vitamin B12 deficiency while concentrations >400 pg/ml confirm absence of vitamin B12 deficiency .Measurement of serum MMA or homocysteine concentrations is a more sensitive and specific approach for screening especially among type 2 diabetic patients with borderline serum vitamin B12 concentrations of 200-400 pg/ml and subtle haematological manifestations. Serum homocysteine and MMA concentrations of 5-15 \u03bcmol/l and <0.28 \u03bcmol/l are considered within the normal range respectively ,44.Among patients with T1DM, there are no clear guidelines regarding screening for vitamin B12 deficiency. However, due to the high prevalence of pernicious anaemia and subsequent vitamin B12 deficiency among T1DM patients reported in most cross sectional studies, it would be pragmatic to screen at diagnosis and then later yearly for 3 years, then five yearly thereafter or in presence of any clinical indication since vitamin B12 deficiency can develop at anytime . ScreeniTreatment of vitamin B12 deficiency does not differ regardless of the aetiology. All patients deficient of vitamin B12 should receive replacement therapy with either oral or parenteral vitamin B12 ,47. BothAmong young patients with T1DM and co-existing vitamin B12 deficiency, replacement therapy with daily intra muscular or oral vitamin B12 in the dose of 100\u03bcg for a week and then monthly is satisfactory. In severe cases, parenteral or oral administration of 1000 \u03bcg/day of vitamin B12 for a week, followed by the similar dose given every week for 1 month and then later monthly is advised .Concomitant folate deficiency should be treated with oral folate replacement in doses of 5 mg daily for 1\u20134 months. Folate administration prior to correcting vitamin B12 deficiency should be avoided because it results into progression and / worsening of the associated neurological manifestations .Vitamin B12 deficiency and the accompanying hyperhomocysteinemia and elevated MMA levels have been documented to cause a distinct sensory polyneuropathy that closely mimics diabetic neuropathy. Worsening of diabetic neuropathy is also noted among patients with co-existing vitamin B12 deficiency .Vitamin B12 replacement has been shown to cause symptomatic improvement among patients with severe diabetic neuropathy. One meta-analysis showed that if used either alone or in combination with vitamin B complex, there was a significant improvement in the somatic symptoms like pain and paraesthesias. Three included studies also noted an improvement in autonomic symptoms with use of vitamin B12 alone .Similar superior positive findings of reduction in pain and paraesthesias were also noted with use of vitamin B12 as compared to nortriptyline in a randomized, single-blind clinical trial done in Iran among 100 patients with diabetic neuropathy. This study was approved by the Ethics review board of the Isfahan University of Medical Sciences, Iran .19. The doses of vitamin B12 in the multivitamin formulations used by the study subjects in this survey were probably inadequate to correct vitamin B12 deficiency.There are no guidelines to address how often patients with T1DM and T2DM should be supplemented with vitamin B12. The optimal supplementation dose of vitamin B12 is also unknown. A recently published follow-up study from the United States of America showed that administration of oral vitamin B12 among type 2 DM patients on long term use of metformin was ineffective in correcting biochemical vitamin B12 deficiencyThis stresses the need of further studies to determine the optimal vitamin B12 supplementation dose and frequency of supplementation among patients with DM. To avert vitamin B12 deficiency especially among adult type 2 diabetic patients on long term use of metformin, it is plausible to adopt a simple and cost effective supplementation approach in diabetes care. A 1000 \u03bcg dose of vitamin B12 given annually would be sufficient to replenish the body\u2019s vitamin B12 stores among this category of patients .Clinical and biochemical vitamin B12 deficiency is highly prevalent among patients with both types 1 and 2 DM. Future large and well designed studies on screening for vitamin B12 deficiency, vitamin B12 supplementation and optimal supplementation dose among type 1 and type 2 diabetic patients are warranted to help guide formulation of guidelines in diabetes clinical care. Annual screening for vitamin B12 deficiency using more sensitive methods like serum homocysteine and methylmalonic acid concentrations  and supplementation should be adopted among diabetic patients with specific risk factors of vitamin B12 deficiency.The authors declare no competing interests.Both authors equally contributed to the development of the concept and manuscript, critically read and approved the final manuscript."}
+{"text": "Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (\u223c30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA. Embryonic development in mammals begins from the female and male interaction which leads to the oocyte fertilization. After 5 to 6 cell divisions inside the zona pellucida, the blastocyst undergoes its development conducing to the implantation in the uterine tissue. The external cells of the blastocyst develop into the placenta, a pivotal organ which allows immune tolerance, bidirectional foeto-maternal exchanges and crucial synthesis of gestational hormones Amnionless gene (AMN) in patients affected by RSA but no causal mutations could be identified et al. described a statistical association between the p.Val617Phe mutation of the Janus kinase 2 protein and RSA At present, although hundreds of mutant mouse models with reproductive phenotypes have been generated Mus spretus SEG/Pas genome fixed at homozygous state on Mus Musculus C57Bl6/J (B6) genomic background. Using IRCS animals we have previously shown that 3 QTL of embryonic lethality mapped on a unique spretus fragment in 3 strains, 66H-MMU13, 66H-MMU1 and 135E. The first, Led1 in 66H-MMU13 strain on the MMU13 (\u223c2.6 Mb) comprised between the rs120693734 and D13Mit47 polymorphic genetic markers. The second, Led2 in 66H-MMU1 was analyzed in the present study and the third, Led3 located on MMU19 in 135E strain encompassing a unique Spretus fragment of 8 Mb located between D19Mit49 and D19Mit137 markers. The 66H-MMU1 strain, which encompasses a unique spretus chromosomal fragment located on MMU1 is affected by high levels of embryonic death (24.6%). This strain encompasses a QTL of embryonic lethality (named Led2) spreading on 32 Mb and containing 215 genes (143 annotated and 72 predicted) In recent years, in order to overcome these constraints we created an original mouse model of interspecific recombinant congenic strains (IRCS) which permit to localize chromosomal regions associated with complex phenotypes (Quantitative Trait Loci or QTL) Led2. For this purpose, we created 15 substrains from 66H-MMU1 animals, which encompass distinct overlapping spretus fragments. Using in vivo high frequency ultrasonography to follow the embryonic development, we used an approach of type \u201cphenotype/genotype association\" to refine this QTL of embryonic death. We identified, into the Led2 QTL, one region of approximately 6 Mb, Led2minA, which has a main effect on the rate of embryonic death. In addition, we pointed out a second region, Led2minB, which could also have a small effect on the phenotype, although statistically not demonstrated.Here, we present a thorough genetic dissection of Procedures for handling and experimentation were conducted in accordance with the policies of the Paris Descartes University, the Cochin Institute and the Guidelines for Biomedical Research Involving Animals. The experiments were approved by the departmental veterinary services of Paris .Mus musculus (C57BL6/J) and Mus spretus SEG/Pas . The design of these crosses was reported in a previous work The 66H-MMU1 strain was created at the Pasteur Institute (Paris) by successive crosses of the two parental species spretus segment present in the 66H-MMU1 genome. Primer microsatellites were retrieved from the Mouse Genome Informatic website (MGI) website of the Jackson Laboratory (www.informatics.jax.org). PCRs were performed using Taq DNA Polymerase (New England Biolabs). PCR products were loaded in a 2% Nusieve, 2% agarose gel .DNA was extracted from mouse tail fragments by a standard procedure. Eight new microsatellites located on MMU1  were genotyped in order to precise the boundaries of the The gestation was obtained by crossing each IRCS female with a C57/BL6 male. Each female was used one time to collect phenotypic data from the primo-gestation. For each group one female per gestation and the number of animals studied is always >4. C57/BL6 females from the control group were crossed with C57/BL6 males. Substrain\u2019s phenotyping was carried out at the small animal imaging facility of the Cochin Institute using high frequency ultrasonography . Eight to 12 weeks IRCS females were mated with C57BL/6J males, during a period of 2.5 days. Then, female mice were anesthetized with 1.5% of isoflurane in order to achieve ultrasound examination . Briefly, a chemical hair remover was used to eliminate abdominal hair. Ultrasonographic contact gel was used to ensure contact between the skin surface and the transducer. Body temperature, electrocardiographic and respiratory profiles were monitored using ultrasound device\u2019s integrated heating pad and monitoring device . Examinations were performed using 2 different high frequency probes depending on the size of the embryos: a 60 MHz transducer for early stages of development (RMV708) and a 40 MHz (RMV704) transducer for late developmental stages.in vivo, three ultrasonographic examinations were performed at three time points (between E7 and E14). During each examination, we assessed the number of implanted embryos in each uterine horn as well as their status  was assessed. For each gestation, the embryonic lethality rate was calculated as the number of resorbed embryos in both horns reported to the total number of implanted embryos.In order to follow the gestation Females from IRCS and B6 strains were crossed with B6 males. Each pregnant female was subjected to ultrasonographic examinations in order to precisely determine the embryonic developmental stage. Female mice were euthanized by cervical dislocation and tissues were taken at E12.5. Total RNA of uterine tissue from six mice of the IRC substrain of interest was extracted using TRIzol Reagent  in accordance with the manufacturer's instructions. Similarly, six B6 were used to extract total RNA. In order to duplicate microarray hybridizations of samples from uterine tissues, two pools of three RNA extractions were created for both IRCS and B6 animals. One microgram of RNA from IRC substrains of interest and the B6 controls was used for hybridization on a NimbleGen expression array. cDNA synthesis, DNA end-labeling, hybridization, scanning, and data normalization were performed at the genomic/transcriptomic platform of the Cochin institute. The average fluorescence values for each transcript were collected chromosome per chromosome for each analyzed strain. To evaluate gene expression modifications in IRC strains, these fluorescence levels  were divided gene per gene by the corresponding ones from B6 which were taken as a reference. The results of the gene expression were deposited at GEO (NCBI) with the accession reference GSE32460.After RNA preparation, the total RNA was treated with DNase I (Invitrogen Life Technologies) for 10 min at room temperature followed by inactivation with EDTA (Sigma-Aldrich). Total uterus RNA was reverse transcribed to obtain cDNA using M-MLV Reverse Transcriptase  following manufacturer\u2019s protocols.Cyclophiline A expression. Primer sequences are listed in Quantitative PCR was carried out using fast SYBR Green Master Mix (Applied Biosystems) and a real time PCR system  according to standard PCR conditions. To validate the primers used in qRT-PCR, four pairs of primers were tested for each gene and four housekeeping genes were also tested to choose the reference gene  samples was evaluated by t-test using the Bonferroni-corrected levels. As we used 7 substrains, a p value <0.007 (0.05/7) was considered as significant. Statistically significant results are labeled as follows in all figures: *: p<0.05; **: p<0.01; ***: p<0.001.Led2 QTL spretus fragment carried by the 66H-MMU1 substrain. However, a uncertainty of \u223c6.4 Mb existed at the proximal boundary of this QTL since the interval comprised between D1Mit134 (80.6 Mb) and D1Mit50 (87.0 Mb) markers corresponds to this distance and the breakpoint is located somewhere between these two markers. Indeed, D1Mit134 and D1Mit50 allele markers are of B6 and spretus natures respectively (http://www.pasteur.fr/recherche/unites/Gfons/ircs/ircshome.htm). In an attempt to precise the position of the breakpoint, we genotyped 8 novel markers located on this region that permitted to reduce the recombination region to a 2.5 Mb interval comprised between 84.5 Mb and 87 Mb  caused by the 1 strain . Among 1in vivo ultrasonography as previously described spretus origin (B6/SEG), while the uterus was homozygous SEG/SEG for the same spretus fragment. The control group was obtained by crossing males and females of the B6 strain. A total of 97 gestations (31 and 66 of B6 and IRCS types respectively) were analyzed. For each gestation, we counted the number of implanted and resorbed embryos during three ultrasonographic examinations. There was no correlation between embryonic death and the position inside the womb, which suggests that the death of one embryo did not have deleterious repercussions on the contiguously implanted structures  shared a large spretus region (>84.5 Mb to 90.5 Mb) with the R6 strain (which does not display the embryonic resorption phenotype) and the rest (until <100.3 Mb) is also shared with the other strains  which are not affected. This configuration suggests that two spretus regions, shared by R3 and R5 strains and not present together in the other strains, seem to be necessary to explain the apparition of the phenotype in R3 and R5. We defined a first spretus subfragment called Led2minA which encompasses D1Mit50 to D1Mit305 region (>84.5 Mb to <90.5 Mb) and a second region called Led2minB located at the rs3692309 marker (>92.5 Mb to <100.3 Mb) (see gray boxes in spretus regions (Led2min) are separated as in R6 (that contains Led2minA only) or in R4, R10, R13 or R14 (Led2minB only) the phenotype of embryonic death is absent. The presence of the two spretus regions seems indispensable to permit the manifestation of the phenotype, it\u2019s the case for R3 and R5  bearing these two spretus regions. The results of statistical t-tests are shown in Led2minA QTL. By contrast, the comparison between R6 and R3 (or R5), did not statistically indicate the presence of Led2minB QTL. However the embryonic death rates of R3 and R5 both have a tendency to be higher than that of R6  and R3 (or R5) was 17%\u201319% which is comparable to 15%\u201317% difference between B6 (with no spretus regions) and R3 (or R5) also suggesting a nil or very small effect of Led2minB. In consequence this result did not support the presence of an epistatic interaction between Led2minA and Led2minB regions, but a Led2minB additive effect could be revealed by increasing sample size of this \u201cQTL\" representative strains. For this raison, the genes present in these two regions, Led2minA and Led2minB are listed in In order to refine at of R6  suggestispretus fragment). Conversely, we performed reverse crosses (\u2640B6 \u00d7 \u2642IRCS), giving a heterozygous placenta for the genes of the fragment, but a B6 homozygous uterus. In this situation, the would-be disorders ought to find their origin exclusively from a placental-fetal/embryonic defect, caused by the spretus state of the MMU1 fragment, but not from a B6 womb defect. In this optic we realized the cross \u2640B6 with \u2642R3 (IRCS group2). We observed that the mean of embryonic resorption rate (\u00b1SEM) was 0.07\u00b10.04 and not significantly different from the control  whereas the inverse crossing, leading also to a heterozygous foeto-placental complex implanted in homozygous spretus uterus (R3), produced a significantly higher embryonic resorption rate   and homozygous uterine alleles (SEG/SEG) within the same genomic region (=\u200a0.001) . From thspretus fragment or the two Led2min regions were considered  . These results were highly reproducible since they showed strong correlations between experimental duplicates (r\u200a=\u200a0.967 for B6 and 0.983 for R3). Thus, for subsequent analysis, we took the average of both values for each transcript. We first focused on transcripts with fluorescence levels higher than 100 AUF, we assumed that values under this threshold were very close to background signals and, with this threshold, we selected 18,085 transcripts . We considered a gene as differentially expressed if a two-fold difference of expression (up or down) was observed. Consequently, 3,436 (19% of the expressed uterine genes) transcripts were modified in R3 uterus when compared to those expressed in B6 . A similnsidered .Led2minA QTL region by quantitative RT-PCR. As shown in In order to validate the differential gene expression obtained by the microarray analysis, we checked 7 genes of the Mus musculus and Mus spretus species, we listed the genes of the Led2min QTL corresponding to these criteria and thus potentially involved in embryonic resorption. Finally we considered the transcripts with an expression level >500 AUF and either exhibiting a deregulation  and the presence of non synonymous polymorphisms between Mus musculus and Mus spretus (provided by SANGER database: http://www.sanger.ac.uk/) (Considering that the uterine dysfunction can take its origin from a deregulation of the gene expression or/and non-synonymous coding polymorphisms accumulated during independent evolutive processes of .ac.uk/) .Then, we searched whether deregulated transcripts could be grouped into functional clusters using DAVID database In the reproductive processes, as in others, hundreds of genes interact into subtle regulatory networks, and this complexity does not permit to easily identify the molecular factors of dysfunctions leading to infertility cases. Moreover, when our interest is turned towards the human clinic, the study of factors involved in reproductive defects is particularly challenging due to obvious ethical constraints, which rends obligatory the use of animal models. However although hundreds of mutant mouse models with infertility/hypofertility phenotypes have been generated Mus spretus genome fixed on a Mus musculus B6 genetic background spretus segments. Mus musculus and Mus spretus diverged \u223c2 million years ago meaning that the association of their two genomes has the potential to lead to genetic incompatibilities spretus origin located on MMU1. This QTL was called Led2 and has been mapped to an interval of 32 Mb which contains 215 genes In the present work we used a mouse model including interspecific recombinant congenic strains (IRCS). The originality of this whole model is based on the presence of a small homozygous fragments of Led2 QTL. Each recombinant substrain females were crossed with B6 males, resulting in a fetus/placenta complex with heterozygous B6/SEG genes (at the Led2 locus) and uterine homozygous spretus genes (at the Led2 locus). During each gestation, the substrains were phenotyped in vivo by ultrasonography. This non-invasive technology, based upon a high frequency ultrasound device in vivo real time high resolution observations of embryonic development Led2minA and Led2minB) of approximately 6 Mb each, present together in spretus version only in R3 and R5 strains. In the other recombinant substrains which have not the phenotype, the one or the other of the region is present but not the two regions together. So we defined the first reduced spretus region called Led2minA which encompasses D1Mit50 to D1Mit305 region (>84.5 Mb to 90.5 Mb) and the second called Led2minB located at the rs3692309 marker (>92.5 Mb to <100.3 Mb). Our statistical analysis succeeded in proving the presence of Led2minA QTL responsible for a main effect on embryonic death but it failed it for Led2minB. However, notable differences between the embryonic death rates of certain strains (R6 compared to R3 or R5) led to suppose that this latter region could also have a small effect in the phenotype. Taken together, these data did not support the presence of an epistatic interaction between Led2minA and Led2minB.To accomplish the fine mapping of this QTL, we generated recombinant substrains from 66H-MMU1 by backcrosses, each of them presenting a unique sub-fragment of the spretus fragment were preferentially modified (\u223c40%). This concentration of deregulated genes located on the spretus fragment has already been reported in a previous study of our group performed on testis transcriptome Mus musculus and Mus spretus are frequent since they appear, in average, every 100 bp. When located on the promoter regions of spretus origin, these nucleotide substitutions could modify the transactivation/transrepression properties of transcription factors of C57BL/6J nature, thus modifying the spretus gene expressions. Additionally, dysfunctions leading to embryonic death could result from non-synonymous coding polymorphisms, accumulated during evolution in the spretus genome. These phenomena should be originated from evolution of separated genomic regions that produces transcription factors/DNA (\u201ctranscriptomic shock\") and/or protein-protein (\u201cproteomic shock\") incompatibilities Reverse crosses using IRCS Group 2 males and B6 females revealed that the genes expressed at heterozygous state in the placental tissues are not deleterious for the gestation. Therefore, we deduced that the high rate of embryonic death occurring during the gestation resulted from dysfunction of genes expressed in the uterine tissue. This is in accordance with the normal embryonic development observed in group 2 IRCS females. Then, we carried out a microarray analysis searching to identify uterine deregulated genes in IRCS animals from Group 2. Although we observed deregulated genes located in all chromosomes (19%) we noticed that those situated on the Led2minA QTL and applying filters from bioinformatics databases, bibliography and our own results, we propose a selection of 7 genes  as putative actors of the embryonic death. These genes play a role in VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathway. Their effects could be reinforced by a small participation of genes situated on Led2minB region and which could act in the same signaling pathways .Focusing on genes of the Trip12, Psmd1, Cops7b and Usp40 from Led2minA and Asb1 from Led2minB are involved in protein degradation process through the ubiquitin-proteasome pathway. Trip12 exerts a ligase activity related to ubiquitination Usp40 functions as a deubiquitination enzyme in the same degradation pathway Asb1 is a member of the ankyrin repeat and SOCS box (ASB) family. These family proteins interact with Cul5-Rbx2 to form E3 ubiquitin ligase Psmd1 is a component of the 26S proteasome. Cops7b is a subunit of the eight-subunit heteromeric Cop9 signalosome complex. Genetic invalidations of some Cop9 subunits have been associated with developmental defects of post-implantation embryos Usp40 functions as a deubiquitination enzyme in the same degradation pathway Asb1 is a member of the ankyrin repeat and SOCS box (ASB) family. These family proteins interact with Cul5-Rbx2 to form E3 ubiquitin ligase Led2minA Ncl gene and Led2minB Ramp1 and Col6a3 genes are involved in angiogenesis. Ncl encodes nucleolin and treatment of endothelial cells with anti-nucleolin antibody induces apoptosis of these cells Ramp1 gene, RAMP1 (receptor activity modifying protein) forms a functional receptor for CALCA  which is a proangiogenic growth factor in the human placental development and plays a critical role in embryonic development and fetal growth Collagen typeVI a3 gene, COL IV is a main endometrial extracellular matrix component, and an abnormal increased deposition of collagen might impair uterine function, possibly by interfering with vascularization or retarding remodeling events at implantation In the same manner, Cab39  and Eif4e2 from Led2minA and Traf3ip1 from Led2minB, participate in the mTOR signaling pathway, a regulatory step of protein synthesis and growth. Cab39 effect has been described upstream of mTOR activation while Eif4e2 is a downstream signaling target involved in translation initiation. Interestingly, Mtor genetic disruption in mice leads to early embryonic death Traf3ip1  mutant mice are not viable. Traf3ip1 mutant mouse line was generated and the enlarged mutant cell size in culture was associated with elevated basal mTOR pathway activity Finally, in vivo approach of the embryonic development on a mouse IRCS model to refine a chromosome 1 region (Led2) responsible for embryonic death. The present study succeeded in fine-mapping Led2minA QTL which has a main effect on the embryonic death (about 30%) and pointed out a second region Led2minB which could have a minor effect on the same phenotype. Collecting and analyzing experimental, bioinformatics and literature data on the expression and function of genes present in the two regions (Led2minA and Led2minB), we propose 7 genes from Led2minA that could be related with the phenotype. It appears that the vascularization could be the common denominator at these categories of genes involving angiogenesis and the fluidity of the extracellular matrix. The actual identification of the gene(s) involved in this phenotype will necessarily pass through further molecular approaches. An important outcome of this study is the possibility to evaluate novel promising candidates of RSA in humans We used an Table S1Sequences of used real time RT-PCR primers.(DOC)Click here for additional data file."}
+{"text": "We recently reproduced in the SIV 8 PFU of ALVAC-SIV at weeks, 0 (V0), 4 (V2), and with both ALVAC SIV and the SIVgp120 envelope proteins, adjuvanted with ALUM at week 12 (V3) and 24 (V4) and analyzed the profile of gene expression at 24 hours after each vaccination. In parallel, we studied the phenotypical and functional changes in the NK cell subsets after vaccination by multi parametric flow cytometry.In here, we immunized, by the intramuscular route, 6 RM with 10Microarray analysis in the PBMC after V1 and V2 showed the up regulation of anti viral  and IFN responsive genes and a down regulation of pro-inflammatory genes. Significant alteration in the gene expression profile was also observed after the gp120/Alum boost (V3). Interestingly, NK cells associated genes were up regulated after V3 vaccination. These finding paralleled our results observed by FACS analysis that demonstrated an increased frequency of NK22 cells at mucosal sites. These NK22 cells expressed CCR6 (a gut homing marker) and are thought to play a role in mucosal immunity. Similarly, vaccination also increased the frequency of NKG2A+ cells that were either cytotoxic (CD107a+) or cytokine producing (IFNg+).Thus, the ALVAC SIV/gp120 vaccine regimen induces a significant activation of NK cells and other innate immune responses. Given that this vaccine platform has conferred some degree of protection from HIV infection in humans, understanding the role of innate immunity in protection from SIVmac251 may guide our effort in the development of novel vaccine strategies against HIV."}
+{"text": "The human immunodeficiency virus type 1 (HIV-1) infection occurs by binding to CD4+ receptor and chemokine receptor 5 (CCR5) or the CXC chemokine receptor (CXCR4). A mutation in the CCR5 gene, 32 base pair deletion consequences a truncated protein that is not expressed on the cell surface. The deletion confers resistance to HIV-1 infection and slows the progression of AIDS in HIV infected individuals. The worldwide distribution of CCR5delta 32 polymorphism was congregated by retrieving the data from literature and genotyping new population samples. A comprehensive resource of frequency data for CCR5delta 32 polymorphism in different population samples was created.The data for different populations was obtained from literature. In order to investigate the genetic variation in CCR5 gene in new population, we analyzed 257 healthy control individuals. We examined the CCR5 32 base pair deletion (CCR5-\u039432) by conventional polymerase chain reaction (PCR).The genotype frequency distribution of CCR5 in new population was found to be  in healthy control.The allele frequency of CCR5-\u039432 observed for new population is 1% which is compared with the other populations. The polymorphism CCR5-\u039432 is primarily found in European population. Compared to our data, frequency of delta 32 deletion is observed at high frequency in European populations. Our data of delta 32 deletion is significantly different from Caucasians (p<0.00000001), Africans (p<0.01458) and Europeans (p< 0.00000001)."}
+{"text": "For a therapeutic HIV-1 vaccine, it should be considered that the immune system has been confronted with a certain viral sequence and mounted CD8 T cell responses specifically towards the infecting virus. One question is whether the immune system of HIV-infected individuals can create a new response towards a variant epitope in the case of vaccination. To study this in a comparable setting, we addressed the question how frequently a new CD8 T cell response can be generated after the occurrence of a viral escape mutation in its recognized epitope in a population not selected for a certain HLA allele.19 HIV-infected untreated subjects were sampled longitudinally  of these. Peptide titration assays revealed 12 (48%) viral escape mutations with 2 de-novo responses (17%). Here the de-novo response showed less effector functions than the original CD8 T cell response. In addition we identified 5 (20%) shifts in immunodominance. None of the subjects with adaptation to the changing virus carried the HLA alleles B27, B57 or B*5801.Our results show that CD8 T cell responses can adapt to the mutations of HIV. However it was limited to only 28% (7 out of 25) of cases in a cohort not expressing protective HLA alleles."}
+{"text": "We have in an epidemiological study identified a group of individuals with multiple asthma symptoms (MSA) that reflect a more severe and uncontrolled disease. An altered innate immunity and adaptive immunity, and effects of microorganisms may play a role in the development of both CRS and asthma. The innate immune system use Toll-like receptors (TLRs) to recognize microbes and activate defense mechanisms within minutes of microbial invasion which is followed by an antigen-specific response by the adaptive immune system. Thus, innate and adaptive immune cells are sequentially activated and reciprocally regulate one another. Glycogen synthase kinase-3\u03b2 (GSK3\u03b2) has been demonstrated as a regulator of both innate and adaptive immunity. GSK3\u03b2 was shown to modulate inflammatory responses through TLR-mediated production of pro-inflammatory cytokines and inactivation of GSK3\u03b2 (phosphorylated GSK3\u03b2) lead to a reduced IL-12 production, which was suggested to skew the balance towards a Th2 response.To determine the degree of expression of TLRs 2, 4, 7 and 9 on monocytes as well phGSK3\u03b2 using flow cytometry. We hypothesize that the co-existence of CRS in patients with asthma and especially patients with MSA has an increased TLR expression involved in the innate immune system.Participants were selected from an epidemiological cohort, the West Sweden Asthma Study. Clinical parameters as well as fresh peripheral blood cells were obtained from one group of non-asthmatic subjects with CRS (CRS) and four different groups of asthmatics: 7 subjects with MSA (MSA), 7 subjects with other asthma (OA), 7 subjects with MSA and CRS (MSA/CRS) and 9 subjects with OA and CRS (OA/CRS). These five groups were compared to a control group consisting of 10 healthy subjects without asthma or CRS.+ monocytes compared to controls as well as the expression of phGSK3\u03b2 and CD14. Individuals in the combined MSA/CRS group showed increased expression of CD14 and TLR2, but increased expression of TLR4 was only found in the CRS group.Individuals with MSA or CRS only, showed significantly increased TLR2, TLR7 and TLR9 expression (rMFI) on CD14The upregulated expression of TLRs in the MSA group compared to control group suggest a higher susceptibility towards an altered immune response that might reflect the degree of severity. Furthermore, the increased phosphorylation of GSK3\u03b2 may indicate a switch in the immune response from a pro-inflammatory to a dysregulated Th2 response."}
+{"text": "As microcystin structure has a significant impact on transporter selectivity, it is potentially possible to develop analogs with even more pronounced OATP1B3 selectivity and thus enable their development as anticancer drugs.Microcystins are potent phosphatase inhibitors and cellular toxins. They require active transport by OATP1B1 and OATP1B3 transporters for uptake into human cells, and the high expression of these transporters in the liver accounts for their selective hepatic toxicity. Several human tumors have been shown to have high levels of expression of OATP1B3 but not OATP1B1, the main transporter in liver cells. We hypothesized that microcystin variants could be isolated that are transported preferentially by OATP1B3 relative to OATP1B1 to advance as anticancer agents with clinically tolerable hepatic toxicity. Microcystin variants have been isolated and tested for cytotoxicity in cancer cells stably transfected with OATP1B1 and OATP1B3 transporters. Microcystin variants with cytotoxic OATP1B1/OATP1B3 IC Microcystis, Oscillatoria, Planktothrix, Nostoc, and Anabaena. They can be considered to be among the best studied cyanobacterial secondary metabolites l-amino acids situated at positions 2 and 4 of the MC backbone. Related compounds are the nodularins, found in Nodularia sp., which also contain Adda but are cyclic pentapeptides instead of heptapeptides (Microcystins (MCs) are cyclic heptapeptides produced by several cyanobacterial genera such as peptides  [57]\u2013+ were isolated in the ion trap, fragmented by collision induced dissociation (CID) using argon as collision gas  to 45.0 kHz), and separated in the TOF analyzer. MS/MS scans were averaged and converted to the mzXML format using the vendor\u2019s software and evaluated using the software mMass  1H-NMR spectra of all isolated compounds can be found in The structures of the isolated MCs have been determined by high-resolution tandem mass spectrometry 3H]-BQ123, a substrate for both transporters. Further, the OATP1B3 clones demonstrated increased uptake of [3H]-CCK-8, a specific substrate for OATP1B3 but not OATP1B1, while the OATP1B1 clones did not show uptake of [3H]-CCK-8. In addition, RKO and HeLa cells stably transfected with an empty expression vector were used as controls. For cytotoxicity assays, the cells were plated in triplicate in 96-well microtiter plates in a medium containing 5% fetal bovine serum at densities of 1000 cells/well. After 24 hours, medium containing MC structural variants was added to the cells. After another 3 days, cell survival was determined with a sulforhodamine-based assay as we have previously described 50 was calculated from the dose response curve as the concentration of drug that produced a 50% decrease in the mean absorbance compared to the untreated wells and reported as the average of at least three independent determinations performed in triplicate using Prism software.Primary hepatocytes are not suitable for studying microcystin toxicity and transport, as OATP transporters are downregulated within hours upon placing cells in culture File S1Details on the Isolation of the Microcystin Congeners.(PDF)Click here for additional data file.File S21H-NMR spectra of all isolated compounds. Raw NMR and MS data of these compounds are available free of charge via the Internet at http://dx.doi.org/10.6084/m9.figshare.880755.Annotated tandem HRMS and (PDF)Click here for additional data file."}
+{"text": "Braun et al. discovered an error in the SAS code of their paper \u201cVariability of Urinary Phthalate Metabolite and Bisphenol A Concentrations before and during Pregnancy\u201d [Environ Health Perspect 120:739\u2013745 (2012)]. The error resulted in the reported absolute concentrations of phthalate metabolites and BPA being approximately 50% higher. The authors mistakenly used the median value from the EARTH study men [specific gravity (SG) = 1.025] rather than women (SG = 1.015) in their SG calculations. Thus, the reported concentrations in Table 2 (columns 2 and 3), Table 4 (columns 2 and 3), and Figure 1, as well as in the Supplemental Material, were higher than if the median SG for this group of women had been used. The authors note that this error did not alter the statistical analyses or their interpretations or the conclusions of their paper.The authors regret the errors."}
+{"text": "Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine domains . AggregaInducible control and SCA7 models expressing full-length ATXN7 with 10 (FLQ10) or 65 glutamines (FLQ65) were used. The autophagic activity was analyzed by measuring the p62 level and by monitoring the LC3 levels in the presence or absence of the lysosomal inhibitor bafilomycin A. Protein aggregation was analyzed by the filter trap method and protein-protein interactions by co-immunoprecipitation and western blot. Cellular toxicity was determined by the WST-1 assay.We found that mutant ATXN7 causes autophagic dysfunction by altering the cytoplasmic activity of p53 resulting in an increased p53-FIP200 interaction, co-aggregation of p53-FIP200 with mutant ATXN7 and ultimately disruption of the key autophagy regulating FIP200-ULK1 complex. Furthermore, we show that treatment with a p53 inhibitor, or a blocker of ATXN7 aggregation, can restore the soluble levels of FIP200, as well as increase the autophagic activity and reduce mutant ATXN7 toxicity.We have identified a novel p53-mediated mechanism by which aggregating polyQ disease proteins can disrupts autophagic activity and showed that this inhibition contributes to polyQ toxicity. An increased understanding of the molecular mechanism by which autophagy inhibition occurs in neurodegenerative disease is of importance if safe and efficient therapeutic approaches based on autophagy stimulation should be developed."}
+{"text": "The aim of our study was to determine the correlation between activated clotting time (ACT) and APTT values in patients receiving unfractionated heparin (UFH) for renal replacement therapy (RRT).A retrospective analysis was made of case notes and laboratory data of 39 critically ill patients who were on UFH for RRT over a 1-year period. There were 183 paired APTT and ACT measurements done at the same time (29 patients). APTT was done at the laboratory and ACT was done at the bedside using an ACTALYKE monitor . Target APTT and ACT ranges for UFH during RRT were 45 to 55 seconds (control 27 to 32 seconds) and 250 to 270 seconds (control 180 to 220 seconds). Datasets were divided into three groups and the correlation coefficient (Pearson's) was calculated using SPSS software.Mean APTT was 129.5 \u00b1 68.29 (range 25.6 to 360) seconds and mean ACT was 234.6 \u00b1 47.02 (range 125 to 387) seconds. APTT and ACT values were divided into three datasets in a 3 \u00d7 3 table. There was no correlation between APTT and ACT values (kappa score being 0.12). There were more above-range APTT values (140/183) against above-range ACT values (36/183). See Table Our data demonstrate that monitoring of anticoagulation with UFH using ACT cannot be recommended."}
+{"text": "Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In  Saccharomyces cerevisiae to respond to nutrient availability and stress The Target Of Rapamycin (TOR) and Ras/cAMP/Protein Kinase A (PKA) signaling pathways enable The activity of PKA is controlled by intracellular cAMP HXT7 encodes a high affinity hexose transporter and its transcription is induced by low levels of glucose or a non-fermentable carbon source and Hxt7 localizes to the plasma membrane. However, in response to glucose abundance, nitrogen starvation or rapamycin treatment HXT7 transcription is repressed and Hxt7 is degraded in the vacuole HXT3 encodes a low affinity hexose transporter that is actively expressed in glucose abundance, but repressed Hexose transporters are regulated at the transcriptional and post-translational levels to allow yeast to adapt to varying nutrient concentrations in the environment. If conditions become unfavorable for the expression of a specific transporter gene, the cell must repress its transcription and degrade the remaining transporter. This degradation occurs via endocytosis and proteolysis in the vacuole. For example, vid/gid mutants are sensitive to the presence of rapamycin in the media VID/GID genes increases in the presence of non-fermentable carbon sources The Vid/Gid proteins play an important role in the yeast\u2019s adaptation to different nutrient conditions. These proteins assemble into a multi-component complex termed the Vid30 complex (Vid30c) Here we further investigate the link between the Vid30c and the regulatory mechanisms that govern hexose transporter (Hxt) turnover. We expand the known function of the Vid30c in the nitrogen starvation and rapamycin-induced turnover of Hxt7 HXT3 to GFPMET25pro) for the chromosomal fusion of the methionine-repressible MET25 promoter (MET25pro) to the 5\u2032 end of HXT3CUP1pro-GFP) for the chromosomal fusion of the copper-inducible CUP1pro-GFP cassette to the 5\u2032 end of HXT7VID30 with hphMX6PGK1pro) for the replacement of the native VID28 promoter with the constitutively active PGK1 promoter. Following transformation, the correct integration events were verified by PCR. It is important to note that HXT7 is a duplicated gene in the yeast genome with HXT6 being its counterpart. The PCR confirmation of HXT7 tagged strains therefore involved the use of an upstream primer in the upstream region of HXT7 that is unique to HXT7. BYtor1-1 was generated as previously described All the yeast strains used in this study are isogenic to BY4742 and listed in RAS2 and YCp50-VAL19RAS2VAL19RAS allele on Hxt turnover. Yeast strains used to monitor Hxt3-GFP localization and degradation were pre-cultured in synthetic complete media  to generate biomass. Cells were washed and transferred to synthetic media (0.17% YNB without amino acids and ammonium sulfate) containing 2.5% glucose and 0.5% ammonium sulfate. Amino acids were added to complement auxotrophic requirements. Following a three hour incubation to stimulate HXT3 expression, cells were imaged or harvested for protein extraction (time zero). The remaining culture was harvested, washed and transferred to synthetic media (0.17% YNB without amino acids and ammonium sulfate) containing 2% ethanol and 0.5% ammonium sulfate. The 2% ethanol media was used to provide conditions in which glucose repression was alleviated; we will here after refer to its effect as \u201cglucose starvation.\u201d Amino acids were added to complement auxotrophic requirements and/or suppress expression from the MET25 promoter. Samples were subsequently collected at the indicated times and used for fluorescence microscopy or protein extraction.YCp50, YCp50-et al. (2008) with the following exceptions: (1) the four hour pre-shift incubation was performed in raffinose with ammonium media containing 100 \u00b5M CuSO4 to stimulate GFP-HXT7 expression from the CUP1 promoter; (2) Cell samples were collected (time zero), and the remaining cells were washed twice with sterile water and resuspended in fresh raffinose with ammonium media devoid of CuSO4 followed by rapamycin treatment.Yeast strains used to monitor GFP-Hxt7 localization and degradation were cultured as described in Snowdon The monitoring of the subcellular localizations of the Hxt-GFP fusion proteins were performed by preparing slides directly from the indicated cell cultures followed by immediate analysis using the 100\u00d7 objective lens of a Nikon Eclipse E600 microscope. Images were recorded using a Coolsnapfx monochrome CCD digital camera (Roper Scientific) and processed using Metamorph .Harvested cells were resuspended in lysis buffer , added to 0.3 g glass beads and vortexed for two minutes. Lysates were centrifuged at 5,000 rpm for 3 minutes to remove cell debris. Supernatants were collected and the protein concentrations determined using the DC Protein Assay (Biorad) according to the manufacturer\u2019s recommendations. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Mouse anti-GFP (Roche) and rabbit anti-Aldehyde Dehydrogenase (ADH)  were used as primary antibodies. ADH was used as the internal control to confirm equal amounts of protein in each lane as previously described HXT3HXT3-GFP were grown in glucose media to activate the expression of HXT3, and Hxt3-GFP was monitored upon a switch to ethanol as the sole carbon source. In glucose (time zero) HXT3 transcription was activated and Hxt3-GFP was visible on the plasma membrane in all the strains tested  renders PKA constitutively active Val19 would impact the turnover of Hxt3-GFP in a shift to ethanol. Using the MET25pro-HXT3-GFP strain, our fluorescence microscopy and western analysis showed that the native RAS2 allele supported the internalization of Hxt3-GFP at 3 hours and almost complete degradation in the vacuole 6 hours after an ethanol shift, while Ras2Val19 stabilized Hxt3 in the plasma membrane even after 6 hours in ethanol media with little, if any, internalization and degradation visible throughout the entire time course , the internalization of Hxt3-GFP in rim15\u0394 cells was markedly delayed when the cells were shifted to ethanol; the protein was still visible in the plasma membrane and abundantly present after 6 hours in ethanol  as it functions as an E3 ubiquitin ligase responsible for the ubiquitylation and subsequent degradation of FBPase and Mdh2 when these enzymes are no longer needed ID genes  and 2. Ctor1-1 allele prevented the endocytosis and degradation of Hxt7 in response to rapamycin treatment and the activation of the Ras/cAMP/PKA pathway is known to suppress Tor deficiencies Val19 or lacking BCY1 supports this hypothesis. Similarly, the rapamycin-induced internalization of Hxt7 is largely prevented in cells expressing Ras2Val19. Furthermore, in rich nutrient conditions in the absence of stress, both TORC1 and PKA prevent cell cycle arrest by inactivating Rim15, the kinase that promotes entry into G0Several signaling pathways enable the molecular response to the presence or absence of nutrient abundance in the environment. The Ras/cAMP/PKA pathway facilitates the cellular response and proliferation when glucose is abundant rsp5-1ts mutant, confirming Rsp5 as an essential E3 ubiquitin ligase in Hxt3 turnover. We could not use the tsrsp5-1 allele in the turnover of Hxt7 as the strain could not tolerate rapamycin treatment during the turnover experiment (data not shown). We therefore used the less dominant rsp5-3ts allele to study Hxt7 turnover. Hxt7 was clearly stabilized in the plasma membrane, but some internalization was observed. These observations implicate two E3 ubiquitin ligases, Rsp5 and the Vid30c, in the nutrient-mediated endocytosis and degradation of Hxt3 and Hxt7. Nutrient transporters have been shown to be direct targets for Rsp5-mediated ubiquitylation, suggesting a more indirect function for the Vid30c where it does not directly ubiquitylate the target Hxt. It is therefore of great importance to identify the precise role the Vid30c in Hxt turnover. Investigation into the signaling surrounding Hxt turnover has implicated the Ras/cAMP/PKA pathway and Rim15 in both unique turnover events. The strikingly similar retention of Hxt3 and Hxt7 in the plasma membranes of the vid28\u0394vid30\u0394 double mutant and cells expressing Ras2Val19 could serve as the foundation to investigate a potential functional link between the Vid30c and the Ras/cAMP/PKA.Rsp5 is an essential E3 ubiquitin ligase responsible for the ubiquitylation and subsequent endocytosis of nutrient transporters Figure S1Deletion of components of the Vid30c causes a slight increase in HXT3 transcription. BY4742 (WT), vid30\u0394, gid2\u0394, vid28\u0394, gid8\u0394, gid9\u0394 (A) and vid28\u0394vid30\u0394 (B) expressing HXT3-GFP were cultured in glucose media as described in the methods (time 0). Following a switch to ethanol media, samples were collected at the indicated times and analyzed by northern blot analysis. Membranes were probed for GFP and ACT1 as a loading control.(TIF)Click here for additional data file.Figure S2The turnover of Hxt7 is dependent on Rsp5. BY4742 (WT) and rsp5-3 (A) and BY4742 (WT), art3\u0394, art4\u0394, art6\u0394 and art8\u0394 (B) expressing HXT7-GFP were cultured in raffinose media as described in the methods, time 0. After treated with rapamycin, samples were collected at the indicated times and analyzed by fluorescence microscopy (Top) and Western analysis with anti-GFP antibodies (Bottom). Identical blots were also probed with anti-ADH antibody as a loading control. (C) BY4742 (WT), art3\u0394, art4\u0394, art6\u0394 and art8\u0394 expressing HXT3-GFP were cultured in glucose media as described in the methods, time 0. Following a switch to ethanol media, samples were collected at the indicated times and analyzed by fluorescence microscopy.(TIF)Click here for additional data file.Table S1Yeast strains used in this study.(DOCX)Click here for additional data file."}
+{"text": "Ischemic preconditioning is a neuroprotective mechanism whereby a sublethal ischemic exposure is protective against a subsequent lethal ischemic attack. We previously demonstrated that SIRT1, a nuclear localized stress-activated deacetylase, is vital for ischemic preconditioning neuroprotection. However, a recent study demonstrated that SIRT1 can also localize to the mitochondria. Mitochondrial localized SIRT1 may allow for a direct protection of mitochondria following ischemic preconditioning. The objective of this study was to determine whether ischemic preconditioning increases brain mitochondrial SIRT1 protein levels and to determine the role of PKC\u025b and HSP90 in targeting SIRT1 to the mitochondria. Here we report that preconditioning rats, with 2 min of global cerebral ischemia, induces a delayed increase in non-synaptic mitochondrial SIRT1 protein levels which was not observed in synaptic mitochondria. This increase in mitochondrial SIRT1 protein was found to occur only in neuronal cells and was mediated by PKC\u03b5 activation. Inhibition of HSP90, a protein chaperone involved in mitochondrial protein import, prevented preconditioning induced increases in mitochondrial SIRT1 and PKC\u03b5 protein. Our work provides new insights into a possible direct role of SIRT1 in modulating mitochondrial function under both normal and stress conditions, and to a possible role of mitochondrial SIRT1 in activating preconditioning induced ischemic tolerance. Ischemic preconditioning (IPC) is an innate neuroprotective mechanism in which a mild ischemic stress protects against a subsequent lethal ischemic exposure. IPC activates both early 0-3h) and delayed 24-72 h) windows of protection -3. The eh and del-72 h win+ dependent deacetylases which is implicated as a metabolic sensor of the cell . In. In39].  peptide  8], and, andp = In this study, we demonstrate a novel regulatory mechanism in targeting SIRT1 to the mitochondria which we believe represents an expansion of the mechanisms and targets by which SIRT1 mediates neuroprotection. We also demonstrate that changes in mitochondrial SIRT1 protein levels were dependent upon PKC\u03b5 activation, suggesting a novel interaction between the PKC\u03b5 and SIRT1 signaling pathways.Mitochondrial increases in SIRT1 protein levels following IPC were only observed during the late phase or delayed window of neuroprotection. The increase in mitochondrial SIRT1 protein is also correlated with our previously reported findings of a late phase but not early increase in nuclear SIRT1 activity ,20. ActiAlthough a basal level of SIRT1 protein was observed in all of the mitochondrial preparations investigated, we found that IPC only increased mitochondrial SIRT1 protein levels in non-synaptic mitochondria and in mitochondria from neuronal-only cultures. These results suggest that IPC alters SIRT1 levels primarily in mitochondria localized to neuronal cell bodies. This specific targeting of SIRT1 to a subpopulation of neuronal mitochondria may be explained by our findings that HSP90 is required for mitochondrial import of SIRT1. In the adult rat brain, HSP90 is reported to primarily localize to the neuronal cell body . Gass etWe also demonstrate that the total cellular SIRT1 protein levels were increased following IPC exposure in neuronal but not glial only-cultures which correlates with the observed increases in neuronal but not glial mitochondrial SIRT1 protein levels. We also observed a reduction in nuclear SIRT1 protein levels 48 hrs following IPC exposure. These findings suggest that the increase in mitochondrial SIRT1 protein levels may be the result of both increased SIRT1 expression, as well as, nuclear to mitochondrial shuttling of SIRT1. Our data also demonstrate that SIRT1 is targeted to mitochondria by mechanisms mediated by PKC\u03b5. This is based upon our findings that activation of PKC\u03b5, in the absence of IPC, increases mitochondrial SIRT1 protein levels and deacetylase activity, whereas inhibition of PKC\u03b5 prevents increases in mitochondrial SIRT1 protein levels and deacetylase activity following IPC exposure. The subcellular localization of SIRT1 is known to be regulated by posttranslational phosphorylation and sumoylation ,46,47. IIn summary, it has been demonstrated in both the heart and brain that SIRT1 is an important mediator of IPC-induced ischemic tolerance. Based upon the data presented here, part of SIRT1 protection may stem from the ability of IPC to regulate SIRT1 mitochondrial protein levels, which may allow for a direct regulation of mitochondrial function by SIRT1. Our discovery that SIRT1 is targeted to mitochondria by PKC\u03b5 following IPC may offer new therapeutic directions in the targeting of metabolic dysfunction, which is associated with the pathophysiology of numerous diseases such as ischemia/reperfusion."}
+{"text": "TIMP3 and p16 was analyzed with MS-PCR. Of the 105 investigated tumors 59.1% (62/105) were WHO grade I, 33.3% (35/105) were WHO grade II and 7.7% (8/105) were anaplastic meningiomas (grade III), respectively. The histopathological data correlates with the recurrence rate of the investigated meningiomas. Hypermethylation of TIMP3 was detected in 13.3% of all meningiomas: 10.9% in WHO grade I meningiomas, 25.0% in grade II and 14.3% in grade III meningiomas, respectively. No correlation of TIMP3 hypermethylation with tumor recurrence or WHO grade (p\u200a=\u200a0.2) was observed. Interestingly, deletion of 1p36 emerged as a significant predictor of shorter overall survival , whereas TIMP3 promoter methylation had no significant effect on overall survival . The results of the current study support the finding that the deletion of chromosome 1p is an independent marker of meningioma recurrence and progression (p\u200a=\u200a0.0097). Therefore the measurement of genetic aberrations in meningiomas allows in a combined histological approach a more precise assessment of the prognosis of meningiomas than histopathology alone.Meningiomas are tumors that arise from the coverings of the brain or spinal cord. 5% of the cases turn into malignant forms with aggressive clinical behavior and increased risk of tumor recurrence. One hundred and five patients with meningiomas were operated by open surgery. To investigate predictors of meningioma recurrence in total 124 samples of 105 patients were investigated by iFISH. Dual-probe hybridization was performed to access chromosomal alterations of chromosomes 1p-, 9p- and 22q. Additionally, methylation of  Meningioma is a tumor composed of neoplastic meningothelial cells and accounts for 13 to 26 percent of intracranial tumors. In its sporadic form, it is typically benign and slow growing, appearing mostly in the later decades of life. Histological features allows for the division of meningiomas into three grades: benign meningiomas (WHO grade I), atypical meningiomas (WHO grade II) which represent 4.7 to 7.2 percent of all meningioma\u015f and anaplastic meningiomas (WHO grade III) representing 1.0 to 2.8 percent of all meningiomas. However, more than eight percent of all meningiomas are characterized by aggressive clinical behavior with increased risk of tumor recurrence NF-2 gene NF2 gene on the long arm of chromosome 22 (22q12.2), often present with multiple meningiomas. However this hereditary alteration has little or no influence on progression TIMP3), which appears to be involved in meningioma progression and a high-grade meningioma phenotype TIMP3 hypermethylation seems to be associated with the allelic loss of 22q12. Moreover, hypermethylation of the TIMP3 promoter has been identified as a common reason of decreased TIMP3 expression levels in numerous tumors like secondary glioblastomas, kidney cancer, or pancreatic adenocarcinomas TIMP3p16Conventional cytogenetic analysis of meningiomas often reveals the frequent entire or partial losses of chromosome 22. Extensive studies by loss of heterozygosity (LOH) also have shown frequent allelic losses on the long arm of this chromosome, pinpointing the tumor suppressor locus in the vicinity of the The p16 pathway, which has been found to be altered in more than 80% of human cancers In contrast to other solid tumors, progression of meningiomas is correlated with increasing hypodiploidy, showing characteristic clonal evolutions that mostly include chromosomes 14,18, and 19 and, more rarely, 6 and 10. Structural aberrations are rare, except for the loss of the short arm of one chromosome 1, which appears to be the decisive step for anaplastic growth In 2001, Zang reported a genetic model of tumor progression in meningiomas Since the publication of Ishino and coworkers in 1998 the fluorescence in situ hybridization (FISH) technique has provided new insights into interphase cytogenetics and is applicable now to clinical practice. Therefore the time-consuming cell culture for conventional chromosome analysis is not further necessary. Instead of paraffin embedded tumor samples, also fresh tumor samples that are speckled on microscope slides can be used.TIMP3 and p16 in meningiomas in order to evaluate the impact of these markers for the biological behaviour of meningiomas.In the current study we examined 124 meningioma samples out of 105 prospective meningioma patients for the partial loss on chromosomes 1p, 9p and 22q, using double-target iFISH. The first aim of this study was to analyze if the iFISH results predict malignant behavior and recurrence of meningiomas as well as conventional cytogenetic analysis. The second aim of this study was to examine the promoter hypermethylation of We performed a prospective study on 124 tumor samples from 105 meningioma patients [76 women and 29 men] operated at the Department of Neurosurgery, Saarland University, between January 1997 and December 2010. The average age of the overall patient population at the date of first surgery was 57.2 years [SD \u200a=\u200a13.3 years], for female patients 58.1 years [SD \u200a=\u200a12.0 years], and for male patients 54.8 years [SD \u200a=\u200a16.3 years]. Written informed consent was obtained from each patient participating in the study.Patients were examined in the neurosurgical outpatient department of the Saarland University, either within routine follow-up or upon appearance of neurological symptoms. A recurrence was defined as new evidence of tumor in CT or NMR after previous complete extirpation (Simpson grades 1 and 2) Complete surgical extirpation of the tumor was defined as Simpson grades I and II. This corresponds to a macroscopically complete tumor resection with bipolar coagulation of the dura insertion.Meningioma grade was assessed by a combined histologic All tumors were classified according to the WHO classification of tumors of the nervous system of 2007 In total 124 different probes were investigated from the 105 patients. In 91 tumors (86.6%), only one surgical procedure was performed, in 10 patients (9.5%) 2 surgery procedures, in 3 patients (2.9%) 3 operations due to recurrence, and in 1 patient 4 surgical approaches was necessary.TIMP3.Tissue specimens from tumors were obtained freshly after surgery and dapped on clean microscope slides previously silanized. After Delauney fixation for 30 sec, slides were stored at \u221220\u00b0C until further FISH analysis. The slides were treated with RNAse for thirty minutes at 37\u00b0C and placed three times in 2xSSC for five minutes at RT. Cells were digested in 100 ml 0,01 M HCL with 10 mg pepsin  for 1 min and 45 sec at 37\u00b0C. Slides were dipped in 1xPBS for 5 min, 4%PFA/1xPBS for 10 min for fixation and 1xPBS for 5 min. They were then dehydrated in 70%, 80%, 95% ethanol and air dried. Dual-probe hybridization was performed using locus-specific probes for 1p36, 22q11 and 9p21 . Target and probe were separately denatured for 5 minutes at 73\u00b0C. Target slides were dehydrated in 70%, 80%, 95% ethanol. Then probes were pipette on slides and incubated overnight at 37\u00b0C in a humidified chamber. Stringency wash was performed in 0.4xSSC/0.3%SDS for 2 minutes at 73\u00b0C and 2xSSC/0.1% SDS at RT. Finally, slides were counterstained with DAPI antifade .At least 200 non-overlapping nuclei per sample were counted for evaluation according to Hopman-criteria Cut-offs for alterations was determined by comparison with normal human lymphocytes control samples at 10% for deletions of 1p36, 22q11 and 9p21.Extraction of genomic DNA from snap frozen tumor tissue was performed with standard methods with samples more than 0.3 g and QIA DNA Mini Kit for samples with less than 0.3 g . DNA-Isolation from peripheral blood leukocytes was performed according to standard protocols Sodium bisulfite treatment of 500 ng DNA was performed with the EZ Methylation Gold\u2122 Kit (Zymo Research Corporation) according to the manufacturer's protocol. PCR analyses were performed with 70 to 80 ng of the bisulfite-modified DNA as template using 0.2 \u03bcl (5 U/\u03bcl) of HotStar Taq DNA Polymerase  in 1\u00d7 Qiagen PCR-buffer with 0.25 mmol/l of each dNTP and 0.1 \u03bcmol/l of each primer. The primers used in this study are described in the literature by Herman et al. p16 and TIMP3 by sequencing the regions of CpG-Islands as described in previous studies Also, we analysed methylation status of Comparison of survival times between groups defined by methylation status was performed by Kaplan-Meier curves and with two-sided log rank tests. Univariate and multivariate Cox regression analysis was performed to identify significant predictors for survival and tumor recurrence. Stepwise backward selection based on the AIC (Akaike Information Criterion) was used for variable selection for the multivariate Cox model. Effects in all models were quantified by hazard ratio estimates with corresponding 95% confidence intervals.All p-values were calculated with two-sided tests. Median progression free survival rates were calculated using the Kaplan-Meier method.We obtained ethical approvel from local ethics committee for \u201cThe role of high risk of local recurrence in meningioma as indicated by the genetic progression score\u201d  involving both, Department of Human Genetics, Saarland University and Neurosurgical Clinic, Saarland, University Medical School.Written informed consent was obtained from each patient participating in the study.We performed a prospective study on 124 tumor samples from 105 meningioma patients. The sex ratio was 2.6:1 in favor of females (76 women and 29 men). The average age of all patients at the date of first surgery was 57.2 years, and females were slightly but non-significant older on average (58.1 years vs. 54.8 years), see In 91 tumors (86.6%), only one surgical procedure was performed, in 10 patients (9.5%) 2, in 3 patients (2.9%) 3, and one patient had 4 times a tumor resection.The histological results showed in 62 cases (59.05%) a WHO grade I tumor, in 35 cases (33.33%) a WHO grade II and in 8 cases (7.62%) a WHO grade III. The WHO grade was significantly correlated with tumor recurrence , loss of the chromosomal region 1p36 in 35.2% (37/105) and loss of the chromosomal region 9p21 in 6.67% (7/105).Breaking down the FISH results to the respective WHO grading: monosomy 22 was detected in 45.2% (28/62) of WHO grade I, 48.6% (17/35) in grade II and 75% (6/8) in grade III meningiomas. Univariate as well as multivariate Cox regression analysis revealed no correlation with tumor recurrence , in grade II in 27.1% (13/35) and in anaplastic meningiomas in 87.5% (7/8) of cases. Only 1.6% (1/62) of WHO I meningiomas and 5.7% (2/35) of WHO II meningiomas had a deletion of 9p21. On the other hand 50% (4/8) of anaplastic meningiomas showed this deletion see also .TIMP3 was found in 13.3% (14/105) of all cases. Breaking down the methylation results by the WHO grade, 6 cases (9.68%) belonged to WHO grade I, 7 cases (20%) to WHO grade II and 1 case (12.5%) to the anaplastic meningioma grade III (TIMP3 hypermethylation from grade I (9.68%) to grade II (20%). However due to the small number of cases of anaplastic meningiomas these results turned out to be not significant  on 22q12.3 has been described as a tumorsupressor gene in different tumor entities The gene for the tissue inhibitor of metalloproteinase 3 (TIMP3 gene resulting in silencing the tumorsupressor gene was more frequently detected in meningiomas with loss of 1p. Bello and coworkers found a correlations with loss of the genes THSB1 (47%), TIMP3 (24%), p73 (33%), p14ARF (23%) and p16 (23%). Also meningiomas with loss of 22q demonstrated a higher methylation status in this study p16 in 62% of meningiomas with LOH of NF2 (located on 22q12.2) in contrast to the normal karyotype In previous studies by Bello TIMP3 5\u2032-CpG island region in about 24% of meningiomas, Liu TIMP3 hypermethylation with MS-PCR and direct sodium bisulfite sequencing and revealed TIMP3 methylation in 67% of anaplastic meningiomas but less in atypical (22%) or benign meningiomas (17%).While Bello and colleagues TIMP3 methylation increases from 9.68% in grade I to 20% in grade II meningiomas. However, due to the low rate of the TIMP3 methylation in grade III meningiomas (1/8 case\u015f12.5%) in our study, overall TIMP3 methylation was not significantly correlated with tumor grade .In our present study we found that the rate of patients with TIMP3 gene is significantly associated with a shorter time to recurrence  and was not concordant with the hypermethylation status of p16. Furthermore our results did not confirm the association of hypermethylation of p16 in meningiomas with LOH of the NF2 gene from the previous study by van Tilborg The hypermethylation status of In The significant association of the deletion of 22q with the deletion of one short arm of one chromosome 1 is in line with our previously published results describing the clonal cytogenetic evolution of meningiomas TIMP3 status was associated with a micro deletion on the long arm of chromosome 22, we could not confirm these results. However, it must be given consideration that the monosomy 22 was ascertained by classic cytogenetic analyses in our study, whereas Barski and coworkers used microsatellite analyses using primers who span the TIMP3 region on chromosome 22 Although Barski et al. found in their study that in 20 of 39 investigated meningiomas the methylation of the After chromosome 22 anomalies, aberrations of the short arm of one chromosome 1 are the most frequent alterations detected by cytogenetic analysis of meningiomas In the present study we confirmed these previous results using the high resolution FISH-technology (iFISH) In Our results are in agreement with former cytogenetic investigations which indicated that the deletion of the distal part of the short arm of a chromosome 1 [1p-] is associated with progression in meningiomas In conclusion, in this study we used iFISH that is easily applicable to clinical practice because it is not very time-consuming and can be applied to various clinical materials, including paraffin embedded tumor samples or dapped slides. Consequently, due to the common feasibility of the iFISH method a multimodal approach to meningioma grading, which is in our opinion the most promising for identifying meningiomas with an increased tendency to recur, is now available for almost all patients.TIMP3 inactivation by methylation seems to be also involved in meningioma progression, at least it is associated with a shorter time to recurrence using the multivariate Cox-regression analysis (p\u200a=\u200a0.0475).The Based on the results of this study, the deletion of the short arm of one chromosome 1 is an independent prognostic factor which correlates significantly with increased risk of recurrence. After initial speculation on the role of 1p deletion for tumor recurrence"}
+{"text": "Chlamydomonasreinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX  to form magnesium-protoporphyrin IX , the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that5A7 is missing theCHLI1 gene and at least eight additional functionally uncharacterized genes.5A7 has an intactCHLI2 gene. Complementation with a functional copy of theCHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in5A7. We have identified the firstchli1 (chli1-1) mutant ofChlamydomonas reinhardtii and in green algae. Our results show that in the wild typeChlamydomonas CHLI2 protein amount is lower than that of CHLI1 and thechli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses theCHLI2 gene. Ourchli1-1\u00a0mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis inChlamydomonas,which has never been studied before.The green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants, can grow photo-autotrophically and heterotrophically (it can metabolize exogenous acetate as a carbon source) and possesses a completely sequenced genome. These attributes make it an elegant model organism to study oxygenic photosynthesis and chloroplast biogenesis. In photosynthetic organisms, tetrapyrroles like Chl and heme are essential for energy metabolism (i.e. photosynthesis and respiration). Biosynthesis of Chl and heme occur via a common branched pathway that involves both nuclear- and chloroplast-encoded enzymes in most photosynthetic organisms. In photosynthetic eukaryotes, 5-aminolevulinic acid (ALA) is synthesized from glutamine through glutamyl-tRNA. Conversion of ALA through several steps yields protoporphyrin IX (PPIX), the last common precursor for both heme and Chl biosynthesis. Ferrochelatase inserts iron in the center of PPIX thus committing it to the heme branch of the pathway. Insertion of Mg2+ in PPIX by MgChel leads to Mgproto, the first biosynthetic intermediate in the Chl branch. Magnesium chelatase has three subunits, which are CHLD, CHLH and CHLI. The ATP-dependent catalytic mechanism of the heterotrimeric MgChel complex includes at least two steps: an activation step, followed by the Mg2+ insertion. Activation of MgChel with ATP involves CHLD and CHLI while CHLH is required for the chelation step. CHLI belongs to the AAA+ family of ATPases. Plants have two isozymes of CHLI1 (CHLI1 and CHLI2) which are 70%\u201381% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. Most of the data on CHLI comes from studies onArabidopsis thaliana chli mutants and the functional significance of CHLI1 and CHLI2 has not been studied in green algae. InArabidopsis CHLI2 plays a limited role in Chl biosynthesis because of its lower expression level compared to that of CHLI1. InArabidopsis the CHLI2 protein amount is lower than that of CHLI1. When overexpressed, CHLI2 can fully rescue anArabidopsis chli1chli2 double mutant.The green micro-algachli1-1) mutant ofChlamydomonas reinhardtii (5A7) which possesses an intactCHLI2 gene. Transformation of5A7 with a functional copy of theCHLI1 gene restored Chl biosynthesis. Western analyses show that the CHLI2 protein level is lower than that of CHLI1 in the wild type strain and CHLI2 protein is barely detectable in the mutant strain. In this study, we present our molecular data on the identification of the mutation locus in5A7 and its complementation.We have isolated the first ,5A7/chli1-1 (generated by our laboratory) andchli1-1 rescued transformants (generated by our laboratory) were grown either in Tris-Acetate Phosphate (TAP) heterotrophic media or in Sueoka\u2019s High Salt (HS) photo-autotrophic media. TAP and HS liquid media and agar plates were prepared in the lab using reagents from Fisher Scientific  according to the protocol given in Gorman and Levine (1965) and Sueoka (1960), respectively. The 4A+ strain andchli1-1 rescued transformants were maintained on TAP agar plates and TAP+zeocin  plates, respectively under dim light intensities (10\u201315 \u00b5mol photons m-2s-1) at 25\u00b0C. The final zeocin concentration was 15 \u00b5g/ml). Thechli1-1 mutant (5A7) was maintained in the dark on TAP 1.5% agar plates containing 10 \u00b5g/ml of paromomycin . Liquid algal cultures used for RNA and genomic DNA extractions and protein analyses were grown in 100 ml flasks on the New Brunswick Scientific Excella E5 platform shaker  at 150 rpm in the dark or in the dim light.Escherichia coli clone harboring the pBC1 plasmid  was used for random DNA insertional mutagenesis. This plasmid contains two antibiotic resistance genes:APHVIII andRAmp  and was used as a selection marker for screening ofChlamydomonas transformants.RAmp was used as a selection marker for screening ofE. coli clones harboring the pBC1 plasmid.E. coli was grown in 1 l of Luria Bertani (LB) broth containing 1% tryptone, 0.5% of yeast extract, 1% NaCl and ampicillin [final concentration of ampicillin:100 \u00b5g/ml]. LB reagent was prepared in the laboratory using reagents purchased from Fisher . Ampicillin was purchased from Fisher . The culture was incubated at 37\u00b0C overnight. Plasmid purification fromE. coli cells was facilitated by Qiagen plasmid mega kit according to the protocol given in the technical manual . Once purified fromE. coli, the circular pBC1 vector was linearized with the restriction enzymeKpn1  according to the protocol given in the technical manual. The linearized DNA was purified using a QIAEX II gel extraction kit  according to the protocol given in the technical manual. All agarose DNA gel electrophoresis was visualized by BioRad Molecular Imager Gel Doc XR+ . Transformation of parental strain 4A+ by the linearized pBC1 vector was performed utilizing the glass bead transformation technique described by Kindleet al. (1989) and Dentet al. (2005). Transformants were plated onto fresh TAP agar plates containing 10 \u00b5g/ml paromomycin (TAP+P) in the dark. Single colonies of mutants were picked and transferred onto fresh TAP+P plates using a numbered grid layout. Screening of photosynthetic and pigment deficient mutants was done by visual inspection and monitoring of growth under different light intensities in heterotrophic, mixotrophic and photo-autotrophic conditions.The purified pBC1 plasmid from the DH5\u03b1AmpR .APHVIIIchli1-1 rescued transformants complements and5A7/chli1-1 were grown in TAP liquid media in the dark to a cell density of about 5 \u00d7 106 cells/ml of the culture. Genomic DNA was purified using a phenol-chloroform extraction method. RNA extraction was facilitated by TRIzol reagent from Invitrogen  following the protocol in the technical manual. DNA and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer from Thermo Fisher Scientific . DNase treatment was performed using Ambion\u2019s TURBO DNA-free kit from Invitrogen  following the protocol in the technical manual to remove genomic DNA from the RNA preparation. Generation of cDNA was performed using Life Technologies Superscript III First-Strand Synthesis System from Invitrogen  following the protocol in the technical manual.4A+,et al. (2005). This protocol was implemented with one modification of utilizing a non-degenerate primer (AD2) derived from the original degenerate primer (AD) for TAIL PCR as this non-degenerate primer was giving us optimum yield without generating excess nonspecific products. HotStar Taq Plus DNA polymerase kit reagents  were used for PCR. The PCR reaction mixture consisted of 1\u00d7 PCR buffer, 200 \u00b5M of each dNTP, 1\u00d7 Q-solution, 2.5 units of HotStar Taq Plus DNA polymerase, 60 pmoles of the non-degenerate primer AD2 and 5 pmol of theAPHVIII specific primer. Primers were ordered from IDT . TAIL1 PCR product was diluted 10 and 25-fold and 2 \u03bcl of the diluted TAIL1 PCR product was used for TAIL2 PCR reactions. The TAIL2 PCR product was gel purified using QIAEX II gel extraction kit  according to the protocol given in the technical manual. The purified TAIL2 PCR product was sequenced at the UC, Berkeley DNA Sequencing Facility .TAIL  PCR was implemented, following the protocol of DentChlamydomonas genome database inPhytozome. Amplifications of genomic DNA and cDNA were executed using MJ Research PTC-200 Peltier Thermal Cycler . HotStar Taq Plus DNA polymerase kit  was used for PCR following the cycling conditions given in the Qiagen protocol booklet. Annealing temperature was between 55 and 60\u00b0C depending on the Tm of the primers. Extension time was varied according to the size of the PCR product amplified. Final extension was set at 72\u00b0C for ten minutes. All genomic and reverse transcription PCR products were amplified for a total of thirty-five cycles. 50\u2013150 ng of genomic DNA or cDNA templates were used for PCR reactions. For semi-quantitative RT-PCR usingCHLI1 andCHLI2 gene specific primers, 3 \u03bcg of total RNA was converted into cDNA and then 150 ng of cDNA templates were used for RT-PCR. Sequences of primers used for genomic and RT-PCR are shown inPrimers were designed based on genomic DNA sequences available in theEcoR1 andNde1  according to the protocol given in the technical manual. TheCHLI1 cDNA template was amplified using primers given inNdeI andEcoR1 digested)CHLI1 cDNA and theNdeI/EcoRI double-digested pDBle vector was done using the T4 ligase and 1 mM ATP . Chemically competent (CaCl2 treated)E. coli cells were used for transformation. After transformation,E. coli cells were plated on LB+ampicillin (100 \u00b5g/ml) plates and incubated at 37\u00b0C overnight. Single colonies were picked the next day and plasmids were isolated from these clones. Isolated plasmids were double-digested withEcoR1 andNde1 to verify the cloning of theCHLI1 cDNA. TheCHLI1-pDBle construct from the selected clone was sequenced by the UC, Berkeley DNA Sequencing Facility . Chromas Lite (http://technelysium.com.au/) andBLAST were used to analyze DNA sequences.The pDBle vector  was double-digested with restriction enzymeschli1-1 was performed utilizing the glass bead transformation technique described by Kindleet al. 1989. 2 \u00b5g of the linearizedCHLI1-pDBle was used to complementchli1-1. Transformed cells were plated onto fresh TAP plates containing 15 \u00b5g/ml zeocin (Z) and placed in the dark at 25\u00b0C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potentialchli1-1 rescued transformants. Screening ofchli1-1 rescued transformants was done by visual inspection of green coloration and monitoring growth of light adapted complement strain cells either on TAP in the dark or in the dim light or HS plates under medium light (300 \u03bcmol photons m-2s-1).Complementation of theChlamydomonas cells from different strains grown in TAP in the dark were harvested, washed twice with fresh medium and resuspended in TEN buffer . Protein concentrations of samples were determined by the method of Lowryet al. (1951) with bovine serum albumin as standard. Gel lanes were either loaded with an equal amount of Chl (4 \u03bcg Chl) or with 40 \u03bcg of protein. Resuspended cell suspension was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer  and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smithet al. (1990) using reagents from Fisher . After incubation, the solubilized protein samples were vortexed and spun at a maximum speed of 20,000g in a microcentrifuge for five minutes at 4\u00b0C. The soluble fraction was loaded on a \"any kD\u2122 Mini-PROTEAN\u00ae TGX\u2122 Precast Gel\"  and SDS-PAGE analysis was performed according to Laemmli (1970) using a Page Ruler prestained or unstained protein ladder  at a constant current of 80 V for 2 hours. Gels were stained with colloidal Coomassie Gel code blue stain reagent  for protein visualization.Arabidopsis thaliana CHLI1 mature protein that lacks the predicted transit peptide. This antibody is a gift from Dr. Robert Larkin (Michigan State University). CHLI1 primary antibodies were diluted to a ratio of 1:2,000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase  and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit .Electrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P\u2013PVDF membrane  was carried out for 2 hours at a constant current of 400 mA in the transfer buffer . The CHLI1 polyclonal antibody was raised in rabbit against the full lengthg for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer . Chla andb concentrations were determined by Arnon (1949) equations, with corrections as described by Meliset al. (1987).Cell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer . Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifugation at 10,0005A7 was generated by random insertional mutagenesis of theChlamydomonas reinhardtii wild type strain 4A+ (137c genetic background).5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media  PCR method was used.APHVIII gene. The PCR results confirmed that the 850 bp DNA product contains the 3\u00b4UTR of theAPHVIII gene  is located on chromosome 6.The linearized plasmid pBC1 was used to generate5A7 . To findII gene . Sequenc asUP6 .UP6 (CrUP6 locus with its eight neighboring genesUP4 (Cre06.g306100),UP3 (Cre06.g306150),UP1 (Cre06.g306250),UP2 (Cre06.g306200)CHLI1 (Cre06.g306300),FDX3 (Cre06.g306350),AMT (g7098) andUP5 (Cre06.g306450). It is to be noted that we have named all of these genes arbitrarily for our study except for theCHLI1 andFDX3 genes, which were annotated in theChlamydomonas genome database. Readers are requested to identify these unknown genes by the gene locus number (Cre or g number) in thePhytozome database. PCR analyses with the genomic DNA of 4A+ and5A7 were performed using primers specific to four neighboring genes upstream of theCHLI1 (includingUP6) and four neighboring genes downstream of theCHLI1 locus  are not known. We do not yet know the exact location of the pUC origin (pUC ori) end of the plasmid ;chli1-1 and 4A+.chli1-1 rescued transformants are able to synthesize Chl, are not light sensitive and are capable of photosynthesis (chli1-1 rescued transformants harbor theBle gene (from the pDBle vector) andAPHVIII gene , they can grow both on zeocin and paromomycin media plates unlikechli1-1 and 4A+  that can be loaded in a mini protein gel, was used .chli1-1 rescued transformants with a CHLI1 antibody show that the CHLI1 protein is absent in thechli1-1 mutant but present in thechli1-1 rescued transformants (Arabidopsis CHLI1 antibody detects both the CHLI1 (40 kDa) and CHLI2 (42 kDa) protein inChlamydomonas as theChlamydomonas CHLI2 has about 62% sequence identity to theArabidopsis CHLI1  analyses of steady state tetrapyrrole intermediates will confirm this hypothesis. mutant . Over-acchli1-1 mutant has a deletion of at least nine genes (including theCHLI1 gene). Currently we are investigating the exact insertion point of the pUC ori end of the plasmid in thechli1-1 genome  Is the low abundance of the CHLI2 protein a general effect or is it a specific effect of thechli1-1 due to the specific absence of CHLI1 or due to the absence of both CHLI1 and the near absence of CHLI2 protein?2) Is the total absence of Chl in strainchli1-1 with antibodies raised against any non-photosynthetic and/or photosynthetic protein. If the low abundance of the CHLI2 protein is due to a general effect of the mutation, there will be an overall reduction of different cellular proteins. The second question can be addressed by overexpressing CHLI2 inchli1-1 to see if Chl biosynthesis can occur in the absence of CHLI1 or by silencing CHLI2 in the wild type strain using RNA interference or micro RNA based techniques.The first question can be addressed by performing Western analyses of. InArabidopsis MgPPIX is hypothesized to be a retrograde signal from the chloroplast to the nucleus on the basis of data obtained with mutants that are defective in the NF, induced down-regulation of the transcription of the light harvesting complex protein B(LHCB) expression [gun (genomes uncoupled) phenotype]. InArabidopsis, there are controversies regarding whetherchli1 mutants aregun mutants. To date inArabidopsis, MgPPIX mediated regulation of genes encoding only photosynthetic or chloroplastic proteins, have been documented. InChlamydomonas, hemin and MgPPIX has been shown to induce global changes in nuclear gene expression inChlamydomonas, unlike that inArabidopsis. InChlamydomonas, the above mentioned tetrapyrroles altered expressions of genes encoding TCA cycle enzymes, heme binding proteins and stress response proteins as well as proteins involved in protein folding and degradation (eg. heat shock proteins). Hence the roles of tetrapyrroles in retrograde signaling appears to be distinct in green algae and in higher plants. In summary, in the future ourchli1-1 mutant can be used to clarify the functional role of CHLI1 and CHLI2 in Chl biosynthesis inC. reinhardtii.Norflurazon (NF) causes photo-oxidative damage to the chloroplast by inhibiting carotenoid biosynthesis. I no longer have any comment nor suggestion. The authors have addressed all my queries and have incorporated my suggested recommendations.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this version of the manuscript, the authors have made all of the suggested revisions that I requested. I have no further requests from the authors.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. In this manuscript, the authors identified a Chl deficient mutant ofChlamydomonas reinhardtii by insertional mutagenesis. I have reviewed the manuscript by GrovensteinArabidopsis possesses two CHLI isoforms, CHLI1 and CHLI2, likeChlamydomonas. However, it is not clear whether these isoforms are paralogous or not among these two photosynthetic organisms. The authors should perform phylogenetic analysis to show whether these isoforms are truly phylogenetically related or not.As described in the manuscript,chli1-1 mutant is Chl deficient, it is reasonable to assume that CHLI2 is not functional inChlamydomonas. In my understanding, the expression of CHLI2 is not affected by the mutation ofCHLI1 gene inArabidopsis. The upper band of Fig.11B could be a non-specific band of CHLI1 protein that hides the original band of CHLI2 in Western blot analysis. In this sense, the expression analysis of CHLI2, as well as functional complementation withCHLI2 in the chli1-1 mutant, are apparently necessary for further understanding.Considering theSpecific comments: CHLI1 andCHLI2. As the authors discussed quantities of these transcripts in the Discussion section (p.10), it is necessary to show the levels of housekeeping genes such as ACTIN8 orUbiquitin10 as loading control.In Fig. 7, the authors performed semi-quantitative RT-PCR analysis ofReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors did incorporate the suggestions from version 1 into version 2 of the article.\u00a0 On my end,\u00a0 version 2 is already approved with no more revisions required.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis. Using molecular biology techniques they have provided sufficient evidence that the novel mutant is missing a CHLI1 gene and having an intact CHLI2 gene. Their results also provided evidence that CHLI1 inC. reinhardtii is important in chlorophyll biosynthesis and that the presence of its homolog CHLI2 is not sufficient to make up for the function of CHLI1 in chlorophyll biosynthesis. The growth phenotype analysis of the rescued transformants has shown clearly the role of CHLI1 in chlorophyll biosynthesis. It would be interesting to know what percentage of rescued transformants was in their complementation analysis.\u00a0\u00a0 Overall this is an interesting article. More importantly, this work reports the firstchli1 mutant to be identified inC. reinhardtii, as well as in green algae. Thechli1 mutant will open up new avenues to further explore the functional roles of CHLI1 and CHLI2 in chlorophyll biosynthesis in particular inC. reinhardtii.The title is appropriate for the content of the article. In addition, the abstract represents a suitable summary of this interesting work. The authors were able to successfully isolate a novelSuggested revisions:5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media .\u201d *Figure 1 should be Figure 2. On page 6, sentence 3 \u201cIt is incapable of photosynthesis and is sensitive to light intensities higher than 20 \u03bcmol photons m-2s-1 ,\u201d ---*can be more clearly referred to as Figure 2A instead of Figure 2 only. \u00a0 On page 8, Figures 5A, 5B and 5C mentioned in text actually refers to Figures 6A, 6B and 6C.On page 6, sentence 2 \u201cReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas mutant deficient in chlorophyll biosynthesis using insertional mutagenesis. Through molecular genetic analyses, the authors demonstrated that the deficiency of theCHLI1 gene, which encodes a subunit of Mg-chelatase, caused the chlorophyll-deficient phenotype. AlthoughChlamydomonas has another homologousCHLI2 gene, this mutant failed to accumulate chlorophylls, suggesting the limited function of CHLI2 on chlorophyll biosynthesis. To clarify whether the reduced expression ofCHLI2 caused chlorophyll deficiency, overexpression ofCHLI2 in the mutant should give further insights into the functionality of CHLI2. In addition, it is desirable to determine the levels of chlorophyll intermediates that are correlated to the photosensitivity of the mutant. In this paper, the authors have successfully identified aSuggested revisions:Chlamydomonas andArabidopsis, I am not sure whether this mutant is useful for analysis of the retrograde signaling. Actually, a number of previous papers suggested distinct roles of tetrapyrroles on nuclear gene expression inChlamydomonas . In this sense, the last paragraph of the Discussion section should be largely revised including already known mechanisms inChlamydomonas cells and not be so focused on Arabidopsis.Considering the distinct regulation of chlorophyll biosynthesis and nuclear gene expression betweenReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas reinhardtii deficient in chlorophyll biosynthesis. In this paper, the authors describe the isolation and characterization of a mutant ofCHLI1 gene encodes a subunit of the enzyme responsible for inserting Mg2+ into protoporphyrin IX. Interestingly, the chli1 mutant also showed decreased expression of a homolog of CHLI1 and CHLI2. The authors also rescued the chli1 mutant phenotype using a wildtype CHLI1 cDNA transgene. The authors screened a population of Chalamydomonas that was transformed with an insertional mutagenesis vector. They found a chlorophyll deficient mutant that only grew under heterotrophic conditions. The authors provided convincing evidence that this mutant lacked the CHLI1 gene as well as several flanking genes. Thechli1 mutant from a green alga. This mutant provides a starting point for additional genetic and biochemical analyses of chlorophyll biosynthesis inChlamydomonas. \u00a0This work is important because it is the first report of aSuggested revisions:chli1-1, since it is the first mutant allele isolated. This sets a precedent for naming new alleles as they are discovered. The authors should change their mutant name toInstead of calling the rescued transformants that contain the wildtype CHLI1 cDNA \"complements\", the authors should consider calling them \"rescued transformants\". Some geneticists adhere to a more strict use of the term \"complementation\" and its derivatives where complementation is done by crossing two mutants, and not by genetic transformation of a mutant using a wildtype gene.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard."}
+{"text": "The sterilizing activity of the regimen used to treat multidrug resistant tuberculosis (MDR TB) has not been studied in a mouse model.Mycobacterium tuberculosis (TB) strain H37Rv, treated with second-line drug combinations with or without the diarylquinoline TMC207, and then followed without treatment for 3 more months to determine relapse rates (modified Cornell model).Swiss mice were intravenously inoculated with 6 log10 of Bactericidal efficacy was assessed by quantitative lung colony-forming unit (CFU) counts. Sterilizing efficacy was assessed by measuring bacteriological relapse rates 3 months after the end of treatment.The relapse rate observed after 12 months treatment with the WHO recommended MDR TB regimen  was equivalent to the relapse rate observed after 6 months treatment with the recommended drug susceptible TB regimen . When TMC207 was added to this MDR TB regimen, the treatment duration needed to reach the same relapse rate dropped to 6 months. A similar relapse rate was also obtained with a 6-month completely oral regimen including TMC207, moxifloxacin and pyrazinamide but excluding both amikacin and ethionamide.In this murine model the duration of the WHO MDR TB treatment could be reduced to 12 months instead of the recommended 18\u201324 months. The inclusion of TMC207 in the WHO MDR TB treatment regimen has the potential to further shorten the treatment duration and at the same time to simplify treatment by eliminating the need to include an injectable aminoglycoside. As the leading cause of death from curable infectious diseases worldwide, tuberculosis (TB) is a serious global health issue Mycobacterium tuberculosis (M.tb) is very low (0.06 \u00b5g/ml). In mice intravenously (IV) inoculated with M.tb, TMC207 combined with first-line anti-TB drugs shortened the treatment duration from 6 to 4 months TMC207 (R207910) is a diarylquinoline with a new mechanism of action (inhibition of ATP synthase), and is in clinical development for treatment of both drug susceptible (DS) and MDR TB. Its minimal inhibitory concentration (MIC) against The laboratory has been approved on July 27th, 2004 to carry out animal experiments. Nicolas Veziris who carried the animal experiments has the following license number: 75-1531. We followed the animal experiment guidelines of the Facult\u00e9 de M\u00e9decine Pierre-et-Marie Curie.TMC207 was provided by Johnson & Johnson , while the other compounds were purchased from the following manufactures: isoniazid (H) from Laphal , rifampin (R), pyrazinamide (Z) from Aventis , moxifloxacin (M) from Bayer , amikacin from Bristol-Myers Squibb , and ethionamide from Sigma (France).Mycobacterium bovis BCG and was further diluted with normal saline to obtain a 0.2 mg/ml suspension for mouse inoculation.The H37Rv strain of M.tb was grown on L\u00f6wenstein-Jensen medium (LJ slants). Colonies were subcultured in Dubos broth  for 7 days at 37\u00b0C. The turbidity of resulting suspension was adjusted with normal saline to match that of standard 1 mg/ml suspension of 6 colony forming units (CFU) of M.tb H37Rv.Four hundred seventy female four-week-old Swiss mice were purchased from the Janvier Breeding Center . They were intravenously infected in the tail vein with 0.5 ml of bacterial suspension containing approximately 1.1\u00d710Mice were randomized into ten groups . NegativTreatment began 19 days after intravenous infection (D0) i.e when a large bacterial population had developed. All groups were treated 5 days per week. Drug suspensions were prepared weekly and kept at 4\u00b0C except for TMC207 which was prepared monthly in 20% hydroxypropyl-B-cyclodextrin and kept at 4\u00b0C. Amikacin was given by subcutaneous injection at 150 mg/kg as previously described To assess the extent of M.tb infection and to provide baseline values before initiation of chemotherapy, 10 untreated control mice were sacrificed at day 1 and 20 at day 19 after infection respectively (i.e. day \u221218 and day 0 in relation to the initiation of treatment). Treated mice were sacrificed either at the end of treatment to assess the bactericidal activity or 3 months after the end of treatment to assess the relapse rate (sterilizing activity) .The severity of infection and the effectiveness of the treatments were assessed by survival rate, spleen weight, gross lung lesions  and lung CFU counts.The number of CFU in the lungs was determined by plating homogenized lung suspensions in triplicate on Lowenstein-Jensen (LJ) slants, either diluted for untreated control mice or undiluted for treated mice. In the latter case the whole suspension (3.5 ml) was plated on 15 LJ slants. The CFU count was assessed after 6 weeks of incubation at 37\u00b0C Mean spleen weights were compared using the Student's t test. Proportion of positive mice 3 months after the end of treatment were compared using the Fisher's exact test (FET) to compare relapse rates since samples were small (less than 30 mice).Fify-two out of 440 mice died during the 15 months experiment. All of the 10 untreated mice died between 18 and 94 days after infection. Deaths among treated mice during the first month were assumed to be due to tuberculosis as mice appeared to be severely ill and had continuous weight loss after becoming infected whereas death occurring after the first month were due to gavage or injection accidents . In group 2, 2 mice out of 39 died because of gavage accidents after the first month, in group 3, 2 mice out of 30 died (1 during first month and 1 afterwards), in group 4, 18 mice out of 90 died (6 during the first month and 11 because of gavage or injection accident afterwards), in group 5, 6 mice out of 90 died (5 during first month and one because of a gavage accident), in group 6, 4 mice out of 30 died (3 during first month and one because of gavage or injection accident), in group 7, no mice died, in group 8, 2 mice out of 30 died in the first month, in group 9, 3 mice died (2 during first month and one because of gavage accident) and in group 10, 6 mice out of 30 died (2 during the first month and 4 afterwards).The day after infection (D-18), the mean spleen weight was 115\u00b130 mg, while 18 days later, at the start of treatment (D0), the mean spleen weight had increased to reach 608\u00b1144 mg. During treatment, mean spleen weights decreased significantly in all the groups (p<0.005), compared to the mean spleen weight on D0 (results not shown).The number of gross lung lesions increased from 0 the day after inoculation to ++18 days later at the start of treatment. After three and four months of treatment, the number of lesions decreased progressively. There was no difference in gross lung lesions among treated groups (results not shown).The day after infection, the mean lung CFU count was 5.6 log10. On D0, the mean lung CFU had increased to 7.1 log 10.All mice were culture negative at the end of treatment in all treatment groups except 1 mouse in the JZM group after 4 months (3 colonies), 1 mouse in the AEtMZ group after 9 month (2 colonies) and 1 mouse in the AEtMZ group after 12 months (1 colony).The proportion of mice that relapsed (i.e. became culture positive) 3 months after the end of treatment was determined for each combination .In the group receiving 2 months of RHZ followed by 4 months of RH, 3 out of 28 mice (11%) relapsed with a mean 1.8 log CFU. In the group receiving 2 months of JRZ followed by 2 months of JR, 3 out of 19 mice (16%) relapsed with a mean 2.5 log10 CFU. Among mice treated with the MDR WHO regimen, i.e. 2 months of AEtMZ followed by 4, 7 or 10 months of EtM, the relapse rates were 11/19 (58%) with a mean 2.4 log10 CFU, 8/16 (50%) with a mean 2.0 log10 CFU and 4/18 (22%) with a mean 0.9 log10 CFU. In the group receiving 2 months of JZM followed by 2, 4 or 7 months of JM, relapse rates were 8/20 (40%) with a mean 1.7 log10 CFU, 2/19 (11%) with a mean 0.3 log10 CFU and 5/20 (25%) with a mean 2.2 log10 CFU. Five out of 18 mice (28%) that received WHO MDR regimen combined with J, i.e. 2 months of JAEtMZ followed by 4 months of JEtM, relapsed with a mean 1.5 log10 CFU. Nine out of 20 mice (45%) that received the WHO MDR regimen in which J replaced M, i.e. 2 months of JAEtZ followed by 4 months of JEt, relapsed with a mean 2.3 log10 CFU. Ten out of 20 mice (50%) that received the WHO MDR regimen in which J replaced Z, i.e. 2 months of JAEtM followed by 4 months of JEtM, relapsed with a mean 1.8 log10 CFU. And finally, nine out of 20 mice (45%) that received the 6 months JM combination relapsed with a mean 1.8 log10 CFU and four out of 18 mice (22%) that received the 6 months JZ combination relapsed with a mean 0.9 log10 CFU.Compared to the standard WHO DS regimen (group 2), the relapse rate of the WHO MDR regimen  was significantly worse after 6 and 9 months (p\u200a=\u200a0.0009 and 0.009 respectively) but not after 12 months (p\u200a=\u200a1). When J was added to the WHO MDR regimen (group 6) the relapse after 6 months became equivalent to that of RHZ (p\u200a=\u200a0.23). When M or Z were removed (groups 7 and 8) from this regimen the relapse rate increased again and became significantly worse compared to that of RHZ .Compared to the standard WHO regimen for DS TB , the relapse rate of regimen JZM was significantly worse after 4 months (p\u200a=\u200a0.03) but was equivalent after 6 and 9 months . Even the regimen combining only J and Z (group 10) for 6 months achieved an acceptable relapse rate in this experiment.We have previously shown that the MDR TB regimen combining amikacin, moxifloxacin, ethionamide and pyrazinamide has the highest bactericidal activity in the mouse model, resulting in culture conversion after 9 months of treatment Several factors complicate the extrapolation of mouse data to the human situation. Firstly, the pathology of tuberculosis in mice is different from humans, the spread of the bacilli is haematogenous rather than bronchogenous, tuberculosis develops much faster than in humans (mice die in less than 2 months), and mice do not develop lung cavities Secondly, some dosages used here may not exactly mimic those used in humans. E.g. the 150 mg/kg dose of amikacin generates a Cmax in mice that is approximately four times higher than the one obtained in patients taking a 15 mg/kg dose. On the other hand, the intracellular penetration of aminoglycosides is known to be low and their efficacy is therefore expected to be better in patients with mainly extracellular TB replication compared to mice with mainly intracellularly replication The third limitation is the statistical power of the study. To achieve a power of 80% to detect superiority between regimens, mouse relapse studies with 20 animals per group require an absolute difference of 40% in relapse rates between regimens The last limitation is that MDR TB isolates are by definition resistant to isoniazid and rifampin, but usually have also acquired additional resistance mutations. In this context, the impact of resistance to pyrazinamide on minimal treatment durations may be a very important one. Acquired pyrazinamide resistance is very common in MDR TB patients, and the data summarized in The second aim of this study was to assess the contribution of the diarylquinoline TMC207 to the sterilizing activity of MDR regimens. TMC207 has been shown to improve both the bactericidal and the sterilizing activity of the first line regimen RHZ The slight increase in relapse rate obtained by extending treatment with JM from 6 to 9 months (groups 5B and 5C) is puzzling. Such increase could be due to the fact that too few mice were studied per group (as discussed above in the limitations of the study), to the development of resistance, or to the possibility that the combination of TMC207+M is not active against bacilli persisting after 6 months of treatment. The emergence of resistance was excluded by testing the sensitivity of strains isolated after 9 months. The combination of TMC207+M might indeed not be sufficient to kill all persistent bacilli, but is very efficient if Z is added, and not stopped after 2 months. Indeed, it was recently reported that the regimen JZM resulted in acceptable relapse rates after just 5 months, and complete bacteriological cure (0% relapses) after 6 months of treatment The control group receiving the DS TB regimen JRZ achieved a relapse rate equivalent to that of the 6 months standard WHO regimen RHZ after 4 months. This confirms an earlier study In conclusion, we showed that treatment of mice with a moxifloxacin+pyrazinamide based regimen, as recommended by WHO for the treatment of MDR TB, results in stable cure in 12 months. An entirely oral regimen combining TMC207, moxifloxacin and pyrazinamide could reduce the duration needed to produce stable cure to only 6 months. These treatment duration estimates need to confirmed in clinical trials. Preliminary results of an ongoing clinical trial with TMC207 against MDR TB are encouraging"}
+{"text": "Serological analysis revealed that compared to wild-type littermates (+/YHmgn5), mice with a targeted mutation in the HMGN5 gene (tm1/YHmgn5), had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between tm1/YHmgn5 and their +/YHmgn5 littermates. tm1/YHmgn5 mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), was significantly decreased in tm1/YHmgn5 mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of tm1/YHmgn5 mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.High mobility group nucleosome-binding protein 5 (HMGN5) is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5  High mobility group (HMGN) proteins are ubiquitously expressed in vertebrate cells and are known to affect both chromatin structure and the levels of post-translational modifications to histone tails; two important epigenetic processes involved in the regulation of gene expression tm1/tm1Hmgn3 mice develop glucose intolerance due to disruptions in insulin release tm1/tm1Hmgn1 mice are deficient in DNA repair and also display behavioral abnormalities Genome-wide analysis revealed that the HMGN1 variant binds preferentially to regulatory elements in the genome, such as DNase hypersensitive sites and gene promoters HMGN5 is the most recently discovered member of the HMGN family in vivo were determined through the use of a genetically engineered mouse that carries a targeted disruption in the nucleosome binding region of the protein. Evaluation of blood chemistries of these mice +/yHmgn5 and tm1/yHmgn5 littermates revealed alterations in the expression of glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), two enzymes known to be involved in glutathione metabolism In this study, the biological consequences of the functional loss of HMGN5 tm1 denotes \u201ctargeted mutation #1\u201d. The Hmgn5 gene is located on chromosome X therefore male Hmgn5y/tm1 do not contain an untargeted allele. The targeting vector for generating the conditional to Hmgn5 tm1/tm1 mice was constructed by a recombinogenic cloning strategy Hmgn5 gene was retrieved from the BAC clone into the targeting vector PL253 by recombination in the DY380 bacteria strain. The neo gene with the phosphoglycerate kinase 1 promoter (pGKneo) was employed as a positive selectable marker and the pGK-thymidine kinase cassette was used as a negative selectable marker loxP/Frt-flanked positive selectable marker and the loxP site for conditional deletion of the HMGN5 exons were inserted as described in Hmgn5 gene injected into C57BL/6 blastocysts generated chimeras that transmitted the mutated allele to progeny FLP-mice, and the genomic fragment containing exons II, III, and IV of the Hmgn5 gene was removed by crossing with EIIA-Cre mice. The mice containing the targeted allele were backcrossed into the C57BL/6 background for at least 5 generations. HNGN5 knockout mice were designated tm1/YHmgn5 and their wild-type littermates denoted a +/YHmgn5. Mice were bred in a specific, pathogen-free facility with food and water ad libitum.The nomenclature of the genetically altered mice confirms to the nomenclature recommended by the mouse genome nomenclature committee and is used by the Jackson laboratory. The superscript The immunofluorescence assay was performed as previously described ad libitum. Standard 12-h light/dark cycles were used. Wild type and tm1/YHmgn5 male mice were placed in metabolic cages  for 24 h to collect urine samples on three separate occasions to acclimatize mice, separated by at least 24 h in a traditional cage. At 10\u201312 weeks of age (n\u200a=\u200a13), mice were killed by CO2 asphyxiation, and tissues were harvested and frozen in liquid N2. All animal studies were approved by the National Cancer Institute Animal Care and Use Committee.Food (NIH31 standard chow) and water was provided 18 column  using an ACQUITY UPLC system (Waters Corp.) with a gradient mobile phase comprising 0.1% formic acid and acetonitrile containing 0.1% formic acid. A 0.5 ml/min flow rate was maintained in a 10-min run. The eluent was introduced directly into a Waters Q-TOF Premier mass spectrometer by electrospray ionization operating in either positive (ESI+) or negative (ESI\u2212) ionization mode. For mass spectrometry scanning, the data were acquired in the centroid mode from 50\u2013850 m/z. To confirm the identity of markers, authentic standards were compared with urine samples for retention time and tandem mass spectrometry fragmentation pattern when collision energies ranging from 15\u201335 V were applied.Urine (1\u22365 dilution) was collected and diluted with 62.5% acetonitrile containing 0.5 \u00b5M of the internal standard chlorpropamide and centrifuged at 18,000 g for 20 min at 4\u00b0C to remove precipitated protein and other particulates, and the supernatant was transferred to an autosampler vial. Liver tissue samples were homogenized in a solvent comprised of 50% acetonitrile and HPLC grade water containing 0.5 \u00b5M chlorpropamide as the internal standard. Following homogenization, liver samples were agitated on a shaking platform at 1000 rpm for 20 minutes at 30\u00b0C, centrifuged at 18,000 g for 20 min at 4\u00b0C, and the supernatant was transferred to an autosampler vial. Samples (5 \u00b5l/injection) were subjected to reverse-phase chromatography on a 50\u2212\u00d7 2.1-mm ACQUITY 1.7 \u00b5m BEH Cm/z and retention time. For urine samples, the peak area corresponding to protonated creatinine  was used to normalize the peak areas of other ions in a sample. This procedure minimized differences in analyte concentrations that were due to variations in renal physiology. For liver metabolomics, peak areas were normalized according to tissue weight. Data from ESI+ and ESI\u2212 were combined to generate a data matrix suitable for downstream analysis.The mass chromatographic data were aligned using MarkerLynx software (Waters) to generate a data matrix consisting of peak areas corresponding to a unique Elemental compositions of ions were determined using the METLIN metabolite database established by the Scripps Center for Metabolomics corr values generated by the OPLS loadings scatter S-plot as well as scores group contribution analysis were used to determine those ions that contributed most to the separation between +/YHmgn5 and tm1/YHmgn5 mouse samples.Centroided and integrated chromatographic mass data from 50\u2013850 m/z were processed by MarkerLynx (Waters) to generate a multivariate data matrix. Pareto-scaled MarkerLynx matrices including information on sample identity were analyzed by PCA and OPLS using SIMCA-P+ version 12.0.1 . Ptm1/YHmgn5 mice were diluted (20- to 500-fold) in 50% acetonitrile containing the internal standard chlorpropamide (0.5 \u00b5M) for reversed phase chromatography and alpha-aminopimelic acid (1.0 uM) for HILIC chromatography. The mass spectrometer was operated in MRM mode, and optimal transition energies for each metabolite were monitored using the following m/z transitions: Betaine 118 \u2192 59+; Creatine: 132 \u2192 165+; Creatinine: 114 \u2192 86+; Glutathione: 308 \u2192 179+; Pantothenic Acid: 220 \u2192 116+; Phenylacetylglycine: 192 \u2192 74 \u2212. Each urine metabolite concentration is expressed as micromoles per millimole creatinine. Liver metabolites are expressed as micromoles per milligram tissue weight.Metabolite concentrations were determined using an ACQUITY UPLC system coupled to a Xevo-TQ triple quadrople mass spectrometer (Waters). Chromatography was as described for UPLC-ESI-QTOF-MS analysis for all metabolites with the exception of glutathione, which was performed using a HILIC method using 50\u00d72.1 mm ACQUITY 1.7 um BEH Amide column Gapdh mRNA as the internal control, and statistical analyses were performed using the \u0394Ct values. Primer sequences for gene expression analysis are available on request.The data for total analysis of the transcriptome of livers from wild type and mutant mice is available in the GEO database under accession number GSE39062. For additional verification of selected genes, total RNA was prepared from frozen liver using Trizol reagent  and the RNeasy mini kit  was used for RNA cleanup. cDNA was synthesized from 500 ng total RNA using iScript cDNA synthesis kit . For qPCR analysis, primers crossed exon-exon junctions, and NCBI-BLAST searches confirmed sequence specificity. Fermentas Maxima SYBR Green PCR Master Mix  was used for analysis on an Applied Biosystems  Prism 7900HT system. Relative expression calculated by the \u0394\u0394Ct method using Nuclei from liver samples were isolated as previously described 2 digested with various amounts of DNase I  for 2 min at 37\u00b0C. Digested DNA was incubated overnight with proteinase K  at room temperature, followed by extraction with water-saturated ether. Purified DNA was amplified by qPCR using the ABI Prism 7900HT system. qPCR reactions were performed using SYBR Green master kit (ThermoFisher). The primer sequences used for amplifications are available on request. Two intergenic regions known to be insensitive to DNase I digestion were used as loading controls, and the Ct of the average of both intergenic regions was used as the normalization control (\u0394Ct). The mean Ct of both intergenic regions was subtracted from the Ct value for each amplicon (\u0394\u0394Ct), The values displayed represent the fold difference of DNA recovered relative to the undigested sample (2\u2212\u0394\u0394Ct). For statistical analysis, two way analysis of variance (ANOVA) was used to determine differences between genotypes using the \u0394Ct values.For DNase I digestions, nuclei prepared as described above were washed in buffer A . Samples containing 50 \u00b5g genomic DNA were then diluted in buffer A containing 6 mM CaClHmgn5 gene, located on chromosome X, only the region corresponding to exons II, III, and IV which code for the nucleosomal binding domain of the protein +/yHmgn5 and tm1/YHmgn5 mice, reveal loss of the HMGN5 protein from the nuclei of the genetically altered mice , operating in both positive and negative ionization mode. The mass to charge (m/z) ratio and retention time and the abundance data generated, were subjected to principal components analysis (PCA) and orthogonal projection to latent structures data analysis (OPLS-DA).To determine the mechanism for the mild hepatic dysfunction suggested by the blood chemistry, metabolite changes in liver and urine samples from tm1/yHmgn5 mice from their +/yHmgn5 littermates  in hepatic glutathione and a significant decrease in the urinary phenylacetylglycine (p\u200a=\u200a0.023), a glycine conjugate. In addition, both creatine and betaine, compounds which are synthesized from glycine, were significantly reduced in the tm1/YHmgn5 mice, by 42% and 52% respectively. Urinary pantothenic acid was also reduced compared to wild-type littermates; however, this difference narrowly missed statistical significance (p\u200a=\u200a0.056). In summary, data from the metabolomics screening suggested a defect in glutathione utilization in tm1/YHmgn5 mice, prompting further investigation into the molecular mechanism.Quantification of metabolites was carried out by use of specific ion monitoring with standards and triple-quadrupole mass spectrometry . Hmgn5tm+/YHmgn5 and tm1/YHmgn5 mice, microarray analysis was carried out +/YHmgn5 and tm1/YHmgn5 revealed that loss of HMGN5 altered the expression of 97 genes. Significantly, two of the genes that were most affected were related to glutathione metabolism: glutathione peroxidase 6 (Gpx6), which was the gene with was most down regulated (2\u22124.08) and hexokinase 1 (Hk1) which was down regulated by more than 4 fold (2\u22122.32) in the liver of tm1/YHmgn5 mice. qPCR analysis of RNA obtained from the liver of a new cohort of mice validated the results of the array (Gpx6 is an isoform of a glutathione-dependent enzyme responsible for the neutralization of hydrogen peroxide as well as reducing lipid hydroperoxides Hk1 is an isoform of a pentose phosphate shunt pathway enzyme involved in the generation of NADPH and ultimately the regeneration of reduced glutathione To examine the alterations in gene expression that could lead to the changes in metabolites between the he array . Gpx6 isGpx6 or Hk1 chromatin. Nuclei isolated from the liver of +/Y and Hmgn5tm1/YHmgn5 mice were digested with DNaseI and the amount of undigested DNA in 3 distinct genomic regions of Gpx6 or Hk1 (Gxp6 and amplicons 2 and 3 in Hk1 (Given that HMGN5 can alter chromatin compaction 6 or Hk1 , was qua3 in Hk1 , suggest+/YHmgn5 mice from their mutant tm1/YHmgn5 littermates, while transcriptional analysis and DNase I digestion studies link these changes the altered chromatin structure and expression of Gpx6 and Hk1. Thus, the results provide insights into the biological function of this HMGN variant.This study revealed that loss of HMGN5, a nucleosome binding protein that affects chromatin structure and function, alters the metabolomic profile of liver and urine. Metabolomic analysis of both liver and urine clearly separated the wild-type tm1/YHmgn5 and +/YHmgn5 mice was an elevation in hepatic glutathione concentrations, approximately 41% higher when quantified using triple-quadrupole mass spectrometry. In urine, metabolomic analysis demonstrated that tm1/YHmgn5 mice had significantly decreased concentrations of betaine, creatine, and phenylacetylglycine, all of which are metabolites of the glutathione precursor glycine, a finding that is in agreement with previous results suggesting that glycine is a requisite for maximal glutathione synthesis The most prominent difference between the livers of tm1/yHmgn5 mice did not show any obvious abnormalities in any of these factors we suggest that the observed changes in glutathione levels are due to the altered expression of the Gpx6 and Hk1 genes involved in the utilization and synthesis of glutathione, respectively. Impairments in glutathione utilization by glutathione peroxidases such as GPX6 could account for the mild elevation in the reduced form of glutathione observed. Furthermore, reduced glutathione is regenerated from its oxidized form through an NADPH dependent reaction catalyzed by the enzyme glutathione reductase Hk1 expression could be a consequence of the elevation in glutathione, as sufficient glutathione production could diminish the need to generate NADPH via the pentose phosphate shunt.The level of hepatic glutathione is regulated by a variety of factors such as insulin tm1/YHmgn5 mouse could be due to altered hepatic glutathione metabolism.Deregulation of glutathione metabolism has been implicated in several diseases including liver dysfunction tm1/YHmgn5 mice. Taken together with the previous finding that  tm1/tm1Hmgn1 mice display an impaired ability to repair damaged DNA as well as elevated tumorigenesis tm1/tm1Hmgn3 mice are mildly diabetic In conclusion, this study demonstrates that functional loss of HMGN5 disrupts glutathione metabolism, most likely due to chromatin changes that lead to altered expression of genes encoding two enzymes involved in glutathione utilization and synthesis, resulting in differences in the metabolism of this important thiol. Disruption of glutathione metabolism could lead to the mild hepatic differences between wild type and"}
+{"text": "HEY1-NCOA2 fusion gene was recently detected in 10 of 15 investigated MCs. The fusion probably arises through intrachromosomal rearrangement of chromosome arm 8 q. We report a new case of MC showing a t as the sole karyotypic aberration. Through FISH and whole transcriptome sequencing analysis we found a novel fusion between the IRF2BP2 gene and the transcription factor CDX1 gene arising from the translocation. The IRF2BP2-CDX1 has not formerly been described in human neoplasia. In our hospital\u2019s archives three more cases of MC were found, and we examined them looking for the supposedly more common HEY1-NCOA2 fusion, finding it in all three tumours but not in the case showing t and IRF2BP2-CDX1 gene fusion. This demonstrates that genetic heterogeneity exists in mesenchymal chondrosarcoma.Mesenchymal chondrosarcomas (MCs) account for 3\u201310% of primary chondrosarcomas. The cytogenetic literature includes only ten such tumours with karyotypic information and no specific aberrations have been identified. Using a purely molecular genetic approach a  The classification of sarcomas describes over 50 different histological subtypes Mesenchymal chondrosarcomas (MCs) are rare tumours that account for 3\u201310% of primary chondrosarcomas (HEY1) gene and the nuclear receptor coactivator 2 (NCOA2) gene was detected in 10 out of 15 analysed MCs (67%) According to the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer IRF2BP2) and the caudal type homeobox 1 (CDX1) gene. Based on the recent report by Wang et al (2012) HEY1-NCOA2 gene fusion, finding it in all three.We report the finding of a balanced t as the sole karyotypic abnormality in an MC. The translocation led to a new fusion between the interferon regulatory factor 2 binding protein 2 gene . However, a tumour in the left iliacus muscle was detected, 3 cm in largest diameter. Biopsies revealed a spindle cell tumour of uncertain malignant potential. The patient received chemotherapy for PCNSL according to Abrey et al. The Norwegian Radium Hospital (NRH) is the largest referral centre for Norwegian patients with bone and soft tissue tumours covering a population of 2.6 million. To identify additional patients with a diagnosis of MC, a database search was performed for cases with this disease. Three additional patients (patients 2\u20134) were identified  was retrieved from The Radium Hospital biobank  in 2008). Specific permission to perform RNA analysis/sequencing was obtained from patient 1 after approval by the Regional Ethics Committee for Medical and Health Research Ethics South-East (REC number: 2010/1389A). The entire study was also approved by the institutional review board at the Norwegian Radium Hospital. All data were analyzed anonymously.Fresh tissue from a representative area of the tumour (patient 1) was received and analysed as part of our diagnostic routine. The samples were disaggregated mechanically and enzymatically with collagenase II . The resulting cells were cultured and harvested using standard cytogenetic techniques http://genome.ucsc.edu/). The selected clones (see Fluorescence in situ hybridization (FISH) was performed using probes from bacterial artificial chromosomes (BACs). BACs and fosmid clones flanking and covering the breakpoint positions were selected using the Human Genome Browser at the University of California web site . DNA labelling was done in a nick translation reaction and the synthesized probes were hybridized to previously G-banded slides. All procedures were performed as previously described Representative samples of tumour tissue were frozen and stored at \u221280\u00b0C after surgery. DNA was isolated using Genomic-tip  to obtain pure high molecular weight DNA. RNA was extracted from tumour tissue using the Trizol reagent (Life Technologies) with a homogenizer . The RNA quality was evaluated using the Experion Automated Electrophoresis System . cDNA was synthesized using the iScript kit and random primers (Bio-Rad Laboratories). All procedures were done according to the manufacturers\u2019 recommendations.Sequencing was performed according to the TruSeq paired-end RNA-sequencing protocols from Illumina for Solexa sequencing on a Genome Analyzer IIx with paired end module . 3.5 \u00b5g total RNA was used as starting material for library construction, using the TruSeq RNA Sample Preparation Kit v2 where the steps include poly-A mRNA isolation, fragmentation, and cDNA synthesis before adapters are ligated to the products and amplified to a final cDNA library. Shearing to about 150 bp fragments was achieved using divalent cations under elevated temperature. Approximately 58 million clusters were generated by the TruSeq PE Cluster Kit v2 on the Illumina cBot Cluster Generation System, and 76 base pairs were sequenced, from each side of the fragments, using reagents from the TruSeq SBS Kit v5 .http://genome.ucsc.edu/, accessed May 2012). UniGene clusters were downloaded from National Center for Biotechnology Information  to assist in locating potential gene fusions. Three spanning reads and two split reads were required to call sequence reads a gene fusion.The Illumina software pipeline was used for processing of image data into raw sequencing data (SCS 2.9 and Casava 1.8.2), and only sequence reads marked as \u201cpassed filtering\u201d were used in the downstream data analysis. A total of 91 million reads were obtained. We utilized the fusion discovery software deFuse (version 0.4.3) HEY1- NCOA2 fusion were identical to the primers used by Wang et al www.ncbi.nlm.nih.gov/BLAST/). All cases were tested for expression of a zinc-finger gene suppressor of zeste 12 homolog (Drosophila) (SUZ12) to assess RNA quality.Primers used in PCR were designed with the FastPCR software PCR experiments on genomic DNA were performed using \u223c100 ng DNA as template in 25 \u00b5l PCR reactions using TaKaRa LA Taq following the manufacturer\u2019s recommendations for LD-PCR: 30 cycles of 98\u00b0C for 10 sec (denaturation) followed by 68\u00b0C for 15 min . PCR products were purified using GeneJET PCR purification kit  and sent for Sanger sequencing (GATC Biotech).The cytogenetic analysis of the only tumour (patient 1) from which we got a fresh sample revealed a balanced t as the sole abnormality in all cells analysed . AnalysiHEY1-NCOA2 fusion which was recently identified in 10 of 15 investigated MCs HEY1 exon 4 and NCOA2 exon 13 in cases 2\u20134, identical to the one previously described PDGFR\u03b2) and the large intron 1 of CDX1.All cases  as well as the three archival tumours) were tested for the IRF2BP2-CDX1 transcript involving two coding regions yielded the highest split count value (number of split reads supporting the fusion) of all the predicted fusions and was predicted to be in-frame. We therefore chose to focus on this putative fusion. cDNA PCR experiments with specific primers were run to validate the IRF2BP2-CDX1 fusion. Two distinct bands were identified using the primer IRF2BP2-895F, located in exon 1, in combination with CDX1-771R, located in exon 3 , whereas on chromosome 5 it was in the large intron 1 of CDX1 (chr5\u2236149551799 bp).Next, we wanted to identify the genomic breakpoints of the 1-26970R  we managIRF2BP2 gene also by FISH, we hybridized BAC clones overlapping IRF2BP2 to metaphases obtained from the cultured cells , confirming that this fusion gene is common in MC. In a majority of the well-known translocation-related sarcomas such as myxoid liposarcoma and low-grade fibromyxoid sarcoma, more than one defining fusion variant has been detected. Often one fusion variant is more common than the others IRF2BP2-CDX1 could be such an additional fusion variant identified in a subset of MCs, but only analysis of larger series of tumours can determine the prevalence of the IRF2BP2-CDX1.MCs are rare, and at our institution only five patients (four included in this study) received this diagnosis during the last 25 years. We identified the HEY1-NCOA2-positive MCs all had tumour manifestations detected in bone, whereas the MC showing the t and IRF2BP2-CDX1 fusion originated from soft tissue. Although most common in bone, one-fifth to one-third of MCs do arise in soft tissue HEY1-NCOA2 was first described Of possible interest is the fact that the three Table S1BAC probes used for FISH experiments in case 1.(XLS)Click here for additional data file.Table S2List of fusions suggested by the deFuse algorithm.(XLS)Click here for additional data file."}
+{"text": "ARMS2/HTRA1 genes at the 10q26 locus have been associated with risk of age-related macular degeneration (AMD), with the most significantly associated variants being A69S (rs10490924), del443ins54 (EU427539) and rs11200638. We wished to explore the association of the del443ins54 in two ethnically different populations from India and Australia.The The del443ins54 was screened in a large cohort of ~1500 subjects from these two populations by a combination of PCR-based agarose gel electrophoresis and validated by resequencing. Statistical analysis comprised the calculations of allele, genotype and haplotype frequencies along with their p values and corresponding odds ratios (OR), and 95% confidence intervals (95% CI) and measures of linkage disequilibrium (LD).\u221213; OR=2.80, 95%CI, 2.12\u20133.70) and Australian cohorts . These associations were similar to those previously identified for the A69S and the rs11200638 variant in these populations that also exhibited high degrees of LD (D\u2019 of 0.87-0.99). A major risk haplotype of \u201cT-indel-A\u201d  and a protective haplotype of \u201cG-wild type-G\u201d  were identified in the Indian and Australian cohorts, respectively.The del443ins54 was significantly associated with AMD in both the Indian (p=1.74\u00d710These data provide an independent replication of the association of del443ins54 variant in two different ethnicities, despite differences in allele and haplotype frequencies between them. High levels of LD in both populations limit further genetic dissection of this region in AMD. ARMS2 (rs10490924) and HTRA1 (rs11200638) in multiple populations worldwide [EU427539) that affects the stability of ARMS2 mRNA by the removal of a polyadenylation signal (443 bases) and insertion of a 54bp AU rich element in the 3\u2032-UTR (del443ins54), has also been identified in the 3\u2032 end of the ARMS2 gene as increasing risk of AMD by several fold in both Caucasian [\u22125 to p=8.4\u00d710\u221234) in individuals of European origin [Age-related macular degeneration (AMD) is a complex multifactorial disease and a leading cause of irreversible blindness in the world . A majorARMS2 rs0490924 a1 rs1120038 in mulARMS2 rs490924 anrs10490924) and rs11200638 variants with AMD in both South Indian and Australian cohorts [rs11200638 variants with AMD susceptibility in two ethnically different cohorts from Southern India and Australia.We have previously reported a significant association of the A69S  and normal controls from cohorts in India (n=433) and Australia (n=1054). The detailed methods of clinical diagnosis along with the inclusion and exclusion criteria have been previously reported ,12. AmplPLINK software [rs11200638 variants and the estimated haplotype frequencies and linkage disequilibrium (LD) were assessed with the Haploview software (version 4.2) that uses the EM algorithm [Allele and genotype frequencies were determined by the gene counting method and estimates of Hardy\u2013Weinberg equilibrium were assessed. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated to assess the risk conferred for each variant using the software . Haplotylgorithm .rs11200638 genetic variants. Each variant was in Hardy\u2013Weinberg equilibrium in both the Indian and Australian cohorts (p>0.05). Allele frequencies for each of the risk variants  exhibited a relatively higher frequency (0.60\u20130.63) in the Indian cohort compared to the European cohorts (0.36\u20130.53) but lesser than that reported in Han Chinese (0.73\u20130.77) or the in the Japanese (0.86\u20130.88) populations  and Australian , cohorts (ARMS2) and the rs11200638 (HTRA1) variants in both the Indian and Australian cohorts, respectively  or Australian cohorts .Homozygosity of the indel and the other variants were strongly associated with an increased risk of AMD in both the Indian and Australian cohorts. Combined homozygosities at the A69S and the rs11200638 variants were remarkably high across this 10q26 region with relatively higher values in the Australian  compared to the Indian  cohorts  between the A69S, del443ins54 and  cohorts . Two maj cohorts . Differewo genes .ARMS2 del443ins54 variant in two cohorts, and to the best of our knowledge for the first time among South Indians with AMD. The strong association of the del443ins54 along with the A69S and rs11200638 variants in the Indian and Australian cohorts were consistent with that observed in other populations [These data provide an independent replication of the association of the ulations . HaplotyARMS2 and HTRA1 genes are in high LD in European populations and thus dissecting out the role of one gene over the other has proved difficult. The advantage of undertaking a comparative analysis of genetic variants in populations of differing ethnicities expands the genetic diversity available and may provide the opportunity of identifying a more defined but associated region for further study. The current study highlighted similar degrees of associations across these three variants despite a relatively lower LD between the A69S, del443ins54 and rs11200638 variants in the Indian compared to the Australian cohort. However, it did confirm the presence of stratification differences between ethnicities with the allele frequency of the Indel of del443ins54 in South Indians being higher at 0.63 in cases compared to that in European populations (0.36\u20130.53) but lower than other Asian populations (0.73\u20130.88). The allele frequency in cases is similar to that previously shown by us in the assessment of the A69S (0.63) and the rs11200638 (0.60) variants of the ARMS2 and HTRA1 genes respectively, in the South Indian cohort. Evidence of population stratification has also been observed in AMD studies of the protective CFHR3\u20131 deletion, with the highest frequencies of the deleted allele being present in African populations (16%\u201320%) compared to Asians (<2%) [The ns (<2%) ,16.ARMS2 and HTRA1 gene in AMD is still unclear but functional dissection of the effect of the rs11200638 promoter variant in the HTRA1 gene has revealed that this variant resides within a putative transcription binding site for the factors AP2\u03b1 and SRF (serum response factor) [HTRA1 expression levels revealed consistently higher levels of expression with the AA genotype compared to the GG genotype [rs11200638 variant have revealed no functional effect on HTRA1 expression [The potential role of the  factor) . Initialgenotype . In contpression .ARMS2 del443ins54 indel found decreased ARMS2 expression and almost 3.0 fold increase in HTRA1 expression [ARMS2 risk del443ins54 results in decrease in mRNA transcription levels of the ARMS2 gene, a non risk associated variant (rs2736911) also leads to significantly reduced ARMS2 transcript levels suggesting that ARMS2 protein deficiency alone is unlikely to be pathogenic in AMD [ARMS2 in mitochondrial homeostasis has also been suggested and the biology concerning mitochondrial dysfunction and the effects on age supports this notion [ARMS2 in retinal epithelial ARPE-19 cells and COS7 transfected cells to the cytosol rather than the mitochondria suggesting that ARMS2 may not confer risk to AMD through the mitochondrial pathway [HTRA1 expression are equivocal and further investigations on the functional role of these variants are required.Analysis of the chromosome 10q26 risk haplotype inclusive of the pression . Interesc in AMD . A functARMS2 and HTRA1 gene in this region will require much larger patient cohorts than have currently been assessed, or through the identification of other ethnic populations which show relatively lower levels of LD over this 10q26 region.In conclusion, we provide convincing evidence for the association of the del443ins54 variant with AMD, despite differences in allele, genotype and haplotype frequencies and LD across the 10q26 region reflecting population stratification differences in two different ethnicities. AMD, a complex multi-factorial disease is associated with multiple genomic regions with varying magnitudes of effect and the relevance of genetic associations differ between populations. Further, elucidation of the genetic basis of this disease through the analysis of individuals from different ethnic groups has the potential to provide useful insights into the genetic diversity of risk and protective variants within a gene as well as their contributions to disease. Also, meaningful genetic dissection of the"}
+{"text": "MFD can be used to study formulations of drugs and active agents in oil, water and emulsions.Molecular Fragment Dynamics (MFD) is a mesoscopic simulation technique based on Dissipative Particle Dynamics (DPD). Whereas DPD beads in general may not necessarily be identified with chemical compounds at all the MFD variant uses specific molecules or molecular fragments as its basic 6E6, C10E6, C12E6 and C16E6 at the water-air interface are performed to study their nanoscale structures and surface properties. The simulations of the self-aggregation of the polyoxyethylene alkyl ether surfactants lead to equilibrium nanoscale structures and computationally determined surface tensions which are in agreement with experimental data for different surfactant concentrations [MFD simulations of the nonionic polyoxyethylene alkyl ether surfactants C"}
+{"text": "KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p\u200a=\u200a0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p\u200a=\u200a0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p\u200a=\u200a0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS  and OS  and KRAS was associated with longer DFS . In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.Mutations in multiple oncogenes including  Endometrial cancer comprises about 4% of cancer in women globally, with higher incidence in developed countries. The American Cancer Society estimates endometrial cancer will be the fourth most common cancer diagnosed and the eighth leading cause of cancer deaths in women in 2010 Considerable effort has gone into developing systems to more effectively identify patients with endometrioid endometrial cancer that carry an elevated risk of recurrence so they can be targeted for adjuvant therapies . Those patients that present with extrauterine disease (stage III/IV) carry a high risk of recurrence and progression. The majority of patients (\u223c80%), however, present with tumors clinically confined to the uterus (stage I/II). In these early stage patients, multiple studies have shown that the risk of recurrence is associated with tumor grade, depth of myometrial invasion, occult extension into the cervix and tumor cell invasion of lymphatic vessels (lymphovascular space invasion: LVSI), where high grade is the most widely accepted adverse prognostic marker Since 1988, the International Federation of Gynecology and Obstetrics (FIGO) has recommended full systematic pelvic and para-aortic lymphadectomy as part of staging for endometrial cancer. A new 2009 FIGO staging system has recently been implemented where tumors with no evidence of myometrial invasion are combined with tumors that show invasion to less than 50% of the myometrium and grouped into stage 1A fibroblast growth factor receptor 2 (FGFR2) mutations in 18/115 (16%) endometrioid endometrial cancers FGFR2, CTNNB1, KRAS and PIK3CA mutation in a large, unselected cohort of endometrioid endometrial cancers and to determine the relationship between mutation status and clinicopathologic variables including outcome. Mutations in PTEN were not included in this analysis due to the increased cost associated with sequencing all 9 exons of this tumor suppressor gene. In addition, the high prevalence of PTEN aberration (70%) argued against a possible association with poor prognosis in this tumor type.FGFR2 has been shown to be activated in a number of cancers due to gene amplification All research subjects provided written consent to ongoing protocols 91-507 and 93-0828, approved by the Washington University's Human Research Protection Office continuing Review Committee. The work performed at TGen was determined to be exempt from IRB approval following review and receipt of a Verification of Protections for Human research subjects form signed by Dr Goodfellow and a copy of the blank consent form.Tumor specimens were prospectively collected at the time of hysterectomy (1991\u20132006) for patients treated by the Division of Gynecologic Oncology at Washington University School of Medicine/Barnes\u2013Jewish Hospital. Surgical staging and tumor grade was assigned on the basis of FIGO 1988. Patients who had received preoperative radiation or chemotherapy were excluded from analysis. The prospectively collected clinical and pathologic information was stored in a computerized database. Following their initial treatment, these patients were routinely followed at 3-month intervals for the first 2 years and then at 6-month intervals for at least 3 years. Disease surveillance included physical examination and periodic pap smears. Diagnostic imaging and directed biopsies were performed as clinically indicated. Histological confirmation of all recurrences was performed. Follow-up data were abstracted from clinic charts, hospital records, and the Siteman Cancer Center/Barnes-Jewish Hospital's cancer registry.Patients for whom follow-up data were unavailable or who died perioperatively (within 30 days of hysterectomy) were excluded from the analyses. The study population comprised 466 patients with endometrioid endometrial cancer, 386 of which had disease confined to the uterus (stage I or II).Tissue specimens and blood were obtained at the time of surgery, snap frozen, and stored at \u221270\u00b0C. Tumors were evaluated to select tissues with >66% neoplastic cellularity for DNA preparations. DNA was isolated using proteinase K and phenol extraction or the DNeasy Tissue Kit . DNA was extracted from peripheral blood leukocytes or, when blood was not available, from uninvolved myometrium, as previously described FGFR2, exon 2 of KRAS, exon 3 of CTNNB1, and exons 9 and 20 of PIK3CA were tested for mutations by direct sequencing. PCR primers and conditions are available upon request FGFR2 and KRAS and CTNNB1. For PIK3CA, rare and novel mutations were confirmed to have arisen somatically and common tumor-associated mutations were confirmed in the majority of samples.Exons 7, 8, 10, 13 and 15 of MSI analysis is routinely performed for all tumors. The MSI status and methods used for the majority of the cases reported here have been previously described p-value of 0.05. Statistical analyses were performed using SAS , as well as the cmprsk R (http://biowww.dfci.harvard.edu/~gray) statistical packages for competing risk analysis.The relationship between gene mutation status and covariates was assessed using Fisher's exact test or Student's t-test as appropriate. Overall survival (OS) was defined as the time from date of surgery to death due to any cause. Survivors were censored at the date of last contact. Disease free survival (DFS) was defined as the time from surgery to recurrence or progression. Patients were excluded if they had died within 30 days of surgery. The Kaplan-Meier product limit method was used to estimate OS and DFS. Univariate and multivariate Cox proportional hazard models were fitted to assess the effects of the covariates on OS and DFS, and the proportional hazard assumptions were checked using scaled Schoenfeld residuals FGFR2 ; KRAS and PIK3CA ; and CTNNB1 . Mutation data for all four genes was obtained for 453 cases (97%).The mean age at diagnosis for the 466 cases analyzed was 63.7 years with a mean follow-up time of 70.2 months (0.7\u2013176). The majority of patients presented with early-stage disease (386 or 83% stage I or II) . MutatioFGFR2 mutations in 48/466 (10.3%) tumors  was excluded from analyses because of uncertainty as to whether the sequence change was functionally significant. The most common mutations were S252W  and N550K . All together, 7 mutations affecting 6 codons  accounted for 90% of the mutations identified (We identified ) tumors , includientified . We idenKRAS in 87/464 (19%) samples, including 115 previously investigated cases We identified mutations at codons 12 and 13 in PIK3CA in a total of 104/464 (22%) cases  cases . The majCTNNB1 in 88/454 (19%) endometrioid tumors  compared to microsatellite stable (MSS) tumors  . Similarly, mutations in FGFR2, were significantly more common in MSI positive tumors  compared to MSS tumors  (p\u200a=\u200a0.016). In contrast, mutations in CTNNB1 were significantly less common in MSI positive tumors  compared to MSS tumors . Mutations in PIK3CA were more common in MSI positive tumors  compared to MSS tumors , although this was not significant (p\u200a=\u200a0.08). 158/466 (34%) of tumors were MSI positive. Mutations in FGFR2 and KRAS mutations would occur in a mutually exclusive pattern. Indeed, only 4/87 (5%) KRAS mutation-positive tumors carried a FGFR2 mutation , whereas 44/377 (12%) KRAS mutation negative tumors carried an FGFR2 mutation . To investigate whether the tumors carrying mutations in both FGFR2 and KRAS were polyclonal, DNA from a different portion of the tumor was extracted from archived paraffin tissue and in all four cases both mutations were confirmed.Based on our understanding of receptor tyrosine kinase-MAPK signaling, and our preliminary analysis of 115 endometrial tumors, we anticipated that KRAS and CTNNB1 demonstrated a similar pattern of mutual exclusivity and rarely occurred together. In the 453 tumors sequenced for both genes, 88 and 85 carried mutations in CTNNB1 and KRAS, respectively. Of those tumors with CTNNB1 mutations, only 5/88 (5.7%) carried KRAS mutations, whereas 80/365 (22%) of the CTNNB1-wildtype tumors carried a KRAS mutation . Given CTNNB1 mutations were significantly more common in MSS tumors, we looked for the relationship between KRAS and CTNNB1 mutations in both MSS and MSI tumors. This association was even stronger in those tumors that demonstrated microsatellite stability where 1/71 (1%) CTNNB1 mutation positive tumors carried a KRAS mutation, whereas 44/230 (19%) of the CTNNB1 wildtype tumors carried a KRAS mutation . In contrast, this association was not present in those tumors with MSI as 4/17 (24%) CTNNB1 mutation positive tumors carried an activating KRAS mutation whereas 36/135 (27%) of the CTNNB1 wildtype tumors carried a KRAS mutation.Perhaps the most surprising finding from this cohort is that mutations in FGFR2 and KRAS, and of CTNNB1 and KRAS, no such pattern was seen for FGFR2 and CTNNB1. Specifically 8/88 (9%) CTNNB1 mutation positive tumors carried an FGFR2 mutation, whereas 40/365 (11%) CTNNB1 wildtype tumors carried an FGFR2 mutation. Within the MSS cohort of tumors, 7/71 (10%) CTNNB1 mutation positive tumors carried an FGFR2 mutation whereas 17/230 (7%) of the CTNNB1 wildtype tumors carried an FGFR2 mutation.Surprisingly, given the near mutual exclusivity of FGFR2, KRAS, PIK3CA mutation and age at diagnosis. CTNNB1 mutations were, however, significantly more common in patients diagnosed before age 60  compared to those diagnosed after age 60  . We chose 60 as our age cutoff based on previous data indicating reduced survival in patients >60 FGFR2 mutations were more common in Caucasian/Asian cases  than African American patients , albeit this was not significant (p\u200a=\u200a0.10). PIK3CA mutations were significantly more common in stage I/II tumors  compared to late stage tumors   ; grade 2, 25/149 (17%); grade 3, 4/62 (6%)  and FGFR2 mutations showed a trend towards an association with grade ; grade 2 17/152 (11%); grade 3, 2/65 (3%) (p\u200a=\u200a0.10)  compared to lower grade tumors 84/392, (21%)  as were FGFR2 mutations ; grade 3, 2/65 (3%) .There was no association between ct test) . CTNNB1 \u200a=\u200a0.10) . As wellMutation status for the four oncogenes investigated was not associated with overall survival (OS) in the total cohort of 466 cases. OS was associated with age >60 (p\u200a=\u200a0.0002), advanced stage (III/IV) (p<0.0001), FIGO tumor grade 2 (p\u200a=\u200a0.0014), FIGO grade 3, p<0.0001) and adjuvant therapy (p<0.0001) . MultivaKRAS mutation was associated with longer disease free survival (DFS)  whereas the mutation status of other genes was not significantly associated with DFS. As expected, DFS was associated with higher stage (III/IV) (p<0.0001), FIGO tumor grade 2 (p\u200a=\u200a0.0019) and 3 (p<0.0001) and adjuvant therapy (p<0.0001) in univariate analysis. Multivariate analysis showed that the presence of a KRAS mutation remained significantly associated with longer DFS   although this finding was of marginal statistical significance (CTNNB1 and PIK3CA mutations had no effect on the multivariate model (data not shown). We did not include adjuvant therapy in the multivariate model as analysis indicated it was not independent of stage and grade.The presence of =\u200a0.048) . When FGificance . When boificance . CTNNB1 FGFR2 mutation positivity and grade 2 differentiation showed a trend towards shorter OS . When FGFR2 mutation was analyzed taking into consideration the effects of known prognostic factors variables, it became more significantly associated with OS  , stage II (p\u200a=\u200a0.007) and high tumor grade (FIGO grade 3) (p<0.0001) . Both FG=\u200a0.025) .FGFR2 mutation (p\u200a=\u200a0.019) were significantly associated with shorter disease free survival (DFS) (KRAS mutation showed a trend towards associating with longer DFS (HR\u200a=\u200a0.26 95% CI 0.06\u20131.11 p\u200a=\u200a0.067) whereas CTNNB1 and PIK3CA mutations were not associated with DFS. When each gene was analyzed alone in multivariate analysis of early stage cancers, FGFR2 mutation status remained a significant factor associated with reduced DFS  (KRAS was significantly associated with longer DFS (HR\u200a=\u200a0.23 CI 0.05\u20130.97 p\u200a=\u200a0.045). When both genes were included in the model, FGFR2 remained significant (HR\u200a=\u200a3.03 CI 1.26\u20137.27 p\u200a=\u200a0.013). Kaplan-Meier survival plots showing the relationship between FGFR2 mutation and DFS and OS in early stage cancers are presented in Univariate analysis revealed only high grade (p\u200a=\u200a0.0005); stage II (p\u200a=\u200a0.009); adjuvant therapy (p\u200a=\u200a0.049) and the presence of an al (DFS) . KRAS mu=\u200a0.008)  and KRASHere we show the patterns of mutations in four endometrial oncogenes in the largest cohort of endometrioid endometrial tumors reported to date (n\u200a=\u200a466). Given the large number of tumors in this single institution Washington University School of Medicine cohort, novel insights have been revealed which have not been evident with smaller subsets of tumors or in some cases where disparate evidence had been reported in smaller panels of tumors CTNNB1 and KRAS mutations in this cohort. Although 5 tumors were identified with mutations in both genes the vast majority of tumors only carried mutations in either KRAS or CTNNB1 (p\u200a=\u200a0.0002). This finding was not a reflection of an association with MSI positive and negative tumors because when we looked in only the MSS tumors, the association was even more significant. Only 1% CTNNB1 mutation positive tumors carried a KRAS mutation whereas 19% of the CTNNB1 wildtype tumors carried a KRAS mutation . In most other cancers, mutual exclusivity of gene activation is observed between two proteins that map to the same signaling pathway, which makes intuitive sense, as activation of the same pathway at two different nodes is redundant. Although KRAS and CTNNB1 have very distinct roles in the MAPK pathway and the Wnt/TCF signaling pathway respectively, recent data suggests novel points of pathway crosstalk in some cell types One finding that may have implications for understanding the biology underlying endometrial cancer is the hereto-unrecognized mutual exclusivity of PIK3CA are not associated with poor prognosis In contrast to a previous study, our data suggest that mutations in exon 20 of FGFR2 mutation rate was 10% (48/466). The 10% mutation rate for this large, unselected series is consistent with the mutation rate reported by Dutt et al.  FGFR2 mutations in endometrial cancers we oversampled for cases that had recurred and tumors with microsatellite instability FGFR2 mutation with both defective DNA repair and recurrence in the current unselected cohort.In this single institution series of endometrioid endometrial cancers, the overall A number of clinical and pathologic prognostic factors have been evaluated in the search for markers to more accurately predict risk of recurrence or death for patients with endometrial carcinoma. Past studies have suggested tumor markers p53, p16, estrogen receptor, progesterone receptor and HER2/neu may have clinical utility in endometrial cancer for predicting lymph node metastasis, prognosis and in directing treatment FGFR2 and KRAS have prognostic significance within the cohort of endometrioid endometrial cancers. Our data suggest that FGFR2 mutations occur more often in the well and moderately differentiated endometrioid tumors  compared to undifferentiated tumors and possibly identify the \u201cbad actors\u201d in an otherwise better prognosis histological subgroup. Recent data in an independent cohort of endometrial tumors reported a similar frequency of mutations across G1\u2013G3 tumors FGFR2 with shorter DFS is more significant in the multivariate analyses where the association of high grade with poor prognosis is accounted for, compared to univariate analysis. These findings strongly suggest that the observed effect of FGFR2 is not simply due to the confounding effects of other known prognostic factors, and underscore the likely functional significance of this gene in determining survival.In this study we have identified that KRAS mutation is associated with longer DFS in the total cohort in both univariate and multivariate analysis. In the subset of early stage cases, KRAS mutation was significantly associated with longer DFS in multivariate analysis after adjusting for grade and stage. We can speculate that the pattern of mutual exclusivity of FGFR2 and KRAS suggests that the role of these two genes in endometrial cancer initiation is likely to be through activation of the MAPK signaling pathway. The fact that they have different and indeed opposing effects on disease free survival leads us to further speculate that activation of \u201cnon-MAPK\u201d pathways downstream of FGFR2 is driving the association of this gene with poor prognosis.A novel finding of this present study is that FGFR2 mutation is an independent prognostic marker in patients with early stage endometrioid endometrial cancer suggests that FGFR2 mutation testing could ultimately prove useful in the management of endometrial cancer. Current National Comprehensive Cancer Network (NCCN) guidelines for endometrioid endometrial cancer confined to the uterus recommends more aggressive adjuvant therapy as tumor grade and tumor stage increases, and also where multiple adverse prognostic indicators are present, including lymphovascular space involvement. We envisage that the mutation status of FGFR2 could be used to inform clinical decision making in a similar way to a poorly differentiated histology. Specifically, the presence of an FGFR2 mutation and absence of a KRAS mutation would stratify a patient as having high-risk disease, resulting in a recommendation for more aggressive therapy  that the patient samples are from a single institution, 2) the frequency of recurrence in early stage endometrioid cases is relatively low in this unselected cohort and 3) we had low number of late stage G1 and G2 tumors in this cohort which may have contributed to lack of statistical significance for FGFR2 in the entire cohort. We are currently sequencing the four exons of FGFR2 containing almost all reported mutations in endometrial cancer samples collected as part of the multi-institutional GOG-210 clinical trial \u201cMolecular Staging of Endometrial Cancer\u201d. This cohort also allows the assessment of FGFR2 mutations on endometrial cancer specific survival as well as overall survival, given the extensive clinical annotation of these samples.FGFR2 mutation testing may identify patients whose tumors will be sensitive to FGFR inhibition FGFR2 mutations as an independent prognostic marker in early stage tumors and the eventual identification of an FGFR inhibitor with clinical activity in patients with metastatic endometrial cancer, holds the promise of utilizing anti-FGFR therapies in an adjuvant setting to reduce the risk of recurrence in patients diagnosed with FGFR2 mutation positive endometrial cancer.Preclinical data suggests that FGFR2 was associated with shorter disease free progression and this was significant in patients diagnosed with early stage disease. This finding has clinical significance in that FGFR2 mutation status could function as a starting point in developing a molecular prognostic risk assessment score that could be used to identify patients that may benefit from more aggressive adjuvant radiation and/or chemotherapy following an initial hysterectomy. In the longer term, anti-FGFR agents could be tested in patients with FGFR2 mutation positive tumors to evaluate whether these agents reduce the frequency of recurrence in the adjuvant setting, in addition to the metastatic setting where they are currently being evaluated. As KRAS mutations were associated with reduced recurrence risk in this cohort, our data would suggest that MEK inhibition may not be effective in an adjuvant setting to prevent recurrence.In conclusion, our mutation analysis of four oncogenes frequently mutated in the endometrioid histology of endometrial cancer revealed that mutated Figure S1Kaplan Meier curves for recurrence/progression free survival (A) and overall survival (B) by FGFR2 mutation status in patients with early stage endometrial cancer.(TIF)Click here for additional data file.Table S1Clinicopathological features of endometrial tumors with FGFR2 mutations.aNumbering relative to NM_022970.2 bNumbering relative to NP_075259.2 cThese mutations have been reported previously (8).(DOC)Click here for additional data file.Table S2KRAS Mutations in Endometrial Tumors.(DOC)Click here for additional data file.Table S3PIK3CA Mutations in Endometrial Tumors.#These mutations are novel and do not appear in Cosmic (May 2011).(DOC)Click here for additional data file.Table S4CTNNB1 Mutations in Endometrial Tumors.(DOC)Click here for additional data file.Table S5Frequency of MSI and mutations, according to FIGO stage.(DOC)Click here for additional data file.Table S6Frequency of MSI and mutations, according to tumor grade.(DOC)Click here for additional data file."}
+{"text": "HMGA2 and PLAG1 in 14 benign and 23 malignant thyroid tumors. To address the question if PLAG1 can be activated by HMGA2, the expression of both genes was quantified in 32 uterine leiomyomas 17 of which exhibited an overexpression of HMGA2. All leiomyomas with HMGA2 overexpression also revealed an activation of PLAG1 in the absence of detectable chromosome 8 abnormalities affecting the PLAG1 locus. To further investigate if the overexpression of PLAG1 is inducible by HMGA2 alone, HMGA2 was transiently overexpressed in MCF-7 cells. An increased PLAG1 expression was observed 24 and 48 h after transfection. Likewise, stimulation of HMGA2 by FGF1 in adipose tissue-derived stem cells led to a simultaneous increase of PLAG1 mRNA. Altogether, these data suggest that HMGA2 is an upstream activator of PLAG1. Accordingly, this may explain the formation of tumors as similar as lipomas and lipoblastomas resulting from an activation of either of both genes by chromosomal rearrangements.In pleomorphic adenomas of the salivary glands (PASG) recurrent chromosomal rearrangements affecting either 8q12 or 12q14\u223c15 lead to an overexpression of the genes of the genuine transcription factor PLAG1 or the architectural transcription factor HMGA2, respectively. Both genes are also affected by recurrent chromosomal rearrangements in benign adipocytic tumors as e. g. lipomas and lipoblastomas. Herein, we observed a strong correlation between the expression of  HMGA2 is located within the region 12q14\u223c15 which is frequently affected by chromosomal alterations PLAG1 (Pleomorphic adenoma gene 1) mapping to 8q12 encodes a genuine transcription factor encompassing seven zinc finger domains and a carboxyterminal transactivation domain. PLAG1 is developmentally regulated and highly expressed in certain fetal tissues PLAG1 plays a key role in the development of lipoblastomas IGF-II) promoter and to stimulate its activity The DNA-binding protein high mobility group AT-hook 2 (HMGA2) and the zinc finger protein PLAG1 share a common role in the molecular pathogenesis of certain benign tumors, e. g. of the salivary glands and of adipose tissue. Pleomorphic adenomas are benign tumors of myoepithelial origin most often located in the parotid glands. Based on the existence of clonal chromosomal aberrations cytogenetic subtypes of pleomorphic adenomas can be distinguished HMGA2 are a common finding in lipomas often as a t HMGA2 locus has been described as well HMGA2 has been found to allow a good discrimination between benign and malignant thyroid lesions HMGA2 ranked at the first position followed by Kallikrein 7 (KLK7), Mannose receptor, C type 2 (MRC2), Leucine-rich repeat kinase 2 (LRRK2), and PLAG1Similar but not identical to what is seen in pleomorphic adenomas both genes participate in the genesis of benign adipose tissue tumors. Chromosomal translocations affecting 12q14\u223c15 and targeting HMGA2 and PLAG1 in the molecular pathogenesis of salivary gland adenomas and adipose tissue tumors, we also quantified and compared the expression of HMGA2 and PLAG1 mRNA in thyroid adenomas as well as in papillary and follicular thyroid carcinomas. To further analyze the relationship between these two genes, we also quantified the PLAG1 expression in 32 uterine leiomyomas (UL) with as well as without 12q14 rearrangements. In addition, the PLAG1 expression was quantified in adipose tissue-derived stem cells (ADSCs) upon a stimulation of HMGA2 by FGF1. Furthermore, the MCF-7 breast cancer cell line, which has previously been used as a model in transfection experiments aiming at the functions of HMG proteins PLAG1 can be transcriptionally activated by HMGA2.Because of the apparent relationship of Formalin-fixed, paraffin-embedded tissue samples of 37 thyroid tumors were classified histologically. Fourteen cases were classified as follicular adenomas (FA), eleven tumors were diagnosed as papillary carcinomas (PTC), and four of them were follicular variants (FV PTC). The remaining twelve tumors were follicular thyroid carcinomas (FTC). Cryopreserved tissue samples of 32 uterine leiomyomas that have been analyzed cytogenetically In case of thyroid tumors, RNA was isolated from six 5 \u00b5m sections of FFPE tissues with the RNeasy FFPE kit . RNA from cryopreserved leiomyoma tissues as well as from cultivated cells (ADSCs and MCF-7 cell line) was isolated with the RNeasy Mini kit (Qiagen). Of each sample, 250 ng RNA was used for reverse transcription with M-MLV reverse transcriptase and random hexamers  according to the manufacturer\u2019s instructions.Quantitative real-time RT-PCR was performed with the TaqMan Universal PCR Master Mix on a 7300 Real-Time PCR System (Applied Biosystems) as described elsewhere HMGA2 expression was performed with the HMGA2-specific TaqMan assay Hs00171569_m1 . For the detection of the endogenous control HPRT1, primers 5\u2032-GGC AGT ATA ATC CAA AGA TGG TCA A-3\u2032 and 5\u2032-GTC TGG CTT ATA TCC AAC ACT TCG T-3\u2032 were used in combination with the HPRT1-specific hydrolysis probe 6FAM-CAA GCT TGC TGG TGA AAA GGA CCC C-TAMRA. The PLAG1 expression was quantified with the TaqMan assay Hs00231236_m1 (Applied Biosystems). Reaction conditions were as follows: 2 min at 50\u00b0C and 10 min at 95\u00b0C followed by 50 cycles of 15 sec at 95\u00b0C and 1 min at 60\u00b0C.The relative quantification of the 5 cells/9.6-cm dish. After 24 hours the serum concentration was reduced to 1%. Another 24 hours later the medium was replaced by serum-free medium supplemented with 25 ng/ml of human recombinant FGF1 . Twelve, 24, and 72 hours after growth factor addition, cells were harvested and total RNA was extracted. As controls cells were cultured in medium 199 supplemented with 1% fetal bovine serum (FBS) without FGF1.Human adipose tissue-derived stem cells were obtained and treated as described previously Plasmid pCR3.1 containing the sequence coding for human wild type HMGA2 as well as the empty vector serving as a control . The day before transfection, 150,000 cells were seeded in 6-well plates  in 2 ml medium 199 supplemented with 20% FBS and allowed to attach for 24 h. Transfection complexes were prepared in a total volume of 500 \u00b5l in serum free medium 199 without antibiotics, 2.5 \u00b5g of the respective plasmid DNA and 7 \u00b5l Lipofectamine LTX transfection reagent (Life Technologies). A mock control treated with transfection reagent only as well as a non-treated control were included for each incubation period. During the formation of transfection complexes (25 min at room temperature), old growth medium was replaced by 2 ml fresh medium. Transfection complexes were then pipetted drop-wise to the cells. After incubation for 24 h or 48 h, cells were harvested in 350 \u00b5l RLT buffer (Qiagen) containing \u03b2-mercaptoethanol for RNA isolation using the RNeasy Mini Kit and Qiacube (Qiagen). As a positive control, the expression of IGF2BP2 (synonym: IMP2), which is known to be regulated by HMGA2 The cell line MCF-7 (breast cancer) was maintained in medium 199 (Life Technologies) supplemented with 20% FBS in an incubator at 37\u00b0C and 5% COHMGA2 and PLAG1 expression levels, Spearman\u2019s rank correlation coefficient was calculated using the data of all 37 thyroid tumor samples analyzed. In addition, Pearson\u2019s correlation coefficient was calculated using the relative expression values as well as the \u0394CT values. In case of uterine leiomyomas, the expression levels of both HMGA2 and PLAG1 were classified as high or low based on the \u0394CT values . The agreement between the HMGA2 and the PLAG1 classes was then quantified and tested by the kappa coefficient of agreement.As a measure of association between the relative This study was conducted in accordance with the Declaration of Helsinki HMGA2 and PLAG1 was quantified in 37 thyroid tumors. An adenoma with low expression of both genes was chosen as calibrator for the relative quantification. The highest relative HMGA2 expression observed in 14 follicular adenomas was 17.4, whereas the expression levels in four FV PTC ranged between 23.9 and 156.9. Even higher expression levels ranging between 128.2 and 1207.5 were observed in seven PTC . In contrast, PLAG1 was expressed at levels between 13.9 and 55.9 in six follicular carcinomas overexpressing HMGA2. No overexpression of PLAG1 (0.9) was detected in only one FTC with high HMGA2 expression.The relative expression of 14 cases  and 2. IHMGA2 and PLAG1 expression for all 37 tumor samples and resulted in a high value  indicating that the expression of both genes is strongly correlated in thyroid tumors. For reasons of comparison, Pearson\u2019s correlation coefficient was calculated and also indicates a strong correlation when using the \u0394CT values . Due to the exponential transformation, the linear relationship is less pronounced on the basis of relative gene expression values .Thus, as to the expression level of both genes a strong variation was noted even within the group of malignant tumors. If an alternative overexpression of either gene represents different pathways of tumorigenesis one would expect no or even an inverse correlation. On the other hand a correlation would indicate their involvement in just one pathway. Spearman\u2019s correlation coefficient thus was calculated as a measure of association between the HMGA2 in uterine leiomyomas. Therefore, UL (n\u200a=\u200a32) were chosen to study a presumable effect of HMGA2 on the PLAG1 expression. One myoma with a normal 46,XX karyotype was chosen as calibrator for the qRT-PCR data obtained for both genes.Recurrent rearrangements affecting the chromosomal band 12q14 are known to cause an overexpression of HMGA2 locus according to FISH HMGA2 was detected in 16 cases. In the remaining case without elevated HMGA2 expression despite a cytogenetically visible t (no. 503) FISH revealed no rearrangement of the HMGA2 locus Of 17 UL with known 12q14 rearrangement including case 646 without cytogenetically visible breakpoint but rearranged HMGA2 expression. A hidden rearrangement of HMGA2 was suspected but no evidence for such a rearrangement was obtained by FISH (case 520).Only 1/15 UL without cytogenetically detectable 12q14 aberration also showed an elevated PLAG1 was clearly elevated in all 17 UL which overexpress HMGA2. In 15 UL with low HMGA2 expression, elevated levels of PLAG1 were detected in only two cases . Similarly a high and significant degree of agreement was found between the low/high expression classification of samples obtained by both parameters , see also The expression of HMGA2 in ADSCs, FGF1 was chosen because it is a known inducer of HMGA2HMGA2 mRNA level 24 h after the addition of FGF1. Simultaneously, the PLAG1 expression increased by 2-fold  as well as after transfection with the empty vector. Cells transfected with the vector containing the HMGA2 insert, however, showed an approximately 2.8-fold increase in PLAG1 expression as compared to mock transfected cells 24 h as well as 48 h after transfection , both genes are expressed at higher levels than in follicular adenomas. Follicular carcinomas with high HMGA2 locus and the HMGA2 protein expression has been shown in uterine leiomyomas HMGA2 and PLAG1 is detectable on the mRNA as well as on the protein level HMGA2 and PLAG1 mRNA expression described herein is expected to reflect a correlation at the protein level as well.A correlation between chromosomal rearrangements affecting the PLAG1 in pleomorphic adenomas of the salivary glands occurs also in tumors with 12q14\u223c15 abnormalities lacking 8q12 aberrations PLAG1. In the same study, the PLAG1 expression was investigated in three UL, and two cases were also found to overexpress PLAG1, but no cytogenetic data were available for these three tumors.Besides the typical rearrangements involving chromosomal band 8q12 including the most frequent t, an activation of PLAG1 activation in tumorigenesis other than gene rearrangements. The results presented herein point to HMGA2 as an upstream regulator of PLAG1 and are additionally confirmed by the correlation between the expressions of both genes in uterine leiomyomas. An activation of HMGA2 in UL by 12q14 aberrations is well known PLAG1 and HMGA2 simultaneously. Of the 15 UL showing low HMGA2 levels, 13 also showed low levels of PLAG1. The two remaining cases showed an elevated PLAG1 expression despite a low HMGA2 mRNA expression, thus pointing to mechanisms other than HMGA2 upregulation being responsible for PLAG1 activation. In pleomorphic adenomas of the salivary gland cryptic, intrachromosomal 8q rearrangements have been observed leading to a fusion of PLAG1 with CHCHD7 or TCEA1PLAG1 by promoter swapping. Similar events that escape detection by conventional cytogenetics may have caused the upregulation of PLAG1 observed in two UL without visible rearrangements affecting 8q12. In all 17 UL with elevated HMGA2 levels a concomitant overexpression of PLAG1 was noted. The fact that increased HMGA2 levels were always linked to elevated PLAG1 levels suggests HMGA2 as an activator of PLAG1. Because no chromosomal rearrangements affecting 8q12 were present in these 17 UL, we assumed that elevated HMGA2 levels caused by 12q14 abnormalities trigger the activation of PLAG1. In turn, HMGA2 may exert its stimulating effect on the growth of UL with 12q14 aberrations PLAG1, which was shown to possess transactivating capacity IGF-II.These findings suggest alternative mechanisms of HMGA2 is accompanied by increased PLAG1 expression levels Click here for additional data file."}
+{"text": "The differences that exist between the CD4 count strata at which certain opportunistic infections occur in sub Saharan Africa suggest that targeting prophylaxis at HIV patients in this part of the world based on data generated from developed countries may be one of the reasons why there is high HIV-related morbidity in Africa.The aim of this study was to investigate the changing incidence of some AIDS defining illnesses in Northern and Southern parts of Nigeria, relating them to different CD4 count strata.In this study, sputum samples of 234 HIV positive patients were analyzed for Mycobacterium tuberculosis and Pneumocystis jiroveci while 202 stool samples of HIV patients were analyzed for the presence of opportunistic intestinal parasites. We considered five CD4 strata . Incidence of the various opportunistic infections and their occurrence within each CD4 strata were estimated using simple statistical method. CD4 count of each patient was estimated using the Becton Dickenson FACSCount system.The prevalence of Mycobacterium tuberculosis, Pneumocystis jirovecii, Cryptosporidium parvum, Isospora belli and Cyclospora spp were 10.3%, 41.9%, 30.8%, 24.2% and 4.4% respectively. All the opportunistic infections occurred at higher rates in CD4 counts less than 200cells/ul (p < 0.0001) except for tuberculosis which occurred highest at CD4 counts >200cells/ul (16 out of the 24 positive sputum smears were recovered in patients with CD4 counts >200 cells/ul). Despite pronounced immunosuppression, P. jiroveci was not detected in sputum samples of 8% patients with CD4 count <200 cells/ul.Opportunistic parasites occurred almost exclusively at CD4 count <200 cells/ul. However, 6.4% of these parasites were isolated in patients with CD4 >200 cells/ul. Cryptosporidium parvum (30.8%) was the most frequently encountered opportunistic parasite, followed by Isospora belli (24.2%) and Cyclospora specie (4.4%).Although HIV related opportunistic infections are often reported to occur exclusively at CD4 count <200 cells/ul in patients; the result of this study shows that disparities exist and so, the possibility of opportunistic pathogens must remain in the differential diagnosis of infections in HIV patients independent of absolute CD4 count."}
+{"text": "Asthma is a serious global health problem. Global prevalence of asthma ranges from 1% to 18% of population in different countries.In India, prevalence of asthma is 3% of the population. Major depressive disorder is the most common mood disorder often found to be higher among people with chronic health conditions like asthma. Presence of depression may lead to increased severity of asthma making it an uncontrolled asthma.Our objective was to see prevalence of depression among asthma patients and effect of asthma control on severity of depression.All patients who met the inclusion criteria evaluated with Goldberg\u2019s The General Health Questionnaire (GHQ 28), Bengali Version of Beck Depression Inventory (BDI) and Holmes & Rahe\u2019s Life Event Scale. Severity of asthma and level of asthma control determined as per GINA guidelines. Follow up was done after 3 months and patients were again evaluated with Goldberg\u2019s The General Health Questionnaire (GHQ 28), Bengali Version of Beck Depression Inventory (BDI) and Holmes & Rahe\u2019s Life Event Scale.Among 100 patients 35 (35%) had normal BDI score, mild mood disturbance was found in 23 (23%), borderline clinical depression in 12 (12%), moderate depression in 20 (20%), severe depression in 9 (9%), extreme depression in 1 (1%) patients. Follow up done at 3 months showed 68 patients had controlled and 32 patients had partially controlled asthma. Follow up evaluation with BDI showed 37 (37%) patients had normal score. Mild mood disturbance was found in 24 (24%), borderline clinical depression in 16 (16%), moderate depression in 14 (14%), severe depression in 8 (8%), extreme depression in 1 (1%) patients.There is no significant correlation between severity of asthma and severity of depression .There is also no correlation of asthma control on severity of depression .Depression is highly prevalent among asthma patients. There is no significant correlation between severity of asthma and severity of depression. There is also no correlation of asthma control on severity of depression."}
+{"text": "Tropomyosin has been reported to be responsible for the cross-reactivity between shrimp and dust mites and the measurement of tropomyosin specific IgE was found superior to shrimp extract for the predicting of shrimp allergic reaction. A total of 55 dust mite allergic patients were recruited to analyse the cross-reactivity between shrimp and mites . Tropomyosin specific IgE for shrimps , Anisakis ( rAnis 3 ), house dust mites ( rDer p10 ) and german cockroach ( nBla g7 ) were measured using Phadia diagnotic test. Two recombinant allergens ( rTyr p10 for Tyrophagus putresstienciae and rPer a 7 for American cockroach ) were used to investigate the cross-reactivity . Basophil histamine release (BHR) assay was used to evaluate its biological activities. The results showed that there were 13 patients  sensitive to tropomyosin. The allergen specific tropomyosin were further analysed, there were 69% (9/13) sensitive to rAnis 3, 62%(8/13) sensitive to rDer p10, 54%(7/13) sensitive to nBla g7 and nPen m1. Most patients were sensitive to at least two different species of tropomyosin. Immuno-Dot study showed that there were 8/13 sensitive to rPer a7 and 7/13 sensitive to rTyr p10. Despite the percent inhibition of BHR were similar between Der p and shrimp stimulation the relationship between the tropomyosin sensitive of all species allergens were not correlated.In conclusion, tropomyosin specific IgE can be detected in 23.6% (13/55) of Der p sensitive patients and the percentage of shrimp specific tropomyosin allergy was higher in AD than BA. The poor relationship of the specific IgE between the shrimp specific tropomyosin with other species of allergen indicated that the allergenic component of torpomyosin from all allergen should be generated and used for the definite diagnosis of food allergy."}
+{"text": "Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800\u20131,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences. Transposable elements comprise a substantial portion of many eukaryotic genomes. These mobile fragments of DNA can directly mutate genes through insertions into coding regions but may also affect the gene regulation through nearby insertions. There is evidence that the majority of transposable elements are epigenetically silenced, and in some cases this silencing may spread to neighboring sequences. This spreading of heterochromatin could create a significant fitness tradeoff between transposon silencing and gene expression. The maize genome has a complex organization with many genes flanked by retrotransposons, providing an opportunity to study the interaction of retrotransposons and genes. To survey the prevalence of heterochromatin spreading associated with different retrotransposon families, we profiled the spread of heterochromatin into nearby low copy sequences for 150 high copy retrotransposon families. While many retrotransposons exhibit little to no spreading of heterochromatin, there are some retrotransposon families that do exhibit spreading. Genes located near retrotransposons that spread heterochromatin have lower expression levels. The families of retrotransposons that spread heterochromatin marks to nearby low-copy sequences may have increased fitness costs for the host genome due to their suppression of genes located near insertions. A substantial fraction of most eukaryotic genomes is composed of transposable elements (TEs) agouti and Axin locus in mouse FLCFWATEs could influence neighboring genes by providing regulatory elements or promoters that would alter expression levels or patterns The complex organization of the maize genome, with interspersed genes and TEs The existence of heavily silenced retrotransposons interspersed with genes throughout the maize genome provides ample opportunities for TEs to exert epigenetic regulation on surrounding sequences. We were interested in further documenting the extent of heterochromatin spreading from maize retrotransposons to neighboring sequencings. Genomic profiling of DNA methylation and H3K9me2 found that heterochromatic spreading is only observed for a small number of specific retrotransposon families. These families tend to be enriched in pericentromeric regions of chromosomes. The analysis of haplotypes lacking specific retrotransposon insertions provides evidence that the adjacent heterochromatin is the result of spreading rather than insertion site bias.DNA methylation and chromatin modifications were profiled for low-copy sequences in the maize genome using methylated DNA immunoprecipitation (meDIP) and chromatin-immunoprecipitation (ChIP) with antibodies specific for H3K9me2 or H3K27me3, respectively. The fractions of the genome enriched for DNA or histone modifications were hybridized to a high-density microarray containing \u223c2.1 million long oligonucleotide probes derived from the unmasked, non-repetitive fraction of the maize genome. The probes are spaced every 200 bp in the low-copy portions of the maize genome and can provide a profile for the chromatin state in these regions A large number of class I TEs (retrotransposons) have been identified within the maize genome A subset of the retrotransposon families also exhibit elevated levels of DNA methylation and H3K9me2 in regions more than 200 bp away from their insertion sites. In general, levels of H3K9me2 and DNA methylation were well correlated, but there were some families with different enrichment for these two marks. As expected, there was no evidence for enrichment (or depletion) of the facultative heterochromatin mark, H3K27me3, in regions flanking the retrotransposons . To idenThe analysis of the whole-genome bisulphite sequencing data supports the classifications of different retrotransposon families . Both CGmop1 mutant of the maize Rdr2 gene mop1 mutation on the expression levels of spreading or non-spreading retrotransposon families. Indeed, spreading retrotransposon families include examples of both up- and down-regulation in mop1 mutant individuals relative to wild-type  gene, which contributes substantially to CHG methylation Spreading retrotransposons exhibit higher levels of CHG methylation within the retrotransposon themselves . Spreadiftp://ftp.jgipsf.org/pub/JGI_data/Zea_mays_Mo17/) were aligned to the B73 reference genome sequence. Empty sites were defined as being those as which at least three Mo17 sequence reads cover a low-copy sequence flanking an insertion but do not align to the retrotransposon itself and for which no Mo17 reads cover the junction between the low-copy sequence and the retrotransposon. In total, 668 empty sites were identified for the spreading (both) retrotransposon families and 29 empty sites for the spreading (H3K9) retrotransposon families for which we had DNA methylation data in the unique regions flanking the insertion. The lack of the specific insertion in Mo17 was confirmed at 13 of the 14 empty sites that were tested using site-specific PCR primers to confirm the presence/absence of specific insertions. This suggests that there is a low false-positive rate in the identification of empty sites in Mo17. However, given the low coverage of the WGS data and challenges associated with aligning polymorphic sequences it is likely that many of the true empty sites were not identified in this analysis.The observation that certain families of retrotransposons have high levels of heterochromatic modifications in adjacent regions could reflect insertion site biases for these families or indicate that these families cause local spreading of heterochromatin. Examples of \u201cempty\u201d sites in the Mo17 haplotypes were identified and used to assess whether the high levels of DNA methylation would be observed in these regions when the retrotransposon was absent. Mo17 whole-genome shotgun WGS) sequences (generated by the DOE's Joint Genome Institute (JGI) and downloaded from The level of DNA methylation at the probe nearest to the empty site was used to assess relative DNA methylation levels with (B73) and without (Mo17) each insertion . The lowThe finding that only a subset of maize class I retrotransposon families are associated with local spreading of heterochromatin suggested that there might be intrinsic differences among different retrotransposon families that would explain this variation. We proceeded to characterize these families to ascertain whether there were specific common attributes of spreading families. None of the LINE families exhibit evidence for spreading of heterochromatic marks. RLG (gypsy) families are over-represented among spreading (both) retrotransposon families, while the spreading (H3K9) retrotransposons have more RLC (copia) families than expected . SpreadiThe relative abundance of spreading (both) retrotransposons is higher in the middle of the chromosome than the other families suggesting that these retrotransposons may be enriched in pericentromeric regions . HoweverThe finding that some retrotransposon families exhibit spreading of heterochromatic marks to surrounding sequences while others do not led us to hypothesize that these families may influence expression of nearby genes. RNAseq was used to estimate transcript abundance in three tissues of B73 and Mo17 including the identical leaf tissue samples used for profiling DNA methylation levels. All maize genes were annotated to identify the first retrotransposon 5\u2032 of the transcription start site and to determine the distance between the retrotransposon and the transcription start site. Genes that are located near retrotransposons that exhibit spreading (both or H3K9) have significantly (p<0.001) lower expression levels in all genotypes and tissue examined . This reEpigenetic variation in low-copy sequences can be the result of pure epigenetic changes (no correlation with DNA sequence polymorphisms) or occur in a facilitated or obligatory fashion such that DNA sequence differences contribute to the epigenetic changes There is also evidence that differences in interspecific variation in transposon insertions contributes to gene expression diversity between related species Drosophila retrotransposons contain insulator elements that reduce the spreading of chromatin states bns locus in Arabidopsis have suggested the presence of an active mechanism to prevent the spreading of heterochromatin from retrotransposons Barbara McClintock proposed the concept that transposons could serve as \u201ccontrolling\u201d elements that would influence nearby genes rd leaf tissue of B73 and Mo17 was performed as described [39 \u2013 GSE29099]. Briefly, methylated DNA was immunoprecipitated with an anti-5-methylcytosine monoclonal antibody from 400 ng sonicated DNA using the Methylated DNA IP Kit . For each replication and genotype, whole genome amplification was conducted on 50\u2013100 ng IP DNA and also 50\u2013100 ng of sonicated DNA (input control) using the Whole Genome Amplification kit . For each amplified IP input sample, 3 ug amplified DNA were labeled using the Dual-Color Labeling Kit  according to the array manufacturer's protocol (Roche NimbleGen Methylation UserGuide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3. H3K9me2 and H3K27me3 profiling were performed on three replicates of B73 and Mo17 seedlings using antibodies specific for H3K27me3 (#07-449) and H3K9me2 (#07-441) purchased from Millipore . For each replicate, 1 g of plant material was harvested on ice, rinsed with water, and crosslinked with 1% formaldehyde for 10 minutes under vacuum. Cross-linking was quenched by adding glycine solution to a final concentration of 0.125 M under vacuum infiltration for 5 minutes. Treated tissue was frozen in liquid nitrogen and stored at \u2212800 C until chromatin extraction. Chromatin extractions were performed using EpiQuik Plant ChIP Kit  according to manufacturer's recommendations. Extracted chromatin was sheared in 600 \u00b5l of the EpiQuik buffer CP3F with 5 10-second pulses on a sonicator. To test and optimize sonication conditions, cross-linking was reversed in a sample of sheared chromatin and the resulting products were analyzed on agarose gel. Sonication conditions were optimized to yield predominantly 200\u2013500 bp DNA samples. Chromatin immunoprecipitations, reverse cross-linking, and DNA cleanup was performed using EpiQuik Plant ChIP Kit (Epigentek) according to manufacturer's recommendations. For each genotype, antibody, and replicate, 50\u2013100 ng of input and immunoprecipitated (IP) DNA was amplified with a whole genome amplification kit . The amplification of no antibody control (negative control) was always 5\u201310 fold less efficient confirming specificity of immunoprecipitation. For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit  according to the array manufacturer's protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3. Samples were hybridized to the custom 2.1 M probe array (GEO Platform GPL13499) for 16\u201320 hrs at 42 C. Slides were washed and scanned according to NimbleGen's protocol. Images were aligned and quantified using NimbleScan software (Roche NimbleGen) producing raw data reports for each probe on the array. The histone modification and methylation mutants array data can be obtained from GEO accession (GSE39460). The resulting microarray data were imported into the Bioconductor statistical environment (http://bioconductor.org/). Microarray data channels were assigned the following factors: B73 immunoprecipitation, Mo17 immunoprecipitation, B73 input, or Mo17 input depending on sample derivation. Non-maize probes and vendor-supplied process control probes were configured to have analytical weights of zero. Variance-stabilizing normalization was used to account for array-specific effects. Factor-specific hybridization coefficients were estimated by fitting fixed linear model accounting for dye and sample effects to the data using the limma package www.maizesequence.org. Each probe was only associated with the closest repeat and all probes located within 5 kb of a repeat were retained for further analyses. The probes were assigned based on distance to the retrotransposon and include both upstream (5\u2032) and downstream (3\u2032) sequences together. The distribution of retrotransposons along the length of the chromosome was performed as described in http://genomics.tacc.utexas.edu/data/rte_methylation_spreading/.DNA methylation profiling on three replicates of 3DNA was extracted from the outer tissues of B73 ears whose silks had emerged but had not been fertilized. Sodium bisulfite-treated Illumina sequencing libraries were prepared using a method similar to that of Lister et al Approximately 63M Mo17 454 whole-genome shotgun sequencing reads generated by the DOE's Joint Genome Institute (JGI) were trimmed and aligned to Maize B73 reference genome (AGPv2) and reads aligned uniquely (single loci) were filtered for subsequent analysis. A retrotransposon insertion site was classified as empty if we identified at least 3 WGS reads supporting the site that aligned to the insertion site that included>50 bp of aligned sequence outside of the repeat region in B73 with similarity of \u226594%, relatively short unaligned tails (\u226420 bp), and contained a long overhang of >20 bp that begins \u00b13 bp from the annotated retrotransposon insertion site. PCR primers were designed to amplify the sequence at the \u201cempty\u201d sites using the B73 sequence (which contained the insertion) and the Mo17 sequence (which lacks the insertion) . These srd leaf, embryo, endosperm, and immature ear) for both B73 and Mo17. Samples were prepared at the University of Minnesota BioMedical Genomics Center in accordance with the TruSeq library creation protocol (Illumina). Samples were sequenced on the HiSeq 2000 developing 6\u201317 million reads per replicate. Raw reads were filtered to eliminate poor quality reads using CASAVA (Illumina). Transcript abundance was calculated by mapping reads to the maize reference genome (AGPv2) using TopHat under standard parameters www.iplantcollaborative.org). RPKM values were calculated per gene. All genes within 500, 1000, 2500, and 5000 bases of the closest upstream annotated transposable element (ZmB73_5a) using BEDtools RNA\u2013seq was performed on three biological replicates of four tissues  The efficient enrichment of DNA associated with H3K9me2 was assessed using qPCR. The copia sequence is known to be enriched for H3K9me2 while the GAPC sequence is not associated with H3K9me2 . Primers for these regions were used to perform qPCR using three technical replicates. The percent of input DNA recovered after IP with the H3K9me2 antibody or a noIG control was determined for both sequences. (B) Several regions were selected for validation based on ChIP-chip profiling. Two regions enriched for H3K9me2 (H1 and H2) and four regions with no evidence for H3K9me2  were used to design primers for qPCR. The percent of input DNA recovered by ChIP using an H3K9me2 antibody or a noIG control was determined for three replicates of B73 using these primers. The H1 and H2 sequences were enriched by ChIP while the L1, L2, L3 and L5 sequences showed much lower levels of recovery.(TIF)Click here for additional data file.Figure S2Levels of DNA methylation within retrotransposons. Whole-genome bisulphite sequencing data was used to assess the average level of methylation within retrotransposons. DNA methylation levels within each sequence context  were determined for each family of retrotransposon. The average levels of methylation for elements classified as having spreading of both 5 mC and H3K9me2, spreading of H3K9me2 only and non-spreading were determined and plotted. The error bars indicate standard deviation among retrotransposon families and \u201c*\u201d indicate significant (p<0.001) differences for a group relative to the non-spreading families. The level of internal methylation at CG sites is similar for retrotransposons with and without spreading of heterochromatin although there is a significant difference in spreading (both) relative to non-spreading families. CHG methylation is slightly lower in non-spreading families. The non-spreading families have slightly elevated levels of CHH methylation relative to the other families.(TIF)Click here for additional data file.Figure S3Chromatin modifications in regions flanking maize retrotransposon superfamilies including RIX \u2013 LINE (black), RLC \u2013 copia (red), RLX \u2013 unknown LTR (blue) and RLG \u2013 gypsy (green). We identified probes located in low-copy DNA flanking retrotransposons in maize. The number of probes for each class is indicated within the Figure legend. The average level of DNA methylation (A\u2013B), H3K9me2 (C) or H3K27me3 (D) is shown for the 5,000 bp adjacent to each superfamily. The level of chromatin modifications are based on ChIP-chip experiments and the y-axis represents the average log ratio for the immunoprecipitated samples relative to genomic input DNA.(TIF)Click here for additional data file.Figure S4Profiles of chromatin surrounding spreading (both) retrotransposons. Black lines indicate DNA methylation. Blue lines indicate H3K9. Green lines indicate H3K27 levels. Chromatin values calculated include probes flanking both ends of retrotransposable elements. Copy number of repeat fragments in the B73 reference genome as well as the total number of probes flanking each repeat are displayed. The y-axis provides the distance (in bp) from the retrotransposon insertion.(TIF)Click here for additional data file.Figure S5Profiles of chromatin surrounding spreading (H3K9) retrotransposons. Black lines indicate DNA methylation. Blue lines indicate H3K9. Green lines indicate H3K27 levels. Chromatin values calculated include probes flanking both ends of retrotransposable elements. Copy number of repeat fragments in the B73 reference genome as well as the total number of probes flanking each repeat are displayed. The y-axis provides the distance (in bp) from the retrotransposon insertion.(TIF)Click here for additional data file.Figure S6Similar profiles of DNA methylation enrichment adjacent to retrotransposon families in different tissues and genotypes of maize. The relative level of DNA methylation in each 200 bp bin is shown for each of the 144 retrotransposon families. The red color indicates enrichment for the modification while blue indicates depletion of the mark. Black indicates levels of the modification similar to genome-wide average values. The color intensity is based on the average log ratio of immunoprecipitated DNA relative to input DNA. The retrotransposons are grouped according to whether they show spreading for DNA methylation and H3K9me2, just H3K9me2 or neither of the marks. The profiles are shown for B73 leaf (A), Mo17 leaf (B), B73 endosperm (C) and B73 embryo (D).(TIF)Click here for additional data file.Figure S7Histone modification patterns in different genotypes. The profile of several histone modifications is shown for both B73 and Mo17. The relative level of H3K9(di)- or H3K27(tri)-methylation in each 200 bp bin is shown for each of the 144 retrotransposon families. Modification levels calculated include probes flanking both ends of retrotransposable elements. The red color indicates enrichment for the modification while blue indicates depletion of the mark. Black indicates levels of the modification similar to genome-wide average values. The color intensity is based on the average log ratio of immunoprecipitated DNA relative to input DNA. The retrotransposons are grouped according to whether they show spreading for DNA methylation and H3K9me2, just H3K9me2 or neither of the marks. (A) and (B) display the profiles for H3K9me2 in B73 and Mo17 seedlings, respectively. (C) and (D) display the profiles for H3K27me3 in B73 and Mo17 seedlings, respectively. (E) shows the H3K27me3 profile for immature ear tissue from B73.(TIF)Click here for additional data file.Figure S8Similar levels of H3K9me2 within spreading and non-spreading retrotransposons. The ChIP-chip assay does not provide information on the abundance of H3K9me2 within repetitive regions. In order to assess whether the level of H3K9me2 was similar within retrotransposons with, and without, spreading we designed primers for internal sequences of 10 retrotransposon families including six with spreading (both) and four that were classified as non-spreading. The qPCR protocol described by Haring et al. (2007) was used to assess the percent input DNA recovered by immunoprecipitation. The percent of input DNA recovered after IP with the H3K9me2 antibody or a noIG control was determined for both sequences and the standard deviation is indicated with error bars. There were high levels of H3K9me2 in each of these retrotransposons but there was no significant difference between the spreading and non-spreading retrotransposons.(TIF)Click here for additional data file.Figure S9Small RNA coverage of retrotransposon families. B73 shoot small RNAs (TIF)Click here for additional data file.Figure S10Characteristics of retrotransposon families with spreading of heterochromatic marks. In each of the plots the retrotransposon families are grouped into both (5 mC and H3K9), H3K9 only and non-spreading columns and the superfamily is indicated by the color. The data points are jittered to allow visualization of all families. (A) The genomic copy number of each family is shown using a log-scale. The families with spreading of both marks tend to have higher copy numbers. However, there is an overlap in the range of copy number for families with and without spreading. (B) The total Mb of the B73 genome comprised by each family is shown. (C) The average insertion date for each family is plotted. While the non-spreading families include both young and old retrotransposons the families with spreading of both marks tend to be younger families.(TIF)Click here for additional data file.Figure S11DNA methylation levels in flanking sequences are similar throughout the chromosome. The level of DNA methylation in sequences flanking retrotransposons was determined from bisulphite sequencing data. Only flanking sequences that did not contain any repetitive sequences within 1 kb of the retrotransposon were used. The proportion of methylated cytosines in CG (A) or CHG (B) contexts was determined for 1 kb of low copy sequences flanking spreading (both), spreading (H3K9) and non-spreading retrotransposon families.(TIF)Click here for additional data file.Figure S12Genes near spreading retrotransposons show lower expression than genes near non-spreading retrotransposons. (A) Average RPKM values  for all genes falling within 0.5 kb, 1 kb, and 2.5 kb from the respective class of retrotransposons were determined. (B) Proportion of genes expressed (RPKM>0) in B73 leaf tissue for genes near 5 mc and H3K9 spreading elements, H3k9 spreading only, non-spreading, and no TE nearby. Red asterisks indicate highly significant (p<0.001) difference from the non-spreading elements within the distance classification. Black asterisks indicate significant (p<0.001) difference from genes not near any TE. (C) Average RPKM values in B73 leaf tissue for expressed genes  falling within 0.5 kb, 1 kb, and 2.5 kb from the respective class of retrotransposons were developed. Red asterisks indicate significant (p<0.05) difference from the non-spreading elements (green) within the distance classification. Black asterisks indicate significant (p<0.05) difference from genes not near any retrotransposons (purple).(TIF)Click here for additional data file.Table S1Attributes and classification of retrotransposon families. A list of 145 retrotransposon families with information and statistics about each family in the B73 genotype.(XLSX)Click here for additional data file.Table S2Primers used for empty site validation.(XLSX)Click here for additional data file."}
+{"text": "No clear information on the optimal dosage of Canakinumab in CAPS is available.To analyse the modification of dosage schedule of Canakinumab in CAPS in 12 months of routinely clinical practice.12 patients (9 children and 3 adults) with Muckle-Wells syndrome (3), MWS/CINCA overlap (3) and CINCA (6) were analyzed. Patients were previously enrolled in the CACZ885D2306 trial and studied for the following 12 months.At baseline, 7 patients were treated with the initial dosage of 2 mg/kg  every 8 weeks. In 5 patients  the dosage was 4 mg/kg (or 300 mg) every 8 weeks.During the following 12 months modification of dosage of frequency was performed in 7/12 patients. The 5 patients at higher dosage during the CACZ885D2306 study needed to increase the frequency of administration with a mean frequency of 6 weeks (range 4-8). The mean reason was the presence of mild clinical manifestation and/or persistent elevation of acute phase reactants. In one of these patients the therapy was subsequently discontinued due to persistent disease activity. An increased frequency (6 and 7 weeks) was also performed in 1 MWS and 1 CINCA patient, respectively.In 5 patients the treatment was not modified being effective in the control of the disease.This study confirms the efficacy of Canakinumab in CAPS. However, pediatric patients and those with a more severe phenotype require higher and more frequent dosage than previously described."}
+{"text": "RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A, SRY and DYS14 was performed by real-time PCR.cfDNA was extracted from maternal plasma (n\u200a=\u200a90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.Hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.Use of this assay for the detection of hypermethylated  Traditionally prenatal diagnosis of fetal genetic status has depended on the use of invasive diagnostic tests, either amniocentesis or chorionic villus sampling (CVS), which carry a small but significant risk of miscarriage de novo. NIPD for fetal sex determination can be carried out at an early gestational age for pregnancies known to be at risk of a sex-linked genetic condition Analysis of cffDNA in maternal plasma is now in routine clinical diagnostic use for non invasive prenatal diagnosis (NIPD) where the target fetal sequence is derived from the father or where the allele arises de novo) allele to make a definitive diagnosis. Absence of detection of the paternal target sequence is either indicative of a true negative result or could also be due the lack of amplification of the sequence due to low concentrations of circulating cffDNA or the complete absence of cffDNA in the sample. Amplification of a fetal specific marker that confirms the presence of cffDNA allows a negative result to be more accurately interpreted as either a true or false negative result. The use of several fetal identifiers has been reported including the hypermethylated RASSF1A promoter Currently, clinical applications of NIPD require the detection of the paternally inherited , at the early pregnancy unit (n\u200a=\u200a4), at a routine ultrasound scan (n\u200a=\u200a16) or for an invasive test procedure (n\u200a=\u200a8) at Salisbury NHS Foundation Trust. The study was approved by the South West 1 Research Ethics Committee A (ref 09/H0104/59). Gestational age at blood collection was confirmed by routine ultrasound in all cases.g for 10 minutes and the plasma fraction transferred to a 2 ml centrifuge tube and re-centrifuged at 20,000 g for 10 minutes. The cell free plasma fraction was stored at \u221280\u00b0C. Cell free DNA was extracted from 3 ml plasma using the Circulating Nucleic Acid Kit (Qiagen) following the manufacturer\u2019s instructions and resuspended in 70 \u00b5l AVE buffer.Maternal blood was collected into two 10 ml EDTA tubes and centrifuged at 1600 TseI and 4 U BsmI (methylation insensitive digest) and 2 U BstYI and 2 U BstUI (methylation sensitive digest). Samples were incubated at 60\u00b0C (undigested and methylation sensitive digest) and 65\u00b0C (methylation insensitive digest) for 2 hours. Additional restriction enzymes were then added to the reaction and the samples digested further at 37\u00b0C for 2 hours using no enzyme (undigested control), 8 U EcoRI, 8 U MspI and 4 U HaeIII (methylation insensitive digest) and 8 U EcoRI, 8 U HhaI and 4 U HpaII (methylation sensitive digest). All enzymes were supplied by New England Biolabs. cfDNA from male plasma was digested for each batch of samples analysed and performed as a digest control for the RASSF1A methylation insensitive and methylation sensitive digests. These digests should proceed to completion and therefore no RASSF1A amplification products should be observed following real time PCR. Further information regarding the selection of the restriction enzymes and details of the cleavage sites of the enzymes in the RASSF1A amplicon are given in Restriction digestion reaction and real time PCR conditions were optimised on a separate training set of 90 plasma samples obtained from the SAFE-RAPID sample bank at Great Ormond Street Children\u2019s Hospital prior to commencing this study RASSF1A or SRY , DYS14  and RASSF1A . The cfDNA from male plasma also acts as a digest control for the RASSF1A methylation insensitive and methylation sensitive digests as digestion should proceed to completion and no RASSF1A amplification products should be observed.Samples that were negative for RASSF1A was determined by positive real time PCR amplification plots  and a correct melt profile showing a specific peak at 91.2\u00b0C  required 3 replicates to be positive for analysis to be undertaken. For the methylation insensitive digest (control for complete enzyme digestion of RASSF1A target sequence) complete digestion was assumed to have occurred if no replicates were positive. For the methylation sensitive digest, \u22652 out of 3 replicates were required to be positive to confirm the presence of hypermethylated fetal DNA. Samples with fewer than 2 positive replicates were not considered to have sufficient cffDNA present for reliable interpretation of the SRY result. Nine SRY (or DYS14) replicates were analysed for each patient sample (three from each digest) and the sample was considered to be male if 6 or more replicates showed amplification  and a correct melt profile showing a specific peak at 80.5\u00b0C  and gestational ages ranged from 5\u201323.2 weeks (mean\u200a=\u200a9.1 weeks). All blood samples were received in the laboratory within 72 hours of collection.RASSF1A in the undigested sample indicating that total cell free DNA had been extracted successfully in all cases. Hypermethylated RASSF1A, was amplified in \u22652 out of 3 replicates for the methylation sensitive restriction enzyme digest for 79 samples (88%) indicating the presence of cffDNA. Of these, 45 showed amplification of SRY (\u22656 out of 9 replicates positive) and were male at delivery and 33 had no detectable SRY (0 out of 9 replicates positive) and were female at delivery (including the XX twins). One sample was male at delivery but only showed amplification of SRY in only 3 out of 9 replicates and therefore this result would have been classified as inconclusive despite the detection of sufficient cffDNA. Hypermethylated RASSF1A was amplified in <2 replicates in 11 cases and these would have failed the data analysis parameters for reporting a clinical result. Of these, 10 samples had gestational ages less than 7 weeks 2 days which could explain the low level or absence of cffDNA in the samples SRY (i.e. an inconclusive result) and two samples had 6 replicates positive for SRY. One male sample showed 0 replicates positive for both hypermethylated RASSF1A and SRY and therefore could have been mis-reported as female if the RASSF1A assay had not been used. All samples showed complete enzyme digestion of RASSF1A in the methylation insensitive digest reactions . Eleven female samples showed 0/9 replicates positive for DYS14 but the remainder (n\u200a=\u200a26) showed \u22651 replicate positive  with an average Ct value of 39.2 (SD 1.7). The male samples (n\u200a=\u200a24) had 9/9 DYS14 replicates positive with an average Ct value of 32.5 (SD 1.8). The male sample that had no amplification of hypermethylated RASSF1A or SRY showed 9/9 replicates positive for DYS14 however the mean Ct value was 38.2. RASSF1A, SRY and DYS14 for male and female pregnancies.Samples that had fewer than 9/9 replicates positive for RASSF1A promoter sequences can be used to confirm the presence of cffDNA in diagnostic samples. The RASSF1A promoter sequence is very GC rich and is therefore problematic to fully digest and amplify by real time PCR. We have made several modifications to previously published protocols RASSF1A sequences, monitor that the digestion of the target has gone to completion and also improve the efficiency and interpretation of RASSF1A real time PCR. In preliminary experiments using published protocols we observed that incomplete digestion of RASSF1A was common in plasma samples derived from men and non-pregnant women where RASSF1A should not be observed following digestion with methylation sensitive enzymes  indicating the presence of cffDNA. For these samples the SRY real time PCR results and fetal sex at delivery were 100% concordant indicating that the test is 96\u2013100% accurate with a 95% confidence interval RASSF1A) were observed in most (n\u200a=\u200a10) samples that had a gestational age of 7 weeks or less. This is concordant with a recently published study of a clinical audit of fetal sex determination that showed that discordant results could be obtained if testing is performed before 7 weeks RASSF1A assay should be considered for all samples to ensure that sufficient cffDNA is present to enable confident interpretation of a test result. For example, in this study two samples of gestational age <7 weeks had reportable levels of hypermethylated DNA and 7 replicates positive for SRY analysis and were phenotypically male at birth. Conversely a sample of gestational age 10 weeks 4 days showed unreportable levels of hypermethylated RASSF1A and an inconclusive number of positive SRY replicates. Using digital droplet PCR to quantify fetal load (RASSF1A) and fetal sex (SRY) it has been shown that the Pearson\u2019s correlation coefficient between SRY and RASSF1A fetal loads is 97.3 with the RASSF1A fetal loads measured for some samples being lower than those determined using SRYSRY result but negative RASSF1A result. In all cases where unreportable levels of hypermethylated RASSF1A are obtained we recommend that repeat blood samples should be requested and testing repeated.Using the modified protocol we assessed the utility of the assay for confirmation of cffDNA when undertaking NIPD for fetal sex determination. Hypermethylated DYS14 has been reported as having a higher sensitivity of detection DYS14 amplification although the Ct values for these samples (mean 39.2) were significantly higher than those obtained for male samples (mean Ct 32.5)  . This haRASSF1A is applicable to all pregnancies since is it polymorphism independent and can be performed at the same time as the fetal sex determination on the same aliquot of cffDNA. The SRY and RASSF1A assays have identical reaction conditions and can be amplified in the same plate and therefore results can be reported within 48 hours of receipt of the maternal blood sample. The assay could also be used for NIPD of single gene disorders and fetal RhD status provided that the restriction enzymes do not cut within the target amplicon. Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.This modified protocol for detection of fetal specific hypermethylated Figure S1SRY and RASSF1A real time SYBR green PCR assays.Examples of PCR amplification plots and melting profiles for the (PDF)Click here for additional data file.Figure S2(a) Amplification plots showing the difference in efficiency of three methylation sensitive RASSF1A restriction enzyme digest protocols using cell free DNA extracted from the plasma of a non-pregnant female. (b) Amplification plots showing an example of the difference in efficiency of two methylation sensitive RASSF1A restriction enzyme digest protocols using cell free DNA extracted from the same plasma sample from a pregnant female.(PDF)Click here for additional data file.Figure S3Details of restriction enzyme digests.(PDF)Click here for additional data file."}
+{"text": "The finding that APECED syndrome is due to a failure in negative thymic selection has suggested that central tolerance could play a role also in polygenic autoimmunity and that autoimmune patients should have an increase in RTEs. The aim of this work was to assess the frequency of RTEs, transitional B cells and Th17 lymphocytes in MS.PBLs from 55 MS patients  and 32 healthy controls were analysed by FACS for RTEs, na\u00efve and memory T cells as well as Th17 cells and transitional B cells.+CD24hiCD38hiCD27-)  were found between controls and MS. A significant decrease in the percentage of CD4 effector memory (CD45RA-CCR7-CD27+) , as well as an increase in Th17 cells  was observed in MS vs controls.No differences in the percentage of RTEs (CD45RA+CD31+PTK7+)  or B transitional cells .The results of this preliminary study do not favour a failure in central tolerance in MS but confirm involvement of Th17 cells which are associated with a higher degree of disability."}
+{"text": "AbstractCimex lectularius Linnaeus, 1758 from the Czech Republic and other European countries, hosted on Myotis Kaup, 1829 (4) and Homo sapiens Linnaeus, 1758 (57). The karyotype of all the specimens of Cimex lectularius analysed contained 26 autosomes and a varying number of the sex chromosomes. The number of sex chromosomes showed extensive variation, and up to 20 fragments were recorded. Altogether, 12 distinct karyotypes were distinguished. The male karyotypes consisted of 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 42 and 47 chromosomes. The females usually exhibited the number of chromosomes which was complementary to the number established in the males from the same sample. However, 11 polymorphic samples were revealed in which the karyotypes of females and males were not complementary each other. The complement with 2n = 26+X1X2Y was found in 44% of the specimens and 57,4% samples of bed bugs studied. The karyotypes with higher chromosome numbers as well as individuals with chromosomal mosaics were usually found within the samples exhibiting particularly extensive variation between individuals, and such complements were not found within samples contaning a few or single specimen. The occurrence of chromosomal mosaics with the karyotype constitution varying between cells of single individual was observed in five specimens (4.3%) from five samples. We assume that polymorphism caused by fragmentation of the X chromosome may result in meiotic problems and non-disjunction can produce unbalanced gametes and result in lowered fitness of individuals carrying higher numbers of the X chromosome fragments. This effect should be apparently enhanced with the increasing number of the fragments and this may be PageBreakthe reason for the observed distribution pattern of individual karyotypes in the studied samples and the rarity of individuals with extremely high chromosome numbers. The assumed lowering of the fitness of individuals carrying higher numbers of the X chromosome fragments could affect population dynamics of variable populations.Variation in the number of chromosomes was revealed in 61 samples of  Cimex Linnaeus, 1758 is the best known taxon of the family Cimicidae (Heteroptera) which contains up to 110 described species of haematophagous ectoparasites exploiting mostly bats and birds as hosts .The bed bug, ome e.g. . Variatiome e.g. , 1967 stome e.g. , 2011 exCimex pipistrelli Jenyns, 1839 is known as an obligate parasite of bats which may share its hosts with Cimex lectularius. The karyotype of Cimex pipistrelli is similar to the standard complement of Cimex lectularius but contains a higher number of autosomes  depending on information available from the collectors. Individual sites within a single city are differentiated by numerals . Bugs identified as Cimex lectularius were also collected at four sites PageBreakPageBreakof bat roosts in the Czech Republic and Slovakia. The complete list of the collecting sites is shown in The studied specimens of 010\u20132012 . The kar1-nY where n stands for the additional X chromosomes. The corresponding karyotypes of females were characterized by the formula 2n=26+2X1-n.The chromosome preparations were made from gonads or midgut using the spreading technique described by After withdrawing of tissues for cytogenetic methods, the material was preserved in 96% ethanol and used in parallel molecular studies. Their results have approved the original specimens determination according to morphological characters , 2013. TCimex lectularius analysed contained 26 autosomes and a varying number of the sex chromosomes. The relative length of chromosomes in the complement was successively diminishing from 5.3 to 1.7%. No distinct size groups of chromosomes could be differentiated; however, the largest and the smallest autosomal pair could be usually recognized according to their size. The original sex chromosomes X1X2Y were medium-sized whereas their supposed fragments occurring in the karyotypes with higher chromosome numbers were the smallest elements of the set.The karyotype of all the specimens of Cimex lectularius studied, 12 distinct karyotypes were differentiated  consisted of a single specimen only. The results recorded in individual collecting sites are summarized in 1X2Y) and 2n=30 in females PageBreakPageBreak(2n=26+X1X1X2X2)  . This cot roosts .1-3Y) and 2n=32 in females (2n=26+2X1-3)  . This chPageBreakKaryotype 3 included complements with four X chromosomes; 2n=31 in males (2n=26+X1-4Y) and 2n=34 in females (2n=26+2X1-4) (6+2X1-4) . This co1-5Y) and 2n=36 in females (2n=26+2X1-5)  . It was 1-6Y) and 2n=38 in females (2n=26+2X1-6)   and it w1-7Y) and 2n=40 in females (2n=26+2X1-7)   and it w1-8Y)   and it wPageBreakKaryotype 8 included a complement with nine X chromosomes and 2n=36 in males (2n=26+X1-9Y) (6+X1-9Y)  and it w1-10Y)   and it w1-13Y)  . This kaPageBreakKaryotype 11 included a complement with 15 X chromosomes and 2n=42 in males (2n=26+X1-15Y) (+X1-15Y) . This ka1-20Y)  . This coThe females exhibited the number of chromosomes which was usually complementary to the number established in the males from the same sample. However, 11 polymorphic samples were revealed in which the karyotypes of females and males were not complementary one another. Two females showing karyotypes with odd numbers of X chromosomes  were recorded .PageBreakrespectively). Mosaicism with two and five X chromosome fragments was also revealed in a male from Italy (Venezia 3) .The occurrence of chromosomal mosaics with the karyotype constitution varying between cells of single individual was observed in five specimens  from five samples. A female from Slovakia (Trnava) had two karyotypically different cell types. The complement with 14 X chromosome fragments (2n=40) was found in mesenteron cells, whereas 17 X chromosome fragments (2n=43) were observed in germinal cells from ovarium. In other individuals showing mosaic, variation was recorded between germinal cells derived from gonads only. In a single female from Austria (Melk), the karyotypes with 12 and 14 X chromosome fragments were recorded . A male from Janov in the Czech Republic showed cells with six or seven X fragments . A similar mosaic constitution was recorded in a female from \u010cesk\u00e9 Bud\u011bjovice (4) . The Trnava sample contained five males with different karyotypes  and a female showing a mosaic karyotype with different chromosomal numbers observed in both examined tissues. The Melk sample included a male with 2n=26+X1-10Y, and two females, one with 2n=26+2X1-7 and another with a mosaic karyotype constitution 2n=26+2X1-12/14.The karyotypes with higher chromosome numbers as well as individuals with chromosomal mosaic were usually found within the samples exhibiting particularly extensive variation between individuals. A sample from Liberec contained two males with karyotypes 2n=26+XCimex pipistrelli included 28 autosomes and the sex chromosome trivalent X1X2Y . This represents the highest X chromosome number recorded within Cimicidae, Heteroptera and probably also Insecta.We have obtained certain findings that are at variance with the previously published results. The distribution pattern of incidence of the X chromosome fragments reported by PageBreakThe results obtained in Cimex pipistrelli confirm the previously published data  has already become widely fixed in the extant populations. However, it is not sure that the assumed original fission resulted always in the formation of the same fragments. Similarly, the nature of subsequent fissions producing successively other fragments is not clear and may vary. The karyotypes of females with higher numbers of the X chromosome fragments could be heterozygous with varying constitution of fragments derived from parents. This possible variation cannot be evidenced with the use of classical cytogenetic techniques and molecular approach should be employed in clarifying this question.Therefore, the most plausible explanation of the origin of the supernumerary elements in the bed bug complements remains fragmentation of the X chromosome. This mechanism produces a complicated system of multiple sex chromosomes and it was proposed already in previously published papers  not related to humans (The causes of the origin and maintenance of extensive fragmentation of the X chromosome of bed bugs remain unclear. Populations of bedbugs have been exposed to various insecticides all over the world for decades . Potentio humans .PageBreakThe irregular meiotic division or meiotic drive may enhance segregational variation in the chromosome number in progeny as well as mitotic segregation disturbances may contribute to the origin of mosaics in somatic cells. Mating of individuals of different origin and possibly different genetic constitution may initiate and increase the occurrence of segregation problems. We can assume that non-disjunction can produce unbalanced aneuploid gametes and result in lowered fitness of individuals carrying higher numbers of the X chromosome fragments. This effect should be apparently enhanced with the increasing number of the fragments and this may be the reason for the observed distribution pattern of individual karyotypes in the studied samples and the rarity of individuals with extremely high chromosome numbers. On the other hand, meiotic drive could cause preferential transmission of certain karyotype variants to the offspring. We found an extraordinarily wide extent of karyotype variation between specimens in a few population samples only. We assume that this extreme variation might result from random mixing of individuals of different origin at a single site. Mating between geographically unrelated individuals can easily be imagined in a parasite such as the bed bug transmitted by migrating people. However, it is difficult to explain why these highly variable samples usually included specimens with an extreme karyotype constitution and the highest numbers of the X chromosome fragments. It is obvious that mating of parents with different karyotypes can produce great variety of recombinant complements in offspring, particularly in females. Variation in the number of chromosome fragments may be associated with abnormalities occurring in chromosome segregation during the cell division. The regular course of meiosis in the bed bug may be influenced by the holokinetic nature of chromosomes, completely achiasmatic male meiosis and inverted meiosis of the sex chromosomes. We can only speculate about relationships between the system of transmission of the fragmented sex chromosomes and the unusual features of reproductive biology of bed bugs. The assumed lowering of the fitness of individuals carrying higher numbers of the X chromosome fragments could potentially affect population dynamics of variable populations. It is apparent that more intensive cytogenetic screening combined with data on molecular variation in DNA sequences might shed light to this question."}
+{"text": "Hepatitis C virus roots a chronic liver disease. Currently approved treatment strategy includes administration of alpha interferon and ribavirin combined therapy for 24-48 weeks. One of the predictor of sustained virological response is an early virological response to treatment characterized as rapid response. Hyper variable region 1 (HVR1) of E2 protein is responsible for viral entry and acts as a target for neutralizing antibodies. Any mutation in this region would effect virus interaction with target cell and viral persistence.Thirty one clones of six pre-treatment samples subjected to combination therapy were investigated. Three of the patients were rapid responders  and two were breakthrough responders (BT1 and BT2). Envelope 2 gene was amplified, cloned and sequenced. Amino acid substitution, frequency, composition and antigenic properties of HVR 1 of E2 protein were studied.In both rapid responders (R.R) (14 amino acid sites) and breakthrough responders (BT.R) (13 amino acid sites) half of the amino acid sites were either conserved or resistant to any physiochemical change due to amino acid substitution. It also indicated that average composition of hydrophilic and basic amino acids were comparatively lower in rapid responders than other samples affecting probable interaction of virus with target cells. A central non antigenic region was constant among the breakthrough responders but differed in length significantly among rapid responders reflecting the adaptive nature of HVR1 to the immune response.We observed that although HVR1is quite variable region in HCV 3a patients responding differently to treatment it still maintains its physiochemical properties for its proper functioning and viability. HCV roots a persistent infection which advances towards chronic hepatitis, liver steatosis, cirrhosis and hepatocellular carcinoma -3. It isThe standard therapy for HCV treatment adds up to taking Alpha-interferon 3 MU three times a week  and 1000effect the outcome of treatment. Rapid responders have recently been identified as positive predictors of sustained virological response in Pakistan [In HCV E1 and E2 protein are heterogeneous, especially hyper variable regions which include HVR1, a 27 amino acid sequence (81 nucleotides) ,12. ManyPakistan . TherefoIn HVR1, four amino acids position 2, 6, 19 and 23 were conserved in all the 31 variants. Conserved and variable sites among variants of rapid responders and among variants of breakthrough responders were also analyzed  at amino acid position 21.In breakthrough responders variable sites that maintained their physiochemical properties were amino acid 5 and 8 that were exclusively polar neutral sites. Amino acid 16 was exclusively non polar and hydrophobic, this amino acid is a conserved site in rapid responders as well. Amino acid 27 was also exclusively polar and hydrophilic in breakthrough responders, whereas in rapid responders this site was almost conserved with polar hydrophilic properties and very rare non polar amino acid substitution (1 out of 15 in our case).Amino acid 16 and 27 maintained their physiochemical properties despite of amino acid substitution in breakthrough responders. Amino acid 16 was at conserved site while amino acid 27 maintained its physiochemical properties in rapid responders thus amino acid 16 and 27 can be considered as sites which are resistant to a functional change.Similarly amino acids 9, 20 and 21 maintained their physiochemical properties in rapid responders were either at conserved sites in breakthrough responders or were resistant to physiochemical changes.Five amino acids sites that were conserved sites in both rapid responders and breakthrough responders are 2, 6, 10, 19 and 23. Five sites that were either conserved sites in one of the two groups or that maintained their physiochemical properties in both the groups were 16, 27, 9, 20 and 21. In total ten amino acids sites were exclusively conserved between samples responding differently to the treatment. Remaining seventeen sites were displaying different physiochemical properties.In rapid responders ten amino acid sites were conserved. Four amino acid sites were not conserved but maintained their physiochemical properties. Therefore among rapid responders more than half part of the HVR1 (fourteen amino acid sites) were stable functionally. In breakthrough responders nine amino acids were conserved. Amino acid substitution in four amino acid sites resisted any physiochemical change. Therefore like rapid responders almost half of the region of HVR1 remained stable functionally.Frequency of individual amino acid in HVR1 was compared between samples  than R.R group (17.6%). Acidic amino acids composition was slightly greater in breakthrough responders (2.22%) than in rapid responders (0.23%). . In R2 antigenic region extended from position 6 to 11 and remained constant in all variants of R2. In sample R3 antigenic region started a bit earlier and ended a bit earlier from position 4 to 8  and two breakthrough responders (BT1 and BT2) were compared at amino acid level. Total number of conserved and variable sites remained almost same in both the groups opposing the common assumption that responders harbors less variability than the non responders. Fourteen out of twenty seven amino acid positions were resistant to any change in physiochemical properties in rapid responders. On the other hand half of the amino acids were conserved or resistant to physiochemical shifts in breakthrough responders. Although number of conserved and variable sites was same in both groups their positions varied between rapid responders and breakthrough responders. Ten amino acids sites were exclusively conserved between samples responding differently to the treatment. Half of these sites were either conserved in one of the two groups or retained their physiochemical properties. Other half of the amino acids did not harbor any substitution at all. Another study confirmed that despite of high variability in HVR1, at most of the position hydropathic characters remained conserved . AnotherIt has been already noted that neutral amino acids are more frequent in HVR1 than in common proteins . It has Hydrophilic residues are exposed on the surface and are involved in interactions with other molecules whereas hydrophobic amino acids anchor E2 to core and maintains conformation of HVR1 . In the Most frequent amino acids in HVR1 were glycine (G). threonine (T), serine (S) and alanine (A)  and ribavirin (10 mg/kg per day) for 6 months. Serum samples taken before the onset of treatment were examined. Three patients  showed a rapid response to treatment as defined by negative HCV RNA (<500 IU/ml) after 1 month of treatment. Two patients (BT1 and BT2) showed a breakthrough virological response during treatment followed by reappearance of HCV RNA at the end of treatment. One of the patient showed negative HCV RNA at the end of treatment and was defined as end of treatment response (ETR). The subjects gave informed consent to participate and the study was conducted in accordance with the 1964 Declaration of Helsinki and Guidelines for Good Clinical Research Practice in Pakistan. The study was approved by Ethics Committee of Molecular Virology Division.Hepatitis C viral RNA was extracted from 100 \u03bcl of stored serum of the patients using Gentra RNA isolation Kit  according to the kit protocol. cDNA synthesis of extracted RNA (10 \u03bcl) was done at 37\u00b0C for 50 min and 42\u00b0C for 10 min using 200 units of Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RTase) (Invitrogen) and 8 Units of RNase inhibitor in a 20 \u03bcl of reaction with E2 outer anti sense primer (GTATTACGAGGTTCTCCAAAGC). For nested PCR amplification of entire E2 gene two sets of primers were optimized. The first round of PCR was done with 10 pM of E2 outer antisense primer and with 10 pM of outer sense primer (TCCTGGTAGTGCTGCTGCTA). Four microliter of the first round PCR products was used as a template for second round of nested PCR using inner sense (GAAACCCACGTCACCGGGGGAA) and inner antisense primers (CGCCTCCGCTTGGGATATGAGTAACA). The amplification conditions were same for both PCR rounds: initial denaturation at 94\u00b0C for 2 min followed by 35 cycles consisting of denaturation at 94\u00b0C for 45 sec, primer annealing at 50\u00b0C for 45 sec and primer extension at 72\u00b0C for 3 min followed by a final extension of 10 min. The amplified products were visualized on 1.2% agarose gel stained with ethidium bromide under UV transilluminator after electrophresis. The required bands were excised and DNA was purified from the agarose gel with Silica Bead DNA Gel Extraction Kit (Fermentas Inc. Germany). Purified DNA was suspended in depc treated water and used for cloning and sequencing.Purified E2 amplified products were directly ligated to 50 ng of PCR 2.1 vector . The product was transformed into chemically competent TOP10F' cells by incubating the entire ligation reaction along with competent cells on ice for 30 min then a 1 minute heat shock was given to cells at 42\u00b0C after which the cells were immediately transferred to ice. Then the cells were incubated with 500 ul of Luria-Bertani (LB) medium at 37\u00b0C for 1 hour. Successfully transformed cells were selected on LB agar plate supplemented with 100 \u03bcg/ml ampicillin and 12.4 ug/ml tetracycline. Plates were subjected to overnight incubation at 37\u00b0C. Clones carrying E2 gene were confirmed by colony PCR. 50 ul of culture was centrifuged at 13000 rpm for 2 min. supernatant was discarded and pellet was resuspended in 50 \u03bcl of 1X T.E. resuspended pellet was subjected to heat shock at 100\u00b0C for 10 min followed by centrifugation at 13000 rpm for 2 min. Supernatant that was supposed to contain the plasmid was used as a template for the PCR reactions. These PCR reactions were prepared both with 10 pmol vector-specific primers M13 forward (TAATACGACTCACTATAGGG) and M13 reverse (TAGAAGGCACAGTCGAGG) and with gene specific primers (E2 inner sense and inner antisense). Bands of size approximately 1056 bp with gene specific primers and 1183 with vector specific primers were visualized under UV confirmed successful cloning. Plasmid was isolated from cultures containing E2 clones using a Plasmid Miniprep Kit, (Fermentas Life Science technologies USA) according to manufacturer's protocol. From each patient 5-10 positive clones were randomly selected for sequencing.For each clone, both strands of E2 gene were sequenced with M13 forward and reverse primers by Applied biosystems prism dye termination method according to the manufacturer's instructions . Sequencing was performed on automated sequencer . Sequencing PCR reaction was performed with profile; 96\u00b0C for 15 sec, 50\u00b0C for 10 sec, 60\u00b0C for 4 min repeated 35 times. Labeled DNA was then purified with 2 \u03bcl of 3M Sodium Acetate and 2 \u03bcl of 125 mM EDTA followed by precipitation with 26 \u03bcl 100% ethanol. After 15 min incubation at room temperature DNA was centrifuged for 30 minutes at 2800 rpm at 4\u00b0C. Washing was done with 36 \u03bcl of 70% ethanol. Pellet was air dried and rehydrated in 12 \u03bcl formamide. Pellet was chilled on ice after it was heat shocked at 95\u00b0C for 5 minutes. Air dried pellet was then analyzed using an ABI prism sequencer. A set of 31 variants was analyzed containing 2-10 clones selected from each patient. . Sequences were analyzed with the Chromas LITE programme .http://www.clcbio.com.Amino acid heterogeneity analysis was done by aligning amino acids using Bioedit clustal W programme. These aligned sequences were then analyzed using MEGA version 4.1 . Number Antigenic regions were identified within the HVR1 of Envelope protein by analyzing antigenic profiles of all the variants by the method of Parker et al. .HM590012 to HM590017, HM584119 to HM584121, HM462252, HQ184929 to HQ184935, HQ202715 to HQ202721 and HQ189124 to HQ189130Accession numbers of nucleotide sequences submitted to GenBank are The authors declare that they have no competing interests.MI conceived the study, participated in its design and coordination and gave a critical view of manuscript writing. MA performed and analyzed the results. SA, AH, MI, MA, SB and BK participated in results analysis. All the authors read and approved the final manuscript."}
+{"text": "Mild hypoxia is common after stroke and associated with poor long-term outcome.Oxygen supplementation could prevent hypoxia and improve recovery. A previousstudy of routine oxygen supplementation showed no significant benefit at 7 and12 months. This pilot study reports the effects of routine oxygensupplementation for 72 hours on oxygen saturation and neurological outcomes at 1week after a stroke.Patients with a clinical diagnosis of acute stroke were recruited within 24 hof hospital admission between October 2004 and April 2008. Participants wererandomized to oxygen via nasal cannulae (72 h) or control . Clinical outcomes were assessed byresearch team members at 1 week. Baseline data for oxygen(n\u200a=\u200a148) and control (n\u200a=\u200a141)did not differ between groups.The median (interquartile range) National Institutes of Health Stroke Scale(NIHSS) score for the groups at baseline was 6 (7) and 5 (7) respectively.The median Nocturnal Oxygen Saturation during treatment was 1.4%(0.3) higher in the oxygen than in the control group (p<0.001) during theintervention. At 1 week, the median NIHSS score had reduced by 2 (3) in theoxygen and by 1 (2) in the control group. 31% of participants in theoxygen group and 14% in the control group had an improvement of \u22654NIHSS points at 1 week doubling the odds of improvement in the oxygen group(OR: 2.9).Our data show that routine oxygen supplementation started within 24 hours ofhospital admission with acute stroke led to a small, but statisticallysignificant, improvement in neurological recovery at 1 week. However, thedifference in NIHSS improvement may be due to baseline imbalance in strokeseverity between the two groups and needs to be confirmed in a larger studyand linked to longer-term clinical outcome.ISRCTN12362720; European Clinical Trials Database 2004-001866-41Controlled-Trials.com  Hypoxia is common after acute stroke, affecting up to 63% of patients at sometime after admission invitro studies that oxygen encourages the formation of toxic freeradicals leading to further damage to the ischaemic brain Oxygen treatment is not without side effects There is only one large (n\u200a=\u200a550) quasi-randomized study ofoxygen supplementation for acute stroke, and this suggests that routine oxygentreatment to unselected stroke patients does not reduce morbidity and mortalityThe evidence for oxygen treatment after acute stroke from experimental and clinicalstudies is conflicting, and, unsurprisingly, stroke management guidelines differwhile based on the same sparse evidence In this paper we report on a pilot study examining the effects of routine fixed doseoxygen supplementation at a rate of 2 or 3 L/min dependent on baseline oxygensaturation for 72 hours on oxygen saturation and neurological recovery at 1week.The protocol for this trial and supporting CONSORT checklist are available assupporting information; see This is a randomized controlled single blind pilot study of routine oxygensupplementation after acute stroke. A double blind study could not be used sincemedical staff would know the group allocated due to the presence/absence ofnasal cannulae. Patients were recruited from the University Hospital of NorthStaffordshire (UHNS), UK, a large teaching hospital admitting about 800 patientswith stroke per year. Depending on bed availability, most stroke patients areadmitted to the acute stroke unit within 24 hours of presentation. Patients withan admission diagnosis of stroke or possible stroke were identified by a memberof the stroke research group, who checked the medical admissions unit log bookevery morning and was contactable by the medical admissions team for new strokesin the day. Adult patients with a clinical diagnosis of acute stroke Recognised indications for oxygen treatment such as oxygen saturation onair <90%, acute left ventricular failure, severe pneumonia,pulmonary emboli, and chronic respiratory failure treated with long termoxygen at home.Recognised contraindications to fixed dose oxygen treatment , e.g. type II respiratory failure.Stroke was not the patient's primary clinical diagnosis. Patientswith other serious life-threatening illnesses likely to lead to deathwithin the next few months were also excluded.2) was greater than93% or at a rate of 3 L/min if baseline SpO2 was 93% orless. This dose was based on the results of earlier studies 2 were assessed regularly (at least three times a day) as partof routine clinical care. Those who developed indications for oxygen, or neededa higher concentration of oxygen than the protocol prescribed, were given theconcentration of oxygen they needed by the treating clinician, irrespective ofthe treatment group they were in.Participants were randomized to one of two treatment groups, the oxygen group andthe control group. Participants in the oxygen group were given oxygen at a flowrate of 2 L/min if baseline oxygen saturation , partial anteriorcirculation syndrome (PAC), lacunar syndrome (LAC) and posterior circulationsyndrome (POC) using the Oxfordshire Community Stroke Project (OCSP)Classification 2 every second producinga moving average every 5 seconds using an internal algorithm. Hands wereinspected to ensure that the fingers were warm and well-perfused. Nail varnishwas removed and long fingernails were clipped, where necessary. The pulseoximeter was attached to the wrist and the sensory probe was fitted to the indexfinger. To reduce movement artifacts the hemiparetic side was used for oximetry2<90%) was corrected to a notional 8 h period usingthe formula: T<90 (in minutes) \u200a=\u200a  x 480. The same corrections were usedfor T<80 and T>98.Oxygen saturation and heart rate were assessed using a Pulsox-3I pulse oximeter(Konica Minolta). The instrument records SpOThe protocol was approved by the North Staffordshire Research Ethics Committee on06.10.2004 (ref: 04/Q2604/73). It is registered in the International StandardRandomized Controlled Trial Number Register (ISRCTN12362720) and the EuropeanClinical Trials Database (EudraCT number 2004-001866-41). Written informedconsent was sought from all study participants. Assent from the next of kin wasaccepted if the patient agreed to take part but was unable to give fullyinformed consent. Participants who were incompetent at the time of recruitment,but competent when followed-up at 1 week were asked to confirm consent. Patientswho were recruited to the study by assent and refused consent at 1 week werewithdrawn from the study.Descriptive statistics and counts were performed using Microsoft Excel, MicrosoftOffice XP, Microsoft Corporation, US, and tests of significance (as detailed inthe text) were carried out using SPSS statistical software version 15.0 . Statistical significance was accepted if thep-value was <0.05.Three hundred and one patients were recruited to the study from October 2004 toApril 2008, with a mean recruitment rate of 7 (range 1 to 23) per month; theflow of patients through each stage of the study is shown in Baseline demographic and clinical characteristics, stroke type and severity, andoxygen saturation at randomization were similar in the control and oxygen group.The mean age of patients included in the study was 72.3 (SD 11.6) years, 137(47%) were male, 243 (84%) were independent for activities ofdaily living before the stroke, and 113 (39%) were living alone.Concomitant medical problems included ischaemic heart disease in 71(25%), congestive cardiac failure in 34 (12%), atrial fibrillationin 53 (18%), and chronic obstructive pulmonary disease in 26 (9%)of participants. The majority of patients were normoxic with a mean oxygensaturation of 96.1%  atrandomization. The mean time from stroke onset to randomization was 17\u223612(SD 8\u223642) hh\u2236min. Bamford classification data is included in Inclusion was via fully informed consent in 115 (78%) participants in theoxygen group and 106 (75%) in the control group and by assent in 33(22%) participants in the oxygen group and 35 (25%) in the controlgroup. At the 1 week reassessment, patients who were included via assent wereasked to confirm consent if they had regained competence. One patient in thecontrol group and 1 patient in the oxygen group refused consent at that pointand were withdrawn.2 of less than 90% . Twelve patients (8%) in the oxygen group and 16 patientsin the control group (11%) were prescribed oxygen for clinicalindications during the treatment period (p\u200a=\u200a0.4Fisher's Exact test).Oxygen saturation was assessed 3 times a day during the first 72 hours as part ofroutine clinical practice. In 48 (32%) patients in the oxygen group and52 (47%) patients in the control group one or more readings showed anSpO2of less than 80% and 90% and the number of participants who spentmore than 1 hour with a saturation of less than 90%. The median MeanNocturnal Oxygen Saturation was 1.3% higher in the treatment group thanin controls . A lower proportion of patients inthe oxygen group spent more than 5 min and more than 30 min with anSpO2 of <90% during the recording night . There was no significant difference in the proportion of patientswho spent more than 60 min with an SpO2 of less then 90%, orin the time spent with an SpO2 of less than 80%, since suchvery severe hypoxia was uncommon, and would have been treated as soon asidentified by the clinical team.Pulse oximetry was conducted successfully in 98 patients in the oxygen group andin 100 controls. All parameters of oxygenation were significantly better in theoxygen than in the control group except the time spent with an SpOEighty-nine participants (29%) had no or insufficient oximetry data foranalysis. This equated to missing values of 41 and 48 for the controls andoxygen group respectively. There was no significant difference in theproportion of patients returning no or insufficient oximety data between the twogroups. Participants who did not complete oximetry were older than those whocompleted (74.1 vs. 71.4 years), less likely to have been independent before thestroke (73% vs. 89%), less likely to have full scores on the GCSverbal response item (57% vs. 71%), less likely to be able to walk(8% vs. 17%), and were recruited sooner after stroke onset(15\u223620 vs. 17\u223610 hh\u2236mm). Otherwise there were no significantdifferences in baseline. Further information on oxygen saturation is provided inAnalysis was by intention to treat. Neurological scores improved from baseline to1 week in both groups. At baseline the median (IQR) NIHSS was 6 (7) in theoxygen and 5 (7) in the control group. By 1 week the median NIHSS scores(including deaths) had improved more in the oxygen than in the control group\u22122 (3) for oxygen vs. \u22121 (2) for control, p<0.001 Mann WhitneyU-test). A significant improvement in neurological scores was defined asreduction of 4 or more points in the NIHSS from baseline to week 1. The oddsratio (95% CI) for significant improvement with oxygen supplementationwas 2.9 (1.59\u20135.4). Forty five (31%) participants showed asignificant improvement in neurological score in the oxygen group compared to 18(14%) participants in the control group . There wOxygen treatment might be more effective in older patients with a history ofheart and lung problems, those with lower baseline oxygen saturation, patientswith more severe strokes, or those with an impaired level of consciousness. Itmight also be affected by treatment with oxygen before randomization and theaetiology of the stroke (infarct or haemorrhage). We therefore carried out anexploratory subgroup analysis to identify subgroups which might benefit more orless from oxygen treatment. The odds ratios for significant improvement withoxygen treatment (NIHSS reduction from baseline of 4 or greater) There was no significant difference in the group means for oxygen and controlrespectively for highest systolic blood pressure (167 SD 29 mmHg vs. 167 SD 28mmHg), highest diastolic blood pressure (93 SD 19 mmHg vs. 91 SD 15 mmHg), andheart rate (92 SD 16 BPM vs. 92 SD 19 BPM) during the 72 hours of the treatmentperiod. The highest mean temperature during the first week was similar in bothgroups (37.2% SD 0.6 vs. 37.1% SD 0.7 C). There was no differencein the proportion of patients requiring antibiotics in the first week(n\u200a=\u200a27 (18%) vs. n\u200a=\u200a22(15%) for oxygen and control respectively) or sedative medications(n\u200a=\u200a9 (6%) vs. n\u200a=\u200a9(6%) for oxygen and control respectively).The main finding of this study is that routine oxygen supplementation, given for 72hours at a rate of 2 or 3 L/min, dependent on baseline oxygen saturation leads, to asmall but statistically significant improvement of neurological recovery at 1 week.The odds of improving by 4 or more NIHSS points at 1 week were doubled in the oxygengroup. While statically significant the difference in the improvement in NIHSS issmall and may be due to the non-significant baseline imbalance in stroke severitybetween the two groups (baseline NIHSS was 1 point higher in the oxygen than in thecontrol group). The NIHSS is not designed as an outcome tool, and the relevance of asmall change in NIHSS during the first 7 days on longer-term functional outcome isunclear.However, other studies have shown that baseline NIHSS is a strong predictor ofrecovery and long-term outcome Our data contrasts with an earlier study by Ronning and Guldvog et alA more recent, but small, study by Singhal While we aimed to include a representative sample of all stroke patients admitted tothe hospital, the majority of subjects included had relatively mild strokes. Themedian NIHSS at baseline in this study was 6 and 5 in the oxygen and control groupsrespectively, while the median NIHSS of patients admitted to the local stroke unitis about 8 . The main reason forthis is the requirement of informed consent or assent and the short time betweenadmission and recruitment. While patients with milder strokes can give informedconsent, those with more severe strokes can only be included if assent from the nextof kin can be gained. By the time subjects are identified for the study, relativeshave usually left hospital and are then contacted during visiting times or have totravel back in to discuss the study. Patients with more severe strokes were morelikely to benefit from oxygen in the Ronning and Guldvog study The results of the overnight pulse oximetry showed that oxygen supplementationeffectively improved all parameters of oxygenation. Mean Nocturnal Oxygen Saturationwas 1.3% higher in the oxygen group than in the control group. In two smallerpreparatory studies we have shown that oxygen supplementation at a rate of 2 L/minduring the day with compliance ensured by continuous observation raises oxygensaturation by 2% Oxygen supplementation is simple, cheap, and applicable even in the most basicclinical environments. The results of this study suggest that oxygen given at 2L/min for 72 h may improve neurological recovery at 1 week. The results of thispilot study should be confirmed in a larger fully powered trial, which is nowongoing (The Stroke Oxygen Study) Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file."}
+{"text": "In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.During November-December 2009, we collected leftover serum from a blood bank, a pediatric children's hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers \u226540 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5\u201317 years (53%) and young adults aged 18\u201324 years (47%), while adults aged \u226550 years had the lowest cumulative incidence (11\u201313%) of pH1N1 virus infection.After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults. The 2009 pandemic influenza A (H1N1) virus (pH1N1) was first identified in April 2009 and caused widespread illness in the United States and around the world During the 2009 pandemic, Florida employed a surveillance system that tracked the percentage of Emergency Department (ED) visits for influenza-like illness (ILI) throughout the state. According to surveillance data, Tampa Bay experienced a gradual increase in influenza activity in the spring and summer of 2009, followed by a large fall wave of influenza activity that peaked in late October and decreased steadily thereafter . EstimatSerosurveys, which estimate the prevalence of antibodies to a specific pathogen, can be a valuable tool in determining the proportion of the population infected with a novel virus. Unlike most influenza surveillance, which relies on presentation of clinical illness, serosurveys capture persons that experienced symptomatic or asymptomatic illness, and can provide information on total infections which may be underestimated with traditional surveillance methodologies. However, serosurveys are limited by the sensitivity and specificity of the assay employed to detect antibody titers To date, one published study has reported on the prevalence of pH1N1 antibodies among residents in one region of the United States In November and December 2009, after pH1N1 virus activity in Tampa Bay had peaked , we collWe sought to collect 160 specimens from each of six age groups: <5 years, 5\u201317 years, 18\u201324 years, 25\u201349 years, 50\u201364 years and \u226565 years. Infants less than 6 months were excluded due to the potential for maternal antibody transmission. The required sample size was calculated using relative standard error measurements. We estimated that the lowest seroprevalence for all groups would be among adults aged \u226565 years. At the time of the serosurvey, we estimated that 15% of this age group would be seropositive, requiring a sample size of approximately 160 to maintain a relative standard error less than 20%. We were able to collect at least 160 samples from all ages groups except for children aged <5 years for which we were only able to collect 60 samples. We estimated that the seroprevalence among this age group would be high (30%), and therefore despite the small sample size, would meet the relative standard error criteria of less than 20%.This study was proposed to the Florida Department of Health Institutional Review Board (IRB) and Centers for Disease Control and Prevention (CDC) IRB who considered the investigation as public health response, and therefore not subject to IRB review and approval.Leftover serum specimens for Tampa Bay residents aged \u226516 years were collected from a large blood bank testing facility during a 4-day period from November 30 -December 3, 2009 . For resAntibodies against pH1N1 virus were detected by the hemagglutination-inhibition (HI) assay as previously described using A/California/07/2009 virus Using sera from patients with pH1N1 laboratory-confirmed infections and non-exposed United States residents, a previous study determined that a threshold HI titer \u226540 yielded a sensitivity of 75% and specificity of 97% in determining previous infection with the pH1N1 virus among persons <60 years of age Because serology cannot differentiate between antibodies produced by virus infection and response to vaccination, we developed a simple statistical model to estimate the proportion of seropositive results due to pH1N1 vaccination coverage  . MonthlyOverall, 27% of the study sample had pH1N1 antibody titers \u226540 with an age-standardized seroprevalence of 25% . A cumulThe overall BRFSS/NHFS vaccination coverage estimate for Florida two weeks prior to the time of specimen collection was 9.0%, with the highest vaccination coverage among children aged <5 years (17%) and school-aged children aged 5\u201317 years (15%) . Based oThe proportion of Tampa Bay residents that were estimated to be seropositive due to infection with pH1N1 virus after the peak of the fall wave was 25% . The higBy December 2009, an estimated 30% of Tampa Bay's population had elevated levels of antibodies against the pH1N1 virus (25% from infection and 5.9% from vaccination). Our estimates indicate that half of young adults aged 18\u201324 years and more than half of school-aged children had antibodies to the pH1N1 virus at titers of \u226540 at that time. Thus, the proportion of the Tampa Bay population among these age groups that remained susceptible to pH1N1 virus infection by December 2009 was markedly decreased.Our seroprevalence estimates are similar to previously published studies from Pittsburgh, PA Though the availability of pH1N1 vaccine was still limited by the time of the serosurvey , school-aged children comprised one of the target groups for initial vaccination campaigns, and thus were more likely to receive the vaccine earlier Our survey has several potential limitations. First, we did not have baseline serum specimens prior to the 2009 pandemic for comparison and therefore were not able to test for a four-fold rise in antibody titers or adjust for pre-existing, cross-reactive antibody titers. Previous studies have suggested that cross-reactive antibodies were most common among persons aged \u226560 years In summary, we performed a serosurvey in Tampa Bay to determine the prevalence of antibodies to the pH1N1 virus after the fall wave of the pandemic, and during the early phases of the pH1N1 vaccination campaign in Florida. We adjusted seroprevalence results to account for HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination. Our results provide evidence for substantial immunity against the pH1N1 virus among the Tampa Bay population. Though disease activity decreased after December 2009, vaccination levels continued to increase in Florida; thus the proportion of the population with immunity to the pH1N1 virus by the end of the pandemic was probably higher than the estimates presented herein.Appendix S1Method of seroprevalence adjustment to account for hemagglutination-inhibition (HI) assay sensitivity and specificity.(DOCX)Click here for additional data file.Appendix S2Simple statistical model used to estimate the proportion of seropositive results due to vaccination.(DOCX)Click here for additional data file.Table S1Statistical model to estimate the proportion of Tampa Bay residents with vaccine-induced pH1N1 virus seropositivity in November- December 2009, including all components and equations.1 Estimated from Behavioral Risk Factors Surveillance System (BRFSS) and National pH1N1 Flu Survey (NHFS)2 Vaccine immunogenicity estimates based on published immunogenicity studies 3 Estimated proportion of population with vaccine-induced seropositivity (\u22651\u223640 GMT) \u200a=\u200a (vaccine coverage) x (proportion with \u22651\u223640 antibody response)4 Seroprevalence adjusted for assay sensitivity and specificity. For children and adults aged <65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 97%. For adults aged \u2265 65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 94% 5 Estimated proportion with pH1N1 virus infection prior to vaccination \u200a=\u200a  x (estimated proportion of population with vaccine-induced seropositivity)6 Proportion with vaccine-induced seropositivity not infected prior to vaccination \u200a=\u200a (estimated proportion of population with vaccine-induced seropositivity) minus (estimated proportion of population with pH1N1 virus infection prior to vaccination)7 Proportion infected with pH1N1 virus \u200a=\u200a  minus (proportion with vaccine-induced seropositivity not infected prior to vaccination).(DOCX)Click here for additional data file."}
+{"text": "There was an error in the last paragraph of the Discussion section. The third and fourth sentences should read:\"Interestingly methylation of Trypanosoma brucei H3K76, which is orthologous to histone H3K79 in mammals, is required for replication initiation and overexpression of the Trypanosoma Dot1A methylase results in over-replication[44]. Although this observation suggests that methylation of H3K76 in Trypanosoma achieves the opposite effect than the methylation of H3K79 in mammals, and it is yet unclear whether Trypanosoma H3K76 methylation associates with replication origins, both enzymes seem to be involved in regulating replication re-initiation events and thus might both be involved in processes that mark genomic regions for initiation. \""}
+{"text": "Institutional affiliations 2 and 3 for authors Khalid Belkhir and Nicolas Bierne are incorrect. Affiliations 2 and 3 should be: UMR 5554, ISEM Universit\u00e9 Montpellier II.ST.In addition, the term \"FST\" was incorrectly typeset multiple times in the \"Genetic Diversity and Population Differentiation\" section of the Results. The correct typesetting of this term is F"}
+{"text": "To determine clinical characteristics of patients hospitalized in the United Kingdom with pandemic (H1N1) 2009, we studied 1,520 patients in 75 National Health Service hospitals. We characterized patients who acquired influenza nosocomially during the pandemic (H1N1) 2009 outbreak. Of 30 patients, 12 (80%) of 15 adults and 14 (93%) of 15 children had serious underlying illnesses. Only 12 (57%) of 21 patients who received antiviral therapy did so within 48 hours after symptom onset, but 53% needed escalated care or mechanical ventilation; 8 (27%) of 30 died. Despite national guidelines and standardized infection control procedures, nosocomial transmission remains a problem when influenza is prevalent. Health care workers should be routinely offered influenza vaccine, and vaccination should be prioritized for all patients at high risk. Staff should remain alert to the possibility of influenza in patients with complex clinical problems and be ready to institute antiviral therapy while awaiting diagnosis during influenza outbreaks. In addition, we included infants who had not left the hospital since birth in whom pandemic (H1N1) 2009 had developed. We included transfers from other hospitals when a transfer was for a reason other than influenza and when the history of influenza clearly indicated that it had been acquired at another hospital. FLU-CIN procedures were reviewed and approved by the Ethics and Confidentiality Committee of the National Information Governance Board for Health and Social Care in England for collection, storage, and use of personal data for surveillance purposes.From this source cohort, we defined patients with nosocomial pandemic (H1N1) 2009 as those admitted to a hospital for a reason other than acute respiratory infection in whom respiratory symptoms developed Of 1,520 patients in the FLU-CIN cohort, illnesses in 30 (2.0%) (15 children) met the criteria for nosocomial influenza (Twelve (80%) adults and 14 (93%) children had serious underlying illnesses. The most common illnesses were hematologic malignancy for adults (5), and congenital abnormality or prematurity (7) or malignancy (4) for children.Of the 15 adults, 2 had been admitted for elective surgical procedures; 1 for emergency surgery; and 8 for deterioration of chronic conditions, including complications caused by chemotherapy, malignancy, or transplantation. Two patients were admitted for pancreatitis (1 of whom had underlying myeloma); 1 patient was admitted for obstetric complications, and another patient was admitted for psoriasis. Of 15 children, 3 were admitted for elective procedures, 6 had been in the hospital since birth , 1 was transferred from another hospital, and 5 had acute conditions .None of the patients had received pandemic influenza vaccine. Although 14 adults were eligible because of concurrent conditions, influenza symptoms developed in 11 either before vaccine became available or before they would have seroconverted if vaccinated at the earliest opportunity (vaccine became available in the United Kingdom at the end of October 2009). Four children were eligible because of age and concurrent conditions, and symptoms developed in 3 before vaccine became available or before they would have seroconverted. Only 2 patients (both adults) had received seasonal influenza vaccine.Pseudomonas aeruginosa in an unspecified intravenous line in 1 patient. Twelve patients received antibacterial drugs during their respiratory illness, 2 of whom had Haemophilus influenzae in sputum samples and 1 (co-infected with rhinovirus) who had had a blood culture positive for Klebsiella sp.Twenty-one (72%) of 29 patients (10 children) received antiviral medication as inpatients (data\u00a0were unknown for 1 patient); all initially received oseltamivir as monotherapy. Therapy for 2 patients (nos. 13 and 18) was switched from oseltamivir to zanamivir after 4 days and 10 days, respectively, because drug-resistant virus carrying the H275Y mutation was identified. Administration of antiviral drugs ranged from 0 to 8 days after symptom onset; 12 (57%) of 21 patients who received therapy did so within 48 hours. Sixteen patients were already receiving antibacterial drugs when influenza symptoms began. Two of these patients had a bacterial co-infection: coagulase-negative staphylococci in a blood culture for 1 patient and The most common signs were fever (8 [53%] adults and 12 [80%] children), cough , coryza , and dyspnea (7). Fewer patients had malaise (4); myalgia (3); anorexia, nausea, diarrhea (2 each); and arthralgia, sore throat, headache, vomiting, altered consciousness, sneezing, and rash (a child) (1 each).Median length of hospitalization before onset of influenza symptoms was 11 days for adults (range 4\u201378 days) and 13 days (range 6\u201354 days) for children, excluding infants in a hospital since birth. For infants in a hospital since birth, the interval from birth to onset of influenza signs ranged from 41 to 123 days (median\u00a078 days).Results of chest radiography \u22643 days after onset of influenza symptoms were documented for 8 adults and 5 children (43%). Of these patients, 4 adults and 1 child (38%) had radiologically confirmed pneumonia.>2 organ systems. This level includes all patients with complex conditions requiring support for multiorgan failure (ICU).Level 0 is care given to patients whose care needs can be met through normal ward care. Level 1 care is given to patients at risk for a deteriorating condition or recently relocated from higher levels of care whose needs can be met in an acute-care ward with additional advice and support from the critical-care team. Level 2 care is given to patients requiring more detailed observation or intervention, including support for a single failing organ system and those changing from higher levels of care (high dependency unit). Level 3 care is given to patients requiring advanced respiratory support alone or basic respiratory support and support for Seven adults and 8 children (50%) required level 3 care . One (3%) adult required level 2 care. Six adults required mechanical ventilation and 1 required noninvasive ventilation (data for ventilatory support were unknown for 1 adult). Three children required mechanical ventilation and 1 required noninvasive ventilation. The remaining 4 children who received level 3 care were 3 infants and 1 child, each of whom required a period of close monitoring, but did not ultimately require ventilation. The remaining 7 adults and 7 children required level 0 or 1 care.<7 months, all remaining adults recovered from influenza and were discharged from the hospital.Five (33%) of 15 adults died in the acute-care hospital that provided treatment, 2 within 30 days after symptom onset. Of adults who died, 3 had underlying malignancy  or were immunocompromised and 2 had diabetes (type I and type II respectively). Pandemic (H1N1) 2009 was included in the recorded causes of death for all 5 adults. Although some patients had a prolonged hospital stay of Of 15 children, 3 (20%) were known to have died, although only 1  died at the hospital where surveillance was conducted; acute respiratory distress syndrome/lower respiratory tract infection was stated as a cause of death. Another child, with malignancy, whose death was expected, died at home shortly after discharge. The third child was transferred to another hospital, and cause of death is unknown. All other children recovered from pandemic (H1N1) 2009. Two children remained in the hospital for treatment of their underlying malignancy, and the other children were discharged.\u2013,Although pandemic (H1N1) 2009 produced a generally mild illness, in the United Kingdom, as elsewhere, severe illness developed in a small proportion of relatively young patients who required hospitalization , many cases of nosocomial infection in other patient groups probably have been overlooked, particularly because influenza in these groups is likely to have been milder. Additionally, some patients are likely to have been discharged from a hospital during the incubation period of nosocomially acquired pandemic influenza. Thus, in this case series, detecting such patients would not have been possible.Nosocomial cases in this study occurred equally in adults and children. Consistent with previous findings  (Vaccine against pandemic (H1N1) 2009 became available at the end of October 2009. Assuming a 2-week period for vaccine administration, case-patients in groups at risk with influenza onset dates after November 30, 2009, could have been vaccinated and would have had time to seroconvert (14 days). Using these criteria, we determined that 4 cases (in 3 adults and 1 child) (13%) were potentially preventable by vaccination; 2 of these patients required escalated care, and 1 patient died. Although 72% of patients received antiviral therapy, similar to 75% in the source cohort  2009 in this case series were associated with high rates of illness and death. This finding highlights the need for adherence to infection control guidelines for staff and visitors , staff vaccination, maintenance of clinical suspicion for influenza in areas of high risk, prompt  antiviral treatment for vulnerable patients in whom influenza is possible or likely, and consideration of postponing nonurgent procedures for hematology patients during periods of known high influenza activity. This report demonstrates that nosocomial transmission is a recurrent problem when the prevalence of influenza is high and the total effect of nosocomial influenza is underestimated by outbreak reports alone."}
+{"text": "Malignant peripheral nerve sheath tumours (MPNST) are rare tumours known to occur at high frequency in neurofibromatosis 1 (NF1), but may also occur in other cancer prone syndromes.The North West Regional Genetic Register covers a population of 4.1 million and was interrogated for incidence of MPNST in 12 cancer prone syndromes. Age, incidence and survival curves were generated for NF1.TP53 mutation carriers. However, there were no cases amongst 5727BRCA1/2 carriers and first degree relatives, 2029 members from Lynch syndrome families, nor amongst 447 Familial Adenomatous Polyposis, 202 Gorlin syndrome, nor 87 vHL cases.Fifty two of 1254 NF1 patients developed MPNST, with MPNST also occurring in 2/181 cases of schwannomatosis and 2/895 NF2 patients. Three cases were also noted in TP53 mutations and is confirmed at high frequency in NF1. It appears to be only increased in NF2 amongst those that have been irradiated. The lifetime risk of MPNST in NF1 is between 9\u201313%.MPNST is associated with schwannomatosis and  TP53 mutations[Malignant peripheral nerve sheath tumour (MPNST) are uncommon tumours varying substantially in clinicopathologic features. Previouutations and schwutations. We haveBRCA1, BRCA2, MSH2, MLH1 and MSH6 mutations. The register is highly ascertained for NF1, NF2, FAP, Gorlin syndrome von Hippel Lindau disease[TP53 mutation carriers and an international database for schwannomatosis patients referred into the Manchester laboratory for SMARCB1 mutation analysis. All the registries have been the subject of cancer verification using the regional North West Cancer Intelligence Service (NWCIS). We have previously published risks of cancers in Lynch syndrome[BRCA1 and BRCA2 carriers[MSH2, MLH1, MSH6)[BRCA1/2[The North West Regional Genetic Register covers a region of North West England, based around Manchester, with a population of 4.1 million. The genetic register service covers a number of tumour predisposing syndromes in particular NF1, Neurofibromatosis type 2 (NF2), Familial Adenomatous Polyposis (FAP), Gorlin syndrome, von Hippel Lindau disease and non-syndromic families with  disease. In addisyndrome and canccarriers. The NWCcarriers). As a d1, MSH6) and BRCA[BRCA1/2 as 50% ost April 2010. MPNST incidence curves were derived for NF1 for strict regional residents to avoid ascertainment bias. Five-year survival was determined using Kaplan-Meier curves. NF1 MPNST cases who were identified on the periphery of the region during the study period were included for the survival analysis only. The Mann\u2013Whitney U test and Wilcoxon (Gehan) statistic were used to test between-group differences in age at diagnosis and survival.Death details from the registries were used to calculate survival rates, and death certificates were reviewed to determine cause of death. Follow-up was censored on 1BRCA1/2 carriers and first degree relatives, nor among 2029 Mismatch Repair mutation carriers and first degree relatives from Lynch syndrome families. As the annual rate of MPNST is about 1.25 per million[BRCA1/2 carriers but not yet in Lynch syndrome. There were also no cases amongst vHL, Gorlin or FAP patients although numbers were smaller. The presence of two male cases with MPNST aged 32  and 33\u2009years (lower limb) and one female case of triton tumour aged 5\u2009years amongst carriers of a 524\u2009G\u2009>\u2009A (Arg175His), a 574delC and a 375\u2009G\u2009>\u2009A TP53 mutation respectively suggests a likely association with Li Fraumeni syndrome. The 375\u2009G\u2009>\u2009A mutation affects the invariant splice donor site CG/g\u2009> CA/g in exon 4. The missense and splicing mutation were inherited, but the framshift deletion de novo. There were also a further three benign nerve sheath tumours in three further unrelated TP53 mutation carriers aged 33\u2009years (Vestibular schwannoma) and two paraspinal/extra-dural at ages 50 and 49\u2009years. There were also three cases with schwannomatosis and MPNST. Two of these occurred in a family with a c.846\u2009C\u2009>\u2009G, p.N288K SMARCB1 mutation that we have previously reported as probably pathogenic[SMARCB1 mutation[SMARCB1 mutation analysis was not possible. There were two cases occurring amongst 921 NF2 patients. Both of these have been reported as having occurred after radiation treatment for vestibular schwannoma[The presence of MPNST among the study populations is shown in Table million the lifethogenic. It appemutation. Both hahwannoma,17. No MThere were 52 cases of MPNST amongst our NF1 patients including 43/1059 patients within the strict regional.boundaries of the Cancer Register. Fifteen cases of MPNST have occurred in the strict regional population since 1996 maintaining an incidence rate of above 1 per 1000 NF1 patient per year as 1010 NF1 patients were alive at some point after 1996.Within the strict regional population of 1059 individuals the cumulative risk of MPNST was 11.7% (95% CI 9.7\u201313.7%) by age 70\u2009years (figureThe association between MPNST and NF1 has long been documented-4,18-22.TP53 mutations. The presence of three proven MPNSTs in only 181 schwannomatosis patients is very suggestive of an association. The main difficulty with this association is the previous problems with classifying schwannomatosis[SMARCB1 mutation suggests that this is a real link. It also broadens the tumour spectrum in families with SMARCB1 mutations. There remains a question as to why children with certain SMARCB1 mutations have a very high risk of the highly Malignant atypical teratoid Rhabdoid tumours[In addition to the known link with NF1 this study has confirmed probable links with schwannomatosis and germline omatosis-27. Howe tumours whereas  tumours although tumours,29,30.TP53 mutation carriers with MPNST makes a link with germline TP53 mutations and Li Fraumeni syndrome very likely. It is quite possible that cases of MPNST are buried amongst reports of soft tissue sarcoma[TP53 analysis on MPNST patients. Both the cases of MPNST occurred after the previous report from our group[TP53 mutation was a case of Triton tumour in a three year old[The presence of three confirmed  sarcoma. It is tur group. Nonetheyear old. This reyear old.TP53 mutation, which in addition to an NF1 mutation may cause a very substantial risk of MPNST. Indeed TP53 mutations have been described in the transformation from benign schwannoma to MPNST[TP53 mutation carriers this will need to be confirmed in larger studies.The link between NF2 and MPNST is controversial,17,32-35Up to 50% of malignant peripheral nerve sheath tumours in non NF1 patients harbour NF1 mutations. There iThe poor survival from MPNST particularly in NF1 patients highlights the need for therapies targeted at the main underlying genetic abnormality. Whole genome sequencing is likely to reveal new targets for therapy and the fast reducing cost of such testing may mean that it will be affordable in the clinic within the next 5\u2009years.TP53 mutations as well as those with NF1. Radiation treatment particularly in childhood increases the risk of MPNST in NF1 and may also cause MPNST to occur in NF2.In conclusion MPNST appears to occur at increased frequency in schwannomatosis and in those with germline The authors declare that they have no competing interests.The research and reviews conceived by DGE, initial manuscript and data assessed by DGE, contributions for data on TP53 from JMB and from SMH for NF1. All authors developed the manuscript and approved the final version."}
+{"text": "Acute aortic dissection (AAD) is a potentially fatal condition that requires rapid assessment and treatment. The American Heart Association and American College of Cardiology suggested new guidelines for early diagnosis of AAD in 2010, and so we applied retrospectively that system to our known patients with AAD in a community hospital.We reviewed 166 patients with confirmed AAD regardless of types from January 2000 to April 2013. We evaluated 12 newly proposed risks, based on the new guideline.Abrupt onset of pain was the most frequent symptom (67.4%). 6 patients (3.6%) were grouped under the low risk, 88 patients (53.0%) under the intermediate risk, and 72 patients (43.4%) under the high risk. 90 patients (54.2%) demonstrated a widened mediastinum in the chest X-rays. 3 patients showed a mediastinal widening among 6 patients with low risk. In 7 patients (4.2%), were initially diagnosed with acute myocardial infarction, 3 patients were categorized as intermediate risk group (risk score 1) and the others as a high risk of AAD (risk score 2).The risk score system in new guidelines detected ADD with high sensitivity. But, in addition to applying the new guideline, having high suspicion for AAD enables the early diagnosis to be more accurate."}
+{"text": "Dio2 and Dio3) are differentially regulated by the circadian clock and by changes of the ambient light. The expression level of Dio2 in adult rats (2\u20133 months of age) kept continuously in darkness is modulated by the circadian clock and is up-regulated by 2 fold at midday. When the diurnal ambient light was on, the expression level of Dio2 increased by 4\u20138 fold and a consequent increase of the related protein was detected around the nuclei of retinal photoreceptors and of neurons in inner and outer nuclear layers. The expression level of Dio3 had a different temporal pattern and was down-regulated by diurnal light. Our results suggest that DIO2 and DIO3 have a role not only in the developing retina but also in the adult retina and are powerfully regulated by light. As the thyroid hormone is a ligand-inducible transcription factor controlling the expression of several target genes, the transcriptional activation of Dio2 could be a novel genomic component of light adaptation.Analysis with DNA-microrrays and real time PCR show that several genes involved in the thyroid hormone cascade, such as deiodinase 2 and 3 ( In photoreceptors, light adaptation has been extensively studied and several mechanisms contribute to it: changes of intracellular calcium concentration In the present manuscript, we analyze changes in gene expression occurring during the circadian rhythm and when the ambient light in the circadian rhythm is modified. A microarray analysis identified the gene coding for the DIO2 enzyme as the gene with the largest changes of expression levels. This observation prompted us to investigate whether the observed DNA microarray results could be confirmed with real time PCR and if the corresponding protein levels are also modified after light exposure. Several reports have described a major role of the thyroid hormone cascade during retinal development We measured changes in gene expression in retinas acutely isolated from adult (3 months old) freely moving rats exposed to controlled ambient steady lights. To determine the amount of light impinging on the retina, we performed a similar analysis also in cultivated intact retinas Dio2 was the maximally up-regulated gene after 3 and 6 hours of light and its up-regulation was consistently observed in all replicas. Since DIO2 regulates the availability of the active thyroid hormone, as a consequence, DIO2 regulates the timing of cellular responses to thyroid hormones We performed an initial screening using the DNA microarray technique. We extracted the mRNA from retinas of rats kept overnight in darkness until 7 am (as control) 0 ZT and subsequently retinas of animals that were exposed either for 3 hours (3 ZT) or 6 hours (6 ZT) to a 1000 lux light. From the 31099 probes present in the microarray, we extracted those whose expression level increased by more than 60% in all three replicas . Up-reguStat3 , Ep300 (E1A binding protein p300) and Pax4 (paired box gene 4) involved in retinal transcription Stat3 and Ep300 are involved in the thyroid hormone cascade as downstream transcription factors Three transcription factors were up-regulated both after 3 and 6 hours of light: http://bioinfo.vanderbilt.edu/webgestalt/). Among up-regulated genes we found 11 genes involved in visual functions and eye development: Psen1 (presenilin 1), Crygb , Crygc , Crygd , Grk1 (G protein-coupled receptor kinase 1), Rpgrip1 (retinitis pigmentosa GTPase regulator interacting protein 1), Myo5a (myosin Va), Stat3 , Pax4 (paired box 4). These genes could be involved in the protection of the retina during exposure to bright lights.We performed a gene ontology analysis of up-regulated genes (Gabrr1 (gamma-aminobutyric acid (GABA) receptor, rho 1) and Cacnb2 . Gabbr1 codes for the GABA receptor subunit rho1, one of the subunits particular of GABAC receptors highly expressed in the retina Cacnb2 corresponds to the beta subunit of a voltage gated calcium channel known to modulate the b-wave of the ERG response in dark There were 18 up-regulated genes involved in cell-to-cell communication, synaptic function and transmission of nerve impulse. Among them we found Atp1a1 , Scarb1 , Crh (corticotropin releasing hormone) and Dio2 .Up-regulated genes involved in general cell functions and metabolism were Adra1b , coding for the alpha 1b adrenergic receptor involved in the control of cyclic AMP (cAMP) when epinephrine is present Adra1b could have an important role in light adaptation.Down-regulated genes were those that had a decreased expression level larger than 0.7 and were 23 and 22 after 3 and 6 hours of light exposure for all three replicas , respectDio2. As a consequence, we decided to confirm changes in the expression of genes involved in the production or reduction of thyroid hormone Dio2 and Dio3 with real-time PCR in retinas extracted from freely moving rats kept in different ambient lights.The gene which was consistently and prominently up-regulated after 3 and 6 hours of light was Dio2 increased by about 2 times at ZT 3, ZT 4, ZT 6, ZT 8 and ZT 12. The expression level of Dio2 in rats exposed to the usual light or to an intense light, from ZT 0 increased with time of light exposure, reaching levels 4\u20138 times larger than in control conditions  level of related protein, the expression levels of associated proteins were determined by Western blot  in human WERI-Rb1 cell lines Opn1mw and Opn1sw) Opn1sw and Opn1mwOpn1sw and of Opn1mw was respectively about 30% and 40% lower than at 0 ZT in the dark  to locate the existence of a DR4 element. We found that Atp1b2  contains this exact sequence and Ep300 (E1a binding protein), Ccng1 (cyclin g1) and Cpt1a  contained a sequence that had the two half sites but spaced by 5, 6, 6 base pairs, respectively. TR binding and weak transactivation to sequences with 5 and 6 base pairs between the two half sites has been reported Cpt1a is known to be regulated by T3 Atp1b2 and Ep300, but the two genes Ccng1 and Cpt1a, were up-regulated also by light, as shown in Ccng1 could have a protective role and in cellular survival as reported for other cyclins Cpt1a play a role in retina metabolism Thyroid hormone receptors, TR\u03b1 and TR\u03b2, regulate target gene expression by binding to the T3 response element (TRE) composed of repeated DNA sequences with different configurations. The consensus sequence recognized by nuclear receptors often contains a hexamer AGGTCA known as \u201cthe half site\u201d. TR forms heterodimers with members of the retinoid X receptor (RXR) family to mediate T3 action Sag and Gcap1. These biochemical pathways could be novel components of light adaptation.These results support the notion that light could activate the thyroid hormone cascade, regulating therefore the expression level of its target genes, such as the cone opsins, Dio2 and Dio3 genes and of their corresponding proteins in the adult retina. The genomic analysis of changes in gene expression with DNA-microarrays in the adult retina shows that Dio2 is the most up-regulated gene by diurnal light . The relative concentrations of T4 and T3 and their availability to the nuclear thyroid hormone receptor (TR) are controlled by the local conversion of T4 to T3 catalyzed by the enzyme DIO2, while the enzyme DIO3 inactivates T3 Dio2 and Dio3 exhibit a regulation of gene expression similar to the one described here in the retina. Several genes present in the retina and in the pineal gland show a phase shift with respect to each other Dio2 and Dio3. Dio2 has a role in photoperiodic modulation in seasonal reproduction in the mediobasal hypothalamus In the pituitary gland, The thyroid hormone cascade acts in the regulation of neurodevelopment, possibly by activation and repression of complex gene networks http://www.mybioinfo.info/) to locate the existence of a DR4 element. We found that Atp1b2  contains this exact sequence and Ep300 (E1a binding protein), Ccng1 (cyclin g1) and Cpt1a  contained a sequence that had the two half sites but spaced by 5, 6, 6 base pairs, respectively. TR binding and weak transactivation to sequences with 5 and 6 base pairs between the two half sites has been reported Ep300 has a role in TR function TR regulates target gene expression by binding to the T3 response element (TRE) composed of repeated DNA sequences with different configurations. The consensus sequence recognized by nuclear receptors often contains a hexamer AGGTCA known as \u201cthe half site\u201d. TR forms heterodimers with members of the retinoid X receptor (RXR) family to mediate T3 action Dio2\u2212/\u2212 knock-out mice had significant deficits in thermoregulation and thermogenesis Dio1\u2212/\u2212 and Dio2\u2212/\u2212 knock-out mice had significant deficits in the Morris water maze test indicating dysfunctions not only in learning and memory development but also, possibly, in visual capability Dio3\u2212/\u2212 knock-out mice, almost 80% of cones are lost through neonatal cell death and the amplitude of both the a- and b-wave of the electroretinogram is significantly reduced Dio2 and Dio3 in retinal visual functions.In humans, the majority of patients with dysthyroid eye disease (Graves' disease), an autoimmune disease where the thyroid is overactive, producing an excessive amount of thyroid hormones have developed color vision defects OPN1LW and OPN1MW, i.e. the long (L) and medium (M) cone opsin genes and were identified as transcriptional targets of the thyroid hormone cascade. Also ARR3 , GCAP1, PDE6H (phosphodiesterase 6H) and PDE6C (phosphodiesterase 6C) were found to be similar transcriptional targets. Arrestin, guanylyl cyclase, and phosphodiesterase are proteins involved in the regulation of the cyclic GMP signal transduction pathways in cones and rods Crx, the cone-rod homeobox OPN1LW, Opn1mw, Arr3 and Gcap1 have been identified to be regulated by CrxDio2 and Dio3 have a similar behavior in wild-type mice treated with MMI/perchlorate, treatment causing mice to become hypothyroidic Opn1mw and Opn1sw are down-regulated after 12 hours of light exposure . All rat experiments were carried out according to the Italian and European guidelines for animal care .Dark-adapted Long Evans male adult rats were sacrificed under an infrared light source. The harvested retinas were expelled into 500 \u00b5l of TRI Reagent T9424 (Sigma Aldrich) on ice and stored at \u221280\u00b0C. Culture retinas were prepared as described on the protocol used by Reidel et al Immunolabeling was performed by standard protocols for tissue fixation and processing, using as primary antibody anti-Dio2 sc-98716  and DAPI  for nuclear staining.2) in ice. The total amount of protein was determined by using BCA protein assay kit (Pierce Biotechnology). The homogenate was diluted in a sample buffer (20\u03bcg), run on SDS-PAGE and western blotted using the following antibodies: anti-Dio2 sc-98716 , anti-Dio3 ab82041 . \u03b2-Actin HRP- conjugated A3854 (Sigma-Aldrich) was used as housekeeping control. Signals were detected analyzing the optical density of the spots.Retinas, dissected from light or dark-exposed mice, were homogenized in Lysis buffer . The RNA quality was checked using a bioanalyzer , and the RNA quantity was measured with ND-1000 Nanodrop spectrophotometer. 10 \u00b5g of RNA sample was used for microarray analysis on Affymetrix RAT230_2 GeneChip containing 31099 probes, corresponding to 14181 probes with a gene symbol (Affymetrix). Low level analysis was performed using an Robust Multi-array Average (RMA) algorithm  directly on the scanned images. All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database , as detailed on the MGED Society website Data were organized in matrices \u2018\u2018m\u00d7n\u2019\u2019 . Five samples were considered: a control at 7 am in dark , a sample always kept in dark till 10 am (3Dij), a sample with 3 hours of continuous light also at 10 am (3Lij), and two similar samples at 1 pm . Data were analyzed by considering log2 changes in gene expression in each replica against the control condition C, that is, log2(3Dij/Cij), log2(3Lij/Cij) and log2(6Dij/Cij), log2(6Lij/Cij). Up-regulated genes for each replica were obtained by selecting all genes showing an up-regulation higher than 60%. Down-regulated genes were obtained considering genes with a decrease of expression larger than 0.7. Intersection between the three replicas was performed and presented in 2 inhalation, the eyes enucleated, the lenses removed and the retinas collected in TRI Reagent (Sigma Aldrich). Total RNA from retinas was extracted according to the manufacturer's instructions (Sigma Aldrich). After resuspension in DEPC-treated H2O, RNA was further purified using an RNeasy column (Qiagen) and quantified using an ND-1000 Nanodrop spectrophotometer (Nanodrop Technologies). Total RNA (500 ng) was treated with DNAse I (Invitrogen) to remove any genomic DNA contamination and converted to cDNA using Superscript II reverse transcriptase (Invitrogen). Twenty microliter PCR reaction mixtures contained cDNA, SYBR green master mix (BioRad), H2O, and custom primers designed for each gene of interest. The PCR reactions were performed in an iQ5 thermocycler (BioRad). Each reaction was performed at least in duplicate, and threshold cycles (Ct) were calculated using the second derivative of the reaction. The Ct of each gene was normalized against that of the control reference transcript Gapdh. The variation of Gapdh in Ct between samples obtained from rats exposed to darkness and to light showed no significant difference. For the experiments shown in Gapdh and Hprt. Normalization was performed using the geometrical mean as described by Vandesompele et al \u2212\u0394\u0394Ct method), using the average of dark control set to one Long Evans rats were bred and maintained under a 12 hour light/dark cycle (7 AM:7 PM). For changes of lighting environment, two groups of overnight dark-adapted animals were maintained in either a darkened or a lighted cage. A 60 W bulb was used as an adjustable light source. For each time point at least six animals were sacrificed by COPrimers shown in Table S1Relative abundance of Dio2 and Dio3. From the data presented in (DOC)Click here for additional data file."}
+{"text": "Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature. Influenza A virus continuously mutates while circulating in nature and overcomes host immunity from previous infections, posing great challenges to disease control Highly pathogenic avian influenza (HPAI) H5N1 viruses have now spread through poultry populations in many countries. These viruses have also crossed species barriers to infect different hosts http://www.who.int]). Nationwide vaccination programs and culling strategies have been performed to control the disease, which has greatly contributed to a reduction in outbreaks. But despite these great efforts to control the disease, HPAI H5N1 viruses continue to evolve and cause outbreaks in poultry and human infections in Vietnam.Vietnam is one of the highest frequencies of HPAI H5N1 outbreaks. HPAI H5N1 virus was first identified in Vietnam in 2001 To better understand the molecular and biological properties of H5N1 avian influenza viruses, we selected 15 H5N1 strains isolated from poultry in Vietnam during 2006 and 2007 and sequenced their entire genomes. We performed phylogenetic analyses combining with the sequence data from the Vietnam influenza viruses and other representative viruses available in public databases, and then genotyped the viruses on the basis of their whole genomes. We also assessed the replication and pathogenicity of these viruses in mice. Understanding the molecular and biological features of avian H5N1 viruses will help reveal the potential evolutionary and transmission features of H5N1 viruses, and benefit disease control and pandemic preparedness.The 15 HPAI H5N1 viruses used in this study were isolated from domestic poultry, including chickens, Muscovy ducks, and ducks on farms in Vietnam. Details of these viruses are given in Viral RNA was extracted from allantoic fluid by using TRIZOL Reagent (Invitrogen), and was then reverse-transcribed. A set of fragment-specific primers (primer sequences available on request) was used for the PCR amplification and sequence analysis. The PCR products were purified with the Watson PCR purification kit (Watson) and sequenced by using the CEQ DTCS-Quick Start Kit on a CEQ 8800 DNA sequencer (Beckman Coulter). Sequence data were compiled with the SEQMAN program . Phylogenetic trees were generated with MEGA 4.0 by neighbor-joining (NJ) methods and bootstrap tests  based on the sequences for the open reading frames (ORFs).The nucleotide sequences obtained in this study are available from GenBank (accession nos. JX420123- JX420242).2 and inoculated intranasally with 106.0 EID50 of H5N1 influenza virus in a volume of 50 \u00b5L 1.0 to 106.0 EID50 of virus in a volume of 50 \u00b5L and calculated by using the method of Reed and Muench Groups of eight six-week-old female BALB/c mice  were lightly anesthetized with COThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People\u2019s Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences .To determine the molecular features of H5N1 avian influenza viruses in Vietnam, all eight gene segments of the 15 viruses were sequenced and those sequences were compared with the sequences in public databases of 25 representative influenza viruses from Vietnam and 4 representative viruses from China. The HA genes of these 44 viruses belonged to seven different clades of the WHO influenza (H5N1) nomenclature system , which could be further divided into five different groups on the basis of their evolutionary relationships .Group 1 contained five clade 0 viruses, three of which were isolated from the eggs of Vietnamese waterfowl in 2005 Analysis of the deduced amino acid sequences of the HA genes showed that all of the 15 isolates sequenced had a series of basic amino acids at the HA cleavage site (-RRRKR/\u2212RRKKR-), a characteristic of highly pathogenic influenza viruses in chickens The NA genes of the 15 strains sequenced and DK/VN/208/05 mainly formed three groups . HomologThe PA genes of the 15 sequenced strains mainly formed two groups . Group 1The PB2 and PB1 genes of the 15 strains also formed two groups, similar to those of the PA genes; however, the PB2 and PB1 genes of MDK/VN/22/07 were included in group 1. Similarly, the same trends were seen in the phylogenetic tree for the NS genes of the viruses. The NP and M genes of all of these viruses shared over 97% homology and were considered to belong to one lineage. Mutations at certain positions in the M2 protein, including I26, A27, and N31, were associated with the amantadine resistance of influenza viruses On the basis of genomic diversity, the 15 strains sequenced in this study were divided into five genotypes . Genotyp6EID50 of virus; three mice were euthanized on day 3 p.i. and their organs, including lung, spleen, kidney, and brain, were collected for virus titration in eggs, while the remaining five mice were observed for two weeks for changes in body weight or signs of death. As shown in 6EID50, and determined their 50% mouse lethal doses (MLD50) as described previously 50 values for these five viruses ranged from 2.5\u20133.5log10EID50 (To determine the ability of H5N1 avian influenza viruses to replicate and be pathogenic in mammals, we tested 12 viruses in mice. Groups of 8 mice were inoculated intranasally with 10g10EID50 . These r. The clade 0, calde 3, clade 5, and clade 7 viruses have been detected in poultry or poultry products in Vietnam HPAI H5N1 viruses were detected in Vietnam as early as 2001 Analysis of the 15 viruses sequenced in this study further revealed that the dominant viruses circulating in Vietnam in 2006 and 2007 belonged to clade 1 and clade 2.3.4. A previous study reported that HPAI H5N1 viruses were concentrated in specific geographical regions, with clade 1 viruses mainly in Southern Vietnam and clade 2.3.4 viruses mainly in Northern Vietnam Most Eurasian HPAI viruses isolated since 1997 can replicate in mammals The virulence of influenza virus is determined by multiple gene products and amino acid sites. Several determinant sites in the PB2, PA, HA, NS1, and M1 genes are associated with the virulence of avian influenza viruses in mammals In summary, we characterized the genetic and biological diversity of HPAI H5N1 viruses isolated in Vietnam and uncovered important information that contributes to our comprehensive understanding of these viruses. Influenza viruses spread in nature all year around in tropical regions"}
+{"text": "Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens.Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins.RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane.Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD. Patients with paraneoplastic cerebellar degeneration (PCD) often harbour Yo antibodies which cross-react with antigens in tumours (often ovarian or breast cancer) and Purkinje cells in the cerebellum PCD is characterised by rapid development of pancerebellar symptoms and loss of Purkinje cells Yo antibodies react with a 62 kDa protein (454 amino acids), the cerebellar degeneration-related protein 2  www.proteinatlas.org) shows strong staining in Purkinje cells while the CDR2 antibodies HPA018151 and HPA023870 show moderate and weak staining, respectively. We therefore hypothesise that Yo antibodies could be directed against both CDR2 and CDR2L with CDR2L being the primary target on Purkinje cells. This was supported by the Genevestigator gene expression search engine (www.genevestigator.com), indicating low to medium CDR2 expression potential in the nervous system , and medium to high CDR2L mRNA levels , both based on the human genome 47 k array and 47 samples included.Given the high sequence identity between CDR2 and CDR2L, we asked if Yo antibodies could cross-react with both antigens. The CDR2L specific antibody HPA022015  and approved by the Regional Committee for Medical and Health Research Ethics in Western-Norway, Diagnostic markers of cancer (188.05). The retrospective study of patient records was also approved by the Regional Committee for Medical and Health Research Ethics in Western-Norway and the clinical data were part of a larger retrospective study on clinical correlations with onconeural antibodies . For both the bio-bank and the retrospective study, the regional ethics committee as well as the Ministry of Health and Care Services specifically waived the need to obtain consent , due to the large number of included subjects and high number of deceased subjects. All participants were adults.First, we screened 42 Yo positive sera (with antibodies against CDR2) sent to the Neurology Research Laboratory, Haukeland University Hospital, Bergen, Norway for CDR2L antibodies using a transcription-translation and immunoprecipitation (IP) technique. Subsequently, we screened sera from patients with ovarian (n\u200a=\u200a179) and breast (n\u200a=\u200a114) cancer, as well as 100 blood donors for CDR2L antibodies using the same IP technique. These blood donor sera have previously been examined for CDR2 antibodies www.origene.com, CDR2 sequence transferred from vector RG204900 with C-terminal green fluorescent protein (GFP) to vector PS100001 with C-terminal myc-DDK tag) or CDR2L  were cloned into expression vectors with a T7 promoter. For the IP experiments, two polyclonal rabbit antibodies from Eurogentec (www.eurogentech.com) which were produced against CDR2 (two synthetic peptides corresponding to amino acid 123\u2013138 (EELKSSGQGRRSPGKC) and amino acid 429\u2013443 (CDEQRTKYRSLSSHS) of the human CDR2 sequence) and CDR2L (two synthetic peptides corresponding to amino acid 282\u2013297 (APEADDPQPGRGDDLG) and amino acid 392\u2013407+C (CICRDSSWRDLRGGEEG) of the human CDR2L sequence) were used as reference sera. The antibodies were specific and did not cross react in the IP assay. CDR2 and CDR2L patient antibodies were then tested by the IP technique using the recombinant proteins as antigens Full-length CDR2  or CDR2L  were used to transfect HeLa cells following the standard protocol for Lipofectamine2000 . Transfected cells were fixed and immunocytochemically labelled 24 h after transfection.HeLa cells (ATCC-CCL-2) were chosen as a cell-line with a human background that can easily be transfected with a high reproducibility of the results. Cells were grown in DMEM, supplemented with 10% FCS (Sigma), L-Glutamine  and Pen Strep . cDNA for full-length CDR2 (HeLa cells (ATCC-CCL-2) were grown on poly-L-Lysine coated glass-coverslips (10 mm). Immunocytochemical staining of HeLa cells with patient sera and antibodies against CDR2 and CDR2L was performed as described elsewhere Imaging of HeLa cells was performed with a Zeiss Axiovert M200 fluorescence microscope with a 40x objective and TILL photonics monochromator or Leica TCS SP5 with Blue diode 405, Argon laser 488, Helium/Neon I laser 594, objective HCX PL Apo 63x.Patient sera with CDR2 and/or CDR2L antibodies were tested by immunofluorescence using rat cerebellar tissue or immunoblot using recombinant onconeural proteins including CDR2  , CDR2L   and CDR2L  . The immunoperoxidase staining followed the standard procedures at the Department of Pathology, Haukeland University Hospital.www.gelifesciences.com) and then read in a luminicent image analyzer.We subjected extracts of human cerebellum (150 \u00b5g) as well as lysates of recombinant CDR2 or CDR2L (from the IP test) to a 10% SDS-PAGE and performed Western blotting Among 42 sera previously tested for paraneoplastic antibodies, 36 were positive for both CDR2L and CDR2 antibodies using the IP technique  and all Twenty eight of the 36 CDR2L and CDR2 antibody positive patients had PNS; 27 had PCD  and one had isolated myelopathy. Three patients had no known PNS and no clinical information was available from five. Of the 36 CDR2L and CDR2 antibody positive patients, 18 had ovarian, 5 uterus , 1 colon and 4 breast cancer; in 8 patients we had no information on a possible underlying tumour .We found 8 patients with only CDR2 antibodies, of which 6 were from the 42 routine sera. Two of the 8 patients had neuropathy, 5 had no known PNS and for one patient we had no clinical information. They all had different cancers; pharyngeal, prostate, uterus, colon, lung cancer or astrocytoma . Two CDRIn 7 patients, we found only CDR2L antibodies; 5 of these were from the ovarian cancer and 2 from breast cancer cohort, and none had PNS. All of the CDR2 or CDR2L positive sera had low antibody avidity and none of them were positive by line blot or with immunofluorescence, including antigen retrieval protocol. None of the 100 blood donors were positive for CDR2L or CDR2 antibodies.Three patient sera that were shown to have different Yo antibody composition in IP experiments were chosen for IC labelling of HeLa cells expressing either CDR2-GFP  A1\u20133, buTo determine if CDR antigens in intact cells are available to react with Yo antibodies, we performed a surface staining of HeLa cells transfected with CDR2-myc or CDR2L-myc . Cells wPolyclonal CDR2L antibodies gave a granular cytoplasmic staining of human Purkinje cells, but no staining of these cells was seen by the CDR2 antibodies . NeitherThe polyclonal CDR2 and CDR2L antibodies used for the Purkinje cell staining showed no cross reactivity between the recombinant CDR2 and CDR2L antigens . The CDRWe found that 36 of 42 CDR2 positive patient sera also contained antibodies to CDR2L. The majority of the patients with both CDR2 and CDR2L antibodies had PCD, most often associated with ovarian cancer. The co-existence of these antibodies indicated that both antibodies had high avidity for their antigens. It is known that high avidity antibodies are more likely to be of pathogenic importance since they represent a more long-standing immune response When CDR2L or CDR2 antibodies occurred alone they had low avidity. Of the 15 sera that were positive only for one of these antibodies, only two of 8 CDR2 positive patients had possible PNS (neuropathy) and none with only CDR2L antibodies manifested a paraneoplastic syndrome. This suggests that the co-existence of these antibodies may be of relevance for the development of PNS, especially PCD. Low avidity CDR2 or CDR2L antibodies were all associated with cancer, but probably of most importance in the general tumour immune response and less for the development of PNS.Although patient sera containing only CDR2 or CDR2L antibodies gave negative immunohistochemistry, these antibodies did stain HeLa cells transfected with their respective antigens. This is in line with the assumption that when occurring alone these antibodies have low avidity and mirrors findings with acetylcholine receptor (AChR) antibodies in which transfected cell assays are able to detect low avidity antibodies due to cross reactivity of the antigens on the cell surface We have shown previously that the IP test is more sensitive than both immunohistochemistry and line blot for the detection of paraneoplastic antibodies HeLa cells transfected with CDR2L showed a different distribution of positivity than those transfected with CDR2. Localisation of CDR2L to the plasma membrane suggests a possible biological function similar to other membrane associated antibodies such as AChR and P/Q voltage-gated calcium channel antibodies (VGCC) that have been shown to be of pathogenic importance in myasthenia gravis and Lambert-Eaton myasthenic syndrome The polyclonal CDR2L antibodies showed a granular cytoplasmic staining of the human Purkinje cells, similar to that described for Yo positive sera www.genevestigator.com, probeset 213230_at (CDR2L) and 209501_at (CDR2).The polyclonal CDR2 antibody did not react with human or rat Purkinje cells, even after antigen retrieval. Western blot studies of human cerebellar extract showed weak reactivity with the CDR2 antibody, but strong reactivity with the CDR2L antibodies. These results suggest that CDR2L is more highly expressed in human Purkinje cells than CDR2, findings that are in line with data showing that CDR2L mRNA levels are much higher than CDR2 levels in human cerebellum (biogps.org and www.proteinatlas.org). We have shown previously that CDR2 is present in various tumours regardless of CDR2 antibody status According to the human protein atlas, both CDR2L and CDR2 are found in various human tumours  showed that Yo antibodies can be taken up by cultured rat Purkinje cells. Our results suggest that it is likely that these sera contained both CDR2 and CDR2L antibodies, and that the co-expression of these antibodies is necessary for rat Purkinje cell staining. Moreover, based on our results, it is likely that the Yo antibody staining of the Purkinje cells in these experiments was due to CDR2L and not CDR2 antibodies. Furthermore, as we have shown in transfected cells, CDR2L is membrane-bound and probably therefore can mediate intracellular uptake of CDR2L antibodies.The presence of high avidity CDR2L antibodies in patients with PCD and the presence of CDR2L in Purkinje cells make it tempting to speculate as to whether these antibodies are important in the Purkinje cell death that occurs in PCD Figure S1Surface staining of HeLa cells overexpressing CDR2L-myc. HeLa cells transfected with CDR2L-myc (A1\u20133) were surface-stained with CDR2L antibody  and myc antibody . The surface staining was done without permeabilisation with Triton-x-100 (A1\u20133). Of the z-stack 15 slices were superimposed to show the localisation of CDR2L to the membrane. The overlay shows a strong co-localisation  of both antibodies, indicating that the myc staining was specific for the transfected CDR2L protein. Scale bar 20 \u00b5m.(TIF)Click here for additional data file."}
+{"text": "Bovine papillomaviruses (BPVs) induce hyperplastic and tumoral lesions not only in cows but also in other different animal species. The transforming activity of BPVs is due to its major E5 oncogene. Recent studies have highlighted the role of E5 in cancer development but very little is known about E6 and E7 oncogenes. In this letter we argue for the need of investigating E6 as well as E7 to better understand the role of these two oncogenes during carcinogenesis. Bovine papillomavirus (BPV) is considered as causative agent of cutaneous and mucosal tumors in its natural host . HoweverIn the last decade, the role of BPV major oncoprotein E5 in cell transformation has been largely investigated in naturally occurring bovine and equine tumors. Overall, these studies confirmed the pivotal role of E5 in cancer development supporting the role of the virus -18. IntePv\u2019s contribution to tumorigenesis is \u201cparadigmatically\u201d based on the actions of more than one oncogene. Human Pvs (HPVs) E5, E6 and E7 oncoproteins act synergistically disturbing different cellular pathways and in so doing they contribute to initiation and progression of cancer . MoreoveIn this regard, among BPV\u2019s oncogenes the contribution of the other two oncogenes (E6 and E7) to cancer development is less known. It has been suggested that BPV E7 may cooperate with E5 in inducing cell transformation whereas E6 can downregulate p53 transcriptional activity by interacting with CBP/300 -24. HoweThe author declares that he has no competing interests."}
+{"text": "We recently characterized several new broadly neutralizing monoclonal antibodies (bnMAbs) remarkable for their in vitro potency. A number of these bnMAbs are almost 10 fold more potent than previously isolated bnMAbs such as PG9 and VRC01 and hundred fold more potent than older prototype bnMAbs such as 2G12 and b12. Of the new antibodies, PGT121 is one of the most broad and potent, and recognizes a glycan-dependent epitope. Based on in vitro neutralization potency, we speculated that PGT121 may be protective in vivo at serum concentrations below those previously determined for other broadly neutralizing anti-HIV antibodies.SF162P3 was evaluated using a TZMbl-based neutralization assay. To evaluate the protective potency of PGT121 in vivo, we then designed a protection study in rhesus macaques. Animals were administered intravenously with 3 different doses of antibody  24 hours before being vaginally challenged with a single high dose of SHIV SF162P3 (300 TCID50). Serum levels of antibody and viral loads were determined throughout the study.The neutralization potency of PGT121 against SHIV SF162P3 with an IC50 of about 0.005 \u00b5g/ml. Preliminary data suggest that an administered dose of PGT121 at 1 mg/kg is sufficient to induce sterilizing immunity against a single high dose vaginal challenge of SHIV SF162P3. A lower 0.2 mg/kg dose appears to be partially protective.PGT121 potently neutralizes SHIV PGT121 is one of the most broad and potent neutralizing antibodies isolated to date. Here, we provide evidence that in vivo protection by PGT121 correlates with the high in vitro potency of the antibody. PGT121 can thus mediate sterilizing immunity at concentrations that are significantly lower than previously observed from passive protection studies with bnMAbs and may be readily achievable through vaccination."}
+{"text": "Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n\u200a=\u200a17), genital warts (n\u200a=\u200a43), anal cancer (n\u200a=\u200a6) and cervical neoplasia cells (n\u200a=\u200a5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p\u200a=\u200a0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p\u200a=\u200a0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types. Human papillomaviruses (HPVs) have been demonstrated to cause a diverse range of epithelial lesions. To date, 120 HPV\u2019s have been identified and each of these types are associated with infection at particular anatomical sites Although the majority of HPV related non-malignant diseases are attributable to HPV 6 and 11 Anal cancer is uncommon in the general population, although incidence in the general Australian population is increasing Cervical cancer and its precursors namely high grade cervical intraepithelial neoplasia (CIN2/3) and adenocarcinoma insitu (AIS) are frequently associated with persistent HPV infection of types HPV16 and HPV18 Several key gene regions and their role in malignant transformation have been extensively studied in high-risk HPV types 16 and 18. For example, viral E6 and E7 oncogenes are key mediators in cell transformation through the disruption of tumour suppressor Rb/E2F and subsequent p53 pathways respectively, which are critical in regulating the initiation of DNA replication. Loss of function or disruption of these pathways due to integration of HPV DNA, leading to increased expression of E6 and E7 proteins, has been demonstrated to result in the development of cervical carcinoma Previous studies investigating genetic variation of these gene regions have used standard reference genomes for comparative purposes. These standard reference genomes for HPV6 are denoted prototypic HPV6b, and non-prototypic HPV6a and HPV6vc, and one standard reference genome for HPV11. A recent study based on phylogenetic analysis of complete genomes derived from published HPV6 and HPV11 variants has proposed a new standard nomenclature for HPV6 and HPV11 This study examines the genetic variability of the LCR, E6 and E7 gene regions in both HPV6 and HPV11 across different lesion types to determine whether different disease states resulting from single HPV6 or HPV11 infection cluster into distinct variant groups. In addition, current data on the genetic diversity of HPV6 and HPV11 variants present within the Australian population are very limited. Only one study has been conducted within the Australian population, sequencing only a portion of the LCR for 47 HPV6 or HPV11 RRP isolates All patients provided written informed consent at time of treatment whereby ethics approval was received from: the human research ethics committee at St Vincent\u2019s Hospital, Sydney, Australia for anal cancer biopsies, the health ethics research committee at the Royal Perth Hospital, Perth, Australia for genital wart biopsies, and the human research ethics committee at the Royal Women\u2019s Hospital, Melbourne, Australia for the collection of cervical cells. In addition, ethics approval was obtained from the ethics in human research committee of the Royal Children\u2019s Hospital, Melbourne, Australia for juvenile RRP biopsies whereby written informed consent was provided by either the parent or guardian at time of treatment.Based on single HPV infection status a total of 71 HPV 6 and HPV11 isolates from four different disease states being: RRP, genital warts, anal cancer, and cervical neoplasia cells were included in this study. Formalin fixed, paraffin embedded (FFPE) biopsies collected at the Royal Children\u2019s Hospital, Melbourne between 1964 and 1994 from 17 juvenile RRP patients described previously For FFPE archival tissue, 5 \u2013 10 \u00b5m sections were deparaffinised with 800 \u00b5l histolene, then mixed with 400 \u00b5l absolute ethanol. The tissue was centrifuged at 14,000 \u00d7 g for 2 minutes. The resultant pellet was washed with 1 ml of absolute ethanol, centrifuged for 2 minutes and the supernatant was discarded. The pellet was air dried and subsequently incubated for 4 hrs at 55\u00b0C with 160 \u00b5l tissue lysis buffer  and proteinase K at a final concentration of 1 mg/ml. DNA was then isolated from digested tissue using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics) on the automated MagNA Pure LC extraction system, according to the manufacture\u2019s protocol, with the addition of 33.3 \u00b5g/ml of poly(A) RNA carrier to the lysis buffer which has been demonstrated to improve extraction efficiency. Nucleic acid was eluted into a final volume of 100 \u00b5l.Fresh 1\u20132 mm biopsies were homogenised using MagNA Lyser (Roche Diagnostic) with 250 \u00b5l of Tissue Lysis Buffer (Roche Diagnostics), by two repetitions of 30 seconds at a speed of 7000 rpm in the MagNA Lyser Instrument. Proteinase K was added to a final concentration of 1 mg/ml, and samples were incubated at 55\u00b0C until fully digested. DNA was then isolated from the digested tissue as described above. DNA was isolated from the PreservCyt samples of cervical cells using the MagNA Pure LC DNA Isolation Kit I on the automated MagNA Pure LC extraction system, according to a modified protocol HPV genotyping of all archival FFPE tissues were confirmed by using the INNO-LiPA HPV genotyping test  which has demonstrated increased sensitivity and accuracy for archival tissue DNA positive for HPV6 or HPV11 samples were subject to type and gene specific PCRs. All primers used for genetic variation analysis of HPV6 LCR, E6 and E7 , were de2, 1 U AmpliTaq\u00ae Gold DNA Polymerase and 1X reaction buffer , and 2.5 to 5 \u00b5l of DNA template. Following a denaturation step of 94\u00b0C for 9 min, PCR steps at 94\u00b0C for 30 sec, 55\u00b0C for 20 sec and 72\u00b0C for 30 sec for 45 cycles, followed by an extension step at 72\u00b0C for 10 min, on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Amplicons were run on a 2% agarose gel for visualization, using 1000 base pair HyperLadder IV  for sizing.All PCR reactions were performed in a 50 \u00b5l reaction volume containing 0.5 \u00b5M of each primer, 2 mM of each deoxynucleoside triphosphate, 2.5 mM MgClAmplicons were sent to the Australian Genome Research Facility for purification and Sanger DNA sequencing using a AB3730xl capillary sequencer (Applied Biosystems). To minimise PCR and sequencing errors, sequencing was performed on both forward and reverse stands with predicted sequencing error of 0.01% based on sequencing of control DNA. For regions which did not meet our quality control criteria  confirmation of HPV sequence was carried out by repeating both the forward and reverse strands in duplicate. Sequences were assembled using SeqMan version 8  and aligned against reference genomes using SeaView version 4 The HPV6 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC300093-KC300140), E6 sequences , E7 sequences (KC300189-KC300237).The HPV11 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC329850-KC329871), E6 sequences (KC329872-KC329893), E7 sequences (KC329894-KC329915).Data in categories were evaluated using two-tailed Fisher\u2019s exact test.This study used samples with either HPV6 or HPV11 infection. Of the entire sample set for each lesion type a total of 49 specimens were identified as HPV6 isolates, that is 8 from RRP lesions, 33 genital warts, 4 anal cancer biopsy specimens and 4 cervical cell samples. In addition, a total of 22 specimens were identified as HPV11 isolates, that is 9 RRP lesions, 10 genital warts, 2 anal cancer biopsy specimens and 1 cervical cell sample . For theAll 49 HPV6 isolates were successfully amplified and sequenced across the E6 (nt 102\u2013554) and E7 (nt 530\u2013826) ORFs. For HPV6 E6/E7, a total of 18 single nucleotide variations (13 in the E6 and 5 in the E7 ORF) from the lineage A reference genome were identified . No isolA total of 48 HPV6 isolates were successfully amplified and sequenced across the LCR . Single nucleotide variations from the lineage A reference genome were identified at 45 sites across the LCR. In addition, 5 insertions and 4 deletions were identified . Three vA total of 105 HPV6 LCR sequences were used to perform a comparative phylogenetic analysis, which included 48 Australian isolates from this study, 45 Slovenian All 22 HPV11 isolates were successfully amplified and sequenced across the E6 (nt 102\u2013554) and E7 (nt 530\u2013826) ORFs. Three genomic variants were identified across the E6/E7 ORFs for HPV11 . None ofThe LCR from 22 HPV 11 isolates was successfully amplified and sequenced . Comparative analysis with the HPV11 sublineage A1 reference sequence revealed 13 single nucleotide variations, 1 insertion and 3 deletions in the LCR . A totalComparative phylogenetic nucleotide sequence analysis was carried out on a total of 94 HPV11 LCR sequences. These publicly available sequences included 22 Australian isolates from this study, 63 Slovenian We present one of the largest studies of Australian isolates to date which investigates genetic variation for HPV6 or HPV11 in some of the key HPV gene regions thought to be associated with development of lesions and disease progression in epithelial cells. This study compares genetic variation within various lesion types sampled from the Australian population for the two most prevalent types of low risk HPV associated with these diseases. Analysis of genetic variance revealed slight association with HPV6 and anogenital lesion and HPV11 and RRP with genetic variants HPV 6 B1 lineage being more strongly associated with anogenital lesion.The majority of HPV11 isolates from this study clustered to the variant A2\u20131 for E6 and E7 . This vaThis study identified a series of variations in the E6 and E7 ORFs of the HPV6 genome within the Australian lesions sampled. A total of three amino acid alterations in each of the E6 and E7 ORFs of different HPV6 isolates were found, some of which were unique to a single isolate, while others were found in several or multiple cases. Of these, several unique amino acid alterations were identified: E113D in E6 and E73K in E7. Conversely, amino acid alterations E19K and H50Q in the E6 ORF and F52Y and N88D in the E7 ORF, present in numerous isolates, have been described previously When the prevalence of HPV-type in genital warts and RRP (lesions for which n>10) was considered, a type specific distribution was indicated for genital warts but not RRP. The prevalence of HPV 6 was found to be higher for genital warts (77% HPV6 vs 23% HPV11) in this sample set, as described previously by Variant analysis of the LCR within Australian isolates was completed for both HPV6 and HPV11. In recent years, a growing number of studies have described HPV6 LCR variations in isolates from the nucleotide positions of the lineage A reference genome Genomic regions such as the LCR have been used by previous studies to identify genetic variants as it provides enough genetic variation to differentiate between variants in both HPV6 and HPV11. For this reason, the LCR was selected for comparative phylogenetic sequence analysis between this and previous studies to determine whether genetic lineages previously described This current study also provides unique pre-vaccination data on the genetic variation present in RRP, anal cancer, cervical cells samples for HPV6 and HPV11 within the Australian population. For genital warts, based on age  and sex , at least 88% of these samples would have come from women and men ineligible for publically funded prophylactic HPV vaccination. The remaining 12% of genital warts were derived from women <26 years, whose vaccination status was unknown. The high proportion of genital wart isolates positive for HPV6 or HPV11 suggests that lesions developed either in unvaccinated patients, or in women who were vaccinated with prevalent infection. Australia was the first country to introduce a government-funded, population-based mass vaccination program in 2007 of the female population (12 to 26 years), using the quadrivalent vaccine Garadasil\u00ae  targeting HPV types 16, 18, 6 and 11. It has already seen a reduction in the number of cases of genital warts due to a high coverage of vaccine In summary, we found slight association between HPV6 with anogenital lesions with sublineage B1 being predominate. This is consistent with the findings of other studies and suggests that viral and/or host epigenetic and other host factors (such as those associated with immune response) may play significant roles in disease susceptibility and progression. In addition, larger studies investigating whole genome sequences may shed further light on possible associations with pathogenesis. However, this study makes a significant contribution toward the slowly expanding knowledge about HPV6, and in particular HPV11 genomic diversity and pre-vaccination circulating HPV6/11 variants within the Australian population.Table S1Summary of HPV6 and HPV11 lesion types.(DOCX)Click here for additional data file.Table S2Summary of genetic variation identified across all lesion types for HPV6 and HPV11.(DOCX)Click here for additional data file.Table S3Primers used for amplifying and sequencing the LCR, E6 and E7 ORFs.(DOCX)Click here for additional data file.Table S4HPV6 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 49 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S5HPV6 nucleotide sequence variation in the LCR from 48 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S6HPV11 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S7HPV11 nucleotide sequence variation in the LCR from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file."}
+{"text": "Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances. Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most common Mendelian genetic cause of Parkinson's disease (PD) currently identified Drosophila model of LRRK2 disease Drosophila data would be that steady state mRNA levels of miRNA targets are altered in the presence of mutant LRRK2. To test whether mutations in LRRK2 cause an alteration in basal gene expression we have examined the impact of mutations in three contexts: fibroblast cells cultured from PD patients carrying mutations in LRRK2 , brain tissue from 5 patients carrying the G2019S mutation compared to idiopathic PD and control brain tissue In addition, a recent study has suggested that pathogenic forms of LRRK2 bind directly to the miRNA processing enzyme Argonaute and thus influence mRNA levels in a 2 sterile Petri dishes at 37\u00b0C in 5% CO2 in 2 ml DMEM  until fibroblasts were seen to migrate from the skin explants 2 dishes for culturing. Samples were designated anonymous identifiers following collection. For collection of RNA, cells were plated in 10 cm plates at equivalent passage number (n\u200a=\u200a7) and at equal density. Following 24 hours growth, RNA was extracted using the RNeasy kit (Qiagen) and RNA quality ascertained prior to microarray analysis using RIN analysis (Agilent). Western blot analysis of endogenous LRRK2 expression was carried out by immunoblotting following BCA assay , with 10 \u00b5g of lysate loaded onto 4\u201312% Bis Tris gels (Invitrogen) following denaturation in 4x sample buffer (Invitrogen) supplemented with 5% \u03b2-Mercaptoethanol (Sigma). Proteins were transferred to PVDF membrane (Millipore) and membranes blocked with 5% milk solution in Phosphate buffered saline for 1 hour prior to probing with primary (90minutes) and secondary (60minutes) antibodies. For LRRK2, rabbit monoclonal MJFF2 (Epitomics \u2013 see http://www.pdonlineresearch.org/discussions/lrrk2-antibodies for characterisation of these antibodies) was used at a 1\u22361000 dilution, for \u03b2 Actin mouse monoclonal AC-74 (Sigma) was used at a 1\u22365000 dilution. HRP secondary antibodies were used at a dilution of 1\u22362000 and 1\u223610000 for LRRK2 (rabbit) and \u03b2 Actin (mouse) respectively. Membranes were washed three times in PBS supplemented with 1% tween, and exposed to Supersignal ECL substrate (Pierce). Membranes were exposed to Kodak biomax film and developed on a Fujifilm developer as per manufacturers instructions.Fibroblast cultures were isolated from PD patients with LRRK2 mutations and healthy controls  and Sun Health brain bank  Illumina HumanWG-6 and HumanHT-12, and Affymetrix Exon 1.0 ST Arrays were used according to manufacturer's instructions as previously described We compared gene expression in three systems: an inducible HEK cell model, patient fibroblasts and patient brain samples for wild type and mutant LRRK2. To confirm expression of LRRK2 in these systems, we carried out western blot analysis using anti-V5 and anti-LRRK2 antibodies for the HEK cells and fibroblasts/brains samples respectively . In the Having confirmed the expression of LRRK2 at the protein level in these systems, we carried out genome wide expression analysis. In the HEK cells we compared each cell line induced for 12 h or 24 h to its uninduced controls; no differences in gene expression were seen in either WT or R1441C expressing cells . SimilarWe also examined gene expression in brains of LRRK2 carriers compared to controls and to idiopathic PD . For thiWe considered that simply looking for genes whose steady state expression might miss more subtle patterns in the data and therefore examined overall gene expression by unsupervised hierarchical clustering . SamplesDrosophila and a HEK cell model This study assessed a series of patient fibroblast cells harbouring mutations in LRRK2, brain samples from G2019S carriers and a cell line system over-expressing LRRK2 wild type and mutant transgenes for alterations in gene expression due to the presence of mutant LRRK2. Overall, our data show that any differences in gene expression are lower than the relatively modest cutoffs  used. In turn, this suggests that mutant LRRK2 does not elicit large changes in steady state mRNA levels within the cell under normal growth conditions. This is in contrast to a recent publication studying a series of mononuclear cells carrying the G2019S mutation via the mTOR pathway, and it is plausible that LRRK2 exerts an influence on gene expression following activation in response to cellular stress. With regard to alterations due to miRNA regulation, our data demonstrates no overall impact on mRNA levels. This does not exclude translational regulation as a mechanism through which a putative LRRK2 miRNA pathway could operate, although the recent publication by Guo et al emphasizes that miRNAs in mammalian cells predominantly function by directly impacting on mRNA levels rather than by premature translation termination or inhibiting ribosome formation or binding Our results do not exclude the possibility that gene expression can be differentially altered by mutations in LRRK2 under conditions of stress, for example under starvation conditions, oxidative challenge or widespread cell loss via its kinase activity or through altered recruitment of other co-factors to an active enzymatic complex, aspects of the biology of LRRK2 that are undergoing investigation in a number of laboratories In summary, our data provides evidence from two independent cellular model systems and brain tissue from patients that LRRK2 mutations do not result in large alterations in basal gene expression under normal growth conditions. These data raise the possibility that mutant LRRK2 exerts its pathogenic affect through an alternative mechanism, for example through post translational modification"}
+{"text": "Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene  Bmal1 ClockPer1, Per2, Cry1, Cry2, Nr1d1) upon binding to E-box elements in the promoters of the genes. After complexing with Casein kinase 1\u03b5/\u03b4, the PER/CRY/Casein kinase complex returns to the nucleus and inhibits the action of BMAL1/CLOCK, thus reducing the expression of the period and cryptochrome genes. NR1D1 plays an important role in the generation of rhythmicity through its repression of Bmal1 expression. We Clock is associated with alterations in metabolism. The Clock19\u0394 mutants produce a protein that can bind to BMAL1, but not induce gene expression, resulting in a loss of rhythmic gene expression in peripheral tissues, but not the SCN Clock in certain tissues  are rescued in Clock19\u0394 mutant mice and Clock null mice by a homologue, Npas2Bmal1 null mouse, originally produced by the Bradfield laboratory Clock19\u0394 mutant or Clock null mice, Bmal1 null mice have poorly entrained wheel running behaviour under a normal photoperiod and are arrhythmic in continuous darkness Bmal2 rescues the functions of Bmal1 in the Bmal1 null mouse Bmal1 null mice are infertile The links between circadian rhythms of gene expression, hormone secretion and metabolism have emerged over recent years. For example glucose metabolism changes across the day and night in humans Bmal1 expression on plasma adipokine levels (adiponectin and leptin) and adipose tissue expression of adiponectin (Adipoq), leptin (Lep), resistin (Retn) and visfatin (Nampt) mRNA in young animals (2 months of age), before any confounding pathology was likely to emerge. In addition we report the effects of global loss of Bmal1 expression on glucose, insulin and pyruvate tolerance tests and expression of liver glycolytic and gluconeogenic enzyme mRNA. Finally we investigated the metabolic impact of a high fat diet on body and adipose tissue weight, plasma metabolites, insulin and adipokines in Bmal1 null mice.In this study we report for the first time the effects of loss of Bmal1 null mice ad libitum. The genotypes of the offspring were determined as previously described by PCR of tail DNA The founder Bmal1 null male mice and their wild-type litter mate controls (4 mice per time point) were killed by decapitation at 2 months of age every four hours across 24 hours at 0800 h, 1200 h, 1600 h, 2000 h, 2400 h and 0400 h. Blood was collected into heparinised tubes and plasma harvested for metabolite and hormone assays. Liver and epigonadal fat were rapidly dissected and immediately placed in RNAlater\u00ae  and then stored at \u221220\u00b0C until processing. An additional group of 6 month old male Bmal1 null and wild-type mice (4\u20139 mice per time point) were killed at the same times of day and blood collected.Groups of Bmal1 null and wild-type mice were fed a high-fat diet (22% fat (wt/wt), 0.15% cholesterol, 4.6 kcal/g, SF00-219, Specialty Feeds, Glen Forrest, Western Australia) or the control diet from 3 to 8 weeks of age (n\u200a=\u200a10\u201319 mice of each sex per genotype). Animals were weighed and killed during the mid light period (1400 h), trunk blood collected and the epigonadal/retroperitoneal fat pads, testes/uteri and kidneys were dissected and weighed.To determine the effects of a high-fat diet on plasma hormones and metabolites, male and female Bmal1 null male mice aged 2 and 6 months (n\u200a=\u200a5\u20136) were maintained on the control diet, fasted overnight and injected with glucose  starting 2 h after the lights were turned on as previously described Wild-type and Bmal1 null 2 month old male mice (n\u200a=\u200a6) and female mice (n\u200a=\u200a5\u20137) were maintained on the control diet, fasted overnight and injected with sodium pyruvate  starting 2 h after the lights were turned on as previously described Wild-type and Bmal1 null male and female mice (n\u200a=\u200a6 per gender) aged 2 months were maintained on the control diet and had food withheld for 2 hours before injection of insulin  2\u20133 h after lights on as previously described Wild-type and To investigate the expression of clock and clock controlled genes in the liver and adipose tissue total mRNA was extracted, reverse transcribed and amplified by real time PCR. Some of the primers have been used previously by our group Plasma glucose and free fatty acids were measured enzymatically post hoc analysis using the Bonferroni correction for multiple comparisons. For the body composition, hormone and gene expression data collected across 24 h, the Estimated Marginal Means are reported in the text for the various comparisons. To determine whether hormone and gene expression data were rhythmic, i.e., fitted a sine curve, the data were analyzed using CircWaveBatch; http://hutlab.nl/Hormone, body weight, organ weight and gene expression data were analyzed by univariate ANOVA (SPSS v17), using genotype and time of day as the dependent variables followed by Bmal1 null mice, however, plasma insulin was lower (P<0.02) and adiponectin (P<0.001) and leptin (P<0.001) were higher in Bmal1 null compared to wild-type mice , higher adiponectin  and higher leptin  than wild-type mice.Similar differences in plasma insulin and adipokines between Bmal1 null mice compared to wild-type mice while plasma glucose and free fatty acids were unchanged. These differences persisted in 6 month old mice.In summary, across 24 hours, plasma insulin was lower and adiponectin and leptin were both higher in 2 month old male Bmal1 null and wild-type mice had similar fasting blood glucose (data not shown). The peak glucose levels and area under the curve during the glucose tolerance tests in wild-type and Bmal1 null mice at 2 and 6 months of age were not different (data not shown).At 2 and 6 months of age, male Bmal1 null mice had a trend to decreased blood glucose , but not females.At 2 months of age, administration of pyruvate resulted in increased blood glucose levels in both male and female ype mice . The bloBmal1 null mice were 14% lower than wild-type mice (P<0.005). On the high fat diet, male Bmal1 null mice gained weight such that they were no longer lighter than wild type mice . Male Bmal1 null mice had 58% and 52% more epigonadal fat and 182% and 64% more retroperitoneal fat per gram of body weight than wild-type mice maintained on the normal chow and high fat diet respectively (P<0.001). When the epigonadal and retroperitoneal fad pad weights were combined, male Bmal1 null mice had 1.8 fold more fat than the wild-type mice on the chow diet and 1.5 fold more when on the high fat diet. Testes weight in male Bmal1 null mice were reduced compared to the wild type mice only when maintained on the chow diet (P<0.001) and kidney weight in the Bmal1 null males was reduced compared to wild type on both diets (P<0.01).Following five weeks on the control diet, body weight of the male ype mice . Wild tyBmal1 null mice on a high fat diet were increased when compared to chow fed Bmal1 females and wild type females placed on a high fat diet . Bmal1 null mice on a high fat diet had more retroperitoneal fat compared to high fat diet wild type mice (P<0.001) and there was a trend for increased adiposity compared to wild type for Bmal1 null mice on a chow diet and when comparing Bmal1 null females on a high fat vs chow diet (P\u200a=\u200a0.016 and P\u200a=\u200a0.014 respectively). When the epigonadal and retroperitoneal fad pad weights were combined, female Bmal1 null mice had 2.3 fold more fat than the wild-type mice on the chow diet and 2.3 fold more when on the high fat diet. While showing a trend to smaller uteri in chow fed animals (P\u200a=\u200a0.018), female Bmal1 null mice uteri were not lighter than wild-type mice maintained on the normal chow or high fat diet (P>0.05), nor were kidney weights effected.Body weights of female fat diet , P<0.01.Bmal1 null mice weighed less than wild-type mice when maintained on the chow diet but caught up when placed on the high fat diet, whereas female Bmal1 null mice had comparable body weight on a normal chow diet, but weighed more when maintained on a high fat diet compared to chow fed Bmal1 null mice or high fat fed wild type mice. For both control and high fat diets, Bmal1 null male and female mice had a greater degree of adiposity than wild type mice.In summary, male Bmal1 null mice, however, plasma triglyceride (78% and 72%) and adiponectin (91% and 51%) were increased in Bmal1 null mice on chow diet and high fat diet respectively .When the male mice were killed at mid-light (1400 h), there was no difference in plasma glucose, free fatty acids and insulin between wild-type and ectively ; P<0.01.Bmal1 null mice, however adiponectin (by 63%) and leptin (by 56%) were increased in Bmal1 null mice on chow diet, and plasma triglyceride was increased in Bmal1 null mice on both chow (33%) and high fat diets . The high fat diet did not affect plasma glucose, triglyceride or adiponectin, but increased NEFA in the wild type (72%) and Bmal1 null mice (38%), increased circulating insulin by 77% in wild type mice and increased circulating leptin in both wild type and Bmal1 null mice .When the female mice were killed at mid-light (1400 h), there was no difference in plasma glucose, NEFA and insulin between wild-type and Per2 (\u221230%), Ppar\u03b3 (24%), Nr1d1 (\u221298%), Adipoq (\u221231%), Retn (\u221229%), Nampt (\u221252%), Adipor1 (\u221216%) and Adipor2 (\u221235%) mRNA was decreased (P<0.05), but Lep mRNA was unchanged (P>0.05) in male Bmal1 null mice compared to the wild-type mice , but Retn and Nampt mRNA expression was arrhythmic. Expression of all genes analysed in male Bmal1 null mice was arrhythmic except for Lep mRNA (P<0.05).In the epigonadal fat, overall expression of ype mice . Expressphosphoenolpyruvate carboxykinase 1), Fbp1 , G6pc (glucose-6-phosphatase ), Gck (glucokinase) and Adipor2 mRNA expression did not vary with genotype, whereas Pfkfb3  mRNA was decreased by 25% in male Bmal1 null mice compared to wild type mice  were also analysed (ANOVA) using only the 0800 h and 1200 h time points (light period), the time around which the glucose and insulin tolerance tests were performed. In contrast to the 24 hour analysis, expression of Gck and G6pc were increased 1.7 fold (P\u200a=\u200a0.05) and 1.6 fold (P\u200a=\u200a0.09) respectively in Bmal1 null mice at these times.The gene expression levels for the genes involved in the glycolysis and gluconeogenesis pathways . At 2 and 6 months of age, plasma adiponectin and leptin were higher in Bmal1 null mice than wild-type mice. Although tending to weigh less than the wild-type mice, Bmal1 null mice had a greater degree of adiposity . Adipoq, Nampt and Retn mRNA expression was reduced in Bmal1 null compared to the wild-type mice, but their increased adiposity is consistent with the increased secretion of these adipokines. We were not able to measure plasma resistin or visfatin in this study due to the limited volume of plasma available for assay.In this study we show for the first time that global disruption of gene rhythmicity in both male and female Bmal1 null mice and observe initial stages of loss of metabolic control, which may have been obscured with a more extreme and less physiological diet. When the mice were fed the diet for 5 weeks, the differences in adiposity between the Bmal1 null and wild-type mice was maintained but not increased, suggesting the Bmal1 null mice are not more sensitive to this metabolic challenge. There was a pattern towards a small increase in body weight in Bmal1 null mice in response to 5 weeks on the high fat diet, with minimal change in the wild type mice. This was not unexpected given the moderate challenge of the diet, however both Bmal1 null and wild type mice increased their adiposity by approximately 50%. Both lines showed signs of increasing their plasma free fatty acids and leptin to a similar extent on the diet. In previous studies, the body weight of comparable age Bmal1 null mice has been reported as normal The \u201cWestern diet\u201d (containing 22% fat and 0.15% cholesterol) used in this study was chosen as we wished to challenge the homeostasis of the Bmal1 null mice at 2 and 6 months of age, which is consistent with the decreased glucose stimulated insulin section reported by 3 independent groups Bmal1 null mice. A possible explanation for the normal glucose despite decreased insulin secretion in Bmal1 null mice may relate to the elevated plasma adiponectin which is known to be an insulin sensitising adipokine Clock19\u0394+MEL mutant mice Adipoq mRNA expression, but unlike the Bmal1 null mice, did not show increased adiposity Clock19\u0394+MEL mutant and Bmal1 null mice have adapted to a pancreatic insulin secretory defect by altering the levels of adipokines that modulate insulin sensitivity (adiponectin), to maintain glucose homeostasis. Interestingly, Clock19\u0394+MEL mutant mice had low plasma free fatty acids Bmal1 null mice did not exhibit this phenotype.In this study, plasma insulin  was decreased in male Bmal1 null mice did not express Per2 mRNA rhythmically in the liver in contrast to the wild-type mice. These results confirm the original observations, that disruption of Bmal1 interferes with circadian rhythm generation Pfkfb3, Pck, Fbp1, and Gck) were rhythmic in wild-type, but not Bmal1 null mice. When analysed across 24 hours, the overall expression of these genes was not altered, except for Pfkfb3 mRNA expression, which was reduced. Expression of Pck and Fbp1 mRNA prior to darkness was clearly reduced in the Bmal1 null mice, perhaps reflecting a change in feeding pattern caused by the loss of rhythmicity.Bmal1 null mice have increased pyruvate tolerance was unexpected because a previous study reported that the loss of Bmal1 resulted in decreased pyruvate tolerance when the test was conducted 7 hours after expected lights on (ZT7) Bmal1. More recently Shimba et al.Bmal1 null mice, as compared to those in control mice following a pyruvate challenge test, similar to that observed here. However, Shimba et al.G6pc and Fbp mRNA in the liver was increased in the Bmal1 null mice and that liver glucose-6 phosphate was significantly higher than that of control mice, whereas we found the overall 24 hour expression of Pfkfb3 mRNA to be lower in the Bmal1 null mice, particularly during the 4 hours before lights off. This discrepancy may be explained by the fact that in the Shimba et al. study ad libitum. We found a trend  for higher G6pc mRNA expression in the livers of Bmal1 null mice at 0800 h and 1200 h, the time at which the pyruvate tolerance tests were conducted. Thus these results may explain the increase in glucose conversion in male and female Bmal1 null mice over the wild type mice. The failure of the gene expression data to reach significance in the sub analysis of the 0800 h and 1200 h time points probably reflects the lower power. Future studies on the impact of loss of Bmal1 on gluconeogenesis should carefully control the time of day and feed access that the analyses are conducted and in addition assess possible changes in enzyme activity.The observation in the current study that Bmal1 null mice on normal chow confirms 3 recent reports Clock19\u0394 (C57Bl/6) mice Clock19\u0394+MEL mice Bmal1 null mice is not typical since both adiponectin and leptin are elevated. In the case of Clock19\u0394+MEL mice, adiponectin is elevated but not leptin Adipor1 and Adipor2 mRNA expression was decreased in epigonadal fat. This suggests that there are important modifications in adipose tissue that develop in the absence of Bmal1 or disrupted clock function when the defect is present throughout life. Interestingly, the size of the adipocytes in the wild-type and Bmal1 null mice were not different (data not shown), suggesting that the lack of Bmal1 potentiated adipogenesis.The observation of increased adiposity in Nr1d1 mRNA expression in the epigonadal fat pads in Bmal1 null mice. It is well established that CLOCK/BMAL1 heterodimers induce Nr1d1 mRNA through interactions at an E-box on the promoter Clock or Bmal1 will reduce Nr1d1 mRNA expression and eliminate its rhythmicity. Adipogenesis is characterised by high Nr1d1 mRNA expression Nr1d1 mRNA is induced by PPAR\u03b3 Bmal1 null mice, loss of rhythmic Nr1d1 resulted in arrhythmic Ppar\u03b3 expression but levels of Ppar\u03b3 mRNA remained around the circadian nadir levels. The high degree of adiposity in Bmal1 null mice would therefore suggest that low constitutive expression of Nr1d1 may be sufficient to allow adipogenesis and that its rhythmicity is not critical or that the lipolytic pathways in Bmal1 null mice are also significantly downregulated.In the current study we identified a profound decrease in Bmal1 null mice is different from that found in Clock19\u0394 mutant mice on either C57Bl/6 Clock19\u0394 mutants is one of obesity, hyper-insulinaemia, hyper-lipidaemia, glucose intolerance and increased insulin sensitivity Bmal1 null mice that were maintained on a mixed background because we did not wish to confound the impact of Bmal1 loss with other metabolically important mutations present in inbred strains like the C57/Bl6 mice Bmal1 null mice with a complete lack of central and peripheral rhythmicity would be exacerbated over and above those observed in Clock19\u0394 mutants. Bmal1 null mice do not, however, show evidence of a major metabolic disorder. This is not to say that Bmal1 null mice are normal, since they have a reduced life expectancy ad libitum feeding, Bmal1 null mice appear able to maintain normo-glycaemia, despite hypertriglyceridaemia, hyperlipidaemia and hyperleptinaemia. Interestingly despite this potential cardiovascular risk, Bmal1 null mice have recently been shown to be hypotensive and have lower stress induced alterations in blood pressure Bmal1 null mice appear to have compensated for the long term lack of Bmal1 and/or rhythmicity by altering a range of physiological systems including adipose tissue growth, adipokine secretion and feeding behaviour to maintain glucose homeostasis.The metabolic phenotype of Bmal1 null model do not discriminate between that caused by arrhythmicity and any non-circadian functions of this gene. Nevertheless, there is mounting evidence for a role of circadian rhythms in the regulation of metabolic function, as demonstrated through a variety of knock out and mutant mice models. Furthermore, from our results, it is clear that removal of the Bmal1 gene leads to cellular arrhythmicity of not only clock genes, but also genes involved in key metabolic processes including adipogenesis and gluconeogenesis. We believe therefore, that it is this circadian arrhythmicity, at both the cellular and whole organism level, that leads to the metabolic perturbations observed here, rather than any non-clock function of Bmal1.Our analyses of the metabolic parameters of the Bmal1, and the circadian arrhythmicity this creates, leads to increased adiposity when maintained on both control and high fat diets. Furthermore, the Bmal1 null mice displayed increased plasma adiponectin and leptin levels, despite reduced or normal expression of Adipoq and Lep mRNA respectively. Presumably the 1.8\u20132.3 fold increase in adiposity, and hence the greater number of fat cells available for secretion of these hormones, plays a role in the elevated concentrations in plasma. Bmal1 null mice also displayed an elevated response to pyruvate challenge, which when considered with the normal glucose and insulin tolerance, suggest an increased capacity for gluconeogenesis. Nevertheless, Bmal1 null mice display normal profiles of plasma glucose. Finally the loss of adipocyte and hepatic rhythmic gene expression and down-regulation of key metabolic genes in both tissues emphasises further the regulatory role Bmal1 is able to play. Together these results demonstrate that disrupted circadian rhythmicity leads to changes in adipocyte function and compensatory changes in adipokines involved in the cellular control of glucose metabolism, and provides further support for the role of circadian rhythms in the regulation of metabolic homeostasis.In summary, global loss of Figure S1Blood glucose response to intraperitoneal administration of insulin. Insulin (0.75 IU/kg) was injected into wild-type (open circle) and Bmal1 null (closed circle) mice and blood glucose measured over the subsequent 2 hours. Data are mean \u00b1 SEM (n\u200a=\u200a6); (a) males, (b) females.(TIF)Click here for additional data file."}
+{"text": "An outbreak of oseltamivir-resistant influenza A (H1N1) occurred in a long-term care facility. Eight (47%) of 17 and 1 (6%) of 16 residents in 2 wards had oseltamivir-resistant influenza A virus (H1N1) infections. Initial outbreak response included treatment and prophylaxis with oseltamivir. The outbreak abated, likely because of infection control measures. Outbreaks of influenza virus infection cause illness and death, especially among residents of long-term care facilities (LTCFs). In addition to annual vaccination and infection control measures, antiviral agents for treatment and prophylaxis are useful components for control of influenza outbreaks in LTCFs  and neuraminidase inhibitors (oseltamivir and zanamivir). Circulation of influenza A viruses resistant to both classes of antiviral agents, A (H3N2) to adamantanes and A (H1N1) to oseltamivir, was reported during the 2007\u201308 influenza season  received the 2007\u201308 influenza vaccine. Of the 685 LTCF employees involved in direct patient care, 385 (56%) received the 2007\u201308 influenza vaccine on site.We defined a confirmed case as a positive rapid or reverse transcription\u2013PCR result for influenza virus from January 20 through February 8, 2008, in a resident of the LTCF. Surveillance for new case-patients included obtaining a nasopharyngeal specimen from all residents with new onset of fever or respiratory symptoms or any unusual behavior within 24 hours after illness onset. All specimens were tested by using the QuickVue A and B Influenza Test . A second specimen was obtained from all persons with positive rapid test results and some (57%) from persons with negative results for confirmation of influenza virus infection and virus subtyping by reverse transcription\u2013PCR. Medical records, vaccination records, resident activity, and visitor logs were reviewed.Testing for antiviral drug resistance was conducted directly on clinical specimens by pyrosequencing as described  of 17 residents in ward 1 and 1 (6%) of 16 residents in ward 2 were infected with influenza A viruses (H1N1) that contained the H274Y mutation but did not have markers of resistance to adamantanes or zanamivir.On January 30, high fever developed in a male resident in ward 3 while on the first day of a home visit . He retuCharacteristics of case-patients are shown in the Sequence analysis of the HA1 gene in outbreak influenza A viruses (H1N1) showed identical or nearly identical sequences, differing by only 1 or 2 nt . These vThe attack rate of illness caused by oseltamivir-resistant influenza A viruses (H1N1) in ward 1 was within the range (20%\u201380%) reported for other facility influenza outbreaks (Although we documented a relatively high attack rate in 1 ward (ward 1), and despite resistance to the antiviral agent initially used, the outbreak abated quickly. High annual vaccination rates among residents and relatively high rates among employees (www.cdc.gov/h1n1flu/recommendations.htm). This outbreak underscores the possibility of 2 influenza A viruses, with different antiviral susceptibilities, in the same facility. During a facility outbreak of influenza, providers should consult antiviral recommendations of the Centers for Disease Control and Prevention and obtain influenza virus typing and subtyping to guide appropriate antiviral drug choices.The proportion of circulating influenza viruses resistant to oseltamivir increased from 12% during the 2007\u201308 season to 99% during the 2008\u201309 season in the United States, and new interim guidelines for use of antiviral agents were released in December 2008 ("}
+{"text": "Lynch syndrome is an autosomal dominant cancer predisposition syndrome which is caused by a germline mutation in one of four genes, MLH1, MSH2, MSH6 or PMS2. Individuals with a germline mutation in one of these genes are at increased lifetime risk of colon, endometrial, ovarian, small intestine, renal pelvis and ureter. Less commonly patients may develop biliary tract cancers, gastric and pancreatic cancers, brain tumours, sebaceous adenomas, carcinomas and skin keratoacanthomas.Immunohistochemical staining for the above four mismatch repair (MMR) proteins is routinely performed for individuals with bowel cancer or a related cancer who are suspected of having Lynch syndrome. This helps target genetic testing to the correct mismatch repair gene. The genes involved in Lynch syndrome work together in a DNA repair complex. The MMR proteins form a heterodimer with MLH1 partnering PMS1, PMS2 or MLH3 and MSH2 partnering MSH3 or MSH6. This means that a mutation in MLH1 leads to loss of staining of both MLH1 and PMS2, while a mutation in MSH2 leads to loss of staining of both MSH2 and MSH6. The same is not true however, for mutations in either PMS2 or MSH6, where only the single protein made by these genes is not expressed in the tumour.However, the pattern of staining using immunohistochemistry is sometimes more complex due to coding microsatellites within the MSH6 gene. We present two unrelated cases with a germline PMS2 mutation which had loss of both MSH6 and PMS2, but normal MLH1 and MSH2 on immunohistochemical staining."}
+{"text": "CHRNA4  were genotyped and two-step cluster analysis was used for phenotypic cluster characterization. Haplotype estimation was determined by HapStat module of R 2.0 software. Three different phenotypic clusters were identified and the C3 cluster was characterized by the highest ZSDS and MNWS scores compared to others. Furthermore, lifetime prevalence of major depression was significantly higher in the C3 cluster (p\u200a=\u200a0.019). In genetic association tests, this cluster was also significantly associated with rs3787138 genotypes (p\u200a=\u200a0.004) while haplotype analyses of three SNPs  revealed that the risk for C3 phenotype was almost three times higher in GCC haplotype carriers compared to others (permp\u200a=\u200a0.013). This is the first report on a significant association between CHRNA4 variants and a subgroup of smokers characterized by massive withdrawal symptoms and affective vulnerability. Identification of such a phenotypic cluster can be a pivotal step for further pharmacogenetic studies on ligands of the alpha4 nAChR subunit. Our results suggest that performing cluster analysis in genetic association studies can be proposed for complex disorders.Heterogeneous phenotypes of complex disorders pose a great challenge for genetic association studies and for the development of personalized treatment strategies. Cluster analysis of phenotypic data has been recently proposed as a reliable auxiliary method for such studies. A cohort of 236 treatment-seeking smokers was investigated after overnight nicotine abstinence. Alpha4 nicotinic acetylcholine receptor (nAChR) subunit-related phenotypes were assessed by the Fagerstr\u00f6m Test for Nicotine Dependence (FTND), exhaled carbon monoxide (CO) measurements, the Minnesota Nicotine Withdrawal Scale (MNWS) and the Zung Self-Rating Depression Scale (ZSDS). Seven tag SNPs (single-nucleotide polymorphisms) across  CHRNA4) because among nicotinic acetylcholine receptors (nAChRs) those containing alpha4 subunit have the highest affinity for nicotine, the primary psychoactive component of tobacco CHRNA4 in multiple smoking-related phenotypes including nicotine dependence, withdrawal and affective symptoms, association studies have provided partly inconsistent results. Positive associations between nicotine dependence and CHRNA4 were reported in case-control and family studies CHRNA4CHRNA4 was not significant on smoking cessation outcome in a nicotine replacement therapy (NRT) treatment study Nicotine dependence is the most prevalent psychiatric disorder in the world and it is responsible for the highest preventable mortality in developed countries CHRNA4 is also implicated in the development of mood disorders. Significant associations between CHRNA4 and depression and loneliness were demonstrated in a study of elderly population CHRNA4CHRNA4 and affective phenotype according to these reports Accumulative data support that depressive phenotypes and smoking have a multifaceted relationship  but other factors did not differ from men. Severe depression symptoms (above 48 points on ZSDS) were detected in 7.5% of the sample which is significantly higher than the Hungarian average of current episode of MDD (major depressive disorder) The average number of cigarettes per day was 21.2\u00b18.4, breath CO level was 19.0\u00b18.7 and FTND mean score was 6.3\u00b11.2. Mean MNWS score was 12.01\u00b16.1. Mean Zung Self-Rating Depression scale score was 37.7\u00b17.4 in the total population. Comparing different variables between the two genders we found that women scored significantly higher on ZSDS . C1 was characterized by the lowest MNWS (8.8\u00b13.8) and ZSDS (34.1\u00b15.1) scores and CO levels (15.0\u00b13.6) (p\u200a=\u200a0.019). FTND scores and CPD (cigarettes/day) were also different among the three subgroups identified. Both C2 and C3 had significantly higher FTND score than of C1  but FTND score did not differ between C2 and C3. In line with the highest exhaled CO level in C2, significantly greater amount of CPD was reported in C2 than in C1 . Gender distribution did not differ significantly among the three clusters.Two-step cluster analysis resulted in the strongest model when MNWS, exhaled CO level and ZSDS score were stepped into the model. In this model 3 different clusters were identified with significantly differing variables (5.0\u00b13.6) , Table 25.0\u00b13.6) , Table 25.0\u00b13.6) , Table 2p\u200a=\u200a0.013); and effects of rs3787138 and rs3787140 on MNWS score variance were significant but not robust enough for Bonferroni corrections  (p\u200a=\u200a0.004) while C allele carriers of rs3787140 had almost double the chance to have C3 phenotype with a nominal significance (OR\u200a=\u200a1.67 (95% CI\u200a=\u200a0.69\u20134.04); p\u200a=\u200a0.013). ZSDS, exhaled CO and CPD had no significant relation to SNPs in single marker association models.We tested the effects of all SNPs on dependent variables under five models. Single marker association tests provided three nominally significant results: effect of rs6090378 on FTND score (ctively) . Howeverp\u200a=\u200a0.013, globalp\u200a=\u200a0.054). The highest MNWS score was associated with the GCC (p\u200a=\u200a0.04) and the lowest score with the ATT haplotypes  showed that three haplotypes had greater than 5% frequency in the sample . The frequency of the GCC haplotype was found to be the highest in the C3 cluster  and the model was also significant (permp\u200a=\u200a0.018). Consequently, GLM  analysis revealed that odds ratio for having the C3 phenotype was almost three times higher in subjects with GCC haplotype compared to others  among smokers which is characterized by significantly more severe withdrawal symptoms with robust affective vulnerability; not only average score on the self-reported depression scale (ZSDS) but also lifetime prevalence of major depression were significantly higher in this subgroup. Of note, the size of this cluster is not negligible: about 20% of the study population belonged to this subgroup. Genotype and haplotype association tests showed that this special phenotype is determined by genetic components: the chance of belonging to the C3 phenotype group was almost three times higher in those carrying the GCC haplotype compared to other haplotype carriers.The risk haplotype identified in our study has a component described earlier as a risk allele associated with different phenotypes in multiple studies. These findings suggest that C allele carriers of rs1044396 (middle SNP of our haplotype) were more prone to develop nicotine dependence CHRNA4 yielded significant increase in acetylcholine sensitivity which can be a reasonable explanation for more serious withdrawal symptoms in carriers of hypersensitive genetic variant of CHRNA4 in our sample CHRNA4 exon 5 in emotional processes have also been confirmed The pivotal role of rs1044396 in receptor function was suggested by several authors. Convergent evidence hints that this missense mutation of exon 5 in From a pharmacogenetic point of view, it was demonstrated previously that rs2236196 and also rs3787138  were associated with treatment response to nicotine supplementation therapy and/or varenicline. These results raise the possibility that members of the C3 cluster have altered response to cessation promoting agents which may suggest that smoking cessation studies should not focus on smokers as a whole but rather on phenotypically different subgroups of them CHRNA4 variants Of note, exhaled CO levels were not the highest in the genetically vulnerable cluster in our cohort. Nevertheless, this finding is partly in line with negative results on the association between serum cotinine levels and CHRNA4 variants.In previous studies it has been demonstrated that smokers with a history of depression report more severe withdrawal symptoms CHRNA4 haplotypes on smoking-related traits using a special phenotypic clustering method. With the help of the phenotypic cluster analysis the possible role of CHRNA4 in smoking behavior and pharmacological actions of cessation agents can be nuanced. Further pharmacological studies are required to extend our knowledge on the role of CHRNA4 in the treatment of nicotine dependence. Based on these findings we propose cluster analysis combined with haplotype association test as an alternative method for identifying genetically predisposed part of complex disorders and for pharmacogenomic trials.In conclusion, we identified a clinically remarkable subgroup within smokers characterized by salient withdrawal symptoms and depressive signs. Here we presented a unique approach and studied the effect of Written informed consent was obtained from all participants and the study was approved by the the Scientific and Research Ethics Committee of the Medical Research Council of Hungary (ad.8-303/2009-1018EKU).A cohort of 350 treatment-seeking smokers from 5 Hungarian Cessation Centers was investigated in the present study. The time between the last smoked cigarette and the clinical interview was not a criterion for study entry, but for the current analysis we selected only those individuals who reported an overnight nicotine abstinence (n\u200a=\u200a236). Participants were interviewed by a detailed background questionnaire on smoking-related variables such as quantity and quality of smoking, nicotine content of the cigarette regularly smoked by them and family history of smoking. Nicotine dependence was assessed by the Fagerstr\u00f6m Test for Nicotine Dependence (FTND) CHRNA4 were genotyped with Sequenom MassArray technology and the iPLEX Gold chemistry . In this method allele discrimination is based on primer extension with single mass-modified nucleotides followed by MALDI-TOF mass spectrometry. Genotyping was performed by the Technology Centre, Institute for Molecular Medicine Finland (FIMM), University of Helsinki. Genotyping quality was examined by a detailed QC procedure consisting of success rate checks, duplicated samples and positive and negative control samples. Genotyping was done blinded to the phenotypic data. SNPs within CHRNA4 were selected based on literature and the International HapMap project 2>95%) constructing one haploblock  covering the ploblock  accordinTwo-step cluster method was used for phenotypic cluster analysis based on Ward\u2019s aggregation criterion performed in SPSS 20.0 software. Smoking-related phenotypes, such as FTND score, smoked cigarettes/day (CPD), exhaled CO level, MNWS and ZSDS scores for depressive phenotype were stepped into the model. The strongest model was chosen for genetic association tests (higher than 0.5 of silhouette measure of cohesion and segregation). Haplotype analyses were performed with GLM and HapScore tests under additive model using HapStat modul of R 2.0 software. Rare haplotypes with a frequency less than 5% were excluded from analyses. To avoid false positive results Bonferroni\u2019s correction in single marker association tests and permutation procedures with 1000 random permutations were performed by R 2.0 software similarly to our earlier publication Table S1Significant results of single marker association tests.(DOCX)Click here for additional data file.Table S2Basic characteristics of the investigated SNPs.(DOCX)Click here for additional data file."}
+{"text": "Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H2O2) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H2O2 accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H2O2 in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H2O2 in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H2O2 in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts , chlorosis and necrosis of the leaves.In order to study the mechanisms behind the infection process of the necrotrophic fungus  Even though the importance of H2O2 and antioxidants for the defense against necrotrophic fungal pathogens are well understood it still remains unclear in which cell compartments H2O2 accumulates during necrotrophic fungal infection and how it correlates to subcellular contents of antioxidants. Such information is crucial to understand the compartment-specific mechanisms behind the infection process of necrotrophic pathogens.The infection of plants by necrotrophic pathogens is characterized by the release of phytotoxins, cell wall degrading enzymes and an oxidative burst which is triggered by the pathogen and finally leads to plant cell death. Early visible signs of these events are the appearance of chlorotic regions followed by necrosis Botrytis cinereaBotrytis cinerea infection ROS accumulation has been observed around the penetrated cell walls, at the plasma membrane and inside cells close at and surrounding the necrotic IS Botrytis cinerea infection is due to the production of phytotoxins, cell wall degrading enzymes and the oxidative burst which eventually leads to the death of the cells One of the best studied necrotrophic fungal pathogens is 2O2 during Botrytis cinerea infection in Arabidopsis Col-0 on a high resolution and to correlate it with subcellular ascorbate and glutathione contents.Thus, the aim of this study was to investigate the compartment-specific accumulation of HArabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0) were grown on \u201cNaturahum\u201d potting soil  in growth chambers with 8/16 h day/night photoperiod. Day and night temperatures were 22\u00b0C and 18\u00b0C, respectively, the relative humidity was 60% and the plants were kept at 100% relative soil water content. Light intensity varied between 110 and 140 \u00b5mol m\u22122 s\u22121.After stratification for 4 d at 4\u00b0C seeds of Arabidopsis thaliana Col-0 were placed in petri dishes containing filter paper wetted with sterile water. A 50 \u00b5l drop of a Botrytis cinerea (strain: Bo510) spore suspension was delivered onto the abaxial side of each leaf (approximately 1\u00d7105 spores ml\u22121). Spores were preincubated in 3 g L\u22121 Gamborg's B5 medium  supplemented with 25 mM glucose for 2 h to stimulate germination. Control leaves (CL) were treated likewise but without spores in the medium. Petri dishes were sealed and stored at light conditions described above.Cut leaves of 8 weeks-old Botrytis cinerea. Due to preliminary investigations these time points were chosen as clear changes could not be determined at earlier time points and as cells at the IS showed massive cell death at 96 hpi which made it impossible to obtain adequate ultrastructure for investigations at later time points.Different parameters were investigated for the IS and for the surrounding leaf tissue, which was defined as AIS. Values were then compared to CL, which had been mock-inoculated. As chloroplasts showed the strongest changes in antioxidative capacity the fine structure of chloroplasts was additionally monitored during the infection period. Samples for the different experiments were harvested at the time of the inoculation (0 h), 12, 24, 48, and 96 hpi with the fungal pathogen 2O2 was performed according to Gro\u00dfkinsky et al. Detection of H2) of at least three different leaves for each treatment and time point (see frames in 3) in 50 mM MOPS-buffer (pH 7.2) for subcellular H2O2 visualization. Samples for ultrastructural and cytohistochemical investigations were then transferred into glass vials and fixed for 90 min at RT in the above mentioned solutions. Samples for the visualization of subcellular H2O2 distribution were incubated with CeCl3 solution for 60 min and then fixed in 2.5% glutardialdehyde in 0.06 M S\u00f8rensen phosphate buffer at pH 7.2 for 90 min at RT. For ultrastructural analysis and H2O2 localization samples were then rinsed in 0.06 M S\u00f8rensen phosphate buffer (4 times for 15 min each) and post-fixed in 1% osmium tetroxide in 0.06 M S\u00f8rensen phosphate buffer for 90 min at RT. The samples were then dehydrated in a graded series of increasing concentrations of acetone . Pure acetone was then exchanged for propylene oxide and the specimens were gradually infiltrated with increasing concentrations of Agar 100 epoxy resin  mixed with propylene oxide for a minimum of 3 h per step. Samples were finally embedded in pure, fresh Agar 100 epoxy resin  and polymerized at 60\u00b0C for 48 h. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S ultramicrotome , post-stained with lead citrate (1% dissolved in 0.6 M NaOH) and uranyl-acetate (2% dissolved in aqua bidest) for 15 min. Sections were then observed in a Philips CM10 TEM. For cytohistochemical investigations samples were rinsed in 0.06 M S\u00f8rensen phosphate buffer (pH 7.2) for 4 times 15 min after fixation. They were then dehydrated in increasing concentrations of acetone  at RT for 20 min at each step. Subsequently, specimens were gradually infiltrated with increasing concentrations of LR-White resin  mixed with acetone (90%) for a minimum of 3 h per step. Samples were finally embedded in pure, fresh LR-White resin and polymerized at 50\u00b0C for 48 h in small plastic containers under anaerobic conditions. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S ultramicrotome .Preparation of samples for TEM and immunogold labeling of ascorbate and glutathione was performed as described previously 2O2 was determined with a modified method according to Blokhina et al. 3 were digitized and the percentage of areas covered with CeCl3 precipitates were measured automatically using the software package Cell D with the particle analysis tool . Measurements were performed in different visually identified and manually traced cell structures such as cell walls, mitochondria, plastids, nuclei and the cytosol. A minimum of 40 sectioned cell structures from at least 15 different cells were evaluated for CeCl3-staining. The obtained data were statistically evaluated using Statistica .The accumulation of HChanges in the number of chloroplasts and their inner structures were evaluated according to Zechmann et al. Immunogold labeling of glutathione and ascorbate was done according to Zechmann et al. Micrographs of randomly photographed immunogold labeled sections were digitized and gold particles were counted automatically using the software package Cell D with the particle analysis tool  in different visually identified and manually traced cell structures . For statistical evaluation at least four different samples were examined. A minimum of 20 (peroxisomes and vacuoles) to 60 (other cell structures) sectioned cell structures of at least 15 different cells were analyzed for gold particle density per sample. The obtained data were statistically evaluated using Statistica .Several negative controls were made to support the specificity of the immunogold procedure. Negative controls were treated either with (i) gold conjugated secondary antibody (goat anti-rat IgG for ascorbate and goat anti-rabbit IgG for glutathione) without prior incubation of the section with the primary antibodies, (ii) non-specific secondary antibody (goat anti-rabbit IgG for ascorbate and goat anti-rat IgG for glutathione), (iii) pre-immune serum instead of the primary antibody and (iv) primary antibody against ascorbate and glutathione pre-adsorbed with an excess of reduced and oxidized ascorbate and glutathione, respectively for 2 h prior to labeling of the sections. For the latter a solution containing either 10 mM of ascorbic acid, dehydroascorbic acid, reduced or oxidized glutathione was incubated with or without 0.5% glutardialdehyde for 1 h. Excess glutardialdehyde was saturated by incubation for 30 min in a solution of 1% (w/v) BSA. The resulting solutions were both used in independent experiments to saturate the anti-ascorbate and glutathione antibodies for 2 h prior to its use in the immunogold labeling procedure described above. Labeling on sections treated as negative controls showed no or only very few gold particles bound to ascorbate and glutathione which was similar to previous results obtained by using the same methods in different plant species Botrytis cinerea did not show symptoms before 48 hpi  when compared to CL. In mitochondria glutathione labeling at the AIS was found to be significantly decreased 24 hpi (\u221233%) but showed no significant changes at other time points when compared to CL. At the IS glutathione labeling in chloroplasts was strongly increased 12 hpi (141%), 24 hpi (335%) and 48 hpi (92%). A similar pattern was found in nuclei, peroxisomes and the cytosol where glutathione labeling density was significantly increased at the IS 12 hpi , and 24 hpi , when compared to CL. At the AIS a strong and significant increase of glutathione specific labeling was observed in chloroplasts, nuclei, and the cytosol 24 hpi , 48 hpi , and 96 hpi  when compared to CL. In peroxisomes of the same leaf area density of glutathione labeling was found to be increased 12 hpi (93%), 24 hpi (214%), 48 hpi (227%) and 96 hpi (328%) when compared to CL . GlutathBotrytis cinerea inoculated plants at 12, 24, 48 and 96 hpi. Mock-inoculated plants contained about 17 and 8 chloroplasts in cell sections of the palisade and spongy parenchyma, respectively. At the IS the number of chloroplasts decreased significantly in cell sections of both tissues 48 hpi  when compared to CL  throughout the infection. At the IS this drop occurred earlier and was more severe than at the AIS showing a good correlation with the accumulation of H2O2. Similar results have been obtained by electron paramagnetic resonance within Arabidopsis leaves where a massive depletion of ascorbic acid correlated with an increase in ROS Botrytis cinerea the decrease of ascorbate contents, insufficient activities of related antioxidative enzymes and a general shift towards the oxidative state at the cellular and organelle level was related to disease progress 2O2 observed here prove that the decreasing capacity of the antioxidative system triggers an oxidative burst leading to chlorosis and eventually cell death during susceptible Botrytis cinerea infection in Arabidopsis. This conclusion is supported by changes in glutathione contents at the IS which showed a strong increase 12 and 24 hpi followed by a strong drop 48 and 96 hpi when H2O2 accumulation reached its maximum. Whereas subcellular glutathione contents 96 hpi are similar to what has been reported previously for tomato Botrytis cinereaROS like H2O2 accumulation could be observed by DAB and CeCl3 staining at the IS at 24 hpi similar effects could be found at the AIS at 48 hpi. Additionally, the situation of H2O2 accumulation at the AIS at 96 hpi mimicked the situation at the IS at 48 hpi. Similar effects of the antioxidative response could be observed at these time points as ascorbate contents dropped earlier at the IS when compared to the AIS in most cell compartments. In addition, both the initial increase and the drop of glutathione contents at the final stages of infection happened earlier at the IS than at the AIS in most cell compartments. The reason for the delayed response of the AIS can be explained by delayed symptom development as the fungal hyphae have to establish the infection at the IS first before they can spread into other areas of the AIS. Thus, we can conclude that H2O2 accumulation and the antioxidative response of the AIS lagged behind but mimicked the situation at the IS.Generally, the response of the AIS lagged behind that of the IS. While HBotrytis cinerea2O2 in chloroplasts at the AIS at this time point. This could be due to a shift from reduced to oxidized glutathione despite higher activity of glutathione reductase which was demonstrated for chloroplasts in Botrytis infected tomato plants at this time point and could also be observed in whole leaves Botrytis cinerea infection 2O2 leading to oxidative damage visible in the form of chlorosis at the AIS and subsequently necrosis caused by massive cell dead at the IS 96 hpi.On the subcellular level it is remarkable that the AIS showed a massive accumulation of glutathione in all cell compartments of up to 600% in chloroplasts at 96 hpi when glutathione contents dropped to background levels at the IS. Again, this increase in glutathione contents can be correlated with the up-regulation of genes encoding enzymes responsible for glutathione synthesis found in Arabidopsis infected with Botrytis cinerea also induced severe changes in chloroplast fine structure. Interestingly, plastoglobuli contents were massively increased 24 hpi and 48 hpi at the IS and 48 hpi and 96 hpi at the AIS. These results correlate well with a strong decrease in thylakoid content 96 hpi at the AIS. Plastoglobuli are considered to be an important storage subcompartment of degenerated thylakoid membranes 2O2 inside the chloroplasts triggered thylakoid degradation which resulted in a strong increase in plastoglobuli size until complete disintegration of chloroplasts.Infection of Arabidopsis with Botrytis cinerea interaction with susceptible Arabidopsis plants induced a severe oxidative bust starting in cell walls at 24 hpi at the IS and spread to mitochondria, chloroplasts, nuclei and the cytosol at later stages. Cell walls, mitochondria and chloroplasts seem to be the main origin of H2O2 as an accumulation occurred in these cell compartments much earlier than in the cytosol and nuclei. Early responses (time points before 24 hpi) observed in other plant species in previous studies seem to be rather local either triggered by oxidative burst in epidermis cells or by ROS produced by the fungal hyphae. The breakdown of the antioxidative system indicated by a strong drop in ascorbate and glutathione levels at later stages of infection correlated with the accumulation of ROS which resulted in the development of chlorosis, necrosis and eventually cell death and was associated with severe changes in chloroplast structure and number.Summing up, this study demonstrates that ROS production during Figure S1Light microscopical images of leaf sections from Arabidopsis thaliana Col-0 inoculated with Botrytis cinerea. Images in A and D show sections of CL at the beginning (0 h) and at the end of the experiment (96 h), respectively, at the mock-inoculation site. Images in B, C and E, F show leaves at the AIS and at the IS, respectively, at 48 hpi  and 96 hpi . Whereas fungal structures remain absent in sections of the AIS  hyphae (arrows) on top (48 hpi) and inside (96 hpi) the leaves could be found at the IS. Bars\u200a=\u200a50 \u00b5m.(TIF)Click here for additional data file.Figure S2Transmission electron micrographs showing ascorbate specific labeling at the AIS. Gold particles bound to ascorbate were detected at the AIS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Bars\u200a=\u200a1 \u00b5m. C\u200a=\u200achloroplasts with or without starch (St), CW\u200a=\u200acell walls, ICS\u200a=\u200aintercellular spaces, M\u200a=\u200amitochondria, N\u200a=\u200anuclei, Px\u200a=\u200aperoxisomes, V\u200a=\u200avacuoles.(TIF)Click here for additional data file.Figure S3Transmission electron micrographs showing ascorbate specific labeling at the IS. Gold particles bound to ascorbate were detected at the IS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Note that 96 hpi fungal hyphae (H) containing gold particles bound to ascorbate and only remnants of organelles such as plastids (P) could be found in the degenerated leaf cells (D). Bars\u200a=\u200a1 \u00b5m. C\u200a=\u200achloroplasts with or without starch (St), M\u200a=\u200amitochondria, N\u200a=\u200anuclei, Px\u200a=\u200aperoxisomes, V\u200a=\u200avacuoles.(TIF)Click here for additional data file.Figure S4Transmission electron micrographs showing glutathione specific labeling at the AIS. Gold particles bound to glutathione were detected at the AIS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Bars\u200a=\u200a1 \u00b5m. C\u200a=\u200achloroplasts with or without starch (St), CW\u200a=\u200acell walls, ICS\u200a=\u200aintercellular spaces, M\u200a=\u200amitochondria, N\u200a=\u200anuclei, Px\u200a=\u200aperoxisomes, V\u200a=\u200avacuoles.(TIF)Click here for additional data file.Figure S5Transmission electron micrographs showing glutathione specific labeling at the IS. Gold particles bound to glutathione were detected at the IS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Note that 96 hpi fungal hyphae (H) containing gold particles bound to glutathione and only remnants of organelles such as plastids (P) could be found in the degenerated leaf cells (D). Bars\u200a=\u200a1 \u00b5m. C\u200a=\u200achloroplasts with or without starch (St), CW\u200a=\u200acell walls, M\u200a=\u200amitochondria, N\u200a=\u200anuclei, Px\u200a=\u200aperoxisomes, V\u200a=\u200avacuoles.(TIF)Click here for additional data file.Table S1Arabidopsis thaliana.Total number of gold particles bound to ascorbate and glutathione in mock inoculated  Values are means with standard errors and document the total amount of gold particles bound to ascorbate and glutathione per \u00b5m2 in different cell compartments of mock inoculated Arabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0). n.d.\u200a=\u200anot detected. n>20 for peroxisomes and vacuoles and n>60 for other cell structures. Different lowercase letters indicate significant differences (P<0.05) analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover.(TIF)Click here for additional data file.Table S2Arabidopsis thaliana Col 0.Total number and size of chloroplast fine structures in mock inoculated  Values are means with standard errors and document the relative area of internal chloroplast structures and the size of chloroplast in \u00b5m2 detected by TEM on a longitudinal ultrathin section within the mesophyll of mock inoculated leaves in Arabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0). Different lowercase letters indicate significant differences (P<0.05) analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover. N>20 from at least four different samples.(TIF)Click here for additional data file."}
+{"text": "CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs.Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method.CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component.LOH of the The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior. Testicular germ cell tumor (TGCT) is diagnosed mainly after puberty and is the most frequent malignancy in young adult men51 phase-controlling mechanism centered around the pRB protein. Different cancers seem to have altered different key components of that mechanism, which may be connected with gene activity patterns in different target cells.7The cell cycle regulatory pathway deregulated in almost all human tumors appears to be the G0 and the early G1 phase, hypophosphorylated pRB is complexed with the transcription factor E2F.1, a significant hyperphosphorylation of the pRB by CDK4 and CDK6 in complex with D cyclins  occurs.9The pRB is essential in cell cycle regulation and its function is regulated by phosphorylation. In GCDKN2 locus at chromosomal region 9p21 encodes p16INK4a tumor suppressor protein involved in the RB cell cycle control pathway.1/S phase transition by inhibiting the activity of CDK4 and CDK6. Thus, by inhibiting pRB phosphorylation, p16INK4a can promote the formation of a pRB-E2F repressive transcriptional complex, which blocks cell cycle progression past G1/S restriction point.11The RB1 and inactivating mutations or promoter methylation of the CDKN2A (p16INK4a) tumor suppressor gene, as well as the overexpression of the cyclin D2/CDK4 complex.13In diverse types of cancer the RB pathway becomes deregulated through alterations in one or more of its components. The most common defects of the RB pathway are mutations or deletions of CDKN2A p1INK4a tumCDKN2A and RB1 tumor suppressor genes in TGCTs.The objective of this study was to evaluate the occurrence of the loss of heterozygosity (LOH) of the Fourty TGCT samples (18 seminomas and 22 nonseminomas) were collected from Sisters of Mercy University Hospital and University Hospital Center, Zagreb, Croatia. The samples were formalin-fixed and paraffin-embedded. Clinical and pathological data for 40 TGCTs according to the WHO 2004 classification are shown in For each specimen, 20 \u03bcm paraffin-embedded section was prepared for DNA extraction. In addition, 4 \u03bcm section was stained with hematoxylin-eosin to identify the tumor and normal tissue areas which were removed separately from the microscopic slide, transferred to microtubes and extracted using QIAamp DNA Mini Kit .23 located close to intron 1 of the CDKN2A gene was analyzed in this study.2, 0.2 \u03bcM of each primer , 1x Flexi buffer  and 0.5 U of GoTaq\u00ae Hot Start Polymerase . One hundred nanograms of DNA were used in each PCR reaction. PCR amplifications were carried out in a Eppendorf Mastercycler Personal , with cycling times of 96\u00baC for 5 min (one cycle), then 45 cycles of 96\u00baC for 30 s, 57\u00baC for 45 s, and 72\u00baC for 30 + 1 s. The final step was incubation at 72\u00ba C for 10 min. Amplified DNA fragments were analyzed on silver-stained 15% polyacrylamide gels. LOH of CDKN2A was considered to had occured if one out of two alleles (heterozygous samples) of a gene marker was missing or significantly reduced in comparison to alleles from adjacent normal tissue.A previously described polymorphic microsatellite marker hMp16\u03b1-I1 consisting of a mononucleotide tract of (A)RB1 was detected using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Amplification with RB1 primers 5\u2032-TCCCACCTCAGCCTCCTTAG-3\u2019 and 5\u2032-GTAGGCCAAGAGTGGCAGCT-3\u2032 used in our study produced a 190 bp segment of intron 17.LOH of CDKN2A gene. From 40 TGCTs, 34 (85%) tumors were informative for this polymorphism, 16 (89%) seminomas and 18 (82%) nonseminomas. Our analysis revealed that two (6%) samples showed LOH of hMp16\u03b1-I1 marker. The observed changes were assigned to two nonseminomas  of total TGCTs were heterozygous for this polymorphism; 15 (83%) seminomas and 19 (86%) nonseminomas. LOH was observed in two (6%) samples when looking at the total TGCTs analyzed. Once again the observed allelic losses were assigned to nonseminomas: two samples  is reported to be the most commonly deleted region early in the development of nonseminomas, which may be implicated in their ability to differentiate into various types, for various markers located within this region.20Despite of promoter methylation and mutations being the most common ways of inactivating CDKN2A were found in nonseminomas with a yolk sac tumor component, one sample also having an embryonal carcinoma component. Furthermore, one nonseminoma with the LOH of CDKN2A demonstrated LOH of TP53 gene, and the other showed LOH of the CDH1 gene.21In our study only nonseminomas demonstrated LOH . Both LORB1 gene is often deleted or mutated to an inactive form in a variety of human tumors. Cells of embryonal testes and intratubular germ cell neoplasia (ITGCN) show no expression of pRB, whereas it is expressed in healthy testes during spermatogenesis. The lack of pRB in most TGCTs may, therefore, reflect its deregulation by normal mechanisms in testicular germ cells. However, the lack of pRB may facilitate the transition of those cells to tumor cells of ITGCN and thus contribute to molecular pathogenesis of TGCTs.23The RB1 gene are, along with its mutations, also reported as one of the most common alterations of the RB pathway. Various studies revealed deletions of the RB1 gene region in testicular cancer.et al.RB1 introns 16 and 20, and found LOH in 5% of seminomas and 28% of nonseminomas analyzed within 93% of informative TGCT cases. The location of the RB1 gene is reported to be one of the most commonly involved in allelic imbalance within TGCTs.RB1 in various forms of TGCTs needs to be further elucidated in more detail. Studies also revealed a different pattern of LOH in different histological types of nonseminomas for markers located within the genomic region containing the RB1 gene (13q14), varying from 0% in yolk sac tumor component to 50% in choriocarcinoma.25In contrast, deletions of RB1 gene was found in nonseminomas with an embryonal carcinoma component, and both nonseminomas with LOH of RB1 also demonstrated LOH of the TP53 gene.26In our study, LOH of the CDKN2A, RB1, TP53 and CDH1 in TGCTs may increase their tumorigenic potential by the increased proliferation capacity due to RB1 loss and decreased rate of apoptosis due to TP53 alteration.RB1 are often seen together with alterations of TP53 in variety of different cancers.RB1 and TP53 genes in a cell produces a synergistic effect, which imposes a stronger selective pressure for the cellular transformation. This may also help to explain the high proliferation rate and/or invasiveness of TGCTs with embryonal carcinoma and yolk sac tumor component. A higher incidence of LOH in nonseminomas may provide a clue to their invasive behavior, because for some of the nonseminoma types there seem to be a region of preferential loss , and all of the TGCTs show gain of 12p11\u201312p12 sequences.28LOH of CDKN2A and RB1 inactivation.24However, the low frequency of observed LOHs in this study could be a consequence of genomic instability in above mentioned nonseminomas, rather than the main cause of"}
+{"text": "Several scoring system have been developed with the aim to identify clinical deterioration among hospitalized patients and allocate resources in accordance with the degree of deterioration - most without validation. The aim of the present study was to describe to which degree the system \"Tidlig Opsporing af Kritisk Sygdom\" (TOKS) is able to identify patients who either have or develop severe sepsis or septic shock within 24 after arrival to hospital.A retrospective descriptive study of patients hospitalized with community acquired severe sepsis or septic shock. Patients were identified based at discharge diagnosis ((IDC10 code A40.0-A41.9). Based at a manual evaluation of all patient records patients were included if they within the first 24 hours after arrival to the hospital fulfilled predefined criteria for severe sepsis or septic shock. Vital values registered at arrival to the hospital were identified and used for the present analysis. TOKS score is based at scores for respiratory frequency, saturation, systolic blood pressure, pulse rate, consciousness and temperature. The score range from 0 to 21 with an indication of need for a doctoral evaluation if the score is 3 or higher.335 patients were discharged with a diagnosis of sepsis. 212 fulfilled the criteria for severe sepsis or septic shock within the first 24 hours of hospitalization. One hundred and six (50%) were male, mean age 70.6 years , 103 (49%) had septic shock. Median TOKS score at arrival was 4 (range 0-13). 10/212 (5%) had TOKS=0, indicating no need for measurements of vital values the next 24 hours, 20/212 (9%) had TOKS=1, indicating measurements of vital values every 8 hours, 13/212 (6%) had TOKS=2 indicating control of vital values after one hour, 66/212(31%) had TOKS 3-4 indicating need for evaluation by a junior doctor and 103/121 (49%) had a TOKS score\u22655 indicating need for urgent specialist evaluation.14% of the patients who develop severe sepsis or septic shock within 24 hours after arrival to the hospital had a TOKS score at arrival indicating a need for control of vital values every 8 hours or less."}
+{"text": "The development of methods for the modification of peptides and proteins under benign aqueous conditions has received a great deal of attention from both industrial and academic laboratories.4O-mesitylenesulfonylhydroxylamine and hexamethylphosphorous triamide.Bacillus lentus SBL(S156C) (1) with 2,5-dibromohexanediamide (2) at pH 8, which afforded intermediate dehydroalanine 3, presumably via the transient formation of sulfonium 4  of \"superfolder\" green fluorescent protein (GFP) bearing a single surface-accessible cysteine. We were able to synthesise unusually stable cyclic sulfonium species and to employ these in a novel bioconjugation that is complementary to current dehydroalanine-based technologies for substrates for which dehydroalanine formation is not facile. Although the presence of sulfonium ions in peptides and proteins derived from the alkylation of methionine is well established,C of \"sup6) with 2 under Davis' conditions  MS indicated quantitative conversion to a species with a molecular weight equivalent to that of sulfonium 8 . Indeed, throughout this study, we observed no evidence for the formation of dehydroalanine 7, or products derived therefrom, even upon incubation of sulfonium 8 for prolonged periods (vide infra). The isolation of this sulfonium species as a stable adduct and its reluctance to undergo elimination to give the corresponding dehydroalanine product 7 was noteworthy and we embarked upon further studies to understand and exploit this unexpected mode of reactivity.We treated GFP(S147C)  with 2 u8 was treated with N-methylbromomaleimide (1 equiv).8, whereas 6 reacted cleanly, thus suggesting that 2 had reacted exclusively on cysteine to generate 8 (see the Supporting Information).In order to gain further evidence for the site of reaction between GFP(S147C) and 2,5-dibromohexanediamide, sulfonium 8 was achieved by varying a number of parameters including the number of equivalents of 2, reaction temperature and reaction time  was apparent by LCMS  excess 2 could be readily removed by repeated diafiltration into fresh buffer. Sulfonium 8 was found to be stable in the absence of nucleophiles at 21 \u00b0C for 24 h and at 4 \u00b0C for 1 month; although, significant decomposition was observed at 37 \u00b0C after 4 h (vide supra). Treatment of 8 with \u03b2-mercaptoethanol  afforded bisthioether 10 a  in complete conversion, presumably through ring opening of the cyclic sulfonium  afforded the expected GFP\u2013thioglucose conjugate 10 b as the sole product, with complete conversion after 5 h . We also confirmed that these reactions could be carried out effectively at room temperature (21 \u00b0C) in 5 h, or more rapidly at 37 \u00b0C or with increased concentrations of the nucleophile . Treatment of sulfonium 8 with phenyl selenol (100 equiv) afforded the mixed thio/seleno bisether 10 d in excellent conversion (entry 6). Although the conversion from reaction between 8 and phthalimide to give 10 e was only modest (entry 7), we were delighted to observe clean reaction in high conversion upon treatment of 8 with sodium azide (1000 equiv) to generate azide-labelled GFP 10 f (entry 8).We then evaluated the reactivity of fter 5 h , entry 1In conclusion, we have shown that treatment of superfolder GFP with bis-alkylating agents can be used to prepare highly stable, yet synthetically useful cyclic protein sulfonium adducts. These species undergo clean addition reactions to allow the introduction of a variety of useful chemical motifs. We envisage that the stability of the generated cysteine-derived sulfonium is presumably strongly correlated to the chemical environment of the alkylated cysteine. This new bioconjugation technique could provide a useful entry into multiply functionalised proteins and further work will be directed toward extending these findings to other systems."}
+{"text": "NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5\u2032/3\u2032 untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules  NLGN3, NLGN4 , NRXN1 and scaffolding proteins SHANK2 and SHANK3Autism spectrum disorder (ASD) is a neurodevelopmental disease with complex genetic and clinical heterogeneity. The core symptoms of ASD are impaired social interactions, deficient communication, restricted interests, and stereotyped activity patterns NLGN3 and NLGN4 are the first discovered ASD-associated genes in this pathway NLGN4 gene was identified in two brothers, one with autism and the other one with Asperger syndrome NLGN4 in ASD NLGN3 was detected in two male ASD siblings until now NLGN3 gene, NLGN4 was more frequently discovered to have ASD-related rare functional mutations in coding regions or promoter regions NLGN4 happen in only a small fraction of ASD cases, their impacts on synaptic function provide an important perspective for understanding the pathogenesis of ASD. Besides the coding region, the regulatory region and copy number of NLGN4 are deserved to be studied in ASD since they may affect the expression level of NLGN4.NLGN proteins, expressed highly in brain, are postsynaptic adhesion molecules interacting with presynaptic NRXNs in a calcium-dependent manner SHANK3 in ASD cases SHANK2 in ASD subjects, suggesting the implication of SHANK2 in ASD SHANK proteins contain multiple interaction sites and assemble many molecules, including guanylate kinase-associated proteins (GKAPs), which links to PSD-95 NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. We also examined the CNVs of NLGN4, NLGN3, SHANK2 and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). Moreover, six common single nucleotide polymorphisms (SNPs) within NLGN4 were identified and analyzed for the association with ASD.In this study, we sequenced the regulatory region including the promoter region and 5\u2032/3\u2032 untranslated regions (UTR) and the entire coding region of The study was approved by the Ethics Committee of Shanghai Mental Health Center and the Ethics Committee of the School of Life Sciences, Fudan University. Informed, written consent was obtained from the controls, the parents or guardians of the children.A total of 285 patients with ASD were recruited from Department of Child and Adolescent Psychiatry, Shanghai Mental Health Center, China. All probands met DSM-IV diagnostic criteria for autism, Asperger syndrome or pervasive developmental disorder not otherwise specified (PDD-NOS). Cases were excluded if they had known medical disorders or chromosomal abnormalities. The patients were 246 males and 39 females with a mean age of 7.05 (SD\u200a=\u200a3.36 years). All individuals were Han Chinese.384 unrelated ethnically-matched healthy controls were recruited from Fudan University. There were 311 males and 73 females.NLGN4 gene was acquired from the UCSC Genome Browser (NM_181332). The promoter region (2000 bp upstream of the transcription start site), six exons including the 5\u2032/3\u2032 UTRs and the open reading frame (ORF), and ten exon-intron junctions were sequenced in 285 ASD patients and 384 controls. Totally, 9110 basepair (bp) of untranslated region and 2451 bp of coding region in NLGN4 were sequenced. Genomic DNA was isolated from whole blood using Puregene Blood Core Kit C . PCR primers were designed by PRIMER 3 (http://frodo.wi.mit.edu/primer3/). The information of primers is shown in NLGN4, while those for exons 1, 2, and 4 can amplify the sequences of NLGN4 and NLGN4Y simultaneously due to sequence homology. PCR was performed in a 10 \u00b5l reaction mixture containing 1 \u00b5l 10\u00d7buffer (Mg2+ Plus), 1 \u00b5l dNTP mixture (2.5 mM), 0.2 \u00b5l primers (10 \u00b5M), 0.05 \u00b5l Takara Hotstar Taq (5 U/\u00b5l), and 1 \u00b5l genomic DNA (20 ng/\u00b5l). Thermo cycling conditions consisted of 35 cycles with an initial denaturation at 95\u00b0C for 5 min, followed by denaturation at 95\u00b0C for 30 s, annealing at 58\u00b0C for 30 s, extension at 72\u00b0C for 30 s, and a final extension at 72\u00b0C for 5 min. We carried out all sequencing reaction by BigDye Terminator version 3.1 in ABI 3100 sequencers .The reference sequence of NLGN4, NLGN3, SHANK2, and SHANK3) were measured by a custom-by-design Multiplex AccuCopy\u2122 Kit  based on a multiplex fluorescence competitive PCR principle as described by Du et al. http://genome.ucsc.edu). Three reference genes  were utilized for normalization. Nine target genomic segments within the four genes (two or three segments for each gene) were chosen for the AccuCopy assay. The forward and reverse primers of target segments were provided in POP1, RPP14, and TBX15) and the S/C ratio for each target fragment was first normalized to three reference genes respectively. The three normalized S/C ratios were further normalized to the median value in all samples for each reference gene respectively and then averaged. If one of the three normalized S/C ratios deviated more than 25% from the average of the other two, it was excluded for further analysis.The copy numbers of four genes  analysis of SNPs and the haplotype association were analyzed using Haploview 4.2 software. LD between the SNPs was measured by a pairwise D\u2032 statistic. The structure of the LD block was examined using the method of Gabriel et al. NLGN4 was detected in our cohort. Direct sequencing of the coding region and regulatory region of NLGN4 identified six common SNPs . They were rs5916355 in the promoter region, rs3810688, rs3810686, rs5916269, rs1882260, and rs140700235 in 3\u2032UTR. Five percent of samples were selected randomly for validation by sequencing in the opposite direction. No genotyping error was detected. Association analysis of these six SNPs with ASD did not show significant difference of allele frequencies between ASD patients and controls (Only one synonymous mutation rs7049300 (Thr311Thr) was found in one ASD individual. No non-synonymous mutation in controls . The LD controls . The polNLGN4 ranged between 0.76 and 1.22 in 246 male samples, and ranged between 1.69 and 2.38 in 39 female samples, which indicated unchanged copy number of NLGN4 for all samples  was identified in two brothers with ASD Since the first report of NLGN3 and NLGN4 to ASD in Chinese population so far NLGN3 and NLGN4 genes were screened and twelve intronic SNPs were genotyped for case-control association analysis in 229 ASDs cases and 184 controls in a Chinese cohort NLGN3 gene was associated with ASD . Six SNPs in the introns of NLGN4, different with the ones examined in our study, were analyzed and showed no frequency difference between ASD cases and controls NLGN4 in 285 Chinese ASD cases by comprehensively testing genetic variants including rare mutations in the coding region and regulatory region, copy number variations and common SNPs. No positive results were identified, suggesting NLGN4 gene is not a major susceptibility gene in our ASD cohort.Only one study investigated the susceptibility of NLGN3, SHANK2, and SHANK3 in this cohort. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density and are believed to play an important role in the formation and maturation of dendritic spines. Rare de novo CNVs and nonsense/frameshift mutations in SHANK2 and SHANK3 have been identified in ASD and mental retardation cases SHANK2 in a German cohort of 184 mental retardation cases and a Canadian cohort of 396 ASD probands de novo deletions and a de novo nonsense mutation of SHANK2 were identified in unrelated mental retardation and ASD individuals. Haploinsufficiency of SHANK3 is associated with ASD and mental retardation in human patients and results in altered synaptic plasticity and reduced social behaviors in mice NLGN3, SHANK2 and SHANK3, indicating the limited role of these genes in Chinese ASD patients. Considering that we did not screen these genes for sequence level variants and did not investigate other genes in this pathway like NRXN3 and SHANK1, recently reported to be associated with ASD To investigate the possibility of other genes in neurexin-neuroligin pathway involved in the etiology of ASD, we examined the copy number of NLGN4 or NLGN3 have been reported by different studies involving about 1500 ASD cases, indicating a low frequency (no more than 1%) of rare variants within NLGN3 and NLGN4 in ASD NLGN4 as well as examine the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in Chinese population. Though the results were negative, this study would extend our understanding about the role of these genes in ASD in different populations.One limitation of this study should be considered. Nine missense/nonsense mutations and 3 deletions within In this study, we used the multiplex fluorescence competitive PCR for copy number measurement. This newly developed method has the advantages of multiplex measurement and low measurement variation NLGN4 were identified and studied for the relationship with ASD in our cohort. These SNPs might affect the expression of NLGN4 by binding with transcription factors or microRNA. The SNP rs5916355 in the promoter region of NLGN4 was predicted to be a binding site for transcription factor HFH-2 using the online tool TFSEARCH. Two websites (http://www.microrna.org/microrna/getGeneForm.do and http://www.mirbase.org/search.shtml) were used to predict miRNAs which bind to the 3\u2032UTR sequence of NLGN4 containing five tested SNPs. The variants rs3810688, rs5916269 and rs140700235 were found located within the potential binding sites of has-miR-1293, has-miR-1324 and has-miR-5011-5p, respectively. These SNPs may affect NLGN4 expression by altering the interaction of miRNAs and mRNA. For example, according to the algorithm RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/), the allele A of rs3810688 in the NLGN4 3\u2032UTR has a mismatch with miR-1293 seed region and an altered minimum free energy value , compared with the allele G . Though we did not find any SNP which had significant frequency difference between ASD cases and controls, the validation in more samples and functional study would help us to understand the potential role of these polymorphisms in ASD.Most previously reported studies focused on the mutations in the coding region. In our study, we also pay attention to the genetic variants which may influence the expression level of NLGN4. Six common SNPs in the promoter region and 3\u2032 UTR of NLGN4 in ASD by comprehensive screening of the coding region and regulatory region of NLGN4 for mutations and examining the copy number variations of NLGN4. 285 ASD patients were studied and no missense mutations or deletion/duplication were identified. There was no association between 6 common SNPs in NLGN4 and ASD. These findings showed that NLGN4 may not play an important role in our ASD cohort.In this study, we investigated the role of Table S1The primer and PCR condition of NLGN4 gene.(DOC)Click here for additional data file.Table S2The primers of four genes for multiplex competitive PCR.(DOC)Click here for additional data file."}
+{"text": "Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ity16/Ity16Ank1 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ity16/Ity16Ank1 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ity16/Ity16Ank1, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous +/Ity16Ank1 mice were also more susceptible to Salmonella infection although to a lesser extent than Ity16/Ity16Ank1 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of +/Ity16Ank1 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants. Salmonella infections in humans are responsible for two major diseases, typhoid fever caused by Salmonella Typhi and Salmonella Paratyphi and a diarrheal disease known as salmonellosis caused by several non host specific serotypes including Salmonella Typhimurium and Salmonella Enteritidis. Typhoid is transmitted by a fecal-oral route through contaminated food and water and is endemic in areas of poor water sanitation. It is estimated by the World Health Organization that there are 21 million new cases of typhoid fever each year resulting in approximately 200,000 deaths. In addition, 1 to 5% of patients become asymptomatic chronic carriers serving as reservoirs from which new Salmonella Typhi infections can be transmitted Salmonella infection Salmonella infection Salmonella Typhimurium infection results in a systemic disease with clinical manifestations resembling those found in humans with typhoid fever. The severity of infection depends on a variety of factors including the dose of the inoculum, as well as the interaction of host and bacterial genetic determinants. Several strains of mice show varying degrees of susceptibility to Salmonella Typhimurium infection with the mouse strain 129 substrains showing the most resistance. The study of the natural variation of the host response to infection with Salmonella Typhimurium in spontaneous mouse mutants have identified important innate immune genes having Mendelian contribution to disease susceptibility and contributing to different mechanisms including pathogen recognition (Tlr4P712H in C3H/HeJ), phagosome transport of divalent cations including iron (Nramp1G169D in C57BL/6J and BALB/cJ) or erythropoiesis and iron metabolism (PklrI90N in AcB61 mice) N-ethyl-N-nitrosurea (ENU) mutagenesis to generate novel mutations responsible for increased susceptibility to Salmonella infection. Recessive ENU induced mutations are bred to homozygosity with the use of a three-generation breeding scheme and challenged with Salmonella Typhimurium. In the current screen we have evaluated 216 pedigrees for their susceptibility to infection and identified 5 deviant pedigrees. In this paper, we report the identification of one of these ENU mutants named Ity16 that carries a nonsense mutation in the gene Ank1. Ank1 encodes a red blood cell (RBC) adaptor protein consisting of three major functional domains: an N-terminal membrane binding, a spectrin binding and a C-terminal regulatory domain Ank1, both spontaneous and ENU-induced, have been described to cause hemolytic anemia Ity16/Ity16Ank1 and +/Ity16Ank1 mice have increased susceptibility to Salmonella Typhimurium infection, although in the latter group the increased susceptibility is delayed and milder. The suppression of hepcidin (Hamp) expression and iron overload contribute to their increased susceptibility to Salmonella infection.Due to the limited amount of natural variation found in classical inbred mice, we have used N-ethyl-N-nitrosurea (ENU) given intraperitoneally. G0 males were outcrossed to 129X1 females to create G1 progeny that were subsequently crossed to DBA/2J. Resulting G2 animals were intercrossed to produce the G3 mice that were phenotyped for their susceptibility to Salmonella infection. Hamptm1Svl knock out mice Slc11a1 (129S6.B6*129S2-Hamptm1Svl).All animal experiments were performed under conditions specified by the Canadian Council on Animal Care and the animal use protocol was approved by McGill University Facility Animal Care Committee. Generation 0 (G0) 129S1 males were mutagenized with a single injection of 150 mg per kg of body weight of ty16/Ity16Ank1 and +/+Ank1 mice using 3730xl DNA Analyzer (Applied Biosystems) from the McGill University and Genome Quebec Innovation Centre.The genome scan was performed using a 1441 SNP panel  on 22 mice. Additional genotyping was performed by microsatellite analysis or restriction enzyme digests of SNP markers. Sequencing of the Ank1 gene was performed on cDNA isolated from spleens of Salmonella Typhimurium strain Keller as described by us previously 2 at day 2 and day 6 post infection and spleen, liver and kidney were removed aseptically, weighed and homogenized. Homogenates were diluted in saline and plated on trypticase soy agar (TSA) overnight. Other groups of mice were coinfected with 2500 CFUs of Salmonella Typhimurium strain Keller and 2500 CFUs of \u0394tonB constructed by allelic exchange in serovar Keller Mice were infected with 5000 CFUs of Ank1 transcript (ENSMUST00000121802) and recognized the full length ANK1. The ACTIN antibody and the secondary antibodies for Western detection (anti-rabbit IgG) were purchased from Cell Signaling Technology.Erythrocyte ghost membranes were prepared by osmotic lysis as previously described . PrimarySalmonella Typhimurium and monitored for survival over a period of 14 days. Littermate controls were given 100 \u00b5l of PBS intraperitoneally.Mice were treated with 50 \u00b5g of HAMP  resuspended in 100 \u00b5l of PBS intraperitoneally. Four hours later, mice were infected with 5000 CFUs of Ity16/Ity16Ank1, +/Ity16Ank1 and +/+Ank1 mice and fixed in 10% neutral buffered formalin for 24 hours at 20\u00b0C, then placed in 70% ethanol at 4\u00b0C before processing and embedding . Embedded tissues were sectioned and stained with hematoxylin and eosin or prussian blue.Tissues were collected from Ity16/Ity16Ank1, +/Ity16Ank1 and +/+Ank1 mice were collected by cardiac puncture and analyzed for CBC & differential and reticulocyte counts . Serum was isolated with a serum separator tube (Sarstedt) and analyzed for bilirubin, blood urea nitrogen (BUN), alanine transaminase (ALT) and aspartate transaminase (AST) .Blood samples from Hamp), ferroportin (Slc40a1), Il6, Il1, Ifng, erythropoietin (Epo), heme oxygenase 1 (Hmox1) and growth differentiation factor 15 (Gdf15) expression. Two housekeeping genes: Tbp (TATA box binding protein) and Hprt (hypoxanthine guanine phosphoribosyl transferase) were used. The relative expression of the genes was normalized to the amount of Tbp and Hprt (endogenous reference) and relative to a calibrator (untreated wild type genotype) for each tissue by using the comparative 2(\u2212Delta Delta Ct) method. The primer sequences are provided in Total RNA was extracted from spleen, liver and kidneys with Trizol Reagent  according to manufacturer instructions. cDNAs were synthesized using SuperScript\u00aeII Reverse Transcriptase (InVitrogen). Quantitative PCR was performed on a Chromo4  or StepOnePlus  using SYBR Green (Applied Biosystems) for hepcidin (Data (unless otherwise specified) was analyzed by two-tailed Mann Whitney test and two-way ANOVA using GraphPad Prism 5. Data for qPCR was analyzed in R by ANOVA and Welch two sample t-test Ity16 pedigree is shown in Salmonella infection with 5,000 CFUs and monitored for a period of 14 days. Mortality in the Ity16 pedigree was observed between day 3 post infection and continued until day 11 with 25% of mice succumbing to infection by day 6 (Ity16 G3 mice were plotted according to their genotype at the peak marker (rs32874474) on chromosome 8 (The breeding scheme used to identify the by day 6 . An initby day 6 . DNA samby day 6  that incmosome 8 . All micmosome 8 .Ity16 mice showed they have severe constitutive anemia as demonstrated by low hematocrit levels (25% in mutants compared to 50% in wildtype and heterozygous mice) which decreased to as low as 15% 2 days after infection . Total number of WBC, neutrophils and lymphocytes were significantly higher in Ity16 mutant mice compared to littermate controls before and after infection  met our criteria. ANK1 is primarily known for its structural role in erythrocytes. Ank1 is part of a small gene family which members are adaptor structural components linking lipid membranes to the cytoskeleton showing an essential role in the stability of plasma membranes of many cell types Ank1 gene comprises a total of 44 exons. Several Ank1 splice variants are observed and the full-length isoform encodes for a protein of 1907 amino acids (200\u2212210 kDa). Erythroid Ank1 consists of three major conserved domains including an N-terminal membrane-binding domain, a spectrin-binding domain and a C-terminal regulatory domain containing a death domain motif Ity16 mutant showed a C to T transition at cDNA position 4069 (c.4069C>T) resulting in a nonsense mutation in exon 33 at amino acid position 1357 (p.Gln1357Ter ) (Ity16 mutant mice (Identification of the causative gene for 357Ter ) . This pr357Ter ) . Immunobant mice . A truncIty16/Ity16Ank1 mutant livers and kidneys (Prussian blue positive pigment) compared with wild type and heterozygous littermates in non-infected conditions and in mice at 2 days after infection (Ity16/Ity16Ank1 mutant mice suggest that the rapid turnover of RBC in these mice leads to accumulation of iron in the liver and kidneys (secondary hemochromatosis). In contrast, splenic iron content was markedly decreased in Ity16/Ity16Ank1 mutant mice compared to wild type littermates  and during infection (liver and kidney). In fact, we observed glomeruli with enlarged mesangium in Ity16/Ity16Ank1mice, a pathological change that may be compatible with membranoproliferative glomerulonephritis  in the liver of mutant mice where there is iron overload , ferroportin (Slc40a1) and heme oxygenase 1 (Hmox1) and regulators of Hamp expression  were investigated  (Gdf15)  known to be upregulated during Salmonella infection and to impact on Hamp transcription were measured (Ity16/ity16Ank1 mutant mice appeared to be underresponsive to infection-induced cytokines as measured by very little or no induction of Il1 and Il6 (Ity16/ity16Ank1 mice showed marked increases in Il1 (liver and kidneys) and Il6 (liver) mRNA expression during infection  were significantly more susceptible to infection than mice carrying one (+/\u2212Hamp) or two (+/+Hamp) (Mantel-Cox test P\u200a=\u200a0.0079) wild type allele at Hamp. These results confirm the importance of Hamp during acute systemic model of Salmonella Typhimurium infection.Because of the severe iron overload present in the liver and kidney of stigated  and 7. Ttermates . On the  Ank1+/+  and 7C. termates  and 7C. the mRNA  and protin (Epo)  and grow (Gdf15) . These atermates . In addimeasured . The spl and Il6 . In the nfection . In theshimurium . We showAnk1+/Ity16 heterozygous mice present an intermediate phenotype with respect to susceptibility to infection  when compared to +/+Ank1 and Ity16/Ity16Ank1 littermates (Ank1+/Ity16 mice did not present any sign of anemia (+/Ity16Ank1 mice presented moderate extramedullary hematopoiesis in the spleen (Hamp expression in the liver (Gdf15 (data not shown).termates . Clinicaf anemia  or splenf anemia . Howeverf anemia . In addie spleen  and lowehe liver  and kidnhe liver  that wasAnk1+/Ity16 mice behaved as wild type littermates for most subphenotypes we have measured including different blood parameters  test p\u200a=\u200a0.008) compared to heterozygous mice treated with PBS confirming the importance of HAMP in the host response to Salmonella infection (Early during infection (day 2), rameters  and orgarameters . To undee kidney . At day termates . The inctes  in the gene Ank1 identified in an ENU recessive screen for susceptibility to typhoid-like disease in mice. The mutation consists in a nonsense mutation (p.Gln1357Ter) located in the spectrin binding domain of Ank1. ANK1 protein was not detected in RBC ghosts suggesting that Ity16Ank1 is a null allele although we could not exclude the possibility that low levels of other ANK1 isoforms lacking the N-terminal region may remain undetected. Mice carrying the homozygous allele for Ity16Ank1 present clinicopathological features of human hereditary spherocytosis (HS) which is the most common cause of inherited chronic hemolysis in Europe and North America with a prevalence in population of 1 in 2000 ANK1 gene accounts for about 50% of genetically defined cases of HS and most cases of HS associated with ANK1 mutations We report here the identification of a novel mutation , two additional one were identified in dominant ENU screens for blood cell phenotypes (RBC2Ank1 and E924XAnk1) and the last one, in a dominant screen for resistance to malaria in SJL/J mice (MRI23420Ank1) Ity16, nb, RBC2, E924X or MRI23420 allele at Ank1 present characteristic clinicopathological features of HS including severe anemia, reticulocytosis, splenomegaly with complete effacement of the normal splenic architecture, multiorgan iron overload and low body weight. Embryonic and neonatal lethality is observed and may vary according to the genetic background and the position of the mutation  and survive to at least 6 months of age. In mutant Ank1 mice where there is 100% neonatal mortality, the death was associated with severe clinical signs of jaundice due to massive hemolysis In addition to the mutation described in the current paper, five mutations within the reported . Two of Ity16/Ity16Ank1 mutant mice and other ENU-induced Ank1 mutants (RBC2Ank1 and E924XAnk1) was similar with hematocrit levels varying between 22\u201327%. Reticulocytosis was present in all mutants and significantly different from control mice. Ity16/Ity16Ank1 mutant mice exhibit extensive iron accumulation in several organs including the kidneys, a pathological observation that was not reported in other Ank1 mutants Salmonella infection because of impaired NADPH-dependent respiratory burst activity Salmonella strain deficient in iron uptake grows better in the iron rich environment of Ity16/Ity16Ank1 liver. We do also show that in heterozygous +/Ity16Ank1 mice, higher bacterial load is detected only in tissues where iron accumulation is detected. These results clearly show the importance of iron overload in susceptibility to Salmonella infection in Ity16/Ity16Ank1 and +/Ity16Ank1 mice.The severity of anemia in Ank1 mutant mice and HS patients, the normal life span of erythrocytes in the peripheral blood is substantially shortened and retention of abnormal erythrocytes by the spleen was shown to be the dominant mechanism for their reduced life-span Hmox1. HMOX1 catalyzes the degradation of heme to carbon monoxide, biliverdin that is converted to bilirubin and ferrous iron Ity16/Ity16Ank1 mutant mice, we observed high serum bilirubin levels and iron overload in the liver and the kidneys but not in the spleen. High Hmox1 mRNA levels was observed in the liver and the kidneys but not in the spleen of mutant mice suggesting that the iron overload present in the liver and kidneys resulted from degradation of heme by Hmox1. Low levels of Hmox1 in the spleen of mutant mice are most likely a consequence of splenic macrophage depletion (data not shown) and could explain the observation that there is no iron deposition in the spleen of mutant mice and less bacterial growth considering that hemophagocytic macrophages may provide a survival niche for SalmonellaHmox1 has been recently shown to impair resistance to Salmonella infection in a context of hemolysis through the suppression of the oxidative burst capacity of neutrophils Hmox1 levels in Ity16/Ity16Ank1 mutant mice may contribute to their susceptibility.In Ity16/Ity16Ank1 mutant mice, the excessive iron load in the kidneys most likely results from the high rate of hemoglobin filtration and reabsorption by renal tubular cells. Iron deposition in glomeruli, and proximal and distal tubules of the kidney has been observed in chronic experimental hemosiderosis Ity16/Ity16Ank1 mutants develop a more severe nephropathy compared to heterozygous and wild type animals and are more susceptible to infection as measured by increased in bacterial proliferation upon infection. The higher degree of iron accumulation in liver and kidneys in older animals is most likely responsible for these observations.It has been shown that the kidney plays an important role in iron metabolism. It has also been reported that a significant amount of serum iron is filtered by the glomeruli and is reabsorbed Ity16/Ity16Ank1 mutant mice is an increase in Epo and Gdf15 levels associated with suppression of Hamp expression. Hamp expression is known to be regulated by intestinal iron absorption, iron recycling by macrophages and iron mobilization from hepatic stores by inhibiting iron export through its binding to Slc40a1 causing its internalization Ity16/Ity16Ank1 mice, low Hamp expression had a modest impact on the expression of Slc40a1 in the liver. The synthesis of Hamp is also upregulated in hepatocytes by inflammatory cytokines and inhibited by anemia, hypoxia and erythropoietic activity Epo is detected in the kidneys. Despite these changes to compensate for erythrocyte demand, erythropoisesis is not efficient and led to massive expansion of the erythroid compartment. GDF15 is a member of the TGF\u03b2 superfamily that was shown to negatively regulate the expression of Hamp in vitroHAMP have been observed in \u03b2-Thalassemia and it is thought that erythroid expansion influences the regulation of HAMP expression through systemic release of GDF15 from erythroblasts Hamp in response to ineffective erythropoiesis, as observed in Ity16/Ity16Ank1 mutant mice, clearly exacerbate extramedullary erythropoiesis, tissue iron deposition and splenomegaly. During infection, liver Hamp expression levels remain suppressed and Gdf15, Il1 and Il6 expression increased significantly in Ity16/Ity16Ank1 mutant mice compared to wild type littermates. Gdf15 has been shown to be highly expressed in macrophages stimulated with LPS and to be modulated by several cytokines including IL-1 Gdf15 expression observed during infection in Ity16/Ity16Ank1 mutant mice could be explained by the progression of anemia and expansion of the erythroid compartment, by the high expression of cytokines induced by infection with Salmonella or as a consequence of intracellular iron deprivation due to low levels of Hamp expression.Another hallmark of ineffective erythropoiesis in Salmonella susceptibility in Ity16/Ity16Ank1 differs from that observed in the iron overload disorder, hereditary hemochromoatosis (HFE). Hfe-deficient mice do not lack Hamp, present excessive accumulation of iron in the liver and improve resistance to Salmonella infection Hfe-deficient mice, high lipocalin-2 (Lcn2) levels were observed in the liver and the spleen prior infection and increased resistance to infection in these mice was associated with higher induction of Lcn2 expression that was shown to reduce the availability of iron for Salmonella within macrophages Ity16/Ity16Ank1 mutant mice, the spleen and liver expression of Lcn2 did not differ from that observed in littermate controls prior infection however we did observe a significant increased in the spleen and liver expression of Lcn2 during infection (data not shown). There were no significant differences between genotypes in the spleen in contrast levels of Lcn2 were significantly higher (by a factor of 1.5 LOG) in the liver of Ity16/Ity16Ank1 mutant mice compared to littermate controls and parallel the high bacterial loads detected in this organ. Altogether, these data suggest that high levels of Lcn2 expression in a context of low Hamp expression do not protect Ity16/Ity16Ank1 mutant mice from systemic Salmonella infection.+/Ity16Ank1 mice to infection with Salmonella Typhimurium. In heterozygous mice, there is only one functional copy of Ank1 which may be limiting to confer a normal structure to the RBC membrane and cause a partial loss of membrane surface. Although the half-life of RBC appears not to be affected in +/Ity16Ank1 mice, small increase in osmotic fragility has been reported which may lead to more RBC destruction by the spleen +/Ity16Ank1 mice although the mice were not anemic. The phenotype observed in +/Ity16Ank1 mice may correspond to the human mild form of HS where patients have compensated hemolysis without anemia. As observed in uninfected Ity16/Ity16Ank1 homozygous mice, liver and kidneys Hamp levels were low compared to controls and levels of Gdf15 were increased. Hamp is mainly produced by the liver but it has been found also to be highly expressed in the apical pole of epithelial cells of distal tubules and collecting ducts of the kidney +/Ity16Ank1 mice, the Salmonella susceptible phenotype appears to be expressed predominantly in the kidney where we found significant accumulation of iron and bacteria. Low levels of Hamp expression in +/Ity16Ank1 heterozygous mice at the time of infection appears to contribute to iron accumulation in the kidney and liver and consequently favor bacterial growth. The central role of Hamp in the host response to Salmonella infection was validated using mice deficient for Hamp. These mice are not anemic and present an important iron overload secondary to low level of Hamp a phenotype similar to the one observed in heterozygous mice Salmonella infection (current paper). Finally, direct evidence of the role of Hamp in the host response to Salmonella infection was obtained by treating +/Ity16Ank1 mice with hepcidin. Hepcidin treatment did not improve resistance of Ity6/Ity16Ank1 mutant mice where iron accumulation in tissues is massive however it was clearly beneficial in +/Ity16Ank1 heterozygous mice. We do not know at the moment the exact mechanism of how hepcidin is improving resistance to infection in +/Ity16Ank1 heterozygous mice. Induction of hepcidin through transgenesis in mouse models of hemochromatosis and \u03b2-Thalassemia was shown to alter the pattern of cellular iron accumulation and limit iron overload \u2212/\u2212 mice caused a partial redistribution of iron from the liver to the spleen Of particular interest was the intermediate susceptibility of Hamp expression and iron overload contribute to the susceptibility of Ity16/Ity16Ank1 mutant and +/Ity16Ank1 heterozygous mice to Salmonella infection. This emphasizes the importance of iron metabolism and a role for Hamp in susceptibility to systemic Salmonella infection.Overall, the current study shows that the suppression of Figure S1Histopathologic examination of the spleen, liver and kidney of 7 week old +/+ANK1 wild type, +/Ity16ANK1 heterozygous, and Ity16/Ity16ANK1 mutant mice before infection (day 0). H&E stain of uninfected spleen of wild type (A), heterozygous (E), and Ity16 mutants (I). Prussian blue stain of uninfected spleen, liver and kidney, respectively, of wild type , heterozygous , and Ity16 mutants . All pictures are taken at 200\u00d7magnification. RP\u200a=\u200ared pulp, WP\u200a=\u200awhite pulp.(PDF)Click here for additional data file.Figure S2Progression of lesions in kidney and liver 2 days after Salmonella infection of +/+ANK1 wild type, +/Ity16ANK1 heterozygous, and Ity16/Ity16ANK1 mutant mice aged 7 and 24 weeks using hematoxylin & eosin staining. H&E stain of uninfected kidney of wild type , heterozygous , and Ity16 mutants  at 7 and 24 weeks of age respectively. H&E stain of day 2 post infection liver of wild type , heterozygous , and Ity16 mutants  at 7 and 24 weeks of age respectively. All pictures taken at 200\u00d7magnification.(PDF)Click here for additional data file.Figure S3Prussian blue staining of liver and kidney of 7 week old +/+ANK1 wild type and +/Ity16ANK1 heterozygous mice at day 2 and day 6 post infection with Salmonella Typhimurium. Prussian blue stain of day 2 post infection liver in wild type (A) and heterozygous (D) mice at 200\u00d7magnification. Prussian blue stain of day 6 post infection liver of wild type (B) and heterozygous (E) mice at 200\u00d7magnification. Prussian blue stain of day 6 post infection kidney of wild type (C) and heterozygous (F) mice at 1000\u00d7magnification.(PDF)Click here for additional data file.Table S1Primer sequences used for quantitative PCR. Two housekeeping genes: Tbp (TATA box binding protein) and Hprt (hypoxanthine guanine phosphoribosyl transferase) were used. Hepcidin (Hamp), ferroportin (Slc40a1), interleukin 6 (Il6), inteleukin 1 (Il1), interferon gamma (Ifng), erythropoietin (Epo), heme oxygenase 1 (Hmox1) and growth differentiation factor 15 (Gdf15).(XLSX)Click here for additional data file."}
+{"text": "ANTIS revealed the presence of an appreciable number of sense and antisense peptide pairs within any protein molecule and those portions were designated as antisense homology boxes (AHB).Each peptide should have specific structure determined by its amino acid sequence which may react with its antisense peptide. To generate candidates of complementary peptide (C-pep) reactive to a target amino acid sequence based upon the sense-antisense amino acid relationship. We invented an evolutionary computer program (MIMETIC) that generates C-pep sequences that have a potential to interact with a target peptide .N-terminal alanine generating acetylated PepA (AcPepA).C5a anaphylatoxin is considered to be an effective target for treatment of hyperinflammation since C5a stimulates generation of tumor necrosis factor alpha (TNF\u03b1 and other inflammatory cytokines. Amino acids 37 to 53 of C5a (RAARISLGPRCIKAFTE) is an antisense peptide to AHBpeptides of the C5a receptor (C5aR), and this has been designated PL37. This region of C5a is presumed to be a potential site for C5aR stimulation. Using the computer program MIMETIC, we generated 19 C-peps to PL37. One of the 7 inhibitory C-peps to PL37 which interfered with C5a function was termed PepA(ASGAPAPGPAGP- LRPMF). To improve stability, we modified PepA by acetylation of its AcPepA rescued Cynomolgus monkeys at lethal shock induced by bacterial LPS (4 mg/kg).The excellent therapeutic effect of AcPepA is due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming positive feedback circuit of inflammation."}
+{"text": "Soon after introducing recombinant hepatitis B virus (HBV) vaccine, universal neonatal vaccination became the corner stone of the preventive measures for this potentially life threatening infection2]3]. B[3]. B2][. B[3]. BA level of 10 IU/L of anti hepatitis B surface antibody (Anti HBsAb) is usually considered as a protective level against future infections. Although this level was initially determined in studies about passive prophylaxis of HBV infection, the same level was arbitrary applied to active immunization though this was debated by some researchers 9][10]. [10]. 9]12]13][[13][12][[[13][12]Tosun et al.  in a pubThe coverage of complete vaccination of infants against HBV was estimated 69% in 2008 worldwide5] while while5] Some problems such as maintaining cold chain in transportation and handling of vaccine, improper injection and other technical problems in this context are still real challenges in many countries making effectiveness of vaccination, like HBV which need a stable cold chain, lower than expected in real practice 5]8]9][8][9]5][[9][8][9][8][9]5] which prThe HBV infection rate in children received vaccination after determining by Anti HBcAb positivity might be as high as 0.5% . The impAfter all we still confront with this major question: How long the protective effect of neonatal HBV vaccination persists? Is there a need for booster after complete series of vaccination? If so when this booster should be administered and to whom? Should it be universal like the neonatal vaccination itself or only be given after monitoring or given only to selected groups?Answer to these questions is not easy and there are contradictory evidences in the literature. The debate is whether the reduction in antibody levels is indicating lower immunity or not. An anamnestic response may occur when people who were previously immunized against an antigen are re-exposed to the same antigen 10]26].[26].10][.[26].10]The same group reported the rate of breakthrough infection among vaccines was less than 0.1% . Even ch"}
+{"text": "Since 2003, H5N1 avian influenza strains have become endemic in many countries in Southeast Asia, including Cambodia. In Cambodia alone there have been 26 outbreaks in poultry flocks and 10 human cases with 9 deaths. We have analyzed the genome of a large panel of H5N1 strains isolated from poultry and human between 2004-2010. Several strains have molecular alterations which are predicted to affect sensitivity to neuraminidase inhibitors (NAI), the primary drugs of choice in the treatment of H5N1 infections. In June 2009 the first H1N1 pandemic (H1N1pdm) viruses were detected in Cambodia and have since been the main influenza strains circulating during the epidemic season. The aim of this study was primarily the surveillance of oseltamivir and zanamivir drug resistance in Cambodian H5N1 and H1N1pdm isolates.in vitro assay of neuraminidase (NA) activity, which utilizes the artificial NA substrate 1,2-dioxetane derivative of sialic acid , was used to determine the concentration of drug required to inhibit 50% of NA enzyme activity (IC50).A chemiluminescence-based We have identified a small number of H5N1 outliers with reduced susceptibility to NAIs and have further characterized mutations predicted to affect drug resistance using computer modeling, and recombinant viruses containing these mutations generated by reverse genetics. We have also investigated several previously described resistance mutations in the context of the Cambodian H5N1 virus using reverse genetics. We found no evidence of NAI drug resistance in H1N1pdm viruses in Cambodia.We have monitored NAI sensitivity of H5N1 and H1N1pdm viruses in Cambodia and in general have not found NAI resistant viruses with classical resistance mutations. However, we have identified naturally occurring mutations in H5N1 viruses which reduce the sensitivity to NAIs. The ongoing surveillance and understanding of novel drug resistance mutations is of great importance in the global efforts against influenza disease and pandemics."}
+{"text": "DLK1 and/or RTL1. The up-regulation of PARK7 was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from Park7 (+/+) and Park7 (\u2212/\u2212) mice were used to examine the effect of differential expression of Park7. The Park7 (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (\u2212/\u2212) myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20-40% in Park7 (+/+) myotubes relative to Park7 (\u2212/\u2212). The level of AKT phosphorylation was increased in Park7 (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the Park7 (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of PARK7 in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of  DLK1-DIO3 imprinted gene cluster Delta-like 1 (DLK1) and Retrotransposon-like 1 (RTL1) in hypertrophied muscles Dlk1 exhibited increased muscle mass and myofiber diameter Dlk1 in the mouse resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers Dlk1 in culture was shown to inhibit myoblast proliferation and enhance myotube differentiation Callipyge sheep exhibit postnatal muscle hypertrophy, with higher rates of protein accretion and lower rates of fat deposition compared to normal sheep semimembranosus muscle of callipyge and normal lambs Parkinson Protein 7  expression was up-regulated in hypertrophied muscles. PARK7 encodes a ubiquitously expressed, highly conserved protein that has been shown to be involved in diverse biological processes including oxidative stress response, transcriptional regulation and cell survival modulation. A mutation causing a loss of function of PARK7 was found to be responsible for a recessive early-onset form of Parkinson's disease Park7 was originally identified as an oncogene that transforms NIH3T3 cells in cooperation with the activated Ras gene Microarray analysis of gene expression identified 199 genes that were differentially expressed in Igf1 caused muscle hypertrophy in mice Igf1 receptor impaired muscle growth due to reduced muscle fiber number and size Akt in muscle fibers results in significantly larger fiber size Akt in muscle exhibit rapid skeletal muscle hypertrophy Akt in mice leads to a smaller body size and shorter life span The PI3K/AKT pathway is known to positively regulate muscle growth PARK7 in hyper-muscular animals suggests a hypothesis that increasing PARK7 gene expression can increase muscle growth and protein accretion. Differential expression of PARK7 was examined in this study using Park7 (+/+) and Park7 (\u2212/\u2212) mouse models to investigate the effects of Park7 gene expression in muscle growth and protein accretion in response to IGF1.Though extensively studied, a role for PARK7 in muscle growth had not been reported until recently. In addition to callipyge lambs Semimembranosus and supraspinatus muscle samples were collected from 3 callipyge and 3 normal lambs at 30\u201335 days of age when muscle hypertrophy is detectable by muscle mass. The semimembranosus from the pelvic limb undergoes the largest magnitude of hypertrophy and supraspinatus from the thoracic limb does not become hypertrophied semimembranosus of callipyge (+/C) relative to normal (+/+) lambs. There were no differences between genotypes in the supraspinatus. Elevated levels of DLK1 protein were also confirmed in the callipyge semimembranosus. No detectable DLK1 expression was found in the muscles of normal animals and non-hypertrophied muscles, along with a reduced PARK7 expression in these same animals and tissues.Park7 (\u2212/\u2212) mouse was obtained from Jackson Laboratory and back crossed with C57BL6 mice to generate Park7 (+/+), (+/\u2212) and (\u2212/\u2212) littermate mice. Live animal body weights were monitored and collected from 3 \u2013 6 weeks of age. Mice were euthanized at 6 weeks and carcass (muscle and skeleton) weight and internal organs weights were collected. The phenotypic data from male and female mice was analyzed by linear regression separately to account for gender differences. No significant differences were found between the three genotypes for analysis of live weight by age, carcass weight by live weight, or organ weight by live weight analysis in males or females  mice (Park7 (+/+) and Park7 (+/\u2212) animals, but no PARK7 protein was detectable in Park7 (\u2212/\u2212) muscles.The expression of PARK7 protein in the /\u2212) mice . The expPark7 (+/+) and Park7 (\u2212/\u2212) animals and fused into myotubes in order to model the effect of Park7 levels on growth and myosin expression. The relative sizes of myotubes were determined after 72 hours of differentiation in fusion media (Park7 (\u2212/\u2212) and (+/+) cultures (Park7 (+/+) myotubes were significantly larger  than Park7 (\u2212/\u2212) myotubes  myotubes having about 10% higher myosin expression than Park7 (\u2212/\u2212) myotubes, regardless of IGF1 treatment (Park7 (+/+) myotubes (25 ng/mL) relative to Park7 (\u2212/\u2212) myotubes (100 ng/mL).Primary myoblasts were isolated from on media . The fuscultures . Park7  in Park7 (+/+) myotubes, relative to Park7 (\u2212/\u2212) myotubes  and 30% (p\u200a=\u200a0.0001) respectively in Park7 (+/+) myotubes. IGF1 also stimulated Myh7 and Myh8 mRNA expression, but only at the lower concentration (10 ng/mL) with 23% increase in Myh7 and 10% increase in Myh8 respectively, relative to the treatment without IGF1. At the high concentration of IGF1 (50 ng/mL), Myh7 expression dropped 12% and Myh8 expression dropped 16% compared to the treatment without IGF1. Myh3 was the only isoform measured that showed no genotype effect or IGF1 effect. There was no significant genotype by IGF1 interactions for the four myosin isoforms.Quantitative PCR was used to detect the effect of myotubes . The mRNmyotubes . The addPark7 regulation on myotube size and myosin gene expression, a series of experiments were conducted to assess the activation of AKT by phosphorylation at S473 in presence or absence of Park7. It has been demonstrated that PARK7 can suppress PTEN phosphatase activity, which would have the effect of increasing receptor tyrosine kinase signaling through AKT Park7 genotype on the phosphorylation of AKT. The level of phosphorylation of AKT was elevated in Park7 (+/+) myotubes relative to Park7 (\u2212/\u2212) myotubes in 5% horse serum differentiation media without added IGF1 (Park7 (+/+) myotubes at all levels of added IGF1. To investigate if the activity of PARK7 could be mimicked by inhibition of PTEN function, VO-OHpic trihydrate, a known effective PTEN inhibitor Park7 (\u2212/\u2212) myotubes and eliminated the differences between the two Park7 genotypes (Park7 (+/+) and Park7 (\u2212/\u2212) myotubes.In order to explore the mechanism of ded IGF1 . The phoenotypes . There wenotypes  or RNA ePark7 (+/+) myotubes, the timing and duration of the elevated phospho-AKT was tested. Myotubes were treated with IGF1 for 15 minutes and protein was collected at several time points up to 12 hours. Western blots showed increased phosphorylation of AKT in Park7 (+/+) myotubes (Park7 (+/+) myotubes relative to Park7 (\u2212/\u2212) myotubes with no added IGF1 (Park7 (+/+) myotubes reached its maximum at 45 minutes at 3-fold higher signal intensity than Park7 (\u2212/\u2212) myotubes after removal of IGF1. Park7 (\u2212/\u2212) myotubes had maximum phosphorylation at 90 minutes and the decreases in phosphorylation over time were similar with Park7 (+/+) myotubes, generally maintaining approximately 20% higher signal intensity through 12 hours.Since increased phosphorylation of AKT was found in myotubes . Quantitded IGF1 . The levOverall, the present results indicate that differential expression of the PARK7 protein can result in increased myosin gene expression, protein accretion and myotube growth through an altered response to IGF1/AKT signaling. This study focused on Park7\u2032s effect on AKT phosphorylation due to its prominent role in regulating muscle growth, other reported biological activities in oxidative stress, anti-apoptosis and several regulatory pathways Park7 (\u2212/\u2212) mice are viable and lack obvious developmental abnormalities. No differences were found between Park7 (+/+) and (\u2212/\u2212) animals in vivo when comparing body weight, carcass weight and internal organ weight which indicate that Park7 is not a dominant regulator of live animal growth. These results were consistent with other findings, which indicated no significant differences in body weights between the two genotypes from birth to 12 months The PARK7 expression in callipyge sheep at the transcriptional level in vitro cell culture model was used in this study to investigate the effects of differential expression of Park7 on myogenesis. The in vitro comparison of myotube size showed Park7 (+/+) myotubes had a bigger size and higher myosin expression than Park7 (\u2212/\u2212) myotubes, but no significant change in the fusion index, which indicates Park7 expression affects myotube hypertrophy (increased myofiber diameter), not hyperplasia (increased myofiber number). These results support the proposed models that elevated PARK7 expression is part of the physiological mechanism for increased muscle growth in callipyge lambs and myostatin null mice and cattle A previous study showed elevated Park7 (+/+) myotubes had increased phosphorylation of AKT compared to Park7 (\u2212/\u2212) after IGF1 stimulation, which induced significantly more total sarcomeric protein accretion. Notably, this increase of protein accretion reached saturation at lower IGF1 concentrations in Park7 (+/+) relative to Park7 (\u2212/\u2212) myotubes. The expression of Myh4 showed the largest increases although Myh7 and Myh8 mRNA expression were also elevated in Park7 (+/+) myotubes indicating that both myosin transcription and translation were affected by Park7 expression. Muscle contractile characteristics partially depend on the expression of myosin heavy chain isoforms. The hypertrophied muscles in callipyge sheep have an increased size of fast twitch glycolytic myofibers along with increased MYH4 expression and a decreased frequency of oxidative fibers Akt had been reported to promote myofiber growth with no effect on fiber specification in regenerating skeletal muscle Akt transgene in mice resulted in the hypertrophy of glycolytic myofibers, but not oxidative myofibers Myh4 and the enhanced activation of AKT in the presence of Park7. Interestingly, double-muscled cattle, in which the expression of Park7 gene is also elevated, have an increased proportion of fast-twitch glycolytic fibers as well The PI3K/AKT pathway is one of the primary targets of IGF1 signaling and has been well characterized for its positive regulation on muscle growth Park7 (+/+) and Park7 (\u2212/\u2212) myotubes was eliminated when treated with higher concentrations of PTEN inhibitor, but the total amount of PTEN had not been changed, suggesting PARK7 acts through inhibition of PTEN phosphatase activity, not total PTEN expression. This result was consistent with the findings of Kim et al. Park7 transfected NIH3T3 cells, but no change was detected; indicating the inhibition of PTEN phosphatase activity by PARK7 is not affected by phosphorylation of PTEN. The mechanism by which PARK7 inhibits PTEN phosphatase activity remains unclear. Some studies have proposed that PARK7 can directly bind to PTEN to confer an inactive conformation by forming a disulfide bond between C71and C124 of PTEN upon oxidative stress The differences in phosphorylation of AKT in Park7 in the regulation of skeletal muscle growth. Park7 expression can modulate IGF1/AKT signaling, increase myosin gene expression and protein synthesis and increase myotube size. These results support the hypothesis that elevated expression of PARK7 is part of the physiological mechanism for muscle hypertrophy in callipyge lambs and increased muscle mass in double muscled cattle. Elevated expression of PARK7 in the muscles of callipyge lambs could lead to increased muscle growth in response to the normal IGF1 signaling present in young growing lambs. The prominent increase in Myh4 gene expression in myotubes was also consistent with changes in myosin fiber types in callipyge muscle. DLK1 is one of the causative genes for the callipyge phenotype due to the inheritance of the mutation PARK7 expression could be considered a downstream response to DLK1 signaling. Identifying how PARK7 gene expression is regulated in callipyge induced muscle hypertrophy could provide new mechanisms to increase livestock muscle growth and production efficiency or prevent the loss of muscle mass due to disease or aging.The present study provides a novel insight into a role for Sheep were maintained at the Purdue Animal Sciences Research and Education Center under standard agricultural housing and management. Lambs were euthanized for tissue sampling using three lethal procedures of captive bolt stunning, exsanguination and pneumothorax. This procedure was approved by Purdue Animal Care and Use Committee (protocol #1112000493). All mice were fed with standard chow food and water, and housed at 22\u201323\u00b0C with nesting material with 12 hour light/dark cycle. Cage bedding was changed weekly by professional personnel. Mice were euthanized by cervical dislocation and pneumothorax for dissection of muscle tissue for the growth trial and a source of primary satellite cells. Mice housing and euthanasia was performed according to protocols approved by the Purdue Animal Care and Use Committee (protocol #1112000440).Park7 locus inactivated by gene targeting [Park (\u2212/\u2212)] are available from JAX mice\u00ae . The initial C57BL6 Park7 (\u2212/\u2212) mouse was crossed with C57BL6 Park7 wild type (+/+) to produce heterozygous (+/\u2212) Park7 mice for breeders. Matings of heterozygous Park7 mice were conducted to generate litters of mice with all three possible Park7 genotypes (+/+) (+/\u2212) and (\u2212/\u2212). Twenty females with 4 Park7 (+/+), 12 (+/\u2212) and 4 (\u2212/\u2212) animals and twenty-five males with 4 Park7 (+/+), 11 (+/\u2212) and 10 (\u2212/\u2212) from seven litters were used to analyze live animal growth. Live animal weights were measured at 6 weeks of age to the nearest 0.01 g. The carcass and internal organ weights were measured on a total of 18 males (4 Park7 (+/+), 6 (+/\u2212) and 8 (\u2212/\u2212) animals) and 16 females (4 Park7 (+/+), 8 (+/\u2212) and 4 (\u2212/\u2212) animals) to the nearest 0.0001 g. Carcass weights were measured by removal of head, tail, skin and fur, internal organs and visceral fat of the mouse. The general linear model and genotype specific models for regression analysis are given in Supplementary Mice with the Rplp38) was used as the housekeeping gene control for T\u0394C calculation [T\u0394C \u200a=\u200a (TC of the target gene) \u2013 (average TC of housekeeping genes)]. Primer sequences were listed in \u2212\u0394\u0394CT methodsT \u200a=\u200a (T\u0394C of the treatment sample) \u2013 (T\u0394C of control treatment samples) with no added IGF1 as control treatment and normalized to 1.RNA from cultured cells was extracted and purified using Nucleo Spin RNA II columns . First strand cDNA was synthesized from RNA using random hexamer and oligo dT priming and moloney murine leukemia virus reverse transcriptase  to produce random primed cDNA. Quantitative PCR measurements were performed using the SA Bioscience SYBR Green Supermix  reagents on an iCycler Real-Time PCR Detection System . Each reaction was carried out in 15 \u03bcl reaction volumes of SA Bioscience SYBR Green Supermix with 5 pM of each primer and diluted first-strand cDNA. All cDNA samples were measured in duplicate. Ribosomal protein large protein 38 , minced and digested in type I collagenase and dispase B mixture . The digested muscle pulp was then filtered through a 100 \u03bcm filter  to remove large muscle fiber debris and then plated on culture dishes. In 3 days, the cells together with the small debris were collected and digested with 0.025% trypsin for 10 minutes with agitation. Cells were seeded in growth media  on rat-tail collagen (Roche Applied Science)-coated dishes with preplating on non-coated plates for 45 minutes to deplete fibroblasts as previous described Semimembranosus and supraspinatus muscles were extracted from callipyge and normal lambs at 30\u201335 days. Samples were frozen immediately after dissection and homogenized in RIPA buffer . Samples were centrifuged at 10,000 \u00d7 g for 10 minutes at 4\u00b0C. The supernatant was transferred into ultracentrifuge tubes and centrifuged at 50,000 \u00d7 g for 15 minutes at 4\u00b0C.Proteins were quantified by BCA assay  per manufacturer's recommendation. A total of 60 \u03bcg of protein from each muscle sample was diluted in SDS loading buffer, denatured by heating for 5 minutes in boiling water and loaded in the 6%\u201318% gradient SDS-PAGE gel. The gel was transferred to PVDF membrane by semi-dry transfer using 20% methanol, 40 mM glycine, 0.15% ethanolamine, 0.2% SDS at 180 mA for 1 h 45 min. PVDF membrane was blocked overnight with agitation in 5% sodium caseinate in PBS with 0.1% Tween 20. Antibodies used for blotting were goat polyclonal anti-Park7 , anti-bovine DLK1 , mouse monoclonal anti-tubulin  and secondary antibodies were rabbit anti-goat antibody conjugated HRP, goat anti-rabbit conjugated HRP, or goat anti-mouse conjugated HRP respectively . The blots were incubated in primary antibody 2 hours at room temperature with a dilution of 1\u22361000. After washing 3 times with DPBS, the blots were incubated with secondary antibody with dilution of 1\u223620000. Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific Pierce) was used to detect HRP on immunoblots. The blots were visualized using Gel Logic 2200 PRO (Carestream Molecular Imaging System) with exposure times of 2\u20136 minutes. Carestream Molecular Imaging Software was used to quantify the chemiluminescent signal intensity in arbitrary units. The signal intensities for PARK7 were compared using a t-test without normalization.Primary myotubes were fixed in 4% formaldehyde and permeabilized in 0.5% Triton X-100. Cells were blocked in 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature and stained with 1\u22361000 anti-myosin heavy chain antibody  for 2 hours. Cells were washed in PBS, and then incubated in 1\u22361000 Alexa Fluor 594-conjugated donkey anti-mouse IgG secondary antibodies (Life Technologies) for 1 hour. Finally, cells were mounted with Prolong Gold antifade reagent with DAPI (Life Technologies). The IP Lab software  and Leica DM6000 microscope were used to acquire pictures.The fusion index was defined as the ratio of nuclei within the myotubes (>2 nuclei) to the total number of nuclei. The average number of nuclei per myotube was determined by counting over 500 nuclei from randomly chosen MHC-positive cells. Myotube diameters were measured at 5 points along the entire tube. A total of 150 myotubes were examined for each genotype in the experiment.Primary myoblasts were put into Matrigel coated-96-well plate at a high density with four replicate wells for each genotype by IGF1 treatment combination. The myoblasts were fused into myotubes for 3 days in fusion media with different concentration  of IGF1. The mature myotubes were fixed in 4% formaldehyde in PBS for 5 minutes at room temperature, washed 3 times with PBS and permeabilized with 0.5% TritonX100 for 5 minutes. After washing 3 times with PBS, the cells were blocked for 1 hour in 1% BSA in PBS. Cells were incubated with anti-myosin heavy chain antibody  diluted in 0.1% BSA overnight at 4\u00b0C, and washed 5 times in PBS. Secondary antibody  were diluted in 0.1% BSA and filtered through 0.2 \u03bcM filters. Cells were incubated in secondary antibody for 1 hour and washed 5 times with PBS. The fluorescence signals were read on Tecan Genios Pro  plate reader.Cultured primary myoblasts or myotubes were used to examine AKT phosphorylation. Different concentrations of IGF1 were added into culture for 15 minutes to induce phosphorylation of AKT. For the PTEN inhibitor experiment, different concentrations  of VO-OHpic trihydrate (Sigma-Aldrich) were added into culture and incubated with myotubes for 15 minutes after removal of IGF1. For testing sustained activity of phosphorylation of AKT, IGF1 was removed after 15 minutes incubation with myotubes. The cells were incubated in normal differentiation conditions and cell lysis was collected at the indicated time points . Cells attached to plates were rinsed with ice-cold DPBS, and scraped on ice in cell lysis buffer  and 1 X complete protease inhibitor cocktail. Cells were collected in 1.5 mL microcentrifuge tubes, rotated for 45 minutes, and centrifuged at 12,000 \u00d7 g for 10 minutes at 4\u00b0C to remove insoluble debris.A total of 45 \u03bcg of protein from each cell sample was diluted in SDS loading buffer, and loaded in the 6%\u201318% gradient SDS-PAGE gel. The gel electrophoresis and transfer to PVDF membranes were the same as described above. The PVDF membranes were blocked in 5% non-fat milk in Tris-Buffered Saline (TBS) with 0.1% Tween 20 for 3 hours. Antibodies used for blotting were rabbit polyclonal anti-AKT , anti-phospho-AKT (Ser473) , anti-PTEN . The primary antibody was incubated at 4\u00b0C overnight at a dilution of 1\u22361000. The goat anti-rabbit conjugated HRP was used as secondary antibody and was incubated for 1 hour at 4\u00b0C at a dilution of 1\u223620000. The visualization of blots was the same as described above.Park7 genotypes used in the mouse growth trials are provided in Supplementary All of the statistical analysis presented in this work was conducted using SAS 9.2 software  with assistance from the Statistical Consulting Service at Purdue University. The linear regression models for pairwise comparisons of the three Figure S1Analysis on live body weight by age in Park7 (+/+), (+/-) and (-/-) mice. Twenty females with 4 Park7 (+/+), 12 (+/-) and 4 (-/-) animals and twenty-five males with 4 Park7 (+/+), 11 (+/-) and 10 (-/-) were examined to analyze the live animal growth. Animals live body weights were collected from 3 weeks to 6 weeks old. Live body weights were regressed on age. Equations were given for each genotype. There were no genotype effect on the slopes and intercepts of any regression lines in both male and female.(DOCX)Click here for additional data file.Table S1Regression analysis of growth in Park7 (+/+), (+/-) and (-/-) mice.(DOCX)Click here for additional data file.Table S2Quantitative PCR primers.(DOCX)Click here for additional data file.Checklist S1(DOC)Click here for additional data file."}
+{"text": "Virus isolates from all patients had the H275Y mutation in the neuraminidase gene. One isolate had the I117M mutation. Of the 11 patients, 6 were  The Korea Centers for Disease Control and Prevention asked clinicians to report all patients with suspected cases of drug-resistant pandemic (H1N1) 2009 when these patients showed treatment failure for oseltamivir or had unusually prolonged viral shedding (defined as >5 days after the onset of symptoms)  2009 virus infection. A total of 225 patients (0.03%) died of disease related to pandemic (H1N1) 2009. During this period, physicians in local clinics and tertiary hospitals sent specimens from 67 patients who were suspected of having drug-resistant pandemic (H1N1) 2009 to the Korea Centers for Disease Control; 11 patients (16%) had drug-resistant virus . After cTo investigate whether virus contained genetic markers associated with resistance to antiviral drugs, a conventional genotyping assay (sequencing) was performed, and neuraminidase (NA) and matrix 2 genes were sequenced. All 11 patients had virus with a histidine-to-tyrosine mutation at residue 275 of the NA protein (H275Y); 1 patient also had virus with an I117M mutation . Detaile<59 months of age and 5 had underlying immunosuppressive conditions; only 1 patient was immunocompetent and >59 months of age  2009 virus before the 11 patients were infected. Influenza-like illnesses developed in the 11 patients a median of 2 days (range 1\u20137 days) after the 8 contact persons were confirmed as having pandemic (H1N1) 2009. Five of the 8 contact patients were children; none had an immunosuppressive condition or were given oseltamivir chemoprophylaxis before illness; and 7 of 8 were All 11 patients were initially given the usual dose of oseltamivir (75 mg 2\u00d7/day in adults). After detection of oseltamivir resistance, treatment regimens were as follows: 3 patients were given high-dose oseltamivir (150 mg 2\u00d7/day in adults), 3 patients were given combination therapy ; 3 patients were given zanamivir nasally; and 2 patients continued to receive oseltamivir.Seven of the 11 patients had complications during treatment: 6 had viral or secondary bacterial pneumonia and 1 had acute respiratory distress syndrome . Three p<59 months of age) or immunocompromised. All isolates had the H275Y mutation in the NA protein, and 1 isolate also had the I117M mutation in the same protein.Our nationwide surveillance of drug-resistant pandemic (H1N1) 2009 in South Korea indicated that most patients were children  2009, particularly if there is treatment failure with oseltamivir or prolonged viral shedding is evident.Our finding that oseltamivir resistance developed in immunocompromised patients is consistent with those of recent case reports that described development of oseltamivir resistance in immunosuppressed patients receiving this drug  2009. Clinical trials have reported oseltamivir resistance in <5.5% of children with seasonal influenza  2009 in South Korea. However, our study was based on the nationwide surveillance system for drug resistance among patients with suspected oseltamivir treatment failures, which is different from other surveillance systems, in which all isolated viruses are tested. Thus, we cannot report the prevalence of drug-resistant pandemic (H1N1) 2009 and risk factors for drug-resistant virus infection in South Korea. Further studies are needed to monitor oseltamivir resistance, especially in immunosuppressed or pediatric patients when treatment failure with oseltamivir or prolonged viral shedding occur. Additional studies are also needed to determine proper oseltamivir dosing for children."}
+{"text": "The key steps involved a Wittig reaction between aldehyde 5 and the ylide derived from phosphonium salt 6 to give enyne 17 and condensation of the same ylide with aldehyde 7 to afford enyne 11. Desilylation of 11 followed by hydrozirconation and iodination gave the vinyl iodide 4 and Sonogashira coupling between this compound and enyne 3 provided alkyne 18. Acetonide deprotection, partial reduction and ester hydrolysis then gave resolvin D2 (1).The total synthesis of the endogenous inflammation resolving eicosanoid resolvin D2 ( In 2002, a new family of endogenously generated lipid mediators involved in the resolution of inflammation named the resolvins (resolution phase interaction products) were identified by Serhan and co-workers in the inflammatory exudates of aspirin treated mice \u20133. The rd D1 (2)  derived 1) prevents the adherence of polymorphonuclear leukocytes (PMN) to the blood vessel wall by promoting the shedding of L-selectin from PMNs thus preventing binding to E selectin on the endothelial cell lining of the blood vessel [1) promotes the influx and phagocytic activity of macrophages, facilitating clearance of dead cells and microbial pathogens, allowing resolution of inflammation and infection [2) [RvD2 (d vessel . Furthernfection . This sunfection . Resolvition [2)  has beention [2) . Whilst S,16R,17S)-RvD2 (1) was communicated by Spur in 2004 [1 for a biological study [1) for biological evaluation but without detailed synthetic sequence to follow and given the very high cost [1 we elected to develop an alternative route to provide this important compound and analogues for further biological evaluation. Herein we describe a synthesis of RvD2 (1) which includes full experimental details so that other researchers can produce useful amounts of this important compound as well as novel isomers.The first total synthesis of  is shown in 1 could be secured via a Sonogashira coupling to form the C11\u2013C12 bond followed by partial reduction. This is similar to the endgame of the reported syntheses of 1 [1 could arise from enyne 3 and vinyl iodide 4 which could both be obtained by Wittig extension using the common linchpin phosphorus ylide derived from phosphonium salt 6 [5 and 7 [A retrosynthetic analysis of RvD2 . Removal of the TIPS group with TBAF gave terminal alkyne 12. Alkyne 12 then underwent smooth hydrozirconation utilizing the procedure reported by Negishi [2HCl is generated in situ by reduction of ZrCp2Cl2 with DIBALH in THF. Iodinolysis of the zirconium species then gave the diene iodide 4 in good yield. Selectivity for this process was excellent with only a trace of the regioisomer formed.Treatment of the salt tography . The min Negishi  were ZrC5 began with the production of the known bis-TES ether [13 [t-BuOH/H2O to give diol 14 in reasonable yield. The enantioselectivity and absolute configuration of the secondary alcohol was determined by conversion of diol into the bis-(S)-Mosher ester [1H MMR spectrum indicated the e.r. was 93.7:6.3 and Mosher analysis  is shown in 3 and 4 was very efficient giving the alkyne 18 in good yield. Removal of the acetonide was effected by treatment with HCl in MeOH to give the known triol 19 [1 were similar with those previously reported [20 in 76% yield. A large excess of the Zn reagent was required to obtain a good conversion of 19 into 20. The 1H NMR spectrum (CDCl3 solvent) of RvD2 methyl ester (20) compared well to that reported [1).The completion of the synthesis of RvD2  had physical data identical to that reported [D \u221217.5\u00b0 ). In our hands, both RvD2 methyl ester (20) and RvD2 (1) itself were highly unstable, especially to acid. Prolonged standing in CDCl3 or CD3CN solution or exposure to light caused rapid decomposition and so NMR spectra were obtained quickly. We found that RvD2 methyl ester (20) was not very soluble in CD3CN so spectra were best run in CDCl3 that was filtered through basic alumina immediately prior to use. Spectra for RvD2 (1) were always measured for CD3CN. Even with short exposure to the solvent, we still observed degradation to unidentified compounds. Samples of RvD2 (1) can be stored in EtOH or frozen in DMSO solution but should be used immediately upon thawing. Alternatively, the triol 19 proved more stable than both RvD2 methyl ester (20) and RvD2 (1) and can be stored for longer periods prior to conversion to 1 which should be used rapidly for biological assessment to avoid degradation.The synthetic RvD2  has been completed using a common linchpin Wittig reaction. Using this approach, we were able to prepare sufficient quantities of this important inflammation resolving compound for further biological evaluation.The total synthesis of RvD2 ."}
+{"text": "Amelogenesis imperfecta (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in AMELX. In humans there are two, non-allelic amelogenin genes: AMELX (Xp22.3) and AMELY (Yp11.2). About 90% of amelogenin expression is from AMELX, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (ARHGAP6). We recruited two AI families and determined that their disease-causing mutations were partial deletions in ARHGAP6 that completely deleted AMELX. Affected males in both families had a distinctive enamel phenotype resembling \u201csnow-capped\u201d teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of ARHGAP6 (g.302534_398773del96240), but removed alternative ARHGAP6 promoters 1c and 1d. Analyses of developing teeth in mice showed that ARHGAP6 is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed ARHGAP6 promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of AMELX results in males with a characteristic snow-capped enamel phenotype, and failed ARHGAP6 expression did not appreciably alter the severity of enamel defects when AMELX was absent. ENAM, 4q21) FAM83H, 8q24.3) MMP20, 11q22.3-q23) KLK4, 19q13.4) WDR72, 15q21.3) AMELX, Xp22.31-p22.1) AMELX and AMELY), but the gene on the Y-chromosome (Yp11.2) is expressed at low levels AMELX and AMELY do not undergo homologous recombination, have diverged AMELY deletions. DNA repeat sequences on the Y chromosome apparently create a structural instability that leads to recurrent 3\u20134 Mb deletions inclusive of AMELY, PRKY and TBL1Y, and sometimes PCDH11Y, with no apparent negative selection AMELY deletions varies by population and is generally low, perhaps about 0.6% AMELY deletions have been reported for \u201cnormal populations\u201d, with the extrapolation that the tooth enamel must therefore be normal in these people. To our knowledge no oral photos or radiographs have been published of person lacking AMELY, although two individuals with AMELY deletions were specifically reported to have normal teeth Non-syndromic amelogenesis imperfecta (AI) is a heterogeneous collection of inherited defects in dental enamel formation that includes a multiplicity of enamel phenotypes, patterns of inheritance, and causative genes AMEL) belongs to the secretory calcium-binding phosphoprotein (SCPP) family of genes AMEL, all human SCPP genes are clustered on chromosome 4, which include 5 genes encoding acidic proteins and 16 genes encoding proteins enriched proline and glutamine Amel apparently arose in the proline-glutamine-rich gene cluster and later transposed into intron 1 of Arhgap6 (Rho GTPase activating protein 6) in the opposite orientation, resulting in a nested gene structure. This may have occurred in fish, as Amel is already nested in Arhgap6 in African clawed toads, suggesting that such was the localization of Amel in early tetrapods Amel and Arhgap6) translocated to the sex chromosomes Amelogenin (Arhgap6 (inclusive of Amelx) to the 5\u2032end of Mid1 did not cause any detectable phenotypic or behavioral abnormalities beyond dental enamel defects. The enamel malformations were largely the same as those observed in the Amelx null mice  with the possible exception of a \u201cflattened layer\u201d on the enamel surface Arhgap6/Amelx double null mice relative to the Amelx single nulls, two other Arhgap6 knockouts that retained Amelx showed no enamel phenotype. A knockout deleting only exons 6\u20138 of Arhgap6 resulted in mutant male and female mice \u201cindistinguishable from their wild-type littermates at birth and thereafter\u201d Arhgap6 to Mid1 led to enamel abnormalities but deletion from exon 6 of Arhgap6 to Mid1 did not\u201d Arhgap6 is not essential for normal enamel formation, but might modify the enamel malformation phenotype when Amelx is absent.ARHGAP6 is a GTPase-activating protein of the Rho-GAP family. Mice deleted for a 1.1-Mb genomic region spanning from the first exon of AMELX cause syndromes that include AI as a phenotype HCCS) adjacent to ARHGAP6 and AMELX on Xp. To date, 16 AMELX mutations have been reported that cause non-syndromic amelogenesis imperfecta . A partial AMELX deletion (g.2525_7247del4723) starting in intron 2 and ending in exon 7 retained only the coding region for the amelogenin signal peptide plus 2 amino acids In humans, larger X-chromosome deletions inclusive of ARHGAP6 deletions that remove all of AMELX. In family 1 the deletion is confined to intron 1 and is not predicted to alter ARHGAP6 expression. In family 2 the deletion includes exon 2 of ARHGAP6 and is predicted to eliminate normal ARHGAP6 expression. These naturally occurring gene knockouts are analyzed to see what information can be gained concerning the human enamel phenotype in the absence of AMELX expression and the potential importance of ARHGAP6 in the process of dental enamel formation.In this study we report the characterization of two kindreds with partial The human study protocol and subject consents were reviewed and approved by the Institutional Review Board at the University of Michigan and the Ethics Committee of Istanbul University, Turkey, where one of the families was recruited. Study participants signed appropriate written consents after an explanation of their contents and after their questions about the study were answered. Any minors age 8 or older signed a written assent form after their parent completed a written parental consent for participation of the minor. The animal protocol was reviewed and approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.The proband (III:4) of Family 1 was a 10-year-old dental patient at the University of Istanbul, Turkey. Oral photographs and radiographs were obtained for the proband and his 11-year-old cousin (III:1). The proband of Family 2 (III:7) was an 11-year-old dental patient at the Children's Clinic, University of Michigan School of Dentistry. His family was Caucasian, of Eastern European decent. Oral photographs and radiographs were obtained for the proband (III:7) and his mother (II:4). Tooth H of the proband was extracted for orthodontic considerations, photographed, and examined by scanning electron microscopy.260 and OD280. Genomic DNA (50 ng) was amplified using the Platinum PCR Supermix , and the amplification products were purified using the QIAquick PCR Purification Kit and protocol . The concentration of purified amplicon was estimated by the intensity of its ethidium bromide-stained band on a 1% agarose gel. The DNA sequencing reactions used 3 ng/\u00b5L for each 1000 bp of amplification product size and 1.0 pMol/\u00b5L of oligonucleotide primer and were analyzed using an ABI Model 3700 DNA sequencer  at the University of Michigan DNA sequencing core.Peripheral whole blood (5 cc) or buccal swabs were obtained from the 8 members of Family 1 and 5 members of Family 2. Genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit and protocol  and its quality and quantity were determined by spectrophotometry at ODAMELX exons along with adjoining intron sequences were amplified by polymerase chain reaction (PCR); however, amplification products were only observed for male and female controls\u2013not from either proband . We concluded AMELX was deleted in both probands. Because the families reported no notable medical histories and the clinical phenotype was limited to the enamel layer, we expected the deletions to be relatively small. PCR primer pairs were designed to survey upstream and downstream regions. Eight primer pairs sampled intron 1 of ARHGAP6 from nucleotides 50,924 to 291,746 (NCBI genomic reference sequence NG_012494.1) . PCR amplification products were observed from both probands in all reactions. ARHGAP6 exons 2 through 7 were sampled on the other side of AMELX. Only exon 2 of ARHGAP6 in family 2 failed to amplify . Thus the deleted regions were confined to the 5\u2032 region of ARHGAP6. Additional amplifications narrowed down the locations of the deletions in family 1  and family 2 .The seven GCTAATTATTGGTGGAAAAG and Fa1-11R: GAACAGAGGCAGGCTGTGTC, producing a 2200 bp product. The amplification reactions included a 2 min denaturation at 94\u00b0C followed by 35 cycles at 94\u00b0C for 30 s, annealing at 57\u00b0C for 1 min, extension at 72\u00b0C for 6 min followed by a final extension for 20 min. The products were characterized by DNA sequencing priming with the primers used for amplification as well as Fa1-BPF: TTGCCAATCTGCTTTTTACAG, and Fa1-BPR: TATCCTCTTTGTGGGACAGC.To sequence across the deletion in Family 1 genomic DNA was amplified with F: TGAAAGCTAGAGGGGAAACC and Fa2-10R: TGCCAAGAGTAGCCATTTGA, producing a 1969 bp product. The amplifcation reactions included a 5 min denaturation at 94\u00b0C followed by 35 cycles at 94\u00b0C for 90 s, annealing at 58\u00b0C for 1 min, extension at 72\u00b0C for 3 min, followed by a final extension for 7 min.To sequence across the deletion in family 2, genomic DNA was amplified with Fa2-5F: The primary tooth (H) from the proband of Family 2 was sputter coated with gold for 75 s and then imaged using a Field Emission Gun Scanning Electron Microscope  at the Microscopy and Image Analysis Laboratory at the University of Michigan.Arhgap6 promoters (1A\u20131D) are used during tooth development, day 5 and day 11 developing first mandibular molars were collected from C57BL6 wild-type mice and processed according to the published protocol GCAAGCATCCTCAGTTCCTC; Ex1bF: GCAGTGAAGTAAGGGGACCA; Ex1cF: GTGACTCCTAGGGGACCACA; Ex1dF: AAGACAGCAAAGACACCGAGA) paired with an exon 4-specific reverse primer (Ex4R: GGGATAAGGGCATTCCAAAT). The PCR amplifications included a 5 min denaturation at 95\u00b0C followed by 35 cycles at 94\u00b0C for 30 s, annealing at 58\u00b0C for 30 s, extension at 72\u00b0C for 1 min. In the last cycle the extension was for 7 min. The products were analyzed on a 1% agarose gel stained with ethidium bromide.To determine which of the four The proband (III:4) of family 1 was a 10-year-old Caucasian male from Turkey. His mixed dentition displayed thin enamel that was only slightly more radiopaque than dentin and in some locations secondarily affected by dental caries . The occThe proband's affected male cousin (III:1) at age 12 exhibited enamel defects similar to those of the proband , althougThe proband of family 2 (III:7) was an 11-year-old Caucasian male of Eastern European ancestry with an anterior cross-bite. His late, mixed dentition displayed thin, rough enamel that barely contrasted with dentin on radiographs . The incA primary cuspid  extracted from the proband for orthodontic considerations was examined by scanning electron microscopy . The enaThe mother (II:4) of the proband was less severely affected than any of the male subjects . Her enaAMELX exons amplified with the appropriate primers. PCR primer pairs were designed to survey surrounding DNA sequence within ARHGAP6, the results of which narrowly defined the deleted regions in both families . Ultimately, we were able to amplify across the deletions in both families and determine the DNA sequences at the break points (AMELX deletion (X*X). The 96,240 bp deletion in ARHGAP6 in family 1 (g.302534_398773del96240) was confined to intron 1 of that gene, but deleted all of AMELX. The 52,654 bp deletion in ARHGAP6 in family 2 (g.363924_416577del52654insA) deleted the 3\u2032 end of intron 1 including all of AMELX, exon 2, and a small part of the 5\u2032 end of intron 2.Mutation analyses using genomic DNA from the probands of families 1 and 2 were initiated, but none of the seven k points . The delARHGAP6 expression we analyzed the DNA sequences 5\u2032 to exon 2 on human and mouse ARHGAP6 expressed sequence tags (ESTs). This analysis indicated that ARHGAP6 expression is directed by at least 4 different promoters, each associated with a different exon 1 . We identified one human EST and 4 mouse ARHGAP6 ESTs that contained exon 1a; 5 human and 44 mouse ESTs with exon 1b; one human and 9 mouse ESTs with exon 1c; and one human and no mouse ESTs with exon 1d . The structure of the ARHGAP6 gene showing the positions of the alternative promoters, AMELX, and the deletions in families 1 and 2 is shown in To better assess the possible effects of these deletions on Arhgap6 promoters are used during enamel formation, we isolated mRNA from ameloblasts (by laser capture) and enamel organ epithelia (EOE) of mouse first molars at days 5 (secretory stage) and 12 (maturation stage), amplified them with primers specific for exons 1a through 1d, and analyzed the products on agarose gels stained with ethidium bromide , which is entirely coding, would shift the reading frame and likely cause degradation of mRNA expressed from the truncated Arhgap6 gene in family 2. Thus while both of our families have an X-chromosome lacking the amelogenin gene, the shortened X-chromosome in family 1 likely has a functional ARHGAP6 gene, while the one in family 2 does not.AMELX mutations. Multiple amelogenins are expressed due to alternative splicing AMELX mutations produce a diversity of enamel phenotypes that seem to correlate with the position of the mutation Amelx caused extensive cytotoxicity because the ameloblasts failed to secrete the mutant protein, engorging the endoplasmic reticulum/Golgi apparatus. AMELX avoids this ambiguity and gives a clearer picture of how enamel forms when only trace amounts of amelogenin (from AMELY) are secreted. A remarkable observation in both of our families with AMELX deletions is that a thicker layer of enamel forms on the cusp tips and marginal ridges relative to the lateral tooth surfaces. The presence of this enamel cannot readily be explained by expression from AMELY as mice lack an amelogenin gene on the Y-chromosome, and yet Amelx null mice produce a thin enamel layer Enam and Ambn null mice fail to make an enamel layer Amelogenin is a tooth-specific protein that accounts for almost 90% of the protein in secretory stage enamel AMELX is nested within the large (>400 kb) first intron of ARHGAP6ARHGAP6 ESTs (Hs.435291) out of a total of 3,328,058 human ESTs for normal tissues. ARHGAP6 activates the GTPase activity of RhoA AMELX deletions, but the missing parts of ARHGAP6 varied. The deletion in Family 1 removed ARHGAP6 alternative promoters 1c and 1d. Our RT-PCR analyses showed that these promoters are not active in ameloblasts, so it is unlikely that this deletion affects ARHGAP6 expression in developing teeth. The deletion in Family 2 removed exon 2 of ARHGAP6, which should have precluded expression of the ARHGAP6 protein. It seems likely then that Family 1 had an AMELX defect only, while Family 2 had a combined AMELX and ARHGAP6 defect. The enamel phenotypes in both families were similar with a characteristic snow-capped appearance caused by the deposition of a relatively thick layer of enamel on the cusp tips, the buccal-occlusal and lingual-occlusal cusp slopes, and marginal ridges. The enamel defects were severe, but there was not a complete absence of enamel. The proband of Family 2 seemed to have less contrasting enamel on radiographs and greater surface roughness than the affected members of Family 1. The enamel phenotypes in these two families are remarkably similar given the broader phenotypic variation observed among the many persons with defined AMELX mutations . We suspect that the minor variations in enamel phenotype between our two families reflects the natural range of phenotypic variation in persons lacking AMELX, but the suspicion that an absence of ARHGAP6 is the cause of the increased surface roughness cannot be excluded given the minor differences in enamel phenotype observed in Amelx null and Amelx/Arhgap6 double null mice ARHGAP6 could modify the AMELX null phenotype, ARHGAP6 defects alone apparently do not cause enamel malformations. Arhgap6 null mice showed no enamel defects AMELX mutations are consistently found in human kindreds with X-linked AI, suggesting there is no second X-linked gene that causes AI, as this would lead to the accumulation of unsolved X-linked AI cases without AMELX mutations, which is not observed.Both of our AI families had complete Figure S1AMELX disease-causing mutations.(DOC)Click here for additional data file.Figure S2Tooth H from the proband of family 2 before it was processed for SEM analyses.(DOC)Click here for additional data file.Figure S3PCR amplification of the 7 AMELX exons.(DOC)Click here for additional data file.Figure S4PCR amplifications in intron 1 and exons 2 through 7 of ARHGAP6.(DOC)Click here for additional data file.Figure S5PCR amplifications in family 1.(DOC)Click here for additional data file.Figure S6PCR amplifications in family 2.(DOC)Click here for additional data file.Figure S7More PCR amplifications in family 2.(DOC)Click here for additional data file.Figure S8Mouse (A) and Human (B) ARHGAP6 expressed sequence tags.(DOC)Click here for additional data file."}
+{"text": "C) into pathogenic conformational isoform (PrPSc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrPSc on conversion of PrPC in vitro using PrPSc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrPSc. The smallest most potent prions present in sCJD brains were composed only of\u223c20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.The mammalian prions replicate by converting cellular prion protein (PrP Sc), often forming large amyloid deposits and fibrils. However, the apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases raised several fundamental questions; specifically, whether presumably protease-sensitive forms of PrPSc exist as distinct conformers; and whether they comprise the initial steps in prion replication or are related to the alternative misfolding pathway generating noninfectious aggregates. We investigated the conformational characteristics of protease sensitive conformers of PrPSc and their role in the pathogenesis of sporadic Creutzfeldt-Jakob disease (sCJD). Using two different in vitro prion protein (PrPC) conversion techniques in tandem with biophysical methods, we identified small oligomers of protease sensitive PrPSc present in sCJD brains as the most potent initiators of PrPC conversion. Their concentration and conformational stability determine the distinctly different replication potency of PrPSc in individual isolates of sCJD and each of these characteristics correlates tightly with duration of the disease. These features argue for a broad range of distinct prion strains causing the sCJD and imply that small oligomers of protease sensitive conformers of pathogenic prion protein are encoding incubation time and progression rate of the disease.Mammalian prion diseases were originally characterized by accumulation of protease-resistant prion protein (PrP Sc) present in sCJD brains beyond the evidence that it is variably resistant to proteolytic digestion The yeast, fungal, and mammalian prions determine heritable and infectious traits, and thus behave like proteinaceous genes Sc), often forming large amyloid plaques and fibrils CScC) into pathogenic protease resistant (r) conformational isoform (PrPSc) Sc and apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases Sc and prion infectivity ScSc demonstrated a high seeding (replication) potency of small oligomers of the pathogenic prion protein Sc found invariably in sCJD-infected brains might be composed of such highly potent small oligomers.Mammalian prion diseases were originally characterized by deposits of protease-resistant prion protein  Sc generated in PMCA is as infectious as the original brain derived sample is currently debated Sc in sonication-driven or quaking-induced conversion (QuIC), and yielded protease-resistant aggregates with a PK digestion pattern closely related to original brain PrPScSc, these approaches helped to define some key elements of prion structure Sc used as a seed Early important studies with mouse and Syrian hamster PrPde novo\u201d from recombinant proteins and thus prove in principle that mammalian prions are misfolded proteins Sc and translating them into unique phenotypes of the disease in different prion strains is largely unknown. To analyze the mechanistic and structural aspects of the replication of different sCJD prions and specifically the role of sPrPSc, we employed an in-vitro amplification of brain PrPSc with QuIC and sPMCA. Using recombinant human PrP substrate in QuIC, and Tg mice brains expressing human PrPC(129M) in the sonication-driven sPMCA, both reactions demonstrated inverse correlation between conversion efficacy and the conformational stability of small protease sensitive (s) oligomers of PrPSc. The observed link between duration of the disease and conversion potency of small oligomers of sPrPSc in individual sCJD cases suggests that these conformers encode the progression rate of the disease in different prion strains.Although the PMCA and analogous techniques allowed to create prions \u201c6-fold diluted sCJD seeds with matching codon 129 polymorphism and Type 1 or Type 2 PrPSc with levels of rPrPSc (PrP 27-30) easily detectable on WBs . The WB mobility of unglycosylated fragments of rPrPSc from individual cases of sCJD matched the original Type 1 or Type 2 seeds . The results extend previous observations on high fidelity replication of sCJD PrPSc using Tg mouse brain expressing homologous human PrPCC in transgenic mice remains to be established QuIC generated in QuIC resulted in a major PK-resistant fragment with SDS PAGE mobility corresponding to the mass \u223c16 kDa . We did not observe differences between masses of peptides generated from rhuPrPQuIC seeded with Type 1 PrPSc(129M) or Type 2 PrPSc(129M). As expected ).After four rounds of sPMCA, the Tg brain homogenate substrate responded to 10Sc present in sCJD brain homogenates of 20 patients homozygous for methionine in codon 129 and containing either Type 1 (n\u200a=\u200a10) or Type 2 (n\u200a=\u200a10) PrPSc. Both techniques showed statistically significant inverse correlation of the amplification potency and duration of the sCJD . The tight correlation between amplification indexes obtained with sPMCA and QuIC in individual samples  proved that the observed range of values is highly reproducible with two different techniques. When we separated sCJD samples according WB type of PrPSc, we observed overall higher seeding efficacy of MM1 PrPSc over MM2 PrPSc . However, the trends were not statistically significant due to the broad range and overlapping amplification values. We concluded from these data that PrPSc present in cases classified as MM1 and MM2 sCJD display a continuum of conversion rates.Using CDI, we measured the conversion potency of PrP sCJD andFig. 1BSc that were either treated or not with PK before QuIC, we observed up to 10-fold higher seeding potency of the samples that were not treated with PK . We concluded from these experiments that in QuIC reaction, the total PrPSc is more efficient seed than protease-resistant fragment rPrPSc.Using seeds of Type 1 or Type 2 sCJD PrP with PK Fig. 2. , ) suggests conformational heterogeneity beyond that observed with WBs. Therefore, we used the CDI Sc in patients who were homozygous for methionine in codon 129 of the PRNP gene and demonstrate pure Type 1 or 2 PrPSc on WBs Sc as well as rPrPSc is operational and therefore all digestions with proteinase K (PK) were performed at constant protein/enzyme ratio equivalent to 3 IU/ml (\u223c100 \u00b5g/ml) in 10% brain homogenate containing 1% Sarkosyl for one hour at 37\u00b0C. The protocol for PrPSc digestion, validated in previously published experiments, was selected according to the following criteria: 1) complete digestion of PrPC determined with CDI in control samples; 2) complete shift of the bands of PrPSc to PrP 27-30 on WBs; 3) unequivocal WB differentiation of Type 1 and Type 2 rPrPSc in all tested samples Sc N-terminus with PK was monitored on WBs in all samples.The broad range of amplification indexes within each Type 1 or Type 2 group . Typical examples of dissociation/unfolding curves before and after PK for MM1 and MM2 PrPSc are shown in 1/2 values, from \u223c2.3 to \u223c3.0 M  with PK resulted in rPrPSc with invariably increased conformational stability  uniformly, with one exception, produced rPrPSc with decreased stability (Sc(129M) remaining after proteolytic treatment demonstrated higher overall conformational stability than total PrPSc. We observed the opposite effect of PK leading to less stable Type 2 rPrPSc.Comparing ten sCJD Type 2 cases, we found a broad range of Gdn HClo \u223c3.5 M Fig. 4ASc, we first evaluated the shift in the [Gdn HCl]1/2 values (Table S1). Alternatively, we subtracted the relative fractional change in stability of rPrPSc after PK treatment from the PrPSc values obtained before PK . In contrast to simple shift in the [Gdn HCl]1/2 values, the \u0394Fapp take into account the difference in the slopes of the unfolding curves. Using both shifts in the [Gdn HCl]1/2 and \u0394Fapp values, the overall stability of Type 1 sPrPSc is lower than that of rPrPSc (Sc(129M) and shift in the [Gdn HCl]1/2 values induced by PK both demonstrate that sPrPSc is more stable than rPrPSc in this sCJD group  and MM2 (n\u200a=\u200a10) sCJD samples, and in both sPMCA and QuIC  showed higher amplification rates . Similarly, PrPC present in MM1 and MM2 sCJD cases remained in the upper portions of the tubes . On the basis of calibration with standard proteins  and s\u03c92t value distribution in the sucrose gradient, we estimate that the range of S values of CJD prions is from \u223c20 to >150. The majority of MM1 PrPSc conformers have an S value in the range 110\u2013130. In contrast, the S value for the majority of MM2 PrPSc conformers is \u2265150. We concluded that both MM1 PrPSc and MM2 PrPSc display a broad range of aggregates and that the MM1 PrPSc aggregates are reproducibly smaller that those composed of MM2 PrPSc. The variable low-density flotating fraction of PrPSc in both MM1 and MM2 PrPSc samples suggests that the aggregates are very small, or that some PrPSc exists in a complex with detergent-insoluble low-density lipids Sc have mass 9\u201311\u00d7106 Da and are composed of \u223c380\u2013460 monomers of PrPSc. The most frequently occurring aggregates in MM2 sCJD PrPSc have mass \u226514\u00d7106 Da and are composed of \u2265600 monomers of PrPSc.To investigate the impact of prion particle size on protease sensitivity and amplification, we separated sCJD prion particles according to sedimentation velocity using high-speed centrifugation in sucrose gradient. The sCJD prions present in brain homogenates of twelve sCJD patients with MM1 (n\u200a=\u200a6) or MM2 (n\u200a=\u200a6) PRNP gene polymorphism and WB pattern of PrPSc were separated in 10\u201345% sucrose gradient and collected fractions were analyzed by WBs, CDI, and QuIC. The PrPSc with increasing size of the aggregates in MM1 (n\u200a=\u200a6) as well as MM2 (n\u200a=\u200a6) sCJD cases  trend toward higher PK sensitivity of the fractions containing small oligomers of PrPSc in comparison to the bottom and floating fractions . Cumulatively, the data demonstrate that the conversion potency of PrPSc in individual isolates of sCJD is inversely related to the duration of the disease. Because the length of the duration of a clinically pronounced prion disease is a function of the incubation time, this finding accords with the bioassay data that show a broad spectrum of transmission rates and incubation times of sCJD prions in transgenic mice expressing human PrPC(129M) or human/mouse PrPC chimeras Sc and MM2 PrPSc and broad distribution of values within each group suggests that a continuum of sCJD prions are causing the disease.The distinct conversion efficacies we observed with prion seeds obtained from different cases of MM1 and MM2 sCJD were highly reproducible with both QuIC and sPMCA techniques  in prion infection Sc accumulating in CJD brains consists of sPrPScSc upon inoculation into wild mice Considerable data demonstrate that sPrPSc varied with the incubation time of the disease Previously we found that levels of sPrPSc, rPrPSc, and sPrPSc on the seeding potency of sCJD prions with QuIC and sPMCA. Both methods revealed that the stability of total PrPSc inversely correlated with the amplification index of sCJD PrPSc. Moreover, when sPrPSc proved less stable than rPrPSc, the difference in stability correlated with more efficient amplification. Conversely, when sPrP conformers proved more stable than rPrPSc, we observed the opposite effect-less accumulated PrPSc in both sPMCA and QuIC  of cellular membranes. This association would place both cellular and pathogenic forms of the prion protein into the same compartment and thus support the hypothesis that PrPSc formation occurs within caveolae In this research, we investigated whether small aggregates of PrPSc, in contrast with the lower sedimentation velocity of MM1 PrPSc indicates that MM2 PrPSc forms much larger aggregates and concurs with the pattern of large coarse deposits of Type 2 PrPSc observed with the brain immunohistochemistry in situ, in contrast to the fine  appearance of the immunoreactivity associated with Type 1 PrPScSc may be responsible for the different peptide fragmentation pattern with predominantly 19 kDa fragments of MM2 rPrPSc and 21 kDa in MM1 rPrPSc after PK treatment.The strikingly high sedimentation velocity of MM2 PrPAll procedures were performed under protocols approved by the Institutional Review Board at Case Western Reserve University. In all cases, written informed consent for research was obtained from patients or legal guardians and the material used had appropriate ethical approval for use in this project. All patients' data and samples were coded and handled according to NIH guidelines to protect patients' identities.We selected 20 representative subjects from a group of 340 patients with definitive diagnosis of sCJD. The criteria for inclusion were (1) availability of clinical diagnosis of CJD according to WHO criteria Retrospective charts review was carried out for all subjects, with particular attention to the documented initial cardinal clinical signs of sCJD such as cognitive impairment, ataxia, and myoclonus Sc was classified as (1) Type 1 PrPSc(129M) (n\u200a=\u200a10) and (2) Type 2 PrPSc . Patients lacked pathogenic mutations in the PRNP and had no history of familial diseases or known exposure to prion agents. These cases underwent additional detailed WB analyses of the PrPSc so that we could ascertain the accuracy of their original classification and confirm that the same brain homogenate analyzed by CDI contained pure Type 1 PrPSc(129M) or Type 2 PrPSc(129M).All Type 1\u20132 patients or uncertain cases were excluded from this study. DNA was extracted from frozen brain tissues in all cases, and genotypic analysis of the PRNP coding region was performed as described Coronal sections of human brain tissues were obtained at autopsy and stored at \u221280\u00b0C. Three 200\u2013350 mg cuts of frontal (superior and more posterior middle gyri) cortex were taken from each brain and used for molecular analyses. The other symmetric cerebral hemisphere was fixed in formalin and used for histologic and immunohistochemical purposes.Slices of tissues weighing 200\u2013350 mg were first homogenized to a final 15% (w/v) concentration in calcium-free and magnesium-free PBS, pH 7.4, by 3 75 s cycles with Mini-beadbeater 16 Cell Disrupter . The homogenates were then diluted to a final 5% (w/v) in 1% (v/v) Sarkosyl in PBS, pH 7.4 and rehomogenized. After clarification at 500\u00d7g for 5 min, one aliquot of the supernatant was treated with protease inhibitors . The second aliquot was treated with 50 \u00b5g/ml of proteinase K  for 1 h at 37\u00b0C shaking 600 rpm on Eppendorf Thermomixer  and PK was blocked with PMSF and aprotinin-leupeptin cocktail. Both aliquots were precipitated with final 0.32% (v/v) NaPTA after 1 h incubation at 37\u00b0C as described Both PK-treated and untreated samples were diluted 9-fold in 1\u00d7 Laemmli Buffer  containing 5% (v/v) beta-mercaptoethanol (ME) and final 115 mM Tris-HCl, pH 6.8. Samples were heated for 5 min at 100\u00b0C and \u223c2 ng of PrP per lane was loaded onto 1 mm 15% Polyacrylamide Tris-HCl, SDS-PAGE gels (Bio-Rad) mounted in Bio-Rad Western Blot apparatus. After electro-transfer to Immobilon-P Transfer Membranes , the membranes were blocked with 2% (w/v) BSA in TBS containing 0.1% of Tween 20 (v/v) and 0.05% (v/v) Kathon CG/ICP . The PVDF membranes were developed with 0.05 ug/ml of biotinylated mAb 3F4  followed by 0.0175 ug/ml Streptavidin-Peroxidase conjugate  or with ascitic fluid containing mAb 3F4 (kindly supplied by Richard Kascsak) diluted 1\u223620,000 followed by Peroxidase-labeled sheep anti-mouse IgG Ab  and diluted 1\u22363000. The membranes were developed with the ECL Plus detection system (Amersham) and exposed to Kodak BioMax MR Films (Fisher Scientific) or Kodak BioMax XAR Films (Fisher Scientific).2PO4 containing 0.03% (w/v) NaN3, pH 7.5. Second, aliquots of 20 \u00b5l from each fraction containing 0.007% (v/v) of Patent Blue V (Sigma) were directly loaded into wells of white strip plates prefilled with 200 \u00b5l of Assay Buffer . Finally, the captured PrP was detected by a europium-conjugated \u22121 cm\u22121 and 21640 M\u22121 cm\u22121, respectively. The purified recombinant proteins were dissolved in 4 M GdnHCl and 50% Stabilcoat , and stored at \u221280\u00b0C. The concentration of PrP was calculated from the CDI signal of denatured samples using calibration cure prepared with either recPrP(23-231) for samples containing full length PrPSc or recPrP(90-231) for samples containing truncated rPrPSc (PrP 27-30) after proteinase-K treatment. This separate calibration was necessary due to the \u223c3.5-fold lower affinity of mAb 3F4 with full-length human PrP compared to PrP The CDI for human PrP was performed as described previously Sc was performed as described previously Sc were thawed, sonicated 3\u00d75 s at 60% power with Sonicator 4000 , and the concentration was adjusted to constant \u223c50 ng/ml of PrPSc. The 15 \u00b5l aliquots in 15 tubes were treated with increasing concentrations of 8 M GdnHCl containing 0.007% (v/v) Patent Blue V  in 0.25 M or 0.5 M increments. After 30 min incubation at room temperature, individual samples were rapidly diluted with Assay Buffer  containing diminishing concentrations of 8 M GdnHCl, so that the final concentration in all samples was 0.411 M. Each individual aliquot was immediately loaded in triplicate to dry white Lumitrac 600, High Binding Plates , coated with mAb 8H4, and developed in accordance with CDI protocol using europium-labeled mAb 3F4 for detection The denaturation of human PrPOBS\u2212TRFN)/(TRFU\u2212TRFN) where TRFOBS is the observed TRF value, and TRFN and TRFU are the TRF values for native and unfolded forms, respectively, at the given Gdn HCl concentration Sc is unfolded ([Gdn HCl]1/2), the data were fitted by least square method with a sigmoidal transition model (Equation 1):The raw TRF signal was converted into the apparent fractional change of unfolding (Fapp) as follows app) in the TRF signal is the function of Gdn HCl concentration(c); c1/2 is the concentration of Gdn HCl at which 50% of PrPSc is dissociated/unfolded and r is the slope constant. To determine the impact of protease treatment on the conformational stability of PrPSc, the values of fractional change after PK were subtracted from Fapp values obtained before PK (\u0394Fapp\u200a=\u200aF0\u2212FPK) and then fitted with a Gaussian model to estimate the proportion and average stability of sPrPSc conformers (Equation 2):The apparent fractional change  in 10 mM Sodium Acetate buffer, pH 4.0, was pretreated with 12 mM HCl at 1\u22363.9  ratio for 7.5 min and immediately diluted to final 0.1 mg/ml into the reaction buffer composed of 20 mM NaH2PO4, 130 mM NaCl, pH 6.9, and containing 0.1% SDS, 0.1% Triton X-100, and 1\u22365000 (v/v) N2 . The QuIC was performed with final volume 100 \u00b5l per well in sterile V-bottom, low-binding polypropylene 96-well plate  equipped with a 3 mm diameter PTFE bead  in each well. The aliquots of sCJD brain homogenates were diluted into the complete QuIC reaction buffer to obtain final 10\u22124 dilution of sCJD prions, and the plates were sealed with sterile AxyMat Silicone Sealing Mat . The QuIC was performed in samples seeded with sCJD PrPSc at 55\u00b0C for 20 hrs in Eppendorf Thermomixer  set for 1 min shaking at 1400 rpm followed by 1 min incubation.The QuIC was performed as described To each well containing 100 \u00b5l of QuIC reaction buffer was added 50 \u00b5l of PBS, pH 6.9, containing 3% (w/v) Sarkosyl and Proteinase K  to obtain the final Sarkosyl concentration 1% (w/v) and PrP/enzyme ratio 10\u22361 (w/w). The plates was incubated for 1 h at 37\u00b0C at 1200 rpm on the Eppendorf Thermomixer with 1 min interval. The PK was blocked in each well with protease inhibitors .Sc was replicated using brains of transgenic mice overexpressing human PrP with methionine at position 129 6-fold dilution of the initial sCJD brain homogenate.Sonication-driven PMCA of sCJD samples was performed as described obs were calculated from formula sobs\u200a=\u200ak/ tubes . The ultracentrifugation was performed at 50,000 rpm for 73 min at 5\u00b0C in Optima TL ultracentrifuge  equipped with Beckman SW 55 Ti rotor. These conditions correspond to the adjusted proportionality constant k\u200a=\u200a58.7 and angular velocity \u03c9\u200a=\u200a5236 rad/s. Observed sedimentation coefficients sSc in Gdn HCl before and after PK treatment We investigated the effect of the following demographic and laboratory variables on survival: sex; age at onset; duration of the disease; electrophoretic Type of PrP 27-30; and the concentration and stability of PrPFigure S1C(129M) and with QuIC using recombinant human PrP substrate. (A) Sonication-driven sPMCA with brain PrPC substrate preserves the differences in mobility of unglycosylated PrPSc in three MM1 and three MM2 sCJD PrPSc after four rounds of amplification and final dilution of brain sCJD prions 106-fold. Asterisk signifies residual full length PrP after PK treatment. (B) 16 kDa protease-resistant fragments of rhuPrPQuIC detected after QuIC by WBs developed with monoclonal antibody 12B2 (epitope residues 89\u201393) (21) or 3F4 (epitope residues 107\u2013112) (9). The sCJD seeds were in QuIC reaction diluted 104-fold.A typical amplification of sCJD prions in sPMCA using brain homogenate of Tg mice expressing human PrP(TIF)Click here for additional data file.Figure S2A) The typical results of QuIC seeded with different sCJD prions and low background levels of de novo PrP. (B) The amplification of sCJD PrPSc after four rounds of sPMCA (n\u200a=\u200a20) correlates to a highly significant degree with QuIC (n\u200a=\u200a20). The amplification index is the ratio between the concentration of PrPSc before and after PMCA measured with CDI. The data points are averages of three PMCA experiments, each measured in triplicate with CDI.The time course of QuIC reaction and comparison with sPMCA. ((TIF)Click here for additional data file.Figure S3Sc recorded with both sPMCA and QuIC in MM1 and MM2 sCJD. (A) Amplification index obtained with sPMCA for (red circles) MM1 (n\u200a=\u200a10) and (blue squares) MM2 (n\u200a=\u200a10) sCJD cases. (B) Amplification index obtained with QuIC for (red circles) MM1 (n\u200a=\u200a10) and MM2 (n\u200a=\u200a10) sCJD. In both sPMCA and QuIC, the differences observed between MM1 and MM2 samples are not statistically significant. The amplification index was determined as described in legend for Continuum of amplification indexes of PrP(TIF)Click here for additional data file.Figure S4The CDI demonstrated the same end point concentrations of recombinant PrP substrate in samples with PK treated or untreated seeds at the end of the QuIC reaction. The total (substrate+seed) PrP concentration was measured with CDI in duplicate in six samples at the end of QuIC reaction.(TIF)Click here for additional data file.Figure S5A) elution profile and (B) calibration plot for estimating S20,w and mass. The calibrants were bovine serum albumin , alcohol dehydrogenase , thyroglobulin monomer , and apoferitin  (20). The protein content in each fraction was determined with BCA protein assay .Calibration of sucrose gradient ultracentrifugation with standard proteins. The ((TIF)Click here for additional data file.Figure S6C. The (A) PrPC in the human platelets isolated from blood of healthy donors and (B) in a control brain of the patient with other than prion disease (OND). The distribution of PrPC (green bars), total PrPSc (blue bars), and rPrPSc (red bars) in sucrose fractions was determined with and without PK treatment by CDI. The PrPSc and rPrPSc values oscillating around zero were used to establish the cutoff and baseline sensitivity limit of CDI in each fraction. The bars represent average \u00b1 SEM from CDI performed in triplicate.Sucrose gradient ultracentrifugation of control samples containing only PrP(TIF)Click here for additional data file.Figure S7C (green bars), total PrPSc (blue bars), and rPrPSc (red bars) in sucrose fractions was determined with and without PK treatment by CDI. The bars represent average \u00b1 SEM from CDI performed in triplicate.Fractionation by ultracentrifugation in sucrose gradient and protease sensitivity of samples taken from frontal cortex of individual (left column) MM1 (n\u200a=\u200a6) and (right column) MM2 sCJD (n\u200a=\u200a6) sCJD cases. The distribution of PrP(TIF)Click here for additional data file.Table S1Descriptive statistics of the data and demographics of sCJD cases.(DOC)Click here for additional data file."}
+{"text": "FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts.The repression of Arabidopsis  FLC gene is a repressor of flowering that confers a requirement for vernalization  to promote flowering in spring FLC encodes a MADS box protein that binds to and represses expression of the floral promoting genes FT and SOC1FLC by a plant homeodomain-polycomb repressive complex 2 (PHD-PRC2) mechanism FLC locus. Detailed studies of the timing of changes in FLC mRNA expression and H3K27me3 levels showed that transcription of FLC is repressed and H3K27me3 increases at a region around the transcription start site during the cold FLC transcription FLC expression and reduced repression of FLC after vernalization FLC locus is important in down-regulating its expression pre- and post-vernalization. Experiments to define the parts of the FLC gene mediating the different phases of the vernalization response showed that the promoter and first exon are sufficient to confer repression of a reporter gene during vernalization, but maintenance of repression after return to warm conditions requires the first intron in addition to the promoter and exon 1 FLC gene fragment including approximately 1.8 kb of FLC intron 1 and the remainder of the 3\u2032 end of the gene recruits PRC2 and H3K27me3 in the absence of transcription FLC repression after vernalization.The Arabidopsis HOTAIR, which targets PRC2 to the HoxD locus, and is associated with HoxD silencing in humans FLC locus have been identified. The COOLAIR antisense transcripts originate from a promoter adjacent to the FLC 3\u2032 untranslated region and consist of two classes, terminating at proximal or distal sites  originating from a region within the first intron of FLCCOLDAIR transcript has been shown to interact with PRC2 and its abundance also increases during vernalization. Reduction of COLDAIR transcript levels by RNAi showed that it is not required for the initial repression of FLC but is required for subsequent maintenance of repression.A complex array of non-coding RNAs is transcribed from eukaryotic genomes, the majority of which are of unknown function. Some long non-coding RNAs (lncRNAs) associate with and target protein complexes to regulate gene expression al sites Figure 1COOLAIR antisense transcripts in the initial vernalization-induced repression of FLC and the results of reporter gene studies we used FLC insertion mutants to test the role of these transcripts in FLC repression.As there are apparent contradictions in the proposed role of the FLC promoter and first intron are required for the stable vernalization-mediated down-regulation of a reporter gene we tested the effect of a Ds insertion of approximately 6 kb (flc-20) that separates these regions in the endogenous FLC gene FLC 5\u2032 unspliced transcript as an approximation of transcription rate flc-20 mutant that was not maintained after plants were returned to warm conditions  in both C24 and flc-20 at the end of cold, but this increase is not maintained in flc-20 after return to warm temperatures .As the flc-20 mutant together with previous data from FLC reporter constructs raised the question of whether antisense transcription from the COOLAIR promoter is a requirement for vernalization-induced repression of FLC. To test this we carried out further experiments with T-DNA insertions close to the 3\u2032 end of the FLC gene that disrupt the distal (SALK_092716) or both proximal and distal (SALK_140021) antisense transcripts , with the exception of regions 3\u2032 of the SALK_092716 insertion which had high H3K27me3 under all conditions as seen previously for non-transcribed parts of FLCThe data from the COOLAIR transcripts is not an essential component of vernalization-induced repression of FLC. The observations presented here for these SALK lines are in agreement with previous results showing that sequences consisting of the promoter, exon 1 and intron 1 but not the COOLAIR promoter, are sufficient to confer stable repression on a reporter gene COOLAIR lncRNAs may play a redundant role in regulating FLC expression, our data shows that they are not required for the vernalization response.These data show that the production of COLDAIR lncRNA acting to maintain repression of FLC by recruiting the PRC2 machinery COLDAIR transcript and all show maintenance of repression after vernalization.Our data are consistent with the FLC, we measured 5\u2032 unspliced FLC transcripts in the swn7clf28 double mutant and in the vin3-4 mutant. CLF and SWN encode histone methyl transferases components of PRC2; loss of function of these genes leads to a genome-wide loss of H3K27me3 swn7clf28 plants have increased 5\u2032 unspliced FLC transcript in non-vernalized plants and show a similar fold-repression as ColFRI during 4 weeks of cold exposure  and crossed with ColFRI, a Col line with an active FRI allele FRI allele activates expression of FLC. PCR was used to identify F2 plants in which the active FRI allele and the T-DNA insertions were homozygous. The flc-20 mutant contains a modified Ds element inserted in the first intron of FLCSalk insertion mutants were obtained from the Arabidopsis Stock Center . Primers used are listed in Table S1. PCR were reactions carried out in quadruplicate, quantified using a standard curve of diluted cDNA and normalized to At4g26410 RNA was extracted using Qiagen Plant RNeasy Mini columns with an on-column DNase treatment according to the manufacturer's protocol. cDNA was synthesized using Superscript III (Invitrogen), primed with oligo dT  or with gene specific primers for the Table S1.Chromatin immunoprecipitation was carried out and amplicons for ChIP-qPCR are as described Figure S1H3K27me3 ChIP qPCR across FLC in C24 and flc-20. ChIP qPCR for amplicons 1\u201312 flc-20 plants grown for 12 long days (12LD), 12 long days followed by 4 weeks at 4\u00b0C (12+4V) or 12 long days, 4 weeks at 4\u00b0C followed by 2 long days (12LD+4V+2LD).(TIF)Click here for additional data file.Figure S2H3K27me3 ChIP qPCR across FLC in ColFRI, SALK_092716, SALK_140021 and SALK_131491. ChIP qPCR for amplicons 1\u201312  in ColFRI, SALK_092716, SALK_140021 and SALK_131491 plants grown for 12 long days (12LD), 12 long days followed by 4 weeks at 4\u00b0C (12+4V), 12 long days, 4 weeks at 4\u00b0C followed by 2 long days (12LD+4V+2LD) or 12 long days, 4 weeks at 4\u00b0C followed by 5 long days (12LD+4V+5LD).(TIF)Click here for additional data file.Table S1Oligonucleotide sequences.(XLS)Click here for additional data file."}
+{"text": "Transmembrane roundabout receptor family members (ROBO1\u2013ROBO4) principally orchestrate the neuronal guidance mechanism of the nervous system. Secreted glycoprotein SLITs are the most appreciated ligands for ROBOs. Recently identified ROBO4 is the key mediator of SLIT-ROBO mediated developmental and pathological angiogenesis. Although SLIT2 has been shown to interact with ROBO4 as ligand, it remains an open question whether this protein is the physiologic partner of ROBO4. The purpose of this review is to summarise how reliable SLIT2 as ligand for ROBO4 is, if not what the other possible mechanisms demonstrated till date for ROBO4 mediated developmental and pathological angiogenesis are. We conclude that ROBO4 is expressed specially in vascular endothelial cells and maintains the vascular integrity via either SLIT2 dependent or SLIT2 independent manner. On the contrary, it promotes the pathological angiogenesis by involving different signalling arm(s)/unknown ligand(s). This review explores the interactions SLIT2/ROBO1, SLIT2/ROBO1\u2013ROBO4, ROBO1/ROBO4, and ROBO4/UNC5B which can be promising and potential therapeutic targets for developmental angiogenesis defects and pathological angiogenesis. Finally we have reviewed the ROBO4 signalling pathways and made an effort to elaborate the insight of this signalling as therapeutic target of pathological angiogenesis. Hence, the current available information is not sufficient to delineate the SLIT2/ROBO1/ROBO4 mediated mechanism of pathological angiogenesis. In this review we have summarized the molecular mechanism of developmental and pathological angiogenesis mediated by magic roundabout (ROBO4) as one of the major processes supporting the tumor growth.The neural and vascular networks often undergo the same routes and similar mechanisms of signalling. Many classes of guidance molecules have been characterized to play critical roles during angiogenesis , 2 such Structural organization of magic roundabout shows significant difference with other ROBO members, most notably in the extracellular region . ROBO4 hROBOs, the neuronal guidance receptors, are newly introduced in pathological vascular development. While ROBO1 is expressed in both endothelial cells and other cell types, ROBO4 is expressed specially in vascular endothelial cells including the tumor vasculature , 11, 42.in vitro, and vascular leak in vivo. This happens by blocking the activation of nonreceptor tyrosine kinases  in ROBO4+/+ endothelial cells but not in ROBO4\u2212/\u2212 cells [Since ROBOs are extensively involved in guidance mechanism, they should be expressed in the leading end cells. Contrary to this hypothesis, ROBO4 was found to be transcribed in stalk cells (mature vascular cells) of retinal blood vessels and absent from many of the tip cells that sense and respond to extracellular cues. This suggests that ROBO4 may have a biological role that is unrelated to the guidance mechanisms regulating vascular patterning . Park et/\u2212 cells . These r/\u2212 cells , 46. SLI/\u2212 cells  indicati/\u2212 cells . ROBO4-p/\u2212 cells  and lead/\u2212 cells , 46. TheAnother mechanism, in this context, is the interaction of ROBO4 with UNC5B, a vascular netrin receptor that also counteracts the VEGF signalling . This rein vitro [The N-terminal Ig like domains of ROBO1 and ROBO4 are 42% identical but the residues identified for SLIT2 binding are not conserved in ROBO4 and are replaced by residues that are not compatible with binding of SLIT2. This suggests that ROBO1 has more efficient binding with SLIT2 compared to ROBO4. Park et al.  have demin vitro , 32 suggin vitro , 32, 55.in vitro . Therefoin vitro  which enHence, there are three complexes that can be exploited to target the pathological angiogenesis: SLIT2/ROBO1, ROBO4/UNC5B, and ROBO1/ROBO4. By promoting or inhibiting the affinity and/or association of these axes according to the mechanism involved in a particular system, inhibition of pathological angiogenesis and vascular high permeability can be appreciated as therapeutic target.Danio rerio) demonstrating that ROBO4 plays an essential role during angiogenesis and guides endothelial cells to their target analogous to ROBO1, ROBO2, and ROBO3 during neuronal guidance [We discussed that ROBO4 has endothelial specific expression and SLIT2/ROBO1\u2013ROBO4 signalling inhibits the endothelial cell migration and pathological angiogenesis by counteracting the VEGF signalling. Contrary to this antiangiogenic nature of ROBO4 signalling, there is also evidence supporting that ROBO4 promotes the pathological angiogenesis , 57. Binguidance . Collectguidance  demonstrguidance .\u03b1 (HIF-1\u03b1) leading to VEGF production and angiogenesis [Evidence shows that SLIT2 is not the ligand for ROBO4 because (1) N-terminal ligand binding domain of ROBO4 is not fit for SLIT2 binding; (2) dimerization of cytoplasmic region of ROBO4 is independent of SLIT2 binding ; (3) ROBogenesis . Also, iogenesis . It can In neurons interaction of SLIT2 with ROBO1 induces the recruitment of Rho GTPase activating proteins (srGAPs) to its CC3 domain which increases the intrinsic GTPase activity of CDC42 leading to its inactivation  Figure . LocalizHow does ROBO4 mediate the proangiogenic attraction guidance signalling in endothelial cell? Again as discussed earlier there could be two possibilities: (1) an unknown ligand for ROBO4 and (2) SLIT2 may signal by interacting with ROBO1\u2013ROBO4 heterodimer. There should be a protein interacting with cytoplasmic region of ROBOs, to determine the antiangiogenic or proangiogenic nature of this signalling. One such appreciable protein is srGAP (SLIT-ROBO GTPase activating protein). These proteins are particularly abundant in SLIT responsive regions. Binding of srGAP to CC3 domain of ROBO1 increases the intrinsic GTPase activity of CDC42, which converts the GTP-CDC42 into GDP-CDC42 inactivating CDC42. Reduction of active CDC42 eventually decreases actin polymerization affecting the cell movement . Repulsi(WIP) and syndapin [To understand the mechanism of ROBO4 to promote or inhibit the fundamental events of angiogenesis, it is important to explore the other interacting partner protein(s) with intracellular region of ROBO4, especially proteins involved in actin regulatory machinery. The novel proteins interacting with ROBO4 involved in actin regulatory machinery include the Wiskott-Aldrich syndrome protein (WASP) and neuronal WASP proteins (NWASP) and their regulatory proteins: WASP-interacting protein syndapin . WASP hasyndapin . Glutathsyndapin . The idesyndapin . Thus, RIt is obvious that interaction of activated CDC42 (CDC42-GTP) presumably with other proteins is needed for the recruitment of MENA, WASP, NWASP, and WIP to ROBO4 for the formation of filopodia and migration. One such protein previously implicated in CDC42-GTP mediated filopodia formation is an SH3 domain-containing insulin receptor substrate protein 53 (IRSp53) . IRSp53 Our discussion converges to the point that ROBO4 is a key mediator of pathological angiogenesis and unravelling its mechanism of action may prove it as double edged sword. To unravel its mechanism there are some key questions to be addressed such as the following: (1) What are the physiological partners of ROBO4? (2) Does the choice of partner proteins of ROBO4 depend on microenvironment of cell that determines the proangiogenic or antiangiogenic nature of ROBO4 signalling? (3) What are the factors that regulate the homodimerization or heterodimerization of ROBOs? (4) Syndecans, a conserved family of heparan- and chondroitin-sulfate, are emerging as central players in cell surface interactions. It has been demonstrated that heparan sulfate serves as essential co-receptor in Slit-Robo signalling , 69. DoeConverging points of evidence discussed in this review indicate that ROBO4 has prominent role in regulation of angiogenesis. ROBO4 inhibits the pathological angiogenesis by counteracting the VEGF signalling either via SLIT2 or by interacting with UNC5B. This inhibition mechanism may be dependent on SLIT2-ROBO1 and ROBO1\u2013ROBO4 interactions. ROBO4 signalling also promotes the pathological angiogenesis either via SLIT2/ROBO1\u2013ROBO4 axis or via interaction of ROBO4 with an unknown ligand other than SLIT2. SLIT2 signalling via ROBO1\u2013ROBO4 heterodimerization and srGAP interaction with cytoplasmic region of ROBO1 may be responsible for proangiogenic attraction guidance signalling in endothelial cells. Most of the literature considering therapeutic aspect of ROBO4 is focused on interaction of SLIT2 and ROBO4. In this review we have attempted to explore whether ROBO1\u2013ROBO4 interaction can be targeted for antitumorigenic and antiangiogenic therapy. Since ROBO4 has dual behaviour of promoting and inhibiting angiogenesis depending on cell/tissue type, the therapeutic agents having properties of promoting and inhibiting ROBO4 signalling, respectively, can be used to target tumor angiogenesis according to the cell/tissue type under consideration. Because of its specific expression at the site of neoangiogenesis, ROBO4 can be used as angiogenesis marker. Although many questions still remain to be addressed regarding ROBO4 signalling, its understanding may be a milestone in targeting the pathological angiogenesis."}
+{"text": "DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome.Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36\u2013288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence  At least half of the human genome is composed of repetitive DNA Macrosatellites consist of repeat units ranging from 1\u201312 kb that are arranged in tandem. The number of repeat units is polymorphic in the general population, and an array can be composed of only a few to over one hundred repeat units, and therefore can encompass large genomic intervals. Most macrosatellite arrays are specific to one or two chromosomal locations in the genome, and only a small number have been confirmed and characterized to some extent PLS3), and 296.5 kb proximal to the angiotensin II receptor (AGTR2)(Our interest in macrosatellite repeats came about through examination of chromatin organization on the human inactive X chromosome (Xi). X chromosome inactivation (XCI) is the mammalian form of dosage compensation r (AGTR2). DXZ4 isr (AGTR2), that acHere we report our findings on four aspects of DXZ4 variation: tandem repeat copy number variation, array instability, monomer sequence polymorphism and differences in array expression.in vivo, but would be aligned on top of one another in silico. A comparison of a single DXZ4 monomer sequence (Accession Number HQ659112) against the assembled human genome sequence (hg19) using the UCSC genome browser (http://genome.ucsc.edu/), reveals approximately fourteen \u223c3 kb DXZ4 monomers covering 50 kb, arranged in tandem centered at 115 Mb on the X chromosome. In addition, two partial and one complete monomer reside in an inverted orientation relative to the main array within the immediate distal 70 kb sequence  revealed hybridizing DXZ4 EcoRI fragments of between 150\u2013300 kb by Southern analysis. This translates into arrays composed of between 50 to 100 monomers. We extended this analysis to an additional 22 unrelated individuals of diverse ethnicity. Agarose embedded genomic DNA was digested with XbaI (a restriction endonuclease for which there are no recognition sites within DXZ4) and were then separated by PFGE before transfer to nylon membrane by Southern blotting. As expected, hybridization of the blots with a DXZ4 probe identified two hybridizing signals in female samples and one in males .Next we sought to confirm that DXZ4 is indeed a tandem array of individual 3 kb monomers arranged in a head-to-tail orientation. In order to do this we used two complementary approaches; restriction endonuclease digest analysis of a DXZ4 bacterial artificial chromosome (BAC) clone, and extended DNA fiber fluorescence http://blast.ncbi.nlm.nih.gov/Blast.cgi). Several clones were identified including clone 2272M5 from the human genomic sperm CITB BAC library D (Accession number AQ745776). The BAC clone was obtained and DNA isolated. Given that the BAC clone insert matches DXZ4 sequence at both ends, uninterrupted tandem arrangement of DXZ4 monomers should result in the generation of a predictable pattern of restriction fragments (BamHI cuts twice per monomer and once in the BAC vector (pBeloBAC11). HindIII cuts once per monomer and is the cloning site used for generation of this library BamHI and 3.0 kb HindIII fragment relative to the vector backbone fragment indicated that the BAC contained several DXZ4 monomers. To confirm this, the BAC clone was digested with four different restriction endonucleases that have recognition sites within the vector backbone but not within DXZ4, and the cut DNA was separated by PFGE. For all four digests the resulting fragment was greater than 100 kb . The two cloned fragments were then labeled with different fluorophores and used for FISH. A tandem arrangement of DXZ4 would result in an alternating red-green signal, as was observed . Most variation was accounted for by polymorphism in copy number of repeat units in the three internal microsatellite repeats. The [GGGCC] repeat ranged from 2 to 5 tandem copies, the [CT] repeat ranged from 9 to 16 tandem copies, and the [TAAA] repeat ranged from 8 to 13 tandem copies  The 18 unique monomers shared 99% or greater sequence identity according to pairwise alignments using BLAST  were identified in monomers from 2272M5 , as wellPvuII, separated by PFGE and Southern blotted, showed the same pattern of bands when hybridized with a DXZ4 probe , two diploid female primary fibroblast cultures and one EBV transformed diploid female lymphoblast cell line. Representative examples of the results are shown in Next we sought to investigate the expression of DXZ4 from the Xi and Xa in 10 different female lymphoblast cell lines by RNA FISH with direct labeled probes; five from CEPH family 1331 and five from family 1345. DXZ4 expression was readily detected in all females . The patPreviously, we had shown that all regions of DXZ4 could be detected by RT-PCR Finally, we examined expression of DXZ4 in a panel of complementary DNA (cDNA) samples prepared from 20 different human tissues. RT-PCR analysis showed robust expression of DXZ4 from all sources  indicatiLike DXZ4 Next we sought to examine if inherited DXZ4 alleles showed comparable expression levels. Once again, to ensure expression originated from a single allele we restricted our analysis to males. Due to X-linkage we also restricted our analysis to sons inheriting DXZ4 from their maternal grandfather. As with the size versus expression analysis described above, DXZ4 expression was variable , and inhExploring variation in the human genome is essential to begin to understand how polymorphism impacts gene expression, phenotypic variance and disease susceptibility. Here we report on variation of the X-linked macrosatellite DXZ4.In this study, we confirm that DXZ4 is a polymorphic uninterrupted tandem array, and extend the range of observed alleles to between 12 and 100 head-to-tail 3 kb repeat units. This variability is comparable to that described for other macrosatellites In addition to monomer copy number variation, we report variation in both the internal microsatellite repeats, and numerous SNPs within the unique regions of a monomer. Our analyses indicate that DXZ4 monomers within one array are more similar to one another than they are to monomers in an array from a different individual. This suggests that mutations acquired in a monomer can spread through an array, most likely via complex gene conversion mechanisms as has been described for other satellite DNA Previously we have described several different RNA species originating from DXZ4, with expression from both the Xa and Xi As with expression of DXZ4 from the Xa, DXZ4 expression from the Xi is variable, differing both between cell lines and from one cell to the next within the same line. Using direct RNA FISH, it is also clear that the level of expression of DXZ4 differs from cell to cell within the same cell line; with some cells showing little to no expression whereas others may show DXZ4 localized transcript patterns comparable in size to that of XIST RNA. It is important to note that large diffuse DXZ4 signals were not common, and were only observed in the hTERT and EBV transformed cell lines. However, primary cells were not extensively examined in this study and therefore no significance can be placed on the extent of the DXZ4 signal and cell immortalization. DXZ4 monomers on the Xi are packaged into both euchromatin and heterochromatin CT). The exception was the SST1 array that is expressed in all tissues examined. Here we show that DXZ4 is also expressed at comparable levels in all tissues, and therefore DXZ4 is not a CT loci, distinguishing DXZ4 from two other X-linked macrosatellite repeats CT47 CT family. The significance of DXZ4 expression is unclear. Like SST1, DXZ4 shows no obvious protein coding function. It is possible that expression of DXZ4 is associated with chromatin organization of the array Recently we described the characterization of four autosomal macrosatellite arrays The data we present here extends our knowledge base of DXZ4 and the biology of macrosatellite arrays, providing the basis for formulation of new hypotheses to explore the role of DXZ4 on the X chromosome.Human tissue total RNA was obtained from Clontech (636643). Residual genomic DNA was removed by pre-treating the RNA with DNaseI (Invitrogen) for 20 minutes at room temperature, before heat inactivating the DNaseI at 70\u00b0C in the presence of 2.5 mM EDTA for 15 minutes. cDNA was prepared using 1 ug of total RNA with or without M-MLV reverse transcriptase (Invitrogen) according to the manufacturers instructions.cDNA was amplified using Taq polymerase (NEB) with the following cycle: 95\u00b0C for 2 minutes, followed by 35 cycles of 95\u00b0C 20 seconds, 58\u00b0C 20 seconds, 72\u00b0C 30 seconds. The sequence of oligonucleotides used to amplify DXZ4 cDNA and the anticipated product size are given in the Supporting Information .QRT-PCR was performed on a CFX96 thermocycler and analyzed using the CFX Manager Software (Biorad). The sequence of oligonucleotides used to amplify DXZ4 cDNA are given in the Supporting Information . Amplifiwww.coriell.org/). Cells were maintained according to Coriell recommendations. Culture media (RPMI), fetal bovine serum and supplements were all obtained from Invitrogen corp. Telomerase immortalized cell lines hTERT-RPE1 , hTERT-HME1  and hTERT-BJ1  were all obtained from Clontech and maintained as recommended by the supplier. Fetal lung fibroblast primary cells IMR-90  and WI-38  were obtained from the American Type Tissue Culture Collection (ATCC), and maintained according to supplied instructions.Lymphoblastoid cell lines of CEPH family members and the individuals used in the variation panels were obtained from the Coriell Institute for Medical Research . Cells were pelleted three more times, resuspending each time in 3\u22361 fix. Fixed cells were either stored at \u221220\u00b0C or used immediately for fiber preparation. Cells in 3\u22361 fix were applied to the raised end of a poly-L lysine coated microscope slide propped up on a paper towel. Using a cover glass, the cells were gently drawn down the length of the slide, dragging only the liquid, not touching the glass slide. Cells were air dried for 5 minutes before immersing in 3\u22361 fix for 5 minutes. Fibers were dehydrated through 70% and 100% ethanol for 2 minutes each before air-drying. Fibers were denatured for 5 minutes in 70% formamide, 2\u00d7 SSC at 75\u00b0C before dehydrating for 3 minutes each in cold 70% and 100% ethanol.FISH probes consisted of 449 bp or 550 bp pCR2.1 cloned PCR fragments of DXZ4 that are approximately 900\u20131100 bp apart in a single monomer . Probes Images were collected using a Zeiss Axiovert 200 M fitted with an AxioCam MRm and were managed using AxioVision 4.4 software (Carl Zeiss microimaging). Image files were exported to Adobe Photoshop CS (Ver.8.0) for preparation of figures.A direct labeled Spectrum Green probe of BAC clone 2272M5 was prepared by Nick Translation according to the manufacturers instructions (Abbot Molecular). The probe (1 microgram) was ethanol precipitated along with 25 micrograms of human Cot-1 DNA before resuspending in 0.1 ml of Hybrisol VII . A direct labeled Spectrum Red probe of XIST exon 1 was prepared as described previously DXZ4 sub-region direct-labeled FISH probes were prepared from sub-fragment clones of DXZ4. Sub-fragment clones were prepared by PCR amplifying unique regions of DXZ4  and TA cImaging was performed as described as above.7 cells were resuspended in 1 ml of L-buffer , before mixing 1\u22361 with 1.0% (w/v) molten low-melt agarose (Biorad). The cell mixture was transferred to plug molds (Biorad) with \u223c80 ul of the cell suspension per plug (approximately 1.6\u00d7106 cells/plug). Plugs were allowed to set at 4\u00b0C for 10 minutes before transfer to 10 volumes of L-buffer containing 1% (w/v) sarkosyl and 1 mg/ml Proteinase-K (Roche) and incubating overnight at 50\u00b0C. Plugs were rinsed with water before three washes of one hour each with 50 volumes of TE [8.0]. Plugs were incubated at 50\u00b0C for 30 minutes in 10 volumes of TE [8.0] supplemented with 80 ug/ml PMSF (Roche). Plugs were rinsed once more with water before three additional hour-long washes in 50 volumes of TE [8.0] at room temperature before storage at 4\u00b0C.Approximately 4\u00d710Agarose embedded DNA was digested with the restriction enzymes given in the legend for the appropriate data figures. All enzymes were obtained from NEB. Each plug was first equilibrated in 300 ul of 1\u00d7 digest buffer at room temperature for 20 minutes, before replacement of buffer with 100 ul of 1\u00d7 digest buffer containing 200 units of restriction enzyme. Digests were performed overnight at 37\u00b0C. Plugs were loaded onto a 1.0% agarose gel prepared using pulsed field certified agarose (Biorad) in 0.5\u00d7TBE. The running conditions  were determined by the auto algorithm function of the CHEF Mapper (Biorad). The following conditions were consistent in each case: 0.5\u00d7 TBE, 14\u00b0C, 1.0% agarose. Separation parameters for the Southern blots in the current manuscript were as follows: HindIII to confirm presence of a common 3 kb fragment and insert size determined by PFGE. BAC clone 2272M5 (Acc. No. AQ745776) was selected for subcloning and sequencing. Cloning vector pBluescript II was digested with HindIII before phosphatase treatment using Calf intestinal phosphatase (NEB). BAC 2272M5 was digested with HindIII and fragments subcloned into pBluescript II, before blue-white screening using standard techniques BAC clones were identified that matched DXZ4 at both ends by BLAST using a single DXZ4 monomer sequence (Acc. No. S60754). BACs were obtained from Invitrogen and DNA isolated using the Qiagen plasmid Midi kit. BACs were digested with At the end of the PFGE run, the gel was rinsed with water before staining with ethidium bromide (1 ug/ml) at room temperature for 30 minutes. The gel was washed twice with water for 15 minutes each and an image captured. The gel was then treated with 0.25 M HCl for 15 minutes before denaturing for 30 minutes . DNA was transferred to Hybond-N+  overnight by standard Southern blotting EcoRI digested total genomic DNA.A DXZ4 probe was prepared by PCR amplification of regions of DXZ4 using oligonucleotides listed in the Supporting Information . The PCRHybridization was performed overnight at 60\u00b0C using Expresshyb (Clontech). Blots were washed the following day at 60\u00b0C using two 8-minute washes in 2\u00d7SSC, 0.1%SDS followed by one wash of 8 minutes in 0.2\u00d7SSC, 0.1%SDS. The probe was detected using anti-DIG-alkaline phosphatase, blocking, wash and detection buffers according to the manufacturers instructions (Roche). Signals were detected by exposure to photographic film (Kodak).Figure S1Comparison of DXZ4 hybridization patterns between PvuII and XbaI PFGE. Southern blots of PFGE separated DNA from 11 independent individuals cut with either XbaI or PvuII and hybridized with a DXZ4-DIG probe. Recognition sequences for either restriction endonuclease are not present in the DXZ4 array and therefore give near identical hybridizing patterns. Sizes in kb are given to the right of each blot. The first PvuII site is 24 kb closer to the array on the distal edge accounting for the smaller sized hybridizing fragments.(TIF)Click here for additional data file.Figure S2BAC clone insert size determination. Ethidium bromide stained 1.0% agarose gel showing restriction endonuclease digestion of DXZ4 BAC clone 2272M5 separated by PFGE. Separation performed at 14\u00b0C for 26 hours in 0.5\u00d7 TBE, separating for 20\u2013200 kb on a CHEF Mapper (Biorad). Markers and sizes are indicated, as are the restriction enzymes used that cut in the vector backbone, but not the DXZ4 array. NotI cuts twice in pBeloBAC11, excising the BAC insert, accounting for the smaller fragment size.(TIF)Click here for additional data file.Figure S3Southern blot of PvuII digested DNA from members of CEPH family 1345. Southern blot of PFGE separated DNA from CEPH family 1345 digested with PvuII and hybridized with a DXZ4-DIG probe. The top portion of the blot has been darkened in Photoshop in order to clearly see the 284 kb extra band (top arrow) also observed with XbaI. The middle arrow points to the additional 234 kb band and the lower arrow points to the additional 227 kb band.(TIF)Click here for additional data file.Table S1DXZ4 variation in genome build HG19. Summary of SNPs and microsatellite alleles in complete \u223c3 kb DXZ4 monomers that define the DXZ4 array in human genome build hg19. Coordinates of SNPs are given relative to the reference sequence of subclone 35. Variants that do not appear in BAC 2272M5 are highlighted in red (only first appearance in the table is highlighted). The largest allele of the (CT) microsatellite is highlighted in green.(DOCX)Click here for additional data file.Table S2DXZ4 variation in monomer submitted by Giacalone et al. Summary of SNPs and microsatellite alleles in the single DXZ4 monomer sequence submitted by Giacalone and colleagues (DOCX)Click here for additional data file.PCR Primers S1DNA sequence of oligonucleotide primers used in current study.(DOCX)Click here for additional data file."}
+{"text": "Our study demonstrates that diabetic patients have increased myocardial edema in the infarct segment at an early time point post acute myocardial infarction which persists till the subacute phase. This may lead to deleterious left ventricular remodeling and worse outcome in these patients.Diabetes is associated with worse left ventricular remodeling and poor prognosis in patients post acute myocardial infarction (AMI). The impact of diabetes on microvascular injury parameters including myocardial edema and hemorrhage post AMI is unknown. Our objective was to characterize the evolution of myocardial edema and hemorrhage post AMI in patients with and without diabetes.Sixty patients were enrolled post AMI and underwent cardiac magnetic resonance on a GE Signa Excite, 1.5T scanner with a 8-channel receive coil at 48 hours and 3 weeks. T2 maps were computed from a previously validated cardiac-gated spiral imaging sequence with T2 preparations yielding TEs=2.9,24.3,88.2,184.2ms to assess myocardial edema. The T2* sequence was a multiecho acquisition with 8 echoes (between 1.4 and 12.7ms) acquired at TR=14.6ms. Delayed hyperenhancement was also performed. We retrospectively reviewed and stratified patients into those that did and did not have diabetes (type 1 or type 2).We compared 15 diabetics versus 45 non-diabetics . At this time interval, the mean T2* was equivalent in the IS of both patient groups .Our study demonstrates that diabetic patients have increased myocardial edema in the IS at an early time point post AMI which persists till the subacute phase (3 weeks). The degree of myocardial hemorrhage does not appear to be influenced by diabetes status. Diabetes increases the area at risk during AMI which may lead to deleterious left ventricular remodeling and worse outcome in these patients.Dr. Connelly is supported by a Heart and Stroke Foundation of Canada Phase 1 Clinician Scientist Award. Dr. Wright receives research funding from GE Healthcare. Dr. Dick is supported by the Heart and Stroke Foundation of Canada. This work was supported in part by a Canadian Institutes of Health Research (CIHR) operating grant and the Ontario Research Fund."}
+{"text": "Arterial partial pressure for oxygen and carbon dioxide, and oxygen saturations were measured in the jugular and arterial blood  . The subjects were exposed to hypoxia , inspiration of 100% oxygen (n = 8), atmospheric air (n = 37), hypercapnia  and asked to hyperventilate  with separate controls. By linear regression the contribution from arterial and jugular blood to ScO2 was estimated and R-squared (R2) and root mean square error (RMSE) between ScO2 and the arterial fraction in the reference saturation were calculated . Only data points where SjO2 \u2264 ScO2 \u2264 SaO2 were included. All reference saturations were calculated, e.g., ScapO2 = 0.50 \u00b7 SaO2 + 0.50 \u00b7 SjO2. The following equation, 0 = ScO2 \u2013 [a \u00b7 SaO2 + (a \u2212 1) \u00b7 SjO2], was used to calculate the arterial fraction (a) for which the difference between ScO2 and the reference saturation was zero.Thirty seven subjects [age 27(9) years, height 181(10) cm, mass 79(13) kg, mean  with a RMSE of 4.233. In contrast, ScapO2  had a R2 of 0.606  and also for that ratio the RMSE was the lowest (2.70) . In addition, the mismatch between ScO2 and the reference ratio was more likely to be zero when a more arterial weighted reference was applied  Figure . The cald Figure . Thus, ocO2 values and the calibration ratio  is observed in five different NIRS devices used to determine cerebral oxygenation in healthy subjects exposed to isocapnic hypoxemia . Thus, the mismatch between the calibration ratio and ScO2 is aggravated when only jugular venous oxygen saturation is altered, which indicates that ScO2 accounts for more than 25% arterial blood and that evaluations of reference saturations for NIRS must also involve conditions known to affect arterial oxygen content.Similar to these findings, no eligible mismatch between ScO2 readings (Rasmussen et al., cO2 readings may be variation in skull thickness and amount of cerebrospinal fluid (Yoshitani et al., cO2 because of competitive absorption of light (Madsen et al., The arterial to venous balance within the brain may differ between individuals and explains the inter-individual variation in absolute ScO2 values are not comparable and exhibit large variations when compared to the calibration ratio, NIRS offers a unique non-invasive method for assessment of cerebral oxygen delivery vs. consumption and its clinical utility relies on changes from baseline rather than on absolute values (Murkin et al., cO2 is limited when skin blood flow is affected either by scalp ischemia or administration of sympathomimetic agents that affect skin blood flow (Davie and Grocott, Despite absolute SIn summary, this report suggests that the reference saturations applied when using Invos cerebral oximetry should be weighted more to arterial hemoglobin saturation than accepted by anatomical models.All authors contributed equally to the design, data analysis, and interpretation, drafting the manuscript and critical revision. All authors approved the final version before submission."}
+{"text": "Mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. The large majority of the alterations identified in these genes are point mutations and small insertion/deletion. However, an increasing number of large genomic rearrangements (LGRs) are being identified, especially in BRCA1. To date just a few large genomic rearrangements of BRCA1 gene have been reported in Poland. Technical limitations of conventional PCR-based methods are cause that gross rearrangements can be overlooked. It has been suggested that about 30% of mutations in the BRCA1 gene are missed by standard mutation detection methods. We screened for LGRs in BRCA1 and BRCA2 genes by Multiplex Ligation-dependent Probe Amplification (MLPA) in 200 unrelated patients with strong family history of breast and/or ovarian cancer negative for BRCA1 Polish founder mutation. We identified 3 different LGRs in BRCA1 gene. No large LGRs were detected in BRCA2 genes."}
+{"text": "It was an observational cohort of pregnant HIV positive women ignorant of antiretroviral therapy with CD4 cell count of>350/mm3. Exclusion criteria: highly active antiretroviral therapy prior to pregnancy.Routine CD4 cell count assessment in HIV positive pregnant women completed by non exclusive measurement of the viral load by PCR /ARN in those with CD4 cell count>350/mm3, median: 528 (IQR: 421\u2013625). 145 underwent measurement of viral load by PCR/RNA at a median gestational of 23 weeks of pregnancy (IQR: 19\u201328). Median viral load 4.4log10/ml, IQR (3.5\u20134.9).19/145(13%) had an undetectable viral load of=1.8log10/ml. 89/145(61%) had a viral load of=4 log10/ml and were eligible for maternal triple ARV prophylaxis.Between January and December 2010, CD4 cell count was systematically performed in all pregnant women diagnosed as HIV-infected (n=266) in a referral center of 25 antenatal clinics. 63% (N=170) had CD4 cell count>350/mm3 may require triple antiretroviral for prophylaxis of MTCT. Regardless of cost, such results are conclusive and may be considered in HIV high burden countries for universal access to triple antiretroviral prophylaxis in order to move towards virtual elimination of HIV MTCT.More than 6 in 10 pregnant HIV positive women with CD4 cell count of>350/mm Sub-Saharan Africa continues to record the highest burden of the HIV-1 epidemic, contributing more than 90% in the new cases of perinatally acquired HIV pediatric infections . Mother-3. Maternal baseline serum samples were quantified for HIV-1 RNA load using real Time reverse transcriptase PCR . All the samples were analyzed with the Laboratoire Centre Pasteur in Yaound\u00e9 acting as service provider at a unit cost of 30US$. ZDV was started for all the others non eligible for triple antiretroviral treatment at 14 weeks of pregnancy, while waiting for the result of the viral load.The study population was an observational cohort of pregnant HIV positive women. Pregnant women with<36 weeks of amenorrhea were enrolled in 25 antenatal clinics in the Djoungolo Health District. After opt-out counseling, serial HIV-1/2 algorithm antibody tests were done using Determine HIV-1/2 assay and SD bioline HIV 1/3 3.0  rapid kits on mothers' serum samples. For all HIV positive women, CD4 cell counts were measured in the referral laboratory. The method used a flow cytometer for measuring the percentage of CD4 T cells; an automated blood cell counter helped to measure the total number of lymphocytes. Results were produced in absolute numbers and in percentages. CD4 cell count screening was followed by non exclusive measurement of viral load when CD4 cell counts were>350/mm10 was considered as undetectable viral load. For this descriptive analysis we considered . A confidence interval of 95% was considered as the margin for percentage accuracy.The following qualitative variables were considered: maternal age, WHO clinical stages. Quantitative variables included: CD4 cell count and HIV Viral load. Viral load values were log10 transformed and stored in an Excel sheet form. A threshold of\u22641.8 logAll interventions within this project were implemented under the supervision of the District Office of the Ministry of Public Health. Data collection and monitoring of the intervention was part of the routine activities of the Health District. Free measurement of HIV1 RNA viral load was offered to all the eligible mothers at the referral center where all activities were coordinated by the Center for HAART treatment and the local board in charge of fighting against HIV and AIDS.Mothers\u2032 mean age (SD) was 27.0 years IQR 22.5\u201330 and 15/172(8.7%) were adolescents. All the mothers were classified in WHO clinical stage 1 or 2.3, IQR (442\u2013646) and 68/170 had CD4 cell counts of between 350 and 500/mm3.Median CD4 cell count was 534 cell/mmMaternal viral load and transmission10. The median log10 viral load was 4.4(IQR 3.5\u20134.9). 13%, 95% CI (7.5\u201318) had an undetectable viral load. A viral load of>3log10 was recorded for 107/145(74%); 89/145(61%) had a viral load of\u22654log10 and for 29/145  a very high level of viral load as usually observed during primary acute infection, was noted (\u22655log10).3, making them eligible for maternal triple ARV prophylaxis as required elsewhere. Taking into account that an average of 4 in 10 HIV positive pregnant women are yet to be eligible for HAART because of CD4 cell count of<350/mm3, it may be deduced that among the remaining 6 in 10, at least 4 may require HAART for prophylaxis according to their HIV viral load. Altogether 8 in 10 women at least may be eligible for HAART during pregnancy based upon biological criteria. Such results vindicate the opinion of those who claim that universal access to maternal triple ARV prophylaxis during pregnancy regardless of CD4 cell count is a must and are consistent with the latest WHO recommendations for women not yet in need of treatment for themselves, namely option B [To our knowledge, this is the first study in Cameroon where viral load is systematically determined in pregnant women in order to assess the eligibility to more effective antiretroviral regimen especially at high levels of CD4 cell count. Our key finding is that the level of HIV viral load is\u22654log 10/ml in more than 60% of HIV pregnant women, ignorant of HAART, with CD4 cell count>350/mmIn Sub-Saharan Africa, some countries have opted for triple antiretroviral therapy for all pregnant women according to the level of care . In the 10 in more than 60% newly diagnosed pregnant women in Cameroon with high levels of CD4 cell count. Such results suggest the promotion of the routine measurement of viral load during pregnancy in A option country or the switch to B option for all, in order to ensure larger access to maternal antiretroviral triple prophylaxis. This approach is likely to be more effective in lowering maternal viral load and reducing mother-to-child transmission of HIV.We concluded that antenatal serum HIV-1 RNA viral load, stands above 4log"}
+{"text": "Isolated aneurysm of subclavian or axillary artery is very rarely seen.Eight patients that were operated on between February 1998 and December 2007 due to true aneurysms of the subclavian and axillary arteries were examined. Six of the patients (75%) were male. Median age was 58 (38-73). Two of these patients had subclavian and 6 of them had axillary artery aneurysms. Median diameter of the aneurysm was 46 mm (30-60).Chief complaint was ischemic symptoms in 2 (25%) patients, non-disturbing mass in 3 (38%), feeling of compression in 2 (25%) and bleeding in 1 (12%) patient. Etiological factors include atherosclerosis in 5 patients (62%), a probable connective tissue disorder in 1 patient (13%) and idiopathic in 2 patients (25%). In these last 2 patients no atherosclerotic changes were prominent histopathologically and no other factors were identified; thus considered as idiopathic. One patient with a probable connective tissue disorder had no histopathological diagnosis. But he was phenotypically marfanoid and his past medical history was significant for surgery due to iliac artery aneurysm. Contrasted CT examination revealed no other aneurismatic segment preoperatively. Past medical history of another patient was also significant for surgery of contralateral axillary artery aneurysm. But histopathologic examination revealed atherosclerotic changes. Therefore this patient was not considered as having connective tissue disorder. One patient with ruptured axillary artery aneurysm was operated on emergently with a chief finding of bleeding.One of two cases with subclavian artery aneurysm that we operated on had acute ischemia findings of the upper extremity and the other had a non-disturbing mass. Consistent with the literature, 2 of our cases with axillary artery aneurysm had non-disturbing mass, 2 had feeling of compression, 1 had active bleeding and 1 had findings of acute ischemia."}
+{"text": "Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived \u2212/\u2212/Crb1Rd8/RD8, Cx3cr1\u2212/\u2212/Crb1Rd8/RD8CCl2 and \u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1Rd8/RD8CCl2 mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of  Mononuclear phagocytes  are part of the myeloid cell lineage and are effector cells of the innate immune system. These cells all express CX3CR1 and are thought to be derived from different circulating monocyte populations CX3CR1 as a genetic risk factor for AMD An important role of these two chemokine pathways in AMD has been supported by the identification of variants of the chemokine receptor gene Ccl2, Ccr2 or Cx3c1 have reported that the abnormal trafficking and function of macrophages and microglia in these models is associated with a variable degree of age-related retinal and RPE degeneration Several conflicting studies on single chemokine knockout mice for Ccl2, Ccr2 and Cx3cr1 knockout mice shows that chemokine signalling defects lead to dysfunctional macrophages in the retina. However, the contributory role of these dysfunctional macrophages to photoreceptor or RPE degeneration is not clear. Some groups have not observed significant age-related retinal and RPE degeneration or any spontaneous CNV in Ccl2 knockout mice Ccl2 and Ccr2 single knockout mice Cx3cr1 knockout mice, accumulation of subretinal macrophages/microglia is associated with a marked, progressive age-related retinal degeneration. This suggests that Cx3cr1 signalling may play a more pronounced role for survival of photoreceptors and the RPE, while the role of Ccl2 in this process remains unresolved Ccl2/Cx3cr1 double knockout mice have been described as developing a more accelerated and severe, early onset retinal phenotype with high penetrance of rapid photoreceptor loss and RPE defects, which suggested that the combined knockout of both chemokine signalling pathways might act synergistically and may thus lead to an early onset retinal degeneration The age-related accumulation of subretinal macrophages in Ccl2 and Cx3cr1 signalling pathways may contribute to the development of the early onset retinal degeneration, we aimed to define primary and secondary pathological events during the age-related progression of the degeneration in CCDKO mice between 2 weeks and 22 months of age Ccl2, single Cx3cr1 and double Ccl2/Cx3cr1 knockout mice from the original CCDKO mouse line by backcrossing with C57Bl/6 mice. We housed all these lines under same lighting conditions in the same room to control for environmental factors including pathogen burden and in addition raised some CCDKO mice in darkness from birth. Through these experiments, we identified the primary pathological event in CCDKO mice in the outer nuclear layer of the retina and established the RD8/RD8Crb1 mutation as a third independent autosomal recessive locus as the cause for the early onset retinal degeneration which was not dependent on light. Furthermore, we observed differential modulatory effects of the genetic background, as well as of both chemokine signalling pathways on the manifestation of the early onset retinal degeneration and therefore show that CCDKO mice are not a model for AMD pathology. These finding also highlight a differential modulatory role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling for retinal degeneration.In order to understand the reported phenotypic discrepancies in the different chemokine knockout mouse models and to study how defects in the two Ccl2/Cx3cr1 double knockout mice (CCDKO mice) used in this study were derived from two breeding pairs  that we obtained from the original line as described by Tuo et al. and were thankfully provided by Chi-Chao Chan and Jingsheng Tuo C57Bl/6 background C57Bl/6J OLA Hsd mice  that were imported as young adult mice at 6\u20138 weeks of age and housed in the same animal rooms next to CCDKO mice. For backcrossing experiments, CCDKO mice from our original homozygous line and C57Bl/6J Ola Hsd mice were used as founder animals (F0). Obtained offspring (F1) that were heterozygous for both, the Ccl2 and the Cx3cr1 alleles were interbred to obtain the F2 generation. F2- animals were genotyped for both cytokine loci and interbred with each other to establish new lines for all combination of cytokine genotypes that include homozygous wildtype (+/+/Cx3cr1+/+Ccl2) mice, new single knockout mice for Ccl2 (\u2212/\u2212/Cx3cr1+/+Ccl2) or Cx3cr1 (+/+/Cx3cr1\u2212/\u2212Ccl2) as well as a new double knockout line for both Ccl2 and Cx3cr1 (\u2212/\u2212/Cx3cr1\u2212/\u2212Ccl2).As control animals we used age-matched in vivo procedures, mice were anesthetized by a single intraperitoneal (IP) injection of a mixture of medetomidine hydrochloride , and ketamine (60 mg/kg body weight) in water. Whenever necessary, the pupils were dilated with 1 drop of 1% tropicamide. The animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and under the UK Home Office project licence (PPL 70/1279).For Ccl2 and Cx3cr1 respectively  using primer combinations distinguishing the wildtype and knockout alleles for ectively  [44]. Gequencing .Autofluorescence imaging was performed using a HRA2 scanning laser ophthalmoscope with a 55\u00b0 angle lense as described previously To phenotype offspring during backcrosses for retinal degeneration, we used fundus images focused on the inner retinal layer and evaluated the phenotype of the animals qualitatively using the red free (barrier filter at 500 nm) and the autofluorescent channels of the HRA2. To more quantitatively evaluate the severity of the phenotype, we subsequently counted the number of autofluorescent lesions per fundus image in the autofluorescent images for each animal after these had been processed in Adobe Photoshop CS 2  using the auto level function. These counts include not only the typical disciform autofluorescent lesions from the inferior retina, but also include any autofluorescent signal from the whole fundus image including those from the superior retina.OCT imaging and fundus fluorescein angiography were performed during the same session using either the Spectralis\u2122 HRA+OCT  for OCT imaging or the HRA2 for fundus fluorescein angiography. First OCT images were obtained from the animals using OCT+IR or OCT+AF channels to correlate the retinal position with the obtained two-dimensional optical section obtained by the OCT. Subsequently, the same animals were injected intraperitoneal with 200 \u00b5l of 2% fluorescein in phosphate buffered saline (PBS) and fundus fluorescein angiography images were taken using the autofluorescent channel of the HRA2 equipped with an Argon laser at 488 nm wavelength. Obtained 2-dimensional OCT images and autofluorescent fundus images were exported and processed in Adobe Photoshop CS 2 .Semithin histological morphometric analyses were performed as described previously CCDKO and C57Bl/6 wildtype mice between 2 weeks and 22 months of age were assessed under bright field microscopy using a 100\u00d7 objective and a 10\u00d7 ocular lense. Singular pathological events in the retina (e.g. misplacement of nuclei from the outer nuclear layer toward the inner or outer retina or disorganized photoreceptor columns) and in the RPE  were counted form the superior to the inferior end of the retina in each section. From these individual sums of damages per section we calculated the mean sum of outer nuclear and RPE damage from three sections per animal to obtain a representative measure of outer retinal or RPE damage score per animal similar to a scheme described by Hollyfield et al. and us To evaluate the pathological changes in the retina and the RPE over time sections from age-matched For 3-D analyses retinae fixed in 3% glutaraldehyde and 1% paraformaldehyde buffered to pH 7.4 with sodium cacodylate were rinsed in buffer, and then osmicated, en bloc stained with uranyl acetate and Waltons lead citrate using the method of West et al. Regions of interest (ROI) were cut from resin blocks following normal surveying by LM and TEM of toluidine blue stained semithin sections and unstained ultrathin sections. The isolated ROI's were then superglued onto a Leica cryopin, re-trimmed to place the region of interest within a mesa of height \u223c1 mm and side 0.5 mm and sputter coated with 5 nm gold palladium. Next, the specimen was locked into the specimen holder of the Gatan 3 View ultramicrotome mounted to the opened chamber door of a Zeiss Sigma variable pressure field emission scanning electron microscope and the diamond knife advanced until the full face of the block was being sectioned at 200 nm increments. At this point the microscope was evacuated and the block face imaged using Gatan's low voltage backscatter detector at 2\u20134 kV and chamber pressure of 10\u201330 Pa to suppress charging artifacts. Finally the pixel dwell time, magnification and chamber pressure were optimised to yield a focused ROI and the microtome programmed to automatically cut a maximum of 999\u00d7100 nm thick sections with intervening image acquisitions. In this way a stack of 999 images spanning an axial distance of 99.9 \u00b5ms were digitally acquired at 4096\u00d74096 pixel resolution in Digital Micrograph format. Three-dimensional reconstruction and labeling of the vascular lumen and Bruch's membrane was obtained using the Amira 5.3.3 software .Eyes for retinal and RPE/choroidal flat mounts were briefly fixed in 4% paraformaldehyde (PFA)/PBS before dissection and post-fixed again in 4% PFA/PBS for a total of 1 hour. After blocking with PBS/1% BSA /5% nonspecific goat serum  including 0.3% Triton X-100 for permeabilisation for 1 hour the flat mounts were incubated over night at 4\u00b0C with a 1\u2236500 dilution of anti Iba1 antibody  in blocking solution to label microglia and with primary TRITC-conjugated lectin at a 1\u223610 dilution  to label endothelial cells. After washing with 3\u20134 times with PBS, 1\u2236500 dilution of goat anti-rabbit AlexaFluor 488 nm-conjugated secondary antibody  was used to visualize the Iba1 antibody. After washing three times with PBS, retinal and RPE/choroidal flat mounts were mounted with fluorescence mounting medium containing Hoechst 33342  and images were obtained using a confocal laser scanning microscope . The obtained Z-stack images were processed for 3D data visualization using Imaris software .Statistical analyses were performed using GraphPad Prism 5 for Windows .CCDKO line C57Bl/6 mice between 1 month and 22 months of age. Autofluorescent fundus imaging by scanning laser ophthalmoscopy (AF-SLO) demonstrated faint abnormal autofluorescent fundus lesion at 1 months of age  and the outer plexiform layer (OPL) which corresponded spatially to the disciform autofluorescent fundus lesions in the inferior retina  or in complete darkness (luminescence <0.5 lx). As an additional control group we included wildtype C57Bl/6 mice raised under 12 h/12 h light/dark cycle conditions. Both groups of CCDKO mice showed a very similar inferior localisation of autofluorescent lesions at 8 weeks of age  do not modulate the retinal phenotype in CCDKO mice significantly during the first 8 weeks of life.The inferior localization of the retinal degeneration observed in s of age , while ce groups . We did CCDKO mice we compared the localisation of AF-SLO and OCT lesions (C57Bl6 and supplementary CCDKO).To further characterise the nature of the autofluorescent lesions in  lesions  with thaCCDKO mice demonstrated relatively normal ramified microglia which showed a slightly more swollen cell body than microglia in C57Bl/6 mice at the same retinal layer (white arrows in CCDKO) versus 3A (C57Bl/6)). In the outer retina, however, CCDKO mice revealed microglia cells that have migrated into the outer nuclear layer (CCDKO) and are positioned around circular columns of photoreceptor nuclei dropping out of the ONL  or the inner segment (IS) area (C57Bl6). The observed degenerating columns of photoreceptor cells in CCDKO mice together with the surrounding recruited microglia correspond in location, size and configuration well with the disciform shape of the autofluorescent fundus lesions observed in AF-SLO images  area C57Bl6. CCDKO mice, we examined superior-inferiorly-oriented sagittal semithin sections for all age groups of CCDKO mice and control C57Bl/6 mice. CCDKO mice demonstrate an early mislocalisation of photoreceptor nuclei in the inferior outer nuclear layer at two weeks of age indicating a very early onset of retinal changes in the inferior outer retina . The age-dependent increase in RPE damage in CCDKO mice was significantly higher compared to C57Bl/6 mice indicating a disease related increase of RPE damage in CCDKO mice  bright field microscopy revealed grey spots in the inferior retina of 1 months old CCDKO animals , Cx3cr1 single knockout mice +/+/Cx3cr1\u2212/\u2212Ccl2), and the re-derived Ccl2/Cx3cr1 double knockout mice (\u2212/\u2212/Cx3cr1\u2212/\u2212Ccl2) we established affected and an unaffected mouse lines that either exhibit the typical autofluorescent fundus lesions at the age of 8 weeks or not  allowed us to establish 6 independent chemokine knockout mouse lines. For each chemokine genotype including s or not . To ensus or not . This stCrb1-RD8 mutation in all affected lines, allowed us to subsequently use these lines to assess the differential modulatory effect of the genetic background and the two chemokine loci, Ccl2 and Cx3cr1, on the phenotype of the RD8 retinal degeneration in offspring from the F2 generation. As a quantitative measure for the retinal degeneration phenotype, we decided to use the number of autofluorescent fundus lesions in AF-SLO fundus images at the age of 8 weeks  that these fluorescent fundus lesion are an early in vivo indicator for typical primary pathological events in the outer retina which led to the recruitment of microglia. Thus, assessing the number of these lesions might be a good measure for a potential modulatory effect of the chemokine knockouts on the retinal degeneration phenotype in vivo.The genetic proof of a third autosomal recessive locus by breeding, genotyping and phenotyping of the lines as well as the presence of  8 weeks . We haveCCDKO line showed significantly higher number of autofluorescent fundus lesions than normal C57Bl/6 mice and all other 6 newly established chemokine knockout mouse lines (Ccl2 or Cx3cr1 as well as the newly established \u201caffected\u201d chemokine double knockout mouse (Ccl2\u2212/\u2212/Cx3cr1\u2212/\u2212/RD8/RD8Crb1), which has the same genotype as the original CCDKO line. This clearly indicates that the retinal degeneration caused by a homozygous RD8/RD8Crb1 mutation in these mouse lines is attenuated by an increasing C57Bl/6 genetic background.At 8 weeks of age, the original RD8/RD8Crb1 mutation indeed causes the early onset retinal phenotype since all mouse lines that carry the RD8/RD8Crb1 mutation  that do not carry the RD8/RD8Crb1 mutation  and \u2212/\u2212/Crb1RD8/RD8Cx3cr1 mice (Cx3cr1\u2212/\u2212 (affected) F3) both showed similar numbers of autofluorescent fundus lesions, which was for both lines significantly higher that the number of autofluorescent fundus lesions in \u2212/\u2212/Crb1RD8/RD8Ccl2 mice (Ccl2\u2212/\u2212 (affected) F3.By comparing the affected mouse lines with each other we observed that the re-derived \u2212/\u2212/Crb1RD8/RD8Cx3cr1) or the double chemokine knockout (\u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2) leads to a more pronounced degenerative phenotype of the RD8 mutation than the RD8 mutation in combination with the knockout of Ccl2 (\u2212/\u2212/Crb1RD8/RD8Ccl2) alone. Furthermore this also shows, that there is no significant additive affect of knocking Ccl2 out in addition to Cx3cr1, since the double chemokine knockout of Ccl2 and Cx3cr1 (\u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2) is not significantly increased compared to the single knockout of Cx3cr1 (\u2212/\u2212/Crb1RD8/RD8Cx3cr1) (This suggests that absence of Cx3cr1 in either the single (RD8/RD8) .Ccl2 signalling alone might also have a modulatory effect on the RD8 retinal degeneration, we evaluated littermates from the backcrosses that all carried the RD8/RD8Crb1 mutation but were either homozygous wildtype (+/+Ccl2/RD8/RD8Crb1), heterozygous (+/\u2212Ccl2/RD8/RD8Crb1) or homozygous knockout (\u2212/\u2212Ccl2/RD8/RD8Crb1) for the Ccl2 locus . All offspring with any of the Ccl2 genotype combinations showed a significantly smaller number of autofluorescent lesions in the fundus compared to the original CCDKO mice , littermates from the backcross that carried the homozygous RD8 mutation, but were wildtype (+/+/Crb1RD8/RD8Ccl2) or heterozygous (+/\u2212/Crb1RD8/RD8Ccl2) for Ccl2 showed a significantly higher number of lesions in the fundus, while Ccl2 deficient littermates (\u2212/\u2212/Crb1RD8/RD8Ccl2) show no significant difference compared with all three groups at 8 weeks of age. Therefore this data suggests that Ccl2 does not have a significant effect on the manifestation of the early onset retinal degeneration in mice that carry a homozygous RD8 mutation, although we cannot exclude a mildly protective effect of Ccl2 deficiency.To clarify, whether absence of l2 locus . These aDKO mice . This re alleles . CompareCcl2/Cx3cr1 double knockout (CCDKO) mouse line and to understand whether and how these two chemokine pathways contribute to the retinal degeneration CCDKO mouse line CCDKO line and suggested an involvement of M\u00fcller cells in disease progression CCDKO knockout mouse lines as well as in all re-derived \u201caffected\u201d Ccl2 and Cx3cr1 and Ccl2/Cx3cr1 double knockout mice, is caused by a third independent autosomal recessive locus. Re-derived Ccl2 or Cx3cr1 single knockout lines and re-established Ccl2/Cx3cr1 double knockout mice which do not carry the third locus exhibited no signs of an early onset retinal phenotype. This indicates that neither Ccl2 nor Cx3cr1 deficiency alone nor the combined double knockout of both chemokines leads to a pronounced early onset retinal degeneration. In addition we showed by genomic sequencing that the naturally occurring homozygous RD8 mutation co-segregates with the early onset retinal phenotype in all affected chemokine lines. This naturally occurring RD8 (RD8/RD8Crb1) mutation is a single base pair deletion of a cytosine in exon 9 of the Crb1 gene that causes a frame shift and premature stop codon in the Crb1 protein which lead to a truncated transmembrane and cytoplasmic domain of Crb1 RD8/RD8Crb1 mice leads to very similar phenotypic features as we have observed in CCDKO mice. These include the early onset inferior fundus lesions, a discontinuous outer limiting membrane, an early dropout of nuclei from the outer nuclear layer as well as the subsequent focal age-related retinal dysplasia which is secondarily associated with vascular lesions in the outer retina CCDKO mice is independent from light is consistent with previous data for \u2212/\u2212Crb1 knockout mice. These mice, when raised in complete darkness for 6 months also show a similar degree of retinal degeneration as \u2212/\u2212Crb1 mice raised under normal lighting conditions (100 lux) \u2212/\u2212Crb1 mice CCDKO mice are about 100fold lower than light levels necessary to modulate the phenotype of this type of retinal degeneration. These data suggest, that the inferior location of the retinal degeneration in Crb1 mutant mice  is not due to the influence of light, but due to an unknown factor that restricts the photoreceptor degeneration to the inferior retina. These pronounced similarities in the phenotype as well as the co-segregation of the RD8/RD8Crb1 mutation with the early onset retinal degeneration in the different re-derived chemokine knockout mice indicate that the retinal degeneration observed in original CCDKO mice is the consequence of the RD8/RD8Crb1 mutation and not due to the combined double knockout of Ccl2 and Cx3cr1 as previously reported In this study, we aimed to identify primary and secondary pathological events during the previously described early onset retinal degeneration in RD8/RD8Crb1 mutation as the underlying cause for the early onset retinal degeneration in CCDKO mice enables us to better interpret the observed secondary degenerative events in original CCDKO mice. The two major secondary events observed in the \u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2 mice with age include RPE changes and closely associated vascular lesions in the area of retinal degeneration. The vascular lesions contained tortuous and dilated vessels in the area of retinal degeneration and showed an increasing penetrance of late phase hyperfluorescence in fluorescein angiography which indicates a low grade vascular inflammation and a low grade late vascular leakiness of retinal vessels within the lesions. Using serial block-face scanning electron microscopy (SB-SEM) and 3D reconstruction, we demonstrated that the retinal vessels within the degenerate area break through the RPE layer and come in close contact with Bruch's membrane, but do not penetrate it. This abnormal location of retinal vessels seems to induce a secondary response of RPE cells which migrate along those vessels into the retina and ensheath them within the RPE/vascular lesion. However, the fact that not all aged CCDKO mice showed the occurrence of the late phase hyperfluorescence in the fluorescein angiography suggests that the manifestation of this vascular phenotype is variable and thus likely not a primary consequence of the RD8 mutation, but rather a secondary process dependent on the individual severity of the progression of the retinal degeneration. Based on the clinical observations  as well as on the basis of our ultrastructural observations, that vessels of retinal origin grow underneath the RPE but do not penetrate Bruch's membrane, we propose that the vessels within the RPE/vascular lesions in the \u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2 mice are similar to retinal telangiectasia rather than choroidal neovascularisation, which was reported to occur in about 10\u201315% of the original CCDKO mouse line above the age of 3 months CCDKO mice The identification of the CCDKO mice are also very similar to those in aged RD8/RD8Crb1 mice RD8/RD8Crb1 mice might show similar vascular lesions as described here and do not develop CNV. Our data supports the view that vascular lesions in patients with Crb1 mutations, that show Coat's-like exudates in the inferior region of the retina, are likely to be more similar to retinal telangiectasia with similar vascular features as observed in this study, than choroidal neovascularisation RD8/RD8Crb1 mice as well as in patients with Crb1 mutations. A similar process in which retinal vessels are located adjacent to Bruch's membrane and are covered by RPE cells has been described in late stage retinal degeneration in rhodopsin knockout mice Crb1, but might be more common during late stages of other severe retinal degenerations.Since the late stage secondary changes in CCDKO mice to a C57Bl/6 genetic background attenuates the observed retinal degeneration in CCDKO mice. The data suggest that the genetic background of C57Bl/6 is a strong modulator of the manifestation of retinal degeneration in all affected chemokine knockout mice carrying the RD8/RD8Crb1 mutation and indicates that other genetic factors influence the observed phenotype. The presence of other genetic modifiers is also supported by the high degree of phenotypic variability observed within each affected mouse lines. Our findings are consistent with the original characterisation of RD8/RD8Crb1 mice, which demonstrated that the retinal degeneration is strongly modulated by backcrosses into C57Bl/6, CAST/EiJ or C3HfB6/Ga mice. Interestingly, only the discontinuous outer limiting membrane phenotype in RD8/RD8Crb1 mice remained preserved after backcrossing RD8/RD8Crb1 mutation in \u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2 mice in this study as well.Our data demonstrates that genetic backcrossing of the Ccl2 and/or Cx3cr1 chemokine signalling differentially modulates the severity of the retinal degeneration caused by the RD8/RD8Crb1 mutation at 8 weeks of age. This is supported by the observation that all Cx3cr1 deficient lines that carry the RD8/RD8Crb1 mutation, including the \u2212/\u2212/Cx3cr1\u2212/\u2212/Crb1RD8/RD8Ccl2 and \u2212/\u2212/Crb1RD8/RD8Cx3cr1 mice, exhibit a more severe retinal degeneration compared to the \u2212/\u2212/Crb1RD8/RD8Ccl2 line of the same backcross generation and age. In contrast, Ccl2 deficiency (\u2212/\u2212/Crb1RD8/RD8Ccl2) alone does not significantly influence the retinal degeneration caused by the RD8/RD8Crb1 mutation, although we can not exclude a slight protective effect of it based on the trend for a reduced number of autofluorescent lesions in Ccl2 deficient mice (\u2212/\u2212/Crb1RD8/RD8Ccl2) compared to heterozygous (+/\u2212/Crb1RD8/RD8Ccl2) or wildtype (+/+/Crb1RD8/RD8Ccl2) littermates. Ccl2 deficiency also does not significantly add to the exacerbated retinal degeneration seen in \u2212/\u2212/Crb1RD8/RD8Cx3cr1 mice suggesting that the effects of Ccl2 and Cx3cr1 signalling defects do not act synergistically on the manifestation of the retinal degeneration in RD8/RD8Crb1 mice. A modulatory influence of either of the two chemokine pathways and in particular of Cx3cr1 signalling on retinal degenerations, is further supported by the observation that Iba1+ microglia cells, which express Cx3cr1 in the retina Our data demonstrates that the genetic inactivation of either CCDKO line and in other affected chemokine lines in this study, the defective formation of the outer limiting membrane due to mutations in Crb1 acts as an endogenous trigger for the local upregulation of inflammatory mediators. This hypothesis is consistent with the increased expression of Cx3cl1 in retinal and choroidal vascular endothelial cells, M\u00fcller cells, RPE as well as photoreceptors after inflammatory processes Ccl2 in M\u00fcller cells within affected retinal areas after focal light injury and after retinal detachment Ccl2 as well as Cx3cl1 provide chemotactic cues for the local recruitment of microglia and systemic monocytes to the primary focal lesion site Cx3cr1 deficient mice in combination with the RD8/RD8Crb1 mutation, or may act mildly protective as eventually indicated by the trend towards a slight reduction in lesion size in Ccl2 deficient mice which also carry the RD8/RD8Crb1 mutation.Our findings support the hypothesis that in original Cx3cr1 knockout mice in the brain, where an increased neurotoxicity of brain microglia on surrounding neurons has been observed after systemic inflammation induced by LPS Cx3cr1 deficiency leads to a reduced dynamic behavior of retinal microglia during immune surveillance as well as during injury response Cx3cr1 deficient microglia can be neurotoxic in the retina as well and have the capacity to act as modulators of retinal degeneration as suggested by our observation. Therefore, we hypothesise that the additional neurotoxic effect of Cx3cr1 deficiency on the retinal degeneration caused by the RD8/RD8Crb1 mutation might be due to an impaired migratory capability and a subsequent reduced removal of dying photoreceptors by Cx3cr1-deficient microglia which might result in the production of more neurotoxic inflammatory mediators including Ccl5, TNFa or IL-6, which may negatively affect photoreceptor cell survival, and have all been previously shown to be upregulated in CCDKO mice Cx3cr1 deficiency, Ccl2 deficiency does not significantly modulate the manifestation of the RD8/RD8Crb1 phenotype. This minor role of Ccl2 on the RD8 retinal degeneration seems consistent with our previous observation that Ccl2 deficiency does not lead to a significant associated age-related retinal degeneration despite an accumulation of subretinal, dysfunctional macrophages Ccl2 in this process or by a compensatory effect of other chemokine ligands that can bind to and activate CCR2 \u2212/\u2212/Crb1RD8/RD8Ccl2 mice may suggest a mild protective effect of Ccl2 deficiency, which would be in line with Ccl2's role for the transendothelial migration of pro-inflammatory monocytes across the blood brain barrier Ccl2 and Ccr2 deficiencies for the formation of atherosclerosis that lead to a reduction of the formation of foam cells due to a reduced recruitment of systemic myeloid cells to the site of the lesion in the periphery Further support for this idea comes from convincing evidence for a microglia-mediated neurotoxicity in Ccl2 and Cx3cr1 chemokine signalling for the inherited retinal degeneration caused by the RD8/RD8Crb1 mutation and suggests that these two chemokine pathways not only modulate age-related degenerative processes or acute inflammation in the retina, but also contribute to the pathology of inherited retinal degenerations. Therefore it seems likely that these two chemokine signalling pathways and maybe the innate immune status of myeloid cells might act as a modifier for CRB1 mutations in humans and thus contribute to the high phenotypic variability observed in patients with different CRB1 mutations that either lead to Lebers congenital amaurosis (LCA), early onset childhood retinal dystrophy or juvenile onset retinitis pigmentosa CCL2 levels and reduced expression of Cx3cr1 on non-classical monocytes in aged probands as well as further increased levels of CCL2 in aqueous humor samples from patients with exudative AMD CX3CL1-CX3CR1 signalling to increased CCL2-CCR2 signalling systemically with age which might contribute to an increased susceptibility to retinal degeneration with age and during disease progression of multi-factorial age-related retinal diseases including AMD and diabetes. Understanding further the role and interaction of chemokine signalling pathways for the control of monocytes and microglia cell in degenerative processes of the retina might help to develop more targeted therapeutic approaches to modulate the progression of inherited and other age-related, multi-factorial retinal degenerations.Taken together, our study provides evidence for a differential modulatory role of Movie S1C57Bl/6. Animation of 3D-reconstructions (IMARIS) of confocaol Z-stack images taken from the inferior area of a retinal flat mount of a C57Bl/6 mouse at 2 months of age. The volume of the z-stack  comprises the deep retinal vascular plexus, the outer nuclear layer and part of the inner segments. The deep retinal vascular plexus was labeled with Tritc-lectinB4 and is represented in red, while retinal microglia labeled with Iba1 are shown in green. At the top of the z-stack microglia located at the level of the deep retinal vascular plexus show a normal ramified morphology. These cells are not present in the outer retina. The red and green Imaris surfaces in the area of the inner segments at the bottom of the z-stack represent autofluorescent signals from photoreceptors.(AVI)Click here for additional data file.Movie S2CCDKO. Animation of 3D-reconstructions (IMARIS) of confocaol Z-stack images taken from the inferior area of a retinal flat mount of a CCDKO mouse at 2 months of age. The volume of the z-stack  comprises the deep retinal vascular plexus, the outer nuclear layer and part of the inner segments. The deep retinal vascular plexus was labeled with Tritc-lectinB4 and is represented in red, while retinal microglia labeled with Iba1 are shown in green. At the top of the z-stack microglia located at the level of the deep retinal vascular plexus show thicker cell bodies compared to wildtype. In addition microglia are also observed in the outer retina in distinct columns. These microglia columns are observed in areas were the inner segment autofluorescence (at the bottom of the z-stack) is absent indicating altered photoreceptor positions or drop out.(AVI)Click here for additional data file."}
+{"text": "We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. Premature labor is the leading cause of neonatal mortality, however to date, few effective and broadly applicable interventions are available for the prevention of preterm birth Due to the potential importance of uterine CASP3 activity in regulating gestational length, this study has focused on determining the factors that may regulate uterine CASP3 during pregnancy with the hope that we can ultimately understand the mechanism by which uterine quiescence across is maintained across gestation, allowing pregnancies to be carried to tern. We speculate that by determining the factors that allow the pregnant uterus to transition from a quiescent to a contractile phenotype in normal term labor, that we may gain insight into the mechanisms that are perturbed allowing for the onset of pre-term birth. In this study we have defined that the uterine myocyte endoplasmic reticulum stress response (ERSR) and its related unfolded protein response (UPR) are the likely regulators of uterine myocyte CASP3 activity during pregnancy. Physiological stimuli such as fluxes in protein synthesis, cellular differentiation, hypoxia and glucose deprivation have been described to result in the accumulation of misfolded proteins and perturbation of ER homeostasis resulting in the activation of the ERSR and its adaptive UPR We propose that physiological and potential uterotonic stimuli that the pregnant uterus experiences across gestation may potentiate CASP3 activation through the ERSR thereby maintaining the pregnant uterus in a quiescent state. However, towards the end of pregnancy we suggest that increased activation of the adaptive UPR limits the uterine ERSR, thereby reducing CASP3 apoptotic potential resulting in its gestational decline towards term. Ultimately these events initially limit the uterine myocyte contractile potential from early to mid-gestation through CASP3 activation, however towards term they permit reconstitution of the uterine myocyte contractile architecture.In summary this study examined three potential signaling pathways in the pregnant mouse uterus that may have the capacity to regulate CASP3 activity. Utilizing a gestational series of mouse uteri from E6\u2013E19 we examined the presence of the ERSR in the pregnant uterus at the transcriptional and translational level. The traditional canonical intrinsic and extrinsic pathways, which initiate through transmembrane receptors at the cell surface or through the cytoplasmic release of the mitochondrial protein cytochrome C (CYCS), respectively All chemicals were obtained from Sigma Aldrich, St Louis MO unless otherwise indicated. Recombinant mouse Bid (truncated and full length) (Cat# Pro-644) was purchased from ProSpec-Tany Technogene Ltd.  and recombinant active caspase 8 (CASP8) (Cat# CC123) was obtained from Millipore Inc, .All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Timed-pregnant female CD-1 mice (6\u20138 weeks old) were obtained from Charles River and housed according to IACUC guidelines. Uterine tissues  were harvested between 0800\u20131000 h on E6, E8, E10, E11, E12, E13, E15, E16, E17, E18 and E19. The uterine horn was cleared of all embryonic material and maternal decidua. The isolation of mitochondrial and cytosolic fractions, were performed on fresh uterine tissues, processed immediately after harvest as described below. For immunofluorescence studies, the uteri were fixed in 4% PFA overnight and subsequently embedded in paraffin blocks for sectioning. The remaining uterine tissue was washed in 1\u00d7PBS and flash frozen for subsequent protein and mRNA analysis.2, 5 mM KCL, and 0.1% Triton \u00d7-100) and 1\u00d7protease/phosphatase inhibitor cocktail (Complete Mini EDTA free/PhosStoP)  using an IKA homogenizer . The homogenate was centrifuged at 5000 RPM for 10 min at 4\u00b0C, and the supernatant was retained as the cytoplasmic fraction. The pellet was washed in NE1 and then resuspended in ice-cold NE2 buffer , 500 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA (pH 8.0)) with 1\u00d7protease/phosphatase inhibitor cocktail and was incubated on ice for 30 min, vortexed every 5 min, and centrifuged at 10,000 RPM for 10 min at 4\u00b0C. The supernatant was retained as the nuclear fraction. Our previous published data in the human myometrial cell Cytoplasmic and nuclear protein extracts were prepared from frozen uterine tissue as described previously In order to isolate pure preparations of intact mitochondria and overcome problems associated with mitochondrial rupture in frozen tissues, we used freshly harvested uterine tissue for subcellular fractionation. The fresh mitochondrial and cytosolic fractions  were isolated from uterine tissues using a protocol modified from previously described methods Equal amounts of protein were loaded on NuPAGE pre-cast gradient gels  and transferred on to Hybond-P PVDF membranes . The membranes were blocked in 5% non-fat milk prepared in 1\u00d7 Tris Buffered Saline with Tween-20 for 1 hr at room temperature and then incubated with the primary antibodies overnight. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 5% milk-1\u00d7TBS-T buffer. Immunoreactive bands were visualized using an ECL detection system (ThermoScientific Rockford IL). The concentrations of primary antibodies and their sources are as follows, Cell Signaling Technologies -HSPA5 (#3177 1:1000), DDIT3 (#5554 1: 200), full length and cleaved CASP12 (#2202 1:500), PDI (#3501 1:1000), full length and cleaved CASP3 (#9665 1:1000), COX4I1 (#4850 1:1000), GAPDH (#2118 1:2000), full length and truncated BID (#2003 1:500); Santa Cruz Biotechnology \u2013 CYCS (sc-7159 1:1000), XBP1 (sc-7160 1:500). Thermo Scientific \u2013 NCOA3 (#PA1-845 1:2000). The immunoreactive cytoplasmic, nuclear, cytosolic and mitochondrial bands obtained by immunoblotting were quantified using ImageJ (NIH Bethesda MD) and normalized to PDI, NCOA3, COX4I1 and CYCS, respectively as their protein concentrations were found to remain relatively unchanged in the pregnant mouse uterus across gestation.Paraffin embedded uterine tissues were sectioned at 5 \u00b5m thickness and collected on Superfrost Plus slides . Paraffin sections were deparaffinized in xylene and rehydrated through an alcohol series. For DDIT3 and HSPA5, antigen retrieval was performed by boiling the sections for 15 minutes in 10 mM citrate buffer pH 6.0. For cleaved CASP3 immunohistochemical analysis, uterine tissue sections were incubated with proteinase k (10 \u00b5g/ml) for 10 minutes at room temperature. Tissue sections were then blocked in 1 X PBS containing 5% normal donkey serum and incubated in the primary antibody overnight at 4\u00b0C at the following concentrations DDIT3 1:100, cleaved CASP3 , HSPA5 1:100. Sections were then washed in 1\u00d7PBS and incubated in the fluorescent secondary antibody for 1 hr at room temperature. Donkey anti-rabbit secondary antibodies conjugated to either Alexa flour 488 or Cy3  were used at a dilution of 1\u2236500 in 1\u00d7PBS. The sections were then washed and incubated with 4\u2032,6-diamidino-2-phenylindole (DAPI) to stain nuclei and mounted in gelvatol. Images were collected at 63\u00d7 on a Leica DMRBE  using a Q-Imaging Micro Publisher 5.0 RTV . Serial sections were used for immunoflourescence as differences in antigen retrieval methods for the CASP3 and DDIT3 antibodies precluded dual immunofluorescence analysis. Companion-blocking peptides were utilized to pre-absorb each primary antibody, at each gestational stage utilized in this study, thereby acting as negative controls for auto-fluorescence and false positives.Rplp0 was used as a housekeeping gene as we have found Rplp0 levels to remain stable across gestation in the pregnant mouse uterus Rplp0. Expression data were analyzed using the \u0394\u0394Ct method RNA was isolated from pulverized uterine tissues using the RNeasy Mini Kit  according to the manufacturer\u2019s instructions. Total RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit . Primers were designed using NCBI Primer Blast to obtain amplicons from 50\u2013170 bp. . Statistical analysis of immunoblots and Q-PCR were performed with StatPlus:mac software 2009 version . Data are represented as mean \u00b1 standard error (SE) of the mean. The results were subjected to a one-way ANOVA followed by pairwise comparison (Student-Neuman-Keuls method) to determine differences between groups. Significance was set at P<0.05.Cytoplasmic proteins were isolated from uterine tissues of pregnant mice from gestation E6 to E19  as described in the Materials and Methods. To determine the levels of full length and active CASP3 immunoblotting was performed . As seenThe hallmark of an active extrinsic caspase signaling pathway is the appearance of cleaved CASP8 and tBID Downstream from CASP8 in the extrinsic pathway of caspase activation is the cleavage of the Bcl-2 family protein, BID To examine whether uterine CASP3 activation occurred through the intrinsic caspase activation pathway, we determined whether the pregnant uterine myocytes released the 15 kDa CYCS from the mitochondria to the cytosol indicating a loss of mitochondrial integrity Cytoplasmic and nuclear protein extracts isolated from pregnant mouse uterine tissues from E6\u2013E19 were examined for markers of the ERSR by immunoblotting. The presence and gestational regulation of the transcription factor DDIT3, a marker for activation of ERSR The expression of the chaperone HSPA5, a marker of the adaptive UPR To determine the localization of DDIT3, active CASP3 and HSPA5 in the pregnant mouse uterus across gestation , we performed immunofluorescence analysis on formaldehyde fixed paraffin embedded sections of mouse uterine tissues as described in the Materials and Methods. Immunohistochemical analysis of DDIT3 confirmed its nuclear localization in uterine myocytes of the pregnant mouse uterus  and thouAtf4, Ddit3, Casp12, Atf6 and Hspa5 across gestation in the pregnant mouse uterus was performed utilizing Q-PCR analysis. ATF4 is a transcription factor that is involved in the induction of Ddit3 expression occurring as a result of ER stress Atf4 and Ddit3 transcript did not significantly change through the duration of pregnancy. Levels of full length Casp12 demonstrated approximately a 1.4-fold induction at E6 and E8 in comparison with the rest of gestation but this increase was not statistically significant . Due to poor sensitivity of commercially available antibodies we were unable to perform immunohistochemical or immunoblotting analysis for ATF4 or ATF6.Examination of the mRNA levels of nificant . We alsonificant . Hspa5 eWe hypothesize that activation of the ERSR and the resulting UPR in the uterus are central in the non-apoptotic triggering and management of uterine CASP3 activity during pregnancy. We have previously determined that uterine CASP3 activation during pregnancy may have the capacity to effectively regulate the timing of parturition, through modulation of the uterine myocyte contractile architecture The first indication that ER stress may act as a potential mechanism controlling uterine CASP3 came from the observation that although we had robust CASP3 activity we were unable to detect its mechanism of activation through either the classic intrinsic or extrinsic apoptotic signaling pathways , 3 and 4Atf4, DditT3 and Casp12 in the pregnant mouse did not reveal any significant changes across gestation (Hspa5 mRNA levels across gestation (Atf6, which has been shown to induce Hspa5 expression. Elevated levels of HSPA5 assist in clearing the backlog of accumulated proteins thereby in the context of the pregnant uterus suggest an active resolution of the uterine ERSR occurs towards term. Immunohistochemical analysis seen in Recently an alternate, mitochondrial independent mechanism of CASP3 activation has been described, where CASP3 is cleaved and activated as a direct result of ER resident CASP12 activation. estation , confirmestation . Chaperoestation . We alsoestation  mirroredestation . We alsoin vivo. We speculate that in the instances of pre-term birth an inappropriate ERSR or an inability to host an appropriate UPR may trigger a precocious withdrawal of uterine CASP3 allowing for a premature increase in uterine contractile responsiveness. With the growing recognition of an association of ER stress with human disease and with increased understanding of the fundamental mechanisms regulating ER stress, novel targets for drug discovery and new strategies for therapeutic preventative intervention are beginning to emerge and may ultimately be utilized to reveal indicators for and the resolution of pre-term birth.In summary, we propose that throughout pregnancy the uterine myocyte must monitor, tolerate and adapt to intrinsic and extrinsic uterotonic stimuli"}
+{"text": "Candida isolates from HIV patients with oral candidiasis and to correlate it with the CD4 count of the patients.Oral candidiasis is a clinical predictor for progression to AIDS. Antifungal drug resistance is becoming a major problem with this immunedepleted population. Considering the above facts, the study was conducted to speciate and to determine the susceptibility pattern of the Samples collected from (n=150) the lesion using sterile cotton swabs in HIV patients with oral candidiasis. Isolation and speciation were done by standard mycological procedures. Antifungal susceptibility was determined by Microbroth dilution method, as per the CLSI guidelines. Estimation of CD4+ T lymphocyte of the patients was done by FACS count system.Candida albicans 118 (78%) was the most common species followed by Candida tropicalis 17 (11%), Candida krusei 8 (5%), Candida parapsilosis 6 (4%), Candida glabrata 2 (1%) and Candida guilliermondi 1 (1%) . By microbroth dilution 18 (11.8%) isolates were fluconazole resistant, 23 (15.1%) were itraconazole resistant and all were amphotericin susceptible. Of the 150 patients, 106 (70.6%) had CD4 count <200 cells/ \u00b5l. Azole resistant was more common in patients with CD4 count <200 cells/ \u00b5l.Of the 150 samples, two revealed a mixture accounting for the 152 isolates. Candida albicans is the most frequently isolated species. Non- albicans Candida species are emerging as important pathogens with increasing rates of azole resistance and with increased immunosuppression. This emphasizes the need for speciation and determination of susceptibility pattern of the Candida isolates from HIV patients with oropharyngeal candidiasis."}
+{"text": "Reduction aortoplasty is an alternative method of treatment for ascending aorta aneurysm without involvement of the aortic root especially in high risk patients to decrease the cross clamp time, our study is to evaluate the early results and outcome of longitudinal reduction aortoplasty supported by pericardial patch at our institute.From 2006 till end of 2011, 37 patients with aneurysm of the ascending aorta without involvement of the aortic root underwent longitudinal reduction aortoplasty supported by pericardial patch which was sewed to the lateral wall of the ascending aorta by 5/0 prolene. The average age was 70.9 year \u00b14.2, females were 28 (75.7%), the average ejection fraction was 42.1%. The size of the ascending aorta was less than 55 mm in all patients; all patients underwent other cardiac procedure at the same time. 33 patients (89%) aortic valve replacement, 22 patients (59.5%) aortic valve replacement with CABG, 4 patients (11%) CABG alone, 7 patients (19%) were redo aortic valve replacement. Mean follow-up was 2 year.Preoperative mortality was in 2 cases 5.4%, both because of heart failure because of low ejection fraction preoperatively. One year survival was 91.9%. Reoperation done in 3 cases one because of hematoma collection beneath the pericardial patch after 3 weeks of the primary operation, the other 2 was because of valve complications. No redilataion seen in any patient till the time we did this study.Reduction aortoplasty is safe alternative to the standard replacement of the ascending aorta for patients without involvement of the aortic root with mild to moderate dilatation, can be done either supported with pericardial patch or unsupported, we think that supported aortoplasty may decrease the risk of redilataion and the risk for reoperation, our early results are encouraging."}
+{"text": "An accurate diagnosis of the morphology of the root canal system is a pre-requisite for successful root canal treatment. Frequently, root canals are left untreated because the clinicians fail to identify their presence, particularly in teeth that have anatomical variations or additional root canals. In this report a maxillary lateral incisor with two roots has been described. Many anatomical studies have declared that maxillary incisors always have a single root, while variations in the number of lateral canals and/or position of apical foramen are reported 2]3]4][3][4]2][[4][3][4][3][4]2]. As indiA brief literature review revealed 11 cases reporting maxillary lateral incisors with two roots 9]10][1[10]19][[1[10]1913]14][[14][13][[[14][13][[1[10]19Neville et al. used the term supernumerary roots when describing the presence of additional roots on a tooth compared with the classical description in dental anatomy. The most frequently affected teeth are the permanent molars  from either arch and mandibular cuspids and premolars .Periapical radiography is an essential tool in diagnosing internal anatomy of a tooth. The use of shift cone angle radiographic technique and parallel angle radiograph to identify superimposed roots and overlapping and unidentified canals has been advocated. In this case report, a rare case of maxillary lateral incisor with two roots is described.A 20 year-old male was referred to the clinic for root canal treatment of the maxillary right lateral incisor (#7). The medical history was found to be non-contributory. Patient\u2019s chief complaint was pain in relation to the upper right anterior teeth region. Clinical examination showed a pit over the cingulum region. Tooth also had grade I mobility and was tender both on palpation and percussion. No sinus tract or fistula was found. Radiographic examination using bisecting angle technique revealed widening of periodontal ligament space and well-defined periapical radiolucency around the lateral incisor as well as the central incisor .Based on the clinical and radiographic evidences, the tooth was diagnosed as having chronic apical periodontitis with an acute episode.The tooth was isolated with rubber dam  and anaesthetized with 1:200000 solution of Xylocaine with adrenaline  using an aspirating syringe. The access was created initially with a no. 2 round bur and then the cavity was enlarged to a triangular outline with round end tapered bur TR 13 . A small standardized #06 K-file  was used to negotiate each of the two separate root canals. Conventional working length radiograph was taken . The mesSodium hypochlorite 3%  alternated with 17% EDTA gel  was used for irrigation to facilitate shaping. The canals were then dried with sterile paper points , filled with mixed calcium hydroxide  and 0.2% chlorhexidine  slurry; the coronal cavity was sealed with Cavit G . The access opening for the adjoining central incisor was also performed on the same day with calcium hydroxide and chlorhexidine dressing. A week later, tooth was non-tender to percussion and the canals were dry. The canals were obturated with laterally condensed gutta-percha . Resin based AH Plus  sealer was used for obturation. The central incisor showed exudate, so it was treated again with calcium hydroxide/chlorhexidine dressing.A final radiograph for the lateral incisor  was thenAfter 1 year on follow-up visit, the patient reported complete alleviation of symptoms along with reduction in mobility. The recall radiograph showed resolution of the periapical pathology .When a maxillary incisor presents with two roots or two root canals, conditions such as fusion, gemination, dens in dente, palatogingival or distolingual groove and some variation in the normal development of Hertwig's epithelial root sheath must be considered 27]30].[30].27][.[30].27]Gemination is an anomaly in which the tooth germ divides during the development of the tooth, resulting in the formation of a double crown with single root, and in the case of fusion, the crown of two separate tooth buds fuse during development resulting in a bifid crown with two root canals in one root. In this case clinical examination as well as the pre-treatment radiographs revealed a crown of normal size and shape when compared with the contra lateral side. Therefore a diagnoses of fusion (single larger crown) or germination (fused or joined crown) can be disregarded 30]..30].There are few reports of maxillary lateral incisor with dens in dente and dens invaginatus showing two roots 6]8]9][8][9]6][[9][8][9][8][9]6]11]. In . In 11].Another developmental anomaly, which may appear similar to this case radiographically, is palatogingival or distolingual groove, but clinical examination ruled it out 27]..27].According to Bhasker  normal r"}
+{"text": "Life expectancies (LEs) of patients in UK Collaborative HIV Cohort (UK CHIC) stratified by CD4 count at start of antiretroviral therapy (ART) have been estimated [1] but not gains in years of life in response to ART. We estimated LE associated with attained CD4 count and viral suppression at different durations of ART. Patients in UK CHIC aged > 20 years who started ART in 2000 to 2008 (excluding person who injects drugs) were followed to end of 2010. All-cause mortality was ascertained from clinic notes and by linkage to national records. We used the nearest CD4 count before ART and the last in each of years 1 to 5 of ART and determined whether patients were virally suppressed (HIV-1 RNA < 400 copies/mL) in the past year for those remaining under follow-up. Poisson models were used to estimate mortality rates by sex, age, latest CD4 count  and viral suppression for each duration of ART. Abridged life tables were constructed from age-specific mortality rates to estimate LE for ages 20 to 85 years. Results are presented as the average number of years that will be lived after exact age 35 years. A total of 17,021 patients started ART from 2000 to 2008 of whom 708 (4.2%) died; 3956 (23%) were lost to study follow-up. There was no difference in mortality between those with attained CD4 350 to 499 and \u2265 500. On starting ART, male LE at exact age 35 was 36, 44 and 42  years for attained CD4 < 200, 200 to 349,\u2265350, respectively; after 5 years on ART, it was 22, 42 and 46  years, respectively. Only 17% of patients had CD4 \u2265 350 at ART start, compared with 78% of patients on ART for > 5 years. The difference in LE between suppressed versus unsuppressed patients was around 11 years. The figure shows that both CD4 count and viral suppression contribute to changes in LE. Male patients that increased their CD4 in the 1st year of ART from < 200 to 200\u2013349 or \u2265 350 gained 6 and 11 years of LE to 42 and 48 years, respectively, with similar rises for women. Overall, LE was 4 years greater for those on ART for > 5 years compared with those starting ART. Individuals that attain viral suppression and a CD4 count > 350 within 1 year of ART start have a normal LE with 35-year olds estimated to live to over 80 years on average. LE in patients with CD4 count < 200 beyond 5 years on ART drops by 15 years. Estimated LE may be biased by under-ascertainment of deaths, missing CD4 measurements and extrapolation beyond available data."}
+{"text": "BRCA1 germline mutations were found in a high proportion (14\u201334%) of patients with triple-negative breast cancer (TNBC). BRCA2 was either not analyzed or showed much lower mutation frequencies. Therefore, we screened a group of TNBC patients (n\u200a=\u200a30) of white European descent for mutations in BRCA2 as well as in BRCA1. Cases were unselected for age of disease-onset , family history of cancer and BRCA1 and BRCA2 mutation status. Half of the patients (15/30) showed a family history of breast and/or ovarian cancer. A high frequency of deleterious germline mutations was observed in BRCA2 , and only one case showed a BRCA1 mutation (3.3%). Although the study group was small, these results point to BRCA2 mutations being important in TNBC.Recently,  BRCA1-associated breast cancers is either TNBC, basal-like breast cancer or both. TNBC patients have a relatively poor outcome and cannot be treated with endocrine therapy or therapies targeted to HER2 due to the lack of related receptors Recently, triple-negative breast cancer (TNBC) has been classified as a breast cancer subgroup that is negative for estrogen and progesterone receptors (ER/PR) and receptor 2 of human epidermal growth factor (HER2). TNBC accounts for approximately 15% of all breast cancer cases and seems to be closely related to basal-like breast cancer, which has an expression profile similar to that of normal basal-myoepithelial breast tissue. Approximately 80% of TNBC cases show a basal-like phenotype, and the majority of basal-like breast cancers can be classified as TNBC. Close to 75% of BRCA1 germline mutations in TNBC cases. In a Canadian TNBC cohort (n\u200a=\u200a54) with an age of onset before 41 years and no familial breast cancer aggregation, five cases with mutations of BRCA1 (9%) and only one case with a mutation of BRCA2 (2%) were detected. The entire coding sequence of BRCA1 and the large exons 10 and 11 of BRCA2 were analyzed BRCA1 mutations and none had BRCA2 mutations when fully screened in both genes. Forty-three of these cases were from patients younger than 41 years of age at diagnosis and with no familial aggregation of breast cancer. An additional 30 cases were unselected for family history of breast cancer because they were younger than 31 years old at onset of the disease BRCA1 (n\u200a=\u200a12) and 3.9% for BRCA2 (n\u200a=\u200a3). The entire coding sequence and the exon-intron boundaries were screened in both genes BRCA1 and BRCA2. In all Ashkenazi Jewish breast cancer cases, approximately 10% of patients carry one of three different founder mutations in BRCA1 and BRCA2. In the investigated Ashkenazi Jewish TNBC cohort (n\u200a=\u200a64), 19 BRCA1 (29.7%) and 6 BRCA2 (9.4%) mutations could be detected when screening for the three founder mutations BRCA2 mutations BRCA2 mutation screening approaches BRCA2 mutation frequency in a TNBC cohort of a Southern German population unselected for age of onset and familial aggregation of cancer. If there was a relevant mutation rate in such cases, TNBC would meet the clinical criteria for mutation screening in BRCA2 and should be added to the current guidelines.Recent reports have focussed on All selected patients underwent genetic counselling and gave their written informed consent for genetic testing. The study protocol was approved by the Ethics Committee of the State of Salzburg (Austria).1 mouse antibodies for estrogen (clone 6F11) and for progesterone receptors . For HER2-IHC we used polyclonal rabbit antibodies from clone A0485 .In a monocentric approach executed in Munich, Germany, newly diagnosed cases of individuals with TNBC diagnosed between 2005 and 2010 were selected from the Pathology Unit. Histological samples were classified as TNBC when the following criteria were met: less than 1% of cells demonstrated nuclear staining for estrogen and progesterone receptors, and immunohistochemical staining for HER2 showing a 0, 1-fold, or a 2-fold positive score and a FISH ratio  of less than 1.8 according to the relevant ASCO and CAP guidelines BRCA1 and BRCA2, we used primer pairs and polymerase chain reaction (PCR) protocols published elsewhere DNA extraction from whole blood samples (EDTA) was performed according to standard protocols. To amplify exons and exon-intron boundaries of BRCA1 and BRCA2. The following clinical criteria were evaluated in approximately 1,000 unrelated cases in a German-wide study:In Germany, patients with a 10% mutation probability are eligible for mutation screening in Two women with breast cancer, with one of them \u226450 years, or three women with breast cancer, independent of age.One woman with breast and one woman with ovarian cancer, independent of age, or one woman with breast and ovarian cancer, independent of age, or two women with ovarian cancers, independent of age.One male breast cancer and one woman with breast or ovarian cancer.One woman with breast cancer \u226435 years.One woman with bilateral breast cancer or two primary breast cancers, with first tumour diagnosed before \u226450 years BRCA1 and five  in BRCA2. We detected four known deleterious frame-shift mutations (BRCA1: c.5266dupC (old nomenclature is \u201c5382insC\u201d) which is one of the most common deleterious BRCA1 mutations BRCA2: c.1029delA http://research.nhgri.nih.gov]) and one unknown splice-site mutation (BRCA2: c.476-1G>A) with a clear predictive deleterious effect. One missense mutation in BRCA2 (p.W2626C) was classified as \u201cpredictive deleterious\u201d in prior literature Characteristics of investigated patients are presented in BRCA1 leading to an exchange of methionine for isoleucine at amino acid position 1,652 of the protein . Variant c.5744C>T causes a point mutation of threonine to methionine at amino acid position 1,915 of BRCA2 and was detected in four cases . Within mutation carriers, two out of six cases (33.3%) did not meet the German clinical criteria for BRCA1 and BRCA2 testing. From all TNBC cases, 15 out of 30 (50%) showed an early age of onset or a familial aggregation which are German clinical criteria for mutational screening of BRCA1 and BRCA2. The median age at diagnosis of the total study group was 58 years (n\u200a=\u200a30), for mutations carriers 50 years (n\u200a=\u200a6), and for the remaining patients (n\u200a=\u200a24) not showing deleterious mutations 61 years.In four cases, we detected c.4956G>A in BRCA2 germline mutations in triple-negative breast cancer (TNBC). We detected 6 deleterious BRCA1 and BRCA2 mutations in 30 TNBC patients (20%). The total mutation frequency for both genes is comparable with those described in recent studies BRCA2 germline mutations in our TNBC study group: four cases with previously published deleterious variants, and one splice-site mutation which was not published before (16.7%). The low rate of BRCA2 mutations found in a Canadian sample (1/54\u200a=\u200a2%) was probably the result of assay design given that only exons 10 and 11 of BRCA2 were tested. Additionally, only TNBC cases with an age of disease onset of less than 41 years were selected for the study BRCA2 mutations. No BRCA2 mutations were detected in 73 TNBC cases from the UK. BRCA1 and BRCA2 genes were fully screened, but patients were diagnosed with TNBC before age 41 (n\u200a=\u200a43) and before age 30 (n\u200a=\u200a30). Cases were selected for the UK study only if no familial aggregation was reported BRCA2 mutation frequencies. Interestingly, a similar study of 77 TNBC cases from the United States where cases were unselected for age of onset (average was 51 years) revealed a much lower BRCA2 mutation rate (3.9% vs. 16.7% in our study) The aim of this study was to investigate the role of BRCA1 and BRCA2 mutation carriers  showed that the proportion of TNBC increased with age at diagnosis in BRCA2 mutation carriers, while it decreased in BRCA1 mutation carriers BRCA2 and higher BRCA1 mutations rates in TNBC Recently, data of a multi-national approach containing approximately 7,000 BRCA1 . Analysis with protein prediction software showed some minor effect BRCA2 (rs4987117) was less frequent  in estrogen receptor-positive patients of a study cohort of 117 sporadic breast cancer cases from Poland BRCA1 and c.5744C>T in BRCA2) could therefore be investigated in much larger association studies to evaluate their impact in TNBC tumorigenesis.In four cases, we detected c.4956G>A in BRCA2 mutations in two patients not meeting the current German clinical criteria for BRCA1 and BRCA2 mutation screening. In our cohort of TNBC patients unselected for age at diagnosis and cancer family history, 15 cases  did not meet those criteria. The mutation rate in this subgroup was 13% (2/15). The main requirement for the German clinical criteria for BRCA1 and BRCA2 mutation testing was defined as being at least a 10% a priori probability to carry either a BRCA1 or BRCA2 mutation BRCA1 and BRCA2 mutation screening, and additional cases may be assessed not only if they present with early-onset of the disease or have a familial aggregation of breast and/or ovarian cancer. BRCA1 and BRCA2 mutation carriers are frequently diagnosed with triple-negative breast cancer BRCA1 and BRCA2 mutation screening in TNBC. In contrast, recent guidelines published by the National Comprehensive Cancer Network in the United States are less stringent. For instance, TNBC diagnosed before age 60, and breast cancer cases with a negative family history for cancer younger than 45 years should be tested according to these guidelines. In comparison, the German guidelines recommend testing single cases only if the diagnosis was made before age 35 We identified deleterious BRCA1 and BRCA2 genes in patients diagnosed with TNBC from Southern Germany and unselected for age of disease onset and familial aggregation of this malignant disease. In contrast to recent reports, an unexpected and high mutation frequency was seen in BRCA2  suggesting that this gene may play an important role in the development of TNBC when mutated. Our data suggest that Southern German patients diagnosed with TNBC may qualify for BRCA1 and BRCA2 mutation screening. Larger patient cohorts have to be investigated to validate our findings.In summary, we found a 20% germline mutation rate (6/30 cases) in"}
+{"text": "FoxP2 is widely involved in a number of important physiological and developmental processes. We systematically studied the evolutionary history and functional adaptations of FoxP2 in teleosts. The duplicated FoxP2 genes (FoxP2a and FoxP2b), which were identified in teleosts using synteny and paralogon analysis on genome databases of eight organisms, were probably generated in the teleost-specific whole genome duplication event. A credible classification with FoxP2, FoxP2a and FoxP2b in phylogenetic reconstructions confirmed the teleost-specific FoxP2 duplication. The unavailability of FoxP2b in Danio rerio suggests that the gene was deleted through nonfunctionalization of the redundant copy after the Otocephala-Euteleostei split. Heterogeneity in evolutionary rates among clusters consisting of FoxP2 in Sarcopterygii (Cluster 1), FoxP2a in Teleostei (Cluster 2) and FoxP2b in Teleostei (Cluster 3), particularly between Clusters 2 and 3, reveals asymmetric functional divergence after the gene duplication. Hierarchical cluster analyses of hydrophobicity profiles demonstrated significant structural divergence among the three clusters with verification of subsequent stepwise discriminant analysis, in which FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were classified into Cluster 1, whereas FoxP2b of Salmo salar was grouped into Cluster 2 rather than Cluster 3. The simulated thermodynamic stability variations of the forkhead box domain (monomer and homodimer) showed remarkable divergence in FoxP2, FoxP2a and FoxP2b clusters. Relaxed purifying selection and positive Darwinian selection probably were complementary driving forces for the accelerated evolution of FoxP2 in ray-finned fishes, especially for the adaptive evolution of FoxP2a and FoxP2b in teleosts subsequent to the teleost-specific gene duplication.As one of the most conserved genes in vertebrates,  FoxP2 is a key transcription factor gene in the FoxP subfamily  distribution at a significance level of 0.05. LR represents the ratio of the maximum likelihood that \u03b8\u03bb > 0 to that of \u03b8\u03bb = 0. In addition, the critical amino acids that are responsible for the type I functional divergence can be identified using a reasonable cutoff value (> 1) for the posterior probability ratio based on the hidden Markov model. The coefficient of functional divergence (\u03b8lication . Type I on 1.04) . A statiFoxP2 of Sarcopterygii), Cluster 2 (FoxP2a of Teleostei) and Cluster 3 (FoxP2b of Teleostei) based on the teleost lineage specific FoxP2 duplication in the ancestral 3R-WGD event  show greater divergence than these functional domains, we also tested the functional divergence between Cluster 1 and Cluster 2 in Data set 2 to determine if the two clusters exhibit homogeneous evolutionary rates in an extensive region  were used to discriminate these sequences. With Data set 2, we investigated whether FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were grouped together with Cluster 1 or with Cluster 2. In order to study whether the classification was a remaining trace after a long-term evolutionary history, we also used the three clusters to discriminate hydrophobicity profiles of ancestral sequences from Furthermore, we performed a stepwise discriminant analysis using SPSS software version 20) to evaluate the consistency of the classification in the four cluster trees constructed with each data set. The variable selection method used in the discriminant analysis was Mahalanobis distance. The prior probability for groups was computed according to size. We kept the F values for entry or removal of variables and the covariance matrix for classification as defaults. Coefficients for both the canonical discriminant function and Fisher\u2019s linear discriminant function were computed. Finally we evaluated the classification results by using the percentage of original grouped cases and cross-validated grouped cases correctly classified. In Data set 1, we focused on the rationalization of groupings for FoxP2 of 0 to evalThe three-dimensional (3D) structure of the forkhead domain of human FOXP2 (PDB: 2A07) provides a model to investigate changes in the stability of the local 3D structure in different evolutionary scenarios. We applied FoldX (version 3.0 b5.1), a fast and effective computer algorithm for estimating the effect of mutations on stability , in ordeFoxP2a and FoxP2b in teleosts experienced dramatic evolutionary changes after gene duplication. Therefore, we tested natural selection pressures with Data set 1 based on the corresponding phylogenetic topology tree (l (twice the log likelihood difference) between a model and its nested model was compared to the value of the \u03c72 distribution with degrees of freedom equal to the numerical difference between the free parameters of the two models. The nested model was rejected at a significance level of P < 0.05.The analyses of functional and structural divergence suggest that ogy tree  using thogy tree . Codon sFirst, three pairs of site models were tested on the data sets and compared based on the LRT . Comparip (one tail probability for \u03c72 distribution) value was not the statistically significant (P < 0.05), we neglected Test 2 and positively selected sites.We used branch specific models to detect whether Cluster 1, Cluster 2 and Cluster 3 experienced different natural selection pressures . Due to FoxP2 shared a highly conserved synteny in four tetrapod species: Homo sapiens, Taeniopygia guttata, Anolis carolinensis and Xenopus tropicalis  could be pinpointed in the chromosomal segments in which FoxP2a and particularly FoxP2b were both greatly divergent from their FoxP2 orthologs. FoxP2a in teleosts possessed a 16-exon composition resembling that in human FOXP2  between Cluster 2 and Cluster 3 in Data set 1 was significantly greater than 0 (\u03bb between Clusters 1 and 2 (or 3), the \u03b8\u03bb between Clusters 1 and 3 was greater than that between Clusters 1 and 2. Moreover, a significant functional divergence between Clusters 1 and 2 was detected in Data set 2 , yielded highly consistent inconsistency coefficients and clusters . All copSalmo salar and Gadus morhua were singly classified in these dendrograms. FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were grouped into Cluster 1, however FoxP2 of Rana daunchina was misclassified into Cluster 2 in the trees based on Correlation, Cosine and Euclidean pairwise distances. In addition, some links connecting dissimilar members (i.e. with inconsistency coefficients > 0.8) existed in these three clusters, indicating possible structural heterogeneity in the clusters. Notably, all dendrograms revealed that FoxP2a of Acanthopterygii were dissimilar to FoxP2a of Salmo salar, Gadus morhua and Otocephala. FoxP2a of Takifugu rubripes and Tetraodon nigroviridis showed significant dissimilarity from FoxP2a of other seven acanthopterygian fishes. Amphibian FoxP2 were possibly divergent from other tetrapod FoxP2. Subsequent stepwise discriminant analysis, which correctly classified high percentages of the original grouped cases (100%) and cross-validated grouped cases (97.2%), strongly demonstrated that FoxP2 of Leucoraja erinacea, Lepisosteus oculatus and Rana daunchina belonged to Cluster 1. Additionally, FoxP2b of Salmo salar and Gadus morhua were classified into Cluster 2 and Cluster 3, respectively. Moreover, FoxP2 in the ancestor of Osteichthyes and the ancestor of Actinopterygii, as well as all the ancestral FoxP2 in Sarcopterygii, were grouped into Cluster 1. FoxP2 in the ancestor of Teleostei, FoxP2b in the ancestor of Euteleostei and all the ancestral FoxP2a in Teleostei were classified as members of Cluster 2. All ancestral FoxP2b of Neoteleostei were classified into Cluster 3.According to the cutoff value (0.8) for inconsistency coefficients, the three clusters  were consistently separated from one another in the four hierarchical dendrograms from Data set 1 , althougLeucoraja erinacea and Lepisosteus oculatus were grouped into Cluster 1. The existence of a substantial inconsistency coefficient (> 0.8) in Cluster 2 indicated significant divergence between FoxP2a of Acanthopterygii and that of Salmo salar, Gadus morhua and Otocephala. FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were confirmed to be members of Cluster 1 in subsequent stepwise discriminant analysis. In addition, FoxP2 in the ancestor of Osteichthyes and the ancestor of Actinopterygii were grouped together with all ancestral FoxP2 in Sarcopterygii into Cluster 1. All ancestral FoxP2a in Teleostei were classified into Cluster 2. Furthermore, 100% of the original grouped cases and cross-validated grouped cases were correctly classified.Similarly, analysis of structural divergence on Data set 2 produced consistent results , classifThe simulated ADTE variations of monomers and homodimers in different evolutionary scenarios showed that such ADTE variations were not always in unison . In 53 oThe comparison of Model 3 to Model 0 in both data sets revealed variable natural selection pressures across sites . NotwithSubsequent analysis of branch specific models in Data set 1 demonstrated that Cluster 3 experienced divergent natural selection pressures against both Cluster 1 and Cluster 2 . SpecifiFoxP2a in ancestor of Tetraodontiformes, FoxP2b in ancestor Neoteleostei and FoxP2b in the ancestor of Tetraodontiformes) were found to have experienced accelerated evolution using Test 1 for branch-site models A in Data set 1  likely experienced accelerated evolution . The test of selection pressures on 27 selected sequences in Data set 2, containing exons 6 and 16, produced similar results (not shown). These two exons with teleost-specific inserts were also demonstrated targets of positive natural selection and relaxed purifying selection.Likewise, five ancestral branches in Data set 2  in these chromosomal segments after the tetrapod-teleost split. Although FoxP2a and FoxP2b show remarkable divergence in teleosts, the phylogenetic trees reconstructed with four member genes  of the FoxP subfamily clearly demonstrate that FoxP2a and FoxP2b in teleosts are members of FoxP2 lineage (not shown). In comparison with FoxP2, greater divergence (e.g. exon-composition) of FoxP2b than FoxP2a suggests that FoxP2b evolves faster than FoxP2a after the gene duplication. Although dimerization of FoxP1, FoxP2 and FoxP4 has been demonstrated, which is important for their DNA binding and transcriptional activity [In this report, we identify the teleost-specific activity , it is nFoxP2b in Danio rerio probably results from pseudogenization [FoxP2a of Danio rerio, FoxP2a of two other species in Otocephala (Ctenopharyngodon idella and Pygocentrus nattereri) are phylogenetically determined. However, deletion of FoxP2b in Ctenopharyngodon idella and Pygocentrus nattereri is uncertain because their genomes are not available. The retention of FoxP2b in Euteleostei implies that the gene may conserve some functions of the parental FoxP2 through subfunctionalization or may gain novel functions through neofunctionalization. The linked paralogon shared by FoxP2a and FoxP2b in teleosts indicates that these two duplicated genes were generated in a large-scale genome duplication event, i.e. 3R-WGD event [FoxP2 may occur in polyploid vertebrates, such as Xenopus laevis and Salmo salar [The absence of nization  of the rGD event ,36  rather than the functional domain itself in FoxP2a and FoxP2 are targets of divergent evolutionary rates. Therefore, the functions of FoxP2a may be more conservative than those of FoxP2b in teleosts.The significant functional divergence between FoxP2a and FoxP2b in Data set 1 implies that FoxP2a and FoxP2b probably execute divergent functions in teleosts. The \u03b8Salmo salar groups together with FoxP2a, as well as FoxP2 in the ancestor of Teleostei and FoxP2b in the ancestor of Euteleostei together with all ancestral FoxP2a in Teleostei, implying that structural divergence between FoxP2a and FoxP2b was probably very weak at the early stage of post-duplication. Perhaps the weak structural divergence of FoxP2a and FoxP2b generated functional redundancy of FoxP2b in Otocephala, further leading to the lineage-specific loss of FoxP2b. The significant structural divergence between FoxP2a and FoxP2b seems to coincide with the split of Neoteleostei from Euteleostei, implying more divergent functions between the genes of Neoteleostei than Salmoniformes. Additionally, some teleost lineages show significant FoxP2a structural divergence, likely indicating divergent functional adaptations in the lineages.Hierarchical cluster analysis and stepwise discriminant analysis of hydrophobicity profiles in CC10 [The significantly divergent variations of thermodynamic stability among FoxP2a, FoxP2b and FoxP2 clusters imply divergent functional adaptations of the forkhead domains in FoxP2a, FoxP2b and FoxP2. Intriguingly, the significant destabilizing effects of mutations (E519D and R523M) on both monomers and homodimers suggest that functional divergence of FoxP2 in teleosts possibly began in the pre-duplication phase. It has been demonstrated that the two mutations, as well as 521S, are responsible for the weak repressive activity of the medaka FoxP2a on the lung-specific gene of CC10 . The theFoxP2b lineage has undergone a generally relaxed purifying selection pressure, which is significantly greater than that of the FoxP2a and FoxP2 lineages. The regions flanking the functional domain of the forkhead box, rather than the forkhead domain per se, in the FoxP2a lineage has experienced a milder purifying selection pressure than those in the FoxP2 lineage. Thus the asymmetrical natural selection pressures acting on the FoxP2a, FoxP2b and FoxP2 clusters, particularly between FoxP2a and FoxP2b, likely led to asymmetrical functional divergence among these clusters. Furthermore, natural selection pressures on branches and sites in each cluster show significant heterogeneity, especially positive natural selection acting on several ancestral branches. Notably, positive selection pressure mainly acts on regions flanking functional domains. Therefore, positive selection pressure possibly was the driving force in forming the teleost-specific inserts in FoxP2a and the truncated regions in FoxP2b. It is also notable that FoxP2 in the ancestor of Actinopterygii probably underwent positive selection, suggesting that positive selection played a role in variation during the pre-duplication phase [FoxP2b in Otocephala possibly resulted from excessively relaxed purifying selection on the redundant copy in the fixation phase [FoxP2a and FoxP2b after Neoteleostei branched off from Euteleostei, implying adaptive evolution of these two genes coincided with diversification of Neoteleostei. Our tests of natural selection pressures based on branch specific models strongly suggest the on phase . The poton phase ,58. PosiMus musculus for lung and esophageal development, especially for postnatal lung alveolarization [FoxP2 is critically involved in the development of foregut derived pulmonary organ systems through regulating lung-specific genes, e.g. CC10 and surfactant protein C (SPC).Studies of FoxP2 in both animal models and humans have shown that the pleiotropic gene is widely involved in a series of important physiological activities and developmental processes ,4,17. Inrization ,22,59. TFoxP2a in teleosts have provided only limited information concerning its functions for organ development [FoxP2a in development of CNS with other vertebrates. No FoxP2a expression has been detected in the swim bladder of adult Danio rerio [FoxP2a expression in early (7 days post-fertilization) embryonic development in Maylandia zebra [FoxP2a decreases to a light but visible level in the swim bladder of late larval (20 days post-fertilization) embryos of Mchenga conophorus [Danio rerio [The teleost swim bladder has been considered homologous to the lung on the basis of consistency of developmental blastema and molecular pathways ,29. It helopment ,19,21. Fia zebra . ExpressFoxP2 in foregut derived air-breathing organ, rapid evolution of FoxP2 (FoxP2a and FoxP2b) in teleosts is possibly correlated with adaptive evolution of the swim bladder [Lepisosteus oculatus retains a swim bladder to breathe air and possesses FoxP2 resembling that of tetrapods. The three species of Otocephala  and one species of Protacanthopterygii  are physostomous fishes with a pneumatic duct connecting the swim bladder to the gastrointestine. It has been suggested that the Otocephala-specific loss of FoxP2b possibly results from excessively relaxed purifying selection on the functionally redundant gene. In spite of retention of FoxP2b in Salmo salar, the divergence of FoxP2a and FoxP2b is noticeably weak. Positive natural selection probably acts on FoxP2a and FoxP2b to favor the divergence of the genes in Neoteleostei, which are physoclistous fishes (i.e. swim bladders of these fishes have lost a pneumatic duct). Taken together, the present study provides a useful introduction to studying the functional involvement of FoxP2a and FoxP2b in teleosts.Given the essential functions of  bladder . NotablyInformation S1Species coverage, PCR primers and predicted FoxP2 sequences.(PDF)Click here for additional data file.Information S2FoxP2, FoxP2a or FoxP2b in eight species.Protein-coding genes around  Twenty upstream (with negative order number) and 20 downstream (with positive order number) protein-coding genes.(PDF)Click here for additional data file.Information S3Amino acid or fragment inserts in FoxP2 and FoxP2a of ray-finned fishes.(PDF)Click here for additional data file.information S4Phylogenetic reconstructions based on Data sets 1 and 2.(PDF)Click here for additional data file.Information S5Posterior probability profiles of site-specific rate difference among clusters in Data sets 1 and 2.(PDF)Click here for additional data file.Information S6Hierarchical dendrograms of genes in Data sets 1 and 2.(PDF)Click here for additional data file.Information S7Test of natural selection pressures on Data sets 1 and 2.(PDF)Click here for additional data file."}
+{"text": "The aim of the study was determination of serum Se concentration and identification of genetic variations in genes related to metabolism of selenium as markers of cancer risks for carriers of BRCA1 gene mutation and individuals with susceptibility to unselected breast cancer.Eight genotypes of 4 most common SNPs localised in GPX1, GPX4, TXNRD2 and SEP15 were selected. Genotyping was performed in 27 triplets matched BRCA1 carriers (cases and controls were matched 1:2) as well as on pairs matched 1:1 consisting of 220 unselected breast cancer (BRCA1 excluded). Cases and controls were matched for year of birth (+/-3 years), number and location of cancer among I\u00ba relatives , smoking - the number of pack years (+/- 10%) and adnexectomy.For group of 27 triplets matched BRCA1 carriers, all patients were carriers of one of three Polish founder BRCA1 mutation  and serum was collected 1 \u2013 2 years before tumor diagnosis. Whereas for 220 pairs of unselected breast cancer, serum was collected before treatment but during diagnosis.The following techniques for laboratory analyses have been applied: a) sequencing on ABI310, b) TaqMan analysis (a melting-curve genotyping with fluorescence-labeled probes based on the LightCycler 480 System (Roche Applied Science), c) determination of selenium concentration in serum using ICP MS \u2013 inductively coupled plasma mass spectrometry with \u00b1 5% accuracy (Perkin Elmer).In both studied groups there was observed that the optimal concentration of selenium was in range 100 \u2013 120\u00b5g/l of serum independently on variants in selenoprotein coding genes.If selenium level data were combined with some selenoprotein genotypes a tendency was found that the optimal selenium level for carriers of GPX1 nCC genotype  with genotype nCC in GPX1 gene the optimal concentration of selenium is around 75\u00b5g/l of serum.\u2022 The results of association studies require confirmation by a prospective observation of large groups of patients.\u2022 Investigation on larger number of patients are needed especially valuable may be observations of women with increased risk of cancers."}
+{"text": "HTLV-1 is the ethiologic agent of adult T-cell leukemia (ATL). The viral regulatory protein Tax1 plays a pivotal role in T-cell transformation by deregulating several signalling pathways including CREB, NF-\u03baB and AP-1 leading to the abnormal expression of several cellular proteins involved in cell survival and proliferation. Previous studies in the lab, using the yeast-2-hybrid approach to screen a T-cell library for Tax1 interacting partners identified the cellular Four and a Half Lim domain protein 3 (FHL3) as a possible Tax1 interacting candidate. FHL3 can regulate the actin-cytoskeleton and functions as a transcriptional activator. The aim of this study is to investigate the physical and functional interaction between Tax1 and FHL3. We have found that Tax1 and FHL3 interact both in vitro and in vivo and deletion analysis of FHL3 revealed that each LIM domain of FHL3 can interact with Tax1. Deletion analysis of Tax1 determined that the interaction with FHL3 occurs at both the N and C terminus, namely amino acids 1-50 and a fragment which encompasses the predicted LIM binding domain 207-353aa. We have demonstrated that FHL3 enhances Tax1 mediated LTR transcriptional activation without affecting basal activity. In contrast to this we show that FHL3 down regulates NF-\u03baB activation by Tax1. Using confocal microscopy, we show that FHL3 co localizes with Tax in the nucleus and at the periphery of co transfected cells. In addition we demonstrate that FHL3 induces nanotubules in Cos7 cells and may be capable of transporting Tax1 from one cell to another. Overall our results so far suggest that the interaction between Tax1 and FHL3 alters both the transactivating activity and the sub cellular localization of Tax1 and provide new insights into molecular mechanisms that underlie the oncogenic nature of this HTLV-1 protein."}
+{"text": "Atoh1, which is necessary for differentiation of hair cells, but not for their specification. Loss of several genes will disrupt ear morphogenesis or arrest of neurosensory epithelia development. We previously showed in null mutants that the loss of the transcription factor, Gata3, results specifically in the loss of all cochlear neurosensory development. Temporal expression of Gata3 is broad from the otic placode stage through the postnatal ear. It therefore remains unclear at which stage in development Gata3 exerts its effect. To better understand the stage specific effects of Gata3, we investigated the role of Gata3 in cochlear neurosensory specification and differentiation utilizing a LoxP targeted Gata3 line and two Cre lines. Cre\u2236Gata3f/fFoxg1 mice show recombination of Gata3 around E8.5 but continue to develop a cochlear duct without differentiated hair cells and spiral ganglion neurons. qRT-PCR data show that Atoh1 was down-regulated but not absent in the duct whereas other hair cell specific genes such as Pou4f3 were completely absent. In addition, while Sox2 levels were lower in the Cre:Gata3f/fFoxg1 cochlea, Eya1 levels remained normal. We conclude that Eya1 is unable to fully upregulate Atoh1 or Pou4f3, and drive differentiation of hair cells without Gata3. f/fPax2-Cre\u2236Gata3 mice show a delayed recombination of Gata3 in the ear relative to Cre:Gata3f/fFoxg1. These mice exhibited a cochlear duct containing patches of partially differentiated hair cells and developed only few and incorrectly projecting spiral ganglion neurons. Our conditional deletion studies reveal a major role of Gata3 in the signaling of prosensory genes and in the differentiation of cochlear neurosenory cells. We suggest that Gata3 may act in combination with Eya1, Six1, and Sox2 in cochlear prosensory gene signaling.Hair cells of the developing mammalian inner ear are progressively defined through cell fate restriction. This process culminates in the expression of the bHLH transcription factor  Jagged1 (Jag1), Lunatic fringe (Lfng), Bone morphogenetic protein  SRY-box containing gene (Sox2), Paired box gene 2 (Pax2) and several Fibroblast growth factors  in the prosensory areas of the mouse at E9.5\u201310.5 suggests that prosensory areas are molecularly defined by these factors at or before this time point Atoh1 expression Atoh1 is not necessary for specification of postmitotic precursors Atoh1 in the cochlea results in transformation of some cells into hair cell-like cells, but these hair cells are unstable and are morphologically and physiologically like vestibular hair cells Atoh1 transfection alone is inadequate for restoration of lost hair cells in the mammalian cochlea suggesting that Atoh1 action depends on the proper molecular context to develop organ of Corti-like hair cells as previously suggested for placodal development The mammalian inner ear develops through a cascade of diffusible factors and transcription factors to generate a complex labyrinth of ducts and recesses out of a flat epithelium, the otic placode Gata3 is particularly high in the ventral otocyst, the area of the cochlear anlage Gata3 expression continues in the organ of Corti and spiral ganglion neurons from specification through late postnatal stages The zinc finger transcription factor Gata3 is expressed throughout the early otic placode. Later Gata3 becomes restricted to future proneurosensory regions (except that of the saccule) concomitant with their segregation from non-sensory domains Gata3 null mutant. Moreover, what specific function the later expression of Gata3 has in these processes remains unclear as no inner ear-specific delayed deletion data exist for Gata3.Haploinsufficiency of GATA3 causes human hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome Gata3 deletion mouse models. We utilized both CreFoxg1 and Pax2-Cre to recombine floxed Gata3 in the ear. CreFoxg1 is a knockin Cre that recapitulates the Foxg1 expression from the E8\u20138.5 otic placode Pax2-Cre is a BAC-transgene that does not fully conform to early Pax2 expression We investigated the function of Gata3 at later embryonic stages utilizing two conditional Cre:Gata3f/fFoxg1 mice have an earlier and more profound loss of Gata3 expression and exhibit a cochlear duct completely devoid of neurosensory cells. Atoh1 shows limited expression levels in the cochlea of these mice, suggesting that a specific Atoh1 level is critical for hair cell differentiation. Some prosensory genes such as Sox2 are expressed at lower levels compared to normal, while Eya1 and others are not altered in their expression levels. This suggests that Gata3 is an essential co-factor for neurosensory development. Utilizing f/fPax2-Cre: Gata3 mice resulted in a delayed deletion of Gata3 before differentiation of the organ of Corti. Only patches of the organ of Corti with abnormal hair cells formed in the cochlear duct along with improperly projecting spiral ganglion neurons. This indicates sustained expression of Gata3 is required to fully differentiate hair cells and spiral ganglion neurons of the organ of Corti.Our results show that All animal care and procedures were approved by the University of Iowa IACUC following the guidelines for use of laboratory animals (ACURF 0804066).Gata3 conditional knockout mice, we crossed CreFoxg1 mice Pax2-CreGata3 floxed mice Foxg1 locus as CreFoxg1 and Pax2 promoter with the Cre recombinase inserted as a BAC transgene as Pax2-Cre. Breedings were always conducted with a male expressing Cre and heterozygous for the Gata3 floxed allele and the female being homozygous for the floxed allele and no Cre. A few of the matings included a Pax2-Cre male with a Gata3 LacZ null allele instead of a Gata3 floxed allele LacZ/fPax2-Cre:Gata3 and f/fPax2-Cre:Gata3 ears. Genotyping by PCR analysis of tail DNA was accomplished under standard conditions with Gata3 specific primers . Embryos were collected from pregnant dams with noon on the day of vaginal plug considered embryonic day (E) 0.5. To collect embryos, pregnant dams were anesthetized with 1.25% Avertin at a dose of 0.025 ml/g by intraperitoneal injection. For non-qRT-PCR experiments, embryos were dissected from the uterus and fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) transcardially using a peristaltic pump. The following genotypes were utilized as controls: Pax2-Cre:Gata3wt/wt; CreFoxg1:Gata3wt/wt; Gata3f/wt; Gata3f/f. All images are representative of assessments of at least 3 biological controls which were identical unless noted in the text.To generate in situ hybridization experiments were conducted as described in Whole-mount Whole-mount Immunochemistry was performed as described in Inner ear innervation was labeled with lipophylic (NeuroVue) dyes . Dyes were placed into the cerebellum and caudal hindbrain for selective labeling of vestibulo-cerebellar and vestibulo-spinal nerves as described Primers were created using the Roche Universal Probe library assay center which utilizes Advance Primer3 to calculate optimal size, melting point, and complementarity of primer sets. A list of primers used can be found in The process of staining, imaging, and reconstruction of inner ears has been previously described The mice for scanning electron microscopy were perfused with 4% PFA after sedating with Avertin. Ears were then dissected and fixed in a 2.5% glutaraldehyde, 4% PFA solution. Ears of postnatal mice were decalcified with EDTA. Following osmication in 2% osmium tetroxide in 0.1 Monophosphate buffer (pH 7.4) for 1 hour, the ears were dissected and prepared including removal of the tectorial membrane. The samples were then rinsed with distilled water to remove ions and dehydrated in a graded ethanol series, critical-point dried, mounted on stubs with carbon tape and coated with gold/palladium. Specimens were then viewed with a Hitachi S-4800 Scanning Electron Microscope with 3MeV acceleration. False coloring was accomplished with Adobe Photoshop CS6.Gata3 null ears fail to develop all inner ear neurosensory epithelia except a few saccular hair cells and neurons Gata3 mouse line CreGata3 line has LoxP sites flanking exon4 which contains the primary DNA binding domain. CreFoxg1 is expressed early in ear development Gata3 is proposed to lead to early embryonic lethality. To assess recombination of the floxed allele by Foxg1Cre we used qRT-PCR to assess Gata3 mRNA levels over time. We designed specific Gata3 exon4 primers to detect recombination. Gata3 exon4 mRNA expression was significantly reduced in mutant ears by embryonic day (E) 8.5 compared to control, and continued to decrease with age  were not detectable by qRT-PCR. Eya1 and Notch1, factors that are necessary for prosensory specification and differentiation in the inner ear Gata3 conditional mutant mice. Pax2, known to be necessary for normal cochlear development Gata2 in the absence of Gata3. This upregulation of Pax2 and Gata2 is obviously unable to compensate for the lack of Gata3. Tecta is a gene known to be an integral player in tectorial membrane development Tecta levels is consistent with the lack of tectorial membrane in these mutants. Axin2 is known to be expressed in the cochlear duct but not in the sensory epithelia and its expression remains unaltered We used qRT-PCR to identify potential down-stream genes for cochleae . Care waAtoh1 expression led us to speculate that Gata3 not only plays a role in specification but possibly also in the differentiation of neurosensory cells of the cochlea. This is consistent with differentiating spiral ganglion neurons and organ of Corti hair cells expressing Gata3 during differentiation. To analyze the timing of recombination of Gata3 for specification versus differentiation, we utilized another Cre strain (Pax2-Cre) which like CreFoxg1 is expressed in the otic placode, but somewhat later in development Pax2-Cre may be slightly less efficient than CreFoxg1, possibly because of a relatively delayed or more inefficient recombination The loss of Atoh1 downstream gene expression in hair cells without the complete elimination of Pax2-Cre to recombine the Gata3 floxed allele over time. The level of the Gata3 exon4 was not significantly altered from control ears until E10.5  in the cochlear duct. These spaces were absent in all mutants analyzed. We also analyzed morphology and neurosensory formation using LacZGata3 expression. The heterozygous wt/LacZGata3 ear contains no obvious morphological or embryonic histologic defects Pax2-Cre mutant mouse shows a high level of Gata3-LacZ expression in the cochlea as in the control (CreFoxg1:f/fGata3 mutant mice there was frequently a short curvature at the end of the somewhat longer cochlear duct (N\u200a=\u200a11/14). One canal crista was present in the anterior part of the ear and a transient formation of a posterior canal crista occasionally occurred (N\u200a=\u200a2/7). Unlike the null and CreFoxg1 mutants there was some formation of spiral ganglion neurons. These few remaining neurons were coalesced into a single location near the base of the shortened cochlear duct, and were not present as a \u201cspiral\u201d ganglion along the length of the cochlea as in the control ear , or Gata3 is necessary for Atoh1 function. This is consistent with some bHLH genes in Drosophila requiring the Gata3 homolog, pannier, for both upregulation of these factors and their DNA binding on downstream genes Our results indicate that in the absence or reduced presence of Gata3, some prosensory cells remain, but are not fully specified and lack full upregulation of Gata3 recombination (Pax2-Cre) we were able to partially assess hair cell and spiral ganglion differentiation in the absence of Gata3. While spiral ganglion neurons are completely absent in the CreFoxg1 ear few spiral ganglion neurons form in the Pax2-Cre ear. However, these spiral ganglion neurons do not differentiate properly as is evident by the aberrantly projecting to the sensory patches in the cochlear duct and the cochlear nucleus. There is a curious resemblance between this mutant and the conditional knockout of Neurod1 utilizing the same Cre line Neurod1 and Gata3 mutants have reduced numbers of spiral ganglion neurons, spiral ganglia cell bodies incorrectly migrate away from the ear towards the hindbrain, and have incorrect patterning within the cochlear nucleus. Interestingly both of these mutants have spiral ganglia that project to the correct regions (organ of Corti and cochlear nucleus), but, once there, do not correctly target appropriate cell types. In addition, timing of Gata3 expression has indicated a role in regulating the Insulin-like Growth Factor (IGF) pathway Pax2-Cre). This is interesting since hair cells in the cochlea do not exit the cell cycle before E11.5 With a slight delay in Atoh1 has been shown to be an essential factor for hair cell development in the ear Atoh1 are essential for hair cell development and viability Atoh1 does not result in long lasting and physiologically functional auditory hair cells Sox2 and Eya1/Six1 have been shown to be necessary for bHLH gene upregulation in the ear, how many other factors are needed in addition remains unclear. Until now no factor has been shown to be exclusively necessary for cochlear and not vestibular hair cells, with the former being known to be more susceptible to a number of genetic and pharmacological insults. We provide here evidence that Gata3 may enable Atoh1 mediated hair cell differentiation in particular in the cochlea. This conclusion is consistent with recent data showing that Gata3 enhances the ability of Atoh1 to upregulate downstream genes Eya1, Pax2, Jag1), which remain expressed in the Cre:Gata3f/fFoxg1 cochlea, are unable to drive Atoh1 expression or neurosensory differentiation in the cochlea in the absence of Gata3. Our data therefore establish Gata3 as an essential player in the emerging molecular network needed for neurosensory development.f/fGata3 cochlea suggests that sustained and profound expression of Gata3 is needed for normal development of these structures. The down-regulation of Tecta in the CreFoxg1 mutant mouse nicely fits into this phenotype as Tecta is necessary for tectorial membrane formation Cre) or severely reduced (Pax2-Cre) in Gata3 mutants. The mal-development of hair cell microvilli in the Pax2-Cre:f/fGata3 cochlea suggests that Gata3 is playing a role not only in hair cell specification but also in hair cell stereocilia differentiation. An assessment of genes selectively down-regulated in these hair cells would be beneficial in elucidating which genes are deregulated in the absence of Gata3 during differentiation. Among those genes could be many microRNA regulated genes as the overall development of hair cells is reminiscent of Dicer1 null mice cochlear hair cells The lack of a spiral limbus, the formation of an incomplete tectorial membrane and formation of only microvilli on hair cells in the Pax2-Cre: Gata3 expression early in ear development in Foxg1Cre: f/fGata3 mice abolishes cochlear neurosensory formation. However, the saccule, utricle and a single anterior canal crista form hair cells and receive afferent and efferent innervation, indicating that Gata3 is less critical in vestibular neurosensory development compared to the cochlea. These ears develop a very short cochlear duct, saccular and utricular recesses, but lack semicircular canals. The lack of canal formation could be caused by lack of canal crista known to be necessary for canal growth f/fGata3 mutant where a single toral structure forms despite the presence of up to three cristae. Formation of a short cochlear duct is consistent with findings in other vertebrates that form outgrowth of such ducts without neurosensory formation Knocking out Cre mutant. We find efferent fibers to vestibular epithelia and presumably some may be olivocochlear efferents which may also project with the facial nerve as previously reported Gata3CreFoxg1 and Pax2-Cre are not expressed in these populations, indicating that this is not a cell autonomous effect. Thus with the loss of their original target vestibular and cochlear efferents are able to segregate, and cochlear efferents are able to survive at least a short time without proper targets.Interestingly, we were able to label the olivo-cochlear efferents in the hindbrain in both mutant lines which migrate their soma into the correct periolivary location Gata3 expression in the forming semicircular canals plays an essential role in translating canal crista signals into proper canal growth. Sustained Gata3 expression is not only needed to initiate development of neurosensory cells but its reduction affects the normal differentiation leading to unusual hair cell formation and aberrant spiral ganglion cell projections. Cochlear development thus depends at multiple levels on the continued presence of Gata3 and future work is now needed to elucidate how Gata3 co-operates with an emerging network of other factors to achieve these multiple functions Click here for additional data file."}
+{"text": "To the Editor: Human astroviruses (HAstVs) are a common cause of gastroenteritis in children, the elderly, and immunocompromised persons (\u2013\u2013www.megasoftware.net) by using the neighbor-joining method and a p-distance algorithm. Bootstrap resampling was performed by using 2,000 replicates to demonstrate robustness of grouping. The nucleotide sequences determined in this study were assigned GenBank accession nos. HQ674630\u2013HQ674650.Fecal samples were collected from a cohort of 364 symptomatic children <5 years of age who had diarrhea and were seeking medical care at Abu Homos Hospital in the Nile Delta of Egypt from September 2006 through September 2007. RNA was extracted from 10% fecal suspensions and reverse transcription PCR for astrovirus was performed as previously described (Consensus astrovirus reverse transcription PCR results were positive in 23 (6.3%) of 364 fecal samples. Five common serotypes of classical HAstV were identified constituting 16 (70%) of 23 positive samples; HAstV type I was most prevalent (n = 9). Alignment of the partial amino acid sequences of the ORF2 capsid region indicates that Egyptian HAstV type I strains share 99%\u2013100% identity , panel AFive of the 7 remaining positive samples were most closely related to MLB1 on the basis of the partial ORF2 sequence analysis. Egypt MLB1 samples shared \u224899%-nt identity with each other, and all grouped in 1 phylogenetic cluster , panel BThe 2 remaining HAstV-positive samples were phylogenetically most similar to astrovirus VA2 (VA2) , panel BOur study describes the detection of the recently identified viruses MLB1 and VA2 in a cohort of symptomatic children with diarrhea residing in Egypt. This study expands the geographic range of these viruses to include northern Africa. The consensus primers used in our study were able to detect a higher percentage of positive HAstV serotypes I\u2013VIII than the Mon340/Astman-2 primers ("}
+{"text": "Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. Bupivacaine is an amide type local anaesthetic used in clinical pain management . Althoug\u03b2/S6K1 axis /IC50), with Graph Pad Prism Software (Version 4.0).Cytotoxicity of bupivacaine was evaluated by its effect on cell viability. Accordingly cell lines NIH-3T3, C6, and C2C12 myoblasts were allowed to grow in the absence (control) or presence of bupivacaine at various concentrations over a period of 24\u2009hrs. While the control cells exhibited normal growth characteristics, significant dose-dependent decline in cellular density was observed in bupivacaine treated cells resulting in substantial cell death . The magGrowth inhibition and apoptosis have quite often been associated with dysregulation of signaling pathways with potential to influence S6K1 activity directly or indirectly. We therefore, sought to investigate any such possibility by analyzing activity status of S6K1 in the presence or the absence of the drug. Endogenous S6K1 was immunoprecipitated from NIH-3T3 cells grown in presence or absence of different bupivacaine concentrations for its ability to phosphorylate GST-S6. As seen in Catalytic and linker domain phosphorylations at the activation loop (AL) and hydrophobic motifs (HM) are established determinants of S6K1 enzyme activity. Accordingly their loss is a hallmark of S6K1 inhibition. We therefore sought to ascertain whether inhibition of S6K1 by bupivacaine did indeed correspond with loss of these phosphorylations. As seen in In an attempt to establish that S6K1 inactivation did indeed mediate the growth inhibitory effects associated with bupivacaine, cells transfected with HA-tagged S6K1 wild type and a constitutively active variant T412E-D3E were examined for their ability to resist bupivacaine induced toxicity. mTORC1 is considered to be a major input acting upstream of S6K1 and implicated in T412 phosphorylation of the enzyme and parallel phosphorylations of its other substrate, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Possible inactivation of mTOR pathway suggested by inhibition of S6K1 should therefore be corroborated by its inability to cause phosphorylations in 4EBP1 (Threonine 70). Phosphorylation of 4EBP1 at Threonine 70 site  remainedn = 3) indicated that the extent of Erk inhibition coincided with the inhibition of S6K1 as much as it did with the kinetics of cell proliferation, to suggest that the effect of bupivacaine may be mediated through inactivation of Erk and S6K1.In the absence of mTOR as the mediator of S6K1 inhibition by bupivacaine, it was imperative to examine other signaling pathways that may directly or indirectly influence S6K1 activity in the cell. Since extracellular signal-related kinase/s (Erk) are believed to participate in the events leading to cytotoxicity by anesthetic drugs like bupivacaine, we sought to investigate the activation status of Erks to possibly relate it with observed inhibition of cellular proliferation and S6K1 activity. The activation of Erks was evaluated by immunoblotting with a phospho specific antibody. As seen in The use of local anesthetics like bupivacaine is associated with chondrotoxicity, myotoxicity, and neurotoxicity at variable propensities , 9, 32. We chose NIH-3T3 cells for their restrained response to bupivacaine induced cytotoxicity compared to C2C12 and C6 cell lines, to help us register the dynamics of signaling events responsible for preventing cell growth and proliferation. We demonstrate that the inhibition of cell proliferation by bupivacaine strictly correlated with the inactivation of ribosomal protein S6 kinase 1 and its relief by the use of constitutively active enzyme to suggest a possible dysregulation of events that control cell division, at least in part via protein translation. With long standing implications that bupivacaine may influence protein synthesis by interfering with amino acylation of tRNAs , the relMAPK signaling has a central role in regulating cell growth and proliferation . AccordiOur data suggests that bupivacaine induced inhibition of cell proliferation is mediated at least in part through inactivation of S6 kinase 1. We also demonstrate redundant role of mTORC1 signaling in mediating this effect to suggest the possible involvement of Erk pathway."}
+{"text": "The input nucleus of the basal ganglia, the striatum, processes cortical inputs in two output streams, called the direct and indirect pathways. Medium Spiny Neurons (MSNs) that express dopamine D1 receptors constitute the direct pathway (D1 MSNs) and MSNs that express dopamine D2 receptors constitute the indirect pathway (D2 MSNs). The two populations of MSNs differ in their intrinsic excitability with D2 MSNs more responsive to somatic current injection  to produ"}
+{"text": "Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members.Members of the var transcript and PfEMP1 expression profiles of the generated parasites were investigated. The IgG reactivity by plasma from children living in malaria-endemic Tanzania was tested to parasites and recombinant VAR3 protein. Parasites from hospitalized children were isolated and the transcript level of var3 was investigated.In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies and the Var3 is transcribed and its protein product expressed on the surface of infected erythrocytes. The VAR3-expressing parasites were better recognized by children\u00b4s IgG than a parasite line expressing a Group B var gene. Two in 130 children showed increased recognition of parasites expressing VAR3 and to the recombinant VAR3 protein after a malaria episode and the isolated parasites showed high levels of var3 transcripts.var3 is transcribed and its protein product expressed on the surface of infected erythrocytes in the same manner as seen for other var genes both in vitro and in vivo. Only very few children exhibit seroconversion to VAR3 following a malaria episode requiring hospitalization, supporting the previous conclusion drawn from var3 transcript analysis of parasites collected from children hospitalized with malaria, that VAR3 is not associated with severe anaemia or cerebral malaria syndromes in children.Collectively, the presented data suggest that  Plasmodium falciparum is a major cause of mortality and disease in sub-Saharan Africa. Immunity to malaria in areas with intense transmission is acquired during childhood as a broad repertoire of specific protective antibodies to parasite-derived polymorphic variant antigens present on the infected erythrocyte surface, develops [Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the best characterized variant surface antigen [var genes per parasite genome [var genes, var1 (UPSA), var2csa (USPE), and var3 (aka Type 3) (UPSA) are conserved in their full length in the global parasite population [vars encoding more diverse domain cassettes not spanning the full length genes [Var3 genes have arisen from a recombination between a DBL\u03b6-DBL\u03b5 encoding sequence only found in the 3\u00b4end of var exon1 and an N-terminal DBL\u03b11 sequence. Sequence analysis has shown that only the DBL\u03b6-DBL\u03b5 part of VAR3, which is 99% identical between VAR3 sequences, is unique to the protein sub-family [develops -3. Plasm antigen -6. A sin antigen -11 as th antigen . Members antigen ,14. The e genome , which ce genome ,17. Threpulation -22. Aparth genes . PfEMP1sth genes  and is tth genes . Var3 geb-family .var types expressed in severe malaria [var mRNA species by PCR amplification and sequencing of a short 350 bp DBL\u03b1 tag present in all var genes except var2csa and var3. As this approach does not capture var3 sequences, quantitative PCR has been deployed to investigate var3 transcript levels in patient samples [var genes are associated with severe malaria in children.VAR2CSA binds chondroitin sulphate A (CSA) in the placenta and facilitates the parasite sequestration causing pregnancy-associated malaria. Similarly, organ specific PfEMP1 mediated sequestration has been linked to severe malaria in children , and evi malaria ,30,33-36 samples ,37. The var3 transcript levels in malaria patient samples have shown that var3 transcripts was not associated to any particular syndrome of severe malaria [P. falciparum-infected erythrocytes has never been achieved and evidence presented by Epp et al.[var3 transcription may be regulated differently from the rest of the var genes and expressed in a non-mutually exclusive manner due to an atypical intron activity.Because VAR3 is conserved, belongs to group A PfEMP1 and has an unusual domain structure, this protein could play a particular important role for the parasite and in development of malaria disease. Malaria-endemic populations has been shown to have acquired antibodies that react with recombinant VAR3 protein , suggest malaria . A formapp et al. suggestsvar3 mRNA and VAR3 protein is expressed similar to other PfEMP1s. Furthermore, in this study, evidence for in vivo expression of VAR3 in malaria-sick children is presented.In an effort to unravel some of the mystery surrounding this PfEMP1, parasites of different genetic background were successfully manipulated to express VAR3 on the surface of the infected erythrocytes. Analysis of these parasites suggests that both Plasmodium falciparum clones 3D7 and IT/FCR3 parasites were cultured in O Rh\u2009+\u2009erythrocytes in RPMI1640 supplemented with Albumax II, as previously described [escribed  by repeaet al.[P. falciparum negative controls, human DNA from Danish donors and blank samples, were included in the setup and the amplified PCR products were analysed by agarose gel electrophoresis.Isolate integrity of the cultures was tested using the method previously described by Snounou et al. with theseryl-tRNA synthetase gene. RNA was reverse transcribed from random hexamers, using Superscript II (Invitrogen), according to the manufacturer\u2019s instructions (Invitrogen).RNA was extracted using Trizol (Invitrogen) according to manufacturer\u2019s instructions. Samples were treated with DNase I (Sigma) to digest any genomic DNA and tested in quantitative PCR for contamination, using a primer set, p90, for the var gene of the P. falciparum clone 3D7 and to the endogenous control genes seryl-tRNA synthetase and fructose-bisphosphate aldolase, have been described previously [var genes extracted from the IT/FCR3 genome available at The Wellcome Trust Sanger Institute (UK) [PFI1820w/PFF0020c/IT4var3 (var3Xi) and PFA0015c (PFA0015cXi) transcripts, respectively, were designed as well   -\u2206CtQuantitative PCR was performed on a Rotorgene RG-3000 thermal cycler (Corbett Research), applying QuantiTect SYBR Green PCR Master Mix (Qiagen) with primers at 20 \u03bcM, and internal control genes + erythrocytes and incubating 24 h at 4\u00b0C. Besides the full length VAR3 protein, two full length and four multi-domain PfEMP1 proteins were included in the Luminex assay  and DAPI and laser scanning confocal microscopy performed using a Nikon TE 2000-E confocal microscope with 60x oil immersion objective lens (DIC) as previously described [Indirect immuno-fluorescence assays were performed on live 3D7VAR3/3D7 unselected and FCR3VAR3/IT/FCR3 unselected in order to observe the staining of individual infected erythrocytes with VAR3-antibodies. The staining was done using Alexaescribed .Levels of plasma PfEMP1 domain-specific IgG were analysed as described previously . In briein vitro model parasite lines expressing VAR3, parasites of two laboratory cultured parasite clones, 3D7 and IT/FCR3, were selected with rabbit antiserum against a 3D7 VAR3 (PFI1820w) recombinant protein. The expression of VAR3 on the surface of 3D7VAR3- and FCR3VAR3-infected erythrocytes, respectively, was investigated by flow cytometry and confocal microscopy using rabbit IgG specific for the clone 3D7 VAR3 PFI1820w which they had been selected with. The selected lines, 3D7VAR3 and FCR3VAR3, were recognized by the rabbit serum and stained the infected erythrocytes in a punctuate pattern typical for PfEMP1 [To generate r PfEMP1 ,51 wherevar transcript profiles of the selected 3D7VAR3 and FCR3VAR3 and unselected 3D7 and IT/FCR3 parasite lines were investigated using var gene specific primers in quantitative PCR. The transcript levels of all var genes relative to internal control genes are shown in Figure var3 transcripts, PFI1820w and IT4var3, than the unselected lines and the var3 transcript levels were similar to those observed for var genes in vivo[in-vitro parasite cultures with verified erythrocyte surface expression of PfEMP1 [var3 genes, resulted in selection of parasites expressing two of these genes (PFI1820w and PFF0020c) but not parasites expressing the third (PFA0015c) , with a peak in transcript level at 19 h\u2009PM corresponding to mid ring stages  Figure . A speciite line  and fourite line .var gene, IT4var60, thought to mediate rosetting [var gene, IT4var59  of the acute patient plasma samples. Only two boys, patient A and B, appeared to acquire IgG to VAR3 but not to VAR2CSA expressing parasites after being sick and hospitalized with malaria Figure . An incr primers  . In prechildren -32. The isolates ,37 does  general ,28-30. Ovar3 and the presence in almost every parasite genome, it is striking that only a fifth of 1,342 individuals from malaria-endemic areas in Tanzania showed acquisition of antibodies to this PfEMP1 [The pattern of Tanzanian children\u00b4s acquisition of antibodies to live VAR3-expressing parasites compared to UPSA and UPSB type PfEMP1-expressing parasites indicate a virulence of VAR3 similar to UPSA type PfEMP1. Yet, in spite of the conserved nature of s PfEMP1 . This mas PfEMP1 ,37 and tvar3 transcribing parasites and VAR3 antibodies in malaria patients can be challenged if var3 genes are regulated and expressed abnormally as appears to be the case for var1. Studies of laboratory parasite lines show that var1 is constitutively transcribed throughout the blood stages cycle, while the transcription of other var genes is silenced when parasites develop into schizonts [var1 often lacks or has a truncated exon2 which may cause a corrupted transcription regulation resulting in protein synthesis and host antibody development upon parasite destruction. It remains unclear if VAR1 serves a particular function for the parasite and why var1 is maintained in the population. Indeed, Epp and colleagues [var intron of the PFF0020c var3 gene did not show any transcriptional activity as opposed to other non-VAR3 var introns. Non-coding RNA is transcribed from var introns and is physically associated with chromatin and believed to be involved in the transcriptional regulation of var genes [var3 genes in general could be regulated differently from other var genes. From the present data it does not seem that var3 genes are transcribed and expressed differently from other var genes. In both 3D7VAR3 and FCR3VAR3 var genes other than var3 were silenced and the var3 transcript profile during the intra-erythrocytic life cycle was similar to that of other var genes in other parasite lines [These interpretations of chizonts . In spitchizonts , VAR1 erlleagues  found thar genes . Thus, ite lines ,52 and pAlso the VAR3 expression on the erythrocyte surface appeared similar to that of other PfEMP1. Antibodies raised towards a recombinant 3D7 VAR3 variant successfully selected VAR3 erythrocyte surface-expressing parasites from both 3D7 and IT/FCR3 parasite populations. The surface expression of VAR3 was confirmed by antibody staining in both flow cytometry and confocal microscopy with expression levels and a punctuate pattern characteristic for PfEMP1 expression in knobs. While the VAR3 in the IT/FCR3 line most likely is the only PfEMP1 on the infected erythrocyte surface, it is possible that both PFI1820w and PFF0020c VAR3 PfEMP1s are expressed by the same parasites, as previously seen in 3D7 parasites .var3 is transcribed and the encoded protein expressed on the surface of infected erythrocytes in the same manner as seen for other viable var genes. VAR3-expressing parasites sustain P. falciparum infections in children living in malaria-endemic areas although only found in a fraction of malaria hospitalizations and not associated with severe anaemia or cerebral malaria syndromes. Whether this structurally unique and conserved PfEMP1 confer parasites expressing it with a rare functional specialization remains unclear, but further studies of the cyto-adherence phenotype of the established VAR3-expressing parasite lines may elucidate this.In conclusion, The authors declare that they have no competing interests.CWW carried out molecular biology studies, analysed data and wrote the paper. TL and TGT participated in the design, coordination and analysis of the study and helped to draft the manuscript. LT designed and carried out molecular biology studies, analysed data and helped to draft the manuscript. DCB, SBS, AMM and MA carried out molecular biology studies and revised the manuscript. PAM and JPL participated in the coordination of the study and revised the manuscript. All authors read and approved the final manuscript.Specificvargene primers used for Q-RT PCR.Click here for filePrimers used forPlasmodium falciparumerythrocyte protein 1 full length (FL) and multi-domain expression.Click here for fileVargene transcript levels ofPlasmodium falciparumIT4var59 parasites.Var gene transcript levels of IT4var59 parasites relative to the average of two internal control genes. The average of control gene transcript levels is set to 100. The distribution of var transcripts is also shown (circular diagram). Transcript levels were measured by quantitative PCR using a set of primers that amplify 56 var genes from clone IT/FCR3. The grouping of var genes are as previously described [escribed ,17,23.Click here for fileTranscript levels ofvar3in patient parasite isolates. Transcript levels of var3-DBL\u03b58 in P. falciparum parasites isolated from 130 hospitalized children  and the var subtype transcript levels in patient parasite isolates, A and B. The levels are relative to the average of two internal control genes, which are normalized to 100. Transcript levels were measured by quantitative PCR using a set of 42 subtyping quantitative primers [ primers .Click here for file"}
+{"text": "Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20\u201330% of genes and both marks in 5% of genes. H3K9m3 was detected in 5\u201310% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer. Bladder cancer is the fifth commonest malignancy in the United States with 70, 530 new cases and 14,680 deaths in 2010 Molecular changes in cancer arise from either genetic or epigenetic events. The latter is defined as stable heritable changes in a chromosome without alterations in the DNA sequence We have previously observed changes in DNA methylation and microRNA expression that reflect the molecular biology of UCC and are associated with the clinical phenotype of tumors We performed massively parallel sequencing to determine the distribution of DNA adjacent to H3K9m3 and H3K27m3 in urothelial cells. Each experiment yielded between 7.7 M and 18.9 M reads . The nat4 bases distant. For H3K27m3, the relationship was bimodal with peak enrichment occurring at both 104 and 107 base distances.Recent data suggest that many tissue and cancer-specific differentially DNA methylated regions (T and C-DMRs) occur within 2 kb around CpG islands , filtered for those with differences between the malignant and normal cells  and selected probes with differential hypermethylation in the cancerous lines ). We mapped these probes to their nearest neighbors to identify regions with adjacent (those <200 bp apart) enriched probes. Depending upon stringency, we found between 1,513 (5/5 adjacent probes) and 106 (11/11 adjacent probes) CpG islands around 711 and 40 protein-coding genes, respectively. Of these, 20% and 26% were located within a promoter and the remainder within (66% and 8%) or downstream from the nearest gene. We annotated the 5 mC profile in each cell line for these 68,292 probes using mRNA expression and obtained data for 10,568 genes . When measured an inverse correlation was seen between mRNA expression and 5 mC enrichment for these probes . In general, DNA and histone enrichment were negatively correlated to mRNA expression . This reTo determine specific loci important for urothelial carcinogenesis we identified those with cancer-specific silencing and associated epigenetic modifications. Specifically, we selected genes with one or more repressive epigenetic mark (defined as within the highest 20% of enrichment) that was shared in the two cancer lines and absent in NHU. From these, we chose genes with reduced expression in EJ and/or RT112, and preserved expression in NHU. This strategy identified 239 and 317 genes in EJ and RT112, respectively . Of thesTo evaluate epigenetically mediated gene upregulation in cancer we used the combined dataset  to identify those with DNA hypomethylation and/or loss of enrichment for either histone modification in the cancer cell lines, when compared to NHU. From these we selected genes with cancer specific expression  Here we have produced integrated epigenomic maps for two UCC cell lines and non-transformed normal urothelial cells. The latter are cultured expansions taken from disease free patients that grow as sheets of histologically normal appearing urothelial cells for 7\u20138 passages before senescence. Our experimental data reveal insights into the epigenetic control of gene expression in the biology of UCC and have specifically identified genes potentially involved in urothelial carcinogenesis.At the global level several observations are apparent. Firstly, H3K9m3 and H3K27m3 are located mostly around gene transcription start sites Our data detail the associations between H3K9m3, H3K27m3, CpG islands and DMRs , one would suspect that the pro-carcinogenic pathways associated with 5 mC are those contributing to malignant transformation.To assess the validity of the genes identified using cell lines, we examined their expression in 150 normal and malignant urothelial samples. Our epigenetic panel correctly estimated the expression of 65% of genes and stratified the tissues according to the presence and phenotype of cancer. This is important as previous epigenetic studies using candidate tumor suppressor genes reveal that low-grade non-invasive cancers differ to high grade and invasive UCC One of the most interesting candidates within our panel is the Cellular Apoptosis Susceptibility/CSE1 chromosome segregation 1-like gene (hCAS/CSE1L). This component of the nuclear import pathway is frequently upregulated in cancer and located at 20q13 within a region amplified in malignancy In conclusion, we have mapped repressive epigenetic events in malignant and normal urothelial cells. In genes with low expression we identified associated H3K27m3 and DNA methylation each in 20\u201330% of genes and both marks in 5% of genes. When all genes were analyzed H3K9m3 did not appear to be associated with expression. DNA methylation was more closely related to gene expression in cancerous than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. We identified a panel of genes with cancer specific epigenetic mediated aberrant expression including those with known carcinogenic functions and members potentially mediating a positive epigenetic feedback loop.We analyzed bladder cancer cell lines representing non-invasive and invasive disease  grown in Dulbecco's medium with 10% fetal calf serum, and normal human urothelial (NHU) cells maintained in keratinocyte serum-free medium containing bovine pituitary extract, epidermal growth factor  and cholera toxin Malignant and normal human urothelial cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature. Glycine was added to the final concentration of 0.125 M and the mixture incubated for 5 min to quench unreacted formaldehyde. Cross-linked cells were washed with PBS, lysed in 250 \u00b5l SDS lysis buffer and sonicated . Samples were pre-cleared with 15 \u00b5l of protein G dynabeads (Invitrogen) and 50 \u00b5l supernatant saved as total input control. Rotating samples were immunoprecipitated overnight at 4\u00b0C with antibodies against H3K9m3 , H3K27m3 (Millipore) and rabbit IgG as a negative control . The chromatin:antibody complex was incubated with 20 \u00b5l dynabeads protein G for 1 h at 4\u00b0C, before washing and DNA elution. Cross-links were reverted by overnight incubation with RNAse A at 65\u00b0C before DNA purification using columns . Immunoprecipitated DNA ends were repaired using the PNK and Klenow enzyme, adenosine was added to the 3\u2032 end of the fragments and the DNA was ligated with the adapters. Following ligation, the ChIP DNA was amplified for 17 cycles and fragments around 200\u2013300 bp isolated using electrophoresis in an agarose gel. The purified DNA was used for cluster generation and sequencing analysis using the Solexa 1 G Genome Analyzer according to manufacturer protocols  as detailed elsewhere Methylated DNA immunoprecipitation and tiling CpG island microarrays  (MeDIP-CHIP) were used to determine genome wide distribution of 5 mC Whole genome mRNA expression was determined by microarray  as detailed elsewhere Our analyses identified a panel of genes that appeared to be important in urothelial carcinogenesis. To evaluate their role in human UCC, we extracted expression data from Array Express . all genes and (b). for genes around CpG islands. As can been seen, H3K9m3 appeared more specific to CpG islands than H3K27m3.(PDF)Click here for additional data file.Figure S2Gene expression and histone enrichment. Gene expression was an average of more than 10 fold lower in TSS with H3K27m3 enrichment, compared to those without enrichment Click here for additional data file.Figure S3Epigenetic gene silencing in bladder cancer. Venn diagrams represent number of down regulated genes in EJ and RT112 bladder cancer cell lines, when compared to NHU, associated with each epigenetic mark.(PDF)Click here for additional data file.Figure S4Epigenetic gene upregulation in bladder cancer. Venn diagrams represent number of upregulated genes in EJ and RT112 bladder cancer cell lines, when compared to NHU, associated with each epigenetic mark.(PDF)Click here for additional data file.Figure S5Functional annotation clustering of genes with epigenetic marks in EJ cells. Using gene enrichment pathway analysis we determined clusters of genes for each set marked by an epigenetic modification. The number of gene clusters within each part of the diagram is indicated in these area proportional Venn diagrams.(PDF)Click here for additional data file.Figure S6The expression of genes within our combined epigenetic panel in an external dataset . The median expression of 124 genes is shown when stratified according to the epigenetic traits found in EJ, RT112 and NHU. As shown, genes with predicted epigenetic upregulation had higher expression than those with predicted silencing.(PDF)Click here for additional data file.Figure S7Unsupervised hierarchical clustering stratified malignant and normal urothelial samples according to phenotype. Previously reported microarray data (PDF)Click here for additional data file.Table S1Reads per experiment obtained from massively parallel sequencing.(PDF)Click here for additional data file.Table S2Details of the combined epigenetic gene panel stratified for associated events and mRNA expression. Below each column is the proportion of total events (as a percentage).(PDF)Click here for additional data file.Table S3Gene ontology analysis for each epigenetic mark. The table reveals the GO details for all identified pathways associated with each event.(PDF)Click here for additional data file.Table S4Phenotype specific gene expression in the epigenetically selected panel as determined using gene expression data .(PDF)Click here for additional data file."}
+{"text": "HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5\u201310 mL) were collected prospectively from patients with clinical stages I\u2013IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK\u2212/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45\u2212/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2\u2212 primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2\u2212 primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.Human epidermal growth factor receptor 2 ( Mammographic screening and systemic chemotherapy in the adjuvant and neoadjuvant settings have prolonged survival of patients with breast cancer. However, many patients experience disease relapse and progression despite such treatment. Clinical trials have indicated that a cure can be achieved in a proportion of patients by personalizing their treatments. Yet, the tests needed to facilitate personalized treatment in breast cancer, such as quantification and biomarker assessment of minimal residual disease in blood and bone marrow (BM), have not been fully realized.The occurrence of circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in BM in patients with early or advanced breast cancer is well recognized. These tumor cells most likely play an important role in the complicated process of metastasis Despite early recognition of the potential utility of evaluating CTCs, isolating these cells has remained a challenge because of their extreme rarity in blood. Their estimated frequency is only one cell per 1 billion blood cells It is well recognized that tumor cells undergo epithelial-to-mesenchymal transformation (EMT) as part of the metastatic process BM has long been recognized as an important homing organ for different types of malignant epithelial tumors. The presence of micrometastases in BM at surgery has been shown to be an independent prognostic factor in breast cancer HER2) gene amplification in these cells.The OncoCEE\u2122 (Cell Enrichment and Extraction) microfluidic platform  has been developed and been shown to efficiently capture and detect CTCs Samples of peripheral blood and BM were collected from patients with operable breast cancer in a prospective protocol approved by the institutional review board of The University of Texas MD Anderson Cancer Center . Blood was collected into 8.5-mL vacutainer tubes containing the anticlumping agent CEE-Sure\u2122  and shipped to Biocept's CLIA/CAP\u2013accredited laboratory for processing. BM aspirates (7.5\u201310 mL) were collected from the bilateral anterior superior iliac crests prior to any surgical manipulation of the primary tumor and were put into tubes containing ethylenediaminetetracetic acid. BM specimens were processed at MD Anderson Cancer Center using a standard density-gradient procedure; a portion of each final cell pellet was shipped to Biocept for DTC capture and analysis.g for 10 min yielded final cell pellets, which were subsequently subjected to tumor cell capture within the OncoCEE\u2122 microchannels. Both CTCs and DTCs captured in this way were then subjected to FISH for detection of HER2 gene amplification.Tumor cells were enriched by density-gradient separation using Percoll (blood) or Ficoll\u2013Hypaque (BM) solution. Following separation, both sample types were incubated with Fc blocker (100 \u03bcg/mL) and a 10-antibody capture cocktail  for 30 min at room temperature. The CEE-Sure\u2122 anticlumping reagent was added to the BM samples during the antibody incubation period. Both sample types underwent a wash and centrifugation followed by incubation with biotinylated secondary antibody for 30 min. Three additional washes with phosphate-buffered saline solution/casein buffer and centrifugation at 400 The OncoCEE\u2122 microchannel technology has been described previously For each peripheral blood pellet, captured CTCs were stained with a mixture of pan-CK , anti-CK 7/17 (clone C-46), anti-CK 18 (clone DA/7), and anti-CK 19 (clone A53-B/A2) antibodies labeled with AlexaFluor-488. The CTCs were costained with anti-CD45 labeled with Alexa-594 and then counterstained with DAPI III  for immediate enumeration and localization by microscopic analysis. All CK+/CD45\u2212/DAPI+ cells were classified as CTCs. The precise location (X and Y stage coordinates) of each CTC was recorded, permitting relocalization of cells following FISH for nuclear interrogation. The CTCs were visualized and imaged by an Olympus Bx51 fluorescent microscope  at 200\u00d7 magnification.For each BM pellet, antibody-labeled DTCs were similarly captured by the streptavidin-coated microchannels but were then immediately processed for FISH. Captured DTCs were not subjected to immunostaining to detect CK and/or CD45. All nuclei were counterstained with DAPI III as part of the FISH procedure (described in the next section).HER2 DNA Probe Kit, which consists of two direct-labeled probes (Abbott Molecular) specific to the centromere of chromosome 17 (CEP17-SpectrumGreen) and the HER2 locus (SpectrumOrange). The FISH analysis for CTCs was performed in two steps. First, each CK+/CD45\u2212/DAPI+ cell was relocated and scored for the number of signals corresponding to each probe. All CK\u2212/CD45\u2212/DAPI+ cells (representing possible CTCs lacking CK and CD45 expression) were then scored. This strategy allowed simultaneous scoring of both CK+/CD45\u2212/DAPI+ and CK\u2212/CD45\u2212/DAPI+ CTC phenotypes for the CEP17 and HER2 probes. Because there are no stain criteria for DTCs, all captured DTCs were subjected to FISH.The microchannels were processed for FISH using the FDA-approved PathVysion HER2:CEP17 ratios were recorded, and all cells whose ratio was more than or equal to 2.0 were regarded as positive for HER2 gene amplification.Both CTCs and DTCs were visualized and imaged by the Olympus Bx51 fluorescent microscope at 400\u00d7 and 600\u00d7 magnification. HER2 status of CTCs and DTCs was compared with that of the primary tumor. Discordance in HER2 status was calculated based on the number of patients with HER2+ primary tumors with no evidence of HER2 gene-amplified CTCs or DTCs and those patients with HER2\u2212 primary tumor with, however, the presence of HER2 gene-amplified CTCs or DTCs.The Matched samples of peripheral blood and BM from 70 patients, peripheral blood alone from 25 patients, and BM alone from eight patients with operable breast cancer, were utilized in the study. Of the 95 patients whose blood samples were processed and subjected to CTC capture using the OncoCEE\u2122 microfluidic device, 26 (27.3%) had CK+/CD45\u2212/DAPI+ CTCs , in numbHER2 in 88 of the 95 patients from whom blood was collected for the study. In 70 of these 88 patients, matched specimens of BM could be evaluated for HER2 by FISH. The details of the clinicopathologic features of the primary tumor are summarized in We could perform FISH for HER2-amplified CTCs in 6 of the 88 (6.8%) patients from whom peripheral blood could be tested for HER2 gene amplification status by FISH using the OncoCEE\u2122 device. The HER2:CEP17 ratios in the gene-amplified CTCs ranged from 2.0 to 18.3. HER2 gene amplification was observed only in CK\u2212/CD45\u2212/DAPI+ cells in two patients, while it was detected only in CK+/CD45\u2212/DAPI+ in the remaining four patients. Among these six patients with HER2-amplified CTCs, four had their primary tumor staged T1N0 and two as T3N0. The primary tumor was positive for HER2 gene amplification in only one of these patients. Therefore, while HER2+ CTCs were found in 1 of the 9 HER2+ primary tumor, they were encountered in 5 of the 79 HER2\u2212 primary tumors. The details of the HER2 gene-amplified CTCs and correlation with HER2 status of the primary tumor is summarized in We observed HER2-amplified FISH signals in all the captured cells. HER2 gene amplification by FISH was observed in 17 of 78 (21.7%) patients for whom a BM specimen was available, with HER2:CEP17 ratios ranging from 2.0 to 15.0. The primary breast tumor was positive for HER2 by FISH in 3 of these 17 (17.6%) patients, and all of these patients were staged as T1N0. In the remaining 14 cases of HER2+ DTCs, the primary tumor was negative for HER2 gene amplification. These 14 tumors were staged as T1N0 (n = 7), T1N1 (n = 2), T2N0 (n = 2), T2N1 (n = 1), T2N3 (n = 1), and T3N0 (n = 1). Therefore, HER2+ DTCs were found in 3 of the 11 patients with HER2+ primary tumors and in 14 of the 67 patients with HER2\u2212 primary tumors. The details of the HER2 gene-amplified DTCs and correlation with HER2 status of the primary tumor is summarized in Initially, CK/CD45 staining was attempted on BM specimens, but we observed positive CK staining in cells that were clearly of hematopoietic origin, which indicated frequent false-positive labeling of nonepithelial hematopoietic cells for CK and other antibodies included in the antibody cocktail. Therefore, we could not reliably ascertain the frequency of CK+/CD45\u2212/DAPI+ cells in BM as we could in the peripheral blood. However, we could accurately interpret HER2 status between the primary tumor and CTCs or DTCs included patients in whom the primary tumor was HER2+, but HER2+ CTCs or DTCs were not detected and patients in whom the primary tumor was HER2\u2212, but the CTCs or DTCs were positive for HER2 gene amplification. Overall, 1 of 9 (11.1%) patients with HER2+ primary tumor had HER2+ CTCs and 3 of 11 (27.2%) patients had HER2+ DTCs. Only one patient had concurrent HER2+ CTCs and DTCs. Among patients with a HER2\u2212 primary tumor, 5 of 79 (6.3%) had HER2+ CTCs and 14 of 67 (20.8%) had HER2+ DTCs; one patient had concurrent HER2+ CTCs and DTCs. The overall discordance of HER2 gene amplification including patients with HER2+ primary tumor with HER2\u2212 CTCs and DTCs and HER2\u2212 primary tumor with HER2+ CTCs and DTCs amounted to 15% for CTCs and 28.2% for DTCs.Discordance in the HER2 gene amplification were 15% between primary breast tumor and residual tumor cells in peripheral blood  and 28.2% between primary breast tumor and residual tumor cells in BM . These results confirm that a proportion of patients with breast cancer harbor tumor cells in blood and/or BM, and that the HER2 status of these residual tumor cells can be different from that of the primary tumor.Overall rates of discordance of HER2 status in CTCs and DTCs and their possible role in the personalized treatment of breast cancer is well recognized. Phenotypic characterization of CTCs and DTCs in breast cancer has been reported by previous studies using techniques such as immunofluorescent or immunocytochemical staining to assess HER2 protein overexpression and FISH or polymerase chain reaction (PCR) to evaluate HER2 gene amplification. FISH is considered to be the standard for accurate determination of HER2 status in the selection of patients for trastuzumab therapy because of its low rates of false-negative or false-positive results. Preanalytic and analytic factors yielding inaccurate results can be a major problem with the immunostaining, immunofluorescence, and PCR techniques. Very few studies have reported using FISH for detecting HER2 status of CTCs or DTCs in breast cancer. The current study is one of the largest.The potential utility of evaluating HER2 expression of CTCs and, to a lesser extent, DTCs in breast cancer HER2, MUC1, and GA733-2). Only two small studies have used FISH to evaluate HER2 gene amplification status in CTCs, in both cases following enrichment of EpCAM-expressing cells from peripheral blood by immunomagnetic separation HER2 amplification was regarded as positive for HER2.Several reports have compared HER2 evaluation of CTCs have varied across studies. Studies including patients with locally advanced or metastatic disease reported HER2+ CTCs in 24\u201347% of the patients, with HER2 status discordance rates ranging from 18% to 58%. In patients with operable breast cancer, 7.9\u201329% have had HER2+ CTCs, with discordance rates ranging from 3.7% to 44.0%. The 3.7% discordance rate, which corresponded to the 7.9% frequency of HER2+ CTCs, was reported by Ignatiadis et al. HER2 status of CTCs HER2 in primary breast tumors varies from 92% to 98% in the reported studies HER2 could be related to several factors. True biologic differences between RNA levels and DNA gene amplification, analytic variability of two different testing methods, extent of chromosome 17 polysomy, and dilution of mRNA obtained from tumor with those from other nonneoplastic elements can all lead to discordant results The results of HER2 gene amplification in DTCs of patients with operable breast cancer is perhaps the first in the literature. The four previous reports of HER2 expression in DTCs in patients with operable breast cancer utilized immunofluorescence and immunocytochemical staining with a HER2-specific antibody and RT-PCR for HER2 mRNA evaluation HER2 discordance in DTCs in those studies ranged from 38% to 44%. Our 28.2% discordance is lower than these studies, which again is most likely related to the different techniques used for evaluation of the HER2 status. The lower discordance rate found in our study is unlikely due to the representation of patients who had early-stage disease without evidence of metastatic disease because of possible independent modes of dissemination through the hematogenous and lymphatic routes HER2 gene-amplified DTCs in 20.8% of patients, including 25% of those with a HER2+ primary tumor and 20.8% of those with a HER2\u2212 primary tumor.Our report of HER2+ CTCs and DTCs in patients with a HER2\u2212 primary breast tumor has been reported by all previous studies irrespective of the patient population studied, but the frequency of HER2+ CTCs and DTCs in these patients has varied among these studies. HER2+ CTCs and DTCs were encountered in 19\u201339% of patients with locally advanced or metastatic disease and in 3.7\u201331% of patients with operable breast cancer, respectively. The 6% and 21% rates of HER2+ CTCs and DTCs, respectively, that we encountered in patients with a HER2\u2212 primary tumor fall within these previously reported ranges.The occurrence of HER2+ CTCs or DTCs in HER2\u2212 primary tumors is extensively debated. While the underlying cause of this discordance is not entirely clear, clonal selection of rare HER2+ cells in the primary tumor following treatment is considered to be a possibility in patients with metastatic breast cancer. Genetic instability of the cells constituting minimal residual disease, resulting in acquisition of HER2 in the course of the disease, has been suggested as another possibility. The fact that some of our patients with a HER2\u2212 primary tumor demonstrated HER2+ CTCs and DTCs despite having early-stage disease clearly suggests that clonal selection or acquisition due to genetic instability can be encountered very early in the course of the disease. Meng et al., in their study of CTCs detected by FISH in patients with metastatic breast cancer, found lower ratios of HER2:CEP17 in CTCs than in the corresponding primary tumor. Our results clearly indicate that the extent of amplification in CTCs and DTCs can vary relative to that in the primary tumor.The exact cause of HER2+ DTCs to occur more often than HER2+ CTCs in our patient population . HER2+ CTCs and DTCs occurred simultaneously in only two patients, and more commonly in either blood or BM. These results establish the importance of evaluating both CTCs and DTCs when identifying breast cancer patients with discordant HER2 expression. In our study, discordance in HER2 status between HER2\u2212 primary breast tumors and minimal residual disease in blood or BM was contributed more often by DTCs than by CTCs (28.2% vs. 15%).Notably, none of the previous studies evaluated CTCs and DTCs simultaneously in patients with breast cancer. We found HER2 gene amplification by FISH. Our identification of CTCs and DTCs with HER2 gene amplification using the OncoCEE\u2122 microchannel system needs further validation in early and advanced breast cancer. This system provides a robust platform for detailed phenotypic and genotypic characterization of intact CTCs and DTCs for accurate evaluation of HER2 status. The clinical utility of this platform for determining HER2 gene amplification by FISH in CK-positive and -negative CTCs and DTCs needs to be tested in large, prospective, multi-institutional clinical trials.The OncoCEE\u2122 microfluidic platform has been shown to be highly efficient and sensitive for the capture of CTCs and now, as shown by our results, for DTCs. The inner surface of the microfluidic channels is coated with streptavidin to which any single biotinylated antibody, or any combination of antibodies, can attach. Mononuclear cells isolated from blood and BM were enriched for tumor cells using an antibody cocktail that included EpCAM. This antibody cocktail helped drive tumor cell capture by increasing cell surface antigen densities. As a result, both epithelial tumor cells expressing low levels of EpCAM and cells with a mesenchymal phenotype lacking EpCAM were captured simultaneously from the same sample. Therefore, we recovered both CK-positive and -negative CTCs and DTCs that were suitable for subsequent testing for"}
+{"text": "Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.The Zap1 transcription factor of  The activity of transcriptional regulatory proteins can be regulated by a variety of mechanisms. These include the control of transcription factor expression, stability, subcellular localization, DNA binding activity, and activation domain function. While the activity of many transcription factors is regulated by only a single mechanism, some are regulated at multiple levels. Multiple levels of regulating a single transcription factor allow that factor to respond to different signals. For example, the C/EBP transcription factors are regulated at transcriptional, translational, and post-translational levels Another advantage of multiple regulatory mechanisms controlling the activity of a transcription factor is the combined effects each mechanism can have on the response of that factor to a single stimulus. For example, the yeast Pho4 transcription factor is regulated in response to phosphate status by phosphorylation at four different sites in the protein E. coli, zinc homeostasis is accomplished largely through the transcriptional control of zinc uptake and efflux transporters E. coli cells strive to maintain essentially no free zinc in their cytosol In this report, we address the multiple levels of regulation that control the yeast Zap1 protein in response to zinc status. Zinc is an essential nutrient for all organisms because of its many functions as a structural and catalytic cofactor. Zinc is also potentially toxic to cells when accumulated in high amounts. The essential but potentially toxic nature of this metal necessitates precise homeostatic control mechanisms. In Saccharomyces cerevisiae. The Zap1 transcriptional activator is a central player in the adaptation of these cells to zinc-limiting conditions. Zap1 activates the expression of as many as 80 genes We know much about zinc homeostasis in eukaryotes from studies of the yeast 2H2 zinc fingers (Znf). Znf3-7 are located at the C-terminus (residues 705\u2013880) and comprise the DNA-binding domain. This domain is responsible for specific recognition of Zinc Response Elements, or ZREs, found in one or more copies in Zap1's target promoters 5\u2032-ACCTTNAAGGT-3\u2032 and we have shown previously that Zap1 binds to this palindromic sequence as a monomer and not as a dimer Zap1 is an 880-residue protein containing seven CZinc-Responsive Domain of AD1 (ZRDAD1) that is required for controlling AD1 function AD2) includes both Znf1 and Znf2. Studies to date indicate that zinc regulates AD1 and AD2 directly by binding to ligand residues in their respective ZRDs to repress activation domain function Zap1's two activation domains, AD1 and AD2, are responsible for increasing gene expression in response to low zinc. AD1 lies between residues 207 and 402 and is embedded within a larger region (residues 182\u2013502) termed the DBD) was zinc regulated While regulation of AD1 and AD2 clearly plays an important role in Zap1 zinc responsiveness, other levels of control may also contribute. For example, at the transcriptional level, Zap1 induces its own expression in low zinc via positive autoregulation ZRT1-lacZ reporter is highly induced by zinc limitation and expressed at decreasing levels as the concentration of zinc in the medium is increased (zap1\u0394 cells expressing wild-type Zap1 at a low level from the GAL1 promoter (pZap1WT) showed a similar response. Although this promoter is highly induced by galactose, we grew these cells in glucose where the level of Zap1 expression was similar to the level observed in zinc-replete wild-type cells expressing Zap1 from its own promoter zap1\u0394 cells expressing a mutant form of the protein (Zap1TC) in which AD1 and AD2 function is made constitutive by mutating regulatory residues required for the zinc responsiveness of those domains ZRT1 and ZPS1, two chromosomal targets of Zap1, were assayed under low and high zinc conditions (2+ in the medium available to cells is less than what is found in standard yeast culture media (i.e. YPD or SD) CMD1, encoding calmodulin, was used as a negative control and its levels were not altered by zinc status.In wild-type cells that express Zap1 from its own promoter, a Zap1-regulated TC protein and the GAD-Zap1DBD fusion in vivo dimethyl sulfate (DMS) footprinting to examine occupancy of a Zap1 binding site (ZRE1) in the ZRT1 promoter zap1\u0394 mutant grown in zinc-limiting  (see The observation that the Zap1ion) see . Less thZAP1 mRNA ZAP1 transcriptional autoregulation does indeed cause corresponding changes in Zap1 protein level. Therefore, the apparent loss of ZRT1 ZRE occupancy observed in wild-type cells grown in high zinc could be due to decreased Zap1 DNA binding activity and/or decreased Zap1 protein level resulting from transcriptional autoregulation.Zap1 controls its own transcription via positive autoregulation. Therefore, the effects of zinc on ZRE occupancy in wild-type cells could result solely from changes in Zap1 protein level. To assess this possibility, we first determined the level of Zap1 protein expressed under these conditions by immunoblot analysis  in zinc finger 4 were mutated to alanines This observation was useful because it allowed us to test the importance of DNA binding regulation to overall Zap1 zinc responsiveness. Zap1 was expressed in a zap1\u0394 cells expressing either low or high levels of Zap1 were grown in LZM supplemented with 3, 10, 30 and 100 \u00b5M Zn and tracer amounts of 65Zn to assess total zinc accumulation. After \u223c5 generations of growth in these media, the cells were harvested, washed to remove surface-bound zinc, and assayed for accumulation. As shown in zap1\u0394 cells expressing low levels of Zap1. These results support the hypothesis that DNA binding control is critical for maintaining zinc homeostasis in these cells.These results show that regulation of Zap1 DNA binding is important for the zinc responsiveness of Zap1 transcriptional regulation. To assess its importance to zinc homeostasis, we assayed zinc accumulation in cells with or without DNA binding control. An effect on zinc accumulation would reflect misregulation of the zinc uptake transporters. Wild-type cells and in vivo DMS footprinting and chromatin immunoprecipitation. One limitation of the footprinting method is that it does not definitively show that the protein protecting the ZRE is Zap1 and not some other transcription factor. However, several observations support the conclusion that the methylation protection observed is due to Zap1 binding. First, no protection was detected in a zap1\u0394 mutant and protection was constitutive in a Zap1-overexpressing strain. Second, in vitro studies have shown that purified Zap1 binds to ZREs with high sequence specificity and affinity in vivo.In this report, we have shown that Zap1 DNA binding activity is regulated by zinc status. This conclusion is based on our observations that ZRE occupancy is altered in response to zinc without any changes in the total level of Zap1 or the nuclear localization of the protein. ZRE occupancy was assayed using both ZAP1 gene expression is under positive transcriptional autoregulation. Here we confirm that this transcriptional control does indeed alter Zap1 protein levels.With the addition of DNA binding control, we now know that Zap1 activity is regulated by zinc via multiple mechanisms. Two of these mechanisms involve the regulation of Zap1's two activation domains. Our previous studies showed that zinc controls AD1 and AD2 independently of each other. This regulation likely occurs through the binding of zinc directly to residues within and flanking these activation domains. We hypothesize that zinc binding to these domains folds them into inactive conformations that are incapable of recruiting coactivators to Zap1 target promoters. We have also shown in previous studies that ZRT1-lacZ reporter when we eliminated that mechanism of control by expressing Zap1 at a constant low level from the GAL1 promoter. However, we note that the ZRT1 promoter contains four high affinity ZREs and therefore may be less sensitive to changes in Zap1 level than other promoters with lower affinity binding sites. This hypothesis is supported by our recent observation that Zap1 target genes with lower affinity ZREs tend to be induced only under the most severe zinc-limiting conditions when Zap1 protein levels are highest ZAP1 expression may play a significant role on those promoters.Having so many different mechanisms of zinc responsiveness raises the issue of what each of these mechanisms contributes to Zap1 regulation and zinc homeostasis. With regard to transcriptional autoregulation, we found no obvious effect on regulation of a ZRT1 mRNA levels decreased following zinc treatment of zinc-limited cells, suggesting this is not the case .From our analysis of activation domain and DNA binding domain regulation, it is clear that these post-translational mechanisms are integrated to control the overall response of Zap1 to zinc. When comparing AD1 and AD2 function, we have found that AD1 is responsible for dictating the zinc dose response and induction kinetics of most Zap1 target genes in vitro but these experiments did not support the hypothesis. Using both electrophoretic mobility shift assays and surface plasmon resonance analysis, we have determined that Zn2+ at concentrations as high as 10 \u00b5M does not inhibit ZRE binding by purified Zap1 . This value is much higher than the nanomolar (or lower) levels of free zinc in vitro but this effect was nonspecific and those levels of zinc also interfered with in vitro DNA binding of control proteins  that function well in zinc-replete cells. Thus, inhibition of DNA binding by zinc ions binding directly to the Zap1 DNA binding domain appears unlikely to be the mechanism that operates in vivo. An alternative model is that DNA binding activity is controlled by post-translational modification, such as phosphorylation. This form of DNA binding regulation has been demonstrated for other C2H2 zinc finger transcription factors, such as Adr1 2H2 zinc finger proteins How might zinc control Zap1 DNA binding? We mapped the regulation of DNA binding activity to the Zap1 DNA binding domain alone. The five zinc fingers of the DNA binding domain are all high affinity structural zinc sites and these domains have metal bound even in zinc-limited cells. Our previous studies demonstrated that binding of zinc by each of the five fingers is required for ZRE binding 2. LZM contains 1 mM EDTA and 20 mM citrate as metal buffers to limit zinc availability. It is important to note that even when supplemented with 1000\u20133000 \u00b5M zinc, LZM has lower free Zn2+ available to cells than is found in standard yeast culture media such as SD or YPD. Regulated expression from the GAL1 promoter in glucose-containing media was achieved using the GEV system. The GEV protein contains the Gal4 DNA binding domain, the VP16 activation domain, and the hormone-response domain of the human estrogen receptor. Treatment of GEV-containing cells with \u03b2-estradiol results in increased expression of genes expressed from the GAL1 promoter All yeast strains were grown in either YP medium supplemented with 2% glucose (YPD) or synthetic defined medium with 2% glucose and the appropriate auxotrophic supplements. Limiting zinc medium (LZM) was prepared as previously described ade6 can1 his3 leu2 trp1 ura3), ZHY6 (DY1457 zap1\u0394::TRP1) MATa leu2 trp1 ura3 prb1 pep4 prc1 gal2) ZRT1-lacZ reporter construct used in this study was pGI-1 ZRT1 promoter (\u2212521 to the ATG start codon) fused to lacZ. The construction of pZap1WT (previously referred to as pMyc-Zap11\u2013880), a plasmid containing an N-terminal myc-tagged ZAP1 allele under the control of the GAL1 promoter in the vector pYef2, was described previously TC was generated as previously described (21). pZap1\u039417-700 was made by generating the corresponding open reading frame by overlap PCR and inserting the resulting fragment into BstX1-linearized pZap1WT using homologous recombination. To create pPGK-ZRT1, a cloning strategy was used in which the ZRC1 open reading frame in the construct pPGK-ZRC1  was replaced with ZRT1 using homologous recombination. Primers were designed such that the 5\u2032 primer contained 40 bp of homology to the PGK1 promoter region and 20 bp of homology to the 5\u2032 end of ZRT1 and the 3\u2032 primer contained 40 bp of homology to the ZRC1 terminator and 20 bp of homology to the 3\u2032 end of ZRT1. The resulting PCR product was co-transformed into DY1457 with BamHI-digested pPGK-ZRC1 and transformants selected on SD media for the plasmid URA3 marker. Plasmids were then rescued from DY1457 using standard procedures. The resulting construct containing ZRT1 fused to the PGK1 promoter and ZRC1 terminator was confirmed by DNA sequencing.The yeast strains used in this study were DY1457 (MAT\u03b1 600\u200a=\u200a0.3\u20130.7) in LZM supplemented with the indicated amount of ZnCl2. Cells were treated with DMS (30 \u00b5l/30 ml of culture) for 7 min and then harvested by centrifugation. Isolation of genomic DNA and in vivo footprinting analysis were performed as previously described ZRT1 promoter at positions \u2212481 to \u2212460 relative to the initiation codon. Quantification of band intensities was performed using a Storm 860 PhosphorImager (Molecular Dynamics) with ImageQuant software. Calculation of the fractional protection of residues was performed as follows ZRE/BIstandard)/(BIZRE in zap1\u0394/BIstandard in zap1\u0394)]\u00d7100 where BIZRE and BIstandard refer to the band intensities of the affected ZRE residues and of a control band elsewhere in the primer extension ladder that was unaffected by zinc or ZAP1 genotype. Similar results were obtained when ZRE protection was quantified relative to several different control bands.Thirty ml cultures were grown for 15\u201320 h to mid-exponential phase (AWT) were grown to an A600 \u223c0.5 and then treated with 1% formaldehyde to cross-link protein-DNA complexes. The cross-linking reaction was quenched by adding 125 mM glycine. After two washes with ice-cold PBS, the cells were lysed with glass beads in buffer containing Complete Protease Inhibitor Cocktail (Roche), 1 mM PMSF, and 2 mM benzamidine. Following centrifugation for 10 minutes at 16,000\u00d7 g, the supernatants were incubated with anti-myc antibody at 4\u00b0C overnight and immune complexes isolated with Protein A-Sepharose. The cross-links were reversed in TES and co-immunoprecipitation of specific promoter fragments with myc-Zap1 was assessed by PCR using primers flanking the ZRT1 and ZPS1 ZREs  digestion were resuspended in an 18% Ficoll buffer  and were disrupted using a Dounce homogenizer. Following removal of cell debris by centrifugation at 5000\u00d7 g for 15 min, nuclei and other organelles were separated from the cytosolic fraction by centrifugation at 25,000\u00d7 g for 30 min. The pellet was resuspended in 18% Ficoll buffer before being layered onto 35% Ficoll in the same buffer and the nuclei sedimented at 100,000\u00d7 g for 90 minutes. All buffers during and after the disruption of spheroplasts contained 1 mM PMSF, 1 mM EDTA and the Complete Protease Inhibitor Cocktail (Roche). The protease-deficient strain BJ2168 was required for these experiments due to the extreme sensitivity of Zap1 to proteolytic degradation during sample preparation from wild-type cells. Total protein extracts for immunoblots were prepared by cell disruption and protein precipitation in the presence of trichloroacetic acid as described Protein extracts for immunoblots were prepared by two different procedures. Subcellular fractionation analysis was performed as described previously 32P end-labeled oligonucleotide probe before digestion by S1 nuclease and separation on an 8% polyacrylamide/8 M urea gel. Probes used are listed in RNA was extracted from cells grown to mid-log phase using hot acid phenol extraction. S1 analysis was performed as previously described 600\u200a=\u200a0.3\u20130.7) in LZM supplemented with the indicated amount of ZnCl2. \u03b2-galactosidase activity was measured as described 420\u00d71000)/(min\u00d7ml of culture\u00d7absorbance of the culture at 595 nm).Cells were grown for 15\u201320 h to mid-exponential phase , and then counted using a Wallac 1480 Wizard\u2122gamma counter. Cell-associated 65Zn was then adjusted for specific activity and normalized by converting the culture A600 values to cell number with a standard curve.Cells were grown in LZM medium supplemented with the indicated concentration of ZnCl"}
+{"text": "Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3\u201331 and 66\u2013105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human \u03b2-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.The Lim domain only 2 ( Lmo2 gene, have shown that Lmo2 is necessary for embryonic yolk sac erythropoiesis Lmo2 expression is maintained in erythroid cells but down regulated in the T-cell lineage Lmo2 results in the development of T-cell related diseases; indeed Lmo2 is located at a recurrent site of T-cell acute lymphoblastic leukemia (T-ALL) specific translocation Lmo2 when the gene therapy vector integrated near Lmo2Lim domain only 2 (LMO2) is a critical transcriptional regulator of hematopoiesis. Gene targeting experiments conducted to introduce null mutations in the mouse Hbb), \u03b1-globin (Hba), retinaldehyde dehydrogenase 2, c-kit and erythroid Kruppel-like factor (Eklf) Previous studies have shown that in erythroid cells LMO2 is usually present as part of a complex with the transcriptional regulators, TAL1, E47, LDB1, and GATA1 Hbb locus control region (LCR) Hbb-b1 gene Hbb LCR is in close proximity to the active Hbb genes (Hbb-b1 and Hbb-b2) whereas the intervening 50 kb of DNA sequence containing the embryonic erythroid cell expressed genes is looped out Hba, Shh, TH2, HoxB1 and olfactory receptor genes Transcription is regulated not only by the sequences immediately upstream of gene transcription start sites (TSS) but in many cases by distal regulatory elements (DRE) which can be located up or downstream of the genes they regulate. In fact genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis for several transcription factors has revealed that a significant proportion (40\u201360%) of transcription factor bound regions are located in the intergenic regions of the genome \u226510 kb from a gene TSS Hbb, Igf2/H19 and HoxA, however, CTCF bound regions are generally bound by CTCF in all cell types IGF2-H19 locus and at the developmentally regulated IFNG locus both cohesin and CTCF are required for maintaining higher-order chromatin conformation cis-regulatory elements not occupied by CTCF The chromatin-chromatin looping interactions that regulate cell-type specific gene expression are also present in a cell-type specific manner whereas many of the proteins present at sites of looping interactions are ubiquitously expressed. For example, CTCF participates in intra- and inter-chromosomal looping at individual gene loci including Lmo2 in erythroid cells appears to involve multiple distal regulatory elements. Three alternate upstream promoters have been identified as well as multiple conserved DRE located up to 100 kb upstream of the Lmo2 gene Lmo2 and 1 kb downstream are capable of enhancing reporter gene expression in erythroid cells and in transgenic mice suggesting that strong expression of Lmo2 in hematopoietic cells requires the combined action of upstream DRE and sequences close to the Lmo2 proximal promoter Lmo2 transcription in the context of its endogenous genomic location using the 3C technique. Our results show that multiple upstream DRE interact with the Lmo2 proximal promoter whereas intervening regions are looped away from the proximal promoter. DRE upstream of Lmo2 are bound by multiple transcription factors, p300, and associated with intergenic transcription when Lmo2 is transcribed at high levels in erythroid cells. Interaction between the DRE and the Lmo2 promoter was identified in erythroid cells but not in kidney cells suggesting a link between the looping conformation of the locus and transcriptional regulation of Lmo2. Furthermore, a CTCF and cohesin occupied region upstream of the most distal enhancer (90 DRE) also contacts the Lmo2 proximal promoter region, potentially insulating the neighboring Cell cycle associated protein 1 (Caprin1) gene from interaction with the DRE that enhance Lmo2 transcription.Transcriptional regulation of Lmo2 have been identified in the human genome and confirmed to have enhancer activity in transgenic mice Lmo2 , is located close to the Lmo2 proximal promoter and contains the distal and intermediate promoters as well as the 12 and 25 DRE. The second interaction domain located further upstream of Lmo2, which we term the distal control region (dCR), contains the 70, 75 and 90 DRE as well as a fragment upstream of the 90 DRE. These are similar, though not identical, to clusters I (\u221290 to \u221264) and II (\u221240 to +1) identified by Landry et al. 2009 as enriched in histone H3 acetylation in erythroid cells Next we performed a locus-wide 3C using the ey cells . These fCaprin1 and Lmo2 highlighted the 75 DRE as being bound by multiple transcription factors but did not reveal binding at the 90 and 70 DRE. By contrast our 3C data revealed a broad domain of chromatin-chromatin contacts between the 70, 75 and 90 DRE and the Lmo2 proximal promoter raising questions about the function of this dCR. We did identify additional p300 association at the 70 DRE suggesting that bound transcription factors are present which recruit p300 to this DRE , containing three transcription factor bound DRE , and a proximal interaction cluster (pCR). Furthermore, 3C experiments revealed no significant interactions between the Caprin1 TSS and the DRE suggesting these elements are specific in regulating Lmo2 transcription.Our investigation of chromatin-chromatin interactions throughout the mouse genomic region containing Lmo2 the 75 DRE contained the highest density of transcription factor association as well as p300, RAD21 and a nearby CTCF bound region. Furthermore our 3C data confirmed specific interaction of the 75 DRE with the Lmo2 proximal promoter and interaction of the Lmo2 proximal promoter with the dCR containing the 75 DRE as well as the 90 and 70 DRE. In addition, a CTCF bound region upstream of the 90 DRE is contained in the dCR. Previous studies in circulating erythroid cells of transgenic mice show that the 75 DRE has the strongest enhancer activity in erythroid cells and drives the expression of a reporter gene, cooperatively with the Lmo2 proximal promoter and +1 enhancer element Lmo2 proximal promoter/+1 DRE region to form an erythroid cell specific chromatin loop that includes other regulatory elements and a cluster of CTCF bound regions. This suggests that in the endogenous context elements throughout the dCR coordinately regulate Lmo2 transcription in erythroid cells.Using a combination of ChIP-Seq data for mature erythroid cells and HPC7 hematopoietic progenitor cells we identified multiple transcription factors bound within both the pCR and the dCR. Even considering the HPC7 ChIP-Seq data that showed more transcription factor peaks across the entire region upstream of Lmo2. Interestingly, we did not identify interactions of the 75 DRE with the fragments containing the 25 or 12 DRE. Transgenic analysis by Landry et al. 2009 of these elements did reveal that the 25 and 12 DRE conferred expression in the fetal liver whereas only the 75 DRE conferred expression in fetal liver as well as in circulating blood cells suggesting that these elements have different functional roles in regulating Lmo2 expression. The fact that we did not identify specific interactions between the 75 DRE and the fragment containing the 25 or 12 DRE suggests that the interactions of these two regions with the proximal promoter are mutually exclusive. These mutually exclusive interactions could occur in different sub-populations of cells within the anaemic spleen or the interactions could be dynamic within individual cells with the proximal promoter region alternately contacting the 25\u201312 region and the 70\u201390 region similar to the flip-flop of the Hbb LCR between the \u03b3- and \u03b2-genes We also identified increased interaction throughout a pCR containing the 25 DRE/distal promoter and 12 DRE/intergenic promoter and the +1DRE/proximal promoter in erythroid cells compared to kidney cells, suggesting that this more proximal region also contributes to transcriptional regulation of endogenous Lmo2 proximal promoter; however the question remains as to which factors are mediating these looping interactions. LMO2 itself is one of the regulatory transcription factors bound to the upstream DRE, specifically at 75, 25 and 12, all of which showed increased interaction with the Lmo2 promoter suggesting an important role for the LMO2 complex in chromatin looping. In support of this, a recent study found LDB1 (a member of the LMO2 complex) at regions of chromatin interaction with the LDB1 bound Hbb-b1 promoter Hbb-b1 and the LCR Lmo2 locus. Cohesin (RAD21) is bound within both the dCR and pCR, specifically at the 75, 25 DRE as well as at the proximal promoter suggesting cohesin bound at the upstream DRE supports erythroid cell specific looping interactions with the Lmo2 proximal promoter.We identified erythroid-cell specific chromatin loops between a dCR and the Lmo2 upstream region, all of which occur within erythroid cell specific interacting domains, though the majority of the CTCF sites were not specific to erythroid cells. This is similar to the findings at the Hbb locus where CTCF bound regions, invariant between cell types, formed cell type specific chromatin loops Caprin1 from interacting with the DRE that enhance Lmo2 transcription in erythroid cells. As overexpression of Caprin1 causes inhibition of cell division it is critical to prevent its aberrant upregulation in rapidly cycling erythroid cells Lmo2 locus being supported by cohesin recruited both to CTCF bound regions  as well as at transcription factor and p300 bound enhancers not associated with CTCF .Cohesin is also recruited to CTCF occupied regions Lmo2 dCR is more reminiscent of the human Hbb LCR Lmo2 dCR may function to facilitate the physical interaction of the dCR with the Lmo2 proximal promoter through recruitment of both regions to a shared transcription factory. This co-localization at a shared transcription compartment would then allow transcription factors recruited to the dCR to influence the basal transcriptional machinery recruited to the Lmo2 proximal promoter thereby enhancing Lmo2 transcription.Our analysis indicates that a large portion of the dCR is transcribed at moderate levels in erythroid cells but not in kidney. Whereas short eRNAs have been identified at enhancers in neuronal cells the broad domain of intergenic transcription we observed throughout the Lmo2-Caprin1 locus adopts a tissue-specific conformation in erythroid cells. This tissue specific organization of the locus brings several DRE into proximity with the Lmo2 proximal promoter while excluding the Caprin1 TSS. A proximal control region, immediately upstream of the Lmo2 proximal promoter, contains the 12 and 25 DRE as well as the distal and intermediate promoters and is more closely associated with the Lmo2 proximal promoter in erythroid cells compared to kidney. Furthermore, a distal control region, covering 39 kb and containing three DRE , forms a strong interaction with the Lmo2 proximal promoter and shares many features with the well characterized Hbb LCR. A CTCF bound region upstream of the 90 DRE flanks the dCR and may function as an insulator preventing the interaction of Caprin1 with the erythroid cell specific Lmo2 enhancers.In conclusion, we found that the mouse All experiments were approved by the University Animal Care Committee (UACC) at the University of Toronto and the Bioscience Local Animal Care Committee (LACC).8) from the spleen of mice 5 days after phenylhydrazine treatment. This treatment induces haemolytic anaemia, as a result of which the spleen becomes the major site of red blood cell production Adult globin expressing mature erythroid cells were isolated from C57/Blk6 mice in large numbers (>1\u00d710Lmo2-Caprin1 locus (RP23-76D2) with the Alpha Aortic Actin 2 BAC clone (RP23-2N15) followed by digestion with HindIII. The digested DNA was then ligated, and purified using phenol/chloroform extraction and ethanol precipitation. HindIII restriction enzyme digestion efficiency was confirmed to be between 85 and 95% efficient at several genomic fragments in anaemic spleen and kidney cells  was subsequently used for quantification. PCR products of the ligated fragments were quantified using real-time quantitative PCR (qPCR) on the Bio-Rad CFX-384 cycler. All data points were generated from an average of three-five independent 3C experiments with the qPCR performed in triplicate. Standard curves were generated by 5 fold serial dilution of the 3C control template and run in parallel with 3C experimental samples. The primers used in qPCR are listed in Gapdh or Epn1 were used for normalization of expression levels. The primers used for real-time RT-qPCR are listed in RNA from anaemic spleen and kidney was isolated using TRIzol, according to the manufacturer`s instructions (Invitrogen). The iScript First strand synthesis cDNA kit from Bio-Rad was used for preparation of random-hexamer primed cDNA. Amplification in qPCR was performed on the Bio-Rad CFX-384 cycler using the standard curve method. The reaction mixture contained 2X Bio-Rad iTaq SYBR green mastermix, 0.3 pM of each primer, 1uL cDNA (10 times diluted from a 20 uL of reverse transcription reaction). The conditions for qPCR were as follows: 94\u00b0C for 3 min followed by 40 cycles at 94\u00b0C for 30 s, 62\u00b0C for 30 s. Expression levels of The 3C data were analyzed by two-way ANOVA using Sigma Plot12. Post tests (Holm-Sidak method) were performed to assess significant differences between anaemic spleen and kidney samples at specific genomic locations.ChIP-Seq raw data for GATA1, KLF1, LDB1, TAL1, and MTGR1 Figure S1The Lmo2/Caprin1 region on mouse chromosome 2. Primers used in chromosome conformation capture (3C) and HindIII restriction sites are shown across the Lmo2/Caprin1 region of mouse chromosome 2. Promoters and distal regulatory elements (DRE) are depicted in red and black respectively. Anchor fragments used in the Caprin1, 75 DRE and Lmo2 3C experiments are marked with an asterisk (*). Distal promoter (pP), proximal promoter (pP).(PDF)Click here for additional data file.Figure S2Distal regulatory elements upstream of Lmo2 overlap transcription factor bound regions in HPC7 hematopoietic progenitor cells. The mouse Lmo2-Caprin1 region. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Coloured boxes represent peaks identified from transcription factor ChIP-Seq data from HPC7 hematopoietic progenitor cells obtained from Wilson et al. 2010. Proximal promoter (pP), distal promoter (dP).(PDF)Click here for additional data file.Figure S3The Caprin1 TSS does not interact with distal regulatory elements upstream of Lmo2. Quantitative chromosome conformation capture (3C) was performed to detect chromatin-chromatin interactions between the Caprin1 TSS and distal regulatory elements (DRE). The profile of interactions identified in anaemic spleen (red) and kidney (blue) is displayed. Black box indicates the anchor fragment at Caprin1 and alternating intensities of grey boxes indicate the fragments investigated for interactions. Data points are an average of three independent biological replicates. Error bars depict the SEM, no significant differences were identified throughout this region.(PDF)Click here for additional data file.Figure S4CTCF bound upstream of Lmo2 in different cell types. The mouse Lmo2 upstream region on chromosome 2 is depicted with chromosome coordinates shown at the top. HindIII restriction sites are indicated by blue lines. The two Lmo2 promoters are indicated by red boxes. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Mouse ENCODE ChIP-Seq data from B Ren (Ludwig Inst. for Cancer Research) and M Snyder (Stanford University) for CTCF in different cell types are shown below the DRE. Proximal promoter (pP), distal promoter (dP), murine erythroleukemia cells (MEL differentiated with 2% DMSO), bone marrow (BM), embryonic stem cells (ES-Bruce4), mouse embryonic fibroblasts (MEF).(PDF)Click here for additional data file.Figure S5Restriction digestion efficiency in chromosome conformation capture. Restriction digestion efficiency was between 85 and 95% at several HindIII restriction sites. Lmo2 proximal promoter (pP), Distal regulatory element (DRE), Caprin1 promoter (Caprin1P). \u201cU\u201d denotes a restriction fragment upstream of the indicated element.(PDF)Click here for additional data file.Table S1Coordinates of distal regulatory elements located upstream of the Lmo2 promoter in the mouse genome. Distal regulatory elements are named acording to their distance upstream of the annotated Lmo2 transcription start site overlapping the proximal promoter. Coordinates are given for homology regions identified by BLAT. All fragments were mapped in NCBI m37 mouse assembly (mm9).(PDF)Click here for additional data file.Table S2Coordinates of the Lmo2 proximal and distal promoters in the mouse genome. The coordintes of proximal promoters and distal promoters for the Lmo2 gene in the mouse genome are listed in the table. Coordinates are given for homology regions identified by BLAT. All fragments were mapped in NCBI m37 mouse assembly (mm9).(PDF)Click here for additional data file.Table S3Chromatin immunoprecipitation sequencing data. Transcription factor binding sites have been obtained from three different cell types; differentiated murine erythroleukemia cells (MEL), hematopoietic progenitor cells (HPC7), and GIE-ER4 a GATA1-null erythroblast cell line in which GATA1 activity was restored. CTCF, DNaseI hypersensitivity, p300 and RAD21 data have been obtained from the mouse ENCODE project, sources listed .(PDF)Click here for additional data file.Table S4Relative transcript abundance in intergenic regions upstream of Lmo2.(PDF)Click here for additional data file.Table S5Primers. Specific primers are listed for the chromosome conformation capture (3C) and RT-qPCR analyses. Left primer (L), right primer (R), primers used to test HindIII restriction digestion efficiency are marked as REX.(PDF)Click here for additional data file."}
+{"text": "Gnas transcript at the Gnas cluster has been thought to account for the PatDp(dist2) phenotype. But PatDp(dist2) also have two expressed doses of the paternally expressed Gnasxl transcript. Through the use of targeted mutations, we have generated PatDp(dist2) mice predicted to have 1 or 2 expressed doses of Gnasxl, and 0, 1 or 2 expressed doses of Gnas. We confirm that oedema is due to lack of expression of imprinted Gnas alone. We show that it is the combination of a double dose of Gnasxl, with no dose of imprinted Gnas, that gives rise to the characteristic hyperactive, chunky, oedematous, lethal PatDp(dist2) phenotype, which is also hypoglycaemic. However PatDp(dist2) mice in which the dosage of the Gnasxl and Gnas is balanced (either 2\u22362 or 1\u22361) are neither dysmorphic nor hyperactive, have normal glucose levels, and are fully viable. But PatDp(dist2) with biallelic expression of both Gnasxl and Gnas show a marked postnatal growth retardation. Our results show that most of the PatDp(dist2) phenotype is due to overexpression of Gnasxl combined with loss of expression of Gnas, and suggest that Gnasxl and Gnas may act antagonistically in a number of tissues and to cause a wide range of phenotypic effects. It can be concluded that monoallelic expression of both Gnasxl and Gnas is a requirement for normal postnatal growth and development.Genomic imprinting results in parent-of-origin-dependent monoallelic gene expression. Early work showed that distal mouse chromosome 2 is imprinted, as maternal and paternal duplications of the region  give rise to different anomalous phenotypes with early postnatal lethalities. Newborns with maternal duplication (MatDp(dist2)) are long, thin and hypoactive whereas those with paternal duplication (PatDp(dist2)) are chunky, oedematous, and hyperactive. Here we focus on PatDp(dist2). Loss of expression of the maternally expressed  Gnas cluster Early work showed that certain chromosomal regions can lead to developmental abnormalities when both copies are exclusively maternally or paternally derived. Distal mouse chromosome 2 was one of the first such imprinting regions described, providing evidence that imprinting must affect expression of some genes according to parental origin Gnas cluster can account for much of the phenotype in both MatDp(dist2) and PatDp(dist2). Gnas is a complex imprinted gene cluster with three promoter regions that give rise to protein coding transcripts Nesp, Gnasxl and Gnas. Each of these transcripts has a unique first exon that splices on to a common set of downstream exons . Nesp is exclusively expressed from the maternal allele and Gnasxl from the paternal allele. Gnas is biallelically expressed in most tissues but is preferentially maternally expressed in some Nesp gives rise to NESP55, neuroendocrine secretory protein 55, Gnas to Gs\u03b1, the alpha subunit of the Gs signalling protein and Gnasxl to XL\u03b1s, a variant form of Gs\u03b1 which can also function as the alpha subunit of the heterotrimeric Gs protein. Both XL\u03b1s and Gs\u03b1 can act on adenylyl cyclase to induce cyclic AMP production Gnasxl in offspring result in increased demands on the mother (growth promoting) whereas the effects of maternally expressed Gnas result in fewer demands, in accordance with the conflict hypothesis Misexpression of transcripts at the imprinted am exons . Nesp iGnas cluster is controlled by an imprinting control region (ICR), a germline differentially methylated region called the Nespas DMR that covers the promoter of a noncoding antisense transcript, Nespas Nespas DMR is part of a larger DMR that covers the Gnasxl promoter tm1JopNespas allele , a deletion of the Nespas DMR, results in loss of imprinting of Nesp and Gnas, and down regulation of GnasxlGnas transcript is controlled by a second differentially methylated region, the Exon 1A DMR that lies just upstream of Gnas exon 1 tm1JopGnas allele , a deletion of the Exon 1A DMR, results in loss of imprinted expression of Gnas and so the Exon 1A DMR solely regulates the imprinted expression of GnasNespas DMR must interact with the Exon 1A DMR to control the imprinted expression of Gnas although the mechanism of interaction is not yet clear.Imprinted expression of protein coding transcripts at the Gnasxl but lack expression of Gnas in imprinted tissues and also lack expression of NespGnas in imprinted tissues and two expressed copies of Nesp but lack expression of Gnasxl. PatDp(dist2) mice have greatly diminished expression of Gnas in newborn brown fat consistent with imprinted expression Gnasxl consistent with two expressed doses PatDp(dist2) mice have two expressed copies of Gnasxl results in newborns with severely reduced suckling ability that become inert on the day of birth and most die within within a few days of birth Gnasxl can account for much of the phenotype observed in MatDp(dist2) mice. On the other hand, maternal transmission of a null mutation resulting in loss of Gnas transcript gives rise to neonates with oedema and square shaped bodies, most of which die before weaning Oedsml-matGnas, (Oed), results in gross neonatal oedema and pre-weaning lethality Oed mice) Gnas transcripts in imprinted tissues can account for the oedematous phenotype seen in PatDp(dist2) mice but cannot account for the hyperactivity. Thus hyperactivity may be due to a double dose of Gnasxl alone or to the combined effect of a double dose of Gnasxl and loss of Gnas in imprinted tissues.From studies of targeted deletions and an ENU induced mutation it is evident that both XL\u03b1s and Gs\u03b1 play important roles in growth, behaviour and survival shortly after birth, but NESP55 does not Gnasxl and loss of Gnas gives rise to an early hyperactivity lethal phenotype. Furthermore when the expressed dosage of Gnas and Gnasxl is balanced then PatDp(dist2) are neither lethal nor hyperactive. Thus the dosage of Gnas and Gnasxl needs to be balanced for long term survival and normal activity. Furthermore PatDp(dist2) with balanced dosage but two doses of both Gnasxl and Gnas have normal viability and activity but preweaning growth retardation followed by catch up growth. Thus it may be important to have a single dose of Gnasxl and Gnas for normal development or there may be other genes involved in determining the PatDp(dist2) phenotype.We have used genetic approaches to show the combination of over expression of All mouse studies were carried out in accordance with the guidance issued by the Medical Research Council in \u201cResponsibility in the use of animals in bioscience research (May 2008)\u201d, were approved by the MRC Harwell ethical review committee and carried out under the authority of the UK Home Office Project Licence numbers 30/1518, 30/2065 30/2526, and 30/2642.a, nonagouti locus Gnas cluster.Paternal duplication (PatDp(dist2)) mice were generated by the standard genetic method of intercrossing genetically marked translocation heterozygotes and identifying PatDp(dist2) mice with the aid of markers \u0394Ex1A (T26H +/+ \u0394Ex1A) , homozygous for \u0394Ex1A (T26H \u0394Ex1A/+ \u0394Ex1A), heterozygous for \u0394Nespas (T26H +/+ \u0394Nespas), and heterozygous for both \u0394Ex1A and \u0394Nespas (T26H+\u0394Ex1A/+ \u0394Nespas +). PatDp(dist2) that were either heterozygous for \u0394Ex1A, or homozygous for \u0394Ex1A, or heterozygous for \u0394Nespas, or heterozygous for both \u0394Ex1A and \u0394Nespas were generated by crossing T26H +/++females with either TH26+/+ \u0394Ex1A, or T26H \u0394Ex1A/+ \u0394Ex1A or T26H +/+ \u0394Nespas or T26H+\u0394Ex1A/+ \u0394Nespas+males. For each cross, reciprocal crosses were also set up to generate PatDp(dist2) with wild-type alleles at \u0394Ex1A and/or \u0394Nespas. The genetic background of PatDp(dist2) mice was mixed but with major contributions from C3H/HeH (circa 45%) and the Harwell LL stock (circa 45%). Toe or tail clips were taken from offspring at birth and these served as both biopsies for identification and for genotyping. Offspring were genotyped for paternal duplication, \u0394Ex1A and \u0394Nespas as previously described To generate PatDp(dist2) with altered dosing of Gs\u03b1 and XL\u03b1s we developed translocation stocks that were heterozygous for Mice were classified for oedema, chunky appearance, activity, tail kink, and paddle feet, a paddling motion of the front feet, by daily visual observation from birth. Mice were removed from the box and observed for 2\u20133 minutes.Gnas and Gnasxl and for Actb have been previously described . Transcript levels were measured by phosphoimager analysis and quantified as described previously .Total RNA was extracted and northern analysis carried out as described previously . Riboprobes specific for the unique first exons of Truncal blood from mice up to one week of age was collected using heparinised capillary tubes. Plasma was separated following centrifugation at 750\u00d7g for 3 minutes and stored at \u221280\u00b0C. Noradrenaline was measured in duplicate using an ELISA kit (Labor Diagnostika Nord GmbH & Co.KG) according to the manufacturer\u2019s instructions. Blood glucose was measured using an Alphatrak Veterinary Blood Glucose Monitoring Meter Kit (Abbott) according to the manufacturer\u2019s instructions. Insulin and glucagon were measured using an ELISA kit (Mercodia) according to the manufacturer\u2019s instructions.2.Mice were weighed weekly from birth and from 4 weeks the length from the tip of the nose to the base of the tail was measured weekly. Body mass index was calculated using the following equation: weight (g)/(length (cm))http://empress.har.mrc.ac.uk).At nine weeks the mice were individually housed in metabolic cages for 24 h, during which time they had free access to a known amount of food and water. After 24 h, the amount of food and water consumed was measured along with the volume of urine produced. After housing in metabolic cages the mice were returned to their home cage. For a fuller protocol, see EMPRess . Mice were weighed and then individually housed in the equipment for 24 h, with food and free access to water. After testing, the mice were returned to their home cage. Indirect calorimetry enabled measurement of oxygen consumption, carbon dioxide production, respiratory exchange ratio and heat production.At 17 weeks of age the mice were weighed and given a recoverable anaesthetic prior to scanning using a General Electric Medical Systems Lunar PIXImus II X-ray densitometer , which allows the fat and lean mass and bone density to be calculated. Mice were placed in a heated box to aid recovery; once fully recovered, they were returned to their home cage.t test (two-tailed) was used for assessing the results of the growth and metabolism studies and expression levels on northern blots. P values <0.05 were considered significant.Fisher\u2019s exact test was used for comparisons of the incidence of PatDps and the occurrence of phenotypic features. Student\u2019s Gnasxl and severely reduced expression of imprinted GnasGnasxl and Gnas we utilised two mutants, \u0394Ex1A and \u0394Nespas. \u0394Ex1A is a deletion of the Exon 1A DMR, the region that regulates the imprinted expression of Gnas. On paternal inheritance Gnas is completely derepressed on the paternal allele \u0394Ex1A  show normal survival to birth for they were generated at a frequency of 44.5% (of 231 neonates) which is not significantly different from the expected 50% (P>0.05).The \u0394Nespas mutant allele is a deletion of the promoter and first exon of Nespas, the ICR for the Gnas cluster \u0394Nespas Gnasxl is downregulated to about 24% of wild type levels  has two expressed doses of e levels  and GnasGnasxl and Gnas (\u0394Ex1A were produced to provide PatDp(dist2)2\u22361 with two predicted expressed doses of Gnasxl and one expressed dose of Gnas. In Cross 2 PatDp(dist2) mice homozygous for \u0394Ex1A were generated to produce PatDp(dist2)2\u22362 with two predicted expressed doses of Gnasxl and two doses of Gnas. Cross 3 gave rise to PatDp(dist2) mice heterozygous for \u0394Nespas predicted to have slightly more than one expressed dose of Gnasxl and slightly less than one expressed dose of Gnas, called PatDp(dist2)1\u22361. From Cross 4 PatDp(dist2) arose that were compound heterozygotes for \u0394Ex1A and \u0394Nespas predicted to have slightly more than one expressed dose of Gnasxl and slightly less than two expressed doses of Gnas, called PatDp(dist2)1\u22362. For each cross reciprocal crosses were also set up resulting in PatDp(dist2)2\u22360.Four crosses were set up to generate PatDp(dist2) mice with altered dosing of and Gnas , Table 1\u0394Ex1A were known to express two doses of imprinted Gnas, and PatDp(dist2)2\u22361 and PatDp(dist2)1\u22361 to express one dose of imprinted GnasGnas were found in newborn brown fat from +/\u0394Ex1A and PatDp(dist2)2\u22362 homozygous for \u0394Ex1A indicating that PatDp(dist2)2\u22362 express two full doses of Gnas (Heterozygotes +/ of Gnas .). The incidence of PatDp(dist2)2\u22360 generated using the T26H translocation was not significantly different from that found previously There were no significant differences in the incidence or phenotypes of PatDp(dist2)2\u22360 from the reciprocal crosses and so the data from these were pooled for the Control cross . The inGnas cluster which lie 21cM apart (www.informatics.jax.org) . Thus in Cross 1, the cross to generate PatDp(dist2)2\u22361, recombinants PatDp(dist2)2\u22360 and PatDp(dist2) 2\u22362 were found. In Cross 3, the cross to generate PatDp(dist2)1\u22361, recombinants PatDp(dist2)2\u22360 and PatDp(dist2)0\u22362 occurred. In Cross 4, the cross to generate PatDp(dist2)1\u22362, recombinants PatDp(dist2)2\u22362 arose. All recombinants in Cross 2, the cross to generate PatDp(dist2)2\u22362, will be identical and indistinguishable from non recombinant PatDp(dist2)2\u22362  to birth.It should be borne in mind that the incidence of PatDp(dist2)2\u22361, PatDp(dist2)1\u22361 and PatDp(dist2)1\u22362 is likely to be an underestimate because of recombination between the TH26 breakpoint on chromosome 2 and the ist2)2\u22362 . In the \u0394Nespas that arose in Cross 3 had a normal phenotype and survived for 6 months. This is probably because in such a mouse the expected levels of Gnasxl expression would be nearly 50% of the wild type level and this appears to be sufficient for viability.Interestingly a recombinant PatDp(dist2)0\u22362 homozygous for Gnas and Gnasxl are expected to be in balance and in PatDp(dist2)1\u22362 predicted to have one expressed dose of Gnasxl and two expressed doses of Gnas, there was a remarkable increase in survival with most PatDp(dist2)2\u22362, PatDp(dist2)1\u22361 and PatDp(dist2)1\u22362 reaching weaning (Gnasxl is disadvantageous but overexpression of imprinted Gnas is not.In agreement with earlier work PatDp(dist2)2\u22360 generated using the T26H translocation only survived for a few days  weaning . After wOed mutant, a point mutation in Gnas exon 6 At birth the PatDp(dist2)2\u22360 mice had the typical phenotype . Thus, mGnas, thus PatDp(dist2)2\u22361, PatDp(dist2)2\u22362, PatDp(dist2)1\u22361 or PatDp(dist2)1\u22362 were oedematous 2\u22361, a number were noted to be chunky , indicatGnasxl together with loss of expression of imprinted Gnas gives rise to this phenotype. However, if the double dose of Gnasxl is matched with a double dose of Gnas the chunky phenotype was not seen. For normality the expressed doses of Gnasxl and Gnas need to be balanced 2\u22361, PatDp(dist2)2\u22362, PatDp(dist2)1\u22361 and PatDp(dist2)1\u22362 described as chunky compared to PatDp(dist2)2\u22360. In addition, fewer PatDp(dist2)2\u22362 and PatDp(dist2)1\u22361 were chunky compared to PatDp(dist2)2\u22361 . Overallbalanced .Gnas and Gnasxl have the same predicted expressed dosage in PatDp(dist2)1\u22361 as in wild type. Loss of functional imprinted Gnas in the presence of a single expressed dose of Gnasxl is not associated with hyperactivity in the first few days of life Gnas knockout model Gnasxl and loss of Gnas in imprinted tissues Gnasxl combined with loss of imprinted Gnas expression is responsible for the hyperactivity phenotype.Hyperactivity in PatDp(dist2)2\u22360 although manifested on the day of birth, becomes fully developed by the next day . There wGnasxl together with either loss of, or a single dose of imprinted Gnas gives rise to this phenotype. For normality the expressed doses of Gnasxl and Gnas need to be balanced 2\u22360, are not significantly different in PatDp(dist2)2\u22361 but are reduced in PatDp(dist2)2\u22362, PatDp(dist2)1\u22361 and PatDp(dist2)1\u22362. Furthermore, fewer PatDp(dist2)2\u22362 and PatDp(dist2)1\u22361 exhibited either tail kink and/or paddle feet than PatDp(dist2)2\u22361 . Overallbalanced .Gnas in tissues in which it is imprinted, chunkiness, hyperactivity, tail kink and paddle feet to overexpression of Gnasxl, combined with loss of imprinted Gnas expression.Thus oedema can be attributed to lack of expression of \u0394Ex1A whereas nonduplication littermates of PatDp(dist2)2\u22362 are heterozygous +/\u0394Ex1A and thus have biallelic Gnas expression. We tested neonates for glucose and key hormones involved in energy homeostasis .Studies of metabolism were undertaken in PatDp(dist2)2\u22360 and and PatDp(dist2)2\u22362 and their nonduplication litttermates. The nonduplication littermates of PatDp(dist2)2\u22360 are wild type for eostasis . In PatD\u0394Ex1A the levels of glucose, insulin and glucagon and noradrenaline were not significantly different from wild type indicating that overexpression of Gnas is not associated with altered glucose metabolism.In newborn +/\u0394Ex1A sibs were carried out. In addition studies of +/\u0394Ex1A and their +/+ sibs from a cross of C3H/HeH females to +/\u0394Ex1A males were undertaken. For weight studies between birth and weaning the weights of both sexes were combined as no significant differences in the weights of males and females were found, and after weaning only males were used. Mice were weighed on the day of birth and weekly thereafter.Further studies from birth to adulthood of PatDp(dist2)2\u22362 and their nonduplication +/\u0394Ex1A mice were lighter at birth, 90.1% (P\u200a=\u200a0.021), of the weight of their wild type siblings, indicating prenatal growth retardation. Growth retardation became more pronounced during the first week of life but by weaning +/\u0394Ex1A had started to catch up so that by nine weeks their weight was no longer significantly different from wild type . Similar findings of prenatal and preweaning growth retardation followed by catch up growth has been reported on paternal inheritance of the Ex1A-T-CON allele which also results in loss of imprinted expression of GnasThe weights are shown as a percentage of their wild type siblings . The +/\u0394ld type . Simila\u0394Ex1A/++\u0394Ex1A so all the nonduplication sibs were heterozygous for \u0394Ex1A. PatDp(dist2)2\u22362 did not differ in weight from their nonduplication +/\u0394Ex1A sibs at birth or at one week of age indicating that PatDp(dist2)2\u22362 were also growth retarded and to the same extent as +/\u0394Ex1A. By two weeks of age PatDp(dist2)2\u22362 were even lighter in weight than +/\u0394Ex1A and continued to weigh less than +/\u0394Ex1A until 6 weeks but subsequently did not differ significantly in weight from +/\u0394Ex1A. Thus postnatal growth retardation in PatDp(dist2)2\u22362 peaked at three weeks and was followed by catch up growth (PatDp(dist2)2\u22362 were generated by Cross 2, T26H++/+++ X T26H+p growth .Gnas in +/\u0394Ex1A results in growth retardation before weaning resulting in proportionately smaller mice followed by catch up growth in adulthood. PatDp(dist2)2\u22362 with predicted balanced expressed dosage of Gnas and Gnasxl show a more severe pre-adult growth retardation resulting in proportionately smaller mice followed by catch up growth by 10\u201311 weeks of age and overall increased weight thereafter.Thus loss of imprinting of \u0394Ex1A mice, their wild type sibs, and PatDp(dist2)2\u22362 and their +/\u0394Ex1A sibs was measured weekly from 4 weeks. +/\u0394Ex1A mice were shorter (93.2\u00b10.5% s.e.m of the length of wild type sibs) than their wild type sibs at 4 weeks  but caught up in length by 10 weeks. PatDp(dist2)2\u22362 were even shorter than +/\u0394Ex1A from 4 to 6 weeks  but thereafter did not differ in length from +/\u0394Ex1A.The body length of +/\u0394Ex1A than wild type at 4 weeks  but did not differ from wild type thereafter. No difference in BMI was found between +/\u0394Ex1A and PatDp(dist2)2\u22362 (n\u200a=\u200a10\u201312).Body mass index, BMI was significantly lower in +/\u0394Ex1A and wild type sibs (n\u200a=\u200a4\u201314) or in +/\u0394Ex1A and PatDp(dist2)2\u22362 (n\u200a=\u200a10\u201312) (data not shown).Analysis of 24h food intake at 10 weeks showed no difference in +/\u0394Ex1A and wild type sibs (n\u200a=\u200a5\u201312) or in +/\u0394Ex1A and PatDp(dist2)2\u22362 (n\u200a=\u200a9) (data not shown).The results of DEXA analysis at 17 weeks indicated there were no significant differences in fat or lean mass in +/\u0394Ex1A tended to show increased oxygen consumption and carbon dioxide output in comparison with wild-type littermates and these increases were statistically significant (P<0.03) by 21 weeks of age in +/\u0394Ex1A mice indicative of a raised metabolic rate (At 10 weeks of age +/lic rate .Gnas as oedema was not seen in PatDp(dist2) in which Gnas expression was restored to normal. This agrees with the results from other studies indicating that null or loss of function mutations of Gnas are associated with neonatal oedema Gnas expression was restored to normal Oed even more so with oedema declining somewhat in both genetic conditions before birth in utero could conceivably affect survival to birth and so the restoration of imprinted Gnas expression in PatDp(dist2)2\u22361 and PatDp(dist2)1\u22361 may account for their increased incidence at birth compared to PatDp(dist2)2\u22360.The oedema in PatDp(dist2)2\u22360 is likely to be due to loss of expression of imprinted Gnasxl combined with loss of imprinted Gnas expression. A considerable improvement in the incidence of newborns that were neither hyperactive nor chunky and some improvement in survival to seven days was found when biallelic expression of Gnasxl was combined with normal monoallelic expression of Gnas in PatDp(dist2)2\u22361. However, for full viability and loss of hyperactivity, biallelic expression of Gnasxl needs to be balanced with biallelic expression of imprinted Gnas or monoallelic expression of Gnasxl with monoallelic expression of Gnas. This need for balanced dosage is in accord with observations indicating that maternally expressed Gnas and paternally expressed Gnasxl act antagonistically The other neonatal phenotypes of PatDp(dist2), namely lethality, hyperactivity, paddle feet, chunky appearance, tail kink, can be attributed to biallelic expression of Gnasxl combined with biallelic, and thus overexpression of imprinted Gnas, in either PatDp(dist2)1\u22362 or +/\u0394Ex1A results in normal viability. Normal viability was also recently reported for the Ex1A-T-CON mutant in which Gnas is overexpressed Gnas has less drastic results on phenotype than overexpression of Gnasxl. But in the presence of two expressed copies of Gnasxl it is critical to have two expressed copies of Gnas in order to survive to weaning.Interestingly monoallelic expression of m\u0394Nesp55 neonates Gnasxl and no expressed doses of imprinted Gnas. The hyperactivity in PatDp(dist2)2\u22360 and m\u0394Nesp55 may reflect a central neurological deficit. The finding of raised noradrenaline in PatDp(dist2)2\u22360 together with a recent report showing that Gnasxl is expressed in neonatal muscle Gnasxl KO pups might be at least partly due to the lack of XL\u03b1s in muscle.PatDp(dist2)2\u22360 are hyperactive and hypoglycaemic and both these phenotypes may contribute to their early death. Hyperactivity has also been reported for some m\u0394Nesp55 neonates are also hypoglycaemic and although poor feeding may well contribute to their hypoglycaemia they may, in addition, have a defect in glucose counterregulation Gnasxl and Gnas, glucose metabolism is restored to normal, suggesting that both Gnasxl and imprinted Gnas have a role in neonatal glucose metabolism.PatDp(dist2)2\u22360 are hypoglycaemic with low insulin and raised noradrenaline. Gnasxl proteins to other aspects of the PatDp(dist2)2\u22360 phenotype is as yet unknown.Both Gs\u03b1 and XL\u03b1s have several variants. Thus C-terminally truncated neural specific forms Gs\u03b1N1 and XLN1 of Gs\u03b1 and XL\u03b1s respectively are known Gnas and Gnasxl have roles in the phenotype of PatDp(dist2). Loss of the orthologous GNAS transcript in humans is associated with a number of endocrine and bone disorders including pseudohypoparathyroidism, Albright Hereditary Ostedystrophy and progressive osseous heteroplasia GNAS and pseudohypoparathyroidism type Ib is caused by defective imprinting at the cluster  mice hyperactivity commencing shortly after birth and elevated noradrenaline are also features of raised Gnasxl levels. It would be of interest to ascertain if metabolism and activity in PHP-Ib patients are affected in the early postnatal period and if so, whether elevated GNASXL is responsible.Our data indicate that both Gnas in +/\u0394Ex1A results in postnatal growth retardation followed by catch up growth and can be attributed solely to loss of imprinting, resulting in biallelic expression, of Gnas. The effects on postnatal growth in +/\u0394Ex1A parallel the growth pattern seen in +/Ex1A-T-CON mice which also have loss of imprinting of GnasGnas clearly has a role in growth. Paternally expressed Gnasxl is also known to affect postnatal growth and mutations resulting in loss of functional Gnasxl also result in postnatal growth retardation Gnas and underexpression of Gnasxl results in growth retardation accords with the parental conflict theory that predicts that maternally expressed imprinted genes will be growth inhibiting and paternally expressed imprinted genes will be growth enhancing Gnas and Gnasxl. However PatDp(dist2)2\u22362 mice show a more extreme postnatal growth retardation than seen with overexpression of Gnas in +/\u0394Ex1A. Thus biallelic expression of both Gnas and Gnasxl has resulted in postnatal growth retardation. One interpretation is that it is important to have monoallelic expression of antagonistic Gnasxl and Gnas for normal growth. The advantages of monoallelic expression of both Gnasxl and Gnas must outweigh the costs associated with functional haploidy.We have shown that loss of imprinting of Figure S1Expression of Gnasxl was reduced on paternal inheritance of \u0394Nespas. (A) Northern blot of Gnasxl and \u03b2-actin loading control using 2.5 \u00b5g of poly (A)+ RNA from 15.5-dpc embryos. MatDp(dist2) have no expressed copies of Gnasxl, whilst PatDp(dist2) have two expressed copies, leading to the absence of the 2.5 kb band and the presence of a strong 2.5 kb band respectively. (B) Bar chart showing the Gnasxl expression levels in +/\u0394Nespas (mean \u00b1 s.e.m 24\u00b13.56%) were decreased on comparison to wild-type (+/+) (mean \u00b1 s.e.m 103\u00b19.39%) *P\u200a=\u200a0.005 . The mean \u00b1 s.e.m was calculated for 3 wild-type (+/+) and four +/\u0394Nespas.(TIF)Click here for additional data file.Video S1Hyperactivity in PatDp(dist2). Video of a 6 day old PatDp(dist2) and wild-type littermate showing almost continual activity and rapid movement with paddling of the front feet in the PatDp(dist2) but very little movement in the wild-type. The PatDp(dist2) has the characteristic tail kink or bend, and by 6 days is clearly smaller than the wild-type but oedema and a chunky appearance are no longer evident. The mice have a genetic background that is 50% Mus musculus castaneus and 50% laboratory mouse. On this background there was slightly better survival of PatDp(dist2) to 7 days (10/23) compared with 3/28 on an entirely laboratory mouse background  but no difference in survival to weaning.(MP4)Click here for additional data file.Video S2Hyperactivity in PatDp(dist2). Video of the same PatDp(dist2) and wild-type littermate at 6 days as in Video S2 and the PatDp(dist2) at 5 days of age on its own. The video shows the ability of the PatDp(dist2) to right itself after falling on its back, as well as its continual activity and rapid movement.(MP4)Click here for additional data file."}
+{"text": "Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart\u2019s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican\u2019s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the  During embryonic development, the extracellular matrix plays a central role in restructuring the single heart tube into a mature multi-chambered organ. To date, only two structural components of the extracellular matrix, versican and its binding partner hyaluronan, have been shown to be indispensably required for successful completion of this morphogenetic transition. Complete loss of either of these matrix molecules results in early embryonic lethality with a failure of endocardial cushion formation and a highly dilated myocardium of the primitive heart tube Vcan; formerly Cspg2) consists of 15 exons and spans a region of approximately 100 kb Versican is a chondroitin sulfate proteoglycan that was first identified in fibroblastic extracts heart defect mouse (hdf), the absence of versican protein results in early embryonic lethality hdf allele display severe cardiac defects including absence of pre-valvular endocardial cushions, loss of anterior (second) heart field structures and a thin, dilated myocardium. Similarly, loss of a subdomain of the G1 domain  results in embryonic lethality of homozygous embryos on a congenic and mixed background. However, on the mixed background some embryos survive longer and have ventricular septal defects Vcan\u2019s alternative splice forms at later stages of heart development using a versican alternative splice form deficient mouse model. The Vcan(tm1Zim) mouse, expresses only the V1 and V3 splice forms of versican in the heart and survive embryogenesis Vcan(tm1Zim) deletion have less severe cardiac defects than the hdf homozygous mice. Differential protein expression profiling using isobaric tag reagents for absolute and relative quantitation (iTRAQ) As shown by the (tm1Zim)Vcan hearts were collected from timed pregnant dams and staged according to Theiler This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (AR#1572) was approved by the Institutional Animal Care & Use Committee (IACUC) at the Medical University of South Carolina. Wild-type C57BL6/J and Vcan(tm1Zim) mice were created and characterized as described elsewhere 5\u2032-GAGAGGACAGAAACACCAAG-3\u2032, 5\u2032-ACTGTGGGTCAAATGAACTC-3\u2032 designed to yield a 454 bp product from the mutated allele and a 312 bp product from the wild-type allele. The absence of the V0 and V2 splice forms was independently confirmed in our laboratory by immunohistochemistry and western blot analysis.The To evaluate the whole mouse embryo hearts by optical sectioning, we adapted a confocal fluorescence imaging technique originally used with avian hearts Embryos (E10.5 pc) were dissected from the mouse decidua and extra embryonic tissues and pooled into Earle\u2019s balanced salt solution (Gibco). Hearts were dissected from the embryos and the AV regions were removed, and cut to expose the lumen. The AV explants were placed immediately onto the surface of drained collagen gels with 1 explant per well, and incubated at 37\u00b0C for 3\u20134 hr to allow attachment of the explants onto the collagen gel surface as previously described Hearts dissected from postnatal mice or embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned as previously described Vcan(tm1Zim) mutant and wild-type E13.5 p.c. embryos obtained from multiple litters were removed by micro-dissection, bisected and rinsed with PBS, frozen in liquid nitrogen and then finely crushed in a liquid nitrogen cooled stainless steel mortar and pestle . Protein profiling was performed on the pooled hearts of 5 animals from each group in order to obtain sufficient protein for analysis. The samples were homogenized  in ice cold 20 mM HEPES, pH 8/4\u00b0C, containing 1 M urea in a 1 mL ground glass tissue grinder then subjected to 770\u00d7g for 10 min at 4\u00b0C to remove nuclei and undisassociated tissue. The resulting supernatants were subjected to centrifugation at 150,000\u00d7g for 90 min at 4\u00b0C (Beckman Optima TLX ultracentrifuge with TKS-55 rotor) to obtain a combined urea soluble and insoluble fraction. The protein concentration in the urea soluble fraction was determined by the BCS method  using bovine serum albumin as standard. The samples were reduced and alkylated prior to digestion with LysC and trypsin at a ratio of 1\u223610 Pregnant females at E13.5 post coitus (pc) were anesthetized and euthanized following approved protocols of the Division of Laboratory Animal Resources at MUSC and AAALAC guidelines. Hearts of homozygous Vcan(tm1Zim) mutant E13.5 pc hearts (n\u200a=\u200a5 per group) were labeled with the \u201cisobaric tag reagents for absolute and relative quantitation\u201d (iTRAQ\u2122) . Duplicate samples (90 \u00b5g of protein each) from wild-type and Vcan(tm1Zim) mutant hearts were each labeled with one of the 4 unique isobaric tags  sample 1; 117- Vcan(tm1Zim) sample 2) and all run in parallel through the iTRAQ analysis. This strategy created an internal control for the analysis since the ratio for any one detected protein when compared to the value for the same protein in the duplicate sample should equal 1 (114-wt/116-wt and 115-Vcan(tm1Zim)/117-Vcan(tm1Zim)). The combined samples were subjected to cationic exchange/reversed-phase HPLC fractionation and mass spectrometry analysis for protein identification and quantification. Protein quantification for all of the iTRAQ peptides found was calculated using a GPS software and the statistical model for iTRAQ proteomic analysis Heart preparations from the Wt (wild type), and Vcan(tm1Zim) mutant and wild-type E13.5 p.c. embryos were obtained by microdissection and proteins extracted as described for iTRAQ analysis. The protein concentration in the urea soluble fraction was determined by the BCS method  using bovine serum albumin as standard. Protein samples (20 \u00b5g) from individual hearts were electrophoresed on 4\u201315% SDS\u2013PAGE gels essentially as we have previously described Hearts of homozygous Vcan(tm1Zim) hearts showed an altered external morphology and the presence of heart defects. External examination of the postnatal Vcan(tm1Zim) hearts showed a dilated right ventricular wall that was most pronounced in the subpulmonary infundibulum adjacent to the right atrium (Vcan(tm1Zim) hearts a more rounded external appearance than in wild-type hearts  hearts achieved normal internal septation. However, AV cushions in all the Vcan(tm1Zim) hearts were smaller than those found in the wild-type hearts (see below) and ventricular septal defects were observed  hearts at E13.5 pc showed significant differences in the size of the central AV endocardial cushions of the heart (Vcan(tm1Zim) hearts as determined by AMIRA 3D reconstruction measurements (Vcan(tm1Zim) and wild type littermates (Vcan(tm1Zim) (Vcan(tm1Zim) compared to the wild-type animals (Vcan(tm1Zim) and wild-type animals (Vcan(tm1Zim) leaflets also appeared smaller than those found in the wild-type littermate controls (Vcan(tm1Zim) hearts . Cushion explants from the AV regions of Vcan(tm1Zim) hearts cultured on 3D hydrated collagen gels (EMT assay) Histological three-dimensional (3D) reconstructions of the he heart . Both thurements . Similartermates . The dortermates . In the (tm1Zim) . Whereas(tm1Zim) . In addi animals  The lume animals . In newbcontrols . The denVcan(tm1Zim) mouse. Results confirmed that, as expected, the GAG-\u03b1/exon 7 antibody did not react with the Vcan(tm1Zim) hearts since the polypeptide region containing the epitope recognized by this antibody is missing in the Vcan(tm1Zim) mouse  mouse . Localizm) mouse .Vcan(tm1Zim) hearts (Vcan(tm1Zim) hearts.Immunolocalization of the GAG-\u03b2/exon 8 epitope, found in both V0 and V1 versican, was localized throughout all valvular leaflets in both the wild-type and the ) hearts . HoweverVcan(tm1Zim) extracellular matrix the protein periostin was evaluated in embryonic and adult hearts because it is expressed in a close pattern with versican in developing cushions and adult structures Vcan(tm1Zim) and wild-type hearts. Staining for periostin appeared more abundant and fibrillar in the Vcan(tm1Zim) (Vcan(tm1Zim) hearts, periostin was detected in a similar pattern, but with more intensity in the walls of the aorta (Vcan(tm1Zim) hearts   B,D than(tm1Zim)  A,C endo(tm1Zim) . Stainin(tm1Zim) . In the he aorta . Also, tVcan(tm1Zim) hearts (E13.5 pc) showed a lack of cells positive for \u03b1-sarcomeric actin within the mesenchyme of the right parietal leaflet and reduced amounts in the central mesenchymalized cushions (Vcan(tm1Zim) become muscularized during the late stages of embryonic development through a process of differentiation or myocardial cell invasion Vcan(tm1Zim) E13.5 hearts, cells positive for of \u03b1-sarcomeric actin staining are completely absent within the cushion mesenchyme of the right parietal leaflet (Vcan(tm1Zim) hearts have a diminished level of \u03b1-sarcomeric actin staining in the central regions of mesenchyme formed by the spina vestibule and dorsal mesenchymal protrusion  was used. Protein extractions were made of separately pooled E13.5 pc Vcan(tm1Zim) (n\u200a=\u200a5) hearts and wild-type (n\u200a=\u200a5) hearts. Replicate aliquots of the WT and Vcan(tm1Zim) heart homogenates were labeled with unique isobaric tags for separate analysis. The separate protein fractions were then analyzed by iTRAQ to profile the identities and relative changes in abundance. The replicate aliquots from the same groups allowed for controlled comparisons within each group. Therefore, as an internal control the ratio of protein abundance for each identified protein within the same (Vcan(tm1Zim) or wt) replicate aliquot should yield a value of 1.0. Only those that deviated from a ratio of 1.0 were considered to represent a unique protein fingerprint for Vcan(tm1Zim) hearts compared to wild-type. Identities, relative abundance and statistical analysis of multiple peptides mapping to individual proteins were performed on the data using methods and software previously described To identify some of the resulting differences in cellular protein abundance that results from the loss of the extracellular Vcan(tm1Zim) and WT fractions. A total of 2882 unique peptides were identified, corresponding to 940 proteins. Well over 50% of the proteins identified were represented by two or more unique peptides. Only those proteins with at least two unique peptides are reported. Forty-seven proteins showed significant increases in the mean expression ratio of abundance by more than 1.20-fold in the Vcan(tm1Zim) mouse heart and 74 were decreased by more than 0.85X (Vcan(tm1Zim) hearts was reduced (0.45x) in abundance compared to the wild-type. Other proteins of interest were surveyed in western blots to verify the relative change in abundance detected by iTRAQ. For example, Annexin A6 (4 unique peptides) and Stathmin 1 (9 uniques peptides) together showed a relative decrease (0.85 and 0.73 respectively) in the Vcan(tm1Zim) heart. Also, all 3 peptides mapped to Serpinh1 (Hsp 47) showed an increased abundance (1.55) in the Vcan(tm1Zim). The relative decreases or increase was confirmed by western blots of protein in more than one animal  hearts into standard gene ontology (GO) categories  mice revealed septal defects, changes in the size of some mesenchymalized cushions, dilated right ventricle with dilation of the sub-pulmonary infundibulum and altered integration of the aortic and pulmonary trunks into the AV cushion complex. These combined altered phenotypes result in a failure of the outflow tract to integrate and align correctly with the ventricles during development.Since the initial identification and characterization of versican (tm1Zim) hearts. Closure of the interventricular foramen occurs at approximately E13\u201314 p.c. in the wild-type mice on the C57B6 background. In the E13.5 p.c. hearts, analyzed by AMIRA 3d reconstruction, forming cushions of the dorsal mesenchymal protrusion, septal and aortic leaflets were significantly smaller and did not merge as in wild-type. This reduction in cushion tissue could contribute to the higher incidence of VSD at E13.5, however many of these defects eventually close as was observed shortly after weaning of the newborn mice . The presence of ventricular septal defects have been described in other mouse models of Vcan partial depletion that include the Crtl-1 null, hdf heterozygous and the Vcan subdomain A null mouse (\u03943/\u03943Vcan) (tm1Zim)Vcan exon mouse heart defects with the Crtl1-null and the hdf heterozygote models show this overlap in structural defects (e.g. VSD) (tm1Zim)Vcan reported here. We also know that hdf heterozygous (tm1Zim)Vcan heart have reduced levels (approximately 50%) of versican. Similarly, the expression of the truncated Vcan gene in \u03943/\u03943Vcan is approximately 55% of the wild type, The hdf heterozygote is able to express all splice forms of Vcan from its unaffected allele, the  \u03943/\u03943Vcan expresses an altered form containing the G3 domain and the (tm1Zim)Vcan can only expresses 2 of the 4 alternative splice form variants. Taken together the observations suggest that the VSDs observed in all 3 models are the result of an overall reduction in versican expression and not specifically due to the splice variants expressed.We observed the presence of VSDs in the VcanThe process by which the outflow tract becomes aligned with the ventricles involves the integration of the proximal outflow tract cushion into the central mesenchymalized cushion of the E13.5 p.c. heart. This positions the aortic and pulmonary arterial trunks in proper alignment between the left and right ventricular canals . The mecVcan(tm1Zim) heart show evidence of less myocardialization. The Vcan(tm1Zim) hearts at E13.5 pc showed reduced numbers of cells positive for \u03b1-sarcomeric actin within both these cushions (Vcan splice forms V0/V2. This could be due to 1) a non-permissive matrix environment in the cushion that does not promote or prevents myocardial differentiation and/or invasion of needed precursor cells; 2) inability of the cushion matrix to sustain the myocardial phenotype within the cushion. The loss of Vcan exon 7 containing alternative splice forms V0/V2 may directly result in a \u201cnon-permissive\u201d matrix, or indirectly alter other ECM molecules to interfere with invasion and/or myocardial phenotypic stability. We have shown that there are overall fewer cushion mesenchymal cells in the Vcan(tm1Zim) heart. It should be noted that the time points chosen for this study might not be entirely sufficient to fully understand the mechanisms of the valvular defects. Others have shown that a sufficient threshold level of cushion mesenchyme is essential for myocardialization to occur using in vitro assays that model the process Both the right mural and the central AV mesenchymalized cushions in cushions  & 12. Th(tm1Zim) mutant cushions. Vcan induced changes in periostin distribution may effect the developing valves by altering the structural integrity of the ECM through altered interaction with other molecules such as tenascin-C, fibronectin, collagen and other proteoglycans Vcan(tm1Zim) hearts correlated with protein abundance changes identified in the proteomic results. Among these, a protein that showed changes in abundance by the iTRAQ analysis was LIM domain binding 3 isoform b also known as cypher/ZASP. Cypher/ZASP is a member of the family of proteins containing a PDZ domain at their amino terminus and LIM domains at their COOH terminus. Cypher null mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles Vcan(tm1Zim) and may be related to the observed lower mesenchyme cell density in the endocardial cushions. The versican V1 (exon 8 containing) splice form still present in the Vcan(tm1Zim) down regulates the expression of vimentin in NIH 3T3 cells Because versican is an extracellular matrix molecule, the cellular responses reflected by the differentially expressed proteins are likely due to the cells interaction with an altered ECM. Periostin is an important molecule of the heart cushion extracellular matrix In conclusion, the defects in cardiac phenotype and alterations in protein abundance reported in this study show that the versican splice forms V1/V2 containing exon 7 are important for regulation of cardiac cushion size and the myocardial remodeling required for the proper integration of the outflow tract into the base of the heart.File S1Supplemental iTRAQ report.(PDF)Click here for additional data file.File S2Supplemental GO file.(PDF)Click here for additional data file."}
+{"text": "Our results show that RASSF2, RASSF5C and RASSF10 are aberrantly hypermethylated in MCC to a varying degree and this might contribute to Merkel cell carcinogenesis.Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. RASSFs are a family of tumor suppressors that are frequently inactivated by promoter hypermethylation in various cancers. We studied CpG island promoter hypermethylation in MCC of RASSF2, RASSF5A, RASSF5C and RASSF10 by combined bisulfite restriction analysis (COBRA) in MCC samples and control tissue. We found RASSF2 to be methylated in three out of 43 (7%), RASSF5A in 17 out of 39 , RASSF5C in two out of 26 (8%) and RASSF10 in 19 out of 84 (23%) of the cancer samples. No correlation between the methylation status of the analyzed RASSFs or between RASSF methylation and MCC characteristics (primary  Merkel Cell Carcinoma (MCC) is a rare but aggressive cutaneous malignancy  of the eC-terminally (RASSF1\u20136) or N-terminally (RASSF7\u201310). The functions of the C-terminal or classical members range from apoptosis induction, cell cycle inhibition to microtubule stabilization [Tumor suppressor genes (TSG) are commonly inactivated by promoter hypermethylation in cancer. Methylation occurs on the DNA level at 5' position of cytosines, when found as dinucleotides with guanine. CpGs are overrepresented in the promoter region of TSG forming so called CpG islands. DNA methylation in CpG islands of TSG leads to epigenetic silencing of the according transcript . Wellization . The focus of our current work was on RASSF2, RASSF5A, RASSF5C and RASSF10, due to the fact that epigenetic inactivation of these tumor suppressors of the RASSF family was already reported in different cancer types. We and others showed promoter hypermethylation of RASSF2 ,19,20, ROur aim in the present study was the comparative analysis of RASSF promoter hypermethylation in a set of primary Merkel cell carcinoma and controls. Therefore the promoter regions of RASSF2, RASSF5A, RASSF5C and RASSF10 were analyzed by combined bisulfite restriction analysis (COBRA). We show that the degree of promoter hypermethylation in MCC varies between the RASSFs, but is present for different RASSFs at the same time.Taq1 sites at position 111. For RASSF5A COBRA PCR product is 334 bp after semi-nested PCR with Taq1 sites at position 94, 234 and 260. COBRA PCR product of RASSF5C is 322 bp after nested PCR with Taq1 sites at position 221 and 284. The RASSF10 COBRA PCR product is 241 bp (with Taq1 sites at 50 and 141) or with alternative primer pair 167 bp (with Taq1 sites at 67). A Summary of COBRA PCR products, CpG islands, primer positions and Taq1 restriction sites is shown in The promoter regions of RASSF2, RASSF5A, RASSF5C and RASSF10 were analyzed by CpG plot  to show We used retrospectively sampled Merkel cell carcinomas from the tumor registries of the University of Heidelberg, the University of Halle , DermPath Friedrichshafen, and the Institute of Pathology of Halle . 87 samples of 85 tumors  from 79 patients  were studied . Skin coTissue specimens were deparaffinized by xylene and ethanol treatment. DNA was isolated with a QIAamp DNA extraction kit  after a proteinase K restriction and concentrations of DNA were determined by UV-photospectrometery.2O and followed by 10 min incubation with 5 \u00b5L 3 M NaOH at 37 \u00b0C. DNA was then precipitated with 100% ethanol and 7.5 M ammonium acetate and redissolved in 1 \u00d7 TE buffer. 200 ng were subsequently used for 25 \u00b5L PCR reaction with COBRA primers  1 h at 65 \u00b0C and resolved on 2% TBE gel together with mock digest.Genomic DNA from MCC or control tissue (2 \u00b5g) was bisulfite treated  and incubated overnight at 50 \u00b0C. Then DNA was purified using MSB Spin PCRapace , eluted in 50 \u00b5L HThe RASSF family members RASSF2, RASSF5A, RASSF5C and RASSF10 all contain CpG islands in their promoter region, as analyzed by CpG plot and UCSC Genome browser and as shown in CpG island promoter methylation was analyzed for RASSF2, RASSF5A, RASSF5C and RASSF10 in Merkel cell tumor  and skinin vitro methylated DNA (ivm) was used as positive control. Representative samples are shown for each RASSF promoter analyzed. In case of RASSF2 promoter methylation in MCCs no sample shows digestion products as compared to ivm DNA . The according control tissue also showed a high degree of RASSF5A promoter methylation (43%). RASSF5C is methylated to a degree of 8% in 26 of tumor samples and unmethylated in control tissue. The RASSF10 CpG island promoter methylation was studied in 84 Merkel cell tumor samples of which 19 were methylated. Control tissue (14 samples) was unmethylated at the RASSF10 promoter region. p > 0.05). For details of tumors analyzed regarding MCPyV status and promoter methylation results see The aim of this work was to perform a comparative analysis of CpG island promoter hypermethylation of four Ras-association domain family members in Merkel cell carcinoma. MCC represents one of the most aggressive kinds of skin cancer , of whicThe RASSF members analyzed in this study have in common a frequent promoter hypermethylation of their CpG island in cancer. The RASSF2 transcript was detected in normal tissue , but wasThe RASSF members chosen for analysis were shown to harbor different functional properties. The mouse model suggests a role of RASSF2 in bone development , but alsC-terminal and RASSF10 as a member of the N-terminal family contribute to carcinogenesis through independent mechanisms. In our study we showed that the RASSF2 promoter region was hypermethylated in 7% of cancer samples, but unmethylated in control tissue. With a total of 43 analyzed tumor samples RASSF2 seems to be hypermethylated in only a small subset of samples. RASSF5A was found to be frequently hypermethylated in Merkel cell carcinoma (44%), but also in control tissue (43%). Therefore promoter hypermethylation of RASSF5A seems not to be restricted to MCC. However, previous studies showed that RASSF5A can be epigenetically inactivated by promoter hypermethylation in other cancer types . The RASTo date only limited functional data exist for RASSF10. However it was shown that the RASSF10 promoter was hypermethylated in cancers. e.g., malignant melanoma of the skin . The curThe present study is the first comparative analysis of RASSF promoter methylation in Merkel cell carcinoma. In summary we were able to show different RASSFs like RASSF2, RASSF5C and RASSF10 are tumor specifically methylated at their promoter region in MCC, additional to the earlier reported presence of RASSF1A hypermethylation in MCC . Promote"}
+{"text": "Clinical benefit from CO2 inhalation in patients with craniofacial pain caused by a putative activation of TRPV1 receptor positive trigeminal neurons has also been reported. These effects are probably mediated via an activation of TRPV1 receptor - positive neurons in the nasal mucosa with subsequent central inhibitory effects (such as conditioned pain modulation). In this study, we aimed to examine the effects of intranasal CO2 on a human model of craniofacial pain elicited by nasal application of capsaicin.Nasal insufflation of CO2 or air was insufflated alternatingly into the nasal cavity at a flow rate of 1 l/min for 60 sec each. In the subsequent experiment, all participants were randomized into 2 groups of 24 each and received either continuous nasal insufflation of CO2 or placebo for 18:40 min after nociceptive stimulation with intranasal capsaicin. In both experiments, pain was rated on a numerical rating scale every 60 sec.In a first experiment, 48 healthy volunteers without previous craniofacial pain received intranasal capsaicin to provoke trigeminal pain elicited by activation of TRVP1 positive nociceptive neurons. Then, CO2 on experimental trigeminal pain were only marginal. In the first experiment, CO2 reduced pain ratings only minimally by 5.3% compared to air if given alternatingly with significant results for the main factor GROUP  and the interaction term TIME*GROUP  in the repeated-measures ANOVA. However, these effects were abrogated after continuous insufflation of CO2 or placebo with no significant changes for the main factors or the interaction term.Contrary to previous animal studies, the effects of CO2 could be seen in this human model of TRPV-1 mediated activation of nociceptive trigeminal neurons, utility is limited as observed changes in pain ratings are clinically non-significant.Although mild modulatory effects of low-flow intranasal CO Preliminary data also show efficacy of nasal CO2 insufflation in migraine patients [Phasic nasal insufflation of COn humans  to recorn humans  and chemn humans . However minutes . Likewistentials . These rtentials  upon propatients ,12.2 is a potent vasodilator, the first studies attributed possible therapeutic effects to the vasodilating properties on cerebral vessels. More recently, CO2 has been shown to be a powerful modulator of activated nociceptive trigeminal neurons [2 or air nasally [2 flow rates of 0.8 l/min but not of 0.4 l/min CO2 or air. Based on additional pharmacological experiments the authors concluded that CO2 exerts its antinociceptive, respectively antihyperalgesic effects by activation of mucosal primary trigeminal afferents through a decreased mucosal pH within the nasal cavity.The putative mode of action is uncertain. As CO neurons . Tzabazi nasally . Nocifenin vivo models of migraine [These findings are well in line with the hypothesis that TRPV1 receptor positive trigeminal C and A delta fibres (which are activated by application of capsaicin) may play a relevant role in the pathogenesis of primary headaches such as migraine  althoughmigraine .2 could have some positive effect on acute headache. We therefore aimed to examine the modulatory efficacy of intranasal CO2 on experimental TRPV1-mediated trigeminal pain elicited by intranasal application of capsaicin in healthy volunteers to answer the following questions:In summary, there are some clinical but also pre-clinical data that nasal instillation of CO2 on a numerical rating scale at a flow rate of 1 l/min?1. How painful is prolonged intranasal application of CO2 lead to relevant systemic changes of pH and pCO2 in capillary blood samples?2. Does intranasally applied CO2 modulate pain ratings after intranasal application of capsaicin?3. Does intranasal insufflation of CO2 on TRPV1-mediated trigeminal pain in healthy volunteers. All participants provided written informed consent prior to inclusion into the study. Our study was approved by the local Ethics Committee (protocol number PV3814) and conformed to the Declaration of Helsinki.We conducted a controlled randomized parallel-group study to investigate the effects of intranasal COHealthy volunteers were recruited among medical students at the Medical Faculty of Hamburg University  for 18:40 minutes and rated their pain on a numeric rating scale (NRS) from 0 (no pain) to 10 (worst imaginable pain) every 80 seconds. To assess possible systemic changes in pH and CO2 levels, pH and pCO2 were additionally determined in 10 patients by a capillary blood gas analyses. This was taken from the earlobe before nasal installation of CO2 started and immediately after the last pain rating while the participants were still exposed to CO2.In a pilot study Figure\u00a0 designed2 (1 l/min) and air (1 l/min) alternatingly. If volunteers had already participated in the pilot study, both experimental sessions were separated by at least 6 weeks. Again, subjects rated the magnitude of pain verbally on a numerical rating scale (NRS) ranging from 0 (non-painful) to 10 (worst imaginable pain).In the main study Figure\u00a0, 48 heal2 and air were insufflated for 60 sec each in an alternating fashion via a nasal cannula. During an interval of 20 sec between exposure to either CO2 or air, no gas was applied and the participant was asked to rate the resulting pain on a NRS. After 4 cycles, insufflation was interrupted for 1 min to allow participants to remove excessive nasal discharge. However, subjects were asked not to blow their noses to avoid early removal of the intranasal capsaicin. Thereafter, 4 identical cycles of CO2, respectively air were given and subjects were asked to rate the pain on the NRS.Initially, participants received one puff of capsaicin spray  into the left nostril to trigger TRPV1-mediated trigeminal nociception. After three initial pain ratings on a NRS every 60 seconds  or placebo in 2 subgroups of 24 subjects each.In the subsequent second experiment separated from the first experiment by at least 4 weeks all participants from the first experiment were randomly allocated to receive either CO2 for a total of 18:40 min via a nasal cannula or placebo  and rated the pain on the NRS as in the pilot study.Again, participants received 1 puff of capsaicin spray into the left nostril. Pain ratings on a NRS were noted every 60 sec over the entire duration of this experiment  was administered from compressed gas cylinders with a volume of 10 l. By using a combined pressure reducing valve (200 mbar/4.5 mbar) and flowmeter  a constant flow of 1 l/min was maintained. Medical grade compressed air was obtained from the hospital gas reticulation system and delivered at a flow of 1 l/min by a flow meter . Nasal cannulae  were used to apply both gases locally into both nasal cavities.Medical grade CO2 should only be insufflated nasally and not inhaled due to safety concerns, all subjects were trained before the first experiment. CO2 was applied bilaterally into the anterior nasal cavity, where highest mucosal responsiveness of evoked potentials to short pulses of CO2 could be found [As CObe found , namely One puff of a nasal spray containing 200 \u03bcg of capsaicin (prepared from 1.42 ml capsaicin liquid extract in 10 ml of refined sesame oil) was applied to the left nostril with the head in a stooped position to avoid contamination of the pharynx. Participants were allowed to remove excessive nasal discharge by soaking cotton swaps, but were asked not to blow their noses to avoid early removal of intranasal capsaicin.2 males: 35-48, females: 32-45.Blood gas analyses were collected in from the earlobe in capillary tubes after pretreatment with an ointment containing 5% benzyl nicotinate  for 10 mins. Immediately afterwards samples were tested with a blood gas analyzer . The manufacturer\u2019s reference values for capillary blood gas analysis were as follows: pH 7.35- 7.45; pCO2 or air). Comparison of categorical data (gender) was done by means of the chi square test. In all tests p values <0.05 were considered significant.Descriptive statistics are given as mean values and standard errors of the mean. Differences on mean values were either examined by paired t-tests or by means of a repeated measures analysis of variance (ANOVA). For analysis of pain ratings over time in the pilot study, the factor TIME (pain ratings 1-15) was used, for other comparisons of pain ratings a two-way repeated measures ANOVA with the factors TIME (corresponding pain ratings) and GROUP .2 was given exclusively to 20 healthy subjects , mean pain ratings over time were 0.6 (\u00b1\u20090.06) out of 10 on the NRS. Pain ratings reached their maximum after 1 min of inhalation with 0.8 (\u00b10.21) out of 10 on the NRS . Seven (35%) of the participants indicated that they had not perceived any pain at all.When CO2 nasally or started talking during the training phase, they mostly complained of a highly unpleasant irritation spreading from the nasal cavity into the nasal sinus and the pharynx which led to interruption of the training.If subjects accidentally inhaled CO2 levels (difference between pCO2 after \u2013 pCO2 before insufflation) did not correlate with corresponding pain ratings in the pilot study (Pearson\u2019s bivariate correlation: p\u2009>\u20090.05).Net changes of pCO2 (3.6\u2009\u00b1\u20090.3) and air (3.8\u2009\u00b1\u20090.3) respectively differed significantly (t(47)\u2009=\u2009-2.107; p\u2009=\u20090.041). Likewise, a repeated-measures ANOVA for the factors TIME (pain ratings for insufflation 1 to 4) and GROUP (CO2 or air) yielded no significant main effect for TIME , but significant results for both the main factor GROUP  and the interaction term TIME*GROUP . Mean values are given in Figure For epidemiological details see Table\u00a02 .6\u2009\u00b1\u20090.3 2 or air in experiment 2 (for further details see Table\u00a0Age and gender distribution did not differ significantly between the randomly assigned subgroups receiving either CO2 or air) yielded a significant main effect for TIME , but not for the factor GROUP \u2009=\u20090.089; p\u2009=\u20090.767) or the interaction term TIME*GROUP .A repeated-measures ANOVA with the factors TIME (pain ratings 1 to 17) and GROUP  = 0.15; p\u2009=\u20090.902).There was no baseline difference defined as comparison of the first pain rating between the CO2. Most notably, no patient complained about dyspnea or other clinical side effects. After 20 min of CO2 insufflation, a significant shift of capillary pH and capillary pCO2 readings could be found. pH decreased and pCO2 levels increased significantly , alternating application of CO2 reduced pain ratings only minimally compared to a sham paradigm with air, but this effect was abrogated during continuous insufflation of CO2.Contrary to previous animal studies, the effects of intranasally insufflated COCapsaicin (8-Methyl-N-vanillyl-trans-6-nonenamide) increases release of substance P and simultaneously blocks its reuptake exerting its effects by activation of the TRPV1 receptor . This lein vitro experiments with slices of rat trigeminal nucleus caudalis [It has been shown in caudalis  that actcaudalis ,28. A recaudalis ,30. Accocaudalis . In contcaudalis .Regardless of whether TRPV1 receptor positive V1 neurons are indeed involved in migraine pathophysiology or not, they have been associated with other craniofacial pain syndromes such as dental pain where an upregulation of TRPV1 expression in rat trigeminal ganglia was observed in a model of lipopolysaccharide-induced pulpitis . We note2 insufflation was rated as mildly painful by 65% of patients. We found systemic changes in capillary pH and pCO2 levels but no relevant alterations beyond the normative range and conclude that nasal application at 1 l/min is safe. Insufficient delivery of CO2 into the nasal cavity seems unlikely as changes of pCO2 levels after nasal insufflation did not correlate with pain ratings in our pilot study.CO2 induced unpleasantness respectively pain with vaporized ammonia to establish a robust sham condition were in vain (data not shown). Ammonia induced a more stabbing and - at higher concentrations - unbearable pain in the nasal cavity with variable pain thresholds. The unpleasantness of intranasal CO2 in our sample implies that true blinding is not feasible at flow rates of 1 l/min and above as most patients will notice an unpleasant or painful perception. As clinical effects were negligible in our sample, potential sources of bias such as a relevant placebo effect are unlikely.Our attempts to mimic COintranasal flow rates of 0.6 l/min, blinding seemed to be less problematic in prior studies. Casale et al. [insufflation of CO2 at 0.6 l/min for only 1 minute \u2013 similar to the setup used in the therapeutic studies in migraine patients by Spierings [Inhalation of CO2 was less well tolerated raising doubts about effective blinding in prior studies reporting efficacy of inhaled CO2.At lower e et al.  reportedpierings ,12. Inha2 in our study was small and reached statistical significance only when CO2 and placebo were given in an alternating fashion within the same subject (experiment 1) \u2013 as opposed to application of either CO2 or air only in experiment 2. It may be easier for the participants to sense a subtle analgesic or anti-hyperalgesic efficacy of CO2 if it is given in a contrasting fashion with air as placebo.The antihyperalgesic efficacy of CO2 with flow rates of 0.8 l/min attenuated nocifensive behaviour after sensitization with capsaicin unlike lower flow rates of CO2 (0.4 l/min) or air. In the nasal mucosa CO2 decomposes into protons and carbonate catalyzed by mucosal carbonic anhydrase and activates TRPV1- and ASIC- positive neurons by proton accumulation. Subsequent, central inhibitory effects are proposed such as a widespread inhibition of afferent trigeminal input though inhibitory interneurons, trans-segmental inhibitory control circuits or conditioned pain modulation.As shown by Tzabazis and colleagues in a rat model , nasal i2 or capsaicin resulted in an acidification of culture medium and a consecutive nociceptive activation with CGRP release [2 attenuated CGRP release by pretreatment with capsaicin if cultured under isohydric conditions which prevents extracellular but allows intracellular acidification.Vause and colleagues showed that incubation of cultured trigeminal ganglions with CO release . Similar2 insufflation on non-sensitized skin as compared to air insufflation [2, so that efficacy could have been better in patients with chronic craniofacial pain. Alternatively, CO2 inhalation with a potentially different locus of action could represent a more powerful alternative although tolerability seems to limit feasibility [Tzabazis and co-workers observed less intense and only short-lived antinociceptive or antihyperalgesic effects of COfflation . These fsibility ,10.2 exerts antihyperalgesic effects in animal models but resulted in only minor clinical effects in our human model of trigeminal pain elicited by activation of nociceptive TRPV1 receptors in healthy volunteers. These moderate effects question the clinical utility of intranasal CO2 in TRPV1-mediated pain at flow rates of 1 l/min.In summary, intranasal insufflation of CO2 in either air or oxygen for 5 min [2 mixed with oxygen for 10 min daily which was repeated up to 2 times if the headache was not relieved [2 suggesting a potentially different mode of action. In addition, translating these clinical results to our human model is difficult as the role of the TRPV1 receptor in migraine pathophysiology has been challenged [2 provoked a headache attack or increased headache intensity in 18 of 40 subjects with mainly posttraumatic headache and migraine. CO2-induced headache attacks or aggravation were less intense than headaches triggered by histamine and adrenaline [2 for 6 min [Marcussen and Wolff successfully treated aura symptoms in migraine patients termed as \u201cvasoconstrictor symptoms\u201d by inhalation of 10% COor 5 min . Likewisrelieved . After 3allenged . Furtherrenaline . Likewisor 6 min .2 in primary and secondary headaches is difficult despite promising data from animal experiments. Trials on the efficacy of inhaled CO2 in various headache syndromes are relatively old and yielded ambiguous results. Evidence for the efficacy of intranasal CO2 in migraine has been published in preliminary form.At present, a firm conclusion on the clinical efficacy of CO2 at flow rates of 1 l/min could be seen in a human model of TRPV1 mediated activation of nociceptive trigeminal neurons which is in line with previous studies. While application was safe, clinical utility at low flow rates was limited in our model as the therapy is uncomfortable and changes in pain ratings are therapeutically irrelevant.Only mild modulatory effects of intranasal insufflation of COhttp://www.linde-healthcare-realfund.com).TPJ and RR: no relevant conflicts of interest, AM: has received unrestricted scientific grant support from Linde Gas (RealFund; TPJ conceived of the study, participated in the conception and design, performed the statistical analysis, interpreted and discussed the data and drafted the manuscript. RR participated in the design of the study and data acquisition and helped in the interpretation of it. AM participated in the conception and design of the study, interpreted and discussed the data and drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Short accessory pathway (AP) effective refractory period (ERP) is one of the risk factors in Wolff-Parkinson-White syndrome (WPW). The purpose of study was to evaluate the reproducibility of APERP measurement during a same electrophysiological study (EPS).EPS consisted of 2 APERP measurements performed prospectively in 77 patients for a WPW in control state (CS) at a cycle length of 400 ms (n=76) and after isoproterenol (n=56).In CS, 18 patients (24 %) had the same APERP at both measurements; 41 (54.6 %) had differences from 10 to 40 ms, 17 (22.4 %) had differences > 40 ms. Among 45 patients with initial APERP > 240 ms, 7 had an APERP \u2264 240 ms at 2nd study. Among 31 patients with initial APERP \u2264 240 ms, 5 had an APERP > 240 ms at 2nd study. Pearson's product-moment correlation was 0.75. After isoproterenol, 5 patients (9 %) had the same APERPs; 37 (66 %) had differences from 10 to 40 ms and 14 had differences > 40 ms. Among 38 patients with initial APERP > 200 ms, 12 had an AP ERP \u2264 200 ms at 2nd study. Among 18 patients with initial APERP \u2264 200 ms, 10 had still APERP \u2264 200 ms at 2nd study. Pearson's product-moment correlation was 0.54.There are important variations of APERPs during EPS mainly after isoproterenol infusion. Therefore the values of APERPs should be interpreted cautiously. The prevalence of a typical Wolff-Parkinson-White (WPW) pattern is estimated to be 0.1% . A recenOther factors are discussed such as the presence of multiple accessory pathways and the male gender. Therefore, the measurement of the AP ERP during an electrophysiological study is important. Actually AP ablation is largely indicated and a short APERP might be sufficient to indicate AP ablation. The aim of this study is to evaluate the reproducibility of the measurement of AP ERP during the same electrophysiological study.Seventy seven patients, 56 males (73 %) and 21 females (27 %) aged from 8 to 57 years (mean age 31 \u00b1 12 years), with a ventricular preexcitation syndrome were consecutively included in this study; 46 patients were asymptomatic (60 %), 20 patients had palpitations (27 %), 7 patients had history of unexplained syncope (7 %), 2 had spontaneous documented atrial fibrillation (3 %). Only 2 patients (3 %) had a history of life-threatening arrhythmic events .Accessory pathway was septal located in 51 patients . Three patients had an anteroseptal location (4 %); 19 patients had a left lateral accessory pathway location (25 %) and 4 a right lateral location (5 %).This was a prospective study performed between 2004 and 2010 after informed consent. An electrophysiological study was performed in patients not sedated and after cessation of treatment. Patients were studied by transesophageal route and/or intracardiac route . The claThe disappearance of the pattern of preexcitation indicated when the APERP was reached. The longest A1A2 that fails to conduct at the atria was considered as the effective APERP. This protocol was reproduced again after several minutes to study the reproducibility of the measure of anterograde effective refractory period.The method was used to induce supraventricular tachycardia. The protocol was performed during 80 consecutive electrophysiological studies. Three patients were excluded from this protocol because sustained atrial fibrillation was induced at first programmed atrial stimulation and flecainide injection was required to stop it. In one patient the AP did not conduct in control state and the measurement of APERP was made only after isoproterenol.In the absence of induction of a tachycardia conducted through the accessory pathway at a rate higher than 250 bpm, isoproterenol (0.02 to 10 \u03bcg.min-1) was then infused to increase the sinus rate to at least 130 bpm and the pacing protocol was repeated. APERP was determined twice at a basic cycle length of 400 ms. Isoproterenol was infused in only 56 patients: 9 patients did not require isoproterenol infusion because they had an electrophysiological malignant form in control state with the induction of an atrial fibrillation and short cycle lengths conducted by AP. Two other patients remained in atrial fibrillation without signs of malignancy after second programmed stimulation in control state. Poorly-tolerated orthodromic tachycardia was induced in 5 symptomatic patients during the basal study and catheter ablation was indicated without evaluation after isoproterenol. In 6 children, isoproterenol infusion was poorly-tolerated and programmed atrial stimulation was not repeated.Conduction over the accessory atrioventricular connection was evaluated by the measurement of the maximal heart rate with a 1 to 1 conduction over the accessory connection and the shortest atrial tachycardia cycle length at which there was 1 to 1 conduction over the accessory connection. A short APERP was defined in the present study as less than or equal to 240 ms in control state (CS) and less than or equal to 200 ms after isoproterenol.Results were presented as mean and standard deviation and compared with the paired Student t test. A value of p < 0.05 was considered to be significant. Correlations were performed between 2 determinations of AP ERP and agreement was expressed according to the Pearson's correlation coefficient. It was obtained by dividing the covariance of the two variables by the product of their standard deviations.The mean fastest cardiac rate conducted by the accessory pathway was 214\u00b155 bpm in control state and the ranges were 100 and 300 bpm. The mean sinus cycle length did no differ significantly during both measurements (805\u00b112 vs 819\u00b111msec). The dispersion of the two measurements of APERP is represented on a graphic in Figure 2. The mean value of APERP at the first study was 264\u00b152 ms. Mean value of the second measure was 265\u00b150 ms (NS). However, there were important individual changes of APERP values between both studies.At the first determination, APERP was more than 240 ms (mean 299\u00b1 37) in 45 patients (59 %); mean value was the same in the second study (299\u00b1 37 ms); APERP became less than or equal to 240 ms at second measurement in 7 patients (15.5 %) and remained more than 240 ms in 38 patients (84.5 %). At the first APERP determination, 31 patients (41 %) had an APERP less than or equal to 240 ms: 24 patients had an APERP less than or equal to 240 ms at both studies (77 %); 7 patients had an APERP more than 240 ms at second study (22.5 %). The mean absolute difference between the two trials was 25.7 ms (\u00b125.2 ms). The variations are reported in Twenty-eight measurements (37%) were shorter in the second trial than in the first with a mean difference of 34\u00b124 ms. Thirty measurements (40 %) were shorter in the first trial than in the second, with a mean difference of 34\u00b123 ms. The Pearson's product - moment correlation was 0.75. The mean fastest cardiac rate conducted by the accessory pathway was 265\u00b118 bpm after isoproterenol infusion. The mean cycle length between 2 measurements does not differ significantly (475\u00b112 mesc vs 479\u00b114 ms). The dispersion of the two measurements with isoproterenol infusion was represented on a graphic in The mean value of the first measurement was 223\u00b140 ms. Mean value of the second measurement was 224\u00b148 ms (NS). Individual data differed. At the first APERP determination, 18 APERPs (32%) were less than or equal to 200 ms; 10 measures (56%) were less than or equal to 200 ms and reproducible at 2 measurements; 8 APERPs were more than 200 ms at the second determination (46%). The mean absolute difference between two trials was 34\u00b125.4 ms. Among 38 patients with initial APERP more than 200 ms, 12 measures were less than or equal to 200 ms at the second determination (33%). The individual variations are reported in For 30 patients (53.4 %) the APERP was shorter at the second measurement than at the first, with a mean difference of 30\u00b118 ms. For 21 patients (37.5 %) the first measure of APERP was the shortest with a mean difference of 47.6 \u00b1 28.4ms. The Pearson's product - moment correlation was 0.54.Fourteen patients underwent transesophageal study and later intracardiac study to perform the accessory pathway ablation. The initial mean values of APERP measured at esophageal study were significantly shorter than the values measured at intracardiac study, but other measurements did not differ significantly. The mean variations were similar either in control state (31\u00b129 ms and 30\u00b127 ms) or after isoproterenol (26\u00b116 and 24\u00b115 ms) .The mean age of our population was 31\u00b112 years. In the youngest population (30 years old or less), representing 36 patients, the Pearson correlation coefficient was 0.675 in control state and 0.58 after isoproterenol infusion. In the oldest population (more than 30 years old), representing 36 patients, the Pearson correlation coefficient was 0.78 in control state and 0.46 after isoproterenol infusion. The subgroups according to the age of the patient were too small to verify possible relationships between the age and the variability of APERP.There were no significant differences of APERP according to the location of AP: AP was left-sided in 19 patients and the variations were 27\u00b125 ms in control state and 25\u00b113.5 ms after isoproterenol. AP was septal in 54 patients and the variations were 24\u00b125.5 ms in control state and 35\u00b127 ms after isoproterenol. AP was right lateral side in only 4 patients and variations (42.5\u00b122 ms in control state and 35\u00b121 ms after isoproterenol) can not be interpreted.Among the 2 patients who have presented life-threatening arrhythmic events and 2 other patients with syncope and malignant form of preexcitation syndrome at electrophysiological study; all had a maximal heart rate with a 1 to 1 conduction over the accessory pathway was more than 240 bpm in control state during induced sustained atrial fibrillation. AF stopped spontaneously and the protocol was performed in these patients. Two patients had an APERP of 250 and 260 ms at the first measurement and a short value (210 and 230 ms) at the second measurement. Other patients had a short value (190 ms) at both measurements.We reported important variations of APERPs during electrophysiological study mainly after isoproterenol infusion. This may mean that ERP measurement with isoproterenol cannot be relied upon for assessing risk. The variations could be partially explained by the various conditions of the autonomic nervous system. The vagal and sympathetic activities constantly interact, and modulate the electrophysiological behaviour of the heart under different conditions such as postural changes, effort, and psychological activation ,13. A caFenici and al  studied As showed by Castellanos et al , if the The classical values of 240 ms in controls state and 200 ms after isoproterenol as cut-off for the evaluation of the arrhythmic risk should be discussed and the values of APERP's interpreted carefully in association with other data of electrophysiological study. Male gender, young age, sport, septal accessory pathway (AP), multiple accessory pathways, short AP refractory period, atrial fibrillation (AF) were reported as risk factors of sudden death in WPW syndrome ,18,19. PSome of our studies were performed by transesophageal route. However 14 of these patients had later an intracardiac study and the variations of APERPs were similar. Nanthakumar et al  have comThree patients were excluded after induction of atrial fibrillation at the first measurement, requiring flecainide to stop it. We can not prove the role of autonomic nervous system because no changes in sinus cycle length were found in the present study. We have not studied the reproducibility of the ventricular rate during induced atrial fibrillation, because AF was not always inducible or required flecainide to stop it.Only 3 patients had a spontaneous malignant form. More, the ages of our population are heterogeneous and a difference between younger and older patients can not be excluded. The data are only applicable to non sedated patients. Accessory pathways were mainly septal located probably because the majority of our patients were asymptomatic. Similar data were noted by our group  in a larIn conclusion this prospective study reports important variations of APERPs during electrophysiological study mainly after isoproterenol infusion. The study highlights the limitation of measurement of ERP as a predictor of the future risk. The APERP in patients with WPW should be interpreted with carefulness in association with other data of electrophysiological study. We suggest repeating the measurement in patients complaining of palpitations or syncope to avoid missing short values of APERP."}
+{"text": "In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/\u2212 ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43\u00b119.3 nm and 62\u00b18.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263\u00b160 nm and 48.3\u00b12.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/\u2212) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the  Many cationic peptides, lipids, and polymers have been used to condense siRNA via electrostatic interaction forming ionic complexes with variable size and surface charge, which proved effective in delivering the RNA cargo into the cytoplasm of mammalian cells in vitroin vivo delivery of siRNA required the use of excess cationic carrier to shield and protect the RNA cargo against nucleases leading to non-specific distribution of the formed complexes to the reticular endothelial system  Preclinical investigations showed the potential of small interfering RNA (siRNA) molecules in selectively silencing the expression of the genes implicated in the development of cancer, cardiovascular, neurodegenerative, and infectious diseases indicating their therapeutic potential Poly(amidoamine), PAMAM, dendrimers are a family of water-soluble polymers that is characterized by a unique, highly-ordered, three dimensional, tree-like branching architecture with a large number of primary, secondary, and tertiary amine groups embedded in their structure, which become ionized at physiologic pH conferring a high positive charge density Earlier studies showed that DNA condensation has two kinetic phases starting with an initial rapid binding (within 15 seconds) of DNA to multivalent cations followed by slower structural rearrangement that reaches an apparent equilibrium typically within 1\u20132 hours and exhibit insignificant changes at longer incubation times It is important to note that pDNA molecules exist in solution as long flexible chains with an average length of \u223c1.2 \u00b5m, which allow them to wrap around cationic carriers forming compact particles that resist degradation by DNase enzymes and achieve high transfection In this article, we describe the complexation of anti-GAPDH siRNA molecules with G4 (flexible) and G5 (rigid) dendrimers based on the size and morphology of the formed dendriplexes at different incubation times (20 minutes and 24 hours). We also investigate the stability of the formed dendriplexes upon incubation with RNase V1 enzymes compared to naked siRNA molecules as a function of exposure time. We used atomic force microscopy (AFM) to visualize the morphology of the formed complexes and monitor the attack of RNase V1 enzymes in solution as a function of time. We relied on established AFM protocols used to study the dynamics of DNA binding to PAMAM dendrimers G4  and G5  with ethylene diamine cores were purchased from Dendritic Nanotechnologies, Inc.  as 10% w/v solutions in methanol. G4 and G5 solutions were dialyzed using Slide-A-Lyzer dialysis cassettes with a 7 kDa MWCO  against DI water for 24 hours to remove polymer debris. The aqueous solutions of G4 and G5 were lyophilized and stored at 4\u00b0C till used. Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. .G4 and G5 dendrimers were dissolved in RNase-free water and mixed with 0.7 \u00b5g of anti-GAPDH siRNA molecules dissolved in 1 \u00b5l of RNase-free water at a nitrogen/phosphate  ratio of 2/1. Each mixture was vortexed and allowed to stand at room temperature for 20 minutes or 24 hours before loading onto a 1% w/v agarose gel containing ethidium bromide (EtBr). The gel was immersed in a Tris-acetate-EDTA (TAE) buffer and run at 60 V for 1 hour and visualized under UV exposure .2 where MgCl2 ions would allow weak adsorption of anionic siRNA molecules to the negatively charged mica surface through electrostatic interaction following established protocols All AFM images were acquired using Nanoscope III MultiMode AFM with a sharp nitride lever  in the tapping mode at a 256\u00d7256 pixel resolution. Selected fields were scanned at scan rates ranging from 3\u20134 Hz where each image was acquired within 90 seconds and tapping frequencies ranged from 8\u201310.5 kHz in solution. Images were flattened to account for Z offsets and sample tilts. Tapping set points were selected close to the free oscillation amplitude to reduce forces exerted on the interfacial species. All imaging experiments started with scanning the mica substrate in DI water to confirm the absence of any adsorbed contaminants. Naked anti-GAPDH siRNA was imaged using 30 \u00b5l of siRNA solution (3.92 \u00b5g/ml) in 1 mM PBS containing 2 mM MgClBoth G4 and G5 dendrimers successfully complexed the loaded anti-GAPDH siRNA molecules (0.7 \u00b5g) at an N/P (+/\u2212) ratio of 2/1 via the electrostatic interaction between the cationic amine groups (N) and the anionic phosphate groups (P). The gel image shows that siRNA molecules were retained in the wells after mixing with G4 and G5 dendrimers for 20 minutes and 24 hours indicating rapid condensation of the loaded siRNA molecules by the cationic carriers Figure 1+2 ions and multiple anionic siRNA molecules t\u200a=\u200a0 minutes) (Atomic Force Microscopy (AFM) is a powerful surface analytical technique that is routinely used to image and characterizes objects in the sub nanometer scale Incubation of G4 dendrimers with anti-GAPDH at an N/P ratio of 2/1 for 20 minutes yielded dendriplexes that were visualized using AFM Figure 3In comparison, G4 dendriplexes prepared by mixing of G4 dendrimers with anti-GAPDH siRNA at an N/P ratio of 2/1 for 24 hours appeared as large globular particles with a dense central core surrounded by \u201cloose\u201d network Figure 4Similarly, incubation of G5 dendrimers with anti-GAPDH at an N/P ratio of 2/1 for 20 minutes yielded compact hexagonal dendriplexes that formed a densely packed monolayers on the surface of freshly cleaved mica with an average full width of 62\u00b18.3 nm at half the maximum height and an average height of 1.1\u00b10.05 nm (+2 cations to allow weak electrostatic adsorption of free siRNA and G4/G5 particles to mica's surface following previously published protocols Particles prepared by ionic complexation of PAMAM dendrimers with siRNA molecules were examined using AFM under air-dry conditions &&&&AFM images show that despite the difference in size, number of cationic amine groups, and flexibility of G4 and G5 dendrimers, they formed similar hexagonal particles when incubated for 20 minutes with anti-GAPDH siRNA &5A. IncAFM images show that increasing the incubation time of G4 and G5 dendrimers with siRNA molecules to 24 hours results in formation of ionic complexes that can protect the loaded RNA cargo against enzymatic degradation without using excess cationic dendrimers. Further, the size of the formed complexes can be tuned by controlling the flexibility of the cationic carrier with flexible G4 forming larger particles than the more rigid G5 dendrimers while maintaining the desired enzymatic stability. These findings provide insight on potential formulation strategies to develop enzymatically-resistant complexes with tunable size for enhanced intracellular delivery of therapeutic siRNA molecules without inducing undesirable side effects due to the use of excess cationic carrier."}
+{"text": "LAMTOR2 and LAMTOR3 as genetic risk factors for breast cancer.The late endosomal LAMTOR complex serves as a convergence point for both the RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. Interestingly, both of these signalling cascades play a significant role in the aetiology of breast cancer. Our aim was to address the possible role of genetic polymorphisms in LAMTOR2 (p14) and LAMTOR3 (MP1) in 50 prospectively collected pairs of cancerous tissue and blood samples from breast cancer patients and compared their genetic variability. We found one single nucleotide polymorphism (SNP) in LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) in both tumour and blood samples, but no somatic mutations in cancerous tissues. In addition, we genotyped all three SNPs in 296 samples from the Risk Prediction of Breast Cancer Metastasis Study and found evidence of a genetic association between rs148972953 and oestrogen (ER) and progesterone receptor negative status (PR) (ER: OR\u200a=\u200a3.60 (1.15\u201311.28); PR: OR\u200a=\u200a4.27 (1.43\u201312.72)). However, when we additionally genotyped rs148972953 in the MARIE study including 2,715 breast cancer cases and 5,216 controls, we observed neither a difference in genotype frequencies between patients and controls nor was the SNP associated with ER or PR. Finally, all three SNPs were equally frequent in breast cancer samples and female participants (n\u200a=\u200a640) of the population-based SAPHIR Study.We sequenced the exons and exon\u2013intron boundaries of LAMTOR2 and LAMTOR3 do not seem to play a relevant role in breast cancer. Our work does not exclude a role of other not yet identified SNPs or that the here annotated polymorphism may in fact play a relevant role in other diseases. Our results underscore the importance of replication in association studies.The identified polymorphisms in  LAMTOR3 (MP1) was identified in a yeast two-hybrid screen as a specific binding partner of MEK1 Anchorage of the p14/MP1/MAP kinase pathway to late endosomes is mediated by a small lipid raft adaptor called LAMTOR1 (p18), which directly binds endosomal lipids Progression, proliferation, and hormone independent growth of breast cancer cells is dependent on MAP kinase (ERK) activity The PI3K/AKT signalling cascade is another major player in cancer progression. Mutations in the PI3K subunit p110 are found in roughly 25% of breast cancers It has been recently shown that treatment of breast cancer cells with MEK or mTOR inhibitors and either Doxorubicin or Tamoxifen results in a synergistic response that highlights the advantages of combining classical chemotherapy with targeted adjuvant treatments Interestingly, in 2007, Conrad et al. submitted a patent on LAMTOR3 (Mp1) as a diagnostic and therapeutic target for breast cancer treatment and prevention . In addition, a recent publication from the same group reports that LAMTOR3 (Mp1) is required for the survival of estrogen receptor positive breast cancer cell lines LAMTOR3 and LAMTOR2 in the aetiology of breast cancer.Taking into consideration the above report and recent findings identifying the LAMTOR complex as a convergence point for both the ERK and mTORC1 pathways, we aimed to investigate the potential role of mutations in This study was approved by the ethics committee of the Innsbruck Medical University (study code UN3377).For mutation screening, tissue samples of 50 consecutive breast cancer patients were prospectively collected at the Innsbruck Medical University starting in July 2009. Patients aged 18 or older, who had signed an informed consent, were consecutively included in the study. The following clinical parameters were collected: age; menopausal status; tumour histology; tumour size; tumour grade; lymph node status; oestrogen receptor status; progesterone receptor status; HER2  status; and presence of metastasis.Genomic DNA was extracted from frozen tumour tissue or from peripheral blood collected on EDTA on a BioRobot EZ1 advanced Workstation with the EZ1 DNA tissue or blood kit  and quantified with a NanoDrop spectrophotometer . Amplification and sequencing primes were designed with Visual OMP .LAMTOR2 gene  were amplified in 2 PCR reactions and sequenced with 8 primers  were amplified in 4 PCR reactions and sequenced with 14 primers , 5 \u00b5l of PCR reaction buffer, 250 \u00b5M each dNTP, and 0.25 \u00b5M each primer. The reaction cocktails were heated to 95\u00b0C (2 min) and then put through 35 amplification cycles: 95\u00b0C for 20 s, 55\u00b0C for 20 s, and 72\u00b0C for 60 sec and a final extension phase at 72\u00b0C for 10 min. Fragment MP1-1 was amplified in a total reaction volume of 25 \u00b5l, containing 60 ng of DNA, 0.5 \u00b5l of KAPA HiFi HotStart DNA Polymerase , 5 \u00b5l of 5X KAPA HiFi GC Buffer, 0.5 \u00b5l KAPA dNTP Mix (10 mM each dNTP), and 0.3 \u00b5M each primer. The reaction cocktails were heated to 95\u00b0C (5 min) and then put through 30 amplification cycles: 98\u00b0C for 20 s, 56.7\u00b0C for 15 s, and 72\u00b0C for 45 sec and a final extension phase at 72\u00b0C for 5 min.PCR products were purified using QIAvac vacuum manifolds (QIAGEN) and eluted in 50\u00b5l distilled water according to the manufacturer\u2019s protocol. For cycle sequencing, 3 \u00b5l of purified PCR product were combined with the sequencing master mix  and cycled  for 30 cycles of 30 s at 96\u00b0C, 20 s at 55\u00b0C, and 1 min at 60\u00b0C. Purification of cycle-sequencing products with Sephadex  was performed according to the procedure described in Brandst\u00e4tter et al. xl capillary sequencer with POP-7 and a 36 cm capillary array.Electrophoretic separation was carried out on an ABI3130LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) were genotyped using a TaqMan\u00ae SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in 296 samples of the Risk Prediction of Breast Cancer Metastasis Study. This study is a multicenter study including prospectively collected breast cancer samples from the cities Innsbruck (Austria), Salzburg (Austria) and Meran . Patients aged 18 or older, who had signed an informed consent, were consecutively included in the study. Detailed information on tumour characteristics  and treatment  were collected using clinical and pathological records. The Risk Prediction of Breast Cancer Metastasis Study was approved by the ethics committee of the Innsbruck Medical University (study code AM330a). The genotyping success rates were 97.3% for rs7541, 99.3% for rs2298735, and 97.3% for rs148972953.For studying the effects of the identified variants in a breast cancer sample, one single nucleotide polymorphism (SNP) in For a more detailed analysis, the SNP rs148972953 was genotyped using a TaqMan\u00ae SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in 7,931 samples of the MARIE Study. The MARIE (Mammary carcinoma Risk factor Investigation) study population comprises breast cancer patients who participated in a population-based case-control study conducted in two German study regions (Hamburg and Rhine-Neckar-Karlsruhe) LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) were genotyped using a TaqMan\u00ae SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in female participants of the SAPHIR Study. The Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) is an observational study conducted in the years 1999\u20132002 involving 1,770 healthy unrelated subjects: 663 females from 39 to 67 years of age and 1,107 males from 39 to 66 years of age For studying the frequency of the identified variants in a healthy working population, one SNP in The genotype distribution was used to calculate minor allele frequencies and deviations from Hardy-Weinberg equilibrium were evaluated using a Chi-square test. Due to the low minor allele frequency of rs148972953, heterozygous and homozygous minor allele carriers were assessed combined in comparison to major allele carriers. Differences in allele frequencies between carriers and non-carriers of rs148972953 by oestrogen receptor status, progesterone receptor status, metastasis at baseline and metastasis after treatment were calculated by using a Chi-squared test. Odds ratios with a 95% confidence interval were calculated for estimating the relationship between rs148972953 and oestrogen receptor status (ER), progesterone receptor status (PR) and metastasis Following functional considerations based on the SNP position, the potential effects of the three SNPs were investigated using selected bioinformatic applications LAMTOR2 and LAMTOR3 revealed three SNPs to occur in a sequencing sample of 50 breast cancer patients, but no novel mutations. One SNP was found in LAMTOR2 (rs7541) and two SNPs within LAMTOR3 (rs2298735 and rs148972953)  and cancerous tissue.Sequencing of the exons and exon-intron boundaries of both 8972953) . Our hypLAMTOR2 could possibly affect splicing regulation   of 4%, we investigated whether a healthy working population sample would show different genotype distributions than the breast cancer sample. Therefore, we genotyped all three SNPs in female participants of the SAPHIR population (n\u200a=\u200a640). The allele and genotype frequencies in SAPHIR women were similar to those in our sequencing sample , and as the SNP rs148972953 in  p>0.05; .LAMTOR3 was found to be strongly associated with negative progesterone receptor status (OR\u200a=\u200a4.27 (1.43\u201312.72); For the analysis of genetic association, we combined the samples for sequencing and those in the Risk Prediction of Breast Cancer Metastasis Study since they were collected at the same University Clinic in the same manner. In the combined breast cancer sample, rs148972953 in Encouraged by the findings from the combined breast cancer sample and the bioinformatic analyses we performed a replication in the MARIE Study. The minor allele of rs148972953 was not found to show differential association by oestrogen receptor or progesterone receptor status (ER: OR\u200a=\u200a1.18 (0.69\u20132.02); PR: OR\u200a=\u200a0.97 (0.60\u20131.58); Based on the results of the bioinformatics analysis, we hypothesized that rs148972953 could be associated with tumour metastasis. However, our assumption was not supported in the MARIE Study. Compared to patients carrying the wildtype of rs148972953 polymorphism, carriers of the minor allele of rs148972953 were neither more likely to have metastasis at baseline (OR\u200a=\u200a1.76 (0.62\u20134.96)) nor were they at higher risk of distant disease-free survival (HR\u200a=\u200a0.96 (0.75\u20131.23)) . The genLAMTOR2 (p14) is significantly up-regulated in breast cancer cells. Interestingly, two independent publications also reported an up-regulation of p14 in invasive ductal breast carcinomas LAMTOR3 (Mp1) was also found to be up-regulated in invasive ductal breast carcinomas LAMTOR3 (Mp1) was also reported to exhibit reduced levels in the stroma of invasive breast carcinoma LAMTOR3 (Mp1) was required for the survival of oestrogen receptor positive breast cancer cell lines LAMTOR2 and LAMTOR3 that could contribute to the aetiology of breast cancer by altering any of the above mentioned processes.Bioinformatic analysis of high-throughput cancer microarrays available on the public domain Oncomine LAMTOR2 and LAMTOR3 could be associated with breast cancer progression and metastasis. We additionally expected that several rare instead of common variants would be detected and those rare variants were not expected to be already listed in databases. Therefore, we applied a discovery stage by sequencing the exons of LAMTOR2 and LAMTOR3 in 50 cancerous tissues and the corresponding peripheral leukocyte DNA and observed no differences in the identified mutations between the two tissues. The identified mutations were investigated in two different case samples (Risk Prediction of Breast Cancer Metastasis Study and MARIE cases) as well as two different control samples (SAPHIR women and MARIE controls).We initially hypothesized that somatic, cancer-tissue-specific mutations in LAMTOR3 and both oestrogen and progesterone receptor status and metastasis. The present work does not exclude that the observed polymorphisms may play a role in other disease contexts or that other not yet identified polymorphisms in LAMTOR2 and LAMTOR3 may in fact contribute to breast cancer aetiology. We would also like to emphasize that genetic variation is not the only factor contributing to disease progression. Many regulatory aspects, in particular those controlling protein stability, posttranslational modifications and association with binding partners, play a fundamental role in determining how much and how active a protein actually is. As discussed before, previous publications identified the LAMTOR complex as a convergence point of key signalling pathways: MAPK and mTOR. Due to the well established role of both signalling cascades in breast cancer progression, and the recent implication of LAMTOR3 in oestrogen receptor positive breast cancer, we leave open the possibility of therapeutically targeting the complex as previously proposed by others Despite the promising results in the initial study, the replication study failed to support an association between the SNP rs148972953 in LAMTOR2 nor LAMTOR3 came up as hits in genome-wide association studies (GWAS) with any kind of disease or trait as indexed by the catalogue of published GWAS LAMTOR3 is located, showed signals of associations with upper aerodigestive tract cancers per se is interesting for cancer. Therefore, future studies including a dense map of SNPs from the LAMTOR complex including the regulatory regions might reveal implications of genetic associations of LAMTOR2 and LAMTOR3 with breast cancer.Although the LAMTOR complex was independently demonstrated to be involved in breast cancer The present study constitutes a good example for the necessity and importance of replication in genetic association studies. Small cohorts intrinsically increase the number of false positives that are accepted. Replication in larger populations is therefore fundamental to separate the wheat from the chaff.Figure S1LAMTOR2.Amplification and sequencing strategy of (DOC)Click here for additional data file.Figure S2LAMTOR3.Amplification and sequencing strategy of (DOC)Click here for additional data file.Table S1LAMTOR2.Primers used for amplification and sequencing of (DOC)Click here for additional data file.Table S2LAMTOR3.Primers used for amplification and sequencing of (DOC)Click here for additional data file."}
+{"text": "BMPR1A or SMAD4 are found in 50% of affected individuals. Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease diagnosed upon the presence of epistaxis, visceral arteriovenous malformations (AVM) or mucutaneous telangiectasias. HHT is diagnosed when there are \u2265 3 manifestations and is suspected when there are at least 2 manifestations. Most HHT cases are caused by a germline mutation in ALK1 or ENG, members of the TGF\u03b2 signaling pathway. Approximately 22% of patients with Juvenile Polyposis Syndrome (JPS) due to a SMAD4 mutation have been reported to also have HHT [SMAD4 mutation including those who underwent systematic screening for AVM\u2019s.Juvenile Polyposis Syndrome (JPS) is defined by the presence of \u2265 5 colorectal juvenile polyps or any number of juvenile polyps in an individual with a family history of JPS. Genetic alterations including either point mutations or large rearrangements in SMAD4 mutation were studied by screening affected patients for cutaneous telangiectases and with cardiac bubble ECHO, CAT scan chest, or MRI of brain for other AVMs.JPS patients were identified from a comprehensive polyposis database using Cologene\u00a9 software. Families carrying a germline SMAD4 mutation. These families include 21 affected relatives, 11 men and 10 women, with a current mean age of 36.3 years (range 4 - 70). Fourteen affected relatives, from 6 families, underwent HHT screening . Eleven of 14 (79%) had \u2265 3 HHT manifestations and two of 14 (14 %) had at least 2. In addition, 3 of 7 unscreened affected relatives have presented with at least 2 manifestations of HHT. Of the 24 families that have not had genetic testing or HHT screening one affected family member presented with \u2265 3 HHT manifestations, and two had at least 2 manifestations.Fourteen of 38 JPS families underwent genetic testing. Nine families were identified to have a SMAD4 mutations had clinically diagnosed or suspected HHT. Genetic testing should be performed in all JPS patients. In addition, systematic HHT screening is recommended for JPS patients with SMAD4 mutations.Greater than 90% of our patients with JPS due to"}
+{"text": "MSH2 account for 1-2% of all CRCs and up to 20% of these are large germline deletions [MSH2 deletion of exons 1-6 has been characterized as a North American Founder Mutation (AFM) [Lynch syndrome (LS) is a genetic disorder that accounts for approximately 3% of all colorectal cancers (CRC) . ClinicaMLH1 and MSH2 genes [MSH2 gene. This results in the premature truncation of the MSH2 protein and confirms the diagnosis of LS.A 68-year-old Caucasian male presented to cancer genetics following a second primary diagnosis of infiltrating poorly differentiated adenocarcinoma of the colon. His history included; moderately differentiated invasive adenocarcinoma of the sigmoid colon at age 40; left ureteral carcinoma diagnosed at 54; and a bladder carcinoma diagnosed at age 59. Additionally, colonoscopy revealed multiple adenomatous polyps within a ten year period. Family history is significant for a son diagnosed with colon cancer at age 34, father with gastric cancer, two paternal aunts and paternal grandfather with colon cancer, and German ancestry. Peripheral blood was sent for analysis of the MSH2 mutations are at a higher risk of developing extracolonic cancers than individuals with MLH1 mutations [MSH2 mutations and male carriers have up to a 28% lifetime risk of developing uroepithelial cancers [This case reveals a LS patient with a history of four metachronous cancers. Phenotypic variations exist amongst the different MMR genes causative for LS -7. Indivutations ,6. There cancers . The sev cancers . A bette"}
+{"text": "PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the nullProp1 (-/-Prop1) and the Ames dwarf (df/dfProp1) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.Humans with  PROP1, POU1F1 (PIT1), HESX1, LHX3, LHX4, OTX2, SOX2, SOX3, and GLI2 , and thyroid stimulating hormone (TSH) in addition to overall pituitary hypoplasia Prophet of PIT1 (PROP1) are the most common known causes of MPHD in humans. The hormone deficiencies are similar to those caused by POU1F1 mutations, except that the deficiencies include reduced gonadotropin production requiring sex hormone substitution and there is a strong tendency toward progressive hormone loss leading to lower circulating adrenocorticotropic hormone (ACTH) later in life, requiring glucocorticoid replacement therapy PROP1 and POU1F1 patients is the tendency of patients with PROP1 mutations to undergo apparent degeneration of the pituitary gland during childhood PROP1 mutations are not well understood.Congenital pituitary hormone deficiency in humans occurs with a frequency of approximately 1 in 4000 live births and is caused primarily by mutations in genes important for pituitary development Prop1 action in pituitary development and function. The Ames dwarf (df/dfProp1) and the nullProp1 (-/-Prop1) mouse mutants recapitulate the human MPHD phenotype in that adult mutants are profoundly deficient in TSH, GH, PRL, have low circulating gonadotropins, and pituitary hypoplasia Prop1 mutant mice show that precursor cells fail to colonize the anterior lobe resulting in reduced cell proliferation and enhanced apoptosis after birth leading to hypoplasia that becomes evident in the weeks after birth Prop1 mouse mutants differ from humans with PROP1 mutations in that the hormone deficits are consistently congenital rather than progressive, thyroid hormone and growth hormone replacement are sufficient for fertility, and there is no clear evidence for transient pituitary hyperplasia.Several mouse models have been used to dissect the mechanism of Prop1 deficient mice, although both alleles, df/dfProp1 and -/-Prop1, have the same features when normalized for genetic background PROP1 can have different clinical presentations Prop1 mouse mutants die of respiratory distress. The lack of pituitary TSH results in fetal hypothyroidism, reduced expression of the thyroid hormone inducible transcription factor TTF1 in the lung, and inadequate production of surfactants, known target genes of TTF1. The lungs fail to inflate, causing respiratory distress and lethality Prop1 deficient mice to lethality after weaning. The reason for this juvenile lethality has not been explored.The genetic background exerts a considerable influence on the phenotype of the Prop1 deficient mice, and corticosterone levels are not reduced in newborn mutants PROP1 patients who have been closely followed appear to have evolving hypocortisolism Prop1 mutant mice is not known, it is necessary to investigate pituitary adrenal function in young and old Prop1 deficient mice on a sensitized (B6) genetic background.Corticotroph development does not appear to be affected in the Prop1 deficient mice. In contrast, our results show increased serum ACTH and corticosterone levels in young and old Prop1 mutants. The pituitary-adrenal axis is functional in young adult nullProp1 mice as demonstrated by elevated activity in response to restraint stress. Prop1 mutants have significantly reduced blood glucose levels, as expected for GH deficient animals, which could trigger the activation of the pituitary-adrenal axis. Untreated hypoglycemia can cause mortality in both humans and mice Prop1 mouse alleles we tested on various genetic backgrounds differ from human patients by maintaining elevated pituitary adrenal axis activity through 1 year of age, with no evidence for evolving hypocortisolism.We report no evidence for progressive ACTH loss in juvenile and young adult All mice were housed in a 12-h light, 12-h dark cycle with unlimited access to tap water and Purina 5008 or 5020 chows. All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals, and all experiments were conducted in accordance with the principles and procedures outlined in the NIH Guidelines of the Care and Use of Experimental Animals.Sactm1Prop1 heterozygous null mice, referred to here as Prop1+/\u2212, were generated from R1 (129/Sv x 129/Sv-CP) ES cells by replacing the coding region of exon 1, intron 1, and a portion of exon 2 with cassettes encoding \u03b2-galactosidase and neomycin resistance . The chimeras were mated to C57BL/6J mice (B6)  to generate F1 heterozygous animals and were first analyzed on a mixed F2 C57BL/6J-129S1/SvImJ background (B6/129). The F2 +/Prop1\u2212 heterozygous mice were backcrossed to B6 mice for four generations to establish the +/Prop1\u2212 N4 B6 breeding colony, which is theoretically 93.75% pure B6. Mice used in the study of pituitary-adrenal function were from the N4 B6 genetic background unless expressly stated otherwise. -/-Prop1 mice were determined by PCR as previously described The +/dfProp1 stock is not inbred. It was obtained from Dr. A. Bartke at Southern Illinois University in 1988 and maintained at University of Michigan. This stock was backcrossed to B6 to N4 The DF/B-Young adult mice  were housed individually 12 hours prior the experiment, with special precautions to avoid stress associated with noise and cage handling. The blood samples were collected in the morning (between 9:00am and 10:30am) by retro orbital bleeding in heparinized collection tubes . The retro orbital bleeding was done in less than one minute after initial mouse handling to prevent stress-induced corticosterone release. Animals were subjected to restraint stress for 30 minutes, after which another blood sampling was performed by the same method For ACTH measurements in non-stressed conditions, animals of various ages were anesthetized with metaphane, rapidly decapitated within less than 1 min from the time of initial handling, and blood samples collected.125I RIA kit  according to the manufacturer's protocol http://www.abbottdiabetescare.com). If glucose levels were below the level of detection, an arbitrary number of 19 mg/dL was assigned for the purpose of statistical analysis.ACTH and corticosterone were measured by radioimmunoassay (RIA) in plasma using a Adrenals were collected immediately after euthanizing and rinsed in ice-cold PBS prior to 1 h fixation in 4% paraformaldehyde on ice . Samples were washed in PBS, dehydrated in a graded series of ethanol, and embedded in paraffin. Seven-micrometer sections were prepared and either stained with hematoxylin and eosin. The 20\u03b1-hydroxysteroid dehydrogenase antibody was generously provided by Yacob Weinstein and used at 1:2000-3000 dilution Data were processed and plotted using StatView software , with the exception of the qRT-PCR data that was processed using Microsoft Excel Software. ANOVA  and Fisher's exact test were used to evaluate the data. All data are shown as +/\u2212 1 SEM (standard error of the mean). P-values of less than 0.05 were considered to be statistically significant.Prop1 on several genetic backgrounds. The Ames dwarf mutant, df/df,Prop1 arose spontaneously on a poorly defined genetic background (DF/B), and it carries a missense mutation in the homeodomain, Ser83Pro +/Prop1\u2212, on a mixed background comprised of C57BL/6J (B6) and 129S1/SvImJ (129) Prop1-/- animals exhibited lethargy, wasting, and death between 3 and 7 weeks of age. Death usually occurred within 3-5 days of initial signs of distress. More males were affected than females (p\u200a=\u200a0.03). A similar wasting and lethality phenotype was also observed in 27% (6/22) of compound heterozygotes, Prop1df/-, on a mixed background.We analyzed the viability of two different mutant alleles of Prop1df/+ and 129/B6-Prop1+/\u2212, four times to B6 to be able to compare the phenotypes of the two alleles on a consistent genetic background. We observed identical viability of the homozygous mutants for each allele at two weeks of age: 17.5% Prop1df/df and 19.5% Prop1-/- for each on N4 B6, p\u200a=\u200a0.69 Prop1 mutant alleles.We back-crossed both strains, DF/B-Prop1-/- and normal mice on the N4 B6 background by RIA to address the ability of Prop1 mutant corticotrophs to secrete ACTH .To determine whether the observed post-weaning lethality on the N4 B6 background could arise from evolving hypocortisolism, we examined ACTH and corticosterone production. We analyzed the serum of 3.5 to 5 week old Prop1 mutants exhibit evolving hypocortisolism at older ages we aged Prop1 mutant animals with three different genotypes and genetic backgrounds  to 7\u201312 months old and measured both ACTH and corticosterone. All genotype combinations of Prop1 mutants had significantly elevated ACTH and corticosterone . The fold increase in corticosterone from basal to post-stress measurements is less for Prop1-/- animals compared to the wild type (2-3 fold compared to 11-16 fold). While this could be described as a blunted response, the absolute value of circulating corticosterone following restraint was higher in mutants than normal littermates. Post stress, the corticosterone values for male and female mutants were 504 +/\u2212 29 ng/ml and normal littermates were 373 +/\u2212 18 ng/ml. The post stress values in the 500 ng/ml range may be the maximal response. Thus, there is no evidence for impaired pituitary-adrenal axis function.Stress increases pituitary ACTH release and subsequent corticosterone secretion by the adrenal gland hallenge Fig. 2. -/-Prop mice were compared to +/\u2212Prop1 and wild type to determine the consequence of elevated ACTH on adrenal growth. The absolute size of the adrenal gland is smaller in the Prop1-/- dwarf males compared to wild type. However, the ratio of adrenal weight to body weight is actually increased in the Prop1-/- males compared to wild type . Similar levels of 21-hydroxylase enzyme, which is important for corticosterone biosynthesis Prop1-/- adrenals compared to +/\u2212Prop1 or wild type. These results are consistent with functioning adrenal glands in -/-Prop1 mice.We used Western blotting to evaluate the levels of steroidogenic enzymes in Prop1 deficient mice. Prop1 deficient mice produce very few somatotrophs and lack detectable circulating GH Igf1) Igf1 expression in the Prop1-/- mouse livers compared to wild type (data not shown). Growth hormone is important for metabolism and glucose homeostasis though its role in modulating Igf1 production Prop1 mutant genotypes at several ages (Prop1-/- mice (N4 B6 background) is similar to that of heterozygous littermates and wild types, 140 +/\u2212 14 mg/dL vs. 177 +/\u2212 16 mg/dL, p\u200a=\u200a0.048 , 193 +/\u2212 55 in Prop1df/- (N\u200a=\u200a12), and 211 +/\u2212 36 in healthy Prop1-/- (N\u200a=\u200a12), and 393 +/\u2212 86 in sick Prop1-/- mice, (N\u200a=\u200a7). The very high corticosterone levels support the idea that the wasting phenotype is not due to failure of the pituitary adrenal axis. The elevated levels are consistent with a response to metabolic stress, but it is difficult to determine whether the cachexia is the cause or the effect of severe hypoglycemia.We hypothesized that reduced glucose levels secondary to growth hormone deficiency could cause the elevated basal levels of ACTH and corticosterone in the blood of Prop1 deficiency mice. At 8\u201310 weeks the N4 B6 Prop1-/- mice had lower glucose levels (ave. 117 +/\u2212 4 mg/dL) than controls (178 +/\u2212 10 mg/dL) . It is possible that severe hypoglycemia contributes to the increased susceptibility of Prop1 mutants to lethality on the B6 background, although other differences in metabolism may be responsible. For example, the livers of healthy Prop1 deficient mice resemble livers of normal fasted mice, and sickly mutant livers are more affected (data not shown) Prop1 mutants that survive to adulthood have significantly longer life spans than their normal littermates, like other strains with reduced insulin like growth factor activity Both Prop1 deficient mice are consistent with clinical data from human patients with GH deficiency. Approximately 5% of humans with GH deficiency also had hypoglycemia, and 10% of the hypoglycemic patients died The lower glucose levels we observed in Prop1 deficient mice. In direct contrast to the human MPHD cases with progressive ACTH loss and hypocortisolism, Prop1 deficient mice exhibit elevated ACTH and corticosterone and reduced glucose levels at 6 mo and 1 yr of age. Young adult Prop1 deficient mice respond to restraint stress with further elevation of ACTH, corticosterone and glucose levels, and show no reduction in adrenal content of steroidogenic enzymes, indicating that the pituitary-adrenal axis can react functionally to this challenge. In addition, the adrenals of the Prop1-/- mice are enlarged relative to normal mice when normalized to body weight, as expected for chronic ACTH secretion in rodents and other mammals, including primates Prop1 mutants have even higher corticosterone levels than healthy mutants.We found no evidence for disruption of the pituitary-adrenal axis in PROP1 deficient patients remains elusive. It is tempting to speculate that it arises from depletion of progenitors, but species differences in function are also possible. Genetic background affects the viability of young Prop1 deficient mice, largely due to different responses of target organs to pituitary hormone deficiency. Multiple Prop1 mutant alleles and genetic backgrounds support elevated ACTH and corticosterone levels and lower glucose levels that persist with age. Although mice with MPHD have been invaluable for understanding the molecular basis for human disorders of hormone-deficiency and dwarfism, pituitary growth, and pituitary cell specification, they may be less pertinent for understanding the nature of progressive hormone deficiency that characterizes humans with PROP1 mutations.The basis for the evolving nature of the hormone deficiencies, including hypocortisolism, in human"}
+{"text": "KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P\u200a=\u200a0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09\u20134.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P\u200a=\u200a0.03) and an associated multivariate HR 2.42 (95%CI 1.14\u20132.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P\u200a=\u200a0.02) and a corresponding HR of 3.07 (95%CI 1.33\u20137.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application. Pancreatic ductal adenocarcinoma (PDAC) is the most fatal form of pancreatic malignancy with a 5 year survival of less than 4% KRAS being the most frequently mutated oncogene in pancreatic cancer with a reported frequency ranging between 20 and 100%, it has not been so far utilized in categorization of tumors for clinical purposes KRAS mutations in resected pancreatic cancers with prognosis Over the years somatic mutations have been shown to be legitimate targets for anti-cancer drugs because of casual relationship with tumor formation and maintenance KRAS mutations in pancreatic cancer were based on relatively small tumor numbers that lacked statistical power to determine association with the disease outcome. In order to address the issue of frequency of KRAS mutation in pancreatic cancer and impact of those mutations on disease outcome, we have in this study included a series of fully characterized 171 pancreatic tumors with complete patient data.Most of the earlier reports on The 163 patients with malignant tumors in this study comprised the following: i) 143 ductal adenocarcinomas that also included 5 adenosquamous and 4 anaplastic undifferentiated variants, ii) 16 rare carcinomas that were comprised of 2 acinar cell carcinomas, 2 (microcystic) tubulo-papillary carcinomas, 9 intraductal papillary mucinous neoplasm , 2 solid pseudopapillary neoplasms (Frantz tumors) and 1 cystadenocarcinoma, and iii) 4 papillary (ampulla of Vater) carcinomas. The non-malignant group was composed of 4 benign lesions in the form of serous cystic adenomas (SCA) and premalignant lesions in the form of 1 mucinous cystic neoplasm (MCN) and 3 non-invasive IPMN . All patKRAS gene was standardized using DNA from cell lines with known KRAS mutation. The sensitivity of SSCP, determined by titration experiments, showed that point mutations in tumor samples up to 5% tumor content were detectable. This provided confidence that our inclusion of tumor samples, only if those had at least 10% tumor content (n\u200a=\u200a171), would more than adequately enable the detection of mutations. Another criterion applied for mutation detection was reproducibility. Mutations were scored only when band shifts were reproducible in at least two independent experiments. Repeat experiments using SSCP followed by DNA sequencing were used for confirmation and identification of mutations (KRAS mutations in a random sub-set (n\u200a=\u200a6) analyzed blindly in the reference laboratory of the Institute of Pathology, University Hospital of Heidelberg.Mutation detection for utations . We alsoKRAS gene, we detected 134 mutations in 171 tumors (78%), with 131 mutations in exon 2 and 3 mutations in exon 3 , 3 at codon 83 , followed by solitary tumors with mutations at codon 58 , codon 129 , codon 130  and one tumor had 2 base pair insertion of GG at codon 78 (CTC>CGGTC). Deletions at the 9p21 locus were detected with varying frequency with 17\u201320% in the CDKN2A (p16INK4a) and 26\u201328% within the promoter associated with exon 1\u03b2 of p14ARF transcript.A total of 43 tumors (25%) showed aberrations in the KRAS mutations tended to shorten survival of patients in general  and in all studied sub-categories (except tumor stage T4), however without reaching statistical significance  of 1.87 . The corresponding HR was 1.55  was poorest with median survival time of 13 months compared to 30 months for patients without any mutations in either KRAS or CDKN2A  showed a similar association with KRAS and CDKN2A genes in pancreatic cancer are well documented; however, their influence on disease outcome in patients with exocrine pancreatic tumors has remained unclear. In this study, we observed that KRAS mutation frequency in pancreatic tumors was consistent with ours and other previous European studies that were based on 70\u2013100 tumors and reported a mutation frequency of 72\u201383% KRAS mutations in pancreatic cancer are believed to be the early events in neoplastic transformation. The hypothesis is supported by mice models based on conditional endogenous expression of the mutant KRAS. Those mouse models were developed with an assumption that KRAS mutation is an essential and early somatic genetic alteration in PDAC progression. Similar observations were reported for KRAS mutations in human acinar-ductal metaplasia (ADM) lesions of pancreas KRAS mutations existed only in the lesions associated with PanIN; the isolated ADM lesions were devoid of any KRAS mutation, with possible involvement of two distinct mechanisms, with and without KRAS mutations KRAS mutation may not be essential for human PDAC progression and other low frequency gene mutations could trigger alternate pathways. The pancreatic genome sequencing of 20,661 genes from 24 tumors identified other low frequency gene mutations KRAS, CDKN2A, TP53 and SMAD4 genes in about 30% of pancreatic tumors Mutations in KRAS gene are part of the conserved \u2018G-domain\u2019 (residues 1\u2013165) required for signal transduction. Tumor malignancy depends not on the presence of a KRAS mutation but on the molecular configuration and constituent mutation type KRAS mutation in pancreatic tumors was associated with reduced survival time. Further analysis showed that the association was significant only for G12D sub-type of KRAS mutation. Lack of statistical power owing to low frequency of other mutation types likely precluded observation of the effect on survival. While our data being concordant with the paradigm of distribution of KRAS mutations, we clearly showed that patients, in particular, those harboring G12D mutation in tumors were at a 2-fold increased risk of death compared to those without any KRAS mutation. In an experimental study, the human cell lines with KRAS mutations were classified into KRAS dependent and independent. The \u2018classical\u2019 PDAC categorized as KRAS dependent were shown to be potentially amenable to the directed therapy KRAS has been established as a predictor of resistance to the treatment The codons 12, 13 and 61 of KRAS mutations could also be due to varying abilities to alter the RAS protein. KRAS mutations at codon 12, in general, have been shown to increase resistance to apoptosis and activate AKT/protein kinase B pathway KRAS G12D mutant was shown not only to initiate neoplastic lesions but was also involved in tumor maintenance KRAS is reported to promote widespread colonic epithelia hyperplasia and neoplasia KRAS mutation subtypes and survival. Our previous report on paraffin embedded tumors did not show any association between the presence of KRAS mutations and patient survival; however, there was difference in survival between the patients with different mutation types KRAS mutational status as predictive of survival was also reported in an earlier trial study of Gemctabine and Erlotinib therapy in patients with advanced pancreatic cancer KRAS mutations in the surgically negative resected margins have also been shown to be associated with clinical cancer recurrence, aggressive tumor biology and poor survival KRAS mutations in retroperitoneal margins, in the patients with complete pancreatectomy also showed poor prognosis The association between pancreatic cancer patient survival and specific sub-types of CDKN2ACDKN2A in the present study was in agreement with that reported in the COSMIC database G12DKras and Ink4a/Arf showed enhanced progression of pre-malignant lesions to PDAC KRAS and CDKN2A aberrations were at 2.5-fold higher risk of death than patients without any alterations in the two genes. In a previous study it was shown that 1\u20132 mutations in pancreatic tumors showed a median survival of 23 months compared to 13 months in our present study KRAS mutation KRAS mutations together with loss of heterozygosity on different chromosomal positions The other gene that has been consistently reported to carry high frequency of somatic mutation in pancreatic cancers is KRAS are frequent but not universal in pancreatic tumors and the presence of KRAS mutations in general, and G12D transformation in particular, were indicative of association with poor survival. Our results also showed that concomitant occurrence of KRAS mutations and aberrations in CDKN2A resulted in a sub-group of patients with lowest survival. Our data from this study is suggestive for a case for the prognostic classification of pancreatic cancer patients based on mutational status of KRAS and CDKN2A. However, the results need independent confirmation in additional studies with definite statistical confidence.In conclusion, our results show that mutations in For all samples analyzed, written informed consent was obtained from the patients. The study was approved by the local ethics committee of the University of Heidelberg.Tumor tissues were collected from pancreatic cancer patients during surgery between January 2002 and September 2009, snap-frozen in liquid nitrogen directly after resection and subsequently stored at \u221280 \u00b0C. A total of 171 tumor tissues, that contained at least 10% tumor by H&E staining were analyzed in the present study. The clinical and histopathological characteristics of the patients are given in Three different tissue sections were selected randomly for hematoxylin and eosin (H&E) staining and histological validation. Slides were scanned with the ScanScope GL System  and visualized using the ImageScope Software. For each tissue sample, three pathologists evaluated independently the histology and percentage of normal, tumor and stroma cells . Only saKRAS mutations in codon 12, 13 and 61 were used as controls that included, A549 cell line with G12S (GGT>AGT) mutation; MiaPaCa, G12C (GGT>TGT); SW 1116, G12A (GGT>GCT); SW 620 G12V (GGT>GTT); LS-174, G12D (GGT>GAT); LoVo, G13D (GGC>GAC); HS 766T, Q61H (CAA>CAC). DNA samples from healthy controls were included as negative control.Frozen pancreatic tissue samples were individually cut into 20 \u00b5m thick slices with a cryotome Leica CM 1850 UV at \u221234 \u00b0C. The tissue slices were covered with liquid nitrogen and gently ground by three turns with a micropestle made of polypropylene  that fitted into 2 ml Eppendorf tubes. DNA from tissue slices and from cell lines was extracted using the AllPrep Isolation Kit . DNA from cell lines with known 2, 0.11 mM each dNTP, 1 \u00b5Ci [\u03b1-32P] dCTP, 0.2 \u00b5M each gene specific primer  which contained 30 probes mapping chromosome 9p21 and 9p22, 13 reference probes and 9 internal controls. Reference probes were located in genomic regions with low frequency copy number changes. The hybridization and ligations were carried out as per instructions and fragment analysis was performed on an ABIPRISM 3130xl capillary sequencer. The data were visualized using peak scanner v1.0 software and the exported data was analyzed with Coffalyser software v8 . Calculation of signal ratios was carried out as described by Mistry et al. MLPA was used to detect homozygous deletions at the Of 171 tumors that were analyzed for mutations, 163 were malignant and 8 non-malignant tumors. Of the 163 patients with malignant tumors, survival data were available for 153 patients, of whom 150 patients had malignant tumors of pancreatic origin including ductal adenocarcinomas (n\u200a=\u200a135) and rare carcinomas (n\u200a=\u200a15). The rest (n\u200a=\u200a3) were carcinoma of ampulla of Vater . The KapFigure S1Histomorphological examination of pancreatic tumor tissue sections with Hematoxylin and Eosin stains. Representative photomicrographs of three sections with low, medium and high tumor contents are shown.(TIF)Click here for additional data file.Figure S2Representative SSCP of KRAS codon 12 and codon 61 in pancreatic tumors. (A) The lanes 1\u20134 contain amplified fragments of exon 2 (codon 12) and lanes 5\u20136 contain amplified fragments of exon 3 (codon 61) from tumor DNA samples. The shifted bands seen in lane 1 contain GGT>GAT (G12D) mutation, lane 2 contains GGT>CGT (G12R), lane 3 contains GGT>GTT (G12V) mutation and lane 4 contains tumor DNA without mutation in exon 2. The shifted bands in lane 5 contain CAA>CAC (Q61H) mutation and lane 6 contains tumor DNA without mutation in exon 3. (B) Sequence analysis of a part of exon 2 of KRAS gene (coding strand) with GGT>GAT (G12D) mutation. (C) A part of exon 2 sequence showing GGT>CGT (G12R) mutation. (D) A part of exon 2 sequence showing GGT>GTT (G12V) mutation. (E) A part of the exon 2 showing wild type sequence at codon 12 and codon of KRAS. (F) A part of exon 3 sequence showing CAA>CAC (Q61H) mutation. (G) A part of the exon 3 showing the wild type sequence at codon 61 of KRAS.(TIF)Click here for additional data file.Figure S3Kaplan-Meier survival curves showing difference in overall survival in exocrine cancer patients with and without mutations. (A) Median survival of patients with KRAS mutations was 17 months against 30 months for patients without mutations in the gene. (B) Median survival of patients with KRAS codon 12 GGT>GAT (G12D) mutations was 16 months against 30 months for patients without any mutation in KRAS. (C) Median survival of patients with concomitant alterations in KRAS and CDKN2A genes was 13 months against 30 months for patients without any alterations in both KRAS and CDKN2A.(TIF)Click here for additional data file.Table S1Primer sequences and SSCP conditions for detection of mutations in the KRAS and CDKN2A genes.(DOC)Click here for additional data file.Table S2Mutation frequency by clinic pathology and effect on survival of pancreatic cancer patients.(DOC)Click here for additional data file.Table S3Clinico-pathological details and tumor mutational status of all pancreatic cancer patients.(DOC)Click here for additional data file."}
+{"text": "Human infections were from a virus clade undergoing antigenic drift that showed resistance to adamantanes but sensitivity to neuraminidase inhibitors. An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused >52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans. Highly pathogenic avian influenza viruses of the H5N1 subtype are circulating in eastern Asia with unprecedented epizootic and epidemic effects , neuraminidase (NA), and M2 are essential viral proteins targeted by host antibodies or antiviral drugs such as oseltamivir and rimantadine  What is the genetic diversity of H5N1 viruses involved in human infections? 2) Can the relationship between human and avian H5N1 isolates help explain the source of infection? 3) Do genetic changes correlate with enhanced viral transmissibility in humans? 4) How sensitive are H5N1 isolates to antiviral drugs? 5) What is the antigenic similarity between human H5N1 viruses and current candidate vaccines? and 6) Can candidate vaccine reference stocks be developed in time for an effective public health response?All work involving infectious H5N1 influenza was performed in government-approved biosafety level 3\u2013enhanced containment facilities with experimental protocols in compliance with applicable federal statutes and institutional guidelines. Influenza A (H5N1) viruses isolated in Asia and A/Puerto Rico/8/34 (PR8) (H1N1) were propagated in embryonated chicken eggs or in Madin-Darby canine kidney (MDCK) cells. The African green monkey kidney Vero cell line was from a cell bank certified for human vaccine production.Viral RNA was extracted by using a commercial lysis solution and resin kit and amplified by reverse transcriptase\u2013polymerase chain reaction with specific oligonucleotide primers. Nucleotide sequencing reactions were performed with a cycle sequencing kit and resolved on an ABI 3100 Genetic Analyzer . DNA sequence analysis was performed by using version 10 of the GCG sequence analysis package (Postinfection ferret antisera were prepared as previously described (50) values for oseltamivir and zanamivir were determined by using NA-Star substrate and Light Emission Accelerator IITM  as previously described  of influenza virus PR8 strain , South Mimms, United Kingdom; Saint Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA; and Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. For brevity, the reverse genetics derivation method described represents a consensus of the institutions; minor unpublished protocol details unique to each laboratory were not described and are available upon request. The VN/04x/PR8 reassortant virus was recovered in embryonated eggs and identified in the allantoic fluid by HA assay. The genetic and antigenic properties of the resulting reassortant virus were determined as described previously . These findings are consistent with the epidemiologic data that suggest that humans acquired their infections by direct or indirect contact with poultry or poultry products . The amino acid sequence of the M2 protein of clade 1 viruses as well as of HK/213/03 indicated a serine-to-asparagine substitution at residue 31 (S31N), known to confer resistance to adamantanes (including amantadine and rimantadine)  and have coevolved with the respective HA genes . No evidInfluenza vaccines whose HA are antigenically similar to circulating strains provide the highest level of protection from infection . Viruses,,,,Antigenic analysis of human isolates from 2005 provided evidence of antigenic drift among the most recently circulating H5N1 strains . For exaThe growing H5N1 epizootic in eastern Asia could expand the environmental load of virus and cause more infections in mammals  can be traced back to viruses isolated in 1997 in Hong Kong (clade 3) and from geese in China (goose/Guangdong/96) . The phyThe 2004\u20132005 H5N1 isolates are sensitive to 2 neuraminidase inhibitors that are recommended for prophylactic or therapeutic intervention against human infections with recent H5N1 strains. Rapidly testing potentially pandemic influenza viruses for their susceptibility to licensed drugs is essential to establish appropriate control measures.An effective H5N1 vaccine is a public health priority and the cornerstone for pandemic prevention and control. Reverse genetics approaches allow the rapid production of high-growth PR8 reassortant viruses by engineering a virus with a homologous HA gene lacking the polybasic amino acids associated with high virulence. These candidate H5N1 pandemic vaccine viruses have been made available to vaccine manufacturers to produce pilot lots for clinical trials and are available for possible large-scale manufacturing should the need arise.Genetic and antigenic analyses have shown that, compared to previous H5N1 isolates, 2004\u20132005 isolates share several amino acid changes that modulate antigenicity and perhaps other biological functions. Furthermore, our molecular analysis of the HA from isolates collected in 2005 suggests that several amino acids located near the receptor-binding site are undergoing change, some of which may affect antigenicity or transmissibility. For example, an isolate (VN/JP12-2/05) showed a change from serine to asparagine at position 223 of the HA1 (S223N) that may affect receptor-binding specificity (,The phylogenies of the 8 genomic segments from the clade 1 and 2 isolates from 2004\u20132005 showed that all genes are of avian origin. All H5N1 isolates from both clades belong to 1 of the genotypes recently circulating in Eastern and Southern Asia, e.g., genotypes V and Z (The currently circulating H5N1 viruses were reported to infect domestic or wild captive felids, such as tigers, feeding on infected bird carcasses, and the infected cats can transmit H5N1 to pen mates ("}
+{"text": "RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identifed recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identifed 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the  RB1) which is rate limiting for tumorigenesis [RB1 gene was cloned, researchers have focused on identifying genetic lesions in retinoblastoma that contribute to tumor progression following RB1 inactivation [DEK gene is within the 0.6 Mb minimal region of chromosome 6p22 that is gained in retinoblastoma and there is a significant increase (~2.5 fold) in gene expression in tumors with 6p22 gain (n=5) compared to those without 6p22 gain (n=2) [ with significantly elevated expression (1.7\u20132.2 fold increase) in tumors with 6p gains [Most retinoblastomas are believed to initiate with biallelic inactivation of the retinoblastoma susceptibility gene  lack RB1 mutations. The authors suggested that in those patients, MYCN amplification may be sufficient for retinoblastoma tumorigenesis [Another recurrent focal amplification found in ~9% of retinoblastomas is a region of the genome on chromosome 2p spanning the blastoma  but it iication \u22650 copies While aCGH and cytogenetic studies have contributed to the identification of recurrent chromosomal lesions in human retinoblastoma, higher-resolution platforms (i.e. SNP 6.0 arrays) may help to identify additional candidate oncogenes or tumor suppressor genes that contribute to retinoblastoma progression. However, such analyses could be complicated by chromosome instability because it may be difficult to distinguish driver mutations that contribute to retinoblastoma progression from those regions of the genome that are inherently unstable but do not directly contribute to tumorigenesis. Recent whole genome sequencing of 4 primary retinoblastomas and their matched germline DNA demonstrated that at least some retinoblastomas have relatively stable diploid genomes with few CNVs or somatic nucleotide variations (SNV). TherefoMYCN was the most commonly amplified gene in 8.5% (8/94) of tumors in our retinoblastoma cohort. We also identified focal amplification in OTX2 in 3% (3/94) retinoblastoma samples. In addition to recurrent deletions in RB1, the most common focal deletions were in BCOR in 4% (4/94) of our retinoblastomas. To characterize the relationship between MYCN amplification and RB1 gene inactivation, we analyzed the RB1 gene status in 46 retinoblastomas with sufficient DNA for custom capture Illumina sequencing. Ten of those tumors had no evidence of RB1 SNVs or indels in the coding region. We performed whole genome sequencing (WGS), RB1 immunohistochemistry and fluorescence in situ hybridization (FISH) using probes spanning the RB1 locus on all 10 tumors. One of the tumors had a wild type RB1 gene and expressed nuclear RB1 protein in virtually all the tumor cells. It also had MYCN amplification consistent with previously published data showing that 1.5% of retinoblastomas may initiate by this mechanism [RB1 gene and 3 tumors had focal chromothripsis on chromosome 13 spanning the RB1 locus. This provides a novel mechanism of retinoblastoma initiation and suggests that molecular assays to detect RB1 chromothripsis should be included in future analyses of this important tumor suppressor pathway.In this study, we performed SNP 6.0 analysis of 94 human retinoblastomas and their matched normal germline DNA. These data allowed us to more precisely define the boundaries of recurrent chromosomal gains and losses and to identify recurrent focal lesions in individual genes or small groups of genes. Previous aCGH and cytogenetic studies of human retinoblastoma have identified several recurrent whole chromosome gains and losses including chromosome 16 monosomy (18%) , 10, gaiMDM4 in 45% (43/94) on chromosome 1 has been described previously[In addition to whole chromosome gains and losses, several whole chromosome arms and/or large chromosomal regions (>3Mb) have been found to be gained or lost in retinoblastoma , 12. In reviously\u201315. The  that had statistically significant increase in expression and were more that 2-fold increased in tumors with a 6p gain  . We identified 4 genes on chromosome 6p 05) Fig. . RAB23 iand liver. HCG18 i    and one of them (UPEN-RB-31) had a focal RB1 deletion  so we performed RB1 immunohistochemistry on that sample  and normal adjacent retinal tissue as a positive control. The SJRB011 retinoblastoma had abundant nuclear RB1 protein in virtually all the tumor cells  of retinoblastomas with .5% of reion Fig. . Subsequple Fig. . We usedlls Fig. . To detesis Fig. .RB1, we performed custom capture and Illumina sequence analysis of all 27 RB1 exons in the 46 tumors with sufficient DNA from patients treated at St. Jude Children's Research Hospital  of the tumor and matched normal DNA. Using a paired-end-sequencing approach, we generated 3064 Gb of sequence data for the 10 pairs of samples (germline DNA and tumor DNA); 2793 Gb (91%) were successfully mapped to the reference genome   Table .RB1 gene even though there was evidence of a reduction in RB1 gene copy number and LOH spanning the RB1 locus  had a deletion  had evidence of chromothripsis on chr13 spanning the RB1 gene  had relatively high rates of chromosomal, regional and focal CNVs with more than 20 lesions per tumor. A larger subset (22%) had an intermediate rate with 10\u201320 lesions per tumor and the majority of tumors (60%) had few lesions (1\u20139) or none at all (7%). In addition to the previously reported recurrent focal losses of RB1 mutation and the number of lesions. A much larger study will be required to determine if there are more subtle associations between the clinicopathological features of retinoblastoma and the rate of CNVs.Previously, we performed whole genome sequencing on 4 primary retinoblastomas and their matched germline DNA, as well as an orthotopic xenograft derived from one of the primary tumors that was continuously passaged for 9 months before sequencing . The anaThe relatively higher rate of chromosomal, regional and focal lesions in a subset of tumors (30%) does not necessarily indicate that those retinoblastomas have unstable genomes. Chromosome instability is a dynamic process that cannot be accurately measured at a single time point because it involves the acquisition of sequential chromosomal lesions over time. It is important to distinguish between such dynamic processes that reflect the continuous accumulation of genetic lesions from more acute genomic events such as chromosomal \u201cshattering\u201d called chromothripsis , 25. To RB1 gene and the BCOR gene were the most common deletion events and amplification of the MYCN gene was the most common focal recurrent gain[OTX2 in 3% of retinoblastomas. OTX2 is a homeobox gene that is involved in photoreceptor and retinal pigment epithelium development[OTX2 plays a role in modulating the photoreceptor differentiation program in retinoblastoma in future studies.The overall low rate of CNVs was consistent with the paucity of focal recurrent lesions in genes. As reported previously, we found that inactivation of the rent gain. Here wevelopment, 26\u201328. velopment, 29 and  we found to be altered by more than 2-fold and significantly associated with the copy number alteration. The RAB23 gene is upregulated in retinoblastomas with 6p gain and there is evidence that RAB23 may potentiate Hedgehog signaling in cancer [COX4I1 gene is downregulted in tumors with 16q loss and previous studies have shown that this gene is downregulated in skin cancer [For the most common large chromosomal lesions  we integrated our data on copy number changes with the gene expression data from the same tumors. While there was an overall trend of subtle changes in expression for the genes on those altered regions, only 5 genes n cancer \u201316. Alson cancer . The othDEK, NUP153, E2F3 and TTRAP[DEK, NUP153, E2F3 and TTRAP and additional studies will be required to determine the functional significance of elevated gene expression in human retinoblastoma.The genes that have been previously found to show changes in gene expression that correlate with gain of 6p were MDM4 is found on a region of chromosome 1 that is gained in 45% of retinoblastomas but the gene and protein are increased in virtually all retinoblastomas. Therefore, the lack of a statistically significant association between copy number and gene expression does not necessarily mean that the gene is dispensable for tumorigenesis.One important caveat of our analysis is the possibility that genes involved in tumor initiation and/or progression may be altered in retinoblastoma irrespective of copy number changes. For example, MYCN, BCOR, or OTX2 because we did not have enough samples with both copy number data and gene expression data. A much larger study will be required to determine if those tumors have distinct gene expression signatures and if there is any association between the focal genetic lesion and the expression of those genes.We were not able to perform detailed integrated analysis of gene expression and copy number changes for the less frequent focal lesions such as MYCN amplification and RB1 inactivation to determine if a subset of retinoblastomas can be driven by a single oncogenic lesion (MYCN amplification) without RB1 loss [MYCN in retinoblastoma tumorigenesis was first suggested in 1984 when Lee and colleagues described retinoblastomas with MYCN amplifications [MYCN amplification is excluding all possible mechanisms of RB1 gene inactivation including SNVs, indels, LOH, deletions, translocations and promoter hypermethylation. In our cohort, we identified 10 retinoblastomas that had no evidence of RB1 gene inactivation by sequencing the 27 exons of the gene. Whole genome sequencing of those 10 tumors and their matched normal germline DNA showed that at least 5 had mutations. One had an indel that was missed due to poor coverage of that exon in our custom capture sequencing analysis. One had a deletion and LOH that was also missed in our targeted sequence analysis. Remarkably, 3 of the tumors had complex structural variations that occurred focally on chromosome 13 spanning the RB1 gene locus. These lesions had all the features of chromothripsis and had breakpoints in the RB1 gene leading to loss of protein expression. This is an important discovery because it is the first report of focal chromothripsis as a mechanism of RB1 gene inactivation in cancer. Moreover, it suggests that chromothripsis can initiate tumorigenisis by inactivating a tumor suppressor gene. This type of lesion would be missed by conventional RB1 mutational analysis and this is why it has gone undetected since the RB1 gene was first cloned in 1986 [RB1 sequence because all of the exons are intact in a sample with chromothripsis spanning RB1. Copy number and LOH analysis may also show distributions that are difficult to distinguish from the germline reference because both alleles are present and copy number changes are focal and subtle. Our data suggest that a significant proportion of retinoblastomas (3/10) thought to have wild type RB1 may actually have gene inactivation by chromothripsis. Whole genome sequencing combined with break-apart FISH analysis of the RB1 locus and RB1 IHC can be used to identify this unique subset of retinoblastoma tumors.One of the goals of our study was to explore the relationship between ications . However in 1986 . SpecifiRB1 gene . Among those, only SJRB011 had MYCN amplification and nuclear expression of RB1 protein. The other 3 had reduced RB1 protein expression and all 3 lacked MYCN amplification. There were no known cancer genes mutated in those 3 tumor samples. It is possible that those tumor samples had biallelic promoter hypermethylation and this caused the reduction in protein expression. Unfortunately, we did not have sufficient DNA to perform methylation analysis. It is also possible that there is a novel oncogenic drive that is altered in those tumors that has not yet been characterized and this in turn leads to downregulation of the RB1 protein. A much larger analysis will be required explore the spectrum of genomic lesions that contribute to retinoblastoma initiation in the absence of RB1 gene mutation.It is important to emphasize that in our cohort of 10 retinoblastomas that we analyzed by WGS, FISH and IHC there were 5 tumors that had at least 1 intact Details for tumors samples acquired at St. Jude Children's Research Hospital have been previously described . In addiDetails for the SNP6.0 arrays have been previously described . The SNPRB1 mutation analysis was performed as described previously [RB1, including the flanking intronic regions, and the promoter region were amplified by the polymerase chain reaction (PCR) and Sanger sequenced. Large deletions and rearrangements were detected by quantitative-real time PCR assays designed to measure the copy number of each individual exon of RB1, including the promoter and 3 ' untranslated regions. Quantitative-real time PCR was performed using a StepOne Plus instrument (Applied Biosystems). Experimental plate setup and analysis was performed in StepOne software v 2.1 (Applied Biosystems). A master mix was prepared for each assay following the manufacturer's instructions. The data was analyzed by the comparative Ct method. The Ct values were then compared with the endogenous control (RNase P) and the reference sample to calculate a \u0394\u0394Ct value. Copy number (CN) was then calculated using the formula: CN = 2*2\u2212\u0394\u0394Ct.eviously . All 27 RB1 promoter of tumor samples was analyzed using the Methyl-Profiler\u2122 DNA Methylation qPCR assays following the manufacturer's instructions, with an assay for RB1 gene .Methylation state of the MYCN copy number was detected by a Taqman copy number assay  using quantitative-real time PCR as described above.OTX2 copy number was detected by quantitative real-time PCR using Sybr Green (Applied Biosystems 4472942) and analyzed by Eppendorf Mastercycler ep Realplex2 system  following manual instruction. DNA was isolated from primary tumor and blood as indicated above. Whole genome amplified DNA was used for samples acquired at St. Jude Children's Research Hospital [OTX2 at chr. 14:56339578 and CTNNA3 designed to SNP_A- 2245588 (rs2105702) at chrl0:67396520 as an internal diploid reference based on SNP6.0 data analysis. Primers were designed using Primer3 [Hospital . Copy nu Primer3 , 39 for OTX2: forward \u2014 AACAGGGCTGGTAAAGAG; reverse - GAGTAGTGCCACTCAGCACA. CTNNA3: forward- CAGGTAGGCCAACAAGTCC; reverse \u2014 AAGGTACCTGCCATGTGAATA.RB1 gene in these tumors. Purified BAC DNA from two RB1 3-prime clones (RPH-115122 and RP11-90K7) were labeled with a green-dUTP  by nick translation, and one RB1 5-prime clone (RP11-795F23) was labeled with a red-dUTP . In normal cells that contain normal RB1 gene this probe set produces very tightly linked red and green signals since the probes are separated by only 80kb. This assay was specifically designed to detect any disruption of the RB1 gene occurring between introns 6 and 16. One hundred interphase nuclei from each tumor were scored for the presence of either normal RB1 genes  or disrupted or deleted RB1 genes . Details for FISH protocol have previously been described [The Cancer Center Core Cytogenetic Laboratory received 10 formalin fixed paraffin embedded (FFPE) retinoblastoma tissue specimens in order to determine by FISH if there is a disruption of the Details of IHC protocol have previously been described . IHC wasWhole genome sequencing and exome capture validation has previously been described ."}
+{"text": "Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed \u22658 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with \u22658 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3\u22123q27.3) showed \u22658 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly \u201camplified for chromosome 3q\u201d when showing \u22658 fluorescent 3q signals; 3) trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider  Squamous cell cancer accounts for about thirty per cent of lung cancer The interphase in situ hybridization technique is becoming a routinely available standard molecular assessment requested to reference Pathology Labs, due i.e. to the value of the Her-2 gene in breast, gastric cancers, 1p/19q in oligodendrogliomas or EGFR and ALK genes in lung adenocarcinoma In the actual study we sought to evaluate the subtypes of genotypic abnormalities of the entire chromosome 3 and the distal locus specific 3q that maps the SOX-2 and PI3CA genes in a serie of squamous lung carcinoma Finally, we inizialize a scoring for the assessment of 3q amplification, in order to provide a tool to support the clinical laboratories either for the diagnostic goal either for selection to clinical trials encountering inhibitors targeting the 3q region such as anti- PI3CA or \u2013SOX2 targeted drugs.We used tissue samples from human participants. All tissue blocks have been previously declaired to be available for the purposes of the actual study by the Istitutional Review Board (study conducted according to the principles expressed in the Declaration of Helsinki). Our institutional review board and the ethics committee approved the original human work that produced the tissue samples . All processing in obtaining the material has been performed after a written informed consent.Full name Ethic/Institutional Review Board: Prof. Marco Chilosi, Prof. Aldo Scarpa, Prof. Francesco Calabr\u00f2, Prof. Guido Martignoni, Nucleo Ricerca&Innovazione.Fourty squamous lung carcinomas were recruited from the LUNG VERONA DATABASE files, Italy. All samples per tumour were reviewed by a pathologist expert in the field (MC) and an appropriate sample containing at least 90% neoplastic cells has been selected for the interphase cytogenetic and molecular studies.Immunophenotyping was performed by using \u0394n-p63 , CK5 , CK7 , TTF-1 , Napsin A , and SOX-2 .Interphase cytogenetic fluorescence in situ hybridization analysis was performed using commercially available telomeric specific probe  mapping on chromosome 3q telomere  and a centromeric  probe mapping on 3p11-q11. FISH procedure was developed according to previous reports from our group 5 \u00b5m sections were cut from paraffin-embedded blocks. The procedure of FISH has been performed as detailed in previous manuscript Centromeric probes for chromosomes 3 and locus specific probes for 3q  were used. Each probe was diluted 1\u223610 in tDenHyb2 buffer . Ten microliters of diluted probe were applied to each slide and cover slips were placed over the slides. Denaturation was achieved by incubating the slides at 80\u00b0C for 10 minutes in a humidified box; then hybridization was done at 37\u00b0C for 16 hours. The cover slips were then removed and the slides were immersed at room temperature in 0.5 XSSC for 2 minutes and in 2 XSSC for 2 minutes. The slides were air dried and counterstained with 10 \u00b5l DAPI/Antifade .The slides were examined using an Olympus BX61 (Germany) with appropiate filters for SpectrumOrange , SpectrumGreen , and the UV Filter for the DAPI nuclear counterstain. The signals were recorded with a CCD camera .Fluorescent in situ signals were evaluated on carcinomatous and normal pulmonary adjacent parenchyma.Fluorescent 3q signals was initially scored per each case. These cases were corrected per the score of chromosome 3 signals (LSI 3q/CEP3 >2 \u201camplified\u201d). From 60 to 250 neoplastic nuclei were assessed per tumours, similarly per adjacent normal parenchyma.Genomic DNA was isolated using QIAamp DNA mini kit  and quantified on the NanoDrop Spectrophotometer . The quantities and qualities of the DNA allowed us to continue with the analyses. As reference was used DNA from pooled peripheral blood leucocytes of healthy males. With Agilent Human 44K array format containing around 44 000 oligonucleotide probes, covering coding and noncoding genome region , we screened for copy number alteration 6 tumours . Briefly, 1.5 \u00b5g of tumor and reference DNA were digested, labeled and hybridized according with the Agilent Protocols (version 4). The digested DNAs were labeled by random priming with Cy3-dUTP (reference DNA) and Cy5-dUTP (patient DNA) by use of the Agilent Labelling Kit, after which the labelled DNAs were purified. The resolution of the fragments analyzed varied from several Kb to <1 Mb depending upon the case selected.The array images obtained after scanning (high resolution Agilent scanner) were processed with the Feature Extraction software (version 10.5), and the output data files were analyzed with the Agilent Genomic Workbench. To identify copy number alterations we used Aberration Detection Method 2 (ADM-2) algorithm and to exclude the small variances in the data we set up a custom aberration filter identifying an alterations in copy number if minimum of 8 probes gained or lost to be present and with minimum absolute average log ratio for region to be 0.5. The region with small copy number variations were excluded by comparing and visualizing the copy number variant regions tool of the Genomic workbench software.All carcinomas showed pure squamous morphology ; 11 caseImmunophenotipically p63 and SOX2 were expressed in all cases (100%) . The whole aberrations span about 27.5 Mb. Specifically, findings from oligonucleotide probes spanning the SOX2 and PI3CA zones revealed more relevant gains for the PA3CA in respect to the SOX2 zones, although significant differences were not observed.The remained cases had normal profile. Relevant genomic abnormalities were observed in three cases (+5p).When matching cases analyzied by both FISH and aCGH, only those two cases showing 3q amplification by aCGH scored \u22658 fluorescent at FISH and evidenced a 3q/CEP3 ratio >2. The remaining four cases showed a flat genomic profile at aCGH with focus on the 3q and centromeric regions.false versus true 3q chromosomal amplification in squamous cell lung carcinoma merit consideration at light of emerging trials involving anti-PI3CA and SOX-2 targeted drugs.In our study we observed that 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 3) correction for chromosome 3 is important to identify true 3q amplification; 4) we propose to consider a case \u201camplified for chromosome 3q\u201d when scoring \u22658 fluorescent signals; 5) Amplification of chromosome 3q has been described in squamous neoplastic transformation from different sites In our study we highlighted the findings of three distinctive clustered cytogenetic patterns and propose that correction for chromosome 3 is critical for the determination of true 3q amplification status. Today, there is no definitive consensus about the optimal scoring system for assessing 3q amplification. Due to the fact that the 3q region maps genes, such as SOX2 and PI3CA genes, that may have potential role as novel targets for therapeutics, we believe that the need in proposing guidelines for appropriate evaluation of the 3q region status is important and may have impact to different clinical levels. The absolute 3q copy number or the alternative 3q/CEP3 ratio has to be evaluated appropriately, when the predictivenesss to drug efficacy is going to be determined. In our serie eight cases displayed true amplification  when corrected by chromosome 3. We validated these findings after wide genomic aCGH analysis. False positive 3q amplification is present when absolute copy number of 3q signals ranges from 3 to 7 per nuclei. Wide genomic molecular profiling such as the array comparative genomic hybridization technique may help in appropriate definition of new cut-offs and validate standard ISH results and interpretation. aCGH may distinguish gains of a chromosome due to polyploidy or genetic instability versus true chromosomal amplification.The chromosomal region spanning the 3q encounters many interesting genes At routinely laboratory level, fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue is an increasingly useful technique in the detection of many diagnostic chromosomal abnormalities in different oncological fields Tonon et al. observed and highlighted different degree of gains of chromosomes/genes in term of magnitude such as focal copy number alterations, such as five genes gained in lung carcinomas and others recurrent copy number alterations including high-amplitude amplifications The PI3K gene also set in the 3q chromosomal region. There are ample genetic and laboratory studies that suggest the PI3K\u2013Akt pathway is vital to the growth and survival of cancer cells In our study we did not observe relevant differences in term of magnitude of genome gains when analyzing findings from oligonucleotide probes spanning the SOX2 and PI3CA chromosomal regions.versus true 3q chromosomal amplification in squamous cell lung carcinoma are observed and its impact to trials involving anti-PI3K and -SOX2 targeted drugs is a direct consequence. Correction for chromosome 3 is critical for the determination of true 3q amplification status: the definition of a detailed scoring system and a consensus is mandatory.The clinical impact of the multifaceted genotypic abnormalities, observed in the actual study, merits further investigation. We evidenced that absolute enumeration of chromosomal copies is challenging and the absence of analytical standardisation may limit pre-clinical and clinical studies focusing on the predictiveness of biomarkers mapping on the 3q region. False"}
+{"text": "FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically.Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human  Anterior segment dysgenesis covers a spectrum of disorders affecting the iris, cornea, trabecular meshwork and Schlemm's canal of the eye, which can result in abnormal aqueous humor drainage from the eye leading to raised intraocular pressure and glaucoma FOXC1, PITX2, PITX3, FOXE3, PAX6, MAF, CYP1B1 and LMX1B. Mutations in the FOXC1 gene FOXC1 can all cause iridogoniodysgenesis; as can mutations in the PITX2/RIEG1 gene FOXC1 mutations have a milder average prognosis for glaucoma development than do patients with any one of the known PITX2 mutations Anterior segment dysgenesis (ASD) phenotypes are inherited as autosomal dominant traits with variable expressivity and incomplete penetrance, pointing to a complex etiology FOXC1 may also be involved in the underlying etiology of iridogoniodysgenesis and other eye abnormalities associated with glaucoma. For example, deletion of 6p24-p25 proximal to the FOXC1 locus causes anterior segment abnormalities FOXC1 as the causative gene FOXC1 but may have been monosomic for FOXF2 Genetic evidence suggests that other genes near Foxf2 gene could be involved in anterior segment development and dysgenesis we took advantage of an ENU mutagenised DNA archive Foxf2 mutant lineages. We describe the genetic analysis of an identified Foxf2 mutation and the phenotypic features of the affected animals. These analyses suggest Foxf2 is essential for normal anterior segment development, and that the FOXF2 gene should be considered as an additional candidate for anterior segment dysgenesis in humans.To investigate whether the nearby Foxf2 genomic DNA .The eyes from ten wildtype ) and a lwildtype . The canFoxf2W174R heterozygous mice (16/18) revealed no substantial damage to the optic nerve, retina or cornea (To investigate the effect of the W174R mutation in older mice (6 months), the retina, cornea and optic nerve of heterozygous mice were examined to determine if there was any apparent glaucomatous damage. Histological analysis of 18 mice showed a range of anterior segment defects as previously seen in younger mice. In addition two mice appeared to have bulging eyes that can be associated with raised intraocular pressure. However, on histological investigation there appeared to be extraneous amorphous tissue between the retina and the lens. Histological analysis in the majority of r cornea . In two r cornea . In miceFoxf2W174R embryos displayed iridocorneal defects, E18.5 embryos were examined by histology. Evagination of tissue from the anterior optic cup begins at E14 to form the future iris and ciliary body. At E18.5 finger-like projections of tissue forming the ciliary body processes are clearly visible in wildtype littermates . Previous reports of cytogenetic abnormalities are consistent with the notion that the eye is exquisitely sensitive to both reduced and increased dosage in this chromosomal region. Although FOXC1Foxf2W174R individuals could be the result of a mutation within a Foxc1 regulatory region. However, other evidence to support the involvement of Foxf2 in anterior segment dysgenesis, including the patterning of its ocular expression Foxc1 coding mutations; in combination with the observed physical phenotype - all contribute towards a greatly strengthened candidacy of Foxf2.Data from the characterisation of a 200 kb deletion located 1.2 Mb upstream of Foxf2 caused palate malformations and an abnormal tongue Foxf2 knockout mice subsequently revealed megacolon, colorectal muscle hypoplasia and agangliosis Foxf2W174R mutation. Foxf2 is expressed in the absence of its closest paralogue (Foxf1) in the CNS, ear, and limb buds as well as the eye Foxf2-associated phenotypes.Previous studies have shown that targeted deletion of Foxf2 knockouts Foxf2 expression in the periocular mesenchyme of the developing eye at about E12.5 in situ hybridisation established that there was continued Foxf2 expression from E13 through to adult stages Foxf2 expression at E17 were observed in the developing ciliary body and choroid. These data support the abnormal morphological finding in the developing ciliary body in homozygous Foxf2W174R embryos.The effect on eye development was not examined in previous analyses of Foxf2W174R is a hypomorphic allele, However it is also possible that the differences are due to genetic background and that the mutation causes a complete loss of function. Molecular modelling of FOXC1 in a previous study revealed that a tryptophan residue (Trp152) - the direct homologue of Trp174 in Foxf2, is one of nine critical intramolecular interaction residues that maintain structural integrity of the forkhead winged helix structure Foxf2 would lead to protein instability. Another example of an unstable forkhead transcription factor with a mutation in the DNA binding region is the I87M variant of FOXC1Foxf2W174R mutation and would be consistent with the hypothesis that haploinsufficiency plays a key role in the pathogenesis of Fox associated anterior segment anomalies.The difference in phenotype that was identified between targeted knockout and homozygous missense mutation could suggest that Foxf2W174R mice are variable in eyes from different individuals, recapitulating the variable expressivity observed in human patients with ASD. Schlemm's canal was often smaller than typically seen in wild-type eyes and trabecular meshwork was either missing or was underdeveloped, suggesting abnormal migration of mesenchymal cells into the iridocorneal angle. The ciliary body malformations may affect aqueous humor production and secretion of antioxidant proteins into the aqueous humor FoxF2W174R mice are very reminiscent of those seen in mice that are heterozygous for Foxc1 or Foxc2 mutations Foxc1, Foxc2 and Foxf2 are all expressed in the developing periocular mesenchyme, this suggests that this tissue is particularly sensitive to gene dosage The ocular abnormalities found in FOXE3 gene affect lens development and can be inherited as either an autosomal dominant or recessive trait FOXC2 gene cause lymphedema-distichiasis syndrome FOXC1FOXL2 mutations cause blepharophimosis-ptosis-epicanthus inversus syndrome  Foxg1 , Foxd1Foxn4 Foxf2 gene can be added to this growing list.Despite the high level of conservation in their DNA binding domain, forkhead transcription factors are an extraordinarily diverse group of genes with roles as varied as development, homeostasis, stress response and cell cycle control FOXC1/FOXF2/FOXQ1) in which FOXC1 is separated from FOXF2 by less than 250 kb of genomic DNA, and FOXQ1 is 470 kb proximal of FOXC1. Because duplication and deletions of this region in human disease often contain more than one of these genes, confirmation of pathogenicity has relied on specific mutations in animal models. Although gene knockouts FOXC1 deletions or mutations, no model carrying an additional functional copy of FOXC1 has been developed to explore gain-of function effects seen in interstitial gene duplication events. Since our data provides evidence that Foxf2 in mice is also critically involved in anterior segment development, then duplications or deletions containing both FOXF2 and FOXC1 in patients may contribute to the phenotype. This is supported by clinical observations where interstitial duplication of FOXC1 alone causes an iris hypoplasia phenotype, whereas duplications containing both genes  cause microcornea and ptosis, without iris hypoplasia The 6p25 region contains a forkhead cluster .PCR amplification products from pooled DNA that exhibited evidence of heteroduplexes in their DHPLC profiles, were individually PCR-amplified and screened by DHPLC. The single DNA heteroduplex that was identified was sequenced on both strands to determine the mutation. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and sequencing was carried out using BigDye 3.1 terminator chemistry on an ABI prism 377 DNA sequencer. Sequences were aligned and compared with consensus data obtained from the mouse genome database  and BsrI (wildtype locus) restriction digestion of the exon1c PCR product to distinguish Foxf2W174R heterozygotes from homozgotes and wildtypes. Because the C3H mice carry a Pde6brd1retinal mutation affecting the eye, the identified Foxf2W174R mice were outcrossed to C57BL/6 mice for 2 generations. To exclude rd1 carriers, genotyping was performed with the following two primers F: 5\u2032-ACCTGAGCTCACAGAAAGGC-3\u2032 and R: 5\u2032-GCTTCTAGCTGGGCAAAGTG-3\u2032 as described previously DdeI restriction digest (which cuts the rd1Pde6b mutant locus) and SnabI (wildtype locus) thus allowing differentiation between rd1Pde6b heterozygotes, homozygotes and wildtypes. All subsequent analyses were carried out on mice with only the Foxf2 mutation. Primers for sequencing the Foxc1 gene are in Recovery of the mutant mouse lineage was achieved by Eyes were enucleated and placed in 50% Karnovsky's fixative for 45 minutes. Eyes were then washed 3\u00d730 min in PBS, dehydrated through a graded ethanol series  and then embedded in paraffin wax. Whole eye sections cut sagitally to a thickness of 5 \u00b5m were counterstained with hematoxylin and eosin. Retinal histology was imaged using a digital camera mounted on an Olympus 1\u00d771 microscope."}
+{"text": "There was an error in the text in the \"Cell dissociation and culture\" section of Materials and Methods. The correct text is: \"The chopped tissues were then transferred into 50 ml tube and incubated with collagenase II (5mg/ml) in 37\u00b0C shaker for 60 min.\""}
+{"text": "Pcp4 chromatin and regulates Pcp4 expression in the second arch. Hoxa2 is also sufficient to induce Pcp4 expression in anterior first arch cells, which are Pcp4-negative.Branchial arches are externally visible tissue bands in the head region of all vertebrate embryos. Although initially formed from similar components, each arch will give rise to different head and neck structures. In a screen designed to characterize the molecular control of branchial arch identity in mouse, we identified Pcp4 as a second branchial arch-specific molecular signature. We further show that the transcription factor Hoxa2 binds to  Hoxa2 and Hoxb2; the first arch does not express any Hox genes. Hoxa2 specifies second branchial arch (IIBA) identity: the second arch follows a first arch fate in the absence of Hoxa2, and IIBA-skeletal derivatives are replaced by typical first branchial arch (IBA) skeletal elements in a mirror image configuration Branchial arches are transient, repetitive structures of the vertebrate embryo, in which cells of the cranial neural crest and mesoderm are encapsulated by epithelia. There are five to six pairs of arches in amniotes, labeled according to their position along the embryo antero-posterior axis; the first corresponds to the most anterior arch (lying below the forebrain) and the sixth to the most posterior (close to the developing heart). Each of the arches shares a similar architecture and ability to form skeletal elements, their associated muscles, blood supply and nerves, but has its own molecular identity and contributes to diverse head and neck structures. Early development of the branchial arches is instructed by signaling molecules and transcription factors. Dlx transcription factors regulate proximo-distal patterning within each branchial arch in vitroPcp4-null mice Pcp4 is a small calmodulin (CaM)-binding protein. It affects the rates of association and dissociation of Ca (2+) from CaM and can attenuate the activity of a number of CaM-dependent enzymes, including CaM kinase II Hoxa2, the main determinant of second arch fate, in first arch cells and analyzed changes in global expression. By intersecting the data obtained with related data sets, we uncover novel genes regulated by Hoxa2. In addition, we identify the gene encoding for Pcp4 as highly differentially expressed in first and second arch cells. We show that, while anterior first arch cells are Pcp4-negative, Pcp4 is enriched in second arch cells, and that Pcp4 expression is directly controlled by the transcription factor Hoxa2 in vivo.To understand the molecular control of inter-branchial arch identity, we forced expression of Hoxa2 in Hox-negative IBA cells , distributed in 36 upregulated and 23 downregulated genes  Hoxa2 loss of function mutant IIBA Hoxa2 gain of function and downregulated in Hoxa2 loss of function. This group, which contains genes activated by Hoxa2, is frequently associated with Hoxa2 bound regions (Hoxa2 loss of function mutant (repressed by Hoxa2) are not affected by Hoxa2 overexpression and are seldom associated to Hoxa2 binding events, suggesting these changes may be indirect effects of Hoxa2 absence , which may potentially compensate Hoxa2 loss of function and mask changes in the expression of Hoxa2 targets. Gain of function of Hoxa2 in IBA cells provides therefore a complementary system to identify Hoxa2 direct targets in branchial arches mesenchymal cells. Finally, few genes displayed changes in expression with the same sign in Hoxa2 gain and loss of function  and IIBA cells and instructs a second arch developmental fate  [2], [3]BA cells . Cells ied genes . We idened genes . We inteHoxa2, with low to undetectable level of expression in GFP-positive first arch cells . Interestingly, expression of Pcp4 was also highly different when comparing expression profiles of cells isolated from the first and the second arch . Since Hoxa2 expression is sufficient to induce Pcp4 expression in IBA cells, we asked whether IIBA cells, which are Hoxa2-positive, endogenously express Pcp4. We analyzed the expression of Pcp4 in branchial arch cells using immunofluorescence: Pcp4 was visualized only in IIBA cells, and was not present in cells derived from the IBA  and Hoxa2 mutant (\u2212/\u2212). We found strong Pcp4 expression in wild type IIBA, but no expression in Hoxa2 mutant IIBA  IIBA; b) Hoxa2 is sufficient to induce Pcp4 expression in IBA mesenchymal cells; and c) Hoxa2 is required for expression of Pcp4 in the IIBA, indicate that Hoxa2 is a main regulator of Pcp4 in embryonic development. Additional experiments will be required to understand the function of Pcp4 in the development of the second branchial arch. Local modulation of calcium signal transduction is a key determinant of beak shape Pcp4 by Hoxa2, leading to its expression in the second arch, and the ability of Pcp4 to modulate Ca (2+) signaling suggest that acquiring IIBA identity may partly rely on a differential ability of IIBA cells to control Ca (2+) levels.Taken together, the observations that a) Hoxa2 is strongly bound to Hoxa2 mutant mice were described previously 2. Ecotropic-Phoenix cells were transfected with Fugene and pMYs-IRES-GFP  or pMYs-Hoxa2-IRES-GFP (containing Hoxa2 in front of the IRES), and branchial arch cells were infected using supernatants from Ecotropic-Phoenix packaging cells, supplemented with polybrene at 2 \u00b5g ml/1 final concentration. After 72 hours cells were sorted by retroviral gene expression of fluorescent proteins using flow cytometry. Animal experiments were carried out under ASPA 1986.RNA was extracted from sorted cells using Trizol (Life Technologies). Labeled targets were generated from total RNA obtained in two independent experiments, using the 3\u2032 IVT Express Kit and hybridized to GeneChip Mouse Genome 430 2.0 arrays Branchial arch cells were isolated as described above and grown on 8-well culture slides  for 24 hours, fixed and incubated with Pcp4 antibody (Sigma) diluted 1\u2236200 and visualized with AlexaFluor 488 goat anti-rabbit (Invitrogen).Pcp4 gene ChIP was performed as described +/\u2212Hoxa2 intercrosses were dissected out and snap-frozen in dry ice. After genotyping the embryos, pools were made with the wild type and \u2212/\u2212Hoxa2 branchial arches and total RNA was extracted using Trizol. RT-PCR was performed as described Pcp4 F 5\u2032-ATGAGTGAGAGACAAGTGCC\u2019-3; Pcp4R 5\u2032-CTAGGACTGTGATCCTGCCT-3\u2032, respectively. Whole mount in situ hybridization was performed as described Hoxa2Pcp4 probe, amplified from IIBA cDNA using the primers above.Second branchial arches of E11.5 embryos from Figure S1Differential Pcp4 expression in the first and second branchial arch. Semiquantitative RT-PCR on cDNA isolated from first (I) and second (II) arch. Pcp4 is detected in IIBA and not IBA. Gapdh is a positive control gene; H2O is the negative control, using both Pcp4 and Gapdh primers.(JPG)Click here for additional data file.Table S1Top regulated genes in Hoxa2 gain of function in IBA cells. Genes are ranked using fold changes. The following cut-offs were applied to the probe sets: average fold difference \u22652.5; P value \u22640.05.(XLS)Click here for additional data file.Table S2Microarray data expressed as fold change and p values for the Hoxa2 loss of function (LOF) and Hoxa2 gain of function (GOF) microarray experiments. Affymetrix probesets are only included if they have a fold change >\u200a=\u200a1.75 in either of these experiments. Additional information shows if a gene passes combined filter thresholds of + or \u2212 1.75; 1 indicates yes and 0 indicates no. Also indicated is whether the gene is located close to a Hoxa2-bound region in ChIP-seq binding site (where \u201cclose\u201d is defined as being one of the two closest genes to a ChIP-seq peak).(XLS)Click here for additional data file."}
+{"text": "There are different risk factors or determinants of AKI, and some are related to cardiopulmonary bypass (CPB). In this study, we explored the association between metabolic parameters (oxygen delivery (DO2 and VCO2 levels of each patient were monitored during CPB. Outcome variables were related to kidney function (peak postoperative serum creatinine increase and AKI stage 1 or 2). The experimental hypothesis was that nadir DO2 values and nadir DO2/VCO2 ratios during CPB would be independent predictors of AKI. Multivariable logistic regression models were built to detect the independent predictors of AKI and any kind of kidney function damage.We conducted a retrospective analysis of prospectively collected data at two different institutions. The study population included 359 adult patients. The DO2 level < 262 mL/minute/m2 and a nadir DO2/VCO2 ratio < 5.3 were independently associated with AKI within a model including EuroSCORE and CPB duration. Patients with nadir DO2 levels and nadir DO2/VCO2 ratios below the identified cutoff values during CPB had a significantly higher rate of AKI stage 2 . The negative predictive power of both variables exceeded 90%. The most accurate predictor of AKI stage 2 postoperative status was the nadir DO2 level.A nadir DO2 level during CPB is independently associated with postoperative AKI. The measurement of VCO2-related variables does not add accuracy to the AKI prediction. Since DO2 during CPB is a modifiable factor (through pump flow adjustments), this study generates the hypothesis that goal-directed perfusion management aimed at maintaining the DO2 level above the identified critical value might limit the incidence of postoperative AKI.The nadir DO The ear2) .2 level of 272 mL/minute/m2 during CPB was independently associated with acute renal failure and peak postoperative serum creatinine levels [2 levels < 260 mL/minute/m2 during CPB were associated with increased lactate formation [2 levels and an increased CO2 production (VCO2) during CPB, with critical values settled at a VCO2 > 60 mL/minute/m2 and a DO2/VCO2 ratio < 5.0 [In 2005, in a retrospective series, we demonstrated that a lowest (nadir) DOe levels . Subsequormation  and thatio < 5.0 .2 during CPB falls below a critical value (in the range of 260 to 270 mL/minute/m2), organ dysoxia may be triggered, with consequent tissue acidosis leading to increased VCO2, and that this mechanism may be a determinant of impaired postoperative renal function.These data generate the hypothesis that when the DO2 and VCO2 is still not the standard of care during CPB. The quality indicators used for CPB management are wide-ranging and are mostly 'stand alone' parameters. There is little or no evidence in the literature to suggest that goal-directed perfusion pressure using current quality indicators [2 during CPB is an independent determinant of postoperative AKI is still based on a single-centre observation.Despite this information, routine measurement of DOdicators  influenc2 and VCO2 during CPB might be independently associated with postoperative AKI.In the present dual-centre large series of patients where these values were routinely monitored during CPB, we investigated the hypothesis that DO2 and VCO2 measurement were introduced as part of standard monitoring during CPB in 2009. The primary end point of this study was the determination whether the nadir DO2 value and DO2/VCO2 ratio and the VCO2 peak value during CPB are independently associated with postoperative renal function impairment, which we defined as the peak postoperative serum creatinine value and the presence of AKI according to the AKI Network (AKIN) criteria [2- and VCO2-related variables with general outcome measurements .In this retrospective analysis of prospectively collected data collected at two institutions  DOcriteria . BrieflyThe study was approved by the local ethics committee and the institutional review boards of the two participating institutions , and the need for written informed consent from the patients was waived.Three hundred fifty-nine patients  who had surgery between September 2009 and June 2010 were admitted into the study. Entry criteria were patient age > 18 years undergoing CPB and expected CPB duration > 90 minutes. Exclusion criteria were the presence of chronic renal failure being treated with dialysis and patients who had undergone heart or heart and lung transplantation.The patients were consecutively enrolled at both Institutions. Since only one dedicated software system was available for data collection at each Institution, some patients were not included in cases where two operations were being performed simultaneously on two suitable candidates for enrolment. This criterion accounted for a missing cases rate of 23%. The patients included in the present series were not included in the previous studies dealing with the same experimental hypothesis.2 (< 260 mL/minute/m2) was calculated as 35%. The study was powered to detect a double AKI stage 2 rate in patients with low DO2. Considering the unequal distribution of patients with low and high DO2, an \u03b1 value of 0.05 and a \u03b2 value of 0.20, the following sample size was calculated: (1) 195 patients with high DO2 (65%) comprising 18 patients with AKI (9%) and (2) 105 patients with low DO2 (35%) comprising 18 patients with AKI (17%). The total patient population included in the study was therefore between 300 and 400 patients to account for possible lack of data during the study period.Power analysis was performed before retrospective data collection. On the basis of historical data from the two institutions, the postoperative AKI stage 2 rate was calculated as 12%. The ratio of patients with low DOThe following data were recorded for all patients.1. Preoperative data: Patient demographics, baseline serum creatinine level (mg/dL), estimated creatinine clearance (measured using the Cockcroft-Gault equation), left ventricular ejection fraction (%), comorbidities , baseline haemoglobin (Hb) value (g/dL), logistic EuroSCORE and acute renal failure risk according to a validated risk score .2 (mL/minute/m2), peak VCO2 (mL/minute/m2) and nadir DO2/VCO2 ratio during CPB, peak serum lactate during CPB and number of packed red cell (PRC) units transfused during CPB.2. Perioperative data: Reoperation, type of operation, CPB duration, nadir body temperature (\u00b0C) during CPB, nadir haematocrit (HCT) level (%) (measured at the start of the CPB operation and every 20 minutes thereafter), nadir DO3. Postoperative data: Peak serum creatinine level (mg/dL), peak postoperative change in creatinine level (percentage change from baseline), estimated lowest creatinine clearance, AKI stage defined according to the AKIN criteria, number of PRC units transfused and days spent in the ICU and postoperatively in the hospital.The number of PRC units transfused was considered as a potential risk factor for renal function impairment. Since transfusions may be the consequence rather than the cause of renal dysfunction, we considered the number of PRC units transfused before the onset of the renal dysfunction (perioperatively or soon after surgery).All patients were treated with moderately hypothermic CPB (27\u00b0C to 36\u00b0C). The CPB circuit consisted of an Avant oxygenator , an HVR Evo hard-shell reservoir (Sorin Group) in London and a closed CVR 1200 soft-shell reservoir (Sorin Group) in Gent, Belgium. The hardware consisted of a St\u00f6ckert S5 heart-lung machine (Sorin Group) and a St\u00f6ckert Heater Cooler System 3T (Sorin Group).2 at the outset of CPB and subsequently adjusted according to the patient's core temperature . A Hb value less than 6 to 7 g/dL during CPB was considered the trigger point for PRC transfusion. All patients received tranexamic acid according to the routine protocol of each institution, and no aprotinin was used.Priming solutions consisted of 1,400 mL of Hartmann's solution and 10,000 IU of heparin (London site) and 1,200 mL of gelatine , 15% mannitol (200 mL) and 2,500 IU of heparin (Gent site). No hydroxyethyl starch was used for priming the circuit or as additional fluid during CPB. Cardioplegic solution was a 4:1 ratio of cold blood cardioplegia (St. Thomas's solution) at an induction dose of 1,000 mL, with subsequent 200-mL doses every 20 minutes of (London site) and 800- to 1000-mL single-dose Saint Thomas II crystalloid cardioplegia with additional doses if considered necessary by the surgeon (Gent site). Pump flow was settled at 2.4 to 2.8 L/minute/m2- and VCO2-related measurements were performed using a dedicated software system provided by Dideco (Sorin Group). Data were collected at 10-minute intervals during CPB. Data required to calculate DO2 and VCO2 were arterial Hb (g/dL), pump flow (L/minute/m2), arterial O2 saturation, gas flow into the oxygenator (L/minute) and expiratory CO2 tension (mmHg) measured at the site of the oxygenator exhaled gas port with a Datex-Ohmeda capnograph ), or with a ventilator-integrated capnograph.DO2 and VCO2 were described in a previous article [2 was calculated using the following equation:All blood gas data were corrected for temperature by using standard equations. The equations used to determine DO article . Briefly2 content was calculated as follows:where arterial O2 content (mL/100 mL) = Hb (mg/dL) \u00d7 1.34 \u00d7 Hb saturation (%) + 0.003 \u00d7 O2 tension (mmHg).Arterial O2 was calculated using the following equation:VCOGas volume and flow are expressed as dry standard temperature and pressure. Adequate corrections according to body temperature and pressure saturated conditions were applied.2 level was defined as the lowest DO2 value registered for at least two consecutive measurements, the peak VCO2 value was defined as the highest VCO2 level registered for at least two consecutive measurements and the nadir DO2/VCO2 ratio was defined as the lowest value registered for at least the two consecutive measurements. The normal DO2 and VCO2 values in healthy, awake subjects are usually around 500 mL/minute/m2 and 100 mL/minute/m2, respectively. Under moderate hypothermia, anaesthesia and haemodilution during CPB, these values are always reduced to unspecified values.The nadir DO2, nadir DO2/VCO2 ratio and peak VCO2 during CPB were tested for association with kidney function variables using bivariate linear regression analysis (for peak postoperative serum creatinine) or bivariate logistic regression analysis (for AKI stage 1 or 2 and any kind of AKI). Other possible confounders were tested for associations with kidney function variables using the same technique. The linearity assumption was checked by exploring the decile-based distribution of the AKI stage 2 rate with spline curves. For the independent predictors of AKI, adequate cutoff values were identified using a receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was used to determine the accuracy of the model. The cutoff values were chosen according to Youden's index ). For each cutoff value, the sensitivity, specificity and negative and positive predictive values were measured.Data are expressed as means \u00b1 standard deviation  or as frequencies and percentages . Nadir DOP < 0.1) with AKI stage 2 or any kind of AKI were entered into multivariable stepwise forward logistic regression models for definition of the independent predictors of kidney function impairment to generate odds ratios with 95% confidence intervals. To avoid overfitting of the models, only one independent variable per 10 events was entered into the model. To avoid multicollinearity, variables affected by mathematical coupling were separately entered into different models. In cases of intercorrelation, the best single independent variable was chosen. For all the statistical tests, a P value < 0.05 was considered significant. Data analysis was performed using SPSS 13.0 statistical software .All the factors associated (N = 359), we identified 31 patients (8.6%) with AKI stage 1 and 44 patients (12.2%) with AKI stage 2, for a total of 75 patients (21%) with any kind of AKI. Within the AKI stage 2 group, five patients (1.4%) with AKI stage 3 were included.The demographics, preoperative, operative and postoperative details of the patient population are shown in Table 2, peak VCO2 and nadir DO2/VCO2 ratio during CPB were all associated with renal outcomes and were therefore admitted into the following multivariable analyses. The other perioperative variables were not significantly associated with renal outcomes.The variables listed in Table 2 levels and nadir DO2/VCO2 ratios with postoperative AKI stage 2, we explored the rate of AKI stage 2 outcomes according to the decile distribution of the two independent variables. The graphical analysis of the AKI stage 2 rate according to nadir DO2 level and DO2/VCO2 ratio during CPB , this value was included in the analysis , with slightly lower values for the nadir DO2/VCO2 ratio (0.62) and nadir the HCT level (0.61). The best cutoff values according to the Youden's index were set at a nadir DO2 < 262 mL/minute/m2, a nadir DO2/VCO2 ratio < 5.3 and a nadir HCT < 23.5%.The best AUC was identified for the nadir DO2 value and nadir DO2/VCO2 ratio were separately analysed because of mathematical coupling between the two variables. The various factors identified in the univariate analysis were tested for intercorrelation. There was a significant intercorrelation between centres and EuroSCORE. Centre 1 included patients at significantly (P = 0.001) higher risk for postoperative AKI (EuroSCORE 13.4 \u00b1 17) than centre 2 (EuroSCORE 4.1 \u00b1 2.1). We therefore included the EuroSCORE and not the centre in the multivariable models. Some of the other univariate predictors of AKI are included in the EuroSCORE  and were not included in the model to avoid multicollinearity.The multivariable analysis was performed with AKI stage 2 rate and the rate of any kind of AKI as dependent variables. For each model, nadir DO2 < 262 mL/minute/m2 and nadir DO2/VCO2 ratio < 5.3. After correction for the other explanatory variables, both a nadir DO2 < 262 mL/minute/m2 and a nadir DO2/VCO2 ratio < 5.3 during CPB remained independently associated with AKI stage 2 rate  higher rate of postoperative AKI stage 2 (23.2% vs. 7.4%), with a negative predictive value of 92.5%. The patient group with a nadir DO2/VCO2 ratio < 5.3 during CPB had a significantly (P = 0.001) higher rate of AKI stage 2 (20% vs. 8%), with a negative predictive value of 92%. The patient group with a nadir HCT < 23.5% had a significantly (P = 0.014) higher rate of AKI stage 2 (18.5% vs. 9.4%).The cutoff values identified for the nadir DO2 Figure . The pat2, peak VCO2 and nadir DO2/VCO2 ratio during CPB) with the ICU and postoperative hospital stays using linear regression analysis. We found significant  negative associations between ICU length of stay and postoperative hospital length of stay with nadir DO2 during CPB. Conversely, there was no association of ICU length of stay and postoperative hospital length of stay with the peak VCO2 value and the nadir DO2/VCO2 ratio  and DO2/VCO2 ratio (5.3), and for both these values the negative predictive power exceeds 90%. However, the measurement of CO2-related variables did not increase the accuracy of the DO2-based predictive model. In clinical terms, our data generate the hypothesis that by maintaining the DO2 at a level > 262 mL/minute/m2 during CPB, the likelihood of experiencing a postoperative AKI might be decreased.In the present study, we investigated the role of potentially modifiable factors related to CPB surgery in determining postoperative AKI. Our results demonstrate, in a relatively large series of patients treated at different sites, that a low DO2 as the lowest acceptable DO2 during CPB is associated with significant differences in terms of ICU and postoperative hospital lengths of stay .Moreover, the cutoff value of 262 mL/minute/m2 < 272 mL/minute/m2 during CPB have been associated with an increased rate of acute renal failure [2 have been associated with increased lactate formation [This study confirms some of the critical values previously identified in other studies ,12,13. V failure , and val2 level, O2 consumption cannot be maintained using aerobic energy production, and that to provide energy to the cells, the anaerobic mechanism is activated [2. This mechanism was identified for kidney tissue in 1966 [2 level is around 300 mL/minute/m2. In our study, we identified and confirmed that under anaesthesia and moderate hypothermia, values as low as 262 mL/minute/m2 can be sustained, but below this value kidney function starts to decline.It is well known that below a critical DOctivated ,18. As a in 1966 , the cri2 -related variables did not increase the accuracy of AKI prediction in our series. VCO2 may increase even under normal aerobic conditions, and during CPB this invariably happens during the rewarming phase as a result of increased metabolic needs. Theoretically, if coupled with the DO2 measurement (DO2/VCO2 ratio), the measurement of VCO2 should offer additional advantages for detecting dysoxia, as suggested by other authors who demonstrated that CO2-related variables, in combination with O2-related variables, are good indicators of critical hypoperfusion [2-related variables was detected.The measurement of VCOerfusion ,22. Unde2 supply during CPB might lead to a hypoxic insult to the kidney, as previously hypothesized by other authors who demonstrated the relationship between a low Hb value during CPB and bad renal outcomes [The results of our study, linking postoperative AKI to a condition of 'dysoxia' during CPB, strengthen the hypothesis that a condition of inadequate Ooutcomes -11.2. As such, they have already been identified as independent predictors of postoperative AKI [2 had a marginally higher accuracy in predicting postoperative AKI, as suggested in a previous study [Hb content and HCT level are physiological variables that concur in the definition of the DOtive AKI -11. In ous study , but the2 supply. Under normal physiologic conditions, peritubular capillaries are nourished by efferent glomerular arteries [2 (pO2) levels as low as 25 mmHg [2. In vitro studies have demonstrated that some highly susceptible areas of the kidney are prone to ischemic injury in cases of even slight reductions in renal DO2 [Kidney physiology and function are particularly dependent on Oarteries , which carteries . This efenal DO2 .2 content in cases of acute anaemia is usually compensated by reduced blood viscosity with increased blood flow in the microcirculation and by a compensatory increase in cardiac output. This last mechanism may be impaired during CPB, where pump flow is usually adjusted on the basis of the patient's body surface area and temperature, not the Hb value.Reduced O2 belongs to the field of the modifiable risk factors; and (3) the negative predictive value of the identified cutoff value is very high, suggesting the potential clinical usefulness of these values within a preemptive strategy of renal protection.Our study highlights some concepts related to postoperative AKI prevention in cardiac surgery: (1) CPB is one of the major determinants of AKI, and, in our analysis, both CPB duration and the metabolic variables measured during CPB were independent predictors of AKI; (2) opposite to other determinants of postoperative AKI, DO2. Historically, it has been difficult to deploy CPB goal-directed strategies because of the limitations of parameters such as cardiac index, mean arterial pressure, venous saturation, arterial blood temperature, minimum Hb, partial pressure of CO2 and pO2.Goal-directed strategies are key to improving artificial heart and lung support during CPB. This is especially true, given our more complex population with more comorbidities than those in other studies. An effective goal-directed strategy should be based on proven, clinically significant quality indicators such as DO2 may limit the risk of postoperative AKI. To maintain an acceptable DO2, the pump flow should be adjusted according to the Hb value of the perfusate, not only according to the patient's body surface area and temperature. In other words, the pump should be used to adjust the cardiac output exactly, as it happens as a compensatory physiological mechanism during acute, severe anaemia.The take-away message from our results is that the maintenance of adequate DO2 are likely to occur. The study is not powered to detect other organs' responses to low DO2. Finally, this study was not a randomized, controlled trial comparing a specific strategy (targeted at a DO2 > 262 mL/minute/m2) with conventional CPB management.Our study has limitations. It lacked patients treated under hypothermic conditions < 27\u00b0C, when different limits of critical DO2. This concept should be tested for its clinical effects on kidney function and the function of other organs in an adequately large, randomized, controlled trial.The concept of goal-directed CPB perfusion merits further investigations that are based on clinically significant quality indicators such as DO2 < 262 mL/minute/m2 during CPB is independently associated with AKI stage 2 following cardiac operations.\u2022 A nadir DO\u2022 Other determinants of AKI are patient age, the presence of comorbidities, the type of operation  and CPB duration.2 level is significantly associated with prolonged ICU and postoperative hospital lengths of stay.\u2022 Nadir DO2 during CPB (by reducing haemodilution and maintaining high pump flow) should be tested as a possible preemptive strategy for postoperative AKI in a randomized, controlled trial.\u2022 Goal-directed perfusion aimed at preserving DO2: oxygen delivery; Hb: haemoglobin; PRC: packed red cells; ROC: receiver operating characteristic; VCO2: carbon dioxide production.AKI: acute kidney injury; AUC: area under the curve; CPB: cardiopulmonary bypass; DO2 and VCO2 monitoring during CPB and received honoraria from Medtronic Inc. and Sorin Group for speaking at congresses and symposia. No financial or nonfinancial competing interests exist for any of the other authors.MR is the owner of a patent for DOMR designed the study, performed the statistical analysis and prepared the manuscript. FDS and MB were responsible for data acquisition and interpretation. JM was responsible for data acquisition and interpretation and drafted the manuscript. GVN was responsible for data acquisition and manuscript preparation. TA performed the statistical analysis and designed the study. All authors read and approved the final manuscript."}
+{"text": "Sse1 is a conserved protein that is a noncanonical member of the Hsp70 protein superfamily. Sse1 influences the cellular response to heat stress and has also been implicated in playing a role in the propagation of prions in yeast. Sse1 can seemingly exert its effects in vivo through direct or indirect actions by influencing the nucleotide exchange activity of canonical cytosolic Hsp70s. Using a genetic screen based on the inability to propagate the yeast [PSI+] prion, we have identified 13 new Sse1 mutants that are predicted to alter chaperone function through a variety of different mechanisms. Not only are these new Sse1 mutants altered in the ability to propagate and cure yeast prions but also to varying degrees in the ability to grow at elevated temperatures. The expression levels of chaperone proteins known to influence yeast prion propagation are unaltered in the Sse1 mutants, suggesting that the observed phenotypic effects are caused by direct functional alterations in these mutants. Mapping the location of the mutants onto the Sse1 crystal structure suggests that more than one functional alteration in Sse1 may result in changes in prion propagation and ability to function at elevated temperatures. All Sse1 mutants isolated provide essential functions in the cell under normal growth conditions, further demonstrating that essential chaperone functions in vivo can to some degree at least be detached from those related to propagation of prions. Our results suggest that Sse1 can influence prion propagation through a variety of different mechanisms.The yeast Hsp110 chaperone  Saccharomyces cerevisiae by the Sse1 and Sse2 proteins. SSE1 and SSE2 constitute an essential gene pair in yeast  and are noncanonical members of the Hsp70 superfamily  result in similar phenotypic defects, supporting the hypothesis that Sse1 is an evolutionary vestige of Hsp70  for Hsp70. Sse1 does reflect that of canonical Hsp70s. It consists of a N-terminal nucleotide-binding domain (NBD), a \u03b2-sandwich domain (SBD-\u03b2) and a three helical bundle domain (3HBD or SBD-\u03b1) toward the C-terminus. The Sse1 protein has a compact structure with tight interactions between the NBD and substrate-binding domain (SBD). Unlike Hsp70, the Sse1 SBD-\u03b1 does not form a lid over its binding pocket but instead interacts with the flank of the Sse1 NBD  was amplified from a cDNA clone purchased from Origene . All plasmids constructed in this study were verified by sequencing.Strains and plasmids used and constructed in this study are listed and described in PSI+] was carried out as described  and SUQ5 causes efficient translation read through of the ochre mutation in the ade2-1 allele. Non-suppressed ade2-1 mutants are Ade- and are red when grown on medium containing limiting amounts of adenine due to the accumulation of a pigmented substrate of Ade2. Partial suppression of ade2-1 by [PSI+] allows growth without adenine and eliminates the pigmentation . Cells were transformed with wild-type (WT) or mutant SSE1 alleles and transformants were selected on medium lacking leucine. At this stage all cells (at least 100) were scored for color phenotype on the basis of being white, red or sectored.Effects on  prion  to others having minor effects on color phenotype . As expected, all Sse1 mutants that could not propagate [PSI+] could not grow on medium lacking adenine .Applying the plasmid shuffle technique as described in +] prion , Table 3 adenine . However adenine . The rea of Sse1 . DeletioFes1 and Sse1 have been shown to be NEFs for cytosolic Hsp70s , cells were placed onto YPD to assess color and \u2013ADE \u2013HIS medium to assess the ability to grow on medium lacking adenine. Although the color phenotype on YPD for Sse1 WT or mutant cells harboring the vector or overexpressing FES1 is consistent with presence of Sse1 alone (compare FES1 YPD panels), the ability of some CMY02 Sse1 mutant cells to grow on medium lacking adenine is influenced greatly by the absence of histidine (compare FES1 \u2013ADE panels). Only G616D appears altered in color on YPD by the presence of FES1 overexpression. However, this color change does not correlate with a significant increased ability to grow on \u2013ADE medium  chaperone family expression levels in all the Sse1 mutants. Sse1. Only the P37L mutant appeared to have slightly increased levels of Hsp104 and Ssa, but taking into account previous findings these are unlikely to be the cause of any prion or temperature-related phenotypes . We theSse1 mutants are unable to grow at 39\u00b0. One possible explanation for this phenotype is that such Sse1 mutants are unstable at this temperature. We therefore used Western blotting to assess the stability of Sse1 mutants following exposure to 39\u00b0 for 1 hr and found no difference in stability between any Sse1 mutants compared to wild-type protein (data not shown).As shown in Sse1 protein alone and in complex with cytosolic Hsp70 has been determined . Of the nine mutants identified within the NBD four are predicted to affect ATP binding , three to alter interaction with cytosolic Hsp70 , and three remain unclear  (File S1). The four mutants isolated in the SBD domain are predicted to alter either Sse1 interaction with cytosolic Hsp70 , substrate binding (S440L), or protein\u2212protein interactions (E504K)  , File S1Figure S1 shows an alignment of Sse1 and Sse2. Although these proteins share 76% identity, Sse2 is unable to compensate for Sse1 in terms of [PSI+] prion propagation or growth at higher temperatures  could propagate [PSI+] in the G600 background. We found that both Fes1 and HSPH1 were unable to complement essential Sse1/2 functions in the CMY02 strain . This new role for Hsp110 proteins is conserved across species and provides the first clearly identified protein disaggregation machinery in mammalian cells  in modulating the histidine and/or adenine biosynthetic pathways. Both pathways are part of the \u201csuper-pathway of histidine, purine and pyrimidine biosynthesis\u201d (Saccharomyces Genome Database) and converge on production of the biosynthetic intermediate aminoimidazole carboxamide ribonucleotide, accumulation of which can be toxic to the cell. If Sse1 is involved in modulating this super-pathway then our mutants may be affected in the ability to synthesize either histidine or adenine (or both) and toxic intermediates on this pathway may also be caused to accumulate. The addition of histidine or adenine to growth medium would have the effect of switching off these pathways and therefore suppressing any impaired growth phenotype due to the accumulation of toxic intermediates.Phenotypic analysis of the eratures , Table 3 complex , Table 3henotype . CurrentPSI+] propagation and also heat shock we were surprised to discover that all the Sse1 mutants were unable to efficiently cure the [URE3] prion. In a previous study, Sse1G223D mutant  was unable to cure [URE3] whereas Sse1K69M (can bind ATP but defective in hydrolysis) efficiently cured [URE3]. Thus, it seemed that efficient Sse1 NEF activity is required to cure [URE3]. Our data suggest that this may be an oversimplification. The clear phenotypic differences observed for the Sse1 mutants in respect of [PSI+] propagation and heat shock cannot be explained by a single unifying change in Sse1 function in all mutants. This suggestion is also supported by the location of the mutations on the Sse1 structure. Therefore it appears that a variety of mechanisms that alter Sse1 function can alter the ability to cure [URE3]. However, it should be noted that the ability to cure [URE3] could be influenced by the prion variant that is present in the cells. The [URE3] variants present in the SB34 strain and strains used by Given the variation in the effects of mutants upon . When this residue is mutated to create Sse2Q504E [PSI+] can be propagated albeit not to the same extent as Sse1  was shown to be unable to complement heat shock phenotypes of a sse1 deletion strain constructed in the W303 background  family is well characterized in its ability to modulate prion propagation and how this function can be distinct from roles in the heat shock response (Sse1.The isolation of a set of new"}
+{"text": "DYT1 mutation in Iranian patients affected withprimary dystonia.To determine the frequency of DYT1 gene. DNA extracted from patients\u2019peripheral blood was subjected to PCR-sequencing for exon 5 of the DYT1 gene. The collectionof samples was based on random sampling.In this study, we investigated 60 patients with primary dystoniawho referred to the Tehran Medical Genetics Laboratory (TMGL) to determine thedeletional mutation of 904-906 del GAG in the DYT1 gene (15099 to 15101based on reference sequence: NG_008049.1) was identified in 11 patients (18.33%). Theaverage age of affected patients with this mutation was 13.64 \u00b1 7.4 years.The deletional mutation of 904-906 del GAG in the DYT1 deletional mutation of 904-906 del GAGhas a high frequency in Iranian patients in comparison with other non-Jewish populations.Therefore, this particular mutation may be the main representative of pathogenic DYT1gene for a large proportion of Iranian patients with primary dystonia.It can be concluded that the  DYT1 gene.The DYT1 gene discovered in 1997 is located onchromosome 9 and encodes a protein termed Torsin A. This protein is a member of a superfamilyof ATPases which is a DNA-binding protein withparticular homology to heat shock proteins. It isexpressed in several tissues, in particular in thecentral nervous system (CNS) in the basal ganglia andcerebral cortex. It is also thought to be involvedin cellular trafficking of the dopamine transporterand other membrane-bound proteins (TOR1A) is the only identifiedgene responsible for type 1 dystonia and the 3 bpdeletion (GAG) in exon 5 (15099 to15101 basedon reference sequence: NG_008049.1) is the mostcommon causative mutation. The deletion resultsin the loss of one of a pair of glutamic acid residuesnear the carboxy terminus in a conservedregion of ATP-binding proteins (Torsin A) withunclear function  in exon 5 ofthe DYT1 who referred to the Tehran MedicalGenetics Laboratory (TMGL) were investigatedto determine the deletional mutation of 904-906del GAG in the DYT1 gene. The Ethics Committeeof Pasteur Institute of Iran approved this atudy. Aneurologist diagnosed the type of disorder accordingto the Fahn et al. Dystonia classification criteria suspectedof criteria.DYT1 gene was amplifiedas follows:Utilizing the primers listed in table 1, a 205 bpfragment from exon 5 of the 2, 0.2 mM dNTPs, 10 pM of each of theforward and reverse primers, 0.5 unit Tag DNApolymerase  and 500-1000 ng genomicDNA. Amplification of the 205 bp was monitoredby electrophoresing 10 \u00b5l of the PCR producton 1.5% agarose gel, stained with ethidium bromideand visualized by exposing to UV light.94\u2103 for 5 minutes for initial denaturation followedby 32 cycles at 94\u2103 for 1 minute, 60\u2103 for1 minute and 72\u2103 for 1 minute. Final extensionwas carried out for 5 minutes at 72\u2103. The PCRfinal volume was 62 \u00b5l containing the following:50 mM KCl, 10 mM Tris-HCl at pH=8.3, 50 mMMgClThe sequencing reactions were performed by thechain termination method as described elsewhere, 13 by AQuantitative variables were expressed as means \u00b1SD while qualitative variables were expressed aspercentages.DYT1 gene (205 bp)and DNA sequencing, we analyzed the rate of 3 bpGAG deletional mutation (In this study 60 patients suspected of type 1 dystoniawere selected and after amplifying the specificfragment for exon 5 of the mutation .There were 36 (57%) males and 26 (43%) females.Table 2 summarizes data related to the average ageof the patient population and those with positivefindings for the mutation. Females (10%) had thehighest mutation frequency. The age of the affectedpatients positive for the GAG deletion mutation waslower compared to the total patient population.DYT1 gene in a group of Iranian patientswith PTD was determined by DNA sequencing.This mutation is the most common cause oftype 1 dystonia studied. Its frequency was 18.33%in the studied population of the globus pallidus and pallidotomy. Althoughexperience with this approach is still limited, preliminaryresults in patients with primary generalizeddystonia, especially dystonia , 18. TheDYT1 in Iranian PTD patients compared to other non-Jewish populations is quite outstanding. Therefore,this mutation is responsible for a significant proportionof affected Iranian patients. For genetic diagnosisof the remainder of the patients, more analysesof other genes implicated in primary dystonia, suchas the DYT6 (THAP1) gene, are necessary (The high frequency of the GAG deletional mutationof ecessary , 20."}
+{"text": "The efforts to achieve good glycemic control might damage the quality of life (QoL). This issue has not been elaborated in Indonesia because there was no diabetes-specific quesionnaire available in Indonesian language previously. This study would like to find out whether there is a correlation between glycemic control and QoL of adolescents aged 13-18 years with type 1 diabetes mellitus (T1DM).Cross-sectional study was held on patients who came to The Pediatric Endocrinology Clinic of Cipto Mangunkusumo Hospital or who joinned The Endocrinology Working Group Unit of Indonesian Pediatric Society programs during October to December 2010. Glycemic control was measured by hemoglobin A1c (HbA1c) levels. The audit of diabetes dependent quality of life-teen (ADDQoL-Teen) Indonesian questionnaire was used to assess the QoL.A total of 36 adolescents participated in the study. Only seven subjects had a normal HbA1c level. Median HbA1c level was 8.7 %. Eighteen subjects considered their QoL as good or brilliant, while 14 subjects felt that diabetes had a negative impact. The most severe negative impact was felt on the matter of dietary aspect, but could be managed better by subjects with intensive treatment. There was no correlation between HbA1c level on the last examination and average weighted impact (AWI) score  or between mean HbA1c level within 6 months and AWI score .This study did not show any correlation between glycemic control and QoL of adolescents with T1DM."}
+{"text": "Eleven characterized allergens of Hevea brasiliensis are available as recombinant proteins for in vitro IgE diagnosis. Hev b 2 and Hev b 13 are only suitable as native proteins. By testing patient sera against this panel of recombinant and native allergens of Hevea brasiliensis individual sensitization profiles and the relevance of single allergens can be assessed.Sera of 30 spina bifida patients with specific IgE to NRL were tested for allergen-specific IgE antibodies (sIgE) to natural rubber latex (NRL) and 13 single Hev b allergens using the ImmunoCAP system. The results of the specific IgE values against single allergens were plotted . Minor and major allergens for spina bifida patients sensitized or allergic against NRL could be identified.Regarding all 30 spina bifida patients with sIgE Hev b 1, 2, 3, 5, 6.01 and 13 were identified as major Hev b allergens. In the patients without latex-related symptoms Hev b 2 and Hev b 6.01 were found only in small percentages of the latex-specific IgE response and low frequencies  whereas in patients with latex-related symptoms these allergens were found in high concentrations and frequencies. Hev b 5 represents the allergen with the highest percentage of the latex specific IgE response in all groups of patients, Hev b 1 is the allergen with the highest frequency of sensitization (about 80%) in all groups.Latex extracts for skin prick testing or in vitro allergosorbents should contain the major allergens Hev b 1, 2, 3, 5, 6.01 and 13. In spina bifida patients Hev b 6.01 and Hev b 2 could be useful to distinguish sensitized from allergic patients."}
+{"text": "Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in  Tbx1, Tbx2, Tbx3, Tbx5, Tbx18 and Tbx20 are expressed in the heart and regulate various aspects of embryonic heart development. The importance of unperturbed T-box gene function is highlighted by mutations in mice and human, which are associated with congenital heart disease [TBX20 suffer from diverse cardiac defects, including ventricular septal defects, aberrant valvulogenesis, tetralogy of Fallot and cardiomyopathy [TBX3 result in the ulnar-mammary syndrome in which a subset of patients has ventricular septal defects [The four-chambered mammalian heart develops from a simple linear heart tube by polar elongation, myocardial differentiation and morphogenesis. This complex regionalization of the developing heart is orchestrated by multiple signaling modules and transcriptional circuits. Members of the T-box family of transcription factors are particularly important regulators of myocardial proliferation and patterning. T-box family members myopathy \u20137. Mutat defects .Tbx20 is expressed throughout the first heart field, in a subset of second heart field progenitors and at later stages in the endocardium and derived mesenchyme of the atrioventricular and outflow tract (OFT) cushions and the atrioventricular septum [Tbx20 expression decreases in the chamber myocardium compared with atrioventricular and cardiac outflow regions [Tbx20 die early during development and display short, severely underdeveloped heart tubes that fail to undergo looping [Tbx20 is compatible with both cell-autonomous and non-cell-autonomous defects of this process. Chamber myocardial genes are not activated in Tbx20 deficient hearts while Tbx2, which is normally restricted to the atrioventricular canal (AVC) and OFT, is ectopically expressed throughout the cardiac crescent and linear heart tube [Tbx20 heterozygous mice survive postnatally but show dilated cardiomyopathy, which phenocopies at least a subset of human TBX20 mutant defects [In the mouse, r septum \u201313. Foll regions ,12. Seve looping . Failureart tube . Tbx20 hTbx3 expression is first detected in the inflow tract at the onset of heart looping. Its expression delineates the developing cardiac conduction system, endocardial cushions in the AVC, and the mesenchyme of the OFT [Tbx3 enables development of the cardiac conduction system by restricting cell division and repressing the chamber-specific gene expression program. Tbx3 deficient embryos are developmentally retarded and die at midgestation apparently due to yolk sac deficiencies although Tbx3 mutant hearts are also abnormal [In the mouse heart,  the OFT ,17. Tbx3abnormal ,19. Tbx3abnormal .Tbx20 and Tbx3 cause distinct congenital heart abnormalities, but their overlapping expression in the AVC and outflow tract in the developing heart raises the question of whether they act independently or through transcriptional regulation of common target genes. Tbx20 expression appears ectopically in the apex of the ventricular septum of Tbx3 mutant hearts at E12.5 [Tbx3 expression is markedly down regulated at E9.5 in the AVC of embryos with a conditional deletion of Tbx20 [Tbx20; Tbx3 double homozygous mutants to explore possible genetic interaction between these two genes based on their overlapping expression in the AVC and OFT. With respect to heart development, the double homozygous phenotype was indistinguishable from that of the Tbx20 single homozygous mutant in that heart looping and development was arrested early in development. In addition, a general developmental delay characteristic of Tbx3 homozygous mutants was apparent in the double homozygous mutants, apparently independent of the cardiac phenotype.Thus, when individually mutated, at E12.5  and Tbx3of Tbx20 . HoweverTbx20 locus, Tbx20lacZ [Tbx20-, or a null mutation of the Tbx3 locus, Tbx3tm1Pa [Tbx3-, were intercrossed to produce double heterozygotes, which were intercrossed to generate double homozygotes as well as other genetic combinations. Embryos and weanling mice were genotyped for Tbx20 and Tbx3 by PCR from lysates of yolk sac or tail tip, respectively. The mutant allele of Tbx20 was genotyped with primers: Primer 1 (LoxP F2): 5\u2019-GACTGGAGAGGCCATCAAAA-3\u2019 and Primer 2 (LacZR2): 5\u2019-GTTTTCCCAGTCACGACGTT- 3\u2019; and the wild type allele was genotyped with primers: T20 wt sense: 5\u201d-CCCAAGGAGAAGGAGGCAGCAGAGAAC-3\u2019 and T20 wt antisense: 5\u2019- CGCAAGTATAAAATGGGGGTTCCTGACC-3\u2019. PCR conditions were 3 minutes at 94oC, 30 cycles , and 5 minutes at 72oC. The primers and PCR conditions for Tbx3 were as previously described [All experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health under protocols approved by the Columbia University Institutional Animal Care and Use Committee (protocol No. AC-AAAD3302). Mutant alleles were maintained on a mixed genetic background derived from 129, C57BL/6Tac and ICR mice. Mice heterozygous for a null mutation of the x3tm1Pa , hereaftescribed .in situ hybridization [For timed pregnancies, females were placed with males overnight and checked the following morning for the presence of vaginal plugs. Noon on the day of the plug was designated E0.5. Embryos between E8.5 and E9.25 were dissected out of the uteri and their extra-embryonic membranes in phosphate buffered saline (PBS) with 0.2% bovine serum albumin and were fixed in 4% paraformaldehyde in PBS overnight. Standard procedures were used for whole mount dization .http://www.wessa.net/rwasp_notchedbox1.wasp).Statistical analyses were performed using the Chi-square distribution, Mann Whitney U test and notched box plot analysis , Tbx20-/-; Tbx3+/- (n=29), as well as double homozygous mutant embryos (n=14), were morphologically indistinguishable from one another at any given developmental stage: Starting at the heart looping stage, heart tube formation was retarded, hearts were small, and heart looping failed, resulting in two vertically oriented chamber-like swellings with a characteristic hour-glass shape by E9.25  similar to Tbx3-/- embryos (4/4)  from heterozygous matings were scored for heart looping and other morphological and developmental features. Of these, 126 embryos were generated by double heterozygous intercrosses while 84 embryos were generated by crossing Tbx20by E9.25  & 3, as - hearts \u201315. EmbrTbx3-/- embryos were not observed between E8.5 and E9.25, which is in agreement with previous work [Tbx20+/-; Tbx3-/- embryos at E8.5 and 1 of 6 double homozygous mutant embryos at E9.25   but was upregulated and ectopically expressed throughout the entire Tbx20-/- mutant heart by E9.25. Similar upregulation and ectopic expression of Tbx2 was seen in Tbx20-/-; Tbx3-/- double homozygous mutant hearts, whereas expression in Tbx3-/- hearts was normal .Expression of escribed   of Tbx20-/-, Tbx3-/- or Tbx20-/-; Tbx3-/- embryos compared to stage-matched wild type controls (Tbx5, which is expressed in the atrioventricular and atrial progenitors in an antero-posterior gradient, was slightly reduced in both Tbx20-/- and Tbx20-/-; Tbx3-/- embryos at E8.5 but no difference in expression was evident among the genotypes by E9.25 (We observed no change in expression of Tbx1, Tbx2 and Tbx3 all interact in OFT development with Tbx1 upstream of Tbx2 and Tbx3. Double homozygous mutants for Tbx1 with either Tbx2 or Tbx3 have heart failure more severe than either single mutant alone [Tbx2 and Tbx5 have been shown to repress or activate, respectively the expression of Nppa through competing interactions with the transcriptional co-factor Nkx2-5 [Tbx3 and Tbx20 have also been shown capable of interacting with Nkx2-5 [Tbx20 has been shown to repress Tbx2 expression in the developing heart, as in its absence, Tbx2 is upregulated throughout the chamber myocardium of the heart tube [The DNA binding domain of T-box proteins, as well as T-box binding motifs identified in the promoters of downstream target genes, are highly conserved. This leads to the possibility that these transcription factors have targets in common and could compete or cooperate in transcriptional regulation of specific targets in areas of overlapping gene expression. Furthermore, some T-box proteins can bind DNA as dimers, raising the possibility of heterodimerization. In the developing heart at least seven T-box genes are expressed ,23 and ant alone . At the r Nkx2-5 \u201327. Tbx3h Nkx2-5 ,17. T-boTbx20 and Tbx3 have areas of overlapping expression in the OFT and AVC of the developing heart and thus have the potential to interact either by affecting common downstream target genes or by cross regulation. A potential example of cross regulation was noted in Tbx3 mutant hearts at E13.5-14.5 where Tbx20, which is normally expressed in the base of the developing interventricular septum, appears to be ectopically expressed in the developing conduction system at the apex of the septum where Tbx3 would normally be expressed [Tbx3-expressing tissue in the abnormally developing septum is not clear. On the other hand, Tbx20 regulates Tbx3 in the AVC as deletion of Tbx20 specifically in the AVC leads to a downregulation of Tbx3 expression at E9.5 [xpressed . However at E9.5 .Tbx20 single homozygous mutants: morphologically the double homozygous mutant hearts displayed a vertically oriented hourglass shaped, two-chambered heart with no looping. Molecularly, Tbx2 was upregulated throughout the heart tube and Nppa was severely down regulated in the double homozygous mutants, similar to Tbx20 single mutants, whereas Pitx2 and Tbx5 were unchanged. This phenotype is indistinguishable from the Tbx20-/- phenotype where cardiac development is arrested and chamber differentiation does not occur although the anterior/posterior and left/right axes are established correctly. Because of this early arrest starting at E8.5, cardiac development never reached the stage where phenotypic features characteristic of the Tbx3 mutant heart, such as convergence defects, increased AVC cell proliferation and ventricular septal defects, could be evaluated. Thus, Tbx20 is epistatic to Tbx3 with respect to the early heart phenotype and heart development is arrested at an earlier developmental stage than the Tbx3 phenotype becomes manifest. To further investigate potential genetic interactions in the cardiac conduction system, conditional alleles that allow survival beyond the time of death of the homozygous mutants would be required. For this type of study, the recently developed Tbx3 allelic series, which has been used to demonstrate exquisite dose sensitivity of the cardiac conduction system to levels of Tbx3 in embryos and adults could be combined with a conditional Tbx20 allele to test for genetic interactions throughout development and adult life [Tbx3 homozygous mutant embryos are delayed in their overall development compared with littermates [Tbx20-/-; Tbx3-/- double homozygous embryos that was not seen in Tbx20 single mutants. Thus, developmental delay is associated with the Tbx3 mutant genotype, even though Tbx20 mutants have a more severe and earlier heart abnormality. It was previously postulated that vascular defects and apoptosis in the Tbx3 mutant yolk sac were responsible for this developmental delay [Tbx3 and Tbx20 are expressed in the yolk sac, Tbx20 in the mesoderm layer [Tbx3 in both endoderm and mesoderm [Tbx3 mutants and was not exacerbated in double mutants. Thus the developmental delay associated with the Tbx3 mutant genotype does not appear to be associated with an early yolk sac vasculature phenotype and remains to be elucidated.To investigate the phenotypic consequences of possible interactions between the two genes at the earliest stages of heart development, we produced double mutants using mutant alleles that ablate function and compared the morphological and molecular phenotypes of the single, compound and double mutants. We found no evidence for a genetic interaction in double heterozygotes, which were viable and fertile, or in double homozygotes, which displayed an embryonic heart phenotype indistinguishable from ult life . It has termates . We obseal delay , even thal delay . Both Tbmesoderm , and vasmesoderm . In this"}
+{"text": "Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.Mice lacking the 4 Starting at 48 hpf, the HSCs then seed what will become the hematopoietic stroma of the caudal hematopoietic tissue (CHT) at 3 dpf, followed by expansion and differentiation The second and definitive wave of hematopoiesis begins in the ventral wall of the DA at 24 hpf clochelycatcdx1 and cdx4cdx4 single mutants and cdx1/cdx4 doubly deficient embryos are accompanied by the down-regulation of the hemangioblast and hematopoietic stem cell (HSC) marker scl1 and a number of Hox genes, and normal hematopoiesis can be rescued by the forced expression of hoxa9aSeveral genes have been implicated in the control of successive steps in the formation of blood and the vasculature. In zebrafish, the earliest acting gene to be identified to date is Hoxb4, leads to defects in blood lineages. Hoxb4 and indeed all remaining Hox genes of paralog group 4  display potent HSC-promoting functions Hox and Meis1 genes in normal hematopoiesis is further reflected in their well-documented contributions to human and murine leukemias Hox genes have also been implicated in hematopoietic and vasculogenic processes in mammals Hox clusters, with zebrafish retaining 7 clusters and 47 genes following evolutionary loss hoxb6b, hoxb7a, and hoxa9a are known to regulate primitive hematopoiesis and are required for hematopoietic stem cell formation pbx and meis function strongly compromise primitive and definitive hematopoiesis as well as the vasculature In teleosts, a genome duplication event generated 8 Hoxd4 function show abnormalities of the anterior-most vertebrae, the atlas and axis, but are viable and fertile hoxd4a loss-of-function morphants. We demonstrate a surprising role for zebrafish hoxd4a in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. Defects resulting from hoxd4a deficiency can be rescued by capped mRNA for hoxd4a, meis1.1, scl, and fli1 but not by cdx4. Impaired meis1.1 function following hoxd4a knockdown leads to widespread Hox gene deregulation including the reduced expression of Hox genes previously implicated in hematopoiesis, vasculogenesis and angiogenesis. The cdx4, hoxd4a and meis1.1 genes are spatially and temporally co-expressed in shield-stage embryos within the ventral-most presumptive mesoderm, the site from which fate mapping studies have shown the hemangioblast to arise hoxd4a morphants display decreased meis1.1 expression in ventral-most presumptive mesoderm, and this meis1.1 expression is rescued by prior injection of capped hoxd4a mRNA. Thus, our data indicate that hoxd4a functions at the earliest times and near the top of a regulatory hierarchy directing hemangioblast formation, acting downstream or in parallel to cdx genes, but upstream or parallel to meis1.1. Another consequence of impaired hoxd4a and meis1.1 function is the subsequent deregulation of multiple Hox genes previously implicated in vasculogenic and hematopoietic processes. We conclude that hoxd4a has acquired (or retained) a much more important role in hematopoiesis and vasculogenesis than observed for its murine ortholog Hoxd4. Additionally, our results add to a growing body of evidence defining functions for Hox genes during their early phase of expression at gastrulation, well before they are deployed along the antero-posterior axis proper.Mice lacking All animal work conformed to the Institutional Animal Care and Use Committee (IACUC) guidelines at Nanyang Technological University and was reviewed and approved under protocol number ARF SBS/NIE-A 0144 AZ.fli1:EGFP) Wild-type AB and transgenic . Optimal doses for each MO were tested based on phenotypic effects, and each experiment was performed in parallel with a non-specific MO (standard control MO supplied by GeneTools Inc) injected at the same concentration.Antisense morpholino oligonucleotides (MOs) were obtained from Gene Tools Inc and injected into zebrafish embryos at 1\u20134 cell stage. Splice MOs targeting the 5\u2032 splice site intron-exon junction (splice acceptor) are designated as follows MO1\u22365\u2032-GTT CAC TGT GAA GGA CAA AAT CAC A-3\u2032 and exon-intron junction (splice donor), MO2\u22365\u2032- GCA AAG AGA GTG GAT CTT ACC CGT A-3\u2032. MOs were diluted in Danieau\u2019s buffer (0.4 mM MgSOhoxd4a was generated by PCR using the primers shown in Table S1in File S1 and cloned into pCR\u00aeII-TOPO\u00ae. The mMESSAGE mMACHINE Kit (Ambion) was used to synthesize capped mRNA. All mRNAs used for rescue experiments were assessed over a range of concentrations and the following amounts were chosen as the minimum sufficient to induce rescue: scl1 (100 pg), fli1a (30 pg), meis1.1 (100 pg) and hoxd4a (50 pg). All injections were done at the 1-cell stage.Full-length cDNA for File S1.Total RNA was extracted from embryos at 26\u201328 hpf using the PureLink\u2122 Micro-to-Midi\u2122 Total RNA Purification System (Invitrogen). 1 \u00b5g total RNA was treated with 1 U DNase I (Fermentas) at 37\u00b0C for 15 min and used for reverse transcription with SuperScript\u00ae III First-Strand Synthesis (Invitrogen). Quantitative reverse transcriptase PCR (qRT-PCR) was performed using SYBR GreenER\u2122 qPCR SuperMix (Invitrogen) on a BioRad iCycler iQ5. The data  were normalized against zebrafish \u03b2-actin. The sequences of the oligonucleotides used for qRT-PCR are given in in situ hybridization (WISH) was performed as previously described nkx2.5, a PCR fragment was amplified from 26\u201328 hpf embryonic cDNA using the primers shown in File S1. The reverse primer incorporated a T7 promoter allowing the PCR product to be used directly for probe production. DIG- labeled antisense RNA probes were transcribed from linearized template using T3, T7 or SP6 RNA polymerase (Roche). Probes for hoxb6b and hoxb7a were derived by RT-PCR-mediated amplification from RNA from 26\u201328 hpf embryos using primers described in Wan et al Whole mount 2, 100 mM NaCl, 0.1% Tween 20) for three times (each for 15 min) at room temperature. For alkaline phosphatase staining, the embryos were incubated in NBT/BCIP (Roche) solution for 30 min.Embryos at 72 hpf were fixed with 4% paraformaldehyde in PBS  at room temperature for 30 min, followed by treatment with pre-cooled acetone for 30 min at \u221220\u00b0C. After rinsing with PBS twice (5 min each), the embryos were equilibrated with NTMT buffer  embryos (150 embryos each) were dechorionated manually at 48 hpf, rinsed for 15 min in calcium-free Ringer\u2019s solution and passed five times through a 200 \u00b5l pipette tip to remove the yolk. The embryos were dissociated in 0.25% trypsin and 1 mM EDTA for 60 minutes at 28.5\u00b0C, during which the sample was passed six times through a 200 \u00b5l pipette tip every 10 minutes in order to obtain a single cell suspension. The dissociated cell suspension was centrifuged at 1000 g for 9 min at 4\u00b0C, the supernatant discarded, and the cells resuspended in ice cold 0.9\u00d7 PBS plus 5% fetal bovine serum and passed by gravity through a 40 \u00b5m nylon mesh filter. Flow cytometry was performed on a BD LSII instrument (BD Biosciences). We used wild type zebrafish embryos as a negative control.hoxd4a during zebrafish development was examined by WISH. Maternal transcripts were seen at the 1 cell stage, and zygotic transcripts were readily detected from 3 hpf to 48 hpf . As observed previously hoxd4a is expressed in the hindbrain with an anterior border at r6/7, in neural crest migrating to the future branchial arches, and in the pectoral fin fields  at the 1\u20134 cell stage resulted in severely deformed embryos that died by 7 dpf. However, at a reduced dosage of MO (4 ng), embryos exhibited grossly normal morphology up to 30 hpf but with a developmental delay of approximately 9 h. In all experiments reported below, we compared morphants and control embryos at equivalent developmental stages. In embryos of at least 26 hpf, we routinely assessed the length between the eye and the otic vesicle as suggested previously To understand the function of hoxd4a MO, in situ hybridization revealed a marked reduction of hoxd4a transcripts at 26\u201328 hpf (>26 somites) , nkx2.5  and myod  was unaltered in the morphants, demonstrating that there was no gross defect in the overall patterning of the mesoderm.Following injection of the anti-somites) Fig. 1W.hoxd4a morphants the number of circulating RBCs was considerably reduced (n\u200a=\u200a96/100) (S3), but was strongly rescued by co-injection with capped mRNA for hoxd4a (. Reduced RBC production at 48 hph (hoxd4a mRNA (Video S3) suggestive of vascular defects. A lack of RBCs was also apparent by observation of the heart (File S1). Apart from anemia, pericardial edema and areas of edema over the yolk sac were also observed from 36 hpf (File S1). In addition, the heart rate was also mildly but significantly slower in morphants at 26\u201328 hpf and 48 hpf (File S1), and may have affected definitive hematopoiesis (see Discussion).The onset of blood circulation in zebrafish is at 25 to 26 hpf. In control-injected embryos at 48 hpf, red blood cells (RBCs) streamed normally over the yolk, through the ducts of Cuvier toward the heart, and through the dorsal aorta, posterior cardinal vein and caudal plexus. By contrast, in hoxd4a morphants, we made use of the Tg(gata1:dsRed) transgenic line gata1 promoter drives expression of the Discosoma red fluorescent protein (dsRed) in erythrocytic lineages during primitive and definitive hematopoiesis. While a strong signal was observed in the ICM and PBI of control embryos at 26 hpf, little could be detected in hoxd4a morphants  was analyzed by WISH. Both gata1 and hbbe1 transcripts were diminished in the posterior PLM at 13 hpf (\u223c 8 somites) (vs C\u2013D) and the ICM and PBI at 26\u201328 hpf (File S1), establishing a defect in primitive hematopoiesis. Co-injection of capped hoxd4a mRNA along with the hoxd4a MO significantly rescued the expression of both erythroid markers , specifically implicating hoxd4a in the phenotype.To assess the effect of reduced runx1 and cmyb marking the HSCs runx1 and cmyb in hoxd4a morphants at 26\u201328 hpf (vs C-D) and 48 hpf (vs G-H) was severely down-regulated. The expression levels of markers of primitive and definitive hematopoiesis were quantitated by qRT-PCR and showed a strong reduction in hoxd4a morphants and significant rescue upon co-injection with capped hoxd4a mRNA .In addition to reduced numbers of blood cells, hoxd4a morphants was investigated through the use of the fli1:EGFP transgenic line which expresses enhanced GFP under the control of the fli1 locus in endothelial cells The state of the vasculature in hoxd4a knockdown on vasculogenesis and angiogenesis were also assessed by exploiting the high endogenous levels of alkaline phosphatase characteristic of endothelial cells. In control embryos, intense signals revealed well-formed ISV, subintestinal vessels (SIVs) and vertebral artery (VTA), whereas such vessels were indiscernible or highly reduced in hoxd4a morphants . Similar hemorrhaging has been observed in fli1 morphants hoxd4a morphants.The effects of  and VTA Fig. 4I.hoxd4a knockdown on genetic programs directing vasculogenesis and angiogenesis, we performed in situ hybridization for the endothelial markers fli1 and flk1. Both genes were significantly reduced in the LPM of hoxd4a morphants at 13 hpf (vs C\u2013D), and in the major trunk vessels and ISVs at 26 hpf (File S1), implying that hoxd4a morphants are defective in axial vasculature. Co-injection of capped mRNA for hoxd4a, rescued both fli1 and flk1 expression, establishing a specific role for hoxd4a in the altered gene expression pattern .To evaluate the effect of efnb2a and ephb4ahoxd4a expression . Likewise, the expression of the vein marker ephb4a was reduced at 13 hpf . Results for fli1, flk1, vegf and efnb2a were quantified by qRT-PCR on RNA extracted from 26\u201328 hpf embryos. The results confirm the decreased expression of these genes in hoxd4a morphants, and the ability of co-injected mRNA for hoxd4a to rescue their expression . The impaired expression of scl and lmo2 persisted at 26 hpf (File S1), suggesting that their later independent functions in definitive hematopoiesis were also compromised. Co-injection of capped hoxd4a mRNA rescued scl and lmo2 expression at 13 hpf (File S1).The hemangioblast is the common precursor to the blood and endothelial lineages and is defined by early expression of such genes as hoxd4a MO was co-injected with capped mRNA for either scl or fli1. The expression of scl and lmo2 was rescued in both cases , placing the action of hoxd4a above these early acting effectors of hemangioblast specification To establish the hierarchy between these key regulators, the gata1 and hbbe1 at 13 and 26 hpf was rescued by co-injection of scl and fli1 mRNA , though rescue by fli1 at 13 hpf was incomplete. Expression of the vasculogenic markers fli1 and flk1 was likewise rescued by scl and fli1 mRNA at 13 hpf (File S1). Together, these results reveal that hoxd4a occupies a very high position in the transcriptional hierarchy governing hemangioblast formation or function.Consistent with the above findings, expression of Meis homologs have been implicated in the earliest stages of hematopoiesis and vasculogenesis in mice and zebrafish meis1.1 in hoxd4a morphants.A number of studies have demonstrated major regulatory roles for Hox transcription factors in hematopoietic cell fate decisions meis1.1 expression was significantly reduced in the PLM of hoxd4a morphants (File S1), while at 26\u201328 hpf meis1.1 expression was reduced throughout the embryo  in hoxd4a morphants by co-injection with mRNA for meis1.1. All three markers were strongly rescued at 13 hpf .At 13 hpf, nscripts Fig. 8C.clochecdx1 and cdx4 genes are among the earliest effectors of the hemangioblast lineage cdx1/cdx4 embryos lack midline vasculature and show a complete failure of hematopoiesis hoxd4a morphants, cdx4 expression is unaltered at 13 hpf  and 26 hpf . We conclude that hoxd4a acts downstream or parallel to cdx1/cdx4 but upstream or parallel to meis1.1.Along with cdx4, hoxd4a and meis1.1 are ever temporally and spatially co-expressed so as to directly implement this hierarchy in a cell-autonomous fashion. Prior fate-mapping studies have established that precursors to the PLM hemangioblasts arise from the ventral-most presumptive mesoderm of shield-stage embryos hoxd4a is expressed . These results validate the suggestion that the early loss of meis1.1 function leads to the deregulation of several, though not all, Hox genes, thereby providing a fuller explanation for how the initial impairment of a single Hox gene, hoxd4a, can lead to massive defects in hematopoiesis, vasculogenesis, and angiogenesis.Members of the MEIS family regulate Hox gene expression in both invertebrates and vertebrates e RT-PCR Fig. 10Ahoxd4a near the top of a regulatory cascade directing hematopoiesis, vasculogenesis and angiogenesis in zebrafish embryos  and vasculogenic defects, and impaired expression of multiple Hox genes Hox gene expression is not affected in meis1.1 or meis3 morphants cdx4 expression in hoxd4a morphants, and the hoxd4a morphant phenotype cannot be rescued by the injection of capped mRNA for cdx4. It may be that hoxd4a is an immediate downstream effector of cdx1/4 function. Alternatively, hoxd4a may act in parallel with cdx1 and cdx4 such that their function, but not expression, is compromised. Such a mechanism is implied by recent studies showing that products of murine CDX1 and HOXD4 form functional heterodimers cdx1 and cdx4 in definitive hematopoiesis could not be established with certainty due to major effects in the development of the dorsal aorta in doubly deficient embryos hoxd4a morphants do form distinctly separate DA and PCV, and these vessels are open to circulation. Nonetheless, we did observe impaired expression of the arterial marker efnb2a, suggesting that the DA may not be appropriately specified in hoxd4a morphants, an event that could lead to defective HSC formation. The mildly reduced heart rate of morphants may contribute to decreased efnb2a expression and impaired specification of HSCs due to sub-optimal levels of nitrous oxide (NO) signaling hoxd4a and meis1.1 in this process. Nonetheless, depressed NO signaling cannot explain all of the defects noted here, since the reduced flk1 expression that we observe at 13 and 26 hpf is not an expected consequence of decreased heart rate hoxd4a morphants show reduced efnb2a expression in tissues that should not be affected by NO levels consistent with a direct role for hoxd4a or meis1.1 and similar observations in meis1.1 morphants The phenotype we report here is in some ways closer to that reported for scl, lmo2 and fli1, and the ability of the capped mRNAs for these genes to rescue development. However, other aspects of the phenotype may represent later and considerably more downstream functions. For example, decreased expression of efnb2a points to a defect in arterial specification downstream of hemangioblast formation and function, as has been noted in meis1.1 morphants fli1, vegf and flk1 in these processes. The vegf gene is also known to play a role in the initiation of hematopoiesis Some of the phenotypic defects observed here can be explained by effects on relatively early embryonic events, such as impaired formation and function of hemangioblast precursors, as indicated by the loss of hemangioblast markers gata1 commits hematopoietic precursors to erythropoiesis during primitive hematopoiesis in the ICM. Later in development, runx1 and cmyb are required for myeloid development during definitive hematopoiesis in the equivalent of the AGM hoxd4a knockdown, decreased expression of scl and lmo2 is likely to contribute to a failure in the induction of gata1 and defective primitive hematopoiesis as evidenced by a loss of expression of hbbe1; but loss of scl and lmo2 function would also account for the observed defect in runx1 and cmyb expression and the subsequent failure of definitive hematopoiesis. Interestingly, we observe the expression of hoxd4a at 26\u201328 hpf in the PBI and at 48 hpf in the AGM and caudal vein plexus. The caudal vein plexus is the site of the future CHT hoxd4a might play a direct role in primitive and definitive hematopoiesis apart from its indirect role through meis1.1 earlier in development. Both early and later functions of meis1.1 in hematopoiesis and vasculogenesis are also likely, given very early effects on the expression of hemangioblast markers and later expression of meis1.1 in the ICM as described by others meis1.1 downstream of scl for the induction of primitive hematopoiesis meis1.1 upstream of scl at an earlier time point during hemangioblast specification.Expression of Xenopus, timed interactions between the organizer and non-organizer mesoderm induce Hox expression and pattern the AP axis hoxd4a knockdown could be attributed to defects in gastrulation, two observations argue against this. First, not all the Hox genes we tested were affected, an observation inconsistent with a global gastrulation defect. Second, our demonstration that the expression of markers of paraxial mesoderm (myod), cardiac mesoderm (nkx2.5) and intermediate mesoderm (pax2.1) was unaffected does not support a major impairment of gastrulation movements. Nonetheless, our findings emphasize the importance of Hox gene function in the early peri-gastrulation phase Hox gene expression in vertebrates is implemented in two phases, an early phase in presumptive mesoderm around gastrulation and a late phase in the body proper Hoxd4 function are viable and fertile, and display defects of the anterior vertebral skeleton Hoxd4 null mice, their viability suggests that there is no severe impairment of these processes. By contrast, mutation of the paralogous Hoxb4 gene does lead to hematopoietic defects and some loss of viability, though these mice are still able to complete embryogenesis and many survive to adulthood hoxd4a function has severely deleterious consequences for hematopoiesis, vasculogenesis and angiogenesis. We suggest that this may be due to a dependency of meis1.1 expression on hoxd4a function that was either acquired by teleosts (or simply acquired by zebrafish) or lost in mammals. It may also be that while Hoxb4 has the predominant role in these processes in mammals, evolution has selected for hoxd4a to take on these functions in teleosts. This situation may be partly analogous to that of the midkine orthologs in mice and zebrafish; the two teleost midkine genes are strongly expressed in the adult brain, whereas the mouse ortholog is not Mice lacking File S1Supporting information. Contains supplementary (PDF)Click here for additional data file.Figure S1Patterning and morphology in hoxd4a morphants. (A\u2013H) Knockdown of hoxd4a does not perturb overall patterning of the mesoderm. (A\u2013D) Expression of pax2.1 shows that intermediate mesoderm forms and matures normally in hoxd4a morphants at 13 hpf (B) and 26\u201328 hpf (D) vs controls .  nkx2.5 expression in the precardiac lateral plate mesoderm at 13 hpf is normal in control (E) and morphant embryos (F).  myod expression in paraxial mesoderm is normal in control (G) and morphant embryos (H). Images in C, D, G and H are lateral views with anterior to the left. A, B, E and F show dorsal views with anterior to the top  or left . (I to O) Lateral views of control and hoxd4a-MO-injected larvae at 72 hpf.  Staining of hemoglobin with o-dianisidine reveals areas of hemorrhage such as in the head  and trunk  in some hoxd4a morphants.  Control larvae (K) but not hoxd4a morphants (L) show abundant RBCs passing through the heart (arrowheads). (M\u2013O) Unlike control larvae (M), hoxd4a morphants display pericardial edema and edema over the adjacent yolk . Scale bars equal 100 \u00b5m. (P) The heart rate in morphants at 26\u201328 and 48 hpf was mildly reduced, but in a statistically significant manner as determined by unpaired Student\u2019s t test (p<0.0001). Error bars give standard deviation.(TIF)Click here for additional data file.Figure S2Reduced expression of markers of primitive hematopoiesis gata1 and \u03b2 embryonic globin (hbbe1) in hoxd4a morphants. WISH in control and morphant embryos at 26\u201328 hpf showing the expression of gata1  and hbbe1  in the ICM and PBI (white arrowheads). Normal expression of gata1 and hbbe1  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a . All images are lateral views with anterior to the left. ctrl, embryos injected with a non-specific morpholino. MO, embryos injected with the anti-hoxd4a morpholino. hoxd4a mRNA, embryos simultaneously injected with the anti-hoxd4a MO plus capped mRNA for hoxd4a. Scale bars equal 100 \u00b5m. All images are at the same magnification.(TIF)Click here for additional data file.Figure S3Reduced expression of markers of angiogenesis and venous specification in hoxd4a morphants. WISH in control and morphant embryos at 26\u201328 hpf showing the expression of fli1  and flk1 . Normal expression of fli1 and flk1  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a . White or black dots denote the tips of dorsally sprouting ISVs. Relative to controls , the expression of the arterial marker efnb2a is reduced in morphants at 26\u201328 hpf (I), while the venous marker ephb4a in morphants has recovered (J). Scale bars equal 100 \u00b5m.(TIF)Click here for additional data file.Figure S4Reduced expression of scl and lmo2 in hoxd4a morphants at 26\u201328 hpf. (A\u2013J) Expression analysis of scl  and lmo2  at 26\u201328 hpf. Normal expression of scl and lmo2  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a  All images present lateral views with anterior to the left and dorsal on top. Scale bars equal 100 \u00b5m. All images are at the same magnification.(TIF)Click here for additional data file.Figure S5scl1 and fli1 act downstream of hoxd4a to direct formation of the hemangioblast. All images are of hoxd4a morphants at 26\u201328 hpf previously injected with capped mRNAs for either scl1 or fli1 as indicated on the left. WISH was performed to detect expression of scl1 and lmo2 (A\u2013D), gata1 and hbbe1 (E\u2013H) and fli1 and flk1 (I\u2013L). Scale bars equal 100 \u00b5m. All images are at the same magnification.(TIF)Click here for additional data file.Figure S6Knockdown of hoxd4a results in decreased expression of meis1.1 but not cdx4 at 13 hpf (\u223c8 somites). (A\u2013F) Expression of cdx4 , hoxd4a  and meis1.1  in control  and hoxd4a morphants  at the shield stage. The white arrowheads in C and D denote the hoxd4a anterior expression boundary in the hindbrain. Scale bars equal 100 \u00b5m. All images are at the same magnification.(TIF)Click here for additional data file.Figure S7The expression of multiple hox genes is reduced at 26\u201328 hpf in hoxd4a morphants. Images are dorsal views (A\u2013H) and lateral views (I\u2013P) of embryos taken through in situ hybridization for the indicated hox genes. Relative to control embryos , hox gene expression is reduced in hoxd4a morphants . All embryos were simultaneously probed for krox20a expression in r3 and r5 as in cdx4 expression is unchanged in control (Q) and hoxd4a morphants (R) at 26\u201328 hpf. Scale bars equal 100 \u00b5m. All images are at the same magnification.(TIF)Click here for additional data file.Table S1Primers used for cDNA cloning.(TIF)Click here for additional data file.Table S2Primers for quantitative RT-PCR.(TIF)Click here for additional data file.Video S1Anterior blood flow in control and hoxd4a morphant embryos. Lateral view focusing on the anterior half of a 48 hpf embryo including the region of the heart, future branchial arches and yolk of control embryos and hoxd4a morphants. In particular, note robust streaming of blood cells through the ducts of Cuvier over the yolk in the control, but an almost complete absence of circulation in the hoxd4a morphant.(7Z)Click here for additional data file.Video S2Trunk blood flow in control, hoxd4a morphant and rescued embryos. Lateral view of the trunk at 48 hpf in a control, hoxd4a morphant and rescuant previously injected with capped mRNA for hoxd4a. Circulation is vigorous in control and rescuant embryos, with abundant RBCs flowing caudally along the DA, streaming dorsally through the ISVs, caudally through the DLAV, and rostrally through the caudal vein and PCV. By contrast, the number of blood cells is greatly reduced in morphants and blood cells are unable to transit the truncated ISVs.(7Z)Click here for additional data file.Video S3Tail blood flow in control and hoxd4a morphant. Lateral view of the tail region in control and hoxd4a morphant embryos at 48 hpf. Control embryos display vigorous blood flow through the DA, ISVs, DLAV and caudal vein plexus. By contrast, morphants show a highly reduced blood cell count with individual blood cells moving slowly caudally through the DA and returning sporadically and haltingly through the caudal vein plexus. Blood cells do not transit from the DA to the DLAV along the ISVs, unlike control embryos. The reddish appearance of the tissue in the caudal vein plexus (white arrows) appears to be due to the accumulation of RBCs.(7Z)Click here for additional data file.Video S4Rescue by meis1.1 mRNA of trunk blood flow in hoxd4a morphant embryos. Video first showing vigorous circulation through the blood vessels of a control embryo at 48 hpf, followed by the absence of blood and weak circulation in a hoxd4a morphant. The last clip demonstrates significant rescue of blood cell count and vasculature in hoxd4a morphants rescued by co-injection of meis1.1 mRNA, including the ability of blood cells to traverse from the DA to the DLAV along intact ISVs.(7Z)Click here for additional data file."}
+{"text": "BRCA1 isoforms. Their proportion varies during the cell cycle, between tissues and in cancer suggesting functional importance of BRCA1 splicing regulation around this exon. Although the regulatory elements driving exon 11 splicing have never been identified, a selective constraint against synonymous substitutions  in a critical region of BRCA1 exon 11 has been reported to be associated with the necessity to maintain regulatory sequences.Alternative splicing across exon 11 produces several BRCA1 alternative splicing program. We report that in-frame deletions and translationally silent nucleotide substitutions in the critical region affect splicing regulation of BRCA1 exon 11.Here we have designed a specific minigene to investigate the possibility that this bias in synonymous codon usage reflects the need to preserve the BRCA1 exon 11. Identification of the trans-acting factors involved in regulating exon 11 alternative splicing will be important in understanding BRCA1-associated tumorigenesis.Using a hybrid minigene approach, we have experimentally validated the hypothesis that the need to maintain correct alternative splicing is a selective pressure against translationally silent sequence variations in the critical region of  BRCA1 gene are associated with a high risk of breast and ovarian cancer. Women heterozygous for such mutations have a lifetime risk of up to around 80% of developing breast cancer and up to 40% of developing ovarian cancer Pathogenic mutations in the BRCA1 is known to undergo alternative splicing of a number of its exons, including the large and functionally important exon 11 BRCA1 open reading frame and allow functional protein production.trans-acting splicing factors that recognise and bind specific pre-mRNA sequences. Their expression changes in cancer as well as in different tissues BRCA1 isoforms vary in quantity during the cell cycle and within different tissues The control of the ratio of splicing isoforms produced within a cell requires regulation by BRCA1 spanning the 3\u2032 end of exon 10 and the 5\u2032 end of exon 11 A bias towards the usage of particular codons, rather than their synonymous counterparts, has previously been reported in a critical region of BRCA1 splicing involving exon 11, we developed a minigene incorporating the whole of BRCA1 exons 8 to 11 and part of exon 12. Using this hybrid minigene approach, we have experimentally validated the hypothesis that maintenance of correct alternative splicing is the cause of selection against silent sequence variations in the BRCA1 critical region spanning exon 11.In order to investigate BRCA1 splicing, we constructed a pB1 wild-type minigene that incorporated sequence from BRCA1 exon 8 up to the first 89 nucleotides of exon 12  and D(11q) isoforms  and D(11q); showing comparable outcomes with that of endogenous BRCA1 in non-transfected MCF7 cells  and breast cancer cells MDA-MB-231 (ATCC). All cell lines tested resembled the increase in D11 isoform observed in MCF7 cells (data not shown).Furthermore, a minigene construct containing the genomic sequence from exons 9\u201312 had the same pattern of splicing Another version of the minigene behaved in exactly the same manner when we inserted the entire genomic sequence from exons 9 to 12 (data not shown).BRCA1 splicing. 13 synonymous substitutions were introduced by site-directed mutagenesis  were assayed for their effect on The resulting mutated minigenes were transiently transfected into MCF7 cell lines and splicing analysed as described . MutatioAll three synonymous substitutions at codon 231 appear to alter splicing. These changes lie 23 nucleotides distal to the start of exon 11 and significantly reduce the levels of FL and D(11q) in favour of increased levels of D(11) isoform. This suggests that sequence changes at this site alter the splicing of exon 11, through reduced recognition of the intron 10 acceptor site, resulting in its exclusion from the final mRNA. This finding points towards an important sequence element, such as an exonic splicing enhancer, located around codon 231.Codon 244 lies 60 nucleotides downstream from the start of exon 11 and is 55 nucleotides proximal to the 11q splice donor site. The T>C synonymous substitution at the third position of this codon seems to enhance levels of the D(11q) isoform. A similar effect was also observed in normal mammary epithelial cells (HMEpC supplied by ECACC) (data not shown).Codon 313 is situated 150 nucleotides 3\u2032 (downstream) of the 11q splice donor site. Synonymous mutations in this codon do not appear to alter exon 11 splicing. Codons 195 and 196 are 9 and 6 nucleotides away from the 3\u2032 end of exon 9 respectively and synonymous mutations at these sites would therefore most likely affect splicing of exon 9. Codon 215 lies 23 nucleotides from the 3\u2032 end of exon 10 and so any splicing effects may be expected to affect this exon. Our assay does not show any significant alteration in relative exon 11 splice isoform abundance with synonymous mutations at these sites. We therefore evaluated their effect on skipping of exon 9 and 10. Splicing products revealed presence of four isoforms {FL, D(9), D(10) and D(9&10)} showing comparable outcomes with that of the pB1WT minigene  isoform . The data show several conserved sequences along the \u2018critical region\u2019 (supporting http://sfmap.technion.ac.il/) In order to identify putative splicing regulatory elements in the \u2018critical region\u2019 of BRCA1 exon 11 splicing is unchanged with deletion D1 and D4. Deletion D3 has a weak effect in reducing levels of the D(11q) isoform in favour of D(11). Transfection with the hybrid minigene carrying deletion D2 has the strongest effect, inducing almost complete skipping of exon 11.We then performed a pB1 minigene deletion analysis of the most conserved regions containing putative binding sites for splicing regulatory proteins. The D1, D2, D3 and D4 deletions are highlighted in http://sfmap.technion.ac.il/) and SpliceAid2 (www.introni.it/spliceaid.html) are shown in supporting The putative splicing factors predicted to bind in D2 and D3 by SFmap  isoform to the detriment of the full and D(11)isoforms. However, it is difficult to speculate upon the relevance of this aberrant splicing effect. This is for two main reasons. Firstly, the effect on the splicing ratio is only relatively weak. Secondly, the D(11q) isoform has been shown to play a role in apoptosis Of the 6 codons analysed, only synonymous substitutions at codon 231 and codon 244 affected splicing of Substitutions at codon 231 caused skipping of exon 11 with a marked increase in amounts of the D(11) isoform. Overexpression of this isoform in mouse epithelial mammary cells has been shown to cause atypical duct hyperplasia Substitutions at codons 195, 196, 215 and 313 did not have any discernible effect on isoform levels. However, there may be other ways by which synonymous changes can exert an effect Nevertheless, the possibility that substitutions at these codon positions affect regulatory elements of splicing should not be excluded. In fact protective variants might have occurred during evolution that compensate for aberrant splicing BRCA1 exon 11 variants. We therefore tested three further synonymous variants found in breast cancer patients to investigate their effect on BRCA1 splicing. Our findings show that the mutations c.795T>C and c.825C>T decrease and increase levels of the D(11q) isoform respectively. However, for the same reasons mentioned above it is not possible to classify these variants as pathogenic mutations causing aberrant splicing since the role of D(11q) is not clear. In fact, the D(11q) isoform has been described as both causing apoptosis and causing cancer The c.693G>A substitution at codon 231 has previously been reported to cause exon 11 skipping in a patient with breast cancer Both artificial and natural variants analysed in this study cause multiple splicing effects . Deletion 3, just next to the D(11q) donor site, decreased D(11q) isoform levels, probably due to the loss of a putative binding site for TIA1 (a donor site modulator) predicted by the SpliceAid analysis. NOVA 1 binding was predicted to bind both regions corresponding to deletion 2 and 3. Binding of NOVA1 at the end of an exon and beginning of an intron was proposed to induce exon inclusion We did not observe strong correlation on the splicing effect of nucleotide changes occurring inside the region of deletion 2 (c.732T>C) and deletion 3 (c.825C>T). This suggests that composite regulatory elements of splicing might be present in this region of BRCA1 exon 11 as has been previously suggested for CFTR exon 12 BRCA1-related cancer, it will be necessary to know the roles of each isoform individually and to understand the combined roles of different isoforms both in health and in tumorigenesis. Part of this understanding will require confirmation of which splicing factors are involved in regulating alternative splicing of BRCA1, including those suggested by this study that may be involved in the splicing of exon 11. Knowledge of these mechanisms would provide a framework for the development of new therapeutic agents capable of manipulating BRCA1 splicing and treating BRCA1-related cancers (eg. breast ovarian and prostate cancer). At some level it may be that cancer predisposition is not so much down to whether individual isoforms are simply present or absent but rather that subtle alterations to a complex and nuanced isoform profile are particularly relevant. Such an isoform environment may interact with other cellular pathways to make conditions favourable or otherwise towards tumour development. If this is the case, it will provide an added challenge of complexity to our understanding of tumorigenesis. However, it may also allow novel and innovative approaches to manipulating the cellular environment in order to prevent and treat cancer.In order to fully understand the effects of altered splice isoform ratios in BRCA1 genomic region from exon 8 to exon 12. Introns have been shortened by PCR amplification with oligonucleotides carrying a non complementary tail for specific restriction digestion and subsequent cloning in pCDNA3+.The pB1 WT minigene is shown in Exon 1 of the \u03b1-globin gene is used as the first exon of the minigene as it provides a strong splice donor site at its 3\u2032 end as well as an ATG start codon at its 5\u2032 end.Using specific oligonucleotides and a two-step PCR mutagenesis method Human breast cancer cell lines, MCF7 (ATCC number: HTB-22\u2122), were grown in DMEM medium with 4500 mg/L glucose, pyruvate and L-glutamine supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Cells were incubated at 37\u00b0C in a 5% CO2 atmosphere.Minigene plasmid vector was transfected into MCF7 cell lines using the FuGENE 6 transfection reagent from Roche. 100 \u00b5l of DMEM serum-free medium containing 2 \u00b5g of vector DNA and 3 \u00b5l of FuGENE reagent was incubated for 15 minutes at room temperature before the mixture was added in 6 cm-well cell cultures (50% confluent) in the presence of 10% fetal bovine serum.BRCA1 exon 11 in the pB1 minigene, RT-PCR was performed with 1.5 \u00b5g of total RNA using the pCSrev primer [5\u2032 GCAACTAGAAGGCACAGTCGAGG 3\u2032] to exclusively target only RNA products from the pB1 minigene. Given the large size of exon 11 (3426 nucleotides), the QIAGEN LongRange RT PCR kit was used following the manufacturer's instructions. 1/4 of the resulting cDNA was amplified in a PCR reaction using primers specific for the desired splice isoforms and PCR products were analysed by gel electrophoresis in 1.5% agarose. The forward primer [9\u201310F: 5\u2032 ACTTATTGCAGTGTGGGAGA 3\u2032] hybridises to the junction between exons 9 and 10. Reverse primers are a mixture of one specific for FL [11FLR: 5\u2032 GGAGTCCGCCTATCATTACATG 3\u2032] and one specific for D(11q) and D(11) [12R: 5\u2032 CCAGATGCTGCTTCACCCT 3\u2032]. 11FLR hybridises within exon 11 distal to the 11q splice site. 12R hybridises to proximal exon 12 and overlaps the exon 10/12 junction and also the exon 11q/12 junction.40 hours after transfection, RNA was extracted from cells using the RNeasy-plus kit from QIAGEN following the manufacturer's instructions. To analyse alternative splicing of BRCA1 alternative splicing of exons 9 and 10, RT-PCR was performed on 1 \u00b5g total RNA using random primers with the Promega kit. BRCA1-minigene specific PCR was performed on resulting cDNA using the forward specific primer alpha-8F [5\u2032 GAGGCCCTGGAGAGGAcAA 3\u2032] and the reverse primer 11+81Rev [5\u2032 TCTCAGTGGTGTTCAAATCA 3\u2032]. Alpha-8F hybridises to the junction between exon 1 of the \u03b1-globin gene and exon 8 of BRCA1. The c nucleotide in lowercase represents the insertion made in exon 8 of the pB1 minigene. 11+81Rev hybridises to exon 11 upstream of the donor site producing D(11q).For the analysis of th of PCR products were subjected to \u2018reconditioning PCR\u2019 for 6 cycles In order to eliminate heteroduplexes from mixed-template 1/10BRCA1 exon 11 were predicted using the computational tools SFmap Putative splicing regulatory sequences in The following calculation parameters were chosen for SFmap:Scoring function: COS(WR);Medium stringency ;Window size: 50.Figure S1Minigene splicing assay of BRCA1 exon 9 and 10.A. The pB1 wild type (WT) version of the minigene is shown. PCMV\u200a=\u200apromoter of the pCDNA3 vector. ATG\u200a=\u200astart codon. TAG\u200a=\u200astop codon. +3C\u200a=\u200ainsertion of cytosine as the third nucleotide in exon 8. pA\u200a=\u200apoly A signal. 1\u200a=\u200aexon 1 of the alfa globin gene. BRCA1 exons from 8 to 12 are numbered. The black solid line represents introns. Dotted lines show alternative splicing of exon 9 and 10. B. Detection of BRCA1 splicing isoforms FL (inclusion of exon 9 and 10); D9 (skipping of exon 9); D10 (skipping of exon 10); D9,10 (skipping of exon 9 and 10).(TIF)Click here for additional data file.Figure S2BRCA1 alignments between nine eutherian mammals. The sequence of exon 11 critical region is in blue. Black characters represent the last 6 nucleotides of intron 10. The alternative donor site (5\u2032ss) in exon 11 which gives rise to the \u0394(11q) isoform is in white characters. Red are the nucleotide variations with respect to the human sequence. Ca.\u200a=\u200aCanis_familiaris; Eq.\u200a=\u200aEquus_caballus; Bo.\u200a=\u200aBos_taurus; Ho.\u200a=\u200aHomo_sapiens; Pa.\u200a=\u200aPan_troglodytes; Po.\u200a=\u200aPongo_pygmaeus; Ma.\u200a=\u200aMacaca_mulatta; Mu.\u200a=\u200aMus_musculus; Ra.\u200a=\u200aRattus_norvegicus. Splicing regulatory proteins (predicted with SFmap) putatively binding to the human sequence are shown at the top of each sequence. The one corresponding to splicing regulatory motifs that are most conserved are highlighted. The list of putative sequences for splicing regulatory proteins and relative scores as reported in SFmap are listed.(DOC)Click here for additional data file.Figure S3Splicing factors predicted to bind. The sequence of deletion 2 and deletion 3 region are shown. Splicing factors predicted (by SFmap and/or SpliceAid) to bind these regions are listed.(XLS)Click here for additional data file.Table S1Information about all synonymous variants tested. The table includes the amino acid number, the nucleotide change, the dbSNP rs number, genomic location in hg19 coordinates (Chr 17 position),.PMID (for existing PubMed records) and our interpretation of the effects caused by the variations on different splicing events tested (isoform regulation).(XLS)Click here for additional data file."}
+{"text": "CYP3A locus have been identified, genetic association studies have so far failed to explain a major part of the phenotypic variability. The term \u201cmissing heritability\u201d has been used to denominate the gap between expected and known genetic contribution, e.g., for complex diseases, and is also used here in analogy. In this review we summarize CYP3A4 pharmacogenetics/genomics from the early inheritance estimations up to the most recent genetic and clinical studies, including new findings about SNPs in CYP3A4 (*22) and other genes (P450 oxidoreductase (POR), peroxisome proliferator-activated receptor alpha (PPARA)) with possible contribution to CYP3A4 variable expression.CYP3A4 is the most important drug metabolizing enzyme in adult humans because of its prominent expression in liver and gut and because of its broad substrate specificity, which includes drugs from most therapeutic categories and many endogenous substances. Expression and function of CYP3A4 vary extensively both intra- and interindividually thus contributing to unpredictable drug response and toxicity. A multitude of environmental, genetic, and physiological factors are known to influence CYP3A4 expression and activity. Among the best predictable sources of variation are drug\u2013drug interactions, which are either caused by pregnane X-receptor (PXR), constitutive androstane receptor (CAR) mediated gene induction, or by inhibition through coadministered drugs or other chemicals, including also plant and food ingredients. Among physiological and pathophysiological factors are hormonal status, age, and gender, the latter of which was shown to result in higher levels in females compared to males, as well as inflammatory processes that downregulate CYP3A4 transcription. Despite the influence of these non-genetic factors, the genetic influence on CYP3A4 activity was estimated in previous twin studies and using information on repeated drug administration to account for 66% up to 88% of the interindividual variation. Although many single nucleotide polymorphisms (SNPs) within the  CYP3A5*1 allele and with low CYP3A4 expression . Average microsomal content has been estimated between \u223c60\u2009pmol/mg of microsomal protein  which occurs in white populations at \u223c2\u20139% but at higher frequencies in Africans. This single nucleotide polymorphism (SNP) was initially found to be associated with higher tumor grade and stage in prostate cancer and showed higher nifedipine oxidase activity in human livers  was identified by a systematic screen for SNPs showing allelic mRNA expression imbalance in human liver CYP3A4*1 another 21 alleles are included, comprising at least 21 amino acid changes and 2 frame shift variants. Although this collection is a useful resource for functional data related to the alleles, it only provides limited information on genetic variability. Further valuable information on CYP3A4 SNPs and clinical pharmacogenetics is available on the homepage of The Pharmacogenomics Knowledgebase PharmGKBCYPallele database or PharmGKB they lack cross reference to documented functional impact  to large structural variations like copy number variants and large deletions and insertions, as well as frequency information and haplotype context . While in the HapMap data  contains in total 471 SNPs assigned to the major transcript (ENST00000336411), including NCBI dbSNP database content (build 134). The 1000 Genomes Project to date (accessed September 2012) contributed 129 new SNPs to this collection. Besides 115 intronic variant positions 7 non-synonymous amino acid changes were previously unknown, of which four variants were reported on the CYPallele homepage  and only one was predicted to be deleterious by phenotype prediction tools SIFT and PolyPhen  genes including CYP3A4 in diverse populations will profit enormously from 1000 Genomes and new candidate SNPs influencing CYP3A4 expression and activity will certainly be discovered, although prediction or experimental testing of functional impact of the many novel variants pose a challenging task.For the CYP3A4 expression have been studied only in recent years by single gene or pathway-directed approaches.Regulation of CYP3A4 phenotype expression is enormously complex including influences from networks of nuclear receptors and other transcription factors  is a microsomal flavoprotein and an obligatory electron donator in the microsomal P450 monooxygenase reaction. In contrast to the multiplicity of CYPs, mammals have only a single POR should thus be expected to influence CYP activity. In recent years rare POR missense mutations in humans were discovered that impair POR function and cause disordered steroidogenesis, ambiguous genitalia, and Antley\u2013Bixler syndrome  is common with frequencies ranging from 19 to 37% in all major ethnicities. In recombinant systems the variant retained >50% of the wild type activity toward several CYPs  and constitutive androstane receptor  are important transcription factors in the regulatory network of CYP3A4 constitutive and inducible expression. Both genes are polymorphic and genetic variants have been studied for impact on CYP3A4 transcription  gene] display largely overlapping substrate selectivity  gene to be associated with higher expression of FoxA2 mRNA and its target genes PXR and CYP3A4. Polymorphisms in FoxA2, HNF4\u03b1, FoxA3 (HNF3\u0393), PXR, MDR1, and the CYP3A4 promoter together with sex explained 24.6% of the variation in hepatic CYP3A4 mRNA expression. However the study lacked information on the relevance of these variations for CYP3A4 protein and activity.In search for additional influential genes, Lamba et al.  phenotypARNT), glucocorticoid receptor (GR), progesterone receptor membrane component 2 (PGRMC2), and peroxisome proliferator-activated receptor alpha (PPARA) to be consistently associated with CYP3A4 phenotype in human liver in the multivariate analysis. Validation in an atorvastatin-treated volunteer cohort confirmed decreased atorvastatin-2-hydroxylation in carriers of PPARA SNP rs4253728. Moreover, homozygous carriers of the variant had reduced PPAR\u03b1 protein expression in liver, and shRNA-mediated PPARA gene knock-down in primary human hepatocytes decreased mRNA expression levels of CYP3A4 by more than 50%. Multivariate analysis revealed that two linked PPARA SNPs alone explained \u223c8\u20139% of the atorvastatin hydroxylase activity variation, whereas all genetic and non-genetic factors together accounted for \u223c33% of atorvastatin 2-hydroxylase variation in this liver cohort , whereas all remaining 24 SNPs were described as trans-acting elements, and only one of these (rs12041966 located on chromosome 1) influenced testosterone hydroxylation  for numerous liver-expressed genes The in vivo phenotype is still far away from the expected 60\u201380% of genetic determination. The use of next generation sequencing approaches for the identification of causal variants in CYP3A4 as well as the numerous genes of its many influential pathways may lead to the identification of many more rare and common DNA variants that together account for a sizeable fraction of this variability. The examples of POR and PPARA demonstrate that CYP3A4 variability is at least in part determined by polymorphisms in genes outside the CYP3A4 locus. However, investigating and validating trans-acting factors is much more difficult due to the more indirect nature of interaction which is more likely to be masked by covariates, thus necessitating larger studies, in vitro or in vivo. In addition, mathematical algorithms are needed to combine many genetic variants, some of which contribute only small fractions to the total variability, into practically useful signatures for application on clinical studies and individualized medicine.In conclusion, the predictive power of currently known genetic polymorphisms with relevance for CYP3A4 The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both  Tbx4 and Tbx5, in three processes essential to the development of the respiratory system: the specification of the lung and trachea primordia, the growth and branching of the airways to form the lung, and formation of cartilage rings of the trachea and bronchi. In the absence of Tbx5, only one lung is specified, and no trachea. Both Tbx4 and Tbx5 regulate the process of lung branching by controlling the expression of the secreted growth factor Fgf10 and activation of Fgf10 signaling. In the trachea, both Tbx4 and Tbx5 are important for condensation of cells to form cartilage rings, although this is regulated by a distinct pathway that does not involve Fgf10.Defective development of the mammalian respiratory system can lead to tracheal, bronchial, or pulmonary malformations causing severe consequences at birth or during postnatal life. Studies using mouse genetics have begun to reveal complex regulatory mechanisms that guide the development of the respiratory system, but understanding how disruption of these mechanisms leads to malformations is far from complete. In this study, we analyze the role of two T-box transcription factors,  The development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen. Because the respiratory system is dispensable for embryonic survival in mammals, defects in development of the respiratory system manifest at or after birth. Indeed, abnormal development of the respiratory system in humans is associated with multiple disorders such as tracheal/bronchial atresia, tracheoesophageal fistula, bronchogenic cysts, pulmonary/lobar atresia and pulmonary hypoplasia Nkx2.1 has been identified as the earliest marker of lung endoderm specification. At E9.25 (25\u201328 somites), the primary lung buds appear as ventro-lateral outpouchings of the foregut connected ventrally by the tracheal primordium. The lung buds grow in a ventro-posterior direction and continue to elongate until E11.5. The point of connection of the lung buds is thought to be the origin of the tracheal tube, which separates from the esophagus in a caudal to cranial direction by E11.5 In the mouse embryo, the endodermal foregut tube is patterned by signals from the lateral plate mesoderm leading to specification of the lung and trachea at embryonic day (E) 9.0 (19\u201324 somites). Wnt2, Fgf10, Bmp4, Shh and retinoic acid synthesis genes, have been shown to play important roles in lung specification and branch formation. Complete absence of both Wnt2 and Wnt2b in mesenchyme surrounding the anterior foregut or absence of \u03b2-catenin in the foregut epithelium leads to a loss of specification of lung primordia as seen by the absence of Nkx2.1 expression Fgf10, which is normally expressed in mesenchyme surrounding the epithelial branching tips, form a short trachea but have no lungs Bmp4 signaling by overexpression of Xnoggin leads to a decrease in lung size and irregularly shaped lung lobes Shh null mutant mice have only a rudimentary lung sac due to branching severe branching defect Shh in lung epithelial cells leads to the formation of hypoplastic lungs with reduced branching of the peripheral tubules Genes involved in different signaling pathways, including Sox9 has been implicated as an important regulator of mesenchymal condensation and chondrocyte differentiation Sox9, FGF2, Igf1, Tgf\u03b22 and Bmp2 enhance chondrocyte formation Shh, Sox2, retinoic acid synthesis genes and Fgf signaling pathway genes have been shown to affect cartilage ring formation Fgf10 mutants form a partial tracheal tube in spite of the failure of lung formation Fgf10 leads to defects in tracheal ring formation and that overexpression of Fgf10 between E11.5 and E13.5 disrupts tracheal rings by altering the periodic expression of Shh in the trachea After E11.5, mesenchyme surrounding the dorsal aspect of the trachea differentiates into the trachealis smooth muscle. Mesenchyme surrounding the ventral aspect of the trachea and lateral aspect of the main stem bronchi segments and differentiates into C shaped rings composed of chondrocytes. Ventral tracheal cartilage is formed by migration of cells that undergo mesenchymal condensation Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing chick lung buds and trachea between stages 15\u201321 Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later Tbx1 homozygous null mutants die at birth due to severe heart defects; the lungs are never fully inflated Tbx4 homozygous mutants, lung buds form but the embryos die at E10.5 due to failure of allantois development and the subsequent lack of chorio-allantoic fusion leading to placental insufficiency Tbx5 mutants die around E10 due to defects in heart development Tbx4 and Tbx5, but not Tbx2 and Tbx3, in lung organ cultures results in inhibition of branching and loss of Fgf10 expression Tbx4 function leads to a reduction in Fgf10 expression in lung mesenchyme and inhibits lung bud formation. Ectopic expression of Tbx4 leads to ectopic expression of Fgf10 and Nkx2.1 and lung bud formation in the esophagus. Additionally, ectopic expression of Tbx4 at the boundary between the trachea and the esophagus can lead to lack of separation of these two structures, resulting in a tracheoesophageal fistula Tbx5 mutations cause Holt Oram syndrome characterized by heart and forelimb abnormalities. A single de-novo mutation in TBX5 has been linked to right lung agenesis The T-box transcription factor genes are important during embryonic development. All members of this gene family contain a conserved DNA-binding T-box domain, which binds to a conserved sequence, the T-box binding element, to activate or repress transcription of specific target genes Tbx4 and Tbx5 in lung and trachea development in the mouse, we made use of conditional alleles to bypass early embryonic lethality. We studied three distinct processes, namely 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation. We show that during early stages of development, Tbx5 is important for specification of the lung buds and the trachea. After specification, Tbx4 and Tbx5 interact during lung growth and branching and the regulation of branching is dependent on Fgf10 signaling. Additionally, Tbx4 and Tbx5 interact in the formation of mesenchymal condensations, which ultimately form the tracheal/bronchial cartilage rings independent of Fgf10 signaling.To study the roles of Tbx5 expression is first detected using in situ hybridization (ISH) at E9.0 (24 somites) in the mesenchyme of the lung and trachea primordia, concurrent with Nkx2.1 expression in the ventral foregut epithelium (Tbx4 expression is detected in the lung buds when they first appear a few hours later at E9.25 (28 somites) in a pattern similar to Tbx5 , the foregut tube is devoid of Nkx2.1 expression and the lung buds are not present Nkx2.1 expression  and removal of Tbx4 in addition to Tbx5 did not exacerbate the Tbx5 phenotype , late enough to bypass lethality but well before lung branching begins. Embryos with CreER and different combinations of the flTbx4 and flTbx5 alleles were examined at E12.5 and E13.5. Conditional Tbx4 null lungs were similar to controls at E12.5 but had fewer branching tips at E13.5 . We hypothesize that the variability in phenotype is due to variable extent of recombination of the flTbx5 allele  became cyanotic at birth and died shortly thereafter due to respiratory distress. Unlike the variable lung size in the lung-specific Tbx5 null mutants, the lungs of these mutants were consistently smaller than controls at E13.5  nor Tbx4 single heterozygous lungs show a branching defect, double heterozygous lungs are smaller at E13.5 and E18.5 with a reduced number of branching tips at E13.5. Because these genes are closely related Tbx4 and Tbx5 could physically interact with each other as heterodimers to activate or repress transcription of downstream targets, as has been suggested for other T-box genes Tbx4;Tbx5 double heterozygous lungs are smaller than conditional Tbx4 nulls. Unequal and distinct functions could be explained by domains outside of the T-box. For instance, the C terminal domain of both Tbx4 and Tbx5 has transcription activating capability which has been correlated to the shared limb outgrowth promoting activity of these genes In lung branching morphogenesis,  another . AlthougTbx4 nulls;Tbx5 heterozygous and lung-specific Tbx4 heterozygous;Tbx5 null lungs, reduction of Tbx4 or Tbx5 leads to a severe decrease in branching, a defect in formation of the lobes and failure of lobe separation, while absence of Tbx4 and Tbx5 leads to branching arrest. These results are in line with the published observations that antisense RNAs used against both Tbx4 and Tbx5 inhibit lung branching in culture and that this affect is explained by loss of Fgf10 expression Fgf10 promoter in vitro suggesting that Fgf10 is a direct downstream target Fgf10 and Wnt2 in the lung mesenchyme. The Fgf10 targets Bmp4 and Spry2 show very low levels of expression in the epithelium of Tbx4 and Tbx5-deficient mutants and Etv5, another Fgf10 target, shows greatly reduced expression in the conditional double null lungs, leading to the hypothesis that the Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. Lungs that are triple heterozygous for Tbx4, Tbx5 and Fgf10 are reduced in size compared to Tbx4;Tbx5 double heterozygous lungs, which supports this hypothesis. Lung-specific Tbx4 heterozygote;Tbx5 null lungs are severely retarded but lung size is not affected by further loss of Fgf10, demonstrating epistasis and supporting the hypothesis that Fgf10 lies downstream of Tbx4 and Tbx5. Lack of rescue of branch formation and lack of activation of the Fgf10 signaling pathway by exogenously supplied Fgf10 in culture suggests that there are other factors downstream of Tbx4 and Tbx5 that affect Fgf10 signaling. Although, lungs deficient in Tbx4 and Tbx5 retain the ability to respond to Fgf10 coated beads . The extracellular matrix (ECM) molecules, heparan sulfate (HS) proteoglycans aid in Fgf10-Fgfr2 interactions during lung development Tbx4 and Tbx5-deficient mutants. Inhibition of heparanase decreases submandibular gland morphogenesis in culture due to deficiency of Fgf signaling Tbx4 regulates ECM molecules in the developing allantois, specifically the chondroitin sulfate proteoglycan versican (R. Arora and V. E. Papaioannou unpublished observations), suggesting that ECM might be one of the targets for T-box genes in regulating development of other organs as well.One possibility for such a factor is a mesenchymal signaling molecule that communicates with the epithelium and activates downstream target(s) required for the activation of the Fgf10 pathway. We examined expression of Tbx4 and Tbx5 causes defects in both formation of the tracheal cartilage rings and development of the trachealis smooth muscle indicating interactions between Tbx4 and Tbx5. Tbx4 and Tbx5 heterozygous tracheas have 10\u201311 cartilage rings but Tbx4;Tbx5 double heterozygous tracheas have 6\u20138 cartilage rings, some of which are incomplete. Additional reduction of Tbx4 and Tbx5 in the lung-specific Tbx4 heterozygous;Tbx5 null lungs leads to a complete disruption of cartilage ring formation supporting a genetic interaction between Tbx4 and Tbx5 in trachea formation.Appropriate dorsal smooth muscle development and ventral tracheal/bronchial cartilage development is important for the normal functioning of the trachea. Smooth muscle provides tracheal flexibility and the cartilaginous rings prevent tracheal collapse. Defects in trachea formation may result in tracheomalacia or tracheal stenosis. Reduction of Sox9, a master regulator of the process of chondrogenesis, is expressed normally at E12.5 throughout the ventral mesenchyme in lung-specific Tbx4 heterozygous;Tbx5 null tracheas but at E13.5 these tracheas show a lack of characteristic mesenchymal condensations and reduced Sox9 expression. Either Tbx4 and Tbx5 control of Sox9 expression becomes more sensitive to dosage at E13.5 or Tbx4 and Tbx5 control another factor, possibly an ECM molecule, which is important for formation of mesenchymal condensations and, in the absence of these condensations, there is a down regulation of Sox9 expression  at a concentration of 20 mg/ml in sunflower oil (Sigma) was administered to pregnant females by intraperitoneal injection between 15.30 and 19.30 hours on E8.5 or between 23.00 and 24.00 hours on E9.0.Whole-mount ISH, immunohistochemistry (IHC), immunofluorescence (IF) and ISH on cryosections was performed as described previously For histology embryos were removed from the uterine horns, dissected out of the decidua and fixed in Bouin's fixative (Sigma). After dehydration in ethanol, embryos were embedded in paraffin wax, sectioned at 8 \u00b5m thickness and stained with hematoxylin and eosin (H & E).Alcian blue staining was performed according to standard protocols 2 on Transwell-Col filters (Fisher Scientific) containing 1.5 ml BGJb media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml vitamin C (Sigma) and 2 \u00b5M 4-OH tamoxifen (Sigma). For lung bud culture, lung buds were dissected at E11.5 in phosphate buffered saline (PBS) with 0.1% bovine serum albumin (Sigma) and cultured in media containing DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen) and 1 \u00b5M 4-OH tamoxifen on 3.0 \u00b5m filters (Millipore) or 0.4 \u00b5m Transwell filters (Fisher Scientific). Similar results were obtained using both types of filter; results reported are for experiments with Millipore filters. Where specified, Fgf10 (R&D) was added after 1 day of culture at a concentration of 500 ng/ml. In some experiments heparin beads coated with Fgf10 (100 \u00b5g/ml) or BSA (100 \u00b5g/ml) were placed near the branching tips of the explants after 1 day of culture. Transwell filters were used for the bead experiments.Foregut culture was carried out as described previously E-cadherin RNA probe using ISH. In case of whole mount lungs, lobes were separated and photographed to count the number of branching tips. The cultured lungs were photographed and the branching tips were counted. For some experiments, after E-cadherin ISH, lungs were post fixed in 4% PFA, washed with PBT, dehydrated in 100% methanol, cleared in BABB and then photographed.Lungs were stained with either E-cadherin antibody using IHC or Figure S1flTbx4 and flTbx5. Females carrying embryos with flTbx4, flTbx5 and CreER alleles were injected with either 8 mg tamoxifen at E9.0 and dissected at E12.5  or 7 mg tamoxifen at E8.5 and dissected at E13.5 . fl/flTbx4 was completely excised to mutant \u2212/\u2212Tbx4 at both doses and times  whereas the single conditional allele of fl/+Tbx5 was incompletely excised by injection at E9.0 (E) but completely excised by injection at E8.5 (F). In addition when lungs with the genotype fl/flTbx4; fl/flTbx5; CreER were treated with 1 \u00b5m tamoxifen in culture, the Tbx4 locus was nearly completely excised at the end of 1 day of culture (C) but the Tbx5 locus was only partially excised (G). At the end of a 4 day culture both Tbx4 (D) and Tbx5 (H) loci achieved virtually complete excision. fl, floxed conditional PCR band; WT, wild type PCR band; Mut, excised PCR band.Analysis of excision efficiency of (TIF)Click here for additional data file.Figure S2Tbx4 and Tbx5 does not affect alveolar differentiation.  T1\u03b1 IHC on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. Nuclear fast red was used as a counter stain. A\u2032 and B\u2032 are higher magnification views of boxed regions in A and B.  Prosurfactant protein C (Pro-SPC) IF on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. DAPI was used to stain the nuclei. Scale bars represent 100 \u00b5m.Loss of (TIF)Click here for additional data file.Figure S3Tbx4 and Tbx5 has multiple affects on branching morphogenesis.  H & E sections of lung-specific Tbx4 heterozygous;Tbx5 null lungs (B) show lack of separation of the cranial (cr), medial (m) and caudal (cd) lobes in the right lung as compared to control lungs (A). Black arrowheads point to the space created between the lobes in the control lungs (A) and to the corresponding regions of the mutant lungs (B).  Control lungs stained for E-cadherin at E13 show greater outgrowth (yellow parentheses) of lateral branches (C) than the lung-specific Tbx4 heterozygous;Tbx5 null lungs (D). Scale bars represent 100 \u00b5m.Loss of (TIF)Click here for additional data file."}
+{"text": "Looptail mutant mouse, which harbors a missense mutation in the vangl2 gene, have been essential for studies of planar polarity and linking the function of the core planar cell polarity proteins to other developmental signals. Originally described as having dominant phenotypic traits, the molecular interactions underlying the Looptail mutant phenotype are unclear because Vangl2 protein levels are significantly reduced or absent from mutant tissues. Here we introduce a vangl2 knockout mouse and directly compare the severity of the knockout and Looptail mutant phenotypes by intercrossing the two lines and assaying the planar polarity of inner ear hair cells. Overall the vangl2 knockout phenotype is milder than the phenotype of compound mutants carrying both the Looptail and vangl2 knockout alleles. In compound mutants a greater number of hair cells are affected and changes in the orientation of individual hair cells are greater when quantified. We further demonstrate in a heterologous cell system that the protein encoded by the Looptail mutation (Vangl2S464N) disrupts delivery of Vangl1 and Vangl2 proteins to the cell surface as a result of oligomer formation between Vangl1 and Vangl2S464N, or Vangl2 and Vangl2S464N, coupled to the intracellular retention of Vangl2S464N. As a result, Vangl1 protein is missing from the apical cell surface of vestibular hair cells in Looptail mutants, but is retained at the apical cell surface of hair cells in vangl2 knockouts. Similarly the distribution of Prickle-like2, a putative Vangl2 interacting protein, is differentially affected in the two mutant lines. In summary, we provide evidence for a direct physical interaction between Vangl1 and Vangl2 through a combination of in vitro and in vivo approaches and propose that this interaction underlies the dominant phenotypic traits associated with the Looptail mutation.Experiments utilizing the  Drosophila wing. Taken together, several recent studies indicate that Fz and Dvl proteins form a complex on one side of the hair cell that is opposite to Vangl and Pk proteins on the other Drosophila, vertebrate PCP proteins also coordinate planar polarity between cells so that, as in the case of hair cells, the stereocilia bundles of neighboring cells are oriented in the same direction.Planar polarity is the polarized organization of cells and cellular structures within the plane of a tissue, perpendicular to the apical-basolateral cell axis vangl2 (Entrez Gene ID: 93840) mutant line Looptail (Lp)Lp is a missense mutation of vangl2 resulting in a Serine to Asparagine substitution at amino acid position 464, which is located in the cytoplasmic domain of the protein. In Lp/Lpvangl2 mutants, hair cell stereocilia are properly formed and polarized, yet individual hair cells are frequently misoriented relative to neighboring cells in the cochlea and utricle vangl1 (Entrez Gene ID: 229658) and vangl2 demonstrating functional redundancy of the two homologous genes Lp/Lpvangl2 mutants have craniorachischisis, which is a severe neural tube defect (NTD). The iconic looped tail of the Lp line is due to a milder neural tube phenotype in heterozygous mice. Craniorachischisis is a class of NTD specifically resulting from disrupted planar polarity during convergent extension of the embryo prior to neurulation, and is a common feature of vertebrate PCP mutants. Other mouse lines that develop craniorachischisis and hair cell planar polarity defects include fz3/6 double knockouts CELSR mutants spin cycle and crashscribble mutants ptk7 knockout mice vangl1 and vangl2 have been linked to neural tube defects in humans An early demonstration the functional significance of planar polarity and PCP signaling mechanisms in hair cells came from analysis of the Lp mouse has served as an important animal model for studying vertebrate planar polarity Lp line classified the Lp mutation as having a partially penetrant and dominant phenotype ex vivo, heterozygous tissue develops pathologic characteristics similar to mutant tissue Lp mutation was mapped to vangl2S464N protein stability and expression in Lp/Lpvangl2 mutants Lp as a null allele with a heterozygous phenotype resulting from haploinsufficiency. The Lp line has implicated PCP signaling in a number of developmental events such as axon pathfinding not originally associated with planar polarity Lp and other mutant lines suggest that other signaling pathways, including those functional in cilia, incorporate Vangl2 Lp mutation.The Lp mutation inhibits Vangl2 function by disrupting Vangl2 protein trafficking through the endoplasmic reticulum (ER). Consistent with this hypothesis, mutations in sec24b, which encodes an ER transport protein that is required for Vangl2 movement to the plasma membrane, result in craniorachischisis similar to LpS464N is not loaded into ER vesicles indicating that the mutated region of the Vangl2 cytoplasmic domain may be required for sorting by Sec24b. Furthermore, a second vangl2 point mutant (m1Jus) fails to enter ER vesicles m1Jus/m1Jusvangl2 mice have craniorachischisis similar to Lp/Lpvangl2in vitroLp/WTvangl2 mice is due to haploinsufficiency or if it is a partially penetrant, dominant phenotype as described in the original characterization of the line.One explanation is that the vangl2 knockout mice is more severe than the Lp and m1Jus mutations because remaining elements of vangl2 are not sufficient to encode a membrane spanning protein. Using the sensory hair cells of the inner ear, we compared the mutant phenotype of vangl2 knockout and Lp mutant mice to establish the phenotypic nature of the Lp mutation during hair cell development.We have generated a complementary mouse line in which a single exon encoding the Vangl2 transmembrane domains is disrupted by LoxP site addition and subsequent excision by Cre recombinase. At the chromosomal level, the mutation in these vangl2 knockout (KO) mouse was generated and compared to the established Looptail (Lp) mouse line to determine the effect of the S464N substitution on Vangl2 function. The vangl2 gene was modified by homologous recombination in ES cells to introduce tandem LoxP sites flanking exon 4, which encodes the four Vangl2 transmembrane domains  confirms a loss of Vangl2 protein in \u0394TMs/\u0394TMsvangl2 embryos . Therefore \u0394TMsvangl2 is a complete null allele.To further evaluate Vangl2 function during sensory hair cell development, a  domains . Accurat domains . Followi domains . Since t embryos . A Vangl\u0394TMsVangl2 mice were backcrossed to C57Bl6 for at least 5 generations before experimental intercrosses. On this genetic background the majority of \u0394TMs/\u0394TMsvangl2 mutants exhibited craniorachischisis, a severe NTD  present only with spina bifida, a milder NTD restricted to the posterior neural tube adjacent to the tail (\u0394TMs/WTvangl2 mice (11/99) had the tail loops or kinks that are characteristic of the Lp line. Tail phenotypes were never observed in \u0394TMs/WTvangl2 mice backcrossed for a minimum of 5 generations to the FVB (0/66 heterozygotes) or strain A/J (0/15 heterozygotes) inbred lines. Finally in some \u0394TMs/\u0394TMsvangl2 KOs, incomplete or failed eyelid closure was observed that was similar in appearance to that described for other PCP mutants \u0394TMs/\u0394TMsvangl2 mice than in Lp/Lpvangl2 mutants.vere NTD  that is the tail . Similar\u0394TMs/\u0394TMsvangl2 phenotype was assayed in greater detail in the inner ear because the sensory hair cells have distinct planar polarity that is readily visualized and quantified for direct comparison with the Lp mutant phenotype. The apical surface of a hair cell extends a bundle of stereocilia that is organized in a staircase pattern with the tallest stereocilia adjacent to single kinocilium. Planar polarity is apparent in the shared stereocilia\u2236kinocilium polarity of neighboring cells  of individual hair cells from all embryos using circular histograms  were grouped and analyzed together as the middle rows  Together these experiments suggest that the presence of the Vangl2\u0394TMs/Lpvangl2 mutants than \u0394TMs/\u0394TMsvangl2 KOs is that altered Vangl2S464N trafficking disrupts the distribution of other polarity molecules. A likely candidate is Vangl1 because Vangl1 expression overlaps with Vangl2, and enhanced planar polarity defects are seen in mice lacking both vangl1 and vangl2DrosophilaS464N or Vangl2D255E may disrupt Vangl1 or Vangl2 delivery to the plasma membrane. This hypothesis was tested using a heterologous system in which hemagluttinin (HA) tagged Vangl2 constructs were co-expressed with EGFP-tagged Vangl1 or Vangl2. Specifically these experiments utilized 3XHA-tagged Vangl2, 3XHA-Vangl2S464N and 3XHA-Vangl2D255E constructs characterized by Merte et al. One explanation for the stronger phenotype that occurs in S464N or 3XHA-Vangl2D255E constructs were found predominantly in cytoplasmic compartments , the Vangl1 antibody appears specific for Vangl1 because there are no differences in Western blot analyses between protein lysates collected from WT and \u0394TMs/\u0394TMsvangl2 embryos  was assayed in ir cells , arrows.ir cells . Asymmetely lost  similar vangl2 knockout line and intercrossing those mice with Lp mutants, we have demonstrated the dominant nature of the Lp mutant phenotype during inner ear development. This was completed through a detailed quantification and comparison of the planar polarity phenotypes of hair cells in \u0394TMs/Lpvangl2 and \u0394TMs/\u0394TMsvangl2 mice. In addition we propose a mechanism for the dominant effect of the Lp mutation in which disrupted trafficking of Vangl2S464N alters the distribution of Vangl1, Vangl2 and Pk2. Together these findings will guide interpretation of studies employing the Looptail line, particularly when Looptail mice are used for molecular dissections of the PCP signaling pathway.By generating a novel S464N is due to disrupted trafficking of the mutant protein and its retention in the ER S464N with Vangl1 in a heterologous system also prevents Vangl1 delivery to the cell surface. Since vangl1 and vangl2 regulate planar polarity in many tissues including the inner ear \u0394TMs/\u0394TMsvangl2 vestibular hair cells that maintain apical localization of Vangl1 than in Lp/Lpvangl2 hair cells where apical Vangl1 is lost. We also demonstrate that Vangl2 forms oligomers with Vangl1 and Vangl2 in heterologous cells. This is similar to findings from Drosophila where Van Gogh oligomerizes in vivoS464N protein encoded by the Lp mutation exerts dominant effect on planar polarity by inhibiting the membrane trafficking and localization of Vangl1 and Vangl2, likely leading to their degradation.At a molecular level, the decreased protein stability and cell-surface presentation of Vangl2Lp/WTvangl2 heterozygotes if the presence of Vangl2S464N reduces but does not eliminate all Vangl1/2 protein from the cell surface. By comparison the uterine epithelium of the female reproductive track (FRT) is more sensitive to the Lp mutation than the neural tube. In a recent study where embryonic FRT tissues were cultured in vitro, similar developmental deficits were observed explants from Lp/WTvangl2 and Lp/Lpvangl2 embryos, further demonstrating the dominant effect of LpLp/WTvangl2 heterozygotes that was restricted to OHC3 and was only present in the less developed apical turns  and coding sequence exons located within the homologous arms were sequenced to check for PCR induced mutations. Following electroporation into the TC1 ES cell line and positive selection with G418, homologous recombination was validated in surviving clones by PCR and Southern blot . The vector contained a 4.2 kb 5\u2032 arm that was modified by inserting LoxP and Hind3 sequences into a unique Xmn1 restriction site located in the intron upstream of ern blot . A singlACTB-FlpE) to remove the NeoR gene. The resulting allele (LoxP\u200avangl2) was not viable when homozygosed because the positions of the remaining LoxP sites disrupted vangl2 expression. As a result, LoxPvangl2 was crossed to transgenic mice ubiquitously expressing Cre recombinase (ACTB-Cre) to permanently delete exon 4 in the germ line and produce the \u0394TMsvangl2 knockout line.FlpE recombinase . C57Bl6 and FVB mice were purchased from Charles River Laboratories  and the ACTB-FLPe mice were obtained from S. Dymecki .For general colony maintenance, \u0394TMsVangl2 mice were genotyped using a common 5\u2032 primer (5\u2032ATCACCTCACTTGGCTGGAATAGATG 3\u2032) paired with 3\u2032 primers that were either KO-specific (5\u2032GAAGTTATAAGCTTTGTTCCAG 3\u2032) or WT-specific (5\u2032GGCGAATGGGAGAAAGGCAGAC 3\u2032). Following \u0394TMsvangl2 and Lp experimental intercross, the Lp mutation was genotyped by PCR amplification with primers flanking the mutation followed by DNA purification and sequencing of the amplified product. Lp genotyping primers are (5\u2032ATATTTGGCTGCTGGACCCACCATCC3\u2032) and (5\u2032TGCAGCCGCATGACGAACTTATGTGA3\u2032).Immunofluorescent labeling of auditory and vestibular hair cells was completed using E18.5 inner ears fixed for 2 hours in a solution of 4% paraformaldehyde prepared in Sorenson's phosphate buffer (pH7.4). Utricles and cochleae were subsequently removed, dissected to expose the surface of the sensory epithelia, permeabilized and blocked using blocking solution  supplemented with Triton X-100 to 0.5%. Primary antibodies and phalloidin Alexa488 (Invitrogen A12379) were diluted in blocking solution supplemented with Tween-20 to 0.1%, and incubated with the tissue overnight at 4\u00b0C. Tissue was washed thoroughly with PBS-T  followed by incubation with species-specific, Alexa Fluor (Invitrogen) or DyLight (Jackson ImmunoResearch) conjugated secondary antibodies. Tissue was subsequently washed with PBS-T, mounted using Fluoro-Gel (Electron Microscopy Sciences), and imaged using a Zeiss LSM510 confocal microscope. Image frames were limited to 1.5 microns in the Y-dimension and a stack of images 3\u20136 microns containing the stereocilia and apical surface of the hair cells was collected. A single image was generated by Z-projection using maximum or averaged pixel intensities. For preparations in which the striola region was marked using Gata3 antibodies, a second stack positioned deeper in the tissue was necessary to image Gata3 positive nuclei. The following commercial antibodies were used in this study: Actin (Millipore mAB1501), GFP (Invitrogen A11122), Gata3 (R&D AF2605), HA (Covance MMS-101P),Acetylated Tubulin (Sigma T7451), Vangl1 (Sigma A36455), Vangl2 (Santa Cruz SC46561), Oncomodulin (Santa Cruz SC7446), Pericentrin (Covance PRB432C). The Pk2 antibody was a gift from M. Scott and J. Axelrod (Stanford Univ.) and has been previously characterized For auditory hair cell analyses the organ of Corti was imaged by confocal microscopy at three positions corresponding to 25%, 50% and 75% of the length of the cochlea measured from the base. The orientation of individual hair cells was measured using the ImageJ (NIH) angle measurement tool. The short arm of each angle was drawn from the pericentrin labeled kinocilium and across the center of the hair cell. The long arm of each angle was drawn parallel to three adjacent hair cells within the same row. This yields a measurement range of 0 to 180 degrees in which a perfectly aligned hair cell has a raw measurement of 90 degrees. For those cells determined to have reversed bundles  the orientation was calculated with the formula x\u200a=\u200a(360-y) where x is orientation and y is the measured angle. This calculation and additional analyses were completed using Microsoft Excel. These absolute measurements of auditory hair cell orientation were assembled as a circular histograms using Oriana circular graphing software (Kovach Computing Services). Planar polarity phenotypes were further quantified by averaging the mean absolute deviation of hair cell polarities from an arbitrary reference (defined as 0\u00b0) for each animal as illustrated in For vestibular hair cell analyses the utricular maculae was imaged by confocal microscopy at two positions spanning the Gata3-positive or Oncomodulin-positive striola and images were combined based upon regions of overlap. As outlined in \u0394TMs/\u0394TMsvangl2 KOs, only KOs with craniorachischisis were included in measurements of hair cell planar polarity. All \u0394TMs/Lpvangl2 and Lp/Lpvangl2 mutants collected had craniorachischisis. Hair cells in which the bundle polarity or pericentrin labeling could not be visualized were not included in the auditory or vestibular analyses.See 7 cells with 30 ug plasmid DNA  in a 2 mm gap electroporation cuvette, followed by electroporation at 260 V with 950 uF capacitance and 25 Ohm resistance using a BTX ECM630 electroporation box. MDCK cells were plated into single wells of a Lab-Tek II 8-well Chamber Slide (Thermo Scientific) and incubated 72 hours to allow adherence junctions to form at cell boundaries before the distribution of EGFP-tagged proteins was assayed by immunofluorescent labeling. HEK293 cells were transfected using Lipofectamine reagent as per the manufacturer's recommendation, cultured for 24\u201348 hours and harvested for Western blot analysis. For these experiments a plasmid expressing EGFP-Vangl1 was generated by PCR amplification from mouse cDNA and cloned into the pEGFP-C1 vector (Invitrogen) to generate an amino-terminus EGFP fusion. EGFP-Vangl2 MDCK cells (SIGMA 84121903) were grown in EMEM supplemented with 2 mM L-Glutamine, 1\u00d7 non-essential amino acids, 10% FBS and 1\u00d7 Penn/Strep. HEK293 cells (American Type Culture Collection ATCC-CRL-1573) were grown in DMEM, 10% FBS and 1\u00d7 Penn/Strep. MDCK cells were transfected via electroporation, by combining 1\u00d710For labeling, MDCK cells were fixed with 4% paraformaldehyde prepared in PBS, permeabilized and blocked as described previously. Cells were immunolabeled with anti-HA and anti-GFP antibodies diluted in 1% BSA/PBS followed by Alexa Fluor conjugated secondary antibodies, and then examined by fluorescent microscopy using a Nikon E600 compound microscope. For protein localization assays only cells expressing both HA-tagged and EGFP-tagged constructs were evaluated, and the distribution of EGFP-tagged proteins were scored as membrane associated, cytoplasmic or present in both locations.Protein lysates were prepared from tissues, MDCK or HEK293 cells using a lysis buffer consisting of 25 mM Tris pH 7.4, 1% Triton X-100, 50 mM NaCl and 1\u00d7 protease inhibitor cocktail (SIGMA P8340) and quantified by UV absorption at 280 nm using a NanoDrop. Following SDS-PAGE electrophoresis proteins were transferred to nitrocellulose filters and blotted with standard Western blot techniques using anti-GFP or anti-HA antibodies followed by chemiluminescent detection using BioRad Immun-Star HRP substrate. Immunoprecipitations were completed using Nynal protein G beads (Invitrogen) bound to rabbit anti-GFP or mouse anti-HA antibodies and combined with protein lysates from electroporated MDCK cells. Prior to immunoprecipitation, levels of recombinant protein expression were determined by Western blot analysis and equivalent amounts of recombinant protein were used from each electroporation condition. Target antigens were absorbed at 4\u00b0C, and washed using the manufacturers recommended protocols and solutions, then eluted from the beads using Laemmli 2\u00d7 Gel-loading Buffer with DTT prior to denaturing SDS-PAGE and conventional Western blot analysis.Figure S1Gata3 and Oncomodulin immunolabeling marks the position of the striola in developing mouse utricle. (A) The transcription factor Gata3 (red) is expressed by multiple cells types located in the striola region of the developing utricle and Gata3 immunolabeling can be used to visualize this region. (B) The calcium binding protein Oncomodulin (red) is expressed exclusively by type1 hair cells located in the striola, and Oncomodulin immunolabeling can also be used to visualize this region. (A\u2013B) Phalloidin (green) was used to assay stereocilia bundle orientation (arrows) and map the position of the line of polarity reversal . In mouse, the LPR is located along the lateral border of the striola.(TIF)Click here for additional data file.Figure S2Ectopic expression of tagged-Vangl1 or Vangl2 proteins does not disrupt cell junctions between adjacent MDCK cells. (A\u2013E) The proper formation of adherens junctions between adjacent MDCK cells was determined by immunofluorescent labeling of endogenous E-cadherin (red) following electroporation with EGFP-Vangl1 , EGFP-Vangl2 , 3XHA-Vangl2 , 3XHA Vangl2D255E  or 3XHA Vangl2S464N  constructs. Asters mark transfected cells. Consistent with experiments utilizing E-Cadherin-EGFP Click here for additional data file.Figure S3Specificity of anti-Vangl1 antibody evaluated by Western blot. (A) Western blot against protein lysates from embryonic tissues demonstrate that the anti-Vangl1 antibody does not cross react with endogenous Vangl2 protein. Blots show a prominent band at the predicted 70 Kd mass in Wt and \u0394TMs/WTvangl2 lysates and the labeling pattern is not altered in \u0394TMs/\u0394TMsvangl2 mutant tissue. Blots for Tubulin were used as a loading control. (B) Western blot against cell lysates from HEK293 cells transfected with EGFP-Vangl1 or EGFP-Vangl2 reveal that the anti-Vangl1 antibody can cross-react with recombinant Vangl2 protein when over-expressed. Serial dilutions of transfected cell lysates show the relative affinity of the anti-Vangl1 antibody and demonstrate that it can detect a 10-fold lower concentration of EGFP-Vangl1 than EGFP-Vangl2. The amount of total protein in each lysate was determined by UV absorption measured with a NanoDrop and equivalent amounts were loaded into each lane. The amount of total protein lysate per lane is indicated.(TIF)Click here for additional data file.Table S1Sample size summary for planar polarity phenotypic analysis. The number of experimental and control animals used for quantification of the averaged mean deviation of bundle orientations in vestibular assays and auditory assays completed at each of the three positions along the length of the cochlea. Different numbers of specimen were available at each location because some were excluded based upon dissection or labeling artifacts.(PDF)Click here for additional data file."}
+{"text": "Young women with autoinflammatory diseases on long term IL-1 blockade are increasingly asking about the feasibility, safety and outcomes of pregnancy but few data are available. The FDA classes anakinra as pregnancy risk grade B and rilonacept and canakinumab as grade C. The manufacturers advise that there are no data on the outcome of pregnancy or on excretion into breast milk and that anakinra and canakinumab should only be taken if the benefits outweigh risk; for rilonacept the advice is to avoid in pregnancy. The literature contains only 3 reported cases of use of anakinra in pregnancy \u2013 all in adult onset Stills disease (AOSD) and with successful outcomes.To assess pregnancy outcomes in women who had received anti IL-1 therapies in pregnancy.We indentified women who have been exposed to anti IL-1 agents in completed or planned to complete pregnancies under our care. Data were collected on medication, pregnancy outcome, breast feeding and development.7 cases were identified; 5 completed, 1 first and 1 second trimester pregnancies. The underlying diseases were: 3 CAPS, 1 TRAPS, 1 FMF, 1 idiopathic pericarditis and 1 AOSD. 3 completed pregnancies  were on anakinra from preconception throughout the pregnancy, the 2 current pregnancies (CAPS) were on canakinumab pre conception, 1 switched to anakinra 8 weeks pre conception, the other stopped canakinumab 8 weeks after conception and currently (12 weeks later) is on no treatment. The patient with FMF also had multiple sclerosis with presumed prolonged febrile myalgia refractory to colchicine for which she received 12 weeks of anakinra from 22 weeks. The AOSD patient received anakinra and prednisolone from 22 to 33 weeks. Median maternal age was 30 years (25-38), all were first pregnancies. The 5 completed pregnancies resulted in 5 healthy boys; median gestation 38 weeks (35 to 41). One baby was delivered by caesarean section at 36 weeks for vaginal bleeding (FMF), the others were vaginal deliveries (2 induced); median birth weight 2.59 kg (2.02-3.94), 1 minute APGAR score was 8 in 1 case and 9 in the rest. All were normal on neonatal checks, one had evidence of unilateral reduced hearing at 6 weeks. One was breast fed, the others bottle fed. Follow up data is available on 3 beyond 6 months and their development remains normal.These 5 successful pregnancies more than double the number of known outcomes in anakinra treated mothers and provide reassurance to physicians caring for young women. Nonetheless the numbers remain very small and each pregnancy should be assessed and the risks and benefits of continued therapy individually discussed with the potential parents.None declared"}
+{"text": "The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis. Changes in cytoskeletal architecture and cell-cell adhesion are often observed in cells undergoing neoplastic transformation. Desmosomes are adherens type junctions that are required for cell-cell adhesion, especially in tissues that experience mechanical stress and anchor intermediate filaments (IF\u2019s) leading to the generation of a tissue wide IF network (reviewed in The aberrant over-expression of K8 and K18 has been observed in a number of squamous cell carcinomas irrespective of their origin Metastasis in colon cancer often correlates with an increased expression of the Phosphatase of Regenerating Liver -3 (PRL-3) Plakophilin3 (PKP3) is a desmosomal plaque protein that belongs to the p120 catenin sub family of the armadillo family of proteins and is found in desmosomes in most epithelial tissues with the exception of hepatocytes Previous work from this laboratory has demonstrated that PKP3 loss in HCT116 cells leads to increased neoplastic progression and metastasis The two clones, S9 and S10, are generated using two different shRNA\u2019s suggesting that the phenotypes observed are probably not due to off-target effects of the shRNA To determine if an increase in RNA levels led to the increase in K8 protein in the PKP3 knockdown clones, a reverse transcriptase coupled Real Time PCR analysis was performed. The mRNA levels of K8 and K18 in the PKP3 knockdown clones were not significantly altered as compared to the vector control (pTU6) , therefoTwo major sites of phosphorylation, S73 and S431, have been reported in K8 To further demonstrate that the change in migration and stability of K8 was due to changes in phosphorylation in K8, two phospho-site mutants (S73A and S431A) It has been reported previously that an increase in PRL3 levels leads to a decrease in K8 phosphorylation and an increase in K8 levels To determine whether inhibition of PRL3 could cause a decrease in the levels of K8 in the PKP3 knockdown clones, the cells were treated with either the vehicle control or increasing concentrations of PRL3 inhibitor. The levels of K8 decreased upon inhibition of PRL3 in the vector control and PKP3 knockdown clones suggesting that an increase in phosphorylation of K8 could account for the decrease in K8 stability . HoweverTo determine if there was a change in PRL3 localization in the cell or increased PRL3 localization on K8 filaments upon PKP3 knockdown, the vector control and knockdown cells were stained with antibodies to PRL3. As shown in To determine whether PRL3 shows an increased localization to K8 filaments upon PKP3 knockdown, HCT116 cells were transfected with a GFP-tagged PRL3 construct and stained with antibodies to PRL3. As shown in To determine the effects of K8 phosphorylation on filament formation, the amount of K8 in the soluble and insoluble fractions was measured in the vector control and PKP3 knockdown clones. Keratin solubility assays demonstrated that all the K8 was present in the insoluble fraction in both the vector control and PKP3 knockdown clones, even when a 100 \u00b5g of the soluble fraction was loaded . FurtherTo determine whether the elevated K8 levels were required for transformation upon PKP3 loss, we inhibited K8 expression using RNA interference using previously described shRNA constructs The PKP3 knockdown cells show a decrease in cell-cell adhesion and desmosome size and an increase in cell migration An increase in cell migration is often accompanied by an increase in actin dynamics and the formation of actin dependent structures such as lamellipodia and filopodia  showed high levels of K8 as compared to primary tumors derived from the double knockdown clones (8.21 and 8.28) . PrimaryLoss of the desmosomal plaque protein plakophilin3, leads to an increase in tumor progression and metastasis PKP3 loss leads to an increase in metastasis to the lungs . K8 lossAnother possible explanation for why an increase in K8 levels leads to increased metastasis might be the effect K8 has on cell migration. Loss of K8 in the PKP3 knockdown clones results in an inhibition of migration, reversing the phenotype observed upon PKP3 loss The GFP tagged phosphosite mutants show an increase in stability in comparison to the GFP tagged WTK8. These results suggest that the increase in stability observed upon PKP3 knockdown is not due to an increase in translation, given that the WT and mutant proteins are being expressed from a heterologous promoter with different 5\u2032 and 3\u2032 untranslated regions from the endogenous K8 gene. This is important because plakophilin family members, including PKP3, have been shown to regulate translation PRL3 has been reported to dephosphorylate K8 at both S73 and S431 It has been reported previously that phosphorylation of keratins leads to an increase in solubility and filament network disassembly The requirement for an intact filament network for transformation is supported by the data that knockdown of K18 does not phenocopy K8 knockdown, which might be due to the fact that another type I keratin is present in these cells . The incThe results described herein suggest that PKP3 loss leads to alterations in phosphorylation on K8, due to an increase in PRL3 levels. These result in increased K8 levels which promote increased migration, transformation and metastasis. Loss of K8 in the PKP3 knockdown clones leads to a reversal of these phenotypes, suggesting that an increase in K8 levels is required for the neoplastic progression and metastasis induced upon PKP3 loss. These results in conjunction with our data on the levels of phosphorylated keratin in human oral squamous cell carcinoma ad libitum. Mice were housed in the Individually Ventilated Cage (IVC) system  provided with autoclaved rice husk bedding material available locally. Protocols for the experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the Advanced Centre for Treatment Reserach and Education in Cancer (ACTREC). The title of the project is \u201cMechanisms underlying tumor progression upon PKP3 loss in epithelial cells.\u201d The project approval number is 13/2007 and approval was granted on the 27th of July, 2007.Maintenance of the animal facility is as per the national guidelines provided by the Committee for the Purpose of Control and Supervision of the Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The animals were housed in a controlled environment with the temperature and relative humidity being maintained at 23\u00b12\u00b0C and 40\u201370% respectively and a day night cycle of 12 hrs each . The animals were received an autoclaved balanced diet prepared in-house as per the standard formula and sterile water The cloning of K8 shRNA constructs and the shRNA resistant K8 (GFP K8 Res) has been described elsewhere HCT116 cells, an epithelial cell line derived from a human colorectal carcinoma, were obtained from Dr. Bert Vogelstein  and ATCCThe high salt extraction for keratins was performed as previously described The primary antibodies for PKP3 , \u03b2 Actin , K8 , K18 , phosphospecific antibodies for K8 Serine 73  or Serine 431 , p38 , rac  rhoA  and PRL3 (Abcam 1\u2236500) were used for Western blot analysis. Respective secondary antibodies were used at a dilution of 1\u22361000 (Invitrogen) or 1\u22365000 (Pierce). Protein samples were resolved on a polyacrylamide gel by SDS PAGE and then transferred to a nitro cellulose or PVDF membranes and processed for Western blot analysis as described The HCT116-derived PKP3 knockdown clone S9 and S10 and the vector control were grown on glass bottom plates and co-transfected with constructs expressing YFP K18 and CFP K8. 24 hours post transfection the cells were fed with DMEM lacking phenol red and placed on ice for 10 minutes prior to FRET to minimize mobility of the cells. FRET measurements were done using the acceptor photobleaching method in live cells efficiency\u200a=\u200a(Dpost\u2212Bpost)\u2212(Dpre\u2212Bpre)/(Dpost\u2212Bpost).FRET The nomenclature and equations for FRET calculations and the FRET protocol was obtained from the Centre for Optical Instrumentation Laboratory, Wellcome Trust Center for Cell Biology, University of Edinburgh.Wound healing assays were performed as previously described Staining for K8 was performed as described The knockdown clones were trypsinized and counted. 2,500 cells were plated in 0.4% soft agarose and the cells were maintained in the presence of the relevant antibiotic as previously described 6 cells of the HCT116 derived PKP3 knockdown or K8 PKP3 double knockdown clones were resuspended in DMEM medium without serum and injected subcutaneously in the dorsal flank of 6\u20138 weeks old NMRI Nude (Nu/Nu) 3) was calculated by the formula \u00bd LV2 where L is the largest dimension and V its perpendicular dimension, as previously reported Nude mice experiments were performed as described Four weeks after subcutaneous injection, the mice were sacrificed and the tumor and lung tissues were fixed in 10% formaldehyde (SIGMA) overnight and processed for histology. Five micron sections of paraffin embedded tissue were prepared and hematoxylin/eosin staining and immunohistochemical staining was performed according to standard methods Genomic DNA was purified from paraffin sections as previously described Total RNA was extracted from the HCT116 derived vector control (pTU6) and PKP3 knockdown clones  using Qiagen RNeasy kit and reverse transcribed using the Omniscript RT kit (Qiagen) as per the manufacturers instructions. PCR reactions for PKP3, PRL3 and GAPDH were performed on the cDNA using Taq polymerase (NEB) as per the manufacturers instructions. Real time PCR analysis using TaqMan gene expression assays (Applied Biosystems) were performed using gene specific probes for K8 and K18 from Applied Biosystems. A similar assay for GAPDH was performed to serve as an internal control for normalization. Fold changes in expression of K8 and K18 in the plakophilin3 knockdown clones were calculated relative to the expression levels in the pTU6 vector control cells as described previously Protein extracts were resolved on a ReadyStrip IPG strip (BioRad) pH range 5\u20138 according to the instructions supplied by the manufacturer. The proteins were then resolved in the second dimension on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8.To detect the interaction between K18 and plakophilin3, HCT-116 cells were lysed with EBC buffer  and lysates were immunoprecipitated with anti K18 antibody. The immunoprecipitates were washed with NET-N buffer  for 3 times and run on SDS polyacrylamide gel electrophoresis followed by Western blots with antibodies to PKP3 and K18. The levels of O-linked N-acetylglucosamine (O-GlcNAc) on K8 were determined as previously described t test.Error bars represent standard error. Statistical analysis was performed using two tailed students Figure S1K8 and K18 organization in PKP3 knockdown cells. A. K18 forms a complex with PKP3 in HCT116 cells. EBC extracts prepared from HCT116 cells were incubated with a control antibody (IgG) or antibodies to K18 (K18). The reactions were resolved on SDS-PAGE gels and Western blots performed with antibodies to PKP3 and K18. (WCE\u200a=\u200aWhole cell extract. IP\u200a=\u200aImmunoprecipitation) B. K8 and K18 levels are altered upon PKP3 knockdown. High salt extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) and the PKP3 and K8 double knockdown clone (8.21) were resolved on SDS-PAGE gels and the gel stained with coomassie blue to identify keratin bands. C. K8 and K18 form filaments in the PKP3 knockdown clones. The PKP3 knockdown clones or the vector control, were stained with antibodies against K8 or K18 . D. Vimentin levels are not altered upon PKP3 knockdown. Protein extracts from the vector control (pTU6), the PKP3 knockdown clones  and the double knockdown clones  were resolved on SDS-PAGE gels followed by Western blotting with antibodies to vimentin. None of the clones showed the presence of vimentin. AW13516 cells served as a positive control and a Western blot for actin served as a loading control.(TIF)Click here for additional data file.Figure S2Post-translational modifications on K8 and K18 upon PKP3 knockdown. A. Migration of K18 in two-dimensional gel electrophoresis. Protein extracts from the vector control and PKP3 knockdown clones were resolved in two dimensional gels followed by Western blotting with antibodies to K18. Note that the levels of K18 are higher in the PKP3 knockdown clones but no difference in migration in two-dimensional gel electrophoresis was observed for K18. B. Densitometric analysis of K8 and phospho-K8 expression in the vector control and PKP3 knockdown clones. Expression of K8, phosphor-S73 and phosphor-S431 was normalized to that of \u03b2-actin and densitometry performed on Western blots using ImageJ software. The values for these are shown in the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) as indicated. C. Testing the specificity of K8 phosphospecific antibodies. GFP tagged WTK8 or the phosphomutants (S73A and S431A) were transfected into HCT116 cells. 48 hours post transfection, protein extracts were resolved on SDS PAGE gels followed by Western blotting with either antibodies that recognize K8 or the phosphor-specific antibodies. Note that the phospho-specific antibodies do not recognize the phosphor-mutant constructs. D. p38 levels are not altered in the PKP3 knockdown clones. Protein extracts from the PKP3 knockdown clones or the vector control were resolved on SDSPAGE gels followed by Western blotting with antibodies to p38. Western blots for 14-3-3\u03b3 were performed as a loading control. E. O-linked glycosylation on K8 is not altered in the PKP3 knockdown clones. Protein extracts from the PKP3 knockdown clones or the vector control were immunoprecipitated with antibodies to K8 followed by Western blots with antibodies recognizing O-GlcNAc glycosylation or K8.(TIF)Click here for additional data file.Figure S3PRL3 localization in PKP3 knockdown cells. A. PRL3 localization in PKP3 knockdown cells. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with antibodies to PRL3 and imaged by confocal microscopy. Note that PRL3 levels increase in the PKP3 knockdown clones with an increase in perinuclear localization and a slight increase in staining at the cell border. . B. GFP-PRL3 colocalizes with endogenous PRL3. HCT116 cells were transfected with GFP-PRL3. The cells were fixed and stained with antibodies to PRL-3 and visualized by confocal microscopy. Note that the GFP-PRL3 signal overlaps with the endogenous PRL3 signal. . C. Inhibition of PRL3 results in a decrease in migration. Scratch wound healing assays were performed in the absence (dark grey bars) or presence (light grey bars) of a PRL3 inhibitor. The distance migrated is graphed on the Y-axis. The bar represents an average of three different experiments and the bars represent the standard deviation. Note that PRL3 inhibition resulted in a significant decrease in migration in all cell types. Statistical analysis was performed using two tailed students t test. D. Identification of an shRNA that inhibits PRL3 expression. HEK293 cells were transfected with GFP-PRL3 with the vector control or a vector expressing the PRL3 shRNA. Extracts from untransfected HEK293 cells served as controls. 60 hours post transfection, protein extracts were resolved on SDS-PAGE gels followed with Western blots with antibodies to GFP. Note that GFP-PRL3 expression is observed in the vector control but not in cells transfected with the shRNA expressing vector. Western blots for actin served as a loading control. E. Identification of PKP3 and PRL3 double knockdown clones. The PKP3 knockdown clone S10, was transfected with either the vector control (vector) or a construct expressing an shRNA targeting PRL3 (PRL3 KD). Stable clones were generated followed by Western blotting with antibodies to PRL3. Note that PRL3 levels were comparable between the cells transfected with the vector control and the PRL3 KD construct. Western blots for \u03b2-actin served as a loading control.(TIF)Click here for additional data file.Figure S4K8 and K18 filament formation in PKP3 knockdown clones. A. Localization of phosphorylated K8 in PKP3 knockdown clones. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with phospho-specific antibodies to PRL3 (S73 and S431) and imaged by confocal microscopy. Note that the intensity of the signal for pS73 increases due to an increase in K8 levels in the PKP3 knockdown clones while the intensity of the signal for pS431 remains the same in the PKP3 knockdown clones. . B. The interaction between K8 and K18 is not altered upon PKP3 knockdown. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were transfected with YFP-K18 and CFP-K8. 48 hours post transfection, FRET experiments were performed as described. The region in the white rectangle was bleached in the YFP channel and a corresponding increase was observed in the CFP channel as expected. FRET efficiencies were calculated for 5 different regions in three cells and results plotted in a bar graph. The FRET efficiency for the K18 K8 pair is comparable between the vector control and the PKP3 knockdown clones as shown in the graph.(TIF)Click here for additional data file.Figure S5Characterization of K8 and PKP3 double knockdown clones. A. Generation of PKP3 and K8 double knockdown clones. Protein extracts from the S10 derived K8  knockdown clones or the vector alone (S10P3) or the vector control (pTU6) were resolved on SDSPAGE gels followed by Western blotting with antibodies to PKP3 and \u03b2-actin. B. The PKP3 K8 double knockdown clones or the PKP3 knockdown clone, were stained with antibodies against K8 . C. Protein extracts from the S10 derived K8 (8.21 and 8.28) or K18 knockdown clones (18.22 and 18.23) or the vector alone (S10P3) were resolved on SDSPAGE gels followed by Western blotting with antibodies to K8, K18, and \u03b2-actin. Note that K18 levels are low in the K8 knockdown clones while K8 levels are not altered substantially in the K18 knockdown clones. D. Scratch wound healing assays were performed on the double knockdown clones  or the vector alone (S10P3). At different time points the distance migrated was measured and plotted as shown. The bars represent the mean of three independent experiments and the error bars represent the standard deviation. Statistical analysis was performed using two tailed students t test. Note that the distance migrated is significantly lowered in the double knockdown clones at 10, 15 and 20 hour time points. E. Scratch wound healing assays were performed on the double knockdown clones  or the vector alone (S10P3). At different time points the distance migrated per minute was measured and plotted as shown. The bars represent the mean of three independent experiments and the error bars represent the standard deviation. Statistical analysis was performed using two tailed students t test. Note that the distance migrated is significantly lowered in the double knockdown clones at 10, 15 and 20 hour time points. F. Scratch wound healing assays were performed on the double knockdown clones 8.21, transfected with either GFP alone or GFP K8 res.(TIF)Click here for additional data file.Figure S6Transformation induced upon keratin loss in the PKP3 knockdown clones. A. The S10 derived K18 (18.22 and 18.23) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2\u20133 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. Densitometric analysis of K8 in tumor samples. Protein extracts derived from tumors generated upon injection of either the vector control (S10P3) or the double knockdown clones 8.21 and 8.28) were resolved on SDS PAGE gels followed by Western blotting Expression of K8 was normalized to that of \u03b2-actin and densitometry performed on Western blots using ImageJ software. Note that K8 levels are lower in the double knockdown clones when compared to the vector control.(TIF)Click here for additional data file."}
+{"text": "A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008\u20132009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n\u200a=\u200a447 out of 1306). No significant difference was found between different age groups positive for rotavirus ( The triple layered capsid consists of 2 proteins in the outer shell (VP7 and VP4), the intermediate layer constitutes VP6 while VP2 forms the inner layer enclosing two proteins VP1 and VP3. Based on VP6 capsid gene, the virus has been classified into seven major genogroups while VP7 and VP4 are the basis of a binary-system for further classifying the genogroup A viruses into 27 G- and 35 P- genotypes respectively Diarrheal infections are considered as a significant cause of infant and childhood morbidity and mortality in both developing as well as developed countries Rotavirus strain identification is considered as the key component of epidemiological surveys, disease distribution, genotype prevalence studies, vaccine administration and efficacy monitoring programs. Many past studies have highlighted the significance of continued monitoring of circulating rotavirus strains in order to maintain sufficient population immunity Samples from hospitalized children suspected of rotavirus gastroenteritis were collected as per World Health Organization\u2019s standard case definitions that describe a suspected case as a child <5 years of age, admitted to a designated sentinel hospital for treatment of gastroenteritis while a confirmed case is a suspected case in whose stool the presence of rotavirus is demonstrated by means of an enzyme immunoassay. The study concept and design was approved through the Pakistan\u2019s National Institute of Health Internal Review Board. The samples were collected after informed and written consent from the patient\u2019s parents/guardians.http://whqlibdoc.who.int/publications/2011/9789241502641_eng.pdf).A total of 1306 stool samples were collected from hospitalized patients at \u2018The Children\u2019s Hospital\u2019 Lahore during January 2008 to December 2009. Majority of these samples were collected from children below than 5 years of age who were hospitalized with suspected rotavirus gastroenteritis. The collected stool samples were processed for the detection and confirmation of Rotavirus antigen. ELISA test was performed using the ProSpecT\u2122 Rotavirus Microplate Assay  as per World Health Organization\u2019s recommendations (A subset (20%) of EIA positive samples were transported to Department of Virology, National Institute of Health, Islamabad for rotavirus RNA detection through RT-PCR and further genotype determination on the basis of VP7 and VP4 gene segments using the protocol as described by Gouvea et al 1990 http://megasoftware.net/). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The GenBank accession numbers, country, year of sample collection and respective genotype information has been given where available. The VP7 sequences obtained in this study have been submitted to GenBank under the accession numbers KC896141-KC896157 and the VP4 sequences under accession numbers KC896127-KC896140.Phylogenetic analyses of VP7 and VP4 sequences were performed in comparison to the strains belonging to different geographical regions as retrieved from GenBank. Evolutionary tree and distances (number of base substitutions per site) were generated by Neighbor Joining method with Kimura-2 parameter using MEGA 4.0 (P>0.05) was found between different age groups positive for rotavirus through ELISA; however, high infection rate was observed among children between 12\u201317 and 24\u201359 months during 2008 and 2009 respectively. The rotavirus positive cases appeared to occur throughout the year with peaks during January to April and July to September  for the presence of rotavirus antigen yielded 447 (34.22%) samples positive for group A major inner capsid protein (VP6 antigen) common to all known rotavirus genotypes. The prevalence rates during 2008 and 2009 were 34.12% (n\u200a=\u200a243 out of 712) and 34.34% (204 out of 594) respectively. No significant difference  samples were found positive for rotavirus through PCR. G and P types were found with the prevalence as G1P The genetic sequence of 18 viruses included in this study was determined for the VP7 gene. These samples were randomly selected on the basis of sample quantity and resource limitations which showed that all viruses are highly identical (99\u2013100%) to each other within their respective genotypes; G1, G2 and G9. The G1 strains shared close sequence similarity with the previously reported viruses from Pakistan (JN001862) as well as to those from Belgium  and USA (JN258346). The VP7 sequences of G9 strains were found 100% identical among themselves as well as highly similar level of homology was found with the viruses from South Africa (GQ338887), Russia (FJ447573) and Australia (AY307090). These viruses also hold 98% similarity to previously reported G9 strains  from Faisalabad city of Pakistan (unpublished data). Likewise, the G2 viruses showed 99\u2013100% identity among themselves and a similar genetic closeness was found among the G2 strains from Bangladesh  as well as the already identified G2 strains from Pakistan .The genetic relationships of rotavirus strains from this study were also determined on the basis of VP4 gene. 14 viruses were grouped among three genotypes P Phylogenetic reconstruction revealed clustering of all rotavirus strains from Children Hospital, Lahore with viruses  differences, in the neutralizing epitopes of VP7 and VP4 proteins, between vaccine and the circulating strains. We compared the VP7 aa sequences of Pakistani viruses, constituting the antigenic epitopes with those of the two available vaccines strains (RotaTeq\u2122 and Rotarix\u2122). The VP7 protein contains three antigenic epitopes defined as 7-1a, 7-1b and 7-2 which comprise of 29 amino acid residues from positions 87 to 291 based on rhesus Rotavirus strain numbering (GenBank accession No. AF295303) Analysis of Pakistani G1 strains showed that the 29 amino acid residues across the three antigenic sites are highly conserved when compared to Rotarix\u2122 G1P The VP7 antigenic epitopes of G2 Pakistani viruses showed 3 variations (02 substitutions within 7-1a and 01 change in 7-1b) from the RotaTeq\u2122 G2P Among the G9 viruses presented in this study, the VP7 protein was compared with Rotarix\u2122 G1 strain as well as all constituent strains of RotaTeq\u2122 vaccine (G1-4 with P The VP4 protein of rotavirus contains four antigenic regions defined as 8-1 to 8-4 spanning between aminoacid positions 88 to 196 based on rhesus Rotavirus strain (GenBank accession No. AY033150) When compared to Rotarix\u2122 P A high degree of variation was observed for P Diarrhea is accountable for approx. 5% of all deaths among children below five years of age. Viral pathogens are the most common cause of gastroenteritis in both developing and developed countries Seasonality of rotavirus gastroenteritis in Pakistan has never been determined although the cases have been detected round the year et al.The higher percentage of samples positive for rotavirus was found among the children between 12 and 17 months of age. These findings are slightly variable than the previous study conducted in Karachi city of Pakistan During the last 15 years, very few reports about circulating genotypes of rotavirus are available from Pakistan In the present study, we compared the amino acid motifs constituting the neutralizing epitopes of VP7 and VP4 proteins between the circulating Pakistani rotaviruses and available vaccine strains. Multiple antigenic variations were noticed among both VP7 and VP4 epitopes highlighting the need for detailed antigenic mapping of prevalent rotavirus genotypes in the country. Complete genome sequencing will therefore be required to generate more comprehensive information on molecular epidemiology and evolutionary dynamics of rotaviruses on account of its segmented genome and the possible role of internal genes in immunity The vaccine introduction has significantly reduced the rotavirus disease burden in many countries like Australia, Austria, Belgium, Brazil, El Salvador, Nicaragua, Panama and the United States and both vaccines have been found to carry equivalent efficacy against G1, G3, G4 and G9 strains with P et al reported that most of the currently circulating G1 strains and the Rotarix\u2122 vaccine RRV-S1 strain differ in their antigenic properties and mutations in these antigenic sites may ultimately cause vaccine failure The mutations in the antigenic regions of rotavirus play an important role in the outcome of vaccine response. When compared to the respective prototype strains, our data substantiates the previous findings Despite these variable epidemiological reports, it has been now established that rotavirus vaccines are equally effective against vast diversity of rotavirus genotypes by generating heterotypic immune response. In the recent vaccine trails, both Rotarix\u2122 and RotaTeq\u2122 have been proven as equally effective in regions with high child and adult mortality Our present findings conclude that the rotavirus genotypes circulating in different geographical areas of Pakistan are quite variable and large scale studies must be conducted to calculate the burden of disease as well as the epidemiological understanding of contributing viral genotypes. Similar diversity of prevalent rotavirus strains within the Indian subcontinent including Pakistan has been reported by Miles et al.,"}
+{"text": "Genetic linkage maps have been essential tools to examine the inheritance of qualitative and quantitative traits, to carry out comparative mapping and to provide markers for molecular breeding applications. Linkage maps for species of Eucalyptus have been reported for several pedigrees using different molecular marker technologies . HoweverE.grandis and E.urophylla. Genomic representations of both parents and their F1 offspring were produced with the same complexity reduction method used to prepare the library (PstI/TaqI) generating the \u2018targets\u2019 for hybridizing to the arrays. Microarray imaging, data extraction polymorphism detection and marker scoring were carried out using DArTSoft v.7.44 (http://www.diversityarrays.com/). Polymorphic markers were filtered according to reproducibility, quality parameter (Q) and marker call rate as described earlier [Map construction was carried out using an F1 progeny of 177 individuals derived from a cross between  earlier . An inte earlier using a fA segregation ratio filtering (both for 1:1 and 3:1 markers) together with a relaxed call rate threshold > 50% were initially applied to include the largest number of potentially mappable markers. A total of 4,271 DArT markers segregated 1:1 and 1,572 segregated 3:1. The complete dataset with 6,065 markers  was submitted to a linkage analysis resulting in eleven groups at LOD \u2265 15 with a total of 2,484 markers. A subset of 1,032 markers positioned at high likelihood for ordering (864 DArTs and 168 microsatellites) provided a framework map with 1,176 cM and an average recombination distance between consecutive markers of 1.15 cM. When all the 2,484 mapped markers were fitted, the total recombining genome length increased to 1,303 cM with a resolution of 0.6 cM. A redundancy analysis of the 6,918 DArT probes for which sequences could be obtained resulted in 4,583 unique sequences (66%). The estimated redundancy of 34% represents a useful resource by providing alternative probes for detecting polymorphism in the same genomic region in different individuals. A total of 6,480 probes (93.7%) were confidently mapped onto the reference genome; 4,189 of them (65%) mapped to single positions while the remaining 2,291 probes mapped to a second position with a relatively high suboptimal alignment score and were therefore considered mapped at lower confidence. About 50% of these low confidence mapping  actually mapped to a sequence position containing a repeat element which might explain the mapping result. A total of 438 DArT probes could not be mapped to the current assembly. These DArT probes might further improve the current genome version by including some yet unassembled genome contigs. A total of 1,987 linkage mapped DArT markers for which sequence was available were positioned relative to the 41,208 gene models in the current genome, distributed in 500 kbp bins providing an average of 1.6 \u00b1 2.4 DArT markers for the 34 \u00b1 15 gene models present in each genome bin. Interestingly a total of 4,663 DArT probes (67%) mapped at less than ten basepairs from the nearest gene model and only 76 probes mapped at more than 10kbp. The largest distance from a DArT probe to the next gene was only 156 kbp and a modest although highly significant coincidence across the whole genome was seen between the number of DArT markers and the number of gene models (Spearman rank correlation R = 0.33 p<0.00000).Eucalyptus grandis genome currently available through the JGI Phytozome website. This map, together with other parallel mapping efforts that used this same genotyping platform have provided between 2,000 and 3,000 segregating markers irrespective of species of Eucalyptus. The mapping density achieved with the Eucalyptus DArT microarray provides unprecedented opportunities for comparative mapping across pedigrees, high resolution QTL analysis and molecular breeding applications across species of the genus. We have shown that not only the DArT marker sequences are highly enriched for genes, but that their distribution relative to the predicted gene models provides an extremely efficient tool to specifically tag genes at distances within the extent of LD in typical breeding populations. The use of this standardized high-throughput genotyping platform will therefore be instrumental to implement genome-wide selection strategies and positional cloning efforts in multiple Eucalyptus species.The linkage map presented in this work was used to aid the ordering of contigs during the assembly of the"}
+{"text": "Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation. The complex organization of tissues is established precisely and reproducibly during development. In the vertebrate neural tube, as in many other tissues, the interplay between extrinsic morphogens and intrinsic transcription factors produces spatial patterns of gene expression that delineate precursors for specific cell types. One such transcription factor, Pax3, defines the precursors of all sensory neuron subtypes and distinguishes them from precursors fated to give rise to the motor circuits. To gain insight into the molecular mechanisms by which the spinal cord is segregated into these two functional domains, we analysed the genomic regulatory sequences responsible for controlling Pax3 activity. We identified two regions of the genome, the coordinated activity of which establishes and refines Pax3 activity. We showed that the combination of activating signals from secreted Wnt factors together with Nkx family homeodomain repressors restrict Pax3 activity to the presumptive sensory region of the neural tissue. Subsequently, Pax3 acts to directly potentiate its own transcription and this autoregulation sustains Pax3 expression at later developmental stages. Together, our study reveals the way in which intrinsic and extrinsic signals are integrated by cells and converted into a sustained pattern of gene activity in the developing nervous system. Embryonic development relies on the coordinated and dynamic control of gene expression. This is achieved, in the main, by interactions between transcription factors (TFs) and the genomic cis-regulatory modules (CRMs) associated with regulated genes The specification of progenitor identity in the vertebrate neural tube is a well studied example of developmental patterning By contrast, less is known regarding the specification of sensory interneurons within the dorsal spinal cord Several studies indicate that a genomic interval immediately upstream of the mouse Pax3 promoter is sufficient to direct expression to the neural tube, however this region is not required for Pax3 expression Here we take advantage of lineage tracing analyses in mice and transgenic assays in chick and zebrafish embryos to dissect the molecular mechanism and regulatory logic of Pax3 expression in dorsal neural progenitors. We show that Pax3 expression is refined during neural tube patterning by the temporal activity of distinct regulatory elements. We provide evidence that in addition to Wnt signaling, Nkx family homeodomain (HD) containing repressors are critical for establishing the restricted expression of Pax3. Moreover, we demonstrate that autoregulation and positive feedback is required to maintain Pax3 expression in the neural tube.Rosa26-YFP or Rosa26-Tomato/GFP reporter strains  , all of which were located within the 4the gene . Candidaon (hpf)  before bon (hpf) . Maximumon (hpf) , after won (hpf)  and progon (hpf) , in agrepression . Howeverpression .The genomic interval corresponding to CNE3 is a highly conserved region of a previously defined CNS specific Pax3 CRM, termed both ECR2 In addition to CNE3, our functional assays identified CNE1 as a CNS specific Pax3 enhancer. CNE1 transient transgenic zebrafish exhibited Citrine expression in presumptive dorsal progenitors at 10 hpf  and 12 hWe sought to investigate the molecular basis of CNE3 activity in order to gain insight into the regulation of Pax3 expression in the CNS. The conservation of sequence across 12 vertebrate genomes was used to define 5 statistically enriched 15 bp motifs, predicted to contain the transcription factor binding sites (TFBS) that mediate enhancer activity . AnnotatTCFSiam transgenic zebrafish TCFSiam transgenic embryos assessed between 12 hpf and 18 hpf exhibited activated Wnt signaling within the tailbud and neural tube, however reporter expression was markedly decreased in the spinal cord of 24 hpf embryos . By contrast, deletion of Motif5 resulted in a significant increase in CNE3 activity along the entire D\u2013V axis of the zebrafish spinal cord compare . EctopicTo investigate the binding of putative repressors to CNE3, we performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts from dissected E3 chick spinal cords and a 48 bp DNA probe which spanned Motif3, 4 and 5. Supershift reactions were performed by the addition of antibodies raised against \u03b2-catenin, several candidate HD containing ventral fate determinants and FoxA2 . AdditioTogether, these data suggested that Nkx6.1, or a protein with a similar binding specificity, interacted with Motif5 of CNE3 to mediate transcriptional repression of Pax3 within the developing spinal cord. This is consistent with the observation that the ventral domain of the Pax3 lineage was mutually exclusive with Nkx6.1 expression in transgenic mice assessed at E9.75 . FurtherWe next sought to investigate the molecular basis of CNE1 activity by identifying statistically over represented conserved 15 bp motifs within this \u223c170 bp enhancer . ControlAmongst the TFs that potentially interact with Motif3 , the SoxpairedExamination of the matrix represented by Motif1 revealed a 14 bp sequence similar to a Pax6 paired domain (PD) binding site . We wereWe assessed the ability of both Pax3 and Pax7 to bind Motif1 by EMSA, using a 33 bp DNA probe and in vitro synthesized proteins. These assays revealed that both TFs interacted with this sequence, verifying it as a functional PD binding site  FurthermPrevious binding and structural studies have shown that the PD is comprised of two helix-turn-helix subdomains, commonly termed PAI and RED, that interact with the 5\u2032 and 3\u2032 region of the binding site, respectively engrailed repressor domain to either Pax3 or Pax7 We next sought to assess the ability of Pax3 and Pax7 to induce CNE1 activity in the neural tube. Electroporation of a dominant active protein consisting of the DNA binding domain of human PAX3 fused to the transactivation domain of FOXO1A (PAX3FOXO1A) was sufficient to activate CNE1 mediated transcription in the ventral neural tube of chick embryos at E3 . MoreoveIn this study we provide evidence that the dynamic expression profile of Pax3 within the developing neural tube is achieved by the coordinated action of two distinct regulatory mechanisms, acting through separate CRMs. The combined activity of these enhancers converts transient inductive cues into a sustained domain of gene expression. CNE3 integrates inductive Wnt signaling and the repressive activity of HD transcription factors to initiate expression of Pax3 within the prospective dorsal neural tube. Subsequently, an autoregulatory loop acting via CNE1 is established that maintains Pax3 expression in the absence of activated Wnt signaling and HD mediated repression.I150 BAC stable line has been shown to recapitulate the expression profile of pax3a in the spinal cord Previously, the zebrafish Pax3-GFPThe initiation of Pax3 transcription in response to Wnt signaling appears to be mediated through CNE3, a small highly conserved region of the genomic interval that has previously been identified as ECR2 and IR1 The transcriptional activity of the Wnt pathway decreases in the neural tube as development progresses, as indicated by a downregulation of Wnt reporter transgene expression prior to the peak of Pax3 transcription in progenitors Motif3 within CNE1, comprising Fox and Sox TFBS, is required for the activity of this enhancer across the AP axis of the CNS. The reduction of CNE1 activity in constructs carrying mutations in the HMG box binding site of Motif3, together with the identification of Sox11 and Sox3 binding at CNE1 in neural progenitors, supports a positive role for SoxB proteins on Pax3 expression and might account for the neural specificity of this enhancer Together, these data provide a molecular framework that describes the regulatory logic of Pax3 expression in the developing spinal cord. Expression is initiated by Wnt induction acting through CNE3, however this induction is limited to prospective dorsal regions of neural tissue by the activity of ventrally induced Nkx class proteins . Pax3 prDrosophila development It is notable that at early developmental time points, both CNE1 and CNE3 appear to be active in Pax3 expressing cells. We propose they act in a cooperative manner to establish the Pax3 expression domain, thereby offering increased robustness and precision. For example, CNE1 mediated autoregulation could function not only to increase output from the Pax3 promoter but may also buffer fluctuations in CNE3 mediated transcription. This mechanism is reminiscent of the increased robustness and precision of gene expression achieved by the synergistic activity of multiple CRMs during The acquisition of unique molecular identities within defined progenitor populations requires the translation of transient inductive cues into discrete expression domains. In the ventral neural tube this process is achieved using a transcriptional circuit involving cross-repression between TFs downstream of the ventral morphogen, Shh. This mechanism functions both to establish and maintain expression domains, as well as confer robustness to fluctuating levels of intracellular signaling http://www.genome.ucsc.edu/) and uploaded to the Mulan alignment suite The genomic interval representing the Pax3 locus in each species was defined in the UCSC genome browser (http://www.ensembl.org/). The alignment of enhancer sequences across vertebrate genomes was produced and analysed using ClustalW2 The Human sequence representing each CNE was used as the query sequence for cross-species BLAST searches, performed within the Ensembl genome browser (Zebrafish embryos were collected within 15 minutes of laying according to established procedures and injected with injected with a mixture of plasmid DNA (20 ng/\u00b5l) and Tol2 transposase mRNA (14 ng/\u00b5l). Embryos were maintained at 28.5\u00b0C and sorted on the basis of Citrine expression before fixation with 4% paraformaldehyde (PFA) at the desired stage. Chick assays were performed in Hamburger and Hamilton stage 11\u201313 embryos pax3a (gift from Simon Hughes) were performed as described pairedWholemount in situ hybridizations for Zebrafish experiments were analysed by determining the number of embryos labeled by Citrine expression in spinal cord progenitors as a proportion of the total transgenic population. The activity of enhancer constructs in the chick neural tube was assayed against the presence of \u03b2-Galactosidase antibody staining, which was used as an internal control of transgenesis. The statistical significance of transgenic assays was determined using two-tailed Fisher's exact tests.Radiolabelled DNA probes were produced as described in Figure S1th intron of the Pax3 locus. (A) The full Mulan alignment of the Pax3 locus, summarised in Several functional CRMs are located within the 4(TIF)Click here for additional data file.Figure S2pax3a expression across the AP axis of the CNS and is restricted to the Pax3/7 domain of the dorsal spinal cord.CNE1 activity recapitulates Pax3 expression. (A) CNE1 stable transgenic embryos assessed at 10 hpf exhibit Citrine expression in the developing hindbrain (hb) and presumptive dorsal progenitors (dP) within the lateral regions of the posterior neural plate.  At 24 hpf, CNE1 activity recapitulates (TIF)Click here for additional data file.Figure S3CNE1 is the only p300 bound Pax3 enhancer at E11.5. UCSC genome browser view displaying the binding profile of the enhancer associated transcription co-factor p300 within mouse limb, forebrain and midbrain tissue, prepared at E11.5 (TIF)Click here for additional data file.Figure S4The conservation of CNE3 across vertebrates. ClustalW2 alignment of CNE3 across 12 vertebrate genomes, revealing multiple clusters of nucleotides that are conserved across the phyla. The location of motifs within CNE3, discovered by MEME analysis, is marked within the alignment.(TIF)Click here for additional data file.Figure S5Wnt pathway effectors directly bind CNE3. UCSC genome browser view displaying the binding profile of the Wnt pathway effector, Tcf3, across the Pax3 locus in mouse embryonic stem cells (mESC) (TIF)Click here for additional data file.Figure S6The conservation of CNE1 across vertebrate genomes. ClustalW2 alignment of detailing the conservation of CNE1 across 12 vertebrate genomes. The location of statistically overrepresented motifs within this sequence is marked within the alignment.(TIF)Click here for additional data file.Figure S7M3Mut1 transgenic zebrafish exhibit a reduction in CNE1 activity compared to controls (compare B to D) . A similar reduction in CNE1 activity is observed in M3Mut2 transgenics (compare B to G) . (F) Graphical summary of the effect of HMG binding site mutations upon CNE1 activity in zebrafish embryos. Experiments performed in chick reveal a reduction in CNE1 activity in both M3Mut1 (n\u200a=\u200a3/7) and M3Mut2 (n\u200a=\u200a2/5) electroporations, however this result did not reach statistical significance .Mutation of the HMG box site within Motif3 reduces CNE1 activity. (A) Schematic outlining the mutations introduced into the HMG box site of Motif3. (TIF)Click here for additional data file.Table S1Annotation of conserved TFBS within CNE3.(DOCX)Click here for additional data file.Table S2Annotation of conserved TFBS within CNE1.(DOCX)Click here for additional data file.Table S3List of oligonucleotides used in the study.(DOCX)Click here for additional data file."}
+{"text": "Cardiac complications mainly related to myocardial iron overload (MIO) remain the main cause of morbidity and mortality in thalassemia major (TM). Thalassemia cardiomyopathy is treatable and partly reversible if appropriate chelation therapy is instituted in time. The validated multislice multiecho T2* Cardiovascular Magnetic Resonance (CMR) technique has permitted to quantify segmental and global myocardial iron burden detecting different patterns of iron overload. Aim of our study was to verify the risk of cardiac complications related to different patterns of MIO in a large cohort of TM patients.We considered 812 TM patients for who CMR and cardiac data were collected in a central data base. Three short-axis views  of the left ventricle were acquired using a multislice multiecho T2* sequence. Using a previously validated software the 16 segmental T2* values and the mean global heart T2* value were provided. A conservative cut off of 20 ms was considered the limit of normal for the segmental and global T2* values.We identified 4 groups of patients: group I (17%) with homogeneous MIO , group II (12%) with heterogeneous MIO (some segments with T2*<20 ms and others with T2*\u226520 ms) and global heart T2*<20 ms; group III (29%) with heterogeneous MIO and global heart T2*\u226520 ms; group IV (42%) with no MIO . The percentage of patients with cardiac complications was significantly different in the 4 groups . In particular, the percentage of patients with heart failure was significantly different in the 4 groups . No significant differences were found among groups in the percentage of arrhythmias and pulmonary hypertension. Odds Ratio for cardiac complications was 1.7  for patients with homogeneous MIO vs patients with no MIO. Odds Ratio for heart failure was 2.3  for patients with homogeneous MIO versus patients with no MIO and 2.2  for patients with heterogeneous MIO and global heart T2*<20 ms versus patients with no MIO.Homogeneous MIO predicts a significantly higher risk to develop cardiac complications, especially heart failure, suggesting an intensive chelation therapy in this group of patients.\u201cNo-profit\u201d support by industrial sponsorships  and \u201cMinistero della Salute, fondi ex art. 12 D.Lgs. 502/92 e s.m.i., ricerca sanitaria finalizzata anno 2006\u201d e \u201cFondazione L. Giambrone\u201d."}
+{"text": "The germline mutations in BRCA1/2 genes are the most significant and well characterized genetic risk factors for breast and/or ovarian cancer. Detection of mutations in these genes is an effective method of cancer prevention and early detection. Different ethnic and geographical regions may have different BRCA1 and BRCA2 mutation spectrum and prevalence due to founder effect. The population of Lithuania has over several centuries undergone limited mixing with surrounding populations and is mostly of indigenous Baltic origin, which is different from Slavs. The aim of our study was to asses full BRCA1/2 mutational profile in Lithuanian population.We performed comprehensive mutation analysis of BRCA1/2 genes in 605 unrelated breast and/or ovarian cancer patients (with/without family history) and predictive unaffected patients (with family history) using high resolution melting (HRM) screening (Light Cycler 480/Light Scanner 384) followed by direct sequencing (ABI 3500) and MLPA for large genomic rearrangements (LGRs).Overall, we have identified 25 different mutations (16 in BRCA1 and 9 in BRCA2 genes). Seven frequent pathogenious mutations in BRCA1 gene  comprised 48%, 29%, 7,7%, 3,5%, 2,1%, 1,4% and 1,4% respectively of all BRCA1 mutations; a single BRCA2 mutation (c.658delGT) comprised 43% of all mutations in this gene. Five novel BRCA1  and 4 novel BRCA2 genes mutations  were identified; 3 different LGRs  were found in BRCA1. The most common c.4035delA  appears to be true Lithuanian  founder mutation and haplotype data confirmed ancient origin of this mutation c.a. 62 generations ago.Characterization of BRCA1/2 mutational profile in Lithuania enabled to develop screening protocol using HRM for 8 common BRCA1/2 point mutations, which comprise 88% of all mutations detected in our country. This knowledge will provide more efficient approach for the individualization of genetic testing affordable for all breast/ovarian patients and their relatives."}
+{"text": "They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair , whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found.Inositol 1,4,5trisphosphate (IPPlcd3 (mNabPlcd3) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant mNabPlcd3 alleles are phenotypically normal. However, the presence of one mNabPlcd3 allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the mNabPlcd3 mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells.We characterise a novel mouse mutant with a spontaneously arisen mutation in mNabPlcd3 mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.The  DAG activates protein kinase C, and IP3 causes the release of calcium ions from intracellular stores, which makes PLC enzymes key regulators of intracellular calcium 2, is a signalling mediator in its own right involved in a variety of processes such as phagocytosis, ion channel activity and cell motility Phosphoinositide metabolism provides an essential intracellular signalling system involved in a broad spectrum of key events in organ development and function. Phosphoinositol 4,5 bisphosphate olt1Pas (synonym Del(9Ctdspl-Slc22a14)1Pas) mutation is the spontaneously arisen genetic defect of a recessive mouse mutation formerly called oligotriche (olt), which shows a combination of alopecia and male infertility. Although the Del(9)olt1Pas mutation is a large deletion on chromosome 9 encompassing the genes Ctdspl , Vill (villin-like), Plcd1, Dlec1 , Acaa1b , and a part of Slc22a14 (solute carrier family 22 member 14), the alopecia of the mutant has been attributed to the loss of Plcd1Plcd1 expression was disrupted by targeted gene inactivation Del(9)olt1Pas mutant mice show varying degrees of hair loss  Plcd1 and Plcd3 causes prenatal death in mice due to vasculature defects in the placenta Studies of mice with spontaneous or engineered ablation of the Plcd3 caused by the insertion of an intracisternal A particle (IAP) genome Plcd3 gene, which causes the predominant expression of a truncated PLCD3 protein lacking the PH domain. Mice homozygous for both the Del(9)olt1Pas deletion and the novel mutant mNabPlcd3 gene live for several weeks after birth and show total alopecia.Here, we report on a spontaneous hypomorphic mutation of Del(9)olt1Pas deletion and the novel mutant mNabPlcd3 gene.The hair follicle is a highly complex and dynamic part of the integument which originates from stem cells and undergoes recurring phases of growth (anagen), regression (catagen) and rest (telogen), and produces the hair shaft which contributes to the pelage oltSH (SH for sparse hair), occurred spontaneously in our stock of Del(9)olt1Pas mutant mice olt1Pas mutant mice during hair follicle morphogenesis, which was most pronounced on the ventral surface (Del(9)olt1Pas mutant mice on postnatal day 14 merely the inguinal and medial femoral region showed a pronounced loss of pelage olt1Pas heterozygous males yielded phenotypic offspring of both the Del(9)olt1Pas and the oltSH phenotypes in equal numbers, indicating that one dose of the novel mutant allele had caused the exacerbation of the phenotype of Del(9)olt1Pas homozygous mutants.A novel pelage phenotype, which we provisionally called also see . In phen surface  [47]. Whentrally . The losthe face . CrossinoltSH mutation and the Del(9)olt1Pas mutation, we set up matings of oltSH females with phenotypically normal males descending from oltSH females bred with Del(9)olt1Pas heterozygous males. Among the offspring, we found 10 mice that had not grown any visible pelage and only a few short vibrissae, which we provisionally called oltNH (NH for no hair) (In order to determine the phenotype of mice being double homozygous for the novel no hair) .oltNH mice were also smaller and weighed less than their littermates. On postnatal day 8, the body weight of wild-type mice was 5,7 gr \u00b10,6 gr (n\u200a=\u200a6), of oltSH mice 4,9 gr \u00b10,3 gr (n\u200a=\u200a3), and of oltNH mice 3,8 gr \u00b10,1 gr (n\u200a=\u200a3), and on postnatal day 25, wild-type mice weighed 11,5 gr \u00b11,7 gr (n\u200a=\u200a4), oltSH mice 9,3 gr \u00b10,4 gr (n\u200a=\u200a3), and oltNH mice 6,7 gr \u00b10,8 gr (n\u200a=\u200a3). While Del(9)olt1Pas homozygous mutants and oltSH mice have lived for more than 1 year, almost no oltNH mouse has lived longer than 40 days (n\u200a=\u200a21) (with one exception of 64 days). Thus, the combination of the Del(9)olt1Pas mutation with the novel mutation had marked additive effects on the dorsal pelage, the body mass and the longevity.Apart from the total alopecia, Del(9)olt1Pas pelage phenotype was caused by the lack of expression of the Plcd1 gene oltSH and oltNH phenotypes might involve other members of the Plcd gene family.Since we had previously identified that the oltSH and oltNH mice hybridised with a probe derived from Plcd3 intron 1  and mutant oltNH genomic DNA. Using primers 1046 of Plcd3 exon 2 and 1049 of exon 3, we amplified the expected 320 bp fragment from wild-type genomic DNA, but a 5.4 kbp fragment from oltNH genomic DNA  oltSH genomic DNA was thus caused by the insertion of a BamHI site within the IAP sequences (mNabPlcd3 mutation (Nab for Neuroanatomy Bonn).As this BamHI fragment in wild-type genomic DNA of C3H mice stretches from intron 1 to intron 3 of omic DNA . The genA (Plcd3 . The IAPequences . We refemNabPlcd3 mutation in mice (Del(9)olt1Pas mutation oltSH mice was (Del(9)olt1Pas) \u2212/\u2212, mNabPlcd3 +/\u2212 (n \u200a=\u200a21), while that of phenotypically oltNH mice was (Del(9)olt1Pas) \u2212/\u2212, mNabPlcd3 \u2212/\u2212 (n \u200a=\u200a24), with + referring to the respective wild-type allele. We also identified 8 phenotypically normal mNabPlcd3 \u2212/\u2212 mice that had at least one wild-type allele of Plcd1.Based on the sequence data, we designed a genomic PCR screen to identify the  in mice . In comboltSH or oltNH phenotype lacked expression of Plcd1 mRNA  or to the more centrally localised catalytic domain (IF12\u201315) . The antre found . These ePlcd3 transcripts and PLCD3 protein were also found in phenotypically wild-type mice  hair shafts are straight and penetrate through the pilary canal to the surface, irrespective of being heterozygous olt1Pas (olt\u201d), the oltSH (oltNH (Del(9)olt1Pas mutants 13,5\u00b13% of hair shafts were distorted in the dorsal skin, while in oltSH mutants 66\u00b15,8% of hair shafts were affected, whereas in oltNH mutants all hair shafts were distorted failing to penetrate to the surface (150 hair follicles counted in 3 different biopsies for each mutant). Thus, in the absence of Plcd1 expression, an increasing dosage of the mNabPlcd3 mutation causes more dorsal hair shafts to be defective.The absence of visible pelage in )olt1Pas , \u201colt\u201d, he oltSH  and the H , but some TUNEL positive cells in the inner root sheath  Foxn1, Msx2 and Hoxc13, showed unaltered expression between wild-type and oltSH and oltNH mutants. The expression levels of genes encoding for secreted signalling proteins Pdgfa, Pdgfb, Shh, Bmp2 and Bmp4 were also unchanged (Krtap12-1 (in the hair cuticle) and Crisp1 (in the hair medulla) Crisp1 is expressed in the medulla of the hair shaft in wild-type, but not oltNH mice on postnatal days 6 and 8 olt1Pas mutant and functional Plcd3 due to a genomic insertion of an IAP show total alopecia, weight loss and die during the first two months of life. While Del(9)olt1Pas homozygous mutant mice show a mild predominantly ventral alopecia due to the deletion of the Plcd1 gene mNabPlcd3 mutant allele have no obvious phenotype, which has also been described for mice in which Plcd3 was inactivated by homologous recombination mNabPlcd3 allele described in this report behaves like a hypomorphic mutation of the Plcd3 gene allowing for a limited period of postnatal life. While in oltNH mutants the bulk of Plcd3 transcripts are shorter and immunblots predominantly show a truncated PLCD3 protein in oltNH mice, apparently normal transcripts of Plcd3 can also be amplified by RT-PCR using primers in exon 1 and exon 5 (not shown). Therefore, we cannot exclude that trace amounts of normal transcript, too little to be detected in Northern blots, are expressed from the mutant mNabPlcd3 allele, just sufficient to overcome the placental defects of the double knockout mice. Still, the oltNH mutant offers the unique opportunity to study postnatal functions of Plcd1 and Plcd3.In this report, we demonstrate that mice lacking expression of both functional Del(9)olt1Pas mutant mice, in which the m1NabPlcd3 mutation occurred. Similar IAP insertions with 98% sequence homology to the m1NabPlcd3 IAP have recently been described in various mutant mice m1NabPlcd3 mutation, the IAP is integrated in reverse orientation with respect to the transcription of the Plcd3 gene, it is most likely that it causes its effects by enhancing transcription from cryptic promoters. The Plcd3 transcript variant ENSMUST00000128650 starting in exon 5 would be predicted to have a molecular weight well within the range of the oltNH mutant PLCD3 protein detected in our immunoblots. This truncated PLCD3 protein would have no PH domain, which is important for the targeting of the enzyme to the substrate in the plasma membrane Many spontaneous mouse mutations are the result of insertions of retroviral elements, recently reviewed in Del(9)olt1Pas mutation oltSH and oltNH mutants (this report) exhibit a similar histological aspect of fragile hair shafts, but differ with respect to the distribution of this defect over the body surface. Similar fragility of hair shafts has been found in mice with altered expression of Foxn1 and Hoxc13, which are directly involved in the expression of hair keratins and keratin associated proteins Foxn1 and Plcd1 in the pre-cortical zone of the hair bulb and the down-regulation of Plcd1 in nude mice suggested that Plcd1 has some function downstream of Foxn1Plcd3 encompasses the entire hair bulb during anagen including and exceeding the domain of Plcd1. It is therefore possible that Plcd3 can compensate for some aspect of Plcd1 function. Since we demonstrated that on postnatal day 8 the medulla-specific, Hoxc13-regulated gene Crisp1 is down-regulated in oltNH mice, but not in oltSH mice, some hair shaft-specific genes may depend strictly on the expression of Plcd3. This may imply that in the absence of normal Plcd1 expression, Plcd3 could possibly play a role in the Hoxc13\u2013 Foxq1 axis of gene regulation in the hair medulla Mutant mice homozygous for the Plcd1 develops in the context of an inflammatory response oltNH skin, but also in oltSH mutants, in which at least some hair follicles were apparently in anagen. As the infiltrates in day 12 oltSH skin were associated with hair follicles in premature catagen, the influence of the inflammatory response is possibly very locally elicited and operative, but may contribute to the sustenance of the abnormal hair follicle regression in the mutant.It has previously been suggested that the alopecia of mice with functional inactivation of Plcd1 was shown to exert direct effects on adipocytes. Knockdown of Plcd1 in an adipocyte cell line interfered with lipid accumulation during differentiation, which was also observed in primary cells obtained from Plcd1 defective mice Plcd1 expression. Further studies will reveal whether Plcd3 is also involved in adipocyte differentiation and function in order to explain, why oltNH mice show an even more dramatic reduction in body mass than mice lacking functional Plcd1 alone. Inflammatory infiltrates in oltNH mice were usually found in the subcutaneous adipose tissue near the dermis, where adipocyte precursors are located. Most recently, the stimulatory activity of BMP (bone morphogenetic protein) and PDGFA (platelet-derived growth factor A) secreted by subcutaneous fat cells and their precursors, respectively, has been highlighted with respect to their stimulating activity for hair follicle stem cells and the sustenance of anagen oltNH mice could possibly negatively interfere with the stimulatory activity of this tissue, which could contribute to the curtailment of the growth phase in the mutant hair follicle.Recently, oltNH mutant after birth and ceases on postnatal day 12, while anagen was less shortened in the lower ventral body region of Plcd1 defective Del(9)olt1Pas mutants, where the alopecia of this mutant is most pronounced Plcd3 is expressed in the hair bulb during this critical period, Plcd1 and Plcd3 may possibly play additive roles in the sustained growth during hair follicles morphogenesis after birth.Phospholipase C delta isozymes are associated with signal transduction processes involved in cell cycle regulation and cell proliferation The animals were sacrificed according to \u00a74.3 of the German law for the protection of animals (Tierschutzgesetz) (file number 50.203.2-BN 6/02). The mice were killed by cervical dislocation avoiding unnecessary pain.Del(9)olt1Pas mutant mice (synonym Del(9Ctdspl-Slc22a14)1Pas, formerly called oligotriche, olt) oltSH and oltNH mice were in a mixed C3He/Orl, C3H/HeJ and C3Heb/FeJ background, which we collectively refer to as C3H in this report. The animals were kept in a 12 hour light/dark cycle with food and water ad lib.We have previously described the origin of Skin biopsies were taken from the dorsal mid-thoracic region of mice after killing them by cervical dislocation. The skin biopsies were fixed for 4 hours in Bouin\u2019s solution, dehydrated in increasing alcohol concentrations and embedded in paraffin as described The TUNEL assay was performed on paraffin sections using the Apoptag Red In Situ Apoptosis Detection Kit  according to the manufacturer\u2019s recommendations. Nuclei were stained with DAPI.oltNH mice were analysed histomorphometrically with respect to the length of the hair follicle  and the width of the hair bulb at its widest diameter. 50 measurements of both parameters were taken from each biopsy using the ImageJ software and statistically analysed with the paired t test using PrismGraphPad 4 software.Tissue sections of three different biopsies of dorsal skin of wild-type and PCR experiments were performed in a TProfessional Basic Thermocycler (Biometra). Usually 35 cycles were run with the optimal annealing temperature chosen according to the recommendation of the supplier of Phusion high fidelity DNA polymerase . All primers  and S2 wFor RT-PCR experiments, the cDNAs were synthesised from total RNA  or mRNA  from skin biopsies using PowerScript\u2122 reverse transcriptase from CLONTECH  with an Oligo (dT)15 primer. PCR was performed on templates of cDNA or genomic DNA prepared from tail biopsies  using Phusion high fidelity DNA polymerase  according to the manufacturer\u2019s recommendations.Krtap genes. Gene-specific fragments were amplified from 1 \u00b5g of cDNA with the optimal annealing temperature chosen according to the recommendation of the supplier of Phusion high fidelity DNA polymerase . The number of PCR cycles run was usually 35, or is otherwise given in the Figure legend.The cDNA preparations synthesised from mRNA were used to amplify expressed sequences of genes that have only one exon, e.g. some Gel electrophoresis of RNA, blotting and detection of DIG labelled probe was performed as described previously GGATCCTAATACGACTCAC) had been added at its 5\u2032 end. Antisense cRNA probes were then derived from these PCR products by in vitro transcription in the presence of DIG-labelled dUTPs with T7 RNA polymerase from Roche  To synthesize DIG labelled cRNA probes, RT-PCR products were re-amplified using a modified antisense primer, to which the sequence of the T7 RNA polymerase binding site (Plcd3 mRNA was generated from skin cDNA using primers 511 (exon 11) und 512 (3\u2032UTR) (Plcd1 has been described The cRNA probe for Northern blot hybridisations to  (3\u2032UTR) . DIG labPlcd3-specific cRNA probe was generated as described above from the RT-PCR product synthesised with forward primer 527 (exon 3) and reverse primer 1352 (exon 5) (The 350 b (exon 5)  and the (exon 5) . In situBecause the histochemical stain for alkaline phosphatase activity in Bouin-fixed tissue sections gives a rather brownish colour and is difficult to distinguish from pigmented areas, skin biopsies were taken from C57BL/6J mice that have mostly black eumelanin and the sections were photographed and analysed using a NuanceVX multispectral camera  with the manufacturer\u2019s software. Areas displaying spectral characteristics of eumelanin were pseudo-coloured in black, those displaying spectral characteristics of the specific in situ signal in red.https://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm).Genomic DNA was digested with BamHI (Fermentas), the fragments separated by agarose gel electrophoresis and transferred to positively charged Nylon membrane (Roche) by vacuum blotting using a Model 785 Vacuum Blotter (BIORAD). Hybridisation was carried out in Dig Easy Hyb (Roche) using 100 ng/ml of DIG labelled probe. Washes and detection of bound DIG were carried out as described in the Roche DIG Application Manual for Filter Hybridisations . For hybridisations to  and 573   or the PH domain of PLCD3 (4H 5\u20139), respectively, were supplied by K. Fukami. The hybridoma supernatant was used undiluted. The secondary antibody,  was used at a dilution of 1\u223630.000. Blotting and signal detection were performed as described Figure S1Origin of oltNH mice. Pedigree showing the breeding scheme that led to the discovery of oltNH mice. White symbols represent wild-type mice and blue symbols mice with the phenotype of Del(9)olt1Pas homozygotes. Red symbols represent mice of the oltSH phenotype and yellow symbols are mice of the oltNH phenotype. The symbols merely show the presence of such phenotype in a litter, but do not represent the relative proportion of phenotypes in each litter. The numbers designate the crosses referred to in the following text. All mice in the F1 generation of a Del(9)olt1Pas female mated with a wild-type C3HeB/FeJ male (cross 1) were phenotypically normal. In the F2 generation of cross 2, (56 mice in 7 litters), we found 10 phenotypically Del(9)olt1Pas homozygotes and one female that showed a greater extent of alopecia, which we termed oltSH (for sparse hair). This female was backcrossed with her father (cross 3) resulting in one litter of six, in which 2 of 6 mice showed the oltSH phenotype. When one of these oltSH females was crossed with a known Del(9)olt1Pas heterozygous male (cross 4), the phenotypically altered offspring showed in equal parts the oltSH and the Del(9)olt1Pas homozygous phenotype, suggesting that one dose of a novel mutation could exacerbate the alopecia in Del(9)olt1Pas homozygous mice. When the oltSH founder female of the F2 generation was crossed with one of the phenotypically normal sons in cross 5, we found altogether 52 mice with different phenotypic alterations in the offspring (n \u200a=\u200a121 in 21 litters): the Del(9)olt1Pas mutant phenotype, the oltSH phenotype, but also 10 mice that did not developed any visible pelage and only a few short vibrissae, which we termed oltNH. Further brother-sister matings of this offspring (cross 6) again produced all three mutant phenotypes.(TIF)Click here for additional data file.Table S1List of Plcd3-specific primers. The oligonucleotide sequences are given in 5\u2032 to 3\u2032 orientation; genome coordinates are given according to NCBI37 mm9 July 2007; \u201cPlcd3 genomic\u201d indicates the location of the oligonucleotide sequence in the Plcd3 locus.(DOCX)Click here for additional data file.Table S2List of primers used in. The oligonucleotide sequences are given in 5\u2032 to 3\u2032 orientation. (DOC)Click here for additional data file."}
+{"text": "The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence Arp2/3 complex DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the Arp2 within adult brain is dependent upon testosterone exposure.Castration dramatically reduces and testosterone replacement restores Arp2 expression in hippocampus. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that Arp2 expression in the adult brain is maintained through sustained epigenetic modifications of the Arp2 gene promoter. We find that castration of adult male rats resulted in decreased Arp2 mRNA expression and increased methylation of specific CpG sites within the Arp2 promoter in the hippocampus. Similarly, castration significantly increased estrogen receptor \u03b1 (ER\u03b1)/androgen receptor(AR) mRNA expression and decreased ER\u03b1 promoter methylation within the hippocampus. These changes were prevented by testosterone replacement.This suggests that the Arp2 DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the cytoskeleton systems."}
+{"text": "The purpose of this study was to describe a novel approach for epimyocardial pacemaker implantation under fluoroscopic guidance associated to atrial access through pericardial reflections as an alternative technique for lead implantation in patients with limited venous access.Between June 2006 and November 2011, 15 adult patients underwent epimyocardial atrioventricular pacemaker implantation through a minimally invasive subxiphoid approach and pericardial window. Mean age was 46.4 \u00b1 15.3 years and 9 (60.0%) patients were male. Patients selected for this new surgical approach were not amenable to transvenous lead placement due to: multiple abandoned leads (5), venous occlusion (3), presence of retained lead fragment in the intravascular after previous device extraction (3), tricuspid valve vegetation under treatment (2) and uncorrected intracardiac defects (2).All procedures were successfully performed. There were no perioperative complications and no early deaths. The mean operating time for isolated pacemaker implantation was 231.7\u00b1 33.5 minutes. Lead placement on the roof of right atrium through the transverse sinus was possible in 12 patients and in 3 patients the atrial lead was implanted on the left atrium through the oblique sinus. None of the patients displayed pacing or sensing dysfunctions and all parameters remained stable throughout the follow-up period of 36.8 \u00b1 25.1 months.Epimyocardial pacemaker implantation under fluoroscopic guidance associated to atrial access through pericardial reflections provides a safe, effective and reproducible approach for atrioventricular pacing in patients for whom the transvenous approach is undesirable or not feasible."}
+{"text": "Previously, a quantitative trait loci (QTL) for alcohol preference on chromosome 2 in a C57BL/6IBG (B6) background has been identified. The overlap of two of interval specific congenic recombinant strains (ISCRS) strains reduced the QTL interval into a 3.4 mbp region.By using the keyword alcohol, we identified a total of 39 genetic elements in the region between markers D2Mit56 and D2Mit10. Among these genetic elements, we found seven with potential function in alcohol preference (Table Our current data suggest that the Atf2 and Titin genes are potentially the most alcohol relevant genes. However, further experiments and examination are still needed to confirm their candidacy. Several other candidate genes are also in the process of being identified."}
+{"text": "Reamed interlocking intramedullary nailing is considered the gold standard treatment for complex fractures of the femoral and tibial shaft. There has been some controversies about dynamization of statically locked nails, and some authors recommended routine dynamization for promotion of healing. This study aims to evaluate treatment of complex fractures in tibia and femur with static interlocking intramedullary nail method.In a retrospective study from January 2003 to April 2008, 173 patients with femoral and tibial shaft fracture that were treated with this method were enrolled. No rod was dynamized in our patients.All patients with tibial fractures achieved union without any need for dynamization during 12-18 weeks . Four patients developed delayed union but all achieved union without any intervention. In femoral fracture, all but one patient achieved complete union during 10-30 weeks (mean: 18.3 weeks). One patient developed non-union who was treated by an exchange nailing and iliac bone graft method. No significant complication was observed in our patients.It is not necessary to routinely dynamize nails in tibial and femoral shaft fractures as all fractures united in acceptable alignment without any complication. Open reamed interlocking intramedulary nailing is the preferred treatment option for complex femoral and tibia shaft fractures.2]3]4][3][4]2][[4][3][4][3][4]2].Some autThe aim of this retrospective study was to help resolving this controversy and determine the union rate of complex femoral and tibia shaft fractures treated with static interlocking nailing without dynamization.From January of 2003 to April 2008, 61 patients  with complex closed fracture of tibial shaft and 112 patients  with complex closed fracture of femoral shaft were treated with static proximal and distal interlocking intramedullary nails. All patients developed fractures due to high energy trauma  and all underwent surgery for treatment of fracture soon after a systemic condition that was stabilized. Our tibial fracture patients were 17 to 70 years old (mean: 28.29) and our femoral fracture patients aged from 17 to 75 years old (mean: 26.57). In tibial fracture group, 33 patients had right sided fracture and 28 had a left sided one. In femoral fracture group, 59 patients had right sided fracture and 53 had a left sided one. All patients had comminuted fracture (Winquist type III or IV). In tibial fracture group, 37 patients had fracture in the middle third of tibial shaft; 9 patient had fracture in proximal third and 2 patients had fracture in distal third of tibial. Thirteen patients had segmental pattern of fracture.In femoral shaft fracture group, 66 patients had fracture in middle third; 16 patients in proximal third and 2 patients in distal third and 28 patients had segmental fracture in the femur. In femoral fracture group, 46 patients and in tibial fracture group, 25 patients had other associated injuries that required specific surgical or medical care . All patients underwent static interlocking intramedulary nailing as soon as general condition of patients was stabilized. In all patients, open reduction was performed under general anesthesia and the canals were reamed as large as possible  and an adequate size static interlocking nail was inserted . A proximal interlocking was performed with specific interlocking guides and a distal locking under an image intersifier. Post-operatively, the patients started partial weight bearing ambulation as early as possible . An isometric quadriceps exercise and a range of motion in the knee, hip and ankle were encouraged. The patients were followed at the OPD clinic by the same surgeon at 3-6 week intervals until complete union was achieved. The clinical and roentgenographic signs of healing process were recorded.All patients were followed for at least for 9 months (range: 9-36 months). Fracture union was defined as follows: Clinically there was no tenderness or pain and fracture had no motion and patients were walking without any walking aids. Roentgenographically, solid bridging callus with cortical density connected fracture fragments in at least 3 from 4 cortices. Delayed union was defined as an incomplete healing during a 6 months period after the fracture fixation.All patients with tibial fracture achieved union without any need for dynamization or any surgical intervention during 12-28 weeks (mean: 13.4 weeks). Two patients with tibial fracture developed wound infection that improved with antibiotic treatment. Four patients in tibial fracture group developed delayed union but all of them achieved union completely without any surgical intervention up to 28 weeks. Alignment of tibial fracture was perfect in all patients without any shortening and rotation. In femoral fracture group, all but one patients achieved complete union during 10-30 weeks (mean: 18.3 weeks). There was no significant complication in femoral fracture group. Seven patients with femoral fracture developed delayed union that were completely united by 24-30 weeks after fracture fixation without any surgical intervention. One patient developed non-union that was treated with changing of the nail and iliac crest bone graft after 36 weeks of primary surgery and achieved union by 16 weeks after bone grafting. Alignment of femoral fracture was perfect without any significant shortening and rotation in our patients with femoral shaft fracture.Intramedullary nailing has become the treatment of choice for fractures of the femoral and tibial shaft.2]3]4][3][4]2][[4][3][4][3][4]2]. In compDuring recent years, many authors recommended dynamization for promotion of healing in statically locked intramedullary nails of femoral or tibial diaphyseal fractures.6]9][12[9][126][[12[9]12. In reviIn a retrospective study in Taiwan, the success rate of dynamization was only 54%. In anoth"}
+{"text": "Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast.Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the  Saccharomyces cerevisiae, contains two sets of lysyl-tRNA synthetases (Msk1 and Krs1) that are encoded by nuclear DNA. Krs1 participates in cytoplasmic protein synthesis while Msk1 in mitochondrial protein synthesis. Despite the structures of the Msk1 and Krs1 being different, functionally they are very similar. The yeast Msk1 has a bacterial ancestry, whereas the Krs1 represents the ancestral eukaryotic type Aminoacyl-tRNA synthetases are a heterogeneous family of enzymes responsible for aminoacylating tRNAs with the appropriate amino acids. The budding yeast, Lys (tRK3), it has been proposed that Msk1 plays an essential role in the import of cytosolic tRNALysCUU (tRK1) into mitochondria LysUUU (tRK2) and the mitochondrial DNA encoded tRNALysUUU (tRK3) in vivo and in vitroYeast Msk1 is a dual functional protein. In addition to aminoacylation of mitochondrial tRNAYeast tRK1 can also be imported into human mitochondria in the presence of yeast cytosolic factors and Msk1 KARS partially suppresses the growth defects that are associated with yeast MSK1 deletion. Interestingly, in vivo experiments suggest that import of tRK1 into yeast mitochondria is independent of yeast Msk1.We previously have shown that human mitochondrial and cytoplasmic lysyl tRNA-synthetases are expressed from alternative spliced mRNAs from a single gene MSK1 do not grow on non-fermentable carbon sources as they are unable to aminoacylate mitochondrial tRNALys (tRK3). This results in the inhibition of mitochondrial protein synthesis and loss of mtDNA. Before investigating the ability of human Kars to import tRK1 into yeast mitochondria, we determined whether Kars could substitute for the yeast Msk1 in the aminoacylation of tRK3. To test this, we introduced the human KARS cDNA or yeast MSK1 on a high/low copy plasmids into a yeast strain heterozygously deleted for MSK1 by insertion of a KANMX4 cassette (NS104). The resulting yeast strains were sporulated on fermentable and nonfermentable carbon containing media plates. In initial tests, all four meiotic progeny from diploid strains that contained high copy yeast MSK1 formed colonies on both fermentable and non-fermentable carbon sources. Progeny from the diploid strain that contained high copy human KARS plasmid yielded four spores on fermentable and nonfermentable carbon sources. Out of the four spores, two were larger and wild type for MSK1 gene while other two were smaller and positive for MSK1 deletion (data not shown). Haploid progeny with KARS on a low copy plasmid failed to grow on non-fermentable carbon sources (data not shown). These results suggest that human KARS can substitute yeast MSK1 deletion partially.Yeast cells deleted for KARS or yeast MSK1 under respiring and fermenting conditions. Cells were grown at 30\u00b0C in selective medium, serially diluted and spotted on rich carbon sources (YEPD) and on non-fermentable carbon sources (YEP plates supplemented with 3% glycerol/ethanol). Cells expressing high levels of human KARS partially suppressed the growth defect of msk1\u0394 cells on both fermentable and on non-fermentable carbon sources . This may be due to requirement of Msk1 at elevated temperature for the import of tRK1 We investigated further the growth properties of the haploid progeny of strain NS104 containing high copy plasmids expressing human mitochondrial  sources . The ext1 NS101; . The reaKARS partially complements yeast MSK1 deletion. We hypothesize that the human Kars can be imported into yeast mitochondria and substitute the function of yeast Msk1.Nevertheless, the results show that human in vitro import system with bacterially expressed human Kars to investigate whether human Kars can import tRK1 into yeast mitochondria. Yeast mitochondria isolated from strain D273-10B in vitro transcribed [32P]-labeled tRK1 under standard import conditions and the import efficiency was determined by RNase protection assay. tRK1 was imported into yeast mitochondria in the presence of wild-type yeast cytosol , consistent with previous studies msk1\u0394 strain msk1\u0394 strain (data not shown).Next, we analyzed the distribution of tRK1 in wild type and msk1\u0394 cells could be due to contamination of mitochondria preparation with cytosol. Loss of mitochondrial DNA as a result of MSK1 deletion could change the mitochondrial morphology and result in enhanced association with the cytosolic fraction. However, we have observed decreased levels of mitochondrial-associated tRK1 in a rho0 strain that was generated by ethidium bromide treatment of parental strain NS101 (data not shown). To determine whether tRK1 was localized inside mitochondria or is a cytosolic contaminant, we used several methods that could eliminate cytosolic contamination with little reduction in the endogenous mitochondrial tRNA levels. First, we selectively permeabilized the mitochondrial membranes by digitonin detergent. Low concentrations of digitonin disrupt and partially solubilize the outer mitochondrial membrane as well as removing other membranous structures contaminating the mitochondrial fraction. To determine the concentration of digitonin that is required to reduce the cytosolic contamination, aliquots of mitochondria were treated with increasing concentrations of digitonin prior to centrifugation. Initially, the pellet and supernatant fractions were analyzed for the selective solubilization of specific proteins that serve as markers for the different mitochondrial subfractions. In subsequent experiments, nucleic acids were isolated from the digitonin soluble supernatant and insoluble pellet fractions and specific tRNA species were detected and quantitated by northern analysis.The association of tRK1 with mitochondria isolated from Treatment of mitochondria with 0.05% digitonin solubilized specifically the mitochondrial outer membrane, as shown by the release of the intermembrane space marker protein, CCPO into the supernatant . Higher We determined if treatment of mitochondria with MNase  would reduce the contamination of cytosolic tRNAs. However, mitochondria treated with MNase alone showed contamination of cytosolic RNAs as evidenced by the presence of tRK2  by northern analysis . However, the amount of tRK1 is at least 10-fold greater than other cytosolic tRNAs, in both wild type and msk1\u0394 mitochondria  of mitochondria, and is also involved in the import of tRK1 into mitochondria KARS) cDNA, when present in multiple copies, partially rescues the growth defect of msk1\u0394 cells on non-fermentable carbon sources. We also show that human Kars imports tRK1 into isolated yeast and mammalian mitochondria in the presence of yeast or human cytosolic factors. Our results provide genetic and biochemical evidence that human Kars can perform the role of yeast Msk1. The results presented here demonstrate for the first time that purified recombinant human Kars could complement the role of yeast Msk1 in the import tRK1 into isolated yeast mitochondria or into mammalian mitochondria in vitro.Yeast Msk1 is a dual functional protein, it aminoacylates the tRNAmsk1\u0394 yeast cells msk1\u0394 strain when compared to tRK1 levels in mitochondria isolated from a isogenic wild type strain. We show that association of tRK1 with mitochondria isolated from the msk1\u0394 strain is not due to non-specific contamination of cytosolic fraction. tRK1 is still detected in mitochondria isolated from msk1\u0394 strain and the mitochondria had been incubated with digitonin and MNase (in vivo. Our results differ from a previous study MSK1 and c) different techniques to detect tRNAs. We employed high-resolution northern blot hybridization to detect mitochondrial tRNAs, whereas dot-blot hybridization method was used in previous studies which may not detect small quantities of tRNAs that are associated with mitochondria msk1\u0394 strain lacks the MSK1 gene by genomic PCR. However, we cannot rule out the possibility that the association of tRK1 in mitochondria isolated from msk1\u0394 strain is strain specific.It had been proposed that interaction between tRK1 and pre-Msk1 is absolutely essential for the import of tRK1 into mitochondria, since no tRK1 was detected in mitochondria from nd MNase . It is cin vivo import of tRK1 occurs independently of mitochondrial lysyl-tRNA synthetase. However, we observe that the import of tRK1 into mitochondria requires the presence of Msk1 in vitro. The in vitro import of tRK1 differs from other well-established systems in the requirement of many factors. Cytosolic factors are not required for the import of tRNAs into mitochondria of TetrahymenaTrypanosomaLeishmaniaAla into Arabidopsis thaliana mitochondria. This evidence suggested that amino acyl-tRNA synthetases are indeed involved in the import of tRNAs into plant mitochondria A. thaliana alanyl-tRNA synthetase in yeast cells was not sufficient to import its cognate tRNAAla into the yeast mitochondria Our studies suggest that the in vitro. The precise role of cytosolic factors in the import of tRK1 into mitochondria is not known. It was speculated that probably the cytosolic factors act to stabilize the pre-Msk/tRK1 complex or facilitate the binding of tRK1 complex to mitochondria in vivo studies show the existence of an import mechanism for the import of tRK1 that is not dependent on Msk1.Cytosolic factors are essential as Msk1 is required for the import of tRK1 into isolated yeast mitochondria in vivo. In support of this hypothesis, we find reduced levels of tRK1 in mitochondria isolated from a msk1\u0394 strain when compared to the parental strain. We have also observed that cytosolic factors alone when present in large quantities, slightly stimulates the import of tRK1 into isolated yeast mitochondria in the absence of Msk1 in vitro (unpublished results). Our findings indicate that the in vivo import of tRK1 differs from the in vitro import. Our results also suggest that alternative import pathways are present for tRNA import and yeast can serve as a model system to study the evolutionarily divergent import pathways of tRNAs.The import of precursor proteins or tRNA into mitochondria requires one or more outer membrane receptors MATa/MAT\u03b1 his3\u03941 leu2\u03940 met15\u03940 ura3\u03940), BY4742 (MAT\u03b1 his3\u03941 leu2\u03940 lys2\u03940 ura3\u0394) and msk1\u0394 (MATa msk::KAN his3\u03941 leu2\u03940 met15\u03940 ura3\u03940) strains were obtained from Research Genetics Inc. Heterozygous diploid strain NS104 (MATa/MAT\u03b1 his3\u03941/his3\u03941 leu2\u03940/leu2\u03940 met15\u0394/MET ura3\u03940/ura3\u03940 LYS/lys2\u03940) was constructed by crossing strain BY4742 with msk1\u0394. Strain NS108 is isogenic to NS104 but contains MSK1 on a high copy plasmid (pTEF MSK1 URA3-2\u03bc). Strain NS112 (MATa his3\u03941 leu2\u03940 met15\u03940 ura3\u03940) contains human KARS on URA3-2\u03bc plasmid. Standard yeast genetics and techniques were used BY4741 (KARS) cDNA was amplified by PCR using Thermo polymerase (Ambion) with KARS-A primer (ACTAGTGAATTCATGTTGACGCAAGCTGCT) containing an EcoRI site and KARS-B primer (GGATCGATCTCGAGGACAGAAGTGCCAACTGT) containing a XhoI site. Human KARS and was cloned into the pGEM-T Easy vector to generate plasmid pNS33. pNS33 was digested with EcoRI and XhoI and the KARS ORF was isolated and cloned into a 2\u03bc vector (pTEF-URA3) at EcoRI and XhoI sites MSK1 was amplified using yeast genomic DNA as a template and primers MSK1-A (GGCACGTGACTAGTATGAATGTGCTGTTAAAA) and MSK1-B (GGATCGATAAGCTTTTACTGCCTGTTTACATC) and the PCR product was cloned into pGEM-T Easy vector to create pNS32. A SpeI/HindIII digestion product of pNS32 containing MSK1 was inserted into pTEF-URA to generate pNS34. PCR was performed to check the insertion of KAN marker at MSK1 locus with sense primer MSK1-C (TAGTCTTTTATTCGTGATAAAAGCGAAAAT) and antisense primer MSK1-D (CGTAGCGTAGTTTATTGGTGTAGAGAAAAA). To exclude the possibility of partial deletion of MSK1 gene, PCR was performed with sense primer MSK1-C (TAGTCTTTTATTCGTGAAAAAAGCGAAAAT) and antisense primer MSK1-E (AATACGCAACTCCAAATGGTCGAAGTC CTT).The complete coding sequence of human mitochondrial lysyl-tRNA synthetase (E. coli BL21 (DE3) Codon Plus (RIL) cells and purified to 80% homogeneity as described MSK1) was PCR amplified and cloned into E. coli expression vector pET24d to generate a hexahistidine tag at the carboxyl terminal of synthetase. This construct was expressed in E. coli BL21 (DE3) Codon Plus (RIL) (Stratagene) at 15\u00b0C for 12 hours in the presence of 0.5 mM isopropyl-1-thio-\u03b2-D-galactopyranoside. The histidine tagged soluble pre-protein was partially purified using Talon metal affinity resin (Clontech) and concentrated. The purified Msk constituted approximately 70% of the total purified protein.The human mitochondrial lysyl-tRNA synthetase cDNA cloned in pET 24d vector (Novagen) was expressed in in vitro transcription using T7 polymerase. The tRK1 in vitro transcript was gel purified and stored in TE pH 8.0. tRK1 was labeled with \u03b1-[32P]-ATP at the 3\u2032 end using E. coli terminal nucleotidyl transferase as described 2.A synthetic gene encoding tRK1 downstream from a T7 polymerase promoter was constructed by annealing ten overlapping oligonucleotides and cloning into EcoRI/BamHI digested pUC19 vector. This plasmid was linearized with Bst NI and used as a template for in vitro synthesized [32P]-labeled tRK1 was preincubated with 2 \u00b5g of yeast cytosolic factors with or without purified human Kars or yeast Msk1 on ice for 10 minutes prior to the import assay. Import of tRNA into mitochondria was performed in a volume of 100 \u00b5l containing 100 \u00b5g of mitochondria, 20 mM HEPES-KOH pH 7.2, 0.6 M sorbitol, 4 mM ATP, 1 mM GTP, 5 mM MgCl2, 25 mM KCl and 0.2 pmol of [32P]-labeled tRK1. The import reaction was carried out at 30\u00b0C for 25 minutes. Following import, mitochondria were re-isolated, suspended in import buffer and treated with 2.5 \u00b5g/ml RNases  for 30 minutes on ice to remove non-imported tRK1. Then mitochondria were reisolated and washed three times with SEM buffer . The washed mitochondrial pellet was resuspended in 0.1 M potassium acetate (pH-5.2), 0.5% (w/v) SDS and mitochondrial RNA was extracted using phenol-chloroform as described Mitochondria were isolated from yeast c peroxidase (CCPO) (intermembrane space), TIM23 (inner membrane), delta-1-pyrroline-5-carboxylate dehydrogenase (Put2p) (matrix) and porin (outer membrane).For generation of mitoplast, digitonin was used to permeabilize the outer mitochondrial membrane of mitochondria. Mitochondria were suspended at a concentration of 1 mg/ml in SEM buffer. 250 \u00b5g of mitochondria were incubated for 25 minutes on ice in 250 \u00b5l of SEM buffer containing 0 to 0.2% digitonin. The resulting mitoplasts were harvested by spinning at 14,000 rpm for 10 minutes and the supernatant was saved for further analysis. The mitochondrial pellet was washed once again with SEM buffer and was used either for micrococcal nuclease treatment or for the isolation of mitochondrial nucleic acids. To determine the efficiency of digitonin treatment, 100 \u00b5g of mitochondria were treated as above with digitonin, centrifuged, and the pellet and supernatant fractions separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane . Immunoblots were performed using antibodies specific for cytochrome 2, 250 mM sucrose and 10 mM MOPS pH 7.1. MNase was added to 500 U/ml unless otherwise specified. MNase treatment was performed for 25 minutes on ice with occasional mixing. MNase was then inactivated by the addition of 10 mM EGTA on ice for 5 minutes. The mitochondrial suspension was diluted with SEM buffer containing 2 mM EGTA and centrifuged at 14000 g for 10 minutes. The reisolated mitochondrial pellet was washed one more time in SEM buffer. The resulting pellet was used to isolate total mitochondrial RNA.For the Micrococcal nuclease (MNase) treatment, aliquots (250 \u00b5g) of mitochondria were resuspended in 2 mM CaClcys and tRNAPhe. To detect tRK1, we used the oligonucleotide probe anti tRK1 (GTAGGGGGCTCGAACCCCTAACC) for tRK2, the probe anti tRK2 (GCCGAACGCTCTACCAACTCAGC) for tRK3, the probe anti tRK3 (TAGCTGGAGTTGAACCAAGCATG) for cytosolic tRNAcys, the probe anti tRNAcys (TCAGGATCGAACTAAGGACCAAG) and for cytosolic tRNAPhe, the probe anti tRNAPhe (TGAGAATCGAACTACATGTAAAT).Mitochondrial nucleic acids were isolated from the various samples and were separated on 12% urea-acrylamide gels. The transfer of nucleic acids to the nylon membranes was performed as described"}
+{"text": "Persons who had worked in a receiving laboratory from September 1, 2004, to April 15, 2005, and had performed routine procedures in virology  were eligible for the study. From May 4 to May 19, 2005, we visited the laboratories to interview supervisory personnel regarding routine work-up of samples and to collect blood from study participants for serologic testing.The national reference laboratory for influenza at Robert Koch Institute performed serologic testing for antibodies against A/Singapore/1/57(H2N2) virus by hemagglutination inhibition. We compared antibody titers of laboratory workers who worked with a CAP sample with those who did not. However, this comparison might not show a difference if (silent) virus transmission had occurred among laboratory staff. To exclude this possibility, we also compared titers of workers born before 1969 with those in a group of volunteers from Robert Koch Institute also born before 1969. Titers <10 were assigned a value of 1.>3 symptoms typical for influenzalike illness in the 4 days after having worked with a CAP sample.Of 47 laboratory workers, 18 either declined to participate or were excluded because they did not perform any of the defined routine procedures during the defined period. Thus, 29 (62%) workers were included in the study, of whom 14 (48%) reported having worked with CAP samples. Of these 14 workers, 12 (2 exclusively) transported samples and 11 (2 exclusively) prepared the samples; 9 (1 exclusively) performed antigen testing, 2 (0) performed nucleic acid amplification tests, and 4 (0) performed virus isolation. Fourteen workers (48%) reported no contact with the samples, and 1 (3%) was unsure. None of the 29 participants reported any event that could have led to release of infectious material during the respective time frame, such as broken test tubes or dropped culture plates. Participating laboratories reported that all procedures were performed under appropriate hygienic and safety precautions. No person had <10. Three (21%) of 14 workers exposed to CAP samples and 4 (40%) of 10 who denied exposure had titers >20. All 7 were born before 1969. The geometric mean of titers of all participating workers born before 1969 did not differ significantly from that of the Robert Koch Institute employees  had a titer of 80, three (12%) had titers of 40, two (8%) had titers of 20, and the remaining 18 (72%) had titers ,\u2013In summary, no evidence was found for laboratory infections with the influenza A H2N2 virus. The risk for laboratory-acquired influenza infections is unknown. Severe acute respiratory syndrome coronavirus and Mycobacterium tuberculosis are infectious agents whose transmission characteristics are similar to those of influenza. For severe acute respiratory syndrome coronavirus, laboratory-acquired infections are well documented ("}
+{"text": "The guidance on the optimal choice of respiratory protection for health care workers (HCWs) caring for patients with suspected/confirmed influenza infection requiring non-invasive ventilation (NIV) has remained controversial.We investigated an influenza A outbreak involving 3 HCWs in a medical ward in July 2009. Contact tracing & viral studies were performed to identify the source of outbreak.2 while Patient B developed respiratory failure requiring 100% O2 and was soon transferred to a side room and put on NIV. Virological investigations confirmed that Patient B & the 3 staffs had influenza A/H3 subtype infection, while Patient A had influenza A/H1 infection.Three HCWs ) reported fever and upper respiratory symptoms on July 13. Nasal/throat swabs confirmed influenza A infection. On contact tracing, all 3 reported contact history with 2 patients (Patient A & B) admitted to the ward on July 10 & 11 respectively who were later diagnosed with influenza A. Patient A was on 2L/min OThe nurse had cared for both patients, the intern had performed physical examination for both patients, and the GSA transported both patients to the isolation facilities. They reported good compliance to droplet precautions & hand hygiene and were wearing surgical mask but not mucosal protection during the care of both patients. These 3 were the only staff among 24 that reported working within close proximity (<1 meter) of Patient B while he was on NIV.Patient B was identified as the index case causing this outbreak of influenza A/H3 subtype.Droplet precaution and the use of surgical mask may not offer adequate respiratory protection to HCWs caring for patients on NIV.None declared."}
+{"text": "Structural changes of chromosomes are a primary mechanism of genome rearrangement over the course of evolution and detailed knowledge of such changes in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A, 5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species. Scientists have long understood that chromosome translocation is a major driving force in shaping genomes during evolution Triticum aestivum L., 2n\u200a=\u200a6x\u200a=\u200a42, genomes AABBDD) is well known, with the first of these translocations was from studies of chromosome pairing and gene-based marker locations T. monococcum  and the 4A/7B translocation must have occurred at the tetraploid level as it is also present in T. durum  The presence of the non-homoeologous translocations between chromosomes 4A, 5A and 7B in the hexaploid wheat  (http://www.wheatgenome.org/) hosted by URGI (http://urgi.versailles.inra.fr). The BLASTN algorithm was applied for all analyses using an E value cut-off of 0.0001. Wheat ESTs for individual deletion bins on chromosomes 4A, 5A and 7B were downloaded from http://wheat.pw.usda.gov/GG2/index.shtml. Comparison of bin-mapped ESTs against Brachypodium CDS sequences was performed using the BLAST++ BLASTN algorithm with an E value cut-off of 0.00001.Gene-coding sequences labelled as CDS from Brachypodium orthologs on this chromosome arm could be placed into two sets. Genes in Set 1 had homoeologous sequences on 5AL, 5BL and 5DL, respectively. The pattern of these chromosome arm locations shows that they are from the original 5AL and were not involved in any interchromosomal translocations. Genes in this set were orthologous to genes on two Brachypodium chromosomes, 1 and 4 with Bradi1g03330 bordering the breakpoint  also detected sequences on \u20184AL\u2019, showing that they are duplicated on this chromosome. The reason for their inclusion in this set is that genes flanking them all detected sequences on the three short arms of the homoeologous group 4 chromosomes : increasing gene IDs for those in 4AL-3, decreasing gene IDs for those in 4AL-4 and decreasing gene IDs for those in 7BS-1  ese ESTs  preventiin 7BS-1  and S3. in 7BS-1  on the mPrevious models of the structures of chromosomes 5A and 7B are highly consistent Brachypodium gene Bradi2g54210. However Brachypodium orthologs on either side of this gene failed to detect sequences on each of the three short arms of the homoeologous group 4 chromosomes Click here for additional data file.Table S2Brachypodium orthologs on modern chromosome arm 7BS.(XLSX)Click here for additional data file.Table S3Brachypodium orthologs on modern chromosome arm \u20184AL\u2019.(XLSX)Click here for additional data file.Table S4Brachypodium orthologs on modern chromosome arm \u20184AS\u2019.(XLSX)Click here for additional data file.Table S5Chromosome arm locations of wheat sequences detected by ESTs reported by Miftahudin et al.(XLSX)Click here for additional data file.Table S6Wheat sequences detected by Brachypodium orthologs on either side of Bradi2g54210.(XLSX)Click here for additional data file.Table S7Chromosome arm locations of wheat sequences detected by Brachypodium genes between Bradi1g00227 and Bradi1g00620.(XLSX)Click here for additional data file.Table S8Brachypodium orthologs on the modern chromosome arm \u20184AL\u2019 translocated from the original 7BS.(XLSX)Click here for additional data file."}
+{"text": "The main goal of this ex vivo study was to assess and compare the cellular and electrophysiological effects of two dental biomaterials, white mineral trioxide aggregate (WMTA) and calcium enriched mixture (CEM) cement, on neuronal cell excitability and electrical properties.1 nerve cells. The dental biomaterials were prepared according to the manufacturers' directions and were applied to the bathing media and 0.05 mL of total mixture of each dental material at a distance of 3 mm from the cells.A conventional intracellular current clamp technique was used to study the cellular effects of WMTA and CEM on the excitability, firing and the shape of action potential of neuronal soma membrane of FFindings indicated that exposure to both dental biomaterials shifted the irregular high frequency firing type observed in control conditions to a more regular low frequency firing pattern. Neuronal exposure to WMTA, but not CEM, significantly hyperpolarized the cell resting membrane potential. Both treatments significantly influenced the duration and the amplitude of action potentials. Extracellular application of either CEM or WMTA caused a significant increase in the after hyperpolarization (AHP) amplitude and AHP area, but the potentiating effect of WMTA was more effective than CEM.1 cells and changed the AP characteristics. Both dental biomaterials reduced the neuronal activity possibly through enhancement of K+ outward current. This may possibly explain the positive mechanisms of these biomaterials in regenerative endodontics, though further research is needed for such a conclusion.Treatment with WMTA or CEM resulted in a profound alteration in the firing behaviour of F Biocompatibility and non-toxicity are two important properties required for ideal biomaterials which are used for pulp capping and root-end fillings . Substan2+ has been shown to be an effective pulp capping material 4]5][4][5]3][[5][4][5][4][5]3]. Preservmaterial 8]9]. O[9]. O2+ . O[9]. Omaterial , exhibitmaterial  or pulp material 13]14][[14][2+ h[[14][2+ material . The abimaterial 17]. Des. Des2+ hmaterial [7][8][9]material 11]12][[12][2+ h[[12][2+ material 16]18][[18][2+ h[[18][2+ material 20]21][[21][2+ h[[21][2+ material 23]24],[24],2+ h,[24],2+ 1 neuronal excitability in Helix aspersa using intracellular recording techniques.The aim of the present ex vivo study was to assess and compare the electrophysiological effects of WMTA and CEM on F1 neurones from the right parietal lobe of suboesophageal ganglia of the Iranian garden snail, Helix aspersa. Animal dissection was performed as previously described . Bri. Bri1 neAll research and animal care procedures were performed according to the protocols approved by Shahid Beheshti University of Medical Sciences ethical committee for animal research.Conventional intracellular recordings under current clamp condition were conducted using Axoclamp 2B amplifier . To study the behavior of the neuronal membrane potential, or to understand how a neuron can be excited and inhibited, a current clamp technique is used in which the voltage difference across the cellular membrane is measured while injecting a constant current into the cell. The electrical responses of neuronal cells were recorded with microelectrodes , which were pulled with a vertical puller  and had resistances ranging from 3.5-5 milliohm when filled with 3MKCl. Sampled data were digitized using an A/D converter  and stored on an IBM computer with Chart software for offline analysis. The following quantitative parameters of action potential (AP) were measured using either Chart 6 software  or Minianalysis : The resting membrane potential (RMP), the amplitude of AP, which was defined as the change in voltage from the RMP to the peak of the AP. The duration of the AP and AHP amplitude were measured at half amplitude and from the RMP to the maximum negativity after an AP, respectively. The area under the curve for AHP  of the AP was also measured  and to qWhite ProRoot MTA  was prepared according to the manufacture\u2019s direction: as a mixture of powder (20 mg) and normal Ringer (0.2 mL) in a slurry form . A recen1 neuron was recorded only once in control conditions and after applying consequent treatment. The two experimental groups were independent of each other. After 15 min of control recording, WMTA or CEM was independently added to the extracellular media close to the right parietal ganglion. The recording was continued for 80 min. The maximum cellular effects which occurred between 25 and 35 min after application of either WMTA or CEM were used for analysis.Experiments were conducted on four groups of snail neurons: WMTA treated (n=11), CEM-treated (n=17) and two separate control groups for each experimental group . From each snail, one FThe effect of both dental materials was irreversible upon wash-out for 20 min (data not shown). There were also no significant differences between the two control groups  in any of the electrophysiological parameters; therefore, the data were pooled for remaining analysis and presented as the control group.2+, and, thereby, to affect the amplitude of the AHP. To address this issue, the concentration of Ca2+ in the extracellular Ringer solution was reduced to half (i.e. reduced from 10 mM to 5 mM).Next, a series of experiments were conducted to verify the ability of CEM and WMTA to release CaNumerical results are given as mean \u00b1 SEM, with n being the number of cells on which the measurement was performed. Significant differences between the groups were evaluated using a Student\u2019s t-test or one-way ANOVA and P<0.05 was considered to be significant.1 neurons in normal Ringer. Of these, 36 were from control, 11 from WMTA treated group, 17 from CEM treated group. These cells in control condition were spontaneously active and exhibited an irregular tonic firing pattern  in a separate experiment. Under this condition, a significant reduction in the amplitude of AHP was observed  when compared to the value recorded in the normal Ringer concentration . By contrast, upon addition of either CEM or WMTA to the low Ca2+ Ringer, a significant enhancement in the AHP amplitude of F1 neurons was observed  outward currents underlying AHP in F1 neurons. To test whether release of Ca2+ from WMTA or CEM contributed to the enhancement of AHP amplitude observed in the presence of both dental materials, intracellular recordings were performed in low Ca2+ concentration Ringer before and after either WMTA or CEM application. The present results showed that both compounds have enhancing effect on AHP amplitude, suggesting the possible involvement of Ca2+ release from the applied dental materials. However, the ability of WMTA to release Ca2+ appears to be greater than CEM, possibly due to their different chemical compositions.In the present study, the induced changes in excitability could be due to enhancement of outward potassium current. This hypothesis is supported by a significant increase in the amplitude of AHP which occurred following application of either WMTA or CEM. AHPs following an AP plays an important role in repolarizing the AP and in shaping the discharge properties, including firing frequency and pattern 39]40][[40][39][[[40][39]a et al.  reportednt cells . Based oElectron probe microanalysis results revealed that CEM cement is mainly composed of CaO, P2O5, SO3, and SiO2 45],[45],44],44]le cells 47]48],[48],2+ c,[48],2+ + channels currents play critical roles in determining AP repolarization, duration and frequency 50].[50].+ ch.[50].+ c+ outward current. Application of both dental materials shortened the AP, reduced the firing frequency and enhanced the AHP amplitude. This may possibly have analgesic and regenerative effects, though further research is needed for such a conclusion.For the first time electro-physiological evidence was provided demonstrating the suppressive cellular effects of WMTA and CEM on neuronal excitability. Both dental biomaterials reduced the neuronal activity possibly through enhancement of K"}
+{"text": "Nothapodytes amamianus) is an endangered tree species observed in only Amami Oshima Island located in southern part of Japan. According to the Red list published by Ministry of Environment, it is classified as 1A  and naturally remaining number is only 20. It contains camptotesin which is used for anti-cancer drugs. Kagikazura (Uncaria rhynchophylla) is an medicinal tree species observed widely in Japan and China. It contains alkaloids  which are good for remedy of high blood pressure and dementia. For the purpose of micropropagation and development of basis of useful substance production by cell culture as well as conservation of endangered species, tissue culture procedure was developed for those two species.Wadatsuminoki . Callus proliferation from stem or root segments was observed on the 1/2LP medium containing 0.5uM BAP and 1 uM 2,4-D, this subculture cell line may be used for the possible production of secondary metabolites in vitro.Shoots were induced from stem spine (thorn) of Kagikazura in the 1/2MS medium containing BAP or Zeatin. Regenerated plants were obtained by rooting of these shoots on 1/2MS medium containing 1 uM IBA. Callus induced around the stem segments were continuously subcultured in the fresh 1/2LP medium containing 0.5 uM BAP and 1 uM 2, 4-D. These cell lines can be used for the possible secondary metabolite production and for breeding by somaclonal variation or genetic engineering."}
+{"text": "Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas.Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters  and survival.Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis . Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases.Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment. Amplification and overexpression of Her2 occurs in a significant number of gastroesophageal adenocarcinomas \u20133. RecenThe aim of the present study was to evaluate the relationship between Her2 and HSP90 in gastric carcinomas and carcinomas of the gastroesophageal junction, and its influence on tumor biology and behavior.All patients gave written informed consent for the use of additional molecular analysis at the time of operation. The usage of human archival tissue for molecular analysis was approved by the local Ethics Committee of the Faculty of Medicine of the Technische Universit\u00e4t M\u00fcnchen.We investigated formalin fixed, paraffin embedded (FFPE) archival cancer tissue from 347 patients with primary resected gastric carcinoma and carcinoma of the gastroesophageal junction who underwent surgery between 1995 and 2005 at the Klinikum Rechts der Isar of the Technische Universit\u00e4t M\u00fcnchen (Germany). None of the patients had received pre- or perioperative neoadjuvant treatment.Two hundred twenty-one of the patients were male (63.7%) and 126 female (36.3%), with a median age of 69 years (range: 29 to 100). Median overall survival (OS) of all patients was 19 months (95% CI 14-23 months). Seventy-three tumors (21.0%) were adenocarcinomas of the gastroesophageal junction, and 274 were gastric carcinomas (79%). Most tumors showed an intestinal phenotype . Sixty tumors were mixed type carcinomas according to Lauren (17.3%), 111 showed a diffuse phenotype (32%) and 23 were unclassifiable (6.6%). Tumor grading was G1 (well differentiated) in 1 case (0.3%), G2 (moderately differentiated) in 54 cases (15.6%) and G3-G4 (poorly differentiated) in 292 cases (84.1%). Complete resection was achieved in 197 patients . For the purpose of this study, all tumors were reclassified according to the current UICC TNM-classification . We incl2O2 blocking using 3% H2O2 in aqua destillata and avidin biotin blocking . The sections were then incubated with antibodies for HSP90  and Her2 . Positive and negative controls were included in each reaction.Immunohistochemistry was performed on FFPE tissue. Preparation of tissue microarrays (TMA) was performed as described before, generating triplicate cores from randomly selected tumor areas with a diameter of 1.0 mm each . The parPositive HSP90 staining was defined as cytoplasmic staining of \u226510% of carcinoma cells  and discrepancies were discussed at a multihead microscope to gain a final consent. Only cores with technically unequivocal staining results and sufficient tumor content (>50 tumor cells) were used for final analysis.All cases were also tested for Her2 amplification by fluorescence in situ hybridization (FISH), irrespective of prior immunohistochemical Her2 results. An assay with fluorescence-labeled locus-specific DNA probes for Her2 and chromosome-17 centromeric \u03b1-satellite (Chrombios) was hybridized onto 4 \u03bcm TMA sections as described before ,22. FISH\u00b2 and Fisher\u2019s exact test. Survival analysis was performed using Kaplan-Meier estimates, log rank tests and Cox\u2019s proportional hazards regression analysis. All tests were 2-sided, and the significance level was set at 0.05.For statistical analysis, IBM SPSS 21.0 Statistics statistical software  was used. Associations between immunohistochemical expression patterns, results of FISH analysis and pathological features were given in crosstabs and were evaluated with XThree hundred thirty-six cases were evaluable for membranous Her2 expression and all 347 cases for Her2 amplification. The majority of tumors showed Her2 expression , which was weak in 96 cases , moderate in 43 , and strong in 35 tumor samples  and 21 patients (6.1%) showed Her2 amplification as defined by a Her2/CEP17 ratio \u22652 Table 2.Correlation was strong between Her2 expression and amplification  would have been considered Her2 negative, and 36 (10.7%) as Her2 positive .Of the 323 cases evaluable for HSP90 expression, immunoreactivity was found in 125 tumors (38.7%). Only 6 cases (1.8%) showed a strong reaction against HSP90 versus a weak cytoplasmic staining in the other positive cases , and intestinal phenotype according to Lauren  and Her2 , but not Her2 status according to the FDA/EMEA (p=0.502), Her2 amplification alone (p=0.802) or elevated Her2 copy numbers (p=0.813) were associated with better prognosis in univariate analysis  and 4. AGastric carcinomas and adenocarcinomas of the gastroesophageal junction have been shown to express Her2 in a significant number of cases, rendering it a possible valuable molecule for molecular targeting \u20134,7,20. One interesting finding of our study was the considerable rate of cases which were classified as polysome, i.e. expressing elevated Her2 and CEP17 copy numbers below a Her2/CEP17 quotient >2, which is the recommended definition of Her2 amplification. Most of these cases were Her2 1+ or 2+. In breast cancer, there are several publications, which direct towards this yet unclear issue of Her2/CEP17 polysomy in terms of determination of true Her2 status, and there is increasing evidence that Her2/CEP17 polysomy represents rather a phenomenon of co-amplification than true polysomy \u201339.Studies in breast cancer have also pointed out the limitations of assessment of Her2 status by immunohistochemistry and additional FISH. Immunohistochemistry has been described to lack objectivity producing false-positive or -negative outcomes due to interobserver variability, and both immunohistochemistry and FISH are heavily dependent from technical issues such as fixation and buffering ,41. HoweIn literature there are congruent data about the rate of Her2 overexpressed and amplified tumors in gastric cancer but there are still inconsistent results regarding any prognostic value of Her2 in gastric carcinomas and adenocarcinomas of the gastroesophageal junction ,7. A relOur observation of the prognostic impact of even weak Her2 immunoreactivity was unexpected, especially with regard to the reports of others , but repOur results speak in favor of a questioning attitude towards the assessment of Her2 in gastric and gastroesophageal carcinomas, like it has been adopted for Her2 in breast cancer for almost two decades now .Given the likelihood of increased application of trastuzumab or other Her2 directed agents in gastric and gastroesophageal cancer and the increasing number of publications about Her2 in these tumors there will be clearly a need for an exact definition of \u201cHer2 status\u201d that will cover yet unclear findings, like polysomy, heterogeneity ,28 and cThe stability and maturation of Her2 has been shown to be mediated by so called \u201cmolecular chaperones\u201d belonging to the family of heat shock proteins (HSPs) ,9. HSPs Inhibitors of HSP90 including Geldanamycin and its derivates  have already entered clinical application \u201359. In gWe could verify the postulated association between Her2 and HSP90 expression on the tissue level, and could demonstrate the prognostic role of Her2/HSP90. This points towards a co-regulation of both molecules in vivo. Furthermore, elevated HSP90 levels may render tumors susceptible for anti-HSP90 directed therapy, a prerequisite met by one third of cases of our case collective. Considering recent pharmaceutical advances, either combination therapy with conventional drugs could be a possible approach, or - with regard to the high association with Her2 expression \u2013 also and especially as additional approach to anti-Her2 therapy . AccordiIn summary, we could demonstrate immunoreactivity for Her2 and corresponding gene amplification in a significant subset of gastric and gastroesophageal adenocarcinomas. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity on the one hand and the definition of Her2 status in gastric cancers on the other hand. Moreover, the correlation with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation of these molecules. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.Table S1(XLS)Click here for additional data file."}
+{"text": "NAT2 variant alleles might exist. We analyzed the in vivo acetylation phenotype and genotype in 504 north-American subjects of Caucasian origin. The analyses of the SNPs rs1801280 and rs1799930 allowed the discrimination of five categories with different acetylation status within the study population. These categories are related to the distinct effect of NAT2 alleles on the acetylation status in vivo and to the occurrence of a gene-dose effect. These five phenotype categories, from higher to lower acetylation capacity, correspond to the genotypes NAT2*4/*4, NAT2*4/*5 or *4/*6, NAT2*5/*5, NAT2*5/*6 and NAT2*6/*6 . The NAT2*6/*6 genotype correspond to a phenotype category of very-slow acetylators. The refinement in phenotype prediction may help to identify risks associated to phenotype subcategories, and warrants the re-analysis of previous studies that may have overlooked phenotype subcategory-specific risks.Indirect evidences suggest that acetylation phenotype categories are heterogeneous and that subcategories, related to specific  NAT2 genotype or phenotype) was initially proposed to predict adverse reactions in patients with tuberculosis receiving isoniazid, prior to the concomitant administration of procainamide and phenytoin, and to analyze the role of NAT2 in drug interactions. These effects, together with the role of NAT2 in cancer risk, in non-malignant spontaneous disorders and in drug response and toxicity, make NAT2 a relevant target for pharmacogenomic testing in clinical practice The widespread use of genetic biomarkers as surrogate endpoints aiming to describe risks, exposures, intermediate effects of treatments, and biologic mechanisms is a goal that scientists have long been pursuing. The adoption of any genetic test as a surrogate biomarker requires previous demonstration of its analytical and clinical validity as well as its clinical utility, and increasing the predictive capacity of genetic biomarkers is one of the major problems that we have to solve in order to transfer advances in pharmacogenomics to routine clinical practice. Determination of the polymorphic acetylation  in agreement with previous studies NAT2 genotyping aimed to identify the signature SNPs for alleles corresponding to the NAT2*5, NAT2*6, NAT2*7 and NAT2*14 clusters, that is, rs1801280 (I114T), rs1799930 (R197Q), rs1799931 (G286E) and rs1801279 (R64Q), respectively. Although several NAT2 alleles have been described , the SNPs analyzed in this study identify the vast majority of slow NAT2 variant allele clusters NAT2 haplotype table described elsewhere p value <0.05 was considered significant. When multiple comparisons were made, adjustments for multiple comparisons were carried out according to Bonferroni\u2019s procedure.NAT2*4 containing genotypes are considered a rapid phenotype, and other genotypes a slow phenotype, was equal to 93.7%. We selected 435 individuals with genotypes NAT2*4/*4, *4/*5, *4/*6, *5/*5, *5/*6 and *6/*6 and phenotype/genotype concordance for further analyses. These corresponded to 73 cases and 362 control subjects. Carriers of variant alleles NAT2*14 were not included in the analyses, because these alleles were not present in the study population ; P<0.001). These findings were not influenced by the sex of participants, age, smoking status, pack-years, drinking status, servings of alcohol per week as stated by multivariable linear regression, or by the case-control status , is close to the mean value for carriers of the NAT2*6/*6 genotypes, thus suggesting that the NAT2*7 alleles in homozygosity may confer a very slow acetylation phenotype; although due to the sample size the comparisons of the acetylation phenotype were not statistically significant. NAT2*7.l status . The effin vivo. Indirect evidence from in vitro studies and from clinical association studies suggest that NAT2 variant alleles produce different functional effects, implying heterogeneity within the \u201cslow\u201d acetylator phenotype NAT2*6/*6 allele, thus suggesting that these individuals may constitute a subcategory of \u201cvery slow\u201d acetylators NAT2 variant alleles and in vivo phenotype categories among slow acetylator individuals has been proved so far. Our findings indicate that the NAT2*6 allele cluster is related with the slowest acetylation capacity in vivo with a gene-dose effect, thus demonstrating the occurrence of a category of \u201cvery slow acetylators\u201d with the genotype NAT2*6 in homozygosity. Because of the ethnic origin of the population study, we were unable to dissect the effect of the allele clusters NAT2*7 and NAT2*14; it should, however, be emphasized that these clusters are rare in caucasian populations Differential effects of acetylation status by different slow acetylation alleles have been suggested previously, but to our knowledge they have not been formally evaluated NAT2 variant alleles may vary by substrate or with substrate concentration NAT2*7 allele cause a different effect in the N-acetyltransferase activity towards 2-aminofluorene and to sulfamethazine NAT2 substrates without confirmation with every specific substrate. Nevertheless, our findings in vivo agree with findings obtained in vitro which suggests that the protein level expressed by common NAT2 alleles is NAT2*4>NAT2*5>NAT2*6NAT2 alleles observed with the probe drug caffeine is likely to be relevant to other NAT2 substrates.The effect of NAT2 genotyping and the identification of clinically relevant associations of the new genotype categories with cancer risk, differential treatment response or clinical outcome are beyond the aims of the study. Although this study included patients with cancer of the exocrine pancreas and control subjects, no association of NAT2 genotype categories with pancreatic cancer risk was observed, in agreement with previous studies The aims of this study are to refine the phenotype inference of in vivo is related to different NAT2 genotypes among slow acetylators, and indicate that variations in the acetylation NAT2 status among slow acetylator individuals result from the co-dominant expression of the NAT2*5 and NAT2*6 alleles or haplotypes, whose diplotypes are related to distinct slow acetylation phenotypes. Additional studies are required to go further in the refinement in phenotype inference, particularly in other human populations with different NAT2 allele frequencies. It may be argued that the difference in function between the variants NAT2*5 and NAT2*6, although statistically significant, is a minor difference compared to the function of any genotype containing at least one NAT2*4 allele and therefore that the clinical relevance of this difference may be limited. However, NAT2*6/*6 homozygotes show roughly a 30% reduction on enzyme activity as compared to NAT2*5/*5 homozygotes. For comparison, the reduction on enzyme activity between NAT2*4 heterozygotes (intermediate acetylators) and NAT2*4/*4 homozygotes (rapid acetylators) in this study is 28%. A 30% reduction in activity among individuals who have a very impaired acetylation capacity may have a higher clinical relevance than a comparable reduction among individuals who have a high acetylation capacity. These findings provide a novel framework for evaluating interactions between NAT2 genotype and adverse drug reactions or cancer risk.The findings reported in this study show that acetylation capacity Table S1Details of the genotyping procedures used in the present study.(DOCX)Click here for additional data file.Table S2Comparison of the Acetylation ratios (log AFMU/1X) in healthy subjects with different NAT2 genotypes.(DOCX)Click here for additional data file.Table S3Details of the acetylation ratios of individuals carrying NAT2*7.(DOCX)Click here for additional data file."}
+{"text": "The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections. E6 contributes to the maintenance of the HPV genome. We demonstrate that E6 must inactivate the cellular factor, p53, for the viral genome to be maintained. Significantly, p53, is inactivated in many types of human cancers and because much research has been done on p53, promising new drugs have been identified that can re-activate p53. If such drugs can re-activate the p53 that has been inactivated by E6, then we hypothesize that these drugs could be used to cure patients with persistent HPV infections and thereby reduce their risk of developing HPV associated cancers.Human papillomaviruses (HPVs) infect epithelial tissues. HPVs that infect mucosal epithelia cause infectious lesions in the anogenital tract and oral cavity. HPV infections are normally cleared by the immune system; however, in rare cases, infections can persist for years. Persistent infections by certain HPVs place one at a high risk of developing carcinomas of the cervix, other anogenital tissues, and the head/neck region. These HPVs are responsible for over 5% of all human cancers. For an HPV infection to persist, the viral circular genome must be maintained, i.e. replicated and inherited during cell division. In this study we define the mechanism by which the viral gene  Human papilloma viruses (HPVs) are small, non-enveloped icosahedral viruses that infect epithelial linings of the body and are the causative agents of warts. Infection is thought to arise when a virus particle enters a dividing basal epithelial cell, accessed through a wound in the epithelia, wherein its viral genome is delivered to the nucleus and viral genes begin to be expressed in vitro using short-term replication assays cis element required for papillomavirus DNA replication, i.e. the viral origin of replication (ori), it has been demonstrated that two papillomaviral genes, E1 and E2, are required for the establishment phase for multiple papillomaviruses including bovine papillomavirus type 1 (BPV1) as well as HPV6b, 11, 16 and 18 E6 is required for maintenance of HPV11, 16 and 31 E7 is only required for maintenance of HPV11 and 31, but is dispensable for maintenance of HPV16 and 18 E1\u2227E4 is important for maintenance of HPV16, but is dispensable for maintenance of HPV11, 18 and 31 E1 is required for establishment of HPV16, expression of E1 has been shown recently to be dispensable for maintenance of HPV16 E7 plays a critical role in amplification of HPV16 and HPV18 E6 is essential for robust amplification of HPV18 E1\u2227E4 and E5 have been shown to contribute quantitatively to this phase of viral DNA replication of HPV16 and HPV31 The papillomavirus genome is an 8 kB circular double-stranded DNA that replicates as a nuclear plasmid in three distinct phases referred to as the establishment, maintenance and productive phases Identifying viral and cellular genes of importance to different stages of the HPV replicative life cycle may help define new strategies for treating persistent HPV-infections. In this study, we sought to identify the roles of E6 that are necessary and sufficient for high risk HPV maintenance in the context of the entire HPV genome. The HPV E6 protein consists of approximately 150 amino acids and contains two zinc finger motifs. High risk HPV E6 proteins also contain a PDZ binding domain at the C terminus E7, differs in its requirement for maintenance between different high-risk HPV types Previous studies of E6 mutants within the context of the HPV31 genome indicated that the ability of 31E6 to degrade p53 is required for the maintenance of HPV31 E6 gene to be maintained as an extrachromosomal nuclear plasmid in normal immortalized human keratinocytes (NIKS), cells that retain wild type p53 To test our hypothesis, we analyzed the capacity of the HPV16 genome carrying various mutations in the Full-length clones of HPV16, HPV18 and HPV31 were subjected to site directed mutagenesis to generate mutations within the E6 open reading frame (ORF) as detailed in NIKS  and primary human foreskin keratinocytes (HFKs) have been previously described by and were obtained from Lynn Allen-Hoffman Total genomic or low molecular weight DNA was isolated from cells and subjected to HPV-specific Southern analysis as detailed in To assess the function of p53 (Entrez Gene ID: 7157) in cells, we monitored the responses of cells to actinomycin D. Briefly, cells treated with 0.5 nM\u20135 nM actinomycin D (Sigma) or vehicle (dimethyl sulfoxide (DMSO) (Sigma)) for 24 hours were harvested, fixed in ice cold ethanol, stained with propidium iodide, subjected to flow cytometry using a Becton Dickenson FACSCalibur, and cell cycle profiles analyzed using Flow Jo Version 9.4.11 software. The G1/S ratio was calculated by dividing the percentage of cells in G1 by the percentage of cells in S phase. The magnitude change in the G1/S ratio of control NIKS after vehicle and actinomycin D treatment was compared to the magnitude change in the G1/S ratio of experimental NIKS after vehicle and actinomycin D treatment using the Sen-Adichie test for parallelism in MSTAT version 5.5.1 software. As another means of assessing p53 function, steady state levels of p21 (Entrez Gene ID: 1026) were determined by western blot analysis of cells likewise treated with actinomycin D or vehicle. Further details on these actinomycin D-based experiments are provided in To determine the requirements of E6 in the establishment and maintenance of the HPV16 genome, we introduced various mutations within the E6 gene in the context of the full-length wild type HPV16 genome. To determine if E6 was required for establishment and/or maintenance of the HPV16 genome as a nuclear plasmid, a stop codon at amino acid 7 was introduced by mutating nucleotide 122 of HPV16 from G to T to create the HPV16 E6STOP mutant genome. The HPV16 E6WT and HPV16 E6STOP genomes were excised from their bacterial vector, re-circularized and each was transfected with a plasmid expressing an antibiotic resistance gene into NIKS. Drug resistant colonies arising 2\u20133 weeks after selection were pooled to generate a population of cells (referred to as passage 0) and were further passaged. Total genomic DNA was harvested from the expanded populations of cells at early passages (passage 1 and 2) and analyzed by Southern hybridization using an oligonucleotide probe set specific for HPV16. To determine the presence of mammalian replicated, extrachromosomal HPV16 genomes, total genomic DNA was digested overnight with BamHI plus DpnI or XhoI alone. Using these restriction enzyme digestions, we were able to: 1) discriminate HPV16 genomes that had replicated in the human cells from input transfected DNA because the latter is selectively sensitive to DpnI digestion, 2) determine if the viral genome was being maintained as an extrachromosomal plasmid by looking for the presence of circular viral DNA genomes in the samples cut with XhoI (non-cutter of HPV16), 3) determine if the viral DNA is integrated by looking for non-unit length viral DNA fragments in the samples cut with BamHI (single cutter of HPV16 - data not shown), and 4) estimate copy number of the viral genome. The HPV16 E6WT genome was capable of replicating extrachromosomally in 56% of populations analyzed, but the HPV16 E6STOP genome was deficient in its ability to replicate extrachromosomally based upon the absence of detectable circular viral genomes . These rSince the HPV16 E6STOP genome contains a stop codon at amino acid 7, this should also inhibit expression of E6 splice products, E6*I and E6*II After determining that E6 was required for stable maintenance of the HPV16 genome, we were interested in identifying the activities of 16E6 that are necessary for stable maintenance of HPV16. To this end, we examined the maintenance of HPV16 genomes containing E6 mutants that differed in their ability to reduce p53 steady state levels. Compared to 16E6WT, the 16E6 R8S/P9A/R10T (E6SAT) mutant is deficient for binding p53, decreasing p53 steady state levels, inhibiting p53-dependent transactivation and attenuating a p53 dependent G1/S growth arrest We were also interested in determining if the C terminus of E6, which is involved in binding PDZ domain containing proteins To ascertain if p53 was functional in NIKS harboring the viral genomes as extrachromosomal, nuclear plasmids  or in NIKS harboring viral genomes in the integrated state (HPV16 E6SAT), previously frozen populations of NIKS transfected with these genomes were thawed and individual colonies were isolated and expanded. Southern blot analysis confirmed the genomic status of the HPV16 mutant genomes in these clonal populations . As withGiven this result, we wanted to determine the functional status of p53 in these HPV-positive epithelial cells. p53 function can be tested by measuring the response of cells to actinomycin D. Actinomycin D induces a p53-dependent G1 growth arrest and HPV16 E6 can inhibit this actinomycin D-induced growth arrest p21 and HDM2In order to determine if 16E6's inactivation of p53 is sufficient to account for E6's role in plasmid maintenance, we created clones of NIKS transduced with a dominant negative, deletion mutant form of p53 that encodes only amino acids 1\u201314 and 302\u2013390 of the mouse p53 protein (p53DD) Functional studies were then performed to confirm that p53DD inhibited p53WT in NIKS. We first analyzed the steady state levels of a p53 target gene, p21, to see if p53DD could attenuate p53-dependent transactivation. After plating NIKS and treating cells for 24 hours with vehicle, 0.5 nM actinomycin D or 5 nM actinomycin D, total cell lysates were harvested and steady state levels of p21 were analyzed by western blot. The steady state levels of p21 increased in an actinomycin D dose dependent response in empty vector transduced NIKS, but not p53DD transduced NIKS . We alsoWe next asked if cells expressing p53DD could rescue maintenance of HPV16 E6 mutant genomes that were deficient for stable maintenance in parental NIKS. NIKS transduced with p53DD or NIKS infected with the vector only were transfected with wild type or E6 mutant  genomes and expanded populations were analyzed by Southern hybridization. While empty vector transduced NIKS supported maintenance of only the HPV16 E6WT genome, p53DD transduced NIKS supported maintenance of HPV16 E6WT, E6STOP and E6SAT mutant genomes . Thus, iWe were interested in determining if inactivation of p53 was sufficient to support maintenance of additional high risk HPVs, HPV18 and 31 in the absence of E6. To test this, empty vector and p53DD transduced NIKS were transfected with HPV31 E6WT, HPV31 E6STOP, HPV18 E6WT or HPV18 E6STOP genomes. In empty vector transduced NIKS, HPV31 E6STOP genomes replicated extrachromosomally at early passages  with a drug resistance gene and either the HPV18 E6WT or E6STOP genome. Southern blot analysis of low molecular weight DNA from these cells, taken at 5.5 weeks post transfection when the cells had expanded sufficiently, demonstrated that HPV18 E6WT and E6STOP reproducibly replicated extrachromosomally in HFKs . As obseOur studies demonstrate that HPV16 E6 is required for maintenance of the HPV16 genome as an extrachromosomal nuclear plasmid and that inactivation of p53 by dominant negative p53 (p53DD) is sufficient to support maintenance of the HPV16 genome in the absence of E6 . InactivWhile inactivation of p53 was necessary for maintenance of HPV16 extrachromosomal genomes, decreased steady state levels of p53 were not necessary since the HPV16 E6I128T mutant is maintained extrachromosomally yet is deficient for decreasing p53 steady state levels . While tOur results also demonstrate that the PDZ binding domain of 16E6 is not required for stable maintenance of HPV16 because the HPV16 E6\u0394146\u2013151 (deletion of nucleotides 539\u2013556) does not contain the PDZ binding domain yet is maintained extrachromosomally . Others Consistent with a previous report that HPV31 E6 is required for maintenance of HPV31 as extrachromosomal replicons Our detection of replicated HPV18 E6STOP and HPV31 E6STOP in early passages of NIKS demonstrates that E6 is not absolutely required for the establishment of the HPV18 and HPV31 genome as an extrachromosomal replicon. This is not surprising as it has been previously shown that the papillomaviral E1 and E2 proteins are sufficient to drive replication of plasmids containing the origin of papillomavirus DNA replication Interestingly, ectopic expression of p53 attenuates establishment replication of BPV-1, HPV-16 and HPV-18 origin of replication in cells also expressing the respective E1 and E2 genes but does not affect maintenance of at least the BPV-1 origin of replication Inactivation of p53 not only restored maintenance of HPV16 in the absence of 16E6, but also increased the efficiency of maintenance of HPV16 E6WT genomes: the HPV16 E6WT genome was maintained in 38% of empty vector populations vs 82% of p53DD populations . One intInactivation of p53 may be important to prevent apoptosis or senescence induced by the presence of the HPV genome and consequently E6 may prevent the loss of cells stably retaining HPV genomes. HPV-induced apoptosis or senescence could be triggered by the induction of DNA damage responses (DDR). During establishment of HPV18, Reison et al. demonstrated that the HPV18 genome co-localizes with \u03b3H2AX, a marker of DDR, Alternatively, but not necessarily exclusively, HPV E6 may need to inactivate of p53 in order to attenuate an interferon-mediated innate immune response that could inhibit stable maintenance of HPV. An interplay between p53 and the interferon response pathway has been previously linked to inhibition of Sendai virus and vesicular stomatitis virus replication and may have similar effects on HPV replication Future work will be required to determine the exact reason behind the requirement of p53 inactivation for maintenance of HPV16 and stable maintenance of HPV18 and 31 and to identify additional roles of E6 that may contribute to maintenance of these genomes. Since p53WT negatively impacts establishment, maintenance and amplification of HPV papillomavirus genomes, it will be of interest to determine if p53 inhibits these different stages of replication in similar or different ways. Regardless, our results raise the interesting concept that drugs that can reactivate p53 in HPV-infected cells should be effective at eliminating persistent high-risk HPV infections and thereby reduce the risk of HPV-associated cancers in infected patients.Text S1(DOC)Click here for additional data file."}
+{"text": "The seventh sentence of the sixth paragraph in the Discussion section should have cited references 41 and 52 instead of 5 and 52.The correct sentence should read: Another promising discovery has been the finding that curcumin, derived from the dietary spice turmeric, is an effective inhibitor of A\u03b2 aggregation [41], [52]."}
+{"text": "Resistance in virus-infected stem cell transplant recipients illustrates the need for surveillance. >4 days of oseltamivir therapy. Three of these 4 patients were critically ill. Oseltamivir resistance in 4 (13.3%) of 30 patients who were administered oseltamivir highlights the need for ongoing surveillance of such resistance and further research on optimal antiviral therapy in the immunocompromised.We describe laboratory-confirmed influenza A pandemic (H1N1) 2009 in 17 hospitalized recipients of a hematopoietic stem cell transplant (HSCT)  and in 15 patients with malignancy treated at 6 Australian tertiary centers during winter 2009. Ten (31.3%) patients were admitted to intensive care, and 9 of them were HSCT recipients. All recipients of allogeneic HSCT with infection <100 days posttransplantation or severe graft-versus-host disease were admitted to an intensive care unit. In-hospital mortality rate was 21.9% (7/32). The H275Y neuraminidase mutation, which confers oseltamivir resistance developed in 4 of 7 patients with PCR positive for influenza after  The 5 centers in Melbourne provide most specialist adult hematology services to the state of Victoria (population 5.4 million), and 2 of these centers perform all allogeneic HSCTs for the state. The participating Sydney center is the largest of 2 centers that perform adult allogeneic HSCT for the state of NSW (population 7 million). Approval for this study was obtained from human research ethics committees of each center.>18 years of age hospitalized at 1 of the 5 Melbourne study centers during May 1\u2013August 30, 2009, or hospitalized at the Sydney center during May 1\u2013September 15, 2009; 2) recipient of an HSCT or had an underlying malignancy (hematologic or solid tumor); and 3) had laboratory-confirmed pandemic (H1N1) 2009 virus infection identified by nucleic acid testing (NAT) during their hospital stay. At each center, investigators were directly involved in active surveillance and management of patients with pandemic (H1N1) 2009, allowing cases to be identified. Data were retrospectively abstracted onto standardized case record forms.Patients were included in the study if they had the following characteristics: 1) ,All cases were confirmed in laboratories whose performance is accredited by the National Association of Testing Authorities in Australia. For the Victorian cases, all but 1 case-patient had laboratory confirmation of pandemic (H1N1) 2009 at the Victorian Infectious Diseases Reference Laboratory (VIDRL) with pandemic (H1N1) 2009\u2013specific NAT by using reverse transcriptase\u2013PCR (RT-PCR) as described , and descriptive statistics were summarized with proportional outcomes. Nosocomial acquisition was defined as the development of symptoms attributable to pandemic influenza after 48 hours in the hospital that were not present on admission. Steroid-refractory or grade III\u2013IV graft-versus-host-disease (GVHD) was defined as severe GVHD.,Length of stay was calculated as length of hospital admission or period of confinement after onset of symptoms for those with nosocomial acquisition. Epidemic curves for the 2 states were aligned with epidemic curves of community pandemic influenza activity ascertained from surveillance data  2009, except patient 11, who had multiple myeloma that had been diagnosed 3 years previously. All autologous HSCT recipients had a relapse of malignancy after transplantation, and 7 of these patients were continuing to receive active treatment for malignancy.At the time they sought treatment, all 8 recipients of autologous HSCT were Two patients (26 and 29) who had had recent a diagnosis of malignancy were in remission and continuing on the primary chemotherapy treatment plan. Seven nontransplant recipients had either not shown a response or were deemed by their treating clinicians to be refractory to their current treatments. Patient 17 had an influenza-like illness (ILI) before admission for chemotherapy and autologous HSCT (further details below). Sixteen patients  were receiving corticosteroids before the onset of pandemic (H1N1) 2009 infection. The mean dose in prednisolone equivalents was 40.9 mg/d (range 5.0\u2013156 mg/d).Clinical features at patients\u2019 initial visit coincided with peak activity of pandemic (H1N1) 2009 in the Victorian and NSW communities . PatientMost patients exhibited fever (94%) and cough (91%). Dyspnea, sore throat, and rhinorrhea were reported in 53%, 50%, and 50% of patients, respectively. Sixteen patients (50%) sought treatment within 48 h of symptom onset. Four patients visited the hospital after experiencing prolonged symptoms: for 12 d (patient 4), 14 d (patients 22 and 25), and 21 d (patient 14). Three of these patients had biphasic illnesses with initial prolonged mild upper respiratory tract illnesses and, on examination, had consolidation shown on chest radiograph. Patient 25 had 2 weeks of dyspnea associated with an exacerbation of chronic obstructive pulmonary disease. Patient 16 sought treatment for shock and respiratory failure.<200 cells/mL, and 18 (56%) patients had pneumonia shown on baseline chest radiograph.Diagnosis of pandemic (H1N1) 2009 was confirmed by NAT of bronchial washings of 3 patients . One of them, patient 17, had symptoms of a viral respiratory tract infection for 20 d with negative results for pandemic (H1N1) 2009 virus by RT-PCR of nasopharyngeal swab specimens taken 2 and 16 d before bronchoscopy. All other patients had the diagnosis confirmed on NAT of nasopharyngeal swab samples. Six patients (19%) had a baseline peripheral blood lymphocyte count Thirty patients (94%) were administered oseltamivir therapy. Of these patients, 13 (41%) were administered therapy within 48 hours, and 16 began therapy >48 hours after symptom onset. Duration of illness before receiving oseltamivir was unknown for patient 2. Nine patients received NA inhibitors for >5 days. Three of these patients  had a positive NAT result >5 days after oseltamivir treatment began. Antimicrobial drug treatment was changed to zanamir for patients 1 and 8 after they received initial therapy with oseltamivir (see below). The longest duration of antiviral therapy was 43 d (patient 1). Patients 15 and 31 had uncomplicated infections and were not administered oseltamivir therapy.Staphylococcus aureus bacteremia from an unrelated source. Patient 17, who came to transplant with an ILI, had bronchial washings that were positive for pandemic (H1N1) 2009 by NAT, galactomannan antigen, and Aspergillus spp. by NAT 5 days after transplant. Pulmonary nodules consistent with invasive fungal infection were seen on a high-resolution computed tomography scan. Patient 13 was infected with respiratory syncytial virus, which was detected by multiplex NAT on a nasopharyngeal swab.Four patients had notable co-pathogens identified. Patient 2 had 4 concurrent herpesviridae  detected by NAT in respiratory specimens and blood. Treatment for patient 2 included liposomal amphotericin B and foscarnet. Patient 7 had Ten (31.3%) patients were admitted to intensive care . In eachAll allogeneic HSCT recipients with the following features required admission to intensive care for mechanical ventilation: transplantation within 100 d, severe GVHD, and nosocomial acquisition of pandemic (H1N1) 2009. However, onset of symptoms for patient 1 was day 119 after allogeneic transplantation.Eight of 10 patients admitted to intensive care had evidence of pneumonia on baseline chest radiograph. Patient 1 initially had normal chest radiograph results, despite the acute onset of hypoxia. Patient 24 was transferred to intensive care after 3 days in the hospital, at which point bilateral infiltrates were seen on chest radiograph, and oseltamivir therapy was begun.>1 further positive NAT  after receiving oseltamivir. Five of these patients had a positive NAT after >5 d of oseltamivir therapy. The longest recorded duration of viral shedding during oseltamivir therapy was 28 d (patient 1).Ten patients had repeat NAT testing to determine clearance of viral shedding. Eight had >4 d of oseltamivir therapy. These 4 patients comprised 13.3% of the 30 patients who received oseltamivir. The findings for the 4 patients who were infected with oseltamivir-resistant influenza virus are summarized in The H275Y NA mutation, a substitution known to confer a high level of oseltamivir resistance, was detected in 4 (57%) of 7 patients who had detectable nucleic acid after Patient 12, who survived despite shedding oseltamivir-resistant pandemic (H1N1) 2009 virus, had a biphasic clinical course. She initially stabilized while she was treated with oseltamivir; progressive respiratory failure then developed, which coincided with her recovery from neutropenia before later improvement. The other patient who survived despite shedding oseltamivir-resistant 2009 pandemic (H1N1) virus (patient 20) had an ILI without pneumonia. Of the 2 patients with resistant isolates who died, patient 5 first experienced the apparent resolution of pneumonia but later succumbed to recurrent pneumonia, and patient 1 had persisting pneumonitis despite 36 days of oseltamivir therapy.Patient 24 had multiple positive NATs, none with the H275Y mutant detected. His NAT was positive 4 d after he began oseltamivir therapy, and he died 1 d after his last specimen, a nasopharyngeal swab, was collected.We report a case series of hospitalized cancer patients with influenza A pandemic (H1N1) 2009 virus infection and their outcomes. Patients with hematologic malignancies accounted for 50% of deaths of persons with pandemic influenza in Victoria during the first 3 months of the pandemic  and chronic lymphocytic leukemia (n = 4). There was also a paucity of patients with solid tumors (n = 2). The severity of illness from influenza in patients who have underlying multiple myeloma and chronic lymphocytic leukemia has not previously been widely appreciated, although a high rate of pneumonic illness from influenza has previously been demonstrated at 1 center  2009 has been described in another immunocompromised patient, a renal transplant recipient  2009\u2013specific RT-PCR result after Only 1 of the patients who was in intensive care and had oseltamivir-resistant pandemic (H1N1) 2009 virus survived. The effects of oseltamivir-resistant influenza virus in this patient are uncertain. The deterioration of her condition after initial stabilization may have been related to development of oseltamivir-resistance and persistent influenza viral replication similar to that seen in a recent case report  2009 infection in immunocompromised patients.A high rate of oseltamivir resistance developed in critically ill HSCT recipients, particularly in those who continued to shed pandemic (H1N1) 2009 virus after 4 d of oseltamivir treatment. Consequently, surveillance, infection control measures, and early treatment of those at greatest risk of pandemic (H1N1) 2009 infection may prove more useful than universal chemoprophylaxis during an outbreak with an influenza strain that is not contained in the available influenza vaccine. Continued surveillance for oseltamivir-resistant influenza virus strains is needed, particularly in the immunocompromised.Oseltamivir Resistance in Adult Oncology and Hematology Patients Infected with Pandemic (H1N1) 2009 Virus, Australia"}
+{"text": "Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that  A critical stage in vertebrate sex determination is entry of the bipotential gonadal primordium into a developmental pathway leading to ovary or testis  and RA-degrading enzymes (i.e. members of the Cyp26 P450-cytochrome family) determines the precise spatial-temporal distribution of RA [Cyp26b1 expression leads to degradation of RA and protects germ cells from entering into meiosis in developing testes, while female-specific down-regulation of Cyp26b1 expression allows RA to induce germ cells to enter into meiosis in embryonic ovaries [Cyp26b1 expression in embryonic mouse testes, or addition of CYP inhibitors to wild-type embryonic testes, induced germ cells to express the pre-meiotic marker Stra8 (stimulated by retinoic acid-8), which is required for the initiation of meiosis in mammals, followed by the expression of the early meiotic marker Sycp3 , and the down-regulation of the pluripotent stem cell marker Pou5f1  [Recent studies showed that in mouse, retinoic acid (RA) in females or its absence in males -- regulated by the RA-destroying Cyp26 Cytochrome P450 enzyme family -- reinforces germ cell fate by controlling the sex-specific initiation of meiosis  ,24,35\u201345Aldh1a2 paralog Aldh1a1 has been reported in somatic cells of embryonic mouse testes [In mice, expression of the RA-synthesizing enzyme Aldh1a2 in the embryonic mesonephros (but not in the gonads) as females initiate meiosis led to the suggestion of a source-sink regulatory system. According to this model, RA synthesized in the mesonephros enters the neighboring gonad and causes germ cells to enter meiosis in embryonic ovaries, which lack the RA-degrading enzyme Cyp26  have been lost in zebrafish and other teleosts, and that this gene loss has altered the functional evolution of the surviving aldh1a paralogs [RA plays a role in the onset of meiosis not only in mammals, but also in other tetrapods, including birds and amphibians \u201349. Whetparalogs \u201357. WhetTo address these questions and to test the hypothesis that meiotic control mechanisms in mouse apply to zebrafish, we performed a comprehensive genomic and expression analysis of the genetic machinery that regulates the synthesis and degradation of RA during gonadogenesis, and studied genetic markers for meiosis and somatic cells of the gonad to investigate the role of RA in the onset of meiosis and sexual fate determination in zebrafish. Results revealed shared underlying regulatory themes between zebrafish and mammals but important genomic and developmental differences in the mechanisms of RA-regulated gonadogenesis and sex determination.Cyp26 paralogs: Cyp26a1, Cyp26b1, and Cyp26c1. Recent studies in mouse [cyp26b1 occurs during gonad development in zebrafish as it does in tetrapods.Tetrapods and zebrafish have three in mouse ,24, chicin mouse ,49 and uin mouse  implicatyp26b1, as well as the other two gene family members cyp26a1 and cyp26c1, in adjacent histological sections of developing zebrafish gonads by in situ hybridization (ISH)  [cyp26b1 was detected only in a few scattered cells in undifferentiated gonads at 15 dpf  and in the retina of 15 dpf juveniles. These results revealed that, in contrast to tetrapods, in which cyp26b1 is the major regulator of RA availability during early gonad development, cyp26a1 is the only paralog up-regulated in developing zebrafish gonads during the time of sexual differentiation of the gonads.To address this question, we investigated expression patterns of con (ISH) . Zebrafimination , became mination , and wasmination . This re- 31dpf) ,58\u201360. Ut 15 dpf , and in analyzed , althougCyp26 gene before the two rounds of vertebrate genome duplications ; alternatively, it might be that zebrafish gene orthologies had been incorrectly assigned. To rule out the possibility of error in orthology assignments, we examined gene orthologies by first investigating the phylogenetic relationships between zebrafish Cyp26a1 [The apparent convergence of the roles of Cyp26a1 in zebrafish and Cyp26b1 in mouse could have either of two explanations: first, it might represent a case of independent subfunctionalization of gene roles possessed by the ancestral  Cyp26a1 , Cyp26b1 Cyp26a1  and Cyp2 Cyp26a1  (origina Cyp26a1 ) and the Cyp26a1 \u201367. Phyl Cyp26a1 .Cyp26 genes would have been produced in VGD1 and VGD2 [Cyp26 genes, raising the possibility that different VGD paralogs were lost in the teleost and tetrapod lineages.Phylogenetic analyses can sometimes give erroneous orthology assignments in cases of reciprocal gene loss after ancient gene duplications \u201370; for and VGD2 , but zebCyp26 genes in zebrafish and mouse , and Cyp26a1 and Cyp26c1 are adjacent and transcribed in the same orientation on Mmu19, consistent with the origin of Cyp26a1 and Cyp26c1 by tandem duplication rather than by genome duplication. In teleosts, however, the three Cyp26 paralogs are on three different chromosomes (i.e. cyp26a1 on zebrafish (Danio rerio) linkage group 12 (Dre12) and on medaka (Oryzias latipes) chromosome 19 (Ola19); cyp26b1 on Dre7 and Ola18; and cyp26c1 on Dre17 and Ola15). Genomic analysis at the chromosomal level using circleplots [Cyp26b1 genomic neighborhood connected to teleost cyp26b1 neighborhoods; and the mouse Cyp26a1/Cyp26c1 neighborhood connected to teleost neighborhoods that harbor cyp26a or cyp26c1, respectively  surrounding nd mouse  and medand mouse . In mouscleplots  revealedectively . Local aith Dysf , cyp26a1th Pde6c , and cyp Tbc1d12 . These rcyp26a1 in Dre12 and cyp26c1 in Dre17 revealed that these two regions are indeed paralogons  event , which encode RA synthesizing enzymes. Because no robust genetic marker for sex is yet available for zebrafish [vasa [in situ hybridization analyses on adjacent gonadal sections from animals representing three key stages of gonad development: i) bipotential gonads (20 days post-fertilization (dpf)) when gonads are sexually undifferentiated and can become either ovary or testis  trar testis ; and iiialdh1a2 was expressed throughout most cells of the ovary-like gonad of juveniles, but was not expressed in the largest germ cells, which were probably developing oocytes judging by their morphology , which is the only other remaining aldh1a paralog in zebrafish due to the loss of aldh1a1 early in the evolution of the teleost lineage [amh was already expressed, but in only a few scattered somatic cells . Analysis by double fluorescent in situ hybridization in sections of bipotential gonads resolved this question, showing that cyp26a1 was expressed in somatic cells but not in germ cells labeled by vasa  showed broad expression of th sexes . Sex assxes ; green arrowhead in eIB); yellow arrowhead in cyp26a1 in late stage IB is compatible with a potential role of Cyp26a1 in arresting oocytes during meiosis. Expression of cyp26b1 and cyp26c1 was not detected at this crucial stage for gonad fate decision in ovaries or testis  continued to express th sexes . In maleis cysts , judgingli cells , A\u2019. In  oocytes . These rcyp26a1  against dead end (dnd), a gene essential for germ cell survival [vasa verified total depletion of germ cells in dnd-MO-injected animals  cannot be discarded  [Pou5f1(Oct4) in germ cells entering meiosis and the up-regulation of the early meiotic marker Sycp3  [To learn whether the sexually dimorphic expression pattern of zebrafish  gene 8) \u201380, whic gene 8) ,44,45,81otein 3) , which iotein 3) .in silico screening of the zv9 version of the zebrafish genome database [Tetraodon, fugu, medaka) as well as the basally diverging spotted gar genome did not identify Stra8. We did, however, find an ortholog for Stra8  in the genome of the basally diverging chordate amphioxus Branchiostomafloridae, and in the elephant shark [stra8 was present in stem chordates and stem gnathostomes, but was lost secondarily in the ray fin fish lineage [Stra8 in teleosts suggests that if RA plays a role in the regulation of the initiation of meiosis in teleosts, it does not act through Stra8 as it does in mammals [To our surprise, database  by BLASTnt shark , support lineage  at least lineage . Future  mammals .Sycp3 by in situ hybridization in adjacent sections showed that all zebrafish analyzed at the bipotential stage  showed germ cells expressing sycp3 throughout the gonad, while cells expressing cyp26a1 were mostly restricted to the dorsal surface , Cyp26b1 expression up-regulates in males but down-regulates in females [Cyp26b1 in gonadal somatic cells is at a time and place appropriate to protect germ cells from RA, and thereby to prevent oogenesis in males by retarding Stra8 expression and postponing the onset of meiosis until postnatal stages [Stra8 in germ cells, and promotes the onset of meiosis and oocyte development, thereby reinforcing the female pathway  and are entering meiotic arrest in diplotene. This observation, together with the fact that cyp26a1 expression disappears late in oocyte development at stage IV when meiosis resumes, is compatible with the Cyp26a1-mediated degradation of RA in oocytes that prevents the progression of meiosis and maintains meiotic arrest. This finding is consistent with results in mouse, in which RA prevents meiotic arrest in testes of 13.5 dpc embryos [cyp26a1 and the pluripotent marker pou5f1 (oct4). The convergence of cyp26a1 and pou5f1 expression in zebrafish oocytes at meiotic arrest is compatible with the proposed roles of Pou5f1 and Cyp26b1 in mouse germ cells in promoting survival and preventing apoptosis [Interestingly, we observed that zebrafish express ogenesis . In zebrg factor . In fema embryos . Interespoptosis ,86, mechpoptosis .fgf9 gene in zebrafish and other teleosts [wnt4 genes [Overall, this work provides the knowledge base on RA metabolic gene expression necessary for the design of experiments that alter RA signaling in zebrafish developing gonads to test the model stemming from our results for the role of RA signaling in the progress of meiosis. In addition, further work is required to understand the relationship of RA signaling to the Fgf9/Wnt4 seesaw that helps regulate gonad fate in mouse ,94\u201398. Eteleosts ,100 and t4 genes , genomicAB strain zebrafish were used in all experiments. Animals were reared and collected under standard conditions and were handled in accordance with good animal practice. The University of Oregon Institutional Animal Care and Use Committee approved all animal work .dead end as described [dnd MO-injected embryos were fixed at 24 hours post-fertilization to confirm the presence or absence of germ cells by whole-mount in situ hybridization using vasa probe as described [To obtain animals lacking germ cells, wild-type zebrafish embryos were injected at the 1-2 cell stage with antisense morpholino oligonucleotide  directed against escribed . Siblingescribed .In situ hybridization experiments on zebrafish cryosections were performed as described [aldh1a2 and aldh1a3 were made as described [amh probe was made as described and used as a somatic cell marker to distinguish differentiating testes and ovaries when morphological features were not clear [vasa was made from its 3\u2019 end as described [cyp26a1 (nucleotides 507-1848 of NM_131146), cyp26b1 (nucleotides 518-1548 of NM_212666), cyp26c1 (nucleotides 198-1533 of NM_001029951), pou5f1 (oct4) (nucleotides 705-1482 of NM_131112), and sycp3 (nucleotides 265-884 of NM_001040350) were cloned in TOPO vector (Invitrogen) and used to synthesize DIG-labeled riboprobes (Boehringer Mannheim). The protocol for the three-color fluorescent in situ hybridization is described in wiki.zfin.org/display/prot/3+color+Fluorescent+in+situ+on+sections.escribed . Adjacenescribed ; amh proot clear ; and proescribed . FragmenTetrapods: Homo sapiens ; Mus musculus ; Gallus gallus ; Teleosts: Danio rerio ; Takifugurubripes ; Gasterosteusaculeatus ; Oryzias latipes ; Cephalochordates: Branchiostomafloridae ; CYP26A1 and CYP26C1 in Mmu19 ; cyp26a1 in Ola19 ; cyp26b1 in Ola18 ; and cyp26c1 in Ola15 . Gene loci that are close to each other may appear to overlap as a single connecting line in circle-plots due to the selected graph resolution, and lines based on best reciprocal blast hits that were not significantly different were not considered. Clusters of conserved synteny were created by coupling results from the reciprocal best hit BLAST pipeline with the use of a 100-gene sliding-window analysis that links chromosome segments with conserved synteny . Circleerred by ). Each gFigure S1Cyp26 orthologies between zebrafish and medaka were supported by clusters of conserved synteny, which extends conclusions from zebrafish to other teleost models.A: cyp26a1 in Dre12 and Ola19; B: cyp26b1 in Dre7 and Ola18; and cyp26c1 in Dre17 and Ola15. Cyp26 orthologs have been labeled with larger fonts, and names of gene neighbors can be surfed in the high-resolution pdf electronic files.(PDF)Click here for additional data file.Figure S2cyp26a1 and tetrapod cyp26b1 are not orthologs.Comparative genomic analysis of synteny conservation between mouse and medaka supports conclusions from comparison of zebrafish and mouse , ruling out the possibility of reciprocal gene loss in different lineages, and supports the notion that teleost B: cyp26b1 in Mmu6 and Ola18; B: Cyp26a1 in Mmu19 and Ola19; and Cyp26c1 in Mmu19 and Ola15. Large fonts label cyp26 orthologs and names of gene neighbors are legible in the high-resolution pdf electronic files.(PDF)Click here for additional data file."}
+{"text": "SLC2A9 gene having the largest effect identified so far. Gene-gene interactions (epistasis) are largely unexplored in these GWA studies. We performed a full pair-wise genome scan in the Italian MICROS population (n\u200a=\u200a1201) to characterise epistasis signals in SUA levels. In the resultant epistasis profile, no SNP pairs reached the Bonferroni adjusted threshold for the pair-wise genome-wide significance. However, SLC2A9 was found interacting with multiple loci across the genome, with NFIA - SLC2A9 and SLC2A9 - ESRRAP2 being significant based on a threshold derived for interactions between GWA significant SNPs and the genome and jointly explaining 8.0% of the phenotypic variance in SUA levels (3.4% by interaction components). Epistasis signal replication in a CROATIAN population (n\u200a=\u200a1772) was limited at the SNP level but improved dramatically at the gene ontology level. In addition, gene ontology terms enriched by the epistasis signals in each population support links between SUA levels and neurological disorders. We conclude that GWA epistasis analysis is useful despite relatively low power in small isolated populations.Genome-wide association (GWA) studies have identified a number of loci underlying variation in human serum uric acid (SUA) levels with the  Serum uric acid is the final oxidation product of purine metabolism in humans. High serum uric acid (SUA) levels can lead to gout and is associated with cardiovascular diseases and diabetes SLC2A9 gene are the most significant genetic risk factors associating with low fractional excretion of SUA and explain 5.3% of the SUA level variance in women and 1.7% of the SUA level variance in men in the VIS population SUA level is a complex trait that is affected by environmental (e.g. diet and excessive body weight) and genetic factors with heritability estimates of 60\u201387% One possible source of the unexplained variation in SUA levels is gene-gene interaction (epistasis) The Italian MICROS cohort was recruited from the villages in South Tyrol DNA samples were genotyped with Illumina Infinium HumanHap300v1/v2 or HumanCNV370v1 SNP bead microarrays and analyzed using the BeadStudio software. Quality control of the genotype data was performed for each cohort using the R/GenABEL package (Version 1.4.3) rntransform function that is implemented in the GenABEL package performing quantile normalisation of residuals from a generalized linear model analysis. The normalised SUA levels were then analysed using a linear mixed model to correct for polygenic effects and relatedness using the polygenic function in the GenABEL package and the resultant environmental residuals  were used as the trait to test for association In each individual cohort the raw SUA levels were corrected for age, sex and BMI and normalised using the mmscore function in the GenABEL package. The consensus GWA threshold of 7.3 (\u2212log10(5.0E-08)) was applied to identify GWA significant SNPs 1SNP and 2SNP, the following genetic models are used to detect epistasis where genotypes of each SNP  were fitted as fixed factors y is the trait of interest, \u03bc is the model constant, 1SNP (or 2SNP) is a fixed factor with three levels, 1*SNP2SNP is the interaction term, e is the random error term. The F ratio test of Model 1 against Model 3 is for the whole pair effect including interaction . The F ratio test of Model 1 against Model 2 is for the interaction between the two SNPs . SNP pairs with missing joint genotype classes  were not evaluated to reduced the risk of inflation of the type I error rate. P values were calculated based on the F distribution with relevant degrees of freedom and transformed in the \u2212log10 scale . We applied the same \u2212log10 scaled thresholds for both the pairF and intF tests to control the type I error rate A single SNP based GWA scan was performed in each population using a score test method (based on the additive model) implemented in the 10 scale) were derived based on Bonferroni correction for multiple tests, i.e. the 5% nominal P value corrected by the number of pair-wise tests. Considering 300,000 SNPs, a SNP-genome scan and a full pair-wise genome scan perform 3.0E+05 and 4.5E+10 association tests respectively, thus the genome-wide threshold is 11.95 (\u2212log10(0.05/4.5E+10)) for the pair-wise genome scan. SNP-genome scans have been used to test epistasis for genome-wide significant signals specifically to increase the power of detection 10(0.05/3.0E+05/N) if there are N GWA significant SNPs.Genome-wide significance thresholds  were retained. The retained results were evaluated using the predefined thresholds to identify genome-wide significant epistatic signals. Each SNP in the retained results was annotated to the nearest gene within a window of 20 kilobases flanking the SNP based on the physical distances to either the start or end of transcription of genes (the distance is set to zero if the SNP is within a gene) without considering linkage disequilibrium (LD). A full pair-wise genome scan was also performed in the CROATIAN population as above to prepare input for GO enrichment analyses (see below).A full pair-wise genome scan was performed in the MICROS population and SNP pairs with a certain interaction signal  was used to claim significant replication for an epistatic pair (i.e. both replicated SNPs were exactly the same as the epistatic SNPs) because only one test was performed. When an epistatic pair was not replicated (e.g. due to missing genotype classes), we also tested the interactions between each of the adjacent SNPs of the first epistatic SNP and that of the second. In that case, the nominal threshold corrected by the actual number of tests was used to claim significant replication.polygenic function was also used to calculate the proportion of the phenotypic variance in SUA levels explained by epistatic pairs in the MICROS population. Because the quantile normalised SUA levels were based on the quantiles of the raw SUA levels, we used standardized SUA levels for variance calculation ). The standardized SUA levels were fitted into the full mixed model including polygenic effects and the identified SNP pairs. The difference of residual variance from a value of 1 is the proportion of phenotypic variance explained by the SNP pairs included.The 10Ppair>6.5 and \u2212log10Pint>6.5) in each population and used the epistatic genes annotated from them as the target for the GO enrichment analysis to mine biological meanings from epistatic signals that were less significant. The GO terms enriched (P<1.0E-03) by the epistatic genes in each population were compared to identify replicated GO terms and then epistatic genes shared by each pair of replicated GO terms. The shared epistatic genes in the replicated GO terms were investigated further for a) associated biological functions or diseases and b) epistatic SNP pairs involved and their replication.A GO enrichment analysis was conducted for each of the MICROS and CROATIAN populations using the running mode of \u201cTwo unranked lists of genes\u201d in GOrilla SLC2A9 gene. The GWA scan for the CROATIAN population also identified seven genome-wide significant SNPs which were exactly the same as those in MICROS. Thus the genome-wide threshold of 7.62 (\u2212log10(0.05/3.0E+05/7) was used for SNP-genome scans. The inflation factor \u03bb (computed by regression in a quantile-quantile (QQ) plot, The mean SUA level and phenotypic correlation between SUA level and BMI were similar across the MICROS, VIS and KORCULA cohorts . The polpairF and intF tests performed on chromosomes 3 and 4  to illustrate why the two tests are needed (pairF test (e.g. \u2212log10Ppair>11.95 may pick up SNP pairs with very weak interactions (e.g. \u2212log10Pint<3), whereas using only the intF test (e.g. \u2212log10Pint>7.62) could pick up those with weak whole pair effects when both SNPs had small marginal effects (e.g. \u2212log10Ppair<6)  had missing joint genotype classes and hence were ignored in this study. We plotted the P values of all the e needed . Using oPpair<6) . The QQ ir tests  showed ant tests  showed a10Ppair>4.7 and \u2212log10Pint>3.2, 212933 in total) retained from the full pair-wise genome scan . However, we found a big cluster of SNP pairs with strong whole pair effects  but weak to high interactions . This cluster of SNP pairs all involved the SLC2A9 gene as one would expect. Two pairs: rs12130085 (NFIA) \u2013 rs737267 (SLC2A9) and rs737267 (SLC2A9) \u2013 rs9316212 (ESRRAP2) were significant based on the threshold of 7.62 for SNP-genome scans , in total 1326 SNP pairs involved 2063 unique SNPs of which 1148 (55.6%) were annotated to 910 unique genes, i.e. 1.5 pairs per gene. In contrast, 17 out of the 1326 SNP pairs involved the SLC2A9 gene including 4 GWA significant SNPs rs733175, rs737267, rs13131257 and rs13129697  and rs737267 \u2013 rs17064136 (adjacent to rs9316212) were 1.35 and 1.32 respectively. Combining the MICROS and CROATIAN data did not make the two significant pairs stronger (\u2212log10Pint was 2.32 and 2.19 respectively) which is in line with the replication results. The two less significant SLC2A9 epistatic pairs also missed the SNP level replication in the CROATIAN population  in MICROS (910 genes from 1326 pairs) and CROATIAN (984 genes from 1260 pairs) were tested for GO enrichment. The GO terms enriched by epistatic genes in MICROS showed a complicated relationship among biological functions  and highlighted the importance of glutamate receptor activity  (SLC2A9 \u2013 LRRC16A (interaction between two SUA candidate genes) (SLC2A9) \u2013 rs4085921 (GPC6) and rs737267 (SLC2A9) \u2013 rs2302558 (CLEC16A). All the 49 epistatic gene pairs involved SLC2A9 and of these 5 involved interactions with other shared epistatic genes: ERC2, GPC6, CTNND2, CNTNAP2 and PTPRD. However, most of the 49 replicated gene pairs had a relatively weak interaction signal (\u2212log10Pint<5).Epistatic genes annotated from the less significant SNP pairs  . Surprise genes) . Two genSLC2A9 gene may be an important epistatic locus interacting with multiple loci across the genome more frequently than expected by chance (SLC2A9 epistatic pairs (NFIA \u2013 SLC2A9 and SLC2A9 \u2013 ESRRAP2) were significant in SNP-genome scans and jointly explained 8.0% of the phenotypic variance in SUA levels where 3.4% was explained by their interaction components. However, caution should be taken in light of the billions of tests performed, the potential overestimation of the variance explained and the limited replication of the two pairs. The NFIA (nuclear factor I/A) gene is known as a cellular transcription DNA replication factor and its haploinsufficiency is associated with a central nervous system malformation syndrome and ureteral and renal defects NFIA was responsible for celiac disease ESRRAP2  is not yet known to have any related functions.Using a full pair-wise genome-wide association scan we generated a profile of epistasis in SUA levels in the Italian MICROS population. No epistatic SNP pairs reached the Bonferroni adjusted threshold for the pair-wise genome scan, which was not a surprise because of the relatively small population size. Nonetheless, we found that the y chance  and S1. SLC2A9 gene is known to interact with sex, age and dietary patterns SLC2A9 is a class II glucose/fructose transporter as well as high-capacity/low-affinity urate transporter with two isoforms expressed in basolateral and apical membranes of proximal renal tubular cells respectively SLC2A9 polymorphism was demonstrated further in two recent studies linking SUA levels with human cognitive aging SLC2A9 were involved in epistatic interactions, which adds another dimension of studying the SLC2A9 polymorphism and provides new clues of the genetic mechanism underlying SUA levels.The SLC2A9 interacting genes had clear neuronal influence  and/or membrane functions (SLC2A9 and genes that interacted with it (8 out of 910 unique genes in MICROS). The epistatic genes encoding glutamate receptors , schizophrenia and bipolar disorder , Alzheimer's disease , Parkinson's disease (DLG2), sclerosis , and diabetes .The impact of SUA levels on human cognitive dysfunction has been investigated in recent years unctions , S2, S3.Statistically detecting and replicating an epistatic pair of SNPs is far more challenging than for a single SNP signal for a number of reasons. Whereas a single SNP association is dependent on strong LD between the marker SNP and causative variant, detection of epistasis requires strong LD for both loci. Small allele frequency changes (e.g. 10%) between detection and replication populations can lead to a dramatic loss of power in replication. Such small allele frequency changes are not uncommon across the small and/or isolated populations. The issue of missing joint genotype class in replication populations  increases the difficulty of replication, especially when the epistatic pair for test has a rare genotype class  \u2013 rs4085921 (GPC6) was replicated at all three levels, rs737267 (SLC2A9) \u2013 rs2302558 (CLEC16A) at the SNP and gene levels but not the GO level as CLEC16A is not associated with an enriched GO term. Both GPC6 (glypican 6) and CLEC16A  are associated with multiple sclerosis The lack of statistical replication at the SNP level may be not too surprising considering that the epistatic signals detected in one isolated sample were to replicate in another isolated sample (CROATIAN) or a less isolated but smaller sample (i.e. SOCCS). Even when all the retained SNP pairs were considered regardless, we just found two SNP pairs (out of 212933) were replicated at the SNP level. When testing for replication at the GO level, we found 13 GO enriched terms (>50%) were replicated. Clearly replication improved dramatically as the level changes from SNP to GO annotation. A high level replication (i.e. gene or GO/pathway) does not guarantee a SNP level replication but is valuable to understand the biology of interest. Therefore, we recommend testing for replication of epistasis signals at all three levels. For example, rs737267 (10Pint of 6.5 used here) could be chosen as long as the big cluster of SLC2A9 pairs was mostly excluded Click here for additional data file.Figure S2The joint genotype \u2013 phenotype maps for two genome-wide significant epistatic pairs.(TIF)Click here for additional data file.Figure S3Gene component ontology terms enriched by epistatic genes in the MICROS population.(TIF)Click here for additional data file.Figure S4Gene component ontology terms enriched by epistatic genes in the CROATIAN populations.(TIF)Click here for additional data file.Table S1SLC2A910Ppair>6.5 and \u2212log10Pint>6.5) in MICROS. involved epistatic pairs (\u2212log(PDF)Click here for additional data file.Table S2Epistatic genes in the replicated GO terms shared by the MICROS and CROATIAN populations.(PDF)Click here for additional data file.Table S3Epistatic pairs with at least one shared GO gene and replicated in MICROS and CROATIAN.(PDF)Click here for additional data file."}
+{"text": "Women with low grade squamous intraepithelial lesions (LSIL) at cervical cancer screening are currently referred for further diagnostic work up despite 80% having no precancerous lesion. The primary purpose of this study is to measure the test characteristics of 3q26 chromosome gain (3q26 gain) as a host marker of carcinogenesis in women with LSIL. A negative triage test may allow these women to be followed by cytology alone without immediate referral to colposcopy.A historical prospective study was designed to measure 3q26 gain from the archived liquid cytology specimens diagnosed as LSIL among women attending colposcopy between 2007 and 2009. 3q26 gain was assessed on the index liquid sample; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were measured at immediate triage and at 6\u201316 months after colposcopic biopsy. The sensitivity of 3q26 gain measured at immediate triage from automated and manually reviewed tests in 65 non-pregnant unique women was 70%  with a NPV of 89% . The sensitivity and NPV increased to 80%  and 98% , respectively, when only the automated method of detecting 3q26 gain was used.3q26 gain demonstrates high sensitivity and NPV as a negative triage test for women with LSIL, allowing possible guideline changes to routine surveillance instead of immediate colposcopy. Prospective studies are ongoing to establish the sensitivity, specificity, PPV and NPV of 3q26 gain for LSIL over time. Human papillomavirus (HPV) and telomerase overexpression have been recognized as independent carcinogenic factors for which the Nobel Prize in Physiology or Medicine was awarded in 2008 and 2009, respectively. Harald zur Hausen defined the link between human papillomavirus (HPV) infection and cervical cancer; and Elizabeth Blackburn, Carol Greider and Jack Szostak detailed the function of telomeres and the enzyme telomerase. Cells that are becoming cancerous or are cancerous express telomerase over-abundantly, allowing gains in the chromosomes\u2019 telomere length. Testing for gains in human chromosome length of HPV infected cells may identify a potential biomarker for cervical cancer screening.The primary screening method for cervical cancer has been the morphologic characteristics of HPV infection in the epithelial cells of the uterine cervix. The Bethesda System, (TBS-2001) Cocktail high risk (HR) HPV testing, such as Hybrid Capture 2 (HC2) is positive in 76\u201380% of LSIL cytology samples The purpose of this research is to establish the test characteristics of sensitivity, specificity, positive and negative predictive values of the gain of the chromosome 3q26 region (3q26 gain) for predicting which women with LSIL cytology do not have CIN 2/3 at colposcopy.We designed a historical prospective study of women who had presented to the colposcopy clinics at Truman Medical Center, the teaching hospitals of the University of Missouri Kansas City School of Medicine, Kansas City Missouri, USA. To be eligible for inclusion, we reviewed the electronic medical record (EMR) for those women whose index liquid cytology LSIL sample had been archived, who had a colposcopically directed biopsy and who was not pregnant at the time of cytology or biopsy sampling. We were granted ethics approval, and consent waiver, from both the University of Missouri-Kansas City School of Medicine Institutional Review Board (#09-70X) and the Truman Medical Centers\u2019 Privacy Board to use the de-identified data.Clinicians collected the index samples in the SurePath\u2122 system  as part of routine cervical surveillance. The specimens were pelleted and resuspended for cytology processing for immediate clinical diagnosis. No other sampling was done on the specimen. The remainder of the resuspended sample was refrigerated at 2\u20134\u00b0C until it was used for 3q26 gain testing. The Bethesda System was used to report the cytology diagnoses , atypical squamous cells of undetermined significance (ASCUS), LSIL, high grade squamous intraepithelial lesion (HSIL)). CIN nomenclature was used to report the histologic diagnosis from the biopsy specimen .The cytology specimens were mailed at ambient temperature to the Ikonisys laboratory where each was transferred to a 15 mL tube and centrifuged for 6 minutes at 1200 RPM. The supernatant was decanted and the pellets resuspended in 5 mL of KCl for 15 minutes at 37\u00b0C. Two milliliters Carnoy\u2019s Fixative was added and the samples centrifuged for 6 minutes at 1200 RPM. The supernatant was discarded and the pellet resuspended in 10 mL of Carnoy\u2019s fixative and incubated overnight. Samples were centrifuged for 6 minutes at 1200 RPM, the supernatant discarded and the pellet resuspended in an appropriate volume of Carnoy\u2019s Fixative. Slides were prepared manually and incubated at 55\u00b0C for 10 minutes. Prior to hybridization, slides were immersed in 2X SSC for 2 minutes at 73\u00b0C and treated with 1% pepsin for 10\u201315 minutes at 37\u00b0C. Slides were then incubated in 1X PBS for 2 minutes and fixed in 2% formalin for 5 minutes, at room temperature. Slides were washed in 2X SSC for 2 minutes at room temperature and dehydrated in an ethanol series .The slides were hybridized using the oncoFISH cervical probe kit . Briefly, 1 \u00b5L of 3q26 probe, 1 \u00b5L of CEP 7 probe, 1 \u00b5L distilled water and 7 \u00b5L of hybridization buffer were mixed and applied to the slide. A 22 mm round glass cover slip was placed over the probe and air bubbles were removed. Slides were transferred to a ThermoBrite , denatured at 76\u00b0C for 5 minutes and hybridized overnight at 37\u00b0C. Following hybridization, slides were washed in 2X SSC, 0.3% NP-40 at 73\u00b0C for 2 minutes then in 2X SSC, 0.1% NP-40 at room temperature for an additional 2 minutes. Slides were counterstained with DAPI  for 3\u20134 minutes at room temperature and dehydrated in an ethanol series. Ten microliters of antifade and a 22\u00d750 mm cover slip were applied and slides were placed at \u221220\u00b0C for at least 15 minutes prior to analysis.We analyzed 65 slides using an automated system developed specifically for rare-cell detection and analysis . The entire sample was scanned using a 20\u00d7 objective and both the total number of nuclei and number of nuclei with >2 signals for 3q26 were determined. The nuclei were ordered based on the number of 3q26 FISH signals, and up to 800 nuclei with the highest number of 3q26 FISH signals were imaged using a 40\u00d7 objective. Nuclei were enumerated for both 3q26 and control centromeric 7 FISH probe signals to determine 3q26 gain. The test was positive if two or more cells with more than four 3q26 FISH signals were detected .Fourteen cytology samples were unable to be read by automated scanning for the 3q26 gain signal. These slides had less than 50 nuclei imaged at high magnification (40\u00d7), and they had an increase in background autofluorescence that could confound the signal to noise ratio. Therefore, following scanning, these fourteen slides were reviewed for 3q26 gain manually, while maintaining blinding to the subjects\u2019 clinical information.The power analysis was calculated with Statistica software 81 LSIL cervical specimens from unique non-pregnant women were collected between May 2007 and January 2009 and stored at 2\u20134\u00b0C. Of these 81, 73 women had colposcopically directed biopsies on average 36 days from their index cytology (standard deviation (SD): 18). Of these 73 specimens, 65 provided sufficient archived material to run the 3q26 gain test resulting in 65 women with complete index cytology, colposcopically directed biopsy and index 3q26 gain status . Of thesThe average age of the study population was 32 years (SD 9.6 yrs) ranging from 17\u201359 years. Most of the women in the study were African-American (51%) or Caucasian (40%). The women had a mean gravidity and parity of 2.6 (SD 1.9) and 2.0 (SD 1.6), respectively, and 40% of the women had had a prior abnormal Pap. Ten of the 65 women (15%) had CIN 2/3 at colposcopy with the remaining having CIN 1 or normal tissue .Test characteristics were calculated for 3q26 gain status determined by both automated slide scanning and manual slide review. Seven of the 10 women with CIN 2/3 were positive for 3q26 gain on the index cytology for a sensitivity of 70% . The specificity of the 3q26 gain test was 91% ; the positive predictive value (PPV) was 44%  and the negative predictive value (NPV) was 89%  .Recalculation of the 3q26 gain test characteristics after exclusion of the manually reviewed slides does not substantially change the four test characteristics of 3q26 gain . There wOf secondary interest, sixteen women whose colposcopically directed biopsy was CIN 1 returned for repeat cytology 6\u201316 months  from their index cytology. None of the women had progressed to HSIL in that time frame for a non-evaluable sensitivity and a 0% positive predictive value. Twelve of the women regressed to normal cytology, one of whom had had a gain in the 3q26 chromosome on her index cytology, representing a false positive 3q26 gain test. None of the four women whose LSIL persisted had a 3q26 gain on her index cytology, making the specificity of the 3q26 gain test for regression or persistence at a 10 month average follow up 94%  and the negative predictive value 100% .In addition, four of the women with biopsy confirmed CIN 2/3 had follow up cytology 6 months (mean 203 SD 16) after their LEEP. All LEEP specimens had negative resection margins. All 6 month follow up cytologies were reported as NILM. Two of the women who showed a 3q26 gain at index cytology showed no 3q26 gain at the post-LEEP follow up cytology.HPV infections create the morphologic effects seen to codify a LSIL entity. Cells must exhibit nuclear enlargement as well as a defined, perinuclear clear area demarcated by a dense cytoplasmic ring (koilocytes); there may be nuclear smudging and binucleation. The nuclear contour of a LSIL cell may be smudged or granular or slightly irregular with hyperchromasia but the chromatin is evenly distributed. The nuclear enlargement seen in LSIL is at least three times that of the normal intermediate cell along with an increased nuclear to cytoplasmic ratio. These changes are most often seen in the superficial and intermediate cells, in sheets or individually While only 2.5% of all women screened for cervical cancer will have LSIL cytology, these women constitute nearly 50% of all the referrals to colposcopy The natural history of LSIL cytology supports an observational management guideline if the sensitivity and negative predictive value of a triage test is high enough to provide reassurance that the LSIL lesion will not progress to CIN 2/3 before the next screening interval. LSIL lesions caused by oncogenic HPV types regress, on average, in 14 months, ranging from 9\u201319 months, while those caused by low risk, benign, types regress, on average, in half that time Positive triage tests to improve the diagnostic accuracy of cytology have included testing for oncogenic HPV types, quantitating viral load, measuring integration and quantifying markers of cell cycle aberration, such as p16ink4a. These triage tests need high specificity and positive predictive value for identifying women whose abnormal cytology hides a true cancer precursor, balanced with an acceptable rate of false positive tests. The results of these positive triage tests would change clinical management from passive surveillance to requiring further diagnostic work up such as referral to colposcopy. Randomized controlled trials show high risk HPV testing, as a triage test after LSIL, to have specificities less than 50% for predicting those women with CIN 2+ disease where CIN 2+ is defined as CIN 2 or 3 or cervical cancer A negative triage test would be one whose negative/normal results would modify current aggressive clinical guidelines to allow observation without clinical intervention until the next screening interval without missing a significant portion of diseased women. A negative triage test is one with a high sensitivity for cancer and cancer precursors in addition to a negative predictive value exceeding 97% A promising new negative triage test for women with LSIL cytology has been the gain of chromosome length of 3q26. Prior studies report greater than 80% sensitivity for the detection of CIN 2/3 from a LSIL population Prior studies using gains in 3q26 amplification showed increasing proportions of 3q26 gain among women with increasingly more severe cervical cytology and histology fulfilling the biological plausibility and temporal association characteristics needed for this triage test to be clinically relevant in cervical cancer screening Our results show a sensitivity of 80% and a NPV of 98% for automated scanning for 3q26 gain among women with LSIL cytology at immediate colposcopy; and 100% NPV at the 6\u201316 month follow up visit after CIN1 biopsy. These excellent negative triage test properties are balanced by reassurance of not missing diseased women as seen from the high specificity of 90% and the PPV of 44%, nearly three times the prevalence rate of CIN 2/3 disease in our study population. These test characteristics are sufficiently high to warrant further prospective investigation of this test as a negative triage tool to change clinical guidelines to conservatively follow LSIL women negative for 3q26 gain instead of the current guidelines of immediate referral to colposcopy.Other clinical states that may benefit from a host-based negative triage test, such as 3q26 gain, include those women with atypical glandular cells of undetermined significance (AGC) The limitations of our study include the historical prospective trial design which relies on the integrity of archived liquid cytology material for 3q26 gain testing; and an unknown disease state in women whose LSIL smears were censored from analysis due to incomplete records of a colposcopic endpoint or interval surveillance screens.A prospective trial in larger numbers of women with LSIL from the general population of wide age ranges is ongoing. This study may determine a more robust characterization of the 3q26 gain test for reassuring women and physicians of the lack of need for immediate colposcopy after a LSIL Pap test."}
+{"text": "Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with  SOD1 gene are responsible for 20\u00a0% of FALS and up to 7\u00a0% of SALS cases .Analysis of variance showed that Dctn1-mRNA and Dctn3-mRNA expression was dependent on both the mutation and the clinical stage only in the frontal cortex [F(3.86)\u00a0=\u00a02.98, Glued) which mediates dynein\u2013dynactin interaction is a critical component of the whole dynein/dynactin complex. It was found that a single base-pair change leading to a glycine-59-serine (p.G59S) substitution in glycine-rich domain of this subunit accelerated motor neuron degeneration in humans and mice which resembled ALS [DCTN1 exons and exon\u2013intron boundaries in 286 samples diagnosed with neurodegenerative diseases  concluded that pathogenic mutations in DCTN1 are rare. Lately Fujiwara and Morimoto [Glued and microtubule stabilization cooperatively suppress axon degeneration.Dynein/dynactin complex is involved in axon maintenance, removal of damaged organelles, vesicles, misfolded and aggregated proteins from axons to the cell body . Many pobled ALS , 34. Vilbled ALS  after seMorimoto  discoverIn the present study we found that in individual SALS subjects, DCTN1-mRNA expression was higher in the motor than in the sensory brain cortex. In our earlier studies conducted on the same SALS subjects, we found that kinesins-mRNA expression was also higher in the motor cortex . In the The comparison of SALS and control cases indicated lower values of DCTN1 expression (on the mRNA and protein level) in both SALS cortexes and also in the spinal cord. Those results were supported by immunoexpression, which indicated that DCTN1 protein in the preserved SALS motoneurons revealed mild immunoreactivity and was less intense than in control material\u2014the difference was especially evident in the spinal cord anterior horns. Our results are with agreement with the studies of Ikenaka et al. , who obsDifferently to DCTN1, in the brain cortex of SALS cases the expression of DCTN3 was similar in both SALS cortexes and it didn\u2019t differ from the expression in control cortexes (on both mRNA and protein levels). In the spinal cord the expression of DCTN3 was much lower in SALS than in control tissues. In immunohistochemistry DCTN3 was hardly seen in all SALS tissues but was much better visible in controls.Mutations in the gene encoding SOD1 cause 2\u20133\u00a0% of all ALS cases and induce motor neuron degeneration in transgenic mice . Mutant All motor proteins transport their cargoes on tracts formed by microtubules. Microtubules assembly, stabilization and organization into bundles depend on tau protein . Tau alsDync1h1) mutations present phenotype similar but milder than SOD1G93A mice but in contrast to SOD1 mice their life span remains almost unchanged [Dync1h1 mutations in pathogenesis of motor neuron degeneration is controversial [Dync1h1 and SOD1G93A mutation [Cra1 mice with dynein heavy chain (nchanged , 15, 45.oversial , 47. Accoversial , Cra1 heoversial . There wmutation . All thoDync1h1 mutations defective retrograde transport is partially corrected and the life span of SOD1 mice extended [Dync1h1 mutation also attenuates motor neuron degeneration in SOD1G93A mice [In mice with double SOD1G93A and extended . Accordiextended  increase93A mice . Zhang e93A mice  suggeste93A mice . In the 93A mice , 45, 49 Dync1h1 mutation itself has slight negative effect on dynactin subunits expression but positively affects the changes caused by SOD1G93A mutation.It can be concluded that in SALS and SOD1-related ALS the impairment of dynein-mediated retrograde axonal transport and degeneration of motor neurons may be related to dynactin subunits deficiency and subsequent disruption of the whole dynein/dynactin complex structure and function. The"}
+{"text": "The aim was to compare two novel endotracheal tubes (ETT), Mallinckrodt TaperGuard  cuff) and KimVent Microcuff , with conventional Portex  in leakages across cuffs (microaspiration) under simulated clinical situations. It has been shown that globular PVC cuffs protect poorly against leakages due to microchannels formed from infolding of redundant cuff material . We hypo2O), normal lungs (PEEP 5 cmH2O) and acute respiratory distress syndrome (PEEP 10 cmH2O), and disconnection from the ventilator with and without spontaneous breathing effort. Spontaneous breathing was simulated with a respiratory gas exchange simulator. Suction was applied at 200 cmH2O sustained for 3 minutes at the Murphy eye. Each scenario was repeated with cuff pressures (Pcuff) 10, 20 and 30 cmH2O maintained by a Pcuff maintenance device.Each ETT was inserted into a silicone cylinder of 2 cm wide inclined at 35\u00b0. Then 20 ml water was added above the cuff and leakage measured every minute under five different simulated clinical conditions: mechanical ventilation for acute severe asthma (positive end-expiratory pressure (PEEP) 0 cmH2O in the presence of suction irrespective of Pcuff . Leakage under these scenarios can be reduced in TG and prevented in MC by Pcuff \u226520 cmH2O (Figure PT leaked grossly in all scenarios without PEEP and at PEEP 5 cmH2O whenever PEEP was lost. The effect of ETT type on the incidence of VAP warrants further investigation.Microcuff outperformed the others in preventing microaspiration, while Portex leaked grossly even at a recommended Pcuff of 20 to 30 cmH"}
+{"text": "PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency.Mutations in genes encoding cationic trypsinogen (ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls.We analyzed all 27 In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups.ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.We did not find an association between heterozygous  PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) can be identified CFTR mutations too enhance the susceptibility for idiopathic chronic pancreatitis Chronic pancreatitis (CP) is an inflammatory disease characterized by destruction of pancreatic parenchyma that can result in permanent impairment of both exocrine and endocrine pancreatic function ATP8B1 (formerly designated as FIC1) ATP8B1 mutations.ATP8B1 deficiency is an autosomal recessive disease characterized by mutations in n\u200a=\u200a316) and France (n\u200a=\u200a191). In the German patients, the diagnosis of CP was based on two or more of the following findings as described previously: presence of a typical history of recurrent pancreatitis, pancreatic calcifications and/or pancreatic ductal irregularities revealed by endoscopic retrograde pancreaticography or by magnetic resonance imaging of the pancreas, and pathological sonographic findings The study was approved by the local ethics committee of the Technische Universit\u00e4t M\u00fcnchen and the ethical review committee of the Universit\u00e9 de Bretagne Occidentale. All study subjects gave their written informed consent for genetic analysis. For this study, 507 patients with hereditary or idiopathic chronic pancreatitis were included. The patients originated from Germany  and French (n\u200a=\u200a285) origin. Whenever a non-synonymous variation in the coding sequence was encountered all 1,027 controls were sequenced for the exon involved.The control group consisted of 1,027 unrelated healthy individuals of German .Genomic DNA of peripheral blood leukocytes was extracted routinely. Primer pairs for PCR were designed to amplify all 27 coding exons, with flanking intron-exon boundaries of To detect nucleotide sequence changes of potential relevance to clinical phenotypes, we analyzed the sequence output of the patient cohort for variants that resulted in amino acid changes, nonsense variants or deletions/insertions. Exons with one of these alterations were subsequently analyzed in the control population by DNA sequencing.http://www.ncbi.nlm.nih.gov/entrez, reference sequence NM_005603.4). The A of the ATG start codon was used as nucleotide +1. The mutations are described according to the nomenclature recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen).The reference sequence was derived from GenBank , all possibly associated in literature with episodic ATP8B1 deficiency. The second patient had two variants , both described in patients with the progressive form of ATP8B1 deficiency. Interestingly both these patients presented with chronic pancreatitis and had no signs of liver disease whatsoever.In our cohort of 507 CP patients we identified 19 different d change . Five (pATP8B1 variations in CP patients did not significantly differ from that in controls (p\u200a=\u200a0.5). Furthermore, in the control population we detected 8 additional non-synonymous variations, of which 5 had not been described before (Those exons in which a non-synonymous variant was detected in CP patients 13 exons) were also sequenced in our control cohort of 1,027 subjects. No alteration was significantly overrepresented in the patient group. Also the combined frequency of these non-synonymous exonic d before . In addid before  and 3. T exons weATP8B1 seemed a plausible candidate gene for chronic pancreatitis due to its high expression in the pancreas, its supposed general function in membrane organization and the finding that 2 out of 10 individuals affected with ATP8B1 deficiency had chronic pancreatitis ATP8B1 variants and hereditary or idiopathic chronic pancreatitis when comparing 507 patients and 1,027 controls.At the outset of our investigations, ATP8B1 variants. We could not experimentally verify independent inheritance as no genomic material was available from parents or unaffected family members. However if these patients were indeed compound heterozygous, these genotypes are predicted to result in an ATP8B1 deficiency phenotype. Especially the p.E665X and p.A1208fs mutations change the structure of ATP8B1 significantly and can cause PFIC. Yet these two CP patients did not have any signs of liver disease or extrahepatic features of ATP8B1 deficiency other than pancreatitis. ATP8B1 deficiency without liver disease has been described before, suggesting that reduced penetrance of the liver phenotype can indeed be seen We did identify two CP patients with two or three non-synonymous ATP8B1 variants in health and disease. For example p.D70N was previously suggested to contribute to the etiology of intrahepatic cholestasis of pregnancy (ICP) as 3/182 ICP patients harbored this variant and none of 120 controls In addition, our data do contribute to a better understanding of the role of rare heterozygous ATP8B1 variants and hereditary or idiopathic chronic pancreatitis. However it suggests that pancreatitis might be the first or sole symptom of ATP8B1 deficiency. Furthermore earlier suggestions of an involvement of ATP8B1 variants in ICP might have been due to a chance effect.In conclusion, our investigation did not reveal an association between heterozygous"}
+{"text": "S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of  Cells are routinely challenged by changes in growth conditions that perturb protein homeostasis. The action of protein quality control (PQC) machinery is essential to maintain levels of non-native proteins within a tolerable range Hsp40s represent a large family of Hsp70 co-chaperones that are essential regulators of the ATP hydrolytic cycle of Hsp70 and target Hsp70 to specialized machineries and cellular locations To define Hsp70-dependent steps in triage decisions that lead to protein degradation in the eukaryotic cytosol we expressed in yeast a terminally misfolded and short-lived chimeric GFP fusion protein (slGFP). SlGFP has an N-terminal domain that is too short to fold into a stable conformation fused to tandem GFPs, so its fate can be monitored visually and biochemically. Therefore, study of slGFP degradation provides a valuable approach to define chaperone dependent steps in protein degradation without having to consider interpretations related to folding.We report that Hsp70 cooperates with Sis1 and the PQC E3 ligase Ubr1 to mediate proteasomal degradation of slGFP. Interestingly, attenuation of Sis1 or Ubr1 activity lead slGFP to accumulate in a Triton X-100-soluble state and be packaged into protein handling centers that are visualized as cytosolic puncta. The slGFP that accumulated in puncta when Sis1 activity was low was subsequently degraded in a proteasome dependent manner. Yet, in the absence of Ubr1, puncta localized slGFP was relatively stable. The Sis1/Hsp70 system and Ubr1 cooperate in degradation of a terminally misfolded cytosolic protein. Cells compensate for saturation of the Sis1/Ubr1 E3 machinery via storage of degradation competent protein assemblies in cytosolic puncta. Terminally misfolded proteins that accumulate in PQC puncta can subsequently be degraded in a process that requires Ubr1.Yeast strains and plasmids are listed in 2O, and boiled for 15 min in denaturing lysis buffer . Cell lysates were precleared at 3,000 rpm for 3 min and the protein concentration of supernatants were normalized. Normalized lysates were diluted in sample buffer  then analyzed by SDS-PAGE and western immunoblotting for the indicated proteins.Yeast cultures expressing the indicated proteins were treated as described in the text. To inhibit protein translation, cultures were treated with 200 \u00b5g/mL cycloheximide and aliquots were removed at indicated times. Cells were lysed by alkaline pretreatment Yeast strains expressing the indicated proteins were lysed by glass bead disruption in Buffer A . Cell extracts were precleared at 3,000\u00d7 G for 3 min at 4\u00b0C. The supernatant was saved and protein concentrations assessed with a BioRad protein determination kit. Protein concentrations were normalized between samples to approximately 3 mg/mL and 300 \u00b5g of protein was incubated with the indicated antisera for 1 hour at 4\u00b0C then incubated with Protein G beads (50% slurry preblocked with BSA) for 30 min at 4\u00b0C. Beads were washed 2\u20133 times with Buffer A then resuspended in sample buffer and analyzed by SDS-PAGE and immunoblotting.2O (+1 mM NaN3 and 20 mM NEM) and lysed by glass bead disruption in Buffer A (+1 mM NEM). Cell extracts were precleared at 3,000 rpm for 3 min at 4\u00b0C, protein concentrations normalized, and GFP immunoprecipitated with anti-YFP antisera and protein G resin using standard methods. Protein G resin was washed three times in Buffer A supplemented with 0.1% SDS. Ubiquitinated slGFP was detected after SDS-PAGE and western immunoblotting of precipitated material for ubiquitin (Covance). Levels of ubiquitinated slGFP were quantified using laser densitometry and ImageJ software (NIH) and normalized as a ratio to the level of slGFP that was immunoprecipitated from the lysate (detected using anti-GFP).Yeast strains expressing slGFP were grown under selection to mid-log phase. Cells were washed with cold HGAL1 promoter for 4 hours before cells were processed for analysis. Cells were visualized with an Olympus IX81 Fluorescence microscope and images processed with Metamorph software. Exposure times and all other settings were standardized across individual experiments unless otherwise noted.Yeast strains expressing the indicated proteins and treated as described in the text were fixed in 3.7% formaldehyde and stored in phosphate buffered saline (pH 7.5) supplemented with 1.2 M sorbitol. Fixed cells were permeabilized and DNA visualized with DAPI as described in Cells were lysed by glass bead disruption in Buffer A (+1 mM DTT) and lysates pre-cleared at 3,000 rpm for 3 min at 4\u00b0C. The supernatant was saved and a quantity of lysate that contained 200 \u00b5g of protein was spun at 100,000\u00d7 G for 30 min at 4\u00b0C. An aliquot was saved prior to the spin to represent the total input. Equivalent volumes from total, supernatant, and pellet fractions were added to 2\u00d7 sample buffer and analyzed by SDS-PAGE and western immunoblotting for the indicated proteins.To analyze how protein degradation and aggregation pathways intersect in the cytosol we examined the degradation pathway of a labile and aggregation-prone cytosolic protein. This protein (herein referred to as slGFP for short-lived GFP) consists of a 126 amino acid N-terminal domain containing several unstructured regions that are enriched in hydrophobic motifs and putative Hsp70/Hsp40 chaperone binding sites  [27], [3ADH1 promoter. Importantly, slGFP expression from this promoter did not impact cell growth although we observed elevation in levels of heat-shock inducible chaperones Ssa1 and Hsp104. However, slGFP did not induce a global heat shock response since the levels of Ydj1 and Sis1 were unchanged  protein requires partner proteins (elongin B & C) and molecular chaperones to fold properly We expected that Sis1 was acting through its partner Hsp70 Ssa1 to accelerate slGFP turnover. To determine if Sis1 action in slGFP degradation is indeed Hsp70-dependent, a mutation was made in the Sis1 J-domain (H34Q) that disrupts a conserved Hsp70 binding motif SIS1 is essential we investigated slGFP degradation in \u0394sis1 complemented with a plasmid in which SIS1 is expressed under control of tetracycline repressible promoter. Sis1 levels in this strain were 85% lower than in an isogenic wild type strain even in the absence of doxycycline, though growth rates were unaffected  suggest that blocking Sis1 function impacts localization of slGFP in the same manner as inactivation of Ubr1 or San1. To explore this further, we evaluated the impact of reducing Sis1 levels on the solubility and degradation of slGFP. As affected . Furtheraffected .\u0394ubr1 or \u0394san1 strain, the slGFP in low Sis1 remained predominantly Triton X-100-soluble . Full-length slGFP is detected by western blot in \u0394ydj1, so slGFP is translated in this strain, but may not be detected by fluorescence microscopy due to its aggregation prior to proper folding. Thus, in contrast to Sis1 action in degradation of slGFP, Ydj1 may be required to chaperone nascent slGFP.Accumulation of Triton X-100 soluble forms of slGFP in puncta in the low Sis1 strain did not appear to result from a general decrease in cytosolic chaperone capacity. This is the case because deletion of the non-essential  60 mins . Ydj1 cockground . We atteUBR1 or SAN1 decreased the rate of slGFP degradation and in each of these instances, slGFP accumulated in Triton X-100-soluble puncta. Therefore, we investigated whether Sis1 function in turnover of short-lived proteins is dependent upon Ubr1 or San1. To test for functional interactions between Sis1 and quality control E3 ligases we first asked if the presence of Ubr1 or San1 is required for Sis1 to accelerate slGFP degradation  to mediate proteasomal degradation of a terminally misfolded protein called slGFP. A decrease in the flux of slGFP through the Sis1/Hsp70/PQC E3 pathway results in the accumulation of slGFP in cytosolic puncta. Triton X-100-soluble forms of slGFP that accumulate in puncta are resolubilized and degraded in an Ubr1 and proteasome dependent manner. Thus, Sis1/Hsp70 and PQC E3s cooperate to clear terminally misfolded proteins from the cytosol. Saturation of this system can lead to storage of degradable protein species in puncta that serve as transient holding depots for detergent soluble assemblies of misfolded proteins When PQC pathways are saturated cells package non-native proteins into several different protein handling centers; the IPOD, JUNQ and peripheral compartment Sis1 is found to function in concert with Hsp70 and PQC E3s to mediate degradation of slGFP. In addition, Sis1 appears to cooperate with Hsp104 to accelerate rates of slGFP degradation, but Hsp104 is not required for normal rates of slGFP degradation. Thus, Sis1 seems to function via Hsp104 independent and dependent mechanisms to facilitate slGFP degradation. Sis1 and Hsp70 Ssa1 are present in complexes that contain Ubr1, and Hsp70 is known to function in substrate selection by PQC E3s To accelerate the rate of slGFP degradation Sis1 may function with Hsp104 in a manner similar to Sis1/Hsp104 function in prion propagation \u0394ubr1 strains is different. SlGFP present in the puncta of the low Sis1 strain is resolubilized and degraded in a proteasome-dependent manner. Yet, in \u0394ubr1 the puncta that contain slGFP are relatively stable and slGFP has a half-life of greater than 90 mins instead of 15 min. These data suggest that slGFP degradation intermediates accumulate in cytosolic puncta when the capacity of Sis1 and Hsp70 to handle non-native proteins is saturated. In addition, it appears that Ubr1 is required for degradation of misfolded proteins that are liberated from the puncta. Further studies are now required to understand the functional interplay between PQC factors that degrade non-native proteins and those that package non-native proteins for reversible sequestration into protein storage depots. A mechanistic understanding of these cellular processes will help define approaches to suppress the proteotoxicity associated with protein conformational disease.The half-life of puncta that contain slGFP in low Sis1 or Figure S1The Domain Structure Of SlGFP. A) Sequence and predicted secondary structure of the N-terminal 120aa of slGFP; C-coiled H-helical S-\u03b2-strand. B) Turnover of slGFP wild type and mutant lacking its nuclear localization sequence (NLS) and nuclear export sequence (NES). C) Fluorescence microscopy of slGFP and NLS/NES mutant.(TIF)Click here for additional data file.Figure S2Depletion Of Sis1 To Undetectable Levels Delays SlGFP Turnover. A yeast strain expressing slGFP as described in (TIF)Click here for additional data file.Table S1Genotypes Of Yeast Strains Used In Study Of The SlGFP Degradation.(DOCX)Click here for additional data file.Table S2Plasmids Utilized In The Study Of SlGFP Degradation.(DOCX)Click here for additional data file."}
+{"text": "Sepsis and its common complication septic shock are generally induced by the action of lipopolysaccharide (LPS) and characterized by peripheral arteriolar vasodilatation that results in hypotension and inadequate tissue perfusion. During sepsis, secretion occurs of large amounts of inflammatory mediators such as nitric oxide (NO), interleukin 1 (IL-1) and TNF\u03b1 that will modulate the inflammatory response. One significant finding in clinics is that men and women respond differently to sepsis, with better prognosis related to women [Male and female (ovariectomized and sham surgery) rats were injected intraperitoneally (i.p.) for three consecutive days with ECP 40 \u00b5g/kg or vehicle. On the third day, after ECP injection, rats receive i.p. injection of 10 mg/kg bacterial LPS or saline solution. Plasma was collected 2, 4 and 6 hours after LPS for NO and cytokine measurements.Administration of LPS increased the NO plasma concentration in males and females . ECP pretreatment decreased the NO concentration in sham females at 4 and 6 hours; conversely, it increased nitrate levels in ovariectomized and in males at 4 and 6 hours. IL-1 plasma concentration was increased in the three groups after LPS administration at 2 and 4 hours and in Sham at 6 hours; ECP pretreatment decreased IL-1 plasma concentration in all groups at 2 hours. LPS administration also increased TNF\u03b1 plasma concentration at 2, 4 and 6 hours in the three groups; ECP pretreatment inhibited the increase of TNF\u03b1 at 2 hours in three groups.Our results indicate that estradiol may have proinflammatory or anti-inflammatory actions depending on the gender and the mediator evaluated; this balance in mediator secretion may be protective and explain in part the better outcomes of woman during sepsis."}
+{"text": "Chromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques.HER2 CISH pharmDx\u2122 Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx\u2122 Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory.Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx\u2122 Kit to both HER2 FISH pharmDx\u2122 Kit and PathVysion HER-2 DNA Probe Kit.The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx\u2122 Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx\u2122 Kit is a reliable chromogenic alternative to fluorescence-based methods.The concordance between results obtained using the recently FDA approved  HER2  amplification is observed in approximately 22% of human breast cancers [HER2  is an important marker for invasive breast cancer. The assessment of the HER2 expression level is routinely done by examining protein expression and/or gene expression levels in formalin-fixed and paraffin-embedded (FFPE) histological sections. Overexpression of HER2 protein and/or  cancers  and has  cancers .HER2 amplification is mostly done by in situ hybridization (ISH) techniques either fluorescence (FISH) [HER2 target sequences in the nuclei of tumor cells is done by fluorescence- or hapten-labeled sequence pairing probes. Implementation of CISH for determination of HER2 amplification in breast cancer has some advantages compared to FISH based detection [Tissue based assessment of HER2 protein expression levels is commonly achieved using immunohistochemistry (IHC), whereas tissue based analysis of e (FISH)  or chrome (FISH) . In ISH etection -9. ChromHER2 FISH techniques. In this paper data is reported from the comparison of 365 breast cancer specimens using a new dual color HER2 CISH method  with two well established and FDA approved HER2 FISH techniques; HER2 FISH pharmDx\u2122 Kit  and PathVysion HER-2 DNA Probe Kit .To implement CISH in the anatomical pathological laboratories for determination of HER2 status in breast cancer the technique needs to be safe and reliable . One wayHER2 CISH pharmDx\u2122 Kit (Dako Denmark A/S), HER2 FISH pharmDx\u2122 Kit (Dako Denmark A/S) and PathVysion HER-2 DNA Probe Kit . Specimens were not individually identifiable and it was impossible to trace the identity of the patients. The study was performed in accordance with the current version of the World Medical Association Declaration of Helsinki and approval from an Institutional Review Board  was granted prior to study start. Evaluation of specimens were performed by three different technologists for the three ISH tests and subsequently reviewed by the pathologists. One pathologist reviewed test results obtained with PathVysion HER-2 DNA Probe Kit and HER2 CISH pharmDx\u2122 Kit with several months in between and another pathologist reviewed test results obtained with HER2 FISH pharmDx\u2122 Kit. Knowledge of test results was not shared between technologists or between pathologists.The study included 365 FFPE invasive breast cancer tissue specimens with known fixation history . The specimens were collected consecutively at a US reference laboratory and the first 304 specimens were included irrespective of HercepTest\u2122  IHC score and additional 61 specimens were included based on a IHC HER2 2+ score as determined by HercepTest\u2122 Serial sections (5 \u03bcm) were cut from each specimen and stained with H&E, HercepTest\u2122 for HER2 protein expresion, HER2 CISH staining was performed according to the manufactures instructions (Dako Denmark A/S) at the US reference laboratory. In short, specimens were subjected to heat-pre-treatment (microwave oven) and pepsin digestion at 37\u00b0C to prepare the tissue for probe hybridization. Denaturation for 5 min at 82\u00b0C and over-night hybridization at 45\u00b0C were performed simultaneously for the HER2/Texas Red labeled DNA probe and the CEN-17/FITC labeled PNA probe using a Hybridizer (Dako Denmark A/S). Specimens were subjected to stringent wash at 65\u00b0C for 10 min before transfer to a CISH wash buffer. The signals from the fluorescent probes were converted to chromogenic signals in an IHC staining reaction performed on an automated platform . The immunohistochemical staining included blocking of endogeneous peroxidase activity, incubation with horseradish peroxidase conjugated anti-FITC and alkaline phosphatase conjugated anti-Texas Red antibodies followed by development of chromogenic signals using red and blue chromogens. The slides were counterstained with hematoxylin and mounted in a permanent mounting medium.HER2 CISH stained slides were interpreted using a bright field microscope with 40\u00d7 and 60\u00d7 objectives. The HER2/CEN-17 ratio was calculated based on the enumeration of 20 nuclei from the invasive tumor area. Based on ratio, the specimens were categorized into amplified (HER2/CEN-17 2.0) or non-amplified (HER2/CEN-17 < 2.0) categories. Specimens with a ratio between 1.8 and 2.2 (borderline cases) were subjected to additional enumeration of 20 nuclei and the ratio was then recalculated for the 40 nuclei to determine if amplification was present or not. Normal cells within the specimen served as an internal control for staining success. Normal cells should exhibit the ratio expected for normal diploid cells with a one to one relationship of red and blue signals.HER2 FISH pharmDx\u2122 was performed according to the manufacturer's instructions at Dako Denmark A/S and FISH using PathVysion HER-2 DNA Probe Kit was performed according to an internally validated procedure at the US reference laboratory.HER2/CEN-17 ratios obtained by CISH and FISH were translated to a HER2 gene status of amplified when the HER2/CEN-17 ratio was higher than or equal to 2.0 or non-amplified when the HER2/CEN-17 ratio was below 2.0.In accordance with FDA approved guidelines for determination of HER2 status All specimens were tested at the US reference laboratory using HercepTest\u2122 as per the manufacturer's instructions to determine the IHC HER2 score.IBM SPSS Statistics were used for statistical work . Agreement calculations were reported with 95% confidence limits based on the binomial distribution using equal tailed Jeffreys prior intervals  as calcuA total of 365 breast cancer specimens were included in this investigation. An overview of the HER2 IHC scores obtained from HercepTest\u2122 staining is provided in Table HER2 amplified and non-amplified test results found by HER2 CISH, HER2 FISH and PathVysion FISH are presented in Table HER2 CISH, 11.4% were amplified by HER2 FISH and 11.0% were amplified by PathVysion FISH (data not shown). Figure HER2 CISH pharmDx\u2122 Kit. In both panels tumor cells having distinctive blue dots are observed corresponding to the reference CEN-17 probe signals  have cluster amplification in which red signals are overlapping, but some single red signals are also visible in some tumor cells.Frequencies of HER2 CISH result and seven specimens lack a HER2 FISH result. Three of these specimens are overlapping and therefore, 348 specimens were eligible for comparison between HER2 CISH and HER2 FISH. Agreement calculations revealed an overall agreement of 98.3%  with positive agreement of 93.2%  and negative agreement of 99.0%  when comparing HER2 CISH and HER2 FISH . Positive agreement was 90.9%  and negative agreement was 98.7%   signals or made them difficult to see. In these cases the blue signals in the normal cells surrounding the tumor cells were clear and distinct and cases could therefore pass the quality control. Therefore, in cases with cluster amplification of blue signals additional caution should be taken during interpretation and results from other test methods such as IHC or FISH should be included before a final HER2 status is given.As indicated in Table  present . Three oHER2 CISH test result (Table HER2 CISH was 97.5% (352/361*100). The success rates for HER2 FISH and PathVysion FISH were 98.4 and 98.9%, respectively.Final success rates were determined after allowing for two staining runs. Of the 13 cases with a missing HER2/CEN-17 ratios was performed by plotting ratios obtained by HER2 CISH as a function of ratios obtained by HER2 FISH  were calculated for all three assays and tabulated for comparison for all the valid specimens  compared to 4:02 for PathVysion FISH (data not shown). This difference was statistically significant using a two-tailed, paired t-test .During the study, the evaluation time for each specimen was recorded for HER2 FISH and HER2 CISH performed in breast cancer specimens [HER2 CISH have not yet been presented. In the current investigation 365 primary breast cancer specimens were included and an overall HER2 status agreement close to 98% is reported when comparing test results obtained by HER2 CISH pharmDx\u2122 Kit to results obtained by HER2 FISH pharmDx\u2122 and PathVysion HER-2 FISH DNA Probe Kit. The study population was enriched for HercepTest\u2122 IHC 2+ specimens because most testing modalities pass on such equivocal specimens to a genetic test. For overall agreement the lower 95% confidence interval limits were at or above 96% in the two comparisons, further stressing the reliability of the HER2 CISH pharmDx\u2122 in this comparison to the two FISH analysis methods.Previously published studies have reported high concordance between pecimens ,15-19, hHER2 amplified or HER2 positive consecutively collected specimens reported in this study by the three ISH assays  and by HercepTest\u2122 IHC (10.5%) seems to be lower than expected based on previously published data [The number of HER2 CISH compared to HER2 FISH and PathVysion FISH with no significant difference in HER2 signal counts between the three assays were observed. This resulted in significantly lower HER2/CEN-17 ratios observed for HER2 CISH. There was no clinical diagnostic impact of this change as the concordance agreement calculations revealed very fine agreement between HER2 CISH and the two FISH assays. In support of the good agreement the analysis of test results for specimens having a HER2/CEN-17 CISH ratio below 3.0 revealed that identical ratios were obtained for HER2 CISH, HER2 FISH and PathVysion FISH. Surprisingly, average copy numbers for both HER2 and CEN-17 were higher for the HER2 CISH method, whereas, HER2 CISH standard deviations seemed to be lower for the average HER2/CEN-17 ratio, HER2 and CEN-17 copy numbers compared to PathVysion FISH. This could be interpreted as the HER2 CISH assay being the most sensitive assay.In the current data analysis a significantly higher average CEN-17 signal counts in HER2/CEN-17 ratios in highly amplified specimens between HER2 CISH and FISH assays (see Figure Also, there was a tendency towards differences between HER2 CISH and PathVysion FISH and as could be expected the average evaluation time for a CISH staining was significantly shorter. This is most likely due to the easy access to the morphological information and hence a faster selection of areas for enumeration. In HER2 CISH a higher number of specimens were reported failed in comparison to staining with PathVysion FISH. This is likely to be due to CISH being a new technique implemented in the US reference laboratory and PathVysion FISH being a well established test method at this site. With respect to HER2 CISH the normal cells in the tissue surrounding the tumor area can be used as internal control securing good staining quality and preventing wrong diagnosis being based on a failed slide.As a parallel study, the slide evaluation time was compared between HER2 CISH and FISH data from 365 different primary breast cancer specimens it is confirmed that the FDA approved HER2 CISH pharmDx\u2122 Kit is a reliable chromogenic alternative to today's FDA approved FISH techniques for HER2 gene status determination in FFPE breast carcinoma specimens.From this study based on JM, UH, SM and AS are employees at Dako Denmark A/S.The clinical trial was planned by UH, AS and JM. Study protocols were written by JM with help from UH and AS. Training of the US reference laboratory was performed by UH and SM. Data analysis was performed by JM and JM wrote the manuscript with help from UH, SM and AS. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/12/3/prepub"}
+{"text": "The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element. In mammals, a small subset of autosomal genes are expressed mainly from one parental allele due to an epigenetic mechanism termed genomic imprinting . These gcis-regulatory elements similar to other imprinted domains . The converted DNA was used for PCR amplification using the two following primers: ECR18-bis-a  with the DMEM plus GlutaMAX medium containing 5% fetal bovine serum and 1% antibiotic-antimycotic (GibcoBRL). On the following day, the cells were transfected with 1 \u00b5g of each reporter construct using 2.5 \u00b5L Lipofectamine 2000 (Invitrogen). Fresh complete media was added 6 hrs post transfection, and total cell lysates were harvested in 100 \u00b5L of 1X reporter lysis buffer 48 hrs post transfection (Promega). The luciferase assay was performed for each promoter construct in triplicate according to the company\u2019s protocol (Promega). We also performed a similar set of transfection experiments using an independent \u03b2-Geo reporter construct to monitor the transfection efficiency. Thus, the initial values from the luciferase assay were normalized with the \u03b2-Gal activity.For promoter assays, we first modified the promoterless \u03b2-Geo reporter  by replaMaterial S1Sequence information for 18 ECRs and oligonucleotides used for 3C and ChIP analyses.(DOCX)Click here for additional data file."}
+{"text": "Ring chromosomes are unusual abnormalities that are observed in prenatal diagnosis. A 23-year-old patient  referred for amniocentesis due to abnormal maternal serum screening result in the 16th week of second pregnancy. Cytogenetic analysis of cultured amniyotic fluid cells revealed out ring chromosome 4. Both maternal and paternal karyotypes were normal. Terminal deletion was observed in both 4p and 4q arms of ring chromosome 4 by fluorescence in situ hybridization (FISH). However deletion was not observed in the WHS critical region of both normal and ring chromosome 4 by an additional FISH study. These results were confirmed by means of array-CGH showing terminal deletions on 4p16.3 (130\u2009kb) and 4q35.2 (2.449\u2009Mb). In the 21th week of pregnancy, no gross anomalia, except two weeks symmetric growth retardation, was present in the fetal ultrasonographic examination. According to our review of literature, this is the first prenatal case with 4p and 4q subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region. Our report describes the prenatal case with a ring chromosome 4 abnormality completely characterized by array-CGH which provided complementary data for genetic counseling of prenatal diagnosis. Autosomal ring chromosomes are uncommon cytogenetic aberrations in prenatal diagnosis. Estimated frequency ranges from 1/27000 to 1/62000 in consecutive newborn and prenatal diagnosis studies . Cases wIn the present study, our aim was to determine whether ring chromosome leads to abnormality in the following weeks of pregnancy or in the postnatal period of fetus which has no gross anomalia in the second trimester of pregnancy, except growth retardation, and the second aim was to manage genetic counselling to family members. For this purpose, we performed classical cytogenetic, molecular cytogenetic (FISH), and array-CGH techniques in amniocentesis and chordosynthesis samples to establish whether any deletion exists in ring chromosome 4 with breakpoints of deleted segments and identify genes involving deletions.A 23-years-old patient with a history of a pregnancy resulted with abortus referred for amniocentesis due to abnormal maternal serum screening test in the 16th week of the 2nd pregnancy. Down syndrome risk was 1,97 according to maternal serum screening test in the 12th week of pregnancy. Cytogenetic analysis of the cultured amniocytes using flask culture method revealed ring chromosome 4 ] . To confThe ring chromosome 4 was characterized by FISH using the 4p and 4q specific subtelomeric probe . FISH study showed deletions at the subtelomeric regions of 4p and 4q on the ring chromosome 4 . WHS criArray-CGH was performed to determine breaking points and involved genes on terminal deletions of ring chromosome 4.CytoSure microarray platform (Oxford Gene Technology) was used for aCGH analysis. This platform has approximately 44,000 oligonucleotide probes. The arrays were scanned on an Agilent G2505B scanner and quantified using Agilent's Feature Extraction software. Data was then normalised using CytoSure visualisation software which uses a standard LOWESS method . SubsequIn the 21th week of pregnancy, no gross anomalia, except 2 weeks symmetric growth retardation, existed in the fetal ultrasonographic examination. Parents were informed about the probable prenatal and postnatal complications of subtelomeric deletion of p and q arms of chromosome 4. However, parents denied to terminate pregnancy.In the present report, prenatal case with 4p16.3 and 4q35.2 subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region was diagnosed.Ring chromosome 4 is an uncommon cytogenetic abnormality that has been rarely diagnosed in the prenatal stage. Up to now, three cases with prenatal diagnosis of ring chromosome 4 were reported in the English literature. In the first case, Sherer et al. reported a severely and symmetrically growth-retarded fetus with microcephaly, hypertelorism, and hypoplastic genitalia with a two-vessel umbilical cord, a characteristic \u201cGreek warrior helmet\u201d facial profile and clubfoot in a 29th week fetus of ring chromosome 4 carrier with 4p15 and 4q35 deletions . In the Among published cases with 4p16 deletions, South et al. reported a case of ring 4 with 1.27\u20131.46\u2009Mb deletion at 4p16.3 presented with significant postnatal growth retardation, mild developmental retardation, and nutritional disturbances. In addition to abnormalities of the first case, the second reported that a case exhibited terminal 4p microdeletion of approximately 1.78\u2009Mb caused complex seizure disorder. No deletion was observed at WHS region in both cases . KhonsarIn the present case, genes that encode 6 zinc finger protein exist on deleted segment of 130\u2009kb at 4p16.3 as shown in According to our knowledge, this is the first de novo ring chromosome 4 case with 4p and 4q subtelomeric deletion without deletion of WHS critical region and that had no fetal anomalia except intrauterine growth retardation determined by second trimester fetal ultrasonographic examination. In the light of literature, genetic counselling was performed to family members by considering genes on deleted segments. The role of FISH and array-CGH on genetic diagnosis and phenotypic correlation in the prenatal evaluation is confirmed."}
+{"text": "In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.Rubisco  catalyses the key reaction in the photosynthetic assimilation of COsensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution.We investigated Rubisco evolution in Amaranthaceae Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco. Observations of significant variation in Rubisco kinetics between plant species 2 and O2 as substrates, and under present-day atmospheric conditions (385 p.p.m. CO2), the carboxylase activity of Rubisco is undersaturated in C3 plants, and the oxygenase activity gives rise directly to the competing process of photorespiration. Photorespiratory rates in C3 plants increase steeply with increasing temperature and give rise to a distinct temperature optimum for net photosynthesis, above which plant yields decline steeply. Increased carbon loss via photorespiration at higher temperatures is attributable mainly to the declining specificity of Rubisco for CO2 relative to O2 (Sc/o). In fact, it has been proposed that the very slow turnover of Rubisco (kcat \u22483 s\u22121) is a direct consequence of the enzyme's particular reaction mechanism, in which Sc/o is maximized by tight binding of the transition-state intermediate kcat and of the large size of the holoenzyme (560 kDa) is that Rubisco comprises up to 50% of soluble protein in photosynthetic tissues and is probably the most abundant enzyme on Earth Rubisco discriminates imperfectly between CO4 photosynthesis or crassulacean acid metabolism (CAM), and in many aquatic organisms, photorespiration is partially or completely suppressed by the operation of an auxiliary CO2-concentrating mechanism. C4 plants initially fix atmospheric carbon in the mesophyll cells using phosphoenolpyruvate carboxylase, an enzyme with a high effective affinity for CO2 (HCO3\u2212 being the true substrate of the enzyme). Further four-carbon compounds  produced by this fixation are transported to the specialized bundle-sheath cells, where CO2 is released and fixed by Rubisco. Rubisco from C4 plants, which experiences \u223c10-fold higher CO2 concentrations in bundle-sheath cells than does the enzyme in C3 plants 2 but a higher kcat (\u22484 s\u22121). Having less specific but faster Rubisco and no photorespiration losses, C4 plants require 60 to 75% less Rubisco to match the photosynthetic capacity of C3 plants 4 plants such as maize, sugarcane and sorghum are among the most productive of all species cultivated agriculturally. Although C4 plants appeared relatively recently in evolutionary terms and constitute only 3% of terrestrial plant species, they are already among the most successful and abundant groups in warm climates and are responsible for about 20% of terrestrial gross primary productivity In terrestrial plants with C4 photosynthesis evolved independently in at least 62 recognizable lineages of angiosperms and represents one of the most striking examples of a convergent biochemical adaptation in plants 4 monocots in the Poaceae, while C4 eudicots have been studied less intensively. The family Amaranthaceae sensu lato (i.e. including Chenopodiaceae) 4 species 4 family among eudicots and the third-largest among angiosperms (after Poaceae and Cyperaceae). C4 photosynthesis evolved at least 15 times within Amaranthaceae 4 photosynthesis and Rubisco. Notably, the Amaranthaceae exceed the Poaceae and Cyperaceae in the diversity of photosynthetic organ anatomy 4 plants that lack Kranz anatomy, with three species having a single-cell rather than the more usual dual-cell C4 system sensu stricto and primarily temperate and subtropical Chenopodiaceae have long been treated as two closely related families (see review in sensu lato (henceforth referred to as Amaranthaceae) constitutes the most diverse lineage of the Caryophyllales. Both C3 and C4 species from this family are adapted to a range of conditions from temperate meadows to the tropics, hot deserts and salt marshes. However, it has been shown that the abundance of C4 Amaranthaceae is correlated with precipitation but not temperature, in contrast to the abundance of C4 Poaceae and Cyperaceae, which is correlated with temperature but not precipitation C4 Amaranthaceae showing different suites of anatomical and biochemical adaptations as well as ecological preferences compared to C4 Poaceae and Cyperaceae, like C4 monocots they possess faster but less CO2-specific Rubiscos than their C3 relatives 4 eudicots and monocots represents a notable example of convergent evolution of enzyme properties in phylogenetically distant groups. However, it is not known whether this functional convergence in Rubisco kinetics evolved via similar or different structural changes in protein dN) and synonymous  mutations along a phylogenetic tree using maximum likelihood and Bayesian frameworks rbcL, which encodes the large subunit of Rubisco that forms the enzyme's active site, and showed that positive Darwinian selection is acting within most lineages of plants 4 lineages of Poaceae and Cyperaceae Flaveria3 and C4 species. However, no specific analysis has yet been made of Rubisco sequence evolution in a large group of C4 eudicots. In this study, we investigate positive selection on the rbcL gene of plants from the Amaranthaceae family and, in particular, focus on coevolution of Rubisco and C4 photosynthesis asking whether positive selection on the rbcL gene occured on branches leading to C4 clades and/or within C4 clades. Finally, we address the following question: which amino-acid replacements were associated with transitions from C3 to C4 photosynthesis in Amaranthaceae, and are these replacements unique to this lineage or shared with C4 monocots and/or Flaveria?Despite CrbcL nucleotide sequences available in GenBank and aligned them. Sequences shorter than 1341 base pairs and sequences with missing data were excluded. The resulting trimmed alignment consisted of 179 rbcL sequences of 1341 base pairs long which represented 94% of the rbcL coding region and corresponded to positions 64 to 1404 of the rbcL sequence of Spinacia oleracea (GenBank AJ400848). The analysed dataset consisted of 95 C3 and 84 C4 species  and synonymous (dS) mutations along a phylogenetic tree dN/dS\u200a=\u200a1), while purifying (negative) or adaptive (positive) selection is expected to deflate (dN/dS<1) or inflate (dN/dS>1) this ratio, respectively. One can use likelihood ratio tests to detect positive selection that affects only a subset of codons in a protein-coding gene, with positive selection indicated by accelerated nonsynonymous substitutions. Models assuming positive selection along all phylogeny or prespecified branches only (e.g. C4 lineages in our case) can be employed within Phylogenetic Analysis by Maximum Likelihood (PAML) framework Positive, neutral, or purifying selection at the molecular level can be inferred by comparing rates of non-synonymous . Further, codeml was used to perform likelihood ratio tests (LRTs) for positive selection among amino acid sites. The tree length value obtained from the model M0 was compared with tree length values obtained from other models to control for consistency among models. We performed two LRTs to compare null models which assume the same selective pressure along all branches of a phylogeny and do not allow positive selection (dN/dS >1) with nested models which do allow it dN/dS \u22641 with the M2a model . The second LRT, M8a-M8, compares the M8a model which assumes a discrete beta distribution for dN/dS, which is constrained between 0 and 1 including a class with dN/dS \u200a=\u200a1 with the M8 model which allows the same distribution as M8a but an extra class under positive selection with dN/dS >1.We used the 4 branches only where positive selection was allowed only on branches leading to C4 clades. The second was the A model for all C4 branches where positive selection was allowed on branches leading to C4 clades and branches within C4 clades. The A1-A LRT compares the null model A1 with the nested model A. Both the A1 and A models allow dN/dS ratios to vary among sites and among lineages. The A1 model allows 0< dN/dS <1 and dN/dS \u200a=\u200a1 for all branches, and also two additional classes of codons with fixed dN/dS \u200a=\u200a1 along prespecified foreground branches while restricted as 0< dN/dS <1 and dN/dS \u200a=\u200a1 on background branches. The alternative A model allows 0< dN/dS <1 and dN/dS \u200a=\u200a1 for all branches, and also two additional classes of codons under positive selection with dN/dS >1 along prespecified foreground branches while restricted as 0< dN/dS <1 and dN/dS \u200a=\u200a1 on background branches. C4 lineages were marked as foreground branches.Finally, we performed two branch-site tests of positive selection along prespecified foreground branches dN/dS >1, whereas the M2a, M8 and A models are alternative models which do allow codons with dN/dS >1. The significance of the LRTs was calculated assuming that twice the difference in the log of maximum likelihood between the two models was distributed as a chi-square distribution with the degrees of freedom (df) given by the difference in the numbers of parameters in the two nested models dN/dS values (0.1 and 0.4) to test for suboptimal local peaks. To identify amino acid sites potentially under positive selection, the parameter estimates from M2a, M8 and A models were used to calculate the posterior probabilities that an amino acid belongs to a class with dN/dS >1 using the Bayes Empirical Bayes (BEB) approaches implemented in PAML codeml we used the SLR program which implements \u201csitewise likelihood-ratio\u201d (SLR) method for detecting non-neutral evolution, a statistical test that can identify sites under positive selection even when the strength of selection is low dN/dS \u200a=\u200a1) against an alternative model (dN/dS \u22601). SLR method is a test of whether a given site has undergone selection or not, and the test statistic summarizes the strength of the evidence for selection rather than the strength of the selection itself codeml and SLR.For all LRTs, the first model is a simplified version of the second, with fewer parameters, and is thus expected to provide a poorer fit to the data (lower maximum likelihood). The M1a, M8a and A1 models are null models which do not allow codons with 4 photosynthesis and (2) the presence/absence of particular amino-acid at sites found to be under positive selection along C4 branches in the A model of codeml. For this purpose, we used the phylogeny obtained using RAxML (see above) and performed Pagel's test of correlated (discrete) character evolution 4 branches and Bonferroni correction was performed for simultaneous statistical testing.Closely related taxa are not independent data points and they consequently violate the assumptions of conventional statistical methods Spinacia oleracea, Amaranthaceae) from data file 1RBO We used the published Rubisco protein structure from spinach  to test for suboptimal local peaks produced identical results. LRTs for positive selection p-value <0.00001; 4 clades we used two branch site models  for variation in 4 clades while under relaxed or purifying selection within C3 clades with a posterior probability >0.99 by BEB in the A model for C4 branches. Both sites had only two alternative amino acids in this dataset  between the presence of C4 photosynthesis and the presence of \u2018C4\u2019 amino acids at sites 281 and 309, shown to be under positive selection along C4 branches.Four sites were identified as evolving under positive selection with a posterior probability >0.95 by BEB  dataset . One of  species , but therbcL showed that positive selection is widespread among all main lineages of land plants, but is restricted to a relatively small number of Rubisco amino acid residues within functionally important sites rbcL is under positive selection in particular taxonomic groups As the performance of Rubisco can directly affect plant growth and crop yields, substantial efforts have been made to study its structure and function, with the ultimate aim of trying to improve Rubisco performance 4 photosynthesis has increased the availability of CO2 for Rubisco in numerous independently evolved lineages of C4 plants, including Amaranthaceae, driving selection for less specific but faster enzymes which have both higher KM(CO2) and kcat values 4 branches provided a significantly better fit to the analysed Amaranthaceae dataset than the null model without selection  and hence allows detection only of the most typical substitutions, potentially missing ones that are unique for a particular branch. Other possible explanations are variation in Rubisco kinetic properties not only between C3 and C4 groups of species but also within these groups 3 and C4 plants rbcS genes encoding small subunits have been shown under positive selection in C4FlaveriaInterestingly, both selected residues in C4 Amaranthaceae and C4 Cyperaceae and Poaceae, representing eudicots and monocots with significantly different anatomy and ecological preferences 3 plants are considered as well. Various groups of C3 plants such as some aquatic species and C3 species from cold habitats have faster but less CO2-specific Rubisco compared with their C3 relatives from terrestrial and warm conditions, respectively 3 plants can arrive at the same evolutionary solutions for Rubisco fine-tuning as C4 plants. Indeed, \u2018C4\u2019 amino acids shown for C4 Amaranthaceae in the present study and for C4 monocots and Flaveria previously 3 plants by Kapralov and Filatov 4\u2019 amino acids, 281S and 309I, evolved in parallel in various phylogenetically distant lineages of C3 and C4 plants in which faster but less specific Rubisco was needed.Identical amino-acids in Rubisco of CThe residue 309 is located on the interface of large subunits within a large subunit dimer, while the residue 281 is involved into dimer-dimer interactions . MethionFlaveria changed Rubisco kinetics from \u201cC3-like\u201d to \u201cC4-like\u201d making the enzyme faster but less CO2-specific. Importance of M309I replacement for changes in kinetics of Flaveria Rubisco was predicted using in silico approach similar to one used in the present study in planta by the study of Whitney et al. Effects of A281S replacement on kinetics of land plants Rubisco has not been studied, while recent study by Whitney et al. Continuing population growth creating increasing demand for food, coupled with future climate change and its potentially dire consequences such as biome collapse and crop failure, both call for an improved understanding of mechanisms allowing plant species to adapt the photosynthetic process to a wide range of conditions. Hence, there is a necessity for more phylogeny-based studies of genes encoding Rubisco from various lineages of phototrophs established in different conditions to better understand Rubisco evolution at the molecular level. The integration of phylogenetic and biochemical research is required to study how Darwinian selection has created a range of enzymes with different kinetic and physical properties tailored to function in virtually all ecosystems on our planet. Knowledge of the role of specific residues in Rubisco adaptation to the particular conditions may provide clues for engineering better enzymes suited to contemporary agricultural needs as well as helping to understand what modifications in the enzyme may have been (and perhaps will be) driven by adaptation to different environmental conditions.Table S1List of studied species.(XLSX)Click here for additional data file."}
+{"text": "Mesenchymal stem cells (MSCs) are able to reduce systemic inflammatory response in an experimental sepsis. One of the limiting factors of MSC therapy is a high degree of apoptosis of transplanted cells. On the basis of recently detected receptors for erythropoietin (EPO) on the surface of MSCs we hypothesized that introduction of EPO together with MSCs may increase survival of grafted MSCs and improve the clinical efficacy of cell transplantation.5 allogeneic MSCs (Group 3), 8.5 \u03bcg recombinant EPO-\u03b2 (Group 4), MSCs and EPO in the same doses (Group 5). Surviving animals were euthanized on the fourth day. The morphological study, white blood cell count (WBC) and serum level of IL-1\u03b2, IL-2, IL-6, and TNF\u03b1 measurement were performed.Fifty Wistar male rats were randomized into five groups with 10 animals in each: Group 1 were healthy controls, Groups 2 to 5 were intraperitoneally introduced bacterial LPS 20 mg/kg. Two hours later LPS injection animals received the following intravenous treatment: 4 \u00d7 10The highest WBC was found in the group of combined treatment EPO + MSCs. The serum IL-1\u03b2 level in Groups 2 and 4 was significantly higher than in healthy and treated with MSCs and EPO + MSCs animals. The main results of the morphologic analysis of different tissues in the groups are presented in Table Combined treatment with EPO and MSCs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of endotoxemia in rats."}
+{"text": "In vertebrates, poly(A) binding protein (PABP) is known to exist in five different isoforms. PABPs are primarily cytosolic with the exception of the nuclear PABP (PABPN1), which is located in the nucleus. Within the nucleus, PABPN1 is believed to bind to the poly(A) tail of nascent mRNA and along with cleavage and polyadenylation specificity factor (CPSF) define the length of the newly synthesized poly(A) tail.The cellular role of PABP1 has been extensively studied over the years; however, the function of other PABPs remains poorly defined. In order to understand the role of PABPN1 in cellular mRNA metabolism and it\u2019s interrelation with other PABPs, we depleted PABPN1 using RNAi in HeLa and HEK293 cells. Our results show that PABPN1 depletion did not have any effect on the poly(A) tail length, nuclear export of mRNA, mRNA translation, and transcription. Rather, PABPN1 depletion resulted in a compensatory response as observed by increased level of PABP5 and nuclear accumulation of PABP4. In addition, PABP4 was associated with the poly(A) tract of pre-mRNA and CPSF in PABPN1 depleted cells. Nevertheless, PABPN1 depletion significantly affected cell survival as evidenced by an increase in apoptosis markers: phosphorylated p53 and PUMA and as judged by the expression of ER stress marker GRP78.Our results suggest that although function of PABPN1 may be compensated by nuclear translocation of PABP4 and perhaps by increase in the cytoplasmic abundance of PABP5, these were not sufficient to prevent apoptosis of cells. Thus PABPN1 may have a novel anti apoptotic role in mammalian cells. Mammalian nuclear poly(A) binding protein (PABPN1) is a highly conserved nuclear RNA binding protein with specificity towards the poly(A) tract of eukaryotic mRNAs. It consists of one typical RNA recognition motifs (RRM) domain with consensus RNP1 and RNP2 motifs in the central region of the polypeptide, and an arginine rich C- terminal domain Results of biochemical studies suggest that the main cellular function of PABPN1 is to stimulate the elongation of poly(A) tract of eukaryotic mRNA, and control its length Xenopus laevis suggests that both PABP1 and PABP4 are required for their normal development In contrast to the presence of only one nuclear poly(A) binding protein, vertebrates express several cytoplasmic PABPs with conserved RNA binding motifs Deletion of yeast pabp2 gene, which is the homolog of mammalian PABPN1, leads to extension of poly(A) tract of mRNAs The Stealth RNAi sequence used for silencing PABPN1 expression was the same as described earlier 2. Approximately 30% confluent cells, grown on 35 mm dishes were transfected with double stranded si RNA using lipofectamine2000\u2122  according to manufacturer\u2019s instructions. In short, for each 35 mm plate 1 \u00b5l of siRNA  was mixed with 5 \u00b5l lipofectamine2000\u2122 and 500 \u00b5l of Opti-MEM  medium and incubated for 20 min before being added to the culture dish. After 24 h, the medium containing the RNA-lipofectamine complex was replaced by fresh DMEM containing 10% FBS and 1% glutamine. For co-transfection of the cells, 1 \u00b5g of plasmid was mixed with siRNA, and transfection was performed as described above. Cells were harvested between 45\u201380 h after transfection depending on the experiment.HeLa and HEK293 cells were grown in Dulbecco\u2019s Modified Eagle\u2019s medium (DMEM) containing 1% glutamine  and 10% FBS  at 37\u00b0C in presence of 5% CO2HPO4, 2H2O, 1.47 mM KH2PO4, pH 7.4). 200 \u00b5l of cytoplasmic lysis buffer  was added to the plate and incubated for 10\u201315 min on ice. Cells were scrapped off and the cell suspension was passed through a 30 gauge needle until 90% cells were lysed as judged by the presence of cytoplasmic tag free nuclei. Cell lysates were centrifuged at 2500 g for 7 min to obtain the cytoplasmic fraction. The pellet (nuclear fraction) was washed 3 times with the lysis buffer and lysed in (a) 150 \u00b5l of Laemmli buffer , 10% (v/v) glycerol, 5% (v/v) \u03b2-mercaptoethanol, 0.01% (w/v) bromophenol blue) for protein extraction or (b) in 1 ml of Trizol  for RNA extraction.For isolation of nuclear proteins, cells grown on a 35 mm plate were washed three times with PBS  according to manufacturer\u2019s instructions. The quality and quantity of the RNA were determined by 1.5% agarose gel electrophoresis and spectrophotometric measurements respectively. The levels of specific mRNAs were determined by RT-PCR. An aliquot of total RNA (100\u2013500 ng) was reverse transcribed using High Capacity cDNA transcription kit . After the reaction, 2 \u00b5L of the cDNA sample was amplified by PCR in a total master mix  reaction volume of 50 \u00b5L, which included 100 ng of primers specific for diffeent mRNAs  2, 1 mM EGTA, 100 mM NaPPi, 100 mM NaF, and 1% Triton X-100) by gentle sonication using XL2020 Sonicator (Mandel Scientific) at setting 1, pulse 3 (4 s each) to break the cytoplasmic membrane. Cells were then centrifuged at 2500 g for 10 min to separate the nuclear fraction (pellet). The pellet was resuspended in PLC buffer with 1% formaldehyde (Electron Microscopy Sciences) for 10 min at room temperature (RT) to crosslink the RNA and proteins of RNP complexes. The nuclear pellet was then incubated in 0.25 M of glycine, centrifuged and washed three times with PLC buffer. The nuclear pellet was finally resuspended in 400 \u00b5l of chilled PLC buffer supplemented with protease inhibitor (Roche) and again sonicated at a higher setting . Cell debris was removed by centrifugation at 12000 rpm for 10 min. The nuclear extract was incubated overnight with PABPC4 or PABPN1 antibody and rabbit or goat IgG-Agarose beads (Sigma). Following Immunoprecipitation, beads were washed extensively with PLC buffer and the RNA-protein cross-link was reversed by incubation at 70\u00b0C for 45 min in 100 \u00b5L of elution buffer . RNA was then extracted with three volumes of a mixture of phenol/chloroform (50/50). The RNA was precipitated using one volume of ethanol, and left overnight at \u221220\u00b0C. The precipitated RNA was collected by centrifugation at 10,000 g for 10 min at 4\u00b0C and resuspended in RNAse free water. Contaminating DNA was removed from RNA samples by RQ1 RNase-free DNAse (Promega) treatment prior to being reverse transcribed and PCR amplified using \u03b2-actin pre-mRNA primers Immunoprecipitation was done as previously described For immunoprecipitation studies nuclear extracts were prepared as described above and incubated with the designated antibody and appropriate IgG agarose beads for 16 h at 4\u00b0C. The eluted samples and nuclear extracts were subjected to SDS-PAGE and Western blotting.12\u201318 in a reaction containing 0.1 M DTT, 10 mM dNTP, 10 mM ATP, SuperscriptH\u2212 buffer, and 10 U/\u00b5l T4 DNA ligase (New England Biolabs). The resultant mixture was hybridized with oligo dT anchor (5\u2032GCGAGCTCCGCGGCCGCGT12 3\u2032) followed by reverse transcription with RNAseH\u2212 reverse transcriptase (Invitrogen). The cDNA was then PCR amplified with oligo dT primer and \u03b2-actin specific primer (CAC ACA GGG GAG GTG ATA GCA).PAT assay used to determine the poly(A) tail length was done as described previously by Salles and Strikeland 1999 35S]-methionine. For this, the cells were pre-incubated in methionine free DMEM medium  with 10% dialyzed FBS  for 2 h at 37\u00b0C to reduce the pool of endogenous methionine. Samples were incubated with 50 \u00b5Ci [35S]-Methionine  in 1 ml of fresh methionine free DMEM medium with 10% dialyzed FBS  at 37\u00b0C for 30 min. Following incubation, cells were lysed with 200 \u00b5L of Lamelli buffer. The samples were boiled for 5 min and separated by SDS-10% polyacrylamide gel electrophoresis (PAGE). The gel was fixed in 10% trichloroacetic acid (TCA), at 95\u00b0C and allowed to cool down for 5 min at RT. The gel was then washed twice with 10% TCA at 20\u00b0C, and twice with 10% ethanol and finally with 100% ethanol. The gel was then soaked in 1 M Na-Salicylate for 30 min and dried at 50\u00b0C for one hour, and autofluorographed with an X-ray film for the desired time before being developed.Protein synthesis was measured by radioactively labelling cellular proteins with [35S] isotope.10 \u00b5L of each sample was spotted on small strips of filter paper. The filter paper was then placed in 100 mL of 10% TCA, and brought to boil and allowed to cool for 10 min; this process was then repeated three times. The filters were washed twice with 10% TCA at RT followed by 100% ethanol. The filter papers were placed in scintillation vials with sufficient amount of liquid scintillation counting fluid and counted using a liquid scintillation counter at a setting for -Methionine into polypeptides. Results of analyses of protein synthesis by SDS-PAGE followed by auto fluorography of equivalent amount proteins from NT, UTR and Si-PABPN1 transfected cells showed that there was no detectable difference in protein synthesis amongst these samples  tract addition (8) we tested whether the nuclear PABP4 in PABPN1 depleted cells also interacts with CPSF. The results of co-immunoprecipitation studies using PABP4 antibody show that CPSF1 was immunoprecipitated by PABP4 antibody from the nuclear extract of PABPN1-Si treated cells but not from the same of NT cells  G. As poExamination of the presence of apoptotic cells by AO/EtBr and DAPI staining revealed that significantly more cells were apoptotic in the cultures following PABPN1-Si treatment than what was observed for the NT and PABPN1-UTR treated cells. After 72 hours almost 40% of PABPN1 depleted cells were undergoing apoptosis while negligible amount of cells in our control cultures were apoptotic . In addiFurthermore, we measured the abundance of additional markers of cell stress, such as, the constitutive HSC70 and the inducible HSP70 by western blotting with an antibody that recognizes both proteins. The result show that HSC/HSP70 abundance did not increase following PABPN1 depletion. Therefore, PABPN1 depletion produced a distinct ER stress response. In our studies the abundance of eIF2-\u03b1 and GAPDH remained unchanged by PABPN1 depletion suggesting that the increase of phospho p53, Puma and GRP78 was not due to non specific effect of PABPN1 depletion on protein abundance. Taken collectively results of our studies suggest that in spite of the effort of PABP4 and PABP5 to rescue cells from the adverse effect of PABPN1 depletion, it was not sufficient to prevent cell death. Perhaps PABPN1 plays a more critical anti apoptotic function than its previously believed role in poly(A) tail elongation.We tested two different target sequence of PABPN1 mRNA for SiRNA mediated ablation of PABPN1 expression. The first target was within the coding region of mRNA and the second target sequence was in the 5\u2032UTR. Our studies demonstrated that the coding region was an effective target and the 5\u2032UTR was probably not accessible for base pairing with the SiRNA due to presence of extensive secondary structures, therefore, did not produce any effect on PABPN1 expression. Examination of various parameters of cellular mRNA metabolism demonstrated that there were no detectable effects of PABPN1 depletion on mRNA export, mRNA abundance, and mRNA translation. In addition, cellular transcription as judged by the presence of pre-mRNA transcript of \u03b2-actin and localization of splicing factor1 in nuclear speckles was unaffected by PABPN1 depletion. Although it is well accepted that the central role of PABPN1 is to stimulate addition of poly(A) tract we did not observe any shortening of the length of the poly(A) tract. We examined the poly(A) tract length for a specific mRNA such as the \u03b2-actin mRNA, and found that the average poly(A) tract length was approximately 150\u2013200 nucleotides for both control and PABPN1 depleted cells.We have demonstrated that amongst the cytoplasmic PABPs only PABP5 showed a significant increase in abundance in PABPN1 depleted cells. The cellular level of other cytoplasmic PABPs including PABP3 and PABP4 remained unchanged. The significance of the increase of PABP5 abundance is not clear at this time. Since PABP5 is also capable of interacting with eIF4G albeit with a lower efficiency than PABP1 (results not shown), perhaps the increase in PABP5 abundance stimulates protein synthesis in PABPN1 depleted cells as cellular homeostasis. Interestingly, a small but reproducible increase of global protein synthesis was observed following PABPN1 depletion. Beside its ability to bind poly(A), little is known regarding the cellular function of PABP5. It is possible that PABP5 is involved in regulating the metabolism of a specific sub-family of mRNAs (18). As such, it will be important to examine whether translation and/or stability of mRNAs encoding regulators of apoptosis can be modulated by PABP5.Although the abundance of PABP4 did not change in PABPN1 depleted cells, an increase in its nuclear localization was evident following depletion of cellular PABPN1. It was previously shown that UV damage results in nuclear translocation of both PABP1 and PABP4 In spite of the cellular homeostasis by increasing PABP5 abundance and nuclear translocation of a related poly(A) binding protein almost 40% PABPN1 depleted cells were apoptotic within 72 hours. Recent evidences show that beside poly(A) addition PABPN1 has other vital functions in RNA metabolism , such as, processing of snoRNAs (20) and selection of cleavage and polyadenylation site (21). It was shown recently that depletion of PABPN1 in mammalian cells leads to a switch to the use of the proximal polyadenylation site for a large number of cellular mRNAs resulting in transcripts with shorter 3\u2032 untranslated region (UTR). Shortening of the 3\u2032 UTR results in the loss of micro RNA target sites, therefore, PABPN1 is important for 3\u2032UTR mediated repression of gene expression (21). It is likely that PABP4 only compensates some but not all the cellular functions of PABPN1.PUMA gene We showed here that in response to PABPN1 depletion p53 was phosphorylated in serine 46 which is known to activate p53 for inducing expression of PUMA. We demonstrated PUMA expression is activated in PABPN1 depleted cells. Phosphorylation alters the structural conformation of p53 leading to its stabilization and activation Figure S1Dose response of PCR reaction for different mRNAs. 250 ng of total RNA from non-transfected (NT) Hela cells was reverse transcribed as described in (TIF)Click here for additional data file."}
+{"text": "Understanding the dynamics of influenza transmission on international flights is necessary for prioritizing public health response to pandemic incursions. A retrospective cohort study to ascertain in-flight transmission of pandemic (H1N1) 2009 and influenza-like illness (ILI) was undertaken for 2 long-haul flights entering Australia during May 2009. Combined results, including survey responses from 319 (43%) of 738 passengers, showed that 13 (2%) had an ILI in flight and an ILI developed in 32 (5%) passengers during the first week post arrival. Passengers were at 3.6% increased risk of contracting pandemic (H1N1) 2009 if they sat in the same row as or within 2 rows of persons who were symptomatic preflight. A closer exposed zone  increased the risk for postflight disease to 7.7%. Efficiency of contact tracing without compromising the effectiveness of the public health intervention might be improved by limiting the exposed zone. The emergence of pandemic influenza A (H1N1) 2009 in Mexico and the United States, with rapid spread to Europe, Asia, and the Pacific, is testament to the ease of spread of infectious disease across the globe (\u2013Mycobacterium tuberculosis (\u2013Reports documenting spread of disease during airline flight are limited (A retrospective cohort study designed to determine exposure risk to known pandemic (H1N1) 2009 virus was undertaken for 2 long-haul flights that entered Australia the weekend of May 23\u201324, 2009. Flight 1 was chosen after identification of 6 passengers with confirmed pandemic (H1N1) 2009 infection within 24 hours after flight arrival from the United States. Flight 2 was chosen after identification of a confirmed case of pandemic (H1N1) 2009. This flight came from an area that lacked community transmission. Passenger details were obtained through collection of Health Declaration Cards and comparing the cards to flight manifests obtained from the airlines.>1 symptom  within 7\u201314 days before the flight or during the flight or <7 days after arrival. The time periods were put in place to help determine when passengers were most likely to have contracted their ILI. Passengers were excluded who indicated bacterial infection  or regular health issues, such as migraines. All but 4 passengers reporting symptoms had confirmation of ILI status from a qualified health professional.The definition of ILI was broad to capture as many persons as possible within the dataset. Passengers were asked to self-report development of any of the following signs or symptoms: fever, cough, sore throat, headache, runny nose, muscle aches, diarrhea, and lethargy. ILI was defined as Self-identification of other health conditions that were considered potential concurrent conditions for purpose of this study included obesity, diabetes mellitus, immunosuppression, asthma, chronic lung disease, and pregnancy. Seat location, concurrent condition status, and contraction of disease were compared. Ethics approval was given by the Australian Government Department of Health and Ageing Ethics Committee and the Australian National University Human Research Ethics Committee.Surveys were distributed to passengers 3 months after flight arrival. The survey asked about influenza-like symptoms, symptom onset time, concurrent conditions, antiviral prophylaxis and treatment, isolation or quarantine dates, other potential exposure to ILI before and after the flight, contact with health professionals after the flight, and details of testing for pandemic (H1N1) 2009 virus. Two reminders were sent to improve the response rate of the study.As a triangulation method, all passenger names, passenger sex, disease onset dates, and postal codes were cross-checked against those of passengers with known pandemic (H1N1) 2009 cases that were notified to national authorities for 1 month after flight arrival to verify information received from the survey responses and identify additional cases. Travel details were verified for pandemic (H1N1) 2009 case-patients identified through national notification. Contact tracing through public health authorities also identified ILI case-patients who had negative laboratory test results for pandemic (H1N1) 2009 virus.The increased risk of passengers contracting either laboratory-confirmed pandemic (H1N1) 2009 or an ILI (including pandemic [H1N1] 2009) was separately estimated by dividing the number of persons with the illness by the number of susceptible persons in the contact zones as described. For pandemic (H1N1) 2009, only passengers sitting in the economy class were considered exposed because of the location of persons displaying symptoms preflight or during the flight and the sectional layout of the aircraft.Of the 738 passengers on the 2 flights, 319 (43%) responded to a questionnaire; 143 (18%) passengers could not be contacted. Cross-checking of mandatory notifications of pandemic (H1N1) 2009 against all passengers on both flights found 2 additional pandemic (H1N1) 2009 infection cases while also confirming symptom data on survey responses. Contact tracing by public health authorities found 5 additional passengers with ILI who had negative test results for pandemic (H1N1) 2009.>1 potential concurrent conditions . Limited analysis demonstrated that the concurrent condition did not make these persons more susceptible than other passengers to contracting an ILI.No passengers who had positive test results for pandemic (H1N1) 2009 had underlying health conditions considered to make them more susceptible to influenza; however, 5 of 32 passengers reporting ILI postflight had Flight 1, an Airbus A380, embarked from Los Angeles and arrived in Sydney on May 24, 2009, carrying 445 passengers. Of the 188 (42%) passengers who responded to a survey, 169 (90%) were Australian residents. Response rate varied with class of travel, with 11 (79%) of 14 first class passengers, 40 (56%) of 71 business class passengers, 19 (59%) of 32 premium economy class passengers, and 117 (36%) of 327 economy class passengers responding.Combined results from the survey and disease notification data sources identified 8 passengers who had an ILI at the beginning of the 14-hour flight. For 4 of these passengers, pandemic (H1N1) 2009 infection was later laboratory confirmed; for 1, pandemic (H1N1) 2009 infection was confirmed as negative; 3 passengers were not tested. ILI symptoms developed in 2 other passengers during the flight, and pandemic (H1N1) 2009 was confirmed in both.<7 days after arrival in Australia. Of these, 2 passengers had laboratory-confirmed pandemic (H1N1) 2009 infection, 15 had illness confirmed as negative for the pandemic virus, and 7 were not tested. Most passengers experienced onset of symptoms <3 days after flight arrival; however, 6 passengers did not state exact date of disease onset (Twenty-four passengers were identified as developing ILI symptoms se onset .Self-reporting of symptoms from passengers did not distinguish between different causes of ILI . Fever wTwenty (83%) of 24 passengers in whom an ILI developed postflight sat in aisle seats . This seSome clustering of cases was seen with the potential for spread of either ILI or pandemic (H1N1) 2009 from passengers who were infectious before and during the flight . The pasIn the cabin section of rows 66\u201377 was a cluster of passengers with symptoms before boarding who were later found to have positive test results for pandemic (H1N1) 2009. ILI symptoms developed in 7 passengers after the flight. In 2 of these passengers, pandemic (H1N1) 2009 was laboratory confirmed; for 4, ILI was confirmed as negative for pandemic (H1N1) 2009; and 1 person (seat 75G) was not tested.Similarly, in the cabin section of rows 50\u201364, symptoms developed in 2 passengers during the flight or on the day of arrival (seats 52C and 58B); these passengers were found to be pandemic (H1N1) 2009 positive. Symptoms developed in 7 passengers after flight arrival; 5 of whom had negative test results for pandemic (H1N1) 2009, 2 (seats 54F and 62D) were not tested.>3 days after flight arrival.We examined the risk of contracting pandemic (H1N1) 2009 infection postflight to all susceptible passengers seated in the economy section of the aircraft. Health authorities contacted 145 passengers on flight 1 for quarantine and prophylactic treatment after potential exposure to pandemic (H1N1) 2009  2009 by sitting in those seats was 1.4% . If the contact zone was modified to sitting in the same row as or within 2 rows either side of passengers who had preflight symptoms (rows 67\u201372) and not those in whom symptoms developed during the flight, the risk of contracting pandemic (H1N1) 2009 postflight increased to 3.6% (95% CI \u20131.3% to 8.6%). No passengers were detected as acquiring pandemic (H1N1) 2009 from either of the passengers with symptoms that developed during the flight; however, 2 passengers in the same section of the aircraft who responded to the survey indicated having had ILI symptoms but not being tested for pandemic (H1N1) 2009.The current zone for contact tracing is defined by passengers in the same row as and within 2 rows either side of the index case-patient. A closer zone forming a square delimited by 2 seats in front, 2 seats behind, and 2 seats on either side of the index case-patient could be prescribed  passengers responding to a survey regarding the potential for contracting an ILI during their flight, 114 (87%) were Australian residents. Public health authorities were not alerted to the potential for passengers carrying pandemic (H1N1) 2009 virus on this flight until 6 days after arrival. They were alerted after mandatory notification of the virus infecting 1 passenger.Survey data showed that 1 passenger was identified as having symptoms consistent with an ILI before the flight and that symptoms developed in 2 additional passengers on the flight. The passenger who was symptomatic before the flight was not tested for pandemic (H1N1) 2009; of the passengers whose symptoms developed during the flight, 1 was not tested and 1 (adult in seat 33D) was tested 6 days after flight arrival. At that time, the test returned a negative result.Six passengers were identified from the survey as having ILI symptoms within 7 days after flight arrival . Of thes INSERT SHAPE Some clustering of cases was seen, with a potential spread of disease from passengers who were symptomatic during the flight . Transmission of disease between adult and child in 33D could have occurred before or during the flight. With lack of a confirmed index case-patient for pandemic (H1N1) 2009, the increased risk for disease was not calculated. No other passengers on this flight had positive test results for pandemic (H1N1) 2009 infection after triangulation methods with the notifiable diseases database.Of 2 long-haul flights entering Australia within the first month after declaration of a level 5 alert for pandemic (H1N1) 2009, a total of 45 (6%) of 738 passengers on 2 aircraft were identified as having the potential to spread an ILI into the local community. Follow-up confirmed 9 passengers with pandemic (H1N1) 2009 infection; 8 of these were from flight 1.Flight 1, originating from a destination with documented widespread community transmission of pandemic (H1N1) 2009, had the greatest potential for introducing the pandemic virus into the Australian community, with 2% of its tested passengers being confirmed positive for pandemic (H1N1) 2009. Spread of the virus from a region known to have sustained community transmission was to be expected and formed part of the case definition in Australia during the early phases of the pandemic  2009 noted on a long-haul flight to New Zealand in 2009  2009. These ILIs could be caused by different viruses, as seen by Follin et al.  2009 and other ILIs can be spread to a community by passengers who were symptomatic before boarding the aircraft. Four of 9 of passengers in whom pandemic (H1N1) 2009 was diagnosed displayed symptoms preflight. This finding is similar to that in a recent study looking at the travel patterns of patients with pandemic (H1N1) 2009 reported from Singapore, where 25% of patients had symptoms before boarding their flights  2009 varies; therefore, if a passenger did not return the survey or contact medical personnel or health authorities after the flight, some cases may have been missed (Spread of pandemic (H1N1) 2009 and other ILIs occurred in limited zones of the aircraft during international flights into Australia during May 2009. The time required to contact passengers postflight resulted in the potential spread of disease into the community despite guidelines and policies in place to reduce the risk for disease importation. Nonetheless, application of these policies by Australian authorities may have assisted in delaying the importation of identified pandemic (H1N1) 2009 cases during the first month of the recent pandemic. The findings of this investigation suggest that efforts to prevent importation of respiratory diseases into a community and protection of individuals from in-flight exposure to ILI may require changes in international policies of both exit screening of symptomatic passengers preflight and contact tracing of those exposed to an ILI in flight. Further research on transmission of ILI in aircraft and into the effects of exit screening at international airport hubs to restrict travel of passengers with symptoms before flying would be of particular interest for respiratory disease of greater severity than pandemic (H1N1) 2009."}
+{"text": "Dear Editor,I read with great interest the article by Yusuf Yilmaz et al.  publisheAPRI=AST level/ Upper normal limit of AST/Platelet count ( 10(9) / L )\u00d7100The authors suggested that the APRI score can be used as an appropriate non-invasive marker because it showed acceptable accuracy for the assessment of liver fibrosis in patients with CHC and NAFLD, but not in those with CHB.We performed the same study but with a different method. The APRI score was calculated in 35 CHB patients, 14 CHC and 13 autoimmune hepatitis (AIH) patients and in 20 controls 7]8]. T[8]. T7][. T[8]. T"}
+{"text": "To the Editor: Establishment of viremia is a characteristic feature of infection with human parvovirus 4 (PARV4). In northern Europe, PARV4 (human partetravirus) is primarily transmitted by blood-borne routes  were tested. Specimens were collected during January\u2013December 2004 during a trial of intermittent preventive malaria treatment in the rural Afigya Sekyere District, Ashanti Region, Ghana . All samples were analyzed by using 2 real-time PCRs and primers described elsewhere (4 copies/mL (2 test).PARV4 genotype 3 DNA was detected in plasma of 32 (8.9%) of 361 children. Viral load ranged from \u2248200 copies/mL to 3.0 \u00d7 10opies/mL . Median 2\u20131.4 \u00d7 104 copies/mL) and 3 (3.8%) showed viremia at both time points and identical viral nucleotide sequences (time between bleedings 8.7 months for 2 children and 9.0 months for 1 child). However, only short genomic regions  could be amplified and sequenced because of low viral loads. Four children had positive results in the first sample, and 3 had positive results in the second sample.PARV4 viremia status was already known for 78 children 24 months of age , a finding similar to ours for the 3 children. However, negative IgM results in the person with hemophilia suggest that the sampling window might have missed the acute infection.PARV4 viremia was detected in a study in the United Kingdom among 110 PARV4-negative persons with hemophilia screened over 5 years for PARV4 viremia and seroconversion (IgG and IgM) (10, with the higher concentrations in the previous study analyzing EDTA whole blood. Whether these differences were caused by the relatively small number of children included or by the fact that whole-blood samples were compared with plasma samples remains to be clarified. However, our previous hypothesis that prenatal or perinatal transient infection was an unlikely mode of virus acquisition needs to be modified because PARV4 infection in newborns has recently been demonstrated (Comparison of results of our study with those of our previous study (Although we lacked IgM and IgG serologic data to interpret our findings, our study suggests that PARV4 genotype 3 infection might be characterized by viral persistence, reactivation, or reinfection. Additional longitudinal studies, including serologic testing for short intervals, are needed to determine the pathogenesis and potential public health role of PARV4 infection."}
+{"text": "Hsp90b1 is an endoplasmic reticulum (ER) chaperone  (MGI:98817) contributing with Hspa5  (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.floxHsp90b1, MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter . Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle. HSP90B1 is located in the endoplasmic reticulum (ER) where numerous proteins translocate and require adequate folding. If misfolded proteins accumulate in ER, this induces a so-called ER stress and triggers the unfolded protein response (UPR) which induces the expression of several genes including ER chaperones such Hsp90b1 or Hspa5 We took advantage of the line generated by Yang et al. (2007) HSP90b1 was reported to be one of the most abundant protein in oocytes We used RTqPCR to analyze the level of Hsp90b1 and Hspa5 transcripts because it was previously described in somatic or cancer cells that Hspa5, a related major ER chaperone is coordinately regulated with Hsp90b1 floxHsp90b1) From the high level of Hsp90b1 expression it could be hypothesized that HSP90b1 is needed during oogenesis and/or early embryonic development. To test this hypothesis, we generated an oocyte-specific deletion of Hsp90b1 using the transgenic mouse line expressing the cre recombinase under Zp3 promoter (Zp3-cre) and the floxed Hsp90b1 line . Ovary histomorphology appeared grossly normal in MT females with the presence of all the follicular stages and corpus luteum as in WT females  and mutant females  oocytes . The levThird we used fluorescent immunodetection  to determine HSP90B1 presence and localization in WT and MT ovary sections. Immunostaining revealed that this chaperone was normally expressed in oocytes as well as in granulosa cells . The strConsistent with the overall normal ovarian histology, the same number of fully-grown oocytes  . The pupTo better identify at which developmental step MT embryos were arrested, we analyzed the progression of the WT and MT embryos during the first cell cycle. This can be determined by the size and position of both pronuclei corresponding to PN1 to PN5 stages see  [12]. WeThen we collected MT embryos blocked at 1-cell stage at 42\u201344 hours post hCG and stained them to label DNA and tubulin in order to determine at which stage of the cell cycle they were arrested. As described above, Hsp90b1 and Hspa5 are known to be often coregulated and we found that they exhibited the same profile of expression during the transition from egg-to-embryo . TherefoHaving identified the developmental stage requiring HSP90B1, we were then interested in the localization of both ER chaperones to better understand ER function in zygotes. Very little is known about ER organization during the first mitosis of mouse embryos As it was reported that ER dynamics was depending on actin cytoskeleton in addition to microtubule network during mitosis All together, our findings indicated that ER organization and probably dynamics were modified in absence of HSP90B1.Chaperones are important and abundant cellular proteins which protect cells against the consequences of accumulating misfolded proteins. In order to better understand the specific function of some chaperone, gene targeting experiments have been performed  flox/floxHsp90b1) females produced oocytes in which the expression of Hsp90b1 was altered from the primary follicular stage. MT oocytes exhibited a thinner zona pellucida. This was probably due to some default in the trafficking of ZP proteins as revealed by the ZP2 positive structures we identified in oocytes from secondary follicles. Those structures were part of the ER network as indicated by a positive staining by HSPA5 antibodies (data not shown). Zona pellucida defects are important even before fertilization as demontrated by ZP1 or Mgat1 knockout which displayed abnormal folliculogenesis, ovulation and severely reduced fertility Mutant  Regarding the ER chaperones themselves, our data were also in agreement with previous work showing that Hspa5 and Hsp90b1 are coregulated In conclusion, our findings demonstrate that Hsp90b1maternal contribution is involved in ZP protein trafficking during oocyte growth but is not essential to obtain fully grown and MII fertilizable oocytes. In contrast we show that HSP90B1 is critical to complete the first mitosis in murine embryos as its absence provoked a severe disorganization of both the microtubular spindle and the actin\u2013ER network surrounding the spindle. This provides new insights regarding additional HSP90B1 clients whose identification requires further work.All animal work has been conducted according to relevant national and international guidelines. In particular, protocols for animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation .flox/floxHsp90b1 mice were generated previously and are described elsewhere  flox/floxHsp90b1 mice were crossed with transgenic mice carrying zona pellucida 3 (Zp3) promoter-mediated Cre recombinase (Tg(Zp3-cre)93Knw, MGI:2176187). Zp3-cre transgene is specifically expressed in oocytes in primary and further developped follicles, starting on day 5 after birth flox/flox;; Hsp90b1 or Zp3-cre/+flox/del.; Hsp90b1 Control females are either wild-type (WT) or flox/floxHsp90b1.The Oocytes were collected either at the GV stage from ovarian antral follicles. Embryos were collected at the indicated time from the oviducts following superovulation or not, as indicated. Superovulation, oocytes, embryos collection and culture were performed as previously described For histology and immunohistochemistry, mouse ovaries were fixed with Bouin, and histological preparations were performed according to a classical procedure . Immunohistochemistry was performed as described before According to the experiment, oocytes or embryos were stained with the following fluorescent dyes: DNA ; actin ; ER glycoconjugates . Fluorescent staining and immunofluorescence protocols were adapted from previous works  dilution 1\u223650; HSP90b1  dilution 1\u2236500 or , dilution 1\u223650; FITC-conjugated alpha-tubulin , dilution 1\u2236100.For chromosome spread and DNA staining at the metaphase I stage, oocytes were fixed in 1% PFA Confocal microscopy and imaging analysis was performed as described previously GCTACCAGGGCCTTTGAGATGGA -3\u2032.Samples preparation, reverse transcription and quantitative PCR were performed as described in Metchat et al. Data were collected from samples collected from at least three animals. The results are presented as means \u00b1 SEM as indicated. Student t-test and Chi square were used to perform statistical evaluation, with p<0.05 considered as significant."}
+{"text": "CYP2E1 mRNA levels correlated with CBF\u03b2-MYH11 transcript levels and with bone marrow blast counts in all cases. CYP2E1 over-expression correlated positively with NQO1 mRNA levels . By immunohistochemistry, CYP2E1 protein was more frequently expressed in AML with inv(16) compared with other types of AML (p < 0.001). We obtained serial bone marrow samples from two patients with AML with inv(16) before and after treatment. CYP2E1 mRNA expression levels decreased in parallel with CBF\u03b2-MYH11 transcript levels and blast counts following chemotherapy. In contrast, CYP1A2 transcript levels did not change in either patient. This is the first study to demonstrate concurrent over-expression of CYP2E1 and NQO1 mRNA in AML with inv(16). These findings also suggest that a balance between CYP2E1 and NQO1 may be important in the pathogenesis of AML with inv(16). Environmental exposure to benzene occurs through cigarette smoke, unleaded gasoline and certain types of plastic. Benzene is converted to hematotoxic metabolites by the hepatic phase-I enzyme CYP2E1, and these metabolites are detoxified by the phase-II enzyme NQO1. The genes encoding these enzymes are highly polymorphic and studies of these polymorphisms have shown different pathogenic and prognostic features in various hematological malignancies. The potential role of different cytochrome p450 metabolizing enzymes in the pathogenesis of acute myeloid leukemia (AML) in an area of active interest. In this study, we demonstrate aberrant CYP2E1 mRNA over-expression by quantitative real-time polymerase chain reaction in 11 cases of  Benzene is a long established leukemogen implicated in the pathogenesis of myelodysplastic syndromes (MDS) and acute leukemias . AlthougCYP2E1 is a hepatic monooxygenase involved in xenobiotic metabolism. The CYP2E1 gene is associated with the metabolism of many carcinogens. Polymorphisms of CYP2E1 and their possible effects have been reported by others. The CYP2E1*5 allele is associated with a higher risk of de novo acute myeloid leukemia (AML) and acute lymphoblastic leukemia . InThis study provides corroborating evidence implicating benzene in the pathogenesis of AML with inv(16), confirming the findings of others . High leThe strong association between CYP2E1 and AML with inv(16) has implications for pathogenesis. First, this association allows one to infer that AML with inv(16) may be related to exposure to environmental toxins. The elevated expression of CYP2E1 alone would enhance the carcinogenic potential of environmental toxins. The general population is exposed to benzene and its toxic metabolites through gasoline, cigarette smoking, air pollution and dietary intestinal breakdown of proteins . ElevateThe explanation for the elevated levels of CYP2E1 specifically in most cases of AML with inv(16) remains unclear. AML with inv(16) is associated with a favorable outcome with long periods of complete remission ,18. WhilNQO1 gene is located on chromosome 16q22.1, some authors have proposed that inversion (16) may be a primary event leading to disruption of this gene, thereby increasing the likelihood of leukemia [There are limitations to this study, the most important being the lack of NQO1 protein levels to reinforce the mRNA levels in the myeloid system. The results, therefore, need to be confirmed in a larger subset of patients with well characterized polymorphisms in the CYP2E1 and NQO1 genes. Since the leukemia . Furtherin vitro experiments also provide direct evidence of induction of cytochrome p450 and NQO1 by benzene and its analogues/metabolites. Based on this data, we suggest that cases of AML, especially those types associated with recurrent genetic abnormalities, are uniquely associated with an increase in specific types of cytochrome p450 enzymes. This increase appears to be more than just a generalized increase in cytochrome p450 enzyme expression. The increase in specific types of cytochrome enzyme also appears to be unique to each type of leukemia as shown by others, such as specific induction of CYP1A1 in AML with t [The results from our  t  as well  t . It alsoIn conclusion, we have shown simultaneously increased mRNA expression levels of the benzene metabolizing enzymes, CYP2E1 and NQO1, in AML with inv(16). Elevated levels of CYP2E1 mRNA and protein occur more commonly in AML with inv(16) compared with other types of AML. We further suggest that the balance between CYP2E1 (toxin producing) and NQO1 (detoxifying) enzymes may determines the overall carcinogenic potential of the xenotoxins."}
+{"text": "Clinical trials with the newer 2nd generation antihistamines in children under the age of 12 years have been performed previously but further studies are needed in order to show efficacy and safety in the most unfavourable clinical conditions such as persistent allergic rhinitis (PER). Rupatadine oral solution was developed for children with allergic rhinitis in view of its rapid onset of action and its lack of relevant side effects. These advantages were confirmed previously in a phase III study in children 6-11 years.To assess the efficacy and safety of rupatadine (RUP) oral solution in a subgroup of children between 6 and 11 years weighing \u226525 kg with PER.A subanalysis was performed from a previous placebo-controlled study carried out in patients between 6-11 years diagnosed as PER according to ARIA criteria. This analysis included patients with a positive prick test, weight \u226525 kg and basal nasal symptoms score  \u226524 obtained in 4 days throughout the 2-week screening period. Patients were allocated to treatment with either RUP oral solution (1 mg/ml) or placebo during 6 weeks. The dose was 5 ml of oral solution. The main efficacy endpoint was the change from baseline of the nasal (4TSS) and global symptoms (5TSS) score at 4 and 6 weeks of treatment. Furthermore the assessment of children's quality of life at 4, 6 weeks by means of PRQLQ was also evaluated.The subgroup analysed was a total of 266 randomized to rupatadine n=131) or placebo (n=135). Table 31 or plaRupatadine oral solution (1mg/ml) was significantly more effective than placebo in improving nasal symptoms (4TSS) at 4 and 6 weeks. This is the first clinical evidence of a H1-receptor antagonist efficacy in children between 6-11 years over 25 kg with PER."}
+{"text": "Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in  These genes are mostly expressed in complementary patterns in the murine central and peripheral nervous system where they drive the production of distinct neuronal populations Neurog2 and Ascl1 appear respectively as key regulators of glutamatergic (excitatory) versus GABAergic (inhibitory) neuronal fates during telencephalic development Ascl1 in GABAergic cell specification has however proved to be highly time and space dependent Ascl1 is also involved in the development of other neuronal subtypes such as hindbrain serotonergic neurons During development, neural specification leads to the emergence of a large diversity of neuronal subtypes that will serve distinct functions in the adult nervous system. A key issue concerns the nature and action of the molecular cues underlying the acquisition of both generic and specific characteristics of neurons. A small number of basic helix-loop-helix (bHLH) transcription factors are necessary and sufficient for progenitor cell commitment towards a neuronal lineage at the expense of a glial fate and have consequently been qualified as \u201cproneural genes\u201d Ascl1 may also have a more specialized role in the commitment of particular cell subtypes since its expression is restricted to subsets of neuronal progenitors, mostly distinct here again from the Neurog2-expressing ones In the retina, beside its proneural function Ptf1a (pancreas transcription factor 1a), drives inhibitory neuron commitment in the retina, at the expense of a glutamatergic destiny Ptf1a within the retina remain to be investigated. Considering the GABAergic instructive function of Ascl1 in various regions of the brain, and the fact that Ptf1a expression is decreased in the dorsal spinal cord and retina of \u2212/\u2212Ascl1 mice Ptf1a transcriptional network.Our previous studies and others highlighted that the bHLH gene Ascl1 function during Xenopus retinogenesis. We found that Ascl1 is required for GABAergic retinal neuron genesis and sufficient to bias a subset of retinal progenitors towards a GABAergic destiny. Then, we took advantage of the Xenopus ectopic neurogenesis induction paradigm to investigate which genetic interactions contribute to Ascl1 activity as an inducer of neurotransmitter phenotypes. We showed that distinct Ascl1-dependent transcriptional networks sustain the production of GABAergic and catecholaminergic neurons. Together, our data suggest that the GABAergic activity of Ascl1 requires Ptf1a, this latter being a direct transcriptional target of Ascl1.To address these issues, we first analysed pCS2-Ascl1  were obtained by conventional procedures of in vitro fertilization and staged according to Nieuwkoop and Faber GFP mRNA was co-injected as a tracer. Loss of function experiments were performed using already described and validated antisense oligonucleotides morpholinos: Ptf1a-Mo Ascl1-Mo  Immunohistochemistry was performed using rabbit polyclonal anti-GABA , mouse monoclonal anti-GFP  and anti-mouse or anti-rabbit fluorescent secondary antibodies . Cell nuclei were counterstained with Hoechst (Sigma).gad1, VGlut1THtubb2bBrudpysl3Ptf1aAscl1in situ hybridization in situ hybridizations on cryosections Digoxigenin-labeled antisens RNA probes for ornithine decarboxylase (ODC) using Biorad CFX Manager Software. Primers sequences are: tubb2b forward 5\u2032cccgtgccatccttgtggatttt3\u2032, tubb2b reverse 5\u2032gcccagttattgccagcaccactt3\u2032, Ptf1a forward 5\u2032gccgctcaggaaccccaaca3\u2032, Ptf1a reverse 5\u2032ggcagcccgtagtctgggtca3\u2032, ODC forward 5\u2032catggcattctccctgaagtacaagaa3\u2032 and ODC reverse 5\u2032ggacagtggtaggggcaagctca3\u2032.Stage 19 embryos were treated with cycloheximide  during 2.5 hours. Total RNA from 8 embryos was then isolated using the Nucleospin RNA XS kit (Macherey Nagel). Reverse transcription was performed using IscriptcDNA Synthesis Kit (Biorad). RNA quality was evaluated using Experion (BioRad). qPCR reactions were performed in triplicate using SsoFast Eva green Supermix (Biorad) on a C1000 Thermal Cycler . All values were normalized to the level of the reference gene Total protein lysates were prepared from stage 14 embryos and submitted to western blot analysis using an anti-Myc antibody (Sigma) as previously described in situ hybridization signal intensity in the eyes of whole embryos was quantified using Adobe Photoshop CS4.Shown in figures are representative data from one experiment that has been performed at least in duplicate. Fluorescent staining was visualized with a M2 Zeiss microscope. Images were captured with a digital camera AxioCam MRc and AxioVision Rel 7.8 software. The quantification of Xenopus experimentation (authorization 91-29 and 91-28), in accordance with French government policies. The study was conducted under an institutional license (number B 91-471-102 up to 2012 and C 91-471-102 since 2013). The study protocol was approved by the institutional animal care committee, the Direction D\u00e9partementale de la Protection des Populations (license B/C 91-471-102).All animal procedures were conducted under the supervision of several licensed personnel, including the director of research of the CNRS and professor at the university Paris-Sud, with licenses to perform Ascl1 in GABAergic and glutamatergic phenotype acquisition, we performed morpholino (Mo) injections in two cell stage embryos. We examined the effect of Ascl1 loss-of-function by analysing the expression of gad1 (glutamic acid decarboxylase) and VGlut1 (vesicular glutamate transporter 1), which respectively encode the rate-limiting enzyme for GABA biosynthesis and a glutamate transporter expressed in neurons. The expression of a retinal pan-neuronal marker BruAscl1-Mo injected retinas compared to control ones   . Thus, iAscl1 involvement in the specification of neurotransmitter phenotypes, we turned to a gain of function strategy using a glucocorticoid-inducible Ascl1 construct (Ascl1-GR), which avoids affecting early nervous system development. We analysed the fate of Ascl1-overexpressing cells at stage 38. Contrasting with the Ptf1a gain of function, which results in a dramatic overproduction of amacrine and horizontal cells Ascl1 overexpression led to a typical neurogenic phenotype, with early born neurons being increased  expression in stage 24 embryos . We thus decided to pursue our comparative study by monitoring TH labelling after Ascl1, Neurog2, NeuroD1, Atoh7 or Ptf1a mRNA injection. In contrast to Ascl1, none of these genes were able to promote the production of TH-positive neurons, emphasizing again the specific role of Ascl1 in neuronal subtype determination  retained a neurogenic activity, as inferred by their ability to promote the expression of the pan-neuronal marker dpysl3 . This demonstrates that the functional specificity of these two proteins resides in their respective bHLH domains. In addition, we found that the presence of the Ascl1 basic domain in chimeric constructs was necessary and sufficient to trigger TH and gad1 ectopic expression (bN and NHA to NbA for gad1 and TH staining). Conversely, only the chimeric proteins containing the Neurog2 HLH domain had the ability to induce glutamatergic neurons (VGlut1 labelling for AbN and NHA versus NbA). In line with this observation, the two chimeric proteins containing both the basic domain of Ascl1 and the HLH of Neurog2 (AbN and NHA proteins) were able to simultaneously promote gad1, TH and VGlut1 expression. Altogether, these results suggest that the functional specificities of Ascl1 and Neurog2 in neurotransmitter subtype specification do not reside in the same protein domain. GABAergic and catecholaminergic inducing activity of Ascl1 primarily relies on its basic domain while glutamatergic inducing activity of Neurog2 is imparted by its HLH domain.We next examined which Ascl1 domains account for the protein activity in neuronal subtype specification. We used a series of chimeric nstructs  with intnjection  and all bNA by in vivo lipofection . In the absence of CHX, Ascl1 overexpression lead to a strong increase of both tubb2b and Ptf1a expression level, as assayed by RT-qPCR analysis  Ptf1a overexpression could rescue Ascl1 knockdown. We found indeed that gad1 expression was restored to a control level in the retina of Ascl1 morphant embryos overexpressing Ptf1a (Ascl1 acting upstream Ptf1a in the transcriptional network controlling GABAergic neuron genesis in the retina.In order to know whether such transcriptional interaction could also hold true in the retina, we first carefully compared their expression pattern during retinogenesis . Consistng Ptf1a . Altogetin vivo neurogenic assay in the Xenopus epidermis, we found that Ascl1 GABAergic determining activity is instructive, as inferred by its ability to counteract glutamatergic differentiation programs. Finally, we gained insights into downstream genetic networks underlying Ascl1 functions in neurotransmitter subtype specification by showing that its GABAergic activity involves the direct transcriptional regulation of Ptf1a (The molecular bases underlying neurotransmitter subtype determination in the retina remain largely unexplored. As previously shown in the brain and peripheral nervous system of Ptf1a .Ascl1 gain and loss of function analyses in the retina have so far uncovered phenotypes predominantly related to its proneural activity. Ascl1 has indeed been shown to be required for neuronal versus glial fate decisions during late neurogenesis Ascl1-expressing progenitors contribute to all major cell types of the retina with the exception of ganglion cells Ascl1 or other bHLH genes such as Neurog2 or Atoh7 results in apparently similar cell type distribution defects within the Xenopus retina Neurog2 nor Atoh7 exhibit this property. Importantly, our results are consistent with a previous study in chick reporting that, unlike Neurog2, NeuroD1 or Atoh7, Ascl1 misexpression promotes an overproduction of amacrine cells, the major retinal GABAergic cell type Most The strong context-dependency of Ascl1 function in neurotransmitter subtype specification relies on the presence of other regionally expressed transcription factors including bHLH ones Ascl1-dependent ectopic GABAergic neuron production was found to be enhanced by Neurog2 co-expression. This suggests that, in the few locations where both factors are co-expressed in the same cells Ascl1 and Neurog2 lineages are largely distinct, such progenitors expressing both genes have recently been described Neurog2 was identified as a direct transcriptional target of Ptf1a in the chick dorsal spinal cord and cerebellum Ptf1a, Ascl1 and Neurog2 expressions partially overlap Neurog2 might thus participate within these domains in the Ascl1/Ptf1a-dependent GABAergic program. Additionally, Ascl1 and Neurog2 were shown to heterodimerize and function in common transcriptional complexes Ascl1 interactions with other expressed bHLH genes in the retina will surely help understand the combinatorial codes governing neurotransmitter subtype determination.Unexpectedly, Ascl1 expression may have interfered with proper GABAergic differentiation. Our domain swapping experiments led to an opposite conclusion, highlighting the essential role of Ascl1 basic domain in both its catecholaminergic and GABAergic inducing activities. Interestingly, the same was not true for Neurog2, whose ability to induce glutamatergic neurons was found to be imparted by its HLH domain. Although we cannot exclude that Ascl1 or Neurog2 might act through different molecular mechanisms depending on the cellular context, our data suggest that distinct domains within these bHLH factors mediate their functional specificities in neurotransmitter phenotype specification.Previous reports proposed that the HLH domain of murine Ascl1 contains the information underlying its specific activity in neuronal subtype specification Ptf1a and Phox2a/Hand2, respectively. We found in particular that Ascl1 ability to promote gad1-positive neurons requires Ptf1a. Additionally, Ascl1 was able to activate Ptf1a transcription and to be necessary for its retinal expression. In line with such a positive transcriptional regulation, previous data showed that Ascl1 is required for the induction and/or maintenance of Ptf1a during the late phase of dorsal sensory interneuron development Ptf1a might constitute a direct transcriptional target of Ascl1. Consensus binding sites for Ascl1 have previously been described in two conserved Ptf1a enhancer regions but have not proved to be functional Ptf1a regulatory regions.We showed that Ascl1 exerts its GABAergic and catecholaminergic determining activities by controlling distinct genetic cascades involving Altogether, our work demonstrated for the first time a role for Ascl1 in the generation of GABAergic interneurons in the retina and provided insights into the regulatory circuits responsible for its activity in neurotransmitter subtype specification."}
+{"text": "Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1  axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to \u201cdouble\u201d  During development, axons extend along stereotyped pathways to form the precisely ordered neuronal networks critical for the nervous system to function + (ATOH1) progenitor-derived dI1 neurons, a class of dorsal sensory interneurons in the developing spinal cord + dI1 axons: 1) the well-described TAG1/axonin1+ commissural axons The role for temporal guidance cues was first demonstrated for the MATH1Altering the rate at which dI1 axons grow had significant consequences for the development of the dI1 neural circuit. In particular, accelerating dI1 axon growth resulted in turning errors when dI1 axons reached the ventral spinal cord in ovo electroporation of chicken embryos. In these embryos, 1) accelerated early born contralaterally-projecting dI1 axons would have no bilateral axon interactions in the FP and 2) accelerated later born ipsilaterally-projecting dI1 axons would not encounter the opposing population of earlier born contralaterally-projecting dI1 axons as they navigated the ventral spinal cord. We have thus explored whether these two putative bilateral axonal interactions have a critical role in the guidance of dI1 axons, using chicken embryos as a model system. We first developed a \u201cdouble\u201d electroporation technique to examine whether the previously observed defects after unilateral acceleration of one population of dI1 axons can be rescued by the bilateral acceleration of both populations of dI1 axons. Second, we used an in vitro tissue culture assay to assess the effect of unilaterally ablating dI1 neurons on the axon trajectory of the spared population of dI1 axons on the other side of the spinal cord. Neither experiment provided any evidence for a bilateral interaction between dI1 axons, suggesting that the two populations of axons project independently of each other.A second, largely unexplored, source of guidance cues in the ventral spinal cord could come from bilateral interactions between the two populations of dI1 axons projecting from either side of the spinal cord. Selective fasciculation between pioneer axons is common in invertebrates tomato, 0.25 mg/ml Math1::farnesylated (f) Gfp and 0.6 \u00b5g/\u00b5l Math1::BmprII\u0394Lim-Gfp. When two fluorophores are electroporated into dI1 neurons using the Math1 enhancer, there is close to a 100% overlap between the expression of the fluorophores. In no case, did double electroporation alter the number of LHX2/9 dI1 neurons.Hamburger-Hamilton (HH) stages 14/15 White Leghorn chicken embryos (McIntyre Poultry) were injected with two sequential solutions of different combinations of the following expression constructs: 0.25 \u00b5g/\u00b5l Math1::tandem dimer (td) DNA solution #1 was injected into the lumen of the spinal cord and an electric current passed across the embryo using a BTX Electro Square Porator (ECM 830) set at ten 50 ms second pulses of 30 V. The embryo was permitted to settle for 10\u201315 minutes and the positive and negative electrodes were inverted.DNA solution #2 was electroporated into the lumen of the spinal cord using five 50 ms pulses of 30 V. A few drops of 1\u00d7 penicillin/streptomycin/glutamine  were applied to the embryo, and the egg was wrapped in parafilm (VWR) and incubated at 37\u00b0C for 2 days until HH stages 24/25.Gfp or 0.6 mg/ml Math1::BmprII\u0394Lim-Gfp into the developing spinal cord lumen using an electric current generated by five 50 ms pulses of 30 V. The electroporated egg was incubated overnight at 37\u00b0C and then dissected in L15 medium (CellGro) at HH stage 18, before any dI1 commissural axons have reached the FP HH stages 14/15 chicken embryos were electroporated with 0.25 mg/ml Math1::fAll spinal cords were processed to result in fixed \u201copen book\u201d explants embedded in collagen. Antibody staining was as previously described Rabbit: LHX2/9 (pan LH2A/B), 1\u22361000 Mouse: GFP, 1\u22361000 . Species appropriate Cyanine 3 and Fluorescein conjugated secondary antibodies were used (Jackson ImmunoResearch Laboratories).Antibodies against the following proteins were used. + axons, i.e. those that normally turn rostrally before reaching the FP, to the number of contralaterally projecting GFP+ axons, i.e. those that normally turn rostrally after crossing the FP. Fluorescent images were collected on Carl Zeiss LSM510 confocal and Axiovert 200 M microscopes. Images were processed using Adobe Photoshop CS4.These explants were quantified by comparing the number of ipsilaterally projecting GFP+ dI1 neurons, we developed a novel method of independently labeling axons on different sides of the spinal cord by \u201cdouble\u201d in ovo electroporating chicken embryos. In control experiments to establish this procedure, HH stages 14/15 chicken embryos were electroporated twice, first with Math1::tdtomato and then with a mixture of both Math1::tdtomato and Math1::fGfp, reversing the position of electrodes between the injections  ipsilateral dI1 axon population  into dI1 neurons results in their extending axons that grow 40% faster than axons expressing GFP alone in ovo electroporation procedure. To unilaterally accelerate dI1 axons, BMPRII\u0394Lim-GFP was introduced into only one population of dI1s, whereas tomato was introduced bilaterally in all dI1s . This experiment gave similar results to those observed previously + tomato+ ipsilaterally projecting dI1 axons  axon pathway in the spinal cord is guided in part by heterophilic interactions between CAMs in the axons themselves  and in the cells of the FP . TAG1 has a neurite promoting activity in vivo phenotypes observed after blocking axonin1 or NrCAM are only a consequence of interactions between heterophilic CAMs or whether there was also a homophilic component resulting from a bilateral interaction between the different populations of dI1 axons growing within the FP and the ventral spinal cord. The close proximity of dI1 commissural axons extending across the FP and the co-incidence of the ipsilateral and contralateral dI1 axons growing within the ventral spinal cord make such an interaction feasible + commissural axons entering the FP could provide a positive substrate for one another, while the rostral turn taken by the contralateral dI1 axons could provide a scaffold for the rostral turn of the later born ipsilateral dI1 axons (arrow, These studies focused on the contact-mediated interactions between the commissural axons and the cells of the FP and did not examine the role of putative interactions between the opposing populations of commissural axons. Thus, it remained unresolved whether the s arrow, .Nonetheless, we were unable to find any evidence for such bilateral interactions between the opposing populations of dI1 axons. Restoring the bilateral interaction was not sufficient to rescue the defects seen after unilaterally accelerating the rate of dI1 axon growth. Moreover, ablating one population of dI1 neurons did not affect the turning behaviors of the remaining dI1 axons. Taken together, these data suggest that the turning behaviors of dI1 axons in the FP and ventral spinal cord occur independently from one another.Our studies The mechanistic basis for these guidance errors remains unresolved. Temporal guidance cues could permit axons to encounter directional information at the right time in development, such that neural circuits develop in concert with the rest of the embryo. However, as discussed above, many of the key molecular guidance cues are present in the FP prior to dI1 commissural axons reaching the ventral spinal cord. A notable exception is WNT4, which is expressed in the ventral spinal cord by HH stages 27/28 In summary, these studies show a surprising absence of any interaction between the two opposing populations of dI1 axons in the spinal cord as they navigate the ventral spinal cord."}
+{"text": "We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence  Influenza A viruses cause one of the major respiratory infection diseases in humans. The viruses possess a genome consists of eight different RNA segments and the incorporation of all the eight RNA segments is required for the generation of an infectious virus particle. The precise process of how these eight viral RNA segments are co-packaged into progeny virus particles remains undefined due to the limitations of methodology to determine the locations of different vRNA segments in infected cells with single-molecule resolution. In this study, we established an experimental system to examine the localization of different viral RNA segments in an infected cell with high spatial precision. We found that viral RNA belonging to different segments gather together in the cytoplasm which is facilitated by cellular recycling endosomal protein Rab11. Our results supported the idea that eight different viral RNAs likely form a super-complex as they travel to the site for virion incorporation. These findings extend our knowledge on the process of influenza virus genome packaging and suggest a mechanism by which the genome assembly of different viral RNA segments is regulated. The assembly process of different vRNA segments during virus infection can therefore be studied with high resolution. Influenza virus is one of the rare RNA viruses which has a nuclear replication phase The Influenza A virus genome consists of eight negative-sense, single-stranded RNAs. In a virus particle or an infected cell, the viral RNAs exist in the form of viral ribonucleoprotein complexes (vRNPs) with the viral RNA (vRNA) encapsidated by the nucleoproteins (NP) and associate with the polymerase complex It has been suggested that the reassortment of influenza vRNAs happens in the cytoplasm because when the cytoplasm of two different virus infected cells was fused, the segments from the two virus strains were incorporated into progeny viruses in a random manner in situ hybridization assay to visualize vRNAs of different segments in influenza virus infected cells. By using this technique, the degree of colocalization between two different vRNAs can be determined with great spatial precision. By performing smFISH and colocalization analysis on virus infected cells at different time points post infection, we found that the incoming vRNPs remain associated until they are imported into the nucleus. Newly synthesized vRNPs were detected at different locations in the nucleus and the newly exported vRNPs of different identities gather together at later stages of replication when the vRNPs are loaded onto Rab11 positive vesicles. Herein, we have provided evidence that progeny vRNPs of different identities travel together in the cytoplasm. These results likely suggest that different vRNPs are selected into a pre-formed super-complex during their trafficking to the plasma membrane, where budding and genome packaging occur.In this present study, we applied single-molecule sensitivity fluorescence in situ hybridization (smFISH) system for influenza vRNAs to determine their locations in infected cells. Single-molecule sensitivity was obtained using 48 single-fluorophore-labeled short DNA oligos targeting different regions of the same vRNA. The targeted vRNAs were bound by multiple probes at the same time giving high fluorescence intensity signals, allowing them to be distinguished as diffraction-limited spots. The number of spots and the location of their centers in 3-dimensional space can then be determined using - imaging processing programs. When 48 Cy5 labeled probes against the PB2 segment were used, fluorescence spots can be observed in influenza A/Puerto Rico/8/34 (PR8) virus infected cells at 4 hours post infection (hpi) but no spots were detected in the mock infected cells , leptomycin B (LMB) or importazole (IPZ), to disrupt different steps of influenza vRNP nuclear transport. The colocalization efficiency of PB2 and NA vRNAs was then analyzed at 20, 40 and 60 minutes post-infection. In the mock treated cells, both PB2 and NA vRNAs could be detected in the nucleus as early as 20 minutes post infection  to block nuclear import of vRNPs. IPZ has been found to interfere with the interactions between importin-\u03b2 and Ran-GTP, an event critical for the release of imported cargos. Treatment with IPZ, therefore, inhibits the importin-\u03b2 dependent nuclear import of cargo and retains the imported cargos at the rims of the nuclei Since the results of colocalization analysis of PB2 and NA vRNPs at different time points post infection indicated that vRNPs of different identities colocalized in the cytoplasm, cellular factors that may be involved in this process were further investigated. It has been reported that influenza vRNA trafficking depends on microtubules, and it has been observed that vRNAs accumulated at the microtubule-organization center (MTOC) after their export from the nucleus To further confirm that an intact microtubule network is not important for PB2 and NA vRNAs to colocalize, we inhibited the polymerization of microtubules with nocodazole in PR8 virus infected cells. MDCK cells infected with PR8 were treated with nocodazole at 1.5 hpi and the cells were then fixed for hybridization at 6 and 8 hpi. Cells were stained with the Alexa488-conjugated wheat germ agglutinin (WGA) to label the plasma membrane and Golgi apparatus. At 6 hpi, large numbers of vRNAs were observed to accumulate at the juxta-nuclear positions in mock treated cells while in nocodazole treated cells, the vRNAs were spread out in the cytoplasm and accumulated toward the lateral plasma membrane . This obIt has been reported that influenza vRNAs travel to the plasma membrane in a Rab11 dependent manner To further demonstrate the involvement of Rab11 in the colocalization of PB2 and NA vRNAs, A549 cells were transfected with GFP tagged Rab11 in either the wild type or dominant negative forms followed by PR8 virus infection. The degree of colocalization between PB2 and NA vRNAs was compared in cells that expressed the wild type Rab11 (WT-Rab11) and cells that expressed the dominant negative Rab11 (DN-Rab11). The viral proteins hemagglutinin and M2, specifically the cytoplasmic tail of M2, have been reported to be involved in the assembly of budding virions The role of M2 protein was assessed with a similar strategy. Two recombinant viruses in the PR8 virus background were generated In this study, we have analyzed the disassembly and the subsequent assembly of influenza vRNPs segments in virus-infected cells using smFISH. We show that vRNPs of different segments remain associated after their release from the incoming virions and they travel as a package to the nuclear membrane. Newly synthesized vRNPs of different segments do not occupy the same space in the nucleus and they are likely exported individually into the cytoplasm because colocalization of the exported vRNPs of different segments is not observed during the early stages of infection. Different viral RNPs colocalize in the cytoplasm at later stages during infection (6\u20138 hpi in MDCK cells) and they are often found to be associated with Rab11-recycling endosomes. These results provide evidence that vRNPs belonging to different segments follow the same trafficking route and the selection for the correct combination of the eight vRNA segments likely takes place before the vRNPs reach the cell surface .in-vitro transcribed vRNAs suggested that specific RNA/RNA interactions among vRNAs exist and that they may participate in the process of genome selection The detection of vRNPs as early as 20 minutes post infection demonstrates the high sensitivity of the smFISH methodology, which allows analysis of the spatial relationship of the vRNPs during the viral entry process. When uncoating of the incoming virus particles was blocked by increasing the pH in the endosomal compartments, the PB2 and NA vRNAs were found to colocalize with very high efficiency. These results indicate that the colocalized vRNPs originate from the same virions, in accordance to the previous report that influenza virus particles package their eight different vRNAs with high efficiency The replication of vRNAs of cytoplasmic negative-sense RNA viruses has been found to occur in virally induced compartments, such as Negri bodies for rabies virus The Rab11 recycling endosome system has been shown to be involved in the transport of vRNPs from the peri-nuclear regions to the apical plasma membrane The efficient incorporation of influenza viral genome into virions has been linked to the cytoplasmic tail of the virus transmembrane proteins: HA, NA and M2 In conclusion, we have shown with smFISH and colocalization analyses that different vRNA segments in influenza virus-infected cells colocalized in the cytoplasm before they reached the plasma membrane. These findings shed light on the selection process of influenza vRNPs packaging 2. Influenza A/Puerto Rico/8/34 (PR8) and PR8 cH9/1 virus strains were grown in 10-day old embryonic chicken eggs as previously described Madin-Darby canine kidney (MDCK) epithelial cells were maintained in Modified Eagle's Medium (MEM)  supplemented with 10% fetal bovine serum (FBS). Adenocarcinomic human alveolar basal epithelial cells (A549) were grown in Dulbecco's Modified Eagle's Medium (DMEM)  supplemented with 10% FBS. Both cell lines were incubated at 37\u00b0C with 5% COThe reverse genetics method for generating recombinant PR8-HA-GFP-HA virus was as described previously Infection of MDCK cells or A549 cells with influenza viruses was performed as previously described 4 A549 cells were transfected with 1 \u00b5g of GFP-rab11-WT or GFP-rab11-DN plasmid using lipofectamine 2000 reagent according to manufacture protocol (Invitrogen). Cover slips were coated with fibronectin (Sigma) by incubating the cover slips in 50 \u00b5g/ml of fibronectin in PBS for 45 min at RT and rinse them once with PBS. The transfected cells were plated onto the fibronectin coated coverslips and incubated at 37\u00b0C for 24 hours before virus infection.5\u00d710in situ hybridization (FISH) was performed according to published protocols with some modifications 4 cells/well and grown overnight at 37\u00b0C. At certain time points post infection, the cells were washed once with ice-cold PBSM , followed by fixation with 4% paraformaldehyde in PBSM for 10 min at room temperature (RT). After a brief wash with ice-cold PBSM, the cells were permeabilized with 0.5% Triton X-100 in PBSM for 1 min at RT. The cells were then washed with PBSM and incubated in 2XSSC  with 10% formamide for 5 min before hybridization. To detect viral RNAs, 4 \u00b5M of labeled probes in 40 \u00b5l of hybridization buffer , 0.02% RNAse-free BSA, 50 \u00b5g E. coli tRNA, 2XSSC, 10% formamide) was used for each sample. Hybridization was carried out in humidified chambers maintained at 37\u00b0C for 16 hours. The samples were then washed twice with 10% formamide 2\u00d7SSC supplemented with 2 mM VRC for 30 min at 37\u00b0C. Nuclear staining using 0.5 \u00b5g/ml of DAPI was performed afterwards and the coverslips were mounted in ProLong Gold antifade mounting media (Invitrogen). The cured samples were subjected to microscopy examination or were stored at \u221220\u00b0C. For samples used for both immunofluorescence and FISH analyses, cells were first blocked with 1% BSA in PBSM for 1 hour at RT after fixation and permeabilization. The coverslips were then subjected to primary and secondary antibody staining in 1% BSA in PBS followed by another fixation step with 4% paraformaldehyde for 10 min. The cells were then washed once with PBSM and equilibrated with 10% formamide 2XSSC for 10 min before the in situ hybridization procedures.RNA fluorescence Cells were placed on a Zeiss Axioplan2IE microscope equipped with a 100\u00d7, 1.4 numerical aperture (NA) oil-immersion objective (Zeiss) and a Zeiss AxioCam MRm camera. Cells were imaged using 200 nm z-dimension axis steps across a range of approximately 4 \u00b5m.We performed subpixel localization and intensity quantification of the spots using custom-designed MatLab (Mathworks) programs which were previously described Figure S1Specificity of single-molecule sensitivity FISH analysis of influenza viral RNAs. (A) MDCK cells were infected with PR8 virus at MOI\u200a=\u200a5. DAPI signal and Cy5 fluorescence from sm-FISH probes targeting the PB2 vRNAs in mock infected and PR8 virus infected cells at 4 hpi are shown in 2D images constructed using maximum-intensity projections. Scale bar\u200a=\u200a10 \u00b5m. (B) MDCK cells were infected with PR8 virus at MOI\u200a=\u200a5 and hybridized with 48 Cy5 labeled probes targeting the PB2 vRNAs. Histogram of the fluorescence spots intensity at 1 hour post infected is shown. The spot intensity distribution displays a well-defined single peak characteristic of single molecules. Black circles: data; red line: Gaussian distribution fits of the data. (C) MDCK cells were infected with either PR8 or PR8 cH9/1 virus (containing the HA ORF expressing the head region of the H9 subtype) for 6 hours before smFISH was performed using Cy3 labeled probes against the HA1 vRNAs and Cy5 labeled probes against the HA9 vRNAs. Maximum intensity merges of a pair of z-stack images taken in the Cy3 channel  and the Cy5 channel  are shown. DAPI staining (blue) stains the nuclei in the cells. Scale bar\u200a=\u200a25 \u00b5m.(TIF)Click here for additional data file.Figure S2Empirical determination of the maximum distance threshold between the centers of spots which defines colocalization. (A) The distances between the centers of colocalized spots were empirically determined by using two probe sets targeting different regions of the same viral RNA. Two different regions of NA vRNA were bound by two probe sets, one labeled with Cy3 fluorophore and the other labeled with Cy5 fluorophore. Z-stack images were taken in both fluorescence channels and the centers for the Cy3 spots and Cy5 spots were localized in 3D space using the spot detection program. The distances between the Cy3 spots and their nearest-neighboring Cy5 spots were measured. The Distributions of the distances between the neighboring spots of different colors is shown here. The distances between the centers of the Cy3 and Cy5 spots were found mainly within 1 pixel (102 nm) and spread to approximately 2.5 pixels (255 nm). Since electron microscopy analysis suggested the packaged vRNPs range from 80 nm to 100 nm , the nearest neighbor distances measured here likely represent the accuracy of measurement of the same diffraction-limited object in Cy3 and Cy5 channels. We therefore define spots as being colocalized if their centers are within 2.5 pixels. (B) In contrast to colocalizing spots, the distribution of nearest neighbor distances between the NA vRNAs (Cy5 spots) and \u03b2-actin mRNA (Cy3 spots) in infected cells shows that the nearest-neighbor distances range from 1 pixel to 10 pixels (1020 nm), with most of the spots clustered within a range from 3.5 pixels to 6 pixels. The red dashed lines show the 2.5 pixels distance threshold to define colocalization of spots in the quantitative colocalization analysis.(TIF)Click here for additional data file.Figure S3Distribution of vRNPs in the cytoplasm. Maximum intensity merge images of MDCK cells infected with PR8 virus at 2, 3 (A) and 6 (B) hpi. The cells were probed against PB2 vRNAs (green), NA vRNAs (red) and stained with DAPI (blue) to define the nuclear regions. The white arrows show the exported vRNAs detected in the cytoplasm. The red arrows indicate the peri-nuclear accumulations of vRNAs. Scale bar\u200a=\u200a10 \u00b5m.(TIF)Click here for additional data file.Figure S4Growth kinetics of PR8 virus in MDCK cells. MDCK cells were infected with PR8 virus at MOI\u200a=\u200a5. Supernatant of the infected MDCK cells were collected at 0, 1, 2, 4, 7, 10 and 12 hours post infection. The virus particles released into the supernatant were titered using standard plaque assays. The titer of the released virus particles at each time point is shown.(TIFF)Click here for additional data file.Figure S5The colocalization of vRNPs of different identities can be observed at 6 hpi. MDCK cells were infected with PR8 virus at MOI\u200a=\u200a5 and sm-FISH and colocalization analyses were performed for different pairs of vRNPs. Quantification of the colocalization efficiency between the indicated vRNA pairs is shown. Low localization efficiency is observed at 4 hpi for all the vRNA pairs tested while elevated colocalization efficiency of these vRNA pairs is detected at 6 hpi, demonstrating similar kinetics.(TIF)Click here for additional data file.Figure S6Positive control of LMB treatment on MDCK cells. As a positive control for the effect of LMB, we examined the nuclear-cytoplasmic shuttling protein MAP kinase (MEK) (TIF)Click here for additional data file.Figure S7Automatic Segmentation of Microtubules and Rab11 Particles.Top: Microtubule Segmentation Example. Left, representative plane from a deconvolved image stack of microtubules labeled with an antibody against tubulin. Center, mask image showing the output of the automated segmentation algorithm for the plane on the left. Right, Overlay of data (green) and segmentation result (red). Bottom: Rab11 Particles Segmentation Example. Left, Maximum Intensity Projection of an image stack of Rab11 particles labeled with antibody against Rab11. Center, Maximum Intensity Projection of the output of the automated segmentation algorithm for the image stack represented on the left. Right, overlay of data (green) and segmentation results (red). Scale Bar: 10 \u00b5m.(TIF)Click here for additional data file.Figure S8The colocalization of PB2 and NA vRNAs in A549 cells. A549 cells were infected with PR8 virus at MOI\u200a=\u200a5 and sm-FISH and colocalization analyses were performed for PB2 and NA vRNAs (Cy5 labeled PB2 and Cy3 labeled NA vRNAs). Quantification of the colocalization efficiency between the two vRNAs is shown. Low localization efficiency is observed at 4 hpi and 6 hpi while elevated colocalization efficiency is detected at 8 hpi and 10 hpi, demonstrating a delayed kinetics compared to that in MDCK cells (CK cells .(TIF)Click here for additional data file.Text S1Probes sequences used to target vRNA or cellular mRNA in influenza virus infected cells. Sequences for each probe used to target influenza vRNA or cellular mRNA are listed here.(DOCX)Click here for additional data file."}
+{"text": "ECM1 gene located on chromosome 1q21. The aim of the study was to investigate the molecular genetic defect underlying lipoid proteinosis in a consanguineous Pakistani family.Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and mucosal lesions and hoarseness appearing in early childhood that is caused by homozygous or compound heterozygous mutations in the ECM1 gene. To screen for mutations in the ECM1 gene, all of its exons and splice junctions were PCR amplified from genomic DNA and analyzed by SSCP and sequenced directly in an ABI 3130 genetic analyzer.Genotyping of seven members of the family was performed by amplifying microsatellite markers, tightly linked to the ECM1 locus. Sequence analysis of the coding exons and splice junctions of the ECM1 gene revealed a novel homozygous mutation (c.616C > T) in exon 6, predicted to replace glutamine with stop codon (p.Q206X) at amino acid position 206.The results revealed linkage of the LP family to the ECM1 gene for the development of lipoid proteinosis.The finding of a novel mutation in Pakistani family extends the body of evidence that supports the importance of  ECM1, which encodes for the glycoprotein extracellular matrix protein 1. To date, several mutations in the ECM1 gene have been reported in unrelated LP families from different geographical areas. In this study we report a novel nonsense mutation in a consanguineous Pakistani family affected with lipoid proteinosis; and an update of ECM1 gene mutation data base.Lipoid proteinosis LP; MIM 247100) also known as Urbach-Wiethe disease or hyalinosis cutis et mucosae, was first reported by Urbach and Wiethe, in 1929 47100 als. Skin leA consanguineous Pakistani family with autosomal recessive LP was ascertained from Rawalpindi district. Two individuals (ages 15 and 23 years) in the family were affected with the disorder  amplified. Each reaction was carried out in 10 \u03bcl volume containing 1.5 mM MgCl2, 0.6 \u03bcM of each primer, 0.2 mM each dNTPs, 1U Taq DNA polymerase and 1 \u00d7 PCR buffer {16 mM (NH4) 2SO4, 67mMTris-HCI (pH 8.8), and 0.01% of the nonionic detergent Tween-20} . Amplification was performed with an initial denaturation for 5 min at 94\u00b0C, followed by 35 cycles of denaturation at 94\u00b0C for 45 sec, annealing at 55\u00b0C for 45 sec, extension at 72\u00b0C for 45 sec and a final extension at 72\u00b0C for 10 min. The PCR products were separated on 8% non-denaturing polyacrylamide gels stained with ethidium bromide and alleles were assigned by visual inspection.Genomic DNA from seven individuals of the family was genotyped using microsatellite markers tightly linked to the ECM1 gene, 8 sets of primers were used to amplify all coding exons and adjacent splice sites by PCR. PCR products were initially screened for mutations by single stranded conformational polymorphism (SSCP) analysis. For this, aliquots of 10 \u03bcl of each PCR product was mixed with 10 \u03bcl denaturing solution , heated for 7 min at 95\u00b0C in PxE thermal cycler  and chilled quickly on ice for 5 min. Denatured DNA was subjected to 8% polyacrylamide gel electrophoresis (20 \u00d7 20 \u00d7 0.1 cm) containing 7% glycerol and 1 \u00d7 tris-borate EDTA (TBE) buffer at constant 30W for 3.5-4.0 hrs. The gels were stained with ethidium bromide (1 \u03bcg/ml) in 1 \u00d7 TBE buffer for about 5 min and visualized under UV transilluminator gel documentation system . The PCR products with mobility shift were then purified for DNA sequencing using commercially available QIAquick PCR Purification Kit . Direct sequencing was carried out by using Big Dye\u00ae Terminator v3.1 cycle sequencing kit in an ABI 3130 genetic analyzer .For detection of mutation in the The affected individuals had hoarseness of voice, pseudo-solar elastosis of the cheeks and forehead and waxy papules along the margins of eyelids. Progressive thickening and scarring of the skin and mucous membranes, hyperkeratosis with warty papules on the palms and dorsum of the hands, elbow and knee were also observed. Mobility of the tongue was limited with yellow discoloration of the lips. The clinical features of LP were identical in both patients; however, severity was varied, which may be due to difference in age as many of the clinical features of LP only manifest fully with time. Both the affected individuals showed initial symptoms during infancy. The heterozygous parents and siblings revealed no clinical signs and symptoms of LP upon detailed skin examination.ECM1 gene. The markers were fully informative, and the results revealed that the affected individuals were homozygous for the markers suggesting linkage to the ECM1 gene.Genotyping of two affected and five normal individuals of the family Figure  was perfECM1 gene. Mutant allele was found to be co-segregating with the disease phenotype in the family. Both affected individuals  in the family were homozygous for the mutant allele, while their parents  and normal siblings  were heterozygous.The SSCP analysis revealed mobility shift bands in PCR products of exon 6 of the ECM1 gene  in exon 6 of the e Figure . To see ECM1 gene comprises of 10 exons and encodes for the extracellular matrix protein 1. There are four splice variants including ECM1a, ECM1b and ECM1c, encoding proteins of 540, 415 and 559 amino acids, respectively. The recently described fourth splice variant comprises transcription of 71 bp at the 3' end of intron 1 and part of exon 2 to give a truncated 57 amino acid protein [The  protein . It prom protein -14. The ECM1 gene have been described so far in unrelated patients affected with lipoid proteinosis  in exon 6 of the ECM1 gene in a consanguineous Pakistani family. Five ECM1 mutations have been previously reported in unrelated Pakistani LP families (Table Forty six mutations in the ECM1 gene in a Pakistani family extending the mutation spectrum of the gene. The study extends the body of evidence that supports the role of ECM1 gene in the development of lipoid proteinosis. Identification of pathogenic mutations in the ECM1 gene should be helpful to improve genetic counseling and DNA based prenatal diagnosis.We have identified a novel nonsense mutation in exon 6 of the The authors declare that they have no competing interests.1 performed experimental work, AL performed clinical study of the family, MA participated in experimental work, RQ updated mutation database and participated in manuscript preparation, MN2 analyzed data and prepared manuscript, AH designed research plan and analyzed data. All authors read and approved the final manuscript.MN"}
+{"text": "HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing.The purpose was to evaluate and compare 5 different  HER2 testing on tissues scored as borderline cases (2+) found by IHC [HER2 gene amplification assays are commercially available from a multiple vendors using either fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH), where the various tests have differing characteristics  probe. The direct labeling of the different FISH probes from Dako and ZytoVision uses red (TexasRed), orange (Rhodamine), or green (FITC) fluorocrome, while the CISH-based assays give rise to either red, green, or blue chromogenic precipitation. Various strategies for blocking of nonspecific probe binding and detection systems have been implemented into the different HER2 genetic assays. ZytoVison uses repeat-free oligonucleotides and thereby does not need to block repeated sequences  while another system (Dako) has developed alu sequence blocking peptide nucleic acids (PNAs) to lower the background generated from the repeated sequences that are located in the HER2 DNA probe [HER2 IQ-FISH (Dako) reduces the assay time from two days to four hours. This is achieved by breaking the hydrophobic forces in the DNA helix used in stacking [HER2 gene copy number determination in breast cancer [HER2 genetic testing and still has a high sensitivity and specificity during routine diagnostic of breast cancer [HER2 assays in a high throughput routine setting using TMA containing breast cancer tissue and to evaluate if different characteristics between the five HER2 genetic assays could affect the perfomance when using digitalization of the HER2 stained slides before manually scoring on a monitor screen.Human Epidermal growth factor Receptor 2 (HER2) expression is investigated routinely on all breast cancer cases to make the therapeutic decisions for patients with breast cancer. The American Society of Clinical Oncology (ASCO)/College of American Pathologist (CAP) recommendations for HER2 status testing are first immunohistochemical (IHC) staining and secondary to perform genetic d by IHC . Ratio-beristics . The HERNA probe . A newlystacking  with thet cancer . CISH hat cancer . Multiplt cancer \u201311. The The study included 108 consecutive breast carcinomas from patients diagnosed at Herlev Hospital, Denmark, with information of HER2 status performed by IHC.TMAs were constructed from formalin-fixed and paraffin-embedded donor blocks, using a fully automated ATA-27 (Beecher Instrument). The areas of interest at the margin of tumor were marked on H&E stained slides by a pathologist. Four cores of 1\u2009mm or one core of 2\u2009mm in diameters was used per donor block and mounted in a new recipient block. Tissue preparation, microscopy, and subsequent laboratory analyses were carried out as part of the daily routine.HER2 genetic testing was performed using 5 assays: HER2 CISH pharmDx Kit-SK109, HER2 FISH pharmDx Kit-K5331, HER2 IQ-FISH pharmDx-K5731 , ZytoDot 2C SPEC HER2/CEN17 Probe Kit-C-3022-40 and SPEC HER2/CEN17 Dual Colour Probe Kit-Z-2020 . The tests were conducted according to the manufacturers' instructions with minor changes, which are specified below. All samples were treated with pepsin for 8 minutes at room temperature. The last step of the ZytoDot CISH dehydration (3x 30\u2009s in 100% ethanol and incubate 2x 30\u2009s in xylene) was changed to air drying for 30 minutes before mounting. This preserves the red signals which can be faintly stained when using xylene or ethanol and avoids trapping of bubbles underneath the coverslip caused by water or air.The HER2 genetic testing were counted without knowledge of patient outcome, IHC status and results from other HER2 genetic testing. Three separate tumor areas were selected and at least 60 signals (either red or green) from invasive tumor cells were counted [HER2 gene and centromere 17 (CEN17). Scoring criteria used for analysis were nonamplified (<1.8), equivocal (1.8\u20132.2), and amplified (>2.2) according to ASCO/CAP guidelines [HER2 ratios, another 60 signals were scored and the final ratio of the case was calculated from the total number of signals. The final scoring of the reanalysed equivocal HER2 ratios was reported according to the cut-off criteria nonamplified (<2.0) and amplified (\u22652.0).The CISH- and FISH-stained TMAs were scanned using a bright field/fluorescent panoramic scan (3D HisTech) equipped with a 40x dry objective and using single focus layer for CISH TMAs and five focus layers separated by 0.75 microns (z-stacking) for FISH TMAs. The scanned TMA full slide was analysed using Panoramic Viewer and manually scored on a computer monitor. Scores for  counted . All oveidelines . For allHER2 genetic assays and \u03ba statistics [The statistical analysis of the accuracy was performed by calculating the agreement between a constructed consensus assay from the five different atistics .HER2 genetic assays  were investigated, resulting in 540 scoring results. The success rate of FISH and CISH HER2 genetic testing during routine condition was 100%. The scanning success was 97,6% (527 out of 540). Thirteen samples failed; of those FISH accounted for 11 samples and CISH accounted for 2 samples. Failures of FISH scanning were missing autofocus, high background staining, and persistent autofluorescence. Failures of CISH scanning for the two samples were caused by a fingerprint and an air bobble captured underneath the coverslip. These 13 patients were excluded (n = 65 scoring results), resulting in a total cohort of evaluated patients of 95 (n = 475 scoring results). All thirteen scanning failures were located on the 2\u2009mm core TMA's, while at least two of the four cores on the 1\u2009mm core TMA could be scanned and analysed successfully. The mean digital imaging scanning time of CISH using a 40x objective was 29\u2009sec per\u2009mm2, while the FISH stained slides using five extended focus layers for the HER2 and CEN17 filters and a single layer for DAPI were 764\u2009sec/mm2.A total of 108 breast carcinomas were included in the study and 5 different HER2 ratio of CISH and FISH scores, concordance was found in 99% (94/95) of the cases , which support earlier published results [HER2 genetic assays. The concordance within the CISH assays was 97.9% and 99.0%  showed 3.0 HER2/CEN17 ratio amplification. An explanation for this difference is that it may be caused by tumour heterogeneous amplification, as a region on the 2\u2009mm core were clearly HER2 amplified. Case 69, four assays showed amplification and one FISH assay showed nonamplification. The divergent scoring results may be due to tumour heterogeneous amplification, where the correct amplified tumour area was not identified on the 2\u2009mm core. With cases 48 and 84, the scorings were very close to the borderline indicated by HER2/CEN17 ratio amplification close to 2.0. The IHC scores were 2+ and 1+, respectively (Discordant patients . In caseectively . This inHER2 status between dual-colour CISH and FISH analysis is reported as well as a reduction in scoring time and laboratory hands on time [HER2 copy number and the mean HER2/CEN17 ratio were lower than those estimated with FISH but did not result in discrepancy of the final result. The number of HER2 counted signals can be underestimated due to overlapping signals. This discrepancy is only a risk in low amplified cases and in our study it was only seen in heavily amplified HER2 signals.HER2 testing is required to identify of breast cancer patients that may benefit for trastuzumab adjuvant therapy. Significant correlation in  on time , 16\u201318. HER2 analyses is needed. By using TMA, an additional possibility emerges to analyse all patients with regard to both IHC HER2 and HER2 genetic status without considerable increase of cost [Introduction of TMA into a clinical routine laboratory can be a useful technique when a high throughput of  of cost .HER2/CEN17 is homogeneously amplified; however in approximately 10% of the carcinomas, HER2 genetic testing shows unusual signal pattern [HER2 amplification staining patterns. Homogeneously amplified tumors with identical results in scored areas, intratumour heterogeneous amplification/nonamplification areas, in which a minimum of one region was scored as amplified and another scored nonamplified and can be considered as hot spot. A third heterogeneous amplification pattern was illustrated by a single cell with extensive amplification surrounded by cells that are nonamplified. However, little is known about the clinical implication of such patterns, except for one study demonstrating intratumour heterogeneity of HER2 gene amplification to be associated with a shorter disease-free survival [In most tumours, the  pattern , 20. We The different use of DNA and PNA probes together with different blocking reagents against repeated regions did not affect the genetic test results.HER2 genetic testing aimed at identifying the best technique for preparation of digital imaging and scanning. The high level of agreement obtained between CISH and FISH genetic testing with respect to assay performance makes the two techniques equivalent, in a technical perspective. The most important advantage of CISH over FISH is the much faster digitalization of a CISH-stained slide in combination with low failure rate. The success of CISH digitalization is an advantage for a future automatic image analysis of HER2 genetic testing in a routine laboratory.To our knowledge, the first study to analyse the difference between CISH and FISH for high throughput HER2 genetic assays do not have effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing when only analysis of a few patients is required.In conclusion, our results show that the differences between the five"}
+{"text": "The prevalence of coronary artery disease increases with age. A significant proportion of patients commonly referred for investigation of coronary artery disease are often unable to perform exercise testing because of advanced age, and can only be assessed with pharmacologic tests. Stress cardiac magnetic resonance (CMR) imaging is a non-invasive modality used for detection of myocardial ischemia, necrosis and viability.The aim of the present study is to determine the applicability and safety of stress CMR in patients older than 70 years.We reviewed the data of all patients older than 70 years who were referred for stress CMR (1.5 Tesla) from January 2006 to February 2010 to our outpatient center. Standard protocol consisted of: 1) assessment of myocardial function at rest; 2) pharmacological stress induced either by dobutamine  until achieving submaximal heart rate ([220-age] x 0.85), or by adenosine (protocol of 140 \u00b5g/kg/minute during 3 minutes followed by a bolus of 10 ml of gadolinium at 4 ml/second \"first pass\"); 3) assessment of myocardial scar and/or viability by delayed enhancement sequences.Among the 309 patients \u226570 years old referred for a stress CMR, the test could be performed in 297 (96%) patients . Mean test duration was 50\u00b19 minutes. Phamacological stress was induced with dobutamine in 98 (33 %) patients and adenosine in 199 (67 %) patients). The test could not be carried out in 12 (4 %) patients because of claustrophobia (8 patients) and excessive thoracic diameter (4 patients). Among the patients who underwent stress CMR, target heart rate was not reached in 13 (4%) patients. Side effects included one case of sustained supraventricular tachycardia and one case of a transient severe hypotension. In 4 patients gadolinium contrast was not injected due to severe renal insufficiency. No other complications occurred. No ischemia or infarction was found in 170 patients (58%), while isolated ischemia was found in 20 patients (7%) and ischemia in the presence of an infarction in 34 patients (12 %). Infarction without ischemia was found in 67 patients (23%).Our data shows that stress CMR performed in elderly ambulatory patients is safe and well tolerated. Myocardial ischemia and/or infarction could be confirmed in 42% and excluded in 58% of patients in less than one hour exam."}
+{"text": "RIFLE provides standardized criteria for defining acute kidney injury (AKI) . It is bAn observational study for 6 months in ICU patients admitted \u226548 hours. For the first 7 days we calculated daily the number of patients diagnosed with AKI-0 using the four AKI definitions.One hundred and one patients (39%) had a known premorbid sCr. Mean age and APACHE was respectively 64 13) and 22 (7). Figure 3 and 22 The early diagnosis of AKI is significantly reduced when urine output criteria are neglected in the RIFLE definition, and also when baseline sCr is estimated. This may significantly impact the assessment of biomarker performance."}
+{"text": "S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in  Direct RNA interaction sites of the studied protein could most likely not be mapped by such an approach. However, it could detect RNA elements which are located in tertiary structure (but not necessarily in primary structure) in proximity to the MNase fusion protein and therefore reveal characteristics of the actual folding state of the analyzed RNP  to an optical density of 1 (OD600) were harvested by centrifugation at room temperature (3000 g) and were washed once in 20 ml ice cold water and twice in 1 ml ice cold buffer AG200 . 1.5 ml of cold buffer AG200 per gram of wet cell pellet were added and the resulting suspension was shaken in the presence of glass beads (1.4 g of 1 mm diameter glass beads per 0.8 ml suspension in a tube of 2 ml volume) on a vibrax mixer (IKA) at 4\u00b0C and maximum speed three times for 5 minutes. The extract was clarified twice by centrifugation for 5 and 10 minutes at 13000 g and 4\u00b0C. Protein concentrations of the clarified extracts were determined by Bradford protein assay (Bio-Rad Laboratories). MNase was activated by incubation of the extracts at 22\u00b0C in the presence of 8 mM calcium chloride. To stop the reaction, extract volumes corresponding to 200 \u00b5g of proteins were added to 500 \u00b5l ice cold buffer AE+  and samples were frozen at \u221220\u00b0C. Subsequently, RNA extraction by hot acidic phenol treatment was performed as described in 32P-labelled probes  equilibrated in buffer AG200 containing 0.5% Triton X-100 and 0.1% Tween20. After one hour incubation at 4\u00b0C the matrix was transferred into a poly prep chromatography column (Bio-Rad Laboratories) and washed three times with 2 ml and one time with 10 ml AG200 containing 0.5% Triton-X100 and 0.1% Tween 20. After an additional wash step with 1 ml of buffer AG200 the affinity matrix was suspended in 1 ml AG200 and split into two parts which were centrifuged for 2 minutes at 2000 g and 4\u00b0C to discard the supernatant. One half of the affinity matrix was suspended in SDS sample buffer and further analyzed by SDS polyacrylamide gel electrophoresis (16% acrylamide gels were supplemented with 4.5 M Urea) and Coomassie staining or Western blotting. Total RNA was extracted from the other half and analyzed by agarose or polyacrylamide/TBE gel electrophoresis followed by ethidium bromide staining.Figure S1Oligonucleotides used in this study.(PDF)Click here for additional data file.Figure S2Plasmids used in this study.(PDF)Click here for additional data file.Figure S3Yeast strains used in this study.(PDF)Click here for additional data file.Figure S4Possible mode of action of a nuclease fusion protein incorporated into an RNP. The figure legend in the lower panel gives a description of the symbols used to represent specific structural features of the RNP and the nuclease fusion protein. The left and right part of the figure show two different conformational states of the RNP which might be the consequence of changes in the RNP\u2019s interaction partners. Small arrows and transparent shapes of the linker, the nuclease and nuclease sensitive sites indicate varying local conformations. In the linear RNA sequence the RNA binding sites of the tested protein can be far away from the respective RNA cleavages made by the fused nuclease.(PDF)Click here for additional data file.Figure S5Growth curves of yeast strains BY4741, Y2361, Y2369 and Y2371. Yeast strains Y206 (wildtype BY4741), Y2361 (MNase-rpS13), Y2369 (MNase-rpL5) and Y2371 (MNase-rpL35) were grown overnight in YPD medium and then diluted to an OD600 of 0.02 in 0.2 ml of fresh YPD in a covered 96 well plate. Cells were incubated at 30\u00b0C in a TECAN infinite F500 reader with measurements taken in kinetic cycle mode . Generation times in logarithmic growth phase of strains Y2369 (MNase-rpL5) and Y2371 (MNase-rpL35) were increased less than 15% compared to the one of the wildtype strain Y206 (103 and 104 minutes versus 93 minutes). Generation time of Y2361 (MNase-rpS13) was increased less than 40% (128 minutes versus 93 minutes). Growth measurements using larger culture volumes incubated at 30\u00b0C in Erlenmeyer flasks on a rotary shaker gave identical results in regard to these relative changes in generation times.(PDF)Click here for additional data file.Figure S6MNase fusion proteins of rpS13, rpL5 and rpL35 expressed in yeast strains Y2361, Y2369 and Y2371 get incorporated into ribosomal particles. Cellular extracts of yeast strains which express no MNase fusion protein  or fusions of MNase linked with rpS13 , rpL5  or rpL35  by two consecutive HA tags were used for affinity purification with an anti-HA affinity matrix as described in (PDF)Click here for additional data file.Figure S7Major cleavage events in extracts of strain Y2371 depend on the addition of exogenous calcium ions. A cellular extract of yeast strain Y2371 expressing rpL35 in fusion with MNAse was prepared as described in (PDF)Click here for additional data file.Figure S8rRNA degradation in yeast cellular extracts after addition of exogenous MNase. Cellular extracts of yeast strain Y206 (wildtype BY4741) were prepared in buffer AG200 as described in (PDF)Click here for additional data file.Figure S9Map of 25S rRNA and 5.8S rRNA including positions of oligonucleotides used in this study and the major cuts observed in yeast strains Y2371 and Y2369. Oligonucleotides are represented by red boxes, major cleavage events by blue arrows and resulting rRNA fragments by grey lines.(PDF)Click here for additional data file."}
+{"text": "Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the \u201cpoised\u201d mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.The regulation of variant gene expression in  Plasmodium falciparum maintains its persistence in the vertebrate host at least in part by expression of several variant proteins at the surface of infected erythrocytes. The most important variant antigen is PfEMP-1  which is encoded by approximately 60 members of the var multigene family var genes are expressed per single parasite The human malaria parasite rif family has approximately 160 different intact members in the 3D7 strain annotated in the PlasmoDB databank. Recently, we rif transcription switches apparently faster than transcription of var genes, at least in vitro. Similar to var genes, rif gene transcription and silencing is dependent on the histone deacetylase PfSir2A rif gene loci are associated to histone 3 lysine 9 trimethylation as are other virulence-associated multigene families var, rif, and stevor seem to be controlled by a common factor and overexpression of artificial loci containing a rif promoter suppressed the transcription of var loci var genes, recently gained further support The multigene var gene which is actively transcribed was associated to histones carrying an acetyl group at the lysine 9 of histone 3 (H3K9) and Chookajorn and colleagues var locus active in early trophozoite stage and silenced in schizont stage  was associated with a dimethylated lysine 4 in histone 3 At the molecular level, Lopez-Rubio and colleagues P. falciparum, we addressed the question if dynamic histone modifications which are decisive in var regulation are also associated with active or silent rif loci. For this, the transcription of rif genes was monitored in consecutive cultures and compared to the histone modifications found for each locus in the same parasites.Given the observation of similar histone landmarks in ChIP-on-Chip experiments Var gene loci in their active state are associated with class 3 histones (H3) carrying an acetylation mark at their lysine 9 (H3K9ac), while silenced var loci associate with H3K9me3 rif loci are associated with the same epigenetic landmarks as var loci, we first performed a qRT-PCR-based rif transcript analysis in parasites previously selected for cytoadherence in CHO-CD36, since in this phenotype a number of rif loci were found active with rapid switching after a small number of reinvasions and given the large number of rif genes the analysis was restricted to these loci rif genes after 10 and 20 reinvasions after the last panning procedure. As expected, a number of rif genes were actively transcribed while others were silenced or transcribed at background levels . Noteworthy, the overall intensity of immunoprecipitation varied between loci. In order to show if predominant H3K9-acetylation - expressed as a higher ratio H3K9ac/H3K9me3 \u2013 statistically correlated with higher transcript abundance, we divided the H3K9ac/H3K9me3 ratios after 20 reinvasions by the H3K9ac/H3K9me3 ratios after 10 reinvasions and plotted them against the coefficients of relative transcript quantity from each locus after 20 and 10 reinvasions (2\u200a=\u200a0.5299). The extreme outliers were not considered in the analysis (Pf080138 and PFB0040c). A significant correlation of H3K9ac/H3K9me3 and transcript quantities was also observed when the data were analyzed separately for the 10 and 20 reinvasion material  were reselected for cytoadherence on CHO-CD36 cells. After two selection rounds, trophozoites at 20\u201324 h post reinvasion showed most transcripts from the PFI0025c locus and minor quantities from loci PFD1240w, PFD0070c and PF100397  rif loci which are actively transcribed in trophozoites (20\u201324 h post reinvasion) and/or schizonts (30\u201334 h post reinvasion), we cultivated parasites previously cytoadherence-selected on CHO-CD36 (25 reinvasions after selection), synchronized them and harvested chromatin 20 and 30 h post reinvasion and analyzed for their modification at H3K9 and H3K4 by ChIP. In trophozoites, we detected relatively low rif transcription at the tested loci (below 0.5 relative units), while in schizonts more transcripts accumulated (rif loci showed a tendency to slightly increased acetylation over trimethylation when transcripts were detected (above 0.1 relative units) and there was a significant correlation of transcript quantity with H3K9ac/H3K9me3 ratios in schizonts but not in trophozoites (rif PFB0040c (silent in trophozoites) showed increased acetylation at H3K9 when compared to trophozoites, and the strongly transcribed PF100397 showed accentuated acetylation. The only locus which did not follow the pattern was PFD0070c, which showed very few transcripts in trophozoites and in schizonts, although its locus was strongly acetylated at H3K9. The loci with no detectable transcription in trophozoites or in schizonts followed the pattern of either stronger trimethylation or lower levels of any modification  when we plotted the transcript quantities against the H3K4me2/H3 enrichment ratio. The silent loci PFL2640, PFD1010w, PF080138 and the weakly transcribed locus PFB0040c were not significantly marked  Posttranslational modification (PTM) of histones is a well-known mechanism that is related to the control of gene expression in eukaryotes. It is believed that a \u201chistone code\u201d orchestrates the accessibility of transcription factors to promoter regions through modification of histone tails by methyl, acetyl and phosphate groups, and proteins as ubiquitin and SUMO as reviewed by Berger rif gene transcription activation and silencing, we performed a detailed analysis of a number of rif genes. Since the selective upregulation of rif genes by phenotypic selection is not yet possible as it is for var genes e.g. rif genes and the frequent transcriptional activity of the herein tested loci In order to elucidate mechanisms responsible for var gene locus activity seem at least partially also functional for rif loci: When a significant presence of transcripts for any tested locus was observed, H3K9 acetylation dominated. This mark has not been identified before for active rif genes. Inversely, in the absence of transcripts, loci were either associated with strong tri-methylation of H3K9, in accordance to data published by Lopez-Rubio and colleagues var loci, the modifications are dynamic var genes, one would perhaps expect higher acetylation in \u201con\u201d rif genes of which oligos amplify in the 5\u2032 region of the gene \u2013 in proximity to the promoter region which is supposed to carry most of the acetyl residues rif genes are much shorter than the sometimes 12 kb-var genes. It is possible that the short central 500 bp range in which the oligos were allocated in the different rif genes may not allow to discriminate these positional effects, taking also in account that the immunoprecipitated genomic DNA fragments generated during the ChIP procedure had around 400 bp (data not shown). In order to address the question of positional effects of priming sites in genes analyzed by ChIP, we compared signal differences in the loci PFI0025c and PF100397, using additional specific oligo pairs in the coding sequence. For this experiment, material from the experiment in rif gene  is distant from transcribed rif genes and no specific pattern of transcript quantity and genomic context  following instructions of the manufacturer. The washed RNA pellet was briefly dried at RT and dissolved in water and stored at \u221280\u00b0C until use. About 5 \u00b5g of total RNA was used for cDNA synthesis. Briefly, total RNA was treated 3 times with DNAse I (Fermentas) prior to synthesis to prevent genomic DNA contamination. The treated RNA was reversed transcribed using MuLV-Reverted aid transcriptase reverse (Fermentas) and random hexamer primers following the manufacturers' instructions.9 parasites were crosslinked with 1% formaldehyde and RBC were lysed in PBS supplemented with saponin 0.1%. Following saponin lysis, parasites were resuspended in lysis buffer , supplemented with 1\u00d7 Protease Inhibitor Cocktail . After 30 minutes of incubation, parasites were lysed using a Dounce Homogenisator (100 strokes), centrifuged for 10 minutes at 14.000 rpm/4\u00b0C, and then sonicated using a cell disruptor (Unique Ultrasonic) until resulting DNA fragments had 200\u2013500 bp. At this stage, 10% of the material (\u201cinput\u201d) was retrieved and submitted directly to the reversion of crosslinking (see below). For immunoprecipitations, sonicated chromatin was diluted in ChIP buffer . Chromatin diluted in ChIP buffer was pre-cleared in 50 \u00b5L/mL of a DNA sperm salmon/protein A-sepharose slurry for 2 h/4\u00b0C. Afterwards, chromatin was incubated with 1\u2236250 of anti-H3K9ac, H3K9me3, H3K4me2 (Upstate) and H3 (Abcam). For negative controls (mock samples) we used an anti-IgG mouse, and for calibration of the overall chromatin quantity, similar amounts of material from the \u201cinput\u201d fraction were employed. The chromatin-immune complexes were then washed under stringent conditions. Following this, crosslink was reverted by incubation at 65\u00b0C for 4 h and 0.2 M NaCl. DNA was extracted twice with Phenol/Chloroform/isoamylalcohol, precipitated and resuspended in 20\u201350 \u00b5l sterile water. The results presented are the average of two or three independent immunoprecipitations from different extracts of parasites . The significance of differences between signals was evaluated by Student's T test (two tailed) using GraphPad Prism 5.ChIP was conducted as described in rif transcript sequences and only oligos with perfect primer-to-target specificity were considered. Later, oligonucleotide pairs were selected for global target specificity by ePCR using the 3D7 genome sequence as template. Importantly, the primer performance in qPCRs differed no more than 1 Ct between oligonucleotide pairs used herein and the internal control oligonucleotide pair. PCRs were performed in triplicates and equal amounts of input DNA (0.05\u20130.5 \u00b5g) per imunoprecipitated sample and mock immunoprecipitations (unrelated IgG) were analyzed together with samples. Mock samples presented CTs>35, which was considered as unamplified targets. All rif oligos in each immunoprecipitation were calibrated with the input DNA signal using the formula 2\u2212\u0394Ct\u200a=\u200a(CtoligoAb\u2212Ctoligoinput), where Ab is the respective antibody used for ChIP. The plotted enrichment was calculated as the ratio of immunoprecipitated Ab oligo signal/immunoprecipitated H3 oligo signal. The internal control transcript used for calibration throughout the experiments was locus seryl t-RNA transferase (PlasmoDB No. PF07.0073), previously shown as reliable  and D, schizonts 30\u201336 h p.i. from the same reinvasion cycle as in D. E: Material from trophozoites 20\u201324 h p.i. freshly repanned over CHO-CD36.(TIF)Click here for additional data file.Figure S2Position of oligos used in qPCR and ChIP in the deduced transcript sequence of analyzed rif targets and their genomic context. A) The black bars above and the numbers below the scheme of each rif coding region indicate the initial position of the amplification of the primer. The numbers at the end of each gene indicate the size of the respective coding regions. The genes were divided in the figure according to the rif promoter classification proposed by Joannin et al 2008 C and T means telomeric and centromeric position, respectively. Rif A type genes are believed to encode IRBC-surface displayed RIFINs while type B RIFINs are probably localized to Maurer's clefts rif loci, extracted from http://plasmodb.org (6/15/2011).(PPTX)Click here for additional data file.Figure S3Relative quantities of rif transcripts and chromatin enrichment for H3K9ac, H3K9me3 and H3K4me2 (poised mark) in trophozoite and schizont stages for selected rif loci. A: relative transcript amounts of chosen rif targets in trophozoites and schizonts in 3D7 parasites of the same reinvasion, calculated as above. Chromatin enrichment of H3K9ac and H3K9me3 for trophozoites (B) and schizonts (C). The chromatin enrichment was normalized using the qPCR data from ChIP with anti-H3 and 10% of the input. Significant differences between H3K9 trimethylation and acetylation are depicted by single asterisks, and significant H3K4me2 modification (compared to the H3K9me3 control) are indicated by two asterisks .(TIF)Click here for additional data file.Figure S4Raw results from experiment in  showing the influence of primer localization on the qPCR signal after ChIP at two rif loci. Immunoprecipitated material from the experiment in (PPTX)Click here for additional data file."}
+{"text": "However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 3\u20325 Triiodo L Thyronine . From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TR\u03b2, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions.In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cis\u2013acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer.This is the first report of novel mechanistic insights into the remarkable downregulation of  However, due to conflicting results regarding the clinical correlation between breast cancer and thyroid diseases, any precise association between thyroid status and the pathogenesis of human breast cancer remains elusive The TRs are ligand modulated transcription factors encoded by two genes, TR\u03b1 and TR\u03b2, located on human chromosomes 17 and 3 respectively The effect of thyroid hormone at the cellular level is mediated through TRs, by interacting with thyroid hormone response elements (TREs) in the promoters of the target genes to regulate transcription. Depending on their ability to bind to thyroid hormone, TRs can activate or suppress gene expression in a tissue specific manner through heterodimerisation with retinoid X receptors (RXRs) and interaction with the positive or negative response elements commonly known as TREs in the regulatory regions of target genes SMP30 gene in breast cancer cells in which programmed cell death is deregulated. Additionally, we observed downregulation of SMP30 expression in rat liver by thyroid hormone SMP30, which is an antiapoptotic gene.The role of thyroid hormones in inducing apoptosis in different systems has been well documented in literature . SMP30 was initially identified as a novel protein that is highly expressed in hepatocytes and in renal tubular epithelia, and its amount decreases with aging SMP30 gene, molecular mechanism of its regulation and consequences in response to thyroid hormone treatment in MCF-7 cells. These highly metastatic breast cancer cells are found to have endogenous expression of TRs as well as SMP30. Here, we are providing promising data in support of challenging breast cancer by targeting thyroid hormone receptors which might provide enough supplementary strength to interfere breast cancer metastasis by adjuvant therapies such as Selective estrogen receptor modulators (SERMs).The mechanism underlying thyroid hormone induced regulation of SMP30 level in breast cancer cells as well as the role of thyroid hormones against breast cancer cells has not been addressed. In our current study, we have attempted to explore the importance of SMP30 gene expression both at transcriptional and translational level. MCF-7 breast cancer cells express a substantially good amount of SMP30 which is a well established antiapoptotic gene. Here we have examined the effect of thyroid hormone on SMP30 (hSMP30) promoter, we performed an electrophoretic mobility shift assay (EMSA). We have scanned for TREs within 2 kb hSMP30 promoter from transcription start site. Transcription start site of hSMP30 gene was analysed by primer extension analysis as shown in SMP30 promoter we got two TR binding half sites i.e. at 613 bp and 1.2 kbp from hSMP30 transcription start site. The sequence of the former from \u2212637 to \u2212600 was found to be TGACCTTAGGATGTTGGTCAGGCTGGTCTCAAACTCCGAAGGACATTAAAGGGACAATTTCTATGACCTGGTGand that of later from \u22121274 to \u22121235 was . These two DNA fragments were 32P-labeled, incubated with nuclear extract (N.E) of MCF-7 cells - transfected with TR\u03b1, TR\u03b2 and RXR \u03b1 expression vectors. The electrophoretic mobility of the radiolabelled DNA fragments was retarded in presence of nuclear extract suggesting possible interaction between the 32P-labeled DNA fragments and TRs present in the nuclear extract. This binding was later confirmed by competition with 50 and 100 fold molar excess of cold self oligos, mutated and non specific oligos as shown in SMP30 promoter has specific binding sites for TRs.To determine whether there were any TR binding sites within human SMP30 promoter in MCF-7 cells by measuring luciferase activity. We transfected reporter constructs having an oligo representative of the TREs appended to some minimal promoter along with or without expression vectors in MCF-7cells. The minimal promoter (\u2212684 to \u2212455) region cloned in PGL3 basic vector is known as hSMP30 TRE1 and cloned (\u22121290 to \u22121015) promoter fragment is known as hSMP30 TRE2. Luciferase activity of both the reporter vectors in MCF-7 cells having endogenous TRs did not show any significant difference in presence or absence of T3 where as luciferase activity was induced by over expressing TRs along with RXR\u03b1 in absence of ligand and repressed in presence of ligand as shown in SMP30 promoter behaved similarly to those of TSH\u03b1 SMP30 promoter were negatively regulated by T3.We examined the response of T3 on transcriptional activity of hSMP30 TRE2 (in the presence and absence of T3) was decreased as compared with wild type and the changes in the repression ratios were also small as compared with those noted with the wild type shown in SMP30 TRE1 did not show the similar pattern. Repression was still observed although decreased luciferase activity was detected in SMP30 gene similar to earlier observation in case of some other genes i.e CD44 gene The total luciferase expression of mutated construct of hSMP30 TREs by estradiol (E2) stimulation, we did luciferase assays of hSMP30 TREs in presence and absence of E2. We found ligand independent or dependent activation of promoter activity in case of both hSMP30 TRE1 and TRE2.However, in case of thyroid hormone treatment there was downregulation of luciferase activity as shown in To analyze the effect of TRs on hSMP30 TRE1, TRE2 in MCF-7 cells having endogenous TRs did not show any significant difference in presence or absence of T3 where as luciferase activity was induced by over expressing TR\u03b1 in absence or presence of ligand. However there was significant repression found while overexpressing only TR\u03b2 in presence of ligand as presented in SMP30 promoter responds to exogenous expression of TR\u03b2 and this effect is synergized by T3 through further repressing the promoter activity.Luciferase activity of both the reporter vectors h3 is known to recruit HDACs to the thyrotropin releasing hormone (TRH) and thyroid stimulating hormone TSH \u03b2 promoters during ligand dependent negative regulation. Hence, histone deacetylation may also be an important mechanism for the negative regulation of other target genes by T3 [32 and 52].T3 mediated SMP30 repression in MCF-7 cells by referring the following papers SMP30 reporter construct was abolished when TSA was added as shown in We therefore examined the effects of the histone deacetylase inhibitor TSA, on TSMP30 gene expression in MCF-7 cells was analysed both at RNA and protein level by RT-PCR and Western Blot analysis in 3 treatment resulted in repression of SMP30 gene expression by 40% and TSA further repressed SMP30 expression by 20% as here in 3 dependent histone acetylation of SMP30 promoter has an important role in regulation of SMP30 gene expression. The above results also imply that deacetylation did not affect T3 dependent silencing of transcription of the SMP30 promoter, but instead was involved in the ligand independent activation, thus reinforcing the differences in the mechanisms behind TR dependent transcriptional regulation of negative thyroid response element (nTREs) vs positive thyroid response element ( pTREs).Effect of TSA on 3-dependent histone acetylation of SMP30 promoter by ChIP analysis  to nTREs by ChIP assays  SMP30 promoter after T3 treatment involves SRC-1 recruitment.We next investigated the cofactors that participated in the Tanalysis . It was P assays .LocationSMP30 gene by T3. Cotransfection of hSMP30 TRE1 and hSMP30 TRE2 luciferase construct with TRs and RXR\u03b1 expression vectors resulted in increased basal transactivation of SMP30 gene in absence of T3. However, in presence of T3 the promoter activity of SMP30 returned to basal level. siRNA mediated silencing of TR\u03b2, completely abolished the basal transactivation of SMP30  thiazol-2-yl)hydrazone] on SMP30 promoter transcriptional activity. CPTH-2, a known modulator of Gcn5 network 3, presumably by decreasing histone acetylation as shown in  revealed histone acetylation of shown in . These rFinally, we investigated whether induction of apoptotic death in MCF-7 cells by thyroid hormone is mediated through down regulation of SMP30 expression. Compared to untreated control, thyroid hormone treatment enhanced the proportion of MCF-7 cells undergoing apoptosis by 50\u201360% . OverexpSMP30 gene to T3 signaling has been reported in an investigation in mouse model SMP30 gene.Recent findings indicate that epigenetic alterations of the genome such as acetylation/ deacetylation and other modifications of histones prove to be key factors in breast carcinogenesis SMP30 gene expression. In an effort to unravel the underlying mechanism, we performed primer extension analysis to find out the transcription start site of human SMP30 (hSMP30) gene, scanning of hSMP30 promoter for TRE sites followed by gel retardation assays. On the basis of these experiments we confirmed that there are two important putative TREs at 613 bp as (hSMP30 TRE1) and 1.2 kbp (hSMP30 TRE2) from hSMP30 transcription start site. The luciferase assays which were conducted by taking different deletion constructs of hSMP30 promoter validated our findings of the putative TRE sites. Looking at the distinct repression caused by liganded TRs (through T3) in SMP30 gene. Similar to our line of study, Guigon et al (2011) have identified STAT-5 as a downstream key molecule in the aberrant thyroid hormone signaling emerging out of mutated TR\u03b2. As the consequence of this the mice is predisposed to development of mammary tumors SMP30 transcription. The above findings revealed that hSMP30 promoter responds to exogenous expression of TR\u03b2 and this effect was synergized by T3 through further repressing the promoter activity. Silencing of TR\u03b2 in MCF-7 cell line reduced the amount of repression as a result of T3 treatment both in terms of protein as well as promoter activity.In the current study our investigation by flow cytometry revealed that in response to thyroid hormone treatment, apoptosis could be induced in MCF-7 cell line as a result of down regulation of SMP30 TRE2 (in the presence and absence of T3) was decreased as compared with wild type and the changes in the repression ratios were also small as compared with those noted with the wild type. From this it is quite clear that the decrease in the repression might be due to decrease binding affinity of mutated TREs with TRs. However, mutated hSMP30 TRE1 did not show the similar pattern. Repression was still observed although decreased luciferase activity was detected. In summary, these results indicated that high affinity TR binding TREs were not necessary for T3 mediated negative regulation of SMP30 gene. In literature one hypothesis suggests that indirect involvement of TR in T3 mediated negative regulation is through the squelching of coregulators from other transcription factors The total luciferase expression of mutated construct of hSMP30 promoter.The supershift assay performed in presence of TR\u03b1, TR\u03b2 and RXR\u03b1 antibodies indicated that TR\u03b2 has direct binding affinity for both TRE1 and TRE2. However, it might be possible that TR\u03b1, TR\u03b2 and RXR\u03b1 protein forms a heterodimer complex then binds to thyroid response elements of both the sites present in hSMP30 promoter are negative response element in the context of liganded TRs. By observing both the TREs differentially regulated by TRs we would like to further distinguish them on the basis of different cofactors recruitment on those TREs after T3 treatment. Our specific finding of distinct recruitment of SRC-1 on TRE2 due to the enhanced association of SRC-1with TR\u03b2 in response to T3 signaling as evidenced in SMP30 gene expression. Distinct withdrawal of SRC-1 from TRE1 supports the possibility that another coregulator/s with HAT activity may participate in the negative regulation of SMP30 gene expression. Previous studies in SRC-1 knockout mice and TR\u03b2 activation function-2 domain mutant knock in mice suggest that coactivators may be important for negative regulation of genes . In our investigation another interesting but opposite result was observed in the recruitment pattern of HDAC1, HDAC2 as well as NCoR on TRE1 and TRE2. These differential recruitment patterns also make our above argument more logical. Similar kinds of observations were reported for SOD1, Necdin, and CD44 - target gene that they are under negative regulation by T3 . We believe enhanced recruitment of AcH3 locally does not speak for the complete active chromatin structure. The recent reports by Wang et alSMP30 gene by TSA (Trichostatin A) which is an inhibitor of deacetylases. The repression of SMP30 gene by liganded TRs was reversed after treatment with HAT inhibitor. From this observation it was very clear that histone acetylation played a major role in the negative regulation of SMP30 gene by liganded thyroid receptor. It also established the authenticity and importance of our characterisation of two negative thyroid response elements i.e TRE1 and TRE2 of SMP30 promoter. These observations resemble the functioning of other liganded TR mediated negative TREs (nTREs) et al discussed that TR mediated recruitment and basal activation by SMRT and NCoR in absence of T3 and reversal of basal activation by dissociation of corepressors in the presence of T3 SMP30 promoter to T3 signaling may not be analysed in isolation with regard to either TRE1 or TRE2. Rather the repression of SMP30 gene expression is the combined outcome of the response of both the above elements to T3 signaling.On the basis of our results we interpreted that thyroid response elements cannot be categorically classified as absolute positive or absolute negative elements. However by considering acetylation of histone i.e H3 as bench mark for the above classification we concluded that both TRE1 as well as TRE2 in SMP30 promoter. To ascertain the role of SMP30 in the thyroid hormone induced apoptosis of MCF-7 cells, we studied the effect of thyroid hormone after overexpressing and knocking down the SMP30 gene in MCF-7 cells respectively. Overexpression of SMP30 in our study resulted in reversal of thyroid hormone induced apoptosis of MCF-7 cells while knocking down of SMP30 made the cells increasingly susceptible to thyroid hormone induced apoptosis. These findings indicated anti-apoptotic role of SMP30 in MCF-7 cells which is in accordance with earlier reports regarding the role of SMP30 in literature SMP30 gene in MCF-7 cells. Interestingly, this repression was further enhanced as a result of T3 as well as TSA treatment. It is a well established fact that TSA which is a HDAC inhibitor induces apoptosis in different types of cancer et alet al (2011) have shown downregulation of STAT-5 as a result of mutation in TR\u03b2 which was manifested in terms of predisposal of the mice to the development of mammary tumors. Our current study and the emerging importance of thyroid hormone in breast cancer support our claim that why we need to seriously think for a combinatorial therapy by taking both T3 as well as other anticancer drugs including those related to TSA Observations in the present study revealed that thyroid hormone mediated down regulation of SMP30 was direct and accomplished through negative TREs within h2 atmosphere in 37\u00b0C until 70\u201380% confluent. For stimulation with T3, culture medium was removed, the cells were rinsed once with phosphate buffer saline (PBS) and medium containing10% charcoal-stripped fetal bovine serum (CS-FBS) was added and incubation continued for 3 days. T3 (1 \u00b5M), (from Sigma) was diluted in medium and charcoal-stripped 10% fetal bovine serum was added to cells for the times indicated in figure legends.MCF-7and HEK 293T were obtained from National Centre for Cell Sciences, Pune. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) mantained in 5% COSMP30 TRE1 reporter construct was prepared by amplifying human SMP30 promoter from MCF-7 genomic DNA by using hSMP PCR 1F2 and hSMP PCR 1R1 primer sequences are shown in pBlue TOPO TA vector (Invitrogen). Insert was taken out from pBlue TOPO TA vector by HindIII digestion and ligated in to HindIII digested PGL3 Basic vector to obtain hSMP TRE1 reporter construct. Similarly, hSMP TRE2 reporter construct was prepared by amplifying human SMP30 promoter from MCF-7 genomic DNA using hSMP PCR 2F and hSMP PCR 2R1 primer sequences shown in pTARGETtm vector. Insert was taken out from pTARGETtm vector by digesting in Kpn I and Xho I enzyme, ligated in to digested PGL3 basic vector. RT-PCR product of human SMP30 was prepared from MCF-7 cDNA by using hSMP30 EcoRI F and XhoI R primers , RXR\u03b1 expression vector or pCMV vector and 100 ng of pRL-TK control vector were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection 1 \u00b5M T3 hormone was added and treatment of TSA and HAT inhibitor (from Sigma) were carried out accordingly as shown in figure legend. The cell lysates were prepared, and luciferase activities were measured in duplicates in three independent experiments.Transient transfections were carried out using MCF-7 cells and similar experiments were also carried out in HEK 293T cell lines. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1\u00d7103, then cells were harvested for luciferase assays and readings were taken in duplicates in three independent experiments.24 hrs after transfection of reporter plasmid DNA and expression vectors, 200pmole of TR\u03b2 siRNA or nonspecific scrambled siRNA (syntesized from Eurogentec) per well were transfected using oligofectamine according to manufacture's instruction (Invitrogen). After 24 hrs of second round transfection, cells were incubated for an additional 24 hrs in the presence and absence of 1 \u00b5M TSMP30 TRE sites were synthesized as shown in 32P ATP using T4 poly nucleotide kinase and annealed to its complementary unlabelled strand. Nuclear extracts of MCF-7transfected with TR\u03b1, TR\u03b2 and RXR\u03b1 expression vectors (10 \u00b5g) to maintain appropriate concentration of functionally active nuclear receptors were incubated with 20fmoles of radiolabelled oligonucleotide duplex and 1 \u00b5g poly (d I-d C) in 30 \u00b5l reaction mixture containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 5% glycerol for 20 mins at room temperature. In competition experiments, 100fold molar excess of unlabeled self and mutated oligonucleotide duplexes (sequences as shown in Electrophoretic mobility shift assay was performed as described in SMP30 TRE1 (h SMP PCR 1F1 and Xho2 R), TRE2 promoter (h SMP PCR 3F1 and 2R2) and non TRE region. These primer sequences were listed in ChIP assay was performed as previously described Co-Immunoprecipitation (Co-IP) and western blot assays were performed as described SMP30 and GAPDH mRNA expression were determined by quantitative RT PCR using SYBR greensystem (SIGMA). Relative values (mean \u00b1 SD) normalized to GAPDH expression.MCF-7 cells were harvested after 15 hrs of T3 followed by 6 hrs of TSA with or without treatment. RNA was isolated and SMP30 were given in SMP30 was as follow: initial denaturation (94\u00b0C 3 min),  for 30 cycles and additional extension was per`formed at 72\u00b0C for 5 min. The PCR condition for GAPDH as follows: initial denaturation (94\u00b0C 3 min),  for 30 cycles and additional extension was performed at 72\u00b0C for 5 min. Then PCR products were electrophoresed in 1.5% agarose gel.RT-PCR was preformed as described elsewhere Whole cell extracts were prepared from cells and Western blotting was performed as described elsewhere SMP30 gene as (SMP30-FLAG) were incubated in presence or absence of 10 \u00b5M T3 for 24 hrs in culture. Knocking down of endogenous SMP30 expression or non specific scrambled siRNA was followed by incubation of MCF-7 cells with or without 10 \u00b5M T3. After 24 hrs of incubation cells were harvested and assayed for apoptosis using the Annexin V FITC apoptosis detection kit, IMGENEX according to manufacturer's instruction. Cells were analyzed in FACS Calibur Analyzer (Becton Dickinson) by using Cell Quest Pro software.To detect intact, necrotic and apoptotic cells, flow cytometry was performed. MCF-7 cells transfected with mock vector (CMV-FLAG) and vector containing Detection of apoptosis by Annexin-V-FITC detection kit was further confirmed by intracellular staining with PE-conjugated polyclonal antibody against cleaved PARP (BD Biosciences 552933) according to manufacturer's instruction. This was followed by washing with PBS twice and analysis by Flow cytometer. Percentage of population shifting right in the gate represent population of cells showing cleavage of caspase substrate PARP.Two bases of the TREs were mutated as shown in the Figure S1Determination of hSMP30 Transcription start site (TSS). Determination of transcription initiation sites by primer extension analysis. A Lane 1\u20134, sequencing reactions; lane 5, primer extension product of MCF-7 total RNA, lane 6, labeled DNA marker from Promega. The sequence corresponding to the transcription start site has been marked by a line and is complementary to the sequencing reactions shown in the figure. B. A schematic diagram of human SMP30 promoter showing two TREs, CAAT box and transcription start site.(DOC)Click here for additional data file.Figure S2To analyze the effect of T3 on SMP30 Promoter activity in MCF-7 cell in relation to RXR\u03b1. Transient transfections of hSMP30 TRE1, TRE2 were carried out using MCF-7 cells. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1\u00d7105 cells per well in 12 well plates. For transient transfection, 0.5 \u00b5g of reporter plasmid DNA, 0.5 \u00b5g of TR\u03b2 and TR\u03b1 (TRs), RXRA\u03b1expression vector, 100 ng of pRL-TK control vector and only vector to control cells were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection, cells were subjected to overnight treatment with 1 \u00b5M T3 and vehicle to control cells in 10% CS \u2013DMEM. Then cell lysates were prepared and luciferase activities were measured. Values are the mean of three independent experiments \u00b1 SD normalized to Renilla activity. *** P<0.0001difference from vehicle control using ANOVA.(DOC)Click here for additional data file.Figure S3Luciferase activity of SMP30 TREs in HEK293 cell line. Transient transfections of hSMP30 TRE1, TRE2 were carried out using HEK 293 cells. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1\u00d7105 cells per well in 12 well plates. For transient transfection, 0.5 \u00b5g of reporter plasmid DNA, 0.5 \u00b5g of TR\u03b2 and TR\u03b1 (TRs), RXR\u03b1 expression vector, 100 ng of pRL-TK control vector and only vector to control cells were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection, cells were subjected to overnight treatment with 1 \u00b5M T3 and vehicle to control cells in 10% CS \u2013DMEM. Then cell lysates were prepared and luciferase activities were measured. Values are the mean of three independent experiments \u00b1 SD normalized to Renilla activity. ***P<0.0001difference from vehicle control using ANOVA.(DOC)Click here for additional data file."}
+{"text": "Ecology and Evolution 2013; 3(8): pages 2547\u2013256710.1002/ece3.636doi: 4, and for the Sinking Creek form at Locality 31, which is actually polymorphic for GAPDH2 and GAPDH3. We still assert that the allozyme frequencies at Locality 36 may reflect gene exchange with LGF.Table 2 reports incorrect GAPDH allozyme frequencies for the Lemon Gap form at Locality 61, which is actually fixed for GAPDHA corrected version of \u201cThe populations at localities 36 and 73 are fixed or nearly fixed for the same variants at 16 presumptive loci, but exhibit complete differentiation at the remaining six . At each of these diagnostic loci except GAPDH, the population at Locality 36 shares a variant with LGF at Locality 61 (Table"}
+{"text": "ALDH2 genotype on the exposure to locally formed acetaldehyde via the saliva without ethanol ingestion.Acetaldehyde associated with alcoholic beverages was recently classified as carcinogenic (Group 1) to humans based on uniform epidemiological and biochemical evidence. ALDH2  deficient alcohol consumers are exposed to high concentrations of salivary acetaldehyde and have an increased risk of upper digestive tract cancer. However, this interaction is not seen among ALDH2 deficient non-drinkers or rare drinkers, regardless of their smoking status or consumption of edibles containing ethanol or acetaldehyde. Therefore, the aim of this study was to examine the effect of the ALDH2 genotypes of 17 subjects were determined by PCR-RFLP. The subjects rinsed out their mouths with 5 ml of 40 vol% alcohol for 5 seconds. Salivary ethanol and acetaldehyde levels were measured by gas chromatography.The ALDH2 genotypes.Acetaldehyde reached mutagenic levels rapidly and the exposure continued for up to 20 minutes. The mean salivary acetaldehyde concentrations did not differ between For ALDH2 deficient subjects, an elevated exposure to endogenously formed acetaldehyde requires the presence of ethanol in the systemic circulation.Our findings provide a logical explanation for how there is an increased incidence of upper digestive tract cancers among ALDH2 deficient alcohol drinkers, but not among those ALDH2 deficient subjects who are locally exposed to acetaldehyde without bloodborne ethanol being delivered to the saliva. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands. ALDH2) gene is 10-fold that for alcoholics without the mutation [Cancers of the upper digestive tract are often found at an advanced stage, remain difficult to treat and have a high mortality rate. The risk for upper digestive tract cancer for alcoholics who have an impaired ability to eliminate acetaldehyde due to a single point mutation in the mitochondrial aldehyde dehydrogenase 2  of the ALDH2 gene, which contains a single nucleotide polymorphism (SNP) and results in the synthesis of an inactive ALDH2 enzyme [ALDH2*2 genotype, alcohol consumption and upper digestive tract cancer risk found in epidemiological data and the elevated local acetaldehyde exposure of ALDH2*2 carriers who consume alcohol provide convincing evidence for the specific carcinogenic potential of acetaldehyde in the pathogenesis of upper digestive tract cancers. Based on these findings, the International Agency for Research on Cancer (IARC) recently classified acetaldehyde associated with alcoholic beverages i.e. present in alcoholic beverages and/or formed endogenously from ethanol to be a Group 1 carcinogen in humans [ALDH2 is a low 2 enzyme . In addi2 enzyme -14. Unifn humans .In addition to alcohol consumption, smoking is also a generally accepted major etiological factor for upper digestive tract cancers . The mulALDH2 genotype significantly potentiates the risk of upper digestive tract cancer, there seems to be no apparent increased ESCC risk with ALDH2 deficient non-drinkers [ALDH2 genotype and raises the question about whether the ALDH2 genotype only has an effect when systemic ethanol is available.Unlike the case for alcohol ingestion, in which the drinkers ,19. Nevedrinkers . This suALDH2 genotype on the exposure of the upper digestive tract to salivary acetaldehyde when ethanol is only rinsed in the mouth, but not ingested.Our present study follows up on our earlier paper in which 7 ALDH2 deficient individuals and 13 individuals with the functional ALDH2 enzyme ingested 0.5 g/kg of ethanol and their salivary acetaldehyde levels were followed thereafter at 20 minute intervals for a period of 240 minutes . At eachALDH2*2 genotype [20 healthy Eastern Asian volunteers  were recruited into the study. The mean age of the participants was 25.7 years range: 21-38 years) and the mean body mass index (BMI) 20.9 (range: 18.0-27.8). All the participants were of Chinese origin and moderate alcohol drinkers i.e. they consumed <20 drinks/week (men) or <14 drinks/week (women). Half of the volunteers reported having a history of alcohol-related flushing symptoms, which are known to correlate significantly with the -38 yearsThe study was approved by the co-ordinating Ethics Committee, Hospital District of Helsinki and Uusimaa (Finland). Signed informed consent to participate in the study was obtained from each study participant.The participants were instructed to abstain from drinking alcohol for 24 hours before the study visit day and to fast for two hours before giving samples. Smoking was prohibited on the day of the study. Salivary samples which were used for genotyping were collected prior to the ethanol rinsing experiment. For the rinsing procedure the volunteers gave a baseline salivary sample and then rinsed their mouths with 5 ml of 40 vol% alcohol for 5 seconds, after which the oral contents were discharged and salivary samples were collected at 30 s, 2 min, 5 min, 10 min, 15 min and 20 min after discharging. The participants also answered a questionnaire regarding their oral health, alcohol use, smoking, diet and medication.ALDH2 gene (rs671). The PCR protocol included one cycle of 95\u00b0C for 5 min, 40 cycles of 98\u00b0C for 10 s, 60\u00b0C for 30 s, and 74\u00b0C for 45 s and a final cycle of 74\u00b0C for 2 min. A 430-bp DNA fragment that contained the polymorphic site of ALDH2 was amplified by PCR using the forward primer 5\u2032-TCAAATTACAGGGTCAACTGCT-3\u2032 and the reverse primer 5\u2032-GGCTGGGTCTTTACCCTCTC-3\u2032 (Sigma-Aldrich). The PCR reaction required 7.5 \u00b5l of distilled water, 12.5 \u00b5l of 2X Xtreme Buffer, 2.5 \u00b5l of 2 mM deoxyribonucleotide triphosphates, 10 pmol each for the two ALDH2 primers, and 0.5 U of KOD Xtreme DNA polymerase  in a total volume of 25 \u00b5l. PCR products were digested using AcuI according to the manufacturer\u2019s instructions (New England Biolabs Inc.). The 430 bp ALDH2*1 fragment was cut into two fragments of 296 and 134 bp. The ALDH2*2 allele (2*/2*) was not cut. Fragments were analyzed by using gel electrophoresis on a 2% agarose gel. Samples of five randomly selected participants were analyzed twice to assess the reliability of the genotyping protocol.The genotyping protocol used was modified from Hayashida et al. . Whole s50 \u00b5l of 6 M perchloric acid was added to 450 \u00b5l of saliva to stop organic reactions, after which the samples were immediately sealed in 20 ml vials and stored at -20\u00b0C until analysis. Dual or triple parallel samples were collected at each time point whenever possible to confirm analytical reliability. Acetaldehyde and ethanol concentrations were measured by headspace gas chromatography as previously described . 100 \u00b5M Target sample size (6) was calculated for an effect size of 1.5 SD with an alpha level of 0.05 and a power of 0.80 using data from our previous study with ALDH2 deficient subjects . InteracALDH2*2 genotype, four (67%) were smokers. Of the participants without the ALDH2*2 genotype, four (36%) were smokers. The genotyping of one smoker was unsuccessful. No chronic illnesses were reported. Aside from one participant using oral medication for birth control, no regular medications were reported. Antibiotics had not been used for at least 30 days. Two participants (10%) reported following a non-lactose diet. One participant (5%) followed a vegetarian diet. Only one participant (5%) reported consuming products that contain lactic acid bacteria.All of the participants reported brushing their teeth at least twice per day, three participants (15%) also used mouthwashes. Nine participants (45%) were smokers and all reported consuming <20 alcohol drinks/week (men) or <14 alcohol drinks/week (women). Of participants with the ALDH2*1/*1 genotype, five the ALDH2*1/*2 genotype and one participant the ALDH2*2/*2 genotype  and those with the deficient ALDH2*2 allele  found that there were no statistically significant differences in the mean in vivo acetaldehyde levels of the saliva samples for any of the time points . Also, analysis of the area under the curve didn\u2019t show a statistically significant difference between the genotype groups . There were no statistically significant differences in levels of salivary acetaldehyde between male and female subjects or between ALDH2 genotype variants. Also, the interaction between smoking and salivary acetaldehyde concentration was not statistically significant .After the 5 s of oral exposure to 40 vol% alcohol, salivary acetaldehyde concentrations rose quickly and peaked at 2 min , Table 1e points . The intALDH2*1 allele  and those with the deficient ALDH2*2 allele  found that there were no statistically significant differences in the mean in vivo ethanol levels of the saliva samples for any of the time points . There were no statistically significant differences in levels of salivary ethanol between male and female subjects or between ALDH2 genotype variants. Also, the interaction between smoking and salivary ethanol concentration was not statistically significant .Measured ethanol levels peaked at 30 s and thereafter declined rapidly . Detectae points . The intWe have earlier demonstrated that after an oral ingestion of a moderate dose (0.5 g/kg) of ethanol, salivary acetaldehyde levels measured at 20 minute intervals are 2-3 times higher among ALDH2 deficient individuals (n = 7) than in those with functional ALDH2 enzyme n = 13) . In that . In thaOur present results show for the first time that after a brief oral exposure to non-ingested ethanol, the concentration of salivary acetaldehyde of ALDH2 deficient subjects is not significantly higher than that of subjects with normal ALDH2 activity. It should be noted, however, that at the 2 min time point, means of salivary acetaldehyde between the groups showed a near-significant difference (p=0.06) which may become significant should the number of study subjects be considerably higher. At high concentrations of salivary ethanol (mean ranging from 248 to 899 mM) that are seen at the 2 and 5 min timepoints, enzymatic activity of the small salivary glands located in the oral mucosa may also contribute to our findings. At the 10 and 15 min time points salivary ethanol concentrations had decreased to 5-30 mM, a level that is comparable to those found after oral ingestion of alcohol .Km ADH enzymes in the gingiva and the lingual mucosa [No measurable levels of acetaldehyde or ethanol were found from the baseline salivary samples taken before subjects rinsed their mouths with 40 vol% alcohol. This is in accordance with earlier findings showing that without the presence of ethanol or tobacco smoke, normal saliva does not contain measurable levels of acetaldehyde ,24. The l mucosa  and alsol mucosa . In concl mucosa .ALDH2 genotyping protocol in order to decrease the risk of sample contamination. Our study did not include the analysis of other genetic factors involved in ethanol metabolism, such as the ADH1B genotype. This warrants further studies that should focus on the possible role of ADH polymorphisms in the exposure of the oral cavity to ethanol without its ingestion.We used direct PCR in our Acetaldehyde is widely present in the environment and has been found to be mutagenic and carcinogenic in vitro and in animal experiments -29. A reKm ALDH enzymes and the presence of high Km ADH enzymes in the gingiva and the lingual mucosa may further increase the local exposure to carcinogenic concentrations of salivary acetaldehyde [Acetaldehyde in concentrations of 100 \u00b5M and above has been shown to result in an exponential increase in mutagenic DNA lesions . Acetaldaldehyde .H. pylori status combined with high consumption of pickled food has recently been shown to result in a 27-fold risk of noncardia gastric cancer [The official criterion for alcoholic beverages is that they contain 2.8 vol% or more of alcohol and their consumption is systematically followed and used in cancer epidemiology. However, many non-alcoholic beverages and edibles produced by fermentation processes may contain low but significant levels of ethanol in addition to mutagenic concentrations of acetaldehyde ,35,36. Tc cancer . Thus feWhen combined, these results imply that the presence of ethanol in the systemic circulation is a key factor for the increased exposure of the upper digestive tract mucosa to endogenously formed acetaldehyde encountered with ALDH2 deficient consumers of alcohol. Likewise, these findings provide a logical explanation for the epidemiological findings that show that ALDH2 deficiency increases the risk of upper digestive tract cancer for alcohol drinkers, but not for non-drinkers who are exposed to acetaldehyde that is derived from sources that do not associate with the presence of ethanol in the systemic blood circulation ,19. SuchALDH2*2 carriers, but not for ALDH2 deficient non-drinkers and rare drinkers, regardless of their smoking status and possible consumption of edibles that contain ethanol or acetaldehyde. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands every time when they are drinking alcoholic beverages.In conclusion, our present study supports earlier findings that show that the elevated levels of carcinogenic acetaldehyde found in the saliva of ALDH2 deficient individuals appear to be derived from the parotid glands ,13, whic"}
+{"text": "Dear EditorBeta Thalassemia major is a genetic disease with an autosomal recessive pattern and is differentiated by severe microcytic hypochromic hemolytic anemia with hepatosplenomegaly, ineffective erythropoiesis and bone marrow expansion. The \u03b2-glIn the last decade, numerous studies have been published about distribution and frequency of mutations within the HBB gene in some regions of the Iran.5] These These5] We firstly attempted also to fully analyze the beta globin gene in 130 blood transfusion dependent individuals in the two closely located towns, namely Eizeh and Baq-malek, After investigation a cohort of 200 beta thalassemia major patients and more than 1000 carriers in the Khuzestan Province in a previous study,8] We[8] We7][ We[8] We"}
+{"text": "Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer.in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort.AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts  and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression.The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups. Chromosomal instability (CIN) is a key determinant of biological behavior of breast cancer The potential utility of a gene expression based measure of CIN is further emphasized by its complex relationship with histological grade All microarray data sets used in this analysis were normalized by robust multi-array average (RMA) Focusing on histological grade, we evaluated 185 invasive breast carcinomas consisting of 63 grade 1, 62 grade 2 and 60 grade 3 FFPE tissue samples regardless of other pathological features from the Buda M\u00c1V Hospital (1999\u20132002), diagnosed and graded by a single pathologist (J.K.). Recurrence-free survival (RFS) time was defined either by loco-regional relapse or the appearance of a distant metastasis, and whichever shorter if both applicable. The study was a retrospective analysis. General written consent was obtained from all patients at time of surgery. The samples were anonymised for the study. The study was approved by the Ethical Committee of the Semmelweis University (IKEB #7/2008 and #7-1/2008).In line with bioinformatics, the clinicopathological properties of the selected 185 breast cancer patients were analyzed. The mean age of patients was 58.8\u00b112.8 years . Among the histological types, invasive ductal carcinoma was the most common overall (65.9%), but less frequent types of cancer were also included in the analysis (8.1%): 1 papillary, 1 tubular, 1 micropapillary and 3 mucinous carcinomas in the grade 1 group; 1 micropapillary and 1 mucionous in grade 2; and 4 with medullary features, 2 metaplastic and 1 micropapillary carcinomas in grade 3 cancers. Tumor size, frequency of vascular invasion, presence of necrosis, Nottingham Prognostic Index (NPI) and number of relapses showed an increase, while ER, PgR expression and RFS decreased with higher grade . When chFive to ten 5 \u00b5m thick sections were used from each case for RNA purification after assessment of cellularity on HE stained slides (minimum of 70% tumor cell content was required). RNA was extracted with Qiagen FFPE RNeasy kit according to the manufacturer's protocol . High Capacity RNA-to-cDNA kit was used to reverse transcribe 1000 ng of RNA . The Eppendorf epMotion 5070 robotic system was used to transfer samples and reagents to 384-well full-skirted white plates . The qPCR was performed in duplicates with Taqman\u00ae Assays  in Gene Flow cytometry was performed for the analysis of ploidy. Briefly, a 50 \u00b5m section was cut from all the FFPE blocks. A scroll of tissue was placed in a microcentrifuge tube and xylene was added to remove the paraffin wax. The tissue was then serially re-hydrated through 100%, 95%, 70% and 50% ethanol for 5 minutes respectively at room temperature. The tissue was washed twice with distilled water. A suspension of nuclei was made by incubating the tissue in a 0.5% pepsin solution  prepared in 0.9% saline at pH 1.5. Incubation was carried out at 37\u00b0C for 30 minutes. The nuclei were washed once with PBS, stained with propidium-iodine and analyzed using the Calibur 1 FACS mashine and CellQuest software . DNA index was assigned as follows: diploids were \u20181.0\u2019, a tumor with a DNA index greater than 1.10 was classified as aneuploid The assignment of each patient into two cohorts using the CIN4 expression signature was performed in the R statistical environment  using the package Prediction Analysis for Microarrays  as described previously In order to select a more limited set of genes that optimally reflect the CIN70 signature we retrieved expression profiles for the CIN70 signature genes from 10 publicly available breast cancer datasets The CIN70 genes were then ranked by Pearson's correlation coefficients to the CIN score (mean CIN70 expression) within these breast cancer microarray cohorts [supplementary references]. In order to derive a clinically applicable qPCR expression signature for use in FFPE tissue, containing fewer genes with equivalent information reflecting mean CIN70 expression For 5 of the 10 breast cancer microarray cohorts histological grade was also available. The above listed 4 genes were also highly correlated with histological grade (Pearson correlation coefficient above 0.7).Next, we assessed the association individually on data sets between the mean-expression level of CIN4 and clinical outcome across the same 10 cohorts containing expression data from 1928 breast cancers. We observed significant discriminative power  by CIN4 for the stratification of good from poor clinical outcome in all of the breast cancer cohorts [supplementary references]. Therefore, the expression of CIN4 appears almost as efficient at predicting cancer outcome as the extended CIN25 and CIN70 signatures. We were able to compare the performance in silico of the CIN4 signature to a number of previously published predictors of outcome such as CIN25, CIN70 B4GALT3, SLC9A3R2 and PUM1 were previously chosen based on their low variability in gene expression datasets described previously Expression of CIN4 was assessed in a retrospective cohort of 185 patients for which we had FFPE primary breast cancer samples available from Buda M\u00c1V hospital, treated between 1999 and 2002 . Kaplan-In order to define a threshold for CIN4 expression for distinct outcome groups, we trained the PAM algorithm using the continuous CIN4 gene expression signature to discriminate clinical outcome of 63 patients with grade 1 breast cancer compared to the poorer outcome associated with 60 grade 3 breast cancers from within this cohort of 185 patients. Using this CIN4 expression threshold that best distinguished grade 1 compared to grade 3 breast cancers, the PAM defined CIN4 score was established, and we assessed the ability of this CIN4 score to stratify cancer outcome in the remaining 62 patients with grade 2 breast cancer from this 185 patient cohort.Using this threshold, the CIN4 score was able to stratify patients with grade 2 breast cancers into good (44 patients) and poor (18 patients) prognostic groups  .For the identified genes that have been previously described to be of prognostic value, we have evaluated the prognostic power of AURKA, FOXM1, TOP2A, and TPX2 separately. Although, in public breast cancer datasets  all the genes showed strong predictive potential Click here for additional data file.Figure S2In silico comparison of the performance of CIN4 vs. CIN25, CIN70, Ki67, genetic grade, HOXB13:IL17BR index, Genomic Grade Index, the 21-gene recurrence score and NKI70 in the GIS, JBI and JBI1 datasets  (A), and assessing the additive power of CIN4 (B).(PDF)Click here for additional data file.Figure S3Kaplan-Meier curves of Ki67 and mitotic index performances in the tissue samples of grade 2 breast tumors.(TIF)Click here for additional data file.Figure S4Individual prognostic performance of AURKA, FOXM1, TOP2A and TPX2 in breast cancer datasets as compared to CIN4 signature (groups split at median expression).(PDF)Click here for additional data file.Figure S5Correlation of CIN4 and markers used in the determination of immunophenotype. CIN4 and A) ER, B) PR, and C) HER2 expression, and D) HER2/chromosome 17 score determined by FISH.(TIF)Click here for additional data file.Figure S6CIN4 expression in ER-negative and ER-positive tumors.(TIFF)Click here for additional data file.Table S1The public datasets analysed in the study and their supplementary references.(XLS)Click here for additional data file.Table S2The probes used for the qPCR evaluation in the study.(XLS)Click here for additional data file.Table S3Expression of control genes in the breast cancer tissue samples according to grade.(XLS)Click here for additional data file.Table S4Performance of the individual components of CIN4 in tissue samples of grade 2 breast tumors.(XLS)Click here for additional data file.Table S5Correlation of the clinicopathological/prognostic variables with each other in grade 2 breast tumours only. Pearson correlation coefficients displayed, significant correlation: gray highlight.(XLS)Click here for additional data file."}
+{"text": "The precise timing of events in the brain has consequences for intracellular processes, synaptic plasticity, integration and network behaviour. Pyramidal neurons, the most widespread excitatory neuron of the neocortex have multiple spike initiation zones, which interact via dendritic and somatic spikes actively propagating in all directions within the dendritic tree. For these neurons, therefore, both the location and timing of synaptic inputs are critical. The time window for which the backpropagating action potential can influence dendritic spike generation has been extensively studied in layer 5 neocortical pyramidal neurons of rat somatosensory cortex. Here, we re-examine this coincidence detection window for pyramidal cell types across the rat somatosensory cortex in layers 2/3, 5 and 6. We find that the time-window for optimal interaction is widest and shifted in layer 5 pyramidal neurons relative to cells in layers 6 and 2/3. Inputs arriving at the same time and locations will therefore differentially affect spike-timing dependent processes in the different classes of pyramidal neurons. Timing is a central concept in cortical function. At the network level, information is encoded in the spiking of neurons and there is much debate about the level of precision that is important +, K+ and Ca2+ channels Recently it has become clear that the input/output function of pyramidal neurons is also profoundly influenced by the computational properties of the dendritic tree itself + channels followed by a slower Ca2+-dependent component nd component is particularly pronounced and typically drives the soma to fire a burst of APs nd component contributes to further somatic depolarization but does not necessarily trigger axonal firing. The dendritic and axonal spike initiation zones are coupled by the influence of the backpropagating action potential (bAP) that lowers the threshold for the initiation of the dendritic spike. This phenomenon, known as \u201cbackpropagation activated calcium spike firing\u201d (BAC firing) Neocortical pyramidal neurons have a spike initiation zone in the apical dendrite In this paper, we investigated the time window of coincidence detection in L2/3, L5 and L6 pyramidal neurons of the somatosensory cortex in rats using simultaneous dual patch-clamp recordings from the cell body and apical dendrite and we show that all three types of pyramidal neurons have a specific time window for somato-dendritic spike interaction.The study was approved by the Veterinary office of the Canton Bern, Switzerland, permission number 90/08.2PO4, 25 NaHCO3, 1 MgCl2, 2 CaCl2, and 25 glucose; pH 7.4. Parasagittal slices, 300 \u00b5m thick, were cut from the tissue block with a vibratome (Microm) and kept at 37\u00b0C for 30 min and then at room temperature until use.Experiments were performed in somatosensory neocortical slices from postnatal day 28\u201349 Wistar rats (n\u200a=\u200a26) using procedures described previously All experiments were performed at 32.0\u00b10.5\u00b0C. Single pyramidal neurons were identified using infrared Dodt gradient contrast or oblique illumination and a CCD camera . Slices were perfused with the same extracellular solution mentioned above. Recording pipettes were filled with intracellular solution containing the following: 130 mM K-gluconate, 5 mM KCl, 30 mM HEPES, 10 mM Phospho-kreatine, 4 mM MgATP, and 0.3 mM GTP; pH 7.3. The somatic pipette contained in addition 10\u201350 \u00b5M Alexa 594 (Invitrogen), 100 \u00b5M Oregon Green BAPTA-1 , and 0.2% Biocytin (Sigma). Dual whole-cell voltage recordings were performed from the soma and dendrites (6\u201310 and 20\u201340 M\u03a9 pipette resistances respectively) using Axoclamp 2A (Axon Instruments) and Dagan BVC-700A amplifiers (Dagan Corporation). Data were acquired with an ITC-18 board (Instrutech) and custom software written for the Igor environment (Wavemetrics). After recordings, slices were fixed and stained as described previously The dendritic recording was made at least 20 min after establishing the somatic recording to allow intracellular spread of the dyes from the soma. Dendrites were targeted with infrared-scanning gradient contrast (IR-SGC) All statistics were calculated using commercial software . If not otherwise indicated values represent means \u00b1 s.e.m. All data were tested for normality and equal variance. Statistical comparisons of spike thresholds were performed using 2-way repeated measurement ANOVA to test for effects of time versus baseline  or for tinj) was injected into the dendrite  & 2/3 in the somatosensory neocortex of rats , no significant effect of layers  but a significant effect of the interaction between layers and time . Post hoc test showed that the threshold reduction was significantly different for L5 pyramidal neurons compared to L6 and L2/3 for many time points, whereas L6 and L2/3 pyramidal neurons were only different from each other at one time point. The presence of an AP had the greatest effect on L5 pyramidal neurons reducing the threshold by 41\u00b17%. Furthermore, the coincidence detection time window for L5 was extended relative to L6 and L2/3 pyramidal neurons (To compare the time windows for somato-dendritic coupling between the different pyramidal cell classes we normalized the values at the different \u0394t's to the threshold for generating a dendritic spike without an axonal AP . 2-way r neurons .In summary, we found that the coincidence timing curve for the initiation of dendritic spikes in L5 pyramidal neurons was wider than for L6 and for L2/3 pyramidal neurons. L6 and L2/3 pyramidal neurons exhibited similar coincidence detection windows to each other but were narrower than in L5 cells implying these cells require more precise synaptic inputs for this effect. The bAP had the greatest relative effect on dendritic spike generation in L5 neurons however the baseline threshold in L5 neurons was much larger than in L2/3 and L6 neurons . Thus, tWhat are the implications of timing differences between pyramidal cell classes? We predict that processes in the dendritic tree which are influenced by the coupling of bAPs with local dendritic membrane potential such as STDP The active and passive properties of L6, L2/3 and L5 pyramidal tuft dendrites are similar but not identical The functional consequence of coincidence detection in pyramidal neurons depends also on the particular inputs that are associated. The cortical layer of the cell bodies and basal dendrites of pyramidal neurons determines the proximal input In conclusion, we have shown that all pyramidal neurons of the rat somatosensory cortex can associate inputs arriving at their distal and proximal dendritic trees in a limited time window that varies between cell classes. This suggests that pyramidal neurons operate in a similar way on the input which reaches the different cortical layers they are covering."}
+{"text": "Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects.Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Ca The T type Cav3.1 and L type Cav1.3 channels are expressed in the sinus node cells and modulate pacemaker activityCalcium ions play a critical role in muscle function. Voltage-gated calcium channels (VGCCs) govern the depolarization induced Ca1 subunit is the basic structure of the channel, while the \u03b2, \u03b12\u03b4 and/or \u03b3 subunits interact with the \u03b11 subunit and play a modulatory role. Calcium channel blockers (CCBs) are widely used in clinical practice to treat cardiovascular disorders from hypertension to angina pectoris, arrhythmia, Raynaud syndrome, and cerebral vasospasm, etc. The basic effect of CCBs is to inhibit VGCCs by binding to the pore forming \u03b11 subunit and the Cav1.2 channel is the major target of CCBs.VGCCs are composed of multiple subunits. The pore forming \u03b11 subunit of Cav1.2 channelThree classes of small molecule CCBs are currently in clinical use: 1,4-dihydropyridines (DHPs), phenylalkylamines (PAAs), and benzothiazepines (BTZs). They all bind to the \u03b1v1.2 channel were identified to link with antihypertensive outcomeGenetic factors determine drug response taking into consideration many other factors such as age, sex, body weight, and heath status. Pharmacogenomics provides information on the linkage of genetic factors to drug responses and may also provide the basis for the use of safer and more efficient medications to patients. In hypertension, genetic associations with antihypertensive response have been established for diuretics, beta-blockers, ACE inhibitors and angiotensin1 receptor blockers. However, most of the information is lacking in calcium channel blockers. Recently, three single nucleotide polymorphisms (SNPs) of Ca1 subunit of Cav1.2 channelv1.2 channels under physiological and pathophysiological conditions and the influence of such changes on pharmacology. The proteomic structure of Cav1.2 channels could change under pathological conditions due to alternative splicing. The way we view individualized medicine in treating cardiovascular diseases may need to be expanded beyond pharmacogenomics.Alternative splicing is a post-transcriptional modification process. Multiple functional variants could be generated from a single gene. Recently, a large number of alternatively spliced exons have been identified within the pore-forming \u03b1CACNA1C, codes for the \u03b11 subunit and contains 55 exons. At least 19 exons are subjected to alternative splicingThe human Cav1.2 gene, The binding site for CCBs is mainly composed of the transmembrane segments 5 and 6 (S5 and S6) of domains I to IV. By using photoaffinity labeling, antibody mapping, and chimeric study, DHPs were found to bind IIIS5, IIIS6 and IVS6 segments, while IIIS6 and IVS6 are the binding sites for PAAs and DTZs2+ influx through Ca2+ channels in cardiac and vascular smooth muscles. However, there exist variable responses to blockade of Cav1.2 channels by CCBs within the two tissues. For example, vascular smooth muscles are more sensitive to DHPs than cardiac muscles. One obvious reason is that calcium channels in smooth muscle possess a higher binding affinity than in cardiac musclev1.2 channels are locked in an inactivated state which favors the DHP blockThe pharmacological effect of CCBs depends on their inhibition of Cav1.2 channel is generally divided into a cardiac isoform (Cav1.2a) and a smooth muscle isoform (Cav1.2b). Cav1.2a channel is the predominant channel in heart while Cav1.2b channel in smooth muscles. Cav1.2a channel contains the combination of exons 1a/8a/-9*/32/33v1.2b) contained exons 1b/8/9*/32/33Cav1.2b channel is more sensitive to DHP block than Cav1.2a channel which is similar to the observations in native heart and blood vesselsThe Cav1.2 channel activity is also regulated by phosphorylationCav1.2 channels in heart and blood vessels, numerous splice variants are found to be expressed in cardiovascular systemv1.2 channels in arterial smooth muscles relates with the left shifted window currents recorded in native smooth musclesAlthough there exist predominant Cav1.2 channels are crucial for cardiovascular functions as deletion of the gene in mouse leads to embryonic lethalityv1.2 channels was linked to many diseasesv1.2 gene was reported in Timothy syndrome, a disorder characterized by dysfunction in multiple organ systems, including heart, skin, eyes, teeth, immune system and brain2+ ions will result in the lengthening of action potential, leading to cardiac arrhythmia and sudden death. The levels of expression of exon 8 and 8a is different in various organs and tissues and thus the location of the mutations in exon 8 or 8a would determine the severity of the symptoms and the involvement of other organs. CCBs are ideal to treat the patients by reducing the Ca2+ influx from mutant channels.Cav1.2 channels has identified to be altered in cardiovascular disorders. Mutually exclusive exons 31 and 32 are developmentally regulatedet alet alAlternative splicing of Cav1.2 channels highlights a novel way towards individualized medication. Besides SNPs, post transcriptional modification produces Cav1.2 channels with huge variability both in structure and function. Each person could express slightly different splice variants in different tissues. But the functional impact could be enormous. Furthermore, under pathological conditions, the splice patterns can be altered. Such alteration could be variable at different stages of the disease. Thus, each patient could express a signature pattern of Cav1.2 channels generated by alternative splicing. This provides possible targets for individualized medication. However, many questions need to be addressed first and chief of which is how the splicing profile from different organs of a patients can be achieved. The nature of alternative splicing makes it impossible to get such information simply from blood. Also the length of the gene and multiple splicing sites makes it difficult to determine combinatorial profiles for the expression of the many alternatively spliced exons in the full length Cav1.2 channel transcripts. The next obstacle is to select suitable splice variants as targets for drug discovery and development. Most of the current CCBs in use are not designed against one splice variant without affecting others. The understanding of alternative splicing of Cav1.2 channels is far from complete. One example is the hemichannels generated by misspliced exonsv1.2 channels remains mostly unclear.The progress in the study of alternative splicing of Cav1.2 channels to cardiovascular pharmacology and pathophysiology. However, the knowledge in other organs and systems are mostly lacking. For example, the splicing pattern in nervous system is not well studied. Considering the higher expression of Cav1.2 channels in neurons, CCBs in treating nervous system disorders could attract more attention if neuronal specific CCB is discovered one day in the future. In conclusion, we presented another consideration for the development or discovery of drugs against Cav1.2 channels that may be efficacious in the management of cardiovascular disorder.In this review, we discussed the progress in relating alternative splicing of Ca"}
+{"text": "PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients\u2019 muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.Mutations in  PLA2G6 mutations (PLAN) constitutes a heterogeneous group of clinical entities which encompasses infantile neuroaxonal dystrophy , atypical neuroaxonal dystrophy (NAD), idiopathic neurodegeneration with brain iron accumulation including Karak syndrome  ), following a conventional protocol  using suOphthalmologic examinations were occasionally limited because many of the patients were young and because of the cognitive complications of the neurodegenerative disorder. The assessment of optic disk pallor was done using indirect ophthalmoscopy. All patients had visual evoked responses (VEPs) using flashing lights and P100 was the most important wave to identify. All patients had brain computer tomography (CT) and/or magnetic resonance imaging (MRI) [Siemens 1.5 Tesla].vastus lateralis) was undertaken in 4 patients and examined by standard histological techniques. Two glutaraldehyde-fixed muscle specimens were available for electron microscopy (EM) examination and processed as described by Weis et al. ), and malnutrition (requiring feeding through gastrostomy tube) and bed sores in another girl aged 9.8 years (F1 [P1]). Two patients died at ages of 12.8 years (F3 [P1]) and 28 years (F6 [P1]). The causes of death were, respectively, respiratory infection due to H1N1 swine flu and septicaemia following bed sores.Physical examination  revealedAbsent or delayed evoked potentials (VEP) were seen in 5/11 (45%) patients . In the Distal axonal-type sensorimotor neuropathy was evident in 9 (90%) patients who had nerve conduction studies . The compound muscle action potentials (CMAPs) morphology was diphasic and the peak amplitudes were recorded. With the exception of one (F5 [P1]), all patients had either unobtainable or low amplitude CMAPs for the peroneal nerve, recorded from the extensor digitorum brevis (EDB) muscle; while in the upper limbs 3/9 patients (33%) showed normal CMAP amplitudes. The median sensory nerve action potentials (SNAPs) recorded in 9 patients were normal in 7 (77%) and reduced in one (F6 [P2]), while sural SNAPs were within the normal range in 4/10 (40%) patients.Brain imaging  was undeglobus pallidus, seen as reduced signal on T2, FLAIR and/or diffusion weighted image (DWI) sequence in the initial scans , 3.1 years (F1 [P1]) and 3.3 years (F2 [P1]), respectively . His affected brother (F6 [P3]) showed clear features of brain iron deposition on MRI done at a younger age  whereas cerebellar atrophy was shown in all patients.Eight (80%) of the 10 children who had MRI scans showed evidence of increased iron deposition in the al scans  and 4. Oal scans  did not ectively . Repeateectively , showed substantia nigra was revealed by the initial MRI in 6 of 10 (60%) patients at a median age of 10.3 years . Three of the patients with initial normal substantia nigra signal had their MRI at 1.3 year (F2 [P2]), 2.8 years (F4 [P1]), and 3.3 years (F2 [P1]) but no subsequent scans. The fourth patient (F5 [P1]), had his initial MRI at the age of 4.2 years, but showed substantia nigra iron deposition when imaging was repeated 3 years 4 months later (Increased iron in the hs later . With imhs later  and 4.c oxidoreductase (complex II + coenzyme Q + complex III) of 4.0 mU/mg (control range = 8.2-44), and decylubiquinol: cyt c oxidoreductase (complex III) of 41 mU/mg (control range = 54-434). Citrate synthase activity was at the lowest reference value . By contrast, the ratios between enzyme activity of the respiratory chain and citrate synthase were not remarkable. The relatively low activity of all enzymes on protein base, combined with the normal enzyme activity ratio\u2019s, lead us to conclude that there is a relatively low mitochondrial content in the muscle of patient [F6 (P1)], rather than a deficiency of one or more respiratory chain enzymes. To confirm these results we determined the density of the intermyofibrillar mitochondria in the patient\u2019s [F6 (P1)] biopsy and compared it to an age and gender matched healthy individual\u2019s muscle specimen taken from the same location  using electron microscopy. We found that the patient\u2019s muscle showed a significant decrease in the mitochondrial density (0.255 +/- 0.05/\u00b5m2) in comparison to the control (0.379 +/- 0.06/\u00b5m2) (p=0.0001).Histological examination of muscle biopsies from 4 patients revealed non-specific neurogenic features. In contrast, electron microscopy  revealedElectron microscopic analysis of the muscle biopsy (taken at the age of 3 years) of patient F1 (P1)  with INAPANK2 gene for 5 families while all the affected members of these were homozygous for the microsatellite markers flanking the PLA2G6 locus . All affected individuals were homozygous for the identified mutation while parents, for whom the DNA was available, were heterozygotes for the mutations and unaffected siblings were either heterozygotes or homozygous for the wild type allele  the age at onset of the disease manifesting as ataxia, and (ii) the evolution of the disease at the functional level. We expressed the results as a graph with exponential trend curves. A curve of the evolution of the age at onset of ataxia and a second one showing evolution of the age at which patients reach functional Stage 7 (wheelchair bound) were formulated. The disease duration was also considered in the graph . We coulPLA2G6 mutations.The present cohort represents one of the largest collections of patients from one country and the same ethnic background (Arabs) with neurodegeneration associated with In the present study, 6 children   had claslocus caeruleus in the disease, which has been documented pathologically in patients with PLA2G6 mutations ) developed complex partial seizure at 21 years of age, which was confirmed by EEG. Another 2 siblings with INAD had brief tonic seizures with normal EEG in one and non-specific changes in another. Fast rhythms on EEG were observed in 2 patients with classic INAD without clinical seizures . These fOptic atrophy occurs in the majority of children with infantile and atypical NAD whereas nystagmus and strabismus are also common -6,28,29.Neurophysiologic studies revealed features of distal axonal neuropathy in all 4 patients with Karak syndrome phenotype. Also 3 of 5 (60%) children with classic INAD had features of axonal neuropathy on NCS. This is similar to the findings in other cohort of patients with INAD [PLA2G6 in mice (iPLA2b-/-) was documented to lead to the development of cerebellar atrophy by the age of 13 months [corpus callosum thinning) was evident in 2 patients (F1 [P1] and F5 [P1]) and borderline changes were detected initially in one of them (F5 [P1]). Similarly, one of 5 reported patients ) and at 4.2 years for another (F1 [P1]). The latter child had an earlier MRI at 3.1 years which revealed globus pallidus hypointensity on DWI only  showed a relatively low mitochondrial content in the muscle. This is compatible with the observed ultrastructural abnormalities seen by electron microscopy, and is also in agreement with the proposed role of PLA2G6 in protecting the mitochondrial membrane from peroxidation  with classic INAD and F6 [P1] with Karak syndrome phenotype). This membranous material is probably derived from the sarcoplasmic reticulum/myotubular system and could have resulted from the disrupted membrane homeostasis following disruption of iPLAodelling . To the xidation .This is the first time that such a genotype phenotype correlation was highlighted in a cohort of PLAN patients, even in studies which included a group of patients belonging to the same ethnic background. Indeed, in the reported series , there wPLA2G6 gene mutations, five of which are novel. We have shown that the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients who belong to the same ethnic background but, in terms of functional disability, was influenced by the genotype. Nevertheless, cerebellar atrophy is the constant and earliest feature of the disease, and precedes brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. It is noteworthy that variable combinations of sites of degeneration and associated symptoms are often observed in childhood-onset recessive ataxias including epilepsy and abnormal cognition [PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of brain iron accumulation. Considering the age at onset and the functional disability, there is an evidence of genotype-phenotype correlation. However, the wide intra- and inter-familial variability of the disease in physiological, psychiatric and other clinical aspects, cannot be explained only by single homozygous mutations in the PLA2G6 gene. At least, other environmental and/or genetic factors  might probably modulate the disease presentation.In conclusion, we describe the phenotypic and genetic spectrum of 11 patients followed for a maximum period of 17 years and report six underlying ognition -36. ElecVideo S1Patient F2 (P2) with INAD, aged 2.4 years, showing tetraparesis and brisk reflexes (including adductor reflex) despite absence of leg stiffness (note the frog position of the lower limbs).(WMA)Click here for additional data file."}
+{"text": "TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function. TRPM6 and -7 the first molecularly defined components of the mammalian Mg transport machinery. TRPM6 and -7 show the unique functional duality of being an ion channel and a kinase. TRPM7, which is ubiquitously expressed, was initially thought to play a prominent role in intracellular Mg homeostasis, whereas TRPM6 controls systemic Mg homeostasis by regulating Mg transport in the kidney and in the gut Magnesium (Mg), the second most abundant intracellular cation, plays a major role in regulating endothelial function TRPM7 mimics the effects of Mg deficiency in these cells The presence of functional TRPM7 channels in human endothelial cells has been demonstrated TRPM7 stimulate cell proliferation TRPM7 induces cell cycle arrest. In addition to the significant elevation of TRPM7 in the vasculature of MgL mice TRPM7 transcript in HUVEC exposed to shear stress has been described In endothelial cells derived from the umbilical vein (HUVEC), two independent reports have shown that siRNAs transiently silencing To this purpose, it is noteworthy that different types of endothelial cells including HUVEC have very low levels of TRPM7 current which shows no significant increase in response to fluid flow Although scarce, the data reported until now point to a potential regulatory role for TRPM7 in the maintenance of vascular integrity Because increasing evidence suggests that TRPM7 might contribute to the pathophysiology of the vasculature in general and of the endothelium in particular, we explored the modulation of the expression of TRPM7 in human endothelial cells and the effects of its inhibition on some aspects of endothelial function.4in vitro, HUVEC were used at low population doublings . PD were calculated as log2 (number of cells at time of subculture/number of cells plated). We define senescent cells as the culture that do not increase the cell number and remain subconfluent for 2 weeks. We confirmed the senescent phenotype with senescence-associated-beta galactosidase activity assay as described Primary HUVEC isolated from the umbilical vein (American Type Culture Collection) were cultured in M199 containing 10% fetal bovine serum (FBS), 1 mM glutamine, 1 mM penicillin and streptomycin, Endothelial Cell Growth Factor (150 \u00b5g/ml), 1 mM sodium pyruvate and heparin (5 units/ml) on 2% gelatin-coated dishes 2O2 (100 \u00b5M) for 30 min and then part of the samples immediately lyzed while the remaining was maintained in culture for 24 additional h before lysis. In other experiments HUVEC were treated with trolox (10 \u00b5M) In some experiments HUVEC were treated with HTRPM7, we utilized the pGIPZ shRNAmir (0.2 \u00b5g/cm2) (Open Biosystems). The construct was transfected into 3\u00d7105 HUVEC using Arrest-in Transfection Reagent (Open Biosystems). Transfection with Non-Silencing (scrambled) pGIPZ shRNAmir was used as control.To silence For proliferation assays, the cells were seeded at low density in growth medium. At the end of the experiments, the cells were trypsinized, stained with a trypan blue solution (0.4%) and the viable cells were counted using a Burker chamber. In some experiments the cells were treated with 2-aminoethoxydiphenyl borate  or Co(III)-hexaammine (250 \u00b5M) (Sigma Aldrich).in vitro model of wound repair as previously described Migration was determined using an TRPM7 amplification cycle consisted of 1 min at 95\u00b0C, 45 sec at 54\u00b0C and 1 min at 72\u00b0C using 400 nmol/l of each primer in a final volume of 25 \u00b5l. The reaction was stopped after 35 cycles. One fifth of the reaction mix was separated on a 2% agarose gel. The sequences of the TRPM7 primers are the following: sense 5\u2032-CTTATGAAGAGGCAGGTCATGG-3\u2032 and antisense 5\u2032-CATCTTGTCTGAAGGACTG-3\u2032 (size of the amplified fragment: 213 bp). RT-PCR with specific primers for actin was performed to normalize .Cells were lyzed in 1 ml Trizol and RNA was purified. RNA was quantified using Nanodrop ND-1000 spectrophotometer and electrophorezed on a 1% agarose gel containing 2.2 M formaldehyde before reverse transcription. Using the Transcriptor first-strand cDNA synthesis kit (Roche Diagnostics), cDNA was synthesized from 2 \u00b5g of total RNA using oligo-dT and 5 units of reverse transcriptase at 50\u00b0C for 60 min, followed by heating to 85\u00b0C for 5 min. PCR amplification was carried out using 1/50 of the final RT reaction. Each HUVEC were lyzed in lysis buffer . Protein concentration was determined using the Bradford protein assay (Bio-Rad). Cell extracts (100 \u00b5g/lane) were resolved by 8% SDS-PAGE, transferred to nitrocellulose sheets at 100 mA for 16 h, and probed with anti-TRPM7 (Chemicon) and anti-actin (Sigma Aldrich) antibodies. Secondary antibodies were labelled with horseradish peroxidase . The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins. All the results shown were reproduced at least three times and a representative Western blot is shown. Densitometry was performed by the ImageJ software on 3\u20135 different blots.2 solutions. The experiments were performed in triplicate and repeated 5 times with similar results. Data are shown as the mean \u00b1 standard deviation.NOS activity was measured in the conditioned media by the Griess method To evaluate whether the expression of TRPM7 is related to cell growth, we compared proliferating vs quiescent HUVEC for their amounts of TRPM7. On culture plates, HUVEC grow until they form a perfect monolayer. At this stage, cells stop growing and become quiescent. This pattern can be shown by the arrest of thymidine incorporation 3H-Thymidine incorporation assay 10 h after seeding the cells, half of the samples were growth arrested by exposure to a starvation medium. After 48 h, the cells were lyzed and Western blot performed. No significant modulation of the total amounts of TRPM7 was observed in cells rendered quiescent by starvation vs proliferating cells . Similarin vitro passage when compared to early passage, young cells , an inhibitor of the proteasome, bafilomycin (100 nM), which inhibits lysosomal activity, or calpeptin (5 and 10 \u00b5g/ml), a specific m- and \u03bc-calpain inhibitor TRPM7 expression is increased by oxidative stress in monocytes and PC12 cells 2O2 (100 \u00b5M), from which hydroxyl radicals are produced by the Fenton reaction, rapidly and stably increased TRPM7 levels after 30 min and up to 24 h. In addition, the antioxidant trolox (10 \u00b5M) prevented TRPM7 accumulation in HUVEC cultured in 0.1 mM Mg containing medium (We then tried to understand the mechanisms underlying the increase of TRPM7 levels in HUVEC cultured in low extracellular Mg. Since i) low Mg promotes oxidative stress in endothelial cells g medium .To determine whether TRPM7 contributes to the proliferation of endothelial cells, we transiently transfected HUVEC with a specific shRNA or with a non-silencing shRNA sequence as a control. TRPM7 levels were dramatically reduced 24 and 48 h after transfection and increased thereafter . We deteTRPM7 as well as treatment with 2-APB (50 \u00b5M) and Co(III)hexaammine (250 \u00b5M) induced HUVEC migration  is a vasoprotective factor with anti-atherogenic properties ilencing .TRPM7 is an important regulator of Mg homeostasis and adequate concentrations of Mg contribute to maintain normal endothelial functions TRPM7 expression is enhanced by oxidative stress in monocytes In general, the expression of ion channels is rather stable, being conductivity regulated by gating mechanisms linked to signalling cascades. Indeed, in vascular smooth muscle cells exposed to fluid flow, TRPM7 expression does not change and the increase in TRPM7 current is due to its translocation to the plasma membrane In HUVEC cultured in high Mg, the levels of TRPM7 decrease. We hypothesize that this downregulation might protect the cells against Mg overload which would negatively influence TRPM7 channel activity. Indeed, as intracellular Mg increases, TRPM7 activity declines. In addition, too much intracellular Mg would generate imbalances of ion concentrations, in particular interfering with calcium homeostasis Last issue to consider is the role of TRPM7 in regulating HUVEC function.TRPM7 and inhibiting its activity with 2-APB or Co(III)hexaammine. The fact that both pharmacological and genetic inhibition of TRPM7 have no effect on NO production is relevant, since our results with shRNA against TRPM7 are in disagreement with a previous report showing that silencing TRPM7 stimulates NO production in an ERK pathway dependent fashion An adequate production of NO is a marker of endothelial function to the point that in clinical practise the examination of vasodilatation in response to stimuli that release NO is routinely employed to assess endothelial function TRPM7 silencing. In particular, it is worth to note that human microvascular endothelial cells are arrested in the G1 phase of the cell cycle upon silencing TRPM7TRPM7 silencing significantly impairs HMEC motility Both genetic and pharmacological inhibition of TRPM7 induce HUVEC proliferation. To our knowledge, HUVEC are unique in responding with an increase of cell growth to The evidence that TRPM7 inhibition impacts on endothelial cell migration and proliferation might have pathophysiologic significance. Indeed, low levels of TRPM7 might facilitate the re-endothelization of vascular injuries, thus preventing excessive subintimal proliferation of smooth muscle cells and reducing the risk of vascular complications. If this theory proves true, maintaining low levels of TRPM7 might contribute to vascular integrity and, in case of damage, to vascular repair."}
+{"text": "Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of \u03b2Klotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly,  In mammals the fibroblast growth factors (FGFs) play diverse roles in the regulation of many cellular processes ranging from development to survival Previous publications have demonstrated that FGF15/19 and FGF21 bind to the \u03b2Klotho (KLB) isoform of the Klotho family while FGF23 has distinct affinity for the \u03b1Klotho (KL) subtype FGF23 plays a well described role in phosphate metabolism and has not previously been shown to affect energy balance We demonstrate here that on both a molecular and whole organism level there are many similarities in the action of FGF19 and FGF21. While FGF21 showed no direct FGFR binding, FGF19 was able to bind FGFR4 independent of KLB. In functional studies we show in 3T3-L1 fibroblasts expressing KLB, both FGF19 and FGF21 were able to stimulate glucose uptake with similar pharmacodynamic properties. When 3T3-L1 adipocytes were treated with a combination of both FGF19 and FGF21 we saw no additive or synergistic effect. Furthermore, treatment with an inhibitory truncated form of FGF21 (termed \u0394N17) For both in vitro and in vivo studies FGF19, FGF21 and \u0394N17 were generated as previously described All animals were individually housed in a temperature-controlled (24\u00b0C) facility with 12 h/12 h light/dark cycle. Animal protocols in this study were approved by the Eli Lilly and Co. Animal Use and Care Committee (Protocol No. 09012).Male C57Bl/6J mice (n\u200a=\u200a6 per group) (Taconic Farms) were maintained on a calorie-rich diet consisting of 40% fat, 39% carbohydrate, and 21% protein caloric content  and had free access to food and water before randomization by weight. Mice were administered either FGF19 or FGF21 for a period of 7 days via continuous infusion using osmotic minipumps  at the doses specified. Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .Male ob/ob mice (n\u200a=\u200a6 per group) (Taconic Farms) were maintained on a standard chow diet  and had free access to food and water before randomization by weight. Mice were administered either FGF19 (1 mg/kg/day) or FGF21 (1 mg/kg/day) for a period of 7 days via continuous infusion using osmotic minipumps . Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .Male ob/ob mice (n\u200a=\u200a6 per group)  were maintained on a standard chow diet  and had free access to food and water before randomization by weight. Mice were administered with either FGF21, \u0394N17 or a combination of both via daily injection at doses indicated for a period of 3 days after which serum was collected for analysis. Prior to sacrifice and blood collection on day 3 the fasted cohorts were deprived of food overnight. Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .On day 1 of the study, an osmotic minipump  containing 5-bromo-2-deoxyuridine  was implanted subcutaneously into each 9-week-old male C57bl/6J mouse . Each mouse was given daily subcutaneous injections of either phosphate-buffered saline , FGF19 (2 mg/kg/day) or FGF21 (2 mg/kg/day) for 7 consecutive days. At the end of the 7-day study samples of liver were collected from each mouse, placed in 10% neutral-buffered formalin, processed routinely, and embedded in paraffin. Multiple tissue sections were produced from each paraffin block, stained with Hematoxylin & Eosin (H&E), or immunolabeled for BRDU by routine immunohistochemical methods as outlined below. The H&E tissue sections were evaluated routinely for microscopic changes Cellular incorporation of BrdU was detected by digesting deparaffinized tissue sections with 0.1% protease (Sigma Aldrich) and treating the sections with 2N hydrochloric acid. Sections were blocked with CAS BLOCK , incubated with a rat antibody to BrdU , and bound rat antibody was detected with biotinylated rabbit antibody to rat IgG . Tissue sections were quenched with Peroxidase Blocking Solution  and retained biotin was detected with Vectastain Elite ABC kit (Vector Laboratories). Reaction sites were visualized with DAB Substrate-Chromagen System  followed by DAB enhancer . Sections were counterstained with hematoxylin.BiaCore studies were performed on a BiaCore 2000 instrument . Proteins were covalently immobilized on censor chip CM4 using amine coupling according to the manufacturer's protocol. Typically, 100\u201350 response units (RU) were immobilized on individual flow cells of the sensor chip. BSA  was immobilized on flow cell 1 as a negative control. Proteins suspended in HBS-P  were then injected for 30 min at a flow rate of 30 ml/min using the kinject command. KD kinetic constants were calculated by BiaEvaluation 4.1 software using a 1\u22361 Langmuir model.Cells were treated as indicated for 3 h and glucose uptake was assayed as previously described Cells were treated as indicated for 5 min and subsequently lysed. Total ERK phosphorylation was assessed using an AlphaScreen SureFire Phospho-ERK1/2 Assay Kit (Perkin Elmer) according to the manufacturer's instructions and an EnVision Multilabel Microplate Reader Model 2103 (Perkin Elmer) with the AlphaScreen HTS Turbo option was used for signal detection.RNA was isolated from tissues using TRIzol reagent  or by homogenization of frozen samples in Lysing Matrix D shaker tubes  and was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit . Reactions were performed in triplicate on an ABI Prism 7900HT (PE Applied Biosystems) and were normalized to either 36B4 mRNA or 18S rRNA. ssays-on-Demand Gene Expression Products (PE Applied Biosystems) were as follows: hEGR1, Hs00152928_m1; hFGFR1, Hs00915142_m1; hFGFR2, Hs01552926_m1; hFGFR3, Hs00179829_m1; hFGFR4, Hs01106908_m1; hKL, Hs00183100_m1; hKLB, Hs00545621_m1; mFGFR1, Mm00438930_m1; mFGFR2, Mm01269930_m1; mFGFR3, Mm00433294_m1; mFGFR4, Mm01341852_m1; mKL, Mm00473122_m1; mKLB, Mm00502002_m1; rFGFR1, Rn00577234_m1, rFGFR2, Rn01269940_m1; rFGFR3, Rn00584799_m1; rFGFR4, Rn01441815_m1; rKL, Rn00580123_m1.Data are presented as mean \u00b1SEM. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test where appropriate. Differences were considered significant when P\u200a=\u200a<0.05.Prior to testing FGF19 and FGF21 for activity in cell based assays we measured expression of FGF receptors and Klotho subtypes in the cell lines we used via RT-qPCR. We found that the expression of FGFR isoforms and the Klotho co-factors differed greatly between the lines. In 3T3-L1 fibroblasts we saw high levels of FGFR1 in addition to lower expression of FGFR2 and only traces of FGFR3 with no detectable FGFR4, KL or KLB . In Hep3In order to assess the specificity and functional significance of the interaction between FGF19, FGF21, FGF23 and the Klotho family we conducted studies in which we expressed either KL or KLB in 3T3-L1 fibroblasts. We chose 3T3-L1 fibroblasts as neither KL or KLB is natively present in these cells To determine if differentiation from fibroblast to adipocyte alters the response of 3T3-L1 cells to the hormone-like FGFs we treated 3T3-L1 adipocytes with either FGF19 or FGF21 and assessed their effects on ERK phosphorylation and glucose uptake. The expression of FGFRs in these cells is very similar to that which we showed for 3T3-L1 fibroblasts As both 3T3-L1 and Hep3B cells express appreciable amounts of FGFRs we turned to L6 cells to confirm our findings as these cells have been reported previously to have vanishingly low expression of both FGFRs and KLs in vitro as a competitive antagonist and leads to inhibition of FGF21 mediated signaling by binding to KLB and blocking FGF21 mediated receptor activation We have previously demonstrated that N terminally truncated FGF21 (\u0394N17) acts To determine if the inhibition of FGF19/21 signaling we see in vitro translates to effects on metabolic parameters in vivo we examined fed and fasted glucose levels in ob/ob mice treated with FGF21, \u0394N17 or a combination of both. In fasted animals FGF21 reduced glucose, an effect blocked by combination with \u0394N17. Interestingly, we found that in fasted animals, treatment with \u0394N17 partially blocked the reduction in serum glucose one normally observes in fasted animals, suggesting \u0394N17 may interfere endogenous glucose homeostasis in the fasted state . FurtherFGF19 has previously been reported to induce mitogenicity in animals Our group and others have previously reported on the efficacy of FGF21 in the treatment of obesity in animal models In an effort to compare efficacy of the factors in another model of obesity we examined the effects of chronic FGF19 and FGF21 infusion in ob/ob mice. Interestingly ob/ob mice seem to display a differential response to FGF treatment when compared to WT mice. In both the FGF19 and FGF21 treatment groups there was a significant attenuation of body mass accrual over the 7 day treatment period, furthermore, the magnitude of the effect was greater in FGF21 treated animals when compared to FGF19 treatment . FGF19 tThe therapeutic potential of both FGF19 and FGF21 in the treatment of metabolic disorders has been discussed extensively in the literature Our group and others have previously reported that the metabolic activity of FGF21 or lack of thereof is determined by the presence or absence of the cofactor KLB In 3T3-L1/KLB fibroblasts we saw FGF19 and FGF21 mediated signaling and glucose uptake with FGF21 more potent than FGF19. We did not see any effect of FGF23 in the 3T3-L1/KLB cells consistent with previous data showing specificity for KL alone In cells which predominantly express FGFR4 but do not express KLB, FGF19 was active but FGF21 was not. In the presence of KLB, both FGF19 and FGF21 can signal via FGFR4 but FGF19 appears to be significantly more potent than FGF21. This was evident in 3T3-L1 fibroblasts stably expressing FGFR4 in which FGF19 was able to act directly in the absence of KLB and induce glucose uptake, while FGF21 did not have any activity. This finding is important as while previous reports have shown binding of FGF19 to FGFR4 On a side note, while L6 cells have previously been reported to be free of background signaling due to a paucity of FGFR expression We found no synergistic or additive effect on glucose uptake when cells were treated with both FGF19 and FGF21 simultaneously. This indicates that these two factors share a common signaling pathway via which they regulate glucose transport in cell culture.in vivo \u0394N17 is also able to block the glucose lowering action of exogenous FGF21 in both fed and fasted mice. In both fed and fasted ob/ob mice treated with FGF21 we see the usual glucose lowering effect we have reported previously This commonality of the two factors extended to our studies of inhibition using the competitive antagonist \u0394N17 which we have previously shown is effective in inhibiting FGF21 at the receptor activation level in vitro. In the present study we observed that not only does \u0394N17 inhibit downstream FGF21 signaling but also shows a similar efficacy in blocking FGF19 mediated effects. These data support the hypothesis that in cell culture models FGF21 and FGF19 operate by activation of a similar signaling cascade. Furthermore, we go on to demonstrate that in vivo administration of dN17 alone affected plasma glucose but only in the fasted state. Given the KLB antagonistic nature of \u0394N17s mode of action, and the absence of effects on glucose homeostasis in a fed mice treated with the protein, we hypothesize that even though a substantial amount of FGF21 is detected in plasma of fed ob/ob mice, it is likely present in a non-functional form which is unable to interact with endogenous KLB in the manner described previously It is also important to note that As several previous studies have noted mitogenic effects in animal models following treatment with FGF19 and absence of thereof with FGF21, we examined both FGF19 and FGF21 in an in vivo setting. In our hands FGF19 dosing led to a very significant increase in proliferation in the liver while FGF21 had no effect. Our data support earlier work suggesting FGFR4 binding by FGF19 may mediate its mitogenic effects Supporting this hypothesis is the finding that treatment with FGF19 improves glucose tolerance in DIO FGFR4KO mice suggesting activation of FGFR4 is not required in the mediation of at least some of the metabolic effects of this factor To date direct comparisons of FGF19 and FGF21 treatment in animal models have not been conducted. Here we show in DIO mice both FGF19 and FGF21 have beneficial effects in the treatment of metabolic dysregulation. It has been previously demonstrated that in DIO models ranging from rodents to primates that FGF21 treatment is able to correct the abnormal metabolic parameters evoked by prolonged high fat diet feeding Here we show that the metabolic effects of treatment with either FGF19 or FGF21 are almost identical. The main difference noted was increased potency of FGF21 when compared to FGF19 in terms of its effect on weight loss. Other effects such as the glucose lowering component of their action were indistinguishable, supporting the hypothesis of a shared mechanism of action.In conclusion, our study demonstrates that the effects of FGF19 and FGF21 both in vitro and in vivo show a high degree of similarity. This interchangeability between the factors likely results from the ability of both to bind KLB and FGFRs. In mice, treatment with FGF19 and FGF21 both led to amelioration of the obese phenotype with significant improvements in all parameters tested. Our data demonstrate that both in vitro and in vivo FGF19 and FGF21 are able to potently activate the KLB/FGFR complex and that this activation likely mediates the positive metabolic outcomes we observe. Our data lend further support for further investigation of both FGF21 and FGF19 as potential therapies for obese/diabetic humans."}
+{"text": "LIM domain-binding factor 2 (LDB2) gene in this region had the strongest association with body weight for weeks 7\u201312 and with average daily gain for weeks 6\u201312. This GGA4 region was previously reported to contain body weight QTL. GGA1 and GGA18 had three SNP effects on body weight with genome-wide significance. Some of the SNP effects with the significance of \u201csuggestive linkage\u201d overlapped with previously reported results.Chicken body weight is an economically important trait and great genetic progress has been accomplished in genetic selective for body weight. To identify genes and chromosome regions associated with body weight, we performed a genome-wide association study using the chicken 60 k SNP panel in a chicken F2 resource population derived from the cross between Silky Fowl and White Plymouth Rock. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance. A chicken chromosome 4 (GGA4) region approximately 8.6 Mb in length (71.6\u201380.2 Mb) had a large number of significant SNP effects for late growth during weeks 7\u201312. The  Body weight is an economically important trait for broiler chickens. The identification of DNA polymorphisms and causative genes affecting body weight provides necessary molecular information for marker assisted selection and gene based selection to improve quantitative traits Blood samples of chickens were collected from the brachial vein by standard venipuncture procedure #XK622, approved by the Animal Welfare Committee of China Agricultural University.The study population was the China Agricultural University chicken F2 resource population that was produced from reciprocal crosses of Silky Fowl and White Plymouth Rock which consisted of four half-sibling pedigrees. In this study, 278 individuals of three generations were included. Body weights of the 229 F2 animals were measured weekly from birth to 12 weeks of age, average daily weight gains (ADG) were calculated from birth to 6 weeks of age (ADG6) and from 6 weeks to 12 weeks of age (ADG12). Basic statistics of phenotype data are displayed in Genomic DNA extraction from blood was performed with phenol/chloroform method, and DNA concentration was diluted to 50 ng/ul. The quality and concentration of genomic DNA fulfilled the requirements for the Illumina Infinium SNP genotyping platform. Genotyping using the Illumina 60 K Chicken SNP Beadchip was carried out at the Illumina-certified service provider, DNA LandMarks Inc., Canada. Quality control was assessed in GenomeStudio v2008.1 Pairwise linkage disequilibrium (LD) measured by r2 values for the F2 population and the parental breeds  were calculated for each chromosome using PLINK (v1.07) We assessed the F2 population structure using MDS analysis available from the PLINK software. All autosomal SNPs were pruned using the indep-pairwise option, with a window size of 25 SNPs, a step of 5 SNPs, and r2 threshold of 0.2 Genome-wide association analyses were carried out in PLINK. Linear regression analyses for body weights were performed with the first MDS component, sex, batch, and birth weight as covariates. While the statistical model for ADGs included the first MDS component, sex, and batch as covariates. Measures of SNP effects were calculated by the EPISNP2 package (v3.4) 2 values exceeding 0.40. Using this approach, the estimated number of independent SNP markers and LD blocks was 25,941, so that the threshold P-value of the 5% Bonferroni genome-wide significance was 1.92\u00d710\u22126 (0.05/25941). The threshold P-value for the significance of \u201csuggestive linkage\u201d that allows one false positive effect in a genome-wide test \u22125 (1/25941). Empirical genome-wide P-values were obtained from 25,000 permutations for each SNP using the maxT function in PLINK.The threshold P-value of the 5% Bonferroni genome-wide significance was calculated based on the estimated number of independent markers and LD blocks for autosome markers 2<0.2 using the first two MDS components  showed that a chicken  chromosome 4 (GGA4) region was strongly associated with body weight for weeks 7\u201312 and with average daily gain for weeks 6\u201312. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance under the LD conditions , and 19 of these 26 SNP effects reached 5% empirical genome-wide significance from permutation tests  gene had the strongest association with late growth (body weights from 7 to 12 weeks of age and ADG12 from 6 to 12 weeks of age). LDB2 is capable of binding to a variety of transcription factors, and is of vital importance during brain development and blood vessel formation TBC1D1 gene was highly significant for body weight at 12 weeks of age. TBC1D1 was reported to be a candidate gene for obesity in humans TBC1D1 haplotype has been under selection during domestication in broiler chickens LOC769270 gene had strong association with late growth (body weights from 11 to 12 weeks of age and ADG12). LOC769270 is a hypothetical protein coding which was bioinformatically predicted in chicken only.The oculocutaneous albinism II (OCA2) gene had highly significant effects on body weight in weeks 11\u201312. The association between OCA2 and body weight in chicken was the first report in this study but the SNP effect in OCA2 overlapped with a reported body weight QTL region detected in intercrossed lines involving White Plymouth Rock background OCA2 gene is associated with body weight and body size in mouse OCA2 gene could be relevant to growth traits.One SNP on GGA1 in the For early growth traits, only one SNP  on GGA18 had significant association with body weight at 2 weeks of age. The lack of SNP effects on early growth traits could be due to epistatic interaction that may explain more of the genetic variance of early growth than single gene effects \u22125)  . These eglypican 6 (GPC6) gene, glypican 5 (GPC5) gene, and gga-mir-17-92 cluster, and is located within the QTL for bodyweight identified in previous studies using the same F2 population as in this study Two SNPs located at 151 and 152.3 Mb on GGA1 had effects on body weight in weeks 11\u201312 and ADG12. This region harbors Popeye domain-containing protein 1 (POPD1) gene on GGA3 had effects on body weight in weeks 10\u201312 and ADG12, and a polymorphism (Gga_rs15178951) 31 kb downstream of BMP7 gene on GGA20 had effects on body weight in weeks 11\u201312. They overlapped with QTL regions reported by two studies A SNP (Gga_rs14373757) within the A previous study DYNC1I1 located at 23.9 Mb on GGA2 was associated with body weight in week 6 and ADG6, and two SNPs both located within the Opioid-binding protein/cell adhesion molecule-like (OPCML) gene on GGA24 were found to be associated with body weight in week 12 and ADG12. A SNP (Gga_rs14269721) within the gene Cbfa2t2 on GGA20 was in association with body weight in week 12 and ADG12. This is a new QTL identified in this study only.Other previous studies In summary, our GWAS detected 26 SNPs with genome-wise significance and 128 SNPs with the significance of suggestive linkage. Most of these SNPs were reported for the first time. Many of the SNP effects overlapped with previously reported QTL regions, providing evidence towards confirmation of QTL effects. The results are also helpful for identifying the exact QTL locations because of the much improved map resolution of the 60 k SNP panel over the map resolution of microsatellite markers used by most of previous reports on chicken QTL effects.Figure S1Manhattan plot of genome-wide association analysis for body weight traits. The dashed line indicates genome-wise significance of suggestive association , and the solid line declares genome-wise 5% significance with a p-value threshold of 1.92\u00d710\u22126.(PDF)Click here for additional data file.Table S1Associated SNP with genome-wise significance of suggestive association for body weight traits.(XLS)Click here for additional data file."}
+{"text": "There is an error in Figure 4. The correct version of Figure 4 can be seen here: [^] Also, in reference to this correction, in the tenth paragraph of the Results and Discussion section, the sentence \"The absence of any interaction with the P3 side-chain explains the diversity of residues (Q|G|K|N|E) observed at this position in MNV cleavage junctions [14], a feature that is also shared by picornavirus 3Cpro cleavage junctions [29], [30]\" should correctly say \"The absence of any interaction with the P3 side-chain explains the diversity of residues (Q|G|H|N|E) observed at this position in MNV cleavage junctions [14], a feature that is also shared by picornavirus 3Cpro cleavage junctions [29], [30].\""}
+{"text": "To the Editor: After the emergence of pandemic (H1N1) 2009 virus, measures for its control were taken quickly  when a clinical suspicion of pandemic influenza was established  2009 virus had been made in the respondent\u2019s household.>20\u00d7/d. Three workers were vaccinated against seasonal and pandemic influenza, while only 1 was vaccinated against pandemic (H1N1) 2009 alone. None took oseltamivir. Five positive samples were identified (13.8% of the study population) being obtained from four doctors and one nurse, all women. The 4 doctors had signs and symptoms for 24\u201348 hours consisting of fever, general indisposition, and coughing; none of the 4 required hospitalization. The nurse was a woman 26 years of age with no influenza symptoms and with a positive PCR result on week 5. None of these 5 workers had received any influenza vaccination.At the outset, 60 members of the hospital staff volunteered to participate. Those who missed >4 sample tests, or >2 consecutive ones, were considered to have abandoned the study. Of the 36 staff members who completed the study, 27 were women (75%). The participants\u2019 average age was 37 years (CI 95%: 34.8\u201339.4). Sixteen were doctors, 16 were nurses, 2 were nursing auxiliary staff, and 2 were hospital orderlies. During the monitoring period, 5 (13%) subjects exhibited coughing, 7 (20%) had runny noses, 3 (8%) experienced painful swallowing, 6 (16%) had headaches, and 1 (2%) felt generally unwell. Nearly 75% stated they washed their hands with antiseptic lotion Three workers reported that a diagnosis of pandemic (H1N1) 2009 influenza had been made with respect to a member of their household, but none of the workers had a positive PCR result. The distribution of positive PCR results in our hospital during the study is shown in the It had previously been hypothesized that the incidence of asymptomatic cases would be higher than the incidence of symptomatic cases  2009 during the study period.Our study began during the week in September 2009 in which the overall rate of incidence of pandemic (H1N1) 2009 in Spain reached 77.8 cases per 100,000 inhabitants  had been vaccinated against the novel form of the influenza A virus, and none of them had positive PCR results for pandemic (H1N1) 2009 virus. On the other hand, 5 (15%) of workers not vaccinated had a positive PCR result. This finding suggests that, despite the climate of uncertainty concerning the evolution of the influenza outbreak, hospital workers had a greater fear of possible side effects of the vaccine than of the disease itself."}
+{"text": "Cervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US.We examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance.CIN3 cases varied substantially by size (1\u201310 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results.We demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion. The natural history of human papillomavirus (HPV) leading to invasive cervical cancer is well established Reporting CIN3 as a single outcome based on the worst histological diagnosis on the cervix Detailed mapping of the LEEP segments that covers the surface of the entire cervix provides an opportunity to identify and characterize clinical subgroups of CIN3 cases. Thus, we performed a detailed characterization of LEEP specimens to understand predictors of CIN3 heterogeneity and to evaluate the relationship of CIN3 heterogeneity with screening test results in a large population-based study of women with abnormal cervical cancer screening results. Examining the heterogeneity CIN3 cases referred to a colposcopy clinic after abnormal cervical cancer screening may aid in elucidating the biological differences between CIN3 cases, and thereby inform future efforts to reduce unnecessary treatment of CIN3 that are not clinically important.We conducted the analysis in the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED), a large population-based study composed of women referred to the University of Oklahoma Health Sciences Center (OUHSC) for abnormal cervical cancer screening test results. SUCCEED design and methodology, including the details on enrollment, questionnaire data, HPV DNA genotyping, histology, and cytology procedures, have been described in depth elsewhere Participants completed interviewer-administered, standardized questionnaires and provided liquid-based cytology specimens for ThinPrep Pap and HPV genotyping by Linear Array (Roche Diagnostics). OUHSC gynecologists performed colposcopic examination according to routine OUHSC practice. Women were treated by LEEP of the transformation zone, if indicated by ASCCP guidelines Every LEEP specimen was divided into 12 topographically designated sections or segments (a \u201cclockface\u201d depiction of the cervix) for detailed histopathological mapping. According to the SUCCEED study protocol, two segments from each LEEP representing the worst lesion and normal cervical tissue were snap-frozen for molecular studies. The remaining 10 segments were formalin-fixed, paraffin-embedded and analyzed to generate individual histology results for each segment. The study pathologists at OUHSC, masked to HPV genotyping data, determined the histology using CIN terminology. One or more of the following diagnoses were noted for each o'clock segment of the cervix per individual: other, negative/normal, atypical metaplasia, CIN1, CIN2, CIN3, adenocarcinoma in situ, squamous cell carcinoma, and adenocarcinoma. In addition, if the clinician determined that the entire transformation zone or extent of a lesion could not be visualized adequately, endocervical curettage (ECC) and/or a deeper, secondary LEEP (\u201ctop hat procedure\u201d), which removes tissue from higher up in the endocervical canal, were performed. Per common practice, the cases were categorized according to the worst diagnosis for each woman based on the diagnosis of the most abnormal LEEP segment, ECC, and/or top hat.During the study period, 975 women were managed by LEEP Figure 1continuous adjacent LEEP segments with CIN3. In addition, we dichotomized the CIN3 lesion size as a \u201csmall\u201d versus \u201clarge\u201d CIN3 lesion, defined by a cut-off of <3 CIN3 segments versus 3+ CIN3 segments. Similar trends were observed with different cut-points .For our analysis, we defined the diagnosis of each o'clock segment of the cervix by the most severe diagnosis, if more than one diagnosis was noted for an individual LEEP segment. We focused our analysis on the following worst outcomes for each LEEP segment: normal, CIN1, CIN2, and CIN3. A priori, we defined subgroups of CIN3 cases by the presence or absence of CIN1 and CIN2 in conjunction with the presence of CIN3 in any of the LEEP segments. Accordingly, we categorized the CIN3 cases into four subgroups: solitary CIN3, CIN3+CIN2, CIN3+CIN2+CIN1, and CIN3+CIN1. As a sensitivity analysis, we further categorized solitary CIN3 as \u201ctrue\u201d solitary CIN3 cases by excluding cases with CIN1 and/or CIN2 diagnoses in any of CIN3 LEEP segments. We also a priori defined the size of CIN3 by the number of LEEP segments with CIN3 among all LEEP segments analyzed and the number of 2 test. In addition, we tested for trend across ordered groups using the nptrend command, a nonparametric test that is an extension of the Wilcoxon rank-sum test. We also calculated the percent detection of small versus large CIN3 lesions by the diagnostic tests. For all analyses, P-values of \u22640.05 were considered statistically significant. All tests of statistical significance were two-tailed. Analyses were performed using Stata 11.1 .First, we performed a case-case comparison of the four CIN3 subgroups for selected known risk factors of cervical cancer: age at LEEP, length span of sexual activity , parity, OC use, lifetime number of sexual partners, smoking, Pap test history (number in past five years), HPV genotypes  HPV type infections, and presence of HPV16 infections). We also performed a case-case comparison for diagnostic factors based on the following cervical cancer screening tests: cytology results prior to LEEP, biopsy results at LEEP visit, and colposcopic impression results at LEEP visit. In addition to the categorical variables of these screening test results, we dichotomized the screening test results to distinguish low grade from high grade diagnoses: biopsy histology of CIN3+ compared with CIN2 or less, cytology of high-grade squamous intraepithelial lesions (HSIL) or worse (HSIL+) compared with less than HSIL, colposcopic impression of CIN3+ compared with CIN2 or less. Second, we evaluated the differences between categories of CIN3 lesion size with the same factors. We tested for differences between categorical variables and CIN3 subgroups and lesion size using the Pearson \u03c7continuous CIN3 size (data not shown). In addition, we also observed a variable range in number of CIN2 (1\u201310 segments) and CIN1 (1\u20136 segments) present as concomitant lesions among the CIN3 cases (data not shown).Three hundred and nine women were identified as CIN3 cases according to the worst LEEP diagnosis Table 1.We summarized all 309 women included in the analysis in a single circular histogram Figure 3The majority of these women  had CIN lesions of lower grades in other LEEP segments in addition to the CIN3. About half of the heterogeneous lesions were CIN3 cases with CIN2 lesions (n\u200a=\u200a116), followed by CIN3 without additional lesions (n\u200a=\u200a79) and CIN3+CIN2+CIN1 (n\u200a=\u200a79), as well as a small percentage of CIN3+CIN1 (n\u200a=\u200a35) Table 2.The distributions of clinical and pathological characteristics examined and presented in CIN3 size was widely distributed in the study population and ranged from 87 women (28%) with one CIN3 segment involved to 55 (18%) women with CIN3 across five or more CIN3 segments Table 3.continuous CIN3 size (data not shown). Dichotomizing CIN3s by lesion size (<3 vs. 3+), the sensitivity of HSIL cytology at LEEP was 74% for small CIN3s and 86% for large CIN3s  presented with CIN3 lesions in conjunction with lower grades of CIN in different regions on the cervix. We observed a wide range of CIN3 sizes from small focal lesions to extensive CIN3 covering most of the cervix. To better characterize women within the CIN3 diagnostic group, we examined the distribution of risk factors as well as clinical and pathological information from cervical cancer screening tests by CIN3 subgroups and CIN3 lesion size.Some previous studies have suggested that CIN may be more common on the anterior and posterior lips of the cervix than at the lateral angles We also observed that compared to CIN3 cases with CIN2, solitary CIN3 and CIN3+CIN1 cases were more likely to have a larger sized CIN3 lesion. Our data corroborate the model that high-grade pre-cancer grows out from a small lesion possibly surrounded by low grade lesions, such that either CIN3 expands while the CIN1 regresses We sought to understand how clinical and pathological characteristics are related to CIN3 lesion size. The CIN3 lesions observed by McCredie and colleagues in their retrospective study of women with untreated CIN3 who progressed to cancer were large We did not observe associations between other risk factors previously reported to be associated with HPV infection and progression  and CIN3 size, suggesting that these factors are not paramount at the later stages of CIN3 natural history. However, we noted an insignificant trend of less OC use in women with larger CIN3s. We recently observed in the same population that contraceptive methods requiring doctor visits such as OCs are associated with more Pap tests in the previous five years, which could explain this observation (data not shown).In addition, we examined the effect of CIN3 size on cervical cancer screening results and found that larger CIN3 lesions were more likely to be diagnosed as HSIL+ at time of LEEP visit. Similarly, larger CIN3 size was more common among more severe colposcopic impression at time of LEEP visit and with a higher percentage of a CIN3+ biopsy result at the colposcopy visit, demonstrating that larger CIN3 cases are easier to detect by colposcopy. These finding highlight a current dilemma in cervical cancer screening: new screening tests such as HPV DNA detection are more sensitive than the current gold standard of following up cytology with colposcopy and biopsy The main strengths of our study are the large population-based sample of CIN3s and the detailed mapping of LEEP specimens, which allowed for a thorough evaluation of the heterogeneous manifestations of CIN3 cases. While examination of the 12 LEEP segments allowed us to study lesion size in unprecedented detail, a finer resolution would have provided more accuracy since multiple histologic diagnoses could be found even within a LEEP segment. Although the cross-sectional design of our study may be viewed as a limitation, it is not possible to follow CIN3 prospectively. Furthermore, this design permitted the accrual of large numbers of women into the study for studying CIN3 cases with detailed mapping of disease in LEEP specimens. In addition, our analysis was not based on panel adjudication of histology results, but on the community histology diagnosis by a single experienced pathologist.In summary, our data showed that women with CIN3 lesions without concomitant CIN2 or CIN1 lesions were more likely to be older, have longer sexual activity span, and have fewer high risk HPV infections and that larger CIN3 lesions were more common among women infrequently screened, with HSIL or worse cytology, and CIN3 or worse impression in colposcopy. Interestingly, we also observed that in our population, HPV16 positivity was not associated with larger CIN3 lesion size. Although our and others' data suggest that CIN3 lesion size is an important indicator of risk of invasion, lesion size can only be determined post-treatment. We show that HSIL cytology and CIN3 impression in colposcopy with CIN3 biopsy results point to larger CIN3s that most likely have a higher risk of invasion compared to small incipient lesions. While the findings from this study are important, they are not sufficient to establish which CIN3 should be treated. We are now conducting detailed molecular analyses of cervical lesions, using microdissection to permit the molecular evaluation of CIN3 heterogeneity to identify better risk markers for management of CIN3."}
+{"text": "RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations \u2013 predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation  suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease.  GUCY2D (LCA1) RPE65 (LCA2) SPATA7 (LCA3) AIPL1 (LCA4) LCA5 (LCA5) RPGRIP1 (LCA6) CRX (LCA7) CRB1 (LCA8) NMNAT1 (LCA9) CEP290 (LCA10) IMPDH1 (LCA11) RD3 (LCA12) RDH12 (LCA13) LRAT (LCA14) TULP1 (LCA15) KCNJ13 (LCA16) IQCB1MERTKCRXIMPDH1 mutations Leber congenital amaurosis  is a group of retinal dystrophies, which represents the most common cause of blindness in childhood (10\u201318%) GUCY2D, RPE65, CRB1, TULP1, CRX, RPGRIP1, AIPL1) CEP290, IQCB1) The implication of LCA genes in retinal dystrophies of later-onset is not uncommon , North-America (The United States and Canada) and China were considered in this study including 574 LCA probands who have not had disease-causing mutations identified in the known LCA genes, 96 probands affected with autosomal recessive early-onset severe retinal dystrophies (EOSRD), 150 with autosomal recessive retinitis pigmentosa (RP) and 32 with other retinal dystrophies. The ethnicity of patients and diagnoses are shown in RD3 mutations independently. Patients of all cohorts but one were screened by direct sequencing using intronic primers designed to amplify the two RD3 coding exons and intron-exon boundaries. The 313 LCA probands of the University of Iowa Carver College cohort were screened by single strand conformation using as controls 151 normal individuals. PCR, direct sequencing and SSCP conditions, as well as primer sequences are available on request.Samples were collected and screened for A variant was predicted to be pathogenic when it showed familial cosegregation with the disease and absence in control individuals and when it was predicted to be damaging by Align DGVD, Polyphen-2, SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder available through the Alamut Interpretation Software 2.0.RD3 gene. Microsatellite markers distributed over a 12.6 Megabases (Mb) interval spanning the RD3 locus on chromosome 1q32.2-q41 (according to the UCSC Genome Browser GRCh37/hg19 assembly) were used for haplotype analysis. Microsatellite markers included (from telomere to centromere): AFMB347YA5 (D1S2782), AFM281YG1 (D1S471), AFM310VB1 (D1S491), AFM108YA3 (D1S205), AFM179XG5 (D1S414) AFMC011YD5 (D1S2810), AFMB342YG1 (D1S2780), AFM203ZB6 (D1S425), AFMA127WB5 (D1S505), AFM297XC1 (D1S2827), AFM058XG9 (D1S2880). Amplified fragments were electrophoresed on an automatic sequencer  and analyzed using the GeneScan Analysis 3.7 and Genotyper softwares. For each marker, the heterozygote frequency and the size range of alleles were either available from Genethon Linkage Map Haplotype analysis was performed using 4/5 families segregating the c.112C>T mutation of the The degree of linkage disequilibrium and the estimation of the age of the mutation were obtained as described previously Three novel mutations were detected in seven apparently unrelated LCA families . Two outAll three mutations segregated with the disease in families and were neither identified in controls nor reported elsewhere.RD3 mutations. Indirect studies at the RD3 locus were performed on 4/5 families indicate that the two Moroccan families and the Turkish family share a small 1 Mb-long common haplotype that differed in the Libanese family . This variant that changes a basic amino acid into an uncharged residue is predicted to be deleterious by the SIFT, Polyphen-2 and AlignGCVD programs. The change was identified neither in 200 control individuals nor in databases listing SNPs. This change was identified in a heterozygous state in an isolated case born to non-consanguineous parents  mouse rod-cone dystrophy 2 (rcd2) collie RD3 in LCA. Yet, the mutation rate obtained in a very large cohort of LCA cases originating worldwide (7/574 LCA cases excluding other LCA genes) and consistent identification of homozygous mutations in consanguineous families , support the very low frequency of heterozygous RD3 carriers in the general population.Until this report, the implication of RD3 encodes an evolutionary conserved basic protein of 195 amino acids that is preferentially expressed in the retina rd3 mouse which express a truncated unstable rd3 protein resulting from the p.R107* mutation, is consistent with a role of the protein in the trafficking of GC2 in rods as well. As a consequence, like the double GC1/GC2 knock-out, the complete inactivation of both rd3 alleles is expected to preclude cGMP synthesis in both rod and cone outer segments and to hinder recovery of the dark state after light stimulation i.e. when eyes open \u2013 and the degeneration is almost complete by the age of 2\u20134 months rd3 frameshift mutation (p.Pro139Alafs*) of the rcd2 collie rcd2 dogs develops almost normally, by the age of 2\u20132.5 months, the outer segments of both rods and cones are completely lost, with rods degenerating faster than cones rd3 and GC double knock-out mice LCA12 and LCA1. Indeed, the review of the natural history and clinical data of our patients and that of the two original RD3 families LCA1 patients harboring GUGY2D mutations Interestingly, these data which support a common mechanism of photoreceptor degeneration in RD3 mutations identified thus far are expected to truncate the RD3 protein . It is tempting to hypothesize that similar to several other LCA genes, mutations with a milder effect on the protein structure and/or function may be responsible for milder phenotypes; for recent reviews see RD3 mutations. This finding is consistent with the LCA type I-specific involvement of RD3 mutations. Indeed, of the LCA genes, most genes involved in EOSRD and RP account for LCA type II GUCY2DRPGRIP1CEP290RD3 [present study]. Refraction may represent a distinctive feature in LCA type I since patients with GUCY2D mutations consistently have high hypermetropia (>+7) with frequent enophthalmia RPGRIP1, CEP290 or RD3 mutations present with lower or no hyperopia : RD3 mutations and an individual of the general population with no family history of LCA is negligible. Unions between affected persons with LCA and or between LCA and RP patients are not uncommon. The identification of RD3 mutations in one spouse must prompt the search for RD3 mutations, only when the other spouse is affected with LCA type I. Indeed, if the spouse is affected with LCA type II or autosomal recessive RP of later-onset, the genetic counseling can straightaway be reassuring.With respect to genetic counseling, our data that support a dramatically low RD3 carrier frequency in the general population indicate that the genetic risk in the case of the union between an affected person harboring RD3 mutations in large cohorts of patients accurately phenotyped by referent clinicians has major clinical implications. Most LCA genes have demonstrated a preferential or specific retinal expression CEP290 which mutations are either responsible for non-syndromic LCA or a range of syndromic forms of the disease; for review see IQCB1 gene at the NPHP5 locus have been shown to cause a form of Senior-Loken syndrome characterized by a congenital retinal dystrophy associated with a renal dysfunction of highly variable age of onset (1st to 5th decade) RD3, it is worth noting that none of the patients reported here whose ages ranged from 2 to 31 had renal failure, neurological symptoms or intellectual disability. Owing to the retina-specific pattern of expression of RD3, it is likely that the disease may remain restricted to this tissue. In these patients, anxiogenic and expensive extraocular explorations must not be systematically prescribed.The comprehensive survey of a sine qua none condition to maintain living and functional rod and cone cells. Our knowledge regarding the mechanisms of this complex process - which alteration accounts for \u223c17% of LCA cases - are incomplete. In particular, most of the molecular players and disease mechanisms of light-driven trafficking between the inner and outer segments of photoreceptors are unknown. Interestingly, although RD3 alterations are quite uncommon, their description in man and animal models unraveled a crucial protein for GC expression and trafficking in photoreceptors. Further studies designed to identify RD3 partners will hopefully contribute to the deciphering of mechanisms for dark-state recovery and will hopefully allow uncovering some of the yet unidentified LCA causing genes.The recovery of the dark state in the retina through GC-mediated increase in cytoplasmic cGMP concentration in photoreceptor outer segments is RPE65\u2212/\u2212 Briard dogs has paved the way for the development of gene and drug therapy for a broad range of eye disorders and made large dogs the species of choice for these developments; for review see RD3 mutations are uncommon causes of LCA, the availability of naturally occurring rd3 animal models, among which a large dog (collie), it is likely that gene and drug therapy protocols will be developed to treat patients with RD3 mutations.Finally, it is worth remembering that the success of gene replacement and drug therapy to restore vision in naturally occurring"}
+{"text": "African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs. Virulent isolates kill domestic pigs within 7\u201310 days of infection. In chronic cases ASF causes respiratory disorders and in some cases swelling around the leg joints and skin lesions. Domestic pigs can survive infection with less virulent isolates and in doing so can gain immunity to subsequent challenge with related virulent viruses ASF is endemic in many sub-Saharan African countries as well as in Sardinia. In 2007 ASF was introduced into Georgia and from there spread rapidly to neighbouring countries in the Trans Caucasus region, including Southern European Russia Ornithodoros spp. and warthogs in eastern and southern Africa. In many regions the isolates circulating in domestic pigs are genetically more similar.Genotyping of ASFV isolates by partial sequencing of the B646L gene encoding the major capsid protein p72 has identified up to 22 genotypes + T cells since depletion of these cells was shown to abrogate this protection Previous work has shown that pigs are protected from challenge with related virulent isolates following infection with natural low virulence isolates and with virus attenuated by passage in tissue culture or by deletion of genes involved in virulence Despite this early experience in Portugal and Spain, the prospect of developing successful attenuated vaccines have improved as substantial progress has been made in identifying ASFV genes involved in virulence and immune evasion and the complete coding sequences of a number of ASFV isolates are now available 22.1ASFV isolates used in this study have been described previously and included Portuguese isolates of ASFV, OURT88/3  and OURT88/1  2.24 TCID50 of non-virulent ASFV isolate OURT88/3 and boosted intramuscularly 3 weeks later with 104 HAD50 of virulent ASFV isolate of OURT88/1. Pigs were then challenged 3 weeks later with 104 HAD50 of either Benin 97/1 or virulent Uganda 1965 intramuscularly.Pigs used in the first experiment (experiment 1) at IAH Pirbright Laboratory UK were cross-bred pigs, Large White and Landrace, of average weight 20\u00a0kg at the first immunisation. For the second experiment specific pathogen free (SPF) Large White pigs were used from Anses, Ploufragan, France, SPF facility and were of 15\u00a0kg average weight at the first immunisation (experiment 2). For the third experiment (experiment 3) carried out at Anses Ploufragan, France, Large White pigs were obtained from a local high health status farm and the average weight at the first immunisation was 11\u00a0kg. All pigs were maintained at high security facilities throughout the experiment. The first experiment at Pirbright was performed under Home Office licence PPL 70-6369. Experiments at Ploufragan were performed according to the animal welfare experimentation agreement given by the Direction des Services V\u00e9t\u00e9rinaires des C\u00f4tes d\u2019Armor (AFSSA registration number B-22-745-1), under the responsibility of Marie-Fr\u00e9d\u00e9rique Le Potier (agreement number 22-17). Briefly, pigs were intramuscularly inoculated with 102.3ASFV-inoculated pigs were monitored for body temperature and other clinical symptoms and these were recorded and scored according to the clinical scoring system shown in 2.450.Peripheral blood was analysed at different days post-immunisation for the presence of ASFV by quantitative PCR (qPCR) as described previously 2.55 HAD50/ml. Uninfected porcine bone marrow culture supernatants were used as negative control antigen.Development of T cell immune responses to ASFV after immunisation was analysed by IFN-\u03b3 ELISPOT and proliferation assays as described previously The development of ASFV specific antibodies was analysed using a competition ASF ELISA kit (INGENASA PPA3 COMPPAC), and the antibody titre was expressed as log\u00a02 dilution of end point which gives 50% competition.33.1Three experiments were carried out in which pigs were immunised with the non-virulent Portuguese OURT88/3 genotype I isolate followed 3 weeks later by the closely related virulent Portuguese isolate OURT88/1 and then challenged 3 weeks later with either the West African genotype I isolate, Benin 97/1, or the genotype X virulent Uganda 1965 isolate. In the first experiment at Pirbright, 3 immunised pigs and 4 non-immune pigs were challenged with Benin 97/1. In the second experiment at Ploufragan, a total of 12 pigs were immunised and challenged with either Benin 97/1 or virulent Uganda 1965. Ten pigs were prepared as non-immune controls and challenged with either Benin 97/1 or virulent Uganda 1965. As a control for weight gain, an extra group of 5 pigs were included in this experiment. In the third experiment at Ploufragan, a group of 7 pigs were inoculated and 6 of these and 6 non-immunised pigs were challenged with Benin 97/1.7 copies of the virus genome/ml; and up to 8.8 HAD50/ml virus), and died or were euthanized for ethical reasons within 7 days of challenge (7 HAD50/mg tissue) . Virus genome was detected at low copy numbers by qPCR in blood samples from an additional 2 pigs but these were negative by HAD assay. Pigs 1819, 1822 and 1841 were terminated for ethical reasons between day 4 and day 6 post boost with OURT88/1 before the potential development of severe ASF symptoms.In the second experiment of the 12 immunised pigs, 5  developed a transient pyrexia  followin50/ml; pig 1845 had a temperature at day 7 of 40.6\u00a0\u00b0C and the virus genome was detected at 633 copies/ml; and virus at 2 HAD50/ml. The other two pigs challenged with virulent Uganda 1965 isolate showed no clinical signs and no virus was detected in blood by qPCR or HAD assay. Five pigs were challenged with Benin 97/1, two pigs  developed typical ASF , and either died or were terminated within 8 days of challenge  and tissues (virus \u223c7 HAD50/mg of tissue) were detected from all lymphoid tissues in all of the non-immune pigs challenged  showed pyrexia from 2 weeks after the first immunisation . This piUnlike the non-immune pigs, immune pigs challenged increased their body weight during the challenge .3.2Lymphocytes from immunised pigs in experiment 1 were collected at various times post-immunisation and IFN-\u03b3 ELISPOT and proliferation assays were performed with OURT88/3 or Benin 97/1 as antigen. In all 3 pigs, the numbers of ASFV specific IFN-\u03b3 producing cells was rapidly increased after the OURT88/3 inoculation and further increased after the OURT88/1 boost. Both OURT88/3 and Benin 97/1 isolates stimulated lymphocytes from immunised pigs to an approximately equal amount A\u2013C. Low At the termination of the experiment, lymphocytes from these pigs were tested for cross-reactivity stimulated with various ASFV isolates by IFN-\u03b3 ELISPOT assays A. ImmuneIn the second experiment B, lymphoA competitive ELISA based on the p72 major capsid protein was used to measure development of anti-ASFV specific antibodies. The results from analysis of sera collected in experiment 2 and 3 are shown in 4In this study we have demonstrated that experimental immunisation of pigs with a non-virulent ASFV genotype I isolate from Portugal, OURT88/3, followed by a boost with a closely related virulent isolate, OURT88/1, can induce protective immunity in European domestic pigs against challenge from two virulent African isolates of ASFV. These included a genotype I isolate from West Africa, Benin 97/1 and a genotype X isolate from Uganda, virulent Uganda 1965. Overall 85.7% and 100% pigs were protected from Benin 97/1 and Uganda 1965 ASFV challenge respectively. More than 78% of pigs challenged with Benin 97/1 and 50% of pigs challenged with Uganda 1965 were completely protected by not showing any sign of disease or development of viraemia.Phylogenetic analysis of the concatenated sequences of 125 genes conserved between 12 complete genome sequences showed that the OURT88/3 and Benin 97/1 sequences are greater than 95% identical across these genes Ornithodoros erraticus ticks in Portugal and described not to cause clinical signs or viraemia ex vivo, with different ASFV isolates, showed various degrees of cross-reactivity and this correlated well with cross-protection induced in vivo. Good cross-reactivity against genotype X isolate virulent Uganda 1965 (ex vivo assay, all of the pigs immunised and challenged with virulent Uganda 1965 virus were protected. No cross-reactivity to genotype XIII isolate Malawi LIL 20/1 was detected and this correlates with the observation that OURT88/3 and OURT88/1 immunised pigs are not protected from Malawi LIL 20/1 challenge . Taken together these data suggest that this ex vivo, IFN-\u03b3 ELISPOT assay might be a useful tool to assess vaccine efficacy and/or to assess possibility of ASFV isolate-cross-protection.The ASFV OURT88/3 strain was isolated from nda 1965 A was obsAn anti-ASFV antibody response also developed after OURT88/3 immunisation and was boosted after the OURT88/1 inoculation. The anti-ASFV antibody titre was measured by a p72 competition ELISA, however we could not conclude from these experiments whether the level of antibody developed by our immunisation protocol is either sufficient or necessary for protection.OURT88/3 has been used as a vaccine model to identify what is required for inducing ASFV protective immunity in domestic pigs. The observations of adverse effects of OURT88/3 immunisation in some of the pigs vaccinated in France suggest that further attenuation of this isolate by deleting additional genes or possibly changing the dose or route of vaccination may be useful. Secondly, the results from experiment 2 showed that our current protocol did not induce complete protection in all of the pigs immunised with the virulent OURT88/1 boost. This may be due to the genetic background of the pigs as we have previously demonstrated that cc inbred pigs are also not always protected by OURT88/3 from OURT88/1 challenge"}
+{"text": "To analyze the genetic pathogenesis and improve the accuracy of the diagnosis of the disease, this study reported the clinical features of 20 patients with Silver Russell Syndrome (SRS) in the Beijing Children's Hospital and detected the chromosome 11p15 imprinting defects in 16 patients of them.20 SRS cases diagnosed in Beijing Children\u2019s Hospital from 2006 to 2011 were studied retrospectively for clinical manifestations, physical signs, laboratory examinations and respond of GH treatment. We compared with 3 different diagnostic criteria and used the methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) method to detect the chromosome 11p15 imprinting defects in 16 patients of them, meanwhile take 10 normal control.We collected 20 SRS patients over a period from 2006 to 2011 with 3 criteria. The concordance is 90%. Include fifteen males and five females, age range 0.08~12.17yr. The most chief complaint is short, 85%. Then, it is asymmetry (5%), and external genital abnormalities (10%). The clinical characteristics with the frequencies accounted for over 80% included small for gestation age(SGA), postnatal growth retardation, craniofacial dysmorphism, asymmetry and super thin of whole body, extremely limbs, fifth finger clinodactyly, BMI<-2SDS and height was obviously lagging behind the bone age. Six of them had used the growth hormone for 3-months to 12 Months. The growth velocity is from 4cm to 10cm/year. In the 16 patients, 6 patients were found hypomethylation in chromosome 11p15 ICR1, the other one with chromosome 11p15 ICR1 hypomethylation and ICR2 hypermethylation may be the result of the maternal chromosome 11p15 uniparental disomy. Another case had duplication of the maternal chromosome 11p15 fragment. Two cases had good effects of GH treatment, one with chromosome 11p15 ICR1 hypomethylation and the other one was normal.The top 3 clinical features in SRS are \u2460 growth retardation include SGA and/or postnatal. \u2461 Malformation include craniofacial dysmorphism, asymmetry of face and/or limbs, fifth finger clinodactyly. \u2462 Super severe low BMI and height was obviously lagging behind bone age. No laboratory and imagination specificity. Chromosome 11p15 imprinting defect is the major genetic disturbance in SRS, about 50%, and ICR1 hypomethylation is the predominant molecular alteration. The MS-MLPA is a technique for detecting all chromosome 11p15 imprinting defects of SRS. It is useful for the study of the genetic aspects of SRS. The relation between the respond of GH treatment and genetic changes is uncertainty."}
+{"text": "We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2\u2032-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The CaThese results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells. We have previously shown significant alterations in endogenous levels of both glutamic and gamma-aminobutyric acids in particular brain structures after the extremely traumatic water immersion restraint stress (WIRS) experience in rats The hippocampal DG has a unique ability to generate new neurons throughout the entire life of an individual, often referred to as adult neurogenesis Myosin VI (Myo6) is an actin-based molecule responsible for the transport of intracellular cargos including proteins, vesicles and organelles trans retinoic acid (ATRA) as a model for neural progenitor cells These previous studies led us to propose a possible role of Myo6 in mechanisms underlying the aforementioned suppression of BrdU incorporation into the hippocampal DG enriched with neural progenitor cells, in mice with traumatic stress. Therefore, the present series of experiments was intended to examine the role of Myo6 in mediating proliferation for self-replication and differentiation into progeny lineages using murine embryonal carcinoma P19 cells endowed to proliferate and differentiate into neuronal and astroglial lineages in the presence of all-2 and 95% air at 37\u00b0C with a medium change every 2 days.Mouse embryonal carcinoma P19 cells  were cultured in alpha minimal essential medium (\u03b1MEM)  supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL). P19 cells are able to differentiate into a neural lineage in the presence of ATRA  as described previously 5 cells/cm2, followed by culture in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) (GIBCO BRL) with 10% FBS for 24 h and subsequent stable transfection with pSI-Myo6, pSI-GFP and pcDNA3.1 vectors, or pSI, pSI-GFP and pcDNA3.1 vectors, using 2 \u00b5g of DNA and Lipofectamine and Plus reagent  in 0.5 ml of Opti-MEM (Invitrogen) as described previously http://blast.ncbi.nlm.nih.gov) analysis revealed a 96% homology between porcine and murine Myo6 proteins.The full-length coding region of porcine Myo6, which was kindly donated by Dr. Tama Hasson , was inserted into pSI vector using the Ligation High reagent as described previously Reverse transcription-polymerase chain reaction (RT-PCR) analysis was conducted as described previously Western blotting was done by using antibodies against Myo6 , Doublecortin , Synapsin I , neuronal nuclei (NeuN) (Merck Ltd.), microtubules-associated protein-2 (MAP2) (Sigma), glial fibrillary acidic protein (GFAP) (Sigma), Sox2 (Santa Cruz Biotechnology) and \u03b2-tubulin (Sigma) as described previously Five different visual fields each in parallel experiments were chosen at random from each culture well with undifferentiated P19 cells cultured with ATRA for a period of up to 4 days, and examined under a phase contrast micrograph in a blinded fashion, followed by calculation of areas of round aggregates composed of clustered proliferating cells for summation using Scion Image \u03b2 4.02 software  as described previously Stable transfectant cells were cultured in the presence of ATRA for 4 days, followed by re-plating on dishes previously coated with poly-L-lysine for further culture for 2 h and subsequent double staining for DNA with 10 \u00b5g/ml Hoechst33342 (Sigma) and 5 \u00b5g/ml propidium iodide (PI) (Sigma). Cells were observed under a confocal microscope . The number of cells stained with these dyes was individually counted in five different visual fields selected at random in a blinded fashion in order to calculate the percentages of dead cells stained with the membrane-impermeable dye PI out of the total cells stained with the membrane-permeable dye Hoechst33342. Cellular viability was also quantified by the activity of lactate dehydrogenase (LDH) released into the culture medium by an enzyme-coupled color development method as descried previously 5 cells/ml in \u03b1MEM supplemented with 5% FBS and 0.5 \u00b5M ATRA, followed by culture for 4 days under floating conditions favorable for proliferation. These floating cells were harvested and trypsinized, followed by plating onto dishes previously coated with poly-L-lysine at a density of 2\u00d7105 cells/ml in \u03b1MEM supplemented with 10% FBS. They were subsequently cultured for an additional period up to 6 days in the absence of ATRA to induce spontaneous differentiation as shown with murine fetal cortical progenitors 2/95% air at 37\u00b0C.Pluripotent P19 cells have been shown to differentiate into neuronal and astroglial lineages after commitment in the presence of ATRA as described previously 5\u2032-ACGCGT (MluI site) GCAAGAACCCTCACTGGC-3\u2032 and the reverse primer 5\u2032-CTCGAG (XhoI site) AGGCTGCCGGGCTGCGGGCG-3\u2032 from the mouse genome. The Myo6 (-1086 to+210) promoter fragment was cloned into the promoterless pGL-3 basic vector , to create the recombinant plasmid \u20131086/+210 Myo6-Luc.A reporter plasmid with the Myo6 promoter was prepared as follows. The mouse Myo6 promoter was obtained by cloning using the forward primer 5 cells/ml in plates (\u03c6100 mm) previously coated with 0.2% agarose, and cultured in \u03b1MEM supplemented with 10% FBS and 0.5 \u00b5M ATRA. Twenty-four hours after transfection of the reporter plasmid, the cells were exposed to 1 \u00b5M A23187 (Sigma) for 6 h, followed by a medium change, and then cultured for an additional 24 or 48 h. The cells were then lysed for determination of luciferase activity using specific substrates in a luminometer according to the manufacturer\u2019s protocol. Transfection efficiency was normalized against the activity of pure Renilla luciferase Pluripotent P19 cells were transiently transfected with a reporter plasmid containing the promoter region of Myo6 (\u22121086 bp to +210 bp) along with the internal control vector pRL-SV40 (Promega) using the Lipofectamine 2000 reagent. Transfected cells were plated at 1\u00d710t-test or one-way analysis of variance (ANOVA) with the Bonferroni/Dunnett post hoc test.Quantitative data are expressed as the mean \u00b1 S.E. and the statistical significance was determined by the two-tailed Student\u2019s In our previous study To evaluate the mechanism underlying the possible deterioration in proliferative activity, each stable transfectant clone was screened for mRNAs for several cell cycle regulators of the Cyclin family An attempt was thus made to determine whether stable overexpression of Myo6 leads to altered expression of a variety of basic helix-loop-helix (bHLH) genes responsible for the positive and negative regulation of proliferation for self-renewal and/or neuronal differentiation in undifferentiated neural stem cells P19 cells were cultured in the presence of ATRA for 4 days under floating conditions to induce clustered aggregate formation due to self-replication, followed by immunostaining for the neural progenitor cell marker nestin. Cells clustered in round aggregates were immunoreactive for nestin and also exhibited a marked increase in MTT reduction, both indices of cellular proliferation We next examined the effects of Myo6 knockdown on proliferation and differentiation of pluripotent P19 cells using RNA interference techniques. Cells were transfected with Myo6 siRNA, followed by culture with ATRA for 2 days under floating conditions and subsequent determination of endogenous levels of Myo6. A drastic decrease in endogenous Myo6 protein levels in cells transfected with 2 different Myo6 siRNA oligonucleotides compared with those with a control oligonucleotide was invariably seen on Western blotting . QuantifWe then examined the expression of Sox2 in P19 cells treated with Myo6 siRNA for 2 or 4 days in the presence of ATRA under floating conditions. Western blotting analysis clearly revealed a marked, statistically significant increase in endogenous Sox2 protein levels in P19 cells transfected with Myo6 siRNA for 2 and 4 days compared to those exposed to control siRNA  Figure . Cells wTo test the selectivity of the correlation between Myo6 expression and cellular proliferation, the MTT assay was used to determine whether Myo6 siRNA affects cellular proliferation in cultured proliferating cells other than pluripotent P19 cells. In C6 glioma cells cultured for 2 days, marked expression of Myo6 protein was seen in a manner sensitive to knockdown by siRNA . AlthougWe have previously shown a more than twofold increase in Myo6 mRNA expression is induced in P19 cells exposed to the calcium ionophore A23187 The essential importance of the present findings is that proliferation was markedly suppressed in pluripotent P19 cells stably overexpressing the motor protein Myo6 without induction of cell death. Conversely, the current findings that proliferation was significantly promoted in P19 cells by decreased endogenous Myo6 levels due to siRNA transfectionargue in favor of the idea that Myo6 negatively regulates self-replication in pluripotent P19 cells endowed to either proliferate for self-renewal or differentiate into neuronal and astroglial lineages. Taking into consideration the unresponsiveness of different cell cycle regulators and bHLH proneural genes other than Hes5, it is conceivable that downregulation of Hes5 expression is at least in part responsible for the inhibition of proliferation in stable Myo6 transfectants. In mice defective for the activator bHLH factor Mash1, for example, severe loss of neuronal precursors together with disappearance of the Notch signaling target Hes5 is seen in the ventral telencephalon normally enriched for Mash1 during neurogenesis However, the reason why Myo6 overexpression inhibited cellular proliferation without affecting the mRNA levels of several cyclins in P19 cells is still not clear. A future comprehensive analysis should be conducted on mRNA expression by all cyclin family members and relevant kinases to evaluatethe possible involvement of particular cyclin family members in the underlying mechanisms other than those tested here. Although the activator type bHLH factor NeuroD1 plays a pivotal role in the survival and neuronal differentiation of neural stem cells during adult neurogenesis Nevertheless, the exact signaling pathway from stable overexpression of Myo6 to preferential downregulation of Hes5 amongst different repressor and activator bHLH proneural genes examined is not clarified so far. One possible but hitherto unproven speculation is that intracellular Myo6 overexpression promotes the migration of particular cargo proteins and/or organelles required to suppress the transactivation of the Hes5 gene toward the nucleus. For instance, the mammalian target of rapamycin (mTOR) signals are known to be essential for maintenance of neural progenitor status through a mechanism associated with upregulation of Hes5 and Pax6 expression in pluripotent P19 cells cultured with ATRA sv/sv mice defective for Myo6, a significant deficit is seen in internalization of a particular ionotropic subtype of non-N-methyl-D-aspartate receptor (NMDAR) after stimulation In hippocampal neurons from Although Myo6 is highly expressed in cultured astrocytes expressing GFAP rather than in cultured neurons expressing NeuN It thus appears that the motor protein Myo6 selectively inhibits cellular proliferation required for self-replication through a mechanism relevant to downregulation of the repressor bHLH proneural factor Hes5 in undifferentiated P19 cells. Further evaluation of Myo6 expression will be undoubtedly beneficial for the future discovery and development of drugs useful for the prophylaxis, therapy and treatment of a variety of abnormalities and malfunctions seen in patients suffering from different neurodegenerative and neuropsychiatric diseases."}
+{"text": "BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China.Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations  accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations  accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated.A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.In this study, our findings suggest that  BRCA mutation spectrum in Chinese populations are limited BRCA1BRCA1/2 mutational screening using conventional full gene sequencing BRCA1 and BRCA2 mutation screening is rarely reported. In additional to BRCA mutation spectrum, identification of founder mutations in various ethnic groups is also important to improve genetic screening and cancer risk assessment because it makes a more specific approach for molecular testing that targets the founder allele possible, thus resulting in reduced cost of genetic testing and faster turnaround time. The high frequency of founder mutations in a given population provides a large patient cohort not only for robust information regarding penetrance but also accurate assessment of the effectiveness of preventive measures.The incidence of breast cancer in Asia has rapidly increased over the past 10 years and is one of the highest in Hong Kong population BRCA1 and BRCA2 genes using conventional full gene DNA sequencing and identified a recurrent mutation c.3109C>T BRCA1 and BRCA2 mutations in a group of 651 Chinese probands (inclusive of the 119 probands) from Southern China using full gene sequencing and full high resolution DNA melting (HRM) analysis. Our results also prompted us to investigate the usefulness of rapid HRM for screening of the BRCA1 and BRCA2 founder mutations in Chinese population.The Hong Kong Special Administrative Region of the People\u2019s Republic of China is an advantageous location to conduct such studies related to hereditary cancers in the Chinese population where 95% of the population is comprised of Chinese The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants involved in this study. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority West Cluster and other contributing hospitals, Hong Kong.www.HRBCP.org) from 1 March 2007 to 28 Feb 2011, were recruited. This group of 651 probands contained all 119 probands from our previous report BRCA mutations; or (6) they were ovarian cancer patients with a family history of breast cancer. The distribution of the 651 patients into the 6 categories was shown in BRCA counseling and testing, and were consented for genetic testing and blood and tumor collection. Patients who tested positive for a BRCA mutation were asked to help recruit their first-degree relatives, who were also offered testing. Written informed consent was obtained from all participants involved in this study. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority West Cluster and other contributing hospitals, Hong Kong.A total of 651 clinically high-risk breast and/or ovarian cancer patients (probands), referred to the Hong Kong Hereditary and High Risk Breast Cancer Programme  according to manufacturer instructions. Mutation analysis was performed by direct DNA sequencing of all coding exons of BRCA1 and BRCA2 and partial flanking intronic sequences. PCR conditions and primer sequences are available upon request BRCA1 and BRCA2, 41 PCR reactions for BRCA1 and 63 PCR reactions for BRCA2 were developed in our HRM run per each patient sample. Thus, samples were amplified in 384-well plates. LightCycler 480 High Resolution Master kit  was used for HRM analysis in patient samples in LightCycler 480 system (Roche). In brief, each reaction was performed in a final volume of 10 \u00b5l containing 20 ng of DNA, 0.25 \u00b5M of each primer (forward or reverse) and 1x LightCycler 480 HRM Master mix (Roche). The PCR profile was pre-activation at 95\u00b0C for 10 min, followed by 45 cycles of 95\u00b0C for 10 sec, a touchdown from 65\u201353\u00b0C for 30 sec at 2.5\u00b0C/sec and 72\u00b0C for 20 sec. At the end of the PCR cycles, PCR products was denatured at 95\u00b0C for 1 min and rapidly cooled to 40\u00b0C for 1 min. HRM analyses were performed from 60\u00b0C through to 98\u00b0C at a temperature gradient of 1\u00b0C/sec, acquiring 25 data points per \u00b0C. Each sample was run in duplicates for analysis. The analytical methods have been applied previously to our mutation scanning BRCA1 and BRCA2 genes were listed in To cover all exons of BRCA1 and BRCA2 germline mutations from unrelated families were genotyped for allele sharing indicative of a common ancestor. Thus, haplotype analysis was conducted at 6 microsatellite polymorphic loci D17S791, D17S855, D17S1323, D17S1322, D17S1335 and D17S1185 of the BRCA1 gene and at 6 loci D13S289, D13S1699, D13S1698, D13S171, D13S1695 and D13S260 of the BRCA2 gene. Primer sequences of all microsatellite polymorphic markers were listed in BRCA1 on chromosome 17q21.2\u201317q21.31 and a \u223c2.5 Mb region encompassing BRCA2 on chromosome 13q12.3\u201313q13.1. Fluorescently end-labeled primers were used to amplify the microsatellite polymorphic regions. Size fractioning of PCR products were performed on a 3130xl Genetic Analyzer (Applied Biosystems) using the GeneScan 500 LIZ Size Standard and analyzed using the GeneMapper v3.7 software (Applied Biosystems). Haplotype estimations were performed using software program PHASE.Individuals with identical 2 analyses describe any difference in BRCA mutations carriers among all Chinese female patients. The significance of age and BRCA status of patients was determined by Wilcoxon test. Fisher's exact test was used in the analysis of categorical data where expected counts are less than 5. Linear-by-Linear association was used in the analysis of ordinal data. SPSS for Windows Release 16.0  was used to analyze the data; statistical significance and marginal statistical significance were set at P<0.05 and P<0.1 respectively.P values from \u03c7This study included 651 probands , comprised of 611 breast cancer patients,17 ovarian cancer patients and the remaining 23 patients with both breast and ovarian patients. The mean age at diagnosis of breast cancer was 43 years old (range 18\u201382) and that of ovarian cancer was 43 years old (range 19\u201364). All probands were from Chinese ancestry and over 90% were from Guangdong province of Southern China.BRCA1 and BRCA2 of a total of 451 probands out of the 651 patients were conducted. Based on our sequencing results, 69  deleterious BRCA gene mutations were identified. Of the 69 deleterious mutations, 29  were in BRCA1 and 40  in BRCA2. There was no significant difference in the age of breast cancer diagnosis between BRCA carriers and non-BRCA carriers . Although the mean age of breast cancer diagnosis of BRCA2 mutation carriers (mean age 45.7) are slightly higher than that of BRCA1 mutation carriers (mean age 40.4), the difference was not significant . The spectra of all deleterious mutations identified are illustrated in BRCA1 and 17 were in BRCA2. Most of the novel deleterious mutations cause sequence frame-shift, leading to early termination of translation for protein products. In this study, we identified 4 recurrent BRCA1 mutations  accounted for 34.5% (10/29) of all BRCA1 mutations and 4 recurrent BRCA2 mutations  accounted for 40% (16/40) of all BRCA2 mutations. In addition, among our cohort of the 33 male probands, 7  deleterious mutations were found. Intriguingly, all 7 male probands carried BRCA2 deleterious mutations only.Extensive sequence analysis of all coding exons in BRCA1 and BRCA2 gene sequencing, we also developed full HRM assays for rapid screening of BRCA1 and BRCA2 mutations. In our developed full gene HRM assays, there are 38 BRCA1 and 63 BRCA2 HRM reactions per patients were established. In order to validate the testing performance of our in-house developed HRM assays, 8 probands with known BRCA1 deleterious mutations and 12 probands with known BRCA2 deleterious mutation from the 451 patients were analyzed. Those known BRCA mutations were previously identified by our full gene sequencing. In this first validation set, all 24 deleterious mutations and variants identified by sequencing were also detected by our in-house developed HRM assays. Thus, the detection rate for variant detection was 100% when compared to sequencing.Apart from the development of full BRCA1 deleterious mutations and 2 patients with different BRCA2 deleterious mutations. The detailed breakdown of all mutations and variants in the 24 patients was shown in BRCA1 and 1512 BRCA2 amplicons were analyzed (BRCA1 and BRCA2 were 1.2% (11/912) and 1.6% (24/1512) respectively. The calculated sensitivity and specificity for BRCA1 mutation and variant detection was 100% and 98.6% respectively while the sensitivity and specificity for BRCA2 mutation and variant detection was 100% and 98.3% respectively  and 4 recurrent BRCA2 mutations  were identified in our Chinese cohort . For BRCA2, the genotypes of all cases were examined at 6 polymorphic markers (BRCA1 mutation (c.981_982delAT) and 3 recurrent BRCA2 mutations  shared the same haplotype suggesting that these 4 putative founder mutations are derived from a common ancestor (BRCA2 mutation (c.3109C>T) BRCA2 mutations and so far is the highest proportion found in our cohort. For the only BRCA1 founder mutation confirmed, it accounted for 6.9% (2/29) all BRCA1 mutations in this cohort while all the BRCA2 founder mutations accounted for 35% (14/40) of all BRCA2 mutations. Characteristics of all 451 probands with or without BRCA mutations were shown in BRCA mutations have higher frequency of the family history of ovarian cancer than those without BRCA mutations. Furthermore, probands with BRCA mutations have greater average number of family member with breast cancer than those without BRCA mutations.In this study, a total of 4 recurrent e cohort . To dete markers . Haplotyancestor . For theBRCA2 founder mutation (c.7436_7805del370) is not easily detected by HRM. Thus, a panel of 5 HRM assays for the remaining founders was developed (BRCA2 founder mutation (c.3109C>T) from the 200 patients. Taken all together, the overall frequency of BRCA2 (c.3109C>T) founder mutation observed was 1.7% (11/651) among 651 Chinese patients and accounted for 26.8% (11/41) of all BRCA2 mutations in our Southern Chinese cohort. The total of 3 recurrent BRCA2 mutations then accounted for 36.6% (15/41) of all BRCA2 mutations.As some recurrent mutations were confirmed to be founder mutations in the Southern China population, we then rapidly developed HRM screening assays targeting each founder or recurrent mutations so as to further screen our Chinese population. Due to the 370 bp deletion, eveloped  and 2 anBRCA mutations to high-risk families of Chinese population from Southern China. Increasing cancer rates are of great concern to these Asian countries because these countries often have limited health and medical care resources and infrastructures to meet the needs of these patients. Thus, it is important to obtain a better understanding of the causes of breast cancer among Asians and other populations so as to improve prevention and cancer risk assessment efficiently worldwide. To our knowledge, this is one of the larger Chinese cohort studies comprising of 651 probands and comprehensive full BRCA1 and BRCA2 gene sequencing on 451 probands was employed for mutation screening analysis. Our findings revealed that the proportion of BRCA mutation is15.3% in a defined high risk group of Chinese population while Caucasian cohorts are estimated to be 5\u201313% In this study, we report the contributions of BRCA2 mutations (58%), similar to that reported in some Asian studies BRCA2 founder mutations in our cohort was higher when compared to that of the BRCA1 founder mutations. Of note, a similar pattern of a predominance of BRCA2 mutations compared to BRCA1 was recently observed in a study of high-risk Asian-Americans Unlike the Caucasian population, we found that a relative predominance of BRCA mutations which have not been published in the BIC Database of NIH. This proportion is relatively high in our Chinese cohort. Reports have found a high frequency of variants in different ethnic populations BRCA testing have not be widely performed, especially in Chinese population In this study, we identified 29 novel deleterious BRCA1 and three BRCA2 putative founder mutations in our Southern Chinese cohort. Interestingly, we also observed that half of the putative founder mutations (3 of 6) have not been published in the BIC Database , their founder effects are required to be confirmed by larger sample size of unrelated probands. The BRCA1 and BRCA2 recurring mutations that did not share a common haplotype could also be attributable to factors such as age of mutation such that associated alleles are no longer in linkage disequilibrium with microsatellite marker. Founder mutations not only provide population-specific genetic risk assessment, but are also useful in the study of penetrance of BRCA mutation in a specific population Notably, we discovered three Database . Since tBRCA1 c.981_982delAT and BRCA2 c.3109C>T are one of the most common mutations found in Asian countries such as China, Korea and Singapore BRCA1 c.5496_5506del11insA, BRCA1 c.390C>A, BRCA2 c.7480C>T and BRCA2 c.3109C>T, are unique mutations that were not found in either other Asian or even Caucasian populations according to our database search from BIC and HGVS. On the other hands, one recurrent mutation BRCA2 c.2808_2811delACAA was frequently observed in other ethnic populations such as Caucasian, African American, Hispanic and Australia.Based on our recurrent mutations identified in this study, some of them such as BRCA1 and BRCA2 founder mutations detectable by conventional DNA sequencing were also detectable by HRM, giving a high sensitivity for the latter method. The true sensitivity remains to be determined in a larger cohort including unselected Chinese women and men.The discovery of this founder mutation may provide a cost-effective option to rapidly screen a population. Our finding of complete concordance between conventional sequencing data and the HRM output in patients DNA suggests that the HRM technology is ready for use in diagnostic setting. Furthermore, there are several advantages of using HRM over other mutation screening methods: (i) Recent reports showed that the sensitivity and specificity of HRM is better than that of denaturing high-performance liquid chromatography (DHPLC) which is the current gold standard of scanning methods BRCA mutation analysis in a large Southern Chinese cohort and four founder mutations were identified. We then rapidly developed HRM mutation screening assays for those recurrent or founder mutations. The only BRCA1 confirmed founder mutations account for 6.9% of all identified BRCA1 mutations, whereas BRCA2 founder mutations account for 37.5% of all BRCA2 mutations. Our findings indicate that both BRCA1 and BRCA2 mutations account for a substantial proportion of hereditary breast/ovarian cancer in the Southern Chinese population. Identification of a founder mutation and knowledge of its prevalence in the Southern Chinese population provides important information both to genetic counseling and cancer risk assessment, as well as to the development of a cost-effective screening strategy.In conclusion, we conducted an extensive Table S1Distribution of the patients in this study according to the recruitment criteria.(DOC)Click here for additional data file.Table S2Sequences of high resolution melting (HRM) primers for BRCA1 and BRCA2 genes.(DOC)Click here for additional data file.Table S3Sequences of PCR primers for microsatellite polymorphic markers.(DOC)Click here for additional data file.Table S4The breakdown of mutations and variants of BRCA1 and BRCA2 in the 24 patients in the blind validation.(DOC)Click here for additional data file.Table S5Genotype of carriers with BRCA1 or BRCA2 founder mutations and family members without mutations.(DOC)Click here for additional data file."}
+{"text": "Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. Relaxin family peptide receptor (RXFP) 1 and RXFP2 are class A G-protein coupled receptors (GPCR). They are therefore members of the largest gene family in humans  by an unA similar mechanism of action of the LDLa module by the two receptors is implied by the degree of sequence similarity they share. The LDLa modules of RXFP1 and RXFP2 share 60% sequence similarity Figure , while rThe present study aims to further investigate this seeming contradiction, and establish why an ectodomain-swapped chimera of RXFP1 with RXFP2 can signal, while an equivalent LDLa-swapped chimera cannot. Upon scrutiny of the constructs made by Kern et al., it would appear that the point chosen to swap the LDLa modules was 27 residues C-terminally to the final cysteine (Cys40) necessary for LDLa structural integrity for RXFP1. Importantly, the equivalent region of RXFP2 which was replaced in the chimera is seven amino acids shorter Figure , meaningl-glutamine and 1% penicillin/streptomycin (referred to as complete DMEM) in incubators (Thermo scientific) maintained at 37\u00b0C, with 5% CO2 and 85% humidity.Recombinant human gene-2 relaxin (H2 relaxin) peptide was kindly provided by Corthera and human INSL3 was chemically synthesized as previously described . Human e2, 0.5\u2009\u03bcl of Taq (Bioline Velocity) and distilled, de-ionized water to make up to a total of 50\u2009\u03bcl. PCR conditions were 2\u2009min at 98\u00b0C, then 30 cycles of 98\u00b0C (30\u2009s), 58\u00b0C (30\u2009s), 72\u00b0C (15\u2009s) followed by a 7\u2009min extension at 72\u00b0C. The LRR/TM regions of each construct were similarly amplified  at 4\u00b0C overnight or at room temperature for 3\u2009h. The vectors were then transformed into DH5\u03b1 E. coli cells using the heat shock method and resultant colonies were picked and grown before isolating the plasmid DNA using a PureLink\u00ae Quick Plasmid Miniprep Kit. The entire insert of successful clones were sequenced to ensure the desired end product was correct, with no unintentional mutations incorporated.RXFP1, RXFP2, and chimeric constructs were cloned into a pcDNA3.1\u2122/Zeo+ AmpR mammalian expression vector  which contained an N-terminal FLAG tag and a bovine prolactin signal sequence . The chi6 cells, and grown overnight. The following day the cells were washed and resuspended in 1% FBS in phosphate buffered saline (PBS) (Gibco) (FBS/PBS) medium. Alexa 647 labeled anti-FLAG antibody (Invitrogen) was added at a 1:1000 dilution in FBS/PBS and cells were incubated on ice for 30\u2009min, then passed through a filter and sorted using FACS (Becton Dickinson FACS Aria III). Only the top 10% of cells with fluorescence levels significantly higher than the background fluorescence in non-transfected HEK293T control cells were collected. The sorted cells were grown in complete DMEM in the presence of 200\u2009\u03bcg/ml Zeocin until confluent. HEK293T cells stably expressing WT RXFP1 were used as a positive control in the FACS sort.Transient transfections were performed using lipofectAMINE\u2122 2000 (Invitrogen) according to the manufacturer\u2019s instructions. For competition binding assays chimeric receptors were selected for semi-stable expression and compared with cells stably expressing RXFP1 or RXFP2 . Semi-stThe presence of the chimeric receptors at the surface of cells was assessed in triplicate, exploiting the FLAG epitope on their N-termini using the method described previously . Briefly50 values were subjected to one-way ANOVA and uncorrected Fisher\u2019s LSD comparison test.Competition binding assays were performed on whole cells as described previously, using Europium labeled INSL3 (Eu-INSL3)  and Euro50 and maximum response (Emax) values were subjected to one-way ANOVA and uncorrected Fisher\u2019s LSD comparison test.Cells were assayed for cAMP signaling by co-transfection of receptors with a pCRE \u03b2-galactosidase  reporter construct as previously described . Cells wp\u2009<\u20090.01) higher expression than RXFP1 (141.2\u2009\u00b1\u200911.3% of RXFP1 expression)  from WT RXFP1 (pEC50 10.89\u2009\u00b1\u20090.03) . The replacement of the RXFP1 TM domain with that of RXFP2 in RXFP212 restored the H2 relaxin stimulated efficacy to 99.9\u2009\u00b1\u20095.3% Forskolin response   and 8.22\u2009\u00b1\u20090.17 for RXFP121 (p\u2009<\u20090.05)  did not differ significantly from RXFP2.RXFP122 has the TM and LRR domains from RXFP2 and the LDLa module from RXFP1. RXFP121 has the LDLa module and the TM region from RXFP1 and the LRRs from RXFP2 Figure ; Table 22+ binding (The activation mechanisms of RXFP1 and RXFP2 represent a unique paradigm in GPCR functioning, since these are the only two mammalian GPCRs that possess an LDLa module. When found in the LDLR and related receptors, these modules are typically involved in protein\u2013protein interactions, and are thus involved in a variety of interactions both with peptides and other molecules . The evi binding , 20, the binding . Both Ty binding . However50 values and unfortunately there was not sufficient Eu-labeled ligand to perform saturation binding to obtain Kd values. However, we have previously demonstrated that RXFP1 and RXFP2 receptors lacking the LDLa module bind ligand normally (50 values are unlikely to be related to the LDLa module swaps.While Kern et al. only made the RXFP1 chimera with the LDLa module of RXFP2 attached, we sought to characterize both this and the equivalent RXFP2 construct with the LDLa from RXFP1. We hypothesized that such chimeras would be informative in relation to common and distinct mechanisms of activation by the LDLa module. Additionally, we explored the concept that the LDLa is exerting its effect by interacting with the TM domain of the receptor in a specific manner by creating LDLa chimeras with matched TM domains in RXFP121 and RXFP212. The chimeric receptors were all expressed at the cell surface indicating they were folded correctly and able to be trafficked to the cell surface. The RXFP211 chimera had significantly higher cell surface expression as previously demonstrated for the chimera produced by Kern et al. (normally . Hence t50 was similar to what we have previously demonstrated when the key RXFP1 LDLa residue Leu7 is mutated to Lysine as it is in the RXFP2 LDLa module (Emax values may reflect the mismatch of the LDLa with the TM domain resulting in the RXFP2 LDLa module acting like a partial agonist on the RXFP1 receptor. Notably, we have seen similar decreases in Emax with multiple mutations of the RXFP1 LDLa module (Emax values compared to RXFP1 (As anticipated the chimeric receptors did show differences in ligand-mediated cAMP activity compared to the WT receptors. This was most obvious for the RXFP1 chimeras where H2 relaxin demonstrated both decreased potency as well as decreased % maximum Forskolin activity on RXFP211. The shift in pECa module . The deca module , 9. Furtto RXFP1 . The simto RXFP1 , 24. HenIt should also be pointed out that it is highly unlikely that changes in potency or efficacy in the receptor chimeras is related to decreased efficiency of homodimerization of the receptors. Our previous studies on dimerization of RXFP1  and RXFPTaken together, this study has shown that the LDLa modules of RXFP1 and RXFP2, which are unique among GPCRs, behave in a comparable fashion to one another, and that their mechanism of action must therefore be closely related. Additionally it is clear that the length of the linker region between the LRRs and the LDLa module is important for RXFP1 function. This information can be used to further elucidate a model of activation, as we gradually clarify the myriad of different elements that come into play upon binding and activation of these complex receptors. Given that relaxin has been implicated in various pathologies including cancer  and thatThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Purkinje neurons express, in high abundance, a glutamate gated chloride channel commonly known as the Excitatory Amino Acid Transporter subtype 4 (EAAT4). EAAT4 belongs to the family of glutamate transporters, which in mammalian nervous system is responsible for clearing synaptic glutamate . StudiesThe role of the chloride channel in glutamate transporters is only known for the EAAT subtype 5 (EAAT5). On rod bipolar cell axon terminals, EAAT5 activation by glutamate results in membrane hyperpolarization, which consequently inhibits terminal glutamate release . WhetherThe model comprises a single compartment with uniform distribution of AMPA receptors and EAAT4. The EAAT4 model is based on a 16-state kinetic model of EAAT2 . AMPA re"}
+{"text": "Bedside percutaneous dilatational tracheostomy (PDT) is a safe procedure with an acute complication rate of 10 to 15%. Our hypothesis was that having an experienced person performing or supervising the procedure results in extremely low complications with PDT. We formed a tracheostomy team which always included at least a consultant or specialist experienced (at least 25 procedures) in performing the procedure.A retrospective chart review of all patients who had PDT in a multidisciplinary adult medical surgical ICU during November 2008 to December 2010. The patients' demographics, indications for intubation and PDT, early and late complications, date weaned off the ventilator, date of decannulation, discharge from ICU and hospital, and outcome of these patients in the hospital were noted.n = 14 (24%) followed by severe head injury n = 13(23%) and cerebrovascular accident n = 8 (14%). The commonest indication for tracheostomy was airway protection n = 40 (73%) followed by prolonged mechanical ventilation n = 25 (45%). The median duration of intubation before PDT was 11 days (IQ 8 to 18). The median time elapsed between tracheostomy and weaning of ventilator was 1 day (IQ 1 to 3). However, the median time to decannulation was 37 day (IQ 10 to 136). Acute complication of paratracheal insertion occurred in n = 1 (1.8%) patient. No deaths were reported related to the procedure. However, n = 13 (22.8%) patients died during the hospital stay. No procedure was converted to surgical tracheostomy. The median duration between tracheostomy and discharge from ICU was 12 days (IQ 5 to 21). Chronic complication of subglottic stenosis occurred in n = 1 (1.8%) patient.Out of a total of 2,364 admission 57 patients underwent PDT, all with bronchoscopic guidance by an intensivist experienced in PDT (>25 procedures); there were 45 (78.9%) males and 12 (21%) females with the median age of 42 (range 18 to 90) years. The most common admission diagnosis was cardiac arrest PDT is an extremely safe procedure when performed by an experienced intensivist under bronchoscopic guidance. Our low complication rate is due to careful screening and selection of patients and being performed or supervised by an experienced intensivist under direct vision."}
+{"text": "In Cambodia, the highly pathogenic avian influenza (HPAI) A subtype H5N1 virus was detected for the first time in January 2004. From 2004 to 2010, there have been 26 outbreaks in poultry and 10 human cases reported in the country but the origin of these epizootics remains unclear.The phylogenetic relationships among the H5N1 strains were reconstructed by neighbour-joining and Bayesians methods. We analyzed the sequences of all 8 genomic segments of 40 H5N1 Cambodian viruses together with sequences from over 100 isolates from Southeast Asia including Vietnam, Thailand and Laos.All viruses isolated in Cambodia since 2004 belong to clade 1, genotype Z. Based on phylogenetic relationships, HPAI H5N1 virus was probably introduced from Thailand in 2004. In 2005 and 2006, several sublineages emerged in Cambodia and were probably the result of multiple introductions of H5N1 virus from Vietnam where similar strains were detected before outbreaks occurred in Cambodia. Interestingly, in 2006, we observed a north to south spread of the virus following a main road. A new sublineage appeared in summer 2006. Since then, all viruses isolated in Cambodia and South Vietnam clustered into this group, suggesting that this sublineage became endemic in the Southern Indochina peninsula. Other clades which have been imported to neighboring countries by migratory birds  have not been detected in Cambodia.Cambodia is essentially a poultry-importing country. The first poultry deaths were observed in semi-industrial farms that imported broiler and layer parental stocks from a sister company in Thailand, where concomitant outbreaks occurred. Then, multiple introductions of H5N1 viruses most likely occurred through illegal trading in poultry from Vietnam. Our data suggest that the clade 1 H5N1 virus is spreading essentially through poultry trading, particularly along road transportation routes. The role of the migratory birds appeared to be at most limited to local or regional transmission. The mechanisms described here would explain the maintenance and extension of the H5N1 virus within the Cambodia-South Vietnam regions for the last 6 years. This also highlights the persistent risk of H5N1 virus transmission in humans in the region while the new H1N1 pandemic virus that affects millions of humans is also frequently detected in pigs and shows a dangerous propensity to recombine."}
+{"text": "Expansions of the polyglutamine (polyQ) domain (\u226534) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27\u201333) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1\u20134% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31\u201332 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p\u200a=\u200a0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls . ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway. ALS is a late-onset, rapidly progressive and devastating neurodegenerative disorder, which is generally associated with selective degeneration of both upper and lower motor neurons (MNs) in the brain, brainstem and spinal cord. Ten per cent of all ALS cases are inherited and referred to as familial ALS (fALS); the remaining 90% are sporadic (sALS) et al. demonstrated that intermediate-length polyQ expansions are associated with neither Alzheimer\u2019s nor idiopathic Parkinson\u2019s diseases, but with ALS and the Parkinson-plus entity progressive supranuclear palsy et al. as well as Gispert et al. reported that among several other polyQ neurodegenerative disease proteins, only ATXN2 is associated with ALS risk In addition to rare mutations and common SNPs, a recent publication reported that ATXN2 dysfunction influences the TDP-43-dependent toxicity seen in ALS and that the intermediate-length expansions to 27\u201333 triplets in the ATXN2 polyglutamine (polyQ) region act as ALS risk factors in 4.7% of North American patients 8CAA(CAG)4CAA(CAG)8 sequence; expansion of this domain to a size \u226534 triplets with a pure CAG sequence primarily causes autosomal dominant SCA2 et al. to be interrupted by at least one CAA triplet et al. identified ATXN2 expansions in 40 ALS patients to be always interrupted by CAA triplets, and defined a haplotype of two ATXN2 SNPs (rs695871 and rs695872) in common between most cases with 3 CAA interruptions and another haplotype in common between most cases with 1\u20133 CAA interruptions ATXN2 usually contains a repeat structure with 22 or 23 triplets coding for glutamine and the (CAG)This study now aims to investigate the association of the ATXN2 chromosomal region with ALS risk in the Turkish population, considering not only the polyQ repeats, but also common SNPs and haplotype patterns.The Ethics Committee of Bo\u011fazi\u00e7i University approved the use of patient samples for this study. Written informed consent forms were obtained from all patients. Control samples were collected anonymously.A total of 236 Turkish ALS patients (158 sALS and 78 fALS) matching El Escorial Criteria 5\u2032- GGG CCC CTC ACC ATG TCG -3\u2032 and the FAM labeled reverse primer 5\u2032\u2212/56-FAM/CGG GCT TGC GGA CAT TGG -3\u2032. PCR cycles included 5 min. at 95\u00b0C, 30 cycles  and 5 min. at 72\u00b0C. Repeat sizes were determined by GeneScan Analysis  and evaluated independently by two authors (SL and \u00d6\u00d6). The reproducibility of the GeneScan analyses was validated via repeating 25 samples among 236 ALS cases and 45 samples out of 420 controls, corresponding to \u223c10% of both cohorts. A SCA2 positive individual with 41 ATXN2 repeat expansion size was used as an internal control in both PCR amplifications and GeneScan analyses. ALS patients with an ATXN2 expansion were further subjected to DNA sequencing, to assess the presence of CAA interruptions .The ATXN2 triplet repeat was amplified from DNA samples of patients and healthy controls, using polymerase chain reaction (PCR) with the forward primer Fisher\u2019s exact test was applied to evaluate the genetic association of ATXN2 expansion sizes with ALS risk, under both the allelic and genotypic models.http://pngu.mgh.harvard.edu/purcell/plink/) http://www.broad.mit.edu/haploview/haploview) In our independent GWA study (unpublished data) performed earlier, we investigated 733,202 SNPs in 116 out of the above 158 Turkish sALS patients and 109 age- and sex- matched neurologically healthy individuals, using the Illumina HumanOmniExpress SNP array. To examine the association of ATXN2 locus variants with ALS risk in the Turkish cohort under study, we extracted 250 kb genotype data, comprising the ATXN2 locus and the surrounding 50 kb (25 kb from 5\u2032 and 3\u2032 ends) using PLINK software  had a significant p-value of 0.0057 and correlated with an increased ALS risk (OR: 2.23). This risk haplotype was observed in heterozygous state in both Turkish ALS patients with ATXN2 expansions, which were part of the GWA genotyping study. Using permutation analysis, which eliminates false positive data after multiple testing more effectively than Bonferroni corrections We investigated a 250 kb region on chromosome 12q in 116 out of the above 158 Turkish sALS patients, including two of the four ALS patients with ATXN2 expansions. Ten of 28 SNPs were excluded due to low HWE and MAF scores. In single marker analysis, none of the SNPs within the ATXN2 gene by itself showed any significant association with ALS risk, but a trend towards association was observed for the SNP rs2239194 within the SH2B3 gene (p\u200a=\u200a0.063) . On the The first analysis of Turkish ALS patients regarding ATXN2 confirms its role as a risk factor. More importantly, this study identifies a common risk haplotype for ALS, containing the ATXN2 and its neighbouring SH2B3 gene.Saccharomyces cerevisiae via Drosophila melanogaster to Homo sapiensThe initial observation that ataxin-2 acts as a modifier protein of TDP-43 overexpression toxicity as a model of ALS risk was consistent from In our study, novel evidence indicates that ALS risk is impacted by a 15-SNP haplotype block in linkage disequilibrium across the genes ATXN2 and its downstream neighbour SH2B3. Haplotype association became more significant when SNPs from both the ATXN2 and SH2B3 genes were included; this suggests a role of the SH2B3 gene in ALS risk. The SH2B3 protein, also known as LNK, is a member of the SH2B (1\u20133) adaptor protein family. They all contain Src Homology 2 (SH2) domains, pleckstrin motifs and proline-rich regions. Thus, they can bind to phosphatidylinositol-lipid containing membranes and to the phosphorylated tyrosine residues, e.g. of receptor tyrosine kinases, modulating the signal transduction that controls proliferation and growth. They exert strong effects in hematopoiesis This first analysis of Turkish ALS patients on ATXN2, not only confirmed its role as a risk factor in rare cases with intermediate polyQ expansions, but also revealed novel evidence that SNPs across the ATXN2/SH2B3 genomic locus may modulate risk in a substantial fraction of ALS patients. These data need to be validated in large and independent populations. In the light of these findings, our results implicate a genetic  interaction between ATXN2 and SH2B3 genes, therefore we propose that it will be useful to investigate genetic variations in this genomic region of ALS patients.Table S1Data on Turkish ALS patients with previously identified mutations.(DOC)Click here for additional data file.Table S2Association Analysis of 18 SNPs across 250 kb at the ATXN2 Locus.(DOC)Click here for additional data file."}
+{"text": "INK4A/Rb rather than on regulation of p19ARF/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16 Bmi1 is part of the polycomb repressive complex 1 (PRC1), a transcriptional repressive complex that silences genes during embryonic development. PRC1 complexes act through recognition of H3K27Me3 epigenetic tags on histone 3 INK4A and p19ArfINK4A and p19Arf suggested that p16INK4A/p19Arf independent targets are responsible for the severe phenotype in mice Among the Bmi1 target genes are regulators of stem cell self-renewal, partially by repressing the senescence and apoptosis-regulating genes p16INK4A and p19Arf dependent as well as independent oncogenic functions of Bmi1 have been shown In cancer, both p16INK4A/Rb mediated effects rather then through p19ARF/p53 effects.Many reports have shown over expression of Bmi1 in tumors of astroglial, neural or neuroendocrine origin Table 1.All animal experiments have been conducted with approval of the ethical committee of the Netherlands Cancer Institute under references 04.003 B21/04.003 B38ext and 04.003 B23/04.003 B39ext. Genetically engineered mice with predispositions leading to cancer were checked twice every week for the occurrence of tumors. Animals were immediately sacrificed when a tumor was detected. All procedures have been conducted according to the standard operating procedures of the institute.Figure S1A, C and results not shown). The first mouse model is referred to as Bmi1LSL and the latter as Bmi1Lox66/Lox71. Detailed information on the transgenes is available at MGI . Reference numbers are: Bmi1LSL, MGI:4398910, Gt(ROSA)26Sor<tm1Nki>, also named Bmi-CTS, when inbred on a FVB background: NKI strain# 1353, when inbred on a C57BL/6 background: NKI strain# 1656. Bmi1Lox66/Lox71, MGI:4398914, Gt(ROSA)26Sor<tm2Nki>, also named Bmi-CTI, when inbred on a FVB background: NKI strain# 1148, when inbred on a C57BL/6 background: NKI strain# 1657.The cDNA encoding mouse Bmi1 with a downstream internal ribosome entry site (IRES)-eGFP cassette was cloned in the Rosa 26 targeting construct CEB11. This construct contains three transcriptional stop (poly A) signals flanked by LoxP sites to enable removal of this stop cassette upon LoxP-recombination (LSL transgenic strain was subsequently crossed with Actin-Cre (Acre) strain to analyze the phenotype when Bmi1 is activated constitutively throughout the mouse. In addition, transgenic mice were crossed to Glial fibrillary acidic protein promoter driven Cre 2Brn), LoxP, LSL to analyze the contribution of Bmi1 over expression on top of this background. All breeding was done on an FVB background. All tumors were analyzed by the pathologists of the NKI and in case of doubt, dr. Annemiek Rosem\u00f6ller, of the VU Medical Center, Amsterdam was consulted.The Bmi1LSL locus by LoxP recombination in the liver by intravenous injecting adenovirus which expressed Cre . For this, 2\u00d7109 virus particles per mouse (estimated 2% infection of hepatocytes) in a total volume of 100 \u00b5l was injected. Wild type mice were treated simultaneously as a reference. One week before the experiment, the mice were given cyclosporine  to reduce immunosupression of adenovirally infected cells. Rosa reporter mice (R26R) which have a Cre inducible beta galactosidase gene targeted to the Rosa26 locus were used as a control for the viral infection  or 1\u2236200 (in mouse tumors unless otherwise stated)), ACTH (Organon 11150), hGH (DAKO A570), LH , Prolactine (DAKO A569), TSH (DAKO A574), NCAM-1 , CAM 5.2 (B&D 349205), Chormogranine A (DAKO A430), anti-human Ki 67 , anti-mouse Ki 67 , Phospho-Histone H3 (Upstate 06-570), glial fibrillary acidic protein , S-100 protein , synaptophysin , p75 NTR, also named low affinity nerve growth factor (NGF) receptor , F4/80 antigen, a 160 kD glycoprotein expressed by murine macrophages , neurofilament , Keratin-8 . Antibodies were detected by peroxidase staining using the Powervision system (Immunologics) followed by visualization on a Zeiss Axiovert microscope.For electron microscopy, a selected area from paraffin-embedded material was dissolved in 1% osmiumtetroxide in toluene and embedded in epoxyresin LX-112. Light microscopy sections were stained with toluidine blue. EM sections were stained with tannic acid, uranyl acetate and lead citrate and examined in a Philips CM10 transmission electron microscope .Detection of eGFP in recombined transgenic ES cells was done on a Facs scan (BD).bona fide oncogene in non-lymphoid compartments, we analyzed the effects of over expression of Bmi1 in mice. For this, we developed a conditional lox-stop-lox Bmi1 transgene model, further on referred to as Bmi1LSL. In this model, Bmi1 is conditionally over expressed from the ROSA26 locus, under control of a combined CMV and beta-Actin (CAG) promoter. This enables constitutive expression of Bmi1 in the cellular compartment of interest independent of the transcriptional signals that normally regulate Bmi1 . All subsequent steps described below were performed with the Bmi1LSL mice.Bmi1 is a proto-oncogene which was identified by its ability to initiate lymphoid tumors Proper gene targeting was confirmed by Southern blot  with an expected frequency of 25%, p>0.1) and mice that were alive were observed until day E15.5-E18.5 suggesting that a neonatal defect is the reason for the death of the mice. Since Bmi1 deficient mice have strong growth defects, we analyzed growth during embryonic development ; Bmi1LSL mice allowing transgenic expression of Bmi1 in GFAP positive cells, which encompass different types of mature astrocytes as well as neural progenitors. Interestingly, these mice generated tumors with a latency of about one year, which shows that transgenic over expression of Bmi1 is sufficient to generate solid tumors  of which 6 tumors showed intracranial localization. In addition, a mammary tumor and a primitive neuroectodermal tumor (PNET) were found. The intracranial tumors consisted of cells of uniform size with round to oval nuclei with a fine chromatin patterning and a moderate to abundant amount of pale cytoplasm. These tumors are referred to as typical and this was observed in 5 out of 6 cases. In one case we observed an atypical form with anaplastic cells with a pleiomorphic nuclei and a thin rim of pale cytoplasm. This latter form was sometimes observed in combination with the typical form as well. The highly vascularised tumors localized to the pituitary gland/hypothalamus as observed from their caudal localization relative to the brain . To confirm that the tumors were derived from endocrine cells of the pituitary gland, the tumors were subjected to immunohistochemistry using a panel of pituitary hormones. This showed that the typical tumors were positive for adrenocorticotropic hormone  as are commonly observed in hormone producing pituitary adenomas (M and N).The number of Ki67 positive cells  ranged from less than 1% to up to 8.4% comparable to human pituitary specimens that typically show between 1% to 3.8% positivity (MIB index). Cytokeratin 8/18 as stained by the CAM5.2 antibody was negative (1). Additional analysis of neuroendocrine markers (Table S1) showed that the tumors stained positive for the intermediate lobe pituitary marker beta-endorphin (B-END) and synaptophysin (SYN), which are commonly observed in pituitary adenomas NGFR (not shown) and the glandular epithelial marker KER8. The tumor that showed the atypical cytomorphology had a different marker profile consisting of a mosaic pattern of expression of the neuroendocrine markers  mice Human pituitary adenomas are derived from the anterior lobe LSL potentially represses p19ARF/p53 function, we anticipated that GFAP-Cre; Bmi1LSL; RbLox/Lox mice might form medulloblastomas. To test this hypothesis, we crossed GFAP-Cre;Bmi1LSL on a RbLox/Lox background (n\u200a=\u200a12). We analyzed the incidence of tumors and these were histologically classified by two independent pathologists  and these formed medulloblastomas with an early onset as expected . These data indicate that the oncogenic function of Bmi1 in GFAP positive cells depends on p16INK4A/Rb regulation and less on p19ARF/p53 regulation.Combined loss of p53 and Rb mediated by GFAP-Cre in the cerebellum leads to medulloblastoma Lox/Lox;Bmi1LSL n\u200a=\u200a12, GCre;RbLox/Lox n\u200a=\u200a20). Although we did observe transgenic Bmi1 expression  is therefore dominant over inactivation the locus by Bmi1 over expression (which happens in in 40% of the cases).Earlier reports describing pituitary tumors because of Rb loss described a similar localization and histology LSL; Rbox/LoxL mice, shows that the transgenic Bmi1 over expression is insufficient to functionally inactivate the p19ARF/p53 pathway and indicates that the oncogenic role of Bmi1 is primarily dependent on repression of p16INK4A/Rb.Together, our results show that transgenic over expression of Bmi1 is sufficient to generate adrenocorticotropic pituitary tumors and a subset of clinical specimens of pituitary adenomas show high expression of Bmi1. The phenotype of Bmi1 over expression overlaps with the phenotype observed in Rb deficient mice and absence of medulloblastoma in GFAP-Cre; Bmi1Bmi1 is a proto oncogene and it\u2019s over expression has been observed in many tumors of neural and astroglial origin Table 1.INK4A expression by hypermethylation is a common mechanism in human pituitary tumors INK4A locus or, alternatively, points to an incomplete transcriptional repression of the p16INK4A pathway or could simply reflect the efficiency of Cre-mediated recombination events for the alleles used. Since no higher incidence of pituitary tumors was found upon combined Rb loss and Bmi1 over expression, this indicates that the oncogenic function of Bmi1 is dependent on the presence of a functional p16INK4A locus, this is substantiated by the observation that in the latter case, 40% of the tumors are positive in IHC for Bmi1, indicating that the remaining 60% of the tumors have inactivated the Rb allele independent of Bmi1 over expression.The incomplete penetrance and long latency period (1 year) of tumor formation upon Bmi1 over expression suggested that additional genetic or epigenetic defects might facilitate the generation of tumors. Loss of Rb itself has been shown to induce pituitary tumors in mouse models LSL mice in non transformed cells of the pituitary gland of postnatal day 8 mice indicates that that transgenic activation has occured before this time point. The occurrence of pituitary tumors using GFAP-Cre mediated recombination of Rb has been shown before in vitro effects, however, in vivo effects were moderate, reflecting the fact that high activity of p16INK4 and p19ARF are seen in cultures of neural stem cells, thereby increasing the reliance of cells upon Bmi1. In contrast, p16INK4 and p19ARF are not detectably expressed by neural stem/progenitor cells in developing or young adult mice We do not know at which stage GFAP-Cre mediated transgene activation is accomplished in our transgenic model although enhanced expression of Bmi1 in Gcre;Bmi1We show that the tumors in the transgenic mice were localized to the pituitary gland, although this could be shown for large intracranial tumors only because of lack material of earlier neoplastic stages. The majority (n\u200a=\u200a5 out of 6) of the tumors showed a typical cytomorphology and these tumors expressed ACTH. One tumor expressed multiple markers in a mosaic pattern and was referred to as an atypical type Table 3.ARF/p53. Hence, our data point to a Rb mediated function of Bmi1 in our model. The p16INK4A or the p53 pathways are frequently mutated in glioblastoma Transgenic expression of Bmi1 failed to generate medulloblastoma even in the presence of the predisposing deletion of Rb. In contrast, the p53/Rb double deficient mice control mice developed medulloblastoma after half a year as expected In our mouse model, we identified both anterior and intermediate lobe tumors. In contrast, human pituitary adenomas are considered to be derived from the anterior lobe only since all tumors resemble cell types of the anterior lobe hormone producing cell types In conclusion, we show here that Bmi1 transgene over expression is sufficient to drive pituitary tumors and we also show that more than 50% of clinical pituitary adenomas have high expression of Bmi1. Furthermore, Bmi1 initiated tumorigenesis is not enhanced by Rb loss which shows that the oncogenic function is largely dependent on Rb function and not on p53 function, which is further substantiated by the observation that Bmi1 overexpression is not sufficient to repress p53 sufficiently to generate medulloblastomas.Figure S1LSL construct contains a transcriptional stop sequence that can be removed by LoxP recombination and the Bmi1Lox66/Lox71 construct contains two partially mutated LoxP sites that recombine upon Cre expression resulting reversion of the DNA that is flanked by the LoxP sites which results in the formation of one recombined LoxP site that has a low chance of reversal of the recombination process. ES cells that were targeted with the Bmi1LSL construct (B) or Bmi1LOX66/LOX71 construct (C) show eGFP expression after Cre mediated activation of the transgene.(A) Comparison of the two targeting constructs for conditional over expression of Bmi1. Two LoxP recombination methods are used, the Bmi1(TIF)Click here for additional data file.Figure S2LSL mice (middle panel) when compared to a wild type controls (lower panel).Bmi1 immunohistochemistry of postnatal day 8 pituitary glands (upper panel) shows enhanced expression of Bmi1 in Gcre;Bmi1 The Bmi1 antibody was used at a concentration of 1\u223650. Bar is 10 \u00b5m.(TIF)Click here for additional data file.Figure S3Immunohistochemistry shows that Bmi1 transgenic mice generate pituitary tumors that stain positive for (A) beta-endorphin (B-END) and (B) synaptophysin (SYN). The tumor is negative for (C) the neural marker neurofilament (NF) as well as for the astroglial and schwann cell markers (D) GFAP and (E) S100, respectively. No Keratin 8 (KER8) staining was observed (F).(TIF)Click here for additional data file.Figure S4LSL transgenic mice shows expression of PCNA , Ki67  and phosphorylated Histone H3 , respectively.Immunohistochemistry of a typical and an atypical tumor that were generated in GCre;Bmi1(TIF)Click here for additional data file.Table S1LSLP induced tumors.IHC analysis of additional markers in Gcre; BmiP(DOC)Click here for additional data file."}
+{"text": "The utility of the WHO-defined stage 1 and 2 clinical factors used in WHO HIV/AIDS clinical staging in predicting low CD4 cell count has not been established in Uganda. Although the WHO staging has shown low sensitivity for predicting CD4<200cells/mmTo validate the World Health Organisation HIV/AIDS clinical staging in predicting initiation of antiretroviral therapy in a low-resource setting and to determine the clinical predictors of low CD4 cell count in Uganda.3 and 350cells/mm3 was 53.5% and 49.1% respectively. Angular cheilitis, papular pruritic eruptions and recurrent upper respiratory tract infections were found to be significant predictors of low CD4 cell count among participants in WHO stage 1 and 2.Data was collected on 395 participants from the Joint Clinical Research Centre, of whom 242 (61.3%) were classified as in stages 1 and 2 and 262 (68%) were females. Participants had a mean age of 36.8 years (SD 8.5). We found a significant inverse correlation between the CD4 lymphocyte count and WHO clinical stages. The sensitivity the WHO clinical staging at CD4 cell count of 250 cells/mmThe WHO HIV/AIDS clinical staging guidelines have a low sensitivity and about half of the participants in stages 1 and 2 would be eligible for ART initiation if they had been tested for CD4 count. Angular cheilitis and papular pruritic eruptions and recurrent upper respiratory tract infections may be used, in addition to the WHO staging, to improve sensitivity in the interim, as access to CD4 machines increases in Uganda. There have been significant declines in HIV-related morbidity and mortality since the advent of anti-retroviral therapy (ART) Some previous studies have explored the utility of using other predictors of low CD4 cell count to guide initiation of antiretroviral therapy. Other clinical factors, such as anaemia and body mass index have low sensitivities in detecting eligibility for HAART initiation In Uganda, although the availability of CD4 testing is increasing, its access is limited due to cost and a lack of laboratory infrastructure, particularly in rural health facilities. Specifically, CD4 testing is not available in decentralized health units that include district hospitals and health centre IV facilitiesthat serve the largest population of HIV-infected Ugandans.The WHO clinical staging system for HIV/AIDS was developed in 1990 and revised in 2007. It uses clinical parameters to classify subjects into any one of four categories i.e. stage 1 to IV, progressing from primary HIV infection to advanced HIV/AIDS. It is these categories that are used to guide decision making for the management of HIV/AIDS patients where there is limited access to laboratory services 3 to make decisions on ART initiation. However, in Uganda the CD4 cut-off for ART initiation changed from 200 cells/mm3 to 250 cells/mm3 or 350 cells/mm3 for the World health organisation. Previous studies of the utility of the WHO HIV/AIDS clinical guidelines for determining ART eligibility, using the cut-off of <200 cells/mm3 have found the sensitivity to be in the range of 51\u201352% and the specificity to be in the range 68\u201388% Initially the WHO clinical staging for HIV/AIDS was used in line with CD4 cut-offs of 200 cells/mm3 and <350 cells/mm3. Evaluation of the WHO HIV/AIDS staging guidelines in comparison to the newer CD4 cell count cut-offs for ART eligibility is needed to determine the potential level of misclassification. In addition, examination of the individual clinical components of the WHO HIV disease staging is needed because some clinical factors associated with stage I or II illness may be predictive of more advanced immune suppression in Uganda. We postulate that if there are significant clinical factors in stages I and II that are predictive of low CD4 counts, they could improve identification of ART-eligible subjects, thereby reducing the missed opportunities for timely ART initiation. The goal of this study was therefore to determine the diagnostic properties  of the WHO HIV/AIDS clinical staging guidelines at CD4<250cells/mm3 and <350cells/mm3 and to determine the WHO HIV/AIDS Stage I and II clinical factors (symptoms and signs) that are predictive of CD4<250cells/mm3 and CD4<350 cells/mm3.Despite the wide use of the WHO HIV/AIDS clinical guidelines in Ugandan primary health care facilities, they have not yet been evaluated in Uganda against the new CD4 cut-offs for ART eligibility of <250cells/mmTo address the study objectives, we conducted a multi-centre, cross-sectional study from January to April 2007 at three JCRC sites located at Mengo , Jinja  and Kasana . All the three sites offered free HIV/AIDS treatment and care at the time of study recruitment. The study sites were selected for convenience. The number of subjects enrolled from each site was proportional to the number of ARV treatment na\u00efve HIV patients seen at each study site in the previous 3 months. Subjects aged \u226518, known HIV positive, ART treatment na\u00efve, and consented to participate in the study were consecutively enrolled to participate in the study.*T 5 Diff CP, while the CD4 cell count was assessed using TriTEST CD4 FITC/CD8PE/CD3 PerCP (TRUCOUNT) reagent method. Quality assurance procedures are routinely conducted to compare the results to that of external laboratories .Clinicians with training in HIV/AIDS clinical staging administered structured questionnaires to study participants. They recorded the socio-demographic data , and clinical factors used for the WHO HIV/AIDS staging guidelines. The latter were used these to determine the patients' clinical stage \u03b12P(1\u2212P)\u00f7W2 where W is the width of confidence intervals was 1%, Z\u03b1 at 95% confidence interval\u200a=\u200a1.96, with an estimate of 50% of subjects that were expected to be eligible for ART The minimum sample size of 385 subjects for the study was determined using the cross sectional studies with 4Z3 and <350 cells/mm3 and calculated 95% confidence intervals for proportions, using STATA version 10.0 . We estimated the association between CD4<250cells/mm3 and <350 cells/mm3 and stages I and II clinical features using odds ratios at 95% confidence intervals. Variables with odds ratios and p\u22640.2 at bi-variate analysis were included in a multivariate logistic regression model to determine the adjusted odds ratios for the association between clinical stages I & II clinical factors and low CD4 counts.We determined the diagnostic properties of the WHO clinical staging compared to CD4<250cells/mmEthical approval was obtained from the Faculty of Medicine Research and Ethics Committee of Makerere University, the Joint Clinical Research Centre, and the Uganda National Council of Science and Technology. Written informed consent was obtained from each subject before enrolment into the study.Between January and April 2007 a total of 417 known HIV patients were screened. Twenty two (5.3%) participants were excluded from the study due to; age <18 years (n\u200a=\u200a1), prior ART experience (n\u200a=\u200a2) and inaccessible CD4 results (n\u200a=\u200a19). A total of 395 participants were enrolled into the study. The subjects were enrolled from JCRC Mengo (75%), Jinja (14%) and Kasana (11%). The majority (61%) of the 395 subjects were classified as in clinical stages 1 and 2. Over two thirds, (68%) of all enrolled subjects were females. . Three h3 (SD\u200a=\u200a270.93) as compared to male participants with 216.77 cells/mm3 (SD\u200a=\u200a226.08), (p<0.0012), while the mean CD4 counts among the urban-based population was higher at 301.62 cells/mm3 (SD\u200a=\u200a260.57) compared to the rural-based population with CD4 cell count of 206.03 cells/mm3 (248.12), p<0.0016.The median CD4 cell count of those in WHO HIV/AIDS clinical stages 1 and 2 was significantly higher than that of participants in clinical stages 3 and 4.  We found3 and 350 cells/mm3 were 53.5% and 49.1%, while the specificities were 81.1% and 86.8% at CD4 counts of 250cells/mm3 and 350 cells/mm3. The positive predictive values at CD4 cell count of 250cells/mm3 and 350cells/mm3 was 79.1% and 90.2%, and the negative predictive values were 56.6% and 40.9%, respectively. (3 and CD4<350cells/mm3).The sensitivities of the WHO clinical staging at CD4 cell count cut-offs of 250 cells/mmctively.  There we3 while angular cheilitis and recurrent upper respiratory tract infections  that had either angular cheilitis or pruritic eruption had a CD4 cell count of <250cells/mm3. Thus 44 subjects would have been correctly considered eligible for ART if any of the two clinical features had been used, while only 19 of the 63 subjects would have been started on ART earlier than the required time if they were considered. The identification of 44 eligible patients using the supplemental clinical symptoms of angular cheilitis and papular pruritic eruption would have improved the sensitivity of the clinical staging from 53.5% to 73.0% and subsequently reduced the false negative rate from 46.5% to 27%.To assess the effect of the use of angular cheilitis and papular pruritic eruption on the sensitivity, specificity and false negative rate to predict CD4<250cells/mm3 among patients in Stage I and II, a variable \u201cclinical factor present\u201d was generated if any of the patients in Stage II disease had one or both clinical factors of angular cheilitis and recurrent upper respiratory tract infection present.To assess the effect of the use of angular cheilitis and recurrent upper respiratory tract infections on the sensitivity, specificity and false negative rate to predict CD4<350cells/mm3 and therefore would have been eligible for ART initiation, while only 18 of the 81 subjects would have been treated earlier than the required time if it they were considered. The identification of63 eligible subjects using only angular cheilitis and recurrent upper respiratory tract infections would have improved the sensitivity of the clinical staging from 49.1% to 71.5% and subsequently reduced the false negative rate from 50.9% to 28.5%.Sixty three (77.8%) of the 81 subjects that had either angular cheilitis or upper respiratory tract infections on both were found to have CD4 counts <350cells/mm3 and 350cells/mm3, respectively. Our findings replicate the low sensitivities that have been previously found in studies of the WHO staging guidelines conducted in Africa Although the WHO HIV/AIDS clinical staging plays a critical role in guiding primary health workers in initiating antiretroviral therapy 3 and 350cells/mm3 respectively, which was also similar to previous studies using various CD4 cell count cut-offs 3 and at CD4<350cells/mm3 of 79.1% and at 90.2%, respectively; and negative predictive values of 56.6% and 40.9% respectively.We found that the specificity of the WHO HIV/AIDS clinical staging guidelines were at 81.1% and 86.8% at CD4 counts of 250cells/mmThe finding that females had a higher mean CD4 cell count than the males is similar to previous findings that demonstrated that women in stage 1 were less likely to have a lower CD4 cell count compared to the men The urban based population in this study may have had a higher mean CD4 cell count compared to the rural population because of better access to the HIV counselling and testing services leading to earlier HIV diagnosis.3 while angular cheilitis and recurrent upper respiratory tract infections predicted a CD4 cell count <350 cells/mm3. These results are in agreement with a Tanzanian study that found that HIV associated mucocutaeous manifestations may improve the sensitivity of the WHO staging guidelines In our study, we found that angular cheilitis and papular pruritic eruptions were significant predictors of CD4 cell count <250cells/mmWhile the WHO HIV/AIDS clinical guidelines are an affordable method to determine ART eligibility, routine or low cost CD4 T-cell count, as compared with WHO HIV/AIDS clinical staging in a resource limited setting is very cost-effective for sub Saharan Africa However, the availability of the tests and the laboratory personnel is still limited in Uganda. Therefore, determining other clinical factors that are predictive of low CD4 cell count in clinical stages I and II, will be very useful in improving the identification of subjects eligible for ART initiation until low cost CD4 cell count testing is widely available in Uganda. We recommend that similar studies with larger sample sizes be conducted in other developing countries to assess the role of clinical predictors, in addition to WHO staging guidelines in determining equivalents to CD4 counts.3 and 350cells/mm3. Further studies are recommended to assess the association between rare clinical features and CD4 cell counts.Our study had much strength; there was minimal referral bias since this was a multi centre study. Random error was minimised by having an adequate sample size for the common clinical predictors. The study was limited by a lack of sufficient sample size to detect the association between rare clinical conditions and CD4 counts below 250cells/mmWHO HIV/AIDS clinical stage misclassification was minimised by use of experienced clinicians trained in the study protocols and blinding of the clinicians to the CD4 results.Based on the findings of this study, we recommend increased access to CD4 cell count testing machines in resource limited settings. In the interim, more study is needed to confirm the clinical features that we found to be predictive of low CD4 counts. If these results are confirmed, then ART initiation among persons with these clinical features should be started regardless of WHO HIV/AIDS clinical stage. This will go a long way in identifying more patients eligible for ART."}
+{"text": "Studies investigating the outcome of conservative scoliosis treatment differ widely with respect to the inclusion criteria used. Prospective cohort studies are available using the SRS inclusion criteria for studies on bracing ,2. This 34 patients (of 152) fulfilled the SRS inclusion criteria with an average age of 12.06 years (10 \u2013 13 years), average Cobb angle of 31 degrees (25 \u2013 40\u00b0), an average Risser stage of 0,35, average in-brace Cobb angele of 13\u00b0 (= 59% of in-brace correction). There were 17 thoracic, 10 double major, 6 lumbar and 2 thoracolumbar curve patterns. After change of workplace of the second author the patients could not be followed up as planned. Therefore a telephone interview was performed by the first author.28 patients (average age 16.5 years) have been reached, 9 of them were still under treatment. No patient has been operated (Rate of surgery 0%) and only one was not satisfied with cosmetic outcome of treatment.Rate of surgery was far less than reported in recent studies using the same inclusion criteria even when all drop outs where rated as failures ,2.Rate of surgery can be reduced with the help of Ch\u00eaneau braces of the latest standard and satisfactory in-brace correction."}
+{"text": "We discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined.125I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver.We generated mutant mice with a novel Cthrc1 null allele by homologues recombination. Cthrc1 null mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 null mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 null mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with Cthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology. We originally discovered collagen triple helix repeat containing 1 (Cthrc1) in a screen for novel sequences induced in rat carotid arteries upon balloon catheter injury Targeted replacement of the first exon of the Cthrc1 gene by a LacZ reporter gene in mice was reported to demonstrate expression of Cthrc1 in inner ear hair cells Expression analyses at the RNA level using in situ hybridization have identified the sites of Cthrc1 expression during embryonic development. In addition, our studies have also shown that Cthrc1 is expressed by the activated fibroblast of remodeling tissues following injury The pituitary gland is the master endocrine gland, with the anterior pituitary expressing and secreting a variety of hormones and the posterior pituitary releasing oxytocin as well as vasopressin expressed by neurosecretory cells of the hypothalamus. Colloid-filled follicles of the anterior pituitary containing PAS (periodic-acid Schiff reaction) positive material have been reported in several vertebrates including humans Here we generated mutant mice with a novel targeted Cthrc1 null allele and focused on the analysis of their phenotype in adulthood. Monoclonal antibodies were generated against C terminal and N terminal epitopes of Cthrc1, which allowed us to localize Cthrc1 in tissues of a variety of species including pig. Our results demonstrate circulating levels of Cthrc1 in human plasma including expression in the anterior pituitary as well as neurosecretory nuclei of the hypothalamus.Guide for Care and Use of Laboratory Animals. All surgical interventions were performed under general anesthesia with tribromoethanol/tert. amyl alcohol. Human plasma samples were obtained under a protocol approved by the Institutional Review Board of the Maine Medical Center (protocol number 3657).All protocols involving animals were approved by the Institutional Animal Care and Use Committee of the Maine Medical Center (protocol numbers 0905 and 1112) and were in compliance with all applicable regulations and guidelines including the National Institutes of Health 5\u2032-CCACTGGAAACCTCTGGAGTTG-3\u2032 and 5\u2032-AAGTTCACACAAAGGAAGCCCCGC-3\u2032) and the mutant allele (primers: 5\u2032-GTGTGTTTTGAGGTGTGGTCCC-3\u2032 and 5\u2032TGGATGTGGAATGTGTGCGAGG-3). These animals have been backcrossed on the 129S6/SvEv (Taconic) or C57BL/6J background for 11 generations. Cthrc1 null mice for experiments were derived from matings of homozygous mutants. The mutant allele was backcrossed regularly with 129S6/SvEv or C57BL/6J breeder stock to prevent genetic drift. The age of the mice used for specific experiments is indicated in the figure legends.We generated a novel Cthrc1 null allele by replacing exons 2, 3 and 4 with a neomycin cassette using the targeting vector pKO Scrambler NTKV-1905 (Stratagene) . Exon 1 Mice were fed a standard rodent diet  and water ad libitum, and housed with dry cellulose bedding  under a 14 hour daylight-10 hour night cycle. Litter size was determined as the number of pups weaned at the age of 21 days after birth. Growth curves were obtained for Cthrc1 null and wild type mice on the 129S6/SvEv (n\u200a=\u200a5\u201333 per time point).Livers of seven month old mixed gender Cthrc1 null and wild type mice on the C57Bl/6J background were examined to assess histology and lipid content. Lipid storage was determined by image analysis of oil red O stained sections using the same approach as described previously 5\u2032- GACCCCTTCATTGACCTCCACTAC-3\u2032 and 5\u2032-ACATACTCAGCACCAGCATCGC-3\u2032 for Gapdh and 5\u2032-GAATGCCTGAGGGAAATCTTTGAG-3\u2032 and 5\u2032-CGTCTCCTTTTGGGTAATCTGCG-3\u2032 for Cthrc1. The annealing temperature was 55.9\u00b0C and 35 cycles of amplification were performed.Pig tissues were obtained from euthanized animals (6 to 12 months of age) of the Advanced Trauma Operative Management (ATOM) courses conducted at our facility or from a local slaughterhouse. Primer sequences used for RT-PCR of pig cDNA were Rat tissues from three month old male Sprague Dawley rats were kindly provided by Dr. Ren\u00e9e LeClair .Mixed gender wild type and Cthrc1 null mice on the 129S6/SvEv background were used for this assay . Samples were obtained from identical sites of the liver and the right gastrocnemius muscle. Glycogen from 10 mg fresh-frozen tissue was hydrolyzed to glucose in 500 \u00b5l of 2 N HCl for 2 h at 100\u00b0C with vortexing every 30 minutes. Samples were neutralized with 500 \u00b5l 2 N NaOH and 50 \u00b5l 1 M TRIS pH\u200a=\u200a7.6. The glucose hexokinase reagent (ThermoFisher) was used as directed. A standard curve was established with glucose standards covering the range of 0.00312 to 0.1 \u00b5mole. Using GraphPad Prism software, concentrations of glucose in the samples were calculated based on the standard curve. Three different samples from each organ were assayed in duplicates and mean values were calculated and compared using Student\u2019s t-test. The assay was performed four times with separate sets of samples from different mice and similar results were obtained each time. The results of a representative experiment are shown.Body composition was determined as described using a Lunar PIXImus II Mouse Densitometer  For glucose tolerance testing, mixed gender wild type and Cthrc1 null mice on the 129S6/SvEv background  were injected intraperitoneally with 1 g of glucose per kg of body weight after 16 hours of fasting. For insulin stress testing, mixed gender, non-fasted wild type and Cthrc1 null mice on the 129S6/SvEv background  were injected intraperitoneally with insulin . Blood was obtained for glucose level measurements using the One Touch Ultra glucometer at the indicated times.A rabbit monoclonal antibody was raised against a synthetic peptide of the conserved C terminus of Cthrc1 (GDASTGWNSVSRIIIEELP) using the services of Epitomics, Inc. . Clone Vli-55 was suitable for Western blotting and immunohistochemistry on paraffin-embedded, paraformaldehyde-fixed tissues. Rabbit monoclonal Vli-55 was used at 20 ng/ml for immunohistochemistry on paraffin-embedded, formalin-fixed tissue section following antigen retrieval with citrate buffer . Subsequent steps of the immunostaining procedure were executed as previously published For detection of human Cthrc1 in plasma, mouse monoclonal antibodies were raised against a synthetic peptide with sequence of the N terminus of human Cthrc1 (SEIPKGKQKAQLRQRE) using the hybridoma services of Maine Biotechnology Services . Anti-Cthrc1 clones 10G7 and 19C7 detected human Cthrc1 expressed in CHO-K1 cells by indirect ELISA without amplification in the low picogram range. Protein A-purified antibodies were conjugated to magnetic beads  following the manufacturer\u2019s protocol. EDTA plasma was obtained from healthy volunteers. 15 ml of plasma were incubated overnight at 1\u00b0C with anti-Cthrc1 conjugated magnetic beads, and washed extensively with phosphate buffer prior to elution with 0.1 M glycine, pH\u200a=\u200a2.6. The eluate was immunoblotted with HRP-conjugated monoclonal anti-Cthrc1 antibodies following SDS-PAGE under reducing conditions.125I (Perkin Elmer) using iodination tubes (Pierce). Six \u00b5g of radioactive labeled Cthrc1 were infused into adult anesthetized Cthrc1 null mice via the left carotid artery (n\u200a=\u200a3 mice). Blood samples were obtained at indicated times and Cthrc1 levels were determined in a gamma counter. The half-life in circulation was calculated from the clearance curve. SDS-PAGE analysis followed by autoradiography was performed on 1 \u00b5l of plasma obtained thirty minutes after injection of 125I-Cthrc1 to verify its integrity. All tissues were harvested six hours after 125I-Cthrc1 injection following extensive perfusion with lactated Ringer\u2019s solution to remove as much blood from organs as possible. 125I-Cthrc1 per mg wet weight of tissue was measured by gamma counting.An adenovirus was generated expressing rat Cthrc1 with a C terminal myc/6\u00d7His tag. CHO-K1 cells were transduced with this adenovirus and Cthrc1 protein was purified from the conditioned medium with HIS-Select affinity gel (Sigma) following the supplier\u2019s instructions. Silver-stained SDS-PAGE gels demonstrated >95% purity of the purified protein (not shown). A BCA protein assay (Pierce) was used to determine the concentration of the purified protein. Purified Cthrc1 was labeled with HEK293-T and CHO-K1 cells were grown as described and transfected with an expression vector for human Cthrc1 using Fugene6 HD (Roche) Data are expressed as means \u00b1 standard deviation. Student\u2019s t-test was used for all calculations. P\u22640.05 was considered significant.in vivo, we generated a novel Cthrc1 null allele by replacing three of the four exons (exons 2\u20134) with a neomycin cassette (tm1VliCthrc1) (To characterize Cthrc1 function 1tm1Vli) . This mu1tm1Vli) . Mice weTo verify that our Cthrc1 allele is a null mutation, we generated and characterized anti-Cthrc1 antibodies. We succeeded in developing highly sensitive and specific rabbit monoclonal antibodies against the conserved C terminus of Cthrc1. These antibodies allowed us to detect Cthrc1 by immunoblotting and in formalin fixed, paraffin embedded tissue sections by immunohistochemistry .The average litter size was slightly lower for Cthrc1 null mice, however, this did not reach a statistically significant level . Growth Liver histology with staining of sections for carbohydrates with the Periodic Acid Schiff (PAS) stain suggested increased carbohydrate storage in hepatocytes of Cthrc1 null mice on the 129S6/SvEv background. Quantification of the glycogen content in liver tissue with biochemical methods confirmed increased glycogen content in livers from Cthrc1 null mice . MeasureRoutine histology of livers from seven month old Cthrc1 null mice on the C57BL/6J background revealed strikingly larger hepatocytes compared to wild type controls. Overall this resulted in a significantly lower cell density . The livWe undertook a comprehensive immunoblot-based and immunohistochemistry-based survey of Cthrc1 expression in tissues from adult mice, rats and pigs. Expression of Cthrc1 in two month old C57BL/6J mice was detected in the supraoptic nucleus son,  and paraIn wild-type mice older than six months of age, we occasionally observed small accumulations of Cthrc1 immunoreactivity in the anterior pituitary . In all As shown above, certain areas of the brain constitutively express Cthrc1 but we currently have no evidence that Cthrc1 from those sites enters the circulation. In brains from pigs, we found foci of paraventricular cells of the lateral ventricles that expressed Cthrc1  and thesIn the anterior pituitary immunoreactive Cthrc1 was observed in close association with chromophobe cells  suggesti125I-labeled Cthrc1 into the carotid artery of Cthrc1 null mice and obtained the clearance curve shown in Immunoblotting of plasma from Cthrc1 transgenic mice We generated mouse monoclonal antibodies suitable for detection of Cthrc1 by ELISA. These antibodies conjugated to magnetic beads were used to isolate Cthrc1 from 15 ml of plasma. For one sample (#1)  the eluaWe originally discovered Cthrc1 in a screen for genes upregulated in balloon-injured arteries, where it was highly induced in activated adventitial cells. Database searches and sequence alignments revealed that Cthrc1 is a highly conserved and unique gene in chordates with no homologues found in lower species such as flies or worms Yamamoto et al. Here we generated a novel Cthrc1 null mutant mouse and focused on the characterization of its phenotype in adulthood. Unlike the two other reported targeted Cthrc1 mutants In summary, our study identifies Cthrc1 as a novel circulating hormone with metabolic effects. Expression in the hypothalamus, pituitary gland and remodeling tissues are likely to contribute to Cthrc1 plasma levels.125I-Cthrc1 to the liver supports this concept. The Cthrc1 null mutant mice examined here were derived from matings of homozygous null mice. Therefore, we cannot rule out the possibility that some of the metabolic abnormalities seen in the null mutants were due to maternal influences. However, litter sizes of the wild type and the homozygous null matings were comparable and we did not see any evidence of early postnatal failure to thrive on any genotype.While Cthrc1 expression was not detectable in the liver and skeletal muscle of the wild type, examination of these organs in our Cthrc1 mutant mice on the C57BL/6J background revealed excessively fatty livers and increased glycogen levels in skeletal muscle and livers of Cthrc1 null mice on the 129S6/SvEv background. This ultimately led us to consider the possibility that Cthrc1 functions as a hormone. We have currently no data related to the mechanism how Cthrc1 affects these organs but our findings do suggest that hepatocytes and myocytes in vivo may express a receptor for Cthrc1 and the binding of With a sensitive monoclonal anti-Cthrc1 antibody suitable for detection by ELISA we succeeded in demonstrating the presence of Cthrc1 in plasma. Working with plasma we deliberately avoided the use of secondary anti-IgG antibodies because even minimal cross-reactivity with human immunoglobulins could make discrimination between Cthrc1 and the similarly migrating band of the immunoglobulin light chain difficult on a Western blot. As shown in The current study also sheds light on the identity of colloid-filled follicles and the anterior pituitary as a source of Cthrc1. In guinea pigs, the first few colloid follicles of the anterior pituitary are detected at the age of 6 months with an average of just over 4 \u00b5m in size"}
+{"text": "Background. Hepatitis C virus (HCV) genotype 3 is known to cause steatosis (fatty liver) that is more frequent and severe than other genotypes. We previously identified sequence elements within genotype 3 HCV Core domain 3 that were sufficient for lipid accumulation. Aims. We examined various genotype 3 Core domains for lipid droplet localization and compared the lipid droplet binding regions of domain 2 with a genotype 1 isolate. Methods. We generated HCV Core domain constructs fused with green fluorescent protein and performed immunofluorescence to visualize lipid droplets. Results. Constructs containing HCV Core domain 2 are appropriately localized to lipid droplets with varying degrees of efficiency. When compared to genotype 1, there are polymorphisms within domain 2 that do not appear to alter lipid droplet localization. Conclusions. In summary, the differences in a steatosis-associated HCV Core genotype 3 isolate do not appear to involve altered lipid droplet localization. The significant differences with genotype 3 core protein that have been highlighted are the phenylalanine at position 164 and sequence polymorphisms within domain 3. Our results here demonstrate that neither of these differences contribute to significant differences in lipid droplet localization between genotype 3 and genotype 1 isolates. Deletion constructs from genotype 3 Core required domain 2 to localize to lipid droplets and sequence differences between genotype 3 and genotype 1 isolates within domain 2 did not occur within the critical regions for lipid droplet localization.In this study, we sought to further define the differences with HCV Core genotype 3 that contribute to clinical steatosis and These results further separate the phenomena of lipid droplet localization and intracellular lipid accumulation when examining the differences in HCV genotype 3 isolates. Previous studies have focused on regions within domain 2 being important for lipid droplet localization \u201333. In aIn the initial work describing regions within domain 2 that were required for lipid droplet binding, Hope and McLauchlan used deletion mutants of domain 2 and Boulant et al. constructed GFP fusion constructs , 31. OurWhen analyzing the sequence differences in domain 2 between genotype 3 and genotype 1 isolates, several things are notable. One is that all of the sequence differences are within the interhelical region or helix 2. Based on the work of Boulant et al., these 2 regions were absolutely essential for association with lipid droplets . HoweverOur results should be analyzed in the light of certain limitations. First, we used GFP fusion constructs which raises concern about the context of these results. However, our full-length GFP fusion and our domain 2 alone fusion both localized to lipid droplets, which is as predicted based on previously published work. The second limitation is this system lacks the context of other viral proteins and the entire viral life cycle. Again, these results are consistent with previous results using the JFH cell culture system.In conclusion, we have demonstrated that HCV Core genotype 3 exhibits the same requirements for lipid droplet localization as genotype 1 isolates and that sequence differences observed in genotype 3 do not alter the critical residues involved in this process. These results further separate lipid droplet localization from intracellular lipid accumulation in the mechanisms of steatosis formation in HCV genotype 3 infections."}
+{"text": "This reagent reduction strategy can be used for reducing the cost of enumerating CD4 lymphocytes in high-volume laboratories from resource-limited settings.Enumeration of CD4 lymphocytes is essential for the clinical management of HIV-infected patients, but it can be difficult to afford in developing countries. In this study we evaluated a reagent reduction strategy for reducing the cost of enumerating CD4 cell absolute count and percentage using the FACSCalibur flow cytometer (Becton Dickinson). We compared the protocol recommended by the manufacturer with a protocol that used half of the usual amount of CD3/CD4/CD45 monoclonal antibody reagent in 100 samples from HIV-infected patients in a rural hospital in India. The concordance correlation coefficient between the two protocols was 0.976 for CD4 cell count and 0.984 for CD4 cell percentage. We did not find significant bias when performing Deming regression or Bland-Altman analysis. Sensitivity and specificity were 97% and 98.5% for identifying patients with less than 200 CD4 cells/ In 2010, it was estimated that 34 million people were living with HIV/AIDS and more than 90% were living in low and middle-income countries  + b[slope]x). If the value of the y-intercept is significantly different from 0 indicates a constant bias and if the value of the slope is significantly different from 1 indicates a proportional bias. The agreement between the two protocols was assessed by using the Altman-Bland method and concordance correlation coefficients (CCCs) [Statistical analysis was performed using Stata Statistical Software . Bias was estimated using Deming regression . Deming s (CCCs) . \u03bcL  and the mean CD4 lymphocyte count with protocol B was 349\u2009cells/\u03bcL . The mean CD4 lymphocyte percentage with protocol A was 18.4%  and the mean CD4 lymphocyte percentage with protocol B was 18.4% . The study included 100 samples from HIV-infected patients. The mean CD4 lymphocyte count with protocol A was 366\u2009cells/y-intercepts included 0 and the confidence intervals for slopes included 1. The concordance correlation coefficient was slightly higher for CD4 cell percentage than for CD4 cell count. Correlation and Bland-Altman plots are shown in Estimation of bias and agreement between the two protocols are presented in \u03bcL need to initiate prophylaxis against Pneumocystis jirovecii pneumonia and this cutoff is useful for identifying patients in risk of opportunistic infections [\u03bcL is an indication for initiating antiretroviral therapy in patients older than 5 years and having less than 25% CD4 lymphocytes is an indication for initiating antiretroviral therapy in children aged 2 to 5 years [Sensitivities and specificities of protocol B for three clinically relevant cutoffs are presented in fections . In HIV- 5 years .Although the reagent reduction strategy has been successfully evaluated in previous studies , to our Protocol B is able to provide reliable results of CD4 cell count and percentage with half of usual amount of monoclonal antibody reagent. This reagent reduction strategy can be used for reducing the cost of enumerating CD4 lymphocytes with the FACSCalibur system."}
+{"text": "Two of these five loci have been suggested by previous association studies , and this study provides further support for these findings. Using a dataset of post-mortem brain samples assayed for gene expression (n\u200a=\u200a399) and methylation (n\u200a=\u200a292), we identified methylation and expression changes associated with PD risk variants in PARK16/1q32, GPNMB/7p15, and STX1B/16p11 loci, hence suggesting potential molecular mechanisms and candidate genes at these risk loci.A previous genome-wide association (GWA) meta-analysis of 12,386 PD cases and 21,026 controls conducted by the International Parkinson's Disease Genomics Consortium (IPDGC) discovered or confirmed 11 Parkinson's disease (PD) loci. This first analysis of the two-stage IPDGC study focused on the set of loci that passed genome-wide significance in the first stage GWA scan. However, the second stage genotyping array, the ImmunoChip, included a larger set of 1,920 SNPs selected on the basis of the GWA analysis. Here, we analyzed this set of 1,920 SNPs, and we identified five additional PD risk loci (combined  This paper describes the largest case-control analysis of Parkinson's disease to date, with a combined sample set of over 12,000 cases and 21,000 controls. After combining our findings with an independent replication dataset of more than 3,000 cases and 29,000 controls, we found five additional PD risk loci in addition to the 11 loci previously identified in earlier consortium efforts. This successful study further demonstrates the power of the GWA scan experimental design to find new loci contributing to disease risk, even in the context of complex disorders like Parkinson's disease. These new findings provide insights into the etiology of PD and will promote a better understanding of its pathogenesis. LRKK2PINK1SNCAPARK2PARK7SNCA and MAPT as unequivocal risk loci BST1GAKHLA-DRACMSD, STK39, MCCC1/LAMP3, SYT11, and CCDC62/HIP1RSNCA, LRRK2, MAPT, BST1, GAK and HLA-DRUntil the recent developments of high throughput genotyping and genome-wide association (GWA) studies, little was known of the genetics of typical Parkinson's disease (PD). Studies of the genetic basis of familial forms of PD first identified rare highly penetrant mutations in We conducted a two-stage association study. Combining stage 1 and stage 2, the data consist of 12,386 PD cases and 21,026 controls genotyped using a variety of platforms . Stage 1Here, we report the combined analysis for this full set of 1,920 SNPs. This step1+2 analysis identified seven new loci that passed genome-wide significance in the meta-analysis. During the process of analyzing these data and preparing for publication, we became aware that another group was also preparing a large independent GWA scan in PD for publication . Following discussion with this group we agreed to cross validate the top hits from each study by exchanging summary statistics for this small number of loci.To provide further insights into the molecular function of these associated variants, we tested risk alleles at these loci for correlation with the expression of physically close gene  and the methylation status (methQTL) of proximal DNA CpG sites in a dataset of 399 control frontal cortex and cerebellar tissue samples extracted post-mortem from individuals without a history of neurological disorders.p<10\u22123. We submitted the most associated SNP in each region for probe design and follow-up genotyping using the ImmunoChip platform. For each region of interest, we also added four SNPs in high level of linkage disequilibrium (LD) to provide redundancy where the most associated SNP would not pass the Illumina probe design step or the assay for that SNP would fail. To complete the array design we also added all non-synonymous dbSNPs located in known PD associated regions In addition to eleven loci that passed genome-wide significance in stage 1 p<5\u00d710\u22128, \u221210 overall). Taking either the nearest gene (or the strongest candidate when available) to designate these regions, these five loci are 1q32/PARK16STBD1, 7p15/GPNMB, 8p22/FGF20STX1B.On the basis of stage 1+2 results, seven new SNPs passed our defined genome-wide significance threshold . To understand the potential biological consequences of risk variation at this locus we tested whether rs708723 was correlated with either gene expression or DNA methylation status of proximal transcripts or CpG sites respectively (NUCKS1 (p\u200a=\u200a1.8\u00d710\u22127) and RAB7L1 (p\u200a=\u200a7.2\u00d710\u22124). We also found correlations with the methylation state of CpG sites located in the FLJ3269 gene (p\u200a=\u200a3.9\u00d710\u221222).rs708723/1q32 has been previously reported as PD associated PARK16,  but thisectively . We founSTX1B, the proximal gene to the most associated SNP rs4889603 is SETD1A. However, STX1B is located 18 kb upstream of rs4889603 and is a more plausible PD candidate gene STX1B (In the case of 16p11/ne STX1B .FGF20 gene (NCBI build 36.3), for which association with PD has been suggested previously in familial PD samples FGF20 or not remains unclear.The SNP rs591323 in the 8p22 region is located \u223c150 kb downstream of the STBD1 and 7p15/GPNMD have not been previously implicated in PD etiology. We found that the risk allele of rs156429, the most associated SNP in the 7p15 region, is associated in our eQTL dataset with decreased expression of the proximal transcript encoded by NUPL2 (GPNMB itself (The regions 4q21/by NUPL2 . The samB itself . NeitherNMD3 and 8q21/MMP16) showed strong evidence of association in stage 1 and 2 but were not disease associated in the Do et al dataset. Further replication is required to clarify the role of variation at these loci in risk for PD.Two additional loci (3q26/LRRK2 gene The strongly associated G2019S variant in the SNCA locus p>0.01) after conditioning on the main SNP in the region. In contrast, after conditioning on the most associated SNPs rs356182 in the SNCA region, several SNPs remained convincingly associated (p\u200a=\u200a9.7\u00d710\u22128 for rs2245801 being the most significant).The ImmunoChip array design provides some power to detect whether multiple distinct association signals exist at individual loci. Indeed, if a SNP showed an independent and sufficiently strong association in stage 1, it would have been included in stage 2 provided that it was not located in the same 10 kb window as the primary SNP in the region. There is precedent for this in PD, with the previous identification of independent risk signals at the LRKK2Lastly, we performed a risk profile analysis to investigate the power to discriminate cases and controls on the basis of the 16 confirmed common associated variants . For eacPARK16, 8p22/FGF20) implicate regions that had been previously associated with PD risk PARK16 showed convincing evidence of association in the Japanese population FGF20 locus had been previously reported in a study of familial PD STX1B/16p11, STBD1/4q21 and GPNMB/7p15) are new.The combination of GWA scans and imputation methods in large cohorts of PD cases and controls has enabled us to identify five PD associated loci in addition to the 11 previously reported by us. Two of these loci (1q32/p\u200a=\u200a2.2\u00d710\u22123) improvement of our ability to discriminate PD cases from controls.Adding the eleven previously reported common variants PARK16 region with the RAB7L1 and NUCKS1 genes  is in low LD (r2\u200a=\u200a0.21) with rs2301134, the SNP reported in SNCA/4q22. A more exhaustive fine-mapping analysis using either sequencing of large cohorts or targeted genotyping arrays will also be required to fully explore this locus.The ImmunoChip data provide limited resolution for the detection of multiple independent association signals in these regions. A previous study As yet, we do not know which of the variants and which genes within each region are exerting the pathogenic effect. We cannot exclude that some of the currently reported variants are in fact tagging high penetrance, but rare, mutations \u22127, minor allele frequencies at a minimum of 1%, exclusion of first degree relatives, and the exclusion of ancestry outliers based on either principal components or multidimensional scaling analyses using either PLINK Participating studies were either genotyped using the ImmunoChip as part of a collaborative agreement with the ImmunoChip Consortium, or as part of previous GWA studies provided by members of the IPDGC or freely available from dbGaP Single SNP test statistics were combined across datasets using a score test methodology, essentially assuming equal odds ratio across cohorts. In addition, fixed and random effects meta-analyses were implemented in R (version 2.11) to confirm that the score test approximation does not affect the interpretation of the results. We also tested the relevant SNPs heterogeneity across cohorts and no significant heterogeneity was detected .2 value on their genotyping platform and provided us with the following summary statistics: odds ratio, direction of effect, standard error for the estimated odds ratio and one degree-of-freedom trend test P-value.We communicated to our colleagues in charge of the independent study  the seven SNPs listed in Quantitative trait analyses were conducted to infer effects of risk SNPs on proximal CpG methylation and gene expression. For the five replicated SNP associations , all avaTable S1Summary of results for fixed and random effects meta-analysis, as estimates of effect heterogeneity across cohorts and SNP used at the Do et al replication stage.(XLSX)Click here for additional data file.Table S2Summary of the quality control parameters applied to the GWA datasets included in this study.(XLSX)Click here for additional data file.Table S3Complete list of tested QTL associations (expression and methylation).(XLSX)Click here for additional data file.Text S1Membership of the Wellcome Trust Case Control Consortium 2.(DOC)Click here for additional data file."}
+{"text": "LAMA2 gene that encodes the laminin \u03b12 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected based on homozygosity mapping and laminin \u03b12 chain deficiency.Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases.We investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2 gene, a homozygous T > C substitution at position +2 of the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients from the 4 families revealed variable disease severity particularly as regards motor achievement and disease progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases indicating a founder effect of the mutation in all 4 families.A previously described mutation in the LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical  LAMA2 gene encoding the laminin \u03b12 chain , \u03b1-dystroglycan , \u03b1-dystroglycan , perlecan  and collagen VI  were investigated in patient 3. Dystrophin, \u03b1-, \u03b2- and \u03b3-sarcoglycan  were tested in patients 6 and 8.Cryosections were incubated in primary antibodies diluted in PBS for 1-2 h at room temperature or overnight at 4\u00b0C. Slides were washed 5 min \u00d72 in PBS, then secondary antibodies were applied in PBS for 30 min-1 h at room temperature. After washing again 5 min \u00d72 in PBS, coverslips were mounted using ProLongGold with DAPI . Secondary antibodies were either goat-anti-mouse tagged with AlexaFluor488 or goat-anti-rat tagged with AlexaFluor594 (both from Molecular Probes).LAMA2 exons (exon numbering according to the Leiden muscular dystrophy database) [Genomic DNA was extracted from peripheral blood and analyzed by the polymerase chain reaction touchdown method using oligonucleotide primers flanking the intron-exon junctions of all 65 atabase) . AberranTotal RNA was prepared from skeletal muscle using TRI Reagent . The isolated RNA was reverse transcribed using a First-Strand cDNA Synthesis Kit , the resulting cDNA was amplified by reverse transcriptase PCR (RT-PCR) and sequenced using appropriate primers.Haplotype analysis was performed on genomic DNA using microsatellite markers provided by Genethon human genetic linkage map  and flanClinical findings are summarized in Additional file Electrocardiogram (performed in 5 patients) and echocardiogram (performed in 4 patients) were normal; cardiac signs were reported in none of the patients. Respiratory support was necessary in patient 3 who died of respiratory failure at 7 years and in patient 5 who also died of respiratory failure at 16 years. No mental retardation, epilepsy or eye abnormalities were observed. MRI or CT, performed in 8 patients, revealed white matter changes in all cases  showed mild dystrophic features; whereas muscle biopsies from patients 5 (taken at 3 years and 2 months), 6 (taken at 5 years) and 8 (taken at 4 years) showed marked dystrophic features. Immunofluorescence analysis of the laminin \u03b12 chain was only performed in patient 3 (with 3 antibodies) and in patient 8 (1 antibody); this revealed greatly reduced staining intensity with the 3 antibodies in patient 3  revealed abnormal conformers in exon 26. The exon was sequenced and showed a T > C substitution at position +2 of the consensus donor splice site. This mutation has been described previously in two siblings from a consanguineous Saudi family unrelated to ours . Direct Because of the consanguinity in the Saudi families and presumed consanguinity in the Sudanese family, it is likely that all affected siblings are homozygous for this mutation.LAMA2 gene in the 4 index cases indicated an identical mutation-associated haplotype (containing the mutation) between marker D6S407 and intragenic polymorphism G6286A (data not shown), suggesting remote consanguinity and a founder effect in all 4 families. By contrast, the downstream markers  were not identical in the 4 cases.Investigation of flanking and intragenic microsatellite markers of the LAMA2 gene associated with variable clinical phenotype. The main phenotypic differences among our 9 cases regard motor achievement. Patients 1 and 2 from the Sudanese family walked at 4 years; patients 7, 8 and 9 from a Saudi family walked at 3, 3.5 and 4 years, respectively. These 5 patients were still walking at 8, 11, 12 and 13 years when last seen, while patients 3, 4, 5, and 6 from the 2 other Saudi Arabian families never walked.In 3 families from Saudi Arabia and 1 from Sudan we have identified a previously reported homozygous c.3924 + 2 T > C mutation in the CK levels were also variable, being very high  in patients 3, 6, 7, and 8; high  in patients 1, 2, 4 and 5; and normal in patient 9.Features common to all were floppiness in infancy, delayed motor milestones or failure to achieve walking, brain white matter attenuation on MRI or CT (not done in 1 patient) and development of joint contractures/foot deformities. Eye and cardiac abnormalities were not observed. In patients 3 and 5 respiratory compromise was present, and both died of respiratory failure at 7 and 16 years, respectively.LAMA2 mutation as found in our patients (but characterized as 3973 +2 T > C according to the previous LAMA2 nucleotide sequence numbering). Both were mildly affected: the boy at 3.5 years had muscle hypotonia and inadequate head control, but walked at 26 months; his younger sister also had hypotonia from early infancy and poor head control and achieved walking at 3 years 8 months. Both had slightly reduced laminin \u03b12 chain expression as investigated by 2 antibodies recognizing the G domain, and highly reduced expression using an antibody recognizing the N terminal. These results demonstrated for the first time that use of more than one antibody can provide valuable indications as to what domain(s) of the laminin \u03b12 chain may be affected in CMD patients. Thereafter, this procedure became the standard method for staining muscle of patients with CMD and has also been used for prenatal diagnosis.In 1997 Allamand et al.  reportedBy contrast, in our patient 3, analyzed with 3 different antibodies, laminin \u03b12 chain expression was markedly reduced: clearly more so than the cases described in Allamand et al. . He was It is noteworthy that our cases show a much wider clinical spectrum than suggested by the siblings described by Allamand et al. : from thLAMA2 gene, which was unrelated to laminin \u03b12 chain expression (completely undetectable in both cases). Such clinical variability implies the presence of other genetic or epigenetic factors able to influence disease phenotype .LAMA2- associated haplotype. Modifier genes, such as those coding for proteins that interact with laminin \u03b12, and/or epigenetic factors, for instance those involved in regulatory signaling functions, are likely to contribute to the observed phenotypic variability. A drawback of the present study is that muscle biopsies were stained for laminin \u03b12 in only 2 cases and extensive immunohistological characterization was only possible in one case.The data of the present study provide further evidence that the clinical spectrum of MDC1A is more heterogeneous than previously thought; motor achievement and disease progression are particularly variable, making it difficult to formulate prognoses even in patients with an identical LAMA2 gene in our patients, strongly suggest a founder effect. The mutation probably originated in Saudi Arabia since studies on severe childhood autosomal recessive muscular dystrophy (SCARMD), the common form of muscular dystrophy in North Africa and the Arabian Peninsula [http://www.dmd.nl/. The clinical implication is that a laminin \u03b12 chain deficiency in Middle Eastern or Sudanese patients should initially prompt a search for the c.3924 + 2 T > C mutation in the LAMA2 gene.We emphasize, finally, that the identical intragenic polymorphisms and upstream microsatellite markers of the eninsula -21 indiceninsula . FurtherLAMA2-associated haplotype.Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.The authors declare that they have no competing interests.CDB carried out the molecular and microsatellite analysis and drafted the manuscript; EB performed molecular analysis; MAMS evaluated clinical and neurologic features of the patients, collected DNA and muscle biopsies and revised the manuscript critically for important intellectual content; MCM carried out homozygosity mapping and linkage analysis; SAM performed immunochemical evaluation of muscle biopsies and prepared the figures; MZS, MMM and ZAK evaluated neurologic, neurophysiologic and MRI features; CAW and KPC supervised the study and critically revised the manuscript; LM and RM contributed to writing the clinical reports, revised the manuscript critically and provided financial support; MM was responsible for study design, supervised the study and manuscript completion. All authors read and approved the final manuscript.Table S1. Clinical findings.Click here for fileFigure S1. Sequencing of genomic DNA from control (A) and patient (B) showing the T > C transition at position +2 of the consensus donor splice site of exon 26. Direct sequencing of the cDNA revealing a 189 bp in-frame deletion, corresponding to aberrant skipping of the whole of exon 26: (C) control and (D) patient.Click here for file"}
+{"text": "Histone tails play an important role in nucleosome structure and dynamics. Here we investigate the effect of truncation of histone tails H3, H4, H2A and H2B on nucleosome structure with 100 ns all-atom molecular dynamics simulations. Tail domains of H3 and H2B show propensity of  Histone tails are the most common sites of post-translational modifications. Tail modifications alter both inter and intra nucleosomal interactions to disrupt the condensed chromatin structure, thereby playing crucial role in gene access. Here we investigated histone tail functions on the stability of a single nucleosome in atomic detail by selectively truncating tail domains in molecular dynamics simulations. Our study revealed that truncation of H3 or H2A tail results in structural alterations in the nucleosome core whereas truncation of H4 or H2B tail does not. A potential role of H2A C terminal tail in regulating nucleosome stability is discussed. Finally, an  Eukaryotic DNA is organized into nucleosomes The The nucleosomal organization of eukaryotic chromatin presents a physical barrier to DNA access and also acts as a repository of epigenetic marks controlling chromosomal behavior during different periods of the cell cycle Post-translational modifications of histones play a key role in the regulation of gene access in eukaryotes A major challenge in chromatin research is to characterize the effect of tail modifications on nucleosome mobility and stability. Evidence suggests that the modifications may recruit chromatin-binding proteins Truncating the end of the H2A C terminal domain results in a 2.4 fold increase in the nucleosome sliding rate Although progress has been made Simulations were performed on the intact nucleosome and on tail truncated nucleosomes with each of the four types of histone tail  truncated, one at a time. The N-terminal tail domains of H3, H4, H2A and H2B were removed up to residues 26, 17, 11 and 20, respectively, and residues 118\u2013128 were also removed from the C-terminal tail domain of H2A . SimulatThe starting structure of all molecular dynamics (MD) simulations was taken from the For each of the simulations, the water molecules and ions were first energy minimized and equilibrated at 300 K for 160 ps with the solute kept fixed. The whole system was then energy minimized for 10000 steps using the conjugate gradient method and keeping the positions of the protein backbone atoms fixed. Harmonic restraints on the backbone atoms were then relaxed stepwise during 30 ps heating and 160 ps equilibration of the whole system.http://www.nics.tennessee.edu/computing-resources/kraken) generating 1 ns of trajectory took approximately 1700 CPU hours on 504 cores generating about 50 gigabyte of raw data.It was observed that H3 and H2A tail truncation destabilizes the nucleosome structure. To verify these results 2 additional independent simulations of H3 tail-truncated nucleosome and 1 additional simulation of H2A tail-truncated nucleosome were performed. Each of the simulations was 100 ns long, making a total of 8 simulations and 800 ns of combined trajectory. On the Kraken Cray XT5 machine  histone tails adopt disordered conformations with many amino acids having occupancy zero. In the simulation these tail domains primarily remain bound to DNA with a low degree of ordering. The overall secondary structure conservation in histones is plotted in To examine the overall stability of the histones during the 100 ns intact nucleosome trajectory we calculated the RMSD of each histone (excluding tails) from the equilibrated structure . The ploTo quantify the overall effect of tail truncation on the nucleosome structure, we define an order parameter, upper panel).To illustrate how tail truncation affects nucleosome structure ulations . Tail trulations . We callTo locate the structural domain of the H2A monomer that is responsible for the increased RMSD, we computed ajectory . The RMSajectory .The simulations reported here are non-equilibrium simulations each of which can follow a different pathway. However, we found that certain changes involving the H2A(2)Realistic parametrization of force fields for the nucleic acids has been a long-standing problem While analyzing the DNA dihedral parameters we did find that one nucleotide base (Cyt49(J)) is unstacked and the neighboring bases (Gua98(I)-Cyt50(J)) show unusual (non Watson-Crick) base-pairing in the intact nucleosome simulation. However, this did not lead to any unusual fluctuation in the neighboring amino-acid residues. Furthermore, since the DNA backbone and helical parameter values derived from all other base pairs are in agreement with x-ray and simulation data, this may not be artefactual.DNA major and minor groove characteristics are plotted in In the intact nucleosome simulation the DNA RMSD stabilizes around 3.2 \u00c5  Analysis of the trajectories with truncated H3 tails revealed an active role of histone arginines in structural alterations in the H2A(2)Concomitant with the above Arg81 interaction change, there is also a change of interaction of the nearby Arg88 of H2A(2) . WhereasThe change of interaction of arginines upon truncation of the H3 tails is likely a result of local change of electrostatic environment . In the Interestingly, truncation of the H2A tails (simulation 1) causes interaction changes in the same H2A(2)The crystal structure of the nucleosome core particle shows that the histone H2A C terminus lies close to the H3 Truncation of the H3 tail destabilizes contact of the H2A docking domain with the surrounding amino acids . In TablDestabilization of the H2A C terminus upon H3 tail truncation affects the C terminus-DNA contact. In the intact nucleosome simulation the H2A C terminus (Lys118 and Lys119) makes stable contact with the DNA through hydrogen bonding. Upon H3 tail truncation the C terminus switches between states \u2018in contact\u2019 with DNA and \u2018detached\u2019 .Truncation of H2A tails removes H2A C terminus-DNA contacts. This affects the remaining part of the H2A tail differently in the independent simulations : in simulation 1 the H2A docking domain breaks contact with Lys44 and Ile51, in the other only minimal loss of contact with Lys44 is observed . FurtherThe aim of the present study was to provide atomic-level information on interactions within the nucleosome that are altered upon tail truncation. This was accomplished by multiple all-atom MD simulations of intact and tail-truncated nucleosomes, with each trajectory covering a time frame 5 times longer than previously-reported simulations comparing intact and tail-truncated nucleosomes The formation of ractions . The staHyperacetylation of lysine residues neutralizes its positive charge by transferring an acetyl moiety onto the The reason for observing interaction changes for one H2A monomer in H3 tail-truncated simulations is not clearly understood (a comparison of interaction changes between histone monomers is provided in Supplementary in vitro studies The H2A docking domain provides the interaction surface between the histone H3\u2013H4 tetramer and the H2A\u2013H2B dimer . DestabiIn the simulations we observe a correlation between the breaking of contacts of the H2A docking domain with close by amino acid residues and the change of interaction of Arg88 of the H2A(2)e formed . When the formed .We also found that certain changes involving the alteration of sidechain hydrogen bonding of Arg88 are common to all the H3 and H2A tail-truncated simulations Click here for additional data file.Text S2Finding H2A docking domain contacts. Through the contact map analysis we want to find contact residues between the H2A docking domain and its surrounding which also form an interacting pair. To do this it is necessary to verify the contact map based information with visualization in 3D. This is achieved by the Molsurfer program which, in addition to the 2D map, has an interface for viewing in 3D (WebMol) as shown in Fig. S19. In the Figure the 2D contact map is shown the left panel whereas in the right panel the docking domain and its surrounding are shown in 3D in backbone representation. In the program when the cursor is pointed to a grid location on the 2D map the corresponding position is shown on the 3D interface by a red dot. One can then \u2018focus\u2019 (or zoom in) on this red dot to see which residues are forming an interacting pair and are in contact. Furthermore, the residue contacts found using Molsurfer are validated by visualizing the trajectory in VMD.(PDF)Click here for additional data file.Figure S1DNA phosphorous atom B-factors obtained from X-ray crystallography (dotted line) and those computed from the last 50 ns of intact and tail-truncated nucleosome simulations (continuous lines). The B-factors are shown for the two chains of DNA: I and J. The labels under the curves indicate the histone chains and secondary structure elements that make intermolecular contacts with the DNA.(PDF)Click here for additional data file.Figure S2DNA helical parameter fluctuations during intact nucleosome simulation. Average helical parameters with fluctuations (standard deviation) indicated as error bars are compared with those obtained from the crystal structure (1KX5.pdb).(PDF)Click here for additional data file.Figure S3DNA phosphorous atom RMSD versus simulation time for intact nucleosome.(PDF)Click here for additional data file.Figure S4Number of hydrogen bonds between Arg88 of H2A(2) and Glu105 of H3(1) or Ala135 of H3(2) as a function of time for tail-truncated nucleosome simulations. In the H2A tail-truncated nucleosome simulation number 2 no hydrogen bond was formed between Arg88 and Glu105.(PDF)Click here for additional data file.Figure S5The 2D contact map of the H2A docking domain with the WebMol interface for viewing the docking domain and its surrounding in 3D. In the WebMol interface atoms are shown in backbone representation. The interface between the H2A docking domain and its surrounding appears as a mesh.(PDF)Click here for additional data file.Table S1Comparison between 20 nsand 100 ns nucleosome trajectories.(PDF)Click here for additional data file.Table S2Interaction change in histone monomers with respect to key findings.(PDF)Click here for additional data file.Video S1Arg81 interacting with Gln55 and Lys56 of H3(1) and Val107 of H2A(2) in the intact nuclesome simulation.(MPG)Click here for additional data file.Video S2Time course of interaction changes in Arg81 during the H3 tail-truncated simulation.(MPG)Click here for additional data file.Video S3Arg88 making stable hydrogen bonds to Asn94, Gly98 and Val100 of H2A(2) in the intact nucleosome simulation.(MPG)Click here for additional data file.Video S4In the H3 tail-truncated simulation Arg88 sidechain moves towards the DNA and then makes stable hydrogen bonds to Glu105 of H3(1) and Ala135 of H3(2).(MPG)Click here for additional data file."}
+{"text": "Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factors of STEC are Shiga toxin type 1 and/or 2 . We previously reported that intraperitoneal (i.p.) injection of rats in the late stage of pregnancy with culture supernatant from recombinant E. coli expressing Stx2 and containing lipopolysaccharide (LPS) induces premature delivery of dead fetuses. It has been reported that LPS may combine with Stx2 to facilitate vascular injury, which may in turn lead to an overproduction of nitric oxide (NO). The aim of this study was to evaluate whether NO is involved in the effects of Stx2 on pregnancy. Pregnant rats were i.p. injected with culture supernatant from recombinant E. coli containing Stx2 and LPS (sStx2) on day 15 of gestation. In addition, some rats were injected with aminoguanidine (AG), an inducible isoform inhibitor of NO synthase (iNOS), 24 h before and 4 h after sStx2 injection. NO production was measured by NOS activity and iNOS expression by Western blot analysis. A significant increase in NO production and a high iNOS expression was observed in placental tissues from rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body weight and killed 12 h after injection. AG caused a significant reduction of sStx2 effects on the feto-maternal unit, but did not prevent premature delivery. Placental tissues from rats treated with AG and sStx2 presented normal histology that was indistinguishable from the controls. Our results reveal that Stx2-induced placental damage and fetus mortality is mediated by an increase in NO production and that AG is able to completely reverse the Stx2 damages in placental tissues, but not to prevent premature delivery, thus suggesting other mechanisms not yet determined could be involved.Shiga toxin-producing  Escherichia coli (STEC) cause a significant public health risk due to contamination of food and water supplies. Gastrointestinal infection with STEC causes diarrhea and hemorrhagic colitis, and is the leading cause of hemolytic uremic syndrome (HUS), a systemic complication that is attributed to expression of Shiga toxins (Stx) In vivo and in vitro studies have demonstrated that LPS may combine with Stx to facilitate vascular injury Shiga toxin-producing To obtain timed pregnant females, both male and virgin female Sprague-Dawley rats (250\u2013300 g of body weight) from the School of Veterinary Medicine animal facility of the University of Buenos Aires were used.ad libitum and were housed under controlled conditions of light  and temperature (23\u201325\u00b0C). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals of the University of Buenos Aires .Mating was performed by placing females in the cages of males for several days. Day 1 of gestation was determined when sperm was observed in the vaginal smear. Animals received food and water E. coli DH5\u03b1 expressing Stx2 were incubated overnight at 37\u00b0C with shaking at 200 rpm in Luria-Bertani broth (Difco Laboratories) supplemented with 100 \u00b5g/ml ampicillin (Sigma Aldrich Co. USA). Bacterial cells were then removed by centrifugation, and the resultant supernatant (sStx2) was filtered through 0.22 \u00b5m pore size filter units  and assayed for toxicity to Vero cells as previously described 50) corresponds to the dilution required to kill 50% of Vero cells. Stx2 cytotoxicity in sStx2 was approximately 1\u00d7104 CD50/ml corresponding to 400 ng/ml when compared with the cytotoxicity of pure Stx2 on Vero cells. Supernatant from E. coli DH5\u03b1 containing only the plasmid was used as control (sControl). Lipopolysaccharide (LPS) content in the culture supernatant was determined using the HEK-Blue LPS Detection Kit . A sample of 1 ml of supernatant contained 30 ng of LPS (75 ng LPS/\u00b5g of Stx2 protein).The stx2 gene was cloned into pGEM-T-Easy  and recombinant ad libitum.In order to examine the effects of sStx2 on the delivery time and fetal status, pregnant rats were intraperitoneally (i.p.) injected with 0.05\u20131.5 ml of sStx2 containing approximately 0.07\u20132 ng Stx2/g body weight (wt) on day 14\u201316 of pregnancy (late stage). Control rats were injected with the same volume of sControl. The rats were individually housed under controlled conditions of light, humidity, and temperature, with food and water available Animals were observed every 30 minutes for any signs of morbidity , vaginal bleeding, and/or preterm delivery (pups present in the cage) after sStx2 or sControl administration. The beginning of preterm delivery was defined as the delivery of the first pup.Pregnant rats on days 15 of gestation were randomly divided into three groups of five rats each. One group was i.p. injected with 0.5 ml of sStx2 containing 0.7 ng Stx2/g body wt, another with 1.5 ml of sStx2 containing 2 ng Stx2/g body wt, and the last one with 0.5 or 1.5 ml of sControl. All rats were anesthetized and killed by cervical dislocation 12 h after treatment, and the kidneys and placentas were removed.14C]arginine into L-[14C]citrulline was used to quantify NOS activity according to a method previously described 2, and 1 mM DTT (dithiothreitol), and incubated at 37\u00b0C with 10 \u00b5M L-[14C] arginine  and 0.5 mM NADPH (nicotinamide adenine dinucleotide phosphate). After 15 min, samples were centrifuged for 10 min at 10,000 g and applied to a Dowex AG50-X8 column , and L-[14C] citrulline was eluted. L-[14C]Citrulline radioactivity was measured by liquid scintillation counting. Enzyme activity is reported as fmoles of L-[14C]citrulline per mg of protein per 15 min.The conversion of L-arginine into L-[14C]citrulline. We observed a significant increase in NOS activity in placental tissues of rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body wt and killed 12 h after injection  protein was detected as a 130 kDa band by western blot in placental tissues from both control and experimental rats killed 12 h after injection . The expThe increase in NOS activity detected in placental tissues after injection of sStx2 containing 0.7 ng Stx2/g body wt decreased significantly when pregnant rats were treated with 100 \u00b5g AG/g body wt 24 h before and 4 h after Stx2 injection .The treatment with AG also decreased the iNOS protein expression observed in placentas from rats injected with 0.7 ng or 2 ng Stx2/g body wt. Values returned to the same levels as those observed in placentas from control rats . Both NOUteri and fetuses from pregnant rats injected with sStx2 (2 ng Stx2/g body wt) showed intrauterine hemorrhage and fetal death 48 h post injection . TreatmePlacentas from rats injected with 0.7 or 2 ng Stx2/g body wt and killed 12 h post injection presented necrotic areas . In contTreatment with 100 \u00b5g AG/g body wt 24 h before and 4 h after sStx2 injection containing 2 ng Stx2/g body wt did not prevent the premature delivery of dead fetuses. In contrast, all the pregnant rats treated only with AG or sControl delivered normal live pups on days 22 or 23 of gestation. After Stx2-treatment, all rats were able to become pregnant and deliver normal pups at term.Our data show that sStx2 (a combination of Stx2 and LPS) induces a significant increase in NO production and levels of iNOS protein in placental tissues from pregnant rats, suggesting overproduction of NO plays an important role in sStx2-induced placental toxicity and fetal mortality NO is produced from L-arginine under the catalytic control of nitric oxide synthase (NOS). Three NOS isoforms have been identified: the endothelial (eNOS) and neuronal (nNOS) isoforms, which are responsible for basal, i.e. constitutive, NO production, and iNOS, which is activated by a variety of agonists including LPS and tumor necrosis factor (TNF). In contrast to the constitutive NOS isoforms, iNOS is thought to account for the increased NO production seen in pathological states Although there are several clinical studies on the enhanced NO production in patients with HUS Here we found that AG did not prevent premature delivery, thus indicating that other mechanisms not yet determined could be involved. It is well known that Stx may act in concert with LPS to elicit cellular dysfunction In summary, the present results allow us to conclude that the Stx2-induced premature delivery in rats in the late gestational stage is, at least in part, mediated by an increase in NO production resulting from the output of iNOS."}
+{"text": "DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only \u2018healthy\u2019 retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 ( Glaucoma is a complex multifactorial disease, which affects an estimated 70 million people worldwide. It is characterised by the selective and progressive loss of retinal ganglion cells (RGCs) and associated reduction in visual field in vivo imaging in mice expressing Thy1-YFP in RGCs Several lines of experimental enquiry have confirmed that the optic nerve head is a site of early damage. These have been confirmed in human glaucoma, in animals with a scleral lamina  and those with a glial lamina Thy1(YFP) to generate mice that develop spontaneous pigmentary glaucoma a subset of RGCs that express YFP in the cell soma and dendritic tree. We also labelled RGCs DiOlistically using the carbocyanine dyes, DiI and DiO The DBA/2J strain of mice is one of the most widely used in glaucoma research Thy1(YFP) mice were analysed as a control group. All eyes were categorised into 3 cohorts on the basis of optic nerve damage Both eyes from DBA2J.YFP mice at 9.5\u201311 months were assessed by which time they would be expected to manifest retinal damage. Retinas from 1\u20133 month DBA/2J.Retinal ganglion cell identity was confirmed by the presence of an axon running toward the optic disc in the retinal nerve fibre layer. Typically we observed that YFP filled the terminal parts of the dendritic tree; regardless of the intensity of YFP fluorescence, we did not observe points in the dendritic tree that suggested a barrier to the distribution of YFP. In total we analysed 68 YFP positive cells from DBA/2J mice at 9.5\u201311 months and 35 cells from 1\u20133 month old mice. The mean (SD) number of cells imaged per retina was 2.59(2.33).P>0.05) for all parameters tested ). There was also no significant difference in retinal ganglion cell dendritic morphology between retinal ganglion cells in the NOE group verses MOD or SEV, or between MOD and SEV groups  (Representative YFP positive RGCs from glaucomatous and control eyes are shown in  tested) , 4.Thy1 promoter in the transgenic mice and endogenous Thy1 has been shown to be down-regulated in animal models of RGC damage or glaucoma Thy1-driven YFP expression would mask early dendrite changes in DBA/2J glaucoma. Therefore, in a second cohort of mice, we assessed dendrite morphology in RGCs that were labelled DiOlistically with the carbocyanine dyes DiI and DiO. These dyes distribute in the plasma membrane and can be used to label cells regardless of their physiological status Gpnmb+ (D2-+Gpnmb) mice (and their 4 month old counterparts to serve as a control) and 4 month C57BL/6J . A representative panel of labelled cells is shown in +Gpnmb strains and we used this as a check against the possible of labelling bias. Using the Sun classification YFP is driven by the +Gpnmb age and sex match controls as assessed by total dendritic field area , total dendritic length  and Sholl analysis area under the curve (AUC)  . No significant difference  was between any of the control groups; 4 month C57BL/6J vs 4 and 11\u201312 month D2-+Gpnmb. As sectorial damage in the retina during glaucoma has been shown independently by different groups There was a significant decrease in dendritic architecture between DBA/2J eyes with no significant optic damage (NOE) and the D2- P<0.05) , 7. TherThy1(YFP) mice. The differences in dendrite mapping are highlighted by comparison of the Sholl plots for YFP and Diolistics . Although axon damage was not assessed in all regions of the optic nerve, we have shown previously that earliest axonal changes occur near or close to the optic nerve head Our data are consistent with a pathophysiological model in which glaucoma induced dendritic atrophy occurs in RGCs with intact axons. In all labelled cells, axons could be traced to the optic nerve head. Also, these changes occurred in eyes with no significant optic nerve damage  using a CFP fluorescent protein transgene that expresses in RGCsThe maintenance and degeneration of dendritic arbours is a complex process that shows considerable variation throughout the CNS. Current evidence suggests limited scope for remodelling of dendrites and that dendritic pruning is complex. The underlying mechanism appears to depend on whether this is driven by injury of as part of development. The mechanisms underling dendritic atrophy remain unclear in glaucoma. Studies in experimental glaucoma have indicated that the retrograde transport of BDNF Atrophic changes in RGCs are likely to influence the response characteristics of RGCs. This has been explored in the primate glaucoma model using explant preparations which showed reduced contrast sensitivity e.g. in Opa1 mutants) in which dendritic atrophy and mitochondrial beading is a hallmark of early disease Several factors are likely to conspire in dendritic degeneration. Histological studies in human glaucoma have confirmed the presence of varicosities within the axons of retinal ganglion cells These considerations do not rule out the role of exogenous (non-RGC related factors). Macrophage activity, in the form of microglial activation and migration, is an early feature of both experimental and human glaucoma ad libitum. All breeding and experimental procedures were undertaken in accordance with the Association for Research for Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Research. The Institutional Biosafety Committee (IBC) and the Animal Care and Use Committee (ACUC) at the Jackson Laboratory approved this study. The DBA/2J, DBA/2J-Gpnmb+, and DBA/2J.Thy1(YFP) strains have been described in detail elsewhere. C57BL/6J mice from The Jackson Lab facility  were used as an alternative age matched control. All mice were female. We used D2-+Gpnmb mice, a non glaucomatous substrain of DBA/2J that carry a wild type version of the Gpnmb gene responsible for the development of the glaucomatous phenotype. Although these animals develop mild pigment dispersion from the iris as a result of a mutation in the Tyrp1b gene this is not associated with elevated IOP Mice were housed in a 14 h light/10 h dark cycle as previously described with food and water available Gpnmb+, and DBA/2J were injected with 1\u20132 \u00b5l (1 mg/ml) Alexa Fluor 594-Cholera Toxin subunit B conjugate (Invitrogen) into the vitreous using a Hamilton syringe (35-gauge needle). After 48 to 72 hours they were anesthetized and euthanized via 4% PFA cardiac perfusion. Brains were submersion fixed for 24 hours after perfusion, cryoprotected in 30% sucrose overnight, OCT cryoembedded, and sectioned at 50 \u00b5m. Alexa Fluor 594 was visualized using an SP5 confocal microscope (Leica). The entire SC was assessed.Thirty 10\u201312 months old DBA/2J-Gpnmb+, and DBA/2J mice  were killed by cervical dislocation and the eyes quickly enucleated and placed in Neurobasal -A media (Invitrogen) at RT. The eyes were punctured at the limbus and a slit cut in the sclera to remove the cornea and sclera anterior to the ora serrata, along with the lens and vitreous. Three cuts were made in the retina before it was flat-mounted ganglion cell layer up on a cell culture insert (Millipore) and submerged Neurobasal \u2013A media. Retinas were incubated at 37\u00b0C and 4% CO2 ready for DiOlistic labelling using a gene gun. The total time between death and DiOlistic labelling was less than 10 minutes.4 and 12 month female C57BL/6J, DBA/2J-n\u200a=\u200a7) and 9.5\u201311 month female DBA/2J.Thy1(YFP) mice (n\u200a=\u200a16) were killed by cervical dislocation, the eyes quickly enucleated and placed into 4% PFA at RT for 1 h. Retina were removed and dissected as above, mounted onto slides, coverslipped and sealed under Floromount (Sigma Aldrich) ready for imaging.1\u20133 month  from 5 cm with a 3.0 \u00b5m pore size, high pore density, cell culture insert (Becton Dickinson) to block the passage of aggregated tungsten particles. Retinas were then incubated for 30 min to facilitate dye diffusion before being placed in 4% PFA at RT for a further 30 min. Retinal preparations were then mounted retinal ganglion cell side up and coverslipped under Floromount and sealed with nail polish. Images were taken within 48 hours.n\u200a=\u200a28; DBA/2J-Gpnmb+, 4 months n\u200a=\u200a38, 12 months n\u200a=\u200a86; DBA/2J, 4 months n\u200a=\u200a53, 12 months n\u200a=\u200a92; DBA/2J.Thy1(YFP), 1\u20133 month n\u200a=\u200a35, 9.5\u201311 month n\u200a=\u200a68) were obtained with a Zeiss LSM 510 confocal microscope (Carl Zeiss) or Leica TCS SP5 confocal microscope (Lieca) using a 20\u00d7 objective to allow the capture of the entire dendritic tree within a single image. Dendritic morphologies were analysed using ImageJ to measure dendritic field area (measured using the convex polygon tool to join the outer most points of the dendritic tree), an ImageJ plugin, NeuronJ to measure total dendritic length, and a custom Matlab macro to run a Sholl analysis Image stacks (0.1 \u00b5m slice width) of 400 RGCs  and stained with PPD. Typically 30\u201350 sections are taken from each nerve. Homology between sections is considered during grading. Optic nerves were analysed and given 1 of 3 gradings The processing of optic nerves and staining with paraphenylenediamine (PPD) which darkly stains the axoplasm and myelin sheath of dying axons has been reported previously No or early damage (NOE) \u2013 less than 5% axons damaged. This level of damage is seen in age and sex matched non-glaucomatous mice,Moderate damage (MOD) \u2013 average of 30% axon loss,Severe (SEV) \u2013 >50% axonal loss and damage."}
+{"text": "The aim of the study was determination of serum Se concentration and identification of genetic variations in genes related to metabolism of selenium as markers of cancer risks for carriers of BRCA1 gene mutation and individuals with susceptibility to other common unselected cancers .Eight genotypes of 4 most common SNPs localised in GPX1, GPX4, TXNRD2 and SEP15 were selected. Genotyping was performed in 93 affected and 186 unaffected matched BRCA1 carriers as well as on pairs matched 1:1 consisting of 108 breast, 50 ovarian and 105 prostate consecutive cancer patients and healthy controls.The following techniques for laboratory analyses have been applied: a) sequencing on ABI310, b) SimpleProbe or TaqMan analysis (a melting-curve genotyping with fluorescence-labeled probes based on the LightCycler 480 System (Roche Applied Science), c) determination of selenium concentration in plasma using atomic absorption spectrometer AAnalyst600 (Perkin Elmer).In none of studied groups statistically significant differences on cancer risk could be found when serum selenium concentration was assessed as a single factor. However, when selenium level data were combined with some selenoprotein genotypes some strong associations with cancer risk have been identified.The strongest association was found for carriers of SEP15 nGG genotype  and GPX1 nCC/TXNRD2 nGG/SEP15 GG .Results were not statistically significant however tendencies similar to those found in BRCA1 carriers and unselected breast cancer were observed.For four genotypes: GPX1 CC, GPX4 nCC, TXNRD2 GG and SEP15 nGG lower risk (~3 times) was found for selenium level ~90\u00b5g/l."}
+{"text": "TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has beenrecently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as  Chronic myelomonocytic leukemia (CMML) is a rare clonal hematological disorder, characterized by the neoplastic transformation of the hematopoietic stem cell RUNX1, JAK2, EZH2, CBL, TET2, ASXL1 and FLT3 genes TET2 mutations are by far the most frequently event in CMML patients TET2 protein plays an important role in the pathogenesis of CMML. However, data regarding the impact of TET2 mutations on the prognosis of CMML patients is still controversial Several mutations have been found in CMML patients including TET2 mutations as a plausible cause for aberrant epigenetic regulation of gene expression in CMML. Clinical studies further support this hypothesis, as clinical responses in CMML patients to treatment with Decitabine, a de-methylating agent, have been shown TET2 mutations has not been addressed.The TET family proteins  have been shown to catalyze the conversion of 5-methyl-cytosine (mC) to 5-hydroxymethyl-cytosine (hmC), a recently identified epigenetic mark, and participate in the epigenetic regulation of gene expression during embryogenesis and cancer TET2 as well as other mutations with potential epigenetic impact, namely IDH1, IDH2, JAK2 and EZH2.Genome-wide strategies offer a unique and systematic approach to adequately establish functional and biological impact of single gene function alterations. Therefore, the present study was designed to establish a global methylation profiling of CMML and to analyze the association between such DNA methylome and the presence of JAK2V617F mutations was performed by the ARMS technique and mutations of TET2, EZH2, exon 4 of IDH1  and exon 4 of IDH2  by direct sequencing as previously described Bone marrow (BM) aspirates and peripheral blood (PB) mononuclear cells (MNC) (N\u200a=\u200a24) were collected from patients with chronic myelomonocytic leukemia (CMML). Diagnosis of CMML was made according to the WHO) classification system of hematological malignancies Microarray-based DNA methylation profiling was performed with CMML and control samples using the Human Methylation27 Beadchip  according to the instructions of the manufacturer Before proceeding with methylation data analysis, 1092 CpGs located on chromosomes X and Y were excluded to avoid biological biases, due to the known methylation-mediated inactivation of one of the X chromosomes in female individuals. Additionally, we evaluated the detection probabilities  for all CpGs and excluded those with values of p>0.05 in more than 10% of cases. From a total of 26,486 CpGs in autosomal chromosomes, 89 CpGs showed a poor detection p value (i.e. above 0.05) in more than 10% of cases and consequently were eliminated from the study. Finally, 26,397 autosomal CpGs were investigated.T test: significantly differentially methylated CpGs (dMCPG) or differentially unmethylated CpGs (dUMCPG) were identified using a difference of at least 0.34 between mean \u03b2 values of each analysis group  and a false discovery rate (FDR) below 0.05, calculated using permutated t tests or analysis of variance if more than two groups were compared.Initially, a global view on the DNA methylation profile of CMML and healthy PB/BM control samples was obtained using an unsupervised hierarchical cluster analysis including only CpGs with standard deviation (SD) >0.25 among samples  A second bioinformatic analysis approach was carried out using R and Bioconductor A third analysis defined differential methylation according to the following criteria: Genes in CMML samples versus control BM/PB samples were considered as hypermethylated when the mean \u03b2 values of controls were <0.25 and at least a 20% of the CMML samples had a \u03b2 value >0.5. Hypomethylated genes in CMML samples versus control BM/PB samples were considered when the mean \u03b2 values of controls were >0.75 and at least a 20% of the CMML samples had a \u03b2 value<0.5. Raw array data files were deposited in a MIAME compliant database Gene Expression Omnibus (GEO) and are available under the accession number GSE31600.http://www.ensembl.org). Ingenuity Pathway Analysis software was also used to identify deregulated gene networks containing differentially methylated genes between CMML patients and healthy controls . Both GO and Ingenuity pathway analyses were performed integrating the differentially methylated genes between CMML patients and healthy control samples coming from the three strategies utilized .Functional enrichment analysis of Gene Ontology (GO) was performed using differentially methylated genes between CMML and control samples using standard hypergeometric tests http://www.mirbase.org/; and snoRABase, http://www-snorna.biotoul.fr/index.php). Subsequently array genes were annotated as including miRNA or snoRNA using gene symbol ID.Analyzed genes were classified as polycomb repressive complex 2 (PRC2) targets according to the genome-wide mapping of Polycomb target genes in embryonic stem cells (ESCs) provided by Lee et al The methylation level of LAX1, SLC22A12 and VHL genes was analyzed by pyrosequencing technique as previously described Quantification of 5 hmC and 5 mC content in specific regions was performed using the EpiMark 5-hmC and 5-mC analysis kit , according to the instructions of the manufacturer, followed by qPCR. The kit distinguishes 5 mC from 5 hmC by the addition of glucose to the hydroxyl group of 5 hmC utilizing T4 B-glucosyltransferase. Briefly, 1 \u00b5g of genomic DNA was treated with 30 units of T4 B-glucosyltransferase. Glucosylated DNA was digested with 100 units of MspI or 50 units of HpaII or no enzyme (mock digestion) at 37\u00b0C overnight. The MspI and HpaII resistant fraction (in the context of CCGG) was quantified by qPCR using specific primers that covered at least one MspI/HpaII site. Resistance to digestion with the enzyme MspI, which is blocked by glucosilated 5 hmC, translates directly into the level of methylation in 5 hmC while 5 mC levels were obtained by subtracting the percentage of 5 hmC from the resistance to digestion with the enzyme HpaII, which is blocked by 5 mC, 5 hmC and glucosilated 5 hmC. Primers and conditions used to qPCR are described in the Before analyzing the methylation profile of samples from patients with CMML, we compared the methylation profile of bone marrow and peripheral blood samples from healthy donors in order to know whether there were differences between these two control groups. This analysis revealed no significant differences in methylation for any of CpG analyzed in the array, suggesting that there are both samples are equivalent in terms of methylation profile and can be combined.AIM2, CDKN2A, POU4F2 or WT1) and from these CpGs, 156 (79%) where located at CpG islands. In contrast, 62 CpG probes (24%) where hypomethylated in CMML , being 38 (61%) of them located outside of CpG islands.Unsupervised analysis performed on 8 samples obtained from healthy donors and 24 samples from patients with CMML including all probes on the array except the probes located on chromosome X, show that CMML samples cluster together and separated from healthy controls, while all the control samples were grouped together . The comThe 260 differentially methylated CpGs were validated in a second set of samples from a recently published study in which samples from 18 CMML and 9 controls were analyzed using the same methylation array hsa-mir-204 and hsa-mir-153-1) were found to be located in differentially hypermethylated regions and one mirtron (hsa-miR-1231) in a hypomethylated region in CMML samples when compared to healthy donor samples  and 94 snoRNAs (177 probes). The majority of these CpG were found to be located in regions unmethylated in CMML as well as in healthy donor samples (76% miRNA/mirtrons and 86% snoRNAs). Two miRNAs  indicating an enrichment in PRC2 targets among differentially hypermethylated genes in CMML.Previous reports have shown that genes acquiring de novo hypermethylation in cancer are frequently targeted by the polycomb repressor complex 2 (PRC2) in embryonic stem cells (ESCs) As hypermethylation frequently targets genes with dense CpG islands JAK2, UTX, DNMT3a, EZH2 and TET2 are a frequent events in patients with CMML JAK2, EZH2, TET2, IDH1 or IDH2 could be implicated in the epigenetic regulation in CMML. In order to validate our hypothesis, we analyzed the mutations in these genes and their association with the DNA methylation patterns observed in CMML patients.Mutations in epigenetic genes such as JAK2, EZH2, TET2, IDH1 or IDH2 genes in CMML patients are represented in TET2 gene mutations. In 4 patients, a JAK2V617F gene mutation was found while only 1 patient showed a mutation of EZH2. No mutations in IDH1 and IDH2 genes were identified (TET2 (TET2-wt) clustered together and separately from mutated TET2 (TET2-mut), with TET2-wt samples showing a higher number of differentially hypermethylated genes than TET2-mut samples  between TET2-wt and TET2-mut CMML samples, with 13 CpGs being hypermethylated in TET2-mut CMML and 50 hypermethylated in TET2-wt samples  . Indeed CMML patients with TET2-wt belonged to the recently described high or intermediate cytogenetic risks groups TET2-wt CMML patients belong to high or intermediate risk groups while only one of 13 TET2-mut patients) (Comparing DNA methylation profiles between control samples and CMML R\u200a=\u200a2.4) . The levhe array . Interesatients) .EZH2 was found to be mutated only in one patient precluding any further meaningful analysis of the relation between EZH2 and methylation. EZH2 was found not to be methylated in CMML or in healthy controls according to the results of the methylation arrays with \u03b2 values <0.3, indicating that the methylation of the EZH2 promoter region itself was not responsible for the differences in methylation in CMML. JAK2V617F mutations were found in 16.6% of our CMML cohort (4 JAK2V617F and 20 JAK2 wild type) , suggesting that at least in CMML, the presence of JAK2V617F is not related with a specific epigenetic profile. As it was the case for EZH2, differences in methylation of the JAK2 promoter were not related to the different methylation patterns observed in patients with CMML. A \u03b2 value between 0.3 and 0.7 was found in 7 healthy donor samples and 14 CMML samples (2 of them with JAK2V617F), a \u03b2 value >0.7 in 1 CMML and 9 CMML samples with a methylation \u03b2 value <0.3, indicating a hypomethylation of JAK2 in these 9 cases (2 of them with JAK2V617F) .ld type) . Cluster profile . HoweverK2V617F) . The anaIDH1 and IDH2 were found not to be methylated in CMML or in healthy controls according to the results of the methylation arrays with \u03b2 values <0.2 and <0.1 respectively, indicating that the methylation of these two genes were not responsible for the differences in methylation in CMML.TET2 is mutated, has not been analyzed. To determine the 5 hmC content, we specifically analyzed the promoter regions of 3 of the 13 genes hypermethylated in TET2-mut CMML patients. This analysis was performed in PB samples obtained from 2 healthy donors, 8 TET2-mut and 5 TET2-wt CMML patients samples. These genes were chosen based on the fact that only CpG dinucleotides associated to CCGG motifs can be analyzed using MspI-HpaII enzymes. For the LAX1 gene, we analyzed two CpG located 5\u2032upstream (\u221238 and \u2212244 bp respectively) to the CpG analyzed in the methylation array  and another one downstream (58 bp) to the CpG analyzed in the methylation array  the content of 5 hmC varies among different genes regardless of the l status ; 2) chanCDKN2B in 58% of cases with CMML and hypomethylation of C-MYC in 2 patients has been previously reported Despite the use of epigenetic drugs as decitabine, 5-azacitidine or the histone deacetylase inhibitor valproic acid for the treatment of patients with CMML Previous studies have shown that genes frequently targeted by the polycomb repressor complex 2 (PRC2) in ESCs are predisposed for cancer-specific hypermethylation BCR-ABL1 oncoprotein JAK2 gene Ingenuity Pathway Analysis software using genes differentially methylated between CMML and controls identified a specific gene network centered in PLC, JNK and ERK pathways. Mice deficient for PLC-beta3 and Lyn develop a myelodysplastic/myeloproliferative neoplasm similar to human CMML TET2, CBL, RUNX1, RAS, IDH1, IDH2, NPM1, ASXL1, NPM1 or EZH2EZH2 or TET2 have been associated with the prognosis of the disease their role is currently unclear TET2 mutations More than 80% of CMML patients harbor mutations in diverse genes including TET2 induces a decrease in the levels of 5 hmC in myeloid progenitors and a dysregulation of hematopoietic stem cells leading to the development of myeloid malignancies similar to CMML TET2-mut patients would be expected to show an increase in hypermethylation in comparison with TET2-wt (due to the lack of capacity to convert 5 hmC) TET2-wt CMML patients. Hypermethylation could be related to the statistically significant association between TET2-wt patients and the presence of abnormal karyotypes (p<0.01). Indeed, TET2-wt and abnormal karyotype have been described as poor prognostic factors in patients with CMML TET2 participates in the conversion of 5 mC to 5 hmC and thus is required for DNA de-methylation TET2-mut patients has not been analyzed. Our results indicate that hypermethylated genes in TET2-mut CMML patients have a high content of 5 mC as expected from the lack of function of TET2. However, the decrease of 5 hmC and increase of 5 mC do not occur equally in every hypermethylated gene or even in different CpGs of the same gene. On the other hand, genes differentially methylated in TET2-wt could be enriched both in 5 mC or 5 hmC. Some of the hypermethylated genes in TET2-wt CMML have been directly or indirectly related to cell cycle arrest, tumor suppression or myeloid differentiation . Whether these genes are in fact silenced by epigenetic mechanisms should be explored in order to determine their role in the pathogenesis of CMML.Although recent studies have demonstrated that silencing of TET2 protein induces a decrease in the content of 5 hmC in normal and malignant myeloid cells TET2 showed a different methylation profile than patients wild type TET2, and these differences segregate CMML patients in 2 groups with an increase in hypermethylated genes in non-mutated patients; 3) although TET2 mutations induced a decrease in the content of 5 hmC, the analysis of specific genes and CpGs demonstrate a heterogeneous behavior in terms of 5 mC and 5 hmC content. These results along with recent studies that demonstrate the role TET2 in hematopoiesis and the development of myeloid malignancies, suggest that epigenetic changes are not likely the mechanism by which inactivation of the TET2 protein induces myeloid malignancies.From our results, we may conclude: 1) CMML is associated with an abnormal epigenetic profile with 249 genes differentially hypermethylated; 2) CMML patients with mutations in Figure S1Unsupervised analysis including all probes on the array except the probes located on chromosome X. Samples are color coded. The top bar beneath the dendrogram shows CMML and healthy donor samples.(TIF)Click here for additional data file.Figure S2Analysis of 260 differentially methylated CpGs in CMML samples from our study and the study from Ko et al. Hierarchical cluster analysis based on abnormally methylated genes identified using 24 CMML patients and 8 controls, validated in another 18 CMML 9 healthy donor samples from the study of Ko M et al. \u03b2 values are depicted using a pseudocolor scale . Samples are color coded. The top bar beneath the dendrogram refers CMML or healthy donor samples, second bar indicates CMML samples and healthy donor samples of our series and series of Ko et al.(TIF)Click here for additional data file.Figure S3Methylation results obtained by pyrosequencing in CMML patients samples with TET2-mut or TET2-wt. Pyrosequencing results of analyzed CpG loci on the array and hipermethylated in CMML TET2-mut patient samples, corresponding to LAX1, SLC22A12 and VHL genes. In addition one CpG located 5\u2032upstream (\u221238) to the CpG analyzed in the methylation array in the case of LAX1; one downstream CpG (58 bp) in the case of SLC22A12 gene and one CpG downstream (5 bp) in the case of VHL gene were analyzed. The values are expressed as percentage of methylation. Median values of percentage of DNA methylation are indicated and P values were obtained using the 2-tailed T test or U Mann Whitney test.(TIF)Click here for additional data file.Table S1Specific primers and probes corresponding to pyrosequencing analysis.(XLS)Click here for additional data file.Table S2Primers, probes and conditions used to quantification of 5 hmC methylation levels by qPCR. The CCGG location refers to the position of this motif with respect to the analyzed CpG dinucleotide in the methylation array. All primers and probes are in 5\u2032 to 3\u2032 and probes are labbeled with FAM in 5\u2032and TAMRA in 3\u2032. In all of PCR the Tm was 60\u00b0C.(XLS)Click here for additional data file.Table S3Differentially methylathed genes between CMML patient samples and healthy donor samples. Gene Name; Red: Genes hypermethylated in CMML samples; Green: genes hypomethylated in CMML samples. CpG ISLAND; TRUE: probe inside in CpG island; FALSE: probe outside CpG island. POLYCOMB; NO: no target of Polycomb group; YES: target of Polycomb group; UD: unknown. PROMOTER CLASS: HCP: high CpG content; ICP: intermediate CpG content; LCP: low CpG content; UD: unknown. miRNA; NO: no annotated miRNA in gene sequence. snoRNA; NO: no annotated snoRNA in gene sequence.(XLS)Click here for additional data file.Table S4Description of Cariotype and JAK2, TET2 and EZH2 gene mutations of CMML patients. *: High risk Caryotype. **: Intermediate risk Caryotype. WT: wild type sequence. UD: undetermined.(XLS)Click here for additional data file.Table S5Differentially methylathed genes between TET2-wt and TET2-mut CMML patient samples. Gene Name; Red: Genes hypermethylated in TET2-mut respect to TET2-wt CMML samples; Green: Genes hypermethylated in TET2-wt respect to TET2-mut CMML samples. CpG ISLAND; TRUE: probe inside in CpG island; FALSE: probe outside CpG island. POLYCOMB; NO: no taget of Polycomb group; YES: target of Polycomb group; UD: unknown. PROMOTER CLASS: HCP: high CpG content; ICP: intermediate CpG content; LCP: low CpG content; UD: unknown. miRNA; NO: no annotated miRNA in gene sequence. snoRNA; NO: no annotated snoRNA in gene sequence.(XLS)Click here for additional data file."}
+{"text": "Oral lichen planus (OLP) is a common chronic inflammatory mucocutaneous disease. Clinical diagnosis of OLP requires clinical work-up and histologic examination to rule out possible dysplasia and carcinoma. It is possible that oral mucosal viral infections including HPV infection may have a causative role in OLP pathogenesis. The aim of this study was to examine the coincidence of human papilloma virus type 18 and oral lichen planus.This study was a case-control study. Twenty nine paraffinized specimens of previously diagnosed oral lichen planus and 14 paraffinized specimens of nonpathogenic mucosa were studied. Polymerase Chain Reaction (PCR) analyze used for detection of DNA HPV 18. The data were analyzed with SPSS software and Fisher\u2019s exact test was used to find the possible relation between HPV18 infection and oral lichen planus.Nine out of 29 (31.0%) lichen planus samples and one out of 14 (7.1%) controls were HPV 18 positive. No significant correlation (P = 0.128) was observed between HPV18 infection and oral lichen planus.According to the findings there might be a co-incidence of human papilloma virus type 18 and oral lichen planus. Oral lichen planus (OLP) is a chronic immunologic inflammatory mucocutaneous disease.2472, 100 \u03bcM deoxynucleotide triphosphate (dNTP), 200 pmoles of primer and 2.5 U of Taq polymerase (invitrogen). A total reaction of 50 \u03bcl containing 1 \u03bcl template DNA was amplified by incubating at 94\u00b0C for 5 min followed by 40 cycles  and 72\u00b0C for 10 min. After PCR reaction, the electrophoresis was performed on PCR products and the bands were observed using a UV eliminating Transilluminator ,10uminator . Those sThis study included 29 OLP cases with minimum age of 22 years and maximum age of 84 years (mean age of 49.55 \u00b1 13.12) and 14 healthy con-trols with minimum age of 29 and maximum age of 70 years (mean age of 48.5 \u00b1 10.18); 12 of cas-es and 7 of controls were female. Nine out of 29 (31.0%) lichen planus cases and one out of 14 (7.1%) controls were HPV 18 positive . Given tA lot of researchers have found that racial and geographical features may affect viral factors which involve in human diseases. Some viral factors have been studied and proposed as etiologic factors in oral lichen planus. Researchers showed that CD8+ cells cause apoptosis in virally infected cells. So it may be possible to consider a role for viral infections in OLP pathogenesis.125However in the present study, no significant difference was observed between the case and the control groups regarding HPV 18 infection. In 2006, Giovannelli reported that HPV infection can be affected by keratinization, so that keratinized tissue is more resistant to HPV infection.In the current study, no significant relation was observed between HPV /18 infection and OLP. According to the findings, there might be a coincidence of human papilloma virus type 18 and oral lichen planus. Given the fact that HPV 16 is a high risk type, it is suggested that another study be conducted to investigate the relation between HPV16 and other subtypes of HPV and OLP lesions."}
+{"text": "HHIP), 4q22 (FAM13A), and 19q13 . Significant eQTLs were replicated in two independent datasets (n\u200a=\u200a363 and 339). SNPs previously associated with COPD and lung function on 4q31  were associated with the mRNA expression of HHIP. An association between mRNA expression level of FAM13A and SNP rs2045517 was detected at 4q22, but did not reach statistical significance. At 19q13, significant eQTLs were detected with EGLN2. In summary, this study supports HHIP, FAM13A, and EGLN2 as the most likely causal COPD genes on 4q31, 4q22, and 19q13, respectively. Strong lung eQTL SNPs identified in this study will need to be tested for association with COPD in case-control studies. Further functional studies will also be needed to understand the role of genes regulated by disease-related variants in COPD.Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Recent genome-wide association studies (GWAS) have identified robust susceptibility loci associated with COPD. However, the mechanisms mediating the risk conferred by these loci remain to be found. The goal of this study was to identify causal genes/variants within susceptibility loci associated with COPD. In the discovery cohort, genome-wide gene expression profiles of 500 non-tumor lung specimens were obtained from patients undergoing lung surgery. Blood-DNA from the same patients were genotyped for 1,2 million SNPs. Following genotyping and gene expression quality control filters, 409 samples were analyzed. Lung expression quantitative trait loci (eQTLs) were identified and overlaid onto three COPD susceptibility loci derived from GWAS; 4q31 ( Chronic obstructive pulmonary disease (COPD) is the fourth most common cause of death worldwide and is predicted to be the third leading cause of mortality in the world by the year 2030 FAM13A), 4q31 (HHIP), 15q25 (CHRNA3/CHRNA5/IREB2) and 19q13  The number of susceptibility genes for COPD is expanding rapidly with lists tabulated at 57 genes in 2009 The goal of this study is to identify lung expression quantitative trait loci (eQTL) within COPD susceptibility loci identified by GWAS. As part of the lung eQTL consortium, we have recently performed a genome-wide search for eQTLs in 1,111 human lung samples http://www.federa.org).All lung tissue samples were obtained in accordance with Institutional Review Board guidelines at the three sites: Laval University , University of British-Columbia  and Groningen University . All patients provided written informed consent and the study was approved by the ethics committees of the Institut universitaire de cardiologie et de pneumologie de Qu\u00e9bec (IUCPQ) and the UBC-Providence Health Care Research Institute Ethics Board for Laval and UBC, respectively. The study protocol was consistent with the Research Code of the University Medical Center Groningen and Dutch national ethical and professional guidelines ; at Groningen, the lung specimens were provided by the local tissue bank of the Department of Pathology, and at UBC, the lung specimens were provided by the James Hogg Research Center Biobank at St Paul's Hospital. COPD diagnosis and severity were determined according to the GOLD recommendations Study subjects and lung specimens were described previously Genome-wide gene expression and genotyping profiles were obtained using a custom Affymetrix array (see GEO platform GPL10379) and the Illumina Human1M-Duo BeadChip array, respectively. Gene expression data are available through the Gene Expression Omnibus (GEO) repository with the accession number GSE23546. Standard quality controls were performed as described previously and only subjects that passed genotyping and expression quality controls were included in this study with 409, 363, and 339 subjects from Laval, Groningen, and UBC, respectively CHRNA3/CHRNA5/IREB2 locus that we have reported on previously FAM13A), 4q31 (HHIP) and 19q13 . SNPs associated with COPD from previous GWAS were tabulated for the three loci , 9 genes on 4q31  and 45 genes on 19q13 .Lung eQTLs were overlaid onto COPD susceptibility loci identified by previous GWAS except for the 15q25-ree loci . SNPs gecis-eQTLs were those passing Bonferroni correction considering the effective number of independent SNPs and genes tested at each locus. The number of independent variables was determined by using the definition of Li and Ji \u22126 for 4q22 (0.05/(279.64 SNPs\u00d735 probesets), 1.50\u00d710\u22125 for 4q31 (0.05/(128.26 SNPs\u00d726 probesets) and 3.43\u00d710\u22126 for 19q13 (0.05/(246.73 SNPs\u00d759 probesets). Significant eQTLs in Laval dataset were then validated in the UBC and Groningen datasets. P values lower than 0.05 were considered significant in the replication sets.Lung eQTLs were identified using a different model than in our previous genome-wide lung eQTL mapping study PPM1K, GPRIN3, SNCA, MMRN1). Significant linkage disequilibrium (LD) was observed among the 64 SNPs (PPM1K (PPM1K and the direction of the effect was the same in the three cohorts. None of the SNPs previously associated with COPD on 4q22 (2>0.5)  and in modest LD (r2\u200a=\u200a0.53\u20130.69) with COPD SNPs were nominally associated with the expression levels of FAM13A (p\u200a=\u200a4.1\u00d710\u22125). The FAM13A-rs2045517 eQTL was replicated in UBC, but not in Groningen  . These froningen .FREM3, BC029578, HHIP, OTUD4) were involved in the significant eQTLs. Only eQTLs associated with BC029578 (35) and OTUD4 (1) were replicated in the two replication sets. eQTL-SNPs on chromosome 4q31 are subdivided in two strong LD blocks . Two SNPs  previously associated with COPD were found to affect the expression of HHIP. Rs1828591 was the most significant SNP associated with HHIP in the Laval dataset. This eQTL was replicated in UBC, but not in Groningen . 210 eQTLs were validated in both replication cohorts. SNPs associated with gene expression were distributed across four LD blocks  and rs7937 was genotyped in our lung eQTL dataset. Rs7937 was not associated with expression of genes located in this predefined 19q13 locus. The gene most significantly regulated by rs7937 was NUMBL (p\u200a=\u200a0.0187). Three SNPs were regulating the expression of EGLN2, a gene previously associated with COPD. The most significant association with EGLN2 was with rs4803369 (p\u200a=\u200a8.9\u00d710\u22127). Rs4803369 is located at 13,274 bp away from rs7937 and is in modest LD (r2\u200a=\u200a0.33) with the latter. The eQTL results for rs4803369-EGLN2 from the three cohorts are illustrated in On 19q13, 739 SNPs and 95 probesets covering 45 different genes were tested. The expression levels of detected . 174 SNPD blocks . 26 SNPsA/CYP2A6 . These eFAM13A), 4q31 (HHIP) and 19q13 . We identified genetic variants influencing gene expression at each locus and replicated findings in two independent cohorts.The goal of this study was to identify causal variants and genes within susceptibility loci associated with COPD. GWAS have indicated four loci associated with this disease as defined by lung function measures MMRN1. Lung eQTLs on 4q22 were found and validated with four genes: PPM1K, SNCA, PPM1 and GPRIN3 genes. A SNP located in SNCA (rs2035268) has been associated with accelerated FEV1/FVC decline 2 between one of our significant eQTL-SNP and rs2035268 was 0.047. No SNP previously associated with COPD within and near FAM13A2>0.5). The latter were nominally associated with the expression of FAM13A and validated in one replication set. Accordingly, our results provide some support that FAM13A is the COPD causal gene on 4q22.The first study to identify an association between the 4q22 locus and COPD was published in 2010 BC029578 transcript and another associated with OTUD4. This transcript is located between the GYPE and GYPB genes. SNPs regulating BC029578 are distributed across a 400 kb region. Further studies are needed to understand the function of BC029578. eQTLs were also associated with FREM3 and HHIP, a member of the hedgehog-interacting protein family. HHIP has been associated with COPD in three GWAS The 4q31 locus was first associated with COPD and lung function in two studies in 2009 CEACAM21 was associated with COPD susceptibility in a sputum eQTLs study on COPD patients AXL, NUMBL, SERTAD3, B3GNT8, CEACAM4, CYP2G1P, LOC100505495 or ZNF780A to the development of COPD or related phenotypes. Rs7937, a SNP located in RAB4B, EGLN2 and MIA-RAB4B and identified in previous GWAS, was genotyped in our datasets. However, no association was detected between this SNP and the expression level of any gene. The strongest association with a suspected COPD gene is EGLN2-rs4803369. This gene is known to be involved in regulating hypoxia tolerance and apoptosis in cardiac and skeletal muscle. These results support that EGLN2 is a potential causal COPD gene on 19q13.There are many genes present in the 19q13 locus. This locus was recently associated with COPD and so far no replication study has been published eQTLs obtained in this study are derived from non-tumor lung parenchymal samples. As all organs, the lung is multicellular. The cellular heterogeneity constitutes an inherent limitation of our study and will inevitably reduce the power to detect eQTLs. It is known that many eQTLs will be missed by studying heterogeneous tissues FAM13A gene were indirectly (in LD) associated with the mRNA expression levels of FAM13A. On 4q31, the suspected candidate in this region, HHIP, was found to be regulated by SNPs previously associated with COPD. On 19q13, SNPs associated with COPD were consistently associated with the expression level of EGLN2. Further functional studies will be needed to verify the contribution of susceptibility genes in COPD. Strong lung eQTL SNPs will also need to be tested for association with COPD in case-control studies and in additional eQTL mapping studies in other disease-relevant tissues and cell types. This study is an important step forward to better understanding the underlying biology of the COPD susceptibility loci. It also shows the potential of eQTLs in a relevant tissue to leverage the results of previous GWAS and extend their functional meaning to gene expression.In conclusion, we used a large collection of human lung specimens from patients with and without COPD to identify SNPs that regulated gene expression in three COPD susceptibility loci derived from GWAS. Strong lung eQTLs were detected in the three COPD loci. However, the eQTL-SNPs were not necessarily the SNPs associated with COPD. On 4q22, SNPs associated with COPD near the Figure S1Linkage disequilibrium plot of significant SNPs on the 4q22 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2\u200a=\u200a1) and white indicates perfect equilibrium (r2\u200a=\u200a0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Figure S2Linkage disequilibrium plot of significant SNPs on the 4q31 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2\u200a=\u200a1) and white indicates perfect equilibrium (r2\u200a=\u200a0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Figure S3Linkage disequilibrium plot of significant SNPs on the 19q13 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2\u200a=\u200a1) and white indicates perfect equilibrium (r2\u200a=\u200a0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Table S1Significant eQTLs at the 4q22 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file.Table S2Significant eQTLs at the 4q31 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file.Table S3Significant eQTLs at the 19q13 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file."}
+{"text": "Shigella sonnei, we performed multilocus variable number tandem repeat analysis of 1,672 isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three major clonal groups were identified; 2 were globally spread. Type 18 and its derivatives have circulated worldwide in recent decades.To investigate global epidemiology of  Shigella sonnei is the most commonly isolated species among the 4 Shigella species in industrialized countries  and the Pacific region by multilocus VNTR analysis (MLVA) to investigate the global epidemiology of S. sonnei.A total of 26 variable number tandem repeats (VNTRs) have been used to type Isolates were obtained from 50 countries on 5 continents and the Pacific region . Of thes<7 loci among the 26 loci. The 3 large clusters displayed distinct allelic diversity features. Eight loci  had Simpson diversity values >0.5 for >1 of the 3 large clusters (Table). Differences in diversity values >0.3 among the 3 clusters were observed for 9 of the 26 VNTRs. The largest difference was in 2 hypervariable VNTRs (SS1 and SS6). These 2 VNTRs displayed a high degree of allelic diversity in cluster A, but were invariant in cluster B. SS1 was invariant but SS6 displayed a high degree of diversity in cluster C.The high resolving power of MLVA is primarily caused by highly diverse VNTRs  were obtained from patients who acquired infections in Taiwan. Most isolates belonged to an insertion element IS1 interspacer 1 clone  A and C were globally spread, and clonal group B was found in countries in Africa, Asia and Europe only.Cluster D contained 2 isolates obtained in French Guiana and Senegal in 2003. The isolate for singleton E was obtained in Malaysia in 1999.S. sonnei isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three large clonal groups were identified; they displayed distinct allelic diversity features, particularly for 2 hypervariable VNTRs (SS1 and SS6). Clonal groups A and C were globally spread. One MLVA18 type (SS18.2) and several of its SLVs were widely distributed over 5 continents in the past 10 years.Genetic analysis using MLVA presented a simple clonal structure for 1,672"}
+{"text": "Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-\u03b1 (TNF\u03b1). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNF\u03b1 stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2\u2236Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNF\u03b1 for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2\u2236Tie1, in response to acute VEGF treatment or chronic TNF\u03b1, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2\u2236Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal. Induction of Tie1 cleavage by VEGF would be expected to enhance Ang1 activation of Tie2, and this has indeed been demonstrated Cleavage of both Tie1 and Tie2 has been reported to be strongly activated by PMA in endothelial cells VEGF is a key physiological activator of both Tie1 and Tie2 cleavage with similar, though less marked, effects as PMA TNF\u03b1 is also an important activator of Tie1 cleavage in endothelial cells The data presented in We also examined levels of released Tie1 and Tie2 ectodomain at early (30 min) and late (24 h) time points in cells treated with VEGF and TNF\u03b1. As shown in Taking the findings on full-length cellular Tie1 and release of ectodomain together these data indicate that VEGF activates Tie1 cleavage acutely but not chronically, whereas TNF\u03b1 causes Tie1 cleavage acutely and further loss of Tie1 chronically, via effects nt involving cleavage. Furthermore, chronic, but not acute, VEGF treatment stimulates loss of Tie2 whereas chronic TNF\u03b1 treatment elevates cellular Tie2. The net result of these ligands therefore is for VEGF to decrease full-length Tie1\u2236Tie2 ratio acutely and increase the ratio chronically whereas TNF\u03b1 causes a clear decrease in Tie1\u2236Tie2 ratio with increasing time of treatment.The cell-associated fragment of Tie1 remaining after ectodomain cleavage encompasses transmembrane and intracellular domains and is clearly observed as an approximately 45 kDa protein that accumulates in endothelial cells following activation with PMA, VEGF and other stimuli As a physiological activator, VEGF induces a more modest Tie2 cleavage than PMA and it was important to test whether this physiologically relevant stimulus is sufficient to induce appearance of the Tie2 endodomain. Therefore HUVEC were stimulated for 30 min and 24 h with VEGF before analysis for the presence of Tie2 endodomain by immunoblotting . As witht-Butyl-Ester), treated with or without PMA and Tie1 and Tie2 detected in cell lysates by immunoblotting. As shown in It has previously been shown that once formed by ectodomain proteolysis Tie1 endodomain is degraded by a two step process involving release of the intracellular domain from the membrane tethered transmembrane sequence, by \u03b3-secretase activity, followed by proteosomal degradation of the 42 kDa intracellular kinase domain Previously we reported the presence of complexes comprising full-length Tie2 physically associated with Tie1 endodomain in endothelial cells treated with VEGF We and others have shown that loss of full-length Tie1 or cleavage of Tie1 ectodomain enhances Ang1 signalling through Tie2 As shown in In addition to signalling from full-length Tie2 it is possible that the Tie2 intracellular domain fragment detected in cells following ectodomain cleavage could have signalling activity. This could occur in the absence of Ang1 as a result of loss of the Tie2 regulatory ectodomain. Indeed, some other intracellular kinase domains released following ectodomain cleavage of full-length receptor tyrosine kinases, such as ErbB4 and TrkA, appear to be active as judged by their phosphorylation status As shown in In this study we have examined the effect different physiological activators have on relative levels of Tie receptors and Ang1 signalling. Our data shows that cleavage of cellular Tie1 and Tie2 are both regulated by VEGF but with different kinetics. This results in an increase in cellular Tie2\u2236Tie1 ratio early after VEGF stimulation and a decrease in the ratio at later time points. In contrast, TNF\u03b1 differentially affects the receptors by enhancing Tie1 loss whilst increasing Tie2 levels leading to a progressive increase in Tie2\u2236Tie1 ratio in cells treated with this ligand. Furthermore, this modulation of Tie1 and Tie2 by VEGF and TNF\u03b1 affects Ang1 signalling, with a decrease in Tie1 relative to Tie2 enhancing Ang1-induced cellular Tie2 activation The mechanisms responsible for the different time courses of Tie1 and Tie2 cleavage in response to PMA and VEGF are not known. However, it is possible that this reflects involvement of different proteases in cleavage of each of the receptors. Recently matrix metalloprotease-14 (MMP14) has been implicated in mediating Tie2 cleavage and the increase in PMA-activated Tie2 cleavage correlates with induction of increased MMP14 expression The ability of TNF\u03b1 to cause a time dependent increase in Tie2\u2236Tie1 ratio in endothelial cells was found to involve both effects on Tie1 cleavage and effects on expression of Tie1 and Tie2. As shown in Together the findings of the present study show that the molecular balance between cellular Tie2\u2236Tie1 is dynamically regulated by pathophysiologically relevant factors and this balance influences the ability of Ang1 to signal in endothelial cells. These findings identify the Tie2\u2236Tie1 ratio as a key determinant of angiopoietin signalling in endothelial cells. Interestingly, Ang1 is considered to provide a fairly constant maintenance signal to endothelial cells, is present bound to extracellular matrix and is produced by perivascular cells at a relatively constant rate in vivo. A prediction from the cellular findings is that in vivo Ang1 signalling under basal conditions is limited by the responsiveness of the Tie2 receptor, which is determined by the Tie2\u2236Tie1 ratio in the cell, to the pool of perivascular Ang1. Furthermore, short-term increases in local VEGF concentration would enhanced Tie2 responsiveness and increase Ang1 signalling. This would allow the endothelium to maintain a quiescent state due to the presence of perivascular Ang1 without requiring maximal signalling through Tie2 under normal basal conditions whilst having the ability to enhance this pro-quiescent signal by increasing Tie2 responsiveness to Ang1 in the presence of transient increases in VEGF in vivo. Such a mechanism of local regulation of Tie2 responsiveness by VEGF would prevent short-term fluctuations in VEGF from causing vascular destabilization without having to maintain constant maximal levels of Ang1 signalling in the endothelium. The endothelium would retain the ability to respond acutely to VEGF if there was a coincident increase in Ang2. In addition, chronic VEGF elevation would also be expected to result in vessel destabilization as the enhancement of Tie2 responsiveness to Ang1 does not occur under conditions of long-term VEGF treatment. Whilst it is possible to speculate on the functional significance of the cellular effects in vivo it will be important in future work to test these hypotheses and examine the significance of the cellular changes in receptors and signalling in pathophysiological situations in vivo such as during angiogenesis.The focus of the present study has been to examine the effects of VEGF and TNF\u03b1 on the relative levels of Tie1 and Tie2 within the same population of endothelial cells and the effects on Ang1 signalling. Clearly it will be important to determine the functional significance of the effects of VEGF and TNF\u03b1 on Ang1 signalling In conclusion this study has shown the levels of intact Tie2 and Tie1 in endothelial cells are differentially controlled in both the short- and long-term by factors regulating ectodomain cleavage of the two receptors. This results in modulation of the molecular Tie2\u2236Tie1 balance in cells by various stimuli. Furthermore, the molecular balance between these two receptors determines the level of Ang1-induced Tie2 activation in the cell. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.Human umbilical vein endothelial cells (HUVEC) were from Promocell and were maintained in Medium 199 containing 20% foetal calf serum, 5 units/ml heparin, and 50 \u00b5g/ml endothelial cell growth supplement. As indicated in Results, cells were treated with the following concentrations of reagents: PMA, 10 ng/ml ; VEGF, 100 ng/ml ; TNF\u03b1, 100 ng/ml ; Ang1 200 ng/ml , unless otherwise indicated. The \u03b3-secretase inhibitor  was added prior to addition of the PMA and remained in the media throughout treatment. Annealed, purified and de-salted double-stranded siRNA oligonucleotides have been previously detailed Cells were lysed with ice-cold lysis buffer  and lysates centrifuged at 13,000\u00d7 g for 5 min to remove particulate matter.For analysis of whole cell lysates, Laemmli sample buffer containing dithiothreitol, to produce a final concentration of 100 mM, was mixed with cleared lysates and boiled for 5 min before loading equal amounts of protein onto SDS-PAGE and resolving. For immunoprecipitates, supernatants cleared of particulate material by centrifugation at 13,000\u00d7 g for 5 min were immunoprecipitated by the addition of 2 \u00b5g of the indicated antibody for2\u20133 h in the presence of protein-A beads. In some experiments Tie2 and Tie1 extracellular domain was immunoprecipitated from conditioned medium, in these cases medium was conditioned for 24 h and cleared by centrifugation before immunoprecipitation as for cell lysates. In all cases immunoprecipitated proteins were recovered by centrifugation at 13,000\u00d7 g for 5 min and washed 3 times with wash buffer (as lysis buffer but with 0.1% Triton X-100). Proteins were eluted from beads by the addition of Laemmli sample buffer containing 100 mM dithiothreitol and boiling for 5 min before SDS-PAGE. For immunoblotting proteins were transferred to nitrocellulose membranes electrophoretically and membranes probed with Tie1, Tie2 or phospho-Tie2 antibodies as appropriate. Blots were stripped and re-probed with anti-tubulin. Immunoreactive proteins were visualized with peroxidase-conjugated secondary antibodies and chemiluminescent detection Bands on Western blots were quantified by densitometric scanning of films. Graphs were derived from 3 or more independent experiments and data is plotted as means and standard error. Statistical analysis was performed using Student's \u2018t\u2019 test and differences between means judged statistically significant for p<0.05."}
+{"text": "Escherichia coli (ETEC) F4ac is a major determinant of diarrhea and mortality in neonatal and young pigs. Susceptibility to ETEC F4ac is governed by the intestinal receptor specific for the bacterium and is inherited as a monogenic dominant trait. To identify the receptor gene (F4acR), we first mapped the locus to a 7.8-cM region on pig chromosome 13 using a genome scan with 194 microsatellite markers. A further scan with high density markers on chromosome 13 refined the locus to a 5.7-cM interval. Recombination breakpoint analysis defined the locus within a 2.3-Mb region. Further genome-wide mapping using 39,720 informative SNPs revealed that the most significant markers were proximal to the MUC13 gene in the 2.3-Mb region. Association studies in a collection of diverse outbred populations strongly supported that MUC13 is the most likely responsible gene. We characterized the porcine MUC13 gene that encodes two transcripts: MUC13A and MUC13B. Both transcripts have the characteristic PTS regions of mucins that are enriched in distinct tandem repeats. MUC13B is predicated to be heavily O-glycosylated, forming the binding site of the bacterium; while MUC13A does not have the O-glycosylation binding site. Concordantly, 127 independent pigs homozygous for MUC13A across diverse breeds are all resistant to ETEC F4ac, and all 718 susceptible animals from the broad breed panel carry at least one MUC13B allele. Altogether, we conclude that susceptibility towards ETEC F4ac is governed by the MUC13 gene in pigs. The finding has an immediate translation into breeding practice, as it allows us to establish an efficient and accurate diagnostic test for selecting against susceptible animals. Moreover, the finding improves our understanding of mucins that play crucial roles in defense against enteric pathogens. It revealed, for the first time, the direct interaction between MUC13 and enteric bacteria, which is poorly understood in mammals.Enterotoxigenic  Escherichia coli (ETEC) expressing the F4 (previously known as K88) fimbriae is a major cause of diarrhea in neonatal and pre-weaned piglets Enterotoxigenic S  and s . It is assumed that susceptibility towards ETEC F4ac is determined by the intestinal receptor that allows the bacterium to adhere to the intestinal tract or not. The identification of the receptor locus is thus desirable for the pig industry as it would enable us to accurately and efficiently eliminate the susceptible allele from nucleus breeding populations, leading to decreased mortalities caused by ETEC F4ac infection.Three antigenic variants of F4 have been described: F4ab, F4ac and F4ad, of which F4ac is the most prevalent LMLN-S0283 region by recombination breakpoint analysis MUC4MUC13MUC20TFRCin vitro F4ac adhesion phenotypes in specific pig populations have been described MUC13 is the responsible gene for the intestinal receptor conferring susceptibility to ETEC F4ac infection in pigs. We further identified MUC13 markers that are in complete linkage disequilibrium with the resistant causal allele in a broad panel of Western pig populations. The finding allowed us to select for the F4ac resistant animals and would greatly benefit the worldwide pig industry.The locus encoding the intestinal receptor for ETEC F4ac, denoted as F4acR, has been initially mapped to the q41 region on pig chromosome 13 (SSC13) by two independent linkage analyses 3 intercross population 2 and 461 F3 animals were recorded for in vitro F4ac adhesion phenotypes by a microscopic enterocyte adhesion assay as described previously 2 pedigree for 194 microsatellite markers covering the pig genome and performed a whole genome scan. The linkage analysis mapped F4acR to a region of 7.8 cM flanked by SW207 and S0075 in the q41 region on SSC13, which confirmed the previous reports of other investigators To identify loci affecting economically important traits in pigs, we constructed a large scale White Duroc \u00d7 Erhualian FSW207 - S0075 interval on SSC13. A panel of 50 informative markers including 32 microsatellite and 18 SNPs on SSC13 were genotyped on all animals in the White Duroc \u00d7 Erhualian F2 cross. A multipoint linkage analysis showed that the UMNp997\u2013 S0283 interval of 5.7 cM was defined as the most likely region harboring F4acR as the association of this region was 100-fold stronger than that for any other region in the genome . We identified susceptible and resistant haplotypes of founder animals by their complete association with adhesion and non-adhesion phenotypes in the cross, respectively. Recombination events in the candidate region of F4acR were observed in one F2  and one F3 animals . Individual 3314 was a non-adhesive animal and should be homozygous for the resistant allele. In the F4acR region, this animal carried a non-recombinant resistant chromosome from Erhualian founder sows and a recombinant chromosome from White Duroc founder boars. The recombinant SWR2054\u2013 UMNp595 interval was identical to the susceptible haplotype, which thus positioned F4acR downstream of UMNp595  on SSC13  at 140.93 Mb on SSC13  as the SNP. We further performed LDLA analysis for F4acR using the 60K chip data and adhesion phenotype data of the F2 population. The analysis detected the most significant marker (MARC0096736) in the 2.3-Mb region on SSC13  that has also been proposed as a candidate of F4acR by other investigators MUC13 g.28784 T>C was the most significantly associated marker in both Chinese and Western pigs. Especially, this SNP had an accuracy of more than 97% (144 out of 148) distinguishing susceptible and resistant animals in the 148 independent Western pigs  that was highly expressed in the jejunum. As the deduced MUC13 protein lacked the typical PTS region of mammalian mucins in the N-terminus that is enriched in proline, threonine and/or serine, we speculated that pig MUC13 could have another much longer transcript containing the PTS region MUC13 transcripts compared with our previous finding MUC13A (JN613414) and MUC13B (JN613417), share the same 5\u2032UTR of 35 bp, transcription start site and 3\u2032UTR of 1497 bp, but have distinct PTS regions that are rich in tandem repeats spanning approximate 3\u20135 kb (MUC13B is a string of 8 amino acid residues comprising threonine and proline (TPTPTTTP or TPTPTTTL). It is noteworthy that we failed to characterize the exact number and length of repeats of both transcripts, as the repetitive sequences were unsuccessfully amplified or sequenced using the current available technologies possibly due to the complex second structures of the sequences. Nevertheless, Southern blot analysis revealed that the length of the tandem repeat region was approximate 3\u20135 kb long (data not shown).We have previously isolated a 2679-bp cDNA of pig e 3\u20135 kb Figure 4MUC13, we screened 4 pig genomic DNA libraries and identified positive BAC/PAC clones encompassing the MUC13 gene from the libraries. By using the Solexa deep sequencing technology, we obtained the DNA sequences of these clones  and characterized the genomic structure of the porcine MUC13 gene. Each BAC clone contained a single MUC13 gene, corresponding to one of the above-mentioned two transcripts of MUC13  exhibit a high degree of sequence identity at the nucleotide level (>95%), and both consist of 12 exons and 11 introns . The Indel was used as a diagnostic marker for MUC13A and MUC13B alleles for the following analysis. Like the cDNA analysis, we unsuccessfully determined the complete DNA sequence of the PTS regions as the Solexa sequencing technology generated short pair-end reads of 148 bp that can not reveal the definite number of tandem repeats. Sequencing mucin genes has been shown to be technically difficult due to the large size and the repetitive structure of these molecules. For example, the missing sequence information for the PTS region is also encountered for MUC3A, MUC6, MUC7, MUC12 and MUC13 in cattle To determine the complete genomic DNA sequence of pig of MUC13 Figure 4MUC13A and MUC13B transcripts are encoded by a single gene or two separate loci, we developed a genomic qPCR assay to quantify copy numbers of MUC13 in the pig genome. The copy number assay measured the relative copy ratio between MUC13 and the reference GAPDH gene. We performed the assay on 60 representative pigs from Chinese and Western diverse breeds. The assay showed that all tested animals had a single MUC13 gene with the copy number ratio of 1.0 to GAPDH from diverse breeds for the diagnostic Indel marker, and analyzed association of the two MUC13 alleles with F4ac adhesion phenotypes in these pigs. We found that all 124 pigs homozygous for the MUC13A allele from the broad breed panel were resistant (non-adhesive) to ETEC F4ac. Moreover, all 594 susceptible animals carried at least one MUC13B allele  showed the complete (100%) association with the adhesion phenotypes in Western MUC13B homozygous pigs (Table S1). It further supported the MUC13 gene as F4acR.To examine the effect of B allele Table 1.MUC13B is associated with both susceptibility and resistance while MUC13A confers only resistance to ETEC F4ac, we analyzed the O-glycosylated site of the two MUC13 transcripts as the site is presumed to be the binding site of the bacteria O-glycosylated, i.e. addition of many short O-linked glycans, such as N-acetyl-galatosamine , to the peptides of mucins. The O-glycosylation is essential for the function of mucins as it is required to maintain an extended conformation to create a long, filamentous structure. The highly elaborate structures allow mucins to mediate the interactions between epithelia and their surroundings. The abnormal interactions have been implicated in many disease processes including infectious and inflammatory diseases, cancer and metastasis To elucidate why O-glycosylation sites . This indicates that the peptide of MUC13A can not form the proper filamentous structure by the O-glycosylation for the attachment of ETEC F4 fimbriae. It hence explains why MUC13A homozygous animals are all resistant to ETEC F4ac. For MUC13B, it has potential O-glycosylation sites predominantly in the PTS region . Therefore, MUC13B could be heavily or lightly O-glycosylated depending on the variable tandem repeat sequences of the PTS region. This is concordant with the observation that MUC13B is associated with both susceptibility and resistance towards ETEC F4ac.Protein motif/domain analysis showed that MUC13A does not have n sites ,Figure S2O-linked mucin-type sialoglycoproteins of 210\u2013240 kDa MUC13B PTS sequence. These findings give additional strong supporting evidence for the MUC13 gene determining susceptibility/resistance to ETEC F4ac.It has been reported that the intestinal receptor for F4ac is MUC13 did not differ significantly in the small intestine of susceptible and resistant animals . The finding is consistent with the recent report that the expression of MUC13 is not related to susceptibility towards ETEC F4ac MUC13 causative mutation(s), we screened variants in the complete coding region except for the PTS repetitive sequences using RNA of susceptible and resistant animals from both White Duroc and Erhualian breeds. We detected 14 nonsynonymous mutations out of 24 cSNPs. All cSNPs along with 55 intronic SNPs of MUC13 were included in the data set of 188 SNPs that were genotyped on the 292 outbred pigs. To test if these mutations of interest contribute to susceptibility towards ETEC F4ac, we analyzed their association with the adhesion phenotypes in the 292 animals. The protein-altered SNPs occurred in both susceptible and resistance animals, thereby excluding them as the causative mutation.Quantitative RT-PCR analysis showed that the expression level of O-glycolysation sites that are essential for the biological functions of mucins. Hence, variance in the number, length and sequence of the tandem repeats can impact the extent and type of glycosylation and consequently the functions of mucins. For example, the variable tandem repeats in a variety of mucins have been associated with disease susceptibility in humans  MUC13 maps to the 2.3-Mb critical region containing F4acR; (2) MUC13 is proximal to the most significant markers in both GWAS and LDLA analyses based on large scale SNPs scan across the pig genome; (3) Of the 188 SNPs around the critical region, the six most significant SNPs that had 1000-fold stronger association than any other SNP in diverse outbred populations were all located in MUC13 (4) MUC13A allele was completely associated with the F4ac non-adhesion phenotype across diverse pig populations; (5) All susceptible animals from the broad breed panel carried at least one MUC13B allele; (6) MUC13 is perfectly consistent with the known biochemical properties of F4acR, as MUC13B has the unique O-glycosylation region that forms the binding site of bacterium and is rich in threonine and proline while MUC13A does not. Altogether, these data allow us to conclude that the MUC13 gene confer susceptibility/resistance to ETEC F4ac in pigs.We herein described the causality of the MUC13 PTS region that can not be amplified and sequenced using the current technologies.Overall, our findings have important practical consequences and will have immediate impact on pig breeding programs, as they allow the rapid elimination of the susceptible allele and consequently greatly benefit animal welfare and the pig industry. The findings also provide novel insights into the functions of mammalian mucins, as it establishes, for the first time, the direct interaction between MUC13 and enteric bacteria. Further endeavors will be directed to identify causative mutations in the All animal work was conducted according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. The ethics committee of Jiangxi Agricultural University specifically approved this study.3 intercross population, one Western commercial population, one Chinese cultivated population (Sutai) and 15 outbred populations. The intercross population was constructed with two divergent founder breeds: White Duroc and Chinese Erhualian. Two White Duroc boars were mated to 17 Erhualian sows, and 9 F1 boars were then intercrossed with 59 F1 sows avoiding full-sib mating to generate 1912 F2 animals, of which 87 boars and 299 sows were intercrossed to produce 5311 F3 animals. In this study, 755 F2 and 461 F3 animals at day 240 were slaughtered for ETEC F4ac adhesion phenotype recording. The management of the experimental population has been described previously www.iciba.com/strain/, animals of each Chinese breed except for Lantang pigs were collected from at least 3 unrelated sire families (no common ancestry for 3 generations) each with 2 to 4 animals. For the three Western breeds, piglets of each breed were collected from 5 nucleus populations representing 18 (Duroc), 8 (Landrace) and 21 (Large White) sire families. Genomic DNA was extracted from ear tissues using a routine phenol/chloroform way and diluted to a final concentration of 20 ng/\u00b5l.Experimental animals were from one White Duroc \u00d7 Erhualian Fin vitro ETEC F4ac adhesion phenotypes with slight modification as described previously \u22121) at 37\u00b0C for 30 min with gentle shaking. Each brush border was subsequently tested for its adhesion with F4ac by a phase contrast microscopy (Leica). A total of 20 well-separated and intact brush borders were examined in each specimen. In cases where less than four brush borders bound more than two bacteria, an additional 20 brush borders were scored. According to the classification standard proposed by Baker et al. A microscopic enterocyte adhesion assay developed by Baker et al. 2 population as described in Guo et al. 1 boars was amplified with primers listed in Table S2 and amplicons were directly sequenced in a 3130xl Genetic Analyzer (Applied Biosystem) using original primers. Additional microsatellite markers in the critical region were mined from the pig genome assembly (Sscrofa10 at http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9823). The newly developed microsatellite and SNP markers were genotyped for all animals in the intercross F2 pedigree with primers given in Table S3 by using the fluoresce dye labeled primers (for microsatellite), SNapshot (Applied Biosystem) and PCR-RFLP technologies. A multipoint linkage analysis was performed on SSC13 with Allegro version 2 A panel of 194 informative microsatellite markers covering the pig genome was genotyped across the White Duroc \u00d7 Erhualian F2 and F3 animals that carried a recombinant susceptible haplotype from the founder animals in the F4acR region were explored to define the genomic location of F4acR by recombination breakpoints.Haplotypes in the candidate region of F4acR were reconstructed for all tested animals in the White Duroc \u00d7 Erhualian cross using SimWalk2 software 2 intercross on an Illumina iScan System following the manufacture\u2019s protocol. The bead arrays with call rate <85% were excluded for further analyses. Genome-wide association studies were performed on all SNPs with a minor allele frequency (MAF) >0.05 and call rate >95% by GenABEL The PorcineSNP60 BeadChips (Illumina) were used to genotype all animals across the White Duroc \u00d7 Erhualian FLDLA was performed in a haplotype-based approach with the assumption that each current founder population was originated from a history population (K\u200a=\u200a20) after recombination and drift of N generations. The haplotypes of the history population can be reconstructed by a Hidden Marcov model Table S4. A final panel of 188 informative SNP markers (Table S4) in 24 genes was genotyped on the 292 purebred animals with the adhesion phenotypes by iPLEX SEQUENOM MassARRAY platform. SNP genotype calls were filtered and checked manually, and aggressive calls were omitted from the dataset. Associations of SNP markers with F4ac adhesion phenotypes were evaluated with the standard \u03c7-test implanted in BEAGLE Polymorphisms in the responsible region of F4acR defined by recombinant breakpoint analysis were identified by comparative sequencing of genomic DNA of two adhesive White Duroc and two resistant Chinese Erhualian animals using primers given in MUC13 specific primers F1/R1 and F2/R2 (Table S5) were designed from the 5\u2032- and 3\u2032- regions of the previously isolated MUC13 mRNA sequence (NM_001105293). Three BAC and one PAC clones harboring the complete porcine MUC13 gene were identified by PCR screening of 4 genomic DNA libraries constructed from Western MUC13 specific primers. These clones were sequenced at 300\u00d7coverage by the Solexa (Illumina) technology at Beijing Genomic Institute, Shenzhen.2 animals using the Rneasy Fibrous Tissue Mini Kit (Qiagen). The first strand-complementary DNA was synthesized with the SMART RACE cDNA synthesis Kit (Clontech) and the 5\u2032-Full RACE Kit (TaKaRa). To obtain the extended 5\u2032-end of MUC13 cDNA, the first strand cDNA (Clontech) was first amplified with MUC13 nested primers F3/NF3 (Table S5) and universal primers UPM/NUP . To isolate the further 5\u2032-end sequence of cDNA, primers F4/NF4 and F5/NF5 (Table S5) were designed from a conserved region of the first exon of mammalian MUC13 and the extended 5\u2032 cDNA sequence by Clontech RACE. The primers together with 5\u2032RACE Outer and Inner Primers  were used to amplify the first strand cDNA (TaKaRa). Primers F6/R6 (Table S5) were designed to amplify a fragment filling the gap of MUC13A transcript. All RACE PCR products were cloned to pGEM-T Easy Vector (Promega) for sequencing analysis using M13 universal primer. The full-length MUC13 cDNA sequence was obtained by joining the 5\u2032RACE amplicon sequences with the previously isolated cDNA sequence MUC13 genomic DNA sequence was determined by the alignment of the obtained cDNA sequence with the BAC/PAC sequences.Total RNA was extracted from the jejunum of both adhesive and non-adhesive FMUC13 and GAPDH specific amplicons of 437 bp and 368 bp were generated by routine PCR with primers MUC13-FP1/RP1 and GAPDH-FP1/RP1 (Table S6), respectively. The two amplicons were connected to form an 805-bp fragment by bridge PCR using primers MUC13-FP1 and GAPDH-RP1 (Table S6). The fused fragment was cloned into a pGEM-T Easy vector (Promega). Sequence analysis confirmed that the recombinant plasmid clone contained a single copy of MUC13 and GAPDH fragments. The plasmid DNA was used as the reference sample in subsequent genomic qPCR assays, which determined copy numbers of MUC13 in the pig genome.Table S6) were designed for target (MUC13) and reference (GAPDH) genes. The target and reference probes were 5\u2032 labeled with 6-FAM and VIC, respectively. Both probes were 3\u2032 labeled with the minor groove binder non-fluorescent quencher (ABI). The amplification efficiencies of MUC13 and GAPDH were measured and validated by the CT slope method over a fivefold range dilution of the reference DNA. Standard curves were created by plotting the CT values against the logarithm amount of DNA. Genomic qPCR assays were performed using 60 independent animals from Chinese and Western diverse breeds. The 60 animals were classified into 10 groups according to their adhesion phenotypes and genotypes at MUC13A and MUC13B alleles. The target/reference ratios of all samples are normalized by the target/reference ratio of the calibrator sample (the plasmid DNA) using the method described in TaqMan probes and primers . The MUC13A allele was indicated by amplicons of 151 bp, and the MUC13B allele was represented by amplicons of 83 bp. The diagnostic test for the most significant MUC13 marker (g.28784 T>C) in outbred populations was performed using the ABI SNapshot protocol. A 280-bp DNA fragment was amplified with the F8/R8 primer pairs . SNapshot reactions were performed with Multiplex Ready Reaction Mix (Applied Biosystem) and an extension primer (5\u2032-TTT TTT TTT TTT TTT CCA TGT ACA TTT CAG AGT CTG AGG GAT-3\u2032) using an ABI 3130XL Genetic Analyzer (Applied Biosystem).The Indel in intron 2 distinguishing O-glycosylation sites and N-Glycosylation sites were predicted using the DictyOGlyc and NetNGlyc server, respectively Computational analyses were performed to identify the protein domains of MUC13A and MUC13B. Since the exact number of tandem repeat in the PTS region was not known, we initially assumed this repeat number as 10 for the following analyses. To make sure the analyses to be robust, we compared the results from the sequences with repeat number varying from 10 to 100. The protein domains were identified using Pfam Figure S1Detection of the diagnostic Indel marker for MUC13A and MUC13B alleles by PCR analysis. Genomic DNA was amplified with forward (5\u2032-TTC TAC TCT GAT TCC ACA TCA CG-3\u2032) and reverse (5\u2032-TGG TCA TGT CTA GGA CTC TTT GAG-3\u2032) primers. Amplicons of 151 bp and 83 bp indicate the MUC13A and MUC13B alleles, respectively. Lanes 1\u20133, 5, 8, 11 and 12: AB; lane 10: AA; lanes 4, 6, 7 and 9: BB; M: 50 bp marker.(TIF)Click here for additional data file.Figure S2Plots of probabilities indicating the potential O-glycosylation sites in the deduced peptides of MUC13A (upper panel) and MUC13B (lower panel). The positions of amino acids are given on the x-axis. Vertical green lines indicate the probabilities for the O-glycosylation at each residue. The red line indicates the threshold for the predicted O-glycosylation site.(TIF)Click here for additional data file.Figure S3Real-time RT-PCR analysis of MUC13B expression in the small intestine of susceptible and resistant animals from White Duroc and Erhualian breeds. Tissue samples were collected from piglets at the age of 6\u20138 weeks for RNA extraction. Three susceptible and three resistant animals homozygous for MUC13B were sampled from each breed. Real-time PCR was performed in triplicate. MUC13B expression levels normalized with \u03b2-actin are given (mean \u00b1 s.e.). No significant difference was observed in MUC13B expression levels between susceptible and resistant pigs. EHL+: Erhualian adhesive pigs; EHL-: Erhualian non-adhesive pigs; WD+: White Duroc adhesive pigs; WD-: White Duroc non-adhesive pigs.(TIF)Click here for additional data file.Table S1The complete association of MUC13B SNPs with F4ac adhesion phenotypes in Western purebred pigs.(DOC)Click here for additional data file.Table S2Primers for identification of SNP markers in the region of F4acR that were genotyped in the intercross population.(DOC)Click here for additional data file.Table S3The microsatellite and SNP markers in the region of F4acR that were genotyped in the intercross population.(DOC)Click here for additional data file.Table S4Primers for identification of SNP markers in the region of F4acR that were genotyped in outbred populations.(DOC)Click here for additional data file.Table S5Primers for isolation of the full-length cDNA and genomic DNA sequence of the porcine MUC13 gene.(DOC)Click here for additional data file.Table S6Primers for the copy number assay of the porcine MUC13 gene.(DOC)Click here for additional data file."}
+{"text": "HSD11B2) buffers the impact of maternal glucocorticoid exposure by converting cortisol/corticosterone into inactive metabolites. However, previous studies indicate that maternal adversity during the prenatal period can lead to a down-regulation of this enzyme. In the current study, we examined the impact of prenatal stress  in Long Evans rats on HSD11B2 mRNA in the placenta and fetal brain (E20) and assessed the role of epigenetic mechanisms in these stress-induced effects. In the placenta, prenatal stress was associated with a significant decrease in HSD11B2 mRNA, increased mRNA levels of the DNA methyltransferase DNMT3a, and increased DNA methylation at specific CpG sites within the HSD11B2 gene promoter. Within the fetal hypothalamus, though we find no stress-induced effects on HSD11B2 mRNA levels, prenatal stress induced decreased CpG methylation within the HSD11B2 promoter and increased methylation at sites within exon 1. Within the fetal cortex, HSD11B2 mRNA and DNA methylation levels were not altered by prenatal stress, though we did find stress-induced elevations in DNMT1 mRNA in this brain region. Within individuals, we identified CpG sites within the HSD11B2 gene promoter and exon 1 at which DNA methylation levels were highly correlated between the placenta and fetal cortex. Overall, our findings implicate DNA methylation as a mechanism by which prenatal stress alters HSD11B2 gene expression. These findings highlight the tissue specificity of epigenetic effects, but also raise the intriguing possibility of using the epigenetic status of placenta to predict corresponding changes in the brain.Maternal exposure to stress during pregnancy is associated with significant alterations in offspring neurodevelopment and elevated maternal glucocorticoids likely play a central role in mediating these effects. Placental 11\u03b2-hydroxysteroid dehydrogenase type 2 ( In humans, the experience of stress during pregnancy is associated with increased risk of preterm birth, reduced birth weight, and smaller head circumference HSD11B2 gene leads to hypertension, excess mineralocorticoid activity, and increased anxiety-like behavior in adulthood HSD11B1 mutation leads to attenuated negative-feedback of the HPA response to stress and improved cognitive performance in aging In mammals, the placenta serves as a critical interface between maternal and fetal physiology and forms a barrier to maternal glucocorticoids nd\u20133rd trimester of human pregnancy) was found to decrease placental 11\u00df\u2013HSD2 enzymatic activity and decrease mRNA levels of this gene HSD11B2 mRNA levels HSD11B2 mRNA levels have also been found associated with intrauterine growth retardation and pre-term birth There is increasing evidence that maternal adversity during pregnancy may lead to a down-regulation of 11\u00df\u2013HSD2. In rats, chronic restraint stress during gestational days 11\u201320 . All procedures were performed in accordance with guidelines of the NIH regarding the Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee (IACUC) at Columbia University.16 female and 8 male Long Evans rats (purchased from Charles River) were maintained on a 12:12 hour light-dark schedule with white lights on at 0800 h and off at 2000 h and housed 2 per cage (same-sex) in 26\u00d750\u00d722 cm polycarbonate cages in the animal facility at the Department of Psychology, Columbia University. Food and water were available Within the sample of 16 mated females, 12 females became pregnant following the 1-week mating period. At gestational day 14, all pregnant females were singly-housed and assigned to either non-stress  or stress (n\u200a=\u200a6) treatment conditions. Control females were left undisturbed throughout gestational days 14\u201320. Stress females were exposed to restraint stress, through placement in a 19\u00d729\u00d712 cm cage  for 1-hour/day from gestational days 14\u201320. The timing of stress exposure was randomized to prevent habituation. At gestational day 20, pregnant dams were sacrificed 1 hour after restraint stress through rapid decapitation and trunk blood was collected for assay of corticosterone levels. Plasma corticosterone was assayed using an RIA kit  and this assay confirmed elevated levels of corticosterone in stressed compared to control dams .via caesarean section at gestational Day 20. Pups were decapitated and whole brains extracted. Placenta and fetal brains were snap-frozen, and stored at \u221280C until further processing. Cortex and hypothalamus of E20 fetal brains were dissected in a cryostat cooled to \u221220C. Placenta samples were dissected such that a pie slice including both the basal zone and inner labyrinth zone was used. Samples were weighted and homogenized in 700 \u00b5l lysis buffer RLT-Plus (Qiagen) with 0.1% \u03b2-mercaptoethanol using a tissue homogenizer (Omni) for 15\u201320 seconds. To analyze both mRNA and DNA methylation from the same sample, RNA and DNA from fetal cortex, hypothalamus, and placenta samples were isolated simultaneously using a dual RNA/DNA extraction kit (Qiagen). cDNA was synthesized using a reverse transcription kit (Applied Biosystems) according to manufacturer\u2019s protocol. Samples were stored at \u221220C until further processing. Subsequent analysis of gene expression and CpG methylation was conducted using tissue from 1\u20132 offspring each of 4 control and 4 stress dams. Selection was based on average pup weight at the time of sacrifice. Only litters in which average pup weight was greater than 2.5 g were included in this sample and average pup weight did not differ significantly between treatment groups.Feti and placenta were extracted HSD11B2), 11\u03b2-hydroxysteroid dehydrogenase -1 (HSD11B1), DNA methyltransferase-1 (DNMT1), and DNA methyltransferase-3a (DNMT3a) were conducted with the 2ddCT method Tissue  from 2 pups per litter from 4 control and 4 stress dams were included in the gene expression analyses. Relative gene expression was measured by real-time quantitative PCR on a 24-well 7500Fast qPCR thermocycler using SybrFast (Applied Biosystems) with standard amplification and Ct calculation protocols by Applied Biosystems. All primers were designed to span exons and were tested for specificity (single melt curve peak) and efficiency (87\u2013105%). Primer pairs are included in Rattus norvegicus HSD11B2 gene  samples from 1 pup per litter from control and stress dams were run in duplicate and included in the DNA methylation analyses. Purified DNA was analyzed (EpigenDX) for CpG methylation by bisulfite pyrosequencing. Samples were bisulfite converted and PCR amplified . 50 ng of DNA was amplified using a PCR amplification kit (Qiagen) according to standardized protocol  with Sybr DNA loading dye. Immediately after amplification samples were run on a 2% gel and imaged under UV light. Lanes expressing a bright band were identified as male. Multiple SRY loci have been identified in the rat genome In order to determine the sex of pups, DNA purified from placenta of each pup was amplified using primers for All statistics were performed using SPSS . Analysis of average ddCT levels to determine differences in gene expression was conducted with a 2-way ANOVA with tissue type and stress treatment as factors. Tukey\u2019s HSD post-hoc tests were used to compare tissue-specific gene expression levels and two-tailed Student\u2019s t-tests were used to determine significant differences associated with maternal stress. Correlation between DNA methylation levels at specific CpG sites was conducted with two-tailed Pearson correlation coefficients. One-way ANOVA was used to compare DNA methylation between tissue types. Repeated measures ANOVA was used to determine the impact of maternal stress on DNA methylation and Tukey\u2019s HSD was used to compare stress effects at specific CpG sites. Analysis of stress effects on overall levels of DNA methylation averaged across sites was conducted using two-tailed Student\u2019s t-tests. Though sex-specific effects on gene expression and DNA methylation were not observed in this sample, sex was used as a covariate in the analyses. Significance was set at the p<0.05 level.HSD11B2 gene expression indicated a main effect of tissue \u200a=\u200a15.37, p<.001), a main effect of stress \u200a=\u200a5.46, p<.05), and a significant tissue by stress interaction \u200a=\u200a6.66, p<.01). Overall, there were significantly higher levels of HSD11B2 mRNA in the placenta (p<.01) compared to fetal hypothalamus and cortex . Prenatal stress was associated with a significant decrease in HSD11B2 mRNA in the placenta . Overall, levels of DNMT1 mRNA were found to be significantly higher in the placenta compared the fetal hypothalamus and cortex (p<.001) and levels of DNMT3a mRNA were significantly elevated in the hypothalamus compared to the cortex and placenta \u200a=\u200a\u22121.79, p\u200a=\u200a.09). Within the fetal cortex, DNMT1 expression was significantly increased in stressed offspring (t(14)\u200a=\u200a\u22122.24, p<.05; DNMT1 expression in the placenta. Analysis of the de novo methyltransferase DNMT3a, indicated a placenta-specific effect of stress on the expression of this enzyme. Prenatal stress was found to be associated with increased placental DNMT3a mRNA levels (t(14)\u200a=\u200a\u22123.71, p<.01; Analysis of HSD11B2 promoter and region from \u2212378 to +56 is rich in CpG sites and contains several transcription factor binding sites for Sp1 and NF-\u03baB, suggesting that this region would be a likely candidate for regulation of gene transcription by DNA methylation. Moreover, previous studies in rats report environmentally-induced differences in methylation within this region associated with variation in gene transcription HSD11B2 promoter  and increased methylation within the placenta .Analysis indicated main effects of tissue , stress , and a significant tissue by stress interaction  on CpG methylation within the HSD11B2. Stress, anxiety, and depression during pregnancy can have a long-lasting impact on the psychological health of children HSD11B2 within the placenta in late gestation DNMT1 and DNMT3a. Within the placenta, this stress effect was specific to the de novo methyltransferase DNMT3a, whereas in fetal hypothalamus and cortex, prenatal stress induced increased mRNA levels of DNMT1. Consistent with the reduced HSD11B2 mRNA in placenta, we find increased placental CpG methylation within several sites of the HSD11B2 gene promoter associated with maternal stress. In contrast, our analysis indicates decreased DNA methylation at several sites of the HSD11B2 gene promoter in fetal hypothalamic tissue associated with stress and no effect of this prenatal exposure on DNA methylation in the fetal cortex. These findings highlight the tissue specificity of epigenetic effects. However, we do identify several CpG sites within the placenta at which an individual\u2019s DNA methylation levels do significantly predict those observed in the fetal cortex, raising the intriguing possibility of using the epigenetic status of placenta to predict corresponding changes in the brain. Overall, these findings provide novel evidence for the epigenetic regulation of HSD11B2 as a potential mechanism linking maternal stress during gestation, dysregulation of placental gene expression, and neurodevelopmental outcomes in offspring.In the current study we report a robust and tissue-specific effect of maternal stress during pregnancy in rats on mRNA levels of the enzyme HSD11B2 mRNA levels, the stress-induced DNA hypomethylation of this gene that we observe in hypothalamic tissue may be an epigenetic precursor to these buffering effects. Time course analysis of HSD11B2 gene expression and DNA methylation throughout the prenatal stress exposure and into the postnatal period may thus be a key strategy for determining the dynamics of glucocorticoid programming mechanisms.Though placental 11\u00df-HSD2 can function as an enzymatic buffer against the deleterious effects of exposure to maternal glucocorticoids, it is clear that this enzyme can be down-regulated by adverse prenatal experiences HSD11B2 has previously been explored outside the context of studies on maternal stress. In humans and in rats, the promoter and first exon of the HSD11B2 gene  are rich in CpG sites and DNA methylation levels at CpG sites within this region are related to the expression of this gene HSD11B2 through in vitro or in vivo treatment with the DNA methyltransferase inhibitor 5-aza-2\u2032-deoxycytidine has been found to increase HSD11B2 expression HSD11B2, CpG methylation at Sp1 and NF1 recognition sequences prevents binding of these transcription factors and diminishes the transcriptional activity of HSD11B2HSD11B2 promoter methylation and decreased HSD11B2 mRNA has also been observed in neonatal offspring exposed to in utero magnesium deficiency HSD11B2 expression and increased HSD11B2 promoter methylation in kidneys at birth and these epigenetic effects lead to altered transcription factor binding of Sp1 and NF-\u03baB HSD11B2 dysregulation. The sex-specificity of environmentally induced changes in DNA methylation is increasingly evident and indeed epigenetic modifications may be involved in the normal process of sexual differentiation HSD11B2 expression or DNA methylation in the current study, it may be that large samples are needed to detect sex-differences in the epigenetic regulation of this gene  or that sex-differences are more likely to emerge in later development.Investigation of the epigenetic regulation of HSD11B2 levels we have found in response to maternal stress during pregnancy may involve the up-regulation of DNA methyltransferase levels in the placenta of stressed offspring. Our data indicate a placenta-specific increase in DNMT3a mRNA in response to maternal stress. This stress-induced effect has implications for genome-wide epigenetic changes, and may account for the diverse phenotypic outcomes associated with maternal adversity during pregnancy. DNMT1 and DNMT3a are enzymes active throughout the lifespan and thus could potentially serve as a mechanism for long-term epigenetic regulation, though it is possible that DNMT3b (only active early in development) is similarly altered by prenatal stress to mediate HSD11B2 promoter methylation. Our data are consistent with previous findings indicating an up-regulation of DNMTs in the placenta of mice exposed to 1st trimester maternal stress DNMT1 or DNMT3a in mice has been found to produce embryonic and postnatal lethality and widespread epigenetic changes \u2013 particularly amongst imprinted genes \u2013 in embryonic tissues DNMTs and DNMT3L may account for the changing epigenetic and transcriptional profiles in this tissue during the course of pregnancy A candidate mechanism for the increased placental DNA methylation and increased HSD11B2 mRNA, DNMT expression, and CpG methylation within the HSD11B2 gene promoter. Despite stress induced decreases in HSD11B2 CpG methylation in the fetal hypothalamus, we do not find differential expression of hypothalamic HSD11B2 and within the fetal cortex we find stress-induced increases in DNMT1 without corresponding changes in the DNA methylation or expression of HSD11B2. The regulation of HSD11B2 expression is complex and these paradoxical findings suggest that gene regulation in response to prenatal stress is also accomplished by other epigenetic mechanisms, such as chromatin remodeling, as well as regulation by transcription factors known to influence HSD11B2 transcription  promoter in fetal cord blood samples and the degree of CpG methylation detected in these cells predicts salivary cortisol levels of infants at 3 three months of age NR3C1 DNA methylation in blood samples from children/adolescents that were in utero during the stressor e.g. blood, placenta) and target tissues  has yet to be established and will likely vary dependent on the target gene, the nature of the environmental exposure, and the timing of sampling. Our data indicate that though at most CpG sites within the HSD11B2 gene promoter, DNA methylation levels are equivalent across placenta, hypothalamus, and cortex, within-individual correspondence in the methylation patterns between these tissues is limited. Though it may be possible to predict brain DNA methylation from the degree of CpG methylation observed in placenta \u2013 the biological relevance of the epigenetic markers identified in the current study has yet to be established. Future studies are needed both in humans and animals to address the issue of tissue specificity in environmentally-induced epigenetic variation and to elucidate the mechanisms contributing to the concordance and discordance in tissue-specific epigenetic profiles.The translation of"}
+{"text": "SNAP25 gene and has a hyperactive phenotype similar to that of ADHD. Such mice are 3 fold more active compared to their control littermates. Genetic association studies support a role for allelic variants of the human SNAP25 gene in predisposing to ADHD.The Coloboma mouse carries a \u223c2 cM deletion encompassing the SNAP25 gene in 1,107 individuals (339 ADHD trios). To assess the functional relevance of the SNAP25-ADHD associated allele, we performed quantitative PCR on post-mortem tissue derived from the inferior frontal gyrus of 89 unaffected adults. Significant associations with the A allele of SNP rs362990  and three marker haplotypes  were observed. Furthermore, a significant additive decrease in the expression of the SNAP25 transcript as a function of the risk allele was also observed. This effect was detected at the haplotype level, where increasing copies of the ADHD-associated haplotype reduced the expression of the transcript.We performed association analysis across the SNAP25 confers risk to ADHD and reduces the expression of the transcript in a region of the brain that is critical for the regulation of attention and inhibition.Our data show that DNA variation at  Attention deficit hyperactivity disorder (ADHD) is a highly heritable disorder of childhood with significant functional impairment and negative lifetime outcomes across all developmental stages. The disorder is marked by disruption of catecholamine signaling, with mainstay treatments for the disorder targeting the dopamine and noradrenaline transporters and the alpha 2a adrenoreceptor 2+-triggered neurotransmitter exocytosis Synaptosomal-associated protein (SNAP-25) is a presynaptic plasma membrane protein that is specifically and highly expressed in nerve cells. The gene encodes a protein component, which interacts with the membrane associated protein syntaxin, and vesicle-associated membrane protein (VAMP) to form the SNARE complex. This complex interacts with membrane proteins known as synaptotagmin to make up a core complex essential for docking and holding synaptic vesicles at the presynaptic membrane in preparation for CaSNAP25 and the gene encoding phospholipase C beta (PLCB-1) SNAP25 copy) may contribute to the hyperactive behavior of the coloboma. Similarly, NA depletion using DSP-4(N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine hydrochloride) significantly reduced hyperactivity in the Coloboma mouse, supporting the noradrenaline hypothesis The Coloboma mouse bears a semi-dominant mutation (cm/+) in which the heterozygous form results in the mutant type while the homozygous is lethal. The mutation is a \u223c2 Cm deletion encompassing genes including SNAP25 gene might confer susceptibility to ADHD. An initial analysis SNAP25 gene Genetic association studies suggest that allelic variations in the SNAP25 influence expression within the IFG, with decreased SNAP-25 expression (as expected from the Coloboma mouse) associated with the ADHD risk variants.Here we sought to provide additional evidence for a role of SNAP-25 in ADHD by performing dense SNP mapping across the gene in nuclear families with ADHD. To assess the functional relevance of ADHD-associated variants, we performed quantitative PCR (qPCR) of the SNAP-25 transcript in a large sample (N\u200a=\u200a89) of post-mortem brains from non-clinical individuals. Our qPCR work focused on expression of the transcript in human inferior frontal gyrus (IFG), an area of the cortex that has consistently been implicated in ADHD from both structural and functional imaging analyses SD\u200a=\u200a3.67) and were predominantly male (87%). All probands fulfilled DSM-IV diagnostic criteria for ADHD. Of these, N\u200a=\u200a44 (13%) had the inattentive subtype, and N\u200a=\u200a19 (5.6%) had the hyperactive impulsive subtype; the remainder had the combined subtype (81.4%). One hundred and seventy-nine probands (52.8%) had a comorbid diagnosis of oppositional defiant disorder (ODD) and 34 (10%) fulfilled criteria for comorbid conduct disorder (CD). Frequencies of ADHD subtypes and comorbidities were similar across the recruitment sites. We obtained a written consent from the next of kin, caretakers, or guardians on the behalf of the minors/children participants involved in our study. The consent was in accordance with the declaration of Helsinki and the ethical approvals from the University of Queensland, Australia, Trinity College Dublin, Ireland, and the University of Birmingham, UK\u201dA total sample of 1,017 individuals, comprising 339 Caucasian ADHD probands and their parents (full trios) across three similar sub-samples was investigated. The first cohort comprised 185 full trios recruited throughout Ireland from child psychiatric clinics and schools in West County Dublin and from the Hyperactive and Attention Deficit Children's Support Group of Ireland. Consensus diagnoses were made according to DSM-IV ADHD either with or without comorbidity. These diagnoses were based on all available clinical information and the Child Behaviour Checklist (CBCL), the Conners' Parents and Teachers Rating Scales, and the Comprehensive Teachers Rating Scale (ACTeRS) In addition to the ADHD samples, 89 unaffected Caucasian brain samples  were obtained from the Australian Brain Bank. Of these, 63 (71%) were from male subjects. None of these individuals were diagnosed with ADHD or other psychiatric condition. The mean age of the sample was 51.6 years (SD\u200a=\u200a12.2), PH ranged from 5.8\u20137 and the post-mortem interval (PMI) was 28.2 hours (SD\u200a=\u200a14).CTTGAAGCATCCCAGGAAGA and the reverse 5\u2032 GAAGGAAAAATGTTGGGGTTT 3\u2032. The PCR product (214 bp) was then restricted with the enzyme Cac81 and the DNA fragments were fractionated on a 3% agarose gel. The G allele was characterized by 164 bp and 51 bp fragments while the A allele was characterized by 215 bp fragment.Fifteen SNPs were included in the current investigation . SNPs weApproximately 100\u2013200 mg of inferior frontal gyrus (IFG) tissue of each sample was used to extract RNA and DNA using TRIZOL reagent as recommended by the manufacturers (Invitrogen). As PH is considered a good indicator of RNA purity and integrity, this was measured and found to range from 5.8\u20137. The optical density (OD) at 260/280 of RNA preparations ranged from 2\u20132.1 indicating good quality of the RNA preparation.SNAP25) and the reference genes \u03b22micoglobulin (\u03b22M) and beta actin (ACTB) gene primers were designed to amplify DNA regions involving exon 7 and 8 of SNAP25, exons 1 and 2 in \u03b22M and exon 3 and 4 of ACTB. The qPCR primers were designed using the INTEGRATED DNA TECHNOLOGIES Oligo Design Tool, which is freely available at http://www.idtdna.com/scitools/scitools.aspx. The SNAP-25-qPCR forward primer sequence was 5\u2032ATGGATGAAAACCTAGAGCAGG 3\u2032 and the reverse was 5\u2032ACACTTAAC CACTTCCCAGC 3\u2032. The \u03b22M forward primer sequence was 5\u2032 GGCATTCCTG AAGCTGACAG 3\u2032 whereas the reverse was 5\u2032TGGATGAAACCCAGACACATAG 3\u2032. The ACTB qPCR forward primer was 5\u2032ACCACACCTTCTACAATGAGC 3\u2032 and the reverse was 5\u2032 GCGTACAGGGATAGCACAG3\u2032. In this context, \u03b22M and ACTB were used as reference genes as they have stable gene expression in various tissues including brain. A standard Invitrogen procedure was used to synthesize first cDNA strands of the samples. Relative quantification was performed using SNAP25 (target gene) compared to \u03b22M and ACTB (reference genes). PCR cycling was performed on a Roche LightCycler-480. Cycling conditions were 95\u00b0C for 5 min followed by 45 cycles at 95\u00b0C for 10 sec, 60\u00b0C for 10 sec and 72\u00b0C for 20 sec. This was followed by a melting curve cycle at 95\u00b0C for 5 sec and 1 min at 65\u00b0C. mRNA relative was expression quantified using normalized threshold cycles (Ct) of target relative to reference genes.To minimize RNA contamination with the DNA, samples were treated with DNASE-I and cleaned using RNeasy Mini Kit as recommended by manufacturers . Furthermore, to prevent any possible interference by DNA contamination when conducting qPCR, the target (http://www.broad.mit.edu/mpg/haploview) using the method of Gabriel et al. Assessment of Hardy Weinberg Equilibrium for all SNPs was undertaken using parental DNA of the ADHD participants. All genotypes were in Hardy Weinberg equilibrium. Genetic association across a 15 SNP array was performed using the transmission disequilibrium test (TDT). Haplotype analysis to test for the transmission of multi locus haplotypes was performed by applying the default block definition in HAPLOVIEW . Permutation testing  revealed that only rs362990  was associated with ADHD, with a trend towards association for rs6108461 (p-corrected\u200a=\u200a0.059).Fifteen SNPs were examined at the 2\u200a=\u200a8.37, p-corrected \u200a=\u200a0.019, OR\u200a=\u200a1.4). An exploratory haplotype analysis using sliding window of 8 SNPs (\u223c20 kbp) extending from intron 3 (rs6108461) to the 3UTR (rs8636) enhanced the risk association signal . Finally, a haplotype comprised of the alleles GTC in the second haplotype block . This implies that this haplotype may confer protection against the development of ADHD.Although strong LD is apparent across the gene , three dlock see  was less2 change \u200a=\u200a5.6.2%]. Similar results were found for rs6108461 . The strong LD between these SNPs meant that controlling for one removed the effect of the other in the above analyses. Possession of the ADHD risk haplotype (AAC) was also significantly associated with expression of SNAP-25 in the IFG tissue [2 change \u200a=\u200a6.3%]. As expected, increasing possession (0 vs. 1 vs. 2 copies) of the risk haplotype was associated with decreased expression. Conversely, increasing possession of the protective haplotype was associated with increased SNAP-25 expression [2 change\u200a=\u200a7.7%].A significant association between rs362990 and relative expression of the SNAP-25 transcript was observed . SpecifiSNAP25 gene that associate with ADHD are also associated with functional changes in the expression level of the transcript in a region of the brain that is an established pathological locus for ADHD. The current investigation provides further support for the notion that SNAP25 is a genetic risk factor for ADHD. Although nominal associations with two SNAP25 SNPs (rs6108461and rs362990) were observed, only the association with rs362990 survived permutation testing. Haplotype analysis enhanced the association (OR\u200a=\u200a1.62), suggesting that the region between intron 3 and the 3\u2032UTR of SNAP25 may harbor an important susceptibility variant for ADHD.The results of the current study show that DNA variants of the SNAP25 an important candidate gene for ADHD.SNAP-25 is important for axonal growth and synaptic plasticity, which are essential steps for wiring the nervous system. Selective inhibition of SNAP-25 expression prevents neurite elongation of cortical neurons SNAP25 gene variants and ADHD. All these studies have examined multiple SNPs and tested for association of single SNPs and haplotypes. The associated SNPs and their position within the SNAP25 gene are presented in SNAP25; see 2\u200a=\u200a12.7, p-corrected\u200a=\u200a0.003, OR\u200a=\u200a1.62) indicating that this region may harbor a risk/functional variant for ADHD.Our results are consistent with some but not all previous association/linkage studies. Apart from the current investigation, a number of studies have tested for association between SNAP25, several SNPs showed evidence of association with the symptoms of ADHD in a quantitative GWAS SNAP25 markers including rs362562 SNAP25 variations in ADHD.Furthermore, it is important to emphasize that the quartile\u2013quartile (QQ) plot for ADHD genome wide association (GWAS) for SNPs in or near candidate genes SNAP25 variants may increase risk to ADHD. Specifically, the lowered expression of SNAP-25 in regions of the cortex that are critical for attention and inhibition, such as the IFG, may ultimately decrease the efficiency of neurotransmitter release and synaptic function, impairing behavior and cognition and conferring risk to ADHD. It should be noted however that the above hypothesis must be viewed with some caution since the current study did not test the expression of SNAP25 in post-mortem samples derived from individuals with ADHD.Computer simulation SNAP25 mutations introduced to the C terminal of the protein at AA positions 78, 81 and 202 resulted in a near elimination of exocytosis Recent biochemical analyses have shown that SNAP-25 amino acids (AA) 7\u201383 and 141\u2013204 are essential motifs that are spontaneously assembled into helical SNARE complexes with Syntaxin1 and synaptobrevin 2 motifs SNAP25 as a susceptibility locus for ADHD. We hypothesize that the region between intron 3 and the 3UTR of SNAP25 may harbor functional variants that confer risk to ADHD. Finally, we stress the importance of independent replication of our findings preferably in different ethnic samples.In summary, this study provides support for the involvement of"}
+{"text": "The five year survival of juvenile systemic sclerosis (jSSc) is around 95%. Patients, who died in the two retrospective cohorts, died mostly in the first 24 months of disease course. Autologous bone marrow transplantation (ABMT) seems to be a promising therapeutic approach for adult patients ( ASTIS (EU) and SCOT (USA)Trial) with severe disease course. Around 8 patients with jSSc are transplanted according the EBMT registry. Currently no consent based inclusion or exclusion criteria for ABMT in jSSc exists.Aim of the survey was to get a feeling from paediatric rheumatologists, when they would apply autologous bone marrow transplantation as a treatment option.Paediatric Rheumatologist - members of the PRES Juvenile Scleroderma Working Group and participants of the Paediatric Rheumatology E-mail Board were asked via Internet to fill out the survey22 centres responded, all of them were academic centres. BMT would be considered for 14 of the 22 colleagues after nonresponse to cyclophosphamide, 10 of 22 after non response to two DMARDs and 12 of 22 after nonresponse to Rituximab. 19 of 22 would consider transplantation if the CHAQ score \u2264 2 , 20 of 22 if the CHQ is less than 40%. 21 of 22 would think about transplantation if the modified Rodnan skin score is more than 30 and 15 of 22 if the DLCO is less than 50%, 18 of 22 if the WHO functional class is 3, 14 of 22 if the FVC less than 60%, 15 of 22 if the pulmonary arterial pressure more than 40 mm/hg and 11 of 22 if left ventricular ejection fraction is less than 40%.This survey represents an impression, when pediatric rheumatologist would consider ABMT. It is a starting point for a possible evolving ABMT program for this orphan disease"}
+{"text": "To compare the hemostatic effect of sulfur hexafluoride 20% (SF6 20%) with lactated Ringer\u2019s solution for prevention of early postoperative vitreous hemorrhage following diabetic vitrectomy.In a prospective randomized clinical trial, 50 eyes undergoing diabetic vitrectomy were divided into two groups. At the conclusion of surgery, in one group the vitreous cavity was filled with SF6 20% while in the other group lactated Ringer\u2019s solution was retained in the vitreous cavity. The two groups were compared for the rate of early postoperative vitreous hemorrhage.The incidence of vitreous hemorrhage was lower in the SF6 group than the Ringer\u2019s group 4 days , 7 days  and 4 weeks  after vitrectomy.In comparison with lactated Ringer\u2019s solution, SF6 20% had a significant hemostatic effect especially in the early postoperative period after diabetic vitrectomy and reduced the incidence of vitreous hemorrhage. Vitreous hemorrhage is one of the most serious complications of vitrectomy for proliferative diabetic retinopathy. The incidence of this complication ranges from 8% to 75%.7Postvitrectomy vitreous hemorrhage decreases visual acuity and impairs fundus visualization, it interferes with laser photocoagulation and detection of other retinal complications, furthermore it can predispose to cellular proliferation and may also increase intraocular pressure (IOP).Thompson et alRegarding the lack of data on the incidence of vitreous hemorrhage following diabetic vitrectomy using lactated Ringer\u2019s solution, this pilot study was performed to evaluate the hemostatic effect of SF6 20% as compared to lactated Ringer\u2019s solution in the early postoperative period following diabetic vitrectomy.In a prospective randomized clinical trial, 67 eyes of 67 patients undergoing diabetic vitrectomy who met the inclusion criteria were randomly divided into two groups using a random number table. Informed consent was obtained from all patients preoperatively. Indications for vitrectomy were nonclearing vitreous hemorrhage and progressive fibrovascular proliferation unresponsive to panretinal laser photocoagulation. Patients with orthopedic or systemic problems who were unable to maintain prone position, monocular patients, subjects who were obliged to travel by air immediately after the operation, those who required concomitant cataract surgery, as well as patients with rubeosis iridis, extensive tractional retinal detachment with or without rhegmatogenous retinal detachment were excluded. Patients who required silicone oil injection intraoperatively were also excluded from the study. Seventeen eyes were excluded due to intraoperative use of silicone oil. Eventually 50 eyes of 50 patients fulfilled the inclusion and exclusion criteria of the study and were analyzed.All eyes underwent standard three port pars plana deep vitrectomy by two surgeons; complete posterior vitreous detachment was induced and delamination and segmentation of preretinal fibrovascular tissue was performed. We decided to use lactated Ringer\u2019s solution instead of BSS during diabetic vitrectomy in both groups because BSS contains sodium citrate which has anticoagulant properties and may promote vitreous hemorrhage.t-test for mean values; P values <0.05 were considered significant.The extent of vitreous hemorrhage was graded using indirect ophthalmoscopy according to the Diabetic Retinopathy Vitrectomy Study (DRVS) as follows:Fifty eyes of 50 patients were enrolled and randomized to SF6 20% (25 eyes) versus lactated Ringer\u2019s solution (25 eyes). Mean age was 53.8\u00b111.3  years in the SF6 group and 51.4\u00b112.5  years in the Ringer\u2019s group (P=0.417). Seventeen (68%) patients in the Ringer\u2019s group and 13 (52%) patients in the SF6 group were female (P=0.248). The incidence of vitreous hemorrhage was higher in the Ringer\u2019s group at all postoperative visits .Eleven (22%) patients had systemic hypertension preoperatively and were receiving antihypertensive agents. The incidence of vitreous hemorrhage during the follow-up period was 54.5% (6 cases) among hypertensive patients and 43.5% (16 cases) among normotensive patients (P=0.425). Forty-two eyes were phakic of which 3 eyes (6%), including two eyes in the SF6 group and one eye in the Ringer\u2019s group, developed cataracts postoperatively. Mean IOP was 12.13\u00b11.24 mmHg in the SF6 group and 12.16\u00b10.34 mmHg in the Ringer\u2019s group (P=0.951) 4 weeks postoperatively. Forty-one (82%) patients had type II and 9 (18%) patients had type I diabetes mellitus. The incidence of vitreous hemorrhage during the follow-up period was 46.3% (19 cases) in type II diabetic patients and 44.4% (4 cases) in type I patients (P=0.87).Visual acuity was hand motions or worse in 26 (52%) patients and better than hand motions but less than 6/120 in 24 (48%) patients preoperatively with no significant difference between the two groups (data not presented). At final visit, 11 (22%) eyes had visual acuity worse than 6/120, including 7 (28%) eyes in the Ringer\u2019s group and 4 (16%) eyes in the SF6 group; 13 (26%) eyes had visual acuity between 6/120 and 6/60, including 8 (32%) eyes in the Ringer\u2019s group and 5 (20%) eyes in the SF6 group; and 26 (52%) eyes had visual acuity better than 6/60, including 10 (40%) eyes in the Ringer\u2019s group and 16 (64%) eyes in the SF6 group. The difference in postoperative visual acuity between the study groups was not statistically significant (P=0.235).Despite laser treatment for proliferative diabetic retinopathy, the ocular sequelae of diabetes mellitus remain one of the most common indications for deep vitrectomy. Postoperative vitreous hemorrhage is a common complication of diabetic vitrectomy with a reported incidence of up to 75%. Advances in surgical technique and instrumentation including endodiathermy and endolaser photocoagulation have reduced the incidence of this complication to 25%.Various substances have been used to prevent vitreous hemorrhage after diabetic vitrectomy. De Bustros et alKoutsandrea et alYang et al In our study, we employed lactated Ringer\u2019s solution instead of BSS, because the latter contains citrate which can promote bleeding during and after surgery. Contrary to Koutsandrea et al,It summary we may conclude that SF6 20% in the vitreous cavity provides temporary tamponade and reduces the incidence of postoperative diabetic vitreous hemorrhage; disadvantages include the potential for inducing cataracts and prone positioning."}
+{"text": "The Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) recently identified 16 loci robustly associated with fasting glucose, some of which were also associated with type 2 diabetes. The purpose of our study was to explore the role of these variants in South Asian populations of Punjabi ancestry, originating predominantly from the District of Mirpur, Pakistan.SLC30A8 rs11558471 SNP was nominally associated with fasting glucose . Of interest, the MTNR1B rs10830963 SNP displayed a negative \u03b2 value for fasting glucose in our study; this effect size was significantly lower than that seen in Europeans (p\u200a=\u200a1.29\u00d710\u22124). In addition to previously reported type 2 diabetes risk variants in TCF7L2 and SLC30A8, SNPs in ADCY5 (rs11708067) and GLIS3 (rs7034200) displayed evidence for association with type 2 diabetes, with odds ratios of 1.23  and 1.16  respectively.Sixteen single nucleotide polymorphisms (SNPs) were genotyped in 1678 subjects with type 2 diabetes and 1584 normoglycaemic controls from two Punjabi populations; one resident in the UK and one indigenous to the District of Mirpur. In the normoglycaemic controls investigated for fasting glucose associations, 12 of 16 SNPs displayed \u03b2 values with the same direction of effect as that seen in European studies, although only the SLC30A8 rs11558471 SNP was nominally associated with fasting glucose in our study, the finding that 12 out of 16 SNPs displayed a direction of effect consistent with European studies suggests that a number of these variants may contribute to fasting glucose variation in individuals of South Asian ancestry. We also provide evidence for the first time in South Asians that alleles of SNPs in GLIS3 and ADCY5 may confer risk of type 2 diabetes.Although only the  Type 2 diabetes, a disease that is 4- to 6-fold more common in South Asian individuals than Europeans, is characterised by impaired glucose homeostasis resulting from a combination of beta cell dysfunction and insulin resistance. This inability to adequately regulate blood glucose levels is also linked to both micro- and macro-vascular complications. Recently, the Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) identified 16 genetic variants robustly associated with fasting glucose in non-diabetic populations of European origin Informed written consent was obtained from all study participants and the studies were approved by the Birmingham East, North and Solihull Research Ethics Committee (UKADS participants) and the Baqai Institute of Diabetology and Endocrinology Institutional Review Board (DGP participants). UKADS registered clinical trial number; ISRCTN38297969.Participants in the investigation  were froAll subjects  were genotyped for the 16 SNPs using the KASPar method . Genotyping success rates were >96% for each SNP. Approximately 10% of samples were genotyped as blind duplicates resulting in an error rate of <1% for each SNP. Genotype counts in the two study populations are shown in p<0.05) and study-wide significance . Genetic risk scores (GRSs) were constructed to investigate the additive effect of multiple SNPs on fasting glucose levels and type 2 diabetes. For all GRS analyses, only those individuals successfully genotyped at \u226512 SNPs (nmax\u200a=\u200a3231) were included. Firstly an allele count GRS (acGRS) was constructed. For each individual, the average number of risk alleles per successfully genotyped SNP  was multiplied by the total number of SNPs included in the GRS. This produced a GRS approximating a simple risk allele count where all SNPs were successfully genotyped. Two weighted GRSs (wGRS) were also produced, one for the fasting glucose analyses and one for the type 2 diabetes analyses. Risk alleles of each SNP were weighted by a SNP-specific \u03b2-value, obtained from the MAGIC study TCF7L2 and SLC30A8 variants, as these SNPs have previously displayed association with the disease in the UKADS and DGP study populations http://ibgwww.colorado.edu/~pshaun/gpc/) http://hydra.usc.edu/gxe) Deviation from Hardy-Weinberg equilibrium (HWE) in the non-diabetic groups was tested using an exact test implemented in Haploview SLC30A8 rs11558471 variant displayed a nominally significant association with fasting glucose levels  (MTNR1B rs10830963 effect size was markedly lower in the current study compared with the Europeans from the MAGIC study p\u200a=\u200a1.29\u00d710\u22124). The acGRS showed a stronger association with fasting glucose than did the wGRS   . No evidctively) .TCF7L2 and SLC30A8 variants, which have previously demonstrated association with type 2 diabetes in the UKADS/DGP study populations ADCY5 rs11708067  and GLIS3 rs7034200  SNPs conferred risk of the disease in this study  than the acGRS  .None of the 16 studied variants displayed strong evidence for heterogeneity of effect on type 2 diabetes risk between the current study and the MAGIC study. In addition to the is study . The str=\u200a0.001) . The strSLC30A8 rs11558471 variant was nominally associated with fasting glucose levels. Twelve of the 16 SNPs displayed positive \u03b2-values, however, suggesting that a number of these variants may be true determinants of fasting glucose levels in our study populations  have negative \u03b2-values in the current study  was not.In this study we investigated the effects of 16 SNPs on fasting glucose levels and type 2 diabetes risk in two South Asian populations of Punjabi ancestry. Only the ulations , even thnt study , and thiGCK, GCKR, G6PC2 and MTNR1B have been shown to be associated with fasting glucose levels in Indian Asians, with similar effect sizes to those seen in Europeans GCK and GCKR SNPs studied in Indian Asians (rs4607519 and rs1260326 respectively) are in strong linkage disequilibrium (LD) with the SNPs genotyped in this study  with the variant (rs10830963) genotyped in our South Asian cohort and reported as the sentinel MTNR1B SNP in the MAGIC study MTNR1B SNPs and their association with glucose levels in South Asians may be useful in fine-mapping the aetiological variant.It is of interest to note that variants within or near t is low , it is nTCF7L2 and SLC30A8 have previously been associated with type 2 diabetes in our Punjabi populations ADCY5 and GLIS3 were associated with the disease in the current study. To our knowledge, this is the first time that either of these SNPs has been implicated in type 2 diabetes development in a South Asian population. The ADCY5 gene encodes adenylate cyclase 5, an enzyme that catalyses the generation of cAMP, a second messenger vital in a number of biological processes. It has been demonstrated in a large meta-analysis of Europeans that the rs11708067 SNP within ADCY5 is strongly associated (p\u22643.6\u00d710\u22128) with type 2 diabetes, fasting glucose levels and HOMA-B (a measure of \u03b2-cell function) but is not associated with HOMA-IR (a measure of insulin resistance) ADCY5 gene (rs9883204), in LD with the variant investigated in this study, is associated with foetal growth and birth weight in utero, potentially providing a common mechanism linking reduced foetal growth with increased risk of type 2 diabetes. The GLIS3 gene encodes the transcription factor GLIS family zinc finger 3 isoform, a protein that regulates target gene transcription and has been shown to play a key role in \u03b2-cell generation in mice GLIS3 gene lead to a syndrome of neonatal diabetes and congenital hyperthyroidism GLIS3 rs7034200 SNP only displayed a weak association with type 2 diabetes in the MAGIC study TCF7L2 SNP, three of the four variants most strongly associated with type 2 diabetes in the MAGIC study MTNR1B rs10830963, PROX1 rs340874 and GCKR rs780094) have odds ratios of less than one in our South Asian populations Click here for additional data file."}
+{"text": "Several novel susceptibility loci for type 2 diabetes have been identified through genome-wide association studies (GWAS) for type 2 diabetes or quantitative traits related to glucose metabolism in European populations. To investigate the association of the 13 new European GWAS-derived susceptibility loci with type 2 diabetes in the Japanese population, we conducted a replication study using 3 independent Japanese case-control studies.MTNR1B, GCK, IRS1, PROX1, BCL11A, ZBED3, KLF14, TP53INP1, KCNQ1, CENTD2, HMGA2, ZFAND6 and PRC1) with type 2 diabetes using 4,964 participants  from 3 independent Japanese samples. The association of each SNP with type 2 diabetes was analyzed by logistic regression analysis. Further, we performed combined meta-analyses for the 3 studies and previously performed Japanese GWAS data . The meta-analysis revealed that rs2943641 in the IRS1 locus was significantly associated with type 2 diabetes,  and 3 SNPs, rs10930963 in the MTNR1B locus, rs972283 in the KLF14 locus, and rs231362 in the KCNQ1 locus, had nominal association with type 2 diabetes in the present Japanese samples (P<0.05).We examined the association of single nucleotide polymorphisms (SNPs) within 13 loci (IRS1 locus may be common locus for type 2 diabetes across different ethnicities.These results indicate that  Type 2 diabetes is a chronic metabolic disorder characterized by hyperglycemia, variable degrees of insulin resistance, and impaired insulin secretion. The total number of individuals with diabetes mellitus is estimated to be nearly 300 million worldwide, and its prevalence continues to increase in many countries, including Japan. Although the precise mechanisms underlying the development and progression of type 2 diabetes have not been elucidated, it is considered that genetic factors play an important role in the pathogenesis of the disease KCNQ1) locus, ubiquitin-conjugating enzyme E2E 2 (UBE2E2) locus and C2 calcium-dependent domain containing 4A (C2CD4A)-C2CD4B locus with type 2 diabetes Currently, approximately 40 susceptibility loci for type 2 diabetes, mostly discovered through genome-wide association studies (GWAS), have been confirmed in populations of European descent  IRS1) was identified by GWAS on French patients Because many of these loci have also been shown to be associated with type 2 diabetes in other ethnic populations, including Japanese, these loci may be considered convincing susceptibility loci for type 2 diabetes across different ethnicities In this study, we aim to evaluate the contribution of these new susceptibility loci identified in European GWAS to conferring susceptibility to type 2 diabetes in the Japanese.We selected 4,964 individuals, 2,839 cases and 2,125 controls, from 3 independent Japanese samples.st study): DNA samples were obtained from peripheral blood samples of type 2 diabetes patients recruited from the outpatient clinics of the Shiga University of Medical Science and the Kawasaki Medical School , We also examined 716 controls who were enrolled in an annual health check conducted either at the Juntendo University or the Keio University .RIKEN case-control study (1nd study): We selected 724 individuals with type 2 diabetes from the outpatient clinic of the Toyama University Hospital . We also examined 763 controls with HbA1c<6.0%, age\u226550, no family history of diabetes mellitus for the first and second degree relatives .Toyama University study (2rd study): We recruited 485 individuals with type 2 diabetes from the outpatient clinic of the St. Marianna University School of Medicine . We also examined 646 controls, who were enrolled in an annual health check conducted at the St. Marianna University School of Medicine St. Marianna University study  GCKR locus and rs2191349 in the DGKB- TMEM195 locus were shown to be associated with type 2 diabetes in the Japanese We first selected 9 single nucleotide polymorphisms (SNPs) from previous reports, rs2943641 within the locus near the SNP genotyping was performed by the multiplex-polymerase chain reaction (PCR)-invader assay, as described previously http://www.sph.umich.edu/csg/abecasis/CaTS/).We performed the Hardy-Weinberg Equilibrium (HWE) test according to the method described by Nielsen et al ADCY5 locus, and rs7957197 in OASL locus) are monoallelic in the Japanese populations. The genotype distributions of rs13292136 in CHCHD9 showed significant deviation from the Hardy-Weinberg equilibrium proportion in the control group  was consistent with that of a previous study in European populations 2<80), whereas no heterogeneity in effect size (odds ratio) was observed on the remaining loci (data not shown). We could not observe remarkable differences in LD structures around each locus between Japanese and European populations \u2014 may also have some effects. Further studies are required to elucidate the association of these as well as other loci with susceptibility to type 2 diabetes, and to understand the biological significance of these genes and their polymorphisms.In conclusion, these results indicate that the Figure S1Linkage disequilibrium structures for 500 kb region around each SNP locus in JPT and in CEU. Pairwise correlation structure analyzed by Haploview (http://www.broadinstitute.org/haploview/haploview). The plot includes pairwise D\u2032 values from the HapMap release 27.(PDF)Click here for additional data file.Table S1Genotype data for 15 SNPs in the 3 independent Japanese samples.(DOC)Click here for additional data file.Table S2The associations of the 14 SNPs with type 2 diabetes in 3 independent Japanese samples.(DOC)Click here for additional data file.Table S3Power estimation for each SNP locus in the present study.(DOC)Click here for additional data file."}
+{"text": "FYVE  and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress.Mutation of the autophagy gene Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum.A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions.The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation. The eye lens is an avascular encapsulated organ whose function is to focus light on to the retina where visual information is translated into nerve signals and ultimately perception of images by the visual cortex of the brain. Disruption of lens transparency as a consequence of developmental defect or damage to lens cells and their components causes cataract formation \u20133 which One intriguing system that may be important for lens differentiation and resistance to environmental damage is macroautophagy (hereafter termed autophagy) which operates in the degradation and recycling of damaged organelles and proteins in many other tissues . AutophaATG5) did not disrupt lens fiber cell differentiation despite the occurrence of autophagy in lens cells , tuberous sclerosis complex 1 [tsc 1], UV irradiation resistance-associated gene [uvrag], astrocyte-elevated gene-1 [aeg-1], high temperature requirement\u00a0 factor A2 [omi/htrA2], phosphatase and tensin homolog [pten], autophagy related 14 [atg14], Bax-interacting factor 1 [bif1], high mobility group box 1 [hmgb1], v-ral simian leukemia viral oncogene homolog B [ralB], retinoblastoma 1 inducible coiled coil-1/focal adhesion kinase (FAK) family interacting protein of 200 kDa [rb1cc1/fip200], forkhead box O1 [foxO1], forkhead box O3 [foxO3], and PKR-like ER kinase/eukaryotic translation initiation factor 2-alpha kinase 3 \u00a0[perk/eif2alpha3k]), eight genes were involved in expansion of autophagic vesicles , six were genes involved in autophagosome fusion to lysosomes  and eight genes were involved in specific autophagy sub- pathways including mitophagy  and chaperone mediated autophagy , which was present on the microarray but not definitively identified to be expressed, was also analyzed. These genes included the autophagy induction genes beclin 1, atg14, fip200, ralb, the autophagy expansion/closure genes atg4a, atg12, map1lc3b/atg8, the autophagy fusion/degradation genes rab7, fyco1, the mitophagy genes nix/bnip3L, pink1 and the adaptor gene sequestosome 1 (p62). The data confirmed that all of the analyzed genes were expressed in human lens epithelium and fiber cells  in lens fibers suggests that autophagy is a functional process throughout the lens. Since autophagy is a response to stress in many tissues  we examifyco1 causes human cataract [Autophagy has been shown to be important for the development, differentiation and protection against oxidative stress in multiple tissues ,88, howecataract , suggestcataract .As a first step toward examining the potential function of autophagy in the lens, we analyzed the expression levels of autophagy genes in microdissected human lens epithelium and fiber cells. Our analysis revealed the lens epithelium and fiber cell expression of 14 genes involved in autophagy induction, eight genes involved in expansion of autophagy vesicles, six genes involved in autophagosome fusion to lysosomes and eight genes involved in specific autophagy sub-pathways including mitophagy and chaperone mediated autophagy . The datTo date, few reports on the role of autophagy in lens function have been published. Matsui et al.  reportedmap1lc3b [bnip3l/nix [beclin1 [rb1cc1/fip200 [bif-1 [lamp2 [beclin 1 and fip200 were embryonic lethal and atg3 was neonatal lethal. Lamp2 knockout mice were viable but exhibited increased mortality and bif-1, map1LC3b and bnip3l/nix were viable. Only two genes (apart from fyco1) that were identified in the present report have a previously reported lens function; these are fox01 and fox03 [atm), has also been reported to be important for lens resistance to cataract [In addition to the ATG5 mouse knockout, other knockout mice have been made for some of the genes we detected in the lens but unfortunately no lens or eye phenotype has been described for any of the mice. These include map1lc3b , bnip3l/ip3l/nix ,91, becl[beclin1 , rb1cc1/1/fip200 , bif-1 [0 [bif-1 , and lam1 [lamp2 . Of thesnd fox03  which wecataract . It is iIn summary, the present data provide evidence for a significant role for autophagy in lens function. Autophagy and mitophagy are likely important for lens cell removal of damaged proteins that could cause cataract upon their accumulation and the degradation of lens organelles during epithelial cell differentiation into fiber cells. Further studies on the role of autophagy in lens resistance to stress and lens cell differentiation are likely to provide additional insight into our understanding of lens development, maintenance and cataract formation."}
+{"text": "Drosophila, the MSL  complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. We show that SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.In  Spt5 gene that encodes a universally conserved elongation factor. SPT5 closes the RNA polymerase II clamp around the DNA template to prevent pausing or premature termination. We find that the dosage compensation complex genetically and physically interacts with SPT5 on actively transcribed genes providing direct molecular support for the elongation model of dosage compensation.Drosophila males hypertranscribe most of the genes along their single X chromosome to match the output of females with two X chromosomes. It had been difficult to imagine how the MSL dosage compensation complex could impose a modest, but essential, \u223ctwo-fold increase by interacting with hundreds of different factors that control transcription initiation for such a diverse collection of genes. An alternative model proposed that dosage compensation instead acted at some step of transcription elongation common to all genes. We performed a genetic screen for mutations that subtly reduce dosage compensation and recovered mutations in the  Drosophila dosage compensation is widely used as a model system to investigate how transcription is regulated by large scale chromatin modifications roX (RNA on X) RNAs A long-standing puzzle is the biochemical mechanism by which the MSL complex up regulates X-linked genes, each of which is controlled by different transcription factors. An elegant model that solves this problem posits that MSL complex does not act with diverse gene-specific transcription factors to alter initiation, but rather at the elongation step of transcription common to all genes Spt5, a universally conserved transcription elongation factor. We found that SPT5 is required for dosage compensation in males and extensively colocalizes with the MSL complex on the X-chromosome. Moreover, we found that SPT5 and MSL1 directly interact with each other. We propose that SPT5 is required downstream of MSL complex recruitment to stimulate transcription elongation. The identification of SPT5 is strong mechanistic evidence supporting the elongation model of dosage compensation.To search for new factors involved in dosage compensation we performed an unbiased forward genetic screen that relies on a sensitive eye pigmentation reporter of MSL complex activity. This approach was designed to recover heterozygous mutations in genes that are essential for general transcription in both sexes but play an additional role in male dosage compensation. We recovered multiple alleles of roX1 transgenic males is a sensitive reporter of MSL activity roX1 transgenes occasionally land in repressive chromatin, the miniwhite marker is epigenetically silenced so that females have solid white eyes. Males have spotted eyes because the MSL complex binds the autosomal roX1 transgene and locally modifies the chromatin allowing miniwhite expression in a fraction of cells. We have previously described a strategy for isolating mutations that increased local MSL activity in vivo, but may associate with the MSL complex too weakly or transiently to copurify with MSL proteins. Second, our genetic strategy retains a wild type allele of the relevant gene allowing us to capture factors that have additional essential functions. Homozygous mutations in such factors would be lethal to both sexes and thus would have been missed in earlier genetic screens based on the male specific lethal phenotype.The eye color of miniwhite reporter linked to roX1. Mutants that were also recessive lethal to both sexes were placed in complementation groups (roX1 transgenes inserted in distinct repressive locations reasoning that those were more likely to affect dosage compensation rather than the particular silencing factors acting on flanking chromatin (miniwhite gene in our roX1 reporters. We tested the effect of the new mutants on m4In(1)w which displays classic position effect variegation in both sexes. Most of the candidate modifiers of dosage compensation did not affect pigmentation in m4In(1)w wm4  arguing Spt5 and Elongin-C are located . However, when we tested the MGE-3Spt5 mutation Complementation group C was chosen for detailed analysis. Meiotic recombination placed the locus near the polytene bands 56C-F but none of the available chromosome deficiencies uncovered the mutation  located . AvailabroX1 lines just like Spt5 mutations had. We crossed six different roX1 mosaic lines that carry roX1 transgenes in diverse chromatin environments to 190 deficiencies. The idea was that any deficiency that affected the eye coloration of multiple roX1 reporter lines was more likely to affect some aspect of dosage compensation rather than the particular repressive environment surrounding the different inserts. We found that only 10 intervals reduced MSL complex reporter activity . This shows that a 50% reduction in SPT5 levels does not alter the phenotype from hypomorphic white alleles or miniwhite. We conclude that Spt5 mutations dramatically affected the probability that males overcome silencing not because of global reduction of transcription across the genome or the white promoter itself, but rather because SPT5 plays some role in dosage compensation to which our roX1 reporter is responsive.We were still concerned that the entation . We alsoSpt5 affected dosage compensation of the X chromosome in addition to the roX1 eye color reporter transgene. Because SPT5 is essential for most transcription, homozygous null animals die early in development. The viability of Spt5/+ males demonstrates that dosage compensation must be adequate even with reduced SPT5 levels. The same is true for any of the msl/+ heterozygotes. However, males with limiting MSL complex might be more sensitive to reduced levels of SPT5. Males missing either roX1 or roX2 are alive but males missing both roX RNAs have greatly reduced male viability roX1 roX2 double mutant males are completely lethal but can be rescued by an autosomal roX transgene msl1 or mle showed reduced viability when roX1 RNA is also limiting roX1 RNA was limiting consistent with a role in dosage compensation S192, a mutation in the elongation factor ELL did reduce male viability. Others have reported that ELL RNAi lines display male specific lethality consistent with a role in dosage compensation roX1 RNA levels.We used a sensitized genetic background to see if ensation . We assaiability . Howevermsl2 mRNA. Ectopic dosage compensation can be induced in females by artificial expression of MSL2 by the [H83M2] transgene that escapes SXL regulation Spt5 weakens dosage compensation then that might reduce the toxic effects of inappropriate dosage compensation in [H83M2] overexpression females. When [H83M2] females also carried a mutation in Spt5, the female-specific developmental delay was modestly rescued  of most Drosophila genes, with additional binding across the transcribed regions In order to place SPT5 in the dosage compensation pathway, we focused on two of its most intensively studied roles. First, unphosphorylated SPT5 binds to and pauses RNA polymerase over the transcription start site (TSS). Release from this 5\u2032 pause requires phosphorylation by P-TEFb at multiple sites at the C-termini of both RNA polymerase large subunit and SPT5 Spt5 null tissue. However, we can approximate that condition by using elongation inhibitors DRB and flavopiridol that block P-TEFb phosphorylation of RNAPII CTD Ser2 and SPT5 that are necessary for pause-release and entry into elongation The Pause Release model is less appealing because it calls for MSL action at the TSS, when MSL complex is instead predominantly located farther downstream Modified histone H3K36me3 is found within active genes with a 3\u2032 bias similar to the MSL complex. This modification may provide one component of MSL targeting specificity through the MSL3 chromodomain To examine MSL binding at a higher resolution than is possible with polytenes, we turned to ChIP analysis of male S2 cells. We measured MSL1 binding to the 5\u2032 and 3\u2032 ends of two highly validated target genes after treatment with flavopiridol. If the scarcity of MSL complex at the 5\u2032 ends of X-linked genes was caused by released RNAPII/SPT5 complex quickly carrying it into the body of genes, we might be able to trap MSL complex over the TSS by treatment with P-TEFb kinase inhibitors. The Pause Release Model predicts that flavopiridol treatment should cause the MSL signal to accumulate at the 5\u2032 end of genes with a corresponding loss at the 3\u2032 end. However, just as was seen with the polytene experiments, flavopiridol treatment did not alter MSL complex distribution as measured by ChIP . These rroX RNAs with regions of SPT5 in vivo, the data show that SPT5 and MSL complex have the ability to interact via MSL1 PEHE.We tested whether the genetic interactions observed between SPT5 and dosage compensation might arise from direct physical contacts. Early attempts to purify intact MSL complex did not recover SPT5 as a partner suggesting that if such interactions occur, they are transient Spt5, we wondered if the other modifier mutations found in the screen might identify a new class of factors needed for dosage compensation. We were able to map a few modifiers to previously characterized genes. In the case of deficiencies, we tested whether point mutations of candidate genes could recapitulate the effect of deficiency. That approach showed that Chromator was the relevant gene that dominantly suppresses the MSL complex dependent reporter expression in Df(3L)BSC21. CHRO is a chromodomain protein that localizes specifically to the interband regions and is implicated in maintaining chromosome structure Df(2R)vg-C vg-C . This inroX RNAs, and the fact that active MSL complex is tightly associated with transcribed chromatin. Extraction methods strong enough to release soluble MSL complex from chromatin may destroy critical contacts with key partners. Genetic approaches also face limitations. If an important partner performs additional functions beyond dosage compensation, mutations would likely be lethal to both sexes masking its interaction with the MSL complex.It is possible that dosage compensation in Drosophila is entirely a consequence of the known histone modifications carried out by its subunits, H4K16ac (MOF) and H2BK34ub (MSL2). However, if additional factors are required, new approaches may be needed to identify them. Biochemical purification is challenging due to the very large size of the MSL complex, the presence of the noncoding Spt5, a universally conserved transcription processivity factor for RNAPs, in the MSL pathway msl1, mle and ChroWe developed an unbiased forward genetic screen able to detect subtle changes in MSL activity that are not large enough to prevent dosage compensation of the male X, but sufficient to alter a sensitive eye pigmentation reporter. This screen implicated Spt5 mutations on the white eye color reporter was entirely dependent upon the adjacent roX1 locus that can recruit soluble MSL complex to any location in the genome. Spt5 mutations had no effect on white or miniwhite gene expression when not linked to roX1. Spt5 mutations acted on all mosaic roX1 reporter transgenes regardless of the chromatin environment surrounding the inserts. Interactions of Spt5 mutants and gain of function msl1 alleles suggest that SPT5 acts between MSL complex and RNA polymerase. Mutations in Spt5 selectively reduced male viability under limiting roX RNA conditions in a manner comparable to the effect of mle mutations. Additionally mutations in Spt5 partially suppressed the toxic effects of ectopic dosage compensation in females. An independent screen of the Drosophila deficiency collection showed that the Spt5 phenotype is rare. Removing one allele of almost any transcription related factor had no effect on the eye pigmentation levels of mosaic roX1 reporters arguing that dosage compensation is particularly sensitive to SPT5 protein levels. Finally, we found that the most ancient and conserved segment of the MSL1 protein physically binds to two different regions of the SPT5 protein consistent with the largely overlapping patterns of chromatin occupancy across the body of X-linked genes.Multiple lines of evidence support a role for SPT5 in dosage compensation. The effect of In eukaryotes SPT5 along with SPT4 forms the DSIF  hsp70 gene from Drosophila While regulated release from pause was originally described using the highly inducible MSL complex mediated H4K16ac is enriched within the body of genes and drives decondensation of chromatin possibly facilitating easier passage of RNAPII Spt5 in this report, our genetic approach also yielded additional candidates. So far, we have mapped two of these to Chro and Sin3A. CHRO, a chromodomain protein copurifies with the MSL complex While we have focused on the analysis of in vivo genetic strategy.An independent RNAi screen using an MSL complex dependent luciferase expression as a reporter in S2 cells also identified a role for CHRO and SIN3A in dosage compensation in vivo support for the elongation model of dosage compensation by linking the SPT5 elongation factor to the MSL complex Drosophila males have \u223c1.4 fold more transcriptionally engaged RNAPII at the distal ends of X-linked genes as compared to autosomes also supports the idea of increased elongation Our results provide direct Mammals also contain a version of the MSL complex composed of MSL1, 2, 3 and MOF, but apparently lacking a large noncoding RNA component and RNA helicase + GMroX1-58D], [w+ GMroX1-60F], [w+ GMroX1-69C], [w+ GMroX1-75C], [w+ GMroX1-99F][w and + GMroX1-102C]y w roX1Mutagenesis was performed as described Plasmids for bacterial expression of MBP fusion SPT5 protein fragments, SPT5-N (aa 112\u2013393), M (aa 389\u2013733) and C (aa 732\u20131054) were a kind gift from Dr. John Lis Polytene squashes were prepared as described in Detailed protocol can be found in MBP-SPT5N, MBP-SPT5M, MBP-SPT5C, the unrelated protein MBP-MCP (MS2 phage coat protein), GST-MSL1 C-terminal domain fusion protein and GST were expressed and isolated from bacteria. Equivalent molar concentrations of MBP proteins bound to amylose beads were incubated with GST proteins. After three washes with 150 mM NaCl, 0.1% NP40 and 20 mM Tris for 10 mins at 4\u00b0C, proteins were eluted by boiling in SDS-loading buffer and separated on 8% SDS-polyacrylamide gels. Westerns were performed as described Figure S1spt5.A forward genetic screen to identify modifiers of MSL complex activity. (A) Approximately 16,000 males were screened and 48 modifier lines established. (B) Mutations were placed into complementation groups based on recessive lethality. The mutations scored as single hits are recessive lethals, which could be due to the modifier allele or an EMS induced secondary mutation. The mutants scored as viable suppressors lower MSL complex dependent red pigmentation and are homozygous viable. We identified 5 alleles of (TIF)Click here for additional data file.Figure S2Spt5 consistently lower mosaic eye pigmentation independent of autosomal roX1 insertion site. Shown above are flies hemizygous for the [GMroX1]/+ transgene at different positions in the genome shown on the side. Flies on the left are wildtype whereas flies on the right are heterozygous for S14FSpt5. All flies shown are males.Mutations in (TIF)Click here for additional data file.Figure S3Spt5 do not affect the white promoter. Males and female eye pigmentation is shown for the hypomorphic wa and we alleles with and without Spt5 mutations. Although Spt5/+ heterozygous males show dramatic pigment reductions from the dosage compensation [w+GMroX1] mosaic transgenes .Mutations is ansgenes , the sam(TIF)Click here for additional data file.Figure S4Validation of anti SPT5 antibodies. (A) Polytene chromosomes stained with anti-SPT5 antibodies. SPT5 is widely distributed on many sites on the genome. (B) Immunodepletion of anti-SPT5 antibodies with SPT5 fragments- SPT5N, SPT5M and SPT5C results in loss of signal indicating that the antibodies predominantly recognize SPT5. (C) Polytene chromosome spreads prepared from males subjected to 30 min heat shock at 37\u00b0C were stained with anti-SPT5 antibodies. In agreement with previous reports (TIF)Click here for additional data file.Figure S5SPT5 and MSL1 colocalize extensively on the X-chromosome. Polytene chromosome spreads were stained with antibodies against (A) MSL1 (green) and (B) SPT5 (red). As expected of a general transcription elongation factor SPT5 binds all over the genome. (C) Colocalization of MSL1 and SPT5.  A closer look at two regions of the X-chromosome. Arrow heads indicates MSL1 only (no SPT5) binding and arrows indicate SPT5 only (no MSL1) binding.(TIF)Click here for additional data file.Figure S6Treatment with Flavopiridol does not affect H3K36me3. (A) Polytene chromosome spreads prepared from salivary glands treated with either DMSO (control panel) or 500 nM Flavopiridol for 30 minutes. Chromosomes were stained with antibodies against H3K36me3. (B) Ser5P RNAPII and SPT5 colocalize on polytenes. After treatment with Flavopiridol, SPT5 is greatly reduced but low levels of SPT5 are found at polytene bands that are positive for Ser5 Phosphorylated RNAPII. (C) A close-up of Ser5P RNAPII and SPT5 colocalization in control and flavopiridol treated samples.(TIF)Click here for additional data file.Table S1roX1 lines were assayed in the deficiency screen (Bloomington Deficiency collection) and the deficiencies that dominantly suppressed the red pigmentation across more than four different mosaic roX lines are indicated. Candidate genes that have been mapped within those deficiencies are also shown. Only ten intervals (14/190 deficiencies) assayed had an effect on the eye phenotype indicating that a general suppression of transcription does not lower the expression of the dosage compensation reporter. Spt5 would be an eleventh locus, but is not uncovered in the Df collection.Results of Deficiency screen. Six mosaic (DOC)Click here for additional data file.Table S2Spt5 decreases male viability. In the absence of both roX1 and roX2 males are dead. The male specific lethality can be rescued by an autosomal GMroX1 transgene. In this assay roX1-,roX2-; [GMroX1-75C] virgins were crossed to balanced males carrying the indicated mutations. The male progeny of this cross that are roX1-,roX2-/Y;mutation/+;[GMroX1-75C]/+ are compared to the nonbalanced female progeny. All the flies therefore carry only one copy of the indicated mutation. The statistical significance was determined by Fisher exact fit test. Lowering (DOC)Click here for additional data file.Text S1Supplementary materials and methods.(DOC)Click here for additional data file."}
+{"text": "We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic \u2013 reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific  However, it is unknown whether the phenotype is due to change in growth versus morphogenesis.Other recent work in zebrafish has identified additional signaling pathways, Notch and BMP, that interact with Edn1, and these studies fill in gaps in our understanding of arch patterning coming from just Edn1 signaling in isolation edn1 and hand2 mutants to focus on pharyngeal arch size regulation and examine edn1;hand2 double mutants to learn the epistatic relationships between these two genes. We extend the previous results showing edn1-dependent DV expansion, and we find that the expansion is due to growth, i.e., arch increase in size, rather than morphogenetic shape change. Previous work in zebrafish had demonstrated that edn1 functions upstream to hand2 in a positive pathway regulating DV patterning hand2 functions as a negative regulator of edn1-dependent ventral outgrowth. We provide direct evidence that the control of this outgrowth is at least in part by regulation of cell proliferation. Finally, we show that hand2 negatively regulates an anterior morphogenetic cell movement in the ventral first arch. This regulation appears to be independent of edn1 function.In this study we use edn1 or hand2 in zebrafish, developmental patterning of the embryonic pharyngeal arches is disrupted along the DV axis edn1 and hand2 single mutants are essentially as previously described edn1 single mutant. In the hand2 single mutant, a recognizable interhyal is absent, but the symplectic and joint are present. Thus it is in this intermediate region where the phenotypes of the two mutants differ.Previous work has established that with severe loss of function of either edn1 mutant both features are absent. In the hand2 mutant the joint is absent, but in the territory of the retroarticular process we see a locally expanded wedge- or fan-shaped region of cartilage, previously interpreted as being dorsalized As we interpret the first arch phenotypes of both single mutants, we also reach the conclusion that the mutants differ in the intermediate region. The dorsal pterygoid process and upper palatoquadrate cartilages are present in both single mutants. Ventrally in both, Meckel\u2019s cartilage is extremely reduced. The intermediate region in WT forms the joint between the palatoquadrate and Meckel\u2019s elements and a prominent retroarticular region of Meckel\u2019s cartilage adjacent to the joint. In the edn1;hand2 double mutant has not been previously described. The most striking aspect is its overall phenotypic resemblance to the edn1 single mutant. This finding is consistent with previous understanding that the two genes act positively with respect to ventral cartilage development, and that they function on the same genetic pathway edn1 mutant rather than the hand2 mutant. In the second arch, the symplectic and joint are absent. Strikingly, in the intermediate region of the first arch where the single mutants perturb the WT condition in opposite directions  we also see an \u2212edn1 -like reduction, showing that for this phenotype edn1 is epistatic to hand2. This finding indicates that the relationship between the two genes is complex, seemingly in opposite directions for the positive interaction responsible for overall cartilage reduction and for the negative interaction in expansion in the first arch intermediate region (see Discussion).The zebrafish edn1\u2013 hand2 interaction, we examined expression of fli1a:EGFP, a marker of neural crest derived ectomesenchyme, during early pharyngula stages ). At these stages patterning genes under control of edn1, including hand2, are transcriptionally upregulated and likely functional dlx2a expression as a proxy for arch morphology, discovered an edn1 mutant arch phenotype that becomes apparent during this period \u2013 a ventral-specific reduction in the DV extent of the pharyngeal arches, which normally are rapidly extending along this axis edn1 and hand2 single mutants, and the edn1;hand2 double mutant  is similar to that of the edn1 single mutant, and note that the reduction in these mutants is opposite to the expansion in the hand2 single mutant , and high PC1 values (WT and hand2 mutants) are not due to convergence and extension. The data do not show that arch convergence and extension is absent at this stage, only that the four genotypic groups do not differ from one another in this respect. Indeed, following the time course of changes by PCA suggests that the early arches are indeed undergoing convergence and extension, and that the genotype-specific changes occur progressively with time  and the epistasis between the two genes is the same; edn1 is epistatic to hand2.We used phospho-histone H3 labeling to examine proliferation, including senchyme . Double-senchyme . Strikine mutant . Genotype mutant . These fhand2 single mutant and the edn1;hand2 double mutant . We also note that in invitro studies in human cancer cell lines EDN1 promotes cell proliferation as well as inhibiting cell death We find that one edn1 being epistatic to hand2 in the control of early proliferation would place hand2 genetically upstream of edn1, were their interaction within a linear genetic pathway edn1 is molecularly upstream of hand2edn1-target genes Dlx genes function in the intermediate domain under positive control of edn1 as demonstrated by their downregulation in edn1 mutants Dlx genes are negatively regulated by hand2, as demonstrated by their upregulation in hand2 mutants edn1-hand2-Dlx interaction thus provides precedent for our model in Figiure 8A, and indeed it is possible that Dlx genes themselves mediate the regulation of early proliferation that our model requires. Talbot et al. described the expansion of the ventral-anterior region of the first pharyngeal arch in the zebrafish hand2 mutant being accompanied by an expansion of the intermediate domain Dlx genes hand2\u2013 Dlx gene interaction is involved in ventral proliferation is likely possible with currently available methods, but would be difficult because of redundancy of function of Dlx genes  and not well understood. It could well be that some specific instances of the human disorder could involve partial failure of anterior extension.hand2, both in terms of signaling pathways that converge on hand2 and in its role as a focal point in the regulation of downstream effector genes mediating a variety of functions \u2013 patterning Our work not only identifies Edn1 as a growth factor in pharyngeal arch development, but particularly highlights the complexity of regulation involving tf216bedn1Df(Chr01:hand2)s6fli1a:EGFP)zdf1p53\u2212edn1 was identified using forward primer: 5\u2032 GGTGCTCCAGCATCTTTGGGTC3\u2032 and reverse primer: 5\u2032TGTCTGTTCTGACTTACTCTGGTG3\u2032 resulting in a 153 bp product. Digestion with MseI cleaved the PCR product from the mutant allele into 84 and 69 bp fragments. \u2212hand2 was identified by PCR using forward primer: 5\u2032 GCGGACAGTGAAACGTAGACC 3\u2032 and reverse primer: 5\u2032 GCCTTTCTTCTTTGGCGTCTGTC 3\u2032 resulting in a 257 bp product from the WT allele. \u2212p53 was genotyped as described Fish were raised under standard conditions Larvae were fixed at 4 or 6 days postfertilization (dpf) and stained with Alcian Blue for cartilage and Alizarin Red for bone as described Skeletal preparations were imaged on a Zeiss Axiophot 2. Static confocal images were captured on a Zeiss LSM 5 Pascal as previously described fli1a:EGFP expressing ectomesenchyme of the first two pharyngeal arches (PAs) were digitized at the positions shown in fli1a:EGFP. To provide a counterstain showing total cells within the arches . For geometic morphometric analysis, twenty landmarks outlining the es as in , the embfli1a:EGFP were counted.Proliferating cells were detected using anti-phospho-histone H3 antibody. Embryos were manually dechorionated, anesthetized with tricaine, and fixed in 4% paraformaldehyde in PBS overnight at 4\u00b0C. Prior to staining, embryos were washed with PBSTx and blocked in 10% normal goat serum (NGS) in PBDTx  for at least 2 hours. Embryos were incubated with rabbit anti-phospho-histone H3 (Millipore) at 1\u22361000 dilution in blocking solution overnight at 4\u00b0C, washed with PBSTx, then incubated with Alexafluor 546 goat anti-rabbit IgG (Invitrogen) at 1\u2236500 dilution in PBSTx overnight at 4\u00b0C. Samples were visualized by confocal microscopy. Phospho-histone H3 positive cells were detected with Volocity. Regions of interest were set around the PAs and only cells positive for both phospho-histone H3 and TUNEL assay was performed to detect cell death. Embryos were dehydrated in 100% MeOH for 1 hour at -20\u00b0C then rehydrated by sequential incubation with 75%, 50%, and 25% MeOH in PBSTx at \u221220\u00b0C and with PBSTx at RT for 10 minutes each. Embryos were permeabilized by incubation in Proteinase-K (1 \u00b5g/mL) then fixed in 4% PFA for 20 minutes. Embryos were further permeabilized in Permeabilization Solution  for 30 minutes then washed with PBSTx. DNA fragmentation was detected by incubation with terminal deoxynucleotidyl transferase and TMR dUTP for 1 hour at 37\u00b0C (Roche). Embryos were washed in PBSTx then imaged.Figure S1The distinctive pharyngeal arch shapes of WT, edn1 and hand2 mutants arise progressively from 24 hpf to 32 hpf. PCA as in edn1 mutants, and light to dark blue triangles represent hand2 mutants. B. The same plot but showing the means for each genotype-age group. At the 24 hpf time point the three genotypes completely overlap. DV extension, captured by PC1, then increases markedly with developmental age for all genotypes, with \u2212edn1 lagging behind WT and \u2212hand2. The hand2 mutant shows progressive expansion in the ventral-anterior arch 1 region captured by negative PC2, whereas WT and \u2212edn1 show slight change in the opposite direction.(TIF)Click here for additional data file.Figure S2Loss of function of the programmed cell death gene p53 does not rescue the skeletal phenotype of the edn1 mutant. Flat-mount of cartilage and bone stained with Alcian Blue and Alizarin Red. The skeletal phenotypes of the edn1 single mutant and the edn1;p53 double mutant appear identical, whereas phenotypic rescue would be expected if programmed cell death of pharyngeal arch precursor cells accounted for the \u2212edn1 hypoplastic skeleton. Hence the experiment argues against cell death as an explanation for the reduced size of the arches.(TIF)Click here for additional data file.Table S1TUNEL labeling to detect cell death in the pharyngeal arches.(DOCX)Click here for additional data file.Movie S1hand2 negatively regulates anterior flow of ventral arch 1 cells.fli1a:EGFP animals were imaged by time lapse microscopy from 24\u201332 hpf. Movies are confocal projections in which individual cells were manually tracked at each frame (arrow). Cells travel from near the midpoint of arch1 to a location posterior to the stomodeum and the eye in wild types and edn1 mutants . Cells originating at a similar location in hand2 and hand2;edn1 double mutants travel much further, to a position well under the stomodeum and the eye . Scale bar: 50 \u00b5m(MOV)Click here for additional data file."}
+{"text": "Ultrafiltration during intermittent haemodialysis has been associated with reduction in microcirculatory perfusion, as observed with sidestream dark-field (SDF) imaging . This teIn this single-centre, prospective, observational study patients with acute renal failure on CVVH were included after hemodynamic stabilization and written informed consent A fixed dose of net ultrafiltration was calculated for each patient, aiming at a negative total fluid balance of 50 ml/hour. Microcirculatory perfusion was observed with sublingual SDF imaging after 1 hour of zero balance CVVH (T1) and additionally after 1 hour of negative fluid balance ultrafiltration (T2). The primary outcome was a change in microvascular flow index (MFI) between T1 and T2. Data are presented as median (IQR). Differences are calculated with a nonparametric test for paired data.Eleven patients were eligible for the study; one denied informed consent. One patient could not be evaluated due to the unavailability of the research team and in two patients we were unable to obtain images of proper quality. The median APACHE II score was 26 21 to 29); at baseline LOS ICU was 5 (3 to 6) days and fluid balance +7.9 (5.1 to 14.2) l. Hemodynamic and microcirculatory variables are depicted in Table  to 29; aA negative net fluid balance of 50 ml/hour during ultrafiltration in CVVH is not associated with microcirculatory perfusion alterations."}
+{"text": "Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing \u2212/\u2212Taf4 MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in \u2212/\u2212Taf4 MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of \u2212/\u2212Taf4 MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in \u2212/\u2212Taf4 MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth.Collagen 6A3 ( In lox/\u2212Taf4 MEFs the TFIID contains predominantly TAF4, whereas in \u2212/\u2212Taf4 MEFs, TAF4b replaces TAF4 to maintain TFIID integrity and cell viability \u2212/\u2212Taf4 MEFs display TGF\u03b2-dependent autocrine growth and deregulated expression of more than 1000 genes.TAF4 is a subunit of the general transcription factor TFIID. In vertebrates, the TAF4 family comprises a ubiquitously expressed TAF4 protein and a tissue specific paralogue, TAF4b, required for testis and ovary function Contact inhibition is a process that arrests cell proliferation upon cellular contacts under conditions of high density. It is an important mechanism of anti-cancer defence, as tumour cells normally lose this property and grow in an uncontrolled manner. The molecular mechanisms underlying contact inhibition are still poorly understood. A number of recent studies identified the Hippo signalling pathway as a major effector of contact inhibition Loss of contact inhibition is not the only event in oncogenic transformation. Other events are required, such as the activation of the Wnt signalling pathway. This pathway regulates many biological processes, including morphology, proliferation, motility and cell fate. The canonical Wnt pathway involves binding of Wnt proteins to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and resulting in stabilization and nuclear import of \u03b2-catenin. Inappropriate activation of this pathway with accumulation of nuclear \u03b2-catenin is observed in several human cancers It is becoming increasingly recognised that the extracellular matrix (ECM) not only provides a 3 dimensional (3D) matrix for cell growth and organogenesis, but that signals from the ECM play critical roles in cell fate and cell growth \u2212/\u2212Taf4 background, a subpopulation of MEFs loses contact inhibition, resulting in the formation of 3D foci and growth as fibrospheres. The cells forming foci are characterised by activated Wnt signalling and inhibition of Hippo signalling. Strikingly, Col6a3 silencing is sufficient to restore contact inhibition in \u2212/\u2212Taf4 MEFs. Our data show for the first time that Col6a3 plays a role in modulating signalling pathways involved in contact inhibition providing an explanation for the observed association between Col6a3 and cancer. It also suggests that changes in the ratio of TAF4 and TAF4b can play a role in the susceptibility of cells to Col6a3-promoted 3D growth. Finally, we also show that all-trans retinoic acid (ATRA) treatment represses Col6a3 expression thus abrogating premalignant changes in both wild-type and \u2212/\u2212Taf4 MEFs. Our data suggest that ATRA could be a valuable treatment for refractory Collagen VI-associated cancers.Here we show that TAF4 is able to attenuate the growth promoting activities of Collagen VI. In a Taf4lox/\u2212 and C3Taf4\u2212/\u2212 MEFs were derived from genetically modified Taf4lox/\u2212 mouse embryos and have previously been described Taf4 allele was defloxed in the C1 MEFs by expression of the Cre recombinase and loss of TAF4 expression in the C3 MEFs was verified by PCR genotyping and by western blot analysis as described (10). Cells were cultured in Dulbecco's minimal essential media (DMEM) supplemented with 4.5 g/l glucose and 10% foetal calf serum. Cells were treated with 10\u22126 M ATRA dissolved in ethanol (DMSO) as indicated.The C16 of the indicated cells were inoculated and grown for 10 days.For fibrosphere assays, the indicated cells were also grown under non-adherent conditions in bacterial culture plates. 10Immunofluoressence on C1 and C3 cells was performed by standard procedure using following antibodies: COL6A3 , CTNNB1 , YAP1 , TAZ , SOX2 . Immunofluorescence was visualized using a Zeiss Axiophot  microscope equipped with epifluorescence illumination. Confocal microscopy was performed on a Leica SP2 microscope.IWR-1  and XAV939  were used at 1 \u00b5g/mL concentration.RNA-seq was performed as previously described Lentiviral shRNA expression vectors and packaging plasmids were purchased from Sigma-Aldrich. The TRC numbers are indicated in the Supplemental information. Lentiviral particles were generated by co-transfection of shRNA vector together with packaging plasmids into 293T cells. Medium was changed 24 hs after transfection and viruses harvested 48 hs after transfection and used for infection of C3 cells. Infected C3 cells were selected with puromycin resistant and silencing of targeted gene was checked by RT-qPCR.RNA was prepared using Trizol reagent  according to manufacturer's protocol. Reverse transcription was performed with Super Script II reverse transcriptase  as described in manufacture's protocol. Random hexaoligonucleotides were used as primers. qPCR was performed using LightCycler 480 SYBR Green I master mix  on the LightCycler 480 Real-Time PCR System. List of oligonucleotides is supplied in x S1.4 cells were seeded in 10 cm plates. At the indicated times the cells were trypsinised and counted. BrdU incorporation was performed using a BrdU Cell Proliferation Assay kit (QIA58) from Merck/CalBiochem as per the manufacturers instructions. Briefly, the cells were grown for the indicated times with or without RA and then BrdU incorporation for 24 hours was measured by an immuno-colometric assay.For cell counting, 5\u00d710\u2212/\u2212Taf4 MEFs by defloxing of lox/\u2212Taf4 MEFs \u2212/\u2212Taf4 MEFs (hereafter C3) are irregularly shaped and have lost contact inhibition as they readily form three dimensional (3D) foci that are never observed with the lox/\u2212Taf4 MEFs (hereafter C1) (We have previously reported generation of fter C1) . We alsofter C1) .Many types of transformed cells can be grown under non-adherent conditions as spheres such as \u2018mammospheres\u2019 in the case of breast cancer cells We next used RNA-seq to profile the changes in gene expression that occur upon 3D growth. RNA was prepared from low or high-density adherent cultures of C3 cells comprising 3D foci and from fibrospheres. In comparison with non-confluent cells, 669 transcripts were-up-regulated and 714 down-regulated in dense cells . SimilarOntology analysis of the down-regulated transcripts indicated strong enrichment in those involved in cell cycle and cell division consistent with the fact that proliferation is considerably reduced in dense C3 cells or when grown as spheres compared to the rapid growth of the non-dense cultures . In contWe had previously reported that genes of the interferon response were strongly induced in the C3 cells lacking TAF4 compared to the C1 cells. We ascribed this difference to TAF4 inactivation Olfm1 and Spp1, although induced in dense C3 cells, is very much higher in fibrospheres  and laminins (a4 and a5) are strongly induced along with VCAM1, thrombospondin 2, intergrins b8, a2b and a7 and cadherins 13 and 26. Thus, 3D growth involves major changes in cell adhesion and remodelling of the ECM. On the other hand, expression of ospheres .Col6a3 strongly increases in dense C3 cells and is strongly expressed in fibrospheres  whose expression is strongly down regulated in dense cells and fibrospsheres . The ability of TAF4 to counteract the growth promoting effects of high Col6a3 expression can be evaluated by comparing the responses of the two cell types under conditions of dense growth. In TAF4-expressing C1 cells, YAP1 and TAZ are located in the nucleus as the cells proliferate at low density. At high density, Kibra remains highly expressed, while Fat4 expression is strongly induced. This suggests that Hippo signalling is maintained in dense C1 cells to mediate contact inhibition, an idea confirmed by the translocation of YAP1 from the nucleus and the overall down-regulation of TAZ expression in dense cultures.In this study, we show that \u2212/\u2212Taf4 cells, YAP1 accumulates in the nucleus to promote 3D growth. Kibra expression can be induced by YAP1 overexpression through an as yet unknown mechanism In contrast, in the absence of TAF4, the pathways and factors that normally maintain Kibra expression under dense conditions are no longer operative and Kibra expression is repressed attenuating Hippo signalling. Consequently, in dense \u2212/\u2212Taf4 MEFs. In dense \u2212/\u2212Taf4 MEFs, Wnt9a expression is strongly up-regulated while Sfrp2 expression is repressed. The nuclear localisation of \u03b2-catenin in the cells forming 3D foci shows enhanced Wnt signalling in these cells. Also shRNA-mediated Wnt9a silencing or use of chemical inhibitors of Wnt signalling abrogates 3D growth indicating the critical role of the pathway in this process. Interestingly, ChIP-seq has revealed SOX2 Wnt9a gene. This suggests that YAP1 and SOX2 in dense C3 cells may activate Wnt9a expression in a positive feed forward loop to promote 3D growth. This contrasts with the TAF4-expressing MEFs, where Wnt9a expression is not induced under dense conditions, but Sfrp2 expression is strongly induced. Consequently, in dense TAF4-expressing MEFs, Wnt signalling is repressed, the opposite of what is observed in Taf4\u2212/\u2212 MEFs. The loss of TAF4 therefore modifies Wnt9a and Sfrp expression to activate Wnt signalling in conditions of high density to promote 3D growth.Several lines of evidence indicate that Wnt signalling is also involved in 3D growth of Together our results support a model where loss of contact inhibition through diminished Hippo signalling allows the cells to form dense foci, while enhanced Wnt signalling is further required for full 3D growth. It is also interesting to note that high SOX2 expression is seen already in rare nuclei of low-density C3 cells. Thus TAF4 inactivation leads to heterogeneity in the cell population suggesting that it is the SOX2 high population that is competent to generate 3D foci under dense conditions.Taf4\u2212/\u2212 MEFs for 3D growth appears to be associated with high Col6a3 expression. The expression of several membrane and ECM components is strongly induced in dense conditions. Nevertheless, shRNA mediated silencing of Col6a3 alone is sufficient to abolish 3D growth. Further evidence for a critical role of Col6a3 in 3D growth comes from the observation that its expression is down-regulated by ATRA that restores contact inhibition and represses 3D growth. ATRA down-regulates both Col6a3 and to a lesser extent Col6a2, thereby down-regulating holo-collagen VI fibre formation. While the expression of many membrane and ECM components are strongly induced by 3D growth, Col6a3 is one of the few regulated by ATRA and is the most strongly repressed. Moreover, ATRA treatment does not affect expression of known components of the Hippo pathway and may even potentiate Wnt signalling through up-regulation of Wnt9a. These observations, together with the results of Col6a3 silencing, indicate that the major mechanism by which ATRA inhibits 3D growth is through repression of Col6a3. The growth suppressive effect of ATRA on these cells by regulation of ECM components is therefore fundamentally different from that seen in F9 embryonal carcinoma cells, HL60 myeloid cells, or mammary carcinoma cells where RA treatment induces cell cycle arrest, differentiation and under some conditions apoptosis The capacity of Col6a3 silencing stimulates expression of Kibra and re-activates Hippo signalling leading to reduced YAP1 expression. These observations show that Col6a3 plays an active role in modulating expression of growth control genes and are in line with previous results showing that high Col6a3 expression modulates cell and tumour growth. Ovarian cancer cells resistant to cisplatin show a potent induction of the Col6a3 gene and in vivo, high grade tumours express higher levels of Col6a3 than low grade tumours Col6a3 is up-regulated in the stroma of colon tumours Col6a3 can modulate expression of critical regulators of the Hippo and Wnt pathways to promote growth and how it can serve as a target for ATRA mediated suppression of growth.ECM components such as COL6A3 can provide a network that physically facilitates 3D growth. While the reduction in this network in the presence of ATRA may contribute to its ability to repress 3D growth, remodelling the ECM does not appear to be the only role of COL6A3 as it's silencing also dramatically modulates gene expression and signalling. ShTogether the results described here reveal a novel and complex interplay between at least three signalling pathways  that control 3D fibroblast growth. We describe the ability of TAF4 to control expression of critical components of the Hippo and Wnt pathways and a novel role of COL6A3 as ATRA-regulated modulator of 3D growth and as regulator of gene expression.Figure S1Expression and localisation of TAZ and SOX2 in C1 MEFs.A. Immunostaining of non-dense and dense C1 MEFs for TAZ. B. Immunostaining of non-dense and dense C1 MEFs for SOX2 (20\u00d7 magnification). C. Control staining of F9 embryonal carcinoma cells and of hepatocyte cells with SOX2 antibody to demonstrate the specificity of the signal.(PDF)Click here for additional data file.Figure S2Expression and localisation of TAZ, YAP1 and SOX2 in C3 MEFs.A. Immunostaining of non-dense and dense C3 MEFs for TAZ. B. Immunostaining of low density C3 cells with YAP1 and SOX2 antibody (20\u00d7 magnification). Cells expressing low or high levels of SOX2 are indicated by arrows. C Immunostaining of dense C3 MEFs for YAP1 and SOX2 (20\u00d7 magnification). The location of cells growing in a 3D foci is indicated.(PDF)Click here for additional data file.Figure S3A. Effect of RA on C3 cell proliferation.A. Kinetics of cell growth in presence or absence of RA as evaluated by cell counting. B. Assessment of cell division by incorporation of BrdU on cells grown for the indicated periods in presence or absence of RA. C. Results of a representative FACS assay showing the % cells in each stage of cell cycle. D. Clonogenic assays of C3 cells in presence or absence of ATRA or shCol6a3 on wells coated with fibronectin.(PDF)Click here for additional data file.Figure S4Effect of shCol6a3 knockdown on gene expression. RT-qPCR on the indicated genes in C3 cells expressing control shRNA or shRNA directed against Col6a3 grown for 3 or 10 days as indicated.(PDF)Click here for additional data file.Figure S5Expression of TAZ in shCol6a3 knockdown cells.A. Expression of TAZ in low-density shCol6a3 knockdown cells. B Expression of TAZ in high density shCol6a3 knockdown cells.(PDF)Click here for additional data file.Table S1Sequences of primers used for qPCR of the indicated genes on the forward and reverse strands.(DOC)Click here for additional data file.Table S2Excel table of RNA-seq results. Page 1 shows transcripts induced in dense conditions. Shown are, the Ensembl gene ID, the average RPKM expression values under each condition, the fold change and Log2 change values under the indicated conditions, gene name and description. Pages 2\u20134 show the same information concerning transcripts induced in spheres, repressed under dense conditions and repressed in spheres respectively.(XLS)Click here for additional data file.Table S3Ontology analyses of genes whose expression is modified under conditions of dense or 3D growth. Each page shows the analysis of genes differentially regulated under the specified conditions with the indicated ontology terms.(XLS)Click here for additional data file.Table S4Genes regulated by ARTA in C3 MEFs. Pages 1 and 2 show the induced and repressed genes after 12 and 72 hours of ATRA treatment. Shown are the Ensembl gene IDs, gene name, log2 ratios \u2212ATRA/+ATRA 12 hours, \u2212ATRA/+ATRA 72 hours, +ATRA12 hours/+ATRA 72 hours, and gene description.(XLS)Click here for additional data file."}
+{"text": "Central precocious puberty (CPP) could be a phenotype of pathology in the central nervous system. While it is generally accepted that all boys with CPP and girls with CPP at less than 6 years of age should undergo brain imaging as part of the workup, there have been controversies as to the use of brain imaging in girls who develop CPP between 6 to 8 years.To evaluate the prevalence and clinical characteristics of intracranial lesions in patients with central precocious puberty aged 6 to 8 years in a single centre in the past 11 years.Retrospective chart review of girls with CPP and their MRI findings between year 1999 to 2009 in a single centre.One hundred and eighty-eight girls had central precocious puberty in the study period and 157 of them (83.5%) had MRI of the pituitary done as part of the workup. The prevalence of intracranial pathology among girls with CPP aged 6 to 8 years was 20.0% while among all girls with CPP aged less than 8 years, 34 girls (21.7%) were found to have intracranial pathology. These pathologies included: pituitary adenoma (n = 16), pineal cyst (n = 8), Rathke\u2019s cleft cysts (n = 4), arachnoid cyst (n = 1), intra-ventricular cyst (n = 1), venous angioma over the left frontal lobe (n = 1), hydrocephalus (n = 2) and an old infarct over the frontal lobe (n = 1). The two cases of hydrocephalus and the case with an old infarct were known before the onset of CPP. None of the lesions detected required further interventions with surgical removal, chemotherapy or radiotherapy within the follow-up period of 7.2 \u00b1 3.0 years.Brain imaging the girls with CPP in our centre mainly detected benign lesions not requiring any intervention during our follow-up period. Though the current data do not justify a practice of performing routine MRI for girls diagnosed to have CPP at 6 to 8 years, longer follow-up assessment of such lesions detected in childhood may be necessary before concluding on their benign outcome."}
+{"text": "Thymic epithelial cells (TECs) are necessary for normal T cell development. Currently, one transcription factor, Foxn1 is known to be necessary for the progression of fetal TEC differentiation. However, some aspects of fetal TEC differentiation occur in Foxn1 mutants, suggesting the existence of additional transcriptional regulators of TEC differentiation. The goal of this study was to identify some of the additional candidate transcription factors that may be involved in the specification and/or differentiation of TECs during fetal development.Foxg1, Isl1, Gata3, Nkx2-5, Nkx2-6 and Sox2 for further studies. Whole mount in situ hybridizations confirmed the expression of these transcription factors within subdomains of the third pharyngeal pouch from E9.5\u2013E10.5. By E11.5 days Foxg1 and Isl1 transcripts were the only mRNAs from this group of genes detected exclusively within the thymus domain of the third pouch. Based on this initial in situ hybridization analysis, we focused on defining the expression of Foxg1 and Isl1 during multiple stages of thymus development and TEC differentiation. We found that Foxg1 and Isl1 are specifically expressed in differentiating TECs during fetal and postnatal stages of thymus development. In addition, we found differential expression of Islet1 and Foxn1 within the fetal and postnatal TEC population.We identified candidate fetal TEC transcriptional regulators via data and text mining. From our data mining we selected the transcription factors Foxg1 and Isl1 may play a role in the regulation of TEC differentiation during fetal and postnatal stages. Our results also demonstrate heterogeneity of TECs marked by the differential expression of transcription factors, potentially providing new insights into the regulation of TEC differentiation.Our studies have identified two developmental transcription factors that are excellent candidate regulators of thymic epithelial cell specification and differentiation during fetal development. Our results suggest that  Thymic epithelial cells (TECs) are a critical component of the thymic microenvironment. TECs are derived from the endoderm of the third pharyngeal pouch. Despite their essential role in thymus function, our current understanding of fetal TEC specification and differentiation is very limited. For example, we do not know which transcriptional regulators are necessary for the earliest specification of the thymus organ domain within the third pharyngeal pouch. In addition we have very limited knowledge about the transcription factors that regulate the differentiation and function of TECs during fetal and postnatal thymus development. Identifying the factors that regulate these key steps in the development of thymic epithelial cells is a key part of understanding the genetic pathways that regulate thymus organogenesis and function.Gcm2 at E9.5 in the parathyroid domain and Foxn1 at E11.25 in the thymus domain of the 3rd pharyngeal pouch marks the patterning of the pouch into the primordium of the two organs Foxn1 which is first detected at E11.25 Foxn1 is first expressed suggests that additional transcription factors are acting within the pouch at times prior to Foxn1 expression. These factors include the transcriptional regulators that activate Foxn1 within the thymus primoridium.Our current knowledge regarding the earliest events in the specification of the parathyroid and thymus suggests that specification occurs early in third pouch development. Localized expression of Hoxa3, Pbx1, Tbx1, Pax1, Pax9, Six1 and Eya1 as necessary for 3rd pouch development. All of these transcription factors, except for Tbx1 are expressed throughout the 3rd pouch at E10.5 prior to detectable Foxn1 expression Tbx1 is initially expressed throughout the 3rd pouch and becomes restricted to the presumptive parathyroid domain at E10.5 in the pouch endoderm Hoxa3, Eya1, Six1, Pax9, Tbx1 and Pbx1 the homozygous mutants either fail to form the 3rd pouch or exhibit very severe early defects in the formation of both the thymus and parathyroid primordia Previous studies have identified the transcription factors in situ hybridization analysis based on previous expression data, our genetic studies have shown that it is necessary for the normal development of the third pharyngeal pouch Gata3 mutants at E12.5 days indicating an early role for this gene in the development of the pouch A major goal of this screen was to identify candidate developmental transcription factors that play an important role in 3rd pharyngeal pouch endoderm development and/or the differentiation of fetal TECs into functional components of the thymus microenvironment. To enable the characterization of the genes identified in our screen we focused our screen on genes for which well-characterized knockout mice are available. In the case of Gata3, which we chose for detailed in situ hybridization data are limited in terms of resolution, they can be used to suggest candidate genes for in depth characterization. We took advantage of this information to focus our in situ hybridization analysis to a short list of candidate transcription factors that were likely to be expressed in localized domains of the 3rd pouch and/or thymic epithelium. For third pharyngeal pouch expression we visually screened data in the MGI/GXD and Emage in situ hybridization databases and examined published reports describing Cre recombinase expression patterns in situ hybridization of para-sagittal sections in the Genepaint database indicated that most of the genes we had identified as being expressed in the third pouch were also expressed within the thymus at E14.5. This suggested that these transcription factors were excellent candidates for genes involved in thymus development and differentiation. After additional literature mining, we selected Nkx2-5, Nkx2-6, Foxg1, Islet1, Gata3 and Sox2 for further analysis in this study. The screen of in situ database information allowed us to focus on performing a detailed characterization of a small group of genes rather than performing a less detailed large-scale screen.A large amount of published and unpublished mouse developmental gene expression data is available in online databases Foxn1 expression within the thymus domain of the third pouch at E11.25 days are not known. One purpose of our study was to search for transcription factors with regionalized expression patterns in the third pharyngeal pouch endoderm between E9.5\u2013E11.25. We analyzed the expression of our candidate genes in E9.5 and E10.5 somite stage matched wild type mouse embryos by in situ hybridization and 3D reconstruction, focusing on their expression in the pharyngeal pouches. By carefully staging embryos by somite counting we found that embryos at the same somite stage had comparable pouch morphologies as determined by 3D reconstruction of paraffin sections. By comparing the expression patterns of the genes we examined to the Gcm2 expression pattern at E10.5 as a landmark for organ specific domains, we found a surprisingly diverse and complex combination of regionalized expression patterns in the third pouch endoderm.Although pouch endoderm as early as E9.5 is capable of developing into a fully functional thymus when transplanted under the kidney capsule Nkx2-5 and Nkx2-6 expression have detected expression of Nkx2-5 in the pharyngeal endoderm and Nkx2-6 in the pharyngeal pouch endoderm. However, these studies have not described the expression pattern of either gene in the 3rd pouch in detail Nkx2-5 is expressed in a localized domain of the 3rd pouch Nkx2-5 at some point in its history Nkx2-5/Nkx2-6 double mutants Previous studies of in situ hybridization analysis revealed that Nkx2-5 and Nkx2-6 are expressed in localized regions of the third pouch. At E9 days (20\u201323 somites) Nkx2-5 is expressed on the ventral side of the forming third pharyngeal pouch endoderm  indicated that it is expressed in the thymus domain of the E10\u2013E10.5 (30\u201335 somites) 3rd pouch Gata3 are associated with a syndrome of hypoparathyroidism, sensorineural deafness and renal disease Gata3 in development of 3rd pouch derivatives.Previous studies have detected Isl1 during pharyngeal pouch development. At the 20\u201323 somite stage, Isl1 is expressed in the cells on the ventral side of the developing second and third pharyngeal pouches but on the dorsal portion of the first pouch , Foxg1 expression is detected at the ventral tip of the 3rd pouch endoderm .Our analysis revealed a dynamic pattern of endoderm . By 30\u20133al pouch , 2F. Thihe pouch . We alsoNkx2-5, Nkx2-6, Isl1, Gata3 and Foxg1 in the ventral portion of the third pharyngeal pouch suggested that these factors might be expressed in the thymic epithelial cells at later stages. To test this idea we examined the expression of these 5 transcription factors in E11.5 wild type embryos. In the E11.5 third pouch endoderm, the thymus domain can be identified by the expression of Foxn1 in the ventral posterior portion of the pouch, complimentary to the parathyroid domain which is marked by Gcm2 expression The expression of in situ hybridization analysis showed that Nkx2-5, Nkx2-6 and Gata3 are not expressed within the thymus domain of the third pouch at E11.5. In fact, at this stage Nkx2-5 and Nkx2-6 are no longer expressed in any of the pharyngeal pouches  protein, which has been shown to be expressed in medullary thymic epithelial cells and in a subset of the cortical epithelial cells at late fetal and postnatal stages In sections of E16.5 fetal thymus, ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells . To confFoxg1 null embryo (data not shown) In contrast, we found that FOXG1 expression in E16.5 thymus completely co-localizes with FOXN1 in TECs . The antFoxn1 transcript and protein expression are at various levels in medullary thymic epithelial cells high; ISL1low; FOXG1low TECs and FOXN1high; ISL1high; FOXG1high TECs were widely present at these stages  embryos were dissected in DEPC-PBST  and somite number was determined. Embryos within a narrow range of somite stages were pooled in groups of 3\u20135 for processing. Therefore, we refer to individual embryos as being within a range of somite numbers since we cannot accurately count somites after the Nkx2-5, a cDNA plasmid clone was generated by RT-PCR using E11-day mouse total RNA (Clontech). Primers were Nkx2-5-3-F: CTACG GCGTG GGTCT CAATG C and Nkx2-5-3-R: GCGTT AGCGC ACTCA CTTTA ATGG. The transcription template was generated by PCR using the SP6 and T7 promoter primers and using our Nkx2-5 plasmid cDNA as template. The Nkx2-6 probe template was amplified from mouse genomic DNA using primers: T7-Nkx2-6-F: taata cgact cacta tagg ACTGGTACTGGACGGCAAGC and SP6-Nkx2-6-R: attta ggtga cacta taga GCACAGCATCTACGTGGCTA. Isl1 probe template was generated by PCR from an MGC cDNA (accession BC132263) using primers: Isl1F-T7: taata cgact cacta taggT CATCC GAGTG TGGTT TCAA and Isl1R-SP6: attta ggtga cacta tagaT GAATG TTCCT CATGC CTCA. Foxg1 probe template was generated by PCR from an MGC cDNA (accession BC046958) using primers: Foxg1F: AGTTACAACGGGACCACGTC and Foxg1R-T3: aatta accct cacta aagg CCCCT GATTT TGATG TGTGA. The Sox2 probe template was generated by PCR using mouse genomic DNA and primers: Sox2-F: GCCCA TGAAC GCCTT CATGG and T3-Sox2-R: aatta accct cacta aagg C ATGCT GATCA TGTCC CGGA. The Gata3 probe template was described previously Digoxygenin (DIG)-labeled antisense RNA probes were synthesized using standard procedures. All probe templates were generated by PCR reactions using either mouse genomic DNA or cDNA clones as templates. In all cases, probe templates were carefully designed to not include highly conserved sequences to eliminate the possibility of cross hybridization. In the following primer sequences the lower case letters indicate the phage promoters. For Comparisons of pouch morphology in 3D reconstructions from different embryos showed that pouch morphology is comparable between different embryos of the similar somite stage (data not shown). This similarity allowed us to make comparisons between reconstructions of different gene expression patterns. Digital images of serial sagittal paraffin sections from a single embryo were assembled into a three-dimensional (3D) image using the WinSurf 4.3 software. The gene expression positive areas and the third pharyngeal pouch endoderm were traced as separate objects.3Citrate pH 6, 0.05% Tween20) for 30 minutes. After cooling down, slides were washed once with 0.05% PBST (0.05% Triton X-100) and blocked in 10% serum in PBST at room temperature for at least 30 minutes. Primary antibodies were mixed in 1% serum/PBST and incubated at 4\u00b0C overnight. After 3 PBST washes, secondary antibodies diluted in PBST were added and incubated at room temperature for 30 minutes. Slides were then washed and mounted in FluoroGel (EMS). Images were acquired using a Zeiss LSM510 META confocal imaging system.Dissected embryos or postnatal thymus tissue were treated differently for ISL1 and FOXG1 antibody staining. For ISL1 staining, E16.5 embryos were fixed in 4% PFA for 4 hours or postnatal thymus for 1.5 hr on ice. Fixed embryos or tissue were then washed three times in PBS and dehydrated through methanol series and embedded into paraffin blocks. 8 \u00b5m sections were cut on a Leica RM2155 microtome and de-waxed and rehydrated into water. Antigen retrieval was done by boiling the slides in AR buffer . 10 \u00b5m frozen sections were then cut on a Leica CM3050 S cryostat. The sections were blocked and incubated with primary and secondary antibodies as described for ISL1 staining.Foxn1  The mouse anti-ISL monoclonal antibody was from the Developmental Studies Hybridoma Bank . This ISL1 monoclonal was developed by Dr. Thomas Jessell and has a long track record of use in mice"}
+{"text": "CDKN2A) gene that encodes the p16INK4a and p14ARF tumor suppressors, and the isocitrate dehydrogenase 1 (IDH1) gene as potential markers of survival for 40 individuals with NDTMM GBMs (telomerase negative and ALT negative by standard assays), 50 individuals with telomerase, and 17 individuals with ALT positive tumors. The analysis of CDKN2A showed NDTMM GBMs had an increased minor allele frequency for the C500G (rs11515) polymorphism compared to those with telomerase and ALT positive GBMs (p\u200a=\u200a0.002). Patients with the G500 allele had reduced survival that was independent of age, extent of surgery, and treatment. In the NDTMM group G500 allele carriers had increased loss of CDKN2A gene dosage compared to C500 homozygotes. An analysis of IDH1 mutations showed the R132H mutation was associated with ALT positive tumors, and was largely absent in NDTMM and telomerase positive tumors. In the ALT positive tumors cohort, IDH1 mutations were associated with a younger age for the affected individual. In conclusion, the G500 CDKN2A allele was associated with NDTMM GBMs from older individuals with poorer survival. Mutations in IDH1 were not associated with NDTMM GBMs, and instead were a marker for ALT positive tumors in younger individuals.Prognostic markers for glioblastoma multiforme (GBM) are important for patient management. Recent advances have identified prognostic markers for GBMs that use telomerase or the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance. Approximately 40% of GBMs have no defined telomere maintenance mechanism (NDTMM), with a mixed survival for affected individuals. This study examined genetic variants in the cyclin-dependent kinase inhibitor 2A ( Acquisition of a telomere maintenance mechanism prevents telomere attrition and is a hallmark of cancer TP53 mutations are a marker for individuals with a poorer prognosis In glioblastoma multiforme (GBM) all of the telomere maintenance scenarios outlined above occur CDKN2A) that encodes two proteins p16INK4a and p14ARFCDKN2A, a substitution in the 3\u2032 UTR of a cytosine for a guanine base at cDNA nucleotide 500 , is associated with different cancer types. The G500 allele had an increased frequency in melanoma families, and in psoriasis patients with squamous cell carcinoma INK4ap16 expression in sporadic colorectal cancer CDKN2A expression CDKN2B) expression; a gene in close proximity to CDKN2A on chromosome 9p21 and encodes the tumor suppressor p15INK4BOther molecular characteristics of GBMs include loss of the cyclin-dependent kinase inhibitor 2A  were ALT positive by standard techniques i.e. long and heterogeneous telomere lengths by TRF length analysis, the presence of large aggregates of the promyelocytic leukemia (PML) protein and telomere DNA called ALT-associated promyelocytic leukemia (PML) bodies (APBs) in >0.5% of tumor cells, and very low or no telomerase activity in tumor protein lysates by the TRAP assay CDKN2A UTR (rs11515). The C500G genotypes are given in Individuals in each GBM telomere maintenance subgroup were genotyped for the C500G polymorphism in the 3\u2032 CDKN2A mutations were identified in G500 heterozygotes as evident by dHPLC and sequence analysis of all CDKN2A exons and exon/intron boundaries in amplicons of tumor and blood leukocyte extracted DNA. Nine individuals  were heterozygous for the G442A polymorphism that substitutes an adenine for a tyrosine amino acid at amino acid residue 148 in p16INK4a.All individuals with the G500 allele were homozygous for the major allele at two other polymorphic sites in close proximity (C540T and C547G). No CDKN2A sequence variant examined was associated with an increased frequency in the NDTMM group. In the telomerase GBM group the G500 allele frequency was not significantly different to the control population selected with the same ethnicity, and age and sex matched to the GBM cohort.In summary, in GBM the G500 allele and no other Cox proportional hazards regression analysis was performed to test whether the GC and CC C500G genotypes in the NDTMM and telomerase positive groups were independently associated with survival. Variables included in the analysis were age, gender, treatment, and extent of surgery . The C50th to the 75th percentile), which was higher compared to a median age of 58 for C500 homozygotes , p\u200a=\u200a0.04 .In the NDTMM tumor group survival data was available for all 40 individuals. Individuals heterozygous for the G500 allele had a median survival time of 3 months following the initial tumor diagnosis, compared to 8 months for individuals homozygous for the C500 allele . The twoIn the telomerase positive tumor group survival data was available for 44 of 50 individuals. Individuals heterozygous for the G500 allele had a median survival time of 4.9 months following the initial tumor diagnosis compared to median survival time of 6 months for CC homozygotes .INK4ap16 or ARFp14, multiplex PCR was used to estimate the gene dosage of exon 1\u03b1 (INK2A)p16 and exon 1\u03b2 (ARF)p14 relative to an internal \u03b2-globin gene fragment To test if the reduced survival associated with the G500 allele in the NDTMM GBM cohort was attributed to a greater loss of homozygosity or heterozygosity for In the NDTMM group, individuals heterozygous for G500 that had loss of exon 1\u03b1 and 1\u03b2 gene dosage had a median survival of 4 months, which was not significantly different to G500 heterozyotes that retained exon 1\u03b1 and 1\u03b2 homozygosity  with altered survival when adjusted for other variables . The 20 tumor sections selected included ten from G500 heterozygotes typed with loss of INK4ap16 gene dosage using the multiplex PCR analysis and ten tumors from C500 homozygotes typed with no loss in INK4ap16 gene dosage by multiplex PCR analysis. All tumors from C500 homozygotes had 2 red and 2 green signals in cellular nuclei, a result consistent with no loss in INK4ap16 gene dosage (data not shown). Seven tumors from G500 heterozygotes had 2 green signals and no red signals consistent with a loss of both INK4ap16 alleles (data not shown). Three tumors from G500 heterozygotes had 2 green signals and 1 red signal consistent with a loss of one INK4ap16 allele. The results from the FISH analysis confirmed those obtained using PCR.To verify that the NDTMM GBMs had lost the INK4a. The 20 tumor sections selected were those used for the FISH analysis described above. All tumors from C500 homozygotes had p16INK4a positive cells . For IDH2 mutations, one mutation was found. The D177A, amino acid residue substitution occurred in a NDTMM GBM.Tumor DNA from all GBMs was used as the template in PCR reactions that amplified all exons of In summary, the majority of ALT positive tumors contained IDH1 mutations. In NDTMM and telomerase positive GBMs, IDH1 mutations were rare.CDKN2A allele may be an important biomarker for NDTMM GBMs identifying individuals with a poorer prognosis. The C500G genotype was associated with survival independently of age and treatment, which are other factors associated with GBM patient survival Currently there is no prognostic marker for GBM tumors without a defined telomere maintenance mechanism. The G500 TP53 mutations compared to ALT positive tumors (unpublished observations). Moreover, the different G500 allele frequency, the absence of IDH1 mutations, and the different effect of mutant TP53 on survival  on survival mitigates against these \u201cNone\u201d GBMs being miss-classified as telomerase or ALT tumors It is unknown why a considerable portion of GBMs have no currently defined telomere maintenance mechanism. Most tumor types documented without telomerase activity or ALT are low-grade tumors such as grade 1 astrocytomas and papillary thyroid carcinomas INK4ap16 and ARFp14 expression CDKN2A is associated with poor tumor outcome in patients and mice INK4ap16 and ARFp14 transcripts, which may give a greater selection pressure for inactivation by a later DNA deletion CDKN2A was predicted to be affected by the C500G polymorphism The G500 allele as a risk factor for cancer has been most extensively characterized for melanoma CDKN2A coding region or the 3\u2032 UTR of CDKN2A region analyzed. The C500G alleles may affect expression of other genes in close proximity to CDKN2A. Both CDKN2B and the non-coding RNA designated ANRIL are included with CDKN2A in the susceptibility locus associated with glioma and other diseases et al. (2010), the G500 allele reduced CDKN2B and no effect on ANRIL expression The G500 allele was not associated with another sequence variant in the The causative variant may be in linkage disequilibrium with the G500 allele and in a region on chromosome 9 not analyzed in this study.INK4ap16 and ARFp14 function and poorer survival. Interestingly, this was not the case for G500 heterozygotes, as those that had loss of exon 1\u03b1 and exon 1\u03b2 gene dosage had no difference in survival compared to those that retained exon 1\u03b1 and exon 1\u03b2. One explanation for this discrepancy is that the G500 allele may lead to loss of INK4ap16 and ARFp14 transcripts initially, followed by loss of exon 1\u03b1 and exon 1\u03b2 DNA at a latter stage of tumor development. The poorer survival associated with the G500 allele may be attributed in part to an increased age at initial tumor diagnosis for G500 heterozygotes Homozygotes for the C500 allele with loss of exon 1\u03b1 and exon 1\u03b2 gene dosage had a poorer survival compared to those that had retained exon 1\u03b1 and exon 1\u03b2, consistent with an association between loss of IDH1 mutations, a marker for improved prognosis in ALT positive tumors IDH1 mutations and ALT are associated in Glioma remains to be understood.The NDTMM GBMs were not associated with New treatments targeting telomerase positive tumors are being developed CDKN2A G500 allele in the 3\u2032 UTR was more frequent in patients with tumors that have no defined telomere maintenance mechanism and was a prognostic marker for poorer survival. The increased incidence of the G500 allele and the absence of IDH1 mutations provide justification for the NDTMM GBMs being a molecularly distinct subgroup of tumor. Further investigation is required to understand the function of the G500 allele in altered cancer risk. In younger GBM patients with ALT positive tumors, IDH1 mutations predominate. Therefore the molecular signature of telomere maintenance mechanisms may provide additional prognostic information for GBM patients in addition to that for age and treatment received.The The study had ethical approval from the Multi-region Ethics and the Upper South Regional Ethics committees in New Zealand, and written individual informed consent was obtained.The inclusion criterion for the study was a diagnosis of a GBM tumor. One hundred and seven tumors with both paraffin-embedded and frozen tissue available were analyzed. All tumors were collected in New Zealand, and all were debulking samples. Consultant pathologists performed the histopathological tumor diagnoses. All individuals were of New Zealand European ethnicity.Telomerase activity was measured in tumor lysates using the Telomeric Repeat Amplification Protocol (TRAP) method. The TeloTAGGG Telomerase PCR ELISA Plus kit  was used according to the manufacturer's instructions. For identification of ALT positive tumors, measurement of telomere length was made by the terminal restriction fragment (TRF) assay using tumor lysates. The TeloTAGGG Telomere Length Assay Kit  was used according to the manufacturer's instructions. The criteria for ALT by TRF length was that previously described 5\u2032-GTGCCACACATCTTTGACCTCAG-3\u2032) and an anti-sense prime in the 3\u2032 UTR (5\u2032-TGCTTGTCATGAAGTCGACAGCT-3\u2032). The C500G alleles were genotyped in 150 blood leukocyte extracted DNA samples from individuals randomly selected from the general New Zealand population of European ancestry.To genotype the C500G (rs11515) polymorphism CDKN2A were amplified by PCR using blood leukocyte and tumor extracted DNA as the template. The following sets of primers were used to amplify exon 1\u03b1  and 1\u03b2  CDKN2A wild-type DNA (1\u22361) and re-analyzed by dHPLC to allow non-heterozygous sequence variants to be detected.All exons of CDKN2A exon 1\u03b1 (INK4Ap16) and exon 1\u03b2 (ARFp14) using tumor extracted DNA Multiplex PCR as previously described for astrocytomas, was used to assay the gene dosage of In situ hybridization was used to confirm the p16INK4a gene dosage results obtained from the multiplex PCR method, and measured p16INK4a gene dosage in 20 NDTMM GBM paraffin embedded tumor sections using the P16 Detection Probe  and the control chromosome 9 probe deletion probe (D9Z3 Cytocell Aquarius). The in situ hybridization protocol was based on that used in the laboratory for FISH analysis of telomeric DNA 5\u2032-ATGCCATCACTGCAGTTGTAGG-3\u2032 and antisense 5\u2032-CCTTGCTTAATGGGTGTAGAT-3\u2032, all primers used were those described previously To identify tumors with IDH1 mutations all exons of the IDH1 gene were amplified from tumor extracted DNA and sequenced. With the exception of the PCR reaction designed to amplify exon 4, which used the following set of primers, sense INK4a epitope was detected using an anti-mouse p16INK4a monoclonal antibody , and detection of the enzyme linked conjugated system  used DAB. Detection of DAB positive cells used a Leica DM2000 light microscope at 400\u00d7 magnification and Leica Version 3.5.0 Application Suite software .The p16INK4a. The analysis was made using the PHREG Procedure SAS System .The frequency of the G500 allele was compared between GBMs with different telomere maintenance mechanisms using the Fisher's exact test and GraphPad Prism software . In the NDTMM and telomerase GBM groups the age at brain tumor diagnosis was compared between G500 heterozygotes and C500 homozygotes, and used the unpaired Student's t test with 95% CI and Welch's correction to compensate for unequal variance. In the NDTMM and telomerase GBM groups, the loss of homozygosity or the loss of heterozygosity of exon 1\u03b1 or exon 1\u03b2 was compared to tumors that retained exon 1\u03b1 or exon 1\u03b2 homozygosity using the Fisher's exact test and GraphPad Prism software (GraphPad). The frequency of missense mutations in IDH1 was compared between GBMs with different telomere maintenance mechanisms using the Pearson's chi square test and SHEsis software The primary end point was overall survival. Cox proportional hazards regression analysis was performed to assess the GC and CC G500C genotypes as a prognostic marker for survival and was adjusted for other variables age (years), gender, tumor resection , and treatment , loss of p16"}
+{"text": "Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In  Hox genes are essential for the proper organization of structures along the developing vertebrate body axis. These genes must be activated at a precise time and their premature transcription is deleterious to the organism. Early on, Hox gene clusters are covered by Polycomb Repressive protein Complexes (PRCs), which help keep these genes silent. However, the mechanism(s) that selectively recruit PRCs to these particular genomic loci remains elusive. We have used a collection of mutant mice carrying a set of deletions inside and outside the HoxD cluster to try and detect the presence of any DNA sequence of particular importance in this mechanism. We conclude that a range of low affinity sequences synergize to recruit PRCs over the gene cluster, which makes this process very robust and resistant to genetic perturbations. Polycomb) was characterized genetically as a repressor of Drosophila homeotic genes via epigenetic mechanisms such as X-inactivation trithorax families (trxG), displaying opposing functions, was shown to maintain Hox gene expression during the entire life of DrosophilaPolycomb group (PcG) proteins are essential for proper development of most eukaryotic organisms. The founding member . PRC2 carries a methyl-transferase activity that methylates the histone H3 tail at lysine 27, a mark largely associated with gene silencing. While the initial deposition of this post-transcriptional modification is carried through by PRC2, PRC1 maintains this methylated status and compacts chromatin, largely, though not solely Drosophila, PcG group proteins are recruited to chromatin through the recognition of polycomb response elements (PREs). These DNA segments, which were generally identified by forward genetics, must satisfy three criteria: (1) they must bind polycomb when randomly inserted into the genome, (2) they must be able to induce a H3K27me3 domain and (3) they must repress a reporter construct, when associated with them Drosophila PREs are approximately 1.5 kb long in average and are usually located in proximal promoter regions. This is the case of the PREs associated with either engrailedHedgehogpolyhomeoticIn in vivo several kilobases away from their target genes and their identification may be biased by genome wide chromatin immunoprecipitation (ChIP) studies. Recently indeed, it has become clear that only a fraction of polycomb enriched regions are direct targets of PRC, whereas others are indirect, via chromatin looping polycomb enriched regions may not always reflect the genuine presence of polycomb at a particular locus. Instead, they may derive from the three-dimensional organization of the genome, which along with the technology employed, may lead to false positives. Furthermore, although high throughput studies have shown that the binding profiles of polycomb proteins correlate with both the transcription start sites (TSSs) and stalled RNA PolII However, PREs have also been mapped Pho ortholog YY1 Pho and PRC remains controversial In mammals, the understanding of the general mechanism (if any) accounting for the recruitment of PRC2 is lacking too. PRC2 recruitment has been associated with the presence of binding sites for the Hox gene clusters  cells, including the four clusters . In thisHox loci by using an in vivo approach, based on the large number of genomic re-arrangements associated with the HoxD locus Drosophila Bithorax complex where a 300 kb large domain of trimethylated histones involves only few PREs, we show that the mouse HoxD locus implements a mechanism that can compensate for large and systematic deletions within the target DNA interval. These results indicate that PRC2 recruitment at this locus must rely upon a range of cooperating binding sites, rather than upon a few nucleation sites. By using isolated transgenes, we also show that in this particular context, CpG islands (or a high GC content) are not the prime factors in PRC2 recruitment, despite their potential importance for local spreading.We have addressed the question of polycomb recruitment at in vivo, we used a genetic approach of the mouse HoxD gene cluster, which is a main target of Pc silencing in ES cells and adult tissues To study the recruitment of polycomb complexes and the resulting H3K27me3 histone modification HoxD cluster did not seem to affect the binding profiles of PRC proteins when assessed by ChIP-qPCR ) in vivo had no effect upon gene expression within the HoxD cluster del(12)). Here again, we did not score any modification of the H3K27me3 profile throughout the gene cluster. We next scanned the entire locus with a set of adjacent deletions, including either Hoxd8, Hoxd9, Hoxd10, Hoxd11, Hoxd12 or Hoxd13 and found no significant change in polycomb-mediated silencing (data not shown), using H3K27me3 as a proxy for PcG occupancy  and Del(9-12)  . These tH3K27me3 , suggestHox gene cluster were of particular importance to set up a platform for recruiting PRC2. We used a large deletion (del(1-10)), where two-thirds of the anterior part of the cluster were removed including its most anterior gene Hoxd1. Again, the remaining mini-cluster was able to compensate for this significant trimming and the H3K27me3 pattern over the remaining loci was nearly identical to that found in wild type conditions d11Lac), did not significantly affect the presence of these epigenetic marks over the remaining 5\u2032 located Evx2 gene , even though the latter displayed massive amounts of H3K27me3 over the anterior part of the cluster , del(13) and del(10-13), which share the same breakpoints but in various configurations. In this set of deletions, the reconstitution of three different neighborhoods did not modify the methylation patterns.This result indicated that each piece of the gene cluster is rather independent in its ability to recruit PRC2, regardless of what would happen over the neighboring loci. The poor impact of the neighboring sequences upon the coverage of any given HoxD. This intergenic region \u2018i\u2019 is over 13 kb long and maps between Hoxd4 and Hoxd8. Its deletion (del(i)) results in a further concentration of genes by bringing Hoxd1, Hoxd3 and Hoxd4 closer to the centromeric (posterior) side including Hoxd8 to Hoxd13. del(i) did not show any clear difference in the H3K27me3 profile over the remaining parts of the gene cluster (del(i)).Single gene deletions did not markedly change the relative distance between transcription units and hence they may not affect PRC2 recruitment, should several PREs locate near each gene and synergize. We thus modified the distance between two genes by excising the longest gene free DNA segment within i to produce a cluster with a 26 kb large gene-free domain inside. Intriguingly, the duplicated configuration did not exhibit any change on region i, except for a weak gain of signal over the duplicated region suggesting that both copies are covered by H3K27me3. The flanking DNA segments, however, displayed the same H3K27me3 profiles as in wild type brains (del(i), dup(i)) indicating that the mechanism recruiting PRC2 over the mouse HoxD cluster can compensate for modifications in the distance between transcription units, emphasizing once more the robustness of this process.We next looked at the effect of introducing a gene free region within the cluster such as to increase the distance between neighboring genes. We duplicated region HoxD cluster, which contain numerous cis-acting sequences. We deleted a 230 kb large piece of DNA, from eight kb upstream Evx2 to a breakpoint located within the flanking centromeric gene desert (del(R1-R5)-d9Lac) HoxD gene cluster per se, it removed the border of the H3K27me3 domain and hence it reconstituted a neighborhood between heavily H3K27 tri-methylated nucleosomes and nucleosomes not methylated at all.In the absence of any strong and discrete signal for PRC2 recognition and nucleation within the cluster itself, we asked whether Pc proteins may be targeted by elements localized within the regulatory landscapes flanking the HoxD cluster in the developing brain, nor did it induce any leakage over the centromeric DNA from the gene desert (del(R1-R5)-d9Lac). Therefore, as previously reported The deletion of this \u2018epigenetic border\u2019 did not elicit any loss of H3K27me3 marks over the HoxA, HoxC and HoxD clusters. In the case of HoxD, the Evx2 gene is found ca. 10 kb upstream Hoxd13 on the opposite strand. This gene, which is covered by H3K27me3 marks and locates close to the epigenetic border, was however not removed in the del(R1-R5)-d9Lac deletion. Therefore, we analyzed a second deletion, ca. 260 kb large, with the same upstream breakpoint into the gene desert (see above), but with a telomeric breakpoint located between Hoxd10 and Hoxd11. In this del(11-R5)-d9Lac mutant, the entire posterior part of the HoxD cluster was removed including Hoxd11, Hoxd12 and Hoxd13 as well as the Evx2 transcription unit and the epigenetic border.Another mechanism to set the PcG epigenetic borders may involve transcripts encoded by the opposite DNA strand, a feature found in the del(R1-R5)-d9Lac deletion, suggesting that additional transcriptional units encoded by either DNA strands are not necessary for the recruitment of PRC2 at the extremity of the HoxD cluster, nor for the fixation of a sharp epigenetic boundary (del(11-R5)-d9Lac). In both deletions, however, a Hoxd9/Lac transgene was relocated at the breakpoint, raising the possibility that transgenic sequences would interfere with PRC2 recruitment and hence we used a final mutant configuration carrying a ca. 800 kb large deletion including the HoxD centromeric regulatory landscape. This del(Nsi-Atf2) deletion not only removes the 5\u2032 epigenetic border, but also most of the regulatory elements that contact Hoxd genes and impose a chromatin topology to the locus In this configuration, the H3K27me3 profile remained unchanged when compared to the wild type pattern. In particular, the reconstituted epigenetic boundary was similar to that seen with the shorter HoxD cluster towards the new centromeric neighboring sequences did not exceed a 800 bp large interval, which corresponds to twice the average length of the sonicated DNA fragments (data not shown). These results further indicated that the capacity to recruit PRC2 is restricted to Hox genes themselves, without any contribution from the surrounding genomic sequences. To demonstrate this point, we produced a transgenic line containing the Hoxd10 gene, which had inserted into a genomic region of average GC density and poor in H3K27me3 marks. While H3K27me3 was scored on the entire transgene, this histone modification did not spread over flanking nucleosomes, as assessed by ChIP-seq . Therefore, when this transgenic stock was crossed back into a mouse carrying a homozygous deletion of Hoxd10 (TgN/del(10)\u2212/\u2212), the H3K27 trimethylation profile (or the lack thereof-) over Hoxd10 reflected that of the ectopic Hoxd10 copy. The Hoxd10 locus was selected because the CpG island located upstream the promoter (CpG32 from UCSC) could be removed by using FRT sites and the Flip recombinase in vivo, without affecting the transcription start site (TSS). To make sure that no additional CpG islands remained after deletion of CpG32, we deleted another potential short island (CpG26) from our starting transgenic construct.The Hoxd10 null mice to assess their H3K27me3 status in developing forebrains . Similar results were observed when the CpG26 sequences had been removed (TgNd10). Moreover, similar amounts of H3K27me3 were scored when the transcription start site of TgNd10 was deleted, suggesting that the recruitment of PRC2 may be independent of transcription (TgNd10\u2202TSS) TgNd10\u2202CpG).Transgenic animals were crossed with a Cre-deleter strain to adjust copy number to one and various transgenes were thus crossed over rebrains . When th removed , TgNd10.Hoxd10 was used, H3K27me3 marks were no longer detected, even though this construct still contained an annotated CpG island (TgNd10\u22023) and was globally GC-rich. Of note, a transgene containing the same four kilobases together with exon 1 of Hoxd10 showed no recruitment of PRC2 either (TgNd10\u2202PREd10), regardless whether or not the TSS was present (TgNd10\u2202TSS\u2202PREd10), suggesting that neither the TSS, nor the CpG32 are essential for recruiting PRC2 in this configuration. Mapping the insertion sites did not reveal any correlation between the presence of H3K27me3 on the transgenes and their insertion into either a H3K27me3-rich or a GC-rich DNA region. In fact, transgenes were found integrated at least 500 kb away from H3K27me3-rich spots and into DNA segments with rather average GC contents (data not shown). These experiments thus defined a 1.4 kb large DNA segment, containing exon 2 and the 3\u2032UTR of Hoxd10, which was necessary for the deposition of H3K27me3 marks. This DNA segments is referred to as PREd10 below.However, when a four kb large transgene containing only the 5\u2032 sequence upstream Hox clusters are twice as dense in differentiated tissues than in ES cells  were thus used to assess the H3K27 methylation status of distinct electroporated DNA elements, overlapping with the deleted Hoxd10 DNA segment. Various portions of the TgNd10 transgene were first cloned between two homologous arms (Env) flanking the transgenes, in the hope of comparing random and targeted integration sites. However, homologous recombination events were not found.While H3K27me3 marks covering the ES cells , the extHoxd10 fragment, including the TSS and both exons, or the 1.4 kb long PREd10 were introduced into our iPSdel(Hoxd10) cells, they became H3K27 tri-methylated, in agreement with the results obtained using classical transgenesis. More surprisingly, when the 5\u2032 sequence corresponding to that used in TgNd10\u22023 was assayed, H3K27me3 was detected too, in contrast to the results obtained in transgenic mice. We checked the capacity of either the vector backbone, or the PGK-neomycin gene promoter to recruit PRC2, by electroporating the neomycin cassette alone. The PGK promoter is ubiquitous and hence neomycin transcripts were detected in all conditions tested . We verified if this recruitment was influenced by the presence of the DNA homology arms included for a potential recombination at the locus, which contained sequences from both the Hoxd11 and a portion directly 3\u2032 to Hoxd10, which could thus initiate a \u2018spreading\u2019 of H3K27me3 marks over the Env-d10\u22023 fragment. Accordingly, we electroporated d10\u22023 (42% rich in GC) into iPSdel(Hoxd10) without any other surrounding DNA sequences. While H3K27me3 was detected over a multimerized version of d10\u22023, this mark was lost after the CRE recombinase had reduced copy number to one (d10\u22023-CRE). In contrast, when CpG-island free PREd10 (44% rich in GC) was introduced into iPSdel(Hoxd10), H3K27me3 marks were readily scored after CRE-excision of the multimers (d10-CREd10PRE). PRC1/2 subunits were also detected over this exogenous, randomly integrated sequence, suggesting it contains all proper information necessary for PcG recruitment to ectopic sites  and PREd10-600 (43% rich in GC), which were tested as individual transgenes. Unexpectedly, both fragments were decorated by H3K27me3, when introduced into iPSdel(Hoxd10) cells as single copy  displaying a temporal sequence in the establishment of their segmented body plan systematically show a complete clustering of their Hox gene complement, whereas other animals following different strategies (such as cell lineages) usually have broken Hox clusters or even Hox genes scattered throughout the genome  Hox clusters are amongst the genomic loci with the highest GC content.As for many Drosophila PREs, PREHox gene clusters, they do not rule out their potential importance for the spreading or the re-enforcement of the coverage by PRC2 (see below). The existence of CpG islands devoid of PcG, as well as PcG target DNA devoid of CpG islands, such as in the case of the first described mammalian PRE-like sequence regulating the mouse MafB/Kreisler, support this view. Moreover, sequences unable to recruit PRC when present as single copy transgenes may become H3K27me3 when concatamerized, suggesting that larger stretches of GC-high sequences can artificially recruit PRC, a possible explanation to the discrepancies observed between our results and those of others Here again however, our results do not support a high GC content as the major parameter in recruiting PRC2. We show that DNA segments with a GC content similar to the average of the mouse genome (42%) are still able to properly recruit PcG proteins and the deletion of CpG islands from our transgenic constructs did not abrogate the trimethylation of H3K27. While these results suggest that CpG islands are neither sufficient, nor required, for the tethering of PcG proteins in the context of ad minima model whereby H3K27me3 is deposited on a series of low affinity PRC2 interacting sequences, which work synergistically between themselves and together with GC-rich sequences to confer robust silencing over target genes .Hoxd1 and either the site telomeric to Hoxd8 (del(1-i)) or the site centromeric of to Hoxd4 (del(1-4)). The del(i) line was produced by TAMERE between the loxP telomeric of Hoxd8 and the one centromeric to Hoxd4. Genotyping was performed on individual yolk sacs.All stocks of mice were kept as heterozygous and bred to homozygosity. Lines were all described and can be found in previous publications of the Duboule laboratory. Two additional lines were produced by recombination between the loxP site in the second exon of Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was performed as described in Mouse embryonic fibroblasts were derived from heterozygous crosses of E13.5 embryos using standard protocols. Cells were cultured in standard MEF/ES cell culture conditions. MEF/ES media contained DMEM supplemented with 10% FBS and LIF (ES media only). Isolated MEF lines were first genotyped using embryonic tissues and subsequently confirmed with DNA extraction procedures. Passage three MEFs were used for iPS derivation experiments.Oct4, Klf4 and Sox2 were cloned and separated by bacterial 2A sequences, in a single lentiviral backbone (3F). Virus was produced in 293T cells using FuGENE HD transfection reagent  and ultracentrifuged. Induced pluripotent (iPS) stem cells were derived following standard protocols SSEA1, Nanog, Oct4 and Sox2 by immunohistochemistry and western blot, standard protocols), a non-aberrant chromosome count  and re-establishment of bivalent domains .Human 6. Electroporated cells were plated on 10 cm dishes coated with DR4 resistant feeders. G148 selection (200 \u00b5g/ml) (Sigma G8168-10ML) was started 24 hours after electroporation and was continued until individual colonies were picked and genotyping. CRE treatment of iPS cells was done as follows: 3\u00d7105 cells were plated overnight and transduced with a PGK-CRE lentiviral construct at MOI 100. Individual colonies were picked 5 days post-transduction, expanded and genotyped.Electroporation of induced pluripotent stem cells was performed using an Amaxa Nucleofector I and the Lonza mouse embryonic stem cell kit (Lonza VPH-1001). Briefly, 25 \u00b5g of DNA were digested overnight, phenol-chloroform purified and resuspended in 10 \u00b5l H2O. Media was changed 4 hours before electroporation. Cells were washed twice with Mg(2+)-Ca(2+)-Free PBS, trypsinized and aliquoted to 2\u00d710HoxD cluster were spotted with 25-mer oligonucleotides at 15b bp resolution . Fragmentation, labeling and hybridization of ChIPed DNA were done following standard protocols.Affymetrix custom-made tiling arrays covering two megabases surrounding the mouse Rps9) were used to derive mean values. Primers are given in Cells were first disrupted and homogenized using a Polytron (kinematic) before RNA was extracted using the RNeasy Microkit . qRT-PCR was performed with SYBR Green. Two biological replicates, processed in triplicates and normalized to a housekeeping gene  from Affymetrix. Data was exported as plain text using a log2 or \u221210log10 scale for the signal, respectively the p-value. Files were visualized in RChiV, an in-house developed genome browser, which takes into account the deleted segment and normalizes the signal using a sliding window approach.Figure S1HoxD cluster in mutant configurations. (A) ChIP-qPCR profiles of PRC1  and PRC2 (Ezh2 (middle panel) and Suz12 (lower panel)) over the HoxD cluster. The wild type values of six genes (from Evx2 to Hoxd3) found within the H3K27me3 domain are used as positive controls (black). Lnp and Mtx2 are found outside of the H3K27me3 domain and are thus used as negative controls (black). Different mutant configurations are color coded and specified on the top (del(10), del(10-11), del(9-12), del(10-13), del(11-R5)). dN stands for HoxdN. NA refers to the absence of the given DNA segment in the specified allele. NI refers to mutant alleles which where not included in the experiment (Suz12 on both del(10-11) and del(11-R5)).Binding profiles of PRC1 and PRC2 over the (TIF)Click here for additional data file.Figure S2HoxD cluster and flanking gene deserts. (B) H3K27me3 profiles of wild type and deleted animals. Genotypes are specified on the left.Effect of large deletions upon the H3K27me3 profiles. (A) Wild type genomic landscape of the murine (TIF)Click here for additional data file.Figure S3Hoxd10 specific probe . A wild type sample (+/+) was used as control (right panel). The positions of both the restriction sites for PvuII and HindIII and the probe used for southern blot are depicted on the wild type profile in Southern blot of transgenic animals. Southern blot using a robe see . Restric(TIF)Click here for additional data file.Figure S4del(10) embryo .H3K27me3 profiles in pluripotent and terminally differentiated cells. A large (A) or focused (B) view of wild type H3K27me3 profiles from differentiated cells dissected from the embryonic brain (top) compared to pluripotent cells derived from a (TIF)Click here for additional data file.Figure S5Hoxd genes in various constructs eletroporated in iPS cells carrying a deletion of Hoxd10. Lnp is located outside the HoxD cluster and is used as a control for active genes, while Hoxd13 is used as a control for silent genes. Clones and culture conditions are color coded and specified on the top. G418 stands for the presence (+) or absence (\u2212) of the antibiotic. Vector refers to a control cell line.H3K27me3 and RNA profiles in various cell lines. ChIP-qPCR and mRNA expression of control and (TIF)Click here for additional data file.Table S1List of the primers used for RT-PCR, either for ChIP experiments (top) or for RNA dosage (bottom).(DOC)Click here for additional data file."}
+{"text": "Irish ICUs typically have bed occupancy rates approaching 100%, with 75 to 80% being the recommended level [The GICU database was examined from 1 March 2010 to 1 March 2011. The H1N1 pandemic was recognised as a period of strain on the ICU and this period was estimated as 24 December 2010 to 21 January 2011. All ICU readmissions during the same hospital stay were noted. Transfers between GICU, cardiac ICU and theatre recovery were excluded as patients were still being treated by the intensive care team. Patients readmitted after transfer for extracorporeal membrane oxygenation (ECMO) were also excluded.The number of GICU admissions during the period was 422. There were 19 readmissions (readmission rate of 4.6%). However, this rate increased to 8.6% during the period of high activity encompassing the H1N1 pandemic (Figure The annual readmission rate for our unit was acceptable . A clear"}
+{"text": "Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species. The large variety of AAV serotypes with different tropisms provides a gene therapy platform that can be applied in different tissues. For instance AAV serotype 1 (AAV1) can be used to correct lipase deficiency in the muscle Efficacy in patients demonstrates that adeno-associated viral (AAV) vectors can be used for in vivoPresence of low levels of neutralizing antibodies (NAb) towards an AAV serotype will preclude efficient transduction of the target organ in vivo gene therapy in CN patients. In addition, the availability of several suitable vectors does allow switching to another serotype for treating patients with pre-existing NAb towards one of these serotypes Our long term goal is to treat Crigler-Najjar syndrome (CN), familial severe unconjugated hyperbilirubinemia. This recessive inherited severe liver disorder is caused by deficiency of uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) In this study we evaluate the efficacy of scAAV5 and scAAV8 to that of scAAV1 in the Gunn rat. AAV8 and AAV1 both provide efficient correction of hyperbilirubinemia. In contrast, AAV5 does not provide any detectable activity in this rat model. Subsequent studies in different mouse and rat models using other transgene cassettes confirm the lack of efficacy of AAV5 in rat liver rendering proof of concept studies in this species with this clinically relevant serotype impossible.AAV vectors were constructed by replacing the factor IX cDNA with the UGT1A1 cDNA, using the EcoR1 and Bbs1 sites of plasmid scAAV-LP1-hFIXco . This plasmid carries the AAV2 backbone with an intact 5\u2032 terminal resolution site (trs) without the 3\u2032trs analogous, a LP1 promoter consisting of core liver-specific elements from human apolipoprotein hepatic control region (HCR) and the human alpha-1-antitrypsin (hAAT) promoter, a modified SV40 small intron and the SV40 late polyA sequence, as previously described 11 and 9\u00d71011 genome copies per plate. Of the average titers (genome copies/ml) of the purified batches of the vectors were 5.3\u00d71012 for scAAV2/1, 5.3\u00d71012 for scAAV2/5 and 1.9\u00d71012 for scAAV2/8. Because of the double stranded genome, each scAAV vector was calculated as containing 2 copies of ss viral genomes.Recombinant AAV was produced with AAV2 Rep and pseudotyped with capsid from AAV serotype 1, 5 and 8 using the adenovirus-free method described before Gunn rats from our own breeding colony were used for all experiments and fed ad libitum. All animal experiments were performed in strict accordance with the Animal Ethical Committee guidelines of the Academic Medical Center of Amsterdam and the Animal Ethical Committee guidelines of the University of Navarra. The protocol was approved by the Animal Ethical Committee of the Academic Medical Center of Amsterdam and the Animal Ethical Committee of the University of Navarra.11 gc/kg of scAAV-LP1-UGT1A1 vector packaged with each of the viral capsids from AAV1, 5 and 8. For portal injections the rats were anesthetized with an intraperitoneal injection of KAR mix: 4 ml ketamine (100 mg/ml), 2 ml Rompun , 1 ml atropine (1 mg/ml); dose of 0.1 ml/100 g body weight. Under deep anesthesia, the peritoneal cavity was opened and AAV vector resuspended in a maximum volume of 500 \u00b5l of PBS was injected into the portal vein using a 30-gauge needle. The animals were sutured and received the analgesic Temgesic subcutaneously following recovery from KAR mix. For bile collection, rats were anesthetized by intraperitoneal injection of KAR mix as above and bile was collected by cannulation of the bile duct as described Male Gunn rats, 8 to 10 weeks of age, with a weight between 180 and 200 g, received a single intraportal injection of 3\u00d710Luciferase activity is represented as photons/sec and RLU per miligram of total protein respectively.Blood was collected by tail vein puncture under gas anesthesia in pediatric heparin tubes. After spinning down the cells the plasma was stored at \u221280\u00b0C until bilirubin measurement performed at the routine clinical chemistry department.5\u2032-GACGCCTCGTTGTACATCAG-3\u2032; reverse, 5\u2032-CACGCTGCAGGAAAGAATC-3\u2032, using the following conditions 95\u00b0C for 5 min and 45 cycles of 95\u00b0C for 10 s, 60\u00b0C for 10 s, and 72\u00b0C for 12 s. The values were normalized with the rat \u03b2-actin gene, using as a forward primer: 5\u2032 AGCCATGTACGTAGCCATCCA3\u2032 and 5\u2032 TCTCCGGAGTCCATCACAATG3\u2032 as the reverse primer. A standard curve was generated by dilution in rat genomic DNA of the UGT1A1 plasmid. To determined the UGT1A1 mRNA levels in liver the qPCR was performed using the same primers for UGT1A1 as used to determine genomic copies and primers for 18S (Fw: 5\u2032-CGAACCTCCGACTTTCGTTCT-3\u2032 and a Rv: 5\u2032-TTCGGAACTGAGGCCATGAT-3\u2032). Vector genomic copies per diploid genome equivalent (vc/dGE) and UGT1A1 mRNA/18S were calculated using the LinRegPCR software High-molecular-weight DNA from tissues was isolated by a sodium dodecyl sulfate-proteinase treatment and subsequently complexed with silica particles in the presence of guanidinium thiocyanate, as described in Total bilirubin in serum was determined by the routine clinical chemistry department using a standard colorimetric assay. Unconjugated bilirubin and bilirubin conjugates in bile were analyzed and quantified by HPLC as described The results obtained in C57/BL6 mice are statistically significant compared to the obtained in Sprague Dawley and Wistar rats using the nonparametric Mann\u2013Whitney test .11 gc/kg of scAAV-LP1-UGT1A1 vector pseudotyped with serotype 1, 5 and 8. Of these scAAV2/8 achieved the most prominent long term correction  was injected into the tail vein of eight Gunn rats. This also resulted in the presence of vector genomes mainly in liver and spleen, but not in detectable expression of GFP mRNA and protein, which renders this explanation unlikely (data not shown). In mice, the stronger liver tropism with AAV1 than AAV5 can be explained by its receptor, platelet derived growth factor receptor (PDGFR) which is expressed at high levels on hepatocytes AAV is the most promising delivery system for The large difference in efficacy of AAV5 found between mice and rat is unexpected but recently two new species of AAV\u2019s from rat and mouse liver Recently it was reported that in rabbits AAV5 also transduced liver very poorly In conclusion, we showed that scAAV8 and scAAV1 provided an efficient liver transduction in the rat model for CN syndrome, the Gunn rat. Furthermore, our data indicated that proof of concept studies with AAV5 as a vector for correcting liver disorders are not possible in rat models.Data S1Supplementary data.(PDF)Click here for additional data file."}
+{"text": "Expression of the sole CX3CR1 ligand, the membrane-tethered and sheddable chemokine CX3CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX3CR1 for microglia function and the CX3C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX3CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context.Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX Under these conditions microglia might however loose much of their uniqueness and turn into prototype macrophages. This calls for the development of experimental systems that will allow the study of microglia in their unique physiological CNS environment.Microglia are members of the mononuclear phagocyte system alongside other macrophages, monocytes and dendritic cells . Of note, a potential confounding problem is the fact that when using CX3CR1gfp mice for microglia studies, animals are heterozygotes for the chemokine receptor and microglia display due to the haplo-insufficiency considerable less CX3CR1 surface expression  promoter  resulting in impaired cognitive functions  and amyothrophic lateral sclerosis (ALS), as well as neuropathological conditions, such as neuropathic pain and cerebral ischemia.The CX3CR1-deficiency has been introduced into several established murine AD models, including the hTAu  deposits surrounded by activated glia and dystrophic neurites. The CX3CR1-deficient microglia were found to overexpress IL-1\u03b2 and display neurotoxic activity  was reported to depend on microglial release of the lysosomal protease cathepsin S potentially in response to ATP release and P2X7 receptor  (LysM-Cre:GRK2f/+ mice)  and are hence located behind and in front of the BBB, respectively. Spinal cord and DRG are therefore differentially accessible to the influx of monocytes and contribution of monocyte-derived macrophages (see below).One issue complicating the exact assessment of the importance of the CX3C axis in microglia biology is that constitutively expressed membrane-tethered neuronal CX3CL1 seems to provide a tonic inhibitory signal to microglia that keeps these cells in a quiescent \u201csampling\u201d or surveillance mode  origin of microglia. Yet, interpretation of the results obtained was confounded by the facts that first microglia are radio-resistant and thus not replaced by a bone marrow graft, and that second the irradiation compromised the BBB allowing monocyte infiltrates. Revision of these studies taking advantage of CX3CR1gfp mice to mark either the microglia or the blood compartment and introducing cellular exchange in parabionts and CCR2 dependency as a indication of monocyte-derivation  that involves the BBB breakage and substantial monocyte recruitment  of the human estrogen receptor (ER) (Feil et al., In order to overcome the above constraints of current microglia studies, we decided to exploit the high CX3CR1Cre and CX3CR1CreER animals differ considerably with respect to the cells targeted. In CX3CR1CreER animals only cells that express CX3CR1 and hence the CreER transgene will undergo rearrangement at the time of the TAM treatment. In contrast and as best demonstrated in combination with respective reporter mouse strains (Yona et al., 3CR1Cre animals also cells that are derived from CX3CR1+ cells but subsequently silenced CX3CR1 expression will have recombined the loxP-flanked loci. CX3CR1Cre animals therefore report on the history of the cell and can be used for fate mapping studies (Yona et al., Importantly, CX3CR1Cre and CX3CR1CreER mice crossed to animals harboring a floxed YFP reporter gene (Srinivas et al., 3CR1Cre: YFP mice, and only in TAM-treated CX3CR1CreER: YFP mice, more than 95% of both brain and spinal cord microglia were found YFP labeled (Figure 3CR1+ microglia, either straight from their development onwards, using the constitutive Cre system, or in specific time windows, using the inducible Cre system.Analysis of CX3CR1. TAM treatment of CX3CR1CreER mice results accordingly also in gene rearrangement in these cells (Yona et al., 3CR1 expression, they are not targeted by the CX3CR1CreER approach in adulthood (Yona et al., 3CR1+ intestinal macrophages on the other hand loose their gene modifications, as they are progressively replaced by monocytes (Zigmond et al., 3CR1Cre and CX3CR1CreER animals provides a unique tool to probe for the involvement of microglia in CNS development, CNS maintenance and CNS responses to pathological challenges.Of note, certain lymphocyte subsets and myeloid cells, other than microglia express CX3C axis is likely to play a prominent role in these activities. Thus accumulating evidence suggest that neuronal CX3CL1 acts as a critical inhibitory signal retaining microglia in quiescent mode and preventing collateral damage due to microglia hyper-activation. Aside from its biological role, the CX3CR1 chemokine receptor provides us however also with a unique foothold to study microglia in context using state of the art imaging and gene manipulation approaches. The near future is hence likely to provide valuable insight into contributions of these intriguing cells to brain physiology and might pave the way for the development of microglia manipulation for therapeutic purposes.Microglia are the main representatives of the immune system in the healthy brain as such likely to critically contribute to the brain's resistance to pathological challenges. Moreover, recent data highlight the critical involvement of microglia in CNS development and homeostasis. Given its strategic localization at the neuronal/microglial interface the CXThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes. FoxD3 is an important stem cell factor expressed in many types of embryonic cells including neural crest cells. In the embryo, neural crest cells are a type of stem cell that forms diverse derivatives, including nerve cells, pigment cells, and facial structures. To better understand neural crest development and differentiation, we have explored how FoxD3 expression is regulated in these cells. By examining non-coding DNA, we have identified distinct genomic regions that mediate expression of green fluorescent protein (GFP) in a pattern that recapitulates FoxD3 expression. Interestingly, we find two genomic \u201con\u2013off\u201d switches or enhancers, called NC1 and NC2, that drive GFP expression in a pattern that recapitulates FoxD3 expression at different times and places during neural crest development. We find that Pax and Msx proteins turn on both NC1 and NC2 enhancers by directly binding to them. In addition, cranial expression driven by NC1 requires a protein called Ets1, whereas trunk expression of NC2 requires a different protein called Zic1. The results show that FoxD3 in differentially regulated in distinct neural crest cell populations in a manner that is specifically encoded in the genome. These enhancers provide valuable tools for understanding neural crest development in birds and mammals. The neural crest (NC) is a transient population of cells that migrates throughout the embryo and forms many different cell types, including neurons and glia of the peripheral and enteric nervous systems, bone and cartilage of the craniofacial skeleton and melanocytes An important challenge is to establish direct connections within the neural crest GRN. For example, the neural plate border marker, Pax7, is essential for expression of a number of different neural crest specifier genes Xenopus and zebrafish Xenopus ectopic expression at the 8-cell stage increases the expression of neural crest markers, while expression of dominant-negative FoxD3 reduces expression of genes like Snail2, Twist and Ets1 Of the neural crest specifier genes, FoxD3 is one of the first markers of premigratory neural crest in many vertebrate species including mouse, chick, cis-regulatory regions of the critical neural crest gene, FoxD3. Taking advantage of the chick's compact genome and ability to assay putative regulatory regions by electroporation, we have identified two enhancers, NC1 and NC2, that mediate reporter expression in spatially and temporally distinct manners in the chick embryo, and in combination closely recapitulate the endogenous expression of FoxD3. Detailed regulatory analysis shows that initial expression of FoxD3 in both cranial and trunk neural crest requires direct input from neural plate border genes, Pax7 and Msx1/2. These factors function in combination with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene Zic1 to activate the NC2 enhancer. These results not only reveal region-specific enhancer activity in the neural crest, but also expand the neural crest GRN and inform upon direct interactions therein. Conserved between mouse and chick, these enhancers further provide excellent tools for assaying gene regulation and manipulation of neural crest gene expression in amniotes.Despite its important role both in stem and neural crest cells, no regulatory element(s) controlling the onset of FoxD3 expression are known. To define linkages and assess direct regulatory interactions in the neural crest gene regulatory network with particular interest in possible targets of Pax7, we set out to dissect the FoxD3 expression domain includes the neural folds of the forebrain and midbrain. Subsequently, at HH8+, FoxD3 expression expands posteriorly to the hindbrain . FoxD3 is expressed in most premigratory and migratory vagal and trunk neural crest, but is down-regulated in melanoblasts  NC1 and mNC2 constructs were electroporated into chick embryos at gastrula stages. The results show that the patterns of eGFP expression driven by mNC1 and mNC2 were identical to those observed with chick NC1 and NC2 , suggestTo examine the dynamic nature and combined activity of the two enhancers in the migrating cranial neural crest, we co-electroporated NC1 (green) and NC2 (blue) enhancers in combination with a previously identified cranial neural crest Sox10E element (red) Time-lapse movies revealed differential temporal and spatial activity of NC1 and NC2 enhancers. While NC1 activity was present in the premigratory neural crest, the expression it drove in the dorsal neural tube appeared transient in most cells and preceded that driven by the Sox10E enhancer  and NC2 (red) enhancers and observed neural crest formation and migration by time lapse microscopy. Analysis of the movies suggested that there was little overlap between cells showing activity of NC1 and those with NC2 , whereasXenopus, but showed no sequence conservation with zebrafish. Primers were designed to amplify fragments of NC1, which were tested for activity at stages HH9\u201310, corresponding to the time it drove strongest expression. Using this approach, NC1 was reduced to 553 bp (NC1.1) without loss of activity , which reflects transcript levels more accurately than At trunk levels, knock-down of Msx1/2, Pax7 and Zic1 resulted in a significant reduction of endogenous FoxD3 expression , whereasin vivo association of Pax7, Msx1 and Ets1 transcription factors with the NC1 enhancer, we performed quantitative chromatin immunoprecipitation (ChIP) experiments. Cross-linked chromatin isolated from the midbrain dorsal neural tube of HH8\u20139 embryos was immunoprecipitated using Pax7, Msx1 and Ets1 antibodies and ChIP-enriched DNA was used in site-specific qPCR, with primers designed to amplify fragments within the NC1 region. For all three factors, we found significant enrichment of the NC1 region amplicon, expressed as a percent of the total input chromatin, compared to IgG controls , as well as in different subpopulations of the cranial neural crest. The enhancer NC1 is active in premigratory and some migratory cranial neural crest rostral to R3, while enhancer NC2 activity initiates in a single continuous wave caudal to rhombomere 4, including vagal and trunk regions, but also later in a subpopulation of migrating cranial neural crest. In our analysis of the conserved regions within the FoxD3 locus, only these two regions were able to mediate reporter expression in patterns reflecting the distribution of neural crest. The proximity of the NC1 and NC2 enhancers to the FoxD3 coding region, the recapitulation of endogenous FoxD3 expression by the combined activity of the enhancers, and the effect of manipulating upstream regulators on both enhancers and endogenous FoxD3 expression, strongly suggest that NC1 and NC2 act as enhancers regulating endogenous expression of FoxD3 in the neural crest.de novo in actively migrating cranial neural crest cells, where its activity is preceded by that of Sox10E2. NC2 is active in only a few delaminating/emigrating cranial neural crest cells but in a majority of the migrating neural crest population. Interestingly, there is little overlap of NC1 and NC2 activity in the cranial neural crest, whereas both overlap with Sox10E2, which appears to be active in all of the migrating cranial crest population.Comparison of the activity of these two enhancers with the cranial Sox10 enhancer Sox10E2 The minimal overlap in activity of NC1 versus NC2 in cranial neural crest populations raises the interesting possibility that there may be a regulatory switch of enhancers from NC1 to NC2 at the endogenous promoter of FoxD3 when the cells reside within the dorsal neural tube and/or are emigrating. Such competition at the promoter could occur if only a single enhancer can be functional at any given time on the FoxD3 promoter. If this is the case, the very few double labeled cells expressing NC1- and NC2-driven reporter expression may represent perdurance of eGFP protein rather than the actual levels of enhancer activity. The finding that NC1 and NC2 enhancers are active in generally separate cranial neural crest populations further suggests that the cranial neural crest represents a heterogeneous population, even as the cells are delaminating from the neural tube, and that this heterogeneity may be encrypted at the regulatory level.It is intriguing to speculate that the differential activity of NC1 and NC2 in distinct subpopulations may reflect differential cell fate and commitment status of future neural crest derivatives. Consistent with the possibility that NC1 and NC2 activity may reflect commitment to different lineages, NC2 is later active in neural crest-derived dorsal root and trigeminal ganglia, whereas NC1 is active transiently in the branchial arches, but not in peripheral ganglia.The activity of NC2 in the vagal and trunk neural crest recapitulated expression of endogenous FoxD3 in premigratory and migratory neural crest cells. In addition, FoxD3 is retained by a subset of neural crest derivatives histone deacetylase 1 (hdac1) mutant, a severe loss of mitfa positive melanophores can be rescued by partial reduction of FoxD3; suggesting hdac1 is required to repress FoxD3 in melanophores NC2 not only was active in neuronal derivatives, but also directed activity in neural crest cells migrating along the dorsolateral pathway, which are melanocyte precursors that migrate 24 h after the ventrolateral population migrate to the ganglia. Cells on the dorsolateral population do not normally express FoxD3 Xenopus, lamprey and mouse, and provides further support for a conserved gene regulatory network in the neural crest. Pax7 and Pax3 are closely related paralogs which have overlapping expression and function Xenopus, Pax3 is expressed in premigratory neural crest along the neural axis, and Pax7 is restricted to cranial levels (and very weak in Xenopus) Xenopus, mouse and lamprey suggests that Pax3 and/or Pax7 is required for FoxD3 expression and neural crest specification Xenopus, Pax3 is necessary for expression of FoxD3 The current results establish for the first time a direct regulatory connection between the neural plate border genes, Pax7 and Msx1/2, and FoxD3, suggesting it is an immediate downstream target. This confirms and validates previous indirect evidence in XenopusMsx1 has been proposed to lie upstream of Pax3, FoxD3 and Snail2 during neural crest induction in in situ hybridization suggests that Ets1 and FoxD3 are expressed concomitantly in the cranial neural crest. The difference in results between these two studies likely rests in the stages at which the knock-down reagents were effective, with the present results uncovering an earlier role for Ets1.Our data also show that Ets1 is necessary for initial FoxD3 expression since electroporation of Ets1 morpholinos during gastrulation (at HH5) depletes FoxD3 expression at HH9. In contrast, a dominant-negative Ets1 inhibited cranial neural crest migration but did not result in decreased FoxD3 expression XenopusRecent work on the Sox10E2 enhancer showed that Sox10 expression in the cranial neural crest is regulated by Ets1, Sox9 and cMyb Xenopus neural crest Snail2 expression domains Although there is little published information regarding the molecular players are involved in the establishment of the more caudal neural crest populations, the present results implicate the neural plate border specifier, Zic1, as a critical factor in the control of FoxD3 expression at vagal and trunk levels. Zic1 has been shown to be required for FoxD3 expression in The present study expands the number of known direct regulatory interactions in the cranial neural crest gene regulatory network, confirming a direct regulation of FoxD3 by Pax3/7 and Msx1/2, and revealing a previously unknown regulation of FoxD3 by Ets1. We have also identified Zic1 as a key player in setting up the FoxD3 expression domain in the trunk neural crest. Several other genes, like Hairy2, Sox10 and Sox5, have been suggested to regulate FoxD3 expression It is well known that the developmental potential of neural crest cells varies along different levels of the neural axis. Quail/chick chimeras have elegantly demonstrated that both the pathways of migration and derivatives differ depending upon the axial level from which neural crest cells emigrate Our data show that the inputs to FoxD3 in the vagal/trunk region are distinct from those functioning at cranial levels, suggesting a model for region-specific expression of FoxD3 . Whereashttp://ecrbrowser.dcode.org) and comparative analysis tracks of UCSC Genome Browser (http://genome.ucsc.edu/). We analyzed a 160 kb genomic region encompassing the FoxD3 locus up to the first upstream (Atg4C) and downstream (Alg6) of neighboring genes. Regions containing elements found to be highly conserved across most vertebrates including human, mouse and Xenopus were amplified using Expand High Fidelity Plus system  with CH261-166E22 and CH261-100C15  BAC clones as templates and directionally cloned into the ptkeGFP or ptkCherry vectors The genomic region of chicken FoxD3 was compared to other vertebrates using the ECR Browser (Chicken embryos were electroporated at HH4 using previously described techniques in situ hybridization for FoxD3 were performed using previously described procedures in situ hybridization for eGFP was modified using the guidelines in in situ hybridization was performed according to Whole mount and section Some embryos expressing RFP and eGFP were processed and cryosectioned at 14 mm. Select sections were labeled using the HNK-1 antibody (diluted 1/50), secondarily detected using goat anti-mouse IgM Alexa 350 . For whole mount immunostaining we used the protocol described by tggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacgg, for 30 bp substitutions acaagcagaagaacggcatcaaggtgaact and for 20 bp substitutions tggagtacaactacaacagc. Fragments were amplified using primers detailed in http://rvista.dcode.org/) and Jaspar database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl) were used to predict and analyze binding motifs within highly conserved regions. Individual sites were mutated by substituting 6\u20138 adjacent critical base pairs with GFP coding sequence, using fusion PCR and sub-cloning into ptkeGFP. Primers used are listed in Regions of NC1 and NC2 were replaced with eGFP coding sequence using fusion PCR protocol. For 100 bp substitutions, the region of eGFP used was ChIP was performed using chromatin prepared from dorsal neural tube regions of HH8\u201310 (4\u201310 somite) chicken embryos using Ets1 , Pax7 (ab34360. Abcam) and Msx1 antibodies (Sigma M0944) with normal rabbit IgGs  as previously described Figure S1In situ hybridization for FoxD3 showing expression in a dorsal root ganglion (DRG) adjacent to the neural tube (NT) in the trunk. No FoxD3 expression is present underneath the ectoderm where melanocytes are localized. (B) Transverse section through the trunk region similar to that shown in (A). eGFP (green) driven by enhancer NC2 is observed in the DRG as well as underneath the ectoderm (arrows) in presumptive melanocytes on the dorsolateral pathway. (C) Whole mount view of NC2 driven eGFP activity at HH19. Expression can be seen in the dorsal root ganglia (arrow) and melanoblasts (arrowheads). (D) In situ hybridization for eGFP shows expression in DRGs (arrow) and melanoblasts (arrowheads). (E) Transverse section of (D) confirms expression of eGFP in migrating melanoblasts (arrows). (F) NC2 activity can be seen in neural crest cells in the gut at HH27.Expression of NC2 in dorsal root ganglia, melanocytes and enteric nervous system. (A) (TIF)Click here for additional data file.Figure S2Putative transcription factor binding sites in the NC1 core region were subsequently mutated to examine effects on activity. (A) Core region of the enhancer with several binding sites highlighted. Mutation of sites in blue had no effect on the activity of the enhancer; sites in red abolished activity of enhancer when mutated. (TIF)Click here for additional data file.Figure S3Sox9 and HNK-1 expression in neural crest persists after knock-down of Pax7, Msx1/2 and Ets1 morpholinos. (A\u2013C) Embryos in which NC1 enhancer-driven Cherry was depleted via knockdown of Pax7, Msx1/2 and Ets1 see  were ana(TIF)Click here for additional data file.Table S1Mutational analysis of the NC2 enhancer reveals importance of Zic binding sites. Mutation M11, which impairs a Zic binding site, causes complete loss of trunk NC2 activity. Mutations M11, M15, M18 and M20 suppress cranial NC2 activity but only result in a slight reduction of enhancer expression in the trunk.(DOCX)Click here for additional data file.Table S2Primers used for NC1, NC2 deletions and substitutions. Text in capitals indicates enhancer sequence, and text in small letters indicates replacement GFP sequence. To make the mutated constructs, mutated primers were paired with flanking primers NC1.1 or NC2.9, amplified and joined in a fusion PCR reaction using the flanking primers NC1.1 or NC2.9.(DOCX)Click here for additional data file.Table S3Primers used for binding site mutations of NC1. Text in capitals indicates mutated sequence. To make the mutated constructs, primers were paired with flanking primers NC1.1, amplified and joined in a fusion PCR reaction using the flanking NC1.1 primers.(DOCX)Click here for additional data file.Video S1Dynamic regulation of FoxD3 and Sox10 enhancers in the cranial neural crest. Time-lapse movie shows differential temporal and spatial activity of NC1 (green), NC2 (blue) and the Sox10E2 (red) enhancers in a chick cranial slice preparation. NC1 drives expression of the reporter in the premigratory neural crest, preceding Sox10E enhancer activity. NC2 activity was observed in few cells within the neural tube, and few delaminating neural crest cells in which Sox10E2 is also active.(M4V)Click here for additional data file.Video S2Time lapse movie of migrating cranial neural crest cells electroporated with NC1:eGFP and NC2:Cherry. Time-lapse movie shows little overlap between cells with activity of NC1 and NC2. NC1 is active transiently in early neural crest cells, while NC2 seems to be primarily responsible for FoxD3 expression in migratory neural crest at cranial levels.(M4V)Click here for additional data file."}
+{"text": "Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a \u201cproof-of-concept\u201d of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode  Haemonchus contortus is a parasitic nematode of sheep that has a high propensity to develop resistance and is the most widely used model system in which to study anthelmintic drug resistance. Ivermectin is an extremely important drug for parasite control in both humans and animals. Here, we report a novel approach using genetic crossing to transfer a region of the H. contortus genome containing ivermectin resistance genes from resistant strains into a susceptible strain. During our backcrossing approach, we have identified a genetic marker showing evidence of genetic linkage to ivermectin resistance. The susceptible strain we have used is currently having its complete genome sequenced making the information and strains generated here extremely valuable for the identification of ivermectin resistance genes. This work represents an important proof of concept for using genetic approaches to identify genomic regions containing drug resistant genes in parasitic nematodes.The use of drugs (anthelmintics) to control nematode parasites (roundworms) is common in both humans and animals. This has led to the widespread development of drug resistance in livestock parasites and serious concerns regarding its emergence in human parasites.  Parasitic nematode worms are important human and animal pathogens. Human parasites infect well over 1 billion people worldwide and livestock parasites cause major economic production loss to grazing ruminants. Control is dependent on the use of a limited number of anthelmintic drugs and intensive use of these has already led to widespread resistance in livestock parasites Haemonchus contortus is a parasitic nematode of sheep which has a high propensity to develop anthelmintic. It is also one of the most amenable parasitic nematodes to experimental manipulation which, together with recent progress in sequencing its genome makes it a potentially powerful model system to study drug resistance in the strongylid nematode group Unfortunately, parasitic nematodes of humans make extremely difficult experimental subjects and so there is a need to develop model systems to study potential mechanisms of anthelmintic resistance. The genetic basis of anthelmintic resistance is still relatively poorly understood. To date, most research has focussed on the investigation of possible associations between the resistance phenotype and polymorphisms in candidate genes. This approach has been successful in identifying polymorphisms in the isotype-1 \u03b2-tubulin gene as important determinants of benzimidazole resistance in vitro). However, a major limitation of such approaches is that selection in the real world is very different to that applied in the laboratory H. contortus making genetic mapping in this organism a feasible objective in the future The artificial selection of resistance by serial passage and underdosing of susceptible laboratory strains has been undertaken by a number of groups in the past in vitroIn this paper we report the introgression of ivermectin resistance-conferring loci from two different ivermectin resistant strains, into the genetic background of the susceptible genome reference strain MHco3(ISE) All experimental procedures described in this manuscript were examined and approved by the Moredun Research Institute Experiments and Ethics Committee and were conducted under approved British Home Office licenses in accordance with the Animals (Scientific Procedures) Act of 1986. The Home Office licence number is PPL 60/03899 and experimental IDs for these studies were E06/58, E06/75 and E09/36.H. contortus sequencing project at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/H_contortus/). In spite of its inbreeding history it retains high levels of genetic polymorphism H. contortus, were originally isolated from South Africa and Australia respectively. Subsequently they have been experimentally passaged through sheep for a number of years at the Moredun Research Institute, and these versions of the strains are designated as MHco4(WRS) and MHco10(CAVR) respectively throughout the manuscript. These strains were chosen for this work for several reasons: Firstly, their origins were from the field and have subsequently been well characterized in the laboratory The MHco3(ISE) strain 1 female worms derived from each generation of backcross were crossed again with male MHco3(ISE) worms (1 progeny of the first genetic crosses between MHco3(ISE) and the two ivermectin resistant strains MHco4(WRS) and MHco10(CAVR) were designated as MHco3/4 and MHco3/10 respectively. The nomenclature for subsequent backcrosses was MHco3/4.BCn and MHco3/10.BCn, denoting the Moredun Research Institute (M), H. contortus (Hco), the unique numbers allocated to both parental strains (3/4 or 3/10), and the backcross generation  . The passage of these larvae through three more rounds of ivermectin selection and crossing against the ivermectin susceptible isolate (MHco3(ISE)) produced a final fourth backcross generation (MHco3/4.BC4 or MHco3/10.BC4)  susceptible strain with female worms from the resistant strains in the initial cross. Subsequently FE) worms . The F1 3 and 4) . Progeny/10.BC4) .4 male worms from one strain and 50 late L4/adult female worms from the other strain directly into the abomasum of a recipient sheep. In order to produce L4 for transplantation male worm-free donor lambs were orally dosed with between 5,000\u201310,000 L3 of either MHco3(ISE), or the ivermectin resistant strain to be crossed against. H. contortus donors with the ivermectin resistant strains were treated with 0.1 mg/kg of ivermectin  on day 10 or 11 post infection to select for ivermectin resistant progeny prior to transplantation. Donor sheep were euthanased on day 14 post infection and worms were harvested from their abomasa  H. contortus and 50\u2013100 female late L4/immature adult H. contortus were surgically transferred into the abomasa of male worm free recipient lambs, within 2 hours of recovery from the donor sheep.Crosses between two strains were performed by surgically transplanting approximately 50 late L4/immature adult MHco3(ISE) H. contortus and 50 female late L4/immature adult MHco4(WRS), or MHco10(CAVR) H. contortus (or subsequent backcross generations) were introduced into the abomasum in approx 5 ml RPMI, using a 5 mm diameter blunt ended, glass pipette. The purse-string suture was then closed and the surgical incision repaired allowing the completion of surgical transfers within about 2 hours from the recovery of the nematodes from the donor sheep. Sheep were routinely injected with 1 mg/kg meloxicam  for post-surgical analgesia and 7 mg/kg amoxicillin/1.75 mg/kg clavulanic acid  and closely monitored on completion of the surgery. No adverse effects were noted during the course of this study. Eggs were identified in the faeces approx 3 days post surgery and collected daily and coprocultured to produce L3.Recipient sheep were anaesthetised to allow a 10 cm vertical incision to be made through the skin, underlying fascia, muscle and peritoneum, over the right flank, midway between the last rib and pelvis and about 10 cm above the midline. The abomasum was located and partially exteriorised, to enable a 1 cm diameter sub-serosal purse-string suture to be placed. A stab incision was then made in the centre of the purse-string suture, through which 50 male late L4, and MHco3/10.BC4 alongside the three original parental strains used in this study, namely MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Seventy-five parasite na\u00efve lambs were divided equally between the five different strains. For each strain, the 15 lambs were further allocated into three treatment groups of five animals: no treatment, ivermectin (0.2 mg/kg BW) or ivermectin (0.1 mg/kg BW). Lambs were initially allocated randomly to strain and treatment groupings that were subsequently balanced, where needed on the basis of sex and the weight of the animal just prior to the experiment, to ensure that groups were as similar as possible. The lambs were infected with 5,000 H. contortus L3 on day 0. Faecal worm egg counts (FEC) were conducted H. contortus burdens in abomasal saline washings and digests H. contortus recovered from 2% (MHco4 and MHco10) and 10%  sub-samples of the abomasal washings and digests were counted and sexed (only adults were seen), the higher sub-sample volume was examined in the backcross strains due to the smaller numbers of worms present. The percentage efficacies of each anthelmintic treatment were calculated using the equation 100 (1\u2212T/C), where T and C are the arithmetic mean total H. contortus burdens of the treated and control groups respectively A controlled efficacy test (CET) using ivermectin was undertaken on the two separate fourth generation backcross strains, MHco3/4.BCAll microsatellite genotyping, on both bulk and single worm DNA lysates, was performed using the same PCR amplification methods and parameters previously described 3 worms from each generation of the backcrossing procedure , the F1 progeny of the initial genetic crosses (MHco3/4 and MHco3/10) and L3 from the three parental strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR).Bulk worm DNA lysates were made as previously described 3 or adult) worm DNA lysates were prepared from seven strains for more detailed genetic analysis: the three parental strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR); the two backcross strains, MHco3/4.BC4 and MHco3/10.BC4 and the two populations of survivors (0.1 mg/kg ivermectin) of both the backcross strains .In addition to the bulk worm DNA preparations, 30\u201340 individual .The genotyping of parasite populations/strains by amplifying a microsatellite from \u201cbulk\u201d DNA lysates made from a population of worms has been previously described ST values were calculated using Arlequin version 3.11 For the single worm genotype data, Pairwise FH. contortus arithmetic mean burdens of treated and control groups  and MHco4(WRS) strains respectively  and MHco4(WRS) strains were 100, 89, 69, 41 and 0% at 0.1 mg/kg IVM and 100, 92, 93, 37 and 36% at 0.2 mg/kg IVM respectively when compared to untreated controls , MHco3/10.BC1 strains and each backcross generation  but were lost at the second backcross generation (MHco3/10.BC2). These were relatively rare alleles in MHco3(ISE) strain - allele 215 bp present at 3.6% and allele 217 bp present at 11.1% - and therefore their loss could be due to purely stochastic reasons. In contrast, almost all alleles specific to the two ivermectin-resistant strains MHco4(WRS) or MHc010(CAVR), disappear during the backcross procedure and are absent in the MHco3/4BC4 and MHco3/10BC4 strains. There are only two exceptions to this. First, the HcX256 allele 243 bp, which is retained in MHco3/10BC4. However, it was only detected at a frequency of 4.6% in MHco3/10BC4 compared with its original frequency of 38% in the MHco10(CAVR) parental strain and so although not completely eliminated, this allele has undergone a dramatic reduction in frequency during the backcrossing procedure (Supplementary 4 and MHco3/10BC4 strains have a similar genetic background to the MHco3(ISE) parental strain based on bulk genotyping with a panel of 18 microsatellite loci as would be predicted from the backcrossing scheme.The genotyping of parasite populations/strains by amplifying a microsatellite from \u201cbulk\u201d DNA lysates made from a population of worms has been previously described 4 and MHco3/10.BC4) were analysed in more detail by genotyping 30\u201340 individual worms with 9 of the most discriminatory microsatellite markers  and MHco4(WRS) had a high level of genetic differentiation  parental strain (8 loci FST\u200a=\u200a0.0299 and 9 loci FST\u200a=\u200a0.0265 for MHco3/4.BC4 and 8 loci FST\u200a=\u200a0.0045 and 9 loci FST 0.0040 for MHco3/10.BC4). In contrast they show a high level of genetic differentiation from the ivermectin resistant parental isolates (8 loci FST\u200a=\u200a0.1930 and 9 loci FST\u200a=\u200a0.1767 between MHco4(WRS) and MHco3/4.BC4 (ST of 0.3821 and 9 loci FST of 0.3657 between MHco10(CAVR) and MHco3/10.BC4 (4 and MHco3/10.BC4 populations are distinct from the MHco4(WRS) and MHco10(CAVR) strains respectively and cluster with the MHco3(ISE) strain (th generation backcross strains is similar to that of the MHco3(ISE) parental strain.Both the BCo3/4.BC4  and 8 lo3/10.BC4 ). Indeed) strain . This de4 and MHco3/10.BC4 backcross populations are ivermectin resistant based on the CET, they are significantly less resistant than the original MHco4(WRS) and MHco10(CAVR) parental strains. This is not unexpected given the nature of the backcrossing scheme (see discussion below) and means the backcross strains consist of a mixed population of worms of differing ivermectin resistant phenotypes. In order to genetically characterize those worms that were phenotypically resistant to ivermectin at a dose which is 100% effective for the MHco3(ISE) isolate, we infected two sheep each with MHco3/4.BC4 and MHco3/10.BC4, treated with 0.1 mg/kg and harvested and prepared DNA from adult worms that survived this drug treatment. These worms surviving ivermectin treatment were then individually genotyped with the 9 microsatellite markers and FST and PCA analysis was performed initially with 8 loci (loci Hcms8a20 excluded) and subsequently with loci Hcms8a20 included in the analysis (9 loci) (ST\u200a=\u200a0.0379) and genetically divergent from the ivermectin resistant parental strain (8 loci FST of 0.2195 for MHco4(WRS))  parental strain (8 loci FST\u200a=\u200a0.0181) than to the MHco10(CAVR) parental strain (8 loci FST\u200a=\u200a0.3927) (Although the MHco3/4.BC(9 loci) . On the o4(WRS)) . Similar\u200a0.3927) .ST\u200a=\u200a0.2137 vs 8 loci FST\u200a=\u200a0.2195 between the MHco3/4.BC4 ivermectin survivors and MHco4(WRS) strain and 9 loci FST\u200a=\u200a0.3647 vs 8 loci FST\u200a=\u200a0.3927 between MHco3/10.BC4 ivermectin survivors and MHco10(CAVR) (ST\u200a=\u200a0.0487 vs 8 loci FST\u200a=\u200a0.0379 between the MHco3/4.BC4 ivermectin survivors and MHco3(ISE) and 9 loci FST\u200a=\u200a0.1129 vs 8 loci FST\u200a=\u200a0.0181 between the MHco3/10.BC4 ivermectin survivors and MHco3(ISE)  ). ConverST analysis, examination of the allele frequency data derived from the single worm genotyping revealed that for eight of the nine markers, the allele frequency histograms of the ivermectin treatment survivors of strains MHco3/4.BC4 and MHco3/10.BC4 were very similar to the MHco3(ISE) susceptible parental strain and divergent from the resistant parental strains MHc04(WRS) and MHco10(CAVR) respectively  susceptible strain. This latter strain is susceptible to the main classes of anthelmintics and is currently being used as the reference strain for the H. contortus genome sequencing project (http://www.sanger.ac.uk/resources/downloads/helminths/haemonchus-contortus.html). The introgression of resistance genes into this strain was achieved by repeated backcrossing of the MHco4(WRS) and MHco10(CAVR) strains against the MHco3(ISE) strain with the application of ivermectin selection at each backcross. A therapeutic dose of 0.1 mg/kg ivermectin was chosen as an appropriate discriminatory dose for selection because it is 100% effective against the parental MHco3(ISE) strain . This was confirmed by our controlled efficacy test; not a single worm of the MHco3(ISE) strain could be found surviving treatment at this dose rate in any of the five treated sheep  parental strain and highly differentiated from the MHco4(WRS) and MHco10(CAVR) resistant parental strains  and MHco4(WRS) parental strains and hence harbouring resistance conferring loci. Such analyses could include genome-wide polymorphism analysis, RNAseq analysis (to examine expression profiles and coding-region polymorphisms) or targeted analysis of candidate genes. It is important to note that it is likely that the introgressed regions of the MHco4(WRS) and MHco10(CAVR) are relatively large since just four generations of backcrossing have been performed and recombination will have had limited opportunity to break down genetic linkage. Nevertheless analysis of these strains should provide the locations of major ivermectin resistance loci in the H. contortus genome. Further backcrossing, together with improving genomic resources for this parasite, will provide the opportunity to iteratively interrogate these strains to identify the genomic location of resistance loci more accurately.The important point is that backcross strains contain a proportion of individuals that are phenotypically resistant to ivermectin (unlike the MHco3(ISE) parental susceptible strain). The observation that some worms in the backcross strains survive treatment at this dose demonstrates that the resistance-conferring alleles have been successfully introgressed from the parental ivermectin resistant isolates MHco4(WRS) and MHco10(CAVR). Importantly, when individual worms from the MHco3/4.BC strains . This deth generation backcross progeny for both back crosses. This was marker Hcms8a20. Furthermore, when the FST and PCA analysis were performed using the nine most discriminatory markers, each single marker was iteratively excluded to check for distortions of the data due to any effects of single markers. The only marker whose exclusion had any discordant effect on the data was Hcms8a20 (Data not shown). The exclusion of the loci Hcms8a20 from the FST and PCA analysis revealed that MHco3/4.BC4 and MHco3/10.BC4 ivermectin resistant worms (survivors), the susceptible MHco3(ISE) strain and their respective backcross strains were all genetically indistinguishable. The inclusion of the loci Hcms8a20 into the same analysis increased the level of genetic differentiation between these aforementioned strains of worms to the point of statistical significance. Examination of the individual allele frequencies for this marker confirms that the allelic profile was more similar to the resistant parental strains than the MHco3(ISE) parental strain . However, these are present at much higher frequencies in the populations of backcross worms that survive 0.1 mg/ml ivermectin treatment . It is impossible to predict the precise changes in allele frequency one would expect at a single locus during the backcrossing procedure, or following drug selection, when several loci may have differing additive contributions to the overall resistance phenotype. However, the fact that the same locus, Hcms8a20, shows evidence of retention of alleles specific for the parental resistant isolates in both fourth generation backcross strains, together with the dramatic increase in frequency of these in the phenotypically ivermectin resistant worms (relative to the unselected backcross populations) provides strong evidence that this locus is linked to a resistance conferring polymorphism. The fact that different alleles appear to be selected from the two different parental resistant strains is not necessarily surprising. These two strains \u2013 MHco4(WRS) and MHco10(CAVR) - are genetically divergent and originally derived from disparate geographical regions. Consequently, it is entirely possible that a resistance-conferring polymorphism would be genetically linked to different haplotypes of adjacent markers. As the H. contortus genome project progresses it will be interesting to \u201cwalk out\u201d from the Hcms8a20 marker to examine additional linked markers to define the size of the region showing evidence of linkage disequilibrium. Furthermore, we hypothesize that additional loci contribute to the ivermectin resistant phenotype of the MHco4(WRS) and MHco10(CAVR) parental strains. We anticipate that these may be identified as we iteratively interrogate the backcross strains with larger marker panels as they become available form the H. contortus genome sequencing project. Similarly, the backcross strains now represent a powerful genetic resource with which to determine if the various candidate genes identified from other studies contribute to the ivermectin resistance phenotype of the MHco4(WRS) or the MHco10(CAVR) strains.Of the 18 microsatellite markers that were used to monitor the backcrossing procedure, based on the presence and absence of strain-specific alleles, there was only one in which alleles specific to the parental resistance strains were retained in the 4l strain . Indeed,H. contortus. This is a novel approach that provides a powerful adjunct to both candidate gene and whole genome analysis aimed at identifying anthelmintic drug resistance loci. The continued advancement of such genetic approaches, alongside genomic resources for H. contortus, should allow this organisms to be used in an increasingly powerful manner to study the genetic basis of anthelmintic resistance in strongylid nematode parasites.In summary, we describe the introgression of resistance-conferring loci from two independent ivermectin resistant strains into a susceptible reference strain of Figure S1Total worm burden of lambs. Total worm burden of lambs following therapeutic dose (0.1 mg/kg and 0.2 mg/kg) of ivermectin against the three parental isolates and the backcross isolates. Mean worm burden per treatment group indicated by trend lines.(EPS)Click here for additional data file.Figure S2Allele frequency histograms of loci HcmsX256 for H. contortus parental and backcross strains. Allele frequencies resulting from individual worm genotyping of populations of 30 single worms confirm the presence of the MHco10(CAVR)-specific allele, 243 bp at the very low level of 4.6% in the MHco3/10.BC4 strain.(EPS)Click here for additional data file.Figure S3Allele frequency histograms of eight loci for H. contortus parental and backcross strains. Allele frequencies of the microsatellite loci, Hcms27 (A), Hcms36 (B), Hcms40 (C), Hc53265 (D), Hcmc22c03 (E), Hc22193 (F), Hcms3086A (G) and Hc44104 (H) for parental strains , the two 4th generation backcross strains  and 4th backcross generation survivors of ivermectin treatment (0.1 mg/kg ivermectin) .(EPS)Click here for additional data file.Table S1Summary information for the five new H. contortus microsatellite markers. Sequence of repeat and primers used to amplify microsatellite loci.(DOC)Click here for additional data file.Table S2Treatment efficacies based on worm burden. Arithmetic mean (\u00b1SEM) and range of H. contortus counts, sex differentiation of worm burdens and percentage efficacies.(DOC)Click here for additional data file.Table S3Treatment efficacies based on faecal egg count reduction. Arithmetic mean (\u00b1SEM) and range of faecal egg count and percentage efficacy seven days post-treatment.(DOC)Click here for additional data file.Table S4Genetic profiles of backcrossed strains derived from MHco4(WRS) monitored by bulk worm microsatellite \u201cfinger-printing\u201d. Alleles present in the bulk worm preparations of the parental (MHco3(ISE) and MHco4(WRS)), F1 and backcross strains.(XLS)Click here for additional data file.Table S5Genetic profiles of backcrossed strains derived from MHco10(CAVR) monitored by bulk worm microsatellite \u201cfinger-printing\u201d. Alleles present in the bulk worm preparations of the parental (MHco3(ISE) and MHco10(CAVR)), F1 and backcross strains.(XLS)Click here for additional data file."}
+{"text": "Autosomal dominant FMF due to deletion of residue 694 was first described in 3 British families in 2000. It has subsequently been reported in other patients of Northern European ancestry but the phenotype has not been fully characterised.To describe the presenting symptoms, complications and treatment responses of familial Mediterranean fever due to deletion M694.We sought patients with a genetic finding of deletion M694 from our database and reviewed their case records.A total of 19 patients (11 M:8 F) had been found to carry the Del M694 variant. Clinical details were available on 16 patients who had been assessed at our centre. 13 were of white British ancestry, the other 3 were of Irish ancestry. 2 patients gave no relevant family history, 1 was adopted and unaware of any family details, 10 patients (from 5 kindreds) gave a history of similar symptoms in at least 1 relative and the final patient reported that his mother died of renal failure of unknown cause raising the possibility of AA amyloidosis although without suggestive symptoms. 13 patients had symptoms: median age at onset 18 years (range 6-48), median age at diagnosis 48.1 years, median attack duration 2.5 days with a median of 1 attack per month, all described fever with peritonitic abdominal pain, pleuritic symptoms occurred in 4 cases, and erysipelas like erythema rash in 2. 3 patients had an appendectomy and 2 cholecystectomy prior to diagnosis. 5 of 7 symptomatic women reported attacks with menstruation and 3 had partial remissions with oral contraception. 3 patients had presented with AA amyloidosis of unrecognised aetiology of whom only 1 was truly asymptomatic. 2 children detected as part of family screening also denied any symptoms. 11 patients are on colchicine with good clinical and inflammatory responses, median age at starting treatment was 50 years, 2 felt that oral contraceptives provided adequate symptom relief, 1 declined treatment.Familial Mediterranean fever associated with deletion M694 is an autosomal dominant condition in Northern European Caucasians with variable penetrance. Disease onset appears slightly later but symptoms and colchicine responsiveness are very similar to classical autosomal recessive FMF. The high rate of AA amyloidosis (19%) may reflect the very delayed diagnosis and late initiation of colchicine treatment.None declared"}
+{"text": "CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan.Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin,  Complex organisms depend on populations of non-dividing quiescent cells for their controlled growth, development and tissue renewal. These quiescent cells are maintained in a resting state, and divide only when stimulated to do so. Unscheduled exit or failure to enter this quiescent state results in uncontrolled proliferation and cancer. Yeast cells also enter a stable, protected and reversible quiescent state. As with higher cells, they exit the cell cycle from G1, reduce growth, conserve and recycle cellular contents. These similarities, and the fact that the mechanisms that start and stop the cell cycle are fundamentally conserved lead us to think that understanding how yeast enter, maintain and reverse quiescence could give important leads into the same processes in complex organisms. We show that yeast cells maintain G1 arrest by expressing a transcription factor that represses conserved activators (cyclins) and hundreds of other genes that are important for cell division and cell growth. Failure to repress some or all of these targets leads to extra cell divisions, prevents reversible arrest and shortens life span. Many Xbp1 targets are conserved cell cycle regulators and may also be actively repressed in the quiescent cells of more complex organisms. Budding yeast that are grown in rich glucose-containing media and are allowed to naturally exhaust their carbon source undergo a series of changes that enable a significant fraction of the cells, primarily daughter cells, to enter a protective quiescent (Q) state CLN3 is transcribed at the M/G1 border CLN1 and CLN2 cyclins and other genes that trigger budding and DNA replication An important characteristic of all quiescent cells is that they arrest their cell cycle in G1. This requires the G1 to S transition to be stably halted by a mechanism that can be readily reversed when conditions permit. In cycling cells, progression through G1 into the next S phase involves two consecutive waves of G1 cyclin (Cln) expression. UBI4 promoter, prevents G1 arrest and causes loss of viability CLN3 genes Cln3/Cdk activity is rate limiting for the G1 to S transition during exponential growth. Excess Cln3 results in shorter G1 phases and smaller cells, while loss of Cln3 function prolongs G1 and results in larger cells CLN3 is a critical target of repression for G1 arrest and for the transition to quiescence. Rad53 checkpoint activity reinforces this arrest in wild type cells and becomes essential when Cln3 is overproduced. Xbp1 is also important for maintaining G1 arrest. Xbp1 is a repressor of CLN3 transcription XBP1 transcript is induced and it is among the most abundant transcripts in Q cells. Xbp1 binds and represses hundreds of genes, including CLN3 during the post-DS phase of growth. In the absence of Xbp1, cells undergo extra post-DS cell divisions and produce very small cells. These phenotypes are Cln3-dependent. xbp1 mutant Q cells are also defective in the maintenance of and recovery from the Q state. xbp1 Q cells maintain viability, but lose the ability to re-enter the cell cycle. Using Next Generation Sequencing In this work, we demonstrate that G1 arrest is initiated before the diauxic shift (DS), which is when all the glucose has been scavenged from the media. 600) of about 24, but the cell number only doubles once after the DS, which occurs between the 12 and 14 hour time points. We have monitored the DNA content of these cells to determine what fraction of cells are in G1, S and G2/M over this time course. Interestingly, the 12 to 14 hour interval shows the sharpest increase in the percentage of cells in G1. This indicates that the signal to slow proliferation is occurring at or before the DS and cells respond by extending or arresting in G1. Yeast cells spend most of their time in a non-dividing state triggered by nutrient depletion from their environment. Under the conditions we employ see , yeast uCLN3 gene (5XCLN3). This strategy maintains all the regulatory features of the wild type CLN3 gene, while it increases the Cln3 expression level. We first verified that the 5XCLN3 construct produces about five-fold higher levels of CLN3 mRNA than wild type as cells grow from log to stationary phase  By day five, 43% of these cells contained ROS and 31% showed DNA fragmentation, as detected by TUNEL staining  and DNA fragmentation has been associated with DNA damage and replication stress in yeast and metazoan cells staining . ROS wasday five . This in5XCLN3 with rad9, which is a DNA damage-specific checkpoint protein 5XCLN3 showed no toxicity in the absence of Rad9  and continues for 48 hours. It is also present at very high levels in Q cells purified from a seven day old culture. In fact, XBP1 ranks within the top 100 most abundant transcripts in Q cells. CLN3 mRNA levels over this same time course in wild type and xbp1 cells. The initial pre-DS drop in CLN3 mRNA still occurs, but we see a two to three-fold de-repression of CLN3 from 14 to 48 hours in the absence of Xbp1. It then drops to a very low level in Q cells, and that drop is also Xbp1-independent. This pattern suggests that there may be three distinct mechanisms for establishing and maintaining CLN3 repression and that Xbp1 plays a role in maintaining CLN3 repression during post-diauxic growth.Repression of 21 alone . This suCLN3 levels still occurs in xbp1 cells indicates that the initial signaling to slow proliferation is intact. xbp1 cells in G1 is very similar to wild type for the first 18 hours. Direct comparison of the FACS profiles of wild type  also display metabolic oscillations, and peak out of phase with Xbp1. Using microarray and motif search tools PIS1, DOG2, and CDC10 are bound in vivo by Xbp1 after the DS, just like CLN3 , which we also verified to be in vivo binding sites for Xbp1 by chromatin immunoprecipitation  are not significantly affected by the absence of Xbp1 during log phase (8 hours). A few transcripts begin to rise in the xbp1 cells at the DS (14 hours), and this trend continues throughout the time course and in purified Q cells. Very few direct targets are down-regulated. This is consistent with our previous findings that Xbp1 functions as a repressor To show that the repression of these transcripts is Xbp1-mediated and to identify other targets, we used our Next-Generation RNA sequencing se. CLN3 , and allXbp1 expression is induced at 14 hours and remains high across this time course , but botCLN3 and CLN1) that drive the G1 to S transition SWI6NDD1MSS11 and MGA1IME1To look more closely at all Xbp1-mediated repression, we identified transcripts that are derepressed three-fold or greater at each time point . SignifiCLB4, CLB2PCL9After 24 hours of growth, 65 known genes are derepressed in the absence of Xbp1. At this time point, cell wall proteins are highly enriched. These include most of the daughter-specific genes By 48 hours, nearly 10% of all genes (515) are derepressed in the absence of Xbp1. At this time point almost half of the known targeted genes are involved in metabolism and the other large class is involved in cell wall biogenesis. 45 cell cycle genes and 25 transcription regulators are also derepressed at this time point. Only one-third of these derepressed genes are also derepressed in Q cells. Xbp1 affects a more diverse group of genes in purified Q cells. Metabolic genes are the largest class. In addition, 42 genes involved in transmembrane transport, including five glucose transporters are repressed by Xbp1 in Q cells.We also analyzed direct and indirect targets separately. What is striking is that direct and indirect targets are largely in the same pathways. At 18 and 24 hours, mitosis, cell cycle, cell division and cytokinesis are significantly enriched classes in both direct and indirect targets  and nitrogen metabolism . In Q cells, they are highly enriched for ribosomal proteins  and genes involved in monosaccharide catabolism . The ribosome biogenesis and ribosomal protein transcripts are tightly and coordinately regulated in response to nutrient conditions xbp1 cells at 48 hours versus purified Q cells suggests that these are fundamentally different states.The dot plots of In rich glucose-containing medium, yeast cells cease growth and division after about 48 hours due to carbon limitation. The resulting culture is a heterogeneous population of live and dead cells. Most of the daughter cells enter a quiescent state and can be purified due to their increased density CLN3 gene, interferes with Q cell formation, and cells lacking Cln3 produce more Q cells. Cells transitioning to quiescence with excess Cln3 accumulate in G1 more slowly than wild type cells, but they eventually arrest and remain viable due to the activation of the checkpoint kinase Rad53.We are investigating the events that differentiate Q cells from nonQ cells and promote their longevity 5XCLN3 cells, so we conclude that replicative stress, not DNA damage, triggers the checkpoint during the transition to quiescence. We detect delayed G1 arrest and increased ROS accumulation as nutrients become limiting, even in wild type cells carrying rad53-21. This suggests that replication stress occurs and this checkpoint pathway plays a role in restricting cell cycle progression during the wild type transition to quiescence. With excess Cln3, checkpoint function becomes essential and cells lacking it fail to arrest in G1 and undergo apoptosis. Related effects have been observed with excess cyclin E, and other activated oncogenes in higher cells Rad53 is an effector of the DNA damage and replication stress checkpoints CLN3 repression is mechanistically different before and after the DS, and that only its post-DS repression is Xbp1-dependent. The initial drop in CLN3 levels and the halt to S phase that we observe at the DS are Xbp1-independent. Only after the DS, the CLN3 promoter is bound and repressed by Xbp1. Cells lacking Xbp1 resume DNA replication and continue to divide after the DS, and this results in a significant population of very small cells. These phenotypes are Cln3-dependent. These data are consistent with Xbp1 playing a role in maintaining repression of CLN3 and G1 arrest as cells transition from growth to quiescence. However, unlike 5XCLN3, xbp1 mutants are not dependent on the Rad53 replication stress checkpoint for viability. We suspect that either the timing or the extent of derepression of CLN3 by xbp1 could explain the Rad53-independence of these cells. A third possibility is that Rad53 acts in the same pathway and upstream of Xbp1 to restrict cell cycle progression. Rad53 has been shown to increase the level of Xbp1 in response to DNA damage Our data indicate that in vivo binding sites for Xbp1. Binding is only detected after the DS, which explains why these targets were not identified in previous studies. None of the Xbp1 targets identified by genome-wide location analysis xbp1 deletion Xbp1 mutant Q cells remain viable, but they are profoundly delayed in cell cycle re-entry upon re-feeding. They are also short-lived as Q cells, entering an irreversible, senescent state more rapidly than wild type. We have not identified the genes responsible for these phenotypes because our data show that Xbp1 plays a global and continuous repressive role in cells as they transition from a dividing to a non-dividing quiescent state. We have identified 520 targets of Xbp1-mediated repression that contain Xbp1 binding sites in their promoters. All seven that we tested are direct XBP1 mRNA oscillates dramatically in cells that are undergoing yeast metabolic and cell cycle (YMC) oscillations XBP1. YMC oscillations are achieved by growing the cells to maximum density, starving them for glucose, then restoring a limited amount of glucose, which is immediately imported and cannot be detected in the media XBP1. Cell division stops and storage carbohydrates accumulate. This is the quiescence-like phase of the YMC XBP1 is turned off, and it's targets peak. Then, DNA replication takes place and cells divide. The striking parallels between the events associated with the transitions in and out of quiescence, and those associated with the YMC suggest that YMC oscillations may be the result of switching on and off the signal to arrest in G1 and enter quiescence. It also seems likely that the oscillation of XBP1 expression is responsible for the subsequent YMC oscillations of its many targets. One such verified target, CLN3, and many other cell cycle regulated transcripts have been shown to have different peak time or multiple peaks in the synchronized cell cycles induced by the YMC protocol CLN3 and 800 other transcripts is also oscillating during the YMC time course may explain some of those altered peak times.Xbp1 negatively regulates the mRNA levels of 15% of yeast genes during post-diauxic growth. When Xbp1 was ectopically expressed during logarithmic growth, only a small number of Xbp1 targets were identified xbp1 early in the transition to quiescence are largely cell cycle and growth regulators. At 18 hours, even the gene products associated with the cell periphery are largely sensors and regulators, rather then structural proteins. This is true of both direct and indirect targets. One possible explanation is that xbp1 mutant cells continue to divide during this interval. About 20% of Xbp1 targets are cell cycle regulated at the transcript level (data not shown). These transcripts are not made when cells stop dividing, so anything that promotes ectopic cell division would increase the transcription of these genes. However, only one-third of the 600 most cell cycle regulated transcripts It is striking that transcripts derepressed by xbp1 mutant. These were prominent only in the last two time points and they do not show any enrichment for Xbp1 binding sites. This supports the view that Xbp1 functions primarily, if not solely, as a repressor. We expect that the reduced levels of these transcripts are an indirect effect of the many perturbations that arise in xbp1 cells where 15% of genes are expressed at a time when they should be off.We also looked for transcripts that were under-represented in the XBP1 mRNA is among the top 1% highest level transcripts in Q cells. Xbp1 has also been shown to be translationally up-regulated in response to both glucose and amino acid starvation xbp1 mutant Q cells demonstrate the importance of this repression. Xbp1 shares homology within its DNA binding domain with four other S. cerevisiae transcription factors that specify cell fate. Swi4 and Mbp1 associate with Swi6 and serve as activators of mitotic growth Candida family members, Efg1, is critical for biofilm formation, which renders these pathogens drug-resistant PMT1, 2, and 4ECM33SMI1 and FKS1CLN3 were generated by integrating additional copies of CLN3 at four different marker loci using the integrating vectors, pRS303-306 rad53-21 mutant  was crossed into the W303 background above to generate BY6741 and subsequently crossed with the 5XCLN3 strain to generate BY6698. CLN3, XBP1 and RAD9 were deleted with KanMX as described The yeast strains were all derived from W303. The auxotrophic markers were corrected in all strains. The strains carrying five copies of 600) of 0.02 and allowed to grow at 30\u00b0C, shaking at 200 RPM. The diauxic shift was defined as the point at which no glucose was detected in the media, which was determined with glucose detection strips . Quiescent (Q) cells were purified from YEPD cultures that were seven days old using a 25 ml percoll density gradient 600 units loaded that sediment to the bottom nine ml of the gradient. Cell size and cell count was measured on a Z2 Beckman Coulter Counter. All time course data was collected in duplicate or triplicate, averaged and error bars are shown.Reproducible growth curves were obtained by patching cells from fresh plates onto YEP plus 2% glycerol and growing them overnight to eliminate petites. This patch was used to inoculate 5 ml YEPD, then a further 1/50 dilution was made and grown overnight. This culture was used to inoculate 25 ml YEPD in a 250 ml flask to an optical density (ODCell viability was monitored using the FungaLight Yeast Viability Kit (Molecular Probes) according to the manufacturer's protocol and the percentage of live cells was plotted over time. Reproductive capacity was assayed as the ability to resume cell division and produce colonies. Serial dilutions were plated on YEPD plates in duplicate and the percentage of colony forming units (CFU) was plotted, using the CFU from the freshly harvested Q cell sample as 100%. The FungaLight and CFU viability data are averages from at least two independent experiments.7 cells were collected and mixed with Calcofluor white M2R  at a final concentration of 100 \u00b5g/ml. Cells were incubated at room temperature for 15 min in the dark then were washed twice with H2O. The stained cells were examined with a Nikon Eclipse E600 microscope with a Nikon Plan Apochromat 60XA/1.40 oil immersion objective and a UV-2E/C DAPI filter (excitation at 330\u2013380 nm). Photomicrographs of cells were taken on a Photometrics Cascade 512B camera and analyzed with MetaMorph version 6.3r2 software . TUNEL Assay: Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, spun down at 5000 rpm for 5 min and washed once with 0.1 M potassium phosphate 1.2 M sorbitol buffer pH 7.5. Stationary phase cells were first resuspended in 100\u2013200 \u00b5l fresh pretreatment buffer , and then pelleted in a microfuge at 2000 rpm for 3 min at room temperature. These cells were resuspended in 1 M sorbitol and pelleted as before. Cell walls were digested with 50 \u00b5g/ml Zymolyase 100T in 1 M Sorbitol buffer (pH 5.8) for 10\u201345 min at 30\u00b0C. Cells were pelleted at 2000 rpm for 3 min, gently washed and resuspended in 15 \u00b5l potassium phosphate/sorbitol buffer, transferred to 0.1% polylysine-coated wells of an eight well microscope slide and allowed to settle for 20 min at room temperature. The slide was washed twice with phosphate-buffered saline (PBS). Each well was incubated with 40 \u00b5l fresh permeabilization solution (0.1% Triton X-100 in a 0.1% sodium citrate solution) for 2 min on ice, then rinsed with PBS buffer. 15 \u00b5l TUNEL reaction mixture  was added to each well, slides were covered and incubated for 60 min at 37\u00b0C, then rinsed twice with PBS. Cells were observed under the microscope with an FITC filter (excitation at 460\u2013500 nm). 100\u2013200 cells per sample were evaluated.Calcofluor staining of bud scars: Approximately 10For flow cytometry, cells were fixed in 70% ethanol for two hours or overnight, washed once with water, then resuspended in .5 ml 50 mM Tris-HCl (pH 8.0) containing 0.2 mg/ml RNAse A and incubated at 37\u00b0C for four hours. These cells were spun down, resuspended in .5 ml 50 mM Tris-HCl (pH 7.5) containing 2 mg/ml Proteinase K, and incubated at 50\u00b0C for one hour. They were then spun down again and resuspended in .5 ml 50 mM Tris-HCl (pH 7.5) and stored at 4\u00b0C. Before analysis, they were sonicated, pelleted, and resuspended in .5 ml I.0 \u00b5M Sytox Green (Invitrogen). Percent of cells in G1, S or G2/M phase of the cell cycle were quantified with FlowJo V9.6 cells were pelleted, gently washed with PBS, then resuspended in 1 mL PBS. 2.5 \u00b5L of a 10 mM carboxy-H2DCFDA (Invitrogen) stock solution was added and the cells were incubated for 30 minutes at 37\u00b0C. Cells were washed twice with PBS, resuspended in 1 mL 50 mM Tris-HCl pH 7.5. Cultures were then sonicated and 30,000 cells per sample were collected on a Fluorescence Activated Cell Sorter FACScan cytometer  and analyzed using Cell Quest software. FACS parameters were set at excitation and emission settings of 495 nm and 529 nm (filter FL-1), respectively. Average from two experiments is reported.For ROS assays, approximately 1\u00d710600 of cells were collected every 10 minutes, washed with RNA buffer  and frozen for later RNA purification. The levels of CLN3 and ACT1 mRNA were monitored by an S1 nuclease protection assay as previously described CLN3 and ACT1 transcript levels were measured in each sample of wild type and 5XCLN3 cells. The ACT1, though not invariant, was not affected by excess CLN3 so it could be used to normalize the RNA levels between the two strains.To generate enough cells for RNA measurements during growth from log phase to stationary phase, 5 ODNext-Generation RNA sequencing was carried out with RNA prepared as above from log phase cells, purified Q cells, and cells grown in YEPD to log phase (8 hours) the DS (14 hours), then 18, 24 and 48 hours. mRNA expression levels following polyA selection were assayed using the HiSeq 2000 next generation sequencing system from Illumina Saccharomyces cerevisiae W303 reference genome using the Tophat application, a fast splice junction mapper for RNA-Seq reads The W303 reference genome in FASTA format and gene annotations in GFF were obtained from the Wellcome Trust Sanger Institute's SGRP group. Sequences from each read were mapped to the in vivo binding sites. PCR primers used to amplify potential targets as well as an unregulated DNA (IRV) are provided as Supplementary Cells carrying Xbp1 tagged with a Tandem Affinity Purification (TAP) tag Figure S1XBP1 (green), and three of its direct targets: PIS1 (blue), CLN3 (red) and DOG2 (purple), reproduced from the Periodic Transcript Server CLN3, DOG2 and PIS1 in the metabolic oscillation data set XBP1 transcript oscillates out of phase with its targets in cells synchronized with limiting glucose. (A) Metabolic oscillations of mRNA for (TIF)Click here for additional data file.Table S1xbp1. Open reading frame number and classification, gene name and gene description are provided. TRUE indicates genes with Xbp1 binding sites within 800 base pairs of translational start sites. FALSE indicates indirect targets.All transcripts derepressed three-fold or more by (XLSX)Click here for additional data file.Table S2Xbp1 repressed genes after different intervals of growth. (A) Targets containing Xbp1 binding sites or (B) that lack Xbp1 binding sites that are listed based on when they are derepressed, that is after 18, 24, 48 hours of growth or in purified Q cells. Genes are organized by gene ontology (GO) process. Corrected P value and the number of genes in each class are shown in parentheses.(DOC)Click here for additional data file.Table S3xbp1 and wild type cells are normalized and provided as a log base 2 value for xbp1/wild type. Rank is provided as an indicator of the likelihood that the genes are differentially expressed in the two strains at each time.Next Generation RNA sequence data for Xbp1 targets. Data for all transcripts derepressed three-fold or more at one or more time point are provided. Open reading frame number and classification, and gene name are provided. TRUE indicates genes with Xbp1 binding sites within 800 base pairs of translational start sites. FALSE indicates indirect targets. For each target gene at each time point, the data for (XLSX)Click here for additional data file.Table S4PCR primers used in chromatin immunoprecipitations.(DOCX)Click here for additional data file."}
+{"text": "Human parvovirus 4 has been considered to be transmitted only parenterally. However, after novel genotype 3 of parvovirus 4 was found in 2 patients with no parenteral risks, we tested infants in Ghana. A viremia rate of 8.6% over 2 years indicates that this infection is common in children in Africa. Parvovirinae.In 2005, a novel human parvovirus, termed parvovirus 4 (PARV4), was identified in a plasma sample from a patient with symptoms resembling those of acute HIV infection  or HIV   and who did not live close to a river  of 279 samples were positive for PARV4 genotype 3 DNA. At the time of blood collection, no infant had signs of acute infection . Positive samples were found for infants in 7 of 9 studied villages, indicating widespread prevalence. To test whether socioeconomic factors might influence prevalence, we conducted a \u03c7 a river .Two age groups were randomly selected from the cohort: 1) 94 infants with a median age of 14.9 months  and 2) 185 infants with a median age of 24.0 months . Significantly more positive results were seen among infants in the older versus the younger age group . Viral loads in the whole study group ranged from 420 to 56,000 copies/mL of whole blood . Median viral loads did not differ significantly between the age groups .A 746-nt fragment of open reading frame (ORF) 1 and a 558-nt fragment of the ORF2 gene and the noncoding region between them were sequenced. Maximal nucleotide distances from previously known PARV4 sequences were 7%\u20138%. To obtain highly informative sequence datasets for phylogenetic analysis, we concatenated ORF1 and 2 sequences and excluded interfragment recombination by using SimPlot and GARD analysis with the HYPHY package . The novAlthough infection with human parvovirus PARV4 has been considered to be restricted to adults and transmitted parenterally, we found high prevalence of PARV4 genotype 3 in blood of infants in Africa. In agreement with findings for the 2 adults with PARV4 genotype 3 infection  are thus suspected. In conclusion, in Africa, novel PARV4 genotype 3 is prevalent among infants who are most likely not at risk for parenteral exposure."}
+{"text": "The legend for Table 6 should read: \"Table 6. Identified 43 biomarkers of the 99 biomarkers that correlated with height adjusted TKV.\"There were errors in Table 6 and Supplementary Table 2. Correct versions of these tables are available below:Table 6: Supplementary Table 2: Click here for additional data file."}
+{"text": "In the present study, we examined the influence of a histamine H1 receptor antagonist, fexofenadine hydrochloride (FEX) on CC10 production in vitro and in vivo.Uteroglobin (CC10) is well known to be an immuno-suppressive protein secreted from airway epithelial cells after inflammatory stimulation and function in development of allergic disorders. Although histamine H6 cells/ml) were stimulated with 20 ng/ml TNF-\u03b1 in the presence of various concentrations of FEX for 24 h. CC10 levels in culture supernatants were examined by ELISA. Patients with Japanese cedar pollinosis were orally treated with FEX twice a day at a single dose of 60 mg for two weeks during Japanese cedar pollen season (February 2011 to April 2011). CC10 levels in nasal secretions were also examined by ELISA.Nasal epithelial cells (5 x 10The addition of FEX into epithelial cell cultures caused dose-dependent increase in the ability of cells to produce CC10 in response to TNF-\u03b1 stimulation, and the minimum concentration that caused significant increase was 200 ng/ml. Oral administration of FEX for two weeks also increased CC10 levels in nasal secretions from pollinosis patients along with attenuation of clinical symptoms.The ability of FEX to enhance CC10 production may account, at least in part, for the clinical efficacy of the agent on allergic disorders, including allergic rhinitis"}
+{"text": "Lhx2 promoter to regulate Cre recombinase expression in transgenic mice we have been able to define a distinct progenitor cell population in the forebrain solely committed to eye development. Conditional inactivation of Lhx2 in these progenitor cells causes an arrest in eye development at the stage when the optic vesicle induces lens placode formation in the surface ectoderm. The eye-committed progenitor cell population is present in the \u2212/\u2212Lhx2 embryonic forebrain suggesting that commitment to eye development is Lhx2-independent. However, re-expression of Lhx2 in \u2212/\u2212Lhx2 progenitor cells only promotes development of retinal pigment epithelium cells, indicating that Lhx2 promotes the acquisition of the oligopotent fate of these progenitor cells. This approach also allowed us to identify genes that distinguish Lhx2 function in eye development from that in the forebrain. Thus, we have defined a distinct progenitor cell population in the forebrain committed to eye development and identified genes linked to Lhx2's function in the expansion and patterning of these progenitor cells.Progenitor cells committed to eye development become specified in the prospective forebrain and develop subsequently into the optic vesicle and the optic cup. The optic vesicle induces formation of the lens placode in surface ectoderm from which the lens develops. Numerous transcription factors are involved in this process, including the eye-field transcription factors. However, many of these transcription factors also regulate the patterning of the anterior neural plate and their specific role in eye development is difficult to discern since eye-committed progenitor cells are poorly defined. By using a specific part of the  The vertebrate eye is a complex and highly specialised neurosensory organ that converts light (photons) into electro-chemical pulses that the brain can translate into images. The development of the eye proceeds through co-ordinated interactions between tissues of different embryonic origin. Immediately after initiation of gastrulation, the eye field is specified in the anterior neural plate The development of the eye is regulated by a number of signalling pathways active at different time points during morphogenesis Lhx2, Pax6, Rx, Tlx, Six6, Six3, and ETMitf whereas cells in close proximity to the lens placode, receiving Fgf signalling, start to express Vsx2 . Vsx2 represses Mitf expression which specifies the neural retina Mitf expression specifying the RPE cells Pax2Sox2, Pax6 and Six3These patterning morphogens impose their actions by activating a cascade of transcription factors that establish cellular identity and subsequent interactions with the environment. The expression of a combination of transcription factors in cells in the anterior neural plate defines the eye field. These transcription factors are collectively referred to as the eye field transcription factors and include Lhx2 has been shown to be important in the development of the eyes since eye development is arrested at the optic vesicle stage prior to lens placode induction in \u2212/\u2212Lhx2 mouse embryos Lhx2 in eye development has also been revealed by the observation that over-expression of various combination of eye field transcription factors other than Lhx2 only induce ectopic eyes when endogenous Lhx2 expression is induced Lhx2 is expressed in the anterior neural plate prior to the formation of the optic vesicle and the Lhx2 null embryos have severe defects in other forebrain structures, such as the cerebral cortex and the hippocampus, revealing that Lhx2 is also involved in the patterning of the forebrain Importantly, many of the eye field transcription factors have additional function(s) in the forebrain neuroectoderm as revealed by defective forebrain development in the respective homozygous mutant mouse embryos Lhx2 function(s) at various stages of eye development it is necessary to distinguish its function prior to and after this commitment step. We therefore developed a novel Cre transgenic mouse strain, denoted Lhx2-Cre, where Cre expression was regulated by an 11 kb genomic region of the Lhx2 promoter immediately upstream of the transcriptional start. Cre expression was not detected in all Lhx2 expressing cells; it rather defined a progenitor cells in the optic pit of the prospective forebrain at embryonic day 8.25 (E8.25) committed to generate the neural part of the eye. Thus, the Lhx2-Cre mouse strain will be a very useful tool to examine the function of genes prior to and after commitment of cells during eye development and in the anterior neural plate. Conditional inactivation of Lhx2 in the Cre+ cells led to a developmental arrest just prior to formation of the optic cup and a subsequent deterioration of the optic vesicle. The optic vesicle developed further in these embryos compared with the conventional \u2212/\u2212Lhx2 embryos. Moreover, genes important for lens differentiation were induced in the surface ectoderm leading to the formation of a lens placode. However, further development of the lens placode required maintained Lhx2 expression since the lens placode regressed in mutant embryos. Cre expression was detected in the optic vesicle in \u2212/\u2212Lhx2 embryos suggesting that commitment to eye development in the neural plate is independent of Lhx2 expression. Expression of transgenic Lhx2 in the Cre+ cell in the \u2212/\u2212Lhx2 background could partly rescue eye development as only cells of the RPE layer developed in these animals. Furthermore, by analysing the expression pattern of a number of genes putatively regulated by Lhx2 in a different cellular context Lhx2 function also in eye development.Identification of the cells in the early forebrain solely committed to eye development would be an important step in examining the function of a gene prior to and after commitment to eye development. To elucidate the molecular basis for Lhx2 gene is widely expressed in the prospective forebrain including the optic vesicle/cup, neural retina, optic stalk and RPE cells Lhx2 promoter to drive expression Cre recombinase in transgenic mice. We initially used a 5 kb and an 11 kb genomic region immediately upstream of the Lhx2 transcriptional start site to drive expression of the Cre. We crossed the Lhx2-Cre transgenic mice to the ROSA26 Reporter (ROSA26R) mouse strain to generate Lhx2-Cre:ROSA26R double transgenic mice. These mice enabled the detection of expression and functional activity of Cre recombinase and therefore permitted lineage tracing of Cre expressing cells based on \u03b2-Galactosidase  activity Cre in transgenic mice (data not shown). However, when the 11 kb region was used to drive Cre expression in the double transgenic animals we reproducible observed \u03b2-Gal+ cells in the neural part of the eye , whereas very few \u03b2-Gal+ cells could be detected in the developing forebrain where the endogenous Lhx2 gene is highly expressed -Cre transgenic founder mouse strains  until E12.5  flox/floxLhx2-Cre:Lhx2 mice obtained were anophthalmic and histological sections of newborn mice revealed that all eye structures were lacking in the flox/floxLhx2-Cre:Lhx2 mice  we reproducibly noticed a few cells with an inactivated Lhx2 gene in the optic vesicle (data not shown), and at E9.5 (ss24\u201328) the Lhx2 gene has been efficiently inactivated in the entire optic vesicle  mutant since the optic vesicle in the conditional mutant comes in close contact with the surface ectoderm and appears to induce a thickening of the surface ectoderm indicative of lens placode formation    . Bmp4 exss18\u201321) . However vesicle . These rLhx2 mediated function in eye development, we tried to identify novel genes/pathways putatively linked to Lhx2 function. To achieve this we took advantage of a previous global gene expression analysis comparing Lhx2+ stem cells to their Lhx2\u2212 progeny in a different cellular context Lhx2 target genes identified in this screen had gene expression pattern in vivo in various organs that overlapped with that of Lhx2, suggesting partly overlapping mechanisms for Lhx2 function in different tissues. Two of these genes, Enc1 and Nuak1, had an expression pattern consistent with this assumption in eye development. Both were expressed in Lhx2 expressing domains, Enc1 was expressed in the prospective neural retina and lens whereas Nuak1 was expressed in the prospective forebrain, but Nuak1 was excluded from the optic cup , further supporting the idea that Fgf15 is mainly a downstream target of BMP4 signalling in the neural retina. In contrast, Nuak1 expression extended into the domain from where it was excluded in the control when Lhx2 was inactivated , where Cre-mediated recombination induces Lhx2-GFP expression  we could observe a bilaterally located small mass of pigmented cells where the eye is normally located  mice, which is a reliable indicator for functional Lhx2 expression in Z/Lhx2-GFP mice after Cre mediated recombination of the double reporter transgene GFP expression in the neural part of the eye at different developmental stages in all Lhx2-Cre:Z/Lhx2-GFP embryos analysed (n\u200a=\u200a4) . genotype , and allhthalmic . However located , suggestand Pax6 , whereasdetected , further (n\u200a=\u200a4) , suggestLhx2 promoter to regulate the expression of the Cre recombinase we have been able to identify cells in the forebrain solely committed to generate the neural part of the eye, suggesting that these Cre+ cells are the earliest cells committed to eye development. Commitment to this progenitor cell fate is independent of prior Lhx2 expression in the anterior neural plate, and we propose that Lhx2 promote the acquisition of the oligopotent state of this progenitor cell population in addition to its requirement for the subsequent differentiation of the optic vesicle into the optic cup. Eye development progresses further in the conditional mutant compared to the conventional \u2212/\u2212Lhx2 mutant mice since the optic vesicle comes in direct contact with the surface ectoderm and induces a lens placode. However, immediately after the developmental arrest of the optic vesicle in the conditional mutant, both the optic vesicle and the lens placode degenerate leading to a complete lack of these structures. These result reveal that lens development require continuous interactions between the lens placode and the optic vesicle even if the lens placode has acquired many of its molecular characteristics. We have compared the expression pattern of some genes involved in eye development in the optic vesicle and lens placode between the conditional mutant (flox/floxLhx2-Cre-Lhx2 results presented herein) and the conventional mutant  before and after commitment and that the difference is not solely due to the delayed inactivation of the Lhx2 gene in the conditional mutant. We have also identified novel genes putatively coupled to Lhx2 function during eye development that previously have been shown to be linked to Lhx2 function in another stem/progenitor cell system. This approach also allowed us to distinguish the function of Lhx2 in the forebrain to that in eye development.By using a defined part of the Lhx2 mutant has previously shown that Lhx2 is important for the optic vesicle to optic cup transformation Lhx2 is expressed in the entire prospective forebrain prior to optic vesicle formation it is difficult to discern if Lhx2 has a function in the patterning of the forebrain and hence commitment to eye development, or expansion and patterning of the optic vesicle, or both. This is also pertinent to other transcription factors suggested to have a role in eye development such as Pax6, Six3, Six6 and TlxLhx2-Cre transgenic mouse strain that defines the first progenitor cells committed to generate the neural part of the eye in the anterior neural plate it is possible to molecularly define the role of any gene in patterning/commitment to eye development and subsequent expansion/patterning of the eye committed progenitor cells. We could confirm that Lhx2 is required in the optic vesicle to optic cup transformation since development is blocked almost immediately following inactivation of Lhx2 in the optic vesicle. This observation is also in agreement with the finding that induction of eye development by ectopic expression of eye field transcription factors can only occur when endogenous Lhx2 expression is induced Pax2 expression is induced and Mitf expression is transiently induced, whereas this process is completely blocked in the \u2212/\u2212Lhx2 embryos. This observation suggests that Lhx2 is important for both establishing and maintaining the patterning of the optic vesicle. The earliest molecular consequence of inactivation of Lhx2 in the optic vesicle is the down-regulated expression of Six6, whereas expression of most other transcription factors involved in early eye development appears to be unaffected. The down-regulated expression of Six6 is in agreement with the finding that Lhx2 synergises with Pax6 to induce Six6 expression Six6 expression cannot explain the block in eye development since \u2212/\u2212Six6 mice have a relatively mild eye phenotype affecting only the late stages of eye development Lhx2 expression is not required for commitment to the eye-specific progenitor cell since Cre expression is also detected in the distal part of the optic vesicle in \u2212/\u2212Lhx2 embryos. However, expression of transgenic Lhx2 in the eye committed progenitor cells in the \u2212/\u2212Lhx2 background only promote development of RPE cells, suggesting that Lhx2 expression prior to eye commitment is important for the progenitor cell to acquire its oligopotency. Thus, Lhx2 might also regulate the establishment of a fully functional eye progenitor cell.Analysis of the conventional Pax6, which is complicated by the fact that it is expressed in cells both in the optic vesicle and the surface ectoderm/lens placode Pax6 function, at least in mice, have been obtained by performing tissue-specific inactivation of Pax6 in surface ectoderm Pax6 is required cell autonomously in the surface ectoderm for lens development. Furthermore, the developing lens is not necessary for the formation of the neural retina and RPE layer, but is rather required for the correct organisation and localisation of the neuroepithelium of the eye. Our results reveal that lens placode development is also arrested immediately after Lhx2 has been inactivated in the optic vesicle. Thus, continuous interactions between the optic vesicle/cup and the developing lens is required for lens formation although the lens placode has formed and acquired many of its molecular characteristics such as expression of Pax6, Six3 and Sox2There are several hypotheses of how optic vesicle-surface ectoderm/lens placode interactions regulate eye development. Many of these hypotheses are based on the study of Bmp4 and Bmp7 mutant mice have profound eye defects \u2212/\u2212Lhx2 embryos since neither Bmp4 nor Bmp7 are expressed and phosphorylated SMADs, the intracellular mediators of BMP signalling, are not detected in mutant optic neuroepithelium Bmp4 is initiated in the optic vesicle similar to the control animals but is rapidly down-regulated, supporting the notion that Lhx2 function is partially mediated by BMP signalling. This notion is further supported by the down-regulated expression of Fgf15, which is suggested to be a down-stream target of BMP4 signalling Bmp7 expression appears to be unaffected in the conditional mutant and hence lack of Bmp expression cannot solely explain the Lhx2 mutant phenotype. Since Bmp7 has been shown to regulate the expression of Pax2 explains why Pax2 expression is unaffected in the conditional mutant whereas it is absent in the \u2212/\u2212Lhx2 mice Bmps appear therefore to be differently regulated by Lhx2 where both Bmp4 and Bmp7 expression is initiated by Lhx2 but maintained expression of Bmp4 is Lhx2-dependent whereas maintained Bmp7 expression is Lhx2-independent. Moreover, Lhx2 re-expression in the \u2212/\u2212Lhx2 eye committed progenitor cells led to the formation of only RPE cells, which is remarkably similar to the eye phenotype in \u2212/\u2212Bmp7 mice Bmp7 nor Bmp4 is expressed in the \u2212/\u2212Lhx2 optic vesicle Lhx2 in the \u2212/\u2212Lhx2 eye progenitor cells might be partly due to suboptimal expression of the Bmps when Lhx2 expression is turned on in the optic vesicle. Alternatively, the level of transgenic Lhx2 expression might not be sufficient to induce enough BMP expression at the correct time in the optic vesicle.BMP signalling has been shown to be important for eye development since both Lhx2 function by comparing global gene expression in Lhx2+ progenitor cells to their Lhx2\u2212 progeny Lhx2 in various tissues and progenitor cell populations, suggesting that mediators of Lhx2 function partly overlap in different tissues/progenitor cell populations. In this study we identified a number of genes that also overlap with Lhx2 expression during eye development. The gene Enc1  encodes a Kelch-related protein suggested to be important in the organisation and function of the cytoskeleton Enc1 was expressed in the prospective neural retina and the lens placode, and was not detected in these tissues in the conditional Lhx2 mutant. Since Lhx2 is expressed in the neural retina but not expressed in the lens placode it suggests that Lhx2 regulate Enc1 in neural retina by a cell autonomous mechanism and in the lens placode by a cell nonautonomus mechanism. Putative mediators of the cell nonautonomous regulation of Enc1 in the lens placode remains to be elucidated, but could partly include mediators of BMP4 signalling since this signalling pathway has been linked to Lhx2 function in eye development and is important for lens development Lhx2 in eye development is further emphasised by the expression pattern of Nuak1, an adenosine monophosphate-activated protein kinase (AMPK)-related kinase  suggested to be involved in the regulation of ploidy and senescence Nuak1 is normally expressed by the neural ectoderm in the forebrain where Lhx2 is also expressed, but its expression is excluded in the optic vesicle and its derivatives, e.g. prospective neural retina, RPE cells, and optic stalk. However, in the conditional Lhx2 mutant the expression domain of Nuak1 extends into these domains of the developing eye. The most simplistic explanation for this phenotype is that Lhx2 promotes Nuak1 expression in the forebrain neural ectoderm whereas it suppresses Nuak1 expression in eye committed neural ectoderm. Thus, the combined effects of down-regulated expression of Six6, Enc1, Fgf15 and Bmp4 in the optic vesicle, misexpression of Nuak1 in the optic vesicle and down-regulated expression of Enc1 in the lens placode, might partly explain the developmental arrest and degeneration of the eye in the conditional Lhx2 mutants. Lhx2 has been suggested to regulate key determinants of both dorsal and ventral identity Lhx2 function presented here starts to explain how Lhx2 accomplishes that.We have previously identified genes putatively linked to Cre expression is regulated by the promoters of Crx, Rx, Pax6 and Six3 genes directing Cre expression to the developing eye Rx-Cre and the Six3-Cre transgenic mice also reveal Cre expression in neural tissue outside of the eye domain in the forebrain, and the Pax6-Cre and Crx-Cre mouse strains show restricted expression within the developing eye. To our knowledge the Lhx2-Cre mouse strain is the first mouse model where progenitor cells solely committed to generate the neural part of the eye can be identified in the anterior neural ectoderm. Lineage tracing of these cells revealed that they do not contribute to any other cells in the prospective forebrain. The conditional inactivation of Lhx2 in these eye committed progenitor cells also confirmed this assumption since the forebrain appears to be intact, which is in contrast to the \u2212/\u2212Lhx2 embryos that lack several forebrain structures Lhx2-Cre mouse strain will be a very useful tool to elucidate the specific role(s) of any gene in the patterning/commitment of anterior neural plate into eye committed progenitor cells, and the subsequent expansion/pattering of the optic vesicle. Moreover, the role of specific genes in the ability of the optic vesicle to communicate with surface ectoderm and hence induce and promote lens development can also be studied in detail by using the Lhx2-Cre mouse strain.Transgenic mice have previously been generated where The mice were maintained at the animal facility at Ume\u00e5 University and all experiments involving animals were approved by the local Animal Review Board .Lhx2-Cre transgenic construct was generated by using an 11 kb DNA fragment of the Lhx2 promoter, which included the first 36 bp of the Lhx2 coding sequence. The Lhx2 promoter fragment was fused in-frame with Cre recombinase cDNA and a SV40 polyadenylation signal was added. Pronuclear injection of the DNA construct generated two founder lines of which one was chosen for further studies. Generation of ROSA26R mice, \u2212/\u2212Lhx2 mice, flox/floxLhx2 mice and Z/Lhx2-GFP transgenic mice has been described previously floxLhx2 allele were: LOX 5\u2032-GCCAGACTAGCAGACGCTGC-3\u2032 and SDL2 5\u2032-CCACCGGTACTCCTCTTCAGAG-3\u2032. Primers used to identify the Z/Lhx2-GFP transgene were GFPforward 5\u2032-TTCCACCATATTGCCGTC-3\u2032 and GFPreverse 5\u2032-AGAACTTGCCGCTGTTCA -3\u2032. Primers used to identify the Lhx2-Cre transgene were: 1084: 5\u2032-GCGGTCTGGCAGTAAAAACTATC-3\u2032 and 1085: 5\u2032-GTGAAACAGCATTGCTGTCACTT-3\u2032. Primers used to genotype the ROSA26R mice were: Lac3 5\u2032-GGT TGT TAC TCG CTC ACA-3\u2032 and Lac4 5\u2032- CGT TAA AGT TGT TCT GCT TC-3\u2032. The morning of the vaginal plug was considered as E0.5.The 2, 5 mM EGTA, 0.02% NP40 and 0.01% sodium deoxycholate) and subsequently incubated in X-gal buffer  , 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide) over night at room temperature. The reaction was stopped with 3\u00d75 minutes washes with PBS, and sections were mounted in 87% glycerol. Whole-mount in situ hybridisation and in situ hybridisation using DIG labelled probes were performed essentially as previously described Lhx2 , Bmp4 , Pax6 , Bmp7 , Pax2 (IMAGE clone: 40142573), Cre , Rx (IMAGE clone: 5366450), Sox2 (IMAGE clone: 6413283), Otx2 , Six6 , Six3 , Vsx2 (IMAGE clone: 6492679), Mitf (IMAGE clone: 40047440), Enc1  and Nuak1 .Embryos were isolated and fixed in 4% paraformaldehyde (PFA) in PBS at 4\u00b0C. Embryos used for \u03b2-Gal staining were fixed for 30 minutes and embryos used for in situ hybridisation were fixed for 1\u20132 hours. After fixation the embryos were transferred to 30% sucrose in PBS for 24 hours at 4\u00b0C, mounted in Tissue-tek (Sakura) and stored at \u221280\u00b0C. Sectioning (8\u201310 \u00b5m) was performed on a cryostat (Microm HM505E) and collected on superfrost plus slides (Menzel-Gl\u00e4ser). For hematoxylin-eosin staining, tissue sections were incubated in Mayer's hematoxylin solution for 2 minutes, in water for 15 minutes, in eosin solution for 2 minutes, in 95% ethanol for 2\u00d71 minutes, in 99% ethanol for 2\u00d71 minutes and in xylene for 5 minutes. The slides were mounted with DPX mounting media (VWR). For \u03b2-Gal staining, tissue sections were washed for 3\u00d720 minutes in wash buffer , E9.5 (D) and E10.5 (F). \u03b2-Gal staining of sections of a head from Lhx2-Cre:ROSA26R double transgenic embryo at E12.5 derived from two different Lhx2-Cre transgenic founder mouse strains revealing that all neural parts of the eye are \u03b2-Gal+ in both founder mice . \u03b2-Gal staining of a sagittal section of a whole Lhx2-Cre:ROSA26R double transgenic embryo at E9.5 (E) and a transversal section of a head at E10.5 (G). \u03b2-Gal+ cells can be detected in the midbrain at E9.5 , in the olfactory placode (OE) at E10.5  and in cells of the hindbrain at E10.5 . NR, neural retina. RPE, retinal pigment epithelium. OS, optic stalk. Scale bar: A\u2013E and F\u2013G 500 \u00b5m.(TIF)Click here for additional data file.Figure S2Conditional inactivation of Lhx2 in the eye committed progenitor cell population cause anophthalmia. All adult flox/floxLhx2-Cre:Lhx2 animals are anophthalmic (C) whereas the flox/floxLhx2 mice develop normal eyes (A). This phenotype is already manifested at postnatal day 1 since no eye structures can be detected on sections of the head of flox/floxLhx2-Cre:Lhx2 animals (D) whereas flox/floxLhx2 animals develop normal eyes (B). Scale bar: 400 \u00b5m.(TIF)Click here for additional data file.Figure S3Increased number of apoptotic cells in the mutant optic vesicle. Immunohistochemical analysis of coronal sections of control optic vesicle (A) and mutant (flox/floxLhx2-Cre:Lhx2) optic vesicle (B) at E9.5 to detect the presence of activated caspase-3. Scale bar: 100 \u00b5m.(TIF)Click here for additional data file.Figure S4Fgf15 expression is significantly down-regulated in the optic vesicle in the conditional mutant. In situ hybridisation analyses of coronal sections of the optic vesicles in control (A) and mutant (flox/floxLhx2-Cre:Lhx2) embryos (B) at E9.5 to detect Fgf15 expression.(TIF)Click here for additional data file.Figure S5Lhx2 expression is induced following Cre-mediated recombination of the Z/Lhx2-GFP transgene. Schematic representation of the vector used to generate the Z/Lhx2-GFP transgenic mouse strain (upper panel) and the organisation of this vector after Cre-mediated recombination (lower panel). The blue arrows correspond to the mRNA that is generated before and after Cre-mediated recombination of this vector. We utilised an expression system based on the Z/AP double reporter vector developed by Lobe and co-workers \u03b2-Geo  is followed by an expression cassette consisting of the Lhx2 cDNA, an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) cDNA. Thus, cells expressing Cre recombinase will delete the \u03b2-Geo gene and initiate expression of Lhx2 and GFP since the Lhx2-GFP part is placed immediately downstream of the promoter/enhancer. Supplementary reference. 1. Lobe, C., Koop, K., Kreppner, W., Lomeli, H., Gertsenstein, M. and Nagy, A. (1999). Z/AP, a double reporter for cre-mediated recombination. Develop. Biol. 208:281\u2013292.(TIF)Click here for additional data file."}
+{"text": "Unconstrained rigid docking, flexible side chain docking and protein crystal structure determinations reveal a water-mediated hinge binding mode for a series of benzimidazole ligands of the protein kinase CHK2. This binding mode is different from those previously postulated in the literature and may provide a useful approach to selective small molecule inhibitor design. Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature. It has been postulated that selective inhibition of CHK2 could increase the efficacy of genotoxic cancer therapies in a p53 mutant background by modulating resistance pathways and may also be radioprotective to normal p53 wild-type tissues.As part of our in-house drug discovery project, we were particularly intrigued by a published series of benzimidazole CHK2 inhibitors for which two closely related binding modes have been proposed, neither of which appeared entirely consistent with the reported SAR (vide infra).1, 2\u20136, 7, 8 and 9,10, 11 and 12, Literature SAR includes analogues where the benzimidazole scaffold has been modified or replaced  and has In silico docking of small molecules into protein binding sites has become a powerful tool for medicinal chemistry design22.1We divided the 19 publicly available CHK2 protein\u2013ligand crystal structures into two groups. The first comprises crystal structures in which the ligand mimics ADP and interacts with the hinge region of CHK2 through one or two hydrogen bonds between the ligand and residues Glu302 and Met304 A.42 The 2.22CN5) and to NSC1095555 (PDB ID: 2W0J) as representative high resolution parent structures for docking . To validate the suitability of these structures for docking, ligands were removed and re-docked into the empty structures using GOLD.We carried out best practice unconstrained rigid docking using a selected set of 50 potent compounds from the published benzimidazole series .15\u201317 We8\u201312, 2CN5), hydrogen bond interactions with both Glu302 and Met304 were observed for the lowest energy poses of 14 active compounds ; the other 36 compounds did not make these interactions. However, even for the 14 ligands that did bind to the hinge, no additional hydrogen bonds with the protein were identified from the Protein\u2013Ligand Interaction Fingerprint  that form a high number of hydrogen bonds (3 out of a possible 4) (13-induced conformation are the two hydrogen bonds to the hinge (Glu302 and Met304) and an interaction between the carboxylate side chain of Asp368 and the N3 atom of the benzimidazole scaffold  that form the highest possible number of hydrogen bonds (4 out of a possible 4)  A. The thscaffold A. For thsible 4) B. The lie Lys249 B. Dockin8 to 12, into the30-induced conformation provides the optimal compromise of a high number of hydrogen bonds (4 out of a possible 4) adopted by a high number of biochemically active ligands (50%) docked into this ligand-induced protein conformation. Thus, the introduction of side chain flexibility into the docking protocol delivers an optimal binding mode mediated by a conserved water molecule to the hinge region. This binding mode is consistent with the observed experimental SAR (vide infra) and is significantly different from the two closely related binding modes previously postulated in the literature.Considering all possible solutions, these results indicate that the ligand 30-induced conformation with the crystal structure 2W0J  representing different ligand binding modes were obtained from the Protein databank. To optimize the positioning of hydrogen atoms in the ATP binding pocket, each crystal structure was subject to Protonate3D as implemented in MOECHK2 crystal structures  to those on the protein surface (lowest priority) until the limit of 10 was achieved.Crystal and Library in GOLD as described by Lovell et al.The permitted amount of flexibility for the 10 selected residues was defined by the parameters 4.6To identify the protein residues interacting with a docked inhibitor, the resulting protein-inhibitor complexes were analysed using the protein\u2013ligand interaction fingerprint (PLIF) implemented in MOE, a method similar to the SIFt.4.7Each ligand-induced protein conformation was analysed by plotting the trade-off between the number of polar interactions formed by the benzimidazole-5-carboxamide scaffold in the ligand-induced binding mode and the number of docked ligands adopting that particular binding mode . The sel4.8The CHK2 kinase domain  was produced as a GST-fusion protein and purified as previously described.4.919, 30, 44 and 47 for protein\u2013ligand crystallography are described in Compound structures have previously been disclosed although preparative methods have not been described for all compounds.4.1019, 4A9S; 30, 4A9R; 44, 4A9T and 47, 4A9U.Atomic coordinates and structure factors for the crystal structures of ligand-bound CHK2 can be accessed using the following PDB codes:"}
+{"text": "GST is a family of enzymes that are important in protection of the body against oxidative stress.Investigate the association between GSTT1 and GSTM1 polymorphism and hypertension.GSTT1 and GSTM1 genotypes were detected by PCR. The fragments were then analyzed by agarose gel electrophoresis.There is no significant association between GSTT1 & GSTM1 polymorphism and hypertension GSTT1 & GSTM1 polymorphism can be considered a risk factor for hypertension. Essential hypertension is a complex multi-factorial disorder with many genetic, environmental and demographic factors contributing to this disorder. Several experimental and clinical studies have highlighted the role of oxidative stress in development of hypertension 2]3]4][3][4]2][[4][3][4][3][4]2]. GST actThe present study aimed to test the hypothesis that the loss of activity of the enzyme due to a deletion polymorphism in the GSTT1 and GSTM1 may affect the risk of developing hypertension. We have also studied other risk factors for hypertension.The control group consisted of 33 non-hypertensive individuals with mean age 41.7 \u00b1 13.6 years.The case group consisted of 30 hypertensive patients with mean age 40.1 \u00b1 14 years. The study was conducted in the Biochemistry department in Dubai Medical College for girls. All subjects signed an informed consent to participate in the study. The hypertensive patients underwent a standardized evaluation consisting of a questionnaire, physical examination, and laboratory tests. Weight, height, waist and hip circumferences were measured. Body mass index (BMI) and Waist Hip Ratio (WHR) were calculated. Hypertension was defined as blood pressure \u2265 140/90 mmHg or current use of anti-hypertensive medication.Subjects with body mass index (BMI) \u2265 25 kg/m2 were considered positive for obesity as defined by World Health organization. Those with waist circumference (WC) > 88 cm for women and > 95 cm for men or with waist hip ratio (WHR) > 0.8 for women and > 0.95 for men were considered positive for abdominal obesity .Lipids and lipid fraction measurements were performed using routine enzymatic tests (DiaSys Kits) as previously described 9]9]DNA was extracted from white blood cells by a salting-out method .For genotype analysis, the GSTT1 and GSTM1 were amplified by using multiplex polymerase chain reaction (PCR) protocol 12]13].[13].12][.[13].12]Statistical analysis was done with SPSS software version 11.0 . Difference in genotype prevalence and association between case and control group were assessed by the Chi-square test. Correlation coefficient, Odds Ratio (OR) and 95% CI were used to describe the strength of association.The distribution of genotypes of GSTM1 and GSTT1 in cases and control are shown in The results of anthropometric measurements & lipid profile among the cases and control are displayed in Oxidative stress may contribute to the generation and/or maintenanceof hypertension via a number of possible mechanisms 15]16].[16].15][.[16].15]"}
+{"text": "Immunogen development for HIV-1 vaccines can be based on epitope identification of naturally occurring neutralizing antibodies in HIV-1 infected patients. A neutralizing monoclonal antibody, HJ16, was obtained at IRB from a patient recruited at ITM which recognized a new epitope in the CD4bs and neutralized mostly tier 2 strains that were not neutralized by b12[HJ16 resistance was induced by culturing the sensitive replication competent VI1090 (CRFO2_AG) strain (IC50 0.10 \u00b5g/ ml) in increasing amounts of HJ16 on freshly isolated PBMC until a resistant strain was obtained that was able to replicate in almost 200 \u00b5g/ml HJ16. Neutralizing activity was measured using TZMbl and PBMC neutralization assays. Site-directed mutagenesis was carried out to induce the observed mutations into the VI1090 expressing vector.Sequencing of the HJ16 sensitive versus the resistant strain revealed a distinct point mutation where the neutral asparagine was replaced by the negative charged aspartic acid. This N276D point mutation is only seen in 0.69% of the group M strains described in the Los Alamos database . Introduction of this mutation into the HJ16 neutralization sensitive construct rendered the mutated strain resistant to neutralization by at least 100 fold higher HJ16 concentration.Results show that the mutation in the N-linked glycosylation site at N276 has a distinct influence on sensitivity to the HJ16 CD4bs antibody. Although it is disappointing that a single mutation seems to induce resistance it is obvious that this mutation is quite unique and occurs rarely in natural infection rendering this epitope very suitable for further vaccine development."}
+{"text": "In Figure 1, not all of the data is included. The incorrect version of the figure represents miR-26a expression analysis in 20 breast cancer specimens and 15 normal breast tissues. The revised version includes 52 breast cancer specimens and 29 normal breast tissues. Please see the revised version of Figure 1 here:"}
+{"text": "Tightly controlled concentration gradients of morphogens provide positional information and thus regulate tissue differentiation and morphogenesis in multicellular organisms. However, how such morphogenetic fields are formed and maintained remains debated. Here we show that fibroblast growth factor 8 (Fgf8) morphogen gradients in zebrafish embryos are established and maintained by two essential mechanisms. Firstly, Fgf8 is taken up into the cell by clathrin-mediated endocytosis. The speed of the uptake rate defines the range of the morphogenetic gradient of Fgf8. Secondly, our data demonstrate that after endocytosis the routing of Fgf8 from the early endosome to the late endosome shuts down signaling. Therefore, intracellular endocytic transport regulates the intensity and duration of Fgf8 signaling. We show that internalization of Fgf8 into the early endosome and subsequent transport towards the late endosome are two independent processes. Therefore, we hypothesize that Fgf8 receiving cells control both, the propagation width and the signal strength of the morphogen. The family of fibroblast growth factors (Fgfs) is currently believed to consist of 23 structurally related polypeptides controlling a wide range of biological functions Fgf8 is one member of the Fgf family with key inductive functions during development of neural ectoderm, mesoderm and limb formation R447H mutant fruit flies, it has been shown, that depletion in uncoating activity correlates with decreased endocytosis One internalization route for growth factors such as Fgfs is via Clathrin mediated endocytosis (CME) a major mechanism for controlled cargo uptake which can be subdivided into several steps The identity of the uptake mechanism for Fgf8 has been a matter of some controversy Although some aspects of Fgf8 internalization route are understood, the factors that execute key functions during specific steps of Fgf8 endocytosis are still unclear. Furthermore, it is not fully understood how endocytosis regulates Fgf8 signaling and it is also unclear whether the signals emanate from the membrane and/or from internalized ligand-receptor complexes. Therefore, localization of the internalization route and subsequent identification of the signaling compartments is crucial.Here the molecular machinery involved in CME of Fgf8 was characterized and the influences of endocytosis on Fgf8 signaling were examined. Therefore, different steps in internalization \u2013 the clathrin mediated uptake and the transport from early to late endosomes- were analyzed. Firstly, we analyzed whether Hsc70 controls the endocytosis of Fgf8 by regulating CME. We show that knock-down of Hsc70 leads to a reduction in Fgf8 internalization and subsequently to an increase in long-range signaling of Fgf8 in zebrafish. Consistently, overexpression of Hsc70 leads to enhanced internalization of Fgf8 into early endosomes. Furthermore, we find that Fgf8 displays a decreased signaling activity in the early endosome compared to the plasma membrane. Finally we show that Fgf8 signaling is terminated by transport of the ligand to the late endosome. Therefore, we conclude that the receiving cells control Fgf8 signaling at two independent levels: firstly the dynamic of CME is important for regulating the activity range of Fgf8, and secondly the rate of transport from early endosomes to late endosomes determines Fgf8 signaling strength.All zebrafish husbandry and experimental procedures were performed in accordance with the German law on Animal Protection and were approved by Local Animal-Protection Committee  and the Karlsruhe Institute of Technology (KIT).Danio rerio) were maintained at 28\u00b0C on a 14-h light/10-h dark cycle 2O2) and the transgenic zebrafish line Tg(Dusp6:EGFP)pt6, which was received from Michael Tsang.Breeding zebrafish (Hsc70 full length coding sequence was amplified with primer pairs (forward/reverse); 5\u2032-TGG TGG CAC TTT TGA TGT GT-3\u2032/5\u2032-TCC CTC TCT GCA GTC TGG TT-3\u2032 from a zebrafish cDNA library at stage 24 h. The Hsc70 full-length cDNA was cloned in the expression vector pCS2+. The plasmid was then also linearized with NOT1 and transcribed using SP6 Message Machine Kit (Ambion).pCS2+Fgf8 GFP plasmid hsc70 MO1 (5\u2032ATAAAACAGAGATGGATGAAGATGC 3\u2032) and hsc70 MO2 (5\u2032 AGCTGGTCCCTTGGACATTGTGTCA 3\u2032) were used at a concentration of 0.5 mM. A volume of 1\u20132 nl was injected per embryo. The injection of MO oligomers was performed in the yolk cell close to the blastomeres at one to two-cell stage.For transient knock-down of gene expression, Morpholino-antisense oligomers , For transient knock-down, Hsc70 MO at a concentration 0.5 mM, was injected at the one- to two-cell stages. Samples of 30 embryos were collected at 28hpf, homogenized and dissolved in 1% triton lysis buffer. Protein solution was then eluted with Lammli buffer and heated to 95\u00b0C for 5 min and subjected to SDS-PAGE.For immunoblot, a Hsc70 polyclonal antibody  was used at a dilution 1\u223650,000. This was followed by incubation with anti-mouse IgG-HRP  at a dilution 1\u223610,000. Chemiluminescence detection was performed using ECL Western Blotting Substrate Kit (Pierce).6 cells in a 10cm plate. After 24 hours cells were transfected according to the manufacturer\u2019s protocol using Promofectin (Promocell). Briefly two mixtures containing DNA/serum free DMEM and Promofectin/serum-free DMEM were combined incubated for 20 min at RT and added to the cells. 24 hrs after transfection the culture medium was replaced and Bafilomycin A1 was added. 24 hrs later the cells were lysed in sodium dodecyl sulfate (SDS)\u2013sample buffer containing 100 mM dithiothreitol (DTT) and subjected to Western blot analysis. Activated Erk was monitored using an antibody against phosphorylated Erk . For the loading control the membrane was stripped  and reprobed with an Erk antibody (Erk 1 (K-23) Santa Cruz). Blots were stained using the enhanced chemiluminescence system (Thermo Fisher Scientific). The quantification of proteins bands in Western blot analysis was performed using the program ImageJ.HEK 293 cells were cultivated to 70\u201380% confluency before transfection. For transfection of Fgf8 and Hsc70 cells were seeded at 2\u00d710The statistical analysis was performed on three independent experiments. All quantifications are given as mean plus standard deviation.in situ hybridization was carried out as described previously fgf8, erm and pea3, using an RNA labeling and detection kit (Roche). Stained embryos were dissected and mounted in glycerol.Whole-mount mRNA After ISH staining, embryos were then incubated in 25 \u00b5M SYTOX nucleic acid stain (Invitrogen) overnight. After washing in 1\u00d7PBS embryos were mounted laterally for confocal analysis.in situ hybridization experiments. Inhibition of molecular transport from the early to the late endosomes was performed by treatment of embryos with 100 nM bafilomycin A1 (Sigma-Aldrich) with 1% DMSO or 1% DMSO only for a period of 1 hour at 30% epiboly stage and subjected to live imaging or fixed at 75% epiboly stage for in situ hybridization experiments. Texas red Transferrin was purchased from Invitrogen.Inhibition of Fgf receptor-mediated signaling was performed by incubating live embryos in 16 \u00b5M of SU5402  in 1% DMSO or with 1% DMSO only at 30% epiboly stage for a period of 3 hours and fixed at 75% epiboly stage for Zebrafish PAC2 cells were used to study the intracellular localization of Fgf8. The following endosomal markers were used: RFP-Rab5a (human) from Ari Helenius, pCS2+mCherry-dmRab7 from Jim Smith, mRFP-Clathrin from Ernst Ungewickell, dsRed-LAMP1 from Erez Raz. Cells were transfected separately with Fgf8-GFP at a concentration of 1 \u00b5g and Hsc70 transfected along with mRFP-Clathrin, RFP-Rab5a, mCherry-Rab7 at a concentration of 600 ng in DMEM solution. 24 hours post transfection, Fgf8-GFP transfected cells were co-cultured with cells transfected with Hsc70 and the respective endosomal markers.Prior to imaging, embryos were mounted in 70% glycerol. Images were taken with the help of an Olympus SZX16 microscope equipped with a DP71 digital camera using the imaging software Cell A. For confocal analysis, embryos were embedded for live imaging in 1.5% low melting point agarose (Sigma-Aldrich) dissolved in 1x E3 solution at 50% epiboly stage. Live embryos as well as cells were images using 20x and 63x water immersion objective. Confocal image stacks were then obtained using the Leica TCS SP5 X confocal laser-scanning microscope. Images were further processed using Imaris 6 (Bitplane AG).erm and pea3, were then measured using Olympus Cell A software. The area of expression was calculated with respect to the area of the entire embryo. Confocal experiments were quantified using Imaris 6 (Bitplane AG).The areas for Fgf target gene expression erm and pea3 by in-situ hybridization  and PAC2 cells transfected with endocytic markers , late endosomes (72%) and lysosomes 78%; . ActivatWe next analyze the consequence of Fgf8 endocytosis for signaling. We used a cell cultivation assay in which we analyzed effective Fgf8 signaling by quantification of Erk1 and Erk2 phosphorylation levels. Transfection of Fgf8 led to a 12-fold stronger level of Erk1/2 phosphorylation compared to the unstimulated control cell culture . SimultaBased on the data from the cell culture experiments we tested our hypothesis if the transport of Fgf8 along the endocytic route influences the signaling range. First we analyzed the endocytic route of Fgf8 in zebrafish embryos. We found that Fgf8 was taken up into Rab5 positive early endosomes, however, co-localization of Rab5 and Fgf8 was a rare event  supportiAlthough Fgf-Fgfr interaction has been extensively studied, the internalization We hypothesized that the rate of CME alters the intracellular uptake of Fgf8 and therefore determines the extracellular availability of Fgf8 and, hence the signaling range  & 2. Preerm and pea3 positive cells rather than Fgf8 spreading. An increased rate of endocytosis uncoupled from cell migration leads to a decrease of the spreading range In parallel, our experiments suggest that Fgf8 signaling can occur at the cell membrane and to a lesser extend in Rab5 positive early endosomes, which contrasts with other growth factor signaling pathways. As a result of impaired CME by expression of Dynamin2 K44A, activated Egfr shows a prolonged residence at the plasma membrane, which leads to a reduced activity of downstream signaling components such as MAPKs Erk1/2 or the p85 subunit of phosphatidylinositol 3 kinase, PI3 kinase After CME, Fgf8 localizes to early endosomes and late endosomes. Blockage of Fgf8 transport to late endosomes increases signaling. This suggests that - in addition to signaling at the membrane - signaling occurs also from early endosomes. Indeed the hypothesis that Fgf signaling can occur through the entire endocytic route has been postulated already in the 80s Here we conclude that the dynamics of Clathrin-mediated endocytosis of Fgf8-Fgfr complexes regulates the signaling range of Fgf8, whereas the retention time of the activated signaling complex at the plasma membrane and in the early endosome defines signaling strength.Figure S1A. Immunoblot analysis of the expression of Hsc70 protein during knockdown and over expression of Hsc70. Embryos were injected with Hsc70 morpholinos as well as Hsc70 mRNA and samples at 28hpf were subjected to immunoblot analysis. Embryos injected with Hsc70 MOs showed a reduction in the expression of Hsc70 when compared to the expression in wild type samples. Embryos overexpressing Hsc70 showed an increase in the expression of Hsc70 when compared to the expression in the wild type. ISH for Fgf8 expression. (B) Shows the expression of Fgf8 at 75% epiboly stage. Fgf8 expression remains unaltered during overexpression (C), knock-down of Hsc70 (D), and injection of Hsc70 MO and mRNA (E).(TIF)Click here for additional data file.Figure S2Endocytosis of Transferrin. Transferrin is taken up into the endosomes in fish fibroblasts (A\u2013A\u201d). Activation of Hsc70 leads to the formation of clusters of Transferrion, most likely in early endosomes (B\u2013B\u201d).(TIF)Click here for additional data file.Figure S3Injection procedure to measure the distance of Fgf8 distribution. Figure shows the schematic diagram of the experiment performed to analyze the spreading of Fgf8 from its source. (A) Fgf8 GFP DNA was injected into live zebrafish embryos at the one cell stage. (B) Zebrafish embryo at 50% epiboly stage show a mosaic expression of Fgf8. (C) Confocal microscopy analysis of live embryos show Fgf8 expressed in specific cells creating local sources from which the distance travelled by Fgf8 from the source to receiving cells is measured .(TIF)Click here for additional data file.Figure S4Quantification of co-localization studies of Fgf8 with endosomal markers. Transfection of Hsc70 led to the formation of Fgf8 clusters with a diameters over 0.8 \u00b5m. These clusters co-localize similarly strongly in Rab5 positive early endosomes. Three independent experiments were quantified.(TIF)Click here for additional data file."}
+{"text": "PAX6) on human chromosome 11p13 is an essential transcription factor for eye formation in animals. Mutations in PAX6 can lead to varieties of autosomal-dominant ocular malformations with aniridia as the major clinical signs. Known genetic alterations causing haplo-insufficiency of PAX6 include nonsense mutations, frame-shift mutations, splicing errors, or genomic deletions. The purpose of this study was to identify genetic defects as the underlying cause of familial aniridia in a large Chinese family.The paired box gene 6 . Quantitative real-time PCR was applied to verify the abnormal aCGH findings in the proband and to test five other family members.All exons of PAX6 in the proband. The aCGH analysis showed two copies of PAX6 but revealed a 566 kb hemizygous deletion of chromosome 11p13, including four annotated genes doublecortin domain containing 1 (DCDC1), DnaJ homolog subfamily C member 24 (DNAJC24), IMP1 inner mitochondrial membrane, IMMP1Landelongation factor protein 4 (ELP4) downstream of PAX6. Quantitative real-time PCR verified the deletion in the proband and further identified the deletion in a blind fashion in four affected family members but not in the one with a normal phenotype.There were no detectable pathogenic mutations in the exons of PAX6 should be the cause of the familial aniridia in this Chinese family, although two copies of PAX6 are intact. aCGH evaluation should be applied if there is a negative result for the mutation detection of PAX6 in patients with aniridia.The 566 kb hemizygous deletion of chromosome 11p13 downstream of  Eight-five percent of individuals with aniridia inherit this disorder as an autosomal-dominant trait, 13% occur as part of the autosomal-dominant WAGR syndrome , and the remaining 2% occur as part of other disorders, including Peters\u2019 anomaly and Gillespie\u2019s syndrome, in either autosomal-dominant or autosomal-recessive inheritances [Aniridia OMIM 06210 is PAX6) is located on chromosome 11p13 and contains 14 exons encoding a protein (PAX6) with 422 amino acids. PAX6 is a transcriptional factor controlling development of a diversity of organs and tissues  by recognizing specific DNA sequences of its downstream target genes [PAX6 gene primarily cause aniridia due to its haplo-insufficiency [PAX6 represent the major causes of aniridia, genomic rearrangements involving the downstream region of PAX6 were identified in patients with aniridia although both copies of PAX6 are intact in these patients [The paired box gene 6  techniques. To our knowledge this is the first case found in an Asian population and is one of few similar cases with this kind of genetic mechanism [In the present study, we report a genomic microdeletion in the downstream region of echanism ,11,12.A five-generation Chinese family with familial aniridia was recruited from Heilongjiang province, northeastern China. There were 15 affected individuals in this family from which five affected members  and one unaffected individual (IV:4) participated in this study . Ocular PAX6 in the proband (III:4) were amplified and sequenced with the ABI BigDye Terminator Cycle Sequencing kit v3.1,  according to the described protocol [(ELP4) gene within the deleted genomic region, primers within the PAX6 gene, and reference primers within the Oral-facial-digital syndrome 1 protein (OFD1) gene on the human X chromosome. Both ELP4 and PAX6 were assayed as test genes compared with OFD1, and both ELP4 and PAX6 were assayed as test/controls.All exons of protocol . The supprotocol . The digprotocol . IndividThe clinical findings are summarized in PAX6 from the six participants of the family were amplified and sequenced using standard techniques. No intragenic point mutation or deletion could be identified (data not shown).All exons of PAX6  according to HG18 , DnaJ homolog subfamily C member 24 (DNAJC24), IMP1 inner mitochondrial membrane (IMMP1L), and elongation factor protein 4 (ELP4). The remaining 34 CNVs were considered to be benign because these CNVs were identified in healthy individuals documented in the Database of Genomic Variation or in recent publications [The aCGH analysis detected 35 copy number variations (CNVs) in the proband\u2019s genome . One of ications ,18.ELP within the deleted region, which showed one threshold cycle difference between the patient and reference DNA samples for the amplification of the test gene . We used the qPCR method for testing the other five family members in a blind fashion, four affected family members with eye anomalies, and one healthy individual. All four patients were confirmed to carry the deletion, including ELP, but not PAX6. The phenotypic normal individual showed two copies for both ELP and PAX6 (data not shown).The 566 kb hemizygous deletion of chromosome 11p13 in the proband was verified by qPCR using primers targeted to est gene . Two copDCDC1, DNAJC24, IMMP1L, and ELP4. The proximal breakpoint of this deletion is approximately 123 kb from the 3\u2032 end of PAX6. We postulate that this 566 kb heterozygous deletion is the underlying cause of the familial aniridia and acts by disrupting the transcription in one of the two PAX6 alleles, even though the two copies of PAX6 were intact in all individuals investigated in this study.The major finding in this Chinese family with familial aniridia is the presence of a 566 kb heterozygous deletion containing four annotated genes: PAX6 controlling the expression of this gene, if disrupted, leading to aniridia and other eye anomalies. To our knowledge, this is the first case found in Asian patients with aniridia and one of few similar cases with this kind of genetic mechanism.Our finding provides further evidence of the existence of the remote 3\u2032 regulatory elements in the downstream region of PAX6 are apparently free of mutations. 2) Several publications reported similar observations in patients with aniridia, but the chromosomal breakpoint from the 3\u2032 end of PAX6 and the fragment of deletion were different. For example, two aniridia pedigrees have been characterized in which the disease segregates with chromosomal re-arrangements that involve 11p13 but do not disrupt the PAX6 gene since the chromosomal breakpoint is at least 85 kb away from the 3\u2032 end of PAX6 [ELP4 gene region, not involving PAX6, was present in all subjects with aniridia but not in the investigated normal relatives [: metallophosphoesterase domain containing 2 (MPPED2), doublecortin domain containing 5 (DCDC5), DCDC1, DNAJC24, IMMP1L, zinc finger CLS domain containing 3 (DPH4), and ELP4) has been characterized that starts 35 kb from the 3\u2032 end of PAX6 in a patient with aniridia, autism, and mental retardation [DCDC1, DNAJC24, IMMP1L, and ELP4) was found in patients with aniridia [PAX6. Deletions in this region have been shown to abolish PAX6 expression and cause aniridia and other eye anomalies due to loss of enhancers and a downstream regulatory region [Our postulation is based on following evidence: 1) The heterozygous deletion segregated with aniridia in the five affected individuals but not in the unaffected individual, while the exons and splicing regions of elatives . A 1.3 My region ,19,20.DCDC1 gene encodes a member of the doublecortin family, which is highly expressed in testis and fetal brain [DNAJC24 is one of several enzymes involved in synthesis of diphthamide, which is a unique posttranslationally modified histidine found only in translation elongation factor 2 [IMMP1L encodes a peptidase similar to mitochondrial inner membrane peptidase (IMP1), which is one of the catalytic subunits of the IMP complex proteolytically removing the mitochondrial targeting presequence of nuclear-encoded proteins [ELP4 encodes a component of the six subunit elongator complex, a histone acetyltransferase complex that associates directly with RNA polymerase II during transcriptional elongation. Two recent reports indicate that ELP4 is possibly associated with the centrotemporal sharp wave electroencephalogram (EEG) trait in rolandic epilepsy and speech sound disorder [Little information is known about the four annotated genes within the 566 kb deletion in this study. It is also unknown whether these genes are involved in any of the phenotypic features found in these individuals carrying the deletion. The al brain . DNAJC24factor 2 . IMMP1L proteins . ELP4 endisorder .PAX6 exons, copy number variation should be investigated in the \ufb02anking regions of PAX6. We suggest that patients, such as the subjects reported here, should be investigated using high resolution aCGH techniques in a clinical setting if sequencing analyses for PAX6 in patients with aniridia is negative.Submicroscopic copy number variations may play a role in human diseases either by loss of gene expression regulatory elements or by disrupting coding sequences. As well as the point mutations in"}
+{"text": "Inherited factors contribute to the burden of prostate cancer, however the identification of susceptibility genes for prostate caner has been challenging. To establish the contribution of eight founder alleles in three DNA damage repair genes  to prostate cancer in Poland, and to measure the impact of these variants on survival among patients, 3750 men with prostate cancer and 3956 cancer-free controls were genotyped for 3 founder alleles in BRCA1 , 4 alleles in CHEK2 , and 1 allele in NBS1 (657del5). Strong associations were seen for both CHEK2 and NBS1. BRCA1 was not associated with the risk of prostate cancer, NBS1 mutation was associated with poor survival - mortality was significantly worse for carriers of a NBS1 mutation than for non-carriers . We conclude that a founder mutation in NBS1 predisposes to aggressive prostate cancer."}
+{"text": "C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [The GGGGCC G4C2) intronic repeat expansion within ia (FTD) ,2. The m intronicbe toxic .in situ hybridisation probes for G4C2 to detect RNA foci in transfected cells and human brain tissue form C9ORF72 mutation carriers. We assessed the toxicity by cell counting, activated caspase, PARP cleavage and Annexin V expression transfected cells and by TUNEL assay in zebrafish embryos. We probed transfected cells with antibodies to 30 RNA binding proteins and then tested the best candidates by immunohistochemistry in human brain tissues. We used biotinylated G4C2 x72 probes to pull down repeat binding proteins and confirmed binding by western blot.In order to determine whether expanded G4C2 transcripts might be toxic and sequester RNA binding proteins we generated G4C2 repeats with 8x, 38x and 72x and expressed them in a range of neuronal and non-neuronal cell lines, primary neurons and zebrafish embryos. We used fluorescent Cellular toxicity was associated with the nuclear retention of transcripts containing 38x and 72x repeats and the appearance of RNA foci. By placing the repeats 3\u2019 to EGFP we were able to monitor nuclear export and show that transfected neuroblastoma cells, but not HEK cells, declined in number over time due to apoptotic cell death. This was most marked in those showing the greatest nuclear retention and foci, indicating cell type and neuron-specific toxicity of longer G4C2 repeats.In order to explore the sequestration hypothesis we screened antibodies to 30 RNA binding proteins on 72x transfected neuroblastoma cells and demonstrated colocalization of G4C2 RNA foci with three proteins. Using biotinylated 72x RNA we were only able to pull down one of these indicating that it is able to bind G4C2 RNA directly. On probing human cerebellar ALS and FTD tissues we detected a striking colocalization of this protein with 70% of RNA foci.Our observation that longer stretches of G4C2 RNA form neurotoxic foci and bind specific RNA binding proteins is similar to other intronic microsatellite expansion disorders where CUG (DM1) and CCUG (DM2) intronic repeats generate RNA foci in cells, animal models and in patients with myotonic dystrophy."}
+{"text": "Plasmodium falciparum infection, in particular the central role of natural killer (NK) cell-derived interferon gamma (IFN-\u03b3), is becoming increasingly recognised. Recently, it has been shown that IFN-\u03b3 production in response to P. falciparum antigens is in part regulated by killer-cell immunoglobulin-like receptor (KIR) genes, and a study from malaria-exposed Melanesians suggested an association between KIR genotypes and susceptibility to infection. This prompted us to determine and compare the frequencies of 15 KIR genes in Gambian children presenting with either severe malaria (n = 133) or uncomplicated malaria (n = 188) and in cord-blood population control samples (n = 314) collected from the same area. While no significant differences were observed between severe and uncomplicated cases, proportions of individuals with KIR2DS2+C1 and KIR2DL2+C1 were significantly higher among malaria cases overall than in population control samples. In an exploratory analysis, activating KIR genes KIR2DS2, KIR3DS1 and KIR2DS5 were slightly higher in children in disease subgroups associated with the highest mortality. In addition, our data suggest that homozygosity for KIR genotype A might be associated with different malaria outcomes including protection from infection and higher blood parasitaemia levels in those that do get infected. These findings are consistent with a probable role of KIR genes in determining susceptibility to malaria, and further studies are warranted in different populations.The relevance of innate immune responses to  Plasmodium falciparum can result in asymptomatic parasite carriage, an uncomplicated febrile disease or a potentially life-threatening illness. Apart from clinical immunity that gradually develops with repeated exposure Infection with the malaria parasite Natural killer (NK) cells are a key component of innate immunity. They kill their targets (diseased cells) by means of cytotoxic activity P. falciparum displays considerable heterogeneity  rather than cytotoxicity is the major contribution of NK cells to host defence ogeneity , 12 and ogeneity , 14. Thubright and CD56dim expressing cells bright cells produce more IFN-\u03b3 than CD56dim cells, CD56dim cells represent about 80% of the NK cell population dim population bright cells are KIR negative, whereas the majority of CD56dim cells express KIRs that are highly polymorphic  and 2 pseudogenes (KIR2DP1 and KIR3DP1). The expression and function of each of these genes influence the expression and function of other members of the gene family KIR molecules are glycoproteins encoded by a diverse and compact set of genes on chromosome 19. They are expressed on specialised lymphoid cells mainly NK cells and a subpopulation of \u03b3\u03b4 T cells and some memory \u03b1\u03b2 T cells KIRs and their interactions regulate NK cell activity in modulating disease outcomes  and KIR systems , 22 encooutcomes , 26\u201333.  alleles , 35, andP. falciparum infection, only a few studies have investigated the role of KIRs in malaria. A study that compared KIR genotypes in Melanesians with and without malaria parasitaemia found evidence for an increased number of activating KIR genes in parasitaemic individuals KIR genes among Gambian children with severe or uncomplicated forms of malaria, and cord-blood population control samples to assess whether individual KIR genes or genotypes are associated with the occurrence of disease or parasitaemia levels. Given that epidemiological findings have in the past implicated individual HLA class I alleles (e.g. HLA-B*53) Whilst there is accumulating evidence favouring an important role of NK cells in DNA samples were obtained from buccal swabs collected from Gambian children with uncomplicated malaria  and severe malaria .The typing technique used in this study (PCR-SSP) allowed us to detect the presence of inhibitory and activating KIR genes including those found in genotype A. The number of specific genes found at the centromeric end of B genotypes is variable including KIR2DS2, 2DL2, 2DL5, 2DS3 and 2DS5, in addition to the framework genes 3DL3 and 3DP1. However, telomeric B motifs are specifically identified with the presence of KIR3DS1 and 2DS1 genes in addition to 2DL4 and 3DL2 present in most motifs.In contrast to the conserved nature of genotype A in terms of gene content, KIR genotype B is highly polymorphic in gene content. Genotype B is a combination of activating and inhibitory KIR genes at the telomeric end all belong to KIR haplotype A . An opposite effect was observed for heterozygote (c-A/B) carriers . No such differences were seen with telomeric genotypes.We adopted the recent techniques and terminologies used by Cooley et al. lotype A . On the  KIR2DS4 . Analysi KIR2DS4  did not  KIR2DS4 . Howevercarriers , with a vs 12.5%, respectively, P = 0.004; adjusted for ethnicity and multiple comparisons P = 0.012), suggesting that having two copies of haplotype A may be protective against malaria infection. An interesting observation was that none of the 14 children with CM+SRD, considered to be the most critically ill patients, was homozygous for KIR genotype A, while a substantial proportion of children with less severe forms of malaria were homozygous for genotype A . Given the small number of individuals per group, this should be considered an exploratory analysis that encourages further investigations in other populations with larger sample sizes.The proportion of individuals homozygous for KIR genotype A varied significantly amongst the three groups . Whilst HLA-Cw*16:01 frequency  to be lowest amongst children with severe malaria . This observation, however, should be taken with caution given the small number of individuals used in this comparison. None of the HLA-B alleles was found to have any impact on malaria in this population. The frequencies of HLA-B or -C dimorphic groups and subgroups  were similar between groups ]. The frequencies of these two compound genotypes were significantly higher in cases compared with population control , suggesting that carriers of KIR2DL2+C1 and/or KIR2DS2+C1 were more at risk of being infected with malaria parasites than those without any of these genotypes. However, these two genes are in strong linkage disequilibrium (LD) even in African populations (It has been shown that KIR and HLA interact in an epistatic manner to modulate human immunodeficiency virus (HIV) disease outcome ulations , 48 makiKIR genes (I/A ratio) or the carriage of a particular KIR genotype is associated with parasite load measured on admission. When the ratios were grouped into four bins containing similar numbers of individuals, the level of parasitaemia was significantly different (PANOVA = 0.03) showing a significant increase with increasing I/A ratio , and parasitaemia levels declined significantly with increasing B content of the KIR genotype  and in a population control group. HLA class I (B and C) genotyping was performed on a proportion of samples with enough genomic DNA left after KIR typing. Possible KIR\u2013HLA interactions were explored as compound genotypes in individuals expressing KIR molecules together with their putative ligand(s). Consistent with previous studies in other West African populations  compared with the population control group. Amongst the participants with available HLA type individuals carrying either 2DL2 or 2DS2 together with their corresponding ligand (HLA-C group 1) were significantly more frequent in the infected group. Taken together, these observations suggest that these genes could be involved in increased susceptibility to malaria infection caused by P. falciparum. Although a few of our study participants have one without the other, KIR2DL2 and 2DS2 genes are in strong LD in most populations worldwide including those of African origin (KIR gene (2DL2) and its corresponding activating counterpart (KIR2DS2) have been associated with other diseases. Absence of these genes has been associated with resistance to infection with herpes simplex virus type-1 (HSV-1) KIR2DS2 in the absence of its specific ligands has been associated with increased susceptibility to psoriatic arthritis In this study, we determined the presence or absence of 15 ulations , 48, we n origin , 48, 50.KIR genes did not differ significantly between disease subgroups, it is worth noting that prevalence of three activating KIR genes, KIR2DS2, KR2DS5 and KIR3DS1 were highest in the most critically ill children presenting with CM+SRD  contributes to exacerbated inflammatory responses that have been implicated repeatedly in the pathogenesis of severe malaria (Although the proportion of subjects with individual h CM+SRD . While t malaria , 56. ButKIR3DS1 was the least frequent KIR gene in the Gambian population, in line with previous data from other African populations (KIR gene evolution amongst different populations worldwide has suggested that KIR3DS1/L1 has been under positive selection in modern Sub-Saharan African populations, with KIR3DS1 being rare and KIR3DL1 allotypes predominating KIR3DS1, such as reduced susceptibility to malaria or protection from severe forms of malaria, for which our data provide some support. In a study that compared KIR genotypes in Melanesian individuals with and without malaria parasitaemia living in a malaria hyper-endemic area KIR3DS1 positivity was equally distributed in both groups, but KIR3DS1/L1 heterozygosity was significantly associated with being parasite positive.Present in 6.4% and 12.5% of our population control samples and malaria cases respectively, ulations , 50, 55.KIR genes influence susceptibility to malaria. The involvement of genes located at the centromeric part of the KIR locus in modulating the outcome of malaria and in particular the role of KIR2DL2 and KIR2DS2 in the susceptibility to P. falciparum malaria warrant further investigations, and we recommend that larger studies should be performed across different populations to test this hypothesis with additional power. Such studies are needed to complement genome-wide approaches KIR haplotypic diversity.Taken together, our data provide some support for the hypothesis that"}
+{"text": "Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS) or increased (as in dup(15)) levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATP\u03b1, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.The molecular defects associated with Angelman syndrome (AS) and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used  UBE3A transcriptional regulation and imprinting UBE3A gene is primarily responsible for the autism phenotype in individuals with 15q duplication is the fact that paternal duplications, where the UBE3A gene is silent on the duplicated allele, can often be non-pathogenic or do not involve a clear ASD phenotype UBE3A gene is sufficient to produce autism-like behaviors in a mouse model of 15q duplication syndrome lending additional support to the hypothesis that elevation of UBE3A levels in the brain is the primary cause of autism in duplication 15q syndrome Angelman syndrome (AS) is a rare and severely debilitating neurological disorder with an incidence of \u223c1/10\u201320,000 children Ube3a deficient mice show defects specifically in experience dependent plasticity Ube3a deficient and Ube3a over-expressing animals have defects in glutamatergic synaptic transmission Ube3a expression in the brain The UBE3A protein is an E3 ubiquitin ligase  electrophoresis we identified a protein that is a master regulator of actin cytoskeleton remodeling (the Rho-GEF ECT2) which physically interacts with both human and fly Dube3a proteins in vivo and may influence phenotypes related to actin cytoskeletal remodeling Our laboratory has demonstrated that over-expression of human UBE3A protein in the Here we utilize this more quantitative and reproducible preparative liquid phase isoelectric focusing (IEF) cell in combination with traditional 1-D denaturing polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by molecular weight and quantify changes in protein levels dependent on fluctuations in Dube3a levels. This method allows us to resolve aliquots from the same fractions repeatedly until the pH comparisons are complete and the appropriate protein bands have been excised from the gels for mass spectrometry protein identification.Drosophila melanogaster heads expressing either no Dube3a protein (15bDube3a) or high levels of human or fly UBE3A proteins. By analyzing three cellular compartments we were not only able to identify proteins regulated by Dube3a involved in transcription or extracellular cellular signaling, but were also able to identify proteins that shifted from one cellular compartment to another in a Dube3a, and thus possibly ubiquitination, dependent manner. Using this approach we identified 50 proteins potentially regulated by UBE3A and implicating Dube3a involvement in neuronal homeostasis, ATP synthesis/metabolism pathways, as well as actin production or maintenance. The identification of these Dube3a targets is the first step in identifying new pathways regulated by UBE3A in humans that are responsible for the underlying defects in both Angelman syndrome and duplication 15q autism and may reveal new therapeutic targets for the treatment of these disorders.We directly compared nuclear, cytoplasmic and membrane extracts from 15bDube3a makes no Dube3a protein in the homozygous state and has been previously described Dube3a, UAS-Dube3a-C/A, and UAS-hUBE3A lines have been described elsewhere Heatshock-GAL4. The progeny of Heatshock-GAL4 crosses to various UAS transgene lines were subjected to a 37\u00b0C heat shock for 1 hr to induce transgene expression (24-fold increase in Dube3a protein and 37-fold increase in Dube3a-C/A protein) All fly crosses, with the exception of heat shock crosses which were raised at RT, were performed at 25\u00b0C on standard corn meal based media. The complete loss of function mutant Cytoplasmic Protein Extraction Buffer (CPEB from Bio-Rad). Heads were ground in ice cold buffer to release proteins and then the supernatant (cytoplasmic proteins) collected by centrifugation at 3500RPM at 4\u00b0C. The pellet was then re-extracted according to the manufacturers instructions to obtain the nuclear protein extracts. Membrane preparations were made by ultracentrifugation in a sucrose gradient using an established Drosophila protocol Heads were removed from frozen flies using molecular sieves under liquid nitrogen as previously described For each type of extract in each genotype, all protein fractions were visually analyzed across the middle 18 IEF fractions (\u223cpH 4.5\u201310.5) by loading \u223c2 ug of total protein per lane on a 20 well 4\u201312% midi-gel in MOPS buffer (Invitrogen). Samples that were similar in pH to within +/\u22120.1\u20130.5 pH units were then run side by side on a 4\u201312% mini-gel for direct comparison of differentially regulated proteins. To quantify fold change in protein intensity the gels were fixed and stained with quantifiable fluorescent protein stain Sypro Ruby (Invitrogen), scanned under UV illumination using a Syngene G-box imaging system and then analyzed using software to measure intensity differences among protein bands (Syngene). Proteins which were determined to change by at least 2 fold+or \u2013 were then excised from the gels after Silver staining for visualization on a light box or directly cut from the Sypro gels under UV illumination in some cases.Peptides were identified from cut bands by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS) at either the UTHSC, Tufts University or Harvard Mass Spectrometry facility. Only top hits were considered for inclusion in the list and accuracy was assessed by p-value, Z-score or percent coverage. Protein identifications were performed using various parameters to determine confidence in that particular ID depending on the facility used  at the UTHSC Proteomics Core (closed 2007), the Tufts University Proteomics Core (closed 2009) and the Harvard University Proteomics Core.15bDube3a, and allowed to recover for \u223c2 min. and then frozen in liquid nitrogen. Cytoplasmic and nuclear extracts were prepared from fly heads using the ReadyPrep Protein Extraction Kit (Bio-Rad). Samples were resolved on a NuPage 4\u201312% Bis-Tris 1.5 mm Gel using MOPS Buffer (Invitrogen) and transferred to Immobilon-FL PVDF membrane (Millipore). The membrane was blocked with 5% milk, 3% BSA, and 0.1% Tween-20 in PBS. Primary antibodies were used at 1\u22365000: anti-GAPDH , anti-Arginine kinase courtesy of Glen Collier , anti-Eps15 courtesy of Hugo Bellen , and anti-ATP\u03b1 (cat. #: \u03b15) from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA. IR labeled secondary antibodies  were purchased from Li-Cor  and used at a dilution of 1\u223610,000. The blot was imaged and analyzed using the Odyssey Infrared Imaging System . Samples were normalized using the signal from anti-GAPDH as the reference channel and signal intensity was then adjusted based on the calculated normalization factor assigned to each lane in the channel. Normalized intensities were used for statistical analysis from at least three technical replicates.Transgenic and control flies, no more than 3 days post eclosion, were subjected to a 37\u00b0C heat shock for 1 hr, or no heat shock in the case of 680 and no loading control was used since quantification was not possible by this method.Ubiquitinated proteins were purified using a polyubiquitin affinity resin (Thermo Scientific) in accordance with the manufacturer\u2019s instructions. In brief, lysates of the appropriate genotype and cellular fraction were incubated with the resin at 4\u00b0C overnight on a rotating wheel. The resin was then washed and ubiquitinated proteins were eluted in SDS-PAGE sample buffer for Western blot analysis. Antibody concentrations and conditions were identical to those described above except all secondary antibodies were IRIn vitro ubiquitination assays were performed as described 2 and 4 mM of ATP) containing 50 ng of E1 ubiquitin-activating enzyme, 500 ng of E2 ubiquitin-conjugating enzyme (UBCH7), 2 \u00b5g of Dube3a-FLAG fusion protein, and 6 \u00b5g of bovine ubiquitin. The reaction was incubated at 30\u00b0C for 2 h. The reaction was terminated by the addition of SDS-sample buffer, samples were boiled and proteins resolved on a 4\u201312% Bis-Tris 1.5 mm acrylamide gel using MOPS buffer (Invitrogen) and transferred to an Immobilon-FL PVDF membrane for Western blot analysis. The blots were probed with both anti-Ubiquitin (Millipore) or anti-ATP\u03b1 . Imaging and analysis was performed on an Odyssey Infrared Imaging System . The mean intensity values were measured from the bottom of the \u223c100 kDa ATP\u03b1 band to the top of the gel for three independent experiments. These values were converted to a percentage of the value for LANE 2 (Dube3a+E1/E2 proteins) in order to compensate for any Dube3a auto-ubiquitination signal. The percentages were then imported into Prism version 5.0 for statistical analysis (GraphPad).Both Dube3a and ATP\u03b1 were purified from fly head cytoplasmic extracts using affinity purification protocols. N-terminally FLAG tagged Dube3a was purified using EZview\u2122 Red anti-FLAG M2 Affinity Gel to purify the Dube3a-FLAG fusion protein following manufactures instruction (Bio-Rad). ATP\u03b1 protein was purified using anti-ATP\u03b1 monoclonal antibody  through immunoprecipitation with immobilized Protein G beads (Pierce) in accordance with the manufacturers instructions. http://tinyurl.com/7u6s5bh). Although some of the genes have multiple splice variants, all primers were designed as common assays that would detect all splice-forms, if possible. Three technical replicates were performed for each gene and normalized to the expression of the housekeeping TATA-binding protein (TBP) gene. Gene expression was quantified for each cDNA sample in triplicate at 40 ng/reaction using the default cycling parameters of the Roche Light Cycler 480 system. The crossing point (Cp) value for each sample was calculated using Roche absolute quantitation algorithm. The average Cp value among three technical replicates was calculated and the average sample Cp values were normalized for loading by comparison to the average Cp value of TATA-binding protein TBP. The fold change (Fc) in gene expression in fly heads was then calculated by comparing the difference of the normalized mean Cp values between 1118w and the 15bDube3a mutant or between Heatshock-GAL4 and each over-expression construct using the equation Fc\u200a=\u200a(2)Dcp.Total RNA was extracted from \u223c100 ul of fly heads using 200 \u00b5l of RNABee solution (AMS biotechnology). RNA was quantified by spectrophotometry (NanoDrop Technologies) and the integrity of the 18S and 26S rRNA verified using an Agilent 2100 Bioanlyzer (Agilent Technologies). Two \u00b5g of total RNA were used as input for each cDNA reaction synthesized using random hexamer primers via the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Intron spanning primers were designed for all 49 potential targets using the Roche Universal Probe Library . Fixed larvae were then treated with a 10 min incubation in PBS +0.01% Triton-X 100 (PBSTx) followed by a 20 min incubation in a 500 ul solution containing 3 ul Phalloidin594 (Invitrogen) in PBSTx. Larvae were washed in PBST twice and then mounted with Prolong Gold Hardset plus DAPI (Vector Labs) for image capture using a 63X lens under oil immersion on a Zeiss LSM 710 confocal microscope with identical imaging conditions for 1118w and 15bDube3a genotypes. The tile feature was used to capture the entire A2 muscle region from either the left or right side of the animal (overlap 10%). Z-stacks of 6 \u00b5m in depth were used to capture images from the muscle nuclei layer to the top of the muscle surface. A stitched Z-stack section from the middle of this stack was then used for intensity analysis using the Zen image analysis package (Zeiss). Intensity was measured in 150 \u00b5m segments perpendicular to the muscle striations in both muscles 6 and 7 from each of four animals per genotype (n\u200a=\u200a4). Statistical analysis was performed using a Student\u2019s t-test in the Prism 5.0 software package (Graphpad Software).Wandering 3Dube3a and a Dube3a mutant that has a single mutation at the active site eliminating the ubiquitin ligase function of the enzyme 15bDube3a which makes transcript, but no protein Heatshock-GAL4 driver line to induce global high-level gene expression in all tissues. Adult flies no more than 3 days post eclosion were subjected to heat shock to induce transgene expression in the brain and then frozen in liquid nitrogen. In the case of the 15bDube3a mutant line, flies were grown at 25\u00b0C and homozygous 15b/Dube3a15bDube3a flies were used for all experiments. Frozen fly heads were then used to isolate cytoplasmic proteins (soluble fraction), nuclear proteins (re-extracted pellet) and membrane proteins (membrane preparation). Using the fly GAL4/UAS expression system Heatshock-GAL4 alone (top) and Heatshock-GAL4>UAS-Dube3a cytoplasmic fraction proteins . Using this approach we identified 137 bands to cut from these gels comprising some 76 individual proteins identified by mass spectrometry (www.flybase.org) in order to compile a comprehensive list of protein identifications, the cellular fraction in which they were identified, pH, relative fold change, apparent molecular weight as well as database identifiers (see Table S1 for complete details).Proteins from each genotype were extracted as described in the methods section. Proteins from each fraction were then separated by isoelectric point using a liquid phase isoelectric focusing cell (IEF). The actual pH and protein concentration for all twenty fractions from IEF cell were recorded and then each fraction run in a second dimension separation by molecular weight. trometry Table 1.Dube3a. In order to visualize the effects of changes in Dube3a expression on individual proteins identified we constructed a heat map for the fold change of each protein under each condition of Dube3a over/under expression and in cytoplasmic, nuclear and membrane fractions , ATP\u03b1 (CG5670), Cuticular protein 72Ec (CG4784), Fat-body protein (CG17285), Fimbrin (CG8649), and Prolyl endopeptidase (CG5355). Elevated protein expression in the mutant may indicate that these particular targets are de-repressed by loss of Dube3a, and thus could be direct ubiquitin targets degraded by the ubiquitin proteasome system. The most likely direct ubiquitin targets on this list are ATP\u03b1 and ArgK since these proteins have previously been identified as a proteins ubiquitinated in the developing fly nervous system Six proteins increased by >20 fold in various cellular fractions from the Punch and Pebble targets Six proteins only showed differential protein expression in the presence of elevated human UBE3A, which was used previously to identify both Table S2). A heat map analysis of all of the transcripts is illustrated in . Thirteen genes showed a >1.5-fold increase in gene expression when the Dube3a-C/A protein was expressed, indicating that the ubiquitin ligase function of Dube3a is not required for increased expression of these particular proteins. Eleven of these genes also responded in a positive fashion to increased wild type Dube3a levels. Eight of these genes responded in a positive direction to increased expression of Dube3a-C/A and a negative manner to loss of Dube3a (i.e. 15bDube3a), or not at all, including the GTP cyclohydrolase I gene which we previously described Gene expression was analyzed for all 49 proteins identified during the proteomic screen using primer sets common to all splice forms of these genes. Seventeen genes did not demonstrate appreciable changes in gene expression (\u22651.5-fold up or down) under any conditions of varying Dube3a protein levels and one gene, Pro-phenol Oxidase 1, could not be analyzed using several different primer sets  showed a >25-fold increase in gene expression in the 15bDube3a mutants ( and Table S2), while a much smaller 2.6-fold increase in gene expression was observed for the heatshock related gene Heatshock Cognate 4. Since neither the 1118w line or the 15bDube3a line were subjected to heatshock, these results indicate that heatshock proteins may be indirectly regulated by Dube3a through a transcription factor intermediate or that Dube3a itself can confer transcriptional co-repression.Three Heatshock protein encoding genes  we identified significant enrichments  for transcription factor modules shared by these eight genes: Apolipophorins, Crystallin, Eps15, Fasciclin 1, Fat-body protein 1, glyceraldehyde-3-phosphate dehydrogenase, neither inactivation nor afterpotential C and PuTable S2. TFM-Explorer analysis revealed 17 clusters with a significant enrichment for 12 different transcription factor binding sites in the promoter sequences of these eight genes . The top five hits included binding sites for transcription factors glial cells missing , Chorion Factor II , Heatshock Factor , Abdominal B  and Ultrabiothorax .We next searched for the presence of transcription factor binding sites in the eight genes that showed increased transcription when we expressed Dube3a-C/A but either were not affected by or showed decreased expression in Drosophila brain and the developing embryo The glial cells missing transcription factor is both necessary and sufficient for the production of glial cells versus neurons in both the central complex of the adult inset). Finite analysis of the GO terms for each protein indicated that the major cellular functions regulated by these proteins include carbohydrate metabolism (11%) as well as energy metabolism (15%), actin cytoskeletal structure (7%), development (4%), and nucleotide binding (6%)  classification term culled from Flybase for each hit. A general root classification of the proteins we found indicates that 18% of the proteins from the screen are cellular components, 36% are involved in a molecular function and 46% are part of a specific biological process (ing (6%) Figure 3in vivoSeveral proteins on the list of regulated candidates were immediately of interest, as they are specifically known to be involved in neuronal development and activity. These proteins include the dopamine regulatory protein Punch, which we previously showed to be regulated by Dube3a Heatshock-GAL4 induction in these cases since all flies containing Heatshock-GAL4 were subjected to the same heatshock protocol and the control flies contained the Heatshock-GAL4 driver for comparisons of band intensities. In addition, we identified at least one Heatshock protein (Hsp70Ba (CG31449)) that decreased in 15bDube3a mutants as compared to wild type outside the context of the heatshock protocol. We also found on our list 4 proteins that make up the actin cytoskeleton or muscle including Actin5c (CG4027), Actin57B (CG10067), and myosin heavy chain (CG17927) as well as Tropomyosin I (CG4898). Finally, there were 11 proteins involved in more basic metabolic processes such as dehydrogenases, isomerases and kinases. Since the over-expression of human UBE3A only resulted in changes to 6 proteins that were not also regulated by changes in Dube3a, we conducted more in depth functional analysis using just the 43 proteins in the Dube3a regulated set.The next most interesting groups of proteins were Heatshock and Heatshock cognate proteins. The identification of changes in Heatshock proteins is not an artifact of the Table S3). These included an enrichment for nucleotide binding proteins (Es\u200a=\u200a4.1), Heatshock proteins (Es\u200a=\u200a3.8), glycolysis (Es\u200a=\u200a3.7), oxidation/reduction (Es\u200a=\u200a3.3), actin cytoskeleton (Es\u200a=\u200a2.9), homeostasis (Es\u200a=\u200a2.1), actin binding (Es\u200a=\u200a1.8) and mitochondrial oxidative phosphorylation (Es\u200a=\u200a1.6). This same set of 43 proteins was then used as an input list for STRING analysis (http://string.embl.de/) in order to identify relationships among proteins that may reveal protein networks regulated by Dube3a. Confidence relationships among these proteins revealed a tight cluster of 13 proteins, some of which are involved in actin cytoskeleton remodeling and some in cellular trafficking, as well as a wider network comprising 27 proteins including the neurotransmitter regulator Punch (CG9441) and the axonal transport protein shibire (dynamin: CG18102) that are interrelated by direct physical interaction or functional association , Pro-phenol Oxidase 1 (Bc: CG42639), Cuticular protein 72Ec (CG4784) and apolipophorins (Rfabg: CG11064) are involved in fly specific structures and functions and thus do not have any appreciable homology to humans and other model organisms. Our gene ontology and phylogenetic analysis indicates that Dube3a may be a key regulator of multiple proteins that regulate cellular trafficking through actin cytoskeletal proteins requiring ATP synthesis and metabolism to function in both flies and humans.A more formal analysis of these 43 proteins identified from the screen was done using pathway enrichment analysis tools within the Database for Annotation, Visualization and Discovery suite (DAVID) , top). Changes in the expression of Dube3a did cause fluxuations in the normalized intensity levels for ATP\u03b1, but these changes were variable among the four independent technical replicates and did not reach significance .In order to investigate direct effects of Dube3a ubiquitin ligase function on potential protein substrates identified in our screen we looked for changes in protein expression or ubiquitination state under differential Dube3a expression conditions. Although antibodies were available for Eps15, Arginine kinase, and ATP\u03b1 for Western blot analysis, only the Eps15 and ATP\u03b1 antibodies revealed appreciable protein stability/expression changes that were Dube3a dependent  resulted in a decrease in the amount of ubiquitinated ATP\u03b1 detectable in the elution as compared to wild type  head extracts . The addition of purified Dube3a plus E1/E2 proteins did produce a Ub signal slightly above background . The signal in LANE 2 was considered the detection threshold above background (100%) since recombinant UBE3A is known to trans-ubiquitinate itself in vitro, LANE 3) and a strong smear appeared with the addition of Dube3a protein . This experiment was repeated three times using three different purifications. One Way ANOVA analysis with Tukey\u2019s multiple comparison testing among the four groups indicated that there is a significant difference among the groups  and that significantly less poly-Ub is detected in the lane containing no E1/E2 proteins  while significantly more poly-Ub is detected in the lane with all three components  as compared to the control lane which lacks the ATP\u03b1 substrate . These results indicate that ATP\u03b1 may act as a direct substrate for ubiquitination by the E3 ubquitin ligase Dube3a in vitro.To determine if ATP\u03b1 can act as a direct substrate for the Dube3a E3 ubiquitin ligase we performed actin5c, actin57B, Mhc, and Tm1 we investigated the effects of decreased Dube3a levels on larval muscle. Crawling 3rd instar larvae have a large area of easily accessible body wall muscle that can be visualized with the filamentous actin (F-actin) toxin phalloidin. We dissected and fixed four wild type and four 15bDube3a mutant larvae and stained the body muscles with fluorescently labeled phalloidin (Ph594). The mean fluorescent intensity was 37% lower in the 15bDube3a mutants  than in wild type animals and the range of signal intensity was lower as well  of the unique proteins identified by our screen did not change in any cellular fraction in the 15bDube3a mutant and of those that did, only 12 out of 24 proteins (50%) changed intensity by >10-fold validating our prediction that the proteomic analysis of complete loss of function mutants alone would not have revealed the majority of proteins that are actually regulated by Dube3a in a temporal specific manner. More importantly, we have now uncovered sets of proteins involved in the process of actin cytoskeletal dynamics as well as ATP driven metabolic and catabolic pathways which will stimulate new avenues of research in existing fly and mouse models of AS and autism.Changes in 15bDube3a loss of function animals, we also found that Tropomyosin I (Tm1) levels decreased by 3.3-fold in the absence of Dube3a protein (Table S2). This may be the cause of the decrease in filamentous actin we found in larvae since Tm1 physically interacts with and stabilizes actin filaments Dube3a loss of function mutations in flies the authors found that adult flies showed locomotor defects, specifically climbing ability Dube3a deficient larvae and most likely in adult muscle as well, suggesting that muscle defects may also contribute to the locomotor defects observed in these animals. Second, the upstream regulation by Dube3a of actin cytoskeleton through fly Pebble or UBE3A regulation of ECT2 may explain the changes in actin cytoskeletal components we found in our screen since Pbl/ECT2 is a Rho-GEF which is a master regulator of the cytoskeleton. Although we did not identify Pbl by liquid phase isoelectric focusing in the current screen, we previously showed that Pbl/ECT2 physically interact with both fly and human Dube3a/UBE3A proteins Ube3a protein have significant defects in experience dependent synaptic plasticity in the visual cortex Ube3a deficient animals Ube3a can result in a decrease in dendritic spine density on basal pyramidal neurons Although we found that both actin and myosin transcripts were elevated in et al. , and failed axon connections et al. Table 2.15bDube3a loss of function mutant (Table S2). A link between the ubiquitin proteasome system and the heat shock response has been known for some time In this study we show a >25 fold increase in gene expression for Hsp70Ab, Hsp70Ba and Hsp70Bb in a homozygous in vivo, the downstream effects of this ubiquitin modification remain somewhat puzzling since there are no appreciable changes in ATP\u03b1 stability of the protein by Western blot . This growing list of transcriptionally regulated Dube3a genes opens a new avenue of research into the mechanisms by which Dube3a can act as part of a transcription factor complex and which transcription factors bind to Dube3a in the nucleus.We previously showed that Dube3a can regulate dopamine levels in the fly brain through the transcriptional (ubiquitin independent) regulation of GTP cyclohydrolase I (Punch) It should also be noted that we observed changes in the cellular compartmentalization of two proteins as a direct result of over-expression of either wild type Dube3a or the presumed dominant negative form Dube3a-C/A . In the cis regulatory regions responsible for these changes in gene expression. Here we propose a new model for UBE3A regulation in the nervous system that involves not only regulation by direct ubiquitination of target proteins but also transcriptional regulation and perhaps even cellular trafficking.Finally, although we have exhausted this particular approach to the identification of direct protein targets of Dube3a in fly heads, the use of the bio-Ub method will enable a deeper investigation into the identification of Dube3a dependent ubiquitin targets in the fly nervous system. Finding additional transcriptionally regulated Dube3a genes will require a different set of tools including whole genome chromatin immunoprecipitation approaches to identify the Table S1Complete list of proteins identified in our screen, fraction where they were identified, molecular weight of the protein excised from the gel, pH, fold change, CG identifying number and FBgn identifying number.(PDF)Click here for additional data file.Table S2Complete qRT-PCR data with error calculations from triplicate replicates for all genes identified in our screen. Genes with an asterisk (*) were used for transcription factor binding site analysis.(PDF)Click here for additional data file.Table S3value and fold enrichment for a given group in the cluster as compared to the entire set of proteins.DAVID analysis of proteins identified in the screen. Clusters of proteins that act in similar pathways or biological processes are listed along with an enrichment score for the cluster as well as a p(PDF)Click here for additional data file.File S1This Microsoft PowerPoint file contains images of gels used for either direct comparison of experimental and control lanes or to excise a previously identified band for proteomic identification. The band numbers and gel numbers can be found in (PPTX)Click here for additional data file."}
+{"text": "Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies  Spermatogenesis occurs in most male mammals throughout their lifetime. Successful self-renewal of the spermatogonial stem cells underpins this process Mll5 have a post meiotic spermatogenic phenotype mixed lineage leukemias) are structurally and functionally homologous to the Drosophila Trithorax proteins It has been reported that independently generated knockout mouse models of S. cerevisiae SET3; Mll5 was found to be part of the NCOR complex which is believed to be functionally similar to the SET3C complex MLL5, consistent with the hypothesis that they may be part of the same complex Mll5 (KMT2E) was initially assigned to this family in part due to the sequence similarity of its PHD and SET domains to those of Mll. However, recent studies suggest that both human MLL5 and mouse Mll5, and the murine paralog, Setd5, have SET domains that are closer in sequence to the yeast SET3 and SET4 proteins Mll5. Mll5 appears not be essential for embryonic development Recently, our group and two others have described mouse knockout models for Mll5 knockout mice, however male infertility was noted in two of the initial reports set3 (\u0394set3), resulted in normal vegetative growth and development Gametogenesis has not been examined in detail in the in vitro fertilisation techniques that male fertility is impaired in tm1ApaMll5 mice, due to multiple defects in terminal spermatozoa differentiation / maturation. Thus providing further experimental evidence supporting that idea that Mll5 is that functional homolog of yeast SET3. We also show specific deregulation of several important gene transcripts in the testis, which may be putative targets of Mll5 regulation.In this report we show using electron microscopy, video microscopy and Mll5 is described in detail elsewhere tm1ApaMll5 by insertion of a \u03b2-galactosidase  reporter cassette and neomycin resistance cassette under an independent promoter (MC1) in coding exon 3 of the murine Mll5 locus (exon 4 in ENSMUST000000094962) in 129S6 (129SvEv) embryonic stem cells resulting in the deletion of 180 bp of coding sequence. This disrupts the 5\u2032-most coding exon generating a frame shift. This allele (with the resistance cassette in place) was passed to the germline and backcrossed twice to 129S6 wild type mice before intercrossing. The loss of full-length mature Mll5 protein was verified by Western blotting tm1ApaMll5 male and female mice can survive through adulthood but display pleiotropic haematopoietic and maturation defects tm1ApaMll5 intercrosses result in Mendelian ratios of homozygous, heterozygous and wild type pups at embryonic day 16.5, although non-Mendelian ratios were observed later in development, likely due to loss of homozygous pups between birth and weaning secondary to an immune defect The generation of a loss of function allele for tm1ApaMll5 homozygous mating pairs produced offspring, or evidence of pregnancies. To determine whether one or both sexes were affected, we first analyzed reciprocal matings of wild type males with homozygous females and wild type females with homozygous males over a 4-month period. tm1ApaMll5 female mice are fertile but exhibit a possible rearing defect . Heterozygous littermates were able to plug and impregnate female mice with a frequency indistinguishable from wild type  reporter within exon 3 downstream of an internal ribosome entry site (IRES), allowing for expression of the \u03b2-Gal enzyme when the tm1ApaMll5 transcript is synthesized from the endogenous Mll5 promoter. Whole-mount \u03b2-Gal staining confirmed the presence of the knock out allele  and early spermatids (st) (Mll5 in developing germ cells.As current antibodies against Mll5 do not work well on tissue sections (data not shown), we used RT-qPCR, \u03b2-Galactosidase  staining and RNA in-situ hybridization to assay and visualize t allele  and RT-qt allele  in the ttm1ApaMll5 male mice and the expression of Mll5 in the developing germ cells of wild type mice, it was conceivable that loss of Mll5 generated by the tm1ApaMll5 allele might impair spermatogenesis. However, we found no gross morphological differences  from wild type mice (Pearson \u03c72(1)\u200a=\u200a107, p\u200a=\u200a3\u00d710\u221225, The presence of sperm but absence of gross morphological defects in the testis suggested that defects in spermatogenesis might lie in terminal maturation stages and / or capacitation events. We examined periodic acid Schiff (PAS) stained sections of testes and epididymides by light microscopy and observed significant differences between wild type and homozygous ld types . In additm1ApaMll5 mice that were not seen in the wild type testes. When surveyed at similar locations of the caput epididymis , in some cases with several tail assemblies pooled into one cytoplasmic droplet \u200a=\u200a0.33, p\u200a=\u200a0.62; tm1ApaMll5 mice, 41.8% showed apparently normal morphology under high-powered light microscopy. This raised the question of whether morphologically normal sperm were nevertheless impaired for sperm motility capacitation or the ability to penetrate the zona pellucida and fuse with the egg membrane. We therefore decided to test whether the infertility of homozygous tm1ApaMll5 mice could be rescued by sperm capacitated using an in vitro fertilization procedure. The sperm heads could be observed binding to the oocyte in vitro \u200a=\u200a67, p\u200a=\u200a3x10\u221215) and severe impairment of their ability to fertilize eggs from superovulated wild type females when identical numbers of sperm were incubated with wild type eggs  level probeset summarization   and 7.9 fold (p\u200a=\u200a2.4\u00d710\u22127) , while transcript Utx, encoding a H3K27-specific histone demethylase that associates with Mll3/4 complexes at Hox genes Mll5 was lost (Mll is responsible for the maintenance of Hox gene expression ype mice . Hence w\u200a0.0034) . Express altered . In addi.4\u00d710\u22127) , respectanalyses , also vaanalyses . These ge testes . In addiwas lost .Mll5 mouse in functional and molecular detail. In addition to the obvious sequences similarities of the SET domain of Mll5 to SET3/4 at critical residues  as well as somatic (Sertoli) cells of mice Mll5 knockout mice also show the late stage spermatogenic impairment We also show by RT-qPCR and in-situ RNA hybridization that the ype mice . Furtherype mice  very clorm cells . In additm1Apahomozygous Mll5 male mice are infertile , although detection of premature or delayed expression could be overlooked in our expression analyses of whole testes. Corroborating our conclusion, post-meiotic spermatogenesis has also been observed in an independently generated knock out of Mll5tm1ApaMll5 mice, it appears that the loss of Mll5 from developing germ cells results in defects predominantly in the post-meiotic stages of male gametogenesis, although subtle meiotic defects cannot be completely ruled out.Our breeding experiments show with high statistical confidence that nfertile . This isnfertile , apoptos- testes , nor wer- testes  or teste- testes  significd bodies  and sperd bodies  and motid bodies  in the tMll5 -/- binds to but is unable to fertilize oocytes in in vitro capacitation and fertilization experiments   & 4. Fureriments . Such deMll5 by constitutive knockout in a mouse exhibits male infertility due to defects in late stage spermatogenesis or spermiogenesis resulting in spermatozoa, which are unable to fertilize oocytes in vitro as well as in vivo.Thus we conclude that Mll5 is important for normal spermatogenesis as functional inactivation of Mll5 in mice and concordantly found that functional inactivation of Mll5 results in a hematopoietic stem cell defect in vivo, microarray was performed on flow cytometry-sorted murine bone marrow cells to purify hematopoietic stem cells (HSCs) since that was the population of cells which was found to be defective in the Mll5 -/- mice Hoxb2 and Hoxb5Mll5 -/- mice and hence assayed differential expression of genes from the testes of wildtype and Mll5 -/- mice using microarray. Unlike differential expression in hematopoietic stem cells, we were able to find over 900 differentially expressed transcripts between wildtype and Mll5 -/- testes. Several genes were found to be differentially expressed in whole mouse testes from wildtype and Mll5 -/- mice by RT-qPCR analyses on independent batches of mice  and is associated with differentiation of the reproductive organs of plants Tlk2 mRNA is expressed at high levels in testes, leading to speculation about a role in gametogenesis in mammals, as in plants tm1Apamll5 mice, implicating the Ras-signalling pathway Sult4a1, which was up regulated over 2 fold in the testes of tm1Apamll5 mice in the microarray  were maintained as an inbred stock on a 129S6 (129SvEv) genetic background on a high-fat sterile diet. Mating pairs were supplemented with dough diet and sunflower seeds. Genotyping of mice was done by PCR using the primers as described elsewhere All mice were bred and maintained as approved by the University of British Columbia Animal Care Committee (A05-0699) or under the authority of a U.K. Home Office Project License (PPL80/1503). The transgenic mice , incubated with hyaluronidase  and washed in M2 or Human Tubal Fluid (HTF) .For experimental matings, male mice were singly housed overnight before the female was introduced and monitored for the presence of a plug. Mice were euthanized by raising COMll5 RNA probe within exon 3 using the protocol described before Testes from euthanized mice were dissected, fixed in 4% paraformaldehyde and stained overnight at 30\u00b0C with X-Gal. RNA in situ hybridization was performed on 10\u201315 \u00b5m sections of frozen testes using a 150-bp Embryo staining was performed as previously described 5 sperm (diluted to 1\u221225\u00d7106 / ml in capacitation buffer for 30 min before mixing with oocytes) from epididymides and vasa deferentia of wild type (Mll5 +/+) and homozygous tm1ApaMll5 (Mll5 -/-). . For assessment of fertilization, the number of two cell embryos with an extruded second polar body, 24 hours after fertilization was recorded.Female ICR mice were superovulated with an intraperitoneal (i.p.) injection of 1.5U pregnant mare serum , followed 48 hr later by an ip injection of 1.5U human chorionic gonadotropin . Thirteen hours after hCG administration, the superovulated female mice were euthanized and the oocytes were harvested and incubated for 4\u20136 hour with 2.5\u00d7102 for 30 min to allow for capacitation of the sperm. An aliquot was then taken and non-motile sperm was counted using a haemocytometer while another aliquot taken to observe sperm using time-lapse microscopy on a Zeiss Colibri AxioObserver.Z1. A third aliquot was fixed in 4% paraformaldehyde and the total number of sperm counted. The number of non-motile sperm was then expressed as a percentage of the total number of sperm. At least 100 single sperm per mouse were analyzed. For imaging, propidium iodide was used in the mounting media and the sperm heads imaged using a Nikon C1 TE2000E2 confocal microscope with a 63x objective.Testes from euthanized male (n\u200a=\u200a3 per genotype) mice were removed and the epididymides and vasa deferentia dissected into 3 ml of DMEM and incubated at 37\u00b0C / 5% COFreshly obtained tissues were fixed in 2.5% glutaraldehyde in a 0.1 M Cacodylate buffer at pH 7.3 for 2 hr and washed in the same buffer lacking glutaraldehyde three times. Next they were fixed in 1% osmium tetroxide and potassium ferrocyanide 1% in the same Cacodylate buffer for one hour followed three rinses in distilled water before being dehydrated through a graded series of Acetone to 100% starting at 30%. After two changes in propylene oxide they were infiltrated with epon 812 and then embedded in the same epoxy resin. 60 to 70 nm thin sections were viewed in the FEI Tecnai 12 Transmission Electron Microscope.tm1apaMll5 testes using QIAzol\u2122 lysis reagent . RNA was extracted according to the manufacturer's instructions  was set up in 384 well plates on the 7900HT Fast Real-Time PCR system  with the respective probes (Roche) and primers . Relativt test for continuous variables, and Pearson's chi squared test or Fisher's exact test for categorical variables. Results with a 2-sided P-value less than 0.05 were considered significant. For birth rate variables with a Poisson distribution, birth counts conditional on the total births have a multinomial distribution, from which exact p-values were obtained Pairwise comparisons were performed using Student's Figure S1Gametogenesis is grossly normal in homozygous tm1ApaMll5 mice. Representative sections of haematoxylin and eosin (H & E) stained seminiferous tubules in testis of wildtype (A\u2013B) and Mll5 -/- (E\u2013F) mice showing various different cell types. 1 \u2013 spermatogonia; 2 \u2013 spermatocytes; 3 \u2013 round spermatids; 4 \u2013 elongating spermatids; 5 \u2013 Sertoli cells, 6 \u2013 Leydig cells. Representative sections of H & E stained epididymides of wild-type (C\u2013D) and Mll5 -/- (G\u2013H) mice showing (1) coiled tubules of the epididymis which are (2) lined by columnar epithelium and (3) contain mature spermatozoa. The original magnification was X400 for all panels except for C and G, where the magnification was X100.(TIF)Click here for additional data file.Figure S2Representation of Microarray data showing outliers in yellow. Plot of fold change (FC) of transcripts against their respective p-values of the transcriptomes of homozygous tm1ApaMll5  (KO) to Mll5 +/+ (WT) testes as determined by microarray. The yellow dots show the outliers and transcripts with FC>1.05 and p<0.05.(TIF)Click here for additional data file.Figure S3Filtered results from the analysis of the Affymetrix Microarray and status of RT-qPCR validation. Comparisons between genes identified as differentially expressed by microarray and their location on the mouse genome according to Mus musculus, NCBI build 37, 2007-07 from the Core Gene Level analysis between Mll5 -/- (KO) and Mll5 +/+ (WT) testes are shown.(TIF)Click here for additional data file.Figure S4Validation of short listed genes by RT-qPCR. This table shows the transcripts, primers and probes used (SYBR\u200a=\u200aSYBR green used without UPL probes) as well as the relative expression of transcripts in testes of homozygous tm1ApaMll5  [KO] mice vs wild type [WT] mice. Up regulated transcripts are highlighted in red while down regulated transcripts are highlighted in green. P values that are significant are in red text.(PDF)Click here for additional data file.Figure S5Mll5 appears to have similar residues in the SET domain to yeast SET3/4. CLUSTAL 2.0.8 multiple sequence alignment of the SET domains of Mll1(KMT2A) (P552200), Mll4 (O08550), Mll2 (KMT2B) (Q6PDK2), Mll3(KMT2C) (QBR4H), Mll5(KMT2E) (Q3UG20), yeast SET3 (P36124) yeast SET4 (P42948) and Setd7 (Q8VHL1). Conserved structural residues are marked in green and residues important for catalysis in red. Critical residues not conserved are marked in purple.(TIF)Click here for additional data file.Table S1Mll5 -/- female mice are fertile.(DOC)Click here for additional data file.Table S2Mean testes weights from Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Table S3Mean testosterone levels in Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Table S4Apoptosis in Testes from Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Data S1Supplemental data.(DOC)Click here for additional data file.Methods S1Supplemental methods.(DOC)Click here for additional data file.Video S1Mll5 -/- female mice are fertile. Time-lapse video microscopy showing differences in motility of sperm from Mll5 +/+ and Mll5 -/- mice.(MP4)Click here for additional data file."}
+{"text": "UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of  Aberrant epigenetic modifications are a feature of several human diseases, including cancer. While several forms of epigenetic modification are known to exist, so far DNA methylation is the only one shown to directly target DNA and to be frequently aberrant in many tumor types PTEN, CDKN2A/p16INK4A and RASSF1A often have expression reduced through CpG island methylation TSPY, HOXB13 and SYK as novel TSGs in melanoma Melanoma genomics studies have identified a large number of chromosomal loci that show repeated loss of heterozygosity (LOH), highlighting widespread chromosomal instability PPP1R3C  subunit 3C), ENC1 , RARRES1  and TP53INP1 (tumor protein p53 inducible nuclear protein 1), had mRNA levels that were inversely correlated with promoter methylation (>40\u201360% of CpG sites) in 35\u201359% of melanoma cell lines and 6\u201325% of the fresh tumors.In a previous study, we combined 5AzadC treatment with Trichostatin A (TSA), an inhibitor of class I and II histone deacetylase enzymes, and conducted a microarray-based analysis on a panel of melanoma cell lines identifying eight highly \u2018reactivated\u2019 genes (expression fold change >4), not previously known to be epigenetically silenced in melanoma In order to identify additional epigenetically silenced genes implicated in melanocytic neoplasia, we have generated new data from 11 melanoma cell lines using Illumina Infinium Methylation27 arrays PPP1R3C, ENC1, RARRES1 and TP53INP1 that were not previously known to be silenced by DNA methylation in melanoma In our previous study, we used a microarray-based strategy in a panel of 12 melanoma cell lines treated with 5AzadC and TSA as an initial screening approach. Select candidate genes were followed up using the Epityper assay in a much larger panel of melanoma cell lines, as well as a panel of fresh-frozen melanoma samples, normal melanocyte cultures, and cell lines from other cancer types. We identified four genes, In order to use more robust criteria to select the genes epigenetically silenced during the development of melanocytic tumors, we have generated new data on 11 melanoma cell lines using Illumina Infinium Methylation arrays. The Infinium Methylation chips interrogate 27,578 CpG loci covering more than 14,000 genes. We screened 11 melanoma cell lines from our pilot study ADM, ENPP2, RAC2, SERPINE1), genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as \u201corf\u201d , and false positives on the Infinium Methylation chips .These new data were then integrated with previous data-sets of global mRNA expression COL1A2, CRABP2, CRIP1, FAM46B, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D, UCHL1 and VAMP8). The genes were further filtered by validating the differences in their expression levels pre- and post-treatment. Transcript levels of these 12 genes were assessed by qRT-PCR in the 5 cell lines with the highest expression differences before and after treatment. Selecting only the genes with a >4\u2013fold average qRT-PCR expression change and the presence of CpG islands in the promoter gave a final list of 10 genes: COL1A2, CRABP2, CRIP1, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D and UCHL1. While the absolute value of the fold-changes varied somewhat between the two methodologies, there was generally good agreement between the microarray and qRT-PCR results . Importantly, for each of these 10 genes both techniques showed a >4\u2013fold average change in expression after drug treatment.These additional filters resulted in 12 genes remaining for initial follow up (able S1) and subjected to the Epityper assay. Three genes were not considered further as they either failed to give any analysable data, probably due to the high CpG density of the region (CRABP2), or showed no differences in methylation profiles between melanocytes and the melanoma cell lines (LOX and RGC32).We then quantitated the degree of methylation of these 10 candidate genes using mass spectrometry of base-specific cleaved amplification products . These informative CpG sites were then scored in each amplicon defining the CpG island for each gene. Only the CpG sites presenting high methylation ratios (as defined below) in melanoma cell lines were averaged for the final percentage of methylation (% of methylation). This allowed grouping of the melanoma cell lines following their % of methylation: no/low methylation (0\u201320%), medium (20\u201350%) and high methylation (>50%). We then defined the average % of methylation for each gene as the average value across the melanoma cell line panel which was then compared to melanocytes.For each gene, the amplicons included in the analysis were those with different average methylation values of >20% in the entire panel of melanoma cell lines and with <10% methylation in melanocytes . For TNFRSF10D, 5 cell lines showed a 5-fold average increase in expression.Following 5AzadC+TSA treatment, In melanoma cell lines, the average % of methylation for these genes was 24%, 31% and 66% respectively. The methylation levels of these genes in the melanocyte pools were close to background (between 2 to 9%).COL1A2 and THBS1 were both divided into 5 amplicons, while 4 amplicons were designed to cover the 5\u2032UTR and first exon of TNFRSF10D . For each of the three genes, the amplicons closest to the transcription start (within 1000 bp) showed high levels of methylation in the melanoma samples, which inversely correlated with mRNA expression (Table S1). Table S4 summarizes the number of CpG sites scored for the definition of the % of methylation for each gene .The 5\u2032UTR regions around the transcription start sites of COL1A2 mRNA expression, which correlated with a high degree of COL1A2 promoter methylation in 67% of this subset. The other 19 cell lines with detectable COL1A2 mRNA expression all showed <20% of methylation, with the exception of 3 lines . .Over fifty percent (21/40) of the melanoma cell lines had no TNFRSF10D, 31 of 43 (72%) melanoma cell lines had no mRNA expression. Of the 31 lines not expressing TNFRSF10D, 28 (90%) showed >60% of methylation . For THBS1, 15 of 43 (35%) melanoma cell lines had no mRNA expression. Eight of the 15 (53%) lines with no THBS1 mRNA showed a high >50% methylation .For UCHL1 expression was increased by an average of 18-fold in 10 out of 12 cell lines. For the Epityper assay, the region around the 5\u2032UTR and transcription start site of UCHL1 was divided in to 4 amplicons, also covering both exons 1 and 2 . While amplicons 2 and 6 did not show any differential methylation between melanoma cell lines and melanocytes (Table S1), amplicons 4 and 8 showed specific profiles of methylation in melanoma cell lines inversely correlated with mRNA expression (Spearman\u200a=\u200a\u22120.53). A majority (56%) of the melanoma cell lines had no UCHL1 mRNA expression, which correlated with a high degree of UCHL1 promoter methylation in 79% of them . The melanoma cell lines with high UCHL1 mRNA expression have <20% methylation .Following 5AzadC+TSA treatment, Next, we assessed protein levels for THBS1 and UCHL1  by western blot analysis Figure 3COL1A2, THBS1 and TNFRSF10D . THBS1 promoter methylation levels were 3.5 and 2 times higher in melanoma cell lines and tumors respectively.Simultaneously, in the Epityper assay, we included 30 fresh-frozen melanoma tumor samples and found that 13%, 15% and 30% of them respectively were methylated for NFRSF10D Table 1.UCHL1 promoter appeared to be 8-fold more methylated in melanoma cell lines compared to melanocytes, and 4-fold more methylated in the 30 fresh tumors compared to the same control. Since mRNA levels were not assessed in the tumors, we are unable to correlate the proportion of methylation with expression in these samples. The observed methylation rates in tumors are lower than in the melanoma cell lines, as expected due to stromal contamination, which will likely have the effect of decreasing the observed overall percentage of methylation of the DNA assessed.The COL1A2, THBS1, TNFRSF10D and UCHL1. Using the same cut off as for the melanoma cell lines and tumors, the percentage methylation for the COL1A2 and UCHL1 CpGs islands was 65% and 82% in the esophageal cancer cell lines respectively and 92% and 90% in the colon cancer cell lines respectively. While THBS1 was only methylated in the colon cancer cell lines (43% methylation), TNFRSF10D appeared to be the only gene methylated in the glioma lines (33%) . These different cancer types were assessed to determine whether the candidate genes might play a more general role in tumor suppression. Two cell lines from each tumor type were assessed for their methylation status for es (33%) Table 1.COL1A2, THBS1, TNFRSF10D and UCHL1 respectively.The objective of this study was to combine different array platforms to strengthen the identification of novel TSGs inactivated by promoter methylation in melanoma. Selection of candidate genes was based on reduced expression in a panel of melanoma cell lines which correlated with a high methylation profile, and lack of the same observation in melanocyte cultures. Using these criteria we identified and subsequently confirmed four genes silenced by DNA methylation in melanoma. We found 24%, 31%, 66% and 42% of cell lines and 13%, 15%, 30% and 21% of tumors were methylated for et al. and Koga et al.COL1A2 as methylated in 35% (7/20) to 89% (16/20) of melanoma tumor samples respectively. In their systematic methylation profiling of several human cancer cell lines, Paz et al. reported reactivation of THBS1 expression following 5AzadC treatment in all 18 melanoma cell lines analysed et al.TNFRSF10D promoter methylation in 85% of their melanoma cell lines (17/20) and 80% of their fresh melanoma tumor samples (32/40). In their study of the molecular effects of low dose of 5AzadC (Decitabine) on 8 melanoma cell lines, Halaban et al.COL1A2, TNFRSF10D, THBS1 and UCHL1. Here, we further document the link between these genes and melanoma by confirming the correlation between methylation and expression of these genes in a larger panel of melanoma cell lines. In our study, we assessed 45 melanoma cell lines, 30 fresh tumor samples and correlated methylation status and mRNA expression levels to observations made in pooled melanocytes. Furthermore, while the majority of the previous methylation studies were based on the analysis of gene re-expression post-5AzadC treatment but not on assesment of promoter methylation per se, we present here a study design which goes beyond 5AzadC treatment to include a precise CpG methylation profile via the Epityper assay, a sensitive and high-throughput method for DNA methylation analysis.Each of these four genes had previously been linked to melanoma. Muthusamy COL1A2 aberrant promoter methylation has been described in different cancer cells such as breast cancer, medulloblastoma, hepatoma, colorectal cancer et al. describe the possible advantages a decrease in collagen synthesis may confer on cancer cells, including faster cell growth and increased tumorigenic potential Within the large collagen family, collagen type I is the most abundant, structural component of healthy connective tissue and consists of a heterotrimer of two \u03b11 (COL1A1) and one \u03b12 (COL1A2) chains. Cellular p53 negatively regulates COL1A2 through TGF-\u03b2 signalling in normal dermal fibroblasts THBS1  encodes the glycoprotein thrombospondin, which is generally considered a tumor suppressor and mediates cell-to-cell and cell-to-matrix interactions important for platelet aggregation and angiogenesis THBS1 by methylation has been described in several cancer types such as neuroblastoma Down-regulation of TNFRSF10D  is a member of the TNF-receptor superfamily containing an extracellular TRAIL-binding domain and a truncated cytoplasmic death domain. This receptor does not induce apoptosis but has been shown to play an inhibitory role in TRAIL-induced apoptosis TNFRSF10D expression is directly regulated by p53 and regulates cellular chemosensitivity TNFRSF10D promoter hypermethylation has been described as a mechanism of inactivating this gene in several cancer types The protein encoded by UCHL1  inactivation by promoter methylation in melanoma. This gene encodes a peptidase activator of the ubiquitin-dependent protein degradation pathway. Originally identified in neurons and in cells of the diffuse neuroendocrine system, mutations in this gene have been associated with Parkinson disease UCHL1 methylation has been reported in multiple tumors UCHL1 methylation as a biomarker for diagnosis and prognosis of certain tumors et al. recently showed in nasopharyngeal carcinoma cell lines that UCHL1 was a member of the p53/p14ARF/MDM2 complex UCHL1 regulatory region.The current study details COL1A2, THBS1, TNFRSF10D and UCHL1) we identify here as methylated in melanoma, encode components that fit within the p53 ontology pathway. Moreover, two of the genes we had identified in a previous methylation study are also associated with p53 function, either being induced by p53 (ENC1) or interacting with p53 (TP53INP1) Interestingly, all four genes , one common methylated gene, A panel of 12 melanoma cell lines derived from primary cutaneous melanomas or their metastases were used http://www.atcc.org/).The 2 colorectal cancer cell lines Co115 and LIM 2405, esophageal cancer cell lines OE19 and OE33, glioma cell lines T46 and T50 were purchased from the American Type Culture Collection . All samples were run on an Agilent Bioanalyzer  using a DNA 12000 LabChip kit to check for DNA integrity, purity and concentration. 500 ng of genomic DNA from 11 melanoma cell lines and a reference pool of melanocytes derived from several donors were bisulfite treated using an EZ-96 DNA methylation kit  as per the manufacturer's instructions. They were then hybridized to Infinium Methylation BeadChips  containing 27,578 CpG loci covering more than 14,000 genes http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE28356.Percent methylation (beta) was calculated from the ratio of fluorescent signal intensities of the methylated (M) and unmethylated (U) alleles, for each sample at each specific CpG site, using the equation beta\u200a=\u200aMax/[Max+Max+0]\u00d7100. On this scale unmethylated sites are represented by beta values close to zero, while heavily methylated sites show values approaching 100%. We then expressed each site specific melanoma cell line methylation value as a delta difference, compared to that of melanocytes. In this way negative values represented a cell line and site specific decrease in methylation, while positive scores indicated a relative increase in the degree of methylation. The provided manifest file linked individual methylation sites to official gene symbols, which we used to associate the methylation data to that of mRNA expression and demethylation, as described below. The data are MIAME compliant and have been deposited in Gene Expression Omnibus database (These data (GSE7127) were generated as part of previously published studies These data were generated as part of a previously published study Expression profiles generated for each cell line before and after drug treatment (expressed as a fold-change ratio) showed that across the panel of 12 cell lines a total of 8,144 non-redundant genes were re-expressed with >2-fold change after treatment . Genes reactivated in all 12 cell lines were removed from further analysis since they are likely to represent cellular stress response to 5AzadC treatment, or promoter demethylation of genes normally silenced within the melanocytic lineage.This filtering left 3125 genes to be considered from cross-analysis with the Beadarray27 methylation and U133 Plus 2.0 mRNA expression data described above.Fold-change expression profiling data (compared to melanocyte pool), fold-change demethylation data (compared to matching untreated cell line) and Illumina methylation profiling data (delta % methylation compared to the melanocyte pool) for each of the 11 cell lines for which all data were available, were imported into Microsoft Excel and linked via official gene symbol (HUGO Gene Nomenclature Committee). Data filters were then applied to each of the three data types in order to identify genes with clear evidence of methylation  and evidence of reduced expression compared to the melanocyte pool. Genes symbols were filtered to identify those in which at least 2 samples showed \u226560% methylation (Beadarray27) correlated to an average post-demethylation re-expression fold-change of >4 and 4-fold mRNA global decrease across the panel of 11 melanoma lines. From this set of 26 genes, after removing oncogenes , genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as \u201corf\u201d , and the presence of false positives on the Infinium Methylation chips , 16 genes remained for initial follow up . Of these, 12 genes  were subject to further validation as described below.mRNA extraction & expression array data were obtained as previously described The Sequenom EpiTYPER assay is based on in-vitro transcription and base-specific cleavage of a PCR amplicon and the subsequent analysis of the resulting RNA fragments by MALDI-TOF mass spectrometry 5\u2032-AGGAAGAGAG-fw primer-3\u2032, reverse: 5\u2032-CAGTAATACGACTCACTATAGGGAGAAGGCT-rev primer-3\u2032) to allow further in vitro transcription. One microliter of modified DNA was used for the PCR reactions carried out in a total volume of 5 \u00b5l. Unincorporated dNTPs were dephosphorylated by incubation at 37\u00b0C for 40 min in the presence of shrimp alkaline phosphatase (SAP) (Sequenom). Two microliters of this SAP-treated PCR mixture were used as a template in a 7 \u00b5l transcription reaction containing RNase A and T7 polymerase (Sequenom). Transcription and digestion were performed simultaneously at 37\u00b0C for 3 h. After the addition of 20 \u00b5l of H2O and 6 mg of CLEAN resin (Sequenom), 22 nl of the cleavage reactions were dispensed onto silicon chips preloaded with matrix . Mass spectra were collected using a MassARRAY mass spectrometer (Bruker-Sequenom) and analysed using proprietary peak picking and signal-to-noise calculations (Sequenom Epityper v1.0.5).EZ-96 DNA methylation kits  were used for bisulfite treatment of 1 \u00b5g of genomic DNA from 44 melanoma cell lines, 1 nevus cell line, 2 colorectal cancer cell lines (Co115 and LIM 2405), 2 esophageal cancer cell lines (OE19 and OE33), 2 glioma cell lines (T46 and T50), and 30 fresh-frozen melanoma tumors. DNA from pools of melanocytes was used as reference. Each gene promoter was divided into several amplicons . The tarThe relative amount of methylation (% methylation) was determined by comparing the signal intensities between the mass signals of methylated and non-methylated template.For data analysis only unique CpG units (units can contain one or more consecutive CpG dinucleotides) are included. CpG units overlapping with other cleavage fragments in the mass spectrum were excluded from data analysis. CpG methylation ratios were filtered using an uncertainty threshold of 10%. Only data values (CpG methylation ratios) with an estimated error smaller than 10% were included in the analysis. This filtering ensured that only precise data values were used for downstream calculation.For each gene, the amplicons presenting no significant difference in methylation between melanoma cell lines and melanocytes were dropped from the analysis . In each2)/n) of the Spearman coefficient.Spearman test was applied for the correlation between mRNA expression and methylation of the genomic region assessed. A t-test (t\u200a=\u200ar/Sr) was performed to obtain the significance Click here for additional data file.Figure S2UCSC browser for localisation of the amplicons used for the Epityper assays. The alignments show the localisation of the Illumina probes.(PDF)Click here for additional data file.Figure S3Distribution of the melanoma cell lines according to their methylation status for a. COL1A2 mRNA expression, b. TNFRSF10D mRNA expression, c. THBS1 mRNA and protein expression, d. UCHL1 mRNA and protein expression. The melanoma cell lines were grouped following their methylation profiles: high (>50%), medium (20\u201350%) and no/low (0\u201320%).(PDF)Click here for additional data file.Figure S4Epityper results for the COL1A2, THBS1, TNFRSF10D and UCHL1 promoters in melanocytes, 45 melanoma cell lines, 30 fresh melanoma tumors and cell lines from other tumor types . The software uses a color coding to show the range of methylation: red to yellow for 0 to 100% of methylation. While the melanocytes show no methylation across the amplicon, the melanoma cell lines and fresh tumors present different patterns of methylation.(PDF)Click here for additional data file.Table S1Primers for PCR. Each gene promoter was divided into different amplicons. The target regions were then amplified using the primer pairs and annealing temperatures defined by the MethPrimer program.(XLS)Click here for additional data file.Table S2Primers for qRT-PCR.(XLS)Click here for additional data file.Table S3Levels of gene reactivation in a panel of 12 melanoma cell lines post-5AzadC+TSA treatment.(PDF)Click here for additional data file.Table S4Informative CpG sites count. For each gene, only the amplicons presenting a significant methylation profile difference between melanoma cell lines and melanocytes were scored. In each amplicon, only the informative SpG sites were counted for the final % of methylation value for each gene.(PDF)Click here for additional data file."}
+{"text": "GDF9) and bone morphogenetic protein15 (BMP15) mRNA expression in cumulus-oocyte complexes (COCs) of buffalo ovary during in vitro maturation (IVM). GDF9 and BMP15 are expressed specifically in mammalian oocytes and also participate in cumulus-oocyte crosstalk. Quantitative real-time polymerase chain reaction (qRT-PCR) technique was applied to investigate the relative abundance (RA) of GDF9 and BMP15 mRNA transcripts throughout the IVM process. Relative mRNA expression pattern of these specific genes were assessed in oocytes and cumulus cells at 0, 6, 12 and 24 h of in vitro culture. Our results revealed that RA of GDF9 during different hours of IVM showed significant reduction between 0 h and 24 h of maturation in oocytes and BMP15 transcript increased significantly (P<0.05) between 6 h and 12 h and decreased again between 12 h and 24. In cumulus cells, GDF9 remained stable during IVM upto 12 h of maturation and decreased significantly between 12 h and 24 h of maturation. Conversely, significant reduction of BMP15 was observed between 0 h and 6 h, stayed stable upto 12 h and became undetectable at 24 h of maturation. In conclusion, these two genes were differentially expressed during the period of oocyte maturation process and notably, BMP15 expression pattern is associated specifically with the period of cumulus cell expansion.The present study has evaluated the association of growth differentiation factor9 ( GDF9 and BMP15 have been involved in the control of ovulation rate in mammals . This stage specific expression through intra-follicular cascade at the correct time is vital for follicular growth in order to reach the ovulatory phase  play numerous role in ovarian folliculogenesis. In particular, a growing body of evidence in recent years indicated that two famous members of Transforming growth factor-\u03b2 superfamily, Campbell . In thisBubalus bubalis) is a multipurpose livestock animal. It is of particular important production animal due to the source of high quality animal protein in developing countries  technique for mRNA expression of GDF9 and BMP15 genes. Accordingly, the current work will provide important leads for understanding the mRNA transcriptional level of GDF9 and BMP15 genes in oocytes during IVM in case of buffalo.The domestic Asian water buffalo  unless otherwise stated. Disposable plastic wares used were purchased from Nunc, Denmark.et al.  normal saline to the laboratory within 2-3 h. In the laboratory, ovaries were washed five times in modified Dulbecco\u2019s phosphate buffered saline (mDPBS) containing gentamicin. Oocytes were aspirated with a 10 cc syringe using 20 gauge needle into 50 ml conical tubes from follicles (>3 mm in diameter) of buffalo ovaries. The aspirated COCs were graded based on morphological appearance of the cumulus cell investments and homogeneity of ooplasm under stereo zoom microscope as described by Nandi et al.  and the et al.The maturation of oocytes was evaluated under stereomicroscope based on the degree of cumulus expansion  were treated with 0.1 per cent trypsin-EDTA for 2\u20133 min and mechanically denuded of surrounding cumulus cells by repeated pipetting. The cumulus-free oocytes were observed under stereo zoom microscope to ensure that they were free of cumulus cells. The oocytes were washed twice in PBS and transferred to a fresh 1.5 ml micro centrifuge tube with minimum volume of PBS and then frozen at \u221280\u00b0C. After the removal of cumulus free oocytes, the remaining cumulus cells in PBS were transferred to micro centrifuge tube and centrifuged at 300 g for 2 min according to Caixeta et al. . The supin vitro matured oocytes (MO 50), cumulus cells (cells removed from 50\u201375 COCs) using RNeasy Plus Mini Kit  and eluted in 30 \u03bcl RNase free-water. The purity and concentration of isolated RNA was determined by ND1000 spectrophotometer  and purity was assessed using the A260/A280 nm ratio with expected values between 1.8 and 2.0. The cDNA synthesis was carried out with 6 \u03bcl of RNA using Superscript III first-strand synthesis system  and random hexamer primer in a final reaction volume of 25 \u03bcl. The cDNA synthesis reactions were carried at 25\u00b0C for 10 min for annealing, 50\u00b0C for 60 min for extension, followed by heat inactivation of the enzyme at 85\u00b0C for 5 min. Reactions without Reverse transcriptase served as controls. After termination of cDNA synthesis, each RT reactions were diluted with nuclease-free water to a final volume of 50 \u03bcl and stored at \u221280\u00b0C till further use.Total RNA was extracted from pools of immature (IM 75), Bubalus bubalis complete mRNA sequence of GDF-9 (GenBank: FJ529501.2), BMP-15 (GenBank: EF375880.1) and GAPDH (GenBank: GU324291.1). Oligo Analyzer (1.1.2 Version) software was used to determine the Tm, GC per cent, primer loops, primer dimers and primer-primer compatibility. The FastPCR Professional (6.2.45 beta 4 version) has been used for Real time PCR primer designing and the oligonucleotide primers were synthesized from Sigma-Aldrich (USA). The designed primers were optimized prior to quantification experiments using polymerase chain reaction. For these genes, the expected sizes of the products were confirmed by gel electrophoresis on a 2% agarose gel. The primer sequences, expected fragment size of amplified products and genbank accession numbers are shown in Table\u00a0Oligonucleotide primers for qRT-PCR analysis were designed from Real-time PCR was performed to quantify the mRNA transcripts of GDF-9, BMP-15 in oocytes and cumulus cells using SYBR Green JumpStart Taq ReadyMix kit from Sigma-Aldrich, USA. Each cDNA sample was analysed in duplicate using real-time thermal cycler . A non template control (NTC) was prepared using DEPC water without cDNA. Reactions were performed in 20 \u03bcl volumes and loaded in the real-time qPCR 96-well plate (10 \u03bcl/well) and the plate was centrifuged in cooling (4\u00b0C) plate at 560 rpm for 2 min to remove the air bubbles and kept for reaction in real time thermal cycler.GDF9 and BMP15 genes using 10-fold dilutions of the sample and resultant slope of the curve was utilized for the calculation of the quantitative expression of both the target genes. The Ct values were recorded for both the target  and endogenous control genes (GAPDH). The data were accepted only if the NTC had no amplification.The PCR cyclic conditions were 95\u00b0C for 10 min followed by 40 cycles of denaturing at 95\u00b0C for 15 seconds and then annealing  for 1 min. The standard curve was obtained for GAPDH, Data were quantified using the method of relative quantification in qPCR Pfaffl . The thrStatistical analysis of the data was done as per the standard procedures of Snedecor and Cochran . The genTotal of 692 culturable grade oocytes were collected in a batch of experiments and 20\u201325 oocytes were cultured in 100 \u03bcl medium at each time-point to observe the progress of IVM. The COCs morphological changes were found at different time periods of culture  variation in the RA of GDF9 transcripts in cumulus free oocytes at between 0 h and 24 h of maturation. A significant (P<0.05) variation in the RA of BMP15 transcript was observed in cumulus free oocytes of different maturation times. It increased between 6 h and 12 h of maturation and reduced between 12 h and 24 h.The mRNA expression of s Figure\u00a0A. SpecifGDF9 and BMP15 mRNA transcripts in cumulus cells during different hours of IVM were presented in Figure\u00a0GDF9 remained stable upto 12 h of maturation, but decreased significantly at 24 h of maturation. However, BMP15 showed a significant reduction in the RA between 0 h and 6 h, stayed stable upto 12 h and become undetectable at 24 h of maturation.The differential expression pattern of GDF9 and BMP15 play essential roles during follicle maturation through actions on granulosa cells. GDF9 and BMP15 expression are less well characterized especially during IVM of COCs. Oocyte maturation is one of the important stages for the successful production of embryos in vitro. Moreover, it is well known that oocyte developmental potential is a reflection of proper maturation. So understanding the molecular mechanisms regulated during maturation is of great importance to optimize the IVM conditions. Since GDF9 and BMP15 are considered as important oocyte maturation factors during folliculogenesis  between 6 h and 12 h and decreased again between 12 h and 24. This increase in BMP15 transcript may be correlated with the cumulus expansion after 12 h of maturation in vitro. Similar to our finding, Li et al.  during IVM; It showed gradual increase between 6 h and 12 h and decreased again between 12 h and 24. However in cumulus cells, GDF9 remained stable during IVM upto 12 h of maturation and decreased significantly between 12 h and 24 h of maturation. In case of BMP15 significant reduction was observed between 0 h and 6 h, stayed stable upto 12 h and become undetectable at 24 h of maturation. In future, further characterization of these genes from in vivo matured buffalo COCs will help in better understanding of the GDF9 and BMP15 role in oocyte maturation of this species.To conclude, during maturation of COCs"}
+{"text": "With dipyridamol stress cardiac magnetic resonance imaging (DSCMR), we obtain high spatial resolution images that allow us to assess the transmurality of hypoperfusion but the clinical usefulness of this information has not been evaluated.We aimed to asses the usefulness of visual estimate of transmurality in relation to the coronary treeWe reviewed the CMR data base( 2008-2009) to obtain data from patients with positive DSCMR and medical records to know the coronary tree. We visually asses transmurality of hypoperfusion in each of the 17 segments in all patients studied and hypoperfusion was classified as> or <50% of the segment area.We studied, 112 consecutive patients 108 of them had performed. coronary angiography, 66 males(61%), Mean age 66\u00b110 years 5 (4.6%) without significant coronary lesions (PPV: 95%). Patients with hypoperfusion > 50% in any segment (79 patients 73%) had a number significantly higher of affected vessels , greater number of segments with systolic dysfunction induced (SDI) and greater number of territories affected 1,7\u00b10,7 vs 1,4\u00b10,6 p=0.04). The number of segments with hypoperfusion> 50% was significantly higher in patients with SDI 1.the presence of hypoperfusi\u00f3n > 50% indicates greater severity of coronary artery disease (CAD) with more vessels and territories afected.2.The number of segments with severe hypoperfusion is higher in patients with systolic dysfunction induced. 3. The transmurality of hypoperfusion is an indicator of severity of CAD and should be reported routinely"}
+{"text": "Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported.Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from  This proceeds in photosynthetic membranes, called thylakoids, which are densely folded inside chloroplasts and heavily packed with protein-pigment complexes Chlamydomonas reinhardtiiThe recent years had witnessed discoveries demonstrating that environmentally-dependent differential phosphorylation of thylakoid membrane proteins regulates lateral migration Chlamydomonas reinhardtii involves a functional coupling of phosphorylated CP29 protein to PSI 72 or Thr74 and phosphorylation of Lhcb4.2 at Thr78 or Thr806 is known as STN7-dependent stn7 and stn7stn8 mutants lacking STN7 is severely retarded under fluctuating high/low light conditions in comparison with stn8 and wild type plants stn7, stn8 and stn7stn8 plants exposed to high light had yet been reported.The photosynthetic state 1 to state 2 transition in stn7, stn8 and stn7stn8 plants exposed to high light. We mapped the sites of phosphorylation in the membrane proteins, quantified phosphorylation of the PSII core proteins under high light and found high-light- and STN7-dependent phosphorylation of CP29 variants Lhcb4.1 and Lhcb4.2. Using immunoblotting and mass spectrometry we conducted a study on the differences in composition of thylakoid protein complexes separated by blue native gel electrophoresis from the plants exposed to normal or high light. We revealed that high light treatment caused relocation of the CP29 protein from the PSII supercomplexes to PSII monomers and dimers and this movement, as well as the phosphorylation of CP29, was lost in the mutant plants lacking the STN7 protein kinase.In this work we analyzed the phosphoproteome of the thylakoid membranes in Arabidopsis wild type, stn7, stn8 and stn7stn8, deficient in the STN7 and STN8 kinases involved in phosphorylation of several major thylakoid proteins stn7, stn8 and stn7stn8 mutants (72 or Thr74) and Lhcb4.2 (phosphorylation at Thr78 or Thr80), was STN7-dependent  at Thr6. This phosphorylation of CP29 in stn7 was 4 to 5 times lower than in the wild type  stn8 (SALK 060869) stn7stn8\u22122 s\u22121 with a photoperiod of 8 h light and 16 h dark was used and in the case of high light experiments photosynthetic flux of 900 \u00b5mol photons m\u22122 s\u22121 was applied.Arabidopsis thaliana wild type (ecotype Columbia) plants, n-dodecyl-\u03b2-D-maltoside. To separate proteins in the second dimension, single lanes were cut out and incubated with 5% \u03b2-mercaptoethanol in SDS sample buffer for 30 min at room temperature and then subjected to SDS-PAGE using 15% acrylamide gels. For immunoblotting the gels were incubated in blotting buffer  for 30 min before the proteins were transferred to a PVDF membrane . The D1, Lhcb1, PsbS and anti-phosphothreonine antibodies were described previously Thylakoid isolation was essentially done as described in 4HCO3, 10 mM NaF to a final concentration of 2.5 mg of chlorophyll/ml and incubated for 3 h at 22\u00b0C with a sequencing grade-modified trypsin from Promega  at 5 \u00b5g of enzyme/mg of chlorophyll and the released peptides were prepared and analyzed as in stn7 mutant thylakoids were esterified using either d0-methyl alcohol or d3-methyl d-alcohol (Sigma Aldrich). The isotope-labeled peptides were mixed 1\u22361 before phosphopeptide enrichment using IMAC Isolated thylakoids were resuspended in 25 mM NHFigure S1Peptide identification views from MASCOT MS data analyses of phosphorylated peptides sequenced by collision induced dissociation (CID) or electron transfer dissociation (ETD) of their ions in the samples from the high-light-treated plants. The spectra and corresponding lists of singly and doubly charged fragment ions identified in the MASCOT search are shown.(DOC)Click here for additional data file.Figure S2The sums of the tandem mass spectra for CP29 isoforms Lhcb4.1 and Lhcb4.2 and for two core proteins from each of the three complexes: PSII (D1 and D2 proteins), PSI (PsaA and PsaB proteins) and LHCII (Lhcb1 and Lhcb2 proteins) counted in the gel bands corresponding to PSII supercomplex, PSII dimer, PSII monomer and LHCII trimer from plants exposed for three hours to either normal or high light.(DOC)Click here for additional data file.Figure S3stn7, stn8 and stn7stn8 mutant plants exposed for three hours to either normal or high light, as indicated. Immunoblotting was done using antibodies against the D1, Lhcb1 and PsbS proteins, as indicated.Blue native gel separation and analyses of the thylakoid membrane complexes from the (EPS)Click here for additional data file."}
+{"text": "Influenza virus is not known to affect wild felids. We demonstrate that avian influenza A (H5N1) virus caused severe pneumonia in tigers and leopards that fed on infected poultry carcasses. This finding extends the host range of influenza virus and has implications for influenza virus epidemiology and wildlife conservation. Panthera tigris) and two leopards (P. pardus) at a zoo in Suphanburi, Thailand, showed clinical signs, including high fever and respiratory distress, and they died unexpectedly. The animals had been fed fresh chicken carcasses from a local slaughterhouse. At that time many chickens around Suphanburi were dying with respiratory and neurologic symptoms of what was retrospectively identified as H5N1 virus infection (The 2003\u20132004 avian influenza A (H5N1) virus outbreak in Southeast Asia resulted in 24 reports of fatal human cases  due to direct transmission of the virus from birds to humans. During the H5N1 virus outbreak in Thailand in December 2003 . The,,The virus isolates obtained from the tiger and the leopard contained a glutamine at position 222 (226 in H3) and a glycine at position 224 (228 in H3) in HA1, which were also found in other recent H5N1 isolates and which are related to preferential binding to avian cell-surface receptors  resulted in transient virus excretion and a temporary increase in body temperature but did not induce clinical signs of disease (\u2013This report is the first of influenza virus infection causing disease or death in nondomestic felids. Generally, influenza virus is also not considered pathogenic for the domestic cat. Experimental infection of domestic cats in the 1970s and 1980s with influenza A viruses of subtypes H3N2 from humans, H7N3 from a turkey, and H7N7 from a harbor seal (,Our findings in tigers and leopards extend the host range of this virus and, together with the findings in domestic cats ("}
+{"text": "SCL6A4 gene has been the most extensively studied functional polymorphism in psychiatry, providing worldwide evidence of its importance in several disorders, such as OCD.Obsessive compulsive disorder (OCD) is a neuropsychiatric condition that has been shown to be heritable and for which a major gene effect has been reported based on segregation studies1SCL6A4), located on chromosome 17q12in vitro compared with the short form of the 5HTTLPRThe efficacy of selective serotonin reuptake inhibitors (SSRIs) in the treatment of OCD has lead to the hypothesis of a serotonergic dysfunction in this disorderet alet alet alSCL6A4 coding region has not revealed differences in OCD patients8et alA possible association between 5HTTLPR polymorphism and OCD has been studied by some research groups. Billett SCL6A4 gene should always be performed. Family-based association analysis confirmed negative linkage disequilibrium between the 5HTTLPR polymorphism and OCD. TDT and HRR analysis did not reveal preferential \u201cl\u201d allele transmission. These studies did not support McDougle\u2019s initial hypothesiset alIn addition, many association studies have resulted in controversial results because population stratification cannot easily be avoided8et alNew evidence showing that some of the previous results may differ since the involvement of the 5HTTLPR (A/G) gene polymorphism modulating the 5HTTLPR, it may impose to new studies the need to assess this polymorphism. In addition, most association data come from case-control studies that do not assess population stratification instead of family based samples. The study presented in this issue by Tibrewal"}
+{"text": "We also found fewer numbers of circulating Pmels in mice that received Runx2 overexpressing Pmels when compared to mice that received control Pmels. In addition, there was decreased accumulation of Runx2 overexpressing Pmels at the tumor site when compared to the control Pmel . To further interrogate the role of Runx2 in T cells, we assessed the production of IFN-\u03b3 after stimulation in vitro with either plate bound anti-CD3 or gp100 expressing tumor cells. We found that IFN-\u03b3 production was comparable between Runx2 overexpressing Pmels and control Pmels after anti-CD3 stimulation. However, IFN-\u03b3 production was impaired in Runx2 overexpressing Pmels upon stimulation with tumor cells. Furthermore, in vitro characterization also revealed that Runx2 overexpressing Pmels have decreased proliferation and display an apoptotic phenotype. Taken together, our studies suggest that Runx2 regulates apoptosis, proliferation and IFN-\u03b3 production in tumor reactive T cells. Further studies to mechanistically understand these findings are ongoing.Adoptive T cell therapy (ACT) is a promising treatment for melanoma patients with a clinical response rate of about 50%. However, half of patients treated do not respond to this therapy, underlining the need for improvement . One of the limitations of ACT is the poor effector function of transferred T cells influenced by the immunosuppressive tumor microenvironment. In order to identify pathways which may contribute to this observation, we used a murine ACT model in which mice bearing established B16 tumors were treated with Pmel T cells which recognize the melanoma antigen gp100 in the context of H-2Db. Pmel T cells were recovered on day 6 and 13, after transfer, from the tumor and spleen of treated mice and their gene expression patterns were compared. We found that 720 genes were differentially expressed by T cells recovered from the tumor site compared to those recovered from the spleen. Amongst the differentially expressed genes were several transcription factors, including Runx2, Rora, E2F1 and Tcf7. After an initial in vivo screen, Runx2 overexpressing Pmels conferred a worse antitumor effect when compared to the control Pmels (median tumor size 30.7 vs 20.7 mm"}
+{"text": "ASF1 and SNF2 on derepression of the DDR genes in hydroxyurea-treated cells. Collectively, our results show that the DDR genes fall into a class in which Asf1 and SWI/SNF independently control transcriptional induction.The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF. SWI/SNF recruitment to DDR genes however does not require Asf1, and deletion of Snf2 does not affect Asf1 occupancy of DDR gene promoters. A checkpoint engagement defect is sufficient to explain the synthetic effect of deletion of  SWI/SNF is a large multi-subunit enzyme that remodels chromatin by a mechanism requiring its DNA-stimulated ATPase activity Like SWI/SNF, the non-enzymatic histone H3/H4 chaperone Asf1 also regulates the interactions of histones with DNA ASF1 and the genes affected by deletion of components of SWI/SNF PHO5 and HO genes of budding yeast PHO5, SWI/SNF functions in advance of Asf1 to promote activator binding under low phosphate inducing conditions. Subsequent stable association of SWI/SNF with the PHO5 promoter requires Asf1 HO, SWI/SNF recruitment by activator bound to the URS1 upstream promoter element paves the way for Asf1 association at a downstream site, URS2. The latter event is necessary for SWI/SNF recruitment to the downstream URS2 element PHO5 and HO depend on Asf1 and SWI/SNF for induction, and that a SWI/SNF recruitment step at these genes depends on Asf1, strongly suggests an important role for Asf1 in SWI/SNF functions that affect transcription.Microarray analysis of mRNA expression has revealed significant overlap between the genes affected by deletion of RNR3 and HUG1. This regulation has been characterized in cells treated with genotoxins that inhibit replication: hydroxyurea (HU), which causes replication fork pausing by triggering depletion of deoxyribonucleoside triphosphates (dNTPs), and methane methylsulfonate (MMS), which modifies DNA in a way that causes fork blocking. Asf1 promotes derepression of RNR3 and HUG1 upon treatment with HU RNR3 in cells treated with MMS Asf1 and SWI/SNF have also been implicated in the regulation of two well-studied DNA damage response (DDR) genes, HODrosophilaSeparate studies of Asf1 and SWI/SNF have revealed participation of both factors in transcriptional regulation of the DDR genes HUG1 and RNR3 in cells lacking ASF1, SNF2, or both, under conditions of replication stress triggered by growth in the presence of 0.2 M HU . Hydroxyurea treatment of wild type cells elicits robust derepression of HUG1 and RNR3HUG1 is more strongly derepressed than RNR3, and its expression peaks approximately 2 hours after that of RNR3  RNR3 derepression in cells treated with HU requires SNF2 , deletion of ASF1 does not have a strong effect on Snf2 recruitment to RNR3 under conditions of replication stress. We next examined the association of another SWI/SNF subunit, Snf5, with the promoter of RNR3 in wild type and asf1\u0394 cells. Although Snf5 was less readily detected by ChIP than Snf2 (see ASF1 has little effect on Snf5 cross-linking to RNR3 (RNR3 and HUG1 increases after treatment with HU or MMS RNR3 and HUG1 in response to HU is unaffected by deletion of SNF2 (compare WT to snf2\u0394 bars). Collectively, our results support the hypothesis that Asf1 and SWI/SNF can be independently recruited to the promoters of DNA damage response genes during replication stress induced by treatment of cells with HU.At Snf2 see , it appe to RNR3 . Like SWasf1\u0394, snf2\u0394, and asf1\u0394 snf2\u0394 cells grown in rich medium. As expected ASF1 null mutants  asf1\u0394 snf2\u0394 haploid strain generated using one-step gene replacement was compared to the growth rates of the asf1\u0394 and snf2\u0394 single mutants under optimal culture conditions (rich medium with glucose at 30\u00b0C). The asf1\u0394 and snf2\u0394 single mutants grew at similar rates and both were slower-growing than the congenic wild type strain; the asf1\u0394 snf2\u0394 double mutant, however, was slower-growing than either single mutant  to grow after UV irradiation, or in the presence of HU or MMS; under all these growth conditions, cells must cope with elevated structural perturbation of DNA. Consistent with shared functions for Asf1 and SWI/SNF in protecting against replication stress, ve to HU . The pro culture . Therefoasf1\u0394 cells do not have the same MMS sensitivity as snf2\u0394 cells: cells lacking ASF1 are sensitive to MMS (as previously reported) SNF2 are not . SNF2 has no effect on Rad53 phosphorylation state under normal culture conditions . Rad53 is also activated normally in the snf2\u0394 mutant when it is cultured for two hours in 0.2 M HU  asf1\u0394 and snf2\u0394 cultures at two hours after HU addition, but are in the minority in asf1\u0394 snf2\u0394 cultures  SCR1 loading control.Total RNA was isolated by hot phenol extraction Total proteins were prepared by trichloroacetic acid precipitation Tandem affinity purification of protein complexes was performed essentially as described Chromatin immunoprecipitation was performed according to Minard et al., including wash-out of hydroxyurea prior to fixation"}
+{"text": "Respiratory viruses are key in the development of exacerbations in COPD and asthma. We sought to characterise respiratory viruses to develop a model in which airway exposure to virus in addition to chronic allergen resulted in an exacerbated inflammatory and altered functional phenotype.2) of H3N2 treated animals was tested using pulse oximetry.BALB/c mice or human ICAM-1 transgenic mice (BALB/c background) were used in all assays. Animals were intranasally inoculated with either human rhinovirus 1B (HRV1B), human rhinovirus 16 (HRV16) or influenza A/Victoria/3/75 (H3N2). The kinetics of viral replication in naive animals and the lung inflammatory profiles in bronchoalveolar lavage (BAL) were assessed for up to seven days after single inoculations. The effects of HRV1B and H3N2 inoculation were further assessed in a model of chronic lung inflammation: animals were intranasally challenged with house dust mite extract (HDM) for up to seven weeks. Virus challenges were given after a robust HDM allergic phenotype developed. Some groups of animals were also treated with fluticasone propionate during and after the viral inoculation. After cessation of the challenge period, airway hyperresponsiveness (AHR) was assessed using whole body plethysmography, followed by BAL, serum and lung tissue analysis. In addition, the arterial oxygen saturation (POIn vitro cytopathic effect assays did not demonstrate that either HRV1B or HRV16 were able to replicate in lungs of relevant mice. After viral inoculation, a variable inflammatory response was detectable only in HRV1B treated animals up to 24 hours post inoculation. Animals treated with H3N2 developed a mixed inflammatory lung inflammation and a PO2 profile indicative of a viral effect on lung functionality. H3N2 also replicated well in the mouse. Single inoculations of either HRV1B or H3N2 were given to animals already challenged with HDM. There was no indication that addition of HRV1B to existing phenotype altered the HDM induced AHR or inflammatory profile. Combination H3N2/ HDM treatment resulted in an increase in BAL eosinophils and neutrophils, but no change to steroid responsiveness nor changes in PO2 readouts.Strains of human rhinovirus are variably able to elicit lung inflammation but not AHR changes following single exposure to mouse hosts. H3N2 infection resulted in viral replication and a strong inflammatory response, cleared in 7 days following infection. We were unable to demonstrate that rhinovirus infection resulted in an exacerbation type response when given to allergic animals, but steroid sensitive inflammatory exacerbation was noted in HDM/ influenza treated animals."}
+{"text": "GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal \u201cGuthrie\u201d blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the  Permanent childhood hearing loss, which occurs in 1\u20132 per 1000 live births GJB2  gene, the SLC26A4 (pendred) gene and in the mitochondrial DNA at position 1555 (A1555G mutation) and congenital cytomegalovirus (CMV) infection have been considered as the major causes of congenital hearing loss in developed countries Childhood hearing loss is an etiologically heterogeneous condition, caused by environmental factors and/or genetic factors. Despite this heterogeneity, mutations in the The study was approved by institutional ethics review boards .The LOCHI study commenced in 2005, with recruitment completed in 2007. All families with children born in Australia between 2002 and 2007 and who presented for hearing services below 3 years of age at Australian Hearing (AH) were invited to participate. Parents of children enrolled in the LOCHI study provided written, informed consent to participate. All participants were invited to give additional informed consent for molecular testing. Of the 451 eligible children, written consent was obtained for 387 children for molecular testing. There was no significant difference in hearing loss  between the consent group and the non-consent group (p\u200a=\u200a0.605). A total of 364 Guthrie card samples were obtained from the custodian authorities of health records. The sample comprised 280 whose hearing loss was detected via UNHS, 64 who did not have access to UNHS and 20 whose screening status was unknown.In Australia, the hearing of newborns is screened using automated auditory brainstem response (AABR). Infants who do not pass hearing screening are referred for diagnostic audiological assessment. These include tympanometry, middle ear muscle reflexes, otoacoustic emissions (OAEs), click-evoked auditory brainstem response (ABR) testing, and ABR or auditory steady-state responses (ASSR) testing using frequency-specific stimuli. Following diagnosis, children are referred to AH for assessment of hearing and provision of amplification, at no cost to families. Electrophysiological tests results are converted to estimated behavioral thresholds for fitting of hearing aids The hearing thresholds of participants from inception to 44 months of age were retrieved from clinical records held at the AH national database, with written permission from parents. Hearing thresholds at 0.25, 0.5, 1, 2, and 4 kHz for each ear were obtained. These audiometric thresholds were used to characterize evolution of children\u2019s hearing loss over the first 3 years of life as fluctuating, progressive, or stable. In accordance with the audiological protocol of AH, fluctuating hearing loss is defined as a change in hearing threshold of 15 dB or greater at any octave frequency between 0.5 and 4 kHz, but subsequently recover over the period of investigation. Progressive hearing loss is defined as a decrease in 10 dB or greater at two or more adjacent frequencies between 0.5 and 4 kHz or a decrease in 15 dB at one octave frequency in the same frequency range over the period of investigation. Longitudinal audiograms that do not demonstrate the changes specified above are labeled as stable.ANSD is characterized by a dysfunction in neural/brainstem transmission of auditory stimuli in the presence of normal cochlear outer hair cell function. The clinical presentation is an absent or abnormal ABR responses and the presence of otoacoustic emissions or cochlear microphonics.Information about enlarged vestibular aqueduct (EVA) was obtained from clinical records held at the AH national database. The diagnosis of EVA was based on CT scan. Radiographic imaging was not part of routine clinical service offered to children identified with hearing loss.et al. 2; 1 mg/ml Proteinase K; 1% Tween-20) at 56\u00b0C for 1 hour. Following incubation at 100\u00b0C for 7 mins. the samples were rapidly cooled on ice. The samples were centrifuged  and the supernatants frozen for >4 hours at \u221280\u00b0C.Lysates for molecular screening were prepared from Guthrie blood spots, using a modification of the method described by Barbi GJB2 (exon 2) and the splice site mutation in intron 1 (IVS1+1 G>A). Exon 2 PCR products were sequenced in both directions. The IVS1+1 G>A mutation was screened for by Hph1 digestion of a PCR product which spans the splice site. If a single mutation in GJB2 was found, connexin-30 deletions del(GJB6-D13S1830) and del(GJB6d13s1854) were screened for as described by Castillo et al.Lysates were screened for mutations in the coding region of SLC26A4 gene exons was PCR amplified. Following nested PCR, the products were analyzed by High Resolution Melt (HRM) on the Corbett Rotorgene 6000 . Primary PCR products were sequenced when HRM variants were identified.Each of the in silico approaches to assess if unreported changes in the GJB2 and SLC26A4 genes are likely to affect protein function: PolyPhen-2 We used A1555G mutation introduces a HaeIII restriction enzyme site. PCR products were digested with HaeIII and analyzed by agarose gel electrophoresis.The mitochondrial 12S rRNA et al. et al. The presence of CMV DNA was determined in a multiplex, quantitative, real-time PCR assay, using the CMV and CPOL probes published in Sanchez GJB2, two mutations for GJB2, one mutation for SLC26A4 gene, two mutations for SLC26A4 gene, CMV infection, enlarged vestibular aqueduct (EVA), conductive loss) as two-category predictors, and the interactions between etiologies and age. We used two-tailed tests for all analyses and set statistical significance at p<0.05. The statistical analysis was performed using Statistica  and R The results are summarized as means and percentages. To investigate the relationship between etiology and hearing thresholds at 3 years of age (Hypothesis 1), multiple regression analysis was carried out using the four-frequency average hearing loss  as dependent variable and etiology as a predictor variable. To determine the evolution of hearing loss, children were grouped according to the AH national protocols into those with fluctuating, progressive, or stable hearing loss. To consider the relation between progression of hearing loss and etiology (Hypothesis 2), a mixed-effects model was fitted using the longitudinal 4FA HL  hearing level) data as dependent variable, the fixed effects were age (continuous variable), the seven etiologies (one mutation for Cx26 and SLC26A4 mutation databases We use the wording \u201creported mutations\u201d for DNA and protein changes classified as causative mutations in GJB2 or SLC26A4 gene were detected in 31 (9%) children. We did not detect mutations or ambiguous changes or evidence of congenital CMV infection in 198 (54%) children. These findings were supplemented by information retrieved from clinical records and parental reports. The cause of hearing loss remained unknown for 146 children (40%).GJB2 were present in 82 children. Of these, 48 (58.5%) had two known mutations and 35 (41.5%) children had one mutation. The inheritance is in all cases compatible with a recessive inheritance pattern. Details of mutations and the evolution of hearing loss for individual children are shown in GJB2. Of the children with two mutations of Cx26, one child passed hearing screening at birth, but was diagnosed with moderate hearing loss at 19.4 months of age. Another child was diagnosed with auditory neuropathy spectrum disorder (ANSD), and two children had EVA. Ambiguous changes in the GJB2 gene were detected in 24 children, 22 of whom have one mutation and the remaining 2 have two mutations.Reported mutations of SLC26A4SLC26A4 mutation, two of whom also carried one uncertain mutation and four others also carried GJB2 mutations. For 8 other children, one ambiguous change of the SLC26A4 gene was detected. Details of mutations and evolution of hearing thresholds are shown in Reported mutations of SLC26A4 gene are associated with Pendred syndrome  and non-syndromic enlarged vestibular aqueduct . Three children who carried one mutation of SLC26A4 also had EVA, and one child who carried two mutations of SLC26A4 also had EVA. EVA was also found for 4 children who had Cx26 mutations, 1 who had CMV infection, and 8 who had normal results for the four causes tested. The number of children who had EVA could not be ascertained because information was available only for those with imaging results recorded in their clinical files.Mutations in the A1555G mutation was detected in one child. The mutation appeared homoplasmic on agarose gel electrophoresis. The child has bilateral ANSD with profound hearing loss. It is not known whether the child had been exposed to aminoglycosides.The mtDNA SLC26A4 gene. Details of the evolution of hearing loss for children who are CMV positive are presented in Cytomegalovirus infection was detected in 28 children. Of these, one child passed UNHS but was diagnosed with meningitis and hearing loss at 5.1 months of age. A second child with congenital CMV infection also had one mutation of Cx26, and was referred for unilateral hearing loss after screening. One CMV positive child also carried two mutations of the In the regression analysis examining the relation between etiology and hearing threshold levels at 3 years of age, the overall p-level for the regression model was 0.27. GJB2, SLC26A4, A1555G and CMV infection, and established a likely etiologic diagnosis for 166 (45.6%) of the 364 cases tested. Of the causes identified, a genetic cause was present in 138 children (83.1%) and an environmental cause in 28 children (16.9%).The incidence of permanent childhood hearing loss is 1 to 1.2 per 1000 at birth increasing to about 1.5 by 3 years of age et al for infants at a hospital in the US showing that 6% (16 of 256 infants with hearing loss) were CMV positive through urine cultures It has previously been suggested that hearing loss attributable to congenital CMV may be progressive in >50% of cases GJB2 gene accounted for 76.1% of the genetic cases. This frequency of occurrence is higher than the 50% previously reported for congenital, autosomal recessive, nonsyndromic, sensorineural hearing loss in the white population GJB2 mutations was not significantly different from those without the mutations is consistent with previous reports Looking specifically at the genetic causes of hearing loss in our population (n\u200a=\u200a138), mutations in the GJB2 gene was detected in 35 children. The pathogenic effect of these single mutations are uncertain, but we have attributed the hearing losses to thise GJB2 gene changes, because the proportion of children with hearing loss and one detectable recessive GJB2 mutation is significantly higher than that of the general population (approximately 1%) GJB2 gene of 24 children are also ambiguous. We have classified the V27I and E114G changes, when together, as a mutation in accordance with the Connexins and Deafness HomepageGJB2 protein is unable to form functional gap junction channels in silico analysis suggests that is unlikely to cause hearing loss. Although SIFT Sequence analysis predicts that it is \u201clikely to affect protein function\u201d, PolyPhen-2 and SIFT BLink predict it is benign Click here for additional data file.Table S2Genotype-phenotype correlation in children with SLC26A4 mutations and evolution of hearing loss. Children with SLC26A4 mutations with additional diagnostic information regarding enlarged vestibular aqueduct (EVA), auditory neuropathy spectrum disorder (ANSD) and congenital cytomegalovirus infection (CMV) are also indicated.(DOC)Click here for additional data file.Table S3Hearing thresholds at time of diagnosis and evolution in children with hearing loss attributable to congenital CMV infection.(DOC)Click here for additional data file.Table S4PolyPhen-2, SIFT BLink and SIFT Sequence predictions of novel or controversial amino acid changes on GJB2 and SLC26A4 gene product functions.(DOCX)Click here for additional data file."}
+{"text": "We and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients.27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from na\u00efve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS.Analysis of Sanger sequences of subtype C HIV-1 isolates from na\u00efve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from na\u00efve patients.While Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger. We and others From a molecular point of view, it has been demonstrated that the RT KKK nucleotide motif at codons 64, 65, 66 in reverse transcriptase of subtype C HIV-1 appears to lead to template pausing that facilitates the selection of K65R, even in isolates from untreated patients The aim of the present study was to clarify the prevalence of K65R in these subtype C isolates from patients failing on a first line including d4T or AZT. Since we had the prevalence of K65R by the Sanger sequencing method, we investigated K65R variants by ultradeep pyrosequencing (UDPS) in the same samples as those already used for bulk sequencing and sought whether UDPS provided additional knowledge to the Sanger results.Subtype C HIV-1 isolates from Indian patients failing  on a first-line treatment including d4T or AZT plus 3TC plus NVP or EFV were sequenced on RT by the Sanger method, and the sequences were recorded in the Los Alamos database (GenBank JF895621\u2013JF895673). Among these samples, 27 were randomly selected for investigation by UDPS using the Roche GS Junior equipment. RNA extracted previously from the samples was used to amplify a short region of RT with primers including specific sequences for the GS Junior system . The revSince the viral isolates obtained at initiation of first-line therapy were not available, the data obtained by Sanger and UDPS in isolates from patients at failure were compared to those of subtype C isolates from 18 na\u00efve patients. Some of their bulk sequences have been previously published by our group Regarding the 27 isolates from treated patients at failure, the drug resistance mutations (DRMs) according to the French ANRS algorithm and following bulk DNA sequencing (Sanger) are listed in Although UDPS has limitations particularly with regard to polymerization and pyrosequencing errors The Sanger results of isolates from treated patients were as expected with a predominance of M184V, numerous TAMs of pathway1  and DRMs to NNRTIs . As mentioned above, 2 isolates of the series exhibited a K65R substitution.With regard to UDPS results at codon 70, the quantitative data were different from those recorded in na\u00efve patients: 8 isolates exhibiting K70R with Sanger had UDPS K70R values above 34.60%, while 2 samples without K70R with Sanger (470 and 489) had K70R variants at quantities above the <0.4% background observed in na\u00efve patients. We hypothesize that these isolates are undergoing a process of selecting K70R mutations. Regarding the K65R values apart the two samples with K65R by Sanger, the UDPS quantities of K65R variants were low and below those of isolates from na\u00efve patients. Our results are not in accordance with those obtained by another group K65R substitutions are generated in subtype C isolates from na\u00efve patients due to the 64\u201365\u201366 motif. There are some constraints in experienced patients failing on a suboptimal regimen with d4T or AZT plus 3TC plus NVP or EFV. We first hypothesize that 184V, which was the most prevalent mutation observed in our series of treated patients at failure, has dampened the emergence of 65R as noted by others"}
+{"text": "By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the jebLamc2 mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in jebLamc2 mice. This modifier is defined by variations in 1\u20133 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the jebLamc2 mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic  Lamc2) gene to address the possibility of genetic modifiers of JEB. We document the potent impact of differing genetic backgrounds on multiple facets of the JEB syndrome expressed in these mice and show that three neighboring amino acid changes within the non-collagenous domain 4 of the collagen XVII protein strongly modify their disease. The study provides a molecular explanation of how a primary mutation that weakens one component of the cutaneous basement membrane is influenced by normally innocuous allelic variants of another component to affect strength of dermal-epidermal adhesion and consequently, the severity of JEB. This approach may guide the genetic prognosis and diagnosis of human EB disorders.Epidermolysis bullosa (EB) is a group of rare genetic Mendelian disorders that result in mechanical fragility of the skin and mucosal membranes. Junctional EB is a subset caused by mutations that result in cleavage of the dermal-epidermal junction. All forms of EB demonstrate substantial variability in their clinical phenotype that is not readily explained. The possibility of genetic modifiers as the cause of this variability has been difficult to address in humans. We apply a mouse model carrying a hypomorphic allele of the laminin gamma 2 ( Epidermolysis bullosa (EB) is a group of rare heritable disorders that result in mechanical fragility of the skin and mucosal membranes. These disorders are caused by defects in any one of at least 18 genes whose encoded proteins control the integrity of the dermal and epidermal layers LAMA3), laminin \u03b23 (LAMB3), and laminin \u03b32 (LAMC2); in either integrin \u03b16 (ITGA6) or \u03b24 (ITGB4), which form the \u03b16\u03b24 integrin heterodimer; or in COL17A1, which forms the collagen XVII homotrimer COL17A1 and can be subcategorized based on generalized or localized manifestations as well as their clinical presentations ITGA6 or ITGB4 and is classified separately based on the additional phenotype of pyloric atresia JEB typically results from mutations in any one of the three genes encoding subunits of laminin 332, a heterotrimeric macromolecule comprised of laminin \u03b13  as a major genetic modifier of JEB in jebLamc2 mice. Ultrafine recombination mapping limited the causal variations to three neighboring amino acid changes within the non-collagenous domain 4 (NC4) of this protein. This identification of a genetic modifier provides a molecular explanation of how epistasis between a primary mutation that weakens one component of the cutaneous basement membrane, laminin 332, and normally innocuous allelic variants of another component, collagen XVII, affects the strength of dermal-epidermal adhesion and consequently modifies the severity of JEB.While a unifying feature of all subforms of EB is adherence to simple Mendelian patterns of inheritance, considerable phenotypic variation in disease severity can persist even within subtypes of EB and within affected families. Genetic modifiers may account for this incomplete penetrance and the \u201cvastly different\u201d clinical phenotypes that are commonly observed jebLamc2 mutation develop a progressive form of JEB characterized most obviously by blistering and scarring of the ears and tails as the result of separations within the dermal-epidermal BM jebLamc2 allele arose on strain 129X1/SvJ and was backcrossed 10 generations onto four other strain backgrounds: C57BL/6J (B6), DBA/1J (DBA1), FVB/NJ (FVB), and MRL/MpJ (MRL). Cohorts of jeb/jebLamc2 males from each strain were aged, visually inspected weekly, and quantitatively scored for the severity of ear and tail lesions (jeb/jebLamc2 progeny from an F1 cross of the most susceptible (MRL) and most resistant (FVB) strain backgrounds. These results were consistent with co-dominant inheritance - jeb/jebLamc2 F2 and (B6 \u00d7 FVB)-jeb/jebLamc2 F2, with >200 mice per group. Both sets of mice were monitored for the age at which ear lesions were first visually detectable and genome wide scans of their DNAs were performed using single nucleotide polymorphism (SNP) markers distributed through the autosomal and X chromosomes. For analysis, the F2 datasets were combined to increase the statistical power 2 3.9\u00d710\u221211). Three QTLs were significant in multiple regression tests: chr19@42cM; chr11@32cM; and sex by QTL interaction (Sex*chr7@4cM). The sex*chr7@4cM QTL was observed only among the B6/FVB heterozygous mice. The chr11@32cM QTL dropped below the significance threshold if the sex*chr7@4cM QTL was excluded from the model, indicating it being a marginal QTL candidate. The most robust QTL (Prob>chi2 7.96\u00d710\u22127), replicated in both mouse crosses independent of sex and explaining 5.6% of the genetic variance, was located on chr19 with a 95% confidence interval of 34\u201350 cM peaking at 42 cM.Crosses were made between phenotypically disparate strains to map the modifiers. Two groups of F2jebLamc2 homozygotes. Grouping the mice by the D19Dcr40 SSLP marker mapping to \u223c47 Mb on chr19 revealed a strong but incomplete concordance between D19Dcr40 genotype and disease onset , we performed genetic association studies on cohorts of (MRL \u00d7 FVB) FjebLamc2 allele was transferred onto existing chr19 consomic stocks B6.PWDchr19 and B6.A/Jchr19 and a B6.129S1chr19 congenic strain. Ear and tail scores and tail tension test comparisons indicated that the A/Jchr19 and 129S1chr19 alleles moderately attenuated disease and the PWDchr19 allele strongly attenuated disease as compared to the B6chr19 allele  F2 mapping cross in which a chr19 QTL exists but the pattern is inverse to the overall strain phenotype: 129X1 is later onset than DBA1, but the 129X1chr19 allele confers earlier onset  to evaluate haplotype relationships in the region surrounding Col17a1 (Col17a1 genes. cDNAs from 7 of the 8 strains (except 129S1) were also sequenced from exon 1 through most of exon 53. A comprehensive list of polymorphisms among the 8 strains is provided as To broadly evaluate the candidacy of sequence polymorphisms in  Col17a1 . These rCol17a1 predicted to result in nonsense changes or altered splicing patterns. With the exception of PWD, there were no missense changes outside of the candidate 1085 bp interval of Col17a1. Limiting the analysis to the 1085 bp candidate interval defined by the PWD/B6 recombinants identified 3 non-synonymous coding SNPs , 5 synonymous coding SNPs (4 in exon 49 and 1 in exon 50) and 1 intronic SNP that distinguished B6 from PWD (PWDchr19 recombinant strains R03F and R03L (None of the strains showed variation in from PWD . Aside ffrom PWD . They weand R03L . The onl1275 N1277 T1292) shared by B6 and 129X1; Haplo 2 (S1275 N1277 I1292) unique to MRL; Haplo 3 (S1275 S1277 I1292) shared by A/J, 129S1, DBA1 and FVB; and Haplo 4 (G1275 S1277 I1292) unique to PWD (Translation of the three candidate missense SNPs identify amino acid (AA) positions 1275, 1277, and 1292 within the non-collagenous 4 (NC4) subdomain of collagen XVII. Four related haplotypes are evident: Haplo 1  and the 1000 Genomes Project  human databases to assess the potential for coding variants in COL17A1 - in populations that were unbiased in that that did not including cases of documented EB - that may be candidates for imparting EB modifier effects. From the combined EVS+1000GP databases, representing \u223c7600 individuals not known to be affected by EB, missense polymorphisms were identified for 13% (195 of 1497) of the codons of COL17A1. The minority allele frequencies of 135 missense polymorphisms among \u223c6500 individuals (those reported in EVS) are shown in Having identified certain coding variants of mouse COL17A1 mutations reported in EVS, 15% were \u2018possibly damaging\u2019 and 47% \u2018probably damaging\u2019. However, such predictions can be misleading. The most common minority allele, leading to the change T210M, found in 42% of the population and homozygous in 20% of the individuals surveyed, is predicted by Polyphen to be \u2018probably damaging\u2019, but 20% of the population does not have EB. Substitutions for Glycine, Proline and Arginine were most frequent overall . No JEB-associated nonsense mutations were found in the EVS or 1000GP databases, perhaps due to their deleterious effects. However, three known JEB-causing missense transpositions  segregating in the general population at low frequency in a heterozygous state. EVS and 1000GP also record missense variants in 6 of 15 AA positions that overlap with JEB-causing nonsense mutations ; the consequences of this overlap are not known.jeb/jebLamc2 mice. A number of JEB causing mutations have been reported to locate to exons 51 and 52 jebLamc2 mutation and varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on a form of JEB-nH. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we unambiguously show that the gene Col17a1 is a strong genetic modifier of the non-Herlitz form of JEB that develops in jebLamc2 mice. This modifier is explained concisely by variations in 1 to 3 neighboring AA in the NC4 domain of the collagen XVII protein that are normally innocuous but act epistatically to alter the strength of dermal-epidermal adhesion in the context of the jebLamc2 mutation and broadly impact the clinical severity of JEB.We have addressed the prospect of genetic modifiers of a heritable, Mendelian mechanobullous disorder. Using mice homozygous for the jebLamc2 mutation was originally described in the context of the129X1/SvJ genetic background on which the mutation was first detected jebLamc2 homozygotes in the context of multiple genetic backgrounds, a remarkable range of variation in their age of onset and overall severity was apparent. In the most extreme cases, the MRL background presented with maximal, life threatening disease scores by 10 weeks of age, while FVB did not achieve maximum scores until >47 weeks of age. Other strains demonstrated intermediate phenotypes, all supporting a typical, complex genetic model in which several allelically variant loci contribute to overall disease.The pathophysiology of the spontaneously arising UitLamc2 allele. This dichotomy likely reflects a common feature of \u2018monogenic\u2019 disorders: mutations that result in profoundly life threatening consequences will overshadow potentially modulating factors, whereas mutations in disease causing genes that result in more attenuated forms, such as the JEB-nH syndrome of jebLamc2 mice, are more susceptible to the effects of background modifiers.However, a unifying feature was their modes of action. Genetic variables envisioned to impact JEB include: 1) those that integrally contribute to the structural elements of the basement membrane; and 2) those that act secondarily through inflammation or repairing skin lesions . The fact that measurements of the strength of dermal-epidermal adhesion was predictive of disease onset and severity well before the appearance of dermal lesions and that the excellent wound healing strain background, MRL Col17a1: 1) mapping in two F2 crosses (DBA1 \u00d7 129X1- jeb/jebLamc2 F2 and B6 \u00d7 FVB-jeb/jebLamc2 F2) identified a strong QTL overlapping the Col17a1 locus; 2) phenotype to Col17a1 genotype correlations of F2 mice of the most polarized strain backgrounds (MRL and FVB) were highly significant; 3) phenotypic analysis of chr19 congenics and sub-congenics consistently framed Col17a1 as the modifier locus. Limitation of the disease modifying effects to neighboring coding changes within exon 50 of Col17a1 was strongly indicated by fortuitous recombinants from crosses of B6 and B6.PWDchr19 mice that limited the chr19 candidate interval to 1085 bp and included three non-synonymous coding SNPs.The results described herein identify relatively conservative AA changes limited to the NC4 domain of collagen XVII that are able to modulate a JEB syndrome caused by a hypomorphic mutation in the LAMC2 component of the laminin 332 complex. Multiple lines of evidence triangulated to Col17a1 using the Persikov \u2013 Singh algorithm This deep genetic resolution is unusual. PRDM9 is a long-array zinc-finger and chromatin-modifier protein that binds DNA sequences and promotes their meiotic recombination jeb/jebLamc2 mice. Moreover, it is likely that the modulatory effects of these variations are not limited to the cutaneous layer because the PWD chr19 conferred more resistant to non-cutaneous manifestations of JEB, including pulmonary functions and bone mineralization. Thus, seemingly subtle AA changes in the collagen XVII NC4 domain modify multiple pathophysiological manifestations of a JEB syndrome caused by a primary defect in a component of laminin 332.The candidacy of these missense SNPs as the cause of the JEB-modifying chr19 QTL among a number of mouse strains analyzed is strongly supported by sequence comparisons. The missense SNPs predict variation in neighboring AA positions 1275, 1277, and 1292 in the NC4 domain . CompariOur results support a model of functional epistasis in which a mutationally crippled form of laminin 332 permits normally innocuous AA changes to become functionally significant modifiers of JEB. The components of laminin 332 form an extracellular matrix heterotrimer that tethers the epidermal and dermal layers. The heterotrimer is secreted by keratinocytes assembled as N-terminal branching from a central coiled-coil structure with globular domains at its C-terminus. The standard model is that this heterotrimer bridges the lamina lucida and lamina densa by binding of its C-terminal globular domains to membrane bound \u03b16\u00df4 integrin of basal keratinocytes and its N-terminus \u03b23 and \u03b32 arms to collagen VII anchoring fibrils in the dermis COL17A1 gene can by themselves result in clinical manifestations of JEB-nH with varying severity. The carboxyl-terminal half of the collagen XVII extracellular domain has been shown to interact with laminin 332 COL17A1 and LAMB3 has also been reported where the proband was a compound heterozygote for L855X and R1226X COL17A1 nonsense mutations and heterozygous for the R635X recessive LAMB3 nonsense mutation Col17a1 modifiers may not be limited in their effect to diseases caused by mutant forms of laminin 332. In this context, it is of interest that QTL genetic analysis of mice that are experimentally induced to develop a form of EB Acquisita after immunization with a collagen VII polypeptide identified a strong QTL whose position overlaps with Col17a1Col17a1 modifiers of JEB may also be able to impact other forms of blistering diseases.A product of basal keratinocytes, collagen XVII forms a homotrimeric type II transmembrane molecule whose extracellular portion is comprised of 15 COL domains that presumably provide rigidity separated by 16 NC domains COL17A1 gene in the EVS and the 1000GP databases, unbiased by cases of documented EB, identifies numerous missense SNPs, including those that encode AA that map within and around the functionally-documented NC4 domain of collagen XVII. Such candidate modifier SNPs would be likely bypassed by conventional mutation detection-biased methods, but may act epistatically with the disease causing mutation as genetic modifiers. Thus, informed by mouse studies and focusing genetic modifier searches on allelic variants within functionally-important interaction domains of dermal-epidermal adhesion proteins may aid in the genetic prognosis and diagnosis of mechanobullous blistering disorders. Furthermore and as exemplified by this study, the elucidation of modifier loci may provide new clues into the functionally relevant interactions among these proteins that could direct new treatments for EB.The existence of remarkably potent genetic modifiers of JEB in mice raises the possibility that genetic modifiers may also contribute to the spectrum of clinical variation noted for many subforms of EB in humans. At the highest level, the considerable phenotypic and clinical variability of EB is explained by the fact that mutations in at least 18 genes can lead to these mechanoblistering disorders. The second level of variation is caused by the nature of each mutation and its penetrance. The third level, which is the focus of our study, is the extent to which missing heritability is caused by genetic modifiers. Potent modifiers of JEB are evident among strains of laboratory mice despite their simple genetic structure in which public allelic variation prevails because of related heritages and inbreeding. Humans, in contrast, have a much deeper haplotype structure with a spectrum of variation ranging from public to rare alleles and confounding genotype/phenotype associations. This is further complicated by the rarity of human cases for any given EB disease-causing mutation and lack of reliable and comparable phenotype information concerning those affected. Our survey of the All animal protocols were reviewed and approved by The Jackson Laboratory Institutional Animal Care and Use Committee .jeb/jebLamc2 and B6-jeb/jebLamc2 homozygous for the hypomorphic jebLamc2 allele were described previously jeb/jebLamc2, FVB-jeb/jebLamc2 and MRL-jeb/jebLamc2 were produced after backcrossing the 129X1-jebLamc2 allele for 10 generations onto DBA/1J, FVB/NJ and MRL/MpJ strain backgrounds and selecting to be homozygous for a congenic interval including the 129X1-defined jebLamc2 allele framed by markers D1Dcr2 and D1Dcr15 located at 150 and 160 Mb on chr1. jeb/jebLamc2 mice consomic for chromosome 19 were produced by crossing the jebLamc2 allele from B6-jeb/jebLamc2 mice to C57BL/6J-A/Jchr19/NaJ and C57BL/6J-PWD/Phchr19/ForeJ to produce strains B6.A/J Lamc2jeb/jebchr19 and B6.PWD Lamc2jeb/jebchr19. The chr19 congenic strain B6.129S1 Lamc2jeb/jebchr19 was similarly produced by a cross of B6-jeb/jebLamc2 to B6.129(Cg)-tm1DyhPnlip/J mice. The tm1DyhPnlip targeted mutation was originally made using (129X1/SvJ \u00d7 129S1/Sv)F1-+Kitl-derived R1 embryonic stem cells 129S1 Lamc2jeb/jebchr19 mice carry a large 129S1-derived chr19 congenic segment  on chr1 at \u223c150 and 160 Mb, respectively. Primers were also developed to identify both Lamc2 wt and jeb mutant bands to confirm homozygosity. Lamc2Dcr1a F- CCTGTCTCATTTCTGGTAGGCTTT and R-CACACGTCACCACACCTGCT oligonucleotide primers mapping to intron 18, and Lamc2Dcr1b- F-GCGCCAGTCCTCCGATAGA mapping to the retroviral insert were used in a three primer reaction, giving bands of 121 bp for wild-type and 218 bp for jebLamc2. Primers used to identify tm1UitLamc2 wild-type and mutant bands were as previously described All crosses which required identification of P<0.05.Mice were typically scored weekly. Ears were scored from 0 (unaffected) to 6 (severe) based on limited or overall inflammation and ear erosion. Tails were scored from 0 (unaffected) to 6 (severe) based on the generalized loss of epithelial structure, scarring, and scabbing. Time to event survival analysis using Cox proportional hazards model was used to identify strain/genotype differences on ear and tail score across different cohorts of mice. Time to event was defined as age at which subjects first reached an ear/tail score of 4. When multiple levels of strains/genotype existed within a given cohort, risk ratios were calculated across all pairs of strains to determine significant differences among strains/genotypes at P<0.05.Determination of the mechanical force in Newtons necessary to cause the dermal-epidermal cleavage of tail skin was performed on euthanized mice as previously described 2jeb/jebLamc2 and 213 (B6 \u00d7 FVB/NJ) F2jeb/jebLamc2 mice were analyzed in this study. Age of onset in weeks was identified for each animal with censored values for those \u2018unaffected\u2019 by 12 months of age. Genetic cohorts were genotyped for 147 or 136 SNP markers across the autosomal and X chromosomes spaced approximately \u223c13 Mb through the Jackson Laboratory Fine Mapping Service by KBiosciences . Data from the two crosses were combined in Pseudomarker2.02 P values for terms in the multiple regression model were calculated, and terms were dropped sequentially until all of the terms in the model were significant at the 1% level for main QTL effects and 0.1% for interaction effects.For QTL analysis, a combined cross of 243 (DBA/1J \u00d7 129X1) FRadial tail sections were cut with a razor blade, frozen in OCT on dry ice, and then stored at \u221240\u00b0C. Samples were sectioned between 10 and 12 \u00b5m thick onto super frost plus slides . The sectioned tissues were fixed in ice-cold acetone for 10 minutes, washed three times in phosphate-buffered saline, and then allowed to dry. Primary antibodies were added to slides pre-blocked with 3% horse serum for 2 hours at room temperature in a humid chamber. The slides were then washed 3\u00d7 in phosphate-buffered saline, incubated in the dark with fluorescence labeled secondary antibodies for 1 hour, washed 3\u00d7 and cover-slipped using anti-fade gel mounting media . Rabbit anti-mouse collagen VII  diluted 1\u223650 and rat anti-mouse integrin \u03b16  diluted 1\u2236100 were used for immunostaining. Secondary antibodies used at a dilution of 1\u2236250 were goat anti-rabbit FITC and goat anti-rat Alexa Fluor 546 . Imaging was performed using a SP5 Leica confocal microscope at 63\u00d7 .\u22121. Once the mouse was anesthetized, a small incision was made between the third and fifth tracheal rings and a tracheal cannula was inserted and secured in place with suture. Each mouse was ventilated at 200 breaths per minute with a tidal volume of 10 ml kg\u22121 body weight using a flexiVent ventilator . Once the mouse was breathing passively, two consecutive sigh breaths were performed to open the airways and lungs. Sigh breaths were performed throughout the experiment to insure that the airways remained open. Pressure-volume loops, elastance and compliance measurements were recorded and analyzed using SCIREQ flexiVent 5.1 software. Comparisons between cohorts were performed using the Student's two-tailed heteroscedastic t-test.Mice were anesthetized with ketamine/dormitor at doses based on body weight. To prevent the mice from breathing against the respirator, they were treated with pancuronium bromide in NaCl as a muscle relaxant at 0.2 mg kgPeripheral dual-energy X-ray absorptiometry performed using a Lunar PIXImus densitometer  was used to assess bone and non-bone tissue parameters. Bone parameters of mineral density (BMD) and mineral composition (BMC), and non-bone parameters of percent fat and total tissue mass (lean+fats) were determined after head exclusion and analyzed using Lunar PIXImus 2.1 software, as described Col17a1 specific oligonucleotide primers pairs for RT-qPCR , stored at room temperature for 24 hours, and then frozen at \u221240\u00b0C until ready to process. Total RNA was isolated using the standard Trizol reagent method (Invitrogen). Total RNA (500 \u00b5g) was converted to cDNA using the MessageSensor RT kit (Ambion/Applied Biosystems).  RT-qPCR  were desCol17a1 specific oligonucleotide primers using a Qiagen LongAmp PCR kit according to the manufacturer's protocols. Genomic DNA obtained from blood processed similarly was used to obtain intron-spanning sequences. PCR products were magnetic bead purified and sequenced on an Applied Biosystems 3730xl by the JAX DNA Sequencing Service. Sequence data were analyzed using Applied Biosystems Sequencing Analysis software version 5.2 and Sequencher software version 4.10. The Sanger Mouse Genomes Project (sanger.ac.uk) was used as the source of complete genomic sequence comparisons of strains including C57BL/6J, A/J, 129S1/SvImJ, DBA/2J and PWK/PhJ. The Mouse Phenome Database (phenome.jax.org) CGD-MDA1 dataset was utilized to compare strain polymorphism patterns in Col17a1 and its flanking regions. These data were combined with our sequence and SSLP typing data related to 129X1, DBA/1J and PWD to identify all Col17a1 polymorphisms among 8 strains  in mice 10 weeks of age for the same strains . *, p\u22640.05; ***; \u22640.001; ****, \u22640.0001. Statistical significance of tail scoring data is not provided because most groups of mice were euthanized before achieving a score of 4 due to the severity of their ear lesions.Mouse strain background causes substantial variation in the onset and severity of JEB-nH in (TIF)Click here for additional data file.Figure S2PWDchr19 recombinants. * indicates the recombination breakpoints that frame the candidate interval.Gel validation of B6/B6.(TIF)Click here for additional data file.Table S1Col17a1 expression among mouse strains surveyed.RT-qPCR fails to identify transcriptional alterations in tail skin (DOCX)Click here for additional data file.Table S2Col17a1 among relevant mouse strains.Strain relationships of the genomic region including (DOCX)Click here for additional data file.Table S3Col17a1 among the mouse strains used in this study.Sequence-based polymorphisms of (XLSX)Click here for additional data file.Table S4Amino acid polymorphisms of human Collagen XVII based on the EVS and 1000 genome databases.(DOCX)Click here for additional data file.Table S5Col17a1.Localization of a PRDM9 binding site in the 1085 bp recombinant interval of (DOCX)Click here for additional data file.Table S6Chr1 and Chr19 markers used in this study.(XLSX)Click here for additional data file."}
+{"text": "Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments. Over the last 40 years, 5 year survival rates for cancer patients have risen dramatically, but total death rates have remained stubbornly high in vitro tissue culture techniques. 2D tissue culture plastic and 3D extracellular matrices (ECM), as scaffolds for cell growth, provide the cell with very different biochemical and mechanical environments. Cells growing in 3D matrices have mechano-transducers and ECM adhesion proteins globally expressed on their surfaces; however, cells grown in 2D only interact with a solid substrate on their basal surface in vitro systems are poor indicators of in vivo processesAn important aspect of chemoresistance is the role of the tumor microenvironment. Tumor cells exist within a dynamic 3D environment which is not completely replicated by standard in vitro.Many investigations have shown that ECM interacting proteins can be used as predictors of tumor behavior and patient outcomes50 values on the order of 10\u2013100 nM for various isoforms of mTOR and PI3K The phosphatide inositol 3 kinase (PI3K)/AKT pathway is an important intracellular signaling cascade which affects cell growth, migration, protein expression and survival To help understand and quantify the effects of the tumor ECM on cell behavior, we have compared proliferation, migration and protein expression of U2OS osteosarcoma cells between 2D and 3D collagen gels, and investigated the effect of changes in the ECM on inhibition of the PI3K pathway.2. PI103 Experiments were performed on the pediatric osteosarcoma cell line U2OS or breast adenocarcinoma cell line MCF7. Cells were cultured in complete RPMI media supplemented with 10% fetal calf serum and 1% penicillin-streptomycin solution . Cell cultures were maintained in 2D monolayers in a humidified incubator at 37\u00b0C, 5% C02) for 2 hours to allow for complete polymerization. The polymerization time was kept consistent as previous experiments indicated that polymerization time can significantly affect pore size (unpublished data). After polymerization, 100 \u00b5l of media was added on top of the gels.Collagen Type 1 gels were prepared as described previously For cell culture experiments, cells were stained with 5 \u00b5M CMRA (Molecular Probes). Cells in 2D experimental groups were plated on tissue culture plastic while cells in 3D conditions were added to the unpolymerized collagen solution to a final concentration of 100,000 cells/ml for the proliferation assay and 30,000 cells/ml for migration trials. The gels were then polymerized as described above, resulting in cell-embedded collagen gels. Media was replaced with fresh media daily. When applicable, experiments were exposed to 250 nM of PI103. For these experiments, PI103 was added to the collagen solutions and the quenching media. Additional drug was added with each subsequent media change to maintain the initial drug concentration.http://www.micro-manager.org). Confocal images were analyzed using IMARIS 7.3.2 . Cells were analyzed using the spot tracking and surface detection routines. Spot tracking gives the centroid position of each cell at each time point and was used to track cell number and cell movement over time. Surface detection outlined the 3D surface of each cell, and was used for cell size and shape measurements. Cell size was used as an excluding measurement to reject overly small cells from being included in cell number and cell movement measurements.Images were acquired using a DMI600B microscope  and ImagEM EM-CCD Camera  using a spinning disc confocal setup . Imaging was done using Micro-Manager 1.4 Software  at 37\u00b0C for one hour. Cells harvested from standard tissue culture plastic were also treated with collagenase to ensure this treatment did not affect results. Liberated cell suspensions were normalized by cell count and lysed in lysis buffer  for 15 minutes on ice and centrifuged for 10 min at 15,000\u00d7g. Whole cell lysates were then subjected to 10% SDS-PAGE followed by electrotransfer onto PVDF membranes. Membranes were blocked overnight at 4\u00b0C in 5% non-fat dry milk (BioRad) in Tris buffered saline with 0.1% Tween-20  before primary antibody incubation in 0.5% non-fat dry milk in TBST (Santa Cruz) for one hour at room temperature. After washing in TBST, membranes were incubated in secondary (horseradish-peroxidase conjugated) antibodies (AbCam) in TBST for one hour at room temperature. Membranes were visualized with Pierce ECL substrate (Thermo Scientific). All protein bands were normalized to GAPDH content and analyzed via densitometry using ImageJ.in vitro experiments. The cells are also very robust and react well to being embedded in collagen gels.In order to investigate the effects of the ECM on cell behavior the proliferation, migration, protein expression and response to PI3K inhibition of U2OS and MCF7 cells on tissue culture plastic and in Type 1 collagen gels of different collagen concentrations was investigated. U2OS cells were chosen due to their high native PI3K/Akt pathway activity 2, and were therefore seeded at 1000 cells/cm2.Because the 3D culture conditions introduce inherent differences in local cell density, the growth of cells seeded at different densities within collagen gels was analyzed. As can be seen in Next, the effect of the live cell dye on the proliferation of U2OS and MCF7 cells in 3D collagen gels was investigated. The CMRA fluorophore has been used previously without effect on cell behavior The proliferation of U2OS and MCF7 cells was examined in type I collagen gels of varying densities. U2OS cells proliferated significantly slower in 3D collagen gels than in 2D monolayer . Cells g\u22125)   This is This evaluation of the proliferation and migration of U2OS and MCF7 cells clearly shows that these cells behave very differently in 3D collagen gels than on flat 2D substrates. Because the PI3K pathway is critical to multiple cellular processes, the levels of key proteins in the PI3K/Akt pathway were compared between U2OS cells grown in 2D and 3D . U2OS ceCulturing U2OS and MCF7 cells in 3D collagen gels produces very different behavior than on standard tissue culture plastic. Further, in U2OS cells the move to a 3D culture platform yields very different levels of activity in the important PI3K/MTOR pathway. How does this difference in behavior and PI3K pathway activation affect the response of U2OS cells to pharmacological inhibition of the PI3K/mTOR pathway? As can be seen from We performed the same experiment on MCF7 cells to show that these results can be obtained from multiple cell lines . After 8Given the reduction in motility in U2OS cells grown in type I collagen gels in comparison with that in 2D monolayer the effect of PI3K pathway inhibition on cellular motility in these different conditions was also investigated. As can be seen in In an attempt to explain the observed difference in drug sensitivity between the two different collagen concentrations the effect of PI103 treatment on the levels of various phosphoproteins in the PI3K pathway was assessed. As can be seen from in vitro investigations of cell behavior. The gels are easy to create, inexpensive and are conducive to live cell manipulation and imaging. The gels are also customizable, with the ability to control pore size, ligand density and stiffness by either changing the concentration of collagen in vivo and can contribute to many kinds of pathologies.Our study provides quantitative understanding of the role of ECM in altering cellular behavior, in particular the response of PI3K pathway, in 2D and 3D environments. Our data indicates that the presence of a three-dimensional ECM significantly affects the proliferation and motility of U2OS and MCF7 cells and is consistent with emerging research into the role of ECM in cell behavior Our study demonstrates that the presence of a 3D ECM is sufficient to alter the activation of the PI3K/Akt pathway in U2OS cells. This reduced activation was consistent with an observed reduction in cellular proliferation and migration speed in cells growing in 3D matrix. Future investigations are needed to understand how a mechano-chemical signal from the ECM is transduced to intracellular signaling cascades.Previous work has shown that the presence of ECM proteins is sufficient to confer resistance to cancer cells against anti-tumor agents The fact that the ECM is sufficient to alter cell behavior, protein expression and drug response must be taken into account when designing and executing drug candidate validation studies and clinical trials. Currently, the vast majority of novel cancer therapeutics are screened for their efficacy against well-established cancer cell lines on high throughput, plate-based 2D systems"}
+{"text": "In approximately 50% of all malignancies this is achieved by inactivating mutations in the p53 gene. However, in several cancer entities, including melanoma, p53 mutations are rare. It has been recently proposed that tyrosinase related protein 2 (TRP2), a protein involved in melanin synthesis, may act as suppressor of the p53 pathway in melanoma. To scrutinize this notion we analyzed p53 and TRP2 expression by immunohistochemistry in 172 melanoma tissues and did not find any correlation. Furthermore, we applied three different TRP2 shRNAs to five melanoma cell lines and could not observe a target specific effect of the TRP2 knockdown on either p53 expression nor p53 reporter gene activity. Likewise, ectopic expression of TRP2 in a TRP2 negative melanoma cell line had no impact on p53 expression. In conclusion our data suggest that p53 repression critically controlled by TRP2 is not a general event in melanoma. ARF, which binds MDM2 and inhibits its E3 ubiquitin ligase activity Caenorhabditis elegans that secretion of the TRP2 homolog Tyr2 by sensory neurons inhibits p53 expression of germ cells. Moreover, TRP2 knockdown in WM266-4 melanoma cells lead to increased p53 expression sensitizing the cells to cisplatin-induced apoptosis.Advanced melanoma is a cancer that is largely resistant to cytotoxic drugs or irradiation; this had been at least in part attributed to an impaired p53 dependent apoptosis response In this report we further scrutinize a possible role of TRP2 in the regulation of p53 in five different melanoma cell lines and do not find such a relation.Tumor samples from primary and metastatic melanomas were obtained by surgical excision for either therapeutic or diagnostic purposes and had undergone routine histology. The Institutional Review Board of W\u00fcrzburg University Hospital approved all described studies and waived the need for written consent for histochemical analysis of anonymised tumor samples. Generation of the WueMel 45 melanoma cell line was done after written consent from the patient .After anonymization of tissue samples a dermatohistopathologist (C.K.) reviewed slides from all blocks, selecting representative areas of tumor tissue to be cored for generation of TMA as previously described 4 \u00b5m sections of paraffin-embedded tumors and TMA were dried at 56\u00b0C and then treated twice with xylene for 10 min at room temperature. Subsequently, sections were washed twice with absolute ethanol and twice with 70% ethanol followed by one rinse with bi-distilled water. For antigen retrieval, sections were incubated with citrate buffer pH 9.0  for 10 min at 90\u00b0C and rinsed with bi-distilled water. Next, slides were rinsed twice with phosphate-buffered saline (PBS) and thereafter incubated with Blocking Solution  for 10 min at room temperature. After two additional washing steps with PBS for 10 min at room temperature, the monoclonal \u03b1-p53 antibody  or \u03b1-TRP2 antibody  was added to the sections at a predetermined concentration in PBS, followed by an over night incubation at 4\u00b0C. After two 10 min washes in PBS, biotinylated multispecies-specific secondary antibody  was added to the sections for 30 min at room temperature. Slides were then washed twice in PBS/bovine serum albumin, and bound antibodies were visualized using streptavidin-HRP (DAKO K5003) and Vector Vip  as peroxidase substrate according to the manufacturer's guidelines. Finally, the nuclei were stained with hemalaun.The cores of specimens on the tissue microarray (TMA) slides were scored using a semi-quantitative scoring system where staining intensity and the proportion of stained tumor cells is taken into account 2) and the p value were determined applying linear regression analysis comparing p53 and TRP2 histology scores.The relationship between the p53 and TRP2 expression was statistically analyzed using the Prism Graph software 5.0 . The coefficient of determination (ROne melanoma cell line (WueMel45) was generated in our lab from a melanoma metastasis obtained from a patient for therapeutic purposes. This cell line as well as the melanoma cell lines FM88 TCAAGAGTCTGTTAGTTGTTTGAATCACATTTTTTT-3\u20325\u2032-GTGATTCAAACAACTAACAGA, TCAAGAGACAGCATGAAATTGCCAACCTTTTTTTTT-3\u20325\u2032-AAGGTTGGCAATTTCATGCTGT and TCAAGAGTCTGCCGAATCACTGGTGGTTTTTTT-3\u20325\u2032-CCACCAGTGATTCGGCAGA, respectively (the sense strand is given), into the lentiviral vector KH1 CATGAAGCTGCCCACG-3\u20325\u2032- ACAG; exchanged nucleotides displayed in bold) were introduced in the shRNA target sequence using the quick change mutagenesis kit .The shRNA vectors TRP2_shRNA #1, #2 and #3 were obtained by cloning the shRNA sequences Infectious viruses were raised by transfecting HEK293T cells CGACATGCCCGGGCATGT). Following puromycin selection flow cytometry to monitor GFP expression in pGreenFire transduced cells was performed on a FACSCanto . Mean GFP fluorescence intensities were normalized to the relative presence of the reporter constructs in the infected cells as determined by real time PCR The p53 reporter construct pGreenFire lentiviral vector codes for a puromycin resistence and for green fluorescent protein (GFP) under the control of a p53 responsive element . Antibodies to p53  TRP2  and \u03b2-tubulin  were used.Prompted by a report demonstrating an inhibitory effect of TRP2 on p53 in a melanoma cell line 2\u200a=\u200a0,0258) and only borderline-significant  positive correlation was observed. Moreover, of all 52 samples with very weak or no TRP2 staining (histology score of 0 or 1) 17 (33%) also displayed very weak or negative p53 staining (histology score of 0 or 1). On the other hand of all 120 samples with middle to strong TRP2 staining (histology score of 2\u201312) 44 (37%) displayed very weak or negative p53 staining (histology score of 0 or 1). Taken together these analyses reveal, that p53 expression is independent of the TRP2 expression level. Moreover, both proteins are detectable in the majority of samples and therefore expression is not mutually exclusive.Overall p53 expression was detectable (score \u22651) in 99 out of 139  cases of primary melanoma and in 17 out of 33  cases of metastatic melanoma . p53 immTo further scrutinize a possible role of TRP2 in the regulation of p53 we performed a series of TRP2 knockdown experiments addressing the expression and transcriptional activity of p53. Initially, we analyzed the expression of p53 and TRP2 in the melanoma cell lines FM88, M26, MelU, MelJuso, and WueMel45. p53 was present in all five melanoma lines, whereas TRP2 expression was detected by western blot in all cell lines except WueMel45. Three short hairpin RNAs were engineered to suppress TRP2 expression. The efficacy of TRP2 knockdown was confirmed by immunoblot; all three shRNAs strongly reduced TRP2 protein expression in melanoma cell lines and the knockdown was most prominent for TRP2-shRNA_#2 . To thisTo test whether the transcriptional activity of p53 may be altered by TRP2 knockdown, the set of melanoma lines were stably transduced with a reporter gene construct (pGreenFire) coding for GFP under the control of a p53 response element. The mean GFP expression was measured by flow cytometry and normalized for the relative number of incorporated pGreenFire copies as determined by real time PCR. As we have shown previously expression levels of p53 do not necessarily correlate with its transcriptional activity suggesting posttranslational inactivation of p53 Interestingly, TRP2 knockdown by two of the three shRNAs (#1 and #3) showed no impact on p53 reporter gene activity. However, infection with the TRP2-shRNA_#2 led to increased p53 reporter gene activity in FM88, Mel-U and MelJuso but not in M26 and WueMel 45 melanoma cells . TherefoInactivation or at least partial repression of the p53 tumor suppressor pathway is thought to occur in almost all human cancers, and inactivating p53 mutations are the genetic alterations most frequently observed in malignancies Caenorhabditis elegans the TRP2 homolog TYR2 as a protein secreted by neurons and acting paracrine in neighboring germ cells to suppress CEP-1 (p53 homolog in the worm) dependent apoptosis In our study we investigated the role of the TRP2 in p53 regulation prompted by a report of Sendoel and colleagues who identified in"}
+{"text": "Permanent pacemaker implantation is available in Nigeria. There is however no national registry or framework for pacemaker data collection. A pacemaker database has been developed in our institution and the results are analyzed in this study.The study period was between January 2008 and December 2012. Patient data was extracted from a prospectively maintained database which was designed to include the fields of the European pacemaker patient identification code.Of the 51 pacemaker implants done, there were 29 males (56.9%) and 22 females (43.1%). Mean age was 68.2\u00b112.7 years. Clinical indications were syncopal attacks in 25 patients (49%), dizzy spells in 15 patients (29.4%), bradycardia with no symptoms in 10 patients (17.7%) and dyspnoea in 2 patients (3.9%). The ECG diagnosis was complete heart block in 27 patients (53%), second degree heart block in 19 patients (37.2%) and sick sinus syndrome with bradycardia in 5 patients (9.8%). Pacemaker modes used were ventricular pacing in 29 patients (56.9%) and dual chamber pacing in 22 patients (43.1%). Files have been closed in 20 patients (39.2%) and 31 patients (60.8%) are still being followed up with median follow up of 26 months, median of 5 visits and 282 pacemaker checks done. Complications seen during follow up were 3 lead displacements (5.9%), 3 pacemaker infections (5.9%), 2 pacemaker pocket erosions (3.9%), and 1 pacemaker related death (2%). There were 5 non-pacemaker related deaths (9.8%).Pacemaker data has been maintained for 5 years. We urge other implanting institutions in Nigeria to maintain similar databases and work towards establishment of a national pacemaker registry. Bradyarrhythmias are a cause of sudden death in Nigeria, though the precise incidence is unknown. Pacemaker implantation is an accepted intervention which has been shown to improve the quality of life and reduce mortality in patients with bradyarrhythmias. Published experience in Nigeria has shown that implantation rates are low, the main indication for implantation is complete heart block (CHB) and most patients receive ventricular implants . PacemakA pacemaker implantation and follow up service was established in our institution in 2008. The aim of this study was to review our experience by analysis of our pacemaker database.Following patient referral the clinical indication for pacemaker therapy is established from the history and the diagnosis confirmed with a 12 lead ECG (and 24 hour holter if necessary). Cardiac function is assessed with a transthoracic echocardiogram. Pacemaker implantation is performed in a dedicated theatre suite equipped with a fluoroscopic C arm. The implantation team is composed of a surgeon who performs the implantation, a cardiac physiologist who performs the checks of pacemaker parameters, a pacemaker technician to operate the fluoroscope for imaging and a scrub nurse. Monitored parameters are the heart rhythm, heart rate, non-invasive blood pressure and peripheral oxygen saturation. A standard subclavian approach is used after infiltration with local anaesthesia in all cases. Prior to lead fixation the R wave  and pacing threshold are checked. Target values are R wave greater than 6 millivolts (mV), P wave greater than 2 mV, lead impedance less than 1200 Ohms and pacing threshold less than 1 volt (V). Diaphragmatic pacing is checked at 10V. The pacemaker pocket is irrigated with 1g of ceftriaxone, the pacemaker lead connected to the pulse generator and the wound closed in layers. An arm sling is used in all cases to restrict movement of the arm on the operation side (to reduce the risk of lead displacement) and the patient is transferred to the ward.Patients are monitored on the ward for 48 hours to exclude lead displacement. After 48 hours a pacemaker check is done and the patient is given a copy of both the pacemaker implantation report  and paceA Microsoft Access database was designed and has been maintained prospectively since the inception of the programme in January 2008. Data storage covers the fields recommended by the European pacemaker patient identification codes . Sample Of the 51 patients implanted there were 29 males (56.9%) and 22 females (43.1%). Ages ranged from 22-92 years with a mean age of 68.2\u00b112.7 years. Age distribution is as shown in The distribution of clinical indications for pacemaker therapy was syncopal attacks in 25 patients (49%), dizzy spells in 15 patients (29.4%), documented bradycardia with no symptoms in 10 patients (17.7%) and dyspnoea/heart failure in 2 patients (3.9%). The ECG diagnosis was Complete Heart Block (CHB) in 27 patients (53%), second degree heart block (SDHB) in 19 patients (27.2%) and Sick Sinus Syndrome with bradycardia (SSS) in 5 patients (9.8%). There was no patient with atrial fibrillation.The distribution by pacemaker manufacturer was Medtronic (Minneapolis Minnesota USA) in 33 patients (66.7%), Pacetronix (Kolkata India) in 10 patients (19.6%) and St Jude (St Paul Minnesota USA) in 7 patients (13.7%). Pacing modes used were VVI (Ventricular Demand Pacing) in 9 patients (17.6%), VVIR (Rate Responsive Ventricular Demand Pacing) in 20 patients (39.2%), DDD  in 2 patients (3.9%) and DDDR  in 20 patients (39.2%). Overall single chamber ventricular pacing (VVI(R)) was used in 29 patients (56.9%) and dual chamber pacing (DDD(R)) in 22 patients (43.1%) with a progressive annual reduction in the use of ventricular pacing and an increased use of dual chamber pacing over the study period . DistribAt implantation the average R wave obtained was 11.8\u00b14.6 mV while the average P wave obtained was 2.9 + 1.8 mV. The average atrial pacing threshold was 0.65\u00b10.54 V and the average ventricular pacing threshold was 0.59\u00b10.39 V. Average impedance for the atrial leads was 650.2\u00b1191.7 Ohms and was 780.5\u00b1230.2 Ohms for the ventricular leads. Complications seen postoperatively and during follow up were 3 lead displacements (5.9%), 3 pacemaker infections (5.9%), 2 pacemaker pocket erosions (3.9%), and 1 pacemaker related death (2%). Details of complications seen and time interval from implantation when they occurred are shown in At follow up in the pacemaker clinic, 282 pacemaker checks have been done on the 51 patients implanted in our institution. Of these 51 patients, 31 patients (60.8%) still remain under follow up in the pacemaker clinic. For these patients, the follow up period has ranged from 1-60 months with a median follow up of 26 months. The number of clinic visits ranged from 114 with a median of 5 visits.File closure has been done in 20 patients (39.2%). The reasons for file closure were pacemaker removal in 5 patients (25%), non-pacemaker related death in 5 patients (25%), transfer to another hospital in 5 patients (25%), lost to follow up in 2 patients (10%), inability to attend from transportation problems in 2 patients (10%) and pacemaker related death in 1 patient (5%). Of the 5 non-pacemaker related deaths, 4 patients were reported to have suffered myocardial infarctions while 1 patient died of complications of prostate cancer.There are currently 10 centres in Nigeria known to implant pacemakers. There are 3 centres in Lagos and 1 each in Enugu, Ibadan, Abuja, Port Harcourt, Calabar, Ife and Ilorin . To date a centre in Lagos  have pubth World survey of cardiac pacing and implantable cardioverter-defibrillators [th world survey showed that there are slightly more males receiving implants than females  which is similar to our experience. The use of dual chamber pacing in 43.1% of patients in our series is considerably higher than reported in other West African series and reflects the worldwide trend of increased use of dual chamber pacing seen in the 11thth World survey. Similar to the findings of other West African series, most patients in our environment are diagnosed with CHB and about 50% present having had syncopal attacks. This is unlike the pattern in the Western World where 30% or less of patient present with CHB and Sinus Node Dysfunction (SND) is the predominant indication for cardiac pacing [The mean age in our series was 68 years with 56.9% being male and 43.1% female. This is within the mean age range of 65 to 75 years reported in the 11Maintaining a stock of pacemakers locally makes it easier to proceed directly to permanent pacemaker implantation. In this series of 51 patients, only one temporary pacemaker was implanted. This is unlike the earlier experience from Lagos  and thisIn our practice there has been a progressive decrease in the use of single chamber ventricular pacing and an increase in dual chamber pacing over the last 5 years of our experience. In the 1990s initial recommendations urged more use of dual chamber pacing as it was thought that the hemodynamic benefits of AV synchrony would translate into improved longevity, improved quality of life and reduction in strokes . The firIt has been suggested that dual chamber pacing though more expensive may be offset by reduced replacement for pacemaker syndrome and improved quality of life . None ofWe noted that all the pacemaker infections occurred in dual chamber implants. It has been shown that there can be a higher complication rate with dual chamber implants . We willWe have considered the re-use of pacemakers as there is some evidence that it is safe and could reduce costs for patients . AverageOnly 1 pacemaker related death occurred in this series. This occurred in an elderly patient who received a Pacetronix implant (VVI) with a tined, non-steroid eluting endocardial lead. Over a period of 2 years there was a gradual rise in pacing threshold from implantation level of 0.8V to 2.5V. Pacing amplitude had been increased to 5V and the patient advised on a change of implant. She however declined and died suddenly, presumably from sudden failure to capture. This singular experience informed our current practice where we no longer use Pacetronix implants and all pacemaker leads used are steroid eluting and active fixation.Of the 5 non-pacemaker related deaths, 4 were reported as being secondary to myocardial infarctions. Diagnosis of myocardial infarction was made by cardiac enzymes and electrocardiogram changes in 1 patient, and purely from ECG changes in the other 3 patients. The incidence of Ischaemic heart disease (IHD) is known to be on the increase in Nigeria  so the cThere is great variability in implantation rates between different countries as shown in the 11th World survey of cardiac pacing and implantable cardioverter-defibrillators . The larIn the midst of this great variability in the number of pacing centres and implants done per country, it is of great concern that Nigeria was not included in this survey. This stresses the urgency in establishment of a framework for a national registry so that the efforts of various implanting institutions in Nigeria can be captured.A pacemaker implantation and follow up service has been established in our institution and a robust database has been developed and maintained. Early results show that the main indications for implantation are complete heart block and second degree heart block. Use of dual chamber pacing is higher than has been reported from other West African Centres. Continued patient follow up may be able to address questions of pacemaker therapy unique to our environment. Complications rates have been low. Complete follow up information is available for 49 patients (96%). We urge other implanting institutions in Nigeria to maintain similar databases and work towards establishment of a national pacemaker registry."}
+{"text": "Healthcare workers in many countries are recommended to receive influenza vaccine to protect themselves as well as patients. A monovalent H1N1 vaccine became available in Hong Kong in December 2009 and around 10% of local healthcare workers had received the vaccine by February 2010.We conducted a cross-sectional study of the prevalence of antibody to pandemic (H1N1) 2009 among HCWs in Hong Kong in February\u2013March 2010 following the first pandemic wave and the pH1N1 vaccination campaign. In this study we focus on the subset of healthcare workers who reported receipt of non-adjuvanted monovalent 2009 H1N1 vaccine . Sera collected from HCWs were tested for antibody against the pH1N1 virus by hemagglutination inhibition (HI) and viral neutralization (VN) assays.We enrolled 703 HCWs. Among 104 HCWs who reported receipt of pH1N1 vaccine, 54% : 44%\u201363%) had antibody titer \u22651\u223640 by HI and 42% (95% CI: 33%\u201352%) had antibody titer \u22651\u223640 by VN. The proportion of HCWs with antibody titer \u22651\u223640 by HI and VN significantly decreased with age, and the proportion with antibody titer \u22651\u223640 by VN was marginally significantly lower among HCWs who reported prior receipt of 2007\u201308 seasonal influenza vaccine . After adjustment for age, the effect of prior seasonal vaccine receipt was not statistically significant.Our findings suggest that monovalent H1N1 vaccine may have had suboptimal immunogenicity in HCWs in Hong Kong. Larger studies are required to confirm whether influenza vaccine maintains high efficacy and effectiveness in HCWs. In a separate study we investigated antibody seroprevalence in HCWs who reported that they had not received pH1N1 vaccine We recruited HCWs between February 11 and March 31, 2010 in 6 public hospitals comprising the Hong Kong West cluster of the local Hospital Authority, with a total workforce of around 7,000 HCWs in one acute care teaching hospital and five non-acute hospitals Serum samples were stored in a refrigerated container at 2\u20138\u00b0C immediately after collection and delivered to the laboratory at the end of each working day for storage at \u221270\u00b0C prior to testing. Serum specimens were tested for antibody responses to A/California/04/2009 (H1N1) by hemagglutination inhibition (HI) and viral microneutralization (VN) assays using standard methods as previously described The conventional neutralization test for the A/California/04/2009 was carried out in micro-titre plates using neutralization of virus cytopathogenic effect (CPE) in Madin-Darby Canine Kidney (MDCK) cells. Serial serum dilutions in quadruplicate were mixed with 100 tissue culture infectious dose 50 (TCID50) for 2 hours and added to MDCK cells. One hour after infection, serum-virus mixtures were removed and serum free MEM with 2 ug/ml trypsin was added to each well. The plates were incubated and cytopathic effect was observed to determine the highest serum dilution that neutralized \u226550% of the wells. A virus back titration and positive and negative control sera were included in each assay.We compared the differences in the proportion of HCWs with pH1N1 antibody titer \u22651\u223640 both by HI and VN between groups with chi-squared tests or Fisher's exact test and used the phi coefficient to compare results between the two assays. We compared antibody seroprevalence over time in our study with the date of administration of pH1N1 vaccines to HCWs in Hong Kong We recruited 703 HCWs during the study period, and seroprevalence data for the 599 HCWs who reported that they had not received pH1N1 vaccine have been reported elsewhere Around 10% of HCWs in Hong Kong had received pH1N1 vaccine by January 2010 . We founAmong the 101 HCWs without confirmed pH1N1 infection, the proportion of HCWs with antibody titer \u22651\u223640 by VN significantly decreased with older age , and was significantly lower among HCWs who reported receipt of 2007\u201308 seasonal influenza vaccine . In the Influenza vaccination is recommended as the primary prevention measure against infection, and HCWs are often one of the groups targeted to receive vaccine not only for their direct protection but also to indirectly protect vulnerable patients against nosocomial infection While only 42% of HCWs had antibody titers \u22651\u223640 by VN, which is consistent with the findings of another study of 409 HCWs in Japan who were provided with one dose of pH1N1 vaccine, and just 38% achieved antibody titers \u22651\u223640 by HI within 21 days of vaccination Another possible reason for lower antibody response is imperfect effectiveness of vaccine in field condition for the general population. Various controlled trials have shown good immunogenicity of the pH1N1 vaccine Although we did not record time of receipt of pH1N1 vaccine in participants in our study, the majority of HCWs in Hong Kong had received pH1N1 vaccine before mid-January 2010, i.e. at least one month before the start of our study . AntibodIt is important to note several limitations of our study in addition to the relatively small sample size. First, we conducted a cross-sectional seroprevalence study following the first pH1N1 wave, and we did not have baseline (pre-pandemic) data to enable us to compare the pre-pandemic antibody titer with the post-pandemic antibody titer among HCWs Vaccination is the most effective way to protect HCWs and the patients around them from influenza, but uptake of influenza vaccines tends to be low in many countries including Hong Kong. Our study indicated lower antibody response than expected among HCWs after receipt of pH1N1 vaccine than antibody results of other two clinical studies using the same pH1N1 vaccine"}
+{"text": "Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.In the yeast  S. cerevisiae, three chromosomal regions are epigenetically silenced with respect to transcription: the telomeres, the silent mating loci (HMR and HML), and the ribosomal DNA (rDNA) repeats  complex SAS-I and the chromatin-condensing activities of NAD+-dependent histone deacetylase (KDAC) Sir2 (silent information regulator-2) In a eukaryotic cell, selective transcriptional repression or silencing dictates the accessibility of specific chromatin domains by the transcriptional machinery; this results in varying degrees of transcriptional competency across the eukaryotic genome. Euchromatin refers to the chromatin regions at which transcription is generally active, whereas heterochromatin refers to those that are largely devoid of transcriptional activity   has been identified as the only type I PRMT in HMT1 in conjunction with the synthetic genetic array (SGA) methodology to systematically and comprehensively screen for all non-essential yeast genes that interact with HMT1, and generated an HMT1 genetic interaction network based on our findings. Gene ontology (GO) analysis of our SGA data showed that HMT1 interacts with genes encoding various components of the Rpd3L (large) complex. In the Hmt1 loss-of-function mutants, recruitment of Rpd3 to the telomeric boundary region is increased. In mutants carrying both Rpd3 and Hmt1 deletions, increased silencing at the telomere and increased Sir2 recruitment at telomeric boundary region is observed compared to \u0394hmt1 mutants. The Hmt1 loss-of-function mutants display a decrease in the levels of H4K5 acetylation (a known Rpd3 substrate) and an increase in the levels of acetylated H4K16 (a known Sir2 substrate) at the telomeric boundary regions. Finally, mutants lacking either Sir2 or Rpd3 display a decrease in the levels of dimethylated H4R3 at the telomeric boundary regions, albeit to a different degree. Overall, our results indicate that Hmt1 has the potential to influence the recruitment and actions of KDACs in order to promote the maintenance of silent chromatin.In the current study, we used a null allele of HMT1 and other genes, we conducted a synthetic genetic array (SGA) analysis using the \u0394hmt1 mutant as the query strain. This approach allowed us to construct and analyze double mutants in which the \u0394hmt1 mutation was combined with deletions in most of the non-essential genes of S. cerevisiae. Such information was expected to enable us to uncover any role Hmt1 may play within a broad biological network. The sensitivity of our screen was maximized by constructing the \u0394hmt1 query strain from a parental strain (15578-1.2b) developed by the Hartman lab To obtain further insight into the interactions between hmt1  and compiled the known physical interactions into a list. These physical interactions were then superimposed on the HMT1 genetic interaction dataset, and the overlap was graphically displayed  as one of the most enriched among the 123 genetic interactions identified in our SGA screen (Saccharomyces Genome Database (SGD) as ones encoding components of the Rpd3L complex, six displayed negative synthetic genetic interactions  with \u0394hmt1, two displayed positive synthetic genetic interaction (\u0394rpd3 and \u0394ume6) with \u0394hmt1, and the other seven genes fell outside our significance criteria . For \u0394sds3, and \u0394ume6, our follow-up screens indicated that they were false negatives (hmt1 to HMT1 spots for \u0394sds3 and \u0394ume6) as they had the same interaction type in the original SGA screen, but their p-values from the original SGA screen did not pass the cutoff criteria  complex, and they did not display any genetic interactions with \u0394hmt1 (HMT1 and \u0394hmt1 rows for \u0394eaf3 and \u0394rco1). Although \u0394sin3 resulted in a synthetic genetic interaction with \u0394hmt1 in the initial screen, this interaction could not be confirmed by our subsequent SGA analysis (hmt1 suggests that Hmt1 has an influence on the activity of the Rpd3L complex.Using software that identifies the enrichment of specific gene function groups based on annotated gene ontology (GO) terms A screen . Both poA screen  and by sA screen . Of the criteria . In our criteria . Two neganalysis . NeverthHMT1. The catalytic subunit of the Rpd3L complex, Rpd3, has a well-known role in the maintenance of silent chromatin at the S. cerevisiae telomere; mutants lacking RPD3 display enhanced silencing in this region Our SGA screen revealed interactions between genes encoding components of the Rpd3L complex and in vivoPreviously, a complex consisting of Rpd3 and Sin3 was shown to be specifically recruited to create a repressive chromatin domain hmt1 mutant backgrounds. Directed chromatin immunoprecipitation (ChIP) experiments were then performed to determine the effects Hmt1 has on the occupancy levels of Rpd3 at distances of 0.35, 0.6, 1.4, 2.8, and 5 Kb from the telomeric repeats  and loss of silencing in the Hmt1 loss-of-function strains (hmt1 and hmt1(G68R)) as previously reported rpd3 strain (hmt1/\u0394rpd3 and \u0394rpd3/hmt1(G68R). Thus, our results indicate that the \u0394rpd3 phenotype is dominant to the silencing defect caused by mutations in Hmt1.Since Hmt1 and Rpd3 affect the dynamics of silent chromatin formation in opposing manners hmt1, \u0394rpd3, and \u0394hmt1/\u0394rpd3 double mutant (hmt1/\u0394rpd3 double mutant when compared to the \u0394hmt1 mutant (hmt1 (black bars) to \u0394hmt1/\u0394rpd3 (striped bars)). As previously seen, Sir2 recruitment increased minimally within 1 Kb of the telomeric end hmt1 (black bars) to \u0394hmt1/\u0394rpd3 (striped bars), and \u0394hmt1 to \u0394rpd3 (gray bars), regions A and B) and this trend continues in both the \u0394hmt1/\u0394rpd3 double mutant even at a distance greater than 1 Kb from the telomeric end (hmt1 (black bars) to \u0394hmt1/\u0394rpd3 (striped bars) and \u0394hmt1 to \u0394rpd3 (gray bars), regions C, D, and E). Thus, our ChIP data support the results from the silencing assay in which we observed more silencing in the \u0394hmt1/\u0394rpd3 double mutant strains when compared to the single Hmt1 mutant strain.To examine this relationship further at the molecular level, we used directed ChIP to compare the level of Sir2 occupancy in \u0394e mutant . We obserpd3-null mutants display enhanced acetylation of H4K5 hmt1 mutants , not that of \u0394hmt1 (less silenced than the wild-type). This is also supported by the change in Sir2 occupancy level in these \u0394rpd3/\u0394hmt1 double mutants, in which Sir2 recruitment at the telomeric region displays a trend more similar to that of \u0394rpd3 than \u0394hmt1. We note that these results do not distinguish whether such an increase in Rpd3 recruitment is simply a consequence of decreased Sir2 occupancy in the Hmt1 loss-of-function mutants, or the decrease in Sir2 occupancy seen in these mutants are due to an increase in Rpd3 recruitment. While we have not tested the role of Hmt1 in controlling the boundaries of the silenced domains at HMR and HML silent mating type loci, our previous work has demonstrated that Sir2 recruitment in Hmt1 loss-of-function mutants is decreased at the E silencer and immediately upstream of the \u03b12 gene in HML and I silencer of HMRHML locus HM silent mating loci, more experiments must be done in order to elucidate the precise molecular mechanisms underlying this phenomenon at the silent mating loci.Interestingly, it has previously been shown that spreading of the SIR complex at telomeric boundary regions is antagonized by the presence of Rpd3 hmt1 loss-of-function mutants. This line of evidence supports our observation of increased Rpd3 recruitment at these regions in these mutants, as Rpd3 deacetylates acetyl-H4K5 Based on our Rpd3 ChIP data, it is likely that an increase in Rpd3 recruitment to these regions would impact the acetylation state of histones, since Rpd3 substrates include a wide range of acetylation sites within histones H4 and H3 in vitro study of the four acetylatable lysine residues in the N-terminal tail of histone H4 reveals that each acetylated histone H4 has a different ability to act as a substrate for Hmt1/PRMT1 Previous studies in mammalian systems have established that KATs and PRMTs have synergistic activities In conclusion, our data indicate that loss of either Hmt1 or its catalytic activity influences Rpd3 occupancy at the telomeric boundary region, and, as a consequence, the levels of H4K5 acetylation in this region. Given the proposed mechanism for how Rpd3 antagonizes Sir2 action at these regions All yeast strains used in this study are listed in hmt1 query strain. 2) images of each plate were taken with a CCD camera (Bio-Rad) to assess the degree of cell growth for each double mutant. These images were then processed using customized MATLAB code adopted from studies done by Collins et al. Three independent screens were carried out to identify all of the genes that interact \u0394hmt1. These interactions were then scored based on the percentage difference in the colony size when compared to a control query strain containing only deletion library mutation. Based on the results from all three screens, a p-value was assigned to each genetic interaction tested. The following criteria were used to score and filter the raw data for inclusion in the final dataset: 1) the double mutant had a corresponding control mating present on the final selection medium; and 2) any interactions with dubious or putative ORFs were removed; and 3) the double mutant exhibited a growth difference of 50% or more relative to the control, and the p-value was < or \u200a=\u200a0.001. The genetic interaction network was created using Cytoscape The SGA methodology was performed essentially as described previously ChIP procedures were performed as described previously HMT1 or RPD3 were constructed in a published yeast strain with ADE2 integrated at the right telomere of chromosome 5 Null mutations of Figure S1HMT1 interactors from the SGA analysis: A) HMT1 interactors found in the Boone lab study only; B) HMT1 interactors identified in both the Boone lab study and in this study; C) HMT1 interactors found in this study that had negative synthetic interactions; and D) HMT1 interactors found in this study that had positive synthetic interactions. Colonies represents non-query single mutant is marked by a white square and the double mutant (both query and non-query) is marked by a white circle.Tetrad analysis was used to confirm (TIF)Click here for additional data file.Table S1HMT1 as described by the current study. The growth difference ranges from +1 (increasing colony size) to -1 (decreasing colony size) and the p-value for each interaction is also shown. Only genes that passed the p-value criteria are included in this table.The list of all genes that display a genetic interaction with (PDF)Click here for additional data file.Table S2hmt1 query mutants. Query S.D.: standard deviation of \u0394hmt1 query colonies. No. Sets: number of replicates scored. Visual Significance: binary assessment of replicate quality; 1\u200a=\u200agood, 0\u200a=\u200apoor.The unfiltered list of SGA data from three independent screens. Control Average: average colony size for control query mutants; Control S.D.: standard deviation of control query colonies, Query Average: average colony size for \u0394(XLSX)Click here for additional data file."}
+{"text": "PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations.The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women.We evaluated the contribution of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.By analyzing the entire coding region of  PALB2  is a protein that interacts with BRCA2, stabilizing the intranuclear accumulation of BRCA2 proteins at sites of DNA damage . BiallelPALB2 mutations are rare, but have been reportedly associated with increased predisposition to breast and other cancers. In multiple-breast cancer case families, germline PALB2 mutations have been reported in 1.1% (10 out of 920), 2.7% (3 out of 113), 2.0% (1 out of 50), and 0.6% (5 out of 779) of Western European families in the United Kingdom, Finland, French-Canada, and Australasian, respectively ,6,172,6,Multiplex genotyping of seven PALB2 mutations identified in exon 4 in this study and an additional two Asian recurrent mutations were performed using high-throughput Sequenom MassARRAY iPLEX platform . Mutations in other exons were identified after the completion of the genotyping study and were therefore excluded from analyses. All mutations identified were confirmed by direct sequencing in an independent DNA sample.In silico analysis of the effect of missense mutations on protein function was determined using Polyphen-2 (Polymorphism Phenotyping version 2)  and SIFTBRCA1 and 13 in BRCA2. The remaining 122 individuals tested negative for BRCA mutations were analyzed for PALB2 germline mutations by direct Sanger sequencing  and deleterious mutations that were reported to be recurrent in other Asian populations (Chinese PALB2 c.751 C>T and PALB2 c.1250_1251 delAAinsTCT)  require other methods, including in vitro functional characterization [Our study also identified 10 re novel ,26. Of trization  and co-sPALB2 c.1050 delAAinsTCT) [PALB2 c.1050 delAACA) at the same site. Sequence alteration caused by deletion of AACA resulted in mutant SNP that is similar to the interrogated SNP and was therefore regarded as a positive screen. This highlights the limitation of genotyping assay in distinguishing distinct mutations which share a common mutant SNP and therefore, confirmation by conventional sequencing is necessary.Our study uncovered an unusual situation where genotyping of a deleterious mutation (AinsTCT)  revealedPALB2 mutations, 2 have moderate family history with Manchester score of 12 and 14 respectively, whereas one developed breast cancer at late age in the absence of any family history of cancer (Manchester score of 2). Indeed, by contrast to BRCA1 and BRCA2 mutation status, we found that there was no association between PALB2 mutation status and family history of breast, ovarian, prostate or pancreatic cancer (data not shown). This is consistent with other studies that have also shown no familial clustering of breast cancer in PALB2 families compared to non-BRCA carrier families [PALB2 carrier families were not available for co-segregation analysis and therefore it was not possible to determine the increased risk caused by germline mutation of PALB2 in our population.Notably, of the 3 individuals with germline deleterious families . The famPALB2 deleterious mutation carriers in 122 high risk non-BRCA1 or BRCA2 breast cancer patients by DNA sequencing and 1 germline carrier in 1406 breast cancer patients by genotyping. Our data shows that PALB2 germline mutations are rare, and are associated with family history of breast cancer in some, but not all families.In summary, we found 2 germline Table S1Amplification primers used for the sequencing of PALB2 gene.(DOCX)Click here for additional data file."}
+{"text": "App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality  and heart function . In addition cardiac connexins  and sodium channel sub-units  were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people.Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the  App-Runx1 genetic interval. Further molecular characterization revealed an overall perturbation of transcription, a few candidates homologous to human chromosome 21 genes, and changes in the expression of several genes, including cardiac connexins  and sodium channel sub-units , likely contributing to ECG defects. This model provides a unique opportunity to study further the DS heart diseases and to propose therapeutic avenues for treatment of DS cardiac complications observed in adults.Down syndrome is a common cause of birth defects mainly due to heart diseases with an incidence of up to 50%. Nevertheless the pathophysiology of DS cardiac anomalies and complications are not well understood. Here we have been able to demonstrate using a series of DS mouse models that birth defects and changes in ECG, similar to those in humans, are linked to dosage sensitive genes located in the  Homo sapiens) is the most common chromosomal anomaly and cause of intellectual disabilities Down syndrome (DS), caused by trisomy of human chromosome 21 , 17  and 10 . The largest duplication created on the Mmu16, the Dp(16)1Yey, results in a mouse trisomic for the whole region from Lipi to Zfp295 that shows cardiac anomalies 16)65Dn (Ts65Dn) model, which carries a shorter trisomy ranging from Mrpl39 to Zfp295, shows specific cardiovascular malformations associated with post-natal lethality and reduced transmission rate of the Ts65Dn allele at weaning Tiam1-Kcnj6 region To further study the correlation between phenotype and genotype in DS, various mouse models have been created At present, while morphological and histological aspects of the cardiovascular phenotypes observed in DS mouse models during perinatal age have been described, the functional aspects remain unexplored in viable adult animals. Before the development of cardiac imaging, ASD, VSD and AVSD were scored by abnormal electrocardiographic recordings (ECG) in man. CHD could be predicted on this basis in 80% of the cases in DS people. Most characteristic features of CHD-induced changes in ECG are superior frontal QRS axis deviation, first degree block and partial bundle branch block App to Runx1 region and their implications in heart defects and lethality by a subtractive approach. For that we used a partial monosomic model, Ms5Yah, carrying a deletion for the previously mentioned region and we found that compound Ts65Dn/Ms5Yah mice were partially rescued for the early post-natal lethality and some aspect of ECG features. Furthermore re-establishing euploidy in the App-Runx1 region modified gene expression changes, highlighting several pathways involved in cardiac function that were deregulated in the Ts65Dn DS mouse model.The aim of the present study was to explore the cardiac function in the Ts65Dn mouse model. Moreover we wanted to assess the susceptibility to dosage effect of genes within the 2P\u200a=\u200a1.74\u00d710\u22128). Observation of litters showed partial loss of progeny with death in the first 48\u201372 hours and some complete loss of litters either abandoned or cannibalized by Ts65Dn mothers. Heart and great vessels microdissection and cardiac histology analyses showed one out of 18 Ts65Dn dead pups presenting both great vessel and cardiac malformations . Moreover, these ECG features were found to be highly predictive of a Ts65Dn genotype.Since DS patients can show electrocardiographic and functional changes even in the absence of overt CHD, an electrocardiographic investigation of Ts65Dn mice was performed to look at more subtle phenotypes than gross morphological anomalies. The six standard peripheral leads  ECG of wt and Ts65Dn mice are illustrated in \u22124). Noticeable changes in the wave amplitudes were also recorded in Ts65Dn as compared with wt . In the P\u200a=\u200a9\u00d710\u22124). PR was prolonged from 35.3\u00b10.5 ms to 44.6\u00b11.1 ms . QRS wave duration was also increased by 8%. Values were respectively 9.02\u00b10.21 ms in wt and 9.89\u00b10.2 ms in Ts65Dn . QT and QTc were also larger in Ts65Dn (73.0\u00b12.0 ms and 73.0\u00b11.3 ms respectively) than in wt . Since the above-described results point to conduction changes in Ts65Dn as compared to wt, a sub-group of these mice (N\u200a=\u200a8) were treated with the Na channel blocker, flecainide. As reported in The results were at variance in the precordial leads . R wave Lipi to Zfp295Sod1 to Zfp295, nor in the Ts1Rhr carrying a trisomy from Cbr1 to Orf9Mrpl39-Sod1 interval. Thus we developed a partial monosomic model named Ms5Yah carrying a 7.7 Mb deletion on Mmu16 between the App and Runx1 genes  indicating that the region causes lethality due to haploinsufficiency. Interestingly the transmission rate was evaluated at 27.9% at post natal day 1  and 48.3% at embryonic day 18.5  indicating a lethality within this period. Thus we considered the App-Runx1 region as a haploinsufficient region inducing post-natal lethality. We checked whether the Ms5Yah pups were carrying cardiovascular defects as observed in the Ts65Dn newborns using microdissection and histological analyses. No cardiovascular malformations, either in the great vessels or in the intra-cardiac septation could be seen in 18 dead pups. 185 Ts65Dn/Ms5Yah offspring were obtained at weaning from the breeding of Ms5Yah males with Ts65Dn females. The expected ratios were calculated considering the previously described transmission rates obtained for Ts65Dn line and Ms5Yah line. Transmission ratios for Ts65Dn and Ms5Yah alone were not significantly different from the expected ratio . However, the transmission rate for combined Ts65Dn/Ms5Yah alleles was 24.9%, as expected for a Mendelian ratio showing that the Ts65Dn-allele-induced lethality was completely rescued by the monosomy of the App-Runx1 region (\u03c72P\u200a=\u200a4.8\u00d710\u22127). However the transmission rate for the Ms5Yah allele did not reach the expected value showing that Ms5Yah-induced lethality still occurs. Thus the deletion between App and Runx1 genes (Ms5Yah) induced a severe postnatal lethality that is partially rescued by combining Ms5Yah with the Ts65Dn model suggesting effect due to the new chromosomal configuration.The Ts65Dn allele induces early post-natal lethality which has also been described in Ts1Yu mice, a trisomic model for a larger region of Mmu16 extending from x1 genes . The traAs we found that the Ms5Yah monosomy rescued Ts65Dn post-natal lethality, we decided to look at ECG in Ts65Dn/Ms5Yah adult mice to determine whether the combination between these two models had an effect on electrocardiologic pattern. Ts65Dn/Ms5Yah mice showed wave shape anomalies similar to those described above for Ts65Dn mice. However, relative distribution of these features in Ts65Dn/Ms5Yah  showed aP\u200a=\u200a0.014) but not from the Ts65Dn (P\u200a=\u200a0.53).As Ts65Dn mice showed a variable orientation of the QRS axis, this item was also determined for Ts65Dn/MS5Yah mice. Repartition of this axis for wt, Ts65Dn and Ts65Dn/Ms5Yah by quadrants is reported in P\u200a=\u200a0.23 and P\u200a=\u200a0.052) or Ts65Dn (P\u200a=\u200a0.57 and P\u200a=\u200a0.48) mice. QTc was significantly reduced down to 65.5\u00b12.8 ms  in Ts65Dn/Ms5Yah mice. This value was no longer different from the wt value (P\u200a=\u200a0.075). Thus two copies of the App-Runx1 region in Ts65Dn/Ms5Yah mice led to a partial recovery of the Ts65Dn electrocardiographic phenotypes. Wave shape anomalies, wave amplitudes on precordial leads and QTc duration were rescued whereas wave amplitudes on peripheral leads were not and RR and PR were partially rescued as they showed an intermediate duration between Ts65Dn and wt values.In the frontal plane, wave amplitude of Ts65Dn/Ms5Yah mice showed the same trend as Ts65Dn mice when compared with wt, with significant decrease of the S wave amplitude in inferior leads, increase in superior leads and smaller changes in R waves. While the Ts65Dn/Ms5Yah model did not rescue the changes in wave amplitudes in the frontal plane, it completely rescued changes in the sagittal plane  as illustrated in App-Runx1 region to two copies in Ts65Dn mice points at the gene dosage effect related to this interval. Whole genome expression arrays were then performed on adult mice heart samples to determine genes that are dosage sensitive and to observe the deregulations on the whole genome. Wt, Ts65Dn, Ms5Yah and Ts65Dn/Ms5Yah heart samples were analysed using Affymetrix Gene Chip technology. RMA normalized data obtained from Affymetrix Expression Console software were filtered using an expression level threshold above 50 in raw data  and fold change (FC) between wt and transgenic mice >1.2 or <0.8. We measured the expression levels of 25,099 transcripts, representing 22,193 genes. Among those, 9,017 genes (40.6%) had a fluorescence signal above the threshold in wt samples and were considered as expressed genes in adult mouse heart. The ratio of gene expression across chromosomes between aneuploid and wt mice was 1.00\u00b10.002 (ranging from 0.98\u00b10.003 to 1.03\u00b10.005) except for genes located on the monosomic and trisomic intervals located on Mmu16 and Mmu17 for the specific region found in the Ts65Dn minichromosome The rescue of Ts65Dn viability and electrocardiographic phenotypes by reestablishing the post hoc analysis. The clustering on arrays of most significantly deregulated genes  are represented on In order to further analyze gene expression modifications GeneSpring software was used to define the most significantly deregulated genes using one way ANOVA and a Tukey HSD App or downstream of Runx1, or on the Mmu17 centromeric regions of the Ts65Dn minichromosome. These genes were found over-expressed in both Ts65Dn and Ts65Dn/Ms5Yah arrays but not in Ms5Yah arrays. We also found a set of 12 genes located elsewhere whose expression follow the same rationale, 9 overexpressed and 3 down-regulated as shown on Msx2, and only 6 additional genes from different chromosomes that had similar expression variations correlated with changes both in the number of copy of the App-Runx1 region and in the Ts65Dn trisomy. The other groups were mainly composed of genes located outside the above-mentioned Mmu16\u2013Mmu17 regions and were distinguished by their expression patterns. Genes from the third group were differentially expressed in Ts65Dn arrays, while comparable to wt in Ms5Yah and in Ts65Dn/Ms5Yah arrays. 3 genes from this group were found in the proximal region of Mmu17 that is trisomic in the Ts65Dn model and one, Tiam1, was located in the App-Runx1 region but whose expression is not sensitive to dosage in Ms5Yah mice. The expression of genes from the fourth group was modified only in the Ms5Yah model. Among those genes, one gene, Sft2d1, comes from the centromeric region of Mmu17 and 5 genes  are from the Ms5Yah interval. They were all down-regulated. In addition 13 down-regulated genes and 18 up-regulated genes are found outside the aneuploid regions. A fifth group contained 10 genes over-expressed in Ms5Yah and Ts65Dn/Ms5Yah arrays whereas 15 additional genes displayed a more complex expression pattern (groups 6 and 7).Gene expression profiles were categorized in seven distinct groups with respect to genotype-associated expression patterns that are listed in App-Runx1 region that were expressed at a ratio versus wt close to 1.0. As shown in Atp5j, Cldn14, Erg, Pcp4 and Prdm15) and four on Mmu17  were found expressed but not dosage sensitive in heart.Looking more closely at the 109 Mmu16 known protein coding genes present on the Ts65Dn minichromosome, we found that 4 were absent from the chip and 39 were expressed below background level. Among the 66 remaining genes expressed in adult mice heart, 78.8% were up-regulated in Ts65Dn arrays with a mean fold change of 1.36\u00b10.01 (ranging from 1.20 to 1.61). Similarly, of the 43 Mmu17 known protein coding genes located on the Ts65Dn minichromosome, 8 were absent from the chip and 7 were not expressed. Of the 28 Mmu17 remaining trisomic genes expressed in adult mice heart, 78.6% were upregulated in Ts65Dn arrays with a mean fold change versus wt of 1.36\u00b10.02 (ranging from 1.21 to 1.55). Thus the presence of the Ts65Dn minichromosome results in an overall upregulation of genes present in three copies as shown on Usp16, Cct8 and Bach1 (located between App and Runx1 genes) showed fold change versus wt above 1.2 in Ts65Dn, below 0.8 in Ms5Yah and close to 1.0 in Ts65Dn/Ms5Yah samples. Dyrk1a and Sh3bgr (located between Runx1 and Zfp295) showed over-expression (fold change above 1.2) in Ts65Dn and Ts65Dn/Ms5Yah and fold change close to 1.0 (Dyrk1a) and slightly under-expressed (Sh3bgr) in Ms5Yah samples. All these data confirm the results obtained from expression arrays. Thus variation of gene expression was scored and partly verified for several genes which belong to groups of genes misregulated as a consequence of Ms5Yah or of Ts65Dn aneuploidies. Some of them were located outside the aneuploid regions, indicating a genome-wide trans effect on gene expression in Ts65Dn, Ms5Yah andTs65Dn/Ms5Yah mice hearts.Five genes were selected to validate expression levels observed with arrays using quantitative PCR analysis , Table 6eGFP transgenic mouse model whose heart conduction system is labeled with Green Fluorescent Protein (GFP) eGFP/+;Ts65Dn mice affected by clearcut ECG phenotypes were compared to three Cx40eGFP/+ disomic mice showing no ECG anomaly. Left and right ventricles were dissected out as described by Miquerol et al.et al.To go further in the understanding of ECG anomalies, an additional experiment was designed by targeting and measuring expression levels of genes related to conduction anomalies in ventricles and atria separately. Action potential propagation in the cardiac cellular network mostly depends on three factors: geometry , cardiac connexins and ion channels availability, in particular sodium channels Cx40, Cx43, Cx45 and Cx30.2 are chamber- and tissue- specific in the heart Scn5a gene which codes for a pore forming protein and is mutated in human heart conduction diseases  and for which heterozygous mutant mice suffer from abnormal heartbeats and defects in the impulse conduction system and also at Scn10a recently implicated in heart conduction + channel Scn4b was found underexpressed in Ms5Yah mice using affymetrix array and we also investigated the other beta-subunit Scn1bCx40 and Cx43 by 34 and 39% respectively and sodium channels-coding genes Scn5a and Scn10a were also down-regulated by 42% and 31% respectively and Scn1b by 29% , frontal QRS axis dispersion, decrease in right precordial lead amplitude and P wave changes, replicating in part the defects observed in DS people. PR interval prolongation (first degree AV block) observed in Ts65Dn mice includes auricle, AV node and His bundle conduction time. Gross morphology of the His bundle, as deduced from CX40-GFP labeling, does not appear to be changed. The AV node is more likely to account for PR interval prolongation even though enlargement of the auricle, as suggested by P wave amplitude increase in V4 lead and P wave axis change, could also be involved. In DS patients, such AV block and P wave changes have been recorded Cx40, Cx43 and Na channels expression that we observed in the auricle of Ts65Dn mice could account for the first degree AV block supported by the prolongation of the PR interval that includes auricle, AV node and His conduction time. This could also account for the P wave amplitude changes. Enlargement of the auricle as suggested by P wave amplitude increase could also participate to PR prolongation. Both the effect of flecainide and the absence of Na channel expression changes in ventricles do not support a decrease in Na channel availability. In the absence of large patch of fibrosis and of clear-cut change in CX40 distribution in Ts65Dn, f-QRS could be related to local defect in CX40 associated to discrete local increase in collagen. This needs further investigation but could account for the observed loss of Cx40 expression.Most of the observed effects of the Ts65Dn trisomy on ECG are related to conduction known to depend on membrane excitability, intercellular coupling and tissue architecture App-Runx1 fragment in the Ts65Dn model (double transgenic Ts65Dn/Ms5Yah mice) resulted in the rescue of Ts65Dn postnatal lethality, indicating that one or more genes present on this region are responsible for the observed lethality and that Ts65Dn trisomic genes located on Mmu17 are not major players in this phenotype. Our finding, combined with the different observations of CHD and lethality present or absent from other DS models Sod1, Tiam1 and unknown predicted genes such as Gm10789 or Gm2771 that are trisomic in Dp16(2)Yey mice which display CHD but are only in two copies in the Ts1Cje, Dp(16)1Yey/Df(16)2Yey, and Ts65Dn/Ms5Yah models. Sod1 was found decreased in Ms5Yah but not overexpressed in Ts65Dn. On the contrary Tiam1 was found increased in Ts65Dn heart and not significantly downregulated in the Ms5Yah. Thus Tiam1 is a candidate for cardiac defect in Ts65Dn. Tiam1 encodes an ephrin related receptor that influences synapse functions and controls epithelial tight junctions Bach1 and Rcan1 which were found deregulated in Ts65Dn and Ms5Yah arrays but not in Ts65Dn/Ms5Yah samples. Nevertheless in Ts65Dn/Ms5Yah mice the monosomy-induced lethality of the Ms5Yah allele is in part rescued but not as complete as for the Ts65Dn allele. Somehow some genes that are not included in the Tiam1-Cbr overlap between Ms5Yah and the Df(16Tiam1-Kcnj6)Yey/+, but located at the boundaries of the considered region, i.e. in the Mrpl39-Tiam1 or Kcnj6-Zfp295 intervals, must have a major effect on survival of the Ms5Yah mice. However, the CHD observed in Ts65Dn dead pups is lower than that observed in DS patients, indicating that one or more gene outside of the Mrpl39-Zfp295 region are contributing to CHD.Restoring the disomy of the genes present in the App-Runx1 region in double transgenic Ts65Dn/Ms5Yah mice. Reduced heart rate and prolonged QRS and QT observed in Ts65Dn mice were all back to normal in Ts65Dn/Ms5Yah mice, whereas the first degree block (PR) was only partially restored. The essential role of the App-Runx1 region in the appearance of the Ts65Dn electrocardiographic pattern is hence highlighted by the reduced ECG phenotype in Ts65Dn/Ms5Yah mice. The obvious changes recorded in the waves registered by precordial recording between Ts65Dn and Ts65Dn/Ms5Yah and the rescue of the QT phenotype point again to a major contribution of the App-Runx1 region to Ts65Dn ECG phenotypes. However, ECG defects are not completely recovered in the Ts65Dn/Ms5Yah compound animals and are probably induced by complex interactions between genes located in distinct regions of the Mmu16.Many aspects of the ECG phenotypes observed in adult Ts65Dn mice were rescued by re-establishing euploidy of the Kcnj6Rcan1 is clearly involved in the formation of the annulus fibrosis between the auricles and ventricles as well as in the formation of the valvules App-Runx1 region that triggers conduction defects and contributes to the risk of CHD.Recent evidence suggests that the same genes that cause defects in heart development and CHD might be involved in cardiac dysfunction such as abnormal electrical conduction and diminished contractile function App-Runx1 interval whose expression was detected in the heart were down-regulated to 0.68\u00b10.08 expression level compared to wt and 3 genes, Tiam1, Slc5a3 and Mrps6, were not affected by decrease in copy number. For most of those aneuploid genes, expression level returned to normal (1.10\u00b10.1) in Ts65Dn/Ms5Yah double transgenic mice. The overall difference in gene expression levels between the different mouse models can be explained by gene dosage. Our data support the hypothesis that a triplicated Hsa21 causes a 50% increase in trisomic genes expression as a primary dosage effect Atp6j expression is not affected by gene copy number while Tiam1 is more sensitive to increase copy number. In Ts65Dn heart, trisomic genes Sod1, Atp5o, Hcls, Ripply3, Psmg1, and Sh3bgr were not affected by the trisomy while Slc5a3 and Mrps6, back to two copies in Ts65Dn/Ms5Yah mice, were still overexpressed.We compared transcriptional profiles of RNA from adult mouse heart of 2n, Ts65Dn, Ms5Yah and Ts65Dn/Ms5Yah mice in order to determine aneuploid genes that are sensitive to gene dosage and hence might be candidate for the heart phenotypes, and to attempt to correlate observed transcript level differences to pathways impacted by the different aneuploidies. Most of the expressed triplicated/monosomic genes were up-regulated in Ts65Dn, down-regulated in Ms5Yah and expressed at similar levels in wt and in Ts65Dn/Ms5Yah hearts. 73 genes located on the Ts65Dn chromosome were overexpressed in the heart with a ratio versus wt of 1.34\u00b10.15; only 12 were not found deregulated. 19 of those genes were located on the Mmu17 region triplicated in the Ts65Dn App-Runx1 interval. 13 genes were found on the proximal part of the Mmu17 of the Ts65Dn chromosome, 14 trisomic from the Mmu16 and 12 located elsewhere in the genome. With the second group we identified 33 genes of which 26 from the App-Runx1 interval, that were deregulated in both Ts65Dn and Ms5Yah and compensated in Ts65Dn/Ms5Yah double mutant mice. Group 3 encompassed 16 genes specifically deregulated by in Ts65dn hearts while 40 genes specific of the Ms5Yah heart are found in group 4. We also identified 3 additional groups  containing 10, 10 and 7 genes respectively, with expression level affected by combination of aneuploidies. For example group 5 were specific for the Ms5Yah with similar level in the Ts65Dn/Ms5Yah mice. Interestingly genes from the seven groups contribute to pathways related to the observed phenotypes. 25 genes out of 151 are associated with embryonic lethality, growth defect, and premature death (Mouse Genome Database (MGD) November 2011, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, Tfb1m and Vwf) that could be involved in the Ts65Dn perinatal lethality and 9 from group 2 , for which mutation impaired embryonic viability and growth that could contribute to Ts65dn and to the Ms5Yah impaired viability. In addition Amot, Ccne2, Cerk, C1qa and Fzd3 from group 4, could contribute to growth retardation and premature death Grin2b or Psmg1 can still contribute to the perinatal lethality observed in the Ts65Dn/Ms5Yah Tfb1m (group 1) causes abnormal heart development and physiology that could affect the viability of the Ts65Dn/Ms5Yah mice Adam19, Slc8a1/Ncx1 and Rcan1 from group 2 are able to induce various types of heart defects from irregular heartbeat to ASD and VSD Ripply3 and Ccne2 (group 4) loss-of-functions potentially induce VSD App-Runx1 region play an important role in the heart defects and lethality observed in Ts65Dn and suggest some pathways altered in DS heart. Overall the analysis reveals the complexity of the phenotype with several trisomic genes along the Hsa21 working alone or in cooperation to contribute to the whole range of heart defects observed DS. Whole genome expression analysis pointed at some deregulated genes, whose contribution should be further analyzed. These data were obtained from adult trisomic mice, and thus do not give information about expression at the embryonic or postnatal states. We believe that further molecular and electrophysiological studies at postnatal states could thus give important information about genes involved in early postnatal lethality and should confirm or point to new candidate genes.Detailed expression analysis highlighted a list of 151 deregulated genes that were categorized in 7 groups . 82 of tMice were handled with the agreement of the local ethical committee and in accordance with the European Council Guidelines for the Care and Use of Laboratory animals (accreditation 7320). They were housed under a 12 h/12 h light-dark cycle in TAAM-CNRS husbandry at Orl\u00e9ans (France) (certificate C45-234-6) and fed on a standard rodent chow. YH, as the principal investigator in this study, was granted the accreditation 45-31 and 67\u2013369 to perform the reported experiments.16)65Dn (Ts65Dn) mice were purchased from the Jackson Laboratory . They were mated with F1 B6C3B males, in which the B6 are C57BL/6J mice and C3B are sighted C3H/HeH, a congenic line for the BALB/c allele at the Pde6b gene et al.App and Runx1 genes were obtained by in vivo TAMERE App locus using MICER vector tm1YahApp mouse line was generated. tm1YgRunx1 mice were described previously Tg(Pgk1-Cre)1Lni, expressing the Cre recombinase under the control of the early acting phosphoglycerate kinase-1 promoter App-Runx1 region in females gametes and to an offspring carrying the Del(16App-Runx1)5Yah noted here Ms5Yah, monosomic for this region  and Mx1 (gene of interest). Apob forward (CACGTGGGCTCCAGCATT), Apob reverse (TCACCAGTCATTTCTGCCTTTG), Mx1 forward (TCTCCGATTAACCAGGCTAGCTAT) and Mx1 reverse (GACATAAGGTTAGCAGCTAAAGGATCA) primers were purchased from SIGMA-Aldrich, and Taqman MGB probes Mx1 FAM (6-FAM-CCTGGTCGCTGTGCA-MGB-NFQ) and Apob HEX (HEX-CCAATGGTCGGGCAC-MGB-NFQ) from Applied Biosystem. PCR conditions were as follows: (1) 50\u00b0C for 2 min, (2) 95\u00b0C for 10 min, (3) 95\u00b0C for 15 sec, (4) 60\u00b0C for 1 min (steps 3 and 4 were repeated 50 times). Alternatively we used the PCR\u2013based protocol For identification of both Ms5Yah and Ts65Dn alleles, genomic DNA was isolated from tail biopsies using the NaCl precipitation technique. The Ts65Dn allele was identified using a Taqman qPCR protocol with differential analysis of quantity between 5\u2032-ATCCGGGAATGGTCCCTA-3\u2032) specific for the wt allele, one Fwd primer (5\u2032-CAAGCACTGGCTATGCATGT-3\u2032) specific for the Ms5Yah allele and a Ms5Yah/wt Rev (5\u2032-GTTCGTTGCCTGAAGGAGAG-3\u2032) primer common to both alleles. PCR reactions gave wt and Ms5Yah products of 482 bp and 328 bp long respectively.The Ms5Yah allele was identified by PCR using one Fwd primer  for 24 hours and rinsed in PBS. Micro-dissections were realized to assess cardiovascular malformations using a Leica MZFL-III dissecting microscope equipped with a Leica DC200 digital camera. Aortic arches were then removed and hearts embedded in paraffin using a Leica TP 1020 tissue processor. Serial sections between 5 \u00b5m and 8 \u00b5m were fixed on Stick-On coated slides (LABOnord). Slides were dewaxed in xylene, rehydrated and stained in hematoxylin and 0.2% eosin. Images were captured using a Leica M420 brightfield macroscope equipped with a Photometrics Cool Snap digital camera.3) with a 1\u2013500 Hz bandwidth  and continuously monitored on a Gould oscilloscope . Acquisition was routinely performed at 2 kHz . R, S, and RS wave amplitudes and heart rate (HR) were averaged and monitored on line every 10 s. After 1 h of control recording, the 6 peripheral and 4 precordial leads were recorded at 5 kHz for one minute each.Electrocardiogram (ECG) was recorded under urethane anesthesia  as previously described et al. to label the second wave adjoining the QRS complex as a J wave 1/2. Electrical axis was determined from the algebraic sum of the QRS amplitude in aVF and DI. Flecainide  was prepared in distilled water and injected intraperitoneally.HR mode was obtained from HR averaging over 10 s. Off-line analysis of ECG was performed with ECG-auto software (v. 1.5.11 EMKA Technologies). The ECG wave analysis used ECG libraries made up from original tracings. Measurements were performed every 20 sec, and reported values are the average of five measurements made on individual wave complexes taken over the first seconds of each interval. As the mouse ECG displayed no real electrical zero, this was taken as the average of five consecutive points taken between the end of P wave and the beginning of Q (or R) wave (from time 15 ms before Q/R wave beginning) as in infants. We followed Liu P<0.05.Results are given as mean values \u00b1 standard error of the mean (sem). Statistical tests were performed with Sigma Stat software (Systat Software). We used the Student's t-test and ANOVA followed by the Student-Newman-Keuls (SNK) multiple-comparison tests. Proportions were analyzed with the Fisher exact test. Significance was set at For Affymetrix arrays and qPCR validation, hearts were isolated from Ts65Dn, Ms5Yah, Ts65Dn/Ms5Yah and wt control mice (N\u200a=\u200a5 per group) at 5 months of age and flash frozen. For cardiac connexins and sodium channels expression, hearts were isolated from 7 Ts65Dn and 5 wt adult mice at 4 months of age, and ventricles were separated from atria and flash frozen. Total RNA was prepared using Trizol (Invitrogen) according to the manufacturer's instructions. Samples quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies).et al.P<0.1 were selected for clustering analysis. Hierarchical clustering was carried out with Cluster3.0 software using Euclidian distances to calculate the distances between the genes and between the samples. Calculated distances were then clustered by complete linkage clustering. Post-Hoc analysis using GeneSpring gave a list of statistically deregulated genes. Known mammalian phenotypes database  and functional annotation clustering using Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics were performed to estimate the potential impact of deregulated genes in transgenic mice. This latter tool mainly provides typical batch annotation and gene-GO term enrichment analysis to highlight the most relevant Gene Ontology (GO) terms associated with a given genes list Biotinylated cDNAs were prepared from total RNAs and hybridized onto GeneChip Mouse GENE 1.0ST arrays (Affymetrix). Chips were washed and scanned on the Affymetrix Complete GeneChip instrument system generating digitized image data files. Raw data was processed with the Robust Multiarray Average (RMA) algorithm developed by Irizarry Ppia, Gnas, Hprt1, and Pgk1 were selected as reference genes for normalization for both Affymetrix array validation and cardiac connexins and sodium channels gene expression experiments. Primers and Taqman probes were designed using Primer3 software (P<0.05.After DNase treatment with TurboDNA-free (Applied Biosystems), 1 \u00b5g of total RNA was converted to cDNA using Superscript III (Invitrogen) primed with poly d(T) and random hexamers. software . Taqman Figure S1App-Runx1 region (A) on Mmu16 was targeted for in vivo Cre/loxP recombination by inserting a loxP site on App locus in tm1YgRunx1 (B). Recombination using Tg(Pgk1-cre)1Lni led to deletion of the floxed fragment creating Ms5Yah mouse model. Chimeras and monosomic mice were distinguished using Southern Blot genotyping (C) and the deletion was confirmed by CGH arrays (D).Ms5Yah mouse model creation and validation. (TIF)Click here for additional data file.Table S1P<0.1. Genes in pink. orange and blue are genes located respectively within the centromere-Pde10a region on Mmu17. the Mrpl39-Runx1 and Runx1-Zfp295 region on Mmu16 trisomic in Ts65Dn mice. We calculated the mean, the standard error to the mean for the expression ratio and the Student-t-test comparing mutant versus wt and annotated with * if P<0.05. ** if P<0.01 and *** if P<0.001. Overexpressed genes with an expression ratio>1.2 are underlined in red whereas down-regulated gene with a ratio<0.8 are in green. Normal expression ration between 0.8 and 1.2 are underlined in grey.List of deregulated genes in Affymetrix hybridization arrays. This list was obtained by using ANOVA with Bonferroni correction and fixing a threshold of (PDF)Click here for additional data file.Table S2Sequences and Tm of primers and Taqman probes used for qPCR analyses. Genes in bold were used as housekeepers.(PDF)Click here for additional data file."}
+{"text": "The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an  Individuals with chronic HIV-1 infection in absence of antiretroviral therapy (ART) tend to develop cross-neutralizing antibodies over a period of years env clones we mapped determinants in the C2V3 region which were found to modulate neutralization properties of autologous Envs amplified from this patient.In the present study, we examined the relationship between genetic and neutralization properties of autologous HIV-1 Envs that modulated neutralization properties of circulating envelopes obtained from a chronically infected Indian patient whose plasma showed BCN response env clones from plasma of this patient that showed presence of clade C and non-clade C mosaics; however only four functional Envs were obtained that gave detectable infectious titers as Env-pseudotyped viruses. The limited number of env clones obtained from the LT5 plasma showed genetic heterogeneity env clones were found to be functional and gave reasonable infectivity titers. Sequence analysis indicated that out of these four env clones, two were of pure clade C (LT5.J3b and LT5.J7b) and two were B/C recombinants (LT5.J4b and LT5.J12). First we examined the neutralization sensitivity of pseudotyped viruses expressing these four Envs to contemporaneous autologous plasma. As shown in We previously reported Next, we examined the degree of sensitivity of these four LT5 Envs to reagents that target distinct sites in Envs, such as CD4bs , V3 loop (MAbs 3074 and 3869), CD4-induced epitopes/coreceptor binding site (17b MAb) and MPER (MAbs 4E10 and 2F5) To dissect the sequence of the LT5.J4b Env conferring enhanced neutralization, first we swapped different segments between the LT5.J4b (sensitive) and LT5.J12 (resistant) Envs to construct chimeric Envs . PseudotAt this juncture, we wanted to fine map residue/s within C2V3 sequence in LT5.J4b Env to examine if there is/are any specific residue/s that possibly contributed to enhanced virus neutralization. When compared between LT5.J4b and LT5.JJ12 Envs, we found fifteen differences in amino acid residues in C2V3 region (twelve in the C2 and three in the V3 loop). Specific point substitutions were made in the LT5.J12 Env as described in Methods and pseudoviruses expressing mutant Envs were prepared. Interestingly, we did not find any specific substitution conferring sensitivity to LT5.J12 Env . The Q24We next assessed the relationship between presence of unique C2V3 sequence and relative binding of MAbs to Env trimers targeting different regions of Env, which showed enhanced neutralization of LT5.J4b Env. 293T cells were transfected with equal concentrations of Env plasmid DNA and the relative binding of MAbs or CD4-IgG2 to Env trimers expressed on 293T cells was assessed by measuring relative luminescence units (RLU) as described previously Since the unique sequence of the C2V3 region was found to modulate both the neutralization sensitivity and binding of Envs to IgG1b12 and CD4-IgG2 which are the prototypic ligands that bind to CD4bs on Env trimer, we subsequently examined the effect of this modulation on the infectivity and CD4 dependence of these Envs. For this, we tested the infectivity of the Envs expressing wild type Envs and C2V3 chimeric Envs in HeLa RC49 cells, expressing high amount of coreceptor CCR5 but limiting amount of CD4 Next we evaluated whether there was any association between neutralization sensitivity of both LT5.J4b (sensitive) and LT5.J12 (resistant) Envs with ligand-induced gp120 shedding. The MAbs and sCD4 (referred to as ligands here) were mixed with pseudotyped viruses expressing LT5.J4b and LT5.J12 Envs with equal infectious TZM-bl titers in a final concentration that showed 50% neutralization of these Envs (see Methods). These virus-ligand mixtures were incubated at 37\u00b0C for different time periods as indicated , and theSince we found that the LT5.J4b Env that was found to be very sensitive to contemporaneous autologous plasma also showed enhanced sensitivity to IgG1b12 and anti-V3 MAb, the LT5 patient\u2019s plasma showing BCN activity In order to understand if presence of unique mutations in the C2V3 region  modulateIn the present paper, we examined the vulnerabilities in circulating HIV-1 Envs obtained from plasma of a chronically infected patient that showed cross neutralization of several heterologous HIV-1 Env-pseudotyped viruses. In essence, we examined the determinants in Env that conferred escape from neutralization by autologous antibodies. Although we obtained mixture of clade B and C and B/C recombinants from the plasma of this particular patient, the plasma antibodies exhibited bias towards clade C The presence of BCN antibodies in the plasma of the LT5 patient who was chronically infected for several years without any anti-retroviral therapy, possibly accounted for restricting clinical advancement to disease stage. The presence of BCN activity of plasma of the LT5 patient containing mixture of viral subtypes in vivo even in the pressure of autologous neutralizing activity in vivo as suggested by the low CD4 dependence of LT5.J4b Env. Compared to LT5.J4b other Envs exhibited high CD4 dependence for entry and might replicate with slower rate although they are neutralization resistant and possibly might contribute to the survival of sensitive species. The survival of the sensitive viral species could also be the decoy strategy to direct the immune response towards them and to retract the antibody pressure exerted on the resistant species. This phenomenon could possibly be the basis for slow disease progression as far as this particular patient is concerned. Irrespective of the basis of existence, both sensitive and resistant Envs provided an opportunity to dissect the region in the Env that conferred enhanced sensitivity autologous antibodies. The chimeric constructs made by using the LT5.J4b and LT5.J12 Envs revealed that presence of unique C2V3 sequence conferred LT5.J4b Env with enhanced neutralization susceptibility. Though the role of V1V2 in masking the epitopes has been well documented we did not observe any change in neutralization sensitivity to plasma antibodies when this region was transferred from LT5.J4b (sensitive) to LT5.J12 (resistant). The C3 and V4 regions in Env individually or combined was not found to be involved in forming neutralization epitopes. The C3 region contains highly variable \u03b12 helix which was shown by previous studies to modulate the neutralization sensitivity to autologous plasma directly or indirectly by influencing CD4 binding loop in C3 and imparting more flexibility or structural adaptation to the protein in response to neutralizing antibodies et alAmong the four Envs from LT5 plasma LT5.J4b was found to be most sensitive to autologous plasma and MAbs targeting different regions on Env. Since, we were not able to analyze Env variants from plasma of earlier and later time points; the evolution dynamics of LT5 Envs could not be known. However, the sensitive viral species can emerge The sequence conservation of the C2V3 region and that it contains the CD4 contact residues signifies the functional role of this region The glycosylation on the Env trimer is another important factor that influences the neutralization sensitivity. Presence of a PNG at the 386 position shields the neutralizing epitopes in the CD4bs and its removal was previously shown to increase the sensitivity of HIV-1 Envs to IgG1b12 apparently by filling a cavity penetrated by W100 of IgG1b12 Our data provided important information on atypical determinants in the C2V3 region of Env that gave rise to greater exposure of neutralizing epitopes in CD4bs, V3 loop and coreceptor binding sites. Both CD4bs and V3 loop are important regions on gp120 which are highly antigenic and immunogenic in nature Although the specificity of LT5 plasma was partially mapped to CD4bs and V3 loop it is possible that other specificities were also present as the gp120 OD construct used to adsorb IgG1b12 like antibodies may also adsorb antibodies which bind further apart from CD4bs on gp120 outer domain. Multiple antibody specificities were reported in chronic infections E coli competent cells. Similar procedure was followed for site directed mutagenesis except the forward primer of insert and corresponding backbone primer which amplifies in opposite direction introduced single or multiple mutation.Chimeric Env cloning and site-directed mutagenesis was carried out by using tailor-made primers as shown in 4 TZM-bl cells were added into the mixture in presence of DEAE-Dextran  making final concentration 25ug/ml (Sigma Inc.). The plates were incubated in a CO2 incubator for 48 hours and the degree of virus neutralization was assessed by measuring relative luminescence units (RLU) by using BriteliteTM luciferase substrate (Perkin Elmer Inc.) in a luminometer .The neutralization assays were carried out as described earlier + 293T cells for 1 hour at 37\u00b0C and subsequently with goat-anti-human IgG conjugated with horse radish peroxidase . The cells were washed for four times with first blocking buffer  2, 1 mM MgCl2, 20 mM Tris, pH 7.5) The relative interaction of Env with MAbs or CD4-IgG2 was assessed by cell-based binding assay as described before et al.5 RLU/well); no MAbs) was incubated for the indicated time periods at 37\u00b0C in culture medium supplemented with a ligand in the concentration that gives approximately 50% neutralization at 1 hour of incubation. The virus incubated at different time points was then used to infect TZM-bl target cells, and virus infectivity was monitored by luciferase activity at 72 h after infection. Time of addition experiments were conducted to probe the kinetics of virus neutralization by different ligands and to assess the induction of gp120 shedding from the virions. Antibody concentrations were chosen to yield neutralization of approximately 50% after 1 h of pre-incubation to allow for the monitoring of increases in neutralization activity over time. The selection of precise ligand concentration was possible for LT5.J4b as it is sensitive to most ligands tested here but LT5.J12 was resistant at 10 ug/ml, highest concentration tested. For LT5.J12 therefore, the highest concentration was used to assess the gp120 shedding over the period of time.The gp120 shedding was assessed by measuring infectivity of virus incubated at 37\u00b0C as described by Ruprecht Escherichia coli-expressed protein based on the sequence of the HxBc2 strain which binds CD4 and the broadly neutralizing antibody IgG1b12 but not the non-neutralizing antibodies b6 and F105 5 RLU virus was then added in each well and incubated the plate for another 1 hour. After incubation 1\u00d7104 cells were added with 25 ug/ml dextran and incubated the plate for 48 hours. Infection of TZM-bl cells was measured by luciferase activity and the increase in infection in presence of probe was compared with the wells where probe was not added.Adsorption of LT5 plasma was performed by using an outer domain (OD) protein of gp120 The correlations between IgG1b12 and LT5 plasma sensitivities of a panel of 30 Envs Structural models of HIV-1 C2V3 regions were produced with SWISS MODEL homology modeling server in project mode The authors declare that they have no competing interests.Figure S1Neutralization sensitivity of LT5 Envs to VRC01 and 2F5 monoclonal antibodies. Note that all the LT5 Envs showed resistance to VRC01 MAb including the sensitive LT5.J4b Env.(TIF)Click here for additional data file.Figure S2Correlation between IgG1b12 and LT5 plasma sensitivities of a panel of 30 Env pseudotyped viruses .(TIF)Click here for additional data file.Figure S3Construction of chimeric and mutant Envs. (A) Chimeric Envs were constructed using a region exchange strategy and mutant Envs were made by introducing specific nucleotide change in primer. (a) The domain to be transferred for example C2V3 (light blue box) was PCR amplified from the env gene using primers (arrows) that annealed to conserved sequences flanking C2V3 and amplified inward. (b) The recipient Env (open box) plus plasmid vector was PCR amplified using primers (arrows) that annealed to sites adjacent but overlapping (at least 15 base pairs) to those of the C2V3 primers and amplified outward. (c) The two fragments were then mixed with PCR cloning cocktail containing pox virus DNA polymerase which forms cohesive ends in PCR fragments by its 3\u2032\u20135\u2032 exonuclease activity. The cohesive ends anneal and forms circular plasmids with a nick at each strand. The annealed product is directly transformed in competent cells where the nicks are sealed. (B) The PCR fragments after treatment with dry down cloning cocktail. The homologous sequence is annealed to yield circular plasmid. (a) chimeric and (b) mutant Env preparation is shown.(TIF)Click here for additional data file."}
+{"text": "Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4+FOXP3+T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4+T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4+ T cells as a predictor of OSCC recurrence.Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4 While the infiltration of effector lymphocytesisgenerally associated with a good prognosis, the infiltration of other immune cell populations (i.e.Myeloid Derived Suppressor Cells (MDSC) and T regulatory cells (Treg)) isthought to promote tumor progression by restraining tumor immunity and promoting neoplastic cell invasion and metastasis +tumor infiltrating cells. The results indicate that the presence of CD4+ cells expressing FOXP3 in the cytoplasm is associated with a favorable prognosis whereas its nuclear localization correlates with an increasedrisk of recurrence. In light of these results,we propose the use of cFOXP3/nFOXP3 ratio as a prognostic factor in OSCC.In this retrospective case-control study, we examined the prognostic value of FOXP3 with respect to recurrence of OSCC taking into account the subcellular localization of FOXP3 within CD4This study was approved by the University of Miami IRB before initiation.Due to the retrospective nature of the study and the lack of personal identifier in the specimens evaluated the requirement for informed consent was waived by the IRB.We selected specimens from patients who underwent glossectomy (with or without neck dissection) and without prior treatment by either radiation or chemotherapy. Subjects were identified from among those treated at our tertiary referral academic medical center between 1/1/2001 and 12/31/2010 by search of a registry of CPT  codes for glossectomy . Only patients with a diagnosis of SCC of the oral tongue staged T1 or T2  whose surgery was limited to a partial glossectomy with or without neck dissection were included. We excluded patients with a diagnosis of in-situ carcinoma, T3 or T4 disease, involvement of other oral or oropharyngealsubsites, need for any form of mandibulectomy or other more extensive surgical procedure, a prior history of radiation to the head and neck or treatment with chemotherapy, or any history of immunosuppression or immune compromise.Additional criteria required availability of sufficient residual archival paraffin embedded tissue from the surgical resection for experimental analysis and documentation of disease status three years from date of surgery. A total of 392 clinical records were obtained, with 49 meeting inclusion criteria.Patients\u2019 HPV status was determined by the head and neck pathology core at University of Miami using standard IHC protocols to detect p16 of Human papillomavirus.Four \u00b5m sections underwent deparaffinization, rehydratation, and incubation for 30 min at RT in a sodium borhydride solution  to reduce sample autofluorescence. Antigen retrievalwas performed by a 15 min incubation at 95\u00b0C in EDTA antigen retrieval solution pH\u200a=\u200a9 . The slides were then incubated with Image-iT (Invitrogen) for 30 min at RT followed by incubation (1 h at RT) with PBS containing 1% BSA and 0.3% Triton-X100 to permeabilize the tissue and to block non-specific binding. Samples were incubated O/N at 4\u00b0C with the mouse monoclonal, anti-human FOXP3 antibody ab20034  and the goat polyclonal anti-human CD4 antibodyAF-379-NK,  in PBS with 1% BSA. After three washes with PBS, samples were labeled for 1 h at RT with the Alexa-555 conjugated anti-mouse antibody (for FOXP3) and with the Alexa-488 conjugated anti-goat antibody (for CD4). Both antibodies were used at a 1/400 dilution in PBS/BSA (1%). Finally, sections were counterstained in PBS containg 2 mMDAPI (Invitrogen),for 15 min at RT, rinsed with PBS and coverslips were mounted using Biomeda gel mounting media . Slides were analyzed with ZeissAxiovert Microscope and the Zeiss Axiovision LE software  at 20\u00d7magnification or with the SP5 spectral confocal inverted microscope (Leica) at the University of Miami imaging core facility.http://rsbweb.nih.gov/ij/www.cellprofiler.comAreas of interest corresponding to the neoplastic lesion of the tissue were identified by an experienced pathologist in serial section stained with H&E. Microphotographs of 5 random fields at 20X were taken in the corresponding area of the fluorescence labeled sections with a Zeiss Axiovert Microscope. Images for each patient were qualitatively evaluated with ImageJ . Consideration of multivariate models was limited to testing the addition of baseline patient characteristics to the single marker model with the best (highest) AUC. All statistical analysis was performed in SAS\u00ae v 9.3.+ CD4+ cells in HNSCC Different hypotheses have been proposed to explain the inconsistent results obtained by different studies on the predictive role of tumor infiltrating FOXP3To evaluate whether tumor infiltrating Treg can serve as a prognostic marker in this defined patient cohort, paraffin embedded sections of the tumor were stained with hematoxylin and eosin  and serial sections labeled with antibodies specific for human CD4 and human FOXP3 and counterstained with DAPI.The anti-human FOXP3 antibody clone 236A/E7 was selected and used because it targets an epitope between aminoacid 105 and 235 of human FOXP3 +FOXP3+ cells were quantified using a computer-aided method for image analysis  that could limit the power of theanalysis, CD4analysis . Brieflyanalysis  and the analysis , Empiricanalysis .+FOXP3+ cells was evaluated with this method for each patient as well as thedifferences between patients with or without recurrence.Despite the use of a homogenous population, validated antibodies, and a computer aided method for tumor infiltrating Treg enumeration, no association was found between the percentage of CD4+FOXP3+ within the and tumor recurrence ,further suggesting that two well defined subsets of FOXP3+CD4+T cells infiltrate the tumor. Furthermore, while the percentage of cytoplasmicFOXP3+T cells was associated with a favorable prognosis  within the CD4 significantly correlated with tumor recurrence , we evaluated whether the subcellular localization of FOXP3 within the CD4p<0.001, .In silico models of the interaction between regulatory and effector T cells +CD4+T cells , there was a significant positive linear correlationwhen in the 19 cases of diseaserecurrence . This analysis indicated a 1.37-fold increase in Treg cells per unit increase in %cFOXP3+/CD4+ cells  between cases and controls (The relationship between Treg and effector FOXP3controls .2(nFOXP3) and log2(ratio nFOXP3/cFOXP3) ) or 50% decrease (\u2212log2(cFOXP3)) in the marker. Receiver operating curves (ROC) and corresponding area under the curve (AUC) are shown in + CD4+ cells gave the highest AUC, we estimated the effect of this marker after adjustment for each of the baseline characteristics previously considered. None of these covariates were significant although T-stage was marginally significant (p\u200a=\u200a0.097)  .+ cells has been evaluated as a prognostic marker for different human malignancies. Nevertheless, while some studies indicate its association with a negative prognosis, others correlate its expression with a positive prognosis or show no association. Here we show that intracellular localization is an important factor to considerwhen assessing the prognostic importance of FOXP3.In oral squamous cell carcinoma,an elevated number of tumor infiltrating CD4+T cellsexpressingFOXP3 in the cytoplasm are indicative of a favorable prognosis (no recurrence within three years) whereas an high concentration of CD4+ T cells expressing nuclear FOXP3 isstrongly associated with recurrence.Furthermore, the ratio between nuclear and cytoplasmicFOXP3+ CD4+ T cells was significantly better than either localized measure as an indicator of the risk of recurrence in patients with OSCC. Thus, we suggest the exploration of this parameter in other malignancies.Since its discovery, FOXP3 expression within the tumor infiltrating CD4"}
+{"text": "Intracerebral inoculation of transgenic mice failed to demonstrate prion disease transmission. Sc). PrPSc propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrPSc presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP C), which predominantly accumulates in the central nervous system  in 5 phenotypically distinct subtypes  accounts for \u224870% of all cases of sCJD and unquestionably is the most prevalent type of human prion disease , can be sporadic, inherited, or acquired by infection; the sporadic form alone accounts for the great majority of all the cases of CJD  affected by typical sCJDMM1 that was histologically and immunochemically confirmed  were provided by the National Prion Disease Pathology Surveillance Center. The patients were 54, 68, 60, and 69 years old with disease durations of \u22482, 2, 3, and 2 months, respectively  (4 L each time) at 4\u00b0C by using the Pierce Slide-A-Lyzer cassette  (10-kDa cutoff) with 2 additional changes over 2 days.Urine samples from sCJDMM1 patients 1\u20133 collected 2 weeks, 1 month, and 1 week before death, and from 3 healthy controls, were concentrated 100-fold by ultrafiltration with Millipore Centricon Plus 70  (10-kDa cutoff) by using a Beckman  centrifuge at 3,000 \u00d7 g for 5 min. Microsomal fractions (MF) were prepared from the frontal cortex of the sCJDMM1 patients according to Reichl et al. , was centrifuged as above. This supernatant, pooled with the previous one, was recentrifuged at 10,000 \u00d7 g for 7 min at 4\u00b0C. The pellet was discarded and the supernatant was centrifuged at 100,000 \u00d7 g for 1 h at 4\u00b0C. The final pellet, representing the MF, was resuspended in PBS at 10% (wt/vol). Transgenic mice expressed full-length human PrP-129M at wild type level in mouse PrP null background, Tg(HuPrP-129M)0/0Prnp (Tg40) (Brain homogenates (BH) from inoculated mice  were prepared at 4\u00b0C in PBS cleared by centrifugation at 1, 000 \u00d7 MF of sCJDMM1 patient 4 was spiked in 100\u00d7 concentrated and dialyzed normal urine. The sample was 10-fold serially diluted and intracerebrally inoculated into Tg40 mice.MF (100 \u00b5L) of sCJDMM1 patient 3 was spiked into normal urine (100 mL) either before or after 100\u00d7 concentration and dialysis. Subsequently, 30 \u00b5L of the differently processed urine samples, both containing an equivalent amount of 3 \u00b5L of MF, were intracerebrally injected into Tg40 mice. The infectivity titers of the 2 differently processed MF spiked urines were compared on the basis of their mean incubation times in Tg40 mice.Thirty microliters of MF from sCJDMM1 patients 1\u20134 suspended in PBS or in 100\u00d7 concentrated and dialyzed normal urine were intracerebrally injected into Tg40 mice , Table 4Infectivity in MF from sCJDMM1 patients 1 and 4, the latter spiked and diluted in 100\u00d7 concentrated and dialyzed normal urine, was estimated by endpoint dilution by using probit regression analysis . Incubation periods , x-axis 50]) by assuming a Poisson distribution in the response variable (The amount of prion infectivity that is below the threshold of detectability of our bioassay but that might be present in the urine of the sCJDMM1 patients was estimated in infectious unit (IU) per milliliter with 95% confidence interval (CI) . Titers from brain MF of sCJDMM1 patients 2\u20134 were estimated by interpolating the mean incubation times of the inoculated Tg40 mice to the dose-incubation period curve of 6 ID50 per gram of brain tissue equivalent . The histologic examination revealed no lesions (data not shown). These data show that 100\u00d7 concentrated and dialyzed urine is not toxic following intracerebral inoculation into Tg40 mice.The effect on prion infectivity of the concentrated and dialyzed urine when used as carrier was assessed by comparing infectivity titers of MF from cerebral cortex of sCJDMM1 patient 4 diluted either in PBS or in 100\u00d7 concentrated and dialyzed urine. The bioassay analyses showed that using concentrated and dialyzed urine as carrier the infectivity titer of MF was reduced \u224820-fold (1.3 log reduction) , indicat6 ID50/g and 6.9 \u00d7 106 ID50/g, respectively, were found in the 2 differently processed samples, showing that the concentration and dialysis procedure per se had no detectable effect on infectivity.The possible loss of prion infectivity during concentration and dialysis procedures was assessed by bioassay. We compared the infectivity titers of brain MF from sCJDMM1 patient 3 spiked in normal urine before the concentration and dialysis procedure with the infectivity of the same amount of MF spiked in normal urine already concentrated and dialyzed . SimilarSc was detected by Western blot, even after enrichment with NaPTA precipitation , no mice showed any evidence of clinical disease . No PK-rpitation , panel Apitation , panel BOn the basis of these negative results, we estimate that prion infectivity in urine of sCJDMM1 patients to be 0 IU/total volume of inoculated urine, which by Poisson distribution is less than 0.37 IU for patients 1 and 2 and 0\u20130.46 IU for patient 3 (upper limit of 95% CI). However, if we take into account the 100\u00d7 concentration of urine and the finding that 100\u00d7 concentrated and dialysized urine may reduce prion infectivity by \u224820-fold , the estSc was detected by Western blot in the brain even after PrPSc enrichment with NaPTA (data not shown). Thus, the estimated infectivity titer in raw urine of sCJDMM1 by Poisson distribution is 0\u20130.11 IU/mL (95% CI), not dissimilar to the value (0\u20130.24) obtained with the same urine after concentration and dialysis.To directly assess the prion infectivity in urine without concentration and dialysis, we inoculated 33 Tg40 mice with raw urine from sCJDMM1 patient 1. No inoculated mice showed clinical signs of prion disease over their normal lifespan (up to 857 dpi) . Similar5 or 104 dilutions of the brain tissue equivalent depending on whether the MF preparations were inoculated directly or after spiking into concentrated and dialyzed normal human urine.The present study demonstrates that the urine from patients affected by advanced sCJDMM1, the most common sCJD subtype that alone accounts for \u224860% of all human prion diseases, contains either no prion infectivity or an infectivity titer that is below the detection limit of our bioassays. The bioassays were done in Tg mice expressing human PrP-129M (Tg40) following inoculation with urine obtained from patients with sCJDMM1 and a variety of positive and negative controls. In limit dilution experiments, Tg40 mice inoculated with MF preparations obtained from the brains of 3 urine donors with sCJDMM1 had prion disease develop at up to 10,,Sc in urine from CJD patients is not known, this urine PrPSc species might show even higher loss of infectivity in the concentrated and dialyzed urine carrier than the brain PrPSc preparations used in the spiking experiments. To address this concern, we inoculated 33 Tg40 mice with raw urine from one of the 3 donors with sCJDMM1. No recipient mice showed evidence of prion disease suggesting an infectivity ranging from 0.0 and 0.11 IU/per mL (upper limit of 95% CI) as estimated by the Poisson distribution.To enhance the sensitivity of our system, urine samples were concentrated and dialyzed before inoculation. Similar procedures have been used in all the previous studies on prion infectivity of urine ,,, the infSc cannot be excluded, our inability to detect prion infectivity in human urine of patients with sCJDMM1 differs from several recent experimental studies on urine of prion-affected animals. Low prion infectivity has been demonstrated in urine from scrapie-infected hamsters  , only minute amounts of PrPSc have been detected in a few nonneural peripheral organs and tissues such as skeletal muscle and spleen (\u2013,Sc to peripheral organs is much wider and typically involves lymph nodes, tonsil, spleen, portions of the intestinal tract, and the skeletal muscle (,,,The most likely explanation for the discrepancy between our negative results on human urine and the positive findings by bioassay in urine from animals resides in the different locale and mode of formation of the prion agents. In all the published animal experiments, including bioassays and PMCA, the prion disease was induced by intracerebral or oral administration of exogenous prions, whereas we examined urine infectivity in a naturally occurring sporadic human prion disease. In exogenously acquired prion diseases, PrPSc and prion infectivity (,Recent data have proved that feces from hamsters infected with scrapie by the oral route and to a lesser extent through intracerebral and intraperitoneal inoculation, contain a discrete amount of PrPSc are present in urine from patients with sCJD and to assess the presence of infectious prion in urine from patients with every other form and subtype of human prion diseases, our study shows that urine from patients with sCJDMM1, the most common subtype of sCJD, does not contain prion infectivity detectable by our bioassay and suggests that no significant prionuria occurs in this common subtype of human prion disease.Although additional studies are still needed to determine whether minute amounts of prion infectivity or PrP"}
+{"text": "Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies , while NPC2-mutant cells had predominantly smaller lamellar bodies  than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities. Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder marked by excess intralysosomal cholesterol, progressive neurodegeneration and hepatosplenomegaly that has no effective approved treatments nih/nihNpc1 mice demonstrated vacuoles in type I and endothelial cells and foamy alveolar macrophages Npc1 mice has been documented but surfactant was not analyzed The most common mutations associated with NPC disease (95% of cases) are in the NPC1 gene with the affected patients demonstrating a broad clinical spectrum Pulmonary surfactant, produced by type II alveolar cells, performs the critical role of reducing surface tension in the alveoli. Cholesterol is the most abundant neutral lipid in surfactant, contributing 5\u201310% weight by volume, with the rest made up of phospholipids and protein Reports on the morphology of type II cells in lungs of patients or animal models of NPC deficiency describe the pneumocytes to be \u201cunremarkable\u201d The mice and felines were housed under the National Institutes of Health and USDA guidelines for the care and use of animals in research. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all experimental protocols.Mice.nih/nihNpc1 and Npc2 hypomorph mice on a BALB/c background were obtained as a gift from Dr. Peter Lobel. Both types of mice are established models of NPC disease. The Npc1 mouse defect arose due to a spontaneous mutation in mouse chromosome 18 Npc2 mutant mice were produced by Sleat, et al. and contain an aberrant recombination event such that the targeted allele has an additional repeat of intron 1 and replacement of amino acid cysteine 42 with a stop codon Npc1 and Npc2 mice were determined by PCR of genomic DNA from tail tips. The primer pairs have been previously described The felines were housed in the School of Veterinary Medicine of the University of Pennsylvania. NPC1 mutant diseased felines and their normal counterparts were produced from the same line. The NPC1 felines have a spontaneous missense mutation (C955S) in the NPC1 protein which results in lysosomal accumulation of unesterified cholesterol The isolation of type II cells from mice lungs was performed as described previously Lamellar bodies were isolated as described previously Lung tissue from 9\u201314 week old mice was collected, homogenized, and separated using denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (8). Primary antibody against rabbit anti-NPC1 , NPC2 , secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated , and ECL Plus Western Blotting Detection Reagent  were used.Lungs from wild type, NPC1 and NPC2 mutant mice were excised from 3 animals for each experimental group. From each mouse, the left lobe, upper right lobe, and lower right lobe were fixed overnight in a mixture of formaldehyde and glutaraldehyde, and embedded in paraffin. Tissue was cut into 5 \u00b5m sections and stained with hematoxylin and eosin. Random fields of distal airway were collected at 60\u00d7 magnification. Alveolar septal thickness was determined using an overlaid test grid. The alveolar septal thickness was measured as the length of a grid line that extended from one alveolus to an adjacent alveolus. Regions containing large airways or capillaries were not included in this measurement. >200 probe \u201chits\u201d of the alveolar septal thickness were gathered from more than 5 sections per mouse and 15 fields for each experimental condition.. Lungs were cut into 1 mm3 blocks, treated for 2 hours with 2% osmium, dehydrated, embedded in EPON, and polymerized for 48 hours at 60\u00b0C. Blocks were sliced into ultrathin (80 nm) sections in a Leica Ultracut UCT ultramicrotome. Samples were stained with 2% uranyl acetate and 0.5% lead citrate. All samples were imaged on a TEM .Lungs from mice were perfused through the pulmonary artery with 0.1 M sodium cacodylate buffer to remove blood cells. Lungs were excised and fixed by perfusion with 5% glutaraldehyde through the pulmonary artery and trachea. The trachea was removed, and whole lungs were fixed for at least 4 hours. A portion of the lung from a cat were perfused with cacodylate buffer through the bronchi and fixed by immersion in 5% glutaraldehyde4), heated for 20 minutes, and then cooled. Distilled water and ammonium molybdate were added to the sample and vortexed before the addition of reagent A . Samples were boiled for 10 minutes, cooled and colorimetric reading was done at 830 nm.To measure cholesterol, samples were prepared following instructions in the Amplex Red Cholesterol Assay Kit . Protein for each corresponding sample was determined using the Lowry assay and data are presented as \u00b5g lipid/\u00b5g protein 3H] dipalmitoyl phosphatidylcholine ([3H]-DPPC) by the isolated, perfused lung was performed as described previously 3H]-DPPC were prepared by freezing and thawing followed by extrusion under pressure through a 100-\u00b5m pore-size filter. The liposomes  were instilled into the lungs of anesthetized mice; the lungs were removed and placed in a perfusion apparatus under ventilation. After 5 min  or 2 hours, the lungs were lavaged and the lung tissue homogenized. The homogenate was extracted Uptake and degradation of liposomes labeled with [choline-methyl-Type II cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were washed, fixative was quenched in 0.3% glycine, and cells were permeabilized in 0.2% triton for 30 minutes at room temperature. Cells were blocked in 10% BSA with 2% normal goat serum for 1 hour. Primary antibodies were incubated on cells overnight in dilute blocking buffer. The next day, cells were washed and incubated in secondary antibody for 1 hour. Cells were then washed and imaged.t-test or ANOVA. Results were considered statistically significance at P\u22640.05.All data are expressed as mean \u00b1 SEM. Statistical comparisons were performed using SigmaStat . Data were analyzed using a Student\u2019s NPC1 and NPC2 mutant mice have average life spans of 72 days Histological evaluation of the lungs of NPC1 and NPC2 mutant mice using hematoxylin/eosin stained sections was performed to determine pulmonary effects due to loss of cholesterol transport. The micrographs showed a thickening of the intra-alveolar septae with a slight enlargement of the airways in the NPC1 mice . QuantitUpon closer inspection of the alveolar architecture at the electron microscopic level, more pronounced pulmonary abnormalities were apparent. In Within mouse lung capillaries were enlarged polymorphonuclear leukocytes or circulating macrophages filled with vacuolar inclusions were found in both mutant mice, apparent in NPC1 mice 2 to 0.7 \u00b5m2. While some LBs in NPC1 mutant mice showed a distribution similar to wild type, a significant proportion of NPC1 type II cell LBs were enlarged  were similar in size as those of the smaller sized wild type LBs (<0.2 \u00b5m2) and were fairly uniform with none larger than 0.7 \u00b5m2. The mean average size of the LBs of the mice reflected these differences with NPC1 (0.81\u00b10.02 \u00b5m2)>WT (0.43\u00b10.02 \u00b5m2)>NPC2 (0.22\u00b10.01 \u00b5m2), all statistically significantly different  from each other. Interestingly, compared to the number of LBs per cell in wild type alveolar type II cells , NPC2 alveolar type II cells exhibited 65% more LBs  while the LB numbers/cell between WT and NPC1 were approximately the same .It appeared that the lamellar bodies of type II cells from the affected mice differed in size. Thus, a morphometric analysis of lamellar body size of type II cells was performed for the NPC1 and NPC2 mutant lungs and their matching litter mates using electron microscopic images of the type II cells and ImageJ analysis. The size of the LBs of wild type mice were primarily distributed in the range from 0.2 \u00b5menlarged . 42% of An enrichment in the cholesterol content of NPC1 mouse lungs has been documented previously As would be predicted from the lipidosis in the alveolar space observed in the electron microscopic data, there was a marked increase in surfactant phospholipid in both mutant mice. The phospholipid levels of the bronchoalveolar lavage (BAL) were elevated over control mice in the NPC1 mutant (4-fold) and in NPC2 mutant mice (3-fold) , mirroriAnalysis of the bronchoalveolar lavage showed that the NPC1 mutant mouse lung had a mild protein accumulation which was not the case for the NPC2 mutant mice . We furtLipid analysis was performed on lamellar bodies isolated from pooled samples of lung homogenates, either from NPC1 mutants, NPC2 mutants or the wild type counterparts. The data revealed an increase in phospholipid  and cholesterol  content compared to wild type animals , with a 3H-choline-labelled DPPC liposomes were instilled into the lungs of wild type or NPC1 mutant mice. After a two hour perfusion time, there was no significant difference in the endocytosis of the DPPC by the lungs . However, total degradation of 3H-DPPC was reduced by 46% in the NPC1 mice lungs as compared to wild type due to significant decreases in all lipid and aqueous fractions  versus 0.40\u00b10.02 micron2  for wild type feline (P<0.05). Measurement of the lung lipid content  lamellar bodies and lipid-enriched lamellar bodies, surfactant and lung tissue. Thus, both mice and feline animal models are suitable for studies of disease progression and pulmonary complications related to loss of a functioning NPC pathway.The feline NPC1 disorder, which progresses more slowly, is felt to be an excellent model for the human pathogenesis We describe previously uncharacterized dysfunctions in type II cells of the NPC1 and NPC2 mutant mice that may help to clarify their contribution to the elevated surfactant cholesterol phenotype associated with Niemann-Pick type C. First, the surfactant storage organelles, the lamellar bodies, have significantly elevated phospholipid and cholesterol content, which correlate to elevated phospholipid and cholesterol in the surfactant. In support of this data, immunocytochemistry directly demonstrated excess cholesterol accumulated within lamellar bodies of type II cells isolated from the NPC mutant mice. Second, there were anomalies in the lamellar body size in the type II cells from both mutant mice: enlarged lamellar bodies in NPC1 mutant mice, mirrored in the NPC1 mutant felines; and small lamellar bodies in NPC2 mutant mice. The relationship between lamellar body size an pulmonary abnormalities is not yet clear although giant lamellar body formation was associated with impaired surfactant secretion in a mouse model of Hermansky-Pudlak syndrome (HPS) Lamellar body size is an important indication of type II cell health since lamellar body size is altered in a number of genetic diseases such as ABCA3 deficiency and HPS The absence of NPC1 protein in mice negatively affected pulmonary function, where inspiratory capacity, elastance and hysterisivity were increased in the diseased lungs In summary, we have identified defects in surfactant homeostasis in both NPC1 and NPC2-mutant mice including elevated surfactant phospholipid and cholesterol, elevated lamellar body phospholipid and cholesterol, and abnormal lamellar body sizes. Many of these pathologic changes were noted in the NPC1 mutant feline pulmonary system as well. The work underscores the importance of understanding the regulation of cholesterol trafficking to maintain proper pulmonary function. These studies elucidate novel characteristics of lung disease progression in Niemann-Pick C1 and C2, and demonstrate that epithelial cell dysfunctions can be a contributing source for lung disease."}
+{"text": "In vitro, hCG stimulated the expression of Plk2 in granulosa cells, but not Plk3. This induction of Plk2 expression was mimicked by both forskolin and phorbol 12 myristate 13-acetate (PMA). Moreover, Plk2 expression was reduced by inhibitors of prostaglandin synthesis or the EGF pathway, but not by progesterone receptor antagonist (RU486) treatment. At the promoter level, mutation of the Sp1 binding sequence abolished the transcriptional activity of the Plk2 gene. ChIP assays also revealed the interaction of endogenous Sp1 protein in the Plk2 promoter region. Functionally, the over-expression of Plk2 and Plk3 arrested granulosa cells at the G0/G1 phase of the cell cycle. In contrast, the knockdown of Plk2 expression in granulosa cells decreased the number of cells in the G0/G1 stage of the cell cycle, but increased granulosa cell viability. In summary, hCG induced Plk2 and Plk3 expression in the rat ovary. Prostaglandins and the EGF signaling pathway are involved in regulating Plk2 expression. The transcription factor Sp1 is important for Plk2 transcriptional up-regulation. Our findings suggest that the increase in Plk2 and Plk3 expression contributes to the cell cycle arrest of granulosa cells which is important for the luteinization of granulosa cells during the periovulatory period.The highly conserved polo-like kinases (Plks) are potent regulators of multiple functions in the cell cycle before and during mitotic cell division. We investigated the expression pattern of Plk genes and their potential role(s) in the rat ovary during the periovulatory period. Plk2 and Plk3 were highly induced both in intact ovaries and granulosa cells  Drosophila melanogaster polo mutation In females, an acute rise of luteinizing hormone (LH) released from the pituitary  triggers ovulation and induces terminal differentiation of preovulatory granulosa cells to become luteal cells. The LH surge terminates granulosa cell proliferation and initiates a program of luteinization in which the cells stop their division and differentiate into luteal cells Among the five members in the mammalian Plks family, Plk1 has been most thoroughly studied. The major function of Plk1 is promoting mitotic entry by phosphorylation of Cdc25C in vivo expression of Plk2 and Plk3 was dramatically increased after treatment with hCG which was used to mimic the preovulatory LH surge. Moreover, hCG stimulated the expression of Plk2, but not Plk3, in granulosa cell cultures. Given the similar and dramatic induction of Plk2 by hCG both in vivo and in vitro, we further explored the regulatory mechanism of Plk2 expression and the potential role of Plk2 in periovulatory granulosa cells using an in vitro model.Considering the roles of Plk1-4 in regulating cytokinesis, we hypothesized that the LH surge induces these Plks and that their induction is involved in the transition of granulosa cells to luteal cells. In the present study, we investigated the periovulatory expression patterns of the Plk family members. We found that the in vivo and the action of LH in preovulatory granulosa cell cultures.The gonadotropin-primed immature female rat is a well accepted experimental model to examine ovulation as well as ovarian granulosa cell function in vivo. Both Plk2 and Plk3 protein levels increased at 4 h after hCG in the intact ovary  and protein kinase C (PKC) signaling pathways in preovulatory granulosa cells hCG also sets in motion a number of steps which are crucial for follicular rupture and oocyte release including activation of epidermal growth factor (EGF) signaling and induction of progesterone receptors (PGR) and prostaglandin-endoperoxide synthase 2 (PTGS2) To investigate which transcription factors are important for Plk2 transcription, three Plk2 promoter reporter constructs  were transfected into granulosa cells isolated from PMSG-primed immature rats. The transfected granulosa cells were treated with FSK+PMA which mimics the action of an ovulatory dose of LH/hCG in vivo, ChIP assays were performed on chromatin samples extracted from periovulatory granulosa cells. PCR analysis revealed that immunoprecipitation of endogenous Sp1 enriched chromatin fragments containing the Sp1 binding sequence in the promoter region compared to that of normal rabbit IgG at 8 h post-hCG  In response to the LH surge granulosa cells of preovulatory follicles stop proliferation and begin to differentiate into luteal cells. Many cell cycle regulators have been reported to play roles in the process of luteinization, such as p27 in vivo after hCG administration, but only Plk2 is induced by hCG in vitro. The induction of Plk2 is mediated by the PKA and PKC pathways, as well as the hCG-induced activation of EGF signaling pathway and prostaglandins. The transcription factor Sp1 is important for Plk2 transcription. Our novel findings suggest Plk2 and Plk3 may regulate the cell cycle arrest of granulosa cells after the LH surge which is critical for granulosa cell luteinization.In summary, the present study demonstrates that both Plk2 and Plk3 are highly expressed in granulosa cells The experimental protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee and Institutional Animal Care and Use Committee at Zhejiang University and complied with the principles of laboratory animal care.Molecular biological enzymes and molecular size markers were purchased from Toyobo . Trizol\u2122 and pCRII-TOPO Vector were purchased from Invitrogen\u2122 Life Technologies, Inc. . Chemicals and reagents  were all purchased from Sigma Chemical Co.  unless mentioned.Immature female Sprague-Dawley rats were provided by Harlan, Inc. and Zhejiang University Laboratory Animal Center and maintained on a 12L:12D cycle as described previously 6/well and cultured at 37\u00b0C in a humidified atmosphere of 5% CO2. The cells were treated with various reagents  for time points outlined below for each experiment. Each experiment was performed at least three times.Ovaries were collected 48 h after PMSG administration and processed as described previously in vitro and in vivo. Oligonucleotide primers for rat L32 , rat Plk1 , rat Plk2 , rat Plk3  and rat Plk4  were designed using OMIGA 2.0 software  and synthesized by Shanghai Sangon Biological Engineering Technology & Services . The specificity for each primer set was confirmed by electrophoresis of the PCR products on a 2.0% agarose gel. The PCR products were sequenced before using. The melting (dissociation) curve was analyzed using a 7300 Real-Time PCR System  after each real-time PCR. The real-time PCRs were carried out as previously described \u2212\u0394\u0394CT method and normalized to the endogenous reference gene L32.Real-time PCR was used to measure expression of Plk1, Plk2, Plk3 and Plk4 mRNA Western blotting was performed as described previously CAC CCA GCA GGC GCG -3\u2032).Genomic DNA was isolated from rat tail samples using an easy-DNA kit (Invitrogen). 921-bp (\u2212884/+37), 163-bp (\u2212126/+37) and 85-bp (\u221248/+37) fragment of the Plk2 gene were amplified using the primers attached with restriction enzyme sites (KpnI and NheI). Fragments of Plk2 promoter were cloned into the pGL3 basic vector . Site-directed point mutations of the Plk2 promoter were generated using a QuickChange II site-directed mutagenesis kit according to the manufacturer\u2019s protocol (Stratagene). The sequences of the oligonucleotide primers used to generate Plk2 promoter containing mutation (shown in lowercase) are following: mutant  and Renilla luciferase vector (pRL-TK vector) using a Lipofectamine 2000 reagent (Invitrogen). The next day, cells were treated with forskolin  and phorbol 12-myristate 13-acetate  for 4 h. The cells were then harvested to measure firefly and Renilla luciferase activities using a dual-luciferase reporter assay system (Promega). Firefly luciferase activities were normalized by Renilla luciferase activities and each experiment was performed in triplicate at least three times.Granulosa cells isolated from immature rats (48 h after PMSG) were seeded into 96 well plates at 1\u00d710ChIP assay was performed on Sp1 in the Plk2 promoter region using a ChIP kit  as described previously The AdEasy XL adenoviral vector system kit (Stratagene) was used to construct Plk2 and Plk3 recombinant adenovirus. The processes for generating and propagating recombinant adenoviruses were described previously Rat granulosa cells collected at 48 h after PMSG priming were cultured in HyQ MEM-RS medium for 4 h before addition of the adenoviruses. The granulosa cells were exposed to Ad-Plk2, Ad-Plk3 or Ad-GFP at a multiplicity of infection (MOI) of 50 pfu/cell for 2 h. Then the medium was replaced with fresh HyQ MEM-RS medium. Approximately a 70% infection efficiency of GFP-adenovirus in granulose cells was routinely observed. Granulosa cells were collected for total RNA isolation, protein extraction, or flow cytometric analysis after adenovirus exposure for 48 h.Granulosa cells were collected from immature rats 48 h after PMSG administration. Cells were transfected with the specific siRNA against Plk2  or scrambled siRNA (Invitrogen) using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer\u2019s instructions. Four hours later, transfection media were replaced with fresh culture media and the cells were treated with hCG (1 IU/ml) for further 48 h. The cells were collected for flow cytometric analysis or processed to prepare cell lysates for Western blot analyses.6) and incubated at 37\u00b0C for 30 min. Then, granulosa cells were resuspended in propidium iodide (50 \u00b5g/mL) and incubated for 15 min in the dark at 4\u00b0C. A FACS Calibur flow cytometer  at Zhejiang University was used to determine the cell cycle distribution at an excitation wavelength of 488 nm. Histograms of cell cycle were obtained from 3 determinations .To determine the impact of Plk2 and Plk3 on cell cycle kinetics, granulosa cells treated with Ad-Plk2 and Ad-Plk3 adenovirus or Plk2 siRNA as described above. Granulosa cells were suspended using 3% trypsin after culture and then stained for DNA content. Briefly, ribonuclease A was added to cells  for further 24 h. Cell viability was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer\u2019s protocol (Promega) as described previously \u03c1 <0.05 considered significant.All the results are presented as means \u00b1 SEM. Two-way ANOVA was used to test differences in Plk2 and Plk3 expression across time of culture and treatment. One-way and t-test analysis of variance (ANOVA) was used to test differences in Plks mRNA levels among treatments. If the effects of time of culture or treatment were revealed significant, the means were compared by Duncan\u2019s test, with"}
+{"text": "Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3\u2032half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35\u00b0C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression.We have cloned the  TPS1 gene TPS2Saccharomyces cerevisiae or Kluyveromyces lactis mutations in the gene TPS1 cause inability to grow in glucose S. cerevisiaeYarrowia lipolytica with a Ki of 3.5 \u00b5M Y. lipolytica is a dimorphic yeast that separated early from the yeast evolutionary trunk Y. lipolytica is also being used as model to study physiological processes like lipid accumulation Y. lipolytica enzymes S. cerevisiaeY. lipolytica hexokinase to T6P it appeared worthwhile to isolate the TPS1 gene of this yeast and to analyze the effects of its disruption. The isolation of this gene presents also a potential technological interest as in Aspergillus niger the degree of expression of the tpsA gene that encodes T6P synthase, influences the rate of citric acid production Y. lipolytica excretes this acid in some conditions Y. lipolytica has a single gene encoding T6P synthase, that its disruption does not preclude growth in glucose but decreases sporulation efficiency and slows down growth at 35\u00b0C. In addition we report that disruption of YlTPS3 abolishes the increase of trehalose observed during heat shock.Trehalose, a non-reducing disaccharide formed by two glucose units, has important and varied functions in different organisms Y. lipolytica was cultured in a synthetic medium with 0.17% yeast nitrogen base without amino acids and ammonium sulfate  and 0.1% glutamate pH 6. S. cerevisiae was cultured similarly but using ammonium sulfate as nitrogen source. Auxotrophic requirements were added at a final concentration of 20 \u00b5g/ml and 2% glucose was generally used as carbon source. Liquid cultures were shaken at 30\u00b0C. Sporulation medium was based in commercial V8 drink essentially as The yeasts strains used are shown in Y. lipolytica cDNA library under the control of the S. cerevisiae PGK1 promoter in plasmid pFL61 Y. lipolyticaY. lipolytica and in S. cerevisiae. Primers used in PCR reactions are shown in A S. cerevisiae were constructed:The following plasmids for YlTPS1 gene was isolated from a cDNA library S. cerevisiae tps1 strain.pCLF1 carrying the YlTPS1, was constructed as follows. The BamHI fragment from plasmid pAN10 S.cerevisiae ADH1 was inserted into pRS316 BamHI. A 1.5 kb blunt-ended NotI fragment with YlTPS1 from pCLF1 was inserted in the blunt-ended HindIII site of this plasmid.pCLF2, a centromeric plasmid that carries YlTPS3 under the control of the ScADH1 promoter. YlTPS3 (YALI0E31086) is annotated as an intron containing gene. Using the FirstChoice RLM-RACE Kit (Ambion) we checked the correctness of the ATG and the cDNA predicted sequence. Primer design to amplify the cDNA from genomic DNA was based on the fact that the first exon is only 23 bp long. Primer 1006 covers the first exon and the first 19 bp of the second exon; together with primer 1007 produced a PCR product of 3168 bp containing the cDNA of YlTPS3. This product was cloned in the pCR-Blunt vector (Invitrogen) and the resulting plasmid was digested with NotI and SpeI blunt-ended and cloned in pDB20 URA3 marker had been substituted by LEU2. The cDNA of YlTPS3 was sequenced again when introduced in this plasmid.pCLF7 expresses Y. lipolytica were constructedThe following plasmids for Y. lipolytica TPS1 gene and was isolated by screening a Y. lipolytica genomic library YlTPS1 probe.pCLF3 carries a fragment of 5.4 kb that contains the YlTPS1 under the control of its own promoter. It was constructed as follows: the YlURA3 marker in plasmid pCL49 YlLEU2 to give pCL49L. The SphI-BamHI fragment of pCL49L was replaced by a 2.8 kb HpaI-BamHI fragment from pCLF3 bearing the YlTPS1 ORF and 1 Kb of upstream sequence.pCLF4 expresses YlTPS1 (a 1.5 Kb NotI fragment) from plasmid pCLF1 under the control of the YlTEF1 promoter in plasmid pCL49L.pCLF5 carries the coding region of YlTPS1 promoter to lacZ. A 1214 bp DNA fragment that includes the 14 initial amino acids of YlTPS1 was obtained by PCR, using oligonucleotides 1010 and 1011 cloned into pGEM-T easy, digested with NotI and BamHI and inserted into plasmid pINA354B ApaI to direct integration into the YlLEU2 locus. Correct integration was checked by PCR and Southern analysis.pCLF8 carries a fusion of the ScTPS1 obtained by PCR using primers 1012 and 1013 in plasmid pCL49L.pCLF9 carries the coding region of SceI restriction site and a deletion of 658 bp were created. Primers 1001 and 1002 with complementary ends including the recognition site of meganuclease I-SceI, 1000 and 1003 were used. With pCLF3 as template and the mentioned primers two fragments corresponding to the 5\u2032 and 3\u2032regions of the disruption cassette were obtained. These products were used as template with primers 1000 and 1003 in a PCR reaction to obtain a disrupted YlTPS1 copy. The product was cloned into pGEM-T easy, digested with I-SceI and ligated to a 1.2 kb YlURA3 fragment flanked by I-SceI sequences from plasmid pINA-URA3-I-SceI NotI was used to disrupt the chromosomal copy of YlTPS1 gene.In two consecutive PCR reactions a I-YlTPS3 gene was obtained by PCR from genomic DNA and primers 1004 and 1005 and cloned in pGEM-T easy (Promega). The resulting plasmid was digested with XhoI, to remove a fragment of 758 bp, and ligated to a 2.1 kb band containing the YlLEU2 gene from a NcoI digestion of the plasmid pINA62 YlTPS3 was disrupted using a 4996 bp NotI fragment from the previous plasmid. Correct disruption was checked by PCR.A piece of 3611 bp containing the YlNTH1) was obtained by PCR using primers 1008 and 1009 and cloned in pGEM-T easy. The resulting plasmid was digested with NheI and XhoI to substitute an internal 816 bp fragment of the NTH1 gene with a 2.1 kb fragment containing the YlLEU2 gene from plasmid pINA62 NcoI. A 3342 bp NotI fragment from this construction was used to disrupt the chromosomal copy. Correct disruption was checked by PCR and Southern analysis.A fragment of 2049 bp containing YALI0D15598 Y. lipolytica was extracted from flash-frozen cells Y. lipolytica CLIB122 genomic sequence. Total RNA was reverse-transcribed into cDNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). The cDNA levels were then analyzed using a LightCycler 480 from ROCHE and the LightCycler 480 SYBR Green I Master mix (Roche) with each primer at 250 nM. Each sample was tested in triplicate. After completion of the RT-qPCR melting-curve data were collected to verify PCR specificity, the absence of contamination and primer dimers. The gene YALI0F27533 (ARP4) was used to normalize the data.Total RNA from 2, 1 mM EDTA, 10 mM glucose 6-P and 5 mM UDP-glucose. Samples were taken at different time intervals and boiled for 3 minutes to stop the reaction. The UDP formed was measured spectrophotometrically in a coupled assay with NADH, pyruvate kinase and lactate dehydrogenase. A blank without added glucose-6P was run in parallel for each sample. Hexokinase and glucokinase were assayed with glucose and fructose as in 4, 40 mM \u03b2 mercaptoethanol, and 2.6 mM 2-nitrophenyl \u03b2-D- galactopyranoside as described by Wallenfels Cell free extracts were prepared by breaking the yeasts in buffer with glass beads in six cycles of 1 min of vortexing and 1 min on ice. The buffer was 20 mM imidazole pH 7, with the addition of 1 mM DTT and 1 mM EDTA when T6P synthase was assayed. The extract was centrifuged in the cold for 15 min at 13000 rpm in an Eppendorf table top centrifuge and the supernatant used for determination of enzyme activities. T6P synthase activity was determined by a two step method The cultures were harvested by centrifugation, washed with water and frozen until use. Trehalose was extracted with boiling water as described in Yeast cells were quickly filtered through a Millipore AAWPO4700 and snap frozen in liquid nitrogen. The frozen pellets were dropped in boiling ethanol and treated as in Nucleotide accession number.- The sequence of the YlTPS1 cDNA was deposited at the GenBank database with the accession number AJ011032.S. cerevisiae tps1 mutant TPS1 gene from Y. lipolytica. We transformed a S. cerevisiae tps1 mutant strain with a cDNA library from Y. lipolytica under the control of the S. cerevisiae PGK1 promoter  TPS1 in this yeast. Attempts to disrupt YlTPS1 placing the disruption cassette after nucleotides 188 or 406 after the ATG failed, only when it was displaced to nucleotide 710 a correct disruption was obtained  exceeded that of the gene encoding hexokinase  (Lack of growth in glucose of obtained . We attrFC1 gene . Lack ofxokinase . Also enpolytica . Since gactivity . Concentactivity  in contrriments) . No immeY. lipolytica grown in glucose up to stationary phase or in glycerol were below 1 nmol/mg dry weight. A possible explanation for this result could be that the genes encoding the trehalose biosynthetic pathway enzymes were not expressed during growth in glucose, therefore we measured the expression of those genes in Y.lipolytica. In the G\u00e9nolevuress database S. cerevisiae TPS2 and YALI0E31086 shows the highest homology with S. cerevisiae TPS3/TSL1. All these genes were expressed during growth in glucose although the levels of YlTPS2 and YlTPS3 were low when compared to that of YlTPS1 . This treatment was considered a strong one as this yeast does not grow at temperatures over 35\u00b0C. We measured the levels of mRNA corresponding to the genes related with trehalose metabolism after heat treatment . Relative abundance of mRNAs corresponding to YlTPS2 and to YlTPS3 increased 4 and 6 times respectively with the heat treatment. The higher increase of YlTPS3 suggested an important role for this gene in the heat response. A disruption of YlTPS3 abolished trehalose accumulation upon heat treatment raising the question that Tps3 could be another T6P synthase an hypothesis that cannot be ruled out due to the similarity between the YlTPS1 and YlTPS3 sequences. This possibility was made unlikely by the absence of trehalose in a Yltps1 mutant after heat shock and by the lack of complementation of the glucose negative phenotype of a S. cerevisiae tps1 mutant by the YlTPS3 cDNA  shows high similarity in its zinc finger domain to that of Msn2/4 and also binds STRE sequences Y. lipolytica database for genes encoding homologues of ScHSF1 yielded gene YALI0E13948. Levels of mRNA corresponding to those genes increased about 3 times after heat shock (In at shock  consisteY. lipolytica diploids homozygous for the tps1 mutation (CJM 724) were placed in sporulation conditions the sporulation frequency was reduced with respect to that of wild type (CJM 722) or heterozygous TPS1/tps1 (CJM 723) diploids. A similar behaviour in tps1/tps1 diploids in S. cerevisiaeMCK1, a gene that stimulates expression of IME1 which encodes a transcriptional activator of sporulation S. cerevisiae MCK1. We have measured the levels of mRNA corresponding to YALI0D20966 and to the genes implicated in trehalose metabolism both in a wild type diploid and in one homozygous for the tps1 mutation in sporulation conditions. After 8 days on sporulation medium the level of YALI0D20966 mRNA in a wild type was increased about 15 times while in a homozygous tps1/tps1 strain it reached a maximum of 3 fold  in their promoters S. cerevisiae requires the Msn2/Msn4 proteins Y. lipolytica. Hurtado and Rachubinsky in vitro. These authors reported that the levels of MHY1 mRNA were not increased after a heat shock at 35\u00b0C. Our results show that upon a heat shock at 40\u00b0C the levels of MHY1 mRNA increase suggesting that MHY1 may play a role in the regulatory response to this stress. It should be noticed that the high GC content in the Y. lipolytica DNA Heat shock increased the levels of trehalose and changed the levels of mRNA corresponding to Y. lipolytica does not grow at temperatures over 35\u00b0C. The finding that disruption of YlTPS1 impairs growth at this limit temperature suggests that trehalose plays a protective role against the changes produced under this condition C. neoformans and C. gattii show important variations in the effects caused by perturbations of that pathway Y. lipolytica that separated early in evolution from other yeasts Many evidences show that in different organisms the trehalose biosynthetic pathway, in addition to its primary role, has an influence in a variety of processes that range from growth on certain substrates or temperatures, to differences in virulence in pathogens. The targets of the pathway are different depending on the organism and even closely related yeast species like Figure S1Growth of wild type and Yltps1 strains. The strains were grown as described in Yltps1) and CLF279 (Yltps1/pCLF4). A representative curve is shown for each strain.(TIF)Click here for additional data file.Table S1Primers used for DNA cloning.(DOC)Click here for additional data file.Table S2Primers used in RT-qPCR.(DOC)Click here for additional data file."}
+{"text": "EYA1, SIX1 and SIX5 genes have been associated with BOR syndrome. In this study, clinical and genetic analyses were performed in patients with BOR/BO syndrome focusing on auditory manifestations and rehabilitation.Branchio-oto-renal (BOR) or branchio-otic (BO) syndrome is one of the most common forms of autosomal dominant syndromic hearing loss. Mutations in EYA1, SIX1, and SIX5 genes.The audiologic manifestations were reviewed in 10 patients with BOR/BO syndrome. The operative findings and hearing outcome were analyzed in patients who underwent middle ear surgeries. The modality and outcome of auditory rehabilitation were evaluated. Genetic analysis was performed for EYA1 mutations and a large deletion encompassing the EYA1 gene.All patients presented with mixed hearing loss. Five patients underwent middle ear surgeries without successful hearing gain. Cochlear implantation performed in two patients resulted in significant hearing improvement. Genetic analysis revealed four novel EYA1 mutations may add to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population.Auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate after middle ear surgeries. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel  Mutations of the EYA1 gene are found in approximately 40% of patients with BOR syndrome EYA1 gene, mutations in the SIX1 and SIX5 genes have been reported to cause BOR phenotypes, although the pathogenic role of the SIX5 gene has been questioned recently SIX1 mutations have been shown to disrupt the EYA1-SIX1-DNA complexes Branchio-oto-renal (BOR) syndrome (OMIM 113650) or branchio-otic (BO) syndrome (OMIM 602588) is one of the most common forms of autosomal dominant syndromic hearing loss with an incidence of 1\u223640,000 and is responsible for causing 2% of profoundly deaf children EYA1 gene located on chromosome 8q13.3 encodes a transcriptional co-activator required for eye morphogenesis which consists of three isoforms  and four transcript variants (EYA1A\u20131D) as a result of alternative splicing The EYA1 gene have been associated with BOR/BO syndrome, and frameshift or nonsense mutations are the most commonly detected mutations, followed by splice-site and missense mutations EYA1 gene that are not detected by direct sequencing of the coding region EYA1 mutations causing BOR syndrome and most of the mutations are unique to individual families To date, approximately 160 mutations of the et al.et al.Hearing impairment, the most common phenotypic feature of BOR syndrome, is found in various forms among which mixed type of hearing loss is most frequently reported (50%), followed by conductive (30%) and sensorineural type (20%) EYA1, SIX1, and SIX5 genes are reported.Because of the conductive component of hearing loss identified in most cases of patients with BOR syndrome, attempts have been made to improve hearing through middle ear exploratory tympanotomy and ossicular reconstruction. However, there are only several studies dealing with hearing outcome of exploratory tympanotomy in patients with BOR syndrome mostly reported before the identification of genetic causes of BOR syndrome, which demonstrated unsatisfactory results et al.Seven families (10 patients) including one multiplex family showing hearing loss and one or more of the typical features of BOR syndrome were included in this study for clinical and genetic analyses. The clinical diagnosis as typical BOR syndrome was made when the clinical criteria proposed by Chang Serial pure tone audiometries were performed in all patients and the various clinical manifestations regarding hearing loss were carefully reviewed. The type, degree, onset, progressiveness and/or fluctuation of hearing loss were evaluated in each patient. The threshold of pure tone audiometry was defined as the average of thresholds at 500, 1000, 2000, and 3000 Hz. The follow-up period of audiologic evaluations ranged from 3 months to 7.5 years. Speech audiometry and language evaluations were carried out in some of the patients whenever possible.The temporal bone CT scan was performed in all of 10 patients and temporal magnetic resonance imaging (MRI) was available for analysis in 4 patients. The temporal bone CT scan was performed with a 16 multidetector row CT scanner  using a standard temporal bone protocol. Contiguous 0.7-mm scans of the temporal bone were acquired in the axial plane and reformatted coronally with 1.0-mm increments. CT images were performed, digitally stored, and displayed by using the Picture Archiving Communication System (PACS) .MRI was acquired by using a 3.0-T  or 1.5-T system  with a six-channel sensitivity encoding (SENSE) head coil. The targeted parasagittal scan perpendicular to the long axis of the internal auditory canal was obtained with T2-weighted three-dimensional (3-D) turbo spin-echo (TSE) sequence with driven equilibrium RF reset pulse (DRIVE), following routine MR sequences with spin-echo T1- and T2-weighted images. The sequence parameters for the T2-weighted 3-D FSE sequence with DRIVE were as follows: repetition time (TR)/echo time (TE)\u200a=\u200a1500/200 ms, 256 acquisition/256 reconstruction, 15-cm field of view, 1.5-mm section thickness with a 0.75-mm overlap, number of acquisitions\u200a=\u200a2, and the scan time was less than 5 minutes.The morphologies of the cochlea, vestibule, semicircular canals, internal auditory canals, vestibular aqueduct, and middle ear structures were analyzed on temporal bone CT scans. On temporal MRI, abnormalities of the brain, the cochleovestibular and facial nerves, as well as the endolymphatic duct and sac were evaluated.EYA1, SIX1 and SIX5 gene were amplified by Polymerase Chain Reaction (PCR) with specific primers (http://frodo.wi.mit.edu/). PCR with H taq polymerase  proceeded as following cycles: 15 minutes at 95\u00b0C, repeated of 30\u201340 cycles of denaturation at 94\u00b0C for 20 seconds; annealing at * \u00b0C for 40 seconds; extension at 72\u00b0C for 30 seconds (* is depended by melting temperature of primers). Last extension step was performed at 72\u00b0C for 5 minutes. Particularly, exons 1 and 2 of SIX5 were amplified using LA taq , because these regions have high GC contents. PCR products were separated on 1.5% agarose gel. The PCR products were purified using Shrimp alkaline phosphatase  and exonuclease I at 37\u00b0C for 70 minutes and directly sequenced using the Bigdye Terminator v3.1 Cycle Sequencing Kit . Ethanol precipitation was used for purification of sequencing reaction products before running the samples on the 3130xl Genetic Analyzer . The data was analyzed utilizing Sequencing analysis v5.2  and Chromas Pro v1.5 software . Multiple alignments of the analyzed sequences were performed using CLC sequence Viewer v.6.0 software .All exons and exons-intron boundaries of  primers  designedBamHI and EcoRI were inserted into multiple cloning sites between exon A and exon B in pSPL3 or pSPL3b vectors. For in vitro splicing assay, HeLa cells were cultured in DMEM  at 37\u00b0C in 5% CO2 concentration. Prior to transfection, HeLa cells were seeded at a density of 2.7\u00d7106 on a 60 mm culture dish. Hybrid minigenes in pSPL3 or pSPL3b vector were transiently transfected into HeLa cells using Fugene 6 Transfection reagent  at 3 \u00b5L per \u00b5g of DNA, and the transfected cells were harvested 24 hours after transfection. Total RNA was extracted from the transfected HeLa cells using an RNeasy Mini Kit  according to the manufacturer\u2019s protocol. Approximately 1 \u00b5g of total RNA was reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit . The cDNA was used as a template for PCR amplification of pSPL3 or pSPL3b vector-specific primers SD6 and SA2. The size of amplified normal and mutant fragments was confirmed on 1.5% agarose gel by electrophoresis.To analyze the splicing pattern, minigene vector was manufactured to include each exon along with about 300 bp of 5\u2032- and 300 bp of 3\u2032- intronic flanking regions . Amplified wild or mutant type products digested with EYA1 kit  that was used includes 17 probes for 14 of the 18 EYA1 exons  and 14 control probes. MLPA was performed according to the manufacturer\u2019s instructions: denaturation at 98\u00b0C for 5 minutes; stabilization at 25\u00b0C; hybridization at 95\u00b0C for 1 minute and at 60\u00b0C for 16\u201320 hours, stabilization at 54\u00b0C; ligation at 54\u00b0C for 15 minutes, inactivation at 98\u00b0C for 5 minutes, and stabilization at 20\u00b0C. PCR was carried out as follows: 35 cycles of denaturation at 95\u00b0C for 30 seconds; annealing at 60\u00b0C for 30 seconds; extension at 72\u00b0C for 1 minute. Final extension step was performed at 72\u00b0C for 20 minutes. Amplified products containing GeneScan\u2122-500 LIZ\u00ae Size Standard  and Hi-Di Formamide  were separated and quantified by capillary electrophoresis on the 3130xl Genetic Analyzer , and the data were analyzed using GeneMarker software v1.6 .Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to detect copy number variations such as deletions or duplications. The SALSA MLPA probemix P153-A2 EYA1 gene were selected from the NCBI database (www.ncbi.nlm.gov) considering their heterozygosity. PCR with H taq polymerase was performed using fluorescently tagged primers. Two PCR products of different fragment size and 0.1 \u00b5L of GeneScan\u2122-500 LIZ\u00ae Size Standard  were mixed and diluted with 8.9 \u00b5L of Hi-Di Formamide . Final diluted products were separated and detected by using ABI 3130x genetic analyzer. GeneMapper v4.0 software  was used to analyze genotypes for each marker.Four microsatellite markers, D8S1795, D8S1060, D8S1807 and D8S570, in the region of 2 Mb including the et al.Seven families including 10 patients were analyzed. All of the patients were females and their age at the time of diagnosis ranged from 1 to 43 years . The cliResults of renal manifestations were evaluated in 4 of 10 patients. In one patient (patient 9), a tiny cyst was seen in the right renal cortex and mild pelvic dilatation of the right kidney was identified by renal ultrasonography without any evidence of renal dysfunction. The other three patients did not reveal any abnormality on renal ultrasonography or blood testing.SLC26A4 mutations  on normal splicing, each exon and the flanking intronic sequences sufficient to allow splicing were inserted into a pSPL3 or pSPL3b vector. Each vector was transfected into the HeLa cells, and transcribed to mRNA in the cells. All three splice site mutations were found to disrupt the normal splicing . Exons 1EYA1, SIX1 and SIX5) by direct sequencing, and the results were compared to the data acquired in one normal control with normal hearing confirmed by pure tone audiometry. The entire EYA1 gene was suspected to be deleted in patient 8 . The results of the MLPA indicated that patient 8 had a heterozygous deletion encompassing the whole EYA1 gene. To investigate the deleted region including EYA1 in patient 8, genotype analysis using microsatellite markers was performed. Only patient 8 revealed homozygous alleles for all 4 markers, while all 12 normal controls showed heterozygous alleles for at least one of the microsatellite markers  who were found not to carry any mutations in the three genes  which is higher than 40% previously reported by Chang et al.EYA1 gene. In the Korean population, there have been only 3 case reports of patients with BOR/BO syndrome carrying EYA1 mutations, and this study adds to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population SIX1 mutations associated with BOR/BO syndrome, more than 10 mutations have been identified worldwide, while there is only one missense mutation reported in the East Asian population SIX1 or SIX5 mutation was found in this study suggesting the limited role of SIX genes as the cause of BOR/BO syndrome in the East Asian population. Since a large deletion was identified by MLPA in one patient with BOR/BO syndrome, we suggest that additional studies should be performed to identify complex rearrangements or large deletions when no mutation is found by conventional sequencing techniques.In contrast to the western population where large cohort studies have been performed on BOR/BO syndrome, limited information is available concerning genetic mutations of this syndrome in the East Asian population, and a total of 16 mutations in the EYA1 gene was identified in a patient presenting with only mixed hearing loss and enlarged vestibular aqueduct (patient 7) EYA1 gene (patient 8) compared to the patients with intragenic EYA1 mutations.No clear genotype-phenotype correlation could be demonstrated in this study in accordance with previous reports, in which no mutation was found in a patient with all major features of BOR syndrome (patient 9) whereas a splice site mutation of the et al.et al.eya1 in the sensory hair cells continued after birth and after maturation (P16 in mouse), suggesting an additional role for eya1 in the differentiation and/or survival of the inner ear cell populations in particular the sensory cells EYA1 mutations considering the possible role of EYA1 in the maintenance and survival of the hair cells and the supporting cells. Therefore, we believe that the possibility of hearing progression should be explained to the patients with BOR/BO syndrome, and regular auditory tests should be performed in order to treat these patients with proper modality of auditory rehabilitation at an appropriate timing.The progression of hearing loss was variable in patients with BOR/BO syndrome included in this study. Cremers eya1 has been reported to be expressed in the mesenchyme surrounding the cartilage premordia of all three ossicles and also in the epithelium of the tubotympanic recess which later develops into the tympanic cavity at E13.5, meaning that mutation in the EYA1 gene can disrupt normal development of the middle ear in various aspects This study clearly demonstrated the limitation of middle ear surgeries for hearing improvement in patients with BOR/BO syndrome consistent with previous reports EYA1 gene is involved in the development of the spiral ganglion and cochlear hypoplasia often seen in patients with BOR/BO syndrome has been known to be a poor prognostic factor of cochlear implantation, successful outcome in terms of speech and auditory performances could be achieved in our patients after cochlear implantation Since patients failed to gain hearing after one or more middle ear surgeries, most of the patients in this study used bilateral hearing aids. In two of the patients with severe to profound hearing loss who could not benefit from hearing aids, cochlear implantation was performed. In patients with syndromic hearing loss, multiple factors have to be considered regarding auditory rehabilitation, including combined mental retardation and developmental delay in addition to various inner ear malformations. Cochlear implantation in patients with syndromic hearing loss is challenging in both surgical and audiologic aspects. Although good results of cochlear implantation have been reported in these patients, some of the syndromes associated with cochlear nerve deficiency or narrow internal auditory canals such as CHARGE syndrome have shown limited outcome EYA1 gene in Korean patients with BOR/BO syndrome, the mutational analysis of EYA1 should be an integral part of the diagnosis of BOR/BO syndrome in the East Asian population. The characteristic inner ear and middle ear anomalies and mixed type hearing loss may also provide clinical clues to suspect BOR/BO syndrome even in the absence of other typical clinical features. The management of hearing loss and auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate of hearing gain achieved by middle ear explorations in patients with mixed hearing loss. Hearing aids are good options in patients with mild to severe hearing loss, but regular hearing evaluations are needed considering the possibility of progression of hearing loss in order to treat these patients with proper modality of auditory rehabilitation at an appropriate timing. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel EYA1 mutations identified in this study adds to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population and the clinical results of this study may provide evidence for recommending proper means of auditory rehabilitation in patients with BOR/BO syndrome.Considering the high mutation rate of the Figure S1Temporal bone CT and temporal MRI findings of patient 10 demonstrating enlarged vestibular aqueduct in a circular shape. (A\u2013F) Axial view of temporal bone CT shows enlarged vestibular aqueduct (black arrows) observed as a circular shape with a diameter significantly larger than that of the posterior semicircular canal (black arrowhead in (TIF)Click here for additional data file.Table S1Primer information of EYA1, SIX1 and SIX5.(DOC)Click here for additional data file.Table S2Identification of EYA1 deletion by microsatellite marker.(DOC)Click here for additional data file."}
+{"text": "These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca LRRK2 is a \u223c280 kDa protein with two enzymatic domains (Ras of complex GTPase domain and kinase domain) and several protein-protein interaction domains such as an amino terminal leucine-rich repeat domain and a carboxy terminal WD40 domain Despite the importance of LRRK2 for the pathogenesis in various diseases little is known about the cellular function of LRRK2. LRRK2 has been implicated in many different signaling pathways such as membrane trafficking 2+ concentration ([Ca2+]c) being at center stage 2+]c2+ influx via vesicular P2X4 receptors which promotes fusion pore expansion and subsequently facilitates surfactant release During LB exocytosis a sequence of highly regulated steps leads to fusion of exocytic vesicles with the plasma membrane, subsequent opening of a fusion pore and finally content release. Several intracellular signalling cascades stimulate LB fusion with the plasma membrane during the exocytic pre-fusion phase 2+ release from intracellular stores upon stimulation with ATP, which promotes triggering of LB exocytosis. However, using a biochemical approach to quantify surfactant secretion we found that surfactant release to the extracellular space was decreased after stimulation. This was not due to failure of actin coat formation and active extrusion of vesicle contents during the exocytic post fusion phase. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signalling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Within this study we investigated whether morphological changes of LBs observed in LRRK2 -/- mice are also present in LRRK2 -/- rats and whether they affect LB exocytosis and surfactant secretion. We measured single LB fusion events with the plasma membrane using high resolution, real-time fluorescent microscopy. ATII cells are particularly suitable for analysing single exocytotic events due to large size of secretory vesicles and slow, sequential vesicle fusion with plasma membrane. LB exocytosis has been intensively investigated in the past decade and there are several established microscopy methods for detection of single vesicle fusion events Rats were maintained at the central animal facility of the Ulm University according to institutional guidelines for ethical care of animals. All experiments in this study were approved by Regierungspr\u00e4sidium T\u00fcbingen, grant Nr. 833.tm1sage, product number: TGRL4620) as well as age- and gender-matched wildtypes (Long-Evans). The age of animals ranged between 5 and 9 weeks.LRRK2 -/- rats were purchased from SAGE Labs , seeded on chamber slides , and used for experiments for up to 48 h after seeding.Alveolar type II cells were isolated from wt and LRRK2 -/- rats according to the procedure of Dobbs et al. Cells were washed twice in ice-cold DPBS  fixed for 20 min in 4% paraformaldehyde  in DPBS and permeabilised for 10 min with 0.2% saponin and 10% FBS  in DPBS. Cells were subsequently stained with primary (1\u2236300) and secondary (1\u2236400) antibodies in PBS, 0.2% saponin and 10% FBS for 30 min. Images were taken on an inverted confocal microscope  using a 63\u00d7 lens (Leica HCX PL APO lambda blue 63.0\u00d71.40 OIL UV). Images for the blue (DAPI), green (AlexaFluor 488), red (AlexaFluor 568) and far red (AlexaFluor 633) channel were taken in sequential mode using appropriate excitation and emission settings. All used primary antibodies were from Abcam  apart from antibodies against surfactant proteins B and C which were a gift from T. Haller . AlexaFluor phalloidin 568 and all conjugated secondary antibodies were from Invitrogen.2+]c and detection of individual LB fusion events was performed as described previously 2, 2 CaCl2, 5 glucose, 10 Hepes; pH 7.4) and kept in bath solution with 0.5 \u2013 1 \u03bcM of FM 1\u201343. To efficiently induce LB exocytosis ATII cells were treated with various known and potent agonists for surfactant secretion: ATP (100 \u03bcM), PMA (300 nM), a combination of ATP and PMA, or ionomycin (1 \u03bcM) . The combined application of ATP and PMA was used because the previous report showed that the combined application can further increase LB fusion probability 2+]c as described in detail earlier The fura-2/FM1-43 assay for combined measurement of changes in the c\u200a=\u200aKd\u00d7[(R\u2212Rmin)/(Rmax\u2212R)]\u00d7(Sf2/Sb2).We estimated the intracellular free Camin was measured after Ca2+ ionophore ionomycin (20 \u00b5M) was added to a Ca2+ free perfusion solution . Rmax was measured after ionomycin (20 \u00b5M) addition to Ca2+-containing perfusion solution . The proportionality coefficient Sf2 was measured as the maximal 380 nm fluorescence intensity after ionomycin addition to Ca2+-free solution. The second proportionality coefficient, Sb2, was measured as the minimal 380 nm fluorescence intensity after ionomycin addition to Ca2+-containing solution. We corrected both coefficients for the background fluorescence which was measured after MnCl2 quench  at the end of the experiment. We used 224 nM as a Kd value for fura-2 ATII cells were seeded in perfusion chambers  and stained with fura-2 AM (3 \u00b5M for 20 min). Ratio (R) was calculated from 340/380 nm excitation intensities in unstimulated cells in normal experimental bath solution. RATII cells were incubated with 100 nM LysoSensor Yellow/Blue (Invitrogen) at 37\u00b0C for 30 min. Cells were washed with experimental bath solution and imaged on the Zeiss Observer microscope  and 40\u00d7 fluar oil objective using Metafluor software. After background subtraction, LB intensities at 340 and 380 nm excitation were measured in ImageJ and the 340/380 nm ratio was used for pH calculation.10((a\u2212b)/(ratio\u2212b)\u22121), where a, b and c are constants.Calibration of the ratio to pH relation was done as described previously We used Microsoft Excel and GraphPad Prism  for statistical analysis. Unless stated otherwise data are expressed as mean +/\u2212 SEM. n denotes true biological replicates  and non-paired student's t-test was used to assess the statistical differences between wt and LRRK2 -/- cell populations. Unless stated otherwise the number of experiments performed/cells analysed in each biological replicate was kept constant to account for correct weighting of individual biological replicates when generating means. Due to the low number of fusions/cell (on average 1\u20132 per responding cell) and within individual experiments (6\u20138 responding cells) fusion delay histograms were derived from pooled data from 3 to 6 animals. A non-parametric Mann-Whitney test was used to compare the medians of fusion delays after stimulation.The previous histological study of the lungs from LRRK2 -/- mice showed enlarged size and number of LBs in ATII cells Expression and localization of LB markers were not changed. LB membrane marker proteins Lamp1 and ABCa3 transporter were localized to the LB membrane in LRRK2 -/- as well as in wt cells  [38]. SuTo further investigate whether the observed increase in LB size is linked to changes in lamellar body homeostasis we also performed experiments to assess the pH in LBs from wt and LRRK2 -/- cells using the dye LysoSensor. No significant difference (p\u200a=\u200a0.45) between the vesicular pH in LBs from wt animals  and LBs from LRRK2 -/- animals  could be observed. Approx. 150 to 250 LBs were analyzed per animal.3 mediated calcium release from internal calcium stores resulting in a \u201ctriggered\u201d fusion response. PMA on the other hand acts via activation of protein kinase C without affecting intracellular Ca2+ concentrations and enables a more prolonged response to stimulation whereas ionomycin acts as an ionophore and causes massive Ca2+-entry from the extracellular space 2+ (ionomycin) signaling pathways and result in comparable activity responses  increased fusion response compared to wt cells   . When anctively) . These d2+]c using fura-2 while monitoring individual LB fusion events  in LRRK2 -/- cells and 0.49\u00b10.19 \u00b5M (n\u200a=\u200a2) in wt cells, respectively (p\u200a=\u200a0.08) . However animals . Mean pe\u200a=\u200a0.08) .Based on our finding that knock-out of LRRK2 results in a significantly enhanced LB fusion response in ATII cells when stimulated with 100 \u00b5M ATP, we next examined whether this also results in increased surfactant secretion. We analyzed the amount of secreted phospholipids (DPPC is the main component of surfactant) using a recently described enzymatic protocol To further elucidate these conflicting results \u2013 increased number of responding cells but reduced surfactant secretion in LRRK2 -/- cells \u2013 we investigated the potential impact of altered LRRK2 expression on the regulation of surfactant secretion during the post-fusion phase of LB exocytosis. The lipophilic nature of surfactant impedes rapid dispersal in aqueous solution and therefore surfactant does not readily diffuse out of fused LBs following opening of the exocytic fusion pore. We already showed that actin coating of fused LBs and subsequent actin coat compression play a pivotal role in surfactant expulsion from the vesicles Although LRRK2 was suggested to play a role in variety of diseases its cellular role remains elusive. Recent evidence implicated LRRK2 in regulation of secretory vesicle trafficking The original observation on increased LB size was made in LRRK2 -/- mice 2+]c2+ was described as a final trigger for LB fusion in ATII cells, the threshold necessary for fusion in ATII cells is very low (\u223c320 nM), and fusion kinetics correlate with [Ca2+]c kinetics 2+]c is not significantly elevated in LRRK2 -/- cells suggests that the increased rise in [Ca2+]c upon stimulation with ATP is the main effector of the increased fusion response in these cells. It is easily conceivable that in LRRK2 -/- cells the threshold for LB fusion is reached more readily and exceeded for prolonged times and hence more cells exhibit LB fusions upon stimulation with ATP. Such a model is also supported by the observation that in LRRK2 -/- cells the exocytic response following ATP treatment is similar to the response following stimulation that causes maximum elevation of [Ca2+]c and activation of Ca2+-dependent fusion activity (ionomycin). The increase in the rise of [Ca2+]c upon ATP treatment in LRRK2 -/- cells could be due to altered Ca2+ levels in intracellular Ca2+ stores or increased Ca2+ release kinetics. It is unlikely to result from altered Ca2+ extrusion mechanisms as the time-course of the [Ca2+]c decay is unchanged. Effects of LRRK2 on Ca2+ homeostasis and Ca2+ signaling have already been reported in other cell types 2+ homeostasis 2+ release from lysosomal Ca2+ stores 2+ release from intracellular stores. This difference could be due to the different signaling mechanism underlying Ca2+ mobilization in the different systems. In ATII cells ATP elicits Ca2+ release via activation of IP3 receptors 2+ mobilization 2+ release in LRRK2 -/- cells following stimulation with ATP likely facilitates or accelerates fusion of LBs that are already localized close to the plasma membrane 2+]c it barely affects recruitment or trafficking of additional LBs to the plasma membrane for fusion .Stimulation of primary ATII cells with 100 \u00b5M ATP resulted in a significantly increased percentage of cells that responded to stimulation with exocytic activity when LRRK2 expression was ablated. LB exocytosis is exceptionally sensitive to [Ca2+ independent LB fusion activity as PMA induced LB fusion response is unchanged in LRRK2 -/- cells. However, we cannot fully exclude the possibility that PMA already causes a maximum stimulatory effect that cannot be increased upon. It is well established that PMA causes a strong fusion response (i.e. more cells responding within a defined period of time after stimulation) due to the long lasting stimulatory effect. Hence the combined application of ATP and PMA, evoking a Ca2+ increase as well as direct stimulation of protein kinase C, does not necessarily need to exceed the PMA response LRRK2 -/- does not seem to affect Ca2+ signaling remains to be answered.The observed increase in exocytic activity in LRRK2 -/- cells is also in line with observations from cortical neurons where LRRK2 silencing increased fusion kinetics Shin et al showed that LRRK2 silencing in neurons leads to impaired compensatory endocytosis Although ATII cells from LRKK2 -/- rats contain larger LBs and these LBs had an increased propensity for fusion with plasma membrane upon physiological stimulation, the amount of secreted phospholipids following stimulation was lower than in wt cells. Therefore, we tested the possibility that the extrusion of secretory material might be impaired in LRKK2 -/- cells. Surfactant is a poorly soluble substance and the opening of the exocytic fusion pore is not sufficient for efficient surfactant release from the fused vesicle. Actin coating of fused LBs and myosin driven compression of this coat is necessary for active surfactant extrusion 2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP. However, further studies are required to fully elucidate the molecular mechanism how LRRK2 regulates intracellular Ca2+ release in these cells and whether LRRK2 affects surfactant loading of LBs. Results of these studies will also shed some more light on whether the observed effects of LRRK2 on exocytosis and secretion in other cell types are linked to changes in intracellular Ca2+ signaling.In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca"}
+{"text": "Ornithodoros tholozani specimens were tested with a Borrelia flaB-PCR. Results were positive for all patients and 2%\u201340% of ticks. A 7\u2013amino acid gap characterized all 9 sequenced flagellin gene amplicons. By phylogenetic analysis, Israel TBRF Borrelia sequences clustered separately from American and African groups.Blood samples from 18 tickborne relapsing fever (TBRF) patients and  Tickborne relapsing fever (TBRF) is caused by Borrelia species and is transmitted to humans by Ornithodoros soft ticks. Worldwide, a dozen Borrelia species are known to cause this disease , and 88% of the case-patients were infected in caves (TBRF in Israel was first reported by Nicholson (Although American (2 traps in caves and were identified as Ornithodoros tholozani (We designed a genus-specific set of primers (BOR1: 5\u00b4 TAA TAC GTC AGC CAT AAA TGC 3\u00b4 and BOR2: 5\u00b4 GCT CTT TGA TCA GTT ATC ATT C 3\u00b4) that targeted the Borrelia flaB flagellin gene . Plasmids containing inserts were purified and sent for 2-strand sequencing. Direct sequencing of DNA amplified by the BOR1 and BOR2 primers was performed later.Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version 3.1 (Three PCR amplicons (from 1 tick and 2 human samples) were sequenced after cloning, whereas 6 amplicons (from 2 ticks and 4 human samples) were analyzed by direct sequencing. These 9 sequenced samples showed 98%\u2013100% homology between them and could be divided into 3 groups. The same DNA sequence was found in tick TG52 and in blood from 2 patients, HumanBlood2 and HumanBlood4. These 3 sequences had an additional triplet at the position 627. The second group of sequences, which consisted of tick samples TGd1 and CBkc7 and blood samples C1025B, FL1, and HumanBlood3, were identical, with only 3 minor substitutions between them. The third group consisted of the HumanBlood1 sample.All the translated sequenced amplicons showed a very specific signature by the presence of a 7\u2013amino acid (aa) gap at position 216  when comComparison with published flaB protein sequences of TBRF Borrelia showed 88%\u201390% homology with B. duttonii and B. recurrentis, 85%\u201390% with B. crocidurae, 86%\u201388% with B. turicatae, 87%\u201389% with B. hermsii, and 85%\u201388% with B. parkeri. The sequences of the Israel TBRF Borrelia isolated from different samples clearly clustered in a separate group from the American and the African TBRF species .Our results suggest that infection rates differ according to location, despite the small number of ticks tested and the use of pools. BOR1-BOR2 PCR was more sensitive than blood smear examination (100% vs 83%). An identical DNA sequence was found in both tick and patient samples and thus confirms, at the molecular level, the role of O. tholozani as the vector of TBRF in Israel.A signature (7-aa gap) of the flaB flagellin defined the Israeli TBRF sequences as a homologous group different from other TBRF species. Despite the small number of samples studied, a clear polymorphism existed also at the protein level, resulting in 3 local types. This diversity can be explained by the use of direct sequencing of samples rather than through cultivation that reduces the biodiversity of isolates by selecting the most successful in vitro clone.This study opens a new avenue in TBRF Borrelia studies by demonstrating a Middle East cluster in addition to the American and African groups. These results open encouraging perspectives for the better understanding of entomologic, epidemiologic, and bacteriologic aspects of this disease and may contribute to better diagnosis and treatment."}
+{"text": "Despite various immunosuppressive agents, juvenile idiopathic arthritis (JIA) related uveitis often take a serious course that leads to sight threatening complications.To evaluate efficacy of Abatacept in combined treatment of JIA related uveitis.10 children aged 4 \u2013 12 years with anterior uveitis associated with JIA were treated by Abatacept in standard infusions pattern and doses. Indication for using Abataceptwas ineffectiveness of standard therapy of arthritis and/or uveitis. 6 patients were suffering from polyarticular variants, 4 \u2013 oligoarticular JIA. Abatacept was combined with Methotrexate in 9, Sulfasalazin in 1 and low-dose steroids in 4 children. 3 patients were switched to Abatacept from Infliximab due to non-efficacy, 7 received abatacept as a first line biologic. 9 patients had bilateral eye involvement. At the administration of Abatacept severe inflammation was seen in 1, moderate in 7, mild in 2 of the cases. Follow up period ranged from 3 to 11 month (mean 7.6). The main outcome measure was the degree of inflammation.nd \u2013 4th injection. From patients switched from Infliximab to Abatacept 1 achieved remission of uveitis, 1 improved, 1 remained stable. Patients with remission of uveitis diminished or discontinued topical medications. No ocular or systemic adverse effects were observed. Glaucoma or cataract surgery was uncomplicated in all 4 cases.Remission of uveitis in current treatment was achieved in 6 (60%), improvement in the degree of inflammation in 2 (20%) of the cases. The initial response was seen after the 2Administration of Abatacept was effective in 80% of children with JIA related uveitis. Further investigations are required to define clear indications for this treatment in severe uveitis."}
+{"text": "Hox6 paralog group genes have been implicated as key determinants of LMC fate at forelimb levels of the spinal cord, through their ability to promote expression of the LMC-restricted genes Foxp1 and Raldh2 and to suppress thoracic fates through exclusion of Hoxc9. The specific roles and mechanisms of Hox6 gene function in LMC neurons, however, are not known. We show that Hox6 genes are critical for diverse facets of LMC identity and define motifs required for their in vivo specificities. Although Hox6 genes are necessary for generating the appropriate number of LMC neurons, they are not absolutely required for the induction of forelimb LMC molecular determinants. In the absence of Hox6 activity, LMC identity appears to be preserved through a diverse array of Hox5\u2013Hox8 paralogs, which are sufficient to reprogram thoracic motor neurons to an LMC fate. In contrast to the apparently permissive Hox inputs to early LMC gene programs, individual Hox genes, such as Hoxc6, have specific roles in promoting motor neuron pool diversity within the LMC. Dissection of motifs required for Hox in vivo specificities reveals that either cross-repressive interactions or cooperativity with Pbx cofactors are sufficient to induce LMC identity, with the N-terminus capable of promoting columnar, but not pool, identity when transferred to a heterologous homeodomain. These results indicate that Hox proteins orchestrate diverse aspects of cell fate specification through both the convergent regulation of gene programs regulated by many paralogs and also more restricted actions encoded through specificity determinants in the N-terminus.A critical step in the assembly of the neural circuits that control tetrapod locomotion is the specification of the lateral motor column (LMC), a diverse motor neuron population targeting limb musculature.  Hox genes in motor neuron specification and patterns of muscle connectivity are poorly understood. We have found that members of the Hox6 gene paralog group  contribute to diverse aspects of motor neuron subtype differentiation. Hox6 gene activity is required during two critical phases of motor neuron development: first as motor axons select a trajectory toward the forelimb and second as they choose specific muscles to innervate. At the molecular level, these two functions are encoded by distinct peptide domains within Hox proteins. This work indicates that Hox proteins execute their critical functions in motor neurons through intrinsic modules that confer distinct specificities and that these activities are central in the genetic network required for motor neuron differentiation.Coordinated motor behaviors\u2014as complex as playing a musical instrument or as simple as walking\u2014rely on the ability of motor neurons within the spinal cord to navigate towards and establish specific connections with muscles in the limbs. The establishment of connections between motor neurons and limb muscles is mediated through the actions of genes encoding Hox proteins, a large family of transcription factors conserved amongst all metazoans. However, the specific requirements for  The neural circuits that govern locomotor behaviors rely on the establishment of orderly sets of connections between motor neurons (MNs) and their peripheral and central synaptic targets. A critical and early step in the emergence of locomotor circuitry is the selection of specific muscle targets by a diverse array of MN subtypes. Three organizational features of MNs emerge during embryonic development that contributes to the specificity of their connections with target cells. First, MNs that project axons to common peripheral targets are organized into columns longitudinally arrayed along the rostrocaudal axis of the spinal cord Hox genes are expressed by MNs, with subsets of related paralogs functioning at distinct levels of the MN differentiation pathway Hox6, Hox9, and Hox10 genes have been implicated in the early columnar organization of MNs and contribute to the specificity of their initial projections into the periphery Hox genes contribute to the specification of MN pools, in part, through the induction of intermediate transcription factors Within the developing spinal cord, Hox proteins exert central roles in the specification of MN columnar and pool subtypes Drosophila indicate two key mechanisms through which Hox proteins regulate target genes Studies in Hox gene, Hoxc9. Hoxc9 is required for the appearance of thoracic-level MN columnar subtypes including preganglionic column (PGC) and hypaxial motor column (HMC) neurons Hoxc9 all brachial Hox genes are derepressed at thoracic levels, and MNs acquire an LMC fate. This broad repressive activity appears to be mediated by direct interactions of Hoxc9 with multiple sites in the HoxA and HoxC loci. Genome-wide analysis of Hoxc9 binding revealed a consensus binding motif which matches a high affinity Hox/Pbx site (TGATTTAT) identified by several groups through in vitro site selection Some insights into the mechanisms by which Hox proteins regulate target genes in MNs have emerged through analysis of mice mutant for a single thoracically expressed Hox6 and Hox10 genes have been implicated in the initiation of the LMC program at brachial and lumbar levels respectively, through activation of the gene encoding the transcription factor FoxP1 Raldh2The problem of Hox specificity in MNs is particularly relevant at limb levels of the spinal cord, where individual neurons express multiple Hox proteins at the time of their differentiation Hox5 genes (Hoxa5 and Hoxc5) and Hoxc8, respectively; and the actions of these Hox genes are necessary for delineating the rostrocaudal position of MN pools Hox4\u2013Hox8 proteins are thought to promote the intrasegmental diversity of MNs, by defining specific molecular codes for each pool subtype. For example, misexpression studies in chick have provided evidence that Hoxc6 is selectively required for the intrasegmental differentiation of pools within the caudal (Hoxc8+) half of the LMC Hox6 paralog group that determines the early columnar identity of forelimb-innervating MNs contains members that promote motor pool fates.While Hox proteins seem to be critical for LMC specification, it is less clear how they contribute to MN pool diversity. At brachial levels the LMC is broadly divided into rostral and caudal domains by expression of Hox6 genes during MN columnar and pool specification programs. First, what are the specific contributions of the three murine Hox6 genes to MN fate specification? Second, to what extent are the diverse activities of a Hox protein unique, or are they shared amongst gene paralogs within a cluster? Third, are there motifs intrinsic to Hox proteins that subfunctionalize in vivo specificities? To address these questions we analyzed mice in which all Hox6 genes are mutated, as well as employed an in vivo approach to dissect functional domains required for Hox specificity in MNs. We find that although LMC specification is retained in mice lacking Hox6 genes, Hoxc6 has a specific role in promoting MN pool identity and appropriate patterns of limb connectivity. The preservation of LMC fate in Hox6 mutants appears to be mediated by a diverse group of Hox5\u2013Hox8 genes expressed at brachial levels. Dissection of a single Hox protein reveals in vivo specificity relies on motifs that ensure deployment of programs common to all LMC neurons, as well as distinct modules that contribute to MN pool identity.In this study we sought to address several unresolved issues concerning the function and specificity of Hox6 genes in the specification of LMC neurons at brachial levels of the spinal cord. Two Hox6 genes, Hoxa6 and Hoxc6, are selectively expressed by brachial MNs in chick, and can convert HMC and PGC neurons to an LMC fate when misexpressed at thoracic levels Hox6 activities are absolutely required for LMC specification in mice is not known. To begin to answer this question we first analyzed the expression of Hox6 paralogs  at brachial levels near the time of LMC differentiation at embryonic day (e) 11.5. Hoxa6 and Hoxc6 are expressed throughout the brachial LMC, while Hoxb6 is expressed by MN progenitors . Hox6 genes are therefore not required for the generation of MNs as a class or in maintaining Hox expression patterns.To assess the function of and Lhx3 . In addi mutants , likely Hoxa6/c6 mutants. We assessed the expression of Foxp1 and Raldh2, two genes that are induced downstream of Hox proteins Hoxa6\u2212/\u2212Hoxc6+/+, Hoxa6+/\u2212Hoxc6+/\u2212, and Hoxa6\u2212/\u2212 Hoxc6+/\u2212 embryos the number of LMC neurons was similar to wildtype embryos, while both Hoxc6\u2212/\u2212 and Hoxa6\u2212/\u2212Hoxc6\u2212/\u2212 mutants displayed significant LMC losses  subtype, a motor neuron column normally present at thoracic levels Hoxa6/Hoxc6, MNs that fail to acquire an LMC fate revert to an HMC-like identity.We next determined the fate of the LMC neurons that are lost in Hoxb6 in MN progenitors could account for the maintenance of LMC identity in Hoxa6/Hoxc6 mutants we also analyzed mice in which all three murine Hox6 alleles are deleted. We found that in Hoxa6/Hoxb6/Hoxc6 triple mutants FoxP1+/Raldh2+ MNs were present, and LMC numbers were grossly similar to Hoxa6/Hoxc6 double mutants  the most prominently expressed by brachial MNs  Smad1/5/8  Pea3 mutants, motor axons project to the CM but fail to branch and arborize the muscle Hoxc6 mutants  muscle, whereas Scip+ MN pools project along the median and ulnar nerve  mutants \u2013S6D. In  mutants \u2013S6D, sugHoxc6 mutants many RhD+ Scip\u2212 neurons were observed  and the homeodomains of heterologous Hox proteins. Fusion of the Hoxc6 N-terminus to the homeodomain (HD) of the \u201cLMC-neutral\u201d Hox protein, Hoxc4 , activatc levels . Thus thss Hoxc9 . Similarpression . These opression .Hoxc9.Hox proteins are known to contain peptide motifs that confer activation and repression of target genes independent of the homeodomain Foxp1 is regulated by Hox proteins we searched for potential Hox sites within the Foxp1 locus. In silico analysis using the Vista enhancer browser Foxp1 transcription start site. This enhancer (Foxp1/hs1149) is highly conserved amongst vertebrates and drives high levels of expression at limb levels of the spinal cord, and lower levels thoracically , which generally conforms to the Hox/Pbx consensus hs1149 element is bound by Hoxc6 in vivo.We next used gel mobility shift assays to determine if Hox proteins could bind the hs1149 element, as well as an optimized Hox consensus binding site (Fkh250con*) Foxp1/hs1149 and Fkh250con*, although with slightly reduced affinity , possibly because Hoxc9-mediated repression is established prior to the time Hoxc6IM is expressed. These observations suggest that either Pbx interactions are dispensable for LMC specification, or that the LMC neurons produced under these conditions reflect the actions of Hox proteins resident to the thoracic spinal cord whose functions are unmasked through suppression of Hoxc9.Because Hox proteins often require cooperative interactions with Pbx proteins to bind DNA, we also examined the consequences of mutating the YPWM motif (Hoxc6IM: YPWM->AAAM). Surprisingly expression of Hoxc6IM did not alter the capacity of Hoxc6 to repress c levels . BecauseHoxc9 repression domain (N\u039491) and YPWM motif were mutated, but retained the intrinsic activation domain (aa 105\u2013111). While Hoxc6 N\u039491 cooperatively bound with Pbx3 to the hs1149 element and Fkh250con* and activated LMC genes (Hoxc9 repression domain and Pbx interaction motif is the ability of Hoxc6 to promote LMC fate lost. These results suggest that Hoxc6 can promote LMC identity through two distinct mechanisms 1) Pbx-independent restriction of Hoxc9 and 2) Pbx-dependent interactions with Foxp1, activities that are separable  presumably acts by overriding the dampening influence of Hoxc9, perhaps by competing for binding sites. This idea is supported by the observation that Hox5\u2013Hox8 proteins can induce aspects of LMC differentiation without suppressing Hoxc9. During normal development this process is likely played out in two distinct contexts: at the border between brachial and thoracic spinal cord, where many LMC MNs express low levels of Hoxc9 While regions outside the homeodomain that contribute to the specificities of Hox proteins in Hoxc9 is a condition sufficient to promote LMC fates even in the absence of a functional LMC Hox protein. When the Pbx interaction motif is deleted from Hoxc6, Hoxc9 is still repressed and LMC neurons are generated, although Hoxc6 presumably will no longer have a direct impact on activating the LMC program. One plausible explanation for this result is that the suppression of Hoxc9 unmasks the activities of Hox proteins that are endogenous to the thoracic spinal cord that have the capacity to activate high levels of Foxp1 , where \u0394\u0394Ct\u200a=\u200a(CtIP\u2212CtInput)\u2212(CtIgG\u2212CtInput).Chromatin immunoprecipitation was performed as previously described pET-14b plasmids carrying His-tagged constructs were used to transform BL21 pLys bacterial strain and protein expression was induced by 0.5 mM IPTG at room temperature overnight. Bacteria were lysed under native conditions in lysis buffer (50 mM Tris pH 7.5 and 100 mM NaCl) followed by sonication. The supernatant was incubated with Ni-NTA agarose beads at 4\u00b0C for 1.5 hours and washed three times in buffer containing 50 mM Tris pH 7.5, 300 mM NaCl, 20 mM imidazole and 0.5% Igepal CA-630. Recombinant proteins were eluted in 50 mM Tris pH 7.5, 300 mM NaCl, 250 mM imidazole and dialyzed overnight in 50 mM Tris-HCl pH 8 and 150 mM NaCl.AGCTGTGGGACGAGG). Double stranded probes were synthesized using Klenow DNA polymerase. Binding between recombinant proteins and DNA probes was performed in binding buffer containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl2, 20% Glycerol, 2.5 mM DTT, 2.5 mM EDTA pH 8, 250 ng/\u00b5L poly dIdC and 0.1% BSA for 20 minutes at room temperature. For each binding assay equivalent molar amounts (0.5\u20132 pmol/reaction) of recombinant protein were used. Binding reactions were resolved on a non-denaturing acrylamide gel and the IRDye-800 was detected using the Odyssey system (Li-Cor).Oligonucleotides containing putative Hox binding sites were annealed to an IRDye-800 labeled linker . We do not work with a species or procedure, including euthanasia, with which I and those members of my research staff involved in this project are not experienced, without first seeking the advice and instruction of a veterinarian from the Division of Laboratory Animal Resources, consult the Division of Laboratory Animal Resources as circumstances require. To the best of my knowledge, the research does not unnecessarily duplicate previous research with respect to the use of laboratory animals. We comply with all requests for data as may be required by governmental and institutional guidelines. We seek the approval of the Institutional Animal Care and Use Committee on all procedures which involve laboratory animals.Figure S1Hoxa6/c6 mutants at e11.5. (A) Increase in the number of HMC neurons at rostral brachial levels in Hoxa6/Hoxc6 mutants at e11.5. Serial sections from rostral to caudal levels of the LMC are shown left to right. HMC neurons are identified by Hb9+Isl1/2 coexpression, indicated in cyan.  Normal expression of Hoxc4 and Hoxc8 in Hox6 mutants at e11.5. Serial sections along the rostrocaudal axis showing normal expression of Hoxc8 and Hoxc4 in FoxP1+ LMC neurons. HoxA genes are also expressed normally in Hox6 mutants (data not shown).Analysis of MN columnar specification in (PDF)Click here for additional data file.Figure S2Hoxc9 is not derepressed at brachial levels in Hoxa6/Hoxc6 mutants. Serial sections at caudal brachial levels showing that Hoxc9 is normally restricted from FoxP1+ LMC neurons in Hoxc6 and Hoxa6/Hoxc6 mutants. At these levels Hoxc9 is normally expressed in neurons located dorsal to the LMC. Rostral to caudal is shown left to right.(PDF)Click here for additional data file.Figure S3Hox6 mutants. (A) Total number of FoxP1+ LMC neurons in the brachial spinal cord of various Hox6 mutant allele combinations. (B) Levels of FoxP1 protein expression are reduced in rostral brachial regions in Hoxa6/Hoxc6 mutants. Levels were determined by measuring the pixel intensities of FoxP1 nuclear staining.  Decrease in the number of FoxP1+ LMC neurons at brachial levels in Hoxa6/Hoxc6 mutants at e10.5 and e11.5. Images show serial sections along the rostrocaudal axis from left to right. Loss of FoxP1 is prominent at rostral brachial levels (Hoxa5/Hoxc5+ region) of the spinal cord. Approximate position of the Hox5/Hoxc8 boundary is indicated.Analysis of LMC specification in (PDF)Click here for additional data file.Figure S4Hox6 triple mutants. In mice lacking all three Hox6 genes  LMC neurons are still generated in caudal brachial spinal cord, as assessed by FoxP1 and Raldh2 expression. In rostral brachial spinal cord, there is an additional loss in LMC neurons in triple mutants when compared to Hoxb6/c6 double mutants, but essentially phenocopies the LMC loss in Hoxa6/c6 double mutants Click here for additional data file.Figure S5Hox gene. Error bars show s.e.m.Efficiency of LMC induction by Hox4\u2013Hox8 proteins. (A) Examples of Hox electroporations in chick showing similar levels of protein expression to endogenous brachial levels. (B) Quantification of mean pixel intensities of Hox staining in n>40 nuclei of electroporated neurons at brachial and thoracic levels. (C) Quantification of the percentage of electroporated MNs (defined by Isl1/2 expression) that express high levels of FoxP1 at thoracic levels after misexpression of the indicated (PDF)Click here for additional data file.Figure S6Hoxc6 mutants. (A\u2013D) Additional examples of whole mount GFP staining showing defects in motor axon innervation of the cm muscle in Hoxc6 mutants at e12.5 and e13.5. (E) Loss of Pea3+ and retention of Scip+ motor neuron pools at e11.5 in Hox6 mutants. There is a marked decrease in the number of Pea3+ MNs at e11.5 in Hoxa6/Hoxc6 mutants.Motor neuron pool defects in (PDF)Click here for additional data file.Figure S7Hoxc6 mutants. (B) Summary of the distribution of labeled MNs after ulnar injection. In control mice only Scip+ MNs are labeled. In Hoxc6 mutant mice Scip\u2212 MNs are labeled, the position of these labeled MNs extends rostrally, and overlaps with the position of the former Pea3+ MN pool. (C) Serial sections from rostral to caudal showing distribution of labeled MNs after ulnar injections in control and Hoxc6 mutant mice.Analysis of tracer injections into the ulnar nerve in Hoxc6 mutants. (A) Summary of the position and distribution of the Pea3+ and Scip+ MN pools in the caudal half of the lateral motor column (cLMC). Relative positions of the pools in transverse sections are indicated for both control and (PDF)Click here for additional data file."}
+{"text": "MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (tm1aMcph1tm1a/) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that tm1aMcph1tm1a/ mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired tm1aMcph1tm1a/ mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of tm1aMcph1tm1a/ mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive.  Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment in children. As a multifactorial disease, the pathogenesis of OM is complicated. Based on previous research, many factors are thought to contribute to the development and persistence of OM including: environmental factors such as smoking and type of child care; anatomical dysmorphology; Eustachian-tube function; adaptive and innate immune system function; viral and bacterial load; and genetic predisposition. However, the mechanisms underlying OM are still elusive. Heritability estimated from twin studies Mcph1)-deficient (tm1aMcph1tm1a/) mice were found to exhibit hearing impairment as a part of the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the Knockout Mouse Project and the European Conditional Mouse Mutagenesis Program (KOMP/EUCOMM) resource of over 17,000 genes targeted in ES cells In the present study, microcephalin 1  encodes MCPH1, which contained three BRCT domains: one in its N terminus and two in its C terminus. BRCT domains have been found predominantly in proteins involved in cell cycle checkpoint functions and in proteins involved in the DNA damage response tm1aMcph1tm1a/ mouse mutants not only had some expected features such as small skull size and increased micronuclei reflecting genome instability, but also showed some unexpected phenotypes including susceptibility to OM implicating MCPH1 in genetic predisposition to OM. This finding implicates a new molecule in the pathogenesis of OM that is relevant to understanding the underlying mechanisms irrespective of the initial trigger for OM.The broad phenotypic screening of the MGP revealed that All mouse breeding and investigation was carried out with authorization of the UK Home Office. Mice were killed by cervical dislocation and decapitation. All efforts were made to minimize suffering.Mcph1-deficient (tm1a(EUCOMM)WtsiMcph1, abbreviated to tm1aMcph1 in this report) mice carry a knockout-first allele LacZ and neo genes were inserted in intron 3\u20134 of the Mcph1 gene (http://www.knockoutmouse.org/kb/entry/102/), exon 4 of Mcph1 was chosen as the critical exon. The vectors containing the Mcph1 knockout-first allele were electroporated into embryonic stem cells (JM8F6) derived from C57BL/6N mice. Targeted embryonic stem cell lines were selected using neomycin and screened by long range PCR after homologous recombination. The presence of the LoxP site was confirmed by sequencing. Correct integration of the 5\u2032 arm and 3\u2032 arm was confirmed by long range PCR using a universal primer and two genome-specific primers, and the subsequent PCR amplicon was verified by sequencing. The positive stem cells were injected into host mouse blastocysts and were used to generate chimeras containing the targeted allele. Male chimeras with 80\u201390% of targeted cells were bred with C57BL/6Brd-c-BrdTyr females and germ line transmission of the Mcph1 knockout-first allele was confirmed by a series of genotyping PCR analyses (http://www.knockoutmouse.org/kb/25/) using the mouse tissue DNA as template. These heterozygous mice were inter-crossed to expand the colony. The mice were maintained in individually-ventilated cages at a standard temperature and humidity and in specific pathogen-free conditions on the mixed C57BL/6N and C57BL/6Brd-c-BrdTyr genetic background. To genotype animals  and exon 14 . Real-time PCR was performed in quadruplicate for each sample using the probe  in an ABI Prism 7000 (Applied Biosystem). Hypoxanthine-guanine phosphoribosyltransferase (Hprt) was amplified simultaneously  as the internal reference. The relative quantity of Mcph1 RNA was calculated using 2\u2212\u0394\u0394Ct method RNA was isolated from the tissues of middle ear, inner ear and forebrain. Littermates were used . Total RNA was isolated with QIAshredder columns  and RNAeasy mini kit . cDNA was synthesized with normalization of the same original amount of RNA using oligo dT and SuperScrip II (Invitrogen). Primers were designed to amplify part of exon 9\u201313  and xylazine hydrochloride  and subcutaneous needle electrodes were inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip  stimuli at a range of intensity levels in 3 or 5 dB steps. Averaged responses to 256 stimuli, presented at 42.2 per second, were analysed and thresholds established as the lowest sound intensity giving a visually detectable ABR response. The peak-peak amplitude of wave 1 of click-evoked ABRs (P1-N1 amplitude) was measured and plotted as a function of sound level above threshold to produce input-output functions (IOFs). The IOF slope from 0\u201335 dB above threshold was calculated since 0\u201335 dB above threshold covered the more linear range before the function begins to flatten towards a plateau and also to ensure that a similar dB SL (sensation level) range was covered in both wild type and Mice used for recurrent ABR measurements were sacrificed after the last measurement and the anatomy of their middle ears was examined. Briefly, the external ear canals, tympanic membranes, ossicles and middle ear cavities were carefully dissected, examined and imaged. The inner ears were dissected out, fixed in 4% paraformaldehyde and examined following inner ear clearing in glycerol 2 at room temperature for 3 hours. Cochleae were finely dissected in PBS. This was followed by further processing by an osmium-thiocarbohydrazide-osmium (OTOTO) method Inner ears of six mice at postnatal day 4 (three mice each of homozygote and wild type) were fixed by 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 3 mM CaClImmunohistochemistry staining was performed using the Ventana Discovery machine (Roche) and reagents according to the manufacturer's instructions. Paraffin sections from wild type mice at postnatal day 7 (n\u200a=\u200a3) and 4 weeks old (n\u200a=\u200a5) were obtained as described above. The expression of Mcph1 was detected by using Mcph1 antibody .Digital X-ray images are acquired using a Faxitron system MX20 (Faxitron X-ray corporation) in 14-week old mice . Mice were anaesthetised and up to five standard images were taken for each mouse. The entire skeleton morphology was assessed using a standardised protocol capturing 41 parameters. To extract the brains, a vertical incision was made through the scalp and both sides were peeled back. Then a vertical cut was made through the midline of the skull using a pair of scissors and the skull was peeled off with a pair of fine forceps. Brains were then removed using a fine spatula and weighed on a Sartorius TE212, accurate to two decimal places.The prevalence of micronucleated normochromatic erythrocytes (MN-NCE) was determined using a flow cytometric assay of micronucleus formation Salmonella enterica serovar Typhimurium M525 Flow analysis was performed on heparinised blood collected from 16-week old mice . The following parameters were analysed: percentages of total T cells, CD4+ and CD8+ T cells, NKT cells, NK cells, B cells, Granulocytes and Monocytes are presented relative to the total CD45+ WBC population. Percentages of memory CD4 and CD4+CD25+ Treg cells are presented relative to the total CD4+ T cell population. Percentages of memory CD8 and mature IgD+ B cells are presented relative to the total CD8+ T cell and B cell populations respectively. All samples are analysed on a BD LSR II.Escherichia coli  adjuvant. Mice were boosted on days 7 and 21. Serum samples were collected on days 28. Detection of TetC-specific antibodies from sera was performed by ELISA. For the measurements of antibody levels, mouse blood was collected by cardiac puncture, and serum was prepared and stored at \u221220\u00b0C. For antigen specific antibody measurements in mouse serum, Nunc MaxiSorp plates were coated overnight at 4\u00b0C with 2 mg/ml tetanus toxin fragment C recombinant protein (TetC) in 0.1 M Na2HPO4 (pH 9.0), blocked with 3% (w/v) BSA in PBS for 1 h, and incubated with 5-fold serial dilutions of mouse serum in PBS with 1% BSA for 1 h. The plates were developed with anti-mouse Ig HRP-conjugated Abs (Dako), followed by o-phenylenediamine substrate tablets (Sigma-Aldrich) dissolved in water. Absorbance was read at 490 nm and optical densities represented as titres.Recipient mice  were immunized by intranasal inhalation of 30 \u00b5l PBS containing 10 mg TetC  combined with 1 mg heat-labile toxin of Mice underwent ophthalmic screening at 15 weeks of age. They were assessed for gross morphological changes to the eye using a slit lamp (Zeiss SL130) and ophthalmoscope (Heine Omega 500). The eye was examined both undilated and dilated (tropicamide). Images using the slit lamp were collected using a Leica DFC420 camera. The mice were culled under terminal anaesthesia followed by cervical dislocation and six eyes from 3 male homozygous mutants and 3 wild type mice were removed and fixed. Pupil-optic nerve sections were processed with hematoxylin and eosin, and standard images were captured under light microscopy for review Blood was collected at 16 weeks of age into ETDA microvette tubes (Startedt) and analyzed on an analyzer  with ten parameters tested.16-week-old mice were terminally anaesthetized and blood was collected from the retro-orbital sinus into lithium-heparin tubes. The plasma was immediately analyzed on an Olympus AU400 Analyzer with 28 parameters tested.We assessed glucose tolerance in mice fed on a high-fat diet  from 4 weeks of age until 13 weeks of age. At 13 weeks, mice were fasted overnight before a blood sample was taken and glucose was measured using an Accu-Chek Aviva (Roche). A bolus of glucose (2 mg/g) was administered intraperitoneally and blood glucose concentration from the tail tip was measured using Accu-Chek Aviva (Roche) after 15, 30, 60 and 120 min.Mcph1 mutant on a C57BL/6 genetic background. The mutant allele is designated tm1a(EUCOMM)WtsiMcph1, and abbreviated to tm1aMcph1 in this study. The design of the tm1aMcph1 allele and genotyping protocol are illustrated in 2 test, p<0.001). Both male and female homozygous mutants are infertile, similar to other Mcph1 mutants reported previously tm1aMcph1tm1a/ mice (data not shown). Reverse transcription PCR was performed to test the effect of the mutation of Mcph1 on transcription. The homozygous mutants, heterozygous and wild type mice produced bands of expected size and sequencing of the PCR products validated the results. This indicated that there was residual Mcph1 transcript in tm1aMcph1tm1a/ mice. Quantitative real-time PCR revealed the residual transcript of Mcph1 in the homozygous mice is only 1\u20134% of the level compared to the wild type mice and the residual levels vary in different organs . tm1aMcph1tm1a/ mice showed mild to moderate hearing impairment with increases of 10\u201350 decibels (dB) compared to the normal thresholds for both click and pure tone stimuli (6\u201330 kHz)  and +Mcph1+/ (n\u200a=\u200a13) mice showed normal ABR thresholds, whereas elevated thresholds can be detected in tm1aMcph1tm1a/ mice as early as 3 weeks of age (n\u200a=\u200a13). Thresholds were generally stable over time, although there was progressive or fluctuating hearing impairment over time in some mice  above the normal hearing reference range as affected in this study, and the penetrance of hearing impairment in tm1aMcph1tm1a/ mice is around 70% based on this criterion.Hearing impairment was discovered by ABR measurement in 14 week old \u201330 kHz) . To furtome mice . Heterozutations . Mcph1tmtm1a/tm1aMcph1 mice  and +Mcph1+/ (n\u200a=\u200a13) mice showed a transparent tympanic membrane, air-filled middle ear cavity, and normal morphology of ossicles (apart from one heterozygous mouse that had some white secretion in the hypotympanum in the right middle ear cavity). Dissection of tm1aMcph1tm1a/ mice (n\u200a=\u200a13) that had elevated ABR thresholds revealed a range of defects in the middle ear including thickened and white bone forming the bulla instead of the normal thin and transparent bone, retracted tympanic membrane, bubbles present underneath the tympanic membrane, or middle ear cavities filled with clear or cloudy fluid. In addition, two tm1aMcph1tm1a/ mice had an amorphous tissue mass in the middle ear cavity . The effusion was confined within the middle ear cavity and did not appear to extend through the round window. In two out of three 60-week old tm1aMcph1tm1a/ mice, we saw only a few inflammatory cells scattered in the middle ear cavity, but the thickened mucoperiosteum was still present (data not shown). The Eustachian tube had a similar appearance in mutant (n\u200a=\u200a4) and control  mice in serial sections through the tube as it exits the middle ear (data not shown).Histological examination was performed to investigate the pathological change . Hearingtm1aMcph1tm1a/ and wild type mice. DNA was extracted from these and the 16S rRNA gene was amplified via PCR with universal bacterial primers (7f and 1510r). From this, 16S rRNA clone libraries were created for a tm1aMcph1tm1a/ and wild type mouse for each tissue sampled. The predominant phylotype found to be present in the nasopharynx of both mutant and wild type mice was matched through BLAST analysis to a previously-uncultured Streptococcus sequence (ERD01G accession number GQ456229.1) tm1aMcph1tm1a/ mouse. We were then able to culture this bacterium from the mutant middle ear by plating it onto a variety of media under micro-aerophilic conditions, thus confirming its presence within the tissue. The identity of this isolate as Streptococcus bacterium (Strep ERD01G) was confirmed by 16S rRNA PCR .We collected tissue from the nasopharynx, middle ears and external ear canal of tm1aMcph1tm1a/ mice have inner ear defects, scanning electron microscopy (SEM) and temporal bone sections were used to examine cochleae in young pups and adult mice respectively. At postnatal day 4, tm1aMcph1tm1a/ mice showed normal morphology of the upper surface of the organ of Corti by SEM  and there was no evidence of any abnormality of the cochlea in tm1aMcph1tm1a/ mice  in rol mice  indicatiSalmonella Typhimurium challenge, tm1aMcph1tm1a/ mice had a similar change of body weight compared to control mice over a 21 day period following the infection . To test if this alteration to the B cell frequency resulted in any changes in antibody production, we performed a prime boost immunisation with Fragment C of tetanus toxin. However, in agreement with the normal response to infection there was no significant change in antibody level in the tm1aMcph1tm1a/ mice . The only significant difference was an increase in the circulating B cell frequency in female m1a mice .tm1aMcph1tm1a/ mice displayed ocular abnormalities including corneal opacity and vascularisation .Mcph1 (tm1a(EUCOMM)WtsiMcph1, knockout-first design Mcph1 in the homozygous mutant mice to less than 4% of the wild type level. During the standardised phenotypic screen of these mutants, we found an unexpected phenotype: mild to moderate hearing impairment. ABR thresholds were raised uniformly across all frequencies tested and the growth of amplitude of the waveform with increasing sound stimulation above threshold was similar in mutants and controls, both features consistent with a conductive hearing impairment. Subsequent ABR measurement, dissection of the middle ear and histopathology indicated otitis media with effusion was present to varying degrees in the mutant middle ears. We found expression of Mcph1 in the epithelia lining the middle ear consistent with its involvement in otitis media. These findings suggest that tm1aMcph1tm1a/ mice are a model for one form of heritable otitis media and reveal a new molecule involved in the pathogenic pathways underlying otitis media that can be used to unravel the underlying mechanisms irrespective of the initial trigger.We report here a new mouse with a targeted mutation of ABR has been used extensively for assessment of mouse auditory function and can detect moderately-raised thresholds due to middle ear inflammation among many other mechanisms underlying hearing impairment tm1aMcph1tm1a/ mice. Furthermore, round windows appeared intact and the exudate was confined within the middle ear cavity. Most tm1aMcph1tm1a/ mice showed ABR threshold elevations no higher than 40 dB, which is what is expected in conductive hearing impairment and all tested frequencies were affected which is another characteristic of conductive hearing impairment. Furthermore, input-output function analysis suggested that there is no evidence for a recruitment-type effect in the hearing-impaired tm1aMcph1tm1a/ mice, where a steeper slope would be seen and be indicative of recruitment of additional auditory nerve fibers into the ABR wave 1 component with increasing sound levels as a result of sensorineural hearing loss. Thus, the features of hearing impairment seen in the tm1aMcph1tm1a/ mice are consistent with conductive impairment. We noticed a few tm1aMcph1tm1a/ mice had ABR threshold elevation higher than 40 dB  also suggest a more complex explanation, as these cells expressed residual MCPH1 protein but were derived from a patient with microcephaly Very recently, three different MCPH1 mutations previously. One possible explanation for this is that hearing impairment can easily be missed in the mouse. Also, owing to practical difficulties OM or hearing impairment has not been reported in human patients or mouse models with tm1a/tm1aMcph1 mice. Similar to studies of other Mcph1 mutants, we found that Mcph1-deficient mice have defects in DNA damage repair revealed by the increased prevalence of micronucleated normochromatic erythrocytes. Eye abnormalities revealed by gross morphology and histopathology present to varying degrees in the mutants implicating Mcph1 function in vision, but have not previously been reported in MCPH1 patients or mouse models.Besides OM, hearing impairment and smaller brain and skull sizes, we observed other defects in Mcph1 was proposed as a potential tumour suppressor because decreased levels of Mcph1 were detected in several types of human cancer including breast and ovarian cancers tm1a/tm1aMcph1 mutants suggests genomic instability so is consistent with a role in cancer. However, the four available Mcph1 mutant mouse lines have not been reported to show any excess of tumours, although none have been systematically aged and examined appropriately to detect tumours. Furthermore, there is anecdotal evidence that the incidence of cancer in MCPH1 patients is low MCPH1 expression in human cancer cells and increased micronuclei in the mice reported here on the one hand and the lack of reported tumour development in mouse Mcph1 mutants and MCPH1 patients on the other hand may reflect the small numbers of individuals studied appropriately.tm1a/tm1aMcph1 mice carry can produce reporter knockouts, conditional knockouts, and null alleles following exposure to site-specific recombinases Flp and Cre tm1aMcph1tm1a/ mouse could provide useful tools for further research to unravel the underlying mechanism of OM. The discovery of a role for Mcph1 in predisposition to OM expands our knowledge of genetic factors underlying OM. Rapid advances in sequencing technologies have already proved valuable in finding novel OM genes The knockout-first allele which"}
+{"text": "Dear Editor,Happiness has several main components including the emotional component the presence of contentment and affection; the social component- the presence of good social relationships and the ability to receive support; and finally the cognitive component that causes the happy person to interpret information in a special way that leads to joy and optimism.2]3]4][3][4]2][[4][3][4][3][4]2]Consequently, peoples\u2019 assessment of themselves and their lives can include cognitive aspects- like judgments about life satisfaction- or emotional aspects like mood and affect.6]7]8][7][8]6][[8][7][8][7][8]6]14] [14] 13][ [14] 13]Our study demonstrated no difference in happiness and life satisfaction between men and women. There were also no differences in the feeling of well-being between men and women across different educational groups. However, the difference between happiness across various faculties was significant. There was a significant difference between men and women in relation to happiness and socioeconomical, cultural, emotional, and mental conditions. Marital divorce and financial status were independent variables and happiness and life satisfaction were predictive variables; parental education was considered as the control variable. Furthermore, we failed to observe any difference among men and women in relation between happiness and needs, expectations and wishes. The mean\u00b1SD of happiness score in this study was 47.13\u00b114.3 that is comparable to other studies in Iran. In comparison with studies performed after 1990, Argyle and Leu reported a 35.6 percent.["}
+{"text": "WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu-insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub-Saharan Africa was \u223c3.3 times lower than in other populations . Meta-analysis across three European sample sets  detected significant association of the WNK1 AluYb8 insertion with blood pressure . Gender-stratified analysis revealed that this effect might be female-specific , whereas no statistical support was identified for the association with male BP . In leucocytes, the expressional proportions of the full-length WNK1 transcript and the splice-form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts. Hum Mutat 32:1\u20139, 2011. \u00a9 2011 Wiley-Liss, Inc.Mutations in  Essential hypertension is a complex disease promoted by an unfavorable combination of person's life style and heritable factors. It is a significant health risk leading to other cardiovascular and renal diseases. Genetic studies of monogenic, Mendelian forms of hypo- and hypertension have identified \u223c20 rare mutations in blood pressure regulating genes with a strong effect on the phenotype  were purchased from Applied Biosystems, Inc. . Primers and probes for the WNK1 transcripts lacking exon 11 (ex\u221211+12) and both exons 11 and 12 (ex\u221211\u221212) were designed using Primer Express version 3.0 (Applied Biosystems Inc.). Oligonucleotide sequences are given in Relative expression analysis of three The real-time RT-PCRs were performed using Applied Biosystems 7900HT Fast Real-Time PCR system in 96 microwell plates. Target region and endogenous control were amplified in the same well. The experimental conditions for the real-time PCR are given in detail in http://www.langsrud.com/fisher.htm). The significance of the associations between the WNK1 AluYb8 insertion and BP  as a quantitative trait was tested using linear regression (additive genetic model) with age and gender as covariates. Additive genetic model assumes a trend per copy of the minor allele to contribute to the trait or disease susceptibility on genotype categories. Association with the diagnosis of hypertension as a binary trait was assessed by logistic regression adjusted for age and sex. Association tests and calculation of LD between SNP pairs (r2) were implemented in the PLINK software, version 1.04 (http://pngu.mgh.harvard.edu/\u223cpurcell/plink/). The Bonferroni threshold for multiple testing correction was estimated 0.05/9 = 5.56 \u00d7 10\u22123, taking into account the number of tested phenotypes (three) and tested study samples (three). Results were combined in a meta-analysis using the inverse-variance method under fixed-effects model using R, version 2.7.2 .Statistical differences in allele frequencies between populations were calculated using the Web-based Fisher's Exact Test calculator (HPRT). For every study subject six replicate values of relative expression per each alternative WNK1 splice form  was calculated. As each of the genotypes  was represented by three individuals, in total 18 data points were collected per transcript within a genotype group. The most outlier Ct value within the respective genotype group was excluded from the statistical testing. Differences of normalized expression values between alternative genotype groups were estimated by Wilcoxon rank sum test implemented in R software.Normalized expression values of target regions were calculated using Microsoft\u00ae Excel\u00ae-based software Q-Gene [Muller et al., WNK1  and the WNK4 (11 CNRs) genes (WNK4 and six SNPs in the WNK1 gene (DHPLC and/or DGGE assays were designed for screening novel polymorphisms in CNRs of the s) genes . Based oNK1 gene . All butWNK1 CNRs, a novel unreported common indel (\u223c300 bp) was detected (AluYb8 element (288 bp without flanking T nucleotides) into a poly-T tract within WNK1 intron 10,\u223c780 bp upstream from exon 11  studied population samples. Frequency of the AluYb8 insertion in human populations differed based on their geographic affiliation  compared to North-African , European , and Asian . On average, the allele frequency of the WNK1 AluYb8 in Sub-Saharan Africa was \u223c3.3 times lower than in other studied populations (P = 9.7 \u00d7 10\u22129).The d Africa . The geniliation . The proWNK1 intron 10 in 11 chimpanzees, 1 gorilla, and 1 orangutan revealed that the WNK1 AluYb8 insertion has most probably occurred in human lineage. No AluYb8 insertion was detected in the WNK1 intron 10 of the studied primate genomes (WNK1 genomic fragment (exon10/intron10/exon11) amplified from a chimpanzee and from human wild-type as well as AluYb8 insertion carrying chromosomes revealed high conservation of intron 10 , exon 11 (459 bp), and intron 10 , respectively. Overall substitution rate was 0.7 and 1.3% between WNK1 exons and introns, respectively. The uniqueness of the WNK1 AluYb8 insertion in human was supported by a negative result of the BLAST search among available genome sequences.The analysis of the  genomes . Comparantron 10 . The subWNK1 AluYb8 insertion was tested for association with BP in the Estonian HYPEST cohort subjects . The analysis detected significantly higher SBP  and DBP , whereas no statistical support was found for the association in men . We also observed higher WNK1 AluYb8 frequency among HYPEST essential hypertension patients  compared to normotensive controls , we performed Stage 2 replication testing in two independent European samples: the BRIGHT (the British) and the CADCZ (Czech)  improved significantly the support for the association with BP . The pronounced effect of this Alu-insertion on BP in women was confirmed . Detected associations of the WNK1 AluYb8 insertion with SBP in the full sample and with female SBP and DBP remained significant after stringent correction for multiple testing .To confirm the discovery association between the  (Czech) . Stage 1 (Czech) . Meta-anWNK1 AluYb8 was observed in CADCZ hypertensive patients compared to controls (17.1 vs. 15.3%), but not in the BRIGHT cases representing extreme family based hypertension . However, AluYb8 was in strong LD with three previously genotyped WNK1 SNPs in the BRIGHT resource: rs11064527 , rs12816718 , and rs956868  , the strongest association was detected with AluYb8  . Amino ain human . When ash AluYb8 .WNK1 gene codes for a high number of mRNA transcripts and extensive alternative splicing has been described for exons 9, 11, and 12 [Verissimo and Jordan, WNK1 transcript completely lacks exons 11 and 12 [Delaloy et al., AluYb8 insertion on the expression profile of WNK1 alternative transcripts, we quantified the gene transcripts in mRNA extracted from the leucocytes of nine women with alternative genotypes  and ex\u221211\u221212  was upregulated among AluYb8 \u2212homozygous carriers (P = 3.56 \u00d7 10\u22122). We conclude that the carrier status of the AluYb8 insertion may have an impact on the profile in WNK1 transcript in leucocytes.The human enotypes . The stu exon 12 . CompareWNK1 and WNK4 to polymorphism screening in order to identify functional variants potentially contributing to BP determination. We identified a novel human-specific polymorphic AluYb8 insertion in WNK1 intron 10. The AluYb8 insertion belongs to a young Alu subfamily represented with \u223c2,200 copies in the human genome compared to only nine insertions detected in chimpanzee [Gibbons et al., WNK1 AluYb8 insertion in chimpanzee, gorilla, and orangutan . The carriers of the WNK1 Alu insertion had a consistent tendency for higher blood pressure  and no association was detected in men. Consistent with our findings, a sex-specific effect on BP determination was recently shown for a WNK1 SNP in intron 1 (rs10774461), which was also associated with BP only in females [Padmanabhan et al., Alu insertion (rs4646994) located in intron 16 of the ACE (angiotensin converting enzyme) gene [Rigat et al., ACE Alu I/D variant is associated with the hypertension risk only in men and not in women [Higaki et al., Our study identified a significant association between pressure . NotablyWNK1 SNP rs765250 [Newhouse et al., AluYb8 and rs765250 in both samples, which may indicate independent effects of WNK1 intron 1 and intron 10 polymorphisms on BP. Interestingly, strong LD (r2>0.8) between AluYb8 and three previously studied WNK1 SNPs in the BRIGHT study alone  positioned AluYb8 on the WNK1 haplotype, was reported to show borderline associations with SBP and DBP (P\u22649 \u00d7 10\u22122) and a strong association with 24-hr urine potassium (P<1 \u00d7 10\u22124) [Newhouse et al., P-values for the association with BP were the lowest for the AluYb8 insertion compared to SNPs in LD. Previously, rs956868 has been shown to exhibit suggestive effect on ambulatory SBP in Europeans (P<9 \u00d7 10\u22122) [Tobin et al., P<5 \u00d7 10\u22122) [Osada et al., AluYb8: +/+). Functional assays should bring understanding whether the detected association with BP is driven by one primary variant or by a combinatory effect of the haplotype-forming alleles.Previously, BP variation in the BRIGHT and the HYPEST subjects has been associated with the WNK1 AluYb8 insertion to the risk for developing hypertension. The initially observed but not confirmed higher proportion of AluYb8 carriers among hypertensives may have resulted from nonoptimal selection of HYPEST controls (biased to too young), and/or different recruitment strategies of hypertensive patients among studies .The design of the current study did not allow us to draw any conclusion about the contribution of the Alu elements are considered to be neutral residents of the human genome, an inserted copy of an Alu repeat could interrupt structurally or functionally important genomic regions and consequently affect the expression of a locus [Batzer and Deininger, Alu elements may alter gene expression through modulating alternative splicing, RNA editing, epigenetic regulation, and translation regulation [Cordaux and Batzer, Alu insertions have been identified [Belancio et al., AluYb8 insertion in WNK1 intron 10 on the expressional profile of WNK1 alternative transcripts. Splicing is an incompletely understood process carried out by large macromolecular complex spliceosome and directed by numerous regulatory elements located within exonic and intronic sequence [Black, WNK1 intron 10  is remarkably increased by the \u223c300 bp AluYb8 insertion . Thus, we hypothesize that the presence of the AluYb8 insertion may disrupt the spatial intronic structure and/or disarrange the possible splicing regulatory sequences within WNK intron 10. Consequently, it may affect the splicing efficiency of the down-stream exons 11 and 12. As alternative splicing tends to be a tissue and developmental stage specific process [Xu et al., AluYb8 insertion on the expressional profile of WNK1 may vary in different tissues. The current study design was limited to addressing the effect of AluYb8 insertion on WNK1 expressional profile in leucocytes using a small number of samples. Further in vitro and in vivo studies using renal tissues would reveal the potential effect of this Alu-insertion on WNK1 expressional profile in kidneys, where it plays an important role in contributing to the regulation of ion transport.Although a majority of e Black, . The sizAluYb8 insertion in WNK1. This AluYb8 insertion showed significant replicated association with blood pressure and a potential effect on the expressional profile of alternative WNK1 transcripts in leucocytes.In summary, we identified a novel human-specific polymorphic"}
+{"text": "Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefined, and studies have suggested harm from hyperoxia exposure . We aime2 levels higher than 40 kPa [We conducted a prospective observational cohort study in 21 ICUs in Finland between 2009 and 2010. The Utstein Guidelines were used for collecting resuscitation and PCA care data, such as initial rhythm and delay to return of spontaneous circulation (ROSC). Measured arterial blood oxygen values during the first 24 hours from admission to the ICU were divided into the following predefined oxygenation categories: low (<10 kPa), normal (10 to 19 kPa), intermediate (20 to 29 kPa), and high (>30 kPa). Exposure to hyperoxia was defined as paO2 measurements during the first 24 hours was eight per patient. A total of 6% of patients experienced paO2 values higher than 40 kPa at any time during the first 24 hours. Average times spent in each time oxygenation category during the first 24 hours were as follows: low 14%, normal 69%, intermediate 14%, and high 2% of the time. Survivors spent less time in the low band (P = 0.029) and more time in the intermediate band (P = 0.029) compared with nonsurvivors. The median paO2 during the first 24 hours was higher in survivors than in nonsurvivors  but there was no difference in lowest  or the highest paO2 values . In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality.A total of 489 patients were included. The average number of paOIn this multicentre observational study we were unable to define an optimal oxygenation level during PCA care, but hypoxia seemed to be more harmful than hyperoxia. Exposure to hyperoxia was less common than in previous trials, and we were unable to confirm previous findings indicating an association with mortality."}
+{"text": "In the section titled, \"Overall survival and Progression free survival\" within the Results Section the value of overall survival of patients at 3 years was incorrectly described as 68.7%. The correct value is 40.6%."}
+{"text": "To assess the effectiveness and functional capacity with rheumatoid artritis treated with biologic after dose reduction.A retrospective study on a cohort of 13 patients diagnosed rheumatoid artritis treated with anti-TNF  and clinical remission (DAS28 < 2.6) in the last 6 mounths, in which reducing the dosing. Clinical activity was evaluated by clinical activity index (DAS28), for functional capacity was used HAQ . Others variables studied was visual analog scale (VAS). Analyzing before starting dose reduction, at 3 and 6 months.The study includes 13 patients, 12 female, with RF in 90% and erosions in 40%, treated with etanercept 10 and adalimumab 3. More of them (85%) had recieved at least 2 FAMES before biological. Dose reduction in the etanercept group was 2 weekly injections to one injection every 5 days. In the case of adalimumab the pattern used was an injection every 20 days.Of the 10 patients treated with etanercept 4 returned to their usual pattern, as well as 2 of the 3 patients with Adalimumab.The reduction of etanercept and adalimumab doses in patients with rheumatoid arthritis in clinical remission (DAS28 <2.6) appears to be effective, since it manages to keep the DAS28 and the VAS. Not so the HAQ in which if there are significant differences that support that after the patient expresses reduce doses higher score on the test. Although the results 6 of 13 patients (46.2%) return to your regular schedule by mutual self-referred as subjectively feel worse and increase in seizures of exacerbation."}
+{"text": "Sprouty2, a negative modulator of RAS/ERK pathway, is important for regulating Fgf8 morphogenetic signal activity by controlling Fgf8-induced signaling pathways and positional information during early brain development.Early brain patterning depends on proper arrangement of positional information. This information is given by gradients of secreted signaling molecules (morphogens) detected by individual cells within the responding tissue, leading to specific fate decisions. Here we report that the morphogen FGF8 exerts initially a differential signal activity along the E9.5 mouse neural tube. We demonstrate that this polarizing activity codes by RAS-regulated ERK1/2 signaling and depends on the topographical location of the secondary organizers: the isthmic organizer (IsO) and the anterior neural ridge (anr) but not on zona limitans intrathalamica (zli). Our results suggest that  Proper embryonic development requires an accurately orchestrated complex network of interactions between signaling and transcription factors. Secreted signaling molecules (morphogens) organize fields of surrounding cells into molecular patterns and are tightly associated to the concept of positional information. This concept implies that a cell reads its position and determines its developmental fate/response according to a concentration gradient of these extracellular factors Fgf8 is expressed preferentially at the so-called secondary organizers The process of neurulation in vertebrates implies a major morphogenetic step for the initiation of brain regionalization. Localized signaling centers along the tube  and the morphogens emanating from them have a key role in refining the subdivisions of the embryonic brain. Among other morphogens, Fibroblast Growth Factors (FGFs) are a family of structurally related polypeptides with pleiotropic activities and are involved in a signaling system conserved from insects to humans Fgf8 transcription at early neural plate stages causes death of the entire mesencephalic and cerebellar primordia revealing a requirement for FGF8 signal in survival of neural progenitors Sprouty2  did not change. Surprisingly, at this developmental stage we still found a small portion at the most dorsal area of the isthmic constriction where Fgf8, Sef and En2 transcripts were expressed  and negative modulators of Fgf8 signaling such Mkp3 (n\u200a=\u200a4/4) and Sprouty2 were not longer expressed at caudal mesencephalon . In these ablation assays the main signaling receptor for FGF8, FGF receptor 1  or Fgf8 negative feedback modulators Mkp3 (n\u200a=\u200a7/7) or Sprouty2 (n\u200a=\u200a3/3) before any treatment  showed no expression of reatment . The ablerritory . After Berritory \u2019. The stCre/+; FgfR1 flox/flox; Finally, as an attempt to exclude any sensitive receptor mechanism underlying this initial polarizing activity exerted by FGF8b signaling we conducted FGF8b bead implantation assays on ONTCs of mutant embryos where FgfR1 was conditionally inactivated in the midbrain-rhombomere 1 region  during these initial planar instructions of FGF8 signaling. We decided to deprive pharmacologically the E9.5 mouse neural tube from any endogenous secreted molecule to the extracellular space (including FGF8 protein). Brefeldin A (BFA) was chosen for the ability to inhibit protein secretion in mammalian and other eukaryotic cells by interfering with the function of the Golgi apparatus, resulting in dysregulation of membrane traffic in vivo by Immunohistochemistry in whole-mount E9.5 mice with specific antibody against FGF8. The results revealed FGF8 staining either at the neuroepithelial intracellular or at extracellular levels in cryostat sections transverse to the IsO constriction  in the mouse IsO, secrete the morphogen near the lumen of the neural tube but the gradient of secreted Fgf8 protein forms a gradient along the basement membrane.We therefore analyzed the contribution of the FGF8 feedback modulators . Indeed, this signal would probably come from the caudal part of the mesencephalon or the isthmus, needed for normal antero-posterior polarity of the epithelium and therefore would not be directly related to an FGF8 signal.The ability of a rapidly internalized receptor to signal after endocytosis is important to ensure the sufficient duration and intensity of signaling. However, this capacity requires receptors to remain active in endosomes and therefore able to di-phosphorylate ERK FgfR1 in the mesencephalon was not affected during the time of the experiments . However, this powerful approach faces the difficulty of dissecting the function of each modulator because of their redundancy in FGF8 signal modulation Mkp3 expression was concomitantly downregulated in the same domain where phosphorylated ERK1/2 was not immunodetected. On the contrary, expression of Sprouty1 and especially Sprouty2 was maintained. Moreover, during BFA treatment FGF8-bead implantation on caudal mesencephalon maintained ERK1/2 polarized activation, indicating that Mkp3 and probably Sef were not required in the specification of FGF8 differential positional planar induction activity in the mesencephalon. It has been proposed that SPROUTY 1/2 and SEF function synergistically to regulate Gbx2 expression in the anterior hindbrain , caused a mirror-like cerebellar tissue induction rostrally to the ectopic implanted source. Within our results we can conclude that those results were due to the first polarized ERK1/2 activity driven by the FGF8b signal Sprouty1/2) at both sides of the IsO epithelium would maintain basic FGF8 intracellular activity to extend and to equilibrate the long-range distribution of active ERK1/2 along the A-P axis Then, what molecular mechanisms are behind this unbalanced activation of ERK1/2? Mutant mice have been used to understand the function of FGF8 negative feedback modulators in the mouse brain  are predominantly expressed at the ventricular zone in E11.5 mouse embryos Sprouty2 in the isthmus independently of ERK phosphorylation Sprouty2 gene expression pattern was maintained in the absence of ERK1/2 activity provide new horizons of FGF8 function. In conclusion, FGF8 may exert distinct signal responses depending on its cellular localization. These differential planar instructions may allow the segregation of neurogenic and proliferation signaling mechanisms or alternatively facilitate diffusion of FGF8-related activity through the basal lamina during vertebrate neural tube patterning Finally, the immunodetection of FGF8 protein assays in embryos and ONTCs revealed staining domains at the ventricular side and at the basal lamina see also . Interesgion see . In embrTimed pregnant mice were sacrificed by cervical dislocation and embryos were dissected in ice-cold phosphate-buffered saline . The embryonic day (E) 0.5 was the noon of the day of the vaginal plug. The embryonic age was determined more precisely by counting the somites in which at E9.5 ranged between 21 and 29 somite pairs neo/null  background and genotyped as previously described by Trokovic et al., All animal manipulation and experimental procedures were performed accordingly to the directives of the Spanish and European Union governments (Council Directive 86/609/EEC) and approved by the Animal Experimentation Committee of the Institute of Neuroscience UMH-CSIC. Mice from ICR strain were used as wild type. The transgenic mouse strain Fgf8Heparin acrylic beads (Sigma-Aldrich H-5263) were rinsed in PBS and soaked in FGF8b solution  for 1 h at 4\u00b0C. FGF8b-soaked beads were rinsed three times in PBS and thereafter implanted in the neural tube explants cultures as previously described Bafilomycin A1  was used for blocking the lysosomal pathway and so, preventing FGF8 degradation after endocytosis Brefeldin A  was used for blocking release of exocytic vesicles content , dehydrated in an ascending ethanol series, and stored in 70% ethanol at 20\u00b0C before processing. Whole-mount ISH was performed according to Garda et al. 2001 protocol In the case of IHC procedure whole mount embryos where dissected in ice cold PBS and fixed in 4% PFA with phosphatase inhibitor tablets (Roche Diagnostics 04906837001) following companies protocol for 2\u20134 hours before starting procedure. In some cases and after ISH or immunostaining procedure, embryos or ONTCs were immersed in ascending sucrose to 30% concentration and then cut at 12 \u00b5m thick sections ONTCs in a cryostat at -26\u00b0C (Microm-ThermoFischer Scientific) for a cellular analysis.2O2) at 3% for 30 minutes to inactivate the endogenous peroxidase activity. Then after 3 washes in PBS-T, they were blocked with goat serum at 5% and bovine serum albumin  at 2% in PBS-T. Incubation of Rabbit anti-dpERK  was done overnight at room temperature (RT). For the immunodetection in toto of the di-phosphorylated form of ERK1/2, the primary antibody was incubated for three nights at 4\u00b0C. In the case of mouse anti-FGF8b  for 2 nights at 4\u00b0C. Then, several washes in PBS-T were done before 1 hour incubation with Anti-rabbit or Anti-mouse biotinylated secondary antibodies at 1\u2236300 . Afterwards, Avidin-Biotin Complex was added at 1\u2236300 for 1 hour and washed in PBS-T . Colorimetric detection in embryos, ONTCs and tissue sections were incubated with 3,3\u2032-Diaminobenzidine  and 0,003% H2O2. In some cases we used combined protocols of ISH and IHC within the same tissue. Finally for immunofluorescence detection of mouse monoclonal anti-FGF8b in cryostat sections an anti-mouse Alexa-Fluor-594; (1\u2236500: Molecular Probes A-11032) was used for 1 hour at RT. DAPI staining  was used to visualize the nuclei of the cells. After all colorimetric detection, embryos ONTCs were washed several times in PBT. All images were photographed with Leica stereoscope (Leica MZ16FA) or an upright microscope (Leica DM6000B) for the cryostat sections, using a Leica DC500 camera or DCF350 camera for fluorescence images. All pictures were taken using Leica LAS AF software.Whole mounts embryos, ONTCs and cryostat tissue sections were rinsed 3 times in PBS 1\u00d7 with 0,1% Triton (PBS-T) and then incubated with hydrogen peroxide (Hneo/null (Fgf8 hypomorph mice in this paper). We also thank Martine\u017as Laboratory Staff for technical support.We are grateful to Dr. Constantino Sotelo for insightful and helpful suggestions during preparation of the manuscript. We would like to thank specially Drs. Laura Lahti and Joan Galcer\u00e1n for technical suggestions. We thank Dr. Gail Martin form kindly providing the mouse strain Fgf8Figure S1Maintenance of molecular isthmic organizer signal activity in mouse organotypic tissue cultures (ONTCs). Gene expression profile in mouse isthmic organizer by in situ hybridizations in mouse E9.5 ONTCs (A\u2019-D\u2019) in comparison to in toto mice of same age (A-D) after 6 hours of incubation. Mouse brain subdivisions at E9.5 ONTCs are described with the expression of Meis2 in blue compared to Fgf8 in red (A\u2019) genes and one half of the explant. The transversal black dashed lines illustrate the boundaries depicted by the genes on the mouse brain tissue. B-D) FGF8 negative feedback modulators, Sprouty2 (B), Mkp3 (C), and Sef (D). Note the similarities of these genes with respect to that of Fgf8 expression but the wider territory occupancy of their signals when compared to that of Fgf8, arguing indirectly the long range of FGF8 signal activity through the neuroepithelium from organizer centers.(TIF)Click here for additional data file.Figure S2Expression pattern profile of Fgf8 mRNA versus FGF8 protein in mouse E9.5 embryo. An anti-FGF8b immunohistochemistry was made onto 12\u00b5m cryostat longitudinal sections to the isthmus (see drawing) to visualize the intracellular and extracellular FGF8b protein (see arrows for the expansion of the protein in C) and compared with the Fgf8 mRNA domain . Note that the FGF8b protein can detected either at the ventricular side and at the pial side Click here for additional data file."}
+{"text": "TGFB1) T29C and TGF \u03b2 receptor type 1 (TGFBR1) 6A/9A polymorphisms have been implicated in the modulation of risk for breast cancer in Caucasian women. We analyzed these polymorphisms and combinations of their genotypes, in pre menopausal breast cancer patients (N\u200a=\u200a182) and healthy women (N\u200a=\u200a236) from western India as well as in breast cancer patients and healthy women from the Parsi community . Western Indian women were characterized by a higher frequency of TGFB1*C allele of the TGF \u03b2 T29C polymorphism (0.48 vs 0.44) and a significantly lower frequency of TGFBR1*6A allele of the TGFBR1 6A/9A polymorphism  as compared to healthy Parsi women. A strong protective effect of TGFB1*29C allele was seen in younger western Indian women . Compared to healthy women, the strikingly higher frequencies of low or intermediate TGF \u03b2 signalers in patients suggested a strong influence of the combination of these genotypes on the risk for breast cancer in Parsi women . The frequency of low signalers in Parsi healthy women, while comparable to that reported in Europeans and Americans, was three times higher than that in healthy women from western India . These observations, in conjunction with the low incidence rate of breast cancer in Indian women compared to White women, raise a possibility that the higher frequency of TGFB1*29C allele and lower frequency of TGFBR1*6A allele may represent important genetic determinants that together contribute to a lower risk of breast cancer in western Indian women.Transforming growth factor \u03b21 ( Germline mutations in various cancer susceptibility genes can account for only around 10% of all breast cancer cases TGFB1 T29C  and TGFBR1 6A/9A remain the two most extensively studied polymorphisms. In TGFB1 T29C polymorphism, a replacement of Leucine by Proline at position 29 (TGFB1 codon10 T>C) has been shown to result in an increased secretion of the cytokine TGFBR1 gene, giving rise to the TGFBR1*6A allele TGFBR1*9A allele In breast cancer patients, TGFB129*C allele that is associated with higher production of the cytokine, would provide protection in the initial stages by virtue of its anti-proliferative action on mammary epithelial cells but would enhance the risk at later stages by promoting invasion as well as spread of the disease TGFB1*CC genotype at a higher risk of the disease TGFB1*CC genotype in older American women and pre menopausal Japanese women TGBR1 6A/9A polymorphism on the risk for breast cancer TGFBR1*6A allele et alWith regard to the risk for breast cancer, it has been proposed that the presence of TGFBR1 6A/9A gene polymorphisms in pre menopausal women from western India. To that effect the study was restricted to Marathi-speaking (Maharashtrian) subjects from Western India, living within 200 km radius around the state-capital city of Mumbai (who constitute 80% of the population of the state of Maharashtra). In parallel, we have studied these polymorphisms in breast cancer patients and healthy women from the Parsi (Zoroastrian) community. Parsis in India represent a geo-ethnically isolated community Population based data regarding these polymorphisms in Indian women remains limited TGFB1*CC) on the risk for breast cancer, our results reveal trends that are opposite to those reported in Caucasian women. The basis for these observations and their implications has been discussed with due supporting literature.The comparison of genotypes in healthy subjects from the two ethnically distinct communities studied, and the comparisons between patients and healthy subjects within each of the groups have revealed novel trends with significant implications. Further, in terms of the influence of high TGF beta1 producing genotype  pre menopausal women with confirmed diagnosis of breast cancer treated at the Tata Memorial Hospital during 1999\u20132005 were recruited within twelve to eighteen months of diagnosis. Lack of family history for malignant disorders could be confirmed for 75% of the cases. Two hundred and thirty six unrelated, healthy premenopausal Maharashtrian women without family history for cancer were recruited from the community. Parsi women with breast cancer (N\u200a=\u200a48) comprised of those treated in various hospitals in the city. Mean post diagnosis duration for these patients was 4.8\u00b14.2 yrs (Median \u200a=\u200a3), with sixty two percent of the patients recruited within five years of diagnosis. Healthy, unrelated Parsi women (N\u200a=\u200a171) were recruited from the community. Eight Parsi patients as well as ten healthy women volunteers reported a strong family history for cancer.The mean age was lower in healthy Maharashtrian controls than in patients . The available data indicated comparable age at menarche and age at first child birth in the two groups . In ParsTGFB1 T29C polymorphism was performed by PCR\u2013SSP based method, using primers described by Perrey et alInternational Histocompatibility Workshop Group (IHWG) reference panel were used to validate the PCR conditions and as controls in each experiment. Results were further confirmed by DNA sequencing in representative samples. Genomic DNA was amplified using primers, GAGGCCCTCCTACCTTTTG (F) and GCAGCTTGGACAGGATCT (R) as described in the SNP500 cancer database http://snp500cancer.nci.nih.gov/sequencing_assays.cfm?snp_id=TGFB1-01. The amplified products were sequenced by the standard method using big dye terminator kit (ABI) on 3100AVANT Genetic Analyzer (Applied Biosystems).Isolation of DNA for genotyping was carried out as described in our earlier report TGFBR1 6A/9A genotyping, exon 1 of TGFBR1 was amplified using primers described by Kaklamani et al2+, 10% DMSO, 0.1% BSA and 0.5 units of Taq polymerase (Fermentas) along with 1.0 \u00b5M primers. PCR conditions were as follows \u221295\u00b0C for 5 min, 35 cycles of 94\u00b0C for 30 s, 65\u00b0C for 30 s, 72\u00b0C for 30 s, followed by extension at 72\u00b0C for 5 min. PCR products (256/247 bp) were analyzed on 2% agarose gel stained with ethidium bromide. The genotypes were also confirmed by running the products on 10% PAGE and by sequencing of representative samples.For TGFB1*29T and TGFBR1*9A, were taken as reference for the respective polymorphisms. For Maharashtrian subjects, the risk was determined after adjusting for age, and was expressed as odds ratio with 95% confidence interval. In each case, the odds ratios were computed for dominant, additive and recessive models. Further, various combinations of genotypes of these two polymorphisms were categorized into high, intermediate and low signalers TGFB1*CC and TGFBR1*9A/9A genotype combination were identified as high signalers in accordance with the high TGF \u03b2 levels and strong TGFBR1 activity associated with these genotypes. Individuals homozygous for the allele associated with either higher TGF \u03b2 levels or with strong signaling (TGFB1*CC or TGFBR1*9A/9A) were grouped as intermediate signalers, and subjects with the remaining combinations constituted low signalers. The risk was estimated for the latter two groups with high signalers as reference. SPSS software (version 15.0) was used for statistical analysis. Power calculations were performed using Quanto Chi-square test was used to find out if the genotype frequencies in controls are in Hardy-Weinberg equilibrium and also to determine the significance of difference in genotype frequencies between different groups. For various comparisons, two sided p values were calculated using Fisher\u2019s exact test. To estimate the risk for breast cancer, the frequencies of subjects homozygous for the wild type alleles, namely Two hundred and twenty four healthy controls (95%) and one hundred and sixty (88%) premenopausal breast cancer patients from the Maharashtrian community could be genotyped for the two polymorphisms studied. Similarly, one hundred and sixty (94%) healthy Parsis and forty three Parsi patients (95%) could be genotyped and included in the analysis. The genotype frequencies for the two polymorphisms studied were in agreement with Hardy-Weinberg equilibrium in healthy subjects from both the communities.TGFB1*29C) in healthy Maharashtrian women was higher compared to that in healthy Parsi women .The frequency of the variant allele  was noted in Maharashtrian subjects for the dominant model only  and a protective influence of the TGFB1*CC genotype recessively, were not statistically significant  as reported by two groups In Maharashtrian subjects, the observed frequencies for TGFBR1*6A allele in Maharashtrian women compared to that seen in Parsis [2.0% vs 6.8%, respectively; p<0.01; power-67%) is also in contrast to data from Whites, where the frequency of this allele varies between six and eleven percent TGFBR1*6A allele observed in Maharashtrian women in the present study is the lowest reported so far.The strikingly lower frequency of TGFB1*29C allele (45% power), and a reduced risk for the disease especially in the younger age group . These observations are in direct contrast with those from the Breast Cancer Association Consortium TGFBR1*6A allele to the observed effect of the TGFB1*29C allele on the risk for the disease in Maharashtrian women cannot be ruled out.Our findings in Maharashtrian premenopausal women suggest an association between the presence of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian patients. Of the one hundred and twenty two patients where the information was available, the hormone receptor negative tumor bearing subjects were characterized by a strikingly lower frequency of TGFB1*CC genotype compared to the hormone receptor positive group  with adequate power, sixteen thousand (dominant model) and forty six thousand (recessive model) patients and controls would have to be studied TGFB1*T29C polymorphism with the observed allele frequencies in the western Indian population, to detect a 15% change in the risk for breast cancer, nearly four thousand patients and an equal number of healthy controls would be needed to achieve a power of 80% (at \u03b1\u200a=\u200a0.05). Similar computations for TGFBR1*6A allele indicate that for a fifteen percent increment in the risk for breast cancer The associations seen in our study however must be viewed with caution in view of the inadequate power of our study  & 4. TheTGFB1*29C allele and lower frequency of TGFBR1*6A allele in healthy Maharashtrian women was revealed when combinations of these genotypes were analyzed. Categorization of individuals into low, moderate and high TGF \u03b21 signalers based on an approach described by Kaklamani et alvs 10.6%; p<0.01). A comparison of the frequencies of high, intermediate and low signalers among healthy subjects reported in literature  for breast cancer indicated for low and intermediate  in western Indian women suggested by our study and in White women by others In summary, the present study on Figure S1Analysis of association of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian subjects. OR \u2013 Age adjusted odds ratios with 95% CI; n\u200a=\u200a224; **p<0.01; *p\u200a=\u200a0.05 for TGFB1*CC genotype.(TIF)Click here for additional data file.Table S1Distribution of various TGFB1 T29C genotypes with respect to ER/PR status.(DOC)Click here for additional data file."}
+{"text": "Split-hand/foot malformation type 1 is an autosomal dominant condition with reduced penetrance and variable expression. We report three individuals from two families with split-hand/split-foot malformation (SHFM) in whom next generation sequencing was performed to investigate the cause of their phenotype.DYNC1I1 exons recently identified as limb enhancers in mouse studies from their target genes, DLX5 and DLX6. In the second family, X-linked recessive inheritance was suspected and exome sequencing was performed to search for a mutation in the affected proband and his uncle. No coding mutation was found within the SHFM2 locus at Xq26 or elsewhere in the exome, but a 106\u2005kb deletion within the SHFM1 locus was detected through copy number analysis. Genome sequencing of the deletion breakpoints showed that the DLX5 and DLX6 genes are disomic but the putative DYNC1I1 exon 15 and 17 enhancers are deleted.The first proband has a de novo balanced translocation t identified by karyotyping. Whole genome sequencing showed that the chromosome 7 breakpoint is situated within the SHFM1 locus on chromosome 7q21.3. This separates the DYNC1I1 exonic enhancers in normal limb formation in humans.Exome sequencing identified a 106\u2005kb deletion that narrows the SHFM1 critical region from 0.9 to 0.1\u2005Mb and confirms a key role of  DLX5/6); double-knockout mice exhibit severe skeletal abnormalities and die shortly after birth,DLX5 has recently been reported to cause split-hand/foot malformation and hearing loss in a consanguineous family.DLX5/6 coding mutations leading to SHFM1 have been reported to date. Several chromosomal abnormalities have been reported in this region that lead to SHFM1; some do not affect the DLX5/6 genes and are thought to disrupt one or more regulatory elements, or result in a physical separation of such elements from the most likely target genes, DLX5/6. The smallest reported deletion is of 880\u2005kb encompassing SLC25A13 and part of DYNC1I1, but leaving DLX5 and DLX6 intact.Dlx5 and Dlx6 are thought to be regulated by the transcription factor tumour protein p63TP63 mutations cause split-hand/split-foot malformation 4 . An enhancer element with a p63 binding site has been reported within the SHFM1 locus 300\u2005kb proximal to Dlx5/6.3Split-hand/split-foot malformation is a congenital limb abnormality characterised by the absence of one or more median rays or digits that results in cone-shaped clefts of hands and/or feet. It is often accompanied by other limb anomalies including monodactyly, syndactyly, and aplasia or hypoplasia of the phalanges, metacarpals, and metatarsals. The malformation can be isolated or syndromic , OMIM #220600), and the severity can be variable between patients as well as between different limbs of the same individual. The condition is genetically heterogeneous with six loci defined, including the SHFM1 locus 7q21 (OMIM #183600) and SHFM2 locus Xq26 (OMIM #313350). The 7q21 locus contains the candidate SHFM genes distal-less homeobox 5 and 6 , a gene that is not expressed during limb development. DYNC1I1 eExon (exonic enhancer) 15 is marked by an enhancer chromatin signature and physically interacts with the Dlx5/6 promoter regions 900\u2005kb distal to DYNC1I1, specifically in the limb.Slc25a13 (solute carrier family 25 member 13), driving the expression of Dlx5/6 in otic vesicle, forebrain, branchial arch and limb\u2014a pattern that correlates with some but not all SHFM1 phenotypes observed in humans.6Recent studies in mouse and zebrafish models identified novel tissue-specific enhancers that correlate with limb, craniofacial and hearing phenotypes observed in individuals with chromosomal rearrangements.supplementary figure\u00a01A) which showed that the chromosome 7 breakpoint is within 7q21.3, not 7q22. The precise breakpoints were confirmed by Sanger sequencing of PCR products spanning the breakpoints (see online supplementary figure\u00a01B), and the translocation was defined as t. The chromosome 2 breakpoint is located within intron 1 of the ornithine decarboxylase 1 (ODC1) gene encoding the rate-limiting enzyme of the polyamine biosynthesis pathway. A polymorphism in this gene has been associated with colon cancer risk,DLX5/6 genes and 500\u2005kb downstream of the DYNC1I1 exons 15 and 17 identified as exonic enhancers in mice and zebrafish (figure 2).We studied three patients with SHFM from two families. Informed consent was obtained from all participants or their parents. Proband 1 had split-hand/split-foot malformation A affecti tp2.1;q21.3. Biallelic mutations in SLC25A13 cause citrin deficiency, an adult- or neonatal-onset metabolic disorder, while homozygous Slc25a13 knock-out mice show no skeletal defectsDYNC1I1 gene encodes the intermediate chain 1 subunit of the cytoplasmic dynein motor protein complex, the primary motor protein responsible for retrograde axonal transport in neurons. Since this gene is not expressed in the developing limb a partial gene deletion is not predicted to cause split-hand/foot malformation. However, the deleted DYNC1I1 exons include the enhancers identified by Birnbaum et al.supplementary figure\u00a01D). Sanger sequencing of a PCR product spanning the breakpoints confirmed a 105\u2005935bp deletion; NC_000007.13:g.95704812_95810747del (see online supplementary figure\u00a01E). The presence of the junction fragment was confirmed in the two affected individuals and also in four non-penetrant unaffected family members , and genital tissues, and the position of these functional enhancers compared to SHFM1-associated chromosome rearrangements suggests that their disruption might, in some cases, explain the additional clinical phenotypes such as hearing loss and craniofacial defects.SLC25A13 is an enhancer with activity in the otic vesicle , it is possible to accurately resolve translocation or deletion breakpoints. For proband 1 the genome sequencing was more accurate than metaphase chromosome analysis and resulted in a reassignment of the 7q breakpoint to the neighbouring chromosome band. The application of next generation sequencing for precise characterisation of breakpoints is likely to find clinical utility as it can show whether the coding region of a candidate disease gene is disrupted or, as in this study, identify the spatial relationship between regulatory elements and their target genes.et al21The use of paired-end whole genome sequencing to map rearrangement breakpoints to the exact nucleotide has previously been described for just seven patients.In summary, by using exome sequencing copy number analysis and whole genome sequencing to map deletion and translocation breakpoints, our study demonstrates that exonic enhancers recently discovered through mouse and zebrafish models are also critical for limb development in humans."}
+{"text": "This phenotype has precluded analysis of Bmp7 function in the later stage of nephrogenesis. In this study, utilization of conditional null allele of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at desired time points during kidney development, and to discover the novel function of Bmp7 to inhibit the precocious differentiation of the progenitor cells to nephron. Systemic knockout of Bmp7 in vivo after the initiation of kidney development results in the precocious differentiation of the kidney progenitor cells to nephron, in addition to the prominent apoptosis of progenitor cells. We also confirmed that in vitro knockout of Bmp7 in kidney explant culture results in the accelerated differentiation of progenitor population. Finally we utilized colony-forming assays and demonstrated that Bmp7 inhibits epithelialization and differentiation of the kidney progenitor cells. These results indicate that the function of Bmp7 to inhibit the precocious differentiation of the progenitor cells together with its anti-apoptotic effect on progenitor cells coordinately maintains renal progenitor pool in undifferentiated status, and determines the nephron number at birth.The number of nephrons, the functional units of the kidney, varies among individuals. A low nephron number at birth is associated with a risk of hypertension and the progression of renal insufficiency. The molecular mechanisms determining nephron number during embryogenesis have not yet been clarified. Germline knockout of  Over the last decade, the impacts of prenatal conditions on health and disease in later life have been subjects of intense research The embryonic kidney is derived from the intermediate mesoderm and formed by a reciprocal induction between the cap mesenchyme and ureteric bud Bmp7 knockout mice exhibited aplastic kidneys with a few nephrons, leading to perinatal death Bone morphogenetic protein 7 (Bmp7) is a morphogen expressed in the kidney. Germline Bmp7 in the developing kidney were analyzed utilizing +/LacZBmp7 mice Bmp7 is widely expressed in cap mesenchyme, ureteric buds, and some derivatives of cap mesenchyme, podocytes and distal tubules. Given that various types of cells in the developing kidney express Bmp7, coupled with the fact that Bmp7 is a secreted factor, we surmised that the deletion of Bmp7 in any specific cell type might not result in a prominent phenotype. We therefore opted for ubiquitous global ablation of Bmp7 at desired time points as a strategy to define its role in kidney maturation.The expression domains of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at different time points during kidney development. While the knockout of Bmp7 before the initiation of kidney development recapitulates germline Bmp7 knockout mice , the knockout of Bmp7 after the initiation of kidney development allowed us to demonstrate the novel phenotype, precocious differentiation of progenitor cells. We also confirmed that Bmp7 inhibits differentiation of renal progenitor cells utilizing kidney explant culture and colony-forming assay. Taken together, we demonstrated for the first time that Bmp7 maintains nephron progenitor cells in the undifferentiated state, thereby influencing the number of nephrons at birth.In our study, the utilization of the conditional null allele of Bmp7 conditional knockout mice Cre (CreERT2Gt(ROSA)26Sor) mice. We bred +/LacZBmp7;CreERT2Gt(ROSA)26Sor mice with fl/flBmp7 mice to yield LacZ/flBmp7;CreERT2Gt(ROSA)26Sor (Bmp7 knockout) and +/flBmp7;CreERT2Gt(ROSA)26Sor (control) embryos. We knocked down Bmp7 expression through the administration of tamoxifen to pregnant mothers bearing both types of embryos. Because of the toxicity of the systemic expression of CreERT2 on hematological tissues in CreERT2Gt(ROSA)26Sor mice Bmp7 knockout and control embryos carry CreERT2.To analyze the role of Bmp7 in kidney development, we utilized Bmp7 in the kidney was approximately 80% (2). While the proliferation of metanephric mesenchyme was similar between the two genotypes (Bmp7 knockout kidneys (We administered tamoxifen to pregnant mice at embryonic day 12.5 (E12.5), a time when the epithelialization of cap mesenchyme has begun, and analyzed the embryos at E14.5 . Histological analysis revealed a significant reduction in the volume of cap mesenchyme in knockout kidneys (2), which was also confirmed by immunostaining of WT1, a marker for cap mesenchyme (and podocytes) (2). The proliferation of knockout kidneys appeared to be decreased 26SorCreERT2Bmp7 embryos at E12.5 and cultured for 72h in the presence or absence of 4-hydroxytamoxifen (4-OHT). The phenotypes of the explants treated with 4-OHT (knockout explants) were compared to those of contralateral explants treated with a vehicle (ethanol) (control explants). The administration of 4-OHT to wild-type kidney explants did not cause any noticeable changes (data not shown). The knockout efficiency of Bmp7 was approximately 90% in knockout explants .The epithelialization and subsequent differentiation of cap mensenchyme is the first step of nephrogenesis. We hypothesized that the precocious maturation of nephron segments in explants Figure 3We also investigated the effect of Bmp7 on the epithelialization of cap mesenchyme using a colony-forming assay, in which the coculture of single cells from cap mesenchymes of E11.5 kidneys with Wnt4 expressing feeder cells (3T3Wnt4) The addition of Bmp7 to this culture system significantly inhibited colony formation in a dose-dependent manner  of metanephric mesenchyme caused by the reduction of endogenous Bmp7, which is also supported by the reduction of Pax2. What we observed in conditional knockout mice, kidney explants and colony assay in this manuscript is the inhibitory effect of Bmp7 on the epithelialization of the induced metanephric mesenchyme, which is the next step after the induction of mesenchyme.Bmp7 expression at E12.5 permits the initial expansion of progenitor cells and enables us to observe the precocious differentiation of progenitor cells in the absence of Bmp7. This phenotype is novel, and could not be observed in germline Bmp7 null mice due to massive apoptosis of metanephric mesenchyme.The phenotype of germline knockout mice is the massive apoptosis of renal progenitor cells, which precludes the analysis of Bmp7 function on progenitor cell differentiation Bmp7 knockout kidneys is \u201cearlier\u201d or \u201cfaster\u201d remains unproven. However, the results of kidney explants and colony-forming assay showed Bmp7 inhibits epithelialization of mesenchyme, which is the early step of differentiation, and hence, at least, the differentiation in Bmp7 knockout kidneys seems to occur \u201cearlier\u201d, while \u201cfaster\u201d differentiation cannot be denied.Whether the differentiation in During tissue development, tissue-specific progenitor cells divide and populate the progenitor pool. The size of the progenitor pool as well as the final size of the tissues is determined by the balance between proliferation, apoptosis and differentiation of progenitor cells. Both massive apoptosis and precocious differentiation of progenitor cells causes depletion of the progenitor cells and reduces the final size of the tissues , indicating the possible contribution of Bmp7-Smad signaling pathway in the maintenance of renal progenitors. More recently, the maintenance of germ line stem cells by Bmp7 has been demonstrated in mouse embryos as well Interestingly, the maintenance of stem/progenitor cells by Bmp may be an evolutionally conserved mechanism in different tissue contexts. It is widely accepted that orthologues of Bmps, Decapentaplegic (Dpp) and Glass bottom boat (Gbb) maintain germ line stem cells via the phosphorylation of Mad, a homologue of Smad proteins in drosophila ovary and testis Bmp7 knockout mice: maternal, extrarenal, and intrarenal Bmp7. Previous reports demonstrated that maternal Bmp7 traverses the placental barrier and localizes in embryonic kidneys until day 14 of gestation +/LacZ;Gt(ROSA)26SorCreERT2Bmp7or fl/flBmp7, such that the amount of Bmp7 originating from the mother is unaltered by the administration of tamoxifen, excluding the possibility that the fluctuation of maternal Bmp7 levels contributes to the phenotypes in Bmp7 knockout embryos. Given the nature of systemic inducible CreERT2Gt(ROSA)26Sormice, the expression of Bmp7 in other tissues was also reduced after tamoxifen treatment, so that the circulating level of Bmp7 (if present) should be lowered. This might contribute to the phenotypes in Bmp7 knockout embryos to some extent. However, the accelerated differentiation of cap mesenchyme was observed in kidney explant culture, in which the ablation of Bmp7 occurs intrinsically within the kidney, indicating that intrarenal Bmp7 is primarily responsible for the phenotypes.There are several possible sources of Bmp7 that contribute to the phenotype in Bmp7 in distinct cell types manifests different spectrums of defects in the kidney. Thus, the loss of Bmp7 from podocytes results in the reduction of proximal tubules in postnatal kidneys Bmp7 knockout mice (this study) exhibits the accelerated maturation of nephrons. These comparisons indicate that there is a broad range of functions for Bmp7 in different cell types at different stages, and raise the possibility that Bmp7 secreted from one cell population functions locally in an autocrine or paracrine fashion to exert a unique function. This hypothesis is strengthened by a previous report indicating that the prodomain of Bmp7 tethers Bmp7 to the extracellular matrix (ECM) near the site of Bmp7 production and inhibits its passive diffusion Interestingly, the inactivation of Finally, our analysis demonstrates for the first time that Bmp7 plays a critical role in the maintenance of the renal progenitor population in the developing kidney, and thereby determines the final number of nephrons. Further analysis will elucidate the mechanism how intrauterine conditions affect the pathway, and provide a rationale for a therapeutic approach to prevent congenital deficit in nephron number.All animal studies were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University (permission number: MedKyo 12522), and were strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were sacrificed by cervical dislocation by well-trained researchers.Bmp7 conditional allele CreERT2Gt(ROSA)26Sor allele were generated at Regeneron Pharmaceuticals using Velocigene\u2122 technology as described elsewhere +/LacZBmp7 knock-in allele has been described previously +/LacZ;Gt(ROSA)26SorCreERT2Bmp7 mice with fl/flBmp7 mice to yield LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 (Bmp7 knockout) and +/fl;Gt(ROSA)26SorCreERT2Bmp7 (control) embryos. Due to the hematological toxicity of CreERT2Gt(ROSA)26Sor mice CreERT2 allele.The LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 and +/fl;Gt(ROSA)26SorCreERT2Bmp7 embryos.Tamoxifen  was dissolved in a sunflower oil/ethanol (9:1) mixture at 30 mg/ml. For activation of CreERT2, the tamoxifen solution was administered at a concentration of 150 mg/kg by intraperitoneal injection to pregnant mothers bearing The kidneys were fixed in Carnoy\u2019s Solution, and embedded in paraffin. Sections (2 \u00b5m thick) were sliced at every 100 \u00b5m (the diameter of any glomeruli was shorter than 100 \u00b5m) and stained with periodic acid-Schiff (PAS) for routine histological examination. Frozen sections of the kidneys were immunostained as previously described Maturation of glomeruli was analyzed in five (E14.5) or three (E18.5) serial sections  at every 100 \u00b5m of the embryonic kidneys for 7 control embryos and 5 knockout embryos at E14.5, and 4 control embryos and 6 knockout embryos at E18.5.The criteria for the assessment of glomerular maturation are based on the previous publications We counted the number of glomeruli in each stage in the serial sections and showed the sum number in Scoring of proximal tubule cross-sections was performed according to the previous publication Real-time reverse transcription (RT) polymerase chain reaction (PCR) was performed as described previously GAPDH, CCAGAACATCATCCCTGCATC; CCTGCTTCACCACCTTCTTGA,Bmp7, TGTGGCAGAAAACAGCAGCA; TCAGGTGCAATGATCCAGTCCGAPDH gene.Serially diluted cDNA was used to generate the standard curve for each primer, and the PCR conditions were as follows: 50\u00b0C for 2 min, 95\u00b0C for 10 min, then 95\u00b0C for 15 sec, and 60\u00b0C for 1 min for 40 cycles. The results were normalized with the amount of 2.Kidney explant culture was performed as previously described Bmp7 in vitro, one side of E12.5 embryonic kidneys from LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 embryos was treated with 1 \u00b5M 4-OHT (Sigma) dissolved in ethanol, while contralateral kidney was treated with vehicle (ethanol), and cultured for 72 h. Neither LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 explants treated with the vehicle or +/fl;Gt(ROSA)26SorCreERT2Bmp7 explants treated with 4-OHT exhibited any morphological changes. To cancel the effect of individual difference between embryos, the results of LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7kidneys treated with the vehicle were shown as controls in comparison with contralateral kidneys from the same embryos treated with 4-OHT. For immunostaining, cultured kidney explants were fixed in 4% paraformaldehyde, quenched with 50 mM NH4Cl, permeabilized by 0.1% triton X, and stained as previously described To knockout To inhibit Smad signaling in kidney explants, one side of E12.5 embryonic kidneys from wild-type mice were treated with 0.5 \u00b5M dorsomorphin  dissolved in DMSO, while contralateral kidney was treated with vehicle (DMSO), and cultured for 48 h.Immunoblotting was performed as described previously 3 cells/well in 12-well plates and treated with recombinant Bmp7  with or without recombinant Noggin-Fc chimera (R&D systems Inc.) 24 h later. After 7 days of culture, the number of colonies was counted.A colony-forming assay was performed as described previously t-test for two group comparisons, and the ANOVA for more than three.All assays were performed at least three times. Data are presented as the mean \u00b1 standard deviation (SD). Statistical significance was assessed by Student\u2019s Figure S1Systemic knockout of Bmp7 at E10.5 recapitulates the phenotypes of germline Bmp7 knockout mice. Pregnant mothers bearing both Bmp7+/fl;Gt(ROSA)26SorCreERT2 and Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos were administered tamoxifen at E10.5, and sacrificed at E16.5. The Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 (Bmp7 knockout) kidney was smaller and exhibited severe reduction of cap mesenchyme. Scale bars: 100 \u00b5m.(TIF)Click here for additional data file.Figure S2Volume of kidneys and mesenchyme is still maintained in Bmp7 knockout embryos at E14.5, but reduces at E18.5 (related to  and ). The maximum horizontal sectional area of kidneys and the thickness of nephrogenic zone, WT1-positive area were not different between Bmp7 knockout embryos (white column) and controls (black column) at E14.5, but significantly reduced in knockout embryos at E18.5. The mean of the values \u00b1 SD is presented in the graphs  n.s.: not significant.(TIF)Click here for additional data file.Figure S3Accerelated maturation of distal tubules in Bmp7 knockout kidneys at E18.5 (related to). (A) Kidneys were stained with nephrin (green) and THP (red) to label glomeruli and distaltubules, respectively. The volume of THP-positive distal tubule sections in Bmp7 knockout kidneys was comparable to the control kidneys, whereas the number of glomeruli was significantly reduced. (B) The number of THP+ distal tubule cross sections normalized by the number of nephrin+ glomeruli tended to increase in knockout kidneys (white column) compared to control kidneys (black column). Data are represented as mean \u00b1 SD. Three sections were stained for each kidney. The sum of the number of THP-positive distal tubule cross sections was divided by the sum of the number of nephrin-positive glomeruli. The mean of the values from five (control) or six (knockout) embryos is presented in the graph. Scale bars: 100 \u00b5m. n.s.: not significant.(TIF)Click here for additional data file.Figure S4The branching of ureteric buds tends to decrease in Bmp7 knockout kidneys (related to ). Kidney explants were taken from Bmp7+/fl;Gt(ROSA)26SorCreERT2 and Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos at E11.5 and cultured for 48 h with or without 4-OHT. In Bmp7+/fl;Gt(ROSA)26SorCreERT2 embryos, the number of ureteric bud tips of tamoxifen-treated explants was almost equal to vehicle-treated explants. In Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos, although the difference was not significant, the number of ureteric bud tips of tamoxifen-treated (Bmp7 knockout) explants tended to decrease compared to vehicle-treated explants. Data are represented as mean \u00b1 SD (n\u200a=\u200a4). n.s.: not significant.(TIF)Click here for additional data file.Figure S5Smad signaling inhibits the differentiation of cap mesenchyme in the kidney explant culture (related to). Kidney explants were taken from wild-type mice at E12.5 and cultured for 48 h in the presence or absence of a Smad1/5/8 inhibitor, dorsomorphin. In explants treated with dorsomorphin, Jagged1-positive regions were significantly expanded. Scale bars: 100 \u00b5m. Data are represented as mean \u00b1 SD (n\u200a=\u200a8). Immunoblotting of the lysates of kidney explants demonstrated the phosphorylation of Smad1/5/8 was decreased in dorsomorphin-treated explants. Ten micrograms of kidney explants lysate was loaded in each lane. As a positive control, primary kidney cells were stimulated with 100 ng/ml Bmp7 for 1 h. GAPDH was used as a loading control. pSmad1/5/8 denotes phospho-Smad1/5/8.(TIF)Click here for additional data file."}
+{"text": "Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates.Although rapid diagnostic tests (RDTs) for Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle.Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed.pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.The levels of transcription of  The ability to accurately diagnose malaria infections is critical to the control and elimination of this disease Plasmodium falciparum histidine-rich protein 2 (PfHRP2) that circulates in the bloodstream of the patient Many of the RDTs marketed to diagnose malaria target Several factors may contribute to the variable sensitivity reported for PfHRP2-detecting RDTs. These include genetic variation of PfHRP2 between parasites. We have previously shown that while genetic polymorphism in PfHRP2 is extensive, it does not appear to affect RDT detection sensitivity at levels > 200 P/uL P. falciparum. It is encoded by pfhrp2, a subtelomeric gene located on chromosome 7 PfHRP2 is a 60-105 kDa water-soluble protein specific to s-tRNA syn or tRNA) and MAL13P1.209 60S ribosomal subunit protein L18 , are transcribed at a relatively constant rate across the asexual lifecycle of the 3D7 falciparum strain The parasite expresses most of its genes as it invades and develops within the erythrocyte, with at least 60% of the genome transcriptionally active during the intraerythrocytic asexual cycle pfhrp2 and pfhrp3 across the asexual cycle A plausible hypothesis is that the amount of PfHRP produced by the parasite varies between different isolates, and as a consequence different amounts of this target protein would be available for detection by RDTs. PfHPR2 production and release has been characterised in a limited number of strains during the intraerythrocytic life cycle pfhrp2 and pfhrp3, and of abundance of PfHRP protein, in different parasite strains, at several time points over the blood stage life cycle. Further, the potential impact that the variation in protein expression has on RDT detection sensitivity is investigated. Defining reasons for RDT failure is of significant public health importance and will contribute to improving diagnostics for falciparum malaria.In this paper, we report the pattern of transcription of in vitro\u00ae Separation Column (Miltenyi Biotec USA) pfhrp2 and pfhrp3 through the erythrocytic life cycle  in RNase-free tubes then frozen at \u221280\u00b0C. For protein isolation, culture supernatant was removed and red cell pellets were frozen at \u221280\u00b0C until protein extraction.Cryopreserved parasites originating from varied geographic areas  were culfe cycle , where sfe cycle  were useFor each sample, the number and proportion of ring-stage parasites was determined by microscopically counting 500 infected erythrocytes. No gametocytes were observed in these cultures.Total RNA was isolated from each parasite pellet using the NucleoSpin\u00ae RNA II Kit (Macherey-Nagel Germany), following the manufacturer's instructions. A second elution step was added to maximise yield as previously described pfhrp2 fragment alignment of P. falciparum 3D7 (GenBank accession number BM275665), FCBR (GenBank accession X69922) and ITG2 (GenBank accession U69551). The forward primers for both pfhrp2 and pfhrp3 were designed to span both exon 1 and part of exon 2, with the reverse primer binding in exon 2. Primers for pfhrp3 were designed using an alignment of ITG2 (GenBank accession U69552) and FCC1/HN (GenBank accession AF202093). Primer sequences are shown in Total RNA was reverse-transcribed into cDNA using SuperScript\u2122 III Reverse Transcriptase (Invitrogen USA) and random hexamers. The real-time PCR primers were designed based on a pfhrp2 and pfhrp3 were optimised using P. falciparum 3D7 cDNA. The use of 150 nM primers per reaction and a 15 minute enzyme activation period on the MX4000 (Stratagene USA) gave the best reproducibility, and were used in all real-time PCR experiments for this study. Two housekeeping genes, PF07_0073 seryl tRNA synthase (s-tRNA syn or trna) gene and MAL13P1.209 , the 60S ribosomal subunit protein L18 were used as endogenous control transcripts to normalize mRNA levels Real-time PCR conditions for pfhrp2 gave a melting temperature at 76.7\u00b0C while for pfhrp3 it was 77.8\u00b0C. Experiments where non-specific peaks in relation to the dissociation curves were observed were repeated. The mean Ct was determined and used in the \u0394Ct method pfhrp2 and pfhrp3 relative to s-tRNA syn and mal13.All samples were run in triplicate using the ABsolute QPCR SYBR Green Mix (ABGene United Kingdom) according to the manufacturer's instructions. The following cycling conditions were used: 15 minutes at 95\u00b0C for initial denaturation and enzyme activation, followed by 40 cycles of 95\u00b0C for 30 seconds, 57\u00b0C for 40 seconds, and 72\u00b0C for 40 seconds, followed by a final extension of 57\u00b0C for 1 minute. The dissociation curve for Pelleted infected red blood cells were thawed and resuspended in 1% Triton X-100 diluted with phosphate-buffered saline (PBS) and 1/1000 volume of a cocktail of proteinase inhibitors (Thermo Scientific USA). Samples were then frozen (\u221280\u00b0C) and thawed three times, with vortexing every 15 minutes. After protein solubilization the solution was spun at 15 000 rpm for 30 minutes at 4\u00b0C and the supernatant removed for use in protein experiments.A 40 \u00b5L supernatant aliquot was diluted 1\u22365 with PBS-T  to reduce interference from residual hemoglobin. One third volume of 3\u00d7SDS sample loading buffer was added to the extracts, and boiled for 10 minutes prior to loading on an SDS-page gel , run for 1 hour at 60 V then 1.5 hour at 150 V. Proteins were then transferred from the SDS-page gel to a 0.45 \u00b5m PVDF membrane (Invitrogen USA) at 100 V for 1.5 hours. Following transfer, the membrane was blocked overnight in 5% skim milk powder dissolved in 100mL PBS-T. The membrane was then incubated with the primary antibody, PTL3  at 1\u22365000 dilution. Following washing in PBS-T, the membrane was incubated with an anti-mouse polyvalent immunoglobulin-alkaline phosphatase conjugate (Sigma USA) at 1\u22365000 dilution. The signal was detected by CDP Star chemiluminescent substrate (Roche Germany) and exposed to imaging films.A PfHRP2 quantitative antigen-capture ELISA  was used according to the manufacturer's instructions. Positive and negative control wells were included to quantitate protein levels for all samples. 3D7 culture supernatant containing a known concentration of PfHRP2 was used for standard curve construction. All samples were tested as undiluted, 1/100, 1/200 and 1/300 dilutions and run in duplicate. Absorbance was measured at 450 nm and 650 nm using a microplate spectrophotometer (SpectraMax Molecular Devices USA).trna transcription value to account for potential differences in the number of parasites in the sample. The trna values from the transcript dataset for the K1 parasite were then used for this normalisation: normalised PfHRP2sample parasite\u200a=\u200aPfHRP2sample parasite\u00d7(trnasample parasite/trnaK1).The software package SoftMax Pro software (Molecular Devices USA) was employed to quantify the protein levels in ng/mL. Samples whose concentration fell outside the standard curve were further diluted and the test repeated. Calculated PfHRP2 levels were then normalised against the samples own To investigate whether differences in PfHRP protein level results in a difference in the detection limit on RDTs, the ring-stage protein sample (80\u2013100% ring) from parasite strains K1, D6 and Hb3 were tested without dilution, then at 1/100, 1/200, and 1/300 dilutions in 1\u00d7PBS, on three RDTs .The experiment was then repeated using the same number of intact parasitized red blood cells of the three parasite lines at seven doubling dilutions  at 50% hematocrit. The RDTs were read using the WHO Colour Intensity chart for RDTs with a scale of 0-4, based on the intensity of the band colour, 4 being the strongest band colour, 3 moderate colour, 2 weak colour, 1 faint colour, and 0 negative .Spearman Rank Correlation using GraphPad PRISM software  was applied to assess the relationship between transcript and parasite stage, protein and parasite stage, and the relationship between transcription and expression.P. falciparum to be characterised in this work have different asexual replication times. To compare levels of transcription of pfhrp2 and pfhrp3 at the well-recognized asexual stages, we grouped parasites based on the proportion that were ring-stage at different times across the lifecycle. For 5 of the 7 lines studied . A significant positive correlation was observed between the proportion of rings and the level of transcription for both pfhrp2 and pfhrp3 (P < 0.01)  . This wanst trna  or againa , K1 and GA3 (1.0 vs 0.3), and moderate between SJ44 and N70 (1.1 vs 0.9), FCQ33 and FCQ41 (0.5 vs 0.3).Levels of ainst K1 . The ratPfhrp3 is absent in the P. falciparum laboratory clone Hb3 pfhrp2 in this parasite line was lower compared to all other strains tested. In other words, no compensatory increase in pfhrp2 transcription was observed when pfhrp3 was absent. Similarly, the parasite line Dd2 lacks pfhrp2pfhrp3 in this line was the lowest of those tested with no compensatory increase to account for the lack of pfhrp2.Monoclonal antibodies raised against PfHRP2 have been shown to cross-react with PfHRP3 pfhrp2 sequence were compared at predominant ring stage (Group 1), a 1\u20134 fold difference in protein levels was observed between the paired strains . K1 returned strong positive results of 4 for all dilutions except the final dilution where the band intensity was slightly weaker at a score of 3. Hb3 worked only weakly until the second dilution, 10 000 P/\u00b5L with a score of 2.pfhrp2 and pfhrp3 genes, and the levels of the corresponding protein PfHRP, vary between different parasite strains, and whether these variations influence the detection sensitivity of malaria RDTs. Our data indicate that pfhrp2 and pfhrp3 transcription is highest at the ring stage of the intraerythrocytic life cycle, a finding that agrees with the transcription profiles for these genes using microarray analysis for the 3D7 strain which is available in the PlasmoDB database pfhrp2 vary widely between geographically variant strains of the parasite, even for strains with the same pfhrp2 sequence and also between strains with different pfhrp2 sequence.The purpose of this study was to investigate whether the transcription of the PlasmodiaControl of transcription is believed to be under the influence of various regulatory systems, many of which are incompletely understood in pfhrp2, or even identical coding sequences may differ in their 5\u2032 UTR and 3\u2032 UTR sequences, resulting in different levels of transcription.It is clear that transcription patterns vary between genetically distinct parasites pfhrp2 and pfhrp3 transcription.Epigenetic mechanisms may also significantly affect transcription pfhrp2 and pfhrp3 transcripts and PfHRP protein measured in this study constitute the amounts of transcript or protein present at that timepoint, reflecting the combined result of transcription and degradation of the target. For transcripts, post-transcriptional control mechanisms including mRNA stability and decay may contribute significantly to the variation in the amount of transcripts present pfhrp3 may play a role in mRNA stability It should be noted that the levels of pfhrp2 transcripts was always higher than that of pfhrp3. This could be a consequence of the promoter for pfhrp2 being stronger, or as a result of a slower decay of pfhrp2 transcripts. A 5\u2032 flanking region of pfhrp3 has been extensively used in transfection studies as a promoter for reporter gene expression, paired with a 3\u2032 flanking region of pfhrp2 as a terminator sequence pfhrp2 promoter for transfection, thus precluding a comparison of the two promoters. In an earlier study, levels of PfHRP2 to PfHRP3 were compared using immunochemical methods, with results indicating that PfHRP3 protein levels were lower than PfHRP2 In all strains tested in this study, we observed that the level of pfhrp2 and pfhrp3 genes are structurally very similar, with regions flanking the tandem repeats, including untranslated regions, showing up to 90% homology pfhrp2 and pfhrp3 genes encode the histidine-rich amino acid repeats beginning 75\u201390 nucleotides downstream from the start pfhrp2 transcription was lower when pfhrp3 was absent and vice versa. If the products of these two genes have a similar function, it would be expected that one would compensate for the absence of the other. From our data, it may be suggested that these genes have a non-overlapping function. pfhrp2 and pfhrp3 are not linked, occurring as single copy genes on separate chromosomes The The variation in transcription we observed was also reflected by differences in the protein expression levels. Although PfHRP protein expression was more constant throughout the intraerythrocytic life cycle in 5 lines tested, wide variations in the level of protein were observed between 16 strains at ring stage, the stage circulating in patient blood, with up to a 24 fold difference observed between strains. The results are in agreement with RDT results observed using patient samples at 200 P/\u00b5L recently reported from Colombia Importantly, we have shown that parasite strains with higher protein levels have a lower detection limit on commercial RDTs compared to those with lower protein levels. The same phenomenon was observed when the number of parasites was controlled. Although the quantity of HRPs in D6 was less than that in K1, RDTs used to test these lines were strongly positive. When compared to the Hb3 result, which had a low protein level and was not detected at low concentrations, a threshold is observed, below which the level of protein affects RDT sensitivity. Parasites with low levels of protein will be difficult to detect at low parasitemia (\u2264200 P/\u00b5L). This provides further evidence that the level of PfHRP protein is a major determinant of the detection sensitivity of RDTs, and therefore that such variations in protein abundance could contribute to variation in the performance of PfHRP2- detecting RDTs. This consideration is particularly important for testing of patients at low parasitemia, where parasites with lower protein levels may return negative results on malaria RDTs. Likewise, low parasite protein expression may also explain why some RDTs return a negative result at relatively high (>1 000 P/\u00b5L) parasitemia levels. An increased understanding of the factors influencing RDT performance will likely help to improve the performance and use of such malaria RDTs that are of significant public health importance.in vitro culture conditions. Although neither pfhrp2 nor pfhrp3 genes were deleted during culture adaptation, it is unknown whether levels of transcription of these genes, and expression levels of the encoded proteins were affected during the adaptation. Further work using these methods to quantify transcription and expression on additional parasite isolates, and especially testing of field isolates, to determine the extent of variation, and the proportion of parasite isolates that produce low level HRPs would strengthen this observation. The impact of variation in protein level on RDT detection sensitivity in patients also requires further study. Important experimental constraints impede the measurement of PfHRP in field samples. These include the lack of synchrony and inability to control parasitemia levels.It should be pointed out that the parasite lines used in this study have all been adapted to pfhrp2 and pfhrp3 and expression of PfHRP protein levels varies between laboratory strains of the parasite, and demonstrated that the variation in protein levels results in differences in RDT detection thresholds. The outcome of this study provides a possible explanation for reported variation in sensitivity of PfHRP2-detecting RDTs and will assist research aimed at improving malaria RDTs.We have shown here that transcription of Figure S1Transcription of pfhrp2 (A) and pfhrp3 (B) normalised to mal 13 over the intraerythrocytic life cycle (with varying proportion of ring stage).(TIF)Click here for additional data file.Figure S2Western Blot of PfHRP2 for 5 time points, D6 line.(TIF)Click here for additional data file.Figure S3Plot of PfHRP expression level against the proportion of ring stage parasites.(TIF)Click here for additional data file."}
+{"text": "Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from  Bacillus thuringiensis (Bt) kill some major insect pests, but are harmless to vertebrates and most other organisms  X 100%. Because potency is inversely related to LC50, we calculated the potency ratio for modified toxins relative to native toxins as the LC50 of a native toxin divided by the LC50 for a modified toxin [ We calculated resistance ratios as the LCed toxin . We repo For each insect strain, we used the bioassay method described above to test Cry1AbMod, Cry1Ac, and Cry2Ab singly, in pairs, and in a trio, with an aggregate total of 18 sets of concentrations including the control with untreated diet  and 4. O We evaluated potential antagonism or synergism by testing for deviation from the null hypothesis of simple independent action , which a(ab)EXP = S(a)OBS X S(b)OBS(1) S(ab)EXP is the proportion of larvae expected to survive exposure to a combination of toxins a and b, S(a)OBS is the observed proportion of larvae that survived exposure to toxin a, and S(b)OBS is the observed proportion of larvae that survived exposure to toxin b. We calculated expected mortality for larvae exposed to the combination of toxins a and b as (1 - S(ab)EXP) X 100%. We applied the same approach to test for synergism among three toxins: where S(abc)EXP = S(a)OBS X S(b)OBS X S(c)OBS(2) S(abc)EXP is the proportion of larvae expected to survive exposure to a mixture of toxins a, b, and c.where S To calculate expected survival, we first calculated the observed adjusted survival for each toxin tested singly as survival on treated diet divided by survival on untreated diet (control). Survival on untreated diet ranged from 87 to 93% (mean = 90%). All of the results reported for treated diet are based on adjusted survival. 50 of Cry1AbMod). Accordingly, for this exceptional case, we used the mean adjusted observed survival (55%) from the three trials at this concentration conducted in February, July, and September 2010. This is a conservative approach for evaluating synergism, because the high survival (89%) in April 2010 would yield higher expected survival when Cry1AbMod was tested in combination with one or both of the other two toxins (Cry1Ac and Cry2Ab). By excluding the anomalously high survival estimate, we made it less likely that we would conclude synergism occurred in tests with Cry1AbMod and more likely that we would conclude antagonism occurred with tests with Cry1AbMod. The susceptible strain was tested in February 2010 and both the susceptible and resistant strains were tested simultaneously in April 2010. On each date, the sample size was 30 larvae per concentration set for each strain for each of the 18 sets of concentrations . For the susceptible strain, the results for each of three toxins  tested singly at each of two concentrations were similar between dates  in five of six cases (3 toxins X 2 concentrations per toxin). For these five cases, we pooled the data across the two dates to calculate observed adjusted survival for each toxin tested singly. In the exceptional case, which was the susceptible strain tested at 1 \u00b5g Cry1AbMod per ml diet, adjusted survival was 50% in February 2010 vs. 89% in April 2010. The 89% survival is anomalously high, because survival ranged from 50 to 59% (mean = 55%) in the three other independent tests at this concentration . For each of the 22 combinations of toxin concentrations tested (11 combinations tested per strain X 2 strains), we used Fisher's exact test  with 2-tailed probability to determine if a significant difference occurred between the observed and expected numbers of dead and live larvae. For each toxin combination tested against each strain, we also pooled the data across the different concentration sets before performing Fisher's exact test. For example, for BX-R versus Cry1AbMod + Cry1Ac, we pooled the data from the four concentration sets tested (n = 120 for observed response and n = 240 for expected response). This approach increases statistical power and determines if a consistent deviation from independent action occurred across the entire set of toxin concentrations tested for each strain for each toxin combination."}
+{"text": "Neutralizing antibodies are an important component of the humoral immune response directed against viral infections So far the few available anti HIV-1 broadly neutralizing antibodies have a limited breadth and potency against clade C viruses. More than 50% of the HIV-1 infections worldwide belong to clade C. Clade C is the most prevalent subtype in India.A human scFv phage display library was constructed from the peripheral blood mononuclear cells of an HIV-1 Clade C infected Indian patient, having a good titre of serum neutralizing antibodies. Diversity of the scFv library was checked by sequencing and DNAfingerprinting analysis of randomly selected clones from the preselected library. One round of biopanning was done against V3 peptide of clade C and clade B. Single chain fragments were checked for their soluble expression in E. coli HB2151 and purfied using Ni+2 affinity colums. Specificity of the soluble scFvs was checked by indirect ELISA. Single chain fragments were checked for their stability in different agents like 30%DMSO. 4M NaCl, pH 2-11.50 clones were randomly selected after biopanning and they were checked for their binding to V3C and V3B peptides. In phage ELISA 15 out of 50 clones showed binding to both the V3 peptides. A 32kDa band was observed in polyacrylamide gel electrophoresis as predicted. The expressed product was confirmed by Western blot analysis using anti His Tag antibody. Specificity of the purified scFvs was confirmed by their binding to V3 peptides and no reactivity against other unrelated peptides. Further these scFvs displayed a stable binding to V3 peptides in different denaturing agents.This is the first study to generate human anti-V3 scFvs against HIV-1 clade C. Further characterization of these scFvs for their neutralization potential will help identify unique and shared epitopes responsible for neutralization of clade C and non clade C viruses."}
+{"text": "HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide). Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100%) conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2.The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was significantly higher in contrast to its conservancy in HCV genotype 1 and 2. Despite of the lower conservancy in genotype 1 and 2, all the predicted epitopes have important implications in diagnostics as well as CTL-based rational vaccine design, effective for most population of the world and especially the Pakistani Population. Flavivirus, Pestivirus, and Hepacivirus. Members of these genera cause various diseases in humans and other animals such as birds, horses and pigs. The only genera Flavivirus contain more than 70 members including Hepatitis C Virus (HCV), Dengue virus, West Nile virus and tick-borne encephalitis virus . The number of individual bases in the genome i.e. the number of adenine; cytosine, guanine and thymine were calculated from DDBJ database. The molecular weight of proteins, percentage of highly repeated amino acid and the least repeated amino acid in the viral protein was calculated by using sequence and search analysis tool at PIR database  \u00d7 A(2) \u00d7 C(3) \u00d7 D(4) \u00d7 P(5) \u00d7 G(6) \u00d7 R(7) \u00d7 A(8) \u00d7 A(9)Score = P(1) + A(2) + C(3) + D(4) + P(5) + G(6) + R(7) + A(8) + A(9)Where P (1) is score of P at position 1.Only the promiscuous epitopes with score higher than the chosen threshold score were assigned as predicted epitopes for the selected HLA alleles . For thehttp://tools.immuneepitope.org/tools/conservancy/iedb_input). All the epitopes having 77-100% conservancy were selected while rejecting the epitopes having variation at the anchor residues. The anchor residues in the predicted epitopes were highlighted by making it bold. The epitopes that were 100% conserved in the selected proteins of the 3 viral genotypes 1, 2 and 3 were also fully bold. Epitopes with 88/77% conservancy were with single or double amino acid variation respectively and to highlight them bold format was used in the conservancy column against each genotype.All the predicted epitopes of HCV 3a proteins of Pakistani origin were subjected for worldwide conservancy analysis among HCV genotype 1, 2 and 3. 5 sequences against each HCV protein (used for epitope prediction) were retrieved from NCBI randomly. The predicted epitopes of HCV 3a (Pakistani origin) along with 5 selected sequences of individual genotypes  were submitted to epitope conservancy analysis tool available at IEDB database (Asteric sign (*) indicates that one out of five selected sequences either does not respond to epitope conservancy or have conservancy lower then 77%. Double asteric sign (**) indicates that only one sequence responds for 77-100% conservancy to the selected epitope.The Peptides with single or double amino acid variation were analyzed for their hydropathic characteristics or pI value . The pI HCV 3a genome of Pakistani origin comprises 9474 bp with GC content 2622 and 2700 respectively. The GC contents are 12.35% higher then AT contents. The genome encodes a polyprotein that subsequently get fragmented into structural and non structural protein of obvious molecular weight. The envelope protein E2 comprises highest moleculat weight 38755.3 KDa , I (Isoleucine), L (Leucine), M (Methionine), V , W (Tryptophan) and Y (Tyrosine) were mainly the anchor residues for MHC II predicted epitopes and are nonpolar in nature. Total 150 epitopes were predicted against 51 alleles of MHC II Table . The higTotal 69 epitopes were predicted as promiscuous epitopes for MHC I alleles. The anchor residues in case of MHCI are quite varying both in amino acid residues and also in their nature. Mostly represented anchor residues are neutral nonpolar and neutral polar. However, quite small percentage of anchor residues were also acidic polar and basic polar in nature. The highest number of MHC I binding epitopes were represented by NS4b protein comprising 26% of all MHC I predicted epitopes. NFVSGIQYL epitope of NS4b is the best promiscuous binder of highest binding score. NS4b is followed by NS2, E2 and NS3 proteins representing 20.28% (NS2 epitopes) and 11.59% (for E2 and NS3). In case of NS2, 14 promiscuous epitopes were predicted with varying binding efficiency. GSRDGVILL, DGVILLTSL, WAAAGLKDL and LQVWVPPLL are the good binders both in term of score and the HLA allele coverage . E2 predicted epitopes covers 20 to 28 HLA alleles except the PLLHSTTEL epitope that covers only 11 HLA alleles but with highest binding efficiency. NS3 epitopes covers 8 to 25 HLA alleles and were also ranked on the basis of their binding efficiency predicted by the score. The least represented epitopes were by NS5a_1a. It comprises only one epitope (HVKNGSMRL) as predicted promiscuous binders for 16 MHC I binding alleles. The promiscuous binders of MHC I for other proteins were also predicted and summarized in table Out of total 150 predicted MHC II epitopes, 75.33% were (77-100%) conserve in genotype 3  conserve in genotype 3 (Table The modern technique for control of HCV infection is a vaccine preparation that can specifically induce antibody-mediated immunity. The rapid advancements in the computational methodologies and immunoinformatics/immuno-bioinformatics provide new strategies for the synthesis of antigen specific epitopic vaccine against infectious agents such as viruses and pathogens. Epitopic vaccine against HIV, malaria and tuberculosis provided promising results and supported the defensive and therapeutic uses of these vaccines . Thus inBy the use of an efficient CTL based epitope delivery technology; the predicted epitopes could eventually become vaccines in their own or fused as polytopes. The design of the HCV vaccine using conserved epitopes can avoid viral mutation and thus provides more efficient results. The study shows that the predicted epitopes were highly conserved in HCV genotype 3 and also but less conserved in genotype 1 and 2 both for MHC I and MHC II. Moreover, to ensure the viral detection at all stages of its intracellular evolution we have used all the viral proteins. Therefore, the total number of predicted epitopes were also maximized in correspond to the number of covered proteins used for the analysis.HCV: hepatitis C virus; HLA: human leukocyte antigen; MHC: major histocompatability complex; CTL: cytotoxic T lymphocytes.The authors declare that they have no competing interests.AS and SH designed the study. AS performed the immunoinformatics analysis and drafted the manuscript. MI critically reviewed the manuscript. All authors have read and approved the final manuscript."}
+{"text": "Saccharomyces cerevisiae. In this study, we show that Snf1 activity, which requires phosphorylation of the Thr210 residue, is needed for protection against selenite toxicity. Such protection involves the Elm1 kinase, which acts upstream of Snf1 to activate it. Basal Snf1 activity is sufficient for the defense against selenite, although Snf1 Thr210 phosphorylation levels become increased at advanced treatment times, probably by inhibition of the Snf1 dephosphorylation function of the Reg1 phosphatase. Contrary to glucose deprivation, Snf1 remains cytosolic during selenite treatment, and the protective function of the kinase does not require its known nuclear effectors. Upon selenite treatment, a null snf1 mutant displays higher levels of oxidized versus reduced glutathione compared to wild type cells, and its hypersensitivity to the agent is rescued by overexpression of the glutathione reductase gene GLR1. In the presence of agents such as diethyl maleate or diamide, which cause alterations in glutathione redox homeostasis by increasing the levels of oxidized glutathione, yeast cells also require Snf1 in an Elm1-dependent manner for growth. These observations demonstrate a role of Snf1 to protect yeast cells in situations where glutathione-dependent redox homeostasis is altered to a more oxidant intracellular environment and associates AMPK to responses against oxidative stress.The AMPK/Snf1 kinase has a central role in carbon metabolism homeostasis in  Saccharomyces cerevisiae is Snf1, which plays a key role in adaptation of cells to glucose limitation and use of alternative carbon sources The AMP-activated protein kinase (AMPK) family is constituted by protein complexes that participate in metabolic stress responses addressed to maintain cellular ATP levels in eukaryotes snf1 mutant is hypersensitive to sodium and lithium or to hygromicin B In addition to glucose limitation Snf1 also participates in the response of yeast cells to other environmental stresses. Thus, a null S. cerevisiae is an adequate model to study the molecular basis of Se toxicity since this yeast lacks selenoproteins and therefore, Se is not required as growth factor. In S. cerevisiae the more toxic form of Se is selenide. This can be formed from other Se forms such as selenite Selenium (Se) is an essential microelement in human cells present as selenocysteine in selenoproteins Based on the stress effects caused by selenite in yeast cells and on the protective role of Snf1 in defense against a diversity of stresses in addition to glucose depletion, this led us to explore the role of the Snf1 pathway in the response to selenite stress and in a broader perspective, in the response to changes in the redox state of the cell. Our results show that Snf1 activity is required to protect yeast cells against situations that decrease the ratio of reduced versus oxidized glutathione, including selenite treatment. We also demonstrate that such protective role of Snf1 takes place at the cytosol and does not correlate with extensive phosphorylation of Thr210 upon selenite addition. Overall, this study reveals a relationship between Snf1 kinase and redox regulation processes in yeast cells.TRK1 and derives from vector pCM262 SNF1 promoter GLR1 open reading frame under the control of the 7tetO promoter in the centromeric vector pCM189 GLR1 under its own promoter Strains employed in this study (W303 genetic background unless otherwise indicated) are listed in S. cerevisiae cell growth. For glucose starvation conditions, concentration of glucose in the medium was 0.05%. YPGal and YPGly contain respectively 2% galactose or 3% glycerol instead of glucose. When required, YPD medium was supplemented with exogenous iron by addition of 90 \u00b5M BPS  plus 100 \u00b5M FeSO42PO4 was added at 0.2 mM (low phosphate conditions) or 7.3 mM . In the former case, KCl was added up to 7.3 mM final concentration. Media were solidified with 2% agar. Sodium selenite (Sigma) was added at the concentration indicated in each case. Cells were grown at 30\u00b0C, with shaking in the case of liquid cultures.YPD  or synthetic SC medium were usually employed for natMX4 cassette and selection for nouseothricin resistance Standard protocols were used for DNA manipulations and transformation of yeast cells. Single null mutants were generated using the short-flanking homology approach after PCR amplification of the 5) were inoculated initially in each parallel culture.Sensitivity to selenite was determined in plate growth assays by spotting serial 1\u223610 dilutions of exponential cultures onto YPD or SC plates containing sodium selenite, and recording growth after 2 or 3 days of incubation at 30\u00b0C. Growth of several strains in liquid medium under parallel separate treatments was automatically recorded  at one-hour intervals during 24 hours, using individual 0.5 ml cultures in shaken microtiter plates sealed with oxygen-permeable plastic sheets, in a PowerWave XS (Biotek) apparatus at controlled temperature. Identical cell numbers . U-MNUA2 and U-MNUA3 filters were employed respectively for Hoesch and GFP staining. Immunofluorescence experiments to localize HA-tagged Mig1 were done with 3F10 rat anti-HA (Roche) and Alexa488 goat anti-rat (Molecular Probes) and parallel nuclear staining with DAPI.GFP-tagged proteins were visualized with an Olimpus BZ51 fluorescence microscope, after nuclear staining of cell samples with Hoesch .Cellular concentrations of oxidized and reduced glutathione were determined by using the Ellman\u2019s reagent method in culture samples quenched by 5-sulfosalicylic acid S. cerevisiae \u0394snf1 null mutant to selenite stress. The mutant was more sensitive to the agent than wild type cells  participate in Thr210 phosphorylation and activation of Snf1 in response to glucose depletion and other stresses. They act redundantly, although in many cases (for instance in glucose-minus conditions) Sak1 plays the most relevant role s tested . Cells e\u0394snf1\u0394elm1 mutant compared to the single \u0394snf1 and \u0394elm1 mutants. The double mutant displayed additive sensitivity  rescues selenite sensitivity GLR1 and another key gene for GSH metabolism, GSH1 (for L-\u03b3-glutamyl-L-cysteine synthetase) \u0394snf1 cells, and we explored this possibility.The tripeptide glutathione  is an essential thiol redox regulator GLR1 and GSH1 expression in \u0394snf1 than in wild type cells, which may be indicative of more intense alteration in glutathione pools in the mutant  was calculated from the GSH/GSSG concentrations, it began with almost identical values in untreated cultures of both strains but increased \u223c16 mV in wild type cells and \u223c24 mV in mutant cells after one hour of selenite treatment, and only returned to similar values for both strains after 6 hours of treatment , which discards a general protective role of Snf1 activity in oxidative stress conditions. To confirm that Snf1 activity is required to counteract the effects of GSH-oxidizing agents, we showed that the snf1-K84R and snf1-T210A mutations do not complement the DEM-sensitive phenotype of a \u0394snf1 mutant, in contrast to the wild type form  ype form . As in type form , and DEMype form . Also asype form . Finallytoxicity .Summarizing, our results showed that Snf1 activity has a general protective role in yeast cells in situations that lead to glutathione oxidation such as the presence of selenite, DEM or diamide.S. cerevisiae cells upon selenite treatment. Although we observed phosphorylation of Snf1 Thr210 at advanced treatment times, the basal activity of Snf1 is sufficient to protect against the agent, and nutritionally or genetically provoked situations where Snf1 becomes permanently phosphorylated at Thr210 do not provide additional selenite resistance. Since Snf1 phosphorylation levels are mainly regulated by the activity of Reg1 \u0394reg1 mutant is also hypersensitive to the agent (this work), and (ii) a \u0394ppz1 mutant is also unable to grow in the presence of selenite (our unpublished results). Ppz1 is a phosphatase with large homology to PP1-type phosphatases whose roles in the regulation of cation homeostasis and other yeast cell processes have been characterized AMPK/Snf1 responds to metabolic stress in yeast cells, its kinase activity being required for adaptation to glucose limitation and growth in alternative carbon sources We have shown that selenite toxicity and the requirement of Snf1 for protection against it is not related to plasma membrane depolarization effects and/or glucose depletion, and that such protection requires the entry of the agent into the cell mostly through the high affinity mechanism of phosphate uptake, which is the main mediator of selenite entry in glucose-grown yeast cells GLR1 glutathione reductase gene protects yeast cells against the toxic effects of selenite GLR1 in \u0394snf1 cells allows growth of the mutant in the presence of selenite up to similar levels as the wild type. This fact points to the changes (compared to wild type cells) of the redox potential of the GSH/GSSG pair as the cause of the hypersensitivity of \u0394snf1 cells to selenite. Accordingly, yeast cells lacking Snf1 are also hypersensitive to agents such as DEM or diamide, which increase the GSSG pools relative to GSH. In addition, overexpression of GLR1 also allows growth of \u0394snf1 cells at the same level as wild type cells in the presence of DEM. Therefore, the relationship between selenite toxicity and cell protection mediated by Snf1 reflects a more general role of this kinase in sensing and responding to intracellular redox changes due to an increase of oxidized glutathione. Again, both DEM and diamide provoke a late phosphorylation of Snf1, which indicates that the possible alteration of the modulatory mechanisms of Snf1 phosphorylation is not circumscribed to selenite, but extends to a broader range of agents acting on intracellular redox homeostasis.How selenite signaling is related to Snf1 activity? Selenite provokes a reduction of GSH and an increase of GSSG in yeast cells The signaling role of Elm1/Snf1 in inducing a protective response to a sudden increase of the GSSG/GSH ratio does not seem to involve previous functions assigned to Elm1 and Snf1, and as discussed above such role would not require the already characterized nuclear effectors of Snf1. Recent studies point to functions of Snf1 other than those previously characterized as metabolic regulator. Thus, analysis of the yeast kinase-protein interactome using protein microarrays has revealed common targets between Snf1 and the Akl1 kinase involved in cytoskeletal functions Figure S1Location of Elm1 protein upon selenite treatment. Strain AKY516 expressing ELM1-GFP derivative was grown in YPD medium with 4 mM selenite for the indicated times. Cells were immediately visualized by fluorescence and phase contrast microscopy using an Olympus BZ51 apparatus.(TIF)Click here for additional data file.Figure S2\u0394snf1 (Wsnf1), \u0394trk1\u0394trk2 (W3) and \u0394snf1\u0394trk1\u0394trk2 (MML1447). The medium was supplemented with 100 mM NaCl to improve growth of \u0394trk cells.  Growth assays of serial dilutions of wild type (W303-1A) and \u0394snf1 (Wsnf1) cells transformed with vector pCM262 or its derivative pYCp414 overexpressing TRK1, in SC medium with selenite. (B) Growth assays of serial dilutions of wild type (W303-1A) and \u0394snf1 (Wsnf1) cells in YPD medium with the indicated concentrations of glucose plus selenite. (C) Relative amount of glucose-6-phosphate per cell. Samples were taken from wild type (W303-1A) or \u0394snf1 (Wsnf1) cells growing exponentially in YPD medium and treated with selenite for the indicated times. Values  were made relative to the unit value corresponding to untreated wild type cells .Selenite sensitivity is not associated to alterations in plasma membrane polarization or intracellular glucose deprivation.  Growth assays of serial dilutions of the following strains on YPD medium with selenite: wild type (W303-1A), (TIF)Click here for additional data file.Figure S3Expression of PHO89 is induced by selenite under the control of Snf1. Northern blot expression analysis of the indicated genes in wild type (W303-1A), \u0394snf1 (Wsnf1), \u0394pho84 (MML1304) and \u0394snf1\u0394pho84 (MML1401) cells in low or normal phosphate cultures treated with 3 mM selenite. SNR19 was employed as loading control.(TIF)Click here for additional data file.Figure S4Northern blot expression analysis of GSH1 and GLR1 in selenite-treated cells. Exponential cultures of wild type (W303-1A) and \u0394snf1 (Wsnf1) cells in YPD medium were treated with 6 mM sodium selenite for the indicated times. SNR19 was employed as loading control.(TIF)Click here for additional data file."}
+{"text": "We have therefore conducted a bench study in order to measure the effect of different levels of O2 supply and degrees of leakage on delivered FiO2. Ventilator tested: Legendair\u00ae . Thirty-six measures were performed in each four ventilators with zero, 5 and 10 l.min-1 leakage and 1,2,4 and 8 l O2 flow.Long term oxygen therapy improves survival in hypoxemic patients with chronic obstructive pulmonary disease (COPD). Because pressure support ventilation with a home care ventilator is largely unsupervised, there is considerable risk of leakage occurring, which could affect delivered FiO2 decreased significantly with 5 l.min-1 leakage for all O2 flow rates, and with 10 l.min-1 at 4 and 8 l.min-1 O2.FiODuring application of NIV on home ventilators, leakage can dramatically decrease inspired FiO2 making it less effective. It is important to know the FiO2 dispensed when NIV is used for COPD at home. We would encourage industry to develop methods for FiO2 regulation Chronic use of NIV for COPD with controlled FiO2 or SpO2 requires further studys. Non-invasive ventilation (NIV) is now recommended for acute on chronic COPD respiratory distress, whereas its chronic use is more controversial . Statistical analysis was performed with JMP 9.3 . Statistical significance was defined as p < 0.05.The Kruskal-Walis test was used to compare all variables and the Four different ventilators were tested three times each, with 12 measurements of FiO2 for each condition of oxygen supply and leakage. Leakage significantly affected FiO2 for all O2 supply between no leak and 5 liters per minute of leaks. FiO2 decreased significantly with 10 liters per minute of leaks only for 4 and 8 liters O2 supply  and it was constant.2 at home was not reported [The Nocturnal Oxygen Therapy Trial (NOTT) and the Medical Research Council study, using similar inclusion criteria, demonstrated the beneficial effects of LTOT on survival in subjects with COPD and severe resting hypoxemia. The median survival in those using O2 for 15 hours/day was approximately twice that of those receiving no O2 [reported .-1[2 because the O2 supply is constant, as demonstrated here. NIV failure to improve surviving in chronic COPD may be due to this.Although some studies show no improvement in mortality ,6,11, th-1. Neverth2 source or an O2 extractor, FiO2 can be monitored but it is not possible to set. Unfortunately regulation of FiO2 with home ventilators is not yet available. Our study is the first to focus on the affect of leakage on FiO2 during NIV with home ventilators, although such effects have been previously shown with ICU ventilators [2 can vary by up to 30%. With the O2 supply of 8 l.min-1 we found a decrease in FiO2 from 70 to 50% with a leakage of 10 l.min-1.There are no studies of the delivered FiO2 in NIV, the value of which is increased by oxygen supplementation with home ventilators. Thys and Schwartz showed the influence of site oxygen delivery on FiO2 ,17. Althtilators . In seve2. In a recent meta-analyse Chen et al. point out that with an inspiratory positive airway pressure (IPAP) greater or equal than 14 cmH2O the PaO2 decreased [2 decreases, and hypoxemia ensues leading to an increase of minute respiratory intake which in turn results in aspirating room air; if the O2 supply is constant then FiO2 decreases again leading a vicious circle of hypoxemia. This may explain the poor results of treating respiratory failure with NIV in the home.NIV offers an excellent treatment for chronic respiratory insufficiency, but in the home there is no control of leaks or measurement of SPOecreased ; increas2 supply can be triggered by the patient\u2019s SpO2 or the ventilator FiO2. The former is more relevant clinically; the latter is more easily measured. The cost of measuring FiO2 is about 1000 Euros; SpO2 is less expensive but the measurement is subject to artefacts.The OThe design (bench test) limits the impact of the findings. On the over hand we underestimated the leaks, which could reach 30 liters per minute; the FiO2 decrease may be greater in clinical conditions.We purpose to adjunct and O2 turbine witch should adjust O2 flow supply to a SpO2 objective.New home NIV chronic use in COPD study\u2019s should be conducted with this O2 flow control.We propose to add a dedicated O2 turbine allowing automated O2 flow adjustment in order to achieve a predefined SpO2.Thereafter, studies will be mandatory to evaluate effects of this automated O2 flow supply on COPD prognosis.This lung model study demonstrates that during application of NIV on home ventilators, leakage can dramatically decrease FiO2 making it less effective. As consequence, it is essential to know the FiO2 dispensed during NIV at home. Further research should explore the role of the O2 flow control during application of NIV in chronic hypercapnic COPD patients.We hope that industry can address the problems we have highlighted in this study.COPD: Chronicle obstructive pulmonary disease; FiO2: Inspired fraction of oxygen; IPAP: Inspiratory positive airway pressure; LTOT: Long term oxygen therapy; O2: Oxygen; NIV: Non invasive ventilation; NOTT: Nocturnal Oxygen Therapy Trial; PEEP: Positive end expiratory pressure; VA/Q: Ratio: ventilation perfusion ratio.The authors declare no competing interest.GP drove the research and write the article, AY made the statistical analysis; and all the authors contributed to measurements. All authors read and approved the final manuscript."}
+{"text": "Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of B. pertussis strains with a novel allele for the pertussis toxin (ptx) promoter ptxP3 in place of resident ptxP1 strains. The recent expansion of ptxP3 strains suggests that these strains carry mutations that have increased their fitness. Compared to the ptxP1 strains, ptxP3 strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the ptxP3 strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the ptxP3 strains compared to the ptxP1 strains. We provide the first evidence that wildtype ptxP3 strains are better colonizers in an intranasal mouse infection model. This study shows that the ptxP3 mutation and the genetic background of ptxP3 strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of ptxP3 strains with a higher expression of virulence genes contribute to increased fitness. Bordetella pertussis is a human-specific pathogen and the causative agent of whooping cough, or pertussis, which is an acute respiratory disease that is particularly severe in infants. Universal immunization programs have contributed to a significant reduction in the incidence of serious disease and mortality caused by B. pertussisB. pertussis surface proteins have been detected in several countries. Variations in the B. pertussis proteins pertussis toxin (Ptx) and pertactin (Prn) have been shown to affect vaccine efficacy in a mouse model ptxP3) associated with recent epidemics in the Netherlands have emerged in several European countries, replacing resident ptxP1 strains ptxP1 and ptxP3 have been predominating in the Dutch B. pertussis population. The recent expansion of ptxP3 strains in the Netherlands and other countries in Europe, Asia and North and South America suggests that ptxP3 strains carry mutations that have increased their fitness. Laboratory data has shown an increase in Ptx production in ptxP3 strains, and epidemiological data has suggested that the ptxP3 strains are more virulent ptxP1 strains.B. pertussis virulence factors is controlled by the two-component BvgAS sensory transduction system B. pertussis strains under virulent growth conditions. These genes include pertactin, pertussis toxin, filamentous hemagglutinin (FHA), fimbriae, adenlyate cyclase toxin, dermonecrotic toxin and the type III secretion system (TTSS) Mycobacterium tuberculosisThe expression of B. pertussis clinical strains isolated in different countries revealed genomic differences between the strains. Genes present in all of the isolates (core genes) are assumed to be phylogenetically conserved, while the genes that are variably present (variable genes) are proposed to be horizontally acquired or differentially lost within the species. The core and variable genome has been defined for B. pertussis based on microarray studies ptxP type and the gene content, suggesting that strains with different ptxP types form different lineages ptxP1 and ptxP3 lineages are distinguished by a region of 18 genes present in ptxP1 strains, but absent in all of the ptxP3 strains that have been analyzed to date ptxP3 isolates are grouped together and form a separate branch B. pertussis ptxP3 strains differ in other key biological properties from the ptxP1 strains, but it is suggested that the ptxP3 strains are the fitter variants of B. pertussisA recent microarray-based analysis of the gene content of over 170 ptxP3 strains.While variation in genetic content is likely to be relevant in pathogenesis, differential gene expression may also be important. Gene expression profiling using DNA microarray technology provides a fingerprint of the full transcriptome, which allows a detailed comparison of strain-specific differences. In this study, we used genome-wide gene expression profiling, allelic exchange and a murine model of infection to gain insight into the factors involved in the global spread of the B. pertussis strains from different countries isolated between 1949 and 2008 using microarray-based CGH and found a strong correlation between the gene content and the ptxP allele B. pertussis strains isolated in the Netherlands between 1949 and 2008 carrying a ptxP1 (n\u200a=\u200a9) or ptxP3 allele (n\u200a=\u200a5). Strains were grown under Bvg+ conditions in a chemically defined growth medium 590) to confirm logarithmic growth (data not shown). Changes in growth phase-associated gene expression were previously studied using transcriptional profiling of vaccine strain 509 under similar conditions et al.), the optimal harvest point for the RNA samples was estimated at OD\u200a=\u200a0.4+/\u22120.05. This time point was chosen to minimize the specific effects of the growth phase.In a previous study, we analyzed the gene content of Bordetella microarray based on the sequence of B. pertussis (Tohama I strain) and all of the extra genes found in B. parapertussis strain 12822 and B. bronchiseptica strain RB50 B. pertussis strains carrying the ptxP1 allele (n\u200a=\u200a9) and ptxP3 allele (n\u200a=\u200a5) were determined to identify differentially regulated genes. The transcript levels were assessed in all (at least) -three biological replicates of each strain  <0.05; corresponds to P<0.013117). The transcription of 952 genes was significantly upregulated in ptxP3 strains, with 818 core genes. For 134 variable genes, expression was significantly higher in the ptxP3 strains. The transcription of 23 genes was significantly downregulated in the ptxP3 strains, with 19 core genes  B. pertussis strains carrying the ptxP3 allele, suggesting that the ptxP3 strains may be more virulent. The differences in expression between the ptxP1 and ptxP3 strains were significant even though they generally did not differ by more than a factor of 1.5-fold, with the exception of the, BP0500, BP1568, BP2252, BP2254 and BP2315  also showed a slightly higher expression (P\u200a=\u200a0.014172) in the ptxP3 strains, but the difference was slightly above the FDR <0.05 cutoff. The microarray data showed a slightly higher (factor of 1.14\u20131.25-fold) expression of the genes in the ptx-operon. BP3783 (ptxA), BP3784 (ptxB), BP3785 (ptxD), BP3786 (ptxE) and BP3787 (ptxC), the difference in expression of ptxB in ptxP3 and ptxP1 strains with a 1.14 fold change P\u200a=\u200a0.022 FDR\u200a=\u200a0.07, slightly above the FDR cutoff of 0.05 and is there was not shown in ptxP3 strains (not shown).Several virulence-associated genes were differentially expressed in the d BP2315 . InteresMicroarray data were validated by real time quantitative PCR assays on a small selection, 5 (BVG controlled) genes . Real tiptxP3 and ptxP1 mutations and the genetic background (gb) of the strains, we constructed two different isogenic strains that carry the ptxP3 allele in the ptxP1 genetic background (P1 gb:ptxP3) or the ptxP1 allele in the ptxP3 genetic background (P3 gb:ptxP1). The colonization of isogenic and wild type strains was tested in an intranasal mouse model that was previously described ptxP3 strains colonize more efficiently than the wild type ptxP1 strains in the lungs and trachea of mice (P<0.0001). The P3 gb:ptxP1 strains and wild type ptxP3 strains showed similar colonization levels in the lungs and trachea, suggesting that the genetic background of the strain significantly contributes to the observed differences. The P1 gb:ptxP3 strains showed increased colonization in the lungs and trachea than the wild type ptxP1 strains (P<0.0001), which suggests that the ptxP3 allele contributes to the increased colonization. Colonization was decreased in the P1 gb:ptxP3 strains compared to the wild type ptxP3 strains, but the differences were not significantly different in lungs and trachea , were more highly expressed in the ptxP3 strains. Previous studies have suggested that strains with different ptxP types form different lineages B. pertussis strain with the ptxP3 allele is a better colonizer in the intranasal mouse infection model than a B. pertussis strain with a ptxP1 allele  are upregulated (FDR<0.05 and P<0.013117) in the ptxP3 strains. Q-PCR data for a small selection of genes confirmed the higher expression in ptxP3 strains compared to ptxP1 strains, although in two genes the higher expression in ptxP3 strains by Q-PCR was not significantly different. The correlation between results by Q-PCR and hybridization with the microarray were shown to be very good for the 5 genes selected. Differentially expressed genes involved in amino acid biosynthesis (P\u200a=\u200a0.00005), energy metabolism (P\u200a=\u200a0.00049), and regulation (P\u200a=\u200a0.00786) between the ptxP1 and ptxP3 strains were significantly overrepresented. Cummings et al. ptxP1 and ptxP3 strains showed a 37% increase in expression of the virulence-associated genes that form the core regulon in the ptxP3 strains, which indicates that these strains may be more virulent. Of the 21 genes with increased expression in the ptxP3 strains, BP0500 , BP1568 (serotype 3 precursor), BP2252, (bop B), BP2254 (bcrH 1) and BP2315 (vag 8) had expression levels that were more than 1.5-fold higher in the ptxP3 strains. The virulence sensor protein (BvgS) was also slightly, but significantly, upregulated in the ptxP3 strains. Han et al.B. pertussis strains carrying the ptxP3 allele. Significantly increased expression of the ptxP operon was detected by microarray based gene expression even though the difference was less than 1.5-fold  in the trachea and lungs of mice compared to the wild type ptxP1 strain. Multivariate analyses performed by van Gent et al. previously showed that ptxP contributed to differences in colonization ptxP1 and ptxP3 strains form separate lineages as shown by differences in genetic content and DNA sequencing and SNP studies ptxP3 and ptxP1 mutations individually and the genetic background (gb) of the strains, two different isogenic strains were constructed carrying the ptxP3 allele in the ptxP1 genetic background (P1 gb:ptxP3) or the ptxP1 allele in the ptxP3 genetic background (P3 gb:ptxP1). In the mouse model, the strain with the ptxP3 genetic background, but not the ptxP3 allele itself (P3 gb: ptxP1), had a similar colonization ability as the wildtype ptxP3 strain. A significantly higher colonizing ability compared to wildtype ptxP1 strains was observed, suggesting that the genetic background of the ptxP3 strains plays a role in increasing the ability to colonize. Since the colonization ability was similar to the wildtype ptxP3 strain this result may also be interpreted in that the ptxP3 allele itself does not contribute that much to the higher colonizing ablility.In contrast, colonization with the strain with the ptxP3 allele in the genetic background of a ptxP1 strain (P1 gb: ptxP3) showed significantly increased colonization compared to the wildtype ptxP1 strain, which indicates that the ptxP3 allele itself also contributes to increased colonization in the mouse model. To our knowledge, no data on the effects of the genetic background of ptxP3 strains on colonizing ability have been previously published.The biological properties of wild type  of mice . We demoptxP3 strains in the lungs and trachea of mice is in agreement with epidemiological data. Mooi et al. ptxP3 strains in many countries worldwide indicates that ptxP3 increases the fitness of the strain or is linked to other genetic loci that do. These researchers suggest that the ptxP3 mutation confers increased fitness. A study in Sweden also suggested that B. pertussis strains differ in virulence because patients infected with strains of the PFGE profile BPSR11 were hospitalized for longer periods of time ptxP3 strains. We demonstrate that the ptxP3 allele and the genetic background of the strains contribute to increased fitness.The higher colonization of Bordetella pertussis strains used in this study are listed in 590 stored at \u221280\u00b0C. One vial of each strain was used to inoculate a primary 500 ml shake flask containing 200 ml THIJS medium 590 of 1.0\u00b10.2 was reached, a secondary shake flask was inoculated with 10 ml of the culture at OD590\u200a=\u200a1.0. For shake flasks at other optical densities, the volumes were adjusted to ensure that an equal amount of cells were used to inoculate each secondary shake flask. Secondary shake flasks were incubated at 35\u00b0C and 200 RPM until an OD of 1.00\u00b10.05 was reached. The cultures were mixed with glycerol (17% v/v), divided into 10 ml working seedlots and stored at \u2212140\u00b0C.The 590\u200a=\u200a1.00\u00b10.05, 3 secondary shake flasks were started for each strain. The triplicate cultivations were inoculated with 10 ml preculture and grown as described. The initial density was OD590\u200a=\u200a0.050\u00b10.05 for all of the secondary cultivations. Separate samples were collected from each secondary shake flask for RNA isolation and consecutive microarray analysis when the OD590 was 0.4\u00b10.05.For each strain, a preculture was inoculated with 10 ml of the seedlot. Precultures were grown in 500 ml shake flasks containing 200 ml THIJS medium at 35\u00b0C on an orbital shaker at 200 RPM. When the preculture reached ODRNase retarding solution 590\u200a=\u200a1.0 was used. For samples at other optical densities, the volumes were adjusted such that an equal amount of cells was used for each sample. The samples were concentrated by centrifugation and treated with Tris-EDTA buffer containing 0.5 mg/ml lysozymes  for 3 minutes. Total RNA was extracted with the SV Total RNA Isolation System  according to the manufacturer\u2019s protocol. The nucleic acid concentration was adjusted by precipitation, and spectral analysis was used to determine the final nucleic acid concentration and purity. RNA integrity was confirmed with the Bioanalyzer RNA6000 Nano assay  according to the manufacturer's protocol.For fixation of the RNA expression profile, 1 volume of bacterial culture was mixed with 2 volumes of a Bordetella microarrays were constructed using the 8 x 15K format developed by Agilent Technologies . The set of 5,910 60-mer oligonucleotides (60-mer) in which one oligonucleotide corresponds to one gene covered 94% of the genes in the three sequenced Bordetella strains, including B. pertussis Tohama I, B. parapertussis 12822 and B. bronchiseptica RB50. In addition, 98 control probes were included in the microarray, and all of the spots were printed in duplicate (non adjacent). User-defined probes were uploaded through the Agilent eArray Web portal (http://earray.chem.agilent.com/earray/). Additional details on microarray production are available through the ArrayExpress microarray data repository (accession number A-MEXP-1697).Custom pan-A two-color hybridization format was used for microarray analysis. For each biological replicate, RNA extracted from each test strain was used to create Cy5-labeled cDNA, and the (common) reference sample containing equal amounts of RNA from all experimental samples was used to create Cy3-labeled cDNA. The use of a common reference across different cDNA microarray experiments improves the reproducibility of the hybridization signals and allows the gene expression levels from separate experiments to be compared. Total RNA samples were reverse transcribed to cDNA and labeled with Cy3/Cy5 dyes using the Chipshot Indirect Labeling kit (Promega Benelux) according to the manufacturer\u2019s protocol with one modification. A total of 2 \u00b5l of random nonamer primer without oligo-dT primers was used per reaction to reverse transcribe the total RNA. For each hybridization, 300 ng Cy3-labeled cDNA and 300 ng Cy5-labeled cDNA were combined with 5 \u00b5l 10\u00d7 blocking agent and 1 \u00b5l 25\u00d7 fragmentation buffer in a total volume of 25 \u00b5l according to the manufacturer\u2019s protocol (Agilent). Prior to loading on the microarray, the hybridization solution was heated for 3 minutes at 60\u00b0C. Microarray slides were hybridized and treated as described in the Agilent protocols for two-color microarray-based gene expression analysis. Microarray experiment details are also deposited at array express under accession number E-MTAB-1594.ptxP1 strains was calculated as mean +/\u2212 SD for 8 strains and the gene expression for ptxP3 strains was calculated as mean +/\u2212 SD for 5 strains. The relative difference in expression were presented as the ratio of ptxP3 strains to ptxP1 strains.For quantitative PCR the total RNA was treated with DNase I to remove contaminating DNA. The quality of the RNA was evaluated by Agilent Bioanalyzer and Naonodrop spectrophotometry. The RNA was reverse transcribed using random hexamers and oligo d(T). Samples were tested in 384 well format in duplicate with a no-RT control for each sample. Universal Human Reference RNA and the control sample were also tested in duplicate with a no-RT control. The samples were analyzed with 6 assays: 5 target assays and 1 endogenous control, as detailed below. All samples were amplified using the Applied Biosystems Prism\u00ae 7900 Sequence Detection System with standard cycling conditions. Primer sequences for the examined 6 genes are in Bordetella microarrays were analyzed using ImaGene software . Individual arrays were internally normalized between the Cy3 and Cy5 channels by LOWESS normalization. A hybridization ratio logarithm [log2(Cy5/Cy3)] was calculated and the gene expression data were normalized to the expression of a BP0015 DNA-directed RNA polymerase beta chain in each microarray slide to compensate for variations between the slides. The normalized data were further processed using Microsoft Excel and TMEV software from the TM4 suite (TIGR). Biological replicates were analyzed separately for all of the strains. The average expression of each individual gene was calculated for all of the strains carrying the ptxP1 and ptxP3 alleles. P-values were calculated with one\u2013way ANOVA statistical analysis (t-test). The fold change in the ptxP3 and ptxP1 strains was determined as follows: 2 \u2227 (average of expression in ptxP3 strains \u2013 average of expression in ptxP1 strains), if this number, between the brackets, is greater then 0 if not then 1/2 \u2227 (average of expression in ptxP3 strains \u2013 average of expression in ptxP1 strains. All microarray data have been deposited in Array express under accession number E-TAB-1594.The hybridized slides were scanned at a 5 \u00b5m resolution using a ScanArray Gx plus microarray scanner (Perkin Elmer) equipped with ScanArrray express software. The images from Agilent pan-B. pertussis strain with the ptxP1 allele in the genetic background of the ptxP3 strain , we constructed a suicide vector pSS1129\u2013 ptxP1 by PCR amplification of an internal fragment of the ptxP promoter from genomic strain B0213 (Tohama I) using the Xba1F (GCT CTA GAC GCT GCA GTC CAA GGC GGT CGT C) and EcoR1R (GGA ATT CAT CCC GTC TTC CCC TCT GCG TTT TGA TG) primers. The PCR product was cloned into pSS1129 using the Xba1 and EcoR1 restriction sites. Suicide vectors were introduced into B. pertussis  via conjugation using E. coli SM10 as a donor strain. B. pertussis mutants were selected based on growth on BG-agar containing appropriate antibiotics. Removal of the vector sequences was forced by growing the conjugates on BG agar plates with 300 \u00b5g/ml streptomycin. A B. pertussis strain with a ptxP3 allele was constructed in the genetic background of a ptxP1 strain  in a similar manner. The suicide vector pSS1129-ptxP3 was constructed and introduced in B. pertussis . Proper insertion was confirmed by sequencing. The gene content of all of the mutant B. pertussis strains was analyzed using microarray-based CGH analysis. The strains B1917, ptxP3 and B0602, ptxP1 have the most common gene content type for ptxP3 and ptxP1 strain respectively and were therefore good respresentatives for these two groups.To generate a All animal experiments were conducted according to relevant national and international guidelines. Strains were grown on Bordet-Gengou plates and animal experiments were performed as previously described Table S1Microarray gene expression data for all strains analyzed in this study. DNA microarray analysis was used to measure the mRNA levels in ptxP3 strains compared to mRNA levels in ptxP1 strains. Gene expression data were shown for all replicates for each individual strain (ptxP1 strains with a green column head and ptxP3 strains with red column head). The gene expression data were normalized to the expression of a BP0015 DNA-directed RNA polymerase beta chain in each microarray slide to compensate for variations between the slides. The average expression of each individual gene was calculated for all of the strains carrying the ptxP1 and ptxP3 alleles. P-values were calculated with one\u2013way ANOVA statistical analysis (t-test), P-values lower than 0.05 were highlighted in green. The fold change in the ptxP3 and ptxP1 strains were calculated as described in (XLSX)Click here for additional data file.Table S2Summary of all genes significantly up or down regulated in strains carrying the ptxP3 allele compared to strains with the ptxP1 allele. The gene ID, gene name and gene category are shown.(XLS)Click here for additional data file.Table S3Quantitative PCR Primer sequences for genes by Q-PCR in this study. Primer sequences for 6 assays: 5 target assays and 1 endogenous control are shown. The gene IDs for targeted genes are given in the first column.(XLSX)Click here for additional data file."}
+{"text": "Fluid overload has recently been linked to adverse outcomes in critically ill patients, but its impact on the outcomes of cancer patients admitted to intensive care units (ICUs) has not been previously described.A total of 234 cancer patients admitted to the medical ICU in a 6-month period were prospectively evaluated for survival. Univariate and multivariate analyses were used to study ICU admission parameters associated with ICU mortality. Exclusion criteria were ICU stay <24 hours and chronic renal failure on dialysis.Overall mortality was 21%. The mean age of all patients was 62.7 \u00b1 11.6 years and 55% were male. Postoperative care (45%) and sepsis (35%) were the major reasons for admission to the ICU. The mean APACHE II score value at 24-hour ICU was 21.2 \u00b1 6.4 and the mean Karnofsky score before ICU admission was 75.2 \u00b1 17.2. At multivariate analysis, the following variables at ICU admission were significantly associated with ICU mortality in cancer patients: Lung Injury Score >2  and positive fluid balance (for each 100 ml/24 hours) .Fluid overload is independently associated with increased mortality in critically ill cancer patients. Further studies are necessary to determine the impact of positive fluid balance on acute organ dysfunction and overall prognosis of cancer patients."}
+{"text": "There is no evidence that during OLV the EtCO2 or PaCO2 should be kept in the normal range. The aim of the present work was to test whether different ventilatory strategies to maintain EtCO2 or PaCO2 in the normal range during OLV have any impact on arterial oxygenation (PaO2).Physiologically, an approximately 5 to 10 mmHg difference exists between end-tidal carbon dioxide (EtCO2 (n = 50) the OLV was guided by capnography, and the respiratory rate (RR) was adjusted to maintain EtCO2 in the normal range. In GrPaCO2 (n = 50) the OLV was guided by arterial blood gas analysis (ABG) and RR was adjusted to maintain PaCO2 in the normal range. ABG was performed in a supine position after induction and in a lateral decubitus position during DLV and every 15 minutes during OLV. During OLV 5 ml/kg tidal volume with 5 cmH2O PEEP, I:E = 1:2 ratio and FiO2 1.0 was used.Data were obtained from 100 patients undergoing thoracic surgery necessitating OLV. Patients were randomized into two groups. In GrEtCO2 values between groups during DLV and at the 15th minute of OLV. There were significant differences in PaO2 at the 30th and 45th minutes between groups. In GrPaCO2 mean airway pressure and RR was higher, and the inspiratory and expiratory time was shorter than in GrEtCO2.There were no significant differences in PaO2 than permissive hypercapnic OLV.The relatively high RR impairs the emptying of alveoli and results in increased functional residual capacity. So the normocapnic lung-protective OLV results in significantly higher PaO"}
+{"text": "Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus\u2013based vaccine is administered 20\u201330 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection. The filoviruses, Marburg virus (MBGV) and Ebola virus (EBOV), have been associated with sporadic episodes of hemorrhagic fever (HF) in Central Africa that produce severe disease and high mortality rates among infected patients  vaccine vector expressing the MBGV glycoprotein (GP) (VSV\u0394G MBGV GP) shortly after a high-dose MBGV challenge (7 PFU) (7 PFU), and the animal in control group 2 received an equal dose of VSV\u0394G/LassaGPC. The animal in control group 3 was not treated. Blood samples for viral infectivity titration, reverse transcription\u2013PCR (RT-PCR), hematologic analysis, serum biochemical analysis, and immunoglobulin (Ig) G were collected before MBGV challenge and on days 3, 6, 10, 14, and 31\u201335 after MBGV challenge.Animal research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. Fifteen healthy, filovirus-seronegative rhesus macaques (each weighing 4 kg\u20137 kg) were randomized into 2 experimental groups of 6 monkeys per group and 3 control groups of 1 animal per group. All 15 animals were challenged by intramuscular (IM) injection with 1,000 PFU of MBGV (Musoke strain). Approximately 24 h after MBGV challenge, animals in experimental group 1 received a single IM injection of VSV\u0394G MBGV GP  and 2 of the 6 animals treated with VSV\u0394G/MBGV GP 48 h after MBGV challenge  survived . In contys 10\u201312 . Symptomectively .10 PFU/mL developed on day 10 in 1 surviving animal (7) treated 48 h after infection, and RT-PCR showed evidence of MBGV in peripheral blood mononuclear cells of this animal on days 6 and 10. Viremia in plasma was cleared, and the animal showed little evidence of illness by day 14. The serologic response profile of MBGV infection after treatment was evaluated by IgG ELISA. All 7 animals that were treated with VSV\u2206G/MBGV GP and survived infection showed moderate to high levels of IgG by day 14 ; humoral response against MBGV was not detectable in the treated animals that died or in the control animals  showed no change in appearance or behavior that indicated overt illness. Changes in hematologic results and/or blood parameters were observed in 5 of the surviving animals  during the course of the study . Plaque  animals .7 PFU of the VSV ZEBOV GP vaccine, which is consistent with doses used in nonhuman primate studies (This rhesus macaque model represents a worse-case scenario such as an accidental needle-stick exposure of a laboratory worker or first responder to a high infectious dose of a filovirus. Accidents such as these have occurred several times over the past 5 years (50 doses (MBGV infection of humans normally progresses at a slower rate than does MBGV infection of macaques, with case-fatality rates in humans of 23%\u201390% ("}
+{"text": "Platelet factor 4 (PF4) is a 70-amino acid protein belonging to the CXC chemokine family. PF4 is also known as CXCL4. This chemokine is released from Megakaryocytes respond to the presence of tumors by increasing their number in the bone marrow accompanied by increase in the number of platelets in circulation, causing changes in chemokine balance.An attempt was made to get new information about PF4 production in NSCLC and clarify clinical significance of measuring PF4 level and platelet count.Peripheral blood samples were collected from patients with histologically proven early (n=32) and advanced (n=12) stage NSCLC, and healthy individuals (n=12). Complete blood count test to measure concentration of platelets and exclude acute inflammation was used. Plasma PF4 levels were determined by ELISA.Higher levels of PF4  observed in plasma of early stage NSCLC patients compared to advanced stage NSCLC  and healthy individuals  (p<0.02). Thrombocyte level and thrombocytosis found in patients with advanced NSCLC did not correlate with PF4 levels (p<0.01).Many cancers have a complex chemokine network. Plasma chemokines as PF4 are significant in developing theories concerning the biology of NSCLC and are potential key molecules for early detection and treatment of cancer."}
+{"text": "Clostridium difficile infection (CDI) doubled to 50 cases. Cases of ribotype 027 increased 5-fold from 5 to 26. In 2012, multiple interventions were initiated reduce the number of CDI cases.The study was conducted at Bornholms Hospital located on an island in the Baltic Sea. In 2011, cases with Observation rounds during weekdays seeing patients isolated because of diarrhea was introduced February 1, 2012 in 3 medical wards (94 beds). The rounds adjusted incorrect interventions and collected data structured by checkpoints that included:\u00b7 Indication for and duration of isolation\u00b7 Turnaround time for microbiological diagnosis\u00b7 Antibiotic (AB) therapy prior to episode of diarrhea\u00b7 Compliance to isolation precautionsData were censored January 31, 2013.CDI cases decreased to 25 compared to 50 the preceding year .In the study period, 100 patients were isolated resulting in a total of 486 isolation days. In all, 20 tested positive, of these 3 with ribotype 027. CDI accounted for a total of 173 isolation days. Average duration of isolation for patients with negative microbiologicial test results was 4.6 days.Average turnaroundtime was 3.3 days; transportation to laboratory accounted for 2.2 days.55/100 patients had received AB therapy within 2 months prior to the episode; these included all patients with microbiologically verifiedCDI .Compliance with isolation precautions was high throughout the study: proper signage 95%, proper use of protective gear 95%, and proper waste management 95%.Daily rounds to audit implementation of isolation precautions documented strict adherence to guidelines during the study period. This may have contributed to the observed decrease in number of CDI.Only 35% of isolation days were implemented for patients with CDI. Information on prior AB treatment could be a useful decision parameter to implement isolation precuations.Point of Care testing to eliminate transportation time to laboratory across sea could reduce turnaround time.Both actions may reduce the number of isolation days unrelated to CDI.None declared"}
+{"text": "BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrolment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrolment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don't support the previous finding of association between hereditary breast cancer and shorter TL.This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of  Telomeres are hundreds to thousands of nucleotide repeats (TTAGGG) located at eukaryotic chromosome ends Many factors are known to be associated with reduced TL, a primary factor being age with peripheral blood lymphocyte telomeres shortening by, on average, 41 base pairs per year Conflicting results have come from studies investigating links between cancer risk and TL. A recent meta-analysis demonstrated an association between shorter TL and increased cancer risk in studies of bladder cancer, oesophageal cancer, gastric cancer, head and neck cancer, ovarian cancer and overall incident cancers BRCA1 or BRCA2 gene mutations is known to increase breast cancer risk and to a lesser extent prostate cancer risk. A recent paper demonstrated reduced TL in breast cancer patients who carried mutations in the BRCA1/2 genes compared with sporadic breast cancer cases BRCA1/2 mutation carriers with prostate cancer. Preclinical studies have suggested that BRCA2 has a role in telomere stabilisation BRCA1 expression may have an effect on TL BRCA2. Both papers from Delgado-Martinez et al. included larger numbers of cases with mutations in BRCA1 than in BRCA2. In this study, we have analysed TL in two distinct cohorts; a retrospective female cohort of BRCA1/2 mutation carriers and their non-carrier relatives, a proportion of whom have a previous diagnosis of breast cancer, and a prospective male cohort of BRCA1/2 mutation carriers and controls, all cancer free at time of blood donation, a proportion of whom have developed prostate cancer during follow-up. Although BRCA1/2 mutations are predominantly associated with breast and ovarian cancer risk the risk of prostate cancer is increased in males, and TL may be useful for risk stratification. The association between genetic status and TL should not be affected by retrospective or prospective nature of the studies.Both prostate and breast cancer have a heritable component to their aetiology IMPACT and RMH Carrier Clinic Set were approved by the National Health Service, Health Research Authority, National Research Ethics Service, London (reference numbers 05/MRE07/25 and 05/Q0801/7 respectively); all participants gave informed written consent.BRCA1 and BRCA2 mutation carriers and controls from the IMPACT study (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted Screening in BRCA1/2 mutation carriers and controls), an international study set up to evaluate PSA screening in male BRCA1 and BRCA2 carriers. To be eligible, men must be either a BRCA1 or BRCA2 mutation carrier or from a family harbouring the gene mutation but have tested negative themselves (controls). All men were aged between 40 and 69 years with no history of prostate cancer, and no previous biopsy for raised PSA. The recruits gave a blood sample for DNA extraction at enrolment and underwent annual PSA screening with PSA>3.0 ng/ml triggering a diagnostic prostate biopsy. A total of 240 BRCA1 and 207 BRCA2 male mutation carriers plus 218 controls were used in a prospective study of TL. As only four controls developed prostate cancer these were removed from the analyses i.e. the association between TL and prostate cancer risk was not tested in the non-carriers. Smoking status was divided into four categories  with \u2018no\u2019 being the baseline category in the analyses.TL was measured in blood DNA from male BRCA1/2 mutation carriers and controls (non-carrier family members) were recruited to the RMH Carrier Clinic set, in which they donate blood samples for DNA extraction at enrolment. To be eligible, recruits must be of known BRCA1/2 mutation status and d over 18 years. Only samples from female recruits were used for the TL analysis. A total of 131 BRCA1 and 109 BRCA2 female mutation carriers plus 43 controls were used in a retrospective study of TL. One person was found to have mutations in both genes so was included in the analyses as a BRCA1 mutation carrier.(\u22121/slope)) and three reference DNAs were added to each plate. All samples were tested in duplicate. The primer sequences quantifying TL were: 5\u2032 CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT 3\u2032 (TEL_F) and 5\u2032 GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT 3\u2032 (TEL_R) (Integrated DNA Technologies) Using quantitative real-time PCR, relative TL was measured in DNA commercially extracted from peripheral blood lymphocytes. 20 ng of DNA per sample was dried down in 384 plates. Each plate also contained serial dilutions of DNA giving 100ng DNA to 1ng DNA in duplicate to allow for efficiency calculations  which had the advantage that it could be amplified under the same PCR conditions as the TEL, thus reducing the risk of inter-plate variation between the TEL and CON reactions. The 36B4 primer sequences were 5\u2032 CAGCAAGTGGGAAGGTGTAATCC 3\u2032 (36B4_F) and 5\u2032 CCATTCTATCATCAACGGGTACAA 3\u2032 (36B4_R) (Integrated DNA Technologies) 2O were added to each well; samples were left for two hours in the dark at 4\u00b0C for resuspension of DNA. The thermal cycling profile proceeded as follows: 10 minutes at 95\u00b0C followed by 35 cycles of 95\u00b0C for 15 seconds, 54\u00b0C for 2 minutes and 72\u00b0C for 15 seconds as used previously The single copy gene used as a control (CON) was \u2212\u0394CtT/S \u200a=\u200a2. The relative TL was determined by normalizing the ratio (T/S) of each sample to the calibrator DNA to standardise sample values across all reaction plates. For ease of data manipulation the natural log was taken of this value. Three internal controls DNA were run in each plate. For each run, PCR efficiency and internal controls values were monitored.The Ct value, defined as the number of PCR cycles taken for the amplified DNA to cross a predefined threshold, was used to calculate the telomere repeat copy number to single copy gene copy number ratio (T/S) using Linear regression analysis was used in both sets to assess the association between age and TL. Correlation was assessed using Spearman's correlation.BRCA1 and BRCA2 mutation carriers was stratified by gene.Linear regression analyses were used to correlate TL with carrier status. The analyses were adjusted for age at enrolment and smoking status in the IMPACT set, and adjusted for age in the RMH Carrier Clinic set ; analyses were carried out using a robust variance estimation to account for several individuals coming from the same family. The joint analysis of both Hazard ratios (HR) for prostate cancer according to TL in the IMPACT study were estimated using Cox regression, adjusting for smoking status and age at blood draw. Age at enrolment was used as the left-censor age.BRCA1/2 mutation or having a relative with such a mutation. A diagnosis of breast cancer, particularly at a young age, would very often be part of the criteria for genetic testing, and since women with breast cancer prior to recruitment were not excluded it is likely that cases of breast cancer would be overrepresented in the set. HRs from a standard Cox regression approach would be potentially biased, so we instead used a weighted cohort Cox regression analysis BRCA1 and BRCA2 mutation carriers in that age interval. As the study group was small and the majority of individuals (94%) were aged less than 60 years, weights were calculated using the post-1950 incidence rates. The analyses were carried out using a robust variance estimation to account for several individuals coming from the same family and were adjusted for age at blood draw. The joint analysis of both BRCA1 and BRCA2 mutation carriers was stratified by gene and the BRCA1 mutation incidence rates were used to calculate the weights.Women in the RMH Carrier Clinic set were recruited on the basis of having a BRCA1 mutation carriers, BRCA2 mutation carriers and controls; 21 men had prostate cancer. The expected negative correlation between TL in and age was observed among those unaffected by cancer , see Participants' characteristics are summarised in BRCA mutation status and TL, analysing BRCA1 and BRCA2 mutation carriers separately and combined, and found no significant association, see We investigated if there was any association between BRCA1 mutation carriers, 5 developed prostate cancer, and of the 207 BRCA2 mutation carriers, 16 individuals were diagnosed. Statistical analysis showed no association between TL and prostate cancer risk. Hazard ratios (HR), 95% confidence intervals and P-values for the association of telomere lengths with prostate cancer risk are shown in Of the 240 The participants' characteristics of this cohort are summarised in BRCA mutation status and TL, either considering BRCA1 and BRCA2 mutation carriers as separate groups, or as a combined set, see Stratifying by cancer status, we found no significant association between BRCA1/2 mutation carriers with cancer and unaffected BRCA1/2 mutation carriers. Statistical analysis showed no association between TL and breast cancer risk. Hazard ratios (HR), 95% confidence levels and P-values for the association of telomere lengths with breast cancer risk are shown in TL was compared in BRCA1/2 mutation carriers and the largest published study in female BRCA1/2 mutation carriers. We found no association between TL and BRCA1/2 mutation status in either of the two cohorts. Martinez-Delgado et al BRCA1 mutation carriers (n\u200a=\u200a45), BRCA2 mutation carriers (n\u200a=\u200a48) and healthy controls (n\u200a=\u200a276); there was no difference between affected individuals with a family history but no mutation in BRCA1/2 (n\u200a=\u200a105) and the healthy control group. However, it is not clear whether the effect was due to the BRCA1/2 mutation status or their cancer status, or indeed previous cancer treatment, as both chemotherapy and radiotherapy have been shown to affect TL BRCA1/2 mutation carriers to explore any relationship between TL (plus a variety of metabolic and lifestyle factors) and breast cancer risk, and will avoid the compounding factors such as treatment effects which may bias the retrospective studies To our knowledge this is the first published study of TL in male BRCA1/2 mutation carriers are at increased risk of developing prostate cancer and therefore, despite the limitations of screening, an unscreened cohort would be more likely to have cases of undiagnosed cancer with potential for bias.In the male set it is important that the individuals underwent screening for prostate cancer, as men in the general population are not routinely screened and prostate cancer is a condition which can go undiagnosed for many years. BRCA1/2 mutation carriers with prostate cancer versus cancer free BRCA1/2 mutation carriers. This finding is in keeping with Mirabello et al BRCA1/2 mutation carriers BRCA1/2 mutation carriers. It should be noted that in this study there were only 21 cases with cancer and mean follow-up time was 46 months. Within the IMPACT study, recruits undergo biopsy based on PSA screening with a PSA>3.0 ng/ml triggering biopsy. Given the known limitations of PSA screening Within the IMPACT set there was no difference in TL in BRCA1/2 mutation carrier set, we found no significant difference in TL between those with breast cancer and cancer-free controls. There has been a discordance in previously published studies of TL in association with sporadic breast cancer, with some studies showing association and others not BRCA1/2 mutations and individuals with sporadic breast cancer showed an association between shorter telomeres in those affected by hereditary cancer, but not in those affected by sporadic breast cancer. In this study, we compared BRCA1/2 mutation carriers with cancer and BRCA1/2 mutation carriers without cancer, predominantly to investigate the possibility of using TL as a method of risk stratification amongst BRCA1/2 mutation carriers. We found no evidence that TL could be useful in this way.In the retrospective female Supplement S1The IMPACT study: Identification of Men with a Genetic Predisposition to Prostate Cancer: Targeted Screening in BRCA1 and BRCA2 carriers and controls.The IMPACT study collaborators.(DOCX)Click here for additional data file."}
+{"text": "In the vertebrate head, central and peripheral components of the sensory nervous system have different embryonic origins, the neural plate and sensory placodes. This raises the question of how they develop in register to form functional sense organs and sensory circuits. Here we show that mutual repression between the homeobox transcription factors Gbx2 and Otx2 patterns the placode territory by influencing regional identity and by segregating inner ear and trigeminal progenitors. Activation of Otx2 targets is necessary for anterior olfactory, lens and trigeminal character, while Gbx2 function is required for the formation of the posterior otic placode. Thus, like in the neural plate antagonistic interaction between Otx2 and Gbx2 establishes positional information thus providing a general mechanism for rostro-caudal patterning of the ectoderm. Our findings support the idea that the Otx/Gbx boundary has an ancient evolutionary origin to which different modules were recruited to specify cells of different fates. \u25ba Otx2 and Gbx2 segregate placode progenitors of different fates. \u25ba Otx2 and Gbx2 mutually repress each other. \u25ba Otx2 target activation is required for olfactory, lens and trigeminal specification. \u25ba Gbx2 is required for otic placode specification. In the vertebrate head, placodes give rise to crucial parts of the sensory nervous system including the olfactory epithelium, the lens, the inner ear and the sensory neurons of the cranial ganglia . They foGbx2 is first detected within the posterior neuroectoderm, Otx2 becomes restricted anteriorly , where cells of different placodal fates are interspersed ; their ateriorly . Both fateriorly  and thisteriorly . Howeverteriorly .Does a similar mechanism establish regional identity within the PPR? Some neural plate border derivatives depend on Gbx2 and Otx2 function. Gbx2 is required for neural crest cell formation and transcripts are also found in the PPR . In miceHere, we test the hypothesis that Otx2 and Gbx2 provide a cell intrinsic mechanism to establish anterior-posterior positional information in sensory placode progenitors. We show that they mutually repress each other to form a boundary between prospective otic and trigeminal placodes and mediate cell segregation within the PPR. While Gbx2 is required for otic specification, Otx2 is necessary for the specification of the olfactory, lens and trigeminal placodes. Thus, Otx2 and Gbx2 provide a global mechanism for patterning of the embryonic ectoderm and ensure the coordinated development of the central and peripheral nervous system in the head.Gbx2/Otx2 boundary, stage HH7 embryos were processed for double in situ hybridization (ISH) for Otx2 and Gbx2. The gene expression boundary was determined and expressed as a percentage of the hn-pc distance  were incubated at 38\u00a0\u00b0C for 24\u201330\u00a0h until they had reached the appropriate stage ; HH. SmaXenopus embryos were obtained as described previously  or the activator E1A were injected; fusion of these constructs to the glucocorticoid receptor  or fluorescein labeled RNA probes were used. escribed , and NBTed  and to the ophthalmic Pax3+ (opV) trigeminal territory  changes in neural En-1 expression are rarely observed  and Pax2 (stage 16) is reduced  and provides a positive input for otic specifiers. However, once induced maintenance of otic identity is independent of Gbx2 function.Finally, we tested whether Gbx2 is sufficient to impart otic character to cells in the anterior PPR. Otx2 domain , which also inhibits Pax6 in this territory (a stages  and act a stages . After ia stages . The laca stages  is likelerritory . Like GbOtx2 directly binds to the lens-specific FoxE3 enhancer and together with intracellular effectors of Notch signaling activates its transcription (Although Otx2 and Gbx2 are required for early placode specification, neither factor alone is sufficient to endow cells with new regional character or to induce ectopic placodes. This appears to differ considerably from their activity in the neural tube, where ectopic expression of either factor respecifies anterior-posterior identity . Howevercription .Gbx and Otx form a boundary within the Amphioxus ectoderm (Gbx2/Otx2 apposition in Amphioxus, MHB specific genes such as En, Wnt1, FGF8/17/18 and Pax2/5/8 are not restricted to this boundary (Unpg/Gbx and Otd/Otx also negatively regulate one another to form a boundary that positions En and Pax2/5/8 in Drosophila (Gbx2 and Otx2 form a boundary in the annelid Platynereis dumerilii that corresponds to a band of En expression (The development of cranial sensory placodes and neural crest is considered to be a key step in the evolution of the vertebrate head . Like inectoderm  raising boundary , indicatboundary . A Gbx/Oosophila . In addipression . Togethe"}
+{"text": "Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans.Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 . Investigation into people with PRS identifies a mutation within a highly conserved and functional region of DLX5 that may contribute to the development of PRS. This exemplifies how the dog will help us better understand common birth defects. Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects, affecting approximately 1 in 1500 live human births in the United States Pierre Robin sequence  is a heterogeneous and phenotypically variable subgroup of CP that affects 1 in 8500 live human births In an effort to understand the genetic basis of craniofacial birth defects such as PRS, we used a relatively unconventional model organism, the domestic dog. Dogs have naturally occurring birth defects, with inherited orofacial clefts that resemble those observed in humans DNA samples were collected from 14 NSDTRs that had clefts of the hard and soft palate. To identify loci associated with the CP phenotype in the NSDTR, a genome-wide association was performed in 14 CP NSDTR cases and 72 controls across \u223c173,000 SNPs. After quality control, 109,506 SNPs remained. Chi-square analysis of the remaining SNPs identified an associated region on canine chromosome 14 cfa14; . A homozQuantile-quantile (Q-Q) plots and a genomic inflation factor of 1.05 indicate that there is no underlying population stratification . To confUsing microsatellite markers from the cfa14 interval, linkage analysis was performed on a subset of CP NSDTRs with the associated haplotype and available family members (n\u200a=\u200a8). LOD scores were calculated under a fully penetrant recessive mode of inheritance . A signiAll 14 CP NSDTRs had clefts of the hard and soft palate , but theAlthough unavailable for micro-CT imaging, the 2 CP cases without the associated haplotype exhibited a normal jaw relationship with no relative mandibular brachygnathism. Since these dogs are phenotypically different, they were excluded from the rest of the analysis.Located within the interval defined by the genome-wide association study are 21 genes . DLX5 anTranscript analysis was performed in cDNA from cerebral cortex for both DLX5 and DLX6. DLX5 and DLX6 transcripts were Sanger sequenced in one neonatal CP1 NSDTR with the LINE-1 insertion and compared to one neonatal unaffected control NSDTR. No polymorphisms were identified within DLX5. cDNA from the CP1 NSDTR showed both the wildtype DLX6 transcript and a larger transcript, which contained 1281 base pairs of the intronic LINE-1 insertion .In order to quantify the amount of both wildtype DLX6 transcript and the larger mutant DLX6 transcript, real-time PCR of DLX6 from cerebral cortex cDNA was performed in 3 neonatal CP1 NSDTRs and 3 neonatal unaffected control NSDTRs. REST analysis indicated that in CP1 NSDTRs, when compared to control NSDTRs, the DLX6 wildtype transcript was downregulated by a mean factor of 0.252 (p\u200a=\u200a0.069), while the DLX6 mutant transcript was significantly upregulated by a mean factor of 60.033  and 92 patients with nonsyndromic clefting of the lip and palate (NSCLP) identified 7 novel intronic or 3\u2032UTR variants . The intThe screening of 31 patients with syndromic CP identified 4 novel rare variants, two of which were protein coding . These iA naturally occurring animal model for PRS was discovered in twelve cases of CP1 NSDTRs, which exhibit relative mandibular brachygnathia and cleft palate. A genome-wide association study within NSDTRs with CP identified a shared 5.1 Mb homozygous haplotype among 12 CP1 cases with relative mandibular brachygnathia. From the associated interval, DLX5 and DLX6 were selected as regional candidate genes based on their roles in development. Sequencing of these regional candidate genes in NSDTRs identified an intronic LINE-1 insertion within DLX6 that segregates with the phenotype in the breed. This discovery prompted the sequencing of the same genes in a human cohort of CP cases, which identified a damaging missense mutation within the homeobox of DLX5 in a patient with PRS was identified.\u2212/\u2212 and Dlx6\u2212/\u2212) were perinatal lethal and resulted in brain, bone, and inner ear defects, with craniofacial abnormalities including a cleft palate, hypoplastic condylar process, and shortened mandible \u2212/\u2212;DLX6\u2212/\u2212) exhibited more severe craniofacial, inner ear, and bone defects, and were a phenocopy of split hand/foot malformation The regional candidate genes, DLX5 and DLX6, make up a pair of convergently transcribed homeobox containing transcription factors Phenotypic similarity between CP1 NSDTRs, the PRS patient, and mutant mice are observed with the changes in the condylar process, cleft palate, and relatively shortened mandibles. There may be associated condylar hypoplasia in PRS, but this was not investigated in the PRS patient +/\u2212 mice appear normal, closer investigation indicates that they have a decreased bone mineral density DLX5 and DLX6 contain homeoboxes that regulate transcription by binding to specific DNA sequences. Homeoboxes are highly conserved nucleotide sequences of 180 base pairs. Point mutations located within the homeobox give rise to disease at a higher frequency than mutations located within the rest of the gene and often show a dominant effect due to haploinsufficiency \u2212/+; Msx1\u2212/\u2212) \u2212/\u2212; Msx1\u2212/\u2212), mice have no cleft palate.The DLX5 heterozygous missense mutation within the PRS patient does not segregate with a Mendelian mode of inheritance, but this is not expected since PRS is a complex trait. PRS was not apparent in the mother, but she may have exhibited subtle craniofacial abnormalities that went unnoticed. This likely complex inheritance suggests a role for additional, not yet identified loci or environmental factors. Support for this is observed when mice with heterozygous expression of a Dlx5 null allele and targeted inactivation of a transcription factor responsible for clefting in people, Msx1, exhibit cleft palate  and CP1 NSDTRs phenotypes are the result of truncating the same 3\u2032 sequence of the homeodomain LINE elements are transposable elements that comprise 21% of the human genome, 16% of the dog genome, and often insert into intronic regions The LINE insertion disrupts transcription of DLX6 within CP1 NSDTRs and leads to downregulation of wildtype DLX6 transcript when compared to unaffected NSDTRs. As a result,, only 25% of the normal expression levels are produced. The reduced expression of wildtype DLX6 transcript is not enough to prevent CP and the mandibular abnormalities. DLX5 and DLX6 expression levels have been observed to vary based on the timepoint and tissue examined with complex transcriptional regulation from tissue specific enhancers and noncoding RNAs This discovery provides a genetic tool for the NSDTR breeder who wishes to avoid producing cleft palate affected puppies. The LINE insertion identified within the CP1 NSDTRs is unique to a subset of cases with CP within the breed and cannot be used as a tool to prevent against all genetic causes of CP. CP disease heterogeneity is observed in the NSDTR breed as 2 of the 14 CP cases did not possess relatively shortened mandibles and the associated LINE-1 insertion. This is unexpected in a purebred dog breed with few founders where the inbreeding coefficient is 0.26 In summary, identifying a mutation in an animal model with naturally occurring birth defects has enabled the identification of new candidate genes for PRS in people. This supports the continued use of the naturally occurring birth defects found within dogs and their unique genetic background to further our understanding of commonly occurring birth defects in people.The use of samples involving human participants was approved by the institutional review board at the University of Iowa . Informed consent was obtained and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The collection of canine samples used in this study was approved by the University of California, Davis Animal Care and Use Committee (protocol #16892).Blood and tissue samples from NSDTRs with cleft palate (n\u200a=\u200a14), healthy littermates (n\u200a=\u200a24), parents (n\u200a=\u200a11), unaffected NSDTRs (n\u200a=\u200a153), and dogs with cleft palate across 20 breeds (n\u200a=\u200a35) were collected from privately owned animals. When available, tissue was collected at post mortem examination and flash frozen. The evaluations of orofacial clefts were performed by visual inspection of affected dogs. Blood samples from unaffected dogs (n\u200a=\u200a284) across 69 other breeds were collected from the William R. Pritchard Veterinary Medical Teaching Hospital. Genomic DNA was extracted from EDTA whole blood and tissue samples using Gentra Puregene DNA purification extraction kit .Genome-wide SNP genotyping was performed with 14 cases and 72 controls using the Illumina CanineHD BeadChip  with 173,662 markers. Samples had a genotyping call rate of \u226590%. 63,195 SNPs were excluded due to a minor allele frequency of \u22640.05 and 2,994 SNPs were excluded for a missing call rate of \u226410%. Chi-square analysis was performed using Plink High-resolution micro-computed tomography (micro-CT) was used to evaluate craniofacial structures in 4 CP1 NSDTRs that were homozygous for the LINE-1 insertion, and in 3 normal NSDTRs homozygous for the wildtype allele. Samples were imaged at the Center for Molecular and Genomic Imaging (UC Davis). The skulls were kept in zip-lock bags and allowed to warm to the CT scanner temperature (29\u00b0C) inside of a custom plastic holder. CT images were obtained on the Centers MicroXCT-200 specimen CT scanner (Carl Zeiss X-ray Microscopy). The CT scanner has a variable x-ray source capable of a voltage range of 20\u201390 kV with 1\u20138W of power. Samples were mounted on the scanners sample stage, which has submicron level of position adjustments. Scan parameters were adjusted based on the manufacturer's recommended guidelines: source and detector distances were 108 mm and 35 mm, respectively; the manufacturers LE4 custom filter was used for beam filtration; the voltage and power were set to 70 kV and 8W, respectively; and 1600 projections were acquired over 360-degrees with an exposure time of 1.5 s. Images were reconstructed on an isotropic voxel grid with 51.1507 microns per edge. Digital TIFF images were imported into Amira 5.4.5  For all specimens, tridimensional reconstructive (3D) images were generated to assess the spatial relationship of the bones. 3D length measurements were performed using the 3D length tool. Visual inspection of the micro-CT images was performed to identify any abnormalities.Four regional microsatellites were mined from the UCSC genome browser (CanFam 2.0) \u201cVariation and Repeats\u201d database within cfa14: 24.2 Mb\u201329.3 Mb. Fluorescently labeled microsatellite primers were designed  using PrPrimers to amplify the exons, intron/exon boundaries, and regions of high conservation within the intragenic and intergenic regions of DLX5 and DLX6 were designed in Primer3  [55]. PC5\u2032AAGATTGTCAGCAATGCCTCC 3\u2032 and reverse primer 5\u2032 CCAGGAAATGAGCTTGACAAA 3\u2032) in these tissues to ensure that equivalent amounts of cDNA were added. Invitrogen 5\u2032 prime RACE system for Rapid Amplification of cDNA ends kit  was used to sequence the 5\u2032 prime end of DLX6 from embryo cDNA. RACE primers were designed using Primer3  tissue protocols. Adult beagle total RNA was obtained from Zyagen . RNA was synthesized into cDNA using Invitrogen Superscript III First Strand Synthesis System to RT PCR kit . GAPDH was amplified (forward primer  Primer3  [55]. RA5\u2032 ACCATCGCTTTCAGCAAACT 3\u2032). Unlabeled reverse primers specific for the LINE-1 insertion (5\u2032 GCAACTAATATTCGATAAAGCAGAA 3\u2032) and wildtype (5\u2032 CTAGGCCCAGAATTCCTCCT 3\u2032) were designed. The PCR program was as follows: 94\u00b0C for 12 minutes, 35 cycles of 94\u00b0C for 30 seconds, 58\u00b0C for 30 seconds, and 72\u00b0C for 45 seconds, followed by 72\u00b0C for 20 minutes. The wildtype product produced a 171 base pair product and the mutant product produced a 184 base pair product. GeneScan 500 ROX size standards were used and the reaction was analyzed on an ABI 3100 Genetic Analyzer . 96 unrelated unaffected NSDTRs and 284 unaffected dogs across 69 breeds were genotyped for the insertion. Nonsyndromic cleft palate cases (n\u200a=\u200a35) across 20 breeds were genotyped. All genotypes were analyzed using STRand software Genotyping primers were designed using Primer3 http://primer3plus.com/). A shared forward primer (5\u2032aaactcagtacctggcccttc 3\u2032) was designed with reverse primers unique to wildtype (5\u2032ccatatcttcacctgtgtttgtg 3\u2032) and mutant (5\u2032aaactcagtacctggcccttc 3\u2032). Semi quantitative PCR using AmpliTaq Gold DNA Polymerase was performed to test the quality of cDNA and primers, to confirm product size and to check for the presence of genomic DNA contamination. Real-time PCR was performed using the Rotor-Gene SYBR Green PCR Kit  using a 2-step cycle protocol  on the Rotor Gene Q real-time PCR system. Cerebral cortex from 3 neonatal unaffected NSDTRs and 3 neonatal CP1 NSDTRs were run in triplicates with each replicate containing 0.2 ng template cDNA. cDNA was prepared as described above. All data was normalized to the housekeeping gene B2M5 Primer sequences were generated using Primer3Plus  from the US, 59 samples from individuals with nonsyndromic cleft palate (NSCP) from the US and the Philippines, and 46 samples from individuals with cleft palate syndromes. The 46 syndromic samples consisted of 30 samples from individuals with PRS and 16 samples from individuals with additional congenital anomalies including club foot, hearing loss, heart disease, and intellectual disability. 60 unrelated CEPH samples (CEU) were used as controls.Primers were designed with Primer3 to cover the complete gene region of DLX6 and all exons of DLX5 Three SNPs  were genotyped in 362 case-parent trios  with nonsyndromic cleft lip with or without cleft palate (NSCL/P) from the US using TaqMan SNP Genotyping Assays  on the ABI Prism 7900HT, and were analyzed with SDS 2.3 software . The Family Based Association Test (FBAT) in STATA (v12.1) was used to test for association these NSCL/P case-parent trios Table S1\u03b8). Genomic location based on Can Fam 2.0 assembly.Location of microsatellite markers on CFA14, calculated LOD scores, and recombination fraction ((DOCX)Click here for additional data file.Table S2Average lengths of neonatal NSDTRs mandibles. Length measurements of the mandible are taken from the angular process to rostral tip of the mandibular body.(DOCX)Click here for additional data file.Table S3Genes located within associated interval on chromosome 14. Genomic locations are based on the Can Fam 2.0 assembly and refer to chromosome 14 base pair locations. Genes listed were identified using the UCSC genome browser RefSeq gene annotation track.(DOCX)Click here for additional data file.Table S4a LINE insert begins at cfa14.25016704. S \u2013 primers used for sequencing.Primers and annealing temperatures for sequencing of canine samples. Genomic locations are based on the Can Fam 2.0 assembly and refer to chromosome 14 base pair locations. (DOCX)Click here for additional data file.Table S5Primers and annealing temperatures for sequencing of canine samples. Genomic locations are based on the hg19 assembly and refer to chromosome 7 base pair locations. S \u2013 primers used for sequencing.(DOCX)Click here for additional data file."}
+{"text": "Overexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization.in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections.This study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors.For this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two Retesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories. Human epidermal growth factor receptor 2 (HER2) is a member of the family of tyrosine kinase receptors. Overexpression of the HER2 receptor generally results from HER2 gene amplification and occurs in approximately 10% to 20% of primary breast carcinomas  and somein situ hybridization). A 2007 report by an American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) panel has estimated that 20% of HER2 testing might be incorrect [et al. retested tumors treated with trastuzumab in the National Surgical Adjuvant Breast and Bowel Project protocol 31 (NSABP-31) trial which compared the addition of trastuzumab to adjuvant chemotherapy [et al. also showed that the concordance between local and central testing was found for FISH (88.1%), Herceptest (81.6%) or other IHC methods (75.0%). Fewer studies have described the frequency of false-negative HER2 test results. O'Malley et al. reported concordant results between 94.8% to 100% for IHC and 98.5% for FISH for all HER2 negative tumors [The HER2 status of a tumor can be assessed by various methods, several of which have been approved for clinical use, including immunohistochemistry (IHC), FISH, SISH and CISH , Netherlands Cancer Institute (Amsterdam), Diakonessenhuis (Utrecht), Isala Klinieken (Zwolle), Leiden University Medical Center (Leiden) and University Medical Center (Groningen). Tumors from the Academic Medical Center Amsterdam were from patients treated in 2006 and 2007. Patients from the Leiden University Medical Center were treated between 2006 and 2008. Tissue blocks that were used in this study were all acquired during routine patient care. According to Dutch law, these can be freely used after anonymizing the tissues, provided these are handled according to national ethical guidelines . An H & E stained section from each tumor was used to identify an area with invasive breast cancer. From each tumor three cores with thickness of 0.6 mm were collected using the Beecher TMA instrument and inserted in a donor block. Each donor block was stained with the antibodies SP3 , 4B5  and Herceptest . Mono color and dual color SISH was performed with the SISH kit obtained from Ventana using the Benchmark XT.in situ hybridization was performed according to ASCO guidelines [Scoring for IHC and idelines . In brieEach TMA was scored by two pathologists. For 4B5, 51 (4.7%) out of 1,093 results showed a discrepancy between two observers (Cohen's \u03ba = 0.787). For SP3, 37 (3.4%) from 1,077 cases showed a discrepancy (\u03ba = 0.833). For Herceptest, 53 (4.8%) out of 1,107 cases showed a discrepancy (\u03ba = 0.743). For 786 mono color SISH cases, 22 results (2.8%) were discordant between two observers (\u03ba = 0.838). For 914 dual color SISH cases, 43 results (4.7%) were discordant between two observers (\u03ba = 0.671). Significantly discrepant scores between the two observers were reviewed by one observer (TD) to resolve the final score. In order to assess the concordance between mono color and dual color SISH, TMA results from all mono color and dual color SISH tested tumors were compared. Tumors that were equivocal on dual color SISH were not considered discordant with either HER2 non-amplified and HER2 amplified mono color SISH results for the same tumor. All tumors that were discordant between mono and dual color SISH were reviewed and scored again on the TMA. When discordant results existed between mono color and dual color SISH, the IHC results from these discordant tumors were evaluated. Results of the different HER2-antibodies were evaluated by determining the number of cases with discordant results between IHC and mono color SISH: HER2 amplified tumors with 0 or 1+ scores on IHC  and HER2 non-amplified tumors with 3+ IHC scores . Positive predictive values were calculated as the percentage of the total number of SISH amplified cases for all 3+ IHC scores.in situ hybridization in case of a 3+ result. For all other centers, in situ hybridization was performed only in the case of a 2+ result. If HER2 gene amplification was present, the HER2 status was scored as positive. If no HER2 gene amplification was detected, the HER2 status was scored as negative. The final HER2 score on the TMA and the HER2 scores from the report were compared for all tumors. If there was a discrepancy in the HER2 score between the TMA score and the score recorded in the pathology report, a whole tissue block of the breast carcinoma was sectioned and used to perform additional staining and in situ hybridization.HER2 scores were retrieved from the pathology reports supplied by participating centers. Four centers performed HER2 testing on the surgical specimens. The other two centers routinely performed HER2 testing on the pre-operative core needle biopsies (CNB). For almost all cases, the algorithm used to obtain a HER2 score was to perform IHC staining first. When 0 or 1+ staining results were observed, the tumor was regarded as HER2 negative. A HER2 3+ score resulted in a HER2 positive score. However, the two centers that determined HER2 status on CNB also performed A total of 1,210 invasive primary breast carcinomas were included in this study. Complete mono color SISH and dual color SISH scores were obtained for 971 tumors. The remaining 239 tumors had incomplete results, due to folding of the core, loss of tumor material or insufficient amounts of invasive breast cancer for scoring. Using mono color SISH, 881 tumors (91%) were non-amplified (HER2 copy number < 6) and 90 (9%) tumors were amplified (HER2 copy numbers > 6). For dual color SISH, 833 tumors (86%) were non-amplified (HER2 to chromosome 17 probe ratio < 1.8), 20 tumors (2%) were considered equivocal for amplification (1.8 < HER2 to chromosome 17 probe ratio < 2.2) and 118 (12%) tumors were amplified (HER2 to chromosome 17 probe ratio > 2.2). Thirty-two tumors were amplified with dual color SISH while negative with mono color SISH, and two were amplified with mono color SISH but were negative for HER2 amplification with dual color SISH. These 34 tumors were thus considered to be discordant between mono color and dual color SISH. Results from the 34 discordant tumors were revised. At this repeated assessment, 11 tumors initially scored as HER2 amplified with dual color SISH were scored as negative for amplification, eight tumors were scored equivocal for amplification and 11 tumors were again scored as HER2 amplified. At repeated assessment of mono color SISH results, two tumors that were initially scored as negative for amplification were scored as HER2 amplified and one tumor initially scored as positive was scored as HER2 negative. After this revision, the number of discordant results was reduced to seven. All these tumors were amplified with dual color SISH (ratios were between 2.2 and 2.97) while no amplification was found with mono color SISH. HER2 gene copy numbers for these tumors with mono color SISH were two (one case), three (five cases) or four (one case). We compared the IHC results for these discordant cases, and only one case showed 3+ staining for at least one of the antibodies used when dual color SISH showed HER2 gene amplification, but mono color SISH showed no HER2 gene amplification. We decided to use the revised mono color SISH results to determine HER2 gene amplification for this HER2 TMA assessment. Overall, correlation between these two SISH methods when considering amplified and non-amplified tumors was very high after revision  of all 1,210 cases, complete results were obtained with 4B5, SP3, Herceptest and mono color SISH. In 847 tumors of these cases (84.7%) no HER2 protein overexpression was found with SP3, 4B5, Herceptest (0 or 1+) or mono color SISH (HER2 copy number < 6). Sixty tumors showed 3+ staining with all three antibodies and HER2 gene amplification (6.0%). For the remaining 93 complete cases, the final TMA HER2 status was determined as follows: 65 tumors (6.5%) showed 0, 1+ or 2+ IHC scores and were negative for HER2 gene amplification, and were thus judged to be HER2 negative. Twelve tumors (1.2%) showed 3+ or 2+ IHC scores and were positive for HER2 gene amplification and were thus judged to be HER2 positive. For 16 cases (1.6%), there was a true discordance between at least one of the antibodies mono color SISH result .The most frequent discordant result between TMA scores was a false-negative result with the Herceptest (IHC score of 0/1+ with Herceptest and 2/3+ with SP3 and 4B5 and positive SISH) which occurred in eight cases. Tumors stained with Herceptest frequently displayed prominent cytoplasmic and moderate but incomplete membranous staining (1+), while 4B5 and SP3 antibodies displayed complete membranous staining. There were four cases for which all three antibodies showed 3+ staining on TMA, while mono color SISH did not detect HER2 gene amplification. Other discordant results include 0/1+ staining with all three antibodies while mono color SISH showed HER2 gene amplification (three cases) and 3+staining with 4B5 and Herceptest while SP3 and mono color SISH were negative (one case). When Herceptest results were omitted, the number of interpretable cases for 4B5, SP3 and mono color SISH increased to 1,020 and the number of discordant results was reduced to eight (0.8%). In total, 932 tumors were HER2 negative and 80 tumors were HER2 positive when determined with these three methods . Sensitivity and specificity for all centers combined was 98.7% and 99.3%, respectively Table .Of the 20 discordant cases, 13 were scored as HER2 positive in local centers but were negative in our TMA retesting  Table . AnotherFor the remaining discordant cases, whole tumor slides stained with SP3, 4B5 and mono color SISH were evaluated in order to assess the reason for the discordant result. For the 2+, ISH amplified results, complete membranous staining on IHC could be reproduced with the SP3 and 4B5 antibodies in all cases. The local ISH results showed low level HER2 gene amplification (six to ten) in four of six tumors and high level HER2 gene amplification (> 10 copies) in two of six tumors, but all were negative for amplification determined with mono color SISH on both TMA and subsequent testing of the whole tumor. These data indicate that the local IHC result was reliable, but the reason for these results was false-positive local ISH due to the ISH procedure. For the remaining three local 3+ results, 3+ staining could not be reproduced with SP3 and/or 4B5 antibodies on TMA for two tumors and these were both negative for gene amplification on mono color SISH on TMA and on full-sized slides. For these tumors, the reason for discordance was the local IHC procedure leading to false 3+ results. The remaining tumor was 3+ on local slides and was also 3+ on SP3 and 4B5 stained slides, while the mono color SISH result was non-amplified (two to three copy numbers). This tumor thus likely represents a case of protein overexpression without gene amplification . SP3 is another monoclonal rabbit antibody, which has also been compared to the CB11 antibody  and A048In conclusion, we have shown that TMAs can be used to evaluate the quality of HER2 testing in pathology laboratories. This provides a reliable evaluation of HER2 testing performance. In the first assessment performed in this way, we found HER2 testing sensitivity to be 98.7% and specificity to be 99.3%. As we have now established this TMA-based evaluation in combination with full-sized slides for discordant cases, we will also offer this to other laboratories.in situ hybridization; FISH: fluorescence in situ hybridization; H & E: haematoxylin and eosin; HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry; ISH: in situ hybridization; NSABP: National Surgical Adjuvant Breast and Bowel Project; SISH: silver in situ hybridization; TMA: tissue micro-array.ASCO/CAP: American Society of Clinical Oncology/College of American Pathologists; CISH: chromogenic All participating hospitals have received funding for this study from Hoffman-La Roche. The following additional possible financial conflicts of interests have been declared: TJA Dekker received lecture honoraria from Hoffman-La Roche. S Ter Borg received lecture honoraria from Hoffman-La Roche. JE Boers has received travel reimbursements, lecture honoraria and research funding from Hofman-La Roche. MJ van de Vijver has received research funding, lecture honoraria and is member of the pathology advisory board from Hofman-La Roche. E Schuuring has received travel reimbursements, lecture honoraria and occasional honoraria for advisory board member for Hoffman-La Roche, and received lecture honoraria from Abbott .TJAD, STB and GKJH participated in data collection, interpretation and analysis. SM, JW, JEB, ES, JB and VTHBMS participated in the design of the study, included tumor material in the study and scored slides. WEM and JRK participated in writing of the manuscript and data interpretation. MJvdV coordinated the study, participated in its design, data interpretation and writing of the manuscript. All authors have read and approved the manuscript."}
+{"text": "Cryptococcus neoformans to resist oxidative stress is one of its most important virulence related traits. To cope with the deleterious effect of cellular damage caused by the oxidative burst inside the macrophages, C. neoformans has developed multilayered redundant molecular responses to neutralize the stress, to repair the damage and to eventually grow inside the hostile environment of the phagosome. We used microarray analysis of cells treated with hydrogen peroxide (H2O2) at multiple time points in a nutrient defined medium to identify a transcriptional signature associated with oxidative stress. We discovered that the composition of the medium in which fungal cells were grown and treated had a profound effect on their capacity to degrade exogenous H2O2. We determined the kinetics of H2O2 breakdown by growing yeast cells under different conditions and accordingly selected an appropriate media composition and range of time points for isolating RNA for hybridization. Microarray analysis revealed a robust transient transcriptional response and the intensity of the global response was consistent with the kinetics of H2O2 breakdown by treated cells. Gene ontology analysis of differentially expressed genes related to oxidation-reduction, metabolic process and protein catabolic processes identified potential roles of mitochondrial function and protein ubiquitination in oxidative stress resistance. Interestingly, the metabolic pathway adaptation of C. neoformans to H2O2 treatment was remarkably distinct from the response of other fungal organisms to oxidative stress. We also identified the induction of an antifungal drug resistance response upon the treatment of C. neoformans with H2O2. These results highlight the complexity of the oxidative stress response and offer possible new avenues for improving our understanding of mechanisms of oxidative stress resistance in C. neoformans.The ability of the opportunistic fungal pathogen  Cryptococcus neoformans is pathogenic basidiomycetous yeast with a ubiquitous worldwide distribution. It exists primarily as an environmental organism associated with soil and is known to have a particular association with bird guano C. neoformans is also an important opportunistic pathogen that causes invasive fungal infections and is responsible for about 1 million cases and 700,000 mortalities annually Aspergillus fumigatus, Candida albicans, and C. neoformans by host immune cells in vitro and the corresponding respective virulence and avirulence phenotypes of these strains in a murine cryptococcosis model In the mammalian host, cell mediated immunity based on phagocytic cells is crucial to counteract fungal infections. Macrophages, neutrophils and other phagocytic cells generate potent reactive oxygen (ROS) and nitrogen (RNS) species that are toxic to most fungal and bacterial pathogens and cause damage to their DNA, protein and lipids. ROS and RNS are implicated in the killing of fungal pathogens such as C. neoformans is its ability to survive inside phagocytic cells. It not only resists killing by macrophages after phagocytosis, but can continue to replicate by budding within this environment and subsequently exit the macrophage without causing host cell lysis C. neoformans to survive and thrive inside this harsh environment suggests it must have mechanisms not only to neutralize the reactive molecular species to which it is exposed within the macrophages but also to repair the cellular damages caused by the oxidative and nitrosative stresses.An important virulence related trait of C. neoformans, a number of genes coding for enzymes of antioxidant defense systems have been shown to be important for both in vitro oxidative stress resistance and also for in vivo pathogenesis C. neoformans resistance to peroxide stress C. neoformans, deletion of these genes renders them sensitive to oxidative stress, albeit with a less severe phenotype than that of a tsa1\u0394 strain, and decreased survival in macrophage culture conditions C. neoformans also contains two glutathione peroxidases Gpx1 and Gpx2, both of which respond differently to various stressors; with only Gpx2 induced in response to H2O2 generated oxidative stress. Furthermore, both GPX1 and GPX2 deletion mutants were only mildly sensitive to oxidant killing by macrophages and exhibited no effect on virulence in a murine model C. neoformans include a cytosolic copper-zinc superoxide dismutase (Sod1) In C. neoformans experiencing oxidative stress have been reported in which the authors either used fungal cells engulfed by macrophages or grown in the nutrient rich YPD medium and treated with exogenous H2O22O2 in YPD medium, suggesting that the environment has a direct effect on the transcriptional response. This may also be due to the differences in the concentration of ROS released by the exogenous application of stress agent compared to that released inside the macrophages. Gene expression studies of oxidative stress resistance in a number of other fungal organisms such as S. cerevisiae, Schizosaccharomyces pombe and Candida sp have been published 2O2 and the magnitude of the elicited cellular response was highly dependent on the composition of the medium in which the cells were grown and treated. For example, S. pombe triggered different signaling networks mediated either by Pap1 or by Sty1 depending on the concentration of H2O2 used for the treatment 2O2 than the Sty1-mediated pathway and was responsible for inducing an adaptive response. This was shown by an induction of Pap1 with an extracellular concentration of 0.2 mM H2O2, whereas H2O2 concentrations above 0.2 mM failed to trigger this activation.Whole genome microarray studies of S. cerevisiae, the authors reported different degrees of sensitivity of yeast cells to external H2O2 when grown in different media 2O2 for gene induction, even though the same strains grown in the presence of glucose exhibited 70% death when treated with 1.5 mM H2O2. The pathogenic fungi C. glabrata has shown to be resistant to up to 40 mM of H2O2, while C. albicans was found to be sensitive at this concentration of H2O2A. fumigatus, continued to grow during two-hour incubations with 5 mM H2O2 and tolerated up to 30 mM H2O2In a study of oxidative stress and aging in 2O2 inside the phagosome during the oxidative burst is not precisely known, however multiple reports suggest that the effective H2O2 concentration may reach hundreds of micro molar in vivo conditions, we chose to treat C. neoformans cells with H2O2 in a nutrient limited YNB medium at pH 4.0. To stress the cells and avoid the induction of an overwhelming apoptotic death response, cells were treated with a concentration of H2O2 that resulted in the killing of \u223c 15% of initial cell population or lethal dose (LD\u223c15) which was determined via H2O2-generated oxidative stress death curves. These curves dictated the use of 1 mM H2O2 for treatment. By quantitatively measuring the concentration of H2O2 in the culture during the treatment, we discovered that within 30 minutes of incubation, all of the H2O2 was completely degraded from the medium. Therefore, we subjected RNA samples isolated from the cells at 5, 15, 30, 45 and 60 minutes post H2O2 treatment to microarray hybridization. We used a custom designed C. neoformans serotype A array using the predicted ORFs in the H99 (serotype A) C. neoformans genome identified by the Broad Institute genome sequencing project (http://www.broadinstitute.org/annotation/genome/cryptococcus_ neoformans/FeatureSearch.html). To facilitate the identification of underlying biological processes from the gene expression dataset, we generated a new gene ontology gene association file for C. neoformans genome. Herein, we report a global time-resolved genomic expression pattern of C. neoformans and identified over-represented biological processes which may point to potential mechanisms by which the fungus detects and destroys the oxidative stressor as well as repairs and recovers from the damage caused by the oxidative stress.In the context of pathogenic fungi and phagocytosis, the concentration of HC. neoformans serotype A strain KN99\u03b1 was used throughout this experiment. Cells were grown on rich medium, YPD , or minimal medium, YNB, pH 4.0 (6.7 g/liter yeast nitrogen base without amino acids plus 20 g/liter dextrose and 25 mM sodium succinate at pH 4.0). Solid media contained 2% Bacto agar. Antimycin-A from Streptomyces sp. (Cat No A8674), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Cat No -C2920), Salicylhydroxamic acid (SHAM) (Cat No -S607) and hydrogen peroxide solution (Cat No H1009) all were purchased from SIGMA-ALDRICH, St Louis, MO, USA. Estimation of H2O2 was performed using OxiSelect H2O2 assay kit from Cell Biolabs, San Diego, CA, USA following the protocol supplied with the reagent. Three independent experiments were carried out to calculate the standard error which is indicated by the error bars in the figures.650\u200a=\u200a1.5) were treated with various concentrations of H2O2 in a shaking 30\u00b0C incubator. Aliquots were taken at various time points, cells pelleted by centrifugation at 4\u00b0C. Cell pellet was washed two times with cold PBS and finally resuspended in PBS for plating on solid YPD and incubated at 30\u00b0C. Colony forming units (CFU) were counted after 2 days.Exponentially growing cells  C. neoformans KN99\u03b1 were used to inoculate three bioreplicates into YNB pH 4.0 media and incubated at 30\u00b0C with shaking (200 rpm) until the cells were in mid-log phase (OD650 \u223c1.5\u20132.0). Each biological replicate culture was split into two cultures and H2O2 was added to one of each of these two cultures to a final concentration of 1 mM. Cultures were incubated for 60 minutes and sampled at 5, 15, 30, 45 and 60-minute intervals. Sodium citrate  was added to control and test samples to rapidly halt the hydrogen peroxide stress. Cells were collected by centrifugation at 1800 g for 5 minutes and washed once with sterile phosphate buffered saline. The washed cell pellets were flash frozen and lyophilized. Lyophilized cells were stored at \u201380\u00b0C.Two-day-old cultures of 8 cells) was vortexed with 0.5 g of 1 mm glass beads  for 5 min., followed by the addition of 600 uL lysis solution (kit supplied) and vortexed for a further 5 min. Disrupted cells were centrifuged at 16 000 g for 3 min. and the supernatant transferred to a pre-filtration column. Rest of the procedures was carried out as described in the Manufacturer\u2019s protocol until the final step. Isolated RNA was treated with RNase free DNase. RNA was quantified using a Nanodrop ND-1000 . The quality of purified RNA was assessed on an Agilent 2100 Bioanalyzer .RNA was isolated using the Agilent Total RNA Isolation (Protocol for Yeast)  according to the manufacturer\u2019s instructions with the following modifications. The lyophilized pellet  according to manufacturer\u2019s instructions with the following modifications. All reaction component volumes were doubled with the exception of RNA concentration resulting in a final reaction mix volume of 40 uL. After the addition of Stop buffer, to remove any unincorporated dye, the reaction mix volume was brought up to 100 uL with nuclease-free water followed by the addition of 3 volumes of 100% nuclease-free ethanol. This 400 uL reaction volume was then applied to an RNeasy Mini Kit column  and processed according to manufacturer\u2019s instructions . Dye incorporation was quantitated using a Nanodrop ND-1000 (Nanodrop Technologies) by measuring emission wavelength at 570 nm (Cy3) and 670 nm (Cy5). Labeled-RNA concentration and dye incorporation were used to determine the RNA labeling specific activity (pmol/ug). Labeled and unlabeled RNA were combined to adjust the sample specific activity to the empirically determined intensities of 40 and 45 pmol/ug for Cy3 and Cy5 labeled samples, respectively. Unstressed Cy3-labeled and 1 mM HMicroarray hybridization and scanning were preformed according to Agilent Microarray processing specifications .rijk\u200a=\u200a\u03b1+Ai+Gk+(AG)ik+Dl+\u03b1rijk; where yrijk is the logarithm of the rth replicate model-based expression value of gene k at the ith time point with the jth treatment . This model accounts for variance in genes (G), arrays (A), and array plus gene; \u03b1 term accounts for error.The LOESS balanced data from the Feature Extraction was used to assess replication consistency across biological and process replicates using a linear ANOVA model that considers all 13,864 probes on the array and data for all the replicates, to determine the significance of differential expression Archive RNA extracted for the microarray experiments was used to make first-strand cDNA using the First-Strand cDNA synthesis kit for reverse transcription-PCR (Roche). This cDNA was used as template for real-time PCR analysis using SsoFast SYBR green PCR reagents (Biorad) according to the manufacturer\u2019s recommendations. A BioRad CFX96 thermal cycler was programmed with the following two step PCR cycles: 5 s at 98\u00b0C, 5 s at 60\u00b0C, and a plate read was repeated in the second step for a total of 40 cycles. A melting curve was performed at the end of the reaction to confirm a single product. Standard curves were performed for each primer set and efficiencies calculated. The data were normalized to glucose-6-phosphate dehydrogenase cDNA expression included with each experiment.http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/Info.html) were submitted to the GOAnno program of AgBase \u221210. The sequences and annotations from the Uniprot database were filtered to remove sequences that had only annotations with an evidence code of IEA (Inferred from Electronic Annotation) or ND . Additional annotation was obtained by submitting those proteins without a match in the UniProt database to InterProScan \u221220. Gene ontology term enrichment was carried out using a hypergeometic test as implemented in the GOstats program, using a p-value of \u22640.01 as a criteria for significance The predicted protein sequences of H99 serotype A from the Broad assembly 2  for 30 min. At the end of the incubation, cells were rapidly chilled and collected by centrifugation. Cell pellet was lysed in 8 M urea containing buffer and equal amounts of protein per sample were used for immune blot analysis using antibodies specific to ubiquitin proteins . Uniform transfer of proteins across the membrane was verified by staining the membrane with Swift Membrane Stain  before subjecting them to immuno blot analysis.2O2 once it has been added to C. neoformans culture, actively growing yeast cells in YNB, pH 4 medium were treated with 1 mM H2O2. At different time intervals aliquot of the cell culture was removed, cells were separated and the supernatant was used to measure the residual H2O2 in the medium. A concentration of 1 mM H2O2 was rapidly degraded by growing C. neoformans cells and was completely absent in the medium after 30 minutes  were decreased to \u223c40\u201350% of the initial concentration by two hours, clearly indicating that unlike the cells grown in YPD, cells grown in nutrient limited YNB medium are much less efficient in degrading exogenous H2O2. at 30\u00b0C . However of H2O2 . Higher 650\u200a=\u200a1\u20138) in either YPD or YNB medium and treated with various concentrations of H2O2 (2\u20138 mM), the rate of H2O2 breakdown increased as the culture density increased (data not shown). A cell culture grown in YPD to a density of OD650\u200a=\u200a2.7 was able to degrade 4 mM of H2O2 in just 20 min, while those with a density of OD650 \u2265 4.0 took only 10 minutes . A concentration of 0.5 mM H2O2 had no significant effect on cell viability at any time point  model as previously described 2O2 in C. neoformans  at the five minute time point . At 15 mresponse  is consist cells .2O2 treatment in our experiment (treatment in YNB medium) to a previously published microarray dataset by Ko et al in which authors employed YPD medium for growth and treatment with H2O2. Consistent with our present findings on the influence of media composition and treatment conditions on the oxidative stress response we found considerable differences in the gene sets, but a high level of concordance for those genes identified as differentially expressed in both datasets 2O2 degradation, array probe differences, strains used and the intrinsic variation of experiments done in two different laboratories. We believe the high degree of concordance between the overlapping probes strongly validates both experimental approaches.We compared the sets of genes differentially expressed in response to H2O2 treatment followed similar profile to microarray dataset validating the quality of our array. The linear regression analysis of the microarray and qRT-PCR measurements resulted in a correlation coefficient  of 0.94, suggesting that array dataset correlated positively and closely with qRT quantification (data not shown).Microarray results were validated by performing qRT-PCR analyses of six genes  that encompassed a range of expression levels at 30 min time point. The mRNA abundance of these transcripts at 30 min after H2O2 treatment and recovery of the viable cells at two hour time point  enrichment analysis was performed and several biological processes were significantly enriched in our lists at all time points  and S3. me point  emphasiz2O2 causes substantial perturbation of cellular processes.In addition to the above biological processes, a greater number of unique cellular processes significantly affected only at 30 and 45 min time points were identified . They in2O2 treatment  TRR1 coding for thioredoxin reductase (CNAG_05847) CCP1; CNAG_01138) CAT1 (CNAG_0498) and CAT3 (CNAG_00575) 2O2-induced stress and further validating the quality of our array.As expected, our dataset identified oxidation-reduction process as one of the top ten enriched categories affected by Hreatment . Moreovereatment . Some ofC. neoformans genome using our GO database assigned a total of 514 genes to the oxidation-reduction functional category. We discovered that 205 genes exhibited differential expression at one or more time point with a maximum of 124 genes showing altered mRNA levels at 45 min post H2O2 treatment . The sulfiredoxins are critical for oxidative stress resistance in yeast and higher eukaryotes C. neoformans or in other fungal species. A preliminary bioinformatic analysis of a few select genes supported their potential roles during oxidative stress in C. neoformans and these are discussed below.In addition to the core antioxidant response, our microarray data revealed the contribution of several additional gene products in oxidative stress resistance that may provide clues to novel mechanisms of stress resistance. The strong transcriptional regulation of 50 genes for a significant period of time during He stress . For exa2O2 treatment. Bacterial dioxygenases are reported to have either iron-sulphur center or non-heme mononuclear iron as cofactors. Importantly, these enzymes catalyze oxidation of the substrates at the expense of reduced NADPH C. neoformans after H2O2 treatment suggests that this may be a part of protective mechanism of the yeast cells to inhibit NADPH-requiring reactions while cells are coping with oxidative stress.One of the genes that are under persistent transcriptional regulation in response to peroxide stress is CNAG_03238, predicted to belong to dioxygenase subfamily. It is similar to a putative dioxygenase gene from a saprophytic soil borne filamentous bacteria, and shows significant repression at four of the five time points post HS. cerevisiae provides increased resistance to menadione-induced stress Arabidopsis and other higher eukaryotes Another uncharacterized gene, CNAG_01542, is predicted to have domains belonging to taurine catabolism dioxygenase super-family and exhibits increased expression at 30, 45 and 60 minutes. Proteins containing these domains have been demonstrated to be important for sulfonate metabolism, in the synthesis of Fe-S cluster protein family members and their expression in C. neoformans genome, with 272 (\u223c45%) showing differential expression at one or more time points during H2O2 treatment  a majority of the metabolic process related genes were still under transcriptional regulation  as well. Sterol analysis of the flucanazole resistant C. albicans showed defects in ERG2 and ERG3S. cerevisiae ERG2 (CNAG_00854) and ERG3 (CNAG_00519) showed altered expression levels in our microarray dataset  and a homolog of MDR1 (CNAG_00796). All three genes show differential transcriptional regulation, with CDR11 showing consistent up regulation at 15, 30 and 45 minutes and MDR1 exhibiting increased levels at 30, 45 and 60 minutes , and AOX1, encoding an alternative oxidase (CNAG_00162). C. neoformans CCP1 is known to protect against external oxidative stress inducing agents and consistent with that role, its transcription was increased at 30 and 45 minutes . By contrast, yeasts such as C. albicans and S. cerevisiae cells were able to grow normally in the presence of 2.5\u20135 ug/ml of FCCP 2O2 in the presence and absence of FCCP and incubated at 30\u00b0C. The capacity to withstand oxidative stress triggered by H2O2 challenge was markedly decreased by FCCP in a concentration dependent manner  gene family have been implicated in a wide variety of cellular functions including those closely connected with oxidative stress resistance such as respiratory growth, glutathione homeostasis, UV and metal ion resistance and double strand DNA repair UBC gene deletion strains exhibit varying degree of sensitivities to diverse external stress conditions indicating the functional overlap between different UBC gene products 2O2 induced oxidative stress involves protein damage by irreversible oxidation and these oxidized proteins are recycled through the ubiquitin dependent proteasomal pathway in other systems C. neoformans, we treated fungal cells with increasing concentration of H2O2 and subjected the cell lysates to immuno-blot analysis. The blots were probed with ubiquitin specific antibody to quantify the amount of ubiquitin tagged proteins in treated cells to their untreated counterpart. We observed an H2O2 concentration dependent increase in the amount of ubiquitin conjugated proteins in the total cell lysate , deubiquitinating enzyme (GO: 0004843), ubiquitin-dependent protein catabolism (GO: 0006511), protein ubiquitinylation (GO: 0016567), and proteosomal ubiquitin-dependent protein catabolism (GO: 0043161) in the s genome . Of the reatment . In S. cl lysate . Densitol lysate .C. neoformans both for establishing an infection in the host and also for surviving extended periods of time in the environment. The kinetics of H2O2 degradation by cells growing in YNB indicates that actively growing C. neoformans cells have an efficient mechanism to degrade exogenous H2O2. This may involve the activity of functional catalase enzymes reported to be present in C. neoformans2O2 degrading activity in the culture supernatant is consistent with an earlier study reporting the absence of secreted catalase family proteins in C. neoformans genome 2O2 breakdown by cells growing in YPD compared to those grown in YNB was rather surprising and clearly suggests that growth media composition may significantly affects cell\u2019s constitutive redox potential. Moreover, we also demonstrated that cells grown to a higher optical density were able to degrade exogenous H2O2 more rapidly compared to the culture at lower optical density, again emphasizing that the cell culture and treatment conditions may significantly influence the magnitude and intensity of the induced cellular transcriptional response to oxidative stress. Therefore these important experimental parameters need to be considered when comparing different datasets generated for H2O2 mediated stress induced either in the same organism or between different organisms.The transcriptional regulation of protective mechanisms against oxidative stress is critical for 2O2 treatment  at the 15 and 30 minute time points consistent with its established role in oxidative stress and virulence C. neoformans TSA1 is a typical 2-cys peroxiredoxin and its homolog in S. cerevisiae is located in the cytoplasm C. neoformans TSA3 (CNAG_06917) and DOT5 (CNAG_02854) belong to 1-cys peroxidase family and have shown increased expression at both protein and mRNA levels in response to oxidative stress in a previous study C. neoformans TSA3 and DOT5 in S. cerevisiae are located to mitochondria and nucleus respectively and are known to play a major role in oxidative stress resistance C. neoformans is not known and their deletion in C. neoformans did not affect oxidative stress resistance in either in vitro or in vivo conditions TSA3 and DOT5 shown in our dataset is similar to the pattern of expression of the majority of genes of potential mitochondrial origin suggesting that both TSA3 and DOT5 may contribute additionally to oxidative stress resistance in C. neoformans by unknown mechanisms.In the oxidation-reduction category we found interesting gene regulation patterns of thioredoxin family genes. Among the genes of thioredoxin family, we found increased expression of C. neoformans showed differential expression. Increased expression of CAT1 is consistent it being the only functional catalase as determined by the in-gel activity staining of C. neoformans cell extracts CAT1 and CAT3 suggests these genes may be regulated by similar factors, consistent with both belonging to the same phylogenetic clade 2O2 in the medium had been degraded. This suggests that the CAT1 and CAT3 gene products may have additional novel roles during in vitro oxidative stress induced by H2O2.Three of the four known catalase genes in C. neoformans undergoes major metabolic adaptation during growth inside the host 2O2 involves the genes encoding pentose phosphate pathway (PPP) enzymes. A major product of PPP is the production of NADPH that is critical for the function of proteins required for repairing oxidative protein damage. Accordingly the components and function of PPP have been shown to be important for resistance and adaptation to oxidative stress in yeast and higher eukaryotes. In both S. cerevisiae and C. glabrata, H2O2 treatment increased the expression of genes belonging to the pentose phosphate pathway ZWF1) is increased during exposure of C. albicans to nitrosative stress S. cerevisiae, independent deletion strains of pentose phosphate pathway genes such as 6-phospho gluconate dehydrogenase (GND1), D-ribulose-5-phosphate 3-epimerase (RPE1), transketolase 1 and transketolase 2 (TKL1and TKL2), glucose-6 phosphate dehydrogenase (ZWF1) and transaldolase (TAL1) all exhibit increased sensitivity to oxidative stress C. neoformans. Previous studies demonstrated that neither the ZWF1 mRNA nor protein levels were altered during nitrosative stress in C. neoformans. Consistent with this deletion of ZWF1 gene in C. neoformans did not increase their sensitivity to either oxidative or nitrosative stress C. neoformans.In addition to their initial exposure to oxidative stress inside the macrophages, CCP1-mediated mitochondrial electron transport chain to meet the sudden increase in demand for ATP necessary for repairing damaged proteins. Previous serial analysis of gene expression (SAGE) revealed a high abundance of tags corresponding to phopsphophenol pyruvate carboxykinase (PCK1), a main control enzyme for the regulation of gluconeogenesis in the lung-exposed cryptococcal library suggesting that gluconeogenesis is important for fungal survival in the host lung due to the limited availability of glucose C. neoformans cells to H2O2 in the presence of glucose we also observed significant and consistent up-regulation of PCK1 (CNAG_04217) after H2O2 treatment , malate synthase (MLS1), pyruvate decarboxylase (PDC1) or alcohol dehydrogenase (ADH1) during oxidative stress in contrast to their increased expression during growth conditions in the host lung. The up-regulation of the glyoxylate pathway during pulmonary infection conditions has been attributed to the higher amount of acetate present in lung tissues 2O2.Unlike the response of the TCA cycle and the gluconeogenic pathway, genes of the glyoxylate cycle that converts acetyl-CoA to succinate for the synthesis of carbohydrates responded completely differently in our dataset compared to the previously published lung exposed cryptococcal SAGE library C. neoformans during nitrosative stress Peroxide-induced oxidative stress also caused significant perturbation of genes of amino acid biosynthesis pathway . IncreasS. cerevisiae transcriptional up-regulation of CCP1 after H2O2 treatment and its role in stress signaling has been reported to contribute to oxidative stress resistance C. neoformans CCP1 was initially identified by bioinformatic analysis, the AOX1gene was identified by its significant up-regulation in C. neoformans in response to exposure to 37\u00b0C temperature AOX1 as being enriched in the mouse lung library, but not CCP1C. neoformans CCP1 and AOX1are potentially subject to different mechanisms. Moreover, the AOX1 deleted strains exhibited a slight virulence phenotype in a mouse model while CCP1 deleted strains were avirulent. However, both CCP1 and AOX1 independent gene deletion strains exhibited in vitro oxidative stress sensitivity towards H2O2 and tert-butyl hydroperoxide respectively CCP1 and AOX1 observed in our dataset in response to peroxide stress further supports that their transcription is under the control of different signaling mechanisms. The absence of AOX1 up regulation seen in our dataset is similar to the one reported in the previously published microarray analysis AOX1observed in our microarray analysis was in agreement with the results obtained in our laboratory when we subjected RNA samples to RNA seq analysis (unpublished results). The functional consequence of differential expression of CCP1 during H2O2 treatment is dramatic and is reflected in the ability of its specific inhibitor antimycin to completely abolish oxidative stress resistance to H2O2, whereas specific inhibition of AOX1 had little effect on oxidative stress resistance.In C. neoformans in the presence of various stress conditions such as nitrosative stress, heat shock and growth inside the host, all identified genes related to the mitochondria and respiratory chain to be differentially expressed S. cerevisiae and C. albicans2O2 provides evidence that C. neoformans cells undergoes substantial damage by H2O2 and energy dependent repair mechanisms are critical for recovering from oxidative stress. The increased concentration of ubiquitin tagged proteins observed by us during H2O2 treatment . UBI4 dependent ubiquitination has been shown to play a major role in the ubiquitin dependent protein degradation pathway during thermal, cell wall and oxidative stress conditions in S. cerevisiae and C. albicansS. cerevisiae RSP5 (CNAG_05355)  . RSP5 enUBC4 and UBC5 in S. cerevisiae were exceedingly sensitive to stress conditions S. cerevisae upon heat stress S. cerevisiae UBC4 in C. neoformans  and RPT gene  families. Several recent reports in yeast (S. cerevisiae) and mammalian systems indicate that degradation of oxidized proteins may occur either by ubiquitin/ATP-dependent  or ubiquitin/ATP-independent  mechanisms 2O2 treatment indicates that the 19S regulatory particle may play a minor role in maintaining cellular homeostasis during oxidative stress in C. neoformans.Deletion mutants of ubiquitin conjugating enzymes ent time . S. cere2O2 removal, we identified many more genes affected by oxidative stress than were identified in previous studies of oxidative stress in C. neoformans. The pattern of the transcriptional response mirrored the kinetics of peroxide removal and allowed us to infer potential mechanisms for the response as well as the recovery from oxidative stress. We found potential novel mechanisms for the role of mitochondria and expanded our understanding of the role of ubiquitin-dependent proteolysis in recovery from oxidative e stress.By examining gene expression differences over a time course that paralleled the kinetics of HFigure S12O2 breakdown by The affect culture density on HC. neoformans cells. A 4 mM of H2O2 was added to cultures at various densities (OD650) growing in YPD (A) and YNB (B). At various time points samples were withdrawn, cells separated by centrifugation and the supernatant was used for H2O2 estimation. The percentage of residual H2O2 was plotted against H2O2 treatment time. Standard bars reflect standard error calculated from three independent experiments.(TIF)Click here for additional data file.Figure S2Differential expression pattern of C. neoformans genes related to anti-fungal drug resistance upon peroxide stress.(TIF)Click here for additional data file.Table S1Global transcriptional profile of C. neoformans2O2 induced oxidative stress. genome during H(XLSX)Click here for additional data file.Table S22O2 treatment.Gene ontology enrichment analysis of the differentially expressed probes at various time points during H(PDF)Click here for additional data file.Table S3Master list of gene ontology annotation of the differentially expressed probes at various time points.(XLSX)Click here for additional data file.Table S4Differential expression pattern of C. neoformans genes assigned to oxidation-reduction functional category at various time points during exposure to oxidative stress.(XLSX)Click here for additional data file.Table S5List of C. neoformans2O2 induced oxidative stress. differentially expressed genes assigned to oxidation-reduction functional category that exhibited persistent induction or repression for a minimum of three time points during H(PDF)Click here for additional data file.Table S6Differential gene expression profile of the genes belonging to metabolic process functional category with proposed gene names.(XLSX)Click here for additional data file.Table S7Transcript abundance at multiple time points of the genes belonging to C. neoformans2O2 induced oxidative stress. ubiquitin dependent protein catabolic processes during H(XLSX)Click here for additional data file."}
+{"text": "To quantify myocardial T2 value in patients with myocarditis and correlate the distribution of abnormal T2 values with the extent of macroscopic late gadolinium enhancement (LGE).25 patients with myocarditis were retrospectively evaluated for the utility of T2 mapping in diagnosing myocarditis. Patients with elevated troponins, negative coronary angiogram, and atypical LGE were diagnosed as acute myocarditis. Patients with normal troponins and macroscopic LGE at the time of cardiac MRI were diagnosed as remote myocarditis. As per our institutional protocol, T2 mapping sequences were performed in all cases with suspected myocarditis in addition to standard LGE images on 1.5 T scanner . T2 mapping was performed on three short axis images , yielding 16 myocardial segments for analysis (AHA segments). Single 4 chamber view image was obtained in addition. Minimum, peak and mean segmental T2 values were calculated by the first reader. Average segmental T2 values were documented along with documentation of the number of segments with elevated T2 values. The presence or absence of LGE was documented by a second reader blinded to the T2 results. Average segmental T2 values were then correlated with troponin levels at the time of the MRI examination.In patients with acute myocarditis, mean T2 values were elevated in segments showing LGE . The T2 values were also elevated in myocardial segments with no macroscopic LGE (avg 60 msec). On an average, there were 6 additional segments that showed elevated T2 values and no macroscopic LGE. In patients with remote myocarditis, the T2 values were normal in areas of LGE.T2 mapping can be utilized as an objective tool for diagnosing acute myocarditis. In addition, T2 values were normal in patients with normal cardiac enzymes and a typical pattern of myocarditis associated LGE. Given the non contrast nature of this technique, normal T2 values may exclude a diagnosis of acute myocarditis in patients with renal insufficiency.None."}
+{"text": "The emergence of X4 tropic viral strains throughout the course of HIV infection is associated with poorer prognostic outcomes and faster progressions to AIDS than for patients in whom R5 viral strains predominate. Here we investigate a stochastic model to account for the emergence of X4 virus via mutational intermediates of lower fitness that exhibit dual/mixed (D/M) tropism, and employ the model to investigate whether the administration of CCR5 blockers in-vivo is likely to promote a shift towards X4 tropism. We show that the proposed stochastic model can account for X4 emergence with a median time of approximately 4 years post-infection as a result of: 1.) random stochastic mutations in the V3 region of env during the reverse transcription step of infection; 2.) increasing numbers of CXCR4-expressing activated naive CD4+ T cells with declining total CD4+ T cell counts, thereby providing increased numbers of activated target cells for productive infection by X4 virus. Our model indicates that administration of the CCR5 blocker maraviroc does not promote a shift towards X4 tropism, assuming sufficient efficacy of background therapy (BT). However our modelling also indicates that administration of maraviroc as a monotherapy or with BT of suboptimal efficacy can promote emergence of X4 tropic virus, resulting in accelerated progression to AIDS. Taken together, our results demonstrate that maraviroc is safe and effective if co-administered with sufficiently potent BT, but that suboptimal BT may promote X4 emergence and accelerated progression to AIDS. These results underscore the clinical importance for careful selection of BT when CCR5 blockers are administered in-vivo. CCR5 blockers are a promising new class of anti-HIV drugs that act by binding to the CCR5 coreceptor, thereby reducing the number of CD4-CCR5 complexes available for viral binding by HIV and consequently inhibiting the viral entry stage of the infection cycle A key concern with the administration of CCR5 blockers in-vivo relates to the emergence of CXCR4 (X4) tropic virus Over the course of untreated infection, X4 tropic virus generally emerges at later stages of infection stochastic model to account for X4 emergence in-vivo. While previous deterministic modelling of the X4 switch demonstrated that X4 emergence can in principle be accounted for by increased activation of naive CD4+ T cells at later stages of infection stochastic process. We assume that the tropism shift is also driven by random viral mutations during the reverse transcription step of the infection cycle stochastic event, with a median emergence time of approximately 4 years post-infection with considerable variation around this time deterministic models.In the present study we investigate a Our model is employed to investigate the course of HIV infection when maraviroc is administered at early, intermediate or late stages of infection, and also when co-administered with background therapy (BT) of variable efficacies. Previous modelling has only considered the administration of therapy at a single time-point Three uninfected CD4+ T cell subpopulations are modelled : restingM respectively denote the total number of resting naive, activated and resting memory CD4+ T cells at time MA respectively denote the number of activated naive CD4+ T cells (CD4+HLA-DR+CD45RA+) and activated memory CD4+ T cells (CD4+HLA-DR+CD45RO+) in PB. It is assumed in our model that only activated cells may give rise to productively infected cells upon infection The variables The term Here In our model The term In the present model The term The parameters modulated infectivities\u201d of X4 and of D/M for activated memory CD4+ T cells. Since activated memory CD4+ T cells predominately express CCR5 modulated infectivitiesmodulated infectivitiestive see , but theThe term For any HIV virion that enters a cell, it is assumed that viral mutations may occur during the reverse transcription step of infection (see above). It is assumed that the 3 amino acids at positions 11, 25 and 29 in the V3 region of the env gene determine viral tropism The term The course of infection is simulated from age 30 to age 40  to below 200 cells/\u00b5L in 10 years (by age 40) for an individual infected with R5-tropic virus, in whom no D/M and/or X4 tropic virus emerges Viral recrudescence from below detection (50 HIV RNA copies/mL) to approximately 4.5 log10 HIV RNA copies/mL after 4 weeks following cessation of HAART Median time of X4 emergence of approximately 4 years post-PHI not to inhibit D/M strains in order to consider the worst-case scenario in the present analysis, despite previous studies reporting that maraviroc may inhibit some D/M strains not to inhibit X4 viral strains, since these utilize the CXCR4 coreceptor that is not blocked by maraviroc. It is assumed in all simulations that maraviroc has an efficacy of 90%  assuming viral mutations in our model. A single simulation for an individual in whom X4 emerges at approximately 4 years post-PHI is shown in We also performed Monte Carlo sampling over 250 trials for the case of untreated infection  C,D,E,F.Next we investigated whether administration of maraviroc without BT (i.e. BT has efficacy 0%) would promote selection for X4 tropic virus and result in accelerated progression to AIDS. To this aim, we simulated an individual who receives maraviroc as monotherapy at age 32  A,B. Admdeterministic modelling Following selection for X4 tropic virus after administration of maraviroc as monotherapy, total CD4+ T cell exhibit a rapid progression to AIDS within 1 year  A, so thTo further investigate the likelihood of selection for D/M and/or X4 tropic virus following administration of maraviroc, we performed Monte Carlo simulations of maraviroc administration at early, intermediate and late stages of infection respectively corresponding to therapy administration after 1, 3.5 and 6 years post-PHI. We considered scenarios in which maraviroc was applied with BT of moderate efficacy (BT has 90% efficacy) and also with BT of high efficacy (BT has 99% efficacy). Maraviroc was assumed to have an efficacy of 90% in all scenarios.First we investigated whether BT therapy of moderate efficacy (BT has 90% efficacy) would ensure sufficient X4 suppression . When thNext we ran Monte Carlo simulations for the case that maraviroc is administered with BT of high efficacy (BT has 99% efficacy) as shown in stochastic model of X4 emergence to examine whether administration of CCR5 blockers in-vivo is likely to promote X4 emergence, thereby resulting in accelerated progression to AIDS. While previous studies investigated deterministic models of X4 dynamics where X4 emergence was driven by increasing activation of naive CD4+ T cells that express CXCR4 but not CCR5 stochastic model of X4 emergence that explicitly includes CD4+ T cell activation dynamics. In addition to increasing activation of naive CD4+ T cells modelled previously stochastic event, with a median emergence time of approximately 4 years post-infection and a considerable variation in the time when X4 emerges deterministic models.Here we investigated a not result in a single instance of X4 emergence  is safe at early, intermediate and also late stages of infection if administered with sufficiently potent BT (99% efficacy). Monte Carlo simulations of this scenario in the present analysis did ence see . These rThe results of the present analysis are further supported by observations that the virus can find novel ways to bind the CCR5 chemokine coreceptor when CCR5 blockers are administered in-vivo, rather than resorting to a tropism switch Our analysis also indicates that the in-vivo administration of CCR5 blockers as monotherapy  can promote X4 emergence, thereby resulting in accelerated progression to AIDS. These results are in agreement with previous reports from three macaques dually infected with R5 and X4 tropic virus, where administration of the CCR5 blocker CMPD 167 as monotherapy was observed to promote outgrowth of X4 tropic virus not inhibit D/M virus, whereas previous studies reported inhibition of some D/M strains by maraviroc The present conclusion, regarding safety of maraviroc with sufficiently potent BT, is likely to be very robust. We assumed that the infectivities of D/M and of X4 (for activated memory CD4+ T cells) increase following suppression of R5 virus, thereby resulting in increased selection for D/M and X4 viral strains We extend on previous modelling by assuming that mutation from R5 to X4 occurs via intermediates that are of D/M tropism and that are of lower fitness than R5 and X4 The model presented here has a number of limitations. Firstly, our modelling did not include additional cell populations such as macrophages that might contribute to increased selection for X4 virus at later stages of the infection when total CD4+ T cell counts are low, since macrophages provide an additional source of CXCR4-CD4 complexes deterministic models of X4 selection dynamics. The key result of our analysis and modelling is that the administration of maraviroc is safe and that it does not promote X4 emergence if employed with BT of sufficient potency. Our results also indicate that selection for X4 tropic virus may occur if BT of insufficient efficacy is administered with maraviroc. These results highlight the need for careful selection of BT when CCR5 blockers are administered in-vivo, as well as raise the question whether alternative anti-HIV CCR5-targeting treatments like gene therapy In conclusion, we have presented the first stochastic model of in-vivo X4 selection dynamics and investigated whether CCR5 blockers promote X4 emergence resulting in accelerated progression to AIDS. Since X4 emergence is a stochastic event with a median emergence time of approximately 4 years post-infection, and with considerable variation in the time when X4 emerges, the modelling results presented here are more likely to reflect likely clinical outcomes than previously presented Figure S1modulated infectivity\u201d Plot of the \u201c Here (TIF)Click here for additional data file.Figure S2modulated infectivity\u201d Plot of the \u201c Here (TIF)Click here for additional data file.Figure S3Plots of the term  Negative values are not shown above. The function values (TIF)Click here for additional data file."}
+{"text": "Classical modified Look-Locker sequence (MOLLI) can induce a long breath-hold and is prone to cardiac and respiratory motion. Several shorter sequences based on inversion  and saturation recovery (SASHA) of longitudinal relaxation have been proposed for derivation of T1 values or calculation of extracellular volume fraction or lambdas. Despite the validation in T1 gel phantoms, it has not been determined whether these novel sequences provide equivalent information on T1 when performed within the same individual.Twenty-three subjects underwent T1 mapping in a mid-ventricular equatorial short axis slice using above sequences on 3T clinical scanner prior and after gadolinium contrast (0.2 mmol/kg) administration. The images were analyzed using PRIDE tool with in-built automated motion correction. ROIs were drawn conservatively within the septal and lateral myocardium and the blood pool. Comparison of the native T1 values and lambdas were performed and expressed as percentage of mean difference from the values obtained with gold-standard MOLLI sequence.3'5MOLLI shows the nearest approximation to MOLLI derived values with an underestimation of values in native myocardium by 1.2% and lambdas by 11%. T1 values obtained with SASHA were overestimated by 17%, and lambdas by 29%, whereas shMOLLI led to an underestimation of T1 values by 13% and lambdas by 22%.We demonstrate that 3'5'MOLLI provides near identical T1 values and its derivatives.We propose that 3'5'MOLLI is the optimal 'shortened' sequence for clinical derivation of T1 values.NIHR"}
+{"text": "IDH1 mutation appeared to be a strong predictor of clinical outcome, overruling histological malignancy grade [IDH1 mutation alters the cellular metabolism and epigenetic phenotype influencing cellular proliferation. IDH1 mutation infers increased levels of D2HGDH leading to the inhibition of DNA and histone demethylating enzymes, resulting in the glioma-CpG island phenotype [IDH1 mutant cells show alterations in glutamine, fatty acid and citrate synthesis pathways, which all may have their influence on cellular proliferation [Recently heterozygous mutations in the active site of the enzyme isocitrate dehydrogenase 1 (IDH1) were discovered in glioblastomas [cy grade . IDH1 iscy grade . IDH1 muhenotype . Alteredhenotype . In addiferation .IDH1 function affect the glucose metabolism, which may explain the different biological behaviour of tumors with and without the IDH1 mutation [IDH1 mutated samples  and in 39 IDH1 wild-type glioma samples  and in four samples of normal brain (Table\u00a0IDH1 mutated cells seem to compensate for the low production of \u03b1-ketoglutarate by overexpressing D2HGDH and L2HGDH in the cytoplasm. They also overexpress GLUD1, which converts glutamate to \u03b1-ketoglutarate inside the mitochondria. In addition, we found that IDH1 mutated cells overexpress HIF1AN. The HIF1AN gene inhibits HIF1\u03b1. Since HIF1\u03b1 acts as an oxygen sensor that promotes angiogenesis, the formation of dysfunctional tumor vasculature is counteracted in IDH1 mutated cells. Furthermore, the IDH1 mutated cells overexpress the LDHB gene while cells without IDH1 mutation overexpress the LDHA gene. The present results illustrate that tumor cells without IDH1 mutation switch their energy production from a low rate of glycolysis followed by the TCA cycle to a high rate of glycolysis followed by aerobic glycolysis (LDHA up-regulation; Figure\u00a0LDHA and LDHB influences the progression of cancer cells [IDH1 mutation tend to correct their energy production through the TCA cycle by overexpressing LDHB (Figure\u00a0IDH1 mutated gliomas.The changes in mutation . In ordeer cells ,9. It maIDH1 mutation normalize their glucose metabolism, which appears to result in a slower tumor progression. Depending on the IDH1 status of the tumor, specific interference with the glucose metabolism and aerobic glycolysis should therefore be considered for future therapeutic strategies.We conclude that gliomas with All authors declare that they have no competing interests.DM and LB carried out the molecular genetic analyses; SS and PvdS carried out the data analysis and DM and JMK conceptualized and designed the study and wrote the manuscript. All authors read and approved the final manuscript."}
+{"text": "Pknox2 was mainly expressed in the zeugopod domain of the murine limb at E10.5 and E11.5. Misexpression of Pknox2 in the limb bud mesenchyme of transgenic mice led to deformities in the zeugopod and forelimb stylopod deltoid crest, but left the autopod and other stylopod skeletons largely intact. These malformations in zeugopod skeletons were recapitulated in mice overexpressing Pknox2 in osteochondroprogenitor cells. Molecular and cellular analyses indicated that the misexpression of Pknox2 in limb bud mesenchyme perturbed the Hox10-11 gene expression profiles, decreased Col2 expression and Bmp/Smad signaling activity in the limb. These results indicated that Pknox2 misexpression affected mesenchymal condensation and early chondrogenic differentiation in the zeugopod skeletons of transgenic embryos, suggesting Pknox2 as a potential regulator of zeugopod and deltoid crest formation.The TALE (Three Amino acid Loop Extension) family consisting of Meis, Pbx and Pknox proteins is a group of transcriptional co-factors with atypical homeodomains that play pivotal roles in limb development. Compared to the in-depth investigations of Meis and Pbx protein functions, the role of Pknox2 in limb development remains unclear. Here, we showed that  Hox genes  are expressed in restricted domains along the axes of the limb buds in spatial and temporal colinearity, and provide positional information for limb patterning, skeletal condensation and differentiation. Expression of the Hoxd 9-13 genes in the limb bud is activated sequentially from Hoxd9 to Hoxd13 at the posterior border of the limb bud. The Hoxa9-13 genes are activated similarly to the Hoxd9-13 genes. The sequential activation of these genes correlate with the malformations in the limb skeletons of specific Hox mutants. For instance, compound mutants of Hoxa9/d9 have a shorter humerus and a loss of deltoid crest formation in the forelimb stylopod Hox9 (Hox9aabbccdd) mutant mice exhibit severe forelimb defects with a loss of posterior skeletal elements including complete autopod loss and partial zeugopod loss, suggesting a role of Hox9 in forelimb anterior-posterior patterning Hox10 (Hox10aaccdd) results in severe agenesis in the hindlimb styplopod, and defects in the forelimb zeugopod and deltoid crest to a lesser degree Hoxa11/d11 selectively display deformities in the forelimb zeugopod Hox11aaccdd) demonstrate dramatic malformations of the fore- and hindlimb Hoxa13/d13 exhibit malformations in autopod development Hoxa and Hoxd genes results in early arrest of limb outgrowth, with severe truncations in distal elements Hox genes in limb development: Hox9 participates in stylopod specification, Hox10/11 contributes to zeugopod formation and Hox12/13 regulates autopod development.The vertebrate limb bud arises from the lateral plate mesoderm and then develops into three segments  along the proximal-distal (PD) axis through an endochondral ossification mechanism. Limb patterning and morphogenesis are established by diffusible signals originating from different domains of the limb bud, including the Fgf, Wnt and RA signaling pathways Hox genes have a universal role in a variety of developmental processes, the specificities of Hox function are achieved by interactions with co-factors such as the members of the TALE (Three Amino acid Loop Extension) superfamily. The TALE superfamily consists of transcription factors with atypical homeodomains including the Pbx, Meis and Pknox proteins. Similar to the Hox genes, these TALE genes are dynamically expressed in the limb bud. Genetic studies reveal that TALE genes regulate limb patterning and development of the skeletal elements. For instance, Pbx1 is exclusively expressed in the proximal limb bud, whereas Pbx2 is expressed throughout the limb mesenchyme Pbx1-deficient mice have malformations in the proximal limb elements, while double Pbx1/2 mutants exhibit distal limb deformities in addition to the proximal limb defects Meis1 is a specific marker for the proximal domain, especially for the presumptive stylopod skeleton in early limb bud Meis1 gene in the limb bud shifts limb PD patterning and promotes the formation of proximal limb segments Shh expression as well as Hox expression in the regulation of limb development Pknox1/2 are also dynamically expressed in the avian limb bud Pknox1-null mutation is lethal in mice at E7.5 and hypomorphic Pknox1 mutant mice exhibit no obvious alterations in limb development Pknox2 protein in limb development remains unclear.While the Pknox2 genes in limb mesenchyme or osteochondroprogenitor cells in the early limb bud in transgenic mice. Misexpression of Pknox2 in the limb bud mesenchyme resulted in deformities in zeugopod elements and a loss of deltoid crest formation. The malformations in these transgenic mice were correlated with the perturbations of Hoxd10-11 gene expression profiles in the zeugopod elements. Therefore, Hox-dependent patterning alterations underlie, at least in part, the limb zeugopod defect in mice of Pknox2 misexpression.Here, we overexpressed the Pknox2 gene  was cloned into a vector harboring the Prx1 promoter Col2a1 promoter Col1a1-3.6 kb promoter Prx1-Pknox2 transgenic mice: 5\u2032- TCTGGTGGCAGCGAAAGTC-3\u2032; forward oligo for Col2a1-Pknox2 transgenic mice: 5\u2032-AGGGTGTTGTTTAGAATGGGA-3\u2032; forward oligo for Col1a1-Pknox2 transgenic mice: 5\u2032-CACTCCAGTGACAGCACCTCT-3\u2032; reverse oligo for transgenic mice: 5\u2032-ATGGAGGATAGTTCAGGGCTT-3\u2032.The 1.4 kb coding sequence CDS of All the mouse embryonic manipulations comply with the guidelines of the Bioethics Committee of Bio-X Institutes of Shanghai Jiao Tong University (SYSK-SH-2011\u20130112).Mouse embryos at P0 were eviscerated, and their skins were removed. Mice were fixed overnight in 95% ethanol followed by staining overnight in Alcian Blue and Alizarin Red solution (SIGMA) as previously reported in situ hybridization, embryos were sacrificed at various ages, dissected, and fixed in 4% paraformaldehyde (PFA)/PBS at 4\u00b0C overnight. After fixation, the tissues were dehydrated in 100% ethanol and embedded in paraffin. The embedded tissues were cut to generate 8 \u00b5m-thick sections and mounted onto slides. HE staining and Safranin O staining were performed following standard protocols. Whole mount in situ hybridization were performed as described For histology and Pknox2 expression has been detected throughout the limb mesenchyme and is particularly strong in the mesenchyme underlying the ectoderm in the chick limb bud Pknox2 expression during mouse limb bud development. Meis1, Hoxa11 and Hoxd13 are used as markers for the stylopod, zeugopod and autopod, respectively Pknox2 expression was first detected as a stripe in the central region of the hindlimb at E10.5, which was between the Meis1-expression domain and the progress zone  controls, the expression domains of Hoxa10, Hoxd10 and Hoxd11 in Prx1-Pknox2 transgenic mice were anteriorly shifted . Safranin O staining on the sections at E15.5 showed that chondrocyte hypertrophy and bone ossification were delayed in the radius and ulna . Collectively, these results suggest that misexpression of Pknox2 disrupts zeugopod formation at the condensation stage and during early chondrogenic differentiation.To gain insight into the timing and appearance of the defects in and ulna . The choand ColX , which aand ColX . In factBmp7 and BmpR1b double knockout mice have a nearly absent ulna and a shortened and bent radius Pknox2 affects Bmp signaling activity, the protein expression of p-Smad1/5/8 was examined in the ulna and radius of Col2-Pknox2 transgenic embryos at E12.5 via IHC. Interestingly, p-Smad1/5/8 expression levels were markedly decreased in the developing ulna and radius of transgenic mice, whereas p-Smad1/5/8 expression levels were relatively normal in the humerus of transgenic mice and WT controls , which was active as early as the osteoblast progenitor stage. Interestingly, there were no apparent defects in other skeletal elements except for the missing deltoid crest in the forelimbs of the Col1-Pknox2 transgenic mice . Therefore, it is likely that perturbations in Hoxa10/d10 expression profiles could not fully account for the forelimb zeugopod deformities caused by misexpression of Pknox2. Double mutants of Hoxa11/Hoxd11 exhibit deformities in the forelimb Prx1-Pknox2 transgenic mice. Several lines of evidence have demonstrated that Pknox2 could form a heterotrimeric complex with the Hox and PBX proteins in vitroPknox2 forms complex with Hox11 and Pbx1/2 in controlling Hox gene expression. Ectopic expression of Meis1 in the distal limb bud also results in abnormalities in forelimb zeugopod formation Pbx1-independent manner Prx1-Pknox2 mice may result from the combination of Hox10/11 gene activity in zeugopod elements.In our study, misexpression of Prx1-Pknox2 mice were recapitulated to a lesser extent in mice ectopically expressing Pknox2 in osteochondroprogenitor cells. On the other hand, early limb patterning was largely intact in both the Prx1-Pknox2 and Col2-Pknox2 mice, which had relatively normal skeletal segmentation in the proximal-distal axis as well as normal anterior-posterior polarity in the autopod skeletal elements. Only chondrogenic differentiation and ossification were strikingly perturbed in the zeugopod elements of transgenic mice at E12.5. Misexpression of Pknox2 in the limb bud mesenchyme altered the expression of Hoxa10/d10 and Hoxa11/d11 in the zeugopod at E11.5. In fact, 5\u2032 Hoxd (Hoxd 9-13) genes have been shown to function at the mesenchymal condensation stage of skeletal formation Hox11 functions from the condensation stage onwards and programs chondrogenic and endochondral ossification Hoxa10/d10 and Hoxd11 domains and a diminished Hoxa11-expression domain in the posterior side might lead to fewer mesenchymal progenitor cells available for ulna condensation and differentiation. These findings indicate that overexpression of Pknox2 perturbs zeugopod development at the condensation and chondrogenic differentiation stages as a co-factor of Hox proteins.The defects in the Pknox2 misexpression was the decrease of Bmp/Smad signaling in the zeugopod elements. Loss of Bmp7 and Bmpr1b in the mouse limb leads to malformations in the ulna, radius and the deltoid crest, similar to the phenotype observed in Prx1-Pknox2 transgenic mice TGFb2 also results in a slightly shortened zeugopod lacking a deltoid crest and olecranon Prx1-Pknox2 mice could be recapitulated in the Col1-Pknox2 mice but not in the Col2-Pknox2 mice, indicating that Pknox2 misexpression affects deltoid crest formation at the osteoblast differentiation stage. In fact, the deltoid crest is induced by the Bmp4 signal from neighboring tendon cells at E13.5 and then develops through endochondral ossification Pknox2 overexpression. For instance, it has been reported that Hoxa13 directly binds to the enhancer elements of Bmp2 and Bmp7 to regulate their expression Pknox2 misexpression may suppress Bmp/Smad signaling activity during zeugopod development and deltoid crest formation as a co-factor for Hox genes.Another molecular event in mice with Figure S1qRT-PCR analysis of Hox10-11 gene expression in the limbs from the Prx1-Pknox2 embryos. qRT-PCR is performed for Hoxa10, Hoxa11, Hoxd10 and Hoxd11 genes in the limbs from Prx1-Pknox2 embryos at E12.5. No obvious alteration is detected in the expression levels of these genes.(TIF)Click here for additional data file."}
+{"text": "Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus  are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon. The cerebral cortex is populated by two main neuronal classes, pyramidal neurons and interneurons that represent respectively 85% and 15% of cortical neurons.Two distinct, though tightly linked processes, control the development and placement of these neurons within the cortex. On one hand, \u2018spatial patterning\u2019 by which the telencephalon is regionally subdivided into defined morphological and molecular progenitor territories, thereby underlying the generation of future interneurons and pyramidal neurons The proneural genes Ngn2 and Ascl1 are respectively expressed in the dorsal and ventral telencephalon, where they determine the regional identity of neural progenitors During recent years a series of transcriptional targets of Ngn2 and Ascl1 that could mediate their functions have been reported H19 gene is transcribed from the maternal allele only , whereas its imprinted neighbor Igf2 is a paternally expressed gene (PEG) de novo DNA methyltransferases (Dnmt) Parental genomic imprinting is an epigenetic mechanism that restrains the expression of about 100 mammalian genes to one parental allele Ube3a) is linked to Angelman disease and several imprinted loci are linked to Autism Spectrum Disorders (ASD) and to Prader-Willi, two syndromes in which cerebral functions are altered Both MEGs and PEGs are required during mouse embryonic development as uniparental embryos  that express only PEGs or only MEGs, die during early development Here we identify 5 imprinted genes, 2 PEGs  and 3 MEGs  situated at an imprinted locus at 12qF1 Gtl2  was highly upregulated in the dorsal telencephalon of Ngn2 KO mice. First, to shed light on Gtl2 localization and infer about its function in the normal developing telencephalon, we performed in situ hybridizations (ISH) on WT animals. At E12.5, Gtl2 is mostly expressed in postmitotic neurons of the ventral telencephalon where it remains strongly expressed up to E17.5 . From E13.5 onward, Gtl2 starts to be expressed in the nascent cortical plate in the dorsal telencephalon , we observed that the non-coding RNA encoded by the imprinted gene cephalon Fig.1A\u2212CM. m. domesticus and M. m. molossinus, we were able to distinguish between parental alleles and to determine that Gtl2 is maternally expressed in the developing dorsal telencephalon , confirming that Gtl2 is a MEG at this developmental stage in this structure. We then confirmed by ISH . We didn\u2019t detect any changes in Gtl2 expression in the ventral telencephalon at any developmental stages in Ngn2 KO mice (data not shown). This data thus shows that in the developing telencephalon, the imprinted gene Gtl2 encodes for a nuclear RNA expressed mostly in post-mitotic neurons of the ventral and dorsal telencephalon and thalamus .In addition, by using reciprocal hybrid cortex issued from crosses between d by ISH Fig.1C ain Dlk1 gene in Ngn2 KO cortex , Mirg and Rtl1  Fig.2A [To determine whether the absence of Ngn2 selectively affects the Dlk1-Gtl2 locus or whether it also impacts on IGs located at other genomic regions, we measured RNA levels of 14 additional IGs present on 4 imprinted loci. These loci are situated at chromosomes 6 Fig.3A a. To further investigate the link between Ascl1 and IGs at the Dlk1-Gtl2 locus, we performed ISH on Ascl1 KO mice. We confirmed that Ascl1 is directly linked to the expression of at least two imprinted transcripts (Gtl2 and Dlk1) in the ventral telencephalon. As shown in We next thought to identify the molecular mechanism responsible for the overexpression of Dlk1, Gtl2, Rian, Mirg and Rtl1 in the Ngn2 KO dorsal telencephalon. One obvious candidate to test is Ascl1 that is known to be highly upregulated in the cortex of Ngn2 KO animals . FurtherAltogether, this suggests a model in which Ascl1 plays a central role in regulating the expression of IGs of the Dlk1-Gtl2 locus in the developing brain. First, Ascl1 would be necessary for their expression in the normal developing ventral telencephalon. Secondly, the upregulation of the RNAs from the Dlk1-Gtl2 locus in Ngn2 KO animals would be a consequence of the ectopic expression of Ascl1 in the respecified dorsal telencephalon.This study has uncovered a link between neurogenic transcription factors and a set of imprinted transcripts. First, we have observed that the imprinted gene Gtl2 is upregulated in the telencephalon of Ngn2 KO animals Fig.1. GGtl2 is part of the Dlk1-Gtl2 imprinted locus at chromosomal position 12qF1 that contains several additional MEGs and PEGs , Fig.S1); on the other hand their upregulation in Ngn2 KO animals is reduced in Ngn2/Ascl1 double KO animals . For analysis, WT and heterozygous animal were pooled as loss on one allele does not markedly alter phenotype The GFP+/\u2212Ngn2ki or GFP+/\u2212 Ascl1+/\u2212Ngn2ki heterozygous intercrosses. RNA was isolated using RNeasy minikit according to the manufacturer\u2019s instructions (Qiagen). Alternatively, RNA was extracted using RNA now (Ozyme). All RNA samples were treated with DNase (Qiagen or Ambion for RNeasy and RNA now methods respectively). Reverse transcription was performed using N6 primers and MMLV-RT (Promega). Quantitative PCR (qPCR) was performed in duplicate in 384 well plates using 2\u00d7 SybrGreen Mix and a LC480 Real-Time PCR System (Roche). Results are presented as linearized Cp-values normalized to housekeeping genes TBP, Gus2 and Gapdh and the indicated reference value (2-\u0394\u0394Ct). The sequence of primers is available upon request.E13.5 dorsal telencephalons were dissected out from embryos obtained from All steps and solutions for RNA in situ hybridization (ISH) were achieved in RNase free environment. Embryos were perfused intracardiacally with a solution of 4% paraformaldehyde (in PBS) and the brains were post-fixed in the same fixative over night. Brains were then cryoprotected in 30% sucrose (in PBS). ISH was carried out on 10 \u00b5m cryostat sections  using digoxygenin-labeled (Roche) RNA probes as previously described Immunostaining was performed on 20 \u00b5m thick cryosections. Blocking solution consisted of PBS supplemented with 5% horse serum (Invitrogen), 0.3% Triton X-100 (Sigma) and 3% Bovine Serum Albumin . Antibody solution consisted of PBS supplemented with 1% horse serum, 0.1% Triton X-100 and 3% BSA. Dlk1 or Tbr1 antibodies (a kind gift of Charlotte Harken Jensen and Robert Hevner respectively) were incubated overnight at 4\u00b0C and secondary during 2 hours at RT. Nuclei were stained with bisbenzimide . Sections were mounted with glycergel (DAKO).in-situ RNA hybridization and immunofluorescence staining were acquired with an Axioplan2 Zeiss microscope and a Spot RT camera, converted in false colors and overlayed using Adobe Photoshop software.Pictures of the Data are presented as mean + standard error of the mean of at least three biologically independent experiments or of WT and KO embryos. A Mann-Whitney\u2019s test was used for comparing the distribution of data of measurements of RT-qPCR experiments.Figure S1Expression of Gtl2 RNA (ISH) at E17.5 in Ngn2 WT and Ngn2 KO mice. (A-E) Co-labeling with Tbr1 protein (immunohistochemistry) was used to show specific localization of Gtl2 positive neurons in the dorsal telencephalon . Cells that show ectopic expression of Gtl2 mRNA in Ngn2 KO mice are localized in the IZ (red arrows in B and D). Scale bars: 150 \u00b5m.(TIF)Click here for additional data file."}
+{"text": "Diabetic ketoacidosis (DKA) is a potentially life threatening complication of initial presentation with type 1 diabetes mellitus (T1DM). Annual DKA incidence data has been reported in selected populations in many studies, however few studies have reported the trend in DKA incidence over time in an unselected representative childhood population.To determine the annual incidence of DKA at initial presentation with type 1 diabetes mellitus in all children <15 years of age between 2000 and 2009 in the Auckland region.Data from Auckland children with newly diagnosed T1DM between 1 January 2000 and 31 December 2009 were collected from Starbase, the Starship Children\u2019s Hospital diabetes database. T1DM was confirmed by the presence of glutamic acid decarboxylase and/or tyrosime phosphatise-like protein (IA2) antibodies. DKA was defined by international criteria as venous or capillary pH and bicarbonate as mild DKA with pH <7.30 and bicarbonate <15 mmol/l, moderate DKA pH <7.20 and bicarbonate <10 mmol/l and severe DKA ph <7.10and bicarbonate <5 mmol/l.There were 481 children diagnosed with T1DM in the Auckland region  between 2000 and 2009. Over the study period the DKA incidence was highly variable (32 to 63% without a discernable change in incidence over the 10 year period [p=0.11]), thus data are expressed as a means over the study period 2000-2009. There were 47.4% of children in DKA at initial presentation which is very similar to the DKA incidence we reported for 1995-96 of 42%[The incidence of DKA at initial presentation of type 1 diabetes mellitus is much higher in Auckland children compared to other published studies. DKA incidence has plateaued over the past 14 years and occurs far more frequently in young children. We speculate that improved community awareness of the symptoms of T1DM will lead to earlier diagnosis of T1DM and avoidance of DKA and associated sequelae."}
+{"text": "We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer.mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay.ALDH1 mRNA expression was significantly reduced  in malignant tumors compared to normal ovaries and benign tumors. The proportion of ALDH1+ cells was significantly lower in malignant tumors  compared to normal ovaries  and benign tumors . ALDH1+ cells occurred in the stroma and surface epithelium in normal ovary and benign tumors, although surface epithelial expression varied more in benign tumors. Localization of ALDH1 was heterogeneous in malignant tumor cells and little ALDH1 expression occurred in poorly differentiated malignant tumors. In benign tumors the distribution of ALDH1 had features of both normal ovary and malignant tumors. ALDH1 protein expression assessed by IHC, WB and FC was positively correlated (p < 0.01). ALDH1 did not appear to be co-expressed with the CSC markers CD44, CD117 and CD133 by IHC.Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers.These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process. In previous studies we identified aldehyde dehydrogenase 1A1 (ALDH1) as a novel antigen in ovarian autoimmunity associated with unexplained infertility and premature menopause . This prALDH1A1 gene at chromosome 9q21 . The Primer sequences were: ALDH1A1 Forward (5'- TTGGAATTTCCCGTTGGTTA-3') and Reverse (5'- CTGTAGGCCCATAACCAGGA-3'); Actin Forward (5'-CTGTGGCATCCACGAAACTA-3') and Reverse (5'- ACATCTGCTGGAAGGTGGAC -3'). The PCR amplifications were carried out in a 25 \u03bcl reaction volume containing 25 ng of cDNA using Platinum Taq DNA Polymerase (Invitrogen) according to manufacturer's recommendation. The mixture was denatured at 94\u00b0C (3 minutes) followed by 35 cycles at 94\u00b0C (30 seconds) and 54\u00b0C (30 seconds) to anneal and 72\u00b0C (1 minute) for extension followed by a final extension at 72\u00b0C (10 minutes) in a programmable Peltier Thermo Cycler . The PCR products were separated by electrophoresis in a 3% (W/V) agarose gel (Invitrogen) and visualized using ethidium bromide stain . Amplicon from one positive sample each from normal ovary and ovarian serous carcinoma was purified using a QIAquick PCR purification kit  and sequenced at DNA sequencing facility (University of Illinois at Chicago) using an ABI 3100 Genetic analyzer (Applied Biosystems). The amplicon sequences were blasted against the NCBI RefSeq human mRNA database and confirmed with a perfect match for ALDH1A1 gene [GenBank: NM_000689.3]. Quantitative Reverse Transcriptase-PCR (qRT-PCR) was carried out using SYBR green master mix in an ABI 7500 RT-PCR system and analyzed using the \u0394Ct method with human Actin as an internal control according to the manufacturer's recommendation (Applied Biosystems). The \u0394\u0394Ct was determined by subtracting \u0394Ct of each sample from the average \u0394Ct of normal ovary. The differences in ALDH1 mRNA expression levels were calculated as the fold change using the formula 2-\u0394\u0394Ct as previously described  (data not shown).ALDH1 mRNA expression was significantly lower in malignant ovarian tumors (n = 5) compared to normal ovary  and benign ovarian tumors   compared to normal ovaries  and benign ovarian tumors  Figure . There wALDH1 protein was detected as a single band at 55 kDa in all of the ovarian tissues tested by Western blot Figure . DensitoALDH1 immunostaining was observed in various cell types in normal ovary and serous ovarian tumors. In normal ovary, a diffuse ALDH1 staining pattern was observed in the stroma in fibroblasts-like cells and fibrous tissue. In addition, the surface epithelial cells stained intensely although there were occasional cells without stain Figure  and 3B. The staining pattern of ALDH1 in uninvolved areas adjacent to benign serous ovarian tumors was similar to that of normal ovary Figure . In contIn malignant serous ovarian tumors ALDH1 staining varied  compared to normal ovary (22.8 \u00b1 6.4%) and benign tumors (16.3 \u00b1 5.6%). The ALDEFLUOR assay was positively correlated with the proportion of cells expressing ALDH1 by semi-quantitative immunohistochemistry . Overall, the estimation of enzyme activity in ovarian cells was consistent with ALDH1 mRNA and protein expression levels.The mean fluorescence intensity (MFI) was significantly decreased in malignant ovarian tumors  compared to normal ovary  and benign ovarian tumors   was smaller than the proportion of ALDH1 immunostained cells suggesting that not all ALDH1 may be active. This was also observed by Deng et al. [The ALDH1 protein expression and enzyme activity were correlated. However, the proportion of ALDEFLUOR positive cells  malignant ovarian tumors have a higher survival rate than patients with less differentiated (high-grade) tumors . ALDH1 sPrevious studies showed that higher ALDH1 expression in tumor cells is associated with poor clinical outcomes in breast, ,47 lung,Recent observations suggest ALDH1 is a marker for CSCs in various malignancies and that ALDH1 in CSCs is associated with chemoresistance and increased malignant potential ,37,47,48We found that the total ALDH1 expression is significantly reduced in malignant serous ovarian tumors compared to normal ovaries and that expression in benign serous ovarian tumors is similar to normal ovary. ALDH1 was expressed in malignant tumor cells but at a low level and was absent in the more aggressive poorly differentiated malignant tumor cells. The heterogeneity of ALDH1 expression pattern suggests ALDH1 could be used as a novel indicator of prognosis and possibly as an indicator of responses to chemotherapy. Further investigation could facilitate understanding the role of ALDH1 in the ovary and ovarian tumors.The authors declare that they have no competing interests.KP, JL and SE worked to develop the experimental design. KP performed the experiments, statistical calculations and wrote the manuscript. SE facilitated the PCR and gene sequencing, and assisted KP in data analysis. AB developed the immunohistochemistry (IHC) protocols, facilitated tissue collection and assisted in writing the manuscript. MB assisted with tissue collection and IHC tissue processing. JL conceived the study, mentored KP in scientific methods and data analysis and assisted in drafting and finalizing the manuscript. All authors approved the final version of the manuscript."}
+{"text": "ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1\u201312, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64\u2013133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene,  Posterior polymorphous corneal dystrophy (PPCD) is a rare bilateral disorder transmitted as an autosomal dominant trait. Clinically PPCD is characterized by vesicles, bands and polymorphous opacities with pathology at the level of Descemet membrane and the corneal endothelium. Peripheral anterior iris adhesions, iris atrophy, pupillary ectropion and corectopia may also develop. Occasional severe visual disability results from secondary glaucoma or corneal edema VSX1; MIM ID #605020) within this locus was reported as disease-causing in a few PPCD cases COL8A2; MIM ID #120252) located on 1p34.3-p32.3, however only one disease-causing mutation in one family has been described to date ZEB1; MIM ID #189909) mapping to chromosome 10p11.2 were identified as disease-causing in PPCD3 (MIM ID #609141) and it has been estimated that pathogenic changes within this gene account for approximately 25% of all PPCD cases, with a range from 9%\u201345% depending on population studied VSX1 from this genetic interval by a linkage study, and lack of disease-causing changes, implies that an as yet undiscovered gene is causative for PPCD1 The genetic heterogeneity of PPCD is currently known to be represented by three loci on chromosomes 20, 1, and 10 Families affected by rare dominantly inherited disorders are often unrelated, however occasionally they share a chromosomal genomic region implying that the pathogenic mutation arose in a common ancestor In this study we observed that PPCD in the Czech Republic appears to have a remarkably high prevalence. A total of 19 Czech PPCD families, including two previously linked pedigrees The study was approved by the Ethics Committee of General University Hospital in Prague, Czech Republic and conformed to the tenets of the Declaration of Helsinki. All participants signed an informed consent prior to inclusion into the study.Subjects from 19 Czech pedigrees with familial PPCD were examined between the years 1995\u20132010 in the Department of Ophthalmology of the First Faculty of Medicine, Charles University in Prague. Ophthalmologic assessment included visual acuity, slit lamp examination, intraocular pressure measurements and specular microscopy using Noncon ROBO Pachy SP-9000 . Diagnosis of PPCD was based on positive family history and the presence of vesicles and polymorphic opacities at the level of Descemet membrane and the corneal endothelium. Pedigrees were drawn and residency within the Czech Republic of the eldest family member known to suffer from PPCD was noted. Geographic origin of the families was plotted on a map.DNA was isolated from venous blood samples using the Nucleon III BACC3 genomic DNA extraction kit according to manufacturer\u2019s instructions . Genotyping was performed using 11 polymorphic microsatellite markers on chromosome 20 which were fluorescently labeled and amplified by polymerase chain reaction (PCR). Ten microsatellites were commercially available: D20S98, D20S118, D20S114, D20S48, D20S605, D20S182, D20S139, D20S190, D20S106 and D20S107 . A dinucleotide marker used in this study, M189K21, was reported previously To investigate the possibility of a common lineage, haplotypes of affected individuals were constructed based on segregation within the families, and then compared between families. In order to calculate allele frequencies and haplotype frequencies in the population, 55 unrelated Czech population matched controls (110 chromosomes) were also genotyped for each marker.www.dmle.org) was used. The program DMLE+ uses Bayesian estimates of the location of a gene with a mutation affecting a discrete (disease) trait based on the observed linkage disequilibrium at multiple genetic markers. Other parameters are also estimated, such as mutation age To infer the location of a gene responsible for PPCD1 in the population studied and to estimate the age of the mutation (i.e. the time elapsed since the appearance of the common ancestor in the population) DMLE+ (Disease Mapping using Linkage disequilibrium) version 2.3 (http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp). For markers which do not appear on this genetic map  the distance in cM was estimated empirically by interpolation using the standard curve of physical distance. Since DMLE+2.3 does not accept identical distances for more than one marker, where the position of two markers obtained from the Marshfield genetic map was identical  position was recalculated using the standard curve as above  reference sequence (NM_003434 and NM_001083330). Both coding and untranslated regions as well as intron-exon boundaries were screened.Two probands from families 1 and 2, previously shown to be linked to 20p11.2 Array comparative genomic analysis (CGH) was used to evaluate DNA copy number variation (CNV) on chromosome 20  with a median probe spacing of 134 bp. Patient DNA from family 1, previously shown to be linked to 20p11.2 Clinical ascertainment demonstrated bilateral corneal changes consistent with the diagnosis of PPCD in 113 subjects  from 19 families. None of the PPCD families were known to be related to one another. In all families PPCD was consistent with autosomal dominant inheritance pattern, with incomplete penetrance noted for family 18 and 19 Analysis of genetic markers on 20p12.1- 20q12 revealed a common haplotype spanning a region of at least 23 Mb surrounding the centromere and encompassing both the short and long arm of chromosome 20. This full common haplotype was detected in 16 affected members of the two previously linked PPCD1 families (1 and 2) and also in 22 affected members from families 3\u20138 and 12 , however no potential pathogenic changes in the annotated coding and untranslated regions were identified. Since a deletion or duplication of the ZNF133 gene would not be detected by PCR amplification and sequencing, we also performed dense chromosome 20 CGH analysis (Nimblegen) to detect any CNVs in an affected individual from family 1. No microdeletions or duplications on 20p12.1-20p11.23 were detected in this patient.The Identification of 113 affected individuals from 19 Czech PPCD families demonstrates, to the best of our knowledge, the highest reported prevalence of PPCD worldwide. Correlated to the population, at least 1 in 100,000 inhabitants in the Czech Republic are affected with PPCD. Because of the relative rarity of this disorder, a founder effect was suspected as the most likely explanation.In this study we show that 38 affected individuals from nine families, including two previously reported families Affected individuals within Czech PPCD1 pedigrees were observed in all subsequent generations implying that the disorder is highly if not fully penetrant, consistent with other studies Although the fully shared haplotype among members of nine families comprised about 1/3 of chromosome 20 including the centromere and pericentromeric regions, we have calculated that the original mutational event did not occur recently. Since the families originate from one particular geographical area, we assume that they represent a relatively isolated population, which are known to have more extensive linkage disequilibrium than outbred populations The mutation age was estimated to be 1800 years assuming a 20-year generation time. It remains to be elucidated if the disease-causing variant arose in the Czech Republic and is specific for that particular geographical region or if it is an ancient mutation introduced from elsewhere. Once the PPCD1 causing gene is discovered it will be possible to further explore these hypotheses.et al. narrowed the proximal boundary of the PPCD1 genetic interval to D20S182 defining the critical disease physical interval to a 1.8 Mb region, under the assumption that there is no micro-heterogeneity The minimal shared region in families 1\u201312 was observed between D20S48 and D20S139, which corresponds to the locus delineation by linkage analysis we described previously in Czech families ZNF133 which is a transcriptional repressor containing KRAB box and zinc finger domains http://www.corneanet.net/) ZNF133 to be the best positional candidate in the refined PPCD1 region, however we did not detect a pathogenic change within currently annotated coding or untranslated regions of this gene. Similarly, no pathogenic variants were observed for ZNF133 in two other studies using probands from different PPCD families linked to chromosome 20 ZNF133 is disease-causing, the change might be located in regulatory sequences, as yet unidentified exons or deep within the introns of this gene.The strongest candidate disease gene based on our current analysis is ZNF133, is very limited such that splice variants and important elements controlling transcription remain undefined. The causative mutations may lie in these uncharacterised upstream regions, exons, and introns or within as yet undiscovered genes on 20p12.1-20p11.23. Independent analysis of each family, by targeted next generation sequencing of the disease interval defined by recombinations within a single large linked family, would circumvent any assumption of locus homogeneity, and may represent the most comprehensive approach, if complemented with Sanger sequencing of gaps and gene expression studies.The data presented here, taken together with previous linkage studies, candidate gene screening, targeted genomic next-generation sequencing ZEB1 were detected in families 18 and 19 Families 15\u201319 apparently originate from different parts of the country than families 1\u201314 and these families provided an excellent internal control for our analyses. None of these kindreds had the full consensus haplotype and, with the exception of the proband from family 19, they did not share the minimal core haplotype segment found in all 64 affected individuals from pedigrees 1\u201312. Based on these observations, it is likely that PPCD in these families is caused by a different mutation or a different gene. In support of this, disease-causing mutations in ZEB1 changes account for approximately 4% cases, whereas a disease-causing gene at the 20p12.1-20p11.23 locus is likely to be responsible for more than 80% of all PPCD cases. A recent study by Vincent et al. highlights the fact that in some cohorts ZEB1 accounts for a smaller proportion of cases than originally thought We conclude that in the Czech population Clinical implications of our study lie in the fact that PPCD1 seems to be more severe showing a higher percentage of secondary glaucoma and necessity for keratoplasty than PPCD3, which has a direct impact on patient counseling ZEB1 mutations were previously identified in two families , and lack of the PPCD1 haplotype in two families  suggests they may not be associated with the PPCD1 locus. In family 15, the PPCD1 locus is excluded and no mutation was detected in VSX1, COL8A2 or ZEB1 suggesting a novel locus may exist.In summary we have demonstrated that the high frequency of PPCD in the Czech Republic is attributable to a founder mutation located on 20p12.1-20p11.23. The disease gene and causative variant have yet to be identified. We anticipate that approximately 80% of PPCD in the Czech population will be attributed to the mutant allele at the locus on chromosome 20p, however it is also clear that other PPCD genes or alleles are implicated as not all families described in this study share a common founder haplotype. Consistent with this finding Table S1Microsatellite markers used to construct haplotypes. Microsatellite markers on chromosome 20 and their extrapolated genetic positions used for genotyping in the current study. Physical distances were from Ensembl release 57 and sex-averaged genetic distances were from The Marshfield Comprehensive Human Genetic Map.(DOC)Click here for additional data file.Table S2Analysis of the ZNF133 gene. Primers used for amplification and sequencing ZNF133, their melting temperature (Tm), length of each amplified fragment and variations identified in the proband from family 1 (A), proband from family 2 (B) and first degree relative from family 2 (C) are shown. Exon number corresponds to reference sequence NM_003434.(DOC)Click here for additional data file.Table S3Identification of a founder haplotype in Czech PPCD families. Each affected individual is represented by a column, presence of the same allele as the consensus haplotype is indicated by x, and presence of the full common haplotype spanning a region of at least 23 Mb is indicated by o. All 67 affected members from Families 1\u201312 originating from the same geographic area within the Czech Republic shared a conserved chromosomal region between D20S48 and D20S139 (highlighted in bold). In families 15\u201319 originating from other parts of the Czech Republic the core haplotype segment between D20S48 and D20S139 was not shared among affected individuals. Affected members from families 13\u201314 were not available for genotyping.(DOC)Click here for additional data file."}
+{"text": "In humans it has been hypothesised that the 2-oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for tm1a(KOMP)WtsiSlc25a21 despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the tm1b(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 3\u2032 of the target gene, was reduced in homozygous tm1a(KOMP)WtsiSlc25a21 mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous tm1a(KOMP)WtsiSlc25a21 mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms.Homozygosity for  More contemporary approaches include the creation of conditional alleles and targeted gene traps, as well as small hairpin RNA (shRNA) and the use of lentiviral transgenesis, reviewed in lacZ tagged null allele neo cassette in the EUCOMM and KOMP tm1a allele, can interfere with the expression of neighbouring genes Knockout mice are invaluable tools for studying the function of genes both during embryonic development and in the adult. Classically, gene disruption in mice is achieved by replacing a part of the target gene with a selectable marker, e.g. a SLC25A21 has previously been reported to be ubiquitously expressed in all tissues tested Slc25a21. From this, three distinct observations were made. Firstly, ablation of Slc25a21 was not found to cause symptoms of 2-oxoadipate acidaemia in mice; animals homozygous for tm1b(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 null alleles were normal for all parameters tested. Secondly, we report that tm1a(KOMP)WtsiSlc25a21 homozygous mice present with profound dental, orofacial and hearing/middle ear phenotypes caused by off-target effects on the expression of the neighbouring gene Pax9. Finally, the resulting novel, hypomorphic allele of Pax9 opens the door to further studies into how Pax9 is involved in the patterning of the palatal rugae and may represent a novel model of otitis media in the mouse. With the growing resource of knockout first conditional ready targeted ES cells created by the EUCOMM and KOMP initiatives, these results represent a timely reminder that the phenotype observed in the knockout first conditional ready mice should be verified by creating and analysing the deletion allele.Here we report characterization of the allelic series derived from the KOMP targeted allele of 2)]. Sterilised Aspen bedding substrate and standard environmental enrichment of nestlet, cardboard tunnel, and three wooden chew blocks were provided. The light cycle was maintained at 12 h light/12 h dark with lights off at 19:30 hours and no twilight period. Room temperature was 21\u00b12\u00b0C and humidity was regulated at 55\u00b110%. Mice were given food and water ad libitum unless otherwise stated The care and use of all mice in this study were in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986, and were approved by the Wellcome Trust Sanger Institute Ethical Review Committee. All efforts were made to minimize suffering including housing mice in a specific pathogen-free unit at a density of 3\u20135 animals per cage in polysulfone individually ventilated cages  were processed for expression analysis by qPCR. For Slc25a21 a custom FAM-labelled TaqMan assay (Applied Biosystems) spanning the junction of exons 8\u20139, 3\u2032 to the floxed exon  was used. For Pax9 a pre-designed TaqMan assay was used . Details of the assays and methods used are described in E13.5 embryo heads [RNA sequencing was performed using 5 \u00b5g of total RNA from a subset of the above E13.5 embryo head samples [3 homozygotes and 2 wild-types for each allele ] as detailed in tm1a(KOMP)WtsiSlc25a21, tm1b(KOMP)WtsiSlc25a21, tm1c(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21), as well as age, sex and genetic background matched controls, were analysed using the standard Sanger Institute Mouse Genetics Project phenotyping screen Homozygous mice (7M and 7F) from each of the alleles (tm1a(KOMP)WtsiSlc25a21 mice (2M and 2F), and age, sex and genetic background matched controls, were fixed in 10% neutral buffered formalin  for 15\u201320 hrs and processed to wax. 5 \u00b5m sections were stained with haematoxylin and eosin then reviewed by an experienced pathologist.At necropsy, skulls were collected and the teeth (both upper and lower jaw) and palate were reviewed and imaged . A range of 42 additional tissues and organs from homozygous tm1a(KOMP)WtsiSlc25a21 mice  were collected at 18 \u00b5m resolution .Micro-CT (computed tomography) images of skulls from tm1a(KOMP)WtsiSlc25a21 mice aged 4 weeks , 8 weeks , 14 weeks , and 26 weeks , using the methods described in detail in Ingham et al. tm1b(KOMP)WtsiSlc25a21 colony , tm1c(KOMP)WtsiSlc25a21 colony  and tm1d (KOMP)WtsiSlc25a21 colony  were tested at 14 weeks of age.ABRs were recorded in Anaesthetised mice were placed on a heating blanket inside a sound attenuating booth and recording electrodes inserted in the skin to record responses of the left ear. For ABR threshold determination, click (0.01 ms duration) and tone pip  stimuli over a range of intensity levels from 10\u201395 dB sound pressure level (SPL) in 5 dB steps were presented in free-field. Averaged responses to 256 stimuli, presented at 42.2/s, were analysed and thresholds established as the lowest sound intensity giving a visually-detectable ABR response.tm1a(KOMP)WtsiSlc25a21 mice aged 9 weeks  were killed by cervical dislocation and the right external and middle ears were examined in detail using a dissecting microscope for any signs of malformation or inflammation, including: appearance of excess cerumen in the external ear canal; thickening, whitening, sponginess or excess vascularisation of the bulla wall; clarity and vascularisation of the tympanic membrane; presence of fluid or white inflammatory material in the middle ear cavity; and solid masses or bony outgrowths of the middle ear wall or ossicles. In addition, the left side of the head of these mice was fixed in 10% formalin for 48 hours, and decalcified in 10% EDTA diluted in phosphate buffered saline (PBS) for 10 days, dehydrated, embedded in paraffin wax, sectioned at 8 \u00b5m and stained with haematoxylin and eosin.Following ABR testing, LacZ reporter gene wholemount expression analysis was performed on tm1a(KOMP)WtsiSlc25a21 adults [aged 15\u201318 weeks ] as described previously tm1a(KOMP)WtsiSlc25a21 E18.5 embryos  was performed using a protocol based on the Cold Spring Harbour method for Alcian blue/Alizarin Red staining Bone and cartilage staining of A reference range approach was used to assess continuous data, including time course. For categorical data, a Fisher's exact test was used to identify phenotypic variants. More details of both approaches are presented in To assess the ABR data, a one-way Kruskall-Wallis ANOVA on Ranks was used where three genotypes were assessed and Mann-Whitney rank sum test where only homozygote and wild-type animals were assessed. The qPCR gene expression results for mutant mice were compared to littermate wild-type mice using a Student's t-Test assuming two-sample unequal variance. P-values for the RNA sequencing were adjusted for multiple testing with the Benjamini-Hochberg procedure. Pre-weaning lethality was assessed using the Test for One Proportion.tm1a(KOMP)WtsiSlc25a21 mice were generated and characterised on a C57BL/6N;C57BL/6-c-BrdTyr background by the Sanger Mouse Genetics Project. Homozygous tm1a(KOMP)WtsiSlc25a21 mice were under-represented at post natal day 14 (P14), as a result of pre-weaning lethality, with 25 homozygous mutants detected among 304 progeny from heterozygous intercrosses .tm1a(KOMP)WtsiSlc25a21 homozygous mice. At 10 weeks of age 13 out of 14 homozygous tm1a(KOMP)WtsiSlc25a21 mice were found to have an abnormally shortened snout (data not shown). At 16 weeks of age all fourteen homozygous tm1a(KOMP)WtsiSlc25a21 mice were found to have macroscopically visible dental abnormalities ranging from white or translucent to severely hypoplastic lower incisors (tm1a(KOMP)WtsiSlc25a21 adult mice (tm1a(KOMP)WtsiSlc25a21 mice was severely under-developed and that the lower incisors appeared to be lacking the enamel coating (tm1a(KOMP)WtsiSlc25a21 mice. Taken together, the 2D X-ray images, micro-CT and X-gal staining in tooth cavities suggests feeding might be uncomfortable for homozygous tm1a(KOMP)WtsiSlc25a21 mice which may explain their reduced body weight and fat mass.Those homozygous animals surviving to weaning, presented with decreased body weight , fat masincisors  comparedincisors . The molult mice , in partult mice . Microscult mice . Micro-C coating  compared coating . Furthertm1a(KOMP)WtsiSlc25a21 mice. Specifically, they presented with abnormal development of palatal rugae number 3 (tm1a(KOMP)WtsiSlc25a21 mice (tm1a(KOMP)WtsiSlc25a21 mice did not reveal any further craniofacial abnormalities.Further characterisation uncovered craniofacial abnormalities in adult homozygous number 3  comparednumber 3 . Micro-Ctsi mice  comparedtsi mice , this watsi mice . The vomtm1a(KOMP)WtsiSlc25a21 homozygous mice were found to have a hearing impairment as they presented with elevated auditory brainstem response thresholds across all frequencies, with severity of impairment increasing with age (tm1a(KOMP)WtsiSlc25a21 homozygous mice displayed an approximately parallel pattern of threshold elevation across the range of frequency stimuli used, a profile which is typical of conductive hearing loss. tm1a(KOMP)WtsiSlc25a21 homozygous mice over the same age range. At 26 weeks of age, some wild-type mice showed age-related high-frequency hearing loss (indicated by larger standard deviations at 24\u201330 kHz). The thresholds recorded in the tm1a(KOMP)WtsiSlc25a21 homozygous mice at all ages were significantly elevated above age-matched wild-types (p<0.001)  .tm1a(KOMP)WtsiSlc25a21 homozygous mice at 9 weeks of age (n\u200a=\u200a8) along with wild-type (n\u200a=\u200a8) and heterozygous (n\u200a=\u200a11) littermates. Compared to wild-types (tm1a(KOMP)WtsiSlc25a21 mouse in which exudate was not observed in the middle ear also had ABR thresholds only slightly above those seen in wild-type mice. Heterozygotes had normal middle ears.To investigate the morphology of the middle ear, the right temporal bone, containing the entire ear, was dissected from ld-types , most ofld-types . Other ftm1a(KOMP)WtsiSlc25a21 homozygous mice and wild-type controls were sectioned through the middle ear, stained with haemotoxylin and eosin, and examined microscopically. Inflammatory changes and accumulation of exudate consistent with otitis media were observed in six of the eight homozygotes WtsiSlc25a21 homozygous mice contained variable amounts of an amorphous eosinophilic exudate containing cholesterol crystals, foamy macrophages and neutrophils, indicating an active inflammatory response. In these mice, the lining epithelium of the middle ear cavity was variably hyperplastic and the underlying lamina propria was oedematous with congestion of blood vessels (The middle ear cavity of the six affected  vessels . None of vessels .tm1a(KOMP)WtsiSlc25a21 homozygous mice showing little or no effusion one had comparatively normal ABR thresholds, whilst the other had raised ABR thresholds.Of the two tm1a(KOMP)WtsiSlc25a21 homozygous mice; assessment of micro-CT images revealed no change in adults whilst alizarin red/alcian blue stained embryos revealed no changes at E18.5 (data not shown).The tympanic ring size was normal in tm1a(KOMP)WtsiSlc25a21 mice did not reveal any further abnormalities, and in particular, no indication of any inflammation other than in the middle ear was found.Systematic histopathology assessment of a range of tissues from homozygous SLC25A21, causes the human disease 2-oxoadipate acidaemia which is clinically characterised by hypotonia, seizures, motor and developmental delay, cerebellar ataxia and varying severities of intellectual disability tm1a(KOMP)WtsiSlc25a21 mice did not phenocopy this human disease. In particular, lean mass, grip strength and gait, correlates of the human disease manifestation, were all normal for these mice (data not shown), and no seizures were detected.It has been proposed that loss of function of ODC, the protein encoded by tm1a(KOMP)WtsiSlc25a21 was performed on RNA extracted from embryo heads (E13.5). When using the Slc25a21 TaqMan assay which spanned exons 8\u20139, 3\u2032 of the cassette insertion, expression was found to be reduced in homozygous tm1a(KOMP)WtsiSlc25a21 samples  but not completely ablated  removed, tm1c a conditional allele with wild-type function restored, and tm1d the deletion allele where both exon 4 and the lacZ reporter were removed. Expression analysis of each of these Slc25a21 alleles was performed on RNA extracted from embryo heads (E13.5). Slc25a21 RNA expression was found to be ablated in homozygous tm1b(KOMP)WtsiSlc25a21  and greatly reduced in tm1d(KOMP)WtsiSlc25a21 homozygotes  (Slc25a21 in embryos homozygous for tm1c(KOMP)WtsiSlc25a21 were not significantly different from wild-type expression  . As expepression .tm1b(KOMP)WtsiSlc25a21, tm1c(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 alleles. Following heterozygous intercrossing, homozygous animals for all three alleles were detected at the expected Mendelian ratio. Furthermore, body weight, fat mass and auditory brainstem response thresholds  .We proceeded by phenotyping animals homozygous for the \u200a=\u200a0.59)  were alltm1a(KOMP)WtsiSlc25a21 targeted allele were not due to ablation of Slc25a21 function, and speculate that off-target effects due to the selection cassette present in the tm1a allele affected expression of neighbouring genes.These results led us to conclude that the phenotypes observed in mice homozygous for the Slc25a21 (500 kb flanking the target gene) is predicted to contain seven known protein coding genes, one non coding RNA, one novel antisense and one putative processed transcript WtsiSlc25a21 homozygous animals, mice carrying a hypomorphic Pax9 allele had previously been reported as causing reduced viability at weaning The 1 Mb genomic interval containing anscript . Of partPax9 was performed on embryo heads (E13.5) for all four alleles in the Slc25a21 series. Pax9 expression was found to be down regulated [approximately 34% of wild-type Pax9 levels (p\u200a=\u200a1.1E-06)] in homozygous tm1a(KOMP)WtsiSlc25a21 samples WtsiSlc25a21, tm1c(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 samples (Pax9 regulatory element (CNS+6) was located within the targeting construct used to create the tm1a(KOMP)WtsiSlc25a21 allele tm1a(KOMP)WtsiSlc25a21 mice caused the reduction of Pax9 expression.RNA expression analysis of  samples  confirmi samples . A knownSlc25a21. Comparing RNA from wild-type and homozygous tm1a(KOMP)WtsiSlc25a21 E13.5 embryo heads, the only gene in the 1 Mb interval surrounding Slc25a21 that had significantly altered expression levels was Pax9 [q \u200a=\u200a7.10E-06 (data not shown)]. Pax9 was not found to be differentially expressed in homozygous tm1b(KOMP)WtsiSlc25a21, tm1c(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 embryo heads. The low endogenous expression level of Slc25a21 in wild-type mice was below the threshold where significant changes could be detected.RNA sequencing was performed to further explore the impact of the targeting event on the genomic environment around tm1a(KOMP)WtsiSlc25a21 mice were broadly consistent with those reported previously in mice carrying hypomorphic Pax9 alleles Pax9 mutant mice and may represent a novel consequence of Pax9 suppression.The phenotypes we observed in homozygous Slc25a21 allelic series derived from a typical KOMP-CSD allele Slc25a21 expression was not found to phenocopy the human disease 2-oxoadipate acidaemia (OMIM 204750) for which it is a candidate gene Slc25a21 null mice [homozygous for the tm1b (KOMP)Wtsi or tm1d (KOMP)Wtsi alleles], a subset of other clinical symptoms associated with 2-oxoadipate acidaemia, including intellectual disability and urine metabolites, were not assessed by the standard Mouse Genetics Project primary phenotyping pipeline with which these animals were screened Slc25a21 null mice that prevent the cytoplasmic build-up of oxoadipate and the associated disruption in the catabolism of key amino acids such as lysine Slc25a21 for 2-oxoadipate acidaemia is not published and it has been speculated that the disease may be due to defective 2-oxoadipate dehydrogenase, an alternative gene in the same metabolic pathway We report the generation and phenotypic characterisation of the tm1a(KOMP)WtsiSlc25a21 homozygous mice investigated in this study were found to be sub-viable. Those homozygous animals surviving to weaning presented with growth retardation, orofacial abnormalities, brittle, white incisors and hearing impairment. Given the absence of any detectable phenotype in mice homozygous for the Slc25a21 tm1b (KOMP)Wtsi, tm1c (KOMP)Wtsi or tm1d (KOMP)Wtsi derived alleles, we conclude that the phenotypes observed in mice homozygous for tm1a(KOMP)WtsiSlc25a21 were not due to ablation of Slc25a21 function, and propose that off-target effects of the tm1a allele affected expression of a neighbouring gene. The spatial, temporal and quantitatively correct activity of a gene requires the presence of not only intact coding sequence but also properly functioning regulatory control. Interference of the expression of neighbouring genes by the presence of selectable marker cassettes or short DNA sequences lacZ reporter gene used in this KOMP-CSD allele has also been reported cis-regulatory elements has been linked with abnormal phenotypes in both mice The Slc25a21 that was differentially expressed, compared to wild-type, in homozygous tm1a(KOMP)WtsiSlc25a21 embryos was Pax9, an observation confirmed by quantitative PCR. Pax9 is located in a 21.06 kb genomic interval directly 3\u2032 of Slc25a21 and orientated on the opposite strand. Evolutionarily conserved non-coding sequences are present both up-and down-stream of Pax9, some of which have been demonstrated to induce Pax9 expression tm1a(KOMP)WtsiSlc25a21 targeting construct, and therefore a candidate for causing altered Pax9 expression. However, sequencing confirmed the integrity of CNS+6 in the tm1a(KOMP)WtsiSlc25a21 allele. Since homozygous tm1b(KOMP)WtsiSlc25a21 mice, which still have part of the targeting cassette present (\u03b2-actin::neo cassette is the most likely cause of the reduction of Pax9 expression seen in homozygous tm1a(KOMP)WtsiSlc25a21 mice. The findings in this study highlight the need to confirm phenotypes found in animals carrying the knockout first conditional ready [tm1a (KOMP)Wtsi or tm1a (EUCOMM)Wtsi] allele by creating and analysing the derived deletion allele. This is a timely reminder, with direct implications on the 3Rs, given the growing resource of knockout first conditional ready targeted ES cells created by the EUCOMM and KOMP initiatives.At E13.5, RNA sequencing revealed that the only gene within the 1 Mb genomic interval surrounding  present , were foPax family of transcription factors plays a pivotal role in embryonic patterning and disease. The expression of each Pax gene is highly regulated in a temporal and spatial manner Pax9 is expressed mainly in the sclerotome of somites, the pharyngeal pouch endoderm and its derivatives, developing limb buds, and in facial mesenchyme of neural-crest cell origin, including nasal and jaw processes and tooth buds Pax9 in the medial edge epithelium during the critical time points of palate fusion Pax9 is expressed in oesophagus and tongue Pax9 deficiency, homozygous Pax9 knockout mice die shortly after birth and exhibit a wide range of developmental defects Pax9 have been published previously Pax9 mRNA at 7% of control levels, presented with hypoplastic upper incisors lacking enamel, missing lower incisors, unilateral or bilateral absence of the third molar in the upper jaw and the second and third molar in the lower jaw, and the lower molars that were present showed severe attrition. In comparison, the hypomorphic allele with 20% of wild-type Pax9 mRNA expression relative to controls presented with less severely affected dentition including relatively normal upper incisors, hypoplastic lower incisors lacking enamel and unilateral or bilateral absence of the upper and lower third molar. We now report homozygous tm1a(KOMP)WtsiSlc25a21 mice which express wild-type Pax9 mRNA at 34% of control levels and present with an even milder dental phenotype including upper incisors that were commonly maloccluded, which may be due to abnormal snout morphology or the fact that the incisors do not wear against each other. We found fragile, damaged or missing lower incisors which were white or translucent; normally incisors in mice are brownish/yellow due to incorporation of iron-containing pigment in the enamel lacZ activity within the cavities of the lower molars of homozygous tm1a(KOMP)WtsiSlc25a21 mice but not heterozygous mice, confirms that bacterial \u03b2-galactosidase is the cause of the staining, not expression of the lacZ reporter gene within the targeted allele. Molars in the upper jaw were less affected. The more severe findings in the lower jaw are consistent with findings in the other Pax9 hypomorphic alleles The Pax9 hypomorphic lines and no abnormalities were reported Pax9 null mice Only limited assessment of the palate has been published for the previous Pax9 expression levels, extended by the data reported herein, clearly demonstrates that dosage of this gene affects orofacial development and that lower jaw dentition in the mouse is more susceptible to reduced Pax9 expression Pax9 gene dosage required for the formation of individual teeth varies, the posterior tooth in each tooth family being the most sensitive to a reduction in Pax9 levels.This gradated allelic series of tm1a(KOMP)WtsiSlc25a21 mice have a normal sized tympanic ring but display a clear hearing impairment with increased thresholds at all frequencies tested. The moderate hearing impairment seen in homozygous tm1a(KOMP)WtsiSlc25a21 mice is robust and reproducible across different cohorts of mice, tested at ages from 4 to 26 weeks of age. The degree of impairment in mutant mice is progressive from 4\u201314 weeks old, with slower progression to 26 weeks of age. The parallel shifts in audiometric profiles across frequencies at the different ages (tm1a(KOMP)WtsiSlc25a21 mice were filled with fluid of varying viscosity at all ages examined and sections revealed inflammation of the lining of the middle ear. The presence of eosinophilic exudate and the thickening of the mucosa are evident in the homozygous tm1a(KOMP)WtsiSlc25a21 mice, indicating an on-going inflammatory process. Whilst the aetiology of otitis media is complex tm1a(KOMP)WtsiSlc25a21 mice. However, cochlear dysfunction cannot be ruled out especially in the 26 week-old group of both mutants and controls. ABR data from tm1b(KOMP)WtsiSlc25a21, tm1c(KOMP)WtsiSlc25a21 and tm1d(KOMP)WtsiSlc25a21 homozygotes indicate that hearing thresholds are normal.A reduction in the size of the tympanic ring has previously been described in Pax9 mutant mice ent ages  are consSlc25a21 expression was not found to phenocopy the human disease 2-oxoadipate acidaemia (OMIM 204750) for which it is a candidate gene. The phenotypes observed in mice homozygous for tm1a(KOMP)WtsiSlc25a21 were not due to ablation of Slc25a21 function, but instead the presence of the selection cassette affected expression of the neighbouring gene, Pax9. The resulting mutant line confirmed and extended the existing knowledge of Pax9 gene dosage effect on orofacial development and represents a novel model of otitis media that may be due to reduced Pax9 expression.In conclusion, ablation of Figure S1Ensembl view of 1 Mb genomic interval encompassing Pax9 and Slc25a21. Ensembl view of the 500 kb of genomic DNA flanking the 5\u2032 and 3\u2032 end of Slc25a21. The tm1a(KOMP)WtsiSlc25a21 targeting construct (shown in blue) designates exon 4 as the critical exon.(TIF)Click here for additional data file.File S1Supplementary materials.(DOC)Click here for additional data file.Table S1The sequence of primers used for molecular characterisation. Molecular characterisation of the targeting event was performed using a combination of PCR assays. The 5\u2032 to 3\u2032 primer sequence along with the expected product size (bp) is presented.(DOC)Click here for additional data file.Table S2Comparison of the outcomes from automatic data evaluation and manual assessment. Comparison of the outcomes from automatic data evaluation and manual assessment for each parameter included in the dataset is presented. Discrepancies between these two methods of assessment are highlighted, and the rationale behind the manual assessment is provided in each instance there was a discrepancy.(DOC)Click here for additional data file."}
+{"text": "The adaptive response (AR) induced by radiation in human lymphocytes has been reported in a range of 1-20cGy pre-exposure. In this study, we investigated the adaptive response using 5cGy conditioning dose of gamma rays followed by 2 Gy challenging dose in peripheral human lymphocyte cells. Blood samples were taken from 30 female volunteers and this experiment was carried out by delivering 5 cGy gamma radiation followed by 2 Gy of challenging. Consequently, the number of micronuclei (MN) in binuclear lymphocyte cells was counted as an endpoint. The results showed that the mean frequency of micronuclei in binuclear lymphocytes which have received both conditioning and challenge doses are significantly reduced in comparison to those only exposed to 2 Gy  (P< 0.01). The results showed the existence of an  The AR reatment . Adaptivreatment , 4. Adapreatment , 5, 6. Tment (.197 (7) dem(.(1977) , 9 parti(.(1977) , 11 and (.(1977) , 12. Sev(.(1977) . AR is a(.(1977) .In the present study, a comparative study on adaptive response using condition dose 5 cGy in human lymphocytes was performed and the effect of gamma radiation at a dose of 2 Gy on adaptive response was investigated.Experimental designIn this experiment, 30 healthy, non-smoker female volunteers aged 19-35 were selected randomly among (O-RH+) blood group subjects due to the higher frequency of this blood group in Babol.The protocol was approved by the Ethics and Scientific Research Committee of Babol University of Medical Sciences. The volunteers signed a written informed consent letter before enrolling in this study. Gamma radiation at a dose of 5 cGy was selected as condition dose (-). To deliver challenging dose, gamma radiation at a dose of 2 Gy was selected (-). Blood samples (2 ml heparinated venous blood) from each volunteer were aliquoted into 4tubes. One part was considered as control group (CTL group). A second part was considered as condition dose and received 5cGy gamma radiation (COD group). The third part was considered as challenging dose and received 2Gy gamma radiation (CD group) and the fourth part was exposed to 5cGy condition dose plus 2Gy challenge dose (COD+CD group).Peripheral blood lymphocytes cultureEach blood sample aliquot was added to 4.5 ml of complete medium . Phytohemagglutinin (PHA) was added as mitogen to stimulate G0 lymphocytes. As suggested by Fenech et al., PHA addition time was considered as cell culture zero time point  and the Mean micronuclei frequencies in lymphocytes of all four groups are summarized in Mean micronuclei frequency in lymphocytes in group 3 (CD group) was 30.2\u00b1 3.29, whereas, this frequency significantly decreased in group 4(p<0.001). In fact when the cells initially received 5cGy followed by 2Gy of challenging dose (group 4), the mean of micronuclei was 20.46\u00b1 2.13Very low doses of DNA-damaging agents can cause adaptation of the cells against higher doses of the same agents, so the cells are less susceptible to damage by subsequent higher doses of these agents. This phenomenon is called adaptive response (AR). This issue was reported for the first time in prokaryotes by Samson and Cairn in 1977 about alkylating agents , 21 with"}
+{"text": "Environ Health Perspect 118:A491 (2010)] incorrectly stated that \u201cAmong individuals who carried both of the GSTT1 and GSTZ1 genotypes noted above (28% of study participants), those with the highest DBP exposure were at a 1.5 times increased risk of bladder cancer compared with carriers with the lowest DBP exposure.\u201d Among individuals who carried both of the GSTT1 and GSTZ1 genotypes noted above (28% of study participants), those with the highest DBP exposure actually were at a 5.9 times increased risk of bladder cancer compared with carriers with the lowest DBP exposure. Risk was only 1.5 times higher with high versus low DBP exposure among study participants who lacked both genotypes. EHP regrets the error.The November Science Selection article \u201cDisinfection By-products and Bladder Cancer: Common Genetic Variants May Confer Increased Risk\u201d ["}
+{"text": "To distinguish between these possibilities we measured epigenetic marks over four generations in rats exposed to a sustained environmental challenge. Dietary energy was increased by 25% at conception in F0 female rats and maintained at this level to generation F3. F0 dams showed higher pregnancy weight gain, but lower weight gain and food intake during lactation than F1 and F2 dams. On gestational day 8, fasting plasma glucose concentration was higher and \u03b2-hydroxybutyrate lower in F0 and F1 dams than F2 dams. This was accompanied by decreased phosphoenolpyruvate carboxykinase (PEPCK) and increased PPAR\u03b1 and carnitine palmitoyl transferase-1 mRNA expression. PEPCK mRNA expression was inversely related to the methylation of specific CpG dinucleotides in its promoter. DNA methyltransferase (Dnmt) 3a2, but not Dnmt1 or Dnmt3b, expression increased and methylation of its promoter decreased from F1 to F3 generations. These data suggest that the regulation of energy metabolism during pregnancy and lactation within a generation is influenced by the maternal phenotype in the preceding generation and the environment during the current pregnancy. The transgenerational effects on phenotype were associated with altered DNA methylation of specific genes in a manner consistent with induction de novo of epigenetic marks in each generation.Induction of altered phenotypes during development in response to environmental input involves epigenetic changes. Phenotypic traits can be passed between generations by a variety of mechanisms, including direct transmission of epigenetic states or by induction of epigenetic marks  Organisms respond to changes in their environment in a variety of ways, both adaptive and non-adaptive There is particular interest in the way in which socioeconomic change in humans, such as increasing affluence or migration, can produce mismatch between the environment experienced by the fetus or infant, including that based on the maternal phenotype, and the actual future environment, and how this increases risk of non-communicable disease Drosophila allows expression of novel phenotypes Induction of altered phenotypes in the offspring by maternal effects can involve changes in the epigenome de novo in each generation through interactions between the maternal phenotype and the environment during her pregnancy. In addition, because the genetic material of the germ cells which will form the F2 generation develops in the F1 during F0 pregnancy The passage of induced phenotypes between generations can also involve transmission of induced epigenetic change. For example, hypermethylation of the hepatic PPAR\u03b1 and glucocorticoid receptor (GR) promoters has been reported in both F1 and F2 offspring of F0 rats fed a protein restricted diet during pregnancy even though F1 dams were nourished adequately during their pregnancy de novo in each generation through interactions between the maternal phenotype and the environment during her pregnancy. We hypothesised that epigenetic marks which were transmitted directly would remain unchanged between generations, while those which were induced de novo in each generation would differ between generations. To mimic transition between stable environments relevant to human dietary transitions, we increased dietary energy content in the treatment group by 25% and maintained this level for the three subsequent generations, comparing the offspring to a reference group which had been fed on standard chow in the breeding colony for more than ten generations  90 expression in gastrulating embryos in each generation. Together, our findings support the suggestion that transgenerational effects involves induction F0 dams gained approximately 40 g more weight at term compared to F1 and F2 dams  3.7, P<0.0001) . HoweverWeight gain post-partum  2.0, P<0.0001) and food intake  2.0, P\u200a=\u200a0.009) was greater in F1 and F2 dams than F0 . There wThe effect of increased energy intake on the phenotype of the adult offspring was assessed by comparison with the offspring of dams fed a lower energy chow diet and which were themselves fed chow from weaning (CF group). Weight gain  5.8, P\u200a=\u200a0.006) on postnatal day 70 was significantly greater in, but did not differ between, F1, F2 and F3 offspring of dams fed the higher energy diet than CF offspring . Energy Because PEPCK is rate limiting in gluconeogenesis and hence is critical to fasting glucose metabolism, the mechanism underlying the change in gene expression between generations was investigated by measuring the methylation of nine individual CpGs in the PEPCK promoter . Comparede novo differed between generations and thus suggests a mechanism for altered epigenetic regulation. Because Dnmt3a showed marked variation in expression between generations, we investigated the mechanism underlying changes in Dnmt3a expression we measured the methylation status of four CpGs in the Dnmt3a2 promoter which accounts of approximately 50% of the Dnmt3a expression in adult liver and is the predominant isoform in developing tissues The mRNA expression of Dnmt1, 3a and 3b was measured in the liver of both adult non-pregnant and pregnant offspring . Dnmt1 eP<0.0001). There was a non-significant trend (P<0.1) towards lower HSP90 expression in F1 than CF embryos. HSP90 expression was significantly lower in F2 and F3 embryos than in CF and F1 embryos  Act (1986) and was conducted under Home Office Licence number 70\u20136457. The study received institutional approval from the University of Southampton Biomedical Research Facility Research Ethics Committee.\u00b0C. Dams and offspring were weighed and 24 hour food intake recorded at 7 day intervals.Female Wistar rats (about 220 g) obtained from a breeding colony were maintained on standard chow for 14 days and then mated. No male was mated with any of its progeny. All diets were obtained from Test Diet . F0 Dams were fed a diet containing 25% more energy compared to the breeding colony diet from conception and throughout pregnancy (n\u200a=\u200a28 per dietary group) . Dams weA group of day 70 female offspring from dams in the breeding colony where female rats had been fed chow diet (2018S)  over at \u03b2HB and glucose concentrations in plasma were measured as described using a Konelab 20 Real time RTPCR was carried out essentially as described The level of methylation of individual CpG dinucleotides in the PEPCK promoter was measured in regions between 44 and 658 bp upstream from the transcription start site  which hapost hoc test. Measures of changes over time were analysed by ANOVA with time as a repeated measure and maternal generation as a fixed factor with Bonferroni's post hoc test. The results of real time RTPCR analysis were non-parametric and were log10 transformed before analysis by ANOVA. Analysis of the relationship between PEPCK CpG methylation and mRNA expression was by linear regression.Values are shown as mean \u00b1 1 SD. Comparison of single time point data between groups was by 1-way analysis of variance (ANOVA) with maternal generation  or offspring group  as fixed factors, with Bonferroni's"}
+{"text": "Hemimetameric Segmental Shift (HMMS) is defined as a hemivertebral deformation in which two or more hemivertebra exists on both left and right sides of the spine, where the hemivertebras are separated by at least 1 normal vertebra. 3D-CT analysis done by Kawakami et al on congenital scoliosis patients proved the existence of mismatch among the anterior and posterior segments, where they coined this phenomenon as discordant anomaly. The purpose of this study is to analyze the morphology and determine clinical features of HMMS 3 dimensionally.HMMS existed in 32 (6.6%) out of 483 patients diagnosed of congenital scoliosis at the respective institution between the years 1998-2013. Of the 32 there were 16 males and 16 females. Average age at the time of their first visit was 6 years 3 months. 3D-CT imaging was done to 30 patients older than 2 years old, with an average age of 9 years 8 months. Using 3D-CT imaging, these 30 patients were classified with posterior elements.Number of patients for each number of hemivertebra present was 21 patients for 2 hemivertebra, 7 patients for 3 hemivertebra, and 2 patients for 4 hemivertebra. Patients with 2 hemivertebra were most common to have hemivertebra in the thoracolumbar spine, while patients with 3 or more hemivertebra was most common to have hemivertebra in the thoracic spine.Analysis using 3D-CT images classified patients into two categories, where malformation exists at an equal level in anterior and posterior sides (unison HMMS) and malformation existing at nonequal levels (discordant HMMS). 9 patients were classified as unison HMMS where all 9 of these patients had 2 hermivertebra. Average number of malformed vertebra in this group was 4.6. On the other hand, 21patients were classified as having discordant HMMS, where 12 patients had 2 hemivertebra, 7 had 3 hemivertebra and 2 had 4 hemivertebra. Average numbers of malformed vertebras were 6.9 in this group, with greater number of malformed vertebras than in the unison HMMS group.HMMS was categorized into unison and discordant classifications. Discordant HMMS existed among 21 patients out of 30 (70%), where all patients with more than 3 hemivertebra were of this type. Out of the 21 patients with 2 hemivertebra, 12 patients (57%) had discordant HMMS. Operation on HMMS patients should be done with extreme care with careful analysis of bone model and radiological images in order to correctly decide on their spinal levels."}
+{"text": "The role of UHRF1 in regulating bladder cancer metastasis was evaluated in bladder cancer cell. We found that UHRF1 levels are upregulated in most clinical specimens of bladder cancer when compared with paired normal tissues, and UHRF1 expression levels are significantly increased in primary tumors that subsequently metastasized compared with non-metastatic tumors. Forced expression of UHRF1 promotes bladder cancer cell invasion, whereas UHRF1 knockdown decreases cell invasion. Overexpression of UHRF1 increases the methylation of CpG nucleotides and reduces the expression of KiSS1. UHRF1 and KiSS1 expression level is negatively correlated in vivo and in vitro. Knockdown of KiSS1 promotes bladder cancer cell invasion. Importantly, forced expression of KiSS1 partly abrogates UHRF1-induced cell invasion. These data demonstrated that upregulated UHRF1 increases bladder cancer cell invasion by epigenetic silencing of KiSS1.Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), as an epigenetic regulator, plays important roles in the tumorigenesis and cancer progression. KiSS1 functions as a metastasis suppressor in various cancers, and epigenetic silencing of KiSS1 increases the metastatic potential of cancer cells. We therefore investigated whether UHRF1 promotes bladder cancer cell invasion by inhibiting KiSS1. The expression levels of UHRF1 and KiSS1 were examined by quantitative real-time PCR assay  Human bladder cancer ranks second in frequency of genitourinary cancer et al demonstrated that UHRF1 overexpression is associated with the grade and stage of bladder cancer et al showed that UHRF1 is overexpressed in colorectal cancer (CRC) cell lines and clinical specimens UHRF1, also called ICBP90 in humans and Np95 in mice, is a multidomain protein, which is required for epigenetic regulation of gene expression and chromatin modification et al demonstrated that KiSS1 hypermethylation is frequently observed and is correlated with low gene expression, being restored by demethylating azacytidine in bladder cancer cells UHRF1 is a very important regulator of DNA methylation, and aberrant DNA methylation is a frequent epigenetic event in bladder cancer In the study, we tested whether UHRF1/KiSS1 represents a novel pathway regulating bladder cancer cell invasion. Our results revealed that UHRF1 expression is upregulated in primary tumors that subsequently metastasized, and overexpression of UHRF1 promotes bladder cancer cell invasion by epigenetic silencing of KiSS1.Human bladder tissues were obtained with written informed consent from the Beijing Friendship Hospital affiliated to Capital Medical University. The study was approved by the Ethics Committee of Capital Medical University. 47 specimens  of patho\u2212\u0394\u0394Ct. *p<0.05.Total RNA from bladder cancer cell lines and specimens was extracted using Trizol reagent . The RT (reverse-transcription) reaction was performed using an M-MLV Reverse Transcriptase kit (Invitrogen). Real-time quantitative PCR was carried out using a standard SYBR Green PCR Master Mix (Life Technologies) protocol on the StepOne Real-Time PCR System (Applied Biosystems) according to the instructions from the respective manufacturer. \u03b2-actin was used as internal control for mRNA. Respective \u0394Ct values (both UHRF1 and KiSS1) were obtained by normalization to \u03b2-actin. Relative expression was calculated with respect to the control. The results were expressed as 2HindIII-EcoRI fragment containing the UHRF1 cDNA into the same sites in pcDNA3.1. The UHRF1 gene was amplified by PCR using the forward and reverse primers: cccaagcttgggATGTGGATCCAGGTTCGGACCATGGACGGG and ggaattccTCACCGGCCATTGCCGTAGCCGGGGAAG. pcDNA-UHRF1 was transfected into bladder cancer cell lines by using Lipofectamine 2000 (Invitrogen).To overexpress UHRF1, plasmid pcDNA-UHRF1 was constructed by introducing a To inhibit endogenous UHRF1 and KiSS1 expression, bladder cancer cells were transfected with 30 nM indicated indicated siRNA or negative control using Lipofectamine 2000. UHRF1-siRNAs  were purchased from Santa Cruz Biotechnology . The KiSS1-siRNAs  were purchased from Santa Cruz Biotechnology.Cell invasion was examined using Transwell invasion assay with inserts of 8-\u00b5m pore size (Corning Costar) as described previously http://www.urogene.org/methprimer/). The forward primers are GATGGAAGGGGAATAGTTTTATTAGA, and the reverse primers are TACAACTAAAACTCCTTCCACCTACA search for enrichment of CpG in KiSS1 was performed and bisulfite sequencing primers were designed using the CpG Island Searcher online tool  from at least three separate experiments. The differences between groups were analyzed using Student's UHRF1 results in abnormal DNA methylation and cancer metastasis, and UHRF1 expression is correlated with a poor prognosis in several cancers. To assess whether UHRF1 regulates bladder cancer metastasis, we first examined UHRF1 expression in bladder cancer cell lines and cancer tissues. in vitro model to investigate UHRF1 by assaying its expression in RT4 or T24 cells after overexpression or knockdown of UHRF1. To investigate the role of UHRF1 in regulating cell invasion, the bladder cancer cell lines treated with UHRF1-siRNA or pcDNA-UHRF1 were analyzed. We first demonstrated whether RT4 and T24 cells can be used as in vivo . Bisulfite sequencing analysis further showed that UHRF1 overexpression increases the methylation of CpG nucleotides of KiSS1  et al showed that UHRF1 contributes to epigenetic gene silencing in prostate cancer progression UHRF1 overexpression is associated with the tumor stages and predicts poor prognoses in various cancers in vivo. Bisulfite sequencing analysis showed that UHRF1 inhibits KiSS1 expression by increases the methylation of CpG nucleotides of KiSS1. More important, KiSS1 overexpression partly inhibits RT4 cell invasion in UHRF1-overexpressing cells.KiSS1 is a tumor metastasis suppressor gene in several cancers. KiSS1 expression is markedly decreased in invasive bladder tumors compared with their respective normal urothelium Our data showed that upregulated UHRF1 contributes to bladder cancer cell invasion by epigenetic silencing of KiSS1."}
+{"text": "The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out \u2013 miR29ab1b2c \u2013 has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow. Hematopoiesis is based on self-renewing hematopoietic stem cells (HSC) and a stepwise process where lineage restricted progenitors generate mature blood cells . MoreoveThe mechanisms underlying HSCs self-renewal are not completely elucidated. Recently, a new class of noncoding genes was identified, called microRNAs . Recent Here, we show that constitutive deletion of miR29ab1 leads to gradual rarefaction of HSCs, whereas miR29b2c knockout mice are entirely normal. Colony forming assays at different age points showed consistent decrease of the HSCs in the miR29ab1 deficient mice compared to the wild type littermates. Also, miR29 deficient bone marrow transplants failed to repopulate the bone marrow of irradiated wild type mice. Affymetrix analysis identified upregulated mRNAs like DNMT3a and b in the miR29 knockouts vs wild type littermate controls. MiR29 double knockouts exhibit markedly generalized atrophy.This indicates a more general role of the miR29 gene clusters in maintaining the size of viscera through an adequate amount of cellularity.All procedures were performed in accordance with the Ohio State University Institutional Animal Care and Use Committee-approved protocols. Homozygous floxed miR-29ab1 mice C57BL6 strain) were generated as follows: for the targeting construct, two homologous recombination arms were amplified by PCR on 129 SvJ/X1 genomic DNA: a 5\u2032 arm of 4171 bp and a 3\u2032 arm of 3857 bp. The genomic fragment to be deleted of 600 bp, containing the miR-29a and miR-29b1, was amplified the same way and cloned in between two loxP sites in a pFlox vector. The recombination arms together with the floxed genes were all cloned into Gateway vectors (Invitrogen) and then assembled together into a destination vector that represented the targeting vector. 129SvJ/X1 ES cells were electroporated with the targeting vector, and clones were screened by Southern blot. DNA was digested with SacI and labeled with a 3\u2032 probe. One positive clone was identified out of 336 screened. The mutant ES cell clone was injected into C57BL/6 blastocysts, and agouti pups were screened by PCR to verify the generation of heterozygous floxed miR-29ab1 mice. Homozygous floxed miR-29ab1 mice were bred to EIIacre mice acquired from Jackson lab to induce ubiquitous deletion of the cluster  and 3U/ml L-GLUPen/Strep. Cells were plated in triplicate for each mouse and the colonies were scored after 10days.Femur bone marrow was flushed with RPMI medium 1640/20% FBS and collected into 5 ml of RPMI medium 1640/20% FBS with heparin 1%. Cells were grown and assessed for chromosomal deletions, translocations, inversions, and number of metaphases.in vitro and amplified by using the BioArray T7 RNA polymerase labeling kit (Enzo Diagnostics). After purification of cRNAs by the RNeasy mini kit , 20 \u03bcg of cRNA was fragmented at 94\u00b0C for 35 min. Approximately 12.5 \u03bcg of fragmented cRNA was used in a 250-\u03bcl hybridization mixture containing herring-sperm DNA , plus bacterial and phage cRNA controls  to serve as internal controls for hybridization efficiency. Aliquots (200 \u03bcl) of the mixture were hybridized to arrays for 18 h at 45\u00b0C in a GeneChip Hybridization Oven 640 (Affymetrix). Each array was washed and stained with streptavidin\u2014phycoerythrin (Invitrogen) and amplified with biotinylated anti-streptavidin antibody (Vector Laboratories) on the GeneChip Fluidics Station 450 (Affymetrix). Arrays were scanned with the GeneArray G7 scanner (Affymetrix) to obtain image and signal intensities.GeneChip Mouse genome 430 2.0 arrays (Affymetrix), containing probe sets for >45,000 characterized genes and expressed sequence tags, were used. Sample labeling and processing, GeneChip hybridization, and scanning were performed according to Affymetrix protocols. Briefly, double-stranded cDNA was synthesized from total RNA with the SuperScript Choice System (Invitrogen), with a T7 RNA polymerase promoter site added to its 3\u2032 end . Biotinylated cRNAs were generated from cDNAs http://www.ncbi.nlm.nih.gov/geo/info/datasets.html.All Affymetrix profile results were uploaded on GEO, at 6) from 5 miR29ab1 homozygous knockout and 5 wild type littermate donors were administered via tail vein injection after the radiation. The mice were sacrificed 50 days later using CO2 euthanasia chambers (as per the OSU IACUC approved protocol). Bone marrow histological sections and aspirates from the transplanted mice were examined. Specifically, the bone marrow aspirates were used for the evaluation of the bone marrow progenitors and the findings were correlated with the histology and cellularity of the bone marrows.Five 8- to 15-week recipient mice were irradiated with 1.1 Gy administered in 2 fractions to minimize toxicity. The recipient mice were represented by wild type littermate controls of the miR29ab1 knockouts. T cell\u2014depleted BM cells  and failure to repopulate irradiated bone marrows in vivo. This is in line with previous studies that demonstrated that overexpression of the miR29a is implicated in the accelerated self-renewal of HSCs and leads to AML . InteresHowever, our Affymetrix data seem to confirm many of the miR29a and b targets like DNMTs, especially 3a and 3b, as previously shown , and oneBased on the Affymetrix data and previous literature , 7, 10 wTo the best of our knowledge, our work is the first to show a comprehensive role of miR29ab1 and b2c in multiple in vivo models in the maintenance of HSCs and possibly other stem cell types. While preparing our manuscript, another group published a similar work in the journal Blood, using miR29ab1 knockouts from Belgium . We had S1 FileMir29ab1 generation strategy (insert: Southern Blot to identify the mutated clones) ; mir29b2c generation strategy (insert: Southern Blot to identify the mutated clones) ; Real Time for miR29a in the miR29ab1 knockout mouse ; Real Time for miR29b in the miR29ab1 knockout mouse ; Real Time for miR29c in the miR29b2c knockout mouse ; spleens collected from 4 month old 29 ab1 ko and WT mice ; flow cytometry on bone marrow of miR29ab1 knockout mice versus wild type for the antibodies indicated in the table  (Table G); complete blood count for miR29ab1 knockout mice versus wild type (Table H);; cytogenetics for miR 29ab1 knockout failed to show any abnormalities ; Kaplan Meyer survival chart\u2014miR29 double knockouts versus wild types ; histology of miR29 double knockout spleens show virtually absent myeloid lineage .(DOCX)Click here for additional data file.S2 File(XLS)Click here for additional data file.S3 File(PPTX)Click here for additional data file."}
+{"text": "To investigate the incidence of local reactions and systemic reactions of subcutaneous immunotherapy in an Allergy Center in Monterrey, Nuevo Le\u00f3n, M\u00e9xico.We conducted a retrospective chart review of patients receiving subcutaneous immunotherapy in our allergy practice between November 2012 to June 2013 looking at the incidence of local and systemic allergic reactions.A total of 8069 subcutaneous immunotherapy injections were applied between November 2012 to June 2013. Among them, 1071 local reactions (LR) were observed (13.2%). Of the total of LR, 1067 were considered small local reactions  (99.6%) and 4 were considered large local reactions  (0.4%). 32 systemic reactions (0.4%) were observed. These SR included respiratory symptoms and cutaneous symptoms. We used the World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System to classify the SR. 27 SR were classified as grade 1 (84.3%). 3 SR were classified as grade 2 (9.3%), characterized by respiratory symptoms  and responded to an inhaled bronchodilator. 2 SR were classified as grade 3 (6.4%) characterized by respiratory symptoms  and did not respond to inhaled bronchodilator and required epinephrine treatment . Neither grade IV nor V systemic reactions were observed.The prevalence of adverse reactions to subcutaneous immunotherapy observed in our study is similar to the reported worldwide. The most reactions are local, small and self limited. The low prevalence of systemic reactions and the absence of fatalities makes subcutaneous immunotherapy a safe therapy."}
+{"text": "BRCA1/BRCA2 genes in breast and/or ovarian cancer families are point mutations or small insertions and deletions scattered over the coding sequence and splice junctions. Such mutations and sequence variants of BRCA1 and BRCA2 genes were previously identified in a group of Sri Lankan breast cancer patients. Large genomic rearrangements have been characterized in BRCA1 and BRCA2 genes in several populations but these have not been characterized in Sri Lankan breast cancer patients.Majority of mutations found to date in the BRCA1 and BRCA2 large genomic rearrangements. One familial breast cancer patient showed an ambiguous deletion in exon 6 of BRCA1 gene. Full sequencing of the ambiguous region was used to confirm MLPA results. Ambiguous deletion detected by MLPA was found to be a false positive result confirming that BRCA1 large genomic rearrangements were absent in the subjects studied. No BRCA2 rearrangement was also identified in the cohort.A cohort of familial breast cancer patients (N\u2009=\u200957), at risk individuals (N\u2009=\u200925) and healthy controls (N\u2009=\u200923) were analyzed using multiplex ligation-dependent probe amplification method to detect BRCA1 and BRCA2 large genomic rearrangements are unlikely to make a significant contribution to aetiology of breast cancer in Sri Lanka.Thus this study demonstrates that  BRCA1 and BRCA2 tumor suppressor genes cause a hereditary predisposition to breast and ovarian cancer[BRCA1and BRCA2 genes in breast and/or ovarian cancer families are point mutations or small insertions and deletions scattered over the whole coding sequence and the splice junctions. Point mutations and sequence variants in BRCA1[BRCA2[BRCA1 and BRCA2 genes. Such large alterations lead to change in genomic copy number and cannot be detected by conventional methods[BRCA1/BRCA2 mutations[Germ-line mutations in n cancer. At presn cancer. Majorit in BRCA1 and BRCACA1[BRCA2 genes we methods. Rearranutations.BRCA1/BRCA2 rearrangements found in different ethnic groups and populations. The prevalence of BRCA1/BRCA2 genomic rearrangements in Asians is thought to be low. However studies done in these populations are limited. Several deletions and duplications have been reported from Singapore[BRCA1 and BRCA2 large genomic rearrangements in Sri Lankans and this study examined the possibility of such genomic rearrangements in a cohort in which point mutations and sequence variants in BRCA1 and BRCA2 were previously described[There is a difference in the degree of ingapore, Korea[ingapore, Malaysiingapore and Chiningapore. There aescribed,6.BRCA2 gene in any of the subjects studied. However, according to MLPA analysis, one breast cancer patient was detected with an average intra normalized ratio  of 0.64 in exon 6 of BRCA1 gene which was predicted as an ambiguous deletion. An average ratio of 0.64 indicates a reduction in relative peak area of the amplification product by 36%. Figure\u00a0BRCA1. Figure\u00a0BRCA2 gene of one of the familial breast cancer patients. The average ratios were within the 0.7-1.3 range indicating absence of exon deletions or duplications of the BRCA2 gene in all samples analysed.MLPA analysis did not reveal any large genomic rearrangements in BRCA1 exon 6, the average ratio for exon 6 was outside the 95% confidence limits and outside the arbitrary range of 0.7 to 1.3. Although for exon 7, the average ratio exceeded the arbitrary range this was still within the 95% confidence limits. Thus we attempted to confirm the BRCA1/ exon 6 deletion by direct sequencing. The sample was sequenced (both forward and reverse strands) along with a healthy control sample for comparison. Sequence data were analysed using Mutation Surveyor DNA Variant Analysis Software \u2013 Softgenetics against reference sequence of BRCA1 in the basic local alignment search tool (BLAST) published by National Centre for Biotechnology Information (NCBI), USA (accession no. L78833). Sequence data showed no change in the DNA sequence of exon 5, 6 and 7of the patient compared to reference sequence. Data also indicated that the MLPA probe hybridization site was intact.In the patient who showed an ambiguous deletion for BRCA1 and BRCA2 mutations and some were positive for BRCA1/2 mutations. From this cohort, three at risk individuals and two familial breast cancer patients were positive for clearly pathogenic BRCA1 mutations and only six familial breast cancer patients were positive for clearly pathogenic BRCA2 mutations. The same cohort was used for the detection of BRCA1 and BRCA2 genomic rearrangements in the present study.Familial breast cancer patients and at risk individuals were previously investigated for BRCA rearrangements is very important in a population since in some populations the occurrence of large deletions and duplications in either BRCA1 or BRCA2 is substantial. A prevalence of 2.1% for BRCA1 large genomic rearrangements has been detected in Spanish hereditary breast/ovarian cancer families testing negative for point variations and small insertions/deletions in BRCA1 and BRCA2[. BRCA1/2 large genomic rearrangements have shown noticeable founder effect in certain European and American populations. One large genomic rearrangement, BRCA1 exon 9\u201312 deletion, is considered as a mutation in Mexican population[BRCA1 gene is currently identified to represent 14.3% (1/7) of the Finnish population[Detection of and BRCA2. BRCA1/2pulation. In NortArthrobacter luteus (Alu) short stretches of repetitive DNA appear to be the main source of large genomic rearrangements by providing hotspots for unequal homologous recombination[BRCA1 have been frequently recognized within intragenic Alu repeats[BRCA1 pseudogene (\u03a8BRCA1) 30\u00a0kb upstream[BRCA1 gene and account for 8%\u201327% of all BRCA1 mutations. Alternatively Alu sequences are less common in BRCA2 gene, where only few large genomic rearrangements are reported, accounting for 0%\u201311% of all BRCA2 mutations[bination,16. Seveupstream,18. To dutations-21.BRCA1and BRCA2 rearrangements for predisposing to breast cancer in familial breast cancer patients and at risk individuals in Sri Lanka. We did not observe any conclusive large genomic rearrangements of BRCA1 and BRCA2 among the subjects studied. However in other Asian countries like Singapore three novel BRCA rearrangements have been found[BRCA1, and exon 4\u201311 duplication in BRCA2. BRCA1 genomic rearrangement found in Korean population involved exons 13\u201315. This exon 13\u201315 deletion has also been identified in three families with America/French-German, Danish, and Singaporean/Indian ethnicities[BRCA1 (exon 13\u201315 deletion and exon 1\u201314 deletion) and one in BRCA2 (exon 14\u201316 deletion) were detected[The aim of this study was to assess the contribution of en found. These wnicities,22,23. Idetected.BRCA1 and BRCA2 large genomic rearrangements are unlikely to significantly contribute to breast cancer in Sri Lanka. This is the first report on the analysis of BRCA1 large genomic rearrangements in Sri Lanka.According to the findings of the present study BRCA1/2 is explained by BRCA rearrangements showing apparent founder effect in some populations which can be used as diagnostic tools. BRAC Analysis Large Rearrangement Test (BART) is already established in the country like US and also has been introduced as new updates to HBOC (Hereditary Breast and Ovarian Cancer) guidelines by National Comprehensive Cancer Network (NCCN). So BART is especially recommended for the individuals with strong personal and family history of breast and ovarian cancer along with routine BRCA analysis. Under such circumstances, it is so important to undergo large genomic rearrangements analysis by the familial breast cancer patients and their at risk individuals in a particular population as a diagnosis tool for breast cancer.The importance of recognizing the large rearrangements with respect to BRCA1 large genomic rearrangement and did not find any BRCA2 large genomic rearrangement in familial breast cancer patients and at risk individuals in the current study, a large study sample especially including Eurasians and other ethnic groups may reveal novel or reported genomic rearrangements among Sri Lankans.Although we failed to find any conclusive A total of 105 participants  were studied. Mean age at diagnosis was 47.76\u2009\u00b1\u20099.55\u00a0years for familial breast cancer patients.Fourteen familial patients were diagnosed below 40\u00a0years of age. Mean age at the MLPA analysis was 36.88\u2009\u00b1\u200914.95 for at risk individuals. Among the familial cases 34, 17 and 4 patients had one, two and three affected family members respectively. Two patients had 4 affected family members. According to histopathlogical data of familial breast cancer patients, 48 had infiltrating (invasive) ductal carcinoma and data were not recorded for remaining 9 patients. None of the patients had metastasis.The majority of the patients and controls and all at-risk individuals were ethnically Sinhalese. There were no descendents of Europeans. Ethical approval from the Research, Ethics and Higher Degree Committee, Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo and written informed consent from the study participants were obtained prior to the study.BRCA1and BRCA2 genes according to manufacturer\u2019s protocol. The processed data obtained via MegaBACE Genetic Profiler software suite\u00ae v2.2 as well as via ABI GeneMapper\u00ae v4.1 were analyzed by using Coffalyser.Net 01 software.Genomic DNA was extracted using the protocol described by Miller et al. from aliExon 5, 6 and 7 specific primers were designed in order to confirm the predicted ambiguous deletion detected from MLPA data. These primers were able to amplify whole regions of exon 5, 6 and 7 and MLPA probe hybridization site as well as several intronic regions. Resultant PCR products were subjected to direct sequencing using Applied Biosystems\u2122 3500 DX Genetic Analyzer in order to locate the deletion site of exon 6 as well as to confirm the data obtained from MLPA analysis.Authors declare that they have no competing interests.SDS carried out molecular genetic studies, sequence alignment and drafted the manuscript. EHK and KHT conceived and designed the study, helped molecular genetic studies, data analysis and revision of the manuscript. IA and PA provided clinical expertise, recruitment of study participants and supervised clinical data and sample collection. All authors read and approved the final manuscript."}
+{"text": "Background: Human labor is a complex series of cellular and molecular events that occur at the materno-fetal and uterine levels. Many hypotheses have been proposed for the initiation of human labor, one hypothesis suggests that maturation of the fetus releases a signal in the amniotic fluid that will be transmitted to myometrium via the fetal membranes and initiate uterine contractions. There is strong evidence that prostaglandins (PGs) play a central role in initiation and progression of human labor.Objectives: In this study we intended to investigate the expression of prostaglandin F synthase and the prostaglandin transporter in the human fetal membranes and to explore the relationship between cytokines and PGs in the mechanism of human labor.Methods: We used fetal membranes obtained before labor at term and after spontaneous labor at term or preterm to identify the changes in prostaglandin F synthase (AKR1B1) and human prostaglandin transporter (SLCO2A1) proteins in relation to parturition. Using fetal membranes explants we tested the effect of cytokines  on PG production and the concomitant changes in cyclooxygenase-2 (PTGS2), AKR1B1 and SLCO2A1 expression.Results: Expression of PTGS2 and AKR1B1 was upregulated in the fetal membranes in association with term labor while SLCO2A1 was downregulated with advancing gestation and during term labor. Before labor, IL-1 increased the expression of PTGS2, however during labor TNF upregulated PTGS2 and AKR1B1 proteins.Conclusions: The prostaglandin F synthase AKR1B1 is upregulated while prostaglandin transporter is downregulated during term labor. The amnion is more responsive than choriodecidua to stimulation with pro-inflammatory cytokines. The mechanisms of term and preterm labor are different. Labor is the result of strong uterine contractions that lead to expulsion of the fetus to the extrauterine environment. The incidence of preterm births is increasing and represents one of the most challenging clinical problems all over the world. In the United States the preterm birth rate increased from 9.5% in 1981 to 12.7% in 2005; the reported rates of preterm birth in other developed countries and Europe range from 5 to 9% , a relatively unstable compound, by the action of prostaglandin H synthases (PTGS) also known as cyclooxygenase enzymes and then into bioactive PGs by terminal synthases such as TXA-, PGE-, and PGF-synthases. PTGS enzymes have received most of the attention in the literature; many studies have investigated their changes with labor and the effect of blocking their actions in the management of preterm labor  from PGH2 by PGH 9-, 11-endoperoxide reductase, (2) from PGD2 by PGD 11-ketoreductase, (3) from PGE2 by PGE 9-ketoreductase  is liberated from membrane phospholipids principally through cytosolic phospholipase A2\u03b1 is the direct conversion of PGH2 by PGH2 9, 11-endoperoxide reductase; this activity is known as PGF synthase  or preterm (25\u201336 weeks), or had an elective cesarean section before any signs of labor at term. Patients admitted for elective preterm deliveries, due to placenta praevia or preeclampsia, or induction of labor (IOL) at term were also recruited Table . The folPrimary antibodies against AKR1B1 and SLCO2A1 were produced and characterized in house . The rabbit anti-goat was used at 1:1000 dilutions, swine anti-rabbit was used at 1: 1000 and the rabbit anti mouse at 1:200. Cytokines  were added to membranes in the explants to test their effects on PG production.Placental tissues were fixed with formalin for 24 h. Representative blocks of each tissue were taken and processed in the Histopathology Department at the Bristol Royal Infirmary with a Leica JUNG TP 1050 Tissue processor  into paraffin wax and sectioned at 2 \u03bcm thickness.The sections were dewaxed in Histo-Clear and passed through alcohol. Endogenous peroxidase activity was blocked by 1% hydrogen peroxide and then washed in running water. The antigen was retrieved by boiling in a microwave after immersion in 500 ml of citrate buffer solution. After washing with PBS 0.02% Tween, the slides were blocked in a solution containing 1% bovine serum albumin (BSA) plus 1% normal human serum and left at room temperature for 1 h.The primary antibodies were prepared in PBS solution, added directly onto the sections and incubated overnight in a humid chamber at 4\u00b0C, then washed in PBS 0.02% Tween-20 three times each for 5 min. Secondary antibodies were added at room temperature for at least 1 h, and then washed in the same way as the primary antibodies. The slides were incubated with DAB solution for 10 min after which were dipped in water and then counter-stained with Mayer's hematoxylin for 30 s and washed with tap water. The slides were dehydrated in graded alcohol solutions and fixed in Histo-Clear, then mounted using mounting media and covered with coverslips. The primary antibodies were excluded during staining of the slides used as a negative control for the secondary antibodies.Images were obtained by using a Leica DC300 camera  mounted on an Olympus BX40 microscope .The tissues were homogenized on ice with cold RIPA buffer  then centrifuged for 40 min at 16,000\u00d7g and 4\u00b0C. The supernatant was collected and the protein concentration was determined by the bicinchoninic acid protein assay kit .Homogenized samples (50 \u03bcg protein) were separated by SDS-PAGE (12% gel) and transferred onto Immobilon membranes . Membranes were blocked with WesternBreeze (Invitrogen) blocking buffer for 1 h at room temperature, and then incubated with diluted primary antibodies overnight at 4\u00b0C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies  for 2 h at room temperature. Membranes were then washed (three times for 5 min) with washing buffer and prepared for detection by chemiluminescence . PTGS2 protein was recognized by its antibody at 72 kDa whereas AKR1B1 protein was recognized by the antibody at 37 kDa and SLCO2A1 protein at 72 kDa. All detected bands were normalized to their individual RhoGDI bands, and then normalized to the ratio of PTGS2 to RhoGDI of a pooled sample used in all gels to eliminate the variations among different gels.Membranes were manually cut into discs (18 mm in diameter) and were held using silicone rubber rings in the upper chamber of a Transwell system (Costar) in which the original disc had been removed. In this model the choriodecidua faces the upper chamber, and the amnion faces the lower chamber. The mounted explant was then placed in a 12-well tissue-culture plate (Costar)  supplemented with 10% fetal calf serum and antibiotic-antimycotic solution  was added to each chamber. The plates were incubated under 5% CO2 and PGF2\u03b1 production  and sheep anti-PGF2\u03b1  were used as selective antibodies. The inter- and intra-assay coefficients of variation (n = 12) were 16 and 10%, respectively. Briefly, 50 \u03bcl of collected culture medium was placed in a 96-well plate coated with goat anti-rabbit (PGE2) or rabbit anti-sheep (PGF2\u03b1) secondary antibody. A volume of 50 \u03bcl from each the tracer and the respective primary antibody was added to each sample after which the samples were incubated overnight at room temperature. After washing each well, 200 \u03bcl of Ellman's reagent  and 54 mM 5,5'-dithiobis  dissolved in 10 mM phosphate buffer pH 7.4 was added. The plate was incubated on a shaker in the dark at room temperature, the reactions between the bound enzyme tracer and the Ellman's reagent yield a yellow color that can be measured with a photometric plate reader. A standard curve was used ranging from 39 to 5000 pg/ml PG.Measurement of PGEThe data was analyzed with SAS statistical software version 9.1.3. . The groups were analyzed separately, using Two-Way ANOVAs for each group. Pairwise comparisons were made between the control mean and each of the 9 treatment means; multiple comparisons were calculated using Dunnett's procedure.AKR1B1 and SLCO2A1 proteins were clearly expressed in decidual cells Figures . In deciStaining with PTGS2 antibody was found to be strongly positive in the cytoplasm of some large decidual cells while smaller cells showed weak staining; connective tissue of decidua basalis showed no staining Figure . In the In the fetal membranes AKR1B1 was highly expressed in the chorion compared to decidua and there was no expression of this protein in the amnion while a poor staining was detected in the mesenchymal layer of the amnion Figure . The intAll layers of the membranes and decidua parietals stained strongly positive with PTGS2 antibody, the staining was also observed in the mesenchymal layer of the amnion Figure .Using immunoblotting we found an increase in the expression of AKR1B1 protein in fetal membranes collected after term labor whether spontaneous or induced. However, preterm labor was not associated with an increase in the level of AKR1B1 protein Figure . By cont2\u03b1 and PGE2 production from the fetal side , its level was found to be significantly higher in the amnion  after the onset of term labor  in labor. TNF acts during labor to increase AKR1B1 protein level leading to increased PGFParturition must be the result of maturational events in the fetus, probably involving the hypothalamic pituitary adrenal axis, increased sensitivity of the uterus to stimulatory agonists and loss of inhibitory mechanisms in myometrial smooth muscle .Michel Fortier has a patent for methods for the regulation of the prostaglandin F synthase (PGFS) activity of AKR1B1 and uses thereof. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "To compare a model of alternate home videoconference consultations/face to face consultations with regular face to face consultations in young rural patients with Type 1 Diabetes Mellitus (T1DM).A 12 month non randomized controlled trial was performed in 2013 comprising a cohort of children, adolescents and young adults with T1DM from the immediate local region compared with a similar cohort from a region greater than 70 Km away. The local cohort continued with 3 monthly appointments and extra visits as required between appointments. The distant cohort had 6 monthly face to face consultations, alternating with 6 monthly formal videoconference consultations to their homes. Extra visits were also managed via videoconference.Outcome was measured by comparison of HbA1c between the two groups before during and after the intervention. Missed or rescheduled visits were compared. A patient satisfaction survey was performed and logistic issues were described from both the patient and medical team perspective.30 patients (mean age 18.3 years) in the control group (mean HbA1c 8.4%) were matched with 29 patients (mean age 17.2 years) in the intervention group (mean HbA1c 8.3%) (NS). During the intervention period, the glycaemic control in both groups deteriorated slightly  (p=0.31) Upon return to regular 3 monthly appointments, HbA1c was 8.4% (control) and 8.6% (intervention). Missed or rescheduled appointments occurred more in the telemedicine group.Patient satisfaction was strong for 4 measures of convenience  but with major inconvenience accessing HbA1c testing. The major disincentives were lack of personal interaction and more difficulty discussing difficult issues. The major issues for the medical team were reduced ability to read patient\u2019s and parent\u2019s emotions because of technology and less commitment to appointments by some families.Telemedicine consultations to home are well accepted and convenient for rural families and young adults with T1DM but are associated with more difficulty accessing HbA1c tests, more missed appointments and more difficulty reading emotional cues during consultations. Glycaemic control did not improve. Catch up videoconferences between appointments were very well accepted. Replacing face to face consultations with direct home videoconference should be done with caution."}
+{"text": "Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8  exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/\u03b2catenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development.  Sp6 and Sp8 during limb development using compound loss-of-function mutants. Sp6 and Sp8, two members of the Sp gene family, are expressed in the limb bud ectoderm and function downstream of WNT/\u03b2catenin signaling for Fgf8 induction. The analysis of the allelic series shows that the progressive reduction in the dose of Sp6 and Sp8 gene products leads to predictable morphology, from syndactyly, to split hand/foot malformation, oligodactyly, truncation and finally amelia, indicating that these two factors act in a complementary manner. The molecular characterization of the mutant limbs reveal that Sp6/Sp8 are required in a dose-dependent manner for Fgf8 and En1 induction, thereby placing them as an important link between the induction of the AER and the establishment of dorsal-ventral patterning during limb development.In this report we examined the functional roles of  The apical ectodermal ridge (AER), a specialized thickened epithelium at the distal edge of the developing limb bud, is a major signaling center for limb development  mutant a virtually normal AER forms despite disturbed DV patterning En1, which in turn restricts Wnt7a to the dorsal ectoderm Very interesting is the connection between the AER and the establishment of dorsal-ventral (DV) patterning Fgf8. Based on their overlapping patterns of expression and on their individual loss-of function phenotypes, we suspected that these two factors act in a complementary manner in the induction and maintenance of the AER downstream of Wnt/\u03b2catenin Sp6;Sp8 null mutants. We also generated Sp6-null;Sp8-conditional mutants using an Sp8 floxed allele with both the AP2\u03b1Cre and the Msx2Cre deleter lines. Interestingly, mutant embryos that lacked the four Sp6;Sp8 alleles or that retained a single Sp6 allele were tetra-amelic. Initial budding occurred, but Fgf8 was not activated in the limb ectoderm preventing further development. Mutants bearing a single functional copy of Sp8 displayed a split-hand/foot malformation phenotype (SHFM) with dorsalization of the digital tips. The phenotypic data together with the molecular defects identified in mutant limb buds indicate that Sp6 and Sp8 are together absolutely necessary for AER development and DV patterning.Sp6 and Sp8, also known as epiprofin and buttonhead, respectively, are two members of the Sp transcription factor family that have been implicated in AER induction and maintenance Sp6 and Sp8 are expressed in the entire prospective limb ectoderm and progressively become confined to the AER as the limb bud emerges. Loss of function of Sp6Sp8Sp6 mutants and the molecular analysis of Sp8 mutants indicates that early limb buds become progressively dorsalized Sp6 or Sp8 does not interfere with the initial activation of Fgf8 in the AER, several studies have demonstrated that both factors function downstream of Wnt/\u03b2catenin signaling and that Sp8 is able to bind and activate the Fgf8 promoter Fgf8 in the AER Both Sp8 expression is maintained in the absence of Sp6Sp6 is expressed in the absence of Sp8 although at a lower level than normal, and is progressively downregulated in concert with the downregulation of Fgf8 expression  were always tetra-amelic  had proximal-distal (PD) complete, but extremely malformed, limbs  and the mutants that retained a single allele of Sp6  were 100% tetra-amelic and showed similar phenotypes to those described above for the double ubiquitous deletions  with that of the Sp8 null mutant (\u2212/\u2212Sp8) in both forelimbs and hindlimbs  resulted in a forelimb truncated at the elbow while the hindlimbs didn't develop  the phenotype notably improved with truncations at the level of the wrist/ankle associated with the formation of an incomplete digit  the phenotype obtained was SHFM  or when only one functional allele of Sp6 remained , Fgf8 was never detected in the limb ectoderm at any of the stages analyzed  and +/\u2212;Sp8\u2212/\u2212Sp6 embryos at the level of the forelimbs showed an irregular thickening of the ventral ectoderm by E10.5 , before ectoderm .\u2212/\u2212; Sp8\u2212/\u2212Sp6 and +/\u2212;Sp8\u2212/\u2212Sp6 embryos, the initial extended expression of Wnt7a was never restricted to the dorsal ectoderm and its expression persisted covering almost the entire limb ectoderm while En1 expression was not detected in the ventral ectoderm , was sufficient to allow the elaboration of all three segments along the PD axis, although the autopod was characterized by the loss or malformation of central elements creating a SHFM.The presence of a single allele of Fgf8 during limb development in \u2212/\u2212;Sp8+/\u2212Sp6 mutants. This analysis showed that the AER precursors were irregularly specified in the ventral ectoderm. The whole mount in situ hybridization at E10 showed obvious gaps and irregularities in the area in which Fgf8 should be uniformly expressed  Fgf10 produced in the limb mesoderm and signaling through the Fgf receptor 2b (Fgfr2b) expressed within the ectoderm The first phase in the formation of the AER is the induction of AER precursor cells in the limb ectoderm characterized by the expression of Sp6/Sp8 is significantly reduced, Fgf8 is not activated, disregarding initial normal Fgf10 expression and Bmp signaling. Because both Sp6 and Sp8 have been shown to function downstream of Wnt/\u03b2catenin signaling Fgf8 promoter Fgf8 downstream of Wnt/\u03b2catenin signaling in the limb ectoderm , indicate an ongoing role for Sp8 in AER maintenance, further supporting our model.It is known that the Wnt/\u03b2catenin signaling pathway is not only required for AER induction, but also for its maintenance. The limb truncations observed when, in the absence of \u03b2catenin loss-of-function mutants in the limb ectoderm that completely lack any evidence of a morphological AER or ectoderm thickening Fgf8 maintenance of expression Most interestingly, our analysis shows that the complete absence of Sp6 and Sp8 transcription factors does not prevent the initiation of AER morphology confirming the independence between AER function and morphology. This is in high contrast to Bmp4, Msx2Fgf8. Sp6 and Sp8 are necessary for the expression of Fgf8, but Bmp ligands and Msx2 are normally activated in the total absence of Sp6/Sp8. Collectively, these data demonstrate that Sp6 and Sp8 mediate only part of the \u03b2catenin functions in the limb ectoderm, principally the induction of Fgf8.\u03b2catenin is also necessary for the expression of other AER markers  to rescue the phenotype of \u03b2catenin loss-of-function in the limb ectoderm is very likely due to Sp8 not reaching, in these experiments, the minimum level of expression required for Fgf8 induction.Recently, it has been shown that a conserved Wnt-Sp8-Fgf8 genetic cassette is also used to regulate the outgrowth of other body appendages such as the genital tubercle limbless, En1 mutants and on misexpression experiments in chick, it was hypothesized that the expression of En1 in the ventral ectoderm might function to establish a DV interface as a prerequisite for AER induction eudiplopodia, the double Wnt7a;En1 mutant and experiments in chick creating bidorsal limbs During normal development the AER forms at the DV boundary of the limb bud reflecting a tight link between AER formation and DV patterning. Based on the analysis of the Sp6/Sp8 gene dose is perturbed. In the amelic phenotypes, even if the limb does not form, the molecular analysis of the emerging limb buds indicates that they are bi-dorsal as Wnt7a expression is extended along most of the limb ectoderm while En1 is not detected. Interestingly, the failure to activate En1 occurs despite normal expression of Bmp ligands in the limb ectoderm and mesoderm. In the SHFM phenotypes the digital tips display conical nails. In these limb buds the AER is irregularly induced and where maintained it remains flat, broad and immature. This correlates with an extension of Wnt7a expression into the ventral ectoderm and a proximally restricted expression of En1Lmx1b expands into the ventral mesoderm distally explaining the bi-dorsal phenotypic traits in the digits of \u2212/\u2212;Sp8+/Sp6\u2212 mutants, while DV patterning is largely preserved at more proximal levels.Here we report that DV patterning is also disrupted when the En1. Sp family members are known to bind and interact with other transcription factors, including Smads. Thus, we hypothesized that Sp6/Sp8 transcription factors interact/cooperate with Smad proteins downstream of Bmp signaling to mediate En1 activation En1 promoter. Interestingly, the putative En1 promoter exhibits 25 potential Sp binding sites and 12 Smad binding sites that are conserved between human and mouse. Further investigation will be required to clarify this relationship  from the AER using Msx2Cre also results in SHFM \u2212/\u2212;Sp8+/\u2212Sp6 mutants, Bmp4 is still expressed in the remaining AER suggesting that this SHFM phenotype is not caused by the loss of Bmp expression in the AER. In fact, since Bmp signalling is required for the induction of Fgf8, the SHFM phenotype following AER-related Bmp removal can also be explained by an irregular induction of Fgf8.Removal of all known AER-related Bmp ligands  and DLX5 and DLX6 (SHFM type I) have been unequivocally associated with this malformation WNT10B (SHFM type VI) were also identified to be causative for SHFM, although there is some doubt on whether these mutations are sufficient for the phenotype Sp6 and Sp8 genes might be part of the Tp63 network. Indeed, the phenotypes of our mutants are identical, including the DV component, to those recently reported in a new identified human mutation in DLX5Tp63, Dlx5 and Dlx6 have essentially normal expression patterns in the early Sp6/Sp8 mutant limb bud indicates that, if Sp6/Sp8 transcription factors act within the Tp63 network, they function downstream of Tp63 and Dlx factors. Tp63 is necessary for the formation and maintenance of a normal epidermal layer Tp63 results in several abnormalities including limb truncations that are most similar to the Sp8-null phenotype Sp8, but not Sp6 in mice. In any case, the relationship between the Tp63-Dlx and the Sp-Fgf8 regulatory modules, both downstream of Wnt/\u03b2catenin, add an extra level of complexity to limb development that requires further investigation.As previously mentioned, despite the identification of 6 loci involved in SHFM, only Sp6 and Sp8 for limb development as in their complete absence, or substantial reduction, no limbs form. By using a variety of loss-of-function alleles to remove the activity of Sp6 and Sp8 genes, we reveal that these two factors work together and in a dose-dependent manner as necessary mediators for AER development and DV patterning.This study provides compelling evidence for the absolute requirement of Fgf8 and also downstream of Bmp signaling in the induction of En1 establishing a link between proximal-distal and dorsal-ventral patterning.Our study supports a model in which these two factors work together downstream of Wnt/\u03b2catenin signaling in the induction of Sp6 null allele Sp8 null allele Sp8 floxed allele AP2\u03b1CreMsx2Cre lines All animal procedures were conducted accordingly to the EU regulations and 3R principles and reviewed and approved by the Bioethics Committee of the University of Cantabria. Mutant mouse lines were described previously: After removing skin and viscera, mouse embryos were fixed in 95% ethanol. Alizarin Red and Alcian blue skeletal staining was performed according to standard protocols, cleared by KOH treatment and stored in glycerol.Bmp4Dlx5 and Dlx6En1Fgf8Fgf10Lmx1bMsx2Tp63Sp6Wnt7aIn situ hybridization (ISH) was performed in whole-mount and in sections following standard procedures using the previously described Embryonic fore and hind- limb buds were dissected in cold RNAse-free PBS from E10.5 wild type embryos. Total RNA was isolated separately from 3 pools of 8 forelimbs or 8 hindlimbs each. cDNA synthesis was done using standard conditions.Real-time RT-PCR was carried out on an Mx3005P cycler, using the SYBRGreen PCR Master Mix (Invitrogen) and the data were analyzed using the MxPro software (Stratagene). Results were tested statistically performing ANOVA and Student-T test, being statistically significant when p<0.05.Sp6 and Sp8 was normalized to that of housekeeping gene 18sRNA. The primers used (5\u2032 to 3\u2032 orientation) were: Sp6-F: tgctaaccgctgtctgtgg; Sp6-R:ctggtatgtctggagaggttgc; Sp8-F: ttatctccaaggtgcacacg; Sp8-R:gcttgaaccaggactcatacg; 18sRNA-R: ttggcaatgtttcgctc;18sRNA-F: cgccgctagaggtgaaattt.Expression of Detection of cell death was performed in sections of paraffin embedded tissue using terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) with the Apoptag Fluorescein Direct In Situ Apoptosis Detection Kit (Intergen) following the manufacturer's instructions.R26R;Ap2\u03b1Cre double transgenic embryos were fixed for 30 min, rinsed in PBS and incubated in the presence of X-gal as described For detection of \u03b2-galactosidase activity, Immunohistochemistry was performed in paraffin sections using the anti E-cadherin , anti Laminin , anti Tp63  and anti Connexin43  primary antibodies. Antigen retrieval was performed by incubation with proteinase K (10 \u00b5g/ml) for E-cadherin and laminin or with citrate buffer in pressure cooker for Tp63 and Connexin43. Alexa\u00ae488 and TexasRED fluorescently tagged secondary antibodies were used. Vecthasield containing DAPI for nuclear counter staining was used as mounting medium. Confocal images were acquired in a SP-5 laser-scan confocal microscope (Leica Microsystems).En1, Sp6 and Sp8 loci between mouse, human, opossum, chicken and zebrafish was determined using pairwise alignment software . Conserved noncoding regions were further analyzed for potential transcription factor binding sites using AliBaba 2.1  and Sequencher 4.8 (Gene Codes Inc.) informatic software.Conservation of Figure S1Sp6 in the limb ectoderm of Sp8 mutants. Whole mount in situ hybridization for Sp6 in limb buds of Sp8 mutant and control littermates. Stage and genotypes as indicated.Expression of (TIF)Click here for additional data file.Figure S2Sp6;Sp8 mutants. Caudal body skeletal preparations of newborns. Genotypes indicated on the left. In the complete absence of Sp6 and Sp8, the pelvis is reduced to a small rudimentary cartilage element. One single functional allele of Sp6  leads to the formation of a misshaped ileum and ischium. A schematic drawing showing the three hip bones in different colors  accompanies each figure.Pelvic girdle morphology in (TIF)Click here for additional data file.Figure S3Sp6 and Sp8 (putative promoter regions). Multiple pairwise alignments of the Sp6 (A) and Sp8 (B) loci comparing human and the species indicated. Light blue corresponds to the untranslated regions of the gene, dark blue to the coding sequence and pink to noncoding regions with at least 70% conservation. Note that only a portion of the chicken Sp6 coding sequence is present in Genebank. Conserved regions within the first intron and the region 5\u2032 to the transcription start site containing binding sites are enclosed in red boxes . These conserved regions are illustrated (5\u2032\u21923\u2032) below the mVista analysis as lines  and depict the relative positions of potential transcription factor binding sites (see legend within the figure). The motifs used to identify potential binding sites are shown in the boxed insert Analysis 5\u2032 upstream of (TIF)Click here for additional data file.Figure S4Cre reporter activity under the Ap2\u03b1 locus in the pre-limb ectoderm. (A) Lateral and (B) dorsal views of E8.5 embryo showing ROSA26 reporter activity. (C) transversal section of the same embryo at the level indicated in B. ROSA26 activity was detected in the entire ectoderm at E8.5 , including the pre-limb ectoderm (black arrowhead in C) and also in the dorsal neural tube.(TIF)Click here for additional data file.Figure S5AP2\u03b1Cre removal of Sp8 on an Sp6 deficient background. The external aspect  and skeletal preparations of the forelimb  and hindlimb  of newborns are shown for each genotype (genotypes indicated at the top). Note that the phenotypes are similar to those of the ubiquitous deletions shown in (TIF)Click here for additional data file.Figure S6En1 (putative promoter region). Multiple pairwise alignments of the En1 locus comparing human and the species indicated. Light blue corresponds to the untranslated regions of the gene, dark blue to the coding sequence and pink to noncoding regions with at least 70% conservation. Conserved regions within the first intron and the region 5\u2032 to the transcription start site containing binding sites are enclosed in red boxes (numbered 1\u20133). These conserved regions are illustrated (5\u2032\u21923\u2032) below the mVista analysis as lines  and depict the relative positions of potential transcription factor binding sites (see legend within the figure). The motifs used to identify potential binding sites are shown in the boxed insert Analysis 5\u2032 upstream of (TIF)Click here for additional data file."}
+{"text": "STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1\u03b1, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1\u03b1 promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1\u03b1. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. \u03b2-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that  The classic JAK/STAT pathway controls cellular responses to cytokines and growth factors by regulating the expression of nuclear-encoded early response genes . CytokinAlthough in the majority of cases, STAT1 and other STATs must be tyrosine phosphorylated to activate gene expression, reports indicate that there are sets of genes regulated by STAT1, STAT3 and other STATs that do not require these transcription factors to be phosphorylated , 3. UnphSTAT1+/+ and STAT1-/- mice, primarily in the context of immune responses, which involve cytokine activation of this transcription factor. Although there are several studies indicating that STAT3 can directly or indirectly affect cellular metabolism, in vivo  or physical activity as measured on the treadmill [Maximum Speed STAT1+/+ 19.33 \u00b1 1.1 compared to STAT1-/- 19.00 \u00b1 0.65 (m/min) and duration of run STAT1+/+ 13.33 \u00b1 1.1 min compared to STAT1-/- 12.83 \u00b1 0.73 min] were detected between STAT1+/+ and STAT1-/- mice (n = 6 mice per group). Although the treadmill endurance was not different between the STAT1-/- and STAT1+/+ mice it is possible that the STAT1-/- mice have increased spontaneous activity in the dark phase. Although STAT1-/- mice displayed no changes in food intake, they showed increased body fat percentage and decreased lean mass weight .We obtained a complete metabolic profile of 12-week-old enditure  during tquotient . No chanquotient , food ins weight . The con glucose . During quotient . IncreasSTAT1-/- mice , but total amounts of triglycerides were increased in WAT of -/- mice . There w-/-mice . This isSTAT1-/- WAT prompted us to examine if there were abnormalities in the distribution of triglycerides and free fatty acids (FFA) in WAT or liver in STAT1-/- mice. There was little loss of subcutaneous or gonadal fat in fasted STAT1-/- mice compared with STAT1+/+ mice   and release of glycerol . We measol (ISO) . Glycero release . To inte release . Forskolipocytes . This reSTAT1-/- adipocytes was similar compared to STAT1+/+ adipocytes. This suggests that the decreased glycerol release in isolated STAT1-/- adipocytes is downstream of HSL activation.We also examined isoproterenol and forskolin-induced phosphorylation of HSL . PhosphoSTAT1-/- mice led us to examine if there were alterations in expression of the mRNA encoding PGC1\u03b1, an important marker of mitochondrial biogenesis [The elevated energy expenditure observed in ogenesis . PGC1\u03b1 hogenesis .STAT1+/+ liver, PGC1\u03b1 mRNA was increased in STAT1-/- liver suggesting there could be alterations in mitochondrial biogenesis in STAT1-/- livers  and STAT1-/- (STAT1-/-ST) mitochondria  did not correct the decreased oxygen consumption in STAT1-/- mitochondria, localizing the defect in respiration to the ETC rather than the phosphorylation apparatus  assays using STAT1 antisera incubated with liver extracts from fed or fasted wild-type mice , glucose is used to make triglycerides which is packaged into VLDL in the liver. VLDL transports lipids to adipose tissue for storage. In the adipose tissue glucose will be used to synthesize new triglcyerides. In STAT1-/- livers (upper right panel), the number of mitochondria is increased as well as the production of ATP. These mice generate VLDL from glucose and transport the lipids to the adipose tissue. However, the STAT1-/- adipose tissue also have increased re-esterification of triglycerides as well as generation of new triglycerides from glucose resulting in increased lipid stores.In STAT1STAT1+/+ mice, the adipose tissue undergoes lipolysis with the activation of hormone sensitive lipase (HSL-P). Triglyceride is hydrolyzed to glycerol and fatty acids. The glycerol is transported to the liver and used for gluconeogenesis. The glucose is released from the hepatocyte and used by other tissues . The fatty acids from adipose tissue lipolysis are transported to the liver and enter the mitochondria for \u03b2-oxidation. The end product, acetyl CoA is used for ketone body synthesis. In STAT1-/- mice very little glycerol is released during lipolysis because most of the glycerol is re-esterfied with FFA back to triglycerides in adipose tissue due to increased phosphoenolpyruvate carboxykinase (Pck1) and increased glycerol kinase expression data not shown). The STAT1-/-mice have more mitochondria which results in greater fatty acid oxidation and ketone body synthesis.In fasted STAT1-/- mice accompanies increased mitochondrial biogenesis. One mechanism by which STAT1 inhibits mitochondrial biogenesis is by suppressing the expression of PGC1\u03b1. ChIP assays using livers from fed mice demonstrate binding of STAT1 to the promoter of PGC1\u03b1. In contrast, there was no binding detected of STAT1 to the promoter in fasted animals (Increased energy expenditure in  animals . AlthougCytokine-mediated activation of STAT1 under most scenarios requires the protein to be tyrosine phosphorylated to induce transcription. However, there are genes whose activation does not require STAT1 to be phosphorylated . We haveSTAT1-/- mice show increased energy expenditure, they have higher fat mass in WAT and decreased \u03b2-adrenergic stimulated lipolysis. Forskolin-stimulated glycerol release which directly activates adenylate cyclase was also decreased in STAT1-/- mice suggesting that \u03b2-adrenergic receptor coupling is not defective in STAT1-/- adipocytes (STAT1-/- compared with STAT1+/+ adipocytes (STAT1-/- WAT, leads to the re-esterification of glycerol to triglycerides and retention of lipid under fasting conditions [STAT1-/- WAT alters lipid metabolism in the liver. The liver compensates for the lack of FFAs released from the WAT by synthesizing more palmitate in the liver. In the STAT1-/- livers, the newly synthesized FFAs undergo \u03b2-oxidation to generate ATP and thus create a futile cycle. This futile cycle prevents lipid accumulation in the livers of fasted STAT1-/- mice (It remains to be determined whether the actions of STAT1 on energy expenditure in mice are interrelated and coordinated with its actions on the breakdown of triglycerides. It is paradoxical that while ipocytes . Surprisipocytes . There anditions . Activat-/- mice .Complementing our findings of a role of STAT1 in energy metabolism, mitochondria are also involved in a variety of cellular functions which are influenced by STAT1 such as cell cycle regulation, oxygen sensing , 26, and"}
+{"text": "The authors regrew the cell cultures in these panels, as well as the parental strains shown in the same panels of The authors would like to correct The authors confirm that these changes do not alter their findings. The authors have provided the underlying images as Supporting Information.S1 Figbld2-6; BLD2; bld2-6 and pf15; PF15::HA on control medium (A) and 8 \u03bcM Taxol-containing medium (B).Serial dilutions of mutant and rescued strains for (TIF)Click here for additional data file."}
+{"text": "Imprinted genes are an exceptional cluster of genes which are expressed in a parent-of-origin dependent fashion. This allele-specific expression is dependent on differential DNA methylation which is established in the parental germlines in a sex-specific manner. The DNA methylation imprint is accompanied by heterochromatin modifications which must be continuously maintained through development. This review summarises the factors which are important for protecting the epigenetic modifications at imprinted differentially methylated regions (DMRs), including PGC7, ZFP57 and the ATRX/Daxx/H3.3 complex. We discuss how these factors maintain heterochromatin silencing, not only at imprinted DMRs, but also other heterochromatic regions in the genome. Imprinted genes are a specialised group of genes in mammalian genomes which are monoallelically expressed in a parent-of-origin dependent manner. Approximately 100\u2013150 imprinted genes have been identified in both the mouse and the human genome to date \u20135. The dIn addition to differential DNA methylation, gDMRs are also associated with differential histone modifications in accordance with the methylation status \u201310. The To correctly specify imprinted gene expression in the developing organisms, DNA methylation at gDMRs must survive a wave of demethylation which occurs during very early development  Fig.\u00a02)2). MammaPGC7  was first identified as a protein which is highly expressed in primordial germ cells (PGCs) . ExpressThe specificity of this altered methylation was found to be conferred by the preferential localisation of PGC7 to H3K9me2 modified chromatin . The twoThe preferential binding of PGC7 to the maternal-derived genome suggested that the H3K9me2 modification may be important for PGC7 localisation. Consistent with this, PGC7 was found to interact most strongly with H3K9me2 modified histone peptides in in vitro binding assays and a pull-down of endogenous PGC7 demonstrated an enrichment for H3K9me2 . FurtherForced expression of Jhmd2a resulted in strong binding of Tet3 to both maternal and paternal derived genomes and knockout of PGC7 resulted in abnormal Tet3 mediated conversion of 5mC to 5hmC on the maternal-derived genome. Furthermore, recombinant PGC7 was found to directly repress the enzymatic activity of Tet3 in in vitro assays . CombineIn addition to PGC7 a second protein, Zfp57, is known to play an important role in maintaining DNA methylation at imprinted DMRs in early embryos. Zfp57 is one of hundreds of KRAB-ZFPs which are present in mammalian genomes . Each KRChIP-seq revealed that Zfp57 was bound to all known imprinted gDMRs and this binding was specific to the methylated allele . In siliThis process is dependent on Zfp57 recruitment of KAP1 to methylated gDMRs as expressing a KAP1-interaction defective, KRAB-domain deleted Zfp57, is unable to rescue the knockout . KAP1 isDNA methylation does not occur in isolation, but is instead inextricably linked to histone modifications. Three well characterised histone modifications\u2014H3K4me3, H3K9me3 and H3K36me3\u2014are particularly important in the context of genomic imprinting. H3K4me3 is a modification which is highly enriched around gene promoters and is permissive to transcription . At imprIn addition to the specific requirement for H3K4me0, the localisation of DNMT3a/b are also guided by interactions with HP1 , 42 whicIn addition to the ADD domain, DNMT3a/b also harbours a PWWP domain which is able to interact with the H3K36me3 modification \u201353. H3K3The association between transcription and DNA methylation at imprinted gDMRs is further supported by studies on the corresponding non-imprinted allele. Many imprinted gene clusters express at least one long non-coding RNA (lncRNA) which is often arranged in the antisense orientation with respect to the methylated gDMR promoter Fig.\u00a0. ExampleIn addition, several other studies have indicated that the process of transcription may also play a role in this process , 65. ForThe active transcription through the intragenic maternal methylated DMRs poses an additional challenge for the maintenance of methylation at these sites. The position of these imprinted DMRs means that these heterochromatic foci are subjected to transcription, and chromatin modifications must be continuously restored following the passage of RNA polymerase. This process has recently been demonstrated to be dependent on a histone variant, H3.3. Unlike the canonical histones (H3.1/H3.2), which are expressed and deposited only during S-phase, histone H3.3 is expressed throughout the cell cycle and replaces histones which are displaced by transcription , 67. ConTwo major chaperone complexes which deposit H3.3 have been identified. The HIRA complex is the major H3.3 chaperone at euchromatin genic sites , 70 whilIn addition to the imprinted DMRs, a number of other methylated genomic sites are also targeted by Zfp57 and the ATRX/Daxx complex , 34, 69.Similar to IAPs and imprinted methylated DMRs, the 3\u2032 end of zinc finger genes are also enriched for heterochromatin modifications , 83. ThiThe phenomenon of genomic imprinting is therefore dependent on the acquisition of differential methylation in parental germlines followed by the selective maintenance of imprints through development. Sex-specific differences during germline specification results in a differential pattern of DNA methylation in parental genomes at fertilisation. These differentially methylated regions include the imprinted gDMRs but are not exclusive to these sites. The differential methylation at non-imprinted regions is subsequently erased during the pre-implantation period when active and passive demethylation processes occur indiscriminately across the genome, except at regions which are specifically protected.The maternal genome and paternally methylated gDMRs are protected from active Tet3 mediated demethylation via H3K9me2 dependent recruitment of PGC7. Silencing modifications at gDMRs are then further reinforced by the binding of Zfp57 and the KAP1 co-repressor complex which recruits both DNMT3a/b and Setdb1 to catalyse DNA methylation and H3K9me3 respectively. The continuous protection of silencing modifications at gDMRs is further facilitated by the ATRX/Daxx complex which deposits the replication independent H3.3 histone variant. Daxx reportedly interacts with KAP1 and Setdb1 which modifies H3.3 to H3.3K9me3, thus ensuring the faithful protection of heterochromatin modifications at gDMRs, even when subjected to transcription. In addition to the methylated gDMRs, both Zfp57 and the ATRX/Daxx/H3.3 complex also bind other methylated regions such as IAP retrotransposons and the 3\u2032 end of zinc finger genes. Thus, neither the acquisition of differential methylation nor the maintenance processes are exclusive to imprinted gDMRs, and neither process is deterministic for genomic imprinting. Instead, the imprinted gDMRs arise from the intersection of these two pathways and it is this combinatorial interaction which ultimately distinguishes imprinted gDMRS from the rest of the genome."}
+{"text": "Anisakis are common parasites of a wide range of aquatic organisms. Public interest is primarily based on their importance as zoonotic agents of the human Anisakiasis, a severe infection of the gastro-intestinal tract as result of consuming live larvae in insufficiently cooked fish dishes. The diverse nature of external impacts unequally influencing larval and adult stages of marine endohelminth parasites requires the consideration of both abiotic and biotic factors. Whereas abiotic factors are generally more relevant for early life stages and might also be linked to intermediate hosts, definitive hosts are indispensable for a parasite\u2019s reproduction. In order to better understand the uneven occurrence of parasites in fish species, we here use the maximum entropy approach (Maxent) to model the habitat suitability for nine Anisakis species accounting for abiotic parameters as well as biotic data (definitive hosts). The modelled habitat suitability reflects the observed distribution quite well for all Anisakis species, however, in some cases, habitat suitability exceeded the known geographical distribution, suggesting a wider distribution than presently recorded. We suggest that integrative modelling combining abiotic and biotic parameters is a valid approach for habitat suitability assessments of Anisakis, and potentially other marine parasite species.Marine nematodes of the genus  The distribution of marine endohelminth parasites is influenced by a wide range of abiotic and biotic factors. While the development and dispersal of excreted propagules (eggs) is predominantly influenced by physical parameters 9161318192021Anisakis contains nine distinct species, each with different ecological characteristics (e.g. host specificity) and human zoonotic hazardous potential92324252728Owing to the routine application of molecular techniques as diagnostic tools in biodiversity research, it is now accepted that the genus et al.Anisakis spp. based on molecularly identified presence data, which has previously been displayed only on the basis of biogeographical occurrence data in form of so called \u201cdot maps\u201d , available since 2014, standardized data of environmental variables are now available, thus, offering an opportunity to model the geographical distribution of marine speciesAnisakis using updated molecular occurrence data extracted from the scientific literature and a state-of-the-art habitat suitability modelling approach based on environmental parameters (abiotic factors) available to date Anisakis species. A high definitive host diversity means that several of the recorded definitive host species are modelled to find suitable habitat conditions in a certain region.The modelling results for the nine Anisakis species are summarized in Anisakis species, the AUC values are high (above 0.95).\u201cArea under the Receiver Operator Curve (AUC)\u201d values for the Maxent models of the nine Anisakis species as well as the number of environmental variables (6 abiotic variables and a varying number of hosts (biotic variable) depending on the species) used for the modelling. The habitat suitability for five definitive host species  could not be modelled as for these species insufficient (less than 10) or no occurrence records were available from AquaMapsAnisakis species the number of considered definitive host species variables is less than the number of recorded definitive host species  between the modelled habitat suitability of Anisakis species obtained by IS1 and definitive hosts obtained by IS2 were calculated for every combination of parasite and host  and a maximum of rs\u2009=\u20090.94 (A. nascettii \u2013 Mesoplodon layardii). In some cases, positive correlations were obtained, even if no parasite-host-interaction between certain combinations has been documented from the literature so far . Simultaneously, not every documented parasite-host-combination, despite documented host record, yielded positive correlation values .Spearman correlation coefficients  as well as A. typica and two sister-species A. nascettii and A. ziphidarumA. physeteris- complex 6A. simplex-complex and A. physeteris-complex are considered cryptic species, distinguishable only by means of molecular analyses as well as slight morphological differences The genus Anisakis species generated in this study coincides in large parts with results of earlier studies6The distribution (dot maps) of Hot spots of occurrence records were found in the Mediterranean region, in the region of Japan, along the North-American coasts as well as in the waters of the North-Atlantic; areas with extensive fisheries and economically  important fish species. Hot spots of host diversity are not inevitably congruent with these areas, mostly because reliable occurrence data for the definitive hosts usually stem from regions with a good accessibility and a scientific and public interest , which do not necessarily overlap with the areas where intermediate fish hosts are caught.Anisakis simplex s.s. has exclusively been recorded by means of molecular methods from hosts in the northern hemisphere, mainly in the Atlantic and Pacific Ocean as well as in the Western Mediterranean (A. pegreffii (see below), can most likely be explained by increased economic research interests in potential harmful organisms in commercially highly significant and often consumed raw fish species.The close phylogenetic relationship between some of the species is mirrored in similar modelling results using biotic and abiotic variables. erranean . The disAnisakis pegreffi shows a disjunct distribution, with a lack of evidence along the North American West Coast and some additional findings between South America and the Antarctic Peninsula and South Africa and New Zealand.A. berlandi, along the Pacific Northwest Coast, the Southern Ocean, the Weddell Sea and the coast of South Africa is comparable to that of the two closely related sister species, A. simplex s.s. and A. pegreffii. The close phylogenetic relationship of these three species is reflected in a similar distribution pattern nean Sea . A. ziphAnisakis physeteris-complex  in the tropical and subtropical Atlantic, and occasionally in Japan/China and New Zealand (only A. paggiae), reflects their close phylogenetic relationship9A. ziphidarum and A. nascettii, increased host specificity; based on literature clear host preferences occur for kogiid Kogia breviceps and K. sima while A. physeteris is the only type of the complex which additionally parasitizes the sperm whale Physeter macrocephalusKogia breviceps (rs\u2009=\u20090.44\u20130.59). Increased correlation values for definitive hosts which are not in the known host range of the three types  (see The relatively homogeneous appearance of the three representatives of the tus) see  can reprAnisakis. Klimpel and R\u00fcckertHysterothylacium aduncum from the North Sea. Fueled by an increased primary and secondary production, permanent upwelling of nutrients resulted in an accumulation of predators and prey in the vicinity of halo/thermocline fronts, and thus, favouring the transmission of parasites among hosts. H\u00f8jgaardAnisakis simplex eggs varied inversely with temperature and that survival time increased with salinity but decreased with temperature, supporting the hypothesis that Anisakis simplex is rather adapted to (off-shore) pelagic marine environments with high salinity. Both temperature and salinity were identified crucial in the variable importance analyses of this study. The influence of land distance and depths is probably an effect of the individual association of certain definitive host groups to either offshore or inshore marine habitats. Sperm whales, for example, usually inhabit deep sea habitats and tend to migrate in off-shore waters over large distances resulting in the dispersal of parasite propagules (e.g. A. physeteris) in offshore pelagic watersTursiops aduncus and T. truncatus, common hosts of A. typica  impacting the distribution of . typica , are ratAnisakis species quite well. The generally high accordance between the distribution of occurrence data and modelling results is supported by high AUC values. As the AUC values depend on the number of considered occurrence records the AUC values may not be used in order to compare the performance of modelling results between species.The modelled habitat suitability using abiotic environmental parameters and biotic host distribution data reflects the observed distribution pattern for all nine Anisakis and definitive host species occurrences. Occurrence data are always affected by a sampling bias, e.g. species are commonly recorded in specific areas more often than in others due to a larger interest in that region compared to others, resulting in unequal probabilities of records. Hot spots of recorded occurrence and hotspots of actual occurrences may thus differ, which may yield a false representation of the species\u2019 niche reducing the reliability of the modelling results.Sampling bias is one of the issues that has to be considered when modelling species\u2019 distribution using correlative approaches and may have affected our data here, i.e. A. simplex s.s./A. pegreffii) the area with high modelled habitat suitability exceeds the area with recorded presences of the Anisakis species. This could have several causes: Despite suitable habitat conditions, Anisakis species do not occur in these regions which may be due to a potential dispersal limitation of the Anisakis species or the associated host species (e.g. migratory vs. more stationary cetacean definitive hosts). A mismatch could also be caused by limited sampling efforts, i.e., that the Anisakis species occur in these regions with high modelled habitat suitability but no  occurrence has been recorded there to date (sampling bias). A third reason might be that a crucial factor relevant for Anisakis habitat requirements was not considered in the modelling. Overall, it is not clear which of these factors might best explain the \u201coverprediction\u201d.In some regions  or environmental niche models (ENMs), are correlative approaches that model the potential geographical range of a species subjected to various environmental variablesAnisakis species.Here, the maximum entropy niche modelling approach (implemented in the freeware MAXENT)41Anisakis occurrence data from studies that have performed molecular methods to guarantee unambiguous identification to (cryptic) species level . In addition to the presence data that have been included in a former approach described in Kuhn et al.Anisakis, Anisakid) to assess the novel research articles in the field that have been published since 2011 and extract specific locality records. In total, 101 publications were considered in the present model916171822364748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142Habitat suitability modelling was carried out using only Anisakis Anisakis species.The environmental data were loaded at a spatial resolution of 5 arc minutes  and rescaled at a spatial resolution of 1 decimal degree computing the mean. This resolution is in accordance with the resolution of the occurrence records for the Anisakis species with the primary aim to identify those variables that on average contributed most to a Maxent model. In order to identify the most important abiotic factors the Maxent models were run for each of the nine Anisakis species. The Maxent permutation importance (i.e. a measure for the variables\u2019 contribution to the Maxent model) was calculated for each of the 14 abiotic factors and was transformed into ordinal rank scale for each of the nine Anisakis species. The six abiotic factors with the lowest median for all nine Anisakis species were taken as a final subset of abiotic factors in order to model the habitat suitability for the Anisakis species, together with the modelled habitat suitability of all known final host species as biotic factors . This was done in order to reduce the number of predictor variables for the final modelling (integration of abiotic and biotic factors), thus, reducing the risk of overfitting. Based on the same subset of 14 abiotic factors  for the definitive host species were converted into binary data  using the threshold that minimizes the difference between sensitivity and specificityAnisakis species resulting in definitive host diversity maps that show the number of definitive host species for the respective Anisakis species with modelled habitat suitability. For visualization, maps were built using Esri ArcGIS 10.3.In order to evaluate the modelling results, dot maps with the recorded occurrences of each of the nine Anisakis species based on the 14 abiotic variables (IS1) and the modelled habitat suitability for the definitive host species based on the same 14 abiotic variables (IS2) were calculated to identify pairs of definitive host species and parasite species with a similar pattern of modelled habitat suitability. For pairs with a similar pattern of modelled habitat suitability a parasite-host interaction was assumed.Spearman correlation coefficients between the modelled habitat suitability for the different How to cite this article: Kuhn, T. et al. Environmental variables and definitive host distribution: a habitat suitability modelling for endohelminth parasites in the marine realm. Sci. Rep.6, 30246; doi: 10.1038/srep30246 (2016)."}
+{"text": "S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75\u201380% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.Targeted alteration of the genome lies at the heart of the exploitation of  S. pombe has helped it to maintain a prominent position alongside the more extensively exploited budding yeast Saccharomyces cerevisiae, as a powerful model system for the characterisation of the basic facets of eukaryotic cell and molecular biology. This malleability is based upon an extensive repertoire of classical and molecular genetic techniques The genetic malleability of the fission yeast S. pombe highlighted the utility of the colony-colour change resulting from the accumulation of P-ribosylaminoimidazole in ade6 mutants that is then oxidised to a red pigment ade6.M210/ade6.M216 hetero-allelic complementation for the selection and maintenance of diploid strains sup3.5 opal suppressor tRNAser mutation as a marker for selection in an ade6.704 mutant background S. pombe leu1 mutations with the S. cereviaisae LEU2+ gene was initially used to apply existing budding yeast technology to fission yeast S. pombe genome means that it does not direct integration into a specific genomic site. However, when used as a marker to select for site specific integration, multiple integration events can occur LEU2+ gene is barely sufficient for growth at low copy number or that the budding yeast enzyme is less attuned to fission yeast physiology than the native 3-isopropyl malate dehydrogenase enzyme, Leu1. Transposition of the lessons learnt from the exploitation of the budding yeast ornithine decarboxylase URA3+ gene for positive and negative selection ura4+ gene from S. pombe to generate the ura4.d18 allele that is so widely used in the field today ura4+ based vectors ade7, his1, his2, his3, his5, arg3, arg12, lys1, lys2 and tyr1his3+, LEU2+and ura4+ remain the most widely-used markers for selection of multi-copy vectors in common use. Integration vectors that target a particular heterologous locus have been less extensively developed, however the pDUAL series and pJK148 vectors are used widely as they exploit recombination to convert the leucine auxotrophy of leu1.32 to leucine prototrophy to select integration at the leu1 locus ura4.294 to target integration at the ura4 locus Classical genetic analysis the adenine biosynthesis pathway in While these auxotrophic selection markers offer powerful tools, they also create the need to introduce an increasingly complex array of background markers into a strain of interest. Not only is this time consuming but many combinations of deficiencies in amino acid provision compromise a host strain\u2019s fitness on certain media, which may complicate the interpretation of the phenotype arising from the mutation of interest. Furthermore, the sensitivity of the broadly acting TOR signalling network to addition of leucine to the medium S. cerevisiaeS. pombe. Genes conferring resistance to kanamycin/G418, hygromycin B, phleomycin/bleomycin and nourseothricin/ClonNat are highly effective dominant markers in fission yeast Following the highly successful exploitation of antibiotic resistance genes as dominant selectable markers for PCR based tagging and deletion approaches in the budding yeast Although attempts to define the extent of the problem have proved challenging S. pombe (Pku70 and Pku80 respectively) increases targeting efficiency at the fin1+ locus from 5% to 80% (16-fold increase). A modification to transformation procedures enhances transformation frequencies by a further 5\u20138 fold when selecting for antibiotic resistance markers. We show that a PCR cassette that combines the cycloheximide sensitivity of rpl42+ in an rpl42.sP56Q background natMX6ura4+ and 5 fluoro-orotic acid (FOA) natMX6 or kanMX6 as a selectable marker.We now show that the removal of either the Ku70 or Ku80 homologues from E. coli strain DH5\u03b1 was used to propagate plasmids in standard LB medium. The S. pombe leu1.32 his2 h+(IH147) and 972h- (IH5974) strains were grown by standard procedures \u22121 Geneticin , Hygromycin B , Nourseothricin/ClonNat , Phleomycin , and Cyclohexamide  were added to generate final concentrations of 100 \u00b5g/ml in the growth medium where appropriate.6 cells/ml). After harvesting cells were washed with H2O, 0.1 M Lithium Acetate (pH 4.9) and re-suspended in 0.1 M Lithium Acetate (pH 4.9) at 109 cells ml\u22121. After 1 h incubation at 25\u00b0C 1\u20135 \u00b5g DNA and 290 \u00b5l of 50% PEG4000  was added to 100 \u00b5l cell-suspension. After 1h incubation at 25\u00b0C and 15 min heat shock at 43\u00b0C, cells were harvested, washed with H2O and re-suspended in MSL-N or spread to YES.Cells were grown to mid-log phase in YES  using oligo nucleotides BH1 and BH2, creating a NotI site. A 2.32 kb fragment extending from \u2212456 to +752 of the S. pombe leu1+ gene was amplified as a NotI fragment using oligo-nucleotides BH3 and BH4 and cloned into the modified pUC19 vector to create pINT1. An 1.78 kb fragment extending from \u2013516 to +1186 of the S. pombe ura4+ gene was amplified from pURA4 ura4+ sequences with 5\u2032 PstI site and 3\u2032 SacI sites. Oligos BH7 and BH8 were used to amplify pINT1, which was then used as recipient for the amplified ura4+ fragment within the leu1+ open reading frame by Gibson-mediated integration (New England Biolabs) to generate pINTLA that can act as recipient for any PstI/SacI fragment containing promoter-insert-terminator  to introduce HindIII NotI sites at one end and PstI site at the other end. The lys1 3\u2032region was amplified (VS645/VS646) to introduce a KpnI site at one end and EcoRI NotI sites at the other end. Both fragments were cloned HindIII - PstI and KpnI \u2013 EcoRI, respectively into pGEM3. The loxP\u2013ura4 cassette was generated by PCR amplification of the +ura4 gene (VS647/VS648) to introduce KpnI site and a LoxP site on one end and SmaI SacI sites with the LoxP site on the other end. This fragment was then cloned as a KpnI \u2013 SmaI fragment into the +lys1 containing vector to generate pINTK, pINTK81, pINTK41 and pINTK1 by cloning the nmt promoters from pREP81, pREP41 and pREP1 respectively as PstI \u2013 BamHI fragments into pINTK. GFP, CFP and YFP tags were amplified to introduce a BamHI site at the 5\u2032end and NheI SmaI SacI sites at the 3\u2032end. The tags were then cloned BamHI \u2013 SacI into the pINTK81/41/1. The 6His tag was cloned as a BamHI \u2013 NheI fragment generated after annealing of complementary oligonucleotides (VS1482/VS1483) into the pINTK1GFP to generate pINTK1-6His. The sequence of the pINTK vector is presented in The PstI and NdeI sites from hphMX6  and a XmaI/SmaI site from natMX6 (CCCGGG>CCCAGG). The following fragments were amplified using the indicated primers to introduce restriction sites at their termini before being cloned into the vector ZeroBluntTOPO (Invitrogen): one 0.85 kb half of hphMX6 flanked with PstI and NotI HindIII (primers DF1 and DF2); the remaining 0.85 kb fragment flanked by EcoRI and EcoRI NotI (primers DF3 and DF4); natMX6 flanked with SacI and EcoRI (primers DF5 and DF6). The SacI \u2013 EcoRI natMX6 fragment was inserted into pUC19 followed by the EcoRI and Pst1-HindIII hphMX6 fragments to generate pINT*. pINT* was digested PfoI and KpnI, end filled with Klenow polymerase (New England Biolabs) to remove a 189 bp fragment and re-ligated to remove the NdeI site of pUC19 to generate pINT**. PstI SacI sequences containing the nmt promoter - cloning site/tag - nmt teminator cassettes from pREP1, pREP41 and pREP81 based plasmids were then inserted between PstI SacI sites in pINT** to generate the vector series. Because the multi-cloning site of pREP41PkN and pREP81PkN contains a SacI site, the pINTH41PkN and pINTH81PkN were generated by sequential insertion of the appropriate SacI and PstI-SacI fragments from pREP41PkN and pREP81PkN respectively. pINT* was digested with NdeI, end filled with Klenow polymerase (New England Biolabs) and re-ligated to generate pINTHA. Full sequences of the pINTH vectors are presented in The first step in the generation of the pINTH vector was Quickchange (Stragene) silent mutagenesis to remove the SmaI sites of the natMX6 gene in natMX6 cassette were destroyed (the NdeI site in pFA6anatMX6 is outside of the cassette). The natMX6 gene was amplified with the oligonucleotides AG1and AG2 that had 20 nucleotides homology to the ends of the natMX6 cassette and 80 bp homology with the sequences adjacent to the LEU2+ integration site of pREP1. 5 \u00b5g of the natMX6 fragment was transformed alongside 1 \u00b5g of the appropriate LEU2+ based pREP vector that had been linearised by digestion with KpnI that cut inside the LEU2+ marker gene of the relevant vector in host IH147. Plasmids were isolated from two antibiotic resistant leucine auxotrophic colonies with the DNA isolation kit (Flowgen), before 5 \u00b5l was transformed into DH5\u03b1. Restriction mapping and sequencing of DNA from two transformants identified the desired vector. The pREPK series were made in the same way as pREPN, but the co-transformation used a KanMX6 fragment amplified from pFA6a-kanMX6 To generate the pREPN vectors, the Generation of cell extracts and western blotting of these extracts was as described previously goi+) before transforming this new host strain with a fragment whose homology to the genome extends beyond either side of the integration site. As this fragment harbours a goi mutation, positively selecting for marker loss and screening by DNA sequencing identifies the candidate with the desired mutation  in rich YES medium before standard procedures were used to make the cells competent to receive DNA. The DNA fragments that were added to these competent cells were generated by PCR amplification of pFA6a antibiotic resistance deletion vector series templates with the same oligonucleotides being used with each template ura4+ integration  to the fin1+ locus (fin1+ ORF\u201d) and a short fragment with 80 bp regions of homology either side of the stop codon that generated a +.3GFPfin1 fusion sequence by standard PCR amplification +.3GFP\u201dfin1). For the \u201cfin1+ ORF\u201d DNA fragment, a single sample of donor DNA was split into four. One quarter was transformed into a pku70::kanMX6 strain, another into an otherwise isogenic pku70+ strain and the remaining two aliquots into +pku80::ura4 and isogenic pku80+ hosts. As the selectable marker for the \u201c+.3GFP\u201dfin1 fragment was the same geneticin/G418 resistance marker that had been used to delete pku70+ with kanMX6, this +.3GFPfin1 fragment was only transformed into +pku80::ura4 and isogenic hosts. Diagnostic PCR analysis of transformants from each comparison revealed that the efficiency of gene targeting was elevated to between 75 and 80% (at least 16 fold increase) by the removal of either Pku70 or Pku80  and ura4+ . YGRC strain number listed in hph.171k cells was indistinguishable from wild type at all temperatures tested  in both rich YES and minimal EMM2 medium. We then generated the pINTH series of vectors shown in hphMX6 sequence in the genome . After backcrossing, protein samples were prepared from mid-log phase cultures 15 hours after expression from the nmt41 promoter was de-repressed by the removal of thiamine and processed for western blotting. To compare the expression level of the pINTH vectors with that obtained with the pINTL series vectors, the same fin1.KD insert had been cloned into the pINTL41PkN vector before integration into the genome, backcrossing and the production of protein extracts from mid-log phase cultures 15 hours after thiamine removal. The levels of Fin1.KD protein attained following induction of expression from either the leu1 targeted pINTL41PkN or the hph.171k targeted pINTH41PkN construct . As expromoters  is less in vivo gap repair in fission yeast S. pombe, only nourseothricin/ClonNat and Kanamycin/G418 resistance can be used in the minimal medium in which the nmt1 based promoters of the pREP series vectors can be de-repressed, making these the only markers that would be of utility for a pREP based series of vectors. The LEU2+ marker of pREP81 and pREP41 derived plasmids was removed by restriction digestion and the linear vector sequences were co-transformed with a DNA fragment in which the natMX6 or the kanMX6 cassette had been amplified with primers that had 80 bp of homology with either end of the opened vector sequences. Plasmids were re-isolated from two nat + or kan + transformants and the new vector sequenced. As reported previously for this approach Multi-copy plasmids that can be selected for in prototrophs to drive the expression of tagged molecules remain popular. To generate a series of vectors we used S. pombe to understand the signal transduction pathways that control cell division. The tools and methods presented in this paper make a significant contribution to resolving the problem of locus-dependent gene targetting efficiency. Manipulation of all loci has now become routine with the enhancements of transformation efficiency after switching the recovery incubation to an overnight incubation in un-supplemented MSL and the removal of the NHEJ response by deleting either pku70+ or pku80+ (the choice of which deletion to use depends upon the genomic location of the gene of interest to be targeted). We have generated pkuX0::kanMX6, pkuX0::hygMX6, pkuX0::natMX6 strains for greatest flexibility in designing a particular knockout strategy (available from the Yeast Genome Resource Centre Japan (http://yeast.lab.nig.ac.jp/nig/index_en.html)). We note that ura4+ and LEU2+ deleted alleles have been generated in other studies We describe a number of the tools that we have developed to assist our efforts that exploit the molecular genetics of pku70.\u0394 and pku80.\u0394 strains than in wild type strains (data not shown). Why this should be is unclear, however we suggest that pku70+ and pku80+ deletions be used as host strains for manipulations of genes in processes other than TOR signalling, but reverting to targeting in a wild type prototrophic strain should targeting efficiencies prove to be poor.While removal of the NHEJ pathway radically enhances the frequency of gene targeting in our work on cell cycle control, our experience with genes in the TOR signalling pathway has been different as targeting can be less efficient in ura4+ complementation of ura4.d18 in media lacking uracil. We assume that this is due to the inherent differences in the nature of the selection pressure in the two cases. Expression of antibiotic resistance molecules is required to prevent an apparently immediate death from a lethal assault by the antibiotic, whereas the ornithine decarboxylase is required to permit the generation of uracil. Cells will simply remain in a stationary phase until ornithine decarboxylase levels reach the critical threshold to allow them to resume growth and division.We found that the efficiency of integration was radically enhanced by altering the nature of the recovery period that is used to enable the expression of antibiotic resistance markers before they are challenged with selective conditions. Switching a from recovery phase on solid medium to a liquid recovery phase in nitrogen free medium increased transformation efficiencies 5\u20138 fold with antibiotic resistance cassettes. Although the greatest enhancement of transformation efficiencies arose at the 24 hour time point, an overnight incubation is normally sufficient and avoids delays in strain construction. While the MSL-N recovery period was a great benefit when the integrated expression cassette directed the expression of antibiotic resistance markers to counteract the otherwise lethal impact of antibiotics, it had only a modest impact when the selection relied upon Pst1-Sac1 restriction fragment from any existing pREP based vector The generation of three series of integration vectors that allow the expression of wild type, mutant, tagged or un-tagged native molecules from three different loci greatly facilitates the analysis of the impact of mutations on individual molecules or compound interactions of mutant molecules in a protein complex. The pINTXX.A vectors can accept the entire promoter\u2013gene\u2013terminator cassette as a Figure S1DNA sequences of the pINTL series.(DOCX)Click here for additional data file.Figure S2DNA sequence of pINTK.(DOCX)Click here for additional data file.Figure S3DNA sequences of the pINTH series.(DOCX)Click here for additional data file."}
+{"text": "The normobaric oxygen paradox (NOP) is a recent concept that postulates the use of intermittent hyperoxia to stimulate erythropoietin (EPO) production . Hyperox2 <50% were included in this prospective observational study. Patients underwent a 2-hour period of hyperoxia (FiO2 100%). The sublingual microcirculation (sidestream dark-field imaging (SDF)) was evaluated at baseline (t0), 2 hours after hyperoxia (t1), and 2 hours after return to basal FiO2 (t2). SDF monitoring was continuously performed also during the variation of FiO2 for 2 minutes. EPO levels were assayed at baseline and for 2 days.Six patients with hemodynamic stability and mechanically ventilated with FiOP = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 - t0) and the change in EPO .An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure Hyperoxia leads to vasoconstriction that seems to be reversible at hyperoxia cessation. Further data are needed to verify the efficacy of the NOP in stimulating erythropoiesis in the critically ill. There might be a relation between hyperoxia-induced reduction in vessel density and the EPO increase."}
+{"text": "BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild\u2010type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin\u2010fixed paraffin\u2010embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next\u2010generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1  and two BRCA2  mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.The overall prevalence of germline  BRCA1 and BRCA2 tumor suppressor genes play an important role in DNA damage and repair pathways. Germline mutations in these genes are strongly associated with an increased risk of breast and ovarian cancer BRCA1/2 mutation BRCA1/2 mutations among ovarian cancer patients worldwide BRCA1/2 loci, indicating essential role of these genes in ovarian cancer pathogenesis, somatic mutations of these genes are relatively rare finding BRCA1 mutations were reported in 5\u20139% of sporadic ovarian cancer cases, whereas somatic genetic variants of BRCA2 were identified in 3\u20134% of tumors Although 20\u201370% of sporadic ovarian tumors display loss of heterozygosity (LOH) in the BRCA1/2 mutation carriers was higher than in wild\u2010type patients BRCA1/2 mutation status, both germline and somatic. Therefore, complex mutational analysis of BRCA1/2 genes could increase the number of patients who might benefit from PARP1 inhibitors treatment.Recently, many clinical trials for specific therapies targeting cells with defect BRCA signaling pathway are ongoing, that is, with poly (ADP\u2010Ribose) polymerase 1 (PARP1) inhibitors. PARP1 is a member of chromatin\u2010associated polymerases involved in the single\u2010strand breaks repair, a common form of DNA damage BRCA1/2 mutations. Mutational analysis of both genes was performed in 100 formalin\u2010fixed paraffin\u2010embedded tissues (FFPE) tissues from ovarian cancers.In this study, we established the frequency and type of In total, 100 FFPE serous ovarian carcinoma samples were enrolled to the study. All samples were obtained from the files of the Department of Pathomorphology, Medical University of Gdansk and were collected between 2008 and 2012. The histological diagnosis of ovarian serous carcinoma and the tumor tissue content (TTC%) of each sample were evaluated by pathologists. In order to obtain cancer cells from heterogeneous histological samples, tissue macrodissection was performed. The average patient age at diagnosis was 60\u00a0years (range: 36\u201381). Informed consent was obtained from all the patients and the research was approved by local ethics committee.Genomic DNA was extracted from the macrodissected FFPE tissues using Cobas DNA Sample Preparation Kit  according to manufacturer's protocol. Quantity and quality of isolated DNA was determined with NanoDrop 1000 UV Spectrophotometer  and Qubit Fluorometer . In a selected 22 samples, DNA from uterus or peripheral blood samples was isolated by using Cobas DNA Sample Preparation Kit  or Blood Midi kit .BRCA1 and BRCA2 mutation screening was performed using the BRCA Tumor MASTR Plus assay  according to the manufacturer's protocol. MiSeq targeted resequencing 99x  was performed. The read length was pair\u2010end and cut\u2010off of 4% for the Variant Allele Frequency was applied. The median coverage for all samples was 1700.BRCA1/2 mutation was confirmed by bidirectional Sanger sequencing . Finally, in order to determine the somatic or germline status of detected alteration in 22 BRCA1/2 positive tumor samples, mutational analysis of DNA isolated from nonneoplastic cells was performed by PCR followed by Sanger sequencing.The analysis was performed with Sophia DDM software . The presence of the BRCA1/2 variants were identified in 27 (28%) samples. All genetic variants are presented at Figure BRCA1/2 positive cases, 20 (74%) BRCA1 and 7 (26%) BRCA2 mutations were identified. The most frequent alteration was c.5266dupC  in BRCA1, detected in 6.2% (n\u00a0=\u00a06/97) of tumors.From the 100 samples three  were excluded from the analysis due to low quality of the results. In the remaining 97 patients diagnosed with serous ovarian carcinoma, pathogenic BRCA1/2 mutations, DNA extracted from uterus or peripheral blood leukocytes was available for further molecular studies. Within this group, only four (4.1%) mutations were found to be somatic while 18 (18.6%) were also identified in a nonneoplastic cells and were classified as germline variants. In total, 14 (78%) and four (22%) germline mutations were found in BRCA1 and BRCA2, respectively.For 22 patients with pathogenic To our knowledge, six of the 27 (22.2%) variants were not previously reported. These six novel mutations included three deletions, two substitutions, and one duplication. All these genetic variants resulted in a premature stop codon and protein truncation Table\u00a0.BRCA1 gene were more frequently found in patients diagnosed before or at the age of 50 compared to older individuals \u2013 40% (8/20) versus 16% (12/77), respectively. None of the BRCA2 alteration was identified in the group of patients younger than 50\u00a0years old. The mean age of ovarian cancer diagnosis was 54.5 (range: 36\u201376), 58.4 (range: 51\u201376) and 61.9 (range: 40\u201381) years for BRCA1, BRCA2 and wild\u2010type patients, respectively.Mutations in the BRCA1 and BRCA2 mutations. One alteration was pathogenic and a second was previously classified as unknown variant although resulting is premature STOP codon.In the study, three individuals were compound heterozygous for Detailed histopathological and molecular data, including all variants identified in the studied group, are shown in Table S1.BRCA1/2 mutations in serous ovarian carcinoma, the entire coding sequence of these genes was analyzed by next\u2010generation sequencing. To distinguish true alterations from artifacts, the cut\u2010off of 4% for the Variant Allele Frequency was applied. The presence of pathogenic variants has been confirmed by Sanger sequencing.Since personalized target therapy appears more common in modern oncology, evaluation of the highly sensitive and cost\u2010effective analysis accessible for routine diagnostics has become a high priority. Currently, the gold standard for detecting somatic mutations is Sanger sequencing, but its diagnostic yield is relatively low. In addition, the analysis of FFPE tumor material is challenging, because the extracted DNA is typically of poor quality and highly fragmented. Moreover, tumor cells are histologically and genetically heterogeneous and tumor DNA may be contaminated with DNA from nonneoplastic cells. Therefore, implementation of novel, powerful tools enabling the detection of low\u2010level mutations, such as next\u2010generation sequencing should be considered as a standard diagnostic procedure. The application of next\u2010generation sequencing allows to detect somatic mutations that may be present in a low proportion of the total DNA. In this report, we determined the frequency of somatic and germline BRCA1/2 revealed the presence of pathogenic mutations in 28% of 97 studied samples. Among the BRCA1/2 positive cases, the frequency of germline and somatic mutations was 18.6% and 4.1%, respectively. The percentage of identified somatic changes is comparable to previous reports BRCA1/2 germline mutation frequency is higher than the expected frequency in a constitutive ovarian cancer cases (~19% vs. 11\u201315.3%) BRCA1/2 mutations n\u00a0=\u00a025/27; 93%) were classified as high\u2010grade serous ovarian tumors. In contrast, only in two (~7%) low\u2010grade tumors BRCA1/2 mutation was detected  BRCA1/2\u2010positive patients had no family history of breast or ovarian cancer According to the guidelines of the American Society of Clinical Oncology genetic testing for BRCA1 and BRCA2 genes in each individual BRCA1 and BRCA2 mutation carriers was mainly, but not only, described in the Ashkenazi Jewish population BRCA1 splicing alterations (c.4484+1G>A and c.4357+2T>G) and nonsense mutations in the BRCA2 gene , whereas patient #47 carried BRCA1 founder mutation  and a known frameshift change in the BRCA2 gene . According to the BIC database BRCA2) and a frequency . In contrast, Martin et\u00a0al. (2005) described the higher frequency among patients with familial pancreatic cancer indicating its potential pathogenicity BRCA2 was previously detected in two Polish patients with familial aggregation of the disease BRCA1/2 mutations was excluded. Finally, in silico analysis suggest a possible impact of c.10095delinsGAATTATATCT on mRNA splicing.The recommendations of the European Molecular Genetics Quality Network (EMQN) suggest to perform mutational analysis of the both BRCA1 and BRCA2 genes were identified  in all ovarian cancer patients in the Polish population should be performed BRCA1 and BRCA2 may significantly increase the total number of patients who may potentially benefit from targeted therapies with PARP inhibitors. In addition, such a genetic testing would allow to identify more family members with BRCA1/2 mutations.In conclusion, the application of a targeted assay using next\u2010generation sequencing of FFPE samples allowed the identification both somatic and germline L.J. did receive honoraria for advisory activities from AstraZeneca. W.B. did receive honoraria for advisory and consulting functions and educational activities from AstraZeneca.Table S1. All identified variants in the BRCA1/2 genes in the serous ovarian tumors.Click here for additional data file."}
+{"text": "This data in brief describes characteristics of chronic stable comorbid patients who were included in reference range studies of [IGFBP7]\u00b7[TIMP-2] \u201cReference Intervals of Urinary Acute Kidney Injury (AKI) Markers [IGFBP7]\u00b7[TIMP2] in Apparently Healthy Subjects and Chronic Comorbid Subjects without AKI\u201d  We also describe the targeted patient population and the inclusion criteria that were used to determine the reference range of [IGFBP7]\u00b7[TIMP-2] in healthy individuals.2The reference range study was designed to include patients commonly seen in intensive care units of hospitals in the United States The protocols for this investigation were approved by investigational review boards/ethics committees as required by each participating institution. All subjects provided written informed consent. Subjects of \u226521 years age, who provided written informed consent for the study participation, and met the morbidity criteria  were selThe patients were recruited at 6 geographically diverse sites . In the stable chronic comorbid cohort most patients had several comorbidities, with the most prevalent being some type of an endocrine or cardiovascular disorder. In terms of specific comorbidities, as might be expected for the US population, the highest prevalence was hypertension (59.7%) with the other top four being hypercholesterolemia (40.1%), osteoarthritis (28.8%), and diabetes (24.5%)."}
+{"text": "Residual functional tricuspid regurgitation can cause significant symptoms after a successful left heart valve replacement surgery. There is more evidence towards repairing moderate tricuspid valve regurgitation of late.We analysed the fate of Tricuspid valve at the end of five years after a left heart valve replacement irrespective of whether they had undergone a concomitant TV repair or not during the initial surgeryBetween January 2008 and December 2009, 200 patients who had undergone a left heart valve replacement were analysed for the degree of TV regurgitation at the end of five years. 162 patients had undergone a Mitral valve surgery and 38 patients had undergone a double valve replacement. Group I - 40 patients had a concomitant TV repair (Modified de Vaga's annuloplasty) during the primary surgery and Group II 160 patients did not have a concomitant TV repair.In Group I, of the 40 patients, 4 patients (10%) underwent TV repair for moderate TV regurgitation, and all the 4 patients had trivial to mid TV regurgitation. 36 patients underwent TV repair for severe regurgitation, of these patients, 30 patients (83.3%) had trivial to mild residual TV regurgitation, while only 6 patients (16.6%) had moderate to severe TV regurgitation. In Group II, of the 160 patients, 58 patients (36.2%) and 16 patients (10%) had moderate and severe TV regurgitation which was left un-corrected. Of the 58 patients who had moderate TV regurgitation, 20 patients (34.4%) had moderate regurgitation and 4 patients (6.8%) had severe TV regurgitation at the end of five years. Of the 16 patients who had severe TR addressed during the initial surgery, 8 patients (50%) had moderate to severe TV regurgitation at the end of five years.A concomitant Tricuspid valve repair is recommended during a left heart valve replacement, if the degree of Tricuspid regurgitation is moderate. This helps in alleviating the symptoms of residual tricuspid valve regurgitation on long term follow up."}
+{"text": "To evaluate the impact of adding 3D Tomosynthesis to Full Field Digital Mammography (FFDM) in the detection and diagnosis of breast lesions.n = 114/166) of the studied cases. FFDM and 3D tomosynthesis examination was done and imaging findings were evaluated before and after the use of 3D tomosynthesis images.The study included 166 mammograms with indeterminate findings selected from 1600 mammograms. They were classified into two groups: group 1 \u2018Diagnostic mammograms\u2019 of symptomatic women and group 2 \u2018Screening mammograms\u2019. Dense breasts assigned as ACR3 and ACR4 presented 69%  and diagnosis  of breast lesions dramatically improved when 3D tomosynthesis images were considered in the evaluation. The sensitivity, specificity, and accuracy of digital mammography was 60%, 20.7% and 48% have significantly enhanced on applying tomosynthesis to be 94.5%, 74% and 89.7%.Both modalities were compared regarding detection and diagnosis, each individually assessed, using the Pearson Chi Square tests. Detection ."}
+{"text": "KRAS mutant colon cancer was found in 5.3%. No relationship was found between HER2 expression and KRAS status (P = 0.486). The evidence of HER2 positive metastatic lesion and primary colorectal cancer suggest that HER2 assessment might be considered in selected cases when this may help change the therapeutic decision.The aim of this study was to compare human epidermal growth factor 2 (HER2) status in primary colorectal cancer and paired liver or lung metastasis. Gene amplification of HER2 has been intensively evaluated in contemporary oncology, especially in breast and stomach cancer. The knowledge of HER2 status in primary and metastatic sites may be of potential value for therapeutic decision making in metastatic colon cancer. The HER2 status was assessed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 94 colorectal cancer with corresponding liver or lung metastases. HER2 amplification was present in 19 of the 188 (10.1%) of both primary and metastases combined. Four (4.6%) patients showed HER2 amplification in the metastasis and 10 (10.6%) patients showed HER2 amplification in the primary tumor. In 14 cases (14.8%), the HER2 status of the primary lesions was different from that of the associated metastases. The presence of HER2 overexpression in  Colon cancer accounts for 10% of all new cancer diagnoses and 11% of all cancer-related deaths. It is also the fourth most common malignancy worldwide, with \u223c1,000,000 new cases and 500,000 deaths recorded each year HER2 gene amplification and the overexpression of HER2 protein have been inconsistent, with incidence rates ranging from 0% to 83% of primary tumors It is currently unclear whether HER2 is a potential therapeutic target in patients with colon cancer, and HER2 is expressed at a far lower level in this malignancy than in breast cancer KRAS), which occur most frequently in codons 12, 13, and 61, are found in \u223c40% of colon tumors KRAS mutations have emerged as a key negative predictive factor for treatment response in patients receiving cetuximab KRAS colorectal tumors would be responsive to cetuximab. However, it has not been established whether there is a relationship between KRAS and HER2 status.Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog  and immunohistochemistry (IHC). For each case, two tissue sections cut at different levels of the histological block were processed to control for tissue heterogeneity.All colon adenocarcinoma samples were obtained during operations performed at the Gachon University of Science and Medicine, Gil Hospital between January 2006 and March 2010. Among the 456 patients who underwent curative or palliative resection for colon cancer, 116 consecutive patients stage IV disease were randomly selected at the outpatient department for analysis. Complete clinical and pathological information was available for analysis in 94 of these cases. The tumor sections were first selected under the microscope to ensure that they comprised at least 70% neoplastic cells. Formalin-fixed, paraffin-embedded tissue blocks, selected on the basis of quality and representativeness of the sample, were cut into 5-\u03bcm thick) were placed on slides coated with polylysine. After deparaffinization and blocking of endogenous peroxidase, HER2 immunostaining was performed using rabbit anti-human c-erbB-2 as a primary antibody  at a 1:100 dilution. Primary antibody binding was assessed using the Dako Quick-Staining, Labeled Streptavidin\u2013Biotin System (Dako), and this was followed by the addition of diaminobenzidine as a chromogen. HER2 immunoreactivity was evaluated by a single pathologist according to the scoring system described by Hofmann et al. Sections of archived formalin-fixed, paraffin-embedded tissue  in a domestic microwave oven for 10 min, and then subjected to proteolytic digestion using pepsin  at room temperature for 10 min. Hybridization was performed in a hybridizer  at 82\u00b0C for 5 min, and then at 42\u00b0C for 16 h. The probes were based on the Probe Mix , containing a mixture of Texas red-labeled cosmid clones covering 220 kb of the HER2 amplicon and a mixture of fluorescein (fluorescein isothiocyanate)-labeled peptide nucleic acid probes targeted at the centromeric region of chromosome 17. After a stringent wash at 65\u00b0C for 10 min, the slides were mounted with a fluorescence mounting medium containing 4\u2032,6-diamidino-2-phenylindol dihydrochloride and a coverslip was used. The slides were stored at 2\u20138\u00b0C in the dark until evaluation, which was performed within 2 weeks using a fluorescence microscope .\u03bcm thick tissue microarray sections. The slides were heated at 60\u00b0C for 1 h and then rehydrated with 100% xylene (four washes for 3 min each), 100% ethanol (four washes for 3 min each), and running water (5 min). The sections were blocked for endogenous peroxidase activity with 1.5% hydrogen peroxidase in methanol (15 min) and then washed under running water for 5 min. This was followed by digestion with 0.01% bacterial protease type XXIV  in prewarmed 5 mmol/L Tris buffer (pH 7.6) at 37\u00b0C for 10 min and washing under running water for 5 min. The sections were then transferred into Tris-buffered saline and incubated for 30 min with EGFR antibody clone H11  at a 1:200 dilution and incubated in horseradish peroxidase-labeled polymer (Dako) for 30 min according to the manufacturer's instructions. The slides were washed with Tris-buffered saline between incubations. The tissue sections were stained using 3,3-diaminobenzidine as a chromogen (Dako) and counterstained with Mayer's hematoxylin. A glioma section was used as a positive control, and the negative control sections were incubated with negative control rabbit immunoglobulin (Dako) in the absence of primary antibody.Immunohistochemical studies were performed on 5-A modified EGFR expression scoring system was used, based on previously published criteria \u03bcm thickness obtained from a representative paraffin-embedded block were placed on slides without coverslips for microdissection and DNA extraction. Briefly, microdissection was performed under direct observation using an inverted microscope and a sterile needle. Each microdissected sample was directly transferred to an Eppendorf tube that contained digestion buffer . The tubes were then incubated overnight at 56\u00b0C, and this was followed by a 10-min incubation at 95\u00b0C to eliminate any remaining proteinase K activity. Polymerase chain reaction (PCR) was performed in 20-\u03bcL reactions that contained 2 \u03bcL of DNA, 2 \u03bcL of commercial PCR buffer , 2.0 mmol/L of MgCl2, 200 mmol/L of each deoxynucleotide triphosphate, 20 pmol of each primer, and three units of AmpliTaq Gold polymerase (Applied Biosystems). A Uno II Thermoblock  was used for thermal cycling. Initial denaturation at 95\u00b0C for 10 min was followed by 41 cycles of denaturation at 95\u00b0C for 1 min, annealing at 52\u00b0C for 1 min, and extension at 72\u00b0C for 2 min and a final extension step at 72\u00b0C for 10 min. Exon 2 of the KRAS gene was amplified by PCR using intron-based primers in order to investigate the mutational status of KRAS codons 12 and 13, which occur frequently in colon cancer. The forward and reverse oligonucleotide primers used to amplify KRAS exon 2 were 5\u2032-CAT GTT CTA ATA TAG TCA CA-3\u2032 and 5\u2032-AAC AAG ATT TAC CTC TAT TG-3\u2032, respectively.Hematoxylin/eosin-stained sections of 5-The amplified DNA was electrophoresed on a 2% agarose gel for 1 h at 110 V. The amplification products were then purified using a MinElute PCR purification Kit  according to the manufacturer's instructions. The PCR products were then sequenced in both directions using the ABI Prism BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and the same primers as those employed for PCR. The PCR products were finally purified on Centri-Sep Spin Columns (Applied Biosystems) and subsequently run on an ABI Prism 310 automatic sequencer (Applied Biosystems). The data were analyzed using Sequencing Analysis 5.2 Software (Applied Biosystems) \u03ba-coefficient was used to assess the level of agreement between samples, with \u03ba-values between 0.61 and 0.8 considered to indicate a very good agreement. Differences were considered statistically significant when the P-value was \u22640.05. All statistical tests were two sided.Pearson's correlation test was used to compare the HER2 status of metastases assessed by IHC and FISH. The similarity in the HER2 IHC status between primary lesions and metastases was calculated as the ratio of concordant cases to total cases. The HER2 gene copy number was evaluated using FISH in 94 consecutive primary colon adenocarcinomas and their matched liver or lung metastatic lesions  histological specimens (Table HER2-positive metastases that were associated with synchronous metastasis (P = 0.033). In 14 cases (14.8%), the HER2 status of the primary lesions was different from that of the associated metastases. Four (4.6%) patients showed HER2 amplification in the metastasis but not in the primary sample, and 10 (10.6%) patients showed the opposite pattern, with HER2 amplification in the primary tumor but not in the metastasis (HER2 cluster amplification in primary tumors and matched liver metastases. Only five (5.5%) patients showed concordant HER2 expression in primary and metastatic sites.Among the metastatic lesions, tastasis . Figure HER2 protein overexpression was assessed using IHC on histological sections obtained from 94 primary tumors and their matched 94 metastatic lesions obtained by surgical resection. Only 2.1% (2 of 94 cases) of the patients had HER2-positive tumors on IHC, with an immunopositive 3+) reaction in >80% of tumor cells. The total concordance between IHC and FISH was 86.1%  were negative for HER2 expression according to both IHC and FISH, two were positive according to both techniques, and 7 (7.4%) gave equivocal IHC results Table . The fouKRAS mutation revealed that the same mutation was present in the primary tumor and the corresponding liver metastasis in 94 cases . In 12 cases , of which six involved synchronous metastases at diagnosis and six showed metachronous metastases development, we found a discordance in KRAS mutation status between primary tumors and metastases. Five of these patients had a KRAS mutation in the primary tumor but not in the liver metastasis. In three cases, the KRAS mutation differed between the primary tumor and the metastases; one of these patients had a Gly13Asp KRAS mutation, whereas the liver metastasis had a Gly12Ser mutation  had negative EGFR expression in the primary lesions but had at least 1 EGFR-positive metastatic site . No relaAfter its development as a therapeutic target for patients with breast cancer, HER2 has been evaluated as a target for patients with other tumor types. This includes metastatic gastric cancer, for which HER2-targeted therapy resulted in a 37% improvement in overall survival, leading to the approval of trastuzumab by the United States Food and Drug Administration for patients with HER2-positive metastatic lesions Our findings suggest that there is in fact a high level of concordance between the results of IHC and FISH used to assess HER2 status in colon cancer. In a majority of cases, both primary lesions and their corresponding metastases showed the same level and pattern of HER2 expression, indicating that the regulation of HER2 is maintained during metastasis. However, because of the relatively small sample size in this study, we cannot draw any conclusions about the relative HER2 expression in synchronous and metachronous metastases.HER2 amplification status was evaluated by FISH in 94 paired primary and metastatic lesions, revealing a total of 19 amplified and 169 unamplified genes in both sites combined. HER2 amplification was concordant in only 5% of the primary and metastatic lesions. Previous studies showed that, for metastatic breast cancer, the HER2 neutralizing antibody Herceptin\u00ae is only effective in the therapeutic range. In a study by Ramanathan et al. Although we found that HER2 protein is present in colon cancer, only in a few cases was its expression strong enough to consider it as a potential therapeutic target (2+ and 3+). The KRAS mutations are frequently the same in primary lesions and their matched liver or lung metastasis, with a concordance rate of 87.2%. HER2 was overexpressed in colon tumors harboring KRAS mutations in 5.3% of cases (10 of 188 cases). This suggests that trastuzumab is a possible treatment option for patients with colon cancer and KRAS mutation.A further noteworthy finding of this study is that Patients with a metastatic colon tumor that overexpresses HER2 (5%) may benefit from trastuzumab therapy. An understanding of the evolution of gene signatures in colon cancer together with molecular profiling may facilitate the identification of molecular subtypes that can predict which patients will respond favorably to trastuzumab therapy."}
+{"text": "Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in  In plants, DNA methylation occurs in both symmetrical  and asymmetrical (CHH) sequence contexts, where DNA methylation in each of the three contexts is thought to be regulated separately Arabidopsis thaliana. In Arabidopsis, CG, CHG, and CHH methylation are highly enriched within transposable elements, repeat sequence, and the pericentromeric region ArabidopsisMuch of our understanding about DNA methylation machinery and mechanisms in plants is based on research in Arabidopsis have provided substantial opportunities to understand DNA and histone methylation in plants, the Arabidopsis genome may not provide a good model for many of the crop genomes. Arabidopsis have very few transposons that are mostly clustered in pericentromeric regions or other heterochromatic knobs Zea mays genome is \u223c20 fold larger than the Arabidopsis genome and exhibits a complex arrangement of transposons and genes that is observed in many plant species. The majority of maize genes are flanked by transposons Arabidopsis genome Although the genetic and genomic resources available for Arabidopsis we find lower levels of CHH methylation and a distinct relationship of H3K9me2 with CHH methylation patterns. The analysis of different sub-families of transposable elements reveals distinct patterns of CHH and H3K9me2 enrichment for different families. Maize genes contain CHG methylation within the gene body and this is partially attributable to the presence of transposons in introns of >10% of maize genes. The presence of heterochromatin transposons within genes does not appear to restrict expression of these genes. We also identified a subset of maize genes with high levels of CHG methylation or H3K9me2 over the transcription start site (TSS) and find that the majority of these genes are not expressed throughout maize development.The relationship between DNA methylation and other chromatin modifications has not been looked at in great detail in maize. Here we combine WGBS with H3K9me2 ChIP-seq to assess the relationship between DNA methylation and H3K9me2 throughout the maize genome. In comparison to Genomic DNA was isolated from the third leaf of 14-day after planting seedling from the B73 inbred line. Samples were fragmented and ligated with TruSeq-methylated adapters. Bisulfite conversion was performed on five hundred nanograms of adaptor-ligated DNA using the MethylCode bisulfite conversion kit (Life Technologies) according to manufacturer\u2019s guidelines. Converted DNA was split into four reactions and amplified using Pfu Turbo Cx DNA polymerase (Agilent) for four cycles and subsequently pooled. Libraries were sequenced on the HiSeq 2000 (Illumina) for 100 cycles, paired end. Sequencing reads (SRA accession SRP022569) were processed to identify and filter poor 3\u2032 quality and incomplete conversion. Sequences were aligned to the B73 reference genome (AGPv2) using the Bismark aligner v0.7.2;  under thhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to identify and filter poor 3\u2032 quality reads. Sequenced reads were aligned to the B73 reference genome (AGPv2) using Bowtie under standard parameters. Duplicate reads from both the NEBNext and Nextera libraries were removed using SAMtools H3K9me2 profiling was performed on three replicates of B73 seedling using antibodies specific for H3K9me2 (#07-441) purchased from Millipore  according to manufacturer\u2019s recommendations as described in Eichten et al. http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to trim poor bases from 3\u2032 end of the sequences, to remove adapters and to filter very short reads resulted from base and adapter trimming. This was run under the pair-end reads mode using standard parameters. Reads that passed quality control were first mapped to the Filtered Gene Set , and unmapped reads were realigned to the maize reference genome (AGPv2) using TopHat www.iplantcollaborative.org). The \u2018Reads count per kilobase per million mapped\u2019 (RPKM) value was calculated and averaged over the three biological replicates to represent the expression level of each filtered gene. Those expression levels were used to group the genes into five categories: not expressed, and four categories with equal number of expressed genes in B73 seedling tissues. The proportion in each of the five categories were determined for genes that were identified to have specific features, e.g., with >1000 bp TE, having high H3K9me2 or CHG in promoters.RNA isolated from 14-day after planting leaf tissue of the B73 inbred line was prepared for sequencing at the University of Minnesota Genomics Center using the TruSeq library preparation protocol (Illumina). Three independent replicates were included. Libraries were sequenced on the HiSeq 2000. Over 10 million 50bp read pairs were generated for each library. Raw reads (SRA accession SRP018088) were filtered to eliminate poor-quality reads using CASAVA 1.8 (Illumina). High-quality reads were then passed to Trim_glore  and H3K9me2, all the 100 bp windows that have data on all three marks were grouped based on the levels of either CHG or CHH. CHG levels were equally split into 10 groups from 0% to 100%. CHH levels were split into 9 groups, 5 groups from 0 to 5% by an increase of 1%, 3 groups from 5% to 20% by an increase of 5%, and a group of >20%. The CHG and CHH groups were cross-tabulated to give a total of 90 combinations (10 CHG groups * 9 CHH groups), and the average H3K9me2 levels were calculated for each combination and were shown as a heatmap.The average DNA methylation and H3K9me2 levels for each transposon sub-family was calculated. The classification of transposon sub-families were based on Maize TE Consortium Genes with high CHG or H3K9me2 in their promoter region were identified by assessing the levels of CHG or H3K9me2 in the 100 bp window that overlaps the transcription start site. Genes with greater than 88.5% CHG methylation  in the promoter region were defined as having high CHG over the promoter region and genes with greater than 2 standard deviations of H3K9me2 reads above the genome wide average in the promoter region were defined as having high H3K9me2 over the promoter region.To plot DNA methylation or H3K9me2 levels over transposons and their flanking regions, we first determined the distance between the 100 bp windows and transposons from the Maize TE Consortium (ZmB73_5b). Windows upstream of the transposable elements were given a negative distance value and windows downstream a positive distance value. We then identified the closest transposon to each 100 bp window, and kept those windows that are located within the transposons or the 1000 bp flanking regions on either side. For windows overlapping or within transposons, the normalized distance across the element on a scale of 1 to 1000 was determined. The scaled 1000 bp element, together with 2000 bp flanking regions, were then divided into 60 equal bins, 20 bins each for the 1000 bp upstream region, the scaled 1000 bp element, and the 1000 bp downstream region. The average methylation levels of the bins were then determined and plotted on a line graph in R.The absolute distance line plots consist of two parts, the 5\u2032 plot and the 3\u2032 plot, each of which contains 5 kb genomic segments. The 5\u2032 plot contains 2 kb upstream regions of the transcriptional start site (TSS) and 3 kb genic sequences from TSS. The 3\u2032 plot contains 2 kb downstream sequences from the transcriptional termination site (TTS) and 3 kb genic sequences from the TTS. For genes that are less than 3 kb, the actual gene size were used, which means less than 5 kb regions will be used. In other words, the further into a gene, the less number of genes will be included in those plots. To make these plots, the physical distance between genes and nearby 100 bp windows was determined. For the 5\u2032 plot, this distance was determined to be the physical distance between the mid-point of the 100 bp window and the TSS. While for the 3\u2032 plot, it was calculated as the physical distance between the mid-point of each window and the TTS. Windows that are falling within the respective 5 kb genomic regions of a gene were kept for downstream analysis. For each plot, the 5 kb regions were then divided into 100 equal bins, and the average methylation level for each bin across all genes were calculated using R. Finally, the averaged methylation level was plotted against the center of each bin using R.Arabidopsis genome (SRA accession SRA035939) also contains more CG and CHG methylation than CHH methylation  genomes  and class II (TIR elements - DNA intermediate) transposons. While Arabidopsis class II transposons exhibit enrichment for CHH methylation near the TIRs (the bumps in the CHH profile at the beginning and end of TIR elements) this enrichment is not noted in maize. H3K9me2 is enriched over both TIR DNA transposons and LTR retrotransposons in both maize and Arabidopsis (DNA methylation and H3K9me2 are frequently enriched over transposable element sequences. The profile of DNA methylation and H3K9me2 over maize and compared . The maibidopsis . The enrbidopsis .The profiles of DNA methylation and H3K9me2 were examined for a number of sub-families of maize transposons using the classifications from the maize genome annotation Arabidopsis (Arabidopsis are somewhat similar but there are a number of differences. The level of CG and CHG DNA methylation in the 2 kb upstream of the transcription start site (TSS) or 2 kb downstream of the transcription termination site (TTS) is much lower in Arabidopsis than in maize. This is likely a result of many maize genes being flanked by transposons sequences and exemplifies the different chromatin environment surrounding maize genes compared to Arabidopsis genes. Maize genes also tend to be closely flanked by regions of elevated CHH methylation, termed CHH islands by Gent et al. Arabidopsis and maize genes exhibit increased levels of CG methylation in the middle of the transcribed regions relative to the regions near the TSS and TTS. Maize gene bodies also contain noticeable CHG methylation whereas this chromatin modification is not observed within Arabidopsis gene bodies. The H3K9me2 profiles reveal depletion in the regions immediately preceding or following the TSS and TTS in both maize and Arabidopsis  and a subset  are classified as the filtered gene set (FGS). The FGS genes are a subset of putative genes with more evidence for functionality  whereas the WGS genes may include pseudo-genes, misannotated transposable elements, or gene fragments. The CG and CHG DNA methylation profile for the FGS genes shows much greater reductions in DNA methylation levels at the TSS and TTS . In contArabidopsis genes. Separately plotting the levels of CHG for intron and exon regions reveals that much of this gene-body CHG methylation is derived from introns rather than exons  The accession numbers for each of the datasets used in this study is listed. The proportion of 100 bp tiles with varying levels of CG (B), CHG (C) or CHH (D) is shown for all regions, genic regions and TE regions. (E) The distribution of read counts (per 100 bp tile) is shown for all tiles, genic tiles and TE tiles.(TIF)Click here for additional data file.Figure S2Zea mays transposable elements were split by their different sub-families and aligned at their 3\u2032 and 5\u2032 ends. Distance across the transposable elements was normalized to a scale of 1 to 1000. Average percent methylation and H3K9me2 reads at each distance are displayed.Relative distance line plots across sub-families of maize transposable elements. (TIF)Click here for additional data file.Figure S3DNA methylation profiles of different types of maize genes. (A) The relative levels of DNA methylation in each context or H3K9me2 ChIP-seq read counts are plotted for all maize genes (black), the filtered gene set (FGS-red) and the working gene set (WGS-green). (B) Maize genes were split by their classification as either syntenic or inserted (TIF)Click here for additional data file.Figure S4Additional examples of transposable elements located within genes. Genic transposable elements as viewed in Integrated Genomics Viewer (IGV) (TIF)Click here for additional data file.Figure S5Arabidopsis. Maize and Arabidopsis genes were aligned at the 5\u2032 and 3\u2032 ends and CG (A), CHG (B) and CHH (C) DNA methylation levels or H3K9me2 read counts (D) are plotted. The vertical dashed lines represent the 5\u2032 and 3\u2032 ends. The regions within genes are classified as introns (red), exons (black) or introns with TEs masked (blue). (E) The proportion of TEs  located within maize genes that are annotated as TIR, LINE or LTR elements is shown compared to the proportion of all TEs in the maize genome in each of these three classes.Absolute distance line plots of DNA methylation and H3K9me2 over genic space in maize and (TIF)Click here for additional data file.Figure S6Clustering of expression levels for genes with high CHG methylation in promoter regions. Many of these genes show very low levels of expression. There are \u223c80 of these genes with low levels of expression (1-5FPKM) in a large number of tissues. There are only 4 genes with high expression levels (at least 100FPKM). Two of these genes show anther specific expression and the other two exhibit expression in specific leaf tissues.(TIF)Click here for additional data file.Figure S7Clustering of expression levels for genes with high H3K9me2 in promoter regions. The majority of these genes show very low levels of expression. There are 40 of these genes with low levels of expression (1-5FPKM) in a large number of tissues. There are about 10 genes that show high levels of expression (>100FPKM) in at least one tissue. Four of these genes show anther specific expression and four show endosperm specific expression while the last two have leaf-specific expression.(TIF)Click here for additional data file.Table S1Genes with High H3K9me2 and/or high CHG methylation over transcription start site.(XLSX)Click here for additional data file."}
+{"text": "The NIH:OVCAR-3 is a cisplatin refractory cell line established from malignant ascites of a patient with progressive adenocarcinoma of the ovary after combination chemotherapy with cyclophosphamide, Adriamycin, and cisplatin . Thus, ODatasets from the NCI60 were obtained from the Gene Expression Omnibus (GEO) of NCBI  (OVCAR3 Transcriptome analysis of OVCAR3 specific gene expression changes resulted in 160 significant transcripts with a fold change > \u00b12 and an ANOVA derived Benjamini Hochberg adjusted p-value < 0.001. Enrichment analysis using a Hypergeometric test identified 189 PharmGKB Disease terms, 90 Drug terms and 31 KEGG pathways associated with these genes. A union of the disease, drug and KEGG pathway gene lists yielded 14 common genes for the dataset which were unique to OVCAR3 cells versus SKOV3 and CEPI (Table This list of OVCAR3 unique genes, and the resulting interactions graph Figure  represen"}
+{"text": "The aim of this study is to compare the accuracy of The study group consisted of women recalled from breast screening for further assessment of circumscribed masses. Clients underwent co-registered DM and DBT in both MLO and CC projections. Two experienced breast radiologists evaluated DM and DBT images and a consensus decision was reached on the percentage of the margin that was well defined on DM and DBT. The lesions were categorised 1 to 4 as follows: 1 = 0 to 25%, 2 = 26 to 50%, 3 = 51 to 75% and 4 = 76 to 100%.P < 0.0002). There were more cancers categorised as 1 on DBT (21/39 = 54%) compared with DM (20/76 = 26%); Fisher's exact test (P < 0.004).One hundred and twenty circumscribed lesions were evaluated. Data on 118 lesions seen on the MLO view are presented. There were 93 benign lesions and 25 cancers. There was a change in distribution of margin categories between DM and DBT. More lesions were categorised as 3 or 4 on DBT (59/118) compared with DM (18/118). Of the 93 benign lesions, 17 were categorised as 3 or 4 on DM and 57 on DBT. The difference between the two proportions was significant (Increased margin visibility of circumscribed masses by DBT improves the accuracy of mammography interpretation and may decrease the recall rate in mammography screening."}
+{"text": "Hypermobility of the first ray is one causative factor in development and recurrence of Hallux valgus,2],3].,[3].2],.2],A group of 108 women with symptomatic hallux valgus and 37 control women, age 21 to 84 years were measured weight bearing and non-weight bearing IMA and calculated the difference. We measured the 1st ray sagittal range of motion by the EMC device [The average of the difference weight bearing and non-weight bearing IMA in the control group was 1.16 and 3.20 in the hallux vallgus group. If we defined 3.6 as having axial hypermobility, 42% of hallux valgus patients had first ray axial plan hypermobility (Table 1st ray axial hypermobility is another disease group in hallux valgus, so should be considered in treatment of hallux valgus.Clinical experimental study."}
+{"text": "The most frequent reasons for hypercapnic respiratory failure (HRF) in ICUs are COPD and in recent years obesity hypoventilation syndrome (OHS) and obstructive sleep apnea (OSA). Even 15 to 30% of COPD patients also have accompanying OSA. Due to increased upper airway resistance, those patients require higher expiratory pressures (EPAP) during noninvasive ventilation (NIV). In order to prescribe optimal mode and pressures during the ICU stay and at discharge, the intensivist should diagnose the underlying OSA. Portable recording devices have been developed and they were approved at least for the diagnosis in high pretest probability patients with results equal to in-laboratory polysomnography. The aim of this study is to assess whether respiratory polygraph (RPLG) can be used for obtaining diagnostic information of OSA in hypercapnic ICU patients.Patients, with HRF requiring NIV, were included in the study. RPLG studies were conducted under nasal oxygen before NIV, using Philips Respironics Alice PDx\u00ae device, which provides the records of pulse oximetry with derived heart rate; snoring and nasal airflow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic difficulties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI).2. Admission arterial blood gases were as follows (mean \u00b1 SD); pH: 7.33 \u00b1 0.07, PaO2: 74 \u00b1 12 mmHg, PaCO2: 69 \u00b1 11 mmHg, HCO3-: 31 \u00b1 5, O2Sat%: 92 \u00b1 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 \u00b1 6 in patients with OAI >5. Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identified to have OSA.Thirty-one patients with the mean age of 67 \u00b1 9 years were included in the study. Their mean APACHE II score was 16 \u00b1 5 and BMI was 33 \u00b1 9 kg/mAlthough it underestimates AHI, RPLG is important and technically feasible in ICU patients in suggesting the presence of OSA and in providing information for appropriate NIV management."}
+{"text": "Intracranial pressure (ICP) monitoring at our department includes a standardized postural change examination to evaluate its effects on ICP. During these procedures we observed that neck flexion caused an increase in ICP in one patient. To clarify if the observation was a random occurrence or if it could be reproduced, we added neck flexion and extension to the standard examination.All patients undergoing invasive ICP monitoring at our department were included prospectively. The postural change examination consists of eight standard postures including both horizontal and vertical positions. In this abstract we focus specifically on the effect of neck flexion on ICP in the vertical positions. We examined the effect on ICP with the patient sitting upright with a straight back and 1) a straight neck or 2) maximal neck flexion, and the patient sitting bent in \u201clumbar puncture position\u201d with 3) neck flexion or 4) a straight neck. Each posture was maintained for ten minutes. We recorded ICP and demographic data.All 45 patients completed both measurements in the upright sitting position , while 38/45 patients also completed both measurements in \u201clumbar puncture position\u201d . In both positions, flexion of the neck caused an increase in ICP in all patients.In the upright sitting position the median ICP with 1) a straight neck was \u20135 mmHg (range \u201328 to 8 mmHg) while the median ICP with 2) neck flexion was 3 mmHg (range \u201318 to 19 mmHg). The median increase in ICP when flexing the neck was 8.5 mmHg (range 2.4 to 18 mmHg). The increase was highly significant (p < 0.001).The median ICP in the lumbar puncture position was 13 mmHg (range \u201315 to 38 mmHg) and -2.7 mmHg (range \u201328 to 14 mmHg) for respectively 3) neck flexion and 4) a straight neck. The median increase was 15.89 mmHg (range 8 to 25.7 mmHg). This increase in ICP was likewise highly significant (p < 0.001).The results indicate that the position of the neck has a more important influence on ICP than previously presumed. We speculate that the increase in ICP is a result of either compression of the jugular veins or the vertebral canal.Further investigation indicates that the jugular veins indeed are affected by the position of the neck. 10 patients undergoing invasive ICP monitoring and postural change examination have been examined by ultrasound (UL). Primary results shows compression of the jugular veins in response to neck flexion and a correlation with an increase in ICP."}
+{"text": "Antiplatelet agents, such as aspirin and P2Y12 inhibitors, are essential in the secondary prevention of cardiovascular disease . The effBlood used was drawn from healthy donors free from antiplatelet medication. Light transmission aggregometry (LTA) was used to determine the optimal concentration of MRS2179. Platelet aggregation was induced by the addition of incremental concentrations of ADP. The optimal concentration of MRS2179 to inhibit ADP induced aggregation was 20\u00b5M. Thrombus formation in vivo occurs due to the tethering, adhesion and translocation of platelets to von Willebrand Factor (vWF) under arterial shear conditions . To testThe results of this study demonstrate that a concentration of 20\u00b5M of MRS2179 effectively inhibits aggregation. In 13 normal donors 20\u00b5M either completely inhibited ADP induced aggregation or enhanced platelet disaggregation (p<0.05). In preliminary experiments from 3 normal donors assayed there were no significant changes in most of the parameters measured in the dynamic assay. However, platelet translocation velocity in the presence of the P2Y1 antagonist was significantly increased (p<0.05).Selective inhibition of the P2Y1 surface receptor results in a significant decrease in aggregation in the presence of an agonist. Preliminary data using a novel dynamic assay of platelet function suggests that P2Y1 inhibition may be of therapeutic value."}
+{"text": "To the Editor: The Global Polio Laboratory Network (GPLN) and the World Health Organization\u2019s Polio Eradication Initiative (GPEI) accord high priority to detecting all vaccine-derived polioviruses (VDPVs) because they are neurovirulent and have the potential to cause outbreaks of poliomyelitis and establish poliovirus circulation. In patients with immunodeficiency diseases, persistent infections may become established with live oral poliovirus vaccine (OPV) and develop into VDPVs (>6 nucleotide substitutions (Sabin2) or \u226510 nucleotide substitutions (Sabin1 and Sabin3) in the VP1 region. The VDPV assays were found to be 100% specific for all 3 poliovirus types, 100% sensitive for Sabin1 and Sabin3, and 76% sensitive for Sabin2 \u2014that is, withdrawal of Sabin2\u2014beginning in April 2016  on January 2008. Sabin3 was isolated from stool sample 1 (R46064), collected 11 days after onset of paralysis. Stool sample 2, collected on the 13th day after paralysis, was negative for virus. R46064 was reported as \u201cSabin-like\u201d by the intratypic differentiation tests  used in the GPLN in 2008  Table. SR46064 produced Sabin3-like results in the VDPV screening assay. R46064 gave false-negative test results because the isolate had a Sabin3 vaccine sequence in the regions corresponding to the probe and primers of the VDPV assay  Figure 2Type 2 VDPV was found in an immunocompetent girl, 3.5 years of age, in March 2014. Sabin2 was isolated from 2 stool samples collected 5 and 8 days after onset of AFP. Sabin2, isolated from stool sample 1 (R93150), was amplified in the VDPV screening assay (reported as Sabin2-like); the isolate from stool sample 2 (R93152) failed to become amplified (reported for sequencing).VP1 sequencing of R93152 showed 6 nt substitutions; therefore, it was reported as VDPV2. R93150 was also sequenced to find out whether it contained the Sabin2 homotypic mixture. VP1 sequence of R93150 showed 6 nt substitutions and no evidence of mixed bases. Substitution was not found at VP1 nt 427/428, the main target of the VDPV screening assay.Complete genome sequence analysis revealed that both isolates contained reversion of the major attenuating site at nt 481 in the 5\u2032 UTR. The genomes of both isolates showed no recombination. Both isolates showed 15 nt substitutions in the capsid region, when compared with Sabin2  Table; oThe occurrence of VDPV2 and VDPV3 as described above may be rare. However, GPLN laboratories are unlikely to detect VDPV strains that produce false-negative results in the VDPV screening assay. False negatives are of greatest concern to the GPEI because they could impede timely detection of VDPV infections (Nucleotide substitutions in the 5\u2032UTR and capsid region of type 2 and type 3 vaccine-derived polioviruses (VDPV), amino acid changes at neutralizing antigenic sites, and nucleotide sequence alignment of VDPV2 and VDPV3 with corresponding Sabin vaccine virus sequences."}
+{"text": "The associated region contains a previously identified glaucoma gene, ADAMTS10, which was subjected to mutation screening in the coding regions. A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher\u200a=\u200a3.5\u00d710\u221227) with a high carrier frequency (25.3%). The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity. Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes. This study establishes also a new spontaneous canine model for glaucoma research to study the ADAMTS10 biology in optical neuropathy.Primary glaucoma is one of the most common causes of irreversible blindness both in humans and in dogs. Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve, and elevated intraocular pressure is commonly associated with the disease. Glaucoma is broadly classified into primary open angle (POAG), primary closed angle (PCAG) and primary congenital glaucoma (PCG). Human glaucomas are genetically heterogeneous and multiple loci have been identified. Glaucoma affects several dog breeds but only three loci and one gene have been implicated so far. We have investigated the genetics of primary glaucoma in the Norwegian Elkhound (NE). We established a small pedigree around the affected NEs collected from Finland, US and UK and performed a genome-wide association study with 9 cases and 8 controls to map the glaucoma gene to 750 kb region on canine chromosome 20 (p Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve. Elevated intraocular pressure (IOP) is commonly associated with the disease. However, normal tension glaucoma is diagnosed as well contactin 4 (CNTN4) myocilin (MYOC)neurotrohin 4 (NTF4)optineurin (OPTN)WD repeat domain 36 (WDR36)cytochrome P450 1B1 (CYP1B1)latent transforming growth factor beta binding protein 2 (LTBP2)ATP-binding cassette, sub-family C (CFTR/MRP), member 5 (ABCC5) has been associated with PCAG In human, several loci In dogs, POAG and PCAG have been described in several breeds ADAM metallopeptidase with thrombospondin type 1 motif, 10 (ADAMTS10), has been found in a research colony of Beagles with POAG has been suggested as causative Despite the presence of glaucoma in many breeds the genetic etiology of glaucoma is almost completely unknown in dogs. A novel locus was mapped in Dandie Dinmont Terriers In addition to Beagles, Norwegian Elkhounds are also affected with POAG ADAMTS10 gene.We aimed to find the genetic cause of primary glaucoma in NEs in this study to better understand the molecular pathogenesis, to establish a large, spontaneous canine model for POAG research, and to develop a genetic test for breeding purposes. We discovered a novel missense mutation in the Blood samples from 596 NEs, including 16 glaucoma affected dogs from Finland, Norway, Sweden, United Kingdom and United States were collected to the canine DNA bank at the University of Helsinki, Finland with owne\u0155s consent and under the permission of the Animal Ethical Committee of County Administrative Board of Southern Finland (ESAVI/6054/04.10.03/2012). All affected dogs were examined by a certified veterinary ophthalmologist. The clinical diagnoses indicated bilateral primary glaucoma with significantly elevated IOP, between 25\u201386 mmHg , and no detectable underlying cause. In addition, various degrees of optic nerve atrophy and cupping were reported. In addition, a progressive vision loss was detected in the affected dogs leading to complete blindness. Other, secondary clinical signs included lens luxation, corneal stromal edema, Haabs striae, keratitis, vitreal syneresis and retinal degeneration. The average age at the time of diagnosis was 6.5 years.Genomic DNA was extracted from EDTA blood using Chemagic Magnetic Separation Module I (MSM I)  according to the manufacturer\u2019s instructions. DNA from buccal swabs  was extracted using QIAamp DNA Mini Kit (Qiagen).http://www.ncbi.nlm.nih.gov/projects/SNP/).A genome-wide association study (GWAS) was performed using Illumina\u2019s CanineHD BeadChip array  with 9 cases and 8 controls Fig. 1. ADAMTS10, were first sequenced in four NE cases, in four NE controls (unaffected >8 years) and in one unaffected Rough Collie samples. The identified candidate mutation was then validated in 596 NEs, including 7 additional cases. In addition, the mutation was studied in 71 dogs from 17 other breeds affected with POAG, PCAG or PLD and in 115 unaffected dogs from 6 breeds (Table S1).The coding regions of the best candidate gene in the associated region, Table S2) were designed for the ADAMTS10 gene to amplify the coding regions and splice sites by standard PCR (Table S2). PCRs were carried out in 12 \u00b5l reactions consisting of 1.2 U Biotools DNA Polymerase , 1.5 mM MgCl2 , 200 \u00b5M dNTPs , 1 x Biotools PCR buffer without MgCl2 , 0.83 \u00b5M forward and reverse primer  and 10 ng template genomic DNA. Reaction mixtures were subjected to a thermal cycling program of 95\u00b0C for 10 min, followed by 35 cycles of 95\u00b0C for 30 s, 30 s at the annealing temperature and 72\u00b0C for 60 s and a final elongation stage of 72\u00b0C for 10 min. ExoSap purified PCR fragments were Sanger sequenced in our core facility the FIMM Technology Center using ABI 3730xl DNA analyzer . Sequence data analysis was performed using the Sequencher software . Build 3.1 of the canine genome reference sequence was applied in the study.Primer pairs  , but it did not affect the result as two mixed model approaches (GenABEL and GAPIT) that better control for population stratification, gave the same results (data not shown).We established a global sample cohort including glaucoma affected (n\u200a=\u200a16) and unaffected NEs (>570) to map the disease locus, to identify the causative gene and to validate the segregation of the mutation. We performed a GWAS in a small pedigree of 9 cases and 8 controls. Statistical analyses revealed a 750 kb locus on CFA20 with 23 most highly associated SNPs between 53070684 bp to 53816416 bp (phttp://www.genopro.com) suggested a recessive mode of inheritance as the affected dogs are born to unaffected parents and there are multiple affected littermates in some litters.A pedigree constructed around the affected dogs using GenoPro genealogy software (ADAMTS10 (Table S3). Only one out of the five synonymous variants, p.P171P, co-segregated with the non-synonymous variant. However, it was not suspected to be in the exonic enhancer region when analyzed by the ESE-finder Fig. S1) in the metalloprotease domain of ADAMTS10 protein . The breed-specificity of the p.A387T mutation was indicated by excluding it from 71 glaucoma or PLD affected dogs from 17 breeds and from 115 unaffected dogs from six breeds.To gather further evidence for the p.A387T segregation, we genotyped seven additional NE primary glaucoma cases and four obligatory carriers Fig. 1 aADAMT10 gene in the NEs affected with primary glaucoma using genome wide association analysis and candidate sequencing strategies. We mapped the disease to a known canine POAG locus including the ADAMTS10 candidate gene and subsequently identified a missense mutation in the exon 9 of the ADAMTS10 gene. This is consistent with the previously reported POAG phenotype of primary glaucoma in this breed Our study has identified a novel recessive mutation in the \u03b2), which regulates the collagen turnover ADAMTS10 is a secreted glycoprotein The members of the ADAMTS family share the same structural organization including catalytic metalloprotease domain, followed by a disintegrin-like, and cysteine-rich domains, a thrombospondin repeat and a spacer region Fig. 3C.\u03b2ADAMTS10 interacts with fibrillin-1 and localized to fibrillin-1 microfibril bundles ADAMTS10 gene has been previously associated with POAG in a research colony of Beagles. The Beagle mutation is positioned in the cysteine rich domain and is hypothesized to disrupt protein folding leading to instability In dogs, a missense mutation (p.G661R) in the \u03b2, which is known to be elevated in the aqueous humor of glaucoma patients ADAMTS10 and its pathway. Secondary lens luxation diagnosed in the affected NEs may be due to abnormal fibrillin-1 functions as the ADAMTS10 and fibrillin-1 interaction may be impaired causing disruption of the lens ligaments.The NE mutation is different from the Beagles and may result in a different pathogenesis. NE mutation changes a highly conserved residue in the metalloprotease domain, which plays a role in the remodeling of the connective tissue Fig. 3C ADAMTS10 in human WMS patients Dagoneau et al. identified three causative mutations in the metalloprotease domain of ADAMTS10ADAMTS17fibrillin 1 (FBN1) ADAMTS17-mutant dogs ADAMTS10-mutant Beagles ADAMTS10 and ADAMTS17 genes. Further studies are warranted to investigate whether the observed breed- and species\u2013specific differences are due to alternate mutations in the metalloproteinase domains or possible other factors.Mutations in ADAMTS10. This study implicates the significant role of ADAMTS10 in canine POAG by identifying the second mutation in the same gene in dogs. Our affected dogs establish a new model to study ADAMTS10 biology in the microfibrillin theory of the glaucoma In summary, we have discovered the genetic cause of primary glaucoma in the NEs by identifying a missense mutation in Figure S1ADAMTS10 protein alignments. ADAMTS10 sequence alignment between different species. The mutation is located in a highly conserved region across 75 species. The arrow marks the mutated alanine residue.(ZIP)Click here for additional data file.Figure S2Q-Q plots. Identity-by-state (IBS) clustering and CMH meta-analysis (PLINK) were used to adjust for population stratification (A). A mild population stratification was identified in the study cohort by genome wide IBS clustering (adjusted genomic inflation factor \u03bb\u200a=\u200a1.1) (B).(TIF)Click here for additional data file.Table S1Dogs affected with glaucoma of PLD (71 dogs in 17 breeds) and healthy dog (115 dogs in six breeds) were sequenced for the ADAMTS10 mutation.(XLSX)Click here for additional data file.Table S2Primers used for PCR amplification and sequencing of canine ADAMTS10 coding regions.(XLSX)Click here for additional data file.Table S3A total of 10 variants were in the coding and splice site regions of ADAMTS10.(XLSX)Click here for additional data file."}
+{"text": "An MRI audit substudy was conducted of patients who underwent an MRI prior to treatment in Australia and New Zealand as part of the international PETACC-6 trial in locally advanced rectal cancer.82 patients of the 127 Australasian patients from 15 centres had rectal MRI scans reviewed for technique, data included in reports and comparison of reports with blinded central reporting by 2 experienced radiologists.82% performed minimum T2 sagittal and T2 axial oblique sequences. The high resolution T2 sequence parameters varied significantly with only 33% obtaining a voxel size of \u22641.3. The rate of inclusion of relevant findings in the reports was; T3 distance in mm 21%, N stage 84%, CRM status 72%, EMVI status 29% and distance from the puborectalis sling 17%. 31% of reports included all of; T stage with T3 substage, N stage and CRM involvement. 17% of reports included these 3 findings and EMVI. Eleven reports used a template with 82% of these including the first 3 findings. The agreement with central reporters was T stage 76%, N stage 70%, CRM status 57% and EMVI 16%.There is significant variation in scan quality and low rates of inclusion of all clinically relevant findings in rectal MRI reports reviewed for this audit. The authors recommend adoption of routine sequences and template reports to improve scan technique and report accuracy in rectal cancer MRI staging scans across Australia and New Zealand."}
+{"text": "The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley.A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene  Hordeum vulgare L.) is an important model crop for plant breeding and genetics because it is diploid and has a long history of genetics research. With the development of molecular markers, high density linkage maps of the barley have been constructed using multiple populations since the 21st century. Varshney et al.et al.et al.SNPs are the most abundant type of genetic markers and ideal for studying the inheritance of genomic regions2Restriction-site Associated DNA (RAD) markers detect genetic variation adjacent to restriction enzyme cleavage sites across a target genome1uzu, sdw1 and denso have successfully been used in barley breeding program until nowuzu, sdw1 and denso genes are located on chromosome 3HL89111213Dwarfism is a valuable trait in crop breeding, because it increases lodging resistance and decreases damages due to wind and rain7889btwd1 mapped on chromosome 7H. The dwarfing gene btwd1 is non-allelic with the uzu and sdw1, which have widely been used in China. The chromosome location of btwd1 is different from those of the uzu, sdw1/denso, br1, br2 genes and QTL PH-7btwd1 gene has been successfully applied in breeding program by Huazhong Agricultural University. In 2011, new barley cultivar Huadamai 9 was registered in Hubei Province with Huaai 11 as male parent. In 2014, new barley cultivar Huadamai 10 was registered in Anhui, Henan and Hubei Provinces with Huaai 11 as male parent. Both cultivars have high yield and good quality.Huaai 11 is a new source of dwarfism, and consisted of desirable agronomic traits such as shortened stature and early maturitybtwd1 dwarfing gene using a double haploid population derived from a cross of Huadamai 6 and Huaai 11. This information could be useful for position cloning of this gene and developing new varieties for plant breeders using MAS.In order to efficiently use this new germplasm for barley breeding program, we have constructed a high-density genetic map based on SNP genotyping and further mapping of 2. Height of plants before ripening was measured in the field from soil surface to top of the main culm (with the spike). The height was calculated as the mean of twelve plantsA DH population including 122 lines was developed from a cross between a common feed barley cultivar Huadamai 6 and Huaai 11 using anther culture in this study. The DH population and parents were planted on the Experimental Farm of Huazhong Agricultural University, Wuhan, China. The field trials were conducted following a randomized complete block design with three replications in 2006\u20132008. Each of the DH and parental lines were grown in three rows in a plot of 0.6\u2009\u00d7\u20091.5\u2009mThe young leaves from each doubled-haploid (DH) lines and parents were collected and frozen for DNA extraction. The CTAB method was used to extract genomic DNA from about 0.6\u20131.0\u2009g tissues of each accession8btwd1 were from Ren et al.Frozen DNA samples were sent to the Personal Biotechnology Co., Ltd. . The libraries were quantified using Qubit fluorometer (Invitrogen), Agilent 2100 (Agilent Technologies) and real-time quantitative PCR, and then submitted for sequencing on the Illumina HiSeq2000 platform. Each marker was required to have an allele present in at least 85% of DH individuals. Marker genotypes not meeting the minimum thresholds were scored as missing data. The information of SSR markers was as previously describedBefore constructing genetic maps, SNPs were filtered by excluding those had poor quality data. Low quality SNP included those with NormR results <0.2 and SNP with large numbers of missing values (15% or more). Markers showing identical segregation patterns were also excluded, with one marker per co-segregating group retained. The DH population, consisting of 122 individuals, was utilized to construct a genetic map. The input datasets were constructed from 1,894 genotyped SNP markers and 68 SSR markers. The program Joinmap 4.0 was used to calculate the marker order and genetic distance1838,268 polymorphic SNPs were detected between the parental lines Huadamai 6 and Huaai 11, of which 10,367 polymorphic SNPs were detected in the mapping population. All 10,367 polymorphic SNP markers showed segregation within the DH population. Low quality SNP markers excluded as a result of filtering processes comprised of 5,375 markers with inconsistent parental scores, monomorphic and more than 15% missing data. The remaining 4992 polymorphic SNP markers and 153 of the obtained before polymorphic SSR marker met the requirements for use in the construction of a genetic map. After removing some of the co-segregated markers and non-linked markers, the remaining 1894 SNP markers and 68 SSR markers were used to create 7 high-density genetic linkage groups. This high-density genetic linkage map was generated from 1,962 markers covering seven linkage groups with a total map distance of 1,375.8\u2009cM. The averaged distance between two positions across the whole map was 0.7\u2009cM. The number of markers on different chromosomes ranged from 209 on 1H to 396 on 7H. The genetic distance on different chromosome ranged from 145.0\u2009cM on 1H to 230.9\u2009cM on 3H .btwd1, and Huadamai 6 was a two-row common feed barley cultivarbtwd1 controlling plant height in Huaai 11 was positioned between SNP marks 7HL_6335336 and 7_249275418 with genetic distance of 0.9\u2009cM and 0.7\u2009cM on chromosome 7HL, respectively sdw3 gene (2HS)uzu gene (3HL)8122932sdw1/denso geng (3HL)8142934br2 (4H)836ari-e gene (5HL)br1 gene (7HS)836229383922939412283942433839404345383922938394243btwd1 is a novel gene.We have used these SNP markers to map the dwarfing gene btwd1 in Huaai 11 has widely been used in China, and provides barley breeders with a new source for barley genetic improvement. In order to efficiently use this new germplasm Huaai 11 for barley breeding program, we will verify the identified SNP markers associated with btwd1 in the lines developed from Huaai 11. We have also constructed a large F2 population including 16000 lines derived from a cross of Huadamai 6 and Huaai 11. We will fine map dwarfing gene btwd1 using this large F2 population and the SNP markers. In near future, we will clone the dwarf gene btwd1 and verify its function.Utilization of dwarfing genes in barley breeding programs has greatly increased barley yields, particularly in Asia and Europe8btwd1 in barley breeding.In conclusion, we have constructed a high-density barley linkage map using SNP markers derived from the DH population RAD sequencing. Our study provides a valuable genetic resource for molecular markers, map-based gene cloning, MAS and the sequence assembly of the barley reference genome. At the same time, the linked SNP markers identified in the present study can provide a useful marker-assisted selection tool to transfer the dwarfing gene How to cite this article: Ren, X. et al. SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley. Sci. Rep.6, 31741; doi: 10.1038/srep31741 (2016)."}
+{"text": "Scientific Reports5: Article number: 1409710.1038/srep14097; published online: 09162015; updated: 03212016This Article contains an error in the order of the Figures. Figure 6 and Figure 7 were published as Figure 7 and Figure 6 respectively. The correct Figures 6 and 7 appear below as"}
+{"text": "The objective of this article is to report our first experience of CT guided percutaneous thoracic biopsy and to demonstrate the accuracy and safety of this procedure. This was a retrospective study of 28 CT-Guided Percutaneous Needle Biopsies of the Chest performed on 24 patients between November 2014 and April 2015. Diagnosis was achieved in 18 patients (75%), negative results were found in 3 patients . Biopsy was repeated in these cases with two positive results. Complications were seen in 7 patients (29%), Hemoptysis in 5 patients (20%), Pneumothorax in 1 patient  and vaso-vagal shock in 1 patient . CT Guided Percutaneous Needle Biopsy of the Chest is a safe, minimally invasive procedure with high sensitivity, specificity and accuracy for diagnosis of lung lesions. CT Guided Percutaneous Needle Biopsy of the Chest is a minimally invasive procedure frequently indicated for the diagnosis of lung lesions . This te24 patients were included to this retrospective study; they were admitted to the Thoracic surgery department between October 2014 and April 2015. These patients underwent lung biopsies with a core needle biopsy device 16G or 18G) under CT guidance . All patients with peripheral masses suspicious for malignancy and without suspicion of vascular disease were included in this study. The patients were admitted one day before the procedure to the thoracic surgery department and a coagulation test was done . One patient was on oral anticoagulants for atrial fibrillation (Coumadine*), the treatment was replaced with heparin 5 days before the procedure [G or 18G 24 patients  were included in our study. The patients\u2019 mean age was 62 years (43 - 90 years). 22 patients were active smokers. Mean lesion size was 43 mm and the median traversed parenchymal distance was 20 mm. 21 patients had peripheral lung lesions whereas 3 had central lesions. Lesions were solid in 10 patients, solid and necrotic in 11 patients and purely necrotic in 3 patients. Emphysema was seen in 10 patients and pneumothorax was sustained in 2 patients. A total of 27 biopsies were performed in 24 patients. Histopathology results were positive in 18 of 24 patients and negative in 3 patients who underwent a second biopsy. All had malignant lesions. 7 patients in our study developed complications : Hemorrhage in 5 patients \u00ab 20, 8% \u00bb (3 mild and 1 moderate); small volume pneumothorax in 1 patient \u00ab 4,2% \u00bb; vasovagal shock in 1 patient \u00ab 4,2% \u00bb. Alveolar hemorrhage and pneumothorax resolved spontaneously with no need to proceed to other interventions. One patient who received anticoagulation for atrial fibrillation underwent biopsy. Their Warfarin was stopped 5 days before the procedure and replaced with heparin [In our retrospective study on CT Guided Percutaneous Needle Biopsy, diagnostic accuracy rate was 75%. Non diagnostic biopsy rate was 12,5%. Complications were essentially alveolar hemorrhage 20, 8%) and pneumothorax  in a smaller proportion. Spontaneous reabsorption of alveolar hemorrhage and pneumothorax was seen in all patients with no need to proceed to other interventions such as chest drain insertion. Percutaneous CT-guided core needle biopsy is a safe procedure with a high accuracy for diagnosis of lung malignancies [% and pneIn conclusion, CT-guided percutaneous needle biopsy is a safe procedure with a high diagnostic accuracy rate in the diagnosis of malignant lung lesions.Percutaneous thoracic CT guided biopsy is an accurate procedure for diagnosis of lung malignancies;Major complications are pneumothorax and alveolar hemorrhage;CT is a very helpful tool to localize the lesion and achieve the procedure.Percutaneous thoracic CT guided biopsy with 18 G co axial needle is a very safe procedure with lesser complication rate;CT protocol used in this study limits the radiation exposure of patients;The procedure can also be achieved in emphysematous patients without increasing complication rate."}
+{"text": "Formyl peptide-receptor 1 and 2 (FPR1 and FPR2) in mice were identified as receptors with contrary affinity for the PAMP fMLF. Formyl-methionyl-leucyl-phenylalanine is either part of the bacterial membrane and is secreted by the mitochondria of eukaryotic ceslls during apoptosis. Furthermore FPR1 and 2 are described as highly relevant factors for the chemotaxis of immune cells. Their role during the acute liver injury has not been investigated yet.-/-), FPR2 (mFPR2-/-) and wild type (WT) mice were challenged with LPS i.p. for 3 h and 6 h. Liver and serum were sampled for further analysis.Constitutive knockout mice for FPR1 (mFPR1-/- compared to wild type and mFPR2-/- mice. After 6 h, IL-6, TNF-\u03b1 and CXCL1 were significantly higher in mice lacking mFPR1 or 2. Consistent to these findings the numbers of CD11b+ and Ly6G+ immune cells were altered in the livers. The analysis of TLR2 and TLR4 revealed time and genotype specific changes in theirs gene expression. Additionally, the liver in mFPR1- and mFPR2-deficient mice seem to be more susceptible to apoptosis by showing a significant higher number of TUNEL+-cells in the liver than WT-mice and displayed less Ki67-positive nuclei in the liver.Liver transaminases were elevated in all mice 3 h and 6 h post LPS stimulation. Gene expression analysis displayed a reduced expression of the pro-inflammatory cytokines IL-6 and CXCL1 after 3 h in the mFPR1The results suggest a prominent role of FPRs in the regulation of the hepatic inflammatory response after LPS induced liver injury. Deletion of mFPR1 or mFPR2 leads to deregulation of the inflammatory response compared to WT mice, associated with more severe liver injury represented by higher levels of transaminases, apoptotic cells and a reduced regenerative capacity. The inflammatory response after a liver injury is important for the induction of liver regeneration These prior findings suggest a differential role of FPR in the recruitment of the different leucocyte subtypes and who might have different functions divided in between tissue resident and towards injury site recruited cells -/--/-E. coli LPS  and kept for 3 h and 6 h. The mFPR1 Mice were a kind gift from Dr. Philip Murphy of the National Institute for Allergy and Infectious Diseases, NIH Five 8-10 weeks old wild type, mFPR1Mice were sacrificed 3 h and 6 h post LPS-stimulation, blood and liver were removed and preserved for biochemical and immunohistological assays. After stimulation the mice were kept in SII-long-cages with access to food and water ad libitum. Blood was taken retroorbital before sacrifice of the mice. The serum was separated by centrifugation and stored at \u221220\u00b0C until measurement.All experiments were performed in accordance to the German protection of animals act and with permission of the authority of the federal state North Rhine Westphalia. The study protocol was approved by the institutional animal care and use committee , Duesseldorf, Reference number: 84-02.04.2013.A246).Cryopreserved liver tissue was homogenized and RNA was extracted by using the Nucleospin RNA-II Kit . Afterwards 400 ng of total RNA was converted into cDNA by using the Omniscript reverse transcriptase kit . All proceedings were performed according to manufacturers' guidelines.The gene expression analysis was performed using an ABI 7500 Real-Time PCR . Genexpression analysis for the murine (ms) genes IL-6, TNF-\u03b1, CXCL1, TLR2, TLR4, mFPR1, mFPR2 and mFPR3 were performed. The Primers for mFPR1 and mFPR2 were published previously +- or Ly6G+-cells per view field.Formalin fixated paraffin embedded (FFPE) liver samples were cut into 5 \u00b5m strong sections and stained for CD11b  and Ly6G . The primary antibody was used in a dilution of 1\u2236100 and a species specific secondary antibody with a HRP-conjugate was diluted 1\u2236500 to detect the primary antibody. Visualization was performed using 3,3\u2032-diaminobenzidine tetrahydrochloride (DAB) . Antigen retrieval was executed according to manufacturer's instruction. Nuclei were counterstained with Haematoxylin . For each individual animal/genotype 7 pictures were taken in 100 and 200 fold magnification using an Olympus BX51. The 100 fold magnification was used for overview whereas the 200 fold magnification pictures were used for the detailed analysis and counting of the total CD11b+-cells in the BDL-model, cryosections of liver tissue were made and stained with a CD11b-antibody  used in a 1\u2236200 dilution. Visualization was performed using an anti-rat ALEXAFLUOR546 . Fluorescent microscopy pictures were taken by an AxioImager Z1 .For the immunofluorescent staining of CD11bFFPE-liver tissue was cut as described above in 5 \u00b5m strong sections. The TUNEL-staining was made according to the manufacturers' instruction . Visualization was performed using DAB and Nuclei counterstaining was made with Haematoxylin .The analysis of the ubiquitous cell cycle marker Ki67 was performed using the rat-anti mouse Ki67  in a 1\u223650 dilution. The detection was conducted with a secondary, HRP-conjugated anti-rat antibody in a 1\u2236300 dilution. The visualization was performed with DAB . Nuclei counterstaining was performed using Haematoxylin .The datasets were analyzed using the Student\u015b T-test and p-values \u22640.05 were regarded as significant and indicated in the respective graph.-/-, mFPR2-/- mice displayed no initial differences among the different genotypes. After LPS stimulation for 3 h the transaminases were significantly increased in mFPR2-/- mice (86 U/L) in comparison to wild type mice (66 U/L), whereas the mFPR1-/- mice displayed elevation after 3 h LPS administration, but did not reach a level of significant difference (63 U/L). The analysis of the 6 h time point displayed that transaminases were significantly upregulated in the serum derived from mFPR1-/-(296 U/L) or mFPR2-/-(291 U/L) mice compared to control (76 U/L) mice after LPS-treatment  and aspartate-aminotransferase (AST) of WT, mFPR1reatment . The hisreatment .-/- and the mFPR2-/- mice displayed lower number of CD11b+) and the Ly6G+-cells ). Also significantly lower were the CD11b+-cells ) and the number of Ly6g+-cells ) in the mFPR2-/- mice in comparison to WT mice. At the later time point, 6 h after intraperitoneal application of LPS (+-cell infiltrates per view field in comparison to wild type mice (96\u00b16). Likewise to the CD11b+-cells a higher presence of Ly6G+-cells was found in the livers of mFPR1-/\u2014 (160\u00b124) and mFPR2-/\u2014mice  were significantly lower . Taken together different degrees of inflammation occurred in regard to the deficiency of either mFPR1 or mFPR2 as wells as in regard to the temporal progression of inflammation.In order to get a better overview of the different immune cell subtypes infiltrating into the liver, we performed immunohistological straining for the surface markers CD11b and Ly6G. These markers identify two relevant cells types involved in promotion of liver injury. CD11b is known as a marker for activated monocytes whereas Ly6G is known to detect neutrophil granulocytes specifically. The immunohistological analysis of CD11b and Ly6G displays a strong presence of both cell types at 3 h as well as 6 h post LPS-treatment. At the time point 3 h after LPS the mFPR1of CD11b+ and Ly6G whereas the mFPR2-/- mice had a significantly higher expression compared to WT-mice (p<0.01). At 6 h post LPS injection the expression of IL-6 was significantly lower in the wild type animals compared to mFPR1 (p<0.05) and mFPR2-deficient mice (p<0.05). The expression of TNF-\u03b1 .The quantitative analysis of TLR4 and TLR2 expression displayed a highly different pattern. Toll-like receptor 2 gene expression is strongly elevated in all mice strains which underwent LPS-stimulation at time point 3 h. The differences in gene expression are significant between mFPR2periment . The anaperiment  showed aTo understand whether livers lacking mFPR1 and mFPR2 were more susceptible to pro-apoptotic signalling, a TUNEL-assay was performed .+-cells in mFPR1 (1.4%) and mFPR2 deficient mice (2.03%) compared to WT mice (1.2%). At 3 h only mFPR2-/- mice had a significantly higher number of TUNEL+-cells detectable in the liver. At the 6 h time point the amount of TUNEL+-cells was elevated in all mice strains. However, mFPR1-/- mice (2.53%) and mFPR2-/- mice (2.91%) had both significantly more apoptotic cells detectable in comparison to WT mice (1.28%) 6 h after injection of LPS . The mFPR2-/- mice itself displayed also a non-significant higher tendency for the number of TUNEL+-cells in comparison to the mFPR1-/- mice at 3 h and 6 h post LPS administration.The analysis of the 3 h time point revealed a higher percentage of TUNELn of LPS   ese mice  during t+-nuclei in the liver of either mFPR1 or mFPR2-deficient mice in comparison to wild type mice  and mFPR2-/-  displayed a significantly lower number of proliferative cells compared to WT mice  . At 6 h post LPS administration the differences between WT  and mFPR1-/-  were still present but did not reached a level of significance. The significant lower proliferation of mFPR-deficient mice  compared to WT mice was still present at this time point (p<0.05).Liver regeneration is a response to compensate the loss of cellular mass after an injury. To understand whether mFPR1 and mFPR2 might have an impact on liver regeneration we analysed the proliferative capacity of livers lacking either mFPR1 or mFPR2. For this purpose, the ubiquitous cell cycle marker Ki67 was stained at 0 h, 3 h and 6 h post LPS stimulation. The analysis of the staining revealed a lower number of Ki67ype mice . At 3 h Formyl peptide receptors 1 and 2 are known to function as important mediators of chemotaxis of hematopoietic cells -/- mice. The mFPR2-/- mice displayed a significant higher level of ALT in the serum. At the later 6 h time point, both, mFPR1-/- and mFPR2-/-, displayed a significantly higher Level of ALT. For AST a slightly different pattern appears. Wild type mice had the highest levels of AST detectable in the serum compared to mFPR1 and mFPR2-deficient mice. 6 h post LPS the mFPR1 and mFPR2 knockout mice displayed significant higher levels of AST in the serum. These findings support a protective role for formyl peptide receptors during progression of LPS induced liver injury. The histological analysis after LPS-stimulation revealed a differential recruitment of immune cells in a time and genotype dependent manner. The cytokine IL-6 is not only known as a recruiting molecule for immune cells. It is described as one of the main drivers of hepatoprotection during liver injury -/- and FPR2-/- mice 6 h after LPS stimulation. Interestingly mFPR2-/- mice showed a lower tendency regarding the number of Ly6G+-cells visible per view field. The closest explanation for this phenomenon is the link to the neutrophil recruiting cytokine CXCL1 (CXCL1) which showed the same tendency at least on mRNA level at 3 h and at 6 h post LPS-stimulation. Differential roles for mFPR1 and mFPR2 regarding immune cell homing is not excluded for granulocytes and supported by the literature We compared the effect of mFPR1 and mFPR2 deficiency after LPS-stimulation, mimicking a bacterial mediated liver injury. The initial analysis of the transaminases AS and ALT  from the-/- mice was the highest of all mice strains used in this study. The anti-apoptotic capabilities of FPR2/FPRL1 in primary human neutrophils are controversially discussed. Previous observations by Nagaoka et al. revealed that FPR2 is protective together with the P2X7 receptor -/- and mFPR2-/- mice in comparison to WT-mice. Furthermore these findings suggest a specific anti-apoptotic signalling of mFPR2 towards TNF-\u03b1 induced pro-apoptotic signalling. Stimulation experiments using a combination of fMLF and pharmacological inhibitors for p38 and MEK resulted in reduced chemotaxis, adhesion and release of superoxide by neutrophils A further finding of the regulation of the anti-inflammatory response is visible for other PAMP-receptors such as TLR4 and TLR2 . The ana2+-Signalling into the focus of attention and suggests an involvement of Ca2+ induced signalling in the mediation of cell cycle progression The investigation of liver proliferation to compensate the loss of liver mass, displayed an impairment of regenerative capacity in mFPR1 and mFPR2-deficient mice. Both genotypes showed a lower proliferation at 3 h and 6 h post LPS-induced liver injury, suggesting critical involvement of mFPR1 and mFPR2 in liver regeneration. Recent studies of liver regeneration showed that other member of the GPCR family especially the cannabinoid type 1 receptors support these findings. Furthermore, it puts the CaThe analysis of the mRNA expression of mFPR1-3 in the BDL model shows an increase of mFPR1 and mFPR3 gene expression in concordance with the number of infiltrating monocytes in the liver . This fiThe consequences of FPR deficiency in an acute model of liver injury seem to be a loss of anti-apoptotic and anti-inflammatory capabilities. Our findings suggest a pro-survival, anti-inflammatory role during an acute LPS-induced liver injury. This is in concordance with previous findings suggesting a positive role for FPR during the phase of acute injury Furthermore, the role of FPRs in progression of chronic inflammatory liver diseases is not understood and ought to be investigated. Based on recent investigations FPR expression could also be shown on natural killer cells Taken together both receptors are important to maintain a functional response to LPS induced liver injury. Furthermore our data suggests that mFPR1 and mFPR2 might also be involved in processes such as liver regeneration and might also have relevance not only during the acute liver injury, but also during chronic liver injury. Further experiments will provide prove to this hypothesis.Figure S1Immunofluorescent staining for CD11b reveals an increase over time after BDL. CD11b+-cells were visualized using Alexa546. Nuclei were counterstained using DAPI.(TIF)Click here for additional data file."}
+{"text": "DPY19L2 whole coding sequence. Furthermore we showed that the DPY19L2 protein is located in the inner nuclear membrane of spermatids during spermiogenesis and that it is necessary to anchor the acrosome to the nucleus thus performing a function similar to that realized by Sun proteins within the LINC-complex (Linker of Nucleoskeleton and Cytoskeleton). SUN1 was described to be necessary for gametogenesis and was shown to interact with the telomeres. It is therefore possible that Dpy19l2 could also interact, directly or indirectly, with the DNA and modulate gene expression during spermatogenesis.Globozoospermia is a male infertility phenotype characterized by the presence in the ejaculate of near 100% acrosomeless round-headed spermatozoa with normal chromosomal content. Following intracytoplasmic sperm injection (ICSI) these spermatozoa give a poor fertilization rate and embryonic development. We showed previously that most patients have a 200\u00a0kb homozygous deletion, which includes Dpy19l2 knock out and wild type mice in order to identify a potential deregulation of transcripts that could explain the poor fertilization potential of Dpy19l2 mutated spermatozoa.In this study, we compared the transcriptome of testes from DPY19L2 knock out and wild type mice. The transcriptome was carried out using GeneChip\u00ae Mouse Exon 1.0 ST Arrays. The biological processes and molecular functions of the differentially regulated genes were analyzed with the PANTHER software.RNA was extracted from testes from Dpy19l2) were down-regulated. These genes were found to be involved in DNA/RNA binding, structural organization, transport and catalytic activity.A total of 76 genes were deregulated, 70 were up-regulated and 6  contains supplementary material, which is available to authorized users. A recent study supported by the World Health Organization indicates than in 2010, an estimated 48.5 million couples worldwide were unable to have a child after five years I is known to play an important role in male fertility. [Ca2+]I signaling is the primary regulator of sperm flagellum beating and calcium intracellular rise is known to be essential for the acrosome reaction [3 in mouse sperm [Dpy19l2 KO mice two calcium binding proteins are up-regulated : Caps2 and Sgk3  during spermiogenesis. We also observed that several genes encoding proteins involved in transports, and in particular Abca1, involved in the cholesterol efflux, were deregulated. This could also contribute to the poor fertilization potential of the round-headed spermatozoa. Secondary anomalies stemming from the morphological abnormalities of the sperm could also lead to a wide range of protein deregulation as exemplified by the absence of PLCzeta. A proteomic analysis of these deregulations could permit to have a functional view of the extent of the molecular anomalies present in Dpy19l2 KO mice. Further work will permit a better comprehension of molecular mechanism involved in spermatogenesis and in the physiopathology of globozoospermia.We showed that Dpy19l2Additional file 1: Table S1: RNA quantification. (DOC 27 KB)Additional file 2: Table S2: Ratios of transcripts values measured in Dpy19l2 WT and KO mice. (XLS 66 KB)Additional file 3: Table S3: PANTHER output of all deregulated genes in Dpy19l2 KO mice. (XLS 60 KB)"}
+{"text": "Recent evidences suggest an association between unresponsive chronic daily headache (CDH) and idiopathic intracranial hypertension without papilledema (IIHWOP). Diagnosis can be challenging. The CSF opening pressure (OP) cutoff value greater than 200 or 250 mmH2O is debated as the role of transverse sinus stenosis (TSS).To investigate the frequency of IIHWOP and TSS in adult patients with refractory CDH.In a prospective study, patients with refractory CDH were consecutively enrolled. Each participant underwent ophthalmologic evaluation and Optical Coherence Tomography to rule out the presence of papilledema; cerebral MR venography (MRV) to detect TSS; and a lumbar puncture in the lateral decubitus position to measure OP.Among the 23 consecutive patients enrolled, 19  completed the study, 4 female patients dropped out. None of the 19 patients had papilledema. We found a TSS in 11 of 19 cases (58%): bilateral in 3 patients and unilateral in 8 cases. All of 19 cases displayed OP lower than 250 mmH2O (range 102-245) and normal CSF composition. We found a OP greater than 200 mmH2O only in four patients  (17%): two of them achieved an improvement of headache intensity and frequency after 12-18 ml CSF withdrawal; one of them had bilateral TSS.Our preliminary data suggest that IIHOWP may be an underestimated condition. We suggest to keep the 200 mm H2O cutoff value at least until the role of venous stenosis will be clarified.No conflict of interest."}
+{"text": "Being widely used NPs, their toxicity assessment studies help in understanding the adverse effects to the humans. It is likely that NPs exposure to the humans can be through different routes but will finally reach the liver. Therefore, an attempt was made to explore the genotoxicity of TiO2 NPs on human liver cells (HepG2).TiO2 NPs were characterized by transmission electron microscopy (TEM) for their primary size, shape and dynamic light scattering for their size, size distribution and zeta potential in culture medium. Cellular uptake of TiO2 NPs in HepG2 cells was detected using flow cytometry method. Moreover, ultrathin sections of cells were analysed using TEM to visualise the internalisation of TiO2 NPs. The genotoxic potential of TiO2 NPs was assessed by micronucleus assay using the conventional and flow cytometry methods.TiO2 NPs revealed a mean diameter size of 30-70 nm and DLS measurements showed a mean hydrodynamic diameter and zeta potential of 192.5 \u00b1 10 nm and -11.4 \u00b1 1.2 mV, respectively. The electron microscopy and flow cytometry studies for particle internalisation showed a significant cellular uptake of TiO2 NPs in the human liver cells (HepG2). A significant (p < 0.05) induction in micronucleus formation was observed at 20 \u00b5g/ml of TiO2 NPs exposure when compared to control cells. However further treatment to HepG2 with higher concentrations (40 and 80 \u00b5g/ml) showed a decrease in micronucleus formation than 20 \u00b5g/ml.TEM analysis of TiO2 NPs than control.In contrary, the flow cytometric results exhibited a significant concentration dependent induction of micronucleus in HepG2 cells exposed to all concentrations  of TiO2 NPs on prepared slides, which hinders the counting of micronucleus . However, in the flow cytometry analysis, these nanoparticles do not interfere with the optics. Hence, it is proposed that in the case of NPs treatment, flow cytometry based micronucleus assay should be used instead of the conventional method.The difference in the micronucleus frequency obtained from conventional and flow cytometry methods in HepG2 may be due to the accumulation of TiO"}
+{"text": "Acetanaerobacterium and Ruminococcus with relatively high abundance. The characterization of the microbial community corroborated the digestion performance affected at the agitated condition, where lower methane yield and delayed methane production rate were observed. This was further verified by the accumulation of propionic acid in the agitated digester.To investigate the distribution and dynamics of microbial community in anaerobic digestion at agitated and non-agitated condition, 454 pyrosequencing of 16s rRNA was conducted. It revealed the distinct community compositions between the two digesters and their progressive shifting over time. Methanogens and syntrophic bacteria were found much less abundant in the agitated digester, which was mainly attributed to the presence of bacterial genera  The divergent effect of agitation on anaerobic digestion has been reported by some studies, while most of which investigated the conventional physiochemical propertiesClassically, environmental microbial communities are analyzed by construction of 16S rRNA clone libraries and the subsequent sequencing of individual clones. The approach, termed Sanger sequencing, has been applied in Tian et al (2013) to compare the microbial community structures for anaerobic digestion at agitated and non-agitated condition, led to the identification of some major microbial phylotypes in bacteria or archaea domain. Due to the fact that a few numbers of clones can be affordably sequenced, Sanger method has its limitation in revealing the whole complexity of microbial communities and is unlikely to adequately represent the genetic diversity. New development of high-throughput next-generation sequencing technologies (NGS), such as 454 barcoded pyrosequencing, not only eliminates the laborious step of preparing clone libraries, but also makes large scale environmental sequencing cost effective and keeps the bias small 4 production, soluble Chemical Oxygen Demand (sCOD) and Volatile Organic Acids (VOAs) profiles.There were limitations in the previous study Two digesters of 5 L, named digester 1 and 2, were built by modifying Pyrex glass jars \u22121day\u22121. Digester 1 and 2 were then emptied and washed and residual substrates were discarded. In trial 2, both digesters were fed with fresh 0.3 kg (wet weight) of washed sugar beet tailings and re-inoculated with approximate 3 L of its own sludge liquor recovered from trial 1 (Sludge liquors from digester 1 and 2 were not mixed). Trial 2 ended at day 14 (or day 32 cumulatively) when the gas production from both digesters was less than 0.05 L@STP L\u22121day\u22121.Digester 1 was operated under non-agitated condition. Two kilograms of bulking materials  were added into digester 1 along with the feedstock to prevent substrate compaction and floatation. Digester 2 was operated under agitated condition without adding the bulking agent. The digester content was continuously mixed at 180 rpm by using a 50.8 mm \u00d7 9.5 mm PTFE coated polygon bar and a large volume magnetic stirrer (Bel-Art ScienceWare Cool Stirrer). Two experimental trials were carried out. In trial 1, both digesters were inoculated with 3 L inoculum taken from an anaerobic digester that has been digesting with sugar beet tailings for months. Trial 1 ended at day 18 when the gas production from both digesters was less than 0.05 L@STP LTotal Solids (TS) and Volatile Solids (VS) contents were determined for the feedstock sugar beet tailings 4 and CO2) was analyzed using Fisher Model 1200 Gas Partitioner. sCOD concentration was determined using Hach method 8000. VOA analysis was conducted using Shimadzu gas chromatograph (GC-9AM equipped with a flame ionization detector) for acetic, propionic, isobutyric, butyric, isovaleric and valeric acid concentrations Daily biogas production from the digesters was measured by a positive displacement gas meter. Gas composition , digester 1 liquor and digester 2 liquor. Digester liquors were sampled at day 3, 5 and 18 for trial 1 and day 1, 4, 8, 11, 12 and 14 for trial 2 . A total 17 samples were analyzed.Total DNA was extracted and purified by using FastDNA Kit  and PowerClean DNA Clean-up Kit  respectively, according to the manufacturer's instruction. The quality of DNA was verified by agarose gel electrophoresis. Extracted DNA was stored at \u221220\u00b0C until further use.2+, and 200 \u00b5M of dNTP ] and 1 \u00b5L of barcoded primers (100 pmoles each). The amplification protocol was as follows: initial denaturation at 94\u00b0C for 3 minutes, followed by 30 denaturation cycles at 94\u00b0C for 45 seconds, annealing at 50\u00b0C for 30 seconds, and extension at 65\u00b0C for 90 seconds, with a final extension for 10 minutes at 65\u00b0C. The tree replicated PCR products were combined for each sample, purified using the QIAquick PCR Purification Kit  and quantified using on-chip gel electrophoresis with Agilent 2100 Bioanalyzer and DNA Lab Chip Kit 7500.For each sample, the V4 hypervariable region of16S rRNA gene was PCR-amplified using the F515/806R primer set that was designed for accurate phylogenetic placement of a broad range of archaeal and bacterial taxa with few biases Because each sample was amplified with a known tagged primer, an equimassic mixture ampicon from different samples could be sequenced simultaneously. Among the total 17 samples, 9 samples were sequenced simultaneously in the first batch, and 8 samples in the second batch. The quantity of each PCR product was made equal within a batch: 432 ng of each PCR product in batch 1 and 480 ng in batch 2.www.mothur.org/wiki). First, sequences were de-noised and filtered, and chimeric sequences were removed (chimera.unchime) to improve data quality. Second, qualified sequences were clustered to operational taxonomic units (OTUs) defined by a 97% similarity level. Third, diversity analyses and diversity index calculations were performed for Chao1 richness estimation, Good's coverage and rarefaction curves. The variability of community composition between samples was evaluated with Principal Component Analysis (PCA), which is a multivariate ordination method that visually represents distance between samples. More similar communities would be placed closer in the ordination. A similarity matrix of Yue and Clayton (ThetaYC) distances that take into account of both membership and relative abundance was calculated to determine each sample's position in the PCA ordination. Statistical analyses were conducted using the mothur package Batch 1 and 2 were sent to Interdisciplinary Center for Biotechnology Research (ICBR) at University of Florida for pyrosequencing using a 454 GS-FLX sequencer . The raw sequence data were sorted based on the sample specific barcode for batch 1 and 2, respectively, and primer and barcode sequences were then trimmed from the sorted sequences. The trimmed sequences from batch 1 and 2 were then combined and processed through mothur (The sequence data are available from the NCBI Sequence Read Archive (Run SRR1283194).TS and VS contents of sugar beet tailings, loading quantities and packing density were determined for 2 trials and presented in 4 production rate for digester 1 peaked at 0.70 m3 d\u22121 (kg VS)\u22121 on day 5, and 0.34 m3 d\u22121 (kg VS)\u22121 on day 11 for digester 2. Trial 2 was started by flooding digester 1 and 2 with digester liquor left in trial 1. Digester 1 reached higher CH4 production rate during trial 2 than during trial 1 (0.94 m3d\u22121(kg VS)\u22121 on day 4), whereas digester 2 exhibited similar production rate (0.35 m3d\u22121(kg VS)\u22121 on day 7). Both digesters reached their maximal production rate earlier than during trial 1.During trial 1, CH4 yield for digester 1and 2 in both trials were shown in 4 yield of 0.37 m3 CH4 at Standard Temperature and Pressure (STP) (kg VS)\u22121 and digester 2 achieved CH4 yield of 0.24 m3 CH4 at STP (kg VS)\u22121 at the end of trial 1. At the end of trial 2, the CH4 yield was 0.35 m3 CH4 at STP kg VS\u22121 for digester 1 and 0.21 m3 CH4 at STP kg VS\u22121 for digester 2.Cumulative CHProfiles of soluble COD (sCOD) and VOA concentration of digester 1 and 2 were shown in The main VOAs detected in both digesters are acetic, butyric and propionic acids. In digester 1, acetic acid was detected with the highest concentrations among VOAs at the beginning of each trial, reaching a maximum around day 3 to 5. Its concentration decreased rapidly to a negligible amount as methane was produced. Similarly, but to a less extent, butyric acid slightly accumulated at the beginning of both trials and then rapidly disappeared from day 4 to 7. Propionic acid was present at constantly low concentration.In digester 2, the concentration of acetic and butyric acid peaked between day 8 to day 11, and the degradation was delayed compared to that in digester 1. Profiles of propionic acid showed no significant degradation, resulting in an evident accumulation that reached 1000 mg/L in trial 2.A total number of 6,993 sequences with average length of 204 bps was obtained from 14,773 raw sequence reads after the quality improving process. The number of sequences for different samples ranged from 126 to 994. Of the total sequences obtained, 149  represented lineage from archaea domain. Across 17 samples, 1,137 OTUs (defined at 97% sequence similarity level) were identified. All samples had Good's coverage above 70% and the rarefaction curves were provided in The variability of community composition was evaluated by a PCA plot . In the Thermotogales and Petromonas were found dominant in the inoculums, digester 1 and digester 2 in general. At beginning of trial 1, the community composition in digester 1 and digester 2 diversified and shifted greatly from that of the inoculums. Phylotype Bacillaes gained dominance at day 3 but decreased quickly with progression of the digestion. In trial 2, changes in the community composition were less dynamic.1,137 OTUs were taxonomically classified to 117 different phylotypes at similarity threshold of 80%. Top 23 phylotypes with highest relative abundance were selected and analyzed for each sample . The dynAcetanaerobacterium, Ruminococcus and Ruminococcaceae were detected in digester 2 and their relative abundance followed an increasing pattern with development of the digestion. These phylotypes were either not detected or detected at a very low abundance in digester 1. In addition, phylotype Anaerobaculum exhibited a different distribution between digester 1 and 2. While Anaerobaculum gradually developed and reached relative abundance of 15% in digester 1 (after 15 days), it was identified with constantly low abundance in digester 2.Despite the aforementioned similarities, digester 1 and 2 differed from each other in many ways with regard to the community compositions. Phylotype Methanoculleus and Methanosarcina were found abundant among methanogens. In digester 1, the relative abundance of methanogens increased over time, peaking at 5% for trial 1 (day 5) and 8% for trial 2 (day 22), respectively. In contrast, the development of methanogens in digester 2 was slow and the relative abundance never exceeded 1.4% (day 18) in trial 1. It continuously reduced in trial 2 until no significant detection was obtained near the trial end. Selected phyloptyes  suggested a lower microbial diversity as verified by the Chao1 richness estimation (results not shown) in comparison with digester 1. Interestingly, the community evolving between digester 1 and 2 seemed to follow a similar path from day 0 to day 5 but subsequently developed in separate ways to disparate compositions. This could imply the effect of agitation was not instantaneous but rather cumulative.Anaerobaculum has been known to degrade peptide and a limited number of carbohydrates Anaerobaculum-related species being dominant Anaerobaculum seemed to play a role in propionate degradation, even though it has not been recognized as a syntrophic propionate utilizing bacterium. It can be postulated that the low abundance of Anaerobaculum in digester 2 probably resulted in accumulation of propionic acid.16s rDNA sequences reads were compared to the entries of RDP database, and assigned to phylogeneic groups. However, a large portion of sequences were classified to an unclassified phylotype, suggesting the complexity of microbial communities in anaerobic digestion is yet to be characterized. Among identified phylotypes, Methanoculleus and Methanosarcina species. Members of Methanoculleus are hydrogenotrophic methanogens Methanosarcina species are mostly acetoclasic but also able to use H2Methanosarcina were generally more abundant than from Methanoculleus. The relative abundance of Methanosarcina was seen to follow a dynamic that coincided with the digestion performance. The marked increase from day 3 to day 5 in trial 1 and day 1 to day 4 in trial 2 corresponded to the high CH4 production rate and the significant reduction of acetic acid during a similar time frame. Methanosarcina spp. has been reported to have higher growth rates and tolerance to pH changes and could potentially lead to stable methenogensis in anaerobic digestion 4 production and the accumulation of propionic acid in digester 2 particular for trial 2.Archaeal (mostly methanogens) versus bacterial community ratio generally agreed with the reported methanogen proportions ranging from 0.1% to 15% of the total microbial population Acetanaerobacterium, Ruminococcus and Ruminococcaceae were found with high relative abundance in digester 2. Those species were closely related and all belonged to family Ruminococcaceae, which are known to degrade cellulose and produce hydrogen (H2) as one of the fermentation products Ruminococcus species has been widely used for H2 production from a variety of feedstockRuminococcus were reported to produce propionate other than ethanol as fermentation products, which could also lead to propionic acid accumulation Phylotypes 2) production is energetically unfavorable due to proton being a poor electron acceptor. The development of syntrophic communities allows H2 production to become energetically favorable and sustain degradation of organic compounds and production of CH4. Due to that syntrophic metabolism, methanogenic activity has to be suppressed in order to produce free H2 or it would have been readily converted to CH4 in anaerobic digestion. pH control is desirable for H2 production because methanogenic activity drops sharply in an acidic environment 2 at low partial pressure has been reported in anaerobic processes operated at neural or near neutral pH Ruminococcus related bacteria produced free H2 in digester 2 even though it was typically considered energetically unfavorable. Some studies seemed to support this speculation. Rychlik and May investigated the effect of Methanobrevibacter smithii on growth rate, organic acid production and specific ATP activity of Ruminococcus albus in the co-ulture Ruminococcus albus did not receive energetic advantage from co-culturing with the methanogen and the syntrophic metabolism was not preferred. Zhou et al investigated the effect of methanogenic inhibitors on methaneogens and three rumen bacteria Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciensRuminococcus may not always rely on the syntrophic relationship with methanogens to grow and produced free H2 in digester 2. Its concentration was expected to be very low that was not analyzed in biogas composition, but may have exerted enough inhibition on methanogensis as discussed below.In anaerobic digestion, hydrogen could be generated through fermentation of intermediate products as sugars or VOAs Desulfotomaculum, Pelotomaculum and Syntrophomonas were detected in both digester 1 and 2 at low proportion Click here for additional data file.Table S1Substrate characteristic and loading quantities for digester 1 and 2.(DOCX)Click here for additional data file.Table S2Taxonomic composition of digester 1 and digester 2 at the rank of phylum, order and genus.(DOCX)Click here for additional data file."}
+{"text": "Cross talk between Fgf and Hedgehog (Hh) signaling was critical for the specification of GABAergic interneurons and oligodendrocytes. The expression of fgf16 in the forebrain was down-regulated by the inhibition of Hh and Fgf19 signaling, but not by that of Fgf3/Fgf8 signaling. The fgf16 morphant phenotype was similar to that of the fgf19 morphant and embryos blocked Hh signaling. The results of the present study indicate that Fgf16 signaling, which is regulated by the downstream pathways of Hh-Fgf19 in the forebrain, is involved in forebrain development.Fibroblast growth factor (Fgf) signaling plays crucial roles in various developmental processes including those in the brain. We examined the role of Fgf16 in the formation of the zebrafish brain. The knockdown of  The forebrain becomes regionally subdivided into the telencephalon and diencephalon during early embryonic brain development in vertebrates. The telencephalon is further subdivided into the rostrally positioned subpallial  telencephalon and more caudally located pallial  telencephalon. The diencephalon is comprised of the hypothalamus, zona limitans intrathalamica (ZLI), ventral thalamus, dorsal thalamus, and pretectum Fgf8 specifies rostral telencephalic fate and represses caudal telencephalic fate in mice and zebrafish fgf3 in zebrafish affects the expression of genes that have been implicated in the development of the forebrain fgf3 and fgf8 functions revealed that fgf3 and fgf8 possessed a unique and combinatorial function in regional patterning of the forebrain and hindbrain Fgf15 knockout mice demonstrated that Fgf15 repressed rostral telencephalic fate fgf19, which is the Fgf15 orthologue in zebrafish, is known to be essential for development of the ventral region of the telencephalon and diencephalon in zebrafish Fgfs comprise a large family of at least 22 members in vertebrates Fgf16, which was originally identified in the rat heart, is predominantly expressed in the heart at adult stages Fgf16 is expressed in the heart, inner ear and brown adipose tissue during embryonic development in mammals Fgf16 knockout mice have been reported and their phenotypes may potentially be affected by genetic backgrounds. Fgf16 knockout mice on a C57BL/6 background exhibited a decrease in the proliferation of embryonic cardiomyocytes and pathophysiological roles in the postnatal heart, whereas the cardiac phenotype of Fgf16 knockout mice on a 129/B6 background has not yet been examined Fgf16 knockout mice on a Black Swiss background died at approximately E11.5 fgf16 is expressed in zebrafish in the pectoral fin bud and forebrain in addition to the otic vesicle fgf16 knockdown zebrafish embryos indicated that fgf16 is an apical ectodermal ridge (AER) factor that is crucial for pectoral fin bud outgrowth fgf16 morphants display morphological abnormalities in the brain. However, these abnormalities have not yet been elucidated in detail.fgf16 during brain development in zebrafish. Our results demonstrated that fgf16 was critical for cell proliferation in the forebrain and midbrain. fgf16 was also critical for development of the ventral region of the telencephalon and diencephalon, and was implicated in the specification of \u03b3\u2013aminobutyric acid (GABA)ergic interneurons and oligodendrocytes in the telencephalon and diencephalon. On the other hand, fgf16 was not implicated in the specification of tectal and tegmental fates. fgf3, fgf8 and fgf19 have also been shown to be involved in the specification of GABAergic interneurons and oligodendrocytes in the ventral region of the forebrain fgf16 and fgf3, fgf8, and fgf19 in the forebrain.In the present study, we examined the roles of Danio rerio) were maintained, according to The Zebrafish BookZebrafish  or universal control MO (5 ng) was injected into the two-cell embryos of zebrafish. fgf3 MO (10 \u00b5g/\u00b5l) and fgf8 MO (20 \u00b5g/\u00b5l) were injected at a volume of 0.15\u20130.25 nl into the two-cell embryos. fgf19 MO was injected at 10 \u00b5g/\u00b5l into the four central blastomeres of 16-cell embryos.Morpholino oligonucleotides (MOs) were synthesized by Gene-Tools, LLC . MOs were diluted in Danieau buffer fgf16, full-length fgf16 cDNA was amplified by PCR and inserted into the vector pCS2+ fgf16 mRNA was made by invitro transcription using SP6 polymerase . mRNA was diluted to 0.5 ng/\u00b5l with distilled water and injected at a volume of 0.5 nl into 2-cell embryos.To construct Proliferating and apoptotic cells were detected using a rabbit polyclonal anti-phosphorylated histone H3 (H3P) (Upstate Biotechnology) antibody and the DeadEndTM colorimetric detection kit (Promega), respectively Whole mount immunostaining was performed as described previously Cyclopamine  fgf16 was expressed in the pectoral fin bud and also that the knockdown of fgf16 function resulted in the absence of fin bud outgrowth at 5 days post-fertilization (dpf) fgf16 morphants exhibited abnormalities at 5 dpf fgf16 morphants were morphologically distinguishable from the wild type at 24 hours post-fertilization (hpf). fgf16 morphants showed morphological abnormalities in the forebrain at 24 hpf  constriction and exhibited a failure to evaginate laterally in the midbrain at 24 hpf . On the other hand, control MO-injected embryos developed normally during embryogenesis fgf16 RNA with fgf16 MO1 rescued the brain defects caused by fgf16 MO1 (n\u200a=\u200a10/13)  . These rfgf16 is expressed in the brains of zebrafish embryos during 18\u201336 hpf fgf16 has not yet been examined in detail in the brain during neural development. We here examined the spatiotemporal expression pattern of fgf16 in the zebrafish embryonic brain in detail using whole mount insitu hybridization. The expression of fgf16 was first detected in the most ventral part of the anterior telencephalon primordium at 14 hpf  was specifically detected in the mitotic cells in mitotic phase (M-phase) fgf16 morphants was significantly lower than that in wild-type embryos at 24 hpf , B.fgf3, fgf8, and fgf19 have been implicated in patterning events in the zebrafish forebrain fgf16 morphants at 24 hpf. The expression of emx1, which is normally detected in the pallial domain of the telencephalon, was observed in the entire region of the telencephalon in fgf16 morphants (n\u200a=\u200a28/32)      . Further=\u200a15/16) . In cont=\u200a27/31) . On the =\u200a21/21) . The ect=\u200a13/13) . In contfgf16 affected diencephalic specification. In addition to the ventral telencephalon, dlx2 is normally expressed in the ventral thalamus. The expression of dlx2 in the ventral thalamus was reduced in fgf16 morphants at 24 hpf (n\u200a=\u200a27/31)     . On the ctively) . We also=\u200a14/14) . On the =\u200a14/14) . Thus, tfgf16 morphants showed morphological abnormalities in the MHB constriction and midbrain. Therefore, to investigate whether fgf16 was involved in MHB development, we examined the expression of fgf8 in fgf16 morphants at 24 hpf. The expression of fgf8 was detected in the MHB of fgf16 morphants (n\u200a=\u200a27/27)  , which ifgf16 was involved in specification of the midbrain. Otx2 is an important player in the regulation of midbrain patterning otx2 was unaffected in the midbrains of fgf16 morphants at 24 hpf (n\u200a=\u200a13/13) , respectively  . Furtherectively . These rfgf16 MO affected neuronal differentiation in the forebrain, the expression of the basic helix-loop helix (bHLH) proneural gene, ngn1, was analyzed in fgf16 morphants at 24 hpf. The expression of ngn1 was unaffected in the dorsal telencephalon of fgf16 morphants, whereas it was reduced in the diencephalon (n\u200a=\u200a11/11)   . We then=\u200a15/20) . These rgad1 encoding glutamic acid decarboxylase was found to be expressed specifically in GABAergic interneurons fgf16 had any effects on GABAergic interneuron differentiation in the forebrain, gad1 expression was analyzed in fgf16 morphants at 28 hpf. gad1 was expressed in the subpallial telencephalon and nucleus of the tract of the postoptic commissure (nTPOC) in the forebrain fgf16 morphants, the expression of gad1 was severely reduced in both the ventral telencephalon and the nTPOC (n\u200a=\u200a27/28) (fgf16 morphants. GABA-immunoreactive cells were not detected in the forebrains of fgf16 morphants at 3 dpf (n\u200a=\u200a20/20)  (fgf16 was involved in the formation of myelinating oligodendrocytes. PLP (proteolipid protein)/DM20 is a marker of oligodendrocyte differentiation and is expressed in newly formed oligodendrocyte progenitor cells, well before myelination plp was not detected in the forebrains of fgf16 morphants at 4.5 dpf (n\u200a=\u200a12/12) (plp in the hindbrain disappeared in fgf16 morphants at 4.5 dpf (n\u200a=\u200a10/12) (fgf16 morphants at 4.5 dpf (n\u200a=\u200a11/11)  2.2, the postmitotic marker of glutamatergic neurons, was analyzed in fgf16 morphants at 28 hpf. In fgf16 morphants, the expression of slc17a6a was unaffected in both the pallial telencephalon and diencephalon (n\u200a=\u200a14/14)  . We also=\u200a20/20) . Oligode=\u200a14/20) . Further=\u200a12/12) . In addi=\u200a10/12) . The imm=\u200a11/11) , B. Thes=\u200a14/14) . This refgf16 led to abnormalities in the regionalization and generation of specific cell types such as GABAergic interneurons and oligodendrocytes in the forebrain. Hh signaling is critical for regulating the expression of fgf3, fgf8, and fgf19 in the forebrain and that of fgf19 and fgf22 in the midbrain Fgf16 was responsive to Hh signaling. Since the alkaloid cyclopamine completely blocked Hh signaling at the level of Smoothened, which transduces Hh signals, in zebrafish Fgf16 in embryos treated with cyclopamine. In embryos treated with cyclopamine, fgf16 expression was lost in the forebrain at 16 and 25 hpf  (fgf16 expression in the midbrain was lost in embryos treated with cyclopamine (n\u200a=\u200a10/10)  forebrain patterning and promotes the GABAergic neuronal/oligodendrocyte lineage restriction of forebrain stem cells ctively) . Further=\u200a10/10) . All con=\u200a10/10) . These rfgf16 led to abnormalities in the regionalization and generation of specific cell types such as GABAergic interneurons and oligodendrocytes in the forebrain. fgf3 and fgf8 are also involved in the regional patterning and generation of GABAergic interneurons and oligodendrocytes in the forebrain. The inhibition of both fgf3 and fgf8 was shown to result in defects in the expression of genes associated with early patterning functions and the specification of GABAergic interneurons and oligodendrocytes in the forebrain fgf16 was detected later than that of fgf3 or fgf8. Therefore, to examine whether the expression of fgf16 was affected by the inhibition of both fgf3 and fgf8 during forebrain development, we examined its expression in fgf3/8 double morphant embryos at 24 hpf. The expression of fgf16 was unaffected in the forebrains of fgf3/8 double morphant embryos (n\u200a=\u200a22/24) (Fgf3 MO and Fgf8 MO led to a reduction in the expression of fgf16 in the midbrain at 24 hpf (n\u200a=\u200a22/24)   . In cont=\u200a22/24) . This re=\u200a12/14) . Thus, fFgf8 is required for MHB development, and the MHB is crucial for proliferation and patterning in the midbrain fgf8 has not been implicated in growth of the forebrain fgf16 knockdown significantly inhibited cell proliferation and led to a reduction in the size and morphological abnormalities in the forebrain and midbrain. fgf16 morphants showed normal expression patterns of fgf8 in the MHB and had normal MHB-specific characteristics. This result indicated that a decrease in cell proliferation in the midbrains of fgf16 morphants was not due to a defect in the MHB. Thus, fgf16 functions are required to promote cell proliferation in the forebrain and midbrain.Fgf signaling regulates the proliferation and differentiation of specific neuronal cell types in the forebrain and midbrain fgf16 was first detected in the most anterior part of the ventral telencephalon at 14 hpf. fgf16 morphants exhibited the expanded expression of markers for the pallial telencephalon, emx1 and tbr1, and decreased expression of markers for the subpallial telencephalon, dlx2, at 24 hpf. These results suggested the loss of subpallial fate in the telencephalon of fgf16 morphants. Reduced cell proliferation in the telencephalon was observed in fgf16 morphants. Therefore, subpallial cells may be formed in smaller numbers due to reduced cell proliferation caused by the inhibition of fgf16. However, the expanded expression of ngn1 and slc17a6a was not detected in the ventral telencephalon of fgf16 morphants, which suggested that ventral cells in the telencephalon of fgf16 morphants were not formed in smaller numbers. Furthermore, fgf16 knockdown did not appear to transform cell fate specification from subpallial to pallial cells, and did not induce differentiation into dorsal neuronal cell types in the subpallial telencephalon. The ectopic expression of otx2 was detected in the ventral telencephalon of fgf16 morphants. Thus, Fgf16 is involved in patterning of the ventral forebrain, whereas the ventral telencephalon does not develop into the pallium following the inhibition of fgf16.The expression of dlx2 was decreased in the ventral thalamus by the inhibition of fgf16 at 24 hpf, whereas that of shh was unaffected in the ventral region. Furthermore, the expression of pax6a was normally detected in the diencepharon of fgf16 morphants at 24 hpf. These results demonstrated that the ventral thalamus was initially induced in fgf16 morphants. Therefore, fgf16 is necessary for maintaining of the characteristics of the ventral thalamus. In contrast, tectum- and tegmentum-specific characteristics were unaffected in the midbrains of the fgf16 morphants. This result indicated that fgf16 may be involved in regulating cell proliferation, but not dorsoventral patterning during midbrain development. In contrast, fgf16 may be involved in both the establishment of the subpallial telencephalon and ventral thalamus as well as the regulation of cell growth during forebrain development.In the diencepharon, the expression of ngn1 was unaffected in the dorsal telencephalon of fgf16 morphants. Furthermore, slc17a6a expression was also detected normally in the dorsal telencephalon of fgf16 morphants. On the other hand, the expression of isl1 was reduced in the ventral telencephalon, anterior ventral thalamus, and epiphysis, which suggested that fgf16 may be involved in neuronal differentiation in the ventral region, but not the dorsal region in the forebrain. However, slc17a6a expression was detected normally in the ventral thalamus of fgf16 morphants. These results indicated that fgf16 was not required for the specification of glutamatergic neurons in the forebrain.Ngn1 is known to be sufficient for conferring neuronal identity on uncommitted precursors and plays an important role in neurogenesis dlx2 was reduced in the forebrains of fgf16 morphants. Dlx2 was shown to be involved in the specification of GABAergic interneurons and oligodendrocytes in the telencephalon GAD1, when ectopically expressed in cortical explants olig2, expressed in oligodendrocyte precursors, is necessary and sufficient for the generation of oligodendrocytes throughout the neuraxis fgf16 knockdown resulted in a severe reduction of the expression of gad1 and olig2 in the ventral telencephalon and diencephalon. GABA-immunoreactive cells were also lost in the forebrains of fgf16 morphants, which indicated that GABAergic neurons did not fully differentiate in fgf16 morphants. plp expression and CC1 immunoreactivity also disappeared in fgf16 morphants, which suggested that the oligodendrocytes did not terminally differentiate into myelinating cells in fgf16 morphants. These results demonstrated that fgf16 was involved in the specification of GABAergic interneurons and oligodendrocytes in the ventral telencephalon and diencephalon. On the other hand, the knockdown of fgf16 did not strongly stimulate apoptosis in the forebrain. This result suggested that the survival of GABAergic interneurons and oligodendrocytes was unaffected by fgf16. Accordingly, Fgf16 appears to be crucial for the differentiation of GABAergic interneurons and oligodendrocytes, but not for that of glutamatergic neurons in the forebrain.The expression of Shh plays a mitogenic role in the brain and the ectopic expression of Hh target genes causes human cancers such as Basal Cell Carcinoma or medulloblastoma, a granule cell tumor fgf16 morphants as well as Shh mutant mice. Furthermore, the expression of fgf16 in the forebrain and midbrain was markedly reduced by the inhibition of Hh signaling at 16 and 25 hpf. These results indicated that Fgf16 may function downstream of Hh activity in cell proliferation in the forebrain and midbrain. On the other hand, Fgf8 participates in the growth of the midbrain, whereas Fgf3 and Fgf8 are not required for growth of the forebrain fgf3 and fgf8 led to a reduction in the expression of fgf16 in the midbrain, whereas it was unaffected in the forebrains of fgf3/8 double morphant embryos. Thus, fgf3 and fgf8 expressed in the MHB may regulate cell proliferation in the midbrain by activating the expression of fgf16 in the midbrain.olig2fgf16 morphants as well as smu/smo mutants exhibited the suppressed specification of GABAergic interneurons and oligodendrocytes in the forebrain. Hh signaling specifies GABAergic interneurons and oligodendrocytes via fgf3, fgf8, and fgf19 in the ventral forebrain, and this ensures the expression of pan-ventral transcription factors, such as dlx2 and olig2, whereas Fgf19 has distinct functions independent from those of Fgf3 and Fgf8 fgf19 led to a reduction in the expression of fgf16 in the forebrain, whereas the expression of fgf16 was unaffected in fgf3/8 double morphant embryos. This result indicates that fgf16 expression in the forebrain is regulated by Fgf19, but not by Fgf3/Fgf8. Thus, the effects of Hh activity on the differentiation of GABAergic interneurons and oligodendrocytes may be mediated through Fgf19-Fgf16 pathways in the forebrain.In addition to cell proliferation, Hh signaling is required for patterning in the telencephalon and the generation of GABAergic neuronal/oligodendrocyte progenitors from ventral forebrain stem cells via the activation of fgf16 expressed in the developing brain plays crucial roles in brain development. fgf16 is involved in cell proliferation in the forebrain and midbrain. fgf16 is also involved in the development of the ventral region and specification and differentiation of GABAergic interneurons and oligodendrocytes in the forebrain. On the other hand, fgf16 was not required for the specification of tectal and tegmental fates. Furthermore, the expression of fgf16 was dependent on Hh and fgf19. The present results suggest that crosstalk between Fgf16 signaling and Fgf19 and Hh signaling may be crucial for cell proliferation, regionalization, and cell type specification during forebrain development.In conclusion, the present results indicated that Figure S1fgf16 morphants. At 24 hpf, apoptotic cells in the brain of the wild-type (A) and fgf16 MO1-injected (B) embryos were marked via TUNEL. Lateral views with anterior to the left and dorsal to the top.Apoptosis in the brain of (TIF)Click here for additional data file.Figure S2fgf16 morphants.  Dorsal views of wild-type embryos (A) and fgf16 morphants (B), labeled to show CC1/APC immunoreactivity at 4.5 dpf.Oligodendrocyte differentiation in the hindbrain of (TIF)Click here for additional data file."}
+{"text": "Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the  Drosophila atonal gene was first identified in 1993 atonal orthologs, atoh1, is initially expressed in the sensory epithelium to later restrict in nascent hair cells atoh1 results in complete absence or ectopic differentiation of hair cells atoh1 genes are found in the inner ear, which act in two distinct phases of sensory development atoh1b appears in a broad prosensory territory to subsequently get restricted to smaller sensory domains. There, atoh1a is induced by atoh1b to direct the differentiation of hair cells Distinct proneural genes from the atonal basic helix-loop-helix (bHLH) superfamily control cell fate specification in the inner ear. atonal proneural family, neurog1, is used to define the otic neurogenic domain where otic progenitors will acquire a neuronal lineage Hes5 is induced and suppresses hair cell fate while promoting supporting cell fate Hes5 is expressed complementary to Delta1 expressing cells in the neurogenic domain Her4 is one of the zebrafish orthologs of mammalian Hes5 and it has been implicated in the control of primary neurogenesis, brain regeneration and neuronal target innervation her4 expression depends on Notch, while does not in the trigeminal sensory ganglia her4 in the inner ear, thus we aimed to study its function and regulation during neural and sensory specification. Here we show that zebrafish her4 is expressed in the neurogenic and sensory domains and requires neurog1 and atoh1b for its expression, respectively. Moreover, her4 in the neurogenic domain is dependent on Notch, while initial broad induction of her4 in the presumptive sensory domain does not require Notch but atoh1b. However, later on Notch restricts her4 expression to the future sensory maculae. Furthermore, we show that loss of her4 mediates lateral inhibition during neurogenesis, but not during sensorigenesis.Another member of ta52bhineurog11059y83her4 DNA containing 22 bp of 5\u2032 UTR sequence of her4 cDNA is controlling EGFP expression. Homozygous mutant mib and neurog1 embryos were obtained by pairwise mating of heterozygous adult carriers. mib descendant embryos were genotyped after in situ hybridization. Embryos were developed in an incubator at 28.5\u00b0C in system water containing methylene blue and staged after counting somite number. Embryonic stages are given as hours post-fertilization (hpf) at 28.5\u00b0C Zebrafish were maintained at the PRBB Animal Facility under standard conditions. The zebrafish protocols followed the guidelines and were approved by the IACUC, Comit\u00e9 \u00c9tico de Experimentaci\u00f3n Animal-Parc de Recerca Biom\u00e8dica de Barcelona (CEEA-PRBB); approved number JIB-08-1098P2. The zebrafish strains used were the following: wild-type (AB), mibin situ hybridization were performed as previously described atoh1b and atoh1aneurog1her4deltaBSynthesis of antisense RNA and whole-mount her4-MO her4 mRNA transcript with sequence: 5\u2032-ATT GCT GTG TGT CTT GTG TTC AGT T-3\u2032. Her4-MO was injected at concentration 0.025 mM and its efficiency was assessed by the specific loss of GFP signal from the Tg(her4:EGFP)y83 transgenic line atoh1b-MO MOs were obtained from Gene Tools. Embryos were injected at 1-cell stage. The Dechorionated zebrafish embryos were incubated with 50 \u00b5M SU5402 , a potent pharmacological inhibitor of Fgf signalling. Incubations were done at 28.5\u00b0C, starting at 10 hpf until the sacrifice of the animals at 16 hpf. The final solution was done in E3 medium from the 5 mM SU5402 stock solution (kept at \u221220\u00b0C in DMSO). For control treatments, embryos were incubated in an equivalent concentration of dimethyl sulfoxide .Pictures were acquired in a Leica DRM microscope or in Leica MZFLIII stereomicroscope using a Leica DFC300 FX camera and the Leica IM50 software. Adobe Photoshop 7.0.1 software was used for photograph editing.her genes in zebrafish, orthologous to mammalian Hes genes, consists of 21 genes based on data published on Ensembl (Zv9). We analysed the expression of 10 her genes during otic development and only her6, her4 and her9 were found to be expressed in the otic vesicle from 12\u201314 hpf to 24 hpf (data not shown). We have previously demonstrated a role for her9 during otic patterning in restricting neurogenesis to the anterior domain, downstream of retinoic acid (RA) signalling her4 gene. Previous studies have analysed the development of the neurogenic and sensory territories separately, however since both domains develop simultaneously, a complete picture of the development of both territories should be taken into account. Moreover, the sequential phases of Notch activation is still not well defined. Consequently, we have generated a precise map of the expression of her4 at early stages of neuro- and sensorigenesis together with the expression of atoh1b, atoh1a and neurog1. her4 expression is first observed at 12 hpf throughout a broad band of cells just adjacent to the hindbrain. The pattern at this stage is strikingly similar to the one from atoh1b (compare her4 is only detected at the future anterior (utricular) and posterior (saccular) sensory maculae with higher levels of expression than atoh1b .The family of  compare . By 13 h compare . At 16 h compare . atoh1a  onwards . At 20 hr macula  . Note that neurog1 expression begins in the neurogenic domain at 15 hpf but her4 expression is not initiated there until two hours later  compared to heterozygous embryos that retain her4 expression  as well as atoh1b (n\u200a=\u200a5/5) confirming that both have similar regulatory requirements  to assess the possible regulation of her4 by atoh1b. her4 expression precedes the onset of atoh1a in the otic placode, thus we reasoned that atoh1b instead of atoh1a was the best candidate for testing the upstream regulation of her4. Indeed, in atoh1b-MO injected embryos, expression of her4 is abrogated in the anterior and posterior sensory epithelia at 15 hpf .In order to distinguish between these possibilities, we have blocked t 15 hpf ; n\u200a=\u200a5/5 maculae ; n\u200a=\u200a5/5 compare '. The exher4 is a target of atoh1b in the sensory domain and depletion of atoh1b function has no effect on her4 neither on neurog1 expression in the neurogenic domain.In summary, her4 with the proneural gene neurog1 by using the hi1059neurog1 mutant line neurog1-/- mutants obtained from heterozygotic crosses of hi1059neurog1 adults, her4 is not expressed in the neurogenic domain at 24 hpf  line Her4-MO sequence binds specifically to the 5\u2032UTR affecting EGFP translation. In control embryos, strong EGFP is visible in the neural tube, while in 0.025 mM her4-MO injected embryos EGFP expression is completely abolished . In cross-sections, an expansion of neurog1 expression is also observed . As we have previously shown that atoh1b is upstream of her4, this result indicates that, as observed for neurog1, her4 does not feedback on atoh1b.We could expect a similar role of her4 becomes dependent of Notch, most probably participating in Notch-mediated lateral inhibition and repressing the differentiation of sensory progenitors to hair cells. If this hypothesis is true, we expected to find increased numbers of hair cells in her4-MO embryos. Intriguingly, the number of cells expressing atoh1a at 24 hpf and 28 hpf, as a readout of committed hair cells did not increase in her4-MO (atoh1a (n\u200a=\u200a5/13).We have seen above that at later stages  her4-MO  and was her4 in inner ear development and its relationship with proneural genes and Notch signalling. In the neurogenic domain her4 and neurog1 expressions are correlated spatially, with a temporal delay between neurog1 induction and her4, suggesting an intermediate step. The fact that in the neurogenic domain her4 expression depends on Notch, positions Notch as the intermediary pathway that activates her4 downstream of neurog1. Moreover, depletion of her4 leads to an increase in the population of neurons. This is similar to what was previously reported for her4 role in primary neurogenesis her4 nor Hes5 has already been analysed directly in inner ear neurogenesis . The data demonstrate for the first time that in the inner ear, as in the CNS, her4 participates in Notch-mediated lateral inhibition to control the final number of neuronal cells.Here we have explored the role of her4 is regulated differently. In the presumptive sensory territory her4 expression is highly dynamic and identical to atoh1b. It initially encompasses a broad medial territory of the otic placode to progressively restrict to the future anterior and posterior maculae. Initial her4 expression requires atoh1b and Fgf signalling but not Notch, indicating that it cannot be assumed that her4 is always regulated by Notch. Nonetheless, in an intermediate developmental period, in the CMD Notch regulates negatively but not positively her4 in the CMD. Our work thus shows that her4 is not the downstream target of Notch to repress atoh1b expression. her6, another member of her repressors, cannot perform this role since is not expressed in the CMD at 12.5\u201313 hpf y83 embryos and also it resulted in expansion of deltaB expression. One of the most plausible explanations is that contrary to what happens in the neurogenic domain, other her genes compensate for her4 loss in the sensory domain. Her6, a Hes1 ortholog, is expressed exclusively in the sensory domain already from 12.5 hpf Click here for additional data file."}
+{"text": "With gold nanoparticles (AuNPs) is possible to develop nanoscale devices that can interact with chemical and biological systems. The phenomenon explored in these nanosystems is called Localized Surface Plasmon Resonance (LSPR), which promotes electromagnetic wave oscillation electronics on these small metallic structures. It is interesting to note that this resonance is directly linked to the size of the nanoparticles, the nature of the dielectric material and support environment where the device is being studied ,2.3C6H5O7) and Sodium Borohydride (NaBH4). Was expected to demonstrate the viability of these two reducing agents and highlight the potential differences obtained in each of the mechanisms. Since knowledge is the ability of stabilizing citrate ions and the strong reducing action of NaBH4 [This work makes a comparison between results obtained in the synthesis of AuNP's reduction method using a Sodium Citrate NaC6H5O7 an4) 2.5 \u00d7 10-4 M, 5 mL of 1% NaBH4 and 5 mL of Na3C6H5O7 also 1%.In these experiments consisted initially in the preparation of the following aqueous solutions: 100 mL Tetrachloroauric Acid  .4 did not generate nanoparticles with a reasonable size for the LSPR occur. Phenomenon responsible for the peak at 530 nm in the spectrum of sodium citrate (Na3C6H5O7).The UV-Vis spectrophotometry shows the absorption of the samples obtained in experiments, it is noted that the easy synthesis with NaBH4 lot nuclei initiators nanoparticles were generated, but reduced in size. Na3C6H5O7 already has a good ability to stabilize, thus required temperature rise in the synthesis process to increase capacity reduction. Nuclei being formed ions favor the stabilization phase of growth, thus generating nanostructures favorable process RPSL to 530 nm.Due to capacity reduction of NaBH"}
+{"text": "Intratumoral heterogeneity of HER2 expression is common in gastric cancers and pose a challenge for identifying patients who would benefit from anti-HER2 therapy. The aim of this study is to compare HER2 expression in biopsy and resection specimens of gastric carcinoma by immunohistochemistry (IHC) and to find the ideal number of biopsy tumor fragments that can accurately predict HER2 overexpression in the corresponding surgically resected specimen. The HER2 IHC results of 702 paired biopsy and resection specimens of gastric cancer were compared.P = 0.0315 and P = 0.0052). ROC curve analysis and positive predictability showed that 4 fragments should be obtained to minimize the differences in HER2 scores between biopsy and resection specimen.The mean number of biopsy fragments among all cases was 4.3 (range 1\u201311). HER2 was positive in 130 (18.5%) endoscopic biopsies and in 102 (14.5%) gastrectomy specimens. Intratumoral heterogeneity of HER2 was found in 80 (61.5%) biopsies and 70 (68.6%) resection specimens. Out of the 70 surgical specimens with intratumoral heterogeneity, 24 (34.3%) of the corresponding biopsies were categorized as negative (positive conversion). In the 86 (12.3%) discrepant cases, negative conversion was observed in 57 (66.3%) cases and positive conversion in 29 (33.7%). The fragment numbers were significantly correlated with the discrepancy of results and positive predictability (In gastric carcinomas with discrepant HER2 results between biopsy and surgical resection specimens, intratumoral heterogeneity is common with most of them showing positive conversion. To predict HER2 status precisely, at least 4 biopsy fragments containing tumor cells are required. Since its first expression characteristics were described in gastric cancer in 1986, human epidermal growth receptor 2 (HER2) has become an established predictive biomarker in this disease , 4. Receet al [However, determining HER2 expression status in the practical setting presents a few challenges. One challenge is due to tissue sampling in biopsy specimens compared with the resection specimens. To ameliorate diagnostic accuracy and reduce discordance between biopsy and resection specimens, sufficient biopsy material is required. Several studies have compared HER2 results in paired biopsy and resection specimens and showed an overall concordance rate varying between 87 to 96% . Severalet al . have reet al  has propAnother challenge associated with interpreting HER2 status in gastric cancer is the protein's affinity for being heterogeneously expressed. High incidence of HER2 heterogeneity is found in up to 79.3% of HER2-positive gastric cancers within the same tumor  . IntratuOnly patients with primary gastric carcinoma who underwent both preoperative endoscopic biopsy and gastric cancer resection at Samsung Medical Center in Seoul, Korea from January 2013 to December 2014 were selected for this study. Patients who underwent preoperative chemotherapy and/or radiotherapy, or patients who were diagnosed with multiple gastric cancers were excluded. The clinicopathologic characteristics of 702 patients are described in Table A tumor fragment was defined as a piece of tissue containing 10 or more viable tumor cells in an endoscopic biopsy specimen as previously described .IHC for HER2 (PATHWAY HER-2/neu (4B5) rabbit monoclonal antibody, Ventana Medical Systems, Inc., Tucson, AZ) was performed in all cases with a BenchMark XT automated stainer . In operation specimens, we reviewed all hematoxylin and eosin stained tumor sections and selected a representative tumor block for IHC analysis. A gastrointestinal pathologist (KMK) evaluated the staining data with no previous knowledge of clinical or pathological parameters. HER2 IHC was scored according to the recently developed assessment guidelines for HER2-associated gastric cancers . In biopT-test was used to compare the number of total fragments between concordant (between biopsy and resection results) and discordant groups as well as between the biopsy positive and negative groups. Linear regression method was used to associate the difference in HER2 score between biopsy and resection specimen with the number of total fragments. A ROC curve was constructed to find a cutoff value for the number of total fragments. These analyses were conducted using SAS version 9.4  and R 3.1.1  by biostatisticians (CS and JSH).A two-sample n = 616, 87.7%) between biopsy and resection specimens, 86 (12.3%) paired specimens showed discrepant results. Of these discrepant cases, negative conversion (positive in biopsy and negative in resection) was found in 57 (66.3%) cases, and positive conversion was seen in 29 (33.7%) cases. After exclusion of equivocal 2+ cases, [The IHC results of 702 paired biopsy and gastrectomy specimens were analyzed . Actually, for six cases with HER2 3+ in biopsies but negative on operation specimen, number of biopsy fragments were 2 (n = 1), 3 (n = 2), 4 (n = 2) and 5 (n = 1) and all five cases with > 2 biopsy fragments showed intratumoral heterogeneity within biopsy specimens. In those 6 cases, we further performed IHC using all tumor blocks from operation specimen and found 2+ in four cases and 3+ in two cases with positive tumor cells ranging from 1% to 10% of total tumor volume. Moreover, cases with heterogeneity in either biopsy or operation specimens showed higher discrepant results compared to cases without heterogeneity  . Out of 102 HER2-positive operation specimens, 70 cases (68.6%) showed heterogeneity, in which 24 (34.3%) showed discrepancy (positive conversion). Heterogeneity found in biopsy specimens was significantly correlated with its surgical operation specimen (n = 159). This value was termed the \u201cdifference value\u201d. For all paired specimens with a difference value of 0 (no difference between biopsy and resection HER2 status), the mean fragment count was 4.9 (n = 50). For the paired specimens with a difference value of 1, 2, and 3, the mean fragment count was 4.9 (n = 53), 4.3 (n = 47), and 2.9 (n = 9), respectively. Additionally, the number of biopsy fragments was significantly correlated with the difference value (P = 0.0096)  within the HER2-positive group was calculated  Figure . Hence, n = 63) in either biopsy or resection specimen were evaluated. HER2 equivocal (2+) cases were excluded to improve the precision of the calculation. Out of 63 cases, 8 cases showed positive conversion (negative in biopsy and positive in gastrectomy). The number of fragments in all positive conversion cases ranged from 1 to 5 . According to ROC analysis, the ideal cut-off value was 3 with an AUC of 0.8045 (Figure \u2018Positive predictability\u2019 was defined as the positive predictive value of HER2 in biopsy specimens. To assess the positive predictability in relation to the biopsy fragment numbers, fragment numbers within only the HER2 3+ positive group a n = 3 in eith5 Figure . TherefoAccurate prediction of HER2 status in endoscopic biopsies of gastric cancer is essential for accurate tumor characterization and also carries important therapeutic implications. However, the heterogeneity of HER2 overexpression in gastric cancer contributes to false-negative results in cases with limited biopsy material suggesting the necessity of extensive tissue sampling . To our Intratumoral heterogeneity is the major underlying cause of discrepant HER2 results between the biopsy and resection specimens \u201313, 15. In this study, the positive predictability of the biopsy specimen was also calculated. True positive cases were determined as HER2 3+ either in the biopsy or resection specimens. Out of 63 HER2 3+ cases, 12.7% of cases were negative in the biopsy specimen and positive in the resection specimen. Interestingly, all cases with positive conversion showed intratumoral heterogeneity in the surgical specimens confirming that heterogeneity is the major reason for the discordant results. In cases with positive conversion, the number of biopsy fragments ranged from 1 to 5. Endoscopic biopsies with six or more fragments showed 100% HER2 status correlation with the surgically resected specimen. In our statistical analyses, a minimum of 4 biopsy fragments was needed to accurately predict HER2 status in the resected specimens.In summary, due to significant intratumoral heterogeneity, the larger the number of endoscopic biopsy fragments available for HER2 IHC analysis in gastric cancer, the higher the correlation with HER2 status will be in the resection specimen. The discordance increased with smaller numbers of biopsy fragments. This warrants some caution in relying on HER2 IHC findings of endoscopic biopsy specimens alone to determine treatment regimens.Finally, we suggest obtaining at least 4 biopsy fragments containing cancer in endoscopic biopsy for accurate HER2 test and recommend to record tumor fragments number in HER2 IHC pathologic report."}
+{"text": "Since 2003, all nursing staff at HUG follow a mandatory course in infection control in the context of an institutional program. As part of a quality improvement pilot program we implemented a knowledge assessment tool in 2 units of a 300-bed geriatric hospital.We aim to assess the nursing staff\u2019s theoretical knowledge of infection control and its application in everyday practice.During the first period (01-04/2014) all nurses and nursing assistants in the 2 participating units were assessed for their theoretical knowledge of infection control during a 30-minute interview using a predefined questionnaire with 12 items. During the same period adherence to hand hygiene was measured based on the WHO framework. During a second period (4-6/2014) individual and group-level feedback about knowledge scores and hand hygiene adherence was provided. Individuals with suboptimal performance in either domain were targeted for individual training sessions. During a third period (6-12/2014) hand hygiene compliance and the implementation of infection control measures was audited.st phase increased from 59 to 98 %. 14 infection control measures were audited and all fulfilled the predefined criteria for adequacy.21 nurses and 13 nursing assistants were assessed during the 1st period. None remembered to have received training in infection control. 5 caregivers reached the maximum knowledge score and had hand hygiene adherence > 80%. 5 caregivers reached the maximum knowledge score but had a hand hygiene compliance < 50%. 1 caregiver reached low scores both with regard to knowledge and hand hygiene compliance. During the 3rd period overall hand hygiene compliance of 6 randomly selected caregivers having participated in the 1Despite a long institutional culture of patient safety and infection control only 15% of the nursing staff had very good theoretical knowledge of infection control and were able to implement this knowledge into good adherence to hand hygiene. This quality improvement pilot program made the whole team reflect on their practices and made it possible to identify caregivers in need of individualized training.None declared."}
+{"text": "Cell motility is an actin dependent process requiring the formation and extension of lamellipodia or filopodia, and is absolutely essential for many cellular processes, especially for morphogenesis during development. During lamellipodial extension, actin dynamics involve switching between branch formation at the leading edge and proximal severing of existing actin filaments . Actin bWhat is known is that Arp2/3 dependent actin assembly and Cofilin dependent disassembly are coordinately regulated at the leading edge by Coronin 1B, which binds the Arp2/3 complex in a phosphorylation-dependent manner. When dephosphorylated, Coronin 1B inhibits the binding of the Arp2/3 complex to actin, but phosphorylation by protein kinase C at Ser2 reduces its association with the Arp2/3 complex, enabling actin branch formation [Cofilin is also regulated by phosphorylation but, in contrast to Coronin 1B, it is inactivated by phosphorylation, and becomes active through Slingshot phosphatase (SSH) dephosphorylation . The firOur recent study has revealed two new important players in cellular motility. We have found that WISp39 (Waf1 Cip1 stabilizing protein 39), an Hsp90 binding protein we had discovered , coordinUsing biochemical approaches to dissect the interactions of WISp39, phospho-Coronin 1B, SSH and Hsp90, we have demonstrated that WISp39 may be the scaffold that orchestrates the activity of key regulators of actin dynamics  to maintain lamellipodia protrusion and directed motility as shown in Figure We have previously demonstrated that WISp39 interacts with cyclin-dependent kinase inhibitor p21 and Hsp90 in a trimeric complex that stabilizes p21 against degradation . That stOur data support a key role for WISp39 in regulating actin dynamics to sustain directed cell motility. We suggest that WISp39 and its binding partner Hsp90 act as a crucial scaffold that integrates Coronin 1B, SSH and Arp2/3 complex at the leading edge."}
+{"text": "Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene  Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation. Oct4, Nanog, and Sox2, during establishment of EPI cells in the blastocyst Sox2 in the blastocyst are unresolved. For example, several studies reported that Sox2 is restricted to the ICM by the blastocyst stage Sox2 expression in the blastocyst are unknown.To create and use pluripotent stem cells, it is essential to understand the origins of pluripotency during normal development. During mouse blastocyst formation, pluripotent epiblast (EPI) cells are established by two cell fate decisions that segregate pluripotent progenitors from extraembryonic tissues Sox2 expression is patterned, the functional roles of Sox2 in the blastocyst are not yet clear. ES cells cannot be derived from embryos lacking zygotic (Z) Sox2Sox2 is essential for pluripotency. In ES cells, Sox2 is required for the expression of pluripotency genes, such as Oct4 and Nanog, and for the repression of TE genes Sox2 might be required for initial expression of pluripotency genes and repression of TE genes in the ICM. However, the expression of pluripotency and TE genes in Sox2 Z null blastocysts has not yet been examined at the level of individual cells. Moreover, maternal (M) Sox2 is also thought to participate in blastocyst formation, which could partially compensate for loss of Z Sox2. RNAi knockdown of M and Z Sox2 in the zygote was reported to disrupt blastocyst formation Sox2 during development.In addition to the unresolved mechanism by which Sox2 mRNA is enriched in ICM progenitors starting at the 16-cell stage Sox2 is restricted to ICM progenitors. Using immunofluorescence and confocal microscopy, we observed that SOX2 is restricted to nuclei of ICM progenitors at the 16-cell stage and later  analysis of oocytes from fl/fl or delZp3Cre; Sox2 females  and ICM (OCT4) markers was normal in the absence of MZ Sox2  to E3.75  or reduced (1/5 Sox2 null embryos), expression of OCT4 was undetectable (1/5 Sox2 null embryos) or reduced (3/5 Sox2 null embryos), and expression of NANOG was also undetectable (1/1 Sox2 null embryos)  . These ome point . We concSox2 mRNA in ICM progenitors Sox2 in regulating cell fate does not become apparent until late blastocyst stage. It is possible that Sox2 is initially genetically redundant with other pluripotency factors, such as Oct4 or Nanog. Although phenotypes resulting from disruption of multiple pluripotency genes have not yet been reported in mice, there is evidence of genetic redundancy among zebrafish Oct/Sox/Nanog orthologues Fgf4 expression is reduced, but not eliminated, in the absence of either Sox2 or Oct4Fgf4, and possibly other targets, synergistically, as has been demonstrated in pluripotent stem cell lines Here we have examined the roles and regulation of SOX2 in the preimplantation embryo, with the goal of deepening our understanding of the origins of pluripotency during development. We showed that SOX2 is a unique, early marker of ICM progenitors, consistent with the reported early enrichment of Sox2 directly or indirectly. In ES cells, the YAP/TEAD complex been shown to bind upstream of Sox2 to promote its expression Sox2 in TE cells. It will be exciting to address this hypothesis in future studies in addition to examining whether position, polarity, and/or contact regulate Sox2 expression, as has been shown for Cdx2Sox2 and Cdx2 in the embryo. Finally, our observations are also consistent with LATS regulating the activity of an as-yet unidentified transcription factor that promotes expression of SOX2 in ICM progenitors. This hypothesis is also supported by qPCR evidence that Lats1/2 maintains expression of Sox2 in the blastocyst Our evidence suggests that SOX2 and CDX2 are patterned by HIPPO pathway components in parallel , but it Sox2 is activated de novo in the trophoblast lineage postimplantation, where it promotes trophoblast development Sox2 expression postimplantation as well. Investigation of the mechanisms by which Sox2 expression becomes activated in the extraembryonic ectoderm is an exciting opportunity to learn about the origins of trophoblast stem (TS) cells, which are derived from extraembryonic ectoderm Sox2 and Tead4Our study provides insight into the regulation of extraembryonic cell types during preimplantation development. In terms of the TE lineage, we showed that SOX2 is not detected in TE cells during preimplantation, nor is it required for their specification. These observations suggest that expression of While SOX2 does not activate TE gene expression, SOX2 also does not appear to repress TE gene expression in the ICM. This is in contrast to OCT4, which is known to repress expression of TE genes in the ICM and in ES cell lines Nanog null embryos has not yet been reported, in Gata6 null embryos, SOX2 is expressed in all ICM cells Gata6 and Nanog null embryos, with and without manipulations to the FGFR/MEK signaling pathway will help clarify the direct and indirect mechanisms regulating Sox2 expression in the ICM.Our analysis led us to explore the genetic regulation of PE specification as well. We have shown that in the ICM, SOX2 becomes expressed in a salt-and-pepper fashion, similar to NANOG. We have also shown that the salt-and-pepper distribution of SOX2 in the ICM depends on FGFR/MEK signaling, but it is not yet clear whether FGFR/MEK signaling regulates SOX2 expression directly, or whether FGFR/MEK signaling maintains cell fate, which in turn regulates SOX2 expression. Alternatively, NANOG or GATA6 could help regulate SOX2 expression in the ICM. While the expression pattern of SOX2 in Sox2 null embryos do not completely phenocopy Fgf4 null embryos, since NANOG was not upregulated in Sox2 null embryos as it is in Fgf4 null embryos Fgf4 in Sox2 null embryos are sufficient to repress NANOG, as we have shown for Oct4 null embryos Sox2 null embryos as has been observed in Fgf4 null embryos Sox2 null embryos is eventually able to restore expression of PE genes 93Knwtm1HsskTead4tm1.1AralCdx2del+Sox2) were generated by crossing mice carrying tm1.1LanSox2 with tm1(cre)Nagy129-AlplAll animal research was conducted in accordance with the guidelines of the University of California Santa Cruz Institutional Animal Care and Use Committee or by RIKEN CDB and Kumamoto University. The following alleles or transgenes were used in this study: 2. Microinjection of mRNA was performed as described As described previously Embryos were fixed, stained, imaged, and recovered for genotyping as previously described Sox2 MZ null embryos as previously described Sox2 alleles Chimeras were performed using Sox2 (GCGGAGTGGAAACTTTTGTCC and CGGGAAGCGTGTACTTATCCTT), Fgf4 (AGCAGGGGCAAGCTCTTC and GGGTACGCGTAGGATTCG), Oct4 (AGCTGCTGAAGCAGAAGAGG and AGATGGTGGTCTGGCTGAAC), and Actb (CTGAACCCTAAGGCCAACC and CCAGAGGCATACAGGGACAG).RNA isolation and single blastocyst qPCR was performed as previously described Figure S1Sox2, the anti-SOX2 antibody non-specifically detects a cytoplasmic pattern in TE cells, while the signal is specific to ICM at the blastocyst stage. B) SOX2 is detected in an increasing proportion of inside/ICM cells during formation of the blastocyst, with nearly 100% of ICM cells expressing SOX2 by E3.5. C) SOX2 does not colocalize with CDX2 in TE cells of the blastocyst, although SOX2 colocalizes with rare inside cells that express CDX2 , consistent with our prior observations that CDX2 is detected in rare inside cells at this stage Details of SOX2 expression pattern in the early embryo. A) In morulae lacking (EPS)Click here for additional data file.Figure S2Sox2 M null evaluated as a negative control), M Sox2 mRNA is not detectable (*) in individual wild type 2-cell embryos by qPCR , and M SOX2 is not detectable in nuclei of Sox2 Z null embryos at the 16-cell stage . B) At E4.5, neither CDX2 nor EOMES are detected in the ICM of Sox2 null embryos. Bar \u200a=\u200a20 \u00b5m.M Sox2 is not detectable in early embryos, and SOX2 does not repress TE fate in the ICM. A) M SOX2 protein is not detectable in wild type zygotes ((EPS)Click here for additional data file.Figure S3Sox2 acts upstream, and not downstream of Fgf4. A) qPCR analysis of single blastocysts at E3.75 shows that M Sox2 is not required for Fgf4 expression. B) Treatment of control (Sox2 M null or nonmutant) or Sox2 null (MZ or Z null) embryos for 42 h starting at E2.75 leads to complete upregulation of SOX17 and downregulation of NANOG throughout the ICM. C) Quantification of embryos shown in panel B . Bar \u200a=\u200a20 \u00b5m, p-value calculated by t-test; n.s. \u200a=\u200ap>0.05; ANOVA performed for panel C.Z and not M (EPS)Click here for additional data file.Figure S4Sox2 null blastocysts. B) Average ICM cell number for time points examined in Sox2 null blastocysts at the equivalent of E4.25. D) Expression of DAB2 is eventually rescued in Sox2 null implantation-delayed blastocysts. The number of EPI cells is significantly reduced in Sox2 null implantation-delayed blastocysts, relative to wild type. By contrast, the number of PE cells is not significantly reduced in Sox2 null implantation-delayed blastocysts, relative to wild type, until the last time point examined. EPI and PE were defined based on SOX17 expression, since other EPI markers are not detectable in Sox2 null blastocysts at this stage. Bar \u200a=\u200a20 \u00b5m, p-value calculated by t-test, n.s. \u200a=\u200ap>0.05.Evaluation of PE quality in late blastocysts. A) At E4.25, the average proportion of ICM cells in which GATA6 is detected is equivalent between control and (EPS)Click here for additional data file.Table S1Cell numbers detected in wild type embryos harvested at the indicated times (E3.0\u2013E4.5).(DOCX)Click here for additional data file."}
+{"text": "Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle. Somatic cell fusion events are critical for development of multicellular eukaryotic organisms. Postnatal growth and repair also frequently rely on somatic cell fusion events, especially in tissues such as skeletal muscle that contain multinucleated cells. However, the genes and molecular mechanisms underpinning this fundamental cellular process in humans are largely unknown. Multiple cell types including trophoblasts, osteoclasts and skeletal muscle require somatic cell fusion events in order to perform their physiological functions \u20133. The oIn humans, a single skeletal muscle fibre can contain thousands of nuclei . Each nuResearch findings from Drosophila have highlighted that key events in the muscle cell fusion process are cell-cell attraction, adhesion and subsequent actin nucleation at sites of cell-cell adhesion, the latter enabling membrane fusion \u201317. A wiPresent within the human genome are three Kirre homologs termed Kin of Irre like (Kirrel) 1 ,2 and 3 . This faOur primary aim was to assess whether Kirrel3 is present in uninjured and regenerating human skeletal muscle following mild damage-inducing exercises such as plyometric jumping and downhill running. Presence of Kirrel3 in the afore mentioned samples would raise questions about its function in skeletal muscle. Currently nothing is known about Kirrel3 in this tissue, or if it is indeed present in adult human skeletal muscle. While its presence alone would not confirm a role in the human muscle cell fusion process, it would provide initial support for further investigation. It is possible that it could mirror the role of Kirre, its Drosophila homolog, in regulating myoblast fusion events in adult human muscle.Our research findings demonstrate that at least three alternative splice variants of Kirrel3 are present in adult human skeletal muscle. Two of these splice variants have not been previously reported in the published literature. Detection of Kirrel3 mRNA in adult human skeletal muscle using standard PCR was sporadic with occasional detection in uninjured and regenerating skeletal muscle samples. Semi nested PCR increased the detection rate. Such sporadic detection, using standard PCR assessment, demonstrates that in adult human skeletal muscle Kirrel3 mRNA is present at very low levels. At the protein level, Kirrel3 immunoreactive proteins in uninjured and regenerating adult human skeletal muscle were observed. Further work is now required in order to ascertain the function of all Kirrel3 splice variants in human skeletal muscle and to provide more insight into stimuli promoting its expression and its subsequent post-transcriptional regulation.th exon 641 nucleotides 3\u2032 to the missed spliced site in B. The 3\u2032 untranslated regions of Kirrel3 A and B are 1079 and 370 nucleotides respectively.Analysis of the National Centre for Biotechnology (NCBI) gene database highlighted the presence of two human Kirrel3 reference sequences NM_032531.3 (hereafter referred to as Kirrel3 A) containing 3777 nucleotides and NM_001161707.1 (hereafter referred to as Kirrel3 B) containing 2534 nucleotides. Aligning Kirrel3 A and B mRNA transcripts to the human genome via DNA sequence present in the genomic contig NT_033899.8, demonstrated that Kirrel3 A has 17 exons while B has 14 exons , five extracellular Ig domains  and a transmembrane domain (AA 536-558 http://www.cbs.dtu.dk/services/TMHMM/). Five putative N-linked glycosylation sites  are predicted to be present in the extracellular domain of both Kirrel3 proteins. The intracellular domain of Kirrel3 A is predicted to contain an amphysisin SH3 binding domain (AA 759-773 http://scansite.mit.edu/). Also present at its C-terminal end is a Post Synaptic density protein 95, Drosophila discs Large tumour suppressor, zonula occludens (PDZ) binding domain corresponding to the amino acids THV [Kirrel3 A is the larger of the two Kirrel3 proteins with 778 amino acids (AA) while Kirrel3 B has 600 AA. The first 565 AA of Kirrel3 A and B are identical. Domains predicted within this region  demonstrated that they were all variants of Kirrel3  study by our group  were utiSkeletal muscle is made up of multinucleated cells (muscle fibres). The Drosophila homolog of Kirrel3, Kirre is involved in enabling muscle cell fusion events. Hence, Kirrel3 may be a putative muscle cell fusion regulator in humans. There is currently no published data available regarding Kirrel3 in adult human skeletal muscle. Therefore, the primary aim of this research was to assess for the presence of Kirrel3 in adult human skeletal muscle. By using PCR and transcript sequencing, we provide the first evidence that three alternatively spliced Kirrel3 mRNA transcripts are present in adult human skeletal muscle. Results from western blot analysis provide support for the presence of Kirrel3 protein in adult human skeletal muscle.The mRNA data highlight that in adult human skeletal muscle Kirrel3 mRNA expression levels are very low and that using standard PCR may result in false negatives. A nested PCR approach yielded a higher rate of Kirrel3 detection. It will be of interest for future studies to examine Kirrel3 mRNA expression in a model that is known to include a large amount of myogenesis such as can be seen in some muscle pathologies. Primary human myoblast cell cultures would provide a useful system to further investigate Kirrel3 and ascertain its importance to human muscle cell fusion. Murine proprioceptive neurons have been reported to express Kirrel3 , therefo-min [The physical process of transcribing Kirrel3 can be regarded as a specialised event because, compared to the vast majority of other genes within the human genome, Kirrel3 spans a very large genomic region of approximately 580kB. Such a long genome span is likely to impact on the rate of Kirrel3 mRNA transcript production. RNA polymerase II has been reported to be capable of transcribing large human genes at a rate of approximately 4\u00a0kb-min . This ra-min , while t-min .Results from our western blot experiments varied depending on the commercial antibody being used. Each antibody targeted different epitopes with one being extracellular and the other intracellular. The fact two immunoreactive proteins that migrated at approximately 70 and 75\u00a0kDa were detected by both antibodies, appears to support that these are Kirrel 3 proteins rather than breakdown products. However, the predicted molecular weight of Kirrel3A is 85\u00a0kDa therefore the Kirrel proteins within the 70\u00a0kDa range are likely to be one or two of the other Kirrel3 splice variants present in human skeletal muscle. It is possible that they represent one splice variant with the larger having undergone post-translational modification. Immunoprecipitation and mass spectrometry analysis will be useful in determining the identity of these immunoreactive proteins.The proteins detected at 50 and 55\u00a0kDa were detected only when using the Kirrel3 antibody targeting the intracellular domain. This antibody may be recognising partially degraded Kirrel3 proteins or these could be alternative truncated isoforms.For analysis of mRNA, our primers were directed towards the 3\u2032 end region of Kirrel3 (intracellular coding region) and we can only speculate on whether or not alternative splicing may occur further downstream towards the 5\u2032 end (extracellular coding region) that would produce even smaller Kirrel3 protein isoforms. If alternative splicing occurs at the 5\u2032 end this could explain why the extracellular targeting antibody did not detect proteins of similar size as those detected by the intracellular targeting antibody. In future, we suggest that 5\u2032 race experiments should be performed to provide useful information in evaluating this possibility.Our rationale for investigating Kirrel3 in human skeletal muscle was derived from research studies in Drosophila that demonstrated the involvement of two genes, Kirre and Rst, in the muscle cell fusion process during embryonic development . While o-/- murine myoblasts displayed reduced fusion capabilities during in vitro myogenesis [In vitro, the extracellular domain of Kirrel3 is capable of binding to the extracellular domain of nephrin, a type 1 transmembrane protein and member of the immunoglobulin superfamily . Nephrinogenesis .Kirrel3 is part of the Kirrel gene family that contains another two structurally similar members: Kirrel and Kirrel2 . In DrosThe splice variants, Kirrel3 A and B, that are present in adult human skeletal muscle and/or astrocytes are predicted to contain significantly different cytoplasmic domains. Kirrel3 A, but not Kirrel3 B, is predicted to contain a SH3 amphysisin binding domain and also a PDZ binding domain. Such differences are likely to result in the two isoforms having divergent functions. Amphysisin is a protein whose function is not completely understood, however, it is highly concentrated at nerve terminals . The C-eThe PDZ binding domain present in Kirrel3 A may confer Kirrel3 with the ability to alter the polarity of the cell in which it is expressed as the PDZ binding domain of human Kirrel3 is capable of binding to the cell polarity protein partitioning defective 3 (PARD3) . SatelliIn conclusion, the results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 present in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies. Full length sequence information should be obtained on all Kirrel3 mRNA transcripts in order to predict the translated Kirrel3 proteins. Physiological studies should be done to confirm involvement in fusion, or not. Production of isoform specific anti Kirrel3 antibodies will enable identification of the cellular localisation of the different Kirrel3 isoforms. Obtaining such information will aid in our understanding of Kirrel3 and its function in human skeletal muscle.Healthy young men aged between 18-28 years of age volunteered to participate in one of two studies aiming to describe molecular and physiological responses to exercise-induced muscle damage. Participants were informed about the purpose and risks of the study in which they were to participate before signing an informed consent document. The experimental protocols were approved by the Committee for Human Research at Stellenbosch University and the studies were conducted according to the ethical guidelines and principles of the International Declaration of Helsinki.Participants were first taught how to perform the squat jump exercise. Subsequently their maximum squat jump height was measured. For the squat jump exercise intervention participants preformed 100 squat jumps at 90% of their maximum jump height. Jumps were divided into sets of 10 with 1\u00a0minute rest interval between each set. Prior to performing the 100 squat jumps participants exercised at a light to moderate intensity for 5\u00a0minutes on a treadmill.2max, 10% decline) on a motorized treadmill. They were allowed a 2\u00a0min standing rest between bouts and all the participants were able to complete all twelve bouts.The protocol used here was previously described . In brievastus lateralis muscle using a 5\u00a0mm trephine biopsy needle with assisted suction. Baseline biopsies were obtained from participants who had not engaged in any strenuous physical activity 7\u00a0days prior to the biopsy. Biopsies from participants who had performed the plyometric jumping protocol were obtained 4 and 24\u00a0hrs post completion of the protocol. Baseline and 24\u00a0hr biopsies were taken from the right leg while the 4\u00a0hr biopsy was taken from the left leg. Similarly, biopsies from participants who completed the downhill running protocol were taken at baseline and one and two days post downhill running in a consistent manner. Biopsies were frozen in liquid nitrogen cooled isopentane and stored at -80\u00b0C until use.Muscle biopsies were obtained from the 5 in 6 well plates (BD Bioscience) in growth media which contained 89% Dulbecco\u2019s Modified Eagle Medium, 1% N-2 Supplement 100X, 10% Fetal Bovine serum . Once cells had reached 70% confluence they were lysed for RNA and protein isolation. Growth media was removed from wells before 200 ul of Tripure (Roche) was added per well of a 6 well plate and incubated at room temperature for 5\u00a0minutes with occasional gentle agitation. For protein isolation 150 ul of protein lysis buffer was added per well and incubated on ice for 5\u00a0minutes with occasional gentle agitation.Human astrocytes  were seeded at a density of 10Primer set 1 \u2013 Forward primer GCCGACTTCCAGACCATCTA, Reverse primer \u2013 TTTGAGGACCTCCAGCTGTT, Primer set 2 - Forward Primer CGCTATACGGTGGAGACCAT, Reverse Primer Same as primer set 1. Primer set 3 \u2013 Forward primer same as primer set 2, Reverse Primer \u2013 CGCTTTTCCCCCTATCTTTC. Amplicons were excised from the agarose gel, purified and sequenced at the central analytical facility at Stellenbosch University. For GAPDH primer set used was Forward primer \u2013 AATCCCATCACCATCTTCCA, Reverse primer - TGACAAAGTGGTCGTTGAGG.Muscle biopsies were sectioned on a cryostat (Leica Bio systems) at -20\u00b0C to obtain approximately 20\u00a0mg of tissue. Samples were then homogenised on ice in 1\u00a0ml of Tripure (Roche). RNA was isolated according to manufacturer\u2019s guidelines and suspended in TE Buffer and stored at -20\u00b0C until use. For reverse transcription (RT) 1ug of RNA was DNAse treated according to manufacturers (Roche) guidelines. Subsequently the 1 ug of DNAse treated RNA was reverse transcribed using random hexamers in accordance with manufacturers (Roche \u2013 Transcriptor First strand cDNA synthesis kit) guidelines. For PCR a 2 ul aliquot of cDNA corresponding to 50\u00a0ng of RNA was used. Each PCR consisted of a total volume of 25 ul. Primers (Sigma Life Science) were used at a 1 uM concentration and remaining components of PCR followed manufacturers (Roche \u2013 Faststart PCR Master) recommended protocol. For semi nested PCR a 2 ul volume from the initial PCR was used in the second PCR instead of 2 ul of cDNA. Post PCR the 25 ul volume was mixed with 6 ul of loading buffer  and loaded onto a 1% agarose gel which contained sybr safe (Life Technologies) at 1X concentration and electrophoresed alongside a 100\u00a0bp ladder (Life Technologies). Gels were visualised on a Chemidoc MP (Biorad) that was supported with Image Lab software (Biorad). Kirrel3 Primer sets used were as follows (BCA) kit (Thermo Fischer Scientific) using Bovine serum albumin (Roche) as standards. 30 ug of protein lysate was mixed with 5\u00d7 Lamelli buffer  to yield 1\u00d7 and electrophoresed using the Mini Protean Tetra system (Biorad). Post electrophoresis proteins were transferred to a nitrocellulose membrane  via Trans Blot Turbo (Biorad) and membranes were stained with ponceau S to confirm transfer and equal protein loading. Subsequently membranes were washed 3 times for 5mins with 1\u00d7 TBST  and then blocked for 1\u00a0hr at room temp in 1\u00d7 TBST and 5% semi skimmed milk. The intracellular targeting primary rabbit anti human Kirrel 3 antibody (102960 Abcam Cambridge UK) and the extracellular targeting primary sheep anti Kirrel 3 antibody (AF4910 R and D systems Minneapolis USA) were incubated over night at room temp with gentle agitation in 1\u00d7 TBST with 5% semi skimmed milk. For blocking peptide experiment intracellular targeting primary anti human Kirrel 3 antibody was incubated with blocking peptide at a ratio of 1:4 for 3\u00a0hrs at room temperature with gentle agitation. Post primary antibody incubation or antibody and blocking incubation membranes were washed 3 times for 5 mins and subsequently incubated either with goat anti rabbit secondary  at 1:15,000 or donkey anti sheep HRP conjugated secondary ( 6900 Abcam Cambridge UK) at 1:30,000 for 1\u00a0hr at room temp in 1x TBST and 5% milk. Membranes were subsequently washed 6X for 5mins followed by incubation with chemiluminescence detection reagents . Membranes were visualised on a Chemidoc MP (Biorad) which was supported with Image Lab software (Biorad).Muscle biopsies were sectioned on a cryostat (Leica Biosystems) at 12\u00a0\u03bcm to obtain ~20\u00a0mg of tissue. Samples were homogenised on ice in 700 ul of lysis buffer . Protein concentration was measured via a Bicinchoninic acid"}
+{"text": "Seed vigor is an important characteristic of seed quality. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed vigor, including the germination potential, germination rate, germination index and time for 50% of germination, at 4 (early), 5 (middle) and 6 weeks (late) after heading in two years. A total of 24 additive and 9 epistatic quantitative trait loci (QTL) for seed vigor were identified using QTL Cartographer and QTLNetwork program respectively in 2012; while 32 simple sequence repeat (SSR) markers associated with seed vigor were detected using bulked segregant analysis (BSA) in 2013. The additive, epistatic and QTL \u00d7 development interaction effects regulated the dry maturity developmental process to improve seed vigor in rice. The phenotypic variation explained by each additive, epistatic QTL and QTL \u00d7 development interaction ranged from 5.86 to 40.67%, 4.64 to 11.28% and 0.01 to 1.17%, respectively. The QTLs were rarely co-localized among the different maturity stages; more QTLs were expressed at the early maturity stage followed by the late and middle stages. Twenty additive QTLs were stably expressed in two years which might play important roles in establishment of seed vigor in different environments. By comparing chromosomal positions of these stably expressed additive QTLs with those previously identified, the regions of QTL for seed vigor are likely to coincide with QTL for grain size, low temperature germinability and seed dormancy; while 5 additive QTL might represent novel genes. Using four selected RILs, three cross combinations of seed vigor for the development of RIL populations were predicted; 19 elite alleles could be pyramided by each combination. Oryza sativa L.) is one of the most important crops in the world. Recently, improving rice vigor become more important, because the direct seeding method has become increasingly important in many Asian countries due to its lower cost and its operational simplicity Seed vigor is an important characteristic of seed quality, reflecting potential seed germination, seedling growth, seed longevity, and tolerance to adversity Arabidopsis, the transcription factors, such as ABI3, FUS3, LEC2 and LEC1, have been identified as being master regulators of seed maturation Medicago trunculata and ArabidopsisSeed development is a crucial process in the lifecycle of plant, which can be divided into the two stages morphogenesis and maturation Seed vigor has been known as a complex quantitative trait which makes the genetic analysis of seed vigor very difficult According to the theory of developmental genetics, genes are expressed selectively at different growth stages 2\u223610) derived from the cross of indica rice IR26 and japonica Jiucaiqing was employed to map the loci underlying four seed vigor traits, including germination potential, germination rate, germination index and time for 50% of germination. The QTLs with additive, epistatic and QTL \u00d7 development interaction effects for seed vigor were conducted during three maturity stages in rice. Finally, the novel parental combinations for seed vigor were predicted in future rice breeding. The selected RILs and identified QTLs might be used to improve seed vigor by marker assisted selection approach.What happens in the dry maturity developmental process to improve rice vigor is still an open question. In this study, the objectives were to investigate the genetic control of developmental behavior of seed vigor in rice. One RIL population (FOryza sativa L.) varieties, Jiucaiqing (japonica) and IR26 (indica), and their 150 recombinant inbred lines (RILs) (F2\u223610) were used in this study. The 25-day-old seedlings were transplanted into a paddy field at the Experimental Station of Nanjing Agricultural University  on June 20th, 2012 and also 2013. The plants were grown with 17 cm between plants within a row and 33 cm between rows. Filed management was carried out according to the local standard methods Two rice  was computed on the basis of the RIL population through analysis of variance using the formula: B2H \u200a=\u200a G2\u03c3/(G2\u03c3 + e2/n\u03c3), where G2\u03c3 is genetic variance, e2\u03c3 is error variance, and n is number of replicates.The experimental data were analyzed using the SPSS 19.0 software, and the phenotype of two parents was compared with Student's The seeds harvested in 2012 were used for identification of QTLs with additive, epistatic and QTL \u00d7 development interaction effects for seed vigor in rice. The genetic linkage map based on 135 simple sequence repeat (SSR) markers at an average interval of 16.5 cM was constructed by Wang et al. 50 simultaneously and low vigor bulk with low GP, GR, GI and higher T50 simultaneously, were selected at each maturity stage respectively, and each bulk containing DNA from 10 individuals was used for BSA. A total of 168 SSR markers that revealed polymorphisms between Jiucaiqing and IR26 were used to determine the SSR markers associated with seed vigor.The seeds harvested in 2013 were used to test those QTLs detected in 2012 by the bulked segregant analysis (BSA) method. Two extreme phenotypic bulks, including the high vigor bulk with high GP, GR, GI and lower T50 at three maturity stages were selected. Then, the positive alleles of the stably expressed additive QTLs among the selected RILs were analyzed. Furthermore, the plant height and grain weight of the selected RILs were observed. Finally, the best three cross combinations were predicted to improve seed vigor in rice.The best cultivars with maximum phenotypic value and elite alleles might be used to design parental combinations for crop breeding 50) of Jiucaiqing and IR26 and their RIL population were investigated by using seeds harvested at 4 (early), 5 (middle) and 6 (late) weeks after heading, respectively , indicating a large amount of genetic variation in the population. The HB2 of GP, GR, GI and T50 was more than 94% at three maturity stages.The seed vigor , four for GR , five for GI , and five for T50 . The phenotypic variance explained by each QTL ranged from 6.95% to 30.08%. Two major QTLs qGR8.2 and qGI8 (r2>20%) were identified with a LOD score of 6.74 and 4.95 respectively, explaining 30.08% and 29.77% of the phenotypic variation, between RM6976 and RM6845 region on chromosome 8.There were sixteen additive QTLs identified at the early maturity stage , includiqGP3), GR (qGR7), GI (qGI7) and T50 (503.2qT), identified at the middle maturity stage , two for GR (qGR8.1 and qGR12), two for GI (qGI1.2 and qGI7) and one for T50 (501.2qT). Among these QTLs, the qGI1.2 was co-located with 501.2qT between RM6950 and RM5759 region on chromosome 1, and the qGI7 was co-located with qGR7 between RM8261 and RM5426 region on chromosome 7. The phenotypic variance explained by each QTL ranged from 9.93% to 40.67%. One major QTL qGR12 (r2>20%) was identified with a LOD score of 2.73, explaining 40.67% of the phenotypic variation, between RM1880 and RM20 region on chromosome 12.There were eight additive QTLs identified at the late maturity stage , includi50 respectively. The epistatic QTL was detected with only epistatic main effect, while no significant QTL \u00d7 development interaction effect. The phenotypic variance explained by each epistatic QTL ranged from 4.64% to 11.28%, and the phenotypic variation explained by each QTL \u00d7 development interaction ranged from 0.04% to 1.17%.A total of nine epistatic QTLs were identified in joint analysis of three-maturity phenotypic values in 2012 . Of them50. The additive QTL was detected with only additive main effect, while no significant QTL \u00d7 development interaction effect. The phenotypic variance explained by each additive QTL ranged from 5.86% to 9.84%, and the phenotypic variation explained by each QTL \u00d7 development interaction ranged from 0.01% to 1.08%.A total of five additive QTLs were identified in joint analysis of three-maturity phenotypic values in 2012 . Each onTo test those additive QTLs detected in 2012, the SSR markers associated with seed vigor were identified using BSA method in 2013. Two extreme phenotypic bulks, including the high and low vigor bulks, were selected at each maturity stage respectively . A total50 at three maturity stages respectively , which will greatly facilitate the detection of QTLs and QTL \u00d7 development interactions. By comparison, the more additive QTLs were expressed at the early maturity developmental stages followed by the late maturity stage. We found that the expressions of QTL were differential at different maturity stages: the additive QTLs rarely co-localized among the different stages. Only the qGR1, qGI1.1 and 501.1qT on chromosome 1 coincided with qGL-1 for grain length qGI1.2 and 501.2qT on chromosome 1 was similar with the region of qGRL1.1 for grain dimension qSD1 for seed dormancy 503.1qT was on the similar location of qSD3.2 for seed dormancy qGR5 located on the same region of qSD-5 for seed dormancy qLTG-5-1 for seed low temperature germinability 507.1qT is near to Sdr4 for seed dormancy qGL7-2 for grain length qGI7, qGR7 and qGP7 on chromosome 7 was similar with the region of qSD7.1 for seed dormancy 507.2qT was on the similar location of qGL7 for grain length qGI8 and qGR8.2 located on the same region of qSD8 for seed dormancy on chromosome 8 qGI10 and qGR10 located on the same region of qLTG-10 for seed low temperature germinability qGI3, 503qT.2, qGP3, qGP4.1 and qGP5 which indicates that these additive QTLs might be novel genes. With the increase in the number of QTLs identified for seed vigor, the genetic control of seed vigor will be better understood.The additive QTLs which stably expressed over years might play important roles in seed vigor in different environments. Comparing the positions of these 20 stably expressed additive QTLs detected here with other QTLs reported previously, we found that the region of QTL Sdr4 has been cloned and identified as one of the major determinants of dormancy in rice, which affects the expression of several DELAY OF GERMINATION 1-LIKE (DOG1-LIKE) genes Sdr4 expression is positively regulated by OsVP1, a global regulator of seed maturation that is orthologous to maize viviparous 1 (VP1) and Arabidopsis ABI3qGI1.2 and 501.2qT identified in this study was similar with the location of OsVP1 on chromosome 1 OsVP1 during seed maturation. To date, only one a major rice QTL qLTG3-1 for low temperature germinability has been cloned qLTG3-1 is functionally associated with the vacuolation of the tissues covering the embryo, which results in the reduction of the mechanical resistance to coleoptile growth. In this study, one major QTLs qGR8.2 was co-located with qSD8 for seed dormancy qGR8.2 is now in progress to elucidate the molecular mechanism of seed vigor using near isogenic lines (NILs).The exploration of physiological and genetic mechanisms in seed vigor is a highlight of seed science Besides gene functional analysis, the genetic mechanisms of seed vigor can also be well revealed by functional mapping. Functional mapping integrates developmental principle of trait formation into a QTL framework, which can detect the specific QTLs that determine the developmental pattern of a complex trait In a hypothetical cross of two cultivars, the trait values of produced RILs can be predicted by the effects of all the detected loci. The best RIL with maximum value would represent the best cross. To improve seed vigor, all the elite alleles might be pyramided into one cultivar as far as possible. Thus, according to the information of phenotypic values and the allelic effects of the 20 stably expressed additive QTLs in two years, the best three cross combinations for the development of RIL populations were predicted in this study. A total of 19 elite alleles could be pyramided by each combination to improve seed vigor. These results showed that the selected RILs as new materials will be valuable in future rice breeding programs. The identified QTLs will be applicable to improve rice seed vigor by marker assisted selection."}
+{"text": "Extensive description of severe asthma phenotypes has gained much attention in the latest years with the hope of achieving better management of this potentially problematic group of asthmatics. However, all previous studies have focused on patient cohorts, and not random population samples.To dissect the concept of severe asthma, and to determine whether clustered complaints or addressing individual complains, will give more coherent understanding of severity.From a large population based study in West Sweden, a subgroup of 744 people with active asthma was characterised with extensive clinical examinations and detailed questionnaires. In this cohort we defined different parameters of severity as follows \u20131) low lung function as FEV1% predicted below 70%, 2) multi-symptom asthma described day-time symptoms, 3) 2 or more night-time awakenings per month, 4) use of rescue medications 3 or more times per week, 5) any respiratory emergency visits and 6) use of oral steroids regularly or on exacerbations in the last year. We outlined particular subgroups of asthmatics who presented with different extend of overlap of these characteristics and looked into possible risk factors for the separate groups.The group of asthmatics who presented with at least one of severity features comprised of 374 people (50.3% of the active asthma group). The distribution for the individual complaints was day-time symptoms in 196 (36.3%), low lung function in 85 (11.4%), rescue medications in 130 (17.5%), night-time symptoms in 111 (14.9%), emergency visits in 78 (10.5%) and use of oral steroids in 66 (8.9%). When at least two complaints in a different combination were examined, the groups comprised of 2.1 -7.6% of the whole asthma population. The extent of the overlap between more than three complaints revealed tightly agglomerated groups representing 0.5 \u2013 7.9% of all asthmatics. The risk factors that were outlined for the different groups were current smoking and having BMI > 30 with ORs ranging from 1.7 \u2013 4.1 and 1.6 \u2013 2.6, respectively.Further dissecting into the different severe asthma phenotypes in a random population could identify particular influencers for severity, and may more exactly stratify distinct groups that require extensive management."}
+{"text": "ROP occurs in 95% of babies weighting less than 1000 g, all cases that had proceeded to the third stage will cause serious complications or complete blindness [Retrospective analysis of examination of 217 newborn of the risk group. . Due to the development of Respiratory Distress Syndrome all children needed respiratory therapy on the first week of living. The patients were divided into 3 groups according to the kind of respiratory therapy received. 1st group ( n-=86) received noninvasive respiratory therapy with Nosal Continuous Airway Pressure, 2nd group (n=69) \u2013 noninvasive therapy with Biphasic, and 3rd group (n=62) \u2013 invasive respiratory therapy with Artificial Respirating Unit.In 1st group we observed 0 cases of ROP, in 2nd - 3 cases were diagnosed, but all of them had a fast regression afterwards, in 3rd group \u2013 20 children developed ROP, only 15 cases regressed and one child had to go through the laser coagulation of the retina vessels.Changing from invasive to noninvasive forms of respiratory therapy may lead to significant decrease of the risk of ROP."}
+{"text": "To evaluate the efficacy of a 5 day short course of oral prednisolone when added with levocetirizine for management of acute urticaria.Prospective, randomized clinical trial was carried out in a teaching hospital.All patients were asked to evaluate the severity of pruritus on urticarial activity score (UAS).Patients were then given oral prednisolone 30 mg for 5 days and tablet levocetirizine 5 mg twice daily for 6 weeks and only levocetirizine tablet 5 mg twice daily for 6 weeks. Patients\u2019 conditions were reassessed clinically with UAS calculated again 2 days later and again 5 days later.49 patients were enrolled; 24 patients received prednisolone with Levocetirizine and 25 received only Levocetirizine. The two groups had similar UAS at enrollment  , but at 2- and 5- day follow-up the prednisolone group had significantly lower UAS (1.4 and 0.2 versus 3.8 and 2.4 respectively) and greater clinical improvement in rash. Response did not correlate with age, sex, or identification of an allergen. No adverse effects were noted in levocetirizine group. In Prednisolone group two patients complained of gastritis.At the end of 6 weeks, 3 patients from the steroid group and 8 patients from the levocetirizine group continued to get urticarial wheals. The addition of a prednisolone short course improves the symptomatic and clinical response of acute urticaria to antihistamines. Patients with steroids improved more quickly and completely without major adverse effects."}
+{"text": "Dual T1 mapping allows for a comprehensive assessment of myocardial tissue by combining detection of edema in the native scan and quantification of extracellular volume (ECV) after administration of Gadolinium. Recent studies proved the diagnostic value of T1 mapping in different pathologies; the aim of this study was, to evaluate the practicability and robustness of T1 mapping in assessing common pathologies in daily CMR practice.Since October 2012, we investigated 140 patients undergoing clinically indicated CMR examination by perfoming additional T1 mapping sequences which were not used for clinical diagnosis. We used a modified Short Modified Look Locker Inversion Recovery (ShMOLLI) sequence with 3 inversion pulses and a 4-(1)-3-(1)-2 readout pattern. The post Gadolinium scan for extra cellular volume calculation was performed 10 min. after administration of a bolus of 0.2 mmol/kg body weight gadopentetate dimeglumine. Diagnosis was based on clinical information and standard sequences for assessment of myocardial tissue comprising native T2 weighted dark blood turbospin echo (tse) sequences, pre and early post gadolinium T1 weighted tse sequences and inversion recovery spoiled gradient echo sequences for late enhancement. Assessment of T1 relaxation time and ECV values was based on 3 short axis views and 1 long axis view using the AHA/ACC 17-segment model.Native T1 relaxation time and ECV for the different pathologies are summarized in Table T1 mapping and ECV correlated well with myocardial alterations in commonly diagnosed cardiac disorders. It proved reliable and robust in daily clinical practice and allows for a good differentiation between normal findings and pcommon pathological CMR diagnoses. The combined use of native T1 and ECV quantification is promising approach for comprehensive assessment of the myocardium and may improve diagnostic accuracy of CMR in myocardial disease.None."}
+{"text": "Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation. Matrilysin MMP7) is one of the smallest members of the MMP family and is a highly potent metallo-protease which can degrade casein, laminin, fibronectin, collagen III/IV/V/IX/X/XI, type I/II/IV/ V gelatins, elastin and proteoglycans , 2. MMP- is one oDesmosomes are cell-cell adhesion junctions present in both simple and stratified epithelial cells. Desmosomes anchor intermediate filaments in adjoining cells and thus play a central role in the formation of a tissue wide intermediate filament network, allowing cells to survive when they encounter mechanical stress during tissue repair , 20. ThrPlakophilin3 (PKP3) is the most widely expressed plakophilin family member and is ubiquitously present in all the layers of the stratified epithelia and simple epithelia except in hepatocytes . PKP3 foRecent results from our lab have shown that PKP3 loss leads to an increase in PRL3 (Phosphatase of regenerating liver-3) protein levels leading to the dephosphorylation of keratin8 (K8), which results in increased neoplastic progression and metastasis . To deteThe oligonucleotides used to generate the MMP7 shRNA constructs  were cloHCT116, HaCaT and FBM cells  were cultured as described previously , 24, 30.RNA isolated from the FBM derived vector control and PKP3 knockdown cells and HCT116 derived vector control and PKP3 knockdown cells were Cy3 labeled and processed for the Sureprint G3 Human GE 8x60k microarray by single color hybridization. The results obtained from the microarray were analyzed using the Agilent Feature Extraction software. Using normalized signal intensities  obtained from the microarray, the fold changes of genes altered in FBM derived PKP3 knockdown clone (shpkp3-2) has been compared to the vector control clone (vec). Similarly, the HCT116 derived derived PKP3 knockdown clone (shpkp3-2) has been compared to the vector control clone (vec). A functional classification of the differentially regulated genes was performed using GeneSpring GX 11.0 software and gene ontology browser. The significant pathway list for differentially regulated genes was obtained using the GeneSpring GX 11.0 and Biointerpreter software . The data for the FBM derived clones has been deposited in the NCBI GEO database (Accession number GSE61512), while the data for the HCT116 derived clones have been deposited in the NCBI GEO database (Accession number GSE64580). Functional classification of genes altered upon PKP3 loss was performed using the PANTHER Classification System software , 33.(-\u0394\u0394Ct) method [The forward and reverse oligonucleotides used in this study are shown in ) method . A changFor Western blots, the mouse monoclonal \u03b2 actin antibody  was used at a dilution of 1:5000, the mouse monoclonal PKP3 antibody  at a dilution of 1:2000, mouse monoclonal MMP7 antibody  at a dilution of 1:100. Goat anti-mouse secondary antibody (Pierce) was used at a dilution of 1:2500. The blots were developed using Supersignal West Pico Cheminiluminescent Substrate (Pierce) according to the manufacturer\u2019s instructions. Cells were lysed in 1X sample buffer as described  and prot2. Cells were observed by time lapse microscopy and images taken every 10 minutes for 20 hours using the AxioCamMRm Camera (Zeiss) with a 10X phase I objective. Axiovision software version 4.8 (Ziess) was used to measure cell migration. Three independent experiments were performed in triplicates for each clone. Invasion assays were performed as described [5 cells resuspended in 200\u03bcl of serum free media were added to the upper chambers and 400\u03bcl of serum containing media was added in the lower chamber. The inner side of the insert was pre-coated with 5\u03bcg of Matrigel (BD Biosciences). After 24 hours, cell culture inserts were then removed from the wells and the cells attached to the inner side of the insert were removed using cotton buds. The inserts with cells on the outer side of the membrane were fixed with 4% para-formaldehyde, stained with 1% crystal violet and mounted on slides using DPX mountant . Images were taken using Olympus SZ61 stereo microscope using a 10X objective lens. Three independent experiments were performed for each clone.Scratch wound healing assays were performed as described . The plaescribed , 36. Bri2500 cells of the HCT116 based plakophilin3 knockdown clones shpkp3-1 and shpkp3-2, the vector control clone (vec), the shpkp3-2 derived vector control and shpkp3-2 derived double knockdown clones were resuspended in 0.4% soft agarose as described . The celFoxn1nu/Crl) of 6\u20138 weeks old, provided by the ACTREC animal house facility, was used for the study. 1 x 106 cells of the HCT116 based shpkp3-2 derived vector control and double knockdown clones were resuspended in 100\u03bcl of PBS and injected sub-cutaneously in the dorsal flank of mice. Six mice were injected for each clone. Tumor formation was monitored at intervals of 2 to 3 days and tumor size was calculated weekly for 5 weeks using the formula (0.5x LV2) where L is the largest dimension and V its perpendicular dimension [3 was obtained 5th week post-injection for a mouse injected with shpkp3-2+vec.No surgical procedure was involved in the present study and therefore no anesthetic or analgesic was employed during these experiments. During injections, the animals were handled by trained, certified animal technicians and were injected by the in-house veterinarian with minimum distress to animals. Mice were sacrificed 5 weeks post injection. Animals were euthanized as per in-house Standard Operating Procedure (SOP) approved by the attending veterinarian (AV) of the ACTREC animal house facility. Carbon dioxide (CO2), an inhalant euthanasia agent recommended by the Committee for the Purpose of Control and Supervision of the Experiments on Animals (CPCSEA), Government of India, was used for euthanasia of mice. Euthanasia was performed under the supervision of the attending veterinarian and according to the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (2013 Edition). Briefly, a compressed CO2 cylinder was used as a source for carbon dioxide to control the inflow of gas, which was connected to a euthanasia chamber. The mice were kept in the chamber and an optimal flow rate was maintained to fill 20% of the chamber volume. After keeping the mice in the chamber, the CO2 cylinder supply valve was turned on to deliver the gas in the chamber so that animals were exposed to the gas slowly and steadily. After sufficient exposure like for 2 to 3 minutes, mice showed cessation of respiration and heart beats. The chamber was not prefilled with CO2 and was vented out post sacrifice and before the next animal was introduced into the chamber. Thereafter, the mice were removed from the chamber and a cervical dislocation performed to ensure that the mice were dead.BALB/c Nude mice , Ministry of Environment and Forest, Government of India. A controlled environment was provided to the animals with a temperature of 22\u00b12\u00b0C and relative humidity maintained at 40\u201370%. A 12 hours day night cycle was maintained (7:00 to 19:00 day and 19:00 to 7:00 night). The animals were given autoclaved balanced diet prepared in house and sterile water. Individually ventilated Cage system  was used to house mice used in the experiments. These IVCs were provided with autoclaved corn cob as bedding for the mice. Animal euthanasia was done under the guidelines of AVMA as mentioned above using CPCSEA recommended euthanizing agent, carbon dioxide. The Institutional Animal Ethics Committee (IAEC) of the Advanced Centre for Treatment Research and Education in Cancer (ACTREC) approved all the protocols used in this report. The project number for the study is 16/2008 and was approved in November 2008.Previous studies from our laboratory have demonstrated that loss of PKP3 in HCT116, HaCaT and FBM cells leads to an increase in transformation in vitro and increased tumor formation and metastasis in vivo . To idenIn confirmation of previously reported results , 30, a rThe mRNA levels of the Matrix metalloprotease7 (MMP7) gene were found to be up-regulated upon PKP3 loss in all three cell types tested . To detePKP3 loss leads to an increase in cell migration as assayed by scratch wound healing assays . To detePKP3 loss leads to an increase in anchorage independent growth and an increase in tumor formation and metastasis in vivo . To detePrevious data from our laboratory has demonstrated that an elevation in the levels of Keratin 8 is observed in the HCT116 derived PKP3 knockdown clones and that this increase is due to an increase in the levels of the phosphatase of regenerating liver 3 (PRL-3) and inhibition of PRL-3 activity using a chemical inhibitor resulted in a decrease in cell migration . An inhiThe results in this report demonstrate that loss of PKP3 leads to an increase in the levels of MMP7 in three independently derived cell lines of different origins. The increase in MMP7 levels is required for cell migration and invasion in vitro and tumor formation in nude mice as loss of MMP7 in the PKP3 knockdown cells results in a reversal of these phenotypes. The increase in MMP7 mRNA is dependent on the expression of PRL-3, whose levels are elevated upon PKP3 loss . Thus, tMin mice leads to a decrease in tumor incidence. Our work has demonstrated that PKP3 loss leads to an increase in migration, invasion, tumor formation and metastasis and that these functions are dependent upon the increase in MMP7 expression upon PKP3 knockdown. To determine if a correlation exists between MMP7 and PKP3 levels across colon cancers at the transcript level, we analyzed the data sets in the Oncomine database (www.oncomine.org). When PKP3 levels in normal colon samples were compared with that in colon cancer, the levels were found to be unchanged, while MMP7 levels were higher in colon cancer samples. None of the databases present had actually validated the PKP3 levels using real time PCR or reverse transcriptase PCR. To determine if a correlation exists between MMP7 and PKP3 levels across colon cancers at the protein level, we examined the data deposited in the human protein atlas database (http://www.proteinatlas.org/). It was observed that a thorough immuno-histochemical analysis of PKP3 and MMP7 levels in a large dataset of colorectal cancer patients has not been performed to determine if any correlation exists between PKP3 and MMP7 levels. Surprisingly, MMP7 loss leads to an increase in colony formation in soft agar but a decrease in tumor formation in nude mice. These results suggest that the ability of MMP7 to induce the degradation of the extra-cellular matrix in vivo maybe essential for the ability of the PKP3 knockdown cells to form a tumor. Taken together these results suggest that the increase in MMP7 levels observed upon PKP3 loss is required for increased tumorigenesis in cells derived from the colon.MMP7 and membrane type-1 matrix metalloprotease (MT1-MMP) are metalloproteases which are exclusively produced by the epithelial cells of the colon while the rest of these matrix-metalloproteases such as MMP9, MMP11 (stromelysin 3) are produced by the stroma \u201343. MMP7PRL-3 levels are increased in colorectal cancers  and PRL-PKP3 knockout mice show symptoms of severe itching, intercellular edema, neutrophil infiltration, epidermal hyperplasia, hair loss and muscle wasting . BecauseThe results reported here lead to the generation of the following model. PKP3 loss leads to an increase in PRL-3 translation leading to an increase in K8 levels  or an inS1 Table(DOCX)Click here for additional data file.S2 TableCompiled data with complete dataset, differentially expressed genes and functional classification of altered genes observed upon PKP3 knockdown in FBM cell line.(XLSX)Click here for additional data file.S3 TableCompiled data with complete dataset, differentially expressed genes and functional classification of altered genes observed upon PKP3 knockdown in HCT116 cell line.(XLSX)Click here for additional data file.S1 Fighttp://www.pantherdb.org/about.jsp). S1B Fig, Cell type specific alterations in FBM derived PKP3 knockdown clones. Reverse transcriptase PCRs were performed using oligonucleotides specific for SAA4, EPPK1, MOBKL2b, MMP9, NR2F1, \u0394Np63, IGFBP3, ARHGEF5 and GAPDH, in HaCaT, HCT116 and FBM derived PKP3 knockdown clones and the respective vector controls. Expression of GAPDH has been used for normalization. The PCR product size has been indicated in base pairs. S1C Fig, MMP7 loss leads to a decrease in migration. Phase contrast images of wound healing at 0 hours (start) and 20 hours (end of experiment) have been shown.S1A Fig, Functional classification of genes whose expression is altered >=2 folds in FBM derived clones and >=1.5 folds in HCT116 derived clones upon PKP3 knockdown. The mRNA expression profile of the FBM derived vector control (vec) was compared with that of the PKP3 knockdown clone (shpkp3-2). Similarly, mRNA expression profile of the HCT116 derived vector control (vec) was compared with that of the PKP3 knockdown clone (shpkp3-2). Differentially expressed genes which show more than two fold (for FBM) and more than 1.5 folds up-regulation or down-regulation in the PKP3 knockdown clones compared to the vector control clone were selected. The pie charts show functional classification of genes obtained from the microarray data analysis using the PANTHER Classification tool ((TIF)Click here for additional data file.S2 Fig6 cells of the vector control or the double knockdown clones were injected sub-cutaneously into nude mice and allowed to develop tumors. Representative images of mice have been shown. S2B Fig, LCN2 expression is not altered upon inhibition of PRL-3. The HCT116 derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) or the vector control (vec) were treated with either the vehicle control (DMSO) or 10 \u03bcM PRL-3 inhibitor-1 (PRL-3i) for 24 hours. The mRNA prepared from the treated cells was used as a substrate for reverse transcriptase followed by real time PCR reactions using oligonucleotides specific for LCN2. All expression was normalized to the levels of GAPDH. The fold change is graphed on the Y-axis and the clone name is on the X-axis. The standard errors are plotted and student\u2019s t test was performed . Note that LCN2 levels are increased upon treatment with PRL-3 inhibitor. S2C Fig, MMP7 expression does not change upon inhibition of PRL3 activity in HaCaT derived clones. The HaCaT derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) or the vector control (vec) were treated with either the vehicle control (DMSO) or 10 \u03bcM PRL-3 inhibitor-1(PRL-3i) for 24 hours. The cell supernatants were collected and a100\u03bcg of acetone precipitated protein was resolved on 12% SDS PAGE gels followed by Western blotting with antibodies to MMP7. The same blot was stained with Ponceau to indicate equal loading.S2A Fig, Tumor formation is inhibited in the PKP3 knockdown cells upon MMP7 knockdown. 10(TIF)Click here for additional data file."}
+{"text": "Blockade of the sphenopalatine ganglion with OnabotulinumtoxinA injections (SphenoBlock) represents a novel approach for treating intractable chronic cluster headache (iCCH).The aim of this pilot study was to explore the safety aspects and therapeutic potential of SphenoBlock.After signing written confirmed consent ten patients with iCCH were injected with 25 U (n=5) or 50 U (n=5) onabotulinumtoxinA towards the sphenopalatine ganglion in an observational study, approved by the Institutional Review Board, with 6 months follow-up. The procedure was performed with a novel image-guided technique. The primary endpoint was to evaluate safety of the procedure, but change in attack frequency from baseline to week 4, 12 and 24 was also registered.Data for the first 5 patients are presented. One patient experienced intermittent ipsilateral visual deficits lasting 4 weeks. Patient number 5 was a failed injection. Four patients were defined as frequency responders (>50% reduction from baseline) in week 4, 3 patients responded in week 12, and 2 patients in week 24 (Figure SphenoBlock in iCCH shows promising preliminary results and give reasons for cautious optimism for further studies on this low-cost alternative treatment of iCCH."}
+{"text": "CUG repeats result in part from the inactivation of the muscleblind-like (MBNL) proteins. To test the role of MBNL3, we deleted Mbnl3 exon 2 (Mbnl3\u0394E2) in mice and examined the onset of age-associated diseases over 4 to 13 months of age. Accelerated onset of glucose intolerance with elevated insulin levels, cardiac systole deficits, left ventricle hypertrophy, a predictor of a later onset of heart failure and the development of subcapsular and cortical cataracts is observed in Mbnl3\u0394E2 mice. Retention of embryonic splice isoforms in adult organs, a prominent defect in DM1, is not observed in multiple RNAs including the Insulin Receptor (Insr), Cardiac Troponin T (Tnnt2), Lim Domain Binding 3 (Ldb3) RNAs in Mbnl3\u0394E2 mice. Although rare DM1-like splice errors underlying the observed phenotypes cannot be excluded, our data in conjunction with the reported absence of alternative splice errors in embryonic muscles of a similar Mbnl3\u0394E2 mouse by RNA-seq studies, suggest that mechanisms distinct from the adult retention of embryonic splice patterns may make important contributions to the onset of age-associated pathologies in DM1.Myotonic dystrophy type I (DM1) exhibits distinctive disease specific phenotypes and the accelerated onset of a spectrum of age-associated pathologies. In DM1, dominant effects of expanded  DMPK and immediately 5\u2032 of SIX51214Myotonic Dystrophy type 1 (DM1) is a multi-system disorder resulting from the expansion of a CTG repeat sequence located in the 3\u2032 untranslated region of DMPK RNAs encoding expanded CUG repeat sequences (CUGexp) play a central role in the development of key aspects of DM1 pathology678DMPK RNA encoding CUGexp with the MBNL family of proteins and allele specific silencing of SIX5 expression resulting from CTG tract expansion respectively, are sufficient to result in a subset of DM1 cardiac, skeletal muscle, ocular and gonad pathologies10111213141516171819Multiple lines of evidence demonstrate that dominant RNA effects resulting from the expression of mutant CUGexp to aberrantly sequester and functionally inactivate members of the muscleblind (MBNL) family of proteins10118212324Mbnl1\u03943/\u03943 muscle, heart and brain and in Mbnl2\u03942/\u03942 brain2628RNA dominant effects in DM1 have been hypothesized to stem in part from the ability of Clc-1 RNA splice defects has been shown to rescue myotonia in the HSALR DM1 mouse modelInsr) RNA has been implicated in the development of abnormal glucose tolerance in DM1262730Experiments directed at the examination of the causal role of RNA splice errors in the development of DM1 features are limited to myotonia, where reversion of Mbnl3, which is an X-linked gene, to develop Mbnl3\u0394E2 mice38kD) and the retention of a truncated 27\u2009kD Mbnl3 isoform (Mbnl327kD) translated from an ATG codon present in Mbnl3 exon 3, as previously described by us and Poulos and colleagues34Mbnl3\u0394E2 mice demonstrate an accelerated onset of a subset of age-associated phenotypes observed in DM1, over a range of 4\u201313 months of age. Specifically, Mbnl338kD deficits trigger the early onset of abnormal glucose metabolism, elevated insulin levels, cardiac systole dysfunction that progresses to left ventricle hypertrophy, and a high incidence of subcapsular and cortical cataract formation. To test if DM1 specific splice errors contribute to the development of these phenotypes, we studied the splice patterns of twenty RNAs including the Insulin Receptor (Insr), Lim Binding Domain 3 (Ldb3) and Cardiac Troponin T (Tnnt2) RNAs in Mbnl3\u0394E2 mice. DM1-like splice errors are not observed in these RNAs and reversion to the embryonic splice patterns is not observed for the Insr and Tnnt2 RNAs in Mbnl3\u0394E2 skeletal muscle and heart. The modest splice error detected in the Ldb3 RNA in Mbnl3\u0394E2 hearts does not recapitulate the embryonic splice pattern. Thus these data demonstrate Mbnl338kD deficits can cause the accelerated onset of age-associated DM1 pathologies and suggest that mechanisms distinct from splice alterations may contribute to the development of such DM1 phenotypes.In this study we tested the role of the third member of the muscleblind family, MBNL3, in DM1 etiology. We deleted exon 2 of Mbnl3\u0394E2 mice on a 129sv background, in which exon 2 of the X-linked Mbnl3 gene was replaced by a Neomycin expression cassette12Mbnl3 mRNA expression in the adult soleus muscle and in the adult mouse heart, lens and brainTo test the potential effects of MBNL3 deficiency in the development of DM1 pathophysiology we developed Mbnl3+/+ and Mbnl3\u0394E2 mice. Briefly, mice were fasted for 6\u2009hours prior to testing and baseline blood sugar levels were obtained from a drop of tail blood. Subsequently, a bolus of sterile 5% dextrose in saline was injected IP at a dose of 1\u2009g/kg at time zero and blood glucose levels were repeatedly tested for up to 3\u2009hours following the injection. Area under the curve for the glucose tolerance test was calculated using an individual t-test at 4 months of age for male Mbnl3\u0394E2 (n\u2009=\u20093) and Mbnl3+/+ (n\u2009=\u20094) mice. At 4 months of age, there was no statistically significant difference in blood glucose levels . However, a trend towards a shorter time for peak glucose levels to be achieved and elevated blood glucose levels 3\u2009hours post injection was observed in Mbnl3\u0394E2 mice when compared to Mbnl3+/+ controls  and Mbnl3\u0394E2 mice (n\u2009=\u20097) (p\u2009<\u20090.001). Upon dextrose injection, peak glucose levels achieved were both significantly higher and were sustained at higher levels for a longer duration, with blood glucose levels ~57% higher in Mbnl3\u0394E2 mice when compared to control animals, 3\u2009hours post injection (p\u2009<\u20090.013) (Mbnl3+/+ and Mbnl3\u0394E2 mice) adjusting for reads and time .At 7\u20139 months of age, baseline blood glucose levels were significantly different between male <\u20090.013) . At 7\u20139 Mbnl3\u0394E2 mice was due to decreased insulin levels, we measured insulin levels in 8\u201310 month old Mbnl3+/+ and Mbnl3\u0394E2 mice, at 0, 8, 15, 30 and 60\u2009minutes following the intraperitonel injection of dextrose at a dose of 2\u2009g/kg, subsequent to a 16\u2009hour fast as previously describedMbnl3\u0394E2 mice when compared to Mbnl3+/+ mice prior to dextrose injection and the incidence rate ratio (IRR) is significantly different between groups (Mbnl3+/+ and Mbnl3\u0394E2 mice) adjusting for reads and time , demonstrating an 80% likelihood of higher insulin levels in the Mbnl3\u0394E2 mouse group when compared to control mouse group .As aberrant splicing of exon 10a of the 7 months  and 11 m7 months  or by qPMbnl3+/+ and Mbnl3\u0394E2 mice as a function of age. Left ventricular function and chamber dimensions were evaluated in male Mbnl3+/+ and Mbnl3\u0394E2 mice by ultrasound echocardiography at 4 months  and 11 months  of age  and a consequent reduction in left ventricular function manifesting as diminished left ventricle percent fractional shortening , left ventricle ejection fraction  and velocity of circumferential fiber shortening  when compared to Mbnl3+/+ animals.Structure-function evaluation of the heart was carried out in male ) of age . These eMbnl3\u0394E2 mice at four months of age occurs without differences in diastole chamber dimensions. In contrast, at 11 months of age, male Mbnl3\u0394E2 mice exhibit a hypertrophic response, most likely in response to the cardiac systole deficits that manifest at 4 months of age. Specifically, significant increases in end-diastolic dimension , left ventricular mass , posterior wall thickness  and ventricular septal thickness  are observed in this age group. Finally, the E/A ventricular filling ratios are in the normal range suggesting no diastole dysfunction accompanying the hypertrophy. These age-related functional and morphological changes are shown in Left ventricle systole dysfunction in male As each sample set at 4 and 11 months of age are separate, typical corrections for multiple comparisons do not directly apply to these data. Given the small sample size, a statistically significant effect indicates very large effect sizes . Even with multiple comparison corrections, the most relevant outcomes of left ventricle percent fractional shortening at 4 months and left ventricular mass and posterior wall thickness at 11 months are significant.Mbnl3+/+ and Mbnl3\u0394E2 mice at 4 and 11 months of age. The wave shapes and intervals were within the normal range for Mbnl3\u0394E2 mice at these time points (Mbnl3\u0394E2 mice (data not shown).Surface electrocardiograms were recorded in parallel from male e points . No arrhLdb3 and Tnnt2 mutations are known to cause cardiac dysfunction, dilated cardiomyopathy and cardiac hypertrophy37Tnnt2 isoforms in the adult heart is strongly associated with heart failure in humansTnnt2 in transgenic mice results in diminished cardiac efficiencyLdb3 and Tnnt2 in adult hearts, two splice errors previously described in DM133Mbnl3\u0394E2 hearts. Ldb3 shows a modest but significant decrease in the inclusion of exon 11 in Mbnl3\u0394E2 hearts when compared to Mbnl3+/+ hearts by RT-PCR at 7 months . This splice error is not reminiscent of the embryonic Ldb3 exon 11 splicing pattern . Consistent with the results from the skeletal muscle, Insr and Mbnl1 splice patterns are also not altered in Mbnl3\u0394E2 hearts at either age .7 months  and by R pattern . The splMbnl3\u0394E2 hearts, we analyzed splice events in Pdlim3/Alp, Trim55/Murf2, Mapt/Tau, Pdlim5, Sorbs1, Sorbs2, Fhod1, Spag9, Mbnl2, Myom1, Clta, Stx2, Csda, Sirt2, Atp2a1 and Atp11a RNAs, which show the aberrant retention of embryonic splice isoforms in adult Mbnl1\u0394E2/\u0394E2 heartsMbnl3+/+ and Mbnl3\u0394E2 hearts and from E18 Mbnl3+/+ hearts. In 11 month Mbnl3\u0394E2 hearts, none of splice events examined showed significant alterations when compared to 11 month old Mbnl3+/+ hearts  and Mbnl3\u0394E2 (n\u2009=\u20098) lenses, demonstrated a single Grade-I subcapsular cataract in Mbnl3\u0394E2 mice. No Mbnl3+/+ lenses studied at this age showed cataract formation , with ~60% showing advanced Grade-II and Grade-III cataracts as evaluated using LOCS II photographic grading standards. Cataracts in Mbnl3\u0394E2 mice were primarily posterior subcapsular or cortical in origin . Electroretinography studies did not show significant retinal defects in Mbnl3\u0394E2 mice at 13 months of age  is a multi-system disorder, which exhibits the accelerated onset of several age-associated disease phenotypes that include abnormal glucose metabolism, left ventricle systolic deficits, hypertrophy and heart failure, ocular cataracts that originate in the sub-capsular and cortical regions of the lens is a mul4232425262826272829303132hologies . SignifiMbnl3\u0394E2 and Mbnl3+/+ mice. A trend towards elevated glucose levels was observed in Mbnl3\u0394E2 mice at 4 months. At 7\u20139 months of age Mbnl3\u0394E2 mice show aberrant glucose tolerance, with both higher peak glucose levels and sustained elevated glucose levels that did not return to baseline 180\u2009minutes post injection. Overt diabetes was not observed at either age and baseline blood glucose levels were not significantly elevated in Mbnl3\u0394E2 mice when compared to Mbnl3+/+ mice. In DM1 patients an increase in basal blood insulin levels is reported44Mbnl3\u0394E2 mice. An increase in insulin levels was observed subsequent to dextrose injection in Mbnl3+/+ mice but not in Mbnl3\u0394E2 mice. The lack of an increase in insulin levels subsequent to dextrose injection may be due to high, approximately a two fold elevation, in basal insulin levels in Mbnl3\u0394E2 mice. Previous studies have strongly implicated elevated CUG-BP1 levels with aberrant glucose metabolism in DM1 patient cellsDmpk\u2212/\u2212 animals that are maintained on a high fat dietEndocrine abnormalities occur frequently in DM1, with patients having an increased risk of developing diabetes142138kD deficits trigger cardiac systole deficits and ventricular hypertrophy, which can be an indicator of the later onset of heart failure. EKG abnormalities are not however observed in Mbnl3\u0394E2 mice. In contrast, our previous studies have shown that deficits of Dmpk and Six5 result in conduction disorders but not echocardiographic abnormalities. Specifically, P-R prolongation, second and third degree heart block and QRS alterations are observed in Dmpk+/\u2212, Dmpk\u2212/\u2212 and Six5+/\u2212 mice respectively1516Mbnl1\u2212/\u2212 loss results in arrhythmias, cardiac histopathology and sudden death and Lee et al. have further demonstrated that Mbnl1\u2212/\u2212/Mbnl2+/\u2212 mice show both arrhythmias, left ventricular hypertrophy with myocardial fibrosis25In DM1, sudden death is widely considered to result from cardiac arrhythmias and although progressive heart failure is less common, left ventricle systole dysfunction is associated with increased risk of overall mortality and sudden death14714748495047484950Mbnl3\u0394E2 mice do not show significant ERG alterations. However these animals demonstrate an increase in ocular cataracts that are subcapsular and cortical in their origin. In our study subcapsular cataracts were first detected at 7 months of age in Mbnl3\u0394E2 mice and by 12 months of age, 100% Mbnl3\u0394E2 lenses examined showed subcapsular or cortical cataracts, with ~60% showing advanced Grade-II and Grade-III cataracts as evaluated using LOCS II photographic grading standards. This is in contrast to Mbnl3+/+ animals, where only one of eight lenses showed grade 1 subcapsular cataracts at 12 months of age. Previous studies have demonstrated that Mbnl1 loss results in subcapsular dust-like opacities and Six5 deficits increase the incidence of nuclear cataracts1718Six5 deficits to the development of DM1 ocular defects is unclear, because in mice Six5 loss results in nuclear cataracts, which are not prominent in DM1 patients. Taken together these data are consistent with Mbnl338kD and Mbnl1 deficits playing an important role in the development of the posterior subcapsular and cortical cataracts observed in DM1.DM1 patients develop an unusual form of ocular cataracts that are located primarily in the posterior subcapsular and cortical regions of the lens1425338kD deficits play a causal role in the development of a set of age-associated pathologies observed in DM1. Our results support the idea that DM1 pathology results from an aggregate of changes in DMPK, SIX5, CUG-BP1, MBNL1, MBNL2 and MBNL3 that occur downstream of the CTG repeat expansion. The potential synergistic interactions that result from these individual alterations and their relationship to the disease trajectory have however yet to be fully understood.Thus, results described in this study demonstrate that Mbnl3Mbnl3\u0394E2 mice we examined splice site choice in RNAs that have been implicated in the development of DM1 pathology. Interestingly, a deficit of Mbnl338kD did not result in DM1-like splice errors in Insr and Tnnt2 in muscle and heart, two splice abnormalities, which can contribute to glucose intolerance and cardiac dysfunction and hypertrophy32373839Ldb3 exon 11 was however observed in Mbnl3\u0394E2 cardiac muscle. This splice error is not reminiscent of the embryonic splice pattern, where inclusion of Ldb3 exon 11 is enhancedLbd3 splice defect observed in DM1 is an aggregate of two events, with Mbnl1 loss either overriding Mbnl3 effects or Mbnl1 loss producing larger changes than Mbnl3 loss. To more generally examine the role of Mbnl338kD depletion in the development of DM1 like splice errors we studied a range of splice events in the heart, skeletal muscle and lens of adult Mbnl3+/+ and Mbnl3\u0394E2 mice and E18 Mbnl3+/+ embryos. No significant difference was observed for these splice events in adult Mbnl3+/+ and Mbnl3\u0394E2 mice. These results are consistent with the observations of Poulos and colleagues who did not detect splice errors as a consequence of Mbnl338kD depletion in E18 forelimbs using RNA-seq analyses38kD target RNAs could underlie the phenotypes observed in Mbnl3\u0394E2 mice34Mbnl3\u0394E2 miceMbnl3\u0394E2 mice. Deciphering the molecular basis of the accelerated onset of age associated pathologies in Mbnl3\u0394E2 mice should provide important new insights into the molecular mechanisms that contribute to DM1.To test the molecular mechanism that underlies the onset of age-associated phenotypes in All experiments were performed in accordance with the institutional guidelines of both the University of Southern California (USC), and the University of California, Los Angeles (UCLA). The USC protocol was approved by the Institutional Animal Care and Use Committee at the University of Southern California, Los Angeles (Protocol number: 10347). The UCLA protocol (99-028) was approved by the UCLA Office of Animal Research Oversight.Left ventricular (LV) size, mass, wall thickness, ventricular and valve function and blood flow were assessed using methods similar to those previously described56Electrocardiograms were obtained for at least 15\u2009minutes from each mouse under light isoflurane anesthesia by inserting two Pt needle electrodes  under the skin in the lead II configuration. The ECG data were amplified (Grass Technologies) and then digitized for analysis with HEM V4.2 software .All morphometry and histology experiments were carried out primarily as described by Jordan and colleagueset al.Glucose tolerance tests were performed according to a protocol modified from Andrikopoulos Mice were fasted in a clean cage with water but no food for 16\u2009hours prior to collecting blood for baseline insulin level measurementsoC heat therapy pad in a Faraday cage with the visual axis of the eye to be measured directed vertically. A 27\u2009g sub-dermal platinum needle electrode was placed in the lower  eyelid. Another identical (ground) electrode was placed in the  ear. The tip of a carbon-fiber wick electrode was placed in contact with the cornea and all three electrodes were connected to a pre-amplifier. A fiber optic cable was centered vertically over the eye to deliver steady background and/or flashes of light (Grass PS22 photo-stimulator powering a Grass Xenon flash unit) to within several millimeters of the corneal surface. Retinal electrical responses to flash stimuli were captured on a Nicolet Electrovisual Diagnostic System for analyses.For ophthalmic examination of the lens, mice were first lightly anesthetized with intra-peritoneal Ketamine HCl (100\u2009mg/kg) and the pupils were dilated with a topical application of phenylephrine hydrochloride 2.5% and tropicamide 0.5% eye drops. Slit lamp examination of the anterior segment was performed using Haag-Streit B900 slit lamp  employing direct and retroillumination techniques. A digital fundus camera system  was used for red reflex and fundus color imaging to evaluate lens clarity. Images were analyzed based on Lens Opacities Classification System-IITotal RNA was prepared using Trizol  according to the manufacturer\u2019s protocol. cDNA was synthesized from 5\u2009\u03bcg of total RNA using the cDNA synthesis kit . cDNAs (150\u2009ng) were used for splicing and qPCR studies with primers and PCR conditions described in How to cite this article: Choi, J. et al. Muscleblind-like 3 deficit results in a spectrum of age-associated pathologies observed in myotonic dystrophy. Sci. Rep.6, 30999; doi: 10.1038/srep30999 (2016)."}
+{"text": "Coordination of cell growth with nutrient availability, in particular amino acids, is a central problem that has been solved by the implementation of complex regulatory cascades. Although the specific regulatory mechanisms differ between kingdoms and species, a common theme is the use of tRNA molecules as sensors and transducers of amino acid starvation. In many bacteria, amino acid starvation leads to high levels of uncharged tRNAs, a signal for the synthesis of the stringent response\u2019s alarmones, halting transcription of stable RNAs and inducing the synthesis of amino acid synthesis pathways  The first modification is found at position 37 of most tRNAs decoding ANN codons, just before the residue that interacts with the first base of the codon. The second modification is found at position 34, the residue that decodes the 3rd base of the codon, of tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG . Both these modifications are found in tRNALysUUU and affect translation efficiency The anticodon-stem-loop (ASL) of tRNAs drives decoding by interacting directly with the mRNA codon . Modifications of the ASL are the most distinct and chemically complex of all RNA modifications 6A and mcm5s2U are both synthesized by elaborate multimeric complexes that were first thought to be transcription factors. The KEOPS complex  in combination with Sua5 is involved in the synthesis of t6A 5s2U Saccharomyces cerevisiae mutants lacking t6A or mcm5s2U display many similar phenotypes. For example, telomere shortening is observed in the absence of either modification 7785s2U deficient strains. Overexpression of tRNALysUUU suppresses the telomere shortening phenotype Schizosaccharomyces pombe, key regulators, including the two subunits Atf1 and Pcr1 of the central regulator of the core environmental stress response 5s2U levels. More generally, proteins involved in translation initiation and elongation are enriched in AAA codons, and reduced in cells lacking mcm5s2U 126A for efficient translation in yeast, but it has been shown in mammals that a decrease in sulfur modified form of t6A (ms2t6A) on tRNALysUUU leads to lower levels of proinsulin t6A or a mcm5s2U deficient S. cerevisiae derivatives is the GCN2 independent activation of GCN4 156A and mcm5s2U.Another phenotype shared by a t5s2U have previously been made: 1) ELP mutants are sensitive to rapamycin and caffeine SIT4 leads to rapamycin hypersensitivity and resistance to zymocin (a tRNase that recognizes mcm5s2U and cleaves tRNALysUUU leading to cell death) et al.5s2U, suggesting that proper tRNA modification affects Gln3 localization and subsequent activation of the nitrogen catabolite repression (NCR) response. Mislocalization of Gln3 occurs both in elp3\u2206 (removing the mcm5 moiety) and in urm\u2206 (s2 moiety). A link between t6A synthesis enzymes and TOR has also recently been seen in flies. The Glavic group showed that lowering levels of one of the subunits of the KEOPS complex (Bud32) reduces TOR phosphorylation of S6K (SCH9 in yeast) required for TOR dependent regulation of ribosome biogenesis The TOR kinases regulate the balance between protein synthesis and protein degradation in response to nutrient quality and TOR activity is inhibited by low nitrogen conditions, caffeine or rapamycin 6A and mcm5s2U is still far from understood at the molecular level. Are the Gcn4 activation and TOR repression in strains lacking these modifications due to direct effects caused by poor translation of specific proteins or are they part of general stress responses caused by translation inaccuracy and protein misfolding? The reality might lie in a combination of responses as in addition to the targeted effects described above, low mcm5s2U increases levels of proteins involved in proteasomal degradation 2 synthesis proteins in S. cerevisiae are unstable at high temperature and reduced levels of the modification lead to activation of the heat-shock response regulator (Hsf1) through the synthesis of unfolded proteins  6A synthesis genes Bud32 and Kae1 in flies activates the Unfolded Protein Response (UPR) 2i6A in mouse led to an increase of the Endoplasmic Reticulum (ER) stress response How Gcn4 and TOR signaling depend on t6A and mcm5s2U modifications of tRNALysUUU draw on primary metabolism intermediates 5s2U thiolation pathway; sulfur starvation reduces the levels of the Uba4 thiolation enzyme and hence the levels of mcm5s2U in yeast 5s2U levels trigger an adaptive response: 1) reduced protein expression due to general slow-down of translation of lysine rich proteins that are found predominantly in the ribosomal machinery; 2) increased levels of methionine, cysteine, and lysine synthesis proteins Because the synthesis of the t6A and mcm5s2U a delicate exercise that will require combining of classical genetic and biochemical studies with the genome wide bioinformatics, proteomic and profiling studies that are now available. These studies are all the more critical as derivatives of both modifications have been linked to human diseases as defects in the ms2t6A synthesis enzyme have been linked to type 2 diabetes 5s2U The complexity of the responses with the interplay of central regulators such as GCN4 and TOR , make the dissection of the roles of t"}
+{"text": "Approximately 30\u201340% of patients undergo leukemic transformation to acute myeloid leukemia (AML) during the course of their disease.n=5) or to AML (n=36) (5 targeting the hotspots of 31 recurrently (>1%) mutated genes in myeloid malignancies  bone marrow samples from 41 MDS patients before (preprogression) and after progression (postprogression) to a more advanced subtype (L (n=36) . The mutgnancies . Dual-bagnancies . The pro3A total of 99 and 122 mutations across 23 genes were identified in preprogression and postprogression samples, respectively . The numASXL1, TET2, SRSF2, U2AF1, RUNX1 and TP53. ASXL1, encoding an epigenetic regulator, was the top ranking mutated gene with a frequency of 44% in preprogression samples and 46% in postprogression samples . Given that the frequency of ASXL1 mutations in MDS ranges from 11 to 15%, and of SF3B1 mutations from 20 to 28% in unselected studies,6 the mutation data concerning these two genes are clearly strikingly different in our study. It should be noted, however, that the patient cohort used in this present study was highly selected\u2014that is, comprising only patients whose disease had progressed. Our data thus indicate that ASXL1 mutations are strongly associated with MDS cases that show disease progression to AML and conversely that SF3B1 mutations are rarely associated with MDS cases that show disease progression. This finding is consistent both with the status of ASXL1 as a poor prognostic marker in MDS7 and with the strong association of SF3B1 mutation with a good prognosis in MDS and with the low-risk MDS subtype refractory anemia with ring sideroblasts.8The most frequently mutated genes (in >15% of samples) were  samples . In cont9 mutations in splicing factor genes  were mutually exclusive, with the only exception being one case with mutations in both U2AF1 and ZRSR2. Mutations of genes involved in splicing , chromatin modification (EZH2 and ASXL1) and DNA methylation  were present in the preprogression and postprogression samples for almost all cases harboring mutations in these genes, and thus represent early events in the disease course in these cases. Interestingly, age-related clonal hematopoiesis, with the majority of variants occurring in DNMT3A, TET2 and ASXL1, has been shown to be a common condition that is associated with increases in the risk of hematologic cancer.11 Mutations of genes involved in transcriptional regulation  and signal transduction (NRAS and KRAS) were found in the postprogression sample only in the majority of cases, suggesting that these are often late events that may co-operate with early events to drive disease progression . The ASXL1\u2013RUNX1 mutated gene association has been previously shown to be significant in studies on large MDS cohorts.3 We also found that all five preprogression samples in our cohort with ZRSR2 mutations also carried ASXL1 mutations, whereas 13 of 36 preprogression samples without ZRSR2 mutations carried ASXL1 mutations . Co-occurrence of mutations in splicing factor genes and in genes involved in epigenetic regulation has been reported previously;3 however, the ASXL1\u2013ZRSR2 association has not been described. In the postprogression samples, there were five cases with mutations of both NRAS and RUNX1 (compared with two cases in preprogression samples), an association reported as significant in a previous MDS study.3 We have also observed co-occurrence of NRAS and ASXL1 mutations in five postprogression samples (compared with two cases in preprogression samples) in our study. Interestingly, NRas mutation and Asxl1 loss co-operate to drive myeloid proliferation and myeloid leukemia in mice,13 and our data on MDS serial samples support this observation. These co-occurring gene mutations may thus have a role in disease progression in MDS.Emerging data suggest that key differences in disease phenotype can be driven by different combinations of comutated genes in MDS.6 In our study, we found that TP53 mutations were present in all four cases in our cohort with abnormalities of chromosome 5, U2AF1 mutations were present in three of five cases with \u221220/del(20q) and two of four cases carrying SETBP1 mutations had \u22127; these association have been previously reported.6Specific associations between mutated genes and chromosomal abnormalities have also been described in MDS.TP53 showing the largest average VAF fold increase (>50%) in postprogression samples compared with preprogression samples among genes mutated in more than five cases, suggesting that this mutation had a major role in driving disease progression in these patients , suggesting that mutations of this gene occurred mainly in a subclone; four of these subclonal mutations expanded with disease progression and two additional TP53 mutations were present in postprogression samples only. Four NRAS mutations identified in preprogression samples were subclonal (VAF range 7\u201330%); eight NRAS mutations were present in postprogression samples only, suggesting that the emergence of new NRAS mutations during the course of the disease may have a role in disease progression. The VAF of all three subclonal mutations in RUNX1 in preprogression samples (VAF range 12\u201319%) increased with disease progression; in addition, four RUNX1 mutations were found in postprogression samples only, suggesting that in the case of RUNX1 both the emergence of new mutations and the expansion of existing ones during the disease course may be involved in progression to AML.The large majority of NRAS mutation expanded during disease progression, whereas the allele burden of mutations in ASXL1, RUNX1 and EZH2 remained constant. In another case, within a background of mutations in ASXL1, EZH2 and ZRSR2, a mutation in SETBP1 emerged mid-progression and a mutation in NRAS was found at the AML stage only. This shows that more precise information on the mutational profile and subclone evolution during disease progression can be obtained by the analysis of multiple serial samples.An additional serial sample was sequenced for four cases in our cohort . In one This is the first study to investigate the mutational status of a large group of MDS patients showing disease progression by the study of serial samples using a NGS myeloid gene panel. We have determined the frequency and chronology of myeloid gene mutation acquisition during disease progression in MDS, identifying specific mutations that are associated with disease evolution, and illuminating the role of subclone development in MDS progression. These data suggest that there are several genetic paths for MDS progression."}
+{"text": "CYP2C19 are very frequent worldwide, particularly in Asia, raising the possibility that reduced metabolism could be advantageous in some circumstances. The evolutionary selective forces acting on this gene have not previously been investigated.Cytochrome P450 CYP2C19 metabolizes a wide range of pharmacologically active substances and a relatively small number of naturally occurring environmental toxins. Poor activity alleles of CYP2C19 genetic markers from 127 Gambians and on 120 chromosomes from Yoruba, Europeans and Asians (Japanese\u2009+\u2009Han Chinese) in the Hapmap database. Haplotype breakdown was explored using bifurcation plots and relative extended haplotype homozygosity (REHH). Allele frequency differentiation across populations was estimated using the fixation index (FST) and haplotype diversity with coalescent models.We analyzed CYP2C19*2 and *3). REHH was high around CYP2C19*2 in Yoruba  and to a lesser extent in Europeans  and Asians . FST at the CYP2C19 locus was low overall (0.098). CYP2C19*3 was an FST outlier in Asians (0.293), CYP2C19 haplotype diversity\u2009<\u2009= 0.037, p <0.001.Bifurcation plots suggested conservation of alleles conferring slow metabolism (CYP2C19*2 is subject to positive selective forces worldwide. Similar evidence was also found for CYP2C19*3 which is frequent only in Asia. FST is low at the CYP2C19 locus, suggesting balancing selection overall. The biological factors responsible for these selective pressures are currently unknown. One possible explanation is that early humans were exposed to a ubiquitous novel toxin activated by CYP2C19. The genetic adaptation took place within the last 10,000\u00a0years which coincides with the development of systematic agricultural practices.We found some evidence that the slow metabolizing allele  CYP2C19 gene which is situated in the CYP2C gene cluster on chromosome 10 where it is in strong linkage disequilibrium with CYP2C9[CYP2C19 exhibits considerable genetic polymorphism giving rise to profound changes in enzyme activity leading to reduced metabolic capacity [The cytochrome P450 enzymes (hereafter referred to as cytochromes) have a wide range of essential biological functions in humans resulting from oxidation of their substrates. CYP2C19 is a key member of the cytochrome family and is responsible for metabolizing a substantial proportion of pharmacologically important compounds  althoughth CYP2C9. CYP2C19capacity . This \u2018pcapacity ,14. HoweCYP2C19 (19154G/A) which creates an aberrant splice site altering the reading frame of the mRNA and introducing a premature stop codon. This allele, designated CYP2C19*2, results in a non-functional protein [CYP2C19*2 is common in Europe , Africa (0.14), China (0.26) and Japan (0.28) [CYP2C19*3 is also common (0.08) [CYP2C19*3 is rare in Europeans and Africans (0\u20130.01) [CYP2C19*3 is a guanine to adenine mutation at position 636 of exon 4 (636G/A) which creates a premature stop codon downstream resulting in a non-functional protein. The prevalence of these slow metabolizing alleles tends to increase moving east so in some parts of East Asia CYP2C19*2 and *3 allele frequencies rise to 0.71 and 0.13 respectively making the slow metabolizer phenotype predominant [17) , Gambians (0.24), Ethiopians and Swedish (0.18) and is rare in Chinese (0.04) [Early investigations into the genetic basis for the poor metabolizer phenotype revealed a single base pair mutation in exon 5 of YP2C19 194G/A whicn (0.08) . CYP2C19(0\u20130.01) -20. The dominant . CYP2C19402C\u2009>\u2009T ) which ie (0.04) .Because of a potential role in metabolizing unknown environmental substances, alleles that cause a change in function of the enzyme would be likely to give an individual a selective advantage or disadvantage in evolutionary terms. For example if the cytochrome is involved in promoting the elimination of a toxic chemical, a gain-of-function allele might confer evolutionary advantage and would thus be positively selected. Conversely if the enzyme catalysed the formation of a toxic compound from an otherwise non-toxic precursor, then alleles conferring low enzyme activity might be selected.New techniques for identifying such selective pressures on genes in specific populations have been developed leading to significant new insights into human genetic diversity . When a In an extension of this idea, relative extended haplotype homozygosity (REHH) is the ratio of the EHH of the core haplotype compared with the EHH of all other haplotypes on the chromosome. The REHH test compares the length of haplotype identity as a function of frequency around each allele compared to an expected distribution . The degRecent studies have highlighted the importance of infectious agents such as malaria ,27 and vCYP2C19 locus for signatures of evolutionary selection. We focussed specifically on the common non-functional CYP2C19 mutations CYP2C19*2 (19154G/A) and *3 (636G/A), measuring their haplotype frequencies and molecular diversity across populations, aiming to identify signals of recent evolutionary selection that might result from exposure to potentially harmful environmental substances.In view of the key role of cytochrome P450 enzymes in metabolism of environmental compounds, we investigated the CYP2C19*2 in the merged genotypic data from Gambians are shown in Figure\u00a0CYP2C19*2/*3-containing alleles are described in Figure\u00a0Bifurcation plots for the common loss-of-function allele CYP2C19*2 (AGT) suggests some evidence of positive selection, shown by the predominance of thick branches. This haplotype, present at a frequency of 0.15, had an REHH of 2.7 at \u221268.3\u00a0kb from the core region which is supportive of the suggestion of positive selection for CYP2C19*2. Since our genotyping data did not extend further than \u221268.3\u00a0kb from the core haplotype it was not possible to examine REHH over longer stretches of the genome in the Gambian study group.The bifurcation plot for the haplotype carrying CYP2C19*2 AGT-haplotype was present in the Yoruba at a similar frequency to Gambians (0.14) and displayed extended homozygosity evidenced by conservation on the bifurcation plot and an REHH of 8.3 at 133.3\u00a0kb from the core. This implies that the slow metabolism allele can confer an evolutionary advantage and confirms the initial findings in Gambians. The magnitude of the REHH for CYP2C19*2 in Yoruba is similar to that observed for G6PD-202A (glucose-6-phosphate dehydrogenase deficiency) which is thought to confer a 50% reduction in risk of malaria [We therefore examined the HapMap data for the Yoruba to obtain more extensive genomic data from people living in a nearby area of West Africa Figure\u00a0B. The sa malaria ,33.CYP2C19*2 for positive selection in Europeans showed similar patterns to those observed in Yoruba . The bifurcation plot for the Japanese and Han Chinese showed clear breakdown of haplotype homozygosity arising from development of a new mutation, depicted by thinning of the branch , which also codes for an inactive enzyme only found in Asia shows some evidence of extended homozygosity  was 0.016 implying that only 1.6% of human genetic diversity at CYP2C19*2 is a result of genetic differentiation among the four populations studied. The CYP2C19*3 allele in Japanese and Han Chinese had a high FST implying unopposed positive selection in this population. Pairwise FST values are given in Additional file CYP2C alleles screened in the Gambians and HapMap populations.FCYP2C19  may be subject to selective forces in recent human evolution. Slow metabolizing haplotypes of CYP2C19 showed extended homozygosity which results from recent positive selection. We inferred this initially in the Gambian population for CYP2C19*2 and these findings were confirmed by analysis of genotypic data in Yoruba, from a nearby region of West Africa. We observed a similar pattern of haplotype homozygosity in Europeans, however the CYP2C19*2 EHH is more striking in Yoruba suggesting stronger positive selection in the West African population. Interestingly the fast metabolizing CYP2C19*17 was also under positive selection in Europeans. The REHH statistic observed for CYP2C19*2 in Yoruba (REHH 8.3) is of a similar magnitude to that seen in the same ethnic group for G6PD [We found some evidence that non-functional haplotypes of for G6PD . SelectiCYP2C19*2 accounts for the poor metabolizing phenotype in Europeans and in people of African descent, whilst CYP2C19*3 contributes to the poor metabolizer phenotype only in Asia. In Asian populations we found some evidence that CYP2C19*3 is also positively selected. This suggests that the CYP2C19 poor metabolism phenotype confers an evolutionary advantage in Asia in addition to Africa and Europe even though some alleles responsible for that phenotype differ between the three continents.ST values estimated for the CYP2C19 SNPs are lower than the average FST values for autosomal SNPs in worldwide human populations which is approximately 0.123. The CYP2C19 FST values are consistent with the low FST observed for many of the genes involved in immunity such as the major histocompatibility complex (MHC) and beta-globin gene which are under balancing selection [ST is an indication that balancing or species-wide directional selection has taken place at the CYP2C19 locus, in contrast to the force of geographically-restricted directional selection that leads to high FST values. Thus one would infer from the long extended haplotype and low FST for the slow-metabolizer CYP2C19*2 allele that CYP2C19*2 is still evolving and has not yet reached fixation.All Felection . The lowCYP2C cluster in the past have been relatively limited. Vormfelde and colleagues analysed selection signals in CYP2C9[CYP2C9*3 appeared to be under positive selection. There is a high degree of Linkage Disequilibrium (LD) in the CYP2C cluster so it is possible that the effects we saw on CYP2C19 might reflect selection of alleles of CYP2C9 and CYP2C8[CYP2C19 genomic region. CYP2C9*3 was not screened in this project as the variant has not been found in sub Saharan Africa.Investigations of evolutionary selection of alleles in the in CYP2C9 and shownd CYP2C8 however CYP2C19*2 poor functional variant worldwide is in accordance with the recent findings by Pimenoff and colleagues who used a similar technique (EHH) and haplotype structure to describe selection pressure on poor activity variants CYP2C19*2 and CYP2C9*3 in Europeans [CYP2C19*3 to be under selective pressure in Asians and the high activity CYP2C19*17 in Europeans. Poor activity alleles especially are favoured in recent human evolution, being selected in Africa, Europe and Asia. A further analysis of cytochrome gene evolutionary selection in the indigenous American populations would give interesting additional information.Our finding of positive selection for uropeans . Our stuCYP2C19 alleles are likely to have been operating over the last 10,000\u00a0years [The population selection forces acting on 00\u00a0years ,36. An aCYP2C19. The period of selection would be likely to correspond with the end of the most recent glacial age and beginning of the Mesolithic era (Middle Stone Age), from 8500 BC to 4000 BC. Over this period there is substantial archaeological evidence of developing systematic agricultural practices in Asia and the Middle East in contrast to the previous hunting and gathering lifestyle of early humans [It is interesting to speculate what biological forces are responsible for evolutionary selection of inactive alleles of y humans .Fusarium strains that contaminate cereals and grains [The storage of grain and tubers from one growing season to another, which is essential for effective agriculture, would have exposed early humans to a range of novel toxins derived from fungi growing on stored foodstuffs. Activation of mycotoxins by cytochrome P450 enzymes is well-described ,39 and td grains .In addition to mycotoxins, other classes of harmful environmental compounds may have been encountered for the first time as a result of developing agricultural practices. For example aryl hydrocarbons formed from incomplete combustion of carbon are highly toxic when partially activated by CYP2C19 and might be produced by the use of fire to clear land for crops .CYP2 family occurred nearly 400 million years ago with more than 50 gene duplication events [CYP evolution started much earlier >600 million years ago at the same gene locus referred to as the cytochrome P450 genesis locus from where all CYP clans or families emerged [CYP2C19*2/*3[Earlier speculations by Nebert suggested that rapid evolution of drug metabolizing enzyme (DME) genes and receptor genes occurred as a result of the interaction between animals as they moved on to land and the plants that they encountered there. An explosion of new animal genes in the n events . Nelson  emerged . The DME emerged . Such fa2C19*2/*3-44.CYP2C19*2 rs4244285 with the regulation of blood pressure [The physiological functions of CYP2C19, particularly in the synthesis of steroid hormones, could also potentially be important in increasing survival fitness . Gomes apressure . CYP2C19pressure ,49 and vpressure . These fCYP2C19 seems to be persuasive. The magnitude of the evolutionary pressure appears to be similar to that exerted on the human genome by infectious diseases. These environmental and/or physiological forces that shaped the cytochrome repertoire of modern humans have important consequences for drug metabolism in the present day [Whilst the mechanism for positive selection of individuals with poor CYP2C19 activity remains unknown, the evidence for such selection in The selective forces that we observed are temporally linked to the development of systematic agricultural practices by early humans and CYP2C physiological functions although it is not possible to infer a direct causal association from our data. It would be useful to extend these studies to other populations where different agricultural practices might have exposed humans to a different range of potentially harmful environmental chemical compounds. Similar signatures of selection might be found for other cytochrome P450 enzymes that activate known mycotoxins ,38. In aFurther work could examine the co-evolution of cytochromes with other elements of the metabolic pathways involved in detoxification of xenobiotic substances and metabolism of drugs. Some speculative studies indicate that considerable synergisms may exist between certain isoforms of cytochromes and transporter molecules that regulate influx of their substrates into cells . If thisInclusion of longer stretches of the genome in increased numbers of individuals could identify long range haplotypes in cytochrome gene clusters which have been positively selected. These haplotypes delimited by rapidly decaying LD would identify biologically important combinations of alleles across the gene cluster . Such haCYP2C19 that have low activity in the metabolic transformation of xenobiotic compounds it seems reasonable to suggest that some environmental agent would be responsible for exerting this pressure and shaping the cytochrome profile of modern humans. This agent could be a known substrate for CYP2C19 which has a metabolite with unrecognised toxic effects or alternatively a known toxin which is activated by the enzyme by a novel molecular mechanism. Either of these hypotheses might provide a fruitful starting point for further biochemical and toxicological research. Such studies might cast further light on human evolution and could potentially identify substances not previously known to be toxic.Since we have identified some evidence of global evolutionary selective forces in favour of alleles of Many forces shape the topography of the human genome and strong natural selection resulting from infectious diseases is well\u2013recognised. Here we show that environmental chemicals could also exert a similar effect on human evolution.CYP2C19 locus and hence strongly influenced the drug metabolizing profile of modern humans.We speculate that a ubiquitous environmental compound may be rendered toxic by the activity of CYP2C19 and thus early humans with poor CYP2C19 activity had a survival advantage. This genetic adaptation took place within the last 10,000\u00a0years which coincides with the development of systematic agricultural practices. These evolutionary forces, which are of a similar magnitude to those exerted by infectious diseases, could have arisen from exposure to a novel toxin perhaps arising from stored foodstuffs. Selective pressure from this toxin may have driven allelic differentiation at the Eighty-five Gambian blood donors from Sukuta and 42 from Brikama (Western Region), Njaba Kunda and Farafenni (Northern Region) gave informed consent for genetic screening and analysis. The subjects from Sukuta participated in an investigation of nucleotide diversity of the TNF gene region  and the Venous blood was collected in EDTA tubes and deoxyribonucleic acid (DNA) extracted using Nucleon kits BACC3  according to the manufacturer\u2019s protocol. The DNA was quantified using Picogreen (Molecular Probes\u00ae) and the NanoDrop 1000\u2122 spectrophotometer (Thermo Scientific) and stored at \u221220\u00b0C until required.CYP2C19 had been previously characterized in fine detail in the adult study group and all common fast and slow metabolizing alleles identified [\u00ae drug metabolizing assay mixes for SNP genotyping were used to screen functional polymorphisms and amplification refractory mutation system PCR to genotype promoter and intronic polymorphisms in CYP2C19 and CYP2C9 in all the subjects. The 13 SNPs IDs genotyped were:Metabolic phenotype for CYP2C19: rs7067866, rs11568729, rs4417205, rs4986894, rs3758580 (mRNA990 *2A*2B), rs4986893 (mRNA636 *3), rs17884712 (mRNA431 *9), rs4244285 (mRNA681 *2), rs12248560 (*17) and1. CYP2C9: rs1799853 (*2), rs7900194 (*8), rs2256871 (*9), rs28371686 (*11).2. Genotypic data from the two Gambian study groups were pooled to give a sample size of 127 subjects.Ethical approval was obtained from the Medical Research Council (MRC) Scientific Coordinating Committee and the MRC/Gambia Government Joint Ethics Committee . Consent was written in English and explained in the local language of the subjects by an interpreter and the response was documented in English on the consent form.CYP2C19*2 in the Gambian population using SWEEP [CYP2C19*3 was not present in any Gambian participant. Missing allelic data were filled in using fastPHASE version 2.3 [CYP2C19*2 locus with the core haplotype rs4244285-rs4417205-rs3758580 and for CYP2C19*3 where it was present (rs12778026-rs4986893-rs4304692). We generated REHH and bifurcation plots from rs7067866 (96501916) to rs9332198 (NCBI build35 96731487) for the Yoruba, to rs1934967 (96731416) for CEU and to rs9332198 (96731487) for the Han Chinese. These plots were then examined for signals of selective pressure.First we constructed relative extended haplotype homozygosity (REHH) and haplotype bifurcation plots to look for positive selection of ng SWEEP . CYP2C19sion 2.3 . We thension 2.3 . These dST statistic was calculated to summarise allele frequency differentiation between populations using FSTAT 2.9.3 [The FAT 2.9.3 . HaplotyAT 2.9.3 . The inpCYP2C9: Cytochrome P450 2C9; CYP2C19: Cytochrome P450 2C19; EHH: Extended haplotype homozygosity; LD: Linkage disequilibrium; REHH: Relative extended haplotype homozygosity; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interests.RTW, REJ and AW contributed to study conception and design. REJ and FSJ participated in the acquisition of data. RTW, REJ and AW took part in data analysis. REJ and RTW drafted the manuscript. REJ, AW, SOS, KJL and RTW interpreted the results. All authors have read and approved the final version of the manuscript.ST for CYP2C19 alleles across Gambians, Europeans, Japanese and Han Chinese and Yoruba.FClick here for file"}
+{"text": "The transfer of patients in extracorporeal membrane oxygenation (ECMO) from a peripheral hospital to a tertiary center represents a high-risk situation of adverse events . The aimWe collected data for the ECMO Regional Reference Centre Careggi Hospital activity from September 2009 to June 2014. In this study, 57 transfers were examined. The ECMO service is activated by a telephone call from a peripheral hospital. The team is represented by an intensivist, a heart surgeon, a cardiologist, a perfusionist and an intensive care nurse, all previously trained in the management of patients with ECMO. Medical personnel and the necessary equipment are transported by an ambulance and a van, specially designed and equipped for the transfer of patients with ECMO.In this study, 57 patients transferred from the peripheral hospital to the ECMO center were examined; in all cases the ECMO system was implanted in the peripheral hospital (54 venovenous ECMO and three venoarterious ECMO). On average, trails were 271 km \u00b1 304 (round trip) (minimum 14 km to maximum 939 km). The activation time from the call to the ambulance departure from our hospital was an average of 2 hours 27 minutes 13 seconds \u00b1 1 hour 25 minutes 35 seconds. Transfer duration (from activation to return to the ECMO center) was an average of 8 hours 25 minutes 6 seconds \u00b1 3 hours 27 minutes 58 seconds (minimum 3 hours to maximum 16 hours 55 minutes). The stop time  was an average of 3 hours 53 minutes 40 seconds \u00b1 1 hour 6 minutes 35 seconds (minimum 2 hours 5 minutes to maximum 7 hours 30 minutes). Major complications related to malfunctions of the devices during transport were not recorded; in some cases it was necessary to manage minor complications .Some studies have found several complications during transfer of patients in ECMO . In our"}
+{"text": "In Southern Europe Pru p3 is the primary sensitizer of plants fruit and it is responsible of severe reactions. Specific immunotherapy (SIT) brings a new perspective to treat those patients. There is a lack of knowledge regarding cellular responses that include changes in the basophil activation during the IT. We aim to analyse early changes in the basophil response to Pru p 3 and other related allergen (Ara h 9) after the first month of sublingual immunotherapy (SLIT).Forty-six peach allergic patients confirmed by positive specific IgE determined by skin prick test or fresh peach (prick-by-prick), ImmunoCAP IgE and/or a double blind placebo control food challenge with peach. Basophil reactivity was determined by the basophil activation test (BAT) with Pru p 3 and Ara h 9 at two concentrations, 1 and 0.1 \u00b5g/ml, before and after 1month of SLIT.Twenty one patients evaluated (45%) performed anaphylaxis and 25 (55%) urticaria and/or angioedema. The 82,6% showed sensitization to other plant foods proteins and 69,5% showed sensitization to pollens. The BAT was done in 25 patients with first month of SLIT completed. The 28% patients have an increase of Pru p 3 reactivity. The 36% patients showed same reactivity after first month and 36% presented a decreased reactivity to Pru p3. Similar results were obtained for Ara h 9 in those patients.Preliminary result disclosed that percentage of patients who underwent changes in BAT reactivity to Pru p3 and Ara h 9 was similar. There have been no differential clinical pattern in the groups studied after one month of SLIT. The BAT shows a good correlation between both Pru p 3 and Ara h 9."}
+{"text": "To the Editor: Novel highly pathogenic avian influenza (HPAI) viruses ofsubtypes H5N2, H5N8, and H5N1 have recently caused numerous outbreaks in commercialpoultry farms in the United States and Canada . Of 3,300 samples tested, 1sample tested positive for HPAI virus subtype H5  Figure 1Minnesota and Iowa lie within regions where migrating waterfowl spend their breedingseason, and waterfowl densities on commercial poultry farms are particularly high Figure 2Of particular note, outbreaks in poultry were densely concentrated within Minnesotaand Iowa in a spatial pattern inconsistent with the much more geographicallydispersed spread of infection in wild birds. The magnitude and clustereddistribution of poultry outbreaks are suggestive of local spread, rather thanmultiple introductions from passing migratory waterfowl. Genetic analyses havesimilarly shown evidence for concurrent multiple introductions as well as commonsource exposures, and surveys of affected farms have shown that local spread couldbe facilitated by the sharing of equipment by multiple farms or through animalsentering barns  virus, subtype H5, inNorth America"}
+{"text": "SLC4A3 has been shown to cause retinal degeneration in a genetically engineered knockout mouse, and in a naturally occurring form of canine progressive retinal atrophy considered to be the equivalent of retinitis pigmentosa in humans (RP). This study was undertaken to investigate if SLC4A3 coding variants were implicated in human retinal degeneration. SLC4A3 exons were amplified and sequenced in 200 patients with autosomal recessive retinal degeneration who had no known molecular diagnosis for their condition, which included 197 unrelated individuals with suspected RP and three individuals with other forms of retinal disease. Three rare variants were identified that were predicted to be potentially pathogenic, however each variant was heterozygous in a single patient and therefore not considered disease-causing in isolation. Of these three variants, SNP-3 was the rarest, with an allele frequency of 7.06x10\u22125 . In conclusion, no compound heterozygous or homozygous potentially pathogenic variants were identified that would account for recessive RP or retinal degeneration in this cohort, however the possibility remains that the rare variants identified could be acting with as yet undiscovered mutations in introns or regulatory regions. SLC4A3 remains an excellent candidate gene for human retinal degeneration, and with the advent of whole exome and whole genome sequencing of cohorts of molecularly unsolved patients with syndromic and non-syndromic forms of retinal degeneration, SLC4A3 may yet be implicated in human disease.The online version of this article (doi:10.1186/s12952-016-0054-z) contains supplementary material, which is available to authorized users. SLC4A3 , encodes the anion exchanger 3 (AE3) protein, which mediates Cl\u2212/HCO3\u2212 exchange across cellular membranes [Slc4a3 identified SLC4A3 as a candidate gene for human vitreoretinal degenerations based on their findings of blindness and retinal degeneration in knockout mice [Slc4a3\u2212/\u2212 mice at four months of age had no gross retinal abnormalities; However, ERG analysis revealed an inner retina defect from birth (reduced b-wave and flicker amplitudes), leading to phototransduction failure at four months (reduced a-wave amplitude). At 4\u20136 months the number of apoptotic nuclei observed by TUNEL labelling increased. By eight months, pathological signs of photoreceptor degeneration were observed including dense astrocytic processes wrapped around inner retina vessels , small diameter major blood vessels, disorganised astrocytic processes at the optic nerve head and rod bipolar cell dendrites aberrantly sprouted into the outer nuclear layer [embranes , and hasSLC4A3 is associated with a form of naturally occurring autosomal recessive (AR) Progressive Retinal Atrophy (PRA) in the Golden Retriever dog breed, known as GR_PRA1 [In addition, we have previously shown that a mutation in  GR_PRA1 .PRA is widely considered to be the veterinary equivalent of Retinitis Pigmentosa (RP) in humans. RP is the name given to a group of inherited retinal degenerations which affects 1 in 3500 to 4500 people . PhotoreRPE65 have been implicated in AR retinal degeneration (LCA) in dogs [RPGR cause XLPRA [At least 17 naturally occurring dog models with retinal degeneration have been described with equivalent human disease (reviewed in ). These  in dogs , 11 and  in dogs , 13, and in dogs \u201320. Simise XLPRA \u201323 and ase XLPRA \u201326. Genese XLPRA . Importase XLPRA .SLC4A3 have been described in humans: a full-length (SLC4A3fl1) isoform comprised of one non-coding (5\u2019UTR) and 22 coding exons and a cardiac (SLC4A3c) isoform with 18 coding exons [SLC4A3fl1 results in a further isoform (SLC4A3fl2) that differs by 81\u00a0bp. SLC4A3fl1 (Genbank: NM_201574.2) encodes a 1259 amino acid protein  is the shorter version and encodes a protein of 1232 amino acids. In SLC4A3c exon C1 replaces exons one to six of the full-length transcripts, and encodes a smaller protein product of 1034 amino acids [SLC4A3 gene, Ala867Asp, has been associated with idiopathic generalized epilepsy (IGE) in humans, with carriers exhibiting an increased risk of developing IGE [SLC4A3 has not been implicated in human retinal disease. Evidence from the mouse and canine disease models suggest the SLC4A3 gene is an excellent candidate for human retinal degeneration. We therefore screened SLC4A3 in a cohort of human patients with predominantly recessive retinal degeneration currently lacking a molecular diagnosis in order to determine whether mutations in this gene cause a significant proportion of retinal degeneration in humans.Two main isoforms of ng exons . These a SLC4A3fl isoform  SLC4A3fl isoform no acids . A rare ping IGE , but SLCThe recruitment of all patients was part of a study protocol that adhered to the tenets of the Declaration of Helsinki and had received approval from the Moorfields Eye Hospital Research Ethics Committee. Written, informed consent was obtained from all participants prior to their inclusion in this study with parental written consent provided on behalf of any minors involved.SLC4A3 variants. Of these, 192 probands were affected with progressive retinal degeneration, consistent with a diagnosis of either retinitis pigmentosa or cone-rod dystrophy with presentation in adulthood (during or after the second decade). Eight additional patients with various forms of retinal degeneration were also selected for screening. In each of these 8 cases, autozygosity mapping previously conducted at UCL Institute of Ophthalmology  had identified large regions of homozygosity that included the genomic region containing SLC4A3, amongst other genes  [Two hundred affected unrelated individuals ascertained from the clinics of Moorfields Eye Hospital were evaluated for m (ExAC)  was usedSLC4A3 exons , SLC4A3fl2(GenBank: NM_005070.3) and SLC4A3c [SLC4A3 exons were amplified by PCR using HotStarTaq Plus DNA Polymerase (Qiagen) in genomic DNA. PCR products were purified using PCR\u03bc96 filter plates (Millipore). Amplification products were sequenced using BigDye Terminator v3.1 (Applied Biosystems), and sequence product was purified using the Montage SEQ96 Cleanup Kit (Millipore), then run on an ABI 3730 Genetic Analyzer. Sequence traces were assembled, analysed and compared with the human reference sequence (GRCh37) using the Staden Package [Primers for amplification and sequencing of  SLC4A3c  were des SLC4A3c . SLC4A3  Package .The potential pathogenicity of variants identified was assessed with various bioinformatics tools  have previously been identified and have entries in the dbSNP database, and 31 are present in the Exome Aggregation Consortium (ExAC) database. Copy number variants, such as large deletions and insertions within introns, or affecting upstream promoter sequences, were not assessed in this study.Sequencing of all known exons and intron-exon boundaries of SLC4A3 isoforms, while SNPs_2 and _3 affect the full-length and cardiac isoforms  and/or prediction of pathogenicity, three rare variants remained that were predicted to affect exon splicing and/or change the amino acid sequence of the protein Table\u00a0. SNP_1 aSNPs_1  and _2  are non-synonymous SNPs that result in amino acid changes that are predicted to be pathogenic by at least one of the in silico prediction tools  to be potentially deleterious. Three individuals with AR RP carry one copy of the minor allele at one of these loci  was found in the heterozygous state only at a frequency of 8.66x10\u22125. The relatively high frequency of SNP_1 in the control exomes and the observation that it was predicted to be potentially pathogenic by only one of the in silico prediction tools suggests the variant is unlikely to play a role in retinal disease. SNP_2 has a relatively high allele frequency and occurs in the homozygous state in the control cohort. This suggests that this rare variant is also unlikely to cause disease in isolation, even though this variant was predicted to be potentially pathogenic by all four in silico prediction tools. Finally, SNP_3 has a very low allele frequency in the control exome data (8.66x10\u22125), but is predicted to affect exon splicing only. The SNPS predicted to affect exon splicing , human SLC4A3 mRNA is also likely to be undetectable and this was not pursued. In vitro assays could be useful to assess pathogenicity of SNP variants. However, it is difficult to select which variants to test, and the assay to use until more convincing evidence is presented that SLC4A3 is involved in human retinal disease. Such a study would therefore be premature at this stage.All three rare variants were also seen in the control datasets. SNP_1  and SNP_2  were found in the homozygous state in a single control exome each, as well as in the heterozygous state in multiple exomes, at a frequency of 7.89x10SLC4A3 identified in this study are unlikely to cause AR RP in the cohort screened in isolation, we cannot fully exclude these variants or the gene as a candidate for retinal degeneration. Potentially pathogenic rare variants in intronic regions or upstream elements that were not screened for mutations by sequencing could affect exon splicing or regulation of gene expression. In addition, it is possible that disease-causing mutations in this gene are extremely rare and essentially private mutations affecting only one or two affected individuals, as is increasingly the case in consanguineous families with a recessive condition, and that these individuals have yet to be screened. For example, a PRCD gene mutation (p.C2Y) was reported to cause PRA in dogs and RP in one person. However, no other disease-causing mutations were found in PRCD in a further 1240 RP patients screened [PRCD has since been identified in an isolated Muslim Arab village in Northern Israel; This founder mutation was homozygous in all 18 RP-affected individuals, but not in any of the 28 unaffected family members [PRCD [SLC4A3 actually causes a syndromic or non-syndromic form of retinal degeneration in humans that would not be clinically classified as RP. Little is known about the phenotype associated with GR_PRA1 in dogs beyond ophthalmoscopic observations. Clinically the fundus appears identical to other forms of PRA and histological and detailed ERG analysis has not been reported. In addition, the age of onset is difficult to define, although the age at which dogs with GR_PRA1 are diagnosed is typically 6\u20137 years [SLC4A3 knockout mouse in which a selective inner retina defect is followed by photoreceptor degeneration at eight months [SLC4A3. Alvarez and colleagues concluded that their results in the knockout mouse linked aberrant SLC4A3 to vitreoretinal degeneration [SLC4A3 than AR RP, but other phenotypes should not be excluded from future studies.While it appears that the variants in screened . Disease members . While trs [PRCD , 50. FinSLC4A3 has been shown to cause retinal disease in the mouse and dog, making the gene a strong candidate for human retinal disease. Three rare variants predicted to be potentially pathogenic were identified in the SLC4A3 gene in a AR RP cohort, however all three variants were present in the heterozygous state, and therefore not disease-causing in isolation. Nevertheless, we could not discount the possibility that these variants have some role in the disease that we have yet to decipher. The SLC4A3 gene remains an excellent candidate gene for human retinal degeneration and the variants identified will help to build a picture of its potential contribution.Aberrant The recruitment of all patients was part of a study protocol that adhered to the tenets of the Declaration of Helsinki and had received approval from the Moorfields Eye Hospital Research Ethics Committee. Written, informed consent was obtained from all participants prior to their inclusion in this study with parental written consent provided on behalf of any minors involved.Not applicable.The datasets supporting the conclusions of this article are included within the article and its Additional files"}
+{"text": "Electronic health records (EHRs) with poor usability present steep learning curves for new resident physicians, who are already overwhelmed in learning a new specialty. This may lead to error-prone use of EHRs in medical practice by new resident physicians.The study goal was to determine learnability gaps between expert and novice primary care resident physician groups by comparing performance measures when using EHRs.We compared performance measures after two rounds of learnability tests . In Rounds 1 and 2, 10 novice and 6 expert physicians, and 8 novice and 4 expert physicians participated, respectively. Laboratory-based learnability tests using video analyses were conducted to analyze learnability gaps between novice and expert physicians. Physicians completed 19 tasks, using a think-aloud strategy, based on an artificial but typical patient visit note. We used quantitative performance measures , a system usability scale (SUS), and qualitative narrative feedback during the participant debriefing session.There was a 6-percentage-point increase in novice physicians\u2019 task success rate  and a 7-percentage-point increase in expert physicians\u2019 task success rate ; a 10% decrease in novice physicians\u2019 time-on-task  and 21% decrease in expert physicians\u2019 time-on-task ; a 20% decrease in novice physicians mouse clicks  and 39% decrease in expert physicians\u2019 mouse clicks ; a 14% increase in novice mouse movements  and 14% decrease in expert physicians\u2019 mouse movements . The SUS measure of overall usability demonstrated only minimal change in the novice group  and no change in the expert group .This study found differences in novice and expert physicians\u2019 performance, demonstrating that physicians\u2019 proficiency increased with EHR experience. Our study may serve as a guideline to improve current EHR training programs. Future directions include identifying usability issues faced by physicians when using EHRs, through a more granular task analysis to recognize subtle usability issues that would otherwise be overlooked. Health information technology\u2019s (HIT) functionality in clinical practice is expanding and physicians are increasingly adopting EHRs as a result of the financial incentives guaranteed by Centers for Medicare & Medicaid Services (CMS) . MeaningAccording to an EHR user satisfaction survey completed in 2012 by 3088 family physicians, approximately 62% of survey respondents were not satisfied with many of the best-known EHR systems, and EHR vendor support and training were the areas with lowest satisfaction ratings . MultiplPrevious studies have shown the importance of usability evaluation in the EHR adoption and implementation process. Current best practices promote the use of cognitive approaches to examine human-computer interactions in EHR systems\u00a0,48-50. KEHRs with poor usability present steep learning curves for new resident physicians, who are already overwhelmed in learning a new specialty. This may lead to error-prone use of EHRs in medical practice by new resident physicians. Identifying and addressing early barriers in the learning environment can help improve the overall capacity of new physicians and save costs for organizations. The objective of this study was to determine the difference in learnability by comparing changes in performance measures between expert and novice primary care physicians 3 and 7 months after 2 rounds of learnability tests . We analyzed learnability by addressing 2 specific research questions: (1) Do performance measures of expert and novice physicians improve after 3 and 7 months of EHR experience? and (2) Does the learnability gap between novice and expert physician groups change after 7 months of EHR experience?To determine learnability gaps between expert and novice physicians when using EHRs, data were collected through learnability testing using Morae video analysis software (TechSmith). Twelve family medicine and 4 internal medicine resident physicians performed 19 artificial, scenario-based tasks in a laboratory setting. Four types of quantitative performance measures, a system usability scale (SUS), a survey instrument , and a qThis study took place at the University of Missouri Health System (UMHS), which is a 536-bed, tertiary-care academic medical hospital located in Columbia, Missouri. In 2012, UMHS had approximately 553,300 clinic visits and employed more than 70 primary care physicians. The Department of Family and Community Medicine (FCM) runs 6 clinics, while the Department of Internal Medicine (IM) oversees 2 primary care clinics . The HeaThere is presently no evidence-based approach to measure a user\u2019s EHR experience; therefore, novice and expert physicians were distinguished based on clinical training level and number of years using the EHR. This decision was based on a discussion with an experienced physician champion (JLB). This study will examine and confirm if after 1 year of EHR use, resident physicians have gained sufficient skills to be considered an expert . Thus, 1In Round 1, 10 novice physicians and 6 expert physicians participated in the study. Out of the 10 novice physicians in Round 1, 7 were from family medicine and 3 from internal medicine. Of the 10 novice physicians, 6 (60%) were male, 8 (80%) identified their race as white, 1 (10%) identified as Asian, and 1 (10%) identified as both Asian and white. The age of novice physicians ranged from 27 to 31 and the mean age was 28 years. In Round 1, 4 (40%) novice physicians had no experience with an EHR other than the one at UMHS, 2 (20%) had less than 3 months of experience, 1 (10%) had 7 months to 1 year of experience, and 3 (30%) had over 2 years of experience with an EHR other than the one at UMHS. In this study, 5 family medicine and 1 internal medicine expert physicians participated in the study. Of the 6 expert physicians, 5 (83%) were female and all (100%) identified their race as white. In this study, 2 did not provide information on their date of birth and EHR experience and were not included in the calculation of age range, mean age, and EHR experience. The age of expert physicians ranged from 30 to 33 and the mean age was 31 years. In this study, 1 (17%) expert physician had no experience with an EHR other than the one at UMHS, 1 (17%) had 7 months to 1 year of experience, and 2 (33%) had over 2 years of experience with an EHR other than the one at UMHS.Of the 8 novice physicians and 4 expert physicians who participated in Round 1 also participated in Round 2 of the study. A total of 2 novice and 2 expert physicians who participated in Round 1 declined participation in Round 2. Conducting 2 rounds of data collection was a major strength of this study, because it allowed us to measure valid learnability. Out of the 8 novice physicians in Round 2, 5 were from family medicine and 3 from internal medicine. Of the 8 novice physicians, 5 (63%) were male, 8 (75%) identified their race as white, 1 (13%) identified as Asian, and 1 (13%) identified as both Asian and white. The age of novice physicians ranged from 27 to 30 and the mean age was 28 years. In Round 2, 3 (38%) novice physicians had no experience with an EHR other than the one at UMHS, 2 (25%) had less than 3 months of experience, 1 (13%) had 7 months to 1 year of experience, and 2 (25%) had over 2 years of experience with an EHR other than the one at UMHS. Four family medicine expert physicians participated in the study. All 4 (100%) were female and all (100%) identified their race as white. The age of expert physicians ranged from 30 to 33 and the mean age was 31 years. In this study, 1 (25%) expert physician had no experience with an EHR other than the one at UMHS, 1 (25%) had 7 months to 1 year of experience, and 2 (50%) had over 2 years of experience with an EHR other than the one at UMHS. Because of the small sample size, we did not attempt to control for age or gender.Two sets of artificial but realistic scenario-based tasks were used in the study. The tasks were created based on discussion with an experienced physician champion (JLB) and 2 chief resident physicians from both participating departments . When completing Round 1 of the learnability test, resident physicians were given a scenario for a \u201cscheduled follow-up visit after a hospitalization for pneumonia.\u201d When completing Round 2 of the learnability test, resident physicians were given a scenario for a \u201cscheduled follow-up visit after a hospitalization for heart failure.\u201d While different, these 2 scenarios were equivalent in difficulty, workflow, and functionalities used. These scenarios were employed to assess physicians\u2019 use of the EHR with realistic inpatient and outpatient information. We included 19 tasks that are generally completed by both novice and expert primary care physicians. These tasks also met 2014 EHR certification criteria 45 CFR 170.314 for meaningful use (MU) Stage 2 [1. Start a new note (\u00a7170.314[e][2]).2. Include visit information (\u00a7170.314[e][2]).3. Include chief complaint (\u00a7170.314[e][2]).4. Include history of present illness (\u00a7170.314[e][2]).5. Review current medications contained in the note (\u00a7170.314[a][6]).6. Review problem list contained in the note (\u00a7170.314[a][5]).7. Document new medication allergy (\u00a7170.314[a][7]).8. Include review of systems (\u00a7170.314[e][2]).9. Include family history (\u00a7170.314[a][13]).10. Include physical exam (\u00a7170.314[a][4] and \u00a7170.314[e][2]).11. Include last comprehensive metabolic panel (CMP) (\u00a7170.314[b][5]).12. Save the note.13. Include diagnosis (\u00a7170.314[a][5]).14. Place order for chest X-ray (\u00a7170.314[a][1] and \u00a7170.314[e][2]).15. Place order for basic metabolic panel (BMP)  to supplement the performance measures. The SUS is a 10-item survey measured on a Likert scale that provides fairly robust measures of subjective usability and is a widely used, validated instrument in HIT evaluation ,55,66. TTwo rounds of data collection were scheduled to measure learnability by comparing whether participants\u2019 performance measures  improved and if participants experienced fewer usability issues with longer exposure to the system. Learnability pertains to the amount of time and effort needed for a user to develop proficiency with a system over time and after multiple use . The 2 gbetween comparison of 2 within comparisons. Therefore, we measured the difference between the novice and expert resident physician groups and the difference within novice and expert physician groups, 3 and 7 months after EHR training. Comparisons of learnability between the 2 groups were between comparisons. Time-on-task, mouse clicks, and mouse movements were measured while users interacted with the EHR system and performance measures were calculated automatically by the Morae Manager usability analysis software program. Percent task success was calculated by creating subtasks out of each task and then identifying each subtask the physician completed successfully. For example, for Task 8 (Include review of systems) the subtasks created to calculate the task success rate were the following: (1) go to review of systems, (2) add \u201cno chills,\u201d (3) add \u201cno fever,\u201d (4) add \u201cfatigue,\u201d (5) add \u201cdecreased activity,\u201d (6) add \u201cdry mouth,\u201d (7) add \u201cno dyspnea,\u201d and (8) add \u201cno edema.\u201dWe confirmed there were no EHR interface changes between data collection in Rounds 1 and 2 that may have influenced the study and tasks. The recorded sessions were examined using Morae Manager, a video analysis software program that was used to calculate performance measures using markers to identify difficulties and errors the participants encountered. Video analysis took approximately 1.5 hours for each 20-minute recorded session. The first step in the analysis was to review the recorded sessions and label any tasks that were unmarked during data collection. The second step was to divide each of the 19 tasks into smaller tasks to determine the task success rate and identify subtle usability challenges that we may have otherwise failed to notice. Geometric means were calculated for the performance measures with confidence intervals at 95% . PerformGeometric mean values of percent task success rates were compared between the 2 physician groups across 2 rounds  69]. Th. Th69]. In Round 1, the novice physician group achieved a higher success rate than expert physicians for 7 tasks , the same success rate for 7 tasks , and a lower success rate for 5 tasks . In Round 2, the novice physician group achieved a higher success rate for 3 tasks , the same success rate for 15 tasks , and a lower success rate for Task 15.Both novice (6%) and expert physician groups (2%) had equally low task success for Task 7 (Add a medication to your favorites list) in Round 1. However, in Round 2 all physicians in both groups successfully completed Task 7 (100%).Geometric mean values of time-on-task (TOT) were compared between the 2 physician groups across the 2 rounds . There wIn Round 1, the novice physician group spent less time than expert physicians completing 4 out of 19 tasks , the same amount of time completing Task 17, and more time completing 14 tasks . In Round 2, the novice physician group spent less time completing 4 out of 19 tasks , the same time completing Task 18, and more time completing 14 tasks .In Round 1, both physician groups had the longest time spent on Task 7 . However, in Round 2, time on Task 7 decreased by 52% for the expert physician group (87s to 50s) and 29% for the novice physician group (133s to 95s).Geometric mean values of mouse clicks were compared between the 2 physician groups across the 2 rounds . There wIn Round 1, the novice physician group achieved lower mouse clicks than the expert physician group for 7 tasks , higher mouse clicks for 9 tasks , and a comparable number of clicks for 3 tasks . In Round 2, novice physicians used less mouse clicks when completing 6 tasks , the same number of clicks when completing 5 tasks , and more clicks completing 8 tasks .In Round 1, both novice and expert physicians had the highest number of mouse clicks out of all tasks when completing Task 7 (Add a medication to your favorites list). However, in Round 2, the task with the highest number of mouse clicks by expert physicians changed from Task 7 to Task 15 (Place order for basic metabolic panel [BMP]) and novice physicians had the highest mouse clicks when completing Task 14 (Place order for chest X-ray) in Round 2, compared to Task 7 in Round 1.Geometric mean values of mouse movements (the length of the navigation path to complete a given task) were compared between the 2 physician groups across the 2 rounds. There was a 14% increase in novice physicians\u2019 mouse movements between Round 1  and Round 2 . There was also a 14% decrease in expert physicians\u2019 mouse movements between Round 1  and Round 2 . When mouse movements were compared between the physician groups, the novice physician group showed slightly longer mouse movements than expert physicians did across the 19 tasks in both rounds.In Round 1, the novice physicians showed longer mouse movements for 15 of 19 tasks , and shorter mouse movements for 4 tasks . In Round 2, novice physicians used shorter mouse movements in completing 8 out of 19 tasks  and used longer movements completing 11 tasks .In Round 1, novice physicians had the longest mouse movements out of all tasks when completing Task 7 (Add a medication to your favorites list) and expert physicians had the longest mouse movements when completing Task 13 (Include diagnosis). In Round 2, the task with the longest mouse movements by novice physicians was Task 14 (Place order for chest X-ray) compared to Task 7 in Round 1 and expert physicians had the longest mouse movements when completing Task 15 (Place order for basic metabolic panel [BMP]).In Round 1, 5 out of 6 expert physicians and all 10 novice physicians completed the SUS. In Round 2, all 4 expert physicians and all 9 novice physicians completed the SUS. The SUS illustrated that novice physicians ranked the system\u2019s usability at a mean of 69  in Round 1 compared to 68  in Round 2. Experts rated the system\u2019s usability at a mean of 74 (acceptable) in both rounds. A novice physician and 2 expert physicians had a score of 50 (not acceptable) or below. These results may indicate that expert users who have achieved a certain level of proficiency may be more confident using the EHR than novice users. A debriefing session confirmed the overall learnability test experience but did not reveal specific learnability issues. After analyzing the recording, however, it was clear that physicians encountered some difficulties when completing the tasks.Because of space limitations, a second manuscript is in preparation with a full review of the usability themes. Sub-task analysis was instrumental in identifying multiple usability concerns. We identified 31 common and 4 unique usability issues between the 2 physician groups across 2 rounds. Themes were created by analyzing and combining usability issues to form an overarching theme . Five thOur findings show that there were mixed changes in performance measures and expert physicians were more proficient than novice physicians on all four performance measures.P=.01). No statistically significant difference between novice and expert nurses was found when considering only completely solved tasks (P=.08). Our study did not report P values due to the small sample size; however, we observed overall improvement in performance measures for both novice and expert physician groups across 2 rounds. The contradictory results from this study and the study by Kjeldskov, Skov, and Stage, suggest that further research is necessary to draw more definite conclusions about task success between novice and expert physicians.In our study, differences were found between expert and novice physicians\u2019 performance measures across Round 1 and Round 2. A study by Kjeldskov, Skov, and Stage  identifyP<.00) [Alternatively, a study by Lewis et al measured performance of novice health sciences students and a predictive model of skilled human performance when performing EHR tasks using a touchscreen. Novice participants were adults with no prior experience using an EHR touchscreen interface using CogTool. CogTool is an open-source user-interface prototyping tool that uses a human performance model to automatically evaluate how efficiently a skilled user can complete a task. Participants completed 31 tasks commonly performed by nurses and patient registration clerks in an Anti-Retroviral Therapy clinic. The mean novice performance time for all tasks was significantly slower than predictions of skilled use (P<.00) . AlthougPhysicians\u2019 perceptions of the usability of a system may have relations to learnability; that is, physicians may find the system more user-friendly (usability) if the amount of time and effort needed to develop proficiency with the system is shorter (learnability). In our study, the SUS, which measures overall usability, illustrated that there was only a slight change in novice  and expert  physicians\u2019 rankings of the system\u2019s usability. In a study by Haarbrandt et al, primary care providers gave a SUS rating of 70.7  when asked about their perception of a health information exchange system, which was similar to the physicians\u2019 scores in our study. Expert and novice participants found the graphical user interface easy to use; however, they only rated the system as acceptable  measuredThis study had several limitations in terms of the methodology. First, it involved a small sample of physicians; therefore, the sample size may not have been sufficient to obtain statistical significance when reporting quantitative results of learnability. However, the sample size was sufficient when identifying usability issues experienced by participants when interacting with the EHR system. This study was conducted at a health care institution where only 1 EHR system was used and may not be representative of all primary care practice. As such, the study\u2019s findings may have limited generalizability to other ambulatory clinic settings, due to different types of EHR applications and physician practice settings. However, the EHR platform employed in this study is one of the top commercial products with significant market share. Based on data from Office of the National Coordinator for Health Information Technology, Cerner was reported as the primary EHR Vendor by 20% of hospitals participating in the CMS EHR incentive programs, making it the second most implemented EHR in hospitals . Second,Overall, this study identified varying degrees of learnability gaps between expert and novice physician groups that may impede the use of EHRs. Our results suggest that longer experience with an EHR may not be equivalent to being an expert or proficient in its use. The physicians\u2019 interactions with the EHR can be communicated to EHR vendors, to assist in improving the user interface for effective use by physicians. This study may also assist in the design of EHR education and training programs by highlighting the areas  of difficulty that resident physicians face. Resident physicians in primary care are offered extensive EHR training by their institutions. However, it is a great challenge for busy physicians to find time for training. Furthermore, it is an arduous task attempting to meet the needs of users and provide hands-on, on-site support , and evi"}
+{"text": "CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas.The absence of MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR).From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. P<0.05). Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P\u200a=\u200a0.041). High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P\u200a=\u200a0.012) and multivariate analyses (P\u200a=\u200a0.020).There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.Level of  MDM2 and CDK4, which are known to play crucial roles in the control of the cell cycle progression CDK4 amplification has been observed in several malignancies including glioma, breast cancer, lymphoma, melanoma, and sarcoma CDK4 amplification in WD and DD liposarcomas is associated with lower rate of recurrence and favorable prognosis Liposarcomas are the most common type of soft tissue sarcoma accounting for approximately 20% of all soft tissue sarcomas MDM2 and CDK4 amplification in a homogeneous population of patients with WD and DD liposarcomas of the abdomen undergoing complete surgical resection.In this study, we sought to identify factors associated with tumor recurrence and patient survival including the levels of From December 2000 to December 2010, 139 patients underwent surgical resection for liposarcoma at Samsung Medical Center, Seoul, Korea. Among these patients, 101 cases were diagnosed as WD or DD liposarcomas. Retrospective review was performed for consideration of inclusion in the study. Cases referred to our institute from other centers for management of recurrent tumors were excluded from the analysis. Cases were selected for this analysis when complete surgical resections with curative intent were carried out for WD or DD liposarcomas of the retroperitoneum and peritoneal cavity. Complete surgical resection of the tumor was achieved when all of the following three criteria were met: no gross residual tumor in the surgical field as observed by the surgeon (pathologic R0 or R1 status), histologic confirmation of negative surgical margins and no radiologic signs of residual tumor in the first postoperative follow-up abdomen computed tomography (CT) scan, typically done between postoperative 1 to 4 weeks.Common practice for surgical resection of abdominal liposarcomas was wide excision of the mass with combined resection of adjacent viscera when the organ is suspected to have direct tumor invasion by preoperative radiologic evaluation and inspection in the surgical field. A tissue expander was left in the space previously occupied by the tumor when adjuvant radiotherapy was planned.Patients were followed at our outpatient clinic with abdominal CT scans and chest plain x-rays every 3 to 6 months. When local recurrence was suspected, abdominal CT scans were repeated after 1 month or positron emission tomography-CT (PET-CT) was done to confirm the presence of locoregional recurrence or distant metastases. When possible, surgical resection of the recurred tumor mass was attempted. Unresectable tumors or distant metastases to multiple sites were managed with systemic chemotherapy.This research has been approved by the institutional review board at Samsung Medical Center . All data collection and analysis were done anonymously, and written or verbal consent were not provided by the participants of this work. The lack of consent for this study was also approved.MDM2 and CDK4 amplification was analyzed by quantitative real-time polymerase chain reaction (Q-PCR) performed on a PRISM 7500HT Fast Realtime PCR system  by using a HotStart-IT SYBR Green qPCR Master mix . Ten nanograms of target DNA was dispensed into each sample with a final reaction volume of 10 \u00b5l. Each sample was amplified in triplicate. The PCR was carried out as follows: preheating at 50\u00b0C for 2 min and then at 95\u00b0C for 10 min, followed by 40 cycles at 95\u00b0C for 15 s and 60\u00b0C for 1 min.  located at 4q11-q13, and were normalized to normal tissue genomic DNA as a calibrator. The level of amplification for MDM2 or CDK4 was calculated as follows: copy number of the target gene /copy number of the reference gene (ALB). Each gene was considered to have positive amplification when the copy number was greater than 2 times that of the reference gene.Immunohistochemical (IHC) staining for CDK4 was performed on representative paraffin-embedded tumor material from the biopsy cut into 4 \u00b5m-thick sections and placed onto glass slides. For IHC staining, the slides were deparaffinized in xylene. Staining for CDK4 was performed automatically using the Leica Bond Max immunostainer with Bond Polymer Refine Detection Kits and heat-induced epitope retrieval pH 6.0 (Bond max ER1 (EDTA) solution, Australia) for 15 min. The CDK4 expression was analyzed using mouse monoclonal antibody specific for CDK4 protein  P-values were less than 0.05.Chi-square tests  were used to compare categorical variables. Pearson\u2019s correlation was used to measure the correlation between variables. The Cox proportional hazard model was used for survival and risk factor analysis. All analyses were done using IBM SPSS Statistics version 19 program. Results were considered to be significant when One hundred thirty-nine patients were surgically treated for liposarcoma at our center during the study period. Pathologic examination of the resected tumors of 101 patients showed either WD or DD histologic subtype. Ninety-two tumors were found in the retroperitoneum or peritoneal cavity. Fifty-four patients had their first surgical treatment at our center and 48 had complete surgical resection of the tumor. The patient selection process for inclusion in this study is outlined in The 48 patients who had complete tumor resection  were included in this analysis. The median age of patients was 57 years (range 37\u223c78 years). There were 31 WD and 17 DD liposarcomas. Tumors were located in the retroperitoneum in 43 cases, the mesentery in 4 cases and the pelvis in 1 case. Tumor size ranged from 6.8 cm to 60 cm (median 21 cm). The characteristics of the 48 patients and clinicopathological features of the liposarcomas according to histologic type are outlined in p\u200a=\u200a0.021). Microscopic examination of the surgical margin was negative in 11 (35.5%) WD and 3 (17.6%) DD liposarcomas. Tumor recurrence after surgical resection with curative intent was observed in 20 patients ; 11 were WD and 9 were DD liposarcomas . Recurrence was locoregionally limited in all 11 WD cases while 6 of the 9 DD liposarcomas recurred as distant metastases. A significant difference in recurrence pattern was observed between the 2 histologic subtypes (p\u200a=\u200a0.001).p<0.05).Locoregional recurrence was observed in 11 WD and 3 DD liposarcoma cases. The median time interval between surgical resection and the occurrence of the first recurrence was 13.5 months and 11.8 months for WD and DD liposarcomas, respectively. Primary WD liposarcomas expressed variable histologic types in their recurrence lesions; pathologic analyses of the recurrent tumor specimen were WD in 6 cases, DD in 2 cases and pleomorphic in 1 case. All recurrences of primary DD liposarcomas were of the DD histologic subtype. Surgical resection of locoregional recurrence was done in 9 WD and 2 DD cases. Patients underwent an average of 2.0 surgical resections for recurrences .Six cases had distant metastases during their follow-up period . Of thesSurgical resection of the metastatic tumor was carried out in 2 cases and systemic chemotherapy was done in 1 case. Metastatic liposarcoma was the cause of death in 4 patients. Patient survival after metastasis was 22.2% at 24 months.CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). The levels of CDK4 amplification were not different between the two histologic subtypes , and also in each histological subtypes .CDK4 immunohistochemistry results were interpretable in all cases. Positive immunoreactivity for CDK4 was observed in 32 out of 48 cases 66.7%, . HoweverCDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P\u200a=\u200a0.012) and multivariate analyses (P\u200a=\u200a0.020). Disease-specific survival and recurrence-free survival curves of WD liposarcoma patients are shown in CDK4 amplification levels greater than 7.54 (CDK4 high) had significantly inferior recurrence-free survival compared to cases with CDK4 amplification less than 7.54 (CDK4 low). None of the analyzed variables were found to significantly affect survival of DD liposarcoma patients.Predictors of recurrence-free survival of WD liposarcoma are shown in MDM2 and CDK4, belong to distinct amplicons and no consistency exists in the amplified sequences between MDM2 and CDK4. It has been well documented that these two genes are involved in the oncogenesis and progression of WD and DD liposarcomas MDM2 amplification and overexpression is present in most WD and DD liposarcomas, CDK4 amplification is absent in a small proportion of cases CDK4 amplification represent a distinct clinical subgroup with a lower recurrence rate and are more likely to be peripherally located et al. was the fact that the better prognosis observed in liposarcomas without CDK4 amplification may have been confounded by these tumors more often being located in the extremities. Liposarcomas of the extremities are more likely to be resected with sufficient free surgical margin compared to those in the retroperitoneum. The liposarcomas included in the current study were limited to tumors arising in the retroperitoneum and peritoneal cavity. Q-PCR of the tumor specimens revealed varying levels of MDM2 and CDK4 amplification, with 44 cases (91.7%) and 46 cases (95.8%) showing positive amplification of CDK4 and MDM2 genes, respectively. Moreover, WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence and the degree of CDK4 amplification was an independent risk factor for recurrence of WD liposarcoma after complete excision. To the best of our knowledge, this study is the first to describe the correlation between CDK4 amplification and WD liposarcoma recurrence in a quantitative manner. In accordance with the study by Italiano et al., four cases in our series that were negative for CDK4 amplification are free of disease recurrence at median 25 months (range 16\u223c39 months) after surgical resection.Amplification of chromosome 12q13-15 is a typical feature of WD and DD liposarcomas CDK4 to DD liposarcoma progression was presented in a study by Barretina et al., in which shRNA was used to knockdown CDK4 in two DD liposarcoma cell lines. Sustained knockdown of CDK4 led to inhibition of proliferation of the cell lines. In addition, pharmacological inhibition of CDK4 with a selective CDK4 inhibitor (PD0332991) induced G1 arrest in the same two cell lines CDK4 gene. Our study adds further rationale to the current body of evidence that the use of CDK4 inhibitors in DD liposarcoma may prove to be beneficial.In vitro data on the contribution of CDK4 amplification by Q-PCR. These results suggest that translational or post-translational events function in fine tuning the final outcome of amplication and protein expression can be induced by other mechanism rather than amplication. Discrepancies between CDK4 IHC and Q-PCR findings were also observed in a study by Sirvent et al., in which negative staining by IHC for CDK4 was observed despite the presence of amplification by Q-PCR in approximately 40% of cases of WD and DD liposarcomas MDM2 was almost ubiquitously overexpressed in WD and DD liposarcomas and therefore did not affect tumor biology MDM2 Q-PCR analysis results also showed MDM2 amplification in 46 out of 48 cases in our liposarcoma specimens.Immunohistochemical analysis of CDK4 has been shown to be helpful in the differential diagnosis of liposarcomas and benign lipomatous tumors Our group previously analyzed 94 cases of liposarcomas in both the trunk and the extremities and concluded that high grade histologic subtype and positive margin status (microscopic and macroscopic) were independent risk factors for poor survival CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas of the retroperitoneum and peritoneal cavity. Well-differentiated liposarcomas with higher level of amplification of CDK4 (\u22657.54) were more likely to recur after surgical resection. Utilization of Q-PCR for analysis of CDK4 amplification may aid clinicians in the postoperative surveillance and management of patients with abdominal WD and DD liposarcomas.In conclusion, the level of"}
+{"text": "Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NF\u03baB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NF\u03baB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that  Microarray analysis is increasingly being used to advance our understanding of the complex transcriptional responses generated downstream of an experimental perturbation or during a disease state Toll-like receptors (TLR) are a family of pattern recognition receptors (PRR) which recognise highly conserved microbial products. In humans 10 functional TLRs have been described including TLR2 and TLR4 which are expressed on the cell surface and recognise conserved bacterial products A study collectively analysing data from multiple published papers from both human and murine macrophage transcriptional studies identified a group of genes upregulated following both TLR2 and TLR4 stimulation, which were predicted to be regulated by NF\u03baB. Additionally a separate IFN-sensitive response element (ISRE) regulated set of genes expression was seen to be upregulated in the TLR4 (and TLR3) stimulations but not the TLR2 stimulation in vivo TLR4 ligand administration have also previously been undertaken in vivo administration of LPS, it was shown that mRNA expression of proinflammatory chemokines and cytokines  peaked at 2 to 4 hours after LPS administration, whereas the cytokine IL10 was maximal at 6 hours. In this study the transcription factors NFKB1, NFKB2, RELA and RELB were significantly expressed and seen to peak after the cytokines and chemokines. The peak time for transcription factors including the STAT  and IRF genes was at 4 to 6 hours in vivo experiments, studies are limited by ethical and practical considerations.Transcriptomic studies following in vitro studies has the potential advantage over isolated cell populations as these different components may have a differential response to stimulation. Autocrine and paracrine signalling between the differing cell populations may result in a response of the whole system that potentially better reflects the in-vivo response. Additionally whole blood potentially has advantages over PBMC, DCs or monocyte derived macrophages since it can be used in situations where it is not possible to obtain large volumes of blood to derive the isolated cell populations. Previously, In-vitro human whole blood has been used as a model to study TLR ligation, predominantly with measurement of specific cytokine protein levels Whole blood comprises cells of both the innate and adaptive immune system. Therefore the use of whole blood for in vitro global temporal transcriptional response to TLR4 and TLR2 ligation in human whole blood. To better understand this gene transcriptional response we used a variety of bioinformatics approaches to delineate both the temporal response and the key transcriptional differences resulting from TLR2 and TLR4 ligation and demonstrate that in a whole blood system that the response to TLR stimulation can resemble that previously identified in isolated immune cells.The objective of our study was to undertake a detailed comparative analysis of the This study was approved by the Central London 3 Research Ethics Committee (09/H0716/41). All participants gave written informed consent.Six healthy volunteers (self-reported questionnaire); three male, three female; aged 25\u201350 years old; of similar ethnic background were recruited into the study. Sixty ml of whole blood from each volunteer was taken at 9 am into 10 ml Vacutainers with sodium heparin 17 international units/ml (BD Vacutainer).Measured by Celltac Automated Hematology Analyzer  at 0 hour, volunteer\u2019s results listed 2 for 0, 1, 3, 6, 12 or 24 hours at which point the contents of the well were thoroughly mixed with 2 mls Tempus Solution (Applied Biosystems/Ambion) to lyse the cells and stabilise the RNA. Samples stored at \u221280\u00b0C until RNA processing.In 24 well plates , 1 ml of heparinised whole blood was stimulated either in the presence of a final concentration of 200 ng/ml of Pam3CSK4 (Invivogen), 1 ng/ml of LPS  added in a volume of 100 \u00b5l with RPMI-1640 with GlutaMAX (Life Technologies). Media control samples were cultured with the addition of 100 \u00b5l of RPMI-1640 with GlutaMAX. Samples were incubated at 37\u00b0C, 5% COReagents (excepting LPS and Pam3CSK4) including Sodium heparin Vacutainers were tested for endotoxin contamination by Limulus assay and were found to be endotoxin free (<0.03 EU/ml). Pam3CSK4 was tested by the manufacturer and confirmed to be endotoxin free (<0.001 EU/\u00b5g).RNA was isolated using the PerfectPure RNA Blood kit (5-PRIME) according to manufacturer\u2019s instructions. 2.5 \u00b5g of isolated RNA was globin RNA reduced using the GLOBINclear 96-well format kit (applied Biosystems/Ambion) according to the manufacturer\u2019s instructions. Isolated and globin reduced RNA quantity was assessed using either Nanodrop 1000 or Nanodrop 8000 spectrophotometer , RNA quality was assessed using an Agilent 2100 Bioanalyser (Range 6.5\u20139.5) (Agilent technologies). 200 ng of globin reduced RNA was amplified to generate biotinylated amplified antisense cRNA using the Illumina CustomPrep RNA amplification kit (Applied Biosystems/Ambion). 750 ng of cRNA was hybridized to Illumina Human HT-12 V4 BeadChip arrays (Illumina) and scanned on Illumina iScan. GenomeStudio (Illumina) was used to perform quality control and generate signal intensity. Two samples were excluded from further analysis at this stage as they failed quality control measures .th percentile shift algorithm. Per-transcript normalisation was undertaken by normalisation to the median of a defined control group. Transcripts were then filtered out if they were not significantly (p<0.01) different in intensity value compared to the background in at least 10% of all the samples.Raw background subtracted data was processed using Genespring V12.6 (Agilent Technologies) and the following principles were applied to all analyses. After background subtraction low signal values (<10) were then set to a threshold of 10, log2 transformed and per chip normalised using 75p<0.01), followed by a further filtering of transcripts by fold change (FC) in which transcripts were filtered if less than 1.8 FC different between variables of interest. Expression heatmaps were generated within Genespring V12.6. Heatmap clustering was undertaken using Differential distance metric and Wards linkage rule, unless otherwise stated.The resulting transcripts were then subjected to statistical filtering (either One-way ANOVA or 2-way ANOVA) with multiple testing correction (Benjamini-Hochberg Media controls from 2 volunteers had evidence of activation of inflammatory genes by 3 hours of culture . This acThe data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE55375.k-means clustering into 9 clusters using Euclidian distance metric. Number of clusters was chosen by the number of 3rd order branches in the dendrogram from the LPS expression heatmap. Clusters were compared across stimulations using Pearsons Correlation (within Graphpad Prism V6).Within Genespring 12.6 the normalised significant transcript lists were separately clustered by www.ingenuity.com). Significant transcripts identified from GX microarray analysis were uploaded into IPA. For time point analyses these lists were filtered by mean FC (>1.8) compared to media control at the time point.The canonical pathway, gene function annotation and upstream analyses were generated through the use of IPA  From the globin reduced RNA cDNA was synthesised using High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer\u2019s instructions followed by RNase H (Promega) treatment for 30 min at 37\u00b0C. IFNB1, IL1A, IL6, NFKB1, NFKB2, STAT1, STAT2 and IRF7 gene expression were quantified by real-time PCR  using the TaqMan system, and normalised to GAPDH mRNA.Primer probes used were IFNB1 (Hs01077958_s1); IL1A (Hs00174092_m1); IL6 (Hs00985639_m1); NFKB1 (Hs00765730_m1); NFKB2 (Hs010208901); STAT1 (Hs01013996_m1); STAT2 (Hs01013123_m1); IRF7 (Hs01014809_g1); GAPDH (Hs02758991_g1) .Analysis of the media controls alone over time revealed 377 significantly expressed transcripts over time. The peak of this difference was at 24 hours with 281 transcripts more than 1.8FC different compared to the 0 hour samples . These dTo gain insight into the differential temporal gene expression in response to TLR4 and TLR2 ligation we performed a comparative microarray analysis of LPS and Pam3CSK4 stimulated human whole blood and accompanying media controls over a time course. From four healthy human volunteers 1 ml of heparinised whole blood was stimulated with either the TLR4 ligand LPS (1 ng/ml), the TLR2 ligand Pam3CSK4 (200 ng/ml), (concentrations shown to result in a plateau in previous studies within our laboratory), or incubated only with media as a control. RNA was isolated at 0, 1, 3, 6, 12 and 24 hours for each stimulus or control.LPS (TLR4) stimulation induced a greater number of differentially regulated transcripts as compared to Pam3CSK4 (TLR2) and had a higher magnitude of response. For this reason the two stimulations were first analysed independently to generate the significant transcript lists compared to media control over the time course after stimulation. LPS stimulation resulted in the differential expression of 4777 transcripts (mapping to 3571 unique genes in IPA) whereas Pam3CSK4 stimulation resulted in only 1202 differentially regulated transcripts (mapping to 922 unique genes in IPA) . Expressk-means clustering was applied to cluster the significant transcript lists based on their similarity in expression over time for each stimulation separately  were small in terms of numbers of genes which were highly expressed by 1 hour after stimulation with both LPS and Pam3CSK4 and peaked in expression between 3 to 6 hours. 80% of the genes in the P2 cluster were found within the L7 cluster. These common genes were predominantly chemokines and cytokines: C15orf48, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4L1, CCL4L2, CCL8, CCRL2, CXCL2, EBI3, IL1A, IL6 and TNF.The \u201cAcute Phase response signalling\u201d clusters (L4 and P8) were characterised by genes involved with inflammatory response. Approximately 60% of the genes in the P8 cluster were also found within the L4 cluster and these common genes included those involved with the inflammatory response: ORM1, ORM2, HAMP, IRAK2, PI3, PTGES, TNFAIP6, TNIP3. In addition, within the L4 cluster but not the P8 cluster were genes involved with IFN regulation: IFI44L, IFIT1, IFIT3, IFNG, OAS3, OASL.The \u201cProtein Ubiquitination pathway\u201d clusters (L1 and P1) had 104 common genes between stimulations which included heat shock protein genes  and Proteasome PA700/20S genes .The \u201cComplement system\u201d clusters (in both L8 and P5) were characterised by genes that were upregulated, had their highest expression at 24 hours and included the metallothionein genes  which were amongst the highest expressed genes at 24 hours following both LPS and Pam3CSK4 stimulations . ClusterCluster L2, which peaked at 6 hours and was characterised by \u201cActivation of IRF by cytosolic pattern recognition receptors\u201d as the top pathway did not have a similar pathway in the Pam3CSK4 clusters. Examination of the genes within this pathway revealed that there were detected a number of IFN regulated/regulatory genes .Clusters L3, L0, L6 and P4, P7 and P0 were all enriched for immune function pathways although there were no similarities between the stimulations in terms of top significant pathway and kinetic profile. Cluster P3 was enriched for the pathway \u201cUDP-N-acetyl-D-galactosamine Biosythesis II\u201d and there was no similar cluster following LPS stimulation.in vitro LPS stimulation of whole blood.To better understand the temporal response we undertook analysis at each time point using the significant transcript lists. Significantly expressed transcripts at each time point were identified by comparing the mean expression of the differentially regulated transcripts in response to LPS and Pam3CSK4 stimulation at each time point to the media control at that time point and filtering those transcripts which were less than 1.8 FC different to the media control  are shown  from 3 hours onwards and was most significant at 6 hours (p<0.01) at any time point following Pam3CSK4 stimulation.The \u201cIFN signalling\u201d pathway in response to LPS was only significant  combined accounted for approximately 20% of the significantly expressed genes at this time point. There was a large degree of overlap between stimulations of the significant cytokines/chemokines at 1 hour, with all of the 17 significant cytokines/chemokines genes from the Pam3CSK4 list also being identified in the LPS list . FollowiIn order to identify which transcriptional regulators may be responsible for the differences observed in gene expression between LPS and Pam3CSK4 stimulation we undertook upstream analysis of the gene lists at each time point within IPA. Upstream analysis attempts to predict which transcriptional regulator may be responsible for the observed differential gene expression by comparing the genes known to be regulated by a transcriptional regulator (derived from the literature) to those significantly differentially expressed genes identified at each time point from this analysis. It can be seen that the top predicted transcription regulator for both stimulations was the NF\u03baB complex and that it was predicted to be activated from 1 hour onwards .By comparison of the mean mRNA expression of the predicted transcriptional regulators compared to media control it can be seen that the magnitude and temporal response was similar for LPS and Pam3CSK4 stimulation for PPRC1 and SP1 . The preThe NF\u03baB genes REL, RELA, RELB, NF\u03baB1 and NF\u03baB2 were all significantly expressed and present in both LPS 4777 transcript and Pam3CSK4 1202 transcript lists . TranscrIn order to attempt to identify the cytokines that were potentially involved in autocrine/paracrine signalling and subsequent gene expression regulation in response to LPS or Pam3CSK4 stimulation, we again undertook per-time point upstream analysis within IPA. TNF, IL1B and IL1A were predicted as potentially activated cytokines, and this predicted activation was early (by 1 hour) and sustained . The mRNIL12B was predicted to be activated upon LPS stimulation but not Pam3CSK4 and this difference was observed in the mean mRNA expression of the IL12B gene (encoding for IL12p40) which was upregulated in response to LPS compared to media control and not following Pam3CSK4 stimulation . IFN cytk-means clustering, canonical pathway analysis and upstream analysis of both potential transcriptional regulators and cytokines highlighting a difference in IFN signalling following LPS and Pam3CSK4 stimulation.Our data show that as early as 1 hour post stimulation IFN gene expression is seen to be upregulated following LPS but not Pam3CSK4 stimulation. This difference in IFN signalling was further emphasised by the k-means clustering, predicted upstream analysis, and canonical pathway analysis as being differentially activated between LPS and Pam3CSK4 stimulations.IRF and STAT genes had been identified by To test if this difference in expression of IRF and STAT genes resulted in differential expression of IFN regulated genes we used a list of human type 1 IFN regulated genes generated from the Interferome database v2.0. We compared the expression of these type 1 IFN regulated genes between LPS and Pam3CSK4 stimulations. From this it can be seen that LPS stimulation results in a greater number of differentially regulated type 1 IFN genes compared to Pam3CSK4. In addition the magnitude of this differential regulation was much higher in LPS stimulated whole blood compared to Pam3CSK4 stimulated whole blood and the peak of this transcriptional response following both LPS and Pam3CSK4 was at 6 hours. .The similarity in NF\u03baB signalling and difference in IFN signalling following Pam3CSK4 and LPS stimulation is reflected in the IPA canonical pathways. There is similarity in the IPA canonical pathway \u201cNF\u03baB signalling\u201d at 3 hours (the peak of significance for this pathway following LPS and Pam3CSK4 stimulations) following LPS and Pam3CSK4 stimulation. However there is clear difference IFN Signalling canonical pathway at 6 hours  where there is seen to be difference following LPS and Pam3CSK4 stimulations .In vivo LPS stimulated human whole blood data was obtained from the Gene Expression Omnibus  and IRF and STAT transcriptional regulators   . Analysiin vitro upstream analysis and seen to be differentially expressed in-vitro and were also significantly differentially expressed following in vivo LPS administration  seen mainly following LPS stimulation. This significant difference in the IFN response between TLR4 and TLR2 ligation could be seen as early as 1 hour post stimulation in our study, and reinforced in the later time points despite the potentially complex autocrine and paracrine signalling in the human whole blood system.The common early transcriptional response following both LPS and Pam3CSK4 stimulations at 1 hour was characterised by highly expressed cytokines and chemokines  as well as the significant upregulation of transcriptional regulators including the NF\u03baB family  and AP-1/CREB . The gene ZFP36 which encodes for the protein Tristetraprolin was also identified as significantly up regulated by 1 hour which has been shown to act in a post-transcriptional regulatory role by binding to the mRNA of some cytokines and promoting their degradation ex-vivo human blood leukocytes. Slight differences in the timing of peaks observed in our study and these other studies could potentially be explained by the difference in time points sampled or differences in the systems used Following LPS stimulation a differential transcriptional response of IFN regulated/regulatory genes was observed as compared to Pam3CSK4. This difference was detectable as early as 1 hour post LPS stimulation with the significant upregulation of the cytokine genes IFNB1 and IFNG and the transcriptional regulator IRF8 following LPS and not Pam3CSK4 stimulation. The IRF and STAT  genes were seen to be differentially regulated following LPS stimulation and the peak of their expression was at 6 hours. This difference in induction of IRF and STAT transcription regulators following TLR4 and not TLR2 ligation is consistent with previous studies which have compared the temporal transcriptional response following TLR2 and TLR4 ligation in murine DCs and macrophages in vitro transcriptional studies in-vivo stimulation where transcriptional difference peaked between 4 to 6 hours and by 24 hours the transcriptional signature had returned to baseline in vivo compared to in vitro and these differences in expression and persistence of differential expression observed in vitro compared to in vivo could be explained by trafficking/removal of activated immune cells out of the circulation in vivo, which is not possible to be represented in an in vitro whole blood system.Following LPS stimulation the peak of differential transcription compared to media control was at 6 hours in terms of the number of differentially regulated genes. At 24 hours 1443 genes were still significantly differentially regulated. This persistence of differential transcription at 24 hours is observed in human LPS stimulated in vitro stimulated human whole blood for transcriptomic analysis of the early innate immune response has potential advantages over the use of isolated immune cells as whole blood stimulation can be carried out in a laboratory where the expertise or equipment to isolate immune cells from blood is lacking. In addition the volume of blood needed for stimulation is much less than that required to isolate immune cells, meaning experiments can be performed in populations where access to larger volumes of blood is not possible e.g. paediatrics or it could be possible to undertake more stimulations/time points with a given volume. The results however must be interpreted in the context of the complex autocrine/paracrine interactions which occur in a mixed cell culture and therefore stimulation of isolated immune cells will remain advantageous to interrogate in detail a specific transcriptional response.The ability to utilise We have shown that human whole blood can be used to study the early temporal transcriptional response following TLR2 and TLR4 ligation and that the results obtained are comparable to those from isolated murine and human immune cells.Figure S1Activation of samples by culture conditions. (A) Heatmap of expression, clustered by transcripts, shows that 2 individuals (out of 6) media controls show activation (marked with arrows), the genes differentially expressed in these 2 media control samples are similar to LPS, but lower in magnitude. Transcripts identified by normalisation to 0 hour samples, filtering by detection from background, statistical filtering (2 way ANOVA with Benjamini Hochberg p<0.01) and then transcripts retained whose expression was >1.8 FC from another condition. (B) Activation is not due to length of time in transport conditions, nor is it individual specific. The single individual shown here had not previously activated, and was included in final dataset. Blood was collected at time point 0 and left in sealed vacutainers, the vacutainers were opened at one hour intervals and 1 ml human whole blood was either immediately mixed with Tempus solution (labelled as Direct from vacutainer) or plated for 3 hours with either media control (RPMI-1640 with GlutaMAX) or LPS (1 ng/ml) and then mixed with Tempus solution. Reagents and containers (including vacutainers) are endotoxin free (undetectable by Limulus assay - sensitivity <0.03 EU/ml). Heatmap of expression , clustered by transcripts shows that regardless of length of time in vacutainer activation occurred in all media control samples, and is not observed in the direct from vacutainer samples, implying that activation is dependent on culture conditions and is not a function of length of time ex-vivo or length of time spent in the vacutainers. Transcripts identified by normalisation to median of \u201cDirect from vacutainer\u201d samples, filtering by detection from background, statistical filtering (ANOVA with Benjamini Hochberg p<0.01) and then transcripts retained whose expression was >1.8 FC from another condition.(TIF)Click here for additional data file.Figure S2Transcriptional changes in media controls over time.(A) Heatmap of normalised expression values of 377 transcripts which were identified to be significantly differentially expressed overtime  and then transcripts retained whose expression was >1.8 FC from the 0 hour samples. Plotted above the heatmaps is the number of significantly expressed genes (mapped in IPA) that were differentially expressed at each time point by more than 1.8 FC compared to the 0 hour samples. (B) Top ten IPA canonical pathways of the significantly expressed genes at 24 hours, with the \u2013log p value for the pathway and the significantly differentially expressed genes listed for each pathway.(TIF)Click here for additional data file.Figure S3Metallothionein gene expression. (A) Heatmap of averaged Metallothionein mRNA expression over time following LPS or Pam3CSK4 stimulation, values normalised to the median of the 0 hour. Note asynchronous scale.(TIF)Click here for additional data file.Figure S4Interferon regulated genes. Heatmap of averaged expression values of Type 1 Interferon regulated genes (List obtained from Interferome v2.0), normalised to the median of the 0 hour, genes retained if they were expressed greater than 1.8 FC from media control in at least one stimulation in one or more time points (resulting in 1105 genes). Graphed above heatmap is the mean absolute fold change of these Type 1 interferon regulated genes.(TIF)Click here for additional data file.Figure S5Real time PCR. Real time PCR of selected genes following LPS and Pam3CSK4 stimulations and media controls, normalised to GAPDH expression. Mean fold change calculated between media controls and stimulations.(TIF)Click here for additional data file.Table S1Volunteer whole blood composition measured by Celltac Automated Hematology Analyzer  at time point 0 hour.(TIF)Click here for additional data file.Table S2Pearson correlations for k-means derived clusters from .(TIF)Click here for additional data file.File S1Listings of transcripts, LPS k-means clusters from .(XLSX)Click here for additional data file.File S2Listings of transcripts, Pam3CSK4 k-means clusters from.(XLSX)Click here for additional data file.File S3LPS time course data. Listings of transcripts from 4777 significant transcript list whose mean expression was 1.8 FC different to media control at each time point from (XLSX)Click here for additional data file.File S4Pam3CSK4 time course data. Listings of transcripts from 1202 significant transcript list whose mean expression was 1.8 FC different to media control at each time point from (XLSX)Click here for additional data file."}
+{"text": "Type 2 diabetes is a major health problem in the Australian Indigenous population. Aboriginal Community Controlled Health Organisations (ACCHOs) are a primary care setting where there is opportunity to partner with health services to reduce the current evidence-practice gap in the provision of health care for Type 2 diabetes. The aim is to examine the effectiveness of a tailored ecologically-based collaborative model in achieving adherence to best practice clinical guidelines for Type 2 diabetes in ACCHOs. This study will examine whether the model results in improvements in diagnostic testing, monitoring and control of diabetes using reliable objective clinical indicators.ACCHOs across Australia who use the Communicare data management system, have at least one doctor providing health care and use an electronic system for pathology results will be eligible for the study. A cluster randomised wait -control design will be used in 18 ACCHOs (9 intervention and 9 wait control). Cross-sectional measurement of the proportion of eligible patients receiving National Health and Medical Research Council (NHMRC) recommended diabetes diagnostic testing, monitoring and control at each ACCHO will be completed at baseline and follow-up.At baseline the mean proportion of patients from clinics who: 1) received diagnostic testing was 58.5% (SD 23.4%) in intervention clinics and 71.4% (SD 14.5%) in control clinics; 2) appropriate monitoring for type 2 diabetes was 49.1% (SD 12.4%) in intervention clinics and 50.7% (SD 18.4%) in control clinics; and 3) appropriate control was 29.2% (SD 7.1 %) in intervention clinics and 34.2% (SD 8.4%) in control clinics.A significant evidence practice gap exists in this setting with a vulnerable population for type 2 diabetes care. The study has a number of potentially significant outcomes including the provision of a model for engaging ACCHOs in examining performance and improving their implementation of best evidence-practice; the development of resources such as staff orientation manuals; the use of naturally occurring reliable data sets for monitoring and feedback and as a tool for intervention delivery and outcome evaluation which may be generalisable to other populations."}
+{"text": "Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP , but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that  Spns2 gene, affecting the release of a lipid called sphingosine-1-phosphate, which has an important role in several processes in the body. For the first time, we report that this molecular pathway is required for normal hearing through a role in generating a voltage difference that acts like a battery, allowing the sensory hair cells of the cochlea to detect sounds at extremely low levels. Without the normal function of the Spns2 gene and release of sphingosine-1-phosphate locally in the inner ear, the voltage in the cochlea declines, leading to rapid loss of sensitivity to sound and ultimately to complete deafness. The human version of this gene, SPNS2, may be involved in human deafness, and understanding the underlying mechanism presents an opportunity to develop potential treatments for this form of hearing loss.Progressive hearing loss is common in the human population but we know very little about the molecular mechanisms involved. Mutant mice are useful for investigating these mechanisms and have revealed a wide range of different abnormalities that can all lead to the same outcome: deafness. We report here our findings of a new mouse line with a mutation in the  Spinster homolog 2 (Spns2) is a multi-pass membrane protein belonging to the Spns family. Though the functions of Spns1 and Spns3 are largely unknown, Spns2 is known to act as a sphingosine-1-phosphate (S1P) transporter, based upon previous studies in zebrafish and mouse Spns2-deficient mice were initially discovered to be deaf during a large-scale screen of new mouse mutants carried out by the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the KOMP/EUCOMM resource of over 15,000 genes targeted in embryonic stem (ES) cells and aims to generate new mutants and screen them for a wide range of diseases and traits to reveal the function of 160 mutant genes each year Spns2-deficient mice showed early onset of hearing loss that progressed rapidly to profound deafness. This was associated with declining endocochlear potential (EP), which appeared to be the primary physiological defect. At later stages we observed degeneration of sensory hair cells and decreased expression of several key genes required for normal generation of the EP in the lateral wall of the cochlea, but these appeared to be secondary effects. By producing and analysing different conditional knockouts, we established that Spns2 expression was required locally in the inner ear rather than systemically. Our study suggests a vital role for Spns2 and S1P signalling in hearing.Spns2 gene . Quantitative real-time PCR revealed that residual transcript of Spns2 in cochleae, eyes and livers of the homozygous mice was substantially reduced compared to that of the heterozygous and wildtype mice . In other aspects of their phenotype, tm1b/tm1bSpns2 mice were broadly similar to tm1a/tm1aSpns2 mice . tm1a/tm1aSpns2 mice were the first to be available and were used for most experiments in this study, and may be more relevant to human disease because most disease-causing mutations reduce rather than eliminate gene activity.The introduction of a cassette with an additional splice acceptor site is predicted to interrupt normal transcription of the ns2 gene  and geneype mice . In ordetm1a/tm1aSpns2 mice had profound hearing impairment during screening at 14 weeks old by auditory brainstem response (ABR) measurement  suggesting normal vestibular function. ABR measurements were recorded at younger ages to find out the time of onset of hearing loss. Hearing impairment in tm1a/tm1aSpns2 mice can be detected as early as 2 weeks of age at high frequencies .The increase in ABR thresholds preceded degeneration of the organ of Corti suggesting that these were secondary changes rather than the primary cause of the hearing impairment.+ into the endolymph and generation of the endocochlear potential (EP) +-rich extracellular fluid, into the sensory hair cells through mechanoelectrical transduction channels. The lateral wall of the cochlea is composed of the stria vascularis, spiral prominence and the spiral ligament. A defect in the function of any of these components could interfere with the generation of the EP. Therefore, we measured the EP to evaluate the function of the lateral wall. The EP values of the control mice were normal, around 99 to 120 mV, which matched their normal hearing. tm1a/tm1aSpns2 mice had abnormally low EP values of 2 to 41 mV at both P21 and P28 when they were profoundly deaf , associated with irregular layout of marginal cell boundaries  in patches along the length of the cochlear duct, and apparently increased branching , Kv7.1 (Kcnq1), Cx26 (Gjb2), Cx30 (Gjb6), Na+, K+-ATPase (Atp1a1), NKCC1 (Slc12a2) and ZO-1 (Tjp1). In homozygotes aged P14  by crossing the tm1aSpns2 allele to a line expressing Flp recombinase to excise the inserted cassette  mice have profound hearing loss and propose an underlying mechanism: a rapid decline in EP paralleling loss of auditory sensitivity and preceding degeneration of hair cells, suggesting that the primary lesion is in the cochlear lateral wall, the site of EP production and maintenance Spns2 is expressed in hair cells as well as in the lateral wall, we cannot exclude the possibility that disruption of Spns2 function in the organ of Corti also contributed to raised ABR thresholds and hair cell degeneration. Analysis of mice with conditional knockout of Spns2 in hair cells and other cochlear cell types will be useful in dissecting the role of Spns2 further.Here, we report that in vitroSpns2 was expressed in blood vessels of the inner ear including spiral modiolar vessels. Any reduction in local S1P level due to Spns2 dysfunction may weaken vasoconstriction and explain the dilation of strial capillaries in tm1a/tm1aSpns2 mice. The relationship between capillary size and EP value is not unidirectional; both smaller and larger strial capillaries have been reported in different mouse mutants with low EP Spns2-deficient and control animals tm1a/tm1aSpns2 mice. BSA is a medium molecular mass tracer (66.4 kDa), so tracers of different sizes and properties such as Evans blue and cadaverine Spns2 acts as a transporter of S1P Spns2-deficient mice. However, the expression of most of these proteins appeared normal at the time when the EP has already started to decline at P14, suggesting that these are likely to be secondary effects. The morphological changes of marginal cell boundaries and reduction in marginal cell density together with a lack of expression of Kcnq1 in affected cells are also likely to be secondary changes because these features were normal when hearing started to deteriorate at P14 and they did not affect strial permeability. Loss of Kcnq1 expression in marginal cells with expanded luminal surfaces may be a common consequence of strial dysfunction because it has been reported in several different mutants with reduced EP Spns2-deficient mice needs further investigation.We found decreased expression of several proteins critical for normal EP production at 5\u20136 weeks of age in Spns2 expression was consistently in the hair cells and spiral prominence. The function of the spiral prominence is unclear. Two types of voltage-dependent K+ currents are expressed in spiral prominence epithelial cells, which may play a role in the homeostasis of inner ear fluids Pds), and the Pds mutant mouse also shows reduced EP Pds mutant shows a severe early developmental defect of the inner ear with extensive hydrops Spns2 mutants. This indicates that Spns2-deficient and Pds-deficient mice may have different mechanisms underlying the reduced EP and hearing impairment. Dysfunction of the spiral prominence in Spns2-deficient mice may be the main trigger of reduction of the EP and a series of pathological changes in inner ears.The most robust labeling for + recycling and are considered to mediate K+ translocation between the epithelial cell network of the organ of Corti and the fibrocyte network of the lateral wall, and to facilitate ion flow directed towards the stria vascularis One later change we saw in the lateral wall was a localised decrease of Gjb2 and Gjb6 expression in the type II fibrocytes of the spiral ligament located adjacent to the spiral prominence. Type II fibrocytes are important for KS1pr2-null mice are deaf and share some pathological changes with Spns2-deficient mice, such as disorganized cell boundaries of marginal cells, dilated capillaries in the stria vascularis, and degeneration of the organ of Corti S1pr2-null or S1pr2/S1pr3 double null mice, no overt vestibular defects were found in Spns2-deficient mice. Thus, we propose that the Spns2-S1P-S1PR2 signalling axis is important for normal hearing function. A similar Spns2-S1P-S1PR2 signalling axis may exist in bones as both S1pr2-deficient Spns2-deficient S1P is a bioactive lipid and acts as a second messenger intracellularly and as a ligand for cell surface G protein-coupled receptors extracellularly Spns2 function in blood vessel or lymphatic endothelial cells, platelets or red blood cells did not affect hearing, suggesting that systemic loss of Spns2 activity in these tissues does not mediate the hearing loss we see in the tm1aSpns2 mutants. However, when we deleted Spns2 locally in the inner ear using the Sox10-Cre recombinase, the resulting mutants were deaf. Sox10 is expressed throughout the otic epithelium from an early stage of development as well as in cranial neural crest-derived cells, so can effectively drive deletion of exon 3 of the tm1cSpns2 allele in the entire inner ear tm1a/tm1aSpns2 mice is due to local loss of Spns2 function in the inner ear.Systemic disruption of tm1apns2 and Stm1bpns2 homozygous mutants. We did not see anterior eye defects in any of the 5 conditional alleles, consistent with normal anterior eye development in another conditional Spns2;Tie2-Cre mutant mouse Spns2 also plays a role in retinal blood vessels. Our results showed that global Spns2 knockout resulted in a mild phenotype of the retinal vasculature (thin and irregular veins) with decreased pericyte coverage in the central retina which may be related to the widely known role of S1P signalling in angiogenesis Spns2 in these tissues. We also found focal retinal degeneration in these mutant eyes suggesting a role for Spns2 in the photoreceptor and/or retinal pigment epithelium. Taken together, these findings suggest that SPNS2 is not only a candidate gene for involvement in deafness, but also for deaf-blind syndromes.Defects of the anterior eye were only seen in the SSpns2-deficient mice displayed rapidly progressive hearing impairment associated with a rapid decline in the EP between P14 and P21. The mechanism by which Spns2 deficiency leads to decreased EP merits further investigation, but it most likely involves local S1P signalling. Following the early drop in the EP, later changes include reduced expression of key proteins involved in cochlear homeostasis and ultimately sensory hair cell loss. Our findings suggest that Spns2 is a promising candidate gene for human deafness. Furthermore, Spns2-deficient mice may serve as a model to learn more about the role of S1P signalling in auditory function and the mechanism underlying at least one form of strial hearing loss.In summary, we report here that Mouse studies were carried out in accordance with UK Home Office regulations and the UK Animals (Scientific Procedures) Act of 1986 (ASPA) under a UK Home Office licence, and the study was approved by the Wellcome Trust Sanger Institute's Ethical Review Committee. Mice were culled using methods approved under this licence to minimize any possibility of suffering. The mice were maintained in individually-ventilated cages at a standard temperature and humidity and in specific pathogen-free conditions. Either sex was used for this study.Spns2 mutant allele we used carries a promoter-driven cassette designed to interrupt normal gene transcription but flanked by Frt sites to enable its removal and conversion to a conditional allele with a critical exon surrounded by LoxP sites WtsiSpns2, abbreviated to tm1aSpns2 in this study. A schematic of the knockout-first design of tm1aSpns2 allele is shown in tm1aSpns2 was confirmed by a series of genotyping PCR analyses The tm1aSpns2 colony was maintained on a mixed genetic C57BL/6BrdTyrc-Brd;C57BL/6Dnk;C57BL/6N background. tm1a/tm1aSpns2 mice were crossed to Tg(CMV-Cre)Brd/WtsiHprt transgenic mice (on a C57BL/6NTac background) with systemic expression of Cre recombinase to remove the cassette and produce mice carrying the tm1bSpns2 allele /WtsiGt(ROSA)26Sor mice expressing Flp recombinase ubiquitously in which the promoter-driven cassette was excised and exon 3 was retained flanked by LoxP sites 1Wdr) Spns2 in the inner ear and craniofacial neural crest-derived tissues tm1dSpns2 allele was confirmed by co-presence of tm1cSpns2 and Sox10-Cre allele PCR bands. Since S1P is released from different blood cells and endothelial cells, we used mice expressing Tie1-Cre tm1c/tm1cSpns2 mice to produce the conditional knockouts.Spns2 probe was designed to cover the 3\u2032 untranslated region (Applied Biosystem). Hypoxanthine-guanine phospharibosyltransferase (Hprt) was amplified simultaneously  as an internal reference. The relative quantity of Spns2 was calculated using the 2\u2212\u0394\u0394Ct method The organ of Corti, lateral wall , eyes and livers of postnatal day (P)4 homozygous, heterozygous and wildtype littermate mice were dissected in RNAlater (n\u200a=\u200a3 for each genotype). Total RNA was isolated with QIAshredder columns  and the RNAeasy mini kit . RNA was normalized to the same concentration for cDNA synthesis using oligo dT and SuperScrip II (Invitrogen). Real-time PCR was performed in triplicate for each sample using a CFX connect real time PCR machine (BIO-RAD). The Spns2 due to the LacZ gene inserted in the cassette of tm1aSpns2 allele (Spns2 promotor. Inner ears of P10 and P14 heterozygous and homozygous mice (at least three mice of each age group) were dissected out and fixed in 4% paraformaldehyde (PFA) for 45 minutes to 2 hrs. These were washed twice with PBS and decalcified in 10% EDTA until soft. After a PBS wash and immersing in 30% sucrose, inner ears were embedded in Agarose type VII , then mounted using OCT compound ready for cryosectioning at 14 \u00b5m. Sections were treated with Solution A  for 15 mins, then incubated with Solution B (Solution A plus 5 mM K3Fe(CN)6; 5 mM K4Fe(CN)6; 1 mg/ml X-Gal in DMSO) over night at 37\u00b0C. Sections were rinsed in water then counterstained in Fast Red to label nuclei, mounted and examined.X-gal staining can be used to visualise the expression of a allele , downstrtm1aSpns2 mice were used at the standard maximum intensity of 95 dB SPL with free field delivery as shown in Mice were anaesthetised by ketamine hydrochloride  and xylazine hydrochloride  and subcutaneous needle electrodes were inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip  stimuli at a range of intensity levels in 5 dB steps. Averaged responses to 256 stimuli, presented at 42.2 per second, were analysed and thresholds established as the lowest sound intensity giving a visually-detectable ABR response The temporal bones were isolated. The inner ears were dissected out and fixed by 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer with 3 mM calcium chloride at room temperature for 3 hours. Cochleae were finely dissected in PBS. This was followed by further processing using an osmium-thiocarbohydrazide-osmium (OTOTO) method Inner ears  were dissected out and gently perfused with 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M sodium phosphate buffer with 0.8 mM calcium chloride through the round and oval windows and a small hole in the apex then fixed at room temperature for 2 hours. Secondary fixation was in 1% osmium tetroxide in sodium phosphate buffer for 1 hour. After 5 washes in 0.1 M sodium phosphate buffer, inner ears were decalcified in 0.1M EDTA at 4\u00b0C until soft. Then the samples were dehydrated through an ethanol series, staining in 2% uranyl acetate at the 30% ethanol stage, embedded in Epon resin mixed 1\u22361 and then 3\u22361 in propylene oxide for 30 min and infiltrated overnight under vacuum in neat resin. The samples were embedded at 60\u00b0C for 24\u201348 hours. 1 \u00b5m sections were cut through the modiolar plane and stained with toluidine blue for light microscope observation. 60 nm sections were cut on a Leica EM UC6 ultramicrotome, stained in 2% uranyl acetate and aqueous lead citrate and imaged on an FEI Spirit Biotwin 120 kV transmission electron microscope using a Tietz F4.15 CCD.Mice were anaesthetized with 0.01 ml/g body weight of 20% urethane, a tracheal cannula was inserted and the bulla was opened to reveal the cochlea while the body temperature was kept at 37\u00b0C by a feedback-controlled heating pad. A small hole was made in the bony wall of the cochlea over the basal turn of scala media, and a micropipette electrode filled with 150 mM potassium chloride was advanced through the hole and through the lateral wall into the scala media. The potential difference between the scala media and a reference silver/silver chloride pellet under the dorsal skin was recorded The inner ears were rapidly dissected out and fixed in 4% paraformaldehyde at room temperature for 2 hours. The lateral walls were dissected out in PBS for surface preparation. Filamentous actin was visualized by rhodamine phalloidin  at room temperature for 2 hours. Strial capillaries were visualized by Isolectin B4  at 4\u00b0C, overnight in PBS with 10% sheep serum). Samples were mounted with Vectashield Mounting medium  and imaged by confocal microscopy . The numbers of capillary branch points per field (220\u00d7220-\u00b5m fields) in the middle turn  of the stria vascularis  was quantified using image J. Data were presented as a density in a 100\u00d7100 \u00b5m field and statistics analysis was conducted using Mann-Whitney Rank Sum Test, SigmaPlot v12.0. Surface preparations were also used for analysis of Kcnq1 expression, using overnight incubation at 4\u00b0C with goat anti-Kcnq1 polyclonal antibody  followed by washing with PBS and incubation with donkey anti-goat secondary antibody  prior to analysis by confocal microscopy. At least three homozygotes and three controls were used at P14, P28 and 6 months for phalloidin labeling to show marginal cell boundaries, and P14 and 5\u20136 weeks for Kcnq1 expression. The density of marginal cells was measured in the phalloidin-labelled whole mount preparations by counting the number of cells defined by their labeled boundaries in two areas each 100\u00d7100 \u00b5m from the middle turn (40\u201370%) of each cochlea .+, K+-ATPase (\u03b11 subunit) monoclonal , mouse anti- NKCC1 monoclonal  and rabbit anti-ZO-1 polyclonal . Sections were blocked by incubation with 10% sheep serum (with 0.1% TritonX-100 in PBS) for 40 mins. Sections were incubated with appropriate primary antibodies overnight at 4\u00b0C, washed with PBS and incubated with corresponding secondary antibodies at room temperature for 2 hours . After washing with PBS, slides were imaged by confocal microscopy. Three mice of each genotype (tm1a/tm1aSpns2 and +/+Spns2) were used for each antibody at P14 and 5\u20136 weeks old.The cochleae were dissected out and fixed in 4% PFA at room temperature for 2 hours. Cryosections were obtained as described above for X-gal staining. We used the following antibodies: rabbit anti-Kcnj10 polyclonal , rabbit anti-Gjb2 polyclonal , rabbit anti-Gjb6 polyclonal , mouse anti- NaThe inner ears  were dissected and round and oval windows were opened in PBS containing 1 mM calcium chloride. A hole was made in the basal turn leading to the scala media. The membranous labyrinth was perfused for 5 minutes with 400 \u00b5l Sulfo-NHS-LC-Biotin  through the round and oval windows and the hole exposing the endolymphatic compartment. Following a PBS wash, the inner ears were fixed in 4% paraformaldehyde at room temperature for 2 hours, and processed for cryosectioning as described above. The biotin tracer was detected by fluorescein isothiocyanate (FITC)-conjugated streptavidin  incubating at room temperature for 30 min. Samples perfused with PBS alone were used as negative controls.2 inhalation and the auditory bullae dissected out. Whole cochleae were exposed and fixed by removing a small piece of bone at the apex and gently perfusing 4% PFA through the round and oval windows. Cochleae were then immersed in fixative and left on a rotator for 1.5 hours at room temperature. Whole-mounts of stria vascularis were dissected from fixed cochleae, covered with Vectashield Mounting Media  in glass bottom culture dishes (MatTek Corp.) and imaged using confocal laser-scanning microscopy .Mice  were warmed in a 39\u00b0C incubator for 5 minutes and then held in a mouse restrainer so that the tail was accessible and the tail vein visible. A 50 \u00b5l aliquot of 5% (w/v) FITC-conjugated bovine serum albumin , made up in sterile PBS, was injected into the tail vein. The mice were left at room temperature for 45\u201360 minutes to allow the BSA-FITC to permeate all capillaries and to allow for any vascular extravasation. The mice were sacrificed by COMice underwent ophthalmic screening at 15 weeks of age. They were assessed for gross morphological changes to the eye using a slit lamp (Zeiss SL130) and ophthalmoscope (Heine Omega 500). The eye was examined both undilated and dilated . Images using the slit lamp were collected using a Leica DFC420 camera. The mice were culled under terminal anaesthesia followed by cervical dislocation and both eyes from 3 male homozygous mutants and 3 wildtype mice were removed and fixed. Pupil-optic nerve sections were processed, stained with hematoxylin and eosin, and standard images were captured under light microscopy for review For whole mount retinal analysis, heterozygotes and homozygotes were used  and the eyes removed and fixed in 4% PFA. Retinae were prepared and stained as described Figure S1Spns2 expression in the vestibular system, normal gross structure of inner ears and normal organ of Corti at P4. A,B: X-gal staining showed expression of Spns2 in the vestibular system at P10. Labelling (blue) was detected in the cristae  and maculae (utricular macula shown here) . Scale bar: 20 \u00b5m. C,D: Cleared inner ears showed no apparent differences in gross structure between Spns2 homozygous mutants and controls at 4 weeks old. Scale bar: 1 mm. E,F: Scanning electron microscopy showed no abnormalities of the surface of the organ of Corti at P4 in the Spns2 homozygous mutants compared with littermate controls. Scale bar: 10 \u00b5m.(TIF)Click here for additional data file.Figure S2A,B: Confocal images focussed at the level of the basal cell boundaries, visualised by phalloidin staining (red). No obvious change was seen in Spns2 homozygous mutants at 4 weeks old. Scale bar: 10 \u00b5m. C,D: The stria vascularis showed dilated and tortuous capillaries with increased branch points in all five Spns2 homozygous mutants studied at 4 weeks old. Strial capillaries were visualised by isolectin B4 (green). Scale bar: 50 \u00b5m. E,F: Strial hyperpigmentation was pronounced in older mutants. A seven month old Spns2 homozygous mutant (F) had obvious accumulation of pigment in the stria vascularis. Scale bar: 20 \u00b5m.Whole-mount stria vascularis examination. (TIF)Click here for additional data file.Figure S3+/K+-ATPase, NKCC1 and ZO-1 in lateral wall and Kcnj10 in spiral ganglion at 5\u20136 weeks. Na+/K+-ATPase (red) labelling in stria vascularis and type II fibrocytes in Spns2 homozygous mutants (A) was comparable with that of controls (B). Notice absence of Gjb2 labelling in the type II fibrocytes in the mutants. NKCC1 (red) labelling in the Spns2 homozygous mutants was located in stria vascularis and type II fibrocytes and appeared similar in the controls . ZO-1 (green) labelling was present in the basal cells of the stria in both Spns2 homozygous mutants and controls . G,H: Acetylated \u03b1-tubulin (red) labelled spiral ganglion neurons and Kcnj10 (green) labelled satellite cells. Kcnj10 expression in Spns2 homozygous mutants was present and comparable with controls, suggesting that the reduced Kcnj10 labelling observed in the stria Click here for additional data file.Figure S4A: Analysis of the percentage of pericyte coverage of the retinal blood vessels revealed a significantly reduced coverage in the mutants in the central retina , but no significant difference in coverage of the peripheral vessels   between the Spns2 mutant homozygotes and heterozygous controls at P10. B: tm1aSpns2 homozygous mutants displayed open eyelids at birth.Pericyte coverage of retinal blood vessels and open eyelids at birth. (TIF)Click here for additional data file."}
+{"text": "The rate and extent of CD4 cell recovery varies widely among HIV-infected patients with different baseline CD4 cell count strata. The objective of the study was to assess trends in CD4 cell counts in HIV-infected patients after initiation of antiretroviral therapy in Tigray, Northern Ethiopia.A retrospective cross-sectional study was conducted by reviewing medical records of HIV patients who received antiretroviral treatment at twenty health centers in Tigray region during 2008\u20132012. Multi-stage cluster sampling technique was employed to collect data, and the data were analyzed using SPSS version 20.0 software.The median change from baseline to the most recent CD4 cell count was +292 cells/\u03bcl. By 5 years, the overall median  CD4 cell count was 444(263-557) cells/\u03bcl while the median (IQR) CD4 cell count was 342(246-580) cells/\u03bcl among patients with baseline CD4 cell counts \u2264200 cells/\u03bcl, 500(241-557) cells/\u03bcl among those with baseline CD4 cell counts of 201\u2013350 cells/\u03bcl, and 652(537-767) cells/\u03bcl among those with baseline CD4 cell counts >350 cells/\u03bcl. Higher baseline CD4 cell counts and being male were independently associated with the risk of immunological non-response at 12 months. Furthermore, it was also investigated that these factors were significant predictors of subsequent CD4 cell recovery.Patients with higher baseline CD4 cell stratum returned to normal CD4 Cell counts though they had an increased risk of immunological non-response at 12 months compared to those with the least baseline CD4 cell stratum. The findings suggest that consideration be given to initiation of HAART at a CD4 cell count >350 cells/\u03bcl to achieve better immune recovery, and to HIV-infected male patients to improve their health seeking behavior. CD4 cell counts are commonly used markers of HIV disease progression and for starting and monitoring antiretroviral treatment in the absence of viral load ,2. SustaCommencement of ART at a baseline CD4 cell count <200 cells/\u03bcl may lead to a high proportion of patients with acquired immunodeficiency syndrome (AIDS) and a high case fatality rate ,7. A sigTo the best of our knowledge, there are no studies from the study area concerning rates of CD4 cell recovery and rates of immunological non-response to ART among HIV-infected patients. Therefore, we conducted a retrospective study in order to assess the trends of CD4 cell recovery among HIV patients after initiation of antiretroviral therapy in Tigray, Northern Ethiopia.The study was conducted in Tigray region, Northern Ethiopia. Twenty health centers in the six administrative zones of the region were included in the study. A retrospective study was conducted by reviewing medical records of HIV patients aged 18 years or greater who received antiretroviral treatment at twenty health centers in Tigray region, Northern Ethiopia during 2008\u20132012. ART delivery was started in September 2003 as fee service and the free ART program started in March 2005. Pregnant women, seriously ill and diabetic patients were also excluded.The required sample was calculated using a single population proportion formula. The following parameters were taken into account during the calculation of sample size: 50% rate of CD4 cell count recovery among HIV-infected patients (to achieve maximum representative sample size), 95% confidence interval and 5% margin of error. Then the determined sample size, after considering 10% non-response rate, was multiplied by 2 to consider the cluster effect and increase power. Thus, a required sample size of 844 medical records of HIV patients was required for this study.Multistage cluster sampling technique was used to select the different health centers. Hence, twenty health centers were randomly selected proportionally among the sixty health centers addressed by MSH/USAID care and support programme from the six administrative zones found in Tigray region. Study participants from the selected health centers were chosen by systematic random sampling method.A baseline and 6-monthly CD4 cell count, and basic information such as patients\u2019 sex, age, weight and WHO clinical stage were collected from medical records.Data were entered and analyzed using SPSS for Windows, version 20.0. The median (IQR) in the absolute CD4 cell count at baseline and every six months thereafter was determined. Changes in CD4 cell count every six months were also examined and stratified on the basis of baseline CD4 cell count .Categorical variables were summarized as frequencies and percentages while numerical variables with non-normal distribution were summarized as median and IQR. Logistic regression was conducted to examine factors associated with the risks of immunological non-response (defined as failure to attain an absolute CD4 cell count increase from baseline of at least 50 cells/\u03bcl at 12 months of ART). A multivariate least-squares linear regression analysis was employed to assess changes in CD4 cell count on the basis of baseline CD4 cell count and these other covariates. An initial analysis included all covariates. A final multivariate model was computed using a backward stepwise approach, retaining only those variables that were statistically significant. All tests of significance were two-sided, with p<0.05 indicating statistical significance.The study was approved for ethical issues by the Health Research Ethics Review Committee of College of Health Sciences, Mekelle University. Data collection was conducted after further approval of the study by the Tigray regional health bureau and directors of each health centers. The purpose of the study was explained to the study participants and all study participants gave a written informed consent.A total of 841 medical records were reviewed; most (68.7%) of the patients were female. At baseline majority (79.5%) of the patients were less than 45 years old with a mean age (SD) of 36.34 (9.48) years. More than half (56.2%) of the patients were in WHO clinical stage III at baseline. About two-third (64.4%) of the patients were found to have baseline CD4 cell counts less than 200 cells/\u03bcl. The weight distribution shows that nearly three-forth (75.1%) of patients were found to have weight of 40\u201360 Kg .th and 60th months where the median CD4 cell count showed a fall. The median change from baseline to the most recent CD4 cell count was +292 cells/\u03bcl, with the overall median (IQR) CD4 cell count increased to 444(263\u2013557) cells/\u03bcl by 5 years after the start of ART. The overall median (IQR) CD4 cell count in our HIV patients increased by about 2-fold and 3-fold from baseline 152 (98\u2013224) cells/\u03bcl, reaching 300(201\u2013398) and 460(240\u2013704) cells/\u03bcl at 12 and 48 months after initiation of ART, respectively. By 5 years, the median (IQR) CD4 cell count increased to 342(246\u2013580) cells/\u03bcl among patients with a baseline CD4 cell count of \u2264200 cells/\u03bcl, to 500(241\u2013557) cells/\u03bcl among those with a baseline CD4 cell count of 201\u2013350 cells/\u03bcl, and to 652(537\u2013767) cells/\u03bcl among those with a baseline CD4 cell count of >350 cells/\u03bcl. The median (IQR) CD4 cell count of patients with a baseline CD4 cell count of \u2264200 cells/\u03bcl were about doubled and tripled by 12 and 36 months after initiation of ART, respectively.The six monthly changes in the median CD4 cell count after the commencement of ART and the number of patients having CD4 data is plotted in An analysis of the six monthly increase in CD4 cell count demonstrated that, the slope of the plotted overall CD4 cell count increased significantly  until 48 months except between 18 and 30 months, where it slightly increased. The proportions of patients who achieved CD4 cell counts of \u2265500 cells/\u03bcl after receiving 48 months of suppressive ART were 40.8%, 45% and 93.3% among patients with baseline CD4 cell counts of \u2264200, 201\u2013350 and >350 cells/\u03bcl, respectively. The percentages of patients who achieved CD4 cell counts of \u2265750 cells/\u03bcl after receiving 48 months of ART were 19.8%, 25% and 60% among patients with baseline CD4 cell counts of \u2264200, 201\u2013350 and >350 cells/\u03bcl, respectively.Baseline CD4 cell count was evaluated whether it was a risk factor associated with immunological non-response (defined as a CD4 cell increase of <50 cells/\u03bcl at 12 months). The percentages of patients failed to attain an absolute CD4 cell count increase from baseline CD4 cell count of at least 50 cells/\u03bcl at 12 months was 22.7%. The proportions of patients who were immunological non-responders were 17.9%, 28% and 50% among patients with baseline CD4 cell counts of \u2264200, 201\u2013350 and >350 cells/\u03bcl, respectively. Furthermore, logistic regression analysis revealed that baseline CD4 cell count was a significant predictor of immunologic failure. An increased risk of immunological non-response was independently associated with higher baseline CD4 cell counts of 201\u2013350  and > 350 cells/\u03bcl . An increment of <50 cells/\u03bcl was also independently associated with male patients. Male patients had about two times increased odds of risk of immunological non-response compared to female patients  .Multivariate regression analysis shown that baseline CD4 cell count was a significant determinant of subsequent CD4 cell recovery . Two anath and 60th months where the median CD4 cell count showed a slight decline [This retrospective study was carried out to assess the trends in CD4 cell recovery among HIV patients after initiation of ART and the effect of baseline characteristics on CD4 cell count response. Improvements in overall CD4 cell count among the patients was seen over time until year 4 though it declined thereafter. These findings are consistent with the retrospective longitudinal study conducted in eastern Ethiopia where the median CD4 lymphocyte count had improved over the five year period except at the 54 decline . Other s decline . Actuall decline .Immunological response rates were evaluated in patients with different baseline CD4 cell in this survey. These results indicated that patients with baseline CD4 cell counts of \u2264200 cells/\u03bc had greater rates of immunological response compared to those with higher baseline CD4 cell counts. These findings are in line with that of another study where patients with the lowest CD4 counts had similar or greater rates of CD4 cell recovery and a lower risk of immunological non-response . HoweverOn the other hand, the present work reported that patients with lower baseline CD4 cell counts had lower peaks CD4 cell counts. Patients with a baseline CD4 cell count of >350 cells/\u03bcl stratum had CD4 cell counts that returned to almost normal levels. These results are consistent with that of a longitudinal observational study among patients receiving primary HIV care in Baltimore, Maryland which shows only patients with a baseline CD4 cell count of >350 cells/\u03bcl had CD4 cell counts that returned to nearly normal levels though CD4 cell count was increased and maintained in all CD4 cell count strata to 6 years . Other sThese findings indicate that the highest CD4 cell counts were achieved when HAART was started at a baseline CD4 cell count >350 cells/\u03bcl. The CD4 cell count is less likely to return to normal when HAART is started at lower CD4 cell counts, this could be a reason to consider initiating HAART before the CD4 cell count decreases to <350 cells/\u03bcl.In this study, an increased risk of immunological non-response was significantly observed among male patients like another study . Other sIn conclusion, the present findings confirm the previous studies that the degree of CD4 depletion prior to ART initiation is the most consistent determinant of subsequent immune reconstitution. Low baseline CD4 count at entry to an ART programme was associated with increased risks of morbidity and mortality . A meta-"}
+{"text": "Immunosuppression is the hallmark HIV infection associated with gradual loss of CD4+Tcells. The effector immune response is regulated by CD4+CD25+Foxp3+Tcells (Tregs). Enumeration of Tregs frequency showed equivocal results before and here we showed clearly their frequency by flowcytometry.One hundred and eight individuals comprising of 71 HIV positive patients (44 patients undergoing HAART and 27 without HAART) and 37 HIV negative healthy controls. PBMCs were isolated and stained with CD4/FITC, CD25/APC, FoxP3/PE antibodies (BD Pharmingen) and analyzed for Tregs frequency by flowcytometer .Our study showed that healthy controls had a Tregs frequency of 4.1%. When Tregs frequency analyzed among HIV/AIDS patients who are yet to receive HAART we found a significant reduction in Tregs population i.e. 1.2%. Encouragingly this deep plunge in Tregs population was found to be restored and surged upon HAART up to 12.6%. However, these changes in percentages did not alter based on their gender or age or absolute CD4 count of the study population.HAART is known to decrease viral load and improve the patient\u2019s CD4 cells count. Here we report that HAART has improved the Tregs frequency. It is yet to be known that the consequence of resurging Tregs during HIV/AIDS."}
+{"text": "We explored the meaning of variations in tissue oxygen saturation downslope (StO2 target. The slope of the desaturation curve was assessed separately for the first part (StO2 down1) and the last part (StO2 down2) of the curve and the difference between Down2 - Down1 was calculated.In this prospective observational study, NIRS (thenar eminence) was applied every day in 93 patients admitted to the ICU. A VOT was performed using a 40% StO2 Down1 or Down2 between ICU survivors (n = 76) and ICU nonsurvivors (n = 17) over the first 10 days in the ICU, while Down2 - Down1 was higher in ICU nonsurvivors .No significant differences were seen in StOICU nonsurvivors tended to show a flattening in the last part of the desaturation curve during a VOT, suggesting a reduced tissue oxygen extraction. This may depend on microvascular dysfunction and/or cellular hypometabolic status."}
+{"text": "The chronic pain sufferer is currently faced with a lack of objective tools to identify the source of their pain. The overarching goal is to develop clinical [18F]FDG PET/MRI methods to more accurately localize sites of increased neuronal and muscular metabolism or inflammation as it relates to neurogenic sources of pain and to ultimately improve outcomes of chronic pain sufferers. The aims are to 1) correlate imaging findings with location of pain symptomology, 2) predict location of symptoms based on imaging findings alone and 3) to determine whether the imaging results affect current management decisions. Six patients suffering from chronic lower extremity neuropathic pain  have been imaged with a PET/MRI system  from mid thorax through the feet. All patients underwent PET/MR imaging one hour after a injection of 10mCi [18F]FDG. Two radiologists evaluated PET/MR images (one blinded and the other unblinded to patient exam/history). ROI analysis showed focal increased [18F]FDG uptake in affected nerves and muscle (approx 2-4 times more) over background tissue in various regions of the body in 5 of 6 patients at the site of greatest pain symptoms and other areas of the body (SUVmax of Target 0.9-4.2 vs. Background 0.2-1.2). The radiologist blind to the patient history/exam was able to correctly identify side/location of the symptoms in 5 out of 6 patients. Imaging results were reviewed with the referring physician, who then determined whether a modification in the management plan was needed: 1/6 no change, 2/6 mild modification  and 3/6 significant modification."}
+{"text": "To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated  Furthermore, the restricted expression of these TSAs on tumor cells allow for the generation of specific therapeutic agents with little on-target/off-tumor effects. However, many antigens are also expressed on normal tissues to varying degrees, and are termed tumor-associated antigens (TAA). Cellular dysregulation that occurs during the oncogenic process can also lead to upregulation of normal tissue proteins. Whilst still present on the tissue cells of origin, this increased level of protein expression can sometimes be correlated with patient prognosis, and therefore can be identified as a TAA [Tumor cells often display an altered array of proteins that distinguish them from the cells of origin. On occasion, these proteins arise from oncogenic mutations, and are termed tumor-specific antigens (TSA), with expression on the tumor cells and conversely absent on the \u2018normal\u2019 surrounding tissue. These TSAs are generated as a TAA . Such isas a TAA ,3. Moreoas a TAA \u20136.de novo [As Her2 is not exclusively present on breast tissue but is also found in the brain, the lower intestine and lung, immunotherapies directed against the Her2 antigen may also encounter on-target/off-tumor effects resulting in toxicity and autoimmunity . In addide novo .Multiple mouse models of the TAA Her2 have emerged and facilitated the study and development of Her2 therapeutics. Two of the most commonly used promoters used to drive the expression of the Her2 antigen are the mouse mammary tumor virus (MMTV) and the whey acidic protein (WAP) promoter. Whilst both are highly expressed in the mammary gland, expression of the MMTV promoter has also been found in other organs, including the kidneys, lungs, testes as well as in T cells ,10. Simiet al. therefore generated the human Her2 (Her2) transgenic mouse model driven by the whey acidic protein (WAP) promoter [In order to adequately assess potential therapies in which the Her2 antigen could be targeted specifically on tumor cells, leaving the surrounding normal tissues with basal expression unaffected, it was necessary to generate a clinically relevant mouse model. Piechocki promoter . In thispromoter ,19\u201321. T+ tumors. Conversely, Her2 tumors are often rejected in wildtype mice due to the high level of immunogenicity against the Her2 antigen. In addition, DNA vaccination with the Her2 antigen prior to Her2+ tumor challenge induced a robust anti-tumor response, with up to 33% survival of Her2+ mice , indicating these mice were able to overcome tolerance to the Her2 antigen, and successfully mount an immune response. No autoimmunity or off-target responses were recorded in this study [Primary analysis of the Her2 mouse model has demonstrated its tolerance to Her2is study .The tolerance to Her2 tumors in this mouse model has been critical in the development of immunotherapies against the Her2 antigen, particularly in solid established tumor models. The adoptive transfer of T cells genetically modified with a chimeric antigen receptor specific for the Her2 antigen into Her2 tumor bearing mice have demonstrated both tumor regression and prolonged survival in the absence of autoimmunity ,23.+/-) mice display a normal phenotype and are similar to their wild type littermates. However, transgenic mice are often bred in a homozygous state, ensuring the progeny themselves are transgenic and thereby reducing the variability between breeding pairs of heterozygous matings. Attempts at generating homozygous Her2+/+ mice have been unsuccessful, potentially since the inheritance of the two Her2 transgenes in this model resulted in embryonic lethality. As no overt pathology or abnormalities were observed in heterozygous mice, we speculated that it would be unlikely that the inheritance of twice as many Her2 copies would result in this lethality. Rather, we hypothesised that perhaps the integration of the Her2 transgene had interrupted a gene essential for embryonic or fetal development. To answer this, we performed whole genome sequencing (WGS) on Her2+/- mice to determine the integration site of the transgene. Whole genome sequencing revealed the integration of the Her2 transgene had indeed interrupted a gene, Pds5b, whose function is known to be integral in the segregation of chromosomes in both meiosis and mitosis [+/+ mice display greater development defects than previously reported for some Pds5b-/- mice [The generation of transgenic mice often involves the random integration of the desired transgene into the genome. While this integration often has little effect on the normal phenotype of the mice, there may be occasions where transgene integration could potentially disrupt a gene integral for development and survival. Heterozygous Her2 mice  with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin and 100 ug/mL streptomycin (Life technologies). The murine E0771 (LMC variant) breast adenocarcinoma line was kindly donated by Prof. Robin Anderson  and maintained at 37\u00b0C in 10% CO2 in Dulbecco's modified Eagle medium (DMEM), supplemented as above. Both cell lines were retrovirally transduced with the human Her-2 antigen (ERB) as previously described [The murine 24JK fibrosarcoma cell line was kindly donated by Dr. Patrick Hwu   and mainescribed .6 24JKERB or 1 x 105 E0771ERB were injected into Her2+/- mice subcutaneously or 5 x 105 E0771ERB injected into the mammary fat pad. Tumors were measured on days as stated and mice were sacrificed when tumor size reached ethical limit of 150 mm2.1 x 10+ mice were interbred to generate litters consisting of Her2-/-, Her2+/- and Her2+/+ mice. Embryos were harvested from pregnant Her2+/- females from E14 onwards and analysed for signs of malformation and abnormal development. Tail clippings were taken at this time point and analysed for the presence of the Her2 transgene via polymerase chain reaction (PCR).Heterozygous Her2+/- transgenic mice using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturers\u2019 instructions or using phenol:chloroform extraction. The DNA was quantified and purity verified. 500 ng of DNA were fragmented using a focal acoustic device (Covaris S2) and used to prepare libraries with the KAPA Library Preparation Kit for Illumina platforms (KAPABIOSYSTEMS). Libraries were size selected to an average fragment size of 600 bp using the PippinPrep Instrument (SAGE Science). Three indexed libraries were pooled and sequenced across three lanes of an Illumina HiSeq2500 flowcell using High Output chemistry v3 (Illumina).Genomic DNA was extracted from the tails of Her2http://genome.ucsc.edu) and the putative Her2 transgene sequence . As a target for sequence alignment we concatenate the mm10 build of the mouse reference . The seei et al ). Bowtie+/+, Her2+/- and Her2-/- was isolated from tail clippings of E14.5 to E19 pups as previously mentioned. Briefly, genomic DNA was extracted from tissue samples (~1\u20132 mm2) using QuickExtract DNA extraction solution . After adding 20 \u03bcL QuickExtract solution to each tissue sample, the samples were incubated at 65\u00b0C for 20 minutes, then 95\u00b0C for a further 20 minutes. The samples were then diluted 1:10 in sterile deionised water, and 1 \u03bcL was used for PCR. PCR amplification was performed using the following primers; Pds5b Forward 5\u2019 GGACTATTTACAGGAAACGTC 3\u2019, Pds5b Reverse 5\u2019 AGCAAGCCACCAGTAAACG 3\u2019 and Her2 Forward 5\u2019 GTCACAGGGGCCTCATCC 3\u2019. The Her2 forward primer spanned the junction point of insertion of the transgene concatemer into the chromosome, with the first 14 bp annealing to the truncated 3\u2019 end of the transgene, and the final 4 bp annealing to the Pds5b gene at insertion. DNA was amplified with the following conditions; 95\u00b0C for 5 minutes, 95\u00b0C for 30 seconds, 58\u00b0C for 30 seconds, 72\u00b0C for 30 seconds, repeat step 2 to 4 for 35 cycles, 72\u00b0C for 5 minutes. Amplified DNA was analysed on a 1% agarose gel.Genomic DNA of Her2+ tumors have been shown to grow poorly in wild type immunocompetent mice due to the highly immunogenic nature of the antigen. The expression of the human Her2 antigen from birth in the Her2 transgenic mouse models generated a functioning immune system tolerant to the Her2 antigen. As a result, Her2+ tumors were able to sufficiently evade the adaptive immune system and grow without hindrance. To validate tolerance of Her2+/- mice, we first demonstrated the ability of Her2+ tumors to grow in Her2 transgenic mice. The murine sarcoma cell line 24JKERB consistently grew subcutaneously in Her2 transgenic mice , instead of the expected 75%, suggesting perhaps a complete absence of homozygous mice. To further substantiate our hypothesis, we had previously attempted generating homozygous mice through intensive iterative breeding programs however were never able to achieve 100% transgenic progeny. As no overt pathology in terms of development or immune function was noted in heterozygous mice, we hypothesised that perhaps the transgene integration site had interrupted an integral gene. We decided to utilize whole genome sequencing (WGS) to determine the integration site, as it is a fast, efficient and reliable method.The generation of transgenic mouse models often involves the microinjection of the linearised DNA transgene into oocytes at the pro-nuclear stage. For most transgenes, this integration occurs in a non-homologous manner, as often these transgenes are preceded by a highly active promoter thus the transcriptional activity of the surrounding genome (with the exception of silencing epigenetic regulation) is irrelevant to the transgene expression. The Her2 mice were previously generated in such a manner, where the 2.5 kb whey acidic protein (WAP) promoter driving the 4.4kb c-ErbB2 transgene cDNA was digested to create a linearized transgene fragment, which was then microinjected into C57BL/6 oocytes  Fig 2)Fig 2). TCTGGGGATCCTCTAGAGTCGACCTGCAGGCA-TGC) at the 5\u2019 end of the WAP promoter was found using WGS analysis (AGGGGAGGTAACCCTGGCCCCTTTGGTCGG-GGCCCC) between the WAP promoter and Her2 5\u2019 untranslated region (UTR) during our WGS analysis was determined to be homologous to the 5\u2019 UTR of a novel variant of human Her2 cDNA as reported by Yamamoto et al. ([AATAAAGACCCAGGGGGAGAAGCTGGGATCCTCTAGAGTCGACGCATGCAAGCTTNAATGA) at the 3\u2019 region of the Her2 3\u2019 UTR was absent in our WGS analysis. This 61 nucleotide sequence at the 3\u2019 region of the Her2 3\u2019 UTR contained a poly-adenylation sequence, AATAAA (bolded above). As such, the WAP-Her2 transgene found by WGS is lacking an endogenous 3\u2019 poly-adenylation sequence, and it is likely the gene uses a poly-A sequence downstream of the integration site.We observed some non-Her2 sequences using WGS. A 34 nucleotide insert  of the transgenic material into the native DNA of the mice. We performed structural variation analysis on the alignments using Socrates  and usedThe breakpoint detection performed with Socrates predicted 173 fusions in the data. To establish the insertion point we searched for overlap with the transgene sequence within the breakpoint set. There were three fusions that overlapped with the transgene: one from the transgene position 1 to the transgene position 6850, another from chromosome 5 at position 150719804 to the transgene (WAP-Her2) at position 4025, and finally from WAP-Her2 at position 2972 to chromosome 5 at position 150719794. A 10 nt duplication (150719804 to 150719794) was observed at these breakpoints in the host genome. The first fusion was caused by the transgene arranged in a 162 copy concatemer and therefore looping back onto its own start. The latter two breakpoints corresponded to the insertion site of the transgene into the native genome. Using whole genome sequencing, we were able to establish the WAP-Her2 transgene had inserted just 19 nucleotides upstream of exon 3 (150719823) in the Pds5b gene on chromosome 5  or one band for Pds5b . A proportion of embryos produced a single Her2 DNA band, indicating their homozygosity . Her2+/-or Pds5b .+/- mice harboured a large number of copies of the Her2 transgene (162 copies), we hypothesised that a further increase in transgene copies in homozygous Her2+/+ mice (324 copies) was unlikely to produce an additive effect, and thus have very little effect on the overall phenotype in these mice. Instead, we proposed that two copies of the Her2 concatemer present in Her2+/+ mice would completely prohibit normal Pds5b production and function. In addition, we hypothesised that Her2+/+ mice would display a phenotype similar to that of Pds5b-/- mice.The insertion of the WAP-Her2 transgene concatemer was found to lie within an intron (19 nucleotides upstream of exon 3) of the Pds5b gene, presumably disrupting and inhibiting normal transcription and translation of Pds5b. Although the RNA splice acceptor site was retained, the insertion of 162 copies of the transgene, constituting approximately 1.1 Mb, likely prevented effective pre-mRNA processing. As heterozygous Her2-/- models also display a myriad of defects in multiple organs, it is speculated that Pds5b may also play a role in regulating the transcription of a number of genes essential for development and organogenesis [Pds5b  is a homolog of Pds5, a regulatory factor of the cohesin complex. The cohesin complex is essential in dictating accurate chromosomal segregation during sister chromatid cohesion for cell division, in both mitosis and meiosis ,32. Disrogenesis . Furtherogenesis .Pds5b-/- mice have yielded conflicting results, which may be due to varying levels of expression and subsequent penetrance observed in these models [Pds5b-/- mice were found to survive to birth but died shortly after. These mice displayed signs of labored breathing, exhibited respiratory distress and had cardiac abnormalities, all of which may have contributed to their early death [Pds5b-/- mice presented with signs of growth retardation, including facial dysmorphisms, smaller head and limb structures and abnormal cleft palates. However, other studies using Pds5b-/- have observed a more severe phenotype, with embryonic lethality occurring at the late post-implantation stages (E16.5 to E18.5), with no Pds5b-/- mice surviving until birth (0 out of 500) [Pds5b alleles greatly affected embryonic development and resulted in embryonic death, however no centromeric cohesion defects were observed in Pds5b+/- mouse embryonic fibroblasts (MEFs) [While highly conserved, the function and necessity of Pds5b greatly varies between species . Pds5b-e models . In one ly death . The rol of 500) . In thats (MEFs) .+/- x Her2+/- mice to generate litters of Her2+/+, Her2+/- and Her2-/- mice. As developmental defects observed in Pds5b-/- mice were only apparent at E14.5 to E16.5 [+/+ mice and Her2+/- or Her2-/- mice. Her2+/+ mice taken at E14-E15 displayed obvious developmental defects, were severely underdeveloped and appeared much smaller than either Her2+/- or Her2-/- mice  was the most efficient and accurate method for determining the transgene integration site in our studies. Prior to WGS, determining the integration point of a transgene required a combination of multiple assays, each laborious and time consuming. Fluorescent in situ hybridisation (FISH) requires the generation of a sequence specific probe, and can only reveal the location of the transgene on a global genome scale. To determine the exact nucleotide location, TAIL-PCR or primer walking would have to be applied [In the present study, homozygous Her2 applied . FinallyPds5b. The WAP-Her2 concatemer consisted of 162 copies of the transgene, each orientated in a \u2018head to tail\u2019 manner. Taking into account the length of the transgene and the high copy number present, we hypothesise the integration of the WAP-Her2 transgene would have isolated the Pds5b promoter from the coding sequence downstream of the integration site, and thus effectively disrupted accurate transcription of the Pds5b gene. We cannot conclude whether an insertion of this size may have also impacted transcription of the genes flanking the Pds5b gene, and thus potentially have contributed to the observed phenotype. However, the similarities between our mice and other models deficient in Pds5b lead us to hypothesize the main phenotypic characteristics of this model was due to the dysregulated expression of Pds5b.Our results from WGS revealed the WAP-Her2 transgene had formed a long unidirectional concatemer and had integrated at a single position in the genome, upstream of exon 3 of the protein-coding gene, It was interesting to note both the copy number and orientation of the WAP-Her2 transgene in the Her2 tg mice. Whole genome sequencing analysis revealed 162 copies (in heterozygous mice) were present and moreover, that all 162 copies were ligated together and had in fact integrated into one location. Furthermore, the orientation of each transgene in the transgene polymer was observed to be ligated in a unidirectional manner and always present in a 5\u2019 to 3\u2019 orientation. Other reports characterizing transgenic mice models have observed similar results, where the transgene has been found to concatenate into a long unidirectional concatemer, with each transgene present in a \u2018head to tail\u2019 orientation \u201342. ThisPds5b. This confirmed the gene disruption. While further downstream analysis of transcriptional and translational expression of Pds5b, such as RT-PCR or western blot analysis, were not performed, the complete absence of Pds5b at a genomic level in addition to the striking similarities observed between our Her+/+ and other published Pds5b-/- models rendered further analysis of downstream expression beyond the scope of the present study, whose aim was to demonstrate the ability of WGS to provide insight into the difficulty in generating homozygous transgenic mice.We further substantiated the WGS results by using PCR to detect the presence or absence of an intact genomic Pds5 models differs greatly between species. Pds5 null S.cerevisiae indicate it plays an integral role in maintaining cohesion and condensation, and is subsequently vital for accurate cell division [Pds5 in Schizosaccharomyces pombe has shown to have little effect on sister chromatid cohesion [Pds5b in mice show varying phenotypes, with some studies observing prenatal or embryonic lethality while others report survival of null mice up until birth but death shortly after. The discrepancies observed may be a result of the methodology and efficiency of the deletion. Indeed, we observed variance in the severity within the same litter in our study. Similar to findings observed by Losada et al. [Pds5b in our model resulted in embryonic lethality prior to E16.The necessity of Pds5b in organogenesis and development has been difficult to accurately assess. Despite the high level of conservation between species, the role of Pds5b and subsequently the phenotype generated from division ,44. In ccohesion . Furthera et al.  the inte+/+ mice, we observed no overt developmental abnormalities in Her2+/- mice compared to Her2-/- littermates. This suggests that Pds5b is required in a dose independent manner, and that one functional copy of Pds5b is sufficient for normal development. Similar results have been observed in mouse embryonic fibroblasts (MEFs) deficient in one or two alleles for Pds5b [Pds5b allele, however, consistent with what we observe in the Her2+/+ mice, loss of both Pds5b alleles resulted in defective embryonic development leading to prenatal lethality. In addition to the increased frequency of aneuploidy observed in null hepatocytes, Losada et al. hypothesise this early lethality may be due to the inability of null cells to maintain mitotic arrest, resulting in an early exit from mitosis and subsequently a reduction in their proliferative capacity [Pds5b gene, and a similar phenotype of lethality and deformities in homozygous embryos to previous studies of Pds5b-deficient mice. A full analysis of the Pds5b-disrupted mice and comparison to other mouse strains deficient in the Pds5b gene was considered beyond the scope of the current manuscript, and therefore in-depth analyses including centromeric cohesion defects and aneuploidy were not performed, but could be the focus of future studies for investigators of Pds5b biology.In comparison to the severe phenotype observed in Her2or Pds5b . Centromcapacity . The cenPds5b in a dose-independent manner, in addition to the high copy number of Her2 (162 copies) observed in phenotypically normal heterozygous Her2+/- mice lead us to conclude that the interruption of both alleles of Pds5b expression, rather than the increase in Her2 transgene copies (from 162 to 324), resulted in the embryonic lethality of Her2+/+ mice. Other published mouse models have observed the threshold for high transgene expression and subsequent phenotypic deformations to occur at much lower copy numbers (~10\u201330 copies), therefore we predict the additional 162 copies present in homozygous mice may not have contributed greatly to the embryonic lethality [Pds5b transgenic rescue in Her2+/+ mice in future studies.In considering whether twice the number of copies of Her2 could have contributed to the observed embryonic lethality, we suggest that the requirement of ethality ,47. HowePds5b-/- have shown contrasting results to that reported by Losada and what we observe in our study. Milbrandt et al. report no observable defects in chromosome cohesion in Pds5b-/- mice, with null mice surviving to birth [Pds5a and Pds5b, Milbrandt et al. observed early embryonic death, with no double homozygous embryos present at E9.5 [Pds5b-/- mice have shown the expression of Pds5a is independent of Pds5b expression [Interestingly, other models of to birth . These mto birth . Using c at E9.5 . This evpression , indicatde novo, in addition to minimising side effects associated with off-tumor targeting, Her2 immunotherapies that engage the hosts\u2019 immune system must also overcome the issue of self-tolerance.The generation of the human Her2 model has facilitated the development of many Her2 based therapeutics and has proved to be an important tool in immunotherapy. The basal expression of tumor antigens on normal tissue is a major hurdle in immunotherapy, and designing therapeutics that specifically target tumor antigens whilst leaving surrounding tissue unscathed is challenging. The first and only clinical trial involving genetically modified chimeric antigen receptor (CAR) bearing T cells directed towards the Her2 antigen highlighted the importance of on-tumor effects. Low levels of Her2 present on lung epithelial cells were found to trigger the infused CAR T cells, resulting in a cytokine storm and eventuating in the patient\u2019s death . Therefo+ tumor growth, and in addition, allowed for the assessment of autoimmune or on-target/off-tumor effects from therapy. As such, the Her2 model has been utilized extensively in the design and testing of Her2 therapeutics, including those designed to target Her2+ breast cancer stem cells [hi regulatory T cells) [Pds5b gene, a regulator of the cohesin complex and integral for both organogenesis and development. We demonstrate that similar to other Pds5b-/- models, Her2+/+ mice are severely underdeveloped, resulting perinatal death and ultimately their resorption from E16 onwards. Our results further contribute to the understanding behind the Her2 transgenic model.The expression of human Her2 from birth in Her2 mice ensured sufficient immune tolerance to permit Her2em cells , Her2 DNT cells) \u201351 as weT cells) ,23. In tS1 FigThe mouse WAP promoter is highlighted in blue, 5\u2019 untranslated region (UTR) of the human ErbB2 cDNA in orange, open reading frame of the human ErbB2 cDNA in pink with the open reading frame (ORF) translated sequence underneath, followed by the 3\u2019 UTR of the human ErbB2 cDNA in yellow. Underlined = truncation of final copy in inserted concatemer and 14 bp of genotyping primer.(PDF)Click here for additional data file."}
+{"text": "Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. Published January 05, 2016Owing to a production error the published Supplementary files 1 and 2 were incorrect. Supplementary file 1 was published with the legend for Supplementary file 2 and a file from another eLife article was published with the legend for Supplementary file 1.The correct Supplementary files 1 and 2 are available here.Supplementary file 1.\u00a0Includes qPCR primer sequences and UPL probe numbers for RT-qPCR experiments described in the manuscript.Supplementary file 2.\u00a0Indicates the source of all cell lines used, as well as results of mycoplasma testing throughout the course of these studies.The article has now been corrected accordingly."}
+{"text": "Until 2011, the main risk factor for the spread of HIV-1 in Romanian adult population was heterosexual contact. More recently, the number of HIV-1 new cases among IDUs significantly increased. This new subepidemic is characterized by circulation of particular forms of infective strains (CRF14_BG), high prevalence of HCV co-infections and infective endocarditis. Genotypic methods are currently used for testing viral tropism in HIV infected patients. Deep sequencing proved to have much higher sensitivity than population sequencing in detecting minority - CXCR4 tropic viruses. Previous studies suggested that the presence of CXCR4 phenotype at baseline is frequently associated with a faster disease progression. The aim of the study was to evaluate the viral tropism at the moment of HIV diagnostic in IDU patients.[coreceptor] bioinformatic algorithm and subtyping with REGA tool version 2.0.We have analyzed sequences from 19 IDUs that presented low CD4 counts (<200 cells/cmm) and/or CDC stage C when HIV-1 infection was diagnosed. They were compared with strains from 24 heterosexuals diagnosed at the same time with the IDUs were included in the study. RT PCR was performed to amplify the V3 loop. Population sequencing was done using BigDye chemistry and 3500 Genetic Analyzer. Deep-sequencing was performed on the GS Junior 454 sequencing platform and AVA software was used to analyze the output sequences. The tropism prediction was assessed by geno2phenoThe IDU group was mainly infected with recombinant forms: CRF14_BG and recombinants between F1 and CRF14_BG ; the heterosexuals had F1 subtype viruses . CXCR4 tropism was associated with IDUs and in particular with CRF14_BG (p=0.0027). All the CRF14_BG were X4 by population sequencing. Furthermore, when tested with deep sequencing the viral populations of CRF14_BG samples were exclusively X4 (no minority R5 populations). Dual tropic (CCR5 and CXCR4) populations were more frequent in F1 samples isolated from heterosexuals and predicted as X4 by population sequencing. We found concordance between the predictions of the two methods. In the heterosexual group both techniques predicted mainly CCR5 viruses.CXCR4 tropic CRF14_BG viruses were very common in IDUs at baseline. This may contribute to faster disease progression in this population than in heterosexuals infected with the F1 CCR5 tropic strains."}
+{"text": "Supplying \u2212800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen , 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate (\u200a=\u200a4.7 kg CO2 captured).Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (\u223c5). Hydrogen production by biocathodes poised at \u2212600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but \u223c5\u201315 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH \u223c6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm Microbial electrosynthesis is a process by which microbes grow as a biocathode and couple electrical energy to the capture and conversion of CO2 into compounds such as methane or organic acids The ability to capture carbon dioxide  buffered at near neutral pH 2 will maintain the pH close to neutrality and thereby support acetogenesis 2 production by the microbiome, which has implications for H2 and organic acid production. This extends our understanding of how these microbiomes perform electrosynthesis, and suggests a platform for the electrosynthetic production of fuels and chemicals Electrosynthesis of acetate and other short-chain fatty acids from COAn electrosynthetic microbiome enriched from brewery wastewater obtained from Palmetto Brewery  was used in this study 2 CMI-7000 cation exchange membrane . A total of 25 grams of graphite granules (Showa Denko) were used in each chamber for the anode and cathode. A 0.95 cm outer diameter fine extruded graphite rod (Graphite Store) was cut into 3 cm long current collectors wound with 0.81 mm diameter titanium wire (Sigma Aldrich). All carbon electrodes were pretreated by washing in acetone and drying, followed by immersion in 1 M NaOH, and 1 M HCl for 24 hours each with deionized water rinses between each step. Reference electrodes were made with a 1 mm diameter AgCl coated silver wire  immersed in 4 mm glass capillary tube (ChemGlass) containing 3 M KCl saturated with Ag/AgCl and to which a Vycor tip (Koslow) was attached using Teflon heat-shrink tape (BASi). The reference electrodes were immersed in a 7 mm diameter Luggin capillary containing 1 M KCl. The cathode and anode chambers were each filled with 50 mL of media. Reactors were poised at \u2212600 mV vs. the standard hydrogen electrode (SHE) unless indicated otherwise. Chronoamperometry and cyclic voltammetry were recorded using a VMP3 potentiostat and EC-Lab Software (Bio-Logic Science Instruments). Voltammetric sweeps ranged from \u2212800 to 0 mV vs. SHE at 1 mV/sec. Reactors were sparged with 100% carbon dioxide at 15 mL/min, except in graphite rod cathode yield tests. Another larger customized reactor (Adams & Chittenden) was designed with a 20 cm2 cation exchange membrane. The anode and cathode chambers each contained 100 g graphite granules and 100 mL of phosphate buffered media. This reactor was used to test the biocathodes at potentials lower than \u2212600 mV vs. SHE. A total of 10 reactors were examined and the inoculation scheme is depicted in Reactors were customized three-electrode, two-chamber glass cells (ChemGlass) separated by a 2 cmAliquots of media were filtered and analyzed for pH using a pH meter (Mettler-Toledo) and fatty acid content via HPLC (Shimadzu) using the method described in Marshall et al. Samples for SEM were incubated in a 0.1 M sodium cacodylate buffer with 2% gluteraldehyde for 3 hours, then incubated in a 2.5% osmium tetroxide, and finally dehydrated with an ethanol dilution series using 0%, 25%, 50%, 75%, % and 100% at 5 minute intervals. Samples were stored in a desiccator before being sputtered with Au and Pd using a Denton Vacuum sputter coater. Images were mounted on a stage with conductive carbon tape and imaged using a JEOL JSM-5600LV Scanning Electron Microscope.http://www.earthmicrobiome.org/). Sequences are publicly available under the MG-RAST IDs: Reactor 4 electrode 4562455.3, and Reactor 4 supernatant 4562456.3 (http://metagenomics.anl.gov/).Reactor 4 was analyzed for microbial composition at 5 months after inoculation. Electrode attached cells were sampled by collecting graphite granules while planktonic cells were collected by filtering supernatant through a Sterivex filter (Millipore). Both samples were stored in Soil Preservation Solution (MoBio Laboratories) at \u221280\u00b0C. DNA from electrode-attached cells was extracted using a PowerSoil DNA Isolation Kit (MoBio Laboratories). DNA from planktonic cells was extracted using PowerWater Sterivex DNA Isolation Kit (MoBio Laboratories). Primers amplifying the V4 region (F515/R806) of 16S ribosomal sequence was amplified using Golay barcoded primers An acetogenic/methanogenic microbiome sans NaBES can, in certain cases, mitigate the competitive advantage of the favorable thermodynamics and H2 scavenging ability of methanogens due to the commensurate kinetics of H2 utilization between some acetogens and methanogens Acetobacterium spp. have been reported to outcompete certain methanogens when H2 is not limiting 2 was used to lower the electrolyte resistance and maintain the cathode potential at a lower overall applied voltage, and avoid the bicarbonate buffer that is more supportive of methanogenesis. The first test with this medium was done with Reactors 4\u20136 . This likely contributed to the pH change since without the gradient>100 mM acetic acid was needed to lower the pH significantly compared to the lowering of pH by only 30 mM acetate with the gradient. In order to determine if the response of the microbiome was due to the change in pH only, two reactors with active acetogenic microbiomes (Reactors 4 and 5) received fresh media and 50 mM NaBES added to the anode and cathode chambers.  Acetate was produced early in the experiment (13\u201319 mM/day) while hydrogen production settled below 25 mM/day and the pH stayed between 6.5 and 7 . Each ca2 surface area) were incubated in the graphite-granule bed cathode of Reactor 4 for 48 days and exposed to 5 medium exchanges and cycles of acidic pH (<5). The rods were then transferred to serve as a defined surface area biocathode without graphite granules in three independent reactors (Reactors 7\u20139) with 50 ml of phosphate buffered medium plus 50 mM NaCl substituted for NaBES in each chamber.Three graphite rods  suggests that the dead microbiome might have left a redox-active species adsorbed to the cathode. Further examination of the mechanisms of how the electron transfer occurs within the biocathode is beyond the scope of this study, but the results indicate that a living and intact microbiome is necessary for the development of high current and organic acid production by these biocathodes.A CV analysis was performed on all of the biocathodes after their transfer to the new reactors while the catholyte was sparged with 100% COutrality . All thr \u2212800 mV  inset. M \u2212800 mV . Autocla2, sealed, and poised at \u2212600 mV vs. SHE. Productivity for two of the three biocathodes (Reactors 8 & 9) was similar over a 4-day test, but the cathode that generated the least current in the CV analysis (Reactor 7) performed poorly in relation to the others  on the electrodes. Protein densities were comparable at this time (14.3 \u00b1 0.9 \u00b5g/cm2) and the average current density to protein for the three biocathodes was of 6.5 \u00b1 1.2 A/g. The coulombic efficiency for each reactor was similar with nearly 90% to hydrogen early on and over 40% to acetate at the end of the experiment  with a larger membrane and working volume (100 mL) was inoculated with granules from Reactor 4. Relieving the membrane limitation allowed the reactor to be operated at \u2212800 mV vs. SHE. Under these conditions, high hydrogen and organic acid production was achieved as the pH decreased . The hydAcetobacterium woodii has been shown to produce a maximum of 123 mM/day acetate when incubated under a partial hydrogen pressure of 1700 mbar A. woodii would produce 421 mM/day in a nutrient enriched medium stirred at 1200 rpm under 40% hydrogen A. woodii to overexpress phosphotransacetylase and acetate kinase or the four THF dependent enzymes of the Wood-Ljungdahl pathway in order to overcome carbon flux bottlenecks. These mutants produced acetate at 480 mM/day in the same medium and conditions. The current densities and product formation by the electrosynthetic microbiota from the current study indicate that electron flux from an electrode to a microbial community is similar to these rates of hydrogenotrophic acetogenesis, albeit without the energy required to pressurize or stir the reactor. For example, the electrosynthetic microbiome has produced>50 mM/day acetate, while an additional 325 mM/day could potentially be generated based on the electron equivalents for maximum hydrogen production rates observed using the system.Acetobacterium sp., particularly on the electrode, where it comprised 90% of the relative abundance  is that these organisms capture electrons directly from an electrode and synthesize acetate Acetobacterium could possibly be contributing to the productivity of the microbiome, but it is difficult to discern DET to acetogenesis when hydrogen is concomitantly produced in such high amounts. Current densities are sufficiently high at cathodic potentials so that CV scans of the biocathodes are dominated by the hydrogen reaction, and hydrogen forming at the electrode surface is readily visualized bubbling vigorously off of the biocathode surface when the pH is low or after the community has grown/adapted to higher hydrogen production following an acidic pH challenge  transfer may also play an important role in the metabolism of the electrosynthetic microbiome. For example, 3/day hydrogen (1 kg H2 \u2248 1 GGE) production by an electrosynthetic microbiome. High acetate production occurred (up to 3.1 kg/m3/day) when the pH was>5, with 1.5 kg of CO2 captured per kg acetate produced. CV analysis indicated a lowering of the overpotential at the cathode, and current densities as high as 12.2 mA/cm2. Oxygen and autoclave treatment ameliorated the cathodic current. Acetobacterium may solely facilitate these reactions but alternatively, interspecies interactions by the microbiome may be required to produce high levels of hydrogen and organic acids during microbial electrosynthesis.Repeated exposure to acidic pH resulted in up to 2.6 kg/mFigure S1Inoculation scheme of the 10 reactors used in this study. Reactors 1-4 were inoculated from a previous electrosynthetic microbiome. NaBES or NaCl at 50 mM was added to the cathode (C) or anode and cathode (A + C) of each reactor. Reactors 1\u20133 were operated with bicarbonate buffered medium with 50 mM NaBES in the catholyte. They were replenished with fresh media wherein 50 mM NaCl was substituted for the NaBES in the catholyte. Reactor 4 was operated with a phosphate buffered medium with 50 mM NaBES in the catholyte or in both catholyte and anolyte. Reactors 5 and 6 were two replicate reactors inoculated from Reactor 4 and under the same conditions. Yield tests and CV were performed in Reactors 7\u20139 which contained phosphate buffered media with 50 mM NaCl in the anolyte and catholyte and rods initially incubated in Reactor 4. Reactor 10 was the larger membrane reactor inoculated with granules from Reactor 4 and poised at lower potentials in phosphate buffered media with 50 mM NaBES in the anolyte and catholyte.(PDF)Click here for additional data file.Figure S2Replicates of Reactor 1 and the conditions presented in. Biocathodes of Reactors 2 (A and B) and 3 (C and D) were incubated in bicarbonate buffered media with 50 mM NaBES (A and C) or 50 mM NaCl (B and D) in the catholyte and poised at \u2212600 mV vs. SHE.(PDF)Click here for additional data file.Figure S3Transferability and replication of the electrosynthetic microbiome. Granules were transferred from Reactor 4 (A) into Reactors 5 (B) and 6 (C) and exposed to lowered pH in phosphate buffered media containing 50 mM NaBES in the catholyte \u2212600 mV vs. SHE.(PDF)Click here for additional data file.Figure S4Abiotic controls. Hydrogen production (solid lines) in low and high pH (dashed) sterile and sealed reactors. Graphite granule cathodes were poised at \u2212600 mV vs. SHE in phosphate-buffered medium with 50 mM sodium BES and with (blue) or without (red) 100 mM acetic acid.(PDF)Click here for additional data file.Figure S5Inactivation of an active biocathode. Cyclic voltammogram of an active rod biocathode in phosphate buffered medium at pH\u200a=\u200a6.3 with 50 mM NaCl in the anolyte and catholyte, and 100% CO2 sparge (black). The active rod was exposed to sterile flowing air (40 mL/min) for 20 hours and the scan was repeated under 100% CO2 sparge (blue). The O2 inactivated rod was then autoclaved on a gravity cycle for 30 min and the scan was repeated again under 100% CO2 sparge (red). An abiotic sterile control (gray) and the autoclave and O2 inactivation treatments showed far less cathodic current densities than the active biocathode.(PDF)Click here for additional data file.Figure S6Improved production at lower potential. Sequential media replacements (A and B) of phosphate buffer medium with 50 mM BES in the anolyte and catholyte in Reactor 10 poised at \u2212800 mV vs. SHE unless otherwise indicated.(PDF)Click here for additional data file.Figure S7Screen shot of hydrogen gas evolving off of biocathode. Video screenshot of a biocathode in phosphate buffered media poised at \u2212600 mV vs. SHE.(PDF)Click here for additional data file.Table S1Summarized parameters and maximum productivities for the reactors in this study. Maximum productivities are in mM/day, and g/m2/day is in parenthesis for the rods. Presence of NaBES or NaCl in the catholyte or catholyte and anolyte is noted by C or C & A, respectively.(PDF)Click here for additional data file.Movie S1Brief video of the biocathode evolving hydrogen gas at \u2212600 mV vs. SHE.(MOV)Click here for additional data file."}
+{"text": "Gene names Ompk36 and Ompk35 are swapped incorrectly throughout the manuscript. All instances of Ompk35 should appear as Ompk36, and all instances of Ompk36 should appear as Ompk35. Corrected versions of Tables"}
+{"text": "More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1\u2013TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1\u2013TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex. In vivo, TRF2 forms a stable complex with Repressor activator protein 1 (Rap1)  . The Rap1 (Rap1) . While T1 (Rap1) , Rap1 de1 (Rap1)  nor telorecently . All thein vitro  domain mediating homodimerization of TRF2, a flexible linker region comprising Rap1 binding motif and a C-terminal DNA Myb domain  Figure . The myein vitro , no signin vitro ,19. The rap1/trf2 was cloned into pHGWA vector (The cDNA sequences of Rap1 and TRF2 were synthesized by Source BioScience and cloned to pDONR/Zeo vector (Life Technologies) using two sets of primers (Supplementary Table S1) and BP clonase enzyme mix from Gateway technology (Life Technologies). Resulting plasmid pDONR/Zeo A vector  using LR6-tags were expressed in bacterial cells as described elsewhere (Escherichia coli BL21(DE3) and Rap1 in E. coli BL21(DE3)RIPL carrying the vector pHGWA -polyacrylamide gel which was subsequently stained using Bio-Safe Coomassie G 250 (Bio-Rad). Mass spectrometry measurements were used to confirm that proteins were expressed in full length and high purity. Supplementary Figure S1 shows SDS-polyacrylamide gel electrophoresis of TRF2 and Rap1 used in the studies.GGTTAGGGTTAGGGTTAGGGTTAG, G-strand sequence; overhang is in bold italic), denoted as Ov  and 35 bp long (GTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG) denoted as R2 (two telomeric repeats) and R5 (five telomeric repeats), respectively, along with 47 nt long DNA substrate containing 24 nt overhang  buffer was used. Reactions containing the same amount of fluorescently labeled DNA (3 pmol) and increasing amounts of protein TRF2 (3\u201315 pmol) were prepared. DNA was labeled with fluorophore Alexa Fluor 488. The influence of protein Rap1 on DNA-binding affinity of TRF2 was detected in reaction mixtures composed of a constant amount of labeled DNA (5 pmol), a fixed amount of protein TRF2 (10 pmol) and increasing amounts of protein Rap1 (20\u201380 pmol). In both cases, the reactions were supplemented with buffer  to a volume of 15 and 3 \u03bcl of 6\u00d7 loading buffer . Reaction mixtures were incubated for 15 min on ice and then loaded on horizontal 5% (w/v) non-denaturing polyacrylamide gels in 0.25\u00d7TBE buffer. The electrophoresis proceeded at 1 V/cm for 45 min and for an additional 3 h at 2 V/cm at 4\u00b0C. Fluorescently labeled DNA in gels were analyzed with a FLA 7000 imaging system (Fujifilm).Kd) was determined from the curve representing the dependence of FA on the concentration of protein added to the solution in the cuvette. Fitting analyses were carried out using programs SigmaPlot 12 (Systat Software) and DynaFit4  . A dissociation constant (in Ltd.) .Rap1 was labeled with fluorescent dye Alexa Fluor 594 or 488. Subsequently, labeled proteins were separated from free fluorophores by gel filtration using a PD 10 Desalting Column . The experimental instrument setting for fluorescence measurement of Rap1 conjugated with the fluorophore Alexa Fluor 594 was 584 nm and 611 nm with the same width of slits 7 nm for excitation and emission. To measure TRF2 binding to DNA that was conjugated with the fluorophore Alexa Fluor 488, the excitation wavelength was set to 492 nm and emission wavelength to 516 nm with the width of slits 9 nm. The integration time was 3 s. The FA titration experiments were carried out in a 10\u00d74 mm quartz-glass cuvette with chamber for magnetic bar stirrer. FA was measured at 25\u00b0C in the buffer containing 50 mM NaCl and 50 mM sodium phosphate (pH 7.0) if not stated otherwise. All fluorescence measurements were performed on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) with an L-format set up equipped with automatically adjustable polarizers for excitation and emission lights under control of an Origin-based FluorEssence software (version 2.1.6).The contribution of electrostatic interactions was determined from the linear dependence of the binding constants on the increasing concentration of NaCl. The electrostatic component of binding originates from the formation of ion pairs between the cationic amino acid residues of the protein and the negatively charged phosphate groups of DNA or amino acid residues of the other protein. The electrostatic component of binding was determined from the binding constant dependence on ionic strength as described in  and in SKa), reaction stoichiometry (n) and binding enthalpy (\u0394H) were obtained from the fit. Binding free energy (\u0394G) and entropy change (\u0394S) were determined from the equation:ITC experiments were carried on a VP-ITC instrument  at 25\u00b0C. Proteins were diluted in the same buffer 50 mM NaCl and 50 mM sodium phosphate (pH 7.0) and degassed. The cell (1423 \u03bcl) was filled with TRF2 (5 \u03bcM). Rap1 (44 \u03bcM) was added in 20 injections of 10 \u03bcl at 5 min intervals, with a stirring rate 240 rpm. Experimental data were analyzed in Origin 7.0 software using one-site binding model to fit a theoretical titration curve. Binding constant  on GLC and NLC sensor chips (BioRad) in a Phosphate Buffered Saline with Tween 20 (PBST). The detailed conditions of SPR measurements are available in Supplementary Data.6 per M and corresponding dissociation constant 21 nM  were similar in the presence or absence of Rap1 (Supplementary Table S4). Similarly, our SPR data did not provide us with convincing results showing significant effect of Rap1 on TRF2 off-rate constants for fully hybridized duplex DNA nor DNA with 3\u2032 overhang (data not shown). It might be caused by the short length of telomeric DNA that was required for our SPR experiments.To quantify the effect of Rap1 on TRF2 binding affinity to double-stranded DNA, we carried out three FA measurements with different experimental arrangements. In the first arrangement, TRF2 alone was allowed to bind to a fluorescently labeled DNA duplex R2 . In accordance with our EMSA data  in the absence or presence of Rap1 . At first, only TRF2 was allowed to bind DNA. Subsequently, the complex of TRF2 with Rap1 was used in DNA-binding assays. It was revealed that TRF2 binding affinity to telomeric DNA is approximately 5-fold higher in comparison with TRF2 binding affinity to non-telomeric DNA duplex N. In other words, the selectivity, which is defined as the ratio of association constant for protein binding to specific telomeric R2 and non-specific random N DNA, was five . On the other hand, in the presence of Rap1, the binding affinity of TRF2 is more than 8-fold higher to telomeric than to non-telomeric DNA duplexes ; the selectivity was eight. Based on these results, we conclude that TRF2 in complex with Rap1 binds telomeric DNA with nearly 2-fold higher selectivity.Ka) were calculated as reciprocal values of dissociation constants obtained for at least three independent measurements at each salt condition. Logarithms of Ka were plotted against logarithms of salt concentration  interactions, were identified (Supplementary Table S2). The parameter Z revealed that the binding of TRF2 to telomeric DNA resulted in the formation of approximately five ion pairs in average. On the other hand, in the presence of Rap1, TRF2 formed only three ion pairs with DNA. The decreased Z value means that the electrostatic interactions with DNA were less pronounced when the preformed complex of Rap1 and TRF2 bound DNA. The contribution of electrostatic component to overall DNA-binding affinity of TRF2 was calculated from values corresponding to the total binding affinity Ka and non-electrostatic contribution to the binding affinity Kanel. Based on the salt dependence of association constant, the total affinity and corresponding Gibbs free energy of binding was divided into contributions originated from electrostatic and non-electrostatic interactions  than to the full-length protein and therefore induce a more profound displacement of TRF2\u0394B from duplex DNA than in case of full-length TRF2. To test this idea, we allowed either full-length TRF2 or TRF2\u0394B to bind to DNA R2 until about half saturation range was achieved . When Rap1 was added, we observed that significantly more DNA was released from TRF2\u0394B than in case of full-length TRF2. This experiment strongly supports our previous measurements and conclusions about the effect of Rap1 on DNA-binding affinity of TRF2 and the importance of the TRF2 basic domain to non-specific DNA binding.We revealed the molecular mechanism of how Rap1 affects TRF2 binding to DNA by using a combination of quantitative biophysical approaches. The explanation of the origin of selective DNA binding of shelterin protein TRF2 is essential for understanding shelterin functions in genome stability maintenance in humans and mammals. Moreover, our findings could be applied on proteins that bind DNA and participate in gene regulation through selective DNA recognition. The quantitative descriptions include several new observations that provide a more complete understanding of the activity of the critical shelterin subunit TRF2 on DNA, which extents and confirms the previous separate observations of other investigators ,17,18.Kd value for binding of full-length Rap1 and TRF2 is in a very good correlation with the value of Kd for the binding of C-terminal domain of human Rap1 . Additionally, our EMSA experiments have shown that Rap1 does not bind telomeric DNA until Rap1:DNA molar ratio 40:1 . These results are partially contradicting the previous finding that Rap1 binds telomeric DNA directly . The difThe main objective of our study was to quantify how Rap1 affects affinity and selectivity of protein TRF2 to telomeric DNA. We found that, on one hand, the presence of Rap1 increases the selectivity of TRF2 binding to telomeric DNA by 2-fold, but on the other hand, Rap1 decreases TRF2 binding affinity to DNA by approximately 2-fold.Kd which could cause the less pronounced Rap1 effects on total release of DNA in case of EMSA experiments . The observed selectivity increase is in a very good accordance with the results obtained recently from gel retardation assays by Arat and Griffith . Additios Figure . The morA probable explanation of how Rap1 could contribute to the observed reduction in the DNA-binding affinity of TRF2 is a modification of net surface charge after Rap1\u2013TRF2 complex formation. Protein interaction with DNA occurs in two steps. In the first step an electrostatic, non-specific attraction of interacting partners occurs . In the Eventually, we assessed how Rap1 affects TRF2 binding to DNA substrate Ov containing naturally occurring overhang that comprises four telomeric repeats Figure . We obseMoreover, the interaction model takes into consideration dimeric arrangements of TRF2 and Rap1. The sequence selective binding of TRF2 in dimeric form is in agreement with DNA sequence recognition mechanism of other selectively binding proteins that take part in regulatory mechanisms .To test this model we prepared a truncated variant of TRF2 lacking the N-terminal basic domain. In accordance with our hypothesis, when the basic domain is absent, TRF2 should have a significantly lowered DNA affinity. Indeed, when we measured the DNA-binding affinity of TRF2 lacking the basic domain, DNA-binding affinity was decreased to the level of the affinity observed for the binding of TRF2 with Rap1 present . Moreover, we observed that more telomeric double-stranded DNA was released when Rap1 added to TRF2 lacking the basic domain pre-bound to DNA compared to full-length TRF2 . This finding strongly supports our previous measurements and the proposed model of Rap1 effects on DNA-binding affinity of TRF2.et al. have shown by combination of nuclear magnetic resonance, differential scanning calorimetry (DSC) and permanganate probing experiments. It has been suggested by the same laboratory that TRF2 basic domain has evolved to finely regulate TRF2 ability to condense DNA  . The onlin vitro binding studies, one may speculate that Rap1 is needed for efficient directing of TRF2 to its proper binding location at telomeres also in vivo. TRF2 alone could be accumulated in internal chromosomal or peritelomeric regions. After Rap1 binding, Rap1\u2013TRF2 complex might relocate to a single/double-strand junction of telomeric DNA. Although little to no effect on TRF2 telomere binding is observed after Rap1 deletion in both human and mouse cells (Rap1 contribution to the whole shelterin function and signal pathways of the cell is matter of a long discussion ,13,16. Tse cells , it is pse cells . It woulWe conclude that Rap1 serves as a selectivity enhancer of TRF2 with newly found ability to partially remove bound TRF2 from telomeric DNA duplex. The observed Rap1 release activity suggests that Rap1 plays an important role in tuning DNA interactions of TRF2, the central subunit of shelterin protein complex. Our data showing the release of TRF2 from telomeric DNA in the presence of Rap1 suggest that protein Rap1 might prompt the relocation of TRF2 to the preferred single/double-strand junction of telomeric DNA. For the first time here, we used combination of quantitative biophysical approaches to describe and explain molecular origins of Rap1 contribution to selective TRF2 recognition of telomeric DNA. We found that Rap1 neutralizes the electrostatic attraction of TRF2 to DNA. Hence, Rap1 reduces overall DNA-binding affinity of TRF2 in order to improve its binding selectivity toward telomeric DNA. The following studies focused on shelterin protein dynamics using single molecule approaches will be of particular interest as they can reveal relocation dynamics of TRF2 induced by Rap1 and the mechanism of regulation of shelterin binding and distribution on telomeres.Supplementary Data are available at NAR Online."}
+{"text": "The following information is missing from the Acknowledgments section:\"We thank Drs. Gautam Shirsekar and Jiangbo Fan who generated the data shown in Figure 3C, 3D, and 3E.\""}
+{"text": "Several genetic variants for type 2 diabetes (T2D) have been identified through genome wide association studies (GWAS) from Caucasian population; however replication studies were not consistent across various ethnicities. Objective of the current study is to examine the possible correlation of 9 most significant GWAS single nucleotide polymorphisms (SNPs) for T2D susceptibility as well as the interactive effect of these variants on the risk of T2D in an Indian population.Case-control cohorts of 1156 individuals were genotyped for 9 SNPs from an Indian population. Association analyses were performed using logistic regression after adjusting for covariates. Multifactor dimensionality reduction (MDR) analysis was adopted to determine gene\u2013gene interactions and discriminatory power of combined SNP effect was assessed by grouping individuals based on the number of risk alleles and by calculating area under the receiver-operator characteristic curve (AUC).TCF7L2 (rs7903146) and SLC30A8 (rs13266634) with T2D. MDR analysis showed statistically significant interactions among four SNPs of SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) genes. Cumulative analysis showed an increase in odds ratio against the baseline group of individuals carrying 5 to 6 risk alleles and discriminatory power of genetic test based on 9 variants showed higher AUC value when analyzed along with body mass index (BMI).We confirm the association of TCF7L2 and SLC30A8. MDR analysis demonstrates that independently non-significant variants may interact with one another resulting in increased disease susceptibility in the population tested.These results provide a strong evidence for independent association between T2D and SNPs for in  The risk of an individual towards T2D reflects the environmental influence in the background of genetic predisposition. Owing to the complex etiology of the disease progression, the identification of genetic markers has been slow until 2007. The advent of new technology in the form of microarray chips, has led to the development of high throughput genome wide association study (GWAS). Nevertheless, only a few variants in genes such as KCNJ11, PPARG, SLC30A8, and TCF7L2 were reported to be linked with T2D  were genotyped in all the subjects. Genotyping of KCNJ11 (rs5219), FTO (rs8050136) andTCF7L2 (rs7903146) polymorphisms was carried out using Tetra primer amplification refractory mutation system (TETRA-ARMS) and genotyping of SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875), CDKN2A (rs10811661), KCNQ1 (rs2237892) andCDKAL1 (rs7754840) polymorphisms were carried out by polymerase chain reaction followed by restriction fragment length polymorphisms (PCR-RFLP). Random samples were picked and direct DNA sequencing was performed  to confirm the genotype.Gene variants studied basically corresponds to those identified from GWAS of T2D till the year 2010 and also shown in replicative studies for its association with T2D susceptibility in other population. DNA was extracted from peripheral blood using standard phenol-chloroform procedure. A total of 9 SNPs [http://www.ncbi.nlm.nih.gov/projects/SNP/). The categorical data of SNPs for association analysis with T2D was performed by using Pearson\u2019s chi-squared test to detect differences in allele frequencies between cases and controls. Hardy\u2013Weinberg equilibrium test was performed using a \u03c72 goodness-of-fit test to assess genotype frequencies. Association analysis was further confirmed by logistic regression after adjusting for age, sex and BMI as covariates and association results of SNPs with T2D was assessed by using odds ratio (OR) and corresponding 95% confidence interval (CI). In our analysis the most frequent homozygous genotype in the control population was considered as the reference category. Further we evaluated the differences in continuous variables  using Students t-test and data are represented as mean \u00b1 SD. Bonferroni correction has been used to reduce the chances of obtaining false-positive results (type I errors). A P value of less than 0.005 has been kept as a threshold of significance (after Bonferroni correction). All statistical analysis was performed using SPSS version 16 for windows  and SNPstat Software. Population attributable risk (PAR%) which identify what percentage of total risk for T2D is due to genetic effect of the variant was estimated for those SNPs which showed positive association with T2D susceptibility. For allele dosage analysis to acquire the combined information from multiple SNPs, we used allele count model where we summed the number of exact risk alleles carried by each individual in cases and control group.Quanto version 1.2.4 was employed for sample size calculation using minor allele frequency data from dbSNP database  method was employed. MDR is a nonparametric method and therefore no hypothesis concerning the value of any statistical parameter is made. It is also model free, thus no genetic inheritance model is assumed. All interactions and identification of best models were performed using 10 fold cross validation consistency (CVC) and permutation testing was done considering all possible SNP combination. Testing balance accuracy in the context of 10-fold cross-validation was used to assess model quality. An overall best model was selected that had the maximum accuracy in the testing data (i.e. testing accuracy or TA). We also recorded the cross-validation consistency or CVC. This provides a summary of the number of cross-validation intervals in which a particular model was found. Higher numbers indicate more robust results. Those models/SNP combinations with highest testing balanced accuracy (TBA) and CVC was selected as \u2018\u2018best model\u2019. This procedure generates an empirical estimate of the null distribution of testing accuracies and corrects for multiple testing because the same number of models are evaluated in all permutated and real data. All interactions were visualized by constructing an interaction dendrogram according to the method described by Moore et al., 2004 , 27, 28.TCF7L2 (rs7903146) and SLC30A8 (rs13266634) with an allelic OR = 1.46, 95% CI 1.15\u20131.85, p = 0.001 and OR = 1.33, 95% CI 1.10\u20131.60, p = 0.002. No significant association was observed between the remaining 7 loci and T2D in this population (p>0.05). Given that, variants in FTO and HHEX have shown to be associated with BMI, we further examined the association of these polymorphisms with BMI in our NGT control subjects and found that FTO (rs8050136) was significantly associated with BMI  controls matched for age, sex and ethnicity. with BMI .p<0.05 were still included in our allele dosage analysis because these were confirmed T2D risk variants and lack of significance may probably due to lower risk allele frequencies. SLC30A8 (rs13266634) and TCF7L2 (rs7903146) which were found to be associated with T2D in our population. We observed that TCF7L2 (rs7903146) accounts for 18.4% and SLC30A8 (rs13266634) accounts for 30.4% PAR. These two genetic regions contribute to a total of 48.8% population attributable risk (PAR) for T2D (FTO (rs8050136) was removed from analysis.The individual risk variants in our study showed similar effect sizes as compared to other large studies from Caucasian population . Several variants in our study though are not associated with T2D at  for T2D . We asse for T2D . The areSLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) SNPs. This model has a significant TBA of 0.618 and a CVC of 10/10. However it\u2019s worth mentioning that 3 SNPs showing genetic interaction in this model were not associated with increased risk of T2D in univariate analysis (IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661)). The entropy based dendrogram obtained by MDR evidently showed an intricate and a hierarchical pattern of interaction among the gene variants constituting the polygenic basis of the disease (CDKN2A (rs10811661) and FTO (rs8050136), IGF2BP2(rs4402960) and HHEX (rs1111875) loci showed maximum degree of synergy in their interactions and on the contrary, different degree of redundancy (antagonism) was observed between TCF7L2 (rs7903146) and SLC30A8 (rs13266634). SLC30A8 (rs13266634), IGF2BP2 (rs4402960) and HHEX (rs1111875), and CDKN2A (rs10811661) as high and low risk groups along with its statistical interactions. As per the four locus model, individuals homozygous for the wild type allele in SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and individuals homozygous for the mutant allele in CDKN2A (rs10811661) were placed in low risk group while, individuals heterozygous for all four SNPs were placed in high risk group. However, we also observed that individuals heterozygous for at least three of the four alleles were also in high risk group.Consecutively to expand the findings in our data, MDR analysis was applied to detect higher order gene-gene interaction in cases and controls. MDR analysis of the above mentioned variants revealed statistically significant interactions. Best interaction models along with testing accuracy and cross validation consistency are represented in  disease . In partKCNJ11 rs5219) belongs to well replicated biological candidate gene which has shown a clear evidence of its relationship to T2D susceptibility [TCF7L2 (rs7903146) has been shown to have a strongest association with the highest effect size for T2D association across various ethnicities [TCF7L2 (rs7903146) with T2D susceptibility with an effect size of 1.46, 95% CI 1.15\u20131.85, p = 0.001. Sladek et al., 2007 was the first to identify SLC30A8 (rs13266634) as a novel gene variant associated with T2D and since then, many studies have been performed across various ethnic population but the results have been contradictory [p = 0.002.The outcome of GWAS has significantly contributed to the discovery of number of genetic loci linked to T2D \u201319. In ttibility . Until n7L2 rs790146 has bnicities , 29, 30.7L2 rs79046 has beC30A8 rs166634 as SLC30A8 and TCF7L2 as a key gene for T2D susceptibility has been well known, the genetic variants in these genes could account for about ~20% of all T2D cases in the Caucasian population [SLC30A8 and TCF7L2 polymorphisms in Caucasian population was 30.4% and 18.4% respectively which is much higher when compared to our population, and this could be attributed to the higher risk allele frequency in Caucasian population. Further in our allele dosage analysis, we have identified a particular subset of individuals at different risk of disease by weighing against the individuals with smallest number of risk alleles in comparison to those with the majority of risk alleles. This in turn allowed us to identify subgroups of the population with distinctly differing risk for disease. In our population, we were able to distinguish 14.5% of individuals carrying >13\u201314 risk alleles that had four times increased risk of T2D when compared to 1.3% of individuals with >5\u20136 risk alleles. The high risk group also had over thrice the OR for T2D than those with the lower number of risk alleles. Thus, variants are not predominantly discriminative but only explain a small percentage of heritability of T2D. However, instead of focusing on individuals with elevated risk alleles at the population level, the efficacy of genetic test perhaps is better utilized by means of ROC curves. The nine T2D variants though had an insufficient discriminatory ability with an AUC of 0.536; a marginal increase was obtained when it was analyzed with BMI with an AUC value of 0.624.While the significance of pulation , 36. TheSLC30A8, IGF2BP2, HHEX, and CDKN2A genes towards T2D susceptibility by MDR analysis. Among the five models for all the loci tested (nine SNPs of nine candidate genes), the best gene-gene interaction model identified was a four locus model which includes SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661). Individuals heterozygous for at least three out of four loci were shown to cluster in high risk groups. In this model the combination of HHEX (rs1111875) CC homozygotes, SLC30A8 (rs13266634) CC homozygotes, IGF2BP2 (rs4402960) GG homozygotes and CDKN2A (rs10811661) TT homozygotes were associated with reduced risk to T2D. Our study did not show any independent association of IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) with T2D. However in MDR analysis, combined effect of these polymorphisms with SLC30A8 (rs13266634) was associated with increased risk of T2D which suggest a cross talk between these genes in the pathogenesis of T2D. Further, a synergistic interaction among these SNPs suggests a significant role of epistasis in the susceptibility of T2D. However, it is also observed that all the SNPs which were included in this study showed a weak synergistic effect with each other and this suggests the interaction of susceptibility alleles with environmental and life style factors. Nevertheless, the risk allele frequencies of these variants in the tested population were entirely different with those from Caucasian population and thus, the absence of positive association towards T2D in remaining loci points towards the contribution of these polymorphisms to T2D susceptibility is not convincing across different ethnicities or to a particular genetic background. To the best of our knowledge, this is the first report from south Indian population trying to investigate the role of these polymorphisms individually and cumulatively towards T2D susceptibility. Nonetheless, further replication studies are warranted to validate these and newer susceptibility loci which are identified from large scale GWAS in independent samples collected from other ethnic groups.Another major finding of our study was the establishment of gene interaction model between S1 Table(XLSX)Click here for additional data file."}
+{"text": "Wuchereria bancrofti endemic area in Odisha, India. Following screening, 104 consenting adults were randomly assigned to treatment with the standard regimen annually for 24 months (S1), or annually with increased dose (H1) or with increased frequency (6 monthly) with either standard (S2) or increased (H2) dose. Pre-treatment microfilaria counts (GM) ranged from 348 to 459 mf/ml. Subjects were followed using microfilaria counts, OG4C3 antigen levels and ultrasound scanning for adult worm nests. Microfilarial counts tended to decrease more rapidly with higher or more frequent dosing at all time points. At 12 months, Mf clearance was marginally greater with the high dose regimens, while by 24 months, there was a trend to higher Mf clearance in the arm with increased frequency and 800mg of albendazole (76.9%) compared to other arms, . Although higher and/or more frequent dosing showed a trend towards a greater decline in antigenemia and clearance of \u201cnests\u201d, all regimens demonstrated the potential macrofilaricidal effect of the combination. The higher doses of albendazole did not result in a greater number or more severe side effects. The alternative regimens could be useful in the later stages of existing elimination programmes or achieving elimination more rapidly in areas where programmes have yet to start.Although current programmes to eliminate lymphatic filariasis have made significant progress it may be necessary to use different approaches to achieve the global goal, especially where compliance has been poor and \u2018hot spots\u2019 of continued infection exist. In the absence of alternative drugs, the use of higher or more frequent dosing with the existing drugs needs to be explored. We examined the effect of higher and/or more frequent dosing with albendazole with a fixed 300mg dose of diethylcarbamazine in a  In order to achieve global elimination of lymphatic filariasis, it may be necessary to consider alternative approaches to mass treatment using higher or more frequent dosing with the existing drugs used in the programmes. Outside Africa, the drugs used are albendazole and diethylcarbamazine given annually. The current study is the first to examine the use of higher or more frequent dosing in a bancroftian filariasis endemic area outside Africa. Four groups of infected subjects were followed for two years using standard techniques for monitoring drug efficacy in Odisha, India. We showed that higher doses of albendazole, or 6-monthly treatment, were more effective than the standard regimen in producing clearance of microfilaria and reductions in antigenemia. Additionally we found that these regimens were more effective in killing the adult filarial worms in the lymphatics. Importantly, the higher doses used did not increase the risk of side effects. These alternative approaches could accelerate lymphatic filariasis elimination either at the end of existing programmes or to permit new programmes to achieve their goals faster. The Global Lymphatic Filariasis Elimination Programme (GPELF) had its inception in 1998 following agreements between the World Health Organization and the international pharmaceutical companies GlaxoSmithKline (GSK) and Merck to donate supplies of, respectively, albendazole and ivermectin for the programme . AlthougThe initial drug regimens used at the initiation of the Global Programme were based on existing approved doses and did not require extensive additional investigation or drug approval. However no attempts to investigate the effectiveness of alternative regimens, either or use of higher or more frequent dosing were made . A studyW. bancrofti infection. Since the Indian programme uses a single dose of 300 mg DEC for all individuals > 15 years, the dose of DEC remained unchanged [The current study was conducted in India to investigate the effect of increasing the dose of albendazole from 400 mg to 800 mg and /or increasing the frequency of dosing for nchanged . The stuW. bancrofti microfilaremia , who checked completeness of records, compliance with GCP and accuracy of data entry.W. bancrofti endemic area within thirty kilometres of Bhubaneswar in the Khurda district of Odisha was chosen for selection of subjects. Community meetings were arranged to provide information on the importance of the filariasis elimination programme and of the study prior to enrolling individuals for the study. In the filaria endemic villages, night blood was collected by finger prick between 19.00 and 23.00hrs from all individuals between 18 and 55 years of age during a door to door survey. Thick smears from 40\u03bcl blood were examined for microfilaria. Microfilaremic individuals who provided consent were further screened for eligibility. Subsequently, in eligible subjects, an intravenous blood sample (5ml) was collected between 21.00\u201322.00 hours and divided into two aliquots; 2ml with EDTA and 3ml without anticoagulant. From 1ml EDTA blood, the microfilaria count was determined using the Nuclepore filtration technique. Haemoglobin was estimated by automated cell counter ; serum ALT and creatinine were measured by automatic biochemistry analyser  and pregnancy was excluded using a \u03b2-HCG rapid test on early morning urine samples. W. bancrofti (OG4C3) antigen levels were also obtained using an ELISA kit .A Subjects of either sex between 18 to 55 years of age, with microfilaria counts greater than 50/ml. of blood, who accepted the hospitalization and follow up requirements were included in the study. Female subjects who were either pregnant or lactating were excluded as were subjects with serum ALT level of > 30 units/dl, creatinine level > 1.2 mg/dl or haemoglobin level of <10 gm%.Individuals satisfying the inclusion and exclusion criteria and providing a second consent for study inclusion were allocated sequentially to one of four treatment groups according to a pre-determined list generated in blocks of 26 using Prism software (Graphpad Prism 6.0).Ultrasonography of inguinal, scrotal, axillary, thigh and arm lymphatic systems was performed prior to treatment using a Doppler Ultrasonography unit  using a linear high resolution 7\u201312 Mega Hz probe, to record any \u201cfilarial dance\u201d sign.The individuals, in groups of 4\u20136, were then admitted to the hospital of the Kalinga Institute of Medical Sciences, Bhubaneswar, for study drug administration, observation and management of adverse events if required. The drugs were taken orally by the subjects around 8.30am after breakfast, under supervision of hospital staff. Where the subject vomited within 1hr of ingestion, the dose was repeated. The subjects were discharged from hospital after two days and followed at home for up to 7 days post drug intake.A 2x2 factorial design with albendazole at two levels  and number of doses per year at two levels  was used. The resulting four regimens are coded as S1, S2, H1 and H2. For each of the regimens 26 patients were allocated with a total of 104 for the study. These numbers were based on data comparing single and multi-dose regimens for the treatment of lymphatic filariasis , 14. We The four treatment arms were as follows:S1: Diethylcarbamazine citrate (DEC) 300mg plus albendazole 400mg. oral single dose repeated annually.S2: DEC 300mg plus albendazole 400mg. oral single dose repeated six monthly.H1: DEC 300mg plus albendazole 800mg. oral single dose repeated annually.H2: DEC 300mg plus albendazole 400mg. oral single dose repeated six monthly.The drug formulations used for the study were authorized drugs marketed in India as Banocide (DEC) 100mg tablets  and Zentel  400mg tablets .4C3 antigen titre, urine \u03b2 HCG test in females, serum ALT and creatinine were evaluated blind at follow up visits before the subjects received the next drug dose. All subjects were admitted to hospital for drug administration and for 2 days post drug to monitor for side effects / adverse events. Side effects/ adverse events were graded based on a previously used scale [The subjects were followed up every 6 months up to 24 months for evaluation with drug administration as appropriate. Ultrasonography was repeated in those subjects with \u201cfilarial dance sign\u201d detectable adult worm at base line, within 48\u201372 h of the first dose, and at 12 months and 24 months by the consultant ultrasonographer (C H Mohanty) who was unaware of the treatment received by the patients. Microfilarial count, OGed scale  and manaThe clinical and laboratory information were recorded in a predesigned format. The data was entered into Excel and analysed using SPSS version 16 with appropriate tests for parametric and non parametric variables. Correctness of data was ensured by double entry and matching of entered data. The results were expressed as percentage changes and mean reductions at 6, 12 (the primary endpoint), 18 and 24 months. The chi-square test was used to compare the proportions, Student\u2019s t-test for comparing mean differences between two regimens and analysis of variance for comparing all the four group means at 12 months, at 18 months and at 24 months. The net effect of the high levels was quantified using the contrast of a factorial design. The significance level was fixed at 5% level.W. bancrofti endemic area of Khurda district in state of Odisha, India. This covered a rural area of around 150 square kilometres between Bhubaneswar  and Khurda . The population is mostly middle and lower socioeconomic status within an agriculture based economy.The study was initiated in October 2008 and final two year follow ups were conducted in September 2012, enrolling study subjects from 8 villages in the In all, 1751 individuals between 18 and 55 years of age were screened to identify 118 (6.74%) microfilaraemic subjects. 104 individuals who satisfied the inclusion/exclusion criteria and were willing to participate in the study were screened for eligibility. The subjects were randomly allocated to one of the 4 arms of the study and followed up with 6 monthly screening. All 104 individuals (26 in each of the four arms) received DEC + albendazole at baseline in the dosage prescribed for the respective arm. A 40-year-old male allocated to S1 arm died 9 months after the initial treatment following an acute abdominal emergency not considered to be related to the drug or study procedures. The remaining 103 study subjects all completed 24 months follow up.p = 0.259 Student T test). There was a preponderance of males in all 4 treatment groups with approximately 17% females in each group, with most being below 35 years of age. Blood chemistry values were in the normal range in all subjects. The microfilarial density and mean OG4C3 units of the study population at enrolment are provided in p = 0.822). The mean OG4C3 antigen units were also comparable (p = 0.057). Ultrasonography prior to first dose detected adult worm nests with filarial dance sign (FDS) in 13 subjects in each of S1, H1 and H2 arms and 15 in S2 arm (p = 0.219).The age and gender distribution of enrolled subjects (n = 104) was comparable among the four arms (p = 0.021) compared to the standard regimen (S1). At 24 months, Mf clearance was higher in H2 arm (76.9%) compared to other arms,  although this was not statistically significant (p = 0.77). The mean microfilarial density showed a sharp reduction by 6 months from baseline  in all four arms with a slower decline thereafter , produced an extra reduction of 0.74 log units and if albendazole was given at the higher dose (800 mg) (H1 and H2) instead of 400mg, there was an extra reduction of 0.64 log units. Biannual treatment with 800mg (H2) albendazole reduced the Mf count by 1.09 log units which was statistically significant (p = 0.021). It can be concluded that an increase in dose and frequency can reduce Mf counts significantly at the end of 12 months and 24 months compared to the standard regimen (S1).p = 12 M: 0.942, 18 M: 0.94 & 24 M: 0.23). Mean percentage reductions in OG4C3 from individual baseline values are shown in 4C3 antigen clearance was seen to start at 12 months in S1 & H2 arms and at 18 months in S2 and H1 arms and increased over time. At 24 months, OG4C3 clearance ranged from 26.9% to 73.1% (p = 0.007). Since the variance in the antigen units was very large due to wide range of values, logarithmic transformation was used to stabilize variance and one way ANOVA was performed on the log transformed values  if albendazole is given biannually (S2 & H2), instead of annually (S1 & H1) there was an extra mean reduction of 0.66 log units in OG4C3 antigenemia (ii) if albendazole is given as 800 mg (H1 & H2), instead of 400mg (S1 & S2), there was an extra mean reduction 0.31 log units of OG4C3 antigenemia and (iii) if both are given at high level (H2) there is an extra reduction of 0.80 log units in OG4C3At 24 months, (i) If albendazole is given biannually (S2 & H2), instead of annually (S1 & H1) there is an extra mean reduction of 0.53 log units in OG4C3 antigenemia (ii) If albendazole is given at 800 mg (H1 & H2), instead of 400mg (S1 & S2), there is an extra mean reduction of 0.86 log units of OG4C3 antigenemia and (iii) If both are given at high level (H2) there is an extra reduction of 1.38 log units in OG4C3 antigenemia. It can be concluded that increase in dose and frequency reduces OG4C3 log counts at the end of 12 months and 24 months.4C3 clearance was observed later at 18 month follow up  and increased over the subsequent 6 months .Among individuals exhibiting adult worm (USG) at baseline, OGp = 0.669) after the first dose (p = 0.124) or 18 months (p = 0.305) dosing. However, at 12 months, S1 (28%) & H1 (15.4%) annual arms had a higher frequency of adverse events compared to S2 (3.8%) & H2 (3.8%) biannual arms. The adverse events noted were light-headedness (31.7%), fever (22.1%), body ache (4.8%), unsteadiness (4.8%), dizziness (2.8%), drowsiness (2.8%), fatigue (1.9%), chills (1.9%), itching (0.9%), and weakness (0.9%). There was no difference in the pattern of side effects noted in different arms (p>0.05). There were no severe drug related adverse events reported following any treatment, and the events that did occur could be managed conservatively.Following drug administration, the frequency of adverse events was comparable in all four arms  , it is only possible to increase the albendazole dose . Thus th4C3 suggest that there is a significant macrofilaricidal effect at this dose.The primary endpoint for the study was taken as Mf clearance at 12 months after initiation of treatment to be consistent with past studies, but follow up and analysis was continued for a further 12 months to establish whether any differences that might be seen at 12 months were sustained. Microfilarial clearance in all the four arms was progressive over time with a higher rate of decline in the mf prevalence in H2 arm. Although the difference in mf clearance between arms was not statistically significant over the 24 months of follow-up, the decline in mf prevalence in H2 arm gives the impression that if followed further, this arm could attain 100% mf clearance earlier than the other arms. This has the potential to shorten the period required for elimination. This was also indicated by the parallel reduction in microfilarial density where at both 12 and 24 months there was a significantly greater reduction in the high dose twice yearly arm (H2) compared to the other arms. A similar study using high dose twice yearly albendazole and ivermectin in Mali also showed enhanced suppression of microfilaria [Increased adverse events/side effects directly due to the higher dose of albendazole or as a result of interaction with DEC could be a limiting factor to deployment , 18, 19.The results of this study have important implications for GPELF in areas where onchocerciasis is not co-endemic. The annual low dose regimen performed well in this study and clearly remains the treatment of choice for the majority of situations. However, the availability of an alternative DEC-based regimen that uses a higher dose of albendazole with increased frequency could assist programmes to meet their elimination targets. In particular, this regimen may find use in areas/countries where MDA programmes are yet to be initiated, in \u201chot spots\u201d identified during surveillance of ongoing programmes and in arresting transmission within shorter time frames . HoweverS1 Fig(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file."}
+{"text": "IRS1), rs1531343 (HMGA2), rs8042680 (PRC1), rs7578597 (THADA), rs1333051 (CDKN2), rs6723108 (TMEM163), rs163182 and rs2237897 (KCNQ1), rs1387153 (MTNR1B), rs243021 (BCL11A), and rs10229583 (PAX4) in our sample. Further, we showed that risk allele of the strongest T2D associated SNP in our sample, rs757832 (IRS1), is associated with increased level of TG. We observed substantial difference of T2D risk allele frequency between the Mongolian sample and the 1000G Caucasian sample for a few SNPs, including rs6723108 (TMEM163) whose risk allele reaches near fixation in the Mongolian sample. Further study of genetic architecture of these variants in susceptibility of T2D is needed to understand the role of these variants in heterogeneous populations.The large scale genome wide association studies (GWAS) have identified approximately 80 single nucleotide polymorphisms (SNPs) conferring susceptibility to type 2 diabetes (T2D). However, most of these loci have not been replicated in diverse populations and much genetic heterogeneity has been observed across ethnic groups. We tested 28 SNPs previously found to be associated with T2D by GWAS in a Mongolian sample of Northern China (497 diagnosed with T2D and 469 controls) for association with T2D and diabetes related quantitative traits. We replicated T2D association of 11 SNPs, namely, rs7578326 ( However, we observed that association of T2D risk allele  of rs7578326 (IRS1)\u2009 \u2009is associated with the increased level of TG. An elevated level of TG has been implicated as a risk factor of T2D, which likely resulted from the diminished activity of insulin causing inhibition of microsomal TG transfer protein activity [IRS1), namely, rs2972146, is reported to be associated with an elevated level of TG as well [r2 = 0.3753, 1000\u2009G phase I), indicating a possibility that the T2D associated SNP or its proxy could be playing a role in pathogenesis of T2D or its related TG metabolism through IRS1 activity. Further functional work will help to understand the role of this SNP. Since we did not have information on previous medical history of treatment for either high cholesterol or T2D for the patients, it is possible that such treatments, if administered previously, could have prevented us from seeing the effect of SNP association with the diabetes related quantitative traits, including TG.None of the SNPs we tested for association with T2D here has been previously implicated to be associated with lipids based on the NHGRI GWAS catalog (available at activity . The majTMEM163) and rs8042680 (PRC1) have substantial allelic differences in our sample compared to Caucasians and the risk alleles have reached near fixed level in Mongolians. On the other hand, a widely replicated T2D SNP, rs7903146 (TCF7L2), in European populations has substantially low risk allele frequency in the Mongolian sample  and is not associated in our sample of modest size. It is likely that the frequency difference of some SNPs between our sample and European population also contributed to the lack of reproducibility in T2D association in our study.Mongolians are one of the people who reside on the Mongolian Plateau in Asia and have heavily depended on nomadic life styles with harsh environments characterized by low temperature and scarce availability of food sources . AlthougAlthough we note that rs6723108 has a wide range of OR estimate , the OR of 7.7 in our study is substantially greater than what was reported in the original study in an Indian population [ IRS1 to be associated with an increased level of TG. The observation of the remarkable allele frequency difference of the T2D SNPs in our sample compared to Caucasians is important in further identifying causative variants for T2D and understanding the role of these SNPs in development of T2D in different ethnic populations.In conclusion, our association study has confirmed association of several previously identified T2D susceptibility loci in the Mongolian sample. We also identified rs7578326 nearThe SNPs were chosen with following considerations: (1) GWAS SNPs found to be associated with T2D in an Asian sample were given higher priority; and (2) subsequently, SNPs found to be associated with T2D in multiple studies were included. Genotyping of six SNPs was not successful, thus these SNPs were included from further analysis."}
+{"text": "The gut is the largest immune organ and plays a central role in the promotion of systemic inflammatory responses . Perturb7 CFU Bifidobacterium longum, 10 \u00d7 106 CFU Lactobacillus bulgaricus, 10 \u00d7 106 CFU Lactobacillus acidophilus. Colonic tissue and serum samples were collected 24 hours after CLP for ELISA and protein expression analysis by western blotting.Sepsis was induced by cecal ligation and puncture (CLP) in Wistar male rats (8 weeks old). They were pretreated with probiotics or vehicle once a day during 7 days before CLP. The chosen probiotic mixture contained 10 \u00d7 10Our data demonstrate that probiotic pretreatment improved survival of septic rats Figure  and thisOur results show that probiotics pretreatment fulfills a dual function at the intestinal mucosa: in addition to preventing intestinal permeability disruption, it also attenuates proinflammatory cytokine release, diminishing the exacerbate host's reaction to infection and offering a novel prophylactic strategy to sepsis."}
+{"text": "Paramecium we have shown that three conserved proteins FOR20, centrin 2 (CEN2) and centrin 3 (CEN3) participates in this process, with FOR20 and CEN2 being also involved in the transition zone assembly. We established a chronology in basal body assembly: CEN2 is required for FOR20 recruitment, the latter being necessary to recruit CEN3. Our goal now is to integrate others molecules in this cascade.Ciliogenesis is conditioned by a correct positioning/anchoring of the basal body at the cell surface. In Paramecium. OFD1 is a well-studied protein which is involved in human development whose mutations in human males can impair basal body docking. In contrast, only studies in Chlamydomonas indicate that VFL3 could be involved in this phenomenon.We used a combination of electron microscopy, immunocytochemistry, GFP protein tagging and RNAi knockdowns to study the function of OFD1 and VFL3/CCDC61 in Paramecium induces defects in basal body docking, these defects being similar to those observed upon inactivation of FOR20, CEN2 and CEN3; 1) like FOR20 and despite its distal location on anchored basal bodies, OFD1 is recruited early during their assembly; 2) while the recruitments of OFD1 and CEN2 proceed independently, the two molecules are required for the recruitment of FOR20. We also present preliminary results indicating that VFL3/CCDC61 is crucial for maintaining both basal body polarity and positioning and for the recruitment of CEN3, but neither for CEN2 or OFD1.As in human, the depletion of OFD1 in"}
+{"text": "Enterovirus\u00a0genus within the Picornaviridae family. The SVDV genome is composed of a single-stranded RNA molecule of positive polarity. Here, we report the first complete sequence of the coding region of a Spanish SVDV isolate (SPA/1/'93).Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the  Enterovirus genus within the Picornaviridae family. SVDV is the etiological agent of a highly contagious disease of pigs (SVD) and is closely related to the human pathogen coxsackievirus B5 (CVB5) (Swine vesicular disease virus (SVDV) is a member of the 5 (CVB5) . The SVD5 (CVB5) , 3. In t5 (CVB5)  and propThe coding sequence of the Spanish SVDV isolate SPA/1/'93 was compared with other complete coding sequences from different isolates that varied geographically and temporally using Clustal 2.1 . This analysis showed that SVDV SPA/1/'93 shared a higher degree of sequence identity (over 98%) with contemporaneous Italian isolates . The sequence data of the SVDV isolate determined in this study will help future research on the epidemiology and molecular biology of this porcine pathogen.KU291213.The genome sequence of the SVDV SPA/1/'93 has been deposited in GenBank under the accession number"}
+{"text": "Rapid response teams (RRT) are important systems for identifying patients requiring intensive car e. CompliaTo investigate the correlation between the clinical outcomes of septic patients and the elapsed time before transfer to the intensive care unit (ICU).A retrospective descriptive study performed in a large hospital in S\u00e3o Paulo, Brazil, with all patients admitted to the ICU by RRT activation due to suspected sepsis, sepsis, severe sepsis or septic shock from January to December 2011.39 patients were attended by RRT 5 to 20 minutes after activation. Thirty patients (76.9%) were immediately transferred to the ICU. The elapsed time since assessment to transfer to the ICU ranged from 15 to 30 minutes , 30 minutes to 1 hour , 1 to 2 hours  and \u22653 hours . As for the clinical outcome in the ICU, 20 (51.3%) had a clinical improvement, 14 (35.9%) died and 5 (12.8%) had an initial clinical deterioration with subsequent improvement. Clinical improvement or initial deterioration with subsequent recovery occurred mainly among patients transferred to the ICU within 15 minutes to 3 hours. In patients transferred after 3 hours, death was the most frequent outcome.The clinical outcomes of septic patients early transferred to the ICU are better than the outcomes of patients transferred later."}
+{"text": "Current rSO2 technologies assume that the oxygen saturation is a fixed ratio of arterial and venous blood. Cerebral arteries have an oxygenation-related vasoactivity that may change the arterial/venous ratio during hypoxia. Thus, absolute rSO2 accuracy may be less important compared with sensitivity to changes in cerebral rSO2.The purpose of this study was to determine the rate and magnitude of response to hypoxia for three different regional oxygen saturation . Resaturation was induced by rapid change in FiO2 to 1.0. After 5 minutes of SpO2 100%, the process was repeated by desaturation to SpO2 70% and rapid return to SpO2 100%. Cerebral and pulse oximetry data were recorded during the study and the time of each FiO2 change and plateau was recorded. rSO2 levels at 10, 20, 40, 60 and 80% of the total SpO2 response were calculated for each device to assess the rate of rSO2 change. The rate of rSO2 change in seconds and total rSO2 change were compared.Ten subjects completed the study and are included in the analysis. One INVOS sensor (SAFB-SM) was placed on the left side and one Equanox (8000CA) or Foresight (1 July 2007 or 1 July 2005) sensor  was placed on the right side of the forehead for bilateral monitoring. Desaturation was induced by adjusting the inspiratory gas mixture of O2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was significantly faster for INVOS (42 \u00b1 16) compared with Foresight (57 \u00b1 20) (P < 0.05). There was significant difference in total rSO2 change between INVOS (23 \u00b1 4%) and Equanox (15 \u00b1 4%) during desaturation and resaturation (P < 0.005) and between INVOS compared with Foresight (20 \u00b1 5%) (P < 0.05).The rate of rSO2 devices followed the SpO2 slope during desaturation as expected. The differences between the devices in terms of total rSO2 change reached statistical significance. There were also significant differences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change in seconds and the magnitude of absolute change may result in better resolution to detect physiological changes. Clinical studies are required to elucidate the clinical relevance of the differences.All rSO"}
+{"text": "BTB and KCTD11BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12BTB and KCTD15BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of \u03b12\u03b23 loop between KCTD11BTB and KCTD12BTB is sufficient to abolish the ability of KCTD11BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6 The BTB  domain is a widespread structural module that is frequently involved in both self and hetero association . ProteinThe analysis of the molecular organization of these proteins indicates that KCTDs are characterized by a modular organization that couples BTB domains, always located at the N-terminus, to C-terminal regions that are frequently unrelated. Comparative analyses of KCTD sequences have highlighted the possibility to cluster KCTD human paralogs in seven clades (denoted with A to G letters), each composed of proteins involved in similar biological processes . NotablyIn this framework, we have here checked the interaction of Cul3 with two proteins (KCTD12 and KCTD15) which play crucial roles in non-cullin dependent functions. In particular, KCTD12, and the other members of the Clade F (KCTD8 and KCTD16), are integral components and modulators of the GABA(B) receptor . KCTD12 BTB), KCTD11 (KCTD11BTB), and KCTD12 (KCTD12BTB) were expressed in E. coli BL21(DE3) and purified by following previously reported protocols. In particular, KCTD11BTB, which comprises residues 15\u2013116 of the long form of the protein (isoform 2 of the UniProt Code Q693B1-2), was obtained by following the procedure reported in Correale et al. . On the other hand, the elution profiles of the mixtures formed by KCTD12BTB and KCTD15BTB with Cul3NTD do not show the presence of high Mw species (BTB and Cul3NTD (insert of BTB and Cul3NTD (insert of NTD (~ 42 kDa), it can be confidently assumed that the BTB domains of these two KCTD proteins do not form stable complexes with Cul3.An initial analysis of the binding of these BTB domains to Cul3 was assessed by gel filtration, which provides estimates of the molecular masses of the proteins/complexes under investigation through the analyses of the elution volumes. The mixing of the proteins KCTD6 appears . This ob species . Indeed,BTB, KCTD11BTB, KCTD12BTB, and KCTD15BTB with Cul3NTD are shown in BTB and KCTD15BTB with this cullin suggest that no binding occurs  and 25 \u00b1 2 nM  for KCTD6BTB/Cul3NTD and KCTD11BTB/ Cul3NTD, respectively. In both cases, the value of the number of sites of KCTDs tetramers per Cul3NTD is ~ 0.25. This analysis suggests that the two proteins form with Cul3NTD the following complexes (KCTD6BTB\u2013Cul3NTD)4 and (KCTD11BTB\u2013Cul3NTD)4.In order to confirm and to quantify the data obtained from the gel filtration analyses, ITC experiments, which quantitative evaluate protein-protein binding through the analyses of the heat exchanges associated with their mixing. The heat exchanges upon titration of KCTD6g occurs . This obg occurs . The fitBTB/KCTD12BTB (CHIM11/12BTB), in which the \u03b12\u03b23 loop of KCTD11 (residues 42\u201363) was replaced with the residues 61\u201380 of KCTD12, which are predicted to correspond to the \u03b12\u03b23 of this latter protein  is in close to the value expected for the tetramer (62 kDa)  the \u03b12\u03b23 loop and (b) the \u03b14\u03b15 helical hairpin , 19. In  protein . This vaCTD11BTB . Moreove(62 kDa) . These fith Cul3 . The lacanalysis .BTB and CHIM11/12BTB in Cul3 recognition, we performed molecular dynamics simulations on these two proteins focusing the attention on the dynamic properties of the \u03b12\u03b23 loop. The starting models used in the simulations were generated as described in the Materials and Methods section. The analysis of the indicators commonly adopted to check system stability in MD analyses indicates that both structures are stable in the simulation timescale (100 ns). This is clearly indicated by the time evolution of (a) the root mean square deviations (RMSD) of the trajectory structures from the starting model, (b) the gyration radius, (c) the secondary structure, and (d) the total number of hydrogen bonds , whose side chains are involved in conserved electrostatic interactions  (BTB exhibits a higher flexibility. The somewhat reduced mobility of this loop in KCTD11BTB may be ascribed to the presence of a proline-rich fragment (residues 50\u201353 PMPP) in the \u03b12\u03b23 central region of the protein sequence.a)Insights into the local mobility of different protein regions have been obtained by the analysis of the root mean square fluctuation (RMSF) values calculated on C\u2013100 ns) . As a geBTB and CHIM11/12BTB is also confirmed by an essential dynamics analysis carried out by using the procedure developed by Amadei et al. . Interestingly, both proteins recognize Cul3 forming stable complexes, which can be easily purified by gel filtration chromatography, with a 4:4 stoichiometry. The affinity of the BTB domains of these tetrameric KCTDs for Cul3 is comparable to that reported for the pentameric KCTD5BTB [BTB with Cul3 has suggested that the cullin binds at the intersubunit interface of the protein tetramer [BTB recognizes this cullin at the tetramer interface. Collectively, these observations suggest that the inter-subunit interface of both KCTD tetramers and pentamers provide large surfaces for tight Cul3 binding. Current data suggest that in monomeric or dimeric BTB-containing proteins a similar tight Cul3 binding is achieved with the contribution of other structural motifs such as the 3-box [The quantification of the binding of Cul3 to KCTD6KCTD5BTB  and muchin KD of  nM. InteKCTD5BTB . Since ain KD of  nM. InteBTB and KCTD15BTB are unable to bind Cul3. Although our data were derived using recombinant proteins expressed in a bacterial system, to the best of our knowledge  than to KCTD12BTB (42% sequence identity) that is also unable to recognize the cullin. In other words, the sequences of cullin binding and cullin independent KCTDs are not segregated in the evolutionary tree. These considerations indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. In particular, the loss of ability in Cul3 recognition may have occurred in the divergence of KCTD12 from KCTD6 and KCTD15 and then in the separation of KCTD15 and KCTD6.ITC and Gel filtration analyses indicate that both KCTD12also Ref . These omparison  and of tBTB to bind Cul3 indicate that the \u03b12\u03b23 loop is crucial for cullin recognition. It is worth mentioning that present MD studies indicate that the inability of CHIM11/12BTB to bind Cul3 cannot be ascribed to the obstruction of the binding site by the \u03b12\u03b23 KCTD12 sequence. Therefore, in KCTD11 and other Cul3-binding KCTDs the \u03b12\u03b23 loop contributes to Cul3 recognition by forming interactions that directly stabilize the complex. The high variability of this region Click here for additional data file.S2 FigBTB are highlighted in blue and red, respectively. The hotspots for cullin recognition are also highlighted.The sequence number of the first residue of the BTB domain of each protein is reported. KCTD19a and KCTD19b refer to the two BTB domains of this protein. Helices and strands of KCTD5(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 FigThe differentiated motions along the first eigenvector are represented in a film-like fashion. Large movements are displayed by the \u03b12-\u03b23 loop region. An arbitrary color scale (from violet to red) is used to represent the movement. For clarity, the motion of a single chain within the tetramers is shown.(TIF)Click here for additional data file."}
+{"text": "ABCC6) gene (OMIM 603234) are known to cause the rare autosomal recessive disease pseudoxanthoma elasticum (PXE). Recently, we have found that copy number variations (CNVs) in pseudogenes of the ABCC6 gene are quite common. The aim of this study was to investigate the frequency and possible contribution of CNV in ABCC6 and its pseudogenes in PXE. Genomic DNA from 212 PXE individuals were examined for copy number by pyrosequencing and quantitative polymerase chain reaction (PCR) and compared with healthy individuals. The frequency of PXE individuals with any CNV was higher than in healthy individuals. The majority of variation comprised known and possibly new deletions in the ABCC6 gene and duplications of the ABCC6P1 and ABCC6P2 genes. ABCC6 deletions and ABCC6P2 duplications were not observed in 142 healthy individuals. In conclusion, by pyrosequencing and quantitative PCR, we were able to detect known and possibly new deletions in the ABCC6 gene that may have caused the PXE phenotype. Pyrosequencing may be used in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and may affect the PXE phenotype.Single mutations in the ATP-binding cassette transporter ( ABCC6 belongs to a large family of membrane proteins (ABC transporters) that are highly conserved and present in all organisms from bacteria to man  pseudogenes, ABCC6P1 and ABCC6P2 , autosomal recessive disease pseudoxanthoma elasticum , a metabolic disorder characterized by ectopic mineralization of soft connective tissues  . Written informed consent was obtained from all subjects before blood samples were taken. The study was approved by the Norwegian Regional Ethics Committees.DNA from patients diagnosed with PXE was obtained from the PXE International Registry and BioBank. The patients included 212 individuals and were mainly of European descent . Of these, 148 (70%) were female and 64 (30%) were male. PXE phenotypes were categorized according to the five organ systems  and severity  , Chinese (n\u00a0=\u00a024), Middle East (n\u00a0=\u00a020), Mexicans (n\u00a0=\u00a024), and Africans (n\u00a0=\u00a024).Genomic DNA from healthy individuals from the National Institute of General Medical Science (NIGMS) was purchased from the Coriell Cell Repositories . The populations were Caucasians . For absolute copy number determination of ABCC6, a TaqMan\u00ae Copy Number Assay targeting ABCC6 specifically in intron 11 was used . Two pyrosequencing assays were used to determine the relative copy number of ABCC6P1  versus ABCC6 (targeting intron 7 of both genes) and the relative copy number of ABCC6P2  versus ABCC6 and ABCC6P1 . The analyses were performed as described previously  was finally deduced from the TaqMan\u00ae Copy Number Assay and the two pyrosequencing assays. Calculation of the absolute copy numbers of ABCC6 pseudogenes was based on relative quantities (ratios) of the ABCC6 gene compared to the ABCC6 pseudogene(s). When one or more assay(s) deviated with respect to absolute copy numbers , the absolute copy numbers of the three genes were interpreted as the most reasonable.The CNV was analyzed in short specific regions involving exon 2, intron 7, and intron 11 of ABCC6 and ABCC6 pseudogenes, and deletions/insertions in other parts of the genes, or other chromosomal reorganization events, may not be identified.The limitation of this method is that CNV is analyzed in short specific regions of Ps\u00a0<\u00a00.05 were taken as statistical significance.The Fisher's two-tailed exact test was used for testing categorical variables between patients and controls. ABCC6 and ABCC6 pseudogenes were obtained from 207 of the 212 PXE individuals . Five patients were excluded from further analysis because of inconclusive copy number results. Both deletions and duplications were found for ABCC6, ABCC6P1, and ABCC6P2 in the PXE patients. The frequency of individuals with any CNV was higher than in a healthy population of Caucasian (controls)   , we identified a deletion of ABCC6 in 10/169 (\u223c6%) of the uncharacterized alleles (Table S1). For 10 of the 11 analyzed PXE patients with ABCC6 deletions, none or one mutant allele only had previously been identified. TableABCC6 and ABCC6P2 (#1) and three patients had deletions in ABCC6 only (#2). Five patients had smaller deletions (#3 and #4) in ABCC6 that were observed in either one or two of the analyzed regions (Exon 2 and/or intron 7)   in ABCC6 was accompanied by a duplication of ABCC6P1. This duplicated segment of ABCC6P1 may in fact have contributed to the ABCC6 deletion by gene conversion. Mendelian transmission of ABCC6 pseudogene CNV could be demonstrated for a few families. In one family (three siblings) with three copies of ABCC6P2, the PXE manifestation was present in individuals with and without this duplication, however, eye, skin, and gastrointestinal symptoms were more severe for the two individuals with three copies of ABCC6P2 (data not shown).In this study, we demonstrated that pyrosequencing is a fast and convenient method for the detection of CNV involving deletions of the entire or part of the ABCC6, mutational information was limited (Table S1). The functional consequence of having three copies of ABCC6 is unknown. Previously, by in vitro studies, we found that reduced mRNA expression of ABCC6P1 could influence the mRNA expression of ABCC6 , we observed a higher frequency of patients with gastrointestinal bleeding (G1 or G2 according to Phenodex\u2122) in patients with more than two copies of ABCC6P2 (3/6) compared to patients with two or less copies of ABCC6P2 (11/178) . It would be interesting to investigate this association further as the pathophysiological cause of gastrointestinal bleeding in PXE is unknown. No significant correlation was observed for other clinical phenotypes, cholesterol diagnosis, and CNV of ABCC6 and/or ABCC6 pseudogenes.When correlating CNV of ABCC6 gene that may have caused the PXE phenotype. This method may be used in combination with quantitative PCR in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and therefore may affect the PXE phenotype.In summary, by pyrosequencing, we were able to detect known and possibly new deletions in the"}
+{"text": "Partial or complete remission from type 2 diabetes was recently observed after bariatric surgeries. Limited data is available about the possibility of inducing diabetes remission through intensive weight reduction. We retrospectively evaluated diabetes remissions after one year of the Weight Achievement and Intensive Treatment (Why WAIT) program, a 12-week intensive program for diabetes weight management in real-world clinical practice. Among 120 obese patients with type 2 diabetes who completed the program, 88 patients returned for follow-up at one year. Nineteen patients (21.6%) had major improvement in their glycemic control, defined as achieving an A1C <6.5% after one year. Four patients (4.5%) achieved either partial or complete diabetes remission defined as A1C <6.5% and <5.7%, respectively, on no antihyperglycemic medications for one year; 2 achieved partial remission (2.3%) and 2 achieved complete remission (2.3%). At the time of intervention, patients who achieved diabetes remission had shorter diabetes duration (<5 years) and lower A1C (<8%) and were treated with fewer than 2 oral medications. They achieved a weight reduction of >7% after 12 weeks. These results indicate that a subset of obese patients with type 2 diabetes is appropriate for intensive lifestyle intervention with the aim of inducing diabetes remission. Type 2 diabetes has been considered as an incurable chronic disease. Traditionally, the focus of clinical management has been directed toward controlling hyperglycemia and its consequent complications . HoweverSince weight gain plays a vital role in the pathophysiology of type 2 diabetes, especially among genetically susceptible individuals , there iAnother meta-analysis of long-term effects of weight loss on diabetes prevention among obese people showed that intentional weight loss reduces risk of developing diabetes by 25%, while larger weight loss achieved through surgical interventions showed a dramatic risk reduction of about 63% .Bariatric surgery has been reported to improve type 2 diabetes and was shown to be superior to medical intervention .The aim of this study is to evaluate diabetes remission after one year of intensive lifestyle intervention in real-world clinical practice. It also aimed at characterizing those patients who are likely to achieve remission after weight loss.2) with type 2 diabetes enrolled in the Weight Achievement and Intensive Treatment (Why-WAIT) program between September 2005 and June 2008. Why WAIT program is a 12-week multidisciplinary intensive weight management program customized for obese patients with diabetes in real-world clinical practice at the Joslin Diabetes Center in Boston [We retrospectively evaluated obese patients  years at the start of the Why WAIT program. They were treated with a mean of 1.5 antihyperglycemic medications and they lost on average 26.4\u2009lbs (\u00b18.5) (11.1%) by the end of the program and maintained an average weight loss of 26.6\u2009lbs (\u00b120.7) (11.2%) at one year. The mean change in A1C, body weight, BMI, blood pressure, lipid profile, and renal function were shown in Participants who achieved major glycemic improvement at one year had a mean age of 53.3 (\u00b18.5) years, a mean body weight of 237.7 (\u00b129.5) pounds, a BMI of 38.2 (\u00b14)\u2009kg/m2, and a mean duration of diabetes of 1.5 (\u00b10.9) years at the start of the program. They were treated with a mean of 1.25 antihyperglycemic medications and lost on average 25.2\u2009lbs (\u00b17.3) (10.9%) by the end of the program and maintained a mean weight loss of 15.9\u2009lbs (\u00b110.7) (6.9%) at one year.Those who achieved partial or complete remission had a mean baseline body weight of 230.9 (\u00b123.2) pounds, a BMI of 35.6 (\u00b11.7)\u2009kg/mIn this study we demonstrated that major improvement in glycemic control can be achieved and maintained for one year after nonsurgical intensive lifestyle intervention in real-world clinical practice. Partial or complete diabetes remission at one year was achieved in small percentage of patients. Remission was more likely to occur in patients with shorter duration of diabetes, on few oral antihyperglycemic medications and with A1C < 8% at start of intervention. These results may indicate that a subset of obese patients with type 2 diabetes on antihyperglycemic medications is appropriate for intensive lifestyle intervention that aims at inducing diabetes remission and stopping the use of antihyperglycemic medications.Look AHEAD  study also showed that complete remission of type 2 diabetes after intensive lifestyle intervention is possible but rare , 14. ForThe concept of diabetes remission in established patients who are already treated with antihyperglycemic medications is intriguing as it contradicts the traditional concept that type 2 diabetes is a chronic progressive disease characterized by significant loss of beta-cell function (up to 80%) at the time of diagnosis , 17. BerThe other explanation for our findings is that patients with short diabetes duration may have significant beta-cell reserve more than what was originally thought. It is unlikely that diabetes remission occurs if 50% or more of beta-cell function is lost at the time of diagnosis. It is worth mentioning that the United Kingdom Prospective Diabetes Study (UKPDS), which created that concept, was not originally designed to evaluate beta-cell function . The stuThis study also demonstrated several indicators of possible diabetes remission in response to intensive lifestyle intervention. They include short duration of diabetes of <5 years, A1C < 8% at the time of intervention, and treatment with fewer than 2 oral medications with higher weight loss of >7% as predictor of improvement. Surgical weight loss studies also showed that patients with short diabetes duration are more likely to achieve diabetes remission after surgery [The current widely accepted concept is that oral antihyperglycemic medications are needed after failure of lifestyle intervention in controlling hyperglycemia . HoweverThis study has several limitations. It is a retrospective observational study. The study also included heterogeneous group of patients with variable diabetes duration and on variable number of antihyperglycemic medications including insulin. These limitations impacted significantly the rate of remission since it is unlikely that patients with long diabetes duration achieve diabetes remission after significant weight reduction. The Look AHEAD study, despite being a randomized controlled study, was not designed or powered to evaluate diabetes remission . However"}
+{"text": "To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.Tightly regulated CaDOI:http://dx.doi.org/10.7554/eLife.04801.001 The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions.Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.tremblor\u2014that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.Shimizu et al. studied a zebrafish mutant\u2014called tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.A compound called efsevin restores regular heart rhythms in Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms.DOI:http://dx.doi.org/10.7554/eLife.04801.002 Ca2+ influx after activation of the L-type Ca2+ channel in the plasma membrane induces the release of Ca2+ from the SR via ryanodine receptor (RyR) channels, which leads to an increase of the intracellular Ca2+ concentration and cardiac contraction. During diastolic relaxation, Ca2+ is transferred back into the SR by the SR Ca2+ pump or extruded from the cell through NCX1. Defects in cardiac Ca2+ handling and Ca2+ overload, for example during cardiac ischemia/reperfusion or in long QT syndrome, are well known causes of contractile dysfunction and many types of arrhythmias including early and delayed afterdepolarizations and Torsade des pointes  and the mitochondrial Ca2+ uniporter (MCU) serve as primary routes for Ca2+ entry through the outer and inner mitochondrial membranes, respectively  locus encodes a cardiac-specific isoform of the Na+/Ca2+ exchanger 1, NCX1h   . The treillation . In thistre mutant embryos suffer from Ca2+ extrusion defects and manifest chaotic cardiac contractions resembling fibrillation  establish a regular contraction pattern with rhythmic Ca2+ transients  (ncx1h deficient embryos (LB). A 32kD protein species was detected from zebrafish lysate due to its binding ability to efsevinLB and OK-C125LB, an active efsevin derivative conjugated to beads, but not to beads capped with ethanolamine alone or beads conjugated with an inactive efsevin analog (OK-C19LB)  . This mo embryos  and was K-C19LB) . FurtherK-C125LB . Mass spK-C125LB .10.7554/2+ handling. To examine this possibility, we injected in vitro synthesized VDAC2 RNA into tre embryos and found that the majority of these embryos had coordinated cardiac contractions similar to those subjected to efsevin treatment (myl7:VDAC2 transgenic fish in which VDAC2 expression can be induced in the heart by tebufenozide (TBF)  . KnockinO hearts . Convers efsevin . Further efsevin . Similar embryos . These f2+ flux  MEFs where VDAC2 is the only VDAC isoform being expressed  were maintained and bred as described previously  and cloned into pCS2+ or pCS2+3XFLAG. Full length cDNA fragments of zebrafish MCU (Accession number: JX424822) and MICU1 (JX42823) were amplified from 2 dpf embryos and cloned into pCS2+. For mRNA synthesis, plasmids were linearized and mRNA was synthesized using the SP6 mMESSAGE mMachine kit according to the manufacturers manual .tretc318 heterozygotes. Cardiac performance was analyzed by visual inspection on 1 dpf. The tre mutant embryos were identified either by observing the fibrillation phenotype at 2\u20133 dpf or by genotyping as previously described (VDAC2 mRNA and morpholino antisense oligos (5\u2032-GGGAACGGCCATTTTATCTGTTAAA-3\u2032)  were injected into one-cell stage embryos collected from crosses of escribed .tre embryos. 12 embryos collected from crosses of tretc318 heterozygotes were raised in the presence of individual compounds at a concentration of 10 \u00b5M from 4 hpf (tretc318 embryos manifest a chaotic movement resembling cardiac fibrillation with intermittent contractions in rare occasion (tre mutant embryos and NCX1h morphants (>500 embryos).Chemicals from a synthetic library  and fromom 4 hpf . Cardiacoccasion . Compounmyl7:GFP hearts were taken at 30 frames per second. Line-scan analysis was performed along a line through the atria or the ventricles of these hearts  treatment. Human EBs were differentiated for 15 days and treated with 5 mM CaCl2 for 10 min before DMSO or efsevin (5 \u03bcM) treatment. Images of beating EBs were acquired at a rate of 30 frames/s and analyzed by motion-detection software. For calcium recording, the EBs were loaded with 10 \u03bcM fluo-4 AM in culture media for 30 min at 37\u00b0C. Line-scan analysis was performed and fluorescent signals were acquired by a Zeiss LSM510 confocal microscope.The mouse E14Tg2a ESC and human H9 ESC line were cultured and differentiated as previously described . At day tre, and efsevin-treated tre embryos were placed on uncoated, microelectrode arrays (MEAs) containing 120 integrated TiN electrodes . Local field potentials (LFPs) at each electrode were collected for three trials per embryo type over a period of three minutes at a sampling rate of 1 kHz using the MEA2100-HS120 system . Raw data was low-pass filtered at a cutoff frequency of 10 Hz using a third-order Butterworth filter. Data analysis was carried out using the MC_DataTool (Multichannel Systems) and Matlab (MathWorks).2-day-old wild type, 2. For the recording of Ca2+ sparks and transients, the external solution contained 2 mM CaCl2. For Ca2+ transients, cells were field stimulated at 0.5 Hz with a 5 ms pulse at a voltage of 20% above contraction threshold. For all measurements, efsevin was added 2 hr prior to the actual experiment. Images were recorded on a Zeiss LSM 5 Pascal confocal microscope. Data analysis was carried out using the Zeiss LSM Image Browser and ImageJ with the SparkMaster plugin (Murine ventricular cardiomyocytes were isolated as previously described . Cells wr plugin . Cells w4HCO3 (30 min at 56\u00b0C) and 100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were washed with 50 mM NH4HCO3, dehydrated with ACN, and dried down in a Speedvac. Gel pieces were then swollen in digestion buffer containing 50 mM NH4HCO3, and 20.0 ng/\u03bcl of chymotrypsin . Peptides were extracted with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC buffer A .For pull down assays mono-N-Boc protected 2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic ester of efsevin and its derivatives through the amide bond. After removal of the Boc group using TFA, the primary amine was coupled to the carboxylic acid of Affi-Gel 10 Gel . 2-day-old zebrafish embryos were deyolked by centrifugation before being lysed with Rubinfeld's lysis buffer . The lysDanio rerio IPI database v3.45 using the SEQUEST algorithm in the BioWorks software program version 3.3.1 SP1. All spectra used for identification had deltaCN>0.1 and met the following Xcorr criteria: >2 (+1), >3 (+2), >4 (+3), and >5 (+4). Searches required full cleavage with the enzyme, <4 missed cleavages and were performed with the differential modifications of carbamidomethylation on cysteine and methionine oxidation.Extracted peptides were analyzed by nano-flow LC/MS/MS on a Thermo Orbitrap with dedicated Eksigent nanopump using a reversed phase column . The flow rate was 200 nl/min for separation: mobile phase A contained 0.1% formic acid, 2% ACN in water, and mobile phase B contained 0.1% formic acid, 20% water in ACN. The gradient used for analyses was linear from 5% B to 50% B over 60 min, then to 95% B over 15 min, and finally keeping constant 95% B for 10 min. Spectra were acquired in data-dependent mode with dynamic exclusion where the instrument selects the top six most abundant ions in the parent spectra for fragmentation. Data were searched against the In situ hybridization was performed as previously described . DIG-labHeLa cells were transfected with a C-terminally flag-tagged zebrafish VDAC1 or VDAC2 in plasmid pCS2+ using Lipofectamine 2000 (Invitrogen). After staining with MitoTracker Orange (Invitrogen) cells were fixed in 3.7% formaldehyde and permeabilized with acetone. Immunostaining was performed using primary antibody ANTI-FLAG M2  at 1:100 and secondary antibody Anti-Mouse IgG1-FITC  at 1:200. Cells were mounted and counterstained using Vectashield Hard Set with DAPI .2+ indicator preferentially localized in mitochondria, for 1 hr at 15\u00b0C followed by a 30 min de-esterification period at 37\u00b0C. Subsequently, cells were permeabilized with 100 \u00b5M digitonin for 1 min at room temperature. Fluorescence changes in Rhod2  immediately after the addition of Ca2+  were monitored in internal buffer  using a FLUOSTAR plate reader .HeLa cells were transfected with zebrafish VDAC2 using Lipofectamine 2000 . 36 hrs after transfection, cells were loaded with 5 \u00b5M Rhod2-AM (Invitrogen), a Ca2+]c in suspensions of permeabilized cells or imaging of [Ca2+]m simultaneously with [Ca2+]c in intact single cells. Permeabilization of the plasma membrane was performed by digitonin (40 \u03bcM/ml). Changes in [Ca2+] in the cytoplasmic buffer upon IP3 (7.5 \u03bcM) addition in the presence or absence of ruthenium red (3 \u03bcM) was measured by fura2 in a fluorometer  and subsequently transferred to the microscope stage. Stimulation with 1 \u03bcM ATP was carried out in a norminally Ca2+ free buffer. Changes in [Ca2+]c and [Ca2+]m were imaged using fura2 (ratio of ex:340 nm\u2013380 nm) and mitochondria-targeted inverse pericam (ex: 495 nm), respectively . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 2 reviewers.Thank you for sending your work entitled \u201cActivation of VDAC2 regulates mitochondrial Ca2+ uptake and cardiac rhythmicity\u201d for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Jodi Nunnari (Reviewing editor); Rosario Rizzuto (peer reviewer 1). Reviewer 2 remains anonymous.The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. All agree that the work is of high quality and represents a significant advance in our understanding of the role of VDAC in calcium handling and cardiac physiology. A majority of the comments pasted below can be addressed by a careful revision of the manuscript. However, as pointed out by reviewer 2, the experiments do not adequately test the authors' conclusion that Ca2+ is directly transferred between the ER and mitochondria. Additional experimentation, for example examining the effect of addition of a Ca2+ chelator.Reviewer #1:The authors point to mitochondrial Ca2+ buffering as a protective mechanism against erratic electrical activity, and indicates a prominent role of VDAC2 as a \u201cfacilitator\u201d of Ca2+ transfer through MCU channels. In this respect, the authors could consider investigating whether additional, possibly isoform-specific mechanisms add to the role of VDAC2 in Ca2+ permeation across the outer mitochondrial membrane.1) Does VDAC 2 interact with MCU and/or with some of its regulators ?2) Is the expression of MCU and its regulators (\u201cMCU complex\u201d) altered in the tre mutants?3) Does pharmacological or molecular stimulation of VDAC2 affect the expression of the MCU complex?Reviewer #2:The manuscript reports exciting results but needs to be improved in the following ways:1) The results in 2) The authors state \u201c... efsevin restores rhythmic cardiac contraction in tre by potentiating VDAC2 activity.\u201d The authors do not measure VDAC2 activity and thus this conclusion is unnecessary speculation. VDAC2 is not an enzyme and thus its action is not so easily defined in this context. The first part of the last sentence on that page is the appropriate summarizing statement.3) The authors state: \u201cVDAC is an abundant channel located on the outer mitochondrial membrane serving as a primary passageway for metabolites and ions including Ca2+  Rapizzi.\u201c The au4) In addition, the authors state \u201cWe examined whether Ca2+ released from intracellular stores could be directly transported into mitochondria through VDAC2 and whether this process could be modulated by efsevin...\u201d The experiments reported do not distinguish between Ca++ release into the medium and quickly taken up my mitochondria or Ca++ directly traveling between the ER and the mitochondria. That could have been tested in 3C by having a strong chelator in the medium. Thus, with the current data the authors cannot claim evidence for direct transfer. They can claim a more effective uptake of Ca++ into mitochondria after efsevin treatment.Based on the results reported in 5B, the authors conclude that Ca++ has been transported into mitochondria in the permeabilized cells, ignoring the ER. The choice of Rhod2 was presumably based on its affinity for Ca++ and its likely saturation with Ca++ in the ER, so that observed changes are likely due to the mitochondria. The authors make no effort to point this out to the reader. It should be addressed.5) In the conclusions the authors state: \u201cIn this model, VDAC-dependent Ca2+ uptake controls the duration and the diffusion of cytosolic Ca2+ near the Ca2+ release sites...\u201d How, physically, can VDAC control the \u201cduration of cytosolic Ca++\u201d and the \u201cdiffusion of cytosolic Ca++\u201d? VDAC cannot control diffusion nor can it control the duration of diffusion. What I hope the authors want to say is that VDAC2 facilitates Ca++ uptake via MCU thus reducing the local Ca++ concentration more rapidly than otherwise. We appreciate the reviewers\u2019 insightful and constructive suggestions to help us improve our manuscript. We have taken the reviewers\u2019 advice and revised the manuscript accordingly. In addition to clarifying the text, we revised Reviewer #1 questioned whether the expression of MCU complex would be affected by NCX1 mutation or by the molecular stimulation of VDAC2.tremblor hearts with and without efsevin treatment. These findings are presented in We address this question by in situ hybridization. We find that the expression levels of MCU and MICU1 are comparable between wild type and Reviewer#1 also questioned whether VDAC2 interacts with MCU and its regulators.tremblor requires the activity of both VDAC2 and MCU provides genetic evidence supporting a functional interaction between VDAC2 and MCU  MICU1 is expressed in the developing heart  overal VDAC2 , and 3) al VDAC2 . These fReviewer #2 made a few suggestions toThe reviewer thought that the sample Mass Spec image presented inwas too small for inspection, and questioned the identities of peptides detected in our Mass Spec analysis.We thank the reviewer for pointing out this problem. In the original The reviewer also questioned whether we identified any other proteins interacting with efsevin.tremblor and knocking down VDAC2 abolishes the responsiveness of tremblor to efsevin. Together, these findings support VDAC2 as a target of efsevin.In the course of our study, we inspected six sets of data. VDAC2 was the only protein that was not present in the controls but was consistently identified from the efsevin affinity column. Vitellogenin 1 (vtg1) and NADPH quinine 1 (nqo1) were frequently found in both the control and efsevin affinity columns and thus were considered as contaminants. More importantly, our genetic analysis showed that overexpression of VDAC2 recapitulates the rescue effect of efsevin on Reviewer #2 raised a few questions regarding the mitochondrial Ca2+uptake analyses.The reviewer questioned the experimental detail about the mitochondrial Ca2+uptake experiment in HeLa cells.2+ into the samples to make the final Ca2+ concentration as approximately 10 \u03bcM. We have now clarified this information in the Methods and Figure Legend.We apologize for the confusing description. In this experiment, we added CaThe reviewer thought it would be helpful to point out how Rhod2 functions as a mitochondrial Ca2+indicator.We appreciate the reviewer\u2019s concern and include this information in the Methods section.The reviewer thought that \u201cexamine whether Ca2+released from intracellular stores could be \u2018locally\u2019 transported into mitochondria through VDAC2\u201d would be a better description of our analysis.2+ transfer through VDAC2 .We thank the reviewer for the suggestions. We have revised the manuscript accordingly."}
+{"text": "T1 mapping is a rapidly evolving field and is emerging as a novel quantitative tissue characterisation technique with potential applications in fibrosis, oedema, fat, iron and other patho-physiology. The impact of gender, segmental analysis and image piloting is not yet fully explored and has the potential to complicate the interpretation of observed differences.9 healthy volunteers and 7 patients with no evidence/history of disease were scanned on a Siemens Avanto 1.5T scanner and a 32 channel coil using the Siemens modified look locker inversion recovery (MOLLI) WIP version 448B (5:3:3 acquisition with inline motion correction and T1 map generation). All subjects had a 4 chamber, basal and mid ventricular short axis acquisition performed. In all slices, a single, slender ROI was placed in the mid wall of the left ventricular septum with meticulous attention to avoiding partial volume of blood pool. In addition, the mid ventricular short axis slice was segmented  into 6 AHA segments to allow regional T1 analysis.There were 9 females and 7 males, mean age 47. Mean T1 times are displayed in table We present T1 times for MOLLI imaging in healthy individuals and demonstrate 1) significantly higher T1 in females than males 2) segment to segment T1 variation and 3) a trend to longer T1 in the 4 chamber compared to short axis. These results suggest that caution should be used when interpreting absolute T1 values. Differences may be observed by gender, slice orientation and segment. Whether these differences are due to true physiological  or artefactual  differences is an area for further study.No external funding."}
+{"text": "Studies that show frequencies of different orthodontic treatment protocols can be used as valuable parameters in the interpretation of treatment tendency with time. The purpose of this retrospective study was to evaluate all orthodontic treatment planning conducted at the Orthodontic Department at Bauru Dental School, University of S\u00e3o Paulo, Brazil, since 1973, in order to investigate extraction and non-extraction protocol frequencies selected at each considered period.P\u2009<\u20090.05).The sample comprised 3,413 records of treated patients and was evaluated according to the protocol choice, divided into 10 groups: Protocol 0 (non-extraction); Protocol 1 (four first premolar extractions); Protocol 2 (two first maxillary and two second mandibular premolars); Protocol 3 (two maxillary premolar extractions); Protocol 4 (four second premolars); Protocol 5 (asymmetric premolar extractions); Protocol 6 (incisor or canine extractions); Protocol 7 (first or second molar extractions); Protocol 8  and Protocol 9 (agenesis and previously missing permanent teeth). These protocols were evaluated in seven 5-year intervals: Interval 1 (1973 to 1977); Interval 2 (1978 to 1982); Interval 3 (1983 to 1987); Interval 4 (1988 to 1992); Interval 5 (1993 to 1997); Interval 6 (1998 to 2002); Interval 7 (2003 to 2007). The frequency of each protocol was compared between the seven intervals, using the proportion test (The results showed that 10 protocol frequencies were significantly different among the 7 time intervals.The non-extraction protocol frequency increased gradually with consequent reduction of extraction treatments. The four premolar extraction protocol frequency decreased gradually while the two maxillary premolar extraction protocol has maintained the same frequency of indications throughout time. The decision to extract teeth or not and the number of teeth to be extracted can influence the final result of orthodontic treatment, including esthetics, occlusion, satisfaction of patients and their families, as well as the treatment time ,2. For mRetrospective studies \u20138 of extThe data was retrospectively obtained from 3,745 consecutively treated patients from the files of the Orthodontic Department at Bauru Dental School, University of S\u00e3o Paulo, from 1973 to 2007. Patient files  were sequentially evaluated and 332 (8.86%) were excluded, resulting in 3,413 cases in the sample. Exclusion criteria were patient transfer or treatment drop out, preventive and orthopedic treatment without following orthodontic treatment with fixed appliances and cases without complete records.The sample consisted of 1,475 males (43.21%) and 1,938 females (56.79%) treated with an initial mean age of 13.76\u00a0years , divided according to the treatment protocol: Protocol 0 (non-extraction); Protocol 1 (four first premolar extractions); Protocol 2 (two maxillary first and two mandibular second premolar or a variation to three first premolar and one mandibular second premolar extractions); Protocol 3 (two maxillary premolar extractions); Protocol 4 (four second premolar or a variation to three second premolar and one mandibular first premolar extractions); Protocol 5 (asymmetric extractions - three premolars or only one premolar); Protocol 6 (incisor or canine extractions); Protocol 7 (first or second molar extractions); Protocol 8  and Protocol 9 (patients with agenesis or previously missing permanent teeth). The frequency of these protocols were evaluated in seven 5-year intervals similar to Profitt [The study protocol was approved by the Ethics Committee on Human Research of Bauru Dental School, University of S\u00e3o Paulo.P\u2009<\u20090.05. These analyses were performed with Statistica software .The frequencies of each protocol were compared among the intervals with the proportion test . ResultsAll 30 re-evaluated patient records presented complete agreement with the first observation, confirming the high reproducibility of the methodology. Figure\u00a0Because the sample consisted of all treated patients in the Orthodontic Department at Bauru Dental School, from 1973 to 2007, it would not be necessary to apply inferential statistical tests. Even though, to provide more mathematical precision, the Proportion Test was used to evaluate whether there was any significant difference in the treatment protocol frequencies between each time interval. This type of frequency distribution test is strongly influenced by the number of events observed. This explains why, in Table\u00a0In this study, the treatment protocols with (Protocols 1 to 9) and without extractions (Protocol 0) showed great statistically significant variation among the considered intervals  decreased significantly, corroborating the findings of other studies ,6\u20138 (Tab decreaseThe two maxillary premolar extraction protocol (Protocol 3) showed a relatively stable frequency around 10% in most of the evaluated periods (Table\u00a0The therapeutic choice of four second premolar extractions (Protocol 4) demonstrated a much-reduced frequency in all evaluated intervals Table\u00a0. AlthougThe asymmetric extraction protocol of three premolars (Protocol 5 - two maxillary and one mandibular premolar) is indicated in Type 1 Class II subdivision malocclusion treatment . AsymmetProtocols 6, 7, and 8 exhibited low frequencies without significant differences among them Table\u00a0. These fProtocol 9 included all cases with previous dental absences. It was considered that these patients should not be excluded from the study by the fact that similar circumstances happen in daily clinical routine, and should be statistically described. They were placed in a separate group because their previous condition could have influenced the treatment planning. The total mean frequency of these cases was 11.28%, and their frequencies in the several periods showed no statistically significant differences  to 54.55% (2003 to 2007), with consequent reduction of extraction treatments from 85.71% (1973 to 1977) to 45.45% (2003 to 2007).The four premolar extraction protocol frequency decreased gradually from 65.72% (1973 to 1977) to 10.72% (2003 to 2007), while the two maxillary premolar extraction protocol has shown the same frequency of indications in the same time period.The following conclusions are drawn from the study:"}
+{"text": "To the Editor: In 2011, a chicken anemia virus (CAV)\u2013related sequence, designated avian gyrovirus 2 (AGV2), was first identified in serum samples from diseased chickens in Brazil  play a critical role in the transmission of poultry pathogens to humans, we used PCR to investigate the presence of AGV2 in chickens (54 feather shaft samples) from 4 LPMs in Yangzhou and in 178 human blood samples from healthy persons living in Yangzhou. The DNA from the feather shafts and human blood were extracted as previously described . The positive rates for samples from the 4 LPMs tested were 25%, 12.5%, 15.8%, and 20%; the positive rate for the 178 human blood samples was 1.1%. The low positive frequency of AGV2 in human blood detected in this study is consistent with that found by investigation in other countries  with MEGA6 . The 12 <15.8%, and these variants of AGV2 were mainly detected in diseased chickens (Our results demonstrate the presence of AGV2 in LPMs and human blood in mainland China. The amplification and analysis of partial AGV2 sequences was the major limitation in our method. The high homology between sequences identified in LPMs and human blood indicates the LPMs are a potential source for AGV2 in humans. Unlike our 12 conserved AGV2, AGV2 identified by Santos et al. in southern Brazil varied"}
+{"text": "The observed photo-responsivity of ~300\u2009A/W is four orders of magnitude larger than the earlier reported results on this material. Even though the role of 2D surface states responsible for high reponsivity is unclear, the novel and simple micromechanical cleavage (exfoliation) technique for the deposition of Bi2Se3 flakes followed by nanowire fabrication using FIB milling enables the construction and designing of ultrasensitive broad spectral TIs based nanowire photodetector which can be exploited further as a promising material for optoelectronic devices.Recently, very exciting optoelectronic properties of Topological insulators (TIs) such as strong light absorption, photocurrent sensitivity to the polarization of light, layer thickness and size dependent band gap tuning have been demonstrated experimentally. Strong interaction of light with TIs has been shown theoretically along with a proposal for a TIs based broad spectral photodetector having potential to perform at the same level as that of a graphene based photodetector. Here we demonstrate that focused ion beam (FIB) fabricated nanowires of TIs could be used as ultrasensitive visible-NIR nanowire photodetector based on TIs. We have observed efficient electron hole pair generation in the studied Bi Thickness and size dependent interesting and exciting light absorption properties of Bi2Se3 has been recently studied122Se3 has shown enhanced light absorption in the visible region compared to bulk sample2Se3 nanodevices has shown that the polarization of light can control the generation of photocurrents2Se3 have been successfully employed for the photocurrent studies2Se3 nanowires fabricated from high quality exfoliated flakes are largely remained elusive and have not been studied yet.Bi2Se3 nanowires fabricated by a focused ion beam microscope. Significant photocurrent is observed when nanowires are illuminated with visible laser (532\u2009nm) and IR laser (1064\u2009nm). In these nanowire devices, we have observed faster rise and decay times which depend on the laser power and the applied electrical bias voltage. The best photoresponsivity measured among the nanowire devices is in the order of ~300\u2009A/W which is ~10000 times larger than the ultrathin nanosheets of Bi2Se3Here we report the broadspectral photo response of Bi2Se3 flake where surface looks clean and smooth. Note that a thin flake of interest was first localized under FESEM but was never exposed to Ga ion imaging. Green colour rectangle shows the area selected for the milling and the small square was used for the alignment of electron beam and Ga ion beam. FIB milling was performed for the fabrication of nanowire geometry by etching out selected portion of the flake as shown in the 2Se3 nanowire device used for the photocurrent measurement in this study. The linear current voltage relationship shown by the black (light off) and the red (light on) curves in the inset of 232Se3 flakes was observed under high resolution transmission electron microscope (HRTEM) ( (HRTEM)  which is2Se3 nanowire devices were characterized for their time dependent photoresponse properties to broad spectral laser excitations using visible laser  and infrared laser . Prior to any photocurrent measurements, laser illumination was guided perpendicularly over wide area covering the entire device as shown schematically in 2 and ~32\u2009mW/cm2, respectively. A Keithley 2634B source measure unit (SMU) was used for the electrical characterization of the fabricated photodetector devices.The BiIPh) was extracted by subtracting the dark current (Idark) from the measured current in presence of laser light (Ilight) i.e. Iph\u2009=\u2009Ilight-Idark24252Se3 nanowire devices as photo switches or high quality photodetectors.First, we have characterized the photocurrent dynamics of the nanowire photodetector for cyclic exposures of visible light  to study the stability and repeatability of the photocurrent as shown in the The response and decay times are important parameters for any photodetector. We have used following exponential equations to calculate the response and decay times, i.e., 2. These values of 2Se3 nanowire based photodetectors compared to photodetectors made from other novel layered materials like MoS2  and is shown in Evolution of photocurrent is further characterized under the illumination of IR light source (1064\u2009nm) for various applied bias voltages as shown in  ~10\u2009nA,  was obse ~10\u2009nA, . Data in2 but small fluctuation in current is noticed at the photocurrent saturation plateau when the bias voltage is 150\u2009mV. For power densities <5\u2009mW/cm2, no noticeable measurement in photocurrent was found, above this threshold power, significant photocurrent rise is detected which is shown in Iph\u2009~\u2009P\u0398, to fit the data of photocurrent measured at different powers. Consequently, a sub-linear increase is found in the photocurrent with increasing power density for the incident illumination systematically. This increment in linearity is the characteristic feature of a photocurrent indicating minimal contribution from the thermoelectric current as Bi2Se3 is a very good thermoelectric material. Light sensitive, polarization dependent generation and control of photocurrents originating from topological surface states in Bi2Se3 have been studied alreadyPhotocurrent detection in nanowire device due to IR light has been further characterized to check dependency of the photocurrent on the incident power density of the laser while keeping the bias at constant voltage. Photocurrent of about ~2\u2009nA is observed for 5\u2009mW/cmR\u2009=\u2009photoresponsivity (A/W), Iph\u2009=\u2009photocurrent, P\u2009=\u2009Light intensity and A\u2009=\u2009effective area of the nanowire. We have calculated the photoresponsivity for both visible and near infrared wavelengths. The photoresponsivity of Bi2Se3 nanowire as a function of laser power density is plotted in 2. This photosensitivity is a huge improvement  compared to the previous photoresponse reports on ultrathin Bi2Se3 nanosheetsD) and external quantum efficiencies  of it can be exfoliated on the substrate for FIB milling by a simple scotch tape  method. The fabricated Bi2Se3 nanowire device has shown broad spectral response (visible and NIR). The response, rise and decay time, of FIB fabricated Bi2Se3 nanowire device is very fast and excellent photocurrent stability and repeatability have been noticed. The photocurrent showed dependency on the laser power density and applied bias voltages. The high photoresponsivity is observed which is ~4 orders of magnitude improvement as compared to ultrathin Bi2Se3 nanosheetsIn conclusion, the photocurrent dynamics of Bi2Se3 nanowires were made by using scotch tape method and focused ion beam (FIB) microscopy. Prior to deposition of flakes on SiO2/Si, substrates were cleaned with acetone, isopropanol, methanol and treated with oxygen plasma for ~5\u2009min. Bi2Se3 ( 99.999% CAS#12068-69-8) material was procured from company Alfa Aesar. By using micromechanical cleavage (scotch tape method), thin flakes of Bi2Se3 were exfoliated on SiO2/Si substrates having predefined gold pads. This method yields random sizes and thicknesses of the Bi2Se3 flakes which were observed under optical (Olympus) and electron microscope . The devices fabricated were loaded in the probe station setup (Cascade Microtech EPS150TRIAX) which has shield enclosure (EPS-ACC-SE750) for low signal measurements.Devices of Biga, 2Se3  were fab2Se3 samples to reveal microstructural information even up to atomic scale including the reciprocal space. In general, a thin flake-type morphology with a facetted-edge was observed in the microstructure. As an illustrative example, 2Se3 (space group: R2Se3 of 10High resolution transmission electron microscopy (HRTEM model: Tecnai G2 F30 STWIN assisted with field emission gun and with an electron accelerating voltage of 300 kV) was employed on Biinset in . The atoinset in  are dispcrograph . The inscrograph  further How to cite this article: Sharma, A. et al. High performance broadband photodetector using fabricated nanowires of bismuth selenide. Sci. Rep.6, 19138; doi: 10.1038/srep19138 (2016)."}
+{"text": "White matter disease in preterm infants comes along with focal destructions or with diffuse myelination disturbance. Recent experimental work with transgenic mice paves the way for a unifying molecular model for both types of brain injury, placing oxygen sensing by oligodendrocyte precursor cells (OPCs) at the center stage. Mice genetically altered to mimic high local oxygen tension in oligodendroglia lineage cells  develop white matter disease resembling cystic periventricular leukomalacia within the first 7\u2009days of life. Mice in which local hypoxia is mimicked in oligodendroglial cells (via genetic inhibition of HIF decay) display arrested OPC maturation and subsequent hypomyelination, reminiscent of the diffuse white matter disease observed in preterm infants and infants with congenital heart disease. These recent experimental findings on oxygen sensing and myelination are awaiting integration into a clinical framework. Gene regulation in response to hyperoxia or hypoxia, rather than oxidative stress, may be an important mechanism underlying neonatal white matter disease. Brain injury in preterm newborn infants involves both destructive and developmental disturbances . Cystic Evidence of dWMD has been revealed by advanced magnetic resonance imaging techniques, notably diffusion tensor imaging and magnetic resonance spectroscopy imaging, in many very low birth weight preterm infants , 12\u201314. Changes of ambient oxygen tension cause a cellular response orchestrated by hypoxia-inducible factors (HIFs) in almost all mammalian cell types . The lac2 0.1) between day 3 and 11 of life.As intrauterine oxygen tension is difficult to manipulate in experimental animals, researchers at the University of California in San Francisco used mice with either targeted deletions of HIF1\u03b1 and HIF2\u03b1, or lack of VHL-mediated HIF1\u03b1/HIF2\u03b1 decay in the oligodendroglial lineage . Embryon2 0.1 for 6\u201372\u2009h) has recently been shown to induce canonical (\u03b2-catenin-dependent) Wnt signaling alongside increased cell proliferation in the hippocampus of adult mice  show increased white matter vessel densities but myelination deficits are only found in rat pups (2 0.6 for 7\u2009days) but prevented by slowly increasing FiO2 from 0.15 to 0.21 during the first 7\u2009days of life (2 0.1) between day 3 and day 11 of life display reduced cortical gray and white matter volumes and delayed maturation of OPCs  , and theiO2 0.21 , 49. In 2 0.6), postnatal day 3 or day 6 (FiO2 0.6), but not day 10, has nevertheless a deleterious effect on the developing white matter in normal rat pups (2 0.8) at 6\u2009days of life for 48\u2009h continue to show signs of dWMD when examined by diffusion tensor imaging (While experimental hyperoxia in rodents is apt to reproduce several findings of human ROP, exposure to neonatal rats or mice to high oxygen does not necessarily generate the cystic white matter lesions observed with gene-mediated HIF1\u03b1/HIF2\u03b1-suppression in OPCs acting already before birth. While the brain maturation of newborn rats and mice recapitulates many features of the human situation during the last trimester of pregnancy and therefore that of preterm infants born 2\u20133\u2009months early, the newborn rodents used in experiments are physiologically adjusted to the perinatal oxygen surge and thus potentially less vulnerable than human preterm babies. Experimental hyperoxia at birth (FiOrat pups , 50. Gro imaging  that ref imaging . Culture imaging  but it iIn sum, the immature white matter of both human beings and rodents is highly sensitive to altered oxygen tension during a critical developmental window prior to the onset of myelination. Both too much and too little oxygen can cause damage by profoundly altering expression of various genes, unrelated to any oxidative stress. These two conditions may occur sequentially in the same brain, as reduced vessel density after high oxygen subsequently leads to low oxygen supply at a time when demand is on the rise. Meticulous attempts to avoid extremes of oxygen tension in the brains of preterm infants and in infants with congenital heart disease undergoing surgery, possibly aided by directly monitoring cerebral oxygenation , will hoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Sickle cell disease (SCD) is a major health burden in India. The objective of the study was to establish a neonatal screening program and to understand the clinical course of children with SCD in central India.Pregnant mothers were screened for sickle hemoglobin using the solubility test. Babies were screened by high performance liquid chromatography if the mother was positive for sickle hemoglobin. The diagnosis was confirmed by molecular analysis. They received early prophylactic treatment and vaccination. Of 2134 newborns screened, 104 were sickle homozygous (SS), seven had sickle \u03b2-thalassemia  and 978 were sickle heterozygous (AS). The other hemoglobin abnormalities detected included HbS -\u03b4\u03b2 thalassemia-1, HbSD disease-2, HbE traits-5, \u03b2-thalassemia traits-4, alpha chain variants-3 and HbH disease-1.These babies were followed up regularly for hematological and clinical evaluation. Pain, severe anemia requiring blood transfusions and acute febrile illness were the major complications with 59.7, 45.1 and 42.6 cases per 100 person years. Fetal hemoglobin (HbF) levels were inversely associated with vaso-oclussive crisis (VOC) and severe anemia while presence of alpha thalassemia increased the rate of painful events and sepsis. Six early deaths occurred among the SS babies.A systematic follow up of this first newborn SCD cohort in central India showed that 47% of babies presented within 1 year of age. In spite of the presence of the Arab-Indian haplotype many babies had severe manifestations. Studies on the natural history of sickle cell disease (SCD) from Jamaica and the USA have confirmed that the greatest morbidity and mortality occurs between 6 and 12 months and that early identification of affected infants by neonatal screening, careful follow up coupled with relatively simple measures decreased the mortality rate . The sicThis is one of the first efforts to raise a cohort of SCD babies by newborn screening and follow them regularly to record the early clinical and hematological presentation in central India.The study was approved by the Institutional Ethics Committee Review Board- \u201cInstitutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR) (NIIH/IEC/21-2007)\u201d, written informed consent was obtained from all participants and all investigations were conducted according to the principles expressed in the Declaration of Helsinki.Pregnant women were screened for sickle hemoglobin (HbS) using the solubility test at Govt. Medical College, Nagpur after a written informed consent was taken from them. Babies of all the mothers who had a positive solubility test were screened by high performance liquid chromatography (HPLC). Heel prick samples were collected in EDTA after birth or within 7 days of birth after a written informed consent from the parents and all the investigations on the babies and the parents samples were conducted according to the principles expressed in the Declaration of Helsinki. A complete blood count was done on an automated cell counter . Hemoglobin (Hb) analysis was done using automated HPLC on the VARIANT\u2122 Hemoglobin Testing System  using the sickle cell short programme and the \u03b2 thal short programme during follow up. Molecular analysis was done to confirm the sickle and other genotypes . Alpha gThe gestational age at delivery, demographic details and neonatal complications were recorded. The babies with SCD were enrolled at the sickle cell clinic in Nagpur. Vaccination included conjugate vaccine for Haemophilus influenzae type B and 7-valent conjugate pneumococcal vaccine  within 4 weeks of birth. All the babies received oral penicillin V starting at 3 months of age and 23-valent polysaccharide pneumococcal vaccine (Pneumovax) was given after 2 years of age. Clinical crisis were defined according to previous published criteria \u201313. A foIncidence rates are presented as the number of cases per 100 person years. The estimates were based on clinical events and follow up time. Descriptive statistics are presented as percentages and mean \u00b1 standard deviation. The p values are calculated by Fischer\u2019s exact test (two tailed). A p value of 0.05 or less was considered significant.0 thal (CD 15 (G\u2192A), CD 41/42 (-CTTT); 5 babies had S-\u03b2+ thal (IVS 1\u21925 (G\u2192C)], one baby had sickle-delta-beta thalassemia  and two babies had HbSD disease. In addition, five babies with HbE trait, four with \u03b2-thalassemia trait, three with alpha chain variants  and one case of HbH disease were also identified. The cohort of 104 SS babies  was followed up clinically every month for a year and then every 3 months for 3\u20134 years for hematological and clinical evaluation. However, in case of any symptoms they came earlier. 92% of the SS babies belonged to non-tribal communities while 8% belonged to tribal communities.From 2009 to 2012, 10,181 pregnant women were screened and 2134 newborn babies were tested of whom 1040 (48.7%) were normal, 978 (45.8%) were sickle cell traits, 104 (4.9%) were sickle homozygous (SS), seven had sickle \u03b2-thalassemia : [2 babies had S-\u03b275 out of the 104 SS newborns, six of the seven S-\u03b2 thal babies and the two babies with Hb SD disease could be followed up clinically while 29 (28%) were lost to follow up.Sixty two of the 75 SS babies had some clinical complications while 13 babies remained asymptomatic. Among them, 12 babies (19%) presented within 6 months of age, 17 (28%) between 6 and 12 months of age, 25 (40%) between 1 and 2 years of age while 8 babies presented after 24 months. The 2 Hb SD disease babies also presented within the first 6 months of life.The clinical events observed in the SS, S-\u03b2 thal and SD babies are summarised in st clinical event observed. Acute splenic sequestration (ASS) crisis was seen in the first 2\u20133 years of life in two SS babies and one HbSD disease baby. One SS and one HbSD disease baby had three episodes each of sequestration crisis but splenectomy was not advised. Severe anemia requiring transfusions (3 or more/year) was the main clinical event in the two HbSD disease babies while the six sickle \u03b2-thalassemia babies had none of the above complaints. Acute chest syndrome (ACS) was not found in this cohort.Staphylococcus, E-coli and Klebsiella.Eight SS babies had sepsis with the overall incidence of 4.8 per 100 person years  and two Seven SS babies had eight episodes of siezures and three babies had five episodes of stroke at 14, 42 and 30 months with an overall incidence of 3.0 per 100 person years . One babThe hematological and hemoglobin analysis at birth and at the last follow up (3\u20134 years) in SS babies is illustrated in Out of the 150 SS chromosomes, 141 (94.0%) were linked to the Arab-Indian haplotype, six were linked to the Bantu A2 and three to an Atypical1 haplotype (+ - + - + + + + -). Of the six babies having the Arab-Indian/Bantu A2 haplotype, two patients showed a moderately severe clinical course and one baby died due to severe anemia. Of the three babies with the Arab-Indian/Atypical1 haplotype, one could not be followed up and two patients showed a mildly severe clinical course.3.7/\u03b1\u03b1 = 12, -\u03b14.2/\u03b1\u03b1 = 6, -\u03b13.7/-\u03b14.2 = 1). The incidence of clinical events per 100 person years was determined in babies with and without alpha thalassemia (4.2/\u03b1\u03b1.Alpha genotyping was done in 69 of the 75 SS babies. 50 babies (72%) had a normal alpha genotype while 19 babies (28%) had alpha gene deletions (-\u03b1lassemia . InfantsHbF levels in the SS cohort varied from 3.3 to 37.2% (mean 21.4 \u00b1 5.4%). Based on an earlier study,  a cut ofSix SS babies (8%) died during the follow up with the overall mortality rate of 3.65 per 100 person years . In threNeonatal screening helps to recognize the early signs of the disease and minimize morbidity and mortality by early comprehensive care and prophylactic treatment. Our targeted newborn screening approach in central India identified 4.9% of SS babies along with other hemoglobinopathies. The above babies were enrolled at the sickle cell clinic and were given early comprehensive care and prophylactic treatment. Few babies with HbS-\u03b2 thalassemia may have been missed as universal screening was not done.Vichinsky et al demonstrated that the overall mortality rate for American patients with sickle cell anemia diagnosed in the neonatal period was 1.8% compared with 8% for children diagnosed after three months of age . In the Such established newborn screening programs have not been initiated in India although the load of SCD is very high in central India. An initial pilot effort was made in Raipur district in Chhattisgarh in India, on screening few babies , however the affected babies were not followed up . ScreeniAnother recent study on newborn screening among tribal populations in South Gujrat in Western India showed that 21.8% of SCD babies had severe clinical complications before 5 years of age . Our preSince all painful events do not lead to VOC, pain and VOC were considered as separate complications. Pain followed by acute febrile illness and severe anemia with blood transfusion requirements were the major complications observed in this study . PainfulAs noted by Miller et al \u201cThree easily identifiable manifestations of sickle cell disease that may appear in the first two years of life  can help to predict the possibility of severe sickle cell disease later in life\u201d. Severe Acute chest syndrome and pneumonia are very difficult to differentiate in babies and pneumonia being one of the precipitating factors for ACS, this complication in the babies was labelled as pneumonia. ACS followed by ASS and sepsis were the major cause of mortality in Jamaica . ACS wasStaphylococcus, E-coli and Klebsiella. This is in contrast to the CSSCD cohort where infections occurred most commonly due to Streptococcus pneumoniae and Hemophilus influenzae and caused 11 deaths [Infection particularly pneumococcal sepsis is the leading cause of death in children with SCD , 30. Sep1 deaths .Stroke is a feature of early childhood with an incidence of 8% by 14 years of age in the Jamaican cohort . Three oSCD patients with alpha thalassemia had a higher risk of painful events, sepsis and dactylitis and lower incidence of severe anemia . Our fin2 levels at the last follow up was observed due to the presence of HbS adducts which tend to increase the actual HbA2 levels on HPLC. The HbF levels at 3\u20134 years were 21.4 \u00b1 5.4%, majority of the sickle chromosomes being linked to the Arab- Indian haplotype.The hematological profile in the SS patients showed that anemia was apparent from 6 months of age. An increase in the HbAIn conclusion, this is the first newborn SCD cohort in central India to be systematically evaluated for 3\u20134 years. It emphasises the importance of early diagnosis and prophylactic treatment, parental education and the importance of tertiary care centres that should be made available to allow for prompt treatment. We have also shown recently that 75.4% adult SCD patients in India with the Arab Indian haplotype also have severe manifestations . Thus SC"}
+{"text": "WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45\u0394 CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire  WT1 gene are found in most stromal-type tumors, often associated with mutations in the CTNNB1 gene [Wilms tumor (WT), a malignant childhood neoplasm of the kidney, is thought to arise from embryonic renal mesenchyme with impaired nephrogenic differentiation potential. Most tumors have a mixed histology, containing blastema, epithelia and stroma. In the WT variant with a predominating stromal component, heterotypic cells, such as rhabdomyoblasts, fat, cartilage and bone can be found, not normally present in the kidney and likely to be derived from abnormal mesenchymal differentiation. Constitutional or somatic mutations in the NB1 gene .WT1 mutant tumors [WT1 mutant Wilms tumors revealed that these carry biallelic WT1 mutations but no CTNNB1 mutations, whereas the associated tumor cells had CTNNB1 mutations [WT1 mutant tumors have additional mutations in CTNNB1 or WTX, highlighting the importance of activated WNT signaling in tumors with inactive WT1 [CTNNB1 or WTX suggests that the functional loss of WT1 poses a strong selection pressure for additional mutations. This is further supported by our previous description of a patient with a germ line WT1 mutation who developed four tumors with different CTNNB1 mutations, suggesting their independent origin and/or tumor heterogeneity. In addition the same tumor harbored different CTNNB1 mutations in different histological areas [WT1 mutation, the second is the loss of heterozygosity (LOH) in 11p, resulting in loss of the WT1 wild type allele and the third is a CTNNB1 mutation [Intralobar nephrogenic rests (ILNR) occurring early in kidney development can be found as precursor lesions in t tumors . Microdiutations . Most WTtive WT1 ,5,8. Theal areas   exhibits gain of function properties. The mutant protein has lost the wild type WT1 function for sequence specific DNA binding, but facilitates the expression of genes regulating the cell cycle [CTNNB1 gene; the gain of function of the mutant WT1 protein in regulating the cell cycle could explain, why these cells do not need additional mutations in CTNNB1 nor WTX [Most cell lines that we have established from ll cycle . Therefo nor WTX .WT1 mutant Wilms tumor cell lines have a limited life span, similar to normal human mesenchymal stem cells (hMSC). Under in vitro growth conditions they can be cultivated at most for 60 population doublings. Primary cells in culture as well as some tumor cells have a limited life span that limits their use for experimental manipulation. Most biochemical and genetic studies require large cell numbers. Therefore, immortalized cell lines would be very useful for such studies. It has been described that the life span of normal human cells can be extended by introduction of the catalytic subunit of telomerase (TERT) [TERT was described for many different normal cell types, for example glomerular mesangial cells, glomerular endothelial cells, podocyctes, mammary fibroblasts and endothelial cells, airway epithelial cells and hepatocytes [All our previously established e (TERT) . More reatocytes \u201317. All WT1 nested within a heterozygous 11p13 deletion and a CTNNB1 mutation. Genetic analyses of cells derived from the Wilms tumor (Wilms10) showed the same WT1/11p13 alteration but a different mutation in CTNNB1 as compared to the bulk tumor DNA. aSNP/CGH revealed UPD in 3p and 11p15 not extending to the WT1 gene. Through immortalization of this cell line with ts LT and hTERT, we have established the first Wilms tumor cell line with a homozygous deletion of the entire WT1 gene, resulting in a complete lack of WT1. This immortalized Wilms10 cell line should be useful for further exploration of the effect of WT1 loss in genetic and biochemical studies and to further explore the origin and cell fate of Wilms tumors with WT1 mutations.Here we describe a stromal type Wilms tumor with a homozygous deletion of A 2 year-old girl presented with an isolated left renal mass. The tumor was surgically removed upfront and the histology showed a triphasic stromal predominant Wilms tumor with focal anaplasia and p53 over-expression in these anaplastic foci . The tumWT1 gene and CTNNB1 exon3 was analyzed as described before [Tumor DNA was isolated directly from a piece of solid tumor tissue. DNA from blood and cell culture cells was isolated by standard methods. The complete d before .TP53 gene was carried out on a GS Junior System instrument (Roche Applied Science). All sequencing data were generated using the Junior Sequencer Instrument software version 2.7 (Roche Applied Science). Sequence alignment and variant detection were performed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science).P53 mutation analysis was done by Next-generation pyrosequencing using 454 Titanium Amplicon chemistry  according to the manufacturer's instructions . Primer sequences and PCR conditions were kindly provided by A. Kohlmann . Sequencing run of the The tumor was finely minced and a cell culture was established using MSCG medium as described . AccordiTERT, pBABE-hygro-hTERT, Addgene 1773) [TERT from a triple transfected HEK293FT cell line were mixed and used to transduce the primary Wilms 10 cells. After infection, the cells were cultured in G418  for 1 week, followed by hygromycin  for another week to select for successfully doubly transduced cells. Single cell clones were isolated and cultivated at 33\u00b0C initially to maintain the active form of the temperature sensitive LT. After the first confluence, clones were split and cultured at 33\u00b0 and 37\u00b0C. One immortalized clone was selected for further studies and was named imWilms10. The imWilms10 cells continued to grow at 37\u00b0C, whereas normal hMSC immortalized with the same genes did not continue to proliferate at 37\u00b0C. The imWilms10 cells were continuously cultured in medium with alternating hygromycin and G418 for one week each, with a break of selection for one week.To establish an immortalized cell line, the catalytic subunit of human telomerase (hne 1773)  and a none 1773) . The trine 1773) ; dl89-97ne 1773) ; and tsAne 1773) ,23. It wne 1773) . The pZine 1773) . AmphotrThe SNP/CGH array (aSNP/aCGH) analysis of the primary tumor and the tumor cell culture from patient Wilms10 was performed using a 2x400K oligonucleotide microarray . With this array format, copy number changes, as well as copy neutral aberrations, such as uniparental disomy (UPD) can be detected on the same array. The samples were prepared and labeled as described by the manufacturer (Protocol Version 7.3 March 2014). To detect uniparental disomy (UPD), a female gender reference DNA  has to be used as control. To quantify the array data the feature extraction module of Agilent's CytoGenomics software (Version 1.5.1.0) was used and, for visualization, CytoGenomics Version 2.0.6.0.WT1 and heterozygous 11p13 deletion, a high-resolution array was designed (http://earray.chem.agilent.com/earray/). The region of the homozygous WT1 alteration was covered with oligonucleotides at a distance of 100 bp, whereas the probes for the heterozygous deletion had a spacing of 300 bp.To determine the breakpoints of the homozygous For the analysis of tsLT and MEST, proteins were extracted from the cells using RIPA buffer ), separated by SDS-PAGE and transferred to a PVDF membrane. The blot was incubated with a monoclonal antibody against SV40 LT (MAb423) or a monoclonal antibody against MEST .TM antibody array was used. Proteins from Wilms10 and imWilms10 cells cultured at 33\u00b0C were extracted and incubated with the antibody arrays as described by the manufacturer (R&D systems).For the simultaneous analysis of the phosphorylation status of 49 tyrosine kinase receptors a ProteomeProfilerPPARG  was used for the analysis of adipogenesis [Adipogenic and osteogenic differentiation was analyzed using reagents from Lonza and as described by the manufacturer. hMSC cells were used as controls in the same experiments. Non-induced control cells were kept in the respective maintenance media. After a 3 week induction and a maintenance phase, cells were processed for adipogenic differentiation by staining with Oil Red O. In addition cells were cultured for 10 days in the induction and maintenance medium for adipogenesis and total RNA  was extracted at day 0 and 10 for Q-RT-PCR analyses. Assay on Demand for ogenesis . ImmortaFor osteogenic differentiation cells were harvested after 19 days of differentiation by scraping them in the presence of 0.5M HCl and a calcium Liquicolor assay was performed according to the manufacturers instructions .Myogenic differentiation was induced with DMEM/F12 (Gibco) supplemented with 2% horse serum for 12 days and HSMM cells were used as controls. For immunofluorescence analysis cells were seeded in four chamber slides (BD Biosciences) and after growth in induction or control medium for 9 days, cells were stained with a Titin-specific antibody as described before .www.bioconductor.org) was used for quantile normalization of microarrays [For gene expression analyses total RNA was isolated from the Wilms10 tumor cell line and the immortalized cells (imWilms10) cultured at 33\u00b0C using the RNeasy Mini Kit (Qiagen). RNA from two biological replicates was analyzed in all gene expression experiments. RNAs were labeled in the One-Color format as described by the manufacturer (Agilent Technologies) and hybridized to 4x44K\u201dWhole Human Genome Oligo Microarrays \" in the presence of \u201cSpike-In positive controls\u201d (Agilent). The microarray scans were quantified using Agilent Feature Extraction Software (V10.1.1.1). Basic statistical analyses were performed within the 'R' statistical computing environment . The R-library 'Limma'  Bioconduroarrays . To compWT1 (Hs00240913_m1) and IGF2 (Hs01005963_m1) with Brilliant II QPCR Master Mix with Low Rox (Agilent Technologies). The expression levels were normalized with RER1 (Hs00199824_m1) RNA. The reason for using RER1 as calibrator was that this gene was basically never regulated in any of our gene expression studies. The Q-RT-PCRs experiments were run on a Mx3000P Sequence Detection System (Stratagene). Statistical significance of the normalized Ct values (\u0394Ct) was assessed by a t-test.For Q-RT-PCR analyses cDNAs were synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems). The Q-RT-PCR experiments were performed in triplicates using the TaqMan gene expression assay probes from Applied Biosystems for WT1 was found in blood or tumor DNA. Multiple-ligation probe analysis  did not identify copy number variations in the blood DNA   and a smaller deletion at 1p31.1 with a maximal size of 203kb (74946777\u201375185022)   . The het5185022) . Further 12.8Mb)  and anot located .DNA from the established tumor cell line (Wilms10) was also analyzed with aCGH/aSNP and the same 11p13 heterozygous/homozygous deletion was identified . A cytogWT1 gene within a heterozygous 11p13 deletion. The derived cell culture harbored the same alterations as the tumor sample.A custom array covering the heterozygous/homozygous deleted 11p13 segment at a high density revealed the start and endpoint position of the heterozygous/homozygous deletions in the tumor and the established Wilms10 tumor cell line . Fig 1F WT1 gene it was of interest to compare the level of WT1 gene expression in the cell lines with the different WT1 mutations versus the Wilms10 cell line. The genetic characteristics of all established cell lines is shown in WT1 mRNA and that the level varies between cell lines with the lowest, almost undetectable level in Wilms1 cells and the highest level in Wilms3 (WT1 in future studies but this is not within the scope of this work.As this is the first cell line with no genomic DNA covering the n Wilms3 . It willCTNNB1 mutation (CTNNB1 mutation identified in the tumor specimen was not seen in the cell culture. As cultured tumor cells have two copies of chromosome 3 (data not shown) and UPD of 3p  enabling their long term passaging. For this purpose an expression construct encoding a novel triple mutant of LT (U19dl89-97tsA58) was used; this mutant T antigen does not bind to ori-like DNA sequences, does not bind BUB1 and is thermolabile [TERT, immortalized cells were readily established and these were termed \"imWilms10\". The cells were cultured so far for more than 30 passages, corresponding to 90 population doublings. Cytogenetic analyses of immortalized cells revealed a normal karyotype (not shown). The immortalized cell line remained cytogenetically normal for many passages, possibly due to the deletion of the amino acids responsible for BUB1 binding [WT1 and the two deletions on chromosome 1 (not shown).All established Wilms tumor derived cell lines from tumors with molabile ,21,23. W binding . aCGH shTERT expression in these cells was verified by RT-PCR  and 39\u00b0C (nonpermissive temperature) and protein was extracted from these cells. Although the tsLT construct encoded a ts mutant protein a low level was detected at 39\u00b0C . Sequency RT-PCR .TERT and tsLT we established gene expression profiles of cells cultivated at 33\u00b0C where both genes are active (permissive temperature for tsLT) using Agilent whole genome microarrays. It was the aim of this experiment to investigate the impact of the expression of these two genes on the transcriptome of Wilms10 tumor derived cells. This information shows whether the \u201cbiology\u201d of immortalized (imWilms10 cells) is affected. However, it should be noted that 33\u00b0C is a nonphysiological temperature for human cells and they grow very slowly at that temperature. First we compared the expression levels of selected genes that are expressed in early kidney development and/or different compartments of the kidney [WT1 mutant cell lines [FOXD1 is typically expressed in stromal cells and also in all WT1 mutant tumor derived cell lines we have established previously. FOXD1 mRNA. Likewise PAX3 is expressed in stromal cells and ectopically in WT with a myogenic histopathology [WT1 mutant cell lines [MEIS1, OSR1, OSR2 and SIX1 [TERT expression ) are down-regulated in imWilms10 cells  and MEST . The strong down-regulation of IGF2 was confirmed by Q-RT-PCR  and an absolute fold change > 1.5. Under these conditions we detected 270 genes with higher and 212 genes with lower expression levels in imWilms10 cells. To gain insight into the function of these genes we used the MetaCore algorithm \u201cenrichment of pathway maps\u201d. The 10 most significant pathway maps are shown in 10 cells . A complQ-RT-PCR  and the nificant . A complHistone H1C, BARD1, CDC7, CCNE1, MCM3 and CCNA1 gene expression levels were strongly induced in imWilms10 cells . A similarity of Wilms tumor derived cell lines with mutant ur group . FurtherWT1 mutant cells have a differentiation potential for these cell fates. Osteogenic differentiation was analyzed by Alizarin red staining and Wilms10 . This indicates that only a few cells have reached a more terminal osteogenic differentiation state and deposit calcium.A number of genes involved in osteogenic, adipogenic and muscle differentiation are expressed at a low level in Wilms10 cells. Therefore, we tested whether parental Wilms10 cells as all other  Wilms10  and imWi Wilms10 . Therefo Wilms10 . This isTo analyze the cells for muscle differentiation we used immunofluorescence with a Titin antibody. Titin is only produced in different muscle cells . ExpressLPL and PPARG [PPARG but not LPL in comparison to uninduced control cells was observed in Wilms10 cells, indicating a limited adipogenic differentiation potential , 37\u00b0C (semipermissive temperature) and 39\u00b0C (nonpermissive temperature). The imWilms10 cells formed large colonies at 37\u00b0 and 39\u00b0C with an efficiency of 77.2% and 80.7% respectively, whereas at 33\u00b0C only 39.3% of the cells formed small colonies . SimilarOur differentiation experiments showed that the parental Wilms10 cells have restricted potentials for muscle, osteogenic and adipogenic differentiation, while these differentiation potentials are still present in imWilms10, they are even further reduced.WT1 nested within a heterozygous 11p13 deletion. The 228kb homozygous deletion only covers the WT1 gene and the 1Mb heterozygous 11p13 deletion extends from WT1 to HIPK3. In addition, UPD restricted to 11p15.2pter and including the IGF2 gene was identified, suggesting a more distal mitotic recombination in 11p15. The high expression of IGF2 in these cells indicates that the paternal allele was duplicated, whereas the rest of chromosome 11 was derived from both parental alleles. We have thus identified a Wilms tumor with a complete lack of WT1 due to a homozygous deletion. It is highly desirable to establish tumor derived cell lines to develop cell culture model systems for Wilms tumors with deleted WT1 genes. Thus our immortalized cell line (imWilms10) represents the first cell culture model system for Wilms tumors with deleted WT1 genes. The immortalized imWilms10 cells were characterized by the expression of selected kidney marker genes, by gene expression profiling, DNA analysis and cell differentiation experiments and were compared to parental Wilms10 cells.We describe here a Wilms tumor (Wilms10) with an unusual tumor specific homozygous deletion of WT1 mutations. These cell lines harbor two genomic DNA copies of mutant WT1, and all of them have two copies of chromosome 11 (unpublished observation). In most cases, the homozygous mutation of WT1 is due to a mitotic recombination event between the last heterozygous marker in 11q11 and the first homozygous marker in 11p12 (unpublished observation). The high expression of IGF2 and low expression of H19 in these cell lines indicates that the paternal allele is duplicated . In one cell line from a WAGR case with a 11p13 deletion on one allele, the remaining copy of WT1 carries a mutation, i.e. the WT1 mutation is hemizygous; both paternal chromosome 11 alleles are present in this case except for the 11p13 deletion. These cell lines express different levels of mutant WT1 mRNAs, whereas Wilms10 cells do not express any WT1 mRNA . This indicates that the tsLT is indeed functionally temperature sensitive. The complete inactivation of the tsLT occurs at 39\u00b0C, but we did not study the cells further at that non-physiological temperature. However, a low level of the tsLT protein was detected by Western blot analysis of imWilms10 cells cultured at 39\u00b0C, suggesting that inactivation of the tsLT protein is due to a conformational effect. Hardy et al., who studied normal human mammary fibroblasts immortalized with telomerase and a tsLT (HMF3A), also detected the tsLT protein at 37\u00b0 and 39\u00b0C by Western blotting . It is iFoxD1 is highly expressed in cortical and nephrogenic interstitium, in the same compartment that expresses Meis1 [Osr1 was shown to identify multi-potent cells of intermediate mesoderm and its expression becomes restricted during kidney development to an Osr1-dependent nephron progenitor compartment [Osr2, was also shown to be expressed during kidney development [Six1 gene plays a role in limb development and in the developing kidney; it is expressed in uninduced metanephric mesenchyme at E10.5 and in induced mesenchyme at E11.5 [WT1 mutant cell lines and Wilms10 cells and are unaltered in imWilms10 cells. This indicates that these WT1 mutant cells may correspond to an early stage of kidney development before specification of the different lineages and the imWilms10 cells have retained these properties. In addition other genes expressed in kidney cells such as SNPO, CD2AP and NES are not changed by the immortalization process.In order to study whether the immortalized cells retain biological properties of the parental Wilms10 cells we first studied the expression of several characteristic kidney marker genes. Laser capture microdissection of major kidney compartments at mouse embryonic day E15.5 and fluorescence-activated cell sorting of component-specific GFP transgenic mice established a kidney atlas of gene expression . FoxD1 ies Meis1 . Expresses Meis1 . Osr1 wapartment . The relelopment . The Sixat E11.5 . Furtherat E11.5 . These cTERT and the tsLT. Another difference is their state of signaling pathways. Paternal Wilms10 cells showed activation of several signaling pathways, i.e. PDGFR, EGFR, IGF and AXL as revealed by proteome profiler antibody arrays. Upon immortalization, these cells show a down-regulation as well as functional inactivation of several enzyme-linked receptors (RTK). In the case of PDGFRA and PDGFRB decreased phosphorylation is linked to a down-regulation of mRNA expression. In contrast, down-regulation of IGF1R phosphorylation was observed with unchanged IGF1R gene expression levels. As phosphorylation of this receptor is triggered by ligand binding, the almost complete down-regulation of IGF2 gene expression in imWilms10 cells might explain this finding. It is interesting that besides IGF2, expression of another fetal growth factor (MEST), is also reduced to basal levels after immortalization. The down-regulation of IGF2 in imWilms10 cells is remarkable in the context of the paternal duplication of chromosome 11p15 and expression of the gene from both alleles. The down-regulation IGF2 and MEST expression occurs at permissive and nonpermissive temperatures and is therefore independent of tsLT. It therefore likely that telomerase is linked to IGF2 and MEST gene down-regulation in imWilms10 cells, as a function of telomerase in overall chromatin structure regulation and epigenetic mechanisms has been described [IGF2 expression [IGF2 expression in immortalized Wilms tumor cells. This observation suggests that the effect of telomerase is highly cell context dependent and that the expression of fetal growth factors in embryonal Wilms tumor cells is not compatible with the establishment of immortalized cells with an unlimited life span. In summary, the immortalized Wilms tumor cells can proliferate despite a down-regulation of ligands and receptors for several signaling pathways. It will be of interest to study the mechanisms that lead to the activation of these different signaling pathways in the parental Wilms tumor cells and the effects of their down-regulation on the growth of these cells. This should be kept in mind when novel therapeutic approaches targeting of one of these signaling pathways are considered, as the others can still be activated [WT1 mutation in the kidney of mice [One major difference between the Wilms10 cells and imWilms10 cells is the up-regulation of cell cycle genes caused by the expression of hescribed . Note thpression , whereasctivated . However of mice . TherefoWT1 mutant cells. The derived immortalized cells have retained these properties although at a reduced capacity, likely due to the fact that they continue to grow at 37\u00b0C, which is not compatible with differentiation. Therefore the parental Wilms tumor cell line with a novel and unusual chromosome 11 alteration and the immortalized derivative with a homozygous deletion of the entire WT1 gene will be useful for further studies on the function of WT1 in Wilms tumor development, to further explore the origin of WT1 mutant tumors as well as for the analysis of activated signaling pathways. Last not least these cells are valuable as negative controls to test various batches of WT1 antibodies, that are known for their unspecific cross reactivity with many proteins.The primary Wilms10 tumor cells show a limited multilineage differentiation potential for osteogenesis, adipogenesis and muscle differentiation, similar to the other S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S14 Fig(PDF)Click here for additional data file.S15 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file."}
+{"text": "The reported high level of mortality among patients both on clinical care and on Highly Active Antiretroviral Therapy (HAART) in Ghana in recent years has been blamed on an advance presentation of AIDS cases due to delay by patients accessing care at Hospitals. The study hypothesized that HIV and AIDS patients report at the ART clinics with very low CD4 lymphocyte count and determined the CD4 lymphocytes count of HIV positive patient at first presentation in the Ashanti Region of Ghana.This retrospective study reviewed clinical records of 2,971 patients accessing care at 19 ART Clinics in the Ashanti region of Ghana for the period January 2010 to December 2012. The date of first reporting and the level of first CD4 counts were recorded. Socio demographic information was also recorded. Data was analyzed using SPSS version 16.More than half  of all HIV patients reviewed presented with CD4 count of less than 250 cells/mm3. Of this number, 37.7% (615/1631) reported with CD4 count less than 50 cell/mm3, 17.3 %(282/1631) with CD4 count of less than 100 cells/mm3, 45.2% (737/1631) with CD4 count of less than 250 cells/mm3. Almost a quarter  presented with CD4 count between 250 and 350cells/mm3 with 21.2% (630/2971) reporting with CD4 count of 350 cells/mm3 and above of which only 11.1% ( 70/630) came with CD4 greater than 500cell/mm3. In all more than three quarter  of the patients reported with CD4 count of less than 350cell/mm3.The study shows that CD4 lymphocytes count of HIV patients accessing care at Hospitals is very low. Delay reporting might account for this development. Public education on the need to access care at the earliest possible time once tested HIV positive must be intensified. Further study is needed to determine the causes for late presenting at the HIV clinic and address them, since this may account for high mortality among HIV positive patients."}
+{"text": "Tumor-infiltrang immune cells such as metastasis-associated macrophages (MAM), regulatory T (Tregreg) cells, and myeloid-derived suppressor cells (MDSC) are reported to promote establishment of the lethal metasta-c foci and restrict efficacy of cytotoxic drugs or tumoricidal immune responses by natural killer (NK) and CD8+ T cells. Recent studies suggest that these pro-tumor immune cells are accumulated a chemokine network established in the tumor microenvironment. Therefore, blockade of these chemokine signals could improve therapeu-c efficacy of chemotherapy and immunotherapy. Metastatic breast cancer is incurable by current therapies including chemotherapy and immunotherapy. Accumulating evidence indicates that tumor-infiltrating macrophages promote establishment of the lethal metastatic foci and contribute to therapeutic resistance. Recent studies suggest that the accumulation of these macrophages is regulated by a chemokine network established in the tumor microenvironment. In this perspective paper, we elaborate on the chemokine signals that can attract monocytes/macrophages to the site of metastasis, and discuss whether inhibition of these chemokine signals can represent a new therapeutic strategy for metastatic breast cancer. The 5-year survival of patients with metastatic disease drops to 21% whereas that of patients with early-stage breast cancer is 89\u2013100% +CD11b +Ly6C\u2013) that is barely found in the normal lung + macrophages reduces the metastatic tumor burden in the lung In breast cancer mouse models, lung metastatic foci show marked accumulation of a distinct macrophage population (F4/80It has been reported that the recruitment of macrophages to the primary site is promoted by various cytokines and chemokines such as colony stimulating factor-1 (CSF-1), vascular endothelial growth factor (VEGF) and CC-chemokine ligand 2 (CCL2), although the mechanisms underlying macrophage accumulation in the metastasis sites are still largely unknown. We have recently reported that the accumulation of MAMs at the metastatic lung is regulated by chemokine ligands CCL2 and CCL3 and their respective receptors CCR2 and CCR1 2+Ly6Chigh) to the tumor challenged lung. These results indicate that the CCL2-CCR2 signal recruits circulating IMs to the site of metastasis where they differentiate into MAMs and promote establishment of metastatic foci. We have further found that the MAMs isolated from the mouse lung with metastatic foci express much higher level of CCL3 compared with circulating IMs It has been reported that high levels of CCL2 in breast cancer specimens correlate with high number of macrophages in the primary tumors In our breast cancer model, another CCL3 receptor CCR5 is not necessary for the early MAM accumulation observed within 24\u00a0h after tumor injection. However, it is reported that CCR5 is required for macrophage accumulation in the lung foci after 7 days of renal cancer cell injection 3The ultimate objective of macrophage-targeting therapy is withdrawal of tumor-supporting and immunosuppressive microenvironment from the secondary sites by disrupting accumulation and/or function of MAMs. Accordingly, the above-mentioned chemokine signaling molecules are potential targets for the treatment of metastatic breast cancer.Ccl3 expression in MAMs as well as their recruitment following mammary tumor metastasis Results from our breast cancer metastasis model suggest that the inhibition of CCL3 secretion from MAMs is one of the possible strategies as they are a major source of CCL3 among other leukocytes such as neutrophils, T, B, and NK cells in the metastatic lung Ccr1Ccr1\u2212/\u2212\u2212/\u2212\u2212/\u2212\u2212/\u2212 mice are healthy without any overt hematopoietic abnormalities unless challenged with specific pathogens Ccr2Ccr2\u2212/\u2212\u2212/\u2212\u2212/\u2212\u2212/\u2212 mice show a reduced number of circulating monocytes Another possible strategy to suppress MAM accumulation is blockade of CCR1 and CCR2. Several companies have developed small molecule inhibitors against CCR1 or CCR2 for rheumatoid arthritis or multiple sclerosis, and most of them are well tolerated and show no adverse effects Ccr1Ccr1Ccr1 and Ccr2Ccr2Ccr2 deficiency can achieve complete elimination of metastatic tumors in the mouse model (maximum reduction rate was 60%) and model dependent reg) cells that are recruited via different chemokine signals reg cells migrate towards primary ovarian tumors via CCL22-CCR4 signal reg cells respectively Despite this optimism a treatment with single chemokine antagonist will almost certainly not be enough to suppress metastatic tumor growth since neither + T cell responses + monocytes (CD11b+Ly6C+) enhances accumulation of adoptively transferred CD8+ T cells in the primary tumor and thereby augments therapeutic effect of the adoptive T-cell therapy on the tumor growth in a melanoma model Although breast cancer cells also express chemokine receptors including CCR5, CCR7 and CXCR4 that enhance tumor cell invasiveness and metastasis 4reg cells. Interestingly, mouse breast cancer cells used in our metastasis model can promote CCL5 expression in cultured macrophages (unpublished data). Furthermore, GM-CSF produced by human breast cancer cell lines induces CCL18 secretion from cultured macrophages reg cells (Our data suggest that a gradient of CCL2 attracts monocytes/macrophages towards the metastatic tumor microenvironment where they are exposed to high levels of CCL2 and are prompted to secrete another chemokine CCL3. As mentioned above, such a chemokine-induced chemokine secretion is also reported in human monocyte culture systems, i.e., exposure to CCL5 or CCL18 induce secretion of CCL2, CCL3, CCL22, and CXCL8 eg cells . Since tDr. Pollard has patents pending on targeting CCR1 at metastatic sites."}
+{"text": "This paper is aimed to explore the significance of plasma kisspeptin level in diagnosis and therapeutic evaluation through the detection of kisspeption level of girls diagnosed with idiopathic central precocious puberty (ICPP) before treatment and after 6-months of treatment and girls with simple premature thelarche (PT).A total of 70 girls including 24 girls diagnosed with ICPP, 21 girls with PT and 25 normal girls were enrolled. ELISA was adopted to detect plasma kisspeptin level.The kisspeptin level of ICPP group before treatment (1.80\u00b10.13ng/ml)was higher than those of other groups with significantly statistic difference. The kisspeptin level of ICPP group after 6-months of treatment (1.49\u00b10.21ng/ml) was significantly lower than those before treatment (P<0.05).We can conclude that plasma kisspeptin level is related with initiation of pubertal development, and it can be served as important parameter in ICPP diagnosis and therapeutic effect evaluation."}
+{"text": "It was reported recently that male mice lacking brain serotonin (5-HT) lose their preference for females , suggesting a role for 5-HT signaling in sexual preference. Regulation of sex preference by 5-HT lies outside of the well established roles in this behavior established for the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Presently, mice with a null mutation in the gene for tryptophan hydroxylase 2 (TPH2), which are depleted of brain 5-HT, were tested for sexual preference. When presented with inanimate  or animate  sexual stimuli, TPH2-/- males show a clear preference for female over male stimuli. When a TPH2-/- male is offered the simultaneous choice between an estrous female and a male mouse, no sexual preference is expressed. However, when confounding behaviors that are seen among 3 mice in the same cage are controlled, TPH2-/- mice, like their TPH2+/+ counterparts, express a clear preference for female mice. Female TPH2-/- mice are preferred by males over TPH2+/+ females but this does not lead to increased pregnancy success. In fact, if one or both partners in a mating pair are TPH2-/- in genotype, pregnancy success rates are significantly decreased. Finally, expression of the VNO-specific cation channel TRPC2 and of CNGA2 in the MOE of TPH2-/- mice is normal, consistent with behavioral findings that sexual preference of TPH2-/- males for females is intact. In conclusion, 5-HT signaling in brain does not determine sexual preference in male mice. The use of pharmacological agents that are non-selective for the 5-HT neuronal system and that have serious adverse effects may have contributed historically to the stance that 5-HT regulates sexual behavior, including sex partner preference. Lmx1b) or tryptophan hydroxylase 2 (TPH2), respectively, suggested for the first time that 5-HT regulates sexual preference [Lmx1b-/- and TPH2-/- mice lost their preference for mice of the opposite sex and this preference could be reinstated by pharmacological replenishment of 5-HT.Serotonin 5-HT) axons innervate virtually all areas of the nervous system from the B1\u2013B9 clusters of neuronal cell bodies located in the mesencenphalon . In its -HT axonseference . In theseference  Lmx1b-/-Interest in TPH2 was reinvigorated when it was discovered that this enzyme existed in two distinct forms . TPH wasIn the course of developing and maintaining our colony of TPH2-/- mice we have noted, apart from occasional bouts of aggression, relatively normal sexual approach and courtship behavior in mating pairs of TPH2-/- mice. The results of studies suggesting that TPH2-/- mice lose their sexual preference ,19 prompad libitum. All mice were socialized , sexually na\u00efve and between 8\u201314 weeks of age. All behavioral tests were observed and recorded by two independent observers. The Institutional Animal Care and Use Committee of Wayne State University approved the animal care and experimental procedures (Permit Number: A3310\u201301) and all procedures are in compliance with the NIH Guide for the Care and Use of Laboratory Animals.TPH2\u2212/\u2212 mice were generated by deleting exon 1 of the Tph2 gene as described  and wereFilter paper (15.5 \u00d7 26.5 cm) was cut to cover the entire bottom of a small mouse cage (16 \u00d7 27 \u00d7 13 cm). Urine was collected from TPH2+/+ males and TPH2+/+ and TPH2\u2212/\u2212 female mice in estrus. Estrus was confirmed by microscopic examination of vaginal smears and is characterized by clusters of cornified squamous epithelial cells . In ordeSexual interactions between 2 mice. This test was performed to determine male interactions with a single, receptive female. Male mice of either genotype were placed individually into a small test cage (16 \u00d7 27 \u00d7 13 cm) for 30 minutes and immediately thereafter, a TPH2+/+ or TPH2\u2212/\u2212 female in estrus was placed into the test cage with the male. The latency to mount and the number of mounts, attacks and intromissions were counted in a 15-min test period.Sexual interactions among 3 mice. This test aimed to determine whether male mice express a sexual preference when presented simultaneously with a choice between a single male and a single receptive female mouse or a choice between two receptive female mice. Male TPH2+/+ or TPH2-/- mice  were placed individually into a small test cage (16 \u00d7 27 \u00d713 cm) for 30 minutes and then two intruder mice were introduced into the cage simultaneously. In one format, the intruder mice were a male TPH2+/+ mouse and a female TPH2+/+ mouse in estrus. In a second format, the intruder mice were a receptive female TPH2+/+ mouse and a receptive female TPH2-/- mouse. The latency and number of mounts, attacks and intromissions were scored for a 15-min period. In order to differentiate between TPH2+/+ and TPH2\u2212/\u2212 female intruder mice, tails were coded distinctly with a permanent marker. Because of the complexity of the interactions among 3 mice in the same cage simultaneously , we modified the test to restrict access of the resident mouse with the intruder mice by placing the intruders in wire cups as used in the three chamber social approach test in the study of autism-like behaviors in rodents [ rodents ,44,45. TFemales were housed for 21 days with males in the following genotype combinations, with the female of the pairing listed first: TPH2+/+ x TPH2+/+; TPH2+/- x TPH2+/+, TPH2+/- or TPH2-/-, and TPH2-/- x TPH2+/- or TPH2-/-. Matings that would produce litters that were 100% heterozygous were not carried out. We had no experimental plan for the use of excess numbers of TPH2+/- mice, so these matings were not justifiable from the perspective of animal use guidelines. At the end of this two week period, males were removed and from this time forward, females remained singly housed until giving birth or until the passage of an additional 21 days . A mating was scored as fertile only when a litter was born.Male mice were sacrificed by decapitation and the VNO and MOE were dissected and stored frozen at \u221280\u00b0C until assayed. Frozen tissue was disrupted by sonication in 1% SDS at 95\u00b0C and insoluble material was sedimented by centrifugation. Protein was determined by the bicinchoninic acid method and equal amounts of protein (70 \u03bcg/lane) were resolved by SDS-polyacrylamide gel electrophoresis and then electroblotted to nitrocellulose. Blots were blocked in Odyssey Infrared Imaging System blocking buffer  for 1 h at room temperature. Primary antibodies against TRPC2 , CNGA2  or GAPDH  were added to blots and allowed to incubate for 16 h at 4\u00b0C. Blots were washed 3X in Tris-buffered saline with 0.1% Tween 20 and once with 1X PBS to remove unreacted antibodies and then incubated with fluorescent IRDye anti-IgG secondary antibodies (1:4000) in the dark for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence and the relative densities of TRPC2-, CNGA2- and GAPDH-reactive bands were determined by imaging with an CLx Odyssey Image Station  and quantified using ImageJ software (NIH). Expression levels of TRPC2 and CNGA2 were normalized to GAPDH levels.www.graphpad.com.Data from the urine preference test and sexual preference behavior when using 3 mice simultaneously were analyzed by two-way ANOVA and post hoc comparisons were carried out using Bonferroni\u2019s multiple comparison test. Data from sexual preference behavior in one-on-one pairings were carried out using one-way ANOVA and post hoc comparisons were made using Tukey\u2019s multiple comparison test. Tests of mating success among pairings of genotypes were analyzed using a two-sided Fisher\u2019s Exact test. Student\u2019s t-tests were performed to analyze the normalized levels of TRPC2 and CNGA2 expression. Values of p < 0.05 were deemed statistically significant. All statistical analyses were carried out using GraphPad Prism version 6.02 for Windows, GraphPad Software, San Diego, CA, 2,58 = 53.63, p < 0.0001). An effect of male genotype was not present and the urine scent x genotype interaction was likewise not significant. Post hoc comparisons showed a significant preference by TPH2+/+ and TPH2-/- males for the urine scent of either female genotype over that of TPH2+/+ male urine (p < 0.0001 for each). When males of either genotype were given the choice of urine scents from female TPH2+/+ versus female TPH2-/-, a significant preference  was seen such that both TPH2+/+ males and TPH2-/- males displayed a significant preference for the urine scent of TPH2-/- females over that of TPH2+/+ females (p < 0.05 for both). These data show that male TPH2-/- and TPH+/+ mice have the same preference for females when given a choice of male and female inanimate sexual stimuli. Males of both genotypes also show a significant preference for urine scents of TPH2-/- females over scents from TPH2+/+ females.TPH2+/+ and TPH2-/- male mice were exposed to urine samples from TPH2+/+ males versus urine from either a TPH2+/+ or a TPH2-/- female. The results in 3,35 = 8.60, p < 0.0002) for mounting TPH2-/- females over TPH2+/+ females was shown by both TPH2+/+ (p < 0.05) and TPH2-/- (p < 0.01) males. TPH2+/+ and TPH2-/- males did not differ from each other in the number of mounts when paired with a female of either genotype. The latency to mount did not vary significantly in any of these pairings . Male TPH2-/- mice had significantly more intromissions with TPH2-/- females by comparison to TPH2+/+ females (p < 0.05). While TPH2+/+ males showed a trend toward more intromissions with TPH2-/- females versus TPH2+/+ females, this difference did not reach significance. As seen for mounts, TPH2+/+ males did not differ from TPH2-/- males in the number of intromissions with females of either genotype. We carried out male-on-male pairings but found that sexually-directed behaviors such as mounting and intromissions were completely displaced by overt aggression on the part of TPH2-/- males toward their cage partner , so it was not possible to determine male sexual preference in testes-intact TPH2-/- males. Future studies will carry out male-on-male pairings in castrated TPH2-/- mice in an attempt to reduce aggressive behavior [The results from pairings of individual male and female mice are presented in pairings . The numbehavior ,47.1,36 = 17.30, p = 0.0002) and resident male  were significant for the number of mounts. The intruder genotype x resident genotype interaction was not significant. TPH2+/+ resident males mounted the TPH2-/- female significantly more than the TPH2+/+ female (p < 0.01), and while TPH2-/- males showed a trend to greater numbers of mounts on the TPH2-/- female versus the TPH2+/+ female, this effect did not reach statistical significance. Using the approach of Liu and colleagues [We followed the approach of Liu and colleagues  to deter2,40 = 63.40, p < 0.0001). The interaction between intruder x resident genotype was also highly significant . When placed into a cage containing male and female TPH2+/+ intruders within wire cups, male TPH2+/+ (p < 0.0001) and TPH2-/- mice (p < 0.05) showed a significant preference for investigating female TPH2+/+ mice over TPH2+/+ males. Because of the complexity of the interactions among 3 mice in the same cage simultaneously, we modified the test to restrict access of the intruder mice to the resident mouse by placing the intruders in wire cups ,44,45. TIn light of the data above showing that TPH2-/- females in estrus seemed to be preferred over TPH2+/+ females by males of both genotypes Figs.  and 2, wA null mutation in the gene for TRPC2 ,48 or sulmx1b knockout) or with a null mutation in the gene for TPH2 lose their preference for females [The results presented in this paper show clearly that male mice with a genetic depletion of 5-HT retain their preference for females over males and stand in contrast to a recent study reporting that male mice with significant reductions in the number of 5-HT neurons in brain  or when comparing sexually-directed behaviors in one-on-one pairings of mice  is straightforward. In these cases, we observed that TPH2-/- males express a preference for females over males to the same extent as TPH2+/+ males. However, when examining sex preference by offering a resident male the simultaneous choice of an intruder male and an intruder female, this grouping of 3 mice is extremely complex and involves far more than the resident male solely expressing a sex choice between the two intruder mice. The intruder male and female mice in this paradigm are also faced with several active choices and do not remain passive in the cage while the resident male makes a choice between them. Certainly, the resident male approaches and eventually mounts the female or the male intruder, or both. We frequently observed that the intruder male will also mount the intruder female while the resident male stands by. If the sexual advances of the resident or intruder male are rejected by the intruder female, it often provokes an attack by the rejected male on the other male. Finally, the resident male, especially TPH2-/- males, attack the intruder male and female without obvious provocation as seen in the resident-intruder test of aggressive behavior ,41. For The pharmacological tools used to study the role of 5-HT in determining sexual preference and activity are limited in their specificity and may have contributed to the confusing state of affairs in this area for several reasons. First, early studies showing that reductions in brain 5-HT led to increased sexual activity depended on the use of extremely high doses of the TPH2 inhibitor p-chlorophenylalanine (pCPA). This inhibitor indeed lowers brain 5-HT levels but it also leads to large depletions of dopamine and norepinephrine . pCPA alMice lacking the gene for TPH2 are viable and fertile and, for the most part, have normal morphological and physiological characteristics ,24,26,27In summary, we conclude that a genetic depletion of 5-HT from brain does not have an effect on sexual preference or activity. Previous studies that have implicated 5-HT signaling in these behaviors used pharmacological approaches  that lack specificity for the 5-HT neuronal system and which have numerous, serious adverse effects, both of which make it difficult to attribute observed changes in behavior to 5-HT. The use of inanimate sexual stimuli (urine scents) and pairings between single male and female mice showed clearly that TPH2-/- males prefer females over males. This preference appeared to be lost when TPH2-/- and TPH2+/+ males were given the choice of interacting with a male or female intruder mouse simultaneously. When competing, non-sexually related behaviors that were displayed among 3 interacting mice were controlled by placing the two intruder mice in wire cups, the preference of male TPH2-/- mice for females was affirmed. TPH2-/- females were preferred over TPH2+/+ females in most tests but this effect did not transfer into fruitful matings in TPH2-/- mice. Finally, the finding of normal expression of TRPC2 in the VNO and of CNGA2 in the MOE of TPH2-/- males alone supports the conclusion that the sexual preference of TPH2-/- males for females over males is intact."}
+{"text": "Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection . The DBL\u03b1 domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Extensive var gene diversity (DBL\u03b1 domain of PfEMP1) is an important aspect of pathogenesis in parasite lines. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind DBL\u03b1 domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBL\u03b1 domain and mechanism behind heparin-mediated rosette inhibition have not been elucidated.The variant surface antigen PfEMP1 (3D structures and epitopes of DBL\u03b1 domain in 3D7 and in two parasite isolates have been predicted and compared. Docking studies on DBL\u03b1 domains with human GAG receptors (heparin and heparan sulphate) to predict the strength of association between the protein\u2013ligand interactions were performed.The DBL\u03b1 domain structures showed extensive diversity and polymorphism in their binding sites. The linear as well as conformational epitope prediction analysis predicted several B cell epitopes at variable positions in different DBL\u03b1 domain variants . The docThus such an analysis may lead to the development of novel interference strategies to block red blood cell invasion and provide protection against malaria and provide an insight into the complexities of host parasite interactions."}
+{"text": "Recently, we reported that there is significant low field relaxation dispersion in myocardial and fibrotic scar tissue during in vivo T1rho MRI, contributing to contrast enhancement compared to spin echo (T2) MRI. However, we also observed that differences in magnetic field susceptibility between the heart and lung organs and poor RF penetration and field homogeneity in large patients and at 3 T contribute to T1rho quantification errors. Our goal was to determine the influence of static (B0 and B1 field heterogeneity influences T1rho MRI. Tilted rotating frame (TRF) Bloch simulations were performed for a rotary-echo spin lock, varying field heterogeneity and spin lock duration  with random Gaussian noise . Phantom experiments were performed using a homogeneous H2O cylinder (MnCl2 = 0.016%). In vivo experiments were performed in Yorkshire swine (n = 5). Simulated phantom and in vivo images were derived from B0 and B1 maps using the TRF Bloch simulations and were correlated with experimental images at the same spin lock duration.We performed simulations and experiments to explore how B0 field and in the 50 mT/m field gradient in which the B0 field was less than 50 Hz off-resonance. Noticeable variations were observed at 100 Hz off-resonance (100 mT/m gradient). The B0 field in vivo is much less homogeneous than that for the phantom; therefore the T1rho images show much more complex patterns  could result in nutation about an effective field oriented away from the idealized spin lock axis. In vivo results correlated well with simulations and phantoms experiments. Measured T1rho relaxation times in the three identical compartments were constant in the homogeneous Bs Figure . Mean B0Spin lock artifacts were the result of static field heterogeneity predominantly confined to the left ventricular posterolateral wall. Static field heterogeneity should be reduced to less than 10% of the spin lock field amplitude to minimize T1rho quantification errors.The authors gratefully acknowledge support from the National Institutes of Health through awards K99HL108157 and R01HL63904."}
+{"text": "The aims of this study were to evaluate the prevalence of Dog Erythrocyte Antigens (DEA) 1, 4, and 7 in Ibizan hounds, to compare the results with the prevalence of DEA in Spanish greyhounds, and to determine the risk of sensitization following the first transfusion of blood not typed for DEA 1 and the probability of an acute hemolytic reaction following a second incompatible transfusion using untyped DEA 1 blood. DEA 1, 4, and 7 status was determined in 92 Ibizan hounds. Results were compared with the previously reported prevalence in Spanish greyhounds. The risks of sensitization and of a hemolytic transfusion reaction were determined amongst Ibizan hounds and between Ibizan hounds and Spanish greyhounds. The prevalence of DEA 1, 4, and 7 was 75%, 98.9%, and 25%, respectively. There was a significantly higher expression of DEA 1 and 7 in Ibizan hounds than in Spanish greyhounds. The probability of sensitization of a recipient dog to DEA 1 with transfusions amongst Ibizan hounds was 18.5% and between Ibizan hounds and Spanish greyhounds was 13.7%. The probability of an acute hemolytic reaction in each group was 3.5% and 1.9%, respectively. There is a higher prevalence of DEA 1 and 7 in Ibizan hounds than in other sighthounds. There is international standardization of seven canine blood groups as categorized by presence of Dog Erythrocyte Antigen (DEA) 1, 3, 4, 5, 6, 7, and 8 , but nonDEA 1 and 7 are the most important blood types with regard to canine blood transfusions. DEA 1 antigen has recently been classified as a unique DEA epitope with variable surface expression   and is pDEA 7 antigens are reported to occur naturally in between 40 and 72% of the canine population , 15, 16.There is also a high prevalence of DEA 4 antigens 98\u2013100%) in the canine population \u2013100% in  and a heAn ideal blood donor does not have blood antigens of types that commonly cause reactions in unmatched recipients. There is no universally agreed definition of a universal canine donor. The most restrictive definition of the universal canine donor would be a dog negative for DEA 1, DEA 3, DEA 5, and DEA 7 and positive for DEA 4. Since 98% of dogs are positive for DEA 4, the rarity of DEA 4 negative dogs means that this antigen is unlikely to influence donor selection. Some transfusion specialists do not exclude DEA 7 positive dogs from the donor pool. In fact the concept of the universal donor in dogs has long been debated since the tests for DEA 3 and DEA 5 antigens are often not available and the quality of these tests is variable; currently typing sera are available only for DEA 1, 4, and 7 antigens. As previously proposed , univershttp://www.fci.be/en/nomenclature/IBIZAN-PODENCO-89.html).Ibizan hounds are medium-size sighthounds originating from the island of Ibiza. They are traditionally used in the Balearic Islands (and less so in Spain and France) to hunt rabbits and other small game. In recent times this breed has been disseminated worldwide through a variety of adoption programs. The Ibizan hound is classified by the F\u00e9d\u00e9ration Cynologique Internationale (FCI) in Group 5 (spitz and primitive types) and in section primitive type-hunting dogs. This dog is a typical and robust representative of one of the oldest breeds still in existence (The aims of this study were threefold: (1) to evaluate the prevalence of DEA 1, DEA 4, and DEA 7 blood type in Ibizan hounds; (2) to compare the results with the previously published prevalence of DEAs in other sighthounds ; (3) to determine the risk of recipient sensitization following the first transfusion of blood not typed for DEA 1 and the probability an acute hemolytic reaction following a second incompatible transfusion of blood untyped for DEA 1 both amongst Ibizan hounds and between Ibizan hounds and Spanish greyhounds.In this prospective study EDTA-anticoagulated blood samples were collected from 92 healthy owned Ibizan hounds in January 2015. Dogs were between 1-year and 3-year old, 89 were female (96.7%) and 3 were male (3.3%), and all were living on Ibiza Island, Spain.With the owners' consent cephalic blood samples were collected with a 23\u2009G needle  connected to a 2\u2009mL syringe . They were transferred to a 1\u2009mL EDTA-anticoagulated tube and were stored between 4 and 5\u00b0C. All blood samples were collected as part of a study to determine the distribution of blood groups in this breed. This study was conducted according to European legislation (2010/63/EU). In all samples blood typing was performed as described below.DEA 1 status was determined using a commercially available card agglutination technique  according to the manufacturer's instructions and as previously described , 20. TheAnalysis for DEA 4 and 7 antigens was performed by gel column agglutination within microtubes  as previously described  using poAnti-DEA 4 and 7 antibodies were imported and used in this study with the authorization of Italian Health Minister (protocol authorization number 0021278-15/10/2014-DGSAF-DGSAF-P). Blood-typing was performed at the Veterinary Transfusion Unit (REV) of the University of Milan, Milan, Italy.\u03bcL of a 0.8% RBC suspension made by suspending 10\u2009\u03bcL of the RBC pellet in 1\u2009mL of low ionic strength solution  was mixed with 25\u2009\u03bcL of DEA 7 antibodies or with 15\u2009\u03bcL of DEA 4 antibodies in the reaction chamber of saline gel columns. For all samples a negative control column with saline solution was included. The gel columns were incubated at 4\u00b0C for 30 minutes and were then centrifuged in a special gel column card centrifuge  at 80 \u00d7 g for 10 minutes. Finally the gel column cards were evaluated for presence and strength of agglutination. Only results validated by negative controls were included in analysis.Briefly 25\u2009The cards were visually interpreted as follows: (0) negative, when all RBCs were at the bottom of the column; 1+, when very few RBC agglutinates were dispersed in the lower part of the gel, with most RBCs at the bottom of the tube; 2+, when all RBCs were agglutinated and dispersed in the gel; 3+, when some RBC agglutinates were dispersed in the upper part of the gel and most of the RBCs formed a red line on the surface of the gel; and 4+, when all RBCs formed a red line on top of the gel. Results were interpreted as negative if no agglutination or 1+ agglutination was present, whereas \u22652+ agglutination reactions were considered positive.The probability of an Ibizan hound becoming sensitized following the first transfusion of blood that was neither cross-matched nor typed for DEA 1 was calculated using the following formula , 10:(1)%The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using untyped blood from any other dog was calculated using the following formula : (2)%\u2009\u2009DResults were analysed by absolute prevalence analysis. Using contingency tables or Fisher's exact test the prevalence of DEA 1, 4, and 7 and universal donors in Ibizan hounds calculated in this study was compared with a population of 205 Spanish greyhounds  in which the prevalence of DEA 1, 4, and 7 and universal donors  was 54.6%, 100%, 8%, and 46.7%, respectively .P < 0.05.All statistical analysis was performed using statistical software  with significance set at For DEA 1 blood type, 69 (75%) dogs tested positive and 23 (25%) tested negative. For DEA 4 blood type, 91 (98.9%) dogs tested positive  and 1 (1.1%) dog tested negative. For DEA 7 blood type, 23 (25%) dogs tested positive (16 samples showed 3+ agglutination and 7 samples showed 2+ agglutination) and 69 (75%) were negative (no samples showed agglutination). Of the 92 dogs, 16 (17.4%) were DEA 4 positive only  and 1 (1.1%) dog was negative for all DEA. All control samples in the gel columns tested negative (absence of agglutination).P = 0.005 and P = 0.002, resp.). The prevalence of universal donors (DEA 1 and DEA 7 negative and DEA 4 positive or negative) was lower in Ibizan hounds (P = 0.000008) than in Spanish greyhounds. There was no significant difference in DEA 4 prevalence between the 2 breeds.Ibizan hounds had a significantly higher prevalence of DEA 1 and DEA 7 positivity than Spanish greyhounds (The probability of an Ibizan hound recipient becoming sensitized following the first transfusion of blood from an Ibizan hound donor that was not cross-matched nor typed for DEA 1 was approximately 1 in 5 (18.8%). The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using blood untyped for DEA 1 from any other Ibizan hound was 3.5%.The probability of a recipient Ibizan hound becoming sensitized following the first transfusion of blood from a Spanish greyhound that was not cross-matched nor typed for DEA 1 was approximately 1 in 7 (13.7%). The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using untyped DEA 1 blood from Spanish greyhound was 1.9%.There are breed and geographical differences in the prevalence of different blood group antigens , 15. It In this study the prevalence of DEA 1 expression in Ibizan hounds was 75%, which is higher than previously reported in other pure breeds , 15, 16 All 92 Ibizan hounds were blood typed and only one was DEA 4 negative. This prevalence of DEA 4 expression (98.9%) is in agreement with the prevalence in the general canine population , 21.The prevalence of DEA 7-positive dogs in the study population (25%) was similar to that reported in Golden Retrievers in USA and Brazil (25% and 27% resp.)  but highThe prevalence of universal or \u201cideal\u201d donors, that is, dogs positive only for DEA 4 or negative for all DEA, in our population of Ibizan hounds, was 18.5%. This is lower than the prevalence of 57.3% found in greyhounds  and in aIn this study the probability of a recipient becoming sensitized and produced antibodies against DEA 1 following the first transfusion of blood that was neither cross-matched nor typed for DEA 1 was 18.8% 1 in 5) amongst Ibizan hounds and 13.7% (1 in 7) between Ibizan hounds and Spanish Greyhounds. These probabilities were lower than previously reported in dogs from Portugal , in Span amongst The gel agglutination technique has been used for decades. This test is sensitive for the detection of DEA 1 and is suited for screening blood donors in a blood bank program , 20. ThiAnother limitation of this study was that the study population of Ibizan hounds was almost exclusively female, mirroring the environment in which Ibizan hounds live. Hunters run these dogs in mostly female packs, as the female is considered the better hunter. In addition we did not know how closely related the dogs used in this study were. It is possible that a closed population of closely related individuals could bias the prevalence of blood types. Finally DEA 3, DEA 5, and Dal blood types were not tested since relevant antisera were unavailable at the time the study was performed.The population of Ibizan hounds studied here showed a different prevalence for DEA 1 and 7 with respect to previous reports of other sighthounds. Although the risk of sensitization and an acute transfusion reaction following incompatible blood transfusion is low, it remains best practice to blood type and cross-match recipients before transfusion."}
+{"text": "This study was conducted to analyze the changes of gait parameters between two different footwear size groups due to change of angle between heel and sole of the footwear by keeping the same heel height for both groups.A cross-sectional descriptive study design.The effects of the angle between the sole and the heel of heeled footwear on single and double support time, stride duration and toe off plantar flexion of females.SD = 2.5) and forty nine subjects to the footwear size 40 group with 24.4 (SD = 2.4) years mean age. Both groups were given similar type three centimeters height heeled footwear and there was 6 greater angle locate between heel and sole of size 36 footwear than size 40.Two cameras  were used to capture the walking trials and each individual perform three trials. Captured two dimensional walking trials were analyzed with Motion view 8.0 video analysis software. Independent sample t-test in Statistical Package for Social Sciences Statistics version 17.0 (SPSS Statistics 17.0) was used to analyze the data.Hundred (100) female subjects were participated for the study and fifty one (51) subjects were included to the footwear size 36 group with 23.8years mean age (p<0.05) while the single support time and stride duration was not shown a significance (p>0.05).Mean toe off phase plantar flexion and double support time was shown a significant difference (The angle between heal and sole of footwear significantly deviate gait parameters so rather than heel height footwear type it need to consider about shoe length."}
+{"text": "Her2 FISH-amplified cases showed an increasing trend significantly through their corresponding HER2 IHC ordinals by the 2007 and the 2013 criteria, respectively . After excluding equivocal cases, the specificity (100%) and positive predictive value (100%) were unchanged under either the 2007 or the 2013 criteria. The sensitivity (100%), negative predictive value (NPV) (100%), and accuracy (100%) of HER2 IHC were higher under the 2013 criteria than those  under the 2007 criteria. Of the total 49 cases, the number (n\u200a=\u200a4) of HER2 IHC equivocal results under the 2013 criteria was 4-fold higher than that (n\u200a=\u200a1) under the 2007 criteria (8.16% vs 2.04%). Conclusively, if first tested by IHC, the 2013 criteria caused more equivocal HER2 IHC cases to be referred to Her2 FISH testing than the 2007 criteria. That decreased the false-negative rate of HER2 status and increased the detection rates of HER2 positivity in mucinous EOC.The remarkable success of trastuzumab and other newly developed anti-HER2  therapies in breast, gastric, or gastroesophageal junction cancer patients has supported us to investigate the HER2 status and its possible therapeutic implication in mucinous epithelial ovarian cancer (EOC). However, there is currently no standardization of HER2 scoring criteria in mucinous EOC. In this study, we aimed to compare both the assay performance characteristics of the 2007 and the 2013 American Society for Clinical Oncology and College of American Pathologists scoring methods. Forty-nine tissue microarray samples of mucinous EOC from Asian women were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) tests using the 2007 and the 2013 criteria, respectively. The overall concordance between IHC and FISH by the 2007 criteria was 97.92 % (kappa\u200a=\u200a0.921), and that by the 2013 criteria was 100% (kappa\u200a=\u200a1.000). The percentage of  The possible mechanisms of carcinogenesis include activation/amplification of oncogene, inactivation of tumor suppressor genes, inhibition of apoptosis, angiogenesis, and so on. Oncogene amplification causes oncoprotein overexpression and promotes tumor growth. HER2 positivity, in which the HER2 receptor is either overexpressed in the protein stage and/or amplified at the genomic level, has accounted for approximately 20% to 30% of breast cancers and 18% to 35% of mucinous EOCs.9After carefully excluding metastatic mucinous carcinoma and borderline tumors, primary mucinous epithelial ovarian cancer (EOC) makes up approximately 2% to 4% of all ovarian epithelial carcinomas.11 However, there is, so far, no consensus in defining the HER2 positivity in mucinous EOC.12The success experiences of trastuzumab therapy in breast cancer, gastric, or gastroesophageal junction (GEJ) cancer patients and newly developed anti-HER2 drugs encouraged the investigation of anti-HER2 therapy application in other cancers, including mucinous EOC.14 Even though pathology communities adopted the 2007 ASCO/CAP algorithms previously, they will soon be familiar with the new 2013 modified rules all over the world. In this study, we aimed to compare both HER2 assay performance characteristics in mucinous EOC using the 2007 and 2013 ASCO/CAP scoring criteria, respectively.Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are still widely used in assessing the HER2 status of clinical specimens. The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) proposed original guidelines to test the HER2 status in breast cancers in 2007 and amended those guidelines in 2013 after concerns were raised about false-positive and false-negative HER2 assessments.9 All the experimental samples used in this study were de-linked from direct patient identifiers, and the research was conducted according to International Conference on Harmonization guidelines and complied with all applicable regulations for protection of human subjects of research, including review and approval by the Institutional Review Board, Chung-Shan Medical University Hospital, Taichung, Taiwan.The study materials consisted of 49 cases of mucinous EOC; the characteristics of the TMA derivation were described in our previous report.The HER2 immunostains were performed on the fully automated Ventana Benchmark XT autostainer using pathway antiHER2/neu rabbit monoclonal antibody . HER2 IHC score 3+ breast cancer was used as a positive control. Negative controls were obtained by excluding the primary antibody. The slides were mounted with Permount for microscopic examination, and the images were captured by the NIKON ECLIPSE 50i microscope and NIKON DS-Fi1 Digital Camera System for study comparison.Her2 DNA Probe Kit protocol . The dual-color FISH consisting of 2 labeled DNA probes was performed on sections cut from the same tissue microarray (TMA) blocks. The LSI HER2 probe that spans the entire Her2 gene was labeled in Spectrum Orange, and the CEP17  probe was labeled in SpectrumGreen and hybridized to the alpha satellite DNA located at the centromere of chromosome-17 (17p11.1\u2013q11.1). Counting 2 separate fields of at least 20 cells was essential. We calculated the Her2: CEP17 signal ratio by recording the numbers of Her2 gene (red) and chromosome 17 (green) signals from preselected tumor areas. In most cases, tumor cells from matching sites of IHC analysis were scored for the number of red (Her2) and green (CEP17) signals. Signal photos were taken with the NIKON ECLIPSE 80i fluorescent microscope with a PlanFluor oil objective (100\u00d7) using a double band-pass filter that permitted simultaneous green and red colors.The FISH test was performed by the ABBOTT/Vysis PathVysion Her2 FISH tests in this study  assessments, we ran an HER2 control daily in all cases and had 1 pathologist routinely screening the slides.Her2 FISH as the reference standard, the HER2 IHC performance measures were calculated by 2007 and 2013 ASCO/CAP scoring criteria, respectively. Sensitivity was defined as the ratio of HER2 IHC-positive cases among Her2 FISH-amplified patients, specificity was defined as the ratio of HER2 IHC-negative cases among Her2 FISH nonamplified patients, positive predictive value (PPV) was defined as the ratio of Her2 FISH-amplified cases among HER2 IHC-positive patients, negative predictive value (NPV) was defined as the ratio of Her2 FISH nonamplified cases among HER2 IHC-negative patients, as well as accuracy was defined as the ratio of HER2 IHC-positive and Her2 FISH\u2013amplified cases plus HER2 IHC-negative cases and Her2 FISH nonamplified cases among all cases. The overall concordance was defined as the ratio of HER2 IHC-positive and Her2 FISH-amplified cases plus HER2 IHC-negative cases and Her2 FISH nonamplified cases among all nonequivocal IHC cases. Data were analyzed using standard statistical software . All tests were 2-sided and the significance level was 0.05.The consistency between 2007 and 2013 ASCO/CAP IHC results was analyzed by categorized variables using Kappa statistics. The HER2 positivity was defined as having a positive IHC result irrespective of the FISH ratio, plus equivocal or negative IHC result but FISH amplification. Furthermore, we applied the Cochran-Armitage trend test to assess for a trend of positive percentages across the ordinal variables. Regarding Her2 FISH results  by the 2007 and the 2013 ASCO/CAP scoring criteria, they agreed perfectly   by the 2007 criteria but reclassified as having HER2 IHC score 2+  by the 2013 criteria (Table Her2 amplification rates of mucinous EOC (n\u200a=\u200a9/49) were the same in both the criteria (18.37% vs 18.37%); the percentage of Her2 FISH amplification increased significantly in a trend through the ordinals of HER2 IHC results  by either the 2007 or the 2013 criteria, respectively (P\u200a<\u200a0.001 vs P\u200a<\u200a0.001)  cases which were identified based on the 2013 criteria, two cases show FISH amplification and two cases show FISH nonamplification. On the other hand, only one HER2 IHC score 2+ case is identified based on the 2007 criteria and it shows FISH amplification.Under the 2007 and the 2013 ASCO/CAP scoring criteria, we demonstrated that Except for 1 case with a HER2 IHC score 2+  by the 2007 criteria, our data for the relationship between IHC and FISH showed 100% (n\u200a=\u200a7/7) in positive concordance, 97.56% n\u200a=\u200a40/41) in negative concordance, and 97.92% (n\u200a=\u200a47/48) in overall concordance ; whereas except for the 4 cases with HER2 IHC score 2+  by the 2013 criteria, our data for the relationship between IHC and FISH showed 100% (n\u200a=\u200a7/7) in positive concordance, 100% (38/38) in negative concordance, and 100% (45/45) in overall concordance   and PPV (100%) by both the 2007 criteria and the 2013 criteria were similar. The sensitivity under the 2007 criteria was lower than that under the 2013 criteria (87.5% vs 100%), the NPV under the 2007 criteria was lower than that under the 2013 criteria (97.6% vs 100%), and the accuracy under the 2007 criteria was lower than that under the 2013 criteria (97.9% vs 100%)  of positive and equivocal results by HER2 IHC and Her2 FISH tests have been down-adjusted, and the 2013 criteria seemed to be less stringent than the 2007 criteria  had Her2 gene amplified by both the 2007 and the 2013 Her2/CEP17 thresholds, respectively (>2.2 vs \u22652.0), but none (n\u200a=\u200a0/49) had Her2/CEP17 <2.2/<2.0 with Her2 gene copy numbers \u22656.0 by either the 2007 or the 2013 criteria.The Her2 FISH test in the 2013 criteria was defined as cases showing a Her2/CEP17 <2.0 and an average absolute Her2 signal count per cell of \u22654.0 and <6.0 versus the 2007 criteria, which defined equivocal as cases showing a Her2/CEP17 ratio between 1.8 and 2.2 or Her2 signal count per cell of \u22654.0 and <6.0. Our data revealed that no equivocal Her2 FISH cases (n\u200a=\u200a0/49) occurred by both the 2007 and the 2013 criteria.The equivocal The positive HER2 IHC test (score 3+) was defined as circumferential, complete, uniform, and intense staining of >10% of tumor cells in the 2013 criteria versus >30% in the 2007 criteria. Our data revealed that 7 cases (n\u200a=\u200a7/49) with HER2 IHC score 3+ existed by both the 2007 and the 2013 criteria. All of them (n\u200a=\u200a7) showed homogenous strong, complete membrane staining >30%.The equivocal HER2 IHC result (score 2+) was defined as circumferential membrane staining that is \u201cincomplete\u201d and/or weak/moderate and >10% of tumor cells in the 2013 criteria versus \u201ccomplete\u201d membrane staining in the 2007 criteria, or complete and circumferential membrane staining that is intense and within \u226410% of tumor cells in the 2013 criteria versus \u226430% in the 2007 criteria. Our data revealed that the 2013 criteria identified more HER2 IHC equivocal cases  than the 2007 criteria  in all 49 cases  was unchanged under 2007 and 2013 criteria. The alterations from HER2 IHC score 0 to 1+ have no clinical relevance. Both the HER2 IHC score 0 and score 1+ have been regarded as \u201cnegative\u201d result category. Our data revealed that 25 cases remained HER2 IHC score 0 (negative) and none were upgraded to a score of 1+ by either the 2007 or the 2013 criteria. However, 3 in 16 cases with an IHC score of 1+ (negative) by the 2007 criteria would be reclassified as IHC equivocal (score 2+) result by the 2013 criteria, which therefore required alternative FISH testing  cases would be considered to have negative HER2 status without any further testing on the basis of the 2007 and 2013 ASCO/CAP algorithms. As a result, the rigorous 2007 ASCO/CAP criteria potentially diminished the detection rates of HER2 positivity in mucinous EOC patients in comparison with the undemanding 2013 ASCO/CAP criteria.We identified that 2.04%  or none  of HER2-positive patients would be missed if the 2007 criteria were used. In other words, if the HER2 IHC test was applied first, the 2013 criteria can detect 1 case with However, the lenient 2013 guidelines may permit more patients to go through the second round of testing, so as to avoid the possibilities of missing HER2 positive mucinous patients. Thus, it unfavorably increases the cost of examination and extends the reporting date of HER2 status.Her2 testing in this study on the mucinous EOC. The major reasons were: FISH has been shown to be theoretically easier to interpret due to the stability of the DNA target; the interobserver variation was lower because FISH was an objectively quantitative test; and none with Her2 equivocal FISH existed under both 2007 and 2013 criteria in all 49 cases. So that, the assay performance characteristics of HER2 IHC testing by both 2007 and 2013 criteria were assessed and compared  in mucinous EOC, respectively; both the frequencies of Her2 FISH amplifications by 2007 and 2013 ASCO/CAP scoring criteria were equivalent ; the frequency of HER2 IHC equivocal results by 2007 ASCO/CAP scoring criteria  was less than that by 2013 ASCO/CAP scoring criteria ; and 1 case with Her2 FISH amplification was missing, when first tested by IHC under the 2007 criteria.In summary, we demonstrated that both 2007 and 2013 ASCO/CAP scoring criteria agreed excellently in assessing the HER2 IHC (kappa\u200a=\u200a0.903) and When evaluating the HER2 status by IHC first in mucinous EOC, we identified 1 more case with HER2 positivity by the 2013 than that by the 2007 ASCO/CAP guideline scoring criteria. Compared with the 2013 criteria, the more rigorous 2007 criteria resulted in inevitable false-negative IHC, which not only subtracted the opportunity to be referred to FISH testing, but also possibly diminished the detection rates of HER2 positivity in mucinous EOC."}
+{"text": "IGF2BP3 (IMP3) is a mRNA binding protein that regulates IGF2 translation and function during embryogenesis. Because IGF2BP3 is undetectable in adult human tissues except the testis, and increased IGF2BP3 expression has been noted in several cancers, it is considered a cancer testis (CT) protein. IGF2BP3 mRNA expression in colorectal cancers (CRC) has not been well studied. This study's aim was to quantitatively assess IGF2BP3 mRNA expression in CRC and, thus, determine if IGF2BP3 has potential as a vaccine target.Data were collected prospectively from CRC patients in an IRB-approved tissue and data bank. Total RNA was isolated and purified from tumor and normal colonic tissue samples and cDNA synthesized. IGF2BP3 expression was analyzed by quantitative PCR (QPCR). Expression levels of IGF2BP3 in tumors and testis were determined and compared. Tumors with levels greater than 0.1% or more of the testis levels were considered positive. Analysis of IGF2BP3 protein expression by immunohistochemistry (IHC) in tumor and normal tissues was also performed.A total of 84 paired tumor and normal tissue specimens were assessed from patients with Stage 2 and 3 CRC; 43% of tumors had IGF2BP3 mRNA expression levels greater than 0.1 % of that of testis and were considered positive. The median tumor expression level was higher in women (p=0.042). No correlation was found between IGF2BP3 mRNA expression and tumor stage or lymph node involvement. IHC was carried out on paired tumor and normal tissue sections from 46 patients; IGF2BP3 staining was noted in 50% of the tumor sections and in 5% of the normal tissue sections.IGF2BP3 mRNA was over expressed in 43% of the tumors whereas the protein was noted in 50% of samples. No correlation between mRNA expression and disease severity was noted. This protein holds promise as a vaccine target, however, a larger study that assesses a more diverse population of patients (Stage 1-4) as well as a study of preoperative serum samples for auto-antibodies to IGF2BP3 are needed to pursue this concept. Surgery remains the mainstay of treatment for patients with colorectal cancer. The last two decades have seen the development of new chemotherapeutic agents and adjuvant and neoadjuvant treatment regimens that have notably improved the long term outcome of these patients. Despite these advances, over 40% of patients who undergo \u201ccurative\u201d surgery will progress and develop metastatic disease . TherefoFor many years, numerous investigators around the world have been pursuing the concept of cancer vaccines. The idea of harnessing the patient's immune system to recognize and attack tumor cells is very attractive and likely to be associated with minimal toxicity when compared to conventional chemotherapy agents. Much progress has been made over the past 40 years in regards to our understanding of how the immune system functions and also how the tumor evades immune detection or inhibits the immune response. A very important advance was the approval for clinical use of antibodies that block immune checkpoint proteins ,3, whichCancer/testis (CT) proteins are a category of proteins that hold particular promise as vaccine targets because of their restricted expression . These pIGF2 mRNA binding protein 3 (IGF2BP3) is an mRNA binding protein and cancer testis protein that regulates the translation of IGF2. IGF2BP3 and the other IGF2 mRNA binding proteins (IMPs) regulate IGF2 translation, stability and localization by binding to mRNA ,6. IGF2BAlthough prior investigators have assessed a group of colorectal adenocarcinomas for the presence of IGF2BP3 protein via IHC \u201317, a quA total of 84 pairs of normal and cancerous colorectal tissue were assessed in this study. There were 39 males and 45 females; average age was 67.5 +/\u2212 14.4 years. The tumor locations were as follows: right, 42 (51%); sigmoid/rectosigmoid, 24 (29%); rectal, 11(13%); and transverse or descending colon, 7(6%). The study was limited to pathologic stage 2 and 3 tumors; there were 48 stage 2 (57%) and 36 stage 3 (43%) tumors.To determine background values for normal tissues, we evaluated the expression of IGF2BP3 in 22 normal adult tissues using semi-quantitative RT-PCR Figure . IGF2BP3All paired samples underwent quantitative PCR as described in the methods section. Of the 84 tumor specimens assessed via RT-PCR, 43 percent of the tumors met the criteria used for scoring positivity of cancer testis proteins, which comprises having expression levels that were greater than observed in normal colonic mucosa and also greater than 0.1 % of the testes expression level (expression levels over 100 in this study) . When thIHC was carried out on 46 paired tumor and normal tissues specimens that collectively covered the full range of RT-PCR protein expression. Positive IGF2BP3 cytoplasmic staining (1+ to 3+ intensity in >10% cells) was noted in 50% of the tumor specimens and 5% of the normal tissue specimens (1+ to 2+) intensity Figure .No significant differences were found in IGF2BP3 mRNA expression levels based on the stage of tumor or presence of lymph node metastases (n=36). There was a non-significant increase in mean expression levels in stage 3 tumors  versus stage 2 tumors  as well as in node-positive group  versus node-negative group . There was however, a significant increase in median IGF2BP3 expression level in women compared to men (p=0.042).The colorectal cancer specimens utilized for this study were taken from IRB approved tissue and data banks organized, managed, and maintained by the Sections of Colon and Rectal Surgery at New York Presbyterian Hospital, Columbia University Health Science Campus and Mount Sinai Roosevelt Hospital in New York City. Samples of tumor tissue and normal adjacent large bowel mucosa were taken from the resected specimens immediately following resection and were rapidly frozen.Demographic data as well as the location of the cancer, the pathology results and the final tumor stage were obtained from the prospective data base. Tumor and paired normal tissue samples from 84 CRC patients ; were studied. Among the study group 6 paired tissue samples were from patients with mucinous tumors.Tumor and normal mucosal samples were obtained from operative specimens after gross evaluation by a pathologist. The tissue samples were placed in separate standard Cryomolds  which were filled with OCT  compound  and placed in liquid nitrogen. The frozen tissue samples were stored in a \u221280\u00b0C freezer.Prior to being included in this study, sections from candidate frozen tumor and normal colonic tissue blocks were taken at \u221220\u00b0C and assessed histologically to confirm the presence of tumor and the quality of each sample. This analysis was done at the Irving Cancer Center of Columbia University in New York City. Poor quality specimens and those with a paucity of tumor cells were not selected for the study.Total RNA was isolated using Qiazol , and purified using the miRNeasy Kit . Briefly, sections of the frozen tissue block (previously embedded in OCT compound) were cut and placed into a 2ml Eppendorf tube, and then homogenized by TissueLyser II  with 1ml Qiazol added. After the aqueous phase was recovered from the chloroform-derived phase separation, another step of acid phenol-chloroform extraction was performed with a phase lock gel tube . RNA precipitation, column binding, on-column DNase digestion, washing and elution were performed according to kit instructions. RNA integrity was confirmed by agarose gel electrophoresis and the concentration was quantified by measuring OD260nm in BioPhotometer .The cDNA first strand reverse transcription was carried out with the ABI High Capacity RNA-to-cDNA Kit . Briefly, 1ug of total RNA and 10ul of 2X reverse transcription buffer and 1ul of 20X enzyme mix were incubated together in a total volume of 20ul at 37\u00b0C for 60 minutes followed by 95\u00b0C for 5 minutes and held at 4\u00b0C. The synthesized cDNA was stored at \u221220\u00b0C until further use.Comparative quantitative PCR was performed with the Mx3005p real-time PCR machine  using the QuantiTect SYBR Green PCR kit . Briefly; PCR was carried out in 20\u03bcl total volume containing a final concentration of 1x reaction buffer, 300nM forward and reverse primers  were purchased from Clontech laboratories, Inc.  and Ambion, Inc. .TMREDTaq RedyMix  and 10 pmol of each primer were used. Primers used for amplification were compiled from the literature or were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)  Primers were designed to have an annealing temperature around 60\u00b0C and to encompass introns, thus allowing product discrimination in the case of genomic DNA amplification. Primer specificity was confirmed by aligning with the NCBI sequence database using BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi. The amplification protocol used was as follows: precycling hold at 95oC for three minutes followed by 35 cycles of denaturation at 95oC for 15 seconds, annealing for 30 seconds  and extension at 72oC for 30 seconds followed by a final extension step at 72oC for 7 minutes. PCR products were separated on 1.5% agarose gels stained with ethidium bromide.RNA samples  were reversed transcribed in a total volume of 20\u03bcl using Omniscript RT kit  according to the manufacturer 's protocol using oligo(dT)12-18 primer and RNaseOUT . The cDNA was diluted five times with nuclease free water, and 3 \u03bcl of diluted cDNA were used in 25\u03bcl PCR reactions. For amplification, JumpStartWith the quantitative PCR results in hand, we selected 46 of our specimens representing a range of RT-PCR protein values from low to high for IHC in order to assess for the presence of IGF2BP3 protein in the tumor specimens and, thus, confirm the RT-PCR expression level results. The sections were cut at 5 mm-thickness and stained with hematoxylin and eosin (H&E). The slides were then reviewed by two pathologists to confirm the presence of tumor in each section. H&E stained slides were screened for optimal tissue and noncancerous tissue adjacent to tumor tissue. Normal colon tissue sections were also assessed after H & E staining for the presence of normal colonic tissue.Immunohistochemistry was performed on fresh frozen slides  using standard IHC protocol. A fresh frozen sample of pancreatic adenocarcinoma was selected as the positive control. IHC staining was performed on 5 mm-thick sections of fresh frozen samples with purified rabbit anti human IGF2BP3 polyclonal antibody . The slides were incubated in H2O2 solution for 10 minutes at room temperature to block the endogenous peroxidase activity. Antigen retrieval was performed by steamer heating in 10mmol/L citrate buffer (pH 6.0). After epitope recovery, slides were incubated with primary antibody IGF2BP3 at 1:100 dilutions overnight at 4\u00b0C temperatures. Slides were washed and incubated with secondary anti-rabbit IgG  at 1:200 dilution and tertiary streptavidin-peroxidase conjugate . Slides were treated with chromogen \u201cdiaminobenzedine\u201d for antigen detection. Counterstaining was performed with hematoxylin, dehydrated, cleared in xylene, and mounted on coated slides.Immunoreactivity was evaluated independently by two pathologists who assessed the immuno-staining. The percentage of neoplastic cells staining was determined (1 to 100%). IHC staining was considered \u201cpositive\u201d when at least 10% or more of neoplastic cells stained positive on a scale of 1 to 3 . IHC staining of 1+ intensity in less than 10% of neoplastic cells was considered negative, and IHC staining of 2\u20133+ staining in less than10% of cells was considered indeterminate.The demographic and clinical data are expressed as the mean and standard deviation of continuous variables whereas frequencies and percentages were determined for categorical variables. The Fisher exact test (for expected frequency of less than 5) and the Chi-Square test and were utilized for comparison of categorical variables such as the pathologic analysis. The t-test was utilized for comparison of continuous variables. A p value of less than 0.05 was considered statistically significant.The present study is the first to utilize RT-PCR to quantitatively assess IGF2BP3 mRNA expression in a reasonable sized population of colorectal cancers. One earlier study did RT-PCR analyzed a subset of 8 patients out of 203 tumors and demonstrated greater mRNA expression in tumors compared to normal tissues . In the To further assess a prospective cancer testis protein or any other protein as a vaccine target it is useful to assess preoperative plasma or serum specimens for the presence of antibodies to that protein to evaluate the occurrence of spontaneous immune response . The finOf note, in the present study, a non-significant increase in the mean IGF2BP3 mRNA expression levels was noted in the Stage 3 patients when compared to the Stage 2 patients. The same is true regarding the presence of lymph node metastases; a non-significant increase in the mean expression level was noted in the node positive group. Importantly, prior investigators who utilized IHC methods on larger populations (n=165\u2013671) of patients have noted that IGF2BP3 expression was associated with a worse prognosis  \u201317. The When a subset of tissue sections of the tumors in the present study were assessed via IHC, IGF2BP3 protein was noted in 50 percent of the cases. This result is in the same range as the RT-PCR mRNA result of 43% which is reassuring. Also, of note, the 43% RT-PCR result is closer to the IHC result than the 63% result obtained when the less stringent RT-PCR criteria is applied  which support the use of the stricter criteria. Also, our IHC results are in the same general range as the results of several investigators who utilized IHC methods to assess for IGF2BP3. Li, et al in an IHC study of 203 colon cancers reported that 65% of tumors assessed expressed IGF2BP3 whereas Yuan et al, also using IHC methods, found an expression rate of 56% in a population of 165 colorectal tumors . In contSome shortcomings of this current study include the fact that only stage 2 and 3 patients were considered. Further, as mentioned, no data is provided regarding serum titers of anti-IGF2BP3 antibodies for the study population. Also, this report supplies no oncologic outcome data . Thus, there is a need for larger RT-PCR studies that include patients with Stage 1 and 4 disease as well as antibody data and long term outcome data to shed further light on this topic.In conclusion, this study is the first to perform RT-PCR for IGF2BP3 on a sizable population of colorectal cancer patients. Forty three percent of the tumors examined were found to express IGF2BP3 mRNA when compared to RT-PCR results obtained with each patients normal colonic tissue. Half of the subset of tumors that were assessed via IHC were found to express the IGF2BP3 protein. No significant difference in mRNA expression levels were noted for the Stage 3 vs the Stage 2 patients or node positive vs node negative tumors. Tumor expression of IGF2BP3 may hold promise as a tumor marker and/or vaccine target; however, larger studies are needed as well as determination of auto-antibody titers to IGF2BP3."}
+{"text": "Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after  Being one of the few exceptions to Mendelian rules, genomic imprinting is an epigenetic mechanism that allows for a subset of genes to be expressed either from the paternally or from the maternally inherited allele2Cell reprogramming, either oocyte-mediatedIn this article we have developed a Systems Biology approach to study the mechanisms involved in the erasure of genomic imprinting. In particular, we show that the reprogramming of spermatogonial stem cells (SSC), a germ-line committed stem cell type that exhibit an androgenetic imprinting pattern in H19 and Igf2rGt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J), which allows for a homogeneous and replicable expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) in the presence of doxycyclinePou5f1, a key marker for stemness in SSCId4, another marker of SSC, was expressed at a higher level in Gfra1+ compared to Gfra1- cells, but the differences were not statistically significant (ANOVA p<0.05). A marker of differentiated spermatogonia cKitSSC and fetal fibroblast were obtained from a mouse model harboring Yamanaka factors under the control of a doxycycline inducible promoter . Skin chimerism was detected based on the agouti coat color from injected iPSC, over the white coat color of blastocyst donor CD1 strain. All cell lines were able to contribute to chimeras to a certain degree. In terms of skin chimerism, the 3 SSCiPSC lines outperformed fiPSC lines, resulting in a significantly higher number of pups with a high skin chimerism (>40%) than fiPSC based on Chi-square test .Ins2, Lhcgr, Lhx1, Nanos2, Nr5a1, Serpina 5 and Tcf21 (Tet1 and Tet2) and DNA methylation   . Gene On Dnmt3b) . KEGG pa Dnmt3b)  online.  Dnmt3b) . Out of in vivo differentiation, by analyzing the methylation status of H19 ICR in teratomas. In vivo differentiation of iPSC did not affect the methylation levels at H19 ICR, as teratomas produced either by intramuscular or subcutaneous injections revealed similar methylation patterns as the SSCiPSC line from which they were derived  and in 3 iPSC cell lines derived from each resultant cell type were analyzed . As expe10 derived .Tet1 and Tet2 in Gt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J mice by CRISPR/Cas9 systemTet1+Tet2 double knockout (DKO) embryos die in utero likely due to epigenetic defectsTet1 and Tet2 (DKO) and another showing frame-disrupting mutations in Tet2 but an in-frame indel and a non-edited allele in Tet1 (Tet2 KO cells)  . The tese wt SSC A and 4B.in vitro from PGC) resulted in the erasure of the imprinting marks, whereas the fusion with ESC, which also reprogram the B cell genome, does not erase the imprinting marks25Pou5f1 before reprogramming in SSC compared to fibroblastsThe regulation of DNA demethylation at ICR is uncoupled from the global demethylation changes leading to pluripotent states in early embryo. Although imprinting is erased during PGC formation2Pou5f1 locus, and in both cases it seems that there is a conversion of 5\u2009mC to 5\u2009hmC. Tets are the only enzymes known to catalyze 5\u2009mC hydroxylation and both Tet1 and Tet2 are present in EGC and ESC, the two cell types differing in their ability to abolish imprinting marks following cell-fusion based reprogrammingin vitro derivation of pluripotent cells from unipotent GSC  report different patterns of ICR methylation depending on the cell type of origin. Pluripotent stem cells derived from GSC of newborn testis (mGS) lose the imprinting of two paternally methylated ICR H19, Meg3 IG and keep the maternally methylated Rasgrf1, Igf2r and Peg10The notion of a cumulative effect of demethylating agents acting on imprinting erasure rather than a specific mechanism for demethylation at ICR agrees with previous findings in different Systems Biology Approaches. The demethylation of ICR is a late event in cell fusion-based mechanism occurring much more slowly than the demethylation occurring at the in vivo differentiation into teratomas. Genomic imprinting is an epigenetic feature generally overlooked in iPSC and while the imprinting erasure did not impair the differentiation abilities of the SSCiPSC, the functionality of these differentiated cells could be affected. As imprinting has been shown to be affected in other types of iPSC3031A particularly relevant observation is that imprinting erasure was not reversed following Tet2Tet enzymesTet1 and Tet2 were dispensable for SSC reprogramming. A possible explanation for the differences between MEF and SSC reprogramming could be that SSC do not require a mesenchymal-to-epithelial transition, the critical step blocked by Tet deficiencyTet1 and Tet2 are the only Tet members expressed in PGCs or ESCTet1 and Tet2 coincides in time with the conversion of 5\u2009mC to 5\u2009hmC at ICR in PGCs, so they have been suggested to play a role in imprinting erasureTet1 paternal or maternal KO  E9.5\u201310.5 embryos were observed to dysregulate several imprinted genes by hypermethylation of paternal or maternal ICR, respectivelyTet1 and Tet2 DKO embryos are deprived from 5\u2009hmC. These pups were fertile and some of its progeny shows altered imprinting in H19, Mest, Peg3 and Igf2rTet3, not expressed in PGC but expressed later during gametogenesis and spermatogenesis26Tet1 and Tet2 DKO SSC does not lose their imprinting marks following reprogramming to SSCiPSC, suggesting an essential role of these enzymes in the process. Our findings are in agreement with the critical role for Tet enzymes in the erasure of imprinting mediated by cell fusion between B cells and EGC, where Tet1 depleted EGC were unable to accumulate 5hmC and erase the imprinting marks on the B cell genome following fusionTet enzymes in imprinting erasure in our model, we cannot rule out that passive DNA demethylation may have also played a role.As plausible drivers for imprinting erasure, Tets  are the only known enzymes that convert 5-methylcytosine (5\u2009mC) to 5-hydroxymethylcytosine (5\u2009hmC), an intermediate in the 5\u2009mC demethylation process that undergo passive demethylation as cells divideCollectively, these results highlight that imprinting erasure following Yamanaka factors based reprogramming depends on the epigenetic status of the original cell, with iPSC obtained from highly differentiated cells (fiPSC) being more able to retain the imprinting mark than SSCiPSC. This observation proposes that imprinting marks are specially protected from demethylation and that reprogramming at imprinted loci is a late step in genome reprogramming driven by the cumulative action of demethylating agents, with Tet enzymes playing an essential role. In this regard, Tet mediated imprinting erasure does not seem to require a specific demethylation mechanism, in agreement with the lack of consensus target sequences at ICRsGt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J14. Germinal cells were enriched from PND6 testis by differential plating. Briefly, testicular stroma was minced and digested with 1\u2009mg/ml type IA collagenase (Sigma) for 10\u2009min at 37\u2009\u00b0C, followed by digestion with 0.05% trypsin-EDTA (Gibco) for 5\u2009min at 37\u2009\u00b0C. Cell suspension was purified through a 70\u2009\u03bcm filter and plated onto a gelatin coated dish for 2\u2009hours, allowing testicular somatic cells to attach to the plate. Germinal floating cells were subjected to MACS (Miltenyi Biotech) for Gfra1, a SSC membrane markerAll experiments involving vertebrate animals were performed in accordance with the approved guidelines of Beltsville Area Animal Care and Use Committee (BAACUC). All experimental protocols involving vertebrate animals were approved by the Institutional BAACUC committee. Fetal fibroblast and SSC were obtained from the mouse model SSC and fetal fibroblasts were cultured following standard protocols in the presence of 2\u2009\u03bcg/ml Doxycycline hyclate (Sigma). For the first passage, individual colonies were picked up and individually trypsinized to obtain clonal lines. Doxycycline concentration was kept at 2 \u03bcg/ml for the first 5 passages and then was gradually withdrawn . After passage 8, cells were incubated without doxycycline.3 biopsy of the teratomas was collected to determine DNA methylation. As this procedure does not exclude the possibility of host cell contamination, the percentage of contamination was estimated by a PCR with three primers JaeCol1a1Gt(ROSA)tm3Jae/J iPSC derivatives. Comparison of band intensity between the PCR products amplified from teratomas or from standard mixes of known amounts of wt and transgenic DNA estimated an ~5% host cell contamination in the samples used for DNA methylation  and injected subcutaneously. The same number of cells was injected intramuscularly diluted in culture media. One month after injection, teratoma growth was obvious and the mice were sacrificed and the teratomas collected and fixed in 4% paraformaldehyde. Fixed tissues were dehydrated through ethanol washes, paraffin embedded, sectioned and stained in hematoxylin/eosin (American Histolabs). An inner ~1\u2009mmhylation . This cohylation .SSCiPSC and fiPSC were individualized and injected into CD1 blastocysts (10 iPSC/embryo). The injected blastocysts were transferred by uterotubal embryo transfer to pseudopregnant recipients. Three different lines from each cell type were tested, performing 3 to 5 embryo transfers and obtaining 15 to 30 weaned pups per cell line analyzed . Skin chhttp://david.abcc.ncifcrf.gov/), and differentially expressed genes with p-value\u2009\u2264\u20095*10\u22125 and fold change\u2009\u2265\u20091.5 were categorized with respect to Molecular Function, Biological Process and Cellular Component. The annotated genes were also mapped into relevant functional groups in a pathway analysis according to the Kyoto Encyclopedia of Genes and Genomes (KEGG).In order to minimize the contamination with the feeder cells, iPSC at passages 13-14 were purified by MACS for SSEA1 following a protocol similar to the one described for SSC enrichment for Gfra1, but using anti-SSEA-1 (CD15) microbeads (Miltenyi Biotech). The purified cells were used for gene expression and DNA methylation analysis. qPCR was performed as previously describedH19-Igf2200\u2009ng of DNA from fibroblasts, FiPS, SSC, SSCiPS and teratomas were treated with bisulfite to convert all unmethylated cytosine to uracil using EZ DNA Methylation-Direct Kit (Zymo Research) according to the manufacturer instructions. A 422\u2009bp fragment of the 2\u2009kb ICR of in vitro transcription  from the plasmid pMJ920 (Addgene 42234) linearized with BstBI and treated with Antarctic phosphatase (NEB). Single guide RNAs (sgRNA) for Tet1 and Tet2, previously described inin vitro transcription  from g-Blocks containing T7 promoter. Both the Cas9 mRNA and the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted in TE buffer. Cas9 mRNA (100\u2009ng/\u03bcl) and sgRNA (20\u2009ng/\u03bcl) were injected into two cell embryos obtained from Gt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J females. Injected embryos were allowed to develop to the blastocyst stage and transferred to CD1 foster mothers by utero-tubal embryo transfer. Genotyping was performed on DNA extracted from a tail biopsy by using specific primers spanning the target sequence ."}
+{"text": "We studied the rapid changes in electrical properties of lumbar motoneurons between postnatal days 3 and 9 just before mice weight-bear and walk. The input conductance and rheobase significantly increased up to P8. A negative correlation exists between the input resistance (Rin) and rheobase. Both parameters are significantly correlated with the total dendritic surface area of motoneurons, the largest motoneurons having the lowest Rin and the highest rheobase. We classified the motoneurons into three groups according to their discharge firing patterns during current pulse injection . The delayed onset firing type has the highest rheobase and the fastest action potential (AP) whereas the transient firing group has the lowest rheobase and the less mature AP. We found 32 and 10% of motoneurons with a transient firing at P3\u2013P5 and P8, respectively. About 20% of motoneurons with delayed onset firing were detected at P8. At P9, all motoneurons exhibit a sustained firing. We defined five groups of motoneurons according to their discharge firing patterns in response to ascending and descending current ramps. In addition to the four classical types, we defined a fifth type called transient for the quasi-absence of discharge during the descending phase of the ramp. This transient type represents about 40% between P3\u2013P5 and tends to disappear with age. Types 1 and 2 (linear and clockwise hysteresis) are the most preponderant at P6\u2013P7. Types 3 and 4 (prolonged sustained and counter clockwise hysteresis) emerge at P8\u2013P9. The emergence of types 3 and 4 probably depends on the maturation of L type calcium channels in the dendrites of motoneurons. No correlation was found between groups defined by step or triangular ramp of currents with the exception of transient firing patterns. Our data support the idea that a switch in the electrical properties of lumbar motoneurons might exist in the second postnatal week of life in mice. Electrical properties of developing spinal motoneurons have been studied in several species and at different embryonic and postnatal stages  following current pulse stimulation have been well documented in rat spinal motoneurons  of motoneurons has been found in the neonate rat  to 9 (P9), P0 being the first postnatal day. All surgical and experimental procedures are conformed to the European Communities council directive (86/609/EEC) and approved by our ethics committee . Most of the experimental procedures were described previously  artificial cerebrospinal fluid (ACSF). Then, a laminectomy was performed and the spinal cord and brainstem were removed, taking care to preserve sufficient length of L5 ventral root, placed in a recording chamber and superfused with ACSF containing (in mM): NaCl, 130; KCl, 4; MgClTM three-dimensional hydraulic microdrive. Motoneurons were impaled at a depth of 150\u2013450 \u03bcm from the spinal cord surface corresponding to the fifth lumbar segment. Motoneurons were identified by their antidromic action potential (anti AP) evoked following electrical stimulation of the ventral root L5. Intracellular recordings were made either in bridge mode with an output bandwidth of 3.0 kHz or in Discontinuous Current Clamp (DCC) mode, using an Axoclamp 2B amplifier (Axon instruments). Electrode resistance and capacitance were compensated before intracellular recordings. Signals were digitized at 10 kHz by an A/D converter (Digidata 1322 Axon Instruments) and saved on a computer using Clampex 9.2 (Axon instruments).To allow for microelectrode penetration in the spinal cord, the pia was carefully removed medially to L5 ventral root entry, using very fine forceps under binocular control. Fine tip micropipettes for intracellular recordings were made from 1.2 mm filamented glass tubes (Clark Instruments) using a pipette puller . Electrodes were filled with 2 M potassium acetate, and their resistances ranged between 60 and 110 M\u03a9. The microelectrode was positioned to penetrate the L5 spinal segment with an angle of 30\u201345\u00b0 above the horizontal and advanced in the tissue using a NarishigeRin was measured by computing the voltage deflections derived from series of hyperpolarizing and depolarizing constant current pulses  injected into the motoneurons. Measurements were made from the averaged voltage over 50 ms taken from the steady state membrane potential at the end of the pulses. Retained values were the averages of three sets of measurements.Spike potentials were analyzed using the Event Detection/Threshold Search module of Clampfit 9.2. Firing behavior was studied as described previously  if p < 0.01 or (***) if p < 0.005. Results were expressed as means \u00b1 SEM or medians with interquartile range when indicated. Graphical representations were obtained using Origin 7.5 (Origin Lab Corporation), Graph pad Prism 6.0 and Corel Draw 12 .Statistical significance was assessed with the non-parametric Permutation with General Score Exact Test for independent or paired samples or the Fisher exact test (StatXact7. Cytel software). The correlation between two sets of data was evaluated by using Pearson\u2019s correlation test or Spearman\u2019s correlation test (Graphpad Prism 6 or StatXact 7). Two groups of data were considered statistically different (*) if All the procedures for morphological studies were described previously .The labeled motoneurons were reconstructed from serial sections (75 \u03bcm thick) on Nikon microscope equipped with a computer interfaced motorized stage and TM. Our own database of intracellularly recorded and stained motoneurons with Neurobiotin comprises more than 50 postnatal mouse lumbar motoneurons at different ages between P3 and P10. Among them, 32 motoneurons were fully reconstructed in 3D with NeurolucidaTM. In the present work we used 14 motoneurons at both ages .A single motoneuron was described by up to 18.000 data points, which were stored in a database together with fiducial marks  in ASCII format files. Reconstructed cells were visualized and three-dimensionally analyzed using NeurolucidaThe data base for the electrophysiological study comprises 103 motoneurons from mice aged between P3 and P9. Only neurons displaying a stable membrane potential more negative than \u221250 mV with overshooting APs during the whole test procedure were kept for analysis. All the motoneurons were identified by recording either the anti AP evoked by the ventral root stimulation Figure .2, n = 3; P8\u20139: 3212 \u00b1 314 \u03bcm2, n = 9; p = 0.04) as well as the mean diameter of primary dendrites . The total dendritic length increases from P3 to P9 by 22%  and the total dendritic surface area by 20%  and P8\u2013P9 (n = 34). The anti AP has comparable amplitude in the two populations  as well as the resting membrane potential  due to ongoing motor axon myelination during this postnatal period.In this series of experiments, a number of parameters of electrical properties was analyzed in two populations of lumbar motoneurons of different ages  compared to the mean Rin in the P3\u2013P5 population . However, the Rin stabilizes between P8 and P9  to 1.76 \u00b1 0.2 nA (n= 31) between P3 and P8 (Table p = 0.009). The mean rheobase is significantly higher at P6/P7  compared to that in P3\u2013P5. Indeed, a significant and negative correlation exists between the rheobase and the Rin  but no significant correlation was found between the gain and the Rin . The mean rheobase was significantly lower at P9  compared to that at P8 . Thus the progression of the rheobase stopped between P8 and P9 in lumbar motoneurons.As expected, the mean Rin was lower in the oldest motoneurons  and between the rheobase and the total dendritic surface area  confirming that the largest motoneurons in the lumbar cord have the lowest Rin and the highest rheobase also during postnatal development. However, we noticed that some motoneurons with similar total dendritic surface area  may have different Rin ranging from 10 to almost 30 M\u03a9 .In twelve motoneurons intracellularly stained and fully reconstructed, significant correlations were found between the Rin and the total dendritic surface area  together with the maximum depolarizing speed which was accelerated from 123 \u00b1 8.69 mV.ms\u22121 to 149 \u00b1 7.38 mV.ms\u22121 with age (p < 0.01). Thus younger motoneurons have spikes with lower amplitude and slower time course. No changes with age were seen in AHP amplitude and total AHP duration between P3 and P9. However, AHP half-duration was shorter and half decay time faster in the P8\u2013P9 group (Table As summarized on Table n = 12). The distribution of the different firing patterns is illustrated in pie charts in Figure During the development of spinal motoneurons, different patterns of discharge have been previously described , spike half width , max rise slope  and time to peak  of the AP. The maximum decay slope also shows statistically significant difference between the three groups (not illustrated). All spike parameters  indicate that the delayed firing type do have the fastest AP. The AHP parameters did not show significant differences although there is a tendency in the AHP amplitude to be larger in the delayed group .We then analysed the electrical properties of the subgroups. We focused on the three patterns of discharge firing in motoneurons from P6\u2013P9 animals. We analyzed 14 passive and active electrical properties Figure  suggesti Figures . The gaiFiring pattern was further characterized using increasing and decreasing (triangular) slow current ramps as described previously  whereas some motoneurons exhibit a delayed onset firing pattern up to the second postnatal week. A majority of motoneurons have a sustained firing at all ages between P3 and P9. The sustained firing is present in all motoneurons at P9. The results also show that a counter clockwise hysteresis and/or a prolonged sustained firing in response to current ramp (types 3 and 4) emerge at P8\u2013P9 corresponding to the maturation of L type calcium channels in dendrites of mouse motoneurons. Dendrites of postnatal motoneurons mainly elongated during this short period of time. Although most morphological parameters were not significantly different between P3 and P9 . This proportion is rather low compared to that (65%) observed in lumbar motoneurons recorded in slice . They represent up to 50% of motoneurons at P8\u2013P9 but only 10% at P3\u2013P5 Figure .The non-linear behavior may originate from sodium and/or calcium persistent inward currents (Schwindt and Crill, G85R transgenic mouse model of ALS at 3\u20134 months of age. Furthermore they found that this population of motoneurons that disappears in SOD1 adult mice was greatly hyperpolarized, which would favour hypoexcitability. We previously showed that lumbar motoneurons from SOD1G85R mice were hypoexcitable very early during the postnatal period having a higher rheobase and lower gain (Bories et al., G93A mice in which an accelerated maturation of lumbar motoneurons might lead to a different time course in the ALS pathology (Quinlan et al., G85R or SOD1G93A mice are needed to elucidate this question.Our results show that the rheobase, input conductance and gain of motoneurons are the highest in the delayed firing group. These results are in agreement with those suggesting that motoneurons with delayed onset firing pattern correspond to the fast motoneurons (Leroy et al., We found rapid changes in the progression of electrical properties of mouse lumbar motoneurons between P3 and P9 whereas the morphology of dendritic arborization evolves slowly. A change of rheobase and Rin progressions occurs, with the disappearance of transient and delayed onset firing types following direct current pulse stimulation, and the emergence of types 3 and 4 discharge patterns following direct ramp current stimulation. We conclude that a switch might exist in the electrical properties of mouse lumbar motoneurons around P8\u2013P9 during the maturation of motor behaviors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "HTLV-1 Tax protein is usually undetectable in freshly isolated peripheral blood mononuclear cells (PBMCs). After in vitro culture, the provirus is reactivated and Tax protein is detectable only in a proportion of infected CD4+ T cells: the percentage of Tax+ cells is always lower than the proviral load (PVL). To identify and further analyse the latently infected cells we have measured the expression of tumour suppressor of lung cancer 1 (TSLC1). TSLC1 is a member of the immunoglobulin super family that is expressed on all cells with the exception of PBMCs. It mediates cell-to-cell adhesion by either homophilic or heterophilic interactions with other members of the immunoglobulin family and also signals to the actin cytoskeleton. TSLC1 has been shown to be expressed on primary adult T cell leukaemia (ATL) cells and ATL cell lines. We assayed the expression of TSLC1 in the PBMCs of 13 asymptomatic carriers (AC) and 13 HAM/TSP patients by flow cytometry. We found that the percentage of TSLC1+ CD4+ T cells was positively correlated with the PVL (P < 0.0001). To test whether TSLC1+ cells themselves were infected, we flow sorted TSLC1+ CD4+ T cells of 3 ACs and 3 HAM/TSP patients. A median of 95% of TSLC1+ CD4+ T cells carried the provirus. Whilst only 20% of infected CD4+ T cells expressed Tax, a further 47% were identified on the basis of TSLC1 expression. As the host cytotoxic T cell (CTL) response is an important protective factor, we are now testing the hypothesis that the presence of TSLC1 on the surface of infected cells affects its recognition and subsequent killing by CTLs."}
+{"text": "The values of C-reactive protein (CRP) and procalcitonin (PCT) were investigated to determine their effects on postoperative complications in patients with or without systemic inflammatory response syndrome (SIRS).3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis.In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The definition of SIRS includes two or more of the following: temperature >38 or <36\u00b0C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mmn = 83) and patients without SIRS . A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specificity of 70% .All patients were divided post hoc into patients with SIRS  Serum PCT values increased significantly after cardiopulmonary bypass in the SIRS group in comparison with patients without SIRS on postoperative day 1 and remain elevated until postoperative day 5. (2) Serum CRP values also follow a similar pattern, buta CRP threshold value was not obtained to differentiate between postoperative complications in patients with or without SIRS. A PCT threshold value of 2.79 ng/ml on postoperative day 1 is a valuable marker to discriminate between patients with or without SIRS."}
+{"text": "The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms. Phylogenetic analysis of the evolutionary history of MAP kinase genes suggests that vertebrate MAP kinases originated from 3 precursors and have expanded through gene duplication during early vertebrate evolution  kinase pathways are evolutionarily conserved signaling modules that play a key role in transducing extracellular signals into intracellular responses Meloche, . These sErk1 or Erk2 mRNA, suggesting that a single ERK isoform mediates cellular responses in these areas  technology has allowed analysis of the phenotypical consequences of the specific depletion of ERK1 or ERK2 in animals and cells. Erk1\u2212\u2215\u2212 mice develop normally, are viable and fertile, and display no observable phenotype  6.5  also revealed a tight relationship between the extent of development of embryos with different combinations of Erk1 and Erk2 alleles and total ERK1/2 activity in embryonic tissues. As a second approach, we asked whether ERK1 can substitute for ERK2 in mouse embryonic development. We found that ubiquitous expression of an Erk1 transgene fully rescues the placental and embryonic defects observed in ERK2-deficient embryos. ERK1-only mice grow normally, are fertile and do not display any overt phenotype. Expression of transgenic ERK1 also rescued the proliferation defect of ERK2-deficient MEFs and restored normal phosphorylation of a panel of ERK1/2 substrates. Our study provides compelling and definitive evidence for a functionally redundant role of ERK1 and ERK2 kinases during development (Fremin et al., Mek2 at the Mek1 locus rescued the placental phenotype of MEK1-deficient mice (Aoidi et al., We have used complementary genetic approaches to rigorously address the question of ERK1 and ERK2 specificity or redundancy in embryonic development (Fremin et al., Erk1 and Erk2 null mice are attributable to differences in expression levels, with ERK2 being the predominant isoform. The higher expression level of Erk2 in most mammalian tissues can be related to a stronger promoter, although further regulation by post-transcriptional mechanisms cannot be ruled out (Busca et al., Differences in the phenotypes of The above findings underscore the concept that a threshold of global ERK1/2 activity determines developmental progression and phenotypic outcome (Figure Erk1 gene has been lost in all bird lineages and some amphibians, whereas squamates only express ERK1 isoform, despite the presence of both Erk1 and Erk2 genes. The finding that tetrapods can live by expressing only ERK1 or ERK2 provides further demonstration of the functional redundancy of ERK isoforms in animal physiology.The group of Philippe Lenormand recently reported the most detailed analysis of the expression and evolution of ERK1 and ERK2 protein sequences in vertebrates (Busca et al., Jnk1 or Jnk2 gene exhibit distinct phenotypes, suggesting that individual JNK isoforms may serve different signaling functions (Davis, Jnk1\u2212\u2215\u2212 and Jnk2\u2212\u2215\u2212 MEFs proliferate at different rates, a phenotype that has been related to the expression levels of cJun (Tournier et al., The JNK pathway provides another example where different groups have reported contradictory conclusions about the specificity or redundancy of closely related MAP kinase isoforms. JNK1 and JNK2 are ubiquitously expressed in the mouse although their expression levels vary across tissue types. Mice deficient in either s Davis, . In addiJnk2M108G allele was integrated at the endogenous Jnk2 locus by homologous recombination to generate mice expressing analog-sensitive JNK2 kinase. Analysis of MEFs bearing different combination of Jnk1 and Jnk2 alleles revealed that pharmacological inhibition of JNK2 in JNK1 proficient cells caused no change in cJun expression or cell proliferation, contrary to the results obtained in Jnk2\u2212\u2215\u2212 cells. However, both genetic ablation and pharmacological inhibition of JNK2 in Jnk1\u2212\u2215\u2212 cells reduced cJun levels and inhibited cell proliferation. These results demonstrated that JNK1 and JNK2 act redundantly to increase cJun expression and promote cell proliferation. The most likely explanation for the misleading phenotype of Jnk2\u2212\u2215\u2212 cells is that loss of JNK2 leads to increased JNK1 function by a compensatory adaptation mechanism. This adaptation is not observed upon acute inhibition of the kinase and/or in conditions where protein expression is maintained. This study also highlighted the importance of using multiple experimental approaches to interpret the phenotypes of mouse mutants, as discussed above for ERK1 and ERK2.The group of Roger Davis has revisited the proposed negative regulatory role of JNK2 on cJun expression and cell proliferation using a chemical genetic approach (Jaeschke et al., p38\u03b1 and p38\u03b2 mouse mutants exhibit distinct phenotypes. Specifically, loss of p38\u03b1 is embryonic lethal owing to defects in placenta morphogenesis (Adams et al., The p38 MAP kinase pathway regulates numerous cellular processes including adaptation to environmental stress, innate immunity; cell cycle progression and cellular differentiation (Cuenda and Rousseau, p38\u03b1 and p38\u03b2 deficient embryos but were absent in single gene knockouts, indicating that that the two isoforms can compensate for each other with respect to these defects (del Barco Barrantes et al., p38\u03b2 knock-in allele in the p38 knockout background developed to E18.5 and showed rescue of spina bifida and exencephaly defects, but not heart defects. Upon increased dosage from the two additional endogenous p38\u03b2 alleles, the heart anomalies were rescued and, more importantly, some of the animals survived to adulthood, thereby overcoming the lung defects observed in p38\u03b1-deficient animals (del Barco Barrantes et al., The group of Angel Nebreda used a combination of genetic approaches to address the question of the specificity and redundancy of p38\u03b1 and p38\u03b2 isoforms (del Barco Barrantes et al., p38\u03b1 and p38\u03b2 genes are deficient in sex determination due to reduced expression of the testis-determining gene Sry (Warr et al., Further evidence that p38\u03b1 and p38\u03b2 act redundantly comes from work demonstrating that embryos lacking both p38\u03b3 and p38\u03b4 genes has unveiled key roles of p38\u03b3 and p38\u03b4 isoforms in tissue regeneration, innate immune responses, inflammation, and tumorigenesis (Esc\u00f3s et al., The two other members of the p38 MAP kinase subfamily also exhibit overlapping functions. p38\u03b3 and p38\u03b4 share 70% amino acid identity (Cuenda and Rousseau, We have used a combination of genetic approaches together with quantitative analysis of embryonic phenotypes and ERK1/ERK2 activity to demonstrate that ERK1 and ERK2 isoforms are functionally redundant in mouse development and physiology. This conclusion is consistent with the discovery of animal species that express only ERK1 or ERK2 and with studies showing that the two isoforms share similar biochemical properties and substrate specificity. Similarly, by combining multiple experimental approaches, other studies have revealed that JNK and p38 MAP kinase isoforms exert functionally redundant roles. These findings clearly illustrate the importance of using multiple genetic, pharmacological and phylogenetic analyses to define the physiological functions of related signaling proteins.V600E metastatic melanoma and other inhibitors of MAP kinase pathways are undergoing clinical evaluation. It is therefore crucial to determine whether the direct or downstream targets of these inhibitors have specific or redundant functions.The question of MAP kinases redundancy has far-reaching implications. Dysregulation of the ERK1/2, JNK1/2/3, and p38\u03b1/\u03b2/\u03b3/\u03b4 pathways has been causally linked to human congenital syndromes and to a variety of diseases including cancer, arthritis, fibrosis, cardiomyopathies, and neurodegenerative diseases. Small molecule inhibitors of the ERK1/2 pathway have been approved for the treatment of BRAFAll authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Recent studies have shown that RD3 interacts with guanylate cyclases GC1 and GC2 in retinal cell extracts and HEK293 cells co-expressing GC and RD3. This interaction inhibits GC catalytic activity and promotes the exit of GC1 and GC2 from the endoplasmic reticulum and their trafficking to photoreceptor outer segments. Adeno-associated viral vector delivery of the normal RD3 gene to photoreceptors of the rd3 mouse restores GC1 and GC2 expression and outer segment localization and leads to the long-term recovery of visual function and photoreceptor cell survival. This review focuses on the genetic and biochemical studies that have provided insight into the role of RD3 in photoreceptor function and survival.Retinal degeneration 3 (RD3) is an evolutionarily conserved 23 kDa protein expressed in rod and cone photoreceptor cells. Mutations in the gene encoding RD3 resulting in unstable non-functional C-terminal truncated proteins are responsible for early onset photoreceptor degeneration in Leber Congenital Amaurosis 12 patients, the  In the dark the basal catalytic activity of GC in photoreceptor outer segments (OS) is balanced by the basal activity of phosphodiesterase (PDE) to maintain cGMP at a level sufficient for maintaining a significant fraction of cGMP-gated channels in their open state. This allows the influx of Ca0\u2013500 nM . At thisal level . Followixchanger . The redation GC . The incGUCY2D gene encoding human GC1 cause Leber Congenital Amaurosis (LCA) Type 1 (LCA1), a severe early onset retinal dystrophy, and cone-rod dystrophy  and structural organization . They coystrophy .rd3 mouse, rcd2 collie, and LCA12 patients has been shown to play a crucial role in the trafficking of GC1 and GC2 in photoreceptors (Photoreceptor cells are highly polarized neurons with the OS segregated from the rest of the cell by a thin cilium. OS proteins must be efficiently transported from the endoplasmic reticulum (ER) in the inner segment through the cilium since OS turnover every 10 days through the phagocytosis of aged OS membranes by retinal pigment epithelial cells and the addition of new membrane at the base of the OS . The moleceptors . In thisrd3 mouse was first identified as an uncharacterized transcript in an in silico search of retina-specific transcripts and called C1orf36 for Chromosome 1 open reading frame 36 (RD3 consists of 3 exons spanning the 5\u2032 and 3\u2032 untranslated region with the open reading frame comprised of part of exon 2 (amino acids 1\u201399) and exon 3 (100\u2013195). RT-PCR and in situ hybridization confirmed the presence of RD3 in the retina with high expression in the photoreceptors.The gene responsible for retinal degeneration in the frame 36 . The genRD3 gene is highly conserved across vertebrates with the human protein consisting of 195 amino acids and sharing 95% sequence identity with other primates, 86% with mouse and rat, 83% with bovine, 67% with chicken, and 50\u201360% with lower vertebrates including Danio rerio (Zebrafish) and Xenopus tropicalis (Western clawed frog). Computer algorithms indicate that RD3 lacks any known homology domains or transmembrane segments. RD3 is predicted to have a high \u03b1-helix content (~43%), no \u03b2-sheets and considerable disordered regions . There are four conserved stretches of predicted \u03b1-helices (H1\u2013H4) with the first helix consisting of 34 amino acids and the last helix having 39 amino acids. Additional conserved features include a putative coil\u2013coil region between amino acids 22\u201354 and several predicted phosphorylation sites.The RD3 protein encoded by the RD3 has been isolated from retinal homogenates and HEK293 cells and bacteria expressing the recombinant protein . On SDS rd3 mouse was one of the first naturally occurring murine strains found to display early onset rod and cone degeneration  in a strain of collies  mice by western blotting and confocal microscopy 30RK/J strain 4Bnr rd3 mice were similar to age-matched WT mice. Up-regulation of endothelin 2 (EDn2), glial fibrillary acid protein (Gfap), and complement component factor 1 (Cf1) and down-regulation of phosducin (Pdc), gap junction protein \u03b15 (Gja5) and retinal G protein \u2013 coupled receptor (Rgr) genes, however, were observed for the rd3 retina , GC1 and GC2 expressed at levels comparable to that of WT mice and localized normally to the photoreceptor OS layer 30RK/J strain of croscopy . GC1 wasOS layer . CollectThe effect of RD3 expression on the localization of GC was also examined in transfected culture cells . GC1 pri2+ sensitivity of GCAPs. It remains to be determined if RD3 plays a significant role in modulating the activity of GCs during phototransduction. It is possible that RD3 only plays an important role in inhibiting cyclase activity during the trafficking of GCs within the inner segments. At present the quantity of RD3 in photoreceptors remains to be determined, but in in vitro measurements, RD3 at nanomolar concentrations competitively inhibited the activation of GC by GCAP  expressed at levels comparable to WT RD3 and bound GC1. The F100ter mutant had no effect of the cyclase activity, whereas the missense mutations inhibited GC activity to varying degrees. The W6R/E23D and G35R mutations inhibited the cyclase activity at a similar concentration as WT RD3, whereas the R68W, K130M and G57V were less effective. It remains to be determined if these missense mutations in RD3 alter the ability of RD3 to function in GC trafficking in photoreceptors.The effect of the RD3 mutation (F100ter) associated with LCA12 and other possible disease-linked missense mutations on the interaction of RD3 with GC was studied . The F10Figure 2). RD3 may also be involved in the trafficking of GC within the cilium although this remains to be determined.Studies carried out to date suggest that RD3 serves two roles in photoreceptor cells. A primary function of RD3 is to facilitate the exit of GC from the ER and its trafficking to the photoreceptor cilium (rcd2 collie showed a 10 times higher level of cGMP than control retinas during a 2\u20138 week period with only 25% reduction in PDE activity (A second function of RD3 is to inhibit GC catalytic activity . The inhactivity .2+ levels can be toxic to cells (2+ through the cGMP-gated channels and the efflux through the Na/Ca-K exchanger. In the absence of GC in the OS, cGMP will not be produced resulting in permanent closure of cGMP-gated channels and a reduction in intracellular Ca2+ below a threshold required for photoreceptor survival. In this case the mechanism of photoreceptor degeneration in the rd3 mouse and LCA12 patients would be similar to photoreceptor degeneration in the GC1/GC2 knockout mouse and LCA1 patients. Direct comparison of the rate of photoreceptor degeneration in the rd3 mouse and GC1/GC2 knockout mouse is complicated by the large variation in the rate of degeneration observed for different strains of rd3 mice, although in general degeneration in the rd3 mouse appears to be more rapid. Comparison of LCA12 with LCA1 patients is complicated by the presence of functional GC2 in rod photoreceptors of LCA1 patients. However, limited clinical assessment of these patients suggests that LCA12 is more severe (The mechanism by which loss in RD3 causes photoreceptor degeneration remains to be determined. Here we discuss two possible mechanisms. The first mechanism is centered on the role of GC and cGMP in maintaining calcium homeostasis in photoreceptors. It is generally known that too high or too low Cato cells . Calciume severe .rd1 and rd10 mice linked to mutations in the \u03b2-subunit of PDE (A second mechanism involves the role of RD3 in the inhibition of GC activity . In the t of PDE , mice det of PDE , and trat of PDE . Both thThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Considered an epicenter of pandemic influenza virus generation, southern China has recently seen an increasing number of human H7N9 infections. However, it is not the only threat. On 30 November 2013, a human H10N8 infection case was first described in China. The origin and genetic diversity of this novel virus is similar to that of H7N9 virus. As H10N8 avian influenza virus (AIV) was first identified from a duck in Guangdong Province during 2012 and there is also evidence of H10N8 infected dogs in this region, we sought to examine archived sera from animal workers to see if there was evidence of subclinical human infections before the first human H10N8 cases.We studied archived serum samples  collected between May and September 2013 from 710 animal workers and 107 non-animal exposed volunteers living in five cities of Guangdong Province. Study participants\u2019 sera were tested by horse red blood cells (RBCs) hemagglutination inhibition (HI) and microneutralization (MN) assays according to World Health Organization guidelines. The A/Jiangxi-Donghu/346-1/2013(H10N8) virus was used. Sera which have an HI assay \u22651:20 were further tested with the MN assay. Questionnaire data were examined for risk factor associations with positive serological assays. Risk factor analyses failed to identify specific factors associated with probable H10N8 infections.Among the 827 sera, only 21 animal workers had an HI titer \u22651:20 (18 had an HI titer of 1:20 and 3 had an HI titer of 1:40). None of these 21 subjects reported experiencing any influenza symptoms during the three months before enrollment. Among the three subjects with HI titers of 1:40, two had MN antibody titers of 1:40, and one had a MN antibody titer of 1:80 (probable H10N8 infections).Study data suggest that animal workers may have been infected with the H10N8 virus before the first recognized H10N8 human infection cases. It seems prudent to continue surveillance for H10N8 viruses among animal workers. Located in southern China, Guangdong Province is home to some of the world\u2019s largest populations of humans, chickens, ducks and pigs and has been associated with human outbreaks of severe acute respiratory syndrome (SARS) and highly pathogenic H5N1 avian influenza infections. This region of China has been considered an epicenter of novel influenza virus generation . In receAs H10N8 AIV was first identified in a duck from Guangdong Province in 2012  and therThe animal worker sera were collected during the period June to August 2013 during a surveillance program for novel zoonotic influenza virus among animal workers living in five cities of Guangdong Province. Non-animal-exposed participants\u2019 sera were similarly collected during the period May to August from middle school teachers and students in Guangzhou and Foshan cities who were healthy, reported no history of having received an influenza vaccine or having direct contact with swine or poultry, during the six months before enrollment. This study was approved by Guangdong Centers for Disease Control and Prevention and begun in early 2013. Study subjects were screened by telephone call and enrolled by informed consent. Participants completed a questionnaire which collected various relevant data including demographic, recent clinical signs and symptoms, and animal occupational exposure history.Sera were first screened by a horse red blood cells (RBCs) hemagglutination inhibition (HI) assay against influenza virus A/Jiangxi-Donghu/346-1/2013 (H10N8). Sera with HI titers \u22651:20 were further studied with microneutralization (MN) assays against the same virus. It is important to note that horse RBCs show a high proportion of sialic acid \u03b12,3-Gal binding, which is preferential for AIV. It had been observed that the use of horse RBCs significantly increased the sensitivity of detection of HI antibodies in the sera of confirmed H7N9 cases compared with the use of turkey RBCs, thus the World Health Organization recommends that horse RBCs should be used to detect HI antibodies for H7N9 virus infection [50 of virus were added to the serially diluted serum at a 1:1 ratio (V/V) and incubated at 37\u00b0C for one hour. Finally, 0.2\u00a0ml of the virus-serum mixtures was transferred to 96-well monolayer plates and incubated in 5% CO2 at 37\u00b0C for 72\u00a0hours. Three wells were run for every dilution of each serum sample. We observed the pathogenic effects every day and tested the cell supernatants with a HA test to confirm the infection.Both assays were performed according to the World Health Organization guidelines . During Study sera which had titers <1:20 in endpoint serum dilution against H10N8 antigens used in this study were considered to be negative. As there was no established HI titer standard to detect a mild or asymptomatic human H10N8 infection, and the positive-control samples have shown HI antibody titers ranging from 1:40 to 1:1280, in this seroepidemiological study we considered sera with HI titers above 1:40 as having possible evidence of previous H10N8 virus infection but only those sera with both HI and MN titers \u22651:40 as having probable evidence of previous infection with influenza A(H10N8) virus.Archived sera and data from 827 poultry workers, poultry retailers in agricultural-trade markets, swine workers, veterinarians, slaughterhouse workers, zoo workers and non-animal-exposed volunteers were studied Table\u00a0. The totHI titers of \u22651:20 were detected in 21 (2.54%) of 827 study subjects (18 with a HI titer of 1:20 and 3 with a HI titer of 1:40). None of these 21 participants  reported having influenza symptoms during the three months before enrollment. Three of these 21 subjects also had MN antibody titers \u22651:40 against H10N8 AIV  and more than 70 human cases with 50% mortality have since then been reported in mainland China . Since 2Our retrospective serologic study is the first to document serological evidence of human asymptomatic infection with H10N8 influenza virus among animal workers before 30 November 2013 (the time of the first index human H10N8 AIV case ) and mayThe results of the present study suggest that workers occupationally exposed to poultry may be at risk of H10N8 AIV infection. It is reassuring that more animal workers were not found to be seropositive and that these findings are consistent with other studies of H7N9 and H5N1 infections among China\u2019s poultry workers. However, it seems appropriate that more extensive serological investigations regarding asymptomatic or subclinical H10N8 infections should be performed among groups expected to be at high risk .Study data suggest that animal workers may have been infected with the H10N8 virus before November 30, 2013, when the first human H10N8 case was recognized. It seems prudent to continue surveillance for H10N8 viruses among animal workers.This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention."}
+{"text": "Changes in the expression and activity of PDK1 and several AGC kinases have been linked to human disease, including cancer.PIK3CA mutation status.We used immunohistochemical analysis to determine PDK1 expression in 241 tumors from patients with breast cancer in which we had previously analyzed PIK3CA mutation status and PDK1 overexpression.Moderate or high expression of PDK1 was observed in 213 of the 241 cases (88%). There was no correlation between Our findings indicate that PDK1 is independently activated in breast cancer and not only as part of the PIK3CA pathway, suggesting that PDK1 plays a specific and distinct role from the canonical PIK3/Akt pathway and promotes oncogenesis independently of AKT. Our data implicate PDK-1 and downstream components of the PDK-1 signaling pathway as promising therapeutic targets for the treatment of breast cancer. The protein kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a key role in signaling pathways activated by several growth factors and hormones. PDK1 functions downstream of phosphoinositide 3-kinase (PIK3) and activates members of the AGC family of protein kinases such as protein kinase B (Akt), protein kinase C (PKC), p70 ribosomal protein S6 kinases, and serum glucocorticoid-dependent kinase by phosphorylating serine/threonine residues in the activation loop. AGC kinases are known to play crucial roles in the regulation of various physiological processes relevant to metabolism, growth, proliferation, and survival.PTEN +/- mice and the resulting mice with deficient PDK1 levels had a reduced prevalence of tumor development. Subsequent studies demonstrated the role of PDK1 in a variety of different cancers; in particular PDK1 appears to play a decisive role in the development of breast cancer [The first evidence that PDK1 might be a viable target in cancer came in 2005 when Bayascas et al. [PIK3CA displayed a reduced dependence on Akt for tumorigenicity, and instead relied on PDK1-dependent activation of another AGC kinase, SGK-3. Breast cancers are known to be a group of diverse diseases, and cellular heterogeneity has been shown to affect disease-free survival in patients with breast cancer [Increased PDK1 expression has also been reported in 45% of patients with acute myeloid leukemia, and PDK1 seems to be a viable target in head and neck cancer, multiple myeloma, pancreatic cancer, and colorectal cancer -7. Vasudt cancer . For exat cancer . MoleculPIK3CA mutations, but also in the absence of these genetic alterations [There is accumulating evidence that PDK1 is overexpressed in particular cancer settings and activates cancer cell growth and survival independent of Akt signaling. These findings suggest that PDK1 is not just an Akt-activating player, but rather an important oncogenetic regulator and a potential therapeutic target in cancer. Recently, it has been shown that PDK1 regulates anchorage-independent growth, resistance to several anticancer drugs, and tumor formation in breast cancer cells\u2013not only in tumors harboring erations .PIK3CA mutations?This study aimed to answer the following questions: (1) Is PDK1 overexpressed in breast cancer and to what extent? (2) Is there any correlation between PDK1 overexpression and PIK3CA mutations [To address these questions, we examined the phosphorylation status of PDK1 in a group of tumors in which we had previously analyzed utations . Four tiPIK3CA mutations were identified in 15.8% of cases in the same patient collective. There was no correlation between PIK3CA mutation status and PDK1 overexpression.Moderate or high expression of PDK1 was observed in 213 of the 241 cases (88%). The fact that some cases without PIK3CA mutations had a moderate or high expression of PDK1 suggests that PDK1 can be independently activated in breast cancer and not only as part of the PIK3CA pathway. Our results indicate that PDK1 plays a specific role distinct from the canonical PIK3/Akt pathway.PIK3CA mutation status and PDK1 expression in tissues from the same tumor group. The results of our study suggest that the PDK-1 signaling pathway might be a promising therapeutic target for the treatment of breast cancer and that PDK1 expression should be tested in addition to PIK3CA status in patients with breast cancer prior to beginning therapy.To the best of our knowledge this study is the first to compare PDK1: 3-phosphoinositide-dependent protein kinase-1; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha.The authors declare that they have no competing interest."}
+{"text": "Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1\u0394DIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1\u0394DIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1\u0394DIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion complex.Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in  Plasmodium, Toxoplasma, and Eimeria genera, for example, are the etiological agents of malaria, toxoplasmosis, and coccidiosis, respectively. P. falciparum causes the most severe cases of human malaria . Sp. SpTgAMAl groove . TgRON2Dine loop . Importaine loop , and alscontacts . The difTgRON2D3 . Togethein silico docking experiments and corresponding proof of principle invasion inhibition studies [High affinity AMA-RON2 complexes anchor the junction between apicomplexan parasites and host cells and play an important role in invasion , 14, 36. studies , 41\u201344.  studies , 23, 24.T. gondii AMA1 where the flexible region of the DII loop was replaced with a short Gly-Ser linker (TgAMA1 selectivity. Notably, TgAMA1 bound both TgRON2D3 and EtRON2D3 but only the former with high affinity, while TgAMA1\u0394DIIloop showed moderate binding to each tested RON2D3 and bound both TgRON2D3 and EtRON2D3 with high affinity (TgAMA1\u0394DIIloop in complex with EtRON2D3. Notably, this is the first cross-genus AMA-RON2 pair to be structurally characterized (TgAMA1-TgRON2D3 and TgAMA1\u0394DIIloop-EtRON2D3 structures enabled the identification of key anchor points in the AMA1 groove exploited to outcompete the DII loop and bind TgAMA1 with high affinity (PfAMA1 [T. gondii and P. falciparum.Initially, we engineered a form of r linker . Solutioaffinity . To estacterized . A compaaffinity . Intrigu (PfAMA1 , 45. ThePlasmodium, and it is even suggested that a complex between AMA1 and a host surface protein may support a pre-invasion step [TgAMA1 that the \u0394DIIloop construct displays a more permissive binding profile. Furthermore, engineering parasites to express \u0394DIIloop constructs of AMA1 will enable the investigation of potential DII loop dependant signalling events associated with high affinity AMA1-RON2 binary complex assembly and moving junction formation.The biological implications of truncating the DII loop have proven difficult to assess. However, since the presence of the AMA1 DII loop results in a more selective receptor, this substructure may act as a structural gatekeeper to restrict formation of unproductive AMA1 complexes. Thus, a more discriminating AMA1 apical groove would ensure the parasite-host cell junction is competent for invasion because only a cognate RON2 would bind with sufficient affinity to outcompete the DII loop. This gatekeeping function may ultimately serve as a molecular switch to signal the parasite that a functional junction has been formed and invasion can proceed. It follows that an apicomplexan parasite expressing the truncated DII loop form of AMA1 might experience more abortive invasion events resulting from unproductive, non-RON2 based AMA1 complexes that are not linked to junction integrity. The potential for AMA1 to form non-RON2 based complexes has been proposed \u201350, partion step . In thisTgAMA1 and TgAMA1\u0394DIIloop reveal a previously underappreciated role for the DII loop in selectively filtering out ligands otherwise capable of binding in the AMA1 apical groove. Companion structural studies offer important insight into molecular recognition thresholds that likely need to be achieved by ligands able to outcompete the DII loop and form high affinity complexes capable of anchoring the moving junction during invasion of the host cell. While the biological implications of apicomplexan AMA1 DII loop conformational changes remain an intriguing topic for future studies, these results provide a molecular basis for understanding AMA1 selectivity, which can support the development of novel therapies targeting the important AMA1-RON2 invasion complexes.The solution binding studies of several RON2 peptides with"}
+{"text": "Wolfram syndrome 1 (WFS1) is a genetic disorder which has been associated both with impaired early brain development and neurodegeneration. Recent studies have suggested regulation of Ca2+ homeostasis by wolframin (Wfs1) and have demonstrated the involvement of endoplasmatic reticulum (ER) stress in Wfs1 deficiency. Despite the ER dysfunction WFS1 shows several characteristics of pathologies related to mitochondrial dynamics. Therefore our aim was to examine the hypothesis that Wfs1 deficiency could disturb mitochondrial dynamics contributing to impaired neuronal functioning. First we show that Wfs1 deficiency induces mild ER stress leading to Inositol 1,4,5-Trisphosphate Receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn alters mitochondrial trafficking, inhibits mitochondrial fusion and augments mitophagy. The overexpression of the active IP3R fragment restores IP3R-mediated Ca2+ release and corrects all perturbations in mitochondrial dynamics suggesting that these events are causally linked. We further demonstrate that suppressing the expression of two Parkinson disease-related proteins, Pink1 and Parkin, leads to reduced Wfs1 deficiency-induced mitophagy and also to the correction of the fusion-fission dynamics and mitochondrial motility. These data suggest that Wfs1 deficiency may over-activate Pink1 and Parkin pathways. Our most important discovery is that Wfs1 deficiency delays neuronal development and axonal growth in primary rat cortical neurons. According to our data, the link between Wfs1 deficiency and delayed neuronal development appears to be mediated by impaired mitochondrial dynamics because suppression of the Pink1-Parkin pathway corrected also the developmental delay. Our data shed light on the mechanisms of neuronal abnormalities in WFS1 and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases."}
+{"text": "These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei  Antibody-based immunotherapy has improved treatment outcomes in diseases ranging from rheumatologic conditions to cancer, and efforts are underway to optimize these approaches to yield even greater clinical impact. Most immunoglobulins are unable to penetrate into living cells and therefore cannot be used to target key intracellular antigens or pathways, and this has been a critical factor that has limited the expansion of the use of antibodies in molecular therapy. However, a select group of lupus autoantibodies penetrate into living cellsin vitro and in vivo3456An unusual lupus anti-DNA antibody that penetrates cell nuclei without causing any apparent harm to normal cells or tissues, 3E10, has been found to inhibit DNA repair and thereby selectively kill cancer cells that are vulnerable to DNA damage due to BRCA2-deficiencySome cells take up immunoglobulins and immune complexes through Fc-receptor mediated endocytosis810H contains the sequence RGLLLDY, and mutation of the arginine to an aspartic acid residue takes away a positive charge and abrogates DNA binding by 3E10H by mutating an aspartic acid residue to asparagine greatly enhances the affinity of 3E10 for DNAThe ability of 3E10 to bind DNA is critically linked to its ability to penetrate cell nuclei. 3E10 is a cationic antibody, like most anti-DNA antibodies, and this contributes to its ability to bind to negatively charged DNA. For example, CDR3 of the 3E10 VOverall, the previous work strongly suggests that 3E10 scFv penetrates cell nuclei by binding to DNA or its components and then following it into cells through the ENT2 nucleoside salvage pathway. If this hypothesis is correct then 3E10 scFv should not be able to penetrate into cells in the absence of extracellular DNA. Moreover, addition of extracellular DNA would be expected to enhance nuclear uptake of 3E10 scFv. We therefore set out to test our hypothesized model of nuclear penetration by 3E10 scFv by evaluating the impact of the presence of extracellular DNA on efficiency of nuclear uptake of 3E10 scFv as described below.We sought to test the efficiency of nuclear penetration by 3E10 scFv into cells in the absence of extracellular DNA, and for these studies selected the GM02605 human fibroblast cell line because it maintains a high degree of viability (>99%) with minimal cell death even while maintained in culture for several days. With minimal cell death in culture the confounding effects of DNA released by dead cells are minimized. The GM02605 cells were washed with serum free media and then treated with 10\u2009\u03bcM 3E10 scFv for one hour, after which cells were fixed and immunostained for presence of the fragment. Remarkably, 3E10 scFv did not penetrate into most cells. Instead, 3E10 scFv was found only in the nuclei of cells centered around what morphologically appeared to be rare dead cells, with a gradient effect observed with decreased amounts of intranuclear fragment detected with increasing distance from the central dead cell. A representative image demonstrating this effect is shown in To test the hypothesis that a factor released by dead cells enhances nuclear uptake of 3E10 scFv we compared the efficiency of nuclear penetration of the fragment into the GM02605 fibroblasts in the presence or absence of a cell lysate. As shown in We hypothesized that DNA is the critical factor in cell lysate that promotes nuclear uptake of 3E10 scFv. To test this, the GM02605 fibroblasts were treated with 10\u2009\u03bcM 3E10 scFv in the presence of cell lysate that had been passed through a Centricon cellulose filter with a molecular weight cut off of 10,000\u2009kDa to remove DNA content. In contrast to the complete cell lysate, the DNA-depleted lysate did not enhance nuclear uptake of 3E10 scFv , which sTo confirm that extracellular DNA enhances nuclear penetration by 3E10 scFv, we next treated the GM02605 fibroblasts with 10\u2009\u03bcM 3E10 scFv in the presence of purified DNA (0.5\u2009g/L). As shown in in vivo due to an expected higher concentration of extracellular DNA in the tumor vicinity released from dead cells in regions of tumor ischemia and necrosis. To test this, subcutaneous U87 human glioma xenografts were generated in immunodeficient mice, and once tumors grew to size of ~100\u2009mm3 mice were treated with intraperitoneal injection of control buffer or 3E10 scFv. Mice were then sacrificed 4 or 24\u2009hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Four hours after treatment 3E10 scFv was not detected in the cell nuclei of normal tissues including heart, kidney, skeletal muscle, and liver. By contrast, cell nuclei in the tumor xenografts stained positive for presence of 3E10 scFv  grows to confluence in 96-well tissue culture plates with remarkably high viability . Cells were grown in MEM with 15% FCS and washed with MEM without serum before incubation with 10\u2009\u03bcM 3E10 scFv for one hour. Nuclear penetration by 3E10 scFv was then examined by anti-Myc immunostaining as previously describedCOS-7 cell lysate was prepared by subjecting cells to multiple freeze-thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation. DNA-depleted COS-7 cell lysate was prepared by passing the lysate through a Centricon cellulose filter with a molecular weight cut off of 10,000\u2009kDa.Purified calf thymus DNA sheared to an average length of 2000\u2009bp was purchased from Invitrogen .3 mice were treated with intraperitoneal injection of control PBS buffer or 0.8\u2009mg 3E10 scFv in PBS. Mice were sacrificed 4 or 24\u2009hours after treatment, and tumors and selected normal tissues were fixed in formalin and embedded in paraffin. Tissues were then surveyed for nuclear penetration by 3E10 scFv by immunohistochemistry (IHC). Tissue sections were deparaffinized, rehydrated, and incubated at 95\u201399\u2009C for 30\u2009minutes for epitope retrieval. Sections were washed, blocked with peroxidase, and probed with a 9E10 anti-Myc  primary antibody directed at the C-terminal Myc tag in 3E10 scFv followed by additional washes and then incubation with a labeled polymer-HRP secondary antibody . After additional washes color development was performed using DAB followed by counterstaining with hematoxylin. All in vivo studies were conducted in accordance with institutional guidelines. The protocol for the in vivo work was approved by Yale University\u2019s Institutional Animal Care and Use Committee.U87 human glioma subcutaneous xenografts were generated in nude mice as previously describedHow to cite this article: Weisbart, R. H. et al. DNA-dependent targeting of cell nuclei by a lupus autoantibody. Sci. Rep.5, 12022; doi: 10.1038/srep12022 (2015)."}
+{"text": "Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of  Bacillus thuringiensis (Bt) have been used in the field since 1996 and are important tools to control insect pests reducing the use of chemical insecticides [Genetically modified (GM) plants expressing insecticidal proteins from the bacterium cticides  but thoscticides , 4.2, constituting one of the largest areas of agricultural activity in Brazil, as it represents almost 25% of the national territory. Annual cultivation of corn, soybean and cotton in the Cerrado begins in October and extends until June [In 2013, the global area planted with GM Bt plants was 75.9 million ha in different countries . In 2010til June . This arInsects can evolve resistance to Bt toxins in laboratory and field conditions , 9, 10. The area of refuge recommended for the cultivation of GM corn in Brazil, was equivalent to 10% of the total area planted with Bt corn and that it should be located at a distance less than 800 meters from the Bt corn field . FurtherS. frugiperda populations to Cry1Fa Bt corn was previously reported in Puerto Rico [S. frugiperda [S. frugiperda populations to Cry1Fa corn in different regions of Brazil including state of Goi\u00e1s in the Cerrado region was confirmed [Resistance of rto Rico . From 20ugiperda , suggestonfirmed . The resonfirmed .Pectinophora gossypiella on Cry1Ac Bt cotton in India [Busseola fusca on Cry1Ab Bt corn in South Africa [Helicoverpa zea on Cry1Ac Bt cotton in USA [Diabrotica virgifera in Cry3Bb Bt corn in USA [Helicoverpa armigera on Cry1Ac Bt cotton in China [Field-evolved resistance in different insect pests in other regions such as in India , Busseolh Africa , Helicovn in USA  and Heliin China  were alsS. frugiperda population that has evolved resistance to Cry1Fa toxin resulting in the survival of this insect pest in Cry1Fa Bt corn. We analyzed the stability of the resistant phenotype after several generations without selection pressure, studied the cross-resistance of this population to other Cry1A and Cry2A toxins and determined whether Cry1AMod toxins that have been reported as effective for the control of various lepidopteran species resistant to Cry1A toxins [S. frugiperda larvae collected from Brazilian Bt corn fields.The objective of this study was to further analyze the A toxins , 20 coulInsects were collected during 2013 on a farm in the municipality of Cabeceiras in the state of Goi\u00e1s, located in the Cerrado region of West-Central Brazil . The property is about 700 acres where Bt corn, conventional corn and herbicide tolerant soybeans and common beans are grown. Cry1Fa Bt corn had been cultivated for four years and Cry1A.105 corn, Cry2Ab corn and herbicide tolerance corn had been cultivated for the past two years. Insect collection activity and maintenance was authorized by the Ministry of Environment of Brazil (MMA), Instituto Chico Mendes de Biodiversidade\u2014ICMBio, Sistema de Autoriza\u00e7\u00e3o e Informa\u00e7\u00e3o em Biodiversidade-SISBIO Number: 39022\u20131.S. frugiperda larvae were collected in 2013 from Cry1Fa Bt corn variety 30F53H, taken to the insect breeding laboratory at Embrapa Genetic Resources and Biotechnology and maintained individually until pupating in 50 ml plastic cups with acrylic covers containing 10 ml artificial diet [Four hundred ial diet . The pupS. frugiperda originated from mass rearing in Embrapa, and named SfLab. This breeding was implemented in 1988 [The susceptible colony of  in 1988  and the cry1Aa, cry1Ab, cry1Ac, cry2Aa, cry2Ab genes, obtained from HD-1 strain were individually cloned into pHT315 vector [B. thuringiensis serovar. israelensis acrystalliferous strain 4Q7 (Bacillus Genetic Stock Center) [xg for 30 min at 4\u00b0 C. The spore-crystal mixtures were frozen and lyophilized for 18 h in a Labconco Lyphlock model lyophilizer. Crystal protein concentration was measured by using the Bradford reagent from BioRad. Bioassays were carried out by spreading 35 \u03bcl of 10 serial dilutions containing different protein concentrations of Cry1Fa protein or mixture of freeze-dried spore crystals, on the surface of artificial diet previously distributed in 24-well plates. After the protein samples were dried in the diet, one second-instar larvae was placed in each well. We tested 24 larvae per toxin concentration in triplicate, and analyzed ten different concentrations of toxin, making a total of 720 larvae per LC50 value determination. As negative control one plate was left without toxin addition. Three biological replicates were performed. The first reading of mortality was made 48 hours after start of the test, at which time the larvae were transferred to 50 ml plastic cups containing diet without toxin. On the seventh day, the second and final reading was taken [50 was determined using generalized linear models (GLM) with probit link function [50 and the slope of the dose were calculated. This assay is accredited by ISO/IEC 17025. Bioassays were performed with larvae of the F1 generation of SfBt and SfLab during 2013. Bioassays were also performed with larvae from F2 and F8 generations of SfBt colony in 2013 and 2014 respectively, using a diagnostic dose corresponding to 10 times the LC50 dose on the SfLab strain . Bioassays for other Cry and Cry1AMod toxins were performed as described above with larvae from the F8 generation.Bioassays were performed with activated Cry1Fa toxin  and a mixture of spores and crystals from Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, Cry1AbMod and Cry1AcMod produced in Bt transformant strains. The 5 vector  and expr Center) . The Cry Center) . The rec Center)  supplemeas taken , 26. Theas taken , where tanalysis). The conth generation) were used per treatment in three repetitions. Mortality was recorded after 7 days.Bioassays with corn leaves were also analyzed under laboratory conditions. Corn leaves from Cry1Fa corn variety 30F53H were collected during 2013 in the Cabeceiras Cerrado region of Brazil. Leaves from non transgenic plants variety 30F53 were also collected from the same region at the same time. Leaves were taken to the insect breeding laboratory at Embrapa Genetic Resources and Biotechnology and maintained at-20\u00b0C. Frozen leaves were defrost in February 2014 and placed in Petri dishes containing a moisture paper with water. A total of 20 larvae in the second instar from SfLab and SfBt  with 1 mM EDTA and 0.1 mM PMSF. The crystals were separated by sucrose gradient centrifugation [2CO3, 0.2% \u03b2-mercaptoethanol, pH 10.5). The protoxins were activated with trypsin (1:50 w/w)  for 2h at 37\u00b0 C; the reaction was stopped by addition of PMSF . Activated toxins were purified as previously described [The mixture of spores/crystals produced by the recombinant Bt strains was washed three times with PBS buffer , aliquoted and stored at-80\u00b0 C. Enrichment of 4\u20136 fold of ALP enzymatic activity compared to the initial gut homogenate was observed in BBMV preparations.Brush border membrane vesicles (BBMV) from the larval gut were purified according to Wolfersberger et al. , from miThe trypsin activated proteins Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Cry1Fa were biotinylated using the ECL Protein Biotinylation System  kit according to the manufacturer's recommendations. After biotinylation, detection was performed with peroxidase-conjugated streptavidin to confirm the biotin labeling of proteins as previously reported  using stS. frugiperda BBMV were incubated with10 nM of each biotinylated toxin for 30 min at 25\u00b0C in 100 \u03bcl total volume of binding buffer . Unbound toxins were removed by centrifugation . The BBMV containing bound Cry toxins were washed three times in binding buffer and suspended in 15 \u03bcl of 1X PBS and 5 \u03bcl of 4X Laemmli sample buffer . The samples were boiled for 3 min, resolved by SDS-PAGE at 9% and transferred to nitrocellulose membrane as described above. Labeled proteins bound to BBMV were visualized with streptavidin-peroxidase conjugate followed by incubation with SuperSignal chemiluminescence substrate as described above. The optical density of the bands was scanned with ImageJ program and the percentage of each band on the blot was calculated.Binding analyses were performed in February 2014 using BBMV from SfBt and SfLab populations as previously reported . BrieflyS. frugiperda larvae (LC50) from the different populations was determined. 50 value was at least ten times greater in the SfBt population compared with the control population SfLab. The exact LC50 values could not be calculated for the SfBt colony since the highest dose of Cry1Fa that we could assayed was 3500 ng/cm2 and at this concentration the mortality was not enough to determine LC50 values.The Cry1Fa lethal concentration required to kill 50% of 2) 10 times higher than the LC50 obtained with Cry1Fa in larvae of the SfLab colony (The colony SfBt was maintained in the laboratory in the absence of selection pressure for eight generations. Each of these generations was used to determine their susceptibility to a Cry1Fa diagnostic concentration (3,500 ng/cmb colony . At thisIn order to determine whether the level of resistance of the SfBt population to Cry1Fa affects the efficacy of Cry1Fa corn plants, the number of survival larvae of the susceptible SfLab and the Cry1Fa-resistant population SfBt on leaves of Bt corn plants was determined. The larvae of the population SfLab did not survive on leaves from Cry1Fa corn while the F8 generation of SfBt population showed 100% survival. The SfLab and SfBt populations had similar 100% survival on leaves from non-transgenic corn.S. frugiperda populations to other Cry proteins as described in Materials and Methods. The F8 generation of SfBt colony showed LC50 values 30 times higher for Cry1Aa toxin than those of the susceptible SfLab colony (50 values of Cry1Ab and Cry1Ac were 4 and 2.5-fold higher respectively when compared to the values obtained in the SfLab colony. The fiducial limits values for these bioassays do not overlap, indicating that these differences are significant. In contrast, analysis of susceptibility to Cry2Aa and Cry2Ab showed that there are not significant differences in susceptibility to these toxins which showed low or no-toxicity to both SfLab and SfBt insects (S. frugiperda larvae in the Cerrado region of Brazil due to their low toxicity. It is worth to mention that these assays were performed with spore/crystal suspensions. In all cases the same acrystalliferous Bt 4Q7 strain was transformed with the pHT315 vector containing the different cry genes as reported in Material and Methods section. There is evidence in the literature that Bt spores produce virulence factors that could be important for toxicity [Subsequently, we compared the susceptibility of the two b colony . The LC5 insects . These dtoxicity . However50 value of a native toxin divided by the LC50 of the corresponding modified toxin, is very high in the resistant strain since Cry1AbMod show a potency of 8.5 fold higher than the native Cry1Ab toxin and Cry1AcMod of 5.7 fold higher than Cry1Ac against the SfBt population (50 value of a Cry1Fa toxin and of the modified toxins against SfBt (Toxicity assays of Cry1AbMod and Cry1AcMod toxins showed that these toxins are highly active against both the SfLab and the SfBt populations. The Cry1AMod proteins showed an important insecticidal activity against the SfBt population. In the case of the SfLab population Cry1AbMod and Cry1AcMod showed insecticidal effect comparable to that of Cry1Aa . Howeverpulation . It woulnst SfBt  indicate50 of a native toxin divided by the LC50 of a modified toxin (based on data from LCS. frugiperda BBMV is specific [S. frugiperda populations. Previously it was shown that binding of Cry1A and Cry1Fa toxins to specific . We analS. frugiperda BBMV showed that Cry1Fa share binding sites with Cry1A toxins but not with Cry2A [Homologous and heterologous binding competition analysis of Cry1Fa toxin to th Cry2A . We perfS. frugiperda colony with significant resistance to Cry1Fa toxin from individuals collected in a region where Bt corn expressing the Cry1Fa toxin was grown for four years. Recently, a parallel study also reported high resistance levels of S. frugiperda populations to Cry1Fa corn in the same Brazilian region [In this work we report the establishment of a n region . Our datS. frugiperda larvae. It remains to be determined if the SfBt population is capable of surviving in other crops expressing Cry1Ab, Cry1Ac or Cry2Ab toxins. Our data indicated no cross-resistance to Cry2A toxin and no susceptibility to Cry2Ab. Due to the low susceptibility of S. frugiperda to Cry2Aa and Cry2Ab toxins it is expected that GM plants expressing Cry2Ab are not likely to be effective in controlling Cry1Fa resistant insects. Resistance of S. frugiperda to Cry1Fa corn was previously documented in Puerto Rico [S. frugiperda resistant population from Puerto Rico showed a similar cross-resistance pattern to Cry toxins to the SfBt reported here, with low cross-resistance to Cry1Ab and Cry1Ac and no cross-resistance to Cry2Ab toxin [Heliothis virescens [Plutella xylostella [B. thuringiensis var. kurstaki strain that expresses the three Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa toxins while XenTari express Cry1C and Cry1D that are toxic to S. frugiperda [S. frugiperda populations. Here we show that Cry1Aa is highly toxic to the susceptible strain of SfLab (S. frugiperda populations from different Latin American regions showed different susceptibility to selected Bt toxins [The resistance mechanism in SfBt associated with reduced binding of Cry1Fa to BBMV, suggesting that the resistant allele may be affecting expression of a Cry1Fa binding molecule. However, these data remain to be confirmed by further studies for Cry1Fa protein receptors identification. The Cry1Fa resistant population SfBt showed high cross-resistance to Cry1Aa, and low cross-resistance to Cry1Ab and Cry1Ac toxins. The latter proteins are expressed in other transgenic plants used in Brazil, including cotton and soybean, which are also host plants for rto Rico . The S. Ab toxin . These direscens , and NO-lostella . It was lostella . DiPel iugiperda . The higof SfLab . It was t toxins  suggestiH. virescens [Helicoverpa armigera, Helicoverpa zea, Spodoptera exigua [Ostrinia nubilalis and also in S. frugiperda [Competition binding assays have shown that Cry1As and Cry1Fa share binding sites in different Lepidopteran insects such as irescens , Helicova exigua  Ostriniaugiperda , 40. CroS. frugiperda BBMV, but we showed here that these toxins showed low toxicity to SfLab or SfBt and did not compete the binding of Cry1Fa to SfLab BBMV. These data indicate that these toxins would not be an alternative to control Cry1Fa resistant populations. Other examples showing lack of correlation between Cry toxin binding to BBMV and insect susceptibility were previously reported [As previously reported , we founreported , 42. Alsreported \u201346.50 value of Cry1AMod toxins to SfBt is comparable to the LC50 value of Cry1Fa to SfLab population. The increase in potency ratio is higher than the resistance ratio of both Cry1AMod toxins indicating that even though that SfBt show cross resistance to Cry1AMod, these Cry1AMod toxins are effective in countering resistance of S. frugiperda to Cry1Fa.The SfBt population showed a significant cross resistant ratio of 3.1 fold to Cry1AbMod and 5.6 fold to Cry1AcMod toxins . It is iS. frugiperda larvae (S. frugiperda molecule involved in Cy1F toxin binding and oligomerization.In the model of the mechanism of action of Cry1A toxins, it was proposed that binding to a membrane bound cadherin receptor facilitates removal of the N-terminal region of the toxin including helix alpha-1 and part of alpha-2 resulting in oligomerization of the toxin . Cry1AMoa larvae . In thisS. frugiperda from other regions of Brazil should be determined in the future. The data presented here showed the rapid evolution of insect resistance to Bt crops in Brazil and is a warning that in the future a proper use/management of Cry toxins would be needed. Also, we showed that the use of Cry1AMod toxins could provide alternative choices to target S. frugiperda and possibly manage resistance to Cry1F.The analysis of resistance mechanisms and the effectiveness of Cry1AMod toxins in other populations of"}
+{"text": "S2 FigInternal fragments of the p48 , type II restriction endonuclease  and xer1  genes were amplified from M. bovis strain PG45 with appropriate primers and inserted between the NotI and PstI sites of the IRR based oriC plasmid. To promote homologous recombination, the recA gene was amplified from M. gallisepticum strain S6 and cloned between the PstI and SalI cleavage sites of the construct.(PPT)Click here for additional data file."}
+{"text": "CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the rd8/rd8Crb1 mutation, we compared the retinal pathology of rd8/rd8/JCrb1 inbred mice with that of two rd8/rd8Crb1 lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three rd8/rd8Crb1 lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, M\u00fcller and microglia activation and telangiectasia-like vascular remodelling\u2014features that were stable in the inbred, variable in the second, but virtually absent in the third rd8/rd8Crb1 line, even at 12 months of age. This suggests that the rd8/rd8Crb1 mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype\u2013phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.Understanding phenotype\u2013genotype correlations in retinal degeneration is a major challenge. Mutations in  Understanding the genotype\u2013phenotype correlation in retinal degenerations remains a major challenge even for monogenetic diseases. Although the causality of many primary mutations has been convincingly established, the clinical manifestation of even identical mutations can be highly variable. This may be due to additional genetic polymorphisms in the same gene  or in additional genes (genetic modifiers) as well as environmental factors. The combination of all these factors in a patient may influence age of onset, progression and severity of disease as well as the development of particular phenotypic features associated with a primary mutation ,2.CRB1 gene (OMIM #604210). They typically cause a spectrum of autosomal recessive (ar) rod-cone dystrophies that ranges from retinitis pigmentosa (RP12)    to Leber OMIM #60210. Theya (RP12) . Both arlopathy) \u20137. So faCA cases \u20137. This  disease ,6,8.CRB1 is a member of the highly conserved CRB protein family that in mammals comprises two additional members, CRB2 and CRB3 ,10. WhilIn the retina, CRB1 is part of the CRB/Crb complex, which is localized at the outer limiting membrane (OLM) where it sits just apically to the adhesion junctions (AJ) that connect photoreceptors with each other and with M\u00fcller glia cells . The CRBCrb1 alleles exist  that causes a frame shift and premature stop codon resulting in a predicted truncated protein that only consists of the N-terminal extracellular domain  and (2)]. Autofluorescent lesions in C57BL/6 Crd8/rd8rb1 (1) mice were small, weak, reduced in number and located more inferiorly  mice, but with a high degree of variability  mice only small inferior lesions were visible , AF-SLO, optical coherence tomography (OCT) and semithin histology revealed that this pathology was mainly confined to the inferior retina of ice Fig.\u00a0. White/oice Fig.\u00a0A\u2013C. Corrble Fig.\u00a0D that weble Fig.\u00a0D since tble Fig.\u00a0E.FigureCrb1rd8 allele, possibly by altered expression of the predicted truncated CRB1 protein . Crb1 transcripts were significantly reduced to \u223c20% of wild type in all rd8/rd8Crb1 mice, independent of the severity of their phenotypes . Consistent with these data, immunohistochemistry using a C-terminal antibody for Crb1 revealed a weak specific signal across the whole superior and inferior OLM in wild-type retina  mice is macroscopically similar to that of wild-type mice  mice  lines. Also the expansion of the glial component at the OLM seems similar at the inferior and superior OLM within each line, but different between them  per 10 \u00b5m in wild-type mice to 12 \u00b1 3 per 10 \u00b5m (range: 7\u201314) in the superior and inferior retina of \u201318 per 1rd8/rd8Crb1 mice lead us to further investigate an association between photoreceptor death and M\u00fcller and microglia activation  and TUNEL+ photoreceptor nuclei in sections  mice. Without exception, these areas were closely associated with localized activation of microglia  and M\u00fcller glia cell . However, no significant differences in expression of chemokine  or microglia activation marker  were detected in retinas of different rd8/rd8Crb1 lines relative to wild type supporting a localized response of microglia cells to these degenerative events . In all these assessments, rd8/rd8C57BL/6 Crb1 (1) animals appeared similar to wild-type mice with the exception of the rare appearance of subtle aneurysm-like changes at the OPL . Three-dimensional (3D) reconstruction of large inferior lesions revealed how activated M\u00fcller and microglia cells surround several photoreceptor columns at the OPL  lines showed again the prominent irregular lesions in the inferior retina  line still only revealed very few small or even no inferior lesions   mice at 2 and 12 months of age did not show prominent vascular remodelling or microglia activation apart from a rare manifestation of a few small aneurysms at the OPL  . As earlbed Fig.\u00a0. In contrd8/rd8/JCrb1 line, and the common ancestry of the rd8/rd8C57BL/6 Crb1 lines (1) and (2), there is a significant variation in the observed phenotype. We reasoned therefore that a modifying gene or genes may have been segregated between the lines. To test this, we first determined whether the different mouse lines carry any known mutant alleles commonly found in inbred mouse strains (rd1Pde6b (c.1041C > A) allele  (Cpfl3Gnat2 (c.598G>A) ], that has been previously shown to be a genetic modifier for retinal degeneration  allele , two mut.518A>G)  and Gnat.598G>A) , and theneration . None ofrd8/rd8/JCrb1 line for which we observed a higher proportion of C3H  SNPs to be present than in the two other rd8/rd8C57BL/6 Crb1 lines (data not shown) suggesting that there are distinct genetic differences between the inbred rd8/rd8/JCrb1 line and the two rd8/rd8C57BL/6 Crb1 lines (1) and (2) that are of common ancestry. Based on this common ancestry, and the distinct phenotypic differences shown by rd8/rd8C57BL/6 Crb1 line (1) and rd8/rd8C57BL/6 Crb1 line (2), we decided to generate preliminary data to help identify regions in the genome that may contain modifying factors. We therefore performed an \u2018analysis of extremes\u2019 using DNA SNP screen data from animals that are from either of the two related rd8/rd8C57BL/6 Crb1 lines (1) and (2), but show the most divergent (extremes) of the AF-lesion phenotype. Statistical analysis of this dataset identified a significant association between the genotype on a region of chromosome 15 and the phenotype , whereas mice with a high AF-lesion number are either heterozygous or homozygous for alternative SNPs. These data suggest that this region on chromosome 15 may carry one or several genetic modifiers that determine the manifestation of the phenotype associated with the rd8/rd8Crb1 mutation.To provide further insight into a potential genetic basis for the phenotypic variability, we decided to perform DNA SNP screen analysis for animals from the different lines. The SNP data confirmed the inbred status of the  (1) and  that arerd8/rd8Crb1 mutation and present initial genetic data from SNP screen analyses that suggest the existence of genetic modifiers on chromosome 15 that modulate the manifestation of the rd8/rd8Crb1 phenotype in mice. These findings indicate that this mutation alone is necessary, but not sufficient for the manifestation of the inferior retinal degeneration and specific associated features, which were only observed in two of the three evaluated homozygous rd8/rd8Crb1 lines. This study also highlights how pronounced the influence of small genetic background differences can be on the manifestation of this retinal degeneration and presents initial data for a candidate region that may carry the underlying genetic modifier(s). This is an important issue since this mutation has been found in many mouse strains across the world  occurs. NMD is a mechanism that results in the degradation of mRNA transcripts that carry premature stop codons and serves as a quality control to prevent the expression of deleterious mutant proteins  over multiple generations. Furthermore, our observation that the two closely related rd8/rd8C57BL/6 Crb1 lines (lines 1 and 2), still differ significantly in their phenotype suggests that a few genetic factors or even a single factor may be responsible for the modifying effects in the different lines. By using an \u2018analysis of extremes\u2019 on the SNP data obtained from animals with the most divergent phenotypes from the two related rd8/rd8C57BL/6 Crb1 lines (1) and (2), we identified a large region on chromosome 15 that is significantly associated with the phenotype and thus may carry such genetic modifiers.Because of the inferior nature of the degeneration, light was considered to be a good candidate for such an additional initiating factor for this type of retinal degeneration. Yet, this hypothesis was recently excluded by the observation that both, nditions ,20. Sincckground . Our stuC57BL/6 allele, animals with a many lesions are either heterozygous or homozygous for an alternative allele. Therefore, the modifying factor(s) on chromosome 15 may act either as recessive suppressor(s) or as dominant enhancer(s) for the manifestation of the rd8Crb1/rd8 phenotype. However, our data do not allow us to conclude with certainty about the origin of the modifying factor(s) from either of the genetic backgrounds since it could be the result of a spontaneous mutation in any of the alleles. To answer these questions and gain mechanistic insight into the function of these additional genetic factors, a more extensive mapping or QTL project would be required to confirm our initial results and narrow down the genomic region on chromosome 15 which may then result in the identification of the genetic modifying factor(s) for the rd8/rd8Crb1 phenotype.While animals with few retinal lesions are homozygous for one allele, consistent with a Crb1 pathway, or alter processes that define the threshold of M\u00fcller glia or microglia responses. A general innate immune activation during disease progression was indicated by the increased accumulation of subretinal macrophages in all rd8/rd8Crb1 mice during later stages. Additional modulating factors that further alter such inflammatory or gliotic responses may lead to an even more permissive inflammatory and pro-angiogenic environment in the retina and may result in vascular remodelling and additional degenerative features. However, the question of why the inferior retina is preferentially affected by these processes remains unresolved. Nevertheless, it is of particular interest that the localized aneurysms- and telangiectasia-like vascular lesions in rd8/rd8Crb1 mice were clinically similar to retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations and that the manifestation of these lesions in humans also seems to be dependent on genetic modifying factors  was compared with two genetically related homozygous rd8/rd8Crb1 lines obtained from a backcross experiment with C57BL/6J Ola Hsd mice, referred to as rd8/rd8C57BL/6J Crb1 (1) and rd8/rd8C57BL/6J Crb1 (2), respectively  amplification with flanking primers and subsequent sequencing  and ketamine (60 mg/kg body weight). Pupils were dilated with 1% tropicamide. Animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the UK Home Office licence (PPL 70/1279).To minimize environmental differences, all lines were housed under the same 12 h/12 h dark/light cycling. During light, animals were exposed to an illuminance of 38 \u00b1 28 lux , when lids, food and water bottles were in place. Outside the cages 200 \u00b1 136 lux (range 50\u2013500 lux) were measured .As described previously, autofluorescent fundus images and OCT images were obtained using the HRA2 scanning laser ophthalmoscope with a 55\u00b0 angle lens and the Spectralis\u2122 HRA+OCT with a 30\u00b0 angle lens, respectively  . The optThe severity of the phenotype  and the number of subretinal macrophages  were determined by counting respective lesions on images focussed on the superficial retina for pathology or on the outer retina for subretinal macrophages.m sodium cacodylate\u2013HCl (pH 7.4) and processed for embedding in araldite resin . Semithin (0.7 \u00b5m) and ultrathin sections (70 nm) were stained accordingly and imaged by bright field microscopy  or transmission electron microscopy (TEM) with a JEOL 1010 connected to a Gatan Orius CCD camera. Calibrated images were imported into Image J for quantification . After counterstaining with Hoechst 33342  and mounting in fluorescent mounting medium (Dako), images were taken by confocal microscopy . High-resolution Z-stack images were used for 3D reconstruction with Imaris .Tissue was fixed and dissected in 4% PFA, cryoprotected with 20% sucrose and embedded in OCT . Eighteen micrometres of retinal sections or flat mounts were blocked with 1% BSA /5% goat serum  including 3% (flat mounts) or 0.3% (sections) Triton X-100 for permeabilization followed by overnight incubation at 4\u00b0C with respective primary and secondary antibodies  per animal were analysed on an SNP array containing 1449 allele-specific probes from across the genome distinguishing m of each primer and 100 nm of the corresponding FAM-labelled probe  followed by cDNA synthesis with the QuantiTect Reverse Transcription kit (Qiagen). For Real Time PCR analysis, 20 \u03bcl reactions were prepared in triplicates containing the FastStart TaqMan Probe Master Mix  with 5 \u00b5l of template cDNA, 200 nStatistical analyses were performed using GraphPad Prism 5 .HMG onlineSupplementary Material is available at .Department of Health's National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital and Alcon Research Institute. J.W.B.B. is supported by an NIHR Research professorship, C.J.C. is a joint MRC and Fight for Sight Clinical Research Training Fellow (G1100383), P.H. is supported by the German Research Foundation (DFG-He 6175/1-1). S.-M.k.H. is partly supported by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n\u00b0 281234, the Medical Research Council (core support at LMCB) and the Batten Disease Family Association, UK. Funding to pay the Open Access publication charges for this article was provided by RCUK.R.R.A. is partly funded by the"}
+{"text": "Skeletal muscle constitutes approximately 40% of body mass, and age-induced decrease of muscle strength impinge on daily activities and on normal social life in the elderly. Loss of muscle strength has been recognised as a debilitating and life threatening condition also in cachexia in cancer patients and in clinical conditions associated with prolonged bed rest. Skeletal muscle dihydropyridine receptors (Cav1.1) act as Ca2+ channels and voltage sensors to initiate muscle contraction by activating ryanodine receptors, the Ca2+ release channels of the sarcoplasmic reticulum. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum Ca2+ storage protein calsequestrin (CASQ1).We hypothesized that JP45 and CASQ1 form a signalling pathway which modulates Cav1.1 channel activity.We isolated flexor digitorum brevis (FDB) muscle fibres from JP45 and CASQ1 double knock-out mice (DKO) and tested whether there were differences in Ca2+ homeostasis between the different mouse lines.in vitro and in vivo.Our results show that Ca2+ transients evoked by tetanic stimulation in DKO fibres, result from massive Ca2+ influx due to enhanced Cav1.1 channel activity. This enhanced activity causes an increase of muscle strength both We conclude that skeletal muscle contraction is strengthened through the modulation of Cav1.1 channel activity by JP45 and CASQ1."}
+{"text": "ADAM10 inhibition or knockdown enhanced trastuzumab response in na\u00efve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p\u22640.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p\u22640.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p\u22640.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells  and  The HER  family of receptor tyrosine kinases includes four receptors: EGFR, HER2, HER3, and HER4. Several ligands bind to these receptors including heregulin and betacellulin . Ligand Protein shedding via various ADAMs  is important for cell fate determination, migration, and proliferation . ADAM10 in vivo and in HER2 positive breast cancer patients.We have previously shown that ADAM17 mediates HER receptor activation during trastuzumab treatment . Since ATo assess the effect of trastuzumab treatment on ADAM10 expression, BT474 and SKBR3 cells were treated with two doses of trastuzumab for 24 hours. ADAM10 mRNA levels were increased in a dose-dependent manner: a 3.6\u2013fold increase in BT474 cells and a 2.5-fold in SKBR3 after 40\u03bcg/ml trastuzumab treatment, compared to untreated control  (figure in vitro and in vivo.To assess the relevance of this observation in an  vehicle  were sta) figure . Therefototal=8, p\u22640.001) (figure We previously established that the PKB inhibition by trastuzumab  increase) figure . We show) figure . CollectThe upregulation of ADAM10 by trastuzumab treatment at 24h in both cell lines occurred with an increase of betacellulin (ligand for EGFR and HER4) in the media figure , correlaWe therefore proceeded to assess the effect of an ADAM10 inhibitor (ADAM10i) in HER2 positive breast cancer cells. Treatment of SKBR3 and BT474 cells with INCB8765 (specific ADAM10 inhibitor) for 24h led to a decrease in the level of basal figures  and trasIn addition, ADAM10i decreased the basal and trastuzumab induced activation of EGFR, HER4, and HER2 in both cell lines figures , S1C. AlTo further prove the role of ADAM10, we silenced ADAM10 using two different siRNAs (and a combination). The knockdown was first optimized and ADAM17 levels were not affected . ADAM10 In view of the upregulation of ADAM10 during trastuzumab treatment in na\u00efve cells, we hypothesized that ADAM10 might be implicated in acquired trastuzumab resistance. ADAM10 mRNA and protein levels were increased in the resistant cells compared to parental cells figure . In addiWe have shown the role of ADAM10 inhibition and knockdown in na\u00efve and resistant HER2 positive breast cancer cells. Previously, we established that ADAM17 inhibition also had an additive effect with trastuzumab treatment . TherefoWe showed that the upregulation of ADAM10 levels occurred during trastuzumab treatment and upon acquired resistance in HER2 positive breast cancer cells. We hypothesized that ADAM10 expression might be relevant as a biomarker to predict prognosis and trastuzumab response in HER2 positive breast cancer patients. Therefore we stained tumors from HER2 positive breast cancer patients and examples are shown in We assessed ADAM10 levels in patients who underwent a window study as outlined in figure We also compared basal ADAM10 levels with those of the tumors obtained at definitive surgery after neoadjuvant chemotherapy and trastuzumab treatment (8 paired samples). There also was an overall trend for increased ADAM10 expression in the post-neoadjuvant treatment samples , which has a stringent quality control in cell authenticity and has incorporated short-tandem repeat (STR) profiling for cell line validation. Cell culturing and the generation of trastuzumab resistant cells were described previously .ADAM10 expression level was scored semi-quantitatively using immunohistochemistry on tumor samples based on staining intensity and distribution using the immunoreactive score of Remmele and Stegner (IRS) . The scoTissue microarrays (TMA) were provided by Oxford Radcliffe Biobank in compliance with Human Tissue Act 2004 (UK). The window study of HER2 positive breast cancer patients was carried out as depicted in figure Prism 6 software (GraphPad software) was used for statistical analysis. The differences between two means were assessed by t-Test. When investigating one factor, one-way ANOVA was used with Bonferroni's multiple comparison. For two factors, two-way ANOVA with Tukey's multiple comparison  was usedFurther details on materials and methods can be found in the"}
+{"text": "Increased Intra-abdominal pressure is common in critically ill patients  and it mTo assess the role of measuring intra-abdominal pressure in predicting success of spontaneous breathing trial, and whether intra-abdominal pressure can predicit risk of re-intubation in the first 24 hours after extubation.Study included 123 patients who were ventilated due to respiratory failure and were ready for weaning.To be enrolled in the study, patients had to have an improvement or resolution of the underlying cause of acute respiratory failure; adequate gas exchange as indicated by a PaO2 above 60 mm Hg while breathing with an Fi O2 of 0.40 or less with a positive end-expiratory pressure (PEEP) of 5 cm H2O or less; awake patients with a Glasgow Coma Scale score above 13; a temperature below 38\u00b0 C; hemoglobin level above 10 g/dl; and no further need for vasoactive or sedative agents. In addition, the responsible physician had to agree that the patient was in stable condition and ready to be weaned from the ventilator. To measure IAP 50 ml of sterilized normal saline was inserted into the bladder using the Foley catheter and pressure was measured using the Krons technique. Baseline IAP was measured before SBT (IAP1)and was measured at 15 minutes interval for 60 minutes(IAP2-IAP3-IAP4-IAP5)Patients with BMI more than 30 ,abdominal pathology and baseline intrabdominal hypertension were excluded.Out of123 patients,94 were successfully extubated after 60 minutes versus 29 who failed the SBT. Mean IAP at all time intervals was 7.4cmH20 in those who were successfully extubated versus 8.4 cmh20 in those who failed the SBT (p = 0.013). IAP was lower at all time intervals in patients who were extubated than those who failed  Out of the 94 patients who were successfully extubated, 31 patients were reintubated in the first 24 hours. The mean IAP in patients who were reintubated was 7.4 cmH20 versus 7.3 cmH20 in those who were not re-intubated.Intrabdominal pressure is higher in patients who fail the Spontaneous Breathing trial druing weaning from Mechanical Ventilation and can be used to predicit failure. However, Intra-abdominal pressure was not significantly different in patients who were re-intubated in the first 24 hours after extubation."}
+{"text": "Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.Kir7.1 is an inwardly rectifying K Kcnj13 gene is a K+ channel belonging to a group of membrane proteins described as K+-transport type channels of the inwardly-rectifying Kir family. Kir7.1 was first described in 1998 and it presents marked differences in sequence as well as functional properties when compared with those of other members of the Kir channel superfamily [+ concentration and more akin to leak-type K+ channels, which is unusual for a Kir family channel whose other members present strong inward rectification properties, that is to say they allow mainly currents associated with K+ influx into cells [Kir7.1 encoded by the to cells .+/K+-pump. Kir7.1 is present at the basolateral membrane of intestinal epithelial cells, thyroid follicular cells and epithelial cells of proximal and distal convoluted tubule [+/K+-pump at the same location, which is the most usual in polarised cell layers. Interestingly Kir7.1 is expressed at the apical membrane of retinal pigmented epithelium (RPE) and in the choroid plexus, epithelia where exceptionally the pump is also expressed apically [+/K+-pump suggests that Kir7.1 could serve the role of K+ recycling needed to keep up with high rates of epithelial to ion transport [Kir7.1 is present in epithelial tissues and shows a remarkable colocalization with the Nad tubule \u20137. Theseapically ,8\u201310. Thransport . An addiransport . Also inransport .KCNJ13 gene are associated with retinal diseases snowflake vitreoretinal degeneration (SVD) [Kcnj13 expression in the RPE and shown that those photoreceptors apposing RPE cells lacking, but not those expressing Kir7.1, degenerate. This suggests a role of Kir7.1 in the ionic regulation of the confined space between RPE and photoreceptors possibly in relation to lactate transport, perhaps in the way it has been proposed to explain a similar phenotype in mice deficient in the ClC-2 Cl- channel [Mutations in human on (SVD)  and Lebeon (SVD) ,16. The on (SVD) . Recentlon (SVD)  have gen channel .Kcnj13 gene. Homozygous null mutant mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest this channel has an essential role in late palatogenesis. Our work also reveals in addition a second unexpected role in the development and physiology of the respiratory system.In order to study the role of Kir7.1 in epithelial transport we generated a Kir 7.1 deficient mouse by ablating the Kcnj13 gene were generated using the Velocigene method [Kcnj13 gene removes the full coding sequence. Mice with gene-targeted disruption of the Kcnj13 gene encoding Kir7.1 K+ channel , wild-type  and heterozygous (Kcjn13+/-) littermates were used for all comparisons. Heterozygous mice were mated to produce Kcjn13-/-, WT and heterozygous progeny.Mice deficient in the e method  in the CAges of mice analyzed are given as embryonic day (E) or days post conception (dpc), where the presence of vaginal plugs was considered as embryonic day E0.5 or 0.5 dpc. Timed pregnant females (E15.5 to E18.5) were sacrificed by cervical dislocation. Embryos were dissected from the uteri and placed in PBS. Body weight of embryos or newborn pups (P0) was measured in an analytical balance after drying off excess fluid.Kcjn13-/- genotype, and were euthanized first. Mice were individually labelled and decapitated for further study. To obtain new Kcjn13+/- mice, only pups presumed to be of Kcjn13-/- genotype were removed and euthanized. The genotype was confirmed to check for a Mendelian distribution of genotypes.Cages containing pregnant mice were surveyed from 7:00 AM on the computed day of birth (E19.5) and new born pups checked for activity and signs of dehydration. After 2 h the whole litter was removed for study. Pups presenting smaller size, signs of dehydration, failure to suckle or breathing difficult were presumed to be of Kcnj13 exon 2 (5\u2019ATCTTCCCGCTAACCTAT3\u2019), SD rev located in the 3\u2019 untranslated region of the kcnj13 gene (5\u2019 CAGCTTTCTACCCAGGGAGC 3\u2019) and Neo for, located in the selection cassette (5\u2019 TCATTCTCAGTATTGTTTTGCC 3\u2019). Mice were maintained on standard laboratory chow  and water ad libitum, under regulated temperature (22\u201326\u00b0C) and light (12:12-h light-dark cycle) conditions. Housing ad breeding was at the SPF animal facility of the Centro de Estudios Cient\u00edficos (CECs), Valdivia, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Animal procedures were approved by the CECs Animal Care and Use Committee.Tail snip DNA was used for genotyping by PCR using the following 3 primers: MP08 for, located in 5\u2019-ATTCTTCATCTTCCCGCTAACCTA-3\u2019) and RevEx3Kir7.1 (5\u2019-GATTTCCCCAGTGCCTTCTTG-3\u2019). Cyclophilin A is used as housekeeping gene.For the analysis of Kir7.1 expression, total RNA was isolated from dissected lung and brain from WT, heterozygous and null mutant mice in TRIzol following the manufacturer\u2019s instructions. The RNA was quantified and tested for quality using spectrophotometry and 1% agarose gel. Five \u03bcg of total RNA was reverse transcribed at 42\u00b0C for 90 min using random primers and ImProm II kir (Promega) to synthesize single-stranded cDNA. The amplicon of Kir7.1 was obtained by PCR using specific primers: For1Ex3Kir7.1  or days post conception (dpc), where the presence of vaginal plugs was considered as embryonic day E0.5 or 0.5 dpc. Birth took place at E19 (P0). Timed pregnant females (E15.5 to E18.5) were sacrificed by cervical dislocation. Embryos were dissected from the uteri and placed in PBS. Body weight of embryos or newborn pups was measured in an analytical balance after drying off excess fluid.Whole embryos (E14.5 to E18.5), P0 dissected tissues or adult tissues were fixed in 4% paraformaldehyde in PBS for 90 min at room temperature. Then, tissues were dehydrated in 30% (vol/vol) sucrose solution for a week, and cryoprotected with Optimal Cutting Temperature Compound . Cryosections (10 \u03bcm) were mounted on Superfrost/Plus  slides, and stained with hematoxylin and eosin (H&E) for lung morphometry or reserved for detection of \u03b2-galactosidase activity. For immunohistochemistry assays, newborn lungs (P0) or adult mice (2 months old) were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4\u00b0C, washed in PBS, and embedded in paraffin.3Fe(CN)6, 4 mM K4Fe(CN)6, 1.2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% Igepal) overnight at 37\u00b0C. Sections were washed with PBS and then counterstained with eosin. Digital images were collected using an Olympus CX31 microscope and recorded with a digital microscope camera (Mshot MD90).The expression of Kir7.1 channel was observed indirectly through the detection of \u03b2-galactosidase activity. OCT preserved tissues were cut into 10 \u03bcm sections and fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS and incubated in X-Gal staining solution  were treated with 10% hydrogen peroxide, blocked with 2.5% normal horse serum, and incubated with anti-Kir7.1 [[Ten random representative fields per H&E stained sample were acquired with the Mshot MD90 camera and Olympus CX31 microscope. All images at a magnification of 400X were taken under the same exposure settings. The percentage of terminal sac spaces is the proportion of white surface area of each field relative to the total image area.2O and then 1% KOH for 24 h. Clearing was carried out by consecutive changes of glycerol  in 1% KOH every 24 h and the embryos were stored in 80% glycerol/1% KOH. Littermates were used and the results were obtained from at least three mice per genotype.Newborn mice were sacrificed by intraperitoneal injection of anesthetic and the skin and internal organs were carefully removed. The remaining carcass was fixed in 100% ethanol for 24 hours. Fat was removed with acetone overnight. Staining was carried out with Alcian Blue (0.3% Alcian Blue 8GS) and 0.1% Alizarin Red for 3\u20134 days at 37\u00b0C on a rocker. The tissues were washed with deionized H+ channel we generated Kir7.1 deficient mice by ablation of its codifying Kcnj13 gene by the Velocigene method [Kcnj13-/- mutant mice as expected. Heterozygous Kcnj13+/- mice were indistinguishable from their wild type Kcnj13+/+ litter mates in growth and development. Homozygous mutant mice however failed to suckle, were often cannibalised by their mothers and did not survive beyond P0. Because of extensive cannibalism we were initially under the impression most of the null mice died in utero, but cesarean delivery and genotyping revealed a Mendelian distribution of the three genotypes in embryos generated after crossing heterozygous mice . Analysis of embryonic tissue  reporter that is driven by the activity  in the ec tissue  revealedc tissue .Kcjn13-/- tissue are smaller than in the WT or heterozygous tissue. This impression is borne out by the quantification of terminal sac spaces shown in Kcjn13-/- mice survived for up to 12 h after birth and did not appear cyanotic, suggesting an absence of respiratory distress. This is consistent with similar levels of expression of proSp-C (surfactant protein C) in lungs of Kcnj13+/+ and Kcnj13-/- P0 mice (not shown). Lungs dissected from Kcjn13+/+, Kcjn13+/- and Kcjn13-/- P0 mice floated when placed in saline solution suggesting that normal respiration had taken place  to then progressing horizontally towards the midline above the tongue (~14.5 dpc). Fusion of the palatal processes has already occurred by dpc 15.5 [Kcjn13+/+, Kcjn13+/- and Kcjn13-/- mice. Palatal processes that are evident at 13.5 and 14.5 dpc in WT and heterozygous embryos have disappeared at 15.5 dpc, at which time complete sealing of the palate is evident. This was not the case for Kcjn13-/- embryos that show a delayed horizontal growth from E14.5 and lack of fusion at 15.5 and 16.5 dpc. Kcjn13+/+, Kcjn13+/- and Kcjn13-/- newborn mice, with the latter presenting evident cleft palate. Also shown are preparations to reveal bone and cartilage (blue), where it can be seen that palatine (pp) and maxillary processes (mp) are extended to the midline in Kcjn13+/+ and Kcjn13+/- tissues. In the Kcjn13-/- mouse, these processes were absent thus exposing the vomer (v) and presphenoid (ps) bones to result in a complete cleft secondary palate.Another cause of early postnatal lethality in null mouse models is a developmental craniofacial alteration leading to cleft palate  and a reelopment . Kcjn13dpc 15.5 . Fig 5a Kcjn13+/+ and Kcjn13-/- embryos. Kcjn13+/+ and Kcjn13-/-, and already fused at E15.5 in the WT but not in the null mutant mouse. The sites of Ki7.1 expression were identified by \u03b2-galactosidase staining of Kcjn13-/- mice. Kir7.1 expression is absent at 14.5 dpc, a stage at which robust expression is already present in the choroid plexus (see inset). Kir 7.1 expression is evident at E15.5 in the respiratory epithelium as well as that covering the palatal processes but here only in the nasal side. The same is seen at E18.5, where the higher magnification picture corroborates that Kir7.1 expression stops towards the tip of the palatal process. Kcjn13+/- tissue. Expression is also present in the respiratory and olphactory epithelia after birth as seen in the Kcjn13-/- section.We undertook a histological examination of the developing palate in Kcjn13 null mutant mice suffer from early postnatal mortality with pups not surviving further than P0. Our initial impression that Kcjn13-/- mice might not experience normal in utero development were dispelled by examining the distribution of phenotypes of 11.5\u201318.5 dpc foetuses of pregnant Kcjn13+/- females that had been crossed with heterozygous Kcjn13+/- males. The distribution of genotypes was not significantly different from the expected Mendelian distribution of 25% Kcjn13+/-/50% Kcjn13+/+/25% Kcjn13-/-. The process of generation of Kcjn13-/- mouse results in the insertion of a DNA coding a \u03b2-galactosidase whose activity is now under the control of the Kcjn13 promoter and therefore reports the channel tissue distribution. Sites of expression of Kir7.1 thus identified included the brain, meninges and choroid plexus; in the eye the retinal pigment epithelium; the gall bladder, and the cricoid cartilage; the small intestine and the thyroid gland. Most of these locations have been identified before as sites of expression of Kir7.1 [Kcnj13 null mutants and heterozygous and WT mice, with the terminal sac spaces in tissue from the Kcnj13-/- mice significantly smaller from E18.5 through to P0. We do not think, however, that this difference can lead to a lethal phenotype as the surviving P0 mice were able of breathing and were not cyanotic, and lungs had inflated as attested by flotation tests.Spurred by the frequent occurrence of respiratory distress as a cause of perinatal lethality, we looked for Kir7.1 expression in the respiratory system. There is extensive expression of the channel in the respiratory tree, including the epithelia of the trachea, bronchioles and alveoli, where Kir7.1 appears to be associated with type II pneumocytes. Expression of Kir7.1 in the respiratory system arises at E16.5. There was some differences in the developing lung between + channel ENaC whilst secretion relies on Cl- channels such as CFTR or Ca2+-activated TMEM-16 Cl- channels. In both cases the activity of basolateral Na+/K+ pump is also essential, as is the presence of K+ channels required for K+ recycling and to maintain a negative membrane potential compatible with continued transport of Na+ or Cl- [+ channels have also been identified in the alveolar and airway epithelia [What might be the reason for this retardation at the saccular development phase or indeed the function of the Kir7.1 channels in adult respiratory epithelium? It is well known that the transport of ions and fluid across all parts of the respiratory system is vital for gas exchange function. Mucociliary clearance is a key element in normal lung innate immune system function. Clearing the airways of particles including pathogenic organisms occurs through ciliary beat and ion transport and its associated water flow into and from the fluid layer covering all portions of the respiratory tract. The electrolyte transport systems that control the airway fluid layer include absorptive as well as secretory functions. Absorption requires the presence of apically located epithelial Na+ or Cl- ,24. Kir7pithelia  suggestiKcnj13-/- mice we can only conjecture that this might be due to a requirement of the Kir7.1 channel in some aspect of the process of expansion of the intra-sac spaces. Fluid secretion is thought to play a role in lumen expansion in vertebrate organogenesis driving expansion of nascent lumens to form a single tube or a sac with CFTR and Na+/K+-pump being often central drivers of the process [Kcnj13-/- mice.As to the retardation in saccular expansion noticed in  process . There i process . It is t+ channels have been proposed to fulfil diverse functions in lung and airway physiology and unravelling the precise role of Kir7.1 will be a daunting task. In a recent review that summarises the evidence for the presence of more than 40 transcripts for K+ channels in airway and alveolar epithelium, Bardou et al. {Bardou, 2009 7315 /id} use the following understatement: \u201cThe physiological and functional significance of this high molecular diversity of lung epithelial K+ channels is intriguing\u201d. This diversity includes members of all major classes of K+ channels: voltage-dependent Kv channels, leak two-pore domain K2P and inwardly rectifying Kir channels, and functions as diverse as gas exchange and alveolar stability, inflammatory responses, epithelial repair after injury, control of transepithelial fluid and ion transport, Cl- secretion and Na+ absorption. Kir7.1 might contribute to one or more of these functions, perhaps in conjunction with other K+ channels. Discovering its contribution to lung and airway physiology will call for more tissue/cell-directed animal modification to allow the study of Kir7.1 avoiding the lethality of the total gene inactivation presented here.KKcnj13-/- mice. Detailed observation revealed that these mice suffered from complete secondary cleft palate, which is the most probable cause of their early postnatal death [Kcnj13 null mutant embryos. Despite the fact that processes do make midline contact at 14.5 dpc in Kcnj13-/- embryos, fusion has not occurred at 15.5 dpc as it does in WT embryos.Examination of head morphology suggested some sort of subtle craneo-facial malformation in al death . PalatogCleft palate is one of the most common birth defects in humans, it includes syndromic and nonsyndromic forms and is influenced by genetic and environmental factors \u201330. A grDrosophila as a model, these authors show that the fly homologue of Kir2.1 affects development perhaps by interfering with Drosophila decapentaplegic (Dpp) signalling. As Dpp is considered analogous mammalian BMP, they propose that an effect of Kir2.1 activity on the BMP pathway could be responsible for the morphological defects in the Kir2.1 deficient mouse.Mice deficient in the inwardly rectifying Kir2.1 channel die postnatally as a consequence of a complete cleft of the secondary palate . Interes+ fluxes in the palatal shelf epithelium, might be a triggering element in palate sealing. It will be interesting to explore further this possibility perhaps in rescue experiments in vitro. Effects of K+ channels not involving their permeation properties have also been seen to play a role in cancer cell migration [+ channels have often been invoked as central participants in cell migration, proliferation and differentiation processes [The expression of Kir7.1 coincides with the moment of palatal process fusion, i.e. between dpc 14.5 and 15.5. Interestingly it appears that the channel is expressed by epithelial cells covering the dorsal aspect only of the palatal shelves and it might be absent from the point of contact between opposite between processes itself. This contrasts with the localization of TGF-\u03b2 3 that is present in the MEE edge and the tip epithelium of the nasal septum . It is cigration  and thisrocesses , and anyAlthough we lack mechanistic explanations for the effect of Kir7.1 inactivation on palatal formation and on lung development, we provide firm evidence of its involvement in these processes. We hope that this evidence will spur new work that might contribute to our understanding of these important biological processes."}
+{"text": "Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2. Members of aspartyl protease family are known to be associated with some pathological states such as breast cancer and pruritic inflammatory skin disease . Further\u03b2) peptide by BACE1 leading to the preliminary constituent of amyloid plaques in the brains of individuals with the disease ..9].Superimposition of the crystal structures of BACE1 (1FKN) and BACE2 (2EWY) gave a root-mean-square deviation (RMSD) of 1.46 over 373 C-alpha atoms indicating that the structures of BACE1 and BACE2 are very similar to each other . AlthougWe defined the ligand binding site as all amino acid residues within 5\u2009\u00c5 from the ligand. Twenty-eight of amino acid residues in BACE1 and 24 of amino acid residues in BACE2 were identified as ligand binding site. The ligand binding site residues of BACE1 and BACE2 together with their evolutionary trace status are summarized in We mapped the ligand binding sites and their ET status onto the chain A of crystal structures of BACE1 (1FKN) and BACE2 (2EWY) . Two outMembers of aspartyl protease family are known to have a loop structure that covers the active site of enzyme upon binding with the ligand. This structure is known as the flap region, and it is flexible and can adopt different structural conformations upon binding with the substrate. It is assumed that this structure will shield the active site from the solvent . The flaThe last group-specific residue at the ligand binding site is Asn233 of BACE1 substituted with Leu246 of BACE2. The observation indicated that, in BACE2, the binding site at this region is more hydrophobic than the one in BACE1. This difference can be exploited for the design of a selective drug targeting either BACE1 or BACE2.\u03b2). As a result, the design of selective BACE1 or BACE2 inhibitors is rather challenging. Analyzing the sequences of a group of selected BACE1 and BACE2 proteins and mapping the details on known 3D structure reveal the differences between these two groups that would be beneficial for the selective drug design.Designing a drug that could selectively inhibit BACE1 could avoid the unwanted side-effects of also inhibiting other aspartic protease family members including BACE2. BACE1 and BACE2 are close homologs and they are competing against each other for the same substrate (AET analysis on amino acid sequences and protein structures of BACE1 and BACE2 from several mammalian species enabled us to identify the distinctive features of BACE1 and BACE2 amino acid sequences. Mapping the ET analysis onto a known 3D structure of BACE1 and BACE2 revealed that their active sites are well conserved. Four group-specific residues were identified in the ligand binding sites of BACE1 and BACE2. The residues are Pro70, Ile110, Ile126, and Asn233 of BACE1 substituting Lys86, Leu126, Leu142, and Leu246 of BACE2, respectively. These group-specific residues would be the reason for cleavage site selectivity in BACE1 and BACE2 biological function and would be the potential residues for the design of selective and specific inhibitors targeting either BACE1 or BACE2."}
+{"text": "The aim of our breast milk study was to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German cohort. Therefore, the time courses of appearance after ingestion, the potential risk of sensitization to peanuts via breast milk in Germany and, if possible, the way Ara h 2 will be processed \u00ae and an ELISA against digestion resistant Ara h 2 (DRP-Ara h 2)). Natural Ara h 2 was digested by Enzynorm f\u00ae and Kreon\u00ae to mimic the effect of the combined gastric and duodenal digestion in vivo and, subsequently, analysed by N-terminal sequencing and MALDI TOF MS.Of 32 lacating, non-peanut allergic women, breast milk samples were collected at different time points after ingestion of 100 g dry roasted peanuts . Breast milk samples were analysed for peanut protein in SDS-PAGE, Western blot and ELISA  at different concentrations and time points of appearance. To assess the way Ara h 2 is processed in vivo, natural Ara h 2 was digested into several digestion resistant immunoreactive peptides <15 kDa after treatment with Enzynorm f\u00ae and Kreon\u00ae, and a 12 kDa fragment was identified by N-terminal sequencing and mass spectrometry corresponding to the middle part of Ara h 2.Ara h 2 was undetectable using Western blot. Performing the Neogen Veratox ELISAAfter maternal ingestion Ara h 2 is secreted into breast milk in our German cohort in 25 % of the volunteers, individually either rapidly  or delayed (after 8h or 12h) and in different concentrations. To study Ara h 2 or Ara h 2 peptides that survive digestion and pass into human breast milk antibodies against the 12 kDa fragment are now raised for enrichment strategies to characterize these sensitizing or tolerogenic peanut structures in our breast milk samples."}
+{"text": "M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron\u2013sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own.Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron\u2013sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1  \u2022The mitochondrial glutathione redox state, but not the cytoplasmic glutathione redox state, is crucial for oxidant tolerance.\u2022The mitochondrial thioredoxin acts as a back-up antioxidant system for the glutathione system.\u2022Mitochondrial glutathione and thioredoxin play an overlapping role in redox regulation which is important during ageing and oxidative stress conditions. It is an essential metabolite in eukaryotes, and for example, mice deficient in glutathione biosynthesis die rapidly Saccharomyces cerevisiae as a model organism. Initial studies confirmed that GSH is an essential metabolite during normal non-stress conditions GSH1, encoding the first step in GSH biosynthesis, are inviable, but viability can be maintained by the addition of exogenous GSH. Importantly, gsh1 mutants undergo a limited number of cell divisions in the absence of GSH during which they utilize their pre-accumulated stores of GSH and hence the consequences of GSH depletion can be readily followed gsh1 mutant cannot be restored by growth under anaerobic conditions GSH1 also accumulate mitochondrial iron, which is a common phenotype in mutants that are defective in the maturation of cytosolic iron\u2013sulphur (Fe\u2013S) proteins Much progress has been made towards understanding the eukaryotic requirement for GSH using the yeast gsh1 mutants depleted of GSH are sensitive to oxidants The essential requirement for GSH, together with the pleiotropic phenotypes which are observed in mutants depleted of GSH, particularly defects in iron metabolism, has meant that it has been difficult to define the role of GSH as an antioxidant. Whilst M1L mutant to examine the requirement for mitochondrial GSH. We show that it is the mitochondrial form of Glr1, rather than the cytosolic form, which is required for oxidative stress tolerance. Our data indicate that the GSSG:2GSH redox couple plays an essential function as a mitochondrial antioxidant, with the mitochondrial thioredoxin system providing only a minor back-up role. Our findings indicate that in contrast to its apparent minor role in cytoplasmic thiol-redox control, glutathione plays an essential role in mitochondrial thiol-redox regulation.Glutathione reductase (Glr1) is an NADPH-dependent oxidoreductase which converts oxidized GSSG to reduced GSH using reducing power generated by the pentose phosphate pathway. Yeast Glr1 is not essential for normal aerobic growth, but is required for viability during exposure to oxidative stress conditions 22.1S. cerevisiae strains used in this study were isogenic derivatives of W303 (MATa ura3-52 leu2-3 leu2-112 trp1-1 ade2-1 his3-11 can1-100). Strains deleted for the mitochondrial thioredoxin (trx3::kanMX4), cytoplasmic thioredoxins (trx1::TRP1 trx2::URA3), glutathione reductase (glr1::TRP1) and GSH1 (gsh1::LEU2) have been described previously M1L) was made in GLR1 with a C-terminal Flag tag contained on plasmid pRS413 or pRS416. The FET3-lacZ reporter fusion has been described previously [25].The 2.2600=1.0, 0.1, 0.01 and 0.001) onto agar plates containing various concentrations of oxidants. Chronological life span experiments were performed in liquid SCD media supplemented with a five-fold excess of uracil, leucine, tryptophan, adenine and histidine to avoid any possible artefacts arising from the auxotrophic deficiencies of the strains.Strains were grown in rich YEPD medium  or minimal SCD medium . For growth on non-fermentable carbon sources, SGE and YEPGE contained 3% (v/v) glycerol and 1% (v/v) ethanol. Media were solidified by the addition of 2% (w/v) agar. Stress sensitivity was determined by growing cells to stationary phase and spotting diluted cultures  LSRFortessa\u2122 cell analyser and data were analysed using BD FACSDiva 8.0.1 software. The degree of probe oxidation (OxD) was calculated as described previously [28].The redox state of Trx3 was measured by covalent modification with the thiol-reactive probe 4-acetamido-4\u2032maleimidyldystilbene-2,2\u2032-disulphonic acid  as described previously 2.4Arthrobacter luteus) with gentle agitation at room temperature for 30\u201360\u00a0min. Spheroplasting efficiency was determined at regular intervals by measuring the OD600 of small aliquots of cells in 1\u00a0ml of 0.6% (g/v) SDS. Cell homogenization was achieved using a tissue grinder . Protein concentrations were measured using a NanoDrop ND-8000 spectrophotometer. Cell fractionation was verified by Western blot analysis using mitochondrial  and cytosolic (\u03b1Pgk1) specific antibodies. Protein extracts were electrophoresed under reducing conditions on SDS-PAGE minigels and electroblotted onto PVDF membrane (Amersham Pharmacia Biotech). Bound antibody was visualized by chemiluminescence . Lipid peroxidation in mitochondrial fractions was determined using an OxiSelect\u2122 thiobarbituric acid reactive substances assay Kit (TBARS) . Methionine oxidation was detected using \u03b1MetO antibodies .Cellular fractionation into cytoplasmic and mitochondrial fractions was performed essentially as described by Gregg et al. 2.5d-galactopyranoside (ONPG) hydrolyzed per minute per microgram of total protein (U). Values shown are the means of at least three determinations. The gsh1 mutant was grown in minimal SCD media in the presence or absence of 1\u00a0mM GSH.For the determination of \u03b2-galactosidase activity, transformants were assayed essentially as described previously 2.6Experiments were performed according to 2.7p-value less than 0.05.Data are presented as mean values\u00b1SD. Statistical analysis was performed by one-way ANOVA and results were considered statistically significant with a 33.1M1L). The Glr1M1L mutant and wild-type Glr1 were expressed as the sole copies of Glr1 in glr1 mutant cells. Cells were fractionated and Glr1 detected using western blot analysis  to compare the redox potentials in different intracellular compartments glr1 mutant as would be expected due to the lack of Glr1 . These data indicate that the presence of Glr1 improves the growth recovery of a gsh1 mutant, presumably acting to recycle limiting amounts of oxidized GSSG. The addition of 0.1\u00a0\u03bcM GSH only partially restored the growth of the gsh1 glr1 mutant strain containing the Glr1M1L mutant  results in an increase in total glutathione levels which is thought to act as a compensatory mechanism in response to the increased redox load trx1 trx2) and found that OxD values were increased by approximately two-fold for both the cytoplasmic and mitochondrial roGFP2 fluorescent probes under fermentative or respiratory conditions  or expression of the Glr1M1L mutant did not alter the redox state of Trx3. Taken together, these data indicate that in contrast to the cytoplasmic redox systems, the redox states of mitochondrial glutathione and Trx3 are maintained independently and the mitochondrial GSSG:2GSH redox couple glutathione is unaffected by the loss of the mitochondrial thioredoxin.Loss of 3.5GLR1 and TRX3 to test whether there is a requirement for Trx3 when the GSSG:2GSH redox couple becomes oxidized. In agreement with previous observations glr1 GLR1 with glr1 trx3 GLR1). However, loss of Trx3 exacerbated the sensitivity of the glr1 mutant containing empty vector or Glr1M1L to both hydrogen peroxide and diamide stress. This suggests that Trx3 can act as a back-up antioxidant to promote oxidant tolerance, under conditions where mitochondrial glutathione becomes oxidized.Although a complete mitochondrial thioredoxin system comprising a thioredoxin (Trx3) and a thioredoxin reductase (Trr2) has been identified in yeast glr1 and trx3 mutant strains to confirm whether Trx3 has an antioxidant function. All amino acids are potential targets for oxidation, but methionine residues are particularly sensitive forming methionine sulphoxide (MetO) in cells glr1 and trx3 mutants. We found that the levels of mitochondrial MetO are comparable in wild-type, glr1 and trx3 mutants. In contrast, mitochondrial MetO concentrations were elevated in the glr1 trx3 double mutant  which is active as a peroxidase and can protect cells against hydrogen peroxide stress e mutant B. Malonde mutant C. These 3.6GLR1 resulted in a modest decrease in viability during stationary phase, although maximal CLS (~16 days) was unaffected relative to the CLS of wild-type and trx3 mutant strains  to a more oxidized state glr1 mutant strains glr1 mutant has a much greater percentage of oxidized glutathione in both the cytosol and the mitochondria compared with a wild-type strain. Hence, there appears to be sufficient GSH available in a glr1 mutant to maintain iron sulphur cluster assembly, which is proposed to only require trace amounts gsh1 Glr1M1L mutant compared with a gsh1 GLR1 mutant. This emphasizes the particular importance of the mitochondrial GSSG:2GSH redox couple for cell growth and oxidant tolerance, since the cytoplasmic GSSG:2GSH redox couple is maintained at wild-type levels in the Glr1M1L mutant.Previous studies have emphasized the role of glutathione in iron metabolism M1L mutant suggesting that similar cytosolic redox environments are maintained in these strains. The fact that the mitochondrial roGFP2 probe becomes more oxidized in a mutant lacking mitochondrial Glr1 suggests that the cytosolic pool of glutathione is insufficient to protect mitochondria against oxidation following exposure to an exogenous oxidant. It is unclear whether oxidant sensitivity and mitochondrial glutathione oxidation in a glr1 mutant is caused by loss of an antioxidant activity which is normally mediated by glutathione. For example, mitochondrial glutaredoxin (Grx2) and peroxiredoxin (Prx1) require glutathione for their antioxidant activities glr1 mutant might arise due to a general loss of mitochondrial thiol redox regulation, and non-specific oxidation of reactive cysteine residues in mitochondrial proteins.Transport of hydrogen peroxide across cell membranes can be facilitated by transporters such as aquaporins, but once inside cells it should be freely diffusible trx1 trx2 mutant, whereas there is no significant alteration in a trx3 mutant. It is surprising that the mitochondrial glutathione pool is oxidized in a trx1 trx2 mutant since previous studies using GFP-based redox sensors have suggested that the glutathione pools in the cytosol and mitochondrial matrix are not linked glr1 mutant does not affect the redox state of cytoplasmic thioredoxins , and thioredoxin oxidation is only detected in response to severe glutathione depletion Extensive overlaps have been reported between the yeast thioredoxin and glutathione systems TRX3 does not alter the redox state of the cytosolic or mitochondrial roGFP2 fluorescent probes suggesting that loss of the mitochondrial thioredoxin does not impose a redox load on the glutathione system. Mitochondrial thioredoxin is however, known to be more reliant on the glutathione redox couple. Unlike cytoplasmic thioredoxins, the redox state of mitochondrial Trx3 is buffered by the GSSG:2GSH redox couple under oxidative stress conditions trr2), but is accumulated in a trr2 glr1 double mutant under both fermentative and respiratory growth conditions GLR1, since the oxidant sensitivity of a glr1 mutant is exacerbated in the absence of TRX3 and markers of oxidant damage including MetO and MDA accumulate in a glr1 trx3 double mutant. We therefore suggest that mitochondrial Trx3 plays an ancillary or back-up role for mitochondrial glutathione, perhaps under stress or growth conditions where mitochondrial glutathione is oxidized.Loss of mitochondrial GLR1 or TRX3 alone does not significantly alter chronological lifespan. However, in a glr1 trx3 double mutant longevity is significantly reduced. The role of Trx3 in CLS appears to require a complete mitochondrial thioredoxin system since a similar defect in CLS is observed in a glr1 trr2 mutant. This is the first time a thioredoxin system-dependent phenotype has been described for Trx3 since the only other reported phenotypes have been detected in a trr2 mutant and not a trx3 mutant It was originally reported that Trx3 is the physiological electron donor for mitochondrial Prx1, which is unexpected since 1-Cys Prx\u2019s are not thought to form a disulphide which could act as a substrate for thioredoxin 5Thiol redox regulation plays a long-recognized role in the response of cells to oxidative stress conditions. Our current data emphasize the importance of compartmentalized redox regulation when cells are subjected to oxidative stress conditions. Whilst cytosolic glutathione represents the first major pool of thiols which would be a target of oxidation in response to exposure to an exogenous oxidant, it is the mitochondrial glutathione pool which is crucial for oxidant tolerance."}
+{"text": "The images for Figs 7, 9 and 10 appear incorrectly in the published article. The figure captions are correct. Please see the correct Figs"}
+{"text": "Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.SRY (Sex Determining Region Y)-Box 4 or  This is a rare event within the mammalian genome but is common in the plant.\u2022The data provides a modified method for brain cell fixation and immobilisation on glass slides for effective RNA-FISH analysis.\u2022Sox4 natural antisense transcripts, known as Sox4ot1 and Sox4ot2 in the production of Sox4_sir3 in vitro.Comparison of two different \u2022Sox4 gene locus allows clear, concise and easy visualisation of various features defined in the region by using Artemis software.Compilation of all the information within the 11.1Sox4 gene locus. The Sox4 gene locus is featured by multiple overlapping sense and natural antisense transcripts (NATs) In silico data mining and mapping were also carried out to enrich the features within the locus and the detailed information is summarized in a GenBank file format as Supplementary GenBank File. A snapshot of the annotated Sox4 gene locus visualized using Artemis Genome Browser and Annotation Tool www.ensembl.org), Sox4ot1, Sox4ot2, Sox4_sir3, untranslated regions, coding region and exons/introns.The data reported here consists of information related to the The most important information within the Supplementary GenBank File is the mapped FANTOM Paired-End Ditags (PET) sequences. Twelve pairs of PET sequences were mapped to the locus indicating the presence of 6 different NATs. These NATs were named PET1-6 with 4 of them were successfully cloned and further analysed in Ling et al.\u00a01.2Sox4 sense was generally diffused all over the cytoplasm whereas Sox4 NATs were depicted as aggregates within the cytoplasm. Whenever Sox4 NATs aggregates were observed, Sox4 sense aggregates were found at the same loci within the cytoplasm.The data article also describes the results for RNA-FISH experiments performed on cells isolated from different regions of the mouse brain . All celSox4, RNA FISH was performed on cells obtained from P1.5 olfactory bulbs using probes against the Hmbs housekeeping gene  to quantitatively estimate the intensity of bands in Western blotting experiments. Pixels from each band from two independent experiments were calculated by using a fixed rectangular selection approach (T-test (2-tailed) was used to compare PET/pcDNA3 and control groups for any significant differences but none of the p-values were lesser than 0.05.We used ImageJ software  Sox4 gene locus  and PET6 (NAT that overlaps Sox4_sir3 origin site). The overexpression of PET3 and PET6 both did not alter the level of sense transcript expression. As expected, the expression of the Sox4 NAT at region overlapped by PET3 and PET6 were significantly upregulated . Amplicons were analysed using agarose gel electrophoresis to estimate the size of PET6. The analysis showed that there were 2 forms of PET6 NATs, one is unspliced and the other one is spliced . Sanger mall RNA .1.7in situ hybridization experiments are appropriately controlled to avoid misinterpretation of noisy signals. Locked Nucleic Acid (LNA)-in situ hybridization (ISH) for small RNA is usually controlled with a scramble probe, a mutated antisense probe or a sense probe. As the control for Sox4_sir3 LNA-ISH reported in Ling et al.\u00a0Sox4_sir3 probe  to determine the relative levels for various Sox4 sense and NATs expression. All RT-qPCR data presented here were conforming to the criteria described elsewhere"}
+{"text": "Area at risk measurements often rely on T2 weighted images, but subtle differences in T2 may be overlooked with this method. Quantitative T2 mapping may bring us beyond some of the technical limitations associated with T2-weighted images . We hypothesize that T2 quantification can detect differences between salvaged and infarcted myocardium within the AAR in a reperfused model of acute myocardial infarction.Dogs underwent 2 hours of coronary occlusion followed by 4 or 48 hours of reperfusion before imaging. CMR imaging was performed at 1.5T (Siemens) using native T2 mapping, T2-prepared SSFP imaging, and late gadolinium enhancement (LGE). One midventricular slice was chosen per animal for analysis. LGE images were used to define infarcted, salvaged, and remote myocardial ROIs. Another ROI of both the infarcted and salvaged areas was defined as the AAR. Data were analyzed with ANOVA and Bonferroni correction for multiple testing. A p value < 0.05 was considered significant.22 animals were imaged after 4 n = 11) or 48 hours (n = 11) of reperfusion. The signal intensity of the AAR was significantly greater than the remote myocardium on T2-prepared SSFP images, both at 4 hours  of reperfusion and 48 hours . This was also the case with T2 quantification of the AAR compared to remote at 4 hours and 48 hours . Dividing the AAR into infarcted and salvaged myocardium demonstrated that the T2 of salvaged myocardium was significantly longer than remote myocardium at both 4 and 48 hours of reperfusion. The T2 of infarcted myocardium was also longer than remote myocardium (see Figures 1 or 48 hT2 mapping techniques quantitatively differentiated sub-regions within the AAR during the first days of reperfusion. The T2 of salvaged myocardium was significantly higher than remote myocardium after both 4 and 48 hours of reperfusion, though the magnitude of the difference was greater at 4 hours. T2 mapping was also able to distinguish salvaged from infarcted myocardium.Funded by the Intramural Research Program of the National Heart, Lung, and Blood Institute of The National Institutes of Health."}
+{"text": "Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices. Superresolution light microscopy and electron cryo-tomography have revealed12357810111314161718202122232425262728293031323334353637383940414243In this Article we study the most favorable (minimum-energy) lattice architectures, and corresponding modulation of protein function, due to bilayer-mediated elastic interactions between mechanosensitive membrane proteins. A diverse range of integral membrane proteins have been shown to be mechanosensitive2020in vitro145253in vivo, and the physiological significance of tetrameric MscL is a matter of debate14155253in vivo52in vivo14145253in vitro experiments, or varying the temperature. In this Article we take the available MscL structures as our starting point, and consider the lattice architectures and collective functions of clusters of both tetrameric and pentameric MscL, as well as mixtures of tetrameric and pentameric MscL.MscL switches from a closed to an open state with increasing membrane tension204951In vitro and in vivo studies have suggested that bilayer-mediated interactions stabilize large clusters of hundreds of MscL5555Bilayer-mediated protein clustering may be driven by curvature deformations212223242526272829303132333132333435362438394041424320435558a is one-half the hydrophobic thickness of the unperturbed lipid bilayer, and, for generality, we consider\u03c4 to u as well as to area changes. Experiments roughly yield kBT/nm2\u200920where the thickness deformation field Mycobacterium tuberculosisBased on structural data48R captures the size of MscL, \u03b5 is the amplitude of angular undulations, and \u03c9 denotes the orientation of MscL with respect to the x-axis. The observed structure of closed pentameric MscLStaphylococcus aureusin polar coordinates, where u along the bilayer-protein interface in the closed and open states of MscL, where the unperturbed bilayer half-thickness E. coli20436368On the basis of structural data on MscL, the hydrophobic thickness of MscL in the closed and open states has been estimatedWhile the anisotropic thickness deformations due to a few proteins can be obtained by minimizing equation 6543U as a summation over elements,Following the standard finite element discretization procedure, we rewrite the variation of the energy in equation where the element stiffness matrix D is a block diagonal matrix with the lipid bilayer parameters as coefficients. The strain-displacement transformation matrix B combines the DKT shape functions H with the linear triangular shape functions M:are integrated over the local coordinates H are given by Batoz et al.M can be found in standard finite element textbooks\u2014see, e.g., ref. 71Explicit forms of the DKT shape functions d is the center-to-center distance between the two MscL and d row of the interaction energy array by rounding d. For fast evaluation of hard-core steric constraints, we constructed an analogous array for the minimum allowed distances To confirm our predictions of the minimum-energy lattice architectures of tetrameric and pentameric MscL we carried out Monte Carlo simulations with simulated annealing of pair interaction potentials796 Monte Carlo steps, and decreased the temperature linearly starting from around In our simulated annealing Monte Carlo simulations, a single Monte Carlo step consists of one displacement and one rotation trial per MscL on average. We used a unit displacement edom see , we firsd, which are greater than d. For fixed protein shape and orientation, thickness-mediated interactions are most favorable for the smallest value of d allowed by steric constraints on lipid size, d, non-pairwise contributions to thickness-mediated interactions can be For curvature- and fluctuation-mediated interactions it has been suggested232532334359d, and the close-packed hexagonal lattice with Thickness-mediated MscL clustering was studied before4320634849u normal to their bilayer boundaries ."}
+{"text": "Haematological malignancies are associated with treatment releated morbidity and mortality. Intensive chemotherapy and haematopoetic stem cell transplantation has increased treatment releated complications. Transferring a severely ill patients to the intensive care unit for life support is often a difficult decision.To detect risk factors effecting haematological malignancy patients mortality after admission to the medical intensive care unit.This study was performed prospectively in the medical intensive care unit of Erciyes Universty Hospital. History, physical exam and laboratory findings on admisssion,and therapeutic interventions during ICU stay were recorded. The study end point was ICU mortality.32 (%60) of the 53 patients included into this study were male. The average age of the patients was 49 \u00b1 19 years. The hematological diagnosis of the patients were as follows; 19 were AML, 12 were MM, 7 were Non-Hodgin Lymphoma and 6were ALL. The most common reasons for ICU admissions were respiratory failure (%57) and septic shock (%17). The mean time delay starting from deteriation to ICU was 6 hours (range 1-48). APACHE-2 score was 26 \u00b1 8 and the early warning system score was 8 (range 1-14). APACHE-2 score and time delay for ICU admission of nonsurvivors were higher . Serum total cortisol levels were lower in survivors compared to nonsurvivors (p= 0.023). ICU mortality rate was 60%.The mortality rate of haematological malignancy patients rate was high. The time delay for ICU admission and APACHE-2 score were important risk factors for ICU mortality. These patients should be admitted to the ICU as soon as possible when vital signs are detoriated."}
+{"text": "C9orf72 is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) acting through a loss of function mechanism due to haploinsufficiency of C9orf72 or a gain of function mediated by aggregates of bidirectionally transcribed HRE-RNAs translated into di-peptide repeat (DPR) proteins. To fully understand regulation of C9orf72 expression we surveyed the C9orf72 locus using Cap Analysis of Gene Expression sequence data (CAGEseq). We observed C9orf72 was generally lowly expressed with the exception of a subset of myeloid cells, particularly CD14+ monocytes that showed up to seven fold higher expression as compared to central nervous system (CNS) and other tissues. The expression profile at the C9orf72 locus showed a complex architecture with differential expression of the transcription start sites (TSSs) for the annotated C9orf72 transcripts between myeloid and CNS tissues suggesting cell and/or tissue specific functions. We further detected novel TSSs in both the sense and antisense strand at the C9orf72 locus and confirmed their existence in brain tissues and CD14+ monocytes. Interestingly, our experiments showed a consistent decrease of C9orf72 coding transcripts not only in brain tissue and monocytes from C9orf72-HRE patients, but also in brains from MAPT and GRN mutation carriers together with an increase in antisense transcripts suggesting these could play a role in regulation of C9orf72. We found that the non-HRE related expression changes cannot be explained by promoter methylation but by the presence of the C9orf72-HRE risk haplotype and unknown functional interactions between C9orf72, MAPT and GRN.A non-coding hexanucleotide repeat expansion (HRE) in The online version of this article (doi:10.1186/s40478-016-0306-7) contains supplementary material, which is available to authorized users. C9orf72 gene as the major cause for chromosome 9-linked ALS and FTD with or without concomitant motor neuron disease 3 probe (sense foci), or [GGGGCC]3 probe (antisense foci) (Exiqon) as previously described [CD14+ monocytes from three escribed . Brieflyescribed  and usedhttps://www.r-project.org/).All statistical analyses, correlations and plots were performed using the free software environment R . All tissue requests received at the NBB are reviewed by NBB's scientific committee and all material and data collected by the NBB are obtained on the basis of written informed consent. Procedures, information and consent forms of the NBB have been approved by the Medical Ethics Committee of the VU Medical Centre. The use of human brain samples for the work leading to the generation of the CAGEseq dataset 3 was approved by the NIH Office for Human Subjects Research as indicated in C. Blauwendraat, under review in another journal. Controls and disease blood donors gave informal consent and study approval was obtained by the Ethics Committee of the University of T\u00fcbingen.C9orf72 locus and identify possible new transcripts on the sense and antisense strands that contribute to C9orf72 expression, we used publically available CAGEseq expression data generated in the context of the FANTOM5 project [C9orf72 is expressed. We observed C9orf72 was expressed above 1 tpm in the sense strand in 83.3\u00a0% of the samples and in the antisense strand in 22.1\u00a0% of the samples. Particularly, C9orf72 expression was very high in CD14+ monocytes, eosinophils, and neutrophils, a subset of myeloid cells involved in innate and adaptive immunity, from now on referred to as myeloid-high , or lymphoid-derived cells like T and B cells were much lower , NM_18325 (Transcript 2) and NM_1256054 (Transcript 3)  Fig.\u00a0. The C9oile Fig.\u00a0 shows thC9orf72 locus: a TSS on the reverse strand downstream of exons 1A and 1B of the annotated transcripts  with the IPO7 gene encoding for importin 7, a nucleocytoplasmic transport protein [The distinct modes of expression of  protein  when conC9orf72 expression between myeloid cells and CNS, we investigated whether this finding was also true across distinct CNS regions. In addition to the FANTOM5 collection that contains data for a range of CNS regions in a limited numbers of adult donors, we analyzed an additional CAGEseq brain dataset consisting of 49 control libraries generated in our laboratory, with data from seven CNS regions from seven control donors .We next investigated roarrays . To confC9orf72 expression in microglia. Therefore we isolated RNA from commercially available microglia from human fetal brain tissue. Digital PCR experiments  and therefore we excluded it from further analysis. In agreement with our CAGEseq data we observed an increase for AS1 in the C9orf72-HRE patients and AS3 TSSs in the C9orf72-HRE and GRN patients as compared to controls .We then investigated whether and to which extent the newly detected ols Fig.\u00a0. HoweverC9orf72 expression is also reduced in non-HRE FTD patients suggests that the reduction of C9orf72 expression is not entirely dependent on the repeat expansion. Sequence analysis of the GC-rich low-complexity sequence (LCS) adjacent to the HRE did not reveal any DNA variants, including the 10\u00a0bp deletion observed in up to 25\u00a0% of C9orf72-HRE patients [C9orf72 expression; therefore we explored other potential causes. One possible explanation might be that the observed C9orf72 decrease in GRN and MAPT cases is a consequence of the neurodegeneration process. We therefore performed a series of qPCR experiments on cDNA from brain RNA of patients with different neurodegenerative disorders including sporadic FTD, AD, ALS, PSP, PD, HD and MS patients   (data not shown). On average, we observed a three fold increase in the expression of S1\u2009+\u20093a and S1\u2009+\u2009S3b TSSs in the individuals homozygous for the risk haplotype as compared to the individuals carrying the non-risk haplotype and a 1.2 and 1.5 decrease in the expression of S2a and S2b TSSs, respectively.Our eQTL analysis shows that the risk haplotype is associated with higher expression of the ly) Fig.\u00a0 and to lNext we determined by genotyping which samples used in our qPCR experiments on brain samples were carrying the risk haplotype. We identified five individuals homozygous and 34 heterozygous for the risk haplotype and 29 individuals non-carriers of the risk haplotype Table\u00a0.C9orf72 transcripts expression levels determined by qPCR with the concordance to the risk haplotype. In our qPCR experiments we could measure the effect of the risk haplotype C9orf72 transcript 1 and 3 in separate assays differently than with CAGEseq as C9orf72 transcript 1 and transcript 3 share the same TSS. We observed a significant association of the risk haplotype with a higher expression of C9orf72 transcript 3 in agreement with our CAGEseq eQTL findings  but not with transcript 1, with the influence of the risk haplotype on C9orf72 transcript 3 clearly shown in Fig.\u00a0C9orf72 transcript 2 probably because of the small effect and sample size.We then correlated C9orf72 expression in brain tissue, but it does not fully explain the observed C9orf72 reduction in MAPT and GRN patients as it was also detected in cases not carrying the risk allele. Therefore additional mechanisms must exist to explain the observed C9orf72 decrease.Taken together these results suggest that the risk haplotype affects C9orf72 promoter region are associated with down-regulation of C9orf72 mRNA expression in HRE patients [C9orf72 promoter in FTD cases with MAPT and GRN mutations has not been yet investigated.Several studies have shown that hypermethylation and H3 and H4 lysine trimethylation of the patients , 48, 53,C9orf72-HRE cases, two samples were strongly hypermethylated and overall C9orf72 promoter methylation levels were higher in C9orf72 patients as compared to controls, MAPT and GRN mutations carriers  while no significant differences were observed between MAPT cases versus controls and GRN cases versus controls, ruling out a direct influence of C9orf72 methylation level on C9orf72 expression in these patients. We then investigated whether the risk haplotype would influence the methylation level of the C9orf72 promoter in the MAPT and GRN mutation carriers. We did not observe a significant association between the risk haplotype, in the homozygous or heterozygous state and C9orf72 promoter methylation level (data not shown). Our results therefore suggest that C9orf72 promoter methylation level by itself or in combination with the risk haplotype does not account for the C9orf72 reduction observed in MAPT and GRN cases.We therefore performed an HhaI methylation-sensitive digestion assay combined with qPCR as described by Russ and colleagues . Among tC9orf72 reduction observed in MAPT and GRN mutation carriers could also imply a functional interaction within the molecular pathways disrupted by the C9orf72, MAPT and GRN mutations respectively. In this respect the decrease in MAPT transcripts containing exon 10 observed by Prudencio et al in RNAseq data from C9orf72-HRE patients is interesting [C9orf72 in MAPT patients, which would be consistent with a functional interaction.The C9orf72 transcripts 2 and 3, MAPT and GRN. qPCR experiments on cDNAs from the transduced cells showed a clear influence on expression of C9orf72 transcripts 2 and 3 after knock down of MAPT and GRN separately compared to scrambled controls  M17 human neuroblastoma cells with lentiviral constructs expressing shRNAs targeting 9orf72 expression using CAGEseq data across the FANTOM5 collection showed that C9orf72 is highly expressed in myeloid cells, particularly in CD14+ monocytes, in agreement with previous findings based on microarrays [Our analysis of global Croarrays .C9orf72 in CD14+ monocytes (up to seven fold increase) as compared to brain  than the percentage reported for peripheral blood leukocytes (7\u00a0%) [C9orf72 is rather similar to that in the CNS [C9orf72 is much more abundant in CD14+ monocytes.Overall the percentage of RNA foci for the antisense es (7\u00a0%)  and for es (7\u00a0%) , while t the CNS , 47, 55,C9orf72 expression by using CAGEseq data reveals new and interesting features for the C9orf72 promoter and gene locus. A major strength of CAGEseq lies in its ability to distinguish closely spaced TSS usage preference that is often tissue or cell specific. Indeed we observed several novel TSSs at the C9orf72 locus with distinct modes of expression in a subset of myeloid cells and CNS suggesting they have a cell and/or tissue specific function. Interestingly, expression levels of S1\u2009+\u2009S3b TSS for C9orf72 transcripts 1 and 3 show a high correlation with the IPO7 gene encoding for the importin 7 protein, a member of the \u03b2-karyopherin family proteins that mediate nuclear import of ribosomal proteins and export of ribosomal subunits necessary for ribosome biogenesis [C9orf72-IPO7 correlation reflects a functional interaction, but it has been shown that C9orf72 protein isoforms localize to the nuclear membrane in healthy neurons and they interact with components of the nuclear pore complex [C9orf72 transcripts 1 and 3 in the CNS might be physiologically involved in nuclear import and export that would be affected by a decrease in their expression. This potential detrimental effect could be exacerbated in C9orf72-HRE patients by the presence of HRE-containing transcripts, as indicated by several studies highlighting defects in nucleocytoplasmic trafficking as results of HRE expression [Our analysis on global ogenesis . Current complex . It is tpression , 11, 57.C9orf72 functions in CNS, particularly when considering therapeutic efforts aiming at simply decreasing C9orf72 expression to ameliorate RNA foci and DPR proteins aggregates formation.More research is therefore warranted in characterizing C9orf72 expression in C9orf72-HRE patients but also in MAPT and GRN mutations carriers pointing to additional regulatory mechanisms besides the HRE-mediated C9orf72 repression. On control brains we observed an association of the C9orf72-HRE risk haplotype with an increase in expression of C9orf72 TSS for transcript 1 and 3 and decrease in TSS for transcript 2. The association of C9orf72 expression with SNPs in the risk haplotype has been already reported in monocytes but never in brain [C9orf72 transcripts 1 and 3 and we replicated the association of the risk haplotype with the increase of C9orf72 transcript 3 but not with transcript 1 or 2, likely because of the small effect and sample size. At first glance the increased expression of transcript 3 determined by the risk haplotype appears to be in contradiction with the CAGEseq and qPCR expression data showing general reduction of transcript 3 in the C9orf72-HRE and FTD cases with MAPT and GRN mutations compared with controls. However the increase in expression of C9orf72 transcript 3 in individuals carrying the risk haplotype is small. Additionally, approximately half of the GRN and MAPT samples do not carry the risk haplotype.Our CAGEseq data and qPCR results showed a consistent decrease in in brain , 59. In C9orf72 transcript 3 might have consequences in C9orf72-HRE patients. The HRE can be transcribed in the pre-mRNA of abortive and/or mature C9orf72 transcripts 1 and 3 that would accumulate in RNA foci [C9orf72-HRE cases might intensify RNA foci formation although not necessarily DPR proteins aggregation as it has been suggested RAN translation might require transcripts containing the first entire intron as template [Albeit small, the increase in expression of RNA foci , 60. In template .C9orf72 transcript 3 expression are unknown. The C9orf72-HRE occurs on a risk haplotype covering 110\u00a0kb region between MOB3B and C9orf72. The HRE is highly polymorphic and prone to expansion particularly in the context of the risk haplotype [C9orf72-HRE patients [GRN and MAPT mutation carriers used in this study.The molecular reasons underlying the association of the risk haplotype with the increase in patients , 52; howC9orf72 expression. We gathered convincing evidence for the existence of three distinct antisense transcripts head-to-head to C9orf72 that are highly expressed in myeloid cells and to a lower extent in brain tissues in C9orf72-HRE cases as well as in individual without the repeat expansion. Several C9orf72 antisense transcripts have been already detected in intron 1b [C9orf72-AS2 and AS3 transcripts are adjacent but downstream the HRE and they might be involved in C9orf72 regulation.Therefore, additional mechanisms must play a role in regulating ntron 1b , 15 and C9orf72 regulation mainly in the context of the G-rich HRE that would mediate RNA-DNA hybrid formation (R-loops) facilitating RNA polymerase II pausing before efficient termination causing transcriptional stalling and nucleolar stress [C9orf72 sense transcripts and antisense transcript AS3 in brain tissues from C9orf72-HRE patients and therefore it might be relevant to investigate whether the novel antisense transcripts hamper or amplify the HRE antisense effects. In addition increasing evidence shows that antisense transcripts act at nearly every level of gene regulation  and even function simultaneously at multiple steps. It has been shown that antisense expression can regulate transcription by affecting DNA methylation. Several studies, including our own, show that hypermethylation of the CpG-island at the 5\u2032 end of the repeat contributes to C9orf72 repression in a high of C9orf72-HRE carriers, yet down-regulation of C9orf72 is more prevalent and shown among FTD sporadic patients [MAPT and GRN mutation carriers (this study). Very recently, co-localization of para-nucleolar DPR proteins inclusions with heterochomatin and H3K9me2 [C9orf72 antisense AS3 affects histone modifications at the C9orf72 locus not only in C9orf72-HRE but also in MAPT and GRN patients.Gene regulation by antisense transcription is rather intriguing. Antisense transcripts are transcribed from the opposite strand of protein-coding genes. This genomic arrangement immediately suggests that the sense and antisense transcripts might act on each other and increasing evidence corroborates such an hypothesis . Antisenr stress . We obsepatients  and MAPT H3K9me2 , a marke H3K9me2 . In lighC9orf72 expression in myeloid cells and the changes in C9orf72 expression in CD14+ monocytes after exposure to microbial pathogens suggest that C9orf72 might be involved in immune-related processes, in agreement also with the recent findings that in mice is required for normal function of myeloid cells [C9orf72 might play a role in the initial steps of autophagy as suggested by its interaction with FIP200, a component of the ULK1-ATG13 autophagosome initiation complex [The robust id cells . Moreove complex .C9orf72 role in autophagy, neurons differentiated from patients-derived iPSC, are more sensitive to autophagy inhibitors and presented higher p62 levels suggesting autophagy might be compromised [Consistent with a possible promised .The half-life of monocytes in blood is relatively short: approximately 1\u00a0day in mice and 3\u00a0days in human. Once monocytes are stimulated by an inflammatory response they activate pro-survival pathways, migrate to tissues where they mediate direct antimicrobial activity by releasing tumor necrosis factor, chemokines, engage in phagocytosis and differentiate to macrophages through molecular mechanisms that involve autophagy .C9orf72 in the autophagy process it is tempting to speculate that reduced C9orf72 expression might impair autophagy affecting monocyte-macrophage differentiation and thereby hamper host inflammatory responses. Although our findings will need to be replicated in a larger samples size including age-matched controls, our qPCR results on CD14+ monocytes from C9orf7-HRE carriers and from patients with clinical diagnosis of FTD and ALS are in line with this hypothesis. Currently we do not have evidence that monocytes in these patients are impaired but we show that they carry a very high antisense RNA foci burden.While waiting for further research to clarify the exact role of C9orf72 expression. We showed that the decrease in C9orf72 expression is a widespread phenomenon in FTD pathogenesis suggesting C9orf72 plays a more general role in neurodegeneration. Moreover the C9orf72 TSSs profile in CNS and myeloid cells suggests distinct C9orf72 transcripts have tissue specific function and they call attention to a potential C9orf72 role in immune response.In conclusion, our findings strongly imply that several mechanisms acting independently from the HRE, or in a concerted manner, contribute to regulate"}
+{"text": "Solanum lycopersicum) transcription factor SlZFP2 fine tunes ABA biosynthesis during fruit development through direct suppression of ABA biosynthetic genes and it also regulates fruit ripening through transcriptional suppression of the ripening regulator CNR. This indicates that SlZFP2 likely modulates the cross-talk between ABA and ethylene in regulation of fruit development and ripening in tomato. Gene expression analysis using ABA deficient mutants sit and flc as well as the SlZFP2 RNAi lines of high fruit ABA production showed that ethylene biosynthetic genes LeACS1A, LeACS1 and LeACO1 were positively regulated by ABA during early fruit growth. We reason that ABA promotes basal ethylene biosynthesis in system 1 during fruit growth and likely plays a minor role in ripening regulation after the onset of ripening process.The stress hormone ABA not only regulates stress response, but is also required for plant development and growth. Some evidences indicate that ABA plays a pivotal role in the ripening process of non climacteric as well as climacteric fruits. In a recent study, we showed that the tomato ( SlZFP2Solanum lycopersicum zinc finger protein 2HA-SlZFP2an expression cassette of hemagglutinin-SlZFP2 fusion proteinACS1-aminocyclopropane-1-carboxylic acid synthaseACOACC oxidaseCNRCOLORLESS NON-RIPENINGNOTNOTABILISSITSITIENSFLCFLACCASlAO1Solanum lycopersicum aldehyde oxidase 1ABAabscisic aciddpadays post anthesisRNAiRNA interferenceFPKMFragments Per Kilobase of transcript per Million mapped reads.SlZFP2, encoding a single C2H2 zinc finger protein, in tomato fruit development.HA-SlZFP2 under 35S promoter repressed ABA biosynthesis in leaves and fruits, whereas silencing its expression increased ABA production in young fruits at 5 and 10 dpa. We also revealed that SlZFP2 regulates fruit ripening through transcriptional repression of the ripening regulator CNR. Thus, the SlZFP2 pathway likely modulates crosstalk between ABA biosynthesis and the regulatory network of fruit ripening in tomato.ABA is well known for its roles in seed maturation and germination, in addition to its pivotal roles in stress response.NOT, FLC, SIT and SlAO1. Since SlZFP2 is mainly expressed during fruit development, it likely plays an important role in maintenance of the dynamic ABA production post pollination. Indeed, SlZFP2 expression negatively correlates with ABA level during fruit development, for example, SlZFP2 expression was relatively low in anthesis ovaries and 20 dpa fruits when high ABA production was observed . Their expression was not impacted at the onset of ripening process by overexpression or RNAi-mediated repression of SlZFP2. Thus, the gene expression analysis suggests that elevated or repressed ABA biosynthesis by manipulating SlZFP2 expression has little impact on ethylene production at the onset of ripening process.However, the action of LeACS1A, LeACS6, LeACO1, 3 and 4 are responsible for the basal ethylene production.SlZFP2 does not directly regulate the induction of ethylene biosynthesis in system 2. However, we found LeACO3 and LeACO4 expression was increased significantly in the 2 dpa fruits of the representative SlZFP2 RNAi line 207 through transcriptome analysis by RNA-seq. The other ethylene biosynthetic genes LeACS1A, LeACS2 and LeACO1, although the LeACS1A and LeACS2 were expressed at low levels, were also expressed at higher levels in the young fruits (SlZFP2 may regulate ethylene biosynthesis in system 1. Thus, the problematic fruit set observed in these SlZFP2 RNAi lines can be explained by elevated ethylene biosynthesis. Our observation is consistent with early studies that ABA promotes flower and fruit abscission through ethylene biosynthesis.SlZFP2 RNAi fruits, SlZFP2 likely regulates ethylene biosynthesis during early fruit growth through ABA pathway. To test the possibility, we analyzed the expression of ethylene biosynthetic genes in ABA deficient mutants sit and flc. We found that LeACO1 was downregulated in both the 5 and 10 dpa fruits of the 2 mutants; LeACS6 expression was also repressed at 5 dpa (Ethylene is the predominant plant hormone regulating climacteric-fruit ripening. In tomato, two systems of ethylene biosynthesis have been proposed, which basal ethylene production is maintained in system 1 during fruit growth and later its production is increased drastically in system 2 during ripening.SlZFP2 plays at least two roles in regulation of fruit development and ripening (SlZFP2, likely induced by high ABA at anthesis, represses ABA biosynthesis after anthesis, and in turn the decrease in ABA level limits ethylene production during fruit set and early fruit growth. Fine-tuning ABA biosynthesis likely helps to maintain ethylene production at its basal level in system 1 for normal fruit growth. Second, SlZFP2 also prevents CNR expression before the onset of ripening process. However, it remains to be determined whether or not the ABA biosynthesis regulated by SlZFP2 interconnects with the CNR-mediated ripening regulation.Collectively,"}
+{"text": "Background: During March 2009 a novelInfluenza A virus emerged in Mexico. We describe the clinical picture of the pandemicInfluenza A (H1N1) Influenza in cancer patients during the 2009 influenza season.Methods: Twelve centers participated in a multicenter retrospective observational study of cancer patients with confirmed infection with the 2009 H1N1Influenza A virus (influenza-like illness or pneumonia plus positive PCR for the 2009 H1N1Influenza A virus \u00a0in respiratory secretions). Clinical data were obtained by retrospective chart review and analyzed.\u00a0Results: From May to August 2009, data of 65 patients were collected. Median age was 51 years, 57 % of the patients were female. Most patients (47) had onco-hematological cancers and 18 had solid tumors. Cancer treatment mainly consisted of chemotherapy (46), or stem cell transplantation (SCT) (16). Only 19 of 64 patients had received the 2009 seasonal Influenza vaccine. Clinical presentation included pneumonia (43) and upper respiratory tract infection (22). Forty five of 58 ambulatory patients were admitted. Mechanical ventilation was required in 12 patients (18%). Treatment included oseltamivir monotherapy or in combination with amantadine for a median of 7 days. The global 30-day mortality rate was 18%. All 12 deaths were among the non-vaccinated patients. No deaths were observed among the 19 vaccinated patients. Oxygen saturation <96% at presentation was a predictor of mortality .Conclusions: In our cancer patient population, the pandemic 2009 Influenza A (H1N1) virus was associated with high incidence of pneumonia (66%), and 30-day mortality (18.5%). Saturation <96% was significantly associated with death. No deaths were observed among vaccinated patients. Influenza with a mortality rate of up to 28%1. Non-transplant cancer patients can also have a high mortality rate of up to 38%2, being higher in patients with lung, hematological and colorectal cancer, in patients that develop lower respiratory tract infections, and in patients with other co-morbid conditions. In Argentina, seasonal Influenza in onco-hematological patients is associated to a 12% incidence of pneumonia and to a 5% of 30-day mortality3.Seasonal influenza is a known cause of morbidity and mortality among cancer and transplant patients. During influenza season, 20 to 30% of stem cell transplant SCT recipients with respiratory symptoms can test positive forInfluenza A virus, later known as 2009pandemic influenza A (H1N1), emerged in Mexico. The new strain initially spread among travelers to the USA and Canada, and subsequently infected people worldwide4. Clinical presentations ranged from mild symptoms to severe cases that lead to pneumonia and respiratory failure\u2013related deaths.In March 2009 a novelInfluenza A (H1N1) in Argentina were reported in May 2009, in travelers returning from Mexico and the USA. From May to December 2009 there were 11931 cases of confirmedInfluenza A H1N1 in Argentina, 617 deaths, and over 90% of the circulating respiratory viruses in adults were the novelInfluenza A H1N15.The first cases of pandemic16. In these studies, the incidence of pneumonia ranges from 2011 to 52%8, while the reported mortality rate ranges from 0\u201310%16 to as high as 21\u201331%15.Data from different studies on the impact of this new virus in the adult cancer and SCT population are somewhat contradictory. Many studies from different countries were reportedInfluenza A H1N1 continued to circulate .During the 2013 winter season, pandemicpandemic H1N1Influenza during the 2009 influenza season, in patients with cancer and SCT in two cities of Argentina.In this study, we examined the effects and severity ofThis is a multicenter retrospective observational study that included 12 medical centers. From May to August 2009, cancer and SCT patients older than 16 years, who presented a confirmed influenza infection by real-time PCR were included.pandemic Influenza A H1N1 virus were performed on nasopharyngeal swabs or bronchoalveolar lavage samples when available. Either of two PCR protocols were used for detection of thepandemic Influenza A H1N1 virus depending on test availability: the Real-time ready InfluenzaA/H1N1DetectionSet\u00ae Version June 2009  and the PCR protocol used by the WHO .The following data were obtained anonymously: underlying illness, type and date of SCT, whether patients were or were not receiving immunosuppressive treatment, at the time of the influenza diseases, immunization for seasonal influenza, clinical presentation (influenza like or pneumonia), laboratory and radiology results, anti-viral treatment, and outcome. In addition, data on the time between the onset of symptoms and the initiation of antiviral therapy, need for ventilation support, and presence of co-infections were also collected. Hypoxemia was defined as an oxygen saturation value lower than 96%. We diagnosed lymphopenia when the absolute lymphocyte count was less than 1000/\u00b5L. The RT-PCR tests forCategorical variables are shown as percentages and they are compared with the \u03c7\u00b2-distribution test or Fisher test. Numeric variables such as median and range are compared with the Wilcoxon test. The association between baseline variables and events is presented as OR with the 95% CI. In all cases, statistical significance was assumed at a value of p=<0.05.From May to August 2009, 12 centers sent data of 65 cancer patients with 2009 H1N1 virus disease confirmed by positive PCR in BAL (3) or nasopharyngeal wash (62). The median age of the patients was 51 years (range 17 to 81), and 57% were female. The majority of patients (47) had onco-hematological cancer (72%) and 18 (28%) had solid tumors. Cancer treatment included chemotherapy (46), SCT (16), no treatment (2) and surgery (1). History of 2009 seasonal influenza vaccination was present in 19/64 patients (30%). No patient had received influenza chemoprophylaxis. The median time of patients follow up from the onset of symptoms was 61 days, range 5 to 259.Data on overall clinical presentation and outcome are shown in17.Patients started treatment at a median of two days from onset of symptoms (range 0 to 45 days). Sixty eight percent (43/63) of patients started treatment within the 48h after the onset of symptoms. Some patients received combined antiviral treatment because of the potential circulation of seasonal Influenza A H1N1 known to be resistant to oseltamivirMost patients acquired the infection in the community  while 7 (11%) of infections were acquired in the hospital setting despite the implementation of adequate standard precautions and isolation measures during this outbreak. Detailed descriptions of the outcome of patients with community acquired (CAPIA) and nosocomially-acquired (NAPIA) pandemic Influenza A H1N1 infection patients are described inMost  of outpatients required hospital admission. Reasons for patient admission included mainly oxygen desaturation, but, in many cases, patients were admitted because of their severe state of immune suppression and the lack of information about this emergent virus, especially when the patient\u2019s social environment prevented him/her from easy access to medical care.Outpatients who presented with upper respiratory tract symptoms (URTI), had the most benign course since the majority  resolved their infections with antiviral therapy in the outpatient setting, and, among the 8 (42%) who were admitted, none of them required ICU admission or developed signs of pneumonia. The 30-day mortality among CAPIA URTI was 0.Outpatients who presented with pneumonia had a more severe course since almost all of them  were admitted, 15/39 (38%) required ICU, 11/39 (28%) required mechanical ventilation (MV), and the 30-day mortality in this group was of 23% (9/39). The worst prognosis in this group was seen among those who presented with pneumonia and desaturation (25), leading to an admission rate of 100% , and a 30-day mortality of 36%.Patients, who developed NAPIA, belonged to 3 different centers and started having symptoms at median of 20 days after admission (range 2 to 33). This group had the poorest prognosis since the 30-day mortality rate was 43% (3/7).One of three (33%) NAPIA URTI progressed to pneumonia, while none of the 19 patients with CAPIA URTI did. Therefore, the overall progression from URTI to pneumonia was of 4.5% (1/22).The 30-day mortality according to the clinical presentation and setting is best described inAcinetobacter baumanii, and GNR that was only seen in direct examination), 2 pneumonias  and 1 meningitis (Ps. aeruginosa). The median time from the onset of symptoms to the development of a bacterial complication was 11 days, range 0\u201334. Bacterial complications developed only among patients who presented with pneumonia  by the pandemicInfluenza A H1N1. No bacterial complication developed among the 22 patients who presented with URTI.Bacterial complications were documented in 6 (9%) patients and included 3 bacteremias  and bleeding (2) (lung and brain). Most patients presented more than one complication.Non-infectious complications developed in 14 (22%) patients. They included: renal failure (5), respiratory failure (5), shock (3), hypokalemia (3), nonbacterial infections (3)  at onset of symptoms and the delay in treatment were not associated to death or mechanical ventilation. By univariate analysis lack of history of vaccination, and the following baseline characteristics: pneumonia, oxygen saturation <96%, and lymphocyte count <800 cells/\u03bcL, were associated to 30-day mortality and mechanical ventilation. By multivariate analysis only lack of history of vaccination (OR did not apply because none died in the vaccinated group) and baseline oxygen saturation <96%  were associated to mechanical ventilation and death. There might be a bias regarding the apparent benefit of vaccination because in cancer patients, immunization is usually advised when the period of major immunosuppression has finished.Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patientsClick here for additional data file.Copyright: \u00a9 2015 Dignani MC et al.2015Data associated with the article are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).Influenza A H1N1 virus in 65 cancer patients from 12 institutions located in two cities of Argentina. Eleven percent of these infections were nosocomially acquired. Overall we found a high rate of pneumonia (66%) and mortality (18%). The clinical course was less severe in those who presented with an URTI in the outpatient setting in contrast to those who presented with pneumonia and desaturation especially in the hospital setting. We also found that the best predictors of death were oxygen desaturation at presentation and lack of vaccination against seasonal Influenza.Our study shows the clinical course of the infection by the 2009 pandemic20 and it is also higher than the incidence of lower respiratory tract infections (LRTI) caused by the 2009 pandemicInfluenza A H1N1 in the hospitalized general population (40\u201344%)21, in solid organ transplant recipients (23%)16, in HCT recipients (21\u201356%)22 and in patients with hematological malignancies (48%)23. Only one small study that includes 15 confirmed cases of 2009 pandemicInfluenza A H1N1 infection in onco-hematological patients reports a higher incidence of pneumonia (87%)15.The incidence of pneumonia we found is higher than the one reported with seasonal influenza in cancer patients (5\u201344%)Adenovirus, Influenza,Parainfluenza, and RSV (5%)3. However, it is similar to the mortality rate reported in hematological patients with pneumonia byInfluenza,Parainfluenza,Picornavirus and RSV at a US institution (15%)24.The 30-day mortality rate we show in this study is more than three times the one observed in Argentina in the same patient population when looking at infections by other respiratory viruses such as20. The pandemicInfluenza A H1N1 virus is an independent risk factor for progression to LRTI  and hypoxemia  compared with seasonalinfluenza virus in HCT recipients1. In addition, immunosuppression was a main risk factor for early mortality among 337 Argentinean patients admitted to ICU with influenza like illness and respiratory failure that required mechanical ventilation. Mortality was highly associated with refractory hypoxemia25. These data explain our high mortality rate observed among the 17 patients who were admitted to ICU  or among the 12 patients who developed respiratory failure . These values are comparable to those reported in the same type of population22, but are higher than those reported in other populations, which ranged from 0\u201324%29. Indeed, the overall mortality rate observed among Argentinean patients admitted to ICU and requiring mechanical ventilation was 46%25. To further support the high mortality of patients with pandemic 2009 Influenza pneumonia, we identified hypoxemia at onset of symptoms as an independent predictor of mortality.It is well known that seasonal influenza-induced pneumonia is independently associated with mortality after HCT 30, and profound lymphopenia (<100 cell/\u00b5L) was reported as a significant risk factor for requirement of mechanical ventilation and death in HCT recipients infected with seasonal influenza virus30. In our study, having fewer than 800 lymphocytes/\u00b5L at presentation was a predictor for the need for mechanical ventilation and death in a univariate but not in a multivariate analysis. We did not analyze a lower value such as <100 of lymphocytes due to the small number of patients included with this value.Lymphocytopenia has been described as a risk factor for progression from upper to lower viral respiratory tract infection in cancer patientsIt is noteworthy that co-infections or bacterial complications developed in less than 15% of patients.30 and hypoxemia30 when instituted early after onset of symptoms. It was reported that delaying therapy in cancer patients with the pandemicInfluenza A H1N1 virus infection was significantly associated with death16. Early initiation of antiviral therapy in these patients may attenuate the severity of disease27. In our series, antiviral therapy was started early after a median of two days after the onset of symptoms, with a range from 0\u201345 days. We did not find any correlation between days from onset of symptoms to therapy or diagnosis to therapy by univariate or multivariate analysis.Neuraminidase inhibitor therapy appears to be effective in preventing progression to LRTI31. In our series half of the outpatients with URTI remained as such, while the other half was admitted but did not require ICU. There is a possibility that most of the admitted patients could have been managed as outpatients as well.It is known that patients with URTI can be treated as outpatients and can recover completely from their infection20.The global progression from URTI to pneumonia in our study was of 4.5% (1/22). This single patient had a nosocomial infection and died 21 days later with sepsis, respiratory failure and neutropenia. This is according to reports of progression to LRTI that may occur even after one week of symptoms22. Higher doses could also be considered in a setting of profound lymphocytopenia30. All our patients received standard dose of oseltamivir (75 mg po twice a day) for a minimum of 10 days based on data on slower viral clearance30.In contrast, patients with LRTI required hospitalization with a high number of them requiring admission to ICU for ventilation support. The dismal outcome seen in these patients despite treatment with oseltamivir probably indicates that this high-risk group needs to be treated differently from patients with isolated URTI. Some authors have suggested an initial treatment with high dose of oseltamivir and/or combination therapy approaches in the case of respiratory failure32 and pandemic 2009 Influenza A H1N1 infection33 can develop even in the setting of appropriate infection control measures. Seven (11%) of our patients had hospital-acquired influenza. Three of them (43%) died. This mortality rate is higher than the previously reported 13\u201327%, however, the number of patients in our report is too small to make any conclusion.Nosocomial outbreaks of seasonal34. In our study all deaths occurred among the non-vaccinated patients, while there were no deaths among the vaccinated patients. Individuals vaccinated against seasonal Influenza A or with previous seasonal influenza infection may benefit from preexisting cross-reactive memory CD4+ T cells and CD8+ T cells reducing their susceptibilityto Influenza A H1N1 infection or explaining, at least in part, the unexpected mild illness in the community39. Whether the trivalent seasonal Influenza vaccine is protective against the pandemicInfluenza A H1N1 virus in cancer patients is still a matter of debate38.Seasonal influenza vaccination is recommended yearly for all patients with cancer and HSCT recipientsInfluenza A H1N1 infection with a high incidence of hospitalization, severe pneumonia, ICU admission, mechanical ventilation, and 30-day mortality. In our series hypoxemia and lack of vaccination with seasonal trivalent influenza vaccine were predictors of mechanical ventilation requirement and death. A larger study is needed to evaluate the possibility of cross protection with the seasonal influenza vaccination.In conclusion, we report a series of cancer patients with the pandemicThe data referenced by this article are under copyright with the following copyright statement: Copyright: \u00a9 2015 Dignani MC et al.Data associated with the article are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).F1000Research: Dataset 1. Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patients doi:http://dx.doi.org/10.5256/f1000research.5251.d10027640Ethical committee approval was not required in Argentina at the time of the study. This is an interesting article that shows the impact of pandemic 2009 influenza A (H1N1) in a highly vulnerable population for influenza complications such as immunocompromised oncohaematological patients.Did you observe any difference in time elapsed since the initiation of symptoms between those with community acquired pandemic 2009 influenza A (H1N1) who presented with upper respiratory tract infections and those who presented with pneumonia?\u00a0Previous vaccination against seasonal influenza showed\u00a0that\u00a0some degree of preexisting\u00a0immunity to pandemic 2009 influenza A (H1N1) strains exists, especially among\u00a0adults aged >60 years. Have you found any difference in progression from UTRI to pneumonia or to death between those older or younger than 69 years of age?The paper is well written and I would like to make some comments or questions to the authors:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Question 1:Patients with community acquired 2009 Influenza A (H1N1) who presented with URTI (N=19) had symptoms for a median of 2 days (range 1-11). Patients who presented with pneumonia (N=39) had symptoms for a median \u00a03 days (range 1-20).There was a trend towards a longer period with symptoms in patients who presented with pneumonia.Question 2:Patients older than 60 years old who had been vaccinated against seasonal Influenza \u00a0(N=10) had 60% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=4) had 100% rate of pneumonia and 25% mortality.\u00a0Patients younger than 60 years old that had been vaccinated (N=9) had 55% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=31) had 68% rate of pneumonia and 26% mortality.\u00a0Vaccination against seasonal Influenza seemed to have some degree of protection against death in all age groups. Does the report include all patients fulfilling the inclusion criteria at the 12 hospitals?Why was the cut-off for lymphocytopenia chosen as <800? Most previous studies have chosen a lower cut-off.One of several reports on outcome of the H1N1 pandemic in 2009. A couple of issues:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard."}
+{"text": "Sirt1 gene that abolishes SIRT1 catalytic activity. Tumor latency was reduced in animals fed a high fat diet but this effect was not dependent on SIRT1 activity. Resveratrol had little effect on tumor formation except in animals heterozygous for the mutant Sirt1 gene. We conclude that the effects of these dietary interventions on tumorigenesis are not mediated by modulation of SIRT1 catalytic activity.The protein deacetylase SIRT1 is involved in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. Both SIRT1 activity and tumorigenesis can be influenced by dietary fat and polyphenolics. We set out to determine whether dietary modulations of tumorigenesis are mediated by SIRT1 catalytic functions. We introduced a mammary gland tumor-inducing transgene, MMTV-PyMT, into stocks of mice bearing a H355Y point mutation in the  The NAD+ produced in this manner may function as a cofactor for the SIRT1 enzyme The rapid proliferation of cancer cells is associated with a metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, a phenomenon known as the Warburg effect Sirt1 may have the properties of a tumor suppressor gene and several studies have suggested that this may be the case Sirt1 is an oncogene Sirt1 has no effect on oncogenesis In mammals, caloric restriction (CR) can delay the onset of various ageing-related diseases including cancer Sirt1 alleles or contain mutations in the Sirt1 gene, suggesting a role as a non-classical tumor suppressor Sirt1 genes. We report below that both dietary modifications modestly affected the onset of tumorigenesis but were not dependent on SIRT1 activity.A relatively scant amount of clinical data has suggested an oncogenic role for SIRT1 in breast cancer Sirt1Y/+), mice on a mixed 129sv/CD1 background and carrying the tm2McbySirt1 allele. This allele harbours a missense mutation in the Sirt1 gene (H355Y) and encodes a catalytically inactive protein Sirt1 alleles was performed as previously described Male FVB/N-Tg(MMTV-PyMT)634Mul/J mice , were a generous gift from Dr. Bill Muller Guidelines for the Care and Use of Animals established by the Canadian Council on Animal Care with protocols approved by the Animal Care Committee of the University of Ottawa, Ottawa, Ontario, Canada.Animals received food and water ad libitum. Our previous work has shown that the caloric intake is similar between mice of all Sirt1 genotypes 2 asphyxiation. Tumours were removed, weighed and fixed in 10% neutral buffered formalin. Lungs were examined for macroscopically visible metastatic nodules prior to fixation in formalin.Animals were euthanized via COP-values are two-sided. Analysis was performed using Graphpad Prism statistical software .The probability of significant differences was determined by analysis of variance (ANOVA), employing the Kruskal-Wallis test with the Dunn's multiple comparison test. Survival and time-to-detection curves were compared using the LogRank test. Correlation was tested using the Spearman rank test. Data is expressed as mean\u00b1SEM (standard error of the mean) and Sirt1 gene  and studied the emergence of mammary tumors in +/+Sirt1, +/YSirt1, and Y/YSirt1 females. Animals were monitored weekly via digital palpation for the presence of any palpable mass in the mammary glands. It should be noted that no compensatory increase in the mRNA levels of other sirtuin family members, Sirt2-7, was observed in the Sirt1Y/Y animals  consisting of 60% calories from fat or a standard rodent diet (6% fat) from time of weaning until they had reached humane endpoint, generally due to significant tumor burden. HFD decreased tumor latency in both Y/Y mice . The eff reduced . The HFDY/+ mice .Sirt1Y/Y mice weighed 20\u201330% less than their Sirt1Y/+ and Sirt1+/+ counterparts (Sirt1Y/Y mice gained weight faster than their Sirt1+/+ and Sirt1Y/+ littermates. By 70 days of age (approximately 7 weeks on HFD) Sirt1Y/Y mice weighed an average of 10\u201320% more than the Sirt1+/+ and Sirt1Y/+ mice  and animals on the low phytoestrogen control diet (r2\u200a=\u200a0.48) (There was no significant difference in tumor burden for any of the her diet . Tumor b\u200a=\u200a0.48) .Sirt1Y/Y mice were less likely to have macroscopically visible metastatic lung nodules than Sirt1+/+ or Sirt1+/Y mice  has yet to be investigated.In all of the studies described here as well as in those reported previously Figure S1Y/Y mice.There is no compensatory increase in expression of other sirtuin family members in tissues of Sirt1 Sirt1-7 mRNA levels measured in A) liver (n\u200a=\u200a1 per group) or B) brain (n\u200a=\u200a2 per group) of Sirt1+/+ and Sirt1Y/Y mice. Values are expressed as the mean \u00b1 SEM of the threshold cycle (CT).(TIF)Click here for additional data file.Figure S2Weight gain over time.Sirt1+/+, Sirt1Y/+, and Sirt1Y/Y  mice were weighed once weekly and the weights represented here range from one week after commencing the specified diet (approximately 28 days of age) until the final week at which all animals in each group were still alive (70 days of age). A) standard rodent diet. B) high fat diet. C) low phytoestrogen diet. D) resveratrol containing diet. N\u200a=\u200a10 for all groups.(TIF)Click here for additional data file.File S1Materials and Methods: Quantitative Polymerase Chain Reaction (QPCR). Measurement of Sirt1 mRNA levels via QPCR.(DOCX)Click here for additional data file."}
+{"text": "Bipolar disorder is a highly heritable neuropsychiatric disorder affecting nearly 2.5% of the population. Prior genetic studies identified a panel of common and rare single-nucleotide polymorphisms associated with the disease that map to the first intron of the PDE10A gene. RNA sequencing of striatal brain tissue from bipolar and healthy control subjects identified a novel transcript of PDE10A, named PDE10A19, that codes for a PDE10A isoform with a unique N terminus. Genomic sequences that can encode the novel N terminus were conserved in other primates but not rodents. The RNA transcript was expressed at equal or greater levels in the human striatum compared with the two annotated transcripts, PDE10A1 and PDE10A2. The PDE10A19 transcript was detected in polysomal fractions; western blotting experiments confirmed that the RNA transcript is translated into protein. Immunocytochemistry studies using transfected mouse striatal and cortical neurons demonstrated that the PDE10A19 protein distributes to the cytosol, like PDE10A1, and unlike PDE10A2, which is associated with plasma membranes. Immunoprecipitation and immunocytochemical experiments revealed that the PDE10A19 isoform interacts physically with PDE10A2 and, when expressed at elevated levels, interferes with the plasma membrane localization of PDE10A2. These studies illustrate the complexity of PDE10A gene expression in the human brain and highlight the need to unravel the gene's complex and complete coding capabilities along with its transcriptional and translational regulation to guide the development of therapeutic agents that target the protein for the treatment of neuropsychiatric illness. Individuals exhibiting mania experience periods of markedly elated moods, are easily distracted and tend to engage in impulsive and high-risk behaviors.11 We recently queried potential BD-associated loci using a \u2018molecular pathway approach,' performing a case\u2013control study focusing on genes that comprise the cAMP pathway,12 prompted in part by numerous reports showing disrupted cAMP signaling in BD.16 Our results identified a series of common and rare single-nucleotide polymorphisms within the first large intron of the PDE10A gene as associated with BD, which generated great interest given the prominent expression of this enzyme in striatal cells,20 the role of the striatum in coordinating and integrating cortical and dopaminergic inputs23 and the promise of PDE10A as a pharmacological target for brain disorders including schizophrenia and BD.24Genome-wide and case\u2013control association studies have identified numerous susceptibility genes for BD with variants exhibiting modest effect sizes.GMP-stimulated PDEs, Anabaenaadenylyl cyclases and Escherichia coli transcription factor Fhla) domains and hydrolyze both cAMP and cGMP, but have a higher affinity for cAMP.25 The PDE10A gene is currently known to be comprised of 23 exons26 and maps to chromosome 6q26,18 a region previously associated with BD.27 The known isoforms of PDE10A, PDE10A1  were obtained from the Human Brain and Spinal Fluid Resource Center . The samples were de-identified of all the information other than clinical diagnosis, sex, age at death and postmortem interval . For RNAThe 5\u2032 RNA ligase-mediated rapid amplification of complementary DNA (cDNA) ends (RLM-RACE) studies were performed with HC putamen and a primer that bound within exon 4. The resulting 5\u2032 DNA fragments were cloned and sequenced. To determine whether PDE10A transcripts were alternately spliced between exons 4\u201322, total RNA was treated with DNAseI and used for cDNA synthesis primed by random hexamers. PCR primer pairs  were the32 RNA isolation, cDNA synthesis and real-time PCR were performed as described above.Polysomes were isolated from four HC striatal tissue specimens, obtained from the Harvard Brain Tissue Resource Center , a part of the NIH NeuroBioBank , via ultracentrifugation on sucrose gradients.Custom anti-peptide antibodies were generated for each PDE10A isoform by an external contractor. The Scripps Research Institute IACUC approved the production of these antibodies. Full-length PDE10A transcripts with 3\u2032-HA or Flag epitope tags were constructed  and usedThe PDE10A19-HA or PDE10A2-HA enzyme was affinity purified from transfected HEK293 cell lysates using the HA-tagged Protein Purification Kit  according to the manufacturer's instructions. Enzymes from four affinity purifications were pooled and desalted by gel filtration to remove excess phosphate from the samples. PDE10A activity was measured within the linear phase of the enzyme's activity over a 15-min time course using the Cyclic Nucleotide Phosphodiesterase Assay Kit  according to the manufacturer's instructions.33 DIV14\u201318 primary neurons were fixed, incubated with anti-HA Alexa 488-conjugated monoclonal antibody and/or anti-peptide antibodies  and imaged.34 Immunoreactivity intensity profiles across neurites were computed and normalized for neurite diameter differences and background intensity. The images were analyzed using Image J and statistical analyses performed using Excel and/or Prism.Primary neuronal cultures from 0- to 1-day-old C57BL/6J pups were prepared for immunocytochemistry as described.de novo RNA transcript assembly for each of the samples. From this de novo alignment, the annotated PDE10A2 transcript (NM_001130690) was detected in all of the HC samples, determined by obtaining reads specific to exon A1/2.1 and spliced to exon 2  were found linked to the annotated exons 2, 3 and 4 (RNA sequencing (RNAseq) was performed on total RNA isolated separately from the putamen and caudate nucleus of postmortem striatal brain tissue from four individuals diagnosed with BD and four individuals with no reported symptoms of any neuropsychiatric disorder (HCs). On average, 20 million reads were obtained from each sample  and aligo exon 2 , while to exon 2 . Surpriso exon 2 .35, 36 T 3 and 4 . A human 3 and 4 . A TATA We generated and sequenced overlapping RT-PCR (PCR with reverse transcription) amplicons spanning PDE10A annotated exons 4\u201322 to ascertain whether any additional RNA transcripts might be produced by alternative splicing across this region. These efforts failed to reveal any major splice variants that arise from the coding region for these exons . A smallThus, our combined RNAseq and 5\u2032 RLM-RACE data indicate the existence of a novel first exon for PDE10A, a heretofore unknown but predicted protein isoform of the human PDE10A gene, named PDE10A19, and a promoter for the PDE10A gene residing upstream of the novel first exon which may confer different regulatory properties on PDE10A19 expression compared with other PDE10A RNA transcripts. The data further indicate that the N terminus of PDE10A19 is unique but comes into frame with the other PDE10A isoforms at exon 2. Therefore, it is predicted to contain the same GAF binding and catalytic domains found in the PDE10A2 and PDE10A1 isoforms .We queried online protein sequence databases and found that the green monkey and the crab-eating macaque both have predicted proteins that are highly homologous to PDE10A19 . We alsode novo assembly analysis (P=0.0482) and PDE10A2 (P=0.0150) transcripts in the putamen when the data for all the eight subjects were pooled and compared (P=0.0087) and PDE10A1 (P=0.0010), while no significant difference was found in the abundance estimate between the PDE10A2 and PDE10A1 transcripts. We also examined the expression level of the three transcripts between the HC and BD subjects. The PDE10A2 transcript was found to be expressed fivefold less abundantly in the BD putamen samples compared with the HC from the RNAseq data (P=0.0039).To estimate the abundance of the PDE10A19 transcript relative to PDE10A1 and PDE10A2, we counted the number of RNAseq reads residing within the novel exon of the transcript and normalized these reads for exon size and number of reads that mapped to the upstream PDE10A genomic region used for the analysis . The PDEcompared . The PDEWe isolated polysomes from postmortem HC striatal tissue to determine whether the three different mRNAs were loaded into protein synthesis machinery. Tissue samples were lysed in the presence of the translational inhibitor cycloheximide and the cytosolic supernatant was separated on a sucrose gradient via ultracentrifugation. The relative transcript abundances in gradient fractions were measured by quantitative RT-PCR. Messenger RNA for all the three PDE10A isoforms, PDE10A2, PDE10A1 and PDE10A19, was detected in both low molecular weight (free RNA) and high molecular weight (containing polysomes) fractions, suggesting their association with polysomes in striatal neurons .We generated anti-peptide antibodies to the unique N termini of the three different PDE10A isoforms to assay their expression in both human tissue and transfected cells. We tested the anti-peptide antibodies using an indirect ELISA assay to determine how the anti-peptide antibodies bound to their specific antigenic peptide . This asWe tested this hypothesis using IP experiments. Cell lysates expressing the HA-tagged PDE10A19 isoform were immunoprecipitated with anti-PDE10A19 anti-peptide antibody, anti-PDE10A2 anti-peptide antibody, anti-HA antibody and normal rabbit IgG. These IP reactions were fractionated and probed with the anti-HA antibody . This prTo test whether the PDE10A19 protein is expressed in human brain, we performed IP experiments using human striatal tissue. A commercially available PDE10A antibody that targets the conserved carboxyl terminus of PDE10A was used to IP PDE10A proteins from HC 3589 and the immunoprecipitates were probed with the PDE10A19 anti-peptide antibody. As shown in m, respectively.To confirm that PDE10A19 hydrolyzed both cAMP and cGMP and exhibited enzymatic properties similar to other PDE10A isoforms, HA-tagged PDE10A19 and HA-tagged PDE10A2 enzymes were affinity purified from transfected HEK293 cell lysates for use in cyclic nucleotide phosphodiesterase assays. 30 None of the three isoforms were expressed in the nucleus.  that mapped to regions of the genome currently annotated as intronic. Others have reported that up to 64% of the total RNAseq reads map to non-exonic regions of the genome using human brain tissue,38 unlike other tissues.39 Thus, our data along with others indicate that the complexity of gene expression in the human brain is greatly underappreciated. This dictates a strategy that most, if not all, neurogenetic studies that focus on individual genes should begin with a complete structural characterization of the gene of interest to reduce the possibility of false conclusions.Our prior case\u2013control study of 29 different genes comprising the cAMP signaling pathway identified seven common and eight rare nucleotide variants associated with BD that mapped to a 23\u2009kb intronic region of the PDE10A gene.The strategy applied here to the PDE10A gene revealed unknown complexity in RNA transcripts and the spectrum of protein isoforms encoded by them. The 5\u2032 RLM-RACE experiments using HC putamen tissue confirmed the existence of the PDE10A19 transcript and argued that it includes a unique transcriptional start site and regulatory region, since multiple and independent cDNA clones of PDE10A19 terminated in the identical 5\u2032 nucleotide. The expression of PDE10A19 transcripts was measured at levels equal to or higher than the two annotated forms of PDE10A, PDE10A1 and PDE10A2. Thus, the impact of PDE10A19 on the physiology of human striatal neurons cannot be trivial. This critical fact now needs to underlie all studies of how cAMP signaling influences the physiological state of medium spiny neurons.40 As rodents do not express the PDE10A19 isoform, mouse physiology and behavioral studies become difficult to perform. However, the primate-specific presence of PDE10A19 is consistent with the thought that neuropsychiatric disorders like BD and schizophrenia are human-specific diseases with human-specific etiology.A very intriguing observation made was that the genomic sequences encoding the novel N terminus of PDE10A19 is conserved only among primates, and not rodents. This is in contrast to PDE10A1 and PDE10A2, which are conserved isoforms in both primates and rodents. This suggests that the PDE10A19 isoform evolved after the separation of primates from rodents. Species expression differences of PDE isoforms are not unusual; in fact, in the case of the PDE4B4 isoform, while humans and rats have relatively conserved genomic sequences for this isoform, only rats express this protein due to the stop codons positioned in every reading frame of the human nucleotide sequence.30 Therefore, this isoform may be primarily responsible for submembranous PDE activity, including the PDE10A activity at the membranes of dendritic spines. Our data argue that the three human PDE10A isoforms are non-nuclear, in contrast to some studies of rat non-striatal neurons where PDE10A was primarily localized to the nucleus.41 In addition, we show here that PDE10A19 and PDE10A2 interact; and that PDE10A19, when expressed at high levels, can redirect PDE10A2 from a membrane-bound position to a cytosolic one. Thus, the level of expression of PDE10A19 under normal circumstances is predicted to influence the ratio of membrane/cytosolic PDE activity. It also seems possible that mis-expression of PDE10A19 in a diseased state would dysregulate the normal trafficking of PDE10A2, rendering it unable to associate with the plasma membrane and unable to control cAMP signaling at the synapse and/or across cellular membranes. One function of PDE10A activity is to inhibit striatal output by reducing medium spiny neuron excitability.42 If this function is primarily because of PDE10A2 membrane localization, then mis-expression of PDE10A19 would be predicted to redirect PDE10A2 to the cytosol, reducing membrane-associated PDE activity, and increasing the excitable state. This idea that a lack of PDE10A enzyme could cause cAMP signaling disruptions has also been suggested as a cause of the motor and psychiatric deficits seen in Huntington's Disease.44 Competitive inhibitors of PDE10A have been proposed as therapeutic targets to ameliorate psychosis.45 However, if the molecular pathology of psychotic disorders arises from a failure of PDE10A2 to regulate excitability at the membrane, due to mis-expression of PDE10A19, such therapies may be ineffective.Besides providing cytosolic PDE activity, PDE10A19 may modulate cAMP signaling by influencing the subcellular localization of its binding partners. PDE10A2 is only one of the three isoforms investigated in this study that appears capable of interacting with the plasma membrane.47 This multimerization occurs through the GAF-B binding domain of the protein,48 and as this region is conserved between the PDE10A19 and PDE10A2 isoforms, it stands to reason that these two isoforms may interact as a heterodimer. The dimerization of PDE10A2 with PDE10A19 may in some way protect or expose the N-terminal antigenic site of PDE10A2.In addition, we discovered that our PDE10A2-specific anti-peptide antibody failed to recognize PDE10A2 protein when expressed alone, but did so when PDE10A19 was expressed. This lack of recognition can be explained by at least two hypotheses: (1) the PDE10A2 anti-peptide antibody fails to recognize the PDE10A2 N terminus because the N-terminal sequences are cleaved from the protein posttranslationally or (2) PDE10A2 palmitoylation at Cys-11 ref.  or phospOur data reveal that the landscape of PDE10A gene expression and protein function in the striatum is much more complex than originally thought. Our studies identify a novel and reasonably abundant form of PDE10A along with its own promoter. We also show that the expression of this isoform influences the subcellular localization of other isoforms, specifically PDE10A2. The cell biological knowledge of this important family of enzymes is critical for understanding the enzyme's role in contributing to neurospsychiatric diseases such as BD. Will the large intronic region found to be associated with BD consist of regulatory elements responsible for controlling the expression of different PDE10A isoforms? As discussed above, disturbances to this balance of transcript/protein abundance could dysregulate cAMP signaling, perhaps in specific subcellular compartments, and alter behavior. Thus, more studies targeted at understanding how these various isoforms are transcriptionally and translationally regulated within the human striatum are warranted."}
+{"text": "The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI).n = 229), patients on dialysis at ICU discharge (n = 77) and patients for whom Clcr on the last day of ICU was not available (n = 206), 784 patients were included in this analysis. We compared eGFR (MDRD equation) with measured Clcr (based on 24-hour urine collection and corrected for BSA) at ICU discharge for patient groups with different ICU stays. We also evaluated the impact of using the two GFR measurements on the estimation of complete recovery relative to baseline eGFR. Parameters were compared with the paired t test and McNemar's test.From a large RCT's database  we exclu2 (P < 0.0001). eGFR was not significantly different from Clcr in patients with ICU stay <7 days. In patients with ICU stay between 8 and 14 days, eGFR was significantly higher than Clcr  and the difference increased even further in patients with ICU stay over 14 days . The percentage of patients with complete recovery differed significantly when evaluated by eGFR (35.3%) or Clcr (28.7%) (P = 0.007). In patients with ICU stay >14 days, this difference increased to 56.4% by eGFR versus 14.1% by Clcr (P < 0.0001).Amongst the 784 patients with AKI, 456 (58%) reached stage 1, 143 (18%) stage 2 and 185 (24%) stage 3. Mean \u00b1 SD Clcr and eGFR at ICU discharge were respectively 54.5 \u00b1 28 and 76 \u00b1 55 ml/minute/1.73 mEstimated GFR at ICU discharge is significantly higher than the measured Clcr in patients with prolonged ICU stay. This difference can be explained by loss of muscle mass with decreased creatinine production and results in an important overestimation of recovery."}
+{"text": "Ara h 8 is hypothesized to be the panallergen responsible for oral allergy syndrome between birch pollen (Bet v 1) and peanut. We recently determined the crystal structure of Ara h 8. In this work, we probed microarrays of peptides with peanut allergic and peanut sensitized patient sera for IgE and IgG4 reactivity.15-mer peptides that were offset by 5 amino acids were printed to glass. Patient sera was incubated with the slides. IgE and IgG4 binding was detected with combinations of secondary and fluorescently-labeled tertiary antibodies. The linear epitopes identified were mapped on the 3-D structure and compared with those of birch pollen protein Bet v 1.The majority of the Ara h 8 IgE epitopes mapped in this work align with those identified with Bet v 1. Considerably more IgG4 epitopes than IgE epitopes were found. Peanut allergic sera were more reactive with regard to IgE and IgG4 than peanut sensitized sera.Our results support both the hypothesis that Ara h 8 could be contributing to oral allergy syndrome between birch pollen and peanut."}
+{"text": "Intraplexal nerve transfer is defined as nerve transfer of a nerve within the brachial plexus with intact spinal cord connections to a more important injured nerve. For elbow flexion, the most popular one for the upper arm brachial plexus injury is the \u201cOberlin\u201d nerve transfer. Transferring a part of the ulnar nerve to the branch to the biceps (Oberlin 1) and possibly transferring a part of the median nerve to the branch to the brachialis (Oberlin 2) provides promising results for elbow flexion without any permanent deficit of the donor nerve function.In 1998, we published the paper using the Oberlin 1 technique in 32 cases with upper arm brachial plexus injury. Thirty patients had biceps strength of M4 (flexion power ranged from 0.5 to 7 kg) and 1 had biceps strength of M3. All but 1 patient demonstrated signs of recovery of the biceps muscle. No notable impairment of hand function was observed.For shoulder abduction, most nerve surgeons recommended nerve transfers both to the suprascapular nerve and the axillary nerve. In 2003, we described the nerve transfer to the deltoid muscle using the nerve to the long head of triceps both anatomically and clinically in 7 cases. All patients recovered deltoid power against resistance (M4) at the last follow-up evaluation. Useful functional recovery was achieved in all 7 patients; 5 had excellent recoveries and 2 had good results. The average shoulder abduction was 124 degrees. No notable weakness of elbow extension was observed.In 2006, we published the paper of combined nerve transfers for C5 and C6 brachial plexus avulsion injury in 15 cases. All 15 patients achieved useful functional recovery. Ten patients experienced excellent recoveries and 5 were classified as having good results. The mean shoulder abduction was 115\u00b0. The mean shoulder external rotation was 97\u00b0. No patients complained of functional deficit from harvesting of the nerve to the long head of the triceps.In our experience, 40 % of our patients with upper arm typed brachial plexus injury demonstrated winged scapula from serratus anterior weakness. In 2009, we described the nerve transfer to serratus anterior muscle using the thoracodorsal nerve in C5 and C6 brachial plexus avulsion injury in 5 patients. All patients recovered serratus anterior muscle function. Two patients had no winged scapula, whereas 3 patients had mild winged scapula after the surgery at the last follow-up evaluation. The result was excellent for 2 patients, good for 2 patients, and fair for 1 patient. The averaged shoulder abduction was increased to 134 degrees and external rotation was 124 degree. No notable weakness of shoulder adduction was observed.For wrist extension in C5, C6 and C7 brachial plexus injury which is difficult to achieve by tendon transfer, we described the nerve transfer using the branch of median nerve to restore the wrist extension. We used Flexor digitorumsuperficialis branch of median nerve transfer to extensor carpi radialis branch of radial nerve. This technique could restore wrist extension in C5, C6 and C7 brachial plexus root avulsion."}
+{"text": "Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes.The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity  The SNF2 family of chromatin remodelers is known to facilitate different regulatory functions involving chromatin. The INO80, a highly conserved member of the SNF2 family of DNA dependent ATPase shows functional diversity and is implicated in transcription, replication, cell division and DNA repair . The altin vitro DNA binding activity to various degrees, specific targeting and recruitment to nucleosomal DNA, as well as regulation of chromatin remodeling activity is believed to be mediated either by specific interactions between specialized domains of chromatin remodeling complexes with post-translationally modified histones or through the interaction with sequence-specific DNA binding proteins, such as YY1, which recruit different complexes including the INO80 complex [CA][CA][CG] GTCA[GC]CC 3\u2019 with a significant enrichment score of 5.75e-07 . When the T at 6th position is altered to G (M3) there is no interaction, while it is altered to A (M6), the interaction persists at equal molar ratio of 1:1, and is competed out at 1:5, suggesting a decrease in affinity for the mutant oligo. These residues are also among the most conserved residues in the consensus motif. Thus, we confirm the binding of INO80 to its sequence motif and that the binding is specific as seen by competitive EMSA and the INO80 knock-down experiments.To assess the base specificity within the consensus DNA sequence, the consensus motif was altered at various positions and these oligos were used in the competitive EMSA . The mutDrosophila  represents the subset of putative INO80 targets devoid of YY1 binding sites; [INO80(+YY1)] represents the subset of the list having both INO80 and YY1 binding sites in the upstream sequences (2000bp upstream of transcription start site).(TIF)Click here for additional data file.S5 FigQuantitative PCR carried out for expression status of target genes tested for INO80 interaction. The reciprocal of Ct values are plotted. Relative to GAPDH the other genes have low expression.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table5\u2019GCCATCAT3\u2019 (8mer) and 5\u2019CCGCCATNTT3\u2019 (10mer) (www.genecards.org).The numbers of putative sites of interaction of INO80 and YY1 proteins in the regions upstream of protein coding genes were analyzed. The sequences of YY1 motif we used for analysis are: (DOCX)Click here for additional data file.S5 Table(DOC)Click here for additional data file."}
+{"text": "In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2\u2009mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress . In addiIt is noteworthy that approximately 7% of world population is affected by those mutations which have been selected by malaria.It is well known that RBCs respond to oxidative stress with a metabolic response finalized to maximize the production of NADPH and to regenerate the stores of GSH and thioredoxin. In parallel, RBCs also respond by activating tyrosine kinases determining the tyrosine (Tyr) phosphorylation of band 3, the most abundant RBC membrane protein and the major linkage between the cytoskeleton and the lipid bilayer \u201312. In RThe redox regulation of band 3 Tyr phosphorylation apparently involves different components. In a previous report, it has been demonstrated that oxidized band 3 is selectively phosphorylated . Lyn is 2O2 of the inhibitory Cys is 0.005-fold lower than GSH, indicating that, at its normal concentrations, GSH should very effectively protect PTPs from oxidative inhibition [Phosphatases (PTPs) have also been implicated in the phosphorylation of band 3 that follows oxidative stress \u201329 and ihibition , 31. Addhibition ; Lyn acthibition , 26. Mor2O2 and by hemichromes which cause irreversible oxidation.In the present report we performed a series of experiments to gain more information on the mechanisms that are involved in the Tyr phosphorylation of band 3 following a reversible membrane protein oxidation triggered by diamide and H2HPO4, 1.5\u2009mM KH2PO4, 20\u2009mM HEPES, 1\u2009mM MgCl2, and pH 7.4) in 5\u2009mM glucose (PBS glucose) to obtain packed cells. RBCs were suspended at 20% hematocrit in PBS glucose and incubated at 37\u00b0C in 0.5\u2009mM diamide at different incubation times  and then in the presence of different diamide concentrations. Separate experiments were also performed in 5\u2009mM H2O2 or 1\u2009mM phenylhydrazine (PHZ). pH was measured after 180 minutes and adjusted to 7.4 with NaOH. When necessary to avoid tyrosine phosphorylation, RBCs were pretreated with 10\u2009\u03bcM of Syk inhibitor II , for 1 hour at 37\u00b0C in the dark, before oxidant treatment. For all protocols described, untreated controls were processed identically except that the inhibitor was omitted from the incubation. To prevent further phosphorylation of band 3, after incubation we washed the cells with cold buffer and membranes were immediately prepared.Venous blood was drawn from healthy volunteers following informed consent and pelleted at 1000 g for 10 minutes at room temperature. After removal of the buffy coat, RBCs were again pelleted and washed 3 times with phosphate buffered saline   containing a protease and a phosphatase inhibitor cocktail and then washed up to 4 more times in the same buffer (until membranes became white) in a refrigerated Eppendorf microfuge at 25000\u2009g. The preparations were stored frozen at \u221280\u00b0C until use. Membrane protein content was quantified using the CD Protein Assay (Bio-Rad).Membrane proteins were prepared at 4\u00b0C on ice as previously described . Briefly\u00b5g of proteins for analytical gels, 1\u2009\u00b5g of proteins for anti-band 3, and 30\u2009\u00b5g of proteins for anti-phosphotyrosine, anti-phosphoserine, and anti-Syk antibodies were loaded for western blot analysis and separated on 8% of polyacrylamide gel under reducing and nonreducing conditions. SDS-PAGE analysis was conducted by heating the sample for 5 minutes at 95\u00b0C and was run on the Bio-Rad mini-protean 3 setup.To perform one-dimensional electrophoresis, membrane proteins were solubilized in Laemmli Buffer  in a volProteins separated by SDS-PAGE were transferred to nitrocellulose membranes as previously described  and then\u00b5M band 3 concentration. Band 3 concentration was estimated measuring total membrane proteins in packed membranes (approximately 4\u2009mg/mL) considering that band 3 represents approximately 25% of total membrane proteins and a band 3\u2009M.W. of 95.000\u2009Da. Resuspended membranes were incubated for 10 minutes on ice, with 0.1\u2009mM diamide in the presence of increasing concentrations of GSH. The reaction was stopped by washing the solution 3 times with HB. The percentage of oxidized band 3 was evaluated by western blot following nonreducing 8% SDS-PAGE and expressed as percentage of the maximal oxidation measured in the absence of GSH. In the absence of GSH an average of 95.2 \u00b1 4.5% of the band 3 was found present in reducible aggregates with a M.W. >200.000\u2009KDa.RBC membranes were diluted in HB to obtain a 5\u2009\u03bcM Syk inhibitor II  was added to prevent further phosphorylation of band 3. The rate of band 3 dephosphorylation was expressed as PTP activity and as a percentage of maximal activity in untreated RBCs.Erythrocyte PTP activity was measured using phosphorylated band 3 as substrate. Phosphorylated band 3 was obtained treating RBCs with 1\u2009mM diamide. Membranes were prepared and incubated for 10\u2009min at 37\u00b0C with the cytoplasmic fraction of RBCs treated with different concentrations of diamide. 10\u2009We used a simplified method to measure the relative changes of Hb in RBC cultures supernatant; after discarding RBC membranes by centrifugation, lysis was quantified by measuring hemoglobin absorbance at 405\u2009nm in RBC supernatant and expressed in nmoles/mL .RBCs were solubilized in HB containing 1% Triton X-100, centrifuged at 15.000\u2009g at 4\u00b0C. High molecular weight hemichrome aggregates were separated from the supernatant on a Sepharose CL-6B microcolumn. The hemichrome fraction was then diluted and quantified measuring heme absorbance at 560, 577, and 630\u2009nm  and exprRBC membrane proteins were treated in the presence or the absence of 2\u2009mM diamide, solubilized for 10 minutes on ice with 3 volumes of 1% Triton X-100 in HB. After centrifugation in a refrigerated Eppendorf microfuge at 15000\u2009g, supernatants were collected and incubated with anti-mouse anti-band 3 cross-linked to Protein A-Sepharose (1\u2009:\u200910) via bifunctional coupling reagent dimethyl pimelimidate for 2 hours at 4\u00b0C under gentle mixing. Beads were washed three times with 1% Triton X-100 in HB . LaemmliTo obtain the oxidized and nonoxidized cdbd3 fragment, RBCs were incubated with or without diamide (2\u2009mM). Membranes were prepared as described above and cytoskeletal proteins were eliminated incubating the membranes with 0.1\u2009M NaOH at 4\u00b0C (stripped membranes). Cdbd3 was then purified from RBC membranes as previously described . The pur4HCO3/ACN (acetonitrile) (50/50 v/v) and successively dried with pure ACN. The gel slices were rehydrated for 45 minutes at 4\u00b0C in 20\u2009\u03bcL of 5\u2009mM NH4HCO3 digestion buffer containing 10\u2009ng/\u03bcL of trypsin. Excess protease solution was removed and the volume was adjusted with 5\u2009mM NH4HCO3 to cover the gel slices. Digestion was allowed to proceed overnight at 37\u00b0C.Bands were excised from electrophoresis gels and were destained by doing several washes in 5\u2009mM NH\u03bcL of the tryptic digests mixed 1\u2009:\u20091 with a solution of CHCA  . MS analysis of peptides from 1-DE gel bands was performed with a MALDI-TOF micro MX  according to the tuning procedures suggested by the manufacturer. Peak lists were generated with Proteinlynx Data Preparation using the following parameters: external calibration with lock mass using mass 2465.1989\u2009Da of ACTH fragment 18-39 background was subtracted using the adaptive mode, performing deisotoping with a threshold of 3%. The MS spectra were converted into pkl files using Mass Lynx 4.0. Peak lists containing the 20 most intense peaks of the spectrum were sent to MASCOT PMF search (http://www.matrixscience.com/) using a Swiss-Prot database . Search settings allowed one missed cleavage with the trypsin enzyme to be selected, carboxymethylated cysteine as fixed modification and oxidation of methionine as potential variable modification and a peptide tolerance of 50\u2009ppm. Only protein identifications with significant Mascot scores (p < 0.05) were taken in consideration.Samples were loaded onto MALDI target using 1\u20092O2) that is physiologically generated from superoxide anion during methaemoglobin formation and by denatured hemoglobin products [Time dependent phosphorylative changes of the RBC membrane proteins have been measured comparing the effects of (i) diamide, a single electron oxidant that induces disulfide formation , 26, . This is plausibly due to the potent scavenging activity of catalase and glutathione peroxidase in RBCs on H2O2.Diamide caused an intense and transient Tyr phosphorylation of band 3 and of proteins 4.1 and 4.2 though to a lesser extent and Ser phosphorylation changes in additional membrane protein Figures . H2O2 in\u03bb-phosphatase, which was used to remove phosphate groups from blotted proteins (data not shown). Control experiments to exclude a direct oxidant effect of PHZ (2\u2009mM) on isolated membranes revealed no effect on band 3 sulfhydryl groups (data not shown). The lack of short term effects of PHZ on membrane protein phosphorylation is coherent with its specific action on hemoglobin and the slow formation of hemichromes [Conversely PHZ caused a slow phosphorylation response measurable only after 60 minutes of incubation Figures . Tyr phoichromes , 39. A lichromes , 18.\u03bcM) that, due to the buffering effect of cellular GSH, are not expected to exert an effect on protein thiols. We observed a dose-dependent increase of the phosphorylation signal (p < 0.01) while no significant difference has been observed between the amount of cdbd3 immunoprecipitated from oxidized and nonoxidized samples  in forming band 3 (5\u2009\u03bcM) intermolecular disulfide bonds on increasing concentrations of GSH. This experiment showed that at 0.1\u2009mM GSH concentration (20-fold higher than band 3) approximately 40% of band 3 was still oxidized by diamide; at 1\u2009mM GSH concentration (200-fold higher than band 3) approximately 20% of band 3 was still oxidized (\u03bcM) no oxidation of GSH was detectable (data not shown).The characteristic accessibility of the two cysteins 201 and 317 located in the cytoplasmic domain of band 3 has been already demonstrated . To obtaoxidized , indicatErythrocyte PTPs have been implicated in promoting the Tyr phosphorylation of band 3 due to an inhibitory Cys residue located in their catalytic domain , 40.To rule out the possibility that diamide treatment, at concentrations that induce band 3 phosphorylation, may also determine a substantial inhibition of PTPs, we compared the levels of band 3 phosphorylation and PTP inhibition at different diamide concentrations. Differently from the effect of diamide that caused reversible changes, after phenylhydrazine treatment, band 3 oxidation, and its phosphorylation, Syk translocation to the membrane and its phosphorylation increased progressively, paralleling the generation of hemichromes . Also inInterestingly, RBCs rapidly react to oxidative stress through very intense Tyr phosphorylation of band 3, their major integral membrane protein. We previously found that the phosphorylation of band 3 affects its interactions with the cytoskeleton inducing membrane destabilization . This phAnyway, the involvement of Syk kinase of band 3 oxidation and of all the major steps of the pathway such as the mechanism of redox sensing, its transduction, the regulation of Syk activation, and docking to band 3 need to be clarified to envisage the physiological role of this intense redox response characteristic of erythrocytes. To address those issues, the present work has been performed to obtain a series of quantitative data to study (i) the temporal and dose effects of different physiological and nonphysiological oxidants in eliciting the minimal band 3 and Syk modifications, (ii) the role of disulfide cross-linked band 3 in docking Syk, (iii) the buffering effect of GSH on the oxidation of band 3 Cys residues to rule out if band 3 could display activity as redox sensor in intact erythrocytes, and (iv) the relative roles of Syk activation and docking versus PTPs inhibition in the phosphorylation of band 3.2O2. The comparative measurement of band 3 phosphorylation and of PTPs inhibition at different concentrations of diamide revealed that intense phosphorylation can occur at concentrations that minimally inhibit erythrocytes PTPs acting on phospho-band 3. This finding is in accordance with the relatively low reactivity of the Cys residue located in the catalytic site of PTPs [The presented results indicate that band 3 possess highly reactive Cys residues capable of being easily oxidized in the presence of physiological concentration of GSH and that disulfide cross-linked band 3 docks Syk and acts as competitive inhibitor of band 3 phosphorylation. Those results support the observed changes in whole RBCs with very low concentration of a specific sulfhydryl reagent (diamide) or following the formation of minute amounts of hemichromes. In both models band 3 phosphorylation exactly parallels its oxidation. On the other hand erythrocytes seem to be fairly protected by H of PTPs  and with2O2 which induces a transient phosphorylation of band 3 does not cause hemolysis, a persistent phosphorylation of band 3 induced by irreversible hemichromes apparently leads to a severe membrane destabilization. It should be anyway noticed that hemichromes formation was also accompanied by serine phosphorylative changes involving some membrane protein; those phosphorylation changes have been usually considered to cause a decrease of the affinity between some components of the RBC membrane junctional complexes [In the present report, we observed that Syk inhibitors are potent inhibitors of the hemolysis that follows to the generation of hemichromes. Considering that treatment with diamide or Homplexes \u201350 and mConsidering that band 3 phosphorylation may have a function in remodeling the RBC membrane to remove noxious hemichromes , the pre"}
+{"text": "Prolonged post-operative air leak is a recognised complication in patients receiving lung volume reduction surgery (LVRS). Some patients are transferred to a portable flutter-valve bag to facilitate discharge. TissuePatchTM is a synthetic absorbable self-adhesive film which acts as an adjunct to minimise air leak.Our aim was to see whether the use of TissuePatchTM would reduce post-operative air leak and the subsequent need for a drain in LVRS patients.We retrospectively analysed LVRS cases over a two year period performed by a single surgeon to minimise procedural heterogeneity. Patients were divided into two groups; group 1 received Tissue PatchTM as the staple line adjunct and group 2 did not.There were 26 cases in total ; group 1=12 , group 2=13 . The median length of stay was 15 for both groups (p = 0.40). The median duration of air leak was 13 days for group 1 and 18 days for group 2 (p = 0.95). Only 2/12 (16%) in group 1 did not have full resolution of air leak and drain removal prior to discharge and were placed on a portable flutter-valve bag compared to 5/13 (38%) in group 2 (p = 0.64).We have observed a reduced trend in the number of patients being discharged with persistent air leak following LVRS with the concomitant use of Tissue PatchTM. A larger study is indicated which may demonstrate significant results."}
+{"text": "FOXG1), Methyl CpG binding protein 2 (MECP2) and Cyclin-dependent kinase-like 5 (CDKL5) have been studied individually, not much is known about their relation to each other with respect to expression levels and regulatory regions. Here we analyzed data from hundreds of mouse and human samples included in the FANTOM5 project, to identify transcript initiation sites, expression levels, expression correlations and regulatory regions of the three genes.Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Forkhead box G1  mediated silencing of Foxg1 in cerebellum.Our investigations reveal the predominantly used transcription start sites (TSSs) for each gene including novel transcription start sites for Our analyses provide a comprehensive picture of the regulatory regions of the three genes involved in Rett Syndrome.The online version of this article (doi:10.1186/1471-2164-15-1177) contains supplementary material, which is available to authorized users. MECP2), Forkhead box G1 (FOXG1) or Cyclin-dependent kinase-like 5 (CDKL5) genes [MECP2 differs from the phenotype of patients with FOXG1 or CDKL5 mutations, there are some similarities in the clinical profile that overlap with RTT. Classic RTT patients with MECP2 mutations have a normal period of development followed by regression of acquired skills, deceleration of head circumference, epilepsy, hand stereotypies, breathing abnormalities, inability to walk or talk and intellectual disability while patients with atypical RTT may show some but not all features of classic Rett syndrome [FOXG1 are known to cause the congenital variant of Rett syndrome where the initial normal developmental window is absent [CDKL5 mutations are found in patients with severe epilepsy during early childhood that later show features that resemble atypical RTT syndrome [Rett Syndrome (RTT) is a disorder caused by mutations in Methyl CpG binding protein 2 5) genes  Althoughsyndrome . Mutatio genes  AMECP2 is expressed ubiquitously [MECP2 mutations and copy number variations in humans lead to neurological phenotypes such as classic or atypical Rett syndrome and in rare cases Angelman Syndrome, X-linked mental retardation and Autism  suggestiewed in , 17. Theiewed in  and it iiewed in , but thein cells .FOXG1 protein is a brain specific member of the forkhead transcription factor family with a role in transcriptional repression. Similar to other members of the forkhead family, FOXG1 has a defined binding sequence motif , which bCDKL5 protein is a serine threonine kinase, whose expression is low in embryonic stages, but increases in postnatal stages up to postnatal day 15  CDKL5 mREven though these three genes have different expression patterns, distinct functions and specific regulatory targets, their paths appear to intersect. Both MeCP2 and FOXG1 proteins regulate transcription via DNA binding and association with other transcriptional regulators \u20131325, 2, 225, 26MECP2 and CDKL5 genes are expressed ubiquitously, their mutations cause a brain specific phenotype suggesting that their expression level, transcription regulation, or function in brain may be distinct from that in other tissues. We tried to resolve these questions through bioinformatics analyses using the FANTOM5 dataset [Although we have some knowledge of their downstream intersecting functions, we are yet unaware of the common genomic features between these three genes, which may provide insights into their regulation. Importantly, although both  dataset .Mecp2 KO mouse model [FOXG1 in mouse and humans. We show that each of these genes use the same TSS in most tissues and provide information on the expression level of the three genes over development in multiple human and mouse samples. Although we did not find a significant correlation between the expression levels of the three genes in the brain, our genome wide analyses uncovered common transcription factors regulating the three genes, suggesting an additional molecular layer in the pathogenesis of Rett Syndrome. The FANTOM5 CAGE dataset also allowed us to locate putative enhancers regulating the three genes in human  of these genes and study their expression profile in hundreds of mouse and human samples using Cap Analysis of Gene Expression method (CAGE) . In conjse model  and stud et al., ) and usiSingle molecule CAGE profiles were generated from RNA obtained from a collection of 573 human primary cell samples (~3 donors for most cell types) covering most mammalian cell steady states. This data set was complemented with profiles of 250 different cancer cell lines, 152 human post-mortem tissues and 456 mouse samples , or provided under informed consent. All non-exempt material is covered under RIKEN Yokohama Ethics applications (H17-34 and H21-14). Mouse tissue samples were collected as per RIKEN Yokohama institutional guidelines. Mouse primary cells were collected as per our collaborators Institutional guidelines and shipped as either purified RNA or as guanidinium isothyocyanate lysates  which were then purified using the miRNeasy kit (QIAGEN). More detailed information for each specific sample is available in Additional file: http://fantom.gsc.riken.jp/5/data/. All CAGE data has been deposited at DDBJ DRA under accession number DRA000991.All the data published by the Fantom5 project and by this study are available through the Fantom5 portal http://fantom.gsc.riken.jp/5/sstar/). Annotation files were built in the context of the FANTOM5 project with respect to Gencode v10 gene model (human), RefSeq (mouse), CpG islands and TATA box in bed format.We used the FANTOM5 database  to idenhttp://www.r-project.org/).We extracted expression information for each TSSs using the FANTOM5 expression dataset for tissues, cell lines and primary cells in human and mouse . All the values for different genes were added together and compared to expression levels of MECP2.We extracted the CAGE defined promoters associated to the genes whose products form the Histone1 transcripts (TSSs identified were expanded by 500 nucleotides on either side (\u00b1500bp). ChIP seq data from Human and mouse ENCODE were downloaded as bed files and intersected with our expanded TSS using intersectBed .P\u2009<\u20090.05) with any of the three Rett genes promoters were included.To identify enhancers associated with the human Rett genes, we used the CAGE derived enhancer database from Andersson et al. . In shorWe downloaded the whole-genome alignment of the human genome with 45 other vertebrate genomes, and of the mouse genome with 29 other vertebrate genomes, from the UCSC Genome Browser database . From thFOXG1 and 6 TSSs for mouse Foxg1  and 30% (140) of human and mouse samples respectively, suggesting that the expression of this gene was limited to selected tissues  and chr12:50485112..50485144,+;  suggesting that other than the brain, fibroblast cell lines may be useful for in vitro analysis of Foxg1 in mouse  identified 8 TSSs for human FOXG1 isoform and therefore a single TSS. We found 8 TSSs for human FOXG1 expressed over 5 TPM and the 3 TSSs with the highest expression in human brain were located at chr14:29235961..29236008,+ (p1@FOXG1); chr14:29234581..29234601,+ ; and chr14:29236269..29236285,+ (p3@FOXG1), with distances of 317, 1697 and 9 bases upstream of the RefSeq annotated TSS, respectively , which are known to faithfully recognize active transcription initiation sites [Foxg1 by Polycomb Repressive Complex 2 (PRC2) , however its expression was not entirely discordant with that of Foxg1 (data not shown). Analysis of the ncRNA database and manual annotation of UCSC Genome Browser revealed several ncRNAs within a genomic window of 1.5 MB around Foxg1, but none of the listed ncRNAs were detectable in the FANTOM5 CAGE dataset.Silencing by chromatin remodeling proteins such as PRC2 requires a non-coding RNA to mediate chromatin modification . TherefoMECP2, less than 100 bases upstream of the RefSeq annotated start sites of which p1@MECP2/Mecp2 was expressed predominantly in most tissues and p2@MECP2/Mecp2 displayed a stable low level expression in all tissues (expression less than 10 TPM)  and cell lines   and P30. In kidney, the expression of Mecp2 declined after P20 and in liver the expression of Mecp2 appeared to be induced after birth (P00) but remained unstable up to the age of P30 , pituitary cortex (n\u2009=\u20091 at each age) and visual cortex (n\u2009=\u20094 at P15 and n\u2009=\u20093 at P30 and P60). Our data reveal that p1@Mecp2 expression fluctuates in embryonic cerebellum samples but is clearly induced after postnatal day 9  confirming our earlier observation of similarities in expression of the two Cdkl5 promoters in mouse.To investigate the relationship between the expression of all TSSs of each gene, we conducted intra gene correlations and found a high degree of correlation between p1@FOXG1 and p3@MECP2, FOXG1 and CDKL5 genes result in overlapping neurological phenotypes, we additionally investigated the inter gene expression correlations of the three genes in the brain. We first generated heatmaps from all brain sub-regions and brain related primary cells  and in humans we found negative correlation of \u22120.1, suggesting slight discordance of expression of the two genes in brain. Thus, our analyses failed to find mathematically significant evidence of contrasting expression between FOXG1 and MECP2. The two promoters of CDKL5 were also poorly correlated with the FOXG1 promoter expression in brain, while there was a positive correlation (23-49%) between expression of MECP2 and CDKL5 in both species  was located 7kb upstream of the gene. Many predicted enhancers for MECP2 had an average correlation of 0.37, the closest enhancer (53kb) had an expression correlation of 0.43, while the highest correlated enhancer (r\u2009=\u20090.55) was over 408kb distant. For CDKL5, the only identified active enhancer had a low correlation of 0.2 and was located over 245 kb upstream of the gene  were broad in keeping with the CpG islands in their vicinity , FOXP1 (p\u2009=\u20090.03), and NFY (p\u2009=\u20090.01) transcription factors. NFY was also predicted to regulate MECP2 (p\u2009=\u20090.01) and possibly CDKL5 (p\u2009=\u20090.09). Similar analyses in the mouse genome revealed motifs for 21, 5 and 3 TFs within 500 bp of the Foxg1, Mecp2 and the Cdkl5 promoters respectively  were common to all three genes. Calculating the statistical significance of the estimated number of binding sites revealed that in mouse for all three genes the promoter regions were enriched for motifs associated with transcription factor NFY, as well as Sp1  and humans  are clearly devoid of FOXG1 expression at any developmental stage. We further confirmed our observation of the absence of Foxg1 expression in mouse cerebellum through analyses of chromatin signatures from mouse ENCODE. Our investigation revealed enrichment of H3K27me3 in the Foxg1 genomic region, suggesting PRC2 mediated silencing of Foxg1 in the cerebellum. Although H3K27me3 has also been reported to be present at transcriptionally active or poised loci [Foxg1 promoter region in cerebellum but not in the cortex, strongly suggest specific repression of Foxg1 in the cerebellum. A similar examination in liver also revealed H3K27me3 enrichment at the Foxg1 promoter region , weMECP2, MECP2 mnslation  but we dMECP2 gene gives rise to two mRNA isoforms with same transcription start site [MECP2 in humans and mouse in all tissues, we could not allocate two distinct start sites for the two isoforms of MECP2. Based on our data, we were unable to conclude whether p2@MECP2/Mecp2 represented an independent poorly expressed protein coding isoform, a shorter non-coding regulatory ncRNA transcript arising from the vicinity of the main promoter p1@MECP2/Mecp2 or a tissue specific enhancer RNA (eRNA) [MECP2. Almost 25% of all enhancers are expected to transcribe short bi-directional capped transcripts called e-RNAs [MECP2/Mecp2, do not support its identification as an e-RNA for MECP2. The two TSSs for CDKL5 are highly correlated with each other in mouse as well as humans. Based on their similar expression levels and distinct promoter shapes, we propose that they represent two independently regulated transcripts despite their proximity.The art site , 57 and A (eRNA)  for MECPd e-RNAs . Our obsThe comparison between corresponding promoters in human and mouse samples, including the novel promoter p1@FOXG1 in human and pA@Foxg1 in mouse, revealed remarkably similar shapes, suggesting evolutionary conservation in their regulation. The only exceptions were the human p2@FOXG1 and pB@Foxg1 mouse, which due to their distinctive promoter shapes appear to be regulated in a species-specific manner.The recently released ENCODE Histone ChIP seq data , allowedFOXG1 and MECP2 but not CDKL5 in humans and NFY is likely to regulate all three genes in mouse. Further investigation will be needed to experimentally verify these findings nevertheless, it will be of interest to study the expression level and presence of mutations in the common TFs in mutation negative RTT patients.Almost 20% patients of atypical RTT do not have mutations in the three genes. We conducted genome wide TFBS analyses with the aim to discover the common transcription factors likely to regulate the three genes and thus identify shared pathways upstream. Mutations or functional impairment of such common TFs may affect the expression of the three genes, which may result in disease phenotype. Our data predict that TFs NFY and SP1 are likely to regulate MECP2 in brain or neurons, which could have explained the predominantly neurological phenotype seen in patients with mutations in this ubiquitously expressed gene.Our investigations failed to demonstrate brain specific promoter usage or particularly high levels of expression of Our comprehensive analyses of data from the FANTOM5 project reveal novel insights into the common and distinct genomic features of the three genes, which are related not only by disease phenotype, but also in their regulation in a species-specific manner.Additional file 1: Table S1: List of all tissues, cells and cell lines with the TPM expression of the FANTOM5 defined transcription start sites of the three genes shown per sample in sheets 1 and 2, and averaged TPM expression across replicates shown in sheets 3 and 4. (XLS 24 KB)Additional file 2: Table S2: List of RefSeq and FANTOM5 detected transcription start sites in human and mouse. (XLS 24 KB)Additional file 3: Figure S1: Top 15 samples in expression for each of the three genes. Panels a, c and e represent mouse, while panels b,d and f are human samples showing promoter expression in TPM, on X-axis, in various tissues, as labeled on Y-axis. For each gene, the samples with the highest expression of the main promoter  are shown. The expression of the other key promoters in these samples is also shown . (PDF 21 KB)Additional file 4: Figure S2: Silencing of Foxg1 in mouse. UCSC Browser image of the genomic locus for Foxg1 showing ENCODE tracks for DNAse-I hypersensitive sites, active enhancer specific histone mark (H3K27ac), active promoter specific histone mark (H3K4me3) and PRC2 mediated repressor mark (H3K27me3) in mouse cerebellum, cerebrum, whole brain and liver as labeled. Cerebellum samples lack the DNAse-I hypersensitive sites visible in cerebrum and whole brain samples. Cerebellum samples also lack the active promoter mark H3K4me3 seen in cortex, but contain PRC2 repressive histone mark H3K27me3 not seen in cortex at the locus. (PDF 36 KB)Additional file 5: Figure S3: Expression levels of Mecp2 and Cdkl5 during development in heart kidney and liver. The line plots show the fluctuations in expression for the two promoters for Mecp2 and Cdkl5 in heart, (a and d), kidney (b and e) and liver (c and f) in mouse. (PDF 46 KB)FOXG1 (a), MECP2 (b) and CDKL5 (c) expression in TPM across a set of adult, newborn and fetal brain regions is shown as labeled. FOXG1 shows the highest overall expression as well as having higher expression in fetal than in adult samples as opposed to the expression of MECP2 and CDKL5 in the same samples. (PDF 39 KB)Additional file 6: Figure S5: Developmental profile for the 3 genes in human brain. Human Foxg1, Mecp2 and Cdkl5 during development in mouse cerebellum (panel a), mouse visual cortex (panel b) and mouse pituitary gland (panel c). Refer main text for details. (PDF 41 KB)Additional file 7: Figure S4: Expression profile of the three genes in mouse in developing brain tissues. Line plots showing expression of selected promoters of MECP2. Bar charts showing TPM expression of the key promoter of MECP2 and collective total expression of Histone H1 TSSs in brain related cells (panels a and c) and tissues (panels b and d) in mouse (panels a and b) and humans (panels c and d). Histone expression levels appear to be over 100 fold higher than MECP2 in brain related cells suggesting a massive up-regulation of MeCP2 at the level of protein translation. (PDF 39 KB)Additional file 8: Figure S6: Comparison of mRNA levels of Histone H1 and Additional file 9: Table S3: Comparison of the expression of the Histone H1 genes promoters and MECP2 in both human and mouse. (XLS 134 KB)Additional file 10: Table S4: Pearson and Spearman correlations for all TSSs in human and mouse. (XLS 20 KB)Additional file 11: Figure S7: Intra and inter gene expression correlations between the three genes. Expression correlation plots for all other promoter combinations not present in Figure\u00a0Additional file 12: Table S5: Location of enhancer and promoter specific Histone marks in relation to TSSs in mouse. (XLS 20 KB)Additional file 13: Table S6: Locations and correlations of human enhancers to the three Rett genes. (XLS 26 KB)Additional file 14: Figure S8: Locations of active enhancers correlated to the three genes in human samples. UCSC snapshot showing the positions of all eRNA producing human enhancers that are correlated to the expression of the three genes: FOXG1 (a), MECP2 (b) and CDKL5 (c). (PDF 58 KB)Additional file 15: Figure S9: Promoter shapes for all the other promoters. The shapes of all the individual promoters in mouse (a-e) and human (f-m) are shown as labeled. The shapes are drawn from the first nucleotide of the first mapped CAGE tag to the first nucleotide of the last mapped CAGE tag, the y-axis shows the counts in TPM for each position. (PDF 41 KB)Additional file 16: Table S7: List of transcription factors with high binding probability of 0.7 and above to the promoters of the three genes in mouse (A) and human (B) genome. Transcription factors common to the three genes are shown in red. (DOC 48 KB)"}
+{"text": "In breast cancer, the presence of Foxp3 (Tregs)  within tThe goal of our study was to quantify Foxp3 in breast cancer patients from Qatar and correlate with their survival.Expression of FoxP3 was studied in 132 FFPE samples with known clinico-pathological data by immunohistochemistry technique and quantified by modified H-score system by pathologist. Results were analyzed via SPSS.Analysis was carried for 132 patients. Age at time of diagnosis was 49 \u00b110.4 years. 76.2% of the patients showed positive expression of FoxP3. FoxP3 expression was not correlated with patient age or hormone receptors. Expression of Foxp3 positively correlate with better patient survival when compared to negative expression .FoxP3 is expressed on lymphocytes that are present in the tumor microenvironment regardless of breast cancer subtypes. Foxp3 is correlated with better survival."}
+{"text": "The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2\u2009Mb running from 14q32.12 to the telomere. Exome sequencing ( Constitutional UPD is associated with developmental disorders caused by the abnormal expression of imprinted genes, that is, genes that are differentially expressed depending on whether they have been maternally or paternally inherited. By contrast, somatically acquired UPD (aUPD) in cancer is a mechanism by which a pre-existing driver mutation  is converted to homozygosity, thereby providing an additional clonal advantage. aUPD may involve whole chromosomes as a result of non-disjunction or, more commonly, whole chromosome arms or terminal segments as a consequence of mitotic recombination. aUPD cannot be detected by conventional cytogenetics but is readily apparent by the finding of somatically acquired long homozygous tracts without change in copy number by genome-wide single nucleotide polymorphism (SNP) analysis.TET2, EZH2, JAK2, CBL and FLT3, respectively.1 Several other regions of recurrent aUPD have been identified for which the target is unknown, for example, chromosome 14q aUPD (aUPD14q) is seen in myeloid neoplasms2 and is one of the most common abnormalities associated with clonal haemopoiesis in population cohorts of elderly individuals.4 Here we show an unexpected and highly significant parental bias associated with aUPD14q, implicating an imprinted locus at 14q32 as the primary target rather than a specific mutated gene.aUPD is prevalent in myeloid neoplasms: chromosomes 4q, 7q, 9p, 11q and 13q are commonly affected and target mutated 5 and the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS).6 The study was approved by the following ethics committees or review boards: the National Research Ethics Service (UK) Committee South West, the Uppsala Regional Ethical Review Board, the Ethics Committee of the Biomedical Research Foundation of the Academy of Athens, Comitato Etico, Azienda Ospedaliero-Universitaria Careggi, Firenze. Informed consent was obtained according to the Declaration of Helsinki.Our study comprised two major groups of individuals: (i) patients diagnosed with a myeloproliferative neoplasm (MPN) or myelodysplastic (MDS)/MPN according to standard morphological, haematologic and laboratory criteria; (ii) population cohorts of elderly individuals from Sweden, specifically the Uppsala Longitudinal Study of Adult Men7 were obtained from peripheral blood or bone marrow leucocyte DNA using the Affymetrix SNP 6.0  or Illumina Human OmniExpressExome v1.2 platforms . Peripheral blood leucocyte profiles for the Swedish population cohorts were obtained using Illumina 2.5M HumanOmni arrays (Illumina) have also been published.8 Exome sequencing for MPN and MDS/MPN cases was performed using the Agilent SureSelect kit  (Human All Exon 50\u2009Mb) and sequenced using an Illumina HiSeq 2000 (Illumina) at the Wellcome Trust Centre for Human Genetics at Oxford, UK. Exome sequencing for the Swedish cases was performed by SciLifeLab, Stockholm, Sweden.Genome wide SNP profiles for MPN and MDS/MPN cases, most of which have been published previously,MEG3 or NHP2L1 with a common FAM labelled reverse primer and analysed with a 3130xl Genetic Analyser  as described.9 Loss of heterozygosity analysis at 14q32 was performed using microsatellites D14S553, D14S267, D14S1006, D14S542, D14S292 and D14S1007, again using a 3130xl Genetic Analyser. All primer sequences are listed in For methylation analysis, DNA was bisulphited  alongside four healthy controls, amplified using forward primers specific for methylated or unmethylated 11 For each SNP the log R Ratio, a measure of normalised total signal intensity, and B Allele Frequency (BAF), a measure of normalised allelic intensity ratio, were determined using the BeadStudio (Illumina) and pennCNV12 software for Illumina and Affymetrix arrays, respectively. Regions of aUPD were identified by BAF segmentation10 which excluded non-informative SNPs (SNPs with BAF >0.9 or BAF <0.1 and SNPs where the absolute difference in BAF between preceding and succeeding SNPs is >0.6), mirrored BAF at 0.5 and used circular binary segmentation to identify regions with similar allelic proportions. For heterozygous SNPs, the BAF is the proportion of the total signal (A+B) accounted for by one allele (B). In a mixed population of cells, the segmented mirrored BAF value will be a combination of values of 1 and 0.5 for cells with and without aUPD, respectively. Regions of aUPD were therefore defined as a region of allelic imbalance (segmented mirrored BAF >0.56) with neutral copy number (log R ratio ~0) that extended to the telomere.12 For samples with known JAK2 V617F levels, determined by pyrosequencing,13 and BAF for both aUPD9p and aUPD14q aUPD, we were able to calculate both the proportion of cells with aUPD14 and the proportion of cells which were homozygous or heterozygous for JAK2 V617F. This allowed us to infer the likely order of acquisition of aUPD14 and JAK2 V617F , variant allele frequencies were used to approximate BAF and were analysed accordingly to confirm regions of aUPD. The minimally affected region identified in patient E5364  was interrogated in all aUPD14q exomes for rare variants that were either novel or had a minor allele frequency of \u2a7d1% in databases of common variation .Analysis of exome sequencing data was as previously described.20 reads . For varn=21), 1/293 (0.3%) had MDS/MPN, 8/563 (1.6%) had JAK2 V617F negative MPN  and 12/1054 (1.1%) had JAK2 V617F positive MPN, an overall prevalence similar to that identified in other studies of myeloid neoplasms .17 Trisomy chromosome 14 (+14) was seen in 4/293 (1.4%) MDS/MPN cases.2 Of the 1641 individuals \u2a7e70 years of age in the Swedish population-based cohorts, 4 (0.2%) had aUPD14,8 similar to the frequency in elderly individuals reported by other larger studies of cases recruited for a variety of genome-wide association studies.4 Cases with aUPD14q or other chromosome 14 abnormalities in our study are summarised in We analysed in house array data on cases with MPN or MDS/MPN and identified aUPD14q extending to the 14q telomere in 25 cases. Of the UK cases .3 This region does not include FANCM, a variant of which was associated with aUPD14q in a single case.18The region affected by aUPD14q was variable between individuals and there was no difference between cases diagnosed with a haematological malignancy and those picked up in population-based screens . Since tn=7) or +14 (n=1). We focused on the identification of novel variants in the minimal region of aUPD14q , thus capturing both constitutional and somatic mutations that might provide a selective advantage when reduced to homozygosity. No gene was identified with likely causative variants in more than one individual  or +14  consistent with paternal aUPD . Methylation values and BAF in the region of aUPD14q were strongly correlated  . This is the first time to our knowledge that aUPD has been associated with a specific parent of origin effect, a finding that indicates that aUPD14q targets an imprinted locus.Constitutional maternal UPD14q causes Temple syndrome, whereas paternal UPD14q causes Kagami\u2013Ogata syndrome. Both are developmental conditions resulting from aberrant expression of genes in the imprinted +14 n=6; . MEG3 isosome 14 . Samples=0.0001) , suggestn=5), an abnormality that is also seen recurrently in myeloid malignancies and population cohorts of elderly individuals. Like aUPD14q, the target of aUPD22q has not been identified but there is a candidate imprinted locus (NHP2L1) within the affected region.19 Methylation analysis indicated maternal aUPD22q in two cases and paternal aUPD22q in three cases  that had either been analysed by SNP arrays and were known to be negative for aUPD14q (n=48) or were randomly selected without knowledge of their aUPD14 status (n=48). Most cases had methylation levels that were indistinguishable from healthy controls, but three had a clear gain of methylation ,20 components of which are recurrently inactivated in myeloid neoplasia by mutation of EZH2, SUZ12 or EED.21 It is possible therefore that the consequence of aUPD14q might be functional inactivation of PRC2, in which case we would expect aUPD14q and PRC2 mutations to be mutually exclusive. Partial sequence analysis of EZH2 and SUZ12, the most commonly mutated components of PRC2, revealed causative EZH2 mutations in two out of eight aUPD14q cases  revealed no other obvious driver mutations in three out of four individuals from the population cohorts, suggesting that aUPD14q alone may be sufficient to promote clonal haemopoiesis. The fourth individual (PIVUS 931) was positive for JAK2 V617F as well as a TP53 mutation; he was subsequently diagnosed with polycythemia vera following recruitment into the PIVUS study. In contrast, all three cases with diagnosed myeloid malignancy that were exome sequenced had additional somatic driver mutations in SF3B1, EZH2, JAK2 or TET2  this cluster falls with the 11.2\u2009Mb MAR, an interval that contains 121 known protein-coding genes plus the immunoglobulin region, and (ii) despite extensive searches there are no other confirmed imprinted loci within the MAR, or indeed elsewhere on chromosome 14. Furthermore, expression of DLK1-MEG3 has been reported to be deregulated in diverse neoplasms in the absence of chromosome 14 abnormalities, including acute promyelocytic leukaemia27 and myelofibrosis.29 In our study, we did not have suitably stored material for expression analysis of aUPD14q cases; however, expression analysis alone is unlikely to be informative since by definition aUPD would be expected to distort the normal expression of imprinted genes in the affected region whether they were of pathogenetic relevance or not.Our finding of a highly significant parent of origin effect associated with aUPD14q strongly implicates an imprinted locus as the primary target, and that paternal homozygosity for this target provides a growth advantage over cells that harbour both alleles. Del(20q), another somatic abnormality associated with myeloid neoplasms, has been shown to target an imprinted gene clusterMEG3 methylation in our study, 21 had positive values indicative of paternal aUPD. One case had a negative methylation value. We do not have an explanation for this aberrant case, but potentially other mutations may have been present that promoted clonal expansion. We note that aUPD for other regions has not been associated with a specific mutational target in all cases, for example, we found that only 9 of 12 MDS/MPN cases with aUPD7q had EZH2 mutations, with the target of the remaining 3 cases remaining unclear.2Of the 22 aUPD14q cases analysed for 31 aUPD14q is more prevalent in the elderly. Previous studies found aUPD14q in 21/50\u2009222 (0.04%) individuals, rising to 14/15\u2009101 (0.09%) in those >60 years of age,4 an increase that parallels the increase in myeloid neoplasia seen in the elderly. Analysis of longitudinal data estimated a 10-fold elevated risk of developing a haematological malignancy for individuals with any acquired chromosome anomaly,4 and a similar risk was estimated for individuals with somatic mutations in genes known to be associated with myeloid malignancies.31 The most commonly mutated genes in population cohorts were DNMT3A, TET2 and ASXL1, and the finding that most individuals had only a single abnormality suggested that mutations in these genes are often initiating events for clonal haemopoiesis. Inspection of our exome sequencing data for the aUPD14q cases revealed no mutations in known driver genes in three out of four population cohort cases. By contrast, all three cases with diagnosed myeloid malignancy had additional somatic driver mutations. We suggest therefore that aUPD14q may also initiate clonal haemopoiesis and predispose to overt malignancy. However, when we examined cases that had both aUPD14q and JAK2 V617F, it was clear that aUPD14q may be an early event in some MPN cases but a late event in others. Furthermore, we found that aUPD14q tends to arise before JAK2 V617F in essential thrombocythaemia but after V617F in polycythemia vera. This is reminiscent of the finding that mutated TET2 may precede or follow the acquisition of JAK2 V617F in MPN, with the order of acquisition influencing clinical features and stem cell biology.32Similar to other mutations associated with clonal haemopoiesis in population cohorts,"}
+{"text": "There is an error in the data in cell Y47 of the Supplementary Table 2. It should read C*03:04. Please see the corrected Table S2 below.Table S2HLA allele information of ELISpot assay responders and its compatibility with previous report. In total, 79 responses among 14 epitopes were identified. HLA allele information of ELISpot assay responders and its compatibility with previous report of responsible HLA alleles listed in Los Alamos database are shown.(XLSX)Click here for additional data file."}
+{"text": "Previous studies have shown the C1 spinal nerve has sensory neurons. Direct stimulation of the C1 spinal nerve provokes peri-orbital pain in migraine patients. No data exist which can predict a positive outcome for C1 nerve root block. Tenderness over the greater occipital nerve has been shown to predict outcome of GON block. We propose that tenderness over the GON with periorbital referral on exam predicts periorbital referral on direct C1 stimulation and predicts a positive outcome of block.Predict the outcome of C1 spinal nerve block based on exam findings.Review of 23 patients, 21 of whom had chronic migraine and 2 of whom had chronic cluster headache. All 23 have undergone C1 spinal nerve block.Of the 23 patients, 17 (74%) had GON tenderness on exam with periorbital referred pain and 6 (26%) had only occipital tenderness. Both cluster patients did not have periorbital nor orbital pain on palpation of the GON. All 17 with periorbital referred pain on GON palpation had reproducible periorbital pain intraoperatively on direct stimulation of the C1 spinal nerve with fluoroscopic guidance. Of those, 15/17 (88%) had a positive block. Of the 6/23 (26%) with a negative block, only two had periorbital pain reproduced on exam by GON palpation. Both cluster headaches had negative blocks. Neither had intraoperative periorbital or orbital pain on C1 stimulation.Tenderness over the GON with periorbital pain referral during exam predicts positive outcome of C1 spinal nerve block in patients with migraine.No conflict of interest."}
+{"text": "Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod's ability to inhibit endothelial \u201csprouting\u201d d < 35 \u03bcM) to the IIA subdomain of albumin (Sudlow's site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the \u201cenhanced permeability and retention\u201d  effect. Thus, despite plasma levels of < 1 \u03bcM, the EPR effect results in intracellular drug concentrations of 2-3 \u03bcM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC50 ~ 0.5 \u03bcM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.Tasquinimod's potency is facilitated by its reversible binding (K The total plasma membrane associated drug concentration (PMt) is determined by subtracting the ICt from the CAt value.A rapid filtration method was developed and validated to measures the kinetics of tasquinimod cell uptake on a series of human cell lines. To do this, exponentially growing cells in a tissue culture flask are trypsinized, and the number of single cells counted and their average diameter determined electronically using Cellometer AutoT4 . Then multiple replicates (N=3-6) of 107 cells were incubated in 1ml of tissue culture media containing 10% fetal bovine serum (FBS) plus 1\u03bcM 14C-labeled tasquinimod for varying times before the cells were centrifuged rapidly  to remove the drug containing media. The cells were rapidly resuspended in phosphate buffered saline and exposed to pressurized  N2 in a nitrogen cavitation devise  for 5 min before the plasma membranes were lysed by the rapid release of the pressure. This detergent free procedure lyses >98% of the cells without disrupting either mitochondria or lysosomes. The lysate was then centrifuged at 800Gx 2 minutes to restrictively pellet only cell nuclei. The radioactivity of this pellet was counted and used to calculate the total amount of tasquinimod associated with 107 cell nuclei. To determine the nuclear drug concentration, this total nuclear drug amount was driving by the total volume of 107 cell nuclei determined from the average diameter of the nuclei determined electronically using Cellometer AutoT4.To specifically determine the concentration of tasquinimod within the cell nucleus, 10in vitro angiogenic sprouting and tube formation assay was used as described previously [The 3-dimensional (3D) eviously . This 3DAll of the values reported are presented as means \u00b1 SE of representative data generated from 1 of a minimum of 3 independent experiments in which there were a minimum of 5 replicates per data point. Statistical analysis was conducted by a 1-way ANOVA with the Newman- Keuls test for multiple comparisons with significance being p<0.05."}
+{"text": "The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley.Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene  Seed dormancy allows wild barley grains to survive dry summers in the Near East but has been selected against for industrial applications such as beer and whisky production that require quicker germination. Here Sato et al. show that Qsd1 is a major seed dormancy gene in barley and encodes an alanine aminotransferase. Hordeum vulgare) was domesticated ca. 10,000 years ago from its wild progenitor H. vulgare subsp. spontaneum in the Fertile Crescent1Barley (Qsd1) and another in the telomeric region of the long arm (Qsd2) have been identifiedQsd2 was isolated and proved to encode a mitogen-activated protein kinase kinase 3  on chromosome 5H, one near the centromere  and Qsd1/Qsd1 (short dormancy) in a Mendelian fashion , a protein of 495 amino acid residues  with the pANDA vectorQsd1 RNAi knockdown on seed germination. Of the two events, #7 T2 transgenic homozygous positive plants showed <5% germination after 21 days at 21\u2009oC  and all of them were located in the coding region of AK372829 , indicatresidues . For proat 21\u2009oC , while haAT gene .qsd1 allele of wild barley H602   . Transcr1 plants . These tQsd1 alleles were compared with the parents used for Qsd1 mappingQsd1/qsd1 alleles. This SNP is associated with a change from a leucine (L) residue at amino acid position 214 in wild barley H602 to a phenylalanine (F) residue in cultivar Haruna Nijo  was much lower than haplotypes having allele C (n=307). Most of the barleys having the allele G have pedigrees that track malting and brewing applicationsTo further confirm that the SNP alleles were correlated with dormancy in barley grain more generally, the germination scores of 353 cultivated and 14 wild barley accessions were analysed. The germination (low-dormancy) scores were compared among haplotypes of non-synonymous SNPs in AK372829 . Average72829 n=4 was muchQsd1 mutation that resulted in the reduced dormancy time, the genomic regions of Qsd1 in 27 wild and 210 cultivated barley accessions were sequenced  for AlaATs in other plant species and for four barley sequences  of Haruna Nijo  of Qsd1, corresponding to E9, was located on motif 11  was further analysed by RNA in situ hybridization 19 days after flowering. Transcripts were detected in the embryo but not in the endosperm  and mutant Qsd1 (F) alleles , but also from Lebanon (W1585) and Jordan (W1215). The result indicates that the Qsd1 allele is in the lineage from wild barleys of the southern Levant, where the first barley domestication also occurred1The haplotype analyses  showed tnd Korea . The C35Jordan W115. The rqsd1 gene is located almost exclusively in the embryo  rather than the cumulative increase in expression that causes different levels of dormancy.As shown in  Promise  causes aactivity . Howevermination . In thesBrassica napus also controls dormancy and has been implicated in alanine metabolism2O2) to break dormancy in barley seedsAs mentioned above, dormancy and germination are complex processes and dormancy has been linked with redox signalling by reactive oxygen species32qsd1 gene for long dormancy appears to be a mutable gene that might be used for adaptation to different environments and this is consistent with the large variation in qsd1 sequence that exists in nature. For example, the long dormancy qsd1 gene could be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. On the other hand, certain haplotypes which carry the Qsd1 mutation are associated with barley lines that have been developed for industrial uses, such as in the malting and brewing industries. The Qsd1 mutation that was discovered soon after barley domestication has proved to be essential for the transition of barley utilization from food to beverage in human diets and contributes further to the debate as to what extent the development of ancient agrarian societies was driven by the human appetite for flour and bread, or for beer and alcoholDespite these interpretative constraints, it is clear from the genetic data presented here that AlaAT is centrally important in the release of dormancy in barley grain. In practical terms, our data suggest that the Spike samples are harvested at physiological maturity (when the colour of first internode turns to yellow). Spikes are dried 48\u2009h by the dehumidifier at the condition of 30\u2009\u00b0C and 10% RH, and then stored at \u221220\u2009\u00b0C until use. Fifty seeds of each sample were exposed to 25\u2009\u00b0C for dormancy reduction treatment and germinated for 4 days on moistened filter paper in Petri dishes at 25\u2009\u00b0C. Seed non-dormancy was scored as the percentage of germinated seeds. Each germination test was replicated twice.Qsd1 was initially mapped with the 93 doubled haploid phenotype data on a map derived from the cross between the cultivar Haruna Nijo and the wild barley line H602 consisting of 2,890 EST markersQsd1 mapped at the centromeric region of the long arm of chromosome 5H. The method of population development is described in Sato et al.3F1 recombinant chromosome substitution lines (RCSLs) derived from the cross between H602 (donor parent) and Haruna Nijo (recurrent parent) were genotyped and plants heterozygous for a chromosome segment with Qsd1 were selected. A RSCL carrying the long dormancy allele qsd1 and short dormancy alleles for other three QTLs was identified132 plants and 4,792 F3 plants derived from the heterozygous F2 plants were produced to genetically map Qsd1  that showed high sequence co-linearity with the other 10 barley genes (EST2 to EST11). By scoring dormancy . A co-segregated marker with an estimated genotype of Qsd1 was identified in two plants from the F3 population. From each of these two plants, 18 F4 plants were used to confirm the respective genotype of F3 by segregation in the progeny.sd1 ref. . The segsd1 ref. . The dirreatment . Becausereatment , the pladormancy , Qsd1 waQsd1 was used to select BAC clones of the less-dormant parent Haruna NijoHindIII cloning site. The average insert size was 180\u2009kb and the total clone number was 172,800 (6 \u00d7). From the BAC library of Haruna NijoEST4R marker and a contig sequence of 121,395\u2009bp was obtained from another BAC clone (GenBank ID: LC054175). Each shotgun library from the selected clone was sequenced using a 3730xl sequencer (Applied Biosystems). Reads were assembled by phred/phrap software into contigs. The EST sequence was mapped on contig sequences to confirm the target sequence on the BAC clone. The Rice Genome Automated Annotation System (http://ricegaas.dna.affrc.go.jp/) was used for gene prediction on the contig sequence. Sequence polymorphisms between Haruna Nijo and H602 were detected by the alignment of contig sequences by CLUSTALX .The primer set of co-segregated markers with AK372829 was amplified using the forward primer 5\u2032-caccaaagctaggagatgg-3\u2032 and reverse primer 5\u2032-gaacacactgccccaaaagt-3\u2032and cloned into the pENTR/D topo vector (Invitrogen). The construct was recombined (according to the manufacturer's instructions) into the binary vector pANDA modified to contain a GatewayTM cassette down-stream of the maize Ubi-1 promoterAgrobacterium strain EHA101. Immature embryos of cv. Golden Promise were used for Agrobacterium-mediated transformation. Transformation was performed by the protocol of Hensel et al.0 plants which had single copy inserts, as shown by Southern analysis, were examined further. We obtained several independent transgenic lines, and two (ids. #6 and #7) were used for further analysis. Genotypes of T1 plants were determined by the inserts of segregating T2 progeny and each T2 plant from the T1 plants were further genotyped by segregation in the T3 progeny.A 509-bp cDNA region from qsd1/qsd1 allele in the genetic background of cv. Golden Promise. Twenty-three B1F1 plants were genotyped by the Golden Gate Assay (Illumina Co.), using selected 384 genome-wide SNP markers from barley OPA1 (ref. 1F1 plants with the substituted segment were self-pollinated to generate homozygous qsd1/qsd1 substitution lines. Agrobacterium-mediated transformation of the substituted line (id. B1H602GP20-7), using a vector construct carrying a cloned Haruna Nijo Qsd1 genomic DNA  and sequenced. The open reading frame region was digested by SacI and HindIII and ligated into binary vector pBUH3 (ref. 0 transgenic plants. Segregating T1 plants (ids #1 and #4) were produced from single T0 plants and further genotyped for the Qsd1 transgene in the T2 progeny.The recombinant chromosome substitution line was used as a donor of dormant PA1 ref.  to identPA1 ref. . Selecteomic DNA , was conTo measure dormancy levels, transgenic barley lines were grown in a growth chamber with 12\u2009h day length and at 15\u2009\u00b0C. Expression levels were measured with the same method described below. Dormancy levels were measured at the time when the difference of long dormancy and short dormancy samples were maximized.de novo using I-TASSERde novo predictions as templates. Modeller DOPE functions, ProSAThe barley AlaAT amino acid sequences were aligned to the barley AlaAT crystal structureA total of 365 barley accessions preserved at Okayama University were evaluated for seed dormancy for two growing seasons. Nucleotide polymorphisms were detected by sequencing PCR amplicons from target regions. Primer sequences are shown in Qsd1 in 27 wild and 210 cultivated barley accessions are sequenced. Non-redundant haplotypes were collected and aligned according to their similarities with the haplotypes of H602  on a Step One thermal cycler (Applied Biosystems Co.) with initial conditions of 95\u2009\u00b0C, 60\u2009s, 40 cycles of denaturing of 95\u2009\u00b0C, 60\u2009s and extension at 60\u2009\u00b0C for 60\u2009s. The qsd1 gene segment comprising part of the 3\u2032-UTR (300\u2009bp) was amplified from cDNA isolated from immature barley spike using specific primers  at 0, 1, 2, 3, 4 and 5 weeks after flowering were measured for  primers . The PCRLC054174: cv. Haruna Nijo BAC clone HNB550I12 and LC054175: wild barley H602 BAC clone HSP216G11. The data that support the findings of this study are available from the corresponding author upon request.BAC clone sequences described in this paper have been deposited in the DDBJ nucleotide database with accession codes How to cite this article: Sato, K. et al. Alanine aminotransferase controls seed dormancy in barley. Nat. Commun. 7:11625 doi: 10.1038/ncomms11625 (2016).Supplementary Figures 1 - 10 and Supplementary Tables 1 - 8Origin and name of accessions of 27 wild and 210 cultivated barleys."}
+{"text": "There is an error in the sixth sentence of the Abstract. The correct sentence is: Reduced GM volumes in three brain areas including right hOC3v in the collateral sulcus of visual cortex (hOC3vR), left cerebellar vermis lobule 10 (vermisL10) and right cerebellar vermis lobule 10 (vermisR10) were found in patients with schizophrenia.There is an error in the first sentence of the Reduced GM volume in patients with schizophrenia subsection of the Results. The correct sentence is: By using SPM Anatomy Toolbox, we found that GM volumes in three brain areas including left hOC3vR in the collateral sulcus of visual cortex (hOC3vL), left cerebellar vermis lobule 10 (vermisL10) and right cerebellar vermis lobule 10 (vermisR10) were significantly reduced in patients with schizophrenia .The term hOC3vL appears incorrectly throughout the manuscript. The correct term is hOC3vR.S2 Fig(TIF)Click here for additional data file."}
+{"text": "P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of  Plasmodium falciparum malaria cases compared to other countries in the Caribbean region .P. falciparum-infected samples received from Guyana, thirteen were collected from female patients while the rest were from males. All 100 patients reported travelling within the two weeks prior to seeking medical attention for clinical symptoms; the majority had travelled to Cuyuni-Mazaruni (55%) and Potaro-Siparuni (34%) (18S rRNA and msp2 genes.Of the 100 ni (34%) . Three o18S rRNA/msp2-positive samples from Guyana were positive for both pfhrp2 and pfhrp3 genes (pfhrp2 (PF3D7_0831900) and one sample was negative for the 3\u2019 flanking gene PF3D7_0831700  and Brokopondo (19%) districts. Travel information could not be retrieved for 30 samples. Twenty-five samples were removed from final analysis because they failed to meet the inclusion criterion requiring amplification of both 18S rRNA/msp2-positive samples from Suriname were pfhrp2-negative. Two of the eleven patients from whom the pfhrp2-deleted isolates were collected, reported recent travel to Brokopondo district and nine patients had been to Sipaliwini district. All eleven of these pfhrp2-negative isolates were collected in 2011; in eight of these samples, exon 2 of pfhrp2 was intact; three samples had deletions in both exon 1 and 2 of pfhrp2. Seventeen of the 78 isolates (22%) had deleted the 5\u2019 pfhrp2 flanking gene, PF3D7_0831900, while three (4%) had deleted the 3\u2019 flanking gene PF3D7_0831700 (Eleven (14%) of the 78 _0831700 .pfhrp2. Although the majority of samples (67%) were positive for pfhrp2 and its flanking genes, fourteen isolates (18%) had only deleted the 5\u2019 flanking gene, PF3D7_0831900 (pfhrp2-negative isolates with both flanking genes intact (8%); two PF3D7_083900/Pfhrp2 double-negative isolates (4%); and one Pfhrp2/PF3D7_0831700-double negative isolate (3%) (We further examined the parasite isolates for gene deletion patterns around _0831900 . Other date (3%) .pfhrp3-negative  was intact in all 78 samples while one sample (1%) was negative for the gene found downstream of pfhrp3, PF3D7_1372400 (Only three of the 78 isolates (4%) were negative . Two of _1372400 .pfhrp3. We found that 96% of the 78 isolates from Suriname had intact pfhrp3 and flanking genes (pfhrp3-negative samples with intact neighboring genes and one (1%) isolate that was pfhrp3/PF3D7_1372400-negative but PF3D7_1372100-positive .Neutral microsatellite genotyping and cluster analysis were performed to investigate a possible relationship among parasite isolates with deletions in Median joining network diagrams were created using allele length data at seven neutral microsatellite loci in order to evaluate the genetic relationships among the parasite isolates. No distinct clustering of Suriname isolates separately from those collected in Guyana was observed, indicating that the parasites from the two countries may be very similar genetically . This oupfhrp2-negative isolates, which were collected in Suriname in 2011, did not cluster together as may have been expected, indicating that their genetic backgrounds differed from each other and that they likely did not all originate from a single clonal type  district in Suriname, consists of tropical rainforest and borders French Guiana to the east, Guyana to the west and Brazil to the south. The Suriname-French Guiana border region along the Marowijne river is a relatively high malaria transmission region [P. falciparum  parasite transmission. Further studies will be required to determine if the pfhrp2 deleted parasites found in Suriname are genetically related to those found in other South American countries.Taken together, these data indicate that the occurrence of h as 40% ,17. Givest areas . It is tpfhrp2 gene deletion was not found among P. falciparum isolates collected in Guyana, it is intriguing that 41% of the parasites had deleted the 5\u2019 flanking gene, PF3D7_0831900  had to be excluded from our analyses because of poor quality DNA; these samples did not meet our inclusion criteria of being able to amplify pfhrp3-negative isolates in Guyana and only a limited number of pfhrp3 deletions in Suriname. In Peru, a larger proportion of parasites was pfhrp3-negative compared to those that had deleted pfhrp2 [pfhrp2 is located on chromosome 8 while pfhrp3 is located on chromosome 13), and that the biological significance of these gene deletions is not known, the implications for the apparent differences in proportions of pfhrp2 versus pfhrp3 deletions in Suriname compared to Peru is unclear.It was surprising to find no d pfhrp2 . Howeverpfhrp2 or pfhrp3 gene deletions occurred in P. falciparum isolates collected in Guyana. On the other hand, pfhrp2 gene deletions also did not occur in isolates collected between 2009 and 2010 in Suriname, but were detected in samples collected in 2011. However, it should be noted that a very small number of specimens were collected in 2009 and 2010. The outcome from Suriname illustrates the importance of regular monitoring for pfhrp2 and pfhrp3 deletions if PfHRP2-based RDTs continue to be used in the region. Use of non-PfHRP2-based RDTs that target P. falciparum-specific parasite lactate dehydrogenase (pLDH) with either pan species or P. vivax-specific pLDH can also be considered as an alternative test for use in Suriname. Furthermore, given that Suriname, Guyana and French Guiana experience influxes of migrant workers, it is likely that pfhrp2 gene deletions may spread through this mobile population. Therefore, a current sampling of P. falciparum isolates to update the findings reported here should lead to careful consideration given in choosing appropriate RDTs for use in this region.In summary, no S1 Supporting InformationWritten permission for the use and modification of the maps in (PDF)Click here for additional data file."}
+{"text": "CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-\u03b21 and acts as a mediator of TGF-\u03b21 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-\u03b21; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-\u03b21 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-\u03b21 in primary osteoblasts.We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14\u201321), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-\u03b21 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-\u03b21 and CCN2 promoter activity and protein induction by TGF-\u03b21 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-\u03b21. We also demonstrated that TGF-\u03b21 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion.This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-\u03b21 for Src activation, CCN2 induction and collagen XIIa in osteoblasts. CCN2lization , 5 and alization , 6\u201311. Clization . One of lization \u201316, howelization . The siglization .Src is the founding molecule of a family of non-receptor tyrosine kinases that, when activated, are involved in numerous physiological and pathological processes including cell proliferation, survival angiogenesis and matrix secretion \u201320. Src -/- null mice we were the first to demonstrate a novel physiological role for AFAP1 in lactation [Actin filament-associated protein 1  is the prototypical member of a family of three structurally related proteins: AFAP1, AFAP1 like 1 (AFAP1L1), and AFAP1 like 2 . AFAP1 was discovered over two decades ago as a binding partner for oncogenic Src . AFAP1 iactation . These sactation . AlthougConsidering that AFAP1 is an important regulator of Src activity and that Src activity plays a central role in relaying TGF-\u03b21 signaling to induce CCN2 expression and controls osteoblast functions including ECM production, we hypothesized that AFAP1 plays a role in the TGF-\u03b21 signaling pathway and the regulation of Src activity in osteoblasts. Thus, this study characterizes the role of AFAP1 in regulating Src activation and CCN2 induction downstream of the TGF-\u03b21 receptor in osteoblasts.The three stages of osteoblast differentiation in primary osteoblast cultures have been well-characterized and include an initial period of cell proliferation until the cells reach confluency (~day 7), followed by a phase of matrix production and maturation (~day 7\u201314), and ending with a stage of mineralization in which mineral deposition accrues in the matrix (~day 14\u201321). We sought to assess the temporal pattern of AFAP1 expression in differentiating primary osteoblast culture and to determine if TGF-\u03b21 was capable of inducing AFAP1 expression at different time points within the spectrum of osteoblast differentiation. Western Blot analysis revealed that AFAP1 protein expression occurred in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production and maturation (~day 14\u201321), an a further increase in expression during mineralization (~day 21) . This exWe previously showed that in osteoblasts TGF-\u03b21 activates Src and TGF-\u03b21 induction of CCN2 requires Src . Since AWe have previously demonstrated that blocking Src expression/activity impairs CCN2 promoter activation and protein expression in response to TGF-\u03b21 treatment in osteoblasts , 42. To We have previously demonstrated that CCN2 is an essential downstream mediator for the TGF-\u03b21-induced, extracellular matrix (ECM) protein collagen type I in osteoblasts . AdditioAFAP1 is considered as an adaptor protein that directs the activity and the location of cSrc and as an effector protein that crosslinks actin filaments. The function of AFAP1 has been studied mainly in prostate cancer and breast cancer where AFAP1 contributes to the progression of cancer by regulating the adhesion of cancer cells , 44. We CCN2 is an important factor in the induction and control of osteogenesis. CCN2 is highly expressed in active osteoblasts during osteogenesis , during We found that reducing the expression of AFAP1 using AFAP1 siRNA impaired Src activation after TGF-\u03b21 treatment in osteoblasts. This finding is consistent with other previous reports concerning the involvement of AFAP1 in binding to activated forms of Src , 54. In levels of CCN2 were inhibited by the activation of PKC, but stimulated by the inhibition of PKC and tyrosine kinase [Interestingly, PKC isoforms have been previously shown to be involved in TGF-\u03b21 induction of CCN2 in mesangial cells , hepatoce kinase . Future TGF-\u03b21 is known to influence bone metabolism through effects in both osteoblasts and osteoclasts. Specifically, in osteoblasts, TGF-\u03b21 recruits and induces the proliferation of osteoblast precursors and inhibits their apoptosis and can modulate expression of factors that control the formation and activation of osteoclasts . In geneA major function of TGF-\u03b21 during osteoblast differentiation is to stimulate production of the extracellular matrix components that compose osteoid , 73. In In conclusion, this study demonstrates that a normal physiological role of AFAP1 is to mediate the downstream signaling pathway of TGF-\u03b21 to induce CCN2 induction and extracellular matrix protein production, the prominent anabolic pathway in osteoblast. This study further supports the role of AFAP1 as an adaptor for Src to direct the activity of the kinase upon receiving a specific input signal, TGF- \u03b21.No human subjects were used in this study. This study was approved by the IACUC board of The Commonwealth Medical College, Scranton PA. . All animals were handled according to national and international guidelines following the principles in the NIH Guide for the Care and Use of Laboratory Animals  and in accordance with principles established in the Weatherall report.Transforming Growth Factor-\u03b21 (TGF-\u03b21) was purchased from Calbiochem  and reconstituted as 1\u03bcg/ml in 4mM HCl with 0.1% bovine serum albumin. Anti-actin antibody was purchased from Sigma . Anti-CTGF, anti-collagen I and anti-collagen XII antibodies were purchased from Santa Cruz . Anti-Src antibodies (clone GD11) were from BD Bioscience and antibodies specific for active form of Src (clone pY416) were from cell signaling Technologies . CCN2 promoter reporter was constructed as previously described . The genOsteoblasts were transfected with 100 pmol of siRNA specific for AFAP1  or 100 pmol control Luciferase siRNA  using Lipofectamine and Plus reagent  according to the manufacturer\u2019s instructions.The generation GFP AFAP1 fusion protein construct was previously reported . GFP exp2 inhalation followed by cervical dislocation. Neonate rats were euthanized by decapitation.Primary osteoblasts were derived from bone  of neonatal Sprague Dawley rats purchased from Charles River . All animals were handled at the AAALAC approved animal facility in The Commonwealth Medical College , according to the principles in the NIH Guide for the Care and Use of Laboratory Animals  and guidelines established by the IACUC of The Commonwealth Medical College . Adult animals were euthanized by COrd\u20135th digestions of the calvarial pieces. These cells were plated in 100mm dishes  at 5 \u00d7 105 cells/plate in osteogenic media consisting of Earle\u2019s Minimal Essential Medium  supplemented with 10% fetal calf serum , 50\u03bcg/ml ascorbic acid (Sigma) and 10mM \u03b2cglycerophosphate (Sigma). The cells were incubated at 37\u00b0C with 5% CO2 with a change of media every three days until they reached 80% confluence. Cells were sub-cultured under identical conditions for utilization in experiments following the third passage. We have previously shown that these culture conditions result in an enhanced (>90%) population of cells committed to the osteoblast lineage using specific markers of osteoblast differentiation  [Primary osteoblast cultures were obtained using neonatal rats as previously described , 10, 11.Osterix) , 11.Lysate preparation, Immunoprecipitation, Western Blotting and trasfection were preformed as described . Blots w4 cells/well), transfected with CCN2 promoter reporter vector and co-transfected with Renilla  luciferase vector. Following transfection, the cells were serum starved overnight and treated with TGF-nd co-transfected with Renilla . Relative luciferase activity was expressed as a ratio of firefly/renilla luminescence values. All samples were normalized to an untreated (cells only) or mock treated (empty vector or diluent only) control reaction.Luciferase activity was determined using the Dual-Glo luciferase assay  according to the manufacturer\u2019s instructions as described . BrieflyFor all quantitative data, analysis of variance (ANOVA) was employed to evaluate the effect of one variable on two or more independent groups. In the event of a significant group effect, individual pairs of means were compared using the Bonferroni post-hoc test. Data were calculated as mean + SEM, and in some cases, converted to percent of control. A value of p<0.05 was used to determine whether differences were statistically significant."}
+{"text": "Ole e 1 is one of the major allergens from olive tree pollen. Up to date there are no specific studies that evaluate in depth the in vitro responses to this purified allergen. The goal of the study was to thoroughly evaluate the celullar responses to nOle e 1 in allergic rhinitis (AR) and local allergic rhinitis (LAR) patients with sensitization to olive tree pollen (OL) demonstrated by nasal allergen provocation test (NAPT).Twelve subjects with AR  to OL), 12 subjects with LAR , and 12 subjects as control group (CG)  were selected. Basophil activation tests (BAT) with OL and nOle e 1, along with dendritic cell (DC) maturation/proliferation studies in response to nOle e 1 stimulation, were carried out in all subjects. Local ethical committee approved the study.All AR subjects had positive BAT responses to OL and 10/12 to nOle e1 (83%); 8/12 LAR (66.6%) had a positive BAT with OL and 4/12 (33%) to nOle e1, with only one subject of the control group with a positive BAT to both OL and nOle e1 (8%). DC proliferation and maturation were increased in SAR>LAR>CG but with no significant differences .BAT with OL and nOle e1 in LAR group showed sensitivity between 66.6 and 33%, demonstrating specific basophil activation with pollens in patients with LAR. DC proliferation and maturation were demonstrated in SAR and LAR subjects although with no significant differences with CG."}
+{"text": "After multiple discrete introductions of influenza A(H1N1)pdm09 virus into Sri Lanka, the virus was transmitted among humans, then swine. The spread of virus between geographically distant swine farms is consistent with virus dispersal associated with a vehicle used for swine transportation, although this remains unproven. The first known transmission of influenza A(H1N1)pdm09 virus to humans from swine was in 2009. As the virus spread among humans worldwide, it was transmitted from humans to swine repeatedly 1, chaTo understand the molecular epidemiology and spatial and temporal dynamics of spillover events, we compared our data with full-genome sequences of H1N1pdm available in public databases as of August 28, 2013. These include all available full genome sequences from swine H1N1pdm viruses (n = 82), all human H1N1pdm viruses from outside of the USA (n = 957), and 100 randomly selected full genome sequences from 1,500 human H1N1pdm sequences from the United States. Reassorted swine or human viruses containing H1N1pdm virus genes were excluded from this analysis. Our final dataset included the full genomes of 35 human and 26 swine samples from Sri Lanka and a global sample of 1,057 human and 82 swine virus sequences.The single breakpoint recombination and genetic algorithm for recombination detection methods 3 exclIn Sri Lanka, swine H1N1pdm clusters sw1 2009/10) and sw2 (2011) were genetically distinct from each other and from other swine viruses isolated globally, indicating 2 separate introductions to local swine that circulated among swine for 11 and 4 months, respectively, for each cluster. The sw1 and sw2 lineages did not appear to establish long-term sustained transmission within pigs. However, the reduced surveillance of farms during the period 2012\ufffd?\"2013 Table 1 009/10 anTo clarify transmission patterns between affected swine farms in Sri Lanka, we obtained contact patterns by interviewing pig farmers using a structured questionnaire  with apOf the 15 farms on which the common truck was used, swine on 3 (20%) were infected by a sw2 clade virus; on 2 farms on which the common truck was not used, no swine were infected. This association was not statistically significant (p = 1.0), however, given the small numbers of farms investigated. Our findings are consistent with dispersal of sw2 clade viruses in association with the truck to infect multiple farms that were geographically distant, but this remains unproven. It was previously documented that influenza viruses can remain viable for prolonged periods of time in water at a temperature of \ufffd%^28A\ufffdC  10 andDespite widespread inter-farm transmission of sw1 and sw2, our results show that only animals on farm C were infected in both spillover events. Farms A and G, on which swine were infected by sw2 in 2011, appeared not to have had infected swine during 2009\ufffd?\"2010, as shown by both virus isolation and serologic testing Table 1.This study demonstrates natural independent spillover events of H1N1pdm influenza viruses from humans to swine. H1N1pdm viruses appear to be spread by multiple, discrete introductions to swine, after which clonal expansion occurs within the swine. The spread of such virus lineages across multiple farms is consistent with virus dispersal by breaches of external biosecurity measures, including the manner of swine transportation, although this remains unproven given the small sample size. Unlike classical swine influenza, North American triple reassortant, and European avian swine viruses that have persistently circulated among swine for several decades in other countries 15, H1Overview of the swine industry in Sri Lanka and surveillance of human and swine pandemic influenza A(H5N1) viruses."}
+{"text": "Critical Care [2) increased significantly in response to emergency intubation, and the authors suggested that the early normalization of ScvO2 after intubation might not be reliable to reflect successful resuscitation. However, they might have ignored the influence of arterial oxygen tension (PaO2) on ScvO2.We read with great interest the study by Hernandez and colleagues published in cal Care . The stu2. However, a very high PaO2 could significantly influence ScvO2 even if arterial oxygen saturation reaches 100%. Pre-oxygenation may result in a very high PaO2 in the emergency intubation, so PaO2 should be taken as a potentially confounding factor. Very high PaO2 (about 288\u00a0mmHg) has a more significant and consistent effect on ScvO2 than a relevant change in cardiac index (>10%) [2 could increase ScvO2 without increasing oxygen delivery [It is well known that when arterial oxygen saturation is approaching 100%, the increase in oxygen delivery would be limited in response to the increase of PaOx (>10%) , and anix (>10%) . Recentldelivery .2 on ScvO2 in the management of critically ill patients.It is worth paying attention to the impact of PaO"}
+{"text": "DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified.The two oppositely imprinted and expressed genes, DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences.We observed that both DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.Our data support the idea that the main cause of the prevalent downregulation of The online version of this article (doi:10.1186/1868-7083-6-29) contains supplementary material, which is available to authorized users. CDKN1C, which is inactivated alternatively by genetic or epigenetic mechanisms in several human cancers, including urothelial carcinoma [The differential expression of alleles inherited from mother or father at genomic imprinted genes is achieved by epigenetic mechanisms, particularly by differential methylation at regulatory regions designated as differentially methylated regions (DMRs). Imprinted genes regulate growth and other physiological functions during embryonic development, but also in adult tissues. Since several maternally imprinted genes limit growth, they possess tumor-suppressive potential and tend to become inactivated in different types of human cancer . Their iarcinoma , 3.DLK1-MEG3 cluster, is affected by allelic losses or epigenetic changes [Delta-like 1 (DLK1), Deiodinase Iodothyronine Type III (DIO3) and Retrotransposon-like Gene 1 (RTL1 or PEG11) [Maternally Expressed Gene 3 (MEG3), Maternally Expressed Gene 8 (MEG8) and RTL1 antisense (RTL1-AS) [MEG3  and 1.3\u00a0kb upstream of the MEG3 transcription start site (MEG3 DMR) [DLK1 promoter is also relevant for its expression. The IG DMR, which is methylated on the paternal allele and unmethylated on the maternal allele, serves as the initial imprinting control region (ICR) for the entire cluster during early development [MEG3 DMR usually represents the dominant regulatory region [DLK1 and MEG3 is commonly reciprocal, possibly as a consequence of regulatory effects of the MEG3 RNA [In several cancers, a cluster of imprinted genes at 14q32.2, the  changes . This clr PEG11) . The matRTL1-AS) , 10 encoelopment , whereasy region . The expMEG3 RNA .Figure 1DLK1 expression is associated with changes in DNA methylation at this gene and its control regions [MEG3 has been reported to act as a tumor suppressor in a broader range of cancers [MEG3 exert various functions relevant for cancer development and progression, including regulation of growth factors and Notch signaling by DLK1, and regulation of TP53, pRB1 and NOTCH activity by MEG3 [Loss of imprinting in the 14q32 region due to epimutations at the IG DMR or microdeletions has been implicated in a range of diseases including UPD14mat/pat  and various cancers . In rena regions , 14\u201316.  cancers \u201319. Both by MEG3 \u201322.DLK1 and MEG3 are good tumor-suppressor candidates. Indeed, MEG3 has recently been reported to become downregulated in the majority of urothelial carcinomas and to exert tumor-suppressive functions [Urothelial carcinoma is the most common cancer of the urinary bladder. It can be categorized into two subtypes, namely papillary tumors and the more malignant invasive carcinomas, which are characterized by pronounced chromosomal instability , 24. In unctions . HoweverDLK1 and MEG3 was strongly diminished in urothelial carcinoma tissues and cell lines. This finding raises a conundrum as it is difficult to envision how either allelic loss or epitype switching could lead to the concomitant downregulation of these two imprinted and normally inversely expressed genes, which are located less than 100\u00a0kb apart. Indeed, upon closer investigation, we found that inactivation of the two genes is associated with the establishment of a novel epigenetic state in the region, which is distinct from that of either parental allele and is independent of copy number changes in most urothelial carcinoma tissues and cell lines. This epigenetic state involves a characteristic DNA methylation pattern and a strong shift towards repressive histone modifications across three major regulatory regions in this imprinted gene cluster. This mechanism could provide a means to silence both genes despite their normal opposite regulation.Unexpectedly, we found that expression of both DLK1 and MEG3 mRNA by qPCR in urothelial cancer tissues (n = 30) and cell lines compared to benign bladder tissue samples (n = 11) and primary cultured normal urothelial cells. Normal kidney tissue and the hepatoma cell line HepG2 were used as additional positive controls. DLK1 mRNA was significantly reduced in urothelial cancer tissues compared to normal bladder tissue  measured copy numbers varied between 1.7 and 2.2, as normalized to normal diploid leukocytes set at two copies. Of 23 urothelial carcinoma samples (BT), 10 cases displayed increased copy numbers and 5 cases had decreased copy numbers, whereas 8 tumors showed copy numbers in the normal range (1.7 to 2.2). Importantly, gene copy number changes affected all three analyzed loci to the same extent within each sample.Similarly, gene copy number changes affected all analyzed genes to the same extent in urothelial cancer cell lines (UC). Primary urothelial cell cultures (UP) were measured as diploid, as expected. Seven cell lines displayed elevated copy numbers between 2.5 and 3.5 across the analyzed region, for example, the Umuc3 and 639v cell lines. In accord with our results the predicted modal copy numbers are 3 for Umuc3 and 639v . DecreasMEG3 and DLK1 expression was reduced in almost all urothelial cancer tissues and cell lines, irrespective of copy numbers [see Additional file Thus, whereas copy numbers of the 14q region were variously increased or decreased in urothelial carcinoma tissues and cell lines, MEG3 and DLK1 downregulation was associated with changes in DNA methylation, we analyzed the three relevant CpG-rich regulatory regions, the DLK1 promoter, the IG DMR, and the MEG3 DMR, by bisulfite sequencing in selected urothelial cancer tissue and cell line samples  and tumor (n = 23) tissue samples, normal urothelial cells (n = 5) and urothelial carcinoma cell lines (n = 15)  in the majority of urothelial cancer tissues and cell lines , which are retained during bisulfite conversion if methylated, but mutated if unmethylated. Thus, up to five different restriction products are obtained if the sequence is heterogeneously methylated [see Additional file The DNA sequence in the DLK1 and MEG3 expression in urothelial cancer is associated with the acquisition of novel DNA methylation patterns, especially at the DLK1 promoter and the MEG3 DMR that in some cases extend to the IG DMR.Cumulatively, these findings indicate that the concomitant loss of DLK1 and MEG3 mRNA in five urothelial cancer cell lines. Treatment with 5-aza-dC or SAHA individually did not significantly induce MEG3 or DLK1 expression except for 5-aza-dC in the BFTC905 cell line. Combined SAHA/Aza-dC treatment consistently restored DLK1 and MEG3 gene expression to detectable levels [see Additional file To determine to which extent DNA methylation contributes to the silencing of the two genes, we tested the effects of the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (Aza-dC) alone or in combination with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the expression of DLK1 promoter, the IG DMR and the MEG3 DMR in normal urothelial cells, HepG2 cells and seven urothelial carcinoma cell lines , we quantified the H3K4me3 histone modification associated with active genes, the H3K9me3 and H3K27me3 modifications associated with repression, and H4K16ac, a marker of transcriptional competence but also of fixed nucleosomes, at the DLK1 promoter. The IG DMR likewise displayed enrichment of both active and repressive histone modifications, H3K4me3 and H3K9me3, and more strongly H3K27me3. Interestingly, H4K16ac was strongly enriched at both DMRs. At the MEG3 DMR, in agreement with the high expression in normal bladder, the active histone modification H3K4me3 was enriched, whereas repressive histone modifications (H3K9me3 and H3K27me3) were low.Because the variable results of DNA methylation analyses had suggested that the epigenetic state of the 14q32 region changes during culture of normal urothelial cells Figure\u00a0. As expeDLK1 expressing control cell line. In HepG2 cells, active and repressive histone modifications were enriched to comparable extents at the DLK1 promoter and the MEG3 DMR. The active mark H3K4me3 was highly enriched at the IG DMR in HepG2 cells, with higher levels of H3K9me3 compared to H3K27me3, which is the inverse pattern compared to that in normal urothelial cells.HepG2 cells were used as a DLK1 promoter and the MEG3 DMR. As a consequence, these latter two regions assumed similar patterns of histone modifications across the urothelial carcinoma cell lines. Interestingly, in contrast to the H3K4me3 mark, the H4K14ac modification was less severely depleted or even retained in some cell lines, with lowered levels especially at the IG DMR.The most striking difference in the urothelial carcinoma cell lines towards the controls was the severe depletion of H3K4me3 at all three regulatory regions analyzed. In comparison, repressive histone modifications (H3K9me3 and H3K27me3) were generally increased, in particular H3K9me3 at both DMRs, whereas increases in H3K27me3 were more evident at the In summary, the ChIP analyses revealed the predominance of repressive histone modifications and a nearly complete loss of H3K4me3, with partial retention of H4K16ac, a modification characteristic of fixed nucleosomes, across the entire analyzed region in all urothelial carcinoma cells. This finding supports the contention that the region acquires a repressive chromatin state in urothelial carcinoma.DLK1 and MEG3 within this cluster are tumor suppressor candidates in urothelial carcinoma due to their known functions in the regulation of cell growth and development [DLK1 is downregulated by epigenetic mechanisms in renal cell carcinoma [MEG3 was reported to act as a tumor suppressor, too, but in a more consistent manner [DLK1 argue strongly that this cell line retains only a paternal allele.Previous molecular and cytogenetic analyses of urothelial cancers have suggested at least one tumor suppressor gene residing at chromosome 14q32.2 , 27, 32.elopment , 33. Indarcinoma . Howeverarcinoma , 34. Thet manner , 19, 22.t manner . Of notet manner , 16, 30.t manner , 30. WitDLK1 or MEG3 was reported to become deregulated, but not both genes, as we observed in urothelial carcinoma. Unfortunately, many papers do not comment on whether they have investigated the other gene at all. In benign bladder tissues, DLK1 and MEG3 were well detectable with MEG3 being expressed more strongly than DLK1, like in normal kidney, liver and pituitary gland [MEG3 and is required for silencing of the paternally expressed genes such as DLK1. Despite their reciprocal relationship in benign bladder tissue, MEG3 and DLK1 expression were found to be both significantly diminished in urothelial cancer tissues and cell lines. With respect to MEG3, our findings are fully consistent with those of Ying et al. [DLK1 in urothelial carcinoma.In previous reports on other cancer types, either ry gland , 30, 35.ry gland . Most mog et al. . UnfortuDLK1 is expressed from the paternal allele and MEG3 from the maternal allele. Their concomitant downregulation is therefore difficult to explain by allelic loss. Accordingly, we found a range of copy numbers between one and four in urothelial carcinoma tissues and cell lines indicating that both losses and gains of this region are frequent, accounting for the high frequency of apparent \u2018loss of heterozygosity\u2019 in previous reports [DLK1 and MEG3 can indeed not be explained by chromosomal deletions.Importantly,  reports , 26. HowMEG3 expression should be retained or even increased, and conversely, if the gene cluster assumed the paternal state, DLK1 expression should be retained or increased. Likewise, DNA methylation at the regulatory regions should become homogeneous and resemble either the maternal or the paternal pattern. This type of change is exemplified by the HepG2 cell line, which shows strong DLK1 expression associated with a paternal epigenetic state.By a similar argument, we can exclude conventional loss of imprinting or epitype switching as a plausible cause of the concomitant downregulation. If the gene cluster assumed the maternal state, DLK1-MEG3 cluster  and between RTL1 and DIO3  have also been described to be significantly reduced or silenced by DNA hypermethylation in bladder tumor tissues and the cell lines RT4, RT112 and T24 [A clue to the actual mechanism is provided by the observation that the DNA methylation patterns at the three regulatory regions in urothelial carcinoma cells are indeed homogeneous, but are different from both the maternal and paternal patterns in normal bladder and renal tissues. These methylation patterns therefore suggest that a novel repressed epigenetic state is established during urothelial carcinogenesis at the 14q32 gene cluster. Of note, several microRNAs encoded in the  and T24 , 29 sugg and T24 .DLK1-MEG3 cluster in urothelial carcinoma cell lines is also reflected in the predominance of repressive histone modifications such as H3K9 and H3K27 trimethylation  with our experimentally obtained methylation patterns suggest strongly positioned and potentially newly phased nucleosomes in cancer compared to benign urothelial cells  became significantly hypomethylated in cancer cells, while methylation of flanking sites rather increased. It could therefore be interesting to map the nucleosomal positioning in the 14q imprinted gene cluster in normal and cancer cells in future work.The repressed state of the n Figure\u00a0. InteresDLK1-MEG3 cluster in urothelial carcinoma was hampered by the lack of an epigenetically stable normal urothelial cell line. Upon culturing, normal urothelial cells acquire a considerable degree of plasticity, including the ability to differentiate into epidermis-like as well as urothelial-like structures [DLK1-MEG3 cluster appears particularly susceptible to such changes, as reduced expression or silencing of MEG3 has also been observed in normal cell lines originating from other tissues [Meg3, have also been reported during establishment of cell cultures of mouse embryonic fibroblasts [DLK1 and MEG3 that varied between individual urothelial cell cultures. These changes did not extend to the IG DMR and were not as pronounced as in the cancer cell lines and tissues. For that reason, we used freshly isolated, noncultured urothelial cells, which are unfortunately only available in limited amounts, for the chromatin immunoprecipitation experiments.Our study of the epigenetic changes at the ructures . Upon imructures . The DLK tissues , 44. Charoblasts . In our DLK1 and MEG3 expression, overcoming the normally antagonistic regulation of these two imprinted genes. One target of these changes is evidently MEG3, which emerges as a tumor suppressor in many different tissues [et al. [MEG3 alone could be achieved by conventional mechanisms such as allelic loss or by epitype switching. The findings reported here and the observation of others that several smaller RNA species encoded in the cluster are downregulated by DNA hypermethylation [DLK1. The question of which of these changes support tumor progression will therefore have to be addressed by future research.In conclusion, our data suggest that the 14q32 imprinted gene cluster acquires a novel epigenetic state in urothelial cancer that allows the concomitant inactivation of  tissues . In urot [et al.  have demhylation , 29, colThe bladder cancer and benign tissue samples were a subset of those described in previous studies , 47 compAll urothelial cancer cell lines  and the hepatocellular carcinoma cell line HepG2 were cultured in DMEM  supplemented with 10% fetal calf serum . They weDLK1 and TBP in a reaction volume of 25\u00a0\u03bcl. MEG3 primers were described by Kawakami et al. 2006 [TBP was used as a reference gene. Primers and QuantiTect assays are listed in Additional file Total RNA was isolated from subconfluent cell cultures and cell lines or from powdered tissues using the RNeasy Mini or Micro Kit . Two \u03bcg RNA was reverse transcribed using 200 U SuperScriptII reverse transcriptase , with 300\u00a0ng oligo-dT and 25\u00a0ng random hexamer primers in a reaction volume of 20\u00a0\u03bcl. Real-time PCR assays were performed with the ABI7900HT System using the QuantiTect SYBR Green PCR Kit  and QuantiTect primer assays for DLK1 promoter and the MEG3 DMR using primers described in [Total genomic DNA was isolated from subconfluent cell cultures using the Blood and Cell Culture DNA Midi Kit . For DNA methylation analyses, 1\u00a0\u03bcg DNA was bisulfite converted using the EZ DNA Methylation-Gold Kit  following the supplier\u2019s protocol with incubations at 95\u00b0C for 10\u00a0min, followed by 64\u00b0C for 2.5\u00a0h. After purification, the bisulfite converted DNA was analyzed for methylation of the ribed in . PCRs weMEG3 DMR bisulfite-treated DNA samples were used with newly designed primers [see Additional file MEG3 DMR region analyzed by standard bisulfite sequencing.For pyrosequencing analysis of the DLK1, IG DMR and MEG3 DMR as in ChIP analysis [see Additional file GAPDH was used as a reference gene. Gene copy numbers were calculated as the ratio of the measured sequence to the reference gene GAPDH and normalized to the value from leukocyte DNA set as two copies.For gene copy number analysis by qPCR 25\u00a0ng total genomic DNA was used with the QuantiTect SYBR Green PCR Kit  and primers for 5 primary urothelial cells freshly prepared from a ureter after nephrectomy using the True MicroChIP kit . The precipitated DNA fractions were quantified by real-time PCR with the QuantiTect SYBR Green PCR Kit  and primers for the DLK1 promoter, IG DMR and MEG3 DMR. ChIP assays [see Additional file CTCFL was used as a control for a silenced gene with inactive histone modifications (H3K9me3 and H3K27me3), whereas GAPDH was used as control for a highly expressed gene with active histone modifications . The input DNA purified from the sheared unprecipitated chromatin was used to make a standard curve for each gene investigated and each sample was measured in duplicate with less than 12% variation. The 100% enrichment refers to the levels at the respective control genes. The results remained in principle unchanged, if the values were normalized to input DNA directly  was performed on triplicate samples with the ChIP-IT Express Kit  according to the manufacturer\u2019s instructions as previously described  or 105 pTable S2. Copy number changes and relative to gene expression of DLK1 in urothelial cancer cell lines. Figure S1. Additional bisulfite sequencing results in urothelial cultures. Method S1. Treatment with epigenetic inhibitors. Figure S2. COBRA analysis of the DLK1 promoter sequence. Method S2. DNA methylation analysis by COBRA. Figure S3. Effects of treatment with epigenetic inhibitors on DLK1 and MEG3 expression. Figure S4. Comparison between two normalization methods for the ChIP experiment. Figure S5. Bioinformatic prediction of nucleosome positioning at the DLK1-MEG3 locus. (DOCX 1 MB)Additional file 1: Table S1: Primer assays."}
+{"text": "Myocyte pO2 values in normal dogs were calculated from the myocardial deoxymyoglobin (Mb- \u03b4) levels using 1H-spectroscopy (MRS) and were normalized to Mb-\u03b4 obtained after complete LAD occlusion. During Protocol 1 (n\u200a=\u200a6), Mb-\u03b4 was measured during sequential reductions of the oxygen fraction of inspired gas (FIO2) from 40, 21, 15, 10, and 5%, while in protocol 2 (n\u200a=\u200a10) Mb-\u03b4 was measured at FIO2 of 3%. Protocol 3 (n\u200a=\u200a9) evaluated time course of Mb-\u03b4 during prolonged exposure to FIO2 of 5%. Myocardial blood flow (MBF) was measured with microspheres and high energy phosphate (HEP) levels were determined with 31P-MRS. MVO2 progressively increased in response to the progressive reduction of FIO2 that is accompanied by increased LV pressure, heart rate, and MBF. Mb-\u03b4 was undetectable during FIO2 values of 21, 15, 10, and 5%. However, FIO2 of 3% or prolonged exposure to FIO2 of 5% caused progressive increases of Mb-\u03b4 which were associated with decreases of PCr, ATP and the PCr/ATP ratio, as well as increases of inorganic phosphate. The intracellular PO2 values for 20% reductions of PCr and ATP were approximately 7.4 and 1.9 mmHg, respectively. These data demonstrate that in the in vivo system over a wide range of FIO2 and arterial pO2 levels, the myocyte pO2 values remain well above the Km value with respect to cytochrome oxidase, and oxygen availability does not limit mitochondrial oxidative phosphorylation at 5% FIO2.Decrease of ambient oxygen level has been used in myocytes culture experiments in examining the responsiveness to stress secondary to hypoxia. However, none of these studies measure the myocytes oxygenation levels resulting in ambiguity as to whether there is insufficient oxygen delivery. This study examined the hypothesis that at a basal myocardial work state, adequate myocyte oxygenation would be maintained until extremely low arterial pO The unpaired electron spin in the heme-Fe(II) complex of deoxymyoglobin (Mb-\u03b4) extends over the proximal histidyl N\u03b4 proton to cause a chemical shift that produces a characteristic resonance on 1H NMR spectroscopy 2 of approximately 2 mmHg and fell to 80% of the basal value at an intracellular pO2 of 1.8 mmHg 2 during basal conditions and 5% saturated during total coronary occlusion, the intracellular pO2 values for 20% reductions of PCr and ATP were calculated to be approximately 4.4 and 0.9 mmHg respectively 2 where significant decreases in high energy phosphates occurred could have been influenced by a decrease in myocardial oxygen consumption caused by a decrease in coronary flow and perfusion pressure 2 levels. Thus, the true critical PO2 level that leads to a reduction of HEP can be better estimated with graded hypoxia through decrease in FIO2 such as in the present experimental setting.in vivo. Graded reductions of FIO2 were produced to document the threshold level of myocyte oxygenation at which perceptible reductions of the PCr/ATP ratio  occurred, and to examine the effect of progressive reductions of myocyte oxygenation on myocardial high energy phosphate content.Consequently, the present study was carried out to examine the effect of decreasing myocyte oxygenation by graded hypoxia on myocardial high energy phosphate content in the intact heart Studies were performed in 25 adult mongrel dogs of either sex weighing 20\u201327 kg. All experimental procedures were approved by the University of Minnesota Animal Resources Committee. The investigation conformed to the \u201cGuide for the Care and Use of Laboratory Animals\u201d published by the US National Institutes of Health .The dogs were anesthetized with sodium pentobarbital , intubated and ventilated with a respirator with supplemental oxygen to maintain arterial blood gases within the physiologic range. A heparin-filled polyvinyl chloride catheter, 3.0 mm o.d., was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed through the fourth intercostal space and the heart suspended in a pericardial cradle. A heparin-filled catheter (3.0 mm o.d.) was introduced into the left ventricle through the apical dimple and secured with a purse string suture. A similar catheter was inserted into the left atrium through the atrial appendage. A homemade intra-cardiac vein catheter (0.3 mm o.d) was inserted directly into the great cardiac vein for coronary vein blood sampling. A 1.5\u20132.0 cm segment of the proximal left anterior descending coronary artery (LAD) was dissected free and a hydraulic occluder constructed of polyvinyl chloride tubing (2.7 mm o.d.) was placed around the artery. A silicone elastomer catheter (0.75 mm i.d.) was placed into the LAD distal to occluder by the method of Gwirtz 31P and 1H-MRS frequencies were 81 MHz and 200.1 MHz, respectively.Measurements were performed in a 40 cm bore 4.7 Tesla magnet interfaced with a SISCO  console. The left ventricular pressure signal was used to gate MRS data acquisition to the cardiac cycle, while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions 1H-MRS detection of the proximal histidyl N-\u03b4 proton resonance of Mb-\u03b4 has been described in detail 1 value of Mb-\u03b4. Each spectrum is acquired within 6 min . Although the short T1 of Mb-\u03b4 and the fast acquisition prevent gating of data acquisition to the cardiac cycle, signal loss as a result of heart motion was negligible because of the inherently broad line width of the Mb-\u03b4 peak. Although the Mb-\u03b4 resonance is temperature sensitive, the chemical shift of this resonance which appeared at 71\u201372 ppm (relative to H2O), remained virtually constant during the study protocol. No other resonances were detected within a 10 ppm region. In phantom studies we have established that the detection sensitivity for Mb-\u03b4 is essentially flat across the wall of the left ventricle. Therefore, the Mb-\u03b4 resonance reflects average whole wall Mb-\u03b4 without the need for correction for differences in sensitivity in the deeper myocardial layers as is the case for HEP measurements (see below).The method for 31P MR spectra were acquired in late diastole with a pulse repetition time of 6\u20137 seconds. This repetition time allowed full relaxation for ATP and Pi resonances, and approximately 90% relaxation for the PCr resonance. PCr resonance intensities were corrected for this minor saturation. RF transmission and signal detection were performed with a 28 mm diameter surface coil. A capillary containing 15 \u00b5l of 3M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal of the water resonance was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenter. This was accomplished using a spin-echo experiment with a readout profile. The information gathered in this step was also utilized to determine the spatial coordinates for spectroscopic localization. Chemical shifts were measured relative to PCr which was assigned a chemical shift of \u22122.55 ppm relative to 85% creatine phosphate at 0 ppm. Spatial localization across the left ventricular wall was performed with the RAPP-ISIS/FSW method. The technical details of this method including voxel profiles, voxel volume, and the accuracy of spatial localization obtained in phantom studies and in vivo have been published elsewhere 0 gradients and adiabatic inversion pulses to an 18 mm\u00d718 mm column coaxial with the surface coil and perpendicular to the left ventricular wall. Within this volume, the signal was further localized using the B1 gradient to 5 voxels spanning the left ventricular wall from epicardium to endocardium. Each set of spatially localized transmural spectra consisted of a total of 96 scans accumulated in a 10 minute block. Resonance intensities were quantified using integration routines provided by SISCO software. The values for PCr and ATP in each voxel were normalized to those present in the basal state and the PCr/ATP ratio was determined for each voxel. Pi resonances were also measured and ratio of Pi/PCr was calculated.51Cr, 85Sr, 95Nb and 46Sc). Microsphere suspension containing 2\u00d7106 microspheres was injected through the left atrial catheter while a reference sample of arterial blood was withdrawn from the aortic catheter at a rate 15 ml/min beginning 5 seconds before the microsphere injection and continuing for 120 seconds. Radioactivity in the myocardial and blood reference specimens was determined using a gamma spectrometer  at window settings chosen for the combination of radioisotopes used during the study. Activity corrected for overlap between isotopes and for background was used to compute blood flow as ml/g of myocardium/minute.Myocardial blood flow was measured using radionuclide labeled microspheres, 15 \u00b5m in diameter labeled with 4 different radioisotopes  2 during each experimental period can be calculated from the relationship Mb-O2 \u200a=\u200a1 - Mb-\u03b4. The fractional contents of Mb-O2 and Mb-\u03b4 and the temperature corrected [PO2]50 value can then be employed to calculate intracellular PO2 using equation 1. Because of continuing collateral blood flow we assumed that the Mb-\u03b4 resonance measured during total coronary occlusion represented 95% of the total myoglobin, and normalized the other Mb-\u03b4 resonances in relation to that value. We assumed that the fractional Mb-O2 content in the basal state  was \u223c90%. This also follows from calculations using equation 1. At 37\u00b0C, calculated intramyocyte PO2 would be 21 and 46 mm Hg for Mb-O2 saturations of 90% and 95%, respectively. Hence, we assumed that a fractional Mb-O2 content of \u223c90% was present because this value is most compatible with the presence of an oxygen gradient between the capillary and intracellular myoglobin and reported values for coronary venous PO2In equation 1, Mb-O31P and 1H MRS spectra were first obtained under basal conditions.Aortic and left ventricular pressures were measured with fluid filled pressure transducers positioned at mid-chest level and recorded on an 8-channel direct writing recorder . Left ventricular pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and In protocol one, of 6 dogs, baseline measurements were acquired at a FIO2 of 40% which leads to an arterial oxygen saturation of approximately 100%. This was followed by a two minute LAD occlusion. In the meantime the deoxymyoglobin MRS data were obtained for pO2 calculation. After the total occlusion Mb-\u03b4 data were acquired, FIO2 was progressively decreased to 21, 15, 10 and 5% respectively and interleaved P-31 and H-1 spectra obtained during each experimental condition every 4 minutes.in protocol 2, 10 dogs (after baseline data were obtained) were exposed to 3% FIO2 and LAD occlusion, respectively. The P-31 and H1 MRS data were obtained similarly.To examine if more severe hypoxia would produce an association between myoglobin desaturation and reduction of myocardial energetic state and alterations in OXPHOS, 2 would continue to decrease during prolonged hypoxia and whether this would be accompanied by a decrease in myocardial oxygenation level and PCr/ATP, in protocol 3, 9 dogs (after baseline data were obtained) were exposed to prolonged 18 minutes 5% FIO2, with P-31 and H1- MRS, myocardial blood flow and venous O2 measured every 4 minutes. Finally, the LAD was completely occluded  and all measurements were repeated.To examine whether the venous O31P spectra were analyzed as described above. Transmural blood flow distribution was determined from the microsphere measurements. Data were analyzed with one-way analysis of variance for repeated measures. A value of p<0.05 was considered significant. When a significant result was found, individual comparisons were made using the method of Sheff\u00e9.Hemodynamic data were measured from the chart recordings. The hemodynamic, myocardial blood flow, arterial and coronary vein blood gas and myocardial oxygen consumption, myocardial HEP and oxygenation data are summarized in 2 when mean aortic pressure and LV systolic pressure increased with subsequent increase in RPP [2, MVO2 actually increased [2 . However, myocardial HEP remained relatively stable [Hemodynamic measurements during each experimental condition are shown in e in RPP . Even thncreased  and thisy stable . Mb-\u03b4 rey stable .2. The mean aortic pressure and LV systolic pressure increased substantially resulting in a significant increase in rate pressure product [2 increased [2 decreased to 12\u00b10.9, which is significantly lower than the myocardial ischemia induced increase of oxygen extraction. The deoxymyoglobin reached approximately 50% of maximum deoxymyoglobin level [To examine whether more severe hypoxia would produce an association between myoglobin de-saturation and reduction of myocardial HEP, 10 dogs were exposed to 3% FIO product . MVO2 inncreased  again asncreased . Coronarin level .2 would result in continuous reduction of coronary venous pO2 and therefore detectable Mb-\u03b4. Prolonged 5% FIO2 caused a significant increase in LV systolic pressure and subsequent RPP. MVO2 increased as a result of increase in MBF but there was a significant decrease in HEP which was associated with an increase in deoxymyoglobin after 8 minutes exposure. It was evident that when the 5% FIO2 was maintained the venous pO2 continued to decrease. Once it was below 14 mm Hg, the Mb-\u03b4 became detectable.Protocol 3 was performed to examine whether prolonged 5% FIO2 beyond 5% or prolonged exposure to low FIO2 was associated with a decrease in PCr/ATP and increase in Pi/PCr which was associated with a decrease in intracellular PO2. The relationships of PCr and ATP with intracellular PO2 are shown in 2 content in the basal state was 90%. Values for PCr decreased when intracellular PO2 approached 10 mmHg. PO2 values that are associated with significant reductions of HEP have been termed \u201ccritical\u201d with the implication that they reflect the presence of O2 limitation of oxidative phosphorylation. Based on 2 value for 20% and 50% reduction of PCr were approximately 7.4 and 0.6 mmHg respectively. Similarly, the (PO2)80 for ATP was approximately 1.9 mmHg. A (PO2)50 value for ATP could not be determined because this degree of ATP reduction was not achieved. The lower critical PO2 for ATP than for PCr is not surprising because the fall of ATP is buffered by PCr through the creatine kinase reaction.Decreasing FIO1H- Mb-d and 31P- MR spectroscopic measurements can detect the signals of reduction of oxygen delivery to the mitochondria. Prior studies did not measure Mb-d, therefore, no evidence of decrease of oxygen delivery to the mitochondria under these conditions. The findings from the present study demonstrate that the in vivo compensate mechanisms can maintain the normal mtOXPHOS even during extreme low FIO2. The present study has further elucidated the response of the myocardial oxidative phorsphorylation (OXPHOS) regulation to hypoxia. Firstly, it has reiterated the hemodynamic response of the in vivo heart to graded hypoxia as a result of a decrease in the fractional inspired oxygen concentration. Secondly, it has depicted the increase in transmural blood flow during hypoxia, which helps in maintaining a normal mitochondrial OXPHOS. Thirdly, it has defined the relationships between myocyte PO2 and HEP levels in vivo myocardium under conditions where myocardial flow is not limited.The present study examined whether mitochondria lack oxygen under the extreme low FIO2 conditions, and if so, how long it takes the compensation systems to fail. We examined whether the During myocardial hypoxia, the lactate production and glycolysis pathway of ATP production are increased 2 up to 10% [2 caused no changes in systemic hemodynamics up to 6% Our study shows that the mean arterial pressure, heart rate, left ventricular systolic and end diastolic pressure and rate pressure product are maintained with graded reductions in FIOp to 10% . With fup to 10% . These c2 [2 caused a decrease in myocardial blood flow but it was still higher than baseline. This increased myocardial blood flow caused myocardial oxygen consumption to be maintained throughout hypoxia, as opposed to LAD occlusion [The graded hypoxia caused a proportional increase in myocardial blood flow up to 5% FIO2 . This wa2 . Howevercclusion .2). The effect of hypoxia on myocardial HEP has been studied in both of the above mentioned conditions in isolated working rat hearts perfused with Krebs-Henseleit buffer at 25\u00b0C 2 below 1.5 mmHg, with (PO2)80 of 1.1 mmHg and (PO2)50 of 0.5 mmHg 2 while coronary flow rate was maintained constant, and demonstrated that PCr began to decrease at an intracellular PO2 of 2 mmHg, with a (PO2)80 of 1.8 mmHg 2)80 of 4.3\u20134.5 and (PO2)50 of 1.0\u20131.2 mmHg 2 for PCr in the perfused heart. As the critical PO2 in the perfused rat hearts is higher in the graded perfused PO2 setting as compared to graded coronary flow setting, we hypothesized that we may find a similar trend in the in vivo setting. In the present study, we examined the critical PO2 in the vivo setting with graded hypoxia and found that the (PO2)80 is 7.4 mmHg, which is significantly higher than the hypoperfusion setting.Under normal baseline conditions, the myocardium has adequate supply of oxygen for oxidative phosphorylation and oxygen does not seem to play a regulatory role. Even at high cardiac work states, no deoxymyoglobin signal is detected despite a decrease in high energy phosphates 2 required for decrease in HEP in the present study suggests that there were other factors which are affecting the intracellular PO2 in the graded coronary artery stenoses setting. Indeed, the response of myocardial HEP levels to decreased coronary perfusion can be affected by changes in myocardial oxygen demand. Gregg initially described that myocardial oxygen consumption can be influenced by coronary flow and perfusion pressures. Several mechanisms have been proposed to modulate oxygen demand during hypoperfusion. The pressure in the coronary system may distend the ventricles, leading to increased contractility and oxygen consumption by an increase in sarcomere length or ventricular stiffness et al showed that in the range of coronary pressure of 60\u2013160 mmHg, decreases in coronary perfusion pressure in isolated ferret hearts resulted in preload independent decrease of the calcium transient which they hypothesized represented a protective mechanism that minimizes energy demands during low flow ischemia 2 levels. Thus, the true critical PO2 level that leads to a reduction of HEP can be better estimated with graded hypoxia through decrease in FIO2 such as in the present experimental setting. This reveals a (PO2)80 of 7.4 mmHg for PCr and a (PO2)80 of 1.9 mmHg for ATP.The higher PO2 that far exceeds the apparent Michaelis-Menten constant of cytochrome oxidase with respect to O2. It was shown that the critical PO2 at which oxidative phosphorylation began to decrease in isolated mitochondria was approximately 0.01 mmHg, which is more than 100 fold lower than the PO2 at which HEP levels started to decrease in the present study. This could be attributed to inhomogeneities of O2 availability at the cellular level, if the PO2 gradient between the cytosol and the inner mitochondrial membrane was significant. However, mathematical models of O2 diffusion suggest very low perimitochondrial O2 gradient 2 gradient near the sarcolemma, while PO2 values toward the center of the cell are nearly flat et al postulated that since the mitochondrial membrane is impermeable to myoglobin, a significant PO2 gradient can occur because oxygen must diffuse through a myoglobin free volume before it can react with cytochrome oxidase et al have recently shown that in the myocardium, Mb-facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the PO2 falls below 1.77 mmHg 2 is around 10 mmHg, myoglobin facilitated oxygen diffusion does not play a major role but under hypoxic conditions such as in the present study it plays a significant role in oxygen diffusion.Another important question to address is why any oxygen limitation occurs at an intramyocyte PO2, significant loss of PCr occurred at an intracellular PO2 of 7.4 mmHg. Thus, in normal in vivo heart, oxygen availability plays an important role in regulation of oxidative phosphorylation only when mean intracellular PO2 falls below 7.4 mmHg.In conclusion, during graded hypoxia caused by decreased FIO"}
+{"text": "Blastocystis, is one of the most common human intestinal protozoan, which has many conflicting reports on its pathogenic role. Gut conditions which obviously varies in asymptomatic individuals, symptomatic and irritable bowel syndrome (IBS) patients in terms of gut flora, pH, osmotic pressure and water potentials could play an important role in its pathogenicity. The present study is the first study to investigate phenotypic characteristics of Blastocystis sp. ST3 isolated from asymptomatic, symptomatic and IBS isolates.Blastocystis isolates were obtained from four IBS patients (IBS1-4) and four symptomatic patients (S1-4) at a local gastroenterology clinic. Asymptomatic isolates (A1-4) were obtained from a field survey at a local village.A total of 8 Blastocystis showed a very coarse and intensely folded surface. The IBS isolates also exhibited a dense material and a thicker layer of surface coat can be seen compared to asymptomatic and symptomatic isolates.All 12 isolates were determined as subtype 3 (ST3). A1-4 isolates showed the highest peak growth followed by IBS1-4 isolates and S1-4 isolates for the growth profile. Parasites from IBS isolates (IBS1-4) showed the largest diameter with a mean 18.43 \u00b1 2.22 \u03bcm compared to parasites of symptomatic isolates (isolates S1-4) 15.54 \u00b1 3.02 \u03bcm and asymptomatic isolates (isolates A1-4) 11.76 \u00b1 0.82 \u03bcm. The symptomatic isolates (average generation time: 9.87 \u00b1 2.97 h) grew faster than the IBS isolates (average generation time: 7.56 \u00b1 1.06 h) and asymptomatic isolates (average generation time: 5.97 \u00b1 1.52 h). The parasites isolated from IBS isolates showed strong aggregation and clumping, which had seen reduced in parasites of isolates S1-4. No clumping was seen in parasites from A1-4. The outer surface of parasites in IBS isolates showed greater binding affinities towards FITC-labelled Concanavalin A (Con A) than symptomatic isolates and asymptomatic isolates. Scanning electron microscopy showed that in IBS isolates, the surface of .There have been no studies thus far providing evidence for phenotypic variation within a particular subtype. The present study is the first to demonstrate the phenomenon of gut environment facilitating adaptation of parasites possibly for survival leading to phenotypic differences for Blastocystis Blastocystis, the most common human intestinal protozoan 32] and tBlastocystis to be the influencing factors for the parasite\u2019s pathogenicity especially subtype 3 where in Malaysia [Blastocystis ST3 was also found to be the only subtype in 100% of patients suffering from urticaria in Egypt [Blastocystis.Studies have implicated that genotypes of Malaysia  SingaporMalaysia  and USA Malaysia  evidencein Egypt  Blasto-Ain Egypt . Tan et in Egypt  also demThe present study however is the first to demonstrate phenotypic variation within a subtype 3 population from three environments namely asymptomatic, IBS and symptomatic. Hence environment adaptability for survival purposes could be a possible reason for the phenotypic variation to exist.Blastocystis towards the intestinal lining of the gut. Blastocystis lysate and live parasite have been shown to trigger cytopathic effect on Chinese Hamster Ovary cells [The thicker surface coat shown by the ultrastructural study in IBS isolates could influence cytopathic effect of ry cells , 42. Thery cells  as well Blastocystis infected IBS patients [In IBS condition inflammation of intestinal layer could cause leaky gut syndrome which facilitates undigested food substances to pass between the cells of the intestinal epithelial layer to the bloodstream causing damage on the intestinal wall  which inpatients .The present finding has a very important implication. Previously all evidences have been pointing to subtype 3 to be the pathogenic one. However the present study cautions on forming such a conclusion and suggest that ascribing subtype to pathogenicity could be an over generalization. It is evident that gut environment can influence phenotypic expression of even the same subtype. A similar study had been done on assessing the phenotypic characterization of entamoeba hystolytica whereby the interaction between the parasites and host component such as bacterial flora and mucins can trigger the parasites to be more pathogenic by exhibiting their virulences factor .It is beyond the scope of the present study to postulate the details of the gut environment although others have suggested that an unhealthy gut has a complex open ended ecosystem which can be a host for various microorganisms \u201351. ThesBlastocystis.There have been no studies thus far providing evidence for phenotypic variation within a particular subtype. The present study is the first to demonstrate the phenomenon of gut environment facilitating adaptation of parasites possibly for survival leading to phenotypic differences for"}
+{"text": "Please see the corrected Figure S2 here.There is an error in the title for Figure S2. The complete, correct Figure S2 title is: \u201cRelationship between the diet diversity (H\u2019) and the prey consumption specificity  and the prey consumption specificity (PSi) at the territory level (n \u200a=\u200a 71).(DOC)Click here for additional data file.Table S2Explanatory variables used in the GLMMs to assess their potential effect on Bonelli\u2019s eagle productivity, classified either as spatiotemporal parameters, breeding pair parameters or diet parameters.(DOC)Click here for additional data file.Table S3Summary of model parameter estimates and standard error of parameter estimates for each model included in the GLMMs. Models are showed following the same order as in Table 3.(DOC)Click here for additional data file."}
+{"text": "SLD5 is a member of the GINS complex composed of PSF1, PSF2, PSF3 and SLD5, playing a critical role in the formation of the DNA replication fork with CDC45 in yeast. Previously, we had isolated a PSF1 orthologue from a murine hematopoietic stem cell DNA library and were then able to identify orthologues of all the other GINS members by the yeast two hybrid approach using PSF1 as the bait. These GINS orthologues may also function in DNA replication in mammalian cells because they form tetrameric complexes as observed in yeast, and gene deletion mutants of both PSF1 and SLD5 result in a lack of epiblast proliferation and early embryonic lethality. However, we found that PSF1 is also involved in chromosomal segregation in M phase, consistent with recent suggestions that homologues of genes associated with DNA replication in lower organisms also regulate cellular events other than DNA replication in mammalian cells. Here we analyzed the function of SLD5 other than DNA replication and found that it is active in DNA damage and repair. Attenuation of SLD5 expression results in marked DNA damage in both normal cells and cancer cells, suggesting that it protects against DNA damage. Attenuation of SLD5 delays the DNA repair response and cell cycle restoration in normal cells but not in cancer cells. These findings suggest that SLD5 might represent a therapeutic target molecule acting at the level of tumor stromal cells rather than the cancerous cells themselves, because development of the tumor microenvironment could be delayed or disrupted by the suppression of its expression in the normal cell types within the tumor. Cells are constantly exposed to genomic DNA damage caused by internal and external agents such as oxidative stress and UV, respectively. Errors in DNA damage repair can result in cancer cell development SLD5 is a member of the GINS complex composed of PSF1, PSF2, and PSF3. This complex regulates the DNA replication fork in budding yeast We identified a mouse orthologue of PSF1 in a DNA library derived from hematopoietic stem cells during embryogenesis in which this cell population actively proliferates +/\u2212mice, which were healthy and fertile, were born at Mendelian frequency and exhibited normal growth. Moreover, there is no large difference of bone marrow cellularity between wild and SLD5+/\u2212 mice +/\u2212 mouse embryonic fibroblasts (MEFs) to analyze DNA damage repair and cell growth after DNA damage. Moreover, we compared the function of SLD5 in DNA damage repair using siRNA knock-down experiments in cancer cells.High expression of GINS genes has been observed in cancers and a correlation of their level of expression with malignancy has been suggested 2. MEFs were prepared from wild-type (WT) or SLD5+/\u2212 mice at embryonic day (E) 15.5 according to the usual method MEFs, B16 cells (mouse melanoma cells), and colon26 cells (mouse colon cancer cells) were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma) with 10% fetal bovine serum , and penicillin/streptomycin (Sigma) at 37\u00b0C under an atmosphere of 5% COSLD5 expression in B16 and Colon26 cells was transiently knocked down with small interfering RNA (siRNA). Lipofectamine RNAiMAX (Invitrogen) was used for the transfection of plasmid and siRNA into cells, following the manufacture\u0155s protocols, and experiments were done 48 h after transfection. We used two different siRNA oligonucleotides specific for SLD5; target siRNA sequences were: 5\u2032-GGA CCA CAC GGA GAC CCA CUU UAA A-3\u2032 (#1); 5\u2032-GAU GAG CAG AGA GAC UAC GUG AUU G-3\u2032 (#2).3 cells were seeded into each well of a 24-well culture plate  in 500 \u00b5l of medium. After 24 h, the wells were exposed to 1 \u00b5M etoposide for 12 h. After the drug was removed, cells were harvested immediately (0 h). Cell viability was evaluated by trypan blue exclusion. The same number of living cells was resuspended in fresh medium, and cells were cultured for 24, 48 or 72 h. They were then detached by adding 100 \u00b5l Trypsin-EDTA to each well; cells were then washed and re-suspended in 400 \u00b5l of medium. 20 \u00b5l of re-suspended cells were mixed with 20 \u00b5l of 0.4% solution of trypan blue dye (Life Technologies) for 1 min. Cells were immediately counted using a Neubauer microchamber with a light microscope. All counts were done using four technical duplicates of each sample. Means and standard deviations were calculated for each subculture.5\u00d710s (Amersham). The immunoreactive proteins were visualized using the ECL Prime Western Blotting Detection system . The blots were scanned using the imaging densitometer Las-300 mini .Cells were collected and lysed in SDS sample buffer  containing a cocktail of protease inhibitors. Proteins were separated on 10% or 15% SDS-PAGE and transferred onto PVDF membranes. After blocking for 1 h in TBST  containing 2% non-fat dry milk, membranes were incubated with anti-SLD5 , anti-\u03b3-H2AX , anti-Rad51 , or mouse anti-\u03b2-actin antibody in blocking buffer overnight. Membranes were then washed with TBST and incubated with HRP-conjugated anti-rat, rabbit or goat antibodies (1\u223610000) for 1 h. Bound antibodies were detected with ECL kitt test was used for statistical analysis. Differences were considered statistically significant when p<0.05.Results are expressed as the mean \u00b1 SD. Student's +/\u2212 mice +/\u2212 MEFs was approximately half of that in WT MEFs  directed against SLD5 (siRNA B16 and siRNA Colon26). We confirmed that SLD5 expression could be efficiently silenced by specific siRNA in B16 and Colon26 cells but not by scrambled control siRNA (SCR B16 and SCR Colon26) (We predicted that attenuation of SLD5 expression would delay DNA repair and cell cycle restoration in cancer cells as observed in normal cells. However, although the level of Rad51 expression was the same in SCR B16 and SLD5 siRNA B16 cells, and in SCR Colon26 and SLD5 siRNA Colon26 cells before treatment with etoposide, thereafter, a rapid increase of Rad51 was similarly observed in all four cell lines (To assess how the rapid response to DNA damage in SLD5 knocked-down cancer cells affects cell cycle restoration, we enumerated the cells after treatment with etoposide. Cell proliferation itself was not affected by knocking down SLD5 in either siRNA B16 or siRNA Colon26 cells in the presence of control DMSO treatment (It has been reported that SLD5 forms a GINS complex with other moieties such as PSF1, PSF2, and PSF3 and is involved in DNA replication in yeast As described above, the GINS complex is involved in DNA replication; however, GINS components PSF1, PSF2, PSF3, and SLD5 do not always form complexes and may have other functions. For instance, PSF1 regulates microtubule organization in M phase and is involved in chromosome segregation +/\u2212 MEFs compared with WT MEFs for repairing heavily damaged DNA. However, SLD5 attenuation was found to delay Rad51 expression in MEFs, resulting in severe retardation of cell cycle restoration. These findings suggested that SLD5 not only protects against DNA damage but regulates the rapidity of DNA repair. Recently, it has been reported that PSF2, also a member of the GINS complex, is phosphorylated by ATM upon DNA strand breakage DrosophilaIt was to be expected that a longer period of time would be required for DNA repair in cells with worse DNA damage as a result of lack of SLD5. We hypothesized that rapid Rad51 expression would be induced in SLD5Recent studies have shown that tumor growth and metastasis are not determined by cancer cells alone but also by various stromal cells. The stroma constitutes a large part of most solid tumors, and the cancer-stromal cell interaction contributes functionally to tumor growth and metastasis"}
+{"text": "Bacillus thuringiensis is a potent microbial control agent against insect pests. Here, we present the draft genome of the Egyptian strain Btm27 that shows high toxicity toward the cotton leafworm. The genome contains three insecticidal genes cry1Ac9, cry2Ab1, and vip3V that have been implicated in conferring toxicity toward lepidoptera. Bacillus thuringiensis has been successfully used as a biopesticide to control many agricultural pests and insect vectors of human disease  collection as 4AC2.This genome sequence has been deposited in GenBank under the accession no."}
+{"text": "There is growing recognition that epigenetic regulation contributes significantly to the fidelity of biological control. Compromised epigenetic pathways have now been shown to provide indications of transformation and tumor progression; they suggest potential targets for cancer prevention and intervention . A formiPost translational modifications of histone3 lysine4 (H3K4) may constitute epigenetically mediated regulatory signatures that can be informative for the molecular characterization of breast cancer pathology. Both methylation and acetylation of H3K4 are associated with gene activation. Tri-methylation of histone3 lysine4 has been extensively studied and is a well-documented marker for active or poised gene transcription. The enzyme complexes that regulate H3K4me3 have been implicated in tumorigenesis, pointing to the relevance of this epigenetic modification in cancer. Histone acetylation as well as histone acetyltransferases and deacetylating enzymes are recognized as important in tumor progression. However, the acetylation of H3K4 in particular has been less extensively investigated to date.Messier, et al., addressed the coordinated contributions of H3K4me3 and H3K4ac to breast cancer tumorigenesis in three well-established human mammary cell lines that represent a normal-like subtype  and two cancer subtypes, luminal  and basal-like metastatic  [In contrast to methylation and acetylation of H3K9 and H3K27, which activate or repress transcription respectively, methylation and acetylation of H3K4 are both associated with gene activation. Observed increases in H3K4 acetylation marks at specific gene promoters in both the MCF7 and MDA-MB-231 cell lines, compared to normal-like MCF10A cells, suggests that H3K4 acetylation may be an early step in cancer initiation or progression. The global increase in H3K4me3 at gene promoters in MDA-MB-231 cells suggests alterations in the epigenetic landscape that may be functionally related to late stage, metastatic breast cancer.From a mechanistic perspective, the presence of H3K4ac and H3K4me3 at specific gene promoters provides a blueprint for regulatory pathways that are functionally linked to epigenetic control of breast cancer onset and progression. The dynamic acetylation of H3K4 is associated with estrogen-receptor responsiveness in the MCF7 cells and the initial loss of mammary tissue\u2013specific gene expression in MCF7 cells that represent early stage breast cancer. Enhancing the pivotal role of H3K4ac in initial stages of breast cancer tumorigenesis, this epigenetic mark appears to poise genes for expression that leverages continued acquisition and persistence of metastatic properties. In contrast, H3K4me3, with persistence of H3K4ac reflects the epithelial to mesenchymal transition in MDA-MB-231 cells that represent late stage breast cancer. Important mechanistic linkages between histone H3K4ac and H3K4me3 modifications and breast cancer tumorigenesis remain to be established.While H3K4ac is emerging as an informative predictor of pathways that are deregulated during the onset and progression of breast cancer, the full potential for exploitation of this epigenetic modification as a biomarker and therapeutic target remains to be explored. Future studies must further define the enzymes that support acetylation and deacetylation of H3K4. Furthermore, the dynamic balance between H3K4 acetylation and methylation must be elucidated within the context of the full spectrum of histone modifications that contribute to combinatorial control of the epigenetic landscape. This will provide a drastically improved understanding of how epigenetic modifications and chromatin organization regulate mammary epithelial phenotype and gene expression related to transformation and tumor progression\u2014the complexity of chromatin organization that supports breast cancer initiation and progression is increasingly evident. Consequently, the clinical value of epigenetic biomarkers and related therapeutic targets can be expanded by detailed characterization of the regulatory signals responsible for the deposition of specific \u201chistone marks\u201d. Adding to the biological challenge inherent in these analyses, the plasticity observed in post translational histone modifications must also be considered. It will be important to establish the relationships between histone modifications and other parameters of epigenetic control and chromatin organization in breast cancer that include perturbations in inter- and intra-chromosomal interactions -8. Altho"}
+{"text": "BRCA1 and BRCA2 mutation carriers. We conducted fine-scale mapping at 9p22.2 to identify potential causal variants in BRCA1 and BRCA2 mutation carriers. Genotype data were available for 15,252  BRCA1 and 8,211 (631 ovarian cancer cases) BRCA2 mutation carriers. Following genotype imputation, ovarian cancer associations were assessed for 4,873 and 5,020 SNPs in BRCA1 and BRCA 2 mutation carriers respectively, within a retrospective cohort analytical framework. In BRCA1 mutation carriers one set of eight correlated candidate causal variants for ovarian cancer risk modification was identified . These variants were located up to 20 kb upstream of BNC2. In BRCA2 mutation carriers one region, up to 45 kb upstream of BNC2, and containing 100 correlated SNPs was identified as candidate causal . The candidate causal in BRCA1 mutation carriers did not include the strongest associated variant at this locus in the general population. In sum, we identified a set of candidate causal variants in a region that encompasses the BNC2 transcription start site. The ovarian cancer association at 9p22.2 may be mediated by different variants in BRCA1 mutation carriers and in the general population. Thus, potentially different mechanisms may underlie ovarian cancer risk for mutation carriers and the general population.Population-based genome wide association studies have identified a locus at 9p22.2 associated with ovarian cancer risk, which also modifies ovarian cancer risk in  BRCA1 and BRCA2, which account for approximately 24% of the familial risk among first-degree relatives [BRCA1 mutation have a lifetime risk of 35\u201360% with an average age of diagnosis of 50 years [BRCA2, with a lifetime risk of 12\u201325% and an average age of diagnosis of 60 years [BRCA1/2 associated ovarian cancers present as high-grade serous histology in advanced stage [Once age is taken into account, family history is the strongest risk factor for ovarian cancer. Women with a first-degree relative with ovarian cancer are at a 3-fold increased risk of developing the disease, indicating the importance of genetic factors in ovarian cancer predisposition. The most important genes in the context of genetic counseling for ovarian cancer susceptibility are BRCA1 and BRCA2 mutation carriers [BRCA1 and BRCA2 mutation carriers [Basonuclin 2(BNC2) and Centlein (CNTLN). BNC2 is a zinc-finger protein spanning nucleotides 16409503 to 16870706. It is expressed in ovary, testis and the male germ line where it regulates cell cycle progression [CNTLN spans nucleotides 17134982 to 17503923, it is ubiquitously expressed and localises at centrosomes to ensure centrosome function during cell division [Genome wide association studies have identified several common germline variants associated with ovarian cancer risk. The 9p22 locus was first found to be associated with ovarian cancer risk in the general population, and subsequently to be an ovarian cancer risk modifier in carriers ,5. The Scarriers . rs38141BRCA1 and 8211 BRCA2 mutation carriers of European ancestry. We comprehensively characterized the associations of genetic variants in the region with ovarian cancer risk for BRCA1 and BRCA2 mutation carriers.Here, we report the fine-scale mapping of the 9p22.2 locus using data from 15252 BRCA1 and BRCA2 mutation carriers participating in the Consortium of Investigators of Modifiers of BRCA1/2 . Eligib2>0.1 with the SNP that had been identified through the GWAS (rs3814113), and the set of SNPs that tagged all remaining SNPs in the region with r2>0.9. A total of 407 and 401 SNPs that were included on iCOGS in the 9p22.2 region passed QC and were available for the analyses for BRCA1 and BRCA2 mutation carriers, respectively. Imputation of genotypes was based on the phase 3 release of the 1000 Genome Project spanning nucleotides 16407967 to 17407967 (build 37) at chromosome 9 with a buffer region of 500bp, using IMPUTE2 v2 [Genotyping was performed using the iCOGS Illumina array . The quaS rs38141, and theBRCA1 and BRCA2 mutation carriers and all analyses were stratified by country of residence and year of birth. The USA and Canada strata were further subdivided by reported Ashkenazi Jewish ancestry.The primary analysis evaluated the association between each variant and ovarian cancer risk. To account for the non-random sampling of mutation carriers with respect to disease status, the analysis was conducted within a retrospective cohort framework by modeling the likelihood of the observed genotypes conditional on the disease phenotypes as previously described . Each muBRCA1 and BRCA2 mutation carriers. A fixed effect meta-analysis weighted by the inverse variance was conducted for imputed and genotyped SNPs when risk estimates were available in both datasets. For BRCA1 and BRCA2 mutation carriers, logarithms of per-allele HR estimates were used. The Cochran Q test was carried out to assess heterogeneity.Ovarian cancer associations were combined in a meta-analysis between BRCA1 and BRCA2 mutation carriers, each SNP was included in a Cox-regression model conditioned on the most strongly associated variant for each dataset and further adjusting by year of birth, and stratifying by country of residence. This approach has been shown to yield valid tests of association [-8. The most parsimonious model in the conditional analyses was identified using a threshold of p<10\u22124 for retaining SNPs in the model.To assess the number of variants independently associated with ovarian cancer risk in ociation . All SNPr2) with the top SNP higher than 0.1 [The set of potential causal SNPs was defined by those SNPs for which their likelihood ratio relative to the most significant variant was equal or less than 100 and having a pair-wise correlation .BEDTools was used to intersect positions of ovarian cancer risk-associated variants with functional genomic features generated by Coetzee et al  includinEach of the host institutions recruited under ethically approved protocols. A list of the local Institutional Review Boards that provided ethical approval for this study is given in BRCA1 mutation carriers of whom 2,462 were censored at ovarian cancer diagnosis . The correlation between the top SNP and the rs3814113 was 0.56 , given the strong prior evidence of association between SNPs in the region and risk for BRCA1 carriers and in the general population we selected the most significant SNPs as associated with ovarian cancer risk. Results for all SNPs with p<0.01 are presented in A total of 8,211 iagnosis . The assiagnosis . SNP rs3BRCA1 and BRCA2 ovarian cancer associated cancer tumors are high-grade serous . In the meta-analysis, the strongest associated variant was the genotyped SNP rs7046326 with a MAF of 0.25 and 0.24 in BRCA1 and BRCA2 mutation carriers, respectively. It displayed an HR = 0.74 .Since the majority of both  serous , referred hereafter as the \"BRCA1 peak\". These variants clustered in a 20kb region around the transcription start site of BNC2 . The SNPs in this set displayed MAFs of 0.24\u20130.28 and imputation accuracy higher than 0.95 and two out of the eight were genotyped  in BRCA2 mutation carriers r2 = 0.76, All except one (imputed SNP rs139555631) of the likely causal variants in BRCA2 mutation carriers, but was rejected from being potentially causal in BRCA1 mutation carriers.The original GWAS hit, rs3814113, was within the set of the strongest associated SNPs in BRCA1/2 meta-analysis, eleven SNPs were the set of potentially causal variants, which included the eight identified in BRCA1 plus three only present in the BRCA2 set. These eleven variants were highly correlated with the lead SNP of the meta-analysis rs7046326 (r2>0.8). Of note, the set excluded the original GWAS hit rs3814113  for the BNC2 gene in whole blood samples .Intersection of variants exhibiting the strongest associations with genomic features derived from cultured ovarian and fallopian tube cells revealed several SNPs that may be functionally relevant in influencing risk. variants . Althouganalysis , four ofBRCA1 and BRCA2 mutation carriers of European ancestry. We identified a set of variants that provided stronger evidence of association than the original GWAS hit.In this study, we performed fine-scale mapping of the 9p22.2 locus using dense genotype data from the iCOGS array in BRCA1 mutation carriers, one independent set of eight highly correlated (r2>0.8) SNPs could not be excluded as being potentially causal for the reported association with ovarian cancer, designated the \"BRCA1 peak\". The BRCA1 peak covers positions 16847520 to 16891647, which lie within or up to 20 kb upstream BNC2. Of note, the original GWAS hit rs3814113 was excluded from the candidate causal variants in this peak.In BRCA2 mutation carriers, 100 correlated variants (r2>0.4) could not be excluded as potentially causal (\"BRCA2 peak\"). The BRCA2 peak spanned positions 16847520 to 16915021, which are up to 44 kb upstream of BNC2 and more than 200kb upstream of CNTLN. The increased number of variants in this case is most likely due to reduced statistical power, as the number of BRCA2 mutation carriers diagnosed with ovarian cancer was only one quarter of the number of affected BRCA1 carriers. The candidate causal SNPs in the BRCA1 peak were mostly contained within the BRCA2 peak but the strongest associated SNP in BRCA2 was excluded from the BRCA1 peak. The current analysis was underpowered to investigate whether the association in BRCA2 mutation carriers is driven by a different set of genetic variants.For BRCA1 and BRCA2 mutation carriers, the meta-analysis would be expected to increase power for refining the set of potential causal variants. However, the combined analysis of BRCA1 and BRCA2 mutation carriers defined a set of eleven variants as potentially causal, which corresponded to the eleven strongest associated variants in BRCA1. This set excluded rs3814113 that was reported in the ovarian cancer GWAS [BRCA1 carriers only.Under the model of one shared causal variant explaining the association in both cer GWAS . The setBRCA1 mutation carriers and results for the most strongly associated SNPs in samples from the general population.Important differences emerged when we compared the patterns of association in the fine-scale mapping of 9p22.2 between BRCA1 mutation carriers, suggesting that the associations in BRCA1 mutation carriers and in the general population may be driven by different causal variants at the 9p22.2 locus. These results may indicate differences in the underlying causal mechanisms explaining the ovarian cancer associations between BRCA1 mutation carriers and the general population. In support of this possibility, differences in the association patterns with ovarian cancer between BRCA1 and the general population have been reported before. The 4q32.3 locus is associated with ovarian cancer risk in BRCA1 but not in BRCA2 mutation carriers or the general population [Fine-mapping results based on iCOGS data from the Ovarian Cancer Association Consortium indicate that SNP rs3814113 remains the most strongly associated SNP at the 9p22.2 region with serous ovarian cancer, the original GWAS hit . Based on our results, this SNP can be confidently rejected from the set of possible causal variants in pulation . HoweverBNC2 gene, and some candidate causal SNPs are eSNPs for BNC2 in whole blood, they may modulate the expression of BNC2 through similar, or different, mechanisms. The possibility that the BRCA1 association signal may differ from that in the general population adds extra complexity and reinforces the value of fine-scale mapping in different populations. These subtle differences in the patterns of associations depending on the underlying genetic landscape may be difficult to uncover by means other than fine-scale mapping, and thus strengthens the value of this approach for generating hypotheses about the functional basis of different sets of variants.As both signals lie in close proximity to the BRCA1 or BRCA2 mutations have shown that BRCA1 and BRCA2 carriers predominantly develop serous disease [BRCA1and BRCA2 mutation carriers.This study cannot exclude the possibility that the actual causal variants were not included in the set of genotyped or well-imputed variants. However, the iCOGs array included variants specifically for fine-scale mapping of the 9p22.2 locus based on data from the 1000 Genomes Project and therefore the region coverage is expected to be high. The relatively low number of ovarian cancer cases with tumor morphology information did not allow performing stratified analyses by ovarian cancer histological subtype. Studies of ovarian tumours in women with  disease ,22. Of tBRCA1 mutation carriers will assist functional studies to identify the gene/s targeted by these variants. BNC2 is an obvious candidate gene, given that the putative causal variants are located in/around its transcription start site. Identifying more strongly associated variants with ovarian cancer in the 9p22.2 region relative to the initial GWAS hit in BRCA1 and BRCA2 mutation carriers will refine the cancer risks associated with this locus further. These novel variants can be included in polygenic risk scores for ovarian cancer and hence inform the identification of patients at greater risk of disease. The results may also help to deepen our understanding of the biology of ovarian cancer development in BRCA1 and BRCA2 mutation carriers, potentially leading to the development of more effective and personalized treatments.Having narrowed down the potential set of causal variants to only eight SNPs in S1 FigThe colour code indicates the linkage disequilibrium with respect to the variant used for adjustment.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableBRCA1 and BRCA2 mutation carriers, respectively.'T' corresponds to genotyped; 'Info' measures the accuracy of the imputation; 'Ref' and 'Eff' correspond to reference and effector allele, respectively; 'MAF' to minor allele frequency, 'HR' hazard ratio and 'CI' confidence interval. Bold cells correspond to the strongest associated SNP in the indicated dataset. Green and violet text indicates the set of potentially causal variant/s in (PDF)Click here for additional data file.S5 TableBRCA1 and BRCA2 mutation carriers and their meta-analysis, respectively.'T' corresponds to genotyped; 'Ref' and 'Eff' correspond to reference and effector allele, respectively; 'MAF' to minimum allele frequency, 'HR' hazard ratio and 'CI' confidence interval. Bold cells correspond to the strongest associated SNP in the indicated dataset. Green, violet and orange text indicate those SNPs within 100 times likely of being the causal variant/s in (PDF)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file."}
+{"text": "The unconscious trauma patient with a possible unstable spinal injury constitutes a clinical challenge. To protect the unintubated airway, some guidelines ,2 recommWe surgically created a global ligamentous instability between C5 and C6 in five fresh cadavers . Four diCompared to RC, LTP created significantly less movement during lateral bending (p=.037), while H1and H2 had significantly less movement than RC in axial translation (p=.009 and .033). There was a tendency towards LTP and H1 and H2 performing better than RC also for other movements.Our results indicate that in unconscious trauma patients, LTP or one of the two HAINES techniques is preferable to the classic recovery position in the setting of an unstable cervical spine injury."}
+{"text": "G93A mice. One and five CCO terms related to cytoskeleton were described from the spinal cord deregulated genes of 40 days (actin cytoskeleton) and 80 days  old transgene mice, respectively. Also, four terms were depicted from the deregulated genes of sciatic nerve of 60 days old transgenes . Kif1b was the unique deregulated gene in more than one studied region or presymptomatic age. The expression of Kif1b [quantitative polymerase chain reaction (qPCR)] elevated in the lumbar spinal cord (40 days old) and decreased in the sciatic nerve (60 days old) of presymptomatic ALS mice, results that were in line to microarray findings. Upregulation (24.8 fold) of Kif1b was seen in laser microdissected enriched immunolabeled motor neurons from the spinal cord of 40 days old presymptomatic SOD1G93A mice. Furthermore, Kif1b was dowregulated in the sciatic nerve Schwann cells of presymptomatic ALS mice (60 days old) that were enriched by means of cell microdissection (6.35 fold), cell sorting (3.53 fold), and primary culture (2.70 fold) technologies. The gene regulation of cytoskeleton molecules is an important occurrence in motor neurons and Schwann cells in presymptomatic stages of ALS and may be relevant in the dying back mechanisms of neuronal death. Furthermore, a differential regulation of Kif1b in the spinal cord and sciatic nerve cells emerged as key event in ALS.Early molecular events related to cytoskeleton are poorly described in Amyotrophic Lateral Sclerosis (ALS), especially in the Schwann cell (SC), which offers strong trophic support to motor neurons. Database for Annotation, Visualization and Integrated Discovery (DAVID) tool identified cytoskeleton-related genes by employing the Cellular Component Ontology (CCO) in a large gene profiling of lumbar spinal cord and sciatic nerve of presymptomatic SOD1 Amyotrophic Lateral Sclerosis (ALS) is a progressive, rapid and fatal neurodegenerative disease that affects motor neurons of the spinal cord, brainstem, and cerebral cortex  develop clinical and pathological features similar to those seen in human ALS and are considered an excellent model to study the pathogenic mechanisms of the disease  were crossbred and the colony was maintained in a specific pathogen-free environment of the animal facility of University of S\u00e3o Paulo Medical School  as described previously  were used in the experiments. No motor neuron death was seen in those animal ages   and reference (100 ng) were reverse transcribed by the Low-input RNA Linear Amplification kit and then transcribed to Cy3-labelled (samples) or Cy5-labelled (reference) according to the manufacturer's instructions  and to previous descriptions  and scanned (Agilent Microarray Scanner). All steps were performed according to the manufacturer's instructions .The raw data from hybridizations and experimental conditions are available on the Gene Expression Omnibus website under accession numbers GSE50642     v6.7b functional tool (G93A and wild-type groups) were microdissected as described previously . RNA was extracted from the microdissected motor neurons using the PicoPure RNA isolation kit . Linear amplification of RNA was performed following Eberwine's procedure  were rapidly removed and frozen in ice cold isopentane at \u221245\u00b0C and stored at \u221280\u00b0C until use. The labeling procedure was performed according to adaptation of a previous description  and their sciatic nerves were dissected under aseptic conditions. Nerves were then placed in 60 mm dishes containing Leibovitz-15 medium , divested of their epineurial sheaths and chopped into 1 mm pieces. The fragments were then transferred to new 60 mm dishes containing D-10 culture medium [composed by DMEM  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin ] and were maintained there in 5% CO2 at 37\u00b0C for 5 days. The sciatic nerve fragments were then transferred to 30 mm dishes containing 2.5 ml Hanks' Balanced Salt solution , 0.05% trypsin , and 1 mg collagenase . The fragments were kept in that solution for 2 h in 5% CO2 at 37\u00b0C. Tissue fragments were washed with D-10 and dissociated by trituration through a 200 \u03bcl-pipette and a 19-gauge sterile needle. The suspension was centrifuged at 1500 rpm for 5 min at 4\u00b0C and the cells were resuspended in D-10 medium. This step was repeated and cells were passed through a 70 \u03bcm-cell strainer .Schwann cells were isolated by means of flow cytometry sorting from the sciatic nerve explants of 60 days old SOD16 cells were resuspended in 500 \u03bcl of buffer. Flow cytometry dot plot Schwann cell profiles are shown in Figure The cells were centrifuged at 1,500 rpm for 5 min at 4\u00b0C and the pellets were resuspended in PBS containing 10% fetal bovine serum and 0.01% sodium azide, . Sciatic nerve-derived cell suspension was incubated with a fluorescein isothiocyanate (FITC)-conjugated mouse p75NGF receptor antibody  diluted in the buffer solution 1/200) for 1 h at room temperature as mentioned above. The p75NGF receptor labeling was employed in the cell sorting experiments because it is a well-characterized surface marker for Schwann cells  according to the manufacturer's protocol. The quantity (NanoDrop 1000 Spectrophotometer) and quality  of RNA were analyzed as described above. Also, the Schwann cell samples were submitted to PCRs in order to access contamination from other cell types. Protocol and results regarding specificity of separated Schwann cell samples are presented in the supplementary material Figures .G93A and wild-type mice as described above. Nerve pieces were transferred weekly to new 60 mm dishes filled with 1 ml of D-10 for 5 weeks. Dishes were replaced every other day with a fresh medium  according to the manufacturer's protocol. The quantity (NanoDrop 1000 Spectrophotometer) and quality  of RNA were analyzed as described above. Cultured Schwann cell RNA samples were submitted to PCRs in order to access fibroblast contamination; the protocol and results are shown in the supplementary material Figure .Kif1b in the spinal cord (40 days old mice) and sciatic nerve (60 days old mice) and it was the only gene that its product has been described in the context of ALS . Sciatic nerve cDNA was synthesized from 100 ng of total RNA by using a Maxima First Strand cDNA Synthesis Kit  according to manufacturers.Kif1b\u2014Forward 5\u2032-3\u2032: CTGCTAGCCCTTTAAGACTCG; Reverse 5\u2032-3\u2032: AAACTCCTAGACAAACGCTCC; Gapdh\u2014Forward 5\u2032-3\u2032: GAGTAAGAAACCCTGGACCAC; Reverse 5\u2032-3\u2032: TCTGGGATGGAAATTGTGAGG) in a 20 \u03bcl final volume reaction.qPCR reactions were carried out in duplicate by means of the PikoReal-Time PCR System  employing 40 ng cDNA for spinal cord and 15 ng cDNA for sciatic nerve, the DyNAmo ColorFlash SYBR Green qPCR kit  and finally 400 nM of each primer . Gapdh was chosen as a housekeeping gene to normalize the qPCR values because the microarray analysis showed no alteration in the gene expression across samples.The cycling for SYBR reactions was composed by an initial denaturation at 95\u00b0C for 10 min. Templates were amplified by 40 cycles of 95\u00b0C for 15 s and of 60\u00b0C for 30 s. A dissociation curve was then generated to ensure amplification of a single product and absence of primer dimers. A standard curve was generated for each primer pair in order to determine the efficiency of the PCR reaction over a range of template concentrations from 0.032 to 20 ng/\u03bc l, using cDNA synthesized from reference mouse RNA. The efficiency for each set of primers was 100 \u00b1 5%. Gene expressions, which were normalized by The statistical method employed in the microarray analysis is described above , which are shown in Table G93A mice , cytoskeleton part (53 genes), actin cytoskeleton (16 genes), neurofilament cytoskeleton (three genes), and cytoskeleton (76 genes). Those genes are overlapped with the 76 deregulated genes of cytoskeleton category (25 down and 51 upregulated).The DAVID analysis of differentially expressed genes of 40 days old SOD1ce Table . The CCOce Table  in the sG93A mice. Furthermore, the CCO indicated four GO terms related with cytoskeleton, specifically the actin cytoskeleton (43 genes), cytoskeleton part (101 genes), microtubule cytoskeleton (64 genes), and cytoskeleton (146 genes). The 146 genes of the cytoskeleton GO term (74 down and 72 upregulated) are overlapped with all other GO terms  were deregulated in both spinal cord and sciatic nerve of SOD1G93A mice at the presymptomatic ages 40 and 60 days, respectively, and 10 genes  were deregulated in the above regions of 80 and 60 days mice, respectively, as shown by the Venn diagram  were obtained by means of single cell laser microdissection and the sciatic nerve Schwann cells were achieved by means of laser microdissection, flow cytometry cell sorting and cell culture. The levels of cDNA specific cell type marker that demonstrated the enrichment for each cell type obtained by respective technique are shown in the Supplementary Material , S3.Kif1b expression showed an upregulation (1.21 fold) of the gene in spinal cord of presymptomatic 40 days old SOD1G93A mice  was seen in laser microdissected motor neurons from 40 days old SOD1G93A mice  by means of single cell laser microdissection (6.35 fold), cell sorting (3.53 fold), and cell culture (2.70 fold)  Figure , regulatGene regulation of cytoskeleton-related molecules employing microarray technology has been described in the spinal cord and/or microdissected survival neurons from post mortem material of ALS patients  gene was in agreement to our analysis. Also, from 11 deregulated genes of the cytoskeleton and motor activity categories described in microdissected toluidine blue-labeled neurons from the spinal cord of asymptomatic SOD1G93A mice  and Kif1b (upregulation) deregulated genes seen in the spinal cord of 40 days old SOD1G93A mice were in line with descriptions of reduction KIF3A\u03b2 in motor cortices of ALS human and animal model  in the spinal cord of 40 days old presymptomatic ALS mice already indicates a very early presymptomatic event related to cytoskeleton with a possible implication to physiopathological mechanisms of the disease onset. From those genes, only Actg1, Adora, Akt1, App, Dctn1, Kif1a, Sirt2, and Stmn1) or related molecules have been studied in the context of ALS.From the 76 deregulated genes (25 down and 51 upregulated) in the spinal cord of 80 days old presymptomatic ALS mice, only eight genes  of ALS mouse model, thus denoting dynactin impairment as a mechanism in the presymptomatic phase of the disease. Mutation in Dctn1 gene has been associated to motor neuron degeneration in ALS  are both enriched on endoplasmic reticulum and Golgi membranes and are capable to interact with cytoskeleton elements in order to maintain the organelle morphology  in the sciatic nerve of 60 days presymptomatic old SOD1Aif1, Ccnb1, and Mapt in the presymptomatic ALS mice is likely to participate in the early events in the ALS peripheral nerve pathology. Aif1 encodes the allograft inflammatory factor-1 (AIF-1) and AIF-1 positive microglia/macrophages are among the earliest cells to respond to nerve injury , an actin-capping protein for the slow-growing end of filamentous actin  and qPCR analysis  and Schwann cells (downregulation). The results were coincident to those obtained in qPCR and microarray analyses of whole spinal cord and sciatic nerve of presymptomatic ALS mice.It is of substantial importance that the methodology allowed the observation of a differential regulation of Kif1b upregulation in immunolabeled motor neurons was in line to a previous work that employed toluidine blue-enriched putative motor neurons of presymptomatic ALS mice  and sciatic nerve (downregulation) was coincident to that found in the enriched motor neurons and Schwann cells and emerged as an important event in the pathogenic mechanism of ALS.In conclusion, the present work demonstrated cytoskeleton gene regulation as an important occurrence in motor neurons and Schwann cells in the presymptomatic stages of ALS and may be of importance in the dying back mechanisms of neuronal death in the neurodegenerative disease. The differential regulation of Jessica R. Maximino, Gabriela P. de Oliveira, and Chrystian J. Alves performed the experiments. Jessica R. Maximino and Gerson Chadi designed the study and analyzed the results. Gerson Chadi wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Arabidopsis PIKLE and rice CHR729 are identified to play critical roles in the regulation of series of genes involved in developmental or stress responding process. In this review we focus on how plant CHD proteins regulate gene expression and the role of these proteins in controlling plant development and stress response.Chromodomain-Helicase-DNA (CHD)-binding proteins have been characterized in various species as important transcription regulators by their chromatin remodeling activity. However, in plant the function of these proteins has hardly been analyzed before except that  Figure 1). Double chromodomains of human CHD1 are associated with methylated histone H3 lysine 4 (H3K4me) which is a hallmark of active chromatin -binding proteins, which are Snf2 family ATP-dependent chromatin remodeling factors, play important roles in the regulation of gene expression. Sequence analysis indicates that CHD proteins contain double chromodomains and Helicase-like region comprising SNF2_N and Helicase_C domains, which are critical for chromatin remodeling activity of the proteins. Based on the structure and function conservation CHD proteins could be divided into three subfamilies: Chd1 subfamily , Mi-2 subfamily , and CHD7 subfamily . In addihromatin . Yeast Cgulation . In Mi-2gulation . The effproteins .Arabidopsis PKL, which is a CHD3 protein. In recent years two papers reported the function of CHR729, a rice CHD3 protein  and its homologs in rice evolved from CHD3 chromatin remodelers were not considered to be CHD protein as they do not have conserved Helicase_C domain in the helicase-like region (Arabidopsis (CHR5) and rice  and rice . All of these proteins contain single PHD domain and two chromodomains except Arabidopsis CHR7 which lacks PHD domain at N-terminal part of the protein . Phylogenetic analysis using helicase-like region showed that CHR7 was the close homolog of PKL . However, later results show that dCHD3 which is homologous to dMi-2 but lacks one PHD domain is associated with actively transcribed sites as well as dMi-2 . This indicates that CHD3 protein may also positively participate in the process of transcription of series of genes.It has long been found that dMi-2 is involved in the repression of hox genes. The NuRD (Nucleosome Remodeling Deacetylase) complex which contains CHD3 protein exhibits histone deacetylase activity further improving the repressive function of the protein . In rice, CHR729, which is the homolog of Arabidopsis CHR4, was found to bind to H3K27me3 via its PHD domain in vitro. Loss-of-function of CHR729 results in genome-wide decrease of H3K27me3 level .In plant, there are also controversial arguments on the function of PKL. It was initially characterized to repress embryonic identity related genes and later was found to negatively regulate several genes expression. The repression mediated by PKL was found to involve trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. PKL target genes were enriched for H3K27me3 and mutation of PKL leads to the loss of H3K27me3 at these loci , 2012. Re3 level . This inFigure 2). Since many transcription factors were down-regulated in chr729 plants it would be interesting to test if these genes are direct targets of CHR729 to clarify if the protein also participate in the process of transcription  genes which are activated by PKL and CHR7 synergistically . They alcription . As ArabArabidopsis, PKL was found to play important roles in plant development as reviewed recently (pkl showed characteristics of embryonic tissue (LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), FUSCA3 (FUS3) and PHERES1 were derepressed in the mutants  and ARF9 by SOLITARY-ROOT (SLR)/IAA14 requires PKL, also named SSL2 in the study  technique affects plant growth, demonstrating that this gene is also important in controlling plant development  and ABI5 in response to ABA implicating the protein may play a role in osmoic stress (Direct evidence that plant CHD3 proteins are involved in stress response has not been present yet. However, in c stress . PKL andc stress . In ricec stress .Arabidopsis PKL and rice CHR729 have revealed that the proteins contribute to epigenetic regulation of gene expression which involves polycomb mediated H3K27me3 albeit the mechanism remains to be explored. PKL was found to control expression of several developmental genes. Despite direct targets are not clear mutants of CHR729 showed multiple developmental defects suggesting that both genes have effects on plant development. In addition, Arabidopsis CHD3 proteins are also involved in stress response. In light of the idea that H3K27me3 is an epigenetic mark associated with regulation of developmental and stress responsive genes it would be intriguing to disclose how CHD3 proteins and H3K27me3 are related in the matter of repressing gene expression (Arabidopsis respectively. Since distinct molecular function of CHD1 protein has been found in human, fly and yeast it is necessary to analyzed whether the protein in plant functions in the same way or adopt a novel mechanism to activate gene expression. Genetic analysis is also required to show whether plant CHD1 protein play important roles in development or stress response as CHD3 protein.Studies on pression . Plant CThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The reports on polymorphism in the KLK2 gene in relation to prostate cancer are contradictory among American and Korean population. Reports are not available regarding polymorphism in KLK2 gene in relation to prostate cancer in Indian population. The relationship between mutants (T) for wild-type (C) allele substitution of the KLK2, circulating human kallikrein-2 (hK2) levels and prostate cancer risk in Indian population has been examined.Prostate cancer is the third most common cancer among men in the world. The human kallikrein-2 gene (KLK2 gene) contains a mutant (T) for wild-type (C) allele substitution, resulting in a Trp to Arg change on the 250The study was limited to 15 subjects that include seven patients with prostate cancer and six controls who were persons with no cancer. Peripheral blood samples were collected from subject and control. DNA was extracted from these samples using standard protocol. DNA was dissolved in 1X TE buffer and used for PCR reactions for the amplification of 322bp KLK2 gene. Polymerase chain reaction- Restriction fragment length polymorphism (PCR-RFLP) procedure was employed using MspI to detect this polymorphism. The enzyme recognizes the 4bp sequence 5\u02b9CCGG3\u02b9 at codon 226 of KLK2 gene. PCR-RFLP products ware separated in 2% agarose gel and viewed under Gel-doc system.The median age of the eight subjects diagnosed for prostate cancer were 60, out of which 6 are found to be homozygous for CC allele of KLK2 gene, 1 was heterozygous having CT allele and 1 was homozygous for TT allele. From 7 subjects diagnosed negative for prostate cancer 2 were having the CT genotype and 5 were found to have the TT genotype.The study indicates a possible correlation among the C/T polymorphism of the KLK2 gene and circulating levels of hK2 and, in combination, may be predictive for prostate cancer. Though significant association has been found with small subjects, uses of allele marker for prostate cancer need further extensive study with large number of subject."}
+{"text": "A receptors since effects were blocked by iontophoretically application of APV and bicuculline, respectively. In contrast, stimulation of secondary somatosensory cortex did not affect most of the Pr5 neurons; however both cortices inhibited the nociceptive responses in the Sp5C nucleus through activation of glycinergic or GABAA receptors because effects were blocked by iontophoretically application of strychnine and bicuculline, respectively. These and anatomical results demonstrated that the somatosensory cortices projects to Pr5 nucleus to modulate tactile responses by excitatory and inhibitory actions, while projections to the Sp5C nucleus control nociceptive sensory transmission by only inhibitory effects. Thus, somatosensory cortices may modulate innocuous and noxious inputs simultaneously, contributing to the perception of specifically tactile or painful sensations.The sensory information flow at subcortical relay stations is controlled by the action of topographic connections from the neocortex. To determinate the functional properties of the somatosensory corticofugal projections to the principal (Pr5) and caudal spinal (Sp5C) trigeminal nuclei, we performed unitary recordings in anesthetized rats. To examine the effect of these cortical projections we used tactile stimulation of the whisker and electrical stimulation of somatosensory cortices. Corticofugal anatomical projections to Pr5 and Sp5C nuclei were detected by using retrograde fluorescent tracers. Neurons projecting exclusively to Pr5 were located in the cingulate cortex while neurons projecting to both Sp5C and Pr5 nuclei were located in the somatosensory and insular cortices (>75% of neurons). Physiological results indicated that primary somatosensory cortex produced a short-lasting facilitating or inhibiting effects (<5 min) of tactile responses in Pr5 nucleus through activation of NMDA glutamatergic or GABA In fact, hyperalgesia evoked by infraorbital nerve constriction induce an increase of excitability of Sp5C neurons  than S1 neurons . We have studied whether or not a single somatosensory cortical neuron projects to both the Pr5 and Sp5C nuclei and the electrophysiological effect this may have in these pre-thalamic nuclei.ad libitum. All animal procedures were approved by the Ethical Committee of the Autonomous University of Madrid, in accordance with European Community Council Directive 2010/63/UE. Efforts were made to minimize animal suffering as well as to reduce the number of animals used.Electrophysiological experiments were performed on 49 adult Sprague-Dawley rats of either sexes weighing 250\u2013300 g . Anatomical experiments were performed in 11 adult Sprague-Dawley rats (RE-1 to R-11) weighing 230\u2013290 g. Animals were housed under standard colony conditions and food and water were supplied The anatomical pathways linking cortical areas with the Pr5 and Sp5C trigeminal nuclei were studied by injecting or depositing the neuroanatomical fluorescent retrograde tracers Fluoro-Gold  and Fast Blue  respectively in the Pr5 and Sp5C nuclei in rats.n = 11) were anesthetized with an intraperitoneal injection of a mixture of ketamine (70 mg/Kg) and xylazine (5 mg/Kg) with supplementary doses when necessary (35 mg/Kg and 2.5 mg/Kg respectively) and placed in the stereotaxic frame. After appropriate craniotomy, 60 nl of a 4% saline dilution of FlGo was injected in the Pr5 nucleus by means of a 1 \u00b5l Hamilton syringe at stereotaxic coordinates: antero-posterior \u22129.5 from Bregma, lateral 2.8 and vertical 8.8 according to Paxinos Atlas . After a survival period of 1 week animals were anesthetized with an overdose of the same anesthesia and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.3 followed by increasing concentrations of sucrose solutions  in the same buffer. Brains were stored in 30% sucrose for at least 5 days for tissue cryopreservation and frozen sectioned on the coronal plane at 40 \u00b5m; sections were collected in three consecutive ordered series devoted to Nissl staining, cytochrome-oxidase staining, and fluorescent visualization. Sections containing the cerebral cortex of the fluorescent visualization series were studied under a Nikon Axioskop fluorescent microscope. Sections for cytochrome oxidase staining were incubated for 3 h in the complex solution , Cytochrome C (Sigma C-7752) and sucrose (Microbiologie) before being studied under an optical/fluorescent microscope, so both cytochrome and fluorochrome labeled cells could be observed. Series processed for Nissl staining were used for delimiting structures.Animals  using the Tile Scan tool of LAS AF software; samples were analyzed under both lin 405 mm UV and lin Ar 488 mm using a 10x objective. Images are a stack of sections in maximal projection, but neurons were counted in each individual layer.2 and heart rate were monitorized.Data were obtained from urethane-anesthetized (1.6 g/Kg i.p.) Sprague-Dawley rats. Supplemental doses of urethane (0.5 g/Kg i.p.) were given to maintain areflexia. Animals were placed in a Kopf stereotaxic device in which surgical procedures and recordings were performed. The body temperature was maintained at 37\u00b0C; the end-tidal COSingle unit recordings were performed by using tungsten microelectrodes  in the Pr5 nucleus  a macroelectrode (120 \u00b5m diameter bluntly cut insulated nichrome wire) was lowered 0.5 mm from the cortical surface into the somatosensory cortex. The EEG was filtered between 0.3 and 30 Hz and sampled at 100 Hz. EEG was used to test the level of anesthesia and to correlate the trigeminal neuronal firing with the EEG activity.2, 20 ms duration; Picospritzer) delivered at 0.5 Hz through a 1 mm inner diameter polyethylene tube. To avoid complex responses due to deflections of multiple vibrissae, these were trimmed to 5 mm long, allowing reproducible responses to be evoked. RFs were defined by the limits at which stimuli elicited spike responses.Whisker deflection was performed with a brief, electronically gated, air puff  was aimed at the cortex  to locate a site with a vigorous multiunit response to tactile stimulation of the whisker. After detecting the RF in the S1 or S2 cortex, a bipolar stimulating electrode (120 mm diameter blunt cut stainless steel wire) was aimed at the same site as the recording electrode . Single pulses  or barrages of pulses (3 trains of stimuli at 50 Hz during 500 ms repeated each 2 s) were applied to the S1 or S2 cortex through bipolar electrodes  by a Grass S88 stimulator coupled to a photoelectric stimulus isolation unit. The experimental protocol consisted in a period of control tactile stimulation  followed by 5 min of continuous tactile stimulation after the cortical stimulation barrages.A bipolar stimulating electrode was positioned at stereotaxic coordinates in the ventroposterior medial nucleus of the thalamus  in order to identify thalamic projecting neurons. Pulses of electrical current  were applied through the stimulating electrode. Antidromically-activated neurons were identified by a constant response latency to VPM stimulation and the ability to follow high frequency stimulus train (>100 Hz).In some cases small electrolytic lesions  were made at the end of the recording session to identify the stimulating and recording electrodes in Nissl stained brain sections.The activity of Pr5 and Sp5C neurons was evaluated in control conditions and after unilateral application of capsaicin cream (1.6%) on the vibrissa pad, affecting one or two whiskers. The cream did not impede the movement of the whiskers. Topical application of capsaicin cream induces excitation of nociceptors and, consequently, pain lasting several hours  or the glycinergic receptor antagonist strychnine hydrochloride  were applied iontophoretically with a multibarrel micropipette (20 \u00b5m tip diameter). The NMDA receptor antagonist D-2-amino-5-phosponovaleric acid  was also applied iontophoretically. A barrel was filled with 1 M NaCl for extracellular recording and a second micropipette was filled with APV, strychnine or bicuculline. The remaining micropipette, filled with 1 M NaCl solution, balanced the currents. Each barrel of the three-barreled pipette was connected via a silver wire to a channel of a microiontophoresis current generator (WPI current generator). The current generator controlled retention and ejection currents for the drug-filled micropipette. Drugs were ejected with negative (for APV) or positive (for bicuculline and strychnine) current, using a single 10\u201330 s pulse of up to 200 nA. Retaining currents of 10\u201320 nA were used to delay drug leakage from the barrel. Since the current pulse used to inject drugs had a small intensity, they only affected the close area around the multibarrel micropipette.The selective GABAt-test was used for comparisons. All data are shown as mean \u00b1 standard error.Recordings were accepted for statistical analysis when the spike amplitude fluctuations were lower than 10% throughout the experiment. Peristimulus-time histograms  were calculated from 30 stimuli using the Spike 2 software. The mean tactile response was measured from the PSTH as the number of spikes evoked at 0\u201350 ms after stimulus onset divided by the number of stimuli. We considered that neurons responded to tactile stimulation when the cell discharged at least one spike every two stimuli. The autocorrelogram time histogram (ACH) was also calculated to reveal oscillatory activity. A paired In order to establish if there are individual or shared corticofugal projections from S1 and S2 to Pr5 and Sp5C trigeminal nuclei, we have injected two different fluorescent retrograde tracers in both trigeminal nuclei. This anatomical study was performed on 11 rats. The locations of the FlGo injection site in Pr5, as well as the FB deposit in Sp5C were confirmed on the sections reserved for fluorescence study of those trigeminal nuclei. Both injections and deposits were confined to the desired site without signals of diffusion in any case  or both (double labeled neurons) tracers used in the experiment have been studied in the cerebral cortex of the animals. In every case the retrogradely labeled neurons were located in layer V, mainly in the cingulate, S1, S2 and insular (Ins) cortices Figure . Single For the quantitative analysis, single FlGo and FB labeled neurons were counted separately; first double-labeled neurons were counted in all sections and then result were contrasted to the superposition of single labeled neurons in all the layers of the confocal stack. The mean percentages of labeled neurons have been reported as the total FlGo, FB and double-labeled neurons in each cortical area Figure . The meaSingle FlGo labeled neurons were present in all the rostro-caudal levels of the cingulate cortex Figure ; howeverThe percentage of single FB labeled neurons (projecting to Sp5C nucleus) was similar in the S1, S2 and Ins cortices (<2%). This means that most of the cortical neurons that project to the Sp5C nucleus are double-labeled cells that also project to the Pr5 nucleus.n = 104), sampled throughout the entire dorsoventral extension of the nucleus, or from lamina III\u2013V of the Sp5C nucleus (at 800\u20131200 mm deep from the surface n = 70). The Pr5 and Sp5C neurons were silent under spontaneous conditions or displayed a low firing rate (<1 spikes/s) that followed slow EEG activity. Note that spikes tended to occur in the positive wave of the EEG . Eleven out of 14 Pr5 neurons (78%) and 11 of 18 Sp5C neurons (61%) could be activated antidromically. Antidromic spikes were identified by the fixed latency  and the ability to follow stimuli above 100 Hz  or 5.7 \u00b1 0.4 ms in Sp5C neurons  and 5.8 \u00b1 0.6 ms (n = 19) in Pr5 and in Sp5C nuclei, respectively.Electrical stimulation of contralateral S1 cortex with a single stimulus  induced orthodromic discharges of 1\u20132 spikes at latencies of 4.9 \u00b1 0.5 ms in Pr5 neurons  induced long-lasting changes in tactile responses according to the stimulated cortical RF. When cortical and Pr5 RFs were different (unmatched condition) most of the Pr5 neurons  showed a statistically significant decrease in tactile responses after S1 stimulation trains  at 1 min, returning to control levels 5 min after cortical stimulation (1.6 \u00b1 0.3 spikes/stimulus).Multiunit recordings in S2 cortex revealed larger RFs in the whisker pad than in S1 cortex, so the RFs received inputs from several whiskers. Electrical stimulation of contralateral S2 cortex (3 trains of stimuli at 50 Hz during 500 ms repeated each 2 s) in the unmatched condition induced long-lasting inhibition of tactile responses in 69% of Pr5 neurons  1 min after the cortical stimulating train, and control levels were recovered 5 min later  most Pr5 neurons increased their tactile response . Bicuculline strongly increased the spontaneous firing rate of Pr5 neurons  and tactile responses  and tactile responses in 7 Pr5 neurons  also increased the spontaneous firing rate  in the Pr5 nucleus reduced tactile responses in Pr5 neurons  and Sp5C neurons  5 min after capsaicin application  or 2.2 \u00b1 0.19 spikes/stimulus in Sp5C (n = 22) neurons in control conditions. Tactile responses decreased 5 min after capsaicin application to 1.2 \u00b1 0.15 spikes/stimulus (p = 0.03) or 1.3 \u00b1 0.18 spikes/stimulus (p = 0.007), respectively  or S2 (n = 4) cortical stimulation . In contrast, tactile responses were inhibited when the cortical and trigeminal RFs were non-overlapping. Similar effects have been described in the dorsal column nuclei cells  (Rauschecker, An interesting point is that nociceptive modulation could be performed at different levels of the somatosensory pathway. Recent results suggest that nociceptive responses in the Sp5C may be modulated by supraspinal mechanisms such as serotoninergic projections to Sp5C (Okubo et al., Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nu\u00f1ez conception and design of research; Eduardo Malmierca and Irene Chaves-Coira performed experiments and analyzed data; Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nu\u00f1ez interpreted results of experiments and drafted manuscript; Eduardo Malmierca, Irene Chaves-Coira, Margarita Rodrigo-Angulo and Angel Nu\u00f1ez approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Nonalcoholic fatty liver disease (NAFLD) is a risk factor for Hepatocellular carcinoma (HCC), but he transition from NAFLD to HCC is poorly understood. Feature selection algorithms in human and genetically modified mice NAFLD and HCC microarray data were applied to generate signatures of NAFLD progression and HCC differential survival. These signatures were used to study the pathogenesis of NAFLD derived HCC and explore which subtypes of cancers that can be investigated using mouse models. Our findings show that: (I) HNF4 is a common potential transcription factor mediating the transcription of NAFLD progression genes (II) mice HCC derived from NAFLD co-cluster with a less aggressive human HCC subtype of differential prognosis and mixed etiology (III) the HCC survival signature is able to correctly classify 95% of the samples and gives Fgf20 and Tgfb1i1 as the most robust genes for prediction (IV) the expression values of genes composing the signature in an independent human HCC dataset revealed different HCC subtypes showing differences in survival time by a Logrank test. In summary, we present marker signatures for NAFLD derived HCC molecular pathogenesis both at the gene and pathway level. Nonalcoholic fatty liver disease (NAFLD) is a condition where fat deposits in the liver. NAFLD refers to a wide spectrum of liver diseases such as fatty liver (steatosis) and inflammation derived nonalcoholic steatohepatitis (NASH). This condition can advance to fibrosis and cirrhosis producing a progressive, irreversible liver scarring that in the 15% of the cases progress into a liver hepatocellular carcinoma (HCC). Th. Th30]. Five-fold crossvalidation scheme was used because it preserves a reduced bias in comparison with resubstitution, it estimates the error with lower variance and uses less computational time compared to the leave-one-out crossvalidation . The feaTo assess the stability of a feature selection technique, variation in the distribution of features present in the subsets selected under different partitioning of the training/input data was calculated. The measure used to assess the stability of the selected subsets was the Normalized Average Hamming distance (NAHD) , 31 betwThis analysis design where there are five runs of each of the different methods allowed to further explore the produced signatures in each of the algorithms in terms of their gene composition frequency and frequency of the enriched deregulated KEGG pathways. By selecting the minimum amount of genes and overrepresented KEGG pathway which expression patterns maximized the classification performance of the phenotypes in their corresponding classes, each of the feature selection runs in the external five-fold crossvalidation procedure produced a genomic signature of genes and another one of pathways. These expression signatures showed phenotype and sample discrimination capabilities. To provide more robust feature subsets it was made a solution to the instability of the feature selection method based on the frequency aggregation of the five subsets resulting from the five runs of the crossvalidation which is essentially an ensemble solution that can be called rank summation . FinallyBootstrap resampling techniques were used to assess the uncertainty in hierarchical cluster analysis by calculating probability values  for each cluster in the dendrogram that represents the possibility that the cluster is the true cluster. Two types of p-values were available: bootstrap probability (BP) value and approximately unbiased (AU) p-value. In both cases thousands of bootstrap samples were generated by randomly sampling with replacement elements of the data and bootstrap replicates of the dendrogram were obtained by repeatedly applying cluster analysis to them. BP is biased as discussed in \u201339 and mClusters with AU larger than 95% were highlighted by rectangles, which are strongly supported by data as in a cluster with AU p-value > 95%, the hypothesis that \"the cluster does not exist\" is rejected with significance level 0.05 Figs .For the signatures of NAFLD progression microarray samples from different stages of the disease from human and mouse were collected to perform a time course analysis. Using a battery of 14 newly adapted feature selection approaches  robust sInitially raw expression values were used for the 14 supervised clustering feature selection algorithms . Then, tFor the genes composing these signatures enrichment of transcription factor binding sites were explored by the OPPOSUM program using a Fisher exact test (p<0.05)  S1 Tabl. The posBy selecting the orthologous genes using homologene database  the mousVenn diagrams were defined to capture genes that were differentially expressed in human steatosis and NASH when comparing cases and controls . Then we identified the common genes in the prognostic signature present between the differentially expressed genes in human steatosis and NASH.The signature holding the genes determining statistical differences in survival length was further validated by a Logrank test with an independent human HCC dataset having a HBV etiology and for which survival data was available . HierarcIn this study a series of newly adapted feature selection approaches was used to define different robust signatures holding the pathways and genes involved in NAFLD progression as well as a signature of differential survival in HCC common for human and mouse.The NAFLD progression signatures were used to study the pathogenesis of NAFLD derived HCC. Gene expression and pathway deregulation signatures with the genes and pathways that can distinguish different disease stages in human and mice were found using as input 471 genes having twofold regulation or more in 20% of the samples. The signatures produced by the 14 supervised clustering feature selection algorithms described in material and methods were aggregated to generate more robust solutions . WeighteUsing this preprocessed data the 14 supervised clustering and external validation feature selection algorithms were run in order to find signatures which produce the optimal cluster . Both thThe signatures produced by the 14 supervised feature selection methods for raw and smoothed data were aggregated separately and then the resulting pathway signatures were compared with the two feature selection methods which produced the optimal clustering result. It was possible to investigate the functional convergence in these signatures in terms of the overrepresented deregulated pathways whose expression levels can distinguish different disease stages and it was found that the different methods converge in similar functional solutions . The actHepatocyte accumulation of triglyceride is known to be a key component in the development of steatosis and NASH. Hepatic steatosis results from abnormal hepatocyte lipid metabolism that was found to be altered along the disease progression . TriglycThe arachidonic acid metabolism was upregulated in all mouse KO HCC samples and in all human samples  were upregulated in all HCC cases  or posterior refinement selection around the selection solution (y = number of refinement iterations) should be specified. It uses the class vector as input. Stage2: Evaluate the selected gene subset. Stage3a. If the process does not take into account the redundancy of the features (RFE): calculates the sample by sample MI excluding each gene. For each excluded gene defines a coefficient I as the difference of the sum of the sample by sample MI between classes and the sum of the sample by sample MI within groups. Stage 3b1: If the process takes into account the redundancy of the features (RFE_MR): for each gene calculates the average gene pairwise mutual information. Stage3b2: For each gene calculates the Coefficient II value by adding the average gene pairwise MI to the coefficient II. Stage4: Remove the m worst coefficient values and their corresponding genes and expression values. Stage5: Find the minimum error rate along the iterations and get the selected genes. S2 Fig. Flow diagram of the steps performed by the MRMR method. Stage1: As it is a forward search procedure, it starts from an empty set of selected genes. The process is iterative where the number of iterations should be specified and uses the class vector as input. Stage2: Calculate the normalized mutual information of the class vector with the vector containing each gene expression values along the samples. Stage3: a. For each gene calculate the average gene pairwise mutual information. b. For each gene in the subset of selected genes calculate the average gene pairwise mutual information. Stage4: For each gene define a coefficient value by dividing the value of the normalized mutual information with the average gene pairwise mutual information. Stage5: Store the gene having the maximum coefficient value and remove from the matrix the corresponding gene. Stage6: Evaluate. Stage7: Find the minimum error rate along the iterations and get selected genes. S3 Fig. Flow diagram of the GA procedure. Stage 1: The procedure initially creates a number of random variable sets (chromosomes). These variable sets form a population of chromosomes. Each random set is created with an initialization that randomly selects 70 genes from the total 504. Stage 2: Each chromosome in the population is evaluated for its ability to predict the group membership of each sample in the dataset (fitness function). Stage 3: Elitism: select the fittest individual intact for the next generation. Stage 4: The population of chromosomes is replicated. The roulette wheel selection ensures that chromosomes with a higher fitness score will generate a more numerous offspring. Stage 5: The genetic information contained in the replicated parent chromosomes is combined through genetic crossover with a crossover probability (For the parameters see supplementary Parameters in the Genetic Algorithm\u201d supplementary section). The chromosomes are ranked according to their fitness value. Above the crossover probability the best chromosomes are maintained intact for the next generation. Below the crossover probability two randomly selected parent chromosomes are used to create two new chromosomes. This crossover mechanism allows a better exploration of possible solutions recombining good chromosomes. Stage 6: Mutations are then introduced in the new chromosomes generated by crossover randomly with a mutation probability. These mutations produce that new genes are used in chromosomes. Stage 7: The process is repeated from stage 2 until the number of generations exceeds certain threshold (100) and the regression between the population of chromosome\u2019s minimum error rate and the generation is less than 0.05. The cycle of replication (stage 3), genetic crossover (stage 4) and mutations (stage 5) is called generation. S4 Fig. Tree structure where each of the stages of the disease has been clustered in a single cluster. Tree structure where each of the stages of the disease has been clustered in a single cluster using the GS1_clust_FOM algorithm to select the variables used as input in pvclust used to perform hierarchical clustering. S5 Fig. A: Ovalbumin serpin expression along the NAFLD progression.B: Positional gene enrichment. A: Ovalbumin serpin expression along the NAFLD progression. MAT1A_15 and GNMT_ko8 are HCC mice samples where the serpins are overexpressed.B: Positional gene enrichment analysis using PGE program [S6 Fig. 91 human HCC data clustering. Using complete hierarchical clustering using the Pearson correlation as a similarity measure it is possible to distinguish two stable clusters, cluster A and B that show statistical significant differences of survival length using by Kaplan-Meier plots and log-rank statistics analysis. S7 Fig. HNF4 alpha expression (log2 mouse KO vs wild type) in 3 and 8 month GNMT and MAT1A; and 15 month MAT1A . S8 Fig. Expression trend (log2 mouse KO vs wild type) of NAFLD progression genes regulated by HNF4a in 3 and 8 month GNMT and MAT1A; and 15 month MAT1A . S9 Fig. HNF4 alpha expression (log2 disease vs control) in human steatosis and NASH. S10 Fig. Expression trend (log2 mouse KO vs wild type) of NAFLD progression genes regulated by HNF4a in human steatosis and NASH. S11 Fig. Expression trend (log2 mouse KO vs wild type) of biosynthesis of unsaturated fatty acids in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S12 Fig. Expression (log2 mouse KO vs wild type) of stearoyl-CoA desaturase in human steatosis and NASH in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S13 Fig. Expression trend (log2 mouse KO vs wild type) of phenylalanine, tyrosine and tryptophan biosynthesis in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S14 Fig. Expression trend (log2 mouse KO vs wild type) of androgen and estrogen metabolism in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S15 Fig. Expression trend (log2 mouse KO vs wild type) of arachidonic acid metabolism in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S16 Fig. Expression (log2 mouse KO vs wild type) of cyclooxygenase in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S17 Fig. Expression trend (log2 mouse KO vs wild type) of PPAR signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S18 Fig. Expression trend (log2 mouse KO vs wild type) of drug metabolism cytochrome P450 in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S19 Fig. Expression trend (log2 mouse KO vs wild type) of metabolism of xenobiotics by cytochrome P450 in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S20 Fig. Expression trend (log2 mouse KO vs wild type) of toll-like receptor signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S21 Fig. Expression trend (log2 mouse KO vs wild type) of p53 signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S22 Fig. Expression trend (log2 mouse KO vs wild type) of MAPK signaling pathway in human steatosis and NASH; in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S23 Fig. Expression trend (log2 mouse KO vs wild type) of bile acid biosynthesis in 3, 8 month GNMT; and MAT1A KO mice and 15 month MAT1A tumors. S1 Table. Summary of the most established biomarkers in NAFLD. S2 Table. Dunn and FOM indexes of the Signatures of NAFLD progression. Dunn and FOM indexes of the Signatures of NAFLD progression resulting from the 14 different supervised clustering based feature selection methods on smoothed data; Ensemble error rate and stability in terms of Hamming distance of the Signatures of NAFLD progression resulting from the 7 different supervised clustering based feature selection methods that minimise the FOM index on smoothed data. S3 Table. Enriched Transcription Factor binding sites. Enriched Transcription Factor binding sites by means of Fisher exact test (p<0.05) in the signatures of NAFLD progression resulting from the two supervised clustering based feature selection methods which produced the optimal clustering result and the two ensemble signatures from raw and smoothed data. S4 Table. Ensemble error rate and the number of the different feature selection methods used to build survival signature. Ensemble error rate and the number of selected genes resulting from the different feature selection methods used to build the survival signatures common for human and mouse. program  shows th(DOCX)Click here for additional data file."}
+{"text": "Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.Kisspeptin1 (product of the  KISS1 mutation Kiss1 and Kiss1r genetic knockdown models also show significant deficits in pubertal development and reproductive functions Kiss1r is expressed widely in the brain, including on GnRH1 neurons that form the final common pathway regulating the pituitary-gonadal axis KISS1 and its receptor play major roles controlling the reproductive system in vertebrates kiss gene  and the kiss receptor gene  have been identified in the nervous system of teleost fishes, including the species used in the present study \u2013 zebrafish (Danio rerio) Two forms of the Vertebrates express 2\u20133 forms of GnRH, with distinct genes, locations and functions kiss1 and kiss2 mRNAs are expressed during embryonic and larval development Given that zebrafish Brass GnRH3:EMD transgenic zebrafish were maintained in a zebrafish aquarium system on a 14L:10D photoperiod at 28\u00b0C. They were fed twice daily with flake food and live brine shrimp. Sexually mature males and females were maintained in separate tanks until the day before breeding. To control the breeding time, each male-female pair was set in one tank with a divider to separate them. The divider was removed shortly after lights on to allow timed breeding, and fertilized eggs were collected. Embryos were maintained in a 28\u00b0C incubator. All embryos for a single set of experiments were from the same parents.All procedures were carried out in accordance with and approved by the Animal Care and Use Committee of UCLA .YNLNSFGLRY-NH2) and Kiss2 (FNYNPFGLRF-NH2) were synthesized by Bachem Inc. , based on the sequence described by Kitahashi and co-workers Biologically-active, 10-amino acid fragments of zebrafish Kiss1  until they were sacrificed, and then fixed with 4% paraformaldehyde (PFA) at various developmental stages for morphological analysis by confocal microscopy. A comparison between the presence and absence of the chorion made no difference to the outcome of kisspeptin treatments on GnRH3 neuron development (data not shown). This indicates that kisspeptin diffuses through the chorion efficiently. In the study on adult zebrafish, embryos were treated with kisspeptins starting at 5 hpf until 3 dpf, and then raised to adulthood in the zebrafish aquarium facility starting at 5 dpf. Adult brain imaging studies were performed at about 8 months of age. For electrophysiology experiments, following the protocol described by Zhao and Wayne (2012) kiss1 and kiss2, embryos were collected daily from 1\u20137 days post fertilization (dpf) in four replicate experiments. Using RNeasy Plus Mini Kit , total RNA was extracted from homogenates of 20\u201350 zebrafish embryos or larvae collected at different developmental stages. Reverse transcriptase reaction was performed from 0.5 \u00b5g of total RNA using the MultiScribe Reverse Transcriptase . The following is the Reverse Transcriptase (RT) reaction mixture and procedure: 0.5 \u00b5g RNA, 10 \u00b5l of 2\u00d7 RT buffer, 1 \u00b5l of 20\u00d7 RT enzyme mixture and RNAase-free water to a final volume of 20 \u00b5l. The mixture was incubated at 37\u00b0C for 60 minutes and then heat inactivated at 95\u00b0C for 5 minutes.To test the early expression of kiss1 and kiss2 primers were previously described by Kitahashi, and co-workers (2009) Elongation factor 1a (ef1a) was used as an internal reference gene for each tested sample. Each sample was processed for quantitative PCR in triplicate. A cycle threshold (Ct)-based relative quantification with efficiency correction normalizing to ef1a was calculated by the 2\u2212\u0394\u0394Ct method. The relative quantity of kiss1 or kiss2 gene expression was presented as the fold difference to that of 1 dpf embryos. The sequences of the primers used for quantitative RT-PCR are shown in ef1a, kiss1 and kiss2 primer sets were 88%, 100%, and 108%, respectively, as determined by serial dilution experiments.Quantitative real-time PCR was conducted using the SYBR Green PCR Master Mix  following the manufacturer's instructions in an Mx 3000P System . The reaction was composed of the following: 1.5 \u00b5l of the RT reaction, 10 \u00b5l of 2\u00d7 qPCR mixture with the appropriate forward and reverse primers to a final volume of 20 \u00b5l. The final concentrations of the primers in the qPCR reaction were 1.25 \u00b5M. The nucleotide sequences of the qPCR Synaptic vesicle protein 2 (SV2) is a marker of synaptic transmission, including in zebrafish Images were acquired and analyzed with Fluoview software using an upright Olympus microscope . Three sets of experiments were performed: 1) analyze neuron number from the different GnRH3 neuronal populations to assess the effects of kisspeptins on early embryonic development of the GnRH3 neural circuit (18\u201325 hpf); 2) analyze the number of synaptic boutons on the cell bodies and along the neuronal processes of the bilateral clusters of TN-GnRH3 neurons to evaluate the effects of kisspeptins on potential synapse formation during embryogenesis (50 hpf) by means of whole mount immunohistochemical staining with SV2 antibody; 3) analyze the number of hypothalamic GnRH3 neurons from adult zebrafish that were treated with kisspeptins as embryos to determine if early changes continued into adulthood. Embryos were mounted in 0.8% agarose in two positions, lateral-side up  or ventral-side up , then viewed and imaged using water immersion 5\u00d7, 20\u00d7 or 40\u00d7 objectives . Z-stacks of images were taken at the interval of 0.5 \u00b5m.To count the number of boutons, we overlaid the projection images of both green-488 (EMD) and red-594 (SV2-ir) channels of bilateral TN-GnRH3 neuron clusters and their processes taken with 40\u00d7 water objective with 3\u00d7 digital zoom, counted the yellow  spots (>0.1 \u00b5m) on the cell bodies and along the neuronal processes projecting from the bilateral clusters, section by section through the projection stacks To assess whether or not embryonic treatment with kisspeptins had long lasting effects into adulthood, embryos were treated 100 nM Kiss1, 100 nM Kiss2, or control solution (5 hpf- 3 dpf) and maintained in a zebrafish aquarium system for eight months until sacrifice. Adult intact brains were removed and fixed in 4% PFA/10% sucrose in PBS at 4\u00b0C overnight. After fixation they were washed three times with 20% sucrose in PBS and were incubated at 4\u00b0C overnight. The brains then were left in the mixture of 20% sucrose and OCT compound (Tissue-Tek) (1\u22362) again at 4\u00b0C overnight. The next day, the brains were sliced horizontally (50 \u00b5m thickness) using a cryostat (Leica CM1850) at \u221220\u00b0C. Fluoview software was used to image and to count the number of GnRH3-EMD neurons in hypothalamus using confocal imaging . Analysis of numbers of cell bodies was performed by one of the authors (MAL) and another researcher (MF) who were blind to the treatments.Whole-cell patch electrophysiology was conducted as previously described p<0.05.Values are shown as mean \u00b1 SEM. Data were analyzed using Prism 6 . Statistical significances among experimental groups in the imaging studies were determined by one-way ANOVA, followed by the Tukey's multiple-comparison test. Paired t-test was used for analysis of electrophysiological data. Differences were considered significant if kiss1 and kiss2 gene expression from 1\u20137 dpf  kiss2 mRNA increased more dramatically than kiss1 mRNA between 1 and 7 dpf .Using quantitative RT-PCR, we tested the dynamic changes of both  1\u20137 dpf . Both kiOur previous work Our previous study showed that multiple populations of GnRH3 neurons emerged in this transgenic zebrafish model by 30 hpf Our earlier work showed that TN-GnRH3 neurons express synaptic vesicle protein (SV2) on their neural processes during development, suggesting that these neurons are capable of synaptic communication during embryogenesis The functions of GnRH3 neurons are dependent upon their locations in zebrafish kiss1 and kiss2 genes are expressed by 1 dpf, and their mRNA levels increase progressively during embryonic and larval development in zebrafish. These findings corroborate earlier work in zebrafish showing increased kiss1 and kiss2 mRNA expression between 1\u20137 dpf, with peak levels achieved just prior to or during the time of puberty kiss1/2 during zebrafish embryogenesis is similar to what was observed in cultured embryonic brain explants from mice in which Kiss1 was also expressed early in development kiss1 and kiss2 mRNA expression peak at somite stage 19 or 1 dpf before declining to low levels by stage 26 or 2 dpf. The expression of both kiss mRNAs stayed low through 8 dpf We show that Both Kiss1 and Kiss2 stimulated the number of GnRH3 neurons in the developing trigeminal neuron population. This was the only effect of Kiss2 observed on any aspect of GnRH3 neuron biology during early development that we investigated. The trigeminal population of GnRH3 neurons is unusual because it is located in the peripheral nervous system. Trigeminal neurons are part of the sensory system and, in part, mediate the effects of mechanosensory stimuli on escape behavior in zebrafish Recent work from our laboratory showed the expression of SV2 on GnRH3 neurons during early embryogenesis in zebrafish, indicating the potential for synaptic transmission while the GnRH3 neural network was still developing Recent work showed that Kiss1, but not Kiss2, could stimulate spike frequency of TN-GnRH3 neurons in the zebrafish embryo \u2013 but only in those neurons that showed a mature tonic pattern of action potential firing. TN-GnRH3 neurons that showed the more immature bursting pattern of action potential firing were unresponsive to the stimulatory actions of Kiss1 kiss1, kiss2, and their receptors in adult zebrafish. The anatomical data suggests that Kiss2, and not Kiss1, is the primary regulator of the brain-pituitary-gonadal axis in zebrafish The physiology data in the present study is at odds with anatomical data showing localization of mRNAs for kiss1/kiss1r and kiss2/kiss2r in medaka indicated that kiss1 and both kiss1r and kiss2r play critical roles in neurulation and embryonic development, but not kiss2gnrh1 and gpr54-2 expression. However, during gonadal recrudescence, Kiss1 was more potent than Kiss2 in elevating plasma luteinizing hormone levels; while Kiss2 down-regulated gnrh1 and gpr-54-2 expression in uteroOverall, our findings that Kiss1 is the dominant stimulator of GnRH3-neuron morphological (present study) and electrophysiological development In summary, the present morphological findings in embryos show that kisspeptins modulate the development of multiple populations of GnRH3 neurons, suggesting it may play a role as a neurotrophic factor and possibly coordinate maturation of this neural network. The electrophysiology data in adult zebrafish suggests that both Kiss1 and Kiss2 regulate the activities of hypophysiotropic GnRH3 neurons. Kiss1 is an activator, while Kiss2 is an inhibitory RFamide. The functional balance of these two neuropeptides may be crucial in central control of reproduction in zebrafish."}
+{"text": "Atoh1 (Math1) was the first gene discovered in ear development that showed no hair cell (HC) differentiation when absent and could induce HC differentiation when misexpressed. These data implied that Atoh1 was both necessary and sufficient for hair cell development. However, other gene mutations also result in loss of initially forming HCs, notably null mutants for Pou4f3, Barhl1, and Gfi1. HC development and maintenance also depend on the expression of other genes  and several genes have been identified that can induce HCs when misexpressed (Jag1) or knocked out (Lmo4). In the ear Atoh1 is not only expressed in HCs but also in some supporting cells and neurons that do not differentiate into HCs. Simple removal of one gene, Neurod1, can de-repress Atoh1 and turns those neurons into HCs suggesting that Neurod1 blocks Atoh1 function in neurons. Atoh1 expression in inner pillar cells may also be blocked by too many Hes/Hey factors but conversion into HCs has only partially been achieved through Hes/Hey removal. Detailed analysis of cell cycle exit confirmed an apex to base cell cycle exit progression of HCs of the organ of Corti. In contrast, Atoh1 expression progresses from the base toward the apex with a variable delay relative to the cell cycle exit. Most HCs exit the cell cycle and are thus defined as precursors before Atoh1 is expressed. Atoh1 is a potent differentiation factor but can differentiate and maintain HCs only in the ear and when other factors are co-expressed. Upstream factors are essential to regulate Atoh1 level of expression duration while downstream, co-activated by other factors, will define the context of Atoh1 action. We suggest that these insights need to be taken into consideration and approaches beyond the simple Atoh1 expression need to be designed able to generate the radial and longitudinal variations in hair cell types for normal function of the organ of Corti. Pou4f3  were completely deaf, \u201cowing to a failure of HCs to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia\u201d  development and thus could be used to regenerate HCs and restore hearing was born in the late 1990s: Mice with a deletion of the Pou domain gene Pou4f3 relevant for the discussion of the role of Atoh1 (aka Math1) for HC differentiation and maintenance? In the following we will explore that Atoh1 has much in common with Pou4f3 in terms of claims raised as a gene that is \u201cnecessary and sufficient\u201d for HC differentiation  of the cerebellum that are unable to migrate and differentiate and eventually die  such as Ascl1, Neurog1 and, rarely, Atoh1  (Ono et al., In the original paper describing absence of HC differentiation in Atoh1 null mutant mice, some supporting cells stain for the LacZ used to replace Atoh1 (Bermingham et al., Atoh1 expression in inner pillar cells may be counterbalanced by Hes and Hey factors (Doetzlhofer et al., Neurod1 suffices to turn some neurons into HCs expressing Atoh1 and Myo7a (Jahan et al., Using the same LacZ knockin model as previous papers (Bermingham et al., Atoh1 generates a \u201cself-terminating\u201d system that results in loss of Atoh1 after a transient presence of Atoh1 protein (Pan et al., Atoh1 and how long residual Atoh1 protein remains in the cell. Thus, while all cells will see recombination of the LoxP flanked Atoh1, this varies between HCs and thus results in different delay lines of HC precursor apoptosis (Pan et al., Atoh1 is replaced by Neurog1 (Jahan et al., Atoh1. A recently available hypomorph mutant of Atoh1 shows a somewhat similar picture of longitudinal and a less clear radial HC loss (Sheykholeslami et al., More recent data provide yet a more complicated picture of lack of Atoh1 expression on HC and OC differentiation. Using an Atoh1 enhancer to drive Cre that activates the Cre only upon presence of Atoh1 protein combined with floxed Data using inducible Cre expression have complicated this picture even further by showing a rapid and complete loss of all HCs when Cre is induced at different stages of late development (Cai et al., In summary, Atoh1 is, much like Pou4f3, a critical factor for HC differentiation and long term maintenance. Atoh1 is involved in regulating Pou4f3 whereas and its long term expression may be dependent on Atoh1 expression. Further work combining the recently reported hypomorphic allele (Sheykholeslami et al., Why is it important to go beyond the idea of \u201cnecessary and sufficient\u201d for Atoh1 function in the ear? First, while unregulated expression of Atoh1 can convert most ear cells into hair cells (Kelly et al., Second, most HCs generated with Atoh1 treatment alone have limited long term viability. In part this may relate to the progressive loss of Atoh1 in these experiments that may needed to maintain long term Pou4f3 expression (Masuda et al., Finally, while the single gene approach to HC regeneration has been extremely influential to catapult much research forward, it is now time to reflect why this approach has not lived up to its promise. We therefore suggest more complex procedures that recapitulate steps in development of the OC in addition to Atoh1. For example, expressing Eya1, Pax2, Sox2, Jag1, Foxg1, Neurod1, Neurog1, and Gata3 prior to Atoh1 expression may \u201cprime\u201d remaining cells of the OC to respond to Atoh1. Alternatively, combining Atoh1 with downstream essential genes for HC maintenance that are only partially regulated by Atoh1 (Ahmed et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "TPVM is a promising technique for measuring myocardial mechanics  but longK-space is fully sampled with 8 spiral interleaves . Data is acquired at 2 levels of acceleration, acquiring 4 (R2) and 3 (R3) out of the 8 spirals in order to assess the effect of acceleration on the measured velocities. Velocity compensated and encoded data are acquired in consecutive heartbeats, with an initial heartbeat used to collect coil sensitivity information (breath-hold durations are 17 and 13 heartbeats for R2 and R3). Acquired spatial resolution is 1.7 \u00d7 1.7 mm (reconstructed pixel size 0.85 \u00d7 0.85 mm). Retrospective cardiac gating is used to cover the entire cardiac cycle (50 phases reconstructed). Images are passed to the Gadgetron for reconstruction and returned to the scanner for viewing within 1 min 20 s. Basal, mid and apical short-axis slices were acquired in 10 healthy volunteers on a Siemens Skyra 3T scanner.Example images and velocity maps for R2 and R3 are shown in Figure The use of spiral trajectories, non-Cartesian SENSE and the Gadgetron GPU reconstruction framework has allowed the acquisition of high temporal resolution TPVM images within a breath-hold time that is easily achievable in the clinical environment (13 heartbeats), while reconstruction time is short enough to allow viewing at the time of scanning. The acceleration is not affecting peak velocity measurements (comparisons with previous unaccelerated spiral data [NHIR CBRU, Royal Brompton Hospital. HRUK grant RG2584."}
+{"text": "Efficient right ventricular (RV) pumping function requires optimal blood flow dynamics. In the left ventricle (LV), diastolic vortex ring formation distal to the mitral valve (MV) has been reported to be an important mechanism for such blood flow optimization. Earlier work based on computational fluid dynamics (CFD) simulations using simplified RV geometry modeling have reported vortex ring formation in the RV during the early filling phase and its breakdown at the late diastolic phase. However, neither those CFD studies have characterized vortex rings nor have they been confirmed by 4D flow MRI. The purpose of this study was to investigate and characterize the formation of vortex rings during diastolic filling in the RV and to compare them with those of LV in healthy volunteers.Ten healthy volunteers (age: 20 \u00b1 7 years) underwent three-dimensional (3D), time resolved, three-directional velocity-encoded MRI at 3T (Philips). MRI was performed in a 3D isotropic dataset of 4.2 \u00d7 4.2 \u00d7 4.2 mm 3 with whole heart coverage. Retrospective gating with 30 phases reconstructed and velocity sensitivity of 150 cm/s in all directions was used. The Lambda2 (\u03bb2) method was used to extract the 3D vortex structures inside the RV at the phases of early (E) and late (A) filling. The most circular and compact ring was extracted from each phase. The location of a vortex ring was characterized by its longitudinal position (L) and its orientation : project number 11626."}
+{"text": "Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan\u00ae chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame ( Sesamum indicum L.) is one of the first oilseed plants to be used for many different purposes [Sesame  is a powerful alternative method for phytoplasma detection and quantification because it offers elevated detection sensitivity, short analysis time, and high automation capability . Real-tiSesame phyllody, an economically important disease of sesame plants, is a serious threat in regions where peanut witches\u2019 broom (16SrII) ,12,38,39Permissions from the owners of sesame fields for sampling were obtained. No endangered or protected species were involved in the study.Orosius orientalis was identified as the only insect vector of phyllody phytoplasmas in our previous study [Plant and insect samples were collected from naturally infected sesame fields in the Aksu, Aspendos, Batem, Belk\u0131s, Be\u015fkonak, Bo\u011fazkent, Bozova, Cumal\u0131, Denizyaka, G\u00fcndo\u011fdu, Kadriye, Kocayatak, Kovanl\u0131k, and Ta\u015fa\u011f\u0131l locations of Antalya province from June to September 2011 through 2014 . Additious study , the qPCus study ,44,45.Total DNA from samples of both sesame plant and vector insects was extracted by the CTAB method . A 100 m\u00ae qPCR assay. Representative sequences of 16Sr groups I through XIV [\u00ae were designed from the18S ribosomal DNA sequence (AJ236041) to detect sesame plant DNA in qPCR. This sesame DNA specific primer pair and probe were developed to serve as an endogenous control to normalize the DNA quantities and to allow for relative quantification of the phytoplasmas in infected plant tissue. The plant primer pair and probe combination were also used to verify the quality of extracted DNA and the absence of PCR inhibitors. Primer Express software  was used to design qPCR primers and probes. They were synthesized by Metabion International AG .Primers and probes designed in this study are listed in ough XIV ,48,49 , MA , ASHY2 , and AP15  were kindly provided by Dr. Assunta Bertaccini of the University of Bologna in Italy. Extracted DNA from phytoplasma samples was amplified with universal phytoplasma primers by nested PCR [Specificity of qPCR assay for 16SrII and 16SrIX group phytoplasmas was determined by testing against DNA from 4 phytoplasma samples representing 4 different 16Sr groups. The samples of phytoplasma strains PPT . qPCR was performed in 20 \u03bcl reaction volumes, containing 2XTaqMan PCR master mix , 300 nM forward and reverse primers, 150 nM 16SrII LC Red, 16SrIX Texas Red, and Sesame 18S-Hex labeled probes, and 2 \u03bcl of template DNA (10 ng per reaction). The thermal cycling protocol consisted of an initial denaturation at 95\u00b0C for 10 min, followed by 45 cycles at 95\u00b0C for 15 sec and at 60\u00b0C for 1 min. Cycle threshold (Ct) values were calculated and analyzed with the Rotorgene Q series software version 1.7 .\u00ae PCR purification kit  and quantified using a spectrophotometer. The copy number of PCR amplicons was calculated using the following formula: Copy number = (Amplicon amount x Avogadro's number) / (Amplicon size in bp \u00d7 650 x 1 x 109) [8 to 1.8 \u00d7 102 and 1.6 \u00d7 108 to 1.6 \u00d7 102 copies per reaction, respectively, was qPCR-amplified in triplicate to generate a standard curve for each phytoplasma group. qPCR assays were also performed to determine whether PCR inhibitors were present in DNA extracted from sesame plant tissues. Each dilution series of 16SrII and 16SrIX group DNA used for construction of phytoplasma standard curves was added separately to 2 \u03bcl (10 ng per reaction) sesame DNA from healthy tissue to produce the same final concentrations in each reaction and simulate proportions of naturally infected sesame tissue. Standard curves were also created from amplification of these samples containing 16SrII or 16SrIX group DNA and sesame DNA. Absolute quantities of 16SrII and 16SrIX phytoplasma DNA in infected sesame plant and insect tissues were calculated based on standard curves using comparative cycle threshold (Ct) values of sesame field test samples.qPCR assays were used to determine both absolute and relative quantities of 16SrII and 16SrIX group phytoplasmas in sesame plant samples and absolute quantities only in insect samples. DNA extracted from sesame phyllody plant and insect samples was added to a multiplex qPCR reaction mix and amplified to determine the amounts of each phytoplasma in field samples. Negative control (uninfected sesame checked by nested PCR), and non-template control (PCR water) were included in each independent run. Standards were prepared by amplifying 16S rRNA sequences of 16SrII and 16SrIX group sesame phyllody phytoplasmas directly with the primer pair Fu5/Ru3 from DNA extracted from sesame samples infected with only the respective 16Sr group phyllody phytoplasmas in conventional PCR ,45. AmplTo measure relative quantities of 16SrII and 16SrIX group phytoplasma DNA in sesame tissues, relative qPCR using the comparative Ct method was used as described in Rotor-Gene Q series user bulletins . Ten ng of total DNA extracted from field collected sesame tissues was simultaneously amplified with both phytoplasma specific and endogenous control primers (18S rDNA) in the same tubes. The lowest detectable dilutions of 16SrII and 16SrIX determined by inhibition assays were designated as calibrators for phytoplasma group quantification experiments. The quantities of calibrators were regarded as 1 and the quantities of all the other field samples were expressed as n-fold difference relative to the calibrators. Absolute quantities calculated from standard curves were omitted in relative quantification because sample quantities were divided by the calibrator quantity. Quantities of 16SrII and 16SrIX group DNA normalized to the endogenous control sesame DNA and relative to the calibrators were either calculated automatically by the software or manually .\u00ae Green  revealed that the primer pair SPHY-16SrII-IX-F/SPHY-16SrII-IX-R amplified a 136 bp PCR product from all 16Sr group phytoplasma sequences tested  of 16SrII and 16SrIX purified PCR products, and sesame DNA to detect sensitivity limits of the qPCR assay . The inibination . Linear bination .4 pg of DNA. Addition of sesame SEPL-18SDNA-F/R primers and the SESAME probe into phytoplasma primers and probes did not affect specificity. The slope, intercept, correlation coefficient, and efficiency values of standard curves for all assays are given in Internal control primer pair efficiently amplified sesame plant DNA through at least five orders of magnitude of 10-fold dilutions beginning with 1.0 \u00d7 10A qPCR reaction combination of phytoplasmas and sesame primers and probes was used to amplify serial DNA dilutions of the target phytoplasma group (16SrII or 16SrIX) DNA in the presence of sesame plant DNA in inhibition tests. The multiplex assays successfully amplified the same seven 10-fold target 16SrII and/or 16SrIX DNA dilutions and resulted in consistent Ct values through 30 cycles . The sta2 and 1.6 \u00d7 102 copies per reaction, respectively. Amplification efficiencies of the 16SrII (0.98) and 16SrIX (1.0) assay in the presence of sesame DNA (Detection limits of the multiplex qPCR assay for 16SrII and 16SrIX group phytoplasmas in the mixed phytoplasmas and sesame DNA were found to be 1.8 \u00d7 10same DNA  were fousame DNA .DNA extracts from 109 plant and 92 insect samples collected from 15 different locations were tested for the presence of sesame phytoplasmas in multiplex qPCR developed in this study . Amplifi7), had the highest and Bogazkent (1.12 \u00d7 106) lowest absolute quantities (copies) of 16SrII DNA per mg of plant tissue. In relative quantification, the highest and lowest amounts of 16SrII phytoplasma DNA were from the Gundogdu and Belk\u0131s district samples (6) contained the highest amounts of 16SrIX phytoplasma per mg of sesame tissue.Absolute and relative quantities of 16SrII and 16SrIX phytoplasma DNAs were determined for the 94 samples from which sesame phytoplasmas were detected . The sam samples . Absolut5) per mg. Samples from other districts contained 16SrII DNA ranging from 4.26 \u00d7 104 to 4.97 \u00d7 103 copies of DNA per mg insect tissue. Insect samples from the campus greenhouse had high 16SrII DNA quantities (1.63 \u00d7 107 copies per mg of insect tissue) because these insects were continuously fed on sesame plants with phyllody disease in cages for transmission assays.Quantities of 16SrII DNA were also found to be quite low in insect samples. Of all districts sampled, insect samples from Kovanl\u0131k had the highest quantities of 16SrII DNA Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigSpecific detection of 16SrII (A) and 16SrIX (B) group phytoplasmas and housekeeping gene (C).(DOCX)Click here for additional data file."}
+{"text": "Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane\u2013cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in  Arabidopsis thaliana mutational analysis, CESAs are not assumed to work alone. Some proteins were predicted to be associated with the cellulose synthase complex, although their direct interaction has never been demonstrated. One of these proteins is KORRIGAN (KOR1), a membrane-bound endo-1,4-\u03b2-D-glucanase with a single transmembrane domain and two putative polarized targeting signals in the cytosolic tail Pichia pastoris cleaves non substituted but non-crystalline 1,4-\u03b2\u2013linked glucan chains and shows no activity against xyloglucans Agrobacterium tumefaciens and Acetobacter xylinumCellulose is synthesized by a large rosette terminal complex, which comprises at least three different cellulose synthases (CESAs). On the basis of KOR1, two additional genes encoding membrane-anchored endoglucanases (EGase) have also been characterized in A. thalianaKOR1 gene is the most widely expressed membrane-anchored EGase throughout various plant tissue while KOR2 and KOR3 are active in specific cell types. Expression of a GUS reporter gene driven by the endogenous promoters of KOR2 and KOR3 have shown that KOR2 is active in trichomes and floral organs while KOR3 is active in developing root hairs. Microsomal fractioning demonstrated that KOR1 is present in the tonoplast, the Golgi apparatus Besides korrigan (kor1-1) showed defects in some aspect of cell wall loosening in primary cell wall biosynthesis KOR mutations (kor1\u20132) have shown to cause the formation of aberrant cell plates and incomplete cell walls acw1 altered cell wall and rsw2 root swelling) showed abnormal plant morphology, defects in primary cell wall formation, reduced cellulose content, increased pectin synthesis, and aberrant cell division similar to that found in the CESA1 mutant rsw1acw1 mutant grown at 31\u00b0C and shows a 40% reduction in crystalline cellulose content compared to the wild type rsw1) and KOR (rsw2) in comparison to either of the single mutants which demonstrates that cellulose biosynthesis in the primary cell walls of plants requires both a glycosyl transferase and glycosyl hydrolase providing further evidence that KOR has a key role in cellulose deposition in the primary cell wall The dwarf mutant KOR homolog was observed during secondary cell wall deposition in cotton irx2-1 and irx2-2) in the KOR1 genes showed similar phenotypes as the irx mutants, i.e. collapsed xylem cell walls due to reduced cellulose synthesis in the secondary cell wall in the base of mature stems kor1-1 mutants that were reported to be primary cell wall mutants also had severely collapsed xylem cells similar to the irx2 mutants KOR genes from hybrid poplar led to moderate to severe defects in plant growth, an irregular xylem (irx) phenotype commonly associated with KOR and other secondary cellulose-specific mutants in A. thalianaReports have indicated that KOR also plays a role in secondary cell wall development. An accumulation of a Since KOR appears to be associated with cellulose synthesis and direct association has been detected with the primary cellulose synthase protein complex The constructs for the MbYTH system concerning the primary and secondary CESAs were generated as described previously Saccharomyces cerevisiae in the Split Ubiquitin System kit . Interactions were performed according to supplier instructions (DUAL membrane Kit 1) and were tested with KOR1 fused to the C-terminal part of the ubiquitin (Cub) and the transcription factor (bait), whereas the CESA1, 3 and 6 proteins were fused to the N-terminal part of the ubiquitin . The bait and prey constructs were co-transformed into the yeast strain NMY51  according to the provided transformation procedure (DUAL membrane Kit 1). Upon interaction between the bait and the prey the transcription factor (TF) is released into the nucleus where it activates reporter genes allowing the yeast to grow on selective medium lacking leucine and tryptophan (SD med. -L-T), and subsequently grown at 30\u00b0C for three days. To quantify the interactions between different preys 100 colonies of each combination were spotted onto selection medium containing the appropriate amount of 3-ammonium-triazole (3-AT) and grown at 30\u00b0C for three days. The number of spots grown was then counted.The interactions between the CESAs and KORRIGAN1 were assayed with the split-ubiquitin membrane-based yeast two-hybrid CESA genes, KORRIGAN1 as well as the truncated forms of KOR1 were generated through Phusion DNA Polymerase  with suitable primers  have been demonstrated irx) mutants also link KOR1 to cellulose synthesis in the secondary cell wall. To test a possible interaction between KORRIGAN and the secondary CESA proteins  the KOR1 was expressed as bait in combination with the secondary CESA as prey. As a negative control, we tested KOR1 as bait with an unrelated protein AGL5 as prey . The yeast strains failed to grow in multiple repetitions of this experiment, demonstrating that a specific interaction between the bait and prey is required to activate the system , and an extracellular catalytic domain . In ordein vitro . Vice vein vitro .in planta, truncated forms of KOR1 were tested for interaction with the primary  and secondary  CESAs using the BiFC assay. All the primary CESAs, CESA1, 3 and 6, were able to interact with KOR1N and KOR1C truncated proteins  and also with CESA4 and CESA8 from the secondary cell wall  and secondary  CESAs using the BiFC assay. Confirming the in vitro assay results, the primary CESAs, CESA1, 3 were able to interact with the truncated protein KOR1TMD  .in planta. Our results showed that fluorescence was restored when two different fusion proteins (YFP/C-KOR1 and YFP/N-KOR1) were expressed in Nicotiana benthamiana, indicating the formation of homodimers or higher oligomers of KOR1 proteins  only two of the secondary CESAs (CESA4 and 8), suggesting possible specificity for the different CESAs from the primary and secondary cell wall.KORRIGAN1 has shown to play an important role in cellulose biosynthesis as the knockouts out of this gene results in reduced cellulose content. Despite previous studies indicating that some primary CESAs do not co-immunoprecipitate with KOR1 kor1-1 dwarf mutant korrigan, (kor1-1) phenotype results in a severe reduction in crystalline cellulose both in the primary and secondary cell wall kor1-3 and kor1-1 mutants than in controls acw1 mutation effects cellulose accumulation in the cell wall and microfibril formation resulting in 40% reduction in crystalline cellulose content compared to wild type plants.The alterations in cellulose content in the kor1-1 mutant that showed distinct primary cell wall defects also showed signs of severely collapsed xylem similar to the irx2 mutants in the secondary cell wall in vitro and in planta we demonstrate that there is indeed a physical interaction between KOR1 and the CESAs in the secondary cell wall except for CESA7. Other reports have also indicated that KOR plays a role in secondary cell wall development. Co-expression of Populus KOR orthologue with the three secondary cell wall CESA proteins has also been reported Atkor1 orthologue in hybrid aspen (PttCel9A1) causes a decrease in cellulose crystallinity irx2-1 and irx2-2 KOR1 mutants showed reduced cellulose synthesis (30% of the WT) in the secondary cell wall irx mutants. PtKOR1 is also shown to be required for secondary cell wall cellulose formation. RNAi suppression of PtKOR1 revealed a significant decrease in crystalline cellulose content and a reduced secondary cell wall thickness It must also be mentioned that the Interestingly, not all the CESAs in the primary and secondary cell wall have a similar interaction pattern with KOR1. The different reaction between the primary and the secondary cell wall proteins is difficult to explain as the specific functions of the different CESA proteins are not known. As previously mentioned CESA1 was the most efficient interactor in the primary cell wall whereas CESA7 from the secondary cell wall did not interact with KOR1 suggesting that specific CESA isoforms may have unique roles in recruiting KOR1. In other words the binding of KOR1 to the different CESA proteins is specific, as KORRIGAN1 does not bind to all of them. Not only does this imply that the methods used are sensitive enough to specifically determine interactions between these highly homologous proteins, it also indicates that the KOR1 protein has a specific position within the rosette.in vitro, CESA6 and CESA4 show a very weak to no interaction with KOR1TMD in planta suggesting that at least for the two mentioned CESAs the presence of TMD is essential but not sufficient for interaction and would require more than just the TMD. It is interesting to note that some proteins required for cellulose synthesis are found in sterol-rich lipid rafts A more detailed view on the interaction between KOR1 and the CESA proteins using truncated versions of KOR1 revealed that all portions which contain the TMD were able to interact and this led to the conclusion that KOR1 transmembrane domain is required for the interaction with CESAs. These data suggest that KOR1 and CESA associate in the CSC in the plasma membrane, between the TMD of KORRIGAN and the TMDs of the CESA proteins. Surprisingly, despite showing interaction in vitro and in planta supporting the hypothesis that KOR1 makes up a part of the rosette structure. The physical interaction also indicates that KOR1 is directly involved in cellulose biosynthesis, and probably does so in the form of a homodimer or a higher order oligomer. Furthermore, our study showed that the TMD of KOR1 is essential not only in the interaction with the different CESA proteins, but also for KOR1 homodimerization.In conclusion, we have determined that the KOR1 protein interacts with secondary cellulose synthase complex proteins both Figure S1Interactions between the different KOR1 domains and the different CESA proteins using the Membrane-based Yeast Two Hybrid. The bars represent the percentage of yeast colonies grown for 3 days on selective medium at 30\u00b0C. The different CESA proteins were expressed in yeast as prey and the different KOR1 protein domains as bait.(PDF)Click here for additional data file.Figure S2Bimolecular Fluorescence Complementation (BiFC) experiments in tobacco leaf epidermis. Confocal images are presented, showing YFP fluorescence indicating interaction. Tests for interactions between the CESAs and truncated versions of KOR1 are shown (A) KOR1N/CESA1, (B) KOR1C/CESA1, (C) KOR1TMD/CESA1, (D) KOR1N/CESA3, (E) KOR1C/CESA3, (F) KOR1TMD/CESA3, (G) KOR1N/CESA6, (H) KOR1C/CESA6, (I) KOR1TMD/CESA6, (J) KOR1N/CESA4, (K) KOR1C/CESA4, (L) KOR1TMD/CESA4, (M) KOR1N/CESA8, (N) KOR1C/CESA8, (O) KOR1TMD/CESA8, (P) KOR1C/CESA7, (Q) KOR1N/CESA7, (R) KOR1TMD/CESA7. Scale bars\u200a=\u200a100 \u00b5m.(PDF)Click here for additional data file.Table S1Primers used for the MbYTH system.(PDF)Click here for additional data file."}
+{"text": "YHWAE with NUTM2B was identified. Integrated analysis of expression and methylation data identified promoter hypermethylation and low expression of the tumor suppressor gene TCF21 (Pod-1/capsulin/epicardin) in all CCSKs except the case with t. TARID, the long noncoding RNA responsible for demethylating TCF21, was virtually undetectable in most CCSKs. TCF21 hypermethylation and decreased TARID expression were validated in an independent set of CCSK tumor samples. The presence of significant hypermethylation of TCF21, a transcription factor known to be active early in renal development, supports the hypothesis that hypermethylation of TCF21 and/or decreased TARID expression lies within the pathogenic pathway of most CCSKs. Future studies are needed to functionally verify a tumorigenic role of TCF21 down-regulation and to tie this to the unique gene expression pattern of CCSK.Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants  were identified. One tumor with t involving fusion of  Clear Cell Sarcoma of the Kidney (CCSK) comprises approximately 5% of all renal malignancies in children, and is observed most often below 3 years of age , 2. CCSKYWHAE intron 5 (17p13) and NUTM2 intron 1 (10q22), resulting in fusion of YWHAE exon 5 to NUTM2 exon 2  . cD. cDTARIDYHWAE exon 5 and NUTM2 exon 2 [RT-PCR analysis was performed using previously reported primers for 2 exon 2 . Briefly"}
+{"text": "Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for \u03b2III-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube  Genetic reprogramming offers unprecedented opportunities for regenerative medicine . Geneticex vivo; clonal analysis of independent amniocentesis samples indicate that the vast majority of cells do not proliferate , 1% L-glutamine and 1% penicillin/streptomycin solution). Cells were routinely maintained on culture wares pretreated with 1:100 dilution of growth factor reduced matrigel (BD Biosciences). All media components in this work were obtained from Life Technologies unless stated otherwise.5 cells/cm2 for immediate use or cryropreserved with standard methods after 24 h recovery. Conditioned hESC media was prepared by culture of inactivated MEFs in hESC media without bFGF for 24 h, supplemented with 4 ng/ml bFGF and filtered sterilized before use. Feeder-free cultures were maintained in MEF-conditioned hESC media, mTeSR-1 (StemCell Technologies) or Essential 8 (Life Technologies) media. Passaging of PSCs cultured on MEF feeders or in MEF-conditioned media was done by manual microdissection of optimal undifferentiated colonies with a fire-polished glass pipette using a dissecting microscope. Feeder-free cultures were passaged with EDTA as described (The H9 (WA09) line of human embryonic stem cells (hESCs)  and iPSCescribed . The ROC5 target cells were seeded at subconfluent densities \u223c1.4 \u00d7 103 cells/cm2 and transfected the following day with pooled plasmid combinations in equimolar ratios (\u223c0.2 \u00b5g DNA/cm2) with Fugene HD  0.15 \u00b5l/\u00b5g DNA at 8 to 12 h intervals for a total of 3 transfections. Transfected cells were maintained in DMEM 15% for \u223c4 days and then switched to MEF conditioned hESC media supplemented with 2.5 mM valproic acid (Sigma-Aldrich) for \u223c2 weeks after colonies appeared. Independent populations of ChMRC.B1p3 cells were transfected with the 3-vector combination and 7 to 9 colonies were recovered from each population. A single representative colony was selected from each and maintained separately as iChMRC.B1A, iChMRC.B1C, and iChMRC.B1E candidate populations. A population of ChM5p10 cells was transfected with the 2-vector combination, but the population became highly confluent in hESC media within 2 weeks and potential colonies were difficult to identify. The transfected population was passaged with Accutase and replated on MEF feeders. hESC-like colonies emerged within 2 weeks, optimal colonies were pooled and maintained as the iChM5A candidate population. Transfected ChM5p12 cells were maintained for 4 days in growth media, treated with Accutase and passaged to MEF feeders as separate populations; a single hESC-like colony was recovered from one population of transfected cells and maintained as the iChM5B candidate population. Optimal hESC-like candidate colonies and control H9 hESC colonies were passaged as needed to maintain healthy cultures.The episomal vectors  that were used in this work are described in Following the first manual passage of candidate colonies from MEF feeders, residual colony fragments in the primary culture plate were maintained in conditioned hESC media without bFGF for 3 to 5 days to allow colony expansion and then switched to regular hESC media to encourage spontaneous differentiation as the MEF feeders age and pluripotency of the expanding population by the absence of bFGF in hESC media. Rosettes were manually isolated as they emerged and passaged in hESC media to matrigel-treated cover slips for immunostaining. Long term cultures of neural progenitors/stem cells (NSPs) were established as described ; neural Total cellular DNA was isolated with GenePure (Qiagen) or QiaAmp DNA Mini (Qiagen) kits and treated with RNAse to remove RNA. Transgenes or endogenous genes were amplified in reactions containing 100 ng genomic DNA or <1 ng plasmid DNA with GC-rich polymerase (Life Technologies) in 1X Buffer A, 3 \u00b5l of Enhancer and 250 nM of oligonucleotide primers  with tou\u00ae Gene Expression Master Mix (Life Technologies). Amplification rates  of cDNA were assayed in more 2 replicates for each gene. The mean (AVG) and standard error (SE) was calculated with the Descriptive Statistics tool in Excel and normalized to \u03b2-glucuronidase (GUSB) with the \u0394\u0394Ct method described in Applied Biosystems/Life Technologies technical resources.Total cellular RNA was isolated with RNAeasy kits (Qiagen) and contaminating DNA was removed by DNAse treatment. RNA was converted to cDNA using SuperScript First-Strand Synthesis System (Life Technologies) and 1 \u00b5l of 1:4 dilution of cDNA in water was amplified in each reaction. Transcript levels in http://dx.doi.org/10.6084/m9.figshare.1153969.Genomic DNA was processed with an Epitect kit (Qiagen) as directed by the vendor. Amplification products were generated with primers that were specific to converted DNA , purifieCells were cultured in multiwell tissue culture plates on cover glass or in multiwall chamber slides that were pretreated with 1:100 dilution of growth factor reduced matrigel. Samples were fixed and immunostained as described  with antAmniotic cell populations were derived from amniocentesis samples  that werReprogramming targets were selected to reflect the range of cell types in amniocentesis samples and proliferation characteristics that we considered to be important to the efficiency of reprogramming. The ChM5 mixed cell population was highly enriched for fibroblast-like stromal cells and cell proliferation continues in confluent cultures, generating dense stratified cell layers . The ChMReprogramming used combinations of 2 or 3 first generation episomal vectors  that colImmunostaining of transfected populations showed a high frequency of Oct4 positive cells following transfection, but the loss of virtually all immunopositive cells by passage 5 . Haemacy5 cells were serially transfected every 8 to 12 h for 3 transfections in order to increase the number of transfected cells. ChMRC.B1p6 cells were transfected with the 3-vector combination . Cells are fixed and prepared for digital analysis of chromosomes in mitotic figures and recognition of chromosomal abnormalities with suitable software. The results of karyotype analysis here indicated that early passage iChM5Ap14 and iChM5Bp14 cells had a normal female  karyotype without chromosomal abnormalities at a detection limit of 5 Mb  expressed Nanog and Sox2 as well as Oct4. Dual labeling showed that nuclear localized Nanog was correlated with nuclear localized Oct4 (n > 500), with and without colocalized Oct4 and Nanog expression, expressed Sox2  in iChM5A and iChM5B cultures  as a stromal cell control  of the parental ChM5 mixed cell pool is unknown and cannot be tested directly, amniotic stromal and epithelial cells alike show stromal cell traits . Stromal control . TranscrThe promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources, in part because immunologically compatible iPSCs from allogenic sources is the more likely path for clinical applications . Particiin vivo  and can provide critical internal controls in the same culture and within the same field of view. Immunostaining in this case is superior to flow cytometry that cannot discriminate between nuclear and cytoplasmic localization of transcription factors or easily correlate gene expression and changes in cell morphology in differentiating cells.The value of neural rosettes in candidate selection was substantiated by subsequent validation of pluripotency of iChM5 derivatives, including evidence for epigenetic modification of chromatin structure  as expecin vivo  iChM5Ap4-derived rosettes and NSPs. Dissociated rosettes from candidate colonies were immunostained as indicated. Rosette immunostained for nestin is indicated by asterisk (*).  Scale bar, in microns as indicated.(A). Click here for additional data file.10.7717/peerj.668/supp-2Figure S2Phase images of NSPB6p12 showing early stage differentiation by withdrawal of mitogens in confluent culture in top image. Middle and bottom images show induced differentiation of NSPB6p12 cells and control hVMNSPs, respectively, at day 7. Representative of presumptive axonal extensions are indicated by arrows. Scale bar, in microns as indicated.Click here for additional data file.10.7717/peerj.668/supp-3Figure S3This image show immunodetection of Sox2 in a subset of HEK293 cells that were transfected with the 2 vector combination of episomal plasmids. This image shows the vast majority of cells did not show SOX2 expression, likely because they lacked an episomal vector and did not express an endogenous genes.Click here for additional data file."}
+{"text": "Due to concerns raised about Fig 4 and Fig 6, the authors would like to publish the raw, uncropped film for Fig 6 as well as an alternate experiment for Fig 4 which verifies the conclusion of the published image.The authors and editors confirm that these changes do not alter their findings.S1 FileThis file includes the alternate experiment for Figure 4.(PDF)Click here for additional data file.S2 FileThis file includes the original autorad for Figure 6.(JPG)Click here for additional data file."}
+{"text": "Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (\u226510 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of  Blood flow is an important biological regulator and initiates and maintains many events during embryonic development. Shear stress, a mechanical force created by blood flow, is an important factor regulating many physiological functions. Although there is expression of arterial and venous specific genes in the vasculature before the onset of flow , 2, therNotch and Dll4 expression and initiate arterial differentiation. In regions of low VEGF concentrations, however, venous identity prevails [Dll4 and Hey1 are expressed in an arterial specific manner before the onset of flow [Genetic predetermination of arterial identity has been shown to occur through the Notch signaling pathway. Notch receptors and ligands are involved in a plethora of developmental processes including somite coordination , 8, cardprevails , 16. In  of flow . Dll4 isNotch1 just after the onset of circulation in mouse embryos. We found Notch1 expression begins with the onset of flow but is pan-endothelial at the stage where erythroblasts enter circulation. Only by E9.5, after vascular remodeling has occurred, is Notch1 restricted to arteries. Using a technique we previously developed [Notch1 expression was arterial after culture while no expression in either arteries or veins was observed in the embryos with ablated flow, indicating that Notch1 requires flow to be expressed. We investigated the patterns of flow that could regulate Notch1 expression and found the largest increase occurred at low levels of laminar shear stress (1\u20135 dynes/cm2) and with oscillatory flow types (either 0 \u00b1 3 or 2 \u00b1 3 dynes/cm2). Not only could Notch1 expression be upregulated by flow, but the expression of Notch ligands and transcription factors were also induced by exposure to shear stress. We therefore knocked down Notch1 and investigated the effect on flow-induced expression of Notch signaling molecules as well as typical shear-induced genes and downstream targets in arterial-venous differentiation. Flow could induce the expression of approximately half the genes investigated, even in the absence of Notch1. Overall, our results indicate that Notch1 expression occurs after the onset of flow and does not become arterial-specific until remodeling has occurred. While Notch1 is required for the mechanotransduction of some genes, our results indicate that it is not an essential component of mechanotransduction since the flow-induced expression of many genes is not affected by Notch1 knockdown.In this work, we have investigated the expression of eveloped , we ablaIn situ hybridization has been described previously [Notch1 [Notch4 [Hey1 [All procedures were approved by the Animal Care Committees of McGill University and we have followed recommendations of the Canadian Council on Animal Care. eviously  with pro [Notch1 , Notch4  [Notch4 , and Heyh4 [Hey1 . Antibodh4 [Hey1 . BrieflyHuman abdominal aortic endothelial cells  were propagated through passage 5 in complete endothelial cell growth medium MV (PromoCell) supplemented with 1% Penicillin Streptomycin. Cells were expanded on 0.1% gelatin. For flow experiments, HAAEC were seeded at a density of 150 000 cells/mL on a culture slide, coated with 4% rat tail collagen type 1 and grown to confluence (3 days).2 and oscillatory shear stress of 0 to 5 \u00b1 3 dynes/cm2 as indicated. Static slides were cultured in parallel. After 1 hour, total RNA or protein was isolated.A parallel plate flow chamber was designed in-house. The parallel plate flow chamber was connected to a closed-loop perfusion system consisting of a vented media reservoir, a flow dampener, a peristaltic pump and/or a computer-driven syringe pump. The viscosity of the endothelial cell media was measured using a Bohlin CVO 120 HRNF Viscometer  to be 1.02334 cP. Laminar flow was verified by seeding the perfusate with fluorescent microparticles and imaging with a high-speed camera on a fluorescent microscope. HAAEC were exposed to laminar flow at a calculated wall shear stress of 1 to 15 dynes/cm16 primers (Applied Biosystems), and M-MLV reverse transcriptase (Invitrogen). Gene expression was analyzed on an ABI Prism 7900HT Sequence Detector with SA Biosciences RT2 Real-Time SYBR Green master mix (Qiagen). Quantitect Primer Assays (Qiagen) were used for HPRT (QT00059066), 18S (QT00248682), Dll1 (QT00057631), Nrp1 (QT00023009), VEGFR2 (QT00069818) and Notch1 (QT01005109). All other primers were designed in-house . RNA concentration and purity was quantified on a NanoDrop. 1 \u03bcg of RNA was reverse transcribed using Oligo-d(T)in-house . The datNotch1 siRNA (SI00119035) and AllStars Negative Control siRNA (1027280) were obtained from Qiagen. 10nM siRNA diluted in 100\u03bcl OptiMem serum-free medium (Invitrogen) with HiPerfect Transfection Reagent (Qiagen) was added to 150 000 cells pre-seeded on a collagen-coated culture slide in 1mL of OptiMem without Penicillin Streptomycin. After 1h of transfection, 1.3mL complete endothelial cell growth medium was added to each slide and incubated for 48 hours. Transfection media was then replaced with 2mL of complete endothelial cell media and the samples incubated for another 24 hours before performing experiments. qPCR and Western Blot was used to measure knockdown. For western blot, primary antibodies for Notch1 were obtained from EMD Millipore , glyceraldehyde phosphate dehydrogenase (GAPDH) from Santa Cruz Biotechnologies .Data are presented as the mean \u00b1 standard error of the mean (SEM). Results were analyzed using ANOVA with Tukey\u2019s post hoc test.Notch1 expression is absent at 5 somites, before the onset of blood flow , expression is the same as static control (2) in which a complete reversal of flow direction is present; oscillatory flow with a slight reversal present (2 \u00b1 3 dynes/cm2) and purely pulsatile flow with no retrograde component (5 \u00b1 3 dynes/cm2). We find that both flow types in which flow reversal is present (0 \u00b1 3 and 2 \u00b1 3 dynes/cm2) induce an upregulation of Notch1, but that pulsatility without flow reversal does not , which corresponds to the average level of shear stress in the embryo at the onset of circulation but is also a level of shear stress in which we observed increased Notch1 expression. Endothelial cells were exposed to flow for one hour. In the presence of control siRNA, exposing endothelial cells to laminar shear stress led to an upregulation of the genes Klf2 and VEGFR2. The knockdown of Notch1 alone had no effect on the expression of these genes in static conditions, nor did it prevent the shear stress-dependent increase in Klf2 expression. However, shear stress failed to upregulate VEGFR2 by shear stress when Notch1 was knocked down.The role of Notch1 in mechanotransduction has been studied surprisingly little. Shear stress induces Notch cleavage in embryonic stem cell-derived VEGFR2+ cells . In zebr+ cells . Other tDll1, in response to flow. A positive feedback loop for amplification of Dll1 by Notch signaling has not been shown unlike the other ligands tested. We also investigated whether Notch4 would compensate for the loss of Notch1. No changes in Notch4 expression are observed.Masumura et al. reported that Notch intracellular domain (NICD) is cleaved by shear stress . NICD inNrp1, EphrinB2) and two venous genes (EphB4 and CoupTFII) gave the clearest results  and 5 dynes/cm2  induced Notch1 expression to the same extent, explaining why expression is present in both vessels at the onset of flow. Our results also show that the onset of circulation is necessary for this expression to occur. The results are, however, contrary to what has been reported in the zebrafish where ablation of flow resulted in an increase in Notch signaling due to an upregulation of Dll4 [VEGFR2 (KDR) and ephrinB2 expression are not affected by the ablation of flow and that VEGFa expression is inhibited by flow. This is contrary to what is observed in mammalian systems, by us and by many others, where VEGFR2, VEGF and EphrinB2 are all upregulated by flow [Notch1 expression by flow is dependent on the magnitude and type of flow that is present. The levels and patterns of hemodynamic stress present in the zebrafish when circulation begins are likely to be very different than in mammalian embryos.We found that flow was required for the expression of Notch1 during development, but that adult artery physiological levels of flow do not induce Notch1 expression. Embryonic shear stress levels can go as high as 5 dynes/cmand E9.5 , 27), anand E9.5 . Given tand E9.5 , we woul of Dll4 . The res by flow , 42\u201344. in vitro results show that adult artery physiological shear stress levels do not induce Notch1 expression, implying that the continued expression in arteries is not dependent on Notch1 induction by shear stress. Activation of Notch receptors induces a positive feedback loop resulting in the expression of Notch ligands [Notch1 but sustained signaling from flow may not be required once expression has been initiated.Our  ligands , 31. Simin vivo work shows a decrease in Nrp1 expression in the dorsal aorta of Notch1 knockout mice [Nrp1 expression with Notch1 knockdown in vitro. We achieved a 70% knockdown of Notch1 in HAAEC, and residual expression of Notch1 in the endothelial cells might account for this variation. We find that Nrp1 expression remains unchanged after cells with Notch1 siRNA are exposed to laminar flow. This indicates that although Nrp1 and VEGFR2 are upstream of Notch1 in the VEGF-signaling pathway [Notch signaling plays a crucial role in arterial and venous differentiation during development. Although earlier out mice , we obse pathway , there mDll1, Jag1 and possibly Cx40) as well as venous-specific genes (CoupTFII), and others (KLF2). Hence, our results show that Notch signaling can modulate how flow affects endothelial cells, but it does not appear to be an indispensable protein in the process of mechanotransduction.Though many genes involved in arterial-venous differentiation failed to be upregulated by flow after the knockdown of Notch1, there are exceptions both within the set of arterial specific genes ("}
+{"text": "The data information provided in this article relate to our research article \u201cUsing patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts\u201d   Specifications TableValue of the data\u2022Better understanding of soy storage protein allergens may contribute to allergy management strategies.\u2022It may also contribute to the generation of hypoallergenic soybean cultivars.\u2022Provide risk assessment tools for the evaluation and characterization of the allergenicity of novel foods.1The data presented here show the western blot detection of A2 or A3 subunits by soy-sensitive human sera  and ELIS22.1Soy-sensitive human sera used in the western blots and epitope mapping are previously described 2.2Western blotting of human sera was conducted as previously described 2.3http://www.mimotopes.com) via parallel array platform. Quality Control Assurance was provided for both peptide synthesis and biotinylation by reverse phase HPLC (RP-HPLC), and by mass spectrometry (MS) respectively. The biotinylated 12-mer peptides, frame-shifted by three residues were used as per manufacturer\u05f3s instructions (Application/Method PT3013). DMSO was used to resuspend the dry peptides and streptavidin-coated high capacity plates (Pierce #15500) pre-blocked with SuperBlock\u2122 buffer were used to capture the biotinylated peptides. Serum was diluted at 1/50 in TBS-BSA 2% except for Patients 4 (1/100) and 5 (1/50 or 1/100). The secondary mouse anti-human IgE-HRP  was diluted at 1/4000 in TBS-BSA 2%. SureBlue Reserve TM TMB microwell peroxidase substrate  was added to the plate, the reaction was stopped by acidification and colorimetric detection was performed on a Tecan Sunrise microplate reader with Magellan\u2122 data analysis software  at 450\u00a0nm. Each experiment was performed in duplicate. Negative controls were performed using the same protocol, but the addition of human sera was omitted. The data was normalized by calculating the ratio of experimental to negative control and graphed.Two peptide sets representing the mature amino acid sequences of glycinin A2  and A3  were synthesized and biotinylated by Mimotopes  using fixed length patterns"}
+{"text": "EXP50) and 95% (CEXP95) exposure levels. Benzene levels were estimated to pose a significant risk with HQ50 > 1 and HQ95 > 1 for workers exposed to benzene as base estimates for petroleum refinery workers (Scenario 1), petroleum refinery workers evaluated with personal samplers in Bulgarian refineries (Scenario 2B) and evaluated using air inside petroleum refineries in Bulgarian refineries (Scenario 3B). HQ50 < 1 were calculated for petroleum refinery workers with personal samplers in Italian refineries (Scenario 2A), air inside petroleum refineries (Scenario 3A) and air outside petroleum refineries (Scenario 4) in India and Taiwan indicating little possible adverse health effects. Also, HQ95 was < 1 for Scenario 4 however potential risk was evaluated for Scenarios 2A and 3A with HQ95 > 1. The excess Cancer risk (CR) for lifetime exposure to benzene for all the scenarios was evaluated using the Slope Factor and Overall Risk Probability (ORP) methods. The result suggests a potential cancer risk for exposure to benzene in all the scenarios. However, there is a higher cancer risk at 95% (CEXP95) for petroleum refinery workers (2B) with a CR of 48,000 per 106 and exposure to benzene in air inside petroleum refineries (3B) with a CR of 28,000 per 106.The health risk resulting from benzene exposure in petroleum refineries was calculated using data from the scientific literature from various countries throughout the world. The exposure data was collated into four scenarios from petroleum refinery environments and plotted as cumulative probability distributions (CPD) plots. Health risk was evaluated for each scenario using the Hazard Quotient (HQ) at 50% (C Petroleum refineries and petrochemical plants are major sources of Volatile Aromatic Hydrocarbons (VAHs) in the environment . BenzeneWhere possible, the use of benzene in manufacturing processes has been reduced by replacement with less hazardous compounds. Hence, benzene is now generally regarded as almost exclusively a product of petroleum refining . Workers3, but investigations of petroleum refinery workers in Bulgaria found benzene concentrations levels higher than 3 mg/m3 [Occupational exposure limits (OELs) have been introduced by various organizations for the management of benzene exposure. As reported in  benzene  3 mg/m3 . ExposurHealth risk assessment for exposure to toxic pollutants is usually carried out to evaluate the adverse effects using single data points to quantify the risk. However, risk assessment using probabilistic techniques utilizes probability distributions to estimate the risk thereby giving an evaluation of variability ,13,14,15The aim of this study was to collect and collate exposure data for benzene in petroleum refineries environment on a global scale and conduct a risk assessment to evaluate the possible adverse health effects.EXP50) and 95% (CEXP95) cumulative probability of benzene exposure levels. The CEXP50 level gave an evaluation relevant to most of the exposed population while CEXP95 was relevant to the 5% most exposed group. On the other hand, Overall Risk Probability was an estimated value relative to the whole population.The strategy used in this research involved collection and collation of benzene exposure data and guideline values from the scientific literature. Like data sets presented in Data sets for benzene exposure in petroleum refinery environments used in this study were gathered from the scientific literature using various search engines such as Google, Web of Knowledge, PubMed, Toxnet, Medline and Science Direct. Each reference provided one or more sets of benzene measurements, with each set representing measurements for a sampling location, activity or occupation  1,2,10,,20,21,23The health risk was focused on evaluating exposure data on benzene concentrations in the ambient air of petroleum refineries. Only data sets reported as individual concentrations and base estimates concentrations were utilized for consistency since a number of data sets were reported as mean concentrations. These data sets (mean data) were not included in the risk assessment analysis since they cannot be combined and interpreted with the datasets on individual measurements and base estimates.i, ith point; n, total number of data points. The linear regression equations of the CPD plots were usually calculated between approximately 10%\u201390% of the Cumulative Probability distribution since this represents the approximately linear part of the CPD plots when a normal distribution occurs.The data sets for benzene exposure were used to develop Cumulative Probability Distribution (CPD) plots by using Microsoft Excel. CP (%) was calculated from Equation (1):The exposure limits for occupational exposure to benzene from various organizations such as European Commission, OSHA, NIOSH, ACIGH and SWA and Air Quality Guidelines (AQGs) and United Kingdom are summarized in 3, ppm and ppb to a uniform unit of \u00b5g/m3. The data sets that were used to develop CPD plots for exposure to benzene were categorized into Scenarios as outlined below.The data sets were obtained from the publications listed in This scenario represents benzene concentrations collected as base estimate concentrations for retrospective benzene exposures in petroleum industries from studies using similar methods in deriving the base estimates from benzene measurements. The studies were for early 1940 to 1996 for the Australian study ; 1902 toThis scenario was for petroleum refinery workers in different occupation within the petroleum refineries exposed to benzene. The concentrations of benzene in air were collected by the workers wearing personal air sampling pumps. The data sets used in this scenario were obtained from ,11.The data sets were derived from air samples of benzene taken within various work locations inside the petroleum refineries. Measurements of benzene concentration levels were obtained by using air sampling pumps positioned at various locations inside the petroleum refineries. The data sets used in this scenario were obtained from ,2,10.The data sets obtained for this scenario were for emissions of benzene from petroleum refineries to the immediate surroundings giving exposure to people living near the petroleum refineries. Benzene concentrations were obtained around the petroleum refineries at a maximum distance of 2 km by using air sampling pumps at different sampling locations near the petroleum refineries. The data sets used in this scenario were obtained from ,10,22.EXP50 (the median level which represents the main group) and CEXP95 representing the highest exposed group in the population levels of exposure of the population within each scenario. The benzene concentrations (Scenario 1 to 4) were converted from \u00b5g/m3 to \u00b5g/kg/day in terms of Lifetime Average Daily Dose (LADD) using values summarized in The data sets for exposure to benzene were categorized into related Scenarios 1 to 4 see  and convEXP is exposure concentration (\u00b5g/m3); IR, Inhalation Rate (m3/day); EL, Exposure Length (day/day); ED, the Exposure Duration (days); BW, Body Weight (kg); LT, Lifetime (days).The Lifetime Average Daily Doses (LADD) (\u00b5g/kg/day) for exposure to benzene concentrations were calculated for all Scenarios using the default values in EXP50) which represents the main group of individuals and the 95% level (CEXP95) representing the highest exposed group in the population. This highly exposed group occurs at a level of 5% in the population and the median group represents over 50% in the population. Benzene concentrations at CEXP50 and CEXP95 were obtained from the CPD plots (EXP50 and CEXP95 using Equation (3):The HQ method of risk characterization was used to estimate the adverse health effects for exposure to benzene. The USEPA Reference Dose (RfD) derived for benzene was used to estimate the HQ for all Scenarios by using Equation (3). Benzene exposures were estimated at the median level (CPD plots  and conv/kg/day) .Cancer risk is expressed as excess risk of developing cancer over a lifetime of exposure (70 years).EXP50 and CEXP95 in terms of lifetime exposure (LADD) in the various scenarios by using Equation (4):The USEPA inhalation slope factor derived for benzene was used to quantitatively estimate the excess cancer risk at C benzene .The ORP method is based on the use of Overall Risk Probability (ORP) curve. The ORP curve is the plot of exposure exceedence values (1\u2014CP) against the corresponding CP values for dose-adverse effects . A detailed description of overall risk probability in risk assessment has been discussed in . The CPDR2) of 0.97 indicating a normal distribution. At CEXP50, exposure to benzene was higher than NIOSH REL but lower than ACIGH TLV, OSHA PEL, EC LV and SWA OEL. However, at CEXP95 exposure to benzene was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The workers in the highly exposed group reported by the high exposure concentrations in the CPD plots were workers involved in activities such as drum fillers, large terminal operator, gauging, line pigging, rail car loading, refueling, tanker loading and cleaning.The CPD plot as shown in R2) > 0.94 for both CPD plots indicating normal distributions. The CPD plots of Scenario 2A and 2B have almost identical slopes of 65 and 60, respectively. This implies that there was a comparatively wide range of benzene concentration distribution. At CEXP50 and CEXP95 exposure to benzene for Scenario 2A was lower than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. While at CEXP50 and CEXP95 exposure to benzene in Scenario 2B was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The high exposure to benzene was for workers in transport and storage of petroleum products facility, benzene manufacturing plant and ethylbenzene\u2014styrene manufacturing plant.The CPD plots  are for R2) > 0.94 for both CPD plots indicating high level of linearity in the distributions. The slope for Scenario 3C is 40 while Scenario 3A has a slope of 117, indicating a relatively wide range of benzene concentration distribution for both CPD plots. At CEXP50 and CEXP95 exposure to benzene for Scenario 3A was lower than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. While at CEXP50 and CEXP95 exposure to benzene in Scenario 3B was higher than NIOSH REL, ACIGH TLV, OSHA PEL, EC LV and SWA OEL. The high exposure to benzene was for workers in transport and storage of petroleum products facility, benzene manufacturing plant and ethylbenzene\u2014styrene manufacturing plant.The CPD plots in R2) > 0.96 indicating a high level of linearity in the distribution and a normal distribution. The slope for CPD plot ) and the results were summarized in 50 were < 1 for petroleum refinery workers (Scenario 2A), benzene concentrations in air inside the petroleum refineries (Scenario 3A), and benzene concentrations in air outside the petroleum refineries (Scenario 4). This result suggests minimal risk to the majority of the population in these exposure Scenarios . Also, HQ95 was <1 for Scenario 4 suggesting minimal risk to the high exposed group. However, HQ95 were >1 for Scenarios 2A and 3A indicating possible risk to adverse effects. HQ50 and HQ95 for lifetime exposure to benzene for Scenario 1 (base estimates for petroleum refinery workers), 2B (exposure to benzene for petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) were >1 indicating possible adverse health effects for the main group of exposed individuals and the high exposed.The calculated LADD at CEXP50) which represents the main group of exposed individuals and the 95% level (CEXP95) representing the highest exposed group in the population (Scenarios 1 to 4) and the results were presented in EXP50, the excess cancer risk in terms of lifetime exposure to benzene for Scenario 2A (petroleum refinery workers), 3A (benzene concentrations in air inside the petroleum refineries) and 4 (benzene concentrations in air outside the petroleum refineries) are very low in the range of 6 to 18 per 106 as compared to Scenario 1 (base estimates for petroleum refinery workers), 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that is in the range of 590 to 10,000 per 106. On the other hand, at CEXP95, the highly exposed group that occurs at a level of 5% in the population, the excess cancer risk in terms of lifetime exposure to benzene for Scenario 2A (petroleum refinery workers), 3A (benzene concentrations in air inside the petroleum refineries) and 4 (benzene concentrations in air outside the petroleum refineries) are very low in the range of 200 to 460 per 106 as compared to Scenario 1 (base estimates for petroleum refinery workers), 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that is in the range of 10,000 to 48,000 per 106. The cancer risk estimated at CEXP95 is only for 5% of the exposed population. The significance difference in the cancer risk estimated is as a result of higher concentration levels of benzene observed in 2B (petroleum refinery workers) and 3B (benzene concentrations in air inside the petroleum refineries) that was 17 to 1400 and 4.6 to 230 times higher than Scenarios 1 to 4 at (CEXP50) and (CEXP95) respectively.The excess CR was calculated for exposure to benzene at the median level (C6) was obtained for Scenario 1 (base estimates for petroleum refinery workers), 0.011% (110 per 106) for Scenario 2A (petroleum refinery workers), 4.8%  for Scenario 2B (exposure to benzene for petroleum refinery workers), 0.015% (150 per 106) for Scenario 3A (benzene concentrations in air inside the petroleum refineries), 1.7%  for Scenario 3B (benzene concentrations in air inside the petroleum refineries) and 0.009% (110 per 106) for Scenario 4 (benzene concentrations in air outside the petroleum refineries).Cancer risk adverse dose\u2014response relationship are shown in EXP95) and the main group of individuals (CEXP50) in the population, while with ORP all of the exposed population were taken into consideration as shown in Overall the ORP and the CR are in reasonable agreement . The dif50 > 1 and HQ95 > 1 for workers exposed to benzene as base estimates for petroleum refinery workers (Scenario 1), petroleum refinery workers evaluated with personal samplers in Bulgarian refineries (2B) and evaluated using air concentrations inside petroleum refineries in Bulgarian refineries (3B). On the other hand HQ50 were <1 for lifetime exposure to benzene in petroleum refinery workers (Scenario 2A), benzene concentrations in air inside the petroleum refineries (Scenario 3A), and benzene concentrations in air outside the petroleum refineries (Scenario 4) suggesting minimal risk to the majority of the population in these exposure Scenarios. HQ95 was <1 for Scenario 4 suggesting minimal risk to the high exposed group however, HQ95 were >1 for Scenarios 2A and 3A indicating possible risk to human health for the high exposed group. The excess cancer risk for lifetime exposure to benzene for all the Scenarios was evaluated using the Slope Factor method at CEXP50 and CEXP95 and also using the ORP method. The two methods showed a reasonable level of agreement. With the ORP method, workers in petroleum refineries in Scenario 2B were observed to have the highest cancer risk 44,000 per 106 followed by those evaluated with data from air inside the petroleum refineries in Scenario 3B with cancer risk of 17,000 per 106 and base estimates for petroleum refinery workers Scenario 1 with cancer risk of 1700 per 106.Benzene levels were estimated to pose a significant risk with HQ"}
+{"text": "Primary immunodeficiency diseases comprise heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system. The aim of this study was to evaluate the humoral response in children\u2019s patients with recurrent infections.+ for the cases that presented decreased of the serum immunoglobulins.Venous blood samples were collected from 64 children in Pediatric Hospital Professor Heriberto Ferreira Bezerra (HOSPED) - Federal University of Rio Grande do Norte (UFRN), Brazil. The laboratory investigation included measurement of serum IgG, IgM and IgA levels by imunoturbidimetry and the B lymphocyte quantification by flow cytometry with monoclonal anti-CD19Among the 64 children included, 33  were male and 31  were female. The children age ranged from 2 months to 15 years old. According to age, 36 (56%) children were aged 0-5 years; 11 (17%) children were aged 5 years and 1 month to 10 years; 10 (16%) children were aged 10 years and 1 month to 13 years and 11 months and 7 (11%) children were aged 14 years to 15 years. Immunoglobulin concentration below age-appropriate reference values was observed for IgG in 7 (11%) children; for IgM in 3  children and for IgA in 3  children. B lymphocytes were absent in 3  children. The superior respiratory tract infections were the most prevalent in this population.B-cell disorders are the most common type of immunodeficiencies and they are characterized by an increased susceptibility to respiratory tract infections. In this study, 3 patients received the clinical and laboratory diagnostic of X-linked agammaglobulinemia."}
+{"text": "Clinical signs and symptoms of pneumoperitoneum are not specific and abdomen radiography is positive in less than 50% of cases. Ultrasonography (US) accuracy for the diagnosis of pneuperitoneum is still unknown.1) define the accuracy of abdominal US for the diagnosis of pneumoperitoneum; 2) define the accuracy of a \u201c2 scan-fast exam\u201d vs a full abdominal exam; 3) compare accuracy of US and abdomen radiographyStudy patients: 11 consecutive adults with a diagnosis of pneumoperitoneum by CT. Control patients: 11 consecutive adults with severe acute abdominal pain with a diagnosis other than pneumoperitoneum by CT. US examination has been performed with a convex and a linear probe using the following scans: epigastrium, right and left hypocondrium, umbelical area and right hypocondrium. All exams were recorded in a video of 5 seconds and each videos reviewed by 2 radiologists and 2 senior physicians blinded to other imaging studies. The reviewers fulfilled a standardized form signing for each scan either presence or absence of pneumoperitonem signs (enhancement of the peritoneum stripe with ring-down artifacts or \u201ccomet tails\u201d starting from peritoneum). If one of the signs of pneumopeitoneum was present in at least one scan, the patient was considered to have a US diagnosis of pneumoperitoneum. The reviewers also evaluated abdomen radiography for the presence/absence of pneumoperitoneum. CT was considered the gold standard.1) Accuracy of abdomen US was 88.6%. Sensitivities of convex and linear probes were similar (88.6% vs 84.1%), while specificity of convex was lower than linear probe (81.8% vs 95.5%). 2) Accuracy of a \u201c2 scan-fast exam\u201d was similar to global exam (87.5%). 3) Abdominal radiography sensitivity (72.2%) was lower than US while specificity (92.5%) was higher.Abdominal US has a good accuracy. The 2 scan-fast exam has a similar accuracy of the full abdominal exam. US sensitivity is superior to abdominal radiography thus ultrasonography can be a useful imaging modality for the detection of pneumoperitoneum."}
+{"text": "Exsanguination and coagulopathy remain one of the leading causes of preventable trauma related death . Low ionA retrospective cohort analysis was performed on all major trauma patients who had received early blood product in the Emergency Department of a single London Major Trauma Centre over a one year period (January 2013 \u2013 January 2014). Ionised calcium levels were taken from venous blood gases from before and after blood product had been transfused. Excel was used to analyse the data.The study included 60 patients aged between 10 and 92 (mean 40), 46 male (77%) and 14 female (23%). Mechanism of injury was predominantly blunt 48 (80%) and penetrating 12 (20%). Patients received between 1 and 16 units of blood product (mode 2). Mean ISS was 26 (5-50) and overall 30 day mortality was 12%.60% were hypocalcaemic on arrival before receiving any blood product (Mean [Ca] 1.1mmol/L 95% CI 1.08 \u2013 1.13) 89% of patients were hypocalcaemic after receiving blood product (Mean [Ca] 0.95mmol/L 95% CI 0.9 \u2013 1.01). There was a statistically significant difference between ionized calcium levels pre and post blood transfusion. A drop in calcium was seen after receiving just one unit of packed red blood cells, with the average drop being 0.05 mmol/L per unit of blood product received.Trauma patients that have sustained blood loss are at risk of hypocalcaemia. Receiving just one unit of blood product further compounds their hypocalcaemic state and the more units that are given the greater the fall that is seen."}
+{"text": "Helianthus annuus L.) Heat Shock Factor A9 (HaHSFA9) enhanced seed longevity in transgenic tobacco (Nicotiana tabacum L.). In addition, the overexpression of HaHSFA9 in vegetative organs conferred tolerance to drastic levels of dehydration and oxidative stress.We have previously reported that the seed-specific overexpression of sunflower (HaHSFA4a) and HaHSFA9 enhanced all the previously reported phenotypes described for the overexpression of HaHSFA9 alone. The improved phenotypes occurred in coincidence with only subtle changes in the accumulation of small Heat Shock Proteins (sHSP) that are encoded by genes activated by HaHSFA9. The single overexpression of HaHSFA4a in vegetative organs (which lack endogenous HSFA9 proteins) did not induce sHSP accumulation under control growth conditions; neither it conferred thermotolerance. The overexpression of HaHSFA4a alone also failed to induce tolerance to severe abiotic stress. Thus, a synergistic functional effect of both factors was evident in seedlings.Here we found that the combined overexpression of sunflower Heat Shock Factor A4a (in planta function. Our results strongly support the involvement of HaHSFA4a and HaHSFA9 in transcriptional co-activation of a genetic program of longevity and desiccation tolerance in sunflower seeds. These results would also have potential application for improving seed longevity and tolerance to severe stress in vegetative organs.Our study revealed that HaHSFA4a requires HaHSFA9 for  DS10 sequences (a seed-specific promoter). We have also shown that the ectopic overexpression of HaHSFA9 from Cauliflower mosaic virus (CaMV) 35S sequences in tobacco seedlings conferred dramatic resistance of green organs and of whole seedlings to severe dehydration  of PSII after treatments with H2O or with 200 mM H2O2 for 24 h.Click here for fileComparison of the accumulation levels of the tagged HaHSFA4a protein in the 35S:A4a and 355:A9/A4a seedlings. 1D-western analyses using anti-hemaglutinin antibodies.Click here for fileExamples of total protein loading controls for the protein samples analyzed by western blot in this article. Ponceau S stained PVDF membranes.Click here for file"}
+{"text": "Quiescent Interval Single Shot (QISS) has emerged as a robust technique for non-enhanced angiography of peripheral arteries (1). It has been clinically validated with contrast enhanced magnetic resonance angiography (CE-MRA) and digital subtraction angiography (DSA) . Both of these validation studies were performed at 1.5T field strength. Although the initial experience with QISS at 3T using similar imaging parameters as those at 1.5T were promising, venous suppression was inadequate in the thigh and pelvic regions of some patients . In this work, we present a strategy to improve venous signal suppression with the QISS sequence at 3T.The imaging parameters were similar to those reported earlier for QISS at 1.5T (2) with the following exception: the tracking saturation pulse for venous signal attenuation was replaced with an adiabatic inversion pulse (hyperbolic secant). This enables a more homogenous venous suppression at 3T than is possible with regular sinc pulse due to B1 inhomogeneity. The prototype sequence was tested in three volunteers in a 3T system .The use of adiabatic inversion pulse instead of a sinc saturation pulse improved venous signal suppression, as shown in Figure Although QISS has shown robust performance at 1.5T, initial experience at 3T demonstrated suboptimal venous suppression. Although not established, this could be attributed to increased B1 inhomogeneity at 3T. We have demonstrated an approach to improve venous signal suppression at 3T using QISS. More patient studies are required and are underway to test the robustness and consistency of this approach.The primary author is a full time employee of Siemens Healthcare."}
+{"text": "Both GCA1 and GCA2 were loss-of-function gene in low-GCA parent and gain-of-function gene in high-GCA parent, encoding the putative Pseudo-Response Regulators, OsPRR37 and Ghd7, respectively. Overexpression of GCA1 in low-GCA parent significantly increases GCA effects in three traits. Our results demonstrate that two GCA loci associate with OsPRR37 and Ghd7 and reveal that the genes responsible for important agronomic traits could simultaneously account for GCA effects.Artificial selection of high yield crops and better livestock is paramount importance in breeding programs. Selection of elite parents with preferred traits from a phalanx of inbred lines is extremely laborious, time-consuming and highly random. General combining ability (GCA) was proposed and has been widely used for the evaluation of parents in hybrid breeding for more than half a century. However, the genetic and molecular basis of GCA has been largely overlooked. Here, we present two pleotropic QTLs are accounting for GCA of days to heading (DTH), plant height (PH) and spikelet per panicle (SPP) using an F One of the most fundamental functions of living organisms is to pass on to their progeny advantageous traits that ensure positive interactions with their environment, whereas any unfit traits are gradually eliminated by natural environmental pressures. This is the well-known evolutionary theory of natural selectionThe first research regarding the genetic variance of GCA and specific combining ability (SCA) was reported by Griffing 1956) using a diallelic cross-mating design. Since then, it has become possible to produce precise estimates of GCA and SCA6 using a6789Recently, GCA has been used as one of the most reliable ways to predict the performance of hybrids. Several prediction methodologies based on molecular markers and genomic, transcriptomic and metabolic prediction methods have been developed111213et al. identified the genetic loci for combining ability that correspond to agronomic traits through QTL mapping using three testcross populations and a backcross recombination inbred line (BCRIL) in rice. The characteristics of the QTLs for combining ability were found to be similar to those of the QTLs for BCRIL performanceet al. identified several genetic loci of GCA and SCA in maize using four testers from different heterotic groups and introgression lines (ILs)More recent studies have attempted to uncover the genetic basis of GCA by employing a quantitative trait locus (QTL) mapping approach1516GCA1 and GCA2\u2014through the BC3F2 population and a set of nearly isogenic lines (NILs). We revealed that GCA1 and GCA2 encode the putative Pseudo-Response Regulator OsPRR37 and Ghd7, respectively. Furthermore, we confirmed that GCA1 functions as a positive regulator not only of the agronomic traits per se but also of GCA by virtue of the overexpression of GCA1 (GCA1OX) in rice. Additional experiments revealed that GCA1 is located in the nucleus and that the loss of the 234 amino acids at its C terminus does not affect its nuclear localisation. Expression profiling of GCA1 indicated that it was constitutively expressed in all developmental stages and tissues and was highly expressed in leaves, young panicles and stem. Our results revealed that the genes for important agronomic traits could simultaneously account for the GCA effects. These findings provide new insight into the molecular genetic basis of GCA and the relationship between important agronomic traits and the effects of GCA.In the present work, we identified 13 QTLs that account for the GCA of three agronomically important traits associated with rice grain yield by using an F2-based NCII design with five testers. We also finely mapped two major pleotropic QTLs\u20142 population for genetic mapping. To identify the effects of GCA and SCA in the F2 population, variance analysis of GCA and SCA for the three agronomic traits DTH, PH and SPP was carried out on 139 individual plants in the F2 population. The mean squares for the GCA effects from the F2 individuals and testers as well as the SCA effects from progeny of F2 individuals and testers (Testers\u00d7F2) were found to be significant for all the three agronomic traits  exhibited the lowest GCA effects, whereas Teqing (TQ) exhibited the preferred positive GCA effects for three agronomically important traits see  online. c traits . The resc traits . These r2 population. As shown in 2 population ranged from \u221217.8 to 30.6 for DTH, \u221210.1 to 9.2 for PH and \u221222.0 to 15.7 for SPP. These results demonstrated that the values of GCA in the F2 population were typical normal distributions, and the mean values of GCA for the three traits were almost zero, suggesting that the GCA effects are quantitative traits. These results indicated that this F2 population was suitable for further QTL analysis of GCA.Our previous results showed that the GCA effects made a major contribution to the performance of hybrid offspring. To genetically map the GCA loci for the three traits, the GCA values from a segregation population are required as phenotypes for genetic mapping. We first evaluated the GCA effects in 139 individuals derived from the F2 population and 141 molecular markers  were used to construct a linkage map with coverage of approximately 1,763\u2009cM . A total of 13 major QTLs for GCA of the three agronomic traits were identified on five chromosomes based on a cut-off LOD score\u2009\u2265\u20093.0  and GCA2 (BC3F2-GCA2) were developed by continuous backcrossing using GL as the recurrent parent via marker-assisted selection (MAS) with the flanking SSR markers RM3670 and RM1306, respectively  conditions. The DTH of dominant individuals from the BC3F3-GCA1 and BC3F3-GCA2 populations was increased by 23.3 days and 20.9 days compared with those of corresponding recessive individuals under natural long day (NLD) conditions, respectively. Simultaneously, other two traits, PH and SPP, were significantly increased in the dominant individuals of BC3F3-GCA1 and BC3F3-GCA2 populations  and two newly developed InDel markers (ID77 and ID710) were used to screen 3,127 individuals from BC3F2-GCA1 population. A total of 23 recombinant individuals were identified, and their GCA values for three agronomic traits were obtained by crossing to the tester varieties  newly developed were used for genotyping within the 802\u2009kb region. Finally, the location of GCA1 was narrowed to a region of approximately 443\u2009kb extending from S77 to the end of chromosome 7 . The expression patterns of those genes in both parents were obtained by RNA sequencing dataArabidopsis Pseudo-Response Regulator 7 (PRR7). The 8\u2009bp deletion in the OsPRR37 in GL resulted in a 234 amino acid truncation at the C terminus, which led to the loss of the CCT domain, whereas the TQ allele has an entire protein with 742 amino acids  carrying 35S:OsPRR37 were generated. The delayed flowering phenotype was observed in positive transgenic plants. The DTH, PH and SPP of homozygous GCA1OX\u22125 plants were increased by 32.5 days, 23.9 cm and 62.6 spikelets, respectively, under natural long day (NLD) condition compared with negative plants (OsPRR37 in positive GCA1OX lines was significantly higher than that in NIL (GCA1+/+)  and NILs (GCA2+/+ and GCA2\u2212/\u2212), as well as the parents GL and TQ were evaluated using TC progenies derived from five testers  and NIL(GCA2\u2212/\u2212) lines were significantly lower than that in TQ, NIL(GCA1+/+) and NIL(GCA2+/+) lines. These results confirmed that OsPRR37 was the gene corresponding to GCA1. Again, the GCA effects of NIL (GCA2\u2212/\u2212) and NIL (GCA2+/+) supported that Ghd7 corresponds to GCA2. These results indicated that GCA1 and GCA2 significantly increase the GCA of DTH, PH and SPP.To verify whether the candidate gene e plants  online. GCA1+/+) . The GCA testers . The resGCA1, the transgenic plants carrying a GUS reporter gene driven by the GCA1 promoter were generated. The results revealed that GCA1 was constitutively expressed in all tissues and at all developmental stages  plants exhibited a diurnal pattern (GCA1 in NIL (GCA1+/+) increased from dawn to noon and then gradually decreased until midnight, with a peak expression level at noon under NLD conditions. Surprisingly, the overexpression of GCA1 driven by the 35S promoter in GCA1OX lines also exhibited a diurnal pattern, but the diurnal expression pattern was altered and peaked around dusk (at 20:00 for GCA1OX\u22125 and 16:00 for GCA1OX\u22129), with the lowest expression levels observed in the morning (at 8:00 for both GCA1OX lines). These results imply that the mRNA level of GCA1 itself could be regulated by unknown diurnal signal(s).To understand the expression profiles of l stages . The mos pattern . The expAtPRR7, an orthologue of GCA1, has a potential bipartite nuclear localisation signal at the C terminus (amino acids 680 to 696). The localisation of the PRR7-GUS fusion was observed to be nuclear in leek epidermal cellsPrevious research has reported that GCA1 and GCA2\u2014that corresponded to OsPRR37 and Ghd7, respectively, and functioned as positive regulators of GCA for DTH, PH and SPP and these traits per se. Our findings revealed that the genes responsible for GCA effects correspond to the genes controlling important agronomic traits. To take another example in this study, two QTLs for the GCA of DTH were found to be located between RM3431 and RM3438 and between RM584 and RM5815 on chromosome 6. The physical regions represented by these SSR markers are close to the physical positions of the genes Hd1 and Hd3a that control flowering time26Hd1 and Hd3a may be responsible for the QTL loci for corresponding GCA effects. These results suggested that QTLs for GCA are directly associated with QTLs for these important agronomic traits.In this study, we identified 13 QTLs for the GCA of three important agronomic traits that were mainly associated with grain yield in rice. We identified two major pleotropic QTLs\u2014GCA1 and/or GCA2 were widely found in elite rice varieties19212324GCA1 and GCA2 while 93-11 contains GCA2. It is apparently the result of domestic selection during cross-breeding. This pattern explains that high GCA-effect parents could easily pass on the corresponding traits to their hybrid progenies by simultaneously integrating the GCA controlling genes into the genome of hybrid progenies during cross-breeding program. It is well explained why elite varieties could be easily developed from parents with higher GCA effects. Our findings help remove the mystery of GCA that has existed since 1942, even though this trait has been widely used for crop and livestock improvements.Previous studies have reported that GCA1 and GCA2 encode OsPRR37 and Ghd7, respectively, which have both been reported to be associated with agronomic traits for high grain yield and adaptability at different latitudes192123GCA effects have widely been applied by breeders to evaluate breeding parents. The consensus of opinion is that elite breeding parents possess preferred GCA effects, which are characteristics of wide adaptability, high hereditary capacity and elite agronomic traits. Our data show that GCA1+/+) was lower than that of GCA1OX transgenic lines, and the GCA effect of SPP in NIL(GCA1+/+) and NIL(GCA2+/+) was even lower than zero (GCA1+/+) and NIL(GCA2+/+) was partly caused by the extremely high GCA effect of SPP in parent TQ. Alternatively, the negative value of the GCA effect of SPP in NIL(GCA1+/+) does not mean inconsistency between NIL(GCA1+/+) and GCA1OX transgenic lines. As in this respect, the GCA effects of NIL(GCA1+/+) and GCA1OX transgenic lines showed the same trends when comparing to the GCA effects of both NIL(GCA1\u2212/\u2212) or GL. For the other explanation, the GCA difference could be caused by the different expression pattern of GCA1 between NIL(GCA1+/+) and GCA1OX transgenic lines  and LATE ELONGATED HYPOCOTYL (LHY) in Arabidopsis32GCA1 and the nuclear localisation of the GCA1 protein indicated that GCA1 might have a similar function as a circadian-regulated transcription factor in rice. Interestingly, GCA2 also possesses a CCT domain exhibiting a diurnal expression pattern and nuclear localisationArabidopsis PRR5 and target DNA in vivoGCA1 and GCA2 might function as transcription factors of various downstream genes in the nucleus by regulating their diurnal expression patterns or expression levels.Previous studies have reported that 2 population with 139 individuals derived from GL\u00d7TQ was used in this study. Five varieties\u2014ZS, AJ, GL, L6 and Aizizhan (AZ)\u2014were used as testers to cross to individual plants from the F2 population based on the NCII mating design in Lingshui, Hainan Province, in the spring of 2009. The 139 F2 plants were crossed with the five testers to evaluate GCA effects and were planted in Wuhan, Hubei Province, in the summer of 2009. Twenty plants of each testcross (TC) progeny were planted in a 16.5\u2009cm\u2009\u00d7\u200920\u2009cm area following a randomised complete block design with three replicates.Estimation of the GCA effects of elite rice varieties was conducted with a diallelic cross design using the Yuetai B (YB), Zhenshan 97 B (ZS), Aijiaonante (AJ), Guangluai #4 (GL), 93-11(L6) and Teqing (TQ) varieties. An F2 population containing the QTL region of interest were continually backcrossed to GL. The BC3F1 plants were selected by marker-assisted selection (MAS), and the GCA effects of the recombinant plants from BC3F2 were evaluated according to the agronomic traits of the TC progeny , plant height (PH) and spikelets per panicle (SPP) were collected for between 10 and 15 plants from each plot. For fine mapping, the accurate GCA effects of three traits were measured for five plants of non-segregated lines and for between 10 and 15 plants of segregated lines derived from the TC progeny in each replication. Variance analysis and evaluation of the GCA were carried out as previously describedwhere \u2009t-test of the agronomic traits\u2014was performed in SPSS statistics software version 17.0 .The variance analysis and effect evaluation of GCA were processed with DPS software version 7.052 population, including 123 SSRs (http://www.gramene.org/), 14 SNPs and four InDel markers developed from previous genome sequencing data for GL and TQ2 population as described previously3F3 family and the corresponding TC progeny was used to distinguish the genotype of the GCA locus in each recombinant plant of BC3F2.A total of 141 polymorphic molecular markers were used to construct the linkage map using plants from the FGCA1 was confirmed by PCR-based sequencing of the exons in genomic DNA and cDNA obtained by the mRNA reverse transcription of TQ. The CDS  and the 3\u2032UTR (623\u2009bp) were artificially synthesised by Gene-Script  and were inserted into the binary vector pCAMBIA1300. A CaMV 35S promoter was then fused to the 5\u2032 end of the CDS, resulting in the overexpression construct pOsPMP625. To generate the GCA1 promoter-GUS construct, a 1,884\u2009bp promoter region was amplified by PCR and inserted upstream of the 5\u2032 end of the reporter gene beta-glucuronidase (GUS) in the binary vector pCAMBIA1300, designated pOsPMP626. All transgenic plants were generated by introduction into GL through Agrobacterium-mediated transformation.The coding sequence (CDS) of the candidate gene GCA1 and GCA2 were obtained from homozygous BC4F2 plants with more than 90% of the genetic background derived from the GL line, as determined by genome-wide SSR and InDel markers (see GCA1+/+ or GCA1\u2212/\u2212) and NIL (GCA2+/+or GCA2\u2212/\u2212) were determined by using appropriate molecular markers. The homozygous GCA1OX plants from the T1 generation were determined by qPCR as described previouslyGCA1+/+ or GCA1\u2212/\u2212) and NIL (GCA2+/+ or GCA2\u2212/\u2212)], three independent homozygous GCA1OX lines and the parent lines (GL and TQ) were crossed to five tester varieties  at Lingshui, Hainan Province, in the spring of 2014. The GCA effects and the traits of these lines were evaluated.NILs corresponding to different genotypes kers see  online. Actin1 gene was used as an internal control to normalise the data.The uppermost fully expanded leaves from at least three individual plants were harvested at 45 days after germination under natural long day (NLD) conditions. Total RNA was extracted from the pooled leaf materials using TRIzol Reagent  according to the manufacturer\u2019s instructions and then treated with RNase-free DNase I . First-strand cDNA was synthesised using M-MLV reverse transcriptase and oligo (dT) primer  as described by the manufacturer. Quantitative RT-PCR was carried out in a total volume of 10\u2009\u03bcl, including 2\u2009\u03bcl of cDNA template (5- to 10-fold dilutions), 0.5\u2009\u03bcl of 10\u2009\u03bcM gene-specific primer and 5\u2009\u03bcl of SYBR Green qPCR Supermix-UDG with ROX Reference Dye . The qRT-PCR was conducted on a StepOne System (ABI) using the following parameters: 95\u00b0C for 10 min, followed by 40 cycles of 95\u2009\u00b0C for 10\u2009s and 60\u2009\u00b0C for 30\u2009s. The relative gene expression levels were calculated according to the \u0394\u0394CT method described previously4 buffer (pH 7.0), 2\u2009mM K3Fe(CN)6, 2\u2009mM K4Fe(CN)6 and 0.2% Triton X-100 on ice and then incubated with staining solution containing 1\u2009mM X-Gluc at 37\u2009\u00b0C overnight. The coloured tissues were photographed with a camera .A GUS staining assay was performed as described in a previous studyGCA1 and truncated GCA1 (tGCA1) were amplified using appropriate primers based on the sequences in TQ and GL, respectively (GCA1 and tGCA1 were inserted into the pHBT-sGFP(S65T) construct at the C terminal of sGFP without a stop codon between sGFP and GCA1 or tGCA1 to create the in-frame fusion constructs sGFP-GCA1 and sGFP-tGCA1, respectively. Rice protoplasts were used for determining subcellular localisation. Protoplast isolation and PEG-mediated transfection were carried out as described in a previous studyCaMV 35S promoter was co-transformed with sGFP-GCA1 and sGFP-tGCA1. To avoid fluorescence artefacts that could disturb localisation, an optimised concentration of plasmid DNA  was used for the transient expression assay. Subcellular localisation was visualised on an Olympus Fluoview 1000 laser scanning microscope . Triplicates of each transient expression experiment were performed to obtain a robust result.The CDS of full-length ectively . IsolateHow to cite this article: Liu, C. et al.OsPRR37 and Ghd7 are the major genes for general combining ability of DTH, PH and SPP in rice. Sci. Rep.5, 12803; doi: 10.1038/srep12803 (2015)."}
+{"text": "NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load.The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 , NEIL2 , and NEIL3  are structural human homologues of Escherichia coli (E. coli) Nei and Fpg, the genes encoding a DNA glycosylase that initiates the base excision repair (BER) process. These three homologues also have actual functional activities as DNA glycosylases  were collected from the TCGA data portal (n (RSEM) , excludin (RSEM) . The som\u03bcM of 5-aza-dC  for 48\u2009h, as described previously  of the 13 (69.2%) cancer types satisfied the 4 criteria for epigenetic silencing described in activity , these rpression . We nextnk test)  but not Since NEIL1, NEIL2, and NEIL3 have been experimentally shown to have the ability to suppress mutations in human cells and/or in bacterial cells \u201312, the Using data for 13 cancer types from the TCGA database, we revealed that the median somatic total and SNP-type mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level. We also showed that a subset of human cancers exhibited reduced NEIL1 and NEIL2 expression levels and an elevated NEIL3 expression level, and these abnormal expressions of NEIL1, NEIL2, and NEIL3 were associated with the mutation load in cancer. We then showed that the reduced NEIL1 expression level observed in various cancers was due to epigenetic silencing by promoter hypermethylation and that such reduction was an independent predictor of a poor outcome among patients with breast invasive carcinoma. Finally, NEIL3 expression was shown to be correlated with the expression of APOBEC3B, a potent inducer of mutations, possibly explaining why an increased NEIL3 expression level was associated with the somatic mutation load in cancer. Thus, our results suggest that the abnormal regulation of NEIL1, NEIL2, and NEIL3 expression is involved in the development of cancer via an increase in the prevalence of somatic mutations, providing a new and important link between abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 and human cancer.Using a TCGA-based analysis, associations between abnormal NEIL1, NEIL2, or NEIL3 expressions and the somatic mutation load were apparently demonstrated in various cancer types for the first time. The association between reductions in NEIL1 and NEIL2 expressions and the increased number of somatic mutations in cancer is understandable, but the association between an elevation in NEIL3 expression and an increased number of somatic mutations in cancer seems surprising at first glance, since NEIL1, NEIL2, and NEIL3 all have the ability to suppress mutations \u201312. The  NEIL1 gene via promoter hypermethylation using data from the TCGA database. Among them, the epigenetic silencing of NEIL1 expression in HNSCC, lung adenocarcinoma, lung squamous cell carcinoma, colon adenocarcinoma, and rectal adenocarcinoma was consistent with the findings of previous reports [ NEIL1 gene via promoter hypermethylation in these cancer types, we suspect that the epigenetic silencing of the NEIL1 gene via promoter hypermethylation might be the chief mechanism underlying the downregulation of NEIL1 expression in diverse human cancers. Interestingly, in breast invasive carcinoma, which is one of the cancers that shows the epigenetic silencing of NEIL1, a reduction in NEIL1 expression was shown to be an independent predictor of a poor survival outcome. This novel finding may be useful for the management of breast cancer patients, and if this marker is used in conjunction with other prognosis markers, such as the hormone receptor status [In this study, we found 9 cancer types that showed epigenetic silencing of the reports , 26, 27,r status , the man NEIL1 or NEIL2 mutations have been experimentally demonstrated to actually have reduced or absent repair activity [ NEIL1 or NEIL2 mutations are considered to have a reduced capacity to repair mutagenic bases; thus, similar to cancers with a reduced NEIL1 or NEIL2 expression levels, a higher incidence of mutation is likely to occur in the cells, leading to cancer susceptibility. This scenario is compatible with a previous paper reporting a germline NEIL2 variant that is a marker for risk and the progression of squamous cell carcinomas of the oral cavity and oropharynx [ NEIL1 or NEIL2 mutations should clarify the role of NEIL1 and NEIL2 in the prevention of mutations.So far, several forms of germline nonsynonymousactivity , 29, 30.opharynx  and thatopharynx . Future In this study, the 0.5-fold, 0.5-fold, and 2.5-fold values of the median expression value in noncancerous tissue samples of each organ were used as cut-off values to dichotomize the NEIL1, NEIL2, and NEIL3 expression values in the cancer cases, respectively. If an expression level is downregulated or upregulated in a disease, a fold-change value of 0.5 and 2.5, respectively, has been used to dichotomize disease cases in previous reports , 34; theIn conclusion, our study indicates that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are likely to be involved in mutagenesis in human cancer. Since little is known about gene abnormalities identified by whole-exome sequencing data that induce mutations in cancer, our findings regarding these novel mutagenic factors should contribute to our general understanding of human cancer.Supplementary Table S1: Sample size of TCGA dataset used in this study.Supplementary Table S2: Abnormal NEIL1, NEIL2, and NEIL3 expressions in human cancer.Supplementary Table S3: Incidence of the cancer cases showing abnormal NEIL1, NEIL2, or NEIL3 expression.Supplementary Table S4: Associations between reduced NEIL1 expression levels and increased numbers of somatic mutations in human cancers.Supplementary Table S5: Associations between reduced NEIL2 expression levels and increased numbers of somatic mutations in human cancers.Supplementary Table S6: List of DNA methylation sites used for the analysis of epigenetic silencing of the NEIL1, NEIL2, and NEIL3 genes.Supplementary Table S7: Immunohistochemical score of NEIL1 protein in head and neck squamous cell carcinoma.Supplementary Table S8: Cox proportional hazard analysis of potential predictors of a poor prognosis in breast invasive carcinoma patients  using data from the TCGA database.Supplementary Figure S1: Scatter plots of the median NEIL1, NEIL2, and NEIL3 expression levels and the median mutation loads in 13 cancer types.Supplementary Figure S2: Correlations of PSMB2 and YWHAZ expressions with the expressions of other housekeeping genes in various organs.Supplementary Figure S3: Representative results of abnormal NEIL1, NEIL2, and NEIL3 expressions in human cancer.Supplementary Figure S4: Comparison of the total somatic mutation loads between the group showing abnormal NEIL1, NEIL2, and NEIL3 expressions and the other group in various carcinomas, as performed using a box-plot analysis.Supplementary Figure S5: Inverse correlation between DNA methylation at the NEIL1 CpG site and NEIL1 expression in various human cancers.Supplementary Figure S6: Strong positive relationship between NEIL3 expression and APOBEC3B expression in human cancer.Supplementary Figure S7: Impact of reduced NEIL1 expression in conjunction with hormone receptor status or HER2 status on overall survival in primary breast cancer patients."}
+{"text": "We here study a general problem in cellular Ca2+-sensing, namely how similar Ca2+-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca2+-binding sites with Mg2+. All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca2+ with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg2+ either occupied the low affinity Ca2+-binding site or it shifted the affinities for Ca2+-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca2+ than by Mg2+, although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca2+-sensors.Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca One family of Ca2+-binding proteins named neuronal calcium sensor (NCS) proteins are predominantly expressed in neuronal tissue and are involved in diverse intracellular processes22+-binding motifs, of which in most cases three (sometimes only two) motifs can bind micromolar to submicromolar Ca2+. One group of the NCS proteins is expressed in sensory cells and among them the guanylate cyclase-activating proteins (GCAPs) perform an important function in controlling the membrane bound guanylate cyclases (GCs) in retinal rod and cone cells45Calcium sensor proteins mediate signaling processes that respond to changing concentrations of Ca2+-free, Mg2+-bound form GCAPs activate GCs, but they switch to an inhibitory mode, when all Ca2+-binding sites are filled with Ca2+72+ in rod and cone outer segments are linked to changing levels of the intracellular messenger cGMP. After light activation of the photoreceptor cell the intracellular cGMP level is depleted, leading to a shutdown of cyclic nucleotide gated (CNG) channels in the outer segment of the cell. This stops the influx of Ca2+, which is however still extruded by the continuous operation of a Na+/ Ca2+, K+ exchanger leading to a net decrease of cytoplasmic Ca2+. This decrease is sensed by GCAPs which in turn increase the GC activity, leading to re-opening of the CNG-channels and is a necessary step for the recovery of the photoreceptor to the dark-adapted state45678In their Ca2+-sensitivity, impact on catalytic efficiency of the target GC and different structural implications of the N-terminally attached myristoyl group102+-relay mode fashion, where GCAP1 is activated at higher free Ca2+, followed by GCAP2, which becomes active, when Ca2+-levels have fallen to lower levels9102+-relay system seems also to work in zebrafish rod and cone cells, where, however, the system is more complex due to the larger number of GCAP forms132+-binding properties, Ca2+-sensitive GC regulation and spatial-temporal transcription/ expression profiles. Four zGCAPs, namely isoforms 3, 4, 5 and 7 are cone specific1214Bovine and mice photoreceptor cells express two GCAP forms, GCAP1 and GCAP2, which bind to distant regions in the target GC and have different properties with respect to Ca2+-binding sites with Mg2+781016N-myristoyltransferase (NMT) in E.coli15in vivo and in vitro click chemistry in combination with fluorescence microscopy. Revealing that all zGCAPs can exist in a myristoylated and non-myristoylated form we investigated its impact for target regulation and Ca2+-dependent membrane interaction. We further asked whether the presence of physiological Mg2+ can influence the binding of Ca2+ to zGCAPs and how Ca2+-induced conformational transitions in zGCAPs are influenced.Two parameters are of critical importance for the trigger and switch function of NCS proteins in general and GCAPs in particular: the myristoylation status and the occupation of EF-hand Ca2+-dependent membrane binding for all zGCAPs. Further Mg2+ ions control the Ca2+-affinity as well as the Ca2+-induced conformational changes in zGCAPs.Our results indicate that myristoylation has a strong impact on the regulatory properties of two zGCAPs (2 and 5), but it does not facilitate CaGreen-fluorescent protein (GFP) constructs of NCS proteins including all zGCAPs, bovine GCAP2 and recoverin were used to transfect HEK 293 cells, which were also supplemented with azido-dodecyl acid (a myristoyl substitute). This allowed us to incorporate an acyl moiety into an NCS protein in a living cell. Successful attachment of the acyl group was monitored in a subsequent copper (I)-catalyzed azide-alkyne cycloaddition using a biotin alkyne derivative forming a triazole ring. Thus, NCS proteins that were labeled with biotin could be detected via streptavidin coupled to peroxidase. Each transfected cell sample was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, blot transfer and presence of biotin in the protein band was visualized by peroxidase staining . Since GN-terminal attached fatty acyl chain and the C-terminal attached GFP to the protein in transfected cells. 192+-concentration, which had been observed in previous studies and is mainly due to hydrophobic/ electrostatic interactions2122In a second alternative approach we employed a copperless cycloaddition suitable for introducing a fluorescent dye (DIBO-TAMRA-dye) to the fatty acyl group of the NCS proteins in living cells. Thus we were able to colocalize the putative 2+-myristoyl switch, which is typically observed in other NCS proteins like recoverin, neurocalcin \u03b4, VILIP or hippocalcin242526E.coli and purified them afterwards. Principal attachment of the myristoyl group was verified by a click chemistry reaction involving azido-dodecanoic acid and the alkyne derivative of biotin as described above. We then incubated myristoylated zGCAPs and recoverin with isolated photoreceptor outer segment membranes in the absence and presence of Ca2+ , which was tested in living and in disrupted cells. This prompted us to ask, whether zGCAPs can perform a Ca of Ca2+ . No zGCA control .2+] at which the GC activity in the presence of a GCAP molecule is halfmaximal denoted as IC50 value and listed in 2+-sensitivity decreased about 4.7-fold for zGCAP2 and increased 5.7-fold for zGCAP5.Nonmyristoylated zGCAPs exhibit different activity profiles when targeting membrane bound GCs2+ under physiological conditions as it was observed for mammalian GCAP1 and 28. So far no precise values of Ca2+-binding to myristoylated zGCAPs are available and furthermore it is not known, whether Ca2+-binding is affected by physiological concentrations of Mg2+. We used a calorimetric approach (ITC) to analyze the energetics of Ca2+-binding to zGCAPs in the presence and absence of 1\u2009mM Mg2+, which allows us to determine precise values of apparent dissociation constants (KD) for multiple binding sites and the associated changes in binding enthalpy (\u0394H). For each titration Ca2+-free zGCAP was kept in a temperature controlled compartment, in which a series of small volumes of CaCl2 was injected. Representative examples for all zGCAPs are shown in 2 (zGCAP3) and \u221217\u2009kcal per mol of CaCl2 (zGCAP7). Small endothermic responses were only observed with zGCAP1  showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range.All zGCAPs contain four EF-hand motifs in their primary structure, where the first one is suggested to bind no Cah zGCAP1 a,5a. Howccessful . We inteplicable . In all 2+the multiphasic binding isotherm for Ca2+-binding gave a best fit with a two site model for zGCAP1, 2, and 3. This result can be best interpreted as having two high affinity sites filled with Ca2+, but the lower affinity site being occupied by Mg2+ .Size exclusion chromatography of myristoylated zGCAPs revealed that the monomeric form was the dominant species for all zGCAPs except for zGCAP5 and 7. A Ca2+-myristoyl switch operation in zGCAPs  differed significantly among the proteins exhibiting the following order: zGCAP5\u2009>\u2009zGCAP2\u2009>\u2009zGCAP7\u2009>\u2009zGCAP3\u2009=\u2009zGCAP4. No signals were observed for zGCAP1, although the purified protein was functional shown by the GC activation assay.Finally, the maximal amplitudes that were reached at the end of the Ca2+-induced conformational changes, which however have different consequences for the protein hydrodynamic properties indicating differences in the extent of conformational transitions.We conclude from these results that all zGCAPs undergo Ca2+-signaling that result in specific cellular responses can be mediated by rather similar Ca2+-sensor proteins, which are very often expressed in the same cell type. In order to work on these issues we chose to compare zGCAPs representing one subfamily of NCS proteins. These NCS proteins are well suited for a comparative analysis, since previous work has shown 1) that all zGCAPs are expressed in photoreceptor cells in the larval and adult stages of the zebrafish retina1314152+-binding proteins12152+-dependent manner12131516Calcium sensor proteins like the group of NCS proteins are involved in unique patterns of cellular regulatory pathways and therefore mediate various physiological responses including ion channel function, enzyme activity control and cellular trafficking22+-sensitivity of mammalian GCAP1, but less influence on the regulatory properties of mammalian GCAP21029in vitro conditions. We here demonstrate using complementary approaches that the six zGCAPs are myristoylated in cell lysates and in living cells. Further, myristoylation is not involved in reversible Ca2+-dependent membrane association  leading to one group of zGCAPs with high Ca2+-sensitivity  and one with low Ca2+-sensitivities . Myristoylation is catalyzed by a retinal NMT, which is not active before 7 dpf due the myristoylation pattern we reported previously for zGCAP3Our previous work on the six nonmyristoylated zGCAPs revealed that zGCAP1, 2 and 3 display GC-activating response curves that are halfmaximal around 30\u2009nM free  was done exactly as described121515Analysis of purified zGCAP samples by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and determination of GC activity (three to five repetitions) in the presence of zGCAPs as a function of the free [Ca2 stock solution at T\u2009=\u200925\u2009\u00b0C. The titration buffer was decalcified using a self-packed gravity flow Chelex 100 column (Bio-Rad). The remaining Ca2+concentration was determined by a BAPTA absorption assay and was found to range between 30 and 100\u2009nM. All buffers were filtered (0.22\u2009\u03bcm) and degassed at least 1h before use. At least three independent repetitions were made for each titration set, if not stated otherwise.ITC experiments with zGCAP-isoforms were performed on a VP-ITC from MicroCal  exactly as described previously for mammalian GCAP1 variants2+ into decalcified buffer without any zGCAP was performed, but did not show significant heat changes in the recording cell. Each titration was analyzed by a model implemented in the software Origin  assuming either three or two Ca2+ binding sites. The best fitting results out of these models were used to obtain dissociation constants KDapp and enthalpy changes (\u0394H).Reference injections of Ca2+-titration experiments and data analysis were performed as outlined elsewhere30312 of the highest grade available was dissolved in the decalcified buffer to a final concentration of 46\u2009mM. This stock solution was used to obtain Ca2+ samples of 0.4\u2009\u03bcM, 0.7\u2009\u03bcM, 0.9\u2009\u03bcM, 1.1\u2009\u03bcM, 1.6\u2009\u03bcM, 2.5\u2009\u03bcM, 4.8\u2009\u03bcM, 14\u2009\u03bcM, 37\u2009\u03bcM and 46.2\u2009\u03bcM. All buffers were filtered (0.22\u2009\u03bcm) and degassed for at least 1h before use. Immobilization of protein samples was achieved by attaching them to the carboxy-methyl dextran matrix of CM5 sensorchips  via the terminal amino group or via internal accessible lysine residues. Typical immobilization densities ranged from 3.5\u2009ng to 10.5\u2009ng/mm2.For SPR experiments, we used exactly the same decalcified buffer conditions as for the ITC experiments, except that Tween20 was added to a final concentration of 0.005% (v/v).The CaHow to cite this article: Sulmann, S. et al. Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding. Sci. Rep.5, 11228; doi: 10.1038/srep11228 (2015)."}
+{"text": "To describe the allergenic food ingredients present in complementary feeding and to examine their potential association with persistent eczema.n 1537). An allergenic food was determined present, if labelled as an ingredient and not if stated as \u2018may contain\u2019. Eczema was determined if the UK working party diagnostic criteria were satisfied at 6 and 12 months.Prospective data on complementary feeding practices, using a detailed food diary over the first 6-weeks of complementary feeding, were collected as part of Cork BASELINE Birth Cohort Study  were introduced to solids between 17-26 weeks, 18% <17 weeks. At least one allergenic food ingredient was provided to 64% of infants; including cow\u2019s milk (56%), wheat (43%), soy (41%), eggs (10%), fish (8%) and kiwi (7%). Infant breakfast cereals were the main source of exposure to cow\u2019s milk (71%), wheat (92%) and soy (98%). First exposure to an allergen was typically between 17-26 weeks (73%) and 26% were exposed after 26 weeks. Mothers <25 years (84 vs 70%), who were smokers (83 vs 69%) and had no university degree (82 vs 64%) were more likely to give an allergenic food ingredient in the complementary feeding diet . There was no association between exposure to allergenic food ingredients and persistent eczema [OR 0.96 ] or between exposure either before 17 weeks [OR 2.43 ] or after 26 weeks [OR 1.29 ] and persistent eczema, compared with 17 to 26 weeks .Infant breakfast cereals were the main source of cow\u2019s milk, wheat and soy during the first 6 weeks of transition to solid food. Exposure to allergenic food ingredients in the initial stage of complementary feeding had no influence on persistent eczema. Clear guidance on the introduction of allergenic food ingredients in foods labeled as appropriate from 4-6 months is required."}
+{"text": "HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival  compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and  HER2 status in breast cancer is important in the diagnosis of breast breast cancer by pathologists HER2 protein and/or gene amplification is of enormous importance, as adjuvant or neoadjuvant therapy decisions, made on the assays, all rely on the scores that are given by these tests Determination of HER2 gene amplification.The ASCO guidelines approved several test assays for the HER2 status assessment including immunohistochemistry (IHC) and/or in situ hybridization (ISH) technics with different visualization methods as fluorescence (FISH), silver (SISH) or chromogenic (CISH) labeled probes In this study, we addressed the question whether such tumors with discordant results by IHC and SISH represent prognostically relevant subgroups. We are not aware of any previous studies, correlating results of this bright-field assay with patient prognosis Tissue microarrays (TMAs) containing eligible 652 samples from breast cancer patients were selected for this study. Eligibility was decided upon availability of sufficient tumor tissues in the paraffin blocks. The TMAs were constructed previously as described The cohort was composed of 318 primary breast cancer (49%), 26 local recurrences (4%), 258 axillary lymph node metastases (39%) and 50 visceral metastases (8%). For the TMA analysis there were 589 of 652 interpretable samples available.Histologically, there were 280 invasive ductal (88%), 35 invasive lobular carcinomas (11%) and 5 special types (1%). Grading was assessed by applying the modified Bloom and Richardson grading system after Ellis and Elston on the primary tumors and on most of the metastatic and recurring lesions Details of clinico-pathological parameters are depicted in HER2 dual DNA Probe Cocktail with the UltraView SISH Detection Kit (HER2 locus) and UltraView Red ISH DIG Detection Kit (CEP17 locus) were done on the same tissue micro arrays. Reactions mentioned above in both detection systems (except the hybridization step) were carried out on the automatic Benchmark system . The hybridization step (containing the HER2 and CEP17 gene locus on chromosome 17) was carried out manually according to the instruction of the EnzMettrade mark, Nanoprobes  and on the automatic Benchmark ULTRA system . The HER2 protein was visualized as a purple-red (EnzMet) or DAB brown (Inform) membranous stain, the gene loci (HER2) as confluent black aggregates or singular black spots, the CEP17 region with red spots or aggregates For the simultaneous detection of the HER2 protein and HER2 gene copy number, we used a previously described protocol by Tubbs et al and Downs-Kelly et al. The simultaneous detection of the HER2 protein and HER2 gene was originally scored according to the 2008 guidelines on HER2 scoring in breast cancer SISHHER2 signals were scored as follows: the number of signal copies and the ratios (HER2/CEP17) were calculated, gene copies (>6) or cluster formations  were defined as amplified. Similarly, a ratio >2.2 was set as amplified status; a ratio <1.8 was negative, and a ratio of 1.8\u20132.2 was referred to as unequivocal (according the 2008 guidelines) and using ratio >2 and > or equal to 6 HER2 gene copies (in >10% of the tumor cells) for a positive status (according to 2013 guidelines).HER2 protein expression was scored as follows: 0 (no staining), 1+ (weak and incomplete membrane staining), 2+  or 3+ (strong complete homogenous membrane staining in more than 30% of the invasive tumor cells). According to the 2013 ASCO/CAP guidelines, the required percentage (10%) for score 3+ and 2+ were used for the re-evaluation of the results based on the 2008 guidelines . Categorical data were analyzed using Chi- Square test. Spearman log rank, Pearson, Breslow and Tarone Ware correlation were used to correlate HER2 protein expression scores and HER2 gene copy number with and clinic-pathological parameters  and overall survival. Non-parametrical tests as Kendall Tau and Spearman rho were used to test correlation between overall survival and HER2 scores with IHC and SISH.There were 589 interpretable spots containing sufficient amount of invasive carcinomas on the TMAs. Sixty of 589 cases were score 3+ by IHC (10%). Fifty-seven of 60 IHC score 3+ cases showed also amplification by SISH (95%). 519 of 589 cases were negative (score 0/1+) by IHC (88%). 3 of 519 IHC negative breast cancers (score 0/1+) showed amplification by SISH (0.6%). Ten of 589 samples were score 2+ by immunohistochemistry (5%), 6 of these cases displayed amplification by SISH (60%). One of the IHC score 3+ cases showed polysomy of both CEP 17 and HER2 gene (<1%) Table 2.. and There was a significantly shorter overall survival among patients whose tumors were either amplified by SISH (p\u200a=\u200a0.002) or were IHC score 2+ or 3+ (p\u200a=\u200a0.006), when these two parameters were analyzed separately . and Fig 3. and When combining IHC and SISH scores, there was as tendency to shortened overall survival in patients with IHC score 1+ with simultaneous gene amplification, when compared to IHC score 0/1+ tumors without gene amplification , Fig. 4. IHC HER2 scores significantly correlated with hormone receptor status (p\u200a=\u200a0.001) and histological grading (p<0.001) and lacked correlation to pathological tumor stage, nodal status or to the presence of distant metastases.HER2 scores also showed a significant correlation to hormone receptor status (p\u200a=\u200a0.002) and to histological grading (p<0.001), but not to tumor stadium, nodal status or to distant metastases.SISH Original HER2 status was available in 310 primary tumors performed by IHC and FISH technology. There were 6 of 310 (1.9%) discrepant cases with IHC and 7 of 310 (2.2%) discrepant cases with FISH as compared to double IHC/SISH HER2 testing.HER2 gene copy number. Importantly, the simultaneous detection of HER2 protein and HER2 gene copy number facilitates the identification of relevant prognostic subgroups with poor prognosis especially in the IHC score 0/1+ group with amplification and in the IHC score 3+/2+ group without gene amplification.We analyzed HER2 status in a cohort of invasive breast cancers using a double IHC/SISH assay, enabling the simultaneous detection of the HER2 protein expression and the The novel technology of bright field protein and gene detection assay was developed and described in 2004 by Tubbs et al In our TMA-based study, we demonstrate an excellent correlation between IHC score 3+ and gene amplification by SISH using a dual probe for IHC and SISH. Concordance between IHC and ISH technologies in HER2 status assessment in breast cancer has been the subject of several previous studies Here, we demonstrate that the novel bright-field ISH assay enables improved diagnosis in borderline breast cancer cases. Simultaneous assessment of HER2 status by IHC and SISH shows a significant correlation to overall survival. The use of different ISH assay for the assessment of the HER2 status was assessed in several previous works HER2 gene amplification represent prognostically poor subgroups with shortened overall survival Our study confirms existing data that IHC score 2+/3+ tumors as well as tumors with HER2 gene. This amplification can vary in size, especially in IHC scores 3+ cases The underlying mechanism of protein overexpression in breast carcinomas is preferential amplification or polysomy of the As to the real frequency of IHC score/1+ cases having simultaneous amplification, the literature is inconsistent, mostly due to the fact, that IHC score 0/1+ cases do not need to undergo obligatory reflex testing with an ISH technology Several problematic topics in routine HER2 testing prompted the revision of the 2007 ASCO/CAP guidelines on HER2 diagnostic guidelines. Diversities in the interpretation of chromosome 17 aneuosomy and of HER2/CEP17 ratios may lead to false negative and false positive HER2 status, having an immediate impact on therapy options for the given patient. Tumor heterogeneity, biological characteristics and pre-analytic issues have been also shown to influence accuracy in HER2 status assessment HER2 gene status. Concordance in HER2 status between primary tumors and metastastic sites was the subject of several previous studies, confirming a high consistency in cases with only one metastastic site Our cohort contained also samples from metastases but not from the primary lesions. In metastatic lesions, there was only one case with discordant HER2 protein and HER2 gene amplification independently from HER2 protein expression in IHC score 0/1+ cases correlates with tendency to poorer overall survival. Importantly, our data suggest that the double immunohistochemistry and silver labeled in situ hybridization assay identifies a prognostically relevant IHC 2+/3+ breast subgroup without HER2 amplification, which cannot be detected using a FISH alone approach.We provide data that"}
+{"text": "The authors have provided a corrected version of ramA was a replication of the image shown for the wild type strain K. pneumoniae Ecl8. The authors have provided a corrected The authors would like to correct The authors confirm that these changes do not alter their findings. The authors have provided raw, uncropped blots for S1 FigybhT and yrbF promoters. The experimental setup was as described previously in 32P-\u03b3 ATP. Purified RamA (200 nM) and the labelled DNA probes (2 nM) were incubated on ice. All reactions were performed on ice prior to electrophoresis on 7.5% native gel. Lane 1 of each panel indicates the labelled DNA probe only, Lane 2 is the BSA control and Lane 3 contains RamA+DNA. The dried gel was scanned after overnight exposure to the phosphor screen under default settings on the phosphorimager Typhoon FLA7000IP .Raw data phosphor image scan for the EMSA analyses on the (PDF)Click here for additional data file."}
+{"text": "GLIS3 (GLI-similar 3) is a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. Human GLIS3 mutations are extremely rare.GLIS3 mutations.The purpose of this article was determine the phenotypic presentation of 12 patients with a variety of GLIS3 gene mutations were sought by PCR amplification and sequence analysis of exons 1 to 11. Clinical information was provided by the referring clinicians and subsequently using a questionnaire circulated to gain further information.GLIS3 who did not present with congenital hypothyroidism. All patients presented with neonatal diabetes with a range of insulin sensitivities. Thyroid disease varied among patients. Hepatic and renal disease was common with liver dysfunction ranging from hepatitis to cirrhosis; cystic dysplasia was the most common renal manifestation. We describe new presenting features in patients with GLIS3 mutations, including craniosynostosis, hiatus hernia, atrial septal defect, splenic cyst, and choanal atresia and confirm further cases with sensorineural deafness and exocrine pancreatic insufficiency.We report the first case of a patient with a compound heterozygous mutation in GLIS3 phenotype, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. All but 2 of the patients within our cohort are still alive, and we describe the first patient to live to adulthood with a GLIS3 mutation, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.We report new findings within the  KCNJ11 and ABCC8 genes, encoding the Kir6.2 and SUR1 subunits of the pancreatic ATP-sensitive potassium (KATP) channel involved in regulation of insulin secretion, account for about half of cases of PND , Wolcott-Rallison syndrome (EIF2AK3), and pancreatic agenesis   result in the concomitant presentation of PND and congenital hypothyroidism. GLIS3, a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains, maps to chromosome 9p24.3-p23 (OMIM 610192)  and congenital hypothyroidism can result from a number of genetic mutations. Mutations in  610192) , 8. In 2GLIS3, providing additional insight into the clinical features associated with this rare condition.We describe a case series of 12 patients with mutations in www.diabetesgenes.org), from clinical notes, and subsequently by using a questionnaire circulated to referring clinicians to gain further information.The study was conducted in accordance with the Declaration of Helsinki principles with informed parental consent given on behalf of children. Clinical information was provided by the referring clinicians via a neonatal diabetes request form (available at GLIS3 gene mutations were sought by PCR amplification (primer sequences are available on request) and sequence analysis of exons 1 to 11 by comparison with the reference sequence NM_001042413. Exon 1 is noncoding (the 5\u2032 untranslated), and the start codon is located within exon 2.\u2212\u0394\u0394tC method.The effect of coding variants on the protein was investigated in silico using the bioinformatic tool Alamut (Interactive Biosoftware). When PCR amplification failed, suggesting a homozygous deletion, parental samples were investigated by real-time quantitative PCR on an ABI 7900 system (TaqMan assay with SYBR Green detection), and the copy number of exons 1 to 11 was determined by the 2Patients 1 and 10 were analyzed for all of the known neonatal diabetes genes using a targeted next-generation assay . MutatioGLIS3 mutations identified in our case series. Deletions of \u22651 of the 11 exons of GLIS3 were observed in most patients. Patients 1, 5, and 10 harbor missense mutations , affecting highly conserved amino acids located in the DNA binding domain and so are likely to be pathogenic, thus severely affecting the function of the GLIS3 protein. Patient 1 is the first patient reported to be a compound heterozygote for 2 mutations in GLIS3 (a deletion and a missense mutation). Patients 3a and 3b are siblings. GLIS3, showing the mutations described in our patient population.GLIS3 mutation to survive into adulthood (aged 36 years). Patient 6 died from liver failure with marked portal hypertension and esophageal variceal bleeding, and patient 9 died of overwhelming measles sepsis and multiorgan failure at 6 months of age. Nine patients in our cohort were born to parents who were first cousins. Patient 3, who was born to apparently not related parents was described previously by Dimitri et al (GLIS3 mutation born to Caucasian parents who are not related (patient 1) and the sibling of patient 3a (patient 3b). A homozygous deletion was confirmed in patient 6; however, consanguinity was denied by the patient's parents.The clinical features of all of the patients are summarized in ri et al . In thisGLIS3 mutations. Age at diagnosis ranged from birth to 23 days. All patients were insulin treated. Patients were initially treated with insulin at 0.4 to 2.0 U/kg/24 h (patient 4 required 2.0 U/kg/24 h), demonstrating a range of insulin sensitivities. Patient 11 had high insulin sensitivity, leading to recurrent hypoglycemic episodes with very small doses of insulin. Others had labile blood glucose (patients 3a and 3b), and one patient clinically demonstrates insulin resistance, particularly during periods of illness (patient 2). In the first year of life, this patient required 0.5 to 0.7 U/kg of insulin per day. However, during times of intercurrent illness, doses of insulin at 3 to 4 times her normal requirement were required to achieve normoglycemia. Despite erratic blood glucose control with periods of insulin resistance, her glycosylated hemoglobin at 1 year of age was 7.8% (62.0 mmol/mol).PND was the only consistent feature of all of our patients with GLIS3 mutations in the first year of life despite normal levels of free T4, which have since normalized. Patients 6 and 10 had normal thyroid anatomy on ultrasonography, and in comparison with other patients with GLIS3 mutations, TSH responded appropriately to conventional doses of levothyroxine with an appropriate increase in the levothyroxine dose with age. Notably, the postmortem examination of the thyroid gland in patient 6 demonstrated a paucity of colloid as well as extensive perifollicular and interstitial fibrosis, explaining the need for thyroxine despite apparently normal thyroid anatomy on ultrasonography.Apart from patient 1, all patients had with congenital hypothyroidism. This is a cardinal feature of the NDH syndrome described previously in all patients with mutations, 9. Althmutations. SimilarLiver disease was documented in 7 of our 12 patients. The hepatic dysfunction presented concomitantly with renal abnormalities and ranged from hepatitis (patients 3b and 4) to hepatic fibrosis and cirrhosis . Nine patients have anatomical kidney changes. Of these, 7 children showed variable renal cystic dysplasia ranging from an isolated cyst observed in patient 10 and bilateral calyceal calcification in patient 11 to extensive cystic renal dysplasia  . PatientGLIS3. She presented with osteopenia, significantly delayed rib fracture healing, and a marked thoracolumbar scoliosis , which subsequently normalized without treatment. Patient 8 was osteopenic by 6 months and despite vitamin D supplementation, her 25-hydroxyvitamin D3 level was 33.5 nmol/L at 6 weeks of age. Adjusted serum calcium in this patient was 2.0 mmol/L in the first week of life and subsequently normalized to 2.5 mmol/L by 12 days of age without calcium replacement. Alkaline phosphatase peaked at 3 months to 2100 U/L but had normalized by 2 years of age. No fractures were identified in patient 8.Patient 2 was the first patient to be described with skeletal manifestations due to a mutation in coliosis , A and Bcoliosis . PatientGLIS3 mutation to present with a splenic cyst (patient 3b).Malabsorption due to exocrine pancreatic insufficiency as demonstrated by low fecal elastase was a feature in patients 2, 3a, 3b, and 7 in our case series. These 4 patients have been treated with pancreatic enzyme supplementation. We report the first patient with a GLIS3 exon affected as suggested by Senee et al  and a deletion of exons 1 to 4 of GLIS3 , have been described previously; the 7.5-kb transcript is strongly expressed in pancreas, thyroid, and kidney with smaller transcripts predominantly expressed in liver, kidney, heart, and skeletal muscle . We thus speculate that the missense mutation may be a hypomorphic change resulting in a mutated protein possessing some residual function.The variability in phenotype observed between our patients and others  has beenmaller 0.\u20132.0 kb, maller 0.\u20132.0 kb, GLIS3 plays a key role in pancreatic development, particularly in the embryogenesis of \u03b2-cells, which explains why our patients and others with GLIS3 mutations to date have presented with PND (GLIS3 interacts with key regulatory genes in pancreatic embryogenesis including ONECUT1 and NEUROGENIN3 (NEUROG3)  \u201315. GLISINS gene . Therefodiabetes , 18. ThePAX8 and NKX2-1 (TTF1/NK2 homeobox-1 or thyroid transcription factor 1), involved in thyroid cell differentiation and proliferation and subsequent expression of genes encoding for thyroglobulin, thyroid peroxidase, thyrotropin receptor, and the sodium-iodide symporter  leading to a frameshift and likely degradation of the transcript reported by Senee et al  and smaller (0.8\u20132.0 kb) transcripts are expressed in the kidney and smaller (0.8\u20132.0 kb) transcripts are expressed in the liver  were observed in patient 5  (GLIS3 and WW domain containing transcription regulator 1 (WWTR1) overlaps in the kidney, and mutations in both genes result in renal cystic dysplasia with a high glomerular cystic load  . A reductic load , 24. SimGLIS3 mutation to present with sensorineural deafness (patient 3a)  . We now  disease . FurtherSLC1A1 (solute carrier family 1). SLC1A1 is principally expressed in neurons, kidney, and small intestine. Mutations in SLC1A1 are thought to cause dicarboxylic aminoaciduria (In one of our families (patient 2) and in a family reported by Senee et al , the delaciduria  and haveaciduria , 28.GLIS3 gene. Our current study has focused on the clinical manifestations of patients with these mutations, and further in vitro work is required to test the GLIS3 missense variants functionally in biological models.We have presented an extended spectrum of clinical features in relation to patients with mutations in the GLIS3 characteristically present with neonatal diabetes with variable insulin sensitivity and congenital hypothyroidism due to a range of underlying causes. Although mutations in GLIS3 are more common in consanguineous pedigrees, we report 2 patients from apparently unrelated parents with GLIS3 mutations. We also report the first patient with compound heterozygous mutations in GLIS3 with preservation of thyroid function, who is also the first patient reported with a GLIS3 mutation to survive into adulthood. Hepatic and renal disease is common in the patients in our cohort, but the presentation is variable. We report new findings within the GLIS3 phenotype including cardiac disease, hiatus hernia, sagittal craniosynostosis, splenic cystic change, and choanal atresia, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. We report further patients presenting with exocrine pancreatic insufficiency and sensorineural deafness. All but 2 of the patients in our cohort are still alive, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.In summary, patients presenting with mutations in"}
+{"text": "In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a \u2018poised\u2019 state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a \u2018TCCCC\u2019 sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Embryonic stem (ES) cells have the unique ability to self-renew indefinitely as well as to differentiate in response to internal as well as external stimuli69Bivalency of chromatin has therefore become an important property to investigate the functional relevance of a gene through development, and the presence of bivalent genes in human and mouse ES cells has been validated by many studies independently9121314ChIP sequencing raw data for H3K4me3 and H3K27me3 histone marks in murine and human ESCs was obtained from Gene Expression Omnibus (GEO)The one2one orthologous regions between human and mouse  were obtained from Ensembl BioMartFor the clustering of bivalent promoters into four groups, we gathered chip sequencing data for three different forms of RNAPII: RNAPIIS5P, RNAPIIS7P and 8WG16We calculated the CpG density as the ratio of observed to expected CpG countsFor identification of factors enriched at bivalent and H3K4me3 promoters, we used data from 49 and 99 ChIP-seq experiments for several factors in human and mouse embryonic stem cells respectivelyRNA sequencing data in murine ES cellsThe sequence motif enrichment analysis was performed with the command findMotifs.pl from HOMERr)\u2009=\u20090.75, for H3K4me3 r\u2009=\u20090.84), but not across human datasets . There are other factors contributing to the variation between samples, for example ES cells were grown in diverse culture conditions, and using different cell lines as well as various antibodies across datasets  of H3K4me3 and H3K27me3 ChIP sequencing (ChIP-seq) datasets for human ES cells and 8 pairs for mouse ES cells from the Gene Expression Omnibus (GEO) database and the Roadmap Epigenomics Project . After adatasets . We therdatasets . Eight Hdatasets . Adding datasets . There wdatasets .rd of H3K27me3 top promoters in any individual dataset overlapped with HC bivalent promoters   and S7, romoters .We also checked whether the peaks of H3K27me3 and H3K4me3 modifications were present at the same genomic location within a promoter region and found that over 95% of H3K27me3 and H3K4me3 peaks overlapped in each pair of samples at HC bivalent promoters. Both chromatin modifications were indeed present at the same genomic location . We compIn summary, by integrating data from multiple studies we identified HC human and murine bivalent promoters, which could not be identified by simply selecting the top peaks from individual samples. The HC bivalent promoters were highly enriched for developmental regulators compared to non-HC bivalent promoters.Bivalent promoters are known to show variation in their levels of occupancy by RNA polymerase II\u221251) while cluster 4 promoters were enriched for developmental functions such as organ morphogenesis . Cluster 3 promoters contained transcription factors important for specific lineages like haematopoiesis factors Gfi1 and Meis1, whereas cluster 4 contained multiple members of transcription factor families controlling development such as winged helix/forkhead box (Fox) and Hox families.HC bivalent promoters could be classified in four distinct clusters based on the presence of PRC1 components and forms of RNAPII . The firet al. (12) suggested that PRC1 was absent in our PRC1 low bivalent promoter  and PRC1 high (cluster 3 & 4). Ku promoter . Ring1b promoter .In summary, all HC bivalent promoters are occupied by components of both PRC1 and PRC2. There exists a distinct set of metabolic genes (cluster 2) which though bivalently marked has RNAPII (S7P) and is expressed at a higher level than other bivalent genes.RNA polymerase II (PolII) may be present but stalled at the promoters of bivalent genes and short (abortive) transcripts may be detected at their promotersAs bivalent genes are thought to be poised for activation or repression, we hypothesised that these genes might be more likely to be differentially expressed upon perturbation of ES cells. We therefore used a collection of differentially expressed genes upon deletion or over-expression of 91 transcription and epigenetic factors in mouse ES cells, and found that 98% of differentially expressed gene sets by the overexpression of at least one TF significantly overlapped  with bivalent genes, and 89% differentially expressed gene sets by the down-regulation of at least one TF . To chec\u221271) and transcription factor activity ; whereas species-specific promoters were not enriched for the two above terms (\u221216) and glycoprotein  and the human-specific for plasma membrane part  and alternative splicing .To perform a systematic comparison of chromatin status between human and mouse promoters in ES cells, we used 16,639 one-to-one orthologous genes between the two speciesve terms . SpecifiTo check whether the chromatin status across species is reflected in the gene expression status, we focused on five groups of promoters : three gAs shown in the first section, the bivalent status of promoters is primarily determined by the detection of an H3K27me3 modification . CpG isl3738Over 90% of our HC bivalent promoters in ES cells in both species overlap with at least one CGI region, whereas only 8% (37 of 397) of human H3K27me3 only promoters contained a CGI and no mouse H3K27me3 only promoters (none of 152) contained a CGI . Previou41The loss of H3K27me3 in rodents (mouse and rat) compared to human ES cells at many developmental genes has been associated with depletion of CGIs; mouse CGI erosion has been characterised at MYO1G, CLEC4G and MYF6 gene loci with corresponding H3K27me3 lossIn summary, the H3K27me3-only CpG-poor promoters demonstrate that polycomb recruitment does not only depend on CpG density. Although the CpG density largely correlates with H3K4me3 and H3K27me3 profiles across promoters, the loss of CGI on a promoter does not always imply a corresponding loss of the H3K4me3 and/or H3K27me3 modification on that promoter.As both active (H3K4me3-only) and bivalent promoters are CpG-rich, we investigated possible modes of distinction between the two in ES cells. Voigt, Tee, and Reinberg\u2212256). Moreover, the co-repressor c-terminal binding protein 2 (CTBP2), required for PcG recruitment in Drosophila4647\u22123). Four other epigenetic regulators, Utf1, Tet1, Rest and Setdb1 were highly enriched at mouse bivalent regions. Utf1  was recently identified as a component of bivalent chromatin by acting as a buffer against full activation of bivalent genesTo identify factors preferentially binding to bivalent promoters, we calculated the overlap between transcription and epigenetic factor binding sites (peaks) and bivalent promoters. Four out of 49 and eleven out of 99 factors characterised by ChIP-seq preferred bivalent promoters in human and mouse respectively . As expeAs expected, many TFs (33 out of 49 factors in human and 39 out of 99 factors in mouse) were enriched at active (H3K4me3-only) promoters. This included known regulators of pluripotency in ES cells such as Klf4, Esrrb, Oct4, Sox2, and Nanog . Only twde novo motif identification on bivalent promoters by providing active promoter sequences as background in HOMER softwarede novo motif discovery). The \u2018TCCCC\u2019 motif was most similar to the known binding sequence of the Mzf1 transcription factorde novo motif enrichment was performed on active human and mouse promoters using bivalent promoter sequences as background, they were enriched for a \u2018CGGAA\u2019 motif found in 40% of the active promoter sequences, which was not enriched in bivalent promoters. This motif is the most similar to the known motif for Elk1 transcription factor  promoters, but more than H3K27me3 only and latent promoters. Active promoters were preferentially occupied by pluripotency factors. On the other hand, bivalent promoters were enriched for Polycomb factors as well as other chromatin modifiers. The factors enriched at bivalent promoters show very little overlap with the ones enriched at active promoters. These findings are consistent with the observed spatial segregation of transcriptional networks in ES cells where Nanog and Polycomb proteins were shown to occupy distinct nuclear spacesBivalent chromatin domains bearing both H3K4me3 and H3K27me3 modifications have been shown to be a key feature of developmentally regulated genes in ES cells8913149149Bivalent promoters are thought to be poised for rapid activation or inactivation during differentiation13We computed binding profiles of PRC components (PRC1 and PRC2) and various forms of RNA polymerase II at bivalent promoters in murine ES cells. All HC bivalent promoters were marked by Suz12, Jarid2, Ring1b and Cbx7. To note, the PRC2-only group defined byMore than half of high-confidence bivalent promoters were conserved between human and mouse, suggesting the existence of a set of genes bivalently marked across most mammalian ES cells . These g54et al. (2014) recently suggested that bivalency is the default chromatin structure for CpG-rich, G+C-rich DNASince a high density of un-methylated CpG is sufficient for vertebrate polycomb recruitment3839On bivalent promoters, the CpG density and H3K27me3 modification are highly correlated. By performing a cross-species comparison, a small fraction (~5%) of human CpG-rich HC bivalent promoters has the corresponding CpG eroded in the mouse genome, while no CpG-rich bivalent promoters in mouse are eroded in human. This erosion of CpG density was correlated with the loss of H3K27me3 and H3K4me3et al.It is intriguing how bivalent domains are established in ES cells. Voigt De novo motif discovery at HC bivalent promoters identified a \u2018TCCCC\u2019 motif in both human and mouse ES cells which was not enriched at active promoters. This motif was present in about half of the HC bivalent promoters and is similar to the sequence motif of MZF1As active (H3K4me3-only) and bivalent promoters are both CpG rich, it is key to unravel the distinguishing factors between these two groups. In summary, this meta-analysis revealed several novel aspects of bivalency in mammalian ES cells and will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Further work will be aimed at understanding how the HC bivalent promoters identified here are resolved in different cellular lineages during differentiation.How to cite this article: Mantsoki, A. et al. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells. Sci. Rep.5, 16791; doi: 10.1038/srep16791 (2015)."}
+{"text": "The homeodomain transcription factor Hoxa2 interacts with the RING-finger type E3 ubiquitin ligase RCHY1 and induces its proteasomal degradation. In this work, we dissected this non-transcriptional activity of Hoxa2 at the molecular level. The Hoxa2-mediated decay of RCHY1 involves both the 19S and 20S proteasome complexes. It relies on both the Hoxa2 homeodomain and C-terminal moiety although no single deletion in the Hoxa2 sequence could disrupt the RCHY1 interaction. That the Hoxa2 homeodomain alone could mediate RCHY1 binding is consistent with the shared ability all the Hox proteins we tested to interact with RCHY1. Nonetheless, the ability to induce RCHY1 degradation although critically relying on the homeodomain is not common to all Hox proteins. This identifies the homeodomain as necessary but not sufficient for what appears to be an almost generic Hox protein activity. Finally we provide evidence that the Hoxa2-induced degradation of RCHY1 is evolutionarily conserved among vertebrates. These data therefore support the hypothesis that the molecular and functional interaction between Hox proteins and RCHY1 is an ancestral Hox property. Hoxa2 belongs to the extremely well-conserved Hox gene family which includes 39 members in mammals. Mammalian Hox genes are organized in four clusters located on different chromosomes and can be classified in 13 paralogue groups based on their sequence similarities and their relative position within the clusters. Hox genes code for transcription factors which fulfill well-documented functions during embryonic development. In particular, their most spectacular activities are associated to the patterning of the main body axis, limb development and organogenesis .Kip1, polH, CHK2 and c-MYC, are known to be involved in the control of cell proliferation and cell death. Consequently, by influencing the abundance of these targets, RCHY1 has been shown to be a regulator of DNA damage response and cell cycle progression . RCHiewed in , 9. Indeiewed in \u201313. Moreiewed in .In a former study, we reported that Hoxa2 expression correlated with a decrease in RCHY1 protein levels. We provided evidence that the RCHY1 decay induced by Hoxa2 involved the proteasome pathway but in an ubiquitin-independent way. Finally, correlatively to the RCHY1 degradation, Hoxa2 expression was also shown to decrease the ubiquitination of p53, in turn, increasing its abundance ), both terminal domains (Hoxa2HD [139\u2013198]), or the homeodomain (Hoxa2\u0394HD [\u03b4139\u2013198]) were removed  that the HOXA2-RCHY1 interaction mainly takes place in the nucleus (2) that the RCHY1 decay induced by HOXA2 depends on both the 19S and the 20S proteasome particles, (3) that the interaction involves molecular determinants from at least two distinct HOXA2 regions providing contacts which are sufficient but not necessary for the binding of RCHY1 and (4) that some HOXA2 deletion derivatives, though still capable of interacting with RCHY1, have lost the ability to provoke its protesomal degradation. Finally, the results provided in the present study support that the downregulation of RCHY1 provoked by HOXA2 is conserved among orthologues from other vertebrate species and that binding to RCHY1 seems to be generic characteristic of HOX proteins yet the ability to stimulate its protesomal degradation is shared only by a subset of them.WMAA protein mutated in the hexapeptide sequence located in the N-terminal part of Hoxa2. Similarly to Hoxa2\u0394N, the Hoxa2WMAA protein has been shown to induce RCHY1 decay [\u0394N and Hoxa2WMAA mutants lack the hexapeptide sequence but maintain full activity with regards to RCHY1 destabilization, we propose that the Hoxa2-mediated effect on RCHY1 degradation is independent of the transcription activity of Hoxa2 and corresponds to a novel non-transcriptional activity for Hoxa2.The N-terminal part of Hoxa2 seems dispensable to the induction of RCHY1 degradation. Indeed, the Hoxa2 deletion mutant lacking the N-terminal portion of the protein destabilizes RCHY1 to the same extent as the WT Hoxa2. This result could be related to what we previously showed for the Hoxa2Y1 decay . This heY1 decay  and in tY1 decay . As bothKQN-RAA), as we previously reported [Conversely, although all the HOXA2 variants tested so far remain capable of interacting with RCHY1, the ability to induce RCHY1 destabilization is only affected upon deletion of the C-terminal part or the entire homeodomain of HOXA2, or as a consequence of point mutations in the homeodomain .COS-7 cells were transfected with vectors coding for VN(TIF)Click here for additional data file.S2 Fig173RCHY1 coding vector together with the VC155p27Kip1 coding vector. Nuclei were stained with DAPI (blue).COS-7 cells were transfected with the VN(TIF)Click here for additional data file."}
+{"text": "Decreased ability in oxygen delivery and diffusion of the blood vessels and increased oxygen consumption in the rapidly proliferating cancer cells both contribute to reduced oxygen availability in the solid tumors, a major signature of the tumor microenvironment . The oxyHIF is a heterodimeric transcription factor, consisting of an oxygen-regulated \u03b1 subunit and a constitutively expressed \u03b2 subunit. The regulation of HIF-\u03b1 protein levels is a critical determinant of HIF-mediated gene transcription. Our previous study showed that chronic hypoxia causes the selective decay of HIF-1\u03b1 at least in part by HSP70- and CHIP-dependent ubiquitination and proteasomal degradation . The levSLC2A3, GPI, PDK3, HGF, in HeLa cells exposed to chronic hypoxia (1% O2 for 72 hr) [2 for 24 hr) [Moreover, we further found that double knockdown of PRDX2 and PRDX4 selectively increases expression of a subset of HIF target genes, including HIFs have been the attractive targets for cancer therapy. Identification of the specific HIF regulators under conditions of acute and chronic hypoxia may yield the new therapeutic strategies. Indeed, high levels of PRDX2 and PRDX4 are significantly correlated with increased survival of patients with bladder cancer (our unpublished data), suggesting that PRDX2 and PRDX4 may be the new therapeutic targets for treatment of cancer."}
+{"text": "During 2001\u20132014, predominant influenza A(H1N1) and A(H3N2) strains in South America predominated in all or most subsequent influenza seasons in Central and North America. Predominant A(H1N1) and A(H3N2) strains in North America predominated in most subsequent seasons in Central and South America. Sharing data between these subregions may improve influenza season preparedness. During 2002\u20132008, infection with influenza viruses caused 40,880\u2013160,270 deaths each year throughout the Americas  and North America 4,535,508 results  to the Global Influenza Surveillance and Response System  of 8 years in Central America and 2 (17%) of 12 years in South America, when there was 1 southern temperate winter epidemic and a smaller northern temperate winter epidemic, all subregions had 1 annual influenza epidemic that lasted \u22485 months.The predominant influenza A(H1N1) virus strains in South America predominated in 9 of 9 subsequent seasons in Central America and 12  of 13 subsequent seasons in North America . SimilarThe predominant A(H1N1) virus strains in North America predominated in 7  of 9 subsequent seasons in Central America and 10  of 12 subsequent seasons in South America. A(H3N2) virus strains in North America predominated in 8  of 12 subsequent seasons in Central America and 10  of 13 subsequent seasons in South America. Influenza B virus strains in North America predominated in 9  of 12 subsequent seasons in Central America and 7  of 13 subsequent seasons in South America. Virus strains that predominated in North America during 1 season were less likely to predominate in the subsequent season in North America .At least 1 component of the Southern Hemisphere vaccine composition recommendations matched a predominant antigenic characterization in South America in 13  matched the Southern Hemisphere recommendations and 24  matched the Northern Hemisphere recommendations .Our findings suggest that virus strains identified during influenza epidemics in South America typically became predominant in subsequent epidemics in Central and North America. Almost as frequently, virus strains identified during epidemics in North America became predominant in the subsequent Central and South America epidemics. Although strain selection for 1 hemisphere\u2019s vaccine formulation typically occurs before influenza activity is widespread in the opposite hemisphere, health officials have an opportunity to anticipate which influenza virus strains may predominate by observing activity in other subregions. For example, influenza A(H1N1)pdm09 virus predominated in Brazil during 2013  and then aggregated by subregion. These samples may not be geographically representative. Additional data will be needed to determine whether the characteristics of 1 subregion reliably predict influenza epidemics in another. New viral strains that appear might be introduced from outside the Americas (In summary, health officials in North and Central America may find clues about which influenza A virus strains are likely to predominate during an upcoming season by observing which were predominant in South America and vice versa. Our findings underscore the need to share timely and representative specimens with World Health Organization Collaborating Centres. In the future, shorter vaccine production times using novel technology might facilitate matching vaccine composition more closely to circulating virus strains. edit 1 Most commonly identified antigenic characterizations of influenza strains in the Americas during 2002\u20132013."}
+{"text": "Incidence of tuberculous lymphadenitis, accounting for 30 \u2013 40% of extra-pulmonary tuberculosis, has increased in parallel with increase in the incidence of tuberculosis worldwide. The diagnosis becomes more challenging when clinical presentation is suggestive but bacteriological proof is lacking.Mycobacterium tuberculosis by Ziehl neelsen (ZN) staining, culture on a Lowenstein Jensen medium, and cytological examination for findings suggestive of tubercular lymphadenitis. DNA from all the samples was amplified by conventional PCR targeting 123 bp and 240 bp fragments of IS6110 and MPT64 genes respectively and RealTime PCR targeting MPT64 gene only.Sixty lymph node aspirates from suspected cases of tuberculous lymphadenitis were examined for MPT 64 gene in PCR could diagnose 45 cases unlike IS6110 gene which could help in diagnosis of 42 cases (7.5 vs 7%). Whereas PCR gave 13 positivities which were negative by any of the conventional methods, it was negative in 2 samples that were positive on cytology only.Histopathology, ZN staining, and culture gave positivity in 25, 20 and 16 cases respectively; out of which 6 (10%) were diagnosed by positivity in all the three conventional techniques, whereas 36 (60%) were positive by any of these three techniques. Diagnosis could be made by conventional PCR in 43 cases when compared to 45 by the RealTime PCR method (7.2 vs 7.5%) while targeting the Though conventional diagnosis remains the method of choice in tuberculous lymphadenitis alternative diagnostic method such as PCR which are more rapid and reliable looks promising and help well manage patients."}
+{"text": "The reported study  is similChinese Journal of Cancer [\u201cThe clinical value of adjuvant radiotherapy in patients with early stage breast cancer with 1 to 3 positive lymph nodes after mastectomy,\u201d published in the f Cancer .PLOS ONE paper.This previous work was cited as reference number 20 in the PLOS ONE paper, patients receiving treatment from July 1998 to November 2007 were included in the study (79 patients received postmastectomy radiotherapy (PMRT); 618 patients did not receive PMRT). In the Chinese Journal of Cancer paper, patients receiving treatment from January 1998 to May 2007 were included in the study .In the PLOS ONE study [The authors wish to clarify the sampling methodologies. For the earlier study , only paNE study  additionThe authors also note additional differences between the two studies. The clinical value of PMRT in patients with T1-2 breast cancer and 1\u20133 positive axillary lymph nodes (T1-2N1M0) remained controversial following publication of the first study ["}
+{"text": "Cilia formation starts at the mother centriole and requires the activity of the microtubule affinity regulator, MARK4, which promotes axoneme extension during the initial phase of ciliogenesis. The defective axoneme extension in MARK4 depleted cells could be in part rescued by co-depletion of the inhibitory complex CP110/Cep97. Whether MARK4 only influences CP110/Cep97 or influences additional inhibitory components is not known. Interestingly, MARK4 has been recently shown to regulate the position and movement of autophagic vesicles within the cell. Recent studies also revealed that autophagy promotes ciliogenesis by inducing the selective degradation of the centriolar satellite pool of OFD1, an inhibitor of centriole elongation and axoneme extension. Here, we investigated whether MARK4 is functionally linked to autophagy to promote cilia formation. Analysis of RPE1 cells stably expressing YFP-LC3 shows that autophagosomes exhibit a perinuclear clustering upon MARK4 depletion. Quantitative immunofluorescence analysis shows that in MARK4 depleted cells, OFD1 levels at the centriolar satellites are higher than in control cells. This could be reverted by partial knock down of OFD1. By co-depleting MARK4 and OFD1, the cilia loss phenotype of MARK4 knockdown was partially reverted. Our results suggest that MARK4 acts in ciliogenesis by regulating the movement and position of the autophagosomes that degrade OFD1 at the centriolar satelites."}
+{"text": "We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca+2 influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate  Cough is a vagally mediated reflex and a primary defensive mechanism to protect the airway by forceful expulsion of irritant agents from the respiratory tract. Although a protective response, sometimes it becomes excessive and harmful to the airway mucosa leading to compromised quality of life. Cough is broadly divided into acute and chronic persistent cough in vitro and in vivo studies have recently established TRPA1 as a major chemosensory receptor of the airways Activation of sensory nerves innervating anatomical regions implicated in cough reflex, including the larynx, trachea and large bronchi, by exogenous inhaled or aspirated substances or by locally produced endogenous biochemical mediators can produce cough 2 or TRPA1 activators - 4-Hydroxynonenal (4-HNE), reactive oxygen species (ROS), reactive nitrogen species (RNS), 15d-PGJ2 in the esophagus during GERD TRPA1 on sensory nerves detects not only exogenous environmental agents that may be inhaled, but is also activated by known endogenous tussive molecules such as PGE2 and bradykinin produced during tissue inflammation. Additionally, it also senses the inflammatory environment present in inflammatory lung disease, viral/ bacterial infection or extrapulmonary diseases. Literature demonstrates TRPA1 receptor expression in the esophagus and simultaneous presence of inflammatory mediators that are known to be either TRPA1 sensitizers - histamine, bradykinin, PGE+2 influx not only in recombinant TRPA1 expressing/ CHO cells but also in human primary lung fibroblasts and airway epithelial cells that express TRPA1 endogenously. Two potent and selective TRPA1 antagonists (GRC 17536 and compound 7) was able to abrogate TRPA1 agonist (s) mediated functional response in these cells. We also further provide in vivo evidence in guinea pig model that citric acid induced cough response is inhibited by GRC 17536. Since TLR7 and 8 have been implicated in viral pathogenesis, we also evaluated effect of TLR ligands on TRPA1 receptor. We demonstrate that TLR ligands, in particular the TLR4 (LPS), TLR7 (loxoribine) and TLR8 (ssRNA) ligands upregulated the expression of TRPA1 and also enhanced calcium uptake mediated by TRPA1. This response was attenuated by TRPA1 specific antagonist, GRC 17536 and compound 7. Hence, TRPA1 receptor could be a common and crucial molecular mediator behind various chronic cough etiologies.In the present study, we investigated if citric acid, which is used for many years to provoke cough in animals as well as human studies 45Ca+2 , Microscint PS , G418  and FBS .DMEM F-12, BSA and HEPES were purchased from Sigma Aldrich Inc. , MEM from Hyclone . Other reagents were procured from vendors as indicated: 2). For Calcium fluorescence assay, Ca+2, Mg+2 free PBS were used instead of DDB for drug dilution. LPS, AITC, capsaicin, H2O2, and crotonaldehyde were procured from Sigma Aldrich Inc. . ssRNA40 and loxoribine were purchased from Invivogen , 15d-PGJ2 from Enzo life sciences , and citric acid from Merck .Glenmark proprietary TRPA1 antagonists , TRPV1 antagonist (GRC 6211) and compound 7  were synthesized in-house by the discovery chemistry group. A 10 mM stock of the above antagonists was prepared in DMSO. Subsequent dilutions from the stock solution were made in drug dilution buffer (DDB) (DMEM F-12 containing 1.8 mM CaCl2. Chinese hamster ovary (CHO) cells stably expressing human TRPA1 (hTRPA1/CHO), TRPV1 (hTRPV1/CHO), TRPV3 (hTRPV3/CHO), TRPV4  were obtained from American Type Culture Collection  and cultured in MEM supplemented with 10% FBS. Human pulmonary alveolar epithelial cell line A549 (ATCC CCL-185) was also obtained from ATCC and maintained in DMEM F-12 supplemented with 10% FBS. Both the cell types were cultured at 37\u00b0C in humidified air containing 5% COad lib standard guinea pig pellet diet and water containing dietary amounts of ascorbic acid.Male Hartley guinea pigs  were acclimatized in cages  before beginning of the experiments and were maintained on an ad lib standard guinea pig pellet diet and water containing dietary amounts of ascorbic acid. All animal experiments were approved by Institutional Animal Ethics Committee (IAEC) of Glenmark Pharmaceuticals Ltd and the study was carried out in strict accordance with recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals . All animal work was conducted as per CPCSEA guideline to minimize discomfort to animals. Guinea pigs were euthanized humanely with CO2 inhalation at the end of the study.Guinea pigs were housed in cages with a light/dark cycle of 12/12 hours and were maintained on an (-\u0394\u0394CT) Livak method of analysis.Total RNA was extracted from hTRPA1/CHO cells using TRI reagent . 3 \u00b5g of RNA was reverse transcribed using iScript select cDNA synthesis kit  according to the manufacturer's instructions to produce single-stranded cDNA. RT-PCR was performed with an real-time thermal cycler Eppendorf Mastercycler Ep  using iTaq SYBR Green Supermix with ROX kit  with human TRPA1 specific primers: 5\u2032-GATATTGTTAACACAACCGATGGA-3\u2032(sense primer) and 5\u2032-CTTTTATGTCTACTTG GGCACCTT-3\u2032(antisense primer). RT-PCR reaction was carried out for 40 cycles at 94\u00b0C for 30 s, 60\u00b0C for 15 s and 60\u00b0C for 60 s. Data was interpreted using 2+2 influx assay was performed as described earlier 2O. Subsequent dilutions were made in Ca+2, Mg+2 free PBS (pH 7.3). No significant change in pH was observed upto 10 mM citric acid. At 30 mM concentration of citric acid, the pH of the solution dropped to 5.5. TLR ligands - LPS , loxoribine  and ssRNA40  were dissolved in media and added to the cells for 16\u201318 hours. TRP selectivity of GRC 17536 and compound 7 was performed for TRPV1, TRPV3, TRPV4 and TRPM8 using Capsaicin (1 \u00b5M), 2-APB (1 mM), 4\u03b1-PDD (30 \u00b5M) and Icilin (250 nM) respectively, as agonist.Fluorimetric and radiometric CaCough was detected both by pressure change and by sound and recorded with a Buxco cough analyzer  as described earlier in vitro experiments, data was analyzed using GraphPad Prism 3.0 . For in vivo experiment, data from the treatment groups were compared with the control group. Mean values were statistically analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett's Multiple Comparisons Test to evaluate significant differences between groups.For +2 influx in CCD19-Lu, A549 and hTRPA1/CHO cells  at the highest concentration of citric acid. In CCD19-Lu cells maximum Ca+2 influx was observed at 10 mM citric acid concentration followed by a decrease in calcium levels at the next higher concentration (30 mM). In A549 and hTRPA1/CHO cells peak Ca+2 influx was observed at 3 mM citric acid concentration followed by a drop in calcium level at the next higher concentration. Although the citric acid response pattern and the maximum Ca+2 influx response observed in these three cell types are different, the responsiveness to citric acid stimulus in the form of increased Ca+2 influx was a clear trend displayed by these TRPA1 expressing cell types, unlike the lack of any significant response seen with the hTRPV1 expressing CHO cell line (data not shown). The differences in response to Citirc acid within these TRPA1 expressing cells could also be due to differential receptor expression level or cellular background .TRPA1 receptor has been earlier reported to be a general sensor for weak acids which can diffuse across the cell membrane in the protonated form and cause intracellular acidification HO cells . Treatme+2 influx. As shown in +2 influx in all the three cell types tested. The IC50 values for GRC 17536 and compound 7 in the above assays was 8.2 nM and 27.9 nM  +2 influx by GRC 17536 is not restricted to a particular chemical class, we tested GRC 17770, a selective TRPA1 antagonist with potency similar to GRC 17536, but belonging to a chemically diverse series in hTRPA1/CHO cell based assay. GRC 17770 also inhibited citric acid induced Ca+2 influx with an IC50 of 7.19 nM . We next evaluated whether these TLR ligands could potentiate TRPA1 agonist mediated calcium uptake. As demonstrated in 2O2 and 15d-PGJ2 induced 45Ca+2 uptake in hTRPA1/CHO cells  or ssRNA40 (1 \u00b5g/ml) resulted in a 20% and 30% increase in 45Ca+2 uptake over AITC alone  in both the cell types (50. Complete inhibition of 45Ca+2 uptake was observed at the highest concentration of the compound (1 \u00b5M). GRC 17138, another compound structurally close to GRC 17536 but devoid of any functional antagonism on TRPA1/CHO cells was used to rule out the chemical class effect and confirm TRPA1 involvement in the blocking Ca+2 influx. It failed to inhibit LPS, Loxoribine or ssRNA40 enhanced calcium flux in either hTRPA1/CHO or A549 cells up to a high concentration of 10 \u00b5M , no significant protective response was observed. Dextromethorphan at 30 mg/kg dose showed 66% inhibition of cough response.To investigate whether Citric acid mediated cough response involved TRPA1 channel, we studied effect of GRC 17536 in citric acid induced cough model in guinea pigs. Guinea pigs exposed to citric acid challenge showed significant cough response. The animals treated with GRC 17536 showed 79 and 89% inhibition of cough at 60 and 100 mg/kg respectively compared to the vehicle treated animal group . At the Cough is a reflex that helps clear the respiratory tract of environmental irritants, and is often associated with symptomatology of inflammatory airway diseases such as asthma, bronchitis and COPD in vitro evidence that TRPA1 is a direct sensor for citric acid using recombinant TRPA1 overexpressing CHO cells as well as in human primary lung fibroblast cells (CCD19-Lu) and pulmonary alveolar epithelial cell line (A549). We demonstrate functional activation of TRPA1 by citric acid, observed as a concentration dependent increase in Ca+2 influx in these cells. Similar findings demonstrating TRPA1 as a general sensor of weak acids have been published by Wang et al 2 via direct gating of the channel by intracellular protons +2 influx in recombinant hTRPV1/CHO cells with citric acid under similar experimental conditions as used for TRPA1 (data not shown). Higher concentrations of citric acid  elicited a drop in Ca+2 influx presumably due to receptor desensitization. Similar observations have been reported earlier for TRPA1 with AITC as agonist In the present study we provide +2 influx in all three cell types tested with similar potency as reported earlier +2 influx in CCD19-Lu and A549 cells. Since human lung epithelial and fibroblast cells are known to express endogenous levels of both TRPA1 and TRPV1 +2 influx in these cells by GRC 6211 further confirms TRPA1 as a mediator of citric acid induced Ca+2 influx.The specificity of TRPA1 activation by citric acid was further confirmed by using two potent and selective TRPA1 antagonists - GRC 17536 (Glenmark) and compound 7 . GRC 17536 concentration dependently inhibited citric acid induced Cain vitro validation of TRPA1 as a mediator for citric acid mediated Ca+2 influx encouraged us to explore whether the in vitro observation can be translated to in vivo animal models. We demonstrated that GRC 17536 reversed citric acid induced cough response in guinea pigs. A dose dependent decrease in tussive response was observed with GRC 17536. This is the first in vivo evidence demonstrating effectiveness of selective TRPA1 antagonist in citric acid mediated cough. Several other environmental irritants known to directly activate TRPA1 receptor e.g. acrolein, crotonaldehyde etc. have also been shown to provoke cough via TRPA1 receptor in guinea pigs and humans Citric acid has been used to provoke cough in human as well as in animal studies. Extensive 2O2, prostaglandins etc. that are known to be TRPA1 activators 45Ca+2 uptake in presence of TLR ligands and upregulation of TRPA1 gene expression.TRPA1 activation is known to induce the release of a variety of neuropeptides and neurotransmitters, e.g. calcitonin gene-regulated protein (CGRP), Substance P and neurokinin, which causes vasodilation and immune cells recruitment to the site of assault, thereby exacerbating the cellular and tissue inflammatory response. The recruited immune cells secrete a number of inflammatory mediators e.g. hypochlorite, HIn summary, TRPA1 is expressed on the pulmonary C fibers that project into airways and terminate into the mucosa and submucosa of the pharynx, larynx and trachea which are anatomical regions driving cough reflex. TRPA1 is activated by endogenous and exogenous pro-tussive agents that are amply documented in literature and some of these induce cough response in guinea pigs and humans. Asthma, COPD, GERD, PNDS and post viral cough seem to be major diseases with chronic cough manifestation which is untreatable. The causes of cough in these pathologies could be diverse but the common link between them could be the activation of TRPA1 receptor on the airway vagal sensory nerves. The implicated role of vagal TRPA1 receptor in such wide variety of cough etiologies suggests TRPA1 to be a major driver of cough reflex. Further, our work demonstrates antitussive effect of GRC 17536 -a potent selective antagonist of TRPA1 receptor in guinea pig cough model. The pulmonary and vagus afferent control of human airways is generally believed to be similar to other mammals. Thus TRPA1 seems to be a perfect target for development of novel anti-tussive agents for various chronic cough conditions."}
+{"text": "The seasonality of influenza virus infections in temperate climates and the role of environmental conditions like temperature and humidity in the transmission of influenza virus through the air are not well understood. Using ferrets housed at four different environmental conditions, we evaluated the respiratory droplet transmission of two influenza viruses  and concurrently characterized the aerosol shedding profiles of infected animals. Comparisons were made among the different temperature and humidity conditions and between the two viruses to determine if the H3N2 variant virus exhibited enhanced capabilities that may have contributed to the infections occurring in the summer. We report here that although increased levels of H3N2 variant virus were found in ferret nasal wash and exhaled aerosol samples compared to the seasonal H3N2 virus, enhanced respiratory droplet transmission was not observed under any of the environmental settings. However, overall environmental conditions were shown to modulate the frequency of influenza virus transmission through the air. Transmission occurred most frequently at 23\u00b0C/30%RH, while the levels of infectious virus in aerosols exhaled by infected ferrets agree with these results. Improving our understanding of how environmental conditions affect influenza virus infectivity and transmission may reveal ways to better protect the public against influenza virus infections. Influenza viruses display distinct seasonal patterns in temperate climates with peak infection rates occurring in the winter months and little to no influenza virus activity detected at other times of the year . HoweverThere are three generally accepted modes of influenza virus transmission . Aerosolin vitro studies have demonstrated that influenza viruses in droplets and aerosols maintain viability better at low humidity [Temperature and humidity are inextricably linked when considering their effects on evaporation rates and on infectious particle dynamics . Althoughumidity ,17. Animhumidity . The ferhumidity . To moreAll ferret procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. Animal studies were performed in accordance with the IACUC guidelines under protocol #2234MAIFERC: \"Transmissibility of influenza viruses with pandemic potential\".Virus stocks of A/Panama/2007/1999 (H3N2) were propagated in the allantoic cavity of 10 day old embryonated hens\u2019 eggs while A/Indiana/8/2011 (H3N2v) was grown in Madin-Darby canine kidney cells (MDCK) as previously described [3 AH); 23\u00b0C/30%RH (6.2 g/m3 AH); 23\u00b0C/50%RH (10.3 g/m3 AH) and 23\u00b0C/70%RH (14.4 g/m3 AH). Prior to inoculation, ferrets were allowed to acclimatize inside the chamber for at least four days. Baseline temperatures were measured using an implantable subcutaneous temperature transponder  and baseline serum, weight and minute volume (MV) of respiration measurements were collected as described [Male Fitch ferrets 5\u201311 months of age (Triple F Farms), serologically negative by hemagglutination-inhibition (HI) assay for currently circulating influenza viruses, were housed during each experiment in cages placed inside a custom environmental chamber  with HEPA filtration operating at 20 air changes per hour. Experiments were conducted at four environmental settings with consideration for the comfort of the animals: 5\u00b0C/70%RH  and was found to be negligible (<0.05 m/s) for all of the cages. One day post inoculation (dpi) a na\u00efve ferret was placed in a cage adjacent to each inoculated animal. Nasal washes (NW) were collected every other day for up to 11 dpi or days post contact (dpc)  for viruThree inoculated ferrets from each experiment were anesthetized and exhaled aerosol samples were collected and analyzed for size distribution using an aerodynamic particle sizer . Aerosol samples were collected from ferrets immediately after exhalation on 2, 4 and 6 dpi for 15 minutes of closed-mouth, normal breathing and for 5 minutes of induced sneezing as described . Size diExhaled aerosols from inoculated animals were analyzed for infectious virus using a viable two-stage cascade impactor  on 1, 3, and 5 dpi as described with modifications . Aerosol2\u2013103 pfu) directly on prepared impactor plates and placing the impactor assembly inside the environmental chamber at one of the four environmental conditions [The effects of the aerosol collection procedure on influenza virus recovery was evaluated in duplicate by applying a known amount of virus .3.8\u2013105.5 pfu of PN99 or IN11 virus   and allo11 virus . One dpiRegardless of the inoculum, changes in activity levels in infected animals were minor (relative inactivity indexes ranged from 1.0\u20131.2) while anorexia and nasal discharge were the most common clinical signs observed in ferrets housed at any of the controlled environmental settings. Peak temperatures were detected 2\u20133 dpi in inoculated animals and ranged from 1.2\u20132.4\u00b0C above baseline for PN99 virus and 1.9\u20133.3\u00b0C above baseline for IN11 virus . MV of r4.2 to 104.8 pfu/mL in ferrets inoculated with PN99 virus and were 3\u201324 times higher (105.2 to 105.7 pfu/mL) in ferrets inoculated with IN11 virus housed under the respective environmental condition; the greatest difference was observed at the 23\u00b0C/70%RH condition  on 1, 3 and 5 dpi and then quantified for influenza virus by plaque assay as well as real-time RT-PCR. Infectious virus detected at any time point and in either size range in normal breathing samples was \u226410 pfu  and in sThe impact of the aerosol collection procedure on virus recovery was tested for each controlled environmental condition by spiking the collection medium on each impactor stage with known quantities of virus. After air was pulled through the impactor for 15 or 5 minutes, to represent normal breathing and sneezing sampling times, respectively, the collection medium was harvested and processed as described for the ferret samples. PN99 virus RNA recovery from either stage and for the 15 and 5 minute collection times ranged from 54 to 79%, similar to IN11 virus RNA recovery which was 47 to 81% . InfectiThe influence that temperature and humidity have on aerosolized influenza virus and the consequence for transmission remain poorly understood despite the potential impact on the seasonality of influenza epidemics in the temperate regions of the world. Climatic conditions in tropical regions of the world represent a different set of parameters affecting transmission, and furthermore, routes of transmission not involving airborne virus (e.g. direct or indirect contact) demonstrate the complexity of global influenza virus transmission and the multitude of potential mechanisms at play each impacted differently by environmental conditions. Here, we evaluated the transmissibility of two H3N2 influenza viruses (PN99 and IN11) using ferrets housed under diverse environmental conditions and concurrently evaluated the aerosol shedding profiles of infected animals. We compared the two virus groups to each other and then combined them to identify common differences at each environmental condition. Higher levels of IN11 virus were measured in ferret nasal washes and in exhaled aerosol samples compared to PN99 virus but overall, we found little difference in the transmission capabilities of the two viruses at any of the environmental conditions with transmission most frequently observed at 23\u00b0C/30%RH (83%) and least frequently observed at 23\u00b0C/50%RH and 5\u00b0C/70%RH (17%).The viruses chosen for this study were selected because one represents a seasonal H3N2 strain PN99 virus), the other one is a swine-origin H3N2 variant strain (IN11 virus) responsible for a summertime infection ,25 and b9 virus, Any successful transmission event through the air requires that virus pass from an infected host to a susceptible one while maintaining infectivity. As this occurs, aerosols are released from the warm, humid environment of a respiratory tract, through the ambient conditions outside of the host and eventually reach the warm, humid environment of a recipient\u2019s respiratory tract. Depending on the ambient conditions and the solute composition of the aerosol, evaporation and condensation processes may have deleterious effects on any virus present in the aerosols . Using cDetection of influenza virus in exhaled aerosols has been largely limited to the detection of viral genetic material via RT-PCR with few reporting infectious virus detection in humans ,13 or laIn the temperate regions of the world, people spend the vast majority of their time indoors, especially during the winter months where the environment is typically maintained at a warm temperature and low RH . This sc"}
+{"text": "Congenital heart disease (CHD) is the most common congenital defect, affecting 1% of all livebirths. Initial cardiac development and the subsequent maturation of the heart are temporally and spatially regulated by several Bone Morphogenetic Proteins (BMPs). BMP signaling plays an important role in the specification and patterning of the early embryo. Animal model studies have established a strong link between BMPs and cardiac development. At least 6 BMPs  are expressed in the heart where they have both independent and redundant functions. BMP2, 4 & 7 are expressed in the AtrioVentricular cushion and Out Flow Tract (OFT) and helps in their formation. However due to functional redundancy of BMP ligands, relatively little is known about the role of individual BMPs. The association of BMPs with CHD has not been studied so far in human subjects. Here we report the genetic variants in BMP2 (NM_001200.2), BMP4 (NM_001202.3) and BMP7 (NM_001719.2) in individuals with CHD.We screened 190 unrelated probands with CHD and 100 healthy controls by PCR and DNA sequencing for 3 genes.We have identified 6 novel sequence variants in BMP2. Out of which 3  are missense variants. One nonsense variants (K241X) has also been identified in one case having Patent Ductus Arteriosus and is not found in 100 controls. In BMP7, 10 novel sequence variants have been identified, out of which 3  are missense, 3  are in 5\u2019UTR, 1 synonymous change (c.682G>T) and 3  are intronic variants. In BMP4, 3 sequence variants are found, out of which only one novel (c.1695A>C) in 3\u2019UTR region has been found in one individual. Other than these some already reported single nucleotide polymorphisms have also been identified in these 3 genes and some of them are more frequently found in CHD cases. In-silico analyses based on evolutionary, biochemical and structural aspect of the gene revealed the disease causing effect of these variants. Further biochemical analysis would reveal the regulatory circuits involving these cardiac transcription factors and their possible role in pathogenesis of the disease."}
+{"text": "Pharmacogenetic effects of recombinant human growth hormone according to growth hormone receptor (GHR) exon 3 polymorphism (fl vs. d3) were controversial. We investigated growth hormone response in children with growth hormone deficiency (GHD).Total 58 prepubertal children (31 boys and 27 girls) with GHD were enrolled in this study. Subjects were divided to 2 groups according polymorphism , and compared baseline phenotypes and the first year growth response to growth hormone treatment.The distribution of GHR exon 3 isoforms in children with GHD demonstrated that the frequency of fl/fl (82.8%) is higher than that in most of European studies. There was no significant difference in baseline height SDS between 2 groups. Height velocity during the first year of growth hormone replacement therapy tended to be higher in subjects who have d3 allele (fl/d3 and d3/d3), but there was no statistical difference according to genotype.It seemed that d3 allele of GHR exon 3 had no impact on the baseline phenotype and growth hormone response in patients with GHD. Relationship between GH dose and IGF-1% to help fully elucidate the value of IGF-1 testing in GH treatment."}
+{"text": "LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-\u03945-sterols was quite marginal  PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol  and not lanosterol , as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences ( It is nonetheless worth noting that 2-tritio lanosterol and 2-tritio cycloartenol when fed to Sorghum bicolor leaves were converted to cholesterol and sitosterol eventhough cycloartenol was a more effective sterol precursor than lanosterol Plants, algae and some protists synthesize their sterols through a biosynthetic route that contains a pentacyclic steroidal cyclization product of 2,3-oxidosqualene, namely, cycloartenol , being able to isomerize cycloeucalenol into the tetracyclic 4\u03b1-methyl sterol, obtusifoliol Arabidopsis thaliana cpi1-1 mutant indicates that a normal life span cannot be attained with the CAS1 biosynthetic segment only The essential role of CAS1 in sterol biosynthesis and plant development was demonstrated by a series of in planta characterization of CAS1 and LAS1 was restricted to Arabidopsis thaliana. In order to further document dual cycloartenol and lanosterol pathways in higher plants we have isolated a CAS1 from Nicotiana tabacum and studied its function in yeast and in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1 in Nicotiana benthamiana. In this report, we show a strict dependence on CAS1 of tobacco sterol biosynthesis.The co-existence of functional CAS1 and LAS1 in plants requires further studies. So far, Nicotiana orthologs of CAS1 and LAS1. We then cloned a Nicotiana tabacum CAS1 cDNA by recursive PCR using degenerated primers over conserved sequences. Primers used for that cloning procedure are given AtCAS1 and consequently was named NtCAS1 (GenBank accession KM452913). We next identified in the genome of Nicotiana benthamiana (http://solgenomics.net/) two genes whose products had 70% and 58% identity with AtCAS1 and AtLAS1, respectively. Relevant sequences identified in Nicotiana and in other Solanaceae are given in Nicotiana benthamiana with the functionally identified tomato triterpene synthases Arabidopsis thaliana \u03b2-amyrin synthase  in the vicinity of the conserved DCTAE motif implicated in substrate protonation, whereas all LAS1 proteins have a strict requirement for a V481 (erg7 (lanosterol-deficient) mutant led to a current understanding of the catalytic differences in both reactions We cloned by RT-PCR a cDNA fragment from d NbLAS1 , confirmm annuum  shows fer a V481  [31], .(PDF)Click here for additional data file.Table S1Gene nomenclature and protein references used to generate .(PDF)Click here for additional data file.Table S2Primers used for NtCAS1 cloning and for qPCR measurements of CAS1 and LAS1.(PDF)Click here for additional data file.Table S3The Solanaceae OSC signatures and gene references.(PDF)Click here for additional data file.References S1(DOCX)Click here for additional data file."}
+{"text": "To assess the effectiveness of transperineal template guided mapping biopsy (TTMB) in detecting prostate cancer in patients with previous multiple negative transrectal ultrasound guided (TRUS) biopsies of the prostate.From April 2011 to February 2013 22 patients who had previously undergone 2 or more TRUS biopsy with a continuing rising PSA were biopsied using an anatomically guided template. Clinical parameters included age, initial PSA, PSA pre TTMB biopsy and number of previous biopsies. Number of cores sampled, number of cores positive for cancer and Gleason score were assessed. Results: Average age is 61.45 years with average of 2.5 previous biopsies and pre biopsy PSA of 15.5. On average 22.3 cores were sampled in each patient. PSA rose 6.4 ng/ml between initial biopsy and PSA triggering TTMB. Average elapsed time from initial biopsy to TTMB was 3.3 years. Overall 31.8% of patients (n=7) were diagnosed with prostate cancer at TTMB. There were 4 patients found to have Gleason 3+3=6, 1 Gleason 3+4=7 and 4 Gleason 4+4=8.TRUS biopsy is known to miss clinically significant pathology of the prostate. In this single surgeon single institution analysis we examined the efficacy of the TTMB in identifying prostate cancer in patients with multiple previous negative biopsies and a rising PSA, and found over 30% of patients with previous multiple negative biopsies to have clinically significant prostate cancer. This study demonstrates that in the right hands TTMB can be an integral step in the management pathway of patients with suspected prostate cancer."}
+{"text": "The latest guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) to test Human Epidermal Growth Factor Receptor 2 (HER2) in breast cancer after being revised in 2008 underwent a second modification in October 2013. The modification includes changes in cut-offs: 10% strong membranous staining for score 3+ on immunohistochemistry (IHC) (previously 30%) and using the ratio of >2 or absolute gene-copy-number (6 or more) alone or in combination with each other by in-situ-hybridisation technology (previously >2.2 and average copy-number of 6 or more). Hereby we addressed the question, which impact the modified cut-offs had on overall HER2-positivity in a single institution.We prospectively analysed 617 consecutive diagnostic breast-cancer cases which underwent double HER2 testing by immunohistochemistry and fluorescent in-situ hybridisation (FISH), using the modified 2013 ASCO/CAP-guidelines for one year (October 2013\u2013October 2014). Results were compared with HER2-test results on 1,528 consecutive diagnostic breast-cancer cases from two previous years (2011\u20132012), using the 2008 ASCO/CAP guidelines, also tested with IHC and FISH in each case.Between October 2013 and October 2014, overall HER2-positivity was 15.8% (98 of 617 cases were either IHC 3+ or FISH amplified). 79 of 617 cases (13%) were IHC 3+, 96 of 617 cases (15.5%) were FISH amplified. Equivocal cases were seen in 25 of 617 cases (4.1%). 22 of 25 equivocal cases (88%) in 2013\u20132014 were IHC 1+ or 2+. In 13 equivocal cases, there was a repeated IHC/FISH testing: 2 of 13 cases (15%) became FISH amplified, 1 of 13 cases (7.5%) became IHC 3+. In 2011\u20132012, overall HER2-positivity (IHC/FISH) was 13.8% . 185 of 1,528 cases (12%) were 3+ on IHC, 181 of 1,522 cases (12%) were amplified by FISH. Six of 1,528 cases were equivocal by FISH, and interpreted as non-amplified (0.3%).Applying the modified ASCO/CAP guidelines from 2013 resulted in an increase (2%) in overall HER2-positivity rate compared to overall-HER2-positivity rate using the 2008 ASCO/CAP guidelines. The increased positivity rate was mainly due to more FISH-positive cases (3.5% more than until 2013). The high rate of equivocal cases (4.1%) did not contribute to increase in overall HER2-positivity, but resulted in delay in definitive HER2-status. The assessment of HER2 status in breast cancer is a part of routine tests on primary and / or on recurring lesions \u20133. HER2 In this study we compared overall HER2 positivity rate prior to the introduction of the modified ASCO/CAP guidelines from October 2013 with the ones using the new definitions from this time.We prospectively analysed consecutive HER2 diagnostic cases from the period of 2011\u20132012 (n = 1528) and compared with HER2 test results between October 2013 and October 2014 n = 617). All breast cancer cases underwent double HER2 testing by immunohistochemistry and fluorescence in situ hybridisation according to the institutional guidelines as published earlier [17. All bConsecutive patients with the diagnosis of invasive breast carcinoma undergoing HER2 testing at the Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland were analysed. Two periods were evaluated, 2011\u20132012 (two years) and from October 2013 to October 2014 (one year). Data on HER2 status from 2011\u20132012 were partially included in a previous paper, cases with polysomy or equivocal HER2 status were not included in this previous study . In the During the whole period (2011\u20132014), the same laboratory procedure for IHC and FISH was applied as published earlier .Protein expression and gene copy number score were scored using the time current ASCO/CAP guidelines as published earlier , 6.Score 0: no staining.Score 1+: weak and incomplete membrane staining in less than 10% of the invasive tumor cells.Score 2+: weak complete staining of the membrane in more than 10% of invasive cancer cells.Score 3+: strong complete homogenous membrane staining in more than 30% of the invasive tumor cells.HER2 signals were scored as follows: the number of signal copies and the ratios (HER2/CEP17) were calculated: gene copies (> 6) or cluster formations  were counted. A ratio > 2.2 was set as amplified status; a ratio < 1.8 was negative, and a ratio of 1.8\u20132.2 was referred to as equivocal (according the 2008 guidelines) and using ratio > 2.2 and > 6 HER2 gene copies (in > 10% of the tumor cells) for a positive status (according to 2008 guidelines). If ratio was <2.2, the gene copy number was not considered alone as amplified based on the definitions in the recommendations in Tables Protein expression and gene copy number score were scored using the latest ASCO/CAP guidelines published in October 2013 .Score 0: no stain or faint incomplete membrane stain in not more than 10% within the cells. Score 1+: weak and incomplete membrane staining in more than 10% of the tumor surface. Score 2+: Complete intense membrane staining in not more than 10% of the invasive tumor cells or weak/moderate heterogeneous incomplete staining in more than 10% of the invasive tumor cells.Score 3+: strong complete homogenous membrane staining in more than 10% of the invasive tumor cells.HER2 gene copy signals were scored as follows:Positive status was defined either as an average HER2 gene copy numbers of 6 at cases with a HER2/CEP17 ratio of less than 2 or a HER2/CEP17 ratio of 2 or more independently of the average gene copy number.Negative status was defined as average gene copy number of less than 4 with a HER2/CEP17 ratio of less than 2.Equivocal cases were defined as average gene copy number of at least 4 and less than 6 with a HER2/CEP17 ratio of less than 2.Comparison of the overall HER2 positivity rate in two periods with at the time current guidelines (ASCO/CAP 2008 and 2013)  are showOverall HER2 positivity (IHC score 3+ or amplified by FISH) was 13.8% (211 of 1528 cases). 185 of 1528 cases (12%) were 3+ on IHC, 181 of 1528 cases (12%) were amplified by FISH.Six of 1528 cases were equivocal on FISH (0.3%). The one case with IHC score 3+ was considered as HER2 positive. The other five cases underwent repeated IHC/FISH testing on the core biopsy or on a different tumor block, and none of them became amplified or IHC positive.Data are shown in Overall HER2 positivity (IHC score 3+ or amplified by FISH) was 15.8% (98 of 617 cases were either 3+ on IHC or amplified by FISH. 79 of 617 cases (13%) were 3+ on IHC, 96 of 617 cases (15.5%) were amplified by FISH.Equivocal cases by FISH were seen in 25 of 617 cases (4.1%). 22 of 25 equivocal cases (88%) were either scores 1+ or 2+ on IHC. In 13 equivocal cases, there was a repeated IHC/FISH testing: 2 of 13 cases (15%) became amplified by FISH, 1 of 13 cases (7.5%) was 3+ on IHC. The other 10 cases remained negative or equivocal by FISH and score 1+ or score 2+ by IHC. Altogether 3 of 13 (23%) equivocal cases on FISH and IHC became HER2 positive after re-testing. Time to re-testing and to definitive diagnosis on HER2 status required up to 5 weeks .Data are shown in In our study we could demonstrate, that overall HER2 positivity rate in breast cancer increased in 2% after implementing the modified ASCO/CAP guidelines in 2013 on HER2 testing. Our data show, that this increase is mainly due to newly defined cut-offs on FISH positivity, whereas score 3+ on immunohistochemistry did not essentially influence the increase in HER2 positivity rate. Additionally, a relative high percentage of equivocal cases (up to 4%) were observed by using these guidelines. This new phenomenon in our diagnostic HER2 testing, being virtually absent prior to these guidelines, resulted in a considerable delay in the definitive pathology report on HER2 status due to re-testing.There is only limited data on long-term impact of the modified ASCO/CAP Guidelines in the literature, as time passed since the official implementation of these guidelines encompasses around one and half years first \u201313. NeveConsequently to increased IHC score 2+ cases, the need for reflex testing with ISH technology is also arising, resulting in higher workload in ISH, which reportedly can be up to doubling the number of ISH assays when comparing numbers from using the previous guidelines in some recent works , 15. In The question of reproducibility of modified scoring criteria was addressed by Bianchi et al in a recent multicentre study . Based oAlthough the updated guidelines address issue on analytical validity and clinical utility, there is also a recommendation on re-testing in cases with histopathological discrepancies as downgrading in histological grade on excision specimens , 17\u201319. In conclusion it can be stated, that implementing the modified ASCO/CAP Guidelines from 2013 resulted in an increase of 2% in overall HER2 positivity rate in comparison to overall positivity rate by applying the previous ASCO/CAP guidelines. The high rate of equivocal cases (4.1%) did not considerably contribute to the increase in overall HER2 positivity, but resulted in delay of the definitive diagnosis on HER2 status. Further data and experiences from diagnostic HER2 services are needed to be collected and prospectively analysed so that adjustments in the guidelines can be met based on evidence and experience."}
+{"text": "GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that  The Hedgehog (Hh) signaling pathway has long been known to play a critical role in early embryonic development While canonical Hh signaling is mainly mediated by PTCH1 and SMO, recent data has clearly demonstrated a parallel existence of non-canonical Hh signaling pathways in vitro and in vivoAs an alternative to targeting SMO, compounds such as arsenic trioxide have been found to inhibit GLI transcription factors and can inhibit growth of sarcoma or OSA cells that express GLI Spontaneously occurring OSA in the dog very closely models the human disease with striking similarities at the clinical and molecular levels GLI1 and found high expression of GLI1 mRNA (transcript) in 2 out of 3 cell lines (Abrams and D17) relative to osteoblast cells , D17 cells showed a significant reduction of ed cells . We alsoed cells . Importaanalyses .PTCH1 mRNA expression by approximately 60% in D17 cells compared to vehicle DMSO treated and untreated cells . However, GLI1 showed 10\u201315 times higher fold mRNA expression compared to GLI2 mRNA in canine Abrams and D17 OSA cell lines. This suggested that GLI1 might possibly be the major Hh-GLI signaling regulatory transcription factor in Canine OSA. While previous studies have identified GLI1 as the main transcription factor of Hh-GLI signaling pathway in several tumor systems, two independent studies have concluded that GLI2 appears to be of highest relative importance in human OSA Initial experiments demonstrated high constitutive expression of We found that GANT61 was capable of inhibiting cell proliferation in all three canine cell lines but was most effective inhibiting the growth of D17 cells that expressed highest levels of GLI. Importantly, the IC50 value of GANT61 (based on reporter activity) is in the range of 5 \u00b5M PTCH1 compared to osteoblast cells and this correlated with low expression of GLI1 and GLI2 expression in these cells. Importantly, PTCH1 expression was significantly reduced in D17 cells after treatment with GANT61, supporting the notion that PTCH1 mRNA can be regulated by the Hh-GLI pathway in canine OSA cells. These results are in agreement with previous studies showing that GLI1 knockdown decreases PTCH1 expression in human medulloblastoma and glioblastoma cells To further examine the effects of GANT61 on canine OSA cells, we went on to examine expression of downstream target genes before and after treatment with GANT61. The majority of studies investigating downstream target genes of GLI have focused on genes with known binding of the GLI1 consensus sequences within the promoter region. Therefore, we chose to first examine expression of constitutive GLI1 downstream target genes PTCH1 and PAX6, in canine OSA cells. We then went on to determine the effect of GANT61 on the expression of these same target genes. High expression of PTCH1 in Abrams and D17 cell lines initially supported the notion that GLI signaling was active in canine OSA. However, the Moresco OSA cell line showed low expression of PAX6, one of the putative Hh/-GLI signaling downstream target genes, showed high expression in Abrams and D17 canine OSA cell lines as would be expected from GLI1, GLI2, and PTCH1 expression analyses. GANT61 treatment reduced 40% of the PAX6 expression in canine OSA cells which is in agreement with a previous study wherein GLI1 knockdown reduced the expression of PAX6 in human medulloblastoma PAX6 expression in osteocytes suggests that PAX6 regulates sclerostin, an osteocyte marker that inhibits canonical Wnt signaling antagonist PAX6 gene can possess both proto-oncogenic and tumor suppressor characteristics and these may vary among tumor types We also found that Altogether, the results of our studies demonstrate that 1) GLI signaling is present in canine OSA cell lines, 2) GANT61 is capable of altering expression of GLI target genes in canine OSA cells, and 3) inhibition of GLI is capable of reducing proliferation of canine OSA cells. While these findings indicate that Hh-GLI signaling is quite similar between canine and human OSA, several questions remain unanswered. Specifically, our studies have not examined the individual contribution of GLI proteins in canine OSA, whereas, GLI2 appears to be the major driver of Hh/-GLI signaling in human OSA GANT61  was purchased from Calbiochem and dissolved in DMSO at a stock concentration of 5 mg/ml.2 chamber. All cell lines tested and confirmed free from mycoplasma. Canine osteoblast cells were grown in growth medium (Cn417D-500) purchased from Cell Application Inc. under identical conditions.Canine osteoblast cells (Cn406-05) were purchased from Cell Application Inc. Abrams and Moresco cell lines were gifts from Dr. Douglas Thamm, Department of Clinical Sciences College of Veterinary Medicine and Biomedical Sciences Colorado State University Canine OSA cell lines Abrams, D17 and Moresco were plated in triplicate at a density of 700 cells per well in 96-well plates. After 24 hours, culture medium was replaced with fresh 2% FBS medium containing various concentrations of GANT61 at concentrations between 0\u201320 \u00b5M for 4 days. Cell viability was measured by incubation with the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt (MTS) one \u2013solution assay reagent  at 37\u00b0C for 1 h before reading absorbance on a spectrophotometer  at 490 nm. DMSO was used as a vehicle to dissolve GANT61 and for controls.Cell survival was determined by clonogenic assay based on previous publication GLI1, GLI2, PTCH1, and PAX6 was determined by using the StepOne Plus Real-Time PCR system (Applied Biosystem). Taq-Man assays (Applied Biosystem) GLI1 (Assay ID # Cf04230663_m1) GLI2 (Assay ID#4331348), PTCH1 (Assay ID #Cf02690587_m1), and PAX6 (Assay ID#Cf02675240_m1) were used to determine the expression. All transcripts expression was equilibrated with endogenous control HPRT1 (Assay ID#Cf02626256_m1) expression , Abrams, D17 and Moresco cell lines with Tri Regent (Sigma) and chloroform-ethanol method. Then determined the quantity and quality of RNA with the help of Biophotometer (eppendorf). Total 2 \u00b5g of total RNA was converted to cDNA by using High Capacity RNA to cDNA Kit (Applied Biosystem). Transcript expression (mRNA) level of pression , S2, S3.The D17 cell line was treated with 20 \u00b5M GANT61 or vehicle DMSO for 96 hrs and cell lysate was made in lysis buffer. Protein was quantified using a BCA protein Assay Reagent (bicinchonic acid) (Pierce). A total of 40 \u00b5g of protein was loaded per sample in the 7.5% polyacrylamide gels under denaturing and reducing conditions and protein was transferred to nitrocellulose membranes. After transfer of protein, the membrane was probed and incubated overnight at 4\u00b0C with antibodies Rabbit anti-GLI1 (Abcam ab49314), Rabbit anti-GLI2 (Abcam ab26056), Rabbit anti-Patch1(Sigma-Aldrich P0088), Rabbit anti-Pax6 (Abcam ab5790), and Mouse anti-Actin (Santa Cruz sc-56459) in 5% non-fat milk. Then membranes were washed and subsequently exposed to the appropriate HRP-conjugated secondary antibodies for 1 hr. at room temperature. Bands were visualized by the enhanced chemiluminescence (Pierce) in Fluorchem E Imaging system . Constitutive expression levels of GLI1 and GLI2 were performed similarly, in the absence of treatment for Abrams, D17 and Moresco cell lines.All results shown are representative of experiments done in triplicate. Analyses were performed using one-way ANOVA and post-doc Tukey's testing (GraphPad Prism version 5.0). Differences were considered significant with a p<0.05.Figure S1Amplification plot of GLI1, GLI2, PTCH1 and PAX6 RNA expression in canine OSA cell lines: This plot was used to determine the Cycle threshold (Ct)value; PCR Cycle number as X and the mean \u0394Rn  with the quenching dye emission (Q) during the Real Time PCR amplification process) value as Y. The standard \u0394Rn (Y\u200a=\u200a0.05) of exponential phase of amplification was selected to determine the optimal CT value. Less cycle number to reach exponential phase of amplification indicates high copy number of RNA . (A) Amplification plot of GLI1 in Canine osteoblast (CO), Moresco, Abrams and D17. (B) Amplification plot of GLI2 in Canine osteoblast (CO), Moresco, Abrams and D17. (C) Amplification plot of PTCH1 in canine CO, Moresco, Abrams and D17. (D) Amplification plot of PAX6 in canine CO, Moresco, Abrams and D17.(TIF)Click here for additional data file.Figure S2Amplification plot of GLI1, GLI2, PTCH1 and PAX6 mRNA expression in canine cell line D17 after the treatment of GANT61 and DMSO. (A) Amplification plot of GLI1 expression in Canine OSA D17 cell line compared to GANT61 and DMSO (control). (B) Amplification plot of GLI2 expression in D17 cell line compared to GANT61 and DMSO (control). (C) Amplification plot of PTCH1 in canine OSA cell line D17 compared to GANT61 and DMSO treatment. (D) Amplification plot of PAX6 in canine OSA cell line D17 compared to GANT61 and DMSO treatment. GANT61 treated cells showed increased number of cycle to reach exponential phase of amplification compare to DMSO (control) (decreased mRNA copy number in GANT61 treated cells compared to DMSO).(TIF)Click here for additional data file.Figure S3Amplification plot of GLI1 and GLI2 mRNA expression in canine cell line Moresco after the treatment of GANT61 and DMSO. (A) Amplification plot of GLI1 expression in Canine OSA Moresco cell line after the treatment GANT61 and DMSO (control). (B) Amplification plot of GLI2 expression in Canine OSA Moresco cell line after the treatment of GANT61 and DMSO (control). GANT61 treated cells showed increased number of cycle to reach exponential phase of amplification compare to DMSO (control) (decreased mRNA copy number in GANT61 treated cells compared to DMSO).(TIF)Click here for additional data file."}
+{"text": "Heart failure secondary to cardiac siderosis is the prevalent cause of mortality in patients with primary or secondary iron overload. Myocardial iron assessment using T2* has been successfully calibrated against myocardial iron at 1.5T and the black blood (BB) technique has demonstrated high reproducibility such that it has become the clinical gold standard. T1 appears to correlate with T2* in cases of significant iron loading (T2* < 20 ms) but this relationship weakens when T2* is in the normal range. We evaluated this further.69 subjects  comprising 20 healthy volunteers (controls) and 49 patients  referred for routine iron assessment were recruited. The same mid-ventricular short axis cardiac slice was used to acquire both BB T2* images and to generate T1 MOLLI maps for each subject at 1.5T . 20 subjects underwent repeat studies on the same day to evaluate reproducibility. Regions of interest were selected in the septum using CMRtools.2 = 0.696; Figure There was a reasonable correlation between BB T2* and T1 values described by a logarithmic relationship (RIn this analysis, T1 mapping using a MOLLI sequence identified all those individuals with significant iron loading as defined by the current gold standard T2* technique and has excellent reproducibility. As expected, T1 can also identify patients with mild cardiac iron loading that is not considered clinically significant. It is possible that T1 may have a clinical role in non-cardiac conditions with very low levels of iron loading, where factors other than iron affect the T2* signal, however tissue calibration remains lacking at this time. Further work is needed to determine whether there might be clinically significant advantages of T1 over the T2* cardiac technique at 1.5T for assessment of cardiac iron, but there are none at this time.This research was supported by the NIHR Cardiovascular Biomedical Research Unit at Royal Brompton & Harefield NHS Foundation Trust and Imperial College London."}
+{"text": "In the legend of Figure 2 of the original version of this article  the capFigure 2. Spectral characterization of BVMOAf1. Visible spectra of native BVMOAf1 (solid line) and BVMOAf1 after unfolding by 1% SDS and incubation at 80\u00b0C (dotted line). Spectral changes observed upon reduction of BVMOAf1 by excess of NADPH (dashed line).The listing of \u2018(solid line)\u2019 and \u2018(dotted line)\u2019 are incorrectly placed in the text and in fact should be located in reverse. The caption should correctly read as follows:Figure 2. Spectral characterization of BVMOAf1. Visible spectra of native BVMOAf1 (dotted line) and BVMOAf1 after unfolding by 1% SDS and incubation at 80\u00b0C (solid line). Spectral changes observed upon reduction of BVMOAf1 by excess of NADPH (dashed line)."}
+{"text": "Blood sugar control for patients with sepsis remains controversial. We aimed to test the hypothesis that the variation of blood sugar level is associated with patient outcome in this study.A retrospective cohort study on nontraumatic adult patients who visited the ED of a tertiary hospital in 2010 and had a clinical diagnosis of severe sepsis was conducted. Patients with two sets of blood culture ordered by emergency physicians and at least two blood sugar tests results available during the first 48 hours of hospitalization were included. The coefficients of variation  of the blood sugar level were analyzed with multivariate logistic regression models to test the association between in-hospital mortality.Of the 1,537 patients included, most were older than 70 years of age , male (54%), without a diagnosis of severe sepsis (63%) and had a previous diagnosis of diabetes (84%). The initial blood sugar levels of patients with and without previously diagnosed diabetes were 259 \u00b1 9.9 and 154 \u00b1 5.7, respectively (mean \u00b1 SEM). The CoV of the consecutively monitored blood sugar level during the first 48 hours of admission for patients with and without previously diagnosed diabetes were 29.4 \u00b1 0.5% and 21.0 \u00b1 0.5%, respectively. Patients with CoV lower than 10% and higher than 30% tended to have higher mortality rate, compared to patients with 10 to 30% CoV level (11% vs. 12% and 7%, respectively, Figure In this retrospectively cohort study, we found that increased blood sugar variation was associated with worse patient outcome. However, further study is merited to test the possible causal relationship between variation of blood sugar level and patient outcome."}
+{"text": "Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms,  Drosophila as model system to analyze the effect of Topo II depletion on chromosome stability. We show that the chromosomal phenotypes of mutant flies vary with the amount of residual Topo II, ranging from site-specific chromosome breaks, variations in chromosome number (aneuploidy and poliploidy) and dramatic defects in chromosome morphology. The chromosomal phenotypes observed in flies recapitulate all phenotypes seen in Topo II-depleted vertebrate chromosomes, reconciling the phenotypic discrepancies reported in previous studies. In addition, our finding that the Topo II dependent phenotypes vary with the residual amount of the enzyme provides useful information on the possible outcome of cancer therapy with Topo II inhibitors.Type II topoisomerases (Topo II) are enzymes that disentangle DNA molecules during essential cellular processes such as DNA replication, chromosome condensation and mitotic cell division. Topo II is a major component of mitotic chromosomes and it is a well known target for cancer chemotherapy. Topo II inhibitors block the Topo II enzymatic activity leading to extensive DNA damage, which ultimately kills the cancer cell. Thus, investigating the role of Topo II in the assembly and structural maintenance of chromosomes is not only relevant to understand chromosome biology but might also have a translational impact on cancer therapy. Here we used  Drosophila have a single Topoisomerase II (Top2) gene. Notably, both the Drosophila Top2 and each of the human Topo II genes can rescue the phenotype of yeast Top2 mutants, highlighting the strong functional conservation of type II topoisomerases Type II topoisomerases are large ATP-dependent homodimeric enzymes that transiently cleave double stranded DNA, pass a second DNA double helix through the break, and then reseal the break Topo II alpha is a major component of vertebrate mitotic chromosomes Studies in yeast have shown that Top2 is not required for completion of DNA synthesis but plays essential roles in mitotic chromosome condensation and sister chromatid segregation. Failure to decatenate sister chromatids results in anaphase chromatin bridges that cause chromosome breakage during anaphase or cytokinesis Although Topo II beta is not normally able to compensate for Topo II alpha loss, overexpression of Topo II beta in human cells can correct the defects caused by Topo II alpha depletion S. cerevisiae and S. pombe, in which Top 2 mutations cause minimal cell cycle delays Another controversial issue is the existence of a decatenation checkpoint triggered by loss of Topo II activity. The existence of such a checkpoint was suggested by studies in human cells showing that catalytic inhibitors of Topo II such as ICRF-187 or ICFR-193 are able to induce a caffeine-sensitive G2 delay that is dependent on ATR and BRCA1, but apparently independent of the DNA damage checkpoint Drosophila. Early work showed that injection of anti-Top2 antibodies or Top2 inhibitors into live Drosophila embryos result in strong defects in chromosome condensation and sister chromatid segregation at anaphase Drosophila Top2 is involved in homolog pairing in cell cultures Several studies have addressed the role of Top2 in solofuso (suo) gene. 1suo and 2suo mutant spermatocytes exhibit severely defective ana-telophases with extensive chromatin bridges; these telophases give rise to achromosomal secondary spermatocytes that are able to assemble bipolar spindles and divide in the complete absence of chromosomes 1suo and 2suo are weak mutant alleles of the Top2 gene and describe the isolation and characterization of a stronger Top2 mutation. We show that modulation of Top2 function by means of these mutant alleles and in vivo RNAi results in multiple cytological phenotypes including site-specific chromosome aberrations, heterochromatin undercondensation, polyploidy, and complete disruption of chromosome morphology accompanied by a cell cycle arrest. These phenotypes recapitulate most of the phenotypes observed in vertebrate cells and indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell.Surprisingly, the role of Top2 in the maintenance of mitotic and meiotic chromosome structure in living flies has never been investigated. In a previous study we identified viable mutants in the 1suo and 2suo, two viable but sterile mutants that exhibit many chromatin bridges and lagging chromosomes in male meiosis suo was originally mapped to the polytene interval 37C-37F5 uncovered by Df(2L)VA17Df(2L)Exel9043, which contains four genes: CG10237, RanGAP, Hs2st and Topoisomerase II (Top2). Complementation tests revealed that suo mutations are not allelic to either CG10237 or RanGAP. However, sequencing of Hs2st and Top2 in suo mutants did not reveal alterations in the protein coding exons of these genes with respect to those of another stock of the \u201cZuker collection\u201d from which the suo mutants have been originally isolated ; this mutation was initially named 3suo. DNA analysis of 3suo mutants revealed a G-A transition at nucleotide 1,040 of the Top2 coding sequence, corresponding to the 5\u2032 splicing site of the second intron of the gene. This substitution results in a premature stop codon that would lead to a truncated form of the protein. These results indicate that 3suo is allelic to Top2. Thus, we renamed 1, suo2suo and 3suo as suo1Top2, suo2Top2 and suo3Top2, respectively. We note that we analyzed only the protein coding sequences of the suo1Top2 and suo2Top2 mutants but not the introns the UTRs or the 5\u2032 regulatory sequences. Thus, the molecular lesions resulting in these mutations remain to be determined.In a previous study we identified and characterized suo1 see . We isolsuo1Top2 and suo2Top2 homozygous flies are viable but sterile in both sexes suo3Top2 homozygous individuals die during embryonic development, but this early lethal phenotype is due to a second site mutation, as suo3/Df(2L)Exel9043Top2 (henceforth suo3/DfTop2) animals survive until an early third instar larval stage. suo3/DfTop2 larvae are devoid of imaginal discs like strong mitotic mutants suo2/DfTop2 individuals are viable but male and female sterile; suo1/DfTop2 and suo1Top2/suo3Top2 mutant flies are semi-lethal, with a few escapers. Together these results allow the ordering of our Top2 mutations in an allelic series with suo3Top2>suo1Top2>suo2Top2.As previously reported, both Drosophila Top2 suo1Top2 homozygous brains, showed further reduction in extracts from both suo1/DfTop2 and suo1Top2/suo3Top2 larvae, and was virtually undetectable in suo3/DfTop2 extracts ; the small fourth chromosome bivalent sometimes is separated from the three major chromatin clumps but more often associates with the X-Y bivalent. Before meiosis, chromosomes become progressively individualized within each chromatin territory but bivalents remain well separated from each other until metaphase I, when they congress at the center of the cell During prophase I, suo1/DfTop2 primary spermatocytes. To compare mutants and wild type spermatocytes, we stained cells for DNA (with DAPI), tubulin and the centriole marker DSpd-2 Top2 spermatocytes at stages S2 and S3 were not very different from those of wild type (see ref suo1/DfTop2 spermatocyte nuclei displayed an average number of 5.6 chromatin masses per cell (n\u200a=\u200a60) while control cells showed an average of 3.4 masses/nucleus (n\u200a=\u200a60). Notably, in Top2 mutant spermatocytes at stages S4 and S5 chromatin clumps of similar size were often closely apposed, suggesting that homologs were unpaired but remained in the vicinity of one another , mutant spermatocytes displayed 3 compact clumps like wild type cells . In anaphase-like figures, individual chromosomes were also no longer recognizable, and the two presumptive daughter cells were almost invariably connected by a number of entangled and irregularly condensed chromatin threads  showing a mild undercondensation of the heterochromatic regions of the major autosomes but the appearance of euchromatic arms was always normal . To define the type and frequency of CABs elicited by the different Top2 mutant alleles we incubated dissected brains in saline with 10\u22125 M colchicine for 1 hour and treated them with hypotonic solution before fixation. Colchicine arrests mitotic cells in metaphase and hypotonic treatment results in chromosome spreading facilitating CAB scoring. In suo2/Top2suo2Top2 brains the CAB frequency was not significantly higher than that of wild type controls, which exhibit 0.008 CABs per cell  and the third chromosome heterochromatin (46.1%). In addition, mutant males displayed 18\u201319% chromosome exchanges, most of which (\u223c98%) were dicentrics or translocations generated by breaks in the Y chromosome and 3L heterochromatin (Drosophila heterochromatin). In suo1/DfTop2 and suo1/Top2suo3Top2 XX females most of the CABs were isochromatid breaks in region h47 of 3L heterochromatin. In contrast with males, these females did not display chromosome exchanges  involving the Y and 3L heterochromatin. Indeed, as shown in suo1/DfTop2 and suo1/Top2suo3Top2 mutants derive from site-specific breaks generated during the anaphase of the previous cell cycle.An analysis of the frequencies of the various types of rearrangements permits us to pinpoint the anaphase events that led to their formation. As shown in suo1/DfTop2 and suo1/Top2suo3Top2 mutant cells appear to progress almost normally through mitosis despite the presence of CABs, suo3Top2/Df brains displayed a dramatic drop in the MI. In brains stained only with DAPI we were unable to observe clear mitotic figures. We thus analyzed suo3Top2/Df brains stained with DAPI and immunostained for both tubulin and the mitotic H3 phospho-histone, which marks mitotic chromatin Drosophila somatic cells require kinetochore-driven MT growth for correct bipolar spindle formation suo3Top2/Df cells can assemble a normal bipolar spindle suggests that Top2-depleted chromosomes/kinetochores retain the ability to drive MT nucleation.While suo3Top2/Df brain preparations might be the consequence of a checkpoint that prevents cells to progress through the cell cycle and enter mitosis. To ask whether the interphase arrest of Top2-deficient cells is caused by checkpoint activation we examined suo3mei-41; Top2/Df and suo3Top2/Df; tefu/tefu double mutants. mei-41 and tefu are the Drosophila orthologues of ATR and ATM, respectively; the kinases encoded by these genes are involved in the signaling pathway of the S and G2-M DNA damage checkpoints suo3Top2/Df mutants . Thus, neither ATR nor ATM loss was able to rescue the block in mitotic progression in suo3Top2/Df brains.The extremely low MI observed in suo1/Top2suo3mei-41; Top2 and suo1/Top2suo3Top2; tefu/tefu larvae. suo1/Top2suo3mei-41; Top2 brains showed a substantial increase in the frequency of CABs compared to either single mutant (suo1/Top2suo3Top2 mutants. This suggests that downregulation of mei-41 (ATR) function might either increase the frequency of anaphase entanglements generated by Top2 deficiency or inhibit repair of the site-specific chromosome breaks they produce. Previous studies have shown that mutations in tefu (ATM) cause both telomeric fusions (TFs) and CABs suo1/Top2suo3Top2; tefu/tefu brains displayed frequencies of TFs and CABs that appear to be the sum of those observed in suo1/Top2suo3Top2 and tefu/tefu mutants. Thus, we conclude that tefu (ATM) and Top2 function in different pathways that mediate maintenance of chromosome integrity.To obtain further insight into the type of DNA lesions caused by Top2 deficiency we examined brains from e mutant . In doubTop2 mutants analyzed here showed different mitotic phenotypes. suo1/DfTop2 and suo1/Top2suo3Top2 brain cells showed a normal MI and frequent CABs that preferentially involve the Y and the 3L heterochromatin. In contrast, suo3/DfTop2 brains showed a drastically reduced MI and rare metaphases with collapsed and/or shattered chromosomes. suo1/DfTop2 and suo1/Top2suo3Top2 brains contain \u223c60% less Top2 than wild type brains, and Top2 is undetectable in suo3/DfTop2 brains  but basic chromosome morphology was maintained. The analysis of chromosome preparations from colchicine-treated and acetic acid-fixed brains revealed a phenotype that was not previously observed in suo1/DfTop2 or suo3/DfTop2 mutants. Most metaphases were either polyploid (31%) or hyperploid (27%) and showed extensive chromosome breakage  overexpression; an excess of Cap-H2 caused a tremendous axial compaction of all arms of polytene chromosomes Top2 mutants.Examination of salivary glands revealed a role of Top2 in the control of polytene chromosome structure. In polytene preparations from mocenter . In Top2 pattern . In contImitation switch (ISWI), Su(var)2-5 (HP1) and Su(var)3-7 mutants Drosophila chromatin regulation ISWI, Su(var)2-5 and Su(var)3-7 mutations Top2 mutants binds the Msl1 and Mle proteins Top2 mutants also binds the dosage compensation factor Mof ; the small fourth chromosome bivalent is either separated from these chromatin masses or associated with the X-Y bivalent suo1/DfTop2 males was not substantially different from wild type, suggesting that spermatogonial divisions are not severely affected. However, at stages S4 and S5 mutant spermatocytes displayed approximately twice as many chromatin masses as their wild type counterparts. In addition, in most mutant nuclei masses of similar size were closely apposed, suggesting a separation of the homologs within each chromatin territory. In the subsequent stages of spermatocyte growth, the number of chromatin masses in mutant nuclei progressively decreased, so that at prometaphase they displayed 3 compact chromatin clumps like their wild type counterparts.We have shown that mutations in lacO system or fluorescent in situ hybridization (FISH) showed that the major autosomes are tightly paired during the S1 and S2 stages. However, pairing is suddenly lost at the S2/S3 transition; the chromosomes remain then unpaired throughout the rest of meiosis but are included in a common nuclear territory until they condense prior to meiotic division Chromosome behavior during spermatocyte growth and male meiosis has been investigated in previous studies, which revealed a complex pairing mechanism suo1/DfTop2 spermatocytes are very different from those previously observed in Cap-H2 and Cap-D3 mutants. In these mutants the chromatin remains diffuse within the spermatocyte nuclei from stage S4 through S6, indicating that the Cap-H2 and Cap-D3 condensin II subunits are required for the formation of the intranuclear territories that comprise the homologous chromosomes suo1/DfTop2 spermatocytes it is also different from that caused by mutations in genes mediating achiasmate homolog pairing in Drosophila males . Spermatocytes of mutants in these genes display diffuse and slightly expanded chromatin territories during stages S4\u2013S6; at prometaphase they show up to eight distinct chromatin clumps corresponding to unpaired univalents The chromatin organization defects within the prophase nuclei of teflon, MNM and SNM) and Top2 play distinct roles in chromatin organization during spermatocyte growth. As previously suggested, condensins are essential for territory formation and appear to function in opposition to homolog conjunction Drosophila male and female meiosis, both types of meiotic divisions share a common Top2-depedent mechanism to facilitate achiasmate chromosome pairing.Collectively, the available results suggest that condensins (Cap-H2 and Cap-D3), the proteins required for homolog conjunction  with virtually no defects in brain cell mitoses exhibit strong defects in chromosome segregation during both meiotic divisions of males . In wild type female meiosis, the heterochromatic regions of the homologous chromosomes remain connected during prometaphase I by chromatin threads that ensure proper biorientation of achiasmatic homologues; these homologous connections are then resolved at later stages of meiosis allowing chromosome segregation. In Top2- depleted oocytes, heterochromatic regions of chromosomes usually fail to separate during prometaphase and metaphase I, and are often stretched into long protrusions with centromeres at their tips (Hughes and Hawley and references therein). These findings indicate that Top2 is required for resolution of the DNA entanglements that normally connect homologous heterochromatic regions during female meiosis and suggest that the pulling forces exerted by the spindle generate chromatin protrusions. However, the meiotic phenotypes elicited by Top2 depletion in males and females are similar but not identical. While in female meiosis only the heterochromatic regions appear to be affected, in male meiosis both euchromatin and heterochromatin are affected. A higher sensitivity of heterochromatin to Top2 depletion is consistent with our observations on Top2 alleles (suo1/DfTop2 and suo1/Top2suo3Top2) only exhibit chromosome aberrations (CABs), most of which involve specific regions of the Y and third chromosome heterochromatin. Severe RNAi-mediated Top2 depletion results in extensive chromosome breakage involving all chromosome regions with a preference for heterochromatin. Previous studies with pharmacological inhibitors of Topo II have also shown that treatments with these drugs cause CABs, but it is currently unclear to which extent these drugs directly induce DNA lesions, cause DNA damage via Topo II inhibition, or affect DNA stability through other off target effects Top2 mutants of budding and fission yeasts We have shown that relatively weak mutant combinations of Top2 Drosophila mutants? Two observations help answering this question. First, in mutant brain cells not treated with colchicine, 37% of the anaphases displayed chromatin bridges or lagging chromosome fragments generated by severing of the bridges. Second, most CABs observed in colchicine-treated cells were \u201cincomplete\u201d chromosome type aberrations (i.e involving both sister chromatids). Namely, they consisted in broken centric chromosomes not accompanied by a corresponding acentric fragment, in normal chromosome complements with an additional acentric fragment, in Y-3 translocations lacking the reciprocal element, or Y-3 dicentric chromosomes lacking the acentric fragment. As illustrated in Drosophila satellite DNA, which is mainly found in the centric heterochromatin of the X chromosome Drosophila heterochromatin We have shown that a relatively modest reduction of the Top2 level results in many isochromatid breaks and chromosome exchanges (translocations and dicentric chromosomes) that primarily involve 4 regions of the entirely heterochromatic Y chromosome  and a specific region of the 3L heterochromatin (region h47). To the best of our knowledge, previous studies did not detect site-specific chromosome aberrations after inhibition of Topo II function. What is then the mechanism underlying the chromosome damage specificity in weak Top2 RNAi brain cells contain very small amounts of Top2 and exhibit only a few divisions, most of which are hyperploid or polyploid. The few scorable diploid figures almost invariably displayed incomplete aberrations involving the Y or third chromosome heterochromatin, often accompanied by breaks in other chromosomes. We could not assess the presence of incomplete aberrations in polyploid metaphases, most of which displayed many apparent breaks of the centric heterochromatin of the major autosomes. These discontinuities in chromosome structure could be either due to drastic failures of heterochromatin condensation or to real breaks generated by the rupture of chromatin bridges during anaphase. Our results do not permit us to discriminate between these possibilities, but we favor the first.Top2 mutant combinations and Top2 RNAi cells revealed different and apparently contradictory effects on cell cycle progression. In suo1/DfTop2 brains that exhibit a \u223c60% reduction in the wild type Top2 level, the MI was comparable to that of wild type controls, but mutant brains displayed an increase in the frequency of anaphases. These data are consistent with previous studies on Drosophila S2 cells showing that RNAi-mediated depletion of Top2 does not affect the MI and causes only a small increase in the anaphase frequency Our observations on different Top2 RNAi brains and suo3/DfTop brains the MI was reduced by one and two orders of magnitude, respectively. Top2 RNAi brains also displayed many aneuploid and polyploid cells. Polyploidy has been also observed in chicken and human cells lacking Topo II activity, and was attributed either to defects in cytokinesis or to a reentry into interphase following a mitotic arrest (restitution) Drosophila brains can be generated by either restitution or cytokinesis failure  and Top2 RNAi S2 cells Drosophila does not have a decatenation checkpoint that arrests cell cycle in response to loss of Top2 function  or tefu (ATM). Because these kinases are involved in the signaling pathways that mediate most cell cycle checkpoints We found that in Top2 cause a specific alteration of the X chromosome morphology in male polytene chromosomes . This observation was confirmed and extended by Hohl and coworkers Top2 mutants the X chromosome retains the ability to recruit the MSL dosage compensation complex. In agreement with this study, we found that the bloated X chromosomes of Top2 mutants are decorated by anti-Mle, anti-Msl3 and anti-Mof antibodies. However, we have been unable to assess whether the staining intensity is the same as that of a normally condensed wild type X. Thus, it is quite possible that a reduction in Top2 expression partially affects the association of the MSL complex with the male X chromosome as recently suggested We first reported that mutations in Top2 mutants is decorated by antibodies against histone H4 acetylated at lysine 16 (H4K16ac). This post-translational modification is mediated by the Mof histone acetyltransferase, whose association with the X chromosome depends on Mle; a mutation in mle or blocking H4k16 acetylation rescues the X chromosome condensation defects caused by mutations in ISWITop2 is rescued in mof; Top2 double mutants, and that both the bloated X of Top2 mutants and the reconstituted X of mof; Top2 double mutants normally recruit the ISWI protein. These results suggest that loss of Top2 does not affect condensation of the dosage compensated chromatin by inhibiting ISWI recruitment. However, in the absence of Mof-mediated H4k16 acetylation the chromatin compaction functions of Top2 and ISWI are both dispensable. The genetic interaction between Top2 and mof is consistent with the previously shown physical and functional interactions between topoisomerase II and histone deacetylases (HDACs) Previous studies have shown that the X chromosome of polytene nuclei from Top2 mutants. Top2 downregulation in brain mitotic cells by either mutations or in vivo RNAi produced different cytological phenotypes. Moderate Top2 depletion (suo1/DfTop2) did not affect chromosome structure, and produced site-specific chromosome aberrations generated by the rupture of anaphase bridges. Severe Top2 depletion (Top2 RNAi) strongly reduced the MI, and induced heterochromatin undercondensation, extensive chromosome breakage, aneuploidy and polyploidy. Finally, complete (or nearly complete) Top2 deficiency (suo3/DfTop2) caused an interphase block and disrupted chromatid individualization in the rare diving cells. These phenotypes indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell. In addition, they recapitulate most, if not all, phenotypes previously observed in vertebrate cells exposed to Topo II inhibitors or RNAi against Topo II (see above). Thus, our results suggest that the previously observed discrepancies in vertebrate chromosome phenotypes elicited by Topo II downregulation might depend on the type of chromosomes examined (e.g. mitotic vs meiotic), slight differences in Topo II activity, or both.We have shown that meiotic chromosomes are extremely sensitive to Top2 depletion and exhibit drastic defects in chromosome morphology even in weak suo1Top2 and suo2Top2 mutant alleles were previously isolated by a cytological screen of a collection of male sterile mutants induced by EMS in C. Zuker laboratory suo3Top2 was isolated from a collection of about 1,500 lines carrying lethal mutations on chromosome 2, arisen in the Zucker collection of viable mutants CyOTbA, bearing the 1 (Tb1)Tubby dominant transgene Df(2L)Exel9043 was obtained from the Bloomington Drosophila Stock Center. 29Dmei-41FM7-GFP balancer. The 29D; Top2suo1/Top2suo3mei-41 and 29D; Top2suo3/Df(2L)Exel9043mei-41 double mutants were obtained by crossing 29D/FM7-GFP; Top2suo3/CyOTbAmei-41 females to suo1/CyOTbAFM7-GFP/Y; Top2 and to FM7-GFP/Y; Df(2L)Exel9043/CyOTbA males, respectively. Male larvae carrying both mutations were identified based on their non-GFP non-Tb phenotype. 3tefu  and Tb dominant markers. The suo1/Top2suo3; tefu3/tefu3Top2 double mutant was obtained by crossing suo1/CyOGFP; tefu3/TM6CTop2 females to suo3/CyOGFP; tefu3/TM6CTop2 males. Doubly mutant larvae were identified on the basis of their non-GFP, non-Tb phenotype. The 1mof mutant stock was kindly provided by J. Lucchesi. To construct mof; Top2 double mutants we crossed 1/FM7-GFPmof; suo1/CyOTbATop2 females to suo3/CyOTbAFMT-GFP/Y; Top2 males; non-GFP and non-Tb male larvae were then selected for cytological examination of polytene chromosomes. Iswi mutant larvae were generated by crossing 2 sp; +/TB3 CyO, TM6By w; Iswi females to 1 BcIswi/SM5, Cy sp males and recognized for the Bc non-Tb phenotype  were crossed to males carrying the Actin-Gal4 driver. The Oregon R laboratory strain was used as wild type control. All the stocks were maintained at 25\u00b0C on a standard medium. For markers, balancers and special chromosomes details see FlyBase (http://www.flybase.org).For in vivo RNAi-experiments, flies carrying a Extract preparation and Western blotting were performed according to ref. \u22125 M in saline), brains were treated for 8 min with hypotonic solution (0.5% Na Citrate), squashed in 45% acetic acid under a 20\u00d720 mm coverslip, and immediately frozen in liquid nitrogen. To analyze anaphases and assess mitotic parameters, larval brains were disssected in saline, directly squashed without colchicine and hypothonic pretreatment and immediately frozen. The mitotic index (MI) was calculated by determining the average number of mitotic figures per optic field as described previously To analyze the morphology and the integrity of metaphase chromosomes, brains from third-instar larvae were dissected in saline (NaCl 0.7%). After incubation for 1 h with colchicine . Grayscale images were collected separately, pseudocolored and merged.Figure S1Examples of anaphases observed in suo1/DfTop2 brains. The lagging acentric fragments comprise either a pair of banded Y chromosome sister chromatids (arrows) or two paired euchromatic elements (arrowheads), which are probably 3L arms. Heterologous fragments comprising a Y chromatid and a euchromatic element were never observed.(JPG)Click here for additional data file.Figure S2Examples of aberrations generated by chromosome breaks that occurred during the anaphase of the previous cell cycle. Panels (3\u20134) and (3\u20136) and the corresponding diagrams show Y-3 dicentric chromosomes (asterisks) and acentric fragments (arrows) generated by chromosome breaks that occurred in the anaphase of the previous cell cycle (see text and (JPG)Click here for additional data file.Figure S3Top2 RNAi brains contain more residual Top 2 than suo3/DfTop2 brains. The image shown was obtained with a longer exposure of the same blot of (JPG)Click here for additional data file.Figure S4Examples of polytene chromosomes from Top2 mutant and Top2 RNAi males. Note that in suo3/DfTop2 and Top2 RNAi males the X chromosomes no longer exhibit their typical banding pattern.(JPG)Click here for additional data file.Figure S5Iswi binds the X chromosome of male polytene nuclei from Top2 mutant males. Note that Iswi decorates both the poorly condensed X chromosome of suo1/Top2suo3Top2 males and the normally condensed X from mof; suo1/Top2suo3Top2 doubly mutant males; wt, wild type.(JPG)Click here for additional data file."}
+{"text": "The definition of an interval cancer is a cancer diagnosed between scheduled screening rounds. Interval cancer rates are an indication of the effectiveness of a Breast Screening Service in achieving early cancer detection, thus reducing overall mortality secondary to breast cancer, and represent a National Health Service Breast Screening Programme (NHSBSP) standard. The British Society of Breast Radiology (BSBR) Interval Classifications are as follows: 0 = unclassifiable, 1 = normal/benign, 2 = uncertain and 3 = suspicious. An audit was undertaken of interval cancers assigned a classification of 2 or 3 within our Breast Service over a 3-year period with the aim of determining and implementing improved clinical practice in a learning environment.The screening and symptomatic records were collected for all NHSBSP clients over a 3-year period subsequently diagnosed with a classification 2 or 3 interval cancer to establish the interval cancer rate benchmarked against NHSBSP standards and to determine the causal factors within the screening pathway.During the audit period 93,296 women underwent breast screening. The total number of interval cancers was 220. Of these 52 were assigned a 2 or 3 interval cancer classification. The NHSBSP standard of 1.2 interval cancers per 1000 women screened for the first 2 years following screening was not achieved for women screened within the initial 2 years of the audit period.Causal factors were most commonly associated with double mammographic screen reading. Subsequent interval cancers occurring most commonly during an incident screen in women aged 50 to 59 years, 12 to 24 months following NHSBSP screening."}
+{"text": "In the sentences \u201cFor vegans the intake of macro- and micronutrients (including supplements) did not reach the NNR for protein, vitamin D, iodine and selenium.\u201d and \u201cVegans reached the recommended daily intake of energy and fats but did not reach the recommended daily intake of protein (Table 2)\u201d, the statements are incorrect regarding the protein intake in vegans. Protein intake among vegans reaches the Nordic Nutrition Recommendations (NNR) as presented in Table 2.P value from the multiple linear regression testing for difference in means of energy intake between the vegan women and women from the DANSDA study is presented as 0.44. Instead, it should be 0.2 as presented in the text.In Table 2 the In the published Additional file n\u2009=\u200924) and women (n\u2009=\u200924) supplementing with specific nutrients, whereas the actual numbers are 20 and 26 for men and women, respectively, as presented in the corrected Additional file The published Additional file It has come to our attention that there are some errors in the original manuscript .In the sThe changes do not affect our conclusions.Additional file 1: Table S1Sex-stratified macronutrient intake in the vegan and the Danish National Survey of Dietary Habits and Physical Activity (DANSDA) study samples  with the 2012 Nordic Nutrition Recommendations (NNR). (DOC 54\u00a0kb)Additional file 2: Table S2Sex-stratified micronutrient intake in the vegan and the Danish National Survey of Dietary Habits and Physical Activity (DANSDA) study samples  with the 2012 Nordic Nutrition Recommendations (NNR). (DOC 77\u00a0kb)Additional file 3: Table S3Overview of supplement intake among the vegans. (DOCX 23\u00a0kb)"}
+{"text": "There is a dearth of good data from the Emergency Medical Services (EMS) regarding emergencies during a Marathon race. This study aims to document the various emergencies that can occur during the course of a marathon in India. We hope that this study will enable planners to create a database and to improve emergency preparedness for future such events.A total of 10,000 runners in the age group of 18-80 years, participated in the full (42.2 km) marathon, half (21.1 km) marathon and the 5 K run. Both trained and untrained runners took part in the race.th of August from 5:00 AM to 1: 00 PM.Southern Indian city of Hyderabad, on the 24Urban and semi-urban parts of the city with both paved and un-paved roads, maximum recorded temperature during the race was 35\u00b0 Celsius.A prospective descriptive epidemiological analysis of race data of runners presenting with emergencies was done using a standardized patient data collection form given to medical aid providers. Triaging was done according to Canadian triage and acuity scale.Of 10,000 runners, 252 runners required medical assistance during the course of the race.Of all the patients presenting to EMS 248 were Triage priority 3 or 4 requiring urgent or less urgent care\u2022 Exercise related musculoskeletal injuries accounted for 200 (79%) of the total cases\u2022 Dehydration related cramps accounted for 33 (13%) of the cases requiring oral or IV fluid rehydration\u2022 Terrain related blisters and foot injuries \u2013 9 (3.5%) patients\u2022 Vomitings - 4 (1.5%)A total of 4 patients were triage priority 2 requiring Emergent care \u2013 Transported by EMS to the nearest tertiary care hospital.A 24 year old with symptomatic hypoglycaemia, requiring Dextrose infusionA 45 year old male with Altered mental status secondary to Heat exhaustion and hypotension requiring IV fluid resuscitationA 70 year old male with Hypotension and dizziness requiring IV fluid resuscitationA 23 year old girl who collapsed with hypotension and heat exhaustion requiring IV fluid resuscitation.Long-distance running can be a safe and benign sport; however there were serious heat related illnesses requiring hospitalization of runners in this study. A well prepared EMS system is paramount to hosting a marathon in India due to extreme temperatures and mass participation.Consent was taken from the four patients who were triaged as priority 2 and received in-hospital care."}
+{"text": "When tested by the NCI against a range of human cancer cell lines, it was found that panicein A2 exhibits very little antiproliferative activity at 10 \u03bcM \u2013 an observation that is at odds with the earlier report that stated panicein A2 exhibits in vitro cytotoxicity against a number of tumour cell lines.The first total synthesis of the unusual aromatic sesquiterpene panicein A This hy2 (5), an example of a cyclised variant of the panicein structure, is a racemic compound that was first isolated in 1994 from Reniera mucosa alongside its non-cyclised isomer 1, and ten other members of the panicein family [2 (5) and D (2) were reported to exhibit in vitro cytotoxicity against four cancer cell lines  with an ED50 of 5 \u03bcg/mL [1 (4) was active against P388, A549 and MEL20 cell lines (ED50 = 2.5 \u03bcg/mL). The diversity of antiproliferative activities shown by these compounds, and particularly panicein A2 (5), presents them to be attractive synthetic targets.Panicein An family . Panicei2 (5) could be synthesised from the cyclisation and subsequent deprotection of propargyl ether 8. Ether 8 could be formed through the addition of the appropriate phenol to acetylenic alcohol 9, which itself can be derived from aldehyde 10 , giving 10 in 51% yield over two steps  being the double-addition species 19  through a modified Claisen rearrangement [20 in toluene for 48 hours, no desired product 5 was obtained, with a complex mixture of compounds produced  in 80% yield. The NMR data for synthetic panicein A2 (5) matched literature values  exhibited activity against four cancer cell lines with ED50 = 5 \u03bcg/mL \u2248 15 \u03bcM. To further expand upon these promising results, synthetic panicein A2 (5) was tested by the NCI against 57 human cancer cell lines, through their developmental therapeutics program. Interestingly, at the tested concentration of 10 \u03bcM, panicein A2 (5) showed very little activity against most cell lines. The best performance of panicein A2 (5) was against T-47D, a breast cancer cell line, in which it showed a 43% reduction of growth when compared to a control. Two of the cell lines tested by the NCI were the same as those tested against in the original isolation study (A549 and HT29). Our results show that panicein A2 (5) only reduces growth of A549 human lung carcinoma by 14% and had no effect on the HT29 human colon cancer cell line compared to control. These results indicated panicein A2 (5) to have poor antiproliferative activity and the possibility that the originally tested natural material was contaminated with trace amounts of an even more active compound.When isolated, natural panicein A2 (5) has been achieved. This synthesis hinges on key steps involving the addition of phenol 16 to carbonate 18 to provide propargyl ether 8 which was then cyclised through a modified Claisen rearrangement to ultimately give the desired cyclic structure of 5. The correlation of literature values for the isolated natural product and synthetic panicein A2 (5) confirm the structure of the natural product. Of particular note, when synthetic 5 was tested against a broad range of human cancer cell lines, it was found to exhibit very little activity at 10 \u03bcM \u2013 this observation is at odds with the earlier report that stated 5 exhibits in vitro cytotoxicity against a number of cell lines (ED50 = 5 \u03bcg/mL).In conclusion, the first total synthesis of aromatic sesquiterpine panicein AFile 15 and NCI testing results sheet.Experimental procedures, characterisation data of new compounds, NMR comparison tables of natural and synthetic File 21H/13C NMR spectra of synthesised compounds."}
+{"text": "Arabidopsis thaliana was studied by screening a series of accessions (ecotypes) under high Calcium (40 mM CaCl2 ) conditions. The genetic basis of this variation was further investigated by QTL analysis using recombinant inbred lines from Landsberg erecta (Ler)\u00d7Cape Verde Islands (Cvi) cross. Four QTLs were identified in chromosome 1, 2 and 5,and named response to high Calcium (RHCA) 1\u20134. The three QTLs  were further confirmed by analysis of near isogenic lines harboring Cvi introgression fragments in Ler background. Real-time PCR analysis showed that several genes associated with high Ca response including SMT1 and XHT25 have changed expression pattern between Ler and near isogenic lines. These results were useful for detecting molecular mechanisms of plants for high Ca adaption.Natural variation for primary root growth response to high Ca stress in  Ca ions  In spacious karst areas, the toxification of excessive Ca to plants significantly inhibits plant growth, crop production and species distribution Amaranthus hypochondriacus respectively, many of the genes are associated with stress response, transcription regulation and signal transduction Arabidopsis, the mutants of sterol methyltransferase 1 (SMT1) were hypersensitive to to higher Ca concentrations SMT1 could enhance the resistance of plants to high Ca stress. Cyclic nucleotide-gated channel 2 (CNGC2) was identified to play a key role in adaptation to high external Ca condition, the cngc2 mutant showed clearly increased sensitivity to external high calcium, and its transcription profile grown in normal media resembled that from wild type plants grown under elevated exogenous calcium condition cax3 mutants are also marginally sensitive to high external calcium Brassica juncea PCR1 (BjPCR1), a member of the plant cadmium resistance (PCR) protein family in Indian mustard, is an exporter required for the translocation of Ca2+ from the root epidermis to the inner cells and ultimately to the shoot. Root hair-specific expression of BjPCR1 in Arabidopsis causes increased Ca2+ resistance and translocation Several studies have focused on the molecular evens under high Ca condition. Genome-wide analysis displayed that 420 and 199 transcripts were found to be up- and down-regulated under high Ca stress in Arabidopsis for high calcium stress response, towards the long-term goals of developing genetic tools that will facilitate the breeding of more high-calcium-adaptive cultivars.QTL analysis has been employed as a powerful approach to discover molecular mechanisms of diverse plant traits Arabidopsis thaliana used were: Shakdara (Sha), Cape Verde Islands (Cvi), Landsberg erecta (Ler), An-1(N99), Columbia (Col) and Eri. For QTL analysis, we used the Core-Pop set of 161 RIL lines for QTL mapping experiments er/Cvi near-isogenic lines (NILs) were used for the confirmation and fine analysis of QTLs Accessions of PhytoTechnology Laboratories, Kansas, USA) agar (0.6%) medium supplemented with 1% sucrose. The Ca and Cl content in MS basic medium were about 3 mM and 6 mM respectively. After placed at 4\u00b0C for 5 days, the seeds were germinated at 23\u00b0C under illumination by light for 16 h and 8 h dark for 3 days. Subsequently, seedlings were transferred to MS agar (1%) medium supplemented with 1% sucrose with or without 40 and 50 mM CaCl2, 40 mM NaCl, 80 mM KCl and 40 mM Ca(NO3)2 respectively. Then, seedlings were grown vertically under photoperiodic cycles of 16 h of light and 8 h of dark at 23\u00b0C for 10 days. Finally, photographs were taken, and primary root lengths of all plantlets were measured. At least 3 seedlings per line per plate for each treatment were assayed. Three independent experiments with two or more replicates were conducted.Seeds were treated with 70% ethanol for 30 seconds, and then surface sterilized with 0.7% sodium hypochlorite for 15\u201320 minutes. After washing 4 times with steriled water, the seeds were sowed on Murashige and Skoog (MS) salt (er) and Cape Verde Islands (Cvi). These lines were previously described and characterized using amplified fragment length polymorphism (AFLP) and cleaved amplified polymorphic sequence (CAPS) markers http://www.kyazma.nl) using both interval mapping (IM) and multiple-QTL model mapping (MQM) methods, as described in the MapQTL reference manual. A logarithm of the odds (LOD) threshold of 2.4 was applied to declare the presence of a QTL A total of 161 recombinant inbred lines (RILs) were derived from Landsberg erecta (Ler/Cvi near-isogenic lines (NILs) under control and high calcium condition were measured. High Ca sensitive NIL lines were obtained for further analysis. These NIL lines were also described and characterized using amplified fragment length polymorphism (AFLP) and cleaved amplified polymorphic sequence (CAPS) markers To validate the presence and the effect of QTLs, primary root lengths of 92 L2 for 24 hours. And then, total RNA was isolated using the trizol method with TRIzol Reagent . After digestion with DNase I, it was reversely transcribed into cDNA with Oligo(dT)18 primer using SuperScript III reverse transcriptase, and used as template for PCR amplification. Actin2 gene was used as internal references. Real-time PCRs were performed in a Mastercycler ep Realplex apparatus  with SYBR Green Real-time PCR Master Mix . The relative expression levels were determined using 2\u2212\u0394\u0394Ct method 5\u2032-TGCACAAAGATGGAAAGGAG-3\u2032 and 5\u2032-CGGTAACAACTGAATTGCTG-3\u2032 for SMT1 (At5g13710); 5\u2032-CATCTCCTCCTTCTAGCACCA-3\u2032 and 5\u2032-TAGGAAAGGAGCAGAGGGTCT-3\u2032 for At1g21110; 5\u2032-AGATCGGACCAGACCGTGTT-3\u2032 and 5\u2032-CGCTTTCCTCATCGGCATTA-3\u2032 for At1g76680; 5\u2032-CGCGTTCCATAGACTCGAGTA-3\u2032 and 5\u2032-ATGGAACCCTCATCACATCGT-3\u2032 for At5g57550.Five-day-old seedlings were soaked in liquid MS medium with or without 40 mM CaClAll data were subjected to a one-way ANOVA analysis followed by DUNCAN  test using statistical package SAS 9.2, and the results were displayed as means \u00b1 standard deviation. Each data was obtained from at least three independent samples.Arabidopsis response to high calcium stress, we compared six commonly used accessions grown under control, 40 and 50 mM CaCl2 conditions. Typical seedlings after treating 10 days are shown in Arabidopsis accessions was observed. In comparison with the control, the plants growing on high calcium medium showed a reduction of primary root length  restricting the root development of Arabidopsis, we studied the responses of Ler and Cvi to the salt solution containing the same amount of Cl\u2212 as CaCl2 solution 2 treatment, meanwhile the two accessions showed nearly the same primary root length on MS medium  was determined under control and 40 mM CaCl2 conditions after 10 days growth. No significant difference in the primary root length of RILs under control condition was observed. A normal distribution of primary root length under 40 mM CaCl2 treatment was detected, indicating that multiple genetic factors are segregated among the RIL population , RHCA3 (chrom.2) and RHCA4 (chrom.5), explaining total 36.8% of the variation. All the Cvi alleles of the four QTLs contributed to shorter primary root length and the Ler alleles of the four QTLs led to longer primary root length in high Ca treatment. Epistatic interactions among QTL loci were not detected. In addition, the same QTL locations were also detected by performing QTL analysis for the ratio of primary root length of RILs in high Ca treatment to the primary root length in the control condition, confirming the existence of the four QTLs.By combining primary root length data with molecular marker data, QTL mapping was performed for primary root length response to high calcium stress. Four significant QTLs controlling primary root length under high calcium condition were detected on chromosome 1, 2 and 5 respectively , and namer\u00d7Cvi near-isogenic lines (NILs) carrying specific Cvi introgression fragments in a Ler genetic background 2 conditions for 10 days. Three NIL lines were obtained based on shorter primary root lengths than Ler under high Ca condition (RHCA1) and 25.40\u201330.5 Mb (RHCA2) respectively  after treated with 20 mM CaCl2SMT1) and At5g15410 (CNGC2) which play the key roles in response to high Ca stress SMT1, At1g21110 (IGMT3), At1g76680 (OPR1) and At5g57550 (XHT25) showing different expression patterns between Ler and NIL lines were obtained , we compared the DNA sequences of At1g21110, At1g76680 and At5g57550 genes between Cvi and Ler. At1g21110 and At5g57550 show lots of variations especially in promoter regions between Ler and Cvi. There are 15 SNPs and 2 single base-pair deletions in promoter region of At1g21110 and 41 SNPs and 7 single base-pair deletions in promoter region of At5g57550. These differences probably affect the expression of At1g21110 and At5g57550 under different conditions and support that the two genes could be the candidates for QTLs. There are only a few SNPs in At1g76680 between Ler and Cvi, indicating that this gene may not be the candidate for QTLs. The amino acid sequences of three proteins show no any change between Ler and Cvi.Three of the four genes were found to be located in the QTL regions. At1g21110 located at 7.3 Mb on chromosome 1 was in the QTL Arabidopsis accessions were treated with normal and high Ca concentrations in this study. The reduction of primary root length of all six accessions in high calcium treatments is consistent with the report of root length reduction of Ws under high calcium condition Arabidopsis thaliana has evolved some capacities to adapt to high Ca stress and the primary root length of seedlings was a suitable index for detecting different high Ca responsive mechanisms among diverse accessions.In order to detect molecular mechanisms of plant adapted to high calcium stress, six commonly used RHCA1 was located in the region of 6.46\u20137.86 cM on chromosome 1 (er and Cvi accessions RHCA1 might be related to Ca uptake. Other three QTLs were first reported by this research. The QTLs on chromosome 1 and 5 were confirmed by NIL lines (RHCA3 location did not show shorter primary root length than Ler seedlings under high Ca condition (data not shown). It is probable that the high Ca sensitive function of RHCA3 depends on Cvi alleles at other regions that could not be detected (epistasis).The primary root length of seedlings from 161 RILs under high Ca treatment showed obvious normal distribution, which indicating that plant response to high calcium stress was quantitative trait . By QTL mosome 1  overlapsIL lines , but theer and NIL lines under normal and high Ca condition by quantitative real-time PCR analysis  and was mainly expressed in roots and hypocotyls er in high Ca treatment  in Arabidopsis although interaction of this protein with calmodulin (CaM) needs to be confirmed experimentally er and LCN5-16 were both reduced, the high background content of XTH25 in Ler probably alleviated the restriction of root elongation caused by high Ca. The low content XTH25 in LCN5-16 was deficient to defend high Ca stress, which contributed to the short root formation. These results imply that a complex regulatory network integrating JA, CaM and sterol metabolization pathways is involved in response to high Ca stress for Arabidopsis primary root growth.In the study, we showed that four genes had different expression pattern between Lanalysis . SMT1 coondition . The lowondition  probablyreatment  affectedreatment  under hiArabidopsis accessions. The response of Arabidopsis to high Ca environment was participated by multiple QTLs and genes. The molecular mechanisms of underlying the genetic variations found in this work for high Ca response should be investigated in detail by further research. Our results provide foundation and clues for future works, which will facilitate in the comprehension of high calcium response mechanisms.In conclusion, our results illustrate that substantial genetic variation exists for the primary root length under high calcium condition between Figure S1er and Cvi seedlings under MS and 40 mM Ca(NO3)2 conditions.The primary root length of L(TIF)Click here for additional data file."}
+{"text": "Seafood is one of the most important causes of anaphylaxis worldwide and considered to be closely associated with house dust mite allergy due to cross-reactivity of mite trypomyosin (Der p 10) with the tropomyosins from shrimps and molluscs. We assessed the prevalence of sensitization to tropomyosin and other shrimp allergens in seafood-allergic subjects from Central Europe and explored the relationship with house dust mite allergy.Sera from 108 patients  with a convincing history of an adverse reaction to shrimp and/or molluscs and a positive routine test result to any seafood (skin prick test and/or specific IgE) were retrospectively analyzed by ImmunoCAP ISAC\u00ae for IgE antibodies against tropomyosin , shrimp arginine kinase (nPen m 2), shrimp sarcoplasmatic calcium-binding protein (nPen m 4), as well as the major dust mite allergens nDer p /f 1 and rDer p/f 2.67/108 sera (62%) reacted with at least one shrimp allergen in the microarray, whereas 38% were negative despite most of them showing a positive CAP result to crude shrimp or mollusc extracts. Sensitization to Pen m 1 was most prevalent (42.6%) followed by Pen m 4 (25.0%) and Pen m 2 (13.9%). 73% of the patients were monosensitized to only one molecule, mainly to Pen m 1 (65%) or Pen m 4 (63%) while sensitization to Pen m 2 was rarely monovalent (13%) and characterized by mostly low sIgE concentrations. Only 23/67 sera (34.3%) were positive for the dust mite allergens Der p/f 1 or 2. Correlating sensitization profiles with symptom severity after ingestion of seafood revealed that tropomyosin sensitization is regularly associated with systemic reactions while symptoms are often milder in case of sensitization to Pen m 2 and 4. Reactions were most severe on average in those with negative ISAC.Although tropomyosin can be confirmed as an important seafood allergen also in this population from Central Europe, it accounts for only 43% of cases. 38% of seafood-allergic subjects are sensitized to unknown allergens other that Pen m 1, 2 or 4. IgE to Der p/f 1 and 2 is only inconsistently found questioning a close causal relationship between house dust mite sensitization and seafood allergy."}
+{"text": "SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.Spinal Muscular Atrophy is caused by homozygous loss of  SMN1 gene SMN2, determines to some extent the phenotype of the affected individual and is unique to the hominid line The Spinal Muscular Atrophies (SMA) is a phenotypically diverse but genetically very similar group, in that the diseases are all caused by homozygous loss of the SMN2 in humans is unclear in the population as a whole, but in SMA patients SMN2 serves an important function as the remaining SMN expressing gene. It fails to completely compensate for the loss of SMN1 due to aberrant splicing of exon 7 which leads to the production of predominantly truncated transcripts and a corresponding decrease in the amounts of functional protein SMN2 is present in all SMA patients it has been extensively studied and serves as a drug target for drugs that specifically correct splicing of exon 7 and thereby increases amounts of functional SMN protein SMN2)89Ahmb mouse model of SMA The role of Smn1 to that of a human SMN2 and in the process changing as little as possible in the endogenous gene.In order to construct a transgenic pig which resembles the human SMA genotype as closely as possible we chose to study the potential in converting the endogenous pig SMN2 exon 7 is caused by the loss of an exonic splicing enhancer (ESE) due to a +6C>T transition in SMN2 exon 7 relative to SMN1 exon 7, leading to loss of binding of SRSF1 and increased binding of hnRNP A1 due to strengthening of pre-existing exonic splicing silencer (ESS) motifs SMN2, but in pigs the ESE motif is only slightly altered to CAAACAA in the wild type Smn1. This poses the question of whether or not this sequence constitutes an active ESE and if a single Smn2-like +6C>T mutation in porcine Smn1 exon 7 can disrupt the function and result in a porcine Smn2-like gene.The aberrant splicing of human Smn1 gene from genomic DNA by designing primers to amplify individual exons based on publicly available data as well as larger parts of the intronic regions surrounding exon 7 which were not publicly available at the time. Additionally, we performed 5\u2032RACE and 3\u2032RACE in order to validate previous assignment of exons and UTR regions. The resulting Yucatan Smn1 gene sequence has been submitted to the GenBank sequence database under accession number KF585502. Contrary to previously published data Smn1 is composed of not 9 exons but instead 10, and that the last two exons are located much further downstream from exon 7 than in the human SMN1 and SMN2 genes.We began by sequencing the Yucatan mini-pig Smn1 exon 9 begins . The distal hnRNP A1 binding site seems to be strengthened in the pig sequence (aagtga>cagtga). Therefore we tested both the human ISS sequence and a mutated human ISS sequence where both potential hnRNP A1 sites are disrupted by 2A>C mutations, which have previously been shown to disrupt hnRNP A1 binding SMN exon 7 inclusion than the corresponding pig ISS sequence. Insertion of the the mutated human ISS resulted in a very modest decrease in pig SMN exon 7 exclusion indicating that the pig ISS has only a very modest ISS activity  ESE, and much less binding of hnRNP A1 (Smn2-like) pig ESE was only slightly increased, indicating that reduced hnRNP A1 binding does not explain why the pig ESE retains some functionality when the +6C>T mutation is introduced. Importantly, the +6C>T mutated pig ESE bound SRSF1 very poorly, indicating that the loss of ESE activity observed in the PSXN13 construct could be due to loss of SRSF1 affinity (SMN2 (+6C>T) construct relative to SMN1. These results therefore indicate that the SRSF1-binding ESE in human SMN1 exon 7 is retained in pig and several other species with an identical motif .pSXN13 minigenes. To generate pSXN13 constructs we used sense and antisense oligonucleotides with desired sequences, which were mixed 1\u22361, phosphorylated and ligated to the BamHI and SalI sites in the artificial exon within pSXN13 5 Yucatan fibroblasts were seeded in 6 well plates and the following day transfected with 0.8 \u00b5g expression plasmid, either SMN constructs or pSXN13 constructs, using FuGENE6 transfection reagent . Transfections were performed in biological triplicates using fibroblasts from Yucatan minipigs. 48 hours post transfection cells were washed in 1 x PBS-EDTA, lysed by adding 900 \u00b5L TRIzol reagent  and incubated on ice for 10 min prior to RNA extraction according to the manufacturer's instructions (Invitrogen). Purified total RNA was used as template in first strand cDNA synthesis using the Advantage MMLV RT-PCR kit  with an oligo (dT)18 primer. App. 1/10 of the cDNA synthesis product corresponding to 100 ng RNA was used as template in each PCR reaction using Tempase DNA polymerase . For the SMN constructs we used an exon-exon junction spanning primer (CATTCCAGAGAACTGTGGAGGT) and a primer located in SMN1/2 exon 8 (GTGGTGTCATTTAGTGCTGCTC). For the pSXN13 constructs we used a primer located in \u03b2-actin exon 1 (AAGGTGAACGTGGATGAAGTTGGTGGTG) and an exon-exon junction spanning primer (CCCACGTGCAGCCTTTGACCTAGTA). PCR products were separated and visualized on agarose gels containing ethidium bromide (EtBr) on an Epi II Darkroom UVP Transilluminator. Bands were quantitated by optical densitometry using ImageJ 1.47 App. 2.0\u00d710Genomic DNA from Yucatan fibroblasts was extracted and 40 ng used as template in PCR reactions using Pfu polymerase  with primers listed in Smn1 sequence was submitted to GenBank database under accession number KF585502.Amplified PCR products were sequenced on the ABI Prism 3100-Avant . The partially complete Yacatan minipig The 5\u2032RACE and 3\u2032RACE experiments were carried out according to the manufacturer's instructions using the SMARTer RACE kit (BD Biosciences Clontech).SMN1: 5\u2032-GGUUUCAGACAAAAUCAA-biotin, SMN2: 5\u2032-GGUUUUAGACAAAAUCAA-biotin, pigSmn1: 5\u2032-GGUUUCAAACAAAAUCAA-biotin, pigSmn2: 5\u2032-GGUUUUAAACAAAAUCAA-biotin or the ISS motif: ISShum wt: 5\u2032-CCAGCATTATGAAAGTGAA-biotin, ISShum mut: 5\u2032-CCCGCATTATGAACGTGAA-biotin, ISSpig wt: 5\u2032-CCATCATTATAACAGTGAA-biotin, ISSpig mut: 5\u2032-CCATCATTATAACCGTGAA-biotin. Pull-down assays and immunodetection of purified proteins were carried out as previously described We used 3\u2032 biotinylated RNA oligonucleotides spanning either the ESE motif: SMN1 exon 7 orthologue sequences were extracted from the Ensembl database including 20 bp upstream and 30 bp downstream of exon 7.We used Human Splicing Finder Figure S1Smn1 mRNA and the Sus scrofa Smn1-pseudogenes. Capitals in the Smn1 sequence indicate the SMN open reading frame, dashes indicate gaps in the alignment.Alignment of the Sus scrofa (DOCX)Click here for additional data file.Figure S2SMN1 exon 7 and mammalian orthologues. MAFFT multiple alignment of SMN1 exon 7 with spanning intronic sequences and seven mammalian orthologues. Capitals indicate exonic bases, dashes indicate gaps. Asterisks indicate fully conserved bases while periods indicate partially conserved bases.Multiple alignment of (DOCX)Click here for additional data file.Figure S3SMN1 and porcine Smn1. Graphical output of analysis of the ESE region in human SMN1 and porcine Smn1 using Human Splicing Finder. A drop in ESE strength is indicated, as well as gain of a putative ESS in the context of porcine Smn1 relative to human SMN1.Analysis of human (DOCX)Click here for additional data file.Table S1Smn1 gene from genomic DNA from Yucatan fibroblast. The listed primers were used for PCR amplification using genomic DNA from Yucatan fibroblasts as template.Primers used for amplification of the porcine (DOCX)Click here for additional data file."}
+{"text": "IFNL) associate with clearance of hepatitis C virus (HCV) infection. One of these polymorphisms, IFNL4 rs368234815, determines loss or gain of function of the IFNL4 gene by frameshift variation. The very same and a second one, IFNL3 rs4803217, are supposed to impact the expression of IFNL3: while IFNL4 rs368234815 is suggested to modulate IFNL3 transcription, IFNL3 rs4803217 is thought to alter IFNL3 mRNA stability. The latter process is believed to be partially driven by an HCV-induced ectopic expression of myosin heavy chain genes 7B and 7 and their co-expressed microRNAs mir499 and mir208B. These ideas are evidenced by functional investigations on peripheral blood mononuclear and hepatoma cells in culture. Our study aimed at exploring IFNL3 gene expression in clinical samples, i.e., in ex vivo derived liver tissue from patients with chronic hepatitis C (n = 57) and various other diseases (n = 56). By applying an assay designed to specifically quantify IFNL3 and discriminating paralogous IFNL2 transcripts, IFNL3 mRNA expression was not found to differ significantly between chronic hepatitis C and control samples. Among patients with chronic HCV infection, moreover, IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles did not associate with reduced IFNL3 gene expression. Finally, myosin heavy chain genes 7B and 7 and corresponding microRNAs mir499 and mir208B were not found activated in liver in chronic HCV infection. Of note, detectability of MYH7 mRNA related to the procedure of liver biopsy sampling, as tissue obtained by direct punctation of the liver during laparoscopic inspection was less likely to contain MYH7 transcripts than samples acquired by percutaneous punctation. In conclusion, data on ex vivo derived liver tissue samples argue against an attenuating impact of IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles on hepatic IFNL3 gene expression in vivo.Genetic polymorphisms in the region of the interferon-\u03bb genes ( IFNL) genes to be correlated with clearance of infection , cIFNL1-3 transcripts, we recently demonstrated IFNL4 transcripts to be clearly activated in chronic HCV infection in patient livers compared to non-HCV infected controls [IFNL4 protein is enabled by the IFNL4 rs368234815 \u0394G variant, while the IFNL4 rs368234815 TT allele prevents translation by causing a frameshift [IFNL genotypes and HCV clearance [IFNL4 rs368234815 or IFNL3 rs4803217 in African American individuals, a recent study identified IFNL4 rs368234815 to be the primary polymorphism determining viral clearance [IFNL3 in peripheral blood mononuclear cells. Our study, yet, could not reveal any evidence for the proposed role of IFNL4 rs368234815 in hepatic IFNL3 gene expression, neither for IFNL3 3\u2019UTR rs4803217.Different to controls ,21. Genelearance . By takiIFNL3 by HCV in the liver of patients with chronic hepatitis C, the lack of an attenuation of basal IFNL3 transcript expression in IFNL3 rs4803217 or IFNL4 rs368234815 minor allele carriers, and the lack of an induction of myosin genes and their corresponding myomiRs argue against IFNL3 rs4803217 or IFNL4 rs368234815 variations to be causally related to clearance of HCV infection by modulating hepatic IFNL3 expression. Our findings, moreover, point at the importance of complementing functional mechanistic evidence with authentic clinical data to unravel the essential mechanisms for persistence and resolution of HCV infection operating in vivo in man.In conclusion, the lack of an induction of"}
+{"text": "LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGF\u03b2) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGF\u03b2 family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but  \u2022We examine expression, promoter use and enhancers for the fibrillin/LTBP gene family.\u2022Promoter switching was observed for most family members.\u2022Multiple enhancers were identified for all family members.\u2022Family members overlapped in tissue specificity with some unique expression patterns.\u2022A degree of redundancy among family members is possible. In rats and mice the FBN3 gene appears to have degenerated and does not produce a functional mRNA FBN3 is likely to be active since transcripts have been detected (data from http://www.ensembl.org). There are a variable number of annotated LTBP genes across species, from two in fish to four in mammals: LTBP1, LTBP2, LTBP3 and LTBP4. It is possible that one or more of the gene family members take over the role of FBN3 in rats and mice.In vertebrates, including eutherian, marsupial and monotreme mammals, birds, reptiles and fish, fibrillins are encoded by three genes, Fbn1 mRNA is ubiquitous in mesenchymal cell types Fbn2 appears more restricted in expression . A similar pattern was reported for human FBN2FBN3 expression is restricted to embryonic/fetal tissues http://biogps.org; Expression of fibrillin/LTBP family members is principally found in cells and tissues of mesenchymal origin. In mouse, Consistent with their function in mesenchymal cell types, mutations in members of this gene superfamily result in phenotypes that primarily affect connective tissue types (reviewed in FBN1FBN2 (unpublished). However those data included few tissues showing expression of FBN3 in humans. The current FANTOM5 project involves many more tissue and cell types likely to express members of the fibrillin and LTBP families 154700; FBN1 OMIM: 134797), congenital contractural arachnodactyly , primary congenital glaucoma  and cutis laxa with severe systemic abnormalities  The FANTOM  projects co-ordinated by the RIKEN Institute in Japan have provided extensive information on gene expression in human and mouse and allowed the identification and characterization of a large number of gene promoter regions http://fantom.gsc.riken.jp/5/.This work is part of the FANTOM5 project. Data downloads, genomic tools and co-published manuscripts have been summarized at 22.1Five human cell lines and a primary adult derived mesenchymal stem cell culture were used to analyze expression patterns of fibrillin/LTBP gene family members. SAOS-2 (sarcoma osteogenic 2) is a hypotriploid line derived from the osteosarcoma of an 11-year-old Caucasian female and has been in culture since 1973 2.2http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60\u00a0\u00b0C http://www.roche-applied-science.com, GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60\u00a0\u00b0C http://www.roche-applied-science.com, Supplementary Table 1). Human GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7\u00a0days, the cells were fixed with 95% ethanol and 5% acetic acid for 20\u00a0min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5\u00a0h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4\u00a0\u00b0C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1\u00a0h at room temperature. The cells were then washed with PBS, and mounted with ProGold\u00a0+\u00a0DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7\u00a0days, the cells were fixed with 95% ethanol and 5% acetic acid for 20\u00a0min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5\u00a0h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4\u00a0\u00b0C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1\u00a0h at room temperature. The cells were then washed with PBS, and mounted with ProGold\u00a0+\u00a0DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in Supplementary Table 1.2.3http://fantom.gsc.riken.jp/zenbu/). Composite promoters are those where two or more robust peaks occur within 100\u00a0bp of each other \u2212\u00a05The tissues and cells used for the FANTOM5 analysis of transcription initiation sites have been described in a parallel publication 2.4Express3DAll 81 DPI-detected robust promoters for the seven genes were clustered using Biolayout The parallel paper 2.5We downloaded the whole-genome alignment of the human genome with 45 other vertebrate genomes from the UCSC Genome Browser database 33.1We used quantitative reverse transcriptase PCR (qRT-PCR) to examine expression of gene family members in cell lines including H1 and RH1 embryonic stem cells, ADMSC mesenchymal stem cells from adipose tissue, SAOS2 and MG63 osteosarcoma cell lines and NHDF fibroblasts, . The resFBN1 expression was consistently high in ADMSC, NHDF and MG63 cell types but minimal in early ES cells (FBN2 was highly expressed early in embryoid body formation from H1 and RH1 embryonic stem cells and in SAOS2 and NHDF cells (FBN3 was present in both ES cell lines (H1 and RH1), with no expression in the remaining cell types  and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1 (FBN3) and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1  and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The maj21\u00a0=\u00a013.0; p\u00a0=\u00a00.0003) and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The majority of robust peaks were within 100\u00a0bp of another peak, forming 18 composite promoters FBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3\u2032 noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis . NotablyFBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3\u2032 noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis (Supplementary Table 2). Notably, promoters for the three alternative first exons of FBN2 transcript ENST00000508053.1 which has an additional six 5\u2032 non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 (LTBP4 A. \u2014ENST00000545697 and ENST00000204005).Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long FBN2 transcript ENST00000508053.1 which has an additional six 5\u2032 non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 .Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long LTBP1, alternate transcripts with a long or short 5\u2032 UTR have been reported (LTBP1 D.). However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200\u00a0bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5\u2032 end of LTBP1, alternate transcripts with a long or short 5\u2032 UTR have been reported . However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200\u00a0bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5\u2032 end of FBN1 promoter region contained a characteristic pyrimidine (T-rich) stretch, about 70 nucleotides upstream of the transcription start site associated with p1@FBN1 FBN2 promoter was in a C-rich region but the promoter region of the alternative transcript (p14@FBN2 and p18@FBN2) was not in a CpG island and was AT rich. The FBN3 promoter was also pyrimidine rich but shared no sequence homology with the other genes. The region around p1@LTBP1 contained alternating strings of purines and of pyrimidines.Although most promoters were located in CpG islands, there was little conservation of sequence around the promoters. Many of the promoter regions themselves were pyrimidine-rich (on the coding strand). The coding strand of the main FBN2 A. \u2014red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project FBN2 A. \u2014red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project 3.3LTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s)  . All proions see , while aLTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s) . This enhancer was not correlated with the other FBN2 promoters. p12@LTBP2 was uniquely associated with a bidirectional expression peak 50\u00a0kb upstream (Supplementary Table 3). In contrast, p10@LTBP3, p12@LTBP3 and p21@LTBP3 located within the second and third exons of the major LTBP3 transcript were correlated with one of the same predicted enhancers as the major promoter region containing p1@LTBP3. For LTBP4, each promoter tended to be associated with a different enhancer.p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C 3.4The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper http://fantom.gsc.riken.jp/5/sstar/ and To investigate this tissue restricted expression further, we used a number of ontologies http://fantom.gsc.riken.jp/5/sstar/ and Supplementary Table 4). Promoters in one composite cluster within a gene tended to be expressed in tissues associated with the same ontology terms    was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms , the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature   . Similaral cells . p2@LTBPyoblast) . The maied terms . Althougar cord) .FBN3 were strongly expressed in cells associated with ontology terms indicating mesenchymal origin  (Supplementary Table 4). Similar ontology terms were found for the main promoters of FBN2, which was expressed in a range of cells from fetal and embryonic tissues, fibroblasts, osteoblasts, placenta, hair follicle and lens epithelial cells  (Supplementary Table 4). The main composite promoter region of LTBP1  was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms (Supplementary Table 4). Although the promoter regions identified for LTBP2 were associated with some generic mesenchymal ontology terms (such as fibroblast and myoblast), the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature  (Supplementary Table 4).In general, the major promoters of all gene family members other than al cells . p2@LTBPpresumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord)  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflection this expression pattern   . p3@FBN2pression . p2@LTBPal cell) . The higtron 25,  displayeve organ .presumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord) (Supplementary Table 4). p3@FBN2 peaked in hematopoietic cell types, and was associated with ontology terms such as classical monocyte as well as terms reflecting mesenchyme/mesoderm expression (Supplementary Table 4). p2@LTBP2 was also expressed in some hematopoietic cells while p3@LTBP4 was associated with lymphocytes of the immune system  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflecting this expression pattern  (Supplementary Table 4). The high expression promoter of LTBP4, p1@LTBP4, was enriched in ontology terms reflecting an endodermal origin. p14 @FBN2 and p18@FBN2, located in intron 25,  displayed highest expression in testis and both promoters were associated with ontology terms such as testis, male reproductive organ, gonad and external genitalia/reproductive organ (Supplementary Table 4).A number of promoters were expressed in tissues of other origins. Thus p7@FBN1 and p1@LTBP3 were enriched in ontology terms reflecting development of the nervous system   l. Promotanscript  were higus layer .FBN3, p2@FBN3, was almost exclusively expressed in cells of fetal and embryonic origin. This expression pattern was associated with ontology terms such as embryonic stem cell, neuronal stem cell, neuroectodermal cell as well as some terms relating to pigmentation  (Supplementary Table 4l). Promoters of LTBP1 leading to the longer transcript  were highly expressed in embryonic and extraembryonic cell/tissue types including chorionic and amniotic membrane cells, fetal heart, epithelial and endothelial tissue samples. Within this region, p3@LTBP1 had the highest activity, and transcription from this region was found primarily in fetal heart. It was most strongly associated with ontology terms such as extraembryonic cell/structure, mesenchyme, compound organ and membranous layer (Supplementary Table 4).Some promoters were associated with early development. The sole robust promoter for LTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) (LTBP1 B.).Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where dLTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) .Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where different colored peaks (representing trimethylation levels in different cell types) can be seen for different promoters. For example, the composite 3.5Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts FBN3 promoter, p2@FBN3, was in this excluded group as were a promoter for FBN2 and LTBP1, seven for LTBP3 and two for LTBP4. Nine of these 11 were singleton promoters while two were within a composite promoter. The network of associations at P\u00a0\u2265\u00a00.5 is shown in LTBP2 promoters continued to cluster together (Cluster 1) while FBN1 (Clusters 2 and 12), FBN2 (Clusters 3 and 11) and LTBP1 (Clusters 4 and 6) formed two clusters each. p6@LTBP1 clustered with the majority of the FBN1 promoters, indicating an expression pattern more similar to FBN1 than to the other LTBP1 promoters. p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C FBN1 promoters. LTBP3  and LTBP4  formed four clusters each. These results suggest that the most diverse expression patterns were found for promoters of LTBP3 and LTBP4, while LTBP2 had the most similar expression pattern across all promoters.At a Pearson correlation coefficient threshold of 0.5, 11 promoters were excluded from the analysis indicating that their expression pattern was sufficiently different from any other promoter that they did not correlate at this level. The sole LTBP2 promoters and single clusters for FBN1, LTPB1 and LTBP4 .Promoters for LTBP genes tended to fail to cluster or formed clusters with other promoters for the same gene, particularly in the primary cell analysis. p1@LTBP1, p2@LTBP1, p4@LTBP1 and p5@LTBP1 formed a small cluster when all samples were analyzed and p3@LTBP1, p7@LTBP1 and p8@LTBP1 formed a cluster when primary cells were analyzed (Supplementary Table 2). However some LTBP promoters clustered with promoters for mesenchymal genes and occasionally were found with promoters for other members of the fibrillin/LTBP gene family. 3.7FBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3  for FBN3 l. HoweveFBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3 (Supplementary Table 2). However, binding motifs for the transcription factors MAZ, PATZ1, RREB1, SP1 and TFAP4 were detected in the promoter region of FBN3.We used Motif Activity Response Analysis (MARA) LTBP1  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both (Supplementary Table 2).4Members of the fibrillin/LTBP gene family appear to play two roles in the extracellular matrix. Firstly, they contribute structurally to the formation of the matrix. In particular, fibrillin-1 and fibrillin-2 are integral components of the 10\u00a0nm microfibrils which provide strength and elasticity to the matrix In this study we have examined gene expression and regulation of the seven fibrillin/LTBP family members using results from the FANTOM5 CAGE analysis, a genome wide analysis of transcription start sites and bidirectional putative enhancers FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN2, where p14@FBN2 and p18@FBN2 were expressed in reproductive cell types. The transcript associated with these promoters, ENST00000507835.1, covers coding exons 26 to 34 of the full length FBN2 and the polypeptide would consist of eight cbEGF domains with short N- and C-terminal flanking sequences (http://www.ensembl.org). Preliminary examination of the mouse CAGE data from FANTOM5 showed an equivalent promoter exclusively expressed in mouse testis, suggesting that this transcript has a real function, perhaps involved with sequestration of calcium. Further investigation of this novel transcript should reveal more about its role.Promoter switching with a change in tissue specificity was also seen for LTBP1 is documented to have at least two alternative transcription start regions (http//:www.ensembl.org) and this switch in promoters was confirmed in our analysis. One promoter region demonstrated high expression in early development and low expression in mesenchymal cells  while the alternate promoter region  was associated with mesenchyme-related ontology terms and expressed in heart. The major promoters p1@LTBP1 and p2@LTBP1 were associated with the same transcription factor motifs , while the alternative promoter region was associated with a distinct set of transcription factors motifs .Putative enhancers within 500\u00a0kb of the promoter regions were significantly associated with many of the promoters examined. The majority were downstream of the promoter region and some were as close 2\u00a0kb from the promoter. In general many promoters in one gene were correlated with the same predicted enhancers although there were a few examples of promoter-specific enhancers, associated with promoters of unique expression pattern.FBN2 showed similar expression patterns and ontology term enrichment to FBN1. However, FBN1 and FBN2 promoters did not cluster together in the co-expression analyses and there was little overlap of transcription factor motif activity between FBN1 and FBN2 promoters, suggesting that there were subtle differences in their expression patterns and regulation. In particular, FBN2 promoters tended to be associated with the epithelial and pluripotency transcription factor KLF4 FBN1 promoters were associated with mesenchymal transcription factors Cluster analysis of expression from the different promoters highlighted the differences between the gene family members. Members of a composite promoter often clustered together, but few promoters for different genes were found in the same cluster. For example, most promoters for LTBP2 A \u2014blue box and C.), for example, showed significant potential to bind GATA4 , for example, showed significant potential to bind GATA4  or Ltbp4 (through p5@LTBP4). Preliminary analysis of FANTOM5 data for mouse indicates that the equivalent of p3@LTBP1 is strongly expressed in embryonic tissue, especially neuron derived neurospheres, while a region equivalent to p5@LTBP4 is expressed in embryonic stem cells , supporting this possibility.Because its expression is restricted to fetal and embryonic tissue types FBN1, FBN2, LTBP2, LTBP3 and LTBP4 have been associated with connective tissue disease in humans. Transgenic and natural mutation mouse models, available for Fbn1, Fbn2, Ltbp1, Ltbp2, Ltbp3 and Ltbp4 also show phenotypic abnormalities of connective tissue (reviewed in Mutations in human 5This study supports the differential roles of members of the fibrillin/LTBP gene family. We have shown the strong mesenchymal expression of most family members and most promoters and highlighted promoter and enhancer switching associated with changes in tissue specificity. We were unable to find evidence for a single \u201cmaster mesenchyme regulator\u201d but there was considerable overlap in the transcription factor activity associated with these genes. These data aid in better understanding the overall function and activity of the members of the fibrillin/LTBP family.Promoters of the fibrillin/LTBP family members.qPCR primers and antibodies.Promoter characteristics, expression levels, transcription factor associations and expression clusters.Enhancers of fibrillin/LTBP family members.Ontology terms associated with cell types expressing fibrillin/LTBP family member promoters.The following are the supplementary data related to this articlehttp://dx.doi.org/10.1016/j.ymgme.2013.12.006.Supplementary data to this article can be found online at The data in the paper will be freely available.http://fantom.gsc.riken.jp/5/. FANTOM5 enhancers can also be accessed from. http://enhancer.binf.ku.dk.The FANTOM5 atlas is accessible from MRD carried out bioinformatic and molecular genetic analysis, participated in the design of the study and helped to draft the manuscript. RA provided the enhancer analysis. JS and NB supported the analysis and visualization. JKB performed the initial clustering of all CAGE detected promoters. MdH carried out the MARA analysis. HK managed the data handling. ARRF was responsible for FANTOM5 management and concept. KMS conceived this study, participated in its design and coordination, carried out much of the analysis and interpretation, performed the clustering analysis of the promoters and drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes  among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes  were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and GSTM1 null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of ADH1C, MnSOD and GSTM1 can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase ( Alcoholic liver disease (ALD) is one of the fast emerging common causes of chronic liver diseases across the globe. It is the clinical consequences of continuous alcohol over consumption  . AlthougADH) and aldehyde dehydrogenase (ALDH) (particularly ALDH2) in risk of development of ALD have been extensively investigated  at the \u20139 amino acid position of the N-terminal signal sequence , two for ADH1C genes [ADH1C*1 (Arg272Ile350) and ADH1C*2 ]. ADH1B*2 allele codes for a higher activity of the enzyme in ethanol oxidation with increased formation of acetaldehyde and ROS and is highly prevalent among neighbouring East-Asians such as Chinese, Japanese and Korean whereas it is least common among Indians [ADH1B polymorphism [ADH1C*1(rs698), which encodes for alcohol metabolism enzyme with high activity, has consistently found protective in alcoholic individuals in Chinese and Korean population [ADH1B (rs2066701) and two of ADH1C, rs1693425 (synonymous variants) and rs1789920 identified with significant association to the increased risk of ALD than ALC. Intronic variants may show association for being in linkage disequilibrium with a causal variant and also if situated at the beginning of an intron may result in alteration of the protein by affecting the splicing phenomenon of the nascent mRNA. rs1789920 which is present at the beginning of intron 2 of ADH1C may have an important role during the splicing phenomenon.Among the 7 human  Indians \u201326. A reH1C*1 Arg2Ile350 apulation , but foupulation , but faiALDH enzymes, ALDH2  is highly expressed in the liver and stomach and it has a very high affinity for alcohol detoxification by acetaldehyde oxidation [ALDH2 exhibits accelerated risk of progression of gastrointestinal cancers such as gastric cancer, esophageal cancer and colon cancer but its role in the liver, the major organ of alcohol metabolism, is still controversial [ALDH2 gene  but none of them showed sigificant association with ALD among \u201cBengalis\u201d which is similar to the previous observation by Bhaskar et al with six ethnic populations from four linguistic groups in India [Out of nineteen putative functional genes that encodes for xidation ,27,28. Doversial \u201330. In tin India .CYP2E1 and generates acetaldehyde and free radicals to induce progression of liver diseases to cirrhosis. The 5\u2019-flanking region of CYP2E1 gene is highly polymorphic at two non-coding loci, rs3813867 and rs2031920 [CYP2E1*5B is associated with higher transcription of CYP2E1. Significant association of this variant with ALD and other liver diseases has been documented in Oriental [Chronic alcohol intake induces the level and activity of s2031920 . These tOriental  and NortOriental  but not Oriental . These lGSTT1 and GSTM1) and superoxide dismutases. Consistent with the North Indian study [GSTM1 gene and coding variant of MnSOD (rs4880Ala) with lower enzyme activity were observed with significantly higher frequencies among ALD in this study. Association of deletion variant of GSTM1 with ALD has been observed in several studies [GSTM1 plays critical role in liver diseases as supported by our gene-gene interaction data where cumulative effect of this null variant has been observed with GSTT1 null genotype, rs4880 of MnSOD and rs2066701 of ADH1B. Again, Alanine to Valine change in the 9th position of mitochondrial target sequence of the single mitochondrial superoxide scavanger MnSOD which alters its amphiphilic helical structure crucial for transport and processing of mitochondrial protein varies among ethnic groups. Although a controversial data is available in the literature, a very few studies documented Valine as risk factor against a large data available for Alanine as effective variant which spontaneously dismutase superoxide to peroxide and oxygen [TNF-\u03b1 mediated apoptosis whereas Valine variant inhibits entrance of MnSOD to inner membrane and enhances programmed cell death. Furthermore, our logistic regression analysis revealed significant association of null genotype of GSTM1 and rs4880TT of MnSOD with severity of liver diseases.The oxidative stress generated in alcoholics is neutralized by two important enzymes, glutathione-s-transfereases  are independently associated with the development of ALD among Bengali alcoholics. A genetic interaction amongst them also deserves importance in disease development. As two genotypes in oxidative stress response genes were also found in significantly higher frequencies among North Indian alcoholics, these genetic variants may serve as powerful predictor of ALD in advance and hence, these markers can be useful in the management of Indian alcoholics. Further stratification in a new cohort of patients and powerful complementary mechanistic studies to determine the physiological and pathophysiological role of those variants in liver disease development has been initiated which may help to improve diagnostic and therapeutic strategies for ALD patients.In conclusion, the current study confirms that the genetic variants in S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(a)Genotypic association of the Alcohol metabolism and oxidative stress related genes in Indian ALD patients. (b): Distribution of genotypes at different loci, which were either monomorphic or not significant in Bengali population of India.(DOC)Click here for additional data file.S4 Table(a)Interaction between rs2066701 of ADH1B and rs1693425 of ADH1C gene. (b): Interactions between anti-oxidative genes GSTT1, GSTM1 and rs4880 of MnSOD. (c): Inter gene interaction between ALD metabolism and oxidative stress related genes.(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file."}
+{"text": "Two hand hygiene techniques are promoted internationally: the World Health Organisation\u2019s 6 step and the Centre for Disease Control\u2019s 3 step techniques; both of which may be considered to have suboptimum levels of empirical evidence for use with alcohol based hand rub (ABHR).The aim of the study was to to compare the effectiveness of the two techniques in clinical practice.A prospective parallel group randomised controlled trial (RCT) was conducted with 1:1 allocation of 6 step versus the 3 step ABHR hand hygiene technique in a clinical setting. The primary outcome was residual microbiological load. Secondary outcomes were hand surface coverage and duration. The participants were medical and nursing participants (n=120) in a large teaching hospital.The 6 step technique was statistically more effective at reducing the bacterial count 1900cfu/ml  to 380cfu/ml  than the 3 step 1200cfu/ml  to 750cfu/ml  (p=0.016) but even with direct observation by two researchers and use of an instruction card demonstrating the technique, compliance with the 6 step technique was only 65%, compared to 100% compliance with 3 step technique. Further those participants with 100% compliance with 6 step technique had a significantly greater log reduction in bacterial load with no additional time or difference in coverage compared to those with 65% compliance with 6 step technique (p=0.01).To our knowledge this is the first published RCT to demonstrate the 6 step technique is superior to the 3 step technique in reducing the residual bacterial load after hand hygiene using alcohol based hand rub in clinical practice. What remains unknown is whether the residual bacterial load after the 3 step technique is low enough to reduce risk of transmission from the hands and whether the 6 step technique can be adapted to enhance compliance in order to maximise reduction in residual bacterial load and reduce duration.None declared."}
+{"text": "K13 mutations superimposed onto a long-standing background of Pfmdr1 amplification.The pivotal factor leading to the declining efficacy of the artemisinin-based combination on the Thailand\u2013Myanmar border (mefloquine\u2013artesunate) to a clinically unacceptable level is the increasing local prevalence of  Background.\u2003Deployment of mefloquine\u2013artesunate (MAS3) on the Thailand\u2013Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial.Methods.\u2003Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance.Results.\u2003Polymerase chain reaction (PCR)\u2013adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8%  compared with 97.8%  in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%.Conclusions.\u2003The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand\u2013Myanmar border. Plasmodium falciparum malaria in 1985. However, resistance developed rapidly ) compared with admission isolates . In the recurrent isolates, more recrudescent isolates also had K13 mutant alleles compared with reinfections .Pfmdr1 copy numbers were measured in 726 (71.4%) admission isolates and 65 (34.8%) recurrent isolates (Supplementary Table 2). The proportion of infections caused by parasites with multiple (>1) Pfmdr1 copies on admission doubled from 32.4%  in 2003 to 64.7%  in 2013  compared with admission isolates 377/726 . Among these recurrent isolates, more recrudescent isolates also had multiple copies of Pfmdr1 compared with reinfections .s and 65 4.8% recuPfmdr1 gene because these relevant single-nucleotide polymorphisms (SNPs) are rare in Thailand [Pfmdr1 wild-type background [K13 were associated with single-copy Pfmdr1, while K13 mutations were associated with amplified Pfmdr1  .Our study did not characterize the single nucleotide polymorphisms of the Thailand  and meflckground , 28\u201330. K13 and Pfmdr1 was lower in recurrent infections because of significantly lower parasitemia (P = .0004) and thus much lower parasite DNA concentrations.The success rate for genotyping of P < .001, test for trend; Table K13 gene was the strongest risk factor for day 3 positivity, with later year of treatment, higher parasitemia, higher hematocrit, and fever on admission (but not Pfmdr1 amplification) as independent risk factors  were each significantly associated with day 3 positivity (Table Clearance data were available for 957 patients 95.2%). There was a significant increase in the proportion of patients who were parasitemic at day 3  recrudescences and 53 (28.5%) reinfections, with 2 indeterminate results, 1 amplification failure, and 13 missing samples. PCR-adjusted parasitological efficacy at day 42 remained above or close to 90% from 2003 to 2009 but declined sharply thereafter  along with year of recruitment and age  and lowest in patients with multiple copies of Pfmdr1 and any K13 propeller SNP . Cure rates declined markedly in recent years as the prevalence of K13 mutations rose .Cure rates were highest for infections with isolates with single-copy K13 and Pfmdr1 amplification were 69.0 and 41.9%, respectively. The PAF for the 2 factors in combination was 82%. Hence these 2 factors alone explain almost entirely the increased ACT treatment failure rate observed in this population.The PAFs  for Supplementary Materials . There was no association between the presence of K13 propeller mutations  or multiple copies of Pfmdr1  and gametocytemia, either at admission or during follow-up.The proportions of patients presenting with pre-treatment gametocytes or with post-treatment gametocytemia and the associated risk factors are shown in the P. falciparum malaria has had a remarkable therapeutic longevity and a dramatic impact on morbidity and mortality on the Thailand\u2013Myanmar border. When artesunate was introduced in 1991, mefloquine was a failing drug, the incidence of falciparum malaria was rising, and most parasite isolates had multiple copies of Pfmdr1 associated with mefloquine resistance [Pfmdr1 copies were replaced by single copy\u2013containing isolates [Pfmdr1 [P. falciparum parasites with K13 propeller mutations was the definitive event that led to the demise of MAS3. The temporal sequence of K13 selection is informative. The E252Q mutation , associated with moderate slowing of parasite clearance [K13 propeller mutations, conferring greater reductions in parasite clearance rates. The main propeller mutation was C580Y, a polymorphism common in Cambodia [K13 mutation is proportional to the degree of parasite clearance prolongation. It is possible that genetic changes outside the K13 locus have contributed to the emergence of artemisinin resistance and the selection of K13 mutations [MAS3 for the treatment of uncomplicated sistance . After disolates . High cuisolates , 32. In  [Pfmdr1 . The rislearance , predomiCambodia , 15. Theutations . It remaK13 mutations on treatment failure. K13 mutations in admission samples are associated with a failure rate of 21.5%. In combination with multiple copies of Pfmdr1, this rises to 42.2%. The sharp decline in MAS3 cure rates temporally corresponds with the emergence of K13 propeller mutant isolates on a long-standing genetic background of Pfmdr1 amplification. The proportion of treatment failures that can be attributed to K13 mutations and multiple copies of Pfmdr1  is 82%. Hence, these 2 factors alone explain the majority of increase in treatment failure observed. Other parasite genetic factors that our study was not designed to detect or changes in the demographics of the patient population may have contributed to the 18% of unexplained variation in treatment failure.The large body of data presented here strongly support the hypothesis that there is a substantial impact of K13 mutations and Pfmdr1 amplification have a multiplicative (rather than an additive) effect on risk of treatment failure. Synergy between these 2 resistance determinants may help to explain why failure rate declined precipitously in 2009\u2014parasites carrying both markers only became common as K13 mutations rose in frequency. Second, in contrast to the 2 Cambodian studies, we observed multiple K13 mutations in this study. These data demonstrate significant heterogeneity in the impact of different K13 mutations on treatment failure. A mutation outside the K13 propeller (E252Q) increased treatment failure relative to wild-type parasites, but the 3 common propeller mutations had a much greater effect , which has a resistance mechanism that is similar to that of mefloquine, involving fication , 33, 34.despread , 35\u201338. P. falciparum malaria in our treatment centers on the Thailand\u2013Myanmar border was changed from MAS3 to DHA\u2013piperaquine, which is currently highly efficacious in this area but relies increasingly on the piperaquine component. The recent emergence of piperaquine resistance in Cambodia [P. falciparum from the area before the recent and substantial gains in malaria control are reversed.ACTs containing an alternative partner drug should provide relief in the short term. In 2012 the first-line treatment of uncomplicated Cambodia  and assoCambodia , 21, 40 Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author."}
+{"text": "In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 \u201cbypass\u201d strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9 \u2013 the latter the ortholog of human S6 protein kinase 1.In human cells TORC1 mTOR (Significance: A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a \u201cpro-survival\u201d role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved. Heat shock protein 90 (Hsp90) provides a molecular chaperone function essential for the conformational maturation, activation and maintenance of proteins essential for sustaining all of the hallmarks of cancer. As such it is now a prime target for drug development, with several Hsp90 inhibitors currently in cancer clinic trials , 2. Inhitarget of rapamycin) protein kinase , wh, wh11], In this study we found little synergy between the inhibitory effects of rapamycin and the Hsp90 inhibitor radicicol on the growth of yeast, irrespective of whether the cells possessed a stress-inducible form of Hsf1 Fig. . Such fitor1\u0394, fpr1\u0394; Fig. The novelty of this work is that it has provided the first demonstration that Hsp90 inhibitor resistance can be influenced by mutations causing resistance to rapamycin Fig.  and, conTOR1.1, TOR2.1 and fpr1\u0394 rapamycin resistance mutations sensitise cells to Hsp90 inhibitor treatment  and measurement of levels of \u03b2-galactosidase expression were as previously described [fpr1\u0394 containing this HSE-LacZ vector was transformed with additional YCplac111-based LEU2 vectors bearing either the wild type or F43Y mutant forms of the FPR1 gene under native promoter control, then grown on SD minus uracil and leucine prior to measurements of their HSE-LacZ expression.Yeast strains used in this study are listed in Table escribed . Culture5 cells/ml), this dilution being used to set up radicicol and rapamycin or radicicol and caffeine dose response matrixes  in 96-well microtiter plates. After 46h growth at 28\u00b0C the cells were re-suspended by agitation, their absorbance determined at 595 nm with a Multiskan Ascent platereader with correction for the background from the corresponding medium, and the data quantitatively displayed with color using the program Java Treeview 1.1.3 (http://jtreeview.sourceforge.net/).For drug sensitivity assays, overnight YPD cultures were either serially diluted, then pinned onto YPD agar containing the indicated antibiotic and the plates incubated as indicated in the figure legends; or they were inoculated into complete SD medium (1\u00d710"}
+{"text": "Antibodies 14F7 and the corresponding anti-idiotype 1E10  were mistakenly conflated in this review. 14F7 reacts with (Neu5Gc)GM3, was recently humanized . The firThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The authors would like to clarify that some of the blots previously depicted in The correct blots for The authors have provided new versions of Figs The text in the Results sections titled \u201cThe effects of acute Herceptin treatment on HER3 and PKB phosphorylation\u201d and \u201cThe discordant effects of Herceptin on ERK and PKB pathways\u201d has been edited to accommodate the removal of panel E in The corrected text, Figs We found that the downregulation of HER2 receptors was detectable after Herceptin treatment in SKBR3 and BT474 cells, and was associated with an increase in HER2 phosphorylation . Lee-HoeWe analysed the effects of Herceptin on the downstream signalling pathways in HER2 positive breast cancer cells. It was found that the effect of acute Herceptin treatment on phosphorylation of PKB and ERK1/2 was not concordant Figs , in contAcute Herceptin exposure increased EGFR/HER2 and HER2/HER4 dimerisation, correlating with an increase in ERK phosphorylation . In contTherefore, Herceptin treatment decreased PKB phosphorylation due to a decrease in HER3 phosphorylation induced by HER2 downregulation. However, one hour of Herceptin treatment increased ERK phosphorylation as a result of ligand-dependent EGFR and HER4 activation.We found that although Herceptin downregulated HER2 receptors, the remaining cells had persistent and increased HER2 phosphorylation in both SKBR3 and BT474 cells . Since HWe postulated that the increased dimerisation of EGFR, HER3 and HER4 with HER2 was due to activation by their respective ligands. We proceeded to assess the levels of endogenous ligands, using heregulin (ligand for HER3 and HER4) and betacellulin (ligand for EGFR and HER4) as examples. Herceptin treated cells were lysed and endogenous ligand levels were detected using ELISA. We found that Herceptin induced a statistically significant upregulation of heregulin and betacellulin (p = 0.0152 and p = 0.0286 respectively) after 1 hour of Herceptin treatment compared to untreated cells in both SKBR3 and BT474 cells (data on BT474 cells are shown in Thus, Herceptin increased the dimerisation of EGFR, HER3 and HER4 with HER2 as a result of the activation by their ligands."}
+{"text": "Systemic onset Juvenile Idiopathic Arthritis (SoJIA) is a subset of juvenile idiopathic arthritis (JIA) that describes patients with intermittent fever, arthritis and skin rash with complex genetic trait in children less than 16 years old. The etiology of disease has not yet been identified; but because of high level of IL-1\u03b2 in the blood of patients; and relation between its complications, and clinical signs of disease; the only theory is mutation in factors which are involved in the control of production and secretion of this proinflammatory cytokine, like IL-1Ra, IL-1 type II decoy receptor proteins and soluble IL-1R accessory protein (IRAP).The aim of this study was to identify the association between MEFV mutations in exons 2 and 10, and SoJIA disease in 30 Iranian patients.Thirty Iranian children with SoJIA disease and 30 healthy control, were screened for MEFV mutations in exons 2 and 10 by sequencing. All patients were diagnosed according to the classification criteria of EULAR for SoJIA. The control group consisted of 30 healthy individuals  who did not have any history of immune system disorders and other diseases with known genetic or hereditary predisposition.Statistical analysis showed that the MEFV mutations in the patients and control groups were significantly different (p<0.01). The mutations were detected in 17 of 30 patients (56.6%). The respective figure for the control subjects was 1 of 30 (3.3%). Leading mutation was R202Q mutation which detected in 13 patients ( 6 homozygote and 7 heterozygote). Following eight patients had M694V mutation ( 2 homozygote and 6 heterozygote). Three patients had E148Q mutation in heterozygote form.MEFV mutations may be an important cause of high level of IL-1\u03b2 in SoJIA patients. It shows that SoJIA is better to be classified as auto-inflammatory disorder.None declared."}
+{"text": "Computational cardiac modelling has been established as a valuable tool for simulating electrophysiology and electromechanics of the heart , with pr2). The entire left ventricle was covered in both systole and diastole with a total of 12 slices with a spatial resolution of 2.2\u00d72.2x6mm3.Data from one healthy volunteer was acquired using a dual-phase dual-slice stimulated echo acquisition mode (STEAM) method  on a 1.53 and 18ms, respectively. Diastolic and systolic diffusion tensors were mapped into a prolate spheroidal coordinate system [To correct diffusion tensors for material strain, additional 3D tagging data were acquired and incorporated into diffusion tensor calculation  with spae system  for 3D dData across the entire left ventricle were successfully acquired in both systole and diastole. Figure The data presented here provides a comprehensive set of information on microstructure and motion and may serve as realistic input for computational modeling projects.This work is supported by UK EPSRC."}
+{"text": "A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseC\u03b32 (PLC\u03b3-2) gene, corresponding to human PLC\u03b3-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2  and exon 27 of the PLC\u03b3-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLC\u03b3-2 gene.Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.PLC\u03b3-2 gene in the pathogenesis of WG in our case-control study.This report does not reveal any hints for an involvement of the  Staphylococcus aureus is a risk factor for disease exacerbation in WG ]]StaphyloPLC\u03b3-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse + status Pooling of DNA was performed as reported before . PatientIn this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included  and 50 ng DNA.For PCR we used the 'tailed primer PCR' as described before . For ampElectrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile )7]). AIPStatistics for comparisons of allele frequencies of patients and controls was performed as described before [ and 7]]]7]]. CasExons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (, exon 27PJ and SW carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and performed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive.  TGFBR1*6A is a common variant of the type I TGF-\u00df receptor, TGFBR1 [TGFBR1*6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of TGFBR1 (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region. In vitro studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-\u00df growth inhibitory signals [TGFBR1*6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that TGFBR1*6A might act as a tumor susceptibility allele [TGFBR1*6A carriers may have an increased risk of breast, colon and ovarian cancer [TGFBR1*6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls.Transforming Growth Factor Beta (TGF-\u00df) is one of the most potent inhibitor of cell growth . Almost , TGFBR1 . TGFBR1* signals ,5. The an cancer . To testTGFBR1*6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59 TGFBR1*6A heterozygotes and three TGFBR1*6A homozygotes among cases, 62 TGFBR1*6A heterozygotes and 1 TGFBR1*6A homozygote among controls. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67)  but was not significant after adjustment for race and age strata within groups  (Table TGFBR1*6A was significant (p = 0.01).A total of 907 cases and controls were genotyped for BRCA2 gene have an increased prostate cancer risk [BRCA2 mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of TGFBR1*6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that TGFBR1*6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that TGFBR1*6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between TGFBR1*6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high TGFBR1*6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the TGFBR1*6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high TGFBR1*6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that TGFBR1*6A is not associated with an increased risk of bladder cancer [TGFBR1*6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer.Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men . A similcer risk . HoweverDNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board.A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons  constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls.\u00ae GC rich kit . The PCR reaction mixture included 20 ng of genomic DNA in a 10-\u00b5L reaction volume and the following concentration of other reagents: primers (0.25 \u00b5M each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg2+, 0.2 mM dNTPs and 0.16 \u00b5L of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94\u00b0C, then 35 cycles of denaturation for 30 seconds at 94\u00b0C and annealing/extension for 2 minutes at 72\u00b0C, followed by a final extension step of 5 minutes at 72\u00b0C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer  was used for data acquisition. A peak at 115 base pairs corresponded to TGFBR1 allele, whereas a peak at 107 base pairs corresponded to the TGFBR1*6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%.DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech AdvantageTGFBR1 genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic TGFBR1*6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of TGFBR1*6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of TGFBR1*6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20\u201340 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.Distributions of All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript."}
+{"text": "This series of homologous polyhedra is found in endohedral clusters of the group 14 atoms such as the endohedral germanium cluster anions [M@Ge10]3\u2212  and [Ru@Ge12]3\u2212 The next members of this series have been predicted to be the lowest energy structures of the endohedral silicon clusters Cr@Si14 and M@Si16 . The largest members of this series correspond to the smallest fullerene polyhedra found in the endohedral fullerenes M@C28 . The duals of the oblate (flattened) ellipsoidal deltahedra found in the dirhenaboranes Cp*2Re2Bn\u22122Hn\u22122  are prolate (elongated) trivalent polyhedra as exemplified experimentally by the germanium cluster [Co2@Ge16]4\u2212 containing an endohedral Co2 unit.The duals of the most spherical  Similarly bare group 15 element vertices  vertices as well as Co(CO)3 and (\u03b75-C5H5)Ni vertices are valence isoelectronic and isolobal with CH vertices. The duals of these most spherical deltahedra represent a series of most spherical trivalent polyhedra in which the degree 3 vertices correspond to the triangular faces of the most spherical deltahedra. This paper surveys the wide range of cluster structures ranging from molecular endohedral germanium clusters to the smallest endohedral fullerenes based on trivalent polyhedra that are duals of the deltahedra found in polyhedral borane and metallaborane chemistry.The fundamental structural units for polyhedral boranes, including the Briangles . 1,2,3],2,3nHn2\u2212closo deltahedra have the following features:(1)All faces are triangles\u2014hence their designation as deltahedra relating to the shape of the Greek letter delta (\u2206). This feature maximizes the number of edges for a given number of vertices and thus maximizes the connectivity between the vertices.(2)The vertices are as nearly similar as possible thereby providing the best approximation to a sphere rather than a prolate or oblate ellipsoid.The most spherical closo deltahedra can be classified into the following three types:(3)hO) 6-vertex regular octahedron with exclusively degree 4 vertices and go as far as the Dd4 10-vertex bicapped square antiprism with two degree 4 vertices and eight degree 5 vertices. These deltahedra are found in the borane dianions BnHn2\u2212 and the isoelectronic carboranes CBn\u22121Hn\u2212 and C2Bn\u22122Hn 3\u2212  a copper atom is encapsulated by a closo 9-vertex tricapped trigonal prismatic tin or lead cluster [closo 10-vertex Dh4 bicapped square antiprism encapsulating a metal atom was found to be the structural motif for the anionic indium cluster Zn@In108\u2212 found in the intermetallic [8In10Zn as well as in the lead clusters M@Pb102\u2212 found in [K]2[M@Pb10] . [Cv3 tetracapped trigonal prism in the M@Ga1010\u2212 clusters found in the K10Ga10M intermetallics  [104+ in Bi14PdBr16 (=[Pd@Bi10][BiBr4]4), [12]2\u2212 contains a platinum atom encapsulated in a closo regular icosahedron of lead atoms. 4),  have alld atoms. 10 pentagonal prism in the anion Co@Ge103\u2212 of [K]4[Co@Ge10][Co2]\u2022toluene [103\u2212 of [K]3[Fe@Ge10] [dT 28-vertex polyhedron found in the endohedral fullerenes M@C28 . [In view of these considerations the discovery of an outer Ge\u2022toluene  and in tFe@Ge10]  was a ma Th, U).  In this oblatocloso dirhenaboranes Cp*2Re2Bn\u22122Hn\u22122   . 3[Ru@Ge12]\u00b74py [8As4]3\u2212, also found in a [K]+ salt of more complicated stoichiometry [7Bi7]4\u2212  tetraanions \u00b74py ,25 and ihiometry . The larraanions  and is pic beams . The 16-ructures ,30,31.n fullerenes having only pentagonal and hexagonal faces. Even with carbon vertices, such polyhedra become large enough to encapsulate metal atoms. Thus the 28-vertex tetratruncated tetracapped tetrahedron  6\u2212 with an outer Si20 regular (hI) dodecahedron (n2 for n = 4) counting one skeletal electron for each group 14 element vertex and all of the valence electrons of the endohedral atom. This relates to the spherical aromaticity [The M@Cgeometry  and Pu@Cgeometry   have central oblate (flattened) ellipsoidal Re2Bn\u22122 deltahedra ]4[Co2@Ge16]\u00b7en where one of the two isolated and crystallographically characterized isomers has a structure based on a prolate trivalent polyhedron with two hexagonal faces, four pentagonal faces, and four tetragonal faces  ellipsoidal deltahedron such as those found in the hypoelectronic dirhenaboranes leads to a prolate (elongated) trivalent polyhedron. The elongated shapes and relatively large internal volumes of such prolate trivalent polyhedra make them particularly suitable to incorporate a pair of interstitial metal atoms. This is seen experimentally in the .,23.2@Ge1closo borane deltahedra having from 6 to 16 vertices form a series of homologous spherical trivalent polyhedra having even numbers of vertices from 8 to 28. This series of homologous polyhedra is found in endohedral clusters of the group 14 atoms. Thus the smallest members of this series are found in the endohedral germanium cluster anions [M@Ge10]3\u2212  and [Ru@Ge12]3\u2212 with Dh5 pentagonal prism and Dd2 bisdisphenoid dual geometries, respectively. The next members of this series have been predicted to be the lowest energy structures of the endohedral silicon clusters Cr@Si14 and M@Si16  with Dh3 tritruncated trigonal bipyramid and Dd4 bitruncated tetragonal trapezohedron geometries, respectively. The largest members of this series correspond to the smallest fullerene polyhedra. The last member of this series, namely the dT tetratruncated tetracapped tetrahedron dual to the 16-vertex Frank-Kasper deltahedron, is found in the endohedral fullerenes M@C28 , which are the smallest fullerene polyhedra known experimentally. Thus the homologous series of most spherical trivalent polyhedra represent a transition from molecular endohedral clusters of the heavier group 14 elements to the smallest fullerene derivatives. In addition, the duals of the oblate (flattened) ellipsoidal deltahedra found in the dirhenaboranes Cp*2Re2Bn\u22122Hn\u22122  are prolate (elongated) trivalent polyhedra as exemplified experimentally by the germanium cluster [Co2@Ge16]4\u2212 with an endohedral Co2 unit.The duals of the most spherical"}
+{"text": "Due to a Production error the references were numbered incorrectly. Reference 14, \"Singapore\", was split into two references (14 and 15). So, reference 16 \"Taiwan\", should be renumbered as \"15\", reference 17, \"Thailand\", should be renumbered as \"16\", and so on through the rest of the references ."}
+{"text": "Mutations in GJB2 gene are the leading cause of deafness in autosomal recessive inheritance, and the 35delG mutation is the most common in many ethnic groups. Besides the 35delG mutation in homozygosis, the mutation is also found in compound heterozygosis, coupled with other mutations in genes GJB2 and GJB6.To determine the prevalence of 35delG/GJB2 and del (GJB6-D13S1830) mutations in patients with sensorineural hearing impairment in residents from the Espirito Santo state, Brazil.77 unrelated individuals with moderate to profound sensorineural hearing loss were evaluated. The 35delG mutation was studied by PCR / RFLP; and the del (GJB6-D13S1830) mutation was screened by the technique of multiplex PCR.88.3% had normal genotype for the studied mutations, 1.3% were compound heterozygotes, 3.9% homozygotic for the 35delG mutation, 6.5% heterozygotic for 35delG/GJB2. The frequency of 35delG/GJB2 and del (D13S1830/GJB6) alleles in the sample was 7.8% and 0.65%, respectively.The data confirmed the existence of the mutations studied in cases of sensorineural hearing loss in a population from Esp\u00edrito Santo / Brazil. These findings reinforce the importance of genetic diagnosis, which can provide early treatment for children and genetic counseling for the affected families. Deafness is the most frequent sensory deficit in human beings; its reported incidence worldwide ranges from 1:300 to 1:1,000 children.13,Connexins are transmembrane proteins that form cell surface cylindrical hexameric structures which bind to other connexin hexamers in adjacent cells to form intercellular communication channels.,Mutations in three connexin encoding genes, GJB2 (Cx 26), GJB6 (Cx 30), and GJB3 (Cx 31) have been found to cause hearing loss.,,13,The 35delG mutation accounts for most of the mutant alleles (60-85%) in Caucasians, among the DFNB-causing GJB2 gene mutations described so far.The 35delG mutation is a deletion of a guanine base in a sequence of six guanines that extend from nucleotide positions 30 to 35 on the GJB2 gene encoding exon, resulting in a stop codon. This deletion causes the polypeptide to be synthesized incompletely; it contains 12 amino acids, rather than the usual 226 amino acids.Analyses of the GJB2 gene in patients with autosomal recessive inherited deafness, especially because of the 35delG mutation, have shown that about 10 to 50% presented only one mutant allele.22Environmental factors cause about 80% of hearing loss cases in Brazil; the remaining 20% supposedly are of inherited causes.,Estimates of the prevalence of 35delG heterozygotes in several European countries range from 2 to 4% of the normal hearing population.Finding the prevalence of mutations in Brazil may help implement expedited molecular diagnosis tests to inform physicians about therapy, along with genetic counseling for family members of patients. It is therefore essential to learn about the genetic diversity of deafness to design improved proposals for the molecular diagnosis of population groups.The purpose of this study was to estimate the prevalence of the 35delG and D13S1830 mutations of the GJB2 and GJB6 genes in a sample of patients from Espirito Santo (Southeast Brazil), with bilateral prelingual non-syndromic sensorineural hearing loss of unknown causes.A cross-sectional cohort study included 77 unrelated hearing loss patients, of which 38 were male and 39 were female. The age of patients ranged from 1 to 52 years. All resided in the state of Espirito Santo.Patients came from auditory oral schools and hearing loss patient support centers in several regions of Espirito Santo; a clinical history to identify the onset of hearing loss, the presence of other cases in their families, to confirm non-syndromic deafness, and to exclude ambient causes, such as prenatal infection  approved this study (protocol no. 121/2006). A commercial DNA-extraction kit (Puregene DNA Purification Kit - Gentra Systems) was used to extract genomic DNA according to the manufacturer's instructions.A primer pair (F: 5 TCT TTT CCA GAG CAA ACC GC 3 and R: 5 GCT GGT GGA GTG TTT GTT CAC ACC CGC 3) was used to amplify the genomic region at 65\u00b0C annealing temperature for analyzing the 35delG mutation on the GJB2 gene. The resulting 89 bp PCR product was digested with the BstNI restriction enzyme. In the absence of deletions, the enzyme yields two fragments (69 bp and 20 bp); if the deletion is present, the enzyme does not cut the DNA (89 bp). The digested fragments were analyzed in 8% polyacrylamide gel electrophoresis.,The del (GJB6-D13S1830) mutation was tracked using the PCR multiplex technique with three primers  at 62\u00b0C annealing temperature.We originally evaluated 308 subjects with hearing loss; 77 of these had no environmental causes for their clinical picture, and were included in the study (25% of cases).The 77 subjects with idiopathic deafness underwent molecular screening for the 35delG mutation in the GJB2 gene and the del (D13S1830) mutation in the GJB6 gene. The 35delG mutation was found in homozygosis in three patients (3.9% of cases), which established the etiology in these subjects. The 35delG mutation was found in heterozygosis in five subjects (6.5% of cases), but the presence of a single allele did not explain the cause of deafness in these patients. Normal and heterozygotic patients for the 35delG mutation were also investigated for the del (GJB6-D13S1830) mutation; one among 74 subjects had this mutation (1.35% dos cases). The heterozygotic patient for the del (D13S1830/GJB6) mutation was also heterozygotic for the 35delG/GJB2 mutation, which defined the double genetic etiology for this patient. These mutations were not found in 68 patients; other related mutations to Cx26 and Cx30 may be associated with the clinical pictures of these patients. The frequency of the 35delG/GJB2 mutant allele in the sample was 7.8%; the frequency of the del (D13S1830/ GJB6) mutant allele was 0.65%.The genetic heterogeneity of non-syndromic hearing loss makes its molecular diagnosis more complicated, given the number of mutations that have been described in dearness-related genes; additionally, the predominance of each varies significantly in different populations.,,The 35delG mutation in the GJB2 gene is the main cause of genetic deafness in Caucasian populations; it may be found in homozygosis or compound heterozygosis (with other mutations in the GJB2 and GBJ6 genes).,,,,,The frequency of the 35delG/GJB2 mutant alleles may vary in different regions of the world, as follows: United States of America (1.0%); Australia (1.0%); Austria (1.7%); Turkey (1.8%); Portugal (2.2%); Spain (2.5%), France (2.7%), and Italy (2-4%).,,Because of widespread racial mixing in Brazil, the 35delG/GJB2 mutation is not rare. Three studies in the state of Sao Paulo have revealed mutant allele frequencies ranging from 0.97% to 2.24%.A survey in 10 cities in different regions of Brazil revealed the following frequencies for 35delG mutation: North region (2.1%); Southeast region (1.5%); South region (1.2%), and Northeast region (0.8%); these differences were not significant.,,40In the present study, the 35delG/GJB2 mutation and the del (D13S1830/GJB6) mutation were investigated in patients with idiopathic deafness in the state of Espirito Santo, Brazil. Among 77 subjects, nine unrelated individuals had the 35delG/GJB2 mutation; of these, one had the mutation in compound heterozygosis with the D13S1830 mutation in the GJB6 gene. The frequencies of the 35delG/GJB2 and del (GJB6-D13S1830) mutant alleles in the sample were respectively 7.8% and 0.65%. Our data concur with other studies done in the Brazilian population , Table 3Although molecular analysis of hearing loss is not frequent in developing countries, it is essential to investigate GJB2 and GJB6 gene mutations for public health and genetic counseling purposes. The proportion of hearing loss patients because of genetic causes tends to increase as a result of investments and improvements in healthcare systems in developing countries such as Brazil. Establishing the prevalence and the types of mutations that cause non-syndromic hearing loss in Brazil, as was done in this study, may help implement simple and specific models to detect the main mutations causing genetic deafness in this country. The molecular diagnosis leads to accurate genetic counseling for family members and makes it possible to provide early rehabilitation for the children in affected families.Our data confirmed the presence of the 35delG mutation in the GJB2 gene in cases of non-syndromic bilateral moderate to profound sensorineural hearing loss in Espirito Santo, Brazil, a result which concurs with other published findings. The del (GJB6-D13S1830) mutation was found in compound heterozygosis with the 35delG/ GJB2 mutation in one patient. These findings underline the importance of a genetic diagnosis that may clarify the etiology and provide early treatment for children and genetic counseling for their family members."}
+{"text": "TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid\u2010related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic  Our study was aimed to identify genetic mutations in our BPDCN cohort, and compare our findings to published BPDCN cohorts. Our work identified a high frequency of biallelic TET2 mutations and canonical ASXL1 mutations with cooccurrence in a subset of cases.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive type of hematologic malignancy derived from plasmacytoid dendritic cell precursor cells. Next\u2010generation sequencing (NGS) techniques have expanded the understanding of the genetics of BPDCN. Epigenetic pathway genes  and 3 (60%) patients, respectively. Two (40%) patients had both TET2 and ASXL1 mutations. TET2 and IDH2 mutations were mutually exclusive, a pattern consistent with other myeloid neoplasms.Genomic DNA extracted from involved bone marrow or skin biopsies was amplified and subjected to mutation analysis. Cases 1 and 2 were sequenced using the Oncomine Myeloid Research Assay of 40 genes and analyzed with Thermo Fisher Scientific Ion Reporter . Cases 3 through 6 were sequenced using the institutional Myeloid Neoplasm Next\u2010Generation Sequencing panel of 53 genes on Illumina platform . All variants identified were in genes included in both panels. The limit of detection for the reported variants was set at 5% VAF. Among the newly diagnosed BPDCN cases [TET2 mutations spread across the coding exons of the gene  to 5\u2010hydroxymethylcytosine (5\u2010hmC) and promotes DNA demethylation. TET2 mutations are generally loss of function variants (frameshift and nonsense) that could be monoallelic or biallelic and occur throughout the length of the gene. Biallelic TET2 mutations have been reported in 10%\u201330% of MDS and AML patients and approximately 30% of CMML patients [TET2 mutations were founder lesions in 72% of CMML cases in one study [TET2 mutations likely result in a relatively more competitive advantage over single TET2 mutations [TET2 mutant allele dosage has potential theranostic impact for myeloid neoplasms. MDS patients with 1 or more TET2 mutations showed response to hypomethylating agents such as azacitidine (AZA) in some studies [TET2 mutations is associated with increased response to hypomethylating agents [patients . The Bia studies . Higher TET2 loss\u2010of\u2010function was a shared ancestral genetic event in some reported cases before divergent clonal evolution occurred in each neoplasm [TET2 mutations. The type and frequency of TET2 mutations in BPDCN appear similar to those observed in other myeloid neoplasms [TET2 mutations appears significantly higher in BPDCN compared to other myeloid malignancies  variant, with the VAFs ranging from 24% to 28%. ASXL1 encodes a polycomb repressive complex protein with a vital role in chromatin regulation. ASXL1 mutations are commonly frameshift or nonsense, and most are located within or upstream of the catalytic domain, which causes C\u2010terminal truncation of the protein. The ASXL1 G646fs*12 mutation is a hotspot mutation and commonly referred to as the \u201ccanonical\u201d ASXL1 mutation. The ASXL1 G646fs*12 mutation accounts for approximately half of all ASXL1 mutations in AML [ASXL1 mutations in reported BPDCN cases . This study also underwent review in the Case Comprehensive Cancer Center Protocol Review and Monitoring Committee (PRMC) due to the inclusion of cancer patients, to further ensure ethical retrospective review of medical records. All study personnel were certified as having up to date relevant training in research ethics, compliance and safety.Following detailed review of the study, the requirement for patient consent was waived by the IRB due to the minimal risk to the subjects and historical nature of the data.Supplemental InformationClick here for additional data file."}
+{"text": "Dear Editor,1. Several distinct functional modules have been characterized for the 1.4-MDa SAGA complex, including a histone acetyltransferase (HAT) module and a histone deubiquitinase (DUB) module that establish histone modification signatures for transcription activation, the largest subunit TRRAP that directly interacts with transcription activators, the core module that serves as a structural scaffold, and the spliceosome U2 snRNP factors SF3B3 and SF3B52  complex, human SAGA (abbreviated as hSAGA) complex is a well-established transcription coactivator that regulates the transcription of numerous genesHere we report the cryo-EM structure of endogenous hSAGA coactivator complex purified from HEK293 cells at an overall resolution of 3.7\u2009\u00c5  is vacant and potentially available for the association of TBP  of the yeast SAGA complex can recruit the U2 snRNP ATPase Prp5 to the promoter region, and consequently mediating a balance between transcription and pre-spliceosome assembly10. Accordingly, we presume that the incorporation of SF3B3 and SF3B5 into hSAGA complex might play a similar role in the coordination of transcription initiation and pre-mRNA splicing. Further studies will be required to examine the specific roles of SF3B3 and SF3B5 within the context of hSAGA complex by generating mutations that selectively disrupt the attachment of SF3B3 and SF3B5 to TAF6L.SF3B3 and SF3B5 usually serve as the structural components of the SF3B complex in the spliceosome U2 snRNP. Structural superposition with the SF3B complex revealed that TAF6L occupies the surface where the HEAT repeat domain of SF3B1 interacts with SF3B3 and SF3B5, suggesting that the incorporation of SF3B3 and SF3B5 into the two complexes is mutually exclusive (Supplementary Fig. 2, further indicating the possible functional differences between human and yeast SAGA in multiple physiological activities. Future work on the structural mechanism for the regulation of hSAGA on transcription factors and its substrate nucleosomes will help understand how the unique configuration of hSAGA contributes to its functions. Our studies also elucidate the structural basis for the association of the splicing factors SF3B3 and SF3B5 with hSAGA complex and will promote functional investigations on the roles of the splicing factors in hSAGA complex.In summary, our cryo-EM analysis reveals that human SAGA complex adopts an overall architecture distinct from yeast SAGA complex due to the different organization of TRRAP relative to the core module (Supplementary Fig. Supplementary InformationSupplementary Table S3Supplementary Table S4"}
+{"text": "The goal of this review is to summarize the characterization of the wnt11f2 zebrafish mutant and to deliver some new insights concerning its role in skeletal development. In addition to the previously described defects in early development in this mutant as well as craniofacial dysmorphia, we show an increase in tissue mineral density in the heterozygous mutant that points to a possible role of wnt11f2 in high bone mass phenotypes.Wnt signaling is a key regulator of osteoblast differentiation and mineralization in humans and animals, mediated by the canonical Wnt/\u03b2-catenin and noncanonical signaling pathways. Both pathways are crucial in regulating osteoblastogenesis and bone formation. The zebrafish silberblick ( The extracellular Wnt signal stimulates several intracellular signal transduction cascades, including the canonical or Wnt/\u03b2-catenin-dependent pathway and the noncanonical or \u03b2-catenin-independent pathway, which can be divided into the Planar Cell Polarity (PCP) pathway and the Wnt/Ca2+ pathway.2Because of the high degree of conservation among species of the Wnt pathway and its extensive homology with its human homolog, zebrafish mutants represent key animal models to unravel the function of the Wnt signaling pathway in development and bone formation.wnt11f2 mutant, presents a mutation in a gene related to the Wnt noncanonical pathway. This fish is also known as silberblick (slb), and it was first characterized in 1996 in the frame of a large-scale screen for mutants affecting forebrain development by using N-ethyl-N-nitrosourea (ENU) as a potent mutagen to introduce random point mutations in the genome.3 Homozygous wnt11f2 mutant embryos were smaller at 24 hours post fertilization (hpf) than controls and the elongation of the body axis was delayed from the tailbud stage until the early somite stages (10\u201314 hpf), as indicated by a shorter and broadened notochord and an abnormally shaped prechordal plate.The wnt11f2 mutants,4 thus leading to incomplete separation of the optic stalk3 and fusion of the eyes to various degrees depending on the penetrance of the phenotype. For that reason, this mutant was called silberblick (slb), which in German means strabismus. At 5 dpf, the jaw was deformed. The mutants displayed a recessive phenotype with variability.Further characterization revealed that anterior migration of central nervous system cells was impaired in wnt11f2 mutant, then known as slb, was found to encode a homolog of the human WNT11 protein, and was thus initially named wnt11.5 Two mutants, carrying the variants tx226 (c.669G>A p.Trp 223) and tz216 (c.463G>T p.Gly 155) in this gene, were analyzed. In addition, the authors showed that microinjecting wnt11 mRNA into slb mutants was able to rescue their phenotype, confirming that the detected mutations in wnt11 are indeed responsible for the phenotype and suggesting that the tx226wnt11f2 allele represents a loss-of-function mutation rather than a dominant-negative form. Interestingly, microinjection of wnt11 mRNA into wild-type embryos often resulted in misshapen eyes, indicating that the amount of Wnt11 proteins needs to be well controlled for normal development.6 Since then, this mutant fish line has been used extensively to understand the function of Wnt signaling in early development of vertebrates.In the year 2000, the locus that affected the wnt11 homologs had been named incorrectly: the gene previously known as wnt11r (wnt11-related) is the true ortholog to the human WNT11, thus now called wnt11, while the gene previously known as wnt11 is present in birds and other teleosts but not existing in mammals is now called wnt11f2.7 Following the zebrafish nomenclature guidelines, a gene should be named after its mammalian ortholog, however, such confusion occurred before the full zebrafish genome had become available because phylogenetic analyses at that time revealed that the closest human family member to slb was WNT11.7In 2019, genomic analysis revealed that the two zebrafish wnt11f2 mutants; and only one article from 20218 was found using the \u201cwnt11f2\u201d search term. In contrast, the carefully manually curated international zebrafish network ZFIN (zfin.org) currently has 155 citations concerning the wnt11f2 zebrafish gene. This situation clearly reflects the confusion in comparative genetics and disease modeling due to incorrect nomenclature resulting from the presence of duplicate paralogs in zebrafish following the whole genome duplication in teleosts. Consequently, this review specifically aims to clarify the role of the wnt11f2 gene because the publications are not well aligned in just one platform.In PubMed, a simple search with the word \u201csilberblick\u201d resulted in 21 publications published between 1996 and 2013; searching for \u201cwnt11 and zebrafish\u201d identified 99 publications, although not all of them are related to zebrafish and the wnt11f2. In the first part of this review, we describe the morphological and genetic characterization of wnt11f2 zebrafish. In the second part, we add some new findings concerning bone formation using this fish model.Therefore, we focused on the characterization of 9 Several coordinated morphogenetic cell movements take place during gastrulation, including convergence and extension (C&E) movements mainly mediated by the noncanonical Wnt signaling pathway, also known as the PCP pathway. The PCP signaling pathway was first identified in Drosophila, and its cellular and molecular regulation is conserved from Drosophila to mammals.Vertebrate gastrulation is a complex morphogenetic process that organizes the embryo proper into the three germ layers: endoderm, mesoderm, and ectoderm.10 The wnt11f2 is maternally expressed11\u201313 and is one of the immediate early genes activated in mesoderm induction. The wnt11f2 is expressed in the dorsal region of the germ ring at sphere/dome stage (4\u20135 hpf). During gastrulation, wnt11f2 expression extends to the lateral and ventral germ ring while being downregulated in the shield and its axial derivatives. In addition to the germ ring, at the end of gastrulation, Wnt11f2 is expressed in restricted areas of the anterior paraxial mesoderm and anterior lateral neuroectoderm.5Wnt11f2 activity is required for cells to undergo correct C&E movements during gastrulation, as the tx226wnt11f2 mutants present an impairment of the Wnt/PCP signaling leading to a diminished body axis elongation.14During the gastrulation process, mesodermal and neuroectodermal cells move toward the dorsal midline and intercalate with one another, which leads to mediolateral narrowing (convergence) and anterior\u2013posterior lengthening (extension) of the developing embryonic axis.wnt11f2 mutants between 10 and 12 hpf: C&E movements of both mesendodermal and neuroectodermal cells are reduced, which results in a shortened and broadened body axis that can be used to identify homozygous mutants at the end of gastrulation.4 The prechordal plate in the anterior axial mesendoderm appears to be abnormally shaped; the presumptive neural plate in tx226wnt11f2 embryos appears to be broader during gastrulation. The wnt11f2 is required in the nonaxial mesoderm to mediate cell intercalation along the anteroposterior axis that contributes to the extension of the body axis during late gastrulation.4 Shield and cell transplantation experiments performed by Heisenberg et al, where wild-type or mutant shields, or small groups of lineage-labeled nonaxial mesodermal cells were transplanted into tx226wnt11f2 mutant or wild-type embryos, respectively, to assess the movements, and the final location of the transplanted cells revealed that Wnt11f2 activity is required within lateral tissues of the gastrula, where it regulates mediolateral cell intercalations that underlie C&E movements.4A gastrulation phenotype is transiently visible in wnt11f2 embryos are predominantly affected in anterior regions of the gastrula suggests that other genes are involved in regulating C&E movements in more posterior regions. Therefore, in the absence of Wnt11f2, abnormal extension of axial tissue results in cyclopia and other midline defects in the head.5The observation that wnt5b partially overlaps functions with wnt11f2 to regulate C&E movements in lateral domains of the gastrula,15 confirming that both Wnt11f2 and Wnt5b are required during gastrulation for proper morphogenesis rather than cell fate specification. The wnt11f2/wnt5b double mutants show severe C&E cell-movement defects, and wnt5b RNA partially rescues the wnt11f2 mutant phenotype.15 At 3-somite stage, tz216wnt11f2 is required for C&E of both the mesoderm and ectoderm in the anterior and posterior region.8Also, tx226wnt11f2 mutants present slower and less direct migratory movements of these hypoblast cells.16 The authors observed that there is a misalignment in the tx226wnt11f2 hypoblast cells during the orientation process and the direction of movement, leading to less efficient movements of hypoblast cells toward the animal pole.16Wnt11f2 also controls the orientation and the velocity of the hypoblast cell migration in the germ ring at the onset of gastrulation; wnt11f2 mutants is the mitotic divisions in the dorsal epiblast cells, which exhibit less-pronounced animal\u2013vegetal axis polarity relative to control cells in both the epiblast and perpendicular planes, meaning that there is a disruption in the cell division orientation in the mutants.17 In addition, it has been described that Wnt/PCP signaling plays an important role in the migration of the cranial neural crest18; together with other genes from the Wnt/PCP signaling such as Fzd7 and Disheveled, wnt11f2 controls the polarization of the protrusions formed in the neural crest, which allow the cells to migrate in a directed motion.20Another defect observed in the tx226wnt11f2 triggers the local accumulation of Fz7 at cell membranes along with the intracellular mediator Dsh and Wnt11f2 itself, modulating local cell contact persistence to coordinate cell movements during gastrulation.21 To summarize, gastrulation is the first large-scale morphogenetic process to occur during development. This process is in part regulated by Wnt/PCP pathway, and disruptions in the regulation of this pathway caused by a mutation in wnt11f2 in zebrafish cause defects in the mediolateral cell intercalation, the migration of hypoblast cells, the cell division orientation, and in cell adhesion.22The Wnt/PCP pathway also plays a role in cell adhesion. The 23This stage focuses on the primordia of the pharyngeal arches, present at early times but difficult to distinguish individually. In zebrafish, the pharyngula stage starts at around 24 hpf. The embryo is most evidently now a bilaterally organized creature, entering the pharyngula period with a well-developed notochord and a newly completed set of somites that extend to the end of a long postanal tail. The nervous system is hollow and expanded anteriorly.wnt11f2 in the wild-type zebrafish is at the developing somites and otic placodes at 24 hpf and also in the mesoendoderm and in midline structures during zebrafish heart morphogenesis.24 At 24 hpf, in about half of the wnt11f2 mutant embryos, the retinae are not properly separated anteriorly. The forebrain in wnt11f2 mutant embryos, although patterned normally, is broadened and shortened, possibly the result of defective C&E during gastrulation.25 The axonal scaffold appears normal, with the exception of a slight deformation at the anterior\u2013ventral positions owing to the fusion of the eyes.4 It is possible that the major cause of the reduced resolution of bilateral eye fates in this mutant is related to the defect of forebrain morphogenesis.25 At 26 hpf, tz216wnt11f2 mutants have normal tails.26The expression of 4 Although wnt11f2 mutants show reduced medial\u2013lateral cell intercalations in both anterior and posterior mesendodermal domains, the extension of anterior regions seems most severely affected.5At 48 hpf, the eyes are slightly turned inward anteriorly, with no further obvious abnormalities detectable. Heisenberg and N\u00fcsslein-Volhard propose that proper midline morphogenesis is essential for lateralization of the eye position.wnt5b but not wnt11f2tz216 embryos had significantly shorter body axes than control siblings and that wnt11f2tz216/wnt5b double-mutant embryos had the shortest body axes. Similarly, the gut tube was normal in wnt11f2tz216 embryos but slightly enlarged in wnt5b mutant embryos and significantly widened in wnt11f2tz216/wnt5b double-mutant embryos. These data suggest that at 48 hpf, wnt5b but not wnt11f2 is required for elongation of the body axis and formation of the gut tube, but wnt11f2 cooperates with wnt5b to regulate endoderm morphogenesis.8Assessment of the posterior body length at 48 hpf revealed that Zebrafish larvae show a clear and distinct swimming pattern in response to light and dark conditions following the development of a swim bladder at 4 dpf. Cartilage cells are distinctive in branchial arches in these later larval stages. The primordium of the operculum extends posteriorly to cover the first or even the second branchial arch. The first visible bone in zebrafish, the transversely oriented cleithrum, appears at 3 dpf.18 As neural crest cells migrate, they extend polarized protrusions allowing the cells to migrate in a directed motion. The Wnt/PCP elements Wnt11f2, Fzd7, and Disheveled control at least in part the polarization of these protrusions.27 Between 55 hpf and 3 dpf, wnt11f2 is expressed in the pharyngeal arches.28 In addition, wnt11f2 has a complex expression pattern, including expression in head neural crest that might eventually form the anterior basicranium. However, the expression domain is within early paraxial mesoderm, before mesoderm has migrated to reach where the head will form,5 with this observations are possible that genes responsible for severe craniofacial phenotypes might be remote to cartilage development itself.29 At 48 hpf the wnt11f2 is expressed in the lower jaw.30 By 4 dpf, most of the craniofacial cartilage elements of the zebrafish have formed.31 Indeed, the Wnt signaling pathways are known to regulate bone homeostasis.Craniofacial cartilage elements are derived from the cranial neural crest. As mentioned above, in zebrafish, Wnt/PCP signaling plays a role in the migration of the cranial neural crest cells.29 In wnt11f2 mutant zebrafish, the jaw is deformed.3 To understand what is causing this bone and cartilage defect, Sisson et al, in 2015,28 marked the outline of chondrocytes to assess the gross morphology of the cartilage elements. In tz216wnt11f2 fish at 4 dpf, the cartilage elements, especially the ceratohyal and Meckel's cartilage, were greatly deformed. The mutants showed disrupted placement of many of the cartilage elements derived from the premandibular, mandibular, and hyoid arches. However, the stacking of the chondrocytes seemed normal. In the wnt11f2 mutants, the prechordal plate is severely defective, which impairs the extension of axial tissues leading to defects in the head cartilage formation, chondrogenesis occurring near the end of embryogenesis.29Wnt11f2 function in zebrafish cartilage formation is to position the initial bilateral sites of chondrogenesis of the anterior basicranium and specify where migrating neural crest cells will settle down at early stages determining the fate of the cartilage morphology.wnt11f2 mutants deform a bilateral organization in the mutants into a one-dimensional array just along the midline. Wnt11f2 is important to determine the position of the initial normally bilateral sites of chondrogenesis of the anterior basicranium.29 Furthermore, molecular studies reveal that wnt11f2 function in Wnt noncanonical signaling pathway is crucial for embryonic midline development.24The tz216wnt11f2 eye phenotype can vary interindividually, together with the severity of the craniofacial defects. To determine whether the severity of the eye phenotype was related to the cartilage placement phenotype, Marlow et al compared embryos manifesting full cyclopia (class 5) to those with lesser degrees of synophthalmia (classes 2 and 3).32 The authors found a clear correlation between the progression of the eye-fusion phenotype and the disruption of the shape of the cartilage elements, especially the ceratohyal. However, the ability of the chondrocytes to stack was not disturbed. This observation suggests that the craniofacial defect seen in the tz216wnt11f2 mutant fish is due primarily to the eye field separation defect displacing the cartilage elements.Of note, the wnt11f2 signaling in zebrafish cartilage formation, we performed Alcian Blue staining, as described,33 to check the cartilage formation at 10 dpf in tx226wnt11f2 mutants , the wnt11f2 embryos showed normal expression of shh. The primary defect in wnt11f2 may be a reduction in medial\u2013lateral intercalation of cells in the axial mesendoderm. Our results and those of Sisson et al21 confirm that wnt11f2 mutations affect craniofacial development indirectly through cyclopia resulting in incorrect head morphology. There is a link between the degree of severity of cyclopia and the degree of severity of craniofacial cartilage defects.The first description of the fgf8a gene expression  only dorsalized the embryos. Blocking canonical Wnt signaling by injecting a dominant-negative form of Tcf3 (\u0394N-Tcf3) led to ventralization, without rescuing the wnt11f2 phenotype.5 In addition, the authors working with the wnt11f2 morpholino model showed that when they injected disheveled (dvl) protein, found a cardiac phenotype rescue and when they blocked dvl, they found a phenocopy of the wnt11f2 phenotype. These observations confirmed that dvl acts downstream of wnt11f2 in the Wnt noncanonical pathway.24After the discovery that a wnt11f2 truncated protein led to the ror2 was disrupted in zebrafish, a phenocopy of wnt11f2 was also found. Moreover, coexpression of low-dose ror2-TM mRNA together with injection of wnt11 morpholino, led to a more severe C&E and eye defects.38Similar observations were made when wnt11f2 is one of the representative noncanonical Wnts transducing PCP signals through fzd7 receptor.19The Wnt/PCP signaling pathway is required during the gastrulation process, controlling tissue polarity and cell movement by activating RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades. The A summary of the major actors during zebrafish gastrulation can be found in 40 This gradient, in turn, induces a gradient in the Vangl2 phosphorylation end establishment of tissue polarity.41 Similar effects were observed for Wnt5b, with PCP activation involved in Wnt5b-induced cell migration and chondrocyte differentiation.42In bone tissue, the PCP signaling pathway and tissue polarization have been associated with embryonal bone and joint formation, which involves cell migration, elongation, and gradient-dependent differentiation. For example, PCP is crucial during embryonal long bone cartilage elongation along the proximal\u2013distal axis.wnt11f2/wnt5b double-mutant phenotype resembles the phenotype of embryos homozygous for kny, which encodes glypican 4 (gpc4). Coexpression of gpc4 RNA potentiated the activity of Wnt11f2 to rescue the wnt11f2 C&E phenotype.43 Furthermore, wnt11f2/gpc4 double mutants displayed a more severe phenotype than either mutant alone, including complete cyclopia featuring a single eye, which indicates that gpc4 acts as a positive regulator of Wnt11f2. The gpc4 is required for C&E of both the mesoderm and endoderm and interacts with wnt11f2.43 The gpc4 zebrafish mutants present broader and shorter body axis, the mutation is lethal after 5\u20137 dpf, but rescue experiments revealed that gpc4 has a role in regulating cartilage cell polarity, chondrocyte stacking, and endochondral ossification.44The gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia. Gpc4 can physically bind both Wnt5b and Wnt11f2 and regulates the formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells.8The transgenic expression of GFP-Wnt5b or Wnt11 initiate the noncanonical Wnt signaling pathway by binding to fzd2 and 7 receptors to regulate C&E movements in zebrafish.15Wnt11f2 and wnt11 show spatial and temporal patterns of expression compatible with a role during heart-tube assembly and their transcripts are expressed in neural ectoderm and mesoendoderm at 12 hpf. The expression pattern of wnt11 overlaps with that of wnt4a in the floorplate at 16 and 24 hpf and with that of wnt11f2 in the developing somites at 24 hpf. Of note, wnt11 transcripts are also expressed in the heart tube at 24 hpf.24 In studying hair cell orientation, Navajas Acedo et al45 reported a parallel role of PCP and Wnt pathway genes in regulating hair cell orientations in zebrafish neuromasts. Mutation in wnt11 disrupts hair cell orientation in neuromasts. However, heterozygous wnt11 and tz216wnt11f2 double mutants do not exhibit defects in hair cells, indicating that these two paralogs do not interact.wnt11f2 mutant zebrafish is an important animal model that can help in understanding bone formation and the role of Wnt signaling pathways in this process.The 24 The wnt11f2 mutant gastrulation phenotype is completely penetrant, whereas the eye phenotype is much more variable. This is also true for the deformities observed in the cranial cartilage, which were associated with the eye phenotype.Obviously, wnt11f2 is extremely important during gastrulation by enabling correct convergence\u2013extension cell movements by activating the noncanonical Dvl/RhoA pathway.Future research should focus on the direct and indirect partners and signaling pathways that lead to the eye phenotype in homozygous mutants.wnt11f2 is involved in the canonical and noncanonical Wnt signaling pathway, which is associated with cyclopia, and this defect is also observed in case of disruption of Shh and Nodal pathways in zebrafish.46\u201348 Probably there is an interaction between these different signaling pathways in zebrafish, but further investigations are needed.The 49 and as many as 1 in 250 human fetus.50In humans, cyclopia is a rare form of holoprosencephaly, and different forms of holoprosencephaly affect \u223c1 in 15,000 human live births51 Even if Wnt11f2 is absent in human, the noncanonical Dvl/RhoA pathway could be interesting to study to better understand holoprosencephaly. Moreover, wnt11f2 remains an important tool to study Wnt signaling in early development.Holoprosencephaly is commonly due to Shh pathway dysfunction, but the Nodal pathway is also involved.wnt11f2 mutants, although normal in their general development, skeletal morphogenesis, and reproductive capacity, present a significantly more highly mineralized bone skeleton.In this study, we show that the heterozygous 52 With this, we believe that the wnt11f2 zebrafish is a great model to better understand HBM etiology and to screen new bone anabolic drugs.Several rare genetic disorders with skeletal effects, like osteopetrosis and sclerosing bone dysplasia, are associated with generalized bone mineral density increase. Many of the HBM diseases are monogenic disorders related to the Wnt signaling pathway.WNT11 in humans exhibits the opposite phenotype.53 In a model of a WNT11 heterozygous mutant human osteoblast-like cell line generated by CRISPR-Cas9, we showed that the noncanonical pathway acts upstream of the canonical pathway, since in the absence of WNT11, the canonical pathway could not be rescued53 as was also observed in tx226wnt11f2 mutant zebrafish.5 This finding combined with our finding of HBM in wnt11f2 heterozygous zebrafish suggests that the noncanonical Wnt pathway is key to bone differentiation independent of bone patterning. Therefore, the phenotypic characterization of tx226wnt11f2 heterozygous adult would provide some insights into the potential genetic and cellular mechanisms of tx226wnt11f2 function in bone.Interestingly, we have recently shown that the heterozygous loss-of-function mutation of wnt11f2 mutant as a model to better understand HBM phenotype.In conclusion, we show that the noncanonical Wnt pathway acts as an inhibitory factor in bone mineralization in adults, and propose the heterozygous"}
+{"text": "PCV13 was licensed by FDA based on safety and immunogenicity data compared with PCV7, and systematic reviews have shown that PCV13 is effective against acute otitis media, pneumonia, and invasive pneumococcal disease (IPD) in children  to 4 (very low certainty).**Streptococcus pneumoniae was detected in the middle ear fluid of 24% , proportion of participants meeting the serotype-specific IgG value of \u22650.35 \u03bcg/mL (response rate)  evaluated the immunogenicity of PCV15 compared with PCV13 in healthy infants and children  or PCV13  at ages 2, 4, 6, and 12\u201315 months (Two economic models (CDC model and Merck model) that assessed cost-effectiveness compared the use of PCV15 and PCV13 according to the currently recommended PCV13 4-dose series for children aged <2 years  for all children aged 2\u201359 months. In addition, risk-based PCV use is recommended for children aged 60\u201371 months with risk conditions, and persons aged 6\u201318 years with an immunocompromising condition, cerebrospinal fluid leak, or cochlear implant. For all recommendations, PCV13 and PCV15 can be used interchangeably. Interruption of the vaccination schedule does not require reinstitution of the entire series or the addition of extra doses.Infants aged 2\u20136 months. Four doses of PCV (either PCV13 or PCV15) are recommended. The primary infant series consists of 3 doses of PCV. Infants receiving their first dose at age \u22646 months should receive 3 doses of PCV at intervals of approximately 8 weeks . The fourth (booster) dose is recommended at age 12\u201315 months and \u22658 weeks after the third dose  who are medically stable enough to be vaccinated  are recommended. The first 2 doses should be administered with an interval of \u22654 weeks between doses. The third dose should be administered at age 12\u201315 months, \u22658 weeks after the second PCV dose.Children aged 12\u201323 months. When PCV is initiated at 12\u201323 months of age, 2 doses (either PCV13 or PCV15) are recommended, with an interval of \u22658 weeks between doses.Children aged 24\u201371 months. Unvaccinated healthy children aged 24\u201359 months should receive a single dose of PCV (either PCV13 or PCV15). Unvaccinated children aged 24\u201371 months with any risk condition should receive 2 doses of PCV (either PCV13 or PCV15) with an interval of \u22658 weeks between doses. Routine use of PCV is not recommended for healthy children aged \u22655 years who have not yet received a dose of PCV.Children and adolescents aged 6\u201318 years with an immunocompromising condition, cochlear implant, or cerebrospinal fluid leak. If a dose of PCV13 or PCV15 has not been administered previously, a single dose of PCV13 or PCV15 is recommended, regardless of whether the child has previously received PPSV23, even if PCV7 was received.Infants and children aged <24 months. Infants and children aged <24 months who have received \u22651 dose of PCV (either PCV13 or PCV15) should complete the vaccination series with either PCV13 or PCV15 . These children should receive a single dose of PPSV23 at age \u22652 years and \u22658 weeks after the most recent PCV dose . ChildreRevaccination with PPSV23 among children with immunocompromising conditions. Children aged \u22652 years who have an immunocompromising condition should receive a second dose of PPSV23 \u22655 years after the first PPSV23 dose.Recipients of hematopoietic stem cell transplants. Recipients of hematopoietic stem cell transplants are recommended to receive 3 sequential PCV doses followed by a dose of PPSV23 beginning 3\u20136 months after the transplant, as described in the General Best Practice Guidelines for Immunization  and the 23-valent pneumococcal polysaccharide vaccine (PPSV23) are recommended for U.S. children, and the recommendations vary by age group and risk group.On June 22, 2022, the Advisory Committee on Immunization Practices recommended use of PCV15 as an option for pneumococcal conjugate vaccination of persons aged <19 years, according to currently recommended PCV13 dosing and schedules. Risk-based recommendations on use of PPSV23 have not changed.Use of PCV15 as an alternative to PCV13 is expected to further reduce pneumococcal disease incidence in children and adolescents."}
+{"text": "Drosophila melanogasterCyp6t3 gene. Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the \u201cBlack Box\u201d step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, we find that Crispr generated null mutations inCyp6t3are viable and have normal developmental timing. Therefore, we conclude that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene\u2019s expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of \u201coff target\u201d effects. Here we describe the genetic null mutant phenotype of the Drosophila melanogasterhave been identified and their activities ordered within a biosynthetic pathway. . Collectively, these genes are referred to as the Halloween group  and includephantom (phm), disembodied (dib), shadow (sad), shroud (sro),and spook (spo)which were first identified in the embryonic lethal screens of N\u00fcsslein-Volhard, Wieschaus, and J\u00fcrgens as mutants that failed to produce differentiated cuticle . A closer examination of the embryo proper revealed that null mutants in these genes all exhibit a common phenotype characterized by arrest at embryonic stages 15-17 with failures of head involution, dorsal closure, and cuticle production . Subsequent genetic and biochemical analysis demonstrated that each of these gene products acts at a specific step in the biosynthetic pathway and their loss results in failure to synthesize ecdysone, and hence they all produce an identical loss-of-function phenotype.In all Arthropods, the steroid hormone ecdysone (E) acts to control molting and metamorphosis . During the past two decades, many of the enzymatic steps that convert dietary cholesterol to ecdysone in the insect; Ou et al. 2011; Christesen et al. 2017).Despite this progress, there remains uncertainty in the enzymes and reactions involved in an early step of the pathway termed the \u201cBlack Box\u201d. The name arises because the chemical intermediates between the upstream 7-dehydrocholesterol (7dC) and the downstream 5beta-ketodiol have eluded isolation . Nevertheless, the activity of several gene products including Shroud (Sro), Spo/Spok, Cyp6t3 and Cyp6u1 have been suggested to act within the Black Box based on rescue experiments that involve feeding mutant larvae intermediate compounds that are thought to be upstream and downstream of the Black Box enzymes. In these experiments, feeding the downstream intermediate 5beta-ketodiol rescues loss-of-function larvae and enables them to progress through additional molts while feeding the upstream intermediate 7dC does not . , the source of larval ecdysone. The embryonic tissue source of E is still unknown. Strong RNAi knockdown in the PG of any of the Halloween gene products leads to first instar arrest. However, feeding the larvae various intermediates often enables them to progress into the second and third instar stage, provided the intermediate is downstream of the enzymatic block. These types of experiments suggested that the Sro and Spo/Spokenzymes are components of the Black Box.Cyp6t3(FBgn0033697) andCyp6u1(FBgn0033121) genes as encoding possible factors of the E biosynthetic pathway came about differently from classical genetic studies.Cyp6t3was identified as a gene that was specifically up-regulated in the PG in response to loss of the transcription factor DHR4. .DHR4mutants pupariate early likely because of precocious ecdysone production, and simultaneous knockdown ofCyp6t3in aDHR4mutant background suppressed the early pupation phenotype. In addition, RNAi knockdown ofCyp6t3in the PGs of wild type animals resulted in slow growth and large pupae, both of which were suppressed by feeding knockdown larvae ecdysone or 5beta-ketodiol but not 7-dC suggesting that, like Spo/Spok and Sro, this enzyme works at the Black Box step. Because of the low transcript abundance ofCyp6t3, it was also hypothesized thatCyp6t3might be a \u201cbottleneck\u201d in ecdysone biosynthesis, or even rate limiting.Cyp6u1 was identified in a genome wide transcriptome analysis of the PG as a P450 that showed high level expression specifically in the PG, and RNAi knockdown in the PG led to a developmental delay/arrest phenotype which was rescued by feeding ecdysone or 5beta-ketodiol but not 7-dC, again positioning this enzyme at the Black Box step.The identification ofCyp6t3 as an additional means to assess its function duringDrosophiladevelopment. Guide RNAs from both ends of theCyp6t3 cytochrome P450 domain were simultaneously injected into a Cas9 stock (Bestgene Inc.) for the purpose of generating mutations at each individual site as well as potential deletions between the sites. After screening the progeny of the G0 flies, we obtained 3 mutationsCyp6t3[Cr1], a 1 bp deletion at Guide 1, Cyp6t3[Cr2], a 2 bp deletion at Guide 2,andCyp6t3[Cr3], a 1 kbp deletion between Guides 1 and 2 . Translation of these mutated genes yields proteins with different premature stops or a large deletion within the Cytochrome P450 domain of Cyp6t3 . All three mutations are homozygous viable. For this reason alone, we deemedCyp6t3a non-essential gene for ecdysone biosynthesis.Since it has been reported that tissue-specific knockdown of genes can have more severe phenotypes than genetic null mutations , we made Crispr-mediated null mutations inCyp6t3 is not essential for animal survival, we reasoned that it could affect the rate of ecdysone biosynthesis. Previous work has shown that either raising or lowering the basal E biosynthetic rate leads to advanced or delayed timing of pupariation, respectively . To address this issue, we assessed the time to pupariation of variousCyp6t3 mutant combinations . In sum, we find no delay in developmental timing compared to the wild type control suggesting that the basal rate of ecdysone synthesis in theCyp6t3mutants is on par with wild type.Although loss ofCyp6t3 is not an essential component of the E biosynthetic pathway under lab growth conditions should not be surprising since, unlike the other Halloween genes that are highly conserved across all arthropods examined to date,Cyp6t3has been lost in some clades . In addition, as previously noted, the transcript abundance ofCypt6t3is very low by qRT-PCR  and did not make the cutoff in 2 different ring gland-specific transcriptional profiles, the only putative E biosynthetic P450 gene failing to do so . Despite these observations, the regulation ofCyp6t3transcription by DHR4 and its ability to suppress the precociousDHR4mutant phenotype when knocked down in the PG remains intriguing. It would be interesting to determine whether aCyp6t3,DHR[1] double mutant shows the same suppression of precocious pupariation as found when RNAi is used to knockdownCyp6t3in the PG of aDHR[1]mutant. It is also possible that Cyp6t3 is important under specialized growth conditions that modulate DHR4 activity. Another possibility is that Cyp6t3 function is redundant inD. melanogaster, and perhaps other species, with another P450 enzyme. Precedence for the existence of partial redundance in E biosynthetic enzymes in insects is exemplified by the Spo/Spok relationship . Both are thought to function within the Black Box but at different developmental stages, Spo acting embryonically and in female follicle cells while Spok acts in the PG during larval stages. One intriguing candidate for a redundant activity could beCyp6u1. This gene product is also a member of the P450 clade 6 subgroup but is highly conserved across Arthropoda . Examining the phenotype ofCyp6u1 null mutants andCyp6t3;Cyp6u1double mutants might provide insight on the redundancy issue.The fact thatIn summary, our studies do not resolve the enigma of the Black Box reaction mechanism in the E biosynthetic pathway. However, our elimination of Cyp6t3 involvement under standard growth conditions helps reduce the complexity of the necessary components. This should aid in simplifying the design of future studies aimed at understanding the details of the biochemical reactions catalyzed by the Blackbox enzymes and whether their regulation serves as a rate limiting step in E biosynthesis as previously suggested .Crispr mutant generationCyp6t3with no off-target sites. The targets with the PAM are: 5\u2019 GGATCCCG\u00afAACCATAAGCGC CGG 3\u2019 and 5\u2019 GGATCAAA \u00af GACGCAGGGCTC CGG 3\u2019. Sense and antisense oligonucleotides (Oligo Table) for the two guides RNAs were cloned into pU6-BbsI-chiRNA . Both guide RNAs were injected into vas-Cas9 embryos . Single G0 flies were screened by PCR using primerscyp6t3 01Fandcyp6t3 02R(Oligo Table) for the presence of deletions, making them candidates for further analysis. Ten to fifteen individual F1 progeny of G0 flies that displayed multiple deletion bands by PCR (possible Cr mutant/+) were then crossed to aCyO* balancer (BDSC 3198), homozygosed, the DNA region amplified by PCR and then sequenced for the presence of a mutation and its precise location. BothCyp6t3[Cr1] andCyp6t3[Cr2]mutations were obtained from a single G0 fly.The CRISPR Optimal Target Finder  was employed to identify two target sites withinClustal omega analysisused SnapGene Version 6.0.7 software.Developmental timingstinstar larvae started to emerge. 30-35 larvae were transferred to vial food  sprinkled with dry yeast and allowed to grow at 25\u00b0 with a 12-hour light/dark cycle. Pupariation was scored every 2 hours with the data from 4 vials combined due to the speed of pupariation and lack of data points necessary for non-linear regression analysis in Prism .Crosses were set up in cages. Four-hour collections of embryos on yeast-apple juice plates  were aged about 16 hours, until 1Oligo TableFly lines"}
+{"text": "Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections. Plasmodium falciparum rapid diagnostic tests (RDT) that are a cornerstone of malaria control efforts in the high-burden, low-resource contexts in which malaria mortality is most acute  are affecting the accuracy of the RDTs. Infections with pfhrp2 deletions are missed by HPR2-based RDTs much of the time; infections with double deletions (missing both pfhrp2 and pfhrp3 genes) are invisible to RDTs and create false-negative results. Because these deletions represent an existential threat to recent gains made in malaria control, the World Health Organization (WHO) has emphasized the critical need for surveillance (Histidine-rich protein 2 (HRP2) is the primary target of the pfhrp2 and pfhrp3 deletions from the country come from a single report confirming their presence in 3 travelers to Australia . We performed malaria confirmation and speciation of 594 dried blood spot samples by multiplex PCR  underwent genotyping and molecular analysis for deletions in exon 2 of pfhrp2 and pfhrp3   from persons d pfhrp3 . Demograd pfhrp3 . We defipfhrp2 and 20.0% for pfhrp3  of sites. Pfhrp2 deletion rates in South Sudan were as high as or higher than the country\u2019s immediate neighbors, where reported deletion rates from specific sites have varied from 26% in Ethiopia to 19% in Kenya, 6% in the Democratic Republic of the Congo, 3% in Uganda, and <1% in Sudan  among 518 genotyped PCR-positive samples was 15.6% for r pfhrp3 . Double r pfhrp3 . In 7/9 pfhrp2 and pfhrp3 deletions and double deletions , whereas age, MOI, and clinical severity were all predictors for pfhrp3 deletions. Only MOI was a significant predictor of double deletions  could be another way that age, disease severity, and deletion risk interact, because milder disease has previously been associated with pfhrp2 deletion surveillance, the transmission period should be considered (Sample collection in this study occurred at the end of the high-intensity malaria transmission season, when potentially high parasitic diversity but low prevalence could favor spread of gene-deleted organisms, making deletions easier to detect (pfhrp2 and pfhrp3 deletion surveillance. Consequently, the precise prevalence of pfhrp2 and pfhrp3 gene deletions causing false-negative results on RDTs in South Sudan was not generated to assess whether it is within the 5% threshold established by WHO (p-LDH RDT\u2013positive samples, preventing us from evaluating the effects of deletions on malaria diagnoses.This study was limited because it was a secondary analysis of a study of molecular markers of antimalarial drug resistance and did not follow WHO protocols for pfhrp2 and pfhrp3 deletions is critical to designing effective public health strategies for malaria control. This study describes these deletions in a clinical cohort in a country with little previous endemic evidence of pfhrp2 and pfhrp3 deletion. Monoclonal infections were a principal predictor of deletions. We identified high levels of single and double deletions of pfhrp2 and pfhrp3, which if more widely present in this or other regions of South Sudan, could s eriously jeopardize HRP2-based RDT effectiveness moving forward. Future studies should be designed according to WHO protocol to produce precise estimates to measure the risk those deletions pose in South Sudan. Local variation in prevalence suggests the potential for deletion hotspots within the country and should be considered when designing malaria control strategies. Characterizing Plasmodium Falciparum pfhrp2 and pfhrp3 gene deletions in malaria-hyperendemic region, South SudanAdditional information about"}
+{"text": "The sizable transient changes in Ca2+ concentrations are necessary for the activation of signaling pathways, which rely on Ca2+ as a second messenger, including those involved with force generation, fiber type distribution and hypertrophy. However, prolonged elevations in intracellular Ca2+ can result in the unwanted activation of Ca2+ signaling pathways that cause muscle damage, dysfunction, and disease. Muscle employs several calcium handling and calcium transport proteins that function to rapidly return Ca2+ concentrations back to resting levels following contraction. This review will detail our current understanding of calcium handling during the decay phase of intracellular calcium transients in healthy skeletal and cardiac muscle. We will also discuss how impairments in Ca2+ transport can occur and how mishandling of Ca2+ can lead to the pathogenesis and/or progression of skeletal muscle myopathies and cardiomyopathies.In healthy muscle, the rapid release of calcium ions (Ca Innately, Ca2+ has flexibility in its bonding angles and lengths, allowing for an array of potential ligation patterns [2+-binding molecules [2+ signaling becomes apparent as it can be regulated by the location and expression of the Ca2+-binding proteins and the kinetics of the Ca2+-binding site.Calcium  can fluctuate from resting concentrations of 100 nM to values above 1000 nM during tetanus [2+]cyt is elevated above resting concentrations, Ca2+ interacts with two categories of Ca2+ binding molecules: buffers and sensors [2+ sensors elicit a downstream signal when binding occurs. The temporal range of Ca2+ signaling can be as brief as seconds but may also exist on the timescale of days [2+ signaling is effectively diminished when [Ca2+]cyt reverts back to resting concentrations. One strategy for increasing the rate of [Ca2+]cyt decay is through Ca2+ buffering. Buffers act to sequester Ca2+ without being directly incorporated into molecular signaling. The presence of Ca2+ buffers allows for the regulation of Ca2+ diffusion, which can indirectly affect molecular signaling pathways cyt is mediated primarily through the movement of Ca2+ across phospholipid membranes [2+]cyt is through Ca2+ sequestration in the sarcoplasmic reticulum (SR), a membrane bound organelle which surrounds the contractile myofilaments cyt but also in the regulation of the energy expenditure of the cell [2+ is also extruded across the plasma membrane to effectively reduce [Ca2+]cyt during the cardiac cycle cyt can result in the unwanted activation of proteolytic and apoptotic pathways, leading to muscle damage, dysfunction, and even disease. In this review, we discuss the role of Ca2+ transport in the maintenance of healthy muscle as well as the role it can have in the genesis and exacerbation of pathological states.Although Caembranes . Within ilaments . Ca2+ isdrolyzed ,10. SERCthe cell . Within ac cycle . The rol2+ release cyt [2+]cyt results in the immediate activation of SERCA, which acts to pump Ca2+ back into the SR. However, with the continuance of high frequency neural signaling the rate of Ca2+ outflow is greater than the ability for SERCA to re-sequester Ca2+. Consequently, [Ca2+]cyt rises within the myofiber and binds to troponin C resulting in the movement of tropomyosin and thus uncovering the myosin binding site on the thin filaments cyt begins.The RyR is a CaCa2+]cyt . Within ilaments ,17. Withilaments ,19. Musc2+]cyt to a peak is quickly followed by a decay, all within milliseconds [2+]cyt has a negative exponential relationship in which the initial rate of decay is rapid but as [Ca2+]cyt approaches pre-stimulation resting concentrations the rate of calcium uptake is attenuated. Within skeletal muscle tissue, the characteristics of a Ca2+ transient varies across different types of muscle fibers. Baylor and Hollingworth (2003) compared the ICTs of slow fibers from soleus tissue and fast fibers from EDL muscle [2+]cyt during Ca2+ release in the slow and fast fibre types; however, the peak [Ca2+]cyt amplitude was two times greater and the ICT half duration was \u223c1.6 times shorter in fast EDL fibers compared with the slow soleus fibers cyt, PLN dissociates from SERCA and does not affect Vmax cyt is elevated, CAMKII will detect these elevations and respond by phosphorylating PLN, thus relieving its inhibitory effect on SERCA and allowing for faster calcium clearance cyt. MLN, on the other hand, is believed to be another inhibitory regulator of SERCA cyt rises due to SR Ca2+ release, the membrane potential effect becomes negligible, and the direction of the exchanger now changes to the forward mode with Ca2+ being extruded from the cytosol [+] and [Ca2+] can impact the direction of the exchanger, under physiological conditions, the direction is largely driven by the [Ca2+]cyt  to potentially help to overcome Na+-dependent inactivation by increasing the electrostatic potential [+ levels and works independent of the [Na+]cyt levels and (2) secondary or slow blockade that is dependent on [Na+]cyt levels, which enhance the affinity of NCX for H+ cyt during repeated muscle contractions when [Ca2+]cyt is very high cyt [2+ at an optimal ratio of 2 mol Ca2+:1 mol PV [2+ at resting [Ca2+]cyt (<100 nM) [2+ is dependent on the rate of dissociation of Mg2+ from the binding sites [2+]cyt, which is why it is often considered a slow Ca2+ buffer cyt ,111,112.Ca2+]cyt . In contCa2+]cyt . FurtherCa2+]cyt . PV is a1 mol PV ,116. The<100 nM) . The ratng sites . Consequ+ buffer . The binl muscle ,120. Howl muscle . Taken t2+ buffering, there is evidence showing that Ca2+ uptake through the mitochondrial Ca2+ uniporter is important for the maintenance of myocyte homeostasis [2+ uptake in cardiomyocytes does not alter [Ca2+]cyt transients or cardiac contractility, there is evidence that mitochondria can modulate [Ca2+]cyt in fast-twitch skeletal muscle fibers under certain conditions cyt. The first theory posits the lack of dystrophin results in transient tearing of the sarcolemma membrane during contractions. The membrane damage could allow for an influx of extracellular Ca2+ into the myofiber [2+ influx into the myofiber [2+]cyt can increase proteolytic activity and the production of ROS [2+ dysregulation [2+]cyt alone are enough to promote a shift toward a DMD like phenotype [2+]cyt by overexpressing SERCA in dystrophic tissue, which ameliorated the pathology cyt and decreased muscular degeneration and fibrosis cyt is decreased in these cells [2+]cyt cyt ,186.2+ release with HF could be due to defective coupling between the RyR and DHPR, reduced SR Ca2+ content could also contribute. This was assessed in one study using a canine model of HF by assessing the CICR gain, which is the amount of Ca2+ released from the SR for a given level of current density from the DHPR channel and \u03b5, which is the effectiveness of coupling between DHPR activation and SR Ca2+ release cyt and inducing relaxation of the myocardium during diastole. Several studies have shown that SERCA Ca2+ uptake is reduced in failing hearts from both human and animal models cyt, it can actually increase the probability of arrhythmogenic events. In failing hearts, with increasing frequency of stimulation, the intracellular Na+ ([Na+]cyt) levels rise [+/H+ exchanger on the sarcolemma [+/K+ ATPase activity [2+]cyt and SERCA uptake of Ca2+ into the SR would also increase leading to SR Ca2+ overload cyt during E-C coupling must be rapidly lowered to re-establish resting concentrations and induce muscle relaxation, which is primarily accomplished by SERCA-mediated Ca2+ transport into the SR. During short contractions of fast-twitch rodent and amphibian myofibers, the cytosolic Ca2+ buffer PV also contributes to muscle relaxation by rapidly lowering [Ca2+]cyt. In the heart, Ca2+ extrusion from cardiomyocytes through the NCX contributes significantly to ventricular relaxation, and the PMCA and MCU also contribute to a very minor extent. Impairments in Ca2+ transport and dysfunctional Ca2+ homeostasis has consistently been shown to be involved with the pathogenesis and/or the progression of multiple myopathies within skeletal muscle and in HF. A better understanding of the function and regulation of muscle Ca2+ transport proteins can allow for greater insights into skeletal and cardiac muscle physiology and disease. Furthermore, this body of work can lead to novel approaches in the treatment of diseases involving Ca2+ dysregulation.Maintenance of Ca"}
+{"text": "Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18\u201322 weeks.NCT02521974 and NCT03554798) conducted in Panama, infants aged 18\u201322-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model.In two similarly designed clinical trials (10 50% cell culture infectious dose (CCID50) and 76\u00b77% at the 5 log10 CCID50 inoculum levels, with rates of 2\u00b78% for 4 log10 CCID50 and 11\u00b78% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4\u00b75 log10 of 0\u00b7007  indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients.Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5\u02b9 untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47\u00b76% at the 4 logThe data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses.Bill & Melinda Gates Foundation. Vaccination with Sabin oral poliovirus vaccine (OPV) results in robust intestinal and humoral immunity, which are key to the control of poliomyelitis, but the inherent genetic instability of the OPV strains is well documented.With the certification of the global eradication of wild type 2 poliovirus the routine use of the type 2 component of Sabin OPV was ceased globally in 2016. Although this switch has been successful in most regions, waning immunity to type 2 viruses in areas of low vaccine coverage has contributed to increasing numbers of outbreaks of type 2 cVDPV, with consequent increases in cases of paralysis.9Evidence before this studyThe Lancet four clinical studies that demonstrated the safety, tolerability, and immunogenicity of nOPV2 in adults, young children, and infants compared with monovalent Sabin OPV2 (mOPV2). The key driver of a change from mOPV2 to use of the novel vaccine in outbreak settings is its improved genetic and phenotypic stability, which has been described in two published reports in NPJ Vaccines from studies using shed stool samples obtained from adults and children administered mOPV2 or nOPV2. We did not conduct a literature search as the few published reports regarding novel OPV2 have been generated from the novel OPV2 consortium.The widespread transmission of type 2 vaccine-derived poliovirus constitutes a Public Health Emergency of International Concern. A novel type 2 oral polio vaccine (nOPV2) received a WHO emergency use listing in November, 2020, to respond to this crisis based on promising clinical data. We previously reported in two publications in Added value of this studyThis report represents the first rigorous comparison of genetic and phenotypic stability, conducted with samples from infants, a primary target population of nOPV2 use in outbreak settings, completing the assessment of nOPV2 in the previously reported clinical studies. We show that the vaccine viruses shed by infants administered nOPV2 have significantly reduced reversion to virulence compared with polioviruses shed by infants administered mOPV2. We also show the improved stability of the primary attenuation site, domain V, of nOPV2 compared with mOPV2.Implications of all the available evidenceThe data from this key study population provide a crucial demonstration of the better genetic and phenotypic stability of shed nOPV2 strains compared with shed mOPV2 in infants. These data suggest that nOPV2 should be associated with less paralytic disease and potentially a lower risk of seeding new outbreaks, the objective of the development of these new vaccines. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses.Novel OPV2 (nOPV2) was designed to provide similar protection as mOPV2 but with reduced risk of loss of attenuation.14WHO/UNICEF Estimates of National Immunization Coverage data  national coverage of a third dose of OPV was estimated to increase from 72% in 2015 to 86% in 2016 while the control phase 4 study was conducted and was estimated to be 88% in 2018 and 2019 while the phase 2 study was being conducted. In this Article, we used established methodsOur study supplements previous reports from similarly designed phase 4 and phase 2 studiesNCT02521974) conducted in 2015\u201316 before the global switch to bivalent OPV (bOPV) containing only types 1 and 3, in which mOPV2 was administered to 110 healthy infants age 18\u201322 weeks who received three doses of bOPV and one dose of inactivated polio vaccine at least 4 weeks before administration of mOPV2. The second is a phase 2 trial  in 2018\u201319, in which 150 healthy infants age 18\u201322 weeks received at least one oral dose of nOPV2 at least 4 weeks after receiving three doses of bOPV and one dose of inactivated polio vaccine.This study is an analysis of shed virus in samples obtained from two trials. The first is a phase 4 trial  virus.Polio Sabin Mono Two (mOPV2), a type 2 Sabin strain, was manufactured by GlaxoSmithKline Biologicals, Rixensart, Belgium. A dose consisted of 2 drops (0\u00b71 mL) containing no less than 106\u00b70 CCID50 in 1 mL.nOPV2, referred to as nOPV2 candidate 1 (S2/cre5/S15domV/Rec1/HiFi3) in earlier publications,10 CCID50 type 2 virus per g of stool was termed the exploratory endpoint specimen  as previously described.men EES; .15 Stool10 or 5 log10 CCID50 amplified virus from EES or control virus. Virus from EES were amplified in a single round of infection of Hep2C cells under conditions previously shown to be non-selective. These doses were selected for the assay to be sensitive to clinically relevant Sabin-2 reversion. The controls were 20 mice inoculated with Sabin-2 (SO\u2009+\u20092/II) at 6 log10 CCID50 doses and ten mice inoculated with SO\u2009+\u20092/II 5 log10 CCID50 doses. A Sabin-2 molecular construct with reversion at two genome sites (481a\u2192g and 2908a\u2192g) was inoculated into 20 mice at 4 log10 CCID50 dose level as a positive control. Back-titrations of the diluted samples confirmed that inoculum titres were within 0\u00b75 log10 CCID50 of the nominal dose. Inoculated mice were monitored for paresis or paralysis over a 14-day period and assigned a clinical score per established protocol.17The virus titrations for EES and controls, and the mTgmNVT were described previously.50) was determined. For these tests, groups of ten Tg-PVR21 mice were inoculated with five different dose levels of each sample.For nOPV2 samples that induced 40% or more paralysis, the paralytic dose for 50% of mice  and its corresponding confidence interval. We used inverse probability weighting to account for stratified subsampling of M5 EES according to post-vaccination week, as described in the 50 values, averaged across mouse sex, were obtained by inverting the regression models. As an exploratory analysis, a similar model was fitted in R (version 4.0.0) to mouse paralysis data for each vaccine group and dose level to assess the relationship between frequency of mutation at key sites and mouse paralysis. See the A binomial logistic generalised linear regression mixed model with mouse sex as a factor and subject (EES)-specific random effects was fitted with SAS (version 9.4) to mouse paralysis data for each vaccine group separately, yielding estimates of paralysis rate. For comparison of paralysis rate of shed nOPV2 versus shed mOPV2, two additional similar models were fitted that also included a term to estimate the odds ratio of paralysis for the virus shed from nOPV2 recipients relative to mOPV2 recipients. This adjusted odds ratio (aOR) is adjusted for mouse sex imbalance owing to mice excluded from evaluation due to reasons unrelated to the inoculum. The primary inferential comparison between groups was predefined to be based on the model-estimated aOR (nOPV2/mOPV2 ratio) at 4\u00b75 logThe funder of the study had no role in data collection, data analysis, or data interpretation. ASB was an employee of the study funder and was involved in the clinical trial design and writing of the report.Although the global cessation of OPV2 usage in routine immunisation required that the M2 and M5 studies were conducted non-contemporaneously, given the similar vaccination histories of the participants in both cohorts, and overall similar rates of viral shedding among nOPV2 and mOPV2 recipients early after vaccination,NGS was performed on shed virus (two replicates of 10% stool suspension per EES) from 36 EES from the M2 study ranging from 1 day to 28 days after mOPV2 administration. 34 EES were successfully sequenced, with two excluded due to insufficient DNA concentration following NGS library preparation. Data from the key genome sites are shown in NGS was performed on 52 EES (three of 55 EES had insufficient DNA for sequencing) ranging from 2 to 28 days following nOPV2 administration. SNPs in the key modified and known attenuating regions are summarised in Variation was also noted at position 459 in domain IV, corresponding to nucleotide 398 in Sabin-2. 459u\u2192c increased in frequency in later EES and is fixed in a day 28 EES. By contrast to mOPV2 EES, mutations which result in reversion of VP1-143 were observed consistently, with several nOPV2 EES at 50% variant frequency or higher (fixed in at day 28 EES). Changes involved in the reversion of VP1-143 are observed at nucleotides 2969 and 2970 and changes at both positions were sometimes detected within the same sample. The 2969 and 2970 variants are reported as SNPs but could represent dinucleotide polymorphisms. Generally, the data suggest these two locations of mutations might undergo more rapid selection for nOPV2 than mOPV2.No reverting polymorphisms (defined as strengthening of a dinucleotide pairing in the stem regions of domain V) were observed in the novel domain V (nucleotides 468\u2013535) of shed nOPV2 . Low-levTo confirm whether the culture-amplified virus was sufficiently representative for neurovirulence assessment, the frequency of mutation in stool samples was compared with the frequency of mutation in the amplified sample for mOPV2 and nOPV2 . PolymorTo provide a sufficiently robust evaluation of potential neurovirulence of shed virus in mice, preclinical and previous clinical data determined that 36 EES per vaccine (mOPV2 and nOPV2) would provide sufficient power for comparison. No subsampling was therefore necessary for mOPV2 EES, and 36 of the 51 nOPV2 EES  were subsampled with simple random sampling within strata defined by post-vaccination week, focusing on those EES most distal to vaccination .10 CCID50 dose and 76\u00b77% at the 5 log10 CCID50 dose  indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients.For nOPV2, rates of paralysis increased with later EES days  and a doShed mOPV2 virus reverted rapidly and showed high paralysis rates in the mouse model. NGS data support the high levels of reversion observed for the shed mOPV2 virus . Reversi10 dose and 17 of 36 showed 10% or more paralysis at the 5 log10 dose. A single day 28 EES (M5-2-351) showed 40% paralysis at the 4 log10 CCID50 dose and 60% paralysis at the 5 log10 CCID50 dose. This day 28 EES had fixed mutations at nucleotide 123 in cre5, VP1-143 and at nucleotide 459 with additional fixed changes at nucleotide 556 within domain V and in the 3D polymerase .For nOPV2 , six of 50 (50) contain higher levels of 459u\u2192c in domain IV (398u of Sabin-2) and VP1-143 substitutions. Virus from the day 28 EES with fixed mutations at key sites shows the lowest (more virulent) PD50 estimate of 5\u00b73 log CCID50. The PD50 of the day 28 EES aligns with data from a series of virus strains constructed to incorporate key polymorphisms in the nOPV2 background and evaluate their impact on neurovirulence (50 6\u00b73 log), but notably higher than the observed PD50 of 3\u00b75 log for a partially-reverted day 7 shed Sabin-2 virus.10 CCID50 dose, suggesting that PD50 would generally be less than 4 log for these Sabin-2 viruses.nOPV2 EES with higher neurovirulence were evaluated in multi-dose neurovirulence tests to determine the PD50 . In geneirulence  and is s10 CCID50 dose level  consistent with the presence of cre5, domain IV and VP1-143 mutations in the shed virus. These mutations appear to accumulate rapidly in nOPV2 . Sequencing data can be accessed on the Sequence Read Archive database (accession number PRJNA786548). Algorithms for polymorphism selection are described in the methods. R codes for tables and figures specific to this publication are available from the corresponding author upon reasonable request.We declare no competing interests. The National Institute for Biological Standards and Control, University of California, San Francisco, claim intellectual property rights associated with the nOPV2-c1. ET works for the manufacturer of nOPV2. JS and BK are employees of Viroclinics, which is paid by PATH for the conduct of next-generation sequencing and mouse neurovirulence tests. ASB was an employee of the study funder and was involved in study design and writing of the report but had no role in data collection or analysis."}
+{"text": "BRAFV600E mutation represent a unique subset of central nervous system tumors. Targeted therapies including BRAF and MEK inhibitors are now being explored as possible new treatment options.High-grade gliomas (HGG) with BRAFV600E mutations treated with BRAF inhibitors.We report an 18-year-old female with a grade 3 pleomorphic xanthoastrocytoma treated upfront with dabrafenib and trametinib. We also conducted a systematic literature review of patients with HGG and BRAFV600E mutations treated with BRAF inhibitors were retrieved through our systematic review of the literature. Only 1 young patient with an anaplastic ganglioglioma was treated upfront with a BRAF inhibitor with a curative intent. Best response reported with radiation therapy and systemic therapy was a stable disease (SD) for 18 patients (56.3%) and progressive disease (PD) for 9 patients (28.1%). Responses to treatment regimens that included BRAF inhibitors were reported in 31 patients and included 4 complete responses (12.9%), 23 partial responses (74.2%), 2 SDs (6.5%), and 2 PDs (6.5%). Despite local recurrences resected surgically, the patient has been on dabrafenib and trametinib for more than 54 months. Thirty-two patients with HGG and Our patient had durable disease control with dabrafenib and trametinib. Given favorable responses reported in patients with HGG treated with BRAF inhibitors, we believe that upfront targeted therapy is a possible treatment approach that should be studied in the context of a clinical trial. BRAFV600E mutation.BRAF inhibitors appear to be an effective approach for the treatment of HGG with BRAFV600E mutation.BRAF inhibitors should be studied as a possible option for upfront treatment in HGG with BRAFV600E mutation.We reported the evolution of a young patient treated upfront with a combination of BRAF/MEK inhibitors. This approach without prior chemotherapy or radiation has rarely been reported. We conducted a systematic review of the literature and report that responses are more frequent in patients treated with BRAF inhibitors when compared to standard regimens. Results from our review and our clinical experience suggest that clinical trials should investigate the possibility to use BRAF and MEK inhibitors in upfront therapy for HGG with BRAFV600E mutation is the second most frequent mutation in pediatric low-grade glioma (LGG) but the alteration has also been reported in a subset of primary and secondary high-grade glioma (HGG).1 The overall survival of HGG BRAFV600E mutation is more favorable than other subgroups including HGG H3.3 G34 R/V but despite standardized treatment strategies including surgery, radiation therapy, and chemotherapy the prognosis is poor.3 New treatment approaches are currently being investigated including BRAF inhibitors (BRAFi) with or without MEKi for LGG and HGG with BRAFV600E mutation. However, there are currently limited data available on the outcome of patients treated with BRAFi. Following our experience with 1 patient with HGG treated upfront with BRAFi and MEKi, we aimed to better understand the expected outcome in similar patients treated with targeted therapy by conducting a systematic review of the literature.We describe a case report of a young patient with a HGG treated with BRAFi and MEKi.BRAFV600E mutations and treated with BRAFi . Relevant articles were then reviewed in detail and included. Given the nature of the study, no ethical board approval was required. The family and patient gave their consent for this case report.A systematic review of the literature in the PubMed and Embase databases was conducted for original articles on HGG including glioblastoma (GBM), anaplastic astrocytoma, anaplastic ganglioglioma, and grade 3 pleomorphic xanthoastrocytoma (PXA) with th BRAFi .4 ArticlOur patient was diagnosed with epilepsy at 14 years of age when she presented with focal seizures characterized by \u201cd\u00e9j\u00e0 vu phenomenon\u201d followed by an alteration of consciousness. Electroencephalogram showed intermittent dysfunction in the right temporal region. Magnetic resonance imaging (MRI) at diagnosis of epilepsy was normal  and B. S2) including a right-side amygdalohippocampectomy  and trametinib (2 mg PO daily by mouth once daily) was initiated.Treatment was initially well tolerated except for mild dry skin. No cardiac dysfunction was detected. After 16 months of treatment, a grade 1 retinal pigment epithelial detachment with minimal visual acuity impairment was observed. Trametinib was dose reduced by 50% to 1 mg daily.BRAFV600E mutation was confirmed by immunohistochemistry and PCR. Given the slow progression and presence of areas with low-grade features in the recurrence, methylation profiling was requested on both the initial tumor and the recurrence. Both profiles were highly consistent with grade 3 PXA (score: 0.98). RNAseq did not identify a resistance mechanism or significant alteration such as telomerase reverse transcriptase promoter mutations. Copy number variation methylation was very similar between recurrence and diagnosis. Reexamination of primary tumor pathology revealed areas compatible with PXA including scattered eosinophilic granular bodies and xanthomatous change .A total of 23 articles were included. Of those, 21 articles were case reports or small case series.th BRAFi \u20133. Media8 Fourteen (43.8%) had leptomeningeal dissemination at 1 point during their follow-up including 2 patients at diagnosis.Progressive disease (PD) was reported in 29 patients (90.6%) and death was reported in 16 patients (50 %) with a median overall survival of 24 months after diagnosis (range 2\u2013112 months). All patients died of their disease except for 1 patient who suffered from an extensive intracerebral hemorrhage related to vemurafenib according to the authors.20 Best response achieved on systemic therapy before BRAFi  was a stable disease (SD) for 18 patients (56.3%) and PD for 9 patients (28.1%).In terms of treatment, 26  had focal radiation therapy following resection and 10 patients (31.3%) received a second round of focal radiation therapy. Five patients did not receive radiation therapy: 3 patients due to poor neurological status and 2 patients due to young age (1.5 and 4 years old). Most patients also received prior systemic treatment before BRAFi ; 12 patients (37.5%) had received more than 2 lines of systemic therapy (range 1\u20134). Only one young patient with an anaplastic ganglioglioma was treated upfront with a BRAFi with a curative intent.Median time between diagnosis and the start of treatment with BRAFi was 11 months (range 2\u201398 months). The treatment regimens varied: 15 (46.9%) patients were treated with combination of dabrafenib plus trametinib; 7 (23.3%) patients received dabrafenib monotherapy; 7 (23.3%) patients were treated with vemurafenib monotherapy; 2 (6.7%) patients received vemurafenib followed by dabrafenib; 1 (3.3%) patient received combination of vemurafenib plus cobimetinib. Median treatment time with BRAFi was 9 months (range 1.5\u201332 months) and 7 (23.3%) patients were still receiving targeted therapy at the time of reporting. Response was reported in 31 patients and included 4 complete responses , 23 partial responses , 2 SDs (6.5%), and 2 PDs (6.5%).BRAFV600E mutations treated with BRAFi in monotherapy or the combination of BRAFi and MEKi were also reviewed.27 Kaley et al. reported 24 adults with CNS tumors and BRAFV600E mutations treated with vemurafenib including 15 patients with HGG including 6 patients with GBM, 5 with anaplastic astrocytoma, 3 anaplastic gangliogliomas, and 1 HGG not otherwise specified. All patients with high-grade tumors had received prior treatment including radiation therapy (15/15) and most had received chemotherapy (12/15). Using the response evaluation criteria in solid tumors 1.1 criteria, 2 patients had a PR . They reported a median overall survival for GBM and anaplastic astrocytoma of 11.9 months.26 In a recent study by Wen et al., adult patients with BRAFV600E mutation were treated with combination of dabrafenib and trametinib.27 The study included 45 HGG (31 GBM). All patients received prior therapy including radiation therapy followed by chemotherapy or concurrent chemoradiotherapy. They observed an overall response rate (ORR) of 33% with 3 CR and 12 PR. SD was seen in 22% and PD in 42% of patients. Median duration of treatment was 14.9 months for HGG. Median overall survival was 13.7 months for GBM and 45.2 months for other HGG.27Two clinical trials involving patients with HGG and BRAFV600E mutation.We report a case of a teenager with a grade 3 PXA who initially presented with focal seizures. Her first MRI was normal, and the lesion was identified 16 months later when seizures recurred with symptoms of intracranial hypertension. The patient was treated upfront with a combination of dabrafenib and trametinib. We conducted a systematic review of the literature and summarized current data on treatment with BRAFi of HGG with 28 The initial MRI as part of the epilepsy workup usually reveals the tumor but in rare cases, the lesion is not detected. In an adult series of patients with HGG, 4.7% had normal imaging at presentation (9/193).29 MRIs are usually repeated in patients with refractory epilepsy, especially if they were well controlled in the past or if they presented with new neurological symptoms to rule out an ongoing process not initially recognized.In children, only 1%\u20133% of patients with epilepsy have an underlying tumor.20Given the extent of resection, tumor location, and family preference to avoid radiation therapy, we decided to treat this patient upfront with dabrafenib and trametinib without radiation. This approach has rarely been reported. In our systematic review, only 4 patients received upfront BRAFi including 3 patients with poor neurological status precluding the use of radiation and chemotherapy. Only 1 young patient with an anaplastic ganglioglioma was treated upfront with a BRAFi with a curative intent. The patient has been stable for more than 23 months according to the authors.BRAFV600E mutation.Responses to BRAFi in patients with HGG vary in the literature. In our literature review, 27 patients (87.1%) presented a significant decrease in their tumors . This high response rate needs to account for a possible publication bias toward selection of case reports with favorable responses to BRAFi. However, other studies have revealed a high rate of response in HGG with BRAFV600E mutations treated with BRAFi.2 A total of 11 children with HGG were reported . Detailed treatment and disease course were not provided but all patients received prior radiation therapy and 9 received between 1 and 3 lines of systemic therapy. Four patients (36%) responded (1 CR and 3 PRs) and all patients except 1 progressed within 18 months. Median time to progression was 10 months.Nobre et al. reported a large cohort of patients with gliomas and BRAFV600E mutations. Sixteen (84.2%) received upfront radiation therapy followed by BRAFi+/\u2212MEKi and 3 patients underwent biopsy with upfront treatment with BRAFi+/\u2212MEKi. They reported an ORR of 64.3% when including CR and PR. Only 1 patient had PD as the best response. They showed a favorable 18-month progression free survival (PFS) of 83% compared to 42% in the BRAF-mutant historical cohort. They reported a 3-year overall survival (OS) of 82% compared to 44% in the BRAF-mutant historical cohort.30Recently, Rosenberg et al. reported an original series of 19 pediatric patients with HGG and BRAFV600E mutation. They reported an ORR of 56% in patients with pediatric HGG and 38.2% in patients with adult HGG treated with BRAFi+/\u2212MEKi.31Andrews et al also conducted a large systematic review of glioma with BRAFV600E mutations (NCT02684058). After completing at least 1 line of treatment (radiation and/or chemotherapy) patients received a combination of dabrafenib and trametinib. The ORR was 56.1% for the entire cohort and 66.7% for PXA grade 3 (4/6 patients). The 12 months PFS was 44.1% and the OS was 32.8%.32Recently, Hargrave et al. presented the results of the recently completed Novartis trial for pediatric HGG with 33 Therefore, the use of BRAFi might be more effective with at least transient improvement in tumor control, neurological function, and quality of life. Given the small number of patients in our literature review, we did not observe significant differences in response rates between pediatric patients, adolescents/young adults, and adults. It will be interesting to see if upcoming studies report different outcomes based on age.In recurrent HGG, response rates with chemotherapy have rarely exceeded 5%, with an overall survival of 5\u20139 months and progression-free survival of less than 3 months.34 This should be investigated in the setting of a clinical trial. However, given the rarity of HGG with this alteration, it may take several years and the involvement of a large consortium to answer this question. Currently, the Children\u2019s Oncology Group is conducting a study using dabrafenib and trametinib for HGG following local radiation therapy (NCT03919071).It is possible that prior treatments negatively impact the outcome of these patients. Radiation and chemotherapy could induce new mutations facilitating resistance to targeted therapy. Data are currently very limited to support this hypothesis but given the poor outcomes of patients with HGG, new treatment approaches (including upfront targeted therapy) and a modification of sequences of treatment should be considered. Treatment with BRAFi could be initiated at diagnosis and radiation therapy with chemotherapy could be reserved for progression. In some cases, targeted therapy could be continued during radiation and chemotherapy in select patients to avoid the rebound phenomenon.36 The combination of dabrafenib and trametinib was used in the Novartis trial for HGG (NCT02684058).32 Despite limited data, we used this approach to optimize tumor control.In 16 patients (50%) a combination of BRAFi and MEKi were used. The benefit of this combination for HGG have not been determined yet but studies in adults with melanoma have reported a reduction in both death and progression with similar toxicity.BRAFV600E mutation . This is higher than what is generally, reported in GBM where approximatively 25% of patients have metastases within the central nervous system at 1 point during their follow-up.37 It will be important to investigate whether HGG with BRAFV600E mutations are more likely to have leptomeningeal dissemination in future large studies.In our literature review, leptomeningeal dissemination was frequently reported in patients with HGG and 38 The term anaplastic is no longer used for diffuse astrocytoma and it was not possible to know if some of these tumors would be classified differently. Nevertheless, we included case reports of patients with features suggestive of higher-grade lesions that have been historically treated aggressively with resection, radiation, and chemotherapy.Another limitation of this systematic review of the literature is the accuracy and classification of diagnosis. Diagnoses were based on descriptions and not in accordance with the latest WHO classification of CNS tumors.We report a case of grade 3 PXA treated upfront with dabrafenib and trametinib. Despite local recurrences, our patient had a favorable and durable disease control. We reviewed the current literature, and this treatment approach has rarely been reported. Given the favorable response seen in patients with HGG treated with the combination of BRAFi and MEKi, we suggest that upfront targeted therapy treatment is feasible and should be studied in the context of a clinical trial.vdac174_suppl_Supplementary_Figure_S1Click here for additional data file."}
+{"text": "SMN genes. These elements within the SMA genes may play key roles in understanding this early-onset neurodegenerative disease as well as how transposable elements can impact gene expression. Understanding the roles of transposable elements in SMA may provide key insights into other neurodegenerative diseases.Transposable elements are DNA sequences that can move throughout the genome. They play essential roles in gene regulation and function. Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality worldwide. Since transposable elements have been linked to other genetic diseases, we examined the genomes from SMA patients as well as healthy genomes for the presence of transposable elements. We identified distinct transposable elements that may impact gene expression by affecting promoter activity or transcriptional termination of the Survival Motor Neuron 1 (SMN1) gene but retention of its nearly perfect orthologue SMN2. Both genes are highly enriched in TEs. To establish a link between TEs and SMA, we conducted a comprehensive, in silico analysis of TE insertions within the SMN1/2 loci of SMA, carrier and healthy genomes. We found an Alu insertion in the promoter region and one L1 element in the 3\u2032UTR that may play an important role in alternative promoter as well as in alternative transcriptional termination. Additionally, several intronic Alu repeats may influence alternative splicing via RNA circularization and causes the presence of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that could lead to SMN1 exons deletions and, ultimately, SMA. TE characterization of the SMA genomic region could provide for a better understanding of the implications of TEs on human disease and genomic evolution.Transposable elements (TEs) are interspersed repetitive and mobile DNA sequences within the genome. Better tools for evaluating TE-derived sequences have provided insights into the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the  Transposable elements (TEs) are interspersed repetitive DNA sequences with the ability to mobilize in the genome. This mobility is mediated by element-encoded proteins such as DNA transposase or reverse transcriptase and occurs within the genome of virtually all walks of life, including prokaryotes, unicellular and multicellular eukaryotes and even large DNA viruses ,2. TEs cRetrotransposons are divided in two major subclasses based on the presence or absence of a long terminal repeat (LTR) sequence. Long and short interspersed nuclear elements  comprise the two types of non-LTR retrotransposons. The only active, autonomous TE family in humans is the LINE-1 family, although most of these sequences are no longer mobile due to various forms of rearrangements, mutations and 5\u2032-truncation ,8,9. SINInitially considered inert remnants of evolution and so called genomic parasites, TEs are now recognized as important players in genomic evolution, genome organization and gene regulation\u2014due primarily to the advances in genome sequencing and better analysis tools Once co-opted by the host genomes, TEs provide important sources of new regulatory sequences that can act as alternative promoters, tissue specific enhancers, splice sites, polyadenylation signals, insulators, termination sites and transcriptional factor binding sites, thereby altering nearby gene expression in cis ,13,14,15While TEs play a beneficial role in genome evolution, their presence can also be detrimental to the host and cause several problems to normal gene expression and to genome organization, stability and integrity ,8,11,19.Survival Motor Neuron 1 (SMN1) but retention of the paralogous Survival Motor Neuron 2 (SMN2) gene [SMN1 and SMN2 are nearly identical except for 20 single nucleotide differences, with the C to T transition in exon 7 (c.840C > T) being the most functionally relevant difference [SMN1 is located in a highly unstable region of the large arm of chromosome 5 (5q13.2), a region of the genome that is enriched with repeated sequences, pseudogenes and transposable elements [SMN1 and SMN2 are both highly enriched in TEs\u2014especially Alu and L1 repeats\u2014spanning both genes [SMN1 introns have an impact on the regulation of the splicing patterns as two Alu elements can give rise to new alternative SMN exons as well as on circularization events of SMN RNA that result from inverted Alu repeats [SMN1 introns also makes this gene prone to deletion events caused by Alu/Alu recombination events [Attention to the contribution of TEs to neurodegenerative diseases has been rising in the last few years . Spinal N2) gene ,27,28. Sfference . SMN1 iselements ,31,32. Ielements ,31. SMN1th genes . The hig repeats ,34,35. Sn events ,37,38.SMN1 and SMN2 (SMN1/2) genes, have only used the reference gene sequence in their analysis [SMN1/2 loci of whole genome sequences from SMA patients, SMA carriers and healthy individuals. The results of this comprehensive bioinformatic analysis could provide important insights into the potential involvement of TEs in SMA onset as well as help understand the roles of TE dynamics in genome evolution, gene regulation and human disease.Prior studies on the organization of TEs within the analysis . As someSMN1/2 gene sequences from 20 SMA carriers, 22 non-carriers and 37 SMA patients obtained from different sources. Genome sequencing data from the \u201c1000 Genomes Project\u201d cohort [SMN1 and SMN2 copy numbers for the MNDRL cohort were confirmed by digital droplet PCR  that is available in Ensemble.Our sample database consisted of \u201d cohort  were sel\u201d cohort  previous\u201d cohort . The SMAplet PCR  [42,43,4SMN1 reference sequence by Geneious Mapper tool [SMN1 and a consensus sequence of the mapped reads which was used for predicting TE insertions.The raw next generation sequencing (NGS) data obtained were processed and then mapped against per tool  (GeneiouSMN1/2 sequences obtained and the SMN1 and SMN2 Consensus Coding Sequences (CCDS) [Homo sapiens\u201d to ensure that the best cut-off was applied to each model thereby ensuring more accurate predictions of TE location and orientation. Overlapping Dfam matches (nearly perfect overlaps) are automatically removed by Dfam so as to remove model redundancy.All s (CCDS)  isoformss (CCDS) ,48. ThisSMN1/2 sequences and CCDS were aligned using Clustal Omega version 1.2.2 multiple sequence alignment (MSA) program [SMN1/2 transcriptional elements and motifs, including the promoter elements and other regulatory sequences were either described previously [All the TE annotated  program    .SMN1/2 promoter exhibits differences in TE insertion sites and subfamilies present between samples. We hypothesize that the untranslated regions may be subjected to less evolutionary pressure thereby allowing more diversity in TE insertions. With respect to the gene region downstream of exon 7, our first analysis of SMN1/2 gene reference sequence obtained from Ensembl showed several TE insertions belonging to various subfamilies (SMN1/2 is located within the terminator region of the gene and primarily serves as the 3\u2032UTR region of the gene [SMN1 deletion), SMA carriers, non-carriers or healthy genomes, all samples show this L1 insertion inside exon 8  in Dfam showed a L1 element at the 3\u2032 end of the CCDS, indicating that these complete SMN1 transcripts have an imbedded TE sequence derived from the L1MC5a insertion in exon 8 , does not contain this L1 insertion  contains the same L1MC5a insertion in a similar 3\u2032 location to that seen in the longest SMN1 transcript (results not shown). Similar to SMN1, the remaining and more common SMN2 transcripts isoforms  do not exhibit any L1 insertion in their sequence (results not shown).Since SMN1/2 introns are highly enriched in Alu-derived repeats with many of them in an inverted orientation  [SMN expression.While these domesticated TEs may be integrated in the gene regulatory network, the significance of the remaining motifs inside the AluJb sequence remains to be investigated. The TSSs present inside the Alu sequence are tissue-specific and/or developmental stage-specific TSSs, with TSS2 being used as a fetal transcription start site and the use of TSS3 is still unknown . This AllncRNAs) . Future SMN1/2 3\u2032UTR region serves as an alternative terminator for the genes transcription by giving rise to longer transcripts. We will experimentally confirm this hypothesis in future studies using rapid amplification of the 3\u2032cDNA ends (3\u2032RACE) in control and SMA cells to identify alternative 3\u2032UTRs.The majority of human genes use alternative polyadenylation sites that are embedded in TEs, suggesting that these can influence the 3\u2032 end processing of host gene transcripts ,72. L1 aSMN1/2, that functions as the 3\u2032UTR of the gene, is an example of a relatively rare event. Furthermore, the L1 insertion in exon 8 does not belong to the active L1 elements  of the human genome that are composed only of the L1PA1 and L1PA2 subfamilies [Two factors may explain how this L1 element became fixed in this gene region. First, exon 8 has a lower percentage of G + C content 36.4%) when compared with the whole gene region (42.3%). The lower G + C content may have favored an L1 insertion as these elements have a bias towards lower G + C regions of the genome . Second,6.4% whenfamilies . TherefoSMN1 isoform transcripts could explain why these transcripts are less common than their shorter counterparts. Interestingly, weakly expressing genes were found to be rich in LINE insertions what can be explained by the ability of L1 elements to disrupt transcriptional elongation based on the presence of strong polyA signals in their sequences that possibly function as transcriptional terminators [SMN1/2 reported in this study, are only moderately selected against and may provide a gradual mechanism of evolution by which retrotransposons alter the expression profile and influence crucial gene networks in the human genome [3\u2032UTR retrotransposon insertions reduce mRNA expression ,67,79. Tminators . We arguminators ,82. 3\u2032UTn genome ,67,83.SMN2 as it shares the same TE insertional patterns in its sequence with SMN1. Accordingly, the longest SMN2 transcript isoform presents this L1 insertion in its 3\u2032 region, in an identical sequence position as in SMN1 longest transcript. This hints to this retrotransposon having the same alternative terminator role in SMN2 transcription to that in SMN1 transcription. We hypothesize that this alternative terminator role can have implications in SMA severity by reducing SMN2 mRNA expression due to the presence of this L1MC5a element within SMN2. The confirmation of this hypothesis will require further analysis using a combination of experimental and in silico methodologies.Additionally, it is possible that this L1MC5a element has the same transcription terminator function in SMN1 reference sequence explains the high levels of circularization of SMN1/2 transcripts as Alu repeats located in introns 4 and 5 are especially active in this process [SMN1/2 introns may explain how these genes generate several circular RNAs, we believe that circRNA biogenesis occurs in healthy individuals and SMA patients to the same extent. It is possible, however, that dysregulation of these Alu repeats in SMA-affected genomes may lead to an increased formation of circRNAs coded by SMN1. These higher levels of circRNAs formation and the widespread alternative circularization of SMN1/2 pre-mRNA may have a still undiscovered role in SMA onset or may contribute to worse SMA phenotypes, owing to circRNAs ability to interfere with the coding capacity of human genes [SMN1/2 may function as a potential biomarker for the genes\u2019 overall transcriptional/splicing stability since higher circRNAs levels indicate aberrant RNA splicing events that may be linked to SMA.Circular RNAs (circRNAs) are a widely expressed class of non-colinear RNAs generated in a diverse set of eukaryotic organisms . Due to  process ,53,58. P process . Additio process . In futu process . The prean genes ,58,84. ASMN genes, exon 6B [SMN1 occurred among Alu elements of the (S) subfamily supporting the idea that sequence identity between the two elements at a locus\u2014alongside proximity\u2014appear to be proportional to their chances of successful recombination [SMN1/2 canonical exons. In a wider spectrum, Alu and other TEs provide transcriptome diversity and ultimately result in the diversification of the human proteome.The two exonization events within the  exon 6B ,57 and e exon 6B , have bebination ,90,91. TSMN1 gene, we cannot draw definitive conclusions about the involvement of Alu elements in the deletion events in a disease context. Our results show that in the analyzed SMA patients and in the remaining samples, the critical Alu elements responsible for these deletions are also present in the same position and orientation in the SMN1/2 sequence as in the gene reference sequence and as described previously [per se is not the reason for the deletion events. Their presence in SMN1/2 introns is a source of sequence homology that can be responsible for genomic rearrangements and consequently disease in some genomes [de novo mutations in SMN1 and are not inherited from carrier parents [SMN1 are responsible for de novo deletions in germinative cells due to unequal recombination since TE silencing mechanisms are often relaxed in these developmental stages [SMN1/2 genomic region leading to disease-causing deletions of SMN1 exons and potentially whole gene deletions under specific circumstances. Alu elements invasion of SMN1/2 makes these genes very susceptible to Alu-mediated deletions, that have critical consequences to genome stability and host health.Some insertional polymorphisms that were observed in these Alu repeats between the analyzed samples may be the result of normal interpersonal sequence variability. Accordingly, polymorphic Alu elements account for 17% of structural variants in the human genome, clearly establishing a link between individual TE polymorphisms and human genetic variation . Since teviously ,37,38. A genomes . TE reco parents . It is ll stages ,19. Alu SMN genes revealed a pervasive invasion of its sequence by TEs that we believe may severely impact these genes\u2019 regulation structure, expression and overall genomic stability. The several TEs present inside these genes, especially Alu and L1 elements that are highly enriched in the promoter and intronic regions of the gene, seem to play important roles in gene expression, novel exon creation, alternative splicing and deletion events known to lead to SMA. Additionally, a L1 element insertion in the 3\u2032UTR region of the gene is also responsible for a domestication event that gave the gene an alternative terminator, therefore increasing the diversity of SMN1 transcripts and being a prime example of how a TE insertion inside a protein-coding gene can create a gradual mechanism of evolution by which retrotransposons alter the human transcriptome.Our analysis of the SMN1/2 completed in this work serves as a starting point for further investigations on the impacts of TEs in human disease and particularly, their role in SMA onset and severity. While the TEs identified in this in silico analysis were present in both SMN1 and SMN2, it is possible that they may affect the regulation of these genes differently. Future studies will further characterize the effects of these TEs in SMN1 and SMN2 on gene regulation under healthy conditions as well as in SMA.The in silico analysis of"}
+{"text": "B1 has a strong inhibitory effect on the melanoma cell line A357 at 100\u2009\u00b5M (76.15% inhibition).A series of novel triazoloquinolinone and imidazoquinazolinone derivatives were designed and synthesised, and their biological activities against SHP2 protein and melanoma A357 cell line were evaluated in\u00a0vitro. The results show that some target compounds have moderate to excellent inhibitory activity on SHP2 protein and melanoma A357 cell line. Structure-activity relationships (SARs) showed that both imidazoquinazolinone and triazoloquinazolinone derivatives have good SHP2 protein kinase and melanoma cell line A357 inhibitory activity. The results of molecular docking also showed that the cores of imidazoquinazolinone and triazoloquinazolinone have a certain affinity for SHP2 protein at the same time. Compared with SHP244, the target compounds have quite good liver microsomal stability and has more drug potential. The most promising compound"}
+{"text": "To evaluate measurement discrepancies by race between pulse oximetry and arterial oxygen saturation  among inpatients not in intensive care.Multicenter, retrospective cohort study using electronic medical records from general care medical and surgical inpatients.Veteran Health Administration, a national and racially diverse integrated health system in the United States, from 2013 to 2019.Adult inpatients in general care , in Veteran Health Administration medical centers.2) of <88% despite a pulse oximetry (SpO2) reading of \u226592%), and whether rates of occult hypoxemia varied by race and ethnic origin.Occult hypoxemia ) patients; non-Hispanic black patients and Hispanic or Latino patients accounted for 6498 (21.6%) and 1623 (5.4%) pairs in the sample, respectively. Among SpO2 values greater or equal to 92%, unadjusted probabilities of occult hypoxemia were 15.6%  in white patients, 19.6% (18.6% to 20.6%) in black patients , and 16.2% (14.4% to 18.1%) in Hispanic or Latino patients . This result was consistent in SpO2-SaO2 pairs restricted to occur within 5 minutes and 2 minutes. In white patients, an initial SpO2-SaO2 pair with little difference in saturation was associated with a 2.7%  probability of SaO2 <88% on a later paired SpO2-SaO2 reading showing an SpO2 of 92%, but black patients had a higher probability (12.9% (\u22123.3% to 29.0%)).A total of 30\u2009039 pairs of SpO2) and pulse oximetry (SpO2) were obtained, black patients had higher odds than white patients of having occult hypoxemia noted on arterial blood gas but not detected by pulse oximetry. This difference could limit access to supplemental oxygen and other more intensive support and treatments for black patients.In general care inpatient settings across the Veterans Health Administration where paired readings of arterial blood gas (SaO Among critically ill patients, patients with occult hypoxemia detected by arterial blood gas but missed by pulse oximetry have recently been shown to have worse clinical outcomes by detailed analyses\u2014including higher mortality and greater incidence of organ failure\u2014as might be expected given the central role of oxygen delivery in healthy cellular functioning.Pulse oximetry is a ubiquitous technology with applications in both ambulatory and inpatient settings. Despite its widespread use, variation in device accuracy by patient race in critically ill patients has been reported as early as 1990.2 <85%.2 readings of \u226592%, acting on existing data could lead to unnecessary invasive measurement of arterial blood gases or unnecessary expenditures on new pulse oximeters. Finally, while SpO2-SaO2 discrepancies are more common in black patients than in white patients, the stability of such discrepancies within an individual patient is unclear. Thus, if a clinician has documented such a discrepancy (or its absence) and the same patient later shows signs or symptoms compatible with arterial hypoxemia, the next step is unclear: whether to repeat an arterial blood gas measurement, or to assume the same direction and magnitude of the SpO2-SaO2 discrepancy as recently documented.Policy and regulatory interest over the technology has increased,2-SaO2 measurements collected during routine care from inpatients in the Veterans Health Administration from 2013 to 2019.We sought to correct these gaps in this study of medical and surgical inpatients in general care in the Veterans Health Administration, a large and diverse health system serving veterans across the US. We hypothesized that black patients in general care  would have an increased frequency of occult hypoxemia compared with white inpatients in general care. We also hypothesized that a blood gas measurement showing the absence of occult hypoxemia would be associated with a low probability of occult hypoxemia on subsequent arterial blood gases, and that this probability would not vary by race. As in past work, we analyzed spontaneously recorded SpO2 and SaO2 data from the Corporate Data Warehouse for all hospital stays in acute medical and surgical care in the Veterans Affairs Patient Database (VAPD). 2 values and time of pulse oximetry measurement on general care floors are generally entered into the electronic health record by the clinician measuring them; SaO2 values of arterial blood gas and timing of the blood draw come directly from laboratory reporting systems.The nationwide Veterans Health Administration provides comprehensive inpatient and outpatient medical care to US veterans in over 100 hospitals. During the study period, Veterans Health Administration hospitals used one electronic health record system (Computerized Patient Record System), which archived data to a central repository (Corporate Data Warehouse). During the study (2013-19), we extracted SpO2 <88%) with a pulse oximetry reading of SpO2 \u226592% recorded at the same time. This range of SpO2 was selected because clinicians would probably not increase oxygen levels in response to SpO2 \u226592%.2 was included as a continuous variable. All pairs of SpO2 and SaO2 values that were measured within 10 minutes for patients in the VAPD were identified. We excluded SaO2 measurements that were labeled as temperature corrected or calculated, and information on whether the SaO2 reading was measured by co-oximetry was not available. To remove critically ill patients and isolate a sample consisting of patients in general care for these analyses, we excluded pairs of readings that were measured on days when patients were in intensive care or transferred in or out of intensive care. For the same reason, we also excluded pairs of SpO2-SaO2 readings from the rare days when more than two blood gases were drawn. We removed SaO2 and SpO2 values lower than 70% to reduce the possibility of mislabeling a venous blood gas as an arterial blood gas or data entry error, because such values seemed unlikely to be accurate in patients who were not critically ill.We defined occult hypoxemia as a low saturation of arterial oxygen ; therefore, these data were complete for all analyses. For race and ethnic origin, adjusted models that predicted occult hypoxemia included non-Hispanic black (referred to in this article as \u201cblack\u201d), non-Hispanic white (\u201cwhite\u201d), and Hispanic or Latino (\u201cHispanic\u201d), consistent with previous research on racial and ethnic differences of occult hypoxemia being most reproducible between those groups.2-SaO2 reading on that day; \u03c72 and independent t tests were conducted to compare patient characteristics by race and ethnic origin. We fit a multivariable logistic regression model to predict the odds of occult hypoxemia (SaO2 <88% for patients with SpO2 \u226592%). Models were adjusted for patient level characteristics that included black race (v white race), age, sex, patient comorbidities (appendix 1), supplemental oxygen (as a continuous variable), and diagnoses on admission .2 readings to account for non-linearity in the difference between SpO2 and SaO2 values, and we included an interaction term for black race and SpO2 readings to test the hypothesis that the relation between race and occult hypoxemia is different across values of SpO2. Standard errors robust to clusters were used to account for clustering at the patient level.Analyses included all patient days with one or two pairs of SpOv white patients, and separately for Hispanic patients v white patients).Because of the complex non-linearities and interactions in the model, we did not report the specific coefficients for race. Instead, we reported predictive margins and average marginal effects of occult hypoxemia for each category of race included in the model  with SpO2 values \u226592%. The number of predicted instances of occult hypoxemia, and not unique hospital admissions or unique patients, is rounded to the nearest thousand to emphasize to readers that this number is an estimate.We estimated the potential population level burden of occult hypoxemia among general care inpatients in VA hospitals across the system under the simple assumption that the probability of occult hypoxemia was the same in SpO2 value minus the SaO2 value), precision (standard deviation of the differences), limits of agreement (bias \u00b11.96\u00d7standard deviation), and accuracy (root mean square error (ARMS) is the square root of the sum of squared bias and squared precision) by race and ethnic origin for the entire dataset according to previously described methods.2 and SpO2). A root mean square error of \u22642-3% is required by the US Food and Drug Administration (FDA) for initial approval pulse oximetry devices.We calculated bias  and Stata/MP version 16.1 . The analytic code is included in appendix 2, and is available through GitHub. This study followed the STROBE statement  guidelinesNo members of the public were formally involved in the conception of this study, but our interest in the phenomenon of pulse oximetry measurement bias stems from caring for patients from different racial and ethnic backgrounds during the covid-19 pandemic.2-SaO2 readings recorded within 10 minutes of each other during the study period. From these, 33\u2009556 SpO2-SaO2 pairs were excluded, representing patients who received intensive care at any point on that calendar day. We also excluded 204 pairs from patient days with more than two pairs of readings in one day. The cohort included 30\u2009039 SpO2-SaO2 pairs ). SaO2 readings were slightly more common on days when an SpO2 reading was recorded for white patients than for black patients ), but if an SaO2 reading was available on an SpO2 monitored day, no substantial difference by race was seen in whether that reading was close enough in time to be in a pair with a recorded SpO2 reading .Characteristics of included pairs of SpO2-SaO2 pairs by race and ethnic origin at the SpO2 range of 89-100% are summarized in appendix 3. Among all values of SpO2 \u226592%, unadjusted probabilities of occult hypoxemia  were 15.6%  for 2823 pairs in white patients, and 19.6% (18.6% to 20.6%) for 1144 pairs in black patients , and pulse oximeters had better precision in white patients than in black patients , when the paired SaO2 values were used as the gold standard across the entire range of SpO2 values in the data (appendix 5).White patients faced less measurement bias in pulse oximetry than black patients  for 24\u2009009 pairs, absolute adjusted increased probability of occult hypoxemia 4.0% ), for pairs no more than 5 minutes apart (2.6 minutes (1.0-4.0) for 12\u2009603 pairs, 3.7% ), or for pairs no more than 2 minutes apart (1.0 minute (0.2-1.5) for 5305 pairs, 4.6% ; This estimate of racial differences in occult hypoxemia was not sensitive to time between SpO2 readings of 92-100% for black and white inpatients in the study. If occult hypoxemia occurred at the same rate as occult hypoxemia in the SpO2-SaO2 pairs analyzed in the study, then an estimated 573\u2009000 additional instances of occult hypoxemia would have occurred in black patients during the study period and would have been detected if pulse oximeters performed as well in black patients as in white patients.In total, we observed 54\u2009048\u2009788 SpO2-SaO2 measurements recorded on the same day. We aimed to determine whether the difference within SpO2-SaO2 readings from the first pair was associated with the odds of occult hypoxemia in the second pair. In 2-SaO2 differences from the first reading of the day into equal groups by tertiles. These groups ranged from having the lowest SpO2-SaO2 difference (SaO2 reading is 0.1 percentage points lower than or any amount higher than SpO2 reading) to having the largest SpO2-SaO2 difference (SaO2 reading is at least 2.5 percentage points lower than SpO2 reading).We recorded a total of 3016 patient days  that had two pairs of SpO2 of 92% in the second pair of readings were 2.7%  if the first pair was in the group with the smallest SpO2-SaO2 difference, 2.4% (\u22120.1% to 4.8%) if the first pair had an SpO2-SaO2 difference in the intermediate group, but 32.0% (15.2% to 48.8%) if the first pair in the group with the largest SpO2-SaO2 difference. Similar patterns in white patients were seen with SpO2 readings of 98% in the second pair. Probabilities of occult hypoxemia of 2.4% (0.4% to 4.5%) and 2.1% (0.3% to 3.9%) were recorded in the groups with the smallest and intermediate SpO2-SaO2 differences, respectively, but 33.8% (26.1% to 41.50%) if the SpO2-SaO2 difference was in the highest group  probability of hypoxemia. The probability of occult hypoxemia in black patients with the largest SpO2-SaO2 differences on the first pair was as high as in white patients if the SpO2 value in the second pair was 92% (39.6% (11.5% to 67.7%), and even when the SpO2 value was 98% (28.4% (18.5% to 38.2%); The probabilities of occult hypoxemia on the second SpO2-SaO2 readings varied depending on magnitude of the difference in the first pair, SpO2 value in the second pair, and patient race. For example, for white patients who did not show occult hypoxemia in the first pair of SpO2-SaO2 readings of a given day, occult hypoxemia was present in only 2.5% of the second pair of SpO2-SaO2 measurements. Among black patients with two pairs of SpO2-SaO2 readings in a given day and with no occult hypoxemia shown in the first pair, occult hypoxemia was present in 6.5% of the second pair of SpO2-SaO2 measurements. While the two SpO2-SaO2 pairs had, on average, the same difference between SaO2 and SpO2 (mean 0.05 for white patients and 0.15 for black patients), the standard deviation in differences between the pairs was 4.1 for white patients and 4.9 for black patients, indicating that the possibility of wide discrepancy was more common in black patients.To put it another way, the odds of occult hypoxemia on the second pair of SpO2-SaO2 measurements from patients identified as Hispanic (or Latino). Among all pairs with SpO2 readings of \u226592%, the unadjusted probability of occult hypoxemia (SaO2 <88%) was 16.2%  in these patients , and pulse oximeters had similar precision in Hispanic patients (6.6 v 6.7), when the paired SaO2 was used as the gold standard across the entire range of SpO2 in the data (appendix 5). Because of the small sample size, we did not further analyze this subgroup of patients.We also analyzed the results of 1623 pairs of SpO2 readings of 92-100%. Including all readings did not meaningfully change the results (appendix 7). Controlling for clustering within hospitals as a random effect did not meaningfully change the results (appendix 8). Analyses testing for a difference in the observed race differences in measurement bias in surgical and non-surgical patients yielded inconsistent results that were sensitive to subtle parameterization choices in the small (<10% of sample) population of patients in surgical general care with SpO2-SaO2 pairs, but did not change the estimated differences meaningfully in the large majority of patients who were not in surgical care (appendix 9).Focusing on differences between black and white patients, our primary analyses in 2-SaO2 readings, white patients could have some reassurance that a later normal SpO2 reading was unlikely to be associated with a SaO2 reading of <88%; however, less reassurance was available for black patients. The overall prevalence of occult hypoxemia could be considerable and racially biased.This study of inpatients in general care in the Veterans Health Administration indicates a significant difference in the ability of pulse oximetry to detect clinically relevant hypoxemia in patients of different races. Black patients were found to have more occult hypoxemia than white patients. Pulse oximetry readings had greater bias and worse precision among black inpatients in general care than among white inpatients in general care. This greater bias and worse precision meant that on receiving a recent and well correlated pair of SpO2 measurement bias between black and white patients, where the odds of occult hypoxemia were higher in black preterm infants,Our findings accord with the most recent data on racial bias in pulse oximetry focused on more ill patients, often in intensive care.Our results add to the literature by showing that the concerns about worse bias and noise  in black patients being monitored with pulse oximetry are also relevant to patients in general care who often receive no or little supplemental oxygen. They also suggest the possibility that this differential pulse oximeter performance affects Hispanic patients, although those results need additional confirmation. Two independent groups have recently shown that arterial hypoxemia undetected by pulse oximetry is associated with worse clinical outcomes.2 and SaO2 readings. These data indicate that excess occult hypoxemia is present in black patients relative to white patients, regardless of whether the SpO2-SaO2 pairs are measured within 2, 5, or 10 minutes apart, suggesting that time between measurements is not a driver of these results\u2014consistent with other recently published work.Our study looks at concerns about possible differential bias being introduced because of the potential interval between SpO2-SaO2 readings measured on the same day, a well aligned SpO2-SaO2 pair for white patients was associated with low levels of occult hypoxemia on subsequent pairs; such concordance might be reassuring in many clinical scenarios. This concordance was less true for black patients, and these differences should be considered in deciding whether to obtain an arterial blood gas reading in appropriate clinical situations until non-racially biased pulse oximeters are in use.Errors in pulse oximeters could be due to a combination of systematic error or bias, which is reproducible across measurements, as well as random error or noise.2 reading of \u226592% is truly associated with an SaO2 reading of <88%. However, for this non-random arterial blood sampling to explain the apparent differences between racial groups, it would be necessary that clinicians are better at making the bedside diagnosis of occult hypoxemia in black patients than in white patients\u2014and, at the same time, that they order confirmatory tests less often but more accurately in black patients. We are unaware of data that support such an alternative mechanism.This study had several limitations. Arterial blood gases are measured less frequently than pulse oximetry. If clinicians are more likely to obtain an arterial blood gas when they have concerns about the accuracy of pulse oximetry, then the probabilities of occult hypoxemia noted in our study would overestimate the frequency with which an SpOSkin color is not consistently recorded as part of the medical record, so we used race as a surrogate, which might not fully reflect the skin tone diversity within each patient group or other differences that might contribute to pulse oximetry bias. Data on Hispanic patients suggest a similar problem, but the smaller sample size resulted in more imprecise estimates; these data should be considered as hypothesis generating unless replicated. Likewise, the potential instability of estimates of racial differentials in surgical patients warrants additional exploration, although should be taken in the light of other published findings.An additional limitation was that nearly all patients had a military service history. No oximeter brand information was available for this study. However, most commercially available oximeters use similar technology.Black patients under the inpatient care of the US Veterans Administration had excess episodes of undetected hypoxemia in 2013-19 compared with white patients. Other scientists have reported increased morbidity and mortality associated with occult hypoxemia.2) is a method used to non-invasively measure arterial oxygen saturation (SaO2); the accepted most accurate method is by invasive arterial blood gasPulse oximetry (SpO2 <88% despite an SpO2 \u226592%) experience worse organ dysfunction and mortality rates in hospital than those without occult hypoxemiaPatients in intensive care with occult hypoxemia  care in hospital, occult hypoxemia was more common for black patients than for white patients; differences in occult hypoxemia by race from real world data did not change according to differences between the recording of the SpO2-SaO2 readings in a day were unlikely to have it on a second pair of SpO2-SaO2 readings on the same day; black patients had less consistency across different pairs of SpO2-SaO2 measurements on the same day White patients not showing occult hypoxemia on a first pair of SpO2 reading does not show occult hypoxemia in a black patient, an elevated index of suspicion might be warranted in black patients with compatible signs and symptoms until unbiased pulse oximeters are routinely availableThese results suggest that if a recent SaO"}
+{"text": "RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes . The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 10 Phenuiviridae, Phlebovirus) is a clinically important mosquito-borne pathogen causing disease in both humans and ruminants. Although most humans have no clinical sign, others develop flu-like symptoms with headaches, fever, or myalgia  gene  for a neutralizing antibody titer at or above the threshold antibody level for protection.The neutralizing antibody response produced by the different titers of r2segMP12 was compared between the use of a single dose versus a single dose followed by a booster dose. Neutralizing antibodies produced following the r2segMP12 vaccine and the rMP12 parental strain vaccine were also examined. Taken together, these data demonstrated that the double deletion of NSs and NSm genes does not reduce the immunogenicity of the MP12 vaccine strain.The r2segMP12 recombinant vaccine candidate for RVFV was produced in a previously published study as summarized in 3, 104, or 105 plaque-forming unit [PFU]) of infectious viruses in a single immunization or two immunizations  were subcutaneously immunized with one of the following: r2segMP12 vaccine candidate, rMP12 parental strain vaccine, or sterile L-15 media. Animals were randomly assigned into 10 groups of 5, representing 8 experimental regimens, using increasing doses (10izations .n\u2009=\u20095) was included to determine the correlation of neutralizing antibodies produced by different dosages of the r2segMP12 strain. Three groups of mice (n\u2009=\u20095) received a second immunization of the r2segMP12 strain to evaluate the effect of a booster immunization using the dose of the vaccine that produced the highest neutralizing antibody titer after the primary vaccine (105 PFU). In addition to the experimental groups that received the r2segMP12 vaccine candidate, four additional groups (n\u2009=\u20095) were designated as control groups, with two positive control groups receiving the rMP12 vaccine at 105 PFU and two negative control groups receiving an equal volume of sterile culture L-15 media.The regimen of a single immunization administered at an increasing dosage per group . Before the initial immunization, all mice were determined to be healthy and seronegative to RVFV through the analysis of collected serum using PRNT (data not shown). Animals were maintained for 6 weeks after the initial immunization. Mice were immobilized using an isoflurane vaporizer before blood collection. Whole blood samples of 0.1\u2009mL were collected from the lateral saphenous vein from immunized animals at 20 days after initial immunization using a 22\u2009g needle. Serum samples were obtained through centrifugation of coagulated blood at 2,000 PRNT were used to determine which vaccination regimen produced the highest titer of neutralizing antibodies following the protocol previously described . Seroconversion was defined using the cutoff of 1:10 PRNT50 titer, a seropositive threshold commonly used for assessing the neutralizing antibody responses elicited by arbovirus vaccines . Finally, PRNT50 titers of animals receiving only a single dosage of the r2segMP12 vaccine candidate at 105 PFU were compared to animals receiving the rMP12 vaccine at 105 PFU at 42\u2009dpi with a Mann\u2013Whitney test. All tests were performed using the GraphPad Prism (version 8.1.2) program .Using a Kruskal\u2013Wallis test followed by a Dunnett's test 50 > 10). All, but one mouse immunized with the r2segMP12 vaccine candidate seroconverted after a single immunization , demonstrating a dose\u2013response relationship in the vaccine immunogenicity. Importantly, the comparison of immunogenicity with the rMP12 vaccine strain suggests the superior immunogenicity of the r2segMP12 strain. Mice immunized with r2segMP12 at a titer of 105 PFU had a significantly higher neutralizing antibody response compared to mice that received the rMP12 vaccine at the same titer (p\u2009=\u20090.0079).Therefore, the r2segMP12 vaccine candidate was capable of eliciting neutralizing antibody responses in CD-1 mice at dosages between 1050 titers were measured in mice that received varying initial titers of the r2segMP12, followed by a booster at 21\u2009dpi. Animals in groups 6, 7, and 8 . In addition, serum neutralizing titers in four out of five mice immunized with the rMP12 vaccine wane below PRNT50 titer of 10, demonstrating the need for a booster immunization. These data suggest that the r2segMP12 strain is superior to the rMP12 vaccine in eliciting neutralizing antibody responses in mice.Given the observation that a single dose of the r2segMP12 vaccine candidate at 10Collectively, these data suggest that the r2segMP12 strain is immunogenic and can elicit neutralizing antibody responses in CD-1 mice that received one single immunization. In addition, the lack of the NSs and NSm genes ensures the safety, but does not compromise the immunogenicity of the r2segMP12 vaccine strain.Due to the impact of RVFV on both human and livestock health, efforts to prevent and control RVFV have been continuous, however, the limitations of each vaccine, multiple doses required, and expenses to maintain these regimens have made it difficult  (McElroy et al, We conclude that the superior immunogenicity of the r2segMP12 strain warrants its advancement in the process of vaccine development, including challenge protection studies conducted in mice and then sheep, which are the amplifying hosts for RVFV. Future experiments will focus on the characterization of the immune response induced by r2segMP12 and its ability to protect against a lethal RVFV challenge."}
+{"text": "Keap1 mutations regulate Nrf2 activity and lead to chemoresistance in cancers. Yet the underlying molecular mechanisms of chemoresistance are poorly explored. By focusing and genotyping head and neck squamous cell carcinoma (HNSCC) that had available pathologic and clinical data, we provide evidence that Keap1 displays frequent alterations (17%) in HNSCC. Functional loss of Keap1 results in significant activation of Nrf2 and promotes cancer cell growth, proliferation, and elevated cancer stem cell (CSCs) self-renewal efficiency and resistance to oxidative stress. Furthermore, decreased Keap1 activity in these cells increased nuclear accumulation of Nrf2 and activation of the Notch pathway, causing enhanced transcriptional alterations of antioxidants, xenobiotic metabolism enzymes, and resistance to chemotherapeutic treatment. Limiting the Nrf2 activity by either Keap1 complementation or by Nrf2 silencing increased the sensitivity to chemotherapy in Keap1-mutated cells and repressed the CSC self-renewal activity. Our findings suggest that Keap1 mutations define a distinct disease phenotype and the Keap1-Nrf2 pathway is one of the leading molecular mechanisms for clinical chemotherapeutic resistance. Targeting this pathway may provide a potential and attractive personalized treatment strategy for overcoming chemotherapeutic resistance conferred by Keap1 mutations. Head and neck squamous cell carcinoma (HNSCC) is the utmost global health concern and affects >890,000 patients, and over 450,000 HNSCC-related deaths occur every year , 2. To dKeap1, and Nrf2- pathway mutations were found in patients with HNSCC.The Cancer Genome Atlas (TCGA) project has profiled an extensive landscape of somatic genomic alterations in HNSCC . This reKeap1) strongly induces NF-E2-related factor 2 (Nrf2), and acquires malignancy in several types of cancer ). The patient had rapid disease progression and underwent biopsy and genotyping of lung metastasis that revealed Keap1 mutations  tumors out of 24 HNSCC patient tumors sequenced, which is much higher than the TCGA data set. The possible putative reason for the higher incidence of Keap1 mutations in our case is unknown however, we speculate that demographic and genetic makeup may play roles in the higher incidence of Keap1 mutations in HNSCC patients. In this study, we found that all Keap1 mutated tumors exhibited nuclear accumulation of Nrf2 as assessed by immunohistochemical analysis. Keap1 mutations result in the accumulation and activation of Nrf2 and may partly confer the resistance to chemotherapeutic treatment in HNSCC patients and reduce drug-induced ROS production. Importantly, all these mutations involved functionally relevant domains of Keap1 protein, including BTB (c.403\u2009C\u2009>\u2009T and c.1129\u2009G\u2009>\u2009A), IVR domain (c.1111\u2009G\u2009>\u2009A), and KR3 region (c.1766A\u2009>\u2009G) of Keap1, which are responsible for the ubiquitination and binding of Nrf2 [Nrf2 pathway has been proposed to be the leading cause of chemoresistance in several cancers [Nrf2 pathway activation in HNSCC, we sequenced the Keap1 and Nrf2 genes and identified mutations in Nrf2 immunopositive tumors. Interestingly, the Nrf2 mutations involved the Neh2 domain where Keap1 binds. Importantly, we did not detect any mutations in the matched normal tissues, confirming that the mutation is somatic in origin. Furthermore, the overall frequency of mutations  for the Keap1 gene in HNSCC tissues suggests that Keap1 mutations are likely a frequent genetic alteration in HNSCC at least in our case which is much higher than that of data reported in TCGA. In addition, we assessed whether the Nrf2 activation pathway is engaged in mediating chemotherapeutic resistance in HNSCC. We used human HNSCC primary tumor cells and HNSCC cell lines to evaluate the functional relationship between Nrf2 activation and chemotherapeutic resistance. Knockdown of Keap1 by siRNA in HNSCC cells demonstrated enhanced Nrf2 pathway activity, which led to enhanced transcriptional activity thereby rendering HNSCC cells resistant to chemotherapy. We have achieved a comparable result in HNSCC tumor and normal tissue, where the loss of functional Keap1 gene and subsequently increased staining intensity of Nrf2 corroborate the above findings. In concordance with the above findings, as expected, GST, NQO1, and SOD1 enzyme levels and GSH levels were highly significantly elevated in the tumor tissues compared with matched normal individuals. This suggests that upregulation of these Nrf2-dependent genes likely contributed to the resistance to chemotherapy treatment and cell survival. This result is in agreement with previously published results, where high antioxidant capacity increases cell survival and proliferation and protects against oxidants, radiation therapy, and chemotherapies [Loss of ux pumps . In the  of Nrf2 , 47. The cancers , 49. To herapies .Keap1 and Nrf2 overexpression induces many stress resistance genes and can restore cancer cell proliferation. It has previously shown that constitutive activation of Nrf2 contributes to tumorigenesis, ROS detoxification, and modulation of redox state and also contributes to resistance to many anticancer drugs [Keap1 has been identified as a possible mechanism of chemoradiation resistance in many cancers [Keap1 mutations showed more resistance to etoposide and carboplatin than to Keap1 wild-type cells [Nrf2 due to Keap1 loss may confer resistance to cisplatin, a widely used chemotherapy regimen for head and neck cancer, by regulating ROS and cancer stem cell pathways. In concordance with these previous results, our results demonstrated that downregulation of the Nrf2 expression in Keap1 mutated cancer cells or introduction of Keap1 cDNA in Keap1 mutant cells significantly enhances the sensitivity to cisplatin. In analyzing the clinical cases, all four Keap1 mutated HSNCC patients had a history of recurrence within a year all cases were treated with 2 and 3 lines of chemotherapy and exhibited poor clinical response, suggesting therapeutic resistance due to Keap1 mutations as well as the potential existence of CSC populations in these tumors.Loss of er drugs , 50. Los cancers . In the pe cells . Thus, oKeap1 mutations during HNSCC oncogenesis due to the deletion of Keap1 led to the increased self-renewal activity of cancer cells and subsequent therapeutic resistance. Importantly the impact of Keap1 loss leads to increased self-renewal activity and therapeutic resistance compared to Keap1 wild-type cells or Keap1 reintroduced clones which define the greater clinical relevance. Previous reports have demonstrated the molecular mechanisms of cisplatin resistance in cancer cells and cisplatin treatment increased mitochondrial ROS generation [Keap1 or silenced Nrf2 observed increased mitochondrial ROS generation and limited the cell growth in cells treated with cisplatin. This suggests that constitutive Nrf2 activation due to Keap1 loss influences oxidative stress and reduces the cellular damage induced by the increase of ROS, therefore, triggering the resistance of chemotherapy, particularly for cisplatin. Han and colleagues [Nrf2 and Notch signaling in OCSC  cells. Activation of Nrf2 in Keap1 deleted cells resulted in hyperproliferation of squamous epithelial cells and activation of Notch signaling [Nrf2 activation in Keap1 mutated cells, suggesting that Notch also plays a role in mediating the effects of Nrf2 activation in HNSCC cells.Our observation from this study suggests a pivotal role for neration  and trigneration , 54. In lleagues  recentlyignaling . CongrueKeap1, activation of Nrf2 and the Notch signaling pathway promotes cellular metabolic reprogramming that sustains cellular proliferation. This reprogramming feature leads to the clonal expansion of mutant cells thus acquiring self-renewal capacity and triggering resistance pathways and outpacing the Keap1 WT cells and potentially acquiring additional genetic changes and subsequently leading to therapeutic failures. All these features explain the therapeutic failure and adverse and poor survival outcomes conferred by Keap1 mutations in rapidly progressing HNSCC cells. In a recent study, it was reported the Keap1-Nrf2 double mutations in lung cancer leads to poor outcome and severe therapeutic resistance in radiation therapy [Nrf2 mutation and none of the patients had concurrent Keap1-Nrf2 mutations. This may be due to the small number of patients assessed in this study and we cannot rule out that Keap1-Nrf2 dual mutations may have somatic or epigenetic consequences and may play a devastating role in therapeutic failures and poor outcomes, disease recurrence in Keap1-Nrf2-mutant tumors. Thus, future studies should include large numbers of patients\u2019 samples to examine the effects of Keap1 mutation as well as to explore the role of Keap1-Nrf2-dual mutation in chemoresistance.Reviewing all our results, it appears that once HNSCC cells acquire a mutation in  therapy . In our Keap1\u2013Nrf2 alterations are known to play roles in chemotherapeutic resistance particularly cisplatin resistance in HNSCC, surprisingly, the mutation status is not widely used to make a treatment decision in head and cancer. It would be interesting to investigate whether loss of Keap1 and overexpression of Nrf2 status in tumor samples are clinically relevant mechanisms of chemotherapeutic resistance in HNSCC and develop alternative therapy to counter Nrf2 activation. This may improve personalized therapy in a subset of HNSCC patients who are prone to therapeutic resistance.In conclusion, although Supplementary Figure LegendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Reporting summer/Reproducibility checklistOriginal Data File"}
+{"text": "Bacillus vallismortis have been reported to be efficient plant-growth-promoting bacteria as well as inducers of systemic resistance. Here, we report the draft genome sequence of Bacillus vallismortis strain BL01, isolated from the roots of Artemisia lerchiana Web.Some strains of  Bacillus vallismortis can produce metabolites with strong growth inhibition activity against phytopathogenic fungi . Plant roots were disinfected with tap water for 30 s followed by 70% ethanol for 5\u2009min and then 15% H2O2 for 10\u2009min and sterile water for 2\u2009min 5 times and subsequently crushed with a mortar and pestle under sterile conditions. Aliquots of 100\u2009mL of the resulting plant juices were plated onto a 1/20 dilution of tryptic soy agar  plates. The sterility check consisted of aliquots of water from the last rinsing that were plated onto 1/20 TSA according to methods reported previously (de novo assembled using SPAdes v3.14.1 (https://github.com/lh3/seqtk). The contigs were mapped to the Bacillus vallismortis strain Bac111 (GenBank accession number CP033052.1) genome by CONTIGuator v2.7   . A totalPRJNA809498. The raw reads were deposited in the Sequence Read Archive under accession number SRR18107534. The GenBank nucleotide sequence accession number is CP092751.All data are available in the National Center for Biotechnology Information database under BioProject accession number"}
+{"text": "Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with high heterogeneity, rapid progression, and paucity of treatment options. The most effective chemotherapeutic drug used to treat TNBC is doxorubicin (Doxo) which is an anthracycline antibiotic. However, Doxo treatment alters cytosolic calcium dynamics leading to drug-resistance condition. The aim of this study is to capture the alterations in the activity of various calcium channels and pumps during Doxo treatment and their consequences on cytosolic calcium dynamics that ultimately result in drug resistance.In the present study, a mathematical model is proposed to capture the complex dynamical landscape of intracellular calcium during Doxo treatment. This study provides an insight into Doxo remodeling of calcium dynamics and associated drug-resistance effect. The model was first analyzed analytically and then explored through numerical simulation using techniques like global sensitivity analysis, parameter recalibration, etc.2+ channels and pumps might provide efficient chemotherapy in TNBC. It was also observed that the indispensability of calcium influx rate is paramount in the Doxo drug resistance. Finally, three drugs were identified from existing literature that could be used as a combination therapy along with Doxo.The model is used to predict the potential combination therapy for Doxo that can overcome Doxo associated drug resistance. The results show targeting the dysregulated CaThe investigation highlights the importance of integrating the calcium signaling of various calcium regulating compounds for their effective anti-tumor effects deliverance along with chemotherapeutic agents. The results from this study might provide a new direction to the experimental biologists to explore different combination therapies with Doxo to enhance its anti-tumor effect. Breast cancer is the leading cause of cancer-related deaths diagnosed among women \u20133. Tripl3R) channels ER reduction. These channels are known as SOCE. This process is intervened by STIM1 of the ER membrane and is sensitive to ER Ca2+ levels. STIM1 proteins bind to ORAI1, the proteins embedded in the plasma membrane that forms the pore of SOCE and triggers their opening. The inward calcium flux through the SOCE channel is defined by the term \u03b2[Pso] [2+ influx through TRPC1 channel is dependent on the difference between the Ca2+ concentration of extracellular space (ces) and cytosol ([Ca2+]c). Therefore, the Ca2+ influx through TRPC1 channel is modeled as \u03b3 (esc \u2013 [Ca2+]c). We assumed the Ca2+ concentration of extracellular to be a constant as taken by Mirzakhalili et al. [\u03be and \u03b4 respectively [We developed a three-dimensional model using ordinary differential equations (ODEs) to capture the complex dynamical landscape of intracellular calcium during Doxo treatment. The first equation of the model describes the rate of change of cytosolic calcium level. The inward flux of Cat rate \u03b1 . SOCE ism \u03b2[Pso] . As mentm \u03b2[Pso] , the Ca2i et al. . The plaectively .2+ flux into cytosol from ER through IP3R2 channel  describes the fraction of STIM1 proteins binding to ORAI1 proteins to activate the SOCE channel for calcium influx into the cytosol from extracellular space. This fraction adjusts depending upon the changes in ER calcium levels because the diffusion of STIM1 within the ER membrane is a slow process ER, and is represented by Hill function (with Hill coefficient 4), considering affinity ks for [Ca2+]ER [The steady-state function [Ca2+]ER . Based ot > 0 : 0 < [Ca2+]c (t) <, Pso] (t) < 1}, with Result 1. (i) The solution of the system  exists aResult 2. The system  has inte21 > 0 and A1A2 > A3 holds simultaneously , \u03b3 ([Ca2+] influx rate through TRPC1 channel), \u03b4(leaking rate), \u03be([Ca2+] export through NCX), p1 ([Ca2+] influx rate through IP3R2), q1 , p2 (flux rate through SERCA1 channel) were estimated using experimental data points by Bong et al. [2+]ER and [PSO] are also obtained and shown in We explored different literatures and obtained few parameters while the rest of the parameters namely, g et al. . Here weg et al. , see FigE* was satisfied, and the interior equilibrium point E* =  is locally asymptotically stable as the eigenvalues of the characteristic equation is . This follows that CRD shows stable dynamics through damped oscillations. SOCE is an important plasma membrane channel of calcium entry in the cytosol that opens in response to ER calcium depletion. Entry through this channel is mediated by STIM1 proteins that disseminate from the ER membrane and binds to ORAI1 proteins and activates SOCE. IP3R2 channel plays an important role in the exit of calcium from ER into cytosol. The involvement of IP3R2 channel in mediating SOCE has been established earlier thereby influencing cytosolic calcium dynamics [\u03b2 channel\u2019s influx rate) and p1 ([Ca2+] influx rate through IP3R2) and its role in establishing CRD. We varied these two parameters 2-fold up down from their base values and generated the 2D parameter space shown in \u03b2 and setting p1 to its baseline value, the model dynamics changes from stable focus to stable node dynamics.We also observed that for this baseline parameter set  the exisdynamics , 42. To \u03b2 , \u03b3 ([Ca2+] influx rate through TRPC1 channel), p1 ([Ca2+] influx rate through IP3R2), p2 (flux rate through SERCA1 channel), p3 (flux rate through PMCA2 channel), and they were simultaneously and step-wise increased by 25% (Doxo 1), 50% (Doxo 2), and 75% (Doxo 3). The calcium profile generated through elevated parameter ranges was given in Doxo works in a concentration-dependent manner . An alteIn this section, we explored the effect of parameter variations on the system\u2019s dynamics. We first varied single parameters through robustness analysis and observed their effect, and then we performed a global sensitivity analysis (GSA) to capture the effect of multi-parameter variation.\u03be, p1, q3  method, which is based on sampling, for GSA. In our study, we used the LHS method as it compactly differentiates the input parameters . PRCC me\u03b2), maximum Ca2+ influx through TRPC1 channel (\u03b3), Ca2+ export through NCX (\u03be) and calcium concentration in extracellular space (ces) were significantly correlated with cytosolic calcium (see 2+ influx through TRPC1 channel (\u03b3), Ca2+ export through NCX (\u03be) and calcium flux rate (p1) and half saturation constant (q1) of IP3R2 channel, calcium flux rate (p2) and half saturation constant (q2) of SERCA1 channel were significantly correlated with [Ca2+]ER  associated with these calcium channels and pumps in our model , \u03b3 , p1 , p2 , p3 . We picked one parameter from the above and reduced its value by 20%. After that, all other parameters were varied 100% up and down from their respective values (i.e. in the Doxo 3 parameter set) as done in the single parameter variation. Out of the five parameters related to Doxo associated proteins, the reduction of \u03b2  by 20% turned out to be the only parameter that can restore CRD in combination with other model parameters. Except for \u03b2 , reduction in other parameters related to Doxo associated proteins was not sufficient to restore CRD in combination with any other model parameter. The results of double parameter variation in 75% increased case are given in We performed a single parameter variation where each parameter was perturbed 100% up-down from their respective values and we filtered out parameters that retained the Doxo profile to the control profile (CRD). This exercise was repeated for different Doxo treatments where associated parameters were increased by 25% (Doxo 1), 50% (Doxo 2), 75% (Doxo 3). Single parametric variation was able to restore the CRD only in the case of 25% and 50% increase. The results of single parameter variation for the first two cases of Doxo treatments are given in We filtered out those restoration approaches which robustly produced CRD once obtained in our parametric variation range with 90% CI of readout values  used in the recalibration exercise done in the previous section . We downloaded a list of drugs with their corresponding drug targets from Drug Bank database  influx), the proliferation of invasive tumor cells was reduced [Doxo is a broad-spectrum chemotherapeutic drug used in the clinic . Targetinowadays . The in gularity . In a re binding , 66. The binding . SKF9636 reduced . Moreove reduced . This unThe present study focused on the mechanistic viewpoint of reduced sensitivity of TNBC towards Doxo. The investigation highlights the importance of integrating the calcium signaling of various calcium regulating compounds for their effective anti-tumor effects deliverance along with chemotherapeutic agents. Here, we proposed few potential strategies which can be used in combination with Doxo. We further used a drug dataset to identify three drugs from existing literature that could be used individually along with Doxo as a combination therapy. Before ending this article, we would like to mention that our work lacks experimental validation, but the proposed approach delineates the complex nature of Doxo drug-resistance and can help in developing effective therapeutic strategies for TNBC."}
+{"text": "In 2021, 20-valent pneumococcal conjugate vaccine (PCV) (PCV20)  and 15-valent PCV (PCV15) (Merck Sharp & Dohme Corp.) were licensed by the Food and Drug Administration for adults aged \u226518 years, based on studies that compared antibody responses to PCV20 and PCV15 with those to 13-valent PCV (PCV13) . Antibody responses to two additional serotypes included in PCV15 were compared to corresponding responses after PCV13 vaccination, and antibody responses to seven additional serotypes included in PCV20 were compared with those to the 23-valent pneumococcal polysaccharide vaccine (PPSV23) (Merck Sharp & Dohme Corp.). On October 20, 2021, the Advisory Committee on Immunization Practices (ACIP) recommended use of either PCV20 alone or PCV15 in series with PPSV23 for all adults aged \u226565 years, and for adults aged 19\u201364 years with certain underlying medical conditions or other risk factorsPPSV23 has been recommended for use in the United States since the 1980s for adults aged \u226565 years and for younger adults with underlying conditions that increase their risk for pneumococcal disease . In 2012Recent systematic reviews continue to support the effectiveness of PCV13 against invasive pneumococcal disease (IPD)During February\u2013October 2021, ACIP reviewed the epidemiology of pneumococcal disease and considerations for use of PCV15 and PCV20 in adults. The ACIP Pneumococcal Vaccines Work Group (Work Group) evaluated the quality of evidence for PCV15 and PCV20 immunogenicity and safety using the GRADE approach.Pneumococcal disease incidence in adults. During 2018\u20132019, the incidence of all IPD in adults aged \u226565 years was 24 per 100,000 population  . The most frequently reported adverse reactions were injection site pain, fatigue, and myalgia. The rates of serious adverse events (SAEs) within 6 months of vaccination were 2.5% among PCV15 recipients and 2.4% among PCV13 recipients. No SAEs or deaths were considered to be related to the study vaccines . When PCV15 is used, it should be followed by a dose of PPSV23 . When PCV15 is used, it should be followed by a dose of PPSV23.Dosing schedule. When PCV15 is used, the recommended interval between administration of PCV15 and PPSV23 is \u22651 year. A minimum interval of 8 weeks can be considered for adults with an immunocompromising condition, cochlear implant, or cerebrospinal fluid leak to minimize the risk for IPD caused by serotypes unique to PPSV23 in these vulnerable groups (Adults with previous PPSV23 only. Adults who have only received PPSV23 may receive a PCV (either PCV20 or PCV15) \u22651 year after their last PPSV23 dose. When PCV15 is used in those with history of PPSV23 receipt, it need not be followed by another dose of PPSV23.Adults with previous PCV13. The incremental public health benefits of providing PCV15 or PCV20 to adults who have received PCV13 only or both PCV13 and PPSV23 have not been evaluated. These adults should complete the previously recommended PPSV23Coadministration with other vaccines. PCV15, PCV20, or PPSV23 can be coadministered with QIV in an adult immunization program, as concomitant administration  has been demonstrated to be immunogenic and safe. However, slightly lower pneumococcal serotype-specific OPA GMTs or geometric mean concentrations were reported when pneumococcal vaccines were coadministered with QIV compared with when pneumococcal vaccines were given alone  (PCV13) and the 23-valent pneumococcal polysaccharide vaccine (PPSV23) are recommended for U.S. adults. Recommendations vary by age and risk groups.On October 20, 2021, the Advisory Committee on Immunization Practices recommended 15-valent PCV (PCV15) or 20-valent PCV (PCV20) for PCV\u2013na\u00efve adults who are either aged \u226565 years or aged 19\u201364 years with certain underlying conditions. When PCV15 is used, it should be followed by a dose of PPSV23, typically \u22651 year later.Pneumococcal vaccination recommendations were simplified across age and risk group. Eligible adults may receive either PCV15 in series with PPSV23 or PCV20 alone."}
+{"text": "The 12hr panels for EPC cells in PLOS ONE editors have no concerns about the MCF-10A panels in There appear to be similarities between the 12hr, 24hr and 48hr Hoechst panels for MCF-10A cells in Raw data underlying all the results reported in the article are available from the corresponding author, except for FCS files underlying the flow cytometry experiments presented in Figs 4, 5, and S1 FileImages were captured using BD Pathway Bio-imager 435 and each file is a composite of four separate images captured as a 2x2 montage.(ZIP)Click here for additional data file.S2 File(DOCX)Click here for additional data file."}
+{"text": "The in ovo injection of vitamin D3 sources has been shown to enhance immunity as well as histomorphological variables in unchallenged conditions. One of the main diseases affecting poultry production is coccidiosis. Because of this, the current study was designed to determine the effects vitamin D3 (D3) and 25-hydroxyvitamin D3 (25OHD3) alone or together on the inflammatory reaction and small intestine morphology of broilers that were challenged with coccidiosis. In this study, it is shown that the in ovo administration of 2.4 \u03bcg of 25OHD3 alone increased villus length to crypt depth ratio (VCR) with the D3 + 25OHD3 treatment being intermediate two weeks post-challenge (28 day of age). Furthermore, chickens that received of 25OHD3 alone experienced lower plasma nitric oxide concentration as a systemic inflammatory indicator in comparison to all other treatments. It is concluded that the in ovo injection of 2.4 \u03bcg of 25OHD3 at 18 days of incubation can enhance intestinal histomorphology as well as inflammatory reaction of broilers when infected with coccidiosis.Vitamin D3 (D3) and 25-hydroxyvitamin D3 (25OHD3) administration on their inflammatory response and small intestine morphology were evaluated. At 18 d of incubation (doi), a 50 \u03bcL volume of the following 5 in ovo injection treatments was administrated: non-injected (1) and diluent injected (2) controls, or diluent injection containing 2.4 \u03bcg D3 (3) or 2.4 \u03bcg 25OHD3 (4), or their combination (5). Four male broilers were randomly allocated to each of eight isolated replicate wire-floored battery cages at hatch, and birds were challenged at 14 d of age (doa) with a 20x live coccidial vaccine dosage. One bird from each treatment\u2013replicate (40 birds in each of 8 replicates per treatment) was bled at 14 and 28 doa in order to collect blood for the determination of plasma IL-1\u03b2 and nitric oxide (NO) concentrations. The duodenum, jejunum, and ilium from those same birds were excised for measurement of villus length, crypt depth, villus length to crypt depth ratio (VCR), and villus surface area. In ovo injection of 2.4 \u03bcg of 25OHD3 resulted in a reduction in plasma NO levels as compared to all other treatments at 28 doa. Additionally, duodenal VCR increased in response to the in ovo injection of 25OHD3 when compared to the diluent, D3 alone, and the D3 + 25OHD3 combination treatments at two weeks post-challenge (28 doa). Therefore, it can be concluded that 2.4 \u03bcg of 25OHD3, when administrated in ovo at 18 doi, may be used to decrease the inflammatory reaction as well as to enhance the small intestine morphology of broilers during a coccidiosis challenge. In broilers challenged with coccidiosis, effects of in ovo vitamin D Coccidiosis is a parasitic infection that still is considered one of the main diseases affecting the performance of poultry reared under intensive production systems. Gy\u00f6rke et al.  estimate3 (D3) and its metabolite, 25-hydroxyvitamin D3 (25OHD3), have been shown to affect small intestine morphology and the humoral and inflammatory responses of broiler chickens , which is the active form of vitamin D3. Increased levels of 1,25(OH)2 D3 alone, with or without associated changes in VDR, resulted in an increase in breast meat yield [3-injected treatment groups in a companion study, the expression of 1\u03b1-hydroxylase and immune suppressor genes increased 14 d post-challenge in broilers that were in ovo-injected with of 25OHD3 (unpublished data). Therefore, improvements in broiler small intestine morphology during a coccidiosis challenge in response to 25OHD3 alone might have been due to increased intestinal expression of 1\u03b1-hydroxylase and an enhanced immunity. In comparison to Der diets . Additiodiets [3 . The effrs (VDR) . The enzat yield  and imprat yield  and immuat yield  of broilat yield . Coccidiat yield . Additio3 reduced NO production during the coccidiosis challenge. Nitric oxide is an important indicator of on inflammatory response and is produced by the oxidation of L-arginine by the action of NO synthase. Inducible NO synthase is considered more important in immunity and inflammation [Eimeria-infected chickens produced higher serum NO concentrations compared with non-infected controls [3 reduced pro-inflammatory responses and increased anti-inflammatory response genes during a coccidiosis infection in comparison to vitamin D3 at the same level of activity [3 modulates the effects of immunoregulatory T-cells during a coccidiosis infection. Dietary 25OHD3 has been shown to increase the number of CD4 + CD25 + cells in mucosal regions, such as cecal tonsils, of Eimeria-infected chickens [3 has the potential to reduce bird losses due to a compromised immunity caused by a coccidia challenge.In the current study, the in ovo injection of 25OHDammation . The expammation . In addicontrols . Dietaryactivity . In addichickens . These r3 sources. Our findings suggest that coccidiosis has detrimental effects on the small intestine morphology and immunity of broilers. However, the in ovo injection of 2.4 \u03bcg of 25OHD3 reduced NO production and small intestine CD and increased the small intestine VL and VCR of challenged broilers. However, the other in-ovo-injected treatments tested in this study did not exhibit beneficial effects on the small intestine morphology and inflammatory response of the challenged birds. These results therefore indicate that in ovo injection of 25OHD3 may reduce negative effects on the inflammation and small intestine morphology of broilers caused by coccidiosis. These benefits may have been due to the expression of genes that contributed to the improved intestinal development and subsequent health of the broilers. Further research is required to determine the regulatory effects of vitamin D3 sources on immunity and intestinal development during a coccidiosis infection. In conclusion, the aim of this study was to determine the small intestine morphology and inflammatory response of broilers challenged with coccidiosis when birds were injected in ovo with two vitamin D"}
+{"text": "Dugesia japonica. Importantly, we found that knockdown of DjMeis1 inhibits the differentiation of neoblasts into eye progenitor cells and results in an eyeless phenotype with normal central nervous system. Furthermore, we observed that DjMeis1 is required for the activation of Wnt signaling pathway by promoting the Djwnt1 expression during posterior regeneration. The silencing of DjMeis1 suppresses the expression of Djwnt1 and results in the inability to reconstruct posterior poles. In general, our findings indicated that DjMeis1 acts as a trigger for the activation of eye and tail regeneration by regulating the differentiation of eye progenitor cells and the formation of posterior poles, respectively.As a member of TALE family, Meis1 has been proven to regulate cell proliferation and differentiation during cell fate commitment; however, the mechanism is still not fully understood. The planarian, which has an abundance of stem cells (neoblasts) responsible for regenerating any organ after injury, is an ideal model for studying the mechanisms of tissue identity determination. Here, we characterized a planarian homolog of Meis1 from the planarian  Meis1 results in severe limb agenesis [Meis1 and associated co-factors can promote the skin tumorigenesis. Knockdown of Meis1 induces a significant decrease in benign and malignant tumors in mice [Meis1 protein is a member of the three amino acid loop extension (TALE) transcription factors family. Previous studies have demonstrated that Meis1 plays essential roles in the embryonic development across metazoans. For example, Meis1 participates in the embryonic cortical development by promoting neuronal proliferation and migration [agenesis . In addiagenesis , eyes, aagenesis ,5. With agenesis ,7. Upreg in mice . Meanwhi in mice ,10. In gDjwnt1 and \u03b2-catenin, act as promoters for the construction of posterior poles [Djwnt1 can cause planarians that are unable to regenerate tails or regenerate heads in the place of tails. \u03b2-catenin acts as a downstream gene of Djwnt1. Silencing of \u03b2-catenin can result in the reversal of posterior poles and cause planarians to regenerate posterior heads from posterior blastema [notum serves as an inhibitor of Djwnt1 during anterior poles regeneration [Notum RNAi can upregulate Wnt signaling and cause animals to regenerate tails in the place of heads [Djpbx and Djislet have been proven to be positive regulators of Djwnt1 during posterior poles regeneration. Knockdown of Djpbx and Djislet can cause planarians that are unable to regenerate their tails [Planarians can regenerate a complete individual from any fragment of their body ,12,13. Tor poles ,25. Knocblastema . Furtherneration . Notum Rof heads . Recentlir tails ,30. In gMeis1 in planarian Dugesia japonica. We observe that DjMeis1 RNAi planarians cannot form mature eye cells or reconstruct the posterior poles. DjMeis1 RNAi decreases the number of eye progenitor cells and suppresses the expression of Djwnt1. Herein, we propose that DjMeis1 serves as a positive factor in the regulation of the differentiation of eye progenitor cells and the establishment of posterior poles during planarian regeneration.In our work, we identify a homolog of Dugesia japonica based on our previous transcriptome [DjMeis2 and DjMeis3 have been previously reported as SmedMeis and SmedMeis-like in planarian Schmidtea mediterranea  and PC2 ) probes to detect the reconstruction of brain nerves [DjMeis1 RNAi animals was normally reconstructed compared to the control animals at 7 dpa  to examine the regeneration of eye progenitor cells in DjMeis1 RNAi animals [+Djovo cells in DjMeis1 RNAi animals was significantly reduced  to determine whether the posterior wound in DjMeis1 RNAi animals could be healed [DjMeis1 RNAi animals showed a normal expression pattern of LaminB compared to the control groups at 7 dpa  reconstruction. We observed that DjMeis1 RNAi animals failed to regenerate VNC posteriorly, which was consistent with the result that DjMeis1 RNAi animals healed the posterior wound at the amputated site without activating the regeneration of tails  of the head fragments and observed that DjMeis1 RNAi animals regenerated incomplete pharynx or failed to regenerate pharynx  at 7 dpa and found that sFRP1+ cells existed both in anterior and posterior poles in double RNAi animals  in hypoxia [DjMeis1 RNAi planarians displayed normal mitotic activities compared to the control groups  used for the RNAi experiments were synthesized by in vitro transcription as previously described [\u03b2-catenin and DjMeis1 maintained a concentration of 2 \u03bcg/\u03bcL dsRNA. The water treated by DEPC was injected to the control group animals. Head, trunk, and tail fragments were amputated from the animals from the anterior and posterior sites of the pharynx at 24 h after the last injection.All the dsRNA  for 10 min at 37 \u00b0C and fixed in 4% paraformaldehyde for 20 min at room temperature. Then, the animals were hybridized with DIG-labeled probes at 56 \u00b0C for 16-17 h and washed in 2\u00d7 SSC  and 0.2\u00d7 SSC three times for 20 min, respectively. Antibody incubation  and colorimetric (NBT/BCIP) were used for in situ detection. Different concentrations of SSC were prepared by dissolving 20\u00d7 SSC in deionized water.Whole-mount ISH (WISH) was performed as previously described . In eachWhole-mount immunostaining was performed as previously described . In eachEye resection was performed as previously described . In eachBrdU was performed as previously described . In each2O2 in deionized water) was used to bleach the animals under bright light for 2 h. After washing in 1\u00d7 SSC and PBST for 5 min and incubating in Proteinase K (20 mg/mL in PBST) for 10 min at 37 \u00b0C, the animals were fixed in 4% paraformaldehyde for 20 min and washed in PBST for 10 min. Then, the animals were hybridized with DIG-labeled PC2 probe at 56 \u00b0C for 16\u201317 h and washed in 2\u00d7 SSC and 0.2\u00d7 SSC three times for 20 min, respectively. PBST with 5% goat serum and 5% Western Blocking Reagent  was used to block the animals for 5 h. Next, the animals were incubated in 1:500 anti-Digoxigenin-POD  overnight. Finally, the animals were treated with tyramide conjugated to Alexa568  and were observed by NIS element software . Different concentrations of SSC were prepared by dissolving 20\u00d7 SSC in deionized water.Fluorescence in situ hybridization was performed as previously described . In eachp < 0.05.Data were shown as means \u00b1 SD, and statistical analyses were performed by students. One-way analysis of variance (ANOVA) was used to analyze the data of two groups. A statistically significant difference was defined as"}
+{"text": "Moreover, the \u0394atg1\u0394pmk1double mutant resembles the single \u0394pmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of+atg1. Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. Although starvation potently induces autophagy, the basal level of autophagy is maintained even in normal growth conditions, thereby controlling development, cellular metabolism, and proliferation in all eucaryote cells . However, how autophagy organizes cellular signaling networks to exert these functions is not fully understood.Autophagy is an evolutionarily highly conserved mechanism to maintain cellular homeostasis via waste clearance using the lysosomal machinery  phenotype, which recapitulates Pmk1 MAPK signaling inhibition (Methods). The rationale of thevicmutant screening is based on the antagonistic relationship between Pmk1 MAPK and calcineurin, a highly conserved serine/threonine protein phosphatase, in the Cl\u2212homeostasis in fission yeast. Knockout of theS. pombecalcineurin geneppb1+ or inhibition of calcineurin activity by the immunosuppressant FK506, a specific inhibitor of calcineurin, results in hypersensitivity to Cl\u2212. This phenotype associated withppb1deletion was reversed by the inhibition or gene knockout of the components of the Pmk1 MAPK signaling pathways, including Pmk1 MAPK, Pek1 MAPKK, Mkh1 MAPKKK, and Pck2 Protein kinase C . Our previous genetic screening to isolatevic mutants identified upstream activating regulators of MAPK signaling such as geranylgeranyl transferase  and farnesyl transferase .Here, we identified2, whereas knockout of thepmk1+gene makes cells grow much faster in the same condition . \u0394atg1 cells grew faster than the WT cells in the presence of FK506 and 0.09 M MgCl2, although the growth of \u0394atg1 cells was slower than that of \u0394pmk1. The WT, \u0394pmk1 and \u0394atg1 cells exhibited essentially the same pattern of sensitivity and resistance to FK506 and Cl\u2212on EMM, YPD and YES plates, although the sensitivity of the WT cells and \u0394atg1 cells was more enhanced on YES than the other plates. Genetic interaction between \u0394atg1and \u0394pmk1was further examined by constructing \u0394atg1\u0394pmk1double mutant cells. The degree of thevic phenotype in \u0394atg1\u0394pmk1double mutants and \u0394pmk1cells was almost equivalent. These results are consistent with Atg1 working upstream of the Pmk1 pathway. The difference in growth of these strains is almost indiscernible in the absence of FK506 and MgCl2.The growth of the wild-type (WT) cells was significantly inhibited in the presence of the calcineurin inhibitor FK506 and 0.09 M MgClet al.2006). This phenotypic evaluation also led to the identification of an SH3 adaptor protein Skb5 as a negative regulator of Pck2/Pmk1 signaling . As shown in Figure 1B,atg1deletion significantly suppressed the toxicity induced by Pck2 overexpression, although the impact ofatg1deletion on the suppression of the toxicity of Pck2 overexpression was smaller than that achieved bypmk1deletion. Thus,atg1 deletion is likely to ameliorate Pck2-mediated Pmk1 MAPK hyperactivation.To further explore the functional relationship between Atg1 and Pmk1 MAPK signaling, we utilized the cell growth inhibition associated with Pck2 overexpression. Pck2 overexpression in the WT cells leads to Pmk1 MAPK hyperactivation and cytotoxicity, which can be suppressed by the inhibition or knockout of the components of the Pmk1 MAPK pathway  . The degree of thevic phenotype of theseatgmutants was similar to that of \u0394atg1 cells. These results suggest that the autophagy system as a whole may be involved in the Pmk1 MAPK signaling regulation.Next, we confirm if the loss-of-function mutants of other autophagy-related genes also display theet al.2006), as well as the role of autophagosome as a scaffold to facilitate spatial coordination of RAF/MEK/ERK phosphorylation . Our epistasis analysis, showing the nonadditivevicphenotype of \u0394atg1\u0394pmk1double mutant and the resemblance of the \u0394atg1\u0394pmk1double mutant to the \u0394pmk1single mutant, suggests that Atg1 acts upstream of the Pmk1 signaling pathway. Furthermore, suppressioin of the toxicity induced by Pck2 overexpression byatg1deletion suggests that Atg1 acts downstream of Pck2. MAPK signaling cascades consist of a core module of three tiers of protein kinases MAPK, MAPKK, and MAP3K, and often an additional upstream MAP4K. It would be intriguing if Atg1 serves as an additional layer of kinase mediating MAPK signaling activation. Future studies will elucidate the mechanism and the functional significance of the genetic interaction between autophagy and MAPK signaling revealed by our yeast genetic screen. Given the highly conserved nature of autophagy and MAPK signaling in the fate of cell death and proliferation, this study will provide valuable information to understand human diseases associated with aberrant regulation of MAPK signaling and autophagy.In summary, our genetic screen revealed a functional interaction between autophagy-related genes and Pmk1 MAPK signaling in fission yeast. Several studies report functional crosstalk between MAPK signaling and autophagy, including the role of MAPK ERK in the maturation of autophagosomes . FK506 was provided by Fujisawa Pharmaceutical Co. . Standard genetic and recombinant-DNA methods  were used except where noted.vicmutant screeninget al. was analyzed on the YPD plates with or without FK506 and 0.09 M MgCl2by spotting growth assay.The growth of 89 viable kinase knockout strains generated by BimboSpotting growth assay660/ml, from which five 10-fold serial dilutions were prepared and then spotted onto the indicated plates. Plates were incubated at 27\u02daC for 3 to 7 days.Yeast cells were cultured in 20 ml of liquid media at 27\u02daC till mid-log phase and diluted to 0.6 OD"}
+{"text": "The transformation of Cs4PbBr6 nanocrystals into CsPbBr3 takes place in two steps, the first step being the surface modification of the Cs4PbBr6 nanocrystals with Zn2+ ions and the second step being extraction of CsBr by the Zn2+ ions resulting in the formation of composite Cs4PbBr6/CsPbBr3 nanocrystals. The transformed composite nanocrystals were found to have a PL QY exceeding 90% and the shape of the nanocrystals also changed from hexagonal to cubic shaped. Owing to the highly ionic nature of the nanocrystals, complete anion exchange could be also realized using ZnI2 salt. In the case of the iodide post-treated samples, nanorods were obtained which exhibited bright red photoluminescence. Photodetectors based on the ZnI2 treated Cs4PbBr6 NCs were fabricated, and the photodetectors exhibited a high on/off ratio with a fast response time. The excellent optoelectronic properties make this treatment versatile for a wide range of functional optoelectronic devices like light emitting diodes and photovoltaic devices.Herein we demonstrate a facile approach for the synthesis of all inorganic cesium lead halide perovskite nanocrystal composites CsPbX 3  with high quantum yield by post-synthetic modulation of zero dimensional Cs4PbBr6 nanocrystals with ZnX2 salts.Herein we demonstrate a facile approach for the synthesis of all inorganic cesium lead halide perovskite nanocrystal composites CsPbX If all the corners of the lead hexahalide octahedra are shared, then they are electronically coupled in all three dimensions and referred to as 3D perovskites. If all the corners of the lead hexahalide octahedra are not shared, they are referred to as 0D perovskites.25Lead halide perovskite NCs  of the NCs.26\u201333 Different groups have recently synthesized 0D colloidal Cs4PbBr6 NCs and it has been found that these nanocrystals don't exhibit any photoluminescence.25 However, the non-luminescent 0D Cs4PbBr6 NCs can be converted to 3D CsPbBr3 NCs using additives like water,34 PbBr2\u200935 ligands,36 and Prussian blue.37 The non-luminescent nature of Cs4PbBr6 at room temperature is attributed to the origin of structural and point defects.38 Nikl et al. observed the true emission of Cs4PbBr6 at low temperatures (4 K) and the true emission is centered at 375 nm. In the case of thin films at room temperature, Cs4PbBr6 is mixed with traces of the CsPbBr3 phase, which in turn results in the emission at 545 nm. However in the case of nanocrystals, no emission has been observed so far for the pure Cs4PbBr6 nanocrystals.It is extensively recognized that the excellent optical properties in lead halide based perovskite materials come from the high defect tolerance.3 nanocrystals by improving the surface capping. Herein we demonstrate ZnX2 mediated post-synthetic transformation of Cs4PbBr6 nanocrystals (NCs) into CsPbX3 nanostructures through an intermediate composite Cs4PbX6/CsPbX3. We find that this procedure results in a highly reproducible increase of the PL QY of up to 90% for the ZnX2 treatment. The transformation takes place through the surface passivation of Cs4PbBr6 nanocrystals with ZnX2 followed by anion exchange (in the case of ZnI2 and ZnCl2) resulting in the formation of an intermediate Cs4PbX6@CsPbX3 passivated by ZnX2 followed by the extraction of CsBr from the composite nanostructures. Interestingly, as soon the ZnX2 is added to the nanocrystals, excitonic absorption resulting from the Cs4PbBr6 particles passivated with ZnX2 is observed. This is the first time that the true emission of the Cs4PbX6 crystal structure in the nanocrystalline phase was observed at room temperature.Recently it has been shown that addition of appropriate metal halide salts significantly enhances the photoluminescence intensity of CsPbBr4PbBr6 nanocrystals were synthesized according to the hot injection strategy as described in the Experimental section. The Cs4PbBr6 nanocrystal dispersion in hexane is completely transparent and as shown in the TEM images in 4PbBr6 nanocrystals consist of hexagons of around 30 nm in size. The powder diffraction pattern indicated that the nanocrystals crystallized in the rhombohedral Cs4PbBr6 phase in the space group R3c.The Cs4PbBr6 nanocrystals (NCs) were post treated with a ZnX2/hexane solution and used for further characterization. Upon addition of ZnX2  solution, clear color change was observed  in comparison to that in the ZnBr2 treatment was observed. Along with the red shifted absorption, a sharp peak at 550 nm can be seen.The absorption spectra of the ZnX4PbBr6 NCs was taken in a cuvette, different concentrations of ZnBr2 were added to the sample and the absorption spectrum of the ZnBr2 treated NCs was measured. 2/hexane solution added to the cuvette containing NCs. A sharp absorption peak at 425 nm along with a long tail extending up to 510 nm was observed. The absorption intensity of the tail increased gradually and a sharp band edge at 510 nm, which is a characteristic peak of CsPbBr3, was observed. The peak at 425 nm is blue shifted to 420 nm and is masked (though visible) by the strong absorption of CsPbBr3 in this region. Fig. S13 nanocrystals synthesized and the ZnBr2 treated Cs4PbBr6 NCs. A similar concentration dependent study was carried out in the case of ZnI2 treated Cs4PbBr6 NCs. 2 treated NCs with different amounts of ZnI2 added. A sharp absorption at 540 nm is evident even with high amounts of added ZnI2. ZnBr2 treated NC samples were found to be highly stable even after 2 months of maintaining under 70% RH conditions. Fig. S22 treated NC sample and the as-prepared CsPbBr3 NCs after storing under 70% RH conditions.To confirm if the peak observed at 420 nm arises from the sample, an aliquot of the Cs2 treated NCs. 2 treated NCs. No strong PL emission was observed, which is consistent with the previously observed decay of excitonic PL above temperatures of 100 K. Very weak PL emission centered at 515 nm was observed which can be attributed to the CsPbBr3 phase embedded in the PL-inactive Cs4PbBr6 crystalline matrix. With the ZnBr2 post-treated NC solution, strong PL was observed at 518 nm. From the literature, it is known that the excitonic absorption peak of pure CsPbBr3 NCs is located at around 510 nm. So, it is reasonable to speculate that the strong excitonic absorption peak and strong PL peak at 518 nm in 3 NCs, indicating that a transformation might have taken place from Cs4PbBr6 to CsPbBr3.To corroborate the observed absorption behavior, photoluminescence (PL) studies were also carried out on the parent NCs and ZnX2 concentration dependent emission studies. With the addition of ZnBr2, two emission peaks were observed, one at 430 nm and the other at 510 nm, similar to the behavior observed in the absorption spectrum. The emission at 430 nm could be attributed to the 425 nm peak observed in the absorption spectrum and the peak at 510 nm could be attributed to the band edge emission corresponding to the absorption edge shown in We also carried out ZnBr3/Cs4PbX6 composites were formed after the post-treatment with ZnX2. In the case of ZnBr2, the excitonic CsPbBr3 absorption was observed at 510 nm and the absorption peak observed at a lower wavelength could be attributed to the Cs4PbBr6 phase. It has been previously reported that defect states created due to the surface halide vacancies could be responsible for quenching the true emission in Cs4PbBr6 and were only observed at low temperatures (4 K). When the Zn2+ salts were added to the Cs4PbBr6 NCs, the surface halide defect states could be passivated and the true emission from the surface passivated/modified Cs4PbBr6 nanocrystals could be observed at 430 nm. The true emission of Cs4PbBr6 is observed for the first time at room temperature when Zn2+ salts are used to passivate the surface defects; however, the Zn2+ salts also triggered the transformation of 0D Cs4PbBr6 NCs into 3D CsPbBr3 NCs. So, the intensity of the true emission of Cs4PbBr6 NCs gradually decreased as the concentration of the zinc salt is increased. When the concentration of the zinc salt was kept constant at 50 \u03bcL of ZnBr2, the intensity gradually decreased indicating that the whole process takes place through a two-step mechanism. Similar studies carried out using other salts like Cu2+ and Mn2+ didn't result in the enhancement of the PL QY. In the case of Mn2+ a slight increase was observed but Cu2+ didn't show any improvement in the PL QY. Fig. S32 and MnBr2 treated Cs4PbBr6 NCs.Together with the results obtained from the XRD studies and UV-vis and PL spectra, we speculate that CsPbX4PbBr6 NCs show hexagonal shaped particles of around 30\u201335 nm in size similar to the particles reported previously. 4PbBr6 NCs and the ZnBr2 treated samples. When the samples were treated with 50 \u03bcL of ZnBr2, initially hexagonal shaped particles were converted mostly to cubic shaped particles as shown in 3 NCs  plane corresponds to the presence of Cs4PbBr6, indicating the formation of a composite of CsPbBr3/Cs4PbBr6. A plausible two step formation mechanism can be inferred from the observed TEM images. As the Cs4PbBr6 nanocrystals were treated with the zinc halide salts, in the first step Zn2+ ions could passivate the surface of the nanocrystals and then result in the extraction of excess Cs+ ions, and an increase in the dimensionality from 0D to 3D is observed. In the second step we believe that an inside out diffusion of Cs+ ions takes place resulting in the formation of CsPbBr3/Cs4PbBr6 composites.TEM images of the as-synthesized Css Fig. S4. The HR-s Fig. S4 shows a 4PbBr6 NCs were treated with 40 \u03bcL of ZnI2 solution, the shape of the particles changed from hexagonal to rod shaped. As seen in the TEM images shown in 2 solution to 100 \u03bcL the length of the rods increased to nearly 250 nm and the width increased to 20\u201330 nm.When the Cs7/2) and 143 eV (4f5/2) with a spin\u2013orbit splitting of nearly 5 eV, which corresponds to Pb in the 2+ oxidation state. Quantitative XPS analysis indicates that Cs\u2009:\u2009Pb\u2009:\u2009Br = 1.5\u2009:\u20091\u2009:\u20094.2 for untreated Cs4PbBr6 NCs, which is Pb rich in composition. However, for ZnBr2-Cs4PbBr6 NCs, the composition becomes anion-rich (Cs\u2009:\u2009Pb\u2009:\u2009Br = 0.6\u2009:\u20091\u2009:\u20095.5), which was also repeatedly observed  is much higher compared to that in the bromine case  measurements have been used to assess the surface modification of the NCs and to understand the mechanism of PL enhancement. After measurements, all core level spectra were calibrated using the C 1s peak at 285.0 eV. The high-resolution XPS spectrum of untreated NCs for Pb 4f yields 2 peaks as shown in Fig. S5.d Fig. S6. A smalle Fig. S7.2 treated Cs4PbBr6 NCs, we have explored their potential application in optoelectronic devices. To this end, we have fabricated photodetectors by dropcasting the ZnI2 treated NCs in hexane between two fluorine doped tin oxide (FTO) electrodes. 2 treated nanocrystals, the currents obtained were lower compared to those using the ZnI2 treated samples. Fig. S82 treated NCs in the same configuration. This indicates that the performance of the photodetectors could be improved if the length of the nanorods could be made longer than the width of the channel used and also by removing the surface capping ligands by appropriate treatments.Owing to the one-dimensional nature of the nanorods in the case of ZnI4PbBr6 into highly luminescent CsPbBr3/Cs4PbBr6 composites using ZnBr2 treatment. The transformation with ZnBr2 treatment proceeds through a two-step process, where in the first step, zinc doped/modified Cs4PbBr6 NCs are formed which then results in the conversion to CsPbBr3/Cs4PbBr6 nanocomposites. The composite of CsPbBr3/Cs4PbBr6 NCs is found to be very highly luminescent with a PL QY of 90%. In the case of ZnI2 treatment, the post synthetic treatment also resulted in morphological transformation from the hexagonal shaped nanocrystals to nanorods. The length of the rods could be varied depending on the concentration of the nanocrystals and the zinc iodide salt added. In both the iodide and bromide case an intermediate is formed upon treatment with the zinc salt. The intermediate was observed in the time/concentration dependent UV-vis and PL studies, and from the X-ray diffraction analysis, we presume that it is the 0D structure modified/doped by the zinc cations. Photodetectors based on the ZnI2 treated Cs4PbBr6 NCs were fabricated, which exhibit a high on/off ratio with a fast response time. The excellent optoelectronic properties make this treatment versatile for a wide range of functional optoelectronic devices like light emitting diodes and photovoltaic devices.In conclusion, we have developed a simple and convenient post synthetic strategy for the transformation of 0D Cs2CO3, 99.9%), lead(ii) bromide (98%), zinc(ii) iodide (98%), zinc(ii) chloride (99%), oleylamine , oleic acid (90%), and 1-octadecene (90%) were purchased from Sigma Aldrich. Zinc(ii) bromide (98%) was purchased from Alfa Aesar.Cesium carbonate , oleic acid (1 mL), and octadecene (16 mL) were added to a three-neck flat bottom flask and kept under vacuum for 30 min at 120 \u00b0C. After 30 minutes the temperature was increased to 150 \u00b0C and the system was kept under a N2 atmosphere until a clear solution of cesium oleate was obtained.Cesium carbonate , oleylamine (1 mL), oleic acid (1 mL) and lead bromide  were loaded into a three-neck round bottom flask and degassed for 30 min at 120 \u00b0C. The flask was then filled with N2 and oleylamine (1.5 mL) and oleic acid (1.5 mL) were added. After complete solubilisation of PbBr2, the temperature was increased to 165 \u00b0C and finally the as-prepared Cs-oleate (1.2 mL) was injected. The reaction was immediately quenched in an ice-water bath.Lead bromide (207 mg) and octadecene (15 mL) were loaded into a three-neck round bottom flask and degassed for 1 hour at 120 \u00b0C. The flask was then filled with NThe crude solution of nanoparticles was first centrifuged at 6000 rpm for 10 min. After that, the supernatant was discarded and the precipitate was redispersed in hexane solution. Then again, the large particles present in the solution were removed by centrifugation at 6000 rpm for 10 min. The supernatant was discarded and a colloidal solution of nanoparticles was obtained in hexane.\u22121) were treated with the pre-made zinc halide solution. Zinc halide solutions were prepared by dissolving zinc halides  (0.05 M) in the mixture of hexane (2 mL) and oleylamine (40 \u03bcL) at room temperature. Then the required zinc halide solution was added to 1 mL of the Cs4PbBr6 NCs in hexane solution (8 mg mL\u22121) under constant stirring. Excess zinc halide salts are removed by centrifugation and the NCs can be stored in organic solvents like hexane, toluene, etc. for a few months.The NCs dispersed in hexane (\u223c8 mg mL\u03bb = 1.541). PL spectra were collected by using a Horiba FluoroMax spectrometer with 400 nm excitation wavelength, while UV-vis absorption measurement was performed using a UV-vis-NIR spectrometer (PerkinElmer LAMBDA 950). The room-temperature PLQY was calculated according to the following equation38High resolution transmission electron microscope images were obtained using a Tecnai G2 F30 300 kV transmission electron microscope. X-ray diffraction spectra were obtained by using a Rigaku Japan SmartLab X-ray diffractometer  were chosen to be the standards for green and red spectral regions and the standard solutions should be freshly prepared. Attention was paid to keeping the optical densities of the sample and standard below 0.1 at the excitation wavelength.In this equation, QYLaser patterned FTO coated glass with an etched area was used as the photodetector. Before deposition, the laser patterned FTO was cleaned thoroughly by using a soap solution, DI water, acetone, and ethanol for 10 minutes, respectively, by ultra-sonication. Perovskite nanocrystals in hexane were drop cast on the etched area of the FTO coated glass by masking the unetched area with scotch tape. The drop cast substrate was then subjected to vacuum drying. Then the photoresponse was measured under a 617 nm LED light.There are no conflicts to declare.NA-001-C9NA00244H-s001"}
+{"text": "Atomically resolvedvisualization of individual N2 dissociation events elucidatesthe fundamental reactive dynamics of the N2/Ru(0001) systemby providing a detailed understanding of the on-surface dissociationdynamics: the distance and angle between nitrogen atoms from the samedissociated N2 molecule, site specificity and coordinationof binding on terrace sites, and the local evolution of surroundingnanoscopic areas. These properties are precisely measured over a rangeof impinging N2 kinetic energies and angles, revealingpreviously unattainable information about the energy dissipation channelsthat govern the reactivity of the system. The experimental resultspresented in this paper provide insight into the fundamental N2 dissociation mechanism that, in conjunction with ongoingtheoretical modeling, will help determine the role of dynamical processessuch as energy transfer to surface phonons and nonadiabatic excitationof electron\u2013hole pairs (ehps). These results will not onlyhelp uncover the underlying chemistry and physics that give rise tothe unique behavior of this activated dissociative chemisorption systembut also represent an exciting approach to studying reaction dynamicsby pairing the angstrom-level spatiotemporal resolution of an in situ STM with nonequilibrium fluxes of reactive gasesgenerated in a supersonic molecular beam to access highly activatedchemical dynamics and observe the results of individual reaction events.This paper examines the reactive surface dynamics ofenergy- andangle-selected N A fundamentalunderstanding of N2 dissociation onto ruthenium surfacesis therefore of great fundamental, technological, and economic importance.12The interaction of gaseous species onruthenium surfaces has beenstudied extensively due to the importance of ruthenium as a catalystfor a wide variety of applications.2 dissociation onto Ru(0001) is a prototypicalactivated dissociativechemisorption process, and understanding the mechanistic featuresof this would have a considerable impact on the field of heterogeneouscatalysis. Compared to other activated dissociation benchmark systems\u2014namelyH2 on Cu15 or CH4 on transition metals\u2014thathave dissociation probabilities (s0) thatapproach unity at normal incident kinetic energies (EN) much greater than the potential barrier(V*),18 N2 exhibits substantially different adsorptionbehavior, demonstrating s0 \u226a 1at EN \u226b V*.21 Nonadiabatic coupling/tunneling mechanisms28 and energy transfer to surface phonons,30 along with theoretical formulations using only adiabatic treatments,33 have all been proposed to describe the unusual dissociation behaviorof N2 on Ru(0001) with increasing number of degrees offreedom explicitly treated in forming the potential energy surface\u2014someformulations treating several degrees25 and some formulationstreating all six degrees of freedom for the nitrogen molecule.36 Neural networkshave been used to treat all degrees of freedom explicitly using ahigh-dimensional fit of molecule\u2013surface interactions, allowingfor less computationally expensive ab initio molecular dynamics simulationsfor a system in which nonreactive scattering dominates (s0 \u226a 1), making reactivity more arduous to sample.30 Neural networks have also beenused to sample the free energy surface of nitrogen dissociation onRu(0001) showing vibrational entropy of surface atoms add appreciablyto the reaction barrier.37 Further experimentalwork is required to answer questions that remain about the fundamentalreactive surface dynamics of this important system.In addition to its relevance to ammoniasynthesis, N2 on a clean Ru(0001) surface held at room temperatureand 262 K. Atomically resolved visualization of individual dissociationevents at different impinging energies and angles provide a detailedunderstanding of the spacing and angle of nitrogen atoms from thesame dissociated N2 molecule, site specificity of terracebinding, and local, nanoscopic information about the reactive evolutionof the Ru(0001) surface in the low-coverage regime. By monitoringthe nanoscopic evolution of the Ru(0001) surface during exposure toenergy- and angle-selected N2, this work elucidates newinformation about the mechanisms of energy dissipation into the surfaceof this important gas\u2013surface interface and more generallyshowcases how nonequilibrium fluxes of reactant molecules from a supersonicmolecular beam paired with an in-line insitu STM can capture the fundamental dynamics of individualreaction events. In conjunction with ongoing and future theoreticalstudies, observation of individual dissociation events will revealinsight into how energy is dissipated into the surface and may divulgethe role surface phonons and nonadiabatic coupling to electron\u2013holepairs (ehps) play in transferring energy during and immediately afterthe activated dissociation event.In thispaper, we present results detailing the reactive dynamicsof N2 on a clean Ru(0001)surface under ambient conditions is very low (s0 \u2248 10\u201312)40 due to a highactivation barrier that occurs late in the dissociation process andrequires significant stretching of the N2 bond.41 Thermal sticking occurs exclusively at crystal steps due to a 1.5eV difference between the activation barrier at terrace (\u223c1.9eV) and step (\u223c0.4 eV) sites,44 illustrating the role step edges and defects can play in adsorptionon single-crystal model systems.46 Molecularbeam studies47 demonstrate that high impingingkinetic energies of N2 activate dissociation on terracesites, with no dependence on surface temperature. The dissociationprobability increases slowly with increasing N2 kineticenergy and plateaus at s0 \u2248 10\u20132 for kinetic energies much higher than the activationbarrier;21 while molecular beams of N2 seeded inHe and H2 carrier gases demonstrate how vibrational excitationof the impinging N2 increases the dissociation probabilitymarkedly,19 nonthermal plasmas have beenused to populate vibrational and electronic excited states, therebyincreasing reactivity of N2 with Ru-based catalysts.50 Additionally, measured isotope effects for N2 dissociation24 and hydrogenation51 of atomic nitrogen make N/Ru(0001) a suitable system for the studyof nonadiabatic tunneling mechanisms.The dissociation probabilityof N53 in conjunction with theoreticalcalculations,43 have helped elucidate the spatial properties, adsorbate\u2013adsorbateinteractions, and binding structures of adsorbed nitrogen atoms withatomic resolution. At low temperatures, molecular N2 adsorbsto Ru(0001) binding perpendicularly at on-top sites, and molecularlybound N2 desorbs from Ru(0001) at temperatures greaterthan 128 K54\u2014ensuring that adsorbedmolecular N2 will not be found in STM images within thisstudy. STM visualization depicts adsorbed nitrogen atoms as triangulardepressions 5 \u00c5 wide in topographical scans.53 N adsorbates (Nad) occupy the hcp 3-fold hollow siteon terraces52 and bridge site on steps43 of the Ru(0001) surface. Interactions betweenNad are repulsive at nearest-neighbor and second-nearest-neighborsites and slightly attractive at third-nearest-neighbor sites, resultingin an approximate pair potential of hard spheres that blocks the first-and second-nearest-neighbor sites in the low coverage regime at roomtemperature.52 Experimental53 and theoretical1 barriersto diffusion of 0.9 and 1.1 eV, respectively, have been observed forNad on the Ru(0001) surface. This allows adsorbate movementto be frozen out kinetically at moderate temperatures after the dissociationevent occurs.STM studies,24 and inelastic scattering47 of N2 onRu(0001); however, the techniques used heretofore are not directlysuited to investigate energy dissipation during/after dissociativeadsorption. Inelastic scattering provides information about the translationalenergy and quantum vibrational and rotational states of reflectedmolecules, but inelastic scattering notably looks at molecules thathave been scattered from the surface and does not directly probe thereaction dynamics of molecules that adsorb to the surface. Temperatureprogrammed desorption (TPD) provides initial sticking coefficientsfor various N2 beam energies, but each value only representsthe ensemble average of a Boolean value\u2014whether the moleculeadsorbs or not\u2014convoluting a multistep process into one scalar value. Instead of measuring ensemble values, our studycaptures the results of individual dissociation events of a processthat demonstrates an atypical sticking coefficient (s0 \u226a 1 at EN \u226b V*).Molecular beam studies have investigated adsorption2 impinging on Ru(0001) terrace sites.STM images from this study will serve as a benchmark for future computationalmodels to provide insight into how energy is transferred in the highlyactivated dissociation of N2 on Ru(0001). This work providesa deeper understanding of N2 dissociation on rutheniumand in collaboration with theoretical exploration can contribute amore fundamental understanding of activated dissociative adsorptionsystems.The results in this paperrepresent the visualization of individualdissociation events resulting from nonequilibrium fluxes of energy-and angle-selected N57 the instrument is composed of a triply differentiallypumped beamline, a surface characterization/preparation chamber thatcontains Auger electron spectroscopy (AES) and low-energy electrondiffraction (LEED) capabilities, and a scanning probe microscope (SPM)chamber that holds the variable temperature SPM based on the ultrastabledesign of Shuheng Pan, built in collaboration with RHK. The custom-builtPAN STM allows the Ru(0001) surface to be exposed to the supersonicmolecular beam at variable polar angles of incidence with the abilityto move the STM tip micrometers away from the area of interest toavoid blocking the molecular beam and then return the tip after thesurface has been exposed to the molecular beam to reveal the morphologyof the same nanoscopic area both before and after exposure to nonequilibriumfluxes of reactive gases.The results reported in this paper were acquiredutilizing a newUHV instrument that contains both a supersonic molecular beam andSTM/AFM techniques. As reported in previous publications,2/97% He gas mixture through a 30 \u03bcm molybdenumpinhole at pressures from 20 to 100 psi and nozzle temperatures rangingfrom 300 to 1150 K (\u00b15%). The translational kinetic energy ofthe molecular beam at each nozzle temperature was measured using time-of-flight(TOF), and values of 0.8 \u00b1 0.3, 1.1 \u00b1 0.4, and 1.3 \u00b10.6 eV were found for nozzle temperatures of 730, 1000, and 1150 K,respectively. The uncertainty values in these energies represent theFWHM of each energy distribution. The molecular beam flux at the crystalfor all beam conditions was on the order of 1013 N2 molecules cm\u20132 s\u20131. Thekinetic energy values reported represent only translational kineticenergy, and the role of vibrational excitation on spatial distributionsis not directly quantified in this study. A Boltzmann distributionwith nozzle temperatures of 730, 1000, and 1150 K indicates, assuming,with no relaxation during expansion, populations of vibrationallyexcited impinging N2 molecules are 0.97%, 3.4%, and 5.5%,respectively.Supersonic molecular beams were generatedby the expansion of a3% N2 molecular beam exposures ontothe Ru(0001) surfacewere performed with the sample in the SPM chamber, which correspondsto a 4 mm diameter beam spot on the crystal. The sample was eitherheld at room temperature or cryocooled with liquid nitrogen duringimaging/exposures; the temperature of the sample was monitored usinga cryostat thermocouple attached to the STM assembly. The surfacetemperature was held constant between exposure and imaging for eachexperiment. The surface plane could be oriented to achieve an incidentpolar angle from 0 to 45\u00b0 during exposures to the N2 molecular beam.All N2 molecular beam and not as a result of trace thermalizedN2 reflected from the tip/chamber, which was confirmedby the fact that only areas of the surface with direct line of sightto the N2 beam were reacted.Reactive evolution of the surface only arosedue to exposure tothe N\u201310 Torr base pressure)by multiple sputter/anneal cycles, similar to those reported previously.59 The Ru(0001) surface was sputtered at room temperature using 0.5keV Ar+ ions generated by a PHI 04-150 ion gun resultingin a current of 0.1\u20130.5 \u03bcA cm\u20132 onthe sample; the sample was flash annealed by electron beam bombardmentto 1500 K for 10 s after sputtering cycles. The temperature was monitoredusing a Mikron infrared pyrometer (\u03b5 = 0.35) during annealing.Hundreds of cleaning cycles were necessary to produce a clean andordered Ru(0001) surface. An Omicron NGL 10 SPECTALEED with both LEEDand AES capabilities was used to determine if the Ru(0001) surfacewas ordered and free of impurities.The Ru(0001) crystalsused in all experiments were cleaned in the characterization/preparationchamber  at slightly lower temperatures(<1400 K) were used and produced a partially clean surface. Localareas of clean and ordered Ru(0001) surface were produced from theseprocedures, but subsequent STM imaging revealed the presence of Moir\u00e9patterns indicative of monolayer and bilayer graphene formation.60 Subsequent higher energy (3 keV) Ar+ sputtering cycles followed by shorter (5 s) and higher temperature(1500 K) annealing cycles produced a clean, ordered Ru(0001) surface,characterized by AES, LEED, and STM visualization in 0.8Ir0.2 tips.Initially, lower energy(0.5 keV) Ar2. As shown in 53 the 3-fold symmetry of the adsorbate suggests binding at eitherthe fcc or hcp sites, with previous experimental and theoretical studiesindicating binding at hcp sites is most favorable.61Once a clean Ru(0001) surfacewas achieved and characterized, thesurface was exposed to N12 Contraryto a previous STM study,53 these \u201cdiffusion\u201dexperiments were performed multiple times on the room temperatureRu(0001) surface, and no observable diffusion was observed over thecourse of hours. The results can be explained most likely by the useof a tunneling current in this study that is <100 times that usedpreviously,53 thereby minimizing tip interactionswith the adsorbates that can influence their movement upon the surface.To further elaborate that room temperature was sufficient to freezediffusion and rule out small fluctuations in room temperature generatingmarkedly different adsorbate distributions on the surface, the Ru(0001)surface was held at 262 K to probe the effect of temperature changeson adsorbate distributions. As expected, the lower surface temperatureshowed no effects on spatial distributions of adsorbates. Annealingabove 1500 K provides an atomically clean surface for N2 molecular beam exposures, but coadsorbed species such as oxygenwere observed after several hours of STM imaging from residual backgrounddosing. Oad was not observed to promote Nad diffusionat this low coverage limit and is thus not expected to impact thespatial distributions of Nad measured in this study. Notably,the lack of diffusion enables the N2 dissociation eventto be directly investigated at room temperature by measuring the distancebetween and location of the resulting nitrogen atom adsorbates. STMimages of atomic nitrogen adsorbate pairs thus provide direct insightinto the energy transfer of the impinging N2 with the surfaceand the liberated energy during the dissociation event and do not show the effects of random thermal diffusion.STM imaging of the same nanoscopic area revealedno observablediffusion of the nitrogen atom adsorbates 3.12 Cont2 impinging 45\u00b0 to the surface normal, where 1 langmuircorresponds to one impinging N2 for every one Ru surfaceatom. In the low coverage regime, the reactivity of 1.1 eV N2 impinging normal to the Ru(0001) surface does not significantlydiffer from the reactivity of the same energy N2 impinging45\u00b0 with respect to the surface plane, suggesting that reactivityscales with total N2 energy, corroborating results froma previous study that showed molecular N2 adsorption onRu(0001) is only weakly affected by incident angle when N2 translational kinetic energies are >0.4 eV.62 N2 molecules impinging normal and 45\u00b0 tothe surfacewith 1.1 eV translational kinetic energy were measured to have anapproximately 1 in 10000 chance of sticking, which is in qualitativeagreement with previous molecular beam studies.47Shown in 2 kinetic energy and angleaffect these values, is crucial to elucidating how the N2 interacts with the surface and what energy transfer mechanisms governthe reaction. Determination of the intrapair distances and relativeangles of nitrogen pairs was accomplished by reacting at extremelylow coverages (<0.08%) to reduce the total number of nitrogen atompairs such that the identity of the nitrogen atom pairs could be uniquelyassigned. Representative STM topography of the low coverage exposuresis shown in 2 impinging normal to the Ru(0001) surfacewhich clearly highlights individual nitrogen pairs formedfrom singular dissociation events. Misidentification of adsorbatespairs is avoided, as clean surfaces were always observed before reactingwith N2 fluxes (as demonstrated in Uncovering the pair distance(s) and the relativeangle(s) betweentwo adsorbates and the azimuthal direction of the incident molecularbeam, and whether the impinging N2 on Ru(0001) wereperformed with varied translational kinetic energies and impingingangles. Polar coordinates  are used to describethe relative position of two nitrogen adsorbates from the same dissociatedN2 molecule on the Ru(0001) lattice, where \u03c6 is theazimuthal angle between two adsorbates relative to the incident azimuthaldirection of the molecular beam. 2 translational kinetic energies and impinging anglesused in this study.Extremely low coverage exposures of NN = 318.The uncertainty of pair distances is reported as standard deviationthroughout this paper. Intrapair spacing did not change significantlyupon increasing the translational kinetic energy of N2 impingingnormal to the surface from 0.85 eV  to 1.3 eV , indicatingthat there is not a strong coupling of impinging translational energyto intrapair distance for the energies explored herein. The distancesmeasured across all molecular beam energies are extremely large consideringthat values are over 20 times larger than the lattice constant ofruthenium.A scatter plot 6A demons2 on Pt(111),63 Al(111),64 Cu(110),65 Rh(110),66 TiO2(110),67 and RuO2(110)68 leaves oxygenatoms from the same dissociated O2 molecule within severallattice spacings on the surface, i.e., <1 nm apart. An early studyof O2 on Al(111)69 indicateddissociative products may be spaced >80 \u00c5 apart, developingthenotion of hot adatom motion, and more STM work followed showing \u201clowtransient mobility\u201d after dissociation.64 Plasmon-induced dissociation of O2 on Ag(110)resulted in oxygen atoms within several lattice constants as well.70 The large intrapair distances observed for N2/Ru(0001) are not without precedent. Notable systems for far-rangedtransient motion following dissociative chemisorption include O2 on Ag(001),71 in which intrapairdistances were found to be either 2 or 4 nm, and Cl2 onTiO2(110),72 which demonstratesan average intrapair distance of 2.6 nm. A \u201ccannonball mechanism\u201dwas proposed for Cl2/TiO2 in which one atomis ballistically propelled from the surface, thereby overcoming surfacecorrugation to explain the large distances observed; this ballisticmotion that avoids corrugation in the potential energy surface couldsimilarly explain the large intrapair distances observed for N2/Ru(0001) despite large barriers to diffusion and providesdirection for future computational work. However, hundreds of identifiedNad pairs identified in this study and a lack of lone Natoms discourage promoting an abstractive mechanism where one atomdesorbs entirely from the surface. STM tip-induced dissociation ofsmall molecules on single crystalline surfaces has also yielded productsat nonadjacent binding sites with products from CH2I2/Cu(110),73m-iodopyridine/Cu(110),74 and O2/Ag(110)75 separated by several lattice constants and F and CF2 up to 5.3 nm apart after CF3 dissociates on Cu(110).76 These previously studied model systems demonstratethat relatively large intrapair distances do occur for specific systems.Comparisons to other dissociative chemisorption systemshighlightthe significance of these results. Background dosing of Oab initio density functionaltheory (DFT) calculations by Prof. Hua Guo\u2019s research groupare getting underway. Mechanistically, how these large intrapair distancesare achieved despite a large barrier (0.9\u20131.1 eV)53 to diffusion poses interesting questions for future computationalmodeling. Does a purely adiabatic picture describe the N2/Ru(0001) system in which a nonthermalized hot adatom diffuses onthe surface until phonons dissipate energy from the adsorbate intothe bulk? Or do the dynamics sample excited neutral and ionic potentialenergy surfaces leading to changed overall dynamics? Beyond identifyingthe average distance between adsorbates, STM images provide a richspatial understanding of the N2/Ru(0001) system.Note that such large distances observed provide important insightinto elucidating the operative energy dissipation channels for a givensystem. Adiabatic processes will contribute dissipation primarilyvia phonon channels, while nonadiabatic processes on excited statesand including ehps when appropriate can account for interesting dynamics.Preliminary results from STM images showed that nitrogen adsorbate pairs were not observedto preferentially align themselves in any direction on the Ru(0001)lattice. We observe that points are isotopically distributed between\u221290\u00b0 and 90\u00b0 in the scatter plot 6 A, and 2 angles and energies found in 2 molecular beam with respect to the Ru(0001)lattice\u2014confirmed by STM imaging of the nitrogen adsorbates77\u2014critical to determining correlations between the incidentangle of N2 and the resulting nitrogen pair spacing, relativeangle, and binding sites.Analysisthus far has focused on the aggregated results of allimpinging N2 impinging 90\u00b0 and 45\u00b0 to the Ru(0001)surface isshown in The distribution of adatom pairs following exposureto 1.3 eV Nnot an obvious memory function in which the relativeangle (\u03c6) between nitrogen adsorbates is affected by the non-normalcomponent of momentum of incident N2 impinging at 45\u00b0to the surface. Similarly, pairs that were more aligned with the azimuthalincident beam direction  in The scatter plots shown in 2 with 1.3 eV total translationalkinetic energy impinging at both 45\u00b0 and 90\u00b0 to the surface,and average intrapair distances resulting from 1.3 eV impinging normal and impinging at 45\u00b0 are similar as well.This result indicates that under these conditions, momentum alongthe azimuthal direction of the impinging N2 is not stronglycoupled into motion of one Nad along the Ru(0001) surfacefollowing dissociation. Results summarized in 2 impinging at non-normal angles does not resultin an obvious memory function for adsorbate spacings or angular distributions.These results are made more compelling by the fact that the angledN2 exposure  was performed below roomtemperature (262 K) so that any potential random isotropic motionfollowing dissociation would be frozen out.Analogously, 2 impinging 45\u00b0 to the surfacenormal did not demonstrate a markedly different trend in adsorbatedistributions on the surface from other room temperature runs surface with 1.1 eV N2 impinging at 45\u00b0 did not producemarkedly different results from 1.1 eV impinging at 90\u00b0 to thesurface\u2014in either the spacing or the relative angle of thepaired nitrogen atoms.  surfaceto 262 K duringexposure to 1.3 eV Nure runs 8 and 9, 2 activated dissociative adsorption on Ru(0001).STM visualization of individual dissociation events identifies thesite and location of the resulting nitrogen adsorbates with angstrom-levelprecision. The spacing of the adsorbates correlates to the energytransfer mechanisms involved in the dissociation process and willhelp uncover the importance of nonadiabatic excitation of ehps inthese reactive events. Paired with theoretical insight from ongoingcollaborations, these previously inaccessible experimental resultswill help to answer many of the unsolved questions about this unusualactivated dissociative adsorption system.The results of this paper represent deep insight into the fundamentaldynamics of N2 from a supersonic molecular beam impinging on a Ru(0001)single crystal. The insights gathered herein are only possible dueto the prescient pairing of a supersonic molecular beam of highlyenergetic molecules and the angstrom-level visualization of an in situ in-line STM to provide a rich spatial understandingof this highly activated dissociative adsorption system. Our workuniquely characterizes single adsorption events to capture the reactivedynamics of this industrially relevant process. We present distributionsof nitrogen adsorbate pairs resulting from highly energetic N2 impinging onto Ru(0001), resulting in an average spacingof 6.6 \u00b1 2.8 nm between previously bonded nitrogen atoms. Thesedistances are more than 20 times the lattice constant of Ru(0001)and pose a novel opportunity for theoretical formulations to investigatethe importance of electronic friction, adiabatic phonon coupling,multielectronic surface dynamics, and ballistic motion in energy dissipationfollowing activated dissociation events. When discussing the effectof N2 impinging energy and angle on the reactivity in alow coverage limit, our preliminary results suggest there is not anobvious memory function for incident momenta/kinetic energy of impingingN2 on the distance between resulting nitrogen adsorbates.Additionally, the non-normal component of impinging N2 momentumdoes not impact spatial distributions on the surface. Our resultsprovide direct experimental insight into the energy transfer mechanismsand detailed aspects of the dissociation process and help addressimportant and unresolved questions surrounding the fundamentally andtechnologically important activated dissociative adsorption processof N2 dissociation on Ru(0001).We present STM images depicting the productsof individual dissociationevents following nonequilibrium fluxes of energy- and angle-selectedN"}
+{"text": "Sox9 expression pattern that translates into the cartilage rings has remained elusive. Here, we review the molecular regulatory interactions that have been elucidated, and discuss possible patterning mechanisms. Understanding the principles of self-organisation is important, both to define biomedical interventions and to enable tissue engineering.The trachea is a long tube that enables air passage between the larynx and the bronchi. C-shaped cartilage rings on the ventral side stabilise the structure. On its esophagus-facing dorsal side, deformable smooth muscle facilitates the passage of food in the esophagus. While the symmetry break along the dorsal-ventral axis is well understood, the molecular mechanism that results in the periodic  The trachea is a long , almost cylindrical tube that serves as a passage of air to the bronchial system Nkx2.1-expressing lung field on the ventral side of the mouse foregut Bmp4) expression is restricted to the ventral foregut from early stages (E8.5) Sox)2 expression in the ventral foregut Efnb2, which establishes an EPH/EPHRIN boundary that results in the physical separation of tracheal and esophageal cells Nkx2.1 null mice, and endodermal mutants for the BMP type I receptor genes Bmpr1a and Bmpr1b upregulate Sox2 and form a continuous ring of smooth muscle and no cartilage rings Bmp4 from the foregut endoderm from E8.5 and from the mesenchyme by E9.5 does not prevent the ventral expression of Nkx2.1 at E9.5, but by E12.5 Nkx2.1 is absent, and expression of the cartilage marker Collagen Type II Alpha 1 Chain (Col2a1) is not observed The separation of cartilage and smooth muscles domains follows the already established dorsal-ventral polarity. Fibroblastic growth factor (FGF) from the cardiac mesoderm induces the Nkx2.1 expression restricted to the ventral side, and Sox2 and Sonic Hedgehog (Shh) expression higher on the dorsal side (Noggin null) that fail to split the tubes Sox9 as early as E10.5 Acta2, a smooth muscle marker Sox9 or a key smooth muscle gene does not alter the expression domain of the other in the trachea Wls conditional mutants blocks Sox9 expression and results in smooth muscle formation also on the ventral side \u03b2-catenin in either the epithelium (Shh-Cre driven) or mesenchyme (Dermo1-Cre driven) results in loss of mesenchymal expression of the chondrogenic factor Tbx4Shh null mice, the ventral restriction of Sox9 expression is lost, and until E13.5, Sox9 is transiently weakly expressed in a circumferential expression pattern on both the dorsal and ventral sides Shh is expressed more strongly dorsally, overexpression of Shh does not affect the relative cartilage and smooth muscle domains Sox9 and Bmp4 expression around the entire tracheal epithelium Once the trachea has split from the future esophagus, it maintains the dorsal-ventral polarity, with sal side . This poIn summary, the separation of the smooth muscle and cartilage domains along the dorsal-ventral axis is controlled by the already existing embryonic dorsal-ventral polarity. The dorsal-ventral polarity is first induced along the epithelial tube, and later translated to the mesenchyme via diffusible morphogens.Sox9 and type II collagen (Col2a1) expression Col2a1-expressing cells do not transdifferentiate into non-cartilage cells Col2a1-expressing cells condense in the cartilage rings, and the intervening space becomes filled by other mesenchymal cells. The Col2a1 gene encodes the pro-alpha1 (II) chain component of type II collagen, which is primarily found in cartilage. At E11.5, collagen type II is restricted to the lamina propria on the ventral side of the trachea Ptch1 appears to be restricted to the nascent cartilage condensations from E13.5 Shh assumes a periodic pattern on the ventral, but not on the dorsal side of the tracheal epithelial tube Tbx5 disappears from the cartilage condensations Fgf10 expression becomes restricted in between the nascent cartilage condensations, but its receptor Fgfr2b remains uniformly expressed in the epithelial tube Bmp4 emerges.The positions of the future cartilage rings in the ventral tracheal mesenchyme first become apparent between embryonic day (E)12.75 and E13 as periodic patterns in Sox9/Col2a1 expression patterns or tracheal cartilage ring formation, even though the trachea forms with correct dorsal-ventral polarity. This analysis thus necessarily excludes potential core components that are involved also in processes upstream of periodic pattern formation as their contribution to periodic patterning cannot be evaluated by this approach. The following mouse mutants have so far been reported that lack tracheal cartilage rings, even though the trachea forms with correct dorsal-ventral polarity: Shh null Sox9 null Mek1/Mek2 removal Wls removal Rspo2Tg/Tg;Lrp6\u2212/\u2212) tracheal rings were absent on the shortened tracheal structure Dermo1-Cre-driven conditional removal of \u03b2-catenin, a core component of canonical WNT signalling, result in loss of mesodermal Tbx4, impaired mesenchymal growth, and lack of cartilage rings at E16.5 Fgfr2b and Fgf10 null mice have a different phenotype from that reported for human FGFR2 (S351C) in that they develop shorter tracheas with 6\u20138 distorted cartilage rings FgfR2b in FgfR2c heterozygous mouse mutants results in overgrowth of the tracheal rings and absence of noncartilaginous mesenchyme Functional genetics can help to identify the components of the core mechanism as their null mutations should result in the loss of cartilage rings. In the following, we will focus on mutants that do not show any periodic Sox9 and Bmp4 expression in Shh null lung explants, but cartilage formation is then no longer restricted to the ventral side; it has not been reported whether periodic patterns are obtained Bmp4 conditional mutants, Nkx2.1 is restricted to the ventral side at E9.5, but is lost by E12.5, and no Col2a1 expression and cartilage ring formation is observed Bmpr1b and Sox2, and a SHH-driven endodermal conditional knockout of Bmpr1a develop a ventral NKX2.1 domain that forms disorganized isolated cartilage pieces/nodules, but not rings at later stages Bmpr1a and Bmpr1b has so far not been reported.BMP4 and its antagonist NOGGIN can both rescue cartilage formation as well as While perturbations in many other pathways affect tracheal ring formation or tracheal growth, no other pathway has been described that is necessary for cartilage ring formation once the tracheal mesenchyme has emerged Sox9 knockout mice Sox9 removal is stopped at E13.5, then some cartilage nodules are observed by E18.5 in the most proximal part Sox9 expression Sox9 expression appears strongly reduced or absent in Rspo2Tg/Tg mutant tracheal mesenchyme \u03b2-catenin in the mesenchyme of the developing trachea to influence expression of chondrogenic factors including Tbx4, Tbx5, Msx1, Msx2, Sox9, and Col2a1Wnt5a and its receptor Ror2, and ablation of Wnt5a or its receptor Ror2 results in shorter trachea with fewer cartilage rings Wnt7b, expressed by the respiratory epithelium and known to mediate Wnt/\u03b2-catenin signaling, does not affect trachea length or width, but results in incomplete cartilaginous rings Wnt4 does not affect tracheal length, but results in 12 distorted rather than 14 tracheal rings, and results in reduced Sox9 and increased Fgf10 expression at 13.5 SOX9 controls all steps of the cartilage differentiation process, and is a necessary factor for cartilage ring formation such that cartilage rings are absent in mesenchymal Col2a1 expression , while regulating SOX9 transcription factor activity via pSMAD1/5/8 and p38 Sox9 and Col2a expression and cartilage formation Despite its importance for cartilage ring formation, the upstream regulators of the MEK/ERK cascasde have remained elusive. FGFs signal via ERK, and overexpression of Interestingly, upon conditional removal of Myorcardin, the cartilage rings fail to expand towards the dorsal side, and the trachael lumen is reduced Sox9 expression, and SOX9 is essential for cartilage formation. Sox9 is still expressed weakly in Shh and mesenchymal Mek1/Mek2 mutants, but fails to organise into rings. As such, SHH and MEK1/2 are either part of the core mechanism that results in periodic Sox9 patterning, or periodic patterning fails because Sox9 expression is too weak in those mutants. Myocardin, a master regulatory of smooth muscle differentiation, is necessary for the dorsal expansion of the nascent cartilage rings to their characteristic C-shape. But what leads to the periodic Sox9 pattern?In summary, WNT signalling  is essential for mesenchymal Drosophila blastoderm, which were initially accounted to a Turing mechanism, but have since been shown to result from cross-repressive transcription factor cascades downstream of opposing morphogen gradients \u03b2 and either the extracellular matrix (ECM) or TGF-\u03b2 antagonists \u03b2-catenin appears necessary also for tracheal Sox9 expression Bmpr1a and Bmpr1b are not known. Unlike in lung branching morphogenesis Fgf10 mice, if delayed and less uniformly shaped compared to the wildtype Shh expression levels Fgf10 null mice Shh expression. Apart from chemical signalling, cell-cell interactions can also result in Turing instabilities Col2a1-expressing cells EEpi and thickness h relative to a thick, incompressible substrate with modulus EMes results in buckling with wavelength \u03bb, and epithelial thickness, h, that is observed in the trachea EEpi, and mesenchyme, EMes, would need to be similar. However, even if the epithelial folds arise from epithelial buckling, they may well be a consequence rather than a driver of mesenchymal condensations. After all, mesenchymal condensations reduce spatial expansion. Given this wide range of possibilities, more quantitative experimental studies and mathematical modelling are required to delineate the mechanism by which the cartilage rings form.A wide range of chemical and/or mechanical instabilities can result in biological pattern formation. The Swift-Hohenberg equation has been shown to recapitulate the complex tracheal cartilage pattern also at the tracheobranchial juncture, if coupled with a gradient to achieve the correct stripe orientation Despite the simplicity of the pattern and the importance of the structure, tracheal cartilage ring formation remains poorly understood. Conditional mutants in combination with explant cultures, organoids, quantitative imaging, and mathematical modelling may help to unravel this patterning mechanism."}
+{"text": "The corrected mCherry and Gad1, Gad2 mRNA in the VTA and SN in cells with and without active Pax5 expression (Pax5 expression also originate from Pax5-expressing progenitor cells.\u201d The errors appear in print and in PDFs downloaded before December 1, 2022.The authors regret that in the original version of pression , demonst"}
+{"text": "Defects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm's canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively.Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes.Angpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity.Our results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle. Impaired AH outflow increases the intraocular pressure (IOP), which is a significant risk factor for glaucoma, a heterogenous group of ocular diseases that are the major cause of irreversible blindness worldwide.,,,,\u2013\u2013The main exit route for AH, produced by the ciliary processes, drains from the anterior chamber via the trabecular meshwork (TM) to Schlemm's canal (SC) and from there to the aqueous and episcleral veins.\u2013The vascular angiopoietin (Angpt)/Tie signaling pathway consists of three ligands, Angpt1, Angpt2, and Angpt4; the primary Angpt receptor tyrosine kinase Tie2; and an orphan regulatory receptor Tie1.Tie2 deletion as well as Angpt1;Angpt2 double deletion in mice lead to complete absence of SC, highly elevated IOP, and severe glaucoma.,\u2013Tie2 heterozygosity or Angpt1 deletion is sufficient to partially degenerate SC in mice,,,\u2013TIE2 and ANGPT1 loss-of-function mutations who have been diagnosed with glaucoma.,,TIE2 and ANGPT1 variants have been associated with increased IOP and glaucoma in genome-wide association studies (GWASs).\u2013,Compelling evidence from recent years shows that Tie2 activation is essential for both SC development and its maintenance. Angpt2 deletion mice, and ANGPT2 variant loci have been associated with increased IOP in glaucoma patient GWAS analyses.\u2013Angpt2 deletion mice, and administration of Angpt2-blocking antibody has been reported to have no effect on IOP in monkeys or on SC area in mice.However, among the Angpt ligands, the importance of Angpt4 in the iridocorneal angle has not been investigated, and the individual role of Angpt2 has remained somewhat controversial. Kim et al.Angpt4 deletion alone did not affect the SC, its deletion combined with Angpt2 deletion had detrimental effects on both SC morphology and AH outflow, causing glaucomatous changes in the eye. Furthermore, being the first to use congenital Angpt2 deletion in this context, we show that Angpt2 is indeed necessary for normal SC morphology and also for the corneolimbal vasculature. Collectively, our data establish a novel role for Angpt4 in the iridocorneal angle and reveal cooperation between Angpt2 and Angpt4 necessary for full Tie2 activation and SC maintenance.Here, we used genetic mouse models to study the importance of Angpt4 in AH drainage. We show that Angpt4 expression is spatiotemporally distinct form the other Angpts in the iridocorneal angle, and while Angpt2 deletion mice were generated by CRISPR/Cas9-based genome editing.\u2013All animal experiments were performed with the approval of the Finnish Project Authorization Board following national and European Union (EU)\u2013level legislation (EU directive 2010/63/EU) and adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Angpt1 allele (referred as Angpt1fl throughout the text) was ordered from The Jackson Laboratory  under a strain name Angpt1tm2.1Sjm/J (#028925).Rosa26CreERT2 mice expressing tamoxifen-inducible Cre under ubiquitous Rosa26 promoter were kindly provided by the GIE-CERBM . Generation of the Angpt4 knockout allele  has been described previously.Angpt1fl/fl;Angpt4\u2212/\u2212;Rosa26CreERT2 mouse line, and the mice were administered tamoxifen  by oral gavaging starting from 4 weeks of age and continued at a 1-week interval until 8 weeks of age to delete Angpt1 and create an Angpt1del;Angpt4\u2212/\u2212 mouse line. Littermates received the same tamoxifen administrations and were used as controls. Angpt4Cre/+;Rosa26mTmG/+ cell fate mapping and Angpt4LacZ reporter mice have been introduced before.,Floxed ,,Primers used for genotyping the herein introduced mouse lines are presented in the Detailed protocols for immunofluorescence whole-mount and section stainings, X-gal staining, hematoxylin and eosin staining, confocal microscopy, transmission electron microscopy, all image analyses, single-cell RNA sequencing (scRNAseq) data analysis, and quantitative PCR (qPCR) are given in the ,,,,IOP was measured using a Tonolab rebound tonometer . Mice were accustomed to handling prior to the measurements. While measuring (consistently around noon), mice were restrained by an experienced animal caretaker, and IOP values were obtained from two to three sets of six recordings, which were finally averaged from both eyes to obtain a single value for each mouse.t-test was used for comparing two groups after confirming normal distribution and equal variance. Statistical significances are marked in figures as following: *P < 0.05, **P < 0.01, and ***P < 0.001, and all figures represent individual data points and mean \u00b1 SD.Normal distribution of the data was confirmed with the Shapiro\u2013Wilk test for normality performed in Prism 9 software , and equal variance was tested with Levene's test for homogeneity of variance using Origin Pro software . When both assumptions were met, comparisons between multiple groups were performed with one-way ANOVA followed by the Tukey post hoc test, and when variances were unequal (indicated in the figure legends), Welch's ANOVA followed by Dunnett's T3 multiple comparisons test was performed using Prism 9 software. Unpaired two-tailed Student's Rosa26mTmG with Angpt4Cre mice,,Angpt4 promoter by Cre-mediated conversion of continuous tdTomato expression to membrane-targeted green fluorescence protein (GFP) expression. We observed that the Angpt4-driven GFP signal started postnatally: at P5  and P10, flat-mounted corneal limbus samples had only very few, faint GFP+ cells, while at P13, their number and signal intensity were beginning to multiply  directly adjacent to the SC inner wall, as well as into the uveal and corneoscleral meshworks next to the anterior chamber , and Ptprb (protein tyrosine phosphatase receptor type B encoding VE-PTP) were all expressed in the Angpt4-negative vascular and SC endothelial cells  and were thereafter downregulated, in line with previously reported results.,Angpt2 expression was downregulated more gradually than Angpt1  was used to determine an individual expression profile over time for the and Tie2 E\u2013H. Tie2n Angpt1 F\u2013G whileh of age H. This wCollectively, spatiotemporal gene expression data from the cell fate tracing and reporter mice, scRNAseq, and qPCR analyses indicated that Angpt4 is expressed at relatively low levels in the TM cells close to the SC. In our analyses, Angpt4 was not enriched during the postnatal SC development and was partly produced in the same cell types than Angpt1 in adult mice, and the cellular sources of Angpt4 were markedly different from those of Angpt2.Angpt4 deletion on the SC. Since prominent Angpt4 expression appeared postnatally at a time point when the SC is already mostly developed ,\u2013,,+ SC area in 1-year-old Angpt4Cre/Cre (hereafter Angpt4\u2212/\u2212) mice , existence of circulating limbal arteries and veins used as a measure of complexity of the limbal vasculature,+ corneolimbal LV area were changed in any of the genotypes  mice A,\u00a02B, inf 1 year A. qPCR cficiency B. As exp2 allele C, S4D, benotypes C, 4E\u2013G. deletion H. Hence,ANGPT2 has been identified as a risk locus associated with glaucoma,\u2013Angpt2 deletion model has been used in any of the previous mouse studies.,,Angpt2 allele (Angpt2\u2212/\u2212 mice), we utilized the CRISPR/Cas9 methodology to induce a 35-bp deletion in the Angpt2 exon 1. This resulted in a codon frameshift and decreased Angpt2 mRNA expression, and Angpt2 antibody staining confirmed the absence of Angpt2 protein , and no circulating veins were found when 8 Angpt2\u2212/\u2212 and 5 Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 samples were analyzed .To investigate the possible Tie2-dependent mechanism of cooperative action of Angpt2 and Angpt4 on the SC, we performed antibody staining using phospho-Tie2 (pTie2) antibody that has previously been used to indicate Tie2 activation state in the SC.n the SC A,\u00a05B. Sirylation A,\u00a05B, budeletion A,\u00a05B. WhAngpt2 deletion by comparing Angpt2+/\u2212 and Angpt2+/\u2212;Angpt4\u2212/\u2212 mice. While at 12 weeks of age, these mice did not have major differences in the SC morphology , or corneolimbal LVs were seen . Despite the reduction in SC area, aged Angpt2+/\u2212;Angpt4\u2212/\u2212 mice did not display major signs of glaucoma ,Angpt1 is deleted at 8 weeks of age.Angpt1 deletion was induced at 4 weeks of age. This was sufficient to cause a hypomorphic SC and an increase in IOP, but the defects were not as severe as in the abovementioned earlier time-point deletions by others (25% decrease in SC area in our model). Altogether, these studies indicate that SC maintenance is less dependent on Angpt1 and that other Tie2 ligands may be more important for that. In this regard, it was interesting to find that both Angpt1 (our data and previously reported),,+ JCT cells, 10.5% simultaneously expressed Angpt4, being 50% of Angpt4+ JCT cells). The lack of any SC defect in the Angpt1fl/fl;Angpt4Cre/Cre mice, in which Angpt4 promoter\u2013driven Cre expression (starting at P10) would prevent Angpt1 expression from the floxed allele if occurring in the same cells, also supports this observation.An outstanding question regarding the Angpt/Tie2-dependent development and maintenance of the SC is the evident presence of three Angpt ligands to activate the Tie2 receptor in the same vascular structure. According to our data presented in this study and the results of others, Angpt1 is highly expressed at the time of SC development and at lower levels later in life,Angpt4 deletion exacerbated the phenotype of Angpt2 deletion but not Angpt1 deletion might be explained by the result that Angpt2 and Angpt4 are produced in different cellular compartments, making their combination deletion more harmful and difficult to compensate by the remaining ligand Angpt1. In addition, as Angpt1 is the high-affinity and strongly Tie2-phosphorylating agonistic ligand, combination deletion with Angpt4, which is a weaker ligandIn contrast to Angpt1 and Angpt4, Angpt2 was in our studies rather steadily present throughout the lifetime in almost all iridocorneal angle structures, but Angpt2 expression was detected only in the JCT but not in the other TM compartments. We showed that Angpt2 is expressed and essential in the limbal blood, lymphatic, and SC endothelium, suggesting that Angpt2 may also activate Tie2 in an autocrine manner, thereby mechanistically differing from the other Angpt ligands in the iridocorneal angle vascular structures. The notion that Angpt2 deficiency on AH drainage have been conflicting, either showing no effect at all,Angpt1 deficiency.,,Angpt2 deletion alone is sufficient to cause defects in the SC in mice, an observation that goes hand in hand with GWAS reports associating ANGPT2 genetic variants with patients who have increased IOP and glaucoma.\u2013Angpt2\u2212/\u2212 mice are also known to have abnormal retinal vasculature with sprouting defects and persistent hyaloid vessels,,,Angpt4\u2212/\u2212 mice have impaired retinal vein development, leading to inner nuclear layer swelling.Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice.So far, reports of the effect of Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice than in the Angpt2\u2212/\u2212 mice, correlating with the slightly diminished Tie2 phosphorylation state in the SC endothelium. Strikingly, also Angpt2+/\u2212;Angpt4\u2212/\u2212 mice, unlike Angpt2+/\u2212 or Angpt4\u2212/\u2212 mice, had a decreased SC area and Tie2 phosphorylation state, providing further evidence of Angpt4 as a regulator of the SC. The herein proposed model of Angpt2 and Angpt4 acting as Tie2 agonists in the SC endothelium correlates with the established role of Angpt2 as a Tie2 agonist in the lymphatic endothelium,Angpt2 deletion dramatically affected certain areas while other portions were unaffected, suggesting that local changes in the availability of the relatively weaker Tie2 ligands Angpt2 and Angpt4 could also fine-tune Tie2 activation state, providing homeostatic support for the SC after its developmental phase mainly mediated by the strongest Tie2 ligand Angpt1. Considering previous publications,,Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice, it can be concluded that Tie2-dependent AH homeostasis is dependent on a sufficiently high amount of Angpt ligands.Our data intriguingly showed that certain SC phenotypes were more severe in the double-deficient Angpt2+/\u2212;Angpt4\u2212/\u2212 mice, the SC was continuously slightly narrower than in WT, Angpt2+/\u2212, or Angpt4\u2212/\u2212 mice, but it did not have as much such extremely narrow convolutions as the SC in Angpt2\u2212/\u2212 and Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice had, suggesting that Angpt2 is essential for adequate SC morphogenesis, but also Angpt4 contributes to the maintenance of optimal width of the mature canal. Despite the reduction in total SC area and pTie2/Tie2 ratio comparable to Angpt2\u2212/\u2212 mice, Angpt2+/\u2212;Angpt4\u2212/\u2212 mice did not have elevated IOP or significant RGC damage. Likely, the numerous SC narrowing points and convolutions seen in Angpt2\u2212/\u2212 and Angpt2\u2212/\u2212;Angpt4\u2212/\u2212 mice are obstructive and inhibit the proper flow of AH in the SC more than a steadily narrow but otherwise normal SC does.Intriguingly, our data also revealed qualitative differences between the genotypes investigated. In Tie1 and Tie2 and double Angpt1;Angpt2 deletion mice,,,,Angpt1del mice did not show any apparent defects in the limbal vasculature maintenance, and limbal BV defects have not been reported in the early Angpt1 deletion models by others, implying that specifically, Angpt2/Tie/Svep1 interplay would be needed in the corneolimbal vasculature. We also found that Angpt2\u2212/\u2212 mice completely lacked corneolimbal lymphatics, in line with published data on early postnatal deletion of Angpt2 or Tie2 resulting in significant but not complete absence of corneolimbal LVs,Angpt1;Angpt2 deletion mice have also been reported to lack corneolimbal LVs,Angpt1 deletion alone has only minor\u2013Angpt2\u2212/\u2212;Angpt4\u2212/\u2212) or presence (Angpt1del) of both corneolimbal BVs and LVs had no obvious additional effects on IOP in models with comparable reduction in SC area.As another novel finding, we showed that Angpt2 is required for the formation of proper corneolimbal blood vasculature . Interestingly, defects in the limbal vasculature have also been described in ,In conclusion, this is the first study to indicate a role for Angpt4 in the SC maintenance. In addition, we provide evidence of Angpt2 as an essential SC and corneolimbal vasculature regulator. All three angiopoietins, Angpt1, Angpt2, and Angpt4, seem to be derived from varying cellular sources in the iridocorneal angle. Previous studies comparing the primary structures of human and mouse Angpt1, Angpt2, and Angpt4 have revealed that Angpt4 is the least conserved ligand among the angiopoietins."}
+{"text": "Computational clusters were further validated by immunohistochemistry with accuracy in a small subset of neurons. Thus, unbiased cluster analysis using electrophysiological properties is a tool that can enhance current interneuronal subclassifications and can complement groupings based on transcription factor and molecular expression.Spinal cord neurons integrate sensory and descending information to produce motor output. The expression of transcription factors has been used to dissect out the neuronal components of circuits underlying behaviors. However, most of the canonical populations of interneurons are heterogeneous and require additional criteria to determine functional subpopulations. Neurons expressing the transcription factor Shox2 can be subclassified based on the co-expression of the transcription factor Chx10 and each subpopulation is proposed to have a distinct connectivity and different role in locomotion. Adult Shox2 neurons have recently been shown to be diverse based on their firing properties. Here, in order to subclassify adult mouse Shox2 neurons, we performed multiple analyses of data collected from whole-cell patch clamp recordings of visually-identified Shox2 neurons from lumbar spinal slices. A smaller set of Chx10 neurons was included in the analyses for validation. We performed k-means and hierarchical unbiased clustering approaches, considering electrophysiological variables. Unlike the categorizations by firing type, the clusters displayed electrophysiological properties that could differentiate between clusters of Shox2 neurons. The presence of clusters consisting exclusively of Shox2 neurons in both clustering techniques suggests that it is possible to distinguish Shox2 The spinal neuronal circuitry participates in the control of a wide variety of movements, ranging from reflexes to highly sophisticated motor skills  transgenic mice  was sectioned transversely using a vibrating microtome (Leica Microsystems). Slices were next transferred to an artificial cerebrospinal fluid, containing the following (in mM): 111 NaCl, 3 KCl, 11 glucose, 25 NaHCO3, 1.3 MgSO4, 1.1 KH2PO4, and 2.5 CaCl2 at 37\u00b0C for 30 min and then passively equilibrated to room temperature for another 30 min before recording. Dissecting and recording solutions were continuously aerated with 95%/5% O2/CO2.For terminal electrophysiology experiments, adult mice  were anesthetized with ketamine (150 mg/kg) and xylazine (15 mg/kg), decapitated, and eviscerated. Spinal cords were then removed in ice-cold dissecting solution containing the following (in mM): 222 glycerol, 3 KCl, 11 glucose, 25 NaHCO2, 1 glucose, 4 NaCl, 5 ATP, and 0.3 GTP, with pH adjusted to 7.4. In some experiments, biocytin  was included in the patch electrode. Data were collected with a Multiclamp 700B amplifier (Molecular Devices) and Clampex software . Signals were digitized at 20 kHz and filtered at 10 kHz.All recordings were performed at room temperature. Fluorescently labeled (tdTomato) Shox2 and (eGFP) Chx10 neurons were visualized with a 63x objective lens on a BX51WI scope (Olympus) using LED illumination (Andor Mosaic System or Lumen Dynamics X-Cite 120 LED). Patch electrodes were pulled to tip resistances of 5\u20138 M\u03a9 using a multistage puller (Sutter Instruments) and were filled with intracellular solution, which contained the following (in mM): 128 K-gluconate, 10 HEPES, 0.0001 CaClm) was recorded shortly after entering whole-cell mode. Input resistance (Rin) was calculated from the current/voltage slope in response to hyperpolarizing voltage steps in which no voltage-gated current activation was evident. Membrane time constant (\u03c4) was calculated as the time to reach 63% of the maximum depolarization in response to a subthreshold depolarizing current step. Membrane capacitance (Cm) was calculated from the time constant and input resistance (Cm = Rin/\u03c4). Rheobase was the minimal current step required to generate an action potential, applied in intervals of 2\u20133pA. Additionally, we recorded action potential (AP) properties from the first AP observed in the depolarizing current at rheobase  and MATLAB. All results are presented as mean \u00b1 SD. Statistical significance was set at p < 0.05 unless otherwise stated. The distribution of the data was determined by Shapiro\u2013Wilk normality test. The statistical comparisons between Shox2 and Chx10 neurons were performed by Mann-Whitney test or unpaired t-test. Comparisons between clusters were performed by Kruskal-Wallis with Dunn's post-hoc test or repeated-measures one-way ANOVA with Bonferroni post-hoc test. Comparisons between percentages were performed by chi-square test.Data analysis was performed with Clampfit (Molecular Devices) and MATLAB (MathWorks). Statistical tests and p < 0.01 to be correlated. Principal component analysis (PCA) and multidimensional cluster analyses were performed on the 6 parameters obtained that were not highly correlated. PCA was used to reduce dimensionality of the number of variables recorded  Hierarchical clusters were determined by the cosine distance between pairs of observations and consideration of an average for the distance between clusters. This combination was used because it resulted in the highest cophenetic correlation coefficient. The number of clusters was determined considering the cutoff below the maximum inconsistency coefficient for each link. For the k-means and hierarchical cluster analyses, we applied MATLAB algorithms, initially standardizing the data to set a mean of 0 and standard deviation of 1 to be able to compare variables with different units.To determine correlations in between the 12 properties obtained for each cell, we performed a Pearson's linear correlation test and considered values of recorded . To visuThe slices containing biocytin-filled neurons were fixed overnight , and subsequently placed in PBS at 4\u00b0C. To visualize biocytin, the slices were incubated for 2 h at room temperature in DyLight 633 conjugated streptavidin . Then, the slices were washed in PBS (3 x 10 min), permeated with 1% Bovine Serum Albumin (BSA), 5% Donkey Serum (NGS), 0.1% Fish Gelatin, and 0.2% Triton x-100 for 1 h and incubated in sheep anti-Chx10 antibody 1:100  at room temperature for 48 hrs. Slices were washed in PBS (3 x 10 min) and incubated for 2 h at room temperature with rabbit anti-sheep Dylight 488 1:400  followed by a final wash in PBS (3 x 10 min). Slices were then placed on slides within tissue spacers and coverslipped with a Vectashield mounting medium (Vector labs). Sections were scanned using a laser scanning confocal microscope  in stacks of 10 optical sections across approximately 50 \u03bcm at 20x magnification. Images were condensed into maximum projections using the Leica collection software and brightness and contrast were adjusted in ImageJ.p < 0.001) between some passive and active cellular properties (m), capacitance (Cm), fast afterhyperpolarization (fAHP) duration, fAHP amplitude, activation voltage of the persistent inward current (PICon) and frequency-current (F/I) slope  slope . To visuI) slope . The firn = 143) with those of Chx10 neurons (n = 28) (in), larger capacitance (Cm), shorter AP half width, shorter fAHP duration, and more depolarized PIC activation voltages (PIC on) than Chx10 neurons. Resting membrane potential, time constant, rheobase, action potential threshold, the sAHP duration, fAHP amplitude, and F/I slope were similar between the groups. This comparison shows that Shox2 neurons and Chx10 neurons share some electrophysiological characteristics, however, differences are observed between subpopulations.Graphical display of the three first PCAs shows that Shox2 and Chx10 neurons partially overlap. We compared the initial 12 electrophysiological properties of adult Shox2 neurons ((n = 28) . Shox2 nn = 88, 51.5%). Initial doublet neurons  fire a doublet of action potentials at the start of the step  and continue firing throughout the step but at a lower frequency (steady frequency of 3.9 \u00b1 5.1 Hz). Neurons with burst of action potentials at the start of the current step are called initial burst firing neurons . These neurons displayed three or more initial spikes at high frequency (>25 Hz) and were either silent or fired action potentials later in the step but these action potentials were not a regular frequency like the tonic neurons. Lastly, a small number of neurons, delay neurons  fired action potentials after a delay from the beginning of the current step. We next looked at the prevalence of firing types in Shox2 and Chx10 neuronal populations. Of the 143 Shox2 neurons, tonic firing neurons were most common , followed by initial doublet  and initial burst  firing, with delayed firing neurons  being rare , over one third were tonic firing , and few neurons fired with an initial burst  or were delayed firing . Chx10 neurons preferentially displayed initial doublet firing and Shox2 neurons were mostly tonic firing.To identify differences in active and passive cellular properties based on firing type, we classified the 171 Shox2 and Chx10 neurons based on the response to suprathreshold depolarizing current steps. We found four types of responses . NeuronsTo identify the electrophysiological characteristics that corresponded to the different firing types, we performed statistical analyses on both groups considered as a single population , higher capacitance , shorter AP half width , and shorter fast  but longer slow  AHPs. We did not find differences between Shox2 and Chx10 neurons with initial doublet firing patterns. The numbers of delayed and initial burst firing Chx10 neurons were low and precluded from statistical analysis. The electrophysiological properties between neurons classified by type of firing were expected to be different since action potential features and spike frequency rely on these properties. Although firing types were differentially distributed in Shox2 and Chx10 populations, it is not possible to separate Shox2 and Chx10 neuronal populations by firing type since the types are common to both.We also compared properties of Shox2 and Chx10 neurons within each firing group . Considek-means cluster analysis (see Methods) on the set of 171 neurons, which included mainly Shox2 neurons and a small sampling of Chx10 neurons to serve as comparison with a known subdivision with the Shox2 population. The cluster analysis considered the six electrophysiological parameters that were not highly correlated, as described above . Chx10 neurons appear more prevalent in k2 cluster than Shox2 neurons, with 14% of Chx10 neurons but only <5% of Shox2 neurons belonging to that cluster, but this is not significant . The proportion of Chx10 neurons in each of the k3 and k4 clusters (39.2% and 39.9%) is similar to that of Shox2 neurons . The silhouette values for each of the 171 neurons in the k-clusters (n = 8) have negative values which indicate that the neuron is close to its cluster centroid but also close to other cluster centroids. Together, this shows that the k-means analysis . We did not find differences in k3 cluster  between Shox2 and Chx10 populations. The k4 also had equal proportions of Shox2 and Chx10 neurons and the only differences between the Shox2 and Chx10 neurons in that cluster were in AP halfwidth  and fast AHP amplitude  = 67, p = 0.04). Notably, there were less differences between Shox2 and Chx10 neurons from individual k clusters than there were differences in the whole populations of Shox2 and Chx10 neurons. This demonstrates similarity within clusters regardless of the transcription factor expressed.We next performed statistical analysis of the k clusters considering Shox2 and Chx10 neurons separately . In k2, The number of clusters in the k-means analysis here was defined by the elbow rule on a graph of the silhouette values  with more neurons in H6 (n = 39). The 28 Chx10 neurons (n = 2\u201313) but there are no Chx10 neurons in H1 and H2 clusters. The difference in the distribution of the 6 H-clusters was statistically significant when comparing Shox2 and Chx10 populations (p = 0.01). Note that H1 and H2 clusters were composed of Shox2 neurons exclusively and these two clusters were most similar to each other, as seen by the height of the next branch point in the analysis. Although clusters H3-H6 contained both Shox2 and Chx10 neurons, the uneven numbers of Shox2 and Chx10 input neurons (143 vs. 28) should be considered  with depolarized voltage thresholds are characteristic of Shox2+Chx10\u2212 neurons.To identify the electrophysiological characteristics of each H-cluster, we performed statistical analysis on these clusters considering all 171 neurons . Neuronsk-means clusters, there were few differences between Shox2 and Chx10 neurons within any of the hierarchical clusters. These were outnumbered by differences between the clusters, suggesting relatively homogeneous populations within each cluster.We also performed statistical analysis of the H clusters considering Shox2 and Chx10 neurons separately . There wk-means and hierarchical algorithms  and 6 Shox2 neurons expressed Chx10 (Shox2+Chx10+). We next determined which clusters each of the 8 neurons belonged to. Considering k clusters (+Chx10\u2212 were classified in k1 (1 neuron) and k3 (1 neuron) clusters, while Shox2+Chx10+ neurons were classified in k2 (1 neuron), k3 (2 neurons), and k4 (1 neuron) clusters. This matches rather well with predictions from the clustering results (+Chx10\u2212 where classified in H1 (1 neuron) and H2 (1 neuron), the clusters devoid of Chx10 neurons. The Shox2+Chx10+ neurons were in H3 (1 neuron), H4 (2 neurons), H5 (1 neuron) and H6 (2 neurons), all clusters which contain both Shox2 and Chx10 neurons. Thus, these results further support the ability of the H clustering method to distinguish populations of Shox2 neurons.A subset of the Shox2 neurons used for this analysis were recorded with biocytin in the electrode and were recovered for labeling with an antibody to Chx10. In total, we stained 8 neurons to determine Shox2identity . Of thosclusters , we foun results  because  results , we founk-means algorithm was run for 4 populations of neurons while hierarchical clustering analysis defined 6 populations. Interestingly, in each of the two clustering analyses, we found clusters containing exclusively Shox2 neurons, suggesting possible definition of Shox2+Chx10\u2212 populations by electrophysiological properties. Finally, as preliminary validation, Chx10 antibody staining of a small subset of biocytin-filled and recovered Shox2 neurons showed that all (8/8) of the filled and post-processed neurons were appropriately found in the expected hierarchical (H) clusters based on Chx10 presence/absence. The k-means clustering corresponded to the expected in 7/8 cases. Taken together, the data demonstrate that it is possible to classify neurons expressing Shox2 in at least six different subpopulations based on active and passive membrane properties. These subpopulations correspond well with Chx10 absence/presence and may provide further divisions between neurons currently defined with intersectional genetics.In the present study, electrophysiological properties were used to define populations of adult spinal Shox2 interneurons. Since Shox2 neurons overlap partly with Chx10-expressing neurons . One may expect that electrophysiological properties are related to transcription factor expression and inferred function. Thus, there would be clusters that contain each of the possible combinations. If this is the case, the distribution of Shox2 and Chx10 neurons in the clusters identified by the k-means and hierarchical algorithms could potentially be used to predict the type of neurons (Shox2+Chx10\u2212 or Shox2+Chx10+) that correspond to each cluster. For example, the lack of Chx10 neurons in k1, H1 and H2 clusters suggest that these clusters are composed of Shox2+Chx10\u2212 neurons. H5 cluster could also be considered as a putative Shox2+Chx10\u2212 cluster, since 93% of H5 cluster is comprised by Shox2 neurons, suggesting higher influence of Shox2 features than Chx10. As k3, k4, H3, H4 and H6 clusters have similar distributions of Shox2 neurons  as the total proportion of Shox2 neurons sampled (83%), we suggest that these clusters are composed of Shox2+Chx10+ neurons. In contrast, Shox2 neurons constitute 64% of the neurons in k2. Even though this percentage is lower than the total distribution of Shox2, this is not significantly different  and therefore we cannot classify them as Shox2\u2212Chx10+ neurons.A main motivation for the analyses was to determine if there was a way to identify subpopulations within a class of interneurons using electrophysiological properties rather than combinatorial genetics. The neurons expressing Shox2 can be divided into 2 subpopulations based on the expression of Chx10, Shox2\u2212Chx10+ neurons. This is most likely because there is a low number of Chx10 neurons in our analysis which resulted in an underrepresentation of the population of Shox2\u2212Chx10+ neurons. Our primary focus here was the division of the Shox2 population and how it would match up with the subgroupings by transcription factor (Chx10) expression. A future analysis may include a more balanced sample of Chx10 neurons, and we expect that would generate clusters consisting of only Chx10 neurons.We do not have any clusters of Shox2k-mean algorithms, previous studies have compared the output of both clusters and then corrected one or both algorithms  and longer fAHP durations in neurons in the k2 cluster (Shox2\u2212Chx10+ enriched), which corresponds well with the proportion of initial doublet firing neurons in each group. Taken together, the computational clustering analysis performed here exposed potential novel differences between Shox2 populations that allow for hypotheses to be made regarding the characteristics of these populations. It is important to note that the electrophysiological recordings were performed at room temperature, as with most recordings of adult locomotor-related neurons in vitro , the Edward Jekkal Muscular Dystrophy Association Fellowship (DG-R), and NIH T32 NS121768 (SS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "L-valine is one of the essential branched-chain amino acids (BCAAs) required for synthesis of proteins in human body. It promotes muscle growth and tissue repair and is important for immune function. Recent data indicate that BCAAs can activate sirtuins expression and elevate mitochondrial biogenesis and fatty acid oxidation in both adipocytes and myotubes thereby increasing life span. Sirtuins are a conserved family of proteins, play a critical role in maintaining metabolic health by deacetylating many target proteins in numerous tissues, and regulate mitochondrial function and the aging process. Due to multiple effect of sirtuins on aging, we sought to determine whether the addition of valine might enhance sirtuin gene expression. We utilized the C2C12 skeletal muscle cell line grown on physiological normal glucose (100mg/dL) media. The cells were treated with two different concentrations of valine (0.5 and 1.0mM) for different time intervals (18 and 24). Gene expression of sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) isoforms were determined by RT-PCR. The results showed increased expression of the sirtuin gene isoforms after treatment with valine. Relative expression varies with in different isoforms of SIRT1 (v1 and v2) and SIRT2 . Among all, SIRT1 v1 and SIRT2 v1 showed maximum expression as compared to the other isoforms used in the study. Our study showed that adequate supplementation of L-valine enhanced sirtuin gene expression, which may promote healthy muscles and healthy aging."}
+{"text": "Following the publication of this article , the UniThe Fig 6A Control and TM+TUDCA panels  partially overlap.\u25cb Fig 5A GRP78 and NCK1 panels.\u25cb Fig 7A PTP1B and GRP78 panels.Areas indicating splicing and inappropriate image manipulation were detected in the following panels:In addition to these concerns, editorial reassessment of the article raised further concerns with results presented in Figs 5, 6, 8, and 9. Specifically,Additional irregularities were detected in the background of the Fig 5A PTP1B panel.\u25cb The Fig 6A TM+NAC panels  also appear to partially overlap with the respective Fig 6A Control and TM+TUDCA panels .\u25cb Fig 8A, lanes 1\u20134 of the Cyt. NF\u03baB p65 panel appear similar to lanes 1\u20134 of the Cyt. GAPDH panel.\u25cb Fig 9C, lane 4 and lane 9 of the Cyt. Lamin A panel appear similar.Additional similarities were detected between and within panels:The underlying data for this article have not been submitted to the journal for editorial review.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the integrity of these data, the SN agreed with the retraction and apologizes for the issues with the article. EP and JR either did not respond directly or could not be reached."}
+{"text": "Gene variability related to carcinogen activation and detoxification may interfere with susceptibility to head and neck cancer.GSTT1 and GSTM1 null polymorphisms and the risk of head and neck squamous cell carcinoma in cigarette smokers.To investigate the relation between GSTM1 and GSTT1 null genotype frequencies were evaluated by multiplex PCR in A case-control study conducted at the Sao Jose do Rio Preto Medical School, Brazil. GSTT1 null genotype was found in 33.3% of the Experimental Group and 23.3% of the Control Group (p= 0.311). Experimental and Control Groups had GSTM1 null genotype frequencies of 35% and 48.3% (p=0.582). No association between alcohol consumption and GSTT1 and GSTMI null genotypes was found in these groups . There were more men, and alcohol consumption was prevalent in both groups.The oral cavity was the most prevalent tumor site for squamous cell carcinoma. The GSTM1 and GSTT1 genotypes and the development of head and neck squamous cell carcinomas in cigarette smokers.In this study we were unable to show a correlation between  Head and neck neoplasms are responsible for many deaths worldwide, being the sixth cause of death by c\u00e2ncer.GSTT1 and GSTM1 - that code phase II enzymes belonging to the glutathione S-transferases (GSTs) family seem relevant for susceptibility to head and neck squamous cell carcinoma; they detoxify carcinogenic tobacco smoke reactive metabolites.23Various polymorphic genes that code enzymes involved in carcinogen biotransformation have been associated with cancer development.GSTM1 gene is polymorphic in humans, including a null-activity allele (GSTM1-) due to a major genic deletion, and two functional alleles (GSTM1A and GSTM1B). The GSTT1 gene is also polymorphic in humans, and may have a deletion null genotype.The Thus, individual gene variability in the metabolic activation and detoxification process appears to be crucial to head and neck cancer susceptibility.GSTT1 and GSTM1 gene null genotypes in smokers with head and neck squamous cell carcinoma and to compare these frequencies with those seen in smokers without a history of cancer, to identify possible susceptibility biomarkers for head and neck cancer.This study aimed to identify Individuals diagnosed with squamous cell head and neck carcinomas confirmed by histopathology came from the Department of Otorhinolaryngology and Head and Neck Surgery of the Navy Hospital / Medical School of S\u00e3o Jos\u00e9 do Rio Preto and the Arnaldo Vieira de Carvalho Institute, SP. The control group included individuals with no history of neoplastic disease, paired by gender, age, ethnic group and use of alcoholic beverages. All subjects (patients and controls) were smokers. Individuals were included in the study after signing a free and informed consent form, and all information was obtained using a confidential standardized data-collection questionnaire . Information on smoking and alcohol-drinking was limited to a definition of user or non-user. The study was approved by the Research Ethics Committee of the Sao Jose do Rio Preto Medical School (CEP-FAMERP - 5639/2002) and the National Research Ethics Council .Genomic DNA was extracted from peripheral blood according to the Abdel-Rahman et al technique.GSTT1 and GSTM1 genes was simultaneously done by the chain reaction (PCR) according to Abdel-Rahman et al.CYP1A1 gene exon 7 sequence was coamplified to serve as an internal amplification control. PCR products were analyzed in agarose gel at 1.5%, stained with ethydium bromide, and the null genotype  for the GSTT1 and GSTM1 genes was identified by the absence of amplification fragments of 480 base pairs (bp) and 219 bp respectively. Presence of the 312 bp fragment corresponded to the amplified CYP1A1 gene sequence, which was evidence of a successful amplification reaction.The analysis of Demographics were presented as mean \u00b1 standard deviation (SD) or proportions. For the statistical analysis of the genotype frequencies obtained we used the exact Fisher test, with a significance level below 5%.Demography data: 120 individuals were recruited, of which 60 had head and neck squamous cell carcinoma (average age 54.6 \u00b1 8 years) and 60 had no history of neoplastic disease (average age 54 \u00b1 9 years). Men predominated (90% men vs. 10% women) and there were more alcohol drinkers .Primary sites: all head and neck squamous cell carcinoma cases were diagnosed and confirmed by pathology exams. The distribution of the primary tumor site is shown on GSTT1 null genotype [-] was found in 33.3% (20 of 60) of patients and in 23.3% (14 of 60) of controls (P = 0.311). The GSTM1 null genotype [-] was found in 21 (35%) of patients and 29 (48.3 %) of controls (p = 0.582). The combined GSTT1 and GSTM1 gene null genotype was seen in 10% (6 of 60) of patients and in 8.3% (5 of 60) of controls (p = 1.0). The most frequent genotypic combination, considering the presence of an unfavorable genotype (null GST), was GSTT1 [+] / GSTM1 [-] in 31.6% (19 of 60) of patients and 40% (24 of 60) of controls (p = 0.353) (Frequency of polymorphisms: The = 0.353) .Table 2GGSTT1 and GSTM1 null genotypes.Genotypes were grouped by tumor site  and therGSTT1 and GSTM1 null genotypes [-] when we compared alcohol-drinking patients and controls .Statistical analysis did not reveal any relation between alcohol use and Epidemiological data has suggested that alcohol use and smoking are the main risk factors for malignant transformation in head and neck cancers.Studies have shown an association between alcohol drinking and the development of head and neck tumors when taking into account exposure time and the amount of alcohol consumed.The higher number of men in this study corroborates findings by Drummond et al,The most frequent tumor site in our patients was the mouth, which is also the most frequent site for head and neck tumors reported in literature.GSTT1 and GSTM1 polymorphism studies done in Brazilian sub-populations reveal similar frequencies for both genotypes. Rossit et al,GSTT1 and GSTM1 gene null genotypes, respectively. Rossini et al,GSTT1 [-] and 42.1% for GSTM1 [-] in the state of Rio de Janeiro. Our study showed similar frequencies for these genotypes . Higher GSTT1 [-] and GSTM1 [-] polymorphism frequencies were observed by Drummond et al.GSTT1 [-] and 70.5% for GSTM1 [-]).GSTT1 and GSTM1 polymorphism studies done in head and neck carcinomas are contradictory. Various authors have demonstrated an association with the GSTM1 null genotype [-],GSTT1 null genotype [-] was also shown in some studies,GSTM1-enzyme activity was significantly reduced in patients with head and neck carcinoma compared to controls, although it did not depend on the unfavorable GSTM1 genotype, which may suggest that other enzymes participate as regulators.GSTT1 genotype [+] is associated with an increased risk of head and neck squamous cell carcinoma in smokers  and that the GSTT1 null genotype [-] may protect individuals against the development of this cancer. Although usually GSTs are considered detoxification enzymes, for certain specific chemical substrates such as dichloromethane (DCM), conjugation of glutathione with the GSTT1 enzyme may activate an electrophilic component, resulting in mutagenic potential.Interestingly, Evans et alGSTT1 and GSTM1 null genotypes and the development of head and neck squamous cell carcinoma in smokers.This study did not allow us to establish any correlation between"}
+{"text": "F is incorrect, and the correct definition is RF = \u2013CF(CF3)(OCF2CF(CF3))nOCF2CF2CF3. The correct versions of The authors regret that a consistent structure error appears in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."}
+{"text": "The tensile property and electrical conductivity of monolayer membranes were explored. The results showed that PVA with 2 wt.% MWNTs nanofibre membrane has the best conductivity (1.0 \u00d7 10\u22125 S/cm) and tensile strength (29.36 MPa) compared with other fillers. Meanwhile, the combination of multilayer membrane ZnO/Fe3O4/Fe3O4/MWNTs/ZnO showed the highest conductivity (1.39 \u00d7 10\u22125 S/cm). The parallel circuit and calculation of parallel resistance were attempted to demonstrate the conductive mechanism of multilayer membranes, which can predict the conductivity of other multilayer films. The production of multilayer composites that enhance electrical conductivity and improve conductive predictions was successfully explored.Advanced research on improving the performance of conductive polymer composites is essential to exploring their potential in various applications. Thus, in this study, the electrical conductivity of multilayer nanofibre membranes composed of polyvinyl alcohol (PVA) with different electroconductive fillers content including zinc oxide (ZnO), multiwalled carbon nanotubes (MWNTs), and Ferro ferric oxide (Fe Electrospinning is an efficient method of producing nanosized or micro-sized fibres using an electrical field. When millions of fibres are collected on the collector device or rotating disk collector, dry polymer fibres of submicron diameter were deposited and formed nanofiber membranes ,2. In thTextile material that contributes to the electrostatic effect due to friction is not suitable to be worn or used near explosive surroundings, such as at gas stations or explosive handling areas because there could be a high risk of explosion . Therefo3O4 into the polymer matrix [PVA is water-soluble and is easy to form nanofibers by electrospinning, and it has extensive applications due to its biocompatible and biodegradable properties . It is wr matrix ,14,15,16r matrix . With thHowever, most of the electrospun nanofibre membrane studies focus on the production of monolayer and single-added particles or monolayer and multicomponent fillers. Multilayer nanofibre membranes are a common form of arrangement in composites that contribute to outstanding material performance compared with monolayer composites . MultilaIn this paper, we present the electrical conductivity of multivariate and multilayer nanofibre membranes using an improved formula to calculate in parallel circuits by considering the interfacial effects between the adjacent films. Before that, the optimization of different filler contents, tensile properties, and electrical conductivity of the monolayer nanofibre membranes were investigated. A hot-pressing process then prepared the multivariate and multilayer nanofibre membranes with three optimal nanofibre membrane contents (wt.%) following a particular combination.\u22123 centipoises) was obtained from Guangzhou Feng Bai Shun Trade Co., Ltd., Guangzhou, China; nanosized zinc oxide (ZnO) (100 nm) was supplied by Shijiazhuang Baisheng Chemical Co., Ltd., Shijiazhuang, China; nanoscale magnetite or ferro ferric oxide (Fe3O4) (80 nm) were provided by Qinhuangdao Taiji Ring Nanoscale Co., Ltd., Qinhuangdao, China); multiwalled carbon nanotubes (MWNTs) (9.4\u223c16.4 nm) purchased from Qinhuangdao Taiji Ring Nanoscale Co., Ltd., China. Polyvinyl alcohol (PVA)  and mass fractions (0 to 5 wt.%) were prepared, as shown in The PVA polymer was dissolved in distilled water (PVA: distilled water = 1:9), and the mixture was stirred for 12 h at room temperature. The PVA solution was defoamed and loaded into a 15 mL syringe (needle number 21) for the electrospinning. Different electroconductive fillers . The mechanical properties (tensile strength and modulus) of the membrane were then measured with a universal strength testing machine . Five tests for each group of samples (90 mm (length) \u00d7 10 mm (width)) were performed under stoke control at a constant speed of 10 mm/min, and the width of clamps was 30 mm. The optical images for all fabricated nanofibre membranes of PVA with different fillers were observed under a scanning electron microscope (SEM) .3O4  of Fe3O4 compared with ZnO (100 nm). Wang and co-workers explored the in situ composite approach of electrospun PVA/Fe3O4 and found that PVA acted as a stabilizer during the coprecipitation [3O4 by making it compatible and embedded together within the fibre. Hence, less precipitation formed on the matrix fibre. However, an agglomeration of fillers appeared on the nanofibre surface at 4 wt.%, uniformly attached to the matrix fibre as shown in 3O4 particles were agglomerated and had attached to the nanofibre surface [3O4  were used, and the nanofibre membrane micrographs are shown in 0.5 wt.% a. It canThe tensile properties of PVA/ZnO nanofibre membrane with varying mass fractions are shown in 3O4 nanofibre membrane are exhibited in 3O4 and a sudden drop at 5 wt.%. This tensile pattern occurred probably due to the cohesion force that weakened and slippage of Fe3O4 particle among fibres PVA matrix; thus, decrement of tensile strength was observed at the first 1 to 3 wt.%. Meanwhile, at 4 wt.% Fe3O4, the particle cohesion force increased suddenly, similar to the tensile strength and tensile modulus with 15.03 MPa and 187.50 GPa, which increased by 20.26 and 125.01%, respectively, in comparison with the pure PVA membrane [3O4 fillers (more than 5 wt.%) resulted in particle agglomeration, which led to the decrease of its tensile properties. This result is in accordance with previous research, which reported at 1 to 3 wt.% Fe3O4 loading exhibit no significant difference on tensile stress and ultimate strain; however higher value was observed if more than 5 wt.% [The fluctuation results for the tensile properties of the PVA/Fen 5 wt.% . The nonInterestingly, a different pattern of tensile strength and modulus of PVA/MWNTs membrane was observed with continuous increment as the mass fraction of MWNTs fillers (0 to 2 wt.%) increased. Jeong and co-researchers also reported the increment of tensile strength at 1 to 2.5 wt.% MWNTs, which attributed to a higher degree of filler orientation in the wrap nanofibre . The pre2 to 107 Hz) are shown in 2 to 105 Hz. Meanwhile, at approximately 106 Hz, the increment was more obvious where the conductivity exponentially increased until 107 Hz for all samples. Thus, from this finding, the conductivity of monolayer nanofibre membrane of PVA/ZnO was fixing at 107 Hz frequency to investigate the effects of different wt.% of ZnO (1 to 5 wt.%) as interpreted in 7 Hz, the coincident conductivity increased when the filler content was at 1 wt.% and decreased for higher filler contents. However, when the ZnO filler was at 5 wt.%, the conductivity was as higher as 1wt.%. In combination with the SEM micrograph, the uniform distribution of 1 wt.% ZnO in PVA fibrous membranes contributing to the possibility of forming a conductive pathway among the particles. The higher amount of ZnO fillers caused severe agglomeration, owing to the higher content and large specific surface area and interaction force of nanosized particles. In contrast, filler at 5 wt.% was enough to contribute to the conductive pathway. The optimization of ZnO contents in the PVA matrix was at 1wt.% because of its sharp efficiency effect; thus, it was referred to as threshold. In particular, the relative conductivity effect should enormously increase when the concentration of filler particles approaches the conductive threshold [The conductivities of monolayer nanofibre membranes of PVA/ZnO with different wt.% of ZnO (1 to 5 wt.%) at different frequencies (10hreshold . Therefo3O4 mass fractions at different frequencies (102 to 107 Hz) are illustrated in 7 Hz are displayed in 3O4 fillers was the highest from 106 to 107 Hz. The curve of all samples in 3O4 filler played an important role in forming the conductive network. Hence, with prominent particle cohesion force at 4 wt.% Fe3O4, it can be determined that the conductivity threshold and the optimized content for PVA/Fe3O4 were at 4 wt.%.The conductivities of monolayer nanofibre membranes with different Fe2 to 107 HZ). Similar to PVA/ZnO and PVA/Fe3O4 monolayer membranes as showed in 6 Hz until 107 Hz. The continuous increasing trend with the increase wt.% of MWNTs is portrayed in \u22126). The PVA membrane obtained conductive property at 2 wt.% MWNTs due to the conductive nature of carbon nanotubes also contributed to a decrease in internal resistance [3O4, and MWNT in films were 1, 4, and 2 wt.%, respectively.sistance . Finally7 Hz frequency of membranes among different fillers are presented in 3O4 particles in the PVA matrix. Despite this, the lowest result in tensile strength was observed when Fe3O4 filler was used in the membrane. The electrical conductivity at 107 Hz frequency showed a similar trend as tensile strength, which is that the sequence from higher to lower conductivity at 2 wt.% MWNTs, 1 wt.% ZnO and 4 wt.% Fe3O4 membrane. Therefore, the PVA membrane with ZnO was selected to be the surface layer. The MWNTs membrane with the highest conductivity and the lowest Fe3O4 membrane was utilized as the interlayers for more effective comparison.The tensile strength and electrical conductivity comparisons at 103O4/ZnO due to the contribution of higher electrical conductivity of PVA/MWNTs. The four-layer membranes were prepared with two interlayers (Fe3O4 and MWNTs). Finally, the additional Fe3O4 was added to the four-layer membranes. 3O4 membrane possessed lower conductivity, however, the effect on the conductivity of the multilayer membrane was greater than that of the single membrane itself.Multivariate and multilayer nanofibre membranes were produced by different univariate and monolayer electrospun nanofibre membranes through the heat and press process, as presented in n is the resistance of different monolayer membrane. According to R = \u03c1L/S and \u03c1 = 1/\u03c3, the total electrical conductivity of multilayer membranes could be calculated by Equation (2).n is the electrical conductivity of different monolayer membrane. The parallel circuit was drawn into a multilayer membrane, shows in n\u22121\u22c5n is the lost conductivity due to the incompatibility of between adjacent membrane. If the two adjacent films have similar conductive particles, the \u03c3n\u22121\u22c5n is assumed to be zero. The lost conductivity of each adjacent film could be calculated. Finally, the \u03c3ZF,\u03c3ZM, and \u03c3FM were worked out through the theoretical value and actual value. The predicted actual electrical conductivity of the ZnO/Fe3O4/Fe3O4/MWNTs/ZnO membrane based on Equation (3) was 1.335 \u00d7 10\u22125 S/cm, and the error value was 3.96%, lower than 0.05. Therefore, Equation (3) was considered able to predict the multivariate and multilayer membranes. However, incompatibility or interaction between adjacent layers was inferred from the interfacial effects between different conductive films and the diverse conductive mechanisms of different conductive particles. The result from the calculation showed the conductivity of the actual multilayer films was considerably lower than the value calculated by Equation (2). The revised equation (Equation (3)) for the total electrical conductivity of the multilayer membrane was put forward, which revealed the regularity of interfaces.3O4, so the electrical conductivity of ZnO/MWNTs/ZnO is better than ZnO/Fe3O4/ZnO because the decreasing degree of resistance is related to the added resistance in parallel circuits [3O4 has electromagnetic shielding effectiveness [Finally, according to the revised equation, the conductivity of multivariate and multilayer conductive membranes relies on the number of layers, the conductivity of different single films, and the interfacial effects between adjacent films. The characteristic of parallel circuits is that the total resistance is smaller than the minimum parallel resistance. The total resistance decreases when the new resistance is added ,34, thuscircuits . Howevertiveness , and thetiveness ,37. The 3O4 and MWNTs) were successfully produced by electrospinning. A PVA/MWNTs monolayer membrane particularly for 2 wt.% was well dispersed and uniformly embedded within PVA fibre, resulting in the highest tensile strength (30 MPA) and revealing great electrical conductivity (1.0 \u00d7 10\u22125 S/cm). The multilayer film conductivity was successfully estimated using the modified conductivity formula based on parallel circuits. However, the accuracy of the lost conductivity of each adjacent film mentioned in this paper still needs to be investigated.Conductive PVA-based nanofibre membranes with different electroconductive fillers (ZnO, Fe"}
+{"text": "Arabidopsis thaliana seedlings exposed to elevated ambient temperatures. The transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) functions as a central hub in this response and promotes hypocotyl growth by transcriptionally inducing auxin biosynthesis genes . Measurements of hypocotyl length at different time points during the night revealed that hypocotyls of seedlings transferred from 10\u00b0C to 28\u00b0C at the beginning of the night grew less during the night than those of seedlings continuously kept at 28\u00b0C. Conversely, seedlings transferred from 28\u00b0C to 10\u00b0C showed enhanced nighttime hypocotyl growth compared with seedlings that were maintained at 10\u00b0C during both day and night  and its consequence (PIF4 levels) not only depends on the size of the change but also its direction.To further unravel how daytime temperature affects nighttime growth, responses of hypocotyl elongation to different combinations of day and night temperatures were monitored in different mutant backgrounds. Short-term temperature memory was disturbed in mutants of several known thermomorphogenic regulators including PIF4 and another transcription factor, ELONGATED HYPOCOTYL 5 (HY5). Confocal imaging of plants expressing PIF4 and HY5 reporter constructs indicated that nuclear levels of both proteins accounted for 80% of the observed growth rate variability. Using luminescence assays, the authors demonstrated that temperature-induced effects on nuclear PIF4 protein levels were largely driven by changes in PIF4 expression, the authors investigated the involvement of this clock protein in the observed temperature memory. ELF3 undergoes a phase transition in response to warm temperatures, forming speckles in the nucleus via its predicted prion-like domain (elf3 mutant background, indicating that ELF3 functions upstream of PIF4 in establishing short-term temperature memory. This was further supported by the observation that ELF3 speckle formation also showed a hysteretic pattern, with a high sensitivity to increasing temperatures and a much lower responsivity to cooling.As ELF3 is a known regulator of e domain . Murcia Taken together,"}
+{"text": "It was determined via STR profiling that cell line UMSCC2 should have been labeled as HSC3 cells. Like UMSCC2 cells, HSC3 cells are squamous cell carcinoma cells that express wild type EGFR, the primary substrate of PTPRJ/DEP1 investigated in this publication. Therefore, this correction does not affect the results and conclusions of the paper."}
+{"text": "Novel oral poliovirus vaccine type 2 (nOPV2) was used to control an outbreak of type 2 circulating vaccine derived poliovirus (cVDPV2) in Tajikistan, in 2021. We measured seroconversion and seroprevalence of type 2 polio antibodies in children who were reported to have received two doses of nOPV2 in outbreak response campaigns.In this community serosurvey, children born after Jan 1, 2016 were enrolled from seven districts in Tajikistan. Dried blood spot cards were collected before nOPV2 campaigns and after the first and second rounds of the campaigns and were sent to the Centers for Disease Control and Prevention  for microneutralisation assay to determine presence of polio antibodies. The primary endpoint was to assess change in seroprevalence and seroconversion against poliovirus serotype 2 after one and two doses of nOPV2.2 p=0\u00b7010). Total seroconversion after two nOPV2 doses was 77% .228 (97%) of 236 enrolled children were included in the analysis. The type 2 antibody seroprevalence was 26%  before nOPV2, 77%  after one dose of nOPV2, and 83%  after two doses of nOPV2. The increase in seroprevalence was statistically significant between baseline and after one nOPV2 dose , but not between the first and second doses . Seroconversion from the first nOPV2 dose, 67% , was significantly greater than that from the second nOPV2 dose, 44% (20/45; 30 to 60; \u03c7Our study demonstrated strong immune responses following nOPV2 outbreak response campaigns in Tajikistan. Our results support previous clinical trial data on the generation of poliovirus type 2 immunity by nOPV2 and provide evidence that nOPV2 can be appropriate for the cVDPV2 outbreak response. The licensure and WHO prequalification of nOPV2 should be accelerated to facilitate wider use of the vaccine.World Health Organization, Centers for Disease Control and Prevention, and Rotary International. Substantial progress has been made in the past several years to eradicate wild poliovirus. In 2021, only five cases of poliomyelitis caused by endemic wild poliovirus were detected; four from Afghanistan and one from Pakistan\u2014the last two remaining endemic countries.VDPVs result from the use of live Sabin-based oral poliovirus vaccine (OPV), which in rare circumstances regains neurovirulence following prolonged circulation in under-immunised populations.Response to cVDPV2 outbreaks typically includes vaccination campaigns with OPV2-containing vaccines because inactivated poliovirus vaccines (IPV) induce insufficient intestinal mucosal immunity required to prevent infection and halt virus transmission.nOPV2 is a modified version of mOPV2, which was purposefully engineered to be more genetically stable than mOPV2, making it significantly less likely to revert into neurovirulent forms.14Evidence before this studyOn Nov 13, 2020, a novel live type 2 oral poliovirus vaccine (nOPV2) was recommended for use under WHO Emergency Use Listing (EUL). As part of the clinical development, phase 1 and phase 2 clinical trials have been completed for nOPV2 in Belgium demonstrating the safety, tolerability, and immunogenicity of the vaccine. A larger phase 2 study conducted in Panama confirmed the safety, tolerability, and immunogenicity of nOPV2 in the target population for polio outbreak response in children aged 1\u20134 years and infants aged 18\u201322 weeks. Because the authors are part of the research group on nOPV2 development, we did not conduct a formal literature search.Added value of this studyBetween Nov 13, 2020, and Oct 14, 2022, approximately 500 million nOPV2 doses have been administered in outbreak response to circulating vaccine-derived type 2 poliovirus (cVDPV2) in 23 countries. This is the first study to provide estimates of immunogenicity of nOPV2 in an outbreak response setting. We observed that vaccination with nOPV2 induced a strong immune response in children younger than 5 years in Tajikistan, in agreement with previous results from phase 1/2 clinical trials. Of note, the first nOPV2 dose resulted in significantly higher seroconversion rates than the second one.Implications of all the available evidenceWe demonstrate that high coverage campaigns provide sufficient immunity against cVDPV2 to interrupt transmission, supporting the use of nOPV2 under EUL. Phase 3 trials and longer-term evaluation of safety and genetic stability of nOPV2 are needed to receive full licensure and WHO prequalification for nOPV2.In Tajikistan, an outbreak of cVDPV2 was detected in January, 2021. A total of 36 patients with paralytic poliomyelitis have been reported in Tajikistan, with onset of paralysis between Nov 1, 2020, and July 31, 2021 .1 An env16The government of Tajikistan responded to the outbreak by conducting a series of vaccination campaigns: initially with IPV in February, 2021, to provide protection against type 2 paralysis in children who had not previously received IPV; followed by two national campaigns of nOPV2 to interrupt cVDPV2 transmission and targeting all children born after Jan 1, 2016, between May 31 and June 5, 2021, and between June 29 and July 5, 2021 .15 An adIn the routine immunisation programme in Tajikistan, children born before April 30, 2016 received trivalent OPV . From May 1, 2016, the schedule changed to bivalent OPV , which does not provide protection against type 2 poliovirus, and was administered at birth and at age 2, 3, 4, and 12 months, and one dose of IPV at age 4 months. IPV introduction into the routine immunisation schedules in Tajikistan was delayed until June 1, 2018, with the IPV catch-up campaign between Feb 15 and 20, 2021, targeting children born between May 1, 2016, and June 30, 2018. WHO/UNICEF estimates that the polio vaccine immunisation coverage in Tajikistan has been consistently higher than 90% in recent years.To provide rapid evaluation of nOPV2 immunogenicity achieved in vaccination campaign settings, we conducted a serological survey of polio antibodies in children in Tajikistan before and after they received nOPV2 during the two national outbreak response campaigns. The study was conducted in areas that were actively affected by the cVDPV2 outbreak, where exposure to circulating virus could not be controlled.This was a community-based, serological survey carried out between May 1 and July 31, 2021. Children born on or after Jan 1, 2016, residing in one of the following districts were eligible for enrolment.Epi Info (version 7) without replacement: Jaloliddin Balkhi (n=1), Dushanbe (n=10), Faizabad (n=3), Kushoniyon (n=1), Tursunzoda (n=1), Vakhdat (n=4), and Vakhsh  were selected for the study, including the capital city Dushanbe . These dhsh n=1; . ChildreThe study received ethical clearance from WHO (ERC.0003599) and the Committee of Biomedical Ethics, Ministry of Health and Social Protection of the Population, Tajikistan.The study procedures were carried out during three health centre visits. Visit 1 was in the days before the first nOPV2 campaign, visit 2 was 1 month after the first campaign of nOPV2 concluded (just before the second nOPV2 campaign), and visit 3 was 1 month after the second campaign concluded. At the first visit, after obtaining written informed consent from the child's parents or guardians, children were enrolled, and a simple demographic questionnaire was taken, which included age, gender, and poliovirus vaccination history; vaccination history of IPV (through routine immunisation or catch-up campaign) and bOPV (through routine immunisation) was recorded through vaccination cards where available, or parental recall. At the second and third visits, inclusion in the preceding nOPV2 campaign was recorded through parental recall. Trained phlebotomists generated dried blood spots (DBSs) on Whatman 903tm cards using a finger prick technique at each of the three health centre visits for each child. A third subnational nOPV2 campaign was conducted after the last visit, due to continued detection of cVDPV2, but this campaign did not affect the analysis in this study because it occurred after all blood samples were collected and the timeframe was outside of the approved protocol.The DBS cards were sent to the Centers for Disease Control and Prevention in Atlanta, GA, USA and were tested for the presence of poliovirus neutralising antibodies using standard microneutralisation assays.The primary endpoint of the study was to assess change in seroprevalence and seroconversion against poliovirus serotype 2 after one and two doses of nOPV2 administered as part of outbreak response campaigns. The secondary endpoint was to describe seroprevalence against poliovirus serotypes 1 and 3.Seropositivity for each serotype was defined as the reciprocal titre of poliovirus neutralising antibodies of 8 or more. Seroconversion was defined as the change from seronegative to seropositive  in children with antibodies at baseline. Boosting was defined as at least a 4 times increase in reciprocal titres in children that were seropositive at the initial timepoint of comparison. Analyses of seroconversion and boosting are both restricted to children aged 6 months or older at enrolment ; boosting analysis was further restricted to children with initial antibody titres of 362 or less to show a 4 times increase given the maximum reported titre of 1/1448.The total sample size was calculated to be 215 children. We assumed a 30% dropout rate during follow-up (estimated higher due to the COVID-19 pandemic) and laboratory analysis and a baseline seroprevalence of 40% to provide 90% power to detect a change in seroprevalence of at least 15%.2 test is used to determine if there was a statistically significant difference between seroprevalence across different timepoints. For univariate risk analysis of seroconversion, binomial generalised linear models were generated to calculate statistical significance.Seroprevalence, seroconversion, and boosting are presented in percentages with binomial 95% CIs. Median antibody titres are provided with IQR. Pearson's \u03c7WHO employees participated in the study design, data collection, data analysis, data interpretation, and writing of the report.%) were enrolled  at enrolment, with 19 (8%) of 228 children born between Jan 1 and April 30, 2016, who received tOPV instead of bOPV in their routine immunisation . 222 (97At visit 1 (baseline visit), 53 of 204 children (26% [95% CI 20\u201333]) with analysable samples were seropositive against poliovirus type 2 . BaselinAfter one dose of nOPV2 the seroprevalence against type 2 poliovirus increased to 77%  and after two doses of nOPV2 the seroprevalence against type 2 poliovirus increased to 83%  at visit 1, 7\u00b777 (3\u00b717\u201310\u00b750) at visit 2; and 7\u00b717 (3\u00b783\u201310\u00b717) at visit 3. The limits of detection of the assay are 2\u00b750 log2 and 10\u00b750 log2.The reverse cumulative antibody titres against serotype 2 at each of three visits are shown in ; 68\u201383; vs 44%; p= 0\u00b7010). The proportion of children that were boosted between visit 1 and 2 was 58% ; between visit 2 and 3 was 9% , and between visit 1 and 3 was 54% .Rates of seroconversion were calculated for children older than 6 months with three analysable samples (n=182). The proportion of children that seroconverted between visit 1 and 2 was 67% , between visit 2 and 3 was 44% , and between visit 1 and 3 was 77% . There was no significant difference in the rate of seroconversion between boys  and girls  or in children who were reported as receiving a dose of IPV  or those who did not receive IPV . We hypothesise that some children had been immunologically primed by one IPV dose but had not seroconverted and that these children readily responded to the first nOPV2 dose.In our study, we observed that the first nOPV2 dose resulted in significantly higher seroconversion rates than the second dose  and does not correspond with the reported IPV coverage in our study (88%), or with WHO/UNICEF estimates (97%). Type 2 seroconversion after a bOPV schedule with a single dose of IPV administered at 3\u20134 months has been measured between 50% and 80%; therefore, our results suggest that IPV in Tajikistan achieved a lower seroconversion or that the coverage was overestimated.% and doeWe observed that seroconversion rates were higher in the capital city Dushanbe (>90%) than in other districts (around 66%) and that this difference was borderline significant (p=0\u00b7048). This difference could indicate that the cVDPV2 circulation in Dushanbe was more intense than in the other areas surveyed, resulting in higher seroconversion in Dushanbe from a combination of the nOPV2 vaccine and natural infection with cVDPV2. The environmental surveillance site in Dushanbe consistently detected positive samples for cVDPV2 during the study period; however, environmental surveillance was not established elsewhere for comparison. Demographic or health disparities  between the capital city and other districts that affect vaccine performance could also be considered.We demonstrated uniformly high seroprevalence against poliovirus types 1 and 3, indicating successful implementation of routine immunisation with bOPV in Tajikistan . There were also no significant differences in type 1 and 3 seroprevalence rates following nOPV2 vaccination as expected.Our study had some limitations. The study was conducted under extraordinary circumstances in Tajikistan during a peak of the COVID-19 pandemic and active poliovirus outbreak, which affected availability of health-care personnel for this study. The study used simple random sampling and was conducted in areas that were affected by the active cVDPV2 outbreak; therefore, it is possible that individuals might have serologically converted in response to infection with cVDPV2. Although no cases of acute flaccid paralysis were reported in study participants, the study did not monitor shedding of cVDPV2 in participants or household contacts; however, evidence suggests that in areas with active transmission, only a small proportion of children in the community excrete poliovirus at any time.The cVDPV2 outbreak in Tajikistan has been officially closed by WHO and the Tajik Ministry of Health, with the last detected isolate in an environmental sample from Dushanbe on Aug 13, 2021. Our study demonstrates that high coverage campaigns with nOPV2 provide an immune response against cVDPV2 and probably interrupted transmission in Tajikistan, providing evidence that nOPV2 vaccine is an appropriate tool to interrupt cVDPV2 transmission during outbreaks. Phase 3 clinical trials and long-term evaluation of safety and genetic stability of nOPV2 need to be performed, to enable consideration of full prequalification from WHO for the nOPV2 vaccine.Individual participant data will not be made publicly available. Contact the corresponding author for inquiries regarding data.We declare no competing interests."}
+{"text": "In the published article, there was an error. Due to an error during production, references in the text relating some tables were incorrect.Abstract, \u201cMethods.\u201d This sentence previously stated:A correction has been made to \u201cA total of 40 nurses from four primary and secondary hospitals in Jiangxi Province and 100 patients treated with\u2026\u201dThe corrected sentence appears below:\u201cA total of 40 nurses from four primary and secondary hospitals in Jiangxi Province and 80 patients treated with\u2026\u201dSubjects and methods, \u201cPatients,\u201d paragraph 1\u20134. This sentence previously stated:A correction has been made to \u201cA total of 100 patients in need of enteral nutrition treatment admitted from January 2020 to December 2021 were selected as the research objects. These data were obtained from four primary and secondary hospitals.Inclusion criteria: (1) Patient age >18 years; (2) indication of enteral nutrition; (3) nutritional risk screening 2002 (NRS2002) score >3; (4) normal cognitive ability; (5) hospital stay >1 week.Exclusion criteria: (1) The presence of intestinal diseases, such as intestinal ischemia, intestinal obstruction, and intestinal perforation; (2) patients with very severe craniocerebral injury or patients at a terminal stage of disease with a short life expectancy.P > 0.05).\u201dA total of 50 patients from January to December 2020 were selected as the control group, and 50 patients from January to December 2021 were selected as the study group. There were 25 male patients and 25 female patients in the control group, with an age range of 18\u201375 years (average age 43.58 \u00b1 10.53 years). The cases in the control group included 15 cases of cerebral infarction, 12 cases of acute pancreatitis, 12 cases of tumor, and seven cases of craniocerebral trauma. There were 27 female patients and 23 male patients in the study group, with an age range of 18\u201375 years (average age 44.25 \u00b1 9.86 years). The cases in the study group included 13 cases of cerebral infarction, 13 cases of tumor, 12 cases of craniocerebral trauma, and 12 cases of acute pancreatitis. The general data of the two groups were comparable  Patient age >18 years; (2) indication of enteral nutrition; (3) nutritional risk screening 2002 (NRS2002) score >3; (4) normal cognitive ability; (5) hospital stay >1 week.Exclusion criteria: (1) The presence of intestinal diseases, such as intestinal ischemia, intestinal obstruction, and intestinal perforation; (2) patients with very severe craniocerebral injury or patients at a terminal stage of disease with a short life expectancy.P > 0.05).\u201dA total of 40 patients from January to December 2020 were selected as the control group, and 40 patients from January to December 2021 were selected as the study group. There were 21 male patients and 19 female patients in the control group, with an age range of 18\u201375 years (average age 43.58 \u00b1 10.53 years). The cases in the control group included 15 cases of cerebral infarction, 12 cases of acute pancreatitis, 6 cases of tumor, and seven cases of craniocerebral trauma. There were 23 female patients and 17 male patients in the study group, with an age range of 18\u201375 years (average age 44.25 \u00b1 9.86 years). The cases in the study group included 13 cases of cerebral infarction, 3 cases of tumor, 12 cases of craniocerebral trauma, and 12 cases of acute pancreatitis. The general data of the two groups were comparable (The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The first sentence of the subsection Secondary malignancies (in Results) should read as follows: Three patients were diagnosed with a secondary malignancy during follow-up (Table 2).(2)the percentage of patients with seizures specified in the last column should be 21.9 (for n\u2009=\u200928). the total number of patients specified in the last column for Neuroendocrine sequelae should be qualified to include two patients with transient endocrine alterations.the percentage of LGG patients with Grade 0 hearing deficits should be 84.8 (for n\u2009=\u200939).In Table 1, the following corrections were required:"}
+{"text": "To this end, using induced neurons (i3Neurons) derived from human iPSCs lacking MAPK8IP3, we demonstrate that loss of hMAPK8IP3 affects endocytic uptake in neurons but does not affect the proteolytic activity of lysosomes in neuronal cell bodies. Our findings indicate that MAPK8IP3 may be a regulator of bulk endocytosis in neurons and that altered endocytic uptake may play a role in MAPK8IP3-linked neurodevelopmental disorders.Perturbations in endo-lysosomal trafficking pathways are linked to many neurodevelopmental and neurodegenerative diseases. Of relevance to our current study, MAPK8IP3/JIP3, a brain enriched putative adaptor between lysosomes and motors has been previously implicated as a key regulator of axonal lysosome transport. Since  De novo variants in MAPK8IP3, a putative adaptor protein believed to link cargo to dynein and kinesin motors , demonstrate that loss of this protein results in altered axonal lysosome abundance in all these models , also examined the effect of some of these variants on axonal lysosome abundance in C. elegans  derived from iPSCs lacking MAPK8IP3 , they did show defects in endocytic uptake of BSA and Dextran, cargoes that serve as markers for fluid phase endocytosis. The finding that loss of MAPK8IP3 does not adversely affect the proteolytic activity of lysosomes in neuronal cell bodies is as important as its potential role in regulating fluid-phase endocytosis. Given variants of MAPK8IP3 are linked to a neurodevelopmental disorder, identifying more neuronal cellular processes that are regulated by MAPK8IP3 will help us understand the neurodevelopmental pathology associated with the different variants.The study by Platzer and colleagues that identified 13 3Neurons were differentiated from iPSCs as described previously  coated with 0.1 mg/ml poly-L-ornithine (Sigma Aldrich) and 10 \u03bcg/ml mouse Laminin (Gibco). i3Neurons were differentiated for 14 days in Cortical Neuron Culture Medium containing KO DMEM F12 (Gibco) B27 supplement (Thermo Fisher), 10 ng/ml BDNF and NT3 (PeproTech), and 1 \u03bcg/ml mouse Laminin. i3Neurons were supplemented with Cortical Neuronal Culture Medium every 3\u20134 days during differentiation.The JIP3 KO/MAPK8IP3 KO iPSC line was previously generated using CRISPR-Cas9 gene editing  for a period of either 5\u20137 h or 2 h, followed by gentle washes [two times with warm imaging media (IM)]. They were then imaged live in IM at 37\u00b0C using a Zeiss 880 confocal microscope in Airyscan mode with a 60\u00d7 objective (NA 1.4). Healthy i3Neurons were identified using brightfield mode and imaged using the 561 and 488 lasers to capture fluorescence of DQ-Red BSA and BSA-488. Fluorescence intensity for each of the two channels was measured following outlining of individual cells using Image J software and the normalized ratio of DQ-Red BSA to BSA-488 intensity was computed for each cell  to label endo-lysosomes , avacuolar ATPase inhibitor, Bafilomycin A , was used , number of cells (n), statistical test used, and 3Neurons, as read out by fluorescence of DQ-Red BSA normalized to fluorescence of BSA-488 for each cell, revealed that the MAPK8IP3 i3Neurons do not have reduced lysosomal proteolytic activity compared to Control i3Neurons  was altered in these MAPK8IP3 KO i3Neurons compared to Control i3Neurons. We found a similar decrease in Alexa-647 Dextranfluorescence in MAPK8IP3 KO i3Neurons compared to Control i3Neurons  represent loss of function mutations. For instance, the study by Platzer and colleagues which used the CRISPR-Cas9 system to target six conserved positions in C. elegans, found that two of the six human alterations resulted in elevated axonal lysosome density while five affected locomotion  could also result from reduced axonal transport to the soma of cargo-loaded endocytic vesicles. However, similar BSA uptake experiments in the hippocampal neurons suggest that the number of BSA-loaded vesicles in axons is about ten percent of those in dendrites  into cells normally occurs In conclusion, our studies reveal a role for MAPK8IP3 in regulating endocytic uptake in human neurons. Future work will focus on elucidating other molecular machinery that acts in concert with MAPK8IP3 in this pathway, as well as whether MAPK8IP3 variants linked with intellectual disability affect this pathway in neurons.The original results presented in the study are included in the article/AS performed experiments and analyzed data. AS and SG designed experiments and wrote the manuscript. SG helped with data analysis and supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "A sphygmomanometer cuff was inflated to a pressure 30mmHg above diastolic pressure until StO2 reached a plateau and deflated to 0mmHg. Tissue oxygen saturation parameters were divided into resting StO2 (r-StO2) and minimal StO2 (m-StO2) at the end of the venous occlusion test. In patients, the cuff was inflated to a pressure 30mmHg above diastolic pressure for 5 min (volunteers\u2019 time derived) or until a StO2 plateau was reached. Tissue oxygen saturation parameters were divided into r-StO2, m-StO2, and the mean time that StO2 reached ScvO2. The StO2 value at the mean time was compared to ScvO2.Observational study in intensive care unit patients. Tissue oxygen saturation was monitored  with a multiprobe (15/25mm) in the thenar position. A venous occlusion test in volunteers was applied in the upper arm to test the tolerability and pattern of StO2 plateau was 7 \u00b1 1 minutes. We studied 22 patients. The mean time for StO2 equalized ScvO2 was 100 sec and 95 sec (15/25mm probes). The StO2 value at 100 sec  was then compared with ScvO2 (75 \u00b1 6%). The StO2 value at 100 sec correlated with ScvO2  without discrepancy (Bland Altman).All 9 volunteers tolerated the venous occlusion test. The time for tolerability or the StO2 during a venous occlusion test.Central venous oxygen saturation can be estimated from StO Measurement of SmvO2 is an established form of monitoring global DO2/VO2 balance (oxygen extraction) in adult intensive care.Failure of oxygen delivery leads to an increase in the extraction ratio of oxygen from the blood, resulting in a decrease in mixed venous saturation  has been advocated as a simple method to evaluate changes in the relationship between supply and demand oxygen in various clinical settings.,3 Therefore, ScvO2 measurements have become an established form of monitoring systemic tissue oxygenation in critically ill patients.,5 Its measurement requires a central venous line that should reside in the superior vena cava. Thus, blood sampling collected in this location reflects systemic oxygenation mainly from the upper part of the body. Venous oxygen saturation differs among several organ systems since they have different metabolic rates.,7However, the measurement of SmvO2 are not without hazards and can be infeasible in certain scenarios. Therefore, it would be useful to find a noninvasive technique to estimate ScvO2. Near-infrared spectroscopy (NIRS) offers a technique for continuous, noninvasive, bedside monitoring of tissue oxygenation. Near-infrared spectroscopy uses the principles of light transmission and absorption to measure the concentrations of hemoglobin noninvasively in tissues and provides a global assessment of oxygenation in all vascular compartments . It has been used to assess forearm skeletal muscle oxygenation during induced reactive hyperemia in healthy adults, and it produced reproducible measurements of tissue oxygenation during both arterial and venous occlusive events.-11 Using the venous occlusion test (VOT) method, NIRS can be applied to measure changes in tissue oxygen saturation (StO2) by following the changes in the concentrations of oxygenated and deoxygenated hemoglobin (HbO2 and Hb). In this method, a pneumatic cuff is inflated to a pressure above the diastolic and below the systolic pressure for a few seconds. Such pressure blocks venous outflow but does not prevent arterial inflow, unlike the standard vascular occlusion test. As a result, venous blood volume and pressure increase,,12 and the increased pooling of venous blood causes an increase in Hb.However, measurements of ScvO2 value. In view of these observations, the aim of this study was to test the hypothesis that NIRS with VOT could be used to estimate ScvO2 in a population of critically ill patients.Thus, it is reasonable to assume that NIRS will reflect this change by decreasing the StOThis prospective observational study was conducted in the intensive care unit (ICU) of a university hospital with 33 beds of the Erasmus Medical Center - Rotterdam, Netherlands. We enrolled consecutive adult (>18 years) critically ill patients within 24 hours of ICU admission who had undergone initial resuscitation and stabilization. All patients were mechanically ventilated and had a central catheter with the tip placed in the superior vena cava. The ICU has single-person closed rooms, and the ambient temperature in each patient\u2019s room was individually and actively set at 22\u00b0C. None of the patients had elevated bilirubin levels. We also recruited healthy volunteers with no history of receiving any vasoactive medication. The volunteers were instructed not to consume caffeine-containing drinks until after the experiments. The institutional review board approved the study. Each patient (or his or her relative) and healthy volunteer provided written informed consent.2-derived tissue oxygenation was continuously monitored using an InSpectra Tissue Spectrometer Model 650  with a multiprobe (15 and 25mm) over the thenar eminence.StO2 changes during VO. Venous occlusion was performed by arresting forearm blood flow using a conventional sphygmomanometer pneumatic cuff. All volunteers were seated with their arm rested on a table at the heart level, and StO2-derived tissue oxygenation was continuously monitored with the probe over the thenar eminence. The cuff was placed around the upper arm and was inflated to a pressure approximately 30mmHg greater than diastolic pressure until StO2 reached a plateau line or compression was not tolerated. The plateau or tolerance was considered the completion of the occlusion period, and then the cuff was rapidly deflated.Venous occlusion (VO) in healthy volunteers was designed to investigate how long a person can tolerate venous pooling of VO and to study the pattern of StO2 parameters were divided into two components: resting StO2 (r-StO2) and minimal StO2 (m-StO2) at the end of venous occlusion.VO-derived StO2 before initiation of the VOT. All measurements were made using a cooximeter. All patients had to have arterial saturation greater than 92% (Maximo Pulse Oximetry). Following the recording of ScvO2, StO2-derived tissue oxygenation was continuously monitored with the probe over the thenar eminence, and the VO test was then performed by arresting forearm blood flow using a conventional sphygmomanometer pneumatic cuff. The cuff was placed around the upper arm and was inflated to a pressure approximately 30mmHg greater than diastolic pressure for 5 minutes  or until a plateau line on the screen was reached. On the completion of the occlusion period, the occluding cuff was rapidly deflated. VO-derived StO2 parameters were divided into three components: resting StO2 values (r-StO2), the minimum StO2 value at the end of venous occlusion (m-StO2), and the time that StO2 reached the same ScvO2 of the patient (StO2 = ScvO2). The StO2 value at the mean time, when StO2 equals ScvO2, was subsequently compared with ScvO2.A blood sample  was collected from the central line placed in the superior vena cava to measure ScvOThe first measurement was performed within 24 hours of intensive care admission after hemodynamic stability was obtained  every 24 hours thereafter until Day 3.2, m-StO2, 100-StO2 and ScvO2 were linearly related to each other. To compare StO2 and ScvO2, we calculated bias, systemic disagreement between measurements (mean difference between two measurements) and precision (the random error in measuring [standard deviation of bias]).A sample size of 19 patients was estimated for a correlation coefficient of 0.6 . The results are presented as the mean \u00b1 standard deviation, unless otherwise specified. A paired t test was conducted to estimate significant differences after a normality test (Kolmogorov-Smirnov test). Bivariate correlation was used to determine whether r-StO A p value < 0.05 was considered statistically significant .The 95% limits of agreement were arbitrarily set, in accordance with Bland and Altman, as the bias \u00b1 1.96 standard deviations.2 in all volunteers in a linear pattern, and the release of the occlusion was followed by a rapid increase in StO2 (All volunteers (n = 9) tolerated the occlusion test well, and the average tolerance time was 7 \u00b1 1 minutes. The VO using a pneumatic cuff resulted in an immediate decrease in StO in StO2 . Conside2 and VO-derived StO2 (15mm and 25mm) for all patients. In four patients, equalization of StO2 and ScvO2 was never obtained.We included 22 ICU patients . All pat2 15mm and 25mm, respectively. Using 100 seconds as a time reference, we used the StO2 descend line of VO to select the exact StO2 value after 100 seconds of VO (100-StO2). That value was then registered and compared with ScvO2. Table 2 shows ScvO2 and VO-derived StO2 parameters stratified by the StO2 probe type. 100-StO2 was significantly correlated with ScvO2, and Bland Altman analysis revealed relevant agreement for both probes  or m-StO2 . At rest, ScvO2 values were lower than StO2 values but higher than minimal StO2 values (Table 2).We found a significant correlation between ScvO2) after a VO test moderately correlates with ScvO2. In addition, we have shown that the time to equalization of both values was close to 100 sec. This may be a method that can assess peripheral StO2 noninvasively and which can be repeated every few minutes and help to estimate ScvO2 or its trend.,12,14,15. In particular, Yoxall et al. and Wardle et al. two decades ago investigated peripheral venous hemoglobin saturation measured by using near infrared spectroscopy after venous occlusion of the forearm with partial venous occlusion to track changes in the balance between global oxygen demand and consumption in preterm infants and adults.,12,15The main finding of our study is that peripheral venous oxygen saturation  to be used in the patients. We assumed that this time length was reasonable to obtain the equalization of StO2 and ScvO2 in our patients. Similar to Yoxall et al., we have shown that the VOT was safe and feasible.,12The first part of the study evaluated VOT tolerability in healthy volunteers. We observed that the VO test until a StO2 and ScvO2 after 100 sec of the VOT. The mean time required for the StO2 value to equalize with the ScvO2 for most patients value was rather short (~100 sec), much less than the predefined 5 minutes VO duration, which makes the VO test rather accurate and safe. Central venous oxygen saturation and 100-StO2 were well correlated without significant discrepancy for both probes, which strengthens the use of StO2 after a VOT as an estimation of ScvO2.We found a moderate correlation between StO2 and m-StO2 were significantly correlated with ScvO2, but the relationship was somewhat weak. At rest, other investigators have shown a similar finding, particularly in nonseptic patients in whom the oxygen extraction capabilities are preserved.,16 Experimental and clinical studies on hypovolemic shock show that StO2 levels correlate with systemic flow variables.,17-20 However, there is uncertainty regarding this correlation when the patient is septic due to altered oxygen extraction capabilities, as has been shown previously after stagnant ischemia. In fact, half of our patients were septic, which could explain the limited correlation found in our study.Both r-StO2 values were lower than r-StO2, which is explained by two mechanisms. First, the peripheral arterial compartment is intact before stagnant ischemia. Second, critically ill patients, mainly septic patients, do show high StO2 because reduced cellular extraction of oxygen is common.,23 In turn, after the VOT, the m-StO2 values decreased considerably. We speculate that the StO2 falls below the ScvO2 during VOT because StO2 is related not only to oxygen consumption but also to the reactive vasoconstriction that occurs during vascular occlusion. Therefore, the decrease in StO2 is due to two factors: muscle VO2 and reactive vasoconstriction, which induces the sharpest decline in StO2 .Prior to the VOT, ScvO2  were followed by similar changes in 100-StO2 or m-StO2. However, 100-StO2 tracked ScvO2 on Day 2. We could not evaluate this relationship on Day 3 because only two patients were followed on the third day. This result, although imperfect to evaluate acute changes in ScvO2, suggests that the 100-StO2/ScvO2 relation is not affected by the clinical condition of the patient. It could be interesting to study this relationship in patients during acute resuscitation, as our patients were partially or fully resuscitated, as one can see by normal ScvO2 and lactate mean values.We did not evaluate whether dynamic changes in ScvOIt is important to emphasize that the results of tissue saturation found in the thenar muscle of one hand may not be reproducible in the other if the conditions of demand and/or oxygen consumption are different.2 if the patients are to mirror at least the venous saturation of the upper part of the body.The regional or peripheral venous saturation value can only be extrapolated as being SvcO2 after a VOT would rapidly track the changes in ScvO2. It is well known that significant changes in StO2 could occur after an intervention that induces ischemia and reperfusion. Our focus, however, was to assess the relation between StO2 after a VOT and ScvO2 in a single moment. Second, changes in ambient temperature at each patient\u2019s bedside were not measured. However, the ICU consists of one-person closed rooms, and the ambient temperature in each patient room was individually controlled at 22\u00b0C. Last, the duration of the VO test (5 minutes) was based on the volunteers\u2019 results, but we do not know whether it is appropriate in heterogeneous disease conditions. There is no support in the literature to show which method of VOT is superior or more reliable to assess the relation between ScvO2 and peripheral SvO2. In addition, we arbitrarily chose to use the StO2 value after 100 seconds of VOT based on our findings in healthy volunteers. Our strategy must be tested in different settings. Therefore, the results of our study cannot be extended to other studies that have addressed the ScvO2/StO2 relation.This study has some limitations that should be acknowledged. First, measurements during changes in the oxygen extraction ratio were not made in this study to investigate whether StO2 after a venous occlusion test in critically ill patients. We found that ScvO2 and StO2 correlate, but StO2 levels are accompanied by alterations in the peripheral circulation, indicating that StO2 abnormalities are related to regional hemodynamics and macrohemodynamics. Thus, it is not surprising that the correlation between both parameters is imperfect but still of clinical usefulness.We established the usefulness of the monitoring of StOIn conclusion, this study has shown the feasibility of frequent noninvasive measurements of peripheral venous oxygen saturation as an estimation of central venous oxygen saturation after a venous occlusion test. However, it is not yet advisable to recommend predicting absolute values of venous oxygen saturation for any given patient based solely on the noninvasive measurement of tissue oxygen saturation after a venous occlusion test, as the correlation was only moderate. Further clinical studies are required before it is considered a useful adjunct to the clinical monitoring setting."}
+{"text": "Oral squamous cell carcinoma (OSCC) is particularly prevalent in Taiwan. The goal of this study was to determine the clinicopathological role of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) proteins as an indicator of clinical outcomes in OSCC patients. In this study, immunohistochemical (IHC) analysis was used to examine IGF2BP2 protein expression in 244 OSCC patients. We investigated the relationships among IGF2BP2 expression, clinicopathological variables, and patient survival. Our results showed that IGF2BP2 cytoplasmic protein expression was significantly correlated with lymph node metastasis, cancer stage, and patient survival. Kaplan-Meier survival curves revealed that elevated cytoplasmic IGF2BP2 expression levels in OSCC patients were associated with poor overall survival. Moreover, multivariate cox proportional hazard models revealed that cytoplasmic IGF2BP2 expression, T status, and lymph node metastasis were independent prognostic factors for survival. In conclusion, IGF2BP2 protein was found to be a helpful predictive marker for OSCC patients, as well as a possible therapeutic target for OSCC treatment. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck region Insulin-like growth factor (IGF) and IGF-binding protein play a vital role in the premalignant oral lesions and oral cancer IGF2BP2 has been shown to promote tumor growth in cases of solid tumors and leukemia Nonetheless, there is a pressing need to further elucidate the function of IGF2BP2 protein expression and other clinical variables in OSCC. In the current study, immunohistochemical (IHC) analysis was used to examine the expression of IGF2BP2 proteins tissue samples from 244 OSCC patients. We also examined the relationship between IGF2BP2 protein expression and OSCC clinicopathological variables and prognosis. Finally, we sought to identify potential prognostic markers to facilitate the early detection of OSCC.n = 244) were recruited from Changhua Christian Hospital in Taiwan. The most common forms of treatment included tumor removal and radical neck dissection followed by post-operative irradiation. A number of patients also received 5-fluorouracil (5-FU) and cisplatin chemotherapy. This study was also approved by the Changhua Christian Hospital's Ethics Committee in accordance with Institutional Review Board guidelines . Prior to surgery, all OSCC patients provided written informed consent.Patients . On the following day, the TMAs were tested for immune complex using a LASB 2 Kit . The TMAs were stained using aminoethyl carbazole followed by hematoxylin to detect enzyme activity. The experiment involved a positive control (pancreatic cancer tissue as a known positive case) IHC staining was performed in accordance with our previous studies The clinicopathological variables of cytoplasmic IGF2BP2 protein expression and OSCC were assessed using Fisher's exact test or the Chi-Square test. The Kaplan-Meier method was used to create overall survival curves for OSCC patients with low and high cytoplasmic IGF2BP2 protein expression, and the log-rank test was used to estimate cumulative survival rates. The Cox proportional hazard regression model was used to confirm prognostic variables of OSCC using univariate and multivariate analyses after adjusting the stage, tumor size, lymph node metastasis and cell differentiation status. A p-value of <0.05 was used to identify statistically significant results Table The expression of IGF2BP2 in OSCC cancer tissue was examined via IHC staining. As shown in Figure The role of IGF2BP2 in tumor prognosis was elucidated in terms of the relationship between IGF2BP2 expression and the overall survival of OSCC patients. In Kaplan-Meier analysis, the survival curves of OSCC patients with high IGF2BP2 expression were lower than those with low IGF2BP2 expression (p=0.003) using log-rank tests (Figure Univariate and multivariate analysis based on the Cox proportional-hazards model were used to determine the degree to which independent prognostic factors of IGF2BP2 expression affect overall survival of OSCC patients Table . UnivariCancer has become one of the most common causes of mortality among middle-aged and elderly individuals. OSCC is among the most common malignant tumors of the head and neck. The high recurrence and metastasis of the disease pose a severe threat to human health and welfare Note that the specific mechanism underlying OSCC tumorigenesis has yet to be elucidated, and there are currently no reliable early indicators for the diagnosis or prognosis of OSCC in vitro experiments Researchers have reported that IGF2BP2 is elevated in cases of malignancy. IGF2BP2 levels can also use as a prognostic indicator of acute myelocytic leukemia in vitro and in vivo testing, will be required to corroborate our data and assess the efficacy of IGF2BP2 as a therapeutic target.In the current study, we used clinical tissue samples from OSCC patients to characterize the connection between IGF2BP2 and clinicopathologic indicators. High cytoplasmic IGF2BP2 expression was strongly linked to disease stage and survival. The connections between positive IGF2BP2 protein expression and lymph node metastases, as well as between IGF2BP2 and AJCC cancer stage, suggest that IGF2BP2 may play a role in OSCC metastasis Table . Our finIn conclusion, our findings demonstrate that IGF2BP2 protein expression is prevalent in OSCC tissues, and that protein expression levels were associated with histological grade, T status, lymph node metastasis, disease stage, and survival. Our results from 244 OSCC patients show a strong link between IGF2BP2 protein levels and survival rates. Our results also show that IGF2BP2 protein expression could potentially be used as an independent OSCC prognostic predictor and/or therapeutic target for OSCC treatment."}
+{"text": "Special AT-rich sequence-binding protein 2 (SATB2) is a new marker that could identify the colonic origin, but whether its expression is preserved in metastatic colorectal carcinomas (CRCs) remains unclear. This study was designed to investigate SATB2 validity in the identification of CRC either alone or in combination with caudal-type homeobox 2 (CDX2) and/or cytokeratin 20 (CK20). Moreover, we examined the concordance of SATB2 expression in primary CRC and paired metastatic specimen. Immunohistochemical expression of SATB2, CDX2 and CK20 was evaluated in primary CRC, 50 paired metastatic CRC and 80 non-CRC specimens. This study demonstrated that the ideal SATB2 cut-off value for recognising colonic from non-colonic origin was 10%. SATB2 was more sensitive and specific than CK20. However, it was more specific but less sensitive than CDX2. Analysing the combined markers expression, SATB2 and CDX2 combination revealed better sensitivity, specificity and larger area under curve compared to SATB2 alone, CDX2 alone and combined CDX2 and CK20. Moreover, SATB2 was able to retain its expression at the metastatic sites. SATB2 was totally concordant between primary CRC and their paired metastatic sites (concordance rate = 100%) with perfect level of agreement. SATB2 could be considered as an accurate diagnostic marker of primary and metastatic CRC. SATB2 and CDX2 is the best combination serving the highest sensitivity and specificity in detection of CRC. Colorectal cancer (CRC) is the third most prevalent cancer in both men and women and the fourth most prominent cause of cancer-related deaths worldwide . Patholoet al [It is well-established that CRCs consistently express cytokeratin 20 (CK20) and caudal-type homeobox 2 (CDX2) . Howeveret al . Taken tThe special AT-rich sequence-binding protein 2 (SATB2) is a transcription factor that binds to the nuclear matrix attachment region of DNA and hence regulates transcription and chromatin remodelling . SATB2 wThis study was designed to investigate the validity of SATB2 in the identification of CRCs either alone or in combination with CK20 and/or CDX2. Moreover, we examined the concordance of SATB2 expression in primary CRC and their paired metastatic CRCs specimens.This is a retrospective study that was carried out at the Pathology Department, Faculty of Medicine, Tanta University, during the period from April 2019 to December 2021. It included 70 cases of primary CRC as well as 80 cases of non-CRC. In addition, 50 paired metastatic specimens of the primary CRC cases  were obtained so as to analyse the concordance of SATB2 expression between primary and metastatic CRCs. The included non-CRC cases exhibited either an intestinal morphology or were poorly differentiated carcinomas. They were incorporated as follows: lung adenocarcinoma (10 cases), oesophageal adenocarcinoma (3 cases), gastric adenocarcinoma (7 cases), small intestine adenocarcinoma (7 cases), pancreatico-biliary carcinoma (8 cases), hepatocellular carcinoma (8 cases), female genital tract adenocarcinoma (19 cases), prostatic adenocarcinoma (6 cases), breast carcinoma (6 cases) and urinary bladder adenocarcinoma (6 cases). The study was approved by the Institutional Research Ethics Committee (reference # 32895).For primary CRC cases, clinical data as well as tumour-related features  were gathered from the accompanying pathology reports and medical records. Primary CRC cases were graded following the 2-tiered grading system into low and high grades, as recommended by the World Health Organization classification . PatholoFor each studied specimen, areas that adequately represent tumour tissues with appropriate preservation were identified. Necrotic areas and those with crushing artefacts were excluded. Tissue microarray (TMA) recipient blocks (6\u00d74 arrays) were produced using the TMA builder mold . This is followed by insertion of two tissue cores from areas of interest on paraffin blocks of the studied specimens into the holes on the recipient blocks to form TMA Blocks.TM FLEX Target Retrieval Solutions was used reaching 97\u00b0C for 20 minutes. Immunostaining was accomplished using Dako Autostainer Link 48. Antibodies included in this study were SATB2 rabbit monoclonal antibody , CDX2 mouse monoclonal antibody  and CK20 rabbit monoclonal antibody . In brief, slides were kept in Peroxidase-Blocking Reagent for 5 minutes, incubated with primary antibodies for 20\u201330 minutes, horseradish peroxidase polymer reagent for 20 minutes and diaminobenzidine chromogen/substrate working solution for 10 minutes. Lastly, counterstaining with haematoxylin was performed.Sections (5 \u00b5m thick) mounted on positively charged slides were left to dry for 30 minutes at 37\u00b0C. Deparaffinisation and antigen retrieval were carried out using Dako PT Link unit. High pH EnVisionPositive staining was identified as brownish nuclear staining for both SATB2 and CDX2, whereas cytoplasmic and/or membranous staining was considered for positive CK20 expression. For markers scoring, percentages of positive tumour cell were considered regardless of the staining intensity. Receiver operator characteristic (ROC) curve was plotted to identify the best SATB2 cut-off point for diagnosis of CRC. The percentage located close by the point that provides maximum sensitivity and specificity was selected as the cut-off point . CDX2 anData were statistically analysed using the Statistical Package for the Social Sciences software version 23. Categorical variables were expressed as frequencies whereas numerical variables were expressed as mean \u00b1 SD. Accuracy, specificity, sensitivity, positive and negative predictive values (NPVs) were used to assess diagnostic values of the tested markers. The histopathologic diagnosis was considered the gold standard.ROC curve was used to select the best cut-off point for SATB2 through assessing the diagnostic values of different percentages of SATB2 expression. Areas under the ROC curve (AUC) of each marker were calculated; higher AUC values indicate better test performance. For evaluating the level of agreement between marker expression in primary CRC and their paired metastatic specimens, concordance rate and Kappa coefficient (\u03ba) were applied.This study included 70 primary CRC cases with a mean age of 55.47 \u00b1 12.78 years. Forty-five cases (64.3%) were male. Tumours were located in left colon in 31 cases (44%) and exhibited fungating appearance in 26 cases (37%). Conventional adenocarcinoma constituted the predominant histologic type (42 cases (60%)) and high-grade features were identified in 42 cases (60%). Vascular and perineural invasion were detected in 22 cases (31.5%) and 17 cases (24.2%), respectively. Tumours were associated with nodal involvement in 53 cases (75.8%) whereas distant metastasis was present in 24 cases (34.3%). As regard TNM staging, Stage III was the most frequent (29 cases (57%)). Clinicopathologic data of the studied cases are represented in Representative figures for the expression of SATB2, CDX2 and CK20 in primary CRC, paired metastatic CRC and non-CRC cases are demonstrated in p < 0.001). SATB2 had 97% positive predictive value (PPV), 95% NPV and diagnostic accuracy of 96%. The ideal SATB2 cut-off value for distinguishing colonic from non-colonic cases, in this study, was 10%.ROC curve analysis of SATB2 expression alone provided 94.3% sensitivity, 97.5% specificity and 0.975 AUC  but weaker specificity (87.5%) whereas CK20 positivity revealed the lowest sensitivity and specificity for detection of primary CRCs versus non-CRCs (sensitivity 91.4% and specificity 86.5%) as demonstrated in Next, we evaluated whether combinations of these three markers could improve the identification of a CRC origin. It was demonstrated that combining SATB2 and CDX2 revealed better sensitivity (98.5%), specificity (98.8%) and larger AUC (0.998) compared to SATB2 alone , CDX2 alone  and combined CDX2 and CK20 .When combining SATB2 and CK20, the same sensitivity as combined CDX2 and CK20 (97.1%) was obtained, but it was better than that of SATB2 alone (94.3%) and CK20 alone (91.4%). The specificity of this combination was the same as SATB2 alone (97.5%) but better than CK20 alone and combined CDX2 and CK20 .Combining the three markers provided better sensitivity (98.5%) and specificity (98.8%) than combining SATB2 and CK20 (sensitivity 97.1% and specificity 97.5%) and combined CDX2 and CK20 (sensitivity 97.1% and specificity 96.3%). Whereas it had the same sensitivity and specificity of SATB2 and CDX2 combination. Both showed the highest sensitivity (98.5%) and specificity (98.8%) as illustrated in In order to evaluate the concordance of SATB2 expression between primary and metastatic CRCs, achievable paired metastatic specimens (50 cases) were obtained. SATB2 expression was totally concordant between primary CRCs and their paired metastatic specimens (concordance rate 100% and \u03ba coefficient = 1 denoting perfect level of agreement). As regard CDX2 and CK20, two cases lost CDX2 expression (concordance rate 96% and \u03ba coefficient = 0.648 indicating good level of agreement) and three cases lost CK20 expression (concordance rate 94% and \u03ba coefficient = 0.638 reflecting good level of agreement) in the paired metastatic specimens, whereas combining CDX2 and CK20 revealed concordance rate of 98% and \u03ba coefficient of 0.790 denoting excellent level of agreement. Combining SATB2 to either CDX2 or CK20 improved the concordance (concordance rate 100% and \u03ba coefficient = 1 denoting perfect level of agreement) as outlined in CRC metastases often simulate the histological patterns of other adenocarcinomas; differentiation between these metastases can be difficult especially in poorly differentiated adenocarcinomas . There iAnalysing SATB2 expression in primary CRC cases, the current study demonstrated that SATB2 was detected as a brownish nuclear staining in 66 94.3%) primary CRC specimens and only in 2 2.5%) non-CRC specimens. SATB2 sensitivity obtained in this work (94.3%) was close to other studies in which the sensitivity ranged from 90% to 99% % primary, 15\u201318. % non-CRCet al [et al [et al [From ROC curve analysis, in this study, the best SATB2 cut-off value that afforded the highest sensitivity and specificity for identifying the colonic origin was 10%. Lin et al  and De Ml [et al  reportedl [et al  consideret al [The cases included in this work were mostly conventional adenocarcinoma, as well as mucinous and signet ring cell carcinomas. Li et al  focused et al [et al [Considering the validity of the examined markers individually, this study achieved that SATB2 was more sensitive than CK20 but less sensitive than CDX2 in detection of CRC. However, SATB2 was more specific than CDX2 and CK20 similar to the findings of other reports , 26, 28.l [et al  who concCombination of different markers could improve their validity in the identification of the colonic origin. Therefore, this work was extended to investigate the validity of SATB2 in combination with CDX2 and/or CK20 in the diagnosis of CRC. Combining SATB2 with CDX2 or CK20 provided better sensitivity, specificity and larger AUC compared to SATB2, CDX2 and CK20 individually.Moreover, it was found that combining SATB2 with CDX2 provided better sensitivity, specificity and larger AUC than the commonly used combination in practice (CDX2 + CK20). It worth noting that the triple combination of SATB2, CDX2 and CK20 offered the same sensitivity, specificity and AUC as the double SATB2 and CDX2 combination. So, the current work suggested that the double combination of SATB2 and CDX2 was the best combination serving the highest sensitivity and specificity in detection of CRC.et al [et al [et al study [Similar results were detected by Dabir et al  and Salil [et al  who repoal study  reportedTo the authors\u2019 knowledge, this is the first study to analyse the level of agreement of SATB2, CDX2 and CK20, either individually or combined, between primary CRCs and their paired metastatic specimens. In the current study, SATB2 was able to retain its expression in the corresponding metastatic lesions with a perfect level of agreement as all SATB2 positive primary CRC specimens retained SATB2 expression in their metastatic specimens. Whereas either CDX2 or CK20 alone provided only a good level of agreement as CDX2 positivity was lost in two paired metastatic specimens while three metastatic specimens lost CK20 expression. Moreover, adding SATB2 to CDX2 and/or CK20 improved their ability to detect the colonic origin in the metastatic specimens.SATB2 provides high sensitivity and specificity for establishing or ruling out the diagnosis of CRC. The optimal SATB2 cut-off value that afforded the highest sensitivity and specificity for discriminating colonic from non-colonic origin is 10%. SATB2 and CDX2 is the best combination for identifying CRC. Adding SATB2 to CDX2 and/or CK20 improves their ability to detect the colonic origin in metastatic specimens. SATB2 is able to retain its expression at the metastatic sites.The authors have no conflicts of interest to declare.The authors did not receive support from any organisation for the submitted work.The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.MSE: Data collection and interpretation of the histopathologic and immunohistochemical stained slides, captured the figures provided in this study, interpretation of the statistical analysis, drafting and writing the manuscript. DMG: Participated in interpretation of the histopathologic and immunohistochemical stained slides, statistical analysis and its interpretation, drafting and writing the manuscript. YAZ: Study design, participated in interpretation of the histopathologic and immunohistochemical stained slides, revision of the draft of the manuscript. AGN: Study design, supervision of the study, revision of the draft of the manuscript. FAE: Study design, supervision of the study, revision of the draft of the manuscript. All authors read and approved the final version of the manuscript.Protocol was approved by the Research Ethics Committee in Faculty of Medicine, Tanta University (reference# 32895/01)."}
+{"text": "Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3\u2032 (2\u2032), 5\u2032-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR  Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat.The highly conserved  It provides on average 19% of the total calories in our diet and is one of the most important sources of plant protein. Bread wheat is unique among cereal crops in that it contains gluten which enables the production of leavened bread and is a major ingredient in numerous other foods [Wheat is a crucially important food source and one of the most widely grown crops in the world comprising approximately 17% of the world\u2019s arable land and a global production of ~700 million tons , has emerged as a powerful tool for the precise manipulation of eukaryotic genomes including those of important crop plants ,12,13,14Arabidopsis and tobacco [The CRISPR/Cas system was initially shown to efficiently mediate targeted double stranded breaks in model plants such as  tobacco ,16, but  tobacco ,21,22,23 tobacco ,13,14,24Sal1 gene encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3\u2032 (2\u2032), 5\u2032-bisphosphate nucleotidase enzyme activity that is hypothesized to be a plastid-localized oxidative stress sensor [Arabidopsis has shown that mutant plants lacking a functional Sal1 , exhibit drought stress tolerance [Sal1 plants exhibit a reprograming of their metabolism resulting in an increase in osmoprotectant compounds and the stress hormone abscisic acid, which manifests itself as abiotic stress tolerance [Sal1 gene also has been implicated in controlling reactive oxygen species levels and leaf senescence in Arabidopsis [Sal1 expression in wheat using a viral-based expression system has suggested that wheat lacking or with reduced Sal1 activity also exhibits drought tolerance under short-term water stress [The s sensor ,27,28,29olerance . The Salbidopsis . The trar stress .Sal1 in hexaploid bread wheat, CRISPR gene editing was used to introduce mutations within the gene family. The generation of transgenic Bobwhite wheat plants, their molecular characterization including the identification of targeted mutations and the characterization of the growth, morphology and response to abiotic stress in the mutants is presented.To investigate the role of Sal1 is a highly conserved single copy gene in the genomes of the model plants Arabidopsis thaliana and Brachypodium distachyon [Sal1 genes in wheat. stachyon . Modern Sal1: 5ABD, 7AD, and two genes on 4A . The pair of genes on 4A are less than 50 kilobase pairs (kb) apart and are highly similar except for a single base pair deletion in the seventh exon that causes a frameshift and premature stop codon in the 4A-1 allele. Although this is near the end of the coding sequence, the high degree of evolutionary conservation in this region implies that 4A-1 may be a pseudogene. All other Sal1 homeologs appear to encode a functional protein sequence. In Chinese Spring, preliminary analysis found seven different homeologs of Sal1 5D allele covering conserved amino acids in the fourth exon was BLAST searched against several other available wheat genomes , the uncultivated subspecies dicoccoides (accession Zavitan) has three copies on chromosomes 7A, 5A, and only a single functional copy on 4A (4A-2), while the cultivated subspecies durum (cv. Svevo and Kronos) also have 5B and the 4A-1 genes. Triticum spelta (accession PI190962) and Triticum aestivum cv. Norin 61 have the same seven alleles as Chinese Spring, however, most other cultivars of Triticum aestivum have lost the 5A homeolog of Sal1. The recently bred Canadian cultivars CDC Landmark and CDC Stanley have five Sal1 homeologs; they are missing 4A-2 and instead have a 4A-1 homeolog that does not have the single base pair deletion found in other wheat genomes.To investigate the evolution of this small gene family in other wheat varieties, a 203 base pair conserved section of the Chinese Spring  genomes . The AegSal1 homeologs have expression data except 7D, which was not annotated in the available datasets. Moreover, 5D is the most highly expressed gene and 5A has the lowest expression, with others being intermediate    targetinweb tool . These t alleles A, with on plants , mutatioSal1 targeting construct, the guide RNAs are flanked by transfer RNA (tRNA) spacers and are expressed using the switchgrass Ubiquitin1 (PviUbi1) promoter with the nopaline synthase (nos) transcription terminator [Cas9 gene is constitutively expressed under the control of the maize Ubiquitin 1 promoter (ZmUbi1) and nos terminator  assay to identify introduced mutations  and five of these events generated T1 progeny where the mutations were inherited. Three events carried mutations within both CAPS assays . These selected events were then propagated to the T2 and T3 generations for further characterization  in phenotyping experiments.One of the heritable events appeared to be a chimeric TSal1 target site, with the exception of the 7D site that contained a single mismatch with the 5\u2032 gRNA. Several major rearrangements in these two events included the insertion of transgenic DNA  and 6KO2, carried mutations in every sequenced enic DNA . The incmed rice ,44,45. ASal1 5B and 5D alleles, but otherwise carried small indels in most target sites. The T0 plant was notably smaller and less fertile than other transformants, possibly as the result of somaclonal variation or the presence of unique mutations. Unlike 6KO1 and 6KO2, not all Sal1 target sites were mutated in this line; however, all sequenced genes appear to produce a nonfunctional protein. The only mutation in this line that did not result in a frameshift occurred within 4A-1 (which may be a pseudogene) and resulted in the loss of a single highly conserved amino acid that may also compromise function. This mutant line was called MM (for multiple mutant).A third independently transformed event had several missing regions in the Sal1 mutations (based on the CAPS assay screening), but were herbicide sensitive and lacked detectable transgenes. As Cas9 was no longer present to create further mutations, we used these lines to investigate the effect of specific mutations within this portion of the Sal1 gene family. These nontransgenic Sal1 mutant lines were named PM1  and PM2. PM1 has two different mutations in the 7A alleles and the 4A-1 gene targets could not be amplified. The PM2 event was missing both the 4A-1 and 4A-2 target sites and the 7D gene had a 1 bp insertion within the 5D 3\u2032 target site. Thus, the 7A and 5B genes were the only unmutated homeologs in PM2, while PM1 had intact 5B, 5D, 4A-2, and 7D Sal1 genes  and their growth was measured and monitored during development  and a regeneration control line  were not segregating in these plants. Given that PM1 only has target mutations in the 7A and 4A-1 genes, and the PM2 line lacks the phenotype , this suggests that the Sal1 7A homeolog may have a role in stem growth and morphology in Bobwhite wheat. Although this developmental phenotype was consistently observed . Both the mutant and control lines also grew similarly well under well-watered conditions  stayed green longer than the control lines and the soil in their pots appeared to dry out more slowly .Drought in the field most often occurs during wheat flowering and seed development, so we examined the performance of the e slowly . All line slowly . These rSal1 mutant lines, their growth was examined when grown in the same pot as an equal number of wildtype Bobwhite plants, both under well-watered and drought conditions. Under these conditions the plants within the pot are competing for the same resources and should be subjected to the same degree of water stress. The 6K01, 6KO2, MM and PM1 lines co-grown with wildtype Bobwhite in a shared pot had significantly less aboveground biomass than when grown in their own pot, while Bobwhite\u2019s biomass levels were unchanged in shared or single pots , which is an encouraging sign that CRISPR-Cas9 sequence recognition in the research presented was still highly specific.Creating gene knockouts is far more difficult in large, complex polyploid genomes such as bread wheat than in plant model systems. The multiple-targeting CRISPR-Cas9 gRNA strategy we used was successful in creating a variety of heritable Sal1 7A may have an important role in stem growth in Bobwhite. Interestingly, although Sal1 7A is not strongly expressed in shoot apical meristem compared to some of the other homeologs  showed that at least six of the seven alleles were expressed; 7D was not annotated as a gene and there is no evidence of its transcription. The expression data (www.genevestigator.com) on 19 April 2022. A 216 base pair fragment of the T. aestivum Chinese Spring Sal1 5D allele covering Exon 4, including the 5\u2032 gRNA target site, was used to interrogate the high-quality wheat genome sequences available at GrainGenes . This region is highlighted in the Bobwhite Sal1 5D sequence shown in Bioinformatics analysis of the publicly available wheat genome sequence initially revealed seven ion data  was accehttps://primer3.ut.ee/; accessed 5 October 2021; Sal1 covering the three CRISPR/Cas9 target sites [Sal1 alleles and a potential Bobwhite 5A homeolog (which was not detected). Sanger sequence reads were aligned with the Chinese Spring sequence and annotated using Snapgene . Genomic DNA was extracted from Bobwhite wheat leaves using a modified Puregene kit protocol . Chinese Spring genomic sequences were used in Primer3 , and A. thaliana (AT5G63980) proteins were manually trimmed to remove the regions that were not sequenced in the Bobwhite Sal1 homeologs. The protein alignment was performed using Clustal Omega (CLUSTAL O (1.2.4)) at https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed 11 April 2022) with the default parameters. Note that the Sal1 5B sequence had a gap starting at residue 60, since we were unable to amplify and sequence that portion of the Bobwhite 5B sequence, likely due to the presence of a large intron nearby that contained a sequence we could not amplify across.For the lignment , T. aestSal1 transformation construct was constructed using standard restriction enzyme cloning techniques. The map and sequence of the plasmid is shown in Sal1-targeting guide RNAs  flanked by transfer RNA (tRNA) spacers, which are expressed using the switchgrass Ubiquitin 1 promoter and the nopaline synthase transcription terminator. The Cas9 gene is constitutively expressed in wheat using the maize Ubiquitin 1 promoter and nos transcriptional terminator.The CRISPR Ubiquitin 1 promoter and nos terminator controlling the expression of the bar selection gene which confers resistance to the bialaphos herbicide [\u00ae herbicide  diluted in water 1:128 . A paintbrush was used to apply a thin layer of the diluted herbicide to a 2 cm long area of a fully expanded young wheat leaf. A wildtype Bobwhite plant of the same age was painted at the same time as a control. The edges of the painted sections were marked with permanent marker, and the leaves were monitored for 3-5 days. The painted sections of transgenic herbicide-resistant leaves remained green, while wildtype sensitive leaves yellowed and/or became necrotic. Thirty-two independent Bobwhite spring wheat transgenic events carrying the CRISPR/Cas transformation construct were generated from these experiments out of 2,945 bombarded immature embryos.Transformation experiments were performed with a mixture of the CRISPR transformation plasmid construct and the pAHC20 plasmid which carries the maize erbicide . Bobwhiterbicide ,50,51. Ierbicide . After aerbicide ,50,51. Nerbicide  and geno0 wheat plants were screened using a Cleaved Amplified Polymorphic Sequences (CAPS) assay of the 3\u2032 target site  and 8 h nights (minimum 55 \u00b0F). Plants were grown in Sunshine Mix #1 (Sungro Horticulture) in equal-sized pots (9.5 \u00d7 9.5 inches) and watered twice daily with fertilizer water (Peter\u2019s 20-20-20 diluted as per instructions) administered by 2 drip lines per pot. During the first three weeks of growth, plants were sprayed with a baking soda solution twice weekly to control powdery mildew . Sal1 gene expression was normalized relative to the expression of the housekeeping gene CJ705892 [Sal1-specific primers were designed in Primer3 [For RNA experiments, 6 seeds of each line were planted per pot, then thinned to 5 per pot 7 days after planting. The youngest leaf on each plant was harvested 21 days after planting (approximately Zadoks stage 21-22) and snap frozen in liquid nitrogen and stored at \u221280 \u00b0C until processing. RNA was extracted using the Zymo Quick-RNA Plant miniprep kit , and the TURBO DNA-free kit was used to remove DNA . Sample RNA concentrations were measured using a DeNovix spectrophotometer (USA); 3 ng/\u00b5L RNA was used for each qPCR reaction. Next, 10 \u00b5L qRT-PCR reactions were performed with the iTaq Universal SYBR Green One-Step Kit  in an Applied Biosystems Quantstudio 3 Realtime PCR System With 96 well 0.1 mL block  using the standard conditions recommended in the iTaq manual. Control reactions with no reverse transcriptase were run for each sample using the CJ705892 primers to confirm that there was no DNA contamination . Sal1 geCJ705892  using th Primer3 . SamplesSal1 mutant line were planted on one side, and three Bobwhite seeds were planted on the other, then thinned to two plants per side at 12\u201314 days. All pots in an experiment were randomly arranged (interspersed with each other) in a 5ft. \u00d7 5ft. area on a single greenhouse bench and rearranged weekly to minimize bench position effects. Plant growth  was recorded weekly. Stomatal conductance was measured with an SC-1 Leaf Porometer (Meter Group Inc.) multiple times during two independent drought experiments.Wheat was grown in a greenhouse as described in Terminal drought experiments were started 5.5 weeks after planting. At the start of the drought experiments, all pots were hand watered to saturation (maximum soil capacity), then irrigation was halted, and they were not watered again for the duration of the experiment. Two pots were grown per line per experiment, and those placed under the drought treatment was determined by a coin flip. Control plants were watered normally with drip irrigation until their primary stems began senescing, at which point the drip lines were removed and the plants were allowed to naturally senesce. Primary stems were removed intact at harvest; their height was measured with a ruler and internodal diameters were measured with a digital caliper. The total aboveground plant biomass was then harvested into paper bags. Drought treated plants were harvested in the same way once they were fully senesced and dry. Biomass was dried in a drying oven 4\u20135 days at 30 C, then weighed. Afterward, the seeds were threshed and counted by hand and weighed. Statistical significance was determined by ANOVA. Three independent experiments were performed for the shared pot and drought experiments."}
+{"text": "N\u2009=\u200927) including (n\u2009=\u200916) cloth masks (CMs), (n\u2009=\u20097) surgical masks (SMs), and (n\u2009=\u20094) N95 filtering facepiece respirators (FFRs), of which SMs and N95 FFRs taken as a standard for efficiency comparison were all tested against ambient aerosols (PM2.5 and PM10\u2009\u03bcg/m3). The prototype cloth masks (PTCMs) (N\u2009=\u20095) design was tailored, and their performance was assessed and compared with that of standard commercial masks. The filtering efficiency tested against ambient coarse particulates (PM10) ranged from (5% to 34%) for CMs with an average of 16%, (37% to 46%) for SMs with an average of 42%, (59% to 72%) for PTCMs with an average of 65%, and (70% to 75%) for N95 FFRs with an average of 71%, whereas against fine particulates (PM2.5), efficacy ranged from (4% to 29%) for CMs with an average of 13%, (34% to 44%) for SMs with an average of 39%, (53% to 68%) for PTCMs with an average of 60%, and (68% to 73%) for N95 FFRs with an average of 70%, respectively. The efficiency followed the order N95 FFRs\u2009>\u2009PTCMs\u2009>\u2009SMs\u2009>\u2009CMs showing poor exposure reduction potential in CMs and high exposure reduction potential in N95 FFRs and PTCMs. Amendment in existing CMs using eco-friendly cotton fabric with better facial adherence can protect human health from exposure to fine particulates <2.5\u2009\u03bcm and can reduce the risk of micro-plastic pollution caused by polypropylene (PP) facemasks.Inexpensive cloth masks are widely used to reduce particulate exposures, but their use became ubiquitous after the outbreak of COVID-19. A custom experimental setup (semiactive at 5.1\u2009m/s airflow rate) was fabricated to examine the efficiency of different types of commercial facemasks collected randomly from street vendors. The sample ( The bowl-shaped topographic structure in Kathmandu Valley is surrounded by mountains that are impediments to wind movement that retains particulates in ambient air , 2, whicLong-term policies such as shifting to clean energy and short-term policies such as population-level interventions such as the use of respiratory protective devices (RPDs) might be the two effective approaches to reduce particulate exposures and other harmful airborne contaminants , 17, 18.Anecdotal evidence showed that during the Manchurian epidemic, handmade masks of cotton gauze became useful for military barracks and healthcare workers when quality commercial masks were inaccessible , 36. SimVery few studies have been done in Nepal regarding evaluating the efficacy of facemasks. The study carried out by Shakya et al.  evaluateThis experiment was conducted in 2020 from February to March and August to November, consecutively, at the open ground of North Valley School, Kathmandu. As humans are naturally exposed to the ambient atmosphere, the experiment was conducted by extracting natural ambient aerosols in sealed setups .2.5) and coarse particulates (PM10) in real-time concentration (\u03bcg/m3) were extracted at 5.1\u2009m/s (stagnant air flow rate) and tested with two calibrated portable hand-held detectors  simultaneously between filtrate air (with facemask) and nonfiltrate air (without a facemask).This experimental setup was fabricated using a normal plyboard, polyvinyl chloride (PVC) pipe, computer fan, revolutions per minute (RPM) controller, and a 12\u2009V (volt) direct current (DC) charger. Ambient fine particulates (PMN\u2009=\u200927), which includes CMs (n\u2009=\u200916), SMs (n\u2009=\u20097), and N95 FFRs (n\u2009=\u20094), were randomly collected from street vendors. The SMs and N95 FFRs were used as standards in this study for efficiency comparison.Twenty-seven nasopharyngeal masks  fabric or tissue paper as per the user's convenience.10 ranged from 37% to 46%, with the lowest efficacy found on SM4 (37%), and the highest efficacy found on SM6 (46%)  of 54%. The average filtering efficacy of SMs against ambient PM10 was found to be 42%.The filtering efficacy of tested surgical masks (SMs) against PMM6 (46%) . The hig2.5 ranged from 34% to 44%, with the lowest efficacy found on SM4 (34%), and the highest efficacy found on SM6 (44%)  of 56%. The average filtering efficacy of SMs against ambient PM2.5 was found to be 39%.The filtering efficacy of tested SMs against PMM6 (44%) . The hig10 ranged from 5% to 34%, with the lowest efficacy found on CM13 (5%) and the highest efficacy found on CM14 (34%)  of 66%. The average filtering efficacy of CMs against ambient PM10 was found to be 16%.The filtering efficacy of tested CMs against PM14 (34%) . The hig2.5 ranged from 4% to 29% with the lowest efficacy found on CM 13 (4%) and the highest efficacy found on CM 14 (29%)  of 71%. The average filtering efficacy of CMs against ambient PM2.5 was found to be 13%.The filtering efficacy of tested cloth masks against PM14 (29%) . The hig10 ranged from 70% to 75%, with the lowest filtering efficiency found on N95 (i), N95 (ii), and NIOSH respirator (70%) and the highest filtering efficiency found on N95 (iii) (75%) at an airflow rate of 5.1\u2009m/s  of only 25%. The average filtering efficacy of N95 FFRs against ambient PM10 was found to be 71%.The filtering efficiency of N95 filtering facepiece respirators (FFRs) against PM 5.1\u2009m/s . The hig2.5 ranged from 68% to 73%, with the lowest filtering efficiency found on N95 (ii) (68%), and the highest filtering efficiency found on N95 (iii) (73%)  of 27%. The average filtering efficacy of N95 FFRs against ambient PM2.5 was found to be 70%.The filtering efficiency of N95 FFRs against PMi) (73%) . The hig10 ranged from 59% to 72%, with the lowest efficacy found on PTCM4 (59%) and the highest efficacy found on PTCM1 (72%)  of 28%. The average filtering efficacy of PTCMs against ambient PM10 was found to be 65%.The filtering efficacy of prototype cloth masks (PTCMs) against PMM1 (72%) . The hig2.5 ranged from 53% to 68%, with the lowest efficacy found on PTCM4 (53%), and the highest efficacy found on PTCM1 (68%)  of 68%. The average filtering efficacy of PTCMs against ambient PM2.5 was found to be 60%.The filtering efficacy of prototype cloth masks (PTCMs) against PMM1 (68%) . The hig10 were found at 42%, 16%, 65%, and 71%, whereas against ambient PM2.5 average efficiency of SMs, CMs, PTCMs, and N95 FFRs were found at 39%, 13%, 60%, and 70%, respectively  for particulate filtration at the stated airflow rate. The efficiency followed the order N95 FFRs\u2009>\u2009PTCMs\u2009>\u2009SMs\u2009>\u2009CMs showing poor exposure reduction potential in CMs and high exposure reduction potential in N95 FFRs and PTCMs.r) analysis between variables showed a significant positive correlation  between number of mask layers and efficiency (PM10) and correlation  between number of mask layers and efficiency (PM2.5), respectively (Karl Pearson's correlation (ectively .10 and PM2.5 are found to be significantly different from each other (p < 0.01), which rejected the null hypothesis of this study.One-way analysis of variance (ANOVA) test showed that the efficiency of all face masks against PM10 aerosols than PM2.5 due to the size and shapes of the aerosols [The efficacy of nasopharyngeal masks was estimated using different methods and techniques in previous studies, which contradict each other because different studies used different methods and experimental approaches whose findings varied from study to study although studied for the same subject . The appaerosols .Poor facial fit increases the particulate penetration level known as total inward leakage (TIL) , 52 at dThe efficacy of SMs was found better than that of CMs because it has similar surface characteristics as N95 FFRs embedded with a complex network of polypropylene (PP) nanofibers forming web-like structures interconnected with each other . HoweverN95 FFRs, characterized by a complex network of multiple layers of nanofibers forming a web-like structure, melt-blown filters, and better facial adherence over the face , 47, 62 The filtering efficacy of PTCMs against fine particulates ranging from 53% to 68% in our study has a close agreement with the filtering efficacy ranged from 20% to 60% against fine NaCl particles . The aveThese modified reusable CMs featured adjustable ear loops, pockets for installing replaceable filters, a nose pin for better facial fit, and some fabric layers for better filtration of particulate exposures, resulting in an efficacy almost equivalent to N95 FFRs. Such designs can be embraced by people when commercial facemasks are shortened or not available , 66.The filtering performance of CMs was found to have poor exposure reduction potential and is marginally beneficial to human health compared to SMs and N95 FFRs. However, few amendments in inexpensive cloth mask materials and design that fits replaceable filters inside, whether PP filter or tissue paper as per that user's convenience, with the installation of a few accessories like a nose pin and adjustable ear straps for better adherence to the human face resulted in efficacy almost equivalent to standard N95 FFRs. The findings suggest that PTCMs can be potential alternatives to expensive standard masks and can play a pivotal role in reducing harmful ambient particulate exposures. The findings of this study can be helpful for the government to formulate policies and guidelines for better use of eco-friendly facemasks as well as it can also help the public regarding the proper selection of facemasks. Eco-friendly facemasks with better efficacy should be brought into mass production to replace plastic-based facemasks to protect both the environment and human health.In the semiactive measurement experiment setup , the air"}
+{"text": "Glucose transporter 10 (GLUT10) is encoded by the SLC2A10 gene. Our recent investigations have shown that GLUT10 is not only involved in glucose metabolism but also involved in the body\u2019s immune response to cancer cells. However, the role of GLUT10 in tumor prognosis and in tumor immunity has not been reported.We knocked down SLC2A10 and performed transcriptome sequencing to analyse the biological function of GLUT10 and found that GLUT10 may be involved in immune signaling. Then, we studied the expression level of SLC2A10 in cancers by the Oncomine database and Tumor Immune Estimation Resource (TIMER) site. We also evaluated the prognostic potential of SLC2A10 in different cancers using the Kaplan\u2012Meier plotter database and PrognoScan online software. The correlations between SLC2A10 expression and immune infiltrates were analysed by TIMER. In addition, correlations between SLC2A10 expression and gene marker sets of immune infiltrates were analysed by TIMER and Gene Expression Profiling Interactive Analysis (GEPIA). Immunofluorescence staining of cyclooxygenase-2 (COX-2) and GLUT10 in lung cancer tissue and adjacent tissue was performed to confirm our findings from the database research.Knocking down SLC2A10 widely activated immune and inflammatory signaling. SLC2A10 was abnormally expressed in several tumors. The expression level of SLC2A10 was closely correlated with cancer prognosis. Low SLC2A10 expression was related to poorer prognosis and increased malignancy of lung cancer. Lung cancer patients with low expression of SLC2A10 have a much shorter median survival time than patients with high expression of SLC2A10. SLC2A10 expression is closely related to the infiltration of different types of immune cells, particularly macrophages. Both database research and lung cancer sample research revealed that GLUT10 might modulate immune cell infiltration via the COX-2 pathway.By transcriptome experiments, database studies, and human sample studies, we found that GLUT10 is a new immune signaling molecule involved in tumor immunity, especially in the immune cell infiltration of lung adenocarcinoma (LUAD). GLUT10 may modulate the immune cell infiltration of LUAD via the COX-2 pathway. The expression of some glucose transporters is downregulated while others are upregulated to meet the energy needs of tumor cells. Of the 14 glucose transport proteins, the roles of glucose transporter 1 (GLUT1) have been well established in a wide range of cancer types. GLUT1 overexpression was shown to promote the proliferation, invasion, and migration of malignant cells . HoweverGLUT10 is encoded by the SLC2A10 gene, and mutations in SLC2A10 result in hereditary arterial tortuosity syndrome (ATS). To date, the mechanism of ATS is still poorly understood . MeanwhiSLC2A10 is most highly expressed in smooth muscle cells and smooth muscle cell-enriched organs such as the aorta, digestive organs, prostate, and thyroid .To exploThe impact of immune infiltration on tumors has been a particular focus in recent decades. Several established databases, such as SEER and TCGA, with detailed survival and gene expression data are available to investigate the involvement and upstream molecules of immune infiltration in tumors.In this study, we used the public databases Oncomine, PrognoScan, Kaplan\u2012Meier Plotter, TIMER and GEPIA and IFC (immunofluorescence) of lung cancer specimens to further investigate the potential prognostic value and immunological roles of SLC2A10 expression in tumors.22.1This animal experiment protocol was approved by the Animal Research Ethics Committee of Renmin Hospital of Wuhan University. Male Sprague\u2012Dawley rats weighing 150\u2013180\u00a0g were purchased from the Hubei Province Center for Disease Control and Prevention (China). Euthanasia of study animals was performed by removal of vital organs under deep anaesthesia as stated in the euthanasia policy of the UCSF laboratory (POLICY - Euthanasia.pdf (ucsf.edu)). The experiment was carried out according to the following process: Rats were intraperitoneally anaesthetized with 2% pentobarbital sodium , and the aorta was isolated in a sterile environment. The aorta was cleaned of all connective tissue, cut into small pieces and placed in a 6\u00a0cm cell culture dish. Then the cells were inoculated with fresh culture medium  . After culturing in a 37\u00a0\u00b0C incubator with 5% CO2 for 30\u00a0min, the medium was replaced after the cells grew and then replaced every 2\u20133 days. After the VSMCs were fully spread in the culture flask, the original medium was removed, the cells were washed twice with PBS , 1\u00a0mL of 0.5% pancreatin (both from Gibco) was added, the cells were digested at 37\u00a0\u00b0C for 2\u20133\u00a0min, and complete medium was added to terminate digestion. Finally, VSMCs were inoculated in culture plates. Cells from passage 3 were used in the present study .This work was supported by Data will be made available on request.The authors declare no competing interests."}
+{"text": "The authors found that reported rates ofmyocarditis or pericarditis were higher after vaccination with mRNA-1273 compared withBNT162b2 and that for both vaccines, the rate was higher following dose 2 of the primary2-dose series when the interdose interval (the timing between dose 1 and dose 2) was 30days or less. These findings add to the body of knowledge about the association of mRNACOVID-19 vaccination with myocarditis and pericarditis and offer additional insight intothe differential risk between the 2 mRNA COVID-19 vaccine products and the possibleassociation of the interdose interval with risk of myocarditis or pericarditis.Buchan and colleagues4Public health and regulatory bodies have been using passive surveillance systems incombination with data on doses administered, clinical reports, and population-basedelectronic medical record systems to evaluate the association of COVID-19 vaccinationwith myocarditis and pericarditis.4 The evidence gathered to date supportsan association between mRNA COVID-19 vaccination and myocarditis or pericarditis; therisk appears highest for adolescent and young adult male individuals following dose 2,with symptom onset usually occurring within several days of vaccination.7Global vaccine-safety monitoring of adverse events following COVID-19 vaccinationhas been ongoing since COVID-19 vaccines became available in December 2020.1 used aCOVID-19 vaccine registry that captures doses administered and an electronic reportingsystem that collects adverse events following immunizations to further investigate thisassociation. Reported rates of myocarditis or pericarditis were generated by vaccineproduct, age, sex, and dose number as well as by the interdose interval. In summary, thereported rates for myocarditis and pericarditis among male individuals were higher thanthose among female individuals for all but 1 age group (25 to 39 years). This findingwas consistent for both dose 1 and dose 2 of BNT162b2 and mRNA-1273. Reported rates ofmyocarditis or pericarditis were higher following dose 2 for both vaccines. The highestreported rate was observed for male individuals aged 18 to 24 years following dose 2 ofmRNA-1273; the rate in this age group was more than 6 times higher than the ratefollowing dose 2 of BNT162b2. These primary findings are generally consistent withfindings from other surveillance systems across multiple countries.4Buchan and colleagues1 is the finding that a longer timeinterval between dose 1 and dose 2 in the primary mRNA COVID-19 vaccination series maybe associated with a lower risk of myocarditis or pericarditis. The overall reportedrates of myocarditis or pericarditis following dose 2 were higher for both vaccineproducts when the interdose interval was 30 days or less compared with 31 to 55 days and56 days or more. Although the absolute numbers were small, there was a consistentreduction in the rates of myocarditis or pericarditis with increasing intervals betweendoses, with the lowest rates occurring among individuals with interdose intervals of 56days or more. In addition, data from other countries indicate that vaccine effectivenessmay be higher with an interdose interval for mRNA vaccinations of 6 to 8 weeks comparedwith the 3- to 4-week interval that is recommended in the US.5 Therefore, an 8-week interval may be optimal forsome people aged 12 years or older, especially for male individuals aged 12 to 39years.5An important new contribution from the study by Buchan and colleagues1 is the finding that a heterologous second dosewith mRNA-1273  was associatedwith higher reported rates of myocarditis or pericarditis than was a homologous seconddoes with mRNA-1273 . The reasons for andsignificance of this finding are unclear, but it merits further study and replication inother data systems. This finding was not replicated when comparing a heterologous vshomologous vaccine series for BNT162b2; the homologous series had slightly higherreported rates of myocarditis or pericarditis for dose 2.Another new contribution of the study by Buchan and colleagues4 Additional analysis of Vaccine Safety Datalink data indicatedthat the risk of myocarditis or pericarditis was higher for mRNA-1273 compared withBNT162b2.6 A study conducted inDenmark also found a higher risk for myocarditis or pericarditis following mRNA-1273vaccination compared with BNT162b2 when evaluating the risk after dose 2 in maleindividuals aged 12 to 39 years.7 Thecorresponding rates of myocarditis or pericarditis within 28 days of vaccination in theDenmark study was 1.8 per 100 000 vaccinated individuals  for BNT162b2 and 9.4 per 100 000 vaccinated individuals  for mRNA-1273; dose 1 findings were notreported.Studies of myocarditis or pericarditis following mRNA COVID-19 vaccination havetended to focus on younger age groups because of their higher risk as observed inseveral vaccine-safety surveillance systems. Based on an analysis from Vaccine SafetyDatalink, an electronic medical record-based monitoring system in the US, mRNAvaccination was associated with a substantially increased risk of myocarditis orpericarditis in persons aged 18 to 39 years, with the highest risk occurring in the 0 to7 days following dose 2 of mRNA-1273 or BNT162b2.COVID-19 vaccination has prevented substantial morbidity and mortality and hasbeen the most effective primary prevention strategy against COVID-19 infection andserious complications. As the epidemiology of the COVID-19 pandemic continues to evolveand as vaccination programs expand to include younger age groups and additional boosterdoses, vigilance in monitoring for myocarditis or pericarditis and other adverse eventswill be critical to ensuring that public health and regulatory authorities have timelyand accurate safety data to weigh the benefits and risks of vaccination and makeevidence-based recommendations to protect the public and mitigate the pandemic."}
+{"text": "However, when applied to critically ill patients with shock, its measurement may be affected by changes in FiO2 and PaO2 and potential associated O2 diffusion between urine and ureteric or bladder tissue. We aimed to investigate PuO2 measurements in septic shock patients with a fiberoptic luminescence optode inserted into the urinary catheter lumen in relation to episodes of FiO2 change. We also evaluated medullary and urinary oxygen tension values in Merino ewes at two different FiO2 levels.Continuous measurement of urinary PO2 decreases and 31 increases in FiO2. Median pre-decrease FiO2 was 0.36  and median post-decrease FiO2 was 0.30 , p\u2009=\u20090.006. PaO2 levels decreased from 83\u00a0mmHg  to 72  mmHg, p\u2009=\u20090.009. However, PuO2 was 23.2\u00a0mmHg  before and 24.2\u00a0mmHg  after the intervention (p\u2009=\u20090.56). The median pre-increase FiO2 was 0.30  and median post-increase FiO2 was 0.35 , p\u2009=\u20090.008. PaO2 levels increased from 64\u00a0mmHg  to 71\u00a0mmHg , p\u2009=\u20090.04. However, PuO2 was 25.0\u00a0mmHg  before and 24.3\u00a0mmHg  after the intervention (p\u2009=\u20090.65). A mixed linear regression model showed a weak correlation between the variation in PaO2 and the variation in PuO2 values. In 9 Merino ewes, when comparing oxygen tension levels between FiO2 of 0.21 and 0.40, medullary values did not differ  and this was similar to urinary oxygen values .In 10 human patients, there were 32 FiO2 and PaO2 within the context of usual care did not affect PuO2. Our findings were supported by experimental data and suggest that PuO2 can be used as biomarker of medullary oxygenation irrespective of FiO2.Changes in FiOThe online version contains supplementary material available at 10.1186/s40635-022-00479-y. There were 14 episodes of successive increase/decrease in the same patient. Such episodes were successive and conditional on the presence of a PaO2 measurement and the time difference between these episodes was 4.29  hours.We observed 63 episodes of changes in the FiO2 was decreased, PaO2 fell from 83  mmHg to 72  mmHg  while urinary oxygen tension values significantly increased .At higher FiO2 (from 0.21 to 0.60), medullary oxygen tension values were similar  but urinary oxygen tension values increased .For a larger change in FiO2 is affected by changes in systemic oxygenation during routine care of patients with septic shock. As expected, changes of FiO2 resulted in significant changes in PaO2. However, we found no significant differences between PuO2 measured before and after the interventions occurred. We supported our clinical findings with data from an experimental sheep experiment showing that medullary and urinary oxygen tension measurements did not differ within a similar range of FiO2 variation.We conducted an observational study in human septic patients to determine whether PuO2 and PuO2 during cardiopulmonary bypass [2 and PuO2, accounting for\u2009\u2264\u20096% of the variation of PuO2 [To our knowledge, there have been no previous investigations of the relationship between systemic and urinary oxygenation in human patients with septic shock. Ngo et al. addressed this issue in a group of patients undergoing cardiac surgery, finding no significant relationship between PaOy bypass . Importay bypass , 12, 13,2 in response to modest but clinically significant changes in FiO2 and thus PaO2 indicate that renal medullary tissue PO2 was not markedly affected by these clinical maneuvers. Experimental evidence supports the concept that extreme variations in FiO2 and/or PaO2 lead to corresponding changes in the oxygen tension of renal tissue. For example, in anesthetized rats a reduction in FiO2 from 1.0 to 0.1 resulted in a decline in cortical and medullary microvascular PO2 as assessed by dual-wavelength phosphorimetry [2 with variations in FiO2 [2 variation is associated with greater PuO2 response, in particular at 0.60 and 1.00 FiO2.The absence of detectable changes in PuOorimetry . Likewis in FiO2 , 15\u201317. 2 from 0.21 to 1.00 increased cortical and medullary tissue PO2 [2 is less responsive to changes in PaO2 than the renal cortical tissue PO2 [2 and/or PaO2 may not appreciably alter renal medullary tissue PO2. In support of this concept, no appreciable difference was observed in medullary tissue PO2 in our group\u2019s preceding experiment when FiO2 was varied from 0.4 to 0.6 [2 did not vary significantly when FiO2 was varied from 0.21 and 0.30 [2 in patients and can only draw indirect inferences from measurement of PuO2 and consideration of available experimental evidence. However, the most parsimonious interpretation of our current findings is that modest changes in FiO2 and thus PaO2 neither markedly alter renal medullary tissue PO2 in patients with sepsis nor confounded the relationship between medullary tissue PO2 and PuO2.Our ovine study sample derived from a larger experiment comprising 18 healthy sheep undergoing abdominal surgery under total intravenous or volatile anesthesia. In this study, increasing FiOssue PO2 , 18, 19.ssue PO2 . Consequ2 settings in patients with sepsis do not result in significant changes in PuO2. In consonance of these findings, we observed that variations of FiO2 between 0.21 and 0.40 did not alter either medullary or urinary oxygen tension measurements in a sheep experiment. Thus, variations of systemic oxygenation seem unlikely to confound or affect the utility of urinary oxygenation as a biomarker for risk of AKI. Nevertheless, at higher FiO2 (0.60 and 1.00), significantly increased PuO2 values were obtained. One possible explanation is that in our septic patients, the FiO2 gap was far smaller in comparison to the experimental study. Also, one could argue that a type 2 error was present in the observational study which may have been controlled for during the experimental protocol.Our findings suggest that commonly performed adjustments to FiO2 variation in face of PaO2 changes derives from the presence of confounding factors affecting medullary oxygen values. Also, further studies in critically ill patients are needed to elucidate whether sustained differences in oxygen exposure [2, as a consequence of altered FiO2 in routine care of patients with septic shock, is unlikely to be a major confounder of the relationship between renal medullary tissue PO2 and PuO2. In the current study, PaO2 was used as a measure of systemic oxygenation because it reflects the balance between oxygen delivery and consumption. Had SpO2 been used, the accuracy would have been affected by peripheral tissue perfusion, use of vasoactive agents and altered cardiac output. Finally, continuous measurement of PuO2 might be useful for monitoring the impact on renal medullary oxygenation.Additional investigation is needed to explore whether the lack of PuOexposure , 23 infl2 and PuO2 is unlikely to be confounded by changes in FiO2 or PaO2 in the range commonly encountered in the ICU. As such, they provide further support for the use of PuO2 as a clinical surrogate of renal medullary PO2.We evaluated systemic and urinary oxygenation in human septic patients and assisted our proposition with experimental data. Our findings are consistent with previous observations in sepsis  and prov2 where the observed intervention (change of FiO2) was not protocolized. Moreover, controlling for variables such as creatinine or urine output was not feasible due to technical limitations and the limited number of patients. However, we aimed to undertake an exploratory analysis to generate a preliminary hypothesis to guide advanced studies. Moreover, we added data from a sheep experiment where FiO2 variation was protocolized. Also, due to the lower number of measurements in the experimental study, greater heterogeneity was observed. Second, the inclusion of septic patients in our clinical study did not occur in the early stage of resuscitation. On the other hand, the instances of FiO2 change we captured took place in a stable state with lower propensity for PuO2 to be affected by additional confounding effects of interventions intended to optimize oxygen delivery to the tissues. Furthermore, changes in FiO2 performed under stable conditions might have reduced the likelihood of reverse causation or provided mitigation of any potential effect of other interventions. Third, the observational nature of the study may have led to confounding by indication. For instance, the reasons motivating the clinician to change FiO2 settings could have affected the relationship between FiO2 and PuO2. However, a larger degree of FiO2 change would be expected if optimization measures capable of affecting such relationship were in place. Our patients were enrolled in the stabilization phase of sepsis, a time when, in general, only limited interventions are performed to achieve physiologic parameters aiming to prevent organ dysfunction. Finally, we addressed only the variation of systemic oxygenation within the normoxemic range. However, such a normoxemic range is typical in the care of patients in the ICU.Our study has several limitations. First, the clinical component was an observation designed to assess the effects on PuO2 and PaO2 within the context of usual care did not appreciably affect PuO2. Our findings suggest that, within the values reported, PuO2 measured in a clinical and experimental setting is not confounded by changes in inspired oxygen fraction or arterial oxygen tension and that PuO2 can be used as biomarker of medullary oxygenation irrespective of FiO2.Changes in FiOAdditional file 1. Relationship between PaO2 and PuO2 values in each individual study patient.Additional file 2. Box plot illustrating the hourly urinary output before and after a change in FiO2."}
+{"text": "Acute Confusional Syndrome (ACS) is the most common neuropsychiatric complication in COVID-19 infection. Its management is still a challenge because the data and recommendations based on the evidence are limited.To describe the differential characteristics in the management of ACS in patients with COVID-19 pneumonia compared to ACS secondary to other causes.We present a descriptive study that is has been carried out in 62 patients with ACS (26 of them diagnosed with COVID 19 pneumonia), who have required assessment by the liaison psychiatry service of Hospital del Mar between February and April, 2020. The sample was divided in 2 groups (with and without COVID 19 pneumonia). Chi square and Fisher\u2019s tests were used to comparisons.Dexmetomidine (26 vs 0) and olanzapine (13 vs 3) were significantly more frequently used in COVID-19 patients (p< 0 001). A greater number of different antipsychotic drugs were used in COVID 19 patients (2.40\u00b1 1 323 number of drugs), (p<0.0001). Further neuroimaging tests were requested in COVID 19 patients and they received less family support (4) compared to non COVID-19 (22), (p<0.005).ACS associated with COVID-19 pneumonia in the patients in our sample is more difficult to manage than ACS associated with other pathologies, similar to which described in other series. It is associated with a longer duration of confusional symptoms and difficulties for control it.No significant relationships."}
+{"text": "To assess the uptake of cervical screening in patients under Birmingham and Solihull Assertive Outreach Teams; this included a re-audit of patients under the Central Birmingham Assertive Outreach Team.Patients with severe and enduring mental illness are known to have poorer physical health outcomes. In Birmingham and Solihull there are 6 Assertive Outreach Teams. These teams manage patients with a diagnosis of psychosis who have complex needs requiring intensive multidisciplinary input and often struggle to engage with health services. The national cervical screening programme aims to prevent cervical cancer by detecting and treating cervical abnormalities. Acceptable coverage is defined as screening at least 80% of people aged 25\u201349 years within the last 3.5 years and 80% of people aged 50\u201364 years within the last 5.5 years. In 2018 71.4% of women in England and 70.9% in the West Midlands were screened adequately. An audit of 15 patients under the Central Birmingham Assertive Outreach Team in 2014 showed 46.2% had taken up screening, measured in the last 5 years for those aged 50\u201364 years and the last 3 years for those aged 25\u201349 years.A list was obtained of all female patients under the Assertive Outreach Teams with patients excluded if they were under 25 years or over 64 years or if they were known to have undergone a total hysterectomy. All GP practices with eligible patients registered to them were written to requesting the date of the patient's most recent smear test. Cervical screening was classed as in date if carried out in the last 3.5 years for patients aged 25\u201349 years or 5.5 years for patients aged 50\u201364 years.Out of 127 eligible patients, 110 had correct GP details on their record. Responses were received regarding 101 patients, 48 of whom had in date cervical screening (47.5%). Of 58 patients aged 25\u201349 years, 26 had in date cervical screening (44.8%). Of 43 patients aged 50\u201364 years, 22 had in date cervical screening (51.2%).13.4% patients did not have a known GP practice, increasing the risk of multiple poor physical health outcomes. The rates of cervical screening among Assertive Outreach Team patients are similar to the original audit in 2014 and fall significantly below the national standards and averages. These findings, along with the importance of working together to address the need for physical health monitoring in this population, will be communicated with the local Assertive Outreach Teams and GP practices."}
+{"text": "The gender distribution of the study participants should have been reported as 227 girls (60.5%) and 148 boys (39.5%). The article has been corrected.In the Research Letter titled \u201cEvaluation of Anxiety and Depression in a Community Sample of Transgender Youth,\u201d"}
+{"text": "It is sensitive, with a lower limit of detection of 0.2 CCID50/mL , and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log There are two prophylactic vaccines against poliomyelitis caused by the three serotypes of poliovirus: inactivated poliovirus vaccine (IPV) and live oral poliovirus vaccine (OPV). The use of these vaccines has dramatically reduced polio incidence worldwide. However, IPV does not stimulate adequate mucosal immunity to prevent transmission of circulating polioviruses ,2, and OThe Global Polio Eradication Initiative (GPEI) led to the eradication of wild polioviruses of types 2 and 3 , but wilSeveral molecular-based procedures have been developed for detection of OPV strains, including ELISA , reverseThe assay for the presence of Sabin 2 contamination in nOPV2 stocks is simple, rapid, reproducible, sensitive, and has large linearity range suitable for quality control of the nOPV2 vaccine.295K was provided by Dr. Andrew Macadam  and Dr. Raul Andino , and was used as negative control for the Sabin 2 contamination assay. Monovalent bulks of nOPV2 candidate 1 (nOPV2-c1) batches nPOL 2016 and nPOL 2018C, monovalent nOPV2 candidate 2 (nOPV2-c2) batches nPOL 2038C and nPOL 2056C, and drug product nOPV2-c1 batches 2060119C, 2060219C and 2060319C were provided by P.T. Bio Farma (Indonesia) and were tested for the presence of Sabin 2 virus.Batch of US neurovirulence poliovirus reference vaccine (Sabin 2 strain of OPV having GenBank accession number AY184220) and plasmid-containing Sabin 2 genome were used as positive control (Lab. method development inventory at US. FDA). Plasmid-containing nOPV2-c1 genome with mutation E295K by transfecting HEp-2C monolayers with T7 transcripts . A,4. A3,4]10 CCID50/mL was subjected to RNA extraction as described above and the RNA was serially diluted 10-times using 10-fold steps in a total volume of 100 \u03bcL. The original and diluted RNA samples were tested, and the resulting Ct values were averaged (mean) for each dilution and plotted against the respective Sabin 2 titers expressed as log10 CCID50/mL as shown in 50/mL  and had a very broad linearity range of at least 8 log10 (from 8.34 to 0.34 Log10 CCID50/mL). RNA from a sample with the lowest titer analyzed (0.034 Log10 CCID50/mL) was undetectable.To evaluate the linearity and sensitivity of the RT-PCR in absence of nOPV2 background, a Sabin 2 strain with titer of 8.34 Log10 CCID50/mL) and a plasmid containing Sabin 2 genome (0.1 ng/mL) were run as positive controls, while another plasmid containing the nOPV2-c1genome (0.1 ng/mL) and water were used as negative controls. The controls were run in quadruplet repeats, and the test and standard reference samples were tested in triplicates. The results of positive and negative controls are presented in In the same run, Sabin 2 RNA . Briefly, the Sabin 2 detection assay yielded consistent and reproducible results.Three Sabin RNA samples with equivalent viral titers of 2.16, 21.63 and 216.27 CCID10 CCID50/mL was serially diluted in 10-fold steps in nOPV2c1-295 virus suspension with the titer of 8.07 log10 CCID50/mL . All diluted samples were subjected to RNA extraction and then to the assay for detection Sabin 2 contamination in nOPV2c1-295 as separate samples in triplicate repeats. The results showed that the presence of nOPV2 virus with high titer did not interfere with the assay that was able to detect Sabin 2 virus at levels of 0.22 CCID50/mL which correspond to traces of Sabin 2 virus in nOPV2 (2 \u00d7 10\u22127% of Sabin 2 in nOPV2 suspension)  . A linea 8 log10 . All neg 8 log10 , confirm50 to those based on genome copy (GC) numbers we analyzed the spiked samples with qmosRT-PCR as described above and the resulting GC numbers were used to calculate the percentages of the Sabin 2 in the samples  and the percentages of Sabin 2 in samples were calculated as the average percentages of the 18 SNPs in domain V  that are mentioned above  and presented in Vaccines are the most efficient tools for preventing, controlling and even eradicating infectious diseases. However, contamination can occur at various stages of vaccine preparation, resulting from the unintentional introduction of extraneous agents present in raw materials or introduced during the manufacturer\u2019s process.As stated above, the nOPV2 was created by introducing targeted genetic changes to the Sabin 2 strain that further stabilize the attenuated phenotype. Since the original Sabin 2 virus can easily convert to circulating vaccine-derived poliovirus type 2 (cVDPV2), it is important to ensure that batches of nOPV2 do not contain Sabin 2 virus if handled in the same manufacturing facility. Therefore, a sensitive and specific assay for detection of Sabin 2 strain is useful for quality control of the nOPV2 vaccine.The previous molecular methods were designed for specific detection of Sabin strains in clinical and environmental samples but cannot distinguish Sabin strains from their genetically modified nOPV derivatives ,23,24,256 to 101 of the relative CCID50/mL), blank (water) and nOPV2 plasmid DNA and/or nOPV2 RNA as negative controls, and Sabin 2 RNA and/or Sabin 2 plasmid DNA as positive controls. All the samples are tested in triplicates or quadruplicates (for controls) in the same 96-well plates by qosRT-PCR that specifically detects Sabin 2 virus and differentiates it from the nOPV2. The qosRT-PCR run is considered valid if all the following conditions are met: R2 of the standard curve is more or equal 0.95, for three out of four positive control repeats Ct values are less or equal to 40, and for at least three out of four negative control repeats have Ct values undetermined (or Ct \u2265 40). The test sample is positive if at least two out of three repeats have Ct values less than 40. In this work the Ct 40 was chosen as threshold. However, to establish a real Ct threshold needs further work to ensure that the assay is specific and works under different conditions, and in different hands and labs.The following provisional layout and acceptance criteria are proposed: the assay for detection of Sabin 2 contamination in nOPV2 RNA samples are tested in triplicate along with reference\u2013Sabin 2 RNA serially diluted in 10-fold steps  of Sabin 2 virus diluted in nOPV2 suspension, and 198 GC/mL corresponds to\u00a00.396 GC/reaction which is in line with having reached the maximum theoretical limit of one GC detected per reaction. The limit of detection may vary from virus to virus depending upon the GC:CCID50 ratio for each virus. No cross-amplification was observed with nOPV2 and a plasmid that contains nOPV2 genome . No Sabin 2 virus was detected in these lots and the results were confirmed by NGS . Based oIn conclusion, the assay for detection Sabin 2 contamination in nOPV2 stocks described in this communication offers a simple and rapid method for the detection and quantitation of Sabin 2 virus either individually or in the presence of nOPV2 virus. The assay for Sabin 2 contamination in nOPV2 stocks is designed specifically to be applied during manufacture of nOPV2 vaccine, for quality control to detect Sabin 2 virus as a potential contaminant in nOPV2 vaccine."}
+{"text": "FOXC1 and PITX2, account for almost half of known cases, while the genetic lesions in the remaining cases remain unresolved. Given the genetic similarity between zebrafish and humans, as well as robust antisense inhibition and gene editing technologies available for use in these animals, loss of function zebrafish models for ARS have been created and shed light on the mechanism(s) whereby mutations in these two transcription factors cause such a wide array of developmental phenotypes. This review summarizes the published phenotypes in zebrafish foxc1 and pitx2 loss of function models and discusses possible mechanisms that may be used to target pharmaceutical development and therapeutic interventions.Axenfeld\u2013Rieger syndrome (ARS) encompasses a group of developmental disorders that affect the anterior segment of the eye, as well as systemic developmental defects in some patients. Malformation of the ocular anterior segment often leads to secondary glaucoma, while some patients also present with cardiovascular malformations, craniofacial and dental abnormalities and additional periumbilical skin. Genes that encode two transcription factors,  Axenfeld\u2013Rieger syndrome (ARS) is a clinically heterogeneous disorder characterized by ocular anomalies with systemic multi-organ system involvement in some patients. This relatively rare disorder, with a prevalence of 1 in 50,000\u2013100,000 live births ,3, cerebFORKHEAD BOX C1 (FOXC1) and PAIRED-LIKE HOMOEDOMAIN (PITX2). DNA lesions involving PITX2 result in ARS type I, in which patients have fully penetrant ocular phenotypes often observed with craniofacial and dental anomalies. Mutations in FOXC1 result in ARS type III, defined by fully penetrant ocular phenotypes often observed with cardiovascular defects and sensorineural hearing loss. While linkage analysis supports an additional gene causing ARS type II on chromosome 13q14 [CYP1B1 gene have been found in a single family with ARS [CYP1B1 appear to be an extremely rare cause of ARS.ARS is inherited as an autosomal dominant disorder, with the genetic lesion defined in approximately 40% of cases. Mutations or copy number variation (CNVs) in two genes identified through a variety of family-based studies account for ARS with fully penetrant ocular manifestations; me 13q14 , no causme 13q14 . This cowith ARS , and whiwith ARS ,19, mutaDanio rerio) has provided valuable mechanistic insights into ARS disease etiology. The zebrafish genome contains two homologues of the FOXC1 gene denoted foxc1a and foxc1b, arising from an ancient duplication in the teleost lineage, and one homologue of PITX2 (pitx2). Gene expression studies using in situ hybridization have highlighted specific cell types that require the expression of foxc1 and pitx2 for normal ocular development. The use of morpholino based antisense inhibition and genome editing have produced zebrafish strains that mimic ARS phenotypes and have provided novel mechanistic data highlighting downstream genes and signaling pathways that are required for ocular and systemic manifestations of the syndrome. This review will focus on the role of foxc1 and pitx2 in the regulation of genes and signaling pathways that regulate formation of the eye, cardiovascular system, and craniofacial skeleton, as defined by loss of function zebrafish mutant strains or antisense inhibition data.The zebrafish ) as well as the lateral plate mesoderm (LPM) [foxc1a continues to be expressed in the adult zebrafish eye, including the anterior segment and retinal ganglion cell layer [At early stages of development, zebrafish express st cells ,20  . Neural rm (LPM) . Neural rm (LPM) ,24,25. Trm (LPM) ,26,27. All layer , possiblpitx2 gene that encodes two isoforms via alternative splicing that correspond to human PITX2A and PITX2C. The expression of pitx2a is found in partially overlapping domains with foxc1a and foxc1b in developing zebrafish embryos. As early as 24 h post fertilization (hpf), pitx2 expression is observed in the periocular mesenchyme [foxc1a and foxc1b, pitx2 is expressed in the neural crest derived tissues of the pharyngeal arches that contribute to the craniofacial skeleton [pitx2c isoform in the lateral plate mesoderm [pitx2a in the neural crest [The zebrafish genome contains a single senchyme   inhibition, a number of zebrafish models have been reported that recapitulate the phenotypes of foxc1a and foxc1b display underdeveloped or absent anterior segments beginning around 3 dpf [foxc1a/foxc1b zebrafish, which are typically not observed in ARS patients. This discrepancy may be the result of complete loss of Foxc1 function in double homozygotes, compared to heterozygous mutations or copy number variations that define ARS. Heterozygous mutations of both foxc1a and foxc1b in zebrafish do not alter early development of the anterior segment, however, published analyses only describe assays performed up to 6 dpf and thus additional analyses on older heterozygous larvae or adults when the anterior segment has matured, could potentially reveal anterior segment anomalies consistent with ARS. Loss of Foxc1 function in zebrafish also leads to endophenotypes of glaucoma, including reduced number of retinal ganglion cells and thinner optic nerves [The zebrafish anterior segment begins to develop as early as 26 hpf when the lens vesicle detaches from the surface ectoderm . By 3 dand 3 dpf . Additioc nerves  . However, many ARS patients have normal tension glaucoma (NTG), defined as having normal intraocular pressure, and thus other mechanisms are likely involved. For example, a number of studies have demonstrated that foxc1 regulates the formation of the zebrafish hyaloid and retinal vasculature [atonal homolog 7 (atoh7). Atoh7 is required for RCG differentiation during embryonic development [ATOH7 in human populations are associated with persistent hyperplasia of the primary vitreous [atoh7 expression via Foxc1 could account for ocular vascular defects in ARS patients and could influence the development of glaucoma independent of, or in combination with, potential increased IOP caused by the anterior segment dysgenesis. Variants in ATOH7 have also been associated with primary open angle glaucoma [atoh7 as a key contributor of RGC and optic nerve health.Certainly, the observed anterior segment defects in culature ,47. Defeelopment ,49, and vitreous  whereby glaucoma  and endoglaucoma ,53,54, iatoh7, other genetic targets regulated by Foxc1a/Foxc1b in zebrafish have been identified that may shed light on potential mechanisms of glaucoma development. Foxc1a/Foxc1b regulates the expression of FOXO1A/foxo1a, a gene expressed in the zebrafish POM that mediates the response to oxidative stress [foxc1a or foxo1a resulted in aberrant responses to increased oxidative stress and increased cell death in the eye [FOXC1. Foxc1a also regulates the expression of fgf19 [foxo1a and fgf19 have not been identified in ARS or glaucoma patients and thus the dysfunction of either gene alone likely cannot cause overt visual defects, however, their reduced expression due to loss of Foxc1 function, as well as other yet to be identified downstream targets, likely contribute to the complex etiology of RGC loss in FOXC1-attributable ARS patients.In addition to e stress . Manipul the eye , indicatof fgf19 ,56, anotof fgf19 ,58. Mutafoxc1a mutants, as well as foxc1a/foxc1b double mutants, have craniofacial defects consistent with abnormalities in the facial structure of ARS patients. In these patients, hypertelorism (increased space between the eyes) and a prominent forehead are often described. In zebrafish, foxc1a and foxc1b are expressed in the neural crest cells that populate the first and second pharyngeal arches that give rise to anterior jaw structures. Mutation of foxc1a alone results in craniofacial dysmorphism [foxc1a/foxc1b mutants. Phenotypes in double homozygous mutants included under-developed palatoquadrate and hyomandibula cartilages [foxc1a and foxc1b play critical roles in the development of anterior facial cartilages (foxc1a). Analysis of genetic targets downstream of foxc1 revealed both foxc1a and foxc1b regulate Sox9-dependent expression of cartilage specification genes, accounting for reduced jaw structures in these animals. While Foxc1 does not directly regulate sox9 expression, loss of foxc1 paralogs in zebrafish causes a decrease in chromatin accessibility for transcription factors such as Sox9 in chondrocytes [Zebrafish morphism ,37 includrocytes , and thufoxc1a mutants in conjunction with homozygous loss of foxc1b (which survive to adulthood) demonstrates craniofacial abnormalities in adults that include a misshapen head that closely mimics that of ARS patients, as well as mandibular retrognathia and dorsally positioned eyes [FOXC1.Analysis of heterozygous ned eyes . CombineFOXC1 mutations or CNVs that include hypoplastic ventricular outflow tract morphology, dysplastic arcade mitral valve, and atrial septal defect [FOXC1 plays an important role in early heart and cerebral vascular development, with such phenotypes recapitulated in zebrafish loss of foxc1 function models.Cardiac anomalies have been described in ARS patients with l defect ,59. Withl defect \u2014all of wl defect . These cfoxc1a alone or in combination with foxc1b leads to cardiac phenotypes that include hypoplastic myocardium and ventricular outflow tract, as well as defects in cardiac valve formation that are similar to that observed in patients with FOXC1-attributible ARS [foxc1 in heart development, reports differ in their analysis of heart morphology, assay different time points, and study different combinations of mutations. For example, Yue et al. [foxc1a\u2212/\u2212 embryos  formation has been proposed. Such studies show that Foxc1a directly binds to the promoter of nkx2.5 [nkx2.5 in zebrafish foxc1 homozygous mutants provides mechanistic insight into the hypoplastic myocardium observed in ARS patients. Furthermore, genes expressed specifically in the AVC are downregulated in foxc1 homozygous mutants, including notch1b, tbx2b and bmp4, providing additional genetic targets that downstream of foxc1 that may contribute to cardiac defects. While foxc1a is expressed in LPM, the heart also receives a contribution of cells from the neural crest, and thus foxc1 expression in neural crest cells could also influence the presence and severity of cardiac phenotypes observed in ARS patients and zebrafish foxc1 mutants.Utilizing f nkx2.5 , a gene foxc1 function in zebrafish has been undertaken. Combined morpholino inhibition of foxc1a and foxc1b causes cerebral hemorrhages [sox10 positive neural crest cell populations. Furthermore, defects in cerebral angiogenesis [As CSVD in ARS patients increases stroke risk, analysis of cerebral vasculature due to loss of orrhages   with early onset glaucoma (a diagnosis similar to ARS) identified a 748 kb deletion containing a conserved PITX2 regulatory element [pitx2 expression and like mutation of the gene itself, resulted in reduced or shallow anterior chambers. These data clearly demonstrate that mutation of pitx2, or genomic modifications that alter pitx2 expression result in anterior segment defects reminiscent of ARS. Although no direct quantification of optic nerve morphology or RGC number has been undertaken, these data demonstrate that mutant pitx2 zebrafish represent an ideal model for subsequent studies that focus on the mechanism by which glaucoma may result in patients with PITX2-attributable ARS.Like elopment ,33,43  [dkk2 in the anterior segment of the eye [pitx2 mutants. Wnt signaling plays a key role in the development of neural crest cells in part through regulation of foxd3 and sox10 [pitx2 expression in early neural crest cells, and its continued expression in the adult anterior segment [pitx2 is likely required for development and maintenance of the anterior segment in part through a Wnt dependent mechanism.Analysis of genetic targets in the eye demonstrates that Pitx2 deficiency reduces the expression of the Wnt ligands ( wnt10a)  as well  the eye . A role nd sox10 ,62 that  segment , pitx2 ipitx2 depletion demonstrate normal expression of pax6a that is required for anterior segment development [pax6a was observed, it has been shown that pitx2 expression is regulated by Pax6a/b in zebrafish [PAX6/pax6 mutations [foxc1, pitx2 may play an important role in early trophic support of the lens and anterior segment structures early in development.Analysis of additional ocular targets due to elopment . While nebrafish  which mautations . The expfoxc1, disruption of pitx2 using antisense morpholinos or with genome editing induced mutations disrupts pharyngeal arch cartilage and jaw formation. Specific defects were observed in the Meckel\u2019s and ceratohyal cartilages, which were under-developed and positioned abnormally [sox10 positive neural crest cells that line the pharyngeal arches and jaw elements demonstrate reduced cell number [foxc1 and pitx2 depleted zebrafish likely involves overlapping mechanisms of decreased neural crest cell migration and survival.Patients with type I ARS typically have dysmorphic craniofacial features that include maxillary and dental hypoplasia, in addition to the anterior segment ocular phenotypes that define ARS. Similar to normally ,43  [pitx2a and pitx2c) [pitx2a is predominantly expressed in neural crest cells and thus mutations that affect this isoform may result in ocular, craniofacial, and cardiovascular anomalies associated with ARS, while pitx2c is predominately expressed in the lateral plate mesoderm and the heart, which could contribute to cardiac defects observed in some ARS patients. Patients with PITX2-attributable ARS may thus present with or without cardiac defects that may depend in part on the location of the disease-causing mutations [pitx2a) or both isoforms (pitx2a and pitx2c) in order to identify similar defects in fish, and to gain mechanistic understanding of such phenotypes in patients and animal models.Like humans, the zebrafish  PITX2d)  with two pitx2c) . During utations ,68. Mutapitx2c) did not result in cardiac defects consistent with PITX2-attributable ARS in one study, although asymmetric looping and overall morphology were the only phenotypes tested [pitx2c specific mutant strain and uncovered cardiomyopathy with fibrosis (PITX2 as a causative gene for atrial fibrillation [foxc1 regulates expression of nxk2.5 in the lateral plate mesoderm, nkx2.5 is required for maintenance of pitx2 expression in this area [foxc1 and pitx2 in regulating heart development.Mutations in zebrafish that affect both isoforms (or just s tested . A seconfibrosis E\u2013H and aillation ,70. Whilhis area , highligfoxc1 and pitx2 loss of function models provides understanding of the mechanisms that lead to most ARS related phenotypes. Both genes when mutated in zebrafish result in defects consistent with mammalian ARS models and patient phenotypes. Zebrafish ARS mutants have ocular anterior chamber defects, and foxc1 mutants additionally display endophenotypes of glaucoma. Disruption of foxc1 or pitx2 in zebrafish display craniofacial anomalies consistent with ARS, as well as cardiovascular defects that are often observed in patients. pitx2 mutants additionally display tooth hypoplasia that is often observed in Type 1 ARS. Mechanisms involving downstream gene regulation are beginning to be uncovered using homozygous embryos and larvae, however, analysis of adult phenotypes in heterozygous mutants is somewhat lacking in the literature. The continued analysis of such mutants will reveal novel insights into disease mechanisms, and given the utility of zebrafish for translational research, pharmaceutical approaches using high-throughput drug screening for phenotypic rescue provides a path toward testing therapeutic interventions.Analysis of zebrafish"}
+{"text": "Cytokines were determined by multiplex assays and ELISA, and gene expression by qPCR. MALAT1 loss of function was performed in OA patient osteoblasts using locked nucleic acids. The osteoblast transcriptome was analysed by RNASeq and pathway analysis. Bone expression of MALAT1 positively correlated to serum DKK1 and galectin-1 concentrations, and in OA patient osteoblasts was induced in response to IL-1\u03b2 stimulation. Osteoblasts depleted of MALAT1 exhibited differential expression (>1.5 fold change) of 155 genes, including PTGS2. Both basal and IL-1\u03b2-mediated PGE2 secretion was greater in MALAT1 depleted osteoblasts. The induction of MALAT1 in human OA osteoblasts upon inflammatory challenge and its modulation of PGE2 production suggests that MALAT1 may play a role in regulating inflammation in OA subchondral bone.Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1) is implicated in regulating the inflammatory response and in the pathology of several chronic inflammatory diseases, including osteoarthritis (OA). The purpose of this study was to examine the relationship between OA subchondral bone expression of MALAT1 with parameters of joint health and biomarkers of joint inflammation, and to determine its functional role in human OA osteoblasts. Subchondral bone and blood were collected from hip and knee OA patients ( Osteoarthritis (OA) is a degenerative joint disease and a leading cause of pain and disability for which there are currently no approved pharmacological disease modifying therapeutics . CurrentMALAT1) lncRNA modulates the inflammatory phenotype of synovial fibroblasts in the OA synovial joint lining by mediating the production of CXCL8 [MALAT1 lncRNA has now emerged as a central mediator of osteoblast function and bone homeostasis. Expression of MALAT1 is reported to be greater in the bone tissue of patients who exhibit aseptic loosening following a hip replacement [MALAT1 has been demonstrated to inhibit the proliferation of the human osteoblast cell line hFOB 1.19 [MALAT1 sponging of the microRNA miR-30 has been shown to promote the osteoblast differentiation of mesenchymal stem cells by inducing RUNX2 expression [MALAT1 is associated with abnormal osteogenic and adipogenic differentiation of BMSCs in the patients with osteonecrosis of the femoral head [Recently, long non-coding RNAs (lncRNAs) have emerged as novel epigenetic regulators of gene transcription ,14 and oof CXCL8 . Howeverlacement  and its lacement . In vitrFOB 1.19 , and MALpression . Furtherral head .MALAT1 could play a key role in the pathological changes that occur in OA diseased bone. However, currently the expression and functional role of MALAT1 in OA subchondral bone has not been reported. Therefore, the aim of this study was to profile the expression of MALAT1 in the subchondral bone tissue of patients with either knee or hip OA and its relationship to parameters of joint damage and to examine the functional role of MALAT1 in OA patient primary osteoblasts.These data suggest that n = 6), who were undergoing surgery at Russell\u2019s Hall Hospital  or the Royal Orthopaedic Hospital (Birmingham UK). OA patient characteristics are detailed in Following ethical approval , subchondral bone tissue and blood was collected from a total of 17 patients  with end-stage OA comprising of 9 patients with hip OA and 8 patients with knee OA and from neck of femur fracture (NOF#) patients without OA  to remove excess fat. The bone chips were then placed in a T75 flasks with fresh culture media and incubated in at 37 \u00b0C in a humidified atmosphere containing 5% CO2. The media was changed every 3\u20134 days, and the chips were removed after 7\u201314 days when the osteoblasts outgrowth occurred.Primary OA osteoblasts were cultured from OA subchondral bone tissue. The bone was cut into small pieces approximately 2 mmThe serum concentration of 24 cytokines were determined using Luminex multiplex platform (Luminex R&D systems) according to the manufacturer\u2019s instructions, having been diluted to 1:2 in assay buffer.MALAT1 or with a control (NC) LNA. To determine the effect of MALAT1 knockdown on the OA osteoblast transcriptome, total RNA was extracted  24 h following transfection with LNAs. RNA integrity (RIN) was evaluated  with a RIN of >7 deemed of sufficient quality for RNA sequencing analysis using the QuantSeq 3\u2032 kit . The sequenced reads were mapped to the hg38 reference human genome using Star Aligner version 2.5.2b [In vitro loss of function studies were performed using lipofectamine 3000  to transfect primary osteoblasts with two different Locked nucleic acids (LNAs) targeting n 2.5.2b . Differen 2.5.2b .MALAT1 knockdown on the production of PGE2 and osteoprotegrin (OPG) was determined by ELISA according to the manufacturer\u2019s instructions . Alkaline phosphatase (ALP) activity was quantified in osteoblast lysates prepared in RIPA buffer diluted 1:5 with 1 mM MgCl2. In brief, diluted osteoblast lysates were combined with ALP substrate  and incubated at 37 \u00b0C for 15 min. The reaction was stopped with the addition of 0.1\u2009N NaOH and absorbance read at 405 nm on a microplate reader. The degree of osteoblast mineralisation was quantified by staining of mineralised nodules using an alizarin red solution . Following 10\u2009min incubation at room temperature, cells were washed with PBS to remove excess stain, and then incubated in 10% cetyl pyridinium chloride  for 10\u2009min. The supernatant was then collected, diluted 1:10 with the 10% cetyl pyridinium chloride and absorbance read at OD550\u2009nm on a microplate reader.The effect of www.ingenuity.com, accessed on 31 January 2020). Differentially expressed genes  were analysed using a core functional analysis to identify significant canonical pathways and cellular processes. Pathway analysis was performed using the software Ingenuity Pathway Analysis . However, this did not reach significance and there was no significant difference in expression between patients with knee OA and those with hip OA (MALAT1 expression in OA patients with greater disease severity (KL4 and joint space < 1 mm), compared to those patients with KL grade 3 and joint space > 1 mm, there was no significant relationship between MALAT1 expression and disease severity  and galectin-1   . HoweverMALAT1 was induced upon an inflammatory challenge. To this end, primary osteoblasts from n = 3 OA patients were stimulated for either 6 h or 24 h with IL-1\u03b2 (1 ng/mL), and the expression of IL-6 and MALAT1 determined by qPCR. As expected, stimulation of osteoblasts with IL-1\u03b2 induced a significant increase in the expression of the pro-inflammatory cytokine IL-6 within 24 h , compared to non-stimulated control. Similarly, osteoblasts stimulated with IL-1\u03b2 for 24 h exhibited significant increase in the expression of MALAT1 , compared to non-stimulated control  and examined the resulting osteoblast transcriptome by RNA sequencing. Primary OA osteoblasts (n = 3 OA patients) were transfected with either a non-targeting control LNA or one of two LNA duplexes targeting MALAT1. Following 24 h transfection, LNAs targeting MALAT1 induced knockdown of between 60\u201390% in the expression of MALAT1, compared to the control LNA  in osteoblasts transfected with the MALAT1 LNAs, compared to LNA control transfected cells. Of the upregulated transcripts, 80 were protein coding genes (including PTGS2) and 2 were antisense lncRNAs. Of the down-regulated transcripts, 66 were protein-coding genes and 7 were lncRNAs, which comprised of 2 lincRNAs, 3 antisense, 1 sense intronic lncRNA and 1 pseudogene  to identify significantly affected canonical pathways and cellular processes. The most significant canonical pathways affected included phosphatidylcholine biosynthesis, fMLP signalling in neutrophils, NAD biosynthesis, eicosanoid biosynthesis and prostanoid biosynthesis. The significantly affected cellular processes included cell\u2013cell signalling, DNA replication, cellular growth and proliferation and cellular development  or left unstimulated and secretion of PGE2 quantified by ELISA. MALAT1 knockdown was confirmed by qPCR  (Given the finding that knockdown of  by qPCR A. Compar < 0.05) B.MALAT1 would affect the innate function of osteoblasts with regard to bone remodelling by determining OPG production, alkaline phosphatase activity and osteoblast mineralisation. To this end, OA osteoblasts were transfected with either a control LNA or a MALAT1 LNA twice per week for a period of 3 weeks. During the 3-week time course, supernatants were collected to analyse OPG production by ELISA and cells lysed to measure alkaline phosphatase activity. At the end of the 3 weeks, cells were stained with alizarin red to quantify mineralisation. Twice weekly transfection with LNA maintained the significant knockdown in MALAT1 expression of between 65\u201385%  in the subchondral bone tissue of both knee and hip OA patients irrespective of disease severity. On average MALAT1 expression was greater in the bone tissue from patients with greater OA severity, as measured by either KL grade or joint space narrowing. However, it should be noted that all the OA patients in this study had advanced end-stage disease, with radiographic severity determined to be either KL3 or KL4, and most patients having <1 mm joint space. Therefore, we cannot comment on whether expression of MALAT1 would differ in the subchondral bone of patients with early OA. This paper reports the expression and functional role of the long non-coding RNA MALAT1 in OA synovial fibroblasts [MALAT1 expression in OA osteoblasts was induced during the IL-1\u03b2 inflammatory response. This suggests MALAT1 may play an important regulatory role in bone homeostasis under the inflammatory conditions exhibited in OA patients. Of note, although the subchondral bone expression of MALAT1 was not related to the serum concentration of IL-1\u03b2 we did observe positive correlations between MALAT1 expression and the serum concentration of both DKK1 and galectin 1. DKK1 is an endogenous inhibitor of the Wnt/beta-catenin signalling pathway and is implicated in bone development and in the pathological remodelling of bone in both OA and osteoporosis and mediating inflammation-induced bone loss by inhibiting osteoblast differentiation [Similar to previous findings on the role of roblasts , MALAT1 ntiation ,31. In ontiation . In contntiation , and in ntiation . MALAT1 in primary human OA osteoblasts profoundly affected the transcriptomic phenotype with pathway analysis revealing significant activation of pathways that promote the production of inflammatory prostacyclins and eicosanoids. Amongst the most differentially expressed upregulated genes were TNFSF12 (>11-fold upregulated), which encodes TWEAK (TNF-related weak inducer of apoptosis). TWEAK is a known mediator of inflammatory bone remodelling [MALAT1 exhibited a significant >10-fold upregulation in the expression of PTGS2, the gene which encodes for the enzyme COX2 that mediates the production of inflammatory prostaglandins including the putative OA pain mediator PGE2 [MALAT1 compared to control cells. PGE2 acting through the E prostanoid receptors EP2 and EP4 sensitizes nociceptors, possibly by acting synergistically with IL1\u03b2 to induce IL-6 and iNOS expression [MALAT1 expression in OA osteoblasts regulates PTGS2 expression as well as both basal and IL-1\u03b2 induced PGE2 production suggests that MALAT1 may play an important role in regulating inflammatory pain in the bone.LNA-mediated knockdown of odelling , and tarodelling . In additor PGE2 ,38. Indepression  and selepression . The subpression  and bonepression . TherefoMALAT1 knockdown on the osteoblast transcriptome and the acute effect on PGE2 production we did not observe any chronic effect of MALAT1 knockdown on several key osteoblast functions. During a time-period of 3 weeks, we induced sustained MALAT1 knockdown but did not observe any significant difference in the secretion of OPG or in the activity of ALP and ability of the osteoblast to form mineralised bone nodules. This contrasts with previous publications which have implicated MALAT1 in mediating both OPG production in osteoblasts [Despite the effect of eoblasts  as well eoblasts .MALAT1 have been reported to exert a pro-osteogenic function by acting as miRNA sponges [MALAT1 in mediating osteoblast differentiation has been linked to the sponging of several miRNAs including miR-204 [MALAT1 depletion, we did not identify any miRNAs which were altered upon MALAT1 KD. However, the isolation of total RNA using columns would likely have excluded many miRNAs from our sequencing analysis.The underlining molecular mechanisms by which lncRNAs mediate their function is complex and for the majority of lncRNAs remains to be determined. Several lncRNAs, including  sponges . Indeed, miR-204 , miR-30  miR-204  and miR- miR-204 . In our MALTA1 in OA subchondral bone, its induction in osteoblasts upon inflammatory challenge and its functional role in modulating the production of the prostaglandin PGE2 suggest that MALAT1 may play an important role in the development of OA bone pain and inflammation.In conclusion, the expression of"}
+{"text": "Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal  Porphyromonas gingivalis, induces chronic infection of gingiva and tooth loss, and also enhances the risk of cardiovascular diseases such as heart attack [Approximately 10\u201315% of adults suffer from periodontal disease with dental bone loss leading to tooth loss in patients and affecting their quality of life . Publisht attack . Additiot attack . Other st attack ,8. ThereThere have been several reported causes of dental bone loss in periodontal disease which increase the risk of congenital disease. For example, hormone or endocrine disorders might inhibit the repair of collagen fiber in the gingival tissue and cause alveolar bone loss . UnhealtPorphyromonas gingivalis, were injected to create inflammation. In addition, alcohol administration through oral exposure of 10% alcohol in drinking water was provided to observe the effects of dental bony defects in mice. In this study, we demonstrated that under LPS-induced toxicity and alcohol exposure, deficiency in the ALDH2 enzyme played a significant role in dental bone loss. The results from this study may provide important clinical information and a potential therapeutic target for the treatment or prevention of dental bone loss in periodontal disease, especially for the large population of East Asians carrying the vulnerable ALDH2*2 genotype.The role of genetic factors in alcohol-induced dental bone loss has not been explored thus far. The polymorphism of a single amino acid substitution which leads to the loss of enzyme function in the mitochondrial aldehyde dehydrogenase 2 (ALDH2) gene is well known . FollowiThe shRNA sequence of ALDH2 was obtained from the RNAi database . The ALDH2 shRNA vector was purchased from Academia Sinica. Osteoblasts expressing silenced human ALDH2 were developed by transfecting cells with lentivirus carrying the ALDH2 shRNA. The targeted ALDH2 sequence was GCAGATCATTCCGTGGAATTT.2. Dulbecco\u2019s modified Eagle\u2019s medium containing 10% fetal bovine serum (FBS), 100 unit/mL penicillin +100 \u03bcg/mL streptomycin , and 2 mM L-glutamine  were used for culture. Cells were grown to 80% confluence, and then passaged. Osteoblasts expressing silenced human ALDH2 were selected by transfecting cells with lentivirus  carrying the ALDH2 shRNA (multiplicity of infection (MOI) = 10) vector. The MOI is defined as the ratio of infectious agents to infection targets. It is also the ratio of viral particles to the number of target cells in a defined space, such as in a cell culture well. The cell line, designated as ALDH2 knockdown osteoblasts were confirmed with low ALDH2 expression by Western blot analysis using the ALDH2 antibody , Abcam, Cambridge, MA, USA). Osteoblasts transfected with an empty lentivirus were used as the control. Cells at 3\u20134 passages were used for the MTT assay.The human osteoblast (transfected with SV40 large T antigen) cell line (hFOB 1.19) was obtained from Bioresource Collection and Research Center . Cells were cultured at 37 \u00b0C in a humidified incubator with 5% CO4 cells per well in a 96-well microplate (n = 8) in the DMEM culture medium for 7 and 14 days. On Day 7 and Day 14, MTT was added to each culture well at a final concentration of 2 mg/mL followed by an incubation at 37 \u00b0C for 4 h. Then, the medium was harvested and centrifuged at 900 rpm for 5 min and aspirated. The pelleted formazan reaction products were dissolved in 200 \u03bcL of dimehtyl sulfoxide  solution and shaken for 15 min. The optical density of the formazan solution was recorded using an enzyme-linked immunosorbent assay (ELISA) plate reader  at 570 nm. The absorbance was proportional to the metabolic activities and viabilities of the cells.Cell viabilities of both control and ALDH2 knockdown transfected osteoblasts were analyzed by using the assay of 3--2,5-diphenyl tetrazolium bromide  . Both pa3 cells/mL and high, 1 \u00d7 105 cells/mL). The degree of mineralization was observed by phase contrast microscopy from the independent culture wells of the control and ALDH2 knockdown groups.In this assay , both coPorphyromonas gingivalis  under anesthesia and manipulated under a microscopy. The injection was performed twice a week and continued for 6 weeks. The corresponding right side of the upper 1st molar and 2nd molar interdental alveolar bone areas and between the 2nd and 3rd molar interdental areas of all mice were injected with 2 \u03bcL vehicle (endotoxin-free water), twice a week, and continued for 6 weeks as the control. Drinking water with 10% alcohol and standard dry food were administered to all mice. After 6 weeks, all mice were sacrificed for evaluation by micro-CT and histological observation with hematoxylin and eosin staining.The animal use was approved and followed the guidelines of the Laboratory Animal Center at the National Taiwan University College of Medicine, Taiwan. The ALDH2*2 knockin mice representing the ALDH2*2 single amino acid substitution of human ALDH2*2 were generated and obtained from Prof. Daria Mochly-Rosen\u2019s lab, Stanford University. The construction, characterization, and phenotype of the ALDH2*2 mice were as described previously . Four C5-, d1 site) was designated as 100% for comparisons with other distance measurements of bone loss. The percentage of bone loss in the wild-type group of the vehicle injected into the right side of the upper 2nd and 3rd molar interdental areas  was designated as 100% for comparisons with other distance measurements. In addition, 2 \u03bcL (20 \u03bcg) of purified lipopolysaccharide from Porphyromonas gingivalis was injected into the corresponding left side of the upper 1st and 2nd molar interdental areas  and the 2nd and 3rd molar interdental areas  as the treatment groups.Micro-CT imaging protocol was as described in our previous study . To obta-, d1 site. The percentage of bone loss of the right upper jaws between the 2nd and 3rd molar interdental areas of the vehicle-injected ALDH2*2 knockin mice was designated as the ALDH2 LPS-, d2 site group. In addition, the percentage of bone loss of the left upper jaws between the 1st and 2nd molars, and between the 2nd and 3rd molar interdental alveolar areas of the ALDH2*2 knockin mice injected with LPS were designated as the ALDH2 LPS+, d1 site group and ALDH2 LPS+, d2 site group, respectively. Three-dimensional (3D) reconstruction of micro-CT image was used to compare bone loss in the upper jaw of the areas defined above in each animal.Similarly, the percentage of bone loss of the right upper jaws between the 1st molar and 2nd molar interdental alveolar areas of vehicle-injected ALDH2*2 knockin mice was designated as the ALDH2 LPSAfter the examination of micro-CT, all samples were demineralized using 15% EDTA for 4 weeks and embedded with paraffin. The samples were sectioned horizontally (5 \u03bcm thickness), perpendicular to the long axis of the teeth, and stained with hematoxylin and eosin for histological observation. The sections were screened from the cemental enamel junction to the apical area vertically and the top first section with bony structure. The expression of osteoblasts in the area of interest of each mouse was analyzed and the total number of osteoblasts was counted.+, WT LPS-, ALDH2 LPS+, and ALDH2 LPS-), were counted three times under a light microscope  and quantified. Comparison of the average total number of osteoblasts in the ROI areas were calculated by using ImageJ software and presented as the mean \u00b1 standard deviation (SD). Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by post hoc procedure  (p < 0.05 was considered to be significant)The interdental areas between 1st and 2nd molar and interdental areas between the 2nd and 3rd molar in the right upper jaws and left upper jaws were defined as the region of interest (ROI) areas of each animal. The average total number of osteoblasts in the top section with bony structure in the ROI areas of each mice of the four different groups . Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by post hoc procedure  (p < 0.05 was considered to be significant).For in vitro measurement of ALDH2 expression, the results were expressed as means \u00b1 standard error from at least three separate experiments. The statistical analyses were determined by Student\u2019s t-test, To evaluate the effect of human osteoblasts that were transfected by lentivirus carrying the human ALDH2-silencing shRNA, ALDH2 protein expression was analyzed by Western blot using the ALDH2 antibody. Compared with the control osteoblasts transfected with an empty lentivirus, ALDH2 protein expression in the ALDH2 knockdown transfected osteoblasts was reduced  staining was used to quantify the mineralization abilities of the osteoblasts on Day 7 and on Day 14 after transfection using the matrix calcification index, as described by Ratisoontorn et al. . On Day Porphyromonas gingivalis LPS-induced dental bone loss by localized injection of purified LPS (20 ug in 2 uL of endotoxin-free water) to mimic the bacterial infection and inflammation of the upper jaw interdental alveolar bone area on the left sides of the region of interest (ROI) areas in all mice. In addition, drinking water with 10% alcohol was provided every day continuously to mimic the effects of chronic alcohol drinking. We compared the percentage of bone loss in the left side of the upper jaws between the 1st and 2nd, and between the 2nd and 3rd molar interdental areas between the wild-type mice and the ALDH2*2 mice injected with 20 \u03bcg (2 \u03bcL in endotoxin-free water) of purified lipopolysaccharide from Porphyromonas gingivalis and the corresponding animal groups injected with the vehicle control (2 \u03bcL of endotoxin-free water). Three-dimensional (3D) reconstruction of micro-computed tomography (micro-CT) image was used to compare bone loss in the upper jaw of the animals. +), the water-injected wild-type group (WT LPS-), the ALDH2*2 knockin LPS-injected group (ALDH2 LPS+), and the ALDH2*2 knockin water-injected control group (ALDH2 LPS-). There are three upper molar teeth on each side of the upper jaw of each mouse. In -, d1 site) was calculated as 100%. The distances were measured and calculated in all other groups and compared with the wild-type control group, as shown in +) or without LPS injection (WT LPS-) (-) showed similar bone loss at the d1 and d2 sites as the wild-type mice with LPS (WT LPS+) or without LPS injection (WT LPS-) (+) showed 30% higher bony loss at the d1 site and 30% higher bony loss at the d2 site than the corresponding sites of the wild-type animals (WT LPS- and WT LPS+) and those of the ALDH2*2 knockin mice without LPS injection (ALDH2 LPS-) (- and WT LPS+) and those of the ALDH2*2 knockin mice without LPS injection (ALDH2 LPS-) (+) > (ALDH2 LPS-) = (WT LPS+) = (WT LPS-).We used a genetic model of ALDH2*2 knockin mice to evaluate the effect of periodontal bone defect in vivo with age-matched wild-type mice as a control. The ALDH2*2 knockin mice mimics the ALDH2*2 single amino acid substitution of ALDH2*2 mutation in humans . We alsoWT LPS-) . The bonWT LPS-) . HoweverH2 LPS-) . Since dH2 LPS-) . In summTo support our findings, a histological analysis of hematoxylin and eosin (H&E) stained paraffin sections of the upper jaws of all mice was also carried out. We counted the total numbers of osteoblasts in the region of interest (ROI) areas of all specimens from both sides of the upper jaws of the four different animal groups.- and WT LPS+, - and ALDH2 LPS+, +) was significantly diminished as compared with the ALDH2*2 mice without LPS injection  was also reduced as compared with the group without LPS injection  was reduced by 17% as compared with the non-treated group . In the ALDH2*2 knockin mice, the total number of the osteoblasts in the LPS treated group (ALDH2 LPS+) was reduced by 21% as compared with the untreated group . Comparing the effects of LPS injection in the wild-type mice and ALDH2*2 knockin mice, the total number of osteoblasts in the ALDH2*2 knockin mice (ALDH2 LPS+) was only about 80% of that of the wild-type mice  indicating that the ALDH2*2 mutation rendered the mice more susceptible to the Porphyromonas gingivalis LPS toxin. Comparing the wild-type mice and the ALDH2*2 knockin mice without LPS injection, we found that the total number of osteoblasts in the ALDH2*2 knockin mice (ALDH2 LPS-) was only about 72% as compared with that of the wild-type mice (WT LPS-). This indicated that alcohol intake alone, without LPS injection, was able to inhibit bone regeneration potential with the ALDH2*2 genotype, although we did not observe this difference using the technique of micro-CT imaging (+). In this group, the number of osteoblasts was reduced by 33.3% as compared with the wild-type mice that did not receive the treatment of LPS toxin . These findings indicated that under alcohol intake, the ALDH2 mutation by itself may reduce the growth of the osteoblasts and injection of Porphyromonas gingivalis LPS toxin further exacerbated the growth of osteoblasts in the ALDH2*2 knockin mice. imaging . As expeA previous study has revealed a significant association between the ALDH2 Glu504Lys (or ALDH2*2) polymorphism and osteoporosis . The prePorphyromonas gingivalis lipopolysaccharides for 6 weeks under the exposure of 10% alcohol in drinking water. Excess alcohol drinking has been shown to directly cause systemic diseases. Many studies have also indicated that alcohol consumption is a risk factor for periodontitis [Porphyromonas gingivalis [Porphyromonas gingivalis is a well-known periodontal pathogen and is one of the predominant species in the gingival pockets of patients with advanced and severe periodontal disease [Porphyromonas gingivalis has been demonstrated to be able to cause bone resorption and inhibit bone formation as a culprit of chronic periodontitis [Porphyromonas gingivalis to create periodontal inflammation and bony defects. Previous studies have shown that LPS from Porphyromonas gingivalis prevented apoptosis of HL60-derived neutrophils and the signaling of P. gingivalis through toll-like receptor 2 (TLR2) and may account for the inhibitory effect of P. gingivalis LPS on apoptosis, thus, provided a mechanism for the development of destructive periodontal disease [P. gingivalis LPS caused a more severe periodontitis in mice [P. gingivalis LPS could activate the p38 MAPK and NF-\u03baB signaling pathways and induce periodontitis and gingival bone resorption [P. gingivalis LPS is a strong inducer of oral bone resorption and periodontal diseases. In our study, we found that P. gingivalis LPS-injected ALDH2*2 knockin mice had a significantly higher percentage of bone loss as compared with the LPS-injected wild-type mice and both ALDH2*2 knockin and wild-type mice without LPS injection based on micro-CT scan  > (ALDH2 LPS-) > (WT LPS+) > (WT LPS-)  . We noteWe believe that these results are important for periodontitis and warrant confirmation by epidemiological and clinical studies in human populations where ALDH2*2 mutation, alcohol drinking, and poor oral hygiene are common. Such confirmation would benefit future development of precision dental care and treatment or prevention of dental bone loss among East Asians with the ALDH2*2 genotype.Porphyromonas gingivalis LPS-induced toxicity in dental bone loss. The ALDH2 function was suppressed by silencing its gene activity in human osteoblasts. After transfecting cells with a lentivirus vector expressing ALDH2 shRNA, ALDH2 protein level was reduced by 80% as compared with the non-transfected osteoblasts. The ALDH2 knockdown osteoblasts lost their proliferation capability and, concomitantly, had reduced mineralization potential. Using a genetic animal model of ALDH2, we compared dental bone loss in wild-type and ALDH2*2 knockin mice with the injection of purified Porphyromonas gingivalis LPS and the administration of 10% alcohol in drinking water for 6 weeks. The micro-CT scan results indicated that dental bone loss was significantly more serious in the ALDH2*2 knockin mice injected with LPS. We also found that alcohol alone is a risk factor for bony defects in animals carrying the ALDH2*2 mutation based on the histology of stained bony samples, and the ALDH2 mutation together with LPS injection exacerbated the extend bony defects. These results suggest that the genetic factor of ALDH2 mutation interacts with lifestyle factors, such as alcohol consumption and poor oral hygiene such as the oral toxicity of bacterial-induced Porphyromonas gingivalis LPS, and therefore plays a crucial role in the pathology of dental bone loss in periodontal disease. Our findings may have important clinical implications for the dental health of 560 million East Asians carrying the ALDH2*2 mutation.In this study, we investigated the roles of ALDH2, alcohol, and"}
+{"text": "The graph labels for placebo and hydroxychloroquine were mistakenly transposed, the hazard ratio for lopinavir and ritonavir should have been 1.16 instead of 1.17, and the Population section should not have mentioned expected hospital stays. This article has been corrected.1In the Original Investigation titled \u201cEffect of Early Treatment With Hydroxychloroquine or Lopinavir and Ritonavir on Risk of Hospitalization Among Patients With COVID-19: The TOGETHER Randomized Clinical Trial,\u201d"}
+{"text": "The World Health Organization has declared SARS-CoV-2 COVID-19) a pandemic in March 11, 2020 9 a pande during tFourteen children who were victims of sexual abuse consulted the pediatric medical emergency department; this number is 2.3 times the number who consulted during the same period in 2019. Although the rate is increased by 2.3, it certainly remains underestimated due to the difficulties by families of accessing hospitals and social structures ["}
+{"text": "Triglycerides (TGs) are transported in the bloodstream by TG-rich lipoproteins in the form of chylomicrons and VLDLs. The hydrolysis of circulating TGs is rate-limiting for their uptake into tissues and is catalyzed by the enzyme lipoprotein lipase (LPL) . The actet\u00a0al. (APOAI/CIII/AIV gene cluster, while van der Vliet identified APOA5 as the most highly induced gene after partial hepatectomy (APOA5 variants are associated with elevated plasma TG levels (Of this list of proteins, the most elusive LPL regulator is probably APOA5. APOA5 was discovered independently by two groups in 2001. Pennacchio atectomy . Targeteatectomy . The rolHow APOA5 lowers plasma TGs has remained controversial. A report suggested that APOA5 reduces VLDL-TG production . Alternaet\u00a0al. (Here, inspiration may be taken from the angiopoietin-like proteins ANGPTL3, ANGPTL4, and ANGPTL8. These proteins function as inhibitors of LPL and are present in plasma at concentrations that resemble the plasma APOA5 concentration. Although ANGPTLs were initially believed to operate individually, recent findings indicate that ANGPTL3 and ANGPTL4 form complexes with ANGPTL8 , 11, 12.et\u00a0al.  now findet\u00a0al. stumbled upon APOA5 when trying to identify proteins in human serum that associate with immobilized ANGPTL3/8. They also succeeded in immunoprecipitating APOA5 from human serum when using an antibody directed against the ANGPTL3/ANGPTL8 complex. Subsequent biolayer interferometry experiments showed that recombinant APOA5 is able to efficiently bind to the ANGPTL3/8 complex. At the functional level, recombinant APOA5 impaired the ability of the ANGPTL3/ANGPTL8 complex to inhibit LPL. By contrast, APOA5 did not influence LPL inhibition by ANGPTL3 and ANGPTL4 alone or in the absence of any angiopoietin-like proteins. Answering to the original conundrum about the low plasma APOA5 concentration, it was found that the suppression of ANGPTL3/8-mediated LPL inhibition by APOA5 occurred at a molar ratio consistent with the plasma concentrations of APOA5 and ANGPTL3/8.Chen After 15\u00a0years of uncertainty, these new biochemical data represent a potential breakthrough in our molecular understanding of how APOA5 lowers plasma TGs. Whether APOA5 also interferes with the function of ANGPTL3/ANGPTL8 in\u00a0vivo is still unknown. Accordingly, it would be of interest to investigate the effect of overexpression of APOA5 or injection of recombinant APOA5 on postprandial plasma TGs or on plasma TG clearance in mice deficient in ANGPTL3 or ANGPTL8.et\u00a0al., another therapeutic approach could be to mimic the binding of APOA5 to ANGPTL3/ANGPTL8, thereby relieving LPL inhibition by ANGPTL3/ANGPTL8. To enable the design of such a strategy, better insight is needed into the specific domains in APOA5 and ANGPTL3/ANGPTL8 involved in their mutual interaction.This exhilarating new discovery might create potential novel opportunities for the therapeutic targeting of hyperlipidemia, an important risk factor for atherosclerotic cardiovascular disease. Currently, ANGPTL3 is a hot target for the treatment of atherosclerotic cardiovascular disease. Inactivation of ANGPTL3 via antisense oligonucleotides and monoclonal antibodies effectively lowers plasma TGs and LDL-C in mouse models and in human volunteers , 16. BasANGPTLs and APOA5 have followed a very different path since they were first cloned about twenty years. Although the importance of APOA5 in regulating plasma TG levels quickly gained ground, the recognition of the role of ANGPTLs in controlling human plasma lipoproteins took a much more gradual course. Their mutual interaction adds an exciting new twist to a now rapidly evolving field."}
+{"text": "The present study investigated the joint impact of adolescent sport experience and dopamine\u2010related genes  on sport participation in adulthood.Using the National Longitudinal Study of Adolescent Health  data, the hierarchical multivariable logistic regression models for predicting sport participation in wave 3 (around 20 years of age) and wave 4 (around 30 years of age) were conducted separately by gender  and gene (DRD2 and COMT genes).Adolescent sport experience significantly interacted with the number of DRD2 A1 alleles and COMT Met alleles in affecting wave 3 sport participation among male adults. The interaction between adolescent sport experience and DRD2 gene significantly affected wave 4 sport participation in opposite direction to that affected wave 3 sport participation among male participants. Among female participants, there were no significant interaction effects between dopamine\u2010related genes and adolescent sport experience on sport participation in both wave 3 and 4.Since adult sport participation is most likely to be influenced by the joint impact of environmental and genetic factors, it is important to consider gene\u2010by\u2010environment interactions when designing policies or programs to promote adult sport participation. The significant effects of the number of DRD2 A1 alleles on wave 3 sport participation became insignificant if male participants actively participated in sports in adolescence, indicating that adolescent sport experience may overpower the effect of DRD2 genes on sport participation in adults aged around 20 years. Sports participation invariably involves physical activity and inherently includes various enjoyable aspects, such as personal challenge, social interaction, goal achievement, and competition. The International Society for Physical Activity and Health also suggests that sports participation is \u201can investment that works\u201d to increase physical activity and improve health . The wave 2 follow\u2010up survey was conducted 1 year after wave 1 (89% response rate). The wave 3 (77% response rate) and wave 4 (80% response rate) follow\u2010up surveys were conducted 6 and 13 years after wave 1, respectively. More information about the survey design of Add Health can be found elsewhere  or strenuous team sports  in waves 3 and 4. Respondents who participated in individual or team sports more than 4 days per week in each wave were categorized as active participators.Using a salivary DNA collection device , participants provided 2 ml of saliva in wave 4. In order to extract DNA, saliva was packed in the container  and shipped to the University of Colorado, Institute for Behavioural Genetics. Nine (0.06%) were empty and 24 (0.15%) were damaged or leaking during shipping. Further information about Add Health wave 4 candidate gene data is reported in more detail elsewhere  were genotyped on either an Applied Biosystems TaqMan\u00ae OpenArray\u00ae (archived samples) or an Illumina BeadXpress\u00ae GoldenGate\u00ae (non\u2010archived samples) platform for both DRD2 Taq1A SNP rs1800497 in the 30 UTR and COMT val158met SNP rs4680. The DRD2 Taq1A (rs1800497) assay and COMT val158met (rs4680) assay were carried out using a fluorogenic 5\u2032nuclease  method , education . Since addictive behaviors have been shown to be associated with DRD2 and COMT genes, smoking (current smoker), binge drinking (5 or more drinks in a row), and marijuana use (1 or more times) were also used as a covariate when predicting sport participation in adulthood.2.3p\u2010values lower than .05 were considered as significant. Analysis of variance (ANOVA) and Chi\u2010square test were used to compare between active and inactive participators in sports. All the analyses were performed using SAS version 9.4 .The hierarchical multivariable logistic regression models for predicting sport participation in wave 3 (around 20 years of age) and wave 4 (around 30 years of age) were conducted separately by gender  and gene (DRD2 and COMT genes). In model 1, the effect of gene on adult sport participation was examined after controlling for age, education, and addictive behaviors. In model 2, adolescent sport experience was included in model 1 to see if there is any change in the effect of gene on sport participation. In model 3, the interaction between adolescent sport experience and gene was included in model 2 to examine the joint impact of adolescent sport experience and gene on adult sport participation. Wave 3 Sport participation was included as a covariate in all models predicting wave 4 sport participation. Other covariates  were assessed at the same wave as dependent variables. Deviance statistics (\u20102 log likelihood) were used to compare nested models. The 33.1Table\u00a0The characteristics of male and female participants by sport participation in wave 4 are presented in Table\u00a03.2The results of multivariable logistic regression for predicting sport participation in wave 3 (around 20 years of age) are presented in Table\u00a0Table 4To our knowledge, the present study is the first study that investigated the joint impact of adolescent sports experience and dopamine\u2010related genes  on sport participation in adulthood. The results of the present study showed that adolescent sport experience is strongly associated with sport participation not only among adults aged around 20 years but also among adults around 30 years of age. This result is in line with previous findings showing that participating in sports during adolescence increases the probability of sports participation in adulthood (Batista et\u00a0al., The present study found out that adolescent sport experience interacts with the number of DRD2 A1 alleles and COMT Met alleles in affecting sport participation among male adults around 20 years of age. As shown in Figures\u00a0Another interesting finding in this study was that the interaction between adolescent sport experience and DRD2 gene affected wave 4 sport participation in the opposite direction to that affected wave 3 sport participation among male participants. The number of DRD2 A1 alleles did not affect wave 4 sport participation among male participants who were not active participators in sports in adolescence (see Figure\u00a0Among female participants, there were no significant interaction effects between dopamine\u2010related genes and adolescent sport experience on sport participation in both waves 3 and 4. Previous studies have shown that women are more susceptible to socio\u2010cultural norms and perceptions compared to men because their social relationships are characterized by emotional support, self\u2010disclosure, and intimacy Maccoby, . Their hThe present study has several limitations. First, more detailed information on sport participation is needed because there exist many different types of sporting activity. In order to understand the influence of dopamine\u2010related genes on sport participation in more detail, future studies should at least distinguish team sport participation from individual sport participation. Second, since this study used self\u2010reported sport participation, there is a risk of recall bias. Third, since Add Health is a prospective longitudinal study that followed up a nationally representative sample of middle and high school students in the United States, the results of this study may not be generalizable to other countries. Despite these limitations, the present study may contribute to the literature by providing new information on the joint impact of adolescent sport experience and dopamine\u2010related genes on adult sport participation. Our results suggest that adolescent sport experience may overpower the effect of DRD2 and COMT genes on sport participation in male adults aged around 20 years. However, our results also indicate that adolescent sport experience may act as a trigger for increasing sport participation in male adults around 30 years of age, who carry the DRD2 A1 allele. Since adult sport participation is most likely to be influenced by the joint impact of environmental and genetic factors, it is important to consider gene\u2010by\u2010environment interactions when designing policies or programs to promote adult sport participation.https://publons.com/publon/10.1002/brb3.2459The peer review history for this article is available at"}
+{"text": "Glioblastoma (GBM) is an extremely heterogeneous tumor and its different regions are populated with phenotypically distinct types of cancer cells. However, it is still unclear how multiple GBM populations arise from the originally homogenous group of tumor precursor cells. Here we showed that GBM cells from the core and edge of the tumor have different composition of ribosomes due to the alternative RNA splicing of multiple ribosomal genes with highest differences observed for RPL22L1. We found that cells at the edge of the tumor express classical isoform of RPL22L1 (RPL22L1a) while core cells have a novel RPL22L1b isoform. RPL22L1b appears due to low pH condition at the core of the tumor. It allows cells to survive during acidosis, promotes more aggressive phenotype in vivo and correlate with worse patient outcome. Mechanistically, RPL22L1b binds to lncRNA MALAT1 in the nucleus and induces its degradation enhancing stemness of GBM cells. On the other hand, RPL22L1a interacts with ribosomes in cytoplasm and upregulates p53 translation favoring less aggressive edge phenotype of GBM. The splicing switch between RPL22L1 isoforms is regulated by SRSF4 proteins. We identified a small molecule compound that inhibits SRSF4 and impairs splicing of RPL22L1, inducing apoptosis of GBM cells and decreasing tumor growth in vivo. Altogether, our data unraveled the mechanism by which less aggressive edge-like GBM cells acquire more malignant core-like phenotype during tumor growth. It may also explain discrepancies between proteome and transcriptome of GBM cell populations. Targeting this pathway may help to decrease tumor heterogeneity and eliminate therapy resistant cells at the tumor core.Work was supported by the Russian Science Foundation grant 19-44-02027."}
+{"text": "Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by  Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. In an earlier study, we examined the genetic mechanism used by haploid yeast cells to acquire resistance to 2-deoxyglucose. Here, we conducted the same genetic selection on diploid cells. Diploid cells use dominant alleles, haploinsufficiency, gene duplication and aneuploidy to achieve drug resistance. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts. Saccharomyces cerevisiae has been used as a model eukaryotic organism for genetic studies due to its many properties that are advantageous to laboratory research. These advantages include its short generation time, a small and well-annotated genome, robust recombination systems and its ability to stably grow in either haploid or diploid states [The yeast d states . Most geREG1 and duplication of a large segment on chromosome 4 that increased expression of three hexose transporters genes. While many of the genetic mechanisms for the acquisition of 2DG-resistance were utilized by both haploid and diploid cells, the use of a monosomy and haploinsufficiency are options only possible in diploid cells. As has been suggested for other traits in yeast [Genetic studies of the yeast cell\u2019s adaptation and response to the metabolic inhibitor 2-deoxyglucose (2DG) have identified many of the genes required for the maintenance of and release from glucose repression . All of in yeast \u20139, these7 cells were spread onto agar plates containing synthetic complete media with 2% glucose and supplemented with 0.1% 2DG. Plates were incubated for 4\u20136 days until colonies appeared. The frequency at which spontaneous 2DG-resistant colonies appeared was approximately 4\u20136 colonies per 107 cells. These 2DG-resistant candidates were propagated in synthetic complete medium, and cultures were preserved in glycerol stocks at -80\u00b0C. Initial characterization of the 2DG-resistant isolates was conducted by examining growth properties of the candidates on agar plates in the absence or presence of 0.1% 2DG. Candidates which failed to retain significant resistance to 2DG were discarded. Nine of the isolates exhibited normal growth properties and stable resistance to 2DG. Seven of the 2DG-resistant diploid isolates formed small colonies when grown on agar plates lacking 2DG  of yeast AMP-activated protein kinase . One strain (DS9) contained both an aneuploid state (monosomy of chromosome 4 and trisomy of chromosome 2) as well as a 117 kb duplication of a region of the chromosome 4  in DS21 and in the beta subunit gene (GAL83) in DS16  and previously as the glycogen binding domain (GBD). This domain is conserved among all AMPK beta subunits from yeast to mammals [SIP1 and SIP2 genes  demonstrate even greater 2DG resistance but this comes with a heavy fitness cost [REG1 locus confers 2DG resistance.Two of the 2DG resistant diploid strains were heterozygous at the one copy . In DS15 alleles . To testn assays  and 2DG n assays . Cells h p<0.01; . HomozygSeven of the 2DG-resistant diploid strains contained a monosomy of chromosome 4 . These iExamination of the diploid strains exhibiting aneuploidy revealed that six of the 2DG resistant strains  shared the trait of having extra copies of chromosome 8 . In an eDOG1 and DOG2 genes are adjacent to each other and are located on chromosome 8. We next analyzed the diploid strain DS14 which contained a chromosome 1 monosomy and chromosome 8 tetrasomy. DS14 was subjected to sporulation and the viable progeny (haploid spores lacking chromosome 1 are not viable) were sequenced and used to generate diploid strains with 2, 3 or 4 copies of chromosome 8. Chromosome copy number was confirmed by whole genome sequencing and comparison of the normalized median read depth for each chromosome (2n) and the strain with chromosome 8 tetrasomy. The dose dependence of DOG gene copy number and 2DG resistance was further confirmed with haploid strains with 0, 1, 2 or more copies of the DOG1 and DOG2 genes  also located on this chromosome. Increased expression of HXT1 and plasma membrane retention [HXT4, HXT1 and HXT5 genes on one copy of the disomic chromosome 8 with the HIS3 gene to generate the hxt415\u0394::HIS3 allele. Finally, as a negative control we replaced the SIP2 gene on chromosome 7 (sip2\u0394::HIS3) since we knew from earlier studies that this gene deletion did not confer 2DG resistance or sensitivity [1n) reference strain. Deletion of the DOG loci from one of the disomic chromosomes significantly reduced the 2DG resistance while deletion of the HXT cluster and the SIP2 gene had no effect on 2DG resistance. Thus, the extra copies of the DOG1 and DOG2 genes are necessary for the 2DG resistance conferred by the disomy of chromosome 8.To confirm that the etention  had beensitivity . The disREG1 gene, which can by itself confer 2DG resistance via haploinsufficiency (DOG1/2 genes (p<0.001) 2DG resistance  exhibited significant 2DG resistance (HXT7 gene did not by itself confer any significant 2DG resistance (plasmid 4F6). However, cells transformed with plasmid 4G6 containing HXT3 and HXT6 were more resistant to 2DG than were cells transformed with plasmid 4H6 that contains only HXT3.While most of our 2DG resistant strains contained a single alteration that was sufficient to confer 2DG resistance, two strains contained more than one genetic change, each capable of contributing to a 2DG-resistant phenotype. For instance, DS7 contains both a stop codon in the ficiency , and a t/2 genes . Closer /2 genes . Sporulasistance . The genpe cells . Wild type cells  whose insistance . IncreasREG1 gene on chromosome 4  lacking the ability to phosphorylate 2DG are completely resistant to 2DG [Two-deoxyglucose is a potent inhibitor of glycolysis in both yeast and mammalian cells growing on glucose ,25. Yeast to 2DG . Also, gt to 2DG .HXK2 gene that reduce the enzyme\u2019s catalytic activity confer resistance to 2DG [HXK2 gene confer 2DG resistance and not mutations in the other two hexokinase genes in yeast is a puzzle that remains to be solved. Both Hxk1 and Hxk2 enzymes are able to phosphorylate 2DG in vitro [HXK2 gene confer 2DG resistance. Loss of function alleles in the HXK2 gene is a genetic adaptation that has only been observed in haploid cells [HXK2 locus do not have a growth advantage in 2DG. In order to increase the rate of 2DG-6P degradation, both haploid and diploid cells increase the expression of the DOG1 and DOG2 genes by increasing the copy number of chromosome 8. 2DG resistance exhibits a strong positive correlation with DOG gene copy number  are hypersensitive to 2DG whereas cells with an activated allele of SNF1 are resistant [SNF1-G53R allele [GAL83-S224R and SNF4-N177Y alleles. Thus dominant, missense alleles in the genes coding all three subunits of the AMPK heterotrimer can confer 2DG resistance  exhibit modest 2DG resistance without the slow growth phenotype in the absence of 2DG observed in diploid cells homozygous for the reg1\u0394 allele.A second mechanism to activate AMPK signaling is to reduce activity in the PP1 phosphatase, an inhibitor of AMPK activity. The PP1 isoform that dephosphorylates and inactivates yeast AMPK is composed of the catalytic subunit, Glc7, bound to the regulatory subunit, Reg1 ,29. The ss costs . Haploidficiency . DiploidREG1 gene is on chromosome 4 and we suspect that it is the reduced expression of REG1 (3) for disomic states responsible for the phenotypic switching of yeast colony phenotype [Diploid cells with a chromosome 4 monosomy are resistant to 2DG  and show of REG1  that con of REG1 . The rathenotype . We havehenotype ,31.REG1, a PP1 phosphatase subunit, is a key driver of the chromosome 4 monosomy. The great advantage of this genetic strategy is that the chromosome 4 monosomy is freely reversible. Diploid cells can reap the benefits of a chromosome 4 monosomy when 2DG is present and rapidly return to normal ploidy when the 2DG selection pressure is removed . This mechanism for increasing gene expression using homologous retrotransposon sequences has been documented in other studies [DOG genes since the retrotransposons flanking the DOG genes would encompass the centromere on chromosome 8. If this region were duplicated, a dicentric and unstable chromosome would have been generated.Downstream targets of AMPK signaling that are important for 2DG resistance are the glucose transporters. Cells challenged with 2DG increase the endocytosis of the glucose transporters in a process that is promoted by the \u03b1-arrestins Rod1 and Rog3 . AMPK si studies  but woulSaccharomyces cells grown in the laboratory and clinical isolates of Candida from human patients utilize aneuploidy as a means for acquisition of drug resistance [Using selection for 2DG resistance as a model for the selection of drug resistance, our studies show that numerous genetic adaptations are utilized to impact a relatively simple pathway of drug resistance . Both Sasistance . Human csistance . Our stuSaccharomyces Genome Deletion Project [The yeast strains used in this study were all derivatives of S288C. Yeast strains with specific gene deletions were generated in our laboratory or by the  Project  and purc Project .7 cells were spread on agar plates containing synthetic complete media with 2% glucose (g/100ml) and 0.1% 2-deoxyglucose. Plates were incubated at 30\u00b0C for 4\u20136 days, and 2DG-resistant colonies were isolated for further study.Spontaneous mutations that conferred 2DG resistance were selected in the diploid strain MSY1527 . ApproxiThe genomes of the 2DG-resistant strains were analyzed by whole genome sequencing. Genomic DNA was prepared using a glass bead phenol extraction method . SequencPfu polymerase, followed by digestion of the starting plasmid template with the restriction enzyme DpnI [DOG1/2 plasmids were made by inserting the 4.1 kb BamHI\u2014EcoRI fragment  into pRS316 (low copy number) and pRS426 (high copy number) plasmids [Oligonucleotide-directed mutagenesis of plasmids was performed with yme DpnI . Mutagenplasmids .2DG resistance was measured in synthetic complete media with 2% glucose (g/100ml) as previously described in McCartney, Chandrashekarappa (11) using either a titration of increasing concentration of 2DG or multiple replicates of cultures grown from independent colonies with and without 0.1% 2DG.S. cerevisiae mRNA using the kallisto software package [RNA samples were prepared from multiple independent yeast cultures grown on synthetic complete medium using the RNeasy Mini Kit (Qiagen). Sequencing libraries were prepared using the TruSeq Stranded mRNA library method (Illumina). RNA sequences were mapped to  package . Each RN package ,40,42. Rp values are indicated as follows: * p<0.05; ** p<0.01; *** p<0.001.Unless otherwise stated, mean values were calculated from a minimum of three independent measurements, and the error bars represent 1 standard deviation. Statistical significance was determined using the Student t test for unpaired variables with equal variance. In all cases, S1 Fig(A) Multiple sequence alignment of the yeast and human (Hs) beta subunit CBM domains. Position of the seven beta sheets (S1-S7) are indicated. The serine residue corresponding to Gal83-S224, Sip2-S226 and Sip1-S541 is indicated by the asterisks. (B) Three dimensional structure of the Sip2 CBM showing the seven beta sheets and the position of S226 (red mesh) is shown using the structure coordinates in 2QLV.pdb . (C and (TIF)Click here for additional data file.S2 FigHaploid strains RS17, RS18 and RS19 with multiple disomic chromosomes were mated with wild type haploid of opposite mating type (MSY188). After sporulation, the haploid progeny were analyzed for 2DG resistance by replica plating and scored as resistant (R) or sensitive (s). Four tetrads from each cross that showed 2:2 segregation of 2DG resistance were selected and the pooled DNA from resistant and sensitive strains was sequenced. The normalized median read depth for each chromosome of the parents  is plotted along with the resistant and sensitive pools.(TIF)Click here for additional data file.S3 Fig(A) Distribution of whole genome sequence read depth for each chromosome is plotted for four haploid strains with median read depth for each chromosome indicated by the yellow bar. (B) Read depth is plotted as a function of position along chromosome 8 near the DOG1/2 locus for MSY1553 (black) and MSY1560 (blue). The gene map for this region of chromosome 8 is shown above. (C) Distribution of read depth 10 kb before, 10 kb after and across the DOG1/2 locus is plotted for MSY1553 (black) and MSY1560 (blue). The median value is indicated by the yellow bar in the plot and numerically below. (D) Read depth is plotted as a function of position along chromosome 8 near the HXT415 locus for MSY1553 (black) and MSY1562 (blue). The gene map for this region of chromosome 8 is shown above. (E) Distribution of read depth 10 kb before, 10 kb after and across the HXT415 locus is plotted for MSY1553 (black) and MSY1562 (blue). The median value is indicated by the yellow bar in the plot and numerically below.(TIF)Click here for additional data file.S1 TableTranscripts per million mapped reads (tpm) is provided for 5917 yeast open reading frames. Samples S72-S83 are triplicate datasets from wild type diploid cells (MSY1527) and DS10 cells (chromosome 4 monosomy) washed off plates and grown for two hours in SC media with 2% glucose or in the same media with 0.1% 2DG. Samples S132-S147 are quadruplicate datasets from wild type diploid cells (MSY1527) and DS14 cells (chromosome 1 monosomy and chromosome 8 tetrasomy) grown to mid-log in SC media with 2% glucose and two hours after addition of 2DG to 0.1%.(XLSX)Click here for additional data file."}
+{"text": "To measure the rate of patients receiving high dosage antipsychotics.To review the adherence to maximum recommended doses of antipsychotics as per the product information approved by Australian Therapeutic Goods Administration, product information approved by Medsafe  and Therapeutic Guidelines High dose antipsychotics or combination of antipsychotics are associated with significant adverse effects including QTc prolongation, arrhythmias, sudden cardiac death, seizures, increased incidence and severity of adverse effects, longer hospital stay and possibly increased mortality. High dose antipsychotic prescribing may arise as a result of EITHER single antipsychotic drug prescribed at a daily dose above the recommended limit (High Dose single drug) OR More than one antipsychotic prescribed concurrently where the sum of doses given expressed as a percentage of the SPC maximum of each drug exceeds 100% (High-Dose through the prescribing of multiple drugs).The data were gathered from all the drug charts for all patients admitted to HDU and Acute ward on 9th April 2019. The Audit standards were 1) Individual antipsychotic dose should be within recommended limit as 100% and 2) Combined antipsychotics should be within recommended limit as 100%Total number of patients on both the HDU and Acute wards = 33Total number of patients on antipsychotics = 30Number of patients on > 100% of recommended cumulative dosage = 13/30 = 43.3%Number of patients on > 100% maximum limits of regular antipsychotics = 3 = 10%Number of patients on > 100% maximum limits of PRN antipsychotics = 0/30Number of patients on 2 antipsychotic = 18/30 = 60%Number of patients on 3 antipsychotic = 8/30 = 26.6%Number of patients on 4 antipsychotic = 2/30 = 6.6%Out of the 30 patients on antipsychotics, almost half were on more than 100% of the recommended cumulative maximum limits of antipsychotics doses, almost 2/3rds were on 2 or more antipsychotic and a quarter on 3 or more. This can be associated with significant adverse effects including QTc prolongation, arrhythmias, sudden cardiac death, seizures, increased incidence and severity of adverse effects, longer hospital stay and possibly increased mortality. There is a need to review PRN antipsychotics prescribed as a norm, clear documentation and need for a protocol for increased vital sign monitoring for patients on high dose antipsychotic treatment."}
+{"text": "Mutations in BRCA1 and BRCA2 genes are well-established risk factors of breast and ovarian cancer. In our former study, we observed that approximately 6% of unselected ovarian cancer patients in the region of Podkarpacie (South-East Poland) carry BRCA1 causative founder variants, which is significantly lower than in other regions of Poland. Therefore, it is deeply justified to do research based on the sequencing of whole BRCA1 and BRCA2 genes.BRCA1 and BRCA2 genes Next-Generation Sequencing study in all cases.We examined 158 consecutive unselected cases of ovarian cancer patients from the region of Podkarpacie. We performed BRCA1 or BRCA2 pathogenic mutations were found. BRCA1 pathogenic variants were detected in 11 of the 158 (7.0%) ovarian cancer cases. 10 of 11 (91%) detected BRCA1 mutations were founder mutations, detectable with the standard test used in Poland. BRCA2 pathogenic variants were found in 7 of the 158 (4.4%) cases. No BRCA2 pathogenic variants were founder mutations. The median age of patients at the diagnosis of the 18 hereditary ovarian cancers was 57.5 years.Altogether, in 18 of 158 (11.4%) ovarian cancer patients with BRCA1 or BRCA2 gene mutation carriers among patients with ovarian cancer from the Podkarpacie region is comparable to other regions of Poland. However, a significantly higher percentage of BRCA2 gene mutations was observed, that were not detectable with a standard test for detection of founder mutations. Diagnostics based only on testing the BRCA1/2 Polish founder mutations is characterized by relatively low sensitivity in the case of ovarian cancer patients from South-East Poland and should be supplemented by NGS study, in particular of the BRCA2 gene.The frequency of  BRCA1  gene [BRCA1 and BRCA2 mutation frequency and spectrum have been reported [BRCA1 or BRCA2 founder mutation carriers, and a slightly different spectrum of these mutations than in other regions of Poland [The incidence of ovarian cancer is approximately 3,600 cases annually in Poland . It has f Poland .BRCA1 and BRCA2 mutations in unselected ovarian cancer patients from the Region of Podkarpacie with the use of NGS, and establish an optimal algorithm for genetic testing of women diagnosed with ovarian cancer from this region.The aim of this study was to define the prevalence and spectrum of Ovarian cancer cases were identified from patients treated at a clinical base in the Department of Obstetrics and Gynecology of Fryderyk Chopin University Hospital No 1 in Rzeszow, Poland between January 2013 and January 2017. All patients were inhabitants of the South-East region of Poland. The study group consisted of 158 consecutive, newly diagnosed cases of ovarian cancer after surgical treatment, unselected for age or family history. The mean age of diagnosis was 58.5 years (range 22\u201384 years). The reference pathologist reviewed a representative slide from each cancer to confirm the diagnosis. 20% of patients were diagnosed in the I and II clinical stages according to FIGO, and 80% in III and IV. 14.8% of ovarian cancers showed pathological grading G1, 11.1 - G2, and 74.1 \u2013 G3. A cancer family history was obtained during an appointment with a clinical geneticist.BRCA1 and BRCA2 genes Next-Generation Sequencing (NGS) study in all cases.DNA was isolated from 5 to 10 ml of blood. We performed BRCA1 and BRCA2 genes were screened using the SureMASTR BRCA Screen Kit from Agilent Technologies. The SureMASTR BRCA Screen (Agilent Technologies) analyses the full coding regions of these genes. In brief, 50ng (2.5ng/\u00b5l) of genomic DNA  was used to amplify the target genes in a single-tube multiplex reaction. The obtained amplicon libraries were purified and diluted before single-tube universal PCR reaction to tag all amplicons with specific p5 and p7 adaptors. Each tagged amplicon library was purified to remove small residual DNA fragments, and the DNA library concentration was quantified using a Pre-proof High Sensitivity Qubit quantification kit (Life Technologies). Equimolar quantities of the individually tagged amplicon libraries were pooled, and the final sequencing library was normalized to a concentration of 4nM. Sequencing was performed on a MiniSeq platform (Illumina) using the MiniSeq Mid Output Kit, 2\u2009\u00d7\u2009150 cycles, to obtain reads for both strands. All detected pathogenic mutations and variants of unknown significance (VUS) were validated using Sanger sequencing. The conventional Sanger sequencing was performed with the use of BigDye terminator sequencing kit v3.1 (Life Technologies) on the ABI Prism 3130 genetic analyzer (Life Technologies) according to the manufacturer\u2019s protocol.Bioinformatics analysis was performed using the software MASTR Reporter (Agilent Technologies). This analysis included read alignment to the human reference genome (Genome Reference Consortium GRCh37), variant calling, and visualization of the sequence reads. Variants above 40X coverage depth and with a minimum variant allele frequency of 5% for germline analysis are displayed in the software. Further filtering was applied to select germline variants which had a minimum of 100X coverage depth. Each sample passed quality control analysis.The clinical significance and the implications of variants were identified based on the annotations in public archives: ClinVar, Breast Cancer Information Core, Leiden Open Variation Database Online Mendelian Inheritance in Man (OMIM), Human Genome Variation Society (HGVS), and VarSome. Germline variants were classified according to the ACMG Standards and Guidelines for the Interpretation of Sequence Variants .BRCA1 or BRCA2 causative variant was found in 18 of 158 (11.4%) unselected ovarian cancer cases. A BRCA1 mutation was detected in 11 (7.0%) patients. The c.5266dupC mutation was the most common, it was diagnosed in six patients, followed by the c.181T\u2009>\u2009G mutation observed in three patients, and the c.676delT mutation in one patient. All carriers of these 3 mutations were diagnosed previously with the test based on detection of founder pathogenic variants characteristic for the Polish population [BRCA1 c.5346G\u2009>\u2009A mutation carrier (Table\u00a0BRCA2 gene mutation was diagnosed in 7 (4.4%) unselected ovarian cancer cases. None of these BRCA2 mutations was a recurrent mutation characteristic for the Polish population. We also found in 3 patients, variants of unknown significance (VUS), all in the BRCA2 gene . The median age of diagnosis of the 18 hereditary ovarian cancers was 57.50 years (range 41\u201382 years), compared with a median age of diagnosis of 58.77 years (range 22\u201384 years) for the 140 cases without a mutation. However, the median age of diagnosis in BRCA1 carriers was lower than BRCA2 carriers \u2212\u200955.8 vs. 60.1 years, respectively. A BRCA1 mutation was found in 3, and BRCA2 in 1 of 31 (together \u2212\u200912.9%) women diagnosed with ovarian cancer at or under the age of 50 compared to 8 BRCA1 and 6 BRCA2 carriers of 127 (together \u2212\u200911.0%) women diagnosed at a later age. Among the 18 women with ovarian cancer and a BRCA1 or BRCA2 mutation, ten reported a first- or second-degree relative with breast or ovarian cancer (55.5%), and there was only a slight difference between BRCA1 and BRCA2 carriers . A mutation was present in 25.6% (10/39) of ovarian cancer patients with a positive family history and in 6.7% (8/119) of women with a negative family history. A significant family history, defined as a presence of first- or second-degree relative with breast or ovarian cancer, was observed in 24.7% (39/158) of patients with ovarian cancer (Table A pulation . In addiBRCA1 or BRCA2 genes [BRCA1 causative founder variants was observed in about 10-13.5% of ovarian cancer patients [BRCA1/2 genes in the same group of 158 women affected with ovarian cancer and diagnosed 18 (11.4%) BRCA1/2 mutation carriers. The frequency of individual BRCA1/2 mutations observed in ovarian cancer patients is shown in Table\u00a0BRCA1/2 founder mutations characteristic for the Polish population [BRCA1 and 7 carriers of the BRCA2 gene. These 8 mutations were detectable by whole sequencing only. Like in other regions of Poland, the most frequent mutation was the BRCA1 c.5266dupC mutation observed in 30% (6/18) of all carriers and the BRCA1 c.181T\u2009>\u2009G mutation found in 15% (3/18). Other 9 BRCA1/2 mutations were observed in single patients and are rare in the Polish population. In contrary to our former observation the frequency of BRCA1/2 mutation carriers in the group of ovarian cancer patients is only slightly lower than in other regions of Poland. However, we observed a significantly lower frequency of founder mutations, in particular, BRCA1 c.5266dupC and to a lesser extent BRCA1 c.181T\u2009>\u2009G. This phenomenon can be caused in general by the lower frequency of these mutations in South-East Poland. However, it should be noted that in this region the extensive genetic testing of BRCA1/2 genes has been carried out in patients with ovarian and breast cancer, as well as, in healthy patients since the year 2000. The testing focused mainly on the detection of founder mutations. As a result, several hundred families with BRCA1 founder mutations have been diagnosed so far and several hundred prophylactic adnexectomies have been performed. It should be taken into account that these BRCA1 founder mutations carriers were thus protected against ovarian cancer, and therefore, we observe their lower representation among BRCA1/2 mutation carriers who have now developed ovarian cancer.The region of Podkarpacie is located in the South-East corner of Poland, bordering Ukraine and Slovakia. In our previous study, we identified 10 of 158 (6.3%) of unselected cases of ovarian cancer from this region carried one of 13 founder mutations in the A2 genes . This ispulation . In addiBRCA1 mutation was 55.8 years, and of the 7 patients with BRCA2 mutation was 60.1 years. In both groups, the mean age at diagnosis was slightly higher than the observed in BRCA1/2 carriers from other regions of Poland [BRCA1/2 carriers. However, for non-carriers, the mean age at diagnosis was similar in the region of Podkarpacie and the rest of Poland (58.77 vs. 56.2\u201362.3 years) [The mean age at diagnosis in the 11 cases with BRCA1 as well as the BRCA2 gene. It can be explained by the relatively larger number of family members in an average family, in this region. However, the frequency of BRCA1/2 mutation carriers with negative family history is so high (44.5%), both in groups with recurrent founder mutations as well as with non-founder mutations, that we cannot recommend limiting performing the BRCA1 and BRCA2 gene testing based on NGS to cases with a burdened family history only. Also, the ovarian cancer age of onset is not a factor facilitating the qualification for this study. Taking into account our observations, it should be stated that performing a test based on the detection of Polish founder mutations in ovarian cancer patients from the Podkarpacie region is associated with relatively low sensitivity (55.5%). In turn, performing the NGS test in all subsequent patients with ovarian cancer is associated with a significant increase in costs. Application of NGS tests only in familial cases is associated also with low sensitivity of 55.5%. One of the compromise solutions would be to perform a standard genetic test based on the detection of founder mutations in all patients, and then if no mutation is detected, perform NGS in cases with a family history. With this algorithm of procedure, the sensitivity of detecting the BRCA1/2 gene mutation in ovarian cancer patients would increase to 77.8%, at a relatively low cost.We observed strong family history in 10 of 18 (55.5%) mutation carriers, which is slightly more frequent than in other regions \u20138. This BRCA1/2 gene is routinely tested using the NGS method in DNA extracted from tumour cells. It seems, therefore, that the most justifiable algorithm for detecting a germinal mutation in these patients is to start testing BRCA1/2 genes using the NGS method in DNA extracted from tumour cells. Then, if a mutation is found, the test should be performed in the patient\u2019s peripheral blood to verify whether it is a germline or somatic mutation. If germline mutation is confirmed, the study should be extended to other relatives. However, if such a protocol is used, it should be taken into account that up to nearly 10% of cancer patients with negative NGS results performed in DNA isolated from neoplastic tissue cells, in fact, may carry germline mutation [However, taking into account the current diagnostic standards of patients with ovarian cancer in the context of determining the optimal treatment and qualifying patients for treatment with PARP inhibitors, the mutation . Lincolnmutation  indicatemutation , 13; (iimutation \u201317, (iiimutation . TherefoBRCA1 or BRCA2 causative variants. We found a significantly higher percentage of BRCA2 gene mutations, which are not detectable with a standard test for founder mutations. Diagnostics based only on testing the BRCA1/2 Polish founder mutations is characterized by relatively low sensitivity in the case of ovarian cancer patients from South-East Poland and should be supplemented by NGS study, in particular of the BRCA2 gene.Approximately 11% of unselected ovarian cancer patients in the region of Podkarpacie carry a"}
+{"text": "Scientific Reports 10.1038/s41598-022-07099-2, published online 24 February 2022Correction to: This Article contained a typographical error in the Results section.\u201cThe direct cause of both accidents Please check and confirm the edit and layis shown in Table\u00a01, and the local workplace factors and organizational factors shown in Tables 2 and 3 have been clarified according to each SHEL element classification.\u201dnow reads:\u201cThe direct cause of both accidents is shown in Table 1, and the local workplace factors and organizational factors shown in Tables 2 and 3 have been clarified according to each SHEL element classification.\u201dThe original Article has been corrected."}
+{"text": "The data in Table 2 regarding population aged younger than 20 years, population aged 70 years or older, and population density should have been calculated using the same measurement units as in Table 1. Table 1 has also been amended to fix incorrect values that were given for population density. This article has been corrected.In the Original Investigation titled \u201cAssociation of Social and Economic Inequality With Coronavirus Disease 2019 Incidence and Mortality Across US Counties,\u201d"}
+{"text": "In an articleIn Figure 1(A), the strips from HepG2 and Huh7 cell lines have been replaced by new strips. The correct strips in Figure 1(A) are shown below.In Figure 5(C), the colony image of Sorafenib\u00a0+\u00a0DZNep treated Huh7 cells in colony formation assay was accidently misplaced and has been replaced by the correct image now. The correct colony image of Sorafenib\u00a0+\u00a0DZNep treated Huh7 cells is shown in the figure below.In Figure 5(D), the TUNEL staining and DAPI staining image of DMSO treated 7404 cells in TUNEL assay was accidently misplaced, and has been replaced by the correct image now. The correct the TUNEL staining and DAPI staining image of DMSO treated 7404 cells in TUNEL assay in the figure is shown below."}
+{"text": "For the CH4(70%)\u2013H2(30%)mixture, the flame in the porous medium can be modulated by fluctuationsbetween 0 and 30% of steady methane flow without any noticeable flamedestabilization.Fluctuations in thefuel flow rate may occur in practical combustionsystems and result in flame destabilization. This is particularlyproblematic in lean and ultralean modes of burner operation. In thisstudy, the response of a ceramic porous burner to fluctuations inthe flow rate of different blends of methane and hydrogen is investigatedexperimentally. Prior to injection into the porous burner, the fuelblend is premixed with air at equivalence ratios below 0.275. Thefuel streams are measured and controlled separately by programmablemass flow controllers, which impose sinusoidal fluctuations on theflow rates. To replicate realistic fluctuations in the fuel flow rate,the period of oscillations is chosen to be on the order of minutes.The temperature inside the ceramic foam is measured using five thermocoupleslocated at the center of the working section of the burner. The flameembedded in porous media is imaged while the fuel flow is modulated.Analysis of the flame pictures and temperature traces shows that theforced oscillation of the fuel mixture leads to flame movement withinthe burner. This movement is found to act in accordance with the fluctuationsin methane and hydrogen flows for both CH The mixturewas ignited upstream of a quartz tube containing a porous medium,which also provided partial optical access to the flame.39 The flame movement was recorded via a digitalcamera, and the temperature was measured by a thermocouple at thedesignated parameters of the hydrogen\u2013air mixture. Kakutkinaet al.39 found that for a 70% hydrogenmixture, the flame propagates upstream at a distance of 100 mm in2000 s, and the maximum temperature recorded was around 950 K at approximately1750 s under steady-state conditions. Fuel interchangeability wasstudied by Alavandi and Agrawal40 by combustinglean blends of hydrogen-syngas and methane fuel mixtures inside aporous burner. The air flow rate was kept constant for all tests,while the methane concentration was lowered for each test as the hydrogenand carbon monoxide fuel rates were adjusted to produce the requiredthermal power under steady-state conditions. The authors40 reported reduced carbon monoxide and nitrogenoxide emissions at any flame temperature for hydrogen and carbon monoxidemixtures compared to those produced by a pure methane flame.Kakutkinaet al.41 studied pollutantemissions when hydrogen was added to a natural gas mixture insidea porous burner. The burner was first operated with a natural gas\u2013airmixture; once the combustion was stabilized, a gradual addition ofhydrogen was made. The experiments were conducted for 0.3 \u2264\u03d5 \u2264 0.95, 100 \u2264 P \u2264 700and an interchangeable hydrogen concentration of up to 100% withinthe natural-gas and hydrogen fuel composition. Gauthier et al.41 reported that as natural gas is slowly replacedby hydrogen a reduction in carbon monoxide, carbon dioxide, and nitrogenoxides took place. Also, they found that when the hydrogen contentexceeds 80%, the flame becomes unstable.Gauthier et al.34 experimentally examinedthe combustion of a premixed hydrogen\u2013air mixture by varyingthe size of the combustion chamber inside the porous burner. The purposeof the study was to monitor the flame stability with the inclusionand exclusion of porous media in the combustion chamber. The authors34 chose a stainless-steel mesh to represent theporous media, whereby the hydrogen\u2013air mixture was operatedfor different mass flow rates and equivalence ratios. Peng et al.34 found that as the diameter of combustor chamberdiminished, the flame front within the porous media enlarged acrossthe flow direction. Also, with the insertion of porous media, an increasein heat transfer accelerated the combustion process. Inspired by Alavandiand Agrawal,40 Arrieta et al.42 studied the combustion of mixtures of methaneand syngas inside a porous burner. Their experiments were focusedon the emissions of CO and NOx, flame stability responseto assigned thermal power, and the effects of volume fraction of thesyngas mixtures under steady-state conditions. Arrieta et al.42 used methane as the basic fuel for all testswhere the hydrogen to carbon monoxide ratio was varied. It was concludedthat the addition of hydrogen-rich syngas to methane did not impactthe flame stability or temperature profile drastically. Yet, a considerablereduction in CO and NOx emissions was reported.Peng et al.38 Upon introduction of fluctuations, the flamefeatured hydrodynamic motion inside the porous foam. It was observedthat there were certain amplitude and frequency of flow modulationunder which the flame could survive.38 Asthe characteristics of CH4\u2013H2 blendsare quite distinctive to those of CH4\u2013CO2, the burner stability for combustion of methane and hydrogen blendsneeds to be re-examined. This is particularly due to the high reactivityof hydrogen which can significantly affect flame blow-off and extinction.Themost recent work of the current authors showed that an ultraleanmixture of methane and carbon dioxide burning inside a porous foamcould strongly respond to fluctuations in the methane flow rate.4\u2013H2 mixtures subject to inlet flow disturbances under ultraleanconditions. In an attempt to address this shortcoming, the currentwork investigates experimentally the unsteady ultralean hydrogen combustionof different blends of methane and hydrogen.As heat transfer dominates combustion in porous media, the latteris expected to be influenced by variations in the inlet flow. Thisis particularly the case in ultralean combustion in which changesin the temperature can have a profound effect on the flame stability.Nevertheless, as seen in the preceding survey of literature, currently,there is no systematic experimental study on CH22.1A schematicrepresentation of the experimental setup is illustratedin 2.1.12O3 foam (20 ppi\u2014\u03b5 \u22480.47) positioned nearest to the inlet valve preceded by a funnel-shapedSiC ceramic foam (20 ppi\u2014\u03b5 \u2248 0.47). This was followedby a stack of low-density SiC foams (10 ppi\u2014\u03b5 \u22480.72) completing the preheating region. The combustion region (visiblethrough the quartz glass tube) consisted of predominately high-densitySiC foams (20 ppi\u2014\u03b5 \u2248 0.47). Two layers of low-densitySiC foams (10 ppi\u2014\u03b5 \u2248 0.72) were placed near thecenter of the combustion region to provide further flame stabilityas illustrated in The combustion compartment was made up of two separate sections:the combustion and preheating regions. Both regions consisted of porousceramic layers stacked one atop the other vertically. The preheatingsection (designed to reduce the probability of flashback) entailedan Al2.1.2An openingwas created between the internal side of the outer casing and theexternal side of the combustion compartment for water cooling to takeplace. The vacant gap acted as a water reservoir with an integratedinlet and outlet within the porous burner. The water coolant systemdrew cool water from the water source filling the water reservoirfrom the inlet located near the bottom of the burner. Once the reservoirwas full, the water departed from the outlet (located near the topof the burner) warranting a thorough cooling of the stainless-steelburner.2.1.3The mixtureentering the burner consisted of a blend of hydrogen and methane dilutedwith air. Fuel was supplied from the corresponding gas cylinders anddigital mass flow controllers (MFCs) equipped with manually operatedvalves. Moisture was removed from the compressed air with the useof an air filter, and the air was supplied in an arrangement similarto that of the fuel where a quarter-turn hand valve was installedbefore the air MFCs. Flow Vision SC software was obtained from ALICATto vary the mass flow rate of each MFC using a computer. The programmableMFCs from ALICAT Scientific operated under an uncertainty of \u00b10.6%for both air and fuel. A number of MFCs were installed with relevantoperational ranges 1a for fu2.1.42 and \u00b120ppm for CO. A full high-definition video camera (1920 \u00d7 1080)was mounted \u22481.5 m from the burner to record the flame performanceand migration.2.243 was noted to be44 is given byThe equivalenceratio2, and NOx of the cases described in x emissions arealmost negligible. This is to be expected as the current low-temperaturecombustion system strongly suppresses formation of thermal NOx. Once the flame had been stabilized, the concluding temperaturevalues were noted from Pico software.After the flame was stabilized, as describedin Uncertainty errorwas also calculated to ensure adequate accuracyof the experimental procedure during flow modulation. This includedthe uncertainty error for MFCs and thermocouples. To determine this,thermocouple values were used from case 6x where T3 reported the highesttemperature (1384 K). Therefore, the T3 uncertainty error was assumedacross all five thermocouples. Combined with the supplied MFC manufacturererror (\u00b10.6%), the total accumulative uncertainty error in flowmeasurements = \u00b12.7%.2.3x-axis to determine the flame migrationin the y-direction. Every picture was transformedfrom an RGB image to black and white where a brightness criterionwas introduced for flame detection. The luminosity value for eachconcentration of fuel varied thus authenticated by visual validation.As the brightness conditions were fulfilled, the vertical positionof the parameter was determined (i.e. where the flame had been identified)at the specified reference points. The designated values were reducedby filtering out only the top and bottom y-location.These values served to define the upper and lower section of the flameacross all reference points. This procedure reoccurred until all pictureshad been processed by the code. The final top and bottom positionsof the brightness criterion were then plotted with respect to time,illustrating migration of the flame vertically within the porous mediaalong the five reference points over a full oscillatory cycle.An in-depth imageanalysis was carried out to observe the change in flame position.The video recording of an entire oscillation of 60 s was cut out fromthe footage of each case described in 3The burner was operated undera steady state as well as fluctuatingfuel flow. Here, the outcomes of these two modes of operation arepresented separately.3.118 Flame stabilization is most commonly attainedwhen the flame speed matches the upstream flow velocity of air andfuel mixture.2738 have extensivelydiscussed the response of methane and biogas mixtures within the burnershown in 4(90%) + H2(10%) mixture exhibits a larger thermal power when comparedto its counterpart CH4(70%) + H2(30%); whichis predominantly due to its larger concentration of methane as (perunit volume) methane has a higher enthalpy of combustion comparedto hydrogen. Further, CH4(90%) + H2(10%) isable to operate at \u03d5 = 0.25 as hydrogen provides a higher flametemperature than methane alone to preheat the incoming cold reactantsand avoid flame extinction.In orderto achieve stable combustion inside the ceramic foam, a fine balancebetween heat production, heat deficit, and heat recirculation is required.4(70%)\u2013H2(30%) mixture produces the highest temperature(1340 K), followed by CH4 (1291 K) and then biogas (1233K). This is due to the addition of hydrogen to methane, as hydrogenproduces a greater flame temperature when compared to pure methane.It is also noted that the peak temperature for each mixture is detectedat a different thermocouple point as the flame location within theburner differs between the fuel mixtures. In 4(70%)\u2013H2(30%) mixture. The CH4(70%)\u2013H2(30%) mixture shows 85% reduction in carbon monoxide emissions incontrast to CH4. This could be primarily attributed tothe higher flame temperatures of CH4\u2013H2 blends compared to those of CH4 and biogas. High temperaturefacilitates the oxidation of carbon monoxide into carbon dioxide andthus reduces CO emissions.3.24(90%)\u2013H2(10%) cases before superimposing oscillatory disturbancesat the inlet. 4(90%)\u2013H2(10%) mixture subject to an oscillatory disturbance introducedon the methane flow with an amplitude of 10% of its steady value,over a period of 60 s. The thermocouples were used to record the temperatureof each case where the cut-off temperature to detect flashback wasdefined as T = 773.15 K. The temporal variation ofmethane flow leads to a distinct movement of the reaction region withinthe ceramic foam. This flame movement was video recorded \u2013H2(10%) mixture. As a result, during the trough at an amplitudeof 50% of methane oscillation, the equivalence ratio plummets to 0.1413.Due to this drop, the porous burner struggles to retain heat evenafter supply of the excess methane to the combustion chamber. A similarphenomenon was also reported by Habib et al.38 while operating the same burner on biogas. Yet, it was observedthat an amplitude of 30% of the fuel flow rate for biogas was sufficientto incur greater heat loss. Further, for case 3x, an overall temperaturereduction is observed at thermocouples 3, 4, and 5 with time as thepreheating region receives minimal quantities of heat from the combustionregion. As this continuous cycle of heat loss progresses, the substantialreduction in temperature rules out the possibility of sustaining aflame within the pores of the ceramic foam. Yet, a thicker flame isvisible as a result of greater forcing amplitude despite very littleflame motion.46 The heat loss can be monitoredby the reduction in flame visibility in 4(90%)\u2013H2(10%) mixture was subjectedto inlet sinusoidal disturbances. 4(90%)\u2013H2(10%) mixture velocity even with the hydrogen amplitude set as highas 50%. Therefore, it is noted that with the exception of the secondthermocouple, most thermocouples are insensitive to the oscillationsin hydrogen flow with the third thermocouple recording the highesttemperature (1384 K) early on in the experiment. It can be seen thatthe porous burner withstands the introduced inlet disturbances withoutany visible signs of flame flashback or blow-off. A distinct contrastin the temperature trace of the second thermocouple emerges betweenthe flow modulation of methane and hydrogen. The temperature tracein 4(90%)\u2013H2(10%) mixture shows insignificantflame motion during the eighth cycle. It is noted that in this case,the brightness and color of the flame are not as vivid as relatedto methane modulation due to hydrogen flame being nearly invisible.Next, the fuel concentration of hydrogen in theCH4(70%)\u2013H2(30%) mixture when methane flowis modulated with an amplitude of 10%. The starting point before introducingdisturbances at the inlet was selected to be case 1b for additionalsafety. As the amplitude of oscillation is 10% for the methane flowrate, the equivalence ratio increases to 0.3 at the peak point ofthe oscillatory cycle, much greater than the value at which steadyCH4(70%)\u2013H2(30%) mixture operates stably.However, the local flame is choked when the equivalence ratio fallsto 0.25 at the lowest point of the oscillatory cycle. Still, as theoscillatory cycle period is limited to 60 s, the system not only holdsonto the heat but as the methane flow rate augments, the excess enthalpycombustion is fast-tracked. Consequently, in 38 as thesame porous burner was operated solely on a CH4 mixture.However, despite the CH4 mixture having an identical amplitudeof oscillation, it was noted that the flashback occurred due to alonger oscillation cycle (180 s). 4(70%)\u2013H2(30%) mixture; convection amid the fluid and solid occurs and warmsthe cold reactants. Yet, due to the existence of the low porosityceramic foam downstream of the combustion region, conduction is acceleratedin response to significant methane fluctuations. 4(70%)\u2013H2(30%) flame as the amplitude of hydrogen flow is modulated by 10%. 4(70%)\u2013H2(30%)mixture. In this case, the porous burner manages to withstand aroundeight oscillatory cycles (y cycles 11a befor4(90%)\u2013H2(10%)and CH4(70%)\u2013H2(30%) mixtures. This comparisonprovides further clarity to the results reported earlier. 4 modulation amplitude. Similarly, H2 fuel modulation outputs larger temperature in comparison to CH4 modulation without directly responding to the oscillatorypattern. Case 5x and case 6x display a negligible temperature change. 4(70%)\u2013H2(30%) mixture as theflame has moved further upstream with the greater addition of H2. As a disturbance is superimposed on the fuel inlet, thetemperature trace appears to follow a monotonic increase with theincrease of H2 amplitude. However, this pattern is notobserved as the CH4 amplitude was increased. Case 1y andcase 3y show greater temperature readings and eventually result inflashback albeit case 2y which maintains stable operation with a lowertemperature.4(90%)\u2013H2(10%) and CH4(70%)\u2013H2(30%) mixtures.Once again, the flame thickness in this figure was inferred throughthe image processing technique detailed in 4(90%)\u2013H2(10%) mixture, it can be seen that as the methane amplitudeis increased, mid-way, the flame thickness contracts 13% before postinga gradual increase of 4%. An inverse behavior is observed for hydrogenmodulation, as the amplitude increases so do the flame thickness mid-waybut then contracts at the maximum amplitude value. However, for theCH4(70%)\u2013H2(30%) mixture a monotonicincrease in flame thickness is detected for an increase in amplitudefor both methane and hydrogen, approximately, 300% overall. The exactreason for this behavior is not immediately obvious, and it callsfor further experimental and numerical studies.44(90%)\u2013H2(10%) and CH4(70%)\u2013H2(30%) mixtures were incorporated as the fuel. The inlet sinusoidaldisturbances were imposed on the hydrogen and methane flows by programmableMFCs. The thermal response of the system was monitored by using thermocouplespositioned at the center of the burner in different axial locationsand by detecting the flame motion using image processing techniques.It was found that under steady-state conditions, CH4(90%)\u2013H2(10%) could operate as low as \u03d5 = 0.25. Additionally,the CH4(90%)\u2013H2(10%) mixture producedgreater carbon monoxide emissions when compared to the CH4(70%)\u2013H2(30%) mixture. As both mixtures were subjectto unsteady inlet flow of hydrogen and methane, the burner remainedstable for the CH4(90%)\u2013H2(10%) mixturewhen hydrogen was oscillated between 0 and 50% and for methane between0 and 10% over a period of 60 s. Similarly, the burner remained stablefor the CH4(70%)\u2013H2(30%) mixture whenhydrogen flow was fluctuated at 0\u201330% of its steady flow rateand methane fluctuated at 30% for the identical oscillatory period.For both mixtures by in large, it was observed that the flame movementcorresponds to the dynamics of the induced disturbances at the inlet.The flame thickness within CH4(70%)\u2013H2(30%) mixture was proven to be far greater when compared to the CH4(90%)\u2013H2(10%) mixture as the hydrogen andmethane flows were amplified. Lastly, both fuel mixtures were notedto be rather insensitive to hydrogen fluctuation beneath 30% amplitude.A custom-designed porous burner was used in an experimental investigationof ultralean combustion to investigate the burner response to theoscillations superimposed on the fuel flow. The work was motivatedby the possibility of temporal changes in the chemical compositionof hydrogen containing fuels including blends of methane and hydrogen.Such variations can strongly influence flame stabilization and leadto flame extinction/flashback. It is therefore important to analyzethe burner response to imposed oscillations on the fuel streams. CH"}
+{"text": "S3 (currently Appendix S2) of the Supplementary Material erroneously skipped blank, and the subsequent rows 43\u201367 (pages 10\u201311) swapped during the revision process. The correct version (Appendix S2) of the Supplementary Material is provided in this correction.The third cell in Row 42 in Appendix The original article has been corrected.Supplementary file1 (PDF 1202 kb)Below is the link to the electronic supplementary material."}
+{"text": "Cell Death Discovery 10.1038/s41420-018-0125-7, published online 21 November 2018Correction to: The original version of this article unfortunately contained mistakes in Figs. 1 and 2. Preliminary experiments with a new batch of neuro2a cells from atcc suggests that neuro2a cells behave similarly to the mn9d cells used in the manuscript (they are sensitive to Mcl1 inhibition with umi-77 and Mcl1 appears to have the same molecular weight as in mn9d cells). The corrected figures can be found below."}
+{"text": "This article has been corrected: The authors recently found errors inFigure 2B and 5D - the images for the \"KYSE-150\" groupwere incorrect. The authors corrected panels 2B and 5D in Figure 2 andFigure 5 by using representative images from the original sets of experiments.These alterations do not affect the results or conclusions of this work. The authors wouldlike to apologize for any inconvenience caused.Figures 2 and 5 are presented below.New"}
+{"text": "Robo2 resulted in misguidance of dI1i axons, whereas dI1c axons remained unperturbed within the mantle zone and ventral commissure. Further, Robo2 profoundly influenced dI1 cell body migration, a feature that was partly dependent on Slit2 signaling. These data suggest that dI1 neurons are dependent on Robo2 for their organization. This work integrated with the field support of a model whereby canonical Robo2 vs. non-canonical Robo3 receptor expression facilitates projection neurons derived from a common precursor domain to read out the tissue environment uniquely giving rise to correct anatomical organization.Sensory information relayed to the brain is dependent on complex, yet precise spatial organization of neurons. This anatomical complexity is generated during development from a surprisingly small number of neural stem cell domains. This raises the question of how neurons derived from a common precursor domain respond uniquely to their environment to elaborate correct spatial organization and connectivity. We addressed this question by exploiting genetically labeled mouse embryonic dorsal interneuron 1 (dI1) neurons that are derived from a common precursor domain and give rise to spinal projection neurons with distinct organization of cell bodies with axons projecting either commissurally (dI1c) or ipsilaterally (dI1i). In this study, we examined how the guidance receptor, Robo2, which is a canonical Robo receptor, influenced dI1 guidance during embryonic development. Robo2 was enriched in embryonic dI1i neurons, and loss of  The physiological function of the nervous system is dependent on the precise spatial connectivity of a diverse range of neural populations. This mature spatial organization originates from a relatively small number of progenitor domains, raising the broad question of how neurons derived from a common precursor origin and environment elaborate the spatial organization required for their later functional connectivity.During development, patterned multipotent neural stem cells differentially express key transcription factors instructing a cascade of events leading to their differentiation. Developing neurons subsequently migrate and grow in response to cues in their environment in a subtype-specific manner. This is achieved by the differential expression and localization of a small number of ligand/receptor molecular pathways used in various combinatorial codes together with various adapter molecules and gating signaling molecules to elicit specific responses. These broadly evolutionarily conserved pathways include the Slit/Robo, Eph/Ephrin, Semaphorin/plexin, neuropilin and Netrin/DCC, Uncs signaling, in addition to growth factors . Of thesRobo2 gene in mice resulted in misguidance of dI1 cell bodies and axons within the embryonic mantle zone. We further found that the misprojecting axons in Robo2 mutant embryos were dI1i neurons whereas pre-crossing and crossing dI1c neurons appeared unperturbed. This Robo2 phenotype was marginally enhanced by the loss of Robo1 and was partially phenocopied by the loss of Slit2. This indicated that Robo1 and Robo2 were not redundant in this context and that this phenotypic effect was elaborated at least in part through canonical Slit/Robo signaling. Overall, these findings taken together with previous results showing that non-canonical Robo3 controls dI1c axon guidance reveal a mechanism whereby Robo2 and Robo3 are differentially enriched at a key anatomical divergence point and result in the ability of dI1i and dI1c neurons derived from a common precursor origin to respond uniquely to their environment to elaborate the neural organization needed for their latter function.Overall, good progress has been made in understanding how Robo signaling and other molecular pathways promote correct guidance and organization of individual classes of neurons. However, in mammals the molecular logic of how this is gated by neurons derived from a common precursor domain or more broadly from multipotent stem cells remains poorly understood. The phenotypic antagonism between canonical vs. non-canonical Robo signaling could serve as a potential mammalian evolved mechanism to drive anatomical diversity from a common precursor domain; however, this remains to be examined. In two of the major systems used to probe neural diversification, the cortex, and the retina, non-canonical Robo3 signaling does not appear to play a major role in commissural axon formation . TherefoWild type SvEv129, GFPBarhl2, LACZMath1, +/\u2013:Robo2+/\u2013Robo1, Robo2+/\u2013, and Slit2+/\u2013 mice . The following primary antibodies were used: chicken \u03b1-GFP , chicken \u03b1-GFP , rabbit \u03b1-Robo1 antibody produced in this paper , rabbit \u03b1-Robo1 . Z-stack photomicrographs (20\u00d7) were acquired using the Leica SP8 Falcon confocal microscope. Maximum projection images were generated using ImageJ, and the region of interest (ROI) was traced to demarcate dl1i and dl1c populations based on anatomy. The ratio between dl1i/dl1c of Robo1, Robo2, or Robo3 pixel intensity was quantified. Statistical analysis was performed as follows: Using Prism software, a one-way ANOVA Kruskal\u2013Wallis non-parametric test followed by Dunn\u2019s multiple-comparison analysis was performed.E12.5 spinal cord tissue from + neuron migration phenotype, transverse brachial sections of E12.5 +/+:Barhl2GFPRobo2 , +/\u2013:Barhl2GFPRobo2 , \u2013/\u2013:Barhl2GFPRobo2 , Slit+/+:Math1LacZ2 , and Slit\u2013/\u2013:Math1LacZ2 , +/+:Robo2+/+:Barhl2GFPRobo1 , \u2013/+:Robo2\u2013/+:Barhl2GFPRobo1 , and \u2013/\u2013:Robo2\u2013/\u2013:Barhl2GFPRobo1  embryos were immunohistochemically labeled with Barhl2 to label dI1 neurons. From the images collected, a grid of 8 times 14 bins was superimposed on the images (Photoshop) so that the grid fitted the dorso-ventral and medial-lateral extreme of each spinal cord hemi-section analyzed  and \u2013/\u2013:Barhl2GFPRobo2  embryos were immunohistochemically labeled with the Lhx9 antibody. From the images collected, the dorso-ventral position of the main cohort of Lhx9+ neurons was measured, relative to the dorso-ventral length of the spinal cord in the section being examined. This percentage value was then averaged for control embryos which establish an average position of the control Lhx9+ neuron. This was 38% for control embryos (n = 7). A 38% ventral cutoff line was positioned on all the spinal cord images of control and mutant embryos , +/\u2013:Barhl2GFPRobo2 , and \u2013/\u2013:Barhl2GFPRobo2  embryos were immunohistochemically labeled with Robo3 to label commissural axons and GFP to label dI1 neurons and were imaged with a Leica SP8 Falcon confocal microscope. Quantification was performed on the maximal projection of Z-stack images. Within ImageJ, two fixed-size boxes were applied to each image, Box 2 within the ventral mantle zone and Box 1 within the ventral commissure , the pixel density was measured within a defined region where dI1 axons and cell bodies were normally absent or observed at a low frequency in control embryos. In short, the area to be analyzed was first defined to exclude cell bodies on all sections analyzed. First, a rectangular region was defined which was delimited by the midline on the medial side and by the bottom of the floor plate on the ventral side , +/\u2013:Barhl2GFPRobo2 (n = 6), \u2013/\u2013:Barhl2GFPRobo2 , +/+:Robo2+/+:Barhl2GFPRobo1 , \u2013/+:Robo2\u2013/+:Barhl2GFPRobo1 , and \u2013/\u2013:Robo2\u2013/\u2013:Barhl2GFPRobo1  embryos. Values from a single embryo were averaged to generate a value for that embryo. The \u201cn values\u201d refer to the number of embryos analyzed for each genotypic group. Values for each embryo analyzed were averaged (mean) for all embryos of a single genotype. The statistical analysis was performed using GraphPad (Prism) software. The data distribution was non-parametric, and the following statistical test was performed to examine statistical differences: Kruskal\u2013Wallis test followed by Dunn\u2019s multiple-comparison test using Prism software.Photomicrographs of E11.5 were imaged. The region of interest was defined by delimiting the white mater tracts as the outer border and commissural axons tract as the inner boundry respectively. Z-stack photomicrographs (20\u00d7) of E12.5 mouse embryonic spinal cord tissue immunohistochemically labeled with GFP (dI1 neurons) were acquired at subthreshold pixel intensity with respect to GFPwild type or GFPBarhl2 mouse embryos, a previously characterized transgenic mouse line which expresses GFP in both dI1 neuron axons and cell bodies  vs. commissural (Robo2low/Robo3+) dI1 neurons whereas Robo1 was expressed in both dI1i and di1c neurons with a modestly higher expression dI1c compared with dI1i neurons , Robo2 heterozygote (+/\u2013:Barhl2GFPRobo2), and control (GFPBarhl2) embryos.Given the enrichment of Robo2 in dI1i neurons, we next explored the possibility whether the canonical Robo2 receptor influenced dI1 guidance. For this analysis, we crossed the Robo2 mutant and control embryos with a transcription factor delineating dI1 neuron nuclei, Barhl2  and control (LacZMath1) embryos to determine whether a similar phenotype was observed between Slit2 and Robo2 mutant embryos. Equivalent to the analysis for the Robo2 mutant embryos, we analyzed the distribution of Barhl2 nuclei in Slit2 mutant embryos. We found a notable medial shift in the population distribution on Slit2 mutant embryos, consistent with a medial shift in the dI1i neural population  and measured pixel density in defined regions , supporting the notion that the phenotypes were derived from the ipsilateral population. Further, we observed that in the Robo2 mutant embryos, commissural axon projections were projecting relatively normally within the mantel zone at E11.5 and E12.5, suggesting that dIc neurons within the mantle zone remained unperturbed  and GFP+ (dI1 neurons) immunolabeled axons within the mantle zone and ventral commissure using pixel intensity as a proxy for axon density. The ratio of the pre-crossing/crossing , Robo1/2 double heterozygote (+/\u2013:Robo2+/\u2013:Barhl2GFPRobo1), and control (GFPBarhl2) embryos. Using comparable analysis to that of Robo2 mutant embryos, we observed both axon guidance and dI1 neuron migration misguidance phenotypes in Robo1/2 double mutant compared with control embryos  does not produce a mantle zone or ventral commissure commissural axon phenotype unless Robo1 is modulated at the same time  within the mantel zone are gated by the expression and activity of the non-canonical Robo3.1 receptor . This, tRobo2 mutant embryos\u2019 dI1i neurons. This suggested that within dI1 neurons, the evidence points to the phenotype observed in the Robo2 mutant embryos within the spinal cord as a cell autonomous effect.Here we examine how neurons derived from a common precursor origin respond uniquely to the environment utilizing Robo signaling. A cohort of previous work has demonstrated that embryonic axonal sorting in their longitudinal white matter tracts is influenced by Robo1 and Robo2 . These sRobo1 and Robo2 mRNA and protein broadly correlated in dI1i neurons within the mantle zone, suggesting that in contrast to white matter tracts, the regulatory mechanism within the mantle zone may be at the transcriptional level. Several transcription factors are candidates that could potentially regulate this; however, this is likely to be complex transcriptional regulation and has not been determined (The expression of Robo2 (and Robo1) we observed in dI1 neurons within the mantle zone was substantially weaker than that observed in the latter projecting white matter tracts . This motermined .Robo1 did not statistically significantly enhance the Robo2 mutant dI1i axonal phenotype, suggesting that Robo2 alone was sufficient for the observed axonal phenotype. Of note, while not statistically significant, we observed a marginal mean increase (trend) in axonal misprojections in Robo1/2 double mutants compared with Robo2 mutant embryos. This evidence leaves open the possibility that Robo1 may contribute to the projection of dI1i neurons, which could be for example by contributing to a \u201cthreshold\u201d of canonical Robo signaling. We also noted a qualitative but not quantitative dI1i axonal phenotype in Robo1/2 double-heterozygote embryos compared with controls. This modest axonal phenotype Robo1/2 double-heterozygote embryos (two Robo gene copies missing) compared with the strong phenotype in Robo2 mutant embryos (two Robo gene copies missing) suggested that the influence of Robo1 and Robo2 were not equal in dI1i neurons. This suggested that while canonical gene dose may contribute to the guidance, canonical Robos\u2019 influence on dI1i guidance is not mediated by gene dose alone. Taken together, we therefore concluded that Robo2 and Robo1 are not redundant in the context of dI1i axonal projections within the mantle zone and that Robo2 plays the major role. We found a statistically significantly enhanced cell body migration phenotype in E12.5 Robo1/2 double mutant vs. Robo2 mutant embryos, suggesting that in the context of the cell body migration phenotype, Robo1 and Robo2 may be additive. Consistent with our analysis overall, in other contexts, Robo1 and Robo2 can act in a distinct, synergistic, or redundant way depending on the context (We demonstrated that in addition to Robo2, Robo1 was expressed in both dI1i and dI1c neurons, raising the possibility that Robo1 influenced dI1 guidance. We found that loss of  context .This, taken together with other studies, suggests that Robo1 may play a role in commissural axon timing and crossing at the ventral commissure, and here we propose that Robo2 plays a relatively minor role in dI1c neuron guidance within the mantle zone and ventral commissure whereas Robo2 is the major gatekeeper of dI1i guidance.Robo signaling is associated in a wide range of fields including neurodevelopment and neurofunctional disorders in addition to disease processes. Here we have contributed to this body of work demonstrating a neurodevelopmental context in which canonical vs. non-canonical Robos regulate tissue organization. Within the nervous system, understanding how neurons derived from common precursor origin elaborate neural organization has important implications for understanding the assembly of functional connectivity. This study provides an example of how evolution of a gene family permits development of anatomical differentiation/identity from cells derived from a common progenitor pool and initial molecular identity, a concept that is widely applicable throughout different systems and animals. There are a small number of examples of the non-canonical function of Robo receptors in other species . WhetherThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Review Board at the Court of Appeal of Northern Norrland.Robo2 phenotype discovery and preliminary analysis and generated reagents. LG-C designed and performed the Robo1, Robo2 and Robo3 expression quantification, contributed to the migration analysis, characterization of the transgenes and antibodies. CL and CT were involved in an early phase of analysis in the work and generated samples. SW conceived and supervised the work, led the study design, generated the funding, made reagents, performed some experimental work and formal analysis, and wrote the manuscript. All the authors edited, read, and approved the manuscript.MW, MM, LG-C, and CL assembled the figures. MW, MM, and LG-C were responsible for writing parts of the manuscript, experimental design, and performance of experiments. MW performed most of the experiments and analysis in the manuscript and codesigned and performed the phenotype quantifications. MM made the initial The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.Our previous studies showed an association between monoallelic  BRCA2) gene was identified in 1995 and has been shown to play an essential role in DNA double strand break (DSB) repair by homologous recombination (HR) and DNA crosslink repair by the Fanconi anemia (FA) pathway [BRCA2 gene are known to be highly associated with the risk of diagnosis with breast, ovarian, prostate, and pancreatic cancer [BRCA2 999del5 truncation mutation in exon 9 was isolated in a family with several cases of male and female breast cancer [999del5 mutant and expression studies showed that even though the short mutant RNA is produced there is no detectable corresponding protein, indicating BRCA2 heterozygosity [The breast cancer susceptibility 2 , telomere loss (TL), extrachromosomal telomere sequences (ECTS) and frequent telomere sister chromatid exchanges (T-SCE) as seen in cells that rely on the alternative lengthening of telomeres (ALT) in absence of telomerase. These results indicated the important role of BRCA2 in telomere stabilization and protection [BRCA2 999del5 germline mutation and relative short telomere length measured in blood cells were at significantly higher risk of being diagnosed with breast cancer [BRCA2 gene expression may not be enough to fulfill telomere maintenance, indicating BRCA2 haploinsufficiency. Anticipation effects have also been reported from this study cohort where daughters of mother-daughter pairs with the BRCA2 999del5 germline mutation have been shown to be diagnosed with breast cancer on average 10 years younger than the mothers [Studies on the role of the BRCA2 protein have shown the requirement of HR-dependent RAD51 filament formation plus stabilization by BRCA2 and the core FA pathway for efficient protection in replication forks from degradation following short-term replication arrest. FA and BRCA2 defective cells are defective in fork protection and show an increase in genome instability, manifesting chromatid breaks and radial structures on mitotic chromosome spreads ,16. DNA otection . A recent cancer . These r mothers .BRCA2 999del5 breast cancer cases [BRCA2 wild-type allele in the tumor tissue. Similar results have been reported from another study cohort where the absence of BRCA2 locus-specific loss of heterozygosity was observed in 46% of breast tumors [BRCA2 999del5 breast cancer cases with wild-type allelic loss had significantly worse breast cancer specific survival compared to cases that remained the wild-type allele [BRCA2 haploinsufficient phenotype in vivo in breast tissues of BRCA2 mutation carriers exhibiting DNA damage that resulted from failed RS- and DNA damage responses and consequently aneuploidy [The 50 years old Knudson\u2019s two-hit hypothesis suggests that both alleles of a tumor suppressor gene require to be inactivated to cause tumor formation, as was first described in heritable cases with retinoblastoma . We haveer cases ,22 and per cases  had lostt tumors . Of noteeuploidy .Mechanisms shown to respond to RS that could counteract karyotypic diversity and contribute to tumor progression include polymerase theta-mediated end joining (TMEJ) alternative repair pathway at resected DSBs when HR is deficient to repair broken forks ,27. OtheBRCA2 genotypes including two BRCA2 wild-type cell lines, five BRCA2 heterozygous cell lines with the 999del5 germline mutation and three FA-D1 cell lines with biallelic BRCA2 mutations. The originality of this study was to compare all three BRCA2 genotypes in a cell type not associated with BRCA2 hereditary cancer. Results showed increased telomere abnormalities between the BRCA2 genotypes in a stepwise manner, including TL, ITS and ECTS.In the present study, we compared telomere dysfunction in lymphoid cell lines with different BRCA2 999del5 mutation were Epstein-Barr virus transformed . Telomere fluorescence in situ hybridization (FISH) was performed by ready-to-use Cy3-conjugated PNA pan-telomere probe  in mixture with FITC-conjugated PNA pan-centromere probe (Dako) and counterstained with 4\u2032,6-diamidino-2-phenylindole (DAPI) as previously described . The micBRCA2 genotypes were combined and performed as one genotype. A two-tailed Student\u00b4s t-test was used for statistical analysis and p < 0.05 was considered statistically significant. The R (CRAN) software was used for graphical representation.Statistical analyses were performed on chromosomal abnormalities as per metaphase and results presented as an average mean with standard error of mean (SEM) bars. Results from each of the At least 60 metaphases were analyzed from each lymphoid cell line with one exception of the HSC62N cell line . Most ofIn addition to analysis of TL, MTS, ITS and ECTS other chromosomal aberrations were measured including sister chromatid and end-to-end chromosome fusions, radial chromosomal configurations and chromosome fragments without telomere signals. These chromosomal aberrations were, however, not included in the results since average events per metaphase were below one which may affect the significance of the results.+/+BRCA2, BRCA2+/- and BRCA2-/- lymphoid cell lines, and a significant difference was found between the BRCA2+/- and BRCA2-/- genotypes  [TL is likely to result from RS that leads to terminal fork collapse, either at a single sister chromatid strand or at both sister chromatids on chromosome ends. TL was found to be increased among us study . In the subgroup . This in signals . Frequenll lines . TelomerCO-FISH) .BRCA2 deficient cells [BRCA2+/+ and the BRCA2+/- and BRCA2-/- lymphoid cell lines, respectively (BRCA2+/- and BRCA2-/- lymphoid cell lines but no difference was, however, found of the total MTS between the two genotypes. Lack of significance in MTS formation between the BRCA2+/- and BRCA2-/- genotypes may be explained by >50% higher TL of the BRCA2-/- genotype (BRCA2+/- and BRCA2-/- genotypes is, however, a strong indication of BRCA2 deficiency related telomere RS due to G-quadruplex formation.MTS is often referred to as fragile telomeres. They have been suggested to represent a recombination event among inter-/intra-chromatid telomeric sequences or ITS in the proximal regions of telomeres  or restant cells . In the ectively . A signigenotype c. The siBRCA2+/- mammary epithelial cell lines compared to a commercial BRCA2+/+ mammary epithelial cell line and cell lines that rely on the classical ALT-positive mechanism for telomere maintenance [BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines (Most ITS probably result from the formation of DSB during RS that is mediated by targeted telomere insertions , althougntenance . In the ll lines  supportiBRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines (BRCA2 genotypes. Sometimes these ECTS have strong fluorescence signals indicating amplification of telomeric c-circles that have been associated with cancer cells that rely on ALT rather than telomerase for telomere maintenance [ECTS found scattered around chromosome spreads presumably result from terminal fork collapse caused by telomere RS. In this study we found a significant stepwise increase of ECTS between the ll lines  probablyntenance . Recent ntenance ,41.BRCA2 genotypes. This was shown in lymphoid cells that are not known to be prone to BRCA2 related cancer risk. It needs to be kept in mind, however, that apart from the BRCA2 genotype the cell lines have generic individual genomic differences. For this same reason, the two BRCA2+/+ control cell lines used in this study may affect the interpretation. The most ideal setup would be to use a homogenous genetic background of a single cell line edited with the CRISPR/Cas9 gene editing technique for insertion of the BRCA2 9999del5 mutation. This has, however, not yet been successful due to the very low viability of cells with the homozygous mutation. This correlates with the fact that no homozygous individuals have been known to exist although expected under the Hardy\u2013Weinberg equilibrium and allele frequency in the Icelandic population [The results clearly show a gradual increase in telomere defects between the BRCA2 dependent loss of HR on Holliday junction resolvases by using HR-intermediates that suppress mitotic crossing over and preserve the genomic stability [BRCA2 deficient cells is not fully understood and needs to be further investigated.Our and other previous findings have shown unequal T-SCE in BRCA2 deficient cells ,18,30. Ctability . BRCA2 dtability ,46,47,48tability . Both RAtability . HoweverBRCA2 999del5 mutation carriers does not differ from non-carriers in blood cells [BRCA2 999del5 mutation carriers was found to be significantly shorter than among non-carriers. Telomere length was shown to be a modifier of breast cancer risk in BRCA2 999del5 mutation carriers in the same study. Another aspect is that BRCA2 999del5 mutation carriers with breast cancer have been associated with a significantly worse prognosis than non-carriers [BRCA2 mutation carriers might be related to consequences of possible telomere RS before and after breast cancer diagnosis needs to be answered in future studies. Such knowledge could have an impact on the prediction of early cancer development, and therefore, be used to advise the timing of preventive therapies, and targeted therapies of BRCA2 related breast cancer in the future as discussed here above.Telomere length homeostasis in unaffected od cells . After acarriers . How defBRCA2+/+, BRCA2+/- and BRCA2-/- genotypes showing a strong indication of BRCA2 haploinsufficiency in telomere maintenance. Our results from telomere FISH show a clear increase in TL, MTS, ITS and ECTS between the genotypes that are a sign of telomere RS in BRCA2 deficient cells. More research is needed on the possible involvement of the RAD52 related BIR and the POLQ related TMEJ backup pathways in telomere maintenance when BRCA2 is not fully expressed and the distinct roles of the two pathways on telomere maintenance and genomic stability.Taken together, we found a stepwise increase in telomere abnormalities between the"}
+{"text": "FOXF2, encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. FOXF2 is located in a conserved gene cluster containing FOXQ1, FOXF2, and FOXC1. We found that expression of Foxq1 is dramatically upregulated in the embryonic palatal mesenchyme in Foxf2\u2013/\u2013 mouse embryos. We show here that the Foxf2 promoter-deletion mutation caused dramatically increased expression of the cis-linked Foxq1 allele but had little effect on the Foxq1 allele in trans. We analyzed effects of the Foxf2 mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the Foxf2 mutation resulted in significantly increased expression of the Foxq1 and Exoc2 genes located in the same topologically associated domain with Foxf2 but not the expression of the Foxc1 and Gmds genes located in the adjacent chromatin domain. We inactivated the Foxq1 gene in mice homozygous for a Foxf2 conditional allele using CRISPR genome editing and generated +/\u2013(Foxf2/Foxq1) mice with loss-of-function mutations in Foxf2 and Foxq1 in cis. Whereas the \u2013/\u2013(Foxf2/Foxq1) mice exhibited cleft palate at birth similar as in the Foxf2\u2013/\u2013 mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (Foxf2/\u2013/\u2013Foxq1) embryos exhibited distinct effects on the domain-specific expression of several important genes, including Foxf1, Shox2, and Spon1, in the developing palatal shelves compared with Foxf2\u2013/\u2013 embryos. These results identify a novel cis-regulatory effect of the Foxf2 mutation and demonstrate that cis-regulation of Foxq1 contributed to alterations in palatal gene expression in Foxf2\u2013/\u2013 embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in development and disease.Disruption of  The secondary palate separates the nasal cavity from the oral cavity and consists of the bony hard palate anteriorly and muscular soft palate posteriorly . In mammShh mRNAs to the oral side palatal epithelium to control the oral-nasal patterning  and chromatin immunoprecipitation-sequencing (ChIP-seq) mediated genome wide mapping of Foxf2 binding sites to identify direct Foxf2 target genes in the developing palatal mesenchyme , 2020. If growth , we showf growth . Among t embryos . Foxq1 i embryos . Foxq1\u2013/reported . Remarka genomes . In bothch other . Thus, t editing  and our Foxf2tm1Rhc (Foxf2c/c) and Foxf2+/\u2013 mice have been described previously  (50 ng/\u03bcl each) targeting the genomic sequence flanking the Forkhead domain-coding sequence in the Foxq1 gene were co-injected with humanized Cas9 mRNAs (50 ng/\u03bcl) into zygotes of Foxf2c/c mice  were used in this study. Founder mice were crossed to Foxf2c/c mice to generate the Foxf2c/c; Foxq1+/\u2013 mice. Genotypically verified G1 Foxf2c/c; Foxq1+/\u2013 mice were intercrossed to generate Foxf2c/c; Foxq1\u2013/\u2013 homozygotes. In addition, Foxf2c/c; Foxq1+/\u2013 mice were crossed to EIIa-Cre transgenic mice  mice. +/\u2013(Foxf2/Foxq1) mice were intercrossed to generate \u2013/\u2013(Foxf2/Foxq1) homozygous embryos for analyses. For timed mating, noon of the day on which a vaginal plug was identified was designated as embryonic day (E) 0.5. All animal work procedures were performed following recommendations in the Guide for Care and Use of Laboratory Animals by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children\u2019s Hospital Medical Center. This study conformed with the ARRIVE  guidelines for preclinical animal studies.eviously  and werec/c mice . The tarnic mice  to inactin situ hybridization as described previously  of the Foxq1 gene (rs29587452) between the C57BL/6J and 129X1/SvJ mouse strains. Purified PCR products amplified from wildtype, +/\u2013Foxf2, and Foxf2\u2013/\u2013 cDNA samples were digested overnight using AciI  at 37oC. Quantification of the digested and undigested DNA fragments was performed by QIAxcel Advanced using QIAxcel\u00ae ScreenGel software .Whole palatal shelves of E13.5 embryos were manually dissected in ice-cold phosphate buffered saline. Total RNAs were extracted using the RNeasy micro kit . First-strand cDNAs were prepared using the SuperScript III First-Strand Synthesis System , and real-time qPCR was performed using a CFX96 Real-Time System  with conditions recommended by the manufacturer. Relative levels of mRNAs in each sample were normalized to that of t test was used for pairwise comparison. One-way ANOVA followed by Newman\u2013Keuls post hoc test was used to compare all pairs when more than two genotypes were included. P < 0.05 was considered significantly different.For statistical analysis, all results were presented as mean \u00b1 SEM. Student\u2019s 1 (2 (accession number GSE138721) (3 (The whole genome chromosome conformation capture (Hi-C) data and topologically associated domain (TAD) map of mouse embryonic stem cells were retrieved from the 3D Genome Browser1 . The ori1 , and assE138721) . The rep8721)  gene, named 1700018A04Rik, which is transcribed from the opposite DNA strand with the most 5\u2032 transcription start site (TSS) located only about 300 bp from the TSS of Foxf2  that is disrupted in the 129SvEv allele. We designed a pairs of PCR primers flanking SNP rs29587452 and used AciI restriction fragment polymorphism to analyze possible differential expression of the two Foxq1 alleles in the Foxf2+/\u2013 mutant palate shelves  and 494  bp, respectively, spanning the entire Forkhead domain-coding region of the Foxq1 gene were established and used in this study +/\u2013 progeny to C57BL/6J mice to establish the +/\u2013(Foxf2/Foxq1) mouse colony. These +/\u2013(Foxf2/Foxq1) mice allowed us to further investigate allele-specific effects of the Foxf2 mutation on the linked Foxq1 allele by direct quantitative comparison of allele-specific Foxq1 expression between different embryos of distinct genotypes  embryos in comparison with their wildtype littermates. However, the amount of mRNAs produced from the wildtype Foxq1 allele in the +/\u2013(Foxf2/Foxq1) embryos was only about 50% of the amount of Foxq1 mRNAs in the wildtype sample  studies have demonstrated that mammalian genomes are organized into a series of topologically associated domains (TADs), megabase-scale genomic intervals where interactions between enhancers and gene promoters take place more frequently within than across adjacent TADs . Althougcent TAD . RT-qPCRtermates . Expressd either . On the termates . In situ embryos . Analysi embryos  indicatend Foxq1 . Furtherpromoter . Remarkapromoter  showed t tissues . In addi tissues  revealed tissues . Togethe+/\u2013(Foxf2/Foxq1) mice generated \u2013/\u2013(Foxf2/Foxq1) mutant mice with over 80% of the homozygous mutants exhibited complete cleft palate (\u2013/\u2013(Foxf2/Foxq1) embryos at multiple developmental stages by histology or skeletal preparations revealed that craniofacial anomalies, including cleft palate, were similar as the phenotypes of Foxf2\u2013/\u2013 embryos described previously  embryos displayed defects in palatal shelf elevation  embryos as previously reported for Foxf2\u2013/\u2013 mutant embryos  embryos in comparison with wildtype littermates at E13.5  embryos compared with the wildtype littermates  mutant embryos  embryos in comparison with the wildtype littermates by both whole mount in situ hybridization and real-time RT-qPCR assays  mutant embryos  embryos (\u2013/\u2013(Foxf2/Foxq1) embryos in comparison with the wildtype embryos . Interestingly, we also consistently detected moderately increased expression of Foxf1 protein in the posterior region of the palatal shelves in Foxf2\u2013/\u2013 embryos compared with the wildtype embryos (H with G). These results indicate that Foxf2 negatively regulates Foxf1 gene expression in the developing palatal mesenchyme and that the increased expression of Foxq1 in the Foxf2\u2013/\u2013 embryonic palatal mesenchyme partly complemented for Foxf2-mediated regulation of Foxf1 expression. Altogether, although deletion of Foxq1 was ultimately insufficient to rescue the cleft palate defects in the Foxf2\u2013/\u2013 mice, the increased expression of Foxq1 resulting from the cis-regulatory effect of the Foxf2 mutation affected the regulation of multiple important genes in palate development.We previously generated and analyzed elopment . Anothern assays . Our preR assays . In the at E13.5 . The exp embryos . We furt embryos . The FoxFOXF2 is located in an evolutionarily conserved gene cluster containing FOXQ1, FOXF2, and FOXC1  mice carrying null alleles of Foxf2 and Foxq1 in cis, and identified a novel cis-regulation of Foxq1 expression by the Foxf2 mutation during palate development. Our results indicate that Foxq1 affected expression of several previously identified Foxf2-dependent genes in palate development, which needs to be taken into consideration for elucidating the molecular mechanisms involving Foxf2 in regulating palate development.nd FOXC1 . Disruptn humans , making in vitro study suggested that FOXF2 binds to the promoter and represses the expression of FOXQ1 in a breast cancer cell line  embryos. This is possibly due to the relatively low level of expression of Foxq1 in the Foxf2\u2013/\u2013 palatal mesenchyme compared with the amount of Foxf2 mRNAs expressed in the wildtype palatal mesenchyme. On the other hand, previous studies have suggested that FOXF2 and FOXQ1 play opposite roles in controlling epithelial-mesenchymal transition and visceral metastasis in basal-like breast cancer cells  embryos compared with the Foxf2\u2013/\u2013 and wildtype control embryos  embryos  embryos, the increased expression of Foxf1 was insufficient to rescue the cleft palate phenotype or the altered expression of many genes in the posterior palatal mesenchyme that resulted from Foxf2 disruption, suggesting that Foxf2 has distinct molecular functions in palate development that could not be complemented by either Foxq1 or Foxf1.The expression of er cells . Other ser cells . We foun embryos . While t embryos . In thisFoxf2 gene promoter had a cis-regulatory effect on the expression of nearby genes. Our results clearly demonstrate that the increased expression of Foxq1 in the Foxf2+/\u2013 and Foxf2\u2013/\u2013 embryos was due to the cis-regulatory effect. Although \u2013/\u2013(Foxf2/Foxq1) mice exhibited similar cleft palate phenotypes as Foxf2\u2013/\u2013 mice, Foxq1 exerted a regulatory effect on the expression of several previously identified Foxf2-dependent genes in palate development. We show that Exoc2, located about 600 kb upstream of Foxf2, was also significantly upregulated in the developing palatal shelves in Foxf2+/\u2013 embryos and was further significantly upregulated in the Foxf2\u2013/\u2013 embryos. Whether the increase in Exoc2 expression in the palatal tissues in Foxf2+/\u2013 and Foxf2\u2013/\u2013 embryos was solely due to the cis-regulatory effect and whether the altered expression of Exoc2 contributed significantly to the craniofacial and other developmental defects in the Foxf2\u2013/\u2013 mice remain to be investigated. Since many gene knockout studies assigned gene function based on promoter-deletion alleles, our finding of a cis-regulatory effect of the Foxf2 mutation calls for caution in interpretation of results and underlying mechanisms from those alleles. Potential cis-regulatory effects on neighboring genes should also be taken into consideration when analyzing pathogenicity of human gene deletion variants. In addition, since Foxf2 mutant mouse studies have shown that multiple tissues and developmental processes, including pericyte development and maturation of the blood-brain barrier, development of the respiratory and digestive organs, in addition to craniofacial and palate tissues, depend on Foxf2 function (reviewed by +/\u2013Foxf2/Foxq1) mice provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in multiple developmental and disease processes.In summary, this study demonstrates that disruption of the GSE67015. The accession number for the palate Foxf2 ChIP-seq data is GSE137585.Publicly available datasets were analyzed in this study. This data can be found here: The accession number for the RNA-seq data is The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children\u2019s Hospital Medical Center.YL and RJ designed the research. JX and HL conducted the experiments and collected the data. JX and RJ wrote the manuscript. All authors analyzed the data, revised the manuscript, and agreed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "However, there is no consensus with respect to the responsiveness of these O2 resaturation parameters for assessing reactive hyperemia.There is a spectrum of possibilities for analyzing muscle O2 resaturation parameters to assess reactive hyperemia in the microvasculature of a clinical group known to exhibit impairments of tissue O2 saturation (StO2).This study investigates the responsiveness of the most utilized muscle O2 after a 5-min arterial occlusion challenge and the following parameters were analyzed: StO2slope_10s, StO2slope_30s, and StO2slope_until_baseline ; time to StO2baseline and time to StO2max ; \u2206StO2reperfusion ; total area under the curve (StO2AUCt); and AUC above the baseline value (StO2AUC_above_base).Twenty-three healthy young adults, twenty-nine healthy older adults, and thirty-five older adults at risk of cardiovascular disease (CVD) were recruited. Near-infrared spectroscopy (NIRS) was used to assess StO2slope_10s was significantly slower in older adults at risk for CVD compared to healthy young individuals (p < 0.001) and to healthy older adults (p < 0.001). Conversely, time to StO2max was significantly longer in healthy young individuals than in older adult at CVD risk.Only StO2slope_10s may be a measure of reactive hyperemia, which provides clinical insight into microvascular function assessment.Our findings suggest that StO Since NIRS detects changes in oxygenated and deoxygenated hemoglobin in the tissues using absorption of near-infrared light at a specific wavelength,,,Over the past decades, near-infrared spectroscopy (NIRS) combined with a vascular occlusion test (VOT) has been used for assessment of tissue O2 signal follows a downward path during the cuff inflation phase and then StO2 goes rapidly upward immediately after cuff deflation (reperfusion phase).-2 signal during reperfusion enables various StO2 parameters to be calculated, which, in general, are interpreted as measures of microvascular function.2 parameters adopted in previous studies include reperfusion rate ,-2 and the difference between the lowest and highest StO2 value),,-2 baseline, time to StO2 maximum, and AUC above the baseline).,,,Performing a VOT with an NIRS device fitted to the limb of interest, the StO2 resaturation parameters, the lack of standardization of which NIRS parameters are used during the reperfusion phase makes comparisons between studies difficult and/or may even lead to misinterpretation of results. For example, since the time to maximum StO2 parameter is calculated as the time elapsed from release of the cuff to the maximum StO2 value during reperfusion, individuals with higher O2 extraction capacity  might exhibit a longer time to maximum StO2. In other words, in individuals with higher O2 extraction, the distance between the lowest StO2 value reached during cuff inflation and the highest StO2 reached during the cuff deflation is longer, thereby influencing calculation of some NIRS parameters, such as time to StO2 maximum and even area under the reperfusion curve. Therefore, the present study investigates the usefulness of the muscle O2 resaturation parameters most utilized to assess reactive hyperemia in a clinical group known to exhibit impairments in muscle StO2. It was hypothesized that some NIRS parameters might be more sensitive than others (mainly those influenced by muscle O2 extraction) for detecting abnormal reactive hyperemia in a clinical sample.Despite a range of possibilities for interpretation of muscle O-three healthy young individuals, 29 healthy older adults, and 35 older adults at risk of CVD were recruited to participate in the study. For the younger group, inclusion criteria were being healthy (not presenting CVD risk factors) and age < 35 years. For the healthy older adult group, individuals had to be healthy and \u2265 60 years of age. The inclusion criteria for older adults at high risk for CVD were age \u2265 60 years and possessing at least four CVD risk factors, according to a previous study demonstrating that presence of three or more CVD risk factors is related to microvascular dysfunction.,o. 1.489.668).TwentyAfter 12 h fasting, the participants arrived at the laboratory, where they underwent anthropometric measurement  before blood samples were taken. Blood samples were collected only at baseline in order to determine the CVD risk factors  of the participants enrolled on the study. Afterwards, participants rested for 10 min in the supine position on an examination table, followed by assessment of the NIRS parameters for level of muscle oxygen saturation. The experimental procedures were conducted between 8:00 and 10:00 a.m. and participants were recommended to avoid caffeine and foods rich in nitrates and nitrites for 24 h before the laboratory visit.2 saturation (%StO2) was assessed using an NIRS system  connected to a personal computer via Bluetooth for data acquisition (10 Hz). Analogue\u2010to\u2010digital conversion and subsequent analysis of the raw data  was conducted using native software , as previously described by Oliveira et al.,2 was recorded continuously throughout the VOT . The cuff was placed over the arm, 2 cm above the medial epicondyle of the humerus and a cuff pressure of 250 mm Hg was used during the occlusion period  StO2 during baseline  was calculated as the average over the 30 s before cuff occlusion; (ii) the lowest StO2 value reached during cuff inflation (StO2min) and the highest StO2 value reached following cuff deflation (StO2max); (iii) time for the StO2 signal to reach its peak after cuff release (time to StO2max); (iv) time for the StO2 signal to reach the pre-occlusion baseline value after cuff release (time to StO2base); (v) difference between StO2min and the StO2max after cuff deflation (\u0394StO2reperfusion); (vi) the area under the reperfusion curve (AUC) above the baseline (StO2AUCabove_base); (vii) the total AUC over 2 minutes of reperfusion (StO2AUCt); (viii) upslope of StO2 signal over a 10 s reperfusion window immediately following cuff deflation (StO2slope_10s); (ix) upslope of StO2 signal over a 30 s reperfusion window immediately following cuff deflation (StO2slope_30s); and (x) upslope of StO2 signal over the time elapsed between StO2min and reaching pre-occlusion baseline StO2 values  for a specific test . Based on a statistical power (1 \u2013 \u03b2) of 0.80, an effect size of 0.46, and an overall significance level of 0.05, 51 participants would be needed to detect a statistical difference. The effect size was based on a previous study that found d=0.93  when assessing reperfusion rates in older individuals and young participants.One-way ANOVA was used to identify significant differences between the characteristics of the participants at baseline and tissue StOBaseline characteristics of the participants and medications used are shown in 2 saturation parameters are shown in 2slope_10s (P < 0.001), and StO2slope_30s (P < 0.001), and blunted \u0394StO2reperfusion (P < 0.001), StO2AUCabove_base (P < 0.001), StO2AUCt (P < 0.001), StO2max (P = 0.035), and StO2 min (P < 0.001) were observed in older adults at CVD risk compared to healthy young individuals. Additionally, StO2slope_10s was significantly slower (P < 0.001) in older adults at risk for CVD compared to their healthy counterparts. Time to StO2max was significantly slower (P = 0.047) in healthy young individuals than older adults at risk of CVD.Muscle StO2slope_30s (P = 0.003) and blunted \u0394StO2reperfusion (P < 0.001), StO2AUCabove_base (P = 0.006), StO2AUCt (P = 0.001), and StO2 min (P = 0.002) were found in healthy older adults compared to healthy young people. No significant differences in StO2base, time to StO2base, or StO2slope_until_base were found between groups (P > 0.05).Significant slower StO2 resaturation parameters as a measurement of reactive hyperemia in the microvasculature of different populations, the present study investigated the effectiveness of the O2 resaturation parameters most frequently used for evaluating reactive hyperemia in healthy older adults or older adults with CVD risk factors. It was hypothesized that analyzing the effectiveness of the most widespread NIRS parameters for detecting aging and CVD risk factors might help with development of criteria for establishing an appropriate set of muscle StO2 parameters in order to facilitate the choice between different methodologies of NIRS analysis.Given the widespread utilization of NIRS devices and the spectrum of possibilities for analyzing muscle O2 resaturation rate parameters  are largely adopted to assess reactive hyperemia in hypertensive individuals,,,,2slope_10s was significantly slower in older adults with CVD risk factors compared to healthy older adults and to healthy young individuals, suggesting that StO2slope_10s may be a more sensitive NIRS parameter for assessing reactive hyperemia. The StO2slope_10s has typically been adopted to assess O2 resaturation rate, since previous studies have reported that this parameter is correlated with reactive hyperemia measured in the brachial artery,2slope_10s may be a stimulus for FMD response given that FMD is evoked by increasing blood flow after cuff release.,,2slope_10s may be considered an appropriate way to analyze O2 reperfusion rate given the linearity of the StO2 signal over the initial 10 s after cuff release.Muscle O2max may be mainly affected by the degree of muscle O2 extraction (StO2min), since this recovery time parameter measures the time interval between the StO2min  and StO2max . Therefore, individuals with higher muscle O2 extraction capacity  will reach a lower StO2 value during the cuff inflation phase (StO2min), which makes the time to StO2max longer for those individuals with higher O2 extraction capacity.,2max was found in healthy individuals compared to older adults at CVD risk, which may have been the result of a lower StO2 value reached during cuff inflation (StO2min), combined with the higher StO2 value reached during the cuff deflation phase (StO2max).Time to StO2max in individuals with metabolic syndrome2max in individuals with peripheral vascular disease  compared to healthy participants. Interestingly, the groups investigated in their study2 extraction parameters, which may explain the expected longer time to StO2max in individuals with peripheral vascular disease. Therefore, adoption of the recovery time parameters  should be recommended when the clinical population evaluated is not assumed to exhibit impairment in muscle O2 extraction; otherwise, data interpretation and comparison may be rendered incorrect.Previous studies have failed to show significant differences in time to StO2reperfusion and StO2AUCt in healthy older adults and those with CVD risk factors, compared to healthy young individuals. However, no significant difference was observed in these parameters in healthy older adults compared to older adults at risk for CVD, suggesting that \u0394StO2reperfusion and StO2AUCt are not sensitive parameters for detecting the effect of CVD risk factors on reactive hyperemia. The \u0394StO2reperfusion and StO2AUCt parameters can be interpreted as measures of the magnitude of O2 resaturation, since the lowest and highest StO2 value (amplitude) within the reperfusion phase (cuff deflation) are considered for calculating these parameters. However, it is important to note that, in common with time to StO2max, these parameters may also be affected by parameters of muscle O2 extraction during cuff deflation (i.e. StO2min).The current study also demonstrated a significant reduction in \u0394StO2AUCt observed in older adults at CVD risk in comparison to healthy young individuals was eliminated when the level of muscle O2 extraction (StO2min) was standardized by varying occlusion times  across the two age groups. It is likely that differences between \u0394StO2reperfusion and StO2AUCt parameters do not only necessarily reflect impairment in O2 resaturation magnitude (reactive hyperemia), but also in muscle O2 extraction during occlusion .,2 saturation throughout the VOT, assessment of \u0394StO2reperfusion and StO2AUCt could be performed controlling the level of tissue ischemia (varying the occlusion times),2reperfusion and StO2AUCt. Alternatively, AUCabove_base is not affected by muscle StO2min and its utilization might be recommendable.Corroborating this idea, Rosenberry et al.2 saturation. For example, a previous study conducted at our laboratory showed that prolonged occlusion of limb blood flow  causes transient microvascular dysfunction that can be assessed by NIRS\u2010derived reperfusion upslope.,,Of note, the NIRS system is a relevant tool that can be easily used during vascular surgery, as it continuously assesses muscle O,,,,Although NIRS systems are largely utilized to assess oxygenation of the forearm muscles, one limitation of this technique is the discrete volume of a specific muscle (2 to 6 cm) and the superficial area of skeletal muscle that NIRS examines.2slope_10s was the most sensitive NIRS parameter for assessing reactive hyperemia. Additionally, the magnitude of reperfusion  and recovery time  parameters should be interpreted with caution when assessing clinical populations, since muscle O2 extraction may affect those parameters. The results of the present study will be helpful for future studies that investigate the effect of an external factor  on muscle O2 resaturation parameters.In conclusion, the findings of this study suggest that StO"}
+{"text": "Transesophageal echocardiography (TEE) and multidetector computed tomography imaging revealed type 0 calcified bicuspid AV based on the Sievers classification   with an aortic valve (AV) area of 0.74 cm2  small curve wire in the mid LV, balloon aortic valvuloplasty (BAV) was tried using Z-MEDII 23\u2009\u00d7\u200940\u00a0mm  to make it easier to cross the device due to very severe AS and to confirm the valve size in type 0 bicuspid AV. However, the balloon could not cross to AV because of strong resistance at the ascending part to the arch graft and the tortuous thoracoabdominal aorta  and advanced another stiff guidewire through a 5 French pigtail catheter  at the ascending part to the arch graft and the tortuous thoracoabdominal aorta.1 (MP4 7148 KB)Online video 2: Easy crossing of SAPIEN 3 through the aorta and aortic valve.2 (MP4 9670 KB)Below is the link to the electronic supplementary material."}
+{"text": "This 3-phase study involves the conceptualization and design, development and usability testing of a Comprehensive Digital Self-care Support System (CDSSS) named myHESTIA for older adults with multiple chronic conditions (MCC). The objective of this study was to test whether a CDSSS can be developed for those who are dealing with MCC and whether such a system that is specifically developed for older adult patients will enable daily capture of self-care data. Participants for this 3-phase study included: 10 older adults (age>60) and 10 caregivers in Phase 1; 15 Geriatrics clinicians and 25 community-dwelling low-income older adults in Phase 2; and, 10 older adults (age>60) with MCC in Phase 3. Agile method of system development was used for the design and development of the system. The first two phases involved collecting data for designing and developing myHESTIA. The third phase involved small group usability and feasibility testing, in which the participants used myHESTIA trackers for 4 weeks. Results from phase 3 shows daily inputs were possible and the self-reported data shows that it was not at all difficult for older adults to track their symptoms daily. User experience data (n=10) shows overall positive experience along pragmatic (5.8 out of 7), hedonic (4.6 out of 7), sociability (5.5 out of 7) and usability (6.3 out of 7) experience dimensions. Finally, all the participants (n=10) who completed the phase 3 study reported intention to continue using myHESTIA. Results indicate that it is feasible to design a CDSSS for older adults with MCC."}
+{"text": "Streptococcus pneumoniae (the pneumococcus) is commonly carried as a commensal bacterium in the nasopharynx but can cause life-threatening disease. Transmission occurs by human respiratory droplets and interruption of this process provides herd immunity. A 2017 WHO Consultation on Optimisation of pneumococcal conjugate vaccines (PCV) Impact highlighted a substantial research gap in investigating why the impact of PCV vaccines in low-income countries has been lower than expected. Malawi introduced the 13-valent PCV (PCV13) into the national Expanded Programme of Immunisations in 2011, using a 3+0 (3 primary +0 booster doses) schedule. With evidence of greater impact of a 2+1 (2 primary +1 booster dose) schedule in other settings, including South Africa, Malawi\u2019s National Immunisations Technical Advisory Group is seeking evidence of adequate superiority of a 2+1 schedule to inform vaccine policy.A pragmatic health centre-based evaluation comparing impact of a PCV13 schedule change from 3+0\u2009to 2+1 in Blantyre district, Malawi. Twenty government health centres will be randomly selected, with ten implementing a 2+1\u2009and 10 to continue with the 3+0 schedule. Health centres implementing 3+0\u2009will serve as the direct comparator in evaluating 2+1 providing superior direct and indirect protection against pneumococcal carriage. Pneumococcal carriage surveys will evaluate carriage prevalence among children 15\u201324 months, randomised at household level, and schoolgoers 5\u201310 years of age, randomly selected from school registers. Carriage surveys will be conducted 18 and 33 months following 2+1 implementation.The primary endpoint is powered to detect an effect size of 50% reduction in vaccine serotype (VT) carriage among vaccinated children 15\u201324 months old, expecting a 14% and 7% VT carriage prevalence in the 3+0\u2009and 2+1 arms, respectively.The study has been approved by the Malawi College of Medicine Research Ethics Committee , the University College London Research Ethics Committee (Ref: 8603.002) and the University of Liverpool Research Ethics Committee (Ref: 5439). The results from this study will be actively disseminated through manuscript publications and conference presentations.NCT04078997. The study is supported by more than 20 years of routine pneumococcal surveillance.The study design is both pragmatic and robust, offering a methodology that is relevant to diverse settings in the region.By engaging community, institutional and government partners, we have optimised its relevance and increased the likelihood of community uptake.ie, children receiving a 2+1 vaccine schedule who reside in or relocate to a cluster implementing the 3+0 vaccine schedule or vice-versa).Limitations to this study include a risk of contamination between clusters  carriage as a prerequisite for the development of disease but also a key process for developing natural immunity.via human respiratory air droplets. With serotype-specific differences, carriage duration decreases with age, lasting from 2 weeks in adults up to 4 months among children.6\u201314In HICs, routine administration of pneumococcal conjugate vaccines (PCV) through the infant immunisation schedule has contributed to a rapid decline of vaccine serotype invasive pneumococcal disease (VT-IPD) in both vaccinated and unvaccinated populations.29Studies undertaken prior to PCV introduction in The Gambia, Kenya, Mozambique, Malawi and South Africa reported VT carriage prevalence ranging from 28% to nearly 50% among children <5 years old.38Currently, WHO recommends the implementation of the PCV vaccine using either a 3+0 schedule  or a 2+1 schedule (two primary infancy doses at 6 and 14 weeks of age and one booster at 9 months of age or after the first year of life). The WHO further recommends that the decision on which schedule to use be based on the epidemiology of disease in the local setting.40A 2017 WHO Technical Expert Consultation on Optimisation of PCV Impact highlighted a substantial research gap in investigating why the impact of PCV vaccines in low-income countries has been less than expected, underlining the need to define an optimal PCV vaccination schedule that will maximise their benefit in such settings.et alMalawi introduced the 13-valent PCV (PCV13) into the national Expanded Programme of Immunisations (EPI) in November 2011, using a 3+0 schedule , with a three-dose catch-up vaccination campaign among all infants <1\u2009year of age. This introduction has been highly successful, with field studies showing an EPI vaccine coverage exceeding 90%.In this context, the Malawi Ministry of Health (MoH) and the National Immunisations Technical Advisory Group are now seeking evidence of adequate superiority of a 2+1 vaccine schedule to inform a change to Malawi\u2019s current EPI schedule. To this aim, a pragmatic health centre-based evaluation comparing the current 3+0 schedule to a 2+1 schedule will be implemented in Blantyre District, southern Malawi. Two pneumococcal carriage surveys, conducted 18 and 33 months following the implementation of the 2+1 schedule, will have the objective of comparing the effect of the two schedules against carriage reduction among otherwise-healthy children and evaluate their potential to enhance herd immunity.The study is a pragmatic health centre-based randomised evaluation of the direct effect of a 2+1 PCV13 vaccine schedule on pneumococcal carriage in vaccinated infants and the indirect effect on non-vaccine eligible children and high-risk adults.2. Healthcare is delivered through a network of private and government hospitals and 28 government primary health centres, where EPI vaccinations are administered. Queen Elizabeth Central Hospital (QECH) is the government referral hospital providing free medical care to the 1.3\u2009million urban, periurban and rural residents of Blantyre District. Children <5 years account for 16% of the total population.The study will be conducted in Blantyre district, southern Malawi. The District is 240 kmWe will investigate whether a 2+1 PCV13 vaccination schedule (two primary doses at 6 and 14 weeks of age\u00a0+1 booster dose at 9 months of age), compared with the current 3+0 schedule , is superior in reducing VT carriage prevalence among vaccinated children and, consequently, creating a superior herd effect among older children and HIV-infected adults. To assess the effect of a district-wide change from 3+0\u2009to 2+1, this study will address two specific research questions: First, will a 2+1 schedule provide enduring vaccine-induced protection against pneumococcal VT carriage into the second year of life? Second, will the 2+1 vaccine schedule generate stronger herd protection and result in decreased VT carriage in older children and unvaccinated high-risk adults in the general population?The primary objective of the study is to evaluate the direct effect of a 2+1 PCV13 vaccination schedule on VT pneumococcal carriage among children aged 15\u201324 months, 3\u2009years after introducing the 2+1 schedule. This objective will answer the question of whether the 2+1 schedule induces a more enduring direct protection against pneumococcal VT carriage into the second year of life, compared with that observed with a 3+0 schedule.The secondary objectives of the study are to evaluate, 3\u2009years after introducing the 2+1 schedule, (1) the indirect effect of a 2+1 schedule on VT pneumococcal carriage among children aged 5\u201310 years (PCV age-ineligible at time of implementing the 2+1 schedule); (2) the indirect effect of a 2+1 vaccination schedule on VT pneumococcal carriage among HIV-infected adults 18\u201340 years old and on antiretroviral therapy (ART).To address these questions, the Malawi MoH and Blantyre District Health Office (DHO) will randomly select 10 health centres  in which the routine PCV13 schedule will be switched to a 2+1 schedule. An additional 10 health centres will be randomly selected to continue with the current 3+0 schedule but will serve as the direct comparator in evaluating the effectiveness of the 2+1 schedule.This switch to a 2+1 schedule is an initiative led by the MoH and will be implemented within the scope of the routine EPI programme, subject to EPI standard procedures for delivery, monitoring and performance assessment. Implementation of all vaccination activities will be implemented, as per routine practices, by MoH EPI vaccinators through the routine EPI programme. The MoH will monitor completeness of dosing following standard reporting practices within the scope of the EPI. Routine study activities throughout the duration of the 3-year study period will include research nurses providing support and guidance to the EPI vaccination teams through weekly site visits. In addition, research enumerators will monitor patient-retained health passports of a representative sample population of vaccinees to confirm they are receiving the proper vaccine schedule (2+1\u2009or 3+0) assigned to the catchment population of their respective health centre.The pragmatic design of this study has been assessed through the tool The PRagmatic-Explanatory Continuum Indicator Summary 2 (PRECIS-2).To ensure the 2+1 schedule is implemented efficiently, and to limit the risk of confusion during the transition to the 2+1 schedule (including not deferring PCV13 vaccination to a later visit), a 3+1 schedule will be implemented during the first several months at health centres implementing the 2+1 schedule. At these health centres, children who have received either their 10\u2009weeks or 14\u2009weeks PCV13 dose before 2+1 implementation will receive a dose at their 14\u2009weeks (third dose PCV13) and their 40\u2009weeks (fourth dose PCV13). Approximately 6 months after 2+1 implementation, all first-contact vaccine visits (post 2+1 implementation) will be for the scheduled visit at 6\u2009weeks of age and the 3+1 schedule will no longer be required.The study will include two cross-sectional carriage surveys, implemented 18 and 33 months after the switch to 2+1. Carriage surveys will be conducted using a well-established methodology implemented extensively in the setting,Sampling will include:Children 15\u201324 months of age, PCV13-vaccinated  with either the 2+1\u2009or the 3+0 schedule. Evaluation within this age group will allow us to evaluate for our primary objective, evaluating the direct effect of vaccination on carriage. Within each of the catchment areas of the 20 participating health centres, we will conduct a random walk method to systematically identify households with eligible children. A \u2018egg and yolk\u2019 strategy will be applied, defining two geographic sampling perimeters around each health centre. Sampling will prioritise those living within the geographic perimeter closest to the health centre. In the event that recruitment targets are not met, sampling will move into the second geographic perimeter. This strategy will maximise the likelihood of recruiting children who have received their vaccines at the selected health centre, minimising recruitment from buffer zonal borders and therefore minimising risk of contamination .Children 5\u201310 years of age, PCV13-vaccinated with the 3+0 schedule. These children, having received PCV13 at infancy, will be recruited from government schools. Evaluation within this age group will allow us to evaluate for a secondary objective, evaluating the indirect effect of a 2+1 schedule on VT pneumococcal carriage among children aged 5\u201310 years, the majority of whom received PCV13 infancy EPI vaccination using a 3+0 schedule. Six schools will be selected, three located centrally in each of the 3+0 clusters and the 2+1 clusters. Children will be chosen at random from school registers. This component will allow the evaluation of indirect effects of the 2+1 compared with 3+0 schedule among children with waning vaccine-induced immunity.Adults 18\u201340 years of age, HIV-infected on ART and PCV13-unvaccinated. Evaluation within group will allow us to achieve the secondary objective of evaluating the indirect effect of a 2+1 schedule on VT pneumococcal carriage among HIV-infected adults (18\u201340 years) on ART, a population at high risk of pneumococcal disease. Participants will be recruited from the QECH, Lighthouse ART Clinic in Blantyre.To assess the differential effects of 3 vs 2 primary vaccine doses in the first year of life, an additional carriage survey will be implemented at health centres approximately 9 months after 2+1 implementation. A convenience sample of children aged 9\u2009months will be recruited at health centres providing either the 2+1\u2009or 3+0 schedule. This will allow an evaluation of carriage prevalence among children receiving 3 (according to the 3+0 schedule) vs 2 (according to the 2+1 schedule and presenting for their booster dose) primary doses. Additionally, this additional carriage survey will contribute to evaluating the coverage of the 2+1 vaccination schedule and the proportion of children receiving all three PCV13 doses at health centres providing the 2+1 schedule.Inclusion criteria for all individuals recruited includes permanent resident in Blantyre District. Among children 15\u201324 months of age (assigned to either 2+1\u2009or 3+0 schedule), inclusion criteria additionally include: aged between 15 and 24\u2009months, parent/legal guardian providing written informed consent, evidence  of having received a full schedule of PCV13. Among children 5\u201310 years of age, inclusion criteria include: aged between 5\u201310 years, parent/legal guardian providing written informed consent, providing written informed assent (if the child is aged \u22658), and either verbal or documented evidence of having received PCV13. Among adults, inclusion criteria include: aged 18\u201340 years, providing written informed consent, and being HIV-infected and receiving ART.Among children 9 months of age, recruited as part of the safety component, inclusion criteria include: aged 9 months, parent/legal guardian providing written informed consent, and evidence  of having received either a full 3+0 PCV13 vaccine schedule  or both primary doses of PCV13 at approximately 6 and 14 weeks of age .Exclusion criteria for all screened individuals include receiving TB treatment at time of screening, hospitalisation for pneumonia within 14 days prior to study screening and terminal illness. Exclusion criteria for all children include parental/legal guardian not providing consent, not providing assent (for children aged\u00a0\u22658 years), having received antibiotic treatment within 14 days prior to study screening. Exclusion criteria for adults include not providing written informed consent or prior vaccination with a pneumococcal vaccine.S. pneumoniae. VT pneumococcal carriage will be serotyped by latex agglutination. Samples from participants with confirmed pneumococcal carriage will be sent to the UK for assessment of multiple serotype carriage (genomic microarray) and for whole genome sequencing.The intervention will consist of two carriage surveys conducted 18 and 33 months after the 10 randomised health centres switch from a 3+0\u2009to a 2+1 PCV13 schedule. In both surveys, a single NP swab will be collected from each participant. Following previously described WHO-recommended procedures,The primary endpoint of this study will be the difference in VT pneumococcal carriage prevalence among children aged 15\u201324 months, comparing those vaccinated with PCV13 in either a 2+1\u2009or 3+0 schedule, 3\u2009years after implementing the switch to a 2+1 schedule. Additionally, the study will evaluate four secondary outcomes: (1) difference in VT carriage prevalence among children 5\u201310 years old, 18 months and 33 months after 2+1 implementation, (2) VT carriage prevalence among HIV-infected adults aged 18\u201340 years and receiving ART at the time of sampling, (3) VT carriage prevalence among infants aged 9 months, who will have received three primary doses  or two primary doses  prior to the booster dose, 9 months after the implementation of the 2+1 schedule and (4) prevalence of multiple serotype carriage, 18 and 33 months after 2+1 implementationPrior to development of the protocol, key stakeholders were informed of the study, including the study sites  and their surrounding communities , the DHO, MoH and the Ministry of Education. We actively sought and incorporated input from these stakeholders into the study objectives and overall design. Community sensitisation will be further strengthened through a community advisory board.The primary endpoint is powered to detect an effect size of 50% reduction in VT carriage, expecting a 14% and 7% VT carriage prevalence among vaccinated children 15\u201324 months old in the 3+0\u2009and 2+1 arms, respectively. Sample sizes were calculated based on a power of 80% and a statistical significance of 0.05. The calculations accounted for household similarities using an intraclass correlation (ICC) of 0.005 (based on previous experience in this setting) and adjusted for a design effect (dependent on both ICC and cluster size.) of 1.21. Minimum sample sizes needed to achieve the necessary power under these assumptions are shown in To assess the primary and secondary endpoints (1) One child 15\u201324 months of age (2+1\u2009or 3+0) will be recruited from each of 40 households randomly selected within each of the 20 clusters. This is a total 800 vaccinated children per survey . (2) A total of 125 children will be recruited from each of the six schools per survey. This is a total 750 children per survey . (3) A total of 1200 HIV-infected adults will be recruited from the QECH ART Clinic over the course of the 3-year study period. (4) A total of 800 children 9 months of age will be recruited from the vaccination centres 9 months after schedule change .Demographic data and relevant medical history will be collected using password-protected electronic data capturing. Each participant will be assigned a unique participant identification number (PID) at recruitment. This PID will be used in all datasheets and files, and will be linked to the laboratory data, hence, only anonymised data will be used for the analysis. Fully anonymised data will be uploaded daily to a secured on-site server, which is backed up daily to both local and off-site facilities. A logbook containing identifiable information (including name) will be kept separate in a secured location by an authorised member of the study team and will only be accessed by authorised study members. This will allow the study team to recover any missing epidemiological information at a later date  and to facilitate any participants who choose to withdraw consent at any time.Continuous variables will be summarised by means and SD, or medians and IQRs if the distribution exhibits skew. Categorical variables will be summarised by frequency distributions. Direct effects of the PCV13 schedule change on VT carriage will be ascertained by comparing carriage prevalence in children aged 15\u201324 months residing in the recruitment clusters of health centres randomised to the 2+1 schedule and those in recruitment clusters of health centres randomised to the 3+0 schedule. Indirect effects will be evidenced by comparing children in the recruitment clusters of health centres randomised to the 3+0 schedule among older children (5\u201310 years) and HIV +adults. Additionally, data obtained in this study will be compared with those obtained from previous carriage surveys to ascertain any changes in VT carriage prevalence before and after the PVC13 schedule change. Statistical tests will be selected depending on the distribution patterns of the data. Potential confounders and sources of interaction  will be identified by testing the association between variables and VT carriage and included in the multivariable models when p<0.1. Sensitivity analyses will include assessing impact of (1) receiving only one, only two or all three doses PCV; (2) having document-confirmed PCV vaccination or (3) schedule adherence to within 2 weeks of each scheduled dose on VT prevalence and on the VT distribution.The study has been approved by the Malawi College of Medicine Research Ethics Committee , the University College London Research Ethics Committee (Ref: 8603.002) and the University of Liverpool Research Ethics Committee (Ref: 5439).The implementation of the study protocol will be reviewed and monitored by an External Advisory Group, providing oversight of the study activities and advise the study team at the time of the interim and final analyses. The group will include experts from the University of Malawi, College of Medicine, the London School of Hygiene & Tropical Medicine and the Medical Research Council.On completion of the first carriage survey, an interim analysis will be implemented using data obtained on the VT carriage prevalence among vaccinated children 15\u201324 months of age. These results will be used to assess possible adaptation of the second community carriage survey. Three possible scenarios are considered: (1) If convincing evidence of major change in carriage prevalence is demonstrated, immediate action will be discussed with the MoH to move to change the schedule and adapt year-3 sampling; (2) If no major change in carriage prevalence is identified, the study will continue as planned; (3) If carriage prevalence has fallen by more than 30% but does not meet the primary threshold of 50%, the second carriage survey will be brought forward by 6 months. Decisions will be reached in consensus between investigators, the Expert Advisory group, and the MoH.The study will only recruit children whose parents/legal guardians have the capacity to provide informed consent or, in the case of adults, are capable of giving consent. Children who are minors but \u22658 years of age will be required to provide informed assent, in addition to parents/legal guardians providing informed consent. Participants will receive both verbal and written information about the study and will be given the opportunity to ask questions and express their doubts and concerns before accepting to take part. They will also be given time to reflect before they come to a decision. An informed consent and/or assent form will be signed and dated by the participant and a member of the research team. The participant will keep a copy of the document, and a second one will be kept in the study file with the Principal Investigator based in Blantyre, Malawi. Participants will be informed of their right to withdraw consent at any point until study ends without the need to provide a reason and without penalty.Study results will be shared with local stakeholders and published in peer-reviewed journals. Partial results and interim analyses will be shared with the Malawi MoH and other relevant policymakers and decision-making stakeholders. Partial and final findings will be presented at relevant international conferences and meetings. Copies of all published materials and reports will be shared with the research ethics committees and collaborators. We will return to the community partners and work with the community advisory board to report and further disseminate our results into those communities where we worked."}
+{"text": "Bioinformatics (2020) doi: 10.1093/bioinformatics/btz595In the above article, the image currently appearing in the \"Figure 2\" position belongs instead in the \"Figure 1\" position; the image currently appearing in the \"Figure 3\" position belongs instead in the \"Figure 2\" position; the image currently appearing in the \"Figure 1\" position belongs instead in the \"Figure 3\" position. The publisher apologises for the error."}
+{"text": "Following the publication of this article , concernIn the left panel of Fig 1C, the left side of the bands do not appear to align correctly with the right sides of the bands.The Day 5 anti-\u03b2-actin panel of Fig 1Db appears similar to the Day 5 \u03b2-actin panel of The Sox9 panel and the GFP panel of The Day 2 \u03b2-actin panel of The corresponding author has provided the original images underlying Fig 1C and indicated that black/white inversion was performed to improve the presentation of the results. The uncropped underlying figures provided in the The similarity between the Sox9 and GFP panel in The similarity between the western blot panels in Fig 1D and The PLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the corresponding author.S1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(XLS)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(TIF)Click here for additional data file.S6 File(TIF)Click here for additional data file.S7 File(TIF)Click here for additional data file.S8 File(XLSX)Click here for additional data file."}
+{"text": "Exercise improves cognition in the aging brain and is a key regulator of neuronal plasticity genes such as BDNF. However, the mechanism by which exercise modifies gene expression continues to be explored. The repressive histone modification H3K9me3 has been shown to impair cognition, reduce synaptic density and decrease BDNF in aged but not young mice. Treatment with ETP69, a selective inhibitor of H3K9me3\u2019s catalyzing enzyme (SUV39H1), restores synapses, BDNF and cognitive performance. GABA receptor expression, which modulates BDNF secretion, is also modulated by exercise and H3K9me3. In this study, we examined if exercise and ETP69 regulated neuronal plasticity genes by reducing H3K9me3 at their promoter regions. We further determined the effect of age on H3K9me3 promoter binding and neuronal plasticity gene expression. Exercise and ETP69 decreased H3K9me3 at BDNF promoter VI in aged mice, corresponding with an increase in BDNF VI expression with ETP69. Exercise increased GABRA2 in aged mice while increasing BDNF 1 in young mice, and both exercise and ETP69 reduced GABRA2 in young mice. Overall, H3K9me3 repression at BDNF and GABA receptor promoters decreased with age. Our findings suggest that exercise and SUV39H1 inhibition differentially modulate BDNF and GABRA2 expression in an age dependent manner. Exercise has been shown to improve cognition in the aged brain across a range of clinical and animal studies . UnderlyOur lab has focused on elucidating the role of H3K9me3 in cognitive function. We first determined that H3K9me3 is elevated in the hippocampi of aged mice. To investigate if H3K9me3 is involved in cognitive decline, we inhibited the histone methyltransferase (SUV39H1) that catalyzes H3K9 trimethylation. We used a selective SUV39H1 inhibitor called ETP69, a chaetocin A analog developed by Dr. Larry Overman . TreatmeStudies have begun to examine how exercise affects the regulation of neuronal plasticity genes such as BDNF, which enhances learning and memory . BDNF isIn this study, we examined how exercise, SUV39H1 inhibition and age affect H3K9me3 promoter repression and expression of BDNF and GABA receptors. We first separated aged and young mice into sedentary, exercised and ETP69 treatment groups. We then measured levels of H3K9me3 at the promoters of BDNF transcripts I, IV, and VI, and at the promoters of GABBR1 and GABRA2. Lastly, we quantified transcript mRNA to determine the correlation between promoter H3K9me3 levels and total gene expression. Exercise and ETP69 reduced H3K9me3 at BDNF promoter VI in aged mice, corresponding with an increase in BDNF VI expression with ETP69. In young mice, exercise increased BDNF I expression, suggesting an age-dependent change in BDNF regulation. Exercise increased GABRA2 expression in aged mice while both exercise and ETP69 reduced GABRA2 expression in young mice. Overall, H3K9me3 repression at BDNF and GABA receptor promoters decreased with age. Our findings suggest that exercise and SUV39H1 inhibition differentially modulate BDNF and GABRA2 expression in an age dependent manner.n = 21 for each age; Jackson Laboratory) were individually housed with food and water ad libitum, and allowed 1 week acclimation to the vivarium prior to experiments. Lights were maintained on a 12 h light\u2013dark cycle. Mice were sacrificed by carbon dioxide gas and cervical dislocation, and brains were rapidly frozen on isopentane cooled in liquid nitrogen. 1 mm punches of the dorsal hippocampus were collected from 500 \u03bcm slices.All experiments were conducted in accordance with the National Institutes of Health guidelines for animal care and use, and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Male 18 month old and 3 month old C57BL/6J mice (All mice were single housed in either standard cages (sedentary and ETP69 injected mice) or cages with a running wheel (exercised mice). Mice were single housed due to the design of the running wheel apparatus, which allows for only a single animal per cage. Single housing also gave each animal free and equal access to a running wheel . ExercisChIP was performed as described previously based on the protocol from the Millipore Magna ChIP kit . Tissue qRT-PCR was performed as previously described . RNA waspost hoc analysis using Prism software. In all cases, p \u2264 0.05 was considered significant. Excel was used for percent input analysis of ChIP data and Pfaffl analysis of qRT-qPCR data.Results were analyzed using one way ANOVA, Welch\u2019s ANOVA, or two way ANOVA, followed by p = 0.0036; Dunnett\u2019s post hoc test, *p = 0.011 ETP69 vs. sedentary). This suggests that the neuronal activity caused by an OLM task changes the regulation of BDNF promoter regions.We first investigated if ETP69 and exercise affected H3K9 trimethylation levels at BDNF promoter regions. We examined BDNF I, IV, and VI as these regulatory exons are highly expressed in the hippocampus . Our labp = 0.0021 exercise vs. sedentary), displaying a specific and parallel effect of exercise and ETP69 on BDNF promoters. Surprisingly, neither ETP69 nor exercise changed H3K9me3 at BDNF promoter transcripts in young mice . There was no change in BDNF I or IV expression with ETP69 or exercise in aged mice . Exercise significantly elevated BDNF IV levels in comparison to ETP69 treatment, but failed to reach significance relative to sedentary controls . Exercise and ETP69 did not increase BDNF VI levels in young mice (p = 0.24). This is in accordance with previous findings that exercise induces BDNF I and IV expression but not BDNF VI expression in young mice after 3 weeks of exercise . These results suggest that GABA receptor promoters are resistant to epigenetic changes by exercise and global reductions in H3K9me3.We next investigated how the repression and expression of GABA receptors correlate with BDNF, as GABA receptor activation increases BDNF signaling . We prevp = 0.024; Tukey\u2019s post hoc test, ex vs. sed *p = 0.052, ex vs. etp *p = 0.044). In contrast, exercise and ETP69 reduced GABRA2 expression in young mice . ETP69\u2019s effect on GABRA2 is surprising considering it does not reduce H3K9me3 at its promoter, suggesting that a global reduction in H3K9me3 activates a pathway that decreases inhibitory signaling. The exercise-induced increase in GABRA2 in aged mice is supported by our research in aged postmortem brains, suggesting that the need for inhibitory signaling increases in the aged brain . Therefore, exercise and ETP69 regulate GABRA2 expression independently of promoter H3K9me3 in an age-dependent manner.We next asked if exercise and ETP69 changed GABA receptor expression levels and found that the direction of change was highly dependent on mouse age. Exercise significantly increased GABRA2 expression in aged mice (ed brain . There wOur lab previously found that H3K9me3 is increased with age in the hippocampi of aged mice, leading us to ask if promoter bound H3K9me3 was also elevated with age in our study . We compOur findings demonstrate that exercise and SUV39H1 inhibition affect the expression of BDNF exons and GABRA2 in an age dependent manner. Exercise and ETP69 significantly decreased H3K9me3 at BDNF promoter VI in aged mice, corresponding to an increase in BDNF VI expression with ETP69 treatment. Apart from this important exception, exercise and ETP69 did not significantly alter H3K9me3 levels at the promoters of neuronal plasticity genes, although they did regulate mRNA levels. These findings suggest that H3K9me3 promoter repression is not a primary driver of neuronal plasticity gene expression. Age had a strong effect on H3K9me3 promoter binding, as H3K9me3 repression of BDNF and GABA receptors decreased significantly in old mice. Exercise and ETP69 altered neuronal gene expression in an age-dependent manner and reduced H3K9me3 recruitment at the BDNF VI promoter region.Of the promoters we examined, exercise and ETP69 only reduced H3K9me3 at the BDNF VI promoter in aged mice, suggesting that promoter bound H3K9me3 is a largely stable epigenetic modification. This finding differs from previous research showing that ETP69 reduces H3K9me3 at BDNF I following an OLM task . OLM incIn addition to increasing BDNF VI levels in aged mice, exercise also elevated BDNF I expression in young mice. This finding is supported by previous studies in our lab showing that 3 weeks of wheel running increase BDNF I levels in the hippocampi of young rats and mice . There wIntriguingly, exercise and SUV39H1 inhibition significantly affected GABRA2 expression, yet the direction of change was dependent on mouse age. While exercise increased GABRA2 expression in aged mice, ETP69 and exercise both reduced GABRA2 levels in young mice. GABA signaling is reduced in the hippocampus with age, contributing to overexcitation and memory impairment . A studyExercise induced changes in BDNF and GABRA2 expression in our models, yet GABRA2\u2019s role in BDNF regulation remains unclear. In aged and exercised mice, the increase in GABRA2 correlated with an increase in BDNF VI expression, suggesting that GABRA2 may upregulate BDNF in aged models of exercise. In contrast, exercise induced a significant decrease in GABRA2 and an increase in BDNF I expression in young mice. GABRA2 may only be involved in BDNF upregulation in systems where BDNF and GABA signaling is impaired, such as models of aging. Future studies should examine the signaling pathway between GABRA2 and BDNF in models of aging and exercise to determine if GABRA2 differentially upregulates BDNF expression.Although promoter H3K9me3 was largely unaffected by our manipulations, its stability and binding affinity appear to decline with age. With the exception of GABBR1, H3K9me3 binding was greater in young mice than aged mice at the promoters of neuronal plasticity genes. Our findings demonstrate that H3K9me3 at promoter regions decreases with age even as overall H3K9me3 increases, which may indicate a change in H3K9me3\u2019s localization and function . AlthougThis study opens up avenues for future investigation into the regulation of neuronal plasticity genes. The role of GABRA2 in BDNF regulation has yet to be explored, and studies involving GABRA2 inhibition or overexpression could elucidate its effect on BDNF expression. Studies on H3K9me3 localization throughout the lifespan will further suggest how its function changes with age. Our SUV39H1 inhibitor, ETP69, globally reduces H3K9me3 but cannot modify this marker at specific genes. ChIP-sequencing could be used to identify how H3K9me3 binding changes with age and exercise, and RNA-sequencing can determine if these changes are correlated with gene expression. A comprehensive examination of H3K9me3 will clarify its effect on neuronal gene expression and specific sites of pharmacological manipulation.We have demonstrated that H3K9me3 regulates the expression of BDNF VI in aged mice. Reducing H3K9me3 with ETP69 increases BDNF VI expression, thus increasing synaptic plasticity at distal dendrites. Increased age reduces H3K9me3 promoter binding and changes the regulation of BDNF and GABRA2 by exercise and ETP69. Overall, exercise and SUV39H1 inhibition can effectively modify the expression of genes involved in cognition.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.AI-T and NB conceived of the presented ideas. CB and AI-T performed the experiments. DM and MW assisted with experimental design and provided PCR equipment. AI-T analyzed the data and wrote the manuscript. CC reviewed the manuscript and supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Wolbachia can manipulate arthropod reproduction and invade host populations by inducing cytoplasmic incompatibility (CI). Some host species are coinfected with multiple Wolbachia strains which may have sequentially invaded host populations by expressing different types of modular CI factor (cif) genes. The tephritid fruit fly Rhagoletis cerasi is a model for CI and Wolbachia population dynamics. It is associated with at least four Wolbachia strains in various combinations, with demonstrated , predicted (wCer1) or unknown (wCer5) CI phenotypes.The endosymbiont Wolbachia strains wCer1, wCer4 and wCer5, and compared these with the previously sequenced genome of wCer2 which currently invades R. cerasi populations. We found complete cif gene pairs in all strains: four pairs in wCer2 , two pairs in wCer1 (both Type I) and wCer4 , and one pair in wCer5 (Type IV). Wolbachia genome variant analyses across geographically and genetically distant host populations revealed the largest diversity of single nucleotide polymorphisms (SNPs) in wCer5, followed by wCer1 and then wCer2, indicative of their different lengths of host associations. Furthermore, mitogenome analyses of the Wolbachia genome-sequenced individuals in combination with SNP data from six European countries revealed polymorphic mitogenome sites that displayed reduced diversity in individuals infected with wCer2 compared to those without.We sequenced and assembled the draft genomes of the Wolbachia are common in arthropods and affect options for Wolbachia-based management strategies of pest and vector species already infected by Wolbachia. Our analyses of Wolbachia genomes of a host naturally coinfected\u00a0by several strains unravelled signatures of the evolutionary dynamics in both Wolbachia and host mitochondrial genomes as a consequence of repeated invasions. Invasion of already infected populations by new Wolbachia strains requires new sets of functionally different cif genes and thereby may select for a cumulative modularity of cif gene diversity in invading strains. Furthermore, we demonstrated at the mitogenomic scale that repeated CI-driven Wolbachia invasions of hosts result in reduced mitochondrial diversity and hitchhiking effects. Already resident Wolbachia strains may experience similar cytoplasmic hitchhiking effects caused by the invading Wolbachia strain.Coinfections with The online version contains supplementary material available at 10.1186/s12864-021-07906-6. Wolbachia bacteria (Alphaproteobacteria) of arthropods can affect host\u00a0reproduction and fitness, including host immunity, in a multitude of ways . Therefore, wCer2 had four complete sets of cif genes and the largest number of cif modules in this host species.Orthology to verified r5 Table\u00a0. OriginaifB gene , but reacifA maximum likelihood tree comprised 41 cifA orthologues, representative of all five types, and was built on an alignment of 1884 nucleotide sites, of which 1267 were parsimony-informative  and 99.6% (cifB) amino acid similarity with CI inducing cifwPip[T4].Both contiguous nes Fig.\u00a0. wCer4 a96_03720 , which acifwCer4T genes whe et al. . wCer5 iwCer2 gene (E3V96_03405) with an identical amino acid sequence to wmk of wMel (WD0626) which had previously been found to cause MK when highly expressed in transgenic D. melanogaster. No orthologues for this gene were found in wCer4 and wCer5. However, for a wmk homologue in wMel, WD0508, for which transgenic expression did not alter sex ratios in D. melanogaster, orthologues were found in wCer2 (similarity of 89%), wCer4 (94%) and wCer5 (93.6%). No orthologues of wmk or its homologues were found in wCer1.Furthermore, we found a R. cerasi individuals were representatives of three  populations with different Wolbachia infection types  and these individuals did not have wCer2, and the mitogenome of wCer2-infected RcerAS was HT2  between the three mitogenomes , 8 of the 12 sites were polymorphic ; for HT2 individuals (with wCer2) only 10 sites were represented by ddRADseq, and none was polymorphic . The variance in the Euclidean distance within the HT1 group of mitochondrial haplotypes (var\u2009=\u20090.07) was higher than group HT2 (var\u2009=\u20090.001). Furthermore, the PERMANOVA showed strong influence of the wCer1/wCer2 grouping on the distance measures between haplotypes (p\u00a0=\u20090.001), but presence of wCer4 (p\u00a0=\u20090.736) or wCer5 (p\u00a0=\u20090.206) had no effect , wCer5  and wCer2  was almost 60% higher than the number of SNPs between the two wCer1 variants in the same two samples . The consensus sequences for each strain for each individual were calculated by majority consensus with no lower coverage threshold, however the additional variant analysis also showed strain variation within host individuals. The wCer1 of RcerHB showed at least two possible nucleotides (with minimum coverage of 35% of a given variant with minimum coverage threshold of 5 reads) at 16 sites; at 12 of these sites both nucleotides were present in the wCer1 of RcerIZ, and at 10 sites both nucleotides were present in the wCer1 of RcerAS. The overlap of variant sites in RcerIZ and RcerAS was at 11 sites . However, our data indicated at most a single SNP in a transposase gene that was single copy in wCer1 but multicopy in wAu and wMel may be a false positive. For wCer5, the single SNP in the phage gene patatin and nine of the 10 SNPs in the major tail sheath protein gene in RcerAS are true differences (>\u200958% frequency) from the reference sequence, while one heterozygous site may be a false positive. Notwithstanding these possible misassemblies, the number of changes still suggests wCer5 has a greater number of SNPs than wCer1 between RcerIZ and RcerAS.The variation across the ces Fig. , 8 and 9ces Fig.  and 12 wDsimRC50 ) showed wCer2, eight SNPs between the Wolbachia genome of Ccap88.6 and its donor RcerAS  from their donor, but three SNPs were common to the two recipients, and an additional two were common to the Ccap88.6 and D. simulans lines , wCer1 had ~20x coverage, wCer4 had ~2x coverage and wCer5 had ~5x coverage, thereby confirming wCer1 and wCer2 as high titre infections, and wCer4 and wCer5 as low titre infections in this species.We also searched the ddRadseq reads mapped to the wCer1 variants and between the HT1 mitogenomes of RcerIZ and RcerHB (11 SNPs and 17 SNPs respectively), equated to 117x relatively more mutations in the mitogenome (Wolbachia genome is approximately 76x larger than the mitogenome). The wCer1 genome comparison of RcerAS and RcerHB revealed 14 SNPs, the mitogenomes of those samples had 24 SNPs, which equated to 130x relatively more mutations in the mitogenome. The wCer1 genome comparison of RcerAS and RcerIZ revealed 19 SNPs, the mitogenomes of those samples differed by 29 SNPs, i.e. 116x relatively more mutations in the mitogenome.The number of mutations between the Wolbachia strain genomes, wCer1, wCer4 and wCer5, of the European cherry fruit fly, R. cerasi, and analysed these in conjunction with the previously sequenced genome of wCer2  genes that recapitulate CI  gene pair [cifwCer2[T5] gene pair for which only cifB had previously been annotated  subclade, however the wMel-like Type I cifA gene could also play a role. Such a prediction is supported by the demonstrated CifA rescue of CI induced by a similar but non-cognate CifA/B pair  may induce CI that is not rescued by Type I cif due to their dissimilarity. While there is no experimental evidence for CI induction by cifwCer2[T5], Type V cif genes have characteristics of other cif types: (i) the cifA and cifB genes are adjacent, transcribed in the same direction and located in prophage regions; (ii) the domain structure is similar to cifwPip[T4]  but only had 36 and 45% similarity with cifwCer2[T5]. Both cif gene pairs are potentially responsible for CI in wCer4 and one or both are presumably incompatible with the wCer2 cif gene pairs. The substantial divergence between the Type V cif genes in wCer2 and wCer4 suggests that they are likely incompatible. The repertoire of cif genes in wCer4 also indicates this strain may be incompatible with wCer1, due to the absence of Type V cif genes in wCer1.Furthermore, capitata . We founwCer5 can induce CI, the genome sequencing of wCer5 showed a high similarity of cifwCer5[T4] to CI-inducing cifwPip[T4] (over 99.6% amino acid identity), and is therefore likely to induce CI. Furthermore, wCer5 is the only wCer genome with Type IV cif genes in this host species, and unlike the other wCer genomes has no Type I cif genes. Therefore, wCer5 is likely bidirectionally incompatible with wCer1, wCer2 and wCer4, and this could cause issues, e.g. slowing down of a wCer2 invasion between populations that are polymorphic in infection status. One would expect that for invasion to be successful either wCer2 and wCer5 spread should be linked, or wCer5 should already exist at a high enough prevalence in populations that are being invaded.While it has not been demonstrated in crossing experiments that wCer genomes contain orthologues of the wmk gene sequence which can simulate a MK phenotype in transgenic D. melanogaster  [cifBwPip[T4]  cannot be rescued by either cognate cifA or non-cognate cifwMel[T1], so lethality is not strictly through CI   and cifBwPip[T4]  when therough CI , and thisimulans .wCer5 variants is larger than the divergence between the respective wCer1 variants (by 60%). This indicates that wCer5 has been associated with R. cerasi for a relatively longer period than wCer1. Furthermore, the variant analysis showed that strain variants comprised a polymorphic population. For wCer5 there were few variable site overlaps, and therefore more accrued  differences between the two sequenced individuals; in contrast, there were many variable site overlaps for wCer1 variants of the same two individuals. The linkage of wCer1 with HT1 is a clear indication of a more recent invasion of wCer1 than wCer5. No such link for wCer5 with a mitochondrial haplotype was detected, and this could indicate that a previously existing linkage may have broken down as a consequence of the wCer1 invasion in R. cerasi. wCer5 maintains high prevalence in some R. cerasi populations where it routinely co-occurs with wCer1, wCer2 and wCer4  or multiple strains in a single host, and resolve the fitness costs (toxicity or incomplete CI self-rescue) that have been demonstrated for single infections of wCer2 and wCer4 in novel hosts. Furthermore, mitogenomes and Wolbachia genomes from WGS projects can guide and increase resolution of SNP analyses from reduced representation genomic datasets, such as ddRadSeq. This enabled us to link Wolbachia strain infection with mitogenome haplotypes in individuals and clearly demonstrated haplotype variation associated with Wolbachia infections and the more recent acquisition of wCer2.Our analyses of the four R. cerasi: RcerAS from Stillfried, Austria (approximately 40\u2009km NE of Vienna), collected in 2001; RcerIZ from Zafferana in eastern Sicily, Italy, collected in 2001; and RcerHB from Bajna, Hungary (approximately 40\u2009km NW of Budapest), collected in 2000  by microinjection, using a donor population of R. cerasi from Sicily , wNo [T3], wPip [T4] and wStri [T5]. The nomenclature of cif gene pairs has recently been proposed to follow the format of cifwStrain[T1] as an example for a Type I pair [cifA alignment excluded orthologues if they were truncated and did not contain the unannotated N-terminal region, or the catalase-rel or DUF domains, because mutations in any of these essential regions can diminish CI and rescue [cifB gene alignment included orthologues only if they contained the unannotated N-terminal region and the two PDDEXK domains common to Type I and Type IV cifB genes which have both been determined experimentally to induce CI. Maximum likelihood trees were estimated from the gene alignments as described above, using models TPM3\u2009+\u2009F\u2009+\u2009G4 for cifA and TPM3\u2009+\u2009F\u2009+\u2009I\u2009+\u2009G4 for cifB.Orthologues of e I pair . Proteine I pair , furin ce I pair  and the e I pair . The gene I pair  and the e I pair . The cifd rescue . The cifwmk orthologues were identified in the wCer genomes from the orthogroup containing wMel WD0626 (wmk). No orthologues were found in wCer1, but each orthologue from wMel (seven genes), wRec (one gene), wCer2 (eight genes), wCer4 (five genes) and wCer5 (three genes) were codon-aligned in MEGA v7 using Muscle, and the amino acid pairwise distances were calculated.wCer1 variants of three populations , and between wCer5 variants of two populations , were identified by read mapping using CLC Genomics Workbench to the final draft genomes at a similarity of 97% over 97% of the read length, and only properly paired reads were kept. These parameters differed from the parameters used to verify the draft genome sequences because here we wanted to capture strain variation within an individual. For the RcerAS library , reads were competitively mapped to the wCer2 (GenBank Accession No: SOZK01000000) as well as the wCer1 and wCer5 genomes simultaneously to restrict errors, primarily due to wCer2 reads mapping to the wCer1 genome. This problem was likely to occur because wCer1 and wCer2 are both supergroup A strains and wCer2 reads were more abundant than wCer1 reads and would therefore inflate the outcome of variant detection. The RcerIZ library was competitively mapped to wCer1 and wCer5, and RcerHB was only mapped to wCer1. These stringency parameters allowed for polymorphisms to be detected, while minimising off target reads.Polymorphisms between wCer1 variant SNP calling, the RcerAS and RcerHB libraries were subsampled (26 million and 10 million reads respectively) to normalize the number of wCer1 reads against the full RcerIZ library that had 258,194 properly paired reads mapped to wCer1 (average 20x coverage). For wCer5 variant SNP calling, the RcerAS library was not subsampled to the level of RcerIZ, because the low number of reads from RcerIZ  gave very low and sparse coverage (average 4x). The full RcerAS library was used and provided ~18x coverage. In order to aid analysis, variants of wCer2 from two D. simulans lines and one\u00a0C. capitata line carrying single infections of wCer2 [wCer2 from RcerAS subsampled to 87 million reads. To determine the consensus sequences, no minimum read number threshold was applied and the majority (>\u200950% reads) nucleotides were extracted for each Wolbachia strain derived from each library to determine a library specific genome. Alignment of these consensus sequences  generate a library-specific consensus sequence for each strain and (ii) detect variation within individuals for each strain. For of wCer2  were alsg Popart . For varR. cerasi de novo assembly via BLASTn match to the C. capitata complete mitochondrial genome (GenBank Acc: AJ242872). The sequencing reads were mapped at high stringency (97% similarity and 90% length) and the circular genomes closed and verified by mapping at 99% similarity and 95% length. Protein coding genes (PCGs), tRNAs and rRNAs were annotated using Mitos2 [The mitochondrial genome contigs were extracted from each R. cerasi mitochondrial genomes were identified by mapping a subsample of each library to the RcerHB mitochondrial genome at 97% similarity and 97% length. Each library was subsampled to achieve approximately 500-fold coverage of the mitogenome, hence RcerHB , RcerIZ  and RcerAS  were sampled and mapped to the RcerHB mitogenome. Variant SNPs were called with a low frequency cut-off of 1%, and differences between populations were identified when found at >\u200999% frequency.SNPs across the three R. cerasi individuals from six countries  published as part of a population study [Wolbachia genomes  and the mitochondrial genome of RcerHB at 85% of read length and 98% similarity, and only reads specific to a single genome were retained. This low length stringency was chosen so the overhanging barcode of 8 to 10 nucleotides met the parameters. The barcodes were used to identify the samples that mapped to regions on the mitochondrial genome that showed variability. These samples were scored for Wolbachia strain presence by examining mapping coverage over the four Wolbachia genomes. The threshold selected was at least one perfect read over at least five mapped regions of the genome. This threshold meant that low titre strains were reliably detected (even at one-fold coverage over many regions) but eliminated misallocation of reads to a different Wolbachia strain where the genome was incomplete; this could have occurred if wCer5 was present but the conservative approach to its genome assembly resulted in some of its reads mapping to another genome.A ddRADseq dataset representing 192 on study  was downwCer2, if they had wCer4 or wCer5, and by country of origin (with Sicily divided into Sicily West and Sicily East). Adonis of the R package vegan [Mitochondrial SNPs for each of 46 samples were identified from the ddRadseq mapped reads, tabulated and converted to a genind object and a Euclidean distance matrix using adegenet , 89 in Rge vegan  was implAdditional file 1.\u00a0UpSet graph showing shared Wolbachia orthogroups.Additional file 2. PHASTER identification of prophage regions in wCer1, wCer4 and wCer5.Additional file 3. cifA and cifB locus names from Wolbachia strains present in phylogeny.Additional file 4. Mitochondrial genomes and the polymorphic sites between RcerHB (HT1a), RcerIZ (HT1b) and RcerAS (HT2).Additional file 5. Polymorphisms in mitogenomes of three complete mitogenomes of RcerHB (HT1a), RcerIZ (HT1b) and RcerAS (HT2) and an additional 46 samples with SNP representation at 12 of these poymorphic sites from ddRadSeq data. Sample names and location are from Bakovic et al [ic et al , togetheAdditional file 6. PERMANOVA\u00a0of mitochondrial genetic distances between samples grouped by presence of Wolbachia strains.Additional file 7.\u00a0FASTA aligned wCer1 consensus sequences from RcerHB, RcerIZ and RcerAS.Additional file 8.\u00a0FASTA aligned wCer5 consensus sequences from RcerIZ and RcerAS.Additional file 9.\u00a0FASTA aligned wCer2 consensus sequences from RcerAS, DsimRC45, DsimRC50 and Ccap88.6.Additional file 10.wCer1 variant calling. Reference position refers to the wCer1 genome position after the 16 contigs were joined in order. Reads from three libraries: RcerHB (sampled to 10 million reads), RcerIZ  and RcerAS (sampled to 26 milion reads), were mapped at 97% length and 97% similarity, and RcerIZ and RcerAS were mapped competitively to the genomes of wCer1 and wCer5; and wCer1, wCer2 and wCer5, respectively. Variants were called with a minimum cut-off of 35% frequency, so a frequency of 65% for a variant is considered homozygous. Location of SNPs within genes is based on PROKKA annotation, and determination of copy number (single or multiple copy) was based on Orthofinder assessment of orthogroups including wCer1, wAu and wMel genomes.Additional file 11. wCer5 variant calling. Reference position refers to the wCer5 genome position after the 57 contigs were joined in order. Reads from two libraries: RcerIZ  and RcerAS , were mapped at 97% length and 97% similarity, and RcerIZ and RcerAS were mapped competitively to the genomes of wCer1 and wCer5, and wCer1, wCer2 and wCer5, respectively. Variants were called with a minimum cut-off of 35% so a frequency of 65% for a variant is considered homozygous. Location of SNPs within genes is based on PROKKA annotation, and determination of copy number (single or multiple copy) was based on Orthofinder assessment of orthogroups including wCer5, wMeg and wPip genomes.Additional file 12. wCer2 variant calling. Reference position refers to the wCer2 genome position after the 11 contigs were joined in order. RcerAS (sampled to 87 million reads) was mapped competitively to the genomes of wCer1, wCer2 and wCer5, respectively at 97% length and 97% similarity. Variants were called with a minimum cut-off of 35% frequency."}
+{"text": "All the synthesised compounds inhibit HDAC1 and HDAC2 selectively over other isoforms and many inhibit DLD1 and HCT116 cells more effectively than a parent compound. Compounds 8 and 16 inhibit HCT116 cells by activation of the apoptosis pathway.Five pathways involving different ring structures led to generation of fourteen thienylbenzamides ("}
+{"text": "Following the publication of this article , the aut\u25e6 The \u25e6 The \u25e6 The In In In Fig 5D, lane 1 of the \u03b2-actin of MRC5 panel for the ATII-MRC5 experiment is identical to lane 2 of the \u03b2-actin of MRC5 panel for the ATII-MRC5 experiment in Contradictory to the figure legend, the blots presented in Quantitative data files in the Supporting Information tables  Tables iThe authors commented that the issues in In relation to the issues highlighted by the authors in Figs 5D and The authors submitted image data to support the western blots presented in Figs The available quantitative data underlying the results presented in this article are provided in the Furthermore, the authors notified PLOS of the below errors in the Supporting Information tables. Updated Supporting Information tables in which these issues have been addressed are provided with this notice.In In In In In In In PLOS ONE editorial team, and it was concluded that the errors did not impact the results presented in the corresponding graphs. Additionally, as The updated tables were reviewed by the PLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the authors.The S2 File(A) The blots in (TIF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 FileNormalized data for Figs (ZIP)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 TableUpdated (DOC)Click here for additional data file.S8 TableUpdated (DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.S10 Table(DOC)Click here for additional data file."}
+{"text": "NF1 deletions often exhibit more severe clinical manifestations than patients with intragenic NF1 gene mutations, including facial dysmorphic features, overgrowth, severe global developmental delay, severe autistic symptoms and considerably reduced cognitive abilities, all of which are detectable from a very young age. Type 1 NF1 deletions encompass 1.4 Mb and are associated with the loss of 14 protein-coding genes, including NF1 and SUZ12. Atypical NF1 deletions, which do not encompass all 14 protein-coding genes located within the type 1 NF1 deletion region, have the potential to contribute to the delineation of the genotype/phenotype relationship in patients with NF1 microdeletions. Here, we review all atypical NF1 deletions reported to date as well as the clinical phenotype observed in the patients concerned. We compare these findings with those of a newly identified atypical NF1 deletion of 698 kb which, in addition to the NF1 gene, includes five genes located centromeric to NF1. The atypical NF1 deletion in this patient does not include the SUZ12 gene but does encompass CRLF3. Comparative analysis of such atypical NF1 deletions suggests that SUZ12 hemizygosity is likely to contribute significantly to the reduced cognitive abilities, severe global developmental delay and facial dysmorphisms observed in patients with type 1 NF1 deletions.Patients with neurofibromatosis type 1 (NF1) and type 1  NF1 gene and its flanking regions at 17q11.2  17q11.2(28997893-29695563)x1). The centromeric deletion breakpoint is located within NF1-REPa, and the telomeric deletion breakpoint within intron 57 of the NF1 gene, as confirmed by MLPA analysis. The deletion identified in patient 310221 encompasses the NF1 gene, its three embedded genes and the five genes located centromeric to NF1, namely CRLF3, ATAD5, TEFM, ADAP2 and RNF135  are loss-of-function intolerant, as determined by the metric \u2018probability of being loss-of-function intolerant (pLI)\u2019 \u2019 . The pLINeither array analysis nor MLPA analysis of blood and saliva-derived genomic DNA of the patient indicated the presence of somatic mosaicism with normal cells. The deletion of patient 310221 was not identified in the blood of his parents or his healthy sister and is hence considered to have occurred de novo.NF1 deletions have been reported [NF1 deletions can be classified into two groups: The first encompasses very large deletions extending beyond one or both of the boundaries of the 1.4 Mb type 1 NF1 microdeletion region which is flanked by NF1-REPa and NF1-REPc. In total, 31 such large atypical NF1 deletions have been identified, which together constitute group #1 . Mutatifeatures . Since irelation . SUZ12 mutations exhibit an overgrowth phenotype [NF1 deletions. In contrast to the short stature observed in most patients with intragenic NF1 mutations, tall stature in adults and overgrowth during childhood have been reported in patients with NF1 microdeletions [RNF135 gene located centromeric to NF1 within the type 1 NF1 deletion region has been suggested to be associated with the overgrowth seen in patients with NF1 microdeletions [NF1 deletions that do not encompass the RNF135 gene but are associated with the loss of SUZ12 [SUZ12 resulting in SUZ12 haploinsufficiency is likely to contribute to the overgrowth phenotype observed in patients with type 1 NF1 microdeletions. This conclusion is supported by the observation that SUZ12 is not included within the deletion interval in patient 310221 who is small for age. Overgrowth or tall stature was also not reported for other patients with atypical NF1 deletions of group #2  of 21 children with type 1 NF1 deletions, which was significantly more frequent than in the general NF1 population [In addition to ADHD, a high incidence of autism has been observed in patients with NF1 ,99,100. pulation . AutistiNF1 microdeletion-associated phenotype and in particular autism has recently been demonstrated for the cytokine receptor-like factor 3 (CRLF3) gene located within the NF1 microdeletion region. CRLF3 is deleted in patient 310221 reported here  present in addition to pathogenic variants in NF1. Taken together, these findings suggest an essential role for CRLF3 in both human brain development and autism [CRLF3 gene in the patient described here may have contributed to his autistic symptoms. Further analyses of patients with different types of NF1 microdeletion will confirm or refute the possible contribution of the CRLF3 gene to the deletion-associated phenotype.A specific role in the development of the ted here . InducedNF1 deletions of group #2, such as the 698 kb deletion observed in patient 310221, are potentially very informative in terms of genotype/phenotype correlations. However, the possibility that atypical NF1 deletions can be associated with mosaicism with normal cells not harbouring the deletion must be considered. Vogt et al. [NF1 deletions. In 6 of these 10 patients, mosaicism was analysed by FISH which allowed the determination of the proportion of cells harbouring the deletion. In the blood samples of these six patients, the proportion of cells with the deletions was 70%, 75%, 80%, 93%, 96% and 98%, respectively. In three (50%) of the six patients, mosaicism was also detected by MLPA, which is considered to have an intrinsic detection limit of ~10%  in those patients whose NF1 microdeletions encompass this gene. However, further studies are necessary to investigate this in greater detail. To date, genotype/phenotype correlations for group #2 atypical NF1 deletions are limited by the scarcity of clinical details available for many of the reported patients and by limited breakpoint definition due to the use of MLPA instead of microarray analysis. Moreover, information about possible mosaicism with cells not harbouring the deletion is not available for many patients with atypical NF1 deletions. The future analysis of genetically and clinically well-characterized patients with atypical NF1 deletions will serve to further clarify the role of the loss of genes such as SUZ12 and CRLF3 for the NF1 microdeletion-associated phenotype.Group #2 atypical"}
+{"text": "After publication of this article , concernThe authors provided underlying image data for all panels shown in Figs 3 and The raw image data revealed that the pAkt and pERK blot images were spliced to remove data between lanes 4 and 5 on the published Figure 3C see  Files.In regard to the control panels included in Figs 3 and The raw data underlying other results in this article are available from the authors.The authors apologize for the errors in the published article.S1 FileOriginal blot data supporting the results in Figure 3A.(PDF)Click here for additional data file.S2 FileOriginal blot data supporting the results in Figure 3C.(PDF)Click here for additional data file.S3 FileOriginal blot data supporting the results in (PDF)Click here for additional data file.S4 FileOriginal blot data supporting the results in the corrected (PDF)Click here for additional data file.S5 FileAdditional original blot data supporting the results in Figs 3 and (PDF)Click here for additional data file."}
+{"text": "In-stent restenosis and stent thrombosis are the major concerns while choosing a coronary stent. This single-centre, retrospective study evaluated the one and three-year clinical outcomes following implantation of Yukon Choice Flex (YCF) sirolimus-eluting stent. A total of 168 consecutive patients with 217 lesions underwent stenting with YCF stent. The presentation was with acute coronary syndrome in 158 (94%) patients. At 3 years, 9 (5.3%) patients died due to cardiac cause. Myocardial infarction, and definite stent thrombosis occurred in 10 (6%) and 4 (2.4%) patients respectively. Redo stenting and coronary artery bypass surgery was performed in 3 (1.8%) and 1 (0.6%) patient respectively. The use of YCF sirolimus eluting stent was associated with a favourable safety and efficacy profile at one and three-years of follow-up in a high-risk population. Patients who died before PCI or had significant renal dysfunction or sepsis, severe thrombocytopenia or coagulation abnormalities, those who had a delayed presentation with refractory left ventricular failure (LVF) or refractory cardiogenic shock in whom PCI could not be performed, were not enrolled. The study aimed at studying the efficacy and outcomes of patients who underwent PCI with YCF stent at 1 and 3 years.The study protocol conformed to the ethical guidelines of the declaration of Helsinki and was reviewed and cleared by the institute's ethical committee.Clinical diagnosis included 88 (52.4%) patients with ST-segment elevation myocardial infarction (STEMI), 70 (41.6%) patients with non\u2013ST-segment elevation ACS [including non-ST elevation myocardial infarction (NSTEMI) in 42 (25%) patients and unstable angina in 28 (16.6%) patients], and 10 (6%) patients with chronic coronary syndromes . The culStatistical analysis was performed with Statistical Package for the Social Sciences version 26 .3A total 217 YCF stents were deployed in 168 patients. The mean age of the participants were 56.4\u00a0\u00b1\u00a012.68 years. The study population were predominantly male (85.9%). The presentation was with ACS in 158 (94%) of study subjects. The baseline characteristics of patients were summarized in The baseline angiographic characteristics were tabulated in Clinical follow up was performed for all patients for three years. At the end of 3 years, a total of 12 (7.1%) deaths occurred, out of which 9 (5.3%) patients died due to presumed cardiac cause. Other outcomes assessed at the end of 3 years was myocardial infraction in 10 (6%) patients, definite stent thrombosis in 4 (2.4%) patients and target lesion revascularization was needed in 2 (1.2%) patients. Individual outcomes at the end of 1 year and from 1 to 3 years are summarized in Redo PCI was done in 3 patients. Coronary artery bypass graft surgery was done in 1 patient during the study period.4,,PCI is the revascularisation modality of choice in most patients with ACS and suitable coronary anatomy. The odds of the development of stent failure in the form of in-stent restenosis and stent thrombosis are important considerations while selecting a stent.Even though the debate over the efficacy and safety of DES versus BMS is continuing; contemporary data suggest that the benefits of DES outweigh the risks compared with BMS mirroring the current increase in DES usage.,Our study population consisted predominantly of ACS patients. More than half of the patients with ACS had STEMI which was similar to other studies from India and west including the INTERHEART study.In an observational study of YCF stent by Xhepa et\u00a0al, the rates of death, myocardial infarction, definite stent thrombosis and ischemia-driven target lesion revascularization were 2.4%, 1.9%, 0.3% and 11.3% respectively at 1 year.,15We would like to reiterate the fact that the study aimed to study the efficacy of YCF stent and the intermediate and long-term outcomes of patient undergoing PCI with YCF. It was not designed to look at outcomes of ACS patients as such. Still the lower 1 year mortality at 4.2% in our study can be explained by the relatively younger patient powith mean patient age of 56.4\u00a0\u00b1\u00a012.68 years. Much lesser patients had diabetes compared to other larger studies and the mean ejection fraction was higher at 46.3\u00a0\u00b1\u00a09.21% compared to prior studies.A study similar to ours by Liu et\u00a0al which enrolled patients of STEMI and NSTEMI undergoing PCI with DES had an overall mortality rate of 5.6% at 5 years and was comparable to our data of 7.1% mortality at 3 years.The major limitations of our study was the absence of routine angiographic follow up and a scarce use of intravascular imaging for PCI optimization.The mean left ventricle ejection fraction in our study was 46.3\u00a0\u00b1\u00a09.21%, which was higher for a cohort predominantly comprising of an ACS population. The sickest of patients who either died before PCI could be performed or had significant renal dysfunction or sepsis, severe thrombocytopenia or coagulation abnormalities or had a delayed presentation with refractory LVF or refractory cardiogenic shock in whom PCI could not be performed, were not enrolled. One would expect these patients to be having a lower LVEF compared to patients undergoing PCI in our study. As a result, selection bias cannot be ruled out.This study provides 3-year clinical outcome data of Sirolimus-eluting Yukon choice flex stent in a real-world setting and highlights the fact that improved stent design technology will lead to comparable clinical outcomes.In conclusion, the use of YCF sirolimus eluting stent was associated with a favourable safety and efficacy profile at one and three-years of follow-up in a high-risk population.There is no conflict of interest amongst the authors in regards to the present study."}
+{"text": "Better markers of early response to neoadjuvant chemotherapy (NACT) in patients with breast cancer are required to enable the timely identification of non-responders and reduce unnecessary treatment side-effects. Early functional imaging may better predict response to treatment than conventional measures of tumour size. The purpose of this study was to test the hypothesis that the change in tumour blood flow after one cycle of NACT would predict pathological response.In this prospective cohort study, dynamic contrast-enhanced MRI was performed in 35 females with breast cancer before and after one cycle of epirubicin and cyclophosphamide-based NACT (EC90). Estimates of tumour blood flow and tumour volume were compared with pathological response obtained at surgery following completion of NACT.Tumour blood flow at baseline  reduced slightly after one cycle of NACT (0.28 \u00b1 0.18 ml/min/ml). Following treatment 15 patients were identified as pathological responders and 20 as non-responders. There were no relationships found between tumour blood flow and pathological response. Conversely, tumour volume was found to be a good predictor of pathological response  at both baseline (area under the receiver operating characteristic curve 0.80) and after one cycle of NACT (area under the receiver operating characteristic curve 0.81).The change in breast tumour blood flow following one cycle of EC90 did not predict pathological response. Tumour volume may be a better early marker of response with such agents. Typically, a change in tumour size is demonstrated after two or more cycles of NACT2 but several studies have examined functional imaging techniques to assess response after only one cycle.3\u20135 Amongst the functional imaging techniques 18F-FDG PET has been used to assess changes in tumour glucose metabolism following NACT6 and a number of PET studies have looked specifically at breast tumour blood flow with either [15-O] H2O PET7 or dynamic 18F-FDG PET.8 However, repeated PET imaging is impractical for routine clinical use9 and so MRI techniques that measure parameters related to flow, via dynamic contrast-enhanced (DCE) MRI, have also been studied.10 A difference between these MRI approaches and PET imaging of blood flow is the lack of absolute quantification by MRI. Moreover, there has been a lack of consistency in study methodology and the reporting of pathological response in MRI studies over the years.10\u201312Neoadjuvant chemotherapy (NACT) offers an increased rate of breast conserving surgery, can downsize advanced breast tumours to allow surgery and with the addition of human epidermal growth factor receptor 2 (HER2)-targeted therapy (to NACT or adjuvant chemotherapy) can improve survival in HER2-positive breast cancer.13 to measure tumour blood flow at baseline and after one cycle of NACT in order to test the hypothesis that the change in blood flow after one cycle of NACT would predict pathological response as defined according to international recommendations.14 Analysis of the HTR data to estimate blood flow also allowed us to assess related haemodynamic parameters such as blood volume, capillary permeability surface\u2013area product (PS) and interstitial volume and, by using the HSR data, we were able to assess tumour volume.In this study, we used a recently developed interleaved high spatial resolution (HSR)/high temporal resolution (HTR) MRI technique2) and cyclophosphamide (600 mg/m2)] followed by three 3-weekly cycles of docetaxel (100 mg/m2). In patients with HER2 positive tumours, docetaxel was accompanied by trastuzumab, and in some (more recent) cases pertuzumab. Blood samples, taken as standard of care during clinic visits, were used to estimate haematocrit at each MRI visit.In this prospective study consecutive patients, with newly diagnosed primary invasive carcinoma of the breast due to undergo NACT with curative intent, were approached and asked if they were willing to take part. All those agreeing to join the local Research Ethics Committee approved study provided written informed consent after the nature of the procedures had been fully explained. Each underwent a block sequential regimen of NACT: three 3-weekly cycles of EC90  when they were not (assessed using the Shapiro\u2013Wilk test). To determine whether differences existed between the measured parameters, estimates from pR and pNR were compared using the non-parametric Mann\u2013Whitney Between August 2015 and April 2018, 40 female patients  gave written informed consent to enter the study. MRI data were obtained in all 40 patients at baseline (7 [4 - 12] days before the first administration of NACT). However, one patient was unable to tolerate the complete imaging protocol and technical problems in one other (back coil was switched on and off intermittently) precluded the measurement of tumour blood flow. Following a single cycle of EC90, 37 patients were scanned (three patients declined further MRI scans). Technical problems (as previously) affected one further scan, so blood flow estimates were obtained in 36 patients. Paired baseline and cycle one data were available for 35 patients and the results presented below reflect these 35 patients only .The immunohistochemical subtype (IHS) of the 35 tumours were: 2 HER2 enriched (oestrogen receptor and progesterone receptor negative and HER2 positive), 9 triple negative and 24 luminal B (oestrogen receptor and/or progesterone receptor positive and HER2 positive or HER2 negative with high levels of Ki-67). Following the completion of NACT and surgery, pathological analysis revealed 6 patients (17%) had a pathological complete response (RCB-0), 9 patients had minimal residual disease (RCB-I), 12 patients moderate (RCB-II) and 8 patients extensive residual disease (RCB-III). Thus, there were deemed to be 15 pR (43%) and 20 pNR (57%).3 and patients going on to a pR had tumours that were significantly smaller than those that went on to pNR (p = 0.003). There was a small but significant decrease in tumour volume following one cycle of NACT  and tumour volume at baseline and after cycle one were good predictors of pR (area under the ROC curve 0.80 (95% CI 0.66 to 0.95) and 0.81 (95% CI 0.66 to 0.95), respectively). Using a volume threshold at cycle one to predict response failure, 12 patients with tumours of 9 cm3 or larger went on to pNR  and all 15 patients who went on to a pR had tumours smaller than 9 cm3 (100% specificity). This test had a positive predictive value of 100%.Pathological complete response to NACT varied as a function of IHS \u2013 neither of the patients with HER2 enriched tumours, 3/9 (33%) patients with triple negative tumours and 3/24 (12.5%) patients with luminal B tumours had a complete pathological response. Less variation was seen in pR where 1/2 (50%) patients with HER2 enriched tumours showed pR compared to 3/9 (33%) with triple negative tumours and 11/24 (46%) with luminal B tumours. Luminal B tumours that were HER2 positive responded better to NACT: 9/12 (75%) HER2 positive tumours had a pR compared to only 2/12 (17%) HER2 negative tumours. Median enhancing tumour volume at baseline was 4.8 [2.3 to 22.5] cmT1 at baseline (1287 \u00b1 87 ms) showed no change following one cycle of NACT (1285 \u00b1 92 ms) and showed no significant variation as a function of IHS (Tumour blood flow at baseline (0.32 \u00b1 0.17 ml/min/ml) and after cycle one (0.28 \u00b1 0.18 ml/min/ml) showed little variation as a function of IHS  or pathon of IHS .p = 0.007) after one cycle of NACT and this was mirrored in an increase of blood T1 from a baseline value of 1660 \u00b1 64 ms to 1766 \u00b1 109 ms (p = 0.00002) after one cycle of NACT.15Patient haematocrit decreased from a baseline value of 0.41 \u00b1 0.03 to 0.39 \u00b1 0.03  of NACT.7 We did not see this effect in our study following just one cycle of NACT. There was, on average, a decrease in blood flow of 12% but this was subject to considerable variation, with both increases and decreases seen in both pR and pNR (8 who saw a large variability in blood flow response in HER2 negative tumours after one cycle of EC chemotherapy (HER2 positive patients in that study experienced a large decrease in blood flow but they were selectively treated with docetaxel and trastuzumab). The primary hypothesis of this study - that change in blood flow after one cycle would predict pathological response - was not supported by our resultsAt least one previous [15-O] H and pNR . Our res10 was predictive of response. This mirrors results seen in the ACRIN 6657 trial that highlighted the importance of enhancing tumour volume, measured at cycle one, to recurrence-free survival.19 At cycle one of our study, 12 of the 35 tumours were 9 cm3 or larger . These patients could potentially have been spared further NACT since all failed to respond (pNR). Of the remaining 23 patients with tumours smaller than 9 cm3, 15 went on to a pR leaving only 8 non-responding patients. Further work is required to identify a specific imaging phenotype to identify those tumours at the early stages of NACT.Conversely, tumour volume, which was identified as a more reliable marker of response than tumour diameter in a previous review,20 Previous quantitative MRI studies have typically reported parameters such as Ktrans (volume transfer constant) without measuring patient-specific arterial input functions21\u201323 or by measuring the input function inadequately24 . It is well recognised that measurement of the arterial input function is very challenging.11 As in the current study, some reports have highlighted the importance of tumour size after a single cycle of NACT as a predictor of response while tracer kinetic parameters have been less useful21,25; others found neither size nor tracer kinetic parameters useful at this timepoint.22 Although the current study lacks direct validation of the blood flow estimates, the values reported very closely reflect previously published estimates obtained using the \u2018gold-standard\u2019 of [O-15] H2O PET in a similar group of patients.26 Moreover, the lack of variation in tumour blood flow as a function of IHS seen in the PET study26 is similarly seen in our data . Despite the well-established variation in response to NACT of the different IHS of breast cancer, there appears to be very little difference in their haemodynamic characteristics at baseline and no clear patterns of change following one cycle of NACT. The decrease of blood flow seen in responders later in the course of NACT by others7 is not yet apparent after one cycle. Measurements of blood flow may prove to be more revealing when assessing NACT regimens with expected antiangiogenic effects.8This is the first published study to report absolute estimates of breast tumour blood flow and related parameters measured using MRI in response to treatment.T1 measurements plus a few additional DCE acquisitions) and this could be reduced if a faster B1-insensitive T1 measurement sequence were employed. The interleaving of HTR data with the clinical HSR data did not have a significant impact on clinical reporting.13 Perhaps more significant limitations are the small number of patients studied and the post-processing overhead both for tumour volume estimation and tracer kinetic analysis, which currently rely on in-house software. Further work is required to develop user-friendly tailored software for these applications.The additional time required to acquire data for blood flow estimation amounts to less than 10 min  show little variation with tumour IHS at baseline or in response to one cycle of NACT and, contradicting our primary hypothesis, blood flow change following one cycle of NACT did not predict pathological response. In contrast and reflecting the findings of a recent multicentre trial, enhancing tumour volume measured after one cycle of NACT appears to show promise for prediction of pathological response."}
+{"text": "Background and Objectives: The aim of this study was to evaluate longitudinal changes using both upper limb muscle Magnetic Resonance Imaging (MRI) at shoulder, arm and forearm levels and Performance of upper limb (PUL) in ambulant and non-ambulant Duchenne Muscular Dystrophy (DMD) patients. We also wished to define whether baseline muscle MRI could help to predict functional changes after one year. Materials and Methods: Twenty-seven patients had both baseline and 12month muscle MRI and PUL assessments one year later. Results: Ten were ambulant (age range 5\u201316 years), and 17 non ambulant (age range 10\u201330 years). Increased abnormalities equal or more than 1.5 point on muscle MRI at follow up were found on all domains: at shoulder level 12/27 patients (44%), at arm level 4/27 (15%) and at forearm level 6/27 (22%). Lower follow up PUL score were found in 8/27 patients (30%) at shoulder level, in 9/27 patients (33%) at mid-level whereas no functional changes were found at distal level. There was no constant association between baseline MRI scores and follow up PUL scores at arm and forearm levels but at shoulder level patients with moderate impairment on the baseline MRI scores between 16 and 34 had the highest risk of decreased function on PUL over a year. Conclusions: Our results confirmed that the integrated use of functional scales and imaging can help to monitor functional and MRI changes over time. In the last few years, the development of new outcome measures has allowed a better definition of the natural history of Duchenne Muscular Dystrophy (DMD), a progressive, X-linked neuromuscular disorder caused by mutation in the dystrophin gene, affecting one in 3600\u20135000 live male births. The absence of functional dystrophin protein leads to a progressive muscle degeneration, weakness and to a predictable pattern of loss of specific functional milestones ,8,9. While in the past the severity of muscle involvement was mainly assessed by muscle biopsy, over the last two decades there has been increasing attention to the use of non-invasive techniques, such as Magnetic Resonance Imaging (MRI) and spectroscopy (MRS) as markers of muscle pathology and disease progression ,18,19,20The aim of the present study was to evaluate longitudinal changes using both upper limb muscle MRI at shoulder, arm and forearm levels and PUL in ambulant and non-ambulant DMD patients. Furthermore, following evidence from lower limb MRI studies showing that subtle muscle MR changes may precede functional changes in boys with DMD ,29 we alThe study is part of a project aimed at establishing muscle MRI changes in DMD. The study was approved by the Ethics committee of our Institution . Written informed consent was obtained for all the patients who agreed to participate. For the minors, consent was obtained by their parents. As part of this study, we enrolled DMD patients attending their routine follow up clinics between June 2017 and December 2018. The exclusion criteria were related to the impossibility to perform MRI without sedation, therefore excluding very young children or those with severe cognitive or behavioral problems. We also excluded patients with severe joint contractures, pacemakers, respiratory or cardiac problems that would interfere with positioning or performing MRI. The investigations were carried out following the rules of the revised Declaration of Helsinki.Unilateral upper-limb MRI was performed at 1.5T  using a flexible body coil (body SENSE 32 Channel Flex Coil). Subjects lay in the scanner in the head-first supine position, with the dominant upper limb to be imaged lying in a comfortable position on the scanner bed alongside the torso. The upper limb was stabilized using a fabricated thermoplastic splint and sandbags were placed over the forearm and hand to minimize motion. Boys were encouraged to watch a movie or cartoon during scan acquisition and usually a parent and/or a staff member was present in the scan room during scan acquisition. The patients did not receive any sedation and the total examination time was approximately 20 to 30 min.Non contrast-enhanced images were obtained from the dominant upper limb.TSE T1-weighted spin-echo was acquired on axial plane selected in respect to the long axis of the humerus for the shoulder and arm, and in respect of the long axis of the radius for the forearm. The slices were set up to cover the entire extension of the upper limb. Each muscle was evaluated throughout its length.Scan parameters were as follows: TSE T1 .Descriptive analysis was used to identify the muscles that were more frequently affected in the different segments. At shoulder level the muscles examined were: deltoid, supraspinatus, infraspinatus, subscapularis, coraco-brachialis, pectoralis major and minor, teres minor, latissimus dorsi, serratus anterior. At arm level the muscles examined were: biceps brachii, brachialis and triceps brachii. At the forearm level the muscles examined were: supinator, pronator teres, flexor carpi radialis, palmar, flexor digitorum superficialis, flexor carpi ulnaris, flexor digitorum profundus, anconeus, flexor pollicis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor carpi radialis, brachioradialis, extensor pollicis longus.For each segment, we identified the muscles that were more often spared or affected. Although in the present paper we did not aim to quantify the level of involvement, as this will be separately reported, we used a previously reported classification to have a rough estimate of the level of involvement. All muscles MRI scans were assessed for normal or abnormal signal intensity within the different muscles groups and scored using Mercuri classification ,17, as fThree examiners  scored the scans separately with consensus on the scores of over 90%, In the discordant cases the scans were reviewed by all examiners and an agreement was found.The PUL 2.0  includesIn the PUL 2.0 there is a maximum score of 12 for the shoulder level, 17 for the middle level, and 13 for the distal level. A total score can be achieved by adding the three level scores . In order to predict functional changes we reported MRI scores at baseline and PUL scores at follow-up.p < 0.05.Descriptive statistics  were used. T student was used to compare differences in total scores and subtotal scores of each level  between baseline and follow up for both MRI and PUL. The correlation between MRI scores at baseline and PUL scores at follow up for each level was explored with Pearson correlation test. The level of significance was set at Thirty-one patients who had muscle MRI at baseline were enrolled in the study. Twenty-seven of the 31 patients completed the study one year later. Three of the remaining 4 were enrolled in clinical trials and the other one refused to repeat the scan. Ten of the 27 patients were ambulant (age range 5\u201316 years), and 17 non ambulant (age range 10\u201330 years). All the patients were on steroids.p = 0.01) was found between MRI shoulder scores at baseline (between 16 and 34), and PUL shoulder scores at follow up.No significant differences were found at baseline and at follow up for both MRI and PUL in total scores and subtotal scores at each level . No significant correlation was found between baseline MRI total scores, baseline MRI subtotal scores of each level and PUL scores at follow up but for the shoulder level where a moderate correlation ; the total score at follow up ranged between 12.5 and 110 ; MRI changes ranged between 0 and + 14  ; the total score at follow up (1 year later) ranged between 6 and 42 ; PUL changes ranged between \u22129 and +2  (For details At shoulder level 11/27 (41%) patients had stable scores between baseline and 1 year (changes = 0/0.5), 4/27(15%) + 1 point and 12/27 (44%) showed changes equal or more than 1.5.  of patients. Ten of 27 patients (37%) showed a worsening (a higher MRI score than baseline) in at least 2 or more than 2 muscles. The changes were most frequent in the deltoid muscle (10/27) (37%), followed by latissimus dorsi (8/27) (30%). Pectoralis major and minor and coracobrachialis were the muscles that showed less often changes at follow up. Of the 10 patients with baseline MRI shoulder scores lower than 16, five (50%) had higher scores (>1.5) on follow up MRI.Of the 11 patients with baseline MRI scores between 16 and 34, 6 (54%) patients had higher shoulder scores (>1.5) on follow up MRI.Of the 6 patients with baseline MRI scores >34, 1 (17%) had higher scores (>1.5) on follow up MRI.At shoulder level 19/27 (70%) patients had stable scores between baseline and 1 year (changes = 0/\u00b1 1); 5/27 (18%) showed changes between \u22122 and \u22123, and 3/27 (11%) showed changes between \u22124 and \u22125 (For details Ten patients had baseline shoulder MRI scores lower than 16: 9 of the 10 (90%) showed stable and 1 (10%) lower PUL scores.Eleven patients had baseline MRI scores between 16 and 34: 4 of the 11 (36%) had stable and 7 (64%) lower PUL scores.Six patients had baseline MRI scores >34: all 6 had a baseline PUL score of 0 with no possibility to show further loss (For details At arm level 17/27 (63%) patients had stable scores between baseline and 1 year (changes = 0), 6/27 (22%) +1 point, 4/27 (15%) showed changes equal or >1.5.  with a similar proportion of patients with a score of 2 or higher with the brachialis showing the highest number of scores >3.Of the 11 patients with baseline MRI scores equal or lower than 4, 9 (82%) had stable scores on follow up MRI, 2 (18%) had a score of +1 point.Of the 10 patients with baseline MRI scores between 6 and 7.5, 4 of the 10 (40%) had higher scores (>1.5) on follow up MRI.Of the 6 patients with baseline MRI scores >7.5, 5 (83%) had stable scores on follow up MRI, 1 (17%) had a score of +1 point.At Mid-level 17/27 (63%) patients had stable scores between baseline and 1 year (changes = 0/\u00b11), 8/27 (30%) showed changes between \u22122 and \u22123, and 1/27 (4%) with changes below 4 (For details Eleven patients had baseline arm MRI scores lower than 4: 10 of the 11 (91%) had stable and 1 (9%) lower PUL scores.Ten patients had baseline MRI scores between 6 and 7.5: 4 of the 10 (40%) had stable and 6 (54%) lower PUL scores.Six patients had baseline MRI scores >7.5: 3 of the 6 (50%) showed stable, 1 (17%) had + 2 points, and 2 (33%) lower PUL scores  patients had stable scores between baseline and 1 year (changes = 0), 1/27 (4%) + 1 point, 6/27 (22%) showed a changes score above 1.5.  and brachioradialis (52%).Of the 11 patients with baseline MRI scores lower than 6 all (100%) had stable scores on follow up MRI.Of the 11 patients with baseline MRI scores between 6.5 and 36, 4 (36%) had higher scores on follow up MRI.Of the 5 patients with baseline MRI scores more than 36, 2 (40%) had higher scores on follow up MRI (2/5 in supinator and extensors muscles), 1 (2%) had a score of +1.At distal level 26 of the 27 (96%) patients had stable scores at follow up (changes = 0/\u00b1 1), and the remaining one (4%) had changes above 2.  ,22,23,24n = 6), or three (n = 3) domains. MRI changes were more often found at shoulder domain, probably related to the higher number of relatively young ambulant patients in our cohort. Although the numbers were relatively small to allow a meaningful statistical analysis taking into account so many variables, it is of interest that in each domain the changes indicating a more severe impairment mainly occurred in muscles which had mild to moderate impairment (between 19 and 34) at baseline and more rarely occurred in the muscles who were more severely impaired or were completely spared at baseline.The possibility to explore all the domains allowed to define which muscles and domains are at higher risk of becoming progressively involved with increased age. MRI changes of at least two points were found in 15/27 (55%) patients on at least one domain explored, with patients showing changes in two (n = 55%) and an overall mean reduction of 2 points/year, in line with previous studies reporting 12 month changes in larger cohorts [p = 0.01). Smaller functional changes were found at the two ends of the MRI spectrum. In patients with low MRI scores at baseline the low risk of fucntional redution was probably related to the relatively preserved muscles. At the other end of the spectrum, in patients with very high MRI scores indicating severe muscle impairment, the PUL scores were already extremely low at baseline and could therefore not show much further deterioration.The MRI scores at baseline could not always predict the risk of showing functional changes over one year. The PUL total scores showed a decrease in 15/27 (At arm and forearm level MRI did not appear to be equally sensitive to detect PUL changes. These results should however be interpreted with caution because of the limited overall number of patients in our cohort and the relatively low number of non-ambulant patients that may have reduced the possibility to observe more changes in the mid or distal domain. Another limitation of the study is that we only performed a visual analysis and better correlation may have was related to the been obtained by using a quantitative MR approach.Another limitation was that related to the exclusion criteria as MRI cannot be easily perfomed in patients with severe behavioural or cognitive problems or severe contractures.Even with these limitations our results confirm that muscle MRI could represent a reliable biomarker to monitor upper limb muscle involvement also in young DMD patients ,29. Our This information can be useful at the time of designing clinical trials or intervention studies to exclude or select patients or to select the most appropriate imaging protocol. The follow up of these patients and the results of the quantitative analysis, both in progress, are needed to provide more accurate information on longitudinal changes at all levels."}
+{"text": "CHRNA5 is strongly associated with the level of nicotine consumption in humans and manipulation of the expression or function of Chrna5 similarly alters nicotine consumption in rodents. In both humans and rodents, reduced or complete loss of function of Chrna5 leads to increased nicotine consumption. However, the mechanism through which decreased function of Chrna5 increases nicotine intake is not well-understood. Toward a better understanding of how loss of function of Chrna5 increases nicotine consumption, we have initiated efforts to identify genetic modifiers of Chrna5 deletion-dependent oral nicotine consumption in mice. For this, we introgressed the Chrna5 knockout (KO) mutation onto a panel of C57BL/6J-Chr#A/J/NAJ chromosome substitution strains (CSS) and measured oral nicotine consumption in 18 CSS and C57BL/6 (B6) mice homozygous for the Chrna5 KO allele as well as their Chrna5 wild type littermates. As expected, nicotine consumption was significantly increased in Chrna5 KO mice relative to Chrna5 wildtype mice on a B6 background. Among the CSS homozygous for the Chrna5 KO allele, several exhibited altered nicotine consumption relative to B6 Chrna5 KO mice. Sex-independent modifiers were detected in CSS possessing A/J chromosomes 5 and 11 and a male-specific modifier was found on chromosome 15. In all cases nicotine consumption was reduced in the CSS Chrna5 KO mice relative to B6 Chrna5 KO mice and consumption in the CSS KO mice was indistinguishable from their wild type littermates. Nicotine consumption was also reduced in both Chrna5 KO and wildtype CSS mice possessing A/J chromosome 1 and increased in both KO and wild type chromosome 17 CSS relative to KO and wild type B6 mice. These results demonstrate the presence of several genetic modifiers of nicotine consumption in Chrna5 KO mice as well as identify loci that may affect nicotine consumption independent of Chrna5 genotype. Identification of the genes that underlie the altered nicotine consumption may provide novel insight into the mechanism through which Chrna5 deletion increases nicotine consumption and, more generally, a better appreciation of the neurobiology of nicotine intake.The gene  CHRNA5, the gene that codes for the nAChR \u03b15 subunit, nicotine dependence and other nicotine dependence relevant phenotypes (CHRNA5 (rs16969968) exhibited the strongest association with cigarettes per day of all tested SNPs (P = 1.2 \u00d7 10\u2212278) (It has become evident that nicotinic acetylcholine receptors (nAChRs) that contain the \u03b15 subunit play a critical role in the risk for nicotine dependence. Human studies repeatedly have found an association between genetic variants in enotypes \u20137. In faChrna5 in nicotine-related behaviors. For example, a few early studies demonstrated that Chrna5 played a role in sensitivity to the acute effects on nicotine in mice  mice, unlike wildtype controls, did not reduce their responding for i.v. nicotine self-administration as the unit dose increased. At the highest nicotine dose tested, Chrna5 KO mice self-administered five times more nicotine than did their wildtype controls. Similarly, Jackson et al.  often without having a measurable effect on the phenotype itself (Chrna5 KO-dependent nicotine consumption. Chromosome substitution strains (CSS) are a panel of strains that share a common genetic background but differ from one another for a single whole chromosome that comes from a donor strain. A full panel of mouse CSS consists of twenty-two strains, one strain for each of the 19 donor strain autosomes, one each for the donor strain sex chromosomes and one strain harboring the mitochondrial genome from the donor strain  and body weight , respectively. Therefore, the main measure we used to assess nicotine consumption was \u03bcg of nicotine consumed per ml of total fluid drank (\u03bcg/ml) which should minimize any confound caused by strain-dependent differences in total fluid intake and weight.Because the most common measure used to assess oral nicotine intake is dose and dose is dependent upon the amount of fluid intake as well as the weight of the mice, the CSS panel was assessed for whether there was a main effect of strain on either fluid intake or animal body weight across the study. Results indicated main effects of strain on both fluid consumption [Chrna5 genotype impacts nicotine consumption as previously reported (Chrna5 null mutation as well as their wildtype (WT) littermates were tested in an ascending two-bottle choice test for oral nicotine consumption at nicotine concentrations 100, 200, and 300 \u03bcg/ml. Results (Chrna5 genotype on nicotine consumption on a B6 background (P = 0.003) with Chrna5 null mutant mice consuming significantly more nicotine than WT mice. There was no main effect of sex and no genotype by sex interaction. Examination of the entire population of tested CSS mice (P = 5.9 \u00d7 10\u221220) as well as sex (P = 1.6 \u00d7 10\u221210) but no genotype x sex interaction. Consistent with B6 background mice, CSS mice harboring the Chrna5 null mutation, on average, consuming more nicotine than CSS mice that were WT for Chrna5.To confirm that reported , 13, fem Results  confirmeCSS mice  also indChrna5 genotype impacts nicotine intake in the CSS, it was then determined whether individual strain impacted nicotine consumption and/or the effect of Chrna5 genotype on consumption. In order to simplify the phenotype, total \u03bcg/ml of nicotine consumed across the experiment  was used for the analysis. For total \u03bcg/ml of nicotine consumed, main effects of strain (P = 5.02 \u00d7 10\u221235), genotype (P = 1.88 \u00d7 10\u221225) and sex (P = 2.33 \u00d7 10\u221212) as well as a strain x genotype interaction (P = 0.006) were observed  was compared to B6 mice homozygous for the null mutation (B6Chrna5KO) in a within genotype analysis (P = 1.1 \u00d7 10\u221218) and sex (P = 1 \u00d7 10\u22126). Due to the main effect of sex, the CSSChrna5KO strains were individually assessed for the effect of sex on nicotine intake , CSS5Chrna5KO (q < 0.005), and CSS11Chrna5KO (q < 0.005) consumed less nicotine then did B6Chrna5KO mice  and increased relative to B6Chrna5KO for both sexes in CSS17Chrna5KO mice   . IncreasChrna5 null mutation could be specific to the Chrna5 deletion or could be a main effect of the CSS on nicotine consumption . To establish whether the effect of strain on nicotine consumption was specific to mice possessing the Chrna5 null mutation or a general effect on nicotine intake, nicotine consumption was assessed in all of the CSS strains possessing the WT allele of Chrna5 and compared to B6 WT mice (P = 6.7 \u00d7 10\u221213) and sex (P = 1.1 \u00d7 10\u22127). Again, due to the main effect of sex, individual CSS strains were evaluated for significant sex effects (q < 0.05) consumed less nicotine than did B6 WT mice while CSS17 WT (q < 0.05) drank more nicotine than did B6 WT mice  also exhibited a main effect of sex. However, the lack of a significant genotype by sex interaction for any of these strains indicates that the effect of Chrna5 genotype on nicotine consumption did not appreciably differ between female and male animals. For six strains , there was a main effect for both genotype and sex suggesting the possibility that the effect of Chrna5 genotype on nicotine intake might be sex dependent. Among these six strains, four  exhibited modest sex differences in the effect of Chrna5 genotype on intake. Nonetheless, the lack of a genotype by sex interaction suggests no meaningful difference of the effect of Chrna5 genotype on nicotine consumption between female and male mice of these strains.Lastly, CSS were examined individually using a between Chrna5-dependent nicotine consumption as indicated by either a within genotype  and/or between genotype comparison , one strain (CSS17) impacted nicotine intake independent of Chrna5 genotype and one strain (CSS1) altered nicotine intake in both a Chrna5 genotype dependent and independent manner.In summary , amongstChrna5, the gene that encodes the nicotinic acetylcholine receptor subunit \u03b15, significantly increases nicotine consumption  includes the observation that nicotine intake in CSS5Chrna5KO, CSS11Chrna5KO, and male CSS15Chrna5KO mice significantly differs from the reference B6Chrna5KO strain. In all cases, the CSSChrna5KO animals consume less nicotine than B6Chrna5KO mice. In addition, mice that possess the WT allele of Chrna5 in these three strains do not significantly differ from B6 mice harboring the WT allele indicating that the strain effect is specific to the Chrna5 null allele. Further, the effect of Chrna5 genotype on nicotine consumption in CSS5, CSS11, and male CSS15 mice is abolished . Removing CSS9 from this analysis due to potential genetic differences from our CSS9 vs. the JAX CSS9 leads to a significant positive correlation . Using dose (mg/kg/day) as an alternative measure of nicotine consumption in the CSS WT mice led to significant correlations with locomotor activity with  or without  the inclusion of CSS9. In sum, these correlations are suggestive of a genetic overlap between oral nicotine intake and the acute effects of nicotine on locomotor activity with animals less sensitive to the locomotor depressant effects/more sensitive to the locomotor stimulating effects of nicotine consuming greater amounts of nicotine.In the only other study that measured a nicotine response in the C57BL/6J-Chr#and Gill  assessedand Gill  and dataChrna5 genotype in CSS1 mice remains to be determined.Genetic mapping of oral nicotine consumption in mice also has been reported in one previous study . In thisChrna5 deletion on nicotine consumption and one CSS was identified that possesses an allele or alleles that substantially increases nicotine consumption independent of Chrna5 genotype. Future studies with the CSSChrna5KO and CSS WT mice of interest can take advantage of the unique structure of the CSS .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Japanese cedar pollinosis is a major seasonal allergy in Japan, and Japanese cypress pollinosis is a growing concern because the cypress pollen season follows the cedar pollen season and cross-reactivity among allergens occurs between these closely related species. Allergens purified from pollen under unspecified collecting conditions can potentially heterogenous allergens profiles and batch to batch variability, and amino acid sequence variants in allergens possibly exist among trees. Polymorphisms have not been investigated for the cypress pollen major allergens, Cha o 1 and Cha o 2. Our aim was to examine the homogeneity of allergen amino acid sequences. DNA sequences of Cha o 1 and Cha o 2 from pollen collected from Chiba and Ibaraki prefectures and from needles of 47 plus trees located at seed orchards in Chiba Prefecture were examined by amplicon sequencing and amino acid substitutions were deduced. Sequence analysis of the pollen samples revealed that eight and seven residues of Cha o 2 were polymorphic, respectively. Thirteen residues in Cha o 2, including those residues identified in pollen, were deduced to be polymorphic for the plus trees. Cha o 2 expressed by the 47 plus trees included amino acid differences when compared with that of isoallergen Cha o 2.0101. No substitution was deduced in Cha o 1 for pollen taken from the two prefectures. One conservative amino acid substitution was deduced in Cha o 1 for the plus trees. Of the 47 plus trees examined, 38 were deduced to express only the isoallergen Cha o 1.0101 isoform, whereas eight trees were heterozygous and a single tree was homozygous for the non-synonymous mutation, which indicates relative uniformity of Cha o 1. Cha o 2 was found to be a heterogeneous allergen which suggests that studies using pollen from different trees may not give the same results. Cupressus arizonica) and Mediterranean cypress (Cupressus sempervirens) release pollen that cause allergic reactions, and a major allergen from Mediterranean cypress is the most common allergen among 75 allergens found in Italy .\u201331.29\u201331We found 1 and 13 amino acid substitutions in Cha o 1 and Cha o 2, respectively, from plus trees located at nurseries in Chiba Prefecture and pollen collected in Chiba and Ibaraki prefectures. These polymorphisms in Cha o 2 may affect mAb affinity toward Cha o 2 when the antibody against Cha o 2 has been raised against a particular Cha o 2 amino acid sequence where one or more of the 13 substitutions resides in the antibody epitope."}
+{"text": "A novel calcium carbonate cement system that mimics the naturally occurring mineralization process of carbon dioxide to biogenic or geologic calcium carbonate deposits was developed utilizing carbon dioxide-containing flue gas and high-calcium industrial solid waste as raw materials. The calcium carbonate cement reaction is based on the polymorphic transformation from metastable vaterite to aragonite and can achieve >40 MPa compressive strength. Due to its unique properties, the calcium carbonate cement is well suited for building materials applications with controlled factory manufacturing processes that can take advantage of its rapid curing at elevated temperatures and lower density for competitive advantages. Examples of suitable applications are lightweight fiber cement board and aerated concrete. The new cement system described is an environmentally sustainable alternative cement that can be carbon negative, meaning more carbon dioxide is captured during its manufacture than is emitted. The cementing reaction is based on the polymorphic transformation from metastable amorphous CaCO3 and vaterite to aragonite or calcite through a dissolution\u2013reprecipitation process in an aqueous medium. However, limited production scale and mechanical properties (3\u201313 MPa compressive strength) have restricted larger scale applications in the construction industry. Nevertheless, the results obtained by Combes et al. provide promising insights for the development of a nature inspired, synthetic CaCO3 cement system at scale from anthropogenic CO2 with properties suitable for various building materials applications.Inspired by the biological use of CaCOe et al.  and Combe et al. ,16 succe3 cement: CO2, calcium, and alkalinity. CO2 can originate from CO2-containing industrial flue gas, e.g., thermal power plants, chemical plants, and cement kilns. Calcium and alkalinity can come from industrial waste streams that contain both calcium and alkalinity in the form of calcium oxide (CaO), calcium hydroxide (Ca(OH)2), and calcium silicates. Examples of calcium and alkalinity-rich industrial waste streams include: carbide lime sludge, an impure Ca(OH)2 by-product of producing acetylene gas (C2H2) from coal and limestone, slag, a Ca/Mg-rich aluminosilicate glass by-product of iron and steel production, and cement kiln dust, an impure CaCO3/CaO by-product of Portland cement manufacturing that is often recycled in the cement kiln feed [4Cl), ammonium sulfate ((NH4)2SO4), and ammonium nitrate (NH4NO3) have been shown to be effective acids in the production of vaterite from CO2 because they can bring the calcium into solution (Equation (1)), absorb CO2 from a gas stream (Equation (2)), and then be regenerated during the formation of CaCO3 (Equation (3)) [Industrially, three feedstocks are required to produce CaCOiln feed ,18. An aion (3)) ,19,20.23 over calcite and aragonite, precipitation is performed at high reactant concentrations, needed to achieve high supersaturation and kinetic stabilization [2 across the gas\u2013liquid interface, (2) the rate at which CO2 converts into carbonate, and (3) the carbonate\u2013bicarbonate equilibrium. The rate of mass transfer can be increased by increasing the flow rate of gas or by increasing the gas\u2013liquid interface, for example by high shear mixing. Subsequent conversion of CO2 into carbonate can occur through the reaction with water (H2O) or hydroxyl ions (OH\u2212), but because the latter reaction is 107 times faster, higher rates can be achieved by performing the reaction at higher pH. Higher pH also favors higher equilibrium concentrations of carbonate over bicarbonate. Except for high gas flow rates, these strategies also improve the CO2 absorption efficiency.To promote the formation of metastable forms of CaCOlization . The con3 cement system that mimics the naturally occurring mineralization process of CO2 to biogenic or geologic CaCO3 deposits is described [3 cement is produced by capturing CO2-containing industrial flue gas and utilizing a calcium and alkalinity-rich industrial waste stream [2 and energy footprint compared to traditional inorganic binders, such as Portland cement. Furthermore, the CaCO3 cement offers processing and performance advantages in a variety of building materials applications over traditional inorganic binders.Herein, a novel CaCOescribed ,22. The e stream . The res2\u2212 and SO42\u2212, see 2-containing flue gas used was generated from a boiler simulator  burning propane. NH4Cl  was used for solubilizing Ca(OH)2.The carbide lime sludge  contained 25 wt% solids and had silica, alumina, and mixed oxidation state sulfur impurities, e.g., S3 cement, the carbide lime sludge is first solubilized with aqueous NH4Cl then passed through a leaf filter to remove insoluble impurities resulting in an aqueous solution of CaCl2 and ammonia (NH3), see Equation (1). NH3 dissolved in water is in equilibrium with ammonium hydroxide (NH4OH). CO2-containing flue gas (11 vol%) is then contacted with the solution in a three-phase continuous-stirred tank reactor (CSTR) controlled at below 40 \u00b0C (Equation (2)), resulting in the precipitation of CaCO3 cement (Equation (3)). The simulated flue gas was blown from the boiler simulator to the absorber via a 20 cm steel pipe using a 600 cfm roots style blower. Unlike some other carbon capture technologies, the CO2 does not need to be concentrated or compressed to high pressures for storage, which is energy intensive. The CaCO3 cement is then mechanically dewatered by a filter press  and thermally dried in a swirl fluidizer  to a free-flowing powder. The NH4Cl solution is recovered during dewatering and is recycled for the solubilization of additional carbide lime sludge. Insoluble impurities accounted for 7% of the dry mass of the carbide lime sludge and were composed of 70% mixed oxides and 30% carbon, see 3 cement manufacturing process. The CaCO3 cement pilot plant is shown in To produce CaCO3 cement. Measurements were carried out with test samples dispersed in isopropyl alcohol using refractive indexes of 1.378 for isopropyl alcohol and 1.58 for CaCO3.A Horiba LA-950V2 Laser Scattering Particle Size Distribution Analyzer (two light sources: 650 nm wavelength laser and 405 nm wavelength LED) was used to study the particle size distribution of the CaCO3 cement. The loss on ignition (LOI) was determined by measuring the mass loss after heating the samples in an electric muffle furnace  at 950 \u00b0C for 60 min.An ARL QUANT\u2019X Energy Dispersive X-ray Fluorescence Spectrometer  was used to evaluate the chemical composition of the oven-dried raw materials and the CaCO3 cement and the transformed cement pastes. The instrument was operated at 40 kV and 30 mA  using a scan range of 5\u201365\u00b0 2\u03b8, a step size of 0.02\u00b0, and dwell time of 4 s. The resulting spectra were analyzed using Jade 9 software  for qualitative phase composition information and MAUD software for quantitative Rietveld analysis (QXRD) [A X\u2019Pert Pro X-ray Diffractometer  was used to determine the phase composition of the CaCOs (QXRD) .3 cement and the transformed cement paste. The samples were coated with platinum/palladium using a sputter coater  prior to secondary electron imaging.A SU-6600 Field Emission Scanning Electron Microscope (SEM)  was used to collect images of the CaCO3 cement was evaluated at 4 h, 10 h, 1 day, 3 days, 7 days, and 14 days. Paste cubes (50 \u00d7 50 \u00d7 50 mm3) with a solution-to-solid ratio (w/c) of 0.35 were utilized. The solution contained 0.1 M magnesium and strontium chloride. The pastes were mixed according to ASTM C305 and then cast and compressed according to ASTM C109 [Compressive strength development of the CaCOSTM C109 ,26. The 3 cement has a median particle size of 15 with a 4 \u00b5m standard deviation. The D90 and D10 for the CaCO3 cement are 19 and 11 \u00b5m, respectively. The CaCO3 cement. The alumina and silica impurities found in the carbide lime sludge are not found in the resulting cement because they are relatively insoluble impurities that were removed during the filtering process  and NH3 gas. The HCl and NH3 gas are recovered while scrubbing the flue gas. A portion of the HCl gas contacts water on the drying CaCO3, forming hydrochloric acid, and is neutralized by the CaCO3 cement, leaving a residual amount of CaCl2 [3 cement produced is mainly composed of metastable vaterite (>99 wt%) with a minor amount of calcite (<1 wt%), see 3 is deliberately avoided as it is difficult to stabilize and process on an industrial scale. Once the CaCO3 cement is dried, it is stable under ambient conditions. process ; howeverof CaCl2 . The CaC3 cement is the transformation of vaterite (CaCO3) to aragonite (CaCO3) or calcite (CaCO3). Vaterite dissolves and aragonite and/or calcite precipitates. Since the cementing reaction is an aqueous dissolution-reprecipitation process, curing the CaCO3 cement requires high humidity to maintain the transformation of vaterite to aragonite and/or calcite. If the humidity of the curing environment is not controlled, the material will desiccate, and the cementing reaction will cease, similar to Portland cement. Additionally, like Portland cement, the cementing reaction of CaCO3 cement can be accelerated by increasing the curing temperature. 3 cementing reaction and compressive strength development. The ultimate compressive strength of the material is 40 MPa, which is 3\u201313\u00d7 higher than the 3\u201313 MPa compressive strength, previously reported for CaCO3 cement developed for medical applications [3 cement paste does not show strength development through 1 day and takes 14 days to achieve the full strength. By elevating the temperature, full strength can be achieved by 7 or 3 days for 60 and 80 \u00b0C, respectively. Additionally, at elevated temperature, CaCO3 cement pastes have developed enough strength by 4 to 10 h  to be demolded and handled. Fast early strength is important for manufactured building materials with the need to transfer materials between initial and final curing steps, e.g., from pre-curing in molds at lower temperatures to autoclaving in stacks at elevated temperatures.The cementing reaction in CaCOications ,15. At 42, MgSO4, Mg(C2H3O2)2), which are known to inhibit calcite growth [2, Sr(C2H3O2)2), which are known to promote aragonite formation [During the dissolution of vaterite, aragonite is the preferred precipitate over the most thermodynamically stable calcite, as aragonite exhibits an acicular morphology which can lead to an interconnected microstructure. The formation of aragonite can be controlled through the use of magnesium-based additives  and heterogeneous structure. At all curing temperatures, aragonite crystals nucleate on the surface of vaterite particles, then grow outward, which results in a dense and interconnected honeycomb microstructure . This ph3 cement (vaterite) to aragonite in the hardened cement paste. Comparing 3 cement is entirely dependent on the vaterite to aragonite transformation, which is less complex than traditional cements and can be easily accelerated through temperature without negative effects on performance. For instance, at 80 \u00b0C, the CaCO3 cement  has fully transformed to 98% aragonite and 2% calcite by 3 days of curing; whereas, at 40 \u00b0C, the hardened cement paste is composed of 12% vaterite, 87% aragonite and 1% calcite at 14 days of curing.2 on a dry basis with minor impurities such as carbon, silica, alumina, iron, and sulfur species depending on the composition of coal and limestone. Carbide lime sludge is used in China to replace limestone as a raw material for Portland cement production which can reduce CO2 emissions yet increase energy demand depending on the additional energy required to dry the carbide lime [Annual global production of carbide lime sludge was estimated at approximately 25 million tons on a dry basis, where >95% is generated in China . Carbideide lime . However2 could be captured from industrial facilities and approximately 230 million tons of CaCO3 cement could be produced.Iron and steel slag production is, on the contrary, more evenly distributed worldwide. It is estimated that about 510\u2013664 million tons of iron and steel slag were generated annually in 2019 . Iron an3 cement is well suited for building materials applications with controlled factory manufacturing processes that are typically made from inorganic binders, such as Portland cement or gypsum. The CaCO3 cement can provide processing and performance advantages over Portland cement, e.g., shorter production cycles, lighter weight, and flexibility on fillers and reinforcement elements. Furthermore, CaCO3 cement is white, which makes it easy to color and texturize for decorative purposes.With its unique properties, CaCO3 cement is not suitable for structural applications that require mild steel reinforcement but is suited for composite systems development with the flexibility to incorporate a wide range of reinforcement elements for superior toughness and flexural properties. The neutral pore solution of the CaCO3 cement also makes it amenable for use in environmentally sensitive areas and applications that require biocompatibility.Because of its neutral pore solution, CaCO3 cement\u2019s porosity has inherent advantages and disadvantages. The advantage is the ability to formulate lightweight products. For example, suitable potential applications are lightweight fiber cement board and autoclaved aerated concrete due to their manufacture in controlled environments that utilize elevated curing temperatures to accelerate property development. In other applications, the porosity could be a disadvantage, such as concrete in severe freeze\u2013thaw or physical salt attack environments. The durability of CaCO3 cement in a range of situations and applications should be investigated in future research projects.CaCO3 cement manufacturing process  and thermal energy for drying at approximately 130 \u00b0C. Based on pilot plant data, production of CaCO3 cement is calculated to require 2.8 to 3.9 MJ/kg when considering a range of dry and wet raw materials. This compares to the 4.6 to 5.6 MJ/kg energy consumed during Portland cement production, mainly from the calcination of limestone at 900 \u00b0C and the sintering process at 1450 \u00b0C [3 cement from waste feedstocks does not require high temperature processes and can capture and store CO2 from industrial emissions, CaCO3 cement has a significantly lower CO2 footprint compared to Portland cement. The energy and carbon footprint for CaCO3 cement manufacturing will vary for different sites and plant scales due to factors such as electricity mix, fuel sources, and operational efficiency.The CaCO process  requires 1450 \u00b0C . Since p3 cement), the electrical energy consumption is 92 kWh/metric ton of CaCO3 cement, and the natural gas consumption is 3.57 GJ/metric ton of CaCO3 cement. The plant operations that consume the most electricity are carbide lime sludge dissolution and filtration (39%) and CO2 absorption including flue gas compression (38%). 88% of the natural gas is consumed in the drying of the feedstock and product. Using a 2019 California grid carbon intensity of 175 kg CO2/MWh, the electricity results in emissions of 16 kg CO2/metric ton of CaCO3 cement. With an emission factor of 56 kg CO2/GJ, the natural gas combustion produces 200 kg CO2/metric ton of CaCO3 cement [2/metric ton of CaCO3 cement. 3 cement production to the CO2 emitted during Portland cement production. Since every metric ton of CaCO3 cement produced captures 440 kg of CO2 and manufacturing CaCO3 cement releases 216 kg of CO2, CaCO3 cement has a net capture of 224 kg of CO2/metric ton of CaCO3 cement produced. Substituting CaCO3 cement for Portland cement, results in a reduction of 882 kg of CO2/metric ton of cement utilized due to net emissions of 658 kg of CO2 avoided from Portland cement and a net capture of 224 kg of CO2 during the manufacture of CaCO3 cement when utilizing a waste Ca feedstock.For the highest energy consumption case  to heat from 20 to 80 \u00b0C. End of life uses for CaCO3 cement composites would be similar to those of Portland cement composites with the dominant recycling scheme being crushed into aggregate.If curing is conducted at elevated temperature, additional energy input is required to heat the cementitious mixture to the curing temperature. For example, 1 m2 emissions into CaCO3 cement at a pilot scale. The CaCO3 cement gains approximately 40 MPa of strength during its conversion from vaterite to aragonite, which is 3\u00d7 higher than previously reported results. The cement paste hardens and gains strength through the formation of a network of interlocking aragonite needles. The conversion and strength gain can be accelerated by elevated curing temperatures, e.g., full conversion and strength in 3 days at 80 \u00b0C. The CaCO3 cement system is particularly well-suited in applications that can take advantage of the inherent porosity, low pH, or rapid kinetics. The new cement system described is an environmentally sustainable alternative cement that can be carbon negative, meaning more CO2 can be captured during its manufacture than is emitted when utilizing a waste Ca feedstock.We have described a method of converting anthropogenic CO"}
+{"text": "Translational Psychiatry 10.1038/s41398-021-01494-5, published online 05 July 2021Correction to: The original version of this article unfortunately contained a mistake in Table 1. All blank boxes (\u2610) in Table 1 columns 3 and 4 should be replaced with the horizontal solid arrow (\u2192). The remaining dashed horizontal arrow (-->) in the bottom cell of column 3 of Table 1 should be replaced with the horizontal solid arrow (\u2192) as well. We apologize for this error caused by the typesetter. The original article has been corrected."}
+{"text": "Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model. GATA2 is a zinc finger transcription factor expressed widely across a number of tissues including vascular, lymphatic, urogenital, cardiovascular, neural, and hematopoietic systems. It is a master regulator of hematopoiesis with the majority of knockout mice succumbing to death from anemia before embryonic day E10.5 . Its impGermline mutations in GATA2 show phenotypes across all ages. However, are particularly enriched in children and teenagers presenting with myelodysplastic syndrome (MDS) and congenital neutropenia . This hiGATA2 gene termed gata2a and gata2b. Both genes are highly homologous to the human GATA2 gene, particularly in the zinc finger domains where they are 98% and 95% identical at the amino acid level, respectively (gata2a and gata2b show synteny with regions of human Chromosome 3 where GATA2 is located (itga2b:eGFP) zebrafish show that in adult hematopoiesis, both Gata2a and Gata2b are expressed in HSPC in an \u201cor\u201d fashion  stocks were maintained according to standard procedures in UK Home Office approved aquaria (um27gata2a carrying a 10 bp deletion in Gata2a (a gift from Nathan Lawson)7, Tg(lyzC:nfsB-mCherry) (a gift from Stephen Renshaw), la116Tg(kdrl:GFP), la2TgTg(itga2b:GFP) la2Tg .2, 0.33 mM MgSO4) in Petri dishes. Pigment formation was avoided by supplementing E3 medium with phenylthiourea (PTU) (Sigma) 0.2 mM from 24 hpf or removed by bleaching post in situ hybridization by incubating in 5% v/v deionized formamide, 5% v/v H2O2, 0.5x SSC. For live experiments and prior to fixation, embryos were anesthetized with ethyl 3-aminobenzoate methanesulfonate . Embryos and larvae were staged according to Embryos were raised at 28.5\u00b0C in E3 medium  as previously described . SB posiO-dianisidine staining was used to label hemoglobinised erythrocytes in zebrafish larvae as previously described (escribed .la2TgTg(itga2b:GFP) larvae were analyzed by flow cytometry at 3 dpf as described previously , and TOPO cloned into pcDNA3.1V5-His. Forward orientation was confirmed by sequencing. mRNA was synthesized using Ambion mMessage mMachine  Sp6 or T7 ultra kit, respectively.c-myb expression in runx1 in Fixed embryos and larvae were imaged either in 1x PBST or 80% v/v glycerol. Brightfield and epifluorescence images were acquired using a Leica M205 FA fluorescent dissecting stereoscope equipped with a Leica DFC 365 FX camera and LAS 4.0 software. Images were processed using Fiji version v1.49u software . Quantifum27 fish were genotyped by PCR and restriction enzyme (RE) digest using MspA1I, which is abolished by the 10 bp deletion, or Kompetitive allele specific PCR (KASP). Gu5008ata2b genotyping was also conducted using PCR and RE digest with FauI enzyme, which abolished by the deletion, or by KASP. Additional details can be located in Gata2a\u00ae software and similarities assessed using BLOSUM62 matrix. Synteny was assessed using the comparative genomics tools-region comparison on ENSEMBL for each GATA2 gene independently and subsequently for 10 genes upstream and downstream of each.GATA2 (human and zebrafish) protein sequences were aligned using GeneiousTg(itga2b:eGFP); um27/+Gata2a\u00d7 u5008/+Tg(lyzC:mCherry);Gata2b embryos were raised in a single tank and genotyped just prior to analysis to mitigate for any tank specific effects. Adult zebrafish kidneys were dissected from terminally anaesthetized animals, dissociated in PBS with 1% FBS using a gentleMACs tissue dissociator (Milteyni) and analyzed by flow cytometry. Flow analysis was conducted using FlowJoTM.For these experiments a clutch of p < 0.05 and all tests used were two-tailed. Data are presented as mean \u00b1 standard deviation. Data from each independent experiment was normalized to the mean of its wildtype group, and experiments were pooled for analysis. No normality assumptions were made, and data was analyzed by Kruskal-Wallis test with Dunn\u2019s multiple comparisons post hoc test or two-way ANOVA with Sidak\u2019s post hoc test. In um27/um27gata2a embryos scored according to their circulation were tested for equal distribution with contingency Chi-squared test.Statistical analysis was performed using GraphPad Prism version 8.1.2 for OS X software . The probability level for statistical significance was um27gata2a mutant embryos over time. The earliest hemato-vascular progenitors express Lmo2 which is required for expression of downstream erythroid and myeloid progenitors. We analyzed the expression of lmo2 in the anterior lateral plate mesoderm (ALPM) at 10ss (14 hpf) in Gata2aum27 mutants to assess hematopoietic progenitors with myeloid potential. Lmo2 expression in the ALPM was not affected by Gata2a loss indicating Gata2a is not required for anterior hemato-vascular cell specification  around this time, the origin of which is not clearly delineated and thought to be distinct from that of anterior derived macrophages  which emerge around this time, however, we did not assess EMP specifically using other markers. In summary, loss of Gata2a does not impact specification of early hematopoietic progenitors but results in loss of both anterior and posterior cells of the myeloid lineage during primitive hematopoiesis.We next assessed primitive myeloid development in and tail  Scheme fucocytes . l-plastnd trunk . A subserophages . Quantif2aum27/+ . Further mutants . At thisc-myb and runx1. Expression of both runx1 and c-myb was reduced in Gata2a mutants . We further quantified HSPC using epifluorescent imaging of gata2aum27 mutants crossed to Tg(itga2b:GFP) transgenic animals where static GFPlo cells mark HSPC . By contrast GFPlo cells are virtually absent in gata2aum27/um27. We hypothesized that the near absence of stem cells in Gata2aum27 homozygous animals is related to its loss of trunk circulation lo cells  transgenic animals, we noted a proportion of um27/um27Tg(itga2b:GFP); gata2a embryos had GFPlo cells in the CHT and circulating GFPhi thrombocytes  line, a small proportion of um27/um27gata2a mutants show expression of myeloid cells in the CHT suggesting those embryos that escape circulatory defects and develop HSPC that are able to undergo differentiation to mature granulocytes, however, it was not possible to quantify this further in view of the vascular defects.We went on to analyze the effects of Gata2a loss on differentiated myeloid and erythroid cells using SB and observed  quantifim27/um27 . As obseum27/um27 ogata2a-dianisidine stained embryos at 28 hpf . We assessed HSPC numbers further at 72 hpf using the Tg(itga2b:GFP) line crossed to u5008gata2b  cells in the CHT at 60 hpf and 4 dpf. As observed in single heterozygotes, HSPC numbers recover and show no difference between genotypes at this timepoint  as well as in u5008gata2b (in definitive hematopoiesis). Using SB as a readout, we did not observe any difference between the number of granulocytes in +/um27; gata2b+/u5008gata2a double heterozygotes compared to single heterozygotes or WT at 2 dpf  at 12 and 15 months post of age. Blood populations were assess based on forward and side scatter characteristics, with the addition of back-gating GFP and mCherry expressing populations . No chantrophils . Therefo4/i4 (i4 phenotype is a tissue specific knockdown of Gata2a in hematopoietic cells and occurs in homozygous gata2ai4/i4, therefore the level of Gata2a is likely significantly lower than in our mutants that are not viable as homozygotes. Also gata2a i4 mutants have reduced levels of Gata2b, potentially adding additional impact to their phenotype. Thus our data supports the fact that double heterozygous mutants exhibit features of GATA2 deficiency, but that there may be compensatory mechanisms from the other ohnolog that ameliorate some of the phenotypes.We did not, however, observe any leukemias in our double heterozygous mutants, or any overt features of defective lymphatic function as observed in Embergers syndrome. Our phenotype is notably more subtle that that observed in gata2ai4/i4 . We thinWe sought to develop a model of human GATA2 haploinsufficiency using zebrafish to further understand the etiology and spectrum of disease related to effects on developmental hematopoiesis. To do this we proposed analysis of loss of Gata2a and Gata2b alone and in combination in zebrafish would recapitulate a range of phenotypes that may contribute to disease evolution and may further infer structure-function relationships inherent from the sequence similarities and differences between these two highly similar ohnologs. In addition, we hypothesized that we may uncover important redundancies between these and potentially other GATA factors.We showed that both Gata2 genes are syntenic with human chromosome 3, indicating that these genes arose from an ancestral additional round of duplication of this region. The expression pattern of Gata2a and Gata2b genes are disparate with limited overlap during development .um27/um27gata2a mutants have severe vascular defects (um27/um27gata2a homozygotes are capable of recovering and developing HSPCs in a few cases where blood flow was recovered or truncal vasculature remained intact. These observations are consistent with the recovery of HSPC development reported in fish with mutations in a conserved enhancer that drives endothelial expression of Gata2a (u5008/u5008Gata2b mutants have normal vascular development but have a reduction in HSPCs in the aorta from 36 hpf. This defect is no longer evident by 4 dpf in the CHT and notably overall their phenotype is milder than that observed in gata2b morphants (um27/+gata2a heterozygotes, consistently with the role of Gata2a upstream of Gata2b, regulating runx1 and gata2b expression in the hemogenic endothelium (Our studies show that loss of Gata2a or Gata2b result in a reduction of HSPC. Prior studies have shown that  defects  which arf Gata2a . Other rf Gata2a . In our orphants . Interesothelium .um27gata2a mutants shows a role for Gata2a during primitive myelopoiesis while analysis of u5008gata2b mutants show that Gata2b is dispensable for primitive myelopoiesis but is required for definitive myelopoiesis. This phenotype can be observed at 4 dpf and is consistent with decreased myeloid cells in the whole kidney marrow of adult Gata2b mutants (gata2b derived promoter elements that the majority of adult myeloid cells are derived from gata2b+ cells. We propose that the role for Gata2b in myelopoiesis is independent from HSPC production, as experiments on heterozygote +/u5008gata2b fish show normal HSPC numbers as soon as 60 hpf. Additionally, u5008Gata2b mutants exhibit normal definitive erythropoiesis, suggesting that Gata2b is required specifically for granulocyte development. This is consistent with findings of neutropenia in patients carrying GATA2 mutations (Analysis of  mutants . Butko eutations .gata2a is expressed as early as 8ss in the PLM, while gata2b expression is much more spatially restricted and can only be detected from 18ss (Our results show that both Gata2a and Gata2b are required for myeloid and HSPC development, each at different developmental stages. A possible explanation could be the sub-functionalization of these Gata2 ohnologs, by exclusion of territories of expression. Expression analysis shows that rom 18ss . Consistum27/um27 homozygotes to a similar level as Gata2a. Rescue studies in Gata2b morphants conversely could not be rescued by constitutive expression of Gata2a (The differing contributions of Gata2a and Gata2b could also be explained by expression exclusion without substantial changes in protein function. The observed non-synergistic effects on HSPC development in compound heterozygotes suggest that over time, one WT copy of each Gata2 gene is sufficient to compensate the lack of the other. This supports the possibility that the differing phenotypes are a product of expression exclusion rather than different protein function. Further support to this comes from circulation rescue experiments which show that Gata2b overexpression is able to partially rescue blood flow in the trunk of Gata2af Gata2a , althougf Gata2a .um27/+;gata2bu5008/+gata2a animals show defects in the myeloid compartment which we first observe at 12 months of age consistent with features of GATA2 deficiency. However, the range of phenotypes and rate if onset is less florid than an alternative published zebrafish Gata2 disease model (Adult se model , where aIn summary, we describe the sequential and complementary defects resulting from loss of Gata2a and Gata2b in zebrafish during development and in adults, further expanding our understanding of the complex inter-regulation of these GATA factors during hematopoiesis, and their role in developmental hematopoiesis relevant to human GATA2 deficiency.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the UK Home Office.OP designed and performed the experiments, interpreted data, wrote, and edited the manuscript. AL, JR, YH, NK, and HL designed, performed the experiments, and interpreted data. CM analyzed and interpreted the experiments. CU performed the experiments and interpreted data. EP conceived the study, directed the experimental design, provided resources, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The incidence is increasing in elderly, diabetic or immunocompromised patients and can be fatal without \u2018appropriate\u2019 antibiotic therapy.3 This novel study evaluated the use of erythrocyte sedimentation rate (ESR) and pain scores as measures of response to determine the dose and duration of antibiotic therapy in patients diagnosed with NOE over a 4 year period from November 2015.CT, MRI, and technetium 99 bone scans are often performed but poorly correlate to the clinical response to empirical treatment.Patients diagnosed with NOE were evaluated using pain scores (0\u201310/10 where 0\u2009=\u2009no pain and 10\u2009=\u2009worst pain) and ESR at presentation and then at subsequent outpatient visits. Patients received 6 weeks of IV antibiotics  followed by oral ciprofloxacin. Initially ciprofloxacin was prescribed at 750 to 500\u2005mg twice daily and the dose and duration of treatment was personalized according to pain scores and ESR. No ethical approval was required since this was an observational study of standard management of this condition at the Trust.Pseudomonas aeruginosa which in four patients was resistant to gentamicin and in two of these patients also to ciprofloxacin. At presentation pain score was 10/10 in all but three patients (5/10 in one patient and 8/10 in two patients) and ESR values ranged from 31 to 82 (median 66). Both values fell within 1\u20132 weeks of commencing IV antibiotics and after 6 weeks pain scores were 0/10 in eight patients, 3/10 in the others. ESR at this stage fell to 10\u201333 (median 15). One patient developed diarrhoea and stopped ciprofloxacin after only 2 weeks remaining well at 6 months follow-up. Eight patients required 3 to 12 months ciprofloxacin. Four patients required restarting or increasing ciprofloxacin dose to control rising pain scores which reflected an increase in their ESR values but in one of these patients having grown Pseudomonas resistant to ciprofloxacin, two further 6 week courses of piperacillin/tazobactam were required. Three patients currently remain on ciprofloxacin at the lowest dose to keep their pain scores and ESR under controlTwelve patients  aged 49 to 91 years (median 74.5 years) were treated. Five had diabetes mellitus and three had been treated for a malignancy. The time of first symptoms to diagnosis ranged from 3 weeks to 32 months (median 3 months). Six had developed facial nerve palsies; three of whom had additional cranial nerve involvement. All had swabs positive for Pain scores with ESR values are invaluable in determining the activity of NOE thereby personalizing the antibiotic dose and duration in patients with this life-threatening infection."}
+{"text": "RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3\u0394 extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene  GCN5 ortholog acetyltransferase  dependent loci in yeast  methylation pathway, with multiple independent hits in SET1, BRE1, and BRE2. We also show results that known mediators of H3K4 repression are not involved and that the RNA Polymerase III complex remains actively bound at the HMR-tDNA in rpd3\u0394 strains.To address this question in more detail and identify additional potential effectors of this Selected transposon mutant strains isolated in the screen that were further characterized in this study are listed in HMR-ADE2 rpd3 reporter strains DDY3133 and DDY2973  containing 45\u2009\u00b5g/ml adenine  due to increased spreading of silencing that represses the ectopic ADE2 allele at HMR. This sufficient but suboptimal level of adenine is critical to the colony color assay, as lower levels (less than 15\u2009\u00b5g/ml) of adenine activate the ADE2 promoter to overcome silencing and higher levels (over 90\u2009\u00b5g/ml) feedback inhibit the pathway, both leading to white colony growth. Colonies typically were grown for 3 days at 30\u00b0C, then plates were held at 4\u00b0C for 3\u20134\u2009days to obtain optimum pigmentation color prior to photographing on a dissecting microscope. Slow-growing rpd3 hst1 hos2 mutants were grown for 5 days at 25\u00b0C to obtain optimum pigmentation.Parent  DDY2973  were conubstrate . HMR-ADELEU2 marked transposon insertion, and complete cosegregation of Leu+ Kan+ markers with white and Leu\u2212 Kan+ with red colony phenotypes.The yeast transposon mutagenized library was obtained from Mike Snyder (Stanford University), and mutagenesis was performed as described . TransfoSET1, BRE1, and BRE2 was performed by standard yeast genetics procedures by amplifying the LEU2 gene from plasmid pRS405  plates to isolate Leu+ colonies that lost the URA3-marked RPD3 plasmid. These 5-FOARrpd3\u0394 isolates were confirmed for proper SET1, BRE1, or BRE2 gene deletion by PCR on each end (confirmation oligos listed in RPD3 from \u223c240\u2009bp upstream of the ORF to \u223c350\u2009bp downstream with oligos DDO-2155 and DDO-2156 and Q5 polymerase (New England Biolabs), using an RPD3-containing genomic library plasmid (with a 12 kilobase insert) as template. The PCR product was digested with Xho I and Not I then cloned into URA3 vector pRS416 . We previously demonstrated that rpd3 mutants result in spread of silencing beyond the boundary, resulting in repression of ADE2 and growth as red colonies on 15% ade media , as complete loss of silencing  results in a nonmating phenotype. Out of 71 primary white colony isolates, 18 were nonmating. Two randomly selected nonmating isolates were found to have independent transposon insertions within the SIR4 gene (DDY5640 and DDY5641). Transposon insertion sites were determined by the anchor bubble ligation-vectorette PCR method , plus two randomly selected nonmating isolates. Interestingly, most of the identified mutants were in the histone H3K4 methylation pathway, including multiple hits in each BRE1, BRE2, and SET1. An additional insertion in NGG1, which codes for a component of the SAGA coactivator complex resulted in a very light pink and variegated phenotype, suggesting a partial suppression of increased rpd3\u0394 mediated silencing. However, this insertion also resulted in a slow growth phenotype, and it is not clear whether slow growth affects the accumulation of the pigment in this reporter assay. SIR4 gene. Identification of white colony isolates containing insertions within the SIR genes was expected, as they completely abolish HMR silencing. Additional expected insertions were identified in adenine biosynthesis pathway genes, including ADE3, ADE4, ADE5,7, and ADE6, as second adenine pathway mutations are known to suppress the ade2 red colony phenotype .To verify single transposon insertions and cosegregation of the insertion with the white colony phenotype, selected primary isolates were backcrossed to strain DDY814 which contains the rpd3\u0394::KanMX allele grew as red colonies on 15% ade media, and those with both rpd3\u0394::KanMX and Tn:LEU2 insertions in SET1, BRE1, or BRE2 grew as white colonies  maintained the red colony color of the parent strain.As an additional verification of suppression, we directly deleted rpd3\u0394 mediated silencing at HMR and was not simply affecting the ADE2 promoter of the reporter gene or the expression of other ADE pathway genes, we used a silencing-dependent mating assay that relies on the repression of a different promoter , and this phenotype was reversed in a strain containing mutations in both rpd3 and bre2 which became nonmating (DDY5679).As a final verification that mutations in the SET1/COMPASS pathway specifically suppress the increased henotype , DDY282.SET1 pathway has been demonstrated to repress target genes by promoting the recruitment of alternative HDACs, namely Hst3p, or a combination of Hst1p and Hos2p . We also performed Northern blot analysis to assay relative transcription levels of the HMR-tDNA using strains specifically marked with a 19 base pair extension to distinguish HMR-tRNA transcripts from those encoded by the seven other copies of this tRNAThr isoacceptor in S. cerevisiae , consistent with our findings reported here.Enhancement of Sir-protein mediated silencing by loss of SET1 encodes the sole H3K4 methyltransferase activity in S. cerevisiae which is responsible for mono-, di-, and trimethylation of this residue. BRE2 encodes a key structural factor within this complex that is also required for methylation  is a conserved multiprotein complex recruited to chromatin to methylate H3K4 (and possibly other substrates) and contains both Set1p and Bre2p . SET1 enhylation . While ihylation . Interesrpd3\u0394 mediated extended silencing at HMR by mutations that affect the SET1 pathway. rpd3 mutations as suppressors of defective silencing in a catalytically inactive sir2N345A mutant. In that study, restored silencing was mediated by the SIR2 related Sirtuin HDAC Hst3p, as rpd3 hst3 mutants did not suppress the sir2 mutation. We deleted HST3 in our HMR-ADE2 rpd3\u0394 strains, but the resulting strains maintained the red colony phenotype  product of Sirtuin deacetylases which has been implicated in promoting silencing (SIR-mediated silencing can propagate via deacetylation by a nonsirtuin HDAC in the absence of local OAADPR production (HMR. We are currently constructing yeast strains to confirm this potential association by conventional ChIP. Since SIR-mediated silencing in S. cerevisiae is modeled to propagate by sequential Sir2p deacetylation followed by Sir3p and Sir4p binding (HMR-tDNA. The subsequent loss of RPD3 function would then allow Sir2p to promote the propagation of silencing on both sides of the tDNA by attaching to and deacetylating the nucleosomes normally targeted by Rpd3p. Why subsequent spreading of silencing in rpd3 mutants is then dependent on the SET1 pathway remains to be determined.So how does loss of oduction  that weaThese studies are significant to gain a deeper understanding of the interplay and crosstalk of chromatin writers, erasers, and readers  but may All yeast strains and plasmids described in this study are available on request. The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article, its tables and figures and the cited literature."}
+{"text": "A blue shiftof the spectral response of the photoinduced absorbance signal inthe first picosecond after the pump excitation is attributed to thedynamical formation of small polarons with a characteristic time of330 fs. A further important result of our work is that the combineduse of steady-state and ultrafast transient absorption allows us topropose a revised value for the optical gap for ceria (Eog = 4 eV), significantly larger than usually reported.Theultrafast dynamics of excited states in cerium oxide are investigatedto access the early moments of polaron formation, which can influencethe photocatalytic functionality of the material. UV transient absorbancespectra of photoexcited CeO The electrons can be used to promote reductionprocesses while the holes can be employed in oxidations, dependingon whether the catalyst is used as photocathode or photoanode. Theefficiency of these processes strongly depends on carrier lifetimeand mobility.Transitionmetal oxides (TMOs)are candidates for efficient photoelectrochemical catalysts of reactionssuch as water splitting and reduction of CO2, TiO2, and Fe2O3 are good catalysts because of their electronic structure,whichallows transition metal ions to undergo redox cycles quickly and repeatably.However, the use of TMOs in photocatalysis is hampered by the formationof small polarons that affect the lifetime mobility of both chargecarriers.7 A polaron is formed whenthe charge carriers polarize the lattice with the ensuing changesof the charge carrier energy.8 The polaronhas a larger effective mass and smaller mobility than the bare chargecarrier. Polarons are classified into two types depending on the spatialextension of the polarization field: large polarons are spread acrossseveral lattice unit cells while small polarons have the size of asingle or few unit cells. The formation of small polarons has beenobserved in several TMOs such as Fe2O3,9 TiO2,10 NiO,11 and Co3O4.12 The textbook case of the small polaronmodel was proposed to explain electron mobility in partially reducedcerium oxide (CeOx2\u2013).5 In particular, the presence of small polaronsin CeO2 was suggested by the temperature dependence ofthe conductivity of single crystals and supported using the thermopower-conductivityrelation.13 The results were interpretedas being due to an enhanced polarization field following the removalof oxygen atoms from the lattice.16 In this picture, thefunctionality of cerium oxide is linked to the mobility of oxygenions, in turn entangled with carrier mobility via polaron hopping.17CeO6 only recently has theformation of small polaronsin photoexcited states been studied,20 in particular in hemeatite19 and NiO.11 Those works have shown that the small polaronis formed by coupling between photoexcited electrons and the longitudinaloptical (LO) phonons. For Fe2O3, a two-stepprocess was proposed2 that takes placeafter photoexcitation by transferring electron density from oxygento iron atoms. This initial coupling between excited electrons andoptical phonons is followed by the recombination of the phonons withthe hot electrons to form small polarons. The time constant for theprocess was found to be 660 fs in Fe2O319 and 0.3\u20131.7 ps in NiO.11While the transport mechanism of the ground-state polaronis established,2 basedon time-resolved optical absorption measurements. We have taken advantageof the peculiar electronic structure of CeO2, which involvesa valence band composed mainly of oxygen 2p states and a conductionband characterized by the cerium 5d states , we explore the dynamicsof the 4f states after the photoexcitation of CeO2. Weobserved a fast blue shift of the photoinduced transition from Ce4f to the empty Ce 5d band. This behavior is interpreted as a modificationof the band structure induced by small polaron formation  on quartz (SiO2) at room temperatureby evaporating metallic cerium in a partial pressure of oxygen (10\u20136 mbar).26 The film wascharacterized using UV\u2013vis spectrophotometry. The absorbance A was estimated by measuring the fraction of transmittedlight T and of specular reflected light R (A = 1 \u2013 T \u2013 R), neglecting the scattered light. As shown in 2 exhibits a strong absorbance at energies higher than 3.2 eV witha shoulder at about 4 eV (highlighted by the dashed line). The analysisof Ce 3d XPS spectra taken in situ after the MBEdeposition reports a superficial concentration of Ce3+ lowerthan 5%, showing the good stoichiometry of the film (see the We used a 6 nm thick film of CeO see the .A. We probed the system using a visible (2.0\u20133.5eV) or a UV (3.5\u20134.3 eV) supercontinuum. The instrument responsefunction (IRF) has been evaluated in separate experiments to be characterizedby a Gaussian with a FWHM of 70 fs28 , which is far fromthe PB signal, suggests that the PIA slightly shifts to higher energiesin the first picosecond  substrate at roomtemperature by evaporating metallic cerium in a partial pressure ofoxygen (10\u20136 mbar). This procedure, already describedin previous works,26 was used to grow a6 nm film of CeO2 with almost full stoichiometry. The filmthickness was determined by using a cerium evaporation rate measuredby a quartz crystal microbalance. The film stoichiometry was evaluatedby in situ X-ray photoelectron spectroscopy (XPS) by fitting Ce 3dspectra using the procedure proposed by Sk\u00e0la et al.37The cerium oxide film examined in this work was grownby molecularbeam epitaxy (MBE) on a quartz (SiOA by measuring the fraction of transmitted light T and of specular reflected light R (A = 1 \u2013 T \u2013 R), neglecting the scattered light.Steady-state UV\u2013vis spectrophotometrymeasurements wereperformed using a white nonpolarized light source generated by a xenonlamp equipped with an ORIEL-MS257 monochromator and a silicon photodetector(with a 250\u2013750 nm range of detection). We estimated the absorbance 2. In order to generate thewhite light supercontinuum that acts as the probe in the visible range(2.00\u20133.60 eV), a small portion of the amplified fundamental800 nm radiation (\u223c3 \u03bcJ) was focused into a rotatingCaF2 crystal.28 The second harmonicof the amplified fundamental (400 nm) was used to drive the supercontinuumprobe generation in the UV energy range (3.50\u20134.35 eV). Inthe transient absorbance maps presented in this work, the chirp ofthe probe pulse has been corrected. The instrument response function(IRF) has been evaluated in separate experiments to be Gaussian witha FHWM of 70 fs. Further details on the experimental setups are givenelsewhere.27Our setup for the transientabsorption spectroscopy is composedof a femtosecond laser system consisting of a chirped-pulse amplifier seeded by a Ti:Sa oscillator. As a pump,we used a 275 nm (4.5 eV) pulse generated by an optical parametricamplifier seeded by the amplifier. The fluence of the pump pulse wasestimated to be 11 \u03bcJ/cm"}
+{"text": "One-third of menstruating adolescents miss school or sports because of dysmenorrhea.2Features suggestive of secondary dysmenorrhea  should p3Box 1:Onset immediately with menarcheProgressively worsening dysmenorrheaAbnormal bleeding (including irregular bleeding) with painFamily or personal history of renal or other congenital anomalies Midcycle or acyclic painDyspareuniaFamily history of endometriosisNaproxen, ibuprofen and other NSAIDs are equally effective, with a number needed to treat (NNT) of 3 to achieve pain relief in people with primary dysmenorrhea.3Combined oral contraceptives have an NNT of 5 for treating primary dysmenorrhea.7Endometriosis is found in as many as 70% of adolescents who undergo laparoscopy for dysmenorrhea refractory to treatment with NSAIDs and hormonal therapy."}
+{"text": "There is a growing appreciation for individual responses to diet. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed. In this study, we searched for genetic variants underlying differences in the responses to American and ketogenic diets between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains.Fmgq1) , Chr5 at 73.7\u2009Mb  , and Chr7 at 40.5\u2009Mb (Fmgq3) . Analysis of serum HDL cholesterol concentration identified a significant  QTL on Chr1 at 168.6\u2009Mb (Hdlq1). Causal network inference suggests that HDL cholesterol and fat mass gain are both linked to Fmgq1.Genetic mapping of fat and lean mass gain revealed QTLs on Chromosome (Chr) 1 at 191.6\u2009Mb (Fmgq2 and Lmgq1, which are also diet-dependent. Interestingly, Fmgq2 and Fmgq3 affect fat gain directly, while Fmgq1 influences fat gain directly and via an intermediate change in serum cholesterol. These results demonstrate how precision nutrition will be advanced through the integration of genetic variation and sex in physiological responses to diets varied in carbohydrate composition.Strong sex effects were identified at both  Efforts to provide individualized dietary recommendations based on genetic markers have been underway for several years. However, these efforts have largely detected alleles with small-effect sizes that are not clinically actionable \u20133. As wiRecently, our group observed strong mouse strain-specific differences in response to feeding different human-relevant diets \u20136. This To further investigate the genetic origin of differential response to carbohydrate restriction and sensitivity to high-fat diets, we generated an intercross population (F2) between B6 and FVB. All F2s were fed either an American  or a ketogenic  diet and changes to body composition and serum cholesterol concentrations were measured. In this population, we performed genome-wide linkage analysis to elucidate the genetic architecture contributing to differential responses to the specific diets. The data obtained provide evidence for genetic loci that not only directly affect body composition response but also loci that indirectly affect response through differences in serum cholesterol concentration. In addition, significant sex differences in the effects of the identified quantitative trait loci (QTL) were detected.Initially, we screened 6-week-old B6 and FVB mice for their response to American  and ketogenic  diets after a 6-month feeding trial. These strains have previously exhibited significantly different responses to these two diets [For the feeding trials, mice were randomly assigned to one of the two diet groups. Half of the B6, FVB, F1, and F2 mice were placed on American diet  and a half on the ketogenic diet . Researchers were not blinded to diet assignments. All animals were maintained in accordance with Texas A&M University Institution Animal Care and Use Committee guidelines at 22\u2009\u00b0C under a 12-h light cycle. At the end of the feeding trial, mice were euthanized by carbon dioxide asphyxiation, blood was collected, and tissues were harvested and immediately flash-frozen in liquid nitrogen.Echo magnetic resonance spectroscopy (MRI)  was used to measure the fat and lean mass of all individuals. During the initial screen of the B6 and FVB strains, body weight and body composition were measured at a 3-month time point of the 6-month feeding trial. In the F1 population, body weight and body composition were measured at the beginning and end of the 3-month feeding trial. The fat percentage of total body weight was calculated at the 3-month timepoint for B6, FVB, and F1 populations. Fat percentage is the percentage of total body fat mass measured by MRI relative to body weight at the time of the MRI measurement. In the F2 population, body weight and body composition from before and after the 3-month feeding trial allowed for changes in fat and lean mass to be calculated. Fat and lean mass gains were calculated as the difference in fat and lean mass prior to the feeding trial and after 3 months on the assigned diet.In the F2 population, total cholesterol, HDL, and LDL fractions, as well as APOA2 were measured in serum obtained from blood at sacrifice at the end of the feeding trial. Total cholesterol, HDL, and LDL measurements were performed in duplicate using the EnzyChrom AF HDL and LDL/VLDL Assay kit . APOA2 measurements were performed in duplicate in a subset of the F2 population with the highest and lowest serum HDL cholesterol concentration , using the Mouse Apolipoprotein A2 ELISA kit .https://kbroman.org/MUGAarrays/new_annotations.html).The F2 population was genotyped on the Mouse Universal Genotyping Array (MUGA) that includes 7854 SNP markers [Broad-sense heritability for body fat percentage was calculated as the ratio of total genetic variance (variance in the F2 population) to environmental variance (variance in the F1 population).Outliers can have a strong influence on the results of QTL analyses. We observed biologically implausible errors in data that were greater than three standard deviations from the mean and suspect that these reflect the technical errors in the measurements. As such, outliers were defined as individuals with phenotypes that were more than three standard deviations away from the mean for each sex and phenotype in the F2 population. Outliers were set to missing. This procedure was repeated iteratively to discover all outliers in the data. Pearson\u2019s correlation between phenotypes was determined after correcting for sex and diet effects  or highly significant . A LOD drop of 1.5 LOD from the top marker was used to determine the 95% confidence intervals, or support intervals, for each QTL. Linear models using ANOVA was used to check for any interactions between sex and/or diet with the top markers of each QTL. The variance explained by the top markers at each QTL was calculated by dividing the sum of squares of the model including the top marker by the total sum of squares of the model without QTL.The combined model includes all F2s, both sexes, and both diets (y~ sex * diet + marker). QTL peaks with a logarithm of the odds (LOD) greater than thresholds determined by 10,000 permutations were considered genome-wide significant  where P\u2009=\u2009\u03b10.05/(number of tests\u2009\u2013\u2009rank of ith hypothesis +1). A P value <\u20090.05 is considered significant. An initial pathway describing the relationship between T1 and T2 was defined based on the inferred causal networks, and the predicted causal models were compared with the Akaike\u2019s information criterion (AIC) and Bayesian information criterion (BIC). A lower AIC and BIC indicates a better model.For traits with overlapping QTL we made causal inferences in networks based on methods described elsewhere . BrieflyStructural equation modeling was used to illustrate observed and unobserved relationships between the QTL models. The models were refined until all path coefficients were significantly different from zero. Linear models using ANOVA was used to check for the amount of variation explained by each predictor in the structural model. The proportion of variation explained with the predictors modeled was calculated by dividing the sum of squares of the model including the predictor by the total sum of squares without the predictor.B2m was used as a housekeeping gene to correct for starting amounts of cDNA in both gonadal fat and liver. Primers were purchased from Integrated DNA Technologies as custom DNA oligos and sequences are provided in Supplementary Table SRNA from flash-frozen gonadal fat and liver were extracted using the simplyRNA Tissue kit on a Maxwell AS3000 (Promega). cDNA was generated using the Transcriptor First Strand cDNA Synthesis kit (Roche). qPCRs were performed on a LightCycler 480 (Roche) and CFX384 (BioRad) with LightCycler 480 SYBR Green I Master reagent (Roche). P\u2009=\u20090.001, CI\u2009=\u20095.8\u201318.0%; Fig. The fat percentage of B6 males consuming an American diet was 1.77-fold higher compared to B6 males consuming a ketogenic diet , lower lean mass gain than males , and lower serum HDL cholesterol concentration than males .As expected, sex had a profound effect on the phenotypes used for analysis  and had lower serum HDL cholesterol concentration on the ketogenic diet compared to F2s on the American diet .Diet had a significant effect on lean mass gain and serum HDL cholesterol .A significant interaction of sex and diet , Chr5 at 73.7\u2009Mb , and Chr7 at 40.5\u2009Mb   accounts for 3.44% of the variance. Fmgq2 is a highly significant QTL where the FVB allele contributes to lower fat mass gain. The top marker (backupUNC050383757) accounts for 6.3% of the total variance in the F2 population  accounts for 3.4% of the variance in fat mass gain.In the combined analysis that incorporates all F2s on both diets (y~ sex * diet + marker), we detected QTL for fat mass gain on Chr1 at 191.6\u2009Mb  affecting fat mass gain. The interaction effect was specific to males on the ketogenic diet. Males that are homozygous for the B6 allele at Fmgq2 and that carry at least one FVB allele at Fmgq1 gained about 5\u2009g more fat on a ketogenic diet than males homozygous for the B6 allele at both loci   explains 3.3% of the variance in lean mass gain. In males on the ketogenic diet, the top marker (JAX00131700) explains 19.0% of the variation in lean mass gain while it only explaining 1.2% of the variation in females on the ketogenic diet and 8.2% and 4.1% of the variation in males and females on the American diet, respectively.In the combined model that includes male and female F2s on both diets (y~ sex * diet + marker), we detected a significant QTL for lean body mass gain during the 3-month feeding trial (b) Table . Lmgq1 oHdlq1) for serum HDL concentration after the 3-month feeding trial , we detected a highly significant QTL on Chr1 at 168.6\u2009Mb , Ephx1 , Camk1g , and Ppp2r5a   and Cox7b2  , Ephx1 (liver), or Ppp2r5a   and observed no genotype-dependent differences  the presence or absence of carbohydrates in the high-fat diet; (2) the combination of alleles occurring in the study population; and (3) sex. In our experiment, B6 males have a lower percentage of body fat in response to the high fat, no carbohydrate, ketogenic diet, while in contrast, B6 females and FVB males and females do not have a differential response to American and ketogenic diets. This sex-specific response to carbohydrate restriction on ketogenic diet observed in B6 males persists in F1s males, and we observed that the combination of B6 and FVB alleles in the hybrids results in a higher percentage of body fat in response to the feeding trial. The difference in ages at the beginning of the parent and F1 feeding trials might also contribute to the higher percentage of body fat we observed in the F1s. In the F2 population, we observed a very high heritability for fat mass gain in both sexes and were able to identify significant QTLs for fat mass gain at Hdlq1 is significant in both males and females and contains Apoa2. Apoa2 has been described repeatedly for its relationship to serum HDL cholesterol concentration, especially in these two strains [Apoa2 locus affecting serum concentrations of HDL cholesterol was made in an association study of females using an advanced intercross line between B6 and NZB/BINJ [Hdlq1 affects HDL in males and females. We confirmed that FVB alleles at Hdlq1 drive higher concentrations of serum HDL cholesterol and serum APOA2.The QTL for serum HDL cholesterol concentration at  strains \u201314. The NZB/BINJ . Later, NZB/BINJ . B6 carrFmgq1. The close proximity of the Fmgq1 and Hdlq1 QTLs puts them in linkage disequilibrium and this makes their direct and indirect effects difficult to detangle. In general, we would have expected that serum HDL cholesterol concentration would have a negative relationship with fat mass gain given the well-established relationship between serum HDL cholesterol and obesity in humans. It is likely that the two traits are independently linked to Fmgq1 because of the linkage disequilibrium. However, the AIC and BIC scores for the models suggest that differences in serum HDL cholesterol concentration occur upstream of the differences we observe in fat mass gain at Fmgq1.In our causal network of the relationship between fat mass gain and serum HDL cholesterol concentration, we found that serum HDL cholesterol concentration and fat mass gain are independently linked to Fmgq1 and Fmgq2 onto metabolically relevant KEGG pathways revealed candidate genes involved in the biogenesis of cortisol (Hsd11b1), aldosterone (Camk1g), and testosterone (Srd5a3). Cholesterol falls upstream of the production of each of these steroid hormones. Ephx1 resides inside of Fmgq1 and plays a role in the transfer of bile acids to the liver, a process that is critical for the endogenous synthesis of cholesterol. Each of these candidate genes has in common, a relationship with cholesterol. This offers insight into the role these particular genes could play in the causal network we inferred that showed serum HDL cholesterol concentration occurring upstream of fat mass gain. The inferred network might be reflective of the relationship between fat mass gain and one of these candidate genes more so than a direct relationship between serum HDL cholesterol concentration and fat mass gain.Overlaying genes within Fmgq1 and Fmgq2. Our most prominent QTL at Fmgq2 harbors Srd5a3 that converts inactive testosterone to active dihydrotestosterone. We confirmed that F2s that are homozygous for FVB alleles at Fmgq2 express Srd5a3 less than F2s that are homozygous for B6 alleles. At this QTL, FVB alleles also drive lower fat mass gain. This suggests that more active dihydrotestosterone promotes more fat mass gain at Fmgq2. The functional interaction we observed between Fmgq1 and Fmgq2 showed us that male F2s that are homozygous B6 at Fmgq2 and carry at least one FVB allele at Fmgq1 gained the most fat mass. Interestingly, we observed that F2s that carry this interaction have a 1.59-fold increase in expression of Srd5a3 relative to animals that are homozygous B6 at both Fmgq1 and Fmgq2. This provides further evidence that more active dihydrotestosterone promotes more fat mass gain in this population. Ppp2r5a at Fmgq1 has been associated with polycystic ovary syndrome (PCOS) through pathway analysis of genes enriched in genome-wide association studies (GWAS). Ppp2r5a is a member of the oocyte meiosis KEGG pathway that was significantly associated with PCOS, a pathway affected by the hyperandrogenemia that frequently occurs with PCOS [of Ppp2r5a with PCOS makes Srd5a3, and Ppp2r5a primary candidate genes of interest for further investigation.Further, our candidate genes offer insight into the sex-specific interaction in males on the ketogenic diet that we observed between ith PCOS . The assSrd5a3 and active dihydrotestosterone levels. Hsd11b1 at Fmgq1 reversibly converts active cortisol to inactive cortisone. Epidemiological data in humans show that the ratio of androgens to glucocorticoids is critical to metabolic homeostasis and alterations in this ratio can lead to increased incidence of obesity and metabolic syndrome [Camk1g at Fmgq1 is a member of this pathway and people with obesity often experience hyperaldosteronism [Ephx1 at Fmgq1 has previously been associated with other Srd5a isoforms in GWAS of PCOS [Cox7b2 at Fmgq2, has been identified in another GWAS for PCOS [Ephx1 and Cox7b2 and abnormal androgen secretion.Each of the remaining candidate genes has a potential relationship with syndrome . In addisyndrome . Camk1g teronism . Ephx1 a of PCOS , and simfor PCOS . The assHsd11b1 and Srd5a3 does result in noncoding transcript variants that might be of interest in further investigation. These noncoding transcript variants would suggest differences in expression of relevant candidate genes like we observed for Srd5a3. However, we did not observe genotype-dependent differences in expression of Hsd11b1, but did find in the F2 population that mice that are B6/B6 at Fmgq2 and carry FVB/FVB alleles at Fmgq1 express Ppp2r5a 1.61-fold more than F2s that are B6/B6 at both loci.Sequence variation between B6 and FVB result in amino acid changes in EPHX1, CAMK1G, and PPP2R5A, sometimes at multiple locations within the proteins. In HSD11B1 and SRD5A3, no amino acid changes have been documented, but sequence variation in Overall, our data parallel a clinical trial showing a greater fat mass loss among men than women on very low energy, ketogenic diet \u201323. The Fmgq2 and Lmgq1 and serum HDL cholesterol concentration at Hdlq2 and Hdlq3 highlights the importance of sex-specific analyses during the development of individualized dietary guidelines. We identified a known QTL at Hdlq1 for serum HDL cholesterol concentration that harbors Apoa2. Interestingly, the QTLs at Fmgq2 and Fmgq3 affect fat gain directly while Fmgq1 seems to influence fat gain directly as well as via an intermediate physiological change in serum cholesterol related to the strain-specific Apoa2 phenotypes. We have shown that genotypes at Fmgq2 alter the expression of Srd5a3 in a sex- and genotype-specific manner. It remains unclear if these loci explain the differential response to carbohydrate restriction we observed in the initial parent strain screen or if instead, they characterize the increased fat mass gain that we observe after the parental genomes are combined in hybrid and F2 populations. Further investigation of the candidate genes presented here will elucidate their roles in response to carbohydrate restriction in the parent, hybrid, and F2 populations.It is possible that the genetic architecture for fat gain in response to high-fat diets is more complex in females than in males, and as such, the genetic component we are able to detect in our model contributes more to the overall response in males than in females. The observation of these sex-specific QTLs for fat and lean mass gain at Supplementary FiguresSupplementary Tables"}
+{"text": "Inactivation of the tumor suppressor p53 has been generally accepted as a hallmark of tumor. MDM2 and MDMX, the two closely related proteins are considered to be critical for negatively regulating p53 activity through inhibitory binding to and post-translational modification of the p53 protein. We have demonstrated that MDMX facilitates MDM2-mediated p53 ubiquitination and degradation via recruitment of the ubiquitin-conjugating enzyme UbcH5c to the MDM2-MDMX heterooligomers. Here, we discuss our new findings from genetically engineered mouse models and a potential therapeutic strategy. Most p53-activation compounds developed to date offer relatively specific MDM2 inhibition by targeting the p53-binding domain on MDM2, such as nutlin [The p53 stress response is an important regulator of cellular homeostasis, responding to a variety of stressors and eliciting hallmark effects on cell proliferation, apoptosis, and senescence . As suchs nutlin . Howevers nutlin . Howevers nutlin , 10. DruIn vitro experiments have demonstrated a greater affinity for the formation of MDM2-MDMX heterooligomers than either MDM2 or MDMX homooligomers alone [in vitro and in vivo studies.Recent studies from our lab and others\u2019 have demonstrated that MDM2 and MDMX heterooligomerization through their RING domains is essential for p53 control, while the MDM2 E3 ligase function is dispensable for growth and development under unstressed conditions but becomes essential for survival after genotoxic stresses \u201313. Whilrs alone . A propors alone \u201317. Studrs alone . Consistrs alone  and rescrs alone . HoweverER). The ERp53 gene is expressed at physiological levels under control of the native promoter [ER activity can be rapidly switched between WT and knockout states by administration and withdrawal of 4-hydroxytamoxifen (4-OHT). We generated mice expressing inducible ERp53 under various MDM2 and MDMX deletion and mutation backgrounds and studied p53 regulation in mice and MEF cells. Unexpectedly, using the inducible p53ER system we found that in vivo MDMX is essential for p53 degradation, as we found that in the presence of WT MDM2 and absence of MDMX the degradation of p53 is hardly detected [in vivo MDM2 and MDMX can interact with p53 in the absence of each other, but they bind p53 more efficiently as a heterodimer; and that the MDM2-MDMX heterodimerization promotes p53 cytoplasmic localization, where p53 is degraded by the cytoplasmic proteasome. However, we believe the most likely mechanism for MDMX to enable MDM2 E3 ligase activity is its ability to bind UbcH5c and bring it to MDM2 proximity. Previous studies have shown that knockdown UbcH5c in MCF-7 cells increases p53 expression [ER system, we found that MDMX, but not MDM2, interacts with UbcH5c, and that the ability of MDMX to interact with both UbcH5c and MDM2 is essential for MDM2 mediated p53 degradation [A major obstacle of investigating individual functions of MDM2 and MDMX in mice is the early embryonic lethality caused by inactivation of either one while in the presence of p53. To solve this problem we took advantage of a previously developed system , in whicpromoter . The p53detected . Severalpression , suggestradation . We furtradation . Based o"}
+{"text": "Observational pain scales can help identify pain in persons with impaired cognition including dementia who may have difficulty expressing pain verbally. The Pain Assessment in Impaired Cognition-15 (PAIC15) observational pain scale covers 15 important items that are indicative of pain, but it is unclear how likely pain is for persons with each summed score . The goal of our study was to determine sensitivity and specificity of cut offs for probable pain on the PAIC15 against three possible standards. We determined cut offs against (1) self report when able, (2) the established Pain Assessment in Advanced Dementia (PAINAD) cut off of 2, and (3) observer\u2019s overall estimate based on a series of systematic observations. We used data of 238 nursing home residents with dementia who were observed by their physician in training or nursing staff in the context of an evidence-based medicine (EBM) training study, with 137 residents assessed twice. The area under the ROC curve was excellent against the PAINAD cut off (\u25a10.8) at both assessments, but acceptable or less than acceptable for the other two standards. Across standards and criteria for optimal sensitivity and specificity, cut offs at the PAIC15 could be 3 or 4. Guided by self report we recommend PAIC15 scores of 3 and higher to represent probable pain with sensitivity and specificity in the 0.5 to 0.7 range."}
+{"text": "In addition, this study presents a multivariate correlation analysis of experimental parameters. The treatment using RF plasma O2 aimed to increase the hydrophilic character of the raw fabric and adherence of paste-based polymeric on polyvinyl alcohol (PVA) matrix and nickel (Ni), silver (Ag) or copper (Cu) microparticles. The purpose of the research was to develop electroconductive textiles for flexible electrodes, smart materials using a clean technology such as radiofrequency (RF) plasma O2 to obtain a hydrophilic surface with zero wastewater and reduced chemicals and carbon footprint. To achieve the foreseen results, we used advanced functionalization technologies such as RF plasma O2, followed by scraping a thin film of conductive paste-based Ni, Ag or Cu microparticles, and multivariate correlation methods to observe the dependence between parameters involved (dependent and independent variables). Overall, the fabrics treated in plasma with O2 using a kHz or MHz generator and power 100\u2013200 W present an excellent hydrophilic character obtained in 3 min. After RF O2 plasma functionalization, a thin film based on polymeric matrix PVA and Ni microparticles have been deposited on the fabric surface to obtain electroconductive materials.This paper presents a study concerning the preliminary treatments in radiofrequency (RF)oxygen (O Samples A1\u2013A6 have been developed based on 3 weaved structures from 100% cotton yarns: twill 2/2 for samples A1 and A4, twill 3/1 for samples A2 and A5, and plain weave 1/1 with cotton yarns Nm 20/2 on weft for samples A3 and A6.Several raw fabrics presented in 2 to avoid classical cleaning treatments that harm air and water through intensive pollution and high chemical consumption. From 3 \u03a9 and are not dependent on the surface type (hydrophilic or hydrophobic). In the case of the samples A1 and A4 having initial surfaces that are hydrophilic and hydrophobic, respectively, and treated with a conductive paste-based Cu, we can observe that the surface resistances after crosslinking are 1012 \u03a9 and 1010 \u03a9, respectively, which means that sample A1 has an insulator character, and sample A4 has an antistatic character.The purpose of the experiments was to compare the classical alkaline boiling-bleaching followed by conductive paste scraping (samples A1\u2013A3), the classical alkaline boiling-bleaching followed by hydrophobization using NUVA TTC and conductive paste scraping (samples A4\u2013A6) and the conductive paste deposition after functionalization in RF plasma OThe surface morphology was obtained by scan electron microscopy Quanta 200 SEM  using 2000\u00d7 magnification.2 and samples coated with conductive paste-based polymeric matrix and Ni are presented in 2 and RF1 MHz generator and power 200 W in comparison with the sample treated using RF2 kHz generator and power 50 W  microparticles. In -Sample 18 from -Sample 2 from -Sample 10 from The chemical composition was investigated using the ESD (Energy Dispersive Spectroscopy) analysis and elemental mapping overlay for samples 18, 2 and 10  performe1 generator and power 200 W (2 followed by coating with Ni-based paste (2 generator and power 100 W (2 followed by coating with Ni-based paste (2 using RF1 generator and 200 W power) than 2 using RF2 generator and 100 W power).er 200 W  and treaed paste . Figure ed paste  and the ed paste  for samper 100 W  and treaed paste . The proed paste  are becaed paste  on the E2 and 50 W power in comparison with the samples treated in RF plasma using RF1 generator and power 200 W , in frequency (using two different RF generators having the frequencies 13.56 MHz (RF1) and 40 kHz (RF1)).The experiments in RF plasma O2 using the generator RF1 and power 200 W become hydrophilic. In addition, the samples treated in RF plasma O2 using the generator RF2 and power 100 W become hydrophilic.The samples treated in RF plasma O2 using the generator RF2 and power 50 W continued to be hydrophobic.On the other hand, the samples treated in RF plasma OESD and M, Pa, Pv and \u03b4. The correlation coefficients were calculated for all samples preliminarily treated using RF plasma O2 with RF1 generator and power 200 W, RF2 and power 100 W, and RF2 and power 50 W:x, y represents the individual values of the variables x and y;x, y;xs, ys represents the standard deviation of all values x and y.The correlation between surface resistance after free drying or crosslinking (dependent variable) and other independent variables  was investigated by analyzing the correlation coefficient Pearson (1) between R1 and power 200 W, ESD free drying), after free drying at room temperature (18\u201320 \u00b0C) for 20\u201324 h, depending on the independent variables , the correlation coefficients between the RESD free drying and mass (2), air permeability (3), vapor permeability (4) and thickness (5) were calculated.In the case of the fabrics treated in RF plasma using RFhickness , air perhickness , vapor phickness , air perhickness , mass anhickness  and masshickness  to obserESD) after free drying and M, Pa, Pv and \u03b4 (ESD) and air permeability (Pa) and vapor permeability (Pv), it is a direct correlation, and this indicates that a textile fabric having higher values for Pa and Pv surface will generate the increase of surface resistance value. Moreover, between RESD and thickness and mass, it is an insignificant dependence.Analyzing the values of the correlation coefficients between surface resistance  and the black dots represent the appropriate points for RESD free drying depending on mass (M) and thickness (\u03b4).ESD free drying and  and the black dots represent the fitting points for RESD free drying depending on air permeability (Pa) and thickness (\u03b4).ESD free drying and  and the black dots represent the fitting points for RESD free drying depending on vapor permeability (Pv) and thickness (\u03b4).ESD free drying and  and the black dots represent the fitting points for RESD free drying depending on air permeability (Pa) and vapor permeability (Pv).ESD free drying and  and the black dots represent the fitting points for RESD free drying depending on mass (M) and vapor permeability (Pv).ESD free drying and  and the black dots represent the fitting points for RESD free drying depending on mass (M) and air permeability (Pa).1 and power 200 W, ESD after crosslinking), after crosslinking, depending on the independent variables , the correlation coefficients between the RESD crosslinking and mass (6), air permeability (7), vapor permeability (8), and thickness (9) were calculated.In the case of the fabrics treated in RF plasma using RFhickness , air perhickness , vapor phickness , air perhickness , mass anhickness , mass anhickness ) to obseESD) after crosslinking and M, Pa, Pv and \u03b4 (ESD) and vapor permeability (Pv), thickness (\u03b4) and mass (M), the correlation coefficients are negative, and the inverse correlation indicates that in the case of a textile fabric coated with a conductive paste-based Ni, the increase of the values for \u03b4, Pv and M will generate a reduction of the RESD value. However, between RESD and Pa, the correlation coefficient is moderately positive, which means a direct insignificant dependence between these variables.By analysis of the values for correlation coefficients between surface resistance  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on vapor permeability (Pa) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and vapor permeability (Pv).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and vapor permeability (Pv).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and air permeability (Pa).2 and power 100 W, ESD after crosslinking), after crosslinking, depending on the independent variables .In the case of the fabrics treated in RF plasma using RFhickness , air perhickness , vapor phickness , air perhickness , mass anhickness  and masshickness  to obser2 with RF2 generator and power 100 W, the correlation coefficients (10)\u2013(13) have been calculated:In the case of the samples preliminarily treated using RF plasma OESD) and air permeability (Pa), there is a direct correlation, and these variables are in a direct proportionality relationship, and this indicates that the increase of the Pa value generates the increase of the RESD and reduction of the electrical conductivity. In addition, we can observe that between RESD and M, Pv and \u03b4, the correlation coefficients are negative. The inverse correlation indicates that increasing the value of the parameters M, Pv and \u03b4 will reduce surface resistance and increase the conductivity.Analyzing the values of the correlation coefficient ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on vapor permeability (Pv) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and vapor permeability (Pv).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and air permeability (Pa).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and vapor permeability (Pv).In 2 and power 50 W, ESD after crosslinking), after crosslinking, depending on the independent variables .In the case of the fabrics treated in RF plasma using RFhickness , air perhickness , vapor phickness , air perhickness , mass anhickness  and masshickness  to obser2 with RF2 generator and power 50 W, the correlation coefficients have been calculated (14)\u2013(17):In the case of the samples preliminarily treated using RF plasma OESD after crosslinking and M, Pa, Pv and \u03b4  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on vapor permeability (Pv) and thickness (\u03b4).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on air permeability (Pa) and vapor permeability (Pv).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and air permeability (Pa).ESD after crosslinking and  and the black dots represent the fitting points for RESD after crosslinking depending on mass (M) and vapor permeability (Pv).2 using RF1 generator at 13.56 MHz frequency and power 200 W and using RF2 generator at 40 kHz frequency and 100 W power present an excellent hydrophilic character only in 3 min compared to samples treated in RF plasma O2 using the RF2 generator at 40 kHz frequency and power 50 W.In conclusion, the samples treated in RF plasma OMoreover, in the case of samples functionalized in plasma and coated with PVA paste-based Ni particles, it was observed that the samples become conductive after drying at room temperature (18\u201320 \u00b0C) for 20\u201324 h.9 \u03a9 to 1010 \u03a9, and the resurface resistance value is a specific value for antistatic fabrics.In addition, in the samples hydrophobized with NUVA TTC and treated with polymeric paste-based PVA matrix and Cu microparticles, after crosslinking, the surface resistance was increased ten times, from 10ESD after crosslinking and M, \u03b4 and Pv are negative in the case of the samples pretreated in RF plasma O2 using RF1 generator and 200 W power, and this indicates a strong inverse correlation and an inverse proportionality relationship between these variables. However, the correlation coefficient between RESD after crosslinking and Pa is positive, and this shows a direct correlation and direct proportionality between these variables and suggests that increasing the Pa  will generate the increasing of the RESD after crosslinking.Analyzing the correlation coefficients, we can conclude that the correlation coefficients between R2 using RF1 generator and power 200 W and RF2 generator and 100 W power, followed by coating with a thin film based on PVA matrix, organic solvents and Ni microparticles present a good conductivity, reduced surface resistance and can be used for sensors development.The fabrics that are treated in RF plasma OThe development of conductive textiles for flexible electrodes using clean technology such as RF plasma oxygen leads to decreasing the wastewater, chemicals, carbon footprint and pretreatment time."}
+{"text": "Integration has become a major concern for governments, healthcare and aged care systems in many countries. However, the research on and implementation of integrated care in China started relatively late, and there is no review on the needs of older adults with regard to integrated care and the influencing factors. Therefore, this paper aims to provide a scoping review by searching, evaluating, and summarizing the Chinese and international literature on the need for and the factors influencing integrated care for older people. In addition, this review highlights evidence of the gap between China and the world in integrated care.Using a framework proposed by Arksey and O\u2019Malley, a systematic search of 12 domestic and international databases was conducted. Of the 890 original studies retrieved, those that met the established inclusion criteria were screened and scored using the Ekman quality assessment tool. The qualitative description method was used to summarize the demand for integrated care for older adults and the influencing factors.A total of 49 papers were included. These studies were from eleven countries on five continents (most commonly China and the US) and were mostly cross-sectional quantitative studies that surveyed the integrated care needs of older people living in homes/communities or long-term care facilities. The analysis shows that existing research on the integrated care needs of older people in China adopts a single perspective and is inadequate and unsystematic in its assessment; the integrated care needs of older adults and the factors influencing them are multifaceted; and both in China and internationally, the community-home care scenario most consistently meets the needs and expectations of older adults.Although there is no uniform definition of integrated care in China or abroad and each country has its own national definition and system of integrated care, there are certain commonalities regarding the needs of older adults and the factors that influence them across countries. Our research reveals a gap between China and the international community in terms of integrated care. In recent years, the ageing trend has become prominent in China. Compared with European developed countries and the United States, China shows a unique ageing pattern of \u201cAgeing before Affluence\u201d, i.e., the rate of population ageing exceeds the rate of per capita income increase . AdditioYiyang Jiehe\u2019\u2014which means \u201chealth and social care combination\u201d or \u201cmedical and older adults care combination\u201d\u2014has become synonymous with integrated care for older people [5In China, \u2018r people 5. The Ch people 5 and desi people 5.Due to their rapid economic development and the early emergence of ageing problems, some developed countries established an institutionalized, large-scale, and increasingly mature integrated care model for older adults as early as the last century through pilots, practice and extension 8. The moYiyang Jiehe\u2019, which is focused on exploring models of integrated care and their pathways to realization and involves four levels: nursing home, home, community and hospital [2medical-nursing combination\u2019 are people-centred [To achieve the Global Strategy and Plan of Action for Active and Healthy Ageing, the World Health Organization (WHO) published the Integrated Caring for Older People (ICOPE) guidelines in 2017, which aim to provide community, primary and secondary health care providers with evidence-based recommendations for preventing, delaying or reversing physical and mental decline in older people . The WHOospital 2. The WHO-centred . Additio-centred 22.As the WHO-ICOPE has noted, the needs of older adults should be fully understood before providing integrated care services. However, as older people age, their health problems tend to become more chronic and complex, with multimorbidity, the coexistence of multiple chronic conditions, becoming the norm rather than the exception . Older a182627In recent years, research on integrated care in China has focused on the connotations of the model, the existing problems and the realization path, while less attention has been given to the cognition and demand of the older population with respect to integrated care 29. Older30Because the scoping review method is flexible and allows qualitative and quantitative research to be included, a variety of topics can be explored without restriction, such as the needs of patients and caregivers and the determinants of health. It can also be used to identify existing gaps in the literature. These gaps 33 are inThis study adopts the framework proposed by Arksey and O\u2019Malley  and refiThe purpose of this review was noted above. At present, there is no uniform conclusion regarding \u2018integrated care\u2019, either domestically or internationally. For the purposes of this review, we have combined the WHO descriptions  with Leu\u201ca process to overcome fragmentation, based on a person-centred approach that connects the health care system  with other human service systems  to improve outcomes  through different levels and sites of care and service delivery to meet people\u2019s needs\u201d.The review attempts to answer three research questions:In integrated care, what are the specific service needs of older adults?What factors  affect older adults\u2019 needs of for integrated care services?In research and services on integrated care, what are the differences in the needs of older people in China and abroad and their influencing factors? What gaps exist?Published and unpublished (\u2018grey\u2019) literature were identified based on a comprehensive three-step search strategy recommended by the Joanna Briggs Institute (JBI) systematic reviews . In the Table 1 for example search strategies from one database).Chinese search strategy: Five Chinese databases were used, namely, the China National Knowledge Infrastructure (CNKI), Wang Fang Data, VIP Chinese Database (VIP), Chinese Scientific Document Service System (CSCD) and Chinese Social Science Citation Index (CSSCI), were searched. Foreign language search strategy: Seven English databases were used, namely, Web of Science, Elsevier/Science Direct, MEDLINE, PubMed, Springer Link, EBSCO ASP, and The Cochrane Library. A manual search was also performed with Baidu Academic and Google search. We did not limit publication dates or language in the searches. We consulted a scientific librarian for the synonyms and search strategies for each database .Studies were selected through a two-step process using the selection criteria below. Two members of the research team independently reviewed the titles and abstracts of each study according to the literature inclusion criteria to complete the preliminary screening. The two team members then carefully read the full text of the articles and used the exclusion criteria to evaluate and complete the secondary screening. If there were differences of opinion, another researcher on the team was consulted to obtain a consistent final result. In addition, during this review, guided by the PRIMSA statement for systematic review protocols , a flowcThe inclusion criteria were as follows: (1) the needs of older adults for integrated care and the influencing factors were studied, and the relevant research results were summarized; (2) the study involved older adults aged \u226560 years; (3) the service location included in the literature could be any setting, including a medical or health institution, an older adult care institution, a community, or a home; and (4) any type of primary study  was performed.The exclusion criteria were (1) reviews, press releases, commentaries, conference briefs, and science fiction; (2) publications concerning older people\u2019s perceived attitudes (acceptance or lack thereof rather than need) or satisfaction with integrated care; (3) duplicates or literature for which full-text information was unavailable; (4) literature that was less relevant to the content of the study; (5) literature that lacked either of the two themes of demand for or factors influencing integrated care; and (6) literature that was rated as low quality by the Ekman quality assessment tool .Although the scoping review did not have strict requirements for quality control of the literature, this study used the Ekman quality assessment tool to evaluate the quality of the included literature. The tool was initially used to evaluate studies on community health services insurance in low- and middle-income countries  and coveMicrosoft Excel software was used to extract data, and bibliometric methods were applied. The final information included the following three categories: (1) literature information: country, first author, publication year, journal name, journal level, article type, and research method; (2) key content: service venue, integrated care needs and influencing factors; and (3) survey information: survey population and sample size, recruitment and data collection methods, inclusion and exclusion criteria, survey duration, and related information. Charting should be an iterative process in which researchers continually extract data and update the data-charting form .Based on the description of Arksey and O\u2019Malley  and LevaIn this scoping review, we sought information from older adults and their caregivers about integrated care needs and the factors influencing them in older adults. In terms of integrated care needs, we referred to the service content and framework in the \u201cGuidelines for the Management of Integrated Medical Care and Care Institutions \u201d  issued bConsultation and member checking are optional when conducting scoping reviews . HoweverBy applying the search strategy, out of 898 citations retrieved, 110 were duplicates, leaving 788 for title/abstract and full text screening, resulting in 49 articles (24 articles in Chinese and 25 articles in English) included in this review. Of these, 25 are high quality (3 stars) [22540414243444546474849505152535455565758596061563646566676869707172737475767778798081828384The earliest journal article was published by Li  in 2004,2546474955586061626566676971727374757677787981828384404142444552535443574825474955626751536264717375254041424548505557637444594352535625464749555860616265666769717273767778798182838452We also found that the sample size of the included studies ranged from a minimum of 56 people  to a max4241435968592540414243444546485052535455566263646668707374757778824751575859607276808125496165676979After classifying and summarizing the actual integrated care needs of older people in the included literature, 8 categories of needs that make up the integrated care needs of older people emerged: basic life, medical and nursing, rehabilitation, ancillary, psycho-spiritual support, social participation, health education, and welfare and aid.Table 2 for a description of each subcategory).For basic life needs, there are 13 subcategories under this category, mapping 43 articles that cover nearly all the basic needs of older people in their daily lives. Of these needs, self-care, housekeeping and meal preparation were the most commonly identified service needs, covering 31, 25 and 19 articles in that order. Six articles referred to the sensory needs of older people in terms of sight, hearing and communication, while sensory needs and self-mobility were discussed simultaneously in 4 articles. However, both sleep aids and memory were only mentioned in 2 articles. The need for access to medical and nursing needs was a category that emerged most often in the studies reviewed; 17 subcategories and 45 articles were included. Of these, 21 articles explored the home health care needs of older people. Disease diagnosis and treatment (n = 18), regular physical examination (n = 14), skilled nursing (n = 11) and medication (n = 11) are all high-frequency needs. However, both emergency care and skin care were discussed in only 1 article each. In total, 14 articles explored the rehabilitation needs of older people, with 10 additional articles addressing rehabilitation and health care rather than rehabilitation guidance. Within the category of ancillary needs, six sub-categories and 19 articles were found, and a relatively high number of articles explored the four subcategories of transportation (n = 8), shopping (n = 5), older adults care hotline (n = 5), and managing money (n = 5); Among psycho-spiritual support needs, there were a large number of articles on spiritual solace, psychological counselling, and company, representing 12, 15, and 12 articles, respectively. The social participation needs category includes 10 subcategories, with cultural and entertainment service (CES) being the most discussed subcategory with 17 articles included. Among health education needs, both health guidance and health lectures appeared in 5 articles, while the three needs of health guidance, health lectures and health training were mentioned simultaneously in Liu\u2019s  dissertaTable 3).After collating and summarizing the influencing factors obtained through various analytical methods in the included literature, it was found that the factors affecting the integrated care needs of older adults can be divided into six categories: demographic, personal, psychological, family, community, and social. Of these, 33 articles explored demographic factors, and the age (n = 19), gender (n = 11), educational attainment (n = 15), preretirement occupation (n = 7) and marital status (n = 9) of older adults were the main demographic factors that influenced integrated care needs. Among the personal factors, the ones with the highest number of articles were health and economic factors, including 37 articles and 23 articles, respectively. Psychological factors mainly included depression and mental state, and 6 articles were included. Both the family factors and the social factors constitute three subfactors each, with different elements under these subfactors. Family factors were highlighted, as the number of adult children (n = 9) and the living arrangements of older people (n = 9) were discussed more frequently. Studies outside of China identified more caregiver factors (including 3 articles), but only one Chinese study mentioned caregivers . In addiOur scoping review included 49 studies covering the integrated care needs of older people aged 60 and older in 11 countries. We noted a considerable degree of similarity in older adults\u2019 needs and factors influencing across the countries, although there were a few clear differences. The integrated care needs of the older adults in each country comprise eight categories: basic life, medical and nursing, rehabilitation, ancillary, psycho-spiritual support, social participation, health education, and welfare and aid. The factors affecting these needs fall into six categories: demographic, personal, psychological, family, community and social. This review adds an important new perspective to the broader body of knowledge on coordinated person-centred integrated care research . In addiOur review found that many studies outside of China were more multifaceted and holistic in their investigations than those in China. First, in terms of research subjects, several international studies have studied the perspective of older people and their caregivers 43596880,435968556273744857596340535759We also found that foreign studies began to differentiate between met and unmet needs 2552575980 525759At present, empirical research on the need for and willingness to accept integrated care in China is neither up-to-date nor sufficient . The exiIn addition, as China\u2019s integrated care needs assessment of the older population is not yet systematic, resulting in crossover and overlap among medical, nursing, health care and rehabilitation services, many scholars are accustomed to randomly combining these four types of services to conduct research, and there is no unified definition of integrated services . In ChinIn general, basic life care and medical-nursing care are the main focuses of integrated care for older adults. In the category of basic life needs, self-care, housekeeping and meal preparation were the services that emerged most often in the studies included in the review. The main reason for this result is that the deterioration of older adults\u2019 physical functions and decrease in energy make it increasingly difficult for older adults to perform household chores . When fa41424350485940505255497678254042444552542552596343With improved material and economic conditions, the number of people requiring spiritual comfort and psychological counselling has increased . The old5162The factors influencing the need for integrated care in older people are broad and complex, involving several different aspects. Demographic characteristics and personal factors are the main factors influencing the integrated care needs of older people, with family and social factors ranking second, suggesting to some extent that the proportion of older people relying on their children to provide family care has declined. Due to globalization and the pursuit of self-fulfilment, geographical distance and life and work pressures make it difficult for young people to provide financial, emotional and caregiving support for their parents; hence, older Chinese people have changed their caregiving expectations, and the value of traditional filial piety that required absolute obedience from children has changed . Age, ge2Among family factors, older people with fewer children or who live alone show a greater need for integrated care 62, and t5654447Regardless of country or region, as the community and family are the most basic spheres of human life, people prefer to age at home or in the community rather than in an institution 4591. Our45Moreover, our literature search also revealed that there were few studies conducted through \u201cLTC institutions\u201d as care scenarios in China 4772768184,727681A strength of this study is the rigorous and systematic process of the literature search, which was conducted following Arksey and O\u2019Malley\u2019s guidelines . To our The review fulfilled the original objectives, answered the questions posed, and identified and summarized the specific needs of older adults in China and abroad for integrated care and the factors influencing them. There are both similarities and differences in the integrated care needs of older people at home and abroad. We also found a large gap between China and the international community in terms of integrated care and even the overall demand for older adult care services. The current research methods and perspectives on the demand for integrated care among older adults in China are still relatively homogeneous, inadequate and unstructured. The results of this review will not only serve as a reference for domestic and international researchers and policy makers but also clarify the direction of efforts and improvements. In brief, there is a long way to go in providing suitable integrated care for older adults in China.The additional file for this article can be found as follows:10.5334/ijic.5946.s1Appendix 1.Characteristics of the included studies."}
+{"text": "Following the publication of this article , concern\u25cbFig 1D, RKIP panel, between lanes 2 and 3.\u25cbFig 2A DU145 RKIP panel, between lanes 1 and 2, as well as between lanes 2 and 3.\u25cbFig 2A PC3 RKIP panel, between lanes 1 and 2, as well as between lanes 2 and 3.Vertical irregularities suggestive of splice lines were detected in the following panels:In Fig 2D, the PC3 PARP panel appears similar to the first two lanes of the right (DU145) panel, despite representing results obtained from different cell lines.\u25cbThe first two lanes of the middle pY705 STAT3 panel appear similar to the first two lanes of the right pY705 STAT 3 panel. However, the corresponding Actin panel results for these lanes do not appear to match.\u25cbLanes 3 and 4 in the middle panel appear similar to lanes 7 and 8 in the middle panel respectively, despite being used to represent different experimental conditions.\u25cbThe last lane of the middle pY705 STAT3 panel appears similar to the last lane of the right pY705 STAT3 panel, despite being used to represent different experimental conditions.In Fig 4A, the following similarities were detected between lanes presented in the middle panel and lanes presented in the right panel:The authors have not provided the original images underlying the panels of concern.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the integrity of the data, the ELM agreed with the retraction and apologises for the issues with the published article. BB did not agree with the retraction. SY, MD, SC-K, KB, TL, KCY, FA-M, EC, and DC either did not respond directly or could not be reached."}
+{"text": "The large number of pathologies that position CCR5 as a central molecular determinant substantiates the studies aimed at understanding receptor-ligand interactions, as well as the development of compounds that efficiently block this receptor. This perspective focuses on CCR5 antagonism as the preferred landscape for therapeutic intervention, thus the receptor active site occupancy by known antagonists of different origins is overviewed. CCL5 is a natural agonist ligand for CCR5 and an extensively studied scaffold for CCR5 antagonists production through chemokine N-terminus modification. A retrospective 3D modeling analysis on recently developed CCL5 mutants and their contribution to enhanced anti-HIV-1 activity is reported here. These results allow us to prospect the development of conceptually novel amino acid substitutions outside the CCL5 N-terminus hotspot. CCR5 interaction improvement in regions distal to the chemokine N-terminus, as well as the stabilization of the chemokine hydrophobic core are strategies that influence binding affinity and stability beyond the agonist/antagonist dualism. Furthermore, the development of allosteric antagonists topologically remote from the orthosteric site  is an intriguing new avenue in GPCR druggability and thus a conceivable novel direction for CCR5 blockade. Ultimately, the three-dimensional structure elucidation of the interaction between various ligands and CCR5 helps illuminate the active site occupancy and mechanism of action. Staphylococcus aureus infections, cancer and atherosclerosis). In most of these pathologies, CCR5 blockade is a promising therapeutic avenue (S. aureus LukED toxin). Interestingly, both gp120 and LukED pathogenic engagement of CCR5 has been reported not to activate the receptor  (PDB ID: 5UIW)  was usedNext, we modeled on 5UIW the first 15 AA of CCR5 N-terminus from the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated CCR5 N-terminus peptide (PDB ID: 6FGP) . We usedA retrospective structure-guided analysis was conducted to examine the quality and effect the six AA differences between 5P7 CCL5 and CCL 5P12 5M exerted on the full CCR5 model. Structures were visualized and analyzed using the PyMOL software.CCR5 stabilization in an inactive conformation by the use of compounds capable to occupy the active site as antagonists may play a significant therapeutic role in several pathologies Figure\u00a01Among the CCL5 derivatives efficiently blocking CCR5, the most potent HIV-1 entry inhibitors reported to date are CCL5 5P12 5M and CCL5 6P4 5M, a CCR5 antagonist and a superagonist, respectively . Five muThe CCL5 5M derivatives have been generated in the absence of the 3D structural details of the CCL5:CCR5 interaction interface, and were the result of previous findings derived from CCL5 short peptides with anti-HIV-1 activity . The cryThe CCR5 antagonist 5P12 CCL5 has been selected as lead compound for drug development , and, asFurther inspection of the CCR5 binding cavity and the extensive occupancy by 5P7 CCL5 allowed the identification of at least two new positions amenable of amino acid substitution. These substitutions have been tested by the SWISS-MODEL generation of 3D models of the CCL5 mutants that indicated a likely further enhancement in receptor occupancy and therefore a possible increase in the potency of the resulting CCL5 mutants (unpublished).The analysis presented here points to the possibility to improve CCL5 in its interaction with CCR5 by modifying residues distal to the chemokine N-terminus, either by direct receptor affinity increase or by stabilization of the chemokine hydrophobic core.i1 protein, as well as a constitutively activated CCR5 coupled to Gi1, and the 6P4 CCL5:CCR5 complex also in presence of Gi1 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Deltapapillomavirus DNA, the E6 oncoprotein has an important role in cell proliferation and might be related to cancer initiation and promotion. The aim of this study was to present a standardized SYBR Green-based quantitative PCR for detection and quantification of the bovine papillomavirus 2 and 13 E6 oncogenes in urinary bladder samples from cattle. Twenty-four urinary bladders from cattle displaying tumors (n = 12) and normal bladder mucosa (n = 12) were tested by quantitative PCR. Of the 12 urinary bladders with tumors, six presented bovine papillomavirus 2 DNA concentrations ranging from 1.05 \u00d7 104 to 9.53 \u00d7 103 copies/\u03bcL, while two had bovine papillomavirus 13 DNA amplified at concentrations of 1.30 \u00d7 104 to 1.23 \u00d7 104 copies/\u03bcL. The healthy bladder mucosa samples were negative for both bovine papillomaviruses. Once the results were confirmed by conventional PCR and direct sequencing, the quantitative PCR assay developed in this study was shown to be a sensitive and specific tool for detecting and quantifying the E6 ORF of bovine papillomavirus 2 and 13 in a variety of clinical samples. Our findings of identification of bovine papillomavirus 2 and 13 DNA in urothelial tumors from cattle suffering from bovine enzootic hematuria agree with data from previous studies, representing the first detection of bovine papillomavirus 13 DNA in malignant bladder lesions of cattle from Brazil.Bovine papillomavirus types 2 and 13 can induce tumors in both the cutaneous and mucosal epithelia of cattle. These viral types are associated with the development of benign cutaneous papillomas and malignant lesions in the urinary bladders of cattle, with the latter being known as bovine enzootic hematuria. Among the viral oncoproteins encoded by  Pteridium aquilinum) ingestion, which contains mutagenic and carcinogenic principles as quercetin and ptaquiloside. These compounds trigger viral gene expression that results in cell transformation, initiating the progression to malignancy (Bovine papillomaviruses (BPVs) cause benign hyperproliferative lesions of the cutaneous and mucosal epithelia, leading to distressing animal diseases, and considerable economic losses . Bovine lignancy , 3. CasePapillomaviridae family within five different genera: Deltapapillomavirus groups BPVs types 1, 2, 13, and 14; BPVs types 3, 4, 6, 9, 10, 11, 12, 15, 17, 20, 23, 24, 26, and 28 in Xipapillomavirus; BPVs 5, 8, and 25 belong to the Epsilonpapillomavirus genus; BPVs 16, 18, and 22 are classified in Dyokappapapillomavirus; and BPV7, which is the only member of the Dyoxipapillomavirus genus. BPVs 19, 21, and 27 remain unclassified . The total DNA was extracted from fragments of tumors or normal bladder tissues weighing 25 mg by using a Qiagen DNeasy Blood and Tissue kit .To detect the BPV2 and 13 DNA, the nucleotide sequences of the moderately variable E6 gene of these viral types were selected to design two sets of specific primers using Primer Blast software. These primer pairs were able to amplify approximately 120 bp of the E6 ORF . Two pos\u2122 4-TOPO\u00ae vector and transformed into Escherichia coli TOP10 according to the manufacturer's instructions . The recombinant plasmids were extracted using a PureLink Quick Plasmid Miniprep Kit (Invitrogen Life Technologies). To confirm the presence of the E6 gene fragment in the plasmids, direct nucleotide sequencing was performed by using a BigDye Terminator v.3.1 Cycle Sequencing Kit  with the corresponding forward and reverse primers in a 3500 Genetic Analyzer (Applied Biosystems). The recombinant plasmids with E6 fragments were quantified using a Quant-iT dsDNA BR Assay Kit in a Qubit Fluorometer (Invitrogen Life Technologies).The E6 gene fragments of each BPV type were cloned using a pCRTo quantify the E6 genes in BPVs 2 and 13, two qPCR assays were performed using 10 \u03bcL of Platinum SYBR Green qPCR SuperMix (Invitrogen Life Technologies), 20 \u03bcM of each primer, and 0.1 \u03bcL of ROX, in a final volume of 20 \u03bcL in 7500 Fast Real-Time PCR (Applied Biosystems). The following thermal profile was used: polymerase activation at 95\u00b0C for 10 min followed by 40 amplification cycles of 10 s at 95\u00b0C and 1 min at 60\u00b0C each .6 down to 1.14 \u00d7 101 DNA copies/\u03bcL and the BPV13 plasmid containing 1.54 \u00d7 106 down to 1.54 \u00d7 101 DNA copies/\u03bcL were analyzed by conventional PCR. The same dilutions were tested in duplicate three different times to evaluate the coefficients of variation (CVs) of the qPCR. The intra- and interassay CVs for the quantification cycle (Cq) values were calculated. The LOD of the qPCR performed using SYBR Green was compared to the conventional PCR assay using the same primer sets. To test the specificity of both PCR assays, the BPV2 and 13 standard curves were run under optimal assay conditions using appropriate primers (specific BPV2 primers in BPV13 samples and specific BPV13 primers in BPV2 samples). A negative control was also included in all the assays.To determine the limit of detection (LOD) of the assay, six 10-fold serial dilutions of the BPV2 plasmid containing 1.14 \u00d7 10To screen the tissue samples, a conventional PCR was performed that targeted a common sequence of the E5 L2 open reading frames (ORFs) . PCR proR2) of 0.999 (slope = \u22123.488) between the Cq value and the logarithm of the BPV2 copy number (2) of 0.998 (slope = \u22123.698) between the Cq value and the logarithm of the BPV13 copy number (Six 10-fold serial dilutions of BPV2 and 13 plasmids were used to generate a standard curve by plotting the logarithm of the plasmid copy number against the measured Cq values . The BPVy number . The BPVy number . The eff6 down to 1.14 \u00d7 101 DNA copies/\u03bcL of BPV2 and 1.54 \u00d7 106 down to 1.54 \u00d7 101 DNA copies/\u03bcL of BPV13. A quantitative analysis of the conventional PCR identified LODs of ~114 and 154 copies of BPV2 and 13 DNA, respectively. The qPCR identified 11.4 and 15.4 copies of BPV2 and 13 DNA, respectively.The LOD of the conventional PCR and qPCR employing primer pairs designed to detect the E6 oncogene were evaluated by testing six 10-fold serial dilutions of the DNA standards, which contained 1.14 \u00d7 10The positive controls for both viruses were detected using the qPCR assays and the conventional PCRs employing the same primer pairs targeting the BPV E6 oncogene. No cross-reaction was observed when primers specific for the BPV2 E6 gene were tested against BPV13 DNA or vice versa. The reproducibility was analyzed using six 10-fold serial dilutions of a standard for BPVs 2 and 13. The BPV2 qPCR intra-assay CVs ranged from 0.065 to 1.077%, while the interassay CVs ranged from 3.81 to 9.99% . The BPVThrough PCR using the E5L2 primers, amplicons of the expected length, measuring ~250 bp, were amplified from 8 out of 12 bladder tumor samples. The direct sequencing of the resulting E5L2 products demonstrated the presence of BPV2 in six bladder tumor samples and the identification of BPV13 DNA in two samples of bladder tumors. The nucleotide sequence identity for the E5L2 fragments varied from 98.6 to 100% for BPV2 and 99.8 to 100% for BPV13.4 to 9.53 \u00d7 103 copies/\u03bcL, and two bladder samples presented BPV13 DNA at a concentration of 1.30 \u00d7 104 to 1.23 \u00d7 104 copies/\u03bcL. The 12 healthy bladder mucosa samples were negative for both BPVs. Nucleotide sequence analyses of the ~120 bp amplicons confirmed the BPV types in the amplified products.In the analyses of the clinical samples by SYBR Green qPCR as developed in this study, six out of 12 tumor samples presented BPV2 DNA at concentrations ranging from 1.05 \u00d7 10In this study, a new real-time SYBR Green-based qPCR system was developed and tested for the detection and quantification of the E6 ORF of BPV types 2 and 13.The qPCR strategy developed herein presented high sensitivity, specificity, reproducibility, and efficiency for the qualitative and quantitative detection of the E6 oncogene of BPVs 2 and 13 in clinical specimens from affected animals, despite the close phylogenetic relationship among these BPV types . Once thDeltapapillomavirus genus of the Papillomaviridae family, are involved in the benign growth of the cutaneous epithelium as well as in malignant lesions present in the mucosa of cattle. Among Delta-PVs, BPV2 is the viral type most frequently associated with the carcinogenesis of the urinary bladder of cattle who feed on bracken ferns (BPVs 2 and 13, which are classified as members of the en ferns , 12, 16.en ferns . DespiteIn Brazil, where cattle breeding is hampered in several regions due to the endemicity of BEH, previous investigations on the participation of BPV in the etiology of urinary bladder cancer confirmed the presence of BPV2 DNA through conventional specific PCR assays , 20. In Deltapapillomavirus genus, was detected in urothelial tumors from cattle from Italy, mostly accompanied by BPV2 and/or BPV13 DNA in multiple infections, being the BPV14 DNA identified in a single infection in only a tumor sample (In this study, four out of 12 malignant lesions of the urinary bladder, histologically confirmed as carcinomas, tested negative for BPV2 and BPV13 DNA in the novel qPCR assay. In a recent molecular investigation of 39 urothelial tumors from cattle suffering from BEH from southern Italy, for the presence of BPV2 and BPV13 DNA through conventional PCR techniques, five carcinomas also tested negative for both viral types . Once urr sample . Howeverr sample .To the authors, the main limitation of this study was the restricted number of bovine urothelial tumors examined by the novel qPCR system which hampered the elucidation of the frequency of naturally occurring malignant bladder tumors presenting single or multiple infections by BPVs types 2 and 13 in cattle presenting BEH from southern Brazil. However, further investigations aiming to evaluate a larger number of samples from urothelial tumors and healthy urinary bladders for the presence of BPV 2 and BPV13 DNA are needed to be performed in cattle from endemic areas for BEH in Brazil.In conclusion, the new real-time quantitative PCR assay developed to detect and quantify the E6 oncogene in BPVs 2 and 13 may be a useful molecular tool for diagnostic purposes in clinical cattle samples, allowing for epidemiological investigations on the prevalence of these BPV types in diverse geographical areas as well as their association with different clinical outcomes. This assay thus contributes to studies on the pathogenesis of BPV and the consequent development of preventive and curative strategies to control important diseases due to BPV infections that cause severe economic losses in the cattle industry.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because the biological samples employed in the study were collected from slaughtered cattle from a private abattoir.BKA carried out most of the experiments, with the collaboration of AMDA. BKA and ML analyzed the data and wrote the manuscript. AAA and AFA procured funding and supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Copper plays a key role in cancer metastasis, which is the most common cause of cancer death. Copper depletion treatment with tetrathiomolybdate (TM) improved disease-free survival in breast cancer patients with high risk of recurrence in a phase II clinical trial. Because the copper metallochaperone ATOX1 was recently reported to drive breast cancer cell migration and breast cancer migration is a critical factor in metastasis, we tested if ATOX1 expression levels in primary tumor tissue could predict the TM treatment outcome of breast cancer patients at high risk of recurrence. We performed ATOX1 immunohistochemical staining of breast tumor material (before TM treatment) of 47 patients enrolled in the phase II TM clinical trial and evaluated ATOX1 expression levels in relation with patient outcome after TM treatment. Our results show that higher ATOX1 levels in the tumor cell cytoplasm correlate with a trend towards better event-free survival after TM treatment for triple-negative breast cancer patients and patients at stage III of disease. In conclusion, ATOX1 may be a potential predictive biomarker for TM treatment of breast cancer patients at high risk of recurrence and should be tested in a larger cohort of patients. Copper levels are increased in serum and tumor tissue of cancer patients, and copper is required for at least three cancer hallmarks: proliferative immortality, angiogenesis and metastasis . Since fThe copper metallochaperone antioxidant protein 1 (ATOX1) is upregulated in breast, colorectal, uterus and liver tumors, while downregulated in bile duct and pancreatic tumors . ATOX1 rA recent phase II clinical trial tested the therapeutic efficacy of adjuvant tetrathiomolybdate (TM) following chemotherapy in triple-negative breast cancer (TNBC) patients, who showed no evidence of disease (NED), but were at high risk of recurrence. TNBC is characterized by its aggressive nature and lack of targeted treatment leading to a high prevalence of metastatic disease and death, respectively ,8. The pGiven that the reliance on copper trafficking in metastasis might vary across the different molecular subtypes of breast cancer and subsequently influence the impact of copper depletion as a therapeutic strategy, we sought to evaluate ATOX1 as a predictive factor for TM treatment outcome of breast cancer patients at high risk of recurrence using tumor tissue sections and follow-up data from the phase II clinical trial described above.Patients with high risk of recurrent breast cancer were enrolled onto a phase II clinical TM study NCT00195091) between 2007 and 2014. The study was approved by the Institutional Review Boards of Weill Cornell Medicine (IRB 0309006307) and Memorial Sloan Kettering Cancer Center (IRB 21-470). Additional details on the clinical trial design have been reported previously [5091 betwThe ATOX1 immunostaining of breast tumor tissue sections was performed as described previously . ShortlyLog-rank test was used to examine the relationship of ATOX1 staining with the relative probability for EFS. Survival curves were computed according to the Kaplan\u2013Meier method in the SPSS software.ATOX1 expression was evaluated in whole breast tumor tissue from breast cancer patients enrolled in the phase II clinical trial of TM using immunohistochemical staining. Visual inspection of the stained tumor sections indicated ATOX1 expression to vary between different tissue sections from negative to strong staining intensities and to be homogeneous within each tissue section, with comparable ATOX1 staining intensity in inner tumor and tumor border. A more detailed look showed ATOX1 staining in both nucleus and cytoplasm of different cell types, including epithelial cells, fibroblasts and immune cells.We performed a semi-quantitative evaluation of ATOX1 staining intensities in the tumor cells only, considering both nucleus and cytoplasm, using intensity scores ranging from 0 to 3 representing negative, weak, intermediate and high staining intensities, respectively. In addition, we scored the percentage of tumor cells with the strongest ATOX1 staining intensity as a measure for tumor heterogeneity and found a high level of tumor homogeneity, with an average of 79% and 82% considering nuclear and cytoplasmatic ATOX1 levels, respectively. Illustrations for ATOX1 IHC staining and ATOX1 intensity scores are depicted in For analysis, we divided the scoring data into two groups: low ATOX1, which includes negative and weak staining intensities , and high ATOX1, which includes intermediate and strong staining intensities . high = 16 out of 16 (100%)) followed by Luminal (nhigh = 16 out of 18 (88%)) and TNBC subtypes (nhigh = 17 out of 23 (74%)), and we found a similar prevalence of high nuclear ATOX1 levels in the tumor tissue from patients with Luminal, HER2 and TNBC subtypes of disease (nhigh = 12 out of 18 (67%), nhigh = 4 out of 6 (67%) and nhigh = 15 out of 23 (65%), respectively) (high = 19 out of 23 (82.6%)) and IV (nhigh = 19 out of 21 (90.5%)) versus stage II (nhigh = 1 out of 3 (33.3%)) and a similar prevalence of high nuclear ATOX1 levels for patients with disease stages II, III and IV (nhigh = 2 out of 3 (66%)), nhigh = 16 out of 23 (69%) and nhigh = 13 out of 21 (62%), respectively)  A,B. Compctively) C,D.To understand if ATOX1 levels in the cancer tissue relate to TM treatment outcome, we evaluated the correlation between ATOX1 expression levels and EFS in the patients. We found that high ATOX1 levels in the tumor cell cytoplasm, but not ATOX1 in the nucleus, correlated with a trend towards better EFS when considering all breast cancer subtypes  A,B.p = 0.45) (p = 0.018) (n = 3).Deeper investigation of the correlation between ATOX1 expression levels and patient outcome after TM treatment for the different subtypes separately demonstrated that this trend of better EFS for high cytoplasmatic ATOX1 was evident for the TNBC patient cohort, with an EFS of 82% for high ATOX1 versus 67% for low ATOX1 ( = 0.45) A. Howeve= 0.018) B. There Copper depletion with TM is a promising treatment for breast cancer patients. A first phase II clinical trial for TM treatment of breast cancer patients with high risk of recurrence demonstrated an EFS of 71.4% at a median follow-up of 10.3 years . Since AATOX1 mRNA expression levels for the HER2 subtype, followed by no change for the Luminal subtype and lower levels in the Basal subtype, and a trend of increasing ATOX1 mRNA levels with higher stages of disease [Classical immunohistochemistry was used to manually score ATOX1 levels in tumor cells and the staining intensities were grouped into low (intensity scores 0 and 1) and high (intensity scores 3 and 4) ATOX1 levels. ATOX1 expression was found and evaluated for both nucleus and cytoplasm of the tumor cells. The staining results for ATOX1 in tumor tissue sections demonstrated that high ATOX1 levels for the tumor cell cytoplasm were most often found in the HER2 subtype, followed by Luminal and TNBC subtypes; in addition, high ATOX1 levels were more frequent at disease stages III and IV in comparison to stage II. These results are in agreement with our previous results of increased  disease .We found that breast cancer patients at stage III of disease with high cytoplasmatic ATOX1 levels in their primary tumor have a 65% higher EFS chance after copper depletion treatment with TM . Evaluation of the different subtypes showed that TNBC patients with high cytoplasmatic ATOX1 levels had an EFS which is 15% higher than for patients with low cytoplasmatic ATOX1 levels. For the HER2 and Luminal subtypes, no conclusions were drawn due to the low sample size. The ATOX1 correlation in TNBC suggests that, here, ATOX1-dependent processes play a crucial role in cancer progression (such as metastasis), and therefore copper depletion with TM may have a beneficial effect ,11. CoppTaken together, cytoplasmatic ATOX1 has the potential to be a predictive biomarker for TM treatment of breast cancer patients at high risk of recurrence, such that patients with high cytoplasmatic ATOX1 levels in the cancer tissue might benefit from TM treatment. We note that a larger sample size is urgently needed to come to conclusions for the HER2 and Luminal subtypes. However, the larger sample size for the TNBC subgroup implies that ATOX1 may be a predictive biomarker for TM in this cancer subtype. A larger randomized phase II clinical trial of TNBC patients is scheduled to begin in late 2021 supported by the Translational Breast Cancer Research Foundation and the NCI NExT program."}
+{"text": "SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3\u2032 splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes  AC (underlined A is a branch point) among introns, the conserved sequence around branch point in mammals is YUNAY , which is more diverse  in MDS with the aberrant splicing mechanism caused by those mutations and an outline of MDS from a splicing point of view.SF3B1 is one of the components of the SF3B complex that stabilizes U2 snRNP binding to the branch point sequence during pre-mRNA splicing  2,29]. . 29]. SFSRSF2, originally called SC35 , is a meIt has also been demonstrated that MDS-responsible mutations in SRSF2 and U2AF1 cause expansion of R loop ,66. ExpaU2AF1 is originally identified as a component of the U2 snRNP auxiliary factor complex (U2AF) that facilitates association of U2 snRNP to the branch point sequences ,68. U2AFZRSR2 mutations cause abnormal splicing via intron retention of U12-depedent minor introns [The ZRSR2 gene is located on chromosome Xp22.2 and mutated in about 5% of MDS patients, predominantly males . Relativ introns . Humans  introns ,4. Among introns .Most recently, it was demonstrated that impaired minor intron excision by knock-out of ZRSR2 protein enhances hematopoietic stem cell self-renewal, and mutations in minor introns are suggested to be potential cancer drivers . HoweverLUC7L2. LUC7L2 is an ortholog of splicing factor LUC7 which is involved in recruitment of splicing factors. The LUC7L2 protein is assumed to be involved in the recognition of non-consensus splice donor sites in association with the U1 snRNP [In addition to four splicing factors, rare mutations in several other splicing factors were also identified. One of the mutated splicing factors is PRPF8. Studies from the yeast Prp8 protein revealed that PRPF8 protein is an essential factor for splicing and interacts with U5 snRNA to align 5\u2032 and 3\u2032 splice sites in the spliceosome . PRPF8 mU1 snRNP . InteresU1 snRNP . SeveralU1 snRNP . Very raU1 snRNP , which aIn this review, we introduced four major splicing factors mutated in MDS with aberrant splicing caused by mutations. Interestingly, most of the proteins described in this review are involved in 3\u2032 splice site recognition . In addiAlthough some aberrant splicing patterns in dysregulated genes have been identified to be involved in MDS onset as described above, it is still under investigation how different mutations in different splicing factors cause different MDS phenotypes. To date, there seems to be no common gene(s) whose aberrant splicing is responsible for MDS onset caused by mutations in four main splicing factors SF3B1, SRSF2, U2AF1, and ZRSR2. It is assumed that hot spot mutations among them in SF3B1, SRSF2, and U2AF1 do not cause reduction of the encoded proteins, whereas mutations in ZRSR2 reduce functional protein amount. Splicing pattern analyses implicate that common pathways affected by mutations of those factors are epigenetics and signal transduction pathways. These points have to be addressed in future analyses. The approaches from mechanistic analyses of aberrant splicing caused by mutated splicing factors should shed light on research for therapies of MDS by identifying drug targets."}
+{"text": "Our findings provide a novel conceptof using tunable quantum tunneling for highly sensitive broadbandphotodetection in mixed-dimensional van der Waals heterostructures.Graphene-based vander Waals heterostructures are promising buildingblocks for broadband photodetection because of the gapless natureof graphene. However, their performance is mostly limited by the inevitabletrade-off between low dark current and photocurrent generation. Here,we demonstrate a hybrid photodetection mode based on the photogatingeffect coupled with the photovoltaic effect via tunable quantum tunnelingthrough the unique graphene/Bi Variousmethods have been introduced to overcome limitations by enhancingthe light absorption of graphene-based photodetectors, including plasmonicnanostructures8 and quantum dots.9 Although these approaches have unique advantages,their photodetection is limited to a short spectral range becauseof the sharp resonant absorption.11Lightdetection over a wide spectral range, from the visible tothe mid-infrared, has a great potential for numerous photonic andoptoelectronic applications. In this scenario, graphene can providea versatile platform for broadband photodetection due to its gaplesselectronic band structure.13 In graphene-based van der Waals heterostructures, semiconductingtwo-dimensional transition-metal dichalcogenide materials are typicallyused as they exhibit superior light\u2013matter interaction propertiesthat allow for enhanced light absorption. However, the absorptionis limited within the visible spectral range due to their band gap.14 Combining graphene with narrower band gap materialshas been proposed for broader light absorption despite the large darkcurrent.17 Among them, most of the studies using topologicalinsulators focused on the formation of graphene/topological insulatorheterostructures to induce the photogating effect, utilizing the novelproperties of topological insulators such as graphene-like hexagonalsymmetry, direct narrow band gap, and ultrafast photocurrent alongthe surface.19 As a material candidate to formvan der Waals heterostructures with graphene, topological insulatorshave advantages beyond the aforementioned properties. For example,compared to black phosphorus, which is similar to a topological insulatorin terms of band gap and electrical properties such as mobility andcarrier multiplication, the surface oxidation of black phosphorusis related to the degradation mechanism and environmental instability,20 while the surface oxidation of the topologicalinsulators serves to protect the surface states.22 Moreover, since the topological insulators are Dirac fermion materialslike graphene, a Dirac-source field-effect transistor can be realizedwith the graphene/topological insulator heterostructures.23Hybridgraphene systems combined with mixed-dimensional materialsfor high-sensitivity broadband light detection have emerged from recentdevelopments by assembling materials into van der Waals heterostructures.7 However, the photodetectionperformance using this method is highly sensitive to the size, thickness,and quality of the interlayer, which makes the fabrication processchallenging. As an alternative pathway free from introducing an interlayer,the natural oxidation layer rapidly formed on the surface of topologicalinsulators22 in the ambient environment canbe utilized as the tunneling barrier.26 In particular, the thicknessof the oxidation layer of the topological insulators is saturatedwithin a few hours after exfoliation, and the oxidation process issignificantly delayed over time so that a uniform oxidation layercan be obtained over the entire surface.22Recent studies reported the control overthe dark current in graphene-basedphotodetectors by introducing an interlayer  tunnelingbarrier with enhanced photodetectivity.2Se3 heterointerface enablesincorporation of the quantum tunneling effect into the photodetectionmechanism, as evidenced by the transition of charge carrier transportmechanisms from direct tunneling to Fowler\u2013Nordheim (FN) tunnelingand/or thermionic emission. In our device architecture, the photogatingeffect in the graphene/Bi2Se3 heterochannelis coupled to the photovoltaic effect through the rectifying tunnelingjunction. This significantly enhances the photodetection performance,which is fundamentally different from the typical graphene-based photodetectorspreviously reported.4 Accordingly, the normalized photocurrent-to-dark current ratio (NPDR)is enhanced by around an order of magnitude via electrical tuningof tunneling resistance for detection of light covering the majorphotonic spectral region from the visible to the mid-infrared wavelengths.Our work provides a new perspective on both the tunneling dark currentsuppression and the efficient photocurrent generation for variousphotonic and optoelectronic applications.Here, we demonstrate that the naturally formed oxidation layerat the graphene/Bi2Se3 heterostructure . The Eg2 peak intensity mapping imageof the Bi2Se3 flake . On the other hand, the 2D peak intensitymapping image of graphene, as shown in 2Se3 flake .Our Dirac-sourcefield-effect transistor based on a lateral heterochanneland a vertical tunneling junction is realized by the graphene/Bicroscopy 1b and Raayer see S1a. On t2Se3 flake canbe clearly identified by the AFMimages, as shown in S2. The average thicknessesof the graphene and Bi2Se3 flakes, measuredby AFM, are \u223c1.3 and 29.2 nm, respectively (see the Supporting Information for more details on theAFM)29.The graphene layer covering thesurface around the edge of Bi2Se3 (Bi: 5d and Se: 3d) takenafter 24 h from exfoliation. The observed oxidation peaks correspondingto Bi2O3 (at around 26 and 29 eV) and SeO2 (at around 59 eV) represent the existence of the oxidationlayer naturally formed on the Bi2Se3 surface.22 The intensity of the SeO2 peak ismuch lower than that of the Bi2O3 peak, indicatingthat the dominant oxidation layer formed on the Bi2Se3 surface is Bi2O3 rather than SeO2 due to the Se vacancies on the Bi2Se3 surface.32 Note that the oxidation time of 24 h after exfoliationunder ambient conditions is set to utilize the uniform oxidation layerwith the stabilized thickness. Although the formation of the nativeoxidation layer is very fast at the initial stage after exfoliation,22 its thickness is known to saturate since the oxidation process issignificantly delayed over time due to the interplay between surfaceexposure and oxygen incorporation.22 Thethickness of the oxidation layer is estimated as \u223c2 nm (\u00b10.2nm).2613 First, we investigated the current\u2013voltage (I\u2013V) characteristics of the graphene/Bi2Se3 heterojunction by choosing different metalelectrodes (see the 2Se3 heterointerface (definedas graphene-Bi2Se3), the I\u2013V curves exhibit the nonlinear I\u2013V relationship  dueto charge carrier transport through the graphene/Bi2Se3 heterointerface. The asymmetric rectifying behavior indicatesthat the tunneling junction is formed at the interface, and the tunnelingbarrier heights are asymmetric. The hysteresis effect of I\u2013V curves arises from charge trapping atthe interface. For comparison, we also measure a reference graphenetransistor (defined as graphene&Bi2Se3),where both source and drain are applied to the graphene channel thatpartially covers the Bi2Se3 flake (see the Supporting Information for details on the measurementconfiguration with different electrodes). The I\u2013V curves, as shown in Figure S3a, reveal the typical Ohmic behavior in the reference graphene transistor.Thevan der Waals heterostructures can enable versatile functionalitieswith higher performance than each material in the van der Waals heterostructures.2Se3, as shownin IDS\u2013VDS curve(drain\u2013source current IDS as afunction of drain\u2013source voltage VDS) at gate\u2013source voltage VGS =0 V is divided into nine steps, which are marked by Roman numeralsfrom I to IX. Each step represents the transition point, where thetransport mechanism changes and the resistance state switches to differentresistance states. The oxidation layer naturally formed on the Bi2Se3 surface is known to act as a tunneling barrierin contact with graphene.26 The tunneling resistance is closely related to thepotential barrier at the interface and the electronic density of statesin graphene and Bi2Se3. Hence, the shape deformationof the asymmetric tunneling barrier will have a great influence onthe tunneling current across the interface. The energy band alignmentsbefore equilibrium and between each step is drawn in 36 . The detailed descriptions forthe hysteretic I\u2013V characteristicsand charge carrier trapping processes at the graphene/Bi2Se3 interface can also be found in the Supporting Information.38 Further details onthe energy band alignment are fully discussed in the Supporting Information.46To understand the mechanism of asymmetric rectifying and hystereticcharacteristics of the graphene-Bi4 On the contrary, in our device, the photogating effect is coupledto the photovoltaic effect through the rectifying tunneling junctionacross the graphene/Bi2Se3 heterointerface,which is supported by the observation of asymmetric rectifying andhysteretic I\u2013V characteristics,as shown in 49 Some of the photogenerated carriers accumulated atthe trap states can act as an external bias voltage to shift the Fermi-levelof graphene, and the other carriers injected into graphene or Bi2Se3 will contribute to the photocurrent. On theother hand, the photovoltaic effect is driven by separating photogeneratedelectron\u2013hole pairs through the rectifying junction, whichcan be further controlled by tuning the tunneling resistance.7 Before exploring the photogating effect, we first characterizedthe enhanced photocurrent with the photovoltaic effect, owing to therectifying tunneling junction, as shown in Figure S3. Interestingly, under the same condition of light illumination(at a wavelength of 532 nm with a laser power of 10 \u03bcW) focusedonto the same graphene/Bi2Se3 heterojunctionregion, much higher photocurrent is realized through the graphene/Bi2Se3 heterointerface , as compared tothe reference graphene transistor . This is because thephotocurrent generated in the reference graphene transistor is hinderedby the carrier recombination within the graphene channel, while thephotocurrent of the rectifying tunneling junction formed through thegraphene/Bi2Se3 heterointerface is enhancedwith the photovoltaic effect.Most graphene-based photodetectors generallyfocus on one photodetectionmechanism: photovoltaic effect, photogating effect, photo-thermoelectriceffect, and bolometric effect, due to their inherent limitations.VDS in our graphene/Bi2Se3 heterochanneldue to the strong coupling between the photogating and photovoltaiceffects. Here, two different VDS (0.5and 1.5 V) are chosen to define the high and low tunneling resistancestates. Both exhibit high photocurrents, but dark currents are obtainedto be considerably different for a proper comparison. Note that thisis based on the color plots of IPC, asshown in IPC = Ilight \u2013 Idark is the photocurrent, Ilight is the drain\u2013source current underlight illumination, and Idark is the drain\u2013sourcecurrent in the dark. In addition, it is found to be more effectivefor modulating the tunneling resistance by tuning positive VDS along the Bi2Se3 side,due to the lower barrier height for electrons on the graphene sidethan that toward the Bi2Se3 side, as estimatedin VDS = 0.5 V), the direct tunneling from graphene to Bi2Se3 will be substantially blocked by the tunneling barrier underthe dark condition, while the photoexcited electrons in graphene canbe easily injected into Bi2Se3 over the lowbarrier height. On the other hand, at the low tunneling resistancestate (VDS = 1.5 V), the dark current ascribed to theFN tunneling and thermionic emission will exceed the current due tophotoexcited electrons. As a result, the dark current will be obtainedto be extremely lower in the high tunneling resistance state (2Se3 heterojunction at VDS = 0.5 V (at a wavelength of 532 nm with a laser power of 100 \u03bcW).The photocurrent generation is pronounced around the heterojunctionregion especially near the edge of the graphene channel overlappingthe Bi2Se3 flake, confirming the major photocurrentgeneration originating from the heterointerface due to the built-inelectric fields applied across it.We also find that the tunnelingresistance can be significantly tuned by varying ce state 4b than tce state 4c. As shIDS as a function of VGS) of the graphene/Bi2Se3 heterointerfacein the dark and under light illumination at various wavelengths of532, 730, 1550, and 4000 nm with a light power of 10 \u03bcW areshown in VDS = 0.5 V) and VDS = 1.5V). The graphene/Bi2Se3 heterochannel operatesas a field-effect transistor, where the carrier mobility depends onthe tunneling resistance. The high (VDS = 0.5 V) and low (VDS = 1.5 V) tunnelingresistance states lead to the different average carrier mobilitiesof 36.8 and 103.2 cm2 V\u20131 s\u20131 for holes and 12.6 and 64.1 cm2 V\u20131 s\u20131 for electrons at room temperature, respectively.From the shift of VDirac, we estimatedthat the photogating effect is attributed to the trapping of photogeneratedcarriers at the graphene/Bi2Se3 interface (seethe Supporting Information for detailson the photogating effect due to the trapping of photogenerated carriers).As shown in the transfer curves, there are almost no hysteresis effectsfor the VGS sweep, thanks to the Al2O3 top passivation layer,50 implying that charge trapping at the graphene/Bi2Se3 interface only occurs during the VDS sweep. In particular, the tunneling dark current is obtained tobe quite low, as shown in Ilight/Idark). At VGS = 0 V, althoughthe photocurrents (IPC = Ilight \u2013 Idark), asshown in IPC, where Ilight = Idark, does not appearin Ilight to Idark. Unlike thephotogating effect in which one type of photogenerated carriers shouldbe captured in trap states, the photovoltaic effect requires the efficientseparation of created electron\u2013hole pairs. Our results suggestthat the tunneling junction in the graphene/Bi2Se3 heterochannel can be utilized to couple the photogating effect withthe photovoltaic effect to enhance the optical switching ratio bytuning the tunneling resistance.The transfer curves(2Se3 is confirmed by photoresponsivity (R = IPC/Peffective) and photodetectivity . Onthe contrary, the photoresponse characteristics of the reference graphenetransistor  is almost unchangedbetween VDS = 0.5 and 1.5 V. In otherwords, the light absorption in the graphene/Bi2Se3 heterostructure does not guarantee the high photodetectivity. Thishighlights that the charge carrier transport through the graphene/Bi2Se3 tunneling junction is of great importance tocouple the photogating effect with the photovoltaic effect in thegraphene/Bi2Se3 heterochannel for highly sensitivephotodetection.Depending on the tunnelingresistance, the electrically tunable photoresponse of graphene-Biectivity 5b, , whenterface5b can beI) at VDS = 0.5 V and VGS = 0 V is plottedin Supporting Information for details on the relation between light powerand responsivity).514, 6.6 \u00d7 103, 3.8 \u00d7 103, and 1.1 \u00d7 103 W\u20131 (at VDS = 1.5 V) to \u223c2.2 \u00d7 105, 1.1 \u00d7 105, 3.5 \u00d7 104, and 8.0 \u00d7103 W\u20131 (at VDS = 0.5 V) by effectively suppressing the tunneling dark current.The idea of tuning the tunneling resistance for enhancing the photodetectivityoffers a new insight to realize the broadband photodetection by couplingthe photogating effect with the photovoltaic effect.52The dependence of photoresponsivityon light intensity  to FN tunneling and/or thermionic emission at the high biasvoltage (low tunneling resistance state), as described in 2Se3 junction.As an alternative pathway free from an artificial introduction ofan interlayer at the interface, our findings demonstrate a new perspectiveof utilizing the oxidation layer naturally formed at the graphene/Bi2Se3 interface for both dark current reduction andefficient photocurrent generation. We confirmed that the naturallyformed oxidation layer can act as a tunneling barrier. We furtherobserved the tunable tunneling resistance and offered direct evidencefor the asymmetric tunneling barrier heights using the tunneling equations.36 An additional advantage of utilizing the naturally formed oxidationlayer is that this approach is not limited by the size, thickness,and quality of the interlayer so that large-scale devices are achievableas long as Bi2Se3 is large enough, making theentire fabrication process simple. This is similar to the currentsilicon technology, where naturally formed silica plays a key role.Another novelty of our work is describing the transition of chargecarrier transport mechanisms through the graphene/Bi2Se3 interface, which is essential for coupling between photogatingand photovoltaic effects. To the best of our knowledge, the hysteresiseffect of I\u2013V curves in graphene/Bi2Se3 heterojunction is first observed in this work,implying the trapping of charge carriers at the interface. This observationsuggests that the trap-assisted photogating effect can be inducedin the graphene/Bi2Se3 heterochannel by trappingof photogenerated carriers,49 which can be coupled with thephotovoltaic effect to effectively enhance the photocurrent owingto the rectifying tunneling junction.7In other studies, several attempts have been madeto control thedark current by utilizing the ultrathin interlayer such as Ta2Se3 interface,26 we have explored a breakthroughway to improve the photoresponse characteristics of the graphene/Bi2Se3 heterochannel by suppressing the tunnelingdark current and injecting the photogenerated carriers. We have foundthat the tunneling resistance of the graphene/Bi2Se3 heterojunction can be tuned to couple the photogating effectwith the photovoltaic effect in the graphene/Bi2Se3 heterochannel. The transition of charge carrier transportmechanisms through the graphene/Bi2Se3 interfaceis a key signature to modulate the optical switching ratio.7 The practical application to improve the device performance throughthe interface engineering  or the materialcombination , which is beyond the scope of this study, willbe a promising research direction. This study will provide usefulinformation for designing novel photodetectors for highly sensitivebroadband photodetection.Based on the asymmetric tunneling barrier formed at the graphene/Bi2Se3 into van der Waals heterostructures. The Bi2Se3 flakes were mechanically exfoliated from bulkmaterial and transferred onto the highly doped Si substrates witha 285 nm thick SiO2 layer preprocessed with solvent cleaningand O2 plasma treatment. The Bi2Se3 flake surface was naturally oxidized under ambient conditions for24 h. Owing to the rapid surface oxidation of topological insulators,22 there was no need to introducean additional interlayer such as an insulating layer grown by atomiclayer deposition (ALD) or h-BN layer before the graphene transfer.In order to form several heterostructures on each substrate at thesame time, large-area monolayer graphene grown by chemical vapor deposition,purchased from Graphenea, was wet-transferred onto the substrates.42 For the complete and smooth coverage of the graphene layer overthe entire Bi2Se3 flake, we selected the Bi2Se3 flakes with a few tens of nanometer thick,which allowed us to minimize the defects caused by the steep surfacemorphology around the flake edges. Each graphene channel was patternedto a regular shape with electron beam lithography  and reactive ion etching (Oxford Instruments PlasmaLab 80 Plus).The lateral heterochannel was defined to be the patterned grapheneribbon and transferred Bi2Se3 flake, while thevertical tunneling junction was formed in the overlapping regions.The heterojunction area was estimated to range from 50 to 100 \u03bcm2, with an average of 83.7 \u03bcm2. Ti/Au electrodesof 5/75 nm were patterned with EBL and deposited through electronbeam evaporation (MASA IM-9912) followed by a lift-off process. Themetal electrodes on the graphene channel and Bi2Se3 flakes were patterned to be orthogonal and parallel to thegraphene channel, respectively. As a passivation layer, a 10 nm thicklayer of Al2O3 grown by ALD at 150 \u00b0C was used to prevent the surface modification from theadhesion of oxygen or water molecules. After completing the devicefabrication, an optical microscope (Olympus BX60) and Micro-Raman(WITec Alpha 300 RA+) system using 532 nm continuous wave laser wereused to characterize the graphene/Bi2Se3 heterostructure.The graphene covering the Bi2Se3 flake edgewas identified by AFM Dimension Icon (Bruker). XPS was performed onthe large area Bi2Se3 flakes using a KratosAxis Ultra ESCA spectrometer with a monoenergetic Al K\u03b1 (1486.96eV) source. The pass energy was \u223c20 eV, and the X-ray spotsize was \u223c200 \u03bcm. Since X-rays penetrate only the topfew layers of flakes, the XPS is useful for quantitative analysisof the surface chemical composition (the outer few nanometers) regardlessof the flake thickness.Our heterojunctionphototransistor was fabricated by integrating graphene and Bi2Se3 flakes. Thephotocurrent measurements covering from the visible range to near-infraredrange were conducted in the WITec system combined with two sourcemeters.The light beams from continuous wave lasers at 532 nm (WITec focusinnovations), 730 nm (Thorlabs MCLS1), and 1550 nm (Photonetics) werefocused onto the heterojunction through an objective lens . The optical microscopy platform system allowed us to focusthe laser beam on desired positions in the sample. The diameters ofthe light spot were around 0.87, 1.18, and 2.49 \u03bcm, respectively.The photocurrent measurements in the mid-infrared range were conductedin a home-built femtosecond laser-based microscopic system combinedwith two sourcemeters. The duration and repetition rate of the incidentpulse were 230 fs and 2 kHz. The laser at 4000 nm is focused to coverthe graphene/Bi2Se3 heterojunction region bya parabolic mirror. The diameter of the light spot was \u223c20\u03bcm.The sample holder was designed in a sizefitting into a fixingholder in a probe stage. After device fabrication, the device chipswere mounted onto each printed circuit board (PCB) attached to thesample holder and wire-bonded to the PCB. All the electrical measurementswere carried out in an optical microscope (WITec Alpha 300 RA+) ora home-built femtosecond laser based microscopic system with two sourcemeters(Keithley 2400 and 2401) at room temperature under ambient conditions.The gate voltage was applied to the Si substrate, while the sourceand drain voltages were applied to the metal electrodes connectedto the graphene channel or Bi"}
+{"text": "The authors apologize for the original versions of our paper unfortunately contained some incorrect representative images. We used the wrong representative images during figure assembly. The IHC images of 7dCon-Runx2, 10dCon-CD146/COL1A1 and 10dT-Del-Runx2 in Figure The authors confirm that the corrections made in this erratum do not affect the original conclusions. The authors apologize for any consequences that these errors may have caused."}
+{"text": "Our understanding of the renin-angiotensin-system (RAS) has been revolutionized over the last 20 years with the discovery of angiotensin converting enzyme (ACE)2 and its principal product \u2013 angiotensin (Ang)-1\u20137) \u20137 1,2]. Additional convertases such as circulating furin and transmembrane protease serine (TMPRSS2) strengthen viral attachment to membranal ACE2 by modification and cleavage of the S1/S2 spike proteins Viral attachment to membranal ACE2 is followed by viral internalization together with ACE2 and ACE2 degradation, leading to depletion of membranal ACE2 Taken together, high expression of membranal ACE2 facilitates viral homing, but its loss by degradation and shedding leads to Ang-(1\u20137) depletion, promoting the devastating clinical manifestations of COVID-19 disease EBioMedicineACE2 transcript. It is important to consider the existence of alternatively spliced isoforms (For that reason, the study of Nikiforuk et\u00a0al., published in isoforms  and to nAll authors contributed to conceptualization, writing, reviewing, editing and have read and agreed to the published version of the manuscript.The authors declare no conflict of interest."}
+{"text": "APX2 and HSP22) and H3K4me3 following recurrent heat stress. Furthermore, CSN5A is also required for the deposition of H3K4me3 following recurrent heat stress. Thus, CSN5A plays an important role in the regulation of histone methylation and transcriptional stress memory after recurrent heat stress.In nature, plants are exposed to several environmental stresses that can be continuous or recurring. Continuous stress can be lethal, but stress after priming can increase the tolerance of a plant to better prepare for future stresses. Reports have suggested that transcription factors are involved in stress memory after recurrent stress; however, less is known about the factors that regulate the resetting of stress memory. Here, we uncovered a role for Constitutive Photomorphogenesis 5A (CSN5A) in the regulation of stress memory for resetting transcriptional memory genes ("}
+{"text": "The PaO2 and peripheral oxygen saturation (SpO2) are closely related and both are used to monitor oxygenation status. However, SpO2 values cannot be used as an exact substitute for PaO2. The aim of this study in acutely ill and stable patients was to determine at which SpO2 level PaO2 is more or less certain to be in the physiological range.In the context of an ongoing debate on the potential risks of hypoxemia and hyperoxemia, it seems prudent to maintain the partial arterial oxygen pressure . A second cohort of retrospective data of patients who underwent pulmonary function testing was also included . Arterial hypoxemia was defined as PaO2 < 60 mmHg and hyperoxemia as PaO2 > 125 mmHg. The SpO2 cut-off values with the lowest risk of hypoxemia and hyperoxemia were determined as the 95th percentile of the observed SpO2 values corresponding with the observed hypoxemic and hyperoxemic PaO2 values.This is an observational study prospectively collecting data pairs of PaO2 measurements occurred in patients with an SpO2 below 94%, and 95% of hyperoxemic PaO2 measurements occurred in patients with an SpO2 above 96%. Additionally in the 1379 data pairs of the ROP cohort, 95% of hypoxemic PaO2 measurements occurred in patients with an SpO2 below 93%.220 data pairs were collected in the PIA cohort. 95% of hypoxemic PaO2 level marking an increased risk of arterial hypoxemia is not substantially different in acutely ill versus stable patients. In acutely ill patients receiving supplemental oxygen an SpO2 target of 95% maximizes the likelihood of maintaining PaO2 in the physiological range.The SpO PaO2 on the other hand is the parameter of primary interest, since it reflects the balance between oxygen delivery and consumption [2 and PaO2 are closely related, the nature of which is reflected by the oxyhemoglobin dissociation curve [2 or SpO2 cannot be interpreted as an exact substitute of PaO2 [2 and PaO2 [2 measurements [2 is close to or at its maximum value of 100%. Indeed, corresponding PaO2 values for any given SpO2 value may range widely [Non-invasive measurement of SpOsumption , 14. SaOon curve . However of PaO2 , 17. Lowand PaO2  and alteurements \u201324. Alsoe widely .2 of 94\u201398% in adults in healthcare and emergency settings [2 above 96% and advocate a lower SpO2 range [2 was 96% or lower [In order to prevent hypoxemia and hyperoxemia and its possible associated risks, the British Thoracic Society Guideline recommends a target SpOsettings . Others O2 range , 30. A ror lower .2\u2013PaO2 relationship, particularly in acutely ill patients receiving oxygen. In this study, we aim to identify a target SpO2 value, with relative safety margins, resulting in a high likelihood of maintaining PaO2 within a physiological range while administering oxygen [In light of the debate on the trade-off between the risks of arterial hypoxemia and hyperoxemia, it is important to increase our understanding of the SpOg oxygen .2 and SpO2 values in two different patient cohorts. The study was registered at clinicaltrials.gov (NCT02666937). This cohort consists of acutely ill patients admitted to the Emergency Room (ER) and the ICU and is referred to as the Prospective Inpatient Acutely ill cohort (PIA cohort).We performed a prospective observational study collecting simultaneously measured PaOThe medical research ethics committee of the Amsterdam University Medical Centers (Amsterdam UMC) approved the study protocol. The patients in the PIA cohort were informed about the use of their data and could object to their data being used through an opt-out procedure. The medical research ethics committee specifically approved the use of opt-out consent for these patients.2 and SpO2 values. This was a retrospective dataset of patients undergoing pulmonary function testing. This dataset was used to gain more insight into the lower range of SpO2 and PaO2 values in a larger population. This cohort of patients is referred to as the Retrospective Outpatients Pulmonary cohort (ROP cohort). The medical research ethics committee granted a waiver of consent for the use of this data.We also included a second cohort of patients with simultaneously measured PaO2 and PaO2 value.For the PIA cohort, data was collected prospectively from December 2015 to August 2017 in mechanically ventilated ICU-patients and acutely ill patients visiting the ER at the Amsterdam UMC. This cohort consisted of a convenience sample, based on the availability of researchers. Adult patients were eligible for inclusion if they required arterial blood gas analysis (ABGA) as part of routine care. Up to 10 data pairs could be collected per patient with a time interval of at least two hours between samples. A data pair is one single measurement of a combined SpO2 was measured with the use of a Nellcor OxiMax DS-100A  pulse oximeter. Reliability of the SpO2 curve was assessed and defined as a curve showing complete pulsatile tracing of the beat-to-beat arterial pulse wave on the pulse oximeter. On the ICU, PaO2 was measured using a combined blood gas analyzer and CO-oximeter . The samples were analyzed immediately after sampling.Simultaneous with the ABGA the SpO2/min when at risk for insufficient oxygenation, according to local guidelines.Patients with methemoglobinemia (> 1.5%), carboxyhemoglobinemia (> 1.5%) or known hyperbilirubinemia (> 20 \u03bcmol/L) were excluded. Additionally, ICU patients were only included if they had an arterial cannula and required mechanical ventilation, and were excluded when undergoing extracorporeal membrane oxygenation or therapeutic hypothermia. Oxygen was titrated to achieve a stable arterial oxygen saturation of \u2265 92% in the ICU during routine clinical care. ER patients were included upon presentation when they required ABGA. Most ER patients were stabilized with a non-rebreather mask with 15 liter O2 measurement. Per patient between one and four ABGA\u2019s were collected during each pulmonary function test. The pulmonary function test consisted of a cycling protocol without supplemental oxygen. The arterial puncture was performed by a pulmonary function technician in the radial artery. SpO2 was measured with a NONIN Avant 9600  pulse oximeter at the Amsterdam UMC and a NONIN 8000S  pulse oximeter at the Northwest Clinics.For the ROP cohort, a dataset was used containing adult patients performing a pulmonary function test at the Amsterdam UMC between 1995 and 2014 and at the Northwest Clinics between 2016 and 2017. A researcher accessed the database to obtain the retrospective data in July 2017. All patients having performed a test were screened for eligibility and were included if they had had an ABGA and a simultaneous SpO2 of 60 mmHg or lower in a clinical trial of oxygenation targets [2 < 60 mmHg [2 > 125 mmHg [The exact cut-offs for hypoxemia and hyperoxemia are relatively arbitrary and not well defined in literature. For example hyperoxemia is defined in a range from > 120 mmHg  up to >  targets . We ther 60 mmHg , 34, 36 125 mmHg , 37, 38.Data were analyzed using SPSS  and RStudio . Values are presented as mean with standard deviation (SD) unless otherwise stated.2 and PaO2 data were first visually assessed with a scatterplot. We determined a hypoxemic and hyperoxemic SpO2 limit for the PIA cohort. The hypoxemic SpO2 limit was determined as the 95th percentile of the observed SpO2 values corresponding with the observed hypoxemic PaO2. The hyperoxemic SpO2 limit was determined as the 5th percentile of the observed SpO2 values corresponding with the hyperoxemic PaO2 values. For the ROP cohort we only defined a hypoxemic SpO2 limit, since the patients did not receive supplementary oxygen.SpO2 and PaO2 values were transformed such that relation between the SpO2 and PaO2 became linear (ln(1\u2014SpO2/100) and ln(PaO2)). The transformed SpO2 was the dependent variable and the transformed PaO2 was included as a covariate with fixed effect. A two-sided 95% prediction interval was constructed for each specific value of PaO2. The upper and lower limit of the prediction interval and the predicted mean value for SpO2 were then transformed back to the original scale.Since multiple measurements were available for most patients, we constructed a mixed linear model in both cohorts. This included a random effect for each patient. First SpO2 curves, resulting in the inclusion of 220 data pairs in 146 patients. 139 data pairs were collected at the ICU and 81 at the ER. We screened 693 patients in the retrospective database for eligibility in the ROP cohort. Thirteen patients were excluded because of no or unreliable SpO2 measurements, resulting in the inclusion of 1379 data pairs in 680 patients. 1003 data pairs were collected at the Amsterdam UMC, and 376 pairs at the North West Clinic. We screened 152 patients in the PIA cohort for eligibility. One patient was excluded because of hyperbilirubinemia and five were excluded because of unreliable SpO2 < 60 mmHg, and 20.5% had a PaO2 > 125 mmHg. In the ROP cohort 10.7% of the data pairs had a PaO2 value < 60 mmHg.Demographics of the included patients and characteristics of the collected data pairs are shown in 2 and SpO2 measurements for both cohorts. In the PIA cohort 95% of data pairs corresponding to hypoxemia had SpO2 values below 94%, and 95% of data pairs corresponding to hyperoxemia had SpO2 values exceeding 96%. The horizontal red dotted lines in 2 limits. The patients in the ROP cohort have more hypoxemic PaO2 values and did not receive supplemental oxygen. In the ROP cohort, 95% of data pairs corresponding to a hypoxemic PaO2 had SpO2 values below 93%.2 >125 mmHg in the PIA cohort and more measurements with a PaO2 < 60 mmHg in the ROP cohortA multilevel analysis was performed for each cohort, taking into account that multiple measurements could be included per patients see . The cur2 measurements within a certain PaO2 range for each SpO2 level in both cohorts. In the PIA cohort at an SpO2 value of 95%, 94,4% of the data pairs had a PaO2 value between 60 and 125 mmHg. For an SpO2 value of 96% and 98%, the corresponding rates of hyperoxemic values were 3.8% and 17.4%, respectively. In the ROP cohort at an SpO2 value of 93%, 4% of the data pairs had a hypoxemic PaO2 value. For an SpO2 value of 92%, the corresponding rate was 11.5%.2 values of 94\u201396% both hypoxemia and hyperoxemia can be effectively excluded. In the ROP cohort hypoxemia can be excluded with a lower SpO2 target of 93%. The lower PIA and ROP targets excluding hypoxemia are not substantially different. Combining these cut-offs, we recommend an SpO2 target of 95%, which clearly has margins of relative safety. This target SpO2 results in the highest likelihood of physiological PaO2 values for the average patient.In the PIA cohort with SpO2 range between 92\u201395% that indeed resulted in a significant decrease of the number of hyperoxemic episodes, while hypoxemic episodes remained unchanged [2 level excluding hyperoxia of 96% [2 levels above 96% [2 of 98% for acutely ill patients [2 target of 98% may result in more hyperoxemia, without being associated with a meaningful lower risk of hypoxemia.Our findings are supported by data from an uncontrolled study, aiming at a target SpOnchanged . Moreovea of 96% . As for bove 96% . The Bripatients . Our dat2 value below 93% results in a higher risk of hypoxemia, for the PIA cohort this is the case below 94%. The lower limits are quite similar in these different cohorts of acutely ill and stable patients. These lower safe limits are in line with recent clinical guidelines recommending a lower SpO2 limit of 92\u201394% [2 values increases the risk for hypoxemia.In the ROP cohort an SpOf 92\u201394% , 29. Aim2 [2 [2 is not heavily influenced by specific pulmonary morbidity.This observational study has some limitations. Different oximeters were used for the two cohorts and potential differences between measurements can depend on the oximeter used. The Nellcor oximeters, used in the PIA cohort, slightly overestimate SaO2 , 41, whi2 < 60 mmHg [2 > 125 mmHg [2 in a physiological range, defined as between 60 and 125 mmHg [It is important to note, that there is no clear definition of hypoxemia and hyperoxemia available. We therefore pragmatically defined hypoxemia in line with literature and clinical practice as PaO 60 mmHg , 34, 36 125 mmHg , 37, 38.125 mmHg .2 values, since stable patients without pulmonary pathology will hardly ever be hypoxemic. Thus, this limitation is intrinsic to the study question of whether the safe SpO2 margin is different in acutely ill versus stable patients.The PIA cohort consisted of a convenience sample. The patient profiles in the PIA cohort varied, with mechanically ventilated patients at the ICU and patients requiring blood gas analysis at the ER. In both cohorts, the number of patients with hypoxemia was relatively small, because of oxygen administration. The ROP cohort consisted of retrospective data of patients performing a pulmonary function test with a cycling protocol. There are very few alternatives to the ROP cohort to obtain more insight in hypoxemic PaO2 and SaO2 or the accuracy of SpO2 measurements, so we cannot provide separate recommendations about specific conditions, such as sepsis, hypotension, heart failure, or acute respiratory failure. The generalizability of the results of this study should be confirmed with sufficiently powered studies in patients with specific conditions with different oximeters to define how clinical variables might change SpO2 targets. Also the different definitions of hypoxemia and hyperoxemia and clinical outcome variables should be taken into account in future studies.This study did not explore the clinical variables potentially modifying the relation between PaO2-target of 95%, or as close to this level as possible, to avoid the occurrence of either hypoxemia or hyperoxemia in acutely ill patients receiving supplementary oxygen.In conclusion, we recommend an SpOS1 File(XLSX)Click here for additional data file."}
+{"text": "Scientific Reports 10.1038/srep21336, published online 22 February 2016Correction to: This Article contains an error in Figure 5 where the data is incorrect in panel (c). The correct Figure 5 appears below as Figure"}
+{"text": "The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities."}
+{"text": "The pharmacokinetics of 5-fluorouracil (5FU) following its administration via the hepatic artery in conjunction with biodegradable albumin microspheres and angiotensin II have been studied. Peripheral venous concentrations of 5FU are lower and plasma clearance values higher following intrahepatic arterial administration compared with a similar dose administered by intravenous infusion over both 2 h and 24 h. For the 2 h drug infusions, plasma 5FU concentrations following co-treatment with angiotensin II and microspheres via the hepatic artery were intermediate between those of arterial and venous infusions of 5FU alone. There was a trend towards the peak plasma drug concentrations and the area under the plasma concentration-time curve (AUC) being significantly lower following co-treatment with angiotensin II and microspheres compared with intra-arterial and intravenous infusions of 5FU over 24 h. Co-administration of 5FU, angiotensin II and microspheres via the hepatic artery may reduce drug exposure in the systemic compartment and therefore may increase the therapeutic ratio of 5FU administration via the hepatic artery."}
+{"text": "Twelve boys and 10 girls on similar long term remission maintenance treatment for lymphoblastic leukaemia had 79 random assays of their red cell 6 thioguanine nucleotide ( 6TGN ) concentrations performed as an index of cytotoxic activity generated by oral 6-mercaptopurine ( 6MP ). Correlation between the dose of 6MP and 6TGN was statistically significant in the girls  but not in the boys (r = 0.15). Additionally, as a group the boys tolerated more 6MP (P less than 0.05), despite similar prescribing criteria, but this did not result in a higher mean 6TGN concentration or increased myelotoxicity. It appears that girls develop 6MP cytotoxicity at lower doses and more predictably than boys. If so, this may be relevant to the as yet unexplained but marked sex difference in prognosis apparent in some studies."}
+{"text": "EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes ( Considering that only the first pollen tube to reach the micropyle passes its genes to the next generation, it is not surprising that the entire process is very fast. In maize, rehydration and germination of the pollen grain occur within 5 min of deposition on the silk, and pollen tubes grow at rates exceeding 1 cm hMu transposon insertion in EXPB1 (GenBank Accession AY197353), a gene that codes for Zea m 1d, the most abundantly expressed of four Zea m 1 isoforms in vivo and the ability of pollen to achieve fertilization under conditions of pollen competition.The group-1 grass pollen allergens are pollen-specific proteins originally identified by immunologists 40 years ago as the main causative agents of hay fever and seasonal asthma induced by grass pollen Mu) insertions  and wild type plants (EXPB1/EXPB1), as well as heterozygous (EXPB1/expb1) plants by crossing the true breeding plants. Analysis of pollen protein extracts by two-dimensional gel electrophoresis and immunoblotting from EXPB1/EXPB1 and expb1/expb1 plants revealed that overall Zea m 1 production was reduced by 31% in expb1 pollen compared with the overall production of Zea m 1 in EXPB1 pollen  also code for the same isoform From a large library of maize lines bearing Robertson's Mutator  revealed no significant effect of plant genotype on pollen viability . For each of the three maize genotypes  75\u201378% of the pollen stained a deep purple . Tukey pairwise comparisons with adjusted probabilities for multiple comparisons revealed that there was no significant difference in the in vitro growth of pollen tubes from expb1/expb1 and EXPB1/EXPB1 plants but that the pollen from the EXPB1/expb1 plants grew faster in vitro .When pollen from 20\u201322 plants from each of the 3 genotypes were germinated and grown on a medium in Petri plates in vitro  most likEXPB1 and expb1 pollen to achieve fertilization under conditions of pollen competition, we varied the volume (number) of pollen grains from EXPB1/expb1 plants deposited onto the silks of EXPB1/EXPB1 plants. We found that the transmission of expb1 depended upon the volume of pollen grains used in the pollination  , indicating that as the intensity of pollen competition increases, the proportion of seeds sired by expb1-bearing pollen decreases.To determine the ability of <0.0001) . When thin vivo pollen tube growth rates indicate that these differences in the ability to achieve fertilization under competitive conditions are due to differences in pollen tube growth rates. When we examined the silks at 8 h after pollination, we found that silks pollinated with EXPB1 pollen contained a significantly greater number of pollen tubes  at 8 cm below the site of pollen deposition than silks pollinated with expb1 pollen (0.98\u00b10.06) . At 22 h after pollination, we found pollen tubes in 37.5% of the ovaries following pollinations by EXPB1 pollen  and higher plants  (see EXPB1 gene (the most abundant isoform of Zea m 1) has no significant effects on pollen viability or in vitro pollen tube growth rates but has a large effect on in vivo pollen tube growth rates and the ability to achieve fertilization under conditions of pollen competition. Because the pollen competition experiment varied the size of the pollen load produced by heterozygous (EXPB1/expb1) plants rather than using the pollen produced by the true breeding (homozygous) plants, the results are not an artifact of differences in vigor between the true breeding lines. This experimental design, however, does not exclude the possibility that loci distinct from EXPB1 could account for our results. After backcrossing the original line with the Mu insertion to the non-mutator parental line for 3 generations, the resulting plants still contain, on average, 12.5% of the genes from the original Mu insertion line. The vast majority of these genes would randomly segregate into the EXPB1 and expb1 pollen produced by the heterozygous plants used in the pollen competition experiment. Only those genes that consistently cosegregate with expb1 could potentially influence performance of expb1 pollen. Because only 3% of the seeds produced by our largest pollen load contained expb1, another mutation that is responsible for the large effect on pollen performance that we observed would have to lie no more than 3 cM away from the EXPB1 locus to cause this skewed ratio. The EXPB1 locus is located on chromosome 9 which has a genetic distance of approx. 150 cM EXPB1 locus represents <4% of the genetic distance of chromosome 9 in maize (chromosome n\u200a=\u200a10). It would be extremely unlikely to find in this small region a second mutation with the precise phenotype described in this paper.Our findings reveal that a 31% reduction in Zea m 1 caused by a transposon insertion into the EXPB1 does not perform a vital role in pollen development or in the internal growth processes of the pollen tube per se. Previous in vitro studies demonstrated that Zea m 1 loosens the cell walls of silks and other studies showed that Zea m 1 is secreted by pollen upon hydration and tube growth EXPB1 is gametophytically expressed because the performance of pollen from heterozygous plants depended upon whether the pollen carried the EXPB1 or the expb1 allele. If the protein (or the mRNA) from EXPB1 was sporophytically produced  and then moved into the pollen, there would be no difference in the performance of pollen bearing different alleles. A few pollen expressed genes have already been shown to affect pollen tube growth rates in vivo  access to the stylar resources necessary for their growth Group-1 pollen allergens (\u03b2-expansins) have been detected in every grass [Because of the association that we observed between Zea m 1 production and breeding success under conditions of pollen competition, it would be difficult for plant breeders to develop and maintain low allergenic cultivars of turf, pasture, and agricultural grasses. This does not bode well for the 400 million people worldwide who suffer from hay fever and seasonal asthma due to this abundantly expressed \u03b2-expansin  Mu insertion in EXPB1  in the progeny of plants that were both self pollinated and crossed to wild type (EXPB1/EXPB1) silks. Ears were allowed to set seed and progeny of both crosses were screened for the presence of Mu in EXPB1 , cleaned by passing through a series of sieves, and stored separately at \u221280\u00b0C. Approximately 25 mg of maize pollen was extracted in 4 volumes (0.1 mL) of 50 mM sodium acetate, pH 4.5, for 1 h at 4\u00b0C. The extract was centrifuged at 20,800 g for 10 min. Proteins in the supernatant were quantified colorimetrically with the Coomassie Plus\u00ae Protein Assay Reagent  according to the manufacturer's instructions.In order to assess the impact of the These proteins were then subjected to 2-dimensional gel electrophoresis. For the first dimension \u2013 isoelectric focusing (IEF) \u2013 Immobiline DryStrip gels  and IPG buffer (pH 6\u201311) were obtained from GE Healthcare Bio-Sciences Corp. . The gels were rehydrated for 16 h with the rehydration buffer  containing the protein extracts and then were focused in a PROTEAN IEF cell apparatus  at the following program: running temperature: 20\u00b0C; maximum current: 50 \u00b5A/gel; Step 1: 200 V for 30 min (linear ramp); Step 2: 300 V for 30 min (rapid ramp); Step 3: 8,000 V for 150 min (linear ramp); Step 4: 8,000 V for 55,000 Vh (linear ramp). After the completion of IEF, the gels were incubated for 15 min in SDS equilibration buffer  with 10 mg/mL dithiothreitol and then switched into the same buffer containing 25 mg/mL iodoacetamide for another 15 min. For the second dimension, proteins were separated by discontinuous SDS-PAGE in a Criterion Dodeca Cell apparatus  using 12.5% precast gels. 2-D gels were stained for protein with SYPRO Ruby  according to the manufacturer's instructions and quantified using a laser scanner (Molecular Imager FX Pro PLUS from Bio-Rad) and 2-D image analysis software (PDQuest Version 7.3 from Bio-Rad). The protein marker, Mark12 Unstained Standard, for SDS-PAGE, was from Invitrogen Inc. .To identify the \u03b2-expansins, the resulting 2-D gels were then subjected to immunoblot analysis. This analysis was performed in a Bio-Rad Criterion blotter as described by Li et al. Mu insertion into EXPB1 on pollen viability, we collected pollen at anthesis from 20\u201322 plants from each of the three genotypes (EXPB1/EXPB1 (N\u200a=\u200a20), EXPB1/expb1 (N\u200a=\u200a22), expb1/expb1 (N\u200a=\u200a20) plants), stained it with thiazolyl blue to assess membrane integrity\u2014a trait that is highly correlated to germinability in vitro growth of pollen tubes from each of the three genotypes by sprinkling the pollen from 20\u201322 plants of each genotype onto Petri plates containing a maize pollen germination and pollen tube growth media To assess the impact of the Mu insertion into EXPB1 on the competitive ability of pollen in vivo, we experimentally manipulated the intensity of competition between mutant and wild type pollen. Pollen from field grown, heterozygous (EXPB1/expb1) plants was collected, cleaned, aliquoted into 50, 100, 250 and 500 \u00b5L volumes, and sprinkled over virgin silks of true breeding wild type plants . We reasoned that under conditions of intense pollen competition  only the fastest growing pollen tubes would achieve fertilization, whereas under conditions of little or no pollen competition  both the fast and slowly growing pollen would achieve fertilization. A random sample of 30 seeds from each of the 16 ears  was assessed for the presence of the mutant allele (expb1) by PCR.To determine the effect of the EXPB1 and expb1 pollen tubes through maize silks. We pollinated the silks of 16 wild type plants (EXPB1/EXPB1) with pollen from either EXPB1/EXPB1 or expb1/expb1 plants. On eight plants (four from each type of pollination), we removed the silks after 8 h; stained the silks with 0.1% aniline blue for 30 min and examined 10\u201314 silks from the central region of each ear for the presence of pollen tubes within the region from 7.5 to 8.5 cm from the site of pollen deposition using fluorescence microscopy We also directly examined the growth of"}
+{"text": "A study was made of the incidence of p53 mutations in Japanese males with prostate cancer or benign prostatic hyperplasia. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) was used as a primary screening technique with gene sequencing being carried out in positive cases. Two out of 21 prostate cancers (9.5%) were found to have p53 mutations. These were stage B2 and D2 prostate cancers. No abnormalities were found in the remaining cases or benign prostatic hyperplasia. Mutations of the p53 gene would thus appear infrequent in the tumourigenesis of primary prostate cancer."}
+{"text": "The reactivation of U.V.-irradiated adenovirus 2 in HeLa cells is enhanced 8-9 fold if the cells are given a brief hyperthermic shock before infection. Maximum reactivation is achieved by heating for 10 min at 45.5 degrees C and with a delay of 36 h between heating and infection. The induction process requires protein synthesis only during the 3 h period immediately following heating; cycloheximide does not prevent the expression of enhanced reactivation if added to the cells after this time. Heat-enhanced reactivation exhibits properties similar in some respects to radiation-enhanced reactivation and indicates an increased capacity of the heated cells to tolerate DNA damage."}
+{"text": "Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) appears to play an key role as both a virulence factor and as a target of naturally acquired immunity var genes of which there are 60 variants in every genome var sequences is now available from clinical isolates around the world var genes contain a DBL1\u03b1 region var genes to create DBL1\u03b1 sequence tags var genes and the fact that they undergo intra-genic recombination A rapidly growing collection of var genes that supports the use of information present in DBL1\u03b1 sequence tags in making comparisons between the expression levels in different isolates. Analysis of the fully sequenced genome of a single P. falciparum isolate 3D7 suggests that the genomic location of the 60 var genes promotes genetic structuring and the maintenance of genetically distinct sequence types var tag grouping system, though it is based on portion of the DBL1\u03b1 domain .Further support for the cysteine/PoLV groupings comes from recent publications. Trimnell et al. found a good correspondence between cysteine/PoLV groupings of cys2 sequences and groups defined phylogenetically within a globally sampled subset of var genes from 3D7, HB3 and IT4 var gene classification. With the exception of group 6 sequences which were not found in HB3 var genes all sequence groups were represented. In all three genomes cysteine/PoLV group 1 sequences are exclusively found in group A var gene and long genes with >5 domains whereas cysteine/PoLV group 5 are found only in non-group A genes and those with 4\u20135 domains. Cys2 sequence tags (groups 1\u20133) were never found in group C var genes.Kraemer et al. have recently performed an analysis and re-classification of whole Kyriacou et al. used a phylogenetic approach to compare DBL1\u03b1 sequence tags from Mali var genes from South American isolates in support a recent study of Amazonian isolates At a higher level of resolution, the distinct sequence identifier (DSID) see A is a poSince the cysteine/PoLV system of classification is based on commonly occurring sequence features it is hoped that it will useful for initial analysis and annotation, comparison of different geographical regions over time and identification of unusual sequences."}
+{"text": "SLC11A1 is known to link infections, autoimmunity and cancers. A review is presented of the mechanisms by which a balance is maintained between infections caused by pathogens  and disorders resulting from (acute or chronic) inflammation, and of the interactions that determine how the initial innate immune system directs subsequent acquired immune responses in human populations Slc11a1 in mice encodes a polytopic integral 10\u201312 transmembrane protein, which is expressed exclusively in macrophages and polymorphonuclear leukocytes and neurons (This review discusses the role of the solute carrier family 11 member 1 protein (SLC11A1), formerly NRAMP1 , in linking infections, autoimmunity and cancer. Chronic inflammation is caused by a variety of factors including bacterial, viral and parasitic infections, chemical irritants and nondigestible particles. Chronic inflammation is associated with a predisposition to cancer and autoimmunity because the longer the inflammation persists, the higher the risk of associated autoimmunity and carcinogenesis. Immune cells involved in inflammation include leucocytes such as neutrophils, monocytes, macrophages and eosinophils. These cells provide the soluble factors that are thought to mediate the development of inflammation. When inflammation goes unchecked it often leads to chronic inflammation-associated diseases. 2+) levels  levels . Thus, sMycobacterim bovis infection are significantly more acidic than those formed in slc11a1-negative ones .SLC11A1 should lead to apoptosis, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. By contrast, transcriptional repression of SLC11A1 could lead to cell survival. SLC11A1 might influence immune responses to viral vector delivery systems 5 ac (gt)5 ac (gt)9 g  drives high gene expression, while the allele t(gt)5 ac (gt)5 ac (gt)10 g  drives low gene expression , at 0.1%, is similar to that in the African populations  and within intronic regions, thus suggesting that this gene might be differentially regulated in different species. One major difference at this locus between species includes the type and locations of transcription factor binding sites in the 5\u2032UTR of SLC11A1. Of particular importance is the binding site for the activating transcription factor-3 (ATF-3; also known as an AP-1-like element, Homo sapiens, Pan troglodytes and Macaca mulatta, this sequence is absent in Mus musculus. In Rattus norvegicus and Pongo pygmaeus, the second cytosine is replaced by a thymine. ATF-3 is a negative regulator of TLR4 (SLC11A1 but it is unclear whether it is an active ATF-3 binding site in SLC11A1. The formation of Z-DNA structure is known to play an active role in modulating inhibitory chromatin structure (SLC11A1 it should repress transcriptional activation of SLC11A1; conversely, heterodimerization of ATF3 with other transacting factors should promote transcriptional activation of SLC11A1.Agents that activate macrophage together with host SLC11A1 alleles will exert different effects on SLC11A1 function, but how SLC11A1 functions help determine the outcome of infection by modulating the production of these cytokines in not known. Comparative analysis of the  of TLR4 , where iIt could be that transcriptional activation of SLC11A1 leads to apoptosis and cell death while transcriptional repression of SLC11A1 leads to cell proliferation and survival if unchecked could result in cancer and autoimmunity. The ATF-3 putative site in the promoter of SLC11A1 along with other transacting factors might have a regulatory role in modulating the effects of SLC11A1. This review has thus highlighted some of the mechanisms by which SLC11A1 might be mediating, influencing or modulating the outcome of these diseases and disorders in human populations.Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation."}
+{"text": "Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH].The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome.The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2. Hepatitis C virus infects approximately 170 million individuals' world wide ,3. HepatDQ417427], with no definable subtype or indication of a mixed genotype infection with a strain of genotype 4. In light of this fact, clonal analysis of the NS5B region from HC9A98987 was undertaken. 14 clones were sequenced [DQ417439\u2013DQ417452]. A search for similarities with previously published sequences was performed with the program Blast.  and not the expected genotype 2 or 4. The corresponding amino acid sequences analysis indicated that there were only 7 unique isolates, GenBank .Following routine RLPH of specimen HC9A98987 it was noticed that an unusual banding pattern indicative of a putative genotype 2 and 4 mixed infection was evident Figure . This inm Blast.  The resuDQ417428\u2013DQ417433] and (ii) the HVR1 sequences [DQ417434\u2013DQ417438] indicated that these regions of the genome were in fact homologous to viruses within the 2k subtype . Two clones from the core region had synonymous mutations . The HCV 2k  isolate has a relatively high prevalence in the countries of the former USSR. Kalinina et al reported in 2002 the identification of a natural intergenotypic recombinant form of HCV which had homology to 2k at the 5' end of the genome and to 1b at the 3' end of the genome  and a phylogenetic analysis was carried out. Phylogenetic trees were constructed with the NEIGHBOR program in the PHYLIP package and genetic distances were calculated with PRODIST program in the PHYLIP package based on Kimura's distance.  (2k/1b)] was recovered by nested RT-PCR [Table DQ839386\u2013DQ839389] with a minor quasispecies [DQ839390]. Sequence analysis indicated that this region of specimen HC9A98987 is homologous with the reference strain [AY587845]  2k/b] was re] Figure . This isAY587845 (2k/1b), the laboratory investigated whether an additional specimen  from another individual with the same RLPH pattern was also a candidate natural intergenotypic recombinant form. Clonal analysis of the NS2 region was performed on this specimen from the second patient (HC9A99966). The sequences of six clones were determined. All six clones were of identical nucleotide sequence [DQ904446] and were found by sequence analysis to be homologous to the natural intergenotypic recombinant related to HC9A98987, i.e., AY587845 (2k/1b) . Nucleotide sequences reported in this study have been assigned the following accession numbers which were derived from HC9A98987 Genbank: [DQ417427] (5'UTR). [DQ417428 to DQ417433] (Core sequences), [DQ417434 to DQ417438] (HVR1 sequences), [DQ417439 to DQ417452] (NS5B sequences), [DQ839386 to DQ839390] (NS2 sequences). [DQ904446] was derived from the NS2 sequence from sera HC9A99966.Reference strains used in the present study were all obtained from GenBank [Hepatitis C Virus HCVUntranslated Region UTRReverse Line Probe Hybridisation RLPHHuman Immunodeficiency Virus HIVThe author(s) declare that they have no competing interests.IM Carried out NS2 clonal analysis, sequence alignment and phylogenetic analysis. Preparation of manuscriptSH Performed core, HVR1 and NS5B sequence/cloningJL Determined qualitative, quantitative and initial genotype of specimens described herePS Assisted SH in RT-PCR and amplicons cloningOC & EKW Clinicians who manage HCV at Cork University HospitalLJF Supervised project and assisted with analysis and preparation of manuscript."}
+{"text": "The tumour suppressor gene p53 has been found to be mutated or inactivated at high frequency in several common human tumours. We have examined a series of exocrine pancreatic carcinomas for over-expression of mutant forms of p53 by immunohistochemistry with a panel of specific antibodies. We found immunodetectable p53 in 13 of 22 (60%) frozen pancreatic cancers and seven of 13 pancreatic cell lines. One of the antibodies, CM1, recognises p53 in formalin-fixed, paraffin-embedded archival material and using this reagent we found immunodetectable p53 in 28 of 124 (23%) pancreatic cancers. We have successfully demonstrated the presence of point mutations by direct sequencing of genomic DNA extracted from archival tissue showing CM1 immunoreactivity. We conclude that p53 activation is an important event in human pancreatic tumorigenesis and that the CM1 antibody can detect a proportion of cases of overexpression of mutant p53 in archival pathological material."}
+{"text": "A Phase 2 trial was conducted using intramuscular lymphoblastoid interferon , 4 MU per day, in 10 patients with chemotherapy-resistant teratomas. There was stabilisation of disease in 2 patients both of whom were in retrospect considered to have had differentiated teratoma at the time of IFN administration. There was progression of presumed active anaplastic germ cell tumour in 8 patients. One of these patients, a 15-year-old boy with biopsy proven differentiated teratoma has received 2 courses of lymphoblastoid IFN and 1 course of recombinant leukocyte A IFN (Roche Products Ltd.) lasting 5 1/2, 8 and 8+ months respectively. He has had a mixed response in his differentiated tumour which on each occasion has been maintained for the duration that he received IFN. Rising HCG levels during his second course of interferon required additional cytotoxic chemotherapy. Lymphoblastoid IFN does not appear to be active against anaplastic germ cell tumours but both lymphoblastoid and recombinant leukocyte A IFN may be useful in the treatment of unresectable differentiated teratoma."}
+{"text": "The pattern of TGF beta expression and in vitro response to TGF beta has been defined in three ovarian carcinoma cell lines . Marked differences in both mRNA expression and growth responses were detected between the cell lines. All expressed mRNA for TGF beta 3, PEO1 and PEO4 but not PEO14 expressed mRNA for TGF beta 1, whereas PEO14 but not PEO1 and PEO4 expressed TGF beta 2. Growth of PEO14 cells in culture was markedly inhibited by both TGF beta 1 and beta 2. PEO1 cells were inhibited by TGF beta 1, but not TGF beta 2 whilst growth of PEO4 cells were not affected by exposure to either of these peptides. These data indicate that several elements of potential autocrine loops involving TGF beta's are present within ovarian cancer cells."}
+{"text": "Gli3 gene encodes a zinc finger transcription factor and homozygous loss-of-function mutations of Gli3 are lethal. Humans heterozygous for mutations in this gene suffer Greig cephalopolysyndactyly or Pallister-Hall syndromes, in which limb defects are prominent, and mice heterozygous for similar mutations have extra digits. Here we examined whether eye development, which is abnormal in mice lacking functional Gli3, is defective in Gli3+/- mice.Knowledge of the consequences of heterozygous mutations of developmentally important genes is important for understanding human genetic disorders. The Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes.We showed that Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.These findings indicate that heterozygous mutations of  GLI3 allele can cause Greig cephalopolysyndactyly (GCPS) or Pallister-Hall Syndrome (PHS) . Mut and man ,17. Miceorm eyes ,18,19 wh defects . There i defects  and losspression -26. ThusAs shown in Fig. Gli3+/-, Pax6+/- and Gli3+/-;Pax6+/- eyes, immunostained for Pax6 expression, are shown in Fig. The appearances of E14.5 +/- eyes appeared slightly larger than those of wild-type siblings, although their appearances were otherwise normal  was significantly less than that of wild-type eyes     was significantly less than that of wild-type eyes , but not significantly different to that of Pax6+/- eyes. There was much greater variation than among eyes of the other genotypes  showed no retinal dysplasia  Some retinae exhibited no dysplasia but were thinner than those of other genotypes (15/28) 28) Fig. . 2) Some28) Fig.  with arePax6+/-, Gli3+/- and Pax6+/-;Gli3+/- animals. Compound heterozygotes that had retinal dysplasia in the regions measured were excluded. Whole retinal thickness was significantly reduced in Pax6+/-;Gli3+/- animals compared to their wild-type, Pax6+/- and Gli3+/- siblings, all of which had similar thicknesses , the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL) and the ganglion cell layer (GCL). We found that the thicknesses of both the ONL and INL of Pax6+/-; Gli3+/- retinae were significantly reduced compared to those of the other genotypes , the OPL was not discernible; where this layer was present, its average thickness was not significantly different to that of wild-type animals   showed highly dysmorphic and dysgenic lenses, which were not observed in any of the other genotype groups  Fig. , which w28) Fig.  in one Pups Fig. .Pax6+/- eye has been reported to be thinner than normal and the corneal stroma is hypercellular [Gli3+/- animals  was significantly thinner than that of wild-type (n = 16) and Gli3+/- (n = 27) eyes, both centrally and peripherally . A similar result was observed in Pax6+/-; Gli3+/- eyes (n = 25) . Overall, these data suggest that loss of one copy of both Pax6 and Gli3 together does not create a corneal epithelium thinner than that resulting from loss of Pax6 alone.Thicknesses of the corneal epithelium and corneal stroma, centrally and peripherally, were measured for the four genotype groups. There was no difference in both central or peripheral corneal epithelium thickness between wild-type and yes Fig. . As prevPax6+/- or Gli3+/- eyes compared to wild-type eyes . Thus, the mean stromal thickness  of Pax6+/-; Gli3+/- eyes was significantly less than that of wild-type , Pax6+/-  and Gli3+/-  eyes.There was no difference in either central or peripheral corneal stromal thickness in yes Fig. . The perPax6 [Gli3[Pax6 results in various eye defects [Gli3 results in eye defects. Thus, in this study, we set out to characterise the morphology of the Gli3+/- eye. Furthermore, because both Pax6 and Gli3 are expressed in the optic cup/retina, lens and iris during development [Pax6 and Gli3 play mutually co-operative roles in eye formation by studying animals compound heterozygous for mutations in both Pax6 and Gli3.The importance of the transcription factors Pax6 ,18,19 anax6 [Gli3,9,10 in  defects -22, littelopment ,18,27-29Gli3+/- eyes that we examined. Where abnormalities were seen, they were very mild. Gli3+/- eyes had a slightly higher mass than wild-type eyes as well as a thicker OPL compared to wild-type retinae. Retinal folding has been described previously in some neonatal Gli3+/- animals [Gli3+/- eyes are either not detectable or minor.No abnormality was observed in the majority of the  animals . OverallPax6 and Gli3 exhibited unique and more severe eye defects than either Pax6+/- or Gli3+/- animals or than would be predicted by addition of their phenotypes. As previously described [Pax6+/- adult eyes was significantly smaller than that of wild-type eyes. The additional loss of one copy of Gli3 resulted in eyes with a wide range of eye mass, from some eyes with smaller mass than Pax6+/- eyes to some eyes which fell within the wild-type range of eye mass.Animals compound heterozygous for mutations in both escribed , the masPax6+/-; Gli3+/- eyes. Some Pax6+/-; Gli3+/- retinae were thinner than normal but exhibited normal lamination while others were severely dysgenic with considerable laminar dysmorphology. The severe retinal defects found in Pax6+/-; Gli3+/- animals were never seen in either Pax6+/- or Gli3+/- animals. One mechanism by which Pax6 and Gli3 might cooperate during retinal development is through their actions in the Shh signalling pathway. This pathway is critical for normal eye development. Shh from the ventral midline of the early neural plate stage embryo is required for early patterning of the visual system [Gli3 can partially rescue the Shh-null phenotype [Gli3+/- mice) mimics the effects of raising Shh levels. There is evidence that Shh suppresses Pax6 expression [Gli3  might not lower Pax6 levels enough to create morphologically detectable defects if both alleles of Pax6 are functional, levels might fall below a critical threshold in Pax6+/-;Gli3+/- heterozygotes. Furthermore, there is evidence that Pax6 suppresses Shh expression [Pax6+/-;Gli3+/- heterozygotes might allow increased production of Shh, thereby further exacerbating the overstimulation of the Shh pathway. The mechanism by which reduced levels of the repressor form of Gli3 result in reduced Pax6 levels might involve loss of repression of intermediate transcription factors such as Pax2 and Vax1, that themselves repress Pax6 [A range of retinal phenotypes was observed in l system . Later, l system  and has l system ,31. Gli3l system . Lowerinhenotype , suggestpression ,33, and pression -26 and sess Pax6 . These pess Pax6 ,34Pax6+/-; Gli3+/- animals were more severe than those of either Pax6+/- or Gli3+/- animals. Pax6+/-; Gli3+/- animals had highly dysgenic lenses which were never seen in Pax6+/- or Gli3+/- animals. There were particularly high incidences of abnormal contacts involving the iris in Pax6+/-; Gli3+/- animals and, in contrast to the iris hypoplasia observed in Pax6+/- animals, many Pax6+/-; Gli3+/- animals exhibited hyperplastic irises. Together these data suggest not only that the iris is sensitive to levels of Pax6 expression but that normal Gli3 expression may also be required, in conjunction with Pax6, for normal iris development to occur. Pax6+/-; Gli3+/- animals also showed thinning of the corneal epithelium and disorganisation of the corneal stroma, similar to that observed in Pax6+/- animals. Unlike Pax6+/- and Gli3+/- animals, however, the peripheral corneal stroma was significantly thinner than in wild-types. As Gli3+/- animals showed no difference in the thickness of the corneal stroma, the reduction of Gli3 dosage alone does not affect stromal thickness but does so only in combination with reduced Pax6 dosage.Like the defects in the retina, abnormalities in the anterior segment of the Pax6 and Gli3 have eye abnormalities that are either not present in, or are more severe than, those present in animals heterozygous for mutations in either gene. Addition of the phenotypes of Pax6+/- and Gli3+/- animals is insufficient to explain the severe phenotypes of the compound mutants. Our findings suggest that Gli3 and Pax6 cooperate during eye morphogenesis.We have found that animals compound heterozygous for mutations in both Pax6+/-  strain was maintained on a mixed  background as described previously [Gli3+/- strain  was maintained on an inbred CBA/Ca background. Animals wild-type at the Pax6 and Gli3 loci, heterozygous for either mutation (Pax6+/- or Gli3+/-) and compound heterozygous for mutations in both genes  were obtained by crosses between Pax6+/- and Gli3+/- animals. 65 animals from 17 litters were analyzed. All comparisons were between wild type, Pax6+/-, Gli3+/- and Pax6+/-; Gli3+/- siblings. Animals were killed by cervical dislocation; eyes were removed and some were weighed before fixing for histological preparation. All animal procedures were performed in accordance with Home Office (UK) legislation.The eviously . The GliEyes were fixed in either 4% paraformaldehyde or Bouin's fluid overnight before dehydrating and processing to paraffin wax. Bouin's fluid was selected for tissue for morphometric analysis as it causes few artefacts of tissue fixation. Sections were cut at 10 \u03bcm and stained with haematoxylin and eosin. Midsections through the eye containing the optic nerve were identified for morphometric retinal and corneal analysis.Pax6 gene were assessed as described previously [Gli3XtJ allele has revealed a 51.5-kb deletion [Gli3 and mutant Gli3XtJ alleles were identified using the following primers: 580For 5' TACCCCAGCAGGAGACTCAGATTAG-3' and 580Rev 5'-AAACCCGTGGCTCAGGACAAG-3'; C3For 5'-GGCCCAAACATCTACCAACACATAG-3' and C3Rev 5'-GTTGGCTGCTGCATGAAGACTGAC-3' producing products of 193 bp and 580 bp for the wild-type and mutant alleles respectively.Mutations in the eviously . Mappingdeletion . Using adeletion , wild-tyMorphometric analysis of the retinal layers was performed only in eye sections where normal laminar morphology of the retina was maintained. Animals with eyes which were severely dysplastic were not included in this analysis. Transverse eye sections at the level of the optic nerve  were photographed with a digital camera at \u00d740. The thicknesses of the whole retina as well as the thicknesses of the inner nuclear layer (INL), outer nuclear layer (ONL), inner plexiform layer (IPL) and outer plexiform layer (OPL) were measured using ImageTool\u2122. Two separate measurements were taken on either side of the optic nerve Fig.  and averAnalysis of corneal epithelial and stromal thickness was performed essentially as described previously . Serial Slides were microwaved in 10 mM sodium citrate to achieve maximal antigen retrieval before addition of primary antibody. Mouse monoclonal anti-Pax6 antibody (1:100) was obtained from the Developmental Studies Hybridoma Bank . Signal was enhanced using the Dako ABC Kit and visualised with diaminobenzidine (DAB).A 611 base pair fragment comprising nucleotides 560\u20131170 of the mouse Gli3 cDNA (a gift from T. Theil) was PCR amplified and subcloned into a pGEM-Teasy vector (Promega). The plasmid was linearized with SpeI and transcribed with T7 RNA polymerase. Non-radioactive RNA in-situ hybridisation on paraffin wax-embedded sections was carried out using a protocol described previously .GCPS Greig cephalopolysyndactylyDAB diaminobenzidineFGF8 fibroblast growth factor 8GCL ganglion cell layerINL inner nuclear layerIPL inner plexiform layerONL outer nuclear layerOPL outer plexiform layerPHS Pallister-Hall SyndromeShh Sonic hedgehogXt extra-toesPZ and JQ designed and carried out most of the experiments, MC participated in the quantitative analysis, JT carried out in situ hybridizations, IS participated in the design and carrying out of genotyping, DP participated in the analysis and writing of the manuscript."}
+{"text": "We have investigated X-ray survival parameters and repair of potentially lethal damage ( PLDR ) in ten early passage squamous cell carcinoma cell lines derived from patients who were biopsied before initiation of radiotherapy or after radiation therapy failure. Radiosensitivity (D0) ranged from 1.07 to 1.93 (Gy), extrapolation numbers (-n) from 1.17 to 2.14 and PLD recovery at 24 h from 1.4 to 20.3. Despite significant differences in these parameters amongst the cell lines, a firm correlation between radiocurability and any individual radiobiological parameter could not be established. Our data suggest that the mechanisms associated with radioresistance are complex and that any single radiobiological parameter may not predict clinical success or failure."}
+{"text": "The distribution of labelled cells through 5 different mouse tumours was measured after a single injection of [3H]-thymidine [( 3H]-TdR) or [3H]-deoxyuridine [( 3H]-UdR). All the tumours had areas where the percentage of labelled cells  was high and areas where the LI was very low. The total area with a low LI was greater after [3H]-TdR than after [3H]-UdR injection in all 5 tumours. In one of the tumours, carcinoma NT, repeated injections of [3H]-UdR at 2 h intervals caused the areas of high LI to spread, eliminating all areas of low LI in many specimens. When 5-fluorodeoxyuridine (FUdR) was injected, to block de novo DNA synthesis in carcinoma NT, [3H]-TdR was incorporated by many more cells. The LI was increased throughout the tumour and no area had a LI below 20% after FUdR plus [3H]-TdR. After flash-labelling with [3H]-TdR alone, nearly half the tumour had a LI below 20%. We conclude that the labelling seen after FUdR plus [3H]-TdR represented the true distribution of S phase cells in carcinoma NT. Routine flash-labelling with [3H]-TdR or [3H]-UdR left nearly half the S phase cells unlabelled and gave an erroneously low value for the proportion of DNA synthesising cells in the tumour. The results suggest that many tumour cells have very large endogenous nucleotide pools which cannot be flooded by a single injection, even of [3H]-UdR."}
+{"text": "Administration of adenosine triphosphate (ATP) in sinus rhythm identifies dual atrioventricular node physiology (DAVNP) in 75% of patients with inducible slow / fast AV nodal reentrant tachycardia (AVNRT). The incidence of DAVNP following termination of AVNRT with ATP is unknown. Incremental doses of ATP (10-60mg) were administered, first in sinus rhythm and then during tachycardia induced at electrophysiologic study, to 84 patients with inducible AVNRT and to 18 control patients with inducible AV reentrant tachycardia (AVRT) and no electrophysiologic evidence of DAVNP. Study end-points were the occurrence of DAVNP or \u2265 2nd degree AV block following administration of ATP in sinus rhythm and tachycardia termination following administration of ATP during tachycardia. Of the 82 patients with AVNRT who completed the study, 62 (75.6%) exhibited DAVNP following administration of 17.1 \u00b1 9.4 mg ATP in sinus rhythm, while 30 (36.5%) exhibited DAVNP at the termination of AVNRT following administration of 10.6 \u00b1 2.4 mg ATP. The occurrence of DAVNP following the administration of 10 mg ATP in sinus rhythm.was a good predictor (62%) of its occurrence after termination of AVNRT with ATP. The dose of ATP had a strong correlation between the presence of DAVNP following AVNRT termination and the ATP doses needed for tachycardia termination. Of the 18 control patients, none had DAVNP at ATP test during sinus rhythm but 1 (5.5%) showed slight (60 msec) PR jump after termination of AVRT with ATP. In conclusion, DAVNP is present in a relatively high proportion (36.5%) of patients following termination of AVNRT with ATP but is much less frequent (5.5%) in control patients. Thus, findings at termination of tachycardia by ATP may be useful in the noninvasive diagnosis of the mechanism of a paroxysmal supraventricular tachycardia. Atrioventricular nodal reentrant tachycardia (AVNRT) is the most frequently encountered type of regular, paroxysmal supraventricular tachycardia in clinical practice. The typical form, observed in >95% of cases of AVNRT, is due to a reentrant mechanism that involves a slow pathway in the antegrade direction and a fast pathway in the retrograde direction. Adenosine-5'-triphosphate (ATP) is almost always effective for terminating AVNRT usually by blocking the antegrade slow pathway . BesidesThe study group consisted of consecutive patients in whom the effects of the intravenous administration of ATP were tested both in sinus rhythm and during sustained (\u2265 1 min) slow / fast AVNRT induced at electrophysiologic study. All patients were evaluated in the absence of antiarrhythmic drug therapy and patients in whom isoproterenol was required to induce AVNRT were excluded. Most patients were included in another study dealing with the use of a simplified ATP test for the noninvasive diagnosis of dual AV nodal physiology and the assessment of the results of slow pathway ablation in patients with AVNRT .The control group consisted of patients who had inducible sustained AVRT involving a concealed accessory pathway and in whom the effects of the intravenous administration of ATP were tested both in sinus rhythm and during sustained (\u2265 1 min) AVRT. Patients were excluded if they had electrophysiologic evidence of DAVNP defined as sudden prolongation of AH by \u2265 50 msec when shortening the cycle length of atrial pacing or the coupling interval of the atrial extrastimulus by 10 msec. Most of these patients were part of a larger study that assessed the use of ATP in the noninvasive diagnosis of concealed accessory pathway .None of the patients participating in the study had a history of asthma (a contraindication to ATP administration), or was treated with drugs known to interfere with ATP metabolism .This protocol was approved by our Ethical Committee and all patients gave informed consent. The effects of ATP on AV nodal conduction in sinus rhythm were evaluated before the introduction of any electrode catheter into the cardiac chambers in order to avoid any catheter-induced arrhythmia or trauma to the fast or slow pathways . After pDAVNP was considered to be present when at least one of the following events occurred following ATP injection: 1) PR interval increased or decreased by \u226550 ms in 2 consecutive sinus beats; 2) an AV nodal echo beat was observed; 3) AVNRT developed. All 3 criteria were solely based on the analysis of simultaneous 12-lead surface electrocardiogram. Single AV nodal echoes are often difficult to discern without the aid of intracardiac recordings. However, AV nodal echoes would be expected to reset the sinus activity. Therefore, AV nodal echos were considered to be present when, following a sinus complex conducted with an increased PR interval: a) a >70% increment in P-P interval was observed or b) retrograde P waves were seen at the end of the QRS complex. We previously found a good correlation between these electrocardiographic criteria and the diagnosis of DAVNP using intracardiac recordings  reached a study end-point following administration of ATP in sinus rhythm and AVNRT and constituted the study group.Of the 82 study patients, 62 (75.6%) patients exhibited signs of DAVNP following administration of 17.1 \u00b1 9.4 (range 10 - 40) mg ATP. DAVNP occurred after administration of 10 mg ATP in 34 (55%) patients, after 20 mg in 22 (35%) patients and after \u2265 30 mg in 6 (10%) patients . This ocAVNRT was terminated by ATP in all 82 (100%) study patients. This occurred 6 to 25 (mean 16.3 \u00b1 4.9) sec following the ATP bolus. The doses of ATP that terminated the tachycardia were 10 mg and 20 mg in 77 and 5 patients, respectively (mean 10.6 \u00b1 2.4 mg). In all patients, tachycardia terminated due to antegrade block in the slow pathway. DAVNP at the termination of AVNRT was observed in 30 (36.5%) patients  . This oDAVNP after termination of AVNRT was more frequently observed in patients who exhibited DAVNP in sinus rhythm  than in those who did not  but the difference did not reach statistical significance (p=0.08).In the whole group of patients, the dose of ATP necessary for demonstrating DAVNP in sinus rhythm  was greater than that required to show DAVNP after conversion of AVNRT  (p <0.001). However, the time of onset of DAVNP in these 2 groups was similar . Also, the number of conducted beats over the slow pathway following ATP administration was not statistically different in the 2 settings . Among the 26 patients who exhibited DAVNP both in sinus rhythm and after termination of AVNRT, the doses of ATP required were 12.7 \u00b1 6 mg and 10.4 \u00b1 2 mg, respectively (p<0.05). The time of occurrence of DAVNP was shorter after ATP injection in sinus rhythm (14.7 \u00b1 3.3 sec vs 19.3 \u00b1 6.9 sec p<0.05); however, the number of conducted beats over the slow pathway following ATP administration was not statistically different in the 2 settings .There was a strong correlation between the ATP dose needed to demonstrate DAVNP in sinus rhythm and the odds for demonstrating DAVNP following AVNRT termination: 62% (21/34) of the patients who exhibited DAVNP following administration of 10 mg ATP in sinus rhythm, exhibited DAVNP following termination of AVNRT. In contrast, only 18% (5/28) of the patients who required \u2265 20 mg ATP for demonstrating DAVNP in sinus rhythm, subsequently exhibited DAVNP upon termination of AVNRT with any ATP dose (p<0.0005).This group included 18 patients [13 females and 5 males (age 30 \u00b1 14 years)] who had AVRT and no electrophysiologic evidence for DAVNP. Administration of ATP (15.6 \u00b1 7.4 mg) resulted in \u2265 second degree AV block without DAVNP in all patients. A mean dose of ATP (10.7 \u00b1 2.7 mg) terminated AVRT in all patients after 16 \u00b1 5 sec. A slight jump (+ 60msec) in PR interval was noted on 1 beat after tachycardia termination in only 1(5.5%) of the 18 patients.The present study shows that administration of ATP in sinus rhythm reveals the presence of DAVNP in 75.6% of patients with inducible sustained slow / fast AVNRT and in none of the control patients with no electrophysiologic evidence of DAVNP. These results are consistent with those found in our pilot study using ATP (2) and by others using adenosine [This incidence rate of DAVNP upon termination of AVNRT with ATP seems even more remarkable if one takes into account that the study end-point following administration of ATP during AVNRT was tachycardia termination rather than exposure of DAVNP. Since doses of ATP required to achieve DAVNP after drug administration in sinus rhythm were higher than those required for termination of AVNRT, it is tempting to speculate that higher ATP doses during AVNRT might have resulted in a higher incidence of DAVNP after tachycardia termination.As explained elsewhere  administThe occurrence of DAVNP following administration of ATP during AVNRT was more frequently observed in those patients who exhibited DAVNP following ATP administration in sinus rhythm than in those who did not. This difference, however, did not reach statistical significance. In contrast, the occurrence of DAVNP after termination of AVNRT with ATP best correlated (in 62% of patients) with its occurrence following the administration of 10 mg ATP in sinus rhythm.The present study was performed during the course of an electrophysiologic procedure and therefore its results cannot be extrapolated to the clinical setting. When adenosine compounds are administered in the acute management of spontaneously occurring AVNRT, this is generally after several hours of sustained tachycardia compared to 1 min of tachycardia as in the present study. Sympathetic activation resulting from hypotension and stress may be more marked during spontaneous, long-lasting tachycardias than during short-lasting laboratory-induced arrhythmias. Taking in account that adenosine's negative chronotropic effect is enhanced in the setting of adrenergic stimulation , one mayAlthough the diagnosis of AVNRT is usually easy when a 12-lead ECG is available, the actual diagnosis may remain difficult in some instances. In addition, single strip electrocardiographic leads are frequently the only available data, especially when the patient is treated in a mobile care unit. According to our study, the presence of DAVNP following tachycardia termination by ATP would suggest that the mechanism of the tachycardia is actually AVNRT. Caution should prevail, however, since DAVNP can also be present in other types of PSVT. For example, we recently found that DAVNP could be suspected with ATP test in sinus rhythm in 7 (21%) of 33 patients with AVRT ; howeverTermination of induced slow / fast AVNRT with ATP is followed by electrocardiographic evidence of DAVNP in a relatively high proportion of patients. Such a finding may be useful in the noninvasive diagnosis of the mechanism of paroxysmal supraventricular tachycardia."}
+{"text": "A biochemical response index comprising ESR, CEA and CA 15.3 was evaluated in 67 patients with systemic breast cancer treated by chemotherapy; 55 were assessable by UICC criteria and the response index . Marker changes at 2 and 4 months showed a highly significant correlation with the UICC assessed response at 3 and 6 months (P < 0.001); sensitivity 100%, specificity 87%; positive predictive value 85%; negative predictive value 100%. This index was then used to select out truly responsive patients and to prospectively direct their chemotherapy. Twenty-six responding  patients were randomised to discontinue cytotoxics after 6 months and move to maintenance hormones (n = 13) or continue chemotherapy whilst the biochemical markers kept falling or remained within the normal range. Biochemical progression prompted a change of chemotherapy. Continuous chemotherapy in biochemically defined responders was associated with a significant lengthening of remission duration and an improved quality of life and survival. We are now using the index to routinely direct chemotherapy and select out true responders for maintenance chemotherapy."}
+{"text": "Effects of recombinant tumour necrosis factor (TNF) on functional and structural vascular volumes in solid murine Meth A tumours were investigated by injection of Hoechst 33342 and staining for the vascular basement membrane component laminin, respectively. Systemic injection of 3 x 10(4) U TNF caused an initial increase in functional volume in the tumour, but a strong decrease from 1 to 48 h after treatment. Early effects of intralesional treatment were more moderate. Systemic injection of 10(4) U TNF or 0.3 or 3 micrograms lipid A caused a fall in functional volume at 4 h, but a recovery was seen at 24 h. This recovery did not occur after treatment with a combination of 10(4) U TNF and 0.3 micrograms lipid A. Structural vascular volume was not markedly reduced until 24 h after treatment with the high doses of the separate agents and the combination. All effects appeared generally more prominent in the tumour centre than in the borders. Data suggest that TNF induces initially an active hyperaemia that rapidly converts to passive hyperaemia. A prolonged disturbance of tumour blood supply is probably necessary for therapeutic activity. Breakdown of laminin in the vascular basement membrane may be a cause of loss of vascular integrity."}
+{"text": "Epidermal growth factor receptor (EGFR) was studied in ovarian tumours with immunohistochemical (IH) and ligand-binding assay (LBA). Two different monoclonal antibodies  with respect to detecting EGFR with different ligand-binding affinities  were used. When comparing the IH data of MoAbs, 2E9 and EGFR1 a significant correlation was found (2P < 0.0001). Both antibodies stained 77% of the adenocarcinoma samples. The incidence of positivity as well as the mean percentage of stained cells was increased in metastases when compared with primary lesions. In 12.5% overexpression of EGFR (score 3) was noticed in some of the tumour cells. This was not due to amplification of the EGFR gene in any of the 25 ovarian tumours studied (including 6 which showed high expression of EGFR in IH). EGFR was detected in 66% of the adenocarcinomas analysed with LBA. A statistically significant correlation was found between the maximum binding capacities of EGFR obtained from Scatchard plots and the percentage of positive tumour cells determined by MoAb EGFR1 (2P < 0.0001). A weaker correlation was found between the reactivity of MoAb 2E9 and LBA (2P < 0.1). Clinical studies are necessary to determine the possible prognostic impact of EGFR determined with either method, or whether a combination of both will give a better discrimination between high- and low-risk patients."}
+{"text": "CHEK2 1100delC allele has been identified at a particularly high frequency in families with hereditary breast and colorectal cancer.Development of multiple primary tumors is a hallmark of hereditary cancer. At least 1/10 of breast cancers and colorectal cancers occur because of heredity and recently the cell cycle kinase 2, CHEK2 1100delC to the development of such metachronous tumors.We utilized the Southern Sweden population-based cancer registry to identify women with double primary breast and colorectal cancer and sequenced tumor material in order to assess the contribution of the CHEK2 1100delC allele. which was not significantly different (p = 0.26) from the 1% (3/300) carriers identified in the control group.Among the 75 patients successfully analyzed, 2 (2.5%) carried the CHEK2 1100delC is not a major cause of double primary breast and colorectal cancer in Sweden, which suggests that this patient group should not routinely be screened for the CHEK2 1100delC variant.In summary, our data suggest that the  BRCA1 and BRCA2 genes are the major causes of hereditary breast and ovarian cancer, but the underlying genetic defect remains unresolved in the majority of the families with familial or hereditary breast cancer , through ATM phosphorylation [CHEK2 1100delC mutation leads to premature termination and abrogates kinase activity. This variant allele occurs in about 1% of normal populations, but has been identified at increased frequencies (1.2\u20131.9%) in individuals with breast cancer [CHEK2 1100delC mutation [CHEK2 1100delC allele have by some investigators been reported in non-BRCA1/2 families with hereditary breast cancer [CHEK2 1100delC variant does not show clear co-segregation with disease it is considered to be a modifier, and may act in cooperation with yet unidentified high-penetrance genes or together with multiple low-penetrant genes as part of polygenic inheritance.Double-strand DNA breaks lead to activation of the cell cycle checkpoint kinase 2, CHEK2 [GenBank:rylation . CHEK2 pt cancer ,8. An apt cancer . These dCHEK2 1100delC has been reported at frequencies of 1.6\u20132.6% among individuals with colorectal cancer, which is not significantly higher than in unaffected individuals from the population, but these studies may be compatible with a risk of 1.5\u20132.0 for development of colorectal cancer among carriers [CHEK2 1100delC allele has been reported in families with hereditary breast and colorectal cancer where 10/55 Dutch families (18%) carried the mutation [In studies from Finland and the Netherlands the carriers -16. A paCHEK2 1100delC mutation to the development of metachronous cancers of these types.Since development of multiple primary tumors in an individual is a sign of hereditary cancer we utilized a population-based cancer registry to identify women who had developed breast cancer and colorectal cancer in order to assess the contribution of the Ethical approval was obtained from the ethics committee at the Lund University. We utilized the population-based cancer registry of the Southern swedish health care region (which currently has approximately 1.7 million inhabitants). The registry was established in 1958 and because of mandatory cancer registration for clinicians as well as for pathologists the registry is estimated to contain 98% of all cancers diagnosed in this area. All females diagnosed with at least one breast cancer and one colorectal cancer during the time period 1958\u20132000 were identified. Totally 96 patients were identified in whom breast cancer was the first tumor in 64 and colorectal cancer was the first tumor in 25, whereas the remaining 7 patients developed synchronous breast and colorectal cancer. The mean age at diagnosis of the first of tumor was 68 (range 40\u201395) years, breast cancer developed at mean age 70 (range 40\u201395) and colorectal cancer at age 74 (range 47\u201398). After omission of patients who were misclassified or from whom the tumor blocks could not be retrieved, 84 patients remained for analysis.Tumor-containing paraffin-embedded tissue and normal tissue (e.g. resection borders or benign lymph nodes) were retrieved. Fresh sections were obtained and stained with Hematoxylin & Erythrosin in order to verify that tumor tissue was present in the samples. DNA was extracted from 3 \u00d7 10 \u03bcm paraffin sections using treatment with Proteinase K in 65\u00b0C for at least 2 hours and boiled for 10 minutes for enzyme inactivation, whereafter the samples were centrifuged and the aqueous phase was removed for use.CHEK2 exon 10. The primers used were 5'-TGGCAAGTTCAACATTATTCCC-3' (forward) [Mutation analysis was carried out using dHPLC  and samples with aberrant patterns were further analyzed by direct sequencing . Due to homologous sequences primer design is complex for forward)  and 5'-ACHEK2 1100delC mutation was detected in 2 patients (2.5%)  were successfully analyzed. In total, 36 breast cancers and 67 colorectal cancers were analyzed and from 34 patients normal tissue was also analyzed. The majority of the patients developed breast cancer as their first tumor table  and 68 oCHEK2 1100delC in the Southern swedish population dHPLC analysis and direct sequencing was performed from 300 healthy individuals with identification of the variant allele in 3/300 individuals, thus at a population frequency of 1% [In order to determine the expected frequency of the cy of 1% .et al. already in 1972 [CHEK2 1100delC mutation was proposed to represent a low-penetrant breast cancer susceptibility allele [CHEK2 1100delC had been described in hereditary breast and colorectal families, we assessed the contribution of this variant to the development of double primary breast and colorectal cancer in a population-based patient material. Among the 75 patients with metachronous tumors of the breast and the colorectum successfully studied, 2 (2.5%) carried the CHEK2 1100delC mutation compared to 1% in the control group [per se not alone to identify individuals with a high likelihood of being carriers of the CHEK2 1100delC mutation. Although development of double primary tumors may be a sign of heredity it is not enough to recommend genetic analysis, though the family history of cancer should be carefully reviewed. Our findings are in line with previous studies that exclude CHEK2 1100delC as a major contributor to the breast and colorectal cancer phenotype [et al. [CHEK2 1100delC mutation among 24 patients from the US with breast cancer and colorectal cancer. Also, in the study by Meijers-Heijboer et al. that primarily identified the link between CHEK2 1100delC and familial breast and colorectal cancer in Dutch families, the vast majority, 45/55 families, who fulfilled these criteria did indeed not carry this variant [CHEK 1100delC mutation.A subtype of familial breast cancer that includes colorectal cancer was recognized by Lynch y allele . Since dol group . These fhenotype . Huang e [et al.  did not  variant . HoweverCHEK2 1100delC allele in our study, one presented with two separate, synchronous, breast cancers. An increased risk of multiple primary breast cancers (OR 5.7\u20136.5) has been reported in individuals carrying this CHEK2 1100delC allele [CHEK2 1100delC mutation has not only been suggested to act as a low-penetrant susceptibility gene in breast cancer families, but the CHEK2 I157T alteration has been proposed to act as a multiorgan cancer susceptibility allele based on observations of an increased risk for development of breast cancer, colon cancer, prostate cancer, thyroid cancer, and renal cancer in Polish families [et al. who identified an increased risk of renal cancer associated with CHEK2, albeit in carriers of the CHEK2 I157T variant [Among the two women with the C allele ,22. Howefamilies . Both pa variant . HoweverCHEK2 1100delC occurs at a low frequency in Swedish women with double primary breast cancer and colorectal cancer, and thus suggests that development of these two tumor types is not sufficient to recommend mutation analysis of CHEK2.In summary, our findings demonstrate that the The author(s) declare that they have no competing interests.AI conceived of the study, performed the sequencing analysis, interpret data and drafted the manuscript. MB also carried out the sequencing. AB participated in its design and helped to draft the manuscript. MN conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Thirty-four primary, untreated sporadic breast cancers were examined for loss of heterozygosity (LOH) at tumour suppressor loci involved in colorectal cancer: APC/MCC at 5q21 and DCC at 18q21. LOH was identified in 28% informative patients at 5q21 and 31% at 18q21. LOH at 5q21 and 18q21 was compared with allele loss at 17p13 and concurrent LOH at two or more of the loci was noted in 24% of tumours. Expression of a 12 kb DCC mRNA was demonstrated in 14/34 (42%) of the cancers and in all five tumours with LOH at the DCC locus there was an additional 11 kb DCC mRNA. Abnormalities of three loci involved in colorectal cancer  therefore also occur in sporadic breast cancer. The accumulation of such genetic abnormalities may confer a growth advantage important in the development of breast cancer."}
+{"text": "The value of chemotherapy in advanced non-small cell lung cancer (NSCLC) remains contentious. Because of this two separate but very similar trials were set up in Australia and Southampton (UK). Two hundred and one patients with stage IIIb or IV NSCLC were randomly assigned to cisplatin 120 mg m-2 on days 1 and 29 and vindesine 3 mg m-2 weekly x 6 or to no chemotherapy. Both groups were eligible to receive radiotherapy or other palliative treatment as required. Of 188 evaluable patients, 97 received chemotherapy and 91 were in the control arm. Response was assessed between days 42 and 49. Responders continued chemotherapy at the same doses though cisplatin being given 6 weekly x 4 and the vindesine 2 weekly x 12. The overall response rate to chemotherapy was 28%; there were no significant differences according to major prognostic criteria. Although the overall survival of the chemotherapy group (median 27 weeks) was longer than that of the no chemotherapy group (median 17 weeks) this was not statistically significant (log rank P = 0.33). For patients without dissemination (IIIb), median survival was 45 weeks in the chemotherapy arm and 26 weeks in the non-chemotherapy (log rank P = 0.075). Toxicity was universal and frequently severe: of 17 patients discontinuing chemotherapy after one cycle, 13 did so because of unacceptable toxicity. This chemotherapy cannot be recommended as routine treatment. Further phase III studies of chemotherapy in advanced NSCLC should continue to use a no chemotherapy control and should also attempt to measure quality of life, an issue not addressed effectively in this or other recent trials."}
+{"text": "Patients with T2 grade 3 and T3 bladder cancer were randomised to be treated with radiation alone (NO MISO) or with radiation and misonidazole (PLUS MISO). Patients in both groups initially received 40 Gy in 2 Gy fractions (5/week). Patients in the NO MISO arm received a further 20 Gy in 2 Gy fractions (5/week). Patients in the PLUS MISO arm received a further 12 Gy in 6 Gy fractions (1/week). MISO was administered orally (3.0 g m-2) and intravesically (1.0 g in 35 ml of solvent) 4 h and 2 h respectively prior to each fraction of 6 Gy. Fifty-eight patients were randomized of whom 53 are evaluable. There is a minimum follow-up of 5 years in the surviving patients. In the NO MISO and PLUS MISO arms, the complete response rate at cystoscopy at 6 months was 63% and 69%, the 5-year survival rate was 41% and 48% and the 5-year local control rate with bladder preservation was 46% and 36% respectively . These differences are not statistically significant. Two patients had grade 3 RTOG late bowel complications. Both patients were in the PLUS MISO arm, had undergone salvage cystectomy and subsequently required colostomies for bowel obstruction for a 5-year late complication rate (RTOG grade 3) of 9%. In addition, two patients in the PLUS MISO arm developed wound sepsis post cystectomy. We were not able to demonstrate improved results from the use of oral and intravesical MISO in this study. The number of patients entered are relatively low and large differences would have been required to be detected with a power of 0.80. The use of an unconventional radiation fractionation schedule may have resulted in increased bowel morbidity in patients in the PLUS MISO arm who subsequently underwent cystectomy."}
+{"text": "Patients accessing antiretroviral treatment (ART) programmes in sub-Saharan Africa frequently have very advanced immunodeficiency. Previous data suggest that such patients may have diminished capacity for CD4 cell count recovery.Rates of CD4 cell increase were determined over 48 weeks among ART-na\u00efve individuals (n = 596) commencing ART in a South African community-based ART programme.The CD4 cell count increased from a median of 97 cells/\u03bcl at baseline to 261 cells/\u03bcl at 48 weeks and the proportion of patients with a CD4 cell count <100 cells/\u03bcl decreased from 51% at baseline to just 4% at 48 weeks. A rapid first phase of recovery  was followed by a slower second phase . Compared to patients with higher baseline counts, multivariate analysis showed that those with baseline CD4 counts <50 cells/\u03bcl had similar rates of phase 1 CD4 cell recovery (P = 0.42), greater rates of phase 2 recovery (P = 0.007) and a lower risk of immunological non-response (P = 0.016). Among those that achieved a CD4 cell count >500 cells/\u03bcl at 48 weeks, 19% had baseline CD4 cell counts <50 cells/\u03bcl. However, the proportion of these patients that attained a CD4 count 200 cells/\u03bcl at 48 weeks was lower than those with higher baseline CD4 cell counts.Patients in this cohort with baseline CD4 cell counts <50 cells/\u03bcl have equivalent or greater capacity for immunological recovery during 48 weeks of ART compared to those with higher baseline CD4 cell counts. However, their CD4 counts remain <200 cells/\u03bcl for a longer period, potentially increasing their risk of morbidity and mortality in the first year of ART. The World Health Organisation (WHO) estimated that in June 2005 4.7 million people living in sub-Saharan Africa were in urgent need of antiretroviral treatment (ART) . DespitePatients enrolling into ART programmes with very low CD4 cell counts have heightened risk of morbidity and mortality both before and during the initial months of ART -8. MoreoIn this study we have examined determinants of CD4 cell count recovery among patients accessing a community-based antiretroviral programme in South Africa. We focus on the hypothesis that advanced pre-treatment immunodeficiency diminishes the capacity for CD4 cell count recovery during ART as determined by rates of CD4 cell count increase and risk of immunological non-response.We studied patients accessing ART at the Gugulethu Community Health Centre, Cape Town, South Africa . The vasThe first-line ART regimen was comprised of stavudine, lamivudine plus a non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine). The second-line regimen for those failing first-line treatment was comprised of lopinavir/ritonavir, zidovudine and didanosine. Treatment adherence exceeds 90% at one year . All trePlasma HIV-1 load was measured at baseline and 4 monthly during treatment using Versant\u2122 HIV-1 RNA 3.0 branched chain DNA assay  with a lower limit of detection of 50 RNA copies/ml. Blood CD4 cell counts were measured at the same time-points by flow cytometry using FACSCount\u2122 . These assays were all performed in a single nationally accredited laboratory which has rigorous quality assurance procedures.Structured clinical and laboratory records were maintained on all patients screened on entry to the ART programme and this information was transferred on a weekly basis to a computer database. Data were analysed from the start of the programme in September 2002 until data censorship in August 2005. All treatment-naive patients aged over 15 years and with at least a 16-week follow-up time point were included in the analysis. Study of these patients was in compliance with the Declaration of Helsinki and was approved by the Research Ethics Committee of the University of Cape Town; all patients enrolled gave written informed consent.Data were analysed using Stata version 9.0 . We calculated absolute responses in CD4 cell counts during three intervals , as well as rates of CD4 cell increase (cells/\u03bcl/month) during each interval. The first interval was used as an estimate of the initial (phase 1) response to ART. The CD4 cell count responses observed during the latter two intervals (16 to 32 and 32 to 48 weeks after treatment initiation) were very similar, and were combined into a single measure of the second phase of CD4 cell responses (phase 2).We further evaluated CD4 cell responses in the following ways: i) whether patients failed to attain an absolute CD4 cell count increase from baseline of at least 50 cells/\u03bcl at 48 weeks ; ii) whether patients achieved an absolute CD4 count of 200 cells/\u03bcl at the 48 week visit; iii) and whether patients had achieved an absolute CD4 cell count of 500 cells/\u03bcl at 48 weeks (super-responders).In bivariate analyses, medians were compared using Wilcoxon rank-sum and sign-rank tests; proportions were compared using chi-square tests. All statistical tests were two-sided at alpha = 0.05. Separate multiple linear regression models were used to examine factors associated with rates of CD4 cell count increase per month during the first and second phases. Baseline CD4 cell counts were categorised as follows: <50, 50\u201399, 100\u2013149 and >150 cells/\u03bcl. Multiple logistic regression was used to assess factors associated with the binary outcomes of a CD4 cell increase of \u2265 50 cells/\u03bcl and \u2265 100 cells/\u03bcl during the 48 weeks of ART, as well as achieving an absolute CD4 cell count of \u2265 200 cells/\u03bcl during follow-up. Variables were included in multivariate models if they demonstrated a persistent association with the outcome of interest, or if their removal affected appreciably associations involving other variables .Of 698 individuals who commenced ART between September 2002 and April 2005, 596 (85%) had completed a 16-week follow-up visit at the time of data censorship, 34 (5%) were awaiting this appointment, 48 (7%) had died and 20 (3%) were either transferred out or were lost to follow-up. Of the 596 individuals who met the inclusion criteria for the study, 580 (97%) remained within the programme at study censorship, 11 (2%) died and 7 (1%) were lost from the programme.10 RNA copies/ml  and 58% of patients had a viral load >100,000 copies/ml. The median blood CD4 cell count was 97 cells/\u03bcl  and the proportions of patients with CD4 cell counts within the ranges <50, 50\u201399, 100\u2013149 and \u2265 150 cells/\u03bcl were 25%, 26%, 23% and 26%, respectively. Eighty per cent of patients had symptomatic disease, with 53% and 27% of patients having WHO stages 3 and 4 disease, respectively. During follow-up, only 7 (1%) patients switched to the second-line drug regimen.At baseline the median age was 32 years  and 75% were female. The median plasma viral load was 4.88 logVirological responses to treatment were excellent with viral load suppressed <400 copies/ml in \u2265 94% of patients at each of the follow-up time-points Table . As a reThe rate of CD4 cell count increase in the first 16 week period greatly exceeded the rates in both the 16\u201332 week and 32\u201348 week intervals, but the rates did not differ comparing the latter two intervals. Thus, the pattern of CD4 cell count increase was divided into 2 phases: a ra\u2013pid phase 1  and a slower phase 2 .Baseline characteristics and rates of CD4 cell count change in phase 1 and phase 2 did not differ when comparing the results of analyses of all eligible patients with those restricted to subjects who had data for every time-point (n = 292); this was also the case for all subsequent stratified analyses. Use of data from the larger cohort (n = 596) was therefore validated.10 copies/ml the phase 1 CD4 cell slope was 21.3 cells/\u03bcl/month compared to 31.0 cells/\u03bcl/month among those with baseline viral load of >5 log10 copies/ml (P < 0.001).Rates of phase 1 CD4 cell increase were similar across all CD4 cell count strata Figure . SubsequContrary to our initial hypothesis, the rate of phase 2 CD4 cell increase was greater among those with a baseline CD4 cell count <50 cells/\u03bcl compared to those with higher baseline counts Figure . MultivaIn the multivariate model predicting rates of phase 2 CD4 cell increase, factors associated with the response to ART during the first 16 weeks were also included. A lower rate of phase 1 CD4 cell increase and full suppression of viral load at 16 weeks were both strongly associated with greater rate of phase 2 CD4 cell increase. Viral load suppression <400 copies/ml at 16 weeks was associated with a subsequent rate of CD4 cell increase of 6.8 cells/\u03bcl/month compared to 0.7 cells/\u03bcl/month among those whose viral load remained >400 copies/ml (P < 0.001).We next examined whether baseline CD4 cell count was a risk factor associated with immunological non-response (defined as a CD4 cell increase of <50 cells/\u03bcl at 48 weeks). Among those followed to 48 weeks n = 311), the blood CD4 cell count increased by <50 cells/\u03bcl among 30 (9.7%) patients; of these, the viral load was suppressed <50 copies/ml among 22 patients, representing a treatment discordance rate of 7%. Contrary to our initial hypothesis, low baseline CD4 cell counts were not associated with increased risk of immunological non-response. Among patients with baseline CD4 cell counts of <50, 50\u201399, 100\u2013149 and 150 cells/\u03bcl, the proportions of patients who were immunological non-responders were 5%, 4%, 11% and 19%, respectively. Furthermore, in multivariate analyses, an increment of <50 cells/\u03bcl was independently associated with higher baseline CD4 cell count as well as older age, lower baseline viral load, and a viral load >400 copies/ml at any follow-up time-point  achieved an absolute CD4 cell count of >500 cells/\u03bcl. These super-responders were principally characterised by age <40 years and by all having follow-up viral loads persistently suppressed <50 copies/ml and a CD4 cell count of 150 cells/\u03bcl after 16 weeks of ART. This group of patients had a wide distribution of baseline CD4 cell counts, and included among them were 4 (19%) who had baseline CD4 cell counts of <50 cells/\u03bcl. Thus, a low baseline CD4 cell count did not preclude patients from having an excellent immunological response.To our knowledge this is the first analysis to examine the determinants of CD4 cell count recovery among patients receiving ART in resource limited settings. These data indicate that those with baseline CD4 cell counts <50 cells/\u03bcl had similar rates of phase 1 CD4 recovery (0\u201316 weeks) and greater rates of phase 2 recovery 16\u201348 weeks) compared to rates among those with higher baseline CD4 cell counts. Moreover, those with the lowest baseline counts were least likely to be immunological non-responders to ART. Despite these observations, those with baseline CD4 cell counts <50 cells/\u03bcl were nevertheless least likely to attain a CD4 cell count 200 cells/\u03bcl at 48 weeks. Taken together, these results suggest that although patients with very low baseline CD4 cell counts retain capacity for similar or slightly greater rates of CD4 cell count recovery compared to those with higher counts, they are nevertheless likely to require a longer period of time to attain a CD4 cell count threshold of 200 cells/\u03bcl. Thus, a prolonged period below a 'safe' CD4 cell count threshold rather than a diminished rate of immunological recovery is likely to underlie the high rates of morbidity and mortality observed among those with advanced disease during the early months of ART [ weeks coThe findings of this analysis are strengthened by the relatively homogeneous study population receiving treatment at a single facility using standardised clinical protocols. Patients were all ART-na\u00efve and received a standard triple-drug regimen with uniform follow-up time points. Quality-assured laboratory assays were all performed in a single nationally accredited laboratory. In contrast, previous studies of the determinants of CD4 cell count recovery have examined heterogeneous study populations in multiple centres and used diverse treatment regimens. Moreover, these studies included many patients with prior ART exposure -22 and set al.[et al. showed a similar but non-significant trend when comparing patients with baseline CD4 cell counts <200 cells/\u03bcl with those with higher counts [et al. found that CD4 cell increases in the first year of ART were similar comparing those with baseline counts <100 cells/\u03bcl with those with counts 100\u2013199 cells/\u03bcl [The immunological response to ART among those with low CD4 cell counts was excellent with the proportion of patients with a CD4 cell count <100 cells/\u03bcl decreasing from 51% at baseline to just 4% at 48 weeks. However, our most important finding was that in multivariate analysis baseline CD4 cell counts <50 cells/\u03bcl were independently associated with similar rates of phase 1 CD4 cell recovery and greater rates of phase 2 CD4 cell recovery compared to individuals with higher baseline CD4 cell counts. This has not been clearly highlighted in previous publications from Europe and North America although comparison of our data with previous studies is difficult in view of differing cohort compositions. However, this overall observation is consistent with the findings of Bennett et al., and Le cells/\u03bcl .Survival bias could potentially have affected our findings. We have previously shown in this cohort that patients with the lowest baseline CD4 cell counts had a higher risk of death  and immuWe have previously shown that low baseline CD4 count at entry to an ART programme was associated with increased risks of tuberculosis and of mortality during the first year of ART ,8. Resul5 log10 copies/ml had 11-fold greater CD4 count increases than those with lower viral loads. Immune dysregulation and immune cell activation promote sequestration of CD4+CD45Ro+ memory T cells in lymphoid tissue; suppression of viral replication then triggers rapid redistribution of this cell pool and a reduction in apoptotic cell death during the initial weeks of ART [Mycobacterium tuberculosis[Cryptococcus neoformans[Rates of phase 1 and phase 2 CD4 cell increase were similar in magnitude to those previously reported from high-income countries ,20. The s of ART ,27. A poerculosis, Cryptoceoformans that is Greater phase 2 CD4 cell recovery was strongly associated with age as reported elsewhere ,24. SustPatients with the lowest CD4 counts in this setting do not have diminished capacity for immune recovery. Although patients with low baseline CD4 counts have increased risk of acute morbidity and mortality, if such patients survive the initial months of ART and fully suppress the viral load, their chances of immunological recovery are good during the first year. Future studies are required to determine the long-term prospects for immune recovery among patients treated in ART programmes in sub-Saharan Africa.The author(s) declare that they have no competing interests.SDL and RW initiated and designed the study. LM did the statistical analyses. LGB and RW established the study cohort and data collection systems. SDL wrote the manuscript, which RW, LM and LGB all helped to revise.The pre-publication history for this paper can be accessed here:"}
+{"text": "The 5T4 oncofetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. The 5T4 antigen phenotype of 72 colorectal cancers has been compared with the clinical outcome of the patients in order to assess its relationship with prognosis. Forty per cent of tumours were 5T4 positive; the remainder were either unlabelled or exhibited stroma-associated labelling only. There was a significant correlation between 5T4 expression in the malignant cells and unfavourable course of disease (P < 0.001). The 5 year survival with 5T4-positive tumours was 22% compared with 75% for patients with 5T4-negative tumours; median survival was 24 versus > 90 months respectively. Stratified analysis showed that 5T4 antigen tumour positivity was acting independently of each of stage, site of tumour, age or sex. There were significant differences in survival for patients with Dukes' B and C stage carcinomas (P = 0.001 and P = 0.034). The results suggest that in colorectal cancer immunohistochemical assessment of 5T4 expression may be useful in identifying patients at high risk for tumour recurrence and for whom additional treatment strategies might be most appropriate."}
+{"text": "Twenty five hormone manipulated patients with prostate cancer and metastatic bone disease, treated at least 6/12 previously by hormone manipulation, were given intravenous infusions of Disodium Pamidronate (APD) over a 6 month period. Patients received 30 mg weekly for 4 weeks then twice monthly for 5 months. No other treatment was administered during study. Eleven of 17 patients with pain at the start of the study were pain free at the end. Fasting morning calcium excretion and serum osteocalcin fell significantly with Pamidronate (P less than 0.0001) and urine hydroxyproline was lowered in 13/20 evaluable patients at 6 months. Alkaline phosphatase fell in a proportion of patients and five of 17 patients with previously progressive bone scans stabilised (4) or regressed (1) on treatment. Rising acid phosphatase levels were also lowered in five patients. It is concluded that Pamidronate may be effective in palliating bone pain in some patients and has a stabilising influence on abnormally high bone turnover in metastatic prostate cancer. Further controlled studies of the compound are now warranted."}
+{"text": "The expression of 5T4, an oncotrophoblast cell surface antigen was examined in 72 colorectal and 27 gastric carcinomas, with immunoperoxidase technique, on frozen sections. Highly significant association was found between 5T4 expression in the malignant cells and metastatic spread. The results suggest that the appearance of 5T4 molecules in cancer cells reflects a change which may contribute to the development of metastatic potential."}
+{"text": "Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified.Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular CaTo investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay.2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp.Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca The contractile/relaxant state of the airway myocyte is a key determinant of airway calibre thus contributing to bronchoconstriction in diseases such as asthma. Previous studies have demonstrated that the contractile response of airway myocytes is dependent initially upon release of intracellular Ca2+ from the sarcoplasmic reticulum  but tha2+ entry ) in cont2+ entry .2+ stores. Contractile agonists such as acetylcholine, histamine and bradykinin may vary in their ability to differentially activate ROC or SOC although all agonists are known to induce activation of phospholipase C with consequent IP3 mediated Ca2+ release from the intracellular stores. The mechanism underlying signalling for subsequent Ca2+ influx in response to store depletion, and hence refilling of the sarcoplasmic reticulum Ca2+ stores, remains unknown. In the current study we have set out to define the signals for SOC activation in human airway myocytes following both store depletion and agonist activation by spasmogens.SOC activation in many cell types including smooth muscle is known to involve depletion of the intracellular sarcoplasmic reticulum CaCRAC) in T lymphocytes via a mechanism which has been proposed to involve translocation of STIM1 from endoplasmic reticulum like sites to the cell membrane i.HASMs (passage 4\u20135) were plated in black walled, clear bottom 96 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in culture medium (DMEM) supplemented with 10% FCS and 2.5 mM probenecid . Cells were then washed with Hanks' balanced saline solution containing 10 mM Hepes, 2.5 mM probenecid, 0.1 mM CaCl2, 10 mM EGTA, 30 mM HEPES, 3.62 mM CaCl2. External Solution: 140 mM NaCl, 5 mM CsCl, 1 mM MgCl2, 10 mM D-Glucose, 10 mM HEPES, CaCl2 (as indicated). K+ was replaced by Cs+ in both external and internal solutions to block K+ currents and Cl- was replaced by an equal molar concentration of methanesulfonate to minimize Cl- currents. Nifedipine (5 \u03bcM) was included in the external solution. Pipettes were drawn from borosilicate glass and had resistances of 5\u20138 M\u03a9 when filled with internal solution. HASM cells were placed directly into the cell chamber, allowed to settle and then were continuously perfused with external solution at a constant speed of 6 ml/min. Experimental drugs were delivered through a puffer pipette positioned 50 \u03bcm around the cells. Cells were held at a membrane potential of -60 mV and current-voltage relationships were analysed every 5s from voltage ramps from -100 to +100 mV at a rate of 0.5 Vs-1. Currents were filtered at 1 kHz and sampled at 4 KHz. Individual cell current densities were calculated by dividing peak current amplitude at maximum activation of inward current (at -100 mV) by cell capacitance.The conventional whole-cell patch-clamp technique  was emplHASMs grown on coverslips, transfected with either 20 nM scrambled siRNA or 20 nM STIM1 siRNA were fixed with 4% formaldehyde. Cells were permeabilized (0.5% TritonX-100) and blocked with 20% goat serum in PBS for 20 min. Cells were incubated with primary antibody   overnight at 4\u00b0C followed by labeling with Alexa fluor 488 (Molecular probes). Cells were visualized on a Zeiss LS 510 confocal microscope .Averaged data are presented as mean \u00b1 sem. Where appropriate, statistical significance was assessed by unpaired Students T tests or one-way ANOVA followed by the Dunnets test for multiple group comparisons. Data were considered significant at *P < 0.05 or **P < 0.01.Initially we assessed the expression of the two known human homologues STIM1 and STIM2 in cells relevant to airway function figure . Both ST2+]i) in monolayers of cultured human airway myocytes. SOC mediated Ca2+ influx was induced by depletion of the sarcoplasmic reticulum Ca2+ store using a combination of low external Ca2+(0.1 mM) and incubation with the SERCA inhibitor cyclopiazonic acid . Incubation of airway myocytes in low Ca2+ in the presence of CPA resulted in an initial small rise in cytosolic Ca2+ indicative of store depletion. Re-addition of extracellular Ca2+ resulted in rapid influx into the cell through SOCs. siRNA targeted at STIM1 resulted in a dose-dependent reduction of SOC associated Ca2+ influx .We next evaluated the ability of siRNA targeted to STIM1 and STIM2 to inhibit SOC using a fluorescence assay utilising Fluo-4AM, designed to measure changes in intracellular free calcium concentration . SOC currents were induced by voltage ramps at a rate of 0.5 mV\u00b7ms-1 from a holding potential of -60 mV. Inward currents (SOC currents) measured at -100 mV were then compared. Changes in current density of SOC are illustrated in Figure 2+ free buffer). The STIM1 suppressed cells showed a reduced SOC inward current compared with cells treated with scrambled or STIM2 siRNA sequences. We also noticed a change in the reversal potential in the STIM1 suppressed cells towards a more negative potential: the significance of this is uncertain but similar findings on ICRAC currents have been reported using EF-hand mutants of STIM1 in RBL cells [We next used whole cell patch clamp electrophysiology approaches to confirm that siRNA targeted at STIM1 but not STIM2 is able to inhibit SOC activation. SOC currents were activated by reducing external CaBL cells .2+ entry in human airway myocytes are shown in figure 2+ responses to be H1 receptor mediated in these cells. Under conditions of low extracellular Ca2+ the sustained rise in intracellular Ca2+ seen following agonist stimulation is reduced, an effect mimicked by a range of di and tri-valent cations including Ni2+, La3+ and Gd3+ and also by the putative ROC inhibitor SKF96365 [2+ response to agonist. Following pre-incubation with siRNA targeted at STIM1 we observed a significant reduction in the magnitude of Ca2+ influx induced by histamine  declare that they have no competing interests.Samantha Peel performed the mRNA expression and calcium studies, Bo Liu performed the electrophysiology and Ian P Hall wrote the paper. All authors were involved in the design of the studies, discussion of the results and preparation of the final manuscript."}
+{"text": "Accurate chromosome segregation during meiosis and mitosis is essential for the maintenance of genomic stability. Defects in the regulation of chromosome segregation during division predispose cells to undergo mitotic catastrophe or neoplastic transformation. Cohesin, a molecular glue holding sister chromatids together, is removed from chromosomes in a stepwise fashion during mitosis and meiosis. Cohesin at centromeres but not on chromosome arm remains intact until anaphase onset during early mitosis and the initiation of anaphase II during meiosis. Several recent studies indicate that the activity of protein phosphatase 2A is essential for maintaining the integrity of centromeric cohesin. Shugoshin, a guardian for sister chromatid segregation, may cooperate with and/or mediate PP2A function by suppressing the phosphorylation status of centromeric proteins including cohesin. At the molecular level, cohesin mediates sister chromatid cohesion through formation of a proposed \"ring\" structure that entraps chromosomes [Sister chromatid cohesion is established during DNA replication and maintained throughout Gomosomes . The cohomosomes . Upon miomosomes  that is omosomes ,5.During the past several years, the mechanism preventing centromeric cohesin from undergoing cleavage/dissociation during early mitosis as well as meiosis I and early meiosis II has been a subject of intensive investigation ,7. ThrouNature and Developmental Cell have convincingly demonstrated that enhanced dephosphorylation of centromeric proteins including cohesin may be a key mechanism responsible for centromeric cohesion of sister chromatids during meiosis and mitosis [A recent study by the Nasmyth group showed that expression of SA2 mutant resistant to Plk1 phosphorylation results in suppression of premature separation of sister chromatids in Sgo1-depleted cells . This su mitosis -17.Par1B', Ppa2C) are important for accurate chromosome segregation in fission yeast as their deletions results in moderate to severe missegregation of chromosomes; a detailed analysis revealed that chromosome missegregation occurs in meiosis II, but not in meiosis I, in fission yeast with either Par1B' or Ppa2C deletion [Sgo1 deletion. These observations thus suggest that PP2A, like Sgo1, may protect centromeric but not arm cohesion of sister chromatids. Consistent with this notion, fission yeast mutants with deletion of Par1B' or Sgo1 are impaired in retaining centromeric Rec8 following meiosis I whereas deficiency in PP2A activity does not have an effect on expression as well as centromeric localization of Rec8 before the onset of anaphase I [Using affinity protein purification followed by tandem mass spectrometry, the Nasmyth group found that protein phosphatase 2A (PP2A) subunits are the prevalent components that are co-purified in meiotic I cells of both fission and budding yeasts . PP2A isdeletion , a defecaphase I .The mechanism by which PP2A protects centromeric cohesin is fully conserved in mammalian cells. Through a combination of co-immunoprecipitation and mass spectrometry, the Watanabe group identified PP2A as a major component in Sgo1, but not in control, immunoprecipitates , suggestSimilar results regarding PP2A's physical interaction with Sgo1 in human cells are also reported by the Yu group . Co-immuDrosophila, Polo phosphorylates MEI-S332, which is essential for its removal from centromeres at anaphase [In eukaryotes, a hallmark for mitotic entry is the activation of mitotic kinases , resulting in phosphorylation of numerous mitotic targets essential for mediating mitotic progression. Early studies established that Bub1 is upstream of Sgo1 in the regulatory hierarchy ,19. Receanaphase ; (iv) Planaphase  althoughGiven the close involvement of Plk1/Polo in regulating anaphase entry and chromosome segregation, it is relevant to examine how Plk1 regulates PP2A activity, which in turn modulates centromeric cohesion. Interestingly, the Yu group showed that co-depletion of Plk1 and a key PP2A subunit through transfection of specific siRNAs causes retention of Sgo1 at centromeres in human cells, which is correlated with significant reduction of chromosomal missegregation; in contrast, co-depletion of Plk1 and Sgo1 fails to prevent missegregation of chromosomes ,15. ThusSgo1 deletion during meiosis I, implying that the localization of PP2A to the centromeric region depends on Sgo1 [The identification of PP2A as a new component in protecting centromeric cohesion of sister chromatids prompts additional studies determining its position in the regulatory hierarchy. As Sgo1 is primarily localized at centromeres during meiosis or early mitosis whereas PP2A signals are detected throughout the cell, it is possible that Sgo1 may help to recruit a specific form of PP2A to centromeres. This notion is consistent with the fact that most PP2A is not associated with Sgo1 . Support on Sgo1 . On the  on Sgo1 . Consist on Sgo1 .Although it remains not entirely clear how Sgo1 regulates PP2A or vice versa with regard to protection of centrometic coehsin/cohesion during meiosis and mitosis, a simple model is proposed to illustrate the regulatory hierarchy in animal cells (Figure"}
+{"text": "Using two monoclonal antibodies directed against placental alkaline phosphatase (H17E2 and D20L) the immunohistological staining of testicular germ cell tumours was compared with that of a wide range of normal and malignant tissues. All seminomas and malignant teratomas tested gave strong positive labelling with H17E2 but were either negative or only patchily positive with D20L. Neither antibody gave any positive reaction on the normal tissues tested. All other malignancies were negative with both antibodies apart from two cases of ovarian and one case of endometrical cancer (strongly stained by H17E2) and three cases of colonic carcinoma (weakly and patchily stained by both H17E2 and D20L). This indicates that germ cell neoplasms generally express a form of placental alkaline phosphatase recognised by antibody H17E2."}
+{"text": "Hematological and biochemical parameters were evaluated in 31 patients receiving 150 MBq 89Strontium (89Sr) intravenously due to painful skeletal metastases from hormone resistant prostate cancer. Two and 3 months after the injection prostate specific antigen (PSA) had increased by a median of 36% and 100%, respectively, as compared to the pretreatment value whereas alkaline phosphatase (APHOS) had decreased by about 20% (median). The leucocyte and platelet counts were reduced by about 20-35%, without reaching grade greater than or equal to 2 toxicity. Pain relief was reported in 14 of 29 evaluable patients at 2 months and in 11 of 23 patients at 3 months. It is concluded that 89Sr represents a worthwhile therapeutic modality in the palliation treatment of patients with hormone resistant prostate cancer, though the biological significance of frequently increasing PSA and decreasing APHOS is not yet completely understood."}
+{"text": "Fifteen patients with neoplastic meningitis received a single intrathecal injection of between 11 and 60 mCi of a 131I radiolabelled monoclonal antibody (MoAb), chosen for its immunoreactivity to tumour. Major toxicity was manifest as nausea, vomiting and headache (7/15 patients), reversible bone marrow suppression (3/8 patients) and seizures (2/15 patients). Nine patients were evaluable for either a tumour or clinical response. Six of these demonstrated an event-free response that was maintained for periods of between 7 and 26 months."}
+{"text": "The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95\u201399% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.We performed a systematic depletion of the MCM proteins in These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells. The MCM (minichromosome maintenance) 2\u20137 proteins play an important role in DNA replication in eukaryotes. They are involved during initiation where they are needed to form the preRC (pre-Replicative Complex) reviewed . This coS. pombe active helicases have been isolated containing only MCM4/6/7 In archael species which have a single MCM protein, the active complex has been suggested to be a hexamer or double hexamer  . The mitotic index in 3MCM6 was comparable to control cells, while decreases were seen for k1214MCM6 and rl74MCM2 (20% and 26% lower)  . As repo% lower) .3MCM6 cells appeared to progress normally through mitosis (not shown). Despite rl74MCM2 and k1214MCM6 having fewer mitotic cells, no mitotic aberrations were detected, suggesting that their defect was a pre-mitotic delay. For 1dpa however the defect seemed to be due to a metaphase delay (anaphases were absent). While chromosome condensation was unaffected, mitotic chromosomes appeared broken and did not congress to a metaphase plate . We therefore decided that a more systematic approach would be to deplete each MCM protein using dsRNA-mediated interference (RNAi) in Drosophila S2 cells. A 500\u2013600 bp dsRNA region was produced for each cDNA . The combined results of seven such experiments are shown in We also analysed the cell cycle distribution of MCM2-7 depleted cells using FACS analysis. A representative data set is shown in Drosophila MCM8 The lack of effects seen after MCM2-6 depletion could have been due to compensation by a functionally redundant protein. Since a possible candidate for this was MCM8 we targeted To determine whether MCM8 could compensate for the loss of MCM2-6 we performed RNAi simultaneously targeting MCM5 and MCM8. Lower MCM protein levels could result in less MCM at each replication fork or a reduced number of active forks. The amount of PCNA associated with chromatin has previously been used as a measure of the number of active forks in a cell Drosophila cells. It further suggests that S2 cells can tolerate a reduction in the number of active replication forks without a significant effect on the cell cycle distribution or cell viability.Depletion of MCM8 consistently reduced PCNA binding by 30\u201350%. This suggests a role for MCM8 protein in DNA replication in Drosophila S2 cells to efficiently deplete the MCM2-8 proteins. The data produced supports three main conclusions about MCM proteins in this system.We have used RNAi in DrosophilaFirstly, although we could demonstrate specificity of the RNAi depletions, we observed co-instability for certain combinations of MCM proteins. Some of our observations can be explained based on the composition of reported MCM sub-complexes reviewed . TherefoDrosophila S2 cells has little apparent effect on cell survival and DNA replication. This therefore suggests that the MCM paradox\u2013originally observed in Xenopus cell free extracts Drosophila we suggest two other possibilities. Firstly, consistent with what has been suggested for Xenopus it might be that under normal circumstances most of the MCM protein in cells is redundant. We estimate that there are 50\u2013100 MCM complexes per origin (assuming origin spacing of 40\u2013100 kb) in S2 cells. Therefore even cells which have lost 99% of a specific MCM should have enough protein to ensure that most origins have one MCM complex. A single MCM complex per origin may therefore be sufficient to allow a full complement of activated replication forks as measured by PCNA loading. In this case our results support proposed MCM mechanisms involving single or double hexamers Secondly our data suggest that a dramatic reduction in the level of MCM2-6 and 8 in vivo in Drosophila MCM8 does play a role in replication. From our data the exact nature of its role is unclear, however the lack of an effect of the depletion on cdc45 loading suggests that unlike the MCM2-7 proteins it is unlikely to be required for the loading of downstream initiation factors. In addition MCM8 cannot be the MCM2-6 compensating protein since co-depletion of MCM5 and MCM8 does not synergistically affect cell viability or DNA replication.The second possibility is that MCM loss is compensated for by other proteins. We investigated whether MCM8 could perform this function. The decrease in PCNA loading observed on depletion of MCM8 suggests that S. cerevisiaeXenopus extracts MCM7 has also been shown to bind to the Rb protein Drosophila cells, however less efficient depletion of Drosophila MCM7 has been seen to produce the same effect (data not shown). Alternatively in addition to acting as a negative regulator of replication, MCM7 may have a positive regulatory effect on replication. In either case the effect is likely to involve MCM7 directly, rather than occurring as a secondary effect of a replication defect, since similar effects are not observed for other MCM proteins.Finally the differences observed between depletion of MCM7 and MCM2-6 suggest that not all MCM proteins are equivalent. The mechanism behind this differential behaviour is not clear. Although a complex of MCM4/6/7 has been shown to have helicase activity, there is no evidence that MCM7 acts independently. Therefore MCM7 may have additional cellular functions. A role for MCM7 as a damage sensor via the ATR pathway has been suggested by work in human rl74mcm2, 1dpa, 3mcm6, and k1214mcm6 were obtained from the bloomington stock center.all fly stocks (canton s (wild type), MCM antibodies were described previously preparation, visualisation and quantification of mitotic and s-phase indices were as described previously Proteins from SDS PAGE were blotted onto Hybond ECL (Amersham) and developed with Supersignal West Pico (Pierce). Visualisation and quantitation were carried out using an Alpha Innotech gel documentation system.6 cells) and the cells were monitored by cell count, FACS analysis, western blotting and RT-PCR over a period of 7 days.Sequence specific primers each containing a 5\u2032 T7 RNA polymerase binding site were designed for MCM2-8 . The dsRcDNA from 500,000 S2 cells was made using the cell to cDNA kit (Ambion) as per manufacturers instructions with the following modifications: cells were lysed in 100 \u00b5l lysis buffer and the RNAase inactivated by heat treatment. 20 \u00b5l of the lysate were used for the reverse transcription reaction. The equivalent of 5,000 cells (1 \u00b5l of the reverse transcription reaction) was amplified using Megamix blue (Microzone). Amplified fragments were run on agarose gels and visualised with EtBr.Amplified regions were located at the following nucleotide positions relative to the start ATG: MCM2 1381\u20131550; MCM3 1701\u20131869; MCM4 1261\u20131400; MCM5 481\u2013630; MCM6 781\u2013922; MCM7 321-480 and MCM8 311\u2013450.cells were harvested and fixed using ethanol. Immediately prior to use they were treated with 10ug/ml RNase/1mM EDTA and the DNA was stained with propidium iodide. Flow cytometry was carried out on an EPICS Xl (Coulter Beckman) using EXPO 32 adc software.5 cells/ml. DNAse  was added at a concentration of 100 units/ml. The incubation was for 1 hour on ice.Cell fractionation was carried out as described"}
+{"text": "In this study, 44 primary or metastatic human ovarian tumours were tested for allelic deletions on the short arm of chromosome 11. Analysis of 12 polymorphic loci by Southern blotting evidenced loss of heterozygosity (LOH) in at least one locus in 41% of cases. Moreover, two hot spots of deletions were tentatively mapped on 11p13 and 11p15.5. Our results demonstrated that LOH at 11p is a common event in ovarian carcinomas and were indicative of the possible existence in 11p of two oncosuppressor genes involved in ovarian carcinogenesis. The similarity observed with 11p allelic losses in Wilms tumours, clustered in 11p13 and 11p15.5 too, suggests that deletion and possibly inactivation of the same growth regulatory genes (WT genes) could also contribute to development of the malignant phenotype in ovarian carcinomas. Finally, a statistically significant association (P = 0.005) between 11p deletions and hepatic involvement was suggested by the analysis of distribution of 11p LOH relative to different clinical and pathological parameters of the tumour patients."}
+{"text": "Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours . Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance."}
+{"text": "To evaluate the results of salvage conformal radiation therapy (3DC-EBRT) for patients submitted to radical prostatectomy (RP) who have achieved complete PSA response and who have never been treated with hormonal therapy (HT).To present the results of biochemical control, a period free from hormonal therapy and factors related to its prognosis.from August 2002 to December 2004, 43 prostate cancer patients submitted to RP presented biochemical failure after achieving a PSA < 0.2 ng/ml. They have never received HT and were submitted to salvage 3DC-EBRT. Median age was 62 years, median preoperative PSA was 8.8 ng/ml, median Gleason Score was 7. Any PSA rise above 0.2 was defined as biochemical failure after surgery. Median 3DC-EBRT dose was 70 Gy, biochemical failure after EBRT was defined as 3 consecutive rises in PSA or a single rise enough to trigger HT.3-year biochemical non-evidence of disease (BNED) was 71%. PSA doubling time lower than 4 months (p = 0.01) and time from recurrence to salvage EBRT (p = 0.04) were associated with worse chance of biochemical control. Biochemical control of 76% was achieved when RT had been introduced with a PSA lower than 1 ng/ml vs. 48% with a PSA higher than 1 (p = 0.19). Late toxicity was acceptable.70% of biochemical control in 3 years can be achieved with salvage radiotherapy in selected patients. The importance of PSADT was confirmed in this study and radiotherapy should be started as early as possible. Longer follow up is necessary, but it is possible to conclude that a long interval free from hormonal therapy was achieved with low rate of toxicity avoiding or at least delaying several important adverse effects related to hormonal treatment. Radical prostatectomy (RP) is an efficient method of achieving prostate cancer control. The follow-up is based on clinical history, physical exams and following the Prostate Specific Antigen (PSA) kinetics. Of the patients who fail to achieve biochemical control, the principle of selecting the best salvage therapy is based upon determining whether the disease is still on the prostate bed or if it has already spread throughout the body. Several studies are aiming at determining which variables correlate with a higher chance of detecting localized recurrences. In these cases, adopt a potentially curative salvage therapy (radiotherapy) instead of hormonal therapy alone.The problem with many of these studies is that they usually include a broad group of patients who have never reached an indetectable PSA or those who have been previously treated with some sort of hormonal therapy making assessment using PSA difficult to evaluate.The objective of this study is to evaluate the results of biochemical control and to analyze a period free from hormonal therapy after salvage radiation therapy in a selected group of patients who have never been treated before with hormonal therapy and have achieved complete PSA response after RP.The secondary objectives are to evaluate prognostic factors related to the success of the salvage radiation therapy.From August 2002 to December 2004, 79 prostate cancer patients previously treated with radical prostatectomy (RP) were submitted to salvage three-dimensional conformal external beam radiation therapy (3DC-EBRT) due to biochemical failure. Thirty-six patients were excluded from the analysis: those who have not achieved PSA nadir (<0.2 ng/ml) after RP or those who were submitted to hormonal therapy before or during salvage radiation. Forty three patients were eligible. The median age was 62 years (range 50\u201373) and Caucasians were predominant (88.4%). Patients and treatment characteristics are show in Table Six patients (14%) were cT1c, 3 (7%) were cT2a, 2 (4.7%) were cT2b, 1 (2.3%) was cT2c. In 31 patients (72.1%) preoperative staging was not available. Median pre-operative PSA was 8.8 ng/ml (range 3 \u2013 62) and hormonal therapy was not administered to any of then prior to surgery. Twenty-eight patients (65%) had information on the biopsy specimen, the median Gleason score was 6 (range 4 \u2013 8).We defined biochemical recurrence after surgery as a single PSA value greater than 0.2 ng/ml after surgery in men with no evidence of distant metastasis at the time of radiotherapy.After salvage radiotherapy, biochemical failure was based on the ASTRO (American Society of Therapeutic Radiation Oncology) criteria as three consecutive PSA rises or a single rise was high enough to trigger the initiation of hormone therapy.In surgical staging according to 2002 AJCC staging system, 11 patients (25.6%) were pT2a; 3 patients (7%) were pT2b; 7 (16.3%) were pT2c; 21 (48.8%) were pT3a and only 1 patient (2.3%) was pT3b. The median Gleason score was 7 (range 4 \u2013 8). Surgical margin was affected in 23 patients (53.5%). Perineural invasion (PNI) was found in 30 patients (69.8%); There was no information regarding PNI, Lymphatic invasion (LI), Vascular invasion (VI) and intraepithelial neoplasia (PIN) in respectively 16.3%, 28%, 30.2% and 58% . Median PSA before salvage radiotherapy was 0.87 ng/ml (range 0.24 \u2013 7.9). PSA doubling time (PSADT) was calculated for each patient based on logarithmic regression formula and we used at least 2 PSA values separated by 2 months in the 18 months before salvage radiation.At the time of recurrence all patients were submitted to physical examination which included a digital rectal exam, chest radiography, whole body bone scan and a trans-rectal pelvic ultrasonography. Eleven patients (25.6%) presented a nodule in the prostatic bed.\u00ae) using conformal three-dimensional technique. All patients were submitted to a pre-planning set up in a simulator (Acuity \u2013 Varian\u00ae) with retrograde urethrogram to help define the isocenter. A pelvic computed tomography (CT) was then performed to delinement of planning target volume. Twenty patients (46.6%) were treated with 2 planning tumor volumes (PTV): PTV1 included the surgical prostate bed and seminal vesicles bed with margins and the PVT2 included only the surgical prostate bed with margin. This technique was used according to the attending physician preferences based on post-surgical pathological information and pre treatment prostate image characteristics. Nineteen out of 20 patients treated this way had T3 tumors. Median dose to PTV1 was 50.4 Gy (range 46 \u2013 54) and for all patients median dose to the prostate bed was 70 Gy (range 66 \u2013 72). Median dose per fraction was 2 Gy (range 1.8 \u2013 2).Patients were submitted to external beam radiation therapy with a 10 MV linear accelerator  scale.Median interval from RP to biochemical failure was 12 months (range 2 \u2013 39) and median time after failure to salvage 3DC-EBRT was 8 months (range 1 \u2013 52). The median follow-up after radiotherapy was 26 months (range 8 \u2013 41). One (1) patient (2.3%) was lost to follow-up. At the end of data collection no patients had died. Distant metastasis developed in 2 (4.7%) patients and 33 patients (76.7%) were free from biochemical failure. Of these, 28 patients (85%) developed undetectable PSA (<0.1 ng/ml) after a median interval of 3 months (range 1 \u2013 30). Actuarial Biochemical Non-Evidence of Disease (BNED) at 3 years was 70.71%  and median PSADT was 5.25 months (range 1.0 \u2013 16.5). Univariate and multivariate analysis of selected variables are displayed on Table 1 \u2013 7.9 aPSADT lower than 4 months was an important negative prognostic factor to BNED/3-years . Total dose to surgical bed higher than 66 Gy did not result in better BNED/3-years (p = 0.6).By multivariate analysis only a PSADT lower than 4 months was a negative predictive factor for BNED/3-years  and 1 patient (2.3%) presented grade 3 late gastrointestinal toxicity. No grade 4, acute or chronic, toxicity was seen. Table Prostate cancer is an indolent disease and the best way to evaluate disease control after radical treatment is monitoring PSA. It is estimated that about 1/3 of patients with biochemical failure following radical treatment will develop distant metastasis in a period of 8 years .After RP 70% of patients will achieve biochemical control in 10 years -6. HowevSeveral variables have been described as prognostic factors for failure after surgery: histological grade (Gleason score); capsular or seminal vesicles extension; positive lymph nodes and involvement of surgical margins -12. In oSalvage radiotherapy after RP is the only potential curative modality, but several published series have not demonstrated uniform results of BNED (18 to 68%) ,13-27.The most important issue in patients with biochemical failure is to define which patients will benefit from salvage treatment to the prostate bed. Unfortunately, an increase in PSA level after local treatment does not distinguish local recurrence from distant metastasis. Usual image exams or biopsy have not proved yet to be helpful in defining anatomical site of biochemical recurrence ,28,29. TIn our experience BNED in 3 years was 71%, a result that is compatible with results from other institutions ,16,22.Although the follow-up is somewhat short for accurately defining the effects of the salvage therapy on local control or survival, another important result of the salvage radiation treatment that should be considered is the effect on the quality of life by delaying hormonal therapy. Hormonal therapy has been shown to produce deleterious side effects on the bone mass inducing higher chance of fractures on the spine and bones that carry the body weight .The results of the present series of 70% BNED in 3 years will probably reflect on the patients' quality of life although we did not raise data to support such a conclusion.Lately, several published series have pointed out adverse factors that could define patients with lower probability of occult distant metastasis which might result in better patient selection for local salvage treatment. The worst prognostic factors related to salvage radiation up to this moment are: higher Gleason score ,18,24-26In a recent prospective trial from the European Organization for Research and Treatment of Cancer EORTC 22911), Bolla et al published results of a randomized comparison of wait-and-see after RP or immediate postoperative radiotherapy for high risk patients  and has shown that adjuvant radiotherapy results in better progression free and local-regional free survival . In smal911, BollWe performed salvage treatment using conformal three-dimensional radiotherapy with a median dose of 70 Gy (range 66 \u2013 72 Gy) and we have not found correlation between radiation dose and BNED (p = 0.6). Small retrospective series suggest that conformal three-dimensional radiotherapy and doses higher than 64,8 Gy do correlate with better biochemical control ,27, but There is no agreement regarding which volume should be treated in prostatectomized patients. In the EORTC 22911 radiotherapy was delivered using 2 planning target volumes. The first Planning Target Volume (PTV 1) was defined by the anatomical limits of the surgical bed including those of the seminal vesicles followed by a boost in a reduced PTV (PTV2) to the prostate bed . In our Some series have described low rates of complications (RTOG > Grade 3) in patients submitted to salvage radiotherapy, but what is generally postulated is that toxicity is higher when radiotherapy is employed after surgery than with exclusive radiotherapy specially in the genitourinary tract ,27. AschOur data suggest that approximately 70% of biochemical control in 3 years can be achieved with salvage radiotherapy in selected patients and that 66 Gy may be sufficient for disease control. The importance of PSADT was confirmed in our series and radiotherapy should be started as early as possible. Longer follow up is necessary to confirm these results, but at this moment it is possible to conclude that a long interval free from hormonal therapy was achieved with low rate of toxicity avoiding, or at least, delaying several important adverse effects related to hormonal treatment."}
+{"text": "Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to  performing mutation screening, if DNA samples of  sufficient number of family members are available, it  is worthwhile to assign the gene involved in disease  progression by the genetic linkage analysis.PKD1  and five for PKD2 . Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31\u201346 and PKD2 .We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients . We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2.Lod scores indicated linkage to PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in  The dis:173910) . PKD1 acPKD1, is predicted to be a large transmembrane glycoprotein [PKD2 codes for polycystin-2 which functions as a non-selective cation channel that can conduct calcium ions [Polycystin-1, the protein encoded by oprotein ,12. PKD2ium ions ,13. It hium ions . A failuPKD1 is large and most of it is reiterated in the form of homologues genes (HG) at least six times on the same chromosome [PKD1 as classic PCR amplifies both PKD1 as well as HG sequences. Several groups have already successfully screened most of the coding region of PKD1 [PKD1 and PKD2 genes with no clustering in mutation 'hot spots'. The majority of them are found to be private. It is therefore worthwhile to first assign the gene involved in a particular ADPKD family before proceeding with mutation screening. Nevertheless less than half of our ADPKD families fitted to the criteria for performing linkage analysis and only in those families could we preselect the ones suitable for PKD1 or PKD2 screening. Due to a limited budget we have screened only a part of the PKD1 gene and the entire PKD2 gene.Although presymptomatic diagnosis of ADPKD is possible by various imaging methods and is relatively reliable in adult patients, genetic diagnosis is important for pre-symptomatic diagnosis in younger individuals and in cases with no family history of the disease. Mutation screening in ADPKD is a cumbersome and expensive process as romosome . The pre of PKD1 -22. FromWe collected samples of 36 Slovene ADPKD families. Linkage studies were performed in 16 families  where samples of at least three affected members or two affected and two unaffected members were available. Ultrasound examination and confirmed history of ADPKD in the families was required to enter the study. Diagnosis of ADPKD was based on ultrasonographic criteria described by Ravine at al., 1994 . The stuPKD1 and PKD2 associated microsatellites: D16S3252 (KG8), D16S665 (SM6), D16S291 (AC2.5), D16S664 (CW3), D16S663 (CW2), D4S1534, D4S2929, D4S1563 and D4S423. We determined amplified product lengths using capillary electrophoresis. Details about primer pairs, labelling, cycling conditions and capillary electrophoresis procedures have been described previously [Genomic DNA was extracted from white blood cells by the standard salting out procedure . We devepedin.bat accordingly.In addition, we also analysed two intragenic RFLPs, p.A4058V and p.A4091A as described previously . Prior tPKD1 and 0.0001 for PKD2. For PKD1, three liability classes were assumed, corresponding to gene penetrances of 0.64, 0.92, and 0.99 for age groups 0\u201310, 10\u201330 and over 30 years respectively [PKD2, the liability classes assumed were 0.50, 0.85, and 0.95 for age groups 0\u201320, 20\u201330 and over 30 years respectively [A gene frequency of 0.001 was assumed for ectively ,29. For PKD1 exons 23 and 31\u201346 for mutations using direct sequencing. Primer sequences and PCR conditions are available on request. Prior to sequencing, we purified PCR products through QIAquick columns (Qiagen QIAquick PCR Purification Kit). We analysed sequences on a Perkin Elmer Thermocycler 9600 using Ready Big Dye Terminator Cycle Sequencing Kit according to the protocol of the producer (Applied Biosystems).We screened PKD2 exon amplification are available on request. We performed heteroduplex analysis (HA) using Hydrolink Mutation Detection Enhancement  gel solution. Complete PCR products (50 \u03bcl) were mixed with 0.5 \u03bcl 0.5 M EDTA, denatured at 95\u00b0C for 5 min and finally cooled at 37\u00b0C for at least 1 hour. We analysed 5:1 ratio of sample and loading dye by electrophoresis through 25 cm-1 \u00d7 MDE gel with 15% urea at 250 V for 16\u201324 h. Then we stained gels with ethidium bromide and photographed them under UV light. DNA samples exhibiting shifted bands we amplified and sequenced in both directions using ABI Prism\u2122 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. The detected changes we tested for segregation in each family by either HA or direct sequencing.Primer sequences and PCR conditions for PKD1 we further analysed for their site conservation in mouse (GenBank:U70209), rat (GenBank:AF277452), cat (GenBank:AF483210) and puffer fish (GenBank:AF013614) cDNA sequences, using BLAST [[Putative missense changes in ng BLAST . The effg BLAST [.PKD1 by only four out of the five microsatellite markers used (/PKD1/-KG8-AC2.5-CW3-CW2). We excluded SM6 from Lod score calculations as the correct determination of alleles was impossible due to stutter products and appearance of new alleles during pedigree analysis .We calculated linkage to l., 1994 ). In addPKD1  and in two families to PKD2 (pedigrees 10 and 31). Linkage to both genes was possible in seven mostly small families, where the co-segregation is likely to be due to chance . In one of the 16 families analysed (pedigree 42), Lod scores were mostly negative and linkage to neither of the two PKD loci could be assumed. [see Figure PKD loci in this pedigree. Table PKD1 to PKD2 or to either PKD1 or PKD2 were selected for further mutation screening.In six families Lod scores and segregation of alleles indicated probable linkage to PKD1 mutation screening. We selected one patient from each family where calculated Lod scores did not indicate exclusion of linkage to PKD1 (6 families with indicated linkage to PKD1 and 7 families where linkage to both genes was possible) and 20 patients from families where linkage analysis was not possible due to lack of family member samples. We screened the PKD2 gene for mutations on 9 patients from families where linkage to the PKD2 gene could not be excluded (2 families with linkage to PKD2 and 7 families where linkage to both genes was possible). We identified seven likely ADPKD causing changes and eleven polymorphisms in both genes. They are summarised in Table Altogether 33 patients were included in the PKD1. The first change is a silent polymorphism c.8509C>T that was detected in family 02. The second change p.E2771K c.8522G>A is a missense mutation already identified in 4 British families [We found 3 changes in exon 23 of families . The mutfamilies ,33 we prPKD1) we detected is the substitution from valine to methionine c.8675G>A p.V2822M. We could not analyse for segregation because no additional samples from other family members were available. The valine residue is only conserved in the cDNA sequences of the species cat, mouse and rat, but not puffer fish. Our secondary structure analysis with PHDsec [The third change in exon 23  c.11693_11697dup. The predicted consequence of this duplication is most probably termination of protein synthesis 116 amino acid residues after glutamate 3828. Patient 148 has gallstones and she started dialysis treatment at the age of 44 (rapid progressor). Patient's daughter has enlarged kidneys with multiple cysts at the age of 23 and she suffers from hypertension and proteinuria. Blood sample of the patient's daughter was not avaliable therefore we could not confirm segregation of the mutation in the family.In patient 148 of family 41 we found the duplication of five base pairs in exon 41 (PKD1) c.11745+3_5dup. The mutation was described previously in a French patient under different name 11745+2ins3 [In family 38 we found the duplication of 3 base pairs in intron 41 (45+2ins3 . We adapPKD1) c.11820_11845del. The mutation most probably results in protein termination after 80 amino acid residues. Both patients have already rather early started with dialysis treatment: patient 77 at the age of 48 and patient 161 at the age of 44. In patient 77 cysts in the liver and idiopathic dilated cardiomyopathy were detected, while sibling 161 has persistent hepatitis B.In two siblings (patients 77 and 161) from family 20 we identified the deletion of 26 base pairs in exon 42 (PKD1) c.12341A>G p.I4044V [PKD1) c.12838T>C [Previously described polymorphism in exon 44 (p.I4044V  we found12838T>C .PKD1) c.12346+22del [PKD1) c.12124C>T.In two apparently unrelated pedigrees (families 12 and 27) we found previously described intronic deletion in intron 44  we found nonsense mutation in exon 45 (PKD1 c.12772dup. The mutation most probably results in premature protein termination after 20 amino acid residues. The duplication segregates with the disease in the family 11. Patient 37 died at the age of 58 years. He was operated on kidney stones and suffered from liver cirrhosis as well as obstructive icterus. His daughter (23 years) has small cysts in kidneys that were detected at the age of 11.In family 11 [see Figure PKD2 gene we found four changes. In family 10 [see Figure (PKD2) c.916 C>T p.R306X. The mutation was already described in three Bulgarian families [In families  and in tfamilies ,42. We cPKD2 gene. In exon 1 (PKD2) we found the sequence change c.362 C>G p.G121A. This variation substitutes glycine for alanine at the N-terminal region of polycystin 2. The sequence change however does not segregate with the disease in the family 31. In the same family we also observed the presence of two already described polymorphisms c.83G>C, p.R28P in exon 1 and c.844-22 G>A in intron 3 [In family 31 we detected three changes in the intron 3 ,40.PKD1 or PKD2 prior to performing mutation analysis in ADPKD families. Of the 16 families included in linkage analysis, Lod scores and segregation of alleles indicated probable linkage to PKD1 in six families  and linkage to PKD2 was indicated in two families . Linkage to both genes was possible in seven mostly small families, where co-segregation is likely to be due to chance . For one out of the 16 families analysed (pedigree 42), Lod scores were mostly negative and linkage to neither of the two PKD loci could be assumed.As both genes associated with ADPKD are large and mutation screening is time consuming and costly, it is prudent to assess linkage to either PKD1 or PKD2 in patients from families with indicative linkage to either one or the other gene and in patients from families where linkage to both genes was possible. In addition we included patients from families where we could not perform linkage studies due to a lack of samples from family members. In total we screened 33 patients for PKD1 mutations and 9 patients for PKD2 mutations.We consequently performed mutation screening in either PKD1 has been hindered by the high (>95%) sequence identity with homologous genes, raising concerns about PCR primer specificity and hence reliability of the analysis [PKD1 (exons 33\u201346) as well as exons 31\u201332 and exon 23 of the duplicated part. Exon 23 was analysed due to its' proximity to the IVS21 polypyrimidine tract, where clustering of mutations has been proposed [PKD1, the region around the polypyrimidine tract shows a higher frequency of changes compared to other parts of the gene [Detection of mutations in the duplicated part of analysis . We haveproposed  due to fproposed ,46. Alththe gene .PKD1 and one mutation and three polymorphisms were detected in PKD2. Three mutations were described previously; p.E2771K c.8522G>A (PKD1) in four British families [PKD1) in a French patient [PKD2) in three Bulgarian [PKD1) most probably result in premature protein termination.Six mutations and eight polymorphisms were detected in families , c.11745 patient  and p.R3ulgarian  and two ulgarian ,42. FourPKD1 in a sufficient number of control chromosomes is costly, the distinction between disease causing mutations and neutral variants has to rely on family segregation studies. Segregation in family we confirmed for four out of seven identified mutations where samples at least two patients from the family were available. From the other three, one mutation was described previously (c.11745+3_5dup) [As there is no functional assay for ADPKD and analysing +3_5dup) , and froPKD1 c.12772dup, c.11820_11845del and c.11693_11697dup seem to be associated with the more severe clinical course of the disease resulting in ESRD  in the range from 44 to 48 years  as already reported by Bulgarian and Czech studies [On the basis of collected clinical data of the patients which is summarised in Table  studies -42. CompPKD1 and one mutation and three polymorphisms were detected in PKD2. Three mutations have been described previously in British, French, Czech and Bulgarian populations. Four out of 7 mutations are novel and probably private. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of the disease resulting in ESRD  in the age range of 44 to 48 years. Nonsense mutation in PKD2 seems to be associated with cysts in liver and slower progression towards ESRD.In our study group of ADPKD patients six mutations and eight polymorphisms were detected in The author(s) declare that they have no competing interest.KV carried out genotyping and linkage analysis, majority of mutation screening in PKD1, coordinated experimental work and drafted the manuscript. LS carried out part of mutation screening in PKD1 and contributed to the writing of the manuscript. JS and JR performed mutation screening in PKD2 and contributed to the writing of the manuscript. MB, TK, SJ, BL, AA, AS, RD and RH are nephrologists who were responsible for clinical examinations, contacting the family members of the patients, collecting of clinical data and blood samples. PH took part in PKD1 mutation analysis. IZP and JB carried out paternity testing in one of the families. GH wrote two subprograms that helped us to analyse linkage data. RK supervised the research activity, contributed in study design and revised the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "In the LMCE1 study using a single course of megatherapy most of the relapses occurred during the first 2 years after autologous bone marrow transplantation. A second pilot study (LMCE2) was therefore set up using a double harvest/double graft approach with two different megatherapy regimens. Objectives were to test the role of increased dose intensity on response status, relapse pattern and overall survival. Thirty-three patients  with a median age of 53 months at first megatherapy  entered this study. They were cases either with refractory disease in partial response after second line treatment for stage 4 neuroblastoma (n = 25) or after relapse from stage 4 (n = 5) or stage 3 disease (n = 3). All patients received Etoposid and/or Cisplatinum (or Carboplatin) containing treatments before megatherapy. The first megatherapy regimen was a combination of Tenoposid, Carmustine and Cisplatinum (or Carboplatin), the second applied Vincristin, Melphalan and Total Body Irradiation. The first harvest was scheduled 4 weeks after the last chemotherapy, the second 60 to 90 days after megatherapy. All marrows were purged in vitro by an immunomagnetic technique. Median follow up time since first megatherapy is 56 months. Response rates for evaluable patients were 65% (complete response rate: 16%) for megatherapy 1 and 60% (complete response rate: 25%) for megatherapy 2. Considering that only patients with delayed response or relapse were eligible for this pilot study the overall survival was encouraging with 36% at 2 years and still 32% at 5 years. The costs for these survival rates were high in terms of morbidity . Double harvesting may have the disadvantage of delayed engraftments related in part to a disturbance of marrow microenvironment by megatherapy 1. This double megatherapy approach achieved a prolonged relapse free interval  in patients reaching megatherapy 2 and justifies further evaluation of concepts with consecutive dose-escalation."}
+{"text": "A total of 474 adult patients with malignant glioma (astrocytoma) grade 3 or 4 were randomised into an MRC study (BR2) comparing 45 Gy (in 20 fractions over 4 weeks) with 60 Gy (in 30 fractions over 6 weeks) of radiotherapy given post-operatively. Using 2:1 randomisation, 318 patients were allocated the 60 Gy course and 156 the 45 Gy course. Adjuvant chemotherapy was not given. The results show that a 60 Gy course produces a modest lengthening of progression-free and overall survival. They suggest a statistically significant prolongation of median survival from 9 months in the 45 Gy group to 12 months in the 60 Gy group . Over 80% of patients reported no morbidity from the radiotherapy, and there was no evidence of increased short-term morbidity in the higher dose group. Late morbidity was not assessed. A prognostic index defined in a previous MRC study was validated in this new cohort. It identifies a group of patients  with a 2 year survival rate of 28% . It was apparent that the survival advantage to the higher dose was maintained even in the poorest prognostic groups defined by this index."}
+{"text": "Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated floxneoWnt4R26. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. floxneoWnt4; Col2a1-CreR26 double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, floxneoWnt4; Col2a1-CreR26 mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype. Wnts have been shown to be expressed in the developing long bones, suggesting that they may have roles in endochondral bone formation. In the developing chick skeleton, Wnt4 and Wnt9a (previously known as Wnt14) are expressed in joint-forming regions, Wnt5a and Wnt11 in the perichondrium, and Wnt5b in prehypertrophic chondrocytes of the growth plate Wnt4 and Wnt5a can alter chondrogenesis and shorten limb growth, apparently by different mechanisms. Wnt4 accelerates chondrocyte differentiation, whereas Wnt5a inhibits this process Wnt9a misexpression has been shown to induce the initiation of joint formation Wnt9a knockout mice formed joints but had ectopic cartilaginous nodules that was enhanced by loss of Wnt4Wnt4/Wnt9a double mutants also had some limb bone fusions apparently because of an inability to maintain joint cell identity Wnt5b as well as Wnt5a inhibits chondrogenesis in mice, but they appear to act differently. Wnt5a inhibits the transition from resting to proliferating chondrocytes in the growth plate, whereas Wnt5b promotes this transition as well as chondrocyte proliferation Wnt signaling has been implicated in the regulation of early patterning and initial outgrowth of the vertebrate limb bud Lef1 in chondrocytes stimulated chondrocyte maturation as well as replacement of cartilage by bone LDL receptor-related protein 5 (Lrp5) gene that encodes a Wnt co-receptor, showed decreased osteoblast proliferation Lrp5-deficient mice also displayed persistent eye vascularization. These bone and eye phenotypes are similar to the abnormalities associated with osteoporosis-pseudoglioma syndrome in human, caused by mutation of LRP5Wnt signaling components have also been investigated for their roles in skeletogenesis. Frb1, a secreted form of Frizzled that is a Wnt receptor, can function as an antagonist when misexpressed in long bone, causing shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation Wnts appears to vary in different animal models. For example, in addition to the perichondrium of chick, Wnt5a expression was also found at the junction of proliferating and prehypertrophic chondrocytes in the radius and ulna of mice Most studies of Wnt signaling in skeleton development have been restricted to the chick model. However, the expression of Wnt4 expression has also been analyzed during kidney and female reproductive system development. Wnt4 homozygous mutant mice died after birth due to a failure of pretubular cell aggregation, an essential step in the formation of nephrons of the kidney Wnt4 mutant mice with an XX karyotype lacked female-specific genital ducts and developed male-specific genital ducts Wnt4 is initially expressed in joint-forming regions, and then is detected in the region of the joint capsule and surface articular chondrocytes Wnt4 expression in long bones is also detected in hypertrophic chondrocytes Wnt4 is also expressed in forming joints and mesenchyme that will form the joint capsule Wnt4 expression in chick and mouse suggest roles in joint development and chondrocyte hypertrophy. In addition, the restricted pattern of Wnt4 expression in bone-forming tissues suggests that its expression must be precisely controlled to coordinate normal bone and skeleton formation.Wnt4 during skeleton development, we created a conditional genetic system to express Wnt4 during chondrogenesis. To accomplish this, we exploited the ubiquitously expressed Rosa26 locus. The ROSA26 mouse mutant was originally produced by infection of embryonic stem (ES) cells with a ROSA\u03b2-geo retrovirus Rosa26 heterozygotes express \u03b2-galactosidase  reporter activity ubiquitously that initiates during preimplantation development at the morula-blastocyst stage. Examination of serial sections through 9.5 days post-coitus (dpc) Rosa26 heterozygotes demonstrated \u03b2-gal activity in all cells Rosa26 homozygous mutants are viable although they are recovered at a lower than expected frequency Rosa26 locus has been used to ubiquitously or conditionally express various gene products in mice Rosa26 locus to express Wnt4 in a Cre-dependent manner. We placed a drug selection cassette flanked by loxP sites between the Rosa26 promoter and a mouse Wnt4 cDNA, blocking Wnt4 expression at the endogenous Rosa26 locus. Cre expression should delete the blocking drug selection cassette, leading to Wnt4 expression.To study the actions of Wnt4 during endochondral bone formation, we used Col2a1-Cre transgenic mice that express Cre activity in cartilage-forming tissues Wnt4 expression in chondrogenic tissues alters skeletogenesis, resulting in skull abnormalities and dwarfism. These studies indicate that alterations in Wnt4 expression can cause severe skeletal pathologies.To examine the action of Wnt4 in a Cre-dependent manner, potentially in any tissue. We modified the ubiquitously-expressed Rosa26 locus by gene targeting in ES cells  have developed in both the tibiae and femurs of controls . At 9 months of age, the tibiae of mutants (n\u200a=\u200a2) were deficient in bone marrow and were filled with adipocytes in epiphyseal and metaphyseal regions , a member of the Hedgehog gene family, is a key molecule in endochondral ossification Ihh is expressed predominantly in prehypertrophic chondrocytes. Hybridization of Ihh showed no obvious differences in mutant tibiae relative to controls  floxneoWnt4; Col2a1-CreR26 mutants was nearly identical to wild type, although with an altered appearance. floxneoWnt4; Col2a1-CreR26 mutants have an expanded zone of hypertrophic chondrocytes and a smaller zone of proliferating chondrocytes. Overexpression of Wnt4 also causes a decrease in VEGF expression that may result in a reduction of vascularization that in turn leads to delayed formation of primary and secondary ossification centers. The Col2a1-Cre transgene is active in chondrocyte precursors of endochondral bones floxneoWnt4; Col2a1-CreR26 mutants were significantly smaller than controls. The skull is composed of elements derived from both endochondral and membranous bone formation. The alterations observed in the membranous bones of the mutant skulls may be indirect effects of altered endochondral skull bones. All of the skeletal alterations mentioned above likely contribute to the development of the dwarf phenotype.The external morphologies of the Wnt4 is expressed in the developing joint regions and a subset of hypertrophic chondrocytes Wnt4 homozygous mutant mice die within 24 hours after birth due to severe defects in kidney function Wnt4/Wnt9a double mutants show some joint cell identity abnormalities Wnt4 in the skeleton after birth. Such studies will require the generation of a Wnt4 conditional null allele Wnt4 misexpression in chick limbs accelerated chondrocyte maturation; in contrast, Wnt5a misexpression in the same model inhibited chondrocyte maturation Wnt4 misexpression, suggesting that Wnt4 influence on limb shortening may be mediated by \u03b2-catenin Wnt4 signaling may have a role in chondrocyte maturation.Wnt4 in the mouse growth plate may also influence chondrocyte maturation, as shown by reduced zones of proliferating chondrocytes and expanded zones of Col10a1 hybridization and hypertrophic chondrocytes in floxneoWnt4; Col2a1-CreR26 mutants. In addition, in growth plates of floxneoWnt4; Col2a1-CreR26 mutants, the zone of Col2a1 hybridization was significantly smaller yet the Ihh zone essentially unchanged, indicating a decrease in proliferating chondrocytes. The decrease of the zone of proliferating chondrocytes may result from lower rate of proliferation in mutants or a higher percentage of cells exiting the proliferation state. However, an in vitro micromass culture assay has shown that infection by a retroviral-delivered Wnt4 did not decrease the rate of cell proliferation Wnt5a and 5b misexpresssion using the same system. Indeed, recent studies in transgenic mice demonstrated that Wnt5b could promote proliferation of chondrocytes in vivo Wnt4 to decrease the zone of proliferating chondrocytes may represent an enhancement of the endogenous activity of Wnt4, a competition with the activity of Wnt5b that promotes proliferation of chondrocytes, or a mimicking of Wnt5a function that inhibits the transition from resting chondrocytes to proliferating chondrocytes. Moreover, since Wnt4 may accelerate the differentiation of chondrocytes, proliferating chondrocytes with overexpression of Wnt4 may exit the cell cycle rapidly, leading to narrower zone of proliferating chondrocytes.We show that overexpression of floxneoWnt4; Col2a1-CreR26 mutants also had disorganized growth plates. The organized structure of the growth plate is tightly linked between the chondrocytes and the extracellular matrix (ECM) and Wnt actions on cell adhesion have been proposed Wnt4 may interrupt such a relationship, by either changing the cell membrane structure of chondrocytes or altering components of the ECM. Wnt4 has been shown to signal canonically, non-canonically, or through neither pathway, depending upon the experimental context Wnt4-induced dwarfism documented in our study.The floxneoWnt4; Col2a1-CreR26 mutants displayed decreased VEGF expression. VEGF is a key regulator for vascularization and plays an important role during endochondral bone formation, where VEGF couples hypertrophic cartilage remodeling, ossification, and angiogenesis Vegf heterozygous mice die at early embryonic stages 120 isoform can survive to term. Vegf120/120 mutants appeared to have low angiogenesis activity floxneoWnt4; Col2a1-CreR26 mutants. These abnormalities include delayed invasion of vessels into the primary and secondary ossification centers, reduction in mineralization of mutant bone, and expansion of the zone of hypertrophic chondrocytes. Thus, these phenotypes observed in the floxneoWnt4; Col2a1-CreR26 mutants may be caused by overexpression of Wnt4, causing reduced expression of VEGF.The growth plates of floxneoWnt4; Col2a1-CreR26 mutant phenotype. However, Wnt gene family members often exert distinct functions in a particular tissue of the same stage. For instance, infection of chick limbs using a retrovirus carrying Wnt5a or Wnt4 presented distinct effects, with Wnt5a delaying chondrocyte differentiation, whereas Wnt4 accelerated it Wnt5b promoted the transition of resting chondrocytes to proliferating chondrocytes, whereas Wnt5a inhibited it Wnt genes, or endogenously, Wnt proteins may function as antagonists of each other.Although the relationship between Wnt proteins and VEGF during skeletal development has not yet been clarified, Wnt/\u03b2-catenin signaling has been suggested to play a role in activation of VEGF gene expression in benign colonic adenomas in which mutant activated Wnt/\u03b2-catenin pathway is often associated with up-regulated VEGF Wnt4 is an important regulator of female reproductive organ development in mice. The ovaries of female mice lacking Wnt4 were masculinized with indications of Leydig cell differentiation Wnt4 may repress angiogenesis in developing female gonads, blocking the testis differentiation pathway.floxneoWnt4; Col2a1-CreR26 mutants became apparent only after birth. However, skeletal changes were found earlier in development, consistent with the activity of the Col2a1-Cre transgene at the initial stages of cartilage formation Wnt4 at fetal stages may be attributable to limited expression of Wnt4 receptors or an excess of Wnt inhibitors. However, the overt dwarfism of the floxneoWnt4; Col2a1-CreR26 mutants may be the result of VEGF insufficiency. Neonatal mice homozygous for a Vegf 120 isoform allele had 10% shorter tibiae, and slight differences in bone length were detected at 16.5 dpc in comparison to controls floxneoWnt4; Col2a1-CreR26 mutants may have stronger VEGF activity than 120/120VEGF mutants because the phenotypes displayed in 120/120VEGF mutants were more severe than those of RWnt4; Col2a1-Cre mutants. For example, the expansion of the zone of hypertrophic chondrocytes was larger, and the delayed time of formation for the primary ossification center was longer in 120/120Vegf mutants. Thus, it may be reasonable to expect that the shortening of tibial length observed in floxneoWnt4; Col2a1-CreR26 mutants as in Vegf120/120 mutants becomes apparent only after birth.The dwarfism of Wnt4 is expressed during skeleton development. The studies presented here demonstrate that dysregulated Wnt4 expression in chondrogenic tissues leads to skeletal defects and dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. In addition, these studies suggest that pathologies that lead to Wnt overexpression may influence chondrogenic tissues.In summary, Wnt4 cDNA encoding the entire open reading frame was isolated by RT-PCR. RNA from 13.5 dpc gonads from mouse strain 129/EvSvTac was isolated and used to synthesize cDNA. The Wnt4 cDNA was subsequently amplified by two rounds of PCR. The first primer set was: forward 5\u2032-CCGCGCGGCGAAAACCTG-3\u2032 and reverse 5\u2032-CTGTTTAAGTTATTGGCCTTC-3\u2032. The second primer set was: forward 5\u2032-GCCTTGGGATCCCTGCCCCGGGCTGG-3\u2032 and reverse 5\u2032-ACGCAGGCGGCCGCACTAGTCCTAGGCATGGTCA-3\u2032. The final PCR product was subcloned into the BamHI and NotI sites of pBluescript KS(-) and sequenced.The mouse Wnt4 expression cassette into the Rosa26 locus. The expression cassette begins with a splice acceptor sequence (SA), followed by Pgkneo and five polyadenylation sequences flanked by loxP sites (floxneo). The mouse Wnt4 cDNA followed by a bovine growth hormone polyadenylation (bpA) sequence was placed 3\u2032 of floxneo. The expression cassette, SA-loxP-Pgkneo-5pA-loxP-Wnt4-bpA, was inserted into the XbaI site of pR26-1, to generate the gene targeting vector (DT) is present within pR26-1 for negative selection. The targeting vector was linearized with KpnI and electroporated into AB1 ES cells and selected in G418 XbaI and analyzed by Southern blot bpA .4 and 9 month-old mice were sacrificed by COSkeletal tissues were treated with hyaluronidase  to unmask epitopes for immunohistochemistry. Immunostaining was performed using the Vectastain ABC kit , according to the manufacturer's instructions. Rabbit anti-mouse VEGF antibody  was diluted 1\u223620.Mice were injected intraperitoneally with bromodeoxyuridine (BrdU) at 100 mg/g body weight and sacrificed 1 hour after injection. Histological sections of skeletal tissues were prepared and immunostained for BrdU . The proliferation rate was calculated as the number of BrdU-labeled cells divided by the total number of cells in the same microscopic field.35[S]-UTP-labeled antisense or sense RNA probes were prepared. After hybiridzation, samples were exposed for 7 to 40 days and then developed using Kodak D-19 developer and fixer, and counterstained with Hoechst dye.The procedures for RNA in situ hybridization of histological sections were adapted from those described"}
+{"text": "Here, I use two methods to estimate the anthropogenic CO2 storage and uptake for a typically large EEZ .Under the United Nations convention on the law of the sea (1982), each participating country maintains exclusive economic and environmental rights within the oceanic region extending 200 nm from its coastline, known as the Exclusive Economic Zone (EEZ). Although the ocean within each EEZ has a vast capacity to absorb anthropogenic CO2 emissions were absorbed by its own EEZ.Depending on whether the Antarctic territory is included I find that during the 1990s between 30\u201340% of Australia's fossil-fuel CO2 sink' could be used as a disincentive for certain nations to reduce their anthropogenic CO2 emissions, which would ultimately dampen global efforts to reduce atmospheric CO2 concentrations. Since the oceanic anthropogenic CO2 sink has limited ability to be controlled by human activities, current and future international climate change policies should have an explicit 'EEZ' clause excluding its use within national carbon accounts.This example highlights the potential significance of the EEZ carbon sink for national carbon accounts. However, this 'natural anthropogenic CO The ocean has hindered the extent of accelerated climate change and will continue to absorb about 33% of fossil-fuel emissions well into the future [2 sink is different to other carbon sinks in that it directly remediates against climate change by sequestering anthropogenic CO2 on both short and long timescales. The exclusive economic zone (EEZ) is an oceanic zone legally bound to nation states under international law [2 sink be for national carbon accounts. I use Australia as a case study to estimate the EEZ anthropogenic CO2 sink due largely to the detailed accounting information on CO2 emissions from fossil fuels and land-use changes [6 km2 [6 km2).Atmospheric COe future . The oces [6 km2 , which m2 within Australia's EEZ (including the Antarctic territory) for the 1990\u20131999 period to be 2.12 \u00b1 0.7 Pg CO2 (Pg = 1 \u00d7 1015 g) with an annual increase of about 220 Mt CO2/yr , the mean anthropogenic CO2 flux for Australia's ocean is about 175 MtCO2/yr throughout the 1990s. This calculation assumes Australia's EEZ acts in proportion to the global average oceanic anthropogenic CO2 flux. Even though the ocean is relatively homogenous, most studies suggest the Southern Ocean to be the region of highest uptake [2 uptake within the Australian EEZ (175\u2013220 MtCO2/yr) is in agreement with a recent modelling study [2/yr depending on the areal extent of the Australian EEZ.I have calculated the accumulation (storage) of anthropogenic COt uptake . Since A2 uptake is significant when comparing to Australia's CO2 emissions via fossil-fuel usage or land-use  is about 3 times the magnitude of the CO2 source due to land-use changes (655 MtCO2) and about 30\u201340% of the total magnitude of fossil-fuel emissions (4695 MtCO2). The implications of including Australia's EEZ or any other nations EEZ within the framework of global carbon trading would be considerable. Furthermore direct human influence of the EEZ CO2 sink through carbon runoff from land use/irrigation/agricultural practices may also significantly influence national carbon accounts for nations with large EEZs.Australia's EEZ anthropogenic CO6 km2), its EEZ anthropogenic CO2 sink only absorbs less than 3% of its annual fossil-fuel emissions[2 emissions relative to their large potential EEZ CO2 sink. Tonga, Fiji, Samoa, Soloman Islands for example have vast oceanic territories that absorb many times over their annual fossil-fuel CO2emissions. Although Australia emits near the highest amount of anthropogenic CO2 per capita in the world, its relatively low population and vast oceanic territory results in the EEZ carbon sink being highly influential to its national carbon accounting. However, this 'natural anthropogenic CO2 sink' could be used as a disincentive for certain nations to reduce their anthropogenic CO2 emissions, which would ultimately dampen global efforts to reduce atmospheric CO2 concentrations. Along with the fact that the oceanic anthropogenic CO2 sink has little ability to be controlled by human activities, it should be explicitly excluded within current or future climate change policies. The international legality of the EEZ carbon sink and its potential implications requires careful consideration in formulating an equitable future framework for climate policy that aims at reducing atmospheric CO2 levels.Nations with large EEZs (like the USA) aren't necessarily those who will benefit the most from including the EEZ carbon sink in international climate policy, as it depends on the relative amount of EEZ sink in comparison to a nations annual anthropogenic emissions. Despite the USA claiming the worlds largest oceanic territory (~10 \u00d7 10emissions. On the 2. Just as nation states have varying degrees of land coverage they also have varying degrees of EEZ extents. To demonstrate the potential implications of the EEZ anthropogenic CO2 sink, I have roughly estimated the uptake for Australias EEZ which is one of the largest in the world. By comparing the amount of anthropogenic CO2 sequestered by Australias EEZ to Australias CO2 emissions via fossil-fuel/land-use, I show that including the EEZ has significant implications for Australias national carbon accounts and any other nation who maintains a large EEZ. As the EEZ carbon sink may introduce legal grounds for nation states to possibly exploit, which would ultimately dampen efforts to reduce atmospheric CO2 concentrations, current and future international climate change policies should have an explicit 'EEZ' clause excluding its use within national carbon accounts.The global EEZ represents over a quarter of the surface area of the ocean, which undoubtedly acts as important reservoir for sequestering anthropogenic CO2 measurements within Australia's EEZ, to quantify the EEZ anthropogenic CO2 sink I use a recently developed method that exploits a purely transient tracer [2 atmospheric history [2 from 1990 to 1999. Although the method is indirect, the total uncertainty has been quantified to be between 10\u201320% by comparing results from direct temporal CO2 estimates [Due to the lack of temporal COt tracer . The mett tracer  to detert tracer . These w history , alkalin history  to estimstimates  and with"}
+{"text": "Responses both to hyperosmotic stress and to heat shock were compared in 3T3 cells, spontaneously transformed cells (ST3T3) and simian virus 40-transformed cells (SV3T3). Cell adaptation to these stresses was measured in terms of surviving cell viability and plating efficiency, while their induced synthesis of stress proteins was monitored in terms of the presence of mRNA for HSP70, the pattern of polypeptides synthesised and the accumulation of HSP70 detectable by monoclonal antibodies. All three types of cells responded similarly to heat shock in their expression of HSP70 and showed no clear differences in ability to recover. In contrast, both ST3T3 and SV3T3 cells adapted more poorly and much more slowly to hyperosmotic stress (0.5 osM incubation) than did normal 3T3 cells. This different pattern of adaptation to hyperosmotic stress was parallelled by the cells' different expression of a stress protein that could not be distinguished from the heat-induced HSP70 by any of the methods listed above. In view of these findings it seems possible that hyperosmotic treatment might be useful in selectively affecting the survival of tumour cells."}
+{"text": "CDKN2A (encodes p16INK4A and p14ARF) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays. CDKN2A (INK4A/ARF) tumor suppressor locus, which was discovered in this and other laboratories CDKN2A deletions occur early during tumor development INK4a  are reported and also through The Cancer Genome Atlas (TCGA) project Tumors evolve through the continuous accumulation and selection of randomly mutated genes. While sets of advantageous mutations are selected in tumors, neutral or even slightly detrimental mutations may also occur due to genomic instability and genetic drift. Recently, much effort has been expended to identify in primary human cancers point mutations in the exons of cancer-related genes. However, systemic mapping of genomic DNA rearrangements has lagged behind, due to technical difficulties in detecting smaller deletions, tumor heterogeneity, and the necessity to purify malignant from normal cells in situ hybridization (FISH) While point mutations and very small insertions or deletions in genomic DNA can be detected by exon re-sequencing, it can be more difficult to detect gene dosage changes of larger genomic fragments, especially deletions Here we report a novel approach, designated as Primer Approximation Multiplex PCR (PAMP), to enrich small amounts of deleted genomic DNA sequences in the presence of wild type DNA. The genomic locations of the enriched sequences are subsequently decoded by a genomic tiling array and confirmed by sequencing.CDKN2A is located on chromosome 9p21  and MTAP (methylthioadenosine phosphorylase) (not shown) are centromeric and telomeric neighboring genes respectively CDKN2A genomic fragment and was used as template to generate probes (excluding repetitive regions) for printing on the minigenomic tiling array. The frequency of repetitive sequences predicted by RepeatMasker is shown at the bottom of the diagram.The ome 9p21 . It encoCDKN2A deficient DNA. We aimed to take advantage of the fact that a shorter deleted genome sequence should be preferentially amplified compared to a much longer WT sequence using \u201capproximated\u201d flanking primers  and reverse (R1-10) primer group can have multiple primers. Therefore, every PCR group (for example F1-R1) pair becomes multiplex PCR. Therefore, we designated this procedure as Primer Approximation Multiplex PCR (PAMP).INK4A exons 1 and 2) deletion on chromosome 9p21 A, FB, RY and RZ) of primers were used for four PAMP reactions (CDKN2A deficient) or HEK293 (CDKN2A wild type) cell lines. Aliquots of all 4 PAMP reaction products were pooled and labeled for hybridization on an INK4A minigenomic tiling array that covers about 25 kb, including all of the exons of INK4A. As predicted in We reported previously that the Detroit 562 cell line has an approximate 20 kb (including eactions  using geB-RY (array 27) and all products pooling (array 29) produce the same result as shown in In addition, four separate arrays were used to hybridize the individual PAMP products described above. A simple plot of signal intensity ratio of mutant/WT PCR products on the tiling array revealed the genomic location of the breakpoint . This anIn order to pinpoint more precisely the area of deletion, nested PCR with pairs of specific primers was designed according to the earlier PAMP results. The PCR product was labeled for array hybridization, yielding a result very similar to that shown in 5 molecules/\u00b5g of haploid genome). The CDKN2A deleted cell line Detroit 562 was serially diluted with CDKN2A wild type HEK293 as shown in the To mimic the heterogeneous population of cancer and host cells typically found in solid tumors, various amounts of genomic DNA derived from Detroit 562 (mutant) and HEK293 (wild type) were mixed for PAMP and array hybridization. In order to test the sensitivity of our approach, we performed a titration experiment. The total genomic DNA for each assay was kept constant (100 ng). This is equivalent to about 28,000 copies of haploid genome  that rely on the absence of a detectable wild type signal, PAMP directly measures the deleted DNA. Therefore, this approach is much less vulnerable to problems associated with normal cell contamination. The experimental procedure is robust enough to detect deletions in the presence of at least 99.9% wild type sequence contamination, which could not be achieved by other procedures We have developed a general strategy that can be applied for pinpointing the genomic breakpoints in unpurified primary cancers. The amplification and tiling protocol described here allows for simple and precise C. elegansPrimer approximation PCR screening has been a useful tool for isolating deletion mutants in CDKN2A locus with a total of 500 primers in one single PCR reaction through computational simulation, which will be described elsewhere (manuscript submitted). A recent paper reported a successful multiplex PCR with more than 1000 primer pairs through the aid of computational design We used multiplex PCR to reduce the workload and cost for PAMP. We were able to multiplex 28 primers easily in a single PCR reaction. Theoretically, one can cover over 90% of the 0.5 Mb of genomic fragment around CDKN2A breakpoints are specific and unique for each cancer with this locus deleted. A highly sensitive assay, such as real-time PCR, can be designed to monitor the status of cancer progression in the blood or other body fluids. The assay should be very specific because amplification is expected to occur only from deletion-containing DNA due to very long distance between the primers in the wild type genome J recombination may be responsible for creating CDKN2A breakpoints of the two cell lines used in this study as reported by others. Second, the DNA is more stable than RNA although it is easier to map fusion transcript if it exists, such as BCR-ABL. Third, the genomic breakpoints are very likely to be different from each patient and become personalized biomarkers, thereby, reducing the risk of false positive results due to cross contamination. However, this is also the biggest hurdle to overcome. For example, many efforts to improve the PCR amplification range for detecting C-MYC/immunoglobulin translocations have had limited success because the breakpoints are scattered across a more than 300 kb region Using breakpoint sequences as cancer-specific biomarkers to monitor minimal residual diseases has been explored CDKN2A with 1 kb resolution. We are currently working on methods to increase multiplexing and reduce the volume of each PAMP reaction for broader applications.Our approach aims to identify breakpoints within a 1 Mb genomic fragment as FISH or other cytogenetic techniques are available for larger genomic rearrangements and the cost and labor significantly increase when the target region expands. We are able to inexpensively produce a tiling array covering a genomic fragment of 0.5 Mb around the The cell lines described in the paper were obtained from the American Type Culture Collection  and cultured as recommended. The genomic DNA was extracted with DNAzol  following the instructions from the manufacturer.INK4A minigenomic tiling array covering a 25 kb fragment in the CDKN2A locus for proving the concept of our approach. DNA probes were generated by PCR with BAC clone RP11-149I2  as template and avoiding the repetitive genomic sequences that were predicted by RepeatMasker. The PCR products were purified with DNA Clean-up and Concentrator-5 , resuspended in 3\u00d7SSC and printed on poly-L-lysine slides at 0.1 mg/ml along with Human Cot-1 DNA , which is enriched for repetitive sequences, and herring sperm DNA , which was used as nonspecific control. The printing procedure has been described and essentially followed the manual of the DeRisi arrayer with silicon microcontact printing pins  http://derisilab.ucsf.edu/microarray/protocols.html).We created an INK4A exons 1-2 along the CDKN2A locus were synthesized by Integrated DNA Technologies . Groups of forward and reverse primers  were used to generate amplicons from 0.1 \u00b5g of genomic DNA templates in a total of 10 \u00b5l of solution mixing with 10 \u00b5l of Taq 2\u00d7Master Mix . The reaction was assembled at 4\u00b0C in a PCR workstation and transferred to a thermocycler with the block preheated to 94\u00b0C. The cycling conditions were a 3-minute denaturation step at 94\u00b0C followed by 35 cycles at 92\u00b0C for 30 sec, 55\u00b0C for 30 sec and 68\u00b0C for 2.5 minutes with a final extension step at 68\u00b0C for 5 minutes. One \u00b5l of unpurified product was subsequently used as templates for another round of amplification to label the amplicons with the same PCR protocol except that dTTP was replaced by a 4\u22361 mixture of aminoallyl dUTP  and dTTP for probe labeling. The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns, eluted in 9 \u00b5l of sodium bicarbonate (pH 9.0) and coupled with 1 \u00b5l of DMSO dissolved Cy3 or Cy5 NHS esters  for 30 to 60 minutes. The Cy3 and Cy5 labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 \u00b5l of 10 mM Tris-HCl (pH 8.0). Paired Cy3 and Cy5 labeled amplicons were combined with 3.6 \u00b5l of 20\u00d7SSC, 0.5 \u00b5l of Hepes (pH 7.0) and finally 0.5 \u00b5l of 10% SDS. The mixed solution was heated for 2 minutes at 95\u00b0C, cooled to room temperature and hybridized to the minigenomic tiling arrays at 63\u00b0C overnight essentially as previously described A simplified PAMP scheme is shown in 50%-HSI) were used to divide the intensity (GI) of each feature representing genomic probes. Each (GI):(50%-HSI) ratio, the normalized genomic probe signal, was plotted at the Y-axis against the corresponding probe's genomic location at the X-axis to ease data interpretation  were used for 35 cycles of PCR . The two sets of internal primers  were used for the second PCR reaction under the same conditions. A single band about 1 kb in size was excised and extracted with Qiaquick gel extraction kit . The purified product was directly sequenced with internal primers.ment see  and 4. EHs578T cell line: Four sets of uniplex PCR reactions were performed by pairing 2 single forward (FA1 and FA2) and 2 single reverse (RX3 and RX4) primers. The PCR program was the same as that for PAMP. The products were analyzed by agarose gel electrophoresis. A single band from the shortest distance pair (FA2-RX3) was excised and sequenced with an internal primer."}
+{"text": "DEFA1 and DEFA3 genes has been reported. Moreover, the DEFA3 gene has been found to be absent in a significant proportion of control population subjects. CNVs involving immune genes, such as \u03b1-defensins, are possibly contributing to innate immunity differences observed between individuals and influence predisposition and susceptibility to disease.Copy number variants (CNVs) account for a significant proportion of normal phenotypic variation and may have an important role in human pathological variation. The \u03b1-defensin cluster on human chromosome 8p23.1 is one of the better-characterized CNVs, in which high copy number variability affecting the DEFA3 absence in 697 samples from different human populations. The proportion of subjects lacking DEFA3 has been found to vary from 10% to 37%, depending on the population tested, suggesting differences in innate immune function between populations. Absence of DEFA3 was correlated with the region's haplotype block structure. African samples showed a higher intra-populational variability together with the highest proportion of subjects without DEFA3 (37%). Association analysis of DEFA3 absence with 136 SNPs from a 100-kb region identified a conserved haplotype in the Caucasian population, extending for the whole region.We have tested the DEFA3 gene may be suggestive of population-specific selective pressures with potential impact on human health.Complexity and variability are essential genomic features of the \u03b1-defensin cluster at the 8p23.1 region. The identification of population differences in subjects lacking the  DEFB1, DEFA6, DEFA4, DEFA1, DEFT1, DEFA3 and DEFA5) and at least two centromeric clusters of \u03b2-defensin genes  [Defensin genes encode a family of small cationic peptides that act as antimicrobial mediators of the innate immune system . DefensiDEFA1 and DEFA3) and \u03b2-defensin  genes in chromosome 8p23.1 has been well detected and characterized [DEFA1 and DEFA3 gene copies has been reported to range from 4 to 11 in a sample of 111 subjects, the DEFA3 allele being completely absent in 10% of them [DEFA1, DEFT1 and DEFA3 has been replaced by DEFA1A3, following recommendations of Aldred et al, since these genes have been considered as being part of a copy number variant (CNV) region [DEFA1 and DEFA3 alleles in 27 subjects and found between 5 and 14 copies per diploid genome, with DEFA3 being absent in 26% of them [Chromosome band 8p23.1 is known to be a frequent site of chromosomal rearrangements mediated by low copy repeats (LCRs) or segmental duplications (SDs). It has been described that as many as one in four individuals from the general population carry a 4.7 Megabase (Mb) inversion of the region . In addicterized -14. The  of them . Gene noDEFA1 and DEFA3 being considered as members of the same CNV (DEFA1A3), they encode different peptides, HNP-1 and HNP-3, respectively. The mature HNP-1 and HNP-3 peptides differ only in their N-terminal amino acid, due to a single nucleotide difference, C3400A, between the DEFA1 and the DEFA3 genes [DEFA1A3 CNV cluster encode the HNP-2 peptide. The three peptides are constitutively produced by neutrophil cell precursors and packaged in granules before mature neutrophils are released into the blood. During phagocytosis, the defensin-containing granules fuse to phagocytic vacuoles where defensins act as antimicrobial agents [Despite A3 genes . This C3l agents .DEFA3 in samples from different human populations. For this purpose, we used the International Haplotype Map (HapMap) Project collection and a cohort of Spanish healthy individuals.Recent work has shown that CNVs are a major source of genetic variation . IndividWe have analyzed 786 samples from four populations with ancestry in Europe, Africa or Asia (the HapMap collection), including Spanish healthy individuals. The source used for this study was the HapMap collection of 269 samples utilized by the International HapMap Consortium for the study of human genomic variation, initially through the investigation of SNPs and their associated haplotypes , and 180DEFA1 and DEFA3 differs only by a single nucleotide (C3400A), which allows distinguishing between DEFA1 and DEFA3 by HaeIII digestion, since a restriction site for this enzyme is absent in the DEFA3 sequence. All samples had at least one DEFA1 copy, but DEFA3 was absent in several subjects of all populations. DEFA3 was absent in different proportions depending on the population tested, ranging from 10% in the Chinese/Japanese dataset to 37% in the Yoruba samples .The coding sequence of DEFA6, DEFA4, DEFA1, DEFA3 and DEFA5), six \u03b1-defensin pseudogenes  and one \u03b8-defensin pseudogene (DEFT1P)  Figure . Such cl3 Figure .DEFA1A3 CNV, previously reported to be variable in copy number between individuals , but only in four cases where a gain or loss was detected, DEFA3 is absent. Copy number variation in the DEFA1A3 region is reported to be much more common than the variation identified by Redon et al [DEFA1A3 CNV makes it undetectable with BAC arrays. Moreover, the presence of segmental duplications in the region entails a bad SNP coverage of the region by the Affymetrix SNP array, which does not allow an accurate detection of the CNV. Thus, the study of this CNV for association purposes has to be performed by quantitative methods or by the analysis of paralogous sequence variants.HapMap samples have been tested for the presence of CNVs by two different techniques Affymetrix SNP array and BAC array . DEFA1A3on et al . HoweverDEFA1A3 cluster and the single copy gene DEFA5 was chosen for the linkage disequilibrium analysis (based on human genome assembly hg17) . However, only 136 of the SNPs had genotype data in all four populations. Interestingly, almost all genotyped SNPs are located outside the DEFA1A3 cluster  Figure . The Hapr Figure . The absOf the 136 SNPs analyzed in all four populations, 55 were monomorphic in at least one of them . Monomorphic SNPs can be used to measure genetic variability, by analyzing their distribution in the different populations. The Chinese and Japanese groups had the highest proportion of monomorphic SNPs (34%) which was very similar to that observed for Caucasian samples (31%), whereas the Yoruba samples had the smallest number of monomorphic SNPs (24%). This indicates that genetic variability is higher within Yoruba samples, while Chinese/Japanese and Caucasian populations show similar proportions of genetic variability. This higher variability for Yoruba samples is similar to that detected in the HapMap analysis for the whole genome . InteresDEFA3 locus absence results; the Yoruba samples showing highest LD variability and also having the highest proportion of DEFA3 absence.The patterns of linkage disequilibrium (LD) in each population are summarized in Figure DEFA3 is inherited together with neighbor SNPs, an association study was performed using the HapMap data for the 100-kb region including the DEFA1A3 cluster. All the SNPs of the region genotyped in the HapMap project were tested for association with the C3400A PSV, which defines the presence or absence of DEFA3 gene, respectively. No association for any of the genotyped SNPs was found in the Yoruba or Japanese/Chinese populations. However, a significant association was found between absence of DEFA3 and 18 SNPs in the Caucasian samples, under a recessive mode of inheritance .To assess whether e Figure . Associae Figure . Moreovek Figure . This esSeveral studies have recently reported a previously unknown high prevalence of copy number variation in humans . A recenDEFA1A3 and DEFB4/DEFB103A) in the 8p23.1 region have been extensively characterized [DEFA3 allele. In the present work, we have tested the absence of the DEFA3 allele in different human populations, finding significant differences between them, which could be indicative of differences in innate immune function between populations. This is not surprising since the different human population groups have been exposed to different environments regarding infectious agents and other factors. One obvious way by which CNVs result in human phenotypic diversity is by altering the transcriptional levels of the genes which vary in copy number [DEFA1A3 in which variation in DEFA1 and DEFA3 copy number, and DEFA3 absence could underlie variable resistance to infection among individuals. Different selective pressures acting in each geographic region could likely explain population differences in DEFA3 absence.CNVs involving \u03b1- and \u03b2-defensin genes  and DEFA3 (GenBank accession number L12691) genes differing by a single nucleotide. A fragment of 304 bp around C3400A SNP was PCR amplified with fluorescently labelled primers  and digested with HaeIII enzyme. In order to accomplish complete digestion, we used saturating conditions (2.5 U/25 \u03bcl reaction) of the enzyme to digest a short DNA fragment containing only one cutting site. In addition, in all the runs, a DEFA3 negative sample was included, as a positive control of the assay. About 2 \u03bcl of digestion product was added to 10 \u03bcl HiDi formamide containing ROX500 marker (Applied Biosystems) and run on an ABI 3100 capillary system (Applied Biosystems). Peaks were analysed using Genemapper software (Applied Biosystems).A PCR amplification assay followed by restriction enzyme digestion (PCR-RFLP) has been used to discriminate The UCSC Genome Browser  served aDEFA3 absence in different human populations. Genotyping data from HapMap public database [DEFA3 absence using logistic regression models. Odds ratios (OR)and 95% confidence intervals (95% CI) were calculated for eachgenotype compared with the homozygous for the major allele . Analyses were initially done under a codominant inheritance model (three genotypes separated). Then, simplified models were fitted: a dominant model , a recessive model , an overdominant model (homozygous grouped) and a log-additive model . The model with lowest Akaike information criteria was the recessive one (minus twice the log likelihood of the model plus the number of variables in the model) and it was selected for an easy summary of the results. P values were derived from likelihood ratio tests, and a significance level of 5% (two sided) was used for the analyses. All these analyses were performed using the SNPassoc R package [Between groups chi-square test was performed to compare the proportion of database  was used package .Haploblocks were constructed using Haploview program . HaplotyEB carried out the genetic molecular studies, the bioinformatics work and drafted the manuscript. JRG carried out the statistical analysis. NB participated in the bioinformatics work and design of the study. XE conceived the study and participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Results of the association study for the three population groups. The p-values under all inheritance modes tested are shown.Click here for file"}
+{"text": "This study sought to produce monoclonal antibodies (MAbs) which reacted with the MUC2 core protein. Two MAbs [3A2 (IgG1) and 4F1 (IgM)] were produced by immunising female BALB/c mice with gel-formed mucin from the LS174T colon cancer cell line followed by a KLH conjugate of a 29 amino acid synthetic peptide whose sequence was derived from the variable number of tandem repeats (VNTR) region of a MUC2 cDNA clone. The MAbs reacted with synthetic MUC2 VNTR peptides but not synthetic MUC1 or MUC3 VNTR peptides, and showed specific reactivity in Western blotting with a high molecular weight protein produced by the LS174T colon carcinoma cell line. The use of shorter peptides indicated that the minimum peptide epitopes for these MAbs were different. Mab 3A2 reacted with amino acids 5-19 of the MUC2 VNTR by inhibition ELISA but not by direct ELISA, while 4F1 reacted with this peptide in both assays. Furthermore, 4F1 reacted in direct ELISA when a larger (29 amino acid) MUC2-derived peptide was coated onto the assay plate by incubating in carbonate buffer or by drying the peptide onto the assay plate, while 3A2 only reacted when this peptide was coated in carbonate buffer. The different specificity of the MAbs was also illustrated by the reactivity of 4F1 but not 3A2 with partially deglycosylated cystic fibrosis mucin. Immunohistochemical analysis with these MAbs revealed a strong reactivity with lung, gastric and colon tumours relative to normal tissue, with some breast and ovarian tumours also reacting. Both MAbs stained some normal goblet cells in the perinuclear region but not the mucin droplet or secreted mucin, indicating a reaction with immature (poorly glycosylated) mucin in the endoplasmic reticulum and/or golgi, but not with mature (fully glycosylated) mucin. In contrast, tumours showed strong diffuse cytoplasmic staining. 4F1 also showed weak apical cytoplasmic staining in some goblet cells and stained some tumours which showed no reactivity with 3A2. These antibodies should prove useful in the study of MUC2 structure and function, and in the diagnosis of some tumours."}
+{"text": "The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three  transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study."}
+{"text": "Tumour tissue oxygenation has been measured in man during carbogen breathing  using a commercially available polarographic electrode system (Eppendorf p02 histograph). At least 200 tumour measurements in each of 17 patients with accessible tumours were taken before, and subsequently continuously after the commencement of carbogen breathing for periods of 10 to 30 min. In 12 out of 17 patients studied there was a significant increase in median tumour p02 during the first 10 min of carbogen breathing (range 9 to 1800%). There was an initial rapid increase in tumour p02 which was maintained until 8 to 12 min, but then decreased throughout the subsequent treatment period. Although there was a reduction in the proportion of point measurements < or = 10 mmHg in 11 out of 13 patients, during carbogen breathing, measured points of < or = 2.5 mmHg were only eliminated in three out of 11 tumours. The time course has implications for the planning of clinical trials utilising radiotherapy with carbogen breathing."}
+{"text": "Cdk2/Cdk4 double knockouts has indicated that these two Cdks are required to phosphorylate Rb during late embryogenesis. The lack of Rb phosphorylation is progressive and associated with reduced E2F-inducible gene expression. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis. However, proliferation in early embryogenesis appears to be independent of Cdk2 and Cdk4. We discuss these observations and propose molecular mechanisms that establish the requirement for Cdk2 and Cdk4 at the G1/S transition. We are considering that the balance between proliferation and differentiation is disturbed, which affects especially heart development and leads to embryonic lethality in Cdk2-/-Cdk4-/- mutants. We also discuss the specific functions of Cdk4 and Cdk6, which ironically do not compensate for each other.Progression through the mammalian cell cycle is associated with the activity of four cyclin dependent kinases . Knockout mouse models have provided insight into the interplay of these Cdks. Most of these models do not exhibit major cell cycle defects revealing redundancies, and suggesting that a single Cdk might be sufficient to drive the cell cycle, similar as in yeast. Recent work on  Cdk2 knockout mice, though Cdk2 was considered to be a unique kinase bound to cyclin E, regulating S phase initiation and progression. This perplexing observation has been quickly addressed by further in vivo analysis demonstrating that Cdc2, which was previously demonstrated to control G2/M, is also able to bind cyclin E and compensates for Cdk2 in S phase [Cdk4 and Cdk6 does not affect cell cycle initiation and progression, suggesting that Cdk2 compensates for the lack of cyclinD dependent kinases [Cdk2-/-Cdk6-/- mice display similar phenotypes as Cdk6 or Cdk2 single knockout mice [Cdk2-/-Cdk4-/- mice and provide new models of mammalian cell cycle regulation.Cell cycle regulation plays an essential role in cellular homeostasis and contributes to determine the fate of cells. Most factors influencing the decision, whether to start a new round of division or not, act at the G1/S transition. Mitogenic factors induce expression of cyclin D and therefore stimulate the activities of Cdk4 and Cdk6. The activation of the cyclin D/Cdk complexes is the first step leading to cell cycle entry and is followed by several waves of cyclin expression . Each family of cyclins binds to a specific Cdk, which is active at a specific phase of the cell cycle and contributes to the activation of the next cyclin/Cdk complex. Recent studies in different Cdk knockout mice have challenged this common model of mammalian cell cycle regulation. Single loss of Cdk2, Cdk4, or Cdk6 did not significantly affect cell proliferation in vivo or in vitro [ S phase . SimilarCdk2/Cdk4 double knockout (DKO) mice and for the first time we observed reduced Rb phosphorylation in vivo and in vitro [Cdc2 and cyclin A2. On the other hand, HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes and cell proliferation. This result suggests that Cdk6 and Cdc2 can regulate cell proliferation, but these two kinases might not phosphorylate Rb to full extent, leading to decreased Cdc2 expression. The declining Cdc2 expression acts as a negative loop leading to proliferation defects. The fact that more Cdk6 is bound to cyclin D1, in the absence of Cdk4, is apparently not sufficient to compensate the lack of Cdk2 and Cdk4 (see below). From our experiments, we conclude that Cdk2 or Cdk4 is required, at a certain point, to phosphorylate Rb thereby maintaining higher levels of Cdc2 protein expression. These two G1/S kinases contribute to the activation of G2/M cyclin/Cdk complexes, and doing so, couple the G1/S transition with mitosis. Characterization of the double knockout mice of Cdk2 and Cdk4 revise the picture of the cell cycle, combining features from the classic mammalian model and features from the yeast model. Nevertheless, though we have uncovered the dynamics of this molecular mechanism, we still need to understand why Rb phosphorylation starts to decline only at midgestation.We recently generated in vitro . The decCdk4 or Cdk2 induces relocation of free p27 to Cdk2 or Cdc2 complexes respectively, which affects their activity. Studies with p27 knockout mice have shown that loss of p27 can rescue a normal entry of S phase in Cdk4-null [Cdk2-null MEFs (unpublished data). In Cdk2-/-Cdk4-/- DKO, these inhibitors might be relocated to Cdc2 complexes and therefore, through their inhibition, contribute to the lack of Rb phosphorylation. We tested this hypothesis by knocking out p27 in Cdk2-/-Cdk4-/- mice, but did not observe a rescue of embryonic lethality or proliferation of MEFs [Cdk2-/-Cdk4-/- MEFs but Cdc2 did not restore normal proliferation (unpublished data).The late embryonic lethality of DKOs suggest that Cdk2 and Cdk4 are required to regulate the Rb/E2F pathway only late in development. In agreement with this, cell proliferation in early embryogenesis is comparable in DKO and wild type embryos. Our analysis has shown that Rb phosphorylation decreases progressively after E13.5, and we can only speculate how Rb phosphorylation is maintained in early development. Therefore, we propose four possible models that are not mutually exclusive and can possibly explain our findings Figure  could beCdk2-/-Cdk4-/- embryos is probably linked to the progressive loss of Rb phosphorylation observed at midgestation. So far, we do not know if the heart defect is related to hypophosphorylation of Rb. A similar cardiac phenotype was observed in cyclin D1-/-D2-/-D3-/- triple knockouts [cyclin D-null and Cdk2-/-Cdk4-/- mice. Cyclin D-null mice do not display an Rb defect, however, it cannot be excluded that the Rb/E2F pathway is deregulated in cyclin D-null cardiomyocytes. This pathway plays a major role in cardiogenesis and the levels of free activated E2F is critical for normal cardiac function [Cdk2-/-Cdk4-/- mutants, Rb represses E2Fs, probably interacting with repressors like Jumonji, which then inhibits cardiomyocyte growth. Further characterization of this pathway might help to better understand the complexity of heart development and the relation to the cell cycle players. Studies of Cdk2-/-Cdk4-/- cardiomyocytes will be a good experimental model to determine how the combined loss of Cdk2 and Cdk4 affects the balance between differentiation and proliferation. We need to determine why the cardiogenesis is more sensitive to inactivation of Cdk2 and Cdk4 than differentiation of other cell types.The embryonic lethality in DKOs is most likely associated with cardiac failure. The small size of the nockouts . It is lfunction . Anotherfunction . The inafunction . From E1Cdk2-/-Cdk4-/- mutants are embryonic lethal, whereas Cdk2-/-Cdk6-/- mice develop normally. Focusing on animal growth and control of cell proliferation, several observations suggest that Cdk4 and Cdk6 do not completely compensate for each other in vivo. Cdk4 single knockout males and females display reduced animal size [Cdk6 reduces the size of the females but to a lesser extend than Cdk4 mutation [Cdk4-null but not in Cdk6-null MEFs [Cdk4-/- and Cdk2-/-Cdk4-/- mutants compared to wild type or Cdk2-/-Cdk6-/- mutants. The complete inactivation of Cdk4 and Cdk2 leads to more pronounced lack of proliferation (at least in the hematopoietic linage and in MEFs) and affects cardiac development, thereby inducing embryonic lethality [Studies with double knockout mouse models have pointed out some differences between Cdk4 and Cdk6. Indeed mal size ,4, whileethality .Cdk2-/-Cdk4-/- mice. Other substrates might also be involved, such as Smad3, phosphorylated by Cdk4 and Cdk2 but not yet tested for Cdk6 [At the biochemical level, few differences have been described between Cdk4 and Cdk6 . In vivfor Cdk6 . Smad3 mfor Cdk6 ). All thCdk2-/-Cdk4-/- mouse model will teach us more details about tumorigenesis.Our knowledge of cell cycle regulation has greatly improved through the characterization of knockout mouse models. The overlap of Cdk functions adds more complexity to the in vitro model of the cell cycle (specific Cdk/cyclin complexes for each cell cycle phase). On the other hand, we can consider that all Cdks are redundant, which would result in a model similar to the yeast cell cycle. Our recent results show that the redundancy is not complete and each Cdk might have its own niche. This specificity could be essential for small subpopulations of cells  or affect cell cycle regulation globally. Indeed, Cdk2 and Cdk4 share a common role in the G1/S transition, which couples this phase with mitosis through E2F-inducible gene expression. Embryonic stem cells might proliferate independently of this coupling, and we presented four models to describe how this coupling can take place during embryogenesis. Among E2F-inducible genes, we focused on the role that Cdc2 can play as a kinase to phosphorylate Rb, however we cannot exclude that other E2F-targets are also important. Moreover, these four molecular mechanisms act probably in concert to establish the G1/S checkpoint. This role might be important with regards to cancer cells. Could the combined targeting of Cdk2 and Cdk4 be a valuable approach for cancer therapy? To answer this question, we have to determine if Rb wild type cancer cells require Cdk2 and Cdk4 activities throughout tumor progression. Future experiments with the Cdk: cyclin dependent kinaseDKO: double knockoutES cell: embryonic stem cellsMEF: mouse embryonic fibroblastPK is co-Editor-in-Chief of Cell Division but was not involved in the editorial and peer review process of this manuscript."}
+{"text": "The cytophotometric DNA content and the argyrophilic nucleolar organiser regions (AgNORs) of biopsy specimens taken before undergoing any treatment were examined in 91 surgically treated oesophageal carcinoma cases. There was a significant linear dependence between the mean DNA content and the number of AgNOR per nucleus (AgNOR number) . The DNA distribution pattern and the range of the AgNOR number also showed a significant correlation (P < 0.01). Twenty three of 28 cases with a low AgNOR number (< 4) were then determined to have a diploid pattern (type II), while 17 out of 22 cases with a high AgNOR number (> or = 6) had high ploidy values (type IV). The patients with a type II DNA distribution pattern and a low AgNOR number thus showed a good post-operative course with a 5 year survival rate of 55.2%, whereas no patients survived over 4 years among the 17 cases with both a type IV DNA pattern and a high AgNOR number (P < 0.001). These data thus demonstrate the close relationship between cytophotometric DNA content and AgNOR number and suggest that the combined detection of these two parameters, using biopsy specimens, should be of benefit in making an accurate preoperative evaluation of prognosis for patients with oesophageal carcinoma."}
+{"text": "Fourteen patients with advanced breast cancer were treated with the ribonucleotide reductase inhibitor didox 6 g m-2 given by intravenous infusion over 36 h every 3 weeks. None responded and toxicity was minimal. Possibilities for the more effective use of this agent are discussed."}
+{"text": "The data presented in this article shows the longitudinal analysis of tear fluid cytokine profiles, blood CD4 and CD8 counts and HIV viral load in 34 dry eye patients with HIV infection during the HAART therapy. Clinical samples were collected from HIV patients with dry eye disease at the time of presentation to the clinic (visit 1), three months (visit 2) and 6 months (visit 3) after the presentation. At each time point tear samples were evaluated for 41 cytokines using Luminex bead based multiplex assay and blood samples were tested for HIV viral load and CD4 and CD8 counts. Specifications TableValue of the data\u2022In our earlier reports cytokines IFN-gamma-inducible protein 10 , epidermal growth factor (EGF) and growth-regulated oncogene (GRO) levels were significantly altered in dry eye patients with human immunodeficiency virus (HIV) infection compared to immunocompetent dry eye patients at the time of presentation (visit 1/baseline) to the clinic \u2022Here we report the longitudinal tear cytokine profiling data of HIV patients with dry eye disease over the period of 6 months with two follow ups (3 months each) and its association with the systemic CD4 (cluster of differentiation 4), CD8 counts and HIV viral loads.\u2022This longitudinal data will help researchers to understand the changes in tear cytokines in HIV patients with dry eyes over the time and its association with systemic and viral factors and study further on HIV associated ocular inflammation and its pathogenesis.1The data presented herein was obtained from longitudinal analysis of tear cytokine profiles in 34 dry eye patients with HIV infection at 3 time points . Systemi2The data herein was obtained from HIV patients who had complaints of symptoms related to dry eyes  with a prior informed consent and were evaluated following the guidelines of the International Dry Eye Workshop (DEWS), 2007"}
+{"text": "CYP2C9*30 (A477T) is associated to diminished response to the antihypertensive effects of the prodrug losartan and affected metabolism of other drugs. Here, we investigated molecular mechanisms involved in the functional consequences of this amino-acid substitution. Molecular dynamics (MD) simulations performed for the active species of the enzyme (heme in the Compound I state), in the apo or substrate-bound state, and binding energy analyses gave insights into altered protein structure and dynamics involved in the defective drug metabolism of human CYP2C9.30. Our data revealed an increased rigidity of the key Substrate Recognition Sites SRS1 and SRS5 and shifting of the \u03b2 turn 4 of SRS6 toward the helix F in CYP2C9.30. Channel and binding substrate dynamics analyses showed altered substrate channel access and active site accommodation. These conformational and dynamic changes are believed to be involved in the governing mechanism of the reduced catalytic activity. An ensemble of representative conformations of the WT and A477T mutant properly accommodating drug substrates were identified, those structures can be used for prediction of new CYP2C9 and CYP2C9.30 substrates and drug-drug interactions.Cytochrome P450 2C9 (CYP2C9) metabolizes about 15% of clinically administrated drugs. The allelic variant  CYP2C9 can affect the clinical response of drugs metabolized by CYP2C9, especially those with a narrow therapeutic index and can induce adverse drug reactions (ADR). The rare allelic variant CYP2C9*30 found in Japanese subjects has been associated to diminished response to the antihypertensive effects of the prodrug losartan [Cytochrome P450 2C9 (CYP2C9) is the most expressed member of the human CYP2C family and metabolizes more than 15% of clinically administrated drugs including hypoglycemic agents, anticonvulsants, anticoagulants, nonsteroidal anti-inflammatory drugs (NSAIDs), antihypertensives, diuretic drugs  and sevelosartan , 11. It losartan . Recentllosartan . HoweverCYP2C9*30. Our results highlighted conformational and dynamic changes believed to be involved in the governing mechanism of the catalytic activity reduction and confirmed that protein dynamics affected by missense mutations could play a crucial role in affected regulation of substrate entry, recognition and metabolism of CYP2C9.Here we provide insights into the dysfunctional metabolism mechanism in human CYP2C9.30 based on molecular dynamics (MD), quantum mechanics and docking simulations. Molecular dynamics (MD) simulations combined with energetic analyses have been demonstrated to be a pertinent approach to understand molecular mechanisms of amino-acid substitutions affecting drug metabolism or regioselectivity of CYP \u201318. In tThe A477T mutation present in CYP2C9.30 variant is located in the Substrate Recognition Site 6 (SRS6) (Supplementary The Root Mean Square Fluctuations (RMSF) results confirmed the stability of the WT and mutant A477T structures during the MD simulations. The mean RMSF values of the merged MD trajectories for each of the six systems varied between 1.08 \u00c5 and 1.18 \u00c5. However, the comparison of the atomic fluctuations per residue see  revealedvs 5.6% in the apo mutant MD simulations) and F476 (a HB conserved in 26.3% in the apo WT vs 2.1% in the apo mutant MD simulations). Changes of the HB interactions of Q214 due to the A477 mutation were also observed in the case of bound losartan to CYP2C9 [In order to find noticeable conformational differences of the substrate binding pocket of CYP2C9 between the WT and the A477T mutant, we performed structural clustering of the five merged MD trajectories for each of the studied systems based on the binding site residues see . The stro CYP2C9 . Therefoo CYP2C9 ) and mayHydrophobic interactions between F476 and other hydrophobic residues of the CYP2C9 binding pocket were monitored within a distance of 5 \u00c5 during the MD simulations see . We obseSubstrate positions are crucial for the oxidation reaction. On the contrary, the losartan reached an equilibrium position , remaining in the same subpocket in contact with the heme similarly to its initial docking position in both WT and A477T mutant proteins see . Along tThe RMSD of Cpd I along the MD simulations  showed tWe explored the structure and dynamics of the substrate access channels for the WT and the A477T mutant. Significant changes of the substrate access channels were found to occur in the presence of the mutation A477T. The open channels detected along all MD simulations for the WT and the mutant proteins are summarized in We observed that several channels were open in more than 20% of the MD simulations of the diclofenac-bound state . This observation is in accordance with the large movements of diclofenac observed during the MD simulations suggesting a dynamic behavior of the channels. Similar results have been reported in a previous study  for CYP2The comparison between the apo WT and mutant proteins showed several important changes due to the mutation. Most of the substrate and solvent access channels were more closed in the A477T mutant as compared to the WT. More precisely, the channels 2b, 2f, 5 and S1 were more closed in the apo state of the mutant than of the WT see . The chaWe carried out molecular docking calculations to identify different conformations of the WT and A477T mutant of CYP2C9 properly accommodating the substrates for the catalytic reaction. In order to explore the binding spectrum of CYP2C9, we studied five drugs metabolized by CYP2C9 by molecular docking: diclofenac, flurbiprofen, warfarin, losartan and glimepiride. It has been shown that the metabolism of diclofenac, losartan and glimepiride is strongly affected in the A477T mutant . To takeTen independent docking calculations were performed for each protein centroid structure. The three top-scored substrate poses were taken for the analysis. This resulted in 360 substrate poses in MD generated WT CYP2C9 conformations and 360 poses in MD generated A477T mutant conformations for each of the five drugs. The two small NSAID flurbiprofen and diclofenac were found to bind in different positions in the CYP2C9 binding pocket, while the other ligands showed preferable binding zones. The comparison of the WT and the mutant showed differences for the diclofenac and flurbiprofen docking in the so called \"flurbiprofen-binding\" subpocket corresponding to the catalytic site subpocket close to the heme . These tThen, we focused on the SOM positions regarding the Cpd I oxygen atom, which was included in the docking calculations. We considered that the docking poses showing a distance between the SOM  and the In order to analyze other possible positions of the losartan in the CYP2C9 active site we compared the docking poses of losartan with the recently reported co-crystalized structures, PDB ID 5XXI (WT\u2013losartan bound) and 5X23 (A477T mutant\u2013losartan bound). The most similar positions were found when the losartan was docked into the active site of the centroid number 4 of CYP2C9 WT apo representing 5.76% of the entire MD simulations for WT apo (RMSD = 5.41 \u00c5) and of the centroid number 0 of A477T mutant\u2013losartan bound representing 6.59% of the entire MD simulations for the A477T apo mutant\u2013losartan bound (RMSD = 4.36 \u00c5). These docking poses suggest that losartan can also adopt positions similar to the co-crystalized ones in CYP2C9 WT and CYP2C9 A477T mutant\u2013losartan bound. Although RMSD are not very low, the losartan is similarly oriented in the docked and experimental structures see . HoweverIn the three best WT apo MD derived centroids the residues F476 and L362 adopted different conformations . The resCYP2C9*30. Our data demonstrated that the A477T mutation present in CYP2C9.30 led to increased rigidity of the SRS1 and SRS5 regions and to shifting of the \u03b2 turn 4 of SRS6 toward helix F thus decreasing the substrate access to some protein channels. We observed different behaviors of the substrates diclofenac and losartan during the MD simulations for the WT and the A477T mutant, suggesting that A477T can alter differently the metabolism of various substrates. The overall stabilization of the WT and the mutant structures in the cases of a bound substrate observed for the performed here MD simulations as well as for the available crystal structures demonstrated the importance to study structural and dynamics properties of the apo protein in order to detect molecular mechanisms due to the mutation without screening effects of a bound ligand. Molecular docking of five drug substrates of CYP2C9 into WT and A477T mutant structures generated by MD simulations indicated that some conformations of the A477T mutant were able to correctly accommodate the substrates, however, their number was very low compared to the WT MD conformations. All together these conformational and dynamic changes are believed to be involved in the governing mechanism of the catalytic activity dysfunction. Finally, an ensemble of representative conformations of the WT and A477T mutant properly accommodating the substrates were identified, these structures can be used for future prediction of unknown CYP2C9.1 and CYP2C9.30 substrates and drug-drug interactions.We investigated molecular mechanisms implicated in the altered substrate metabolism in CYP2C9.30 exhibiting < 50% of the WT enzyme activity. MD simulations performed for the active species of the enzyme, Compound I, in apo or substrate-bound states, and binding energy analyses for several drugs revealed molecular mechanisms involved in the defective drug metabolism in carriers of Six crystallographic structures of CYP2C9 were taken from the Protein Data Bank (PDB) and analysed: the native sequence flurbiprofen\u2013bound one (PDB ID 1R9O) , losartaThe initial models of diclofenac-bound and losartan-bound WT and A477T mutant structures were generated using docking with Autodock 4.2 program . The besThe six initial CYP2C9 models  were fully solvated with explicit TIP3P water molecules  and neutThe NAMD2.6 program  was usedTo find the most representative structures of the substrate binding pocket for each of the six systems we merged the five MD trajectories. For the structural clustering, we took 25,000 protein conformations (1 conformation every 10 ps) for each system and used the quality threshold algorithm  of the Vwww.chemaxon.com). Interestingly, ChemAxon predicted two different pKa values for losartan, 4.29 or 8.0, starting from two different smile structures downloaded from BindingDB and DrugBank, respectively. We analyzed possible protonation of the losartan molecules co-crystallized with CYP2C9 in the PDB structures ID 5X23 and 5XXI and decided to consider the protonated state of the losartan, which corresponds better to the losartan environment in the binding sites. Gasteiger atom charges were added using the AutoDockTools package. Molecular dockings were carried out with the Autodock 4.2 program [Five drugs known to be CYP2C9 substrates were docked into the crystal CYP2C9 structures PDB IDs: 1OG5 and 4NZ2 with restored native residues, 1R9O, 5XXI, 5X23, as well as into the initial models of the WT or the mutant A477T and into 240 representative conformations selected after the MD simulations for the six studied systems. The heme was kept in the intermediate Cpd I state. Drug protonation states were calculated at pH 7.4 with the major microspecies option of the ChemAxon package Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file."}
+{"text": "Sarcophilus harrisii) predominantly affecting their facial regions. DFT1\u2019s cellular origin is that of Schwann cell lineage where lesions are evident macroscopically late in the disease. Conversely, the pre-clinical timeframe from cellular transmission to appearance of DFT1 remains uncertain demonstrating the importance of an effective pre-clinical biomarker. We show that ERBB3, a marker expressed normally by the developing neural crest and Schwann cells, is immunohistohemically expressed by DFT1, therefore the potential of ERBB3 as a biomarker was explored. Under the hypothesis that serum ERBB3 levels may increase as DFT1 invades local and distant tissues our pilot study determined serum ERBB3 levels in normal Tasmanian devils and Tasmanian devils with DFT1. Compared to the baseline serum ERBB3 levels in unaffected Tasmanian devils, Tasmanian devils with DFT1 showed significant elevation of serum ERBB3 levels. Interestingly Tasmanian devils with cutaneous lymphoma (CL) also showed elevation of serum ERBB3 levels when compared to the baseline serum levels of Tasmanian devils without DFT1. Thus, elevated serum ERBB3 levels in otherwise healthy looking devils could predict possible DFT1 or CL in captive or wild devil populations and would have implications on the management, welfare and survival of Tasmanian devils. ERBB3 is also a therapeutic target and therefore the potential exists to consider modes of administration that may eradicate DFT1 from the wild.Devil Facial Tumour 1 (DFT1) is one of two transmissible neoplasms of Tasmanian devils ( Sarcophilus harrisii) belongs to the Dasyuridae family, it is a carnivorous marsupial that is extinct on mainland Australia and now found only on the island of Tasmania. Superficial dermal cutaneous lesions of wild Tasmanian devils can be found commonly in the form of skin sores [The Tasmanian devil  A pilot study of thirty-five Tasmanian devils differing in age, sex and geographic location were selected  to compa\u00b0C. Serum samples were retrieved from the frozen archive and thawed at room temperature immediately before analysis.Blood samples from Tasmanian devils  were col\u00b0C. Sections were deparaffinised, rehydrated and stained using Jung autostainer XL (Leica) for Haematoxylin  and Eosin, dehydrated cleared and mounted in CV Mount .Tasmanian Devil tissues were fixed in 10% Neutral Buffered Formaldehyde  for 24 hours and selected tissues were cassetted and processed overnight using a standard 15 hour overnight procedure in the TP1050 tissue processor . Tissues were orientated on the EG1160 (Leica), embedded in paraffin wax  and sectioned at 3 microns using Leica RM2245 microtome and adhered to microscope slides  for 20 minutes at 60\u00b0C for 8 minutes using a Pascal pressure chamber  then cooled to 20\u00b0C. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide  in methanol  for 30 minutes. Detection of primary antibodies was achieved using Mach1 Universal HRP-Polymer detection kit . Protein block (Background Sniper BS966L10) was applied for 20 minutes prior to application of primary antibodies. Monoclonal rabbit anti-human ERBB3  was diluted 1:50 with antibody diluent  and applied to both devil tumour and normal devil control tissues at room temperature for 30 minutes. Negative control was omission of primary antibody with buffer substitution. Universal HRP-polymer was applied for 30 minutes (MRH538L10) followed by 1 drop of Betazoid DAB Chromogen 3,3 Diaminobenzidine (BDB900G) in 1ml of substrate buffer (DB900) applied for 4 minutes. Tris buffered saline  was used to rinse between all steps. Slides were rinsed, stained with Carazzi\u2019s Haematoxylin for 5 minutes, washed for 3 minute in tap water, dehydrated, cleared and mounted in CV mount. Sections were viewed under light microscopy using Olympus BX41  and selected areas were photographed using an Olympus digital camera (DP20).Archival Tasmanian devil tissues and tumours were sectioned at 3 microns, floated onto Superfrost plus slides (Menzel Glaser) and subjected to standard deparaffinisation and rehydration techniques using an automated stainer (Leica). Antigen retrieval in tissue sections was conducted in citrate buffer at pH 6.0  at 120\u00b0C. The supernatant was removed and wells were washed 4 times with 300 uL of 1X wash solution using an Immunowash 1575 . One hundred microliters of prepared biotinylated anti-ERBB3 was added to each well and the assay plate incubated for 1 hour at room temperature. The assay plate was washed as described after which 100 uL of prepared HRP-streptavadin conjugate was added to each well and the assay plate incubated for 45 minutes at RT. The assay plate was again washed as described and 100 uL of TMP substrate was added and the plate incubated for 30 minutes at room temperature in the dark, after which 50 uL of stop reagent was added to each well. The absorbance of each well was measured at 450 nm using a Tecan Infinite M200 microplate reader .Serum ERBB3 levels were measured using the RayBio anti-human ERBB3 ELISA Kit  according to manufacturer\u2019s instructions. Briefly, serum samples were diluted 1/5 in Assay Diluent A and 100 uL of standard or diluted sample were added in duplicate to wells of a 96 well assay plate and incubated for 24 hrs at 4The ELISA standard curve was plotted using Prism v5  and results for each serum interpolated and corrected for dilution. The significance of differences in serum ERBB3 between groups was determined using a Kruskal-Wallis test with Dunn\u2019s Multiple Comparison utilizing Prism v5 .DFT1 histology  and HaemSerum ERBB3 levels are shown in Fifteen Tasmanian devils without neoplasia  were studied with an average serum ERBB3 of 32 pg/ml. Collectively, CHD Tasmanian devils serum ERBB3 levels ranged from <30\u2013663 pg/ml which could be considered representative of the reference range for Tasmanian devils. Wild caught devils 6 and 7 were unremarkable and had serum ERBB3 levels <30 pg/ml however devil 9 (220 pg/ml) and devil 10 (92 pg/ml) both recorded skin abscesses. The ERBB3 levels in the CHDD group  ranged from <30\u2013220 pg/ml all had a small isolated dermatopathy such as abscess (devil 9), pyogranuloma (devil 10), skin tag with associated inflammation (devil 11) and small focus of dermatitis (devil 12) all recorded a low serum ERBB3 levels of <92 pg/ml. The CHJD  approximately 10 months old had an unremarkable clinical history that indicated serum was collected for a health check only, reflected in the low serum ERBB3 level of <30 pg/ml.2 containing capped at one devil per 100 m2.Further assessment of data and clinical history  revealedWe noted that skin injuries were commonly recorded although no abnormality was noted for devil 1, alopecia bilaterally around the hind limbs and flank was present on one mother due to her 3 pouch young (devil 2) and multiple puncture wounds were present on the remainder . Given that these devils were otherwise clinically healthy it would suggest that skin wounds caused by biting may contribute to some elevation in the serum ERBB3 of Tasmanian devils. There is also the possibility that simply being a Tasmanian devil living in a free range enclosure as opposed to wild populations may in itself be contributory to elevation in serum ERBB3 due to more frequent devil-devil engagement. Our results indicate that Tasmanian devils without injuries or an isolated skin lesion have serum ERBB3 levels <30 pg/ml whereas Tasmanian devils with multiple injuries or large abscesses have serum ERBB3 levels ranging from 92\u2013663 pg/ml. Together, these results suggest that cancer-free Tasmanian devils have a serum ERBB3 range of <30\u2013663 pg/ml.All devils with DFT1 were wild caught and all subjected to field autopsy with most serum samples reaching the laboratory within one to three days. We assessed the available clinical history  includinWe included Tasmanian devils with cutaneous lymphoma (CL) in the study for two reasons. Firstly, they were non-DFT1 devils with a severe skin condition that can affect the facial regions and secondly, the disease presentation of alopecia, excoriation and thickened plaques is distinct from DFT1 . Our resThe capture and detection of antibody in our ELISA assay is selective for the extracellular domain (ECD) of transmembrane ERBB3 in serum or plasma, thus ERBB3\u2019s ECD is cleaved and shed from the plasma membrane would be a natural assumption. In contrast the ERBB3 receptor is internalised, although very slowly, for negative regulation and inactivation \u201371 utiliDefective internalisation, recycling and degradation of cell surface proteins and ligands is an emerging feature of cancer . It is tAs well as functional transmembrane forms, secreted soluble forms of Epidermal Growth Factor Receptors have been well documented for ERBB1 \u201384, ERBBThe antigenic peptide used for this assay is located within the N-terminal domain of the full length ERBB3 protein. Full length ERBB3 translates into a 180 kDa protein whereas ERBB3 transcripts, created by intron read through and alternative polyadenylation signals result in serum ERBB3 isoforms translating into various proteins ranging in size from 22\u201375 kDa . SecreteThe correlation of serum levels with disease severity and progression would be the foundation of a good biomarker  as well;ERBB3 is crucial to the sequential transition from precursor to immature and finally mature Schwann cells where ERBB3 is down-regulated as myelination proceeds . The aduDespite evidence for multiple resistance mechanisms for existing therapeutic targeting of ERBB1/2 \u2013141 numehttp://www.utas.edu.au/news/2015/10/16/19-world-first-trial-of-tasmanian-devil-vaccine-begins-in-the-wild/). Confidence that immunisation can be successful stems from research showing that Tasmanian devils have a competent immune system [TM.However, managing wildlife disease is considerably more difficult than human disease because of limited data, the effect of the disease on the host and the transmission of disease within a dynamic population makes it difficult to model . Previoue system , 198\u2013200e system , 201. Ane system . Develope system  however,e system , 204, ERe system \u2013207 or be system  includine system  all showe system . Veterine system  the onlyRecent investigations reveal that the tumour microenvironment of metastatic DFT1 expressed B7-H1 and DFT1 cell lines could upregulate B7-H1. Immune-Our research has highlighted ERBB3 as a potential therapeutic target however treatment of Tasmanian devils with DFT1 with therapeutic regimes such as chemotherapy and radiotherapy are impractical. However, a combinatorial approach using therapeutic cancer vaccines including inactivated allogenic DFT1 cancer vaccine, ERBB3 monoclonal antibody, ERBB3 Peptide or xenogeneic vaccine in combination with anti-immune checkpoint blockade therapy would be easier to implement in the field as well as providing a sustained immunological response against DFT1.ERBB3 had previously avoided scrutiny due to its kinase inactivity; however, ERBB3 has now been the subject of intense investigation over the past decade and is now recognised as a potent partner of the epidermal growth receptor family. ERBB3 upregulation during developmental, dedifferentiation and regenerative processes encapsulates the Schwann cell\u2019s inherent plasticity and imparts certain characteristics of malignant transformation advantageous to transmission of DFT1. Our pilot study has shown for the first time that ERBB3 is consistently expressed immunohistochemically and that ERBB3 is also elevated in the serum of Tasmanian devils with advanced DFT1 and cutaneous lymphoma. Therefore, our research indicates that serum ERBB3 has the potential to be employed as a biomarker of DFT1 or CL in Tasmanian devils to assist conservationists in the management and welfare of Tasmanian devils and species survival. The simplicity of the ELISA Serum ERBB3 methodology is easily incorporated into routine laboratory batch testing and equally applied to include rapid turnaround of results for urgent cases. Extension of this research is necessary to include greater numbers of healthy Tasmanian devils both with and without visible injuries, devils with large and small DFT1 lesions as well as pre-clinical DFT1. This will firmly establish the normal reference range for serum ERBB3 from which potential pre-clinical DFT1 may be identified. In addition, ERBB3 is now recognised as a therapeutic target and therefore the potential exists to consider modes of administration in addition to existing whole cell vaccination such as ERBB3 monoclonal antibody, peptide or xenogeneic vaccines including checkpoint inhibitors. A combinatorial immunotherapeutic approach will enhance cytotoxic destruction, provide long term immunity from DFT1 and therefore eradicate this transmissible tumour from the wild.S1 Fig(DOCX)Click here for additional data file."}
+{"text": "The transcription factors GATA4, GATA5 and GATA6 play important roles in heart muscle differentiation. The data presented in this article are related to the research article entitled \u201cGenome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis\u201d   Specifications TableValue of the data\u2022GATA4, 5 and 6 are important regulators of heart muscle differentiation (cardiomyogenesis).\u2022These data identify genes differentially regulated by each of these GATA factors during cardiomyogenesis.\u2022These data also identify genes that are regulated by these factors in common during this process.\u2022These data will help us understand the molecular mechanisms that govern the function of these factors and therefore improve our knowledge about the gene regulatory network involved in cardiomyogenesis.\u2022The analysis of gene ontology (GO) terms associated with cardiogenic GATA-regulated genes provides the different biological processes in which these factors are involved in during normal development and homeostasis.1The data represents RNA-Seq performed on cardiogenic samples, single GATA4, 5 and 6 depleted samples as well as samples with GATA4, 5 and 6 triple depleted, described in the related research article gata4, gata5 or gata6 . Sheet 2: The 361 genes that are exclusively reduced by gata4 knockdown (see sox7 and sox18 (highlighted in red). Sheet 3: represents GO biological process terms associated with all the 1480 genes that are reduced in gata4 knockdown  according to the manufacturer's instruction. 50fg of RNA for Activin were injected. Xenopus gata4 splice morpholino, gata5 splice morpholino Activin \u03b2 B DNA constructs for mRNA synthesis have been described previously 2.2Xenopus laevis embryos were obtained as previously described 2.3Xenopus laevis genome (version 9.1) using HiSat2 At least 30 explants were used for RNA preparation with a previously described protocol"}
+{"text": "This study clearly shows the potential that oxygen sensors possess in terms of assisting in the optimised development of commercial RTE salad products.Optical oxygen sensors were used to ascertain the level of oxygen consumed by individual salad leaves for optimised packaging of ready-to-eat (RTE) Italian salad mixes during refrigerated storage. Seven commonly found leaves in Italian salad mixes were individually assessed for oxygen utilisation in packs. Each leaf showed varying levels of respiration throughout storage. Using the information obtained, an experimental salad mix was formulated (termed Mix 3) which consisted of the four slowest respiring salad leaves\u2014Escarole, Frisee, Red Batavia, Lollo Rosso. Mix 3 was then compared against two commercially available Italian salads; Mix 1  and Mix 2 . Optical sensors were used to non-destructively monitor oxygen usage in all mixes throughout storage. In addition to oxygen consumption, all three salad mixes were quality assessed in terms of microbial load and sensorial acceptability. In conclusion, Mix 3 was found to consume the least amount of oxygen over time, had the lowest microbial load and was most sensorially preferred ( More recently, the use of high O2 atmospheres  have been used as an alternative technique to low O2 equilibrium modified atmosphere (3% O2) [2, as well as CO2, within the pack relates to the metabolic state of the produce [The growth in the ready-to-use vegetable market ~10% p.a) has been largely due to increasing demand consumers for fresh, healthy and convenient foods 0% p.a ha. Consumes i.e., >0% O2 hav2 sensors have been shown to provide valuable information in food packaging applications [2 detection to monitor O2 levels in different forms of food packaging provides ample information as to the package environment conditions of a food product throughout storage and shelf life. Problems associated with packaging and containment failures can be instantly observed post packaging using this technology [2 sensors in specific food applications, such as: MAP cheese [sous vide products [Optical Oications ,9,10. Thchnology . ResearcP cheese , vacuum P cheese , MAP andP cheese , cooked P cheese , MAP andP cheese , as wellproducts . 2 sensors have not been employed to monitor O2 levels in retail packs of fresh produce and have never been evaluated as a technology to assist in the optimised development of commercial RTE salad products. Therefore, the objective of this study was to employ the use of non-destructive O2 sensing technology to ascertain the level of O2 consumed by individual salad leaves so that the optimised packaging of RTE Italian salad mixes might be determined. An increase in the O2 gas fill of such MAP salads is also used as an alternative to traditional 3%\u20135% O2.To date, the use of O2, 5%\u201310% CO2 and 85%\u201390% N2) and monitored for O2 levels over time. In order to appropriate the correct level of O2 to a mixed salad product, it was necessary to understand the individual O2 requirement for each salad leaf within the product mix. Individual salad leaves were packaged using a higher O2 level  to assess the true O2 levels utilised by each leaf and subsequent salad mixes during the shelf life period. Mixes of salad leaves were also prepared as described; totalling 170 g, with approximately 25% of each leaf and contents are described in 2, 5%\u201310% CO2 and 85%\u201390% N2) without salad fill and monitored for oxygen over time. This was undertaken to assess the level of permeation of gases using these commercial packaging materials over a 14 day period. The level of permeation was negligible as sample free packs were seen to hold initial gas fill within <0.5% O2, showing poor permeability.Salad leaves used in this study were grown in Ireland (and in close proximity to the processing facility where product packaging was undertaken) and consisted of the following salad leaf varieties: Escarole, Frisee, Radicchio, Lollo Rosso, Cos, Iceberg and Red Batavia. Salad leaves undergo treatments such as washing and shredding by manufacturer prior to packaging. Approximately 170 g of each salad leaf was packaged individually for assessment, using an Ishida SE multi head weigher  and stored at 4 \u00b0C. A Sandiacre Novus 350  was used to form, fill and seal samples in orientated polypropylene/low density polyethylene laminate films (220 \u00d7 290 mm) of 45 micron thickness with modified atmospheres. Control RTE salad mixes were produced commercially with normal packaging conditions  was used in conjunction with O2 sensors allowing for regular non-destructive monitoring of oxygen within packs. The Mocon OpTech-O2 device allows for reliable and accurate O2 readings from 0.001% to 25% O2 in 0.5 s. The device causes excitation of the phosphorescent dye embedded in the optical oxygen sensor and is quenched by molecular oxygen. Calibration of instrumentation was carried out using a Platinum CalCard allowing for instant calibration before each set of measurements were taken. O2 readings were taken at day 0, 3, 5, 7 and 10 for commercially prepared MAP RTE salads, individual salad leaves and modified salad mixes. The final measurement was taken on day 10 of storage as no further O2 drop was observed and samples had exceeded microbial limits.Oxygen sensors were prepared using Pt-OEPK  spotted on Durapore  paper using Gilson P100 pipette  and cut to a size of 5 mm in diameter. These sensor materials were then placed on 3 cm Avery price marking stickers  for adhesion to packaging films. A Mocon OpTech-OMicrobial testing was carried out during refrigerated shelf-life assessment of individual salad leaves and RTE Italian salad Mixes 1, 2 and 3 on sampling days 1, 3, 5, 7 and 10. Total viable counts (TVC) were carried out to determine microbial numbers (colony forming units (cfu)/g samples) in accordance with ISO standard method (ISO 4833:2003)  using toA sensory panel made up of 26 members was recruited in University College Cork, Ireland. Panellists were selected based on their availability on test days and were regular consumers of RTE salad products. All panellists had previous experience in carrying out sensory analysis. A list of descriptors for sensory analysis was selected and these are presented in versus PC 2 is presented; other PC\u2019s did not yield additional information. To derive significance indications for the relationships determined in the quantitative APLSR; regression coefficients were analysed by jack-knifing  was used to process the mean data accumulated from the 26 panellists during the sensory evaluation. Principal component (PC) 1 -knifing .2 modified atmosphere conditions consisting of <5% O2. This practice concurs with similar reported practices [2 levels were monitored over time. From 2 provided in such packs may be insufficient for optimised product respiration, as by day 3 of storage, less than 0.5% O2 remained within packs. This move towards an anoxic environment leads to quality-linked deteriorative processes within the pack [In commercial Irish applications, Italian mixed RTE salad leaf products are packaged in low Oractices . In thesthe pack ,22.2 requirement for each salad leaf within the product mix. Consequently, individual salad leaves typically used within Italian-styled RTE mixed salad leaf products  were packaged individually within MAP formats  to ensure adequate O2 was available for respiration) containing a pre-fitted optical O2 sensor. The individual salad leaf packs were monitored over seven days for O2 levels to ascertain O2 consumption by each salad leaf type. 2 consumed by each salad leaf type during storage . The O2 differential between leaves showed that after seven days, samples ranged in oxygen utilisation by up 3.2% O2. For example, the Radicchio salad leaf was found to consume the greatest amount of O2 over the seven days of storage (15.79%), whereas the Lollo Rosso leaf was determined to consume the least amount of O2 (12.47%) over the same period of time. The consumption of O2 in packs appears to slow after day 7, at which the product is spoilt.It was necessary to understand the individual O2 required by individual salad leaves to respire adequately over a seven day storage period was far greater than the typical <5% O2 provided in typical commercial MA packaging applications. This insufficient level of O2 utilised in MAP could lead to anaerobic respiration with the production of undesirable metabolites and associated physiological disorders [The data generated in this study showed that the amount of Oisorders . 2 utilisation can be categorised in a table representing each salad leaf in the order of increasing respiratory levels. 2 consumption by individual salad leaves in order of increasing O2 utilisation on storage days 3, 5 and 7. Escarole, Lollo Rosso and Frisee were consistently the slowest respiring salad leaves while Cos, Radicchio and Iceberg were consistently found to utilise the greatest amount of in-pack O2. Individually packed leaves underwent microbial (TVC) testing.It is apparent that each leaf respires at different rates under the same conditions. The level of OEach individually packed salad leaf was measured by total viable count (TVC) assay. Testing was carried out on day 1, 3, 5, 7 and 10 to establish the microbial load in colony forming units per gram of sample. 2 over 10 days and had the second lowest log TVC value. Results can also be ranked in terms of which salad leaf has the lowest microbial load in order to understand the difference in microbial quality across all samples. This can be seen in In the case of Radicchio, a correlation between highest level of oxygen utilisation of all leafs and microbial counts at day 7 are observed. The inverse of this can be seen with Lollo Rosso, where it was found to consume the lowest level of O2 utilised by each salad leaf , an experimental salad mix was formulated using the four lowest respiring leaves determined by the O2 sensor previously. This Italian-styled formulation (Mix 3) and two similar commercially available formulations (Mixes 1 and 2) were modified atmosphere packaged using the same packaging conditions, in the same manufacturing plant, using salad materials grown and processed in the same manner. All three salad mixes were produced in volume and a shelf-life evaluation study undertaken. Oxygen measurements were taken throughout product storage and optical O2 sensor readings showed that Italian salad Mix 3 had the lowest O2 consumption of all three salad mixes  of all seven leaf types assessed previously and commonly found in Italian-style mix leaf salads. Salad Mix 2 was also comprised of the Cos leaf, which was also found to be amongst the highest O2 consumers (13.33%) of all leaves assessed. Consequently, the use of Radicchio and other high respiring leaves in mixes ultimately results in a final product that consumes greater amounts of O2. Therefore, it was essential to provide a modified atmosphere with enough O2 to allow sufficient O2 for respiration irrespective of what leaves selected. It is important to recognize that extremely low O2 levels or excessively high CO2 levels can result in the generation of off-flavours or visible tissue damage [Based on the results obtained by monitoring the level of Oad mixes . After sns Mixes  and 2 wee damage . 2 over time. By day 7, Mix 3 was found to still maintain acceptable limits of <log 6 [2 over time appears to result in a product that has a lower microbial count than that of one which consumes more.All three salad leaf mixes were assessed for microbial quality during refrigerated storage . Microbif <log 6 ,23, wherp \u2264 0.01), flavour (p \u2264 0.05) and acceptability (p \u2264 0.01) were significantly and positively correlated. Day 7 data contrasted significantly with salad samples found to be negatively correlated with overall appearance, flavour and acceptability, along with a positive correlation for wilting appearance (p \u2264 0.01) and non-crispy texture (p \u2264 0.01). As for the individual salads, it was apparent that salad Mix 3 was favoured amongst panellists over salad Mixes 1 and 2, where overall appearance and acceptability were significantly correlated (p \u2264 0.01) and descriptors such as wilting appearance and non-crispy texture were found to be negatively correlated (p \u2264 0.05) showing a true preference for salad Mix 3. This selection appears to be attained by the use of Red Batavia instead of Radicchio. Panellists were asked if any individual leaf was found to be particularly undesirable and in this case 16 of the 26 panellists (61.5%) found the Radicchio leaf to be undesirable within the salad mix. The Radicchio leaf is understood to be a spicier and a more bitter intense leaf [2 (21%) in packaging did not appear to have an adverse affect on browning of the salad leaves. Data derived by Jack-Knifing uncertainty testing for individual salad leaves find no significance in LSB or LEB. Salad Mixes 1, 2 and 3 were assessed for sensory acceptability over the seven day storage period . Panellists were made aware of relevant descriptors  and ratinse leaf  which mo2 sensor in determining the oxygen respiration levels shows a novel and effective tool in understanding the O2 levels used in respiring salads leaves packaged in modified atmosphere conditions. This valuable information let the development of a product with a longer, more stable shelf life and a product more acceptable to consumers.With the understanding that Mix 3 was found to be the most desirable in terms of sensory analysis, the formulation of a mix with a lower microbial load and slower respiration rate compared well to common commercially produced salad mixes. The use of an optical O2 utilised by individual salad leaves was successfully achieved using optical O2 sensors. Information gathered using O2 sensors showed that an O2 level in excess of 10% is required to provide salad leaves with enough O2 to comfortably respire within a typical shelf-life period of seven days. The apparent lack of O2 within such packs has been previously shown to cause many deleterious quality processes. The use of optical O2 sensors showed that a salad mix can be optimised by understanding specific product requirements, like that for oxygen, and subsequently, careful product selection; thereby assisting in extending product shelf-life and promoting quality.The ability to non-destructively assess the level of O"}
+{"text": "Systemic therapy for cutaneous squamous cell carcinoma is typically reserved for rare cases of metastatic disease or lesions which cannot be adequately managed with local therapies. The relevant published literature is comprised only of case series and reports. Herein the authors present two cases of neglected locally advanced cutaneous squamous cell carcinoma not initially amenable to local therapies, which achieved dramatic partial responses to palliative Cisplatin and Docetaxel, facilitating sequential palliative radiotherapy. These reports add to the limited literature supporting the use of platinum-based therapy in locally advanced and metastatic cutaneous squamous cell carcinoma. They are the first describing the use of Cisplatin and Docetaxel in this setting. Non-melanomatous skin cancers are the most common forms of human malignancy, with more than 1 million cases expected to be newly diagnosed in the United States in 2009 . The majThe authors here aim to contribute to the literature by reporting two cases of locally advanced and metastatic cutaneous SCC treated with sequential palliative chemotherapy and radiotherapy.2 and Docetaxel 55 mg/m2 every 3 weeks. He tolerated 12 cycles extremely well with only mild myelosuppression, oral mucositis and tinnitus. The chest lesion showed an early and sustained dramatic response to therapy  due to myalgias, oral mucositis and 2 episodes of febrile neutropenia requiring hospitalization and G-CSF support. The visible lesion showed an early and sustained significant response to therapy : 1 of these then had surgery alone, 2 had radiotherapy alone and 1 had both, resulting in 2 patients having no evidence of disease (NED) at 13 and 22 months post-chemotherapy respectively, 1 having NED after further surgery and radiotherapy required for a local recurrence at 8 months post-chemotherapy, and 1 dying of disease at 19 months post-chemotherapy. Seven of the 13 patients achieved a partial response (PR): 2 of these then received no local therapy, 3 had radiotherapy alone, and 2 had surgery and radiotherapy, resulting in 4 patients dying of their disease at 12, 11, 8 and 4 months post-chemotherapy respectively, and 3 having NED after 12, 11 and 10 months post-chemotherapy respectively. One of the 13 patients had no response to chemotherapy, received subsequent interferon and died of disease 10 months post-chemotherapy. The last of the 13 patients progressed through chemotherapy, received subsequent retinoids and died of disease 4 months post-chemotherapy. Overall then, of 13 evaluable patients, 6 had NED and 7 had died of their disease at the time of manuscript preparation.Sadek et al.  reported2/d for 4 days and Bleomycin 20 mg/d for 4 days via continuous infusion. The first patient received a reduced dose of Bleomycin (15 mg/d). Cycles were repeated every 3 weeks. The first patient achieved a CR, the second a PR, and the third progressed through chemotherapy. All underwent subsequent surgery within 3 weeks. No recurrences had been recorded during short follow-up periods of between 2 - 9 months.Denic  reported2 and Doxorubicin 50 mg/m2 every 3 weeks for up to 4 cycles. Seven of the 12 patients received chemotherapy alone; 2 of these 7 achieved a CR and had NED at 12 and 4 months from the initiation of chemotherapy respectively, 2 achieved a PR (1 for 3 months before dying of progressive disease and 1 for 6 months before developing progressive disease), and 3 patients had no response to chemotherapy . One of the 12 patients received chemotherapy followed by surgery which resulted in a PR after chemotherapy and a CR of 11 months\u2019 duration following surgery. The remaining 4 of 12 patients received chemotherapy followed by radiotherapy: 2 had no response to chemotherapy but achieved CRs of 32 and 30 months post-radiotherapy respectively, 1 achieved a CR after chemotherapy that persisted for 13 months post-radiotherapy and 1 achieved a CR after chemotherapy that persisted for 11 months post-radiotherapy until that patient died of comorbid lung cancer.Guthrie et al.  also rep2 on day 1 and 5-Fluorouracil 1 g/m2/d via 4-day continuous infusion. Cycles were repeated every 3 weeks. Patients received between 2 - 6 cycles. The first patient with metastatic disease achieved a CR and had NED 24 months following chemotherapy. The second patient achieved a PR and developed progressive disease 3 months following chemotherapy. Two patients with locally advanced disease achieved CRs; one was lost to follow-up 3 months following chemotherapy, and the other died of a myocardial infarction but had NED 13 months following chemotherapy. Two patients with locally advanced disease achieved PRs; one achieved a subsequent CR following consolidative surgery and the other refused chemotherapy after 2 cycles and outcome data was not provided. One patient with locally advanced disease had no response to 6 cycles of chemotherapy and died of extensive locoregional disease and a CVA.Khansur and Kennedy  reported2/week). The patient recurred one month later, but achieved a second CR after 7 additional cycles before developing progressive disease requiring 2 cycles of palliative 5-Fluorouracil (600 mg/m2/d) for 5 days every 2 weeks. The patient progressed and died of disease 1 month later. Alternatively, Wollina et al. [2 on days 1 - 13 and subcutaneous interferon-\u03b1-2a 3 x 3 million units 3 times weekly for 3 cycles).Other options for systemic treatment include retinoids and interferon-alpha. Lippman et al.  reporteda et al.  describeTo the authors\u2019 knowledge the present reports are the first describing the use of Cisplatin and Docetaxel in the management of locally advanced or metastatic cutaneous SCC. The rationale for their use was similar to that employed within other reported series; the agents have proven efficacy for SCC in other disease sites , they ar"}
+{"text": "Plasmodium falciparum protein, Pfs25, produced from the baculovirus expression system. Pfs25 is a malaria transmission-blocking vaccine candidate, with a compact and complex structure including 22 cysteines. This supplementary data is related to the research \u201cDisulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate\u201d  [1]. In brief, Pfs25 was digested with trypsin/Lys-C and derived peptides separated by High Performance Liquid Chromatography (HPLC) and analyzed by mass spectrometry (MS) by MSE fragmentation. The theoretical peptides and their respective masses along with disulfide bond locations with linked peptides are presented here alongside the mass spectrometry analysis. The raw mass spectrometry data is made available through the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.This article contains the peptide data obtained while performing disulfide bond mapping of the recombinant  Specifications TableValue of the data\u2022The derived peptides and mass spectrometry data is provided here for further details from the disulfide bond analysis of Pfs25.\u2022The disulfide bond locations and linked peptides are discussed alongside the mass spectrometry analysis here and data made accessible to the scientific community.\u2022Pfs25 is a compact and complex 17.9\u202fkDa protein with 22 cysteines (11 disulfide bonds) that has presented difficulty in prior disulfide bond analysis\u2022A method was developed to map the disulfide bonds of a complex and compact protein, which may be applicable to other proteins, an important step in recombinant protein development for vaccines.1The Pfs25 disulfide bond mapping peptides are discussed in further detail in this manuscript to further support the elucidation of disulfide bonds of Pfs25 as discussed in Utilizing Biopharmalynx 1.3 the mass spectral data was analyzed and compared to the theoretical peptides to obtain the localization of the 11 disulfide bonds present in the recombinant Pfs25. The disulfide bond locations and linked peptides  masses are presented in 210 and Cys24. The remaining ten fragment ions were consistent with constituent peptides  were consistent with an internal disulfide bond linkage between Cyspeptides , Fig. 5.795 to Cys113 linkage. Four fragment ions  were specific to the linkage between T14 and T16 and confirmed the linkage of Cys115 to Cys129. A further 21 fragment ions were consistent with the linkage of T12, T14, and T16 and remaining 11 fragments consistent with constituent peptides  were specific to the linkage between T12 and T14 and confirmed the Cyspeptides , Fig. 6.8137 and Cys148. Ten fragments were consistent with constituent peptides  and a 2489 UV/Vis Detector  set at 214\u202fnm. An XBridge  BEH 300 C18  was used at a column temperature of 37\u202f\u00b0C and gradient with 0.1% Triflouroacetic acid (TFA) in purified water (Mobile Phase A) and 0.1% TFA in acetonitrile (Mobile Phase B) as described in 10.3E mode using MassLynx v4.1 . RAW MS files have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.MS analysis was done with a QTOF Premier mass spectrometer  equipped with an electrospray source as described in 10.4The mass spectral data was analyzed using BiopharmaLynx 1.3  as described in"}
+{"text": "ESCC) and to provide valuable information for the identification of potential diagnostic biomarkers in ESCC. Tumor samples of ESCC patients retrieved from The Cancer Genome Atlas were divided into Grade 1 , Grade 2  and Grade 3  groups in accordance with the clinical record of the tumor grade of ESCC patients. The genes associated with tumor grade were identified. The signaling pathways of identified genes were enriched from the Kyoto Encyclopedia of Genes and Genomes (KEGG). The diagnostic value of candidate genes was assessed by receiver operating characteristic analysis. We used the GSE23400 dataset generated from the Gene Expression Omnibus to examine the expression levels of candidate genes in ESCC tissues compared to matched mucosa tissues. In total, 440 genes positively correlated with tumor grade and 882 genes negatively correlated with tumor grade were identified. There were 310 differentially expressed genes (DEGs) between G1 and G2, 184 DEGs between G2 and G3, and 710 DEGs between G1 and G3. There were 1322 genes associated with tumor grade that were significantly enriched in pathways in cancer and the phospholipase D signaling pathway. Cyclin\u2010dependent kinase inhibitor 1A, golgin A7 family member B and transforming growth factor B1\u2010induced anti\u2010apoptotic factor 1 (TIAF1) had potential diagnostic value for discriminating ESCC patients with G1 from those with G3. TIAF1 was significantly down\u2010regulated in ESCC. The results of the present study comprise useful groundwork with respect to determining the tumorigenesis mechanism in ESCC and discovering potential diagnostic biomarkers for ESCC.The present study aimed to identify the genes associated with the histologic tumor grade of patients with esophageal squamous cell carcinoma ( ARNT2aryl hydrocarbon receptor nuclear translocator 2AUCarea under the curveBCRbiospecimen core resourceBDKRB2bradykinin receptor B2BEBarrett's esophagusCA12carbonic anhydrase 12CABC1coenzyme Q8ACDKN1Acyclin\u2010dependent kinase inhibitor 1ADEGdifferentially expressed geneEACesophageal adenocarcinomaECesophageal carcinomaESCCesophageal squamous cell carcinomaGEOGene Expression OmnibusGERDgastroesophageal reflux diseaseGNA15G protein subunit \u03b115GOLGgolginHCChepatocellular carcinomaJUPjunction plakoglobinKEGGKyoto Encyclopedia of Genes and GenomesLAD1ladinin 1LAMB3laminin subunit \u03b23NSCLCnonsmall cell lung cancerPHLPPPH domain and leucine rich repeat protein phosphatasePKP1plakophilin 1PPARperoxisome proliferator activated receptorPRSS8protease, serine 8ROCreceiver operating characteristicTCGAThe Cancer Genome AtlasTIAF1transforming growth factor B1\u2010induced anti\u2010apoptotic factor 1TMEM108transmembrane protein 108TNMtumor, node and metastasisTSStissue source siteEsophageal carcinoma (EC) is a common malignancy of digestive tract carcinoma worldwide, with an estimated 455 000 new cases and 400 000 deaths each year Smoking, a low intake of vegetables and fruits, being overweight, obesity, chronic gastroesophageal reflux disease (GERD) and Barrett's esophagus (BE) are risk factors in EAC The histologic tumor grade of ESCC is segregated into Grade 1 (well\u2010differentiated), Grade 2 (moderately\u2010differentiated) and Grade 3 (poorly\u2010differentiated) according into the World Health Organization classification. Tumor grade is an independent prognostic factor of EC PHLPP2PRSS8) with hypermethylation is significantly decreased in ESCC, which predicts a shorter overall survival of patients with ESCC. Demethylation of PRSS8 contributes to the inhibition of tumor progression, including cell proliferation, migration and cell cycle arrest It is acknowledged that the intersection of etiological factors, including dysregulated genes, aberrant expression of micorRNAs and abnormal DNA methylation, contributes to the initiation and development of ESCC. Over\u2010expression of miR\u2010483\u20103p results in a poor prognosis for patients through promoting ESCC progression as a result of targeting EI24 The Cancer Genome Atlas (TCGA) database. We aimed to provide the groundwork with respect to determining the tumorigenesis mechanism in ESCC, as well as discovering potential diagnostic biomarkers.In the present study, bioinformatics analyses were performed to identify the dysregulated genes and pathways correlated with histologic tumor grade based on the expression profiling of ESCC in The present study used sequence\u2010related data from ESCC tissues from the TCGA database. ESCC tissues and clinical metadata of eligible ESCC patients were collected by Tissue Source Sites (TSSs), such as the University of Alabama, the Technical University of Munich and the University of Kansas Medical Center. After a preliminary pathological review, TSSs deliver ESCC tissue samples and metadata to the Biospecimen Core Resource (BCR). Next, the BCR verifies the quality and quantity of the pathological diagnosis of ESCC tissues. The RNA is then extracted from ESCC tissues and also by BCR for genomic characterization and high\u2010throughput sequencing. Sequence\u2010related data are deposited in the TCGA database A total of 188 esophageal squamous cell carcinoma patients with clinical records (collected from 26 June 2012 to 28 January 2015) were available in the TCGA database. The tumor grade of ESCC samples is recorded, which was divided into five grade groups, such as GX (unknown), G1 (well\u2010differentiated), G2 (moderately\u2010differentiated), G3 (poorly\u2010differentiated) and G4 (undifferentiated), in accordance with the World Health Organization classification.The inclusion criteria of patients were patients: (a) with a subtype of esophageal squamous cell carcinoma; (b) without a history of other malignancy; (c) without a history of neoadjuvant treatment; (d) for whom the expression profiling of mRNA was available; and (e) for whom the record of histologic tumor grade was G1\u2013G3. In the present study, ESCC patients were separated into G1, G2 and G3 groups in accordance with the recorded tumor grade. Level 3 mRNA sequence data of ESCC patients were downloaded from the TCGA data portal, which is based on UNC Illumina Hiseq_RNASeqV2.rP < 0.05 was considered statistically significant.Those mRNAs with a 0 reads count were excluded from the study. A linear by linear association test rrThe significant correlations between expression levels of genes and tumor grade were visualized via a Box\u2010plot analysis in P < 0.05 was considered statistically significant.The genes associated with tumor grade were identified. To clarify the expression specificity of those genes, the significance analyses of differentially expressed genes between G1 and G2 groups, between G2 and G3 groups, and between G3 and G3 groups were subjected to Tukey's honest significant difference genecodis3 P < 0.05 was considered statistically significant.To obtain insights into the signaling pathways of genes associated with the tumor grade of ESCC, KEGG pathway enrichment was performed using ARNT2), GOLGA7B, transforming growth factor B1\u2010induced anti\u2010apoptotic factor 1 (TIAF1), cyclin\u2010dependent kinase inhibitor 1A (CDKN1A), peroxisome proliferator activated receptor (PPAR)G and laminin subunit \u03b23 (LAMB3) in ESCC via the pROC package in r.To assess the diagnostic value of candidate genes in ESCC (G1 and G3 groups), the ROC curve was deciphered and the area under the curve (AUC) was calculated to assess the diagnostic value of aryl hydrocarbon receptor nuclear translocator 2 , which is a freely public data repository that archives microarray, next\u2010generation sequencing and other forms of high\u2010throughput functional genomics data submitted by the research community.To examine the expression levels of candidate genes in ESCC tissues, the microarray expression profiling of ESCC tissues and adjacent nontumor tissues were obtained from GEO database (GSE23400 (53 ESCC versus 53 adjacent nontumor mucosa tissues) were incorporated in the present study.The microarray expression profiling of ESCC issues was searched and the inclusion criteria of datasets required that: (a) the dataset was generated from the mRNA expression profiling of ESCC patients; (b) both the ESCC and adjacent nonumor mucosa tissues samples were available in the dataset; (c) the sample size of dataset was greater than 50; and (d) sequence data of the candidate genes were available in the dataset. Finally, P\u2010value indicating the difference between two group was calculated. P < 0.05 was considered statistically significant.Box\u2010plot analyses were performed to describe the expression of candidate gene both in ESCC and matched mucosa tissues. The A total of 83 ESCC patients were incorporated into the present study based on the exclusion criteria. In total, 16, 47 and 20 patients with ESCC were assigned to the G1, G2 and G3 groups, respectively. There were 20 531 genes available in the mRNA sequence data of the ESCC tumor based on the Illumina Hiseq_RNASeqV2 in TCGA database. After trimming the 6026 genes with a 0 reads count, 14 506 genes were included in the present study.TMEM108), C3orf47 and coenzyme Q8A (CABC1) were the top three genes positively correlated with tumor grade, whereas ladinin 1 (LAD1), TIAF1 and FGF11 were the top three genes negatively correlated with the tumor grade of ESCC patients. The respective expression status of genes in the G1, G2 and G3 groups is shown in Fig. TMEM108 and C3orf47 were positively correlated with tumor grade and eight genes, including LAD1, TIF1, FGF11, carbonic anhydrase 12 (CA12), G protein subunit \u03b115 (GNA15), junction plakoglobin (JUP), plakophilin 1 (PKP1) and PPARD, were negatively correlated with the tumor grade of ESCC patients.Correlations between the expression of genes and tumor grade of ESCC patients were analyzed by a linear by linear association test. Eventually, 1322 genes associated with tumor grade were identified, including 440 genes positively correlated with tumor grade and 882 genes negatively correlated with tumor grade Table . As showIn total, 1322 genes associated with the tumor grade of ESCC were significantly enriched in 14 signaling pathways, including pathways in cancer (hsa05200), the phospholipase D signaling pathway (hsa04072), small cell lung cancer (hsa05222) and gastric acid secretion (hsa04971)  were identified as being differentially expressed genes. There were 310, 184 and 710 differentially expressed genes, respectively, identified in G1 versus G2, G2 versus G3 and G3 versus G1 , between the G2 and G3 groups (G2 versus G3) and between the G1 and G3 (G3 versus G1) groups was analyzed. Genes showing a significant difference (G1 Table . As showTIAF1 (0.862), GOLGA7B (0.869), LAMB3 (0.812), ARNT2 (0.819) and CDKN1A (0.878) was greater than 0.8; the AUC of GOLGA1 (0.762) and PPARG (0.766) was less than 0.8. CDKN1A, GOLGA7B and TIAF1 had a larger AUC than the other four candidate genes. The sensitivity and specificity of CDKN1A was 90% and 68.8%  compared to G3 (poorly\u2010differentiated) and significantly up\u2010regulated in G1 compared to G2. In the GSE23400 dataset, it was significantly down\u2010regulated in ESCC tissues compared to matched mucosa tissues. In addition, TIAF1 could discriminate between ESCC patients with well\u2010differentiated ESCC from those with poorly\u2010differentiated ESCC. The results of the present study indicate that TIAF1 might function as a tumor suppressor in the development of ESCC and might also be a potential biomarker for ESCC diagnosis. TIAF1 contributes to the progress of Alzheimer's disease and tumor GOLGA7B and GOLGA1 in ESCC patients with G3 (poorly\u2010differentiated) were significantly lower than those with G2 (moderately\u2010differentiated) and G1 (well\u2010differentiated). GOLGA7B locates at chromosome 10 and encodes GOLGA7B. GOLGA1 locates at chromosome 8 and encodes golgin A1. The exact roles of GOLGA7B and GOLGA1 remain unknown. GOLGAl is reported to be stably expressed in 17 tissues samples, including breast, esophagus, lung, pancreases, colon, rectum and duodenum GOLGA2 encodes golgin A, another member of golgin family, which is required for efficient tracking in the secretory pathway at the Golgi complex In vivo mice model experiments show that down\u2010regulation of GOLGA2 suppresses lung cancer tumorigenesis by inhibiting angiogenesis and cell invasion GOLGA7B and GOLGA1 were identified as novel genes associated with the tumor grade of ESCC. The biological roles of GOLGA7B and GOLGA1 in ESCC are unknown. Further investigation is necessary to clarify the mechanism of GOLGA7B and GOLGA1 with respect to the initiation and development of ESCC through in vitro and in vivo experiments.In the present study, the expression levels of ARNT2, PPARG, PPARD, FGF11, JUP, CDKN1A, bradykinin receptor B2 (BDKRB2) and LAMB3, overlapped from the 56 genes mentioned above, and 702 differentially expressed genes between the G3 and G1 groups and the top 50 genes negatively/positively correlated with tumor grade were obtained.A total of 56 genes associated with the tumor grade of ESCC was significantly enriched in pathways in cancer Table . Eight kLAMB3 encodes laminin subunit \u03b13 and belongs to laminin family of extracellular matrix glycoproteins (secreted molecules) that have a heterotrimeric structure consisting of \u03b1, \u03b2 and \u03b3 subunits. LAMB3, along with LAMA1, LAMA3 and LAMC2, was enriched in pathways involved in cancer. Figure LAMBA3 was significantly down\u2010regulated in ESCC with G3 (poorly\u2010differentiated) compared to those with G1 (well\u2010differentiated) and G2 (moderately\u2010differentiated). However, the expression status of LAMB3 in GSE23400 was in agreement with the previous studies. The expression level of LAMB3 is reported to be up\u2010regulated in ESCC compared to normal tissues and LABM3 expression is correlated with the depth of invasion and malignancy ARNT2 and PPARG showed positive correlations with the tumor grade of ESCC and were up\u2010regulated in G3 (poorly\u2010differentiated) compared to G1 (well\u2010differentiated). ARTN2, which encodes aryl hydrocarbon receptor nuclear translocator, is a transcriptional factor related to adaptive responses PPARG encodes peroxisome proliferator activated receptor \u03b3, which is a member of PPAR subfamily of nuclear receptor. PPARG was significantly down\u2010regulated in ESCC tissues compared to matched mucosa tissues, for which the results were not compatible with our bioinformatics analyses showing that PPARG was significantly up\u2010regulated in ESCC with G3 (poorly\u2010differentiated) compared to those with G1 (well\u2010differentiated) and G2 (moderately\u2010differentiated). However, the expression status of PPARG in GSE23400 was in agreement with previous studies. PPARG is reported to function as an oncogene in ESCC and the activation of PPARG suppresses cell proliferation and induces cell apoptosis of esophageal cancer cells by inhibiting the TLR\u20104 dependent mitogen\u2010activated protein kinase pathway CDKN1A, also known as CDKN1 and P21, encodes cyclin\u2010dependent kinase inhibitor 1A. It contributes to regulate cell\u2010cycle progression by inhibiting the activity of cyclin\u2010cyclin\u2010dependent kinase 2/4. In the present study, the expression level of CDKN1A was negatively correlated with the histological grade of ESCC. It was significantly down\u2010regulated in ESCC patients with G3 (poorly\u2010differentiated) compared to those patients with G1 (well\u2010differentiated). Decreased expression of nucleostemin suppresses cell proliferation of esophageal carcinoma by up\u2010regulating CDKN1A CDKN1A, GOLGA7B and TIAF1 was greater than 0.85, indicating that CDKN1A and GOLGA7B might be potential biomarkers for discriminating ESCC patients with G1 (well\u2010differentiated) from those with G3 (poorly\u2010differentiated).The dysregulated genes correlated with the tumor grade of ESCC were identified by bioinformatics analyses based on the TCGA database in the present study. To access the discriminatory ability of those dysregulated genes between ESCC patients with G1 and those with G2, the counts of candidate genes originating from 188 ESCC patients incorporated into our in silicon analysis in the TCGA database were used to depict the ROC curve. The ROC analyses showed that the AUC of GSE23400 dataset was used in our analyses. The expression level of TIAF1, LAMB3 and PPARG was statistically different between ESCC tissues and matched mucosa tissues; however, the expression levels of GOLGA7B, GOLGA1, ARNT2 and CDKN1A were not statistically different between ESCC and matched mucosa tissues. There is heterogeneity of the expression status of candidate genes between the TCGA and GSE23400 datasets. Two confounding factors might contribute to the the contradictory results of the expression status of candidate genes in ESCC between the these datasets. First, different sequencing platforms were used. The mRNA expression profiling of the GSE23400 dataset was based on an Affymetrix Human Genome U133A Array and an Affymetrix Human Genome U133B Array and the mRNA expression profiling of the TCGA dataset was based on RNA\u2010sequencing. Different sequencing platforms might lead to heterogeneity between the TCGA and GSE23400 datasets. Second, there were different procurement countries for the ESCC samples available in the respective datasets. The procurement countries of ESCC samples in the TCGA dataset included Netherlands, USA, Russia, Ukraine and Canada, whereas the ESCC samples in the GSE23400 dataset were obtained from patients who underwent surgery in China.To detect the expression levels of candidate genes in ESCC tissues, the in vivo and in vitro experiments. Second, the expression levels of candidate genes in ESCC patients compared to matched mucosa tissues and in ESCC patients with well\u2010, moderate\u2010 and poor differentiation need to be validated in a large sample size of ESCC patients in future studies. Third, the diagnostic value of CDKN1A, GOLGA7B and TIAF1 needs to be validated by use of a large cohort in clinical practice.The present study comprises a preliminary analysis for the identification of genes associated with the tumor grade of ESCC. There are limitations to our study. First, several candidate genes associated with the tumor grade of ESCC were identified, although the biological roles of corresponding genes with respect to cell differentiation, migration and adhesion of ESCC were not explored through CDKN1A, GOLGA7B and TIAF1 have potential diagnostic value for discriminating ESCC patients with G1 (well\u2010differentiated) from those with G3 (poorly\u2010differentiated).Taken together, the association between dysregulated genes and the tumor grade of ESCC was analyzed based on the metadata of patients retrieved from the TCGA database. In total, 1032 dysregulated genes associated with the tumor grade in ESCC were identified and these genes were significantly enriched in the phospholipase D signaling pathway, as well as in pathways involved in cancer and small cell lung cancer. CL designed the experiment. CL and JX analyzed the data. JX and CL collected patient information. JX and CL drafted the manuscript.Table S1. The full list of genes associated with the tumor grade in ESCC.Click here for additional data file.Table S2. The identified differentially expressed genes in G1 versus G2, G2 versus G3 and G3 versus G1.Click here for additional data file."}
+{"text": "Longevity assurance homolog 2 of yeast LAG1 (LASS2) has been reported to act as an important tumor suppressor in the development of human cancers. However, little is known about the prognostic value of LASS2 in hepatocellular carcinoma (HCC) . In the present study, we analyzed correlation between LASS2 and TGF-\u03b21 levels, and evaluated their prognostic values in HCC patients. We first analyzed the expression of LASS2 and TGF-\u03b21 in two independent cohorts  using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis revealed a positive correlation between the expression of LASS2 and TGF-\u03b21. HCC Patients with low expression of LASS2 and TGF-\u03b21 had shorter overall survival (OS) and time to recurrence (TTR) than patients with high expression of LASS2 and TGF-\u03b21. Furthermore, combination of LASS2 and TGF-\u03b21 was an independent and significant risk factor for OS and TTR. In conclusion, low expression of LASS2 and TGF-\u03b21 contributes to the aggressiveness and poor prognosis of HCC, and may represent a novel prognostic biomarker for HCC patients. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, the third major cause of cancer-related death worldwide245Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), which is also known as tumor metastasis suppressor gene (TMSG1) or ceramide synthase 2 (CerS2), is the gene identified from a human liver cDNA library by our laboratory13Transforming growth factor-\u03b21 (TGF-\u03b21) plays biphasic functions in tumor tumorigenesis, having a growth inhibitory effect at early stages, but at later stages enhancing the malignant conversion. The TGF-\u03b21 signaling could cross-talk with multiple intracellular signaling in tumor cell. Generally, TGF-\u03b21 signal is transduced from plasma membrane to the nucleus via serine/threonine kinase receptor type I (T\u03b2R-I) and type II (T\u03b2R-II) and their downstream effectors, smads. Moreover, TGF-\u03b21 signal is also transduced via Smad-independent pathways through activation of mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF-\u03baB) and phosphatidylinositol 3-kinase (PI3K)161718202123182627We enrolled a test cohort consisting of 184 HCC patients and a validation cohort of 118 HCC patients, who had undergone curative resection between 2008 and 2013. Using immunohistochemical staining, we examined expression of LASS2 and TGF-\u03b21 in the two independent cohorts, and analyzed their coexpression. The observed LASS2 staining pattern in HCC tissues was both plasma membranous and cytoplasmic. The level and extent of staining varied from weak focal to extensive strong expression. For TGF-\u03b21 staining, TGF-\u03b21 was primarily localized in the cytoplasm of tumor cells, which were producing TGF-\u03b21 . Based oAmong the 184 HCC patients of test cohort, 64 (34.8%) and 100 (54.3%) cases had high expression of LASS2 and TGF-\u03b21, respectively. We performed further analyses to determine the clinicopathological significance of LASS2 and TGF-\u03b21 in HCC. The expression level of LASS2 was negatively correlated with the tumor size, tumor differentiation, and TNM stage . In addiWe determine the prognostic value of LASS2 and TGF-\u03b21 in the test cohort of HCC patients. OS and TTR were evaluated using the Kaplan-Meier method. Our results showed that the median OS time was 46.5 (95% CI: 39.7\u201353.3) months for HCC patients with low expression of LASS2 and 58.4 (95% CI: 51.9\u201364.9) months for HCC patients with high expression of LASS2  and TTR , whereas high LASS2 high TGF-\u03b21 group had the longest OS  and TTR  . CollectTo evaluate whether the expression levels of LASS2 and TGF-\u03b21 were independent prognostic value for HCC patients in the test cohort, univariate and multivariate analyses using a Cox regression model were applied. As shown in We observed similar prognostic results of LASS2 and TGF-\u03b21 in another independent cohort consisting of 114 HCC patients after surgery. Among the 114 HCC patients of validation cohort, 40 (35.1%) and 53 (46.5%) cases had high expression of LASS2 and TGF-\u03b21, respectively. Kaplan\u2013Meier analysis showed that low LASS2 group had the shorter OS  and TTR , whereas high LASS2 group had the longrer OS  and TTR  .Furthermore, we found that patients with low TGF-\u03b21 expression also had shorter OS in vitro through interacting with vacuolar ATPase3213Hepatocellular carcinoma is one of the most aggressive cancers featured with high rate of metastasis and mortality. Although several molecular biomarkers have been reported to have clinical significance for predicting HCC prognosis2930Our previous study showed that knockout of LASS2 significantly upregulated the expression of TGF-\u03b21 in DEN-induced liver tumourigenesis of miceTGF-\u03b21 particularly achieves its tumor suppressive effect through the inhibition of proliferation and the induction of apoptosis or senescense in the early stage-tumors18In the present study, we provided the evidence that high level of LASS2/TGF-\u03b21 expression predicted a favorable OS and TTR rate for HCC patients. The 1-, 3- and 5-year OS rates of HCC patients with high level of LASS2 or TGF-\u03b21 expression were remarkably higher than those of HCC patients with low levels of LASS2 or TGF-\u03b21 expression, respectively. Accordingly, the 1-, 3- and 5-year TTR rates of HCC patients with high level of LASS2 or TGF-\u03b21 expression were significantly lower than those of HCC patients with low levels of LASS2 or TGF-\u03b21 expression, respectively. As combination of multiple markers might yield more information for predicting clinical outcome of HCC patients, combination of LASS2 and TGF-\u03b21 expression were therefore used as a predictor of clinical outcome. In both test cohort and validation cohort, HCC patients with both high expression of LASS2 and TGF-\u03b21 had the most favorable OS and TTR rates, whereas those patients with both low expression of LASS2 and TGF-\u03b21 had the poorest OS and TTR rates. Besides, we used univariate and multivariate analyses to further study the prognostic value of LASS2 and TGF-\u03b21, the results showed that combination of LASS2 and TGF-\u03b21 was significant and independent prognostic factor for HCC patients, but either LASS2 or TGF-\u03b21 was not.In conclusion, our study demonstrated that high levels of LASS2 expression and TGF-\u03b21 expression have a positive relationship with prognosis of HCC patients. The combination of LASS2 and TGF-\u03b21 was more sensitive than either of them alone with respect to OS and TTR, and could be used as a new independent prognostic marker of HCC patients.This study involved two independent cohorts of patients with HCC: test cohort (184 HCC patients) and validation cohort (118 HCC patients), obtained from Eastern Hepatobiliary Surgery Hospital  between January 2003 and April 2006. For suspected cases with elevated serum AFP level (>20\u2009ng/ml as positive), computed tomography (CT) and/or magnetic resonance imaging (MRI) were used to verify tumor recurrence. No patient received either radiotherapy or chemotherapy before the surgery, and no other cancers co-occurrence. The overall survival (OS) was defined as the length of time between the surgery and death or the last follow-up examination. The time to recurrence (TTR) was calculated from the date of tumor resection until the detection of tumor recurrence, death or the last observation. Tumor stage was defined according to the American Joint Committee on Cancer  TNM staging systemFor the use of clinical materials for research purposes, prior patients\u2019 consents and approval were obtained from the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine and EHBH of the Second Military Medical University. Written informed consent was obtained from all subjects for publication of this study. All experiments were performed in accordance with approved guidelines of Shanghai Jiao Tong University School of Medicine.HCC tumor tissues were resected from the patients, and then were fixed with 10% formalin and embedded in paraffin before prepared for tissue microarray. Briefly, 1-mm cores were taken from intratumoral tissue and serial sections (4-\u03bcm thick) were placed on slides coated with 3-aminopropyltriethoxysilane. The immunohistochemistry analysis was conducted as described previously2 analysis or two-tailed Student t test. Kaplan-Meier analysis was used to assess survival. Log-rank tests were used to compare survival of patients between subgroups. Multivariate analyses were performed by multivariate Cox proportional hazard regression model. Data were presented as mean \u00b1 SEM. Differences were considered to be statistically significant for p\u2009<\u20090.05.Differences among variables were assessed by \u03c7How to cite this article: Ruan, H. et al. Co-expression of LASS2 and TGF-\u03b21 predicts poor prognosis in hepatocellular carcinoma. Sci. Rep. 6, 32421; doi: 10.1038/srep32421 (2016)."}
+{"text": "Avian influenza continues to circulate and remains a global health threat not least because of the associated high mortality. In this study antibody persistence, booster vaccine response and cross-clade immune response between two influenza A(H5N1) vaccines were compared. Participants aged over 18-years who had previously been immunized with a clade 1, A/Vietnam vaccine were re-immunized at 6-months with 7.5 \u03bcg of the homologous strain or at 22-months with a clade 2, alum-adjuvanted, A/Indonesia vaccine. Blood sampled at 6, 15 and 22-months after the primary course was used to assess antibody persistence. Antibody concentrations 6-months after primary immunisation with either A/Vietnam vaccine 30 \u03bcg alum-adjuvanted vaccine or 7.5 \u03bcg dose vaccine were lower than 21-days after the primary course and waned further with time. Re-immunization with the clade 2, 30 \u03bcg alum-adjuvanted vaccine confirmed cross-clade reactogenicity. Antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available.Trial Registration: ClinicalTrials.gov NCT00415129 The first influenza pandemic of the 21st century in 2009 was caused by a novel influenza A(H1N1) strain that was first recognized in Mexico  as adjuvant [The booster study, presented here, was conducted in 2 phases. A cohort of participants at one study site (n = 154) received a booster dose of the 7.5 \u03bcg (H5N1) clade 1 vaccine, 6 months after having received the primary immunization course with the 7.5 \u03bcg (H5N1) clade 1 vaccine, either without adjuvant or with 30 \u03bcg alum. At a later stage and in 2 of the 3 remaining sites, subjects who had been primed with two doses of the 30 \u03bcg alum-adjuvanted vaccine, were invited to participate at the booster study which implied that they received one booster immunization with a high dose (30 \u03bcg) (H5N1) clade 2 alum-adjuvanted A(H5N1) vaccine at month 22 after the primary immunization . FurtherIn order to assess antibody persistence all participants were invited to undergo a blood test at 6 months after starting the study. Thereafter, all participants who did not receive a booster vaccine at 6 months were invited to have a blood test to assess antibody persistence at 15 and 22 months.In one study site, parallel to the 22-month clade 2 booster immunization an additional 50 A(H5N1) vaccine-na\u00efve adults, aged between 18 and 60 years, were recruited to receive a primary course of the same clade 2 vaccine that was used as booster. These participants received 2 doses of the vaccine given three weeks apart.Adults who had previously participated in the primary study were invited to take part in the follow-up and booster phase . The folThe study was approved by the institutional ethics review boards based in each participating Belgium institution  and the National Health Service Oxford ethics committee for the Oxford site., The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all participants before enrolment in this part of the study. The trial is registered with Clinicaltrials.gov: NCT00415129.Up to 30 ml of blood was sampled to assess antibody persistence at 6, 15 and 22-months in the different groups of participants as described above. For those who received booster immunizations at 6 months, blood was sampled prior to and 21 days after immunization. For those who received the 22-month booster immunization, blood was sampled just prior to booster immunization and at 7 and 21 days after immunization. The 7-day blood was taken to look for evidence of early response suggestive of immunological memory. Finally those immunized with a two-dose schedule of the clade 2 vaccine as a primary course had blood sampled prior to each immunization and 21 days after the second dose.3), propagated in embryonated eggs. The dose of Al(0H)3, used as the adjuvant, was 600 \u03bcg/dose (expressed as the content of Al3+). Both formulations were presented in ready-to-use multi-dose vials from which 0.5 ml were withdrawn and injected intramuscularly into the deltoid muscle using a standard syringe and 25 G, 25 mm needle.The vaccines were inactivated, split-virion preparations of vaccine strains; clade 1: A/Vietnam/1194/2004/NIBRG-14 (H5N1) (7.5 \u03bcg HA without adjuvant) or clade 2: A/Indonesia/5/05-RG2 (H5N1) (30 \u03bcg with Al(OH)After each immunization participants were monitored for 30 minutes for immediate reactions. Participants kept diaries in which they recorded any solicited (prelisted) local or systemic reactions that occurred up to 7 days after immunization. Specifically participants were asked to record injection site induration, injection site ecchymosis, temperature, malaise and shivering for 3 days after vaccination. Participants were also asked to record any other adverse event that occurred up to 21 days after immunization. Serious adverse events were collected throughout the study. Participants were followed up between 7 and 28 months (12 months for the group receiving the clade 1 A(H5N1)/Vietnam booster; 22 months for participants who took part in the primary phase of the study only; 28 months for participants who received the clade 2 A/Indonesia booster at 22 months and 7 months for 50 additional participants primed with the clade 2 A(H5N1N)/Indonesia.Serum samples were used for antibody titration against one of the vaccine strains (clade 1 or clade 2) using haemagglutination inhibition (HI) and seroneutralization (SN) assays.The HI assay reflects the ability of the specific anti-influenza virus antibodies to inhibit haemagglutination of erythrocytes by influenza virus as previously described . BrieflyThe SN assay is based on the ability of antibodies to inhibit the infection of Madin-Darby canine kidney (MDCK) cell culture, as previously described . BrieflyIn all assays samples were tested in duplicate and the titer analyzed was the geometric mean of the duplicates, expressed as the reciprocal of dilution. Samples without detectable antibody activity were assigned the titer of half the assay detection limit. These analyses were performed under blinded conditions at the Public Health England laboratories .www.clinicalstudydatarequest.com. If further assistance is required then the corresponding author is to be contacted.Sample size for the additional participants receiving a primary vaccination series of the clade 2 vaccine A(H5N1)/Indonesia, was arbitrarily set at 50 subjects. The sample size required to determine the booster response to clade 2 A(H5N1)/Indonesia was deemed to be lower than the initial cohort size, as only half the original study population were eligible to continue in the study, i.e. those who received the higher dose vaccine during the primary phase. Accordingly, participants at centre 4 were not invited to take part in the booster phase of the study. The minimal data sets underlying the findings are available to interested researchers who in the first instance may request access to anonymized patient-level data and clinical study documents at The study was descriptive and no formal statistical comparisons between groups have been made. The results are presented with 95% confidence intervals of point estimates calculated using normal approximation for quantitative data and exact binomial distribution (Clopper-Pearson method) for proportions. The population analyzed for all the timepoints presented here are the full analysis set. A per-protocol analysis was pre-designated for the A(H5N1) vaccine na\u00efve group receiving the primary course with clade 2 vaccine, however there were no protocol deviations in this group therefore the per protocol and full analysis set were equivalent. For the groups that received either booster vaccination the full analysis set is defined as all those who received the booster immunization. The analysis included all data available at each timepoint.HI data are expressed as GMT\u2019s in accordance with criteria based on those defined for the HI method by the committee for Medicinal Products for Human Use (CHMP) for seasonal influenza vaccines which are applied to pandemic vaccines in absence of specific alternative criteria. These criteria are: a seroprotection rate (% with HI titers \u2265 40 dil-1) of over 70%; a rate of seroconversion or significant titer increase  of over 40% and an increase in geometric mean titer ratio (GMTR) between post and pre vaccination titer by over 2.5. In adults over the age of 60 these criteria are modified to a seroprotection rate of over 60%, seroconversion rate or significant increase in titer of over 30% and a GMTR of over 2 . It is rAnalyses were performed by the Sanofi Pasteur Biostatistics Department, Marcy I\u2019Etoile, France, using SAS software version 8.2 ) and were independently validated by the Oxford Vaccine Group, University of Oxford, using SAS version 9.3 (M.V.).At 6-months after the start of the primary phase of the study 587 out of the original 600 participants remained eligible to continue in the study . For theIn total 33 serious adverse events (SAEs) have been reported in this phase of the study, that is up to 42 days after the start of the study. SAEs that occurred in the primary phase are reported in the primary phase publication . There wUp to 21 days after the booster dose of the 7.5 \u03bcg clade 1 A(H5N1)/Vietnam vaccine the percentage of adults aged between 18 and 60 years reporting at least one solicited reaction was 50% in those primed with the high dose adjuvanted vaccine and 35.9% in those primed with the low dose non-adjuvanted vaccine. In adults aged over 60 years at least one solicited event was reported by 19.5% and 27.8% in the high dose and low dose priming groups respectively.Up to 21 days after booster immunization with the high-dose alum-adjuvanted clade 2 A/Indonesia vaccine 74.6% of adults aged between 18 and 60 reported at least one solicited injection site reaction and 42.4% at least one solicited systemic reaction whilst 36.8% and 33.3% of those aged over 60 reported at least one solicited injection site and systemic reaction, respectively.Most common reactions in both groups were pain at the injection site, headache and malaise which started within 3 days of vaccination. Unsolicited reactions that started after 21 days were considered not related to the vaccine. A break down of the type of reactions in the first 7 days after immunization is given in In the na\u00efve group (18\u201360 years old) who received a primary course of the clade 2 A(H5N1)/Indonesia vaccine no SAEs were reported. Overall 72% of participants experienced a solicited reaction within 21 days of immunization. Injection site pain and headache were the most commonly reported reactions in the first 7 days after immunization .\u226532) was 6.2% in the 18 to 60 years group and 14.4% in the over 60 years group (At 6 months after primary immunization with the 7.5 \u03bcg clade 1 A(H5N1)/Vietnam vaccine the percentage of individuals who had antibody levels above the threshold for seroprotection (defined as HI antibody titer rs group .In the group who had received the 30 \u03bcg adjuvanted clade 1 A(H5N1)/Vietnam vaccine, 6.2% of participants aged 18 to 60 and 18.9% of participants aged over 60 had HI titres > 32. Again these levels decreased with time .Booster response to 7.5 \u03bcg clade 1 A/Vietnam vaccine at 6 months .The response to this booster vaccine at 6 months after the primary immunisation as measured by HI antibody did not meet any of the CHMP criteria in either of the two age groups.Booster response to 30\u03bcg with adjuvant clade 2 A/Indonesia vaccine at 22 months .The booster response was tested against both clade 1 and 2 viruses. At 7 days after the booster immunization the GMTR was above the threshold required by the CHMP (fulfilling 1 out 3 requirements) in the 18 to 60 year group against both clade 1 and clade 2 strains but not in the over 60 age group.By 21 days after immunization in the 18 to 60 year age group 2 out of 3 CHMP criteria (seroconversion rate and GMTR) were fulfilled when tested against the clade 2 virus and a single criterion (GMTR) was met when tested against the clade 1 virus. In the over 60 year group the only CHMP criterion met was the GMTR for both of the viral strains tested.Primary Response to clade 2 vaccine .In the na\u00efve group who received the clade 2 vaccine only, prior to immunization there was no evidence of prior immunity to the clade 2 A/Indonesia virus. Twenty-one days after a single dose of vaccine one CHMP criterion (GMTR over 2.5) was met. This did not change 21 days after the second immunization. The demographics of the booster and na\u00efve group are given in the supplementary material.This study investigated the potential usefulness of a prime-boost vaccination strategy using two different influenza A(H5N1) strains to induce cross clade protection. The data show that a heterologous booster vaccine strain can induce cross-clade reactivity. Despite the fact that the primary vaccination schedule had induced antibody responses that met CHMP criteria in some groups, 6 months after the booster dose antibody levels had waned below the CHMP thresholds established for primary vaccination. It is important to stress here that no CHMP criteria exist for the duration of antibody persistence and the extent of cross-clade reactivity of influenza vaccines. It may not be indicated to appreciate the value of this prime-boost strategy using a possibly non-appropriate scoring system .Few reports are available on antibody persistence with influenza A(H5N1) vaccines exceding a 6 month period whereas a pandemic threat can persist for several years. Lin et al., (2009) reported that 6-months after immunization with low dose, alum-adjuvanted, whole virion A(H5N1) vaccine antibodies were only present in 4.8% to 20.8% of adults aged under 60 years and thus failed to meet any CHMP at this time point . In contIn the present study participants were followed for 22 months following the first immunization, which is the longest follow-up period reported. Six months after primary vaccination the magnitude of HI titres differed more by age group than by primary vaccination group . In the younger age group 6% of participants had HI titres detected at 6 months, irrespective of the type of vaccine administered. In the older age group 14.4% and 18.9% had detectable HI titres with the low- and high-dose vaccine, respectively. This trend continued for 22 months after immunization and may reflect the effect of prior priming via natural exposure or seasonal influenza vaccine in the older group. In fact even at baseline, prior to A(H5N1) immunization, 16% of the older age group had elevated HI titres; similar results have been found by other investigators .Vaccine-induced cross-clade immune response is defined as the ability to induce immune responses against pathogen strains that are genetically distinct from the strain(s) used to produce the vaccine . PracticThe main study limitation stemmed from the lower than expected antibody responses to the low dose vaccine described in the primary phase of the study  this meaIn conclusion antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available. Further criteria to assess this strategy are needed.S1 Fig(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file."}
+{"text": "To secure elimination, further monitoring is warranted to detect any use of tOPV or monovalent OPV type 2 (mOPV2).The Global Polio Eradication Initiative (GPEI) has made substantial progress since its launch in 1988; only 37 wild poliovirus type 1 (WPV1) cases were detected in 2016, the lowest annual count ever. Wild poliovirus type 3 has not been detected since November 2012, and wild poliovirus type 2 was officially declared eradicated in September 2015. This success is attributable to the wide use of live oral poliovirus vaccines (OPVs). Since 2001, numerous outbreaks were caused by the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation  comprises 146 World Health Organization (WHO)\u2013accredited poliovirus laboratories in 92 countries located in the six WHO regions , and enterovirus surveillance . These surveillance systems ensure sensitive and timely detection of circulating polioviruses worldwide. Whereas AFP surveillance has been the standard surveillance system for poliovirus since the beginning of the GPEI, recently, existing environmental surveillance for poliovirus has been expanded  were achieved in all countries. From January to April 2016 , 46 countries were reporting PV2 detected by GPLN laboratories from specimens from persons with AFP or their contacts and sewage samples . From MaField investigations in response to detection of PV2 after the switch found breaches in OPV2 withdrawal with evidence of continued inadvertent use of tOPV in India (During the pre-switch period (January\u2013April 2016), PV2 was detected through both AFP and environmental surveillance; after the switch, PV2 was detected primarily through environmental surveillance  Figure . In counTo provide evidence concerning the origin and significance of circulating PV2, on August 1, 2016, GPLN laboratories began to refer all PV2s detected from all sources for genetic sequencing. Isolation of Sabin-like poliovirus with zero or few nucleotide differences from Sabin2 by GPLN laboratories were instrumental in 1) identifying continued use of tOPV in some countries post-switch and in 2) confirmation of three post-switch cVDPV2 outbreaks caused by genetically related cVDPVs that began circulating before the switch.Virologic monitoring through AFP cases and sewage samples indicate that withdrawal of a live vaccine, OPV2, used in routine immunization programs and mass immunization campaigns, was successfully accomplished by the GPEI. Some evidence of limited use of tOPV after the global tOPV to bOPV switch was found; however, 1 year after OPV2 withdrawal, PV2 has been isolated only in the few areas where mOPV2 has been used in response to detection of cVDPV2 isolates. By expanding the preexisting surveillance network to include environmental surveillance for polioviruses during the last 5 years, GPLN successfully detected VDPV2 emergences and outbreaks to allow GPEI to respond in a timely manner. AFP and environmental surveillance with laboratory testing for poliovirus by GPLN will continue to play a long-term, critical role in ensuring polio eradication  to respond to this outbreak. Nigeria and Pakistan also have circulation of WPV1, and WPV1 circulation continues in Afghanistan.Reintroduction of live PV2-containing vaccine through the use of 19 mOPV2 immunization campaigns to interrupt VDPV2 transmission in six countries , from May 2016 to Mar 2017 has disrupted the goal of interrupting PV2 transmission globally after the switch. The GPEI has established a mOPV2 advisory group, which advises WHO about each use of mOPV2, after an in-depth review of risk assessments conducted after any VDPV2 event or outbreak detection. In countries where no type 2-containing vaccine has been used after the switch, only three countries  have reported VDPV2 detection since September 2016.Environmental surveillance for polioviruses detected the majority of PV2 from September 2016 to March 2017. Detection and sequencing of polioviruses isolated from sewage samples is difficult because these isolates often represent complex mixtures of viruses. Despite these challenges, further expansion of environmental surveillance is needed to maintain the high level of vigilance required to detect and respond to any type 2 poliovirus from all sources in the future, including breaches in containment in facilities retaining or still working with PV2 materials, including WPV2.PV2s were tracked in both human specimens and sewage samples using a newly designed molecular diagnostic assay and algorithm developed by CDC , which was rapidly and efficiently implemented in GPLN laboratories in 2016. PV2 detection and genetic sequencing has been essential for the following: 1) providing evidence of continued use of tOPV after the withdrawal of this vaccine in April 2016; 2) identifying and following up unusual patterns of PV2 detection or circulation that signal gaps in herd immunity against PV2; and 3) classifying VDPV2s as either circulating viruses (cVDPV2s) or originating from immunodeficient persons (iVDPV2s), or of ambiguous origin (aVDPV2s)  has made substantial progress since 1988; in 2016, only 37 wild poliovirus (WPV) type 1 (WPV1) cases were detected, the lowest number ever recorded. WPV type 2 has been eradicated, and WPV type 3 has not been detected since 2012. To reduce the risk for paralysis from infection with vaccine-derived polioviruses (VDPVs), in April 2016, all 155 oral poliovirus vaccine (OPV)\u2013using countries switched from trivalent OPV (tOPV) to bivalent OPV (bOPV), containing vaccine virus types 1 and 3.After the withdrawal and destruction of tOPV, the GPEI devised mechanisms to monitor disappearance of type 2 polioviruses (PV2s) in human populations and the environment. Enhanced environmental surveillance and provision of clear guidance to the Global Polio Laboratory Network has allowed timely, accurate, and comprehensive detection of PV2 by examining approximately 208,000 stool specimens and sewage samples. Preceding the tOPV to bOPV switch (January\u2013April 2016), 43 countries reported detection of PV2; during January\u2013March 2017, the number of countries reporting PV2 had declined to seven.To prevent paralysis caused by VDPVs, elimination of vaccine viruses from the environment will be critical. Lessons learned from surveillance for PV2 after the global synchronized withdrawal of the PV2 component from vaccines have resulted in development of standardized procedures for investigation of PV2 detection in humans and the environment, and handling PV2 in diagnostic laboratories. These lessons will guide the elimination of OPV1 and OPV3 once eradication of polio has been certified."}
+{"text": "In patients with ST elevation myocardial infarction (STEMI), myocardial tissue injury is not restricted to the territory supplied by the culprit artery but it affects also the remote myocardium supplied by unaffected arteries.(1) The aim was to investigate the T1 and T2 characteristics in infarcted and remote myocardium comparing it with normal reference standard.30 patients  with STEMI and successful revascularisation by percutaneous coronary intervention were included. Each subject underwent clinical CMR at 1.5 T with T2 and T1 mapping (MOLLI) pre and post contrast (equilibrium contrast technique for extracellular volume (ECV) quantification) within 48hours of presentation. The T2 and pre and post contrast T1 values were evaluated in each of the 16 AHA myocardial segments.Out of 480 myocardial segments, 143 were affected and the rest unaffected or remote. The mean native T1 and the mean ECV in the remote myocardium was higher than the reference standard (1054 \u00b1 65 msec vs 950 \u00b1 21 msec and 0.32 \u00b1 0.06 vs 0.25 \u00b1 0.04). (2) However, the mean native T1 and the mean ECV of the remote myocardium was significantly lower compared to infarcted myocardium . In addition the mean T2 of the remote myocardium was significantly lower compared to infarcted myocardium .This is the first study looking at the impact of STEMI on remote myocardium via non-invasive tissue characterisation by advanced CMR relaxometry technique. Our study highlights that the remote myocardium is also affected following STEMI when compared to normal. Albeit the myocardial tissue injury in the infarcted territory is significantly greater than remote myocardium. These findings may have significant future implications in the treatment of STEMI, including targeting the remote myocardium."}
+{"text": "LPP locus. The lesions occurred in the foot by presenting one lump in the plantar soft tissue, and three lesions were detected in the calcaneus and in the navicular bone. All tumors showed the double immunophenotype of epithelial markers and S100 protein expression. No rearrangement of the EWSR1 and FUS loci was detected as reported in myoepitheliomas. However, molecular karyotyping detected an unbalanced rearrangement of the LPP locus, not involving the HMGA2 locus, which is the most frequent translocation partner observed in benign mesenchymal tumors such as lipomas  and pulmonary chondroid hamartoma.We report a case of multiple myoepithelioma with synchronous bone and soft tissue tumors, associated with a new genomic alteration of the  Myoepithelial tumors (METs) of soft tissue and bone are rare tumors of uncertain histogenesis. The first deep tumor was described in the retroperitoneum , followeHistologically, METs are made of a homogeneous population of myoepithelial cells. They could be considered as a part of a continuum with mixed tumors when ductal differentiation is present. They may harbor chondroid and bone differentiation as observed in classical mixed tumors. The diagnosis of MET requires the coexpression of both epithelial markers and S100 protein , 9. TheyEWSR1(22q12) gene fusions have been detected in half of METs arising in deep soft tissues and in up to 70% cases of intraosseous MET [EWSR1 are described: POU5F1(6p21.33) (16%), PBX1 (16%), PBX3, ZNF444, ATF1, KLF17, and NFATC2 [EWSR1-POU5F1 more often occurs in children and young adults while the EWSR1-PBX1 occurs in middle-aged adult patients [EWSR1-PBX1 rearrangement presents a bland sclerotic appearance or clear cell morphology with a diffuse EMA staining. However, none of the EWSR1-rearranged tumors show the presence of ductal or glandular differentiation or cartilage/bone matrix formation [FUS(16p11) gene has also been reported in rare cases of MET arising in deep soft tissue as well as in bone [KLF17 and POU5F1. These FUS-rearranged tumors also lack ductal differentiation [A different genetic pattern distinguishes METs arising in the skin from those in deep soft tissue and bone. eous MET . Severald NFATC2 \u201316. The patients , 14. Theormation . Rearran in bone , 17. Twontiation .PLAG1 (8q12.1) and HMGA2 (12q14.3) rearrangements are the most common genetic events in pleomorphic adenomas [PLAG1 is also found in MET [PLAG1 gene rearrangement [adenomas . PLAG1 id in MET , 20. In angement . All tumLPP unbalanced rearrangement without EWSR1 and FUS alterations in both soft tissue and bone lesions.We report an unusual observation in a 52-year-old man of a multifocal MET without obvious ductular differentiation and harboring a new A 52-year-old patient complained of pain and swelling of the foot. MRI and plain radiography demonstrated a main lesion in the calcaneus, two others in the navicular bone, and a last one in the plantar soft tissue . The maiThe tumor of the calcaneus measured 4.4\u2009\u00d7\u20093.4\u2009cm , and theImmunohistochemistry demonstrated an intense and diffuse staining of plasmacytoid cells for the broad-spectrum cytokeratin, S100 protein Figures . There wThese results were in favor the diagnosis of a MET. Based on the multiplicity of localizations, a transtibial amputation was decided by the local multidisciplinary committee. Although lesions were multiple and some pictures were suspicious for vascular emboli, the patient had no recurrence or distant metastasis 2 years later. The last follow-up detected small lung lesions, which remained stable and were considered as aspecific.Conventional karyotyping detected the same abnormal complex pseudodiploid clone in the soft tissue tumor as in the bone tumor .EWSR1 and FUS. FISH detected no rearrangement of those loci. Other differential diagnoses were excluded by FISH: extraskeletal myxoid chondrosarcoma (NR4A3) and alveolar soft part sarcoma (TFE3).FISH experiments were performed to look for rearrangement of genes known to be altered in MET arising in deep soft and bone tissues, LPP gene, probably the 8 first exons (Molecular karyotyping detected 2 main deletions on the long arm of chromosome 3 located at 3q22.1-3q26.2 (37Mb)  and 3q27st exons . Three oLPP locus  [KMT2A(11q23) in a secondary acute leukemia [BCL6(3q27) through a 3q27 interstitial deletion in a primary central nervous system lymphomas [Three other A1(6p21) , and in leukemia  and BCL6ymphomas .LPP remains unclear, while the role of the partner gene seems to be crucial. Several lines of evidence suggest that HMGA2 truncation occurring in the most common t fusion gene is implicated in lipomagenesis. However, the HMGA2-LPP fusion protein retains the transactivation functions of two LPP LIM domains which might contribute to the mesenchymal tumorigenesis by directly affecting transcriptional regulation processes [The oncogenic role of rocesses .LPP locus suggests that carboxy-terminal LIM transactivating domains may contribute to the chimeric protein with the aminoterminal part of an unidentified gene. Although not rearranged by FISH, we cannot exclude a cryptic insertion of the 5' part of HMGA2 within the LPP locus. The only candidate locus for a gene fusion with LPP may be TRPS1 (8q23.3) whose 3' part is deleted. TRPS1 is known to be associated with tumorigenesis, metastasis, and angiogenesis in several tumors, including osteosarcoma [LPP-partner gene by 3' RACE-PCR or RNASeq.In the present case, the suspected breakpoint within the osarcoma . UnfortuPLAG1 and HMGA2 have been described in pleomorphic adenomas (mixed tumors) of salivary gland [PLAG1 rearrangements were mainly identified in a subset of cutaneous and superficial soft tissue MET tumors, often displaying ductal structures and considered as mixed tumors [PLAG1 has not been tested because of the absence of ductular differentiation in the tumor.No ductal differentiation was present although membranous EMA staining was focally detected. The stroma was focally myxoid but without a mesenchymal cell population as observed in mixed tumors. Recurrent genetic abnormalities involving ry gland . PLAG1 rd tumors . PLAG1 hLPP (3q27-3q28) locus in synchronous tumors presenting epithelioid features. The partner gene remains to be characterized. Analysis of the LPP locus should be performed by FISH on MET without EWSR1 or FUS rearrangements and pleomorphic adenomas of salivary gland without PLAG1 and HMGA2 aberrations to define the recurrence and the tumor characteristics associated with this new alteration.We describe here a new rearrangement of the"}
+{"text": "HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, \u03941-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.We characterized an aquaporin gene  These proteins facilitate the transport of water and small uncharged molecules across biological membranes  was reduced by antisense technology, the NtAQP1-downregulated tobacco plants showed reduced root hydraulic conductivity and wilting phenotypes under water stress  have also been reported, implying the complexity of PIP function in plants  is one of the most agronomically cultivated crops; it is more adaptable to drought, salinity and cold than other cereal crops  in yeast and Arabidopsis and characterized these lines to understand the functions of the barley PIP gene under high salt and high osmotic stress conditions.In this study, we overexpressed barley Hordeum vulgare cv. NP21) cDNA was prepared using superscriptTM III reverse transcriptase , and total RNA was extracted with TRIzol\u00ae Reagent . A 873 bp-length HvPIP2;5 coding sequence (GenBank Accession number: AB377270.1) was cloned into TA cloning vector pTOPO2.1  using gene specific primers  at the EcoRI site and named pYES2: HvPIP2;5. For plant transformation, HvPIP2;5 coding sequence was cloned into a standard plant binary vector pCAMBIA2301. The resulting overexpression construct was named pCAMBIA2301-35S:HvPIP2;5.Barley  and FY3 strain only were used in stress assays. The yeast strain FY3 was transformed with a pYES2 empty vector or pYES2:HvPIP2;5 recombinant vector by lithium acetate method .The For stress analysis, transformed yeast cells were propagated in SC-U medium containing 2% galactose for 12 h and cell density was adjusted to 1.0 of OD600 followed by serial dilutions. Yeast cell were spotted on YPD medium supplemented with PEG (4%) or NaCl (200 mM), respectively. Plates were maintained at 30\u00b0C and growth was monitored after 2 days.Agrobacterium tumefaciens GV3101 harboring pCAMBIA2301-35S:HvPIP2;5, Arabidopsis Columbia-0 plants were transformed via floral dipping method  media and allowed to germinate at 22\u00b0C and 60% relative humidity with a 16/8 h light-dark photo cycle after 3 day stratification at 4\u00b0C.For germination analysis, the seeds of WT and homozygote Final concentration of 100\u2013200 mM NaCl  or 10\u201320% PEG (for high osmotic stress) was supplemented to the MS media for stress administration. Hereby salt stress or osmotic stress indicates high salt stress or high osmotic stress, respectively. Emergence of cotyledons was used as a germination criterium. The number of germinated seeds was expressed as a percentage of total number of seeds planted after 7 days. For root elongation analysis, vertically grown seedlings with 1\u20131.5 cm long root were transferred onto a MS vertical plate supplemented with or without stress agents (200 mM NaCl and 20% PEG). Root length was measured 15 days after transfer. Root length of seedlings under stress conditions was expressed as a percentage of their respective controls grown on normal MS medium. All stress analysis experiments were conducted three times and each contained 3 biological repeats. For drought test with soil-grown plants, 4 week old seedlings were subjected to drought stress by withholding water supply for 21 days and then re-watered. For salt and osmotic stress, 3 week old plants were irrigated with either half strength MS liquid (control) or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. The chlorophyll, proline, and malondialdehyde (MDA) content in control or stress treated plants were determined by the methods reported previously by Lichtenthaler , Bates eHvPIP2;5 OE lines were excised and treated either with 200 mM NaCl or 20% PEG or deionized water after measuring the fresh weight. After 24 h of incubation, the tissues were weighed again for their turgid weight and then dried completely to obtain dry weight. The root RWC was calculated by the following formula; RWC (%) = [(fresh weight \u2212 dry weight)/(turgid weight \u2212 dry weight)] \u00d7 100  staining and hydrogen peroxide by 3, 3'diaminobenzidine (DAB) staining was performed according to methods previously described by Rao and Davis  or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. Contents of superoxide and hydrogen peroxide were estimated by methods previously described by Elstner and Heupel  and SagiPlants were treated with water for control or 300 mM NaCl for 6 h or 20% PEG for 6 h, and total RNAs were extracted using RNeasy Plant mini Kit . cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit , and resulting cDNAs were used for semi-quantitative RT or qRT-PCR experiments for selected genes. Primer pairs used in experiments are shown in Supplementary Table P \u2264 0.05).All statistical comparisons between variances were determined by ANOVA  and least significant differences (LSD) between variants were calculated using Statistix 8.1 computation software. Statistically significant mean values were denoted as * (HvPIP) genes and examined their expressions under salt and osmotic stress conditions , one of the HvPIP2 genes that possesses abundant transcripts and demonstrates down-regulation of gene expression under salt and osmotic stresses . HvPIP2;5 protein shares 81% identity with Arabidopsis PIP2;5 protein . As shown in Supplementary Figure PIP2;5 orthologs . As expected, HvPIP2 proteins showed high degree of homology among them, and HvPIP2;5 was located in the same clade as HvPIP2;1 with 85% identity to HvPIP2;1 , while yeast cells with HvPIP2;5 displayed growth until 10\u22126 dilution under the same condition  lines were selected. PCR analysis confirmed the presence of the HvPIP2;5 gene in transgenic plants (data not shown) and semi quantitative RT-PCR for HvPIP2;5 expression analysis demonstrated that transgenic lines were overexpressing HvPIP2;5 gene  and 20% PEG (high osmotic stress) was first tested for two independent  Figures . Also, H Figures .HvPIP2;5 OE lines under salinity and osmotic stresses compared to WT. The relative root length of HvPIP2;5 OE lines was significantly higher in the presence of 200 mM NaCl (59\u201362% vs. 34%) or 20% PEG (47\u201350% vs. 28%) compared to that of WT  was measured from the root tissues of WT and C Figure . LikewisC Figure .HvPIP2;5 OE lines under high salt (200 mM NaCl) and high osmotic stress (20% PEG). Compared to WT, the HvPIP2;5 OE lines showed significantly weaker NBT staining and less 2O2 content revealed lower amounts of H2O2 in the OE lines than in WT after NaCl treatment  are generated in plants under drought, salt, and temperature stresses. Using nitro blue tetrazolium (NBT) staining for superoxide and diaminobenzidine (DAB) staining for hydrogen peroxide, we measured the levels of ROS in WT and  Figures . Similar Figures . HoweverHvPIP2;5 OE lines than in WT, which implicates lesser membrane damage in HvPIP2;5 OE lines  is an end product of lipid peroxidation of cell membrane lipids and a good indicator of oxidative damage  and superoxide dismutase (SOD) in WT and s Figure , the levs Figure . InteresHvPIP2;5 OE lines under 100 and 200 mM NaCl conditions. GR and APX are major enzymes for the ascorbate-glutathione cycle which is an important component of the ROS scavenging system in plants  and ascorbate peroxidase (APX) in s Figure . The acts Figure .HvPIP2;5 OE lines under salt and osmotic stresses is possibly related to enhanced activities of ROS scavenging enzymes.Taken together, these results indicated that reduction of oxidative stress in 1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) in WT and HvPIP2;5 OE lines. Real time PCR analysis revealed that gene expression levels of P5CS1 and P5CS2 were higher in HvPIP2;5 OE lines than in WT under salt and osmotic stresses  in both yeast and Arabidopsis improved tolerance to high salt and high osmotic stresses   overexpression brought about reduced osmotic stress tolerance in Arabidopsis and tobacco (Jang et al., AtPIP1;2, rice RWC3, and tobacco NtAQP1 share approximately 80% sequence identity. Despite this, overexpression of AtPIP1;2 in tobacco has caused reduced stress tolerance (Aharon et al., RWC3 overexpression in rice and NtAQP1 overexpression in tomato have shown enhanced tolerance under drought and salt stress, respectively (Lian et al., pip2;2 mutants display defects in hydraulic conductivity despite the expression of a very close homolog AtPIP2;3 which shares >96% homology, demonstrating that close aquaporin homologs could not function redundantly even within the same plant (Javot et al., While HvPIP2;5 and AtPIP2;5 share high sequence homology, there are differences in gene regulation. AtPIP2;5 expression levels remain low in Arabidopsis and are only up-regulated by drought and cold (Jang et al., HvPIP2;5 is one of the highly expressed PIPs in barley which is down-regulated by osmotic stress (Katsuhara et al., PIP2;5 in Arabidopsis and barley, and may be attributed to contrasting stress responses in HvPIP2;5 and AtPIP2;5 overexpressors.Although, Another explanation for the contrasting results might lie in the difference in protein sequence between HvPIP2;5 and AtPIP2;5 proteins. Amino acid sequence differences are mainly found in the N-terminus which is expected to be exposed on the cytosol side (Walz et al., HvPIP2;5 overexpressing Arabidopsis, up-regulation of P5CS2, high levels of proline, and increased activities of SOD, CAT, GR, and APX with reduced levels of ROS under drought and salt stress conditions. Particularly, up-regulation of the key proline biosynthetic P5CS genes coincided with increased levels of osmoprotectant proline under salt stress (Figure HvPIP2;5 overexpressing plants seemed to result, at least in part, from increased expression of proline biosynthetic genes and elevated activities of ROS scavenging enzymes. Similar to our findings, the overexpression of wheat TaAQP7, one of the closest homologs of HvPIP2;5, in tobacco enhanced drought tolerance in correlation with decreased levels of MDA and H2O2 and increased activities of SOD and CAT enzymes (Zhou et al., HvPIP2;5 overexpression might sensitize transgenic plants, making the overexpressors react faster to osmotic stress signals and eventually induce enhanced stress defense. Thus, one might speculate that HvPIP2;5 aquaporins in transgenic plants might activate osmotic and salt stress sensing or upstream steps in signaling pathways to induce better stress-tolerance mechanism than WT under stress conditions. Consistent with our speculation, it has been suggested that aquaporins may be part of an osmotic stress signaling cascade (Maurel et al., We found, in s Figure . Prolines Figure . Thus, eHvPIP2;5 can improve tolerance to salt and osmotic stresses when overexpressed in yeast and Arabidopsis. Our results contrast with a previous AtPIP2;5 overexpression study where osmotic stress sensitive phenotypes in AtPIP2;5 overexpressing plants were reported (Jang et al., In conclusion, we have shown that HA, BhL, and SKP designed the experiments; HA, JPA performed the experiments; GR, LS, BhL, and SKP advised the research; HA, BhL, and SKP discussed the results and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "This study investigated the potential of a newly proposed scattering foil free (SFF) electron beam scanning technique for the treatment of skin cancer on the irregular patient surfaces using Monte Carlo (MC) simulation. After benchmarking of the MC simulations, we removed the scattering foil to generate SFF electron beams. Cylindrical and spherical phantoms with 1 cm boluses were generated and the target volume was defined from the surface to 5 mm depth. The SFF scanning technique with 6 MeV electrons was simulated using those phantoms. For comparison, volumetric modulated arc therapy (VMAT) plans were also generated with two full arcs and 6 MV photon beams. When the scanning resolution resulted in a larger separation between beams than the field size, the plan qualities were worsened. In the cylindrical phantom with a radius of 10 cm, the conformity indices, homogeneity indices and body mean doses of the SFF plans (scanning resolution = 1\u00b0) vs. VMAT plans were 1.04 vs. 1.54, 1.10 vs. 1.12 and 5 Gy vs. 14 Gy, respectively. Those of the spherical phantom were 1.04 vs. 1.83, 1.08 vs. 1.09 and 7 Gy vs. 26 Gy, respectively. The proposed SFF plans showed superior dose distributions compared to the VMAT plans. Advanced radiotherapy techniques such as intensity modulated radiation therapy (IMRT) as well as volumetric modulated arc therapy (VMAT) enable conformal delivery of a prescription dose to deeply seated target volumes while sparing dose to normal tissues \u20134. Howevet al. suggested using scattering foil free (SFF) electron beams for MERT ., 13.et a\u00d7 10 cm2 . Further2 generally resulted in a slightly better plan quality than did the beams with a field size of 1 \u00d7 1 cm2. For both cylindrical and spherical phantoms, target conformity as well as body mean dose with the field size of 4 \u00d7 4 cm2 were better than those with the field size of 1 \u00d7 1 cm2 when the scanning angle was equal to or less than 10\u00b0 for all phantom sizes. Because fine resolution scanning takes a long beam delivery time, the scanning angle of 10\u00b0 with a field size of 4 \u00d7 4 cm2 seems optimal considering both plan quality as well as treatment time.We tried to find optimal parameters for the SFF scanning technique. In the results, the plan quality was good when the scanning resolution was small enough to result in full overlap between the scanning beams. At the very least, the scanning intervals should be smaller than the field size of the scanning beam to ensure good plan quality. If the scanning resolution was larger than the field size of the scanning beam, drastic degradation of the plan quality was observed. If the scanning resolution was fine enough for full overlap between beams, the field size of 4 \u00d7 4 cmTreatment time for the proposed technique was not calculated in this study. The SFF scanning technique synchronized with gantry rotation and couch movement in order to deliver electron beams perpendicular to the patient surface while maintaining the SSD might result in long treatment times. However, we hypothesize that the increased beam output of the SFF beam may partially counteract the increased treatment time. Although the treatment time of the proposed technique is potentially long, the SFF scanning technique provides value due to its considerable dosimetric advantages in comparison with other radiotherapy techniques for the treatment of extensively developed skin cancer on irregular patient surfaces.In this study, we used virtual cylindrical and spherical phantoms with radii from 5 cm to 15 cm because the average radius of a 2-months-old female\u2019s head is 5.9 cm and the average radius of Caucasian adult male\u2019s head is 10.08 cm . As show2, beam output increased by 21 times and bremsstrahlung contamination decreased by 7 times compared to the 6 MeV electron beam with scattering foil. With MC simulations, we demonstrated that the SFF scanning technique with 6 MeV electrons can reduce mean dose to normal tissue by 260% in a spherical phantom that is comparable to the average size of a Caucasian adult male\u2019s head (10 cm radius) while keeping same target coverage, as compared to the VMAT technique. The target conformity and homogeneity of the SFF scanning technique were also better than those of VMAT.By removing scattering foil in the treatment head of 6 MeV electron beam with a field size of 2 \u00d7 2 cmS1 TableThe benchmarking results of the simulated 6 MeV electron beam are shown.(XLSX)Click here for additional data file.S2 TableThe 6 MeV electron beams with a field size of 1 cm by 1 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S3 TableThe 6 MeV electron beams with a field size of 2 cm by 2 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S4 TableThe 6 MeV electron beams with a field size of 4 cm by 4 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S5 TableThe 6 MeV electron beams with a field size of 10 cm by 10 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file."}
+{"text": "A previous multi-locus lineage (MLL) analysis of SSR-microsatellite data of old olive trees in the southeast Mediterranean area had shown the predominance of the Souri cultivar (MLL1) among grafted trees. The MLL analysis had also identified an MLL (MLL7) that was more common among rootstocks than other MLLs. We here present a comparison of the MLL combinations MLL1 (scion)/MLL7 (rootstock) and MLL1/MLL1 in order to investigate the possible influence of rootstock on scion phenotype.A linear regression analysis demonstrated that the abundance of MLL1/MLL7 trees decreases and of MLL1/MLL1 trees increases along a gradient of increasing aridity. Hypothesizing that grafting on MLL7 provides an advantage under certain conditions, Akaike information criterion (AIC) model selection procedure was used to assess the influence of different environmental conditions on phenotypic characteristics of the fruits and oil of the two MLL combinations. The most parsimonious models indicated differential influences of environmental conditions on parameters of olive oil quality in trees belonging to the MLL1/MLL7 and MLL1/MLL1 combinations, but a similar influence on fruit characteristics and oil content. These results suggest that in certain environments grafting of the local Souri cultivar on MLL7 rootstocks and the MLL1/MLL1 combination result in improved oil quality. The decreasing number of MLL1/MLL7 trees along an aridity gradient suggests that use of this genotype combination in arid sites was not favoured because of sensitivity of MLL7 to drought.Our results thus suggest that MLL1/MLL7 and MLL1/MLL1 combinations were selected by growers in traditional rain-fed cultivation under Mediterranean climate conditions in the southeast Mediterranean area.The online version of this article (doi:10.1186/s12898-017-0114-3) contains supplementary material, which is available to authorized users. Map3 content (2.0\u201357.7%)  Table\u00a0. It has ne soils . We thusne soils . Support3 described above, that some environmental parameters have a differential influence on oil quality in GG1 vs. GG2 trees. The decreasing abundance of trees belonging to GG1 (i.e. common MLL1 grafted on MLL7) along an aridity gradient  on the MLL7 rootstock was governed by two opposing forces. On the one hand, growers in the past may have chosen to graft the common cultivar (MLL1) on MLL7 in order to enhance crop performance under certain environments. As MLL7 was used for grafting much more commonly than other MLLs , 27. As"}
+{"text": "Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1\u20139 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells  HDAC11 has a single lysine deacetylase domain surrounded by a short N- and C-terminus1. This lysine deacetylase domain is shared by all zinc-dependant HDACs (HDAC1-11) and is predicted to catalyse the removal of acetyl groups from acetylated lysine residues. HDAC11 is similar to both class I  and class II  zinc-dependent HDACs3. As there is little justification for assigning HDAC11 specifically to class I or class II HDACs, it is considered exclusively as the first and only \u2018class IV\u2019 HDAC identified to date4. The catalytic activity of HDAC11 is inhibited by HDAC inhibitors such as Mocetinostat, Vorinostat, Panobinostat and Quinostat at nanomolar concentrations6. Recently there have been reports of an inhibitor predicted to specifically target HDAC117.Histone deacetylase 11 (HDAC11) is the most recently identified member of the HDAC familyHDAC11 is specific to certain tissues, including the central nervous system1. Compared to the other HDACs, Hdac11 mRNA is particularly abundant in the rat brain with a peculiar pattern of expression in the hippocampus where it is most concentrated in the CA1 (Cornu Ammonis 1) region8. HDAC11 appears to be closely related to cell proliferation/differentiation as its expression is mutually exclusive with proliferative marker Ki-67 and its expression increases as neural cells differentiate in vitro11. Overexpression of HDAC11 in mouse fibroblasts inhibits their proliferation12. The Hdac11 gene and its chromosomal region are associated with variability in regional brain volume of mice14 and humans15. Additionally, multiple reports suggest associations between HDAC11 and malignant disease21. Two studies have implicated HDAC11 as a negative regulator of cell cycle component DNA replication factor Cdt1 (chromatin licensing and DNA replication factor 1)23. Whereas other studies have shown a role for HDAC11 in the response of antigen presenting cells25 and the differentiation of neural cells26. Watanabe et al., demonstrated that knockdown of HDAC11 inhibits dendrite growth of neurons generated in the adult mouse hippocampus26. This appeared to result from a regulatory role upstream of FEZ1 (fasciculation and elongation protein zeta 1), as they show that HDAC11 deacetylates BUBR1 (Budding uninhibited by benzimidazole 1-related protein kinase), relieving its inhibition of CDC20/APC (cell division cycle 20/anaphase-promoting complex), allowing that to ubiquitinate FEZ1 and therefore its degradation.The expression of 28. As previous studies report a role for HDAC11 in cell proliferation and neural differentiation, the aim of this study was to examine the nervous system in a previously uncharacterised Hdac11 knockout mouse. Hdac11KO/KO mice were born at Mendelian ratios and brain morphology appeared normal suggesting that HDAC11 does not have a critical role during development. Accordingly, in vitro studies on neural cell lines generated from Hdac11KO/KO mice showed normal proliferation patterns. However, we found that the expression of Fez1 was decreased in both proliferating and differentiating neural cell cultures. We further observed specific decrease in Fez1 gene expression in the hippocampi of adult Hdac11KO/KO mice. This suggests that HDAC11 has an age-dependent, brain region specific function in regulating FEZ1, a gene associated with schizophrenia. This is important because it demonstrates that the regulatory role of HDAC11 in the hippocampus is developmentally regulated. Given previous associations between HDAC11, FEZ1 and schizophrenia, a goal of future research should be to examine the performance of homozygous Hdac11 knockout mice in behavioural tests.Knockout mouse models for HDACs 1\u20139 have demonstrated the consequences resulting from complete loss of each individual HDACHdac gene expression levels between proliferating and differentiating neural cells revealed that Hdac11 has the largest increase in expression compared to all the other Hdacs and Sirtuins  and fibroblast growth factor (FGF). Removal of mitogens from the culturing media results in fewer proliferating cells  in Hdac11KO/KOand Hdac11WT/WT neural cell lines  which displays a small decrease  during the differentiation of Hdac11WT/WT cells but an increase  during the differentiation of cells derived from Hdac11KO/KO mice  or differentiating (p\u2009=\u20090.24) conditions  and adult (P60/\u201c2 month old\u201d) mice. This revealed that Fez1 expression was specifically downregulated in the hippocampus of adult Hdac11KO/KO mice  and Plp1 (Proteolipid protein 1) because they had previously been reported to be decreased following loss of Hdac1110. However, we did not see a statistically significant impact on the expression of either of these genes  and certain class II HDACs (Hdac4 and -7)31 which display either complete or partial embryonic/perinatal lethality28. Hdac11 expression increases during the first 2 months of postnatal mouse brain development9. However, we found that the gross anatomy of the brain appeared normal in 2-month-old homozygous Hdac11 knockout mice. Three of the previously generated Hdac knockout mice  displayed only subcellular or age-dependent phenotypes34. Similarly, Hdac11 knockout mice do not display an obvious phenotype but rather have an age dependent brain region specific decrease in Fez1. It is interesting to note that HDAC11 and HDAC6 appear to be recruited to the same protein complex26 and both are reported to regulate CDC20, a component of the pathway that controls FEZ1 ubiquitination35.Fertile, homozygous Hdac11 expression has been reported to impact on the proliferation of fibroblasts in vitro12. Together with reports of roles for HDAC11 in the cell cycle23 and its association with cancer and brain volume20 we predicted that there would be an impact on the proliferation rate of cells derived from the nervous system of homozygous Hdac11 knockout mice. However, we did not find any effect from HDAC11 loss on neural proliferation and these cells also exited the cell cycle when directed. Accordingly, the gross brain morphology of homozygous Hdac11 knockout mice appeared normal suggesting these processes had at least generally been regulated appropriately. One possibility is that HDAC11 has roles specific to cancer cells. Supporting this idea is a report that HDAC11 knockdown selectively impacts carcinogenic cell lines, leaving normal cell lines unharmed20.Partial loss of Hdac11 results in decreased expression of IL-10 (Interleukin 10)24, Mbp (Myelin basic protein) and Plp (Proteolipid protein 1) genes10. Additionally, Deubzer et al. report unpublished results that HDAC11 represses the transcription of Bmp4 (Bone morphogenetic protein 4) in a neuroblastoma cell line20. We found that the expression of oligodendrocyte associated genes Mbp and Plp were expressed at reduced levels in the adult Hdac11KO/KO hippocampus but this was not statistically significant. It is likely that loss of HDAC11 has cell type specific effects on gene expression which should be considered carefully when investigating its function. The previous studies on HDAC11 function had examined macrophages24, oligodendrocyte precursor cell lines10, fibroblast cells12 and a neuroblastoma cell line20. We propose that the Hdac11 knockout mouse line can be used for investigation of tissues and cell types relevant to these experiments.Previous studies have reported that knockdown of 1 where it is understood to regulate gene expression24. Indeed, the results of this study show that HDAC11 is enriched in nuclear fractions. It has been reported that HDAC11 becomes increasingly localised in cytoplasmic compartments during the differentiation of neural progenitors in the hippocampus26. Despite limitations preventing us using HDAC11 antibodies to examine HDAC11 localisation using immunocytochemistry, we did observe HDAC11 expression in cytoplasmic fractions and more specifically in synaptosomes. This provides additional insight into the subcellular localisation of HDAC11 and offers the possibility that it has a regulatory role in the synaptic compartment. Like class IIa HDACs, HDAC11 may shuttle between cellular compartments in response to stimuli. HDAC11 has previously been reported to regulate dendrite development26 and enrichment of HDAC11 at the synapse supports the enticing possibility that regulating its activity will impact synaptic plasticity and memory.HDAC11 was initially reported to be localised to the nucleusFez1 expression in both proliferating and differentiating cells derived from homozygous Hdac11 knockout mice so we investigated this in more detail. Examination of different brain regions revealed that this effect was specific to the hippocampus of adult mice which was surprising given that we also observed decreased Fez1 expression in proliferating neural cells in vitro. This may reflect an acquired identity of these in vitro lines and reinforces the importance of follow up studies on appropriate tissue.We observed decreased 26. Given their results, it would be predicted that loss of HDAC11 activity would increase FEZ1 protein expression. Regrettably, we do not report FEZ1 protein levels in response to loss of HDAC11 as we were unable to detect the expression of FEZ1 using commercially available antibodies. The decreased level of Fez1 transcript in response to loss of HDAC11 may be due to a self-regulatory function of FEZ1. This self-regulatory function may involve interaction with DISC1 (Disrupted in schizophrenia 1)36. Patients with schizophrenia carrying a high risk allele for the disease in the DISC1 gene show reduced FEZ1 expression in the hippocampus37.A previous study showed that FEZ1 ubiquitination is downstream of a pathway activated by HDAC11 activityFEZ1 gene expression in the hippocampus of patients with schizophrenia is particularly relevant as this was seen in the homozygous Hdac11 knockout mice37. The relationship between HDAC11 activity and schizophrenia-associated phenotypes is strengthened by Watanabe et al.\u2019s evidence for a signalling cascade implicating HDAC11, FEZ1 and DISC126. Sheng et al. examined the CA1 and CA2/3 regions of the hippocampus from post-mortem schizophrenia patient tissue38. They identified copy number increases in the HDAC11 gene which resulted in increased HDAC11 expression specifically in the CA2/3 region. In contrast, post-mortem tissue from patients with a different psychiatric disease (bipolar disorder) displayed no difference in the copy number or expression of HDAC11 compared to healthy controls. A more recent study reports an association between schizophrenia and single nucleotide polymorphisms (SNPs) in the human HDAC11 gene39.The report of reduced et al.40. In their review they note that Hdac11 is expressed in the hippocampus and such patterns provide a spatial association with schizophrenia but emphasise that functional inferences cannot be drawn from this. The results of this study demonstrate that Hdac11 knockout mice provide a means to examine the functional association between HDAC11 and schizophrenia-associated FEZ1.The emerging association between HDAC11 and schizophrenia is interesting as it should be considered during the development of therapeutic drugs. The promise of treating schizophrenia by selective inhibition of certain HDACs has previously been discussed in a review by We\u00efwer 41. Furthermore, it would be interesting to investigate the response of Hdac11 knockout mice to phencyclidine, which is reported to result in a variety of phenotypes associated with schizophrenia including hyperlocomotion41. Hdac11 knockout mice may share similar characterisics to Fez1 knockout mice which do not display any anatomical abnormalities but do display a hyperlocomotion phenotype in a variety of tasks42.Our findings are specific to the adult-mouse hippocampus, suggesting that this area will be particularly important to following investigations, particularly for investigating the molecular pathways regulated by HDAC11. Disrupted performance on hippocampal dependent tasks such as the Morris water maze, T-maze and radial arm maze are reported in mouse models of schizophrenia43. HDAC11 may have a more general association with psychiatric disease. Studies have found links between HDAC11, the administration and reinforcement of drugs. An impact on Hdac11 expression is reported in the brain of rodents administered with cocaine44, alcohol45 and methamphetamine47. Incidentally Fez1KO/KO mice have an enhanced response to methamphetamine treatment, at least in part due to increased mesolimbic dopaminergic transmission42. Understanding how these observations converge on HDAC11 may provide an important insight into the neural mechanisms disrupted in a variety of disorders. Furthermore, it is important that we understand the consequences of losing HDAC11 activity as this will be valuable for guiding the development of isoform specific HDAC inhibitors as therapeutics for disease.Genetics is important in the etiology of schizophrenia but it remains difficult to determine the precise causative genetic factors in this complex polygenic disorderMouse Embryonic Stem Cell (mESC) proliferation media was formulated in a basal media of Dulbecco\u2019s modified Eagle medium (DMEM), High Glucose, GlutaMAX\u2122  with 20% embryonic stem cell grade FBS . The media contained 1\u00d7 Non-Essential Amino Acids , 50\u2009\u03bcM beta-mercaptoethanol  and 10\u2009\u03bcg/mL (Leukemia inhibitory factor) LIF .Neural proliferation media was formulated in a NeuroCult\u2122 NSC Basal Medium (Mouse)  with NeuroCult\u2122 NSC Proliferation Supplement (Mouse) . The medium contained 20 ng/mL recombinant mouse epidermal growth factor (rmEGF)  and 10 ng/mL recombinant mouse fibroblast growth factor (rmFGF) .Neural differentiation media (N2B27) is a formulation of 50% DMEM/F-12, GlutaMAX\u2122  and 50% Neurobasal\u00ae Medium . This contained 0.5x N-2 Supplement , 0.5x B-27\u00ae Serum-free Supplement , 0.5x GlutaMAX\u2122 Supplement , 1x Non-Essential Amino Acids  and 50\u2009\u03bcM beta-mercaptoethanol .et al.48 before they were maintained in the neural proliferation media described above.Generation of mESC-derived neural cells was carried out according to Conti 49. The cells were cultured in suspension on untreated plastic in neural proliferation media (see cell culture media). They were passaged every 4 days by dissociation with Accutase\u00ae  and reseeded at a density of 10,000 cells/cm2.The mouse embryonic ganglionic eminence was dissected and cultured according to Chojnacki and Weiss\u2019s protocol2, whereas those for differentiation inducing conditions were seeded at 100,000/cm2. After 24\u2009hours, the cells had adhered to the surface and the media was replaced with the experimental media. For proliferative conditions, the media was replaced with neural proliferation media (see cell culture media). For differentiation inducing conditions, the media was replaced with neural differentiation media (see cell culture media). The media was replaced with fresh media every day until the cells were harvested.For consistency between approaches, all proliferating neural cell lines were seeded as an adhered monolayer on 0.1\u2009mg/mL poly-DL-ornithine  followed by 5\u2009\u03bcg/mL laminin  coated plastic before experimental procedures began. Following a typical passage, proliferating neural cells were dissociated into a single cell suspension ensured by passing the suspension through a sterile 40 \u03bcm cell strainer  into a 50\u2009mL tube. Cells destined for proliferative conditions were seeded at 20,000/cmIncorporation of the thymidine analogue EdU into the DNA of proliferating cells over a 24\u2009hour period was detected using the Click-IT\u00ae EdU Alexa Fluor\u00ae 488 Imaging kit  according to the manufacturer\u2019s instructions.Cells for immunocytochemistry assays were seeded into black walled and optical bottomed 96 well plates . They were fixed for 20\u2009minutes in a 4% formaldehyde solution . Primary and secondary antibodies were prepared in Phosphate buffered solution (PBS)  made with 10% donkey serum , 0.1% Triton-X 100  as described in supplementary Table\u00a0http://imagej.nih.gov/ij/). Cell nuclei were set as regions of interest (ROI) as defined by the nuclear counter stain (i.e. Hoechst 33342). Then the average signal intensity (i.e. Ki-67 and EdU) was measured for each ROI using the Analyze Particles command. A cell was considered positive if the average signal intensity for its ROI was greater than the ROI with the maximum value from staining with the secondary antibody alone (Ki-67 experiment) or differentiation inducing (i.e. proliferation preventing) condition (EdU experiment).Fluorescent images were acquired using an inverted microscope . Analyses of Ki-67 or EdU positive cells were quantified using Fiji/ImageJ  according to the manufacturer\u2019s instructions. TaqMan\u00ae assays  and the other does not . Data were collected on the C100TM Thermal Cycler and CFX96TM Real-Time System (BIO-RAD). Data collection from TaqMan\u00ae Low Density Arrays (TLDA) was carried out on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).Real time PCR was carried out using the 2et al.51. Synaptosome fractions were prepared as described in Valencia et al.52.Nuclear and cytoplasmic fractionation was carried out as described in Davies et al.53. The antibody incubation details are described in Supplementary Table\u00a0Western blotting was carried out as described by Landles Hdac11 knockout mice can be found on the Taconic website (http://www.taconic.com/mouse-model/hdac11-ko-6978). The gene was targeted in C57BL/6 ESCs by deletion of exon 3 leading to a premature stop codon. To identify the genotype, a PCR reaction with the forward primer (TGCTGCCTGTGAGCCACTGC) and reverse primer  were used. The reaction was run on a program of 4\u2009minutes at 95\u2009\u00b0C, before 36 cycles of 25\u2009seconds at 95\u2009\u00b0C, 20\u2009seconds at 60\u2009\u00b0C and 45\u2009seconds at 72\u2009\u00b0C. The program finished with a single final step for 6\u2009minutes at 72\u2009\u00b0C.Details of the Mice were anesthetised with an injection of 100\u2009\u03bcL sodium pentobarbital . The mice were transcardially perfused with 20\u2009mL of PBS followed by perfusion with 20\u2009mL of 10% formalin (i.e. 4% PFA) . After the perfusion was complete, the brain was stored in 10% formalin at 4\u2009\u00b0C overnight before equilibration in 30% sucrose  made in PBS at 4\u2009\u00b0C. The brain was then snap frozen in 2-methylbutane  that had been cooled on dry ice. The brains were sectioned every 30 \u03bcm on a cryostat, cooled to \u221217\u2009\u00b0C. The sections were attached to glass adhesion SuperFrost\u00ae plus slides . They were stained in a 1% cresyl violet , 1% Glacial acetic acid solution for 10\u2009minutes at room temperature (cerebellum and hippocampus images) or 60\u2009minutes at 37\u2009\u00b0C (cortex images). The slides were then dehydrated in increasing amounts of ethanol with only a few seconds incubation in the final 100% ethanol step to avoid removing too much stain. They were then incubated in Xylene  for 15\u2009minutes before mounting with DPX mounting medium .In situ hybridisation was carried out using RNAscope\u00ae Technology (Advanced Cell Diagnostics) according to the manufacturer\u2019s instructions. The Hdac11 probe targeted the region 35-981 of NM-1449.9.2. The Fez1 probe targeted the region 2-1010 of NM-183171.4. To ensure that comparative numbers of cells were included in the quantification, single, individual cells expressing Hdac11 were selected within the region of interest (dentate gyrus of the hippocampus) (see Supplementary Fig.\u00a0Supplementary Information"}
+{"text": "Here we show that mutants of the bacterium Salmonella enterica Serovar Typhimurium LT2 lacking either the s2- or the (c)mnm5-group of (c)mnm5s2U34 grow poorly especially at low temperature and do not grow at all at 15\u00b0C in both rich and glucose minimal media. A double mutant of S. enterica lacking both the s2- and the (c)mnm5-groups, and that thus has an unmodified uridine as wobble nucleoside, is nonviable at different temperatures. Overexpression of 2- or the (c)mnm5-group and of 2-group exaggerated the reduced growth induced by the modification deficiency, whereas overexpression of 5-group did not. From these results we suggest that the primary function of cmnm5s2U34 in bacterial 5s2U34 in 5-group of 5s2U34 in these yeast tRNAs is to improve cognate codon reading rather than prevents missense errors. Thus, although the xm5s2U34 derivatives are universally conserved, their major functional impact on bacterial and eukaryotic tRNAs may be different.In the wobble position of tRNAs specific for Gln, Lys, and Glu a universally conserved 5-methylene-2-thiouridine derivative (xm Although their presence in tRNA is scattered over the entire molecule, there are two positions in the tRNA that are more frequently modified than others\u2013about 50% of nucleosides in position 34 (the wobble position) and about 80% in position 37 (next to and 3\u2019 of the anticodon) are modified  ] . The redborators .elp3, tuc1 double mutant tRNAs specific for Gln, Lys, and Glu lack mcm5s2U34 in their wobble positions and have instead an unmodified U34. Such a double mutant is not viable, but overexpression of hypomodified versions of these tRNAs rescues the double mutant elp3, tuc1 [mnmA, mnmE double mutant, which should also have an unmodified wobble U34 in the corresponding tRNAs, would therefore be potentially nonviable. Since the xm5s2U34 derivatives are universally conserved their function might be similar so that overexpression of bacterial hypomodified tRNAs might, as in yeast, rescue a potential nonviable double mutant mnmA, mnmE. Overexpression of a hypomodified tRNA mnm5s2U34 of 5-group (See above). Plasmid pUST313 harbors the metT operon consisting of genes glnU and glnW encoding the (c)mnm5s2U34 containing glnV and glnX encoding the C34 containing metT/U genes encoding leuW gene encoding mnmA3, tusB27 and tusE30 mutants as well as into the wild type strain. Overexpression of mnmA mutant (data not shown). However, plasmid pUST313 severely reduced the growth of the three independent mutants mnmA3, tusB27 and tusE30 all lacking the s2- group of (c)mnm5s2U34 in 2-deficieint mutants, it was the lack of the s2-group of (c)mnm5s2U in mnmA3 mutant. Sequencing the metT operon of the plasmid present in such a large spontaneously occurring colony revealed that a recombination had occurred between the plasmid encoded metT and metU genes resulting in the loss of genes glnU and glnW, which code for metT/U gene encoding glnV and glnX genes encoding the C34 containing mnmA3 and the tusB27 mutants compared to the almost complete inhibition of growth caused by the 2-group of (c)mnm5s2U34 but not at all in the wild type strain. In summary, overexpression of s2U-deficient mnmA3, tusB27 and tusE30) induced a severe growth reduction that was not observed if this tRNA was not overexpressed.Strains having a mutation in o5UAGLeu . Note, nr degree . Slow grs2UUGGln . The ress2UUGGln . Plasmid2-group of (c)mnm5s2U34 but not in the wild type control. A similar growth reduction was also observed with plasmid pUST314, which does not encode 2-deficiency of (c)mnm5s2U34 in the cell, since a similar growth reduction was not observed in the wild type strain mnm5s2U34 (mnmE13 or mnmG1) inducing a similar mnm5- group deficiency, the growth reduction is likely due to the hypomodified 5-group causes a substantial growth reduction on rich medium and even a more severe growth reduction on minimal medium. We suggest that 5-group induces missense errors. Interestingly, 5-group also increases misreading of GGA (Gly), GAU (Asp) and GAC (Asp) codons [Plasmid pUST313 was introduced to the nm5s2U34 . Since a) codons .valU operon, which consists of the genes valU, valX, valY and lysV, the latter of which is the structural gene for mnmA3 or mnmE13 influence only the structure of the 2-group of mnm5s2U34, suggesting that overexpressing thiol-deficient 2-deficiency of mnm5s2U34 in 2-deficiency of mnm5s2U34 in 5-deficient, suggesting that this deficiency did not induce any increased translational errors such as missense error. This result is indeed consistent with earlier reports showing that mnm5-deficient Plasmid p815 contains the e errors . Howeverll et al . If so, ) codons , 8. Thes2-deficent 5-group reduced growth although much less than s2-deficiency, whereas lack of the mnm5 group of overexpressed The combined results above show that overexpressed smnmA, mnmE we wanted to combine strains harboring both the selectable mnmA16<>cat (CmR) and mnmE17<>Km (KmR) mutations and such a double mutant should have tRNAs specific for Gln, Lys, and Glu with an unmodified wobble uridine (U34). We grew the mnmA16<>cat mutant in rich medium at 37\u00b0C and mixed the cells with the transducing phage P22 grown on the mnmE17<>Km. We then spread the mixture on rich agar plates containing kanamycin to select for the mnmE17<>Km mutation and thereby creating the wanted double mutant mnmA16<>cat, mnmE17<>Km. The KmR transductants were selected at several temperatures to monitor if a double mutant might be viable at another temperature than 37\u00b0C. The donor strain GT8176 (STM3453-2550::Tn10dTc) also contains a selectable  mutation known not to induce any growth defect and this mutation was used to monitor the efficiency of transduction. As recipient we also used strain GT8173 (pmnmA+/ mnmA16<>cat), which contains the same mnmA16<>cat mutation on the chromosome and the complementing plasmid pmnmA+ containing the wild type allele of the mnmA gene. Such strain should behave as the wild type unless the mutation mnmA16<>cat introduces some unknown phenotype, which is not related to the mutation in mnmA gene and therefore not complemented by the pmnmA+ plasmid.To construct a double mutant mnmA16<>cat, mnmE17<>Km was obtained at any temperature clearly demonstrating that such a double mutant is not viable. This result cannot be due to an inefficient transduction, since the transfer of the STM3453-2550::Tn10dTc marker was similar to the transfer frequency of the mnmE17<>Km mutation to the wild type (Compare the number of TcR colonies to the number of KmR colonies in the wild type). Moreover, the transduction frequency of mnmE17<>Km was similar when strain GT8177 (pmnmA+/ mnmA16<>cat) was used as recipient. Note also, that we waited several days in order to allow a very slow growing double mutant to appear but still no such mutant was recovered. Moreover, we did not obtain any external suppressor mutant with mutation counteracting the lack of modification of U34. We conclude that cells having an unmodified wobble uridine of tRNAs specific for Gln, Lys and Glu are not viable on rich medium at temperatures between room temperature (about 20\u00b0C) and 42\u00b0C.R transductants (STM3453-2550::Tn10dTc) shows that the recipient mnmA16<>cat mutant is not able to grow at such low temperature consistent with the results shown in The inability to obtain any transductants at 15\u00b0C when selecting Tc5s2U34 in yeast tRNAs is to improve the efficiency of the cognate anticodon-codon interaction rather than preventing missense errors, since overexpression of hypomodified tRNAs specific for Gln, Lys or Glu counteracts the phenotypes induced by deficiency of either s2, mcm5- or both modifications. However, overexpression of bacterial 2- or cmnm5- deficiency but rather exaggerated the reduced growth of these mutants  defective in the thiolation and two independent mutants (mnmE13 and mnmG1) defective in the synthesis of the side chain  not present in bacteria to reduce missense errors [We  and othe mutants . This grde chain . Moreovehis tRNA . These re errors .5s2U34 derivative. It was thought that the presence of this modification in the wobble position of Gln, Lys and Glu tRNAs would prevent missense errors [mnmA3 and mnmE17 mutants show that these modifications are pivotal for cellular growth  in position 37 and both 2A. The ct6A improves the stacking interaction with the base above (A38) and below (A36) of the anticodon loop but it also makes a cross strand stack to the first base (A) of the codon and thereby stabilizes the weak A-U36 base pairing in the first position [2A, which is present in 6A37 and accordingly it is not essential for viability [5s2U wobble modifications is different in these three tRNAs not only because differences in the primary sequence of the anticodon loop but also because of differences in the modification pattern and in particular the nature of the modifications at position 37 mnm5-group of (c)mnm5s2U34 induces frameshifts by the peptidyl-slippage mechanism (reviewed in [sufA6) does not in any major way influence cellular growth (unpublished observation). Taken together, increased frameshifting as an explanation to the severe growth reduction observed upon overexpression of hypomodified tRNAs is not likely. On the other hand the growth reduction may cause an aberrant folding of several proteins. The nascent polypeptide is folded on the ribosome and a change of the polypeptide synthesis rate may influence the folding of proteins [5s2U34 is to prevent such errors. Moreover, such errors have been observed experimentally [5-group did not reduce growth suggesting no increase missense errors consistent with results obtained earlier [5s2U34 wobble modification have been shown theoretically and experimentally to influence the accuracy of translation besides its effect on the efficiency of cognate codon reading. Therefore, the primary function of the xm5s2U34 modifications may be different in yeast and in bacteria.We have suggested that the reduced growth linked to the overexpression of hypomodified iewed in ) and acciewed in  noticed iewed in . Moreoveproteins . The speproteins , 44. Oveproteins . From thmentally  althoughmentally , 8. Thes earlier , 8, ThusS1 Table(DOCX)Click here for additional data file."}
+{"text": "GLABRA3 gene is a major regulator of trichome patterning in Arabidopsis thaliana. The regulatory regions important for the trichome-specific expression of GL3 have not been characterized yet. In this study, we used a combination of marker and rescue constructs to determine the relevant promoter regions. We demonstrate that a 1 kb 5\u2032 region combined with the second intron is sufficient to rescue the trichome mutant phenotype of gl3 egl3 mutants. Swap experiments of the second intron suggest that it is not sufficient to generally enhance the expression level of GL3. This implies that the second intron contains regulatory regions for the temporal and spatial regulation of GL3. The corresponding GUS-marker constructs revealed trichome-specific expression in young trichomes.The  Arabidopsis trichomes are single epidermal cells that develop on the surfaces of most aerial organs. Trichomes are regularly distributed on rosette leaves, cauline leaves, sepals and the stem without any obvious reference to other morphological structures (TRANSPARENT TESTA GLABRA1 (TTG1) (ructures . The dis1 (TTG1) , the bHL1 (TTG1)  and the 1 (TTG1) , and the1 (TTG1)  and MYB21 (TTG1) , 2005. I1 (TTG1) . The tri1 (TTG1) : The act1 (TTG1) .GL3. In rosette leaves, GL3 in situ hybridization experiments have shown that GL3 is expressed in developing trichomes. The expression analysis of a 2.5 kb 5\u2032-promoter fragment driving the GUS reporter gene revealed a similar expression pattern ]. The 1051 bp 3\u2032 fragment was cloned into the PmeI site of pAMPAT-GW-GL3(5\u2032-1 kb) to create the pAMPAT-GW- GL3(5\u2032-1 kb):LR recombination cassette:(3\u2032-1 kb) vector. All genomic fragments of GL3 were cloned into pDONR201 by BP reactions (Invitrogen). Deletions of single introns within the genomic sequence of GL3 were introduced by PCR based site directed mutagenesis. The entry clone carrying the GL3 gene with the second intron was generated using the following strategy: an entry clone carrying the genomic GL3 was cut with EcoRV and KpnI generating a GL3 fragment that includes the second and third intron of GL3. This fragment was exchanged against the corresponding GL3 fragment without introns in the entry clone carrying the coding sequence of GL3. Thereafter, the third intron was deleted by PCR based site directed mutagenesis. Coding and genomic sequences of GL3 were introduced into pAMPAT-GW-GL3(5\u2032-1 kb):LR recombination cassette:(3\u2032-1 kb) by LR recombination with the respective entry clones to generate the various intron deletion constructs. Plants were transformed using the floral dip method described previously  ecotype. The gl3-3 mutant line has been described previously  and first-strand cDNA was then synthesized from the total RNA (1 \u03bcg) using the RevertAid H Minus 1st strand cDNA synthesis (Thermo) as described by manufacturer\u2019s instruction. Real-time polymerase chain reactions (PCR) contained 1 \u03bcl of primer mix (10 \u03bcM), 1 \u03bcl cDNA template (10-fold dilution),10 \u03bcl 2 \u00d7 SYBR Green master PCR mix and 8 \u03bcl water to a total of 20 \u03bcl. cDNA concentrations in different samples were normalized with reference to GUS stainings were essentially done as previously . After sGL3 fused to GUS reveals trichome specific expression in leaves and that a fusion to the GL3 cDNA can rescue the trichome phenotype in gl3 egl3 double mutants (GL3 fragment including 1kb of the 5\u2032-promoter was sufficient to rescue the gl3 trichome phenotype (pGL3(1 kb):GUS line. We observed ubiquitous GUS expression in young leaves  in the gl3-3 egl3-77439 double mutant. The gl3-3 single mutant shows about 50% reduction in trichome number whereas the egl3-77439 mutant shows a significant reduction of about 10% similar as reported for the egl3 allele in the Ler background :3\u2032-1 kb] such that the coding region can be replaced by recombination . This construct was used to study the rescue ability in gl3 egl3 mutants in the T1 generation. As expected, we found a range of rescue phenotypes in the T1 generation. The average rescue efficiency was used as a reference for subsequent analysis (Table 1). Next, we created a series of constructs each lacking one of the six introns . We found a clear rescue with constructs missing the third, fourth, or fifth intron. The deletion of the first or the sixth intron resulted in a weaker rescue of trichome formation. No rescue was observed in plants carrying the pGL3:GL3 (genomic\u0394intron 2):3\u2032-1 kb construct (Table 1) indicating that the second intron is essential. We therefore focused in the following on the function of the second intron.Our data indicated that the 5\u2032 promoter region and the 3\u2032-1 kb are not sufficient for rescuing the trichome phenotype of GL3:genomic GL3 construct . These lines showed a rescue of the trichome phenotype. Thus, the 3\u2032-1 kb of GL3 is not necessary for trichome rescue.To test the relevance of the 3\u2032-1 kb region we studied the rescue in lines harboring the pGL3 in the leaf, we expressed the GL3 cDNA containing the second intron at its original site under the 1 kb 5\u2032-promoter [pGL3:GL3(Intron 2)] in gl3 egl3 mutants . The majority of T1 lines showed rescue of the trichome phenotype (Table 2). In an attempt to map potential relevant regions in the second intron, we compared two deletion constructs missing either the 5\u2032 125 nt (pGL3:GL3(Intron 2 delta 3-125) or 454 nt at the 3\u2032end [pGL3:GL3(Intron 2 delta 126-579)] of the second intron. Only the construct containing the 125 nt at the 5\u2032end rescued the gl3 egl3 mutant trichome phenotype (Table 2) suggesting that this fragment contains all regulatory sequences. In a next step, we assessed whether the position of the intron 2 is important. Toward this end we placed intron 2 in front of the 5\u2032 promoter in both directions . Neither construct was able to rescue the gl3 egl3 trichome mutant phenotype (Table 2) indicating that intron 2 does not act as a transcriptional enhancer element. This suggested to us that its position in the transcribed region is important for its function. One well-characterized regulatory mechanism that requires the intron within the transcribed sequence in its correct orientation is intron mediated enhancement (IME) . The first intron of UBQ10 resulted in a 2- to 10-fold higher GL3 expression as compared to wild type Col . Insertion of the first intron of the Cor15a gene lead to wild-type levels or up to about two-fold increased GL3 expression . By comparison, constructs containing intron 2 or the first 125 nt of intron 2 could enhance GL3 expression up to three-fold . However, neither the UBQ10 nor the Cor15a constructs rescued the trichome phenotype (Table 2).In order to demonstrate that the second intron together with the 1 kb 5\u2032-promoter fragment is sufficient for the transcriptional regulation of nt (IME) . To addrGL3 expression by replacing intron 2. It is therefore conceivable that intron 2 is important for the proper regulation of the temporal and spatial expression of GL3.These results suggest that it is not sufficient to merely increase the GL3 is sufficient for complete rescue. To study the expression pattern mediated by this construct we fused the GUS marker gene directly after the second intron. As the signal levels were very low when using X-Gluc as a substrate, we used the more sensitive magenta-Glc-A as a substrate . In addition we noted weak expression in epidermal pavement cells in young leaves . In older leaves with young trichome stages at the leaf bases and mature trichomes at the tip of the leaf trichomes exhibited much stronger expression then the mature trichomes . Low levels of GL3 were maintained during further leaf growth but disappeared in fully mature leaves .Our analysis revealed that a 1 kb promoter fragment combined with intron 2 in the transcribed region of ubstrate . We deteGL3 gene are important for the transcriptional regulation in the context of trichome development. While previous data suggested that the 1 kb 5\u2032 sequences together with 1 kb 3\u2032 sequences might be sufficient for rescuing the trichome phenotype  binding sites in the second intron of GL3 (Supplementary Figures Arabidopsis lyrata, Capsella rubella, and Arabis alpina (Supplementary Table This raises the question, how and by which factors intron 2 is regulated to mediate trichome specific expression. One likely scenario would be the regulation by trichome patterning genes, in particular eriments . A possipression . This repression . In contt of GL1 . AlthougGL3 in Arabidopsis. For future studies of the temporal and spatial regulation of GL3 it will be helpful that we could map one relevant region down to a fairly small fragment of only 125 nt containing conserved binding sites.Therefore, these binding sites are potentially relevant for the regulation of AF, BZ, SH, MP, and AS designed, planned and performed the experiments and analyzed the data. MH supervised the project and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JB and handling Editor declared their shared affiliation, and the handling Editor states that the process met the standards of a fair and objective review."}
+{"text": "We propose ways to experimentally characterize and control important parameters of the model catalyst\u2014the coverage of the ceria layer, the influence of the Cu substrate, and the density of surface defects on ceria, particularly the density of step edges and the density and the ordering of the oxygen vacancies. The large spectrum of controlled parameters makes ceria on Cu(111) an interesting alternative to a more common model system ceria on Ru(0001) that has served numerous catalysis studies, mainly as a support for metal clusters.An important part of fundamental research in catalysis is based on theoretical and modeling foundations which are closely connected with studies of single-crystalline catalyst surfaces. These so-called model catalysts are often prepared in the form of epitaxial thin films, and characterized using advanced material characterization techniques. This concept provides the fundamental understanding and the knowledge base needed to tailor the design of new heterogeneous catalysts with improved catalytic properties. The present contribution is devoted to development of a model catalyst system of CeO The bulk lattice parameter of cubic cerium dioxide is 0.54 nm which corresponds to the [1 0 \u22121] lattice vector length in the CeO2(111) plane of aCeO2 = 0.382 nm. Thus the expected aCeO2/aCu ratio is 1.50 indicating very good lattice matching with negligible strain (<0.6%) for the observed (1.5 \u00d7 1.5) commensurate superstructure. The absence of rotational domains in the LEED pattern indicates very good ordering of the layer. Epitaxial growth and practically negligible lattice mismatch made the preparation of very thin continuous CeO2 film using reactive vapor deposition feasible and it opened a new promising field of model studies of cerium oxide surfaces.The LEED diffraction pattern presented in 2(111)/Cu(111) very thin films for mimicking the cerium oxide single-crystal surface depends on the substrate-oxide interaction that can strongly influence the chemical properties of the ceria/Cu systems as demonstrated in many studies of Cu-ceria inverse catalysts. DFT + U calculations of systems consisting of Cu atoms supported by stoichiometric and reduced CeO2(111) surfaces show that Ce3+ species are always present underneath the Cu particles supported by stoichiometric and reduced ceria (111) surfaces [2 interface leading to the full reduction of the first ceria monolayer underneath the supported Cu particles. Therefore the emerging question concerning the physicochemical properties of the CeO2(111)/Cu(111) thin films was related to the ceria-copper interaction and the extent to which this interaction determines the properties of ceria/Cu(111). Scanning Tunneling Microscopy (STM) and ab-initio calculations allowed to determine the unusual properties of the first ceria monolayer in contact with the Cu(111) substrate showing finite size effects when the limiting thickness of the oxide monolayer and the proximity of the metal substrate cause significant rearrangement of charges and oxygen vacancies compared to thicker and/or bulk ceria [Suitability of the CeOsurfaces ,38. The lk ceria . This relk ceria .d3/2-3d5/2 spin-orbit-split doublets  representing different 4f configurations in the photoemission final state and arising from 4f hybridization in both the initial and the final states [f0 signal at 917 eV, together with an f1 peak (889 eV) which is less intense than the f2 peak (882.5 eV), is evidence of the formation of CeO2 oxide [3+ state. In order to estimate the Ce3+ state concentration the spectra must be decomposed to elementary doublets. However this is not a simple task because of the ambiguity of background subtraction , choice of elemental peak shape including asymmetry and insufficient spectrometer resolution in general [The main property of ceria in chemical reactions is the release and the uptake of lattice oxygen to/from the reaction atmosphere. Upon leaving the ceria lattice, the neutral O atom leaves behind two electrons that localize on two Ce atoms occupying the 4f state of Ce . The chal states . The appO2 oxide ,44. Two  general . A typic3+ and Ce4+ contributions with very high sensitivity using so called Resonance Photoelectron Spectroscopy (RPES) [Employing tunable radiation of a soft X-ray synchrotron photoemission beamline, resonance effects in the Ce 4d\u20134f photoabsorption region can be used to distinguish between Cey (RPES) ,46.x is shown in 3+ (4f1) and Ce4+ (4f0) valence states. At 115 eV there is no resonance. As we proposed in [i.e., by obtaining so called resonance enhancement DCe3+ or DCe4+, see 3+/DCe4+ was proposed and is used as a parameter sensitively indicating the degree of reduction of cerium oxide surface. Besides the high sensitivity to small concentrations of Ce3+ the energy of detected photoelectrons in the range of 100 eV guarantees the highest surface sensitivity of the RPES the Ce 4d\u20134f signal.This method is based on tuning the photon energy in the proximity of the resonant energy where a resonant enhancement of the Ce 4f photoemission can be observed . A serieposed in  the densi.e., perfect stoichiometry of the ceria film.The resonance spectra shown in Scanning Tunneling Microscopy (STM) represents a primary research tool for investigating morphology of nanostructured ceria and metal-ceria samples yielding an indispensable input for the advanced structure-property studies. Local information of the morphology of the model catalysts combines favorably with the information on their electron and chemical state obtained by space-averaging experimental techniques. STM imaging provides information on densities of atomic step edges , densitiAdjustable morphology and degree of reduction represent desirable properties of model oxide substrates for heterogeneous catalysis  prepared2(111) on Cu(111) with deterministically controlled density of atomic steps [A range of bottom-up experimental approaches that allow preparation of oriented thin films of CeOic steps  and the ic steps ,49 has bic steps .2(111) film, i.e., on using metallic Ce as a homotypical reducing species [2O3 on Cu(111) [2O3 represents the ultimate reduction of ceria that is difficult to obtain by other methods practically used for reducing ceria samples. The extremely sensitive RPES reveals no Ce4+ contribution (at 3.56 eV) in the Ce2O3 layer after reaction (the uppermost spectrum in 2O3 [For obtaining a broad range of reduction of ceria layers on Cu(111) we developed a method based on physical vapor deposition of metallic Ce onto a stoichiometric CeO species ,49. We d Cu(111) ,49 as we Cu(111)  substrat2(111) by metallic Ce, LEED measurements reveal surface reconstructions in the reduced ceria that can be characterized as 1 \u00d7 1, (\u221a7 \u00d7 \u221a7) R19.1\u00b0, (3 \u00d7 3), and 4 \u00d7 4  R19.1\u00b0 reconstruction, the CeO1.67 phase for the (3 \u00d7 3) reconstruction, and the cubic bixbyite c-Ce2O3(111) phase for the (4 \u00d7 4) reconstruction  truction B. Beside2O3 thin films shown in 2 . However, the contraction of the lattice constant of ceria upon oxidation causes cracking of the ceria layer revealing up to 2% of the Cu substrate on reoxidized Ce2O3/Cu(111) samples .2(111) films of different thickness were grown on the Cu(111) substrate, typically at 520 K in 5 \u00d7 10\u22125 Pa of O2. The LEED diffraction pattern shows no contribution of Cu(111) spots for the film equivalent thickness above 2.5 ML. The resonance photoelectron spectroscopy at the Ce 3d\u21924f resonance has been used as an efficient tool for determining the surface stoichiometry of cerium oxide. Discontinuous CeO2(111) layers exhibit a higher concentration of defects than continuous layers grown at the same conditions. The 5 ML continuous films can be prepared with practically Ce3+ free surfaces; on the other hand concentration of Ce3+ site and oxygen vacancies can be tailored by combining growth at constant and variable temperature. We can obtain independent control of coverage and step density of the ceria layers on Cu(111) and prepare ceria layers with adjustable density of the surface steps.Heteroepitaxial ultra-thin CeO2 thin film on Cu(111) with deposited metallic Ce yields a highly ordered layer of cubic bixbyite c-Ce2O3(111). The surface structure of the layer corresponds to bulk-terminated c-Ce2O3(111). It contains ordered vacancy clusters, each consisting of four oxygen vacancies. The surface exhibits a very characteristic and sharp (4 \u00d7 4) LEED pattern relative to CeO2(111), allowing easy experimental identification. We suggest that the c-Ce2O3(111) film is a unique model experimental system for highly reduced ceria surfaces. It provides an atomically well-defined surface exposing exclusively Ce3+ ions and a high density of oxygen vacancies with a precisely defined environment. A stepwise reduction of CeO2(111) by metallic Ce leads to different surface reconstructions in the reduced ceria that can be characterized as (\u221a7 \u00d7 \u221a7) R19.1\u00b0 and (3 \u00d7 3) surface structures corresponding to samples with ceria stoichiometry CeO1.71 and CeO1.67.It was shown that interfacial reaction of a stoichiometric CeOThe high degree of control of the basic morphological and chemical properties of thin film ceria on Cu(111) presented in this article make them versatile substrates for present and future model catalytic experiments revealing the most important information about the relations between the morphology, stoichiometry, electronic structure and chemical reactivity in ceria based catalysts."}
+{"text": "RDM16 and STA1 regulate differential usage of exon/intron in RNA directed DNA Methylation pathway (RdDM)  vs RDM16, WT vs STA1, and RDM16 vs STA1. Also we included the alignment of MORC6 protein to the ATPase-C family members that have conserved three ATP binding sites and conserved Mg2+ binding sites in the spliced exon.This article contains data on  Specifications TableValue of the data\u2022The data from article [5] shows the expression profiles of the genes that contain at least one alternative splicing event in different conditions. This information will be useful for other researchers to understand the regulation of gene expression by alternative splicing.\u2022MORC6 protein to the ATPase-C family simplifies the mechanism by which splicing factor RDM16 regulate the MORC6.Alignment of \u2022This data provides the information of the genes that are affected in RdDM pathway by knockdown of RDM16 and STA1 splicing factors. This data will help other researcher to validate the findings of the exon/intron level analysis in RdDM pathway.1vs RDM16, WT vs STA1, and RDM16 vs STA1 respectively. The color key is given with MORC6 protein to the ATPase-C family members that have conserved three ATP binding sites at 8, 11 and 14th position of the alignment. There are few more ATP binding sites at 55\u201365, 104\u2013107, 123\u2013125, 166\u2013169 but may not be contributing in the ATP binding since co-factor binding site is only available in the protein sequence that is coded by exon4 in MORC6 (region highlighted in yellow).MORC6 protein to the ATPase-C family members that have conserved Mg2+ binding site at 11th position of the alignment. Highlighted (yellow color) query sequence shows the protein sequence that is coded by exon4 in MORC6. ASP (D) and ASN (N) are essential amino acid for Mg2+ binding but do not contribute in it 2GSE44635. The alignment of the reads were done using TopHat2 pipeline via featurecount function in Rsubread package MORC6 protein to ATPase-C family members was done using ClustalX software [3]The experiment contains RNA-Seq samples in three conditions; WT (wild type), mutant RDM16 and STA1. The raw data were downloaded from Gene Expression Omnibus (GEO) with accession number Color key used in expression profiles of genes in different contrasts ."}
+{"text": "The dysfunction of neuregulin 1 (NRG1) is one of the plausible hypotheses for the pathogenesis of schizophrenia. The neuregulin 1 (NRG1) is located on chromosome 8p, as suggested by multiple linkage studies. The aim of this study is to clarify the contribution of polymorphisms of the neuregulin 1 (NRG1) with schizophreniaAfter informed consent was obtained, 100 schizophrenia patients and 100 control subjects were enrolled in this study. All subjects were administered the Diagnostic Interview for Genetic Studies (DIGS)  by a research assistant with extensive training in this interview. Blood samples were collected in anonymously identified 10-ml Vacutainer tubes (Becton Dickinson). DNA was prepared by a modified SDS/Proteinase K procedure . We genotyped polymorphism neuregulin 1 (NRG1) with the PCR-RFLP methods. The PCR products were digested by restricted enzyme.We observed a significant association between the polymorphism neuregulin 1 (NRG1) and the schizophrenia (Chi-Square Test P= 0.0449).The NRG1gene was originally identified as a susceptibility gene for schizophrenia by using a combination of a linkage and association approaches based on microsatellite markers and then using SNPs after microsatellite at risk haplotypes were identified. We found there is the frequency of the polymorphism of neuregulin 1 (NRG1) was significantly increased in schizophrenia patients. This allelic association suggests that the functional polymorphism neuregulin 1 (NRG1) may play a role in susceptibility to schizophrenia. Further study with larger sample sizes is required."}
+{"text": "In this paper, stress wave behaviors of two-dimensional buckyball (C60) lattice are investigated based on square close packing and hexagonal close packing. We show that the square close packed system supports highly directional Nesterenko solitary waves along initially excited chains and hexagonal close packed system tends to distribute the impulse and dissipates impact exponentially. Results of numerical calculations based on a two-dimensional nonlinear spring model are in a good agreement with the results of molecular dynamics simulations. This work enhances the understanding of wave properties and allows manipulations of nanoscale lattice and novel design of shock mitigation and nanoscale energy harvesting devices.Orderly arrayed granular crystals exhibit extraordinary capability to tune stress wave propagation. Granular system of higher dimension renders many more stress wave patterns, showing its great potential for physical and engineering applications. At nanoscale, one-dimensionally arranged buckyball (C We have established a semi-empirical nonlinear spring (NS) model to accurately describe nanoscale solitary waves at low temperature (10\u2009K), as a simplification of intermolecular interaction as well as an analogue to Nesterenko solitary wave. Substituting buckyballs with carbon nanotubes , we have demonstrated that 1D single-walled carbon nanotube (SWNT) system serves as a highly effective and reusable energy absorber60-SWNT hybrid system, having achieved a quantitative tuning of nanoscale solitary waves with good precisionGranular materials, due to their intrinsic discreteness, randomness and interaction diversity, exhibit a wide range of distinguished physical phenomena in mechanics, electromagnetism as well as quantum mechanics from macroscale to nanoscale123456891011121314151619202122232627282930313233343536373839404143444546474849505152535455565758scp) and the hexagonal close packing (hcp) system. In Sec. 3, we put forward a theoretical model to describe 2D problem. In Sec. 4, we present the results of MD simulations and numerical calculations based on the 2D NS model of the two packing modes. Finally, in Sec. 5, we present some concluding remarks and possibilities for future investigations.In this study, we investigate stress wave behaviors in 2D buckyball system via molecular dynamics (MD) simulation, as an extension of our previous work on 1D nanoscale lattice. As coherent macroscale studies suggest, 2D constructions render more types of wave patterns, suggesting a very promising potential for stress wave-related applications. The presentation of our work is structured as follows. In Sec. 2, we first introduce the setup of investigated system, including the square close packing , as is illustrated in 60 molecules equals to the equilibrium spacing d0\u2009=\u200910.05\u01fa60 molecule on one edge away from the corners is selected as an impactor to generate a stress wave with an initial velocity of 2000\u2009m/s, a moderate impacting velocity for studying nanoscale impact dynamics. The impacting direction is determined by an impacting angle \u03b8, defined as the angle between the velocity and the y axis  for 3\u2009ps, which is a sufficient time duration in this study  is the potential function of two interacting C60 molecule with respect to distance r; ui is the displacement vector from the initial position of ith C60 molecule; eij is the unit vector pointing from ith C60 molecule to jth C60 molecule.where scp and hcp arrangements all interactions of a C60 molecule with 8 surrounding molecules are taken into consideration  particle velocity magnitude vM distributions at given instants; (b) particle velocity amplitude vAmp distributions; (c) comparison between MD simulation results and numerical calculation results, i.e. solutions of scp and hcp configurations.We choose two impacting angles to study the impact responses of nanoscale lattice, i.e. scp configuration under two different impacting angles are presented in Particle velocity magnitude distributions at given instants and particle velocity amplitude distributions of scp system is highly directional where the major portion of wave energy transmits through initially excited chains. In fact, waves traveling in initially excited chains are solitary waves, whose shape and wave speed remain constant as they travel . For buckyball system, Ln\u22483a, consistent with the simulation results. It is smaller than 5a in Hertzian chains with very small initial compression due to higher nonlinearity. Above analysis further confirms that this solitary wave obeys Nesterenko\u2019s theory.where hcp configuration under two different impacting angles are presented in Particle velocity magnitude distributions at given instants and particle velocity amplitude distributions of hcp configuration tends to be directional, the amplitude decays as the wave travels and the input energy tends to be distributed. The reason is that in hcp system adjacent row and column will be affected by moving particles regardless of their velocity directions and thus energy is distributed to two or more surrounding particles. This pattern will continue spreading to more and more particles and energy or particle velocity amplitude is gradually decreasing. Therefore, hcp configuration of C60 molecules can serve as a nanoscale protective device.As can be seen, although stress wave propagation in scp system is shown in The comparison between MD simulations and numerical calculations of 60 molecules and it is not surprising due to the atoms vibrating about the equilibrium positions instead of being absolutely static after stress wave passes through. Therefore, the position or time of a C60 molecule when it interacts with other surrounding molecules is actually random. In addition, the parameters of NS model are obtained in 1D system where two degrees of freedom are eliminated, which may not be utterly suitable for 2D situation in this study. Due to error accumulation, the precision of model prediction decreases as the length of the force chain increases, and therefore, precisely predicting the velocity or acceleration of C60 molecule with a complex stress wave path can be fairly difficult. This also explains why the predictions on scp configurations are usually more accurate than hcp configuration on the condition that traveling distances of the stress waves are similar.Although the agreement between the results of MD simulations and numerical calculations is generally good, it can be observed that there appears a little discrepancy between simulations and model predictions for the velocity and acceleration curves of some selected Chcp configuration, a 30\u00b0 observation line is defined .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function\u201d  [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access \u2013 the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study \u201cDeletion of  Specifications TableValue of the data\u2022IDO1, IDO2 or TDO2 gene.The experimental approach and data allow comparative evaluation of baseline nosepoking and licking behaviors as percentages of total corner visit frequency, and nosepoke or lick frequency in every visit, of mice with or without a functional \u2022IDO1, IDO2 or TDO2 genes.The data are valuable for understanding behavioral adaptation to the introduction of fixed drinking sessions (i.e. availability of water as a reward) by mice deficient in the \u2022The data make clear that the genetic background of different mice may influence adaptative behavior to the drinking sessions more than the gene modifications tested, emphasizing that studies with GKO mice must employ WT mice of the identical strain.\u2022It is shown that mice deficient in IDO1, IDO2 or TDO2 enzymes exhibit different cognitive changes compared to their WT equivalents, which is relevant to understanding the role of the kynurenine pathway of tryptophan metabolism in behavioral adaptation.1IDO1 (IDO2 (TDO2 (IDO1 mice (TDO2 (The current data show distinct behaviors of WT and GKO mice deficient in the IDO1 , Fig. 7,O1 (IDO2 , Fig. 8 O2 (TDO2 , Fig. 9 DO1 mice  as well ce (TDO2  mice to ce (TDO2 , to provce (TDO2 .2The data involve the same set of animals and methods as described"}
+{"text": "The changes in serum prostate-specific antigen (PSA) concentrations can be used as a prognostic factor in patients undergoing maximum androgen blockage for metastatic prostate cancer. A total of 149 patients followed in our department were classified into 4 groups on the basis of PSA changes: group 1, those with normalisation of PSA levels within the first 3 months; group 2, those with normalisation of PSA between months 3 and 6; group 3, those with a decrease in PSA, but not reaching normal range; group 4, those with no decrease. The gleason scores and the number of bone metastases were also compared between the groups. Again time to progression in patients with Gleason scores 5-7 (grade 2) and over 7 (grade 3) whose PSA levels decreased between first and 3rd months (mean 21.2 months) were significantly longer than the patients with same gleason scores whose PSA levels decreased between 3rd and 6th months (mean 13.4 months) (p < 0.001). The decrease in PSA level is more important than gleason scores in determining the time to progression. Early normalisation of PSA delays the time to progression and when combined with gleason scores, PSA is an important prognostic factor in predicting the success of the therapy."}
+{"text": "However, only two scaffolds containing single separate prmt1 exon with high GC contents are present in the current chicken genome assembly. Besides, prmt1 exons are scattered in separate small scaffolds in most avian species. Complete prmt1 gene has only been predicted from two falcon species with few neighboring genes. Crocodilians are considered close to the common ancestor shared by crocodilians and birds. The gene arrangements around prmt1 in American alligator are different from that in birds but are largely conserved in human. Orthologues of genes in a large segment of human chromosomal 19 around PRMT1 are missing or not assigned to the current chicken chromosomes. In comparison, prmt8, the prmt1 paralogue, is on chicken chromosome 1 with the gene arrangements downstream of prmt8 highly conserved in birds, crocodilians, and human. However, the ones upstream vary greatly in birds. Biochemically, we found that though prmt1 transcripts were detected, limited or none PRMT1 protein was present in chicken tissues. Moreover, a much higher level of PRMT8 protein was detected in chicken brain than in mouse brain. While PRMT8 is brain specific in other vertebrate species studied, low level of PRMT8 was present in chicken but not mouse liver and muscle. We also showed that the ADMA level in chicken was similar to that in mouse. This study provides the critical information of chicken PRMT1 and PRMT8 for future analyses of the function of protein arginine methyltransferases in birds.Nine protein arginine methyltransferases (PRMTs) are conserved in mammals and fish. Among these, PRMT1 is the major type I PRMT for asymmetric dimethylarginine (ADMA) formation and is the most conserved and widely distributed one. Two chicken  NG, NG dimethylarginine (ADMA) belong to type I  for protein extract preparation. The protein concentration in each fraction was determined using the BCA reagent (Pierce) with bovine serum albumin as the standard protein.prmt1 forward (5' GGAGCTGCCCGTGGAGAA 3') and mouse prmt1 reverse (5' GAGAAGCCGGTCCTCTTGTG 3\u2019), mouse prmt8 forward . First strand cDNA was synthesized from 3 \u03bcg of total RNA by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed with the primer set: mouse AGCAAGTGGTGACCAATGCCTG 3') and mouse prmt8 reverse (5' GGACATAGTCGTTGCGCTGGAT 3'), chicken prmt1-1 forward (5' GGAGATGCTGAAGGATGAGG 3') and chicken prmt1-1 reverse (5' GACGATCTTGACGGCGTAAT 3'), chicken prmt1-2 forward (5' GGCGAACTGCATCATGGA 3') and chicken prmt1-2 reverse (5' TTGGCAGCAAACATGCATAG 3'), chicken prmt8 forward (5' TCCCAGACTCCTCAGCCTAC 3') and chicken prmt8 reverse (5' AGGATTCCTGTGCCACTTCC 3'). The cDNA of glyceraldehyde 3-phosphate dehydrogenase (gapdh) was amplified with the primer set: mouse gapdh forward (5' AACTTTGGCATTGTGGAAGG 3') and mouse gapdh reverse (5' ACACATTGGGGGTAGGAACA 3'), and chicken gapdh forward (5' GTGAAAGTCGGAGTCAACGG 3') and chicken gapdh reverse (5' ACAGTGCCCTTGAAGTGTCC 3') as controls. Amplified cDNA products were resolved by agarose electrophoresis and visualized by ethidium bromide staining.Nuclei were collected by centrifugation at 17, 530 xg at 4\u00b0C for 20 min. Histones were acid extracted by shaking in 0.2 M sulfuric acid for 1 h at 4\u00b0C. After centrifugation, histones were precipitated with ethanol at \u221220\u00b0C overnight, washed once with ethanol, and resuspended in distilled water.2, 1 mM PMSF, 1 mg/ml aprotinin, 1 \u03bcg/ml pepstatin, 1 \u03bcg/ml leupeptin) and homogenized with a Teflon pestle with about 10 strokes. The fractionation scheme was illustrated in Brain washed with PBS was cut into small slices, re-suspended in 3\u00d7 (vol/wt) homogenization buffer . The membranes were blocked with 5\u20137% skimmed milk powder in TTBS  for 30 min, incubated with primary antibodies  from Millipore/Upstate or 1:1000 for anti-PRMT1 from Abcam , 1:2000 for anti-PRMT8 from Millipore or from Abiocode; 1:10000 for anti-PRMT3 and 1:2000 for anti-FMRP from abcam; 1:500 for anti-hnRNP A2/B1 and 1:2000 for anti-GAPDH from Santa Cruz) overnight, washed three times in TTBS, then incubated with secondary antibody for 1 h. Chemiluminescent detection was performed using the Supersignal kit (Pierce) or VisGlow Chemiluminescent Substrate, HRP .prmt1 genes are present in peregrine falcon  and saker falcon , whose genomes are assembled with more than 100-fold coverage and have about 16,200 genes predicted [prmt1 orthologue is present in chicken but missed in the current chicken genome assembly. We then used the human or falcon prmt1 as the query to conduct nucleotide BLAST against the chicken or avian NCBI nr database. Even though there were few target hits, among them were two chicken cDNA entries (CR524297 and BX930603). We further assembled full-length chicken cDNA sequences with available EST and TSA sequences to fill the ends. The predicted sequences are consistent with two splicing variants with or without an alternative exon at the 5\u2019 region between the 1st and 2nd constitutive exons  could detect signals in brain, liver, and muscle. Besides, with the primer set encompassing the alternative exon (prmt1-2), two RT-PCR products corresponding to the two splicing variants could be detected . A T/C single nucleotide polymorphism is present at nucleotide from the ATG start codon. We also used the chicken prmt1 cDNA sequence to conduct nucleotide BLAST of the chicken genome. In chicken, two hits corresponding to exon 1 (chrUn_Scaffold23446) and exon 8 (chrUn_Scaffold23978) were identified from the newly released Gallus_gallus-5.0. They are separate small scaffolds (2017 bp and 1863 bp) of unknown chromosomes with high GC content (0.7297 and 0.6334) and long GC stretches.We then designed primers according to the assembled sequences to amplify the whole coding region of prmt1 cDNA as the query to BLAST avian genomes to detect more avian prmt1 genes. Forty-three hits from twenty-two avian species could be identified with the default parameters. The identities of the nucleotide sequences are mostly higher than 90%. The only target with the sequence identity lower than 85%  is from prmt8. Of the rest forty-two targets, thirty correspond to a single exon of prmt1 and twenty-one are in scaffolds shorter than 1000 bp, indicating that they are from poorly assembled chromosomal fragments. Among the hits, 17 contain exon 8 and none contain exon 4, the smallest constitutive exon  was detected in common canary Serinus canaria. Though exon 3, 4 and 6 may be missed in the scaffold, partial sequences of exon 3 can be identified in another scaffold. We then manually assembled the canary prmt1 based on these sequence. We also assembled and re-annotated the prmt1 from four scaffolds  from American crow Corvus brachyrhynchos and three scaffolds from Tibetan ground tit Pseudopodoces humilis  together with available predicted mRNA sequences , a crocodilian with the best assembled genome, to trace the syntenic block arrangement in the common ancestors of archosaurs. Fifteen genes are in the same assembly with prmt1 in alligator as shown in rcn3 is a direct prmt1 downstream neighbor with the same opposite direction in falcons.As indicated in the previous sections, most of the avian ng prmt1 . We thenPRMT1 is on chromosome 19, the chromosome that has the highest gene density and contains the majority of the missing blocks in the assembled avian genomes. The prmt1 gene in alligator clusters with seven orthologous genes directly upstream of human PRMT1 in the NOSIP-PRRG2-PRR12-RRAS-SCAF1-IRF3-BCL2L12-PRMT1 order , another model reptile. Most of the orthologues of human PRMT1 neighbors are present on chromosome 6 of green anole. The upstream genes nosip-prrg2-prr12-rras-scaf1-irf3 are linked and five downstream genes cpt1c-tsks-ap2A-fuz-med25 are clustered together as in human  with tbc1d17, its human orthologue is a few genes downstream of the cpt1c-tsks-ap2A-fuz-med25 cluster. The bcl2L12 gene connected with prmt1 in both human and alligator is located upstream of the rcn3 and med25 clusters in near proximity in green anole.Missing chicken genes were reported to be clustered in syntenic blocks in lizard and human. To follow the possible chromosome rearrangements near in human . The rcnprmt8, the vertebrate paralogous gene of prmt1. From \u201cgene\u201d search in PubMed, thirty-one prmt8 genes are annotated in birds and the prmt8 gene is assigned to chromosome 1 in five different species including chicken, Japanese quail (Coturnix japonica), Great Tit (Parus major), turkey  and zebra finch (Taeniopygia guttata). The avian PRMT8s also share very high sequence identities (~99%). Basically, the prmt8 genes are in well assembled segments in avian genomes and the arrangements can be obtained in numerous species. We illustrated the syntenic gene arrangement around prmt8 in chicken  that could well recognize zebrafish PRMT1 as shown in our previous study , detecteFor different tissues, the amount of GAPDH and beta-actin varies as detected by the antibody and thus were difficult to be used as loading controls. Nevertheless, coomassie blue staining showed the similar amount of protein loading of the chicken, mouse or zebrafish samples. The same tissue showed similar protein expression patterns whether they were from chicken or mouse .For PRMT8, the western blot results were consistent with the RT-PCR results. Interestingly, the PRMT8 level in chicken brain was much higher than that in mouse brain. Besides, weak PRMT8 protein signals were detected in chicken liver and muscle . In mousSince PRMT1 is the major type I PRMT in other vertebrates but the expression level of chicken PRMT1 is low as shown in the previous section, we analyzed the levels of asymmetric dimethylarginine (ADMA) containing proteins formed by the catalysis of type I PRMTs in chicken. The levels of ADMA-containing proteins in chicken were close to that in mouse as detected by western blot analyses with ADMA-specific antibodies . For braBirds retain type II and type III PRMTs catalyzing the formation of symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). In comparison, we also analyzed the expression pattern of SDMA and MMA containing proteins with specific antibodies . In geneSpecifically, an intensive signal of the molecular mass around 20 kDa was detected in chicken but not mouse brain by all three methylarginine-specific antibodies . A majorWe further detected the arginine methylation status of specific PRMT1 substrates in chicken. PRMT1 is an epigenetic modifier catalyzing the methylation of the arginine 3 (R3) residue of histone H4 to form H4R3Me2a . We prepin vitro methylation reactions, the methylation signals catalyzed by the tissue extracts were very low, with brain extract contained the highest PRMT activity. In case that there were inhibitors or certain factors in the total extracts that might interfere the assays, we further used a scheme similar to that had been used to fractionate porcine brain [We would like to determine the PRMT activities in chicken. By ne brain  to fractWe also compared the distribution of PRMTs in the fractions . PRMT1 sprmt3 transcripts in both chicken and mouse and observed that high expression level is high in brain and low in liver and muscle. We also detected the expression of PRMT3 in these brain fractions. PRMT3 in mouse and chicken was mainly detected in the S4 fraction. The result is consistent with the reports that PRMT3 exists only in the cytosol of mammalian cells [We had examined the levels of an cells  and methan cells . The disWe finally detected some markers to verify the fractionation. Fragile X mental retardation protein (FMRP) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) showed the highest expression in S4. Besides, histone H3 was only detected in the P1 fraction, consistent with the nuclear distribution. These results confirmed the successful fractionation of the brain extracts.prmt1 gene encoding the most conserved PRMT in birds. At first, we considered that this gene is missing in birds because we could not identify the gene in chicken genome, the earliest assembled and annotated avian genome assembly. In a previous study of conserved syntenic clusters of protein coding genes missing in birds, a block with genes just downstream of human PRMT1 was recorded as a deletion group, and some genes just upstream of PRMT1 were listed as missing genes in chicken in close proximity to missing syntenic blocks [prmt1 was deleted in chicken. However, there are reports that genes previously considered missing in birds have been identified in avian species especially the recently sequenced and assembled genomes of saker falcon, perigrine falcon and ground tit [prmt1 gene was annotated in two falcons. We thus carefully examined the avian and chicken nucleotide databases and identified a few cDNA and EST targets encoding PRMT1. We assembled the sequences and predicted two chicken prmt1 variants with high sequence identities with the human and alligator PRMT1. RT-PCR analyses confirmed the chicken prmt1 expression. Besides, we identified two separate small contigs for separate prmt1 exons, both with high GC contents. It is consistent with the study by Hron et al that a subset of avian genes with high GC content and long GC stretches are underrepresented in genomic databases and thus are hidden but not missing in birds [prmt1 gene is present in chicken but genomic fragments containing the gene were poorly recovered and assembled.In this study, we focused on the c blocks . We thusound tit . Indeed,in birds . Thus thprmt1 gene is fragmented and only single and few exons are present in short scaffolds in most avian genomes. We also tried to assemble prmt1 in other avian species with the limited sequences available. Extremely high sequence identities within avian PRMT1s and with human or alligator PRMTs indicate that PRMT1 is highly conserved in amnions.Further BLAST analyses against avian genomes in the databases showed that the prmt1 in birds , reptiles  and mammals (human). Only the rcn3 gene linked to prmt1 in alligator is conserved in falcon. On the other hand, the gene arrangements near prmt1 in alligator is very close to that in human. A syntenic block of eight genes upstream and another syntenic block containing five genes downstream of PRMT1 are conserved in human and alligator. In human, the RCN3 gene downstream of PRMT1 in alligator moved upstream of the NOSIP-PRMT1 block and flipped its direction. Interestingly, most of the orthologues of alligator and human PRMT1 neighbors are present on chromosome 6 in green anole, but the prmt1 gene is assigned to a separate long contig. Besides, the bcl2l12 gene connected with prmt1 in both human and alligator is located upstream of the rcn3 and med25 clusters in near proximity in green anole. Thus the rcn3 and bcl2l12 genes together with prmt1 are the major breakpoints for chromosome translocations through evolution from the common ancestor to birds, mammals, and green anoles.The slowly evolved crocodilians are proposed to be close to the common ancestor of the archosaurs group containing crocodilians, birds and the extinct dinosaurs. We compared gene arrangements neighboring RCN3 remained in the missing gene list [rcn3 exists in falcons just behind prmt1, thus is present in birds. Though the missing gene list in chicken is shrinking, it is apparent that genes near human PRMT1 in a large syntenic block on human chromosome 19 are underrepresented in chicken and many avian genomes currently. Human chromosome 19 has the highest gene density with 1,461 protein-coding genes and 321 pseudogenes [In the recent Gallus_gallus-5.0 genome assembly with 1911 protein coding genes added, some human PRMT1 neighbors previously consider missing  have beeene list . Neverthudogenes . An attrPRMT1, CARM1 (PRMT4) is also on human chromosome 19. PRMT1 and CARM1 locate on chromosome 19q13.3 and 19p13.2 respectively and both locations are reported to have syntenic blocks deleted in birds [carm1 can be predicted in eagle with correct synteny but does not BLAST align to chicken [carm1 is predicted in 50 different avian species but no in Galliformes. Nevertheless, we could amplify chicken carm1 cDNA by RT-PCR with the primer sequences conserved in other avian species. Thus carm1 is not missing in chicken and its expression and function in birds require further analyses.Besides in birds . CARM1 iin birds . As repo chicken . From geprmt1 paralogous gene prmt8. Unlike prmt1 with no chromosomal assignment, prmt8 is well assembled in alligator and some avian species. The gene is located on chicken chromosome 1 with neighbors annotated. Genes neighboring prmt8 in alligator are in a similar arrangement in a syntenic block downstream of prmt8 in the avian species we examined. However, the direct neighboring genes upstream of prmt8 are completely different in different avian species and alligator  in different avian lineages in the recent 150 million years. In contrast, their neighbors and arrangements between crocodiles and human are highly conserved since 320 MYA.The amniotes divided into the ancestral lineages of mammals and reptiles about 320 million years ago (MYA). Archosaurs containing crocodilians and birds is one of the two major clades in reptiles diverged about 280 MYA. Birds originated from a theropod lineage more than 150 MYA . As alliprmt1, why and how prmt1 is regulated at the posttranscriptional or translational level requires further experiments.We conducted RT-PCR and western blot analyses to confirm the expression of PRMT1and PRMT8 in chicken as well as to compare the PRMT expression in chicken and mouse. To our surprise, it appears that the expressed protein level of PRMT1 in chicken is lower than that in mouse, but the level of PRMT8 is higher than that in mouse. While PRMT1 is the major type I PRMT in almost all vertebrate species, in chicken we could barely detect PRMT1 protein expression in the tissues we examined. The antibodies we used could recognize zebrafish PRMT1. Compared with chicken PRMT1, zebrafish PRMT1 is less conserved with human PRMT1 that is the immunogen of the antibodies. It is thus unlikely that the weak/none chicken PRMT1 signals was due to poor antibody recognition of chicken PRMT1. Because we could detect the mRNA expression of chicken in vitro methylation analyses revealed that fractions containing the highest type I PRMTs activity in mouse and chicken were not in accord with any PRMT expression patterns we examined. Though GAR with glycine and arginine rich sequences from human fibrillarin protein is a typical type I PRMT substrates, we could not exclude the possibility that type II and III PRMTs might still recognize the protein and catalyzed the reaction.Previous analyses of PRMT1 knockdown/knockout animals or cells all showed a significant decrease of ADMA-containing proteins as detected by western blot analyses using ADMA-specific antibodies , 34, 35.For other PRMT to complement low PRMT1 levels in chicken, it has to function in the same subcellular compartments as other vertebrate PRMT1. Fractionation of the brain extracts from either chicken or mouse showed highly concentrated PRMT3 in a specific fraction consistent with the previous reports of cytoplasmic distribution of PRMT3  with theprmt8 RNA, as well as protein signals, could be detected in chicken liver and muscle, indicating putative de-repression of prmt8 expression in these chicken tissues. PRMT8 had been shown to methylate histone H4 in vitro [PRMT8 is a minor brain-specific paralogue of PRMT1 in other vertebrates . Howeverin vitro , 36. Thoin vitro , there win vitro , 38. PRMin vitro  and had in vitro . Thus itin vitro . We coulprmt1 and showed that its coding sequence is highly conserved with that of other amniotic PRMT1. However, the chromosomal localization of chicken prmt1 remained undetermined and the PRMT1 protein expression level we detected was low. It is not known why chicken maintained the prmt1 gene but expressed a low level of this critical PRMT. Instead, prmt8 that is on chromosome 1 in chicken expressed at a high level. Chicken thus appears to be a unique natural system with prmt1 knocked down and PRMT8 overexpression. Our studies provide the basic bioinformatic as well as biochemical information for further investigation in birds to elucidate the critical functions of protein arginine methylation conserved and not conserved in vertebrates.In summery, we determined the cDNA sequence of chicken S1 Table(XLSX)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 Fileprmt1 from chicken, ground tit, saker falcon, peregrine falcon, canary and crow are aligned with human and alligator PRMT1 by ClustalW.The predicted protein-coding sequences of assembled (PDF)Click here for additional data file.S1 FigExpression of PRMT1 protein in chicken and mouse tissues analyzed by western blots analysis. Proteins were prepared from chicken and mouse liver, muscle and brain tissues. For PRMT1 analyses, chicken tissue extracts (75 \u03bcg) and mouse tissue extracts (25 \u03bcg) were examined using anti-PRMT1 (Millipore/Upstate 07\u2013404).(TIF)Click here for additional data file.S2 Fig(A) Fractionation of the brain was conducted as described in . Basical(TIF)Click here for additional data file."}
+{"text": "Platelet activation and aggregation are key elements in the development of coronary atherosclerosis. Recent studies have shown that the two polymorphisms of platelet ADP receptor P2RY12 (haplotypes H2 and 34T) are associated with increased platelet aggregation and atherothrombotic risk. It was shown that these polymorphisms promote reduced body response to antiplatelet therapy.We investigated the association of P2RY12 gene polymorphisms with aspirin resistance in patients with coronary artery disease (CAD).This case-control study included 100 cases with CAD (mean age 57.6 \u00b1 2.8 years) treated in the cardiology department of the city hospital Semey, Kazakhstan, 90 of whom suffered from myocardial infarction. The control group (n = 100) were healthy people without a history of CAD, matched on sex and age. Genotyping of polymorphisms H1/H2 in P2RY12 gene was performed by PCR. Statistical analysis was performed using SPSS v.19.0.p = 0.0036 and p = 0.0001 in cases and controls, respectively). Genotype H2 was associated with risk of CAD with aspirin resistance . We found significant differences in the distribution of the mutant genotype H2 between CAD patients with aspirin resistance and healthy controls .The distribution of H1/H2 genotypes P2RY12 was 42%, 34%, and 24%, respectively, in cases and 42%, 58%, and 0%, respectively, in controls. All allele frequencies were consistent with the Hardy Weinberg equilibrium (We found an association of H2 haplotype in P2RY12 gene with aspirin resistance in patients with CAD. However, in order to obtain definitive conclusions about the role of genetic variants with the development of aspirin resistance in patients with CAD, there is a need for further research with a larger sample size as well as the use of selective thromboxane receptor antagonists for studying functional effects of genetic variants."}
+{"text": "Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. In this report, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the transport system. Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC). However, malignant cells frequently circumvent DFMO therapy by up-regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and a polyamine transport inhibitor (PTI) provide the basis for a combination therapy leading to effective intracellular polyamine depletion. We have previously shown that the pattern of uptake of a series of polyamine analogues in a  For both Ant444 (6) and Triamide444 (9) the EC50 values  were 10 to 15-fold lower than the concentration of Ant44  used in the assays. Maximum protection from Ant44 was observed at 10 \u03bcM 6 and 5 \u00b5M 9, respectively. These activity profiles are similar to Trimer44 (7) which is an effective transport inhibitor in mammalian Chinese Hamster Ovary (CHO) and L3.6pl cells (8) was a less effective PTI in the Drosophila model with an EC50 of 144 \u03bcM and gave maximum protection at 300 \u03bcM (Addition of Ant444 (nt44 (5) a,b. Theipl cells c 40]. I. I6) andt 300 \u03bcM d. These 5) on mammalian cells and Drosophila leg imaginal discs by competing for binding and transport via the PTS [5) were evaluated in Assay 1  and Triamide444 (9) are 3.6 \u03bcM and 2.8 \u03bcM, respectively (6 and 9 were approximately 12\u201315 times better than spermidine in inhibiting the toxicity of Ant44 (5).Compounds containing recognizable polyamine sequences should be able to compete for access to the polyamine receptor on the cell surface. Our previous work has shown that spermidine is able to inhibit the toxicity of Ant44  inhibition of imaginal disc development with an EC50 value of 19.7 \u03bcM and afforded complete protection at 40 \u03bcM  and Triamide444 (9) were 5-fold and 7-fold lower than spermine respectively, demonstrating that these compounds are more efficient at competing for access to the PTS than either of the native polyamines spermidine or spermine. The data for Ant444 (6) and Triamide444 (9) are similar to Trimer44 (7). In contrast, Triamide44 (8) . Spermine\u2014a native tetraamine\u2014was more effective than spermidine in blocking Ant44 (at 40 \u03bcM b. The EC5) in imaginal discs. Concentrations of up to 1 mM putrescine had no effect on the inhibition of imaginal disc development by Ant44 (5) (5), which is a triamine analogue. Therefore, the inability of putrescine to rescue cells from Ant44 (5) could be due to differences in relative binding affinity. An alternative interpretation is that Ant44 (5) is imported into the cell via a polyamine transporter which does not recognize putrescine. Indeed, the existence of multiple polyamine transporters with different affinities and selectivity for the native polyamines has been suggested in mammalian cells [In contrast to spermidine and spermine, the native diamine, putrescine, was unable to rescue the inhibition of Ant44 (nt44 (5) . One int6), Trimer44 (7) and Triamide444 (9) are all considerably more effective than either of the native polyamines spermidine or spermine in competing with Ant44 (5) for access to the PTS. Because putrescine could not rescue Ant44 (5) toxicity in disc development, no comparisons can be made for this native diamine.In conclusion, Ant444 (5), which accessed cells via the PTS (6), Trimer44 (7), Triamide44 (8) and Triamide444 (9) could prevent the rescue of DFMO-treated imaginal disc development by exogenous native polyamines. Essentially, we asked if these compounds could effectively compete with native polyamines for access to the PTS in DFMO-treated imaginal discs.Assay 1 tested the ability of candidate PTIs to block the toxicity of Ant44 . Data for these experiments are shown in Drosophila model assay were much higher (200\u2013500 \u03bcM) than those observed in mammalian cells (around 1 \u03bcM). The higher doses are likely due to the fact that unlike cell culture, imaginal discs are an intact epithelial tissue surrounded by extracellular matrix, which may impede polyamine access to the PTS.Each of the three native polyamines were evaluated for their ability to rescue the development of leg discs treated with DFMO. Addition of 500 \u03bcM putrescine to the culture medium resulted in a significant increase , Trimer44 (7) and Triamide444 (9) were each found to be non-toxic to imaginal disc development at 100 \u03bcM, whereas Triamide 44 (8) was non-toxic at 300 \u03bcM.As with Assay 1, it was important to use a non-toxic dose of each PTI compound because in Assay 2 a toxic PTI would generate a false positive. To avoid introducing this bias, non-toxic concentrations of the PTI compounds were determined and used in both assays. In a series of control experiments, Ant444 (6), Trimer44 (7) and Triamide444 (9) (6), Trimer44 (7) or Triamide444 (9) is significant, our data suggest that Trimer44 (7) is less effective than Ant444 (6) or Triamide444 (9) in inhibiting the uptake of putrescine. Consistent with earlier studies, 100 \u03bcM or 300 \u03bcM Triamide44 (8) was unable to compete with putrescine for access to the Drosophila PTS  a. Imagine444 (9) a. Likewie444 (9) a. Additie444 (9) a. While hila PTS a.6), Trimer44 (7) and Triamide444 (9) were all able to significantly inhibit import of spermidine. In the presence of 10 mM DFMO and 200 \u03bcM spermidine imaginal disc development decreased from 39% to 11% in the presence of 100 \u03bcM Ant444 and to 13% in the presence of 100 \u03bcM Trimer44 (8) failed to inhibit import of spermidine even at 300 \u03bcM  did not reduce uptake of spermine, whereas 100 \u03bcM Trimer44 (7) significantly reduced imaginal disc development from 67% to 34%. Triamide444 (9) showed even greater ability to reduce spermine uptake reducing imaginal disc development from 60% to 15% (8) was unable to inhibit import of spermine even at 300 \u03bcM concentration.Finally, we tested the ability of the PTIs to inhibit import of spermine in the presence of 10 mM DFMO and 200 \u03bcM spermine. As shown in % to 15% c. Thus, 6), Trimer44 (7) and Triamide444 (9) have similar EC50 values for protection against toxicity of Ant44 (5) and a similar concentration of full protection against Ant44 (5) (6) is better at blocking uptake of putrescine, Ant444 (6) and Trimer44 (7) show similar abilities to block uptake of spermidine and Triamide444 (9) is the most potent of the PTIs at blocking spermine uptake. These findings suggest that the PTIs have different specificities for the polyamine transport systems active in the presence of DFMO. In this regard, there may be a basal and DFMO-stimulated PTS in Drosophila. The basal PTS is assessed via the Ant44 assay (Assay 1), whereas the DFMO-stimulated PTS is assessed via Assay 2 (8) in these assays is consistent with the inability of this compound to block the toxicity of Ant44 (5)  a\u2013c, theynt44 (5) . Ant444  Assay 2 . The poont44 (5) d and sug6) and Trimer44 (7) are effective PTIs and showed different specificities towards putrescine, spermidine and spermine respectively, a combination of these inhibitors was used to block the rescue of DFMO treated leg discs by a mixture of all of the native polyamines. As shown in 6) or Trimer44 (7) alone was unable to significantly inhibit the rescue of DFMO-treated discs by the exogenous native polyamine cocktail. In contrast, a combination of 50 \u03bcM Ant444 (6) and 50 \u03bcM Trimer44 (7) significantly inhibited rescue by native polyamines even though the amount of each PTI was reduced by half compared to experiments when only one PTI was used. This result suggests that a combination of polyamine transport inhibitors will be more effective in inhibiting the import of all three native polyamines than individual inhibitors dosed alone. In the next experiments, we further examined our finding that the PTIs have different specificities for the PTS. Specifically, we asked if a cocktail of PTIs was more effective than individual PTIs in inhibiting rescue of DFMO-treated imaginal discs in the presence of all three native polyamines. In our prior experiments, we studied the effects of individual native polyamines, however, all three polyamines are present in vivo. For example in circulating red blood cells, the levels of putrescine, spermidine and spermine were found to be 3, 55 and 35 pmol/mg protein respectively . Because6), Trimer44 (7) and Triamide444 (9) towards native polyamines and the ability of a cocktail of PTIs to inhibit transport more effectively than individual PTI\u2019s suggests the existence of multiple polyamine transport systems in Drosophila as has been observed in unicellular organisms [6) shows a greater ability to inhibit uptake of putrescine, whereas Trimer44 (7) is more effective in inhibiting uptake of spermine (5)  , consistDrosophila imaginal disc assay as an early and inexpensive system in which to evaluate compounds targeting the mammalian PTS. There are several advantages to our approach. First, mammalian cell culture is not a natural cellular environment because cells lack cell-cell contacts and extracellular matrix, both of which are factors influencing drug accessibility to cells in vivo. In contrast, the imaginal disc assay tests the effects of medicinal compounds on cells in a more natural environment. Second, inexpensive early animal model testing of promising compounds can reduce the time it takes successful compounds to reach the clinic by up to fifty percent. Mice are more expensive to use in the early stages of drug development where most compounds will fail, therefore a cheaper system such as our Drosophila assay is useful. Third, experiments in mice can only be performed on a small scale, whereas we can assay relatively large numbers of imaginal discs, typically more than 100 per assay. Our work reinforces the value of the Drosophila and mammals. Our work suggests that this is the case. In previous work, we compared the uptake of nine polyamine analogs in mammalian CHO and L1210 cells and Drosophila imaginal discs [5) and N1-(3-aminopropyl)-N4-(anthracen-9-ylmethyl)butane-1,4-diamine (Ant43) gain entry to mammalian cells via the polyamine transport system as evidenced by spermidine competition experiments and greatly reduced uptake in CHO-MG cells, which lack a functional transport system [Drosophila imaginal discs, suggesting that they do not utilize the transport system to gain access to cells in either system. In addition, Trimer44 (7) has previously been shown to be an effective inhibitor in mammalian cells, whereas Triamide44 (8) was not [Drosophila assay. Of course, the imaginal disc assay is only useful to understand mammalian transport if the polyamine transport system is similar in al discs . Two of t system . In imag was not  and thesDrosophila imaginal disc assay has added to our knowledge of polyamine transport inhibitors. We show that two compounds that exhibit toxicity in mammalian cell culture, Ant444 (6) and Triamide444 (9), are non-toxic in the Drosophila assay and are effective PTIs with activity profiles similar to that of Trimer44 (7). The reduced toxicity of Ant444 and Triamide444 in Drosophila may due to a lower effective concentration of these compounds reaching the cell surface due to the presence of intact cell-cell adhesions and extracellular matrix. We also provide activity data for the PTIs against all three native polyamines  whereas most mammalian cell culture studies focus on spermidine uptake. This approach revealed differences in the ability of each PTI to inhibit uptake of individual polyamines suggesting the existence of multiple transport systems. This view is further reinforced by our finding that a mixture of two PTIs is more effective than either PTI alone at inhibiting uptake of a cocktail of all three native polyamines.Use of the i values for several of these compounds in terms of competing with 3H-radiolabeled spermidine for the putative cell surface receptors in L1210 murine leukemia cells. The L1210 Ki values for putrescine, spermidine and spermine are 208.2, 2.46 and 1.34 \u03bcM, respectively [i values for Ant44 (5), Ant444 (6) and Trimer44 (7) are 1.8 \u03bcM, 0.05 \u03bcM and 0.49 \u03bcM, respectively [i value of Triamide44 (8) was not determined in L1210 cells, a comparative study of the Trimer44 and Triamide44 compounds in human L3.6pl pancreatic cancer cells revealed Ki values of 36 nM and 398 nM, respectively [In this study, we assayed the ability of PTIs to inhibit the rescue of DFMO treated imaginal discs in the presence of exogenous polyamines. This approach is clinically relevant in that many tumors circumvent DFMO treatment via upregulation of their polyamine transport systems. Our previous work indicates that the PTIs inhibit polyamine uptake. Our data are consistent with the reported Kectively . The L12ectively ,43. Althectively , suggesti values of Ant44, Ant444 and Trimer44 suggest that these compounds compete with the native polyamines for uptake. For example, Ant44 (a triamine) has a L1210 Ki value of 1.8 \u03bcM and provides a fluorescent molecule with similar affinity for the polyamine transport system as the native polyamines spermidine (L1210 Ki = 2.46 \u03bcM) and spermine (L1210 Ki = 1.34 \u03bcM). We speculate that in order to be successfully imported, compounds must bind and release from the cell surface receptors. The Ki values of the native polyamines (spermidine and spermine) suggest that Ki values in the low \u03bcM range are optimal for these binding and releasing properties. The related Ant444 compound 6 (a tetraamine) has a significantly lower L1210 Ki value (51 nM) indicating high affinity for the cell surface receptors. Using confocal microscopy, we have demonstrated that this higher affinity of Ant444 was observed as a compound which could not be washed off the surface of L1210 cells by phosphate buffered saline (PBS). In contrast, the triamine Ant44 could be readily washed off the surface of L1210 cells by PBS and appeared to have improved uptake past the cell membrane [5:48 h L1210 IC50 = 0.3 \u03bcM) compared to Ant444 (6:48 h L1210 IC50 = 7.5 \u03bcM) [The low Kmembrane . This da 7.5 \u03bcM) . In summ6) and Triamide444 (9) are effective PTIs expands our understanding of the chemical rules governing an effective PTI design. Inhibitors presenting diamine arms, like Triamide44 (8), are ineffective transport inhibitors. In contrast, compounds containing a higher number of charges in their polyamine arms such as Trimer44 (7) and Triamide444 (9) are effective PTIs. In this regard, N1-substituted triamine and tetraamine analogues can be used to design efficient ligands and inhibitors of polyamine transport. Our work and previous studies suggest that presentation of at least three or more positive charges is necessary for efficient competitive binding to the PTS. Our finding that Ant444 , was recently shown to behave in a similar manner as AMXT-1501 (11) both in its ability to shrink tumors in vivo as well as to beneficially modulate the immune response [Drosophila model can be used to pre-screen PTIs prior to more expensive testing in mouse models. Having a cheap model system for early animal testing will reduce the time from conceptual PTI design to future validation in clinical trials. A combination therapy using DFMO and a PTI has shown promise in cancer growth inhibition ,49. Whil11, 12 , which iresponse . Thus, t"}
+{"text": "Toxoplasma gondii is an opportunistic protozoan apicomplexan and obligate intracellular parasite that infects a wide range of animals and humans. Rhoptry proteins 5 (ROP5), ROP16, ROP18 and dense granules 15 (GRA15) are the important effectors secreted by T. gondii which link to the strain virulence for mice and modulate the host\u2019s response to the parasite. Little has been known about these molecules as well as GRA3 in type Chinese 1 strains that show polymorphism among strains of archetypical genotypes. This study examined the genetic diversity of these effectors and its correlated virulence in mice among T. gondii isolates from China.I/III), and the other 4 as type II (ROP16II). The strains investigated may be divided into four groups based on GRA3 amino acid alignment, and all isolates of type Chinese 1 belong to the \u03bc-1 allele except Wh6 which is identical to type II strain.Twenty-one isolates from stray cats were detected, of which 15 belong to Chinese 1, and 6 to ToxoDB #205. Wh6 isolate, a Chinese 1 strain, has an avirulent phenotype. PCR-RFLP results of ROP5 and ROP18 presented few variations among the strains. Genotyping of GRA15 and ROP16 revealed that all the strains belong to type II allele except Xz7 which carries type I allele. ROP16 amino acid alignment at 503 locus demonstrated that 17 isolates are featured as type I or type III  contains supplementary material, which is available to authorized users. Toxoplasma gondii is an obligate intracellular protozoon that can infect a broad spectrum of vertebrate hosts including humans. The infection with T. gondii in domestic animals causes abortion and leads to great economic losses in livestock production. In humans, Toxoplasma infection usually does not lead to obvious clinical symptoms and signs [Toxoplasma infection, however, may become activated in immunocompromised individuals, causing severe or life-threatening disseminated toxoplasmosis such as encephalitis or lethargy [Toxoplasma infection [Toxoplasma infection [Toxoplasma infection [nd signs . Latent lethargy . Recent nfection  and morenfection . Long-tenfection . Additionfection .Toxoplasma contains only one species, Toxoplasma gondii. However, genotyping results of the strains collected from human and animals around the world show a rich genetic diversity [T. gondii genotypes varies greatly with geographical locations [T. gondii database (http://toxodb.org/toxo). In North America and Europe, T. gondii has three archetypal clonal lineages known as types I, II and III which exhibit remarkable phenotypic differences [ToxoDB#9) dominates in the ten types identified [In the biological taxonomy, the genus iversity  which poocations . So far ferences . Comparaentified \u201319, whicToxoplasma gondii has evolved a number of strategies to subvert its host\u2019s immune responses. Previous studies indicated that ROP16I/III carried in types I and III strains induces alternatively activated macrophage (M2) in host innate immunity to Toxoplasma infection, modulating host signaling pathways and producing virulence in mice [I, while all strains (100%) isolated from meat were avirulent for mice. Interestingly, we found that all the Chinese 1 isolates, including virulent Wh3 and avirulent Wh6 strains, shared the genes of GRA15II with type II strains and ROP16I/III with types I or III strains [rop16I/III deficient Wh3 strain (data unpublished) did not result in a remarkable attenuation of virulence in mice, suggesting that ROP16I/III in the parasite with GRA15II background is not closely associated in the virulence of Chinese 1 strains. in mice . Alvarez in mice  analyzed strains . We obseT. gondii identified the rhoptry kinase ROP18 and rhoptry pseudokinase ROP5 as virulence factors of the three archetypal clonal lineages. They function together to block innate immune mechanisms activated by IFN-\u03b3 in murine hosts [Previous genetic mapping of crosses between clonal type I, II, and III strains of ne hosts \u201326. Howene hosts  and playne hosts . Our prene hosts , suggestT. gondii in mice.Consequently, we analyzed the polymorphism of rhoptry proteins of ROP16, ROP18, ROP5 and dense granule proteins of GRA15 and GRA3 of type Chinese 1 strains to (i) explore the characteristics of the effectors that are associated with host immunity subversion and mouse virulence among the strains and (ii) to reveal the polymorphism-related pathogenesis of Female Swiss Webster (SW) mice (specific pathogen free) aged 6 to 8\u00a0weeks were obtained from the Biomedical Research Institute of Nanjing University, China. The mice were treated in compliance with the Care and Use of Laboratory Animals of the National Institutes of Health. Ethical permission was obtained from the Institutional Review Board of the Institute of Biomedicine at Anhui Medical University (Permit No. AMU26\u2013081108).T. gondii strains were extracted from the ascitic fluid of mice infected with each isolate using a QIAamp\u00ae DNA Mini kit  following the manufacturer\u2019s protocols. Genomic DNA stocks were stored at -80\u00a0\u00b0C until analyzed.Genomic DNA of the GRA15, ROP16 and ROP18 PCR-RFLP protocols were followed in accordance with a previous study . ROP5 alROP16 nucleotides 1424\u20131614  and the T. gondii strains was tested based on the previous mouse bioassay data. The virulence difference between Wh3 and Wh6 strains has been noted in long-term serial mouse passages. Here mice were infected with 1000 tachyzoites by intraperitoneal injection and observed for 30\u00a0days post-infection. Toxoplasma gondii strains that caused 0\u201329%, 30\u201379% and 80\u2013100% mortality of mice were considered non-virulent, intermediate and virulent, respectively [Virulence of We assessed the mouse mortality of type Chinese 1 and ToxoDB #205 strains in combination with the data of previous studies. Mice were intraperitoneally infected with 1000 tachyzoites and mouse survival was observed for 30\u00a0days post-infection. The results showed that all of these strains presented a virulent phenotype in mice except for the Wh6 isolate which has the only avirulent phenotype [rop18 allele 2 except for Xz7 strain which carries rop18 allele 1 .rop5 allele 7 was found to be a unique genotype which could not be digested by FspBI.ROP5 is the major determinant of acute virulence in mice and was found to have allele 5 in all Chinese 1 strains but allele 7 in ToxoDB#205 and allele 5 in Xz8 strains  of ROP16 and found that all strains of Chinese 1 and strains of Xz7 and Xz8 of ToxoDB #205 are characterized by carrying leucine at position 503 while Xz9, Xz37, Xz39 and Xz40 strains of ToxoDB#205 presented serine. Additionally, amino acid of ROP16 at position 486 in Chinese 1 and ToxoDB#205 strains are homologous to that of archetypal type II strains except for Xz7 and Xz8 strains that are identical to archetypal type I strain, as shown in Table\u00a0gra3 full length of nucleotides was amplified in the strains investigated. The coding sequences were aligned, followed by construction of the phylogenetic tree. We found that the sequences could be categorized into 5 types, the archetypal types I, II and III and \u03bc-1 and \u03bc-2 . Amino acid alignments revealed 96% or 99% positivities of ROP18 of the Chinese 1\u00a0Wh3 strain in comparison with that of GT1 or ME49, respectively, suggesting that sequence variations of ROP18 indeed exists but was not able to be identified by the PCR-RFLP analysis in the present study.T. gondii strains. The results revealed that the ROP18 and ROP5 gene allele types could be used to predict strain virulence in mice. ROP18 has 4 alleles; alleles 1 and 4 show association with a virulent phenotype, while allele 2 and 3 strains show a low virulence in mice [2 and ROP55 show a virulent phenotype in mice except the Wh6 strain. This result is inconsistent with the previous report using RFLP [2 with ROP182 and ROP17 [Shwab et al.  analyzed in mice . However in mice . Strainsing RFLP , suggestnd ROP17 , and/or II in type II strains mediates NF-kB nuclear translocation, drives macrophages towards a classically activated phenotype (M1) and promotes host innate immunity against Toxoplasma infection [I/III of types I and III has the ability to phosphorylate both STAT3 and STAT6 which results in the alternative activation of macrophages (M2), facilitating replication of parasites within the host cells [GRA15nfection . Meanwhist cells , 34, 35.II at position 503 in type II strains  is serine (ROP16 S503) which has no phosphorylation activity of STAT3/6 kinase. The types I and III strains , however, are featured with ROP16 L503 (ROP16I/III) instead of S503 (ROP16II). ROP16I/III is able to phosphorylate STAT3/6, drive macrophage to M2 polarization and help the parasite with its multiplication in macrophages [Toxoplasma infection. Interestingly, in the present study, we found that the amino acid residue of ROP16 at position 503 is leucine  in all of the isolates of type Chinese 1 and some isolates  of ToxoDB#205 identified in China mainland. Additionally, we noted that all of the strains of type Chinese 1 and some of ToxoDB#205, regardless of their mouse virulence, carry GRA15II, which is identical to GRA15II in type II strains [T. gondii that have both key effectors of ROP16I/III and GRA15II implicates the unique mechanism of the host immune response and pathogenesis in Chinese 1\u00a0T. gondii infection.Yamamoto et al.  have shorophages . This ma strains . The chaDense granule protein 3 (GRA3) is known to be secreted by the parasites after invasion and appears at the parasitophorous vacuole membrane (PVM) . It playToxoplasma [Furthermore, a more comprehensive analysis of type Chinese 1 strains is needed at the genomic DNA and transcriptome level. The development and application of CRISPR-Cas9 technology will enable us to precisely manipulate target genes to extend the power of reverse genetics in xoplasma .T. gondii from China. We demonstrated that the majority of those isolates are featured with the phenotypic ROP16I/III of type I and type III strains and GRA15II of type II strains. This strongly suggests that the characterized polymorphism of the crucial effectors of ROP16 and GRA15 in Chinese 1 strains may result in a significantly different outcome of Toxoplasma infection through the subversion of the host\u2019s innate immunity. Furthermore, the Wh6 strain contains type II GRA3 which is unique in Chinese 1 strains and may lead to phenotype differences. Additionally, the present results indicate that genotyping of the loci including ROP5 and ROP18 with a PCR-RFLP strategy that is globally used may be inadequate to predict the virulence of Chinese 1 isolates of T. gondii.The present study examined sequence variations of ROP5, ROP16, ROP18, GRA3 and GRA15 genes in 21 Chinese 1 and ToxoDB#205 isolates of Additional file 1: Figure S1.. Analyzing the full length of GRA15, the result shows that all Chinese 1 and ToxoDB #205 strains (but not Xz7 strain) were identical to the type II strain, which has\u00a0an 84 aa deletion from position 519 to 602. (TIFF 3957 kb)GRA15 translations alignmentAdditional file 2:GRA3 translations alignment. Aligning the full length of GRA3 translations of those strains, shows that Xz7 is the only one which has a longer length compared with the others. GRA3 of Wh6 and Xz8 strains are homologous to that of type II. The amino acid sequence from 131 to 180 is the mutation cluster region constituting a major part of all mutations. (TIFF 4457 kb)"}
+{"text": "Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a potent oncogene overexpressed in various solid and haematological tumors and its high expression levels are associated with decreased expression of several TSGs including p16INK4A, BRCA1, PPARG and KiSS1. Using its several functional domains, UHRF1 creates a strong coordinated dialogue between DNA methylation and histone post-translation modification changes causing the epigenetic silencing of TSGs which allows cancer cells to escape apoptosis. To ensure the silencing of TSGs during cell division, UHRF1 recruits several enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 to the right place at the right moment. Several in vitro and in vivo works have reported the direct implication of the epigenetic player UHRF1 in tumorigenesis through the repression of TSGs expression and suggested UHRF1 as a promising target for cancer treatment. This review describes the molecular mechanisms underlying UHRF1 regulation in cancer and discusses its importance as a therapeutic target to induce the reactivation of TSGs and subsequent apoptosis.Epigenetic silencing of tumor suppressor genes (TSGs) through DNA methylation and histone changes is a main hallmark of cancer.  N. N105]. promoter . We havepromoter . In accopromoter . EGCG  that targets the SRA domain with subsequent impact on DNMT1/UHRF1 interactions and decrease in global DNA methylation ."}
+{"text": "Kcnj10 mutations identified in both mouse and human cohorts. However, genetic analyses of epilepsy phenotypes in mice to date have been carried out as acute studies in seizure-naive animals or in Mendelian models of epilepsy, while humans with epilepsy have a history of recurrent seizures that also modify brain physiology. We have applied a repeated seizure model to a genetic reference population, following seizure susceptibility over a 36-d period. Initial differences in generalized seizure threshold among the Hybrid Mouse Diversity Panel (HMDP) were associated with a well-characterized seizure susceptibility locus found in mice: Seizure susceptibility 1. Remarkably, Szs1 influence diminished as subsequent induced seizures had diminishing latencies in certain HMDP strains. Administration of eight seizures, followed by an incubation period and an induced retest seizure, revealed novel associations within the calmodulin-binding transcription activator 1, Camta1. Using systems genetics, we have identified four candidate genes that are differentially expressed between seizure-sensitive and -resistant strains close to our novel Epileptogenesis susceptibility factor 1 (Esf1) locus that may act individually or as a coordinated response to the neuronal stress of seizures.Epilepsy has many causes and comorbidities affecting as many as 4% of people in their lifetime. Both idiopathic and symptomatic epilepsies are highly heritable, but genetic factors are difficult to characterize among humans due to complex disease etiologies. Rodent genetic studies have been critical to the discovery of seizure susceptibility loci, including  Epilepsies in humans are highly heritable syndromes that are modified by complex interactions between genes and the environment. In the US, \u223c2.2 million people will develop some form of epilepsy during their lifetime .Identification of gene by environment interactions for individuals with epilepsy is limited by complex disease etiologies and the inability to identify at risk populations prior to disease onset. Consequently, the majority of epilepsy genome-wide association studies (GWAS) carried out to date compare affected populations to seizure-naive controls rather than following epilepsy progression in a population from a seizure-naive to an epileptic state . ExperimPreclinical epilepsy models have implicated multiple gene mutations as contributors to seizure disorders. This success is due in part to strong genetic influences that segregate among rodent models of seizure susceptibility . UnfortuSzs1 locus  that may mitigate the effects of Szs1. This hypothesis is supported by interactions between Szs1 and our novel epileptogenesis susceptibility locus on chromosome 4; Epileptogenesis susceptibility factor 1 (Esf1). Systems genetics analysis of the Esf1 region has uncovered a coregulated set of expression QTL (eQTL) that have been implicated in neurological stress responses that are now also implicated in epileptogenesis.We have utilized a repeated-flurothyl-induced seizure model delivered to the Hybrid Mouse Diversity Panel (HMDP) to identify genetic susceptibility factors that modify epileptogenesis (n = 8); BTBR +Ltpr3tfT/J ; BXD#/TyJ ; BXD##/RwwJ ; C57BL/6J ; C57BL/10SNJ ; C57BL/10J ; C57BL/6NJ ; C57BLKS/J ; CAST/EiJ ; CBA/J ; CE/J ; DBA/2J ; FVB/NJ ; LG/J ; MRL/MpJ ; NOD/ShiLtJ ; NZW/LacJ ; PWD/PhJ , and Sm/J . Male and female mice were obtained from the Jackson Laboratories  and (1) were acclimated to the animal facility for 1 wk before seizure testing commenced, or (2) were further bred at the Wadsworth Center and exposed to the repeated-flurothyl seizure model. Mice were housed on a 12 hr light\u2013dark cycle with ad libitum access to food and water.The rationale for using the HMDP and its advantages with respect to statistical power and genetic resolution have been previously described (n = 150) from this cohort were genotyped with the Mouse Universal Genotyping Array  and mapping  was performed using R/qtl  ether diluted in 95% ethanol] infused via a syringe pump onto a gauze pad suspended at the top of the chamber at a rate of 100 \u03bcl/min . Once a P < 4.4 \u00d7 10\u22126  were isolated from 11- to 12-wk-old B6 and D2 seizure-naive or seizure-exposed mice 90 min after completion of the repeated-flurothyl model as in Brains .Actb ; Per3 ; Park7 ; Camta1 ; and Vamp3 . Primer sequences are available upon request or from the manufacturer (www.idtdna.com/site/order/qpcr/predesignedassay). Real-time 5\u00d7 HOT FirePol qPCR master mix purchased from Mango Biotechnology was used to perform PCR reactions according to the manufacturer\u2019s recommendations (Solis BioDyne) using a 7500 Fast Real-Time PCR system (Applied Biosystems). All qPCR reactions were quantified using the relative standard curve approach based on the manufacturer\u2019s recommendations. Candidate genes Camta1, Park7, Per3, and Vamp3 were normalized to \u03b2 actin prior to determining B6:D2 expression ratios that exceeded a P value <0.05 assuming unequal variance between the populations.Relative RNA expression levels were quantified using the following Primetime premade 5\u2032 nuclease assays from Integrated DNA Technologies: t-tests using JMP version 10.0 (SAS Institute). Groups were defined for interaction studies between Szs1 and Esf1 based on their genotype at single nucleotide polymorphisms (SNPs) rs8259388 for Szs1 and rs13478053 for Esf1. Correlational and eQTL mapping tools using BXD strains are freely available using GeneNetwork (http://www.genenetwork.org).Statistical analyses evaluating genetic interactions between GSTs by trial were carried out by either one-way ANOVA or Student\u2019s Esf1 were selected using the tools available on GeneNetwork. We initially used expression data from the UTHSC BXD Aged Hippocampus data generated from Affymetrix mouse gene 1.0 ST exon level arrays  for correlational analyses with retest GST values. Additional datasets used to confirm these analyses included GN72 provided by Genome Explorations Inc. for the NIAAA as part of an SBIR grant to Dr. David Patel; GN46 that was generated by the UTHSC-SJCRH cerebellum transcriptome profiling consortium; and published datasets GN206 and GN281  or the UTHSC Genome Browser (http://ucscbrowserbeta.genenetwork.org).Cis-eQTLs adjacent to http://www.genenetwork.org/).All seizure threshold data are available from the GeneNetwork website under accession numbers 18963\u201318981 , based on their average daily GST scores of 275 sec for the most sensitive groups (D2 and FVB mice) compared to 350 sec for the intermediate groups . We calculated the slope of induction phase GSTs  as a quantitative surrogate for the kindling effect seen previously (P < 0.0001) between strains . As shown in Calmodulin Binding Transcription Activator 1 (Camta1) gene  gene .Bis1, congenic analyses performed by others in conjunction with our mapping results exclude the retest association from overlap with this locus (Epileptogenesis susceptibility factor 1 (Esf1); a novel seizure susceptibility locus that is dependent on prior seizure exposure to manifest its effects.Chromosome 4 GWAS associations were detected at two distinct time points and locations during the repeated-flurothyl seizure model. We first detected a locus with maximal effect (\u2212log p 2.29 e\u221207) at rs32914632 near 138 Mb on chromosome 4 in the 6-d GST data , while rSzs1 and Esf1 effects in our model may indicate an interaction between these loci driven by repeated seizures. We tested for interdependency between the two genetic states by segregating BXD strains based on their genotype at Szs1  to compare respective GSTs at either end of the seizure protocol (GST1 and retest GST). As expected, the average response between B6 and D2 Szs1 groupings was significantly different for GST1 (P = 0.001), but not for retest GST , but not GST1 response. Notably, when the B6 and D2 Szs1 subsets were further divided based on their genotypes at Esf1, we only observed significant differences between the D2-Szs1/B6-Esf1 compared to the D2-Szs1/D2-Esf1 subset   were detected within four genes: Camta1, Period 3 (Per 3), Parkinson protein 7 (Park 7), and Vesicle Associated Membrane Protein 3 (Vamp3)  . These cfor Esf1 .cis-eQTL and Esf1, we correlated the expression values for each probe set with retest GST values. Most probe sets with LRS over 25 were significantly correlated (P < 0.05) with retest GST  were observed in two Camta1 probes present in exons 7 and 10 of Camta1 (To better define the relationship between these test GST . Exons felations . The mosf Camta1 .cis-eQTLs were found in other expression datasets using distinct arrays and brain tissues (cis-eQTL (selected based on a LRS score of 15 or greater within 2 Mb of a given locus) trended toward tissue-specific patterns. For example, while Camta1 and Park7 had cis-eQTL in hypothalamus  were observed between B6 and D2 hippocampus in the seizure-naive set for Camta1, Park7, and Per3, but not in cerebellar mRNA. In mice undergoing repeated-flurothyl seizures, there was an induction of Park7 and Vamp3 in B6 cerebellum that did not occur in D2 cerebellum. However, only Camta1 expression in hippocampus was significantly and differentially expressed between B6 and D2 mice after seizures.We confirmed the presence of differential expression between B6 and D2 inbred strains using qPCR. We compared control cerebellum and hippocampus from age-matched B6 and D2 animals as well as similar sets that had undergone the full flurothyl induction protocol, collecting tissue 90 min after the last seizure. As shown in This work demonstrates a novel preclinical approach to discover gene by environment interactions that mediate epileptogenesis. While rodent genetic studies have uncovered important seizure mechanisms among mammals  could influence Szs1 effects, either by suppression of an existing Szs1 mutation, like the reported Kcnj10 mutation, or via alterations in other genes in this region. Multiple QTL affecting a variety of neurological traits in mice are present on distal chromosome 1 (Szs1 effects on seizure susceptibility.While association of the Kcnj10) have been observed in both human temporal lobe epilepsy and experimental epilepsy models (r2 = \u22120.71) could be due to suppression of Kcnj10 activity in subsequent seizure trials. Reductions in Kcnj10 activity may be a common feature of repeated seizures that are mediated by uncharacterized mechanisms of epileptogenesis. In our model, suppressing both normal and mutant alleles of Kcnj10 as a means of seizure adaptation would consequently remove the Szs1 effects from associations after the initial seizure. The mechanisms responsible for these effects may be within the Szs1 interval or could be trans-acting factors that modify the effects of Szs1. While environmental confounds like animal age could contribute to seizure progression, all animals in our study were 6\u20137 wk old at the onset of the analysis. The presence of significantly different rates of kindling among HMDP strains suggests genetic factors mediate this activity within the HMDP. However, poor correlation between HMDP strain haplotypes within Szs1 and kindling in our data indicates that the uncharacterized kindling modifier and Szs1 are likely unlinked.Reductions in astroglial Kir4.1 channels , whose inhibition is reported to result in epileptiform activity (Significant SNP associations reappear on day 6 in the induction phases that correspond with the timing of the GST plateau effect seen in B6 mice (Esf1 is the predominant effect controlling seizure susceptibility with the top five associations all occurring in Camta1.Seizure-driven epileptogenesis is predicted to continue into the incubation phase of the repeated-flurothyl model due to a period of spontaneous seizures where B6 mice have as many as six seizures a day remitting to less than one seizure a day by the end of our model . When reKcnj10 segregating in the HMDP are associated with initial seizure effects that diminish over time, which may also be true in humans with epilepsy, raising the question of the nature of the association of Kcnj10 with epilepsy. A possibility is that the effect of Kcnj10 deficiency on seizure susceptibility is twofold. Its primary effect on hyper-excitability is mitigated by repeated seizures, but its effects on baseline K+ buffering in the interictal period remains abnormal. Such imbalances in K+ would be amplified in the context of repeated seizures leading to imbalances in downstream signaling cascades, in particular, Ca2+ signaling that has been shown to be altered by the presence of functional Kir4.1 channels (Complex networks of neuronal adaptation are activated by seizures (Szs1, which is likely due in part to deficits in Kcnj10, has a significant impact on retest GST when these mice are further segregated based on their Esf1 genotype, while those carrying a B6 Szs1 haplotype have reduced effect on Esf1 outcome. Thus, differences in Szs1 could prime the ultimate outcomes that are observed in Esf1, possibly through an imbalance in steady-state K+ levels that exacerbate alternative mechanisms of epileptogenesis either within astrocytes or through effects on neuronal networks.We show that strains carrying D2 alleles of cis-eQTL around Esf1 was previously associated with other neuronal complex traits, including stress response (Per3. We also found differential baseline expression in Per3 in B6 and D2 mice (Camta1 expression differences.Utilization of BXD strains was important to our project in two ways. First, it added increased power in our association analyses that would not have been possible with inbred strains alone . The pre D2 mice , dependicis-eQTL adjacent to Esf1 was also observed in the hippocampus of B6 and D2 parental strains by qPCR. While three of the four candidate loci showed significant differences in mRNA prior to seizures, only Camta1 maintained significant differences after completion of the repeated-flurothyl model. The differential expression of these loci was consistently higher in B6 mice indicating that a global regulatory control may impact this effect. Thus, our observation that the two SNPs rs369709 and rs3697097 that account for all of the cis-eQTL with LRS over 20 map to a region proximal to Esf1 could provide a good place to compare these genomes for transcriptional elements that may facilitate this kind of coordinated differential transcription.Confirmation of the differential expression of Esf1 have plausible ways they could contribute to epileptogenesis. As mentioned above, Per3 has previously been implicated in neuronal stress response and other gene family members have been implicated in induced seizures in mice (Park 7 protects against oxidative stress and may protect neurons from death (Vamp3 is a downstream target of MeCP2 that mediates SNARE-dependent exocytosis of glutamate (Each of these potential candidates for Camta1 is an excellent candidate for epileptogenesis susceptibility. In addition to having the five most significant SNPs associated with retest GST, and the best correlation with eQTLs in our system genetics data, it has several known functions that make it a good candidate from a biological perspective. First, calmodulin is a well-known mediator of postictal brain activity and has been implicated in brain adaptation to chemical (+ buffering capacity that is an aspect of our model. Finally, mutations in CAMTA1 are associated with multiple neurological phenotypes, including seizure susceptibility in humans (chemical  and elec"}
+{"text": "TET1 as one of the differentially methylated regions with gene-body hypermethylation. Herein, we characterize mechanisms that control TET1 gene activity at the transcriptional level. We show that treatment of CLL cell lines with 5-aza 2\u00b4-deoxycytidine (DAC) results in the activation of miR26A1, which causes decrease in both mRNA and protein levels of EZH2, which in turn results in the decreased occupancy of EZH2 over the TET1 promoter and consequently the loss of TET1 expression. In addition, DAC treatment also leads to the activation of antisense transcription overlapping the TET1 gene from a cryptic promoter, located in the hypermethylated intronic region. Increased expression of intronic transcripts correlates with decreased TET1 promoter activity through the loss of RNA Pol II occupancy. Thus, our data demonstrate that TET1 gene activation in CLL depends on miR26A1 regulated EZH2 binding at the TET1 promoter and silencing of novel cryptic promoter by gene-body hypermethylation.The Ten-eleven-translocation 1 (TET1) protein is a member of dioxygenase protein family that catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. TET1 is differentially expressed in many cancers, including leukemia. However, very little is known about mechanism behind TET1 deregulation. Previously, by characterizing global methylation patterns in CLL patients using MBD-seq, we found  TET2, while less is known about TET1 and 3 [TET1 was identified as a fusion partner of mixed lineage leukemia (MLL) gene in AML patients [TET1 coding sequences in hematologic cancers [Aberrant DNA methylation profiles are well documented epigenetic alterations that are implicated in several disorders including hematopoietic malignancies. The DNA methylation pattern across the genome is a culmination of a balance between DNA methylation and demethylation, which is brought by dynamically regulated functional interplay between DNA methyl transferases (DNMTs) and Ten-Eleven Translocation protein family (TETs) . Active T1 and 3 , 6. TET1patients . TET1 hapatients \u201311. TET1patients . Since c cancers , its exa cancers .Chronic lymphocytic leukemia (CLL) is a common incident in the west and characterized by diverse clinical behavior and heterogeneous disease course . Recent Recently, our comprehensive methylation analysis by Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq), identified several differentially methylated regions, including promoter elements and gene-body regions . It is vTET1 gene-body (Intronic region) but not its promoter region showed hypermethylation in CLL patient samples compared to normal healthy sorted B cell samples. Both IGHV mutated and unmutated CLL prognostic groups showed significantly hypermethylated specific peak in the TET1 intronic region compared to normal sorted B cell sample   compared to normal B cell samples   Figure . Consist) Figure . No sign) Figure .TET1 gene showed significant differential expression in CLL samples (n= 96) compared to normal sorted B cells (n =9)  Figure . We furts Figure . As showTET1 gene expression, we wanted to explore whether it has any functional link to TET1 expression. For this purpose, TET1 expression levels were analyzed in four leukemic cell lines: two CLL cell lines (HG3 and MEC1) and two MCL cell lines (GRANTA 519 and Z138) and all four cell lines express TET1 gene   , indicats Figure  and 3C. 2 Figure . We nexte Figure . Taken tFrom the published evidence it is known that promoter methylation correlates with gene repression  while thTET1 promoter. As shown in Figure Based on the promoter activity data of the HMR, we proceeded further to check for the presence of any intronic transcripts in the HMR region. To detect intronic transcripts encoded by the HMR region, the total cDNA was synthesized using DNase 1 treated total RNA and RT-qPCR analysis was performed using primers spanning, downstream and upstream of the HMR region. To check if amplification is from genomic DNA contamination, mock cDNA samples without RT enzyme were used as controls for all the respective cDNA samples. As shown in Figure TET1, which encode antisense transcripts spanning across the HMR of its sense counterpart TET1. We next wanted to investigate whether induction of cryptic promoter activity has any functional connection to the TET1 promoter activity upon DAC treatment. To this end, ChIP assay was performed on TET1 promoter TSS site, HMR region and downstream to HMR region using antibodies against RNA Pol II on DAC treated and untreated MEC1 cell line samples. Interestingly, following DAC treatment, the occupancy of RNA Pol II at the TET1 promoter decreases while at the HMR downstream region the RNA Pol II occupancy increases  was amplified containing the predicted cryptic promoter region and assayed for the promoter activity using luciferase assays. As shown in Figure Genes are regulated at the levels of transcriptional initiation, transcription elongation, pre-mRNA 3\u2019 processing and mRNA degradation. These gene regulatory steps further controlled by complex and coordinated multi-level cross talks between transcription factors, chromatin remodelers and long and small noncoding RNAs , 30. In TET1 gene-body [TET1 gene-body hypermethylation correlates with TET1 gene activation, we wanted to understand the functional role of gene-body hypermethylation in TET1 gene expression. DAC treatment to induce demethylation across the genome has been widely used to explore the functional role of DNA methylation in gene expression. Thus by treating four different CLL cell lines with DAC, we have shown that demethylation of hypermethylated TET1 gene-body leads to the activation of methylation dependent cryptic promoter. Both strand-specific RT-PCR and promoter luciferase assays clearly demonstrated that cryptic promoter encodes antisense transcripts, which span across the TET1 promoter. Activation of cryptic transcription across the TET1 promoter correlates with the loss of RNA Pol II occupancy and TET1 gene silencing, indicating that antisense cryptic transcription may be regulating TET1 gene expression in part by occluding the transcription initiation machinery from the TET1 promoter. Thus our data is consistent with the functional role of gene-body methylation in repressing aberrant cryptic promoter activity. Interestingly, we observed presence of many SINE and LINE Alu repeats located in the HMR region where the intronic transcripts are expressed. It would be interesting to know the role of these transcribed repetitive elements in TET1 expression.Gene-body hypermethylation has lately been recognized as a transcriptional regulatory step in regulating alternative splicing  and crypene-body . Since TmiR26A1, EZH2, in the TET1 gene expression. Previously, miR26A1 has been implicated in the regulation of TET1 [TET1 promoter in the UCSC genome browser utilizing the ENCODE/Broad Institute ChIP-seq datasets, revealed presence of EZH2 peaks at the TET1 promoter in several cell types. Thus we wanted to investigate the functional interplay between EZH2, TET1 and miR26A1. Previously, we documented that miR26A1 is hypermethylated in CLL samples compared to normal B cells [miR26A1 can regulate TET1 at the transcriptional level via modulating the levels of EZH2 at the TET1 promoter. Interestingly, in this context EZH2 acts as an activator in the TET1 gene regulation, which is in contrast to its widely accepted function as transcriptional repressor via catalyzing H3K27me3 modification. Previously, in a few instances EZH2 has been implicated in the gene activation function which is independent of histone methyltransferase activity [miR26A1 directly regulates TET1 at the post-transcriptional level or via regulating EZH2 occupancy at the TET1 promoter there by interfering with TET1 activity indirectly at the transcriptional level. According to our data  treatment as shown in the Figure miR26A1, decrease in EZH2 levels and induced expression of intronic transcripts at HMR region resulting in reduced RNA Pol II occupancy at the TET1 promoter, leading to reduced TET1 expression levels. Thus our data demonstrate a novel regulatory loop between miR26A1-EZH2 and TET1 and thus providing an explanation for consistent upregulation of TET1 and EZH2 in CLL patients while miR26A1 is hypermethylated. Hence, our study opens up a new avenue in the further exploration of regulatory connection of EZH2 and TET1.Finally, a schematic representation of overall data of this study is shown in Figure Total 40 CLL patient samples were included in the present study. All the peripheral blood mononuclear cells (PBMCs) CLL samples were collected from different hematology departments in the western part of Sweden after written consent had been obtained. 5 DNA samples of CD+19 sorted normal B cells from healthy age matched controls were purchased from 3H biomedical . All samples were diagnosed according to recently revised iWCLL criteria , showingDNA was extracted from CLL PBMC samples using DNA extraction kit  according to the manufacturer's protocol. RNA extractions were done using miRNeasy Mini Kit  along with on column DNase1treatment  to remove any residual genomic DNA. The total cDNA synthesis was performed using Superscript III FS synthesis supermix kit  according to the manufacturer's protocol.TET1 promoter TSS  . All the primer sequences used for Pyrosequencing assay are listed in The hypermethylated region (HMR) peak from obtained from MBD seq data  is 687 bThe expression levels of TET1 and EZH2 genes were analyzed with Taqman gene expression assays  . For the expression of intronic transcripts, 7 sets of custom primers were designed using Primer 3 software, spanning the intronic region between Promoter TSS and HMR on TET1 gene and the RT-qPCR analysis was done using Power SYBR Green PCR master mix . Differences in expression were calculated using the \u0394\u0394Ct method. All the primer sequences and the product size of the amplified region were listed in the Strand specific PCR is performed using specifically designed 5\u2019GSP (Gene specific Primer) and 3\u2019GSP for synthesizing gene specific cDNA from DNase1 treated RNA samples. The 5\u2019GSP specifically binds to antisense strand and the 3\u2019GSP binds to the sense strand. After cDNA synthesis, to quantify the strand specific cDNA synthesis we used two sets of primers which are located downstream to the 5\u2019GSP and 3\u2019GSP. The PCR product size and the primer sequences used were listed in In order to identify the promoter activity of HMR and downstream cryptic promoter regions, we constructed several PGL3 dual-luciferase reporter vectors  containing the target sequence for checking the promoter activity. Luciferase activity was determined 48 h after transfection as described earlier , by use ChIP was performed with the shearing module kit  and one-day ChIP kit , according to the manufacturer's instructions. Western blot analysis was performed using total cell lysates lysed from transfected CLL cell line samples. The details of ChIP assay, western blot analysis along with the Antibodies used are described in Supplementary Materials and Primer sequences are provided in"}
+{"text": "Saccharomyces cerevisiae and was later found to be involved in many aspects of cell functions and many biological and pathological processes. The role of MAP4K4 in immunity, inflammation, metabolic and cardiovascular disease has been recognized. Information regarding MAP4K4 in cancers is extremely limited, but increasing evidence suggests that MAP4K4 also plays an important role in cancer and MAP4K4 may represent a novel actionable cancer therapeutic target. This review summarizes our current understanding of MAP4K4 regulation and MAP4K4 in cancer. MAP4K4-specific inhibitors have been recently developed. We hope that this review article would advocate more basic and preclinical research on MAP4K4 in cancer, which could ultimately provide biological and mechanistic justifications for preclinical and clinical test of MAP4K4 inhibitor in cancer patients.The serine/threonine kinase MAP4K4 is a member of the Ste20p (sterile 20 protein) family. MAP4K4 was initially discovered in 1995 as a key kinase in the mating pathway in  MAP4K4, also known as HGK  or NIK  is a serine/threonine (S/T) kinase that belongs to the mammalian family of Ste20 protein kinases due to their shared homology to the budding yeast kinase Ste20p . Ste20 fMAP4K4 contains ~1200 amino acids with a molecular mass of ~140\u00a0KDa , 8. The As summarized in Table\u00a0Despite different localizations of their catalytic domains (N-terminus vs. C-terminus), mammalian Ste20 kinases share similar features in the kinase domain . In geneTaking the global phosphoproteomics approach and by comparing the corresponding SILAC (Stable Isotope Labeling by Amino acids in Cell) ratios of EGF  stimulation and erlotinib (EGFR inhibitor) treatment in lung adenocarcinoma cells, a prior study identified two serine sites S648 and S708 in the middle domain of MAP4K4 as EGFR  signaling-dependent phosphorylation sites . Serine The C-terminal domain of MAP4K4 contains a citron-homology domain (CNH) that appears to determine MAP4K4 association with other factors . For insTaken together, current evidence, as summarized above, strongly supports that the MAP4K4 kinase activity can be positively or negatively regulated by upstream kinases. The identities of the kinases remain largely unexplored. If multiple kinases are involved, it is highly likely that the selection within a repertoire of candidate kinases is context-dependent, depending on the cell type, the nature of the external stimuli, and the cell state. The biochemical and biological consequences of phosphorylation could also be context-dependent. In addition to negatively or positively regulating MAP4K4 kinase activity, phosphorylation may also determine MAP4K4 subcellular localization and substrate-selection.MAP4K4 gene contains a nearby p53 binding sites cluster downstream of the promoter and six potential p53 binding sites in the first intron, four of which are confirmed by chromatin immunoprecipitation (ChIP) experiments [A recent study points to a possibility that in T cells of type 2 diabetes patients, mRNA level of MAP4K4 might be affected by enhanced methylation of CpG islands in its promoter region , suggesteriments , 29. IndDespite the fact that evidence from genetic studies using mouse model is still lacking, emerging evidence from preclinical and patient association studies strongly suggests that MAP4K4 may play an important role in many types of cancer and could serve as a novel actionable target for cancer treatment. The first evidence suggesting a role of MAP4K4 in cancer came from observations that MAP4K4 is highly expressed in 40 of the NCI-60 human tumor cell lines and can modulate cellular transformation, adhesion and invasion . In thisLittle information exists regarding how MAP4K4 is involved in cancer. As shown in Fig.\u00a0In addition to above mentioned candidate downstream mediators of MAP4K4, MAP4K4 also participated in the regulation of other cancer-related signaling pathways or factors including insulin pathway, hippo signaling (LATS1/2 and YAP/TAZ) and mTOR/AMPK , 47\u201352. How MAP4K4 regulates its downstream effectors or signaling mediators remains largely unexplored. Since MAP4K4 is a kinase, one would expect that the primary job of MAP4K4 is to phosphorylate its substrate. Indeed, MAP4K4 can directly phosphorylate TRAF2 at serine 35 to promote its degradation in T cells . HoweverAs summarized in Table\u00a0Current evidence supports but not yet provides sufficient biological and mechanistic justification for MAP4K4 as a novel cancer therapeutic target. Evidence definitely linking MAP4K4 to the development and progression of any types of cancer is still lacking. To this end, it is essential to examine the impact of genetic manipulation of MAP4K4 in mouse models of cancer. When interpreting results from experiments using MAP4K4 knockout mice (whole-body or tissue-specific knockout), potential redundancy and functional compensation among MAP4Ks should be taken into consideration. Since small-molecule inhibitors of MAP4K4 are available, in order to eventually use these inhibitors in clinic, future studies should attempt to test their tumor prevention and antitumor activity in mouse models of cancer.Currently there is no sufficient information suggesting in which type of cancer MAP4K4 inhibitor can be used as a novel promising therapy. Target overexpression is an overrated predictor of efficacy since it may also represent a cellular attempt to limit unbridled growth, unless functional results from genetic manipulation of MAP4K4 in that particular tumor model are available, it is difficult to predict cancer types responsive to MAP4K4 inhibitor treatment. It is possible that MAP4K4 could promote tumor development and progression in certain types of cancer and functions as a tumor repressor in other types of cancer, or plays different roles at different stages during tumor development and progression. Therefore it is crucial to gain a thorough understanding of how MAP4K4 is involved in a particular type of cancer functionally and mechanistically before conducting clinical testing of MAP4K4 inhibitor in cancer patients.No information is available about whether and how MAP4K4 is involved in resistance to standard cancer therapy. If MAP4K4 contributes to cancer development and progression, it is highly likely that MAP4K4 can also be involved in treatment resistance. Therefore in addition to examine the potential anti-tumor activity of MAP4K4 inhibitors as a standalone therapy, it is also important to test if MAP4K4 inhibitors can be used in combination to overcome resistance to chemotherapy, radiation therapy, targeted therapy and immunotherapy.Detailed molecular understanding of how MAP4K4 is involved in cancer biology is essential for firmly establishing MAP4K4 as a target for that particular type of cancer. Crucial to this effort is to identify key upstream regulators and downstream effectors including substrates of MAP4K4. To develop efficient methods to block MAP4K4, it is also crucial to understand how MAP4K4 functionally interacts with Ste20 family members.Cancer remains a largely incurable disease, indicating an urgent and unmet need for novel effective therapeutic approaches. Identifying a novel cancer therapeutic target that could be amenable to pharmacologic intervention is challenging. To this end, we believe that a better understanding of biological functions and underlying mechanisms of MAP4K4 in cancer could have far-reaching implications for new directions in cancer therapy."}
+{"text": "There are many single-gene causes of steroid-resistant nephrotic syndrome (SRNS) and the list continues to grow rapidly. Prompt comprehensive diagnostic testing is key to realising the clinical benefits of a genetic diagnosis. This report describes a bespoke-designed, targeted next-generation sequencing (NGS) diagnostic gene panel assay to detect variants in 37 genes including the ability to identify copy number variants (CNVs).This study reports results of 302 patients referred for SRNS diagnostic gene panel analysis. Phenotype and clinical impact data were collected using a standard proforma. Candidate variants detected by NGS were confirmed by Sanger sequencing/Multiplex Ligation-dependent Probe Amplification with subsequent family segregation analysis where possible.NPHS1 or NPHS2.Clinical presentation was nephrotic syndrome in 267 patients and suspected Alport syndrome (AS) in 35. NGS panel testing determined a likely genetic cause of disease in 44/220 (20.0%) paediatric and 10/47 (21.3%) adult nephrotic cases, and 17/35 (48.6%) of haematuria/AS patients. Of 71 patients with genetic disease, 32 had novel pathogenic variants without a previous disease association including two with deletions of one or more exons of Gene panel testing provides a genetic diagnosis in a significant number of patients presenting with SRNS or suspected AS. It should be undertaken at an early stage of the care pathway and include the ability to detect CNVs as an emerging mechanism for genes associated with this condition. Use of clinical genetic testing after diagnosis of SRNS has the potential to stratify patients and assist decision-making regarding management. Patients with nephrotic syndrome (NS) suffer a breakdown of the glomerular filtration barrier at the level of the podocyte leading to massive proteinuria, hypoalbuminaemia and oedema.COL4A3, COL4A4 or COL4A5 and may present with proteinuria (some with FSGS on biopsy), and more commonly haematuria.COL4A3 and COL4A4 have been identified in association with FSGS, together with thin basement membrane nephropathy but without the extrarenal features of AS.Pathogenic variants in single genes affecting podocyte function are a common cause of SRNS, reported in up to 29.5% of a predominantly childhood cohort.COL4A3, COL4A4, COL4A5).The proportion of single-gene cases identified inversely correlates with the age of onset with 69.4%\u2013100% of congenital-onset disease reported as having a genetic aetiology.Timely genetic testing can considerably alter patient management and facilitate a greater understanding of the genetic complexity of the condition.We have developed a clinically\u00a0approved gene panel test for 37 SRNS and collagen-related genes  using a In total, 302 patients were referred with informed consent for diagnostic gene panel analysis over a 26-month period. The diagnostic test has been formally assessed for validity, and socio-legal/ethical implications by the UK Genetic Testing Network and UK National Health Service (NHS) commissioners through the gene dossier process, and was undertaken in an accredited UK NHS Laboratory. Data presented pertain only to anonymised auditing of routine diagnostic testing; therefore, this study was not subject to ethical approval.The sequencing and bioinformatics protocol is described in brief with further details as 10.1136/jmedgenet-2017-104811.supp1Supplementary file 110.1136/jmedgenet-2017-104811.supp2Supplementary file 2cis or trans) and segregation. CNVs in patients with single heterozygous variants in recessive genes were identified by CONTRAWhere possible, relative testing was undertaken using Sanger sequencing to determine phase  being issued in 22 calendar days.An average gene coverage of 99.26% coding sequence at a minimum read depth of 30\u00d7 was achieved on a typical 12-patient run. The per-gene coverage is shown in Targeted gene panel testing of all 302 patients identified 71 (23.5%) with a likely genetic cause for disease. The genetic diagnostic rate among the group with SRNS was 21.2%, including 44/209 (21.1%) paediatric and 10/46 (21.7%) adult nephrotic cases . In patiThe spectrum of pathogenic variants is summarised in NPHS1, WT1 and NPHS2 in 12 (4.7%), 11 (4.3%) and 7 (2.7%) patients, respectively. In the Alport group, the variants were all in collagen genes: COL4A3, COL4A4 and COL4A5 in 5 (14.3%), 2 (5.7%) and 10 (28.6%) patients, respectively. Of note, five SRNS/FSGS patients had LP variants in COL4A3, COL4A4 and COL4A5  including a single novel LP COL4A3 variant, c.698G>A, p.(Gly233Glu), in a patient with a dominant family history of FSGS (patient 4) which tracked with disease in an affected brother. Among patients with SRNS/FSGS who were found to have genetic disease (excluding those with collagen variants), there was an autosomal-dominant mode of inheritance in 16/41 (36.6%) of those with disease onset\u00a0\u226418 years compared with 6/7 (85.7%) of those\u00a0>18 years.The most frequently\u00a0detected LP variants in the SRNS group (n=255) were in NPHS1 and NPHS2; and 5.8% each in INF2, MYH9, PLCE1 and PTPRO. Of the 71 patients with likely genetic disease, 11 cases had 12 additional VUS in genes other than the main causative one for that patient, most frequently collagen genes in 41.7% and WT1 in 16.7%. Overall, of the 64 recorded VUS, 31.3% were in collagen genes, 6.3% in NPHS2, 6.3% in NPHS1 and 6.3% in MYH9.In addition to the patients with LP variants, a further 40 patients had one or more VUS .NPHS2 exon 2 in combination with a paternally\u00a0inherited c.1032delT variant. The c.1032delT variant has been previously reported as the most frequent pathogenic variant in NPHS2 in Poland (Kashubian region).ACTN4 (1 patient), COL4A3 (2), COL4A4 (1), COL4A5 (10), INF2 (2), LAMB2 (2), NPHS1 (5), NPHS2 (2), SMARCAL1 (1), TRPC6 (1) and WT1 (3).Among the 71 patients with a genetic cause for disease, 32 had variants without a previous disease association including 26 with one or more novel variants absent from population databases. Two patients had gene deletions of one or more exons detected by CNV analysis . PatientINF2 LP variants were detected in two adult-onset NS patients: p.(Tyr50Asp) (patient 25) and p.(Leu165Arg) (patient 26). Both were missense variants affecting highly conserved residues within the diaphanous inhibitory domain of the INF2 protein consistent with the previously\u00a0reported spectrum of disease-causing variants. Segregation supports pathogenicity, p.(Tyr50Asp) co-segregating with disease in five affected family members and p.(Leu165Arg) present in one affected family member and absent in two unaffected family members. An additional sensory neuropathy phenotype previously reported in 12.5% of INF2 casesNovel WT1 variants were identified in the known hotspot region (exons 6\u20139)WT1 exon 8 (patient 65) was associated with age of onset of 30 years and was inherited from an affected father who was diagnosed in his 30s. Although WT1 is normally associated with childhood-onset disease, these findings are consistent with a previously described biphasic childhood and adulthood presentation of variants in WT1.Two novel missense patient 6 was assoNPHS1 or NPHS2 that has been previously published as disease causing. No CNVs were identified. The finding of these variants may be incidental. However, given the low incidence of SRNS, the young age of onset  and the low frequency/absence of the variants in databases of subjects without known renal disease it is possible that there are additional NPHS1/NPHS2 variants in unsequenced intronic or promoter regions which may act in combination to cause the phenotype in these patients. These findings were reported as compatible with the phenotype but insufficient to make a diagnosis.In six clinically affected SRNS cases (patients 112\u2013117 in online\u00a0sNPHS1. Patient 112 developed SRNS under the age of 4 years with a heterozygous p.(Asp105Asn) variant previously reported in a Japanese patient with CNS where a second variant was not detected.NPHS1 or other genes in the panel. Patient 117 had a single heterozygous missense variant in NPHS2: c.872G>A, p.(Arg291Gln) previously reported as pathogenic in the homozygous/compound heterozygous state. Patient 117 also had a novel single heterozygous variant in NPHS1: c.2512C>A, p.(Pro838Thr) which is not reported in population databases and prediction tools suggest is deleterious.Three of these patients  with early-onset SRNS were heterozygous for previously\u00a0reported rare pathogenic variants in NPHS2 p.(Arg138Gln) (exon 3) and p.(Leu156Phefs*11) (exon 4) in a compound heterozygous state with the NPHS2 non-neutral polymorphism p.(Arg229Gln). Tory et alNPHS2 is contributing to these patients\u2019 phenotypes.Two further patients 115 and 116) presenting with classical NS had single previously\u00a0reported pathogenic variants in  and 116 NPHS1 (12 patients), LAMB2 (3 patients), NPHS2 (2 patients) and WT1 (1 patient). For cases of SRNS with known age of onset >18 years, the rate was 28.6% (6/21) with LP variants in INF2 (two patients), WT1 (two patients), SCARB2 (one patient) and TRPC6 (one patient).The age of disease onset was known with certainty for 164 (64.3%) of 255 patients referred with a clinical presentation of SRNS and online s10.1136/jmedgenet-2017-104811.supp4Supplementary file 4LAMB2 and NPHS2 pathogenic variants. At least nine family tests are known to have helped inform suitability for donor transplant treatment. Other familial testing has provided diagnoses for relatives and aided in clarifying variant pathogenicity. Other responses confirmed that genetic testing impacts on diagnosis and prognosis after transplant.Physicians were asked whether the results of genetic testing would alter immunosuppressive management in patients with SRNS. Responses were obtained in relation to 71 (27.8%) of these patients. The response rate was not high enough to make definitive conclusions, but broadly clinicians reported that results would influence decisions to reduce or stop immunosuppression and one physician indicated that treatment decisions would be made after the gene panel results were known. The diagnostic test result from 67 patients resulted in the subsequent testing of 148 family members including two prenatal tests for Gene panel testing is becoming more and more relevant for screening rare diseases, with greatly improved cost/benefit. The knowledge of the genetic basis of SRNS has expanded considerably over the past five years with several national and international cohorts recently publishedThe frequency of likely\u00a0pathogenic variants among patients with SRNS was 21.2%. This is marginally lower than 24%\u201334% reported in other studies which included predominantly subjects with childhood-onset disease.EMP2, KANK1 and KANK2 have been described in family studies but evaluation in larger cohorts did not identify additional patients with pathogenic variants.KANK2 was included in the gene panel reported here and no likely pathogenic variants were identified. The updated version of the panel includes EMP2, KANK1 and KANK4. Our current recommendation for clinicians is not to use the NGS gene panel test for patients with persistently SSNS unless there are specific reasons to suspect a genetic aetiology.Twelve patients with SSNS were referred for genetic testing and none had a potentially pathogenic variant. Thus far, no purely monogenic causes for SSNS have been identified.In this study, 100% of LP variants identified by NGS meeting diagnostic variant-calling quality parameters have subsequently been confirmed as being present on Sanger sequencing. Sanger confirmation of NGS LP variants is currently in line with best practice ACGS guidance. It also provides a confirmation of sample identity following pooling of samples during the NGS process and establishes a familial test for relatives. As further evidence is collected, confirmatory testing may become redundant.WT1 variant without any manifesting extrarenal features. In addition, variants in secondary genes which may potentially contribute to the phenotype of the patient can be identified by a panel approach.The use of gene panel testing supersedes stepwise screening protocolsGene panel screening identified 32 likely\u00a0pathogenic variants without a previous disease association. Absence or rarity in population data is used as evidence to support pathogenicity; however, it is acknowledged that some ethnicities in this global cohort were either not known or may currently be insufficiently represented in population databases. Where possible, segregation analysis was performed to provide additional evidence to support pathogenicity; however, family members were not always available for testing, reflecting the use of this panel in a clinical setting.The interpretation of variants of unknown significance in a global cohort is also a challenge of panel testing. As well as segregation analysis, future expansion of population databases will allow improved filtering of population-specific variants and functional work may also aid the interpretation of variants.NPHS1 or NPHS2 pathogenic variant detected, suggesting deep intronic or regulatory variants if these genes are truly recessive in mechanism. Future whole-genome sequencing in these patients may help to elucidate a genetic pathogenesis.This study has demonstrated two cases of CNVs present in NS genes; therefore, CNV analysis of NGS data should be routinely undertaken as part of the variant analysis pipeline, together with confirmation using a second method such as MLPA. It is also apparent that there are a number of clinically typical cases with only a single known COQ2, COQ6 or PDSS2, coding for proteins the coenzyme Q10 pathway, may indicate the potential for benefit from treatment with this enzyme.PLCE1 variants who responded to treatment with steroids and ciclosporin.PLCE1 variants as their affected relatives suggest a more complex genotype\u2013phenotype interplay and raise the possibility of spontaneous improvement rather than a true response to medication.WT1 variants have responded to steroids and immunosuppression.WT1, should prompt search for other features of an associated syndrome, such as Frasier syndrome and risk of gonadoblastoma.The timing of testing in relation to disease onset and the speed of genetic reporting are important for clinical utility. It is potentially possible to generate results within 1\u20132 weeks, thereby avoiding diagnostic biopsy in some cases. In certain contexts, earlier testing and more rapid turnaround are important because results may have direct consequences for prenatal testing and patient treatment. Pathogenic variants in The presence of a causative variant in SRNS is associated with a lower risk of post-transplantation recurrence of disease, occurring in 25.8% of patients testing negative for genetic disease compared with 4.5% of those with an identified variant in a European cohortWe report that NGS gene panel testing with bioinformatics analysis for SNVs and CNVs at an early stage after diagnosis of SRNS or suspected AS with results in a clinically relevant time\u00a0scale has the potential to improve patient stratification and the care pathway."}
+{"text": "Wide local excision followed by radiotherapy was offered as an alternative to mastectomy to 93 women on 96 occasions. Complications attributable to the surgery occurred in 4 per cent whilst 37.5 per cent developed complications secondary to the radiotherapy. The complications were generally of a mild nature. Nine patients (9.3%) have developed regional recurrence and seventeen (17.7%) distant metastases with a mean follow up of 53.4 months."}
+{"text": "We document the descriptive statistics and detailed regression outputs for educational attainment and measured 2D:4D ratios, based on the RLMS data . Regression analysis is conducted using STATA 13, gologit2 which is a special code for the generalized ordered logit regression in STATA environment. We provide graphs of differences in means of 2D:4D ratios by educational attainment. Information about the distribution of self-identified nationalities and fields of university degrees of respondents is presented. Specifications TableValue of the data.\u2022Descriptive statistics of national self-identities, major fields, educational attainment and fingers\u2019 2D:4D measurements are helpful in the case of analyzing possible associations between education and 2D:4D.\u2022Detailed generalized ordered logit regressions\u2019 outputs are helpful in understanding of the general patterns of 2D:4D associations at different levels of education obtained. Inclusion of classical predictors (education of parents) is helpful in foreseeing their correlations with different levels of educational attainment in the studied samples.\u2022Graphs of means are helpful in comparison of differences between averaged 2D:4D of individuals with university degree and averaged 2D:4D of individuals who completed lower levels of education.1Measurements were taken by a special team of 2 trained assistants, while other information was taken from the RLMS survey which contains questions regarding an individual\u05f3s socioeconomic characteristics and family background 2See"}
+{"text": "RUNX1 is a member of the RUNX family of transcription factors that also includes RUNX2 and RUNX3 [CDH1 promoter to positively regulate E-Cadherin expression.The functional consequences of RUNX1 loss or gain of expression that contribute to breast cancer development and progression have not been fully elucidated. A role for RUNX1 in suppressing ER oncogenic signaling is suggested by the fact that combined loss of Runx1 with either TP53 or Rb1 leads to hyperproliferation of ER+ luminal cells in the mouse mammary gland . InactivIn apparent conflict with the role of RUNX1 as a suppressor of EMT in ER+ luminal tumors, downregulation of RUNX1 expression in TN tumors inhibits tumor invasion and metastasis . How do In summary, increasing evidence supports a role for RUNX1 in breast cancer progression. Understanding how RUNX1 functions in a cell context-dependent manner will be essential for determining the mechanism(s) by which this transcription factor impacts tumor biology."}
+{"text": "Bifidobacterium lactis HN019 reduces transit time, but its mechanisms of action and effects on motility patterns are poorly understood. The aim of this study was to investigate changes in GI motility induced by an extract of HN019 on distinct patterns of colonic motility in isolated rat large intestine, compared with a known promotility modulator, prucalopride. The large intestines from male Sprague Dawley rats (3\u20136 months) were perfused with Kreb's buffer at 37\u00b0C in an oxygenated tissue bath. Isometric force transducers recorded changes in circular muscle activity at four independent locations assessing contractile propagation between the proximal colon and the rectum. HN019 extract was perfused through the tissue bath and differences in tension and frequency quantified relative to pre-treatment controls. Prucalopride (1 \u03bcM) increased the frequency of propagating contractions (by 75 \u00b1 26%) in the majority of preparations studied (10/12), concurrently decreasing the frequency of non-propagating contractions (by 50 \u00b1 11%). HN019 extract had no effect on contractile activity during exposure (n = 8). However, following wash out, contraction amplitude of propagating contractions increased (by 55 \u00b1 18%) in the distal colon, while the frequency of non-propagating proximal contractions decreased by 57 \u00b1 7%. The prokinetic action of prucalopride increased the frequency of synchronous contractions along the length of colon, likely explaining increased colonic rate of transit in vivo. HN019 extract modified motility patterns in a different manner by promoting propagating contractile amplitude and inhibiting non-propagations, also demonstrating prokinetic activity consistent with the reduction of constipation by B. lactis HN019 in humans.Attention is increasingly being focussed on probiotics as potential agents to restore or improve gastrointestinal (GI) transit. Determining mechanism of action would support robust health claims. The probiotic bacterium  Constipation is a common functional gastrointestinal (GI) disorder affecting 20% of the general population worldwide   and incubated at 37\u00b0C in an anaerobic workstation  containing 10% CO2, 10% H2 and 80% N2 for 48 h. A 50 mL secondary culture was inoculated with 0.5 mL of primary broth (adjusted to an OD600 of 1.5) and incubated anaerobically for 16 h. Two 150 mL secondary cultures were inoculated with 1.5 mL of secondary broth and incubated anaerobically for 16 h to the stationary phase. The bacterial cell culture was harvested and processed into extract under anaerobic conditions, then used in the motility assays freshly each day. Bacterial cells were collected by centrifugation  and resuspended in 5 mL of anaerobic Krebs solution . The bacterial cell pellet was washed twice in Krebs solution, resuspended in 5 mL and incubated on ice for 10 min. The mixture was then sonicated on ice using 20 s pulses with 30 s intervals, power level 2 at 40% duty for 10 min . The sonicated mixture was then centrifuged  to remove the cell debris and the resulting supernatant was ultra-centrifuged to remove any remaining cell membranes  and to prevent excessive frothing of the solution in the motility experiment phase. The final supernatant was a cell-free extract (HN019 extract) that was adjusted to pH 7.4 (with 5 M sodium hydroxide) and diluted 1/10 dilution in Kreb's solution for use in the motility assay.A bacterial extract was used in this study as opposed to live bacteria because ) Espey, . Therefoad libitum.This study was carried out in strict accordance with the recommendations of the New Zealand Animal Welfare Act 1999. The protocol was approved by the AgResearch Limited (Grasslands) Animal Ethics Committee . Male adult Sprague Dawley rats, 3\u20136 months of age, weighing 250\u2013400 g were obtained from AgResearch Ruakura . The rats were housed under a 12 h light/dark cycle, and fed Sharpes Diet 86 . Food and water were available 2, 5% CO2) whilst the colon was gently flushed with Krebs solution to expel fecal pellets and the entire tissue was then mounted in an organ bath (approximately 350 mL capacity), a stainless steel rod (35 cm in length and 2 mm in diameter) was inserted through the lumen of the colon, which was then perfused at 20 mL/min with Krebs buffer at 35 \u00b1 1\u00b0C. The lumen was also perfused with Krebs buffer at 1.5 mL/min which was pumped aborally using a constant flow pump because this provided the pressure required to record consistent propagating contractions. Changes in circular muscle tension were recorded from four sites simultaneously along the length of large intestine, using four custom-made metal hooks anchored 3 cm from both oral and anal ends of the preparation and evenly spaced apart at approximately 4 cm intervals. These hooks were connected via silk thread to force transducers and contractions measured after applying 2 g of tension. Muscle contraction data were recorded using isometric force transducers (MLT0201) connected to an eight-channel bridge amplifier, integrated using PowerLab 8/35 hardware and acquired and analyzed using LabChart 8 software. All recording equipment hardware and software were from ADInstruments Pty Ltd., Bella Vista, NSW, Australia.The protocol for recording motility in isolated intact whole large intestine has recently been described  with one experimental factor (the three treatments) and one repeated factor (four time points) was used to analyze differences in the frequency and amplitude of synchronous contractions within experiments, and also compared with the Krebs treatment control. All analyses were carried out using the R software version 3.2.3. ANOVA assumptions were met through log or square root transformation where necessary. Linear mixed effects models were used with appropriate variance function for modeling heterodasticity.Data were excluded from statistical analysis where they were contrary to the main effects observed: 2/12 preparations for prucalopride and 2/10 preparations for HN019, and are described in the results section.Contractile patterns were recorded from the circular muscle layer at four locations simultaneously along the length of the large intestine , before and after addition of treatment conditions. Motility patterns were quantified with respect to changes in frequency and amplitude of synchronous and non-synchronous phasic contractions, and contractions that occurred only in the proximal colon Figure .n = 8) synchronous contractions occurred between proximal colon and rectum. Although no changes in synchronous contractions were detected over the course of the experiment  over the initial 30 min of exposure and by 75 \u00b1 26% over 30\u201360 min exposure. This occurred without any change in amplitude  to the large intestine resulted in no detectable change in contractile activity over 60 min, yet it was found that within the first 30 min following washout of HN019, the contractile amplitude increased by 55 \u00b1 18% in the proximal colon Figures , 2A,B. PHN019 extract decreased the amplitude of non-synchronous contractions in the proximal colon to 30% lower than that for the corresponding Krebs treatment control at 60 min Figure . The inhThe ratio of non-synchronous to synchronous contractions averaged 5.2:1 across the pre-treatment controls  and was reduced to 1.0:1 after 60 min of prucalopride, and 1.6:1 for HN019 extract post-treatment, yet remained high at 5.0:1 for the Krebs control  . AL was supported by a Palmerston North Medical Research Foundation Summer Scholarship. Experiments in this project were in part conducted in the laboratory of NS and funded by the NH&MRC of Australia (#1067335).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Angiopoietin-1 (Angpt1) is a glycoprotein ligand important for maintaining the vascular system. It signals via a receptor tyrosine kinase expressed on the surface on endothelial cells, Tie2. This receptor can undergo regulated ectodomain cleavage that releases the ligand-binding domain (sTie2) into the circulation. The concentration of sTie2 is increased in a range of conditions, including peripheral arterial disease and myocardial infarction, where it has been suggested to bind and block Angpt1 resulting in vascular dysfunction. Here we use a joint mathematical modelling and experimental approach to assess the potential impact of sTie2 on the ability of Angpt1 to signal. We find that the concentrations of sTie2 relative to Angpt1 required to suppress signalling by the ligand are more than ten\u2013fold higher than those ever seen in normal or disease conditions. In contrast to the endogenous sTie2, an engineered form of sTie2, which presents dimeric ligand binding sites, inhibits Angpt1 signalling at seventy-fold lower concentrations. While loss of Tie2 ectodomain can suppress Angpt1 signalling locally in the cells in which the receptor is lost, our study shows that the resulting increase in circulating sTie2 is unlikely to affect Angpt1 activity elsewhere in the body. In humans there are three angiopoietins, angiopoietin-1 (Angpt1), Angpt2 and Angpt44. Angpt1 is constitutively secreted under normal conditions by perivascular cells4 and has crucial vascular protective effects, acting to maintain endothelial quiescence, suppress vessel inflammation, prevent microvessel regression and inhibit vascular leakage5.The angiopoietins are a group of secreted glycoprotein ligands that act mainly on the vasculature4. This transmembrane protein is expressed on endothelial cells7. Tie2 is activated by Angpt1, stimulating receptor phosphorylation, intracellular signalling and functional effects5. Endothelial cell effects of Angpt1 include suppression of apoptosis, enhancement of cell:cell junction integrity and inhibiting expression of inflammation-associated genes5.The primary receptor for angiopoietins is the receptor tyrosine kinase Tie29. Soluble Tie2 (sTie2) originates from endothelial cells where it is generated by proteolytic cleavage and release of the ectodomain from full-length receptor located at the cell surface9. sTie2 contains the ligand binding domain of the receptor and can bind angiopoietins, preventing them from interacting with cellular Tie2 and thereby inhibiting angiopoietin action9. sTie2 is increased in a number of diseases14. The possibility that increased sTie2 could contribute to vascular pathology by inhibiting Angpt1 protective signalling has been highlighted in peripheral arterial disease, myocardial infarction, vessel permeability in inflammation, organ failure in post cardiac-arrest syndrome and trauma, pulmonary hypertension, and neoangiogenesis19. sTie2 therefore may be an important target for therapeutic inhibition.In addition to cellular Tie2, a soluble form of the angiopoietin receptor has been detected in the circulationsTie2 has the potential to have profound effects on regulating angiopoietin availability and contribute to vascular pathology in diseases in which it is elevated. Despite this we know relatively little about how sTie2 affects the ability of angiopoietins to act on endothelial cells. In this study, therefore, we use computational modelling and experiment to assess the effects of sTie2 on the ability of Angpt1 to bind and activate its cellular receptor.20 for Angpt1 binding in the absence and presence of sTie2  on endothelial cells. To test this prediction we directly examined the effects of sTie2 on the ability of Angpt1 to activate cellular Tie2. Human endothelial cells were challenged with Angpt1, or control vehicle, in the absence or presence of sTie2 Fig.\u00a0, and recie2 Fig.\u00a0. This acie2 Fig.\u00a0. Additioie2 Fig.\u00a0, confirm28. Examples of such ligand-traps include TNF-\u03b1 receptor ectodomain, Etanercept, used in treatment of rheumatoid arthritis29, and the VEGF ligand-trap, Aflibercept, used in a number of pathologies including diabetic macular oedema30. In contrast to physiological soluble receptors, ligand-traps are usually fusion proteins in which the ligand-binding domain is engineered to be presented in a dimeric format28. We were interested to investigate how a dimeric form of sTie2, similar to that which would be used for a ligand trap, compares with monomeric sTie2 in their ability to affect Angpt1 action. As shown in Fig.\u00a0Soluble receptor ectodomains are used as therapeutic ligand-traps to block effects of pathological or inappropriately elevated concentrations of ligand31 and vary little with disease, though a decrease to 14pM has been reported in sepsis17. Circulating sTie2 concentrations in humans have been reported to be in the region of 100\u2013200 pM in normal conditions31, rising as high as 350 pM in severe sepsis17. The molar ratio of sTie2:Angpt1 found in vivo therefore is normally around 3\u20136 rising to 25 in severe sepsis. Such relative concentrations are much lower than those we found to affect the ability of Angpt1 to assemble tetrameric Tie2 complexes in our model and suppress Tie2 activation in our experiments. This suggests that circulating sTie2 is unlikely to influence the ability of circulating Angpt1 to activate cellular Tie2 at the concentrations found in normal and disease states in vivo.Our results show that Angpt1 activation of cellular Tie2 is poorly responsive to sTie2. We found that in order to suppress signalling-competent Tie2 complex formation by 50% sTie2 is required at concentrations in the range of 350\u2013200 fold that of Angpt1 in our model and experiments. Concentrations of Angpt1 in humans under normal conditions are around 30\u201370pMin vivo and not incorporated in our model and experiments could enhance the ability of sTie2 to modify Angpt1 action, although there is no evidence for such factors to date. In fact, in vivo sTie2 is likely to be less effective at suppressing Angpt1 action on endothelium than our data suggest. This is because in vivo Angpt1 is released by pericytes and smooth muscle cells on the abluminal surface of the endothelium, and the ligand is also present in the extracellular matrix around vessels4. Therefore, to access this pool of Angpt1 circulating sTie2 would have to cross the endothelial permeability barrier to access the ligand. In a number of diseases where sTie2 is increased this is accompanied by an increase in Angpt213. As Angpt2 is able to bind Tie23 it is conceivable that where Angpt2 concentrations are increased sufficiently this ligand could compete with Angpt1 for binding to sTie2 and further decrease the ability of sTie2 to modulate Angpt1.It is possible that additional factors present 26. There have also been reports of Tie2 levels varying across different vascular beds32, although others report uniformity in Tie2 expression33. Our simulations show that sTie2 remains relatively ineffective at modulating Angpt1-activation of cellular Tie2 tetramerization even when cellular Tie2 levels are varied over a 100-fold range. Thus we found that increasing cellular Tie2 levels ten-fold further decreased the effectiveness of sTie2 to affect Angpt1 action. Decreasing cellular Tie2 levels in our simulations lowered the concentration of sTie2 required to cause 50% suppression of Angpt1-induced cellular Tie2 activation to 57\u2009nM. Even this lowered concentration of sTie2 corresponds to a molar ratio of more than 300 fold sTie2:Angpt1. Further decreases in cellular Tie2 are likely to increase sensitivity to sTie2, though at such low levels of cellular Tie2 the signal from the cellular receptor will be negligible irrespective of sTie2.Cellular levels Tie2 are known to be increased at sites of angiogenesisin vivo based on the lack of association between circulating sTie2 and disease severity in septic and non-septic critically ill patients34.Our data do not discount Tie2 shedding as an important mechanism regulating endothelial responses to Angpt1. Clearly, proteolytic shedding of cell surface Tie2 would suppress the ability of Angpt1 to signal through Tie2 in cells in which shedding has occurred. Furthermore, it is possible that local very high concentrations of released sTie2 could accumulate abluminally or be present near the surface of endothelial cells undergoing proteolytic Tie2 shedding. In both of these situations Angpt1 actions could be inhibited in the locality of the cells undergoing shedding, although the ability of sTie2 to affect Angpt1 even locally could be blunted where Angpt2 is also locally released and able to compete for sTie2 binding. Furthermore, as discussed, the released ectodomain is unlikely to affect circulating or abluminal Angpt1 action elsewhere in the vasculature, minimizing systemic effects. Others have also questioned whether sTie2 has any systemic effect on Angpt1 action In contrast to natural circulating sTie2, a ligand trap consisting of a dimeric Tie2 ectodomain fusion protein was far more effective at blocking Angpt1-induced cellular Tie2 activation. This likely reflects the increased avidity of the dimeric trap providing it with a significant kinetic advantage relative to monomeric sTie2.In conclusion, this study shows that normal and pathological levels of sTie2 have little effect on Angpt1-induced Tie2 clustering and activation in endothelial cells. These data suggest that therapeutic targeting sTie2 in conditions where the ectodomain is elevated would have little if any effect. However, dimeric sTie2 is able to inhibit Angpt1 activation of endothelial Tie2 at much lower concentrations. Thus in contrast to endogenous monomeric sTie2, the dimeric engineered ectodomain could provide a route for suppression of angiopoietin action.992-Tie2 antibody were obtained from R&D Systems. Human umbilical vein endothelial cells (HUVEC) were maintained in Medium 200 supplemented with Low Serum Growth Supplement and 10% (v/v) foetal bovine serum. Monomeric sTie2 and dimeric sTie2 were purified from medium of transfected HEK-293 cells using the transfection and purification methods we previously described for dimeric sTie235. For monomeric sTie2, cDNA encoding Tie2 ectodomain residues 1\u2013442 (including the secretory leader sequence) was obtained as previously described35 and cloned into the expression vector in frame with a C-terminal hexahistidine tag. cDNA encoding dimeric sTie2 was as described previously35. Following purification protein was dialysed against phosphate buffered saline (PBS) with 10% glycerol and protein concentration determined. Purity was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining and was routinely better than 99%. All other materials were as previously described36.Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY20 for Angpt1-Tie2 binding and corresponding numerical simulations were performed with Matlab (R2015b) SimBiology package and described in the Supplementary Information.The mathematical model based on biochemical kinetics37. Western blots were quantified by densitometric analysis of images. Cellular Tie2 was quantified by comparison of band density with the standard curve constructed from known amounts of recombinant Tie2 blotted and probed on the same membranes. The number of Tie2 molecules at the cell surface was derived from the percentage of total cellular Tie2 that is surface localized and total number of Tie2 receptors in each cell. The percentage of total Tie2 at the cell surface was determined by surface biotinylation as we previously described38 and quantification of biotinylated and non-biotinylated Tie2 by immunoblotting following separation with streptavidin-agarose.The number of Tie2 receptors in HUVEC was determined by immunoblotting. Cells were counted and then lysed in Laemmli sample buffer containing 100\u2009mM dithiothreitol Cleared lysates were heated to 100\u2009\u00b0C for 5\u2009min before resolving proteins by SDS-PAGE. In parallel known amounts of recombinant Tie2 were also resolved by SDS-PAGE. Proteins were transferred to PVDF or nitrocellulose membranes and probed with Tie2 antibody. Immunoreactive proteins were detected with peroxidase conjugated secondary antibody and chemiluminescent detection992-Tie2 antibody. Immunoreactive proteins were detected with peroxidase conjugated secondary antibody and chemiluminescent detection37. Blots were stripped and re-probed with anti-Tie2 or anti-vinculin to assess loading. Bands on Western blots were quantified by densitometric analysis.Prior to ligand treatment HUVEC were incubated in serum-free medium for one hour. As appropriate, and indicated in Results, cells were treated with 0.18\u2009nM Angpt1 in the absence and presence of sTie2 for 15\u2009min. In preliminary experiments we confirmed that at 15\u2009min Angpt1-induced Tie2 phosphorylation had plateaued Supplementary Fig.\u00a0Supplementary Information"}
+{"text": "Cohesins are vital for chromosome organisation during meiosis and mitosis. In addition to the important function in sister chromatid cohesion, these complexes play key roles in meiotic recombination, DSB repair, homologous chromosome pairing and segregation. Egg-laying mammals (monotremes) feature an unusually complex sex chromosome system, which raises fundamental questions about organisation and segregation during meiosis. We discovered a dynamic and differential accumulation of cohesins on sex chromosomes during platypus prophase I and specific reorganisation of the sex chromosome complex around a large nucleolar body. Detailed analysis revealed a differential loading of SMC3 on the chromatin and chromosomal axis of XY shared regions compared with the chromatin and chromosomal axes of asynapsed X and Y regions during prophase I. At late prophase I, SMC3 accumulation is lost from both the chromatin and chromosome axes of the asynaptic regions of the chain and resolves into subnuclear compartments. This is the first report detailing unpaired DNA specific SMC3 accumulation during meiosis in any species and allows speculation on roles for cohesin in monotreme sex chromosome organisation and segregation. Cohesins have also been implicated in promoting nucleolar structure and function16.Cohesins are non-histone proteins of the structural maintenance of chromosomes (SMC) group. Traditionally they are described as the molecular glue that keeps sister chromatids physically attached after DNA replication during both mitosis and meiosis. More recently it has been discovered that cohesins are also central to a number of other fundamental mechanisms in chromosome biology  is the inclusion of the SMC3 subunit.Mitotic cohesin is a multiprotein complex comprising four subunits: SMC3, SMC1\u03b1, an \u03b1-kleisin subunit (Rad21), and a sister chromatid cohesin component . There are also meiosis specific isoforms including SMC1\u03b2, Rad21L and Rec8 (\u03b1-kleisin subunit isoforms), and the sister chromatid cohesin subunit isoform Stromal Antigen 3 (SA3/STAG3). Although many of the meiosis specific functions remain unclear it is known that some meiotic cohesin subunits appear to act in functionally specific combinations15. In mouse, RAD21L is recruited to the chromatin loops of the XY sex body, a pattern similar to the \u0263H2AX repressive histone variant which marks silenced sex chromatin17. Another aspect of meiotic sex chromosome regulation involves an association with nucleoli which has been implicated in their organisation and transcriptional silencing due to the close association at pachytene18. Proteins that localise to the sex chromosomes during pachytene have also been observed to accumulate in nucleoli which may provide some insights into meiotic nucleolar function20. During mouse zygotene, nucleoli form on nucleolar organising regions (NORs) however at pachytene these nucleoli disassociate from the NORs, coalesce and migrate to the sex body until diplotene21. While similar nucleolar-sex chromosome organisation has been reported in multiple eutherian species24, metatherians25 and even invertebrates26, the purpose of this association remains unclear.In meiotic cells, immunostaining for cohesin complexes shows the same localisation as the SC to the extent that some investigations now report the CC to be part of the SC28 with seven known pseudoautosomal regions  that mediate pairing to form an alternating XY 10 sex chromosome chain during meiosis I30. The complexity of monotreme sex chromosomes and their homology to the avian Z raises fundamental questions about the mechanisms underlying meiotic pairing, recombination, evolution of meiotic sex chromosome inactivation (MSCI) and sex chromosome segregation. Our recently published findings indicate that the platypus SC may be different to other mammalian SCs with 3 significantly divergent copies of SYCP3 which are expressed at high levels in platypus testis31. However, there is currently no information about the role of cohesin proteins in platypus meiosis.The platypus carries a highly unusual sex chromosome system comprising 5X and 5Y chromosomesIn the present study we examine cohesin and synaptonemal complex dynamics during meiotic prophase I in platypus, with particular focus on the sex chromosome chain. We discovered a highly dynamic and specific reorganisation of accumulated cohesin associated with the formation of the sex chromosome complex and to a large nucleolar structure. Our finding of temporal and differential cohesin accumulation specifically on asynapsed sex chromosome chromatin loops implicates a possible role for cohesin in sex chromosome organisation at prophase I. Specifically we speculate roles in unpaired DNA-specific functions and/or establishment of configurations or markers for faithful alternate segregation of the 10 sex chromosome elements at anaphase I.In order to investigate the dynamics of SC formation in monotreme prophase I, we used antibodies raised against the central element protein SYCP1 and the cohesin subunit SMC3 to visualise chromosome axial cores. SMC3 is a component of all meiotic cohesin complexes discovered to date and platypus SMC3 is highly conserved with over 99% pairwise identity with human SMC3 at the amino acid level Figure\u00a0. Western32 suggests that this is a large nucleolus.SYCP1 immunostaining showed an expected SC staining pattern in platypus prophase I cells, however the SMC3 antibody detected remarkably strong localised accumulations unlike mouse cells at the same stage Fig.\u00a0. As this5 (Oa_Bb-152P15) and Y2 (CH236-145P9). Y5 and Y2 consistently reside within the regions of SMC3 accumulation, demonstrating that the platypus sex chromosomes experience significantly more cohesin loading in prophase I relative to the autosomes  which also lacks a central element signal (SYCP1). This is because the bulk of the sex chromosome DNA is not homologous and therefore doesn\u2019t undergo SC-mediated synapsis involving SYCP1. As the intense SMC3 accumulations in platypus prophase I nuclei lacked SYCP1 this represented the location of sex chromosomes. This was confirmed by immuno-FISH on meiotic spreads using anti-SMC3 and anti-SYCP1 antibodies followed by two Y chromosome specific BAC probes targeting Y which al34  regions. After SMC3 immunostaining, imaged cell coordinates were recorded prior to a series of DNA FISH experiments using combinations of Xes Table\u00a0. At mid es Table\u00a0. By contes Table\u00a0.Table 1P5 BACs and four X1 BACs, b) X1Y1 [PAR1] and Y4X5 [PAR8], c) X3 and Y3, d) Y3X4 [PAR6] and X2Y2 [PAR3], e) Y2 and Y5. This revealed that the X and Y specific sequences  consistently colocalised with the intense SMC3 domain which lacked central element staining  were positioned either outside or on the periphery of the SMC3 domain and in all cases had associated central element staining  five Xing Fig.\u00a0. In conting Fig.\u00a0.Figure 831 and the absence of platypus MSCI32 currently remain unclear in terms of pairing checkpoints or preparations for metaphase and segregation of the 10 sex chromosomes.This is the first detailed analysis of the organisation at male meiotic prophase I of the most complex known mammalian sex chromosome system. We observed massive and differential recruitment of cohesin specifically to unpaired parts of the sex chromosome chain and this specific and transient cohesin accumulation to the sex chromosome complex coincided with nucleolar association and progressive condensation. There were clear differences between platypus and mouse in the temporal and spatial cohesin accumulation dynamics including the rapid loss of SMC3 from specific regions of sex chromosomes. The relationship between these differences and our recent reports on the highly diverged platypus SYCP338. In some cases the former may simply be due to the fact there is only one chromosomal axis on the sex chromosomes whereas the signal observed between autosomal homologues is created from the close proximity of two axial cores41. Such a phenomenon was also observed for grasshopper B chromosomes42. In addition, generalisations are confounded by differing cohesin subunit loading dynamics occurring on AEs following pachytene38. Interestingly, in grasshopper preleptotene cells the X chromosome lacks SMC3, however by leptotene the levels on the X are similar to the autosomes and by pachytene the loading appears weaker than that observed on the synapsed autosomes39. In contrast, other mammals such as the armadillo have SMC3 signals on the unpaired sex chromosome axial cores which is indistinguishable in intensity from autosomal SMC343. In horse this is more pronounced, where the X shows greater SMC3 recruitment at axial cores than that seen on the autosomes44. Contrasting this are observations in rat where cohesins form scattered foci along the axial core length41. Despite the evolution of species specific variations in cohesin loading, platypus sex chromosomes stand out by the sheer magnitude of axial core SMC3 accumulation relative to autosomes.In platypus we observed a remarkably different XY cohesin recruitment programme in terms of the extent and specificity to unpaired regions which deviates significantly from that described for other species. Although there are exceptions, the majority of studied species show cohesin sex chromosome loading at pachytene as weaker, as for REC8 in mouse, or comparable to autosomes, such as STAG3 or SMC332. The association of the condensed sex chromosome chain with the nucleolus in combination with heavy cohesin loading specifically on the unpaired DNA invites the idea that MSCI may not be required to avoid checkpoint arrest. We are unable to test this hypothesis by inducing autosomal inversions or depleting SMC3 in platypus meiosis therefore rendering these ideas untestable. However, the identification of additional antibodies recognising DDR pathway components and detailed epigenetic profiles on the sex chromosomes in platypus may aid in understanding the observed cohesin dynamics in respect to meiotic progression and possibly segregation. It has been shown that NOR chromosomal positioning dictates pachytene nucleolar localisation during spermatogenesis45 and we observe here a conserved central nucleolar location due to the \u2018interstitial\u2019 NOR on platypus chromosome 6. The fact that in other species such as mouse the nucleolar-sex chromosome association begins later during pachytene may imply a requirement for platypus sex chromosomes association with nucleoli for chaperoning through the critical period of homologue synapsis21. Such a requirement is supported by the observation of Daish and colleagues that Y5 closely and consistently associates with the nucleolus from the earliest pachytene stages32 prior to chain contraction (see below) however this association was highly likely but also showed exceptions at early stages of pachytene as seen in Fig.\u00a046 and also at the mating-type heterochromatic region of fission yeast where it is recruited by Swi6(HP1)47.In platypus early pachytene, the cohesin subunits SMC3 and STAG3 colocalise with autosomal SCs similar to other species however cohesin is then massively recruited to the unpaired sex chromosome axial cores. Only in platypus is the increased level of cohesin enrichment restricted to unpaired regions of the sex chromosomes. This raises the question of what cohesin may be doing differently on the unpaired DNA in terms of function and/or interactions and invites speculation on roles in protecting unpaired DNA from checkpoint surveillance machinery which may otherwise halt meiotic progression or simply one of facilitating condensation of the sex chromatin. We recently showed that therian-like sex chromosome transcriptional silencing (MSCI) evolved after the divergence of monotremes due to absence of repressive epigenetic marks such as \u03b3H2AX and DNA damage repair pathway components on the sex chromosome chain during late pachytene49. Nucleolar association is required for establishment of X chromosome inactivation in somatic cells50 demonstrating strong functional links between heterochromatin, cohesins, the nucleolus, and sex chromosomes, however the significance of this in the context of platypus meiosis remains unclear and currently is functionally untestable.Another area which requires investigation is the mechanism underlying the alternate segregation of the 10 sex chromosomes, an event which may be contingent on the sex chromosome specific accumulation and/or modification of structural complexes incorporating elements of both cohesin and synaptonemal complex components. There are examples to support such speculation in species with achiasmate sex chromosomes which utilise SC structural components to mediate faithful segregation52 which possibly represents an ancestral mode of organising heteromorphic sex chromosomes that predates the advent of sex chromosome specific silencing (MSCI). Interestingly, while monotremes and birds have sex chromosomes that share significant homology53 the heteromorphic chromosomes experience meiosis in males and females respectively. An example where proteins normally function to mediate homologous synapsis having roles in heteromorphic sex chromosome associations is seen in marsupials where SC proteins form a structure called the dense plate. The dense plate appears to mediate and maintain sex chromosome associations in the absence of a PAR54 and therefore further highlights the dynamism and functional flexibility of the meiotic machinery following sex chromosome evolution. To our knowledge however this is the first report showing such a marked association of SMC3 on XY specific chromatin.Our results show that reorganisation of SMC3 distribution coincides with sex chromosome chain condensation during pachytene. In birds the ZW pair undergoes synaptic adjustment and equalisationIn summary, we have shown that organisation of the platypus sex chromosome complex during prophase shows distinct and dynamic cohesin loading that is differentially loaded between paired and unpaired parts of the sex chromosome chain, for which we offer a simplified and functionally speculative model Fig.\u00a0. Key poiThe authors confirm that all experiments were performed in accordance with relevant guidelines and regulations at the University of Adelaide. All experimental approaches were done according to the University of Adelaide biosafety and ethics committee regulations . Platypus samples were collected in 2002  and 2008 (AEC permit no. S-49-2006) at the Upper Barnard River  during the breeding season. Mouse testis were obtained from three week old animals (Swiss white).et al.30. Briefly the testis were placed in a petri dish containing 1 x PBS with 1 x Protease Inhibitor (Roche). The surrounding tunica albuginea was punctured and the testis material was teased apart using needles resulting in the release of meiotic cells and sperm from the ruptured tubules. A pipette was used to break up and flush the remaining material to ensure maximal release of cells. The resulting cell suspension was mixed with DMSO , slow cooled and stored in liquid nitrogen for future use.The method of meiotic sample preparation and storage was the same as previously described by Daish et al.55. Briefly, a 75\u2009\u00b5L aliquot of the testis 10% DMSO suspension was thawed. Either 75\u2009\u00b5L 0.3\u2009M sucrose or 75\u2009\u00b5L of 75\u2009\u00b5M KCl was added to the testis material and left to sit at room temperature for 5\u2009minutes. Next 50\u2009\u00b5L of 1% PFA/0.15% Triton X-100 pH 9.5 was dropped onto slides and spread with a pipette tip. 20\u2009\u00b5L of the testis material/hypotonic solution was then dropped onto the PFA coated slides. Slides were kept in a moist chamber for 2\u2009hours and then washed in 50\u2009mL of 1 X PBS followed by a final wash with water and wetting agent. Slides were air dried and used immediately for immunostaining or stored at \u221220\u2009\u00b0C.Meiotic spreads of testis material were prepared by a method based on that previously described by Peters et al.52. Briefly, slides were blocked in 1 X PBS with 0.5% w/v BSA and 0.5% w/v milk powder three times for five minutes and incubated in a humid chamber with a primary antibody (1:200 dilution) in 1 X PBS/10% w/v BSA for either 2\u2009hours at 37\u2009\u00b0C or overnight at room temperature. Slides were then washed in 1 X PBS three times for five minutes each and blocked in 10% v/v goat serum in blocking buffer for three times for five minutes each and incubated at 37\u2009\u00b0C for 2\u2009hours in a humid chamber with a secondary antibody (1:400 dilution) in 10% v/v goat serum in blocking buffer . Finally slides were washed three times for five minutes in 1 X PBS, dipped in DAPI (1:5000 dilution) for one minute, washed twice with MQ water and mounted with Vectashield (Vecta Laboratories).Immunostaining was carried out as per Schoenmakers 39.To observe the central element we used an antibody  raised against the central element protein; Synaptonemal Complex Protein 1 (SYCP1). We employed the use of an SMC3 antibody  to visualise the axial core in platypus since it has been previously shown that SMC3 recruitment follows synaptic progressionIn Situ Hybridisation (FISH) based on the original protocol described by McDougall et al. (1972) was optimised for PFA fixed meiotic material. Briefly, BAC DNA probes were directly labelled using Klenow and a random 9mer primer overnight with either SpectumOrange or SpectrumGreen 2\u2032deoxyuridine-5\u2032-trisphosphate (Abbott Molecular). To reduce the background signal on slides, labelled probes were subsequently co-precipitated with salmon sperm and sonicated platypus male genomic DNA. Pellets were dissolved in deionised formamide and 2 X hybridisation buffer containing dextran sulfate. Slides containing the PFA fixed meiotic material were dehydrated in an alcohol series (70\u2013100% ethanol) for 5\u2009minutes each, treated with RNase A (100\u2009\u00b5g/mL) for 30\u2009minutes at 37\u2009\u00b0C, pepsin  for 10\u2009minutes at 37\u2009\u00b0C, fixed in formaldehyde  in PBS/50\u2009mM MgCl2, dehydrated in an alcohol series (70\u2013100% ethanol) for 5\u2009minutes each, denatured in 70% formamide/2 X SSC at 70\u2009\u00b0C for 7\u2009minutes for the first FISH and 3\u2009minutes for each subsequent FISH on the same slide, dehydrated (70\u2013100% ethanol) for 5\u2009minutes each and air dried ready for hybridisation with the labelled probe. The probes were applied to the dry slide and covered with a coverslip, sealed with rubber cement and incubated overnight at 37\u2009\u00b0C in a moist chamber. The coverslip was removed and slides were washed three times in 50% formamide/2 x SSC followed by 2 x SSC, once in 2 X SSC at 42\u2009oC for 5\u2009minutes, once at 60\u2009oC in 0.1 X SSC for 5\u2009minutes and finally once at 42\u2009oC in 2 X SSC for 5\u2009minutes. Slides were then stained with DAPI for one minute, washed twice with MQ water and mounted with Vectashield (Vecta Laboratories).A method of Fluorescence http://www.gimp.org/) and ImageJ .Slides were visualised using a Zeiss AxioImager 2.1 microscope equipped with a 10x ocular and 10x, 20x, 63x and 100x objective lenses. Fluorescent tags were visualised using 3 filters: DAPI, for DAPI stained DNA; GFP, for SpectrumGreen and Alexa 488 and; DS red, for SpectrumOrange and CY3. Images were recorded via an Axiocam CCD-camera and Zeiss Axiovision software. Unless otherwise stated, images were captured using the 100x objective lens, appearing as 1000x with the 10x ocular. Images were processed using Axiovision (Zeiss), GIMP 2.8 (Supplementary Data"}
+{"text": "We previously demonstrated the effects of Ankaferd hemostat (AH) on human umbilical vein endothelial cells (HUVECs) in Turkish Journal of Hematology [1]. Endothelial cells adhered to each other within seconds and critical intracellular transcription factors were activated just after the application of AH (5 \u00b5L) to the HUVECs . Rapid vWe further determined that the cellular effects of AH on HUVECs are clinically important in pharmacobiological hemostasis ,2,3. End"}
+{"text": "HOXA10 and HOXB13 revealed association with Epithelial-Mesenchymal Transition (EMT). While HOXA10 expression correlated positively with E-Cadherin and negatively with Vimentin expression, HOXB13 showed the reverse trend. Chromatin immunoprecipitation study in vitro revealed the ability of E7 to increase HOX gene expression by epigenetic regulation, affecting the H3K4me3 and H3K27me3 status of their promoters, resulting from a loss of PRC2-LSD1 complex activity. Thus, besides identifying the deregulated expression of HOX cluster members in HPV16 positive cervical cancer and their association with EMT, our study highlighted the mechanism of HPV16 E7-mediated epigenetic regulation of HOX genes in such cancers, potentially serving as bedrock for functional studies in the future.The Homeobox (HOX) genes encode important transcription factors showing deregulated expression in several cancers. However, their role in cervical cancer pathogenesis, remains largely unexplored. Herein, we studied their association with Human Papillomavirus type 16 (HPV16) mediated cervical cancers. Our previously published gene expression microarray data revealed a significant alteration of 12 out of 39 HOX cluster members among cervical cancer cases, in comparison to the histopathologically normal controls. Of these, we validated seven  by quantitative real-time PCR. We identified decreased HOXA10 expression as opposed to the increased expression of the rest. Such decrease was independent of the integration status of HPV16 genome, but correlated negatively with E7 expression in clinical samples, that was confirmed  Cervical cancer is the fourth leading cancer among women globally with HPV16 infection alone associating with ~50% of the cases \u20138. Such HOX genes are a family of transcription factors that show deregulated expression in a variety of cancers and known as targets of PRC2-complex during embryonic development \u201311. The Since HOX cluster members function as transcription factors, the common thought was that these show enhanced expression in cancers driving carcinogenesis by activating the downstream signalling cascades. However, several studies reported reduced expression of HOX cluster encoded molecules. Aberrant expression of HOX cluster genes is integral to a network of regulatory mechanisms involved in normal adult tissue homeostasis, and maintenance and activation of stem cell self-renewal process, crucial to malignant transformation \u201320. The Therefore, in this study, we focussed on elucidating the expression profile of HOX cluster member genes associated with cervical cancer pathogenesis, and their probable association with the Epithelial Mesenchymal Transition (EMT). We also aimed at identifying the association of HOX cluster expression with viral factors like HPV16 integration status and E7 expression among the cervical cancer cases. Finally, we elucidated the role of HPV16 E7 in epigenetically affecting the expression of HOX cluster genes through concomitant functional abrogation of PRC2-LSD1 complex in cervical cancer cell lines. Towards this, we estimated the H3K4me3 and H3K27me3 status of the promoters of the HOX genes with altered expression.To identify the HOX cluster members with deregulated expression among HPV16 positive cervical cancer cases in comparison to the histopathologically normal control samples (HPV negative and HPV16 positive), microarray data generated on 20 HPV16 positive cervical cancer cases, 11 HPV16 positive histopathologically normal samples and 11 HPV negative histopathologically normal samples (Accession No: GSE67522) was used. The histopathologically normal samples were grouped together for comparison, as the HPV negative and HPV16 positive normal samples did not reveal any differentially expressed genes as described in our previous study .Out of 39 HOX cluster genes, the microarray analysis revealed expression levels of 31 genes. Of these, 12 members of the HOX cluster portrayed significant differential expression among the cervical cancer cases, as compared to the histopathologically normal controls Figure ..We validated the expression profile of seven out of the twelve deregulated HOX cluster members, HOXA10, HOXA13, HOXB13, HOXC8, HOXC9, HOXC11 and HOXD10, using SYBR green based quantitative real-time PCR. For such analysis, we used a sample set of seventy cervical cancer cases  and thirty histopathologically normal cervical samples (11 HPV16 positive and 19 HPV negative). Such analysis, confirmed significant altered expression of all seven HOX cluster members among the cervical cancer cases, in comparison with the HPV negative control samples . Of thesDeregulated expression of several HOX genes in cervical cancer cases prompted us to interrogate the impact of such changes in cervical cancer pathogenesis. Existing reports \u201324, highE-Cadherin expression was significantly decreased among the cervical cancer cases  as compared to the HPV negative control samples. The cervical cancer samples with episomal HPV16 genome showed a reduction by 11.79-fold (p=0.03), while the cervical cancer samples with integrated HPV16 genomes showed a reduction by 6.02-fold (p=0.04) Figure . HoweverExpression of both the EMT markers, E-Cadherin and Vimentin, showed correlation with the expression of the HOX cluster members, HOXA10 and HOXB13 . While Hin vitro, where HOXA10 showed significantly reduced expression  in C33A cells transfected with E7 expressing plasmid, as compared to untransfected C33A cells . However, HPV16 E7 seemed to play an important role in the activation of HOX cluster genes despite the fact that some of the HOX cluster members failed to show any significant correlation with HPV16 E7.The HOX cluster members that revealed altered expression in cervical cancer cases in our study, also show deregulated expression in one or more of the other cancer types. Similar to our observations, HOXA13 shows enhanced expression in oesophageal squamous cell carcinoma, gastric cancer and hepatocellular carcinoma \u201335. HoweThe increased expression of majority of the HOX cluster members was also in line with our hypothesis of HPV16 E7 driven abrogation of PRC2-LSD1 complex activity leading to loss of gene silencing. A significant correlation of HOXA10 and HOXD10 expression with HPV16 E7 expression highlighted the impact of E7 on HOX gene expression. One of the possible mechanism of HOXA10 reduction could be through HPV16 E7-dependent activation of DNMT1 leading to HOXA10 promoter methylation , 41. On in vitro observations could be due to the heterogeneity of cancer tissues as opposed to the homogenous pool of cells in vitro. The gain of H3K4me3 and loss of H3K27me3 marks at the promoters of HOX cluster genes could explain the activated expression of such genes, under the influence of HPV16 E7 expression. McLaughlin-Drubin et al., [Although E7 expression did not show significant correlation with the expression of majority of the HOX cluster members among the cervical cancer samples, E7 overexpression in HPV negative cervical cancer cell line resulted in the activation of HOX cluster genes. Such differences in clinical and  et al.,  had earl et al., . In this et al., .In conclusion, along with identifying altered expression of HOX cluster genes and their association with metastasis in HPV16 related cervical cancers, our study also highlighted the master regulatory role of HPV16 E7 in modulating the expression of HOX cluster genes. Such expression alteration predominantly involved enrichment of H3K4me3 and loss of H3K27me3 marks at the gene promoters. Thus global studies employing ChIP-Seq for identifying the status of H3K4me3 and H3K27me3 marks at the gene promoters, along with RNA-Seq, seem to be important to identify the concomitant changes in the transcriptome brought about by HPV16 E7. This would further identify and confirm the role of HPV16 E7 in the epigenetic regulation of host cellular transcription. Such studies would also help in identifying molecular targets for therapy of HPV-active cervical cancer, which have distinctly different molecular phenotypes from HPV-inactive cervical cancer, as has been identified in a recent report studying TCGA cervical cancer data .The healthy control cervical biopsy samples were derived from married women aged 36 \u2013 70 years (median: 52 years) without any prior history of cervical dysplasia/malignancy, from Calcutta Medical College and Hospital, Kolkata, West Bengal, India and were histopathologically confirmed as normal. The Cervical cancer samples used for this study were derived from married subjects aged 34 \u2013 65 years (median: 52 years) attending a cancer referral hospital . The cervical cancer biopsy samples were histopathologically confirmed as invasive squamous cell carcinomas  categorised using the WHO classification and clinically diagnosed as stage III and above as per FIGO classification. Samples included in the different study groups did not differ significantly in terms of their median age.The samples were collected with informed consent from the participants, approved by the ethical committee for human experimentation of the National Institute of Biomedical Genomics (NIBMG) and frozen post collection. We performed all experiments in accordance with the approved ethical guidelines and regulations. All such clinical samples were tested for their HPV/HPV16 status by PCR based detection as described earlier . Confirm-12 v4 Expression Bead Chip array based platform generated by our laboratory [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67522). This data was used to identify the distinct differences of HOX cluster members at the gene expression level, between the HPV16 positive cervical cancer cases and the histopathologically normal control samples.The microarray data was generated using Illumina HumanHTboratory  and submRNA isolation, reverse transcription and quantitative real-time PCRs were all performed, as described previously . The pri2. C33A cells were transfected with1 \u03bcg of plasmid pcDNA3.1-HPV16 E7 vector generated in our laboratory [HPV negative cell line C33A and HPV16 positive cell line SiHa were cultured in DMEM supplemented with 10% FBS, 50 Units/ml of penicillin and 50 \u03bcg/ml of streptomycin at 37\u00b0C and 5% COboratory , using L3) and incubated at room temperature for 30 mins with agitation. The DNA was reverse cross-linked by adding 12\u03bcl of 5M NaCl per 100\u03bcl elute and incubated at 65\u00b0C overnight, followed by RNase A treatment and purified using PCR purification kit.SiHa and C33A cells were cultured as described above. For the analysis of H3K4me3 and H3K27me3 marks, we used SiHa cells and C33A cells, untransfected or transfected (HPV16 E7 expressing). The cells were cross-linked using formaldehyde at 1% final concentration and incubated at room temperature for 10 mins. The cross-linking was quenched using glycine at a final concentration of 0.125M and incubated at room temperature for 5 mins, followed by washing twice with 1X PBS. The cells were harvested by scraping in 1X PBS (with Protease Inhibitor cocktail) and pelleting the cells by centrifugation at 300xg for 5 mins, at 4\u00b0C. The cells were re-suspended in 1ml of SDS lysis buffer  and incubated on ice for 15 mins. The lysate was sonicated at 30% amplitude, 14 \u00d7 10 secs pulses, 20 secs pauses, followed by centrifugation at 14,000 rpm at 4\u00b0C. The lysate was pre-cleared with 80\u03bcl of 50% slurry of Protein A Agarose beads for 60 mins at 4\u00b0C, followed by centrifugation at 4,000 rpm for 1 min at 4\u00b0C. Specific antibody (5\u03bcg) was added to the supernatant and precipitated overnight at 4\u00b0C with rotation. After overnight rotation, 60\u03bcl of blocked Protein A Agarose beads  were added to the antibody/protein complex, followed by rotation for 60 mins at 4\u00b0C. The beads were pelleted by centrifugation at 1,000 rpm for 1 min at 4\u00b0C, followed by sequential washing with 1X Low Salt Immune Complex wash buffer , 1X High Salt Immune Complex wash Buffer , 1X LiCl Immune Complex wash Buffer  and 1X TE . The beads were eluted with freshly prepared elution buffer  was performed to check for statistical significance of the changes in expression levels, as the \u0394Ct values did not follow normal distribution. Multiple sets of experiments using cell line model were compared using ANOVA for statistically significant differences in expression levels between the experimental categories. All statistical analyses were done using SPSS v16.0. The p values were reported after performing Bonferroni multiple testing corrections."}
+{"text": "The images for S1 FigDkk3 cds. (B) Validation of the Dkk3 exon 2 and exon 3 primer sets using increasing concentrations of 1st strand cDNA primed total RNA isolated from two independent mouse astrocyte preparations. Each data point determined in triplicate. (C) Dkk3 mRNA levels were normalized to GAPDH mRNA. Data shown as (mean \u00b1 SE) from 3 independent experiments.(A) The position of the PCR primer pairs used for amplification of exon 2 and exon 3 shown on the (TIF)Click here for additional data file.S2 FigDkk3 gene. TSS2 is positioned upstream of the forward LoxP site (black box) of the gene and the downstream LoxP site (in black) is positions 35 nt upstream of exon 3. Position of the CFP234 5\u2019RACE primer indicated by arrow. (B) Sequence of the 5\u2019UTR of the Cfp mRNA captured by 5\u2019RACE highlighted in yellow.(A) Map of the insertion of the CFP promoter trap in gene-edited intron 2 of the (TIF)Click here for additional data file."}
+{"text": "Fig 7 contains an error on the x-axis. Fecal testosterone metabolites (fTM) and fecal glucocorticoid metabolites (fGM) values that are plotted on day 2 after an ACTH injection should be plotted on day 1, and the values that are plotted on day 1 should be plotted on day 2. Please see the corrected"}
+{"text": "C9orf72) is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF) complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1) autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings.Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 ( C9orf72; DeJesus-Hernandez et al., Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting motor neurons . The most prominent feature of the protein is the presence of a \u201cDifferentially expressed in normal and neoplastic cells\u201d (DENN) domain  translation of the hexanucleotide expansion  should be pursued. Understanding of how the mutant hexanucleotide expansion of C9orf72 affect neuronal membrane traffic and autophagy would benefit from analyses using appropriate cellular and While the manuscript was in its final stage of review, Chen and colleagues reported a C9ORF72/SMCR8/WDR41-containing complex that also contain ATG101 . A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy. Sci. Adv. 2(9): e1601167). The C9orf72 complex has Rab39B GEF activity, and also regulates Ulk1 levels as well as autophagic activity.BLT conceived and wrote the article.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "We discover that two different HS modifying enzymes, Hs2st and Hs6st1, indeed differentially modulate the properties of emerging Fgf8 protein concentration gradients and the Erk signalling output in response to Fgf8 in living tissue in ex vivo cultures. Both Hs2st and Hs6st1 are required for stable Fgf8 gradients to form as rapidly as they do in wild-type tissue while only Hs6st1 has a significant effect on suppressing the levels of Fgf8 protein in the gradient compared to wild type. Next we show that Hs2st and Hs6st1 act to antagonise and agonise the Erk signalling in response to Fgf8 protein, respectively, in ex vivo cultures of living tissue. Examination of endogenous Fgf8 protein and Erk signalling outputs in \u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 embryos suggests that our ex vivo findings have physiological relevance in vivo. Our discovery identifies a new class of mechanism to tune Fgf8 function by regulated expression of Hs2st and Hs6st1 that is likely to have broader application to the >200 other signalling proteins that interact with HS and their function in neural development and disease.Fibroblast growth factor (FGF) morphogen signalling through the evolutionarily ancient extracellular signalling-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway recurs in many neural and non-neural developmental contexts, and understanding the mechanisms that regulate FGF/ERK function are correspondingly important. The glycosaminoglycan heparan sulphate (HS) binds to FGFs and exists in an enormous number of differentially sulphated forms produced by the action of HS modifying enzymes, and so has the potential to present an extremely large amount of information in FGF/ERK signalling. Although there have been many studies demonstrating that HS is an important regulator of FGF function, experimental evidence on the role of the different HS modifying enzymes on FGF gradient formation has been lacking until now. We challenged  Summary: Regulating the fine structure of cell surface carbohydrates provides a mechanism for fine tuning the formation of morphogen gradients in the embryonic mouse fore-brain. Fibroblast growth factors (FGFs) are secreted signalling proteins that function as morphogens by forming protein concentration gradients emanating from focal sources to elicit dose-dependent outcomes . The bron HS polymer is synthesised by EXT enzymes and modified by enzymatic addition and removal of sulphate groups by heparan sulphotransferases (HSTs) and sulphatases (SULFs), respectively. HS modifying enzymes underwent evolutionary expansion from the origin of multicellular animals suggesting a role in evolving tissue complexity. Mammals have 17 HS modifying enzymes classed into five categories  according to exactly where on the uronic acid\u2013glucosamine disaccharide residue they add or remove sulphate groups. Many HS modifying enzymes are expressed in developing brain, pointing to functional importance in neural development , while Hs6st1 sulphates position 6 of glucosamine (6-O HS sulphation). We turned to the tractable system provided by Fgf8 signalling in the ventricular zone (VZ) of the cortico-septal boundary (CSB) at the developing mouse embryonic day (E)14.5 telencephalic midline to investigate the function of Hs2st and Hs6st1 in Fgf8 gradient formation, and the Erk response to Fgf8 in living tissue. Severe developmental phenotypes map to the CSB when FGF8/Fgf8 or HS are disrupted in humans or mice, indicating their functional importance in this region  from the tissue. According to the model, reaching a steady-state protein concentration gradient is key for the interpretation of the gradient by cells . FGF proelopment . Furtherll lines . Althougs region . Hs2st\u2212/ embryos . This woHs2st and Hs6st1 in Fgf8 gradient formation modulation in vivo is difficult as there are many variables that are not tenable to experimental control, such as the amount of Fgf8 protein emanating from the source. Therefore, we designed an ex vivo bead assay in which we challenged slices of embryonic forebrain in which HS was enzymatically removed (Heparanase treatment) or lacking functional HS modifying enzymes Hs2st (Hs2st\u2212/\u2212 tissue) or Hs6st1 (\u2212/\u2212Hs6st1 tissue) with a constant amount of Fgf8 to probe the formation of a Fgf8 protein gradient through time. The ex vivo assay allowed us to probe Fgf8 gradient emergence in much smaller timescales, that is impossible to assay in vivo, providing a higher temporal resolution of Fgf8 gradient formation.Fgf8 gradient formation involves the production of Fgf8 at the source, followed by Fgf8 transport through the extracellular matrix in the tissue, activation of the signalling pathway through receptor binding of responding cells, and finally Fgf8 protein clearance. The emergence of the Fgf8 protein gradient could be modulated by HS modifying enzymes at any step of the pathway. Dissecting the role of Fgf8 mRNA expression is restricted to the angle between the cerebral cortex and the septum at the CSB, identifying this region as the source of Fgf8 protein in vivo (ex vivo by embedding beads infused with recombinant Fgf8 protein (or BSA control) into the VZ at the CSB angle of telencephalic midline tissue explants (dissected region indicated by red dashed line in [Fgf8]) varies with distance (d) from the Fgf8-bead edge .In E14.5 mouse telencephalon, WT) tissue was challenged with an Fgf8-soaked bead, the concentration of Fgf8 protein ([Fgf8]) is highest closest to the Fgf8-bead and decays with increasing distance from the bead edge ([Fgf8] [expressed as arbitrary units (AU)] in the tissue against distance from the bead at each time-point produces classic decay curves with [Fgf8] falling non-linearly with increasing distance from the bead. Comparison of these curves shows no change over the 1-4\u2005h period, indicating that a steady state gradient is achieved <1\u2005h after introducing the Fgf8-bead into the tissue  gradient over time by removing HS (pre-treating WT tissue with heparanase) or using tissue from \u2212/\u2212Hs2st or \u2212/\u2212Hs6st1 embryos. Representative images of Fgf8 immunofluorescence show that these three conditions resulted in an unstable [Fgf8] gradient that does not achieve the WT steady state between 1 and 4\u2005h  indicating impaired ability for forming a steady state [Fgf8] gradient. There were also significant deviations from the WT [Fgf8] gradient, although here, heparanase-treated, \u2212/\u2212Hs2st, and \u2212/\u2212Hs6st1 experiments showed different effects. Following heparanase treatment, the spatial concentration curve was higher than WT after 1\u2005h and then dropped rapidly, becoming lower than WT by 2\u2005h and barely detectable after 4\u2005h, all of these differences were significant. Both \u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 tissue also formed non-steady-state [Fgf8] gradients. However it is important to note that the [Fgf8] gradient in \u2212/\u2212Hs2st tissue was not significantly different to the WT [Fgf8] gradient after 4\u2005h in culture, although the [Fgf8] gradient fluctuated in the earlier time-points. The spatial concentration curve formed in \u2212/\u2212Hs2st tissue was significantly higher than WT after 1\u2005h, lower than WT after 2\u2005h, and not significantly different to WT after 4\u2005h  (\u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 experiments) (These data demonstrate that the formation of a steady-state eriment) E,I,M,Q, [Fgf8] gradient is determined by three independent factors: [Fgf8] at the source; the amount of Fgf8 protein in the gradient (the amplitude); and the rate at which [Fgf8] decays with increasing distance from the source (the decay). In these experiments [Fgf8] at the source is constant (see below). We next asked whether the HS manipulations contribute to changing the [Fgf8] gradient by modulating its amplitude, decay, or both.The [Fgf8] gradient from the area under the curves in WT . Comparing [Fgf8] amplitude between time-points of each condition, the amplitude of WT tissue remained constant over time with no significant differences, indicating a steady-state amplitude was achieved as early as 1\u2005h of culture . Heparanase treatment resulted in amplitude not significantly different to WT for the first 2\u2005h followed by a significant drop to very close to detection threshold ; the more negative the slope the more rapid the [Fgf8] decay as a function of distance (WT and any HS condition at any time-point (Table\u00a0S1C), indicating that none of the HS manipulations described here have a significant impact on [Fgf8] decay as a function of distance from the source. Interestingly, although the gradient of \u2212/\u2212Hs6st1 was significantly different from WT at the 4\u2005h time-point, neither the [Fgf8] amplitude or [Fgf8] decay , gives [Fgf8] at the source which is the surface of the Fgf8 bead (<1\u2005\u00b5m from bead edge), and there were no significant differences  except between WT and heparanase treated tissue at the 4\u2005h time-point. This indicates the bead [Fgf8] was constant in all conditions and time-points (except heparanase treated cultures after 4\u2005h). This was confirmed by direct quantification of bead [Fgf8] , so bead [Fgf8] was not a variable contributing to differences observed between other conditions and time-points.The decay of a protein gradient is described by different equations depending on both diffusion kinetics and whether the degradation rate, for example clearance of Fgf8 by RME, is linear or non-linear as a function of distance from the source , as opposed to modulating the decay of Fgf8 as a function of distance from the source ([Fgf8] decay). Critically, our analysis found that Fgf8 behaved differently in living \u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 tissue with a much stronger effect in \u2212/\u2212Hs6st1 tissue, indicating that the Hs6st1 plays the dominant role in supressing Fgf8 levels.To conclude, the rapid (<1\u2005h) formation of a steady state WT cultures, pErk forms a gradient with the highest concentration of pErk ([pErk]) closest to the bead ([pErk] gradient after 4\u2005h (red=black>>blue lines in Table S2). Our analysis quantifies both Fgf8 and pErk signals at each distance from the Fgf8-bead, and these values were combined in a [pErk] (y-axis) vs [Fgf8] (x-axis) plot to generate a dose response curve showing the pErk response elicited by different [Fgf8] in the tissue , and the [Fgf8]/[pErk] dose response shows strong de-sensitisation to Fgf8 . The [Fgf8]/[pErk] dose response shows that \u2212/\u2212Hs2st tissue becomes progressively more sensitive to Fgf8 over time  significantly reduces the  to Fgf8 J. In cone points C and thecultures D where t compare I to L. Wex vivo experiments showed us how living tissue reacts when challenged with an implanted bead acting as an experimentally introduced source of Fgf8, likely at non-physiological levels. HS was found to be critical for [Fgf8] gradient formation and the Erk response to Fgf8 as the loss of HS resulted in no detectable [Fgf8] gradient after 4\u2005h in culture and loss of Erk response to Fgf8. Hs2st was found to play a minor role in the maintaining a stable Fgf8 gradient as removal of Hs2st resulted in [Fgf8] fluctuations during [Fgf8] gradient formation before eventually reaching WT [Fgf8] levels, albeit taking a longer time than WT tissue. Meanwhile, Hs6st1 was found to be needed to suppress [Fgf8] during the [Fgf8] gradient formation. The ex vivo experiments also showed that Hs2st antagonises the Erk response to Fgf8, while Hs6st1 is needed to promote Erk response to Fgf8.The ex vivo for the differential control exerted by Hs2st and Hs6st1 on the Fgf8 protein concentration gradient and the pErk response it elicits apply in vivo? In order to address this, we examined the expression of Hs2st, Hs6st1, Fgf8, Fgfr1, and pErk in the CSB region of E14.5 WT, \u2212/\u2212Hs2st, and \u2212/\u2212Hs6st1 embryos. Hs2st and Hs6st1 are expressed at relatively high levels in the VZ at the CSB angle  provided a constant [Fgf8], so the effects we describe can be attributed to perturbations on Fgf8 diffusion and/or degradation. [Fgf8] varies with distance from the source as a power law gradient, a model describing the distribution of a protein diffusing from a source and being cleared from the tissue in a concentration-dependent manner, for example higher Fgf8 concentrations causing increased Fgf8 degradation through an Fgf8-sensing feedback mechanism  or leaving HS intact but losing the function of different HS modifying enzymes (\u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 experiments) all caused significant fluctuations in the gradient amplitude (amount of Fgf8 in the gradient) through time that were not observed in WT cultures. Although the fluctuations in Fgf8 gradient amplitude observed in both \u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 tissue were significant, we observed an initial surge in amplitude at the 1\u2005h time-point in \u2212/\u2212Hs2st, while the surge was only observed in the 2\u2005h time-point in \u2212/\u2212Hs6st1. The Fgf8 amplitude of both mutant tissues reached WT levels after 4\u2005h in culture. This prompted the question of whether the differences we observed in the Fgf8 gradient formation and the Erk response to the Fgf8 gradient due to the loss of Hs2st and Hs6st1 was due to a timing issue of when steady state is achieved, or that Hs2st and Hs6st1 have fundamentally different roles in the process of establishing the Fgf8 gradient and the Erk response. Analyses of the Fgf8 bead cultures for a longer time period would give further insight into this question, however culturing explants with Fgf8 beads for periods longer than our current study is not viable as Fgf molecules have a relatively short half-life. For example, Fgf10  has a half-life of \u223c5\u2005h. This is supported by other studies which performed similar Fgf bead assays using similar time-frames as this study . In contrast to amplitude, the [Fgf8] decay, revealed by the slope of log [Fgf8] vs log distance power function plots, was not significantly altered by removing HS or losing the function of Hs2st or Hs6st1. This indicates that the mechanism regulating the decay of Fgf8 as a function of distance from the source, including feedback mechanisms regulating decay, were not significantly affected by loss of Hs2st or Hs6st1 function. Net [Fgf8] decay is determined both by rates of diffusion and degradation and dissecting the regulation of each component, particularly by Hs6st1 which has the greater effect on Fgf8 concentration, is a topic for future research.The formation of a steady state protein concentration gradient requires the balancing of three factors: concentration at the source; diffusion through the tissue; and degradation in the tissue. In our echanism . We findis study . TherefoWT tissues form a steady-state [Fgf8] gradient in <1\u2005h when challenged with an exogenous Fgf8 bead. While the loss of Hs2st affected [Fgf8] amplitude stability  CSB in addition to higher Erk activation when compared to WT. However, this higher Erk activation was not much higher than the Erk activation in \u2212/\u2212Hs2st tissue. We reasoned that the increase in Erk activation found in \u2212/\u2212Hs6st1 tissue in vivo was due to the increased levels of Fgf8 (\u223cthreefold increase) found in vivo  supports the hypothesis that regulating specific HS sulphation via expression of HS modifying enzymes provides a more nuanced mechanism to control Fgf8 morphogen function than globally regulating HS synthesis. Removing HS effectively shuts down Fgf8 function while removing Hs2st or Hs6st1 play more modulatory roles. Furthermore, in contrast to the promiscuous interaction of HS with all paracrine FGFs, we find that Hs2st and Hs6st1 each have distinct effects on Fgf8 and may well have similarly specific relationships with other FGFs, and some of the >200 HS interacting signalling molecules comprising the heparanome in mammals  as opposed to its modification  and LacZ-IRES-hPLAPHs6st1 (Hs6st1\u2212) mutant alleles were maintained on a CBA background ; goat anti rabbit Alexa Fluor 488 (1/200) (Invitrogen); and Fgf8 staining was detected via TSA Plus Fluorescence Systems (Perkin-Elmer). LacZ staining performed as described previously  and immunofluorescence  were performed on escribed . Primaryeviously . N\u22653 forWT, \u2212/\u2212Hs2st, or \u2212/\u2212Hs6st1 explants, some of which were pre-treated with Heparanase (Sigma) for 2\u2005h at 37\u00b0C, were cultured for 1, 2, or 4\u2005h prior to fixation, sectioning (10\u2005\u00b5m frozen sections) and processing for Fgf8 and pErk immunofluorescence and DAPI counterstaining.Modification of methods described by Fig.\u00a0S1 for illustration of ROI placement) and used to quantify signal intensity at increasing distances from the edge of the bead into the tissue (for Fgf8 and pErk gradient quantification) and in the outer edge of the bead (for Fgf8 only). Data were then combined and graphed using GraphPad Prism 6 (GraphPad). Images from all genotypes of each time point experiment were imaged, processed and analysed in parallel to minimise technical variation for comparison between genotypes of the same time point. Values from BSA control cultures were subtracted to remove background fluorescence. Fgf8 fluorescence in in vivo sections were quantified using IMAGEJ. Quantification of Fgf8 in vivo: mean fluorescence for boxes of 100\u00d7180\u2005\u00b5m drawn at the CSB were obtained for at least 2 sections per embryo with N being number of embryos analysed. WT, N=4; \u2212/\u2212Hs2st, N=3; \u2212/\u2212Hs6st1, N=3.Fluorescent sections were imaged using Leica AF6000 epifluorescence microscope connected to a DFC360 camera on constant exposure settings for three channels, DAPI, Fgf8, and pErk, and analysed using IMAGEJ. IMAGEJ was used to draw 10-\u00b5m wide regions of interest (ROI) on each image  and culture durations  using Kolmogorov\u2013Smirnov (KS) test with Bonferroni correction to compare [Fgf8] and [pErk] gradients and ANOVA followed by Holm-Sidak post hoc test to compare [Fgf8] amplitude, [Fgf8] at the bead and slopes of Fgf8 distribution. The Fgf8 and pErk fluorescence intensity were normalised to the value at the point closest to the bead of the 1\u2005h time point in WT tissue, which was designated as 100%. Statistical comparisons were made between WT, \u2212/\u2212Hs2st and \u2212/\u2212Hs6st1 for Fgf8 fluorescence intensity of in vivo sections using one-way ANOVA followed by Dunnett's post hoc test.Statistical comparisons were made between different tissue conditions ("}
+{"text": "ZEB1 coding region mutations.To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity.The promoter, 5\u2019 UTR, and coding regions of OVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type.OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.Previously identified as the cause of PPCD1, the  ZEB1) on chromosome 10 that likely result in ZEB1 haploinsufficiency. Bas BasOVOL2ZEB1 mutation was not identified were submitted to the UCLA Clinical Microarray Core for array comparative genome hybridization (aCGH) to identify copy number variation (CNV) using a custom Agilent 8x60K array . The custom array was designed for the interrogation of: a 16.7 Mb region (hg19: 17.3 Mb\u2013 34.0 Mb) on chromosome 20 encompassing the refined PPCD1 interval and approximately 2.7Mb (0.54 Mb 5\u2019 and 2.17 Mb 3\u2019) of flanking sequence; and a 5.2 Mb region (hg19: 29.1Mb\u2013 34.3 Mb) on chromosome 10 containing the ZEB1 gene (31.6 Mb\u2013 31.8 Mb) and 5 Mb (2.5 Mb 5\u2019 and 2.5 Mb 3\u2019) of flanking sequence.[Genomic DNA samples from eight PPCD probands in whom a sequence. The arra\u00ae LTX (Life Technologies) according to the manufacturer\u2019s recommendations. To test the impact of the OVOL2 c.-307T>C variant on promoter activity, HCEnC-21T cells were transfected with 250 ng of either a OVOL2 PWT, OVOL2 Pc.-307T>C, or empty (pGL3-Basic) promoter luciferase construct (generously provided by Dr. Alison J. Hardcastle of the University College London).[\u2122 BCA Protein Assay Kit (Life Technologies) and FilterMax F5 microplate reader . Promoter activity was measured using the Dual-luciferase\u00ae Reporter Assay System (Promega) according to manufacturer\u2019s instructions. Each trial was normalized to Renilla firefly luminescence in order to account for variability in transfection efficiency.Immortalized human corneal endothelial cells (HCEnC-21T) were seeded at 50% confluency into 24-well plates. The following day, the cells were transfected using LipofectamineThe probability and corresponding 95% confidence interval of the identified variant, c.-1409G>C, found within the screened probands was estimated based on the observed frequency in the probands and from the observed frequency of same variant in the population assuming binomial distribution of allele frequencies.ZEB1 translational start site in 31 PPCD probands without a ZEB1 coding region mutation.[ZEB1 coding region mutation were identified, we screened the 1 kb upstream of the initiation methionine in these six individuals. The known single nucleotide polymorphism (SNP) c.-803G>C  0.316) was identified in the heterozygous state in two individuals, one of whom also demonstrated a second known SNP ) in the heterozygous state. Of the 37 DNA samples from these individuals without a ZEB1 promoter, 5\u2019 UTR, or coding mutation, 27 had sufficient DNA to analyze the 1.8kb region upstream of the OVOL2 translational start site.We have previously reported the absence of presumed pathogenic variants in the non-coding 1 kb region (containing both the 5\u2019 UTR and promoter) upstream of the mutation. After siOVOL2, one proband (P1) is a member of the family that we mapped to the PPCD1 locus and in which we identified a OVOL2 promoter variant (c.-307T>C) that segregated with the affected phenotype.[OVOL2 in the remaining 26 PPCD probands in whom a ZEB1 truncating mutation was not identified revealed five different heterozygous variants in eight probands, while the remaining 18 probands did not demonstrate any heterozygous variants within either the OVOL2 promoter or 5\u2019 UTR (OVOL2 promoter (6/54 chromosomes corresponding to an observed MAF of 0.111) was double the global MAF of 0.0541, the difference was not statistically significant (p = 0.064). Given this, and as the global minor allele frequency is much higher than the population prevalence of PPCD, the c.-1409G>C variant is very unlikely to be pathogenic. Screening of each of the four OVOL2 coding exons in the 27 probands revealed only one heterozygous variant (c.327C>A), which was identified in three probands. As the c.327C>A variant has a MAF of 0.0403, much higher than the population prevalence of PPCD, this variant is also very unlikely to be pathogenic.Of the 27 probands who had sufficient DNA to perform screening of the henotype., 24 Screr 5\u2019 UTR . Of the OVOL2 was screened in affected and unaffected members of the family with PPCD that was the first to be mapped to the PPCD1 locus on chromosome 20  were identified in five of the eight probands, ranging in size from 134 to 14,007 base pairs  proband, and the second gain present in three (3/8) probands; and a loss in intron 2 of PIGU, which was identified in three (3/8) probands. There were no identified CNVs within OVOL2. In the PPCD3 locus, CNV were identified in eight genomic regions, although none involved ZEB1.CNV analysis within the PPCD1 and PPCD3 loci was performed in eight of the 27 PPCD probands without a se pairs . Three oOVOL2 promoter activity in human corneal endothelial cells. Immortalized human corneal endothelial cells, HCEnC21T, were transfected with a luciferase reporter construct containing either no promoter (empty), an OVOL2 wild-type promoter (OVOL2 PWT), or an OVOL2 promoter harboring the c.-307T>C mutation (OVOL2 Pc.-307T>C). While both OVOL2 PWT and OVOL2 Pc.-307T>C were able to drive the expression of luciferase, OVOL2 Pc.-307T>C produced significantly higher levels of luciferase expression when compared to the OVOL2 PWT  and one family initially diagnosed with autosomal dominant CHED that was mapped to the region on chromosome 20 containing OVOL2, promoter mutations in OVOL2 have been convincingly associated with PPCD1.[The recent identification of entified., 25 We ientified., 18 Althentified. Given thentified. This varth PPCD1., 25OVOL2 or ZEB1 coding region or promoter mutation or CNV involving either gene in 26 PPCD probands indicates that other genes may be associated with PPCD. In the PPCD mouse, a chromosomal inversion flanked by deletions involving Csrp2bp and Dzank1 has been identified in the portion of chromosome 2 that is syntenic to the human PPCD1 locus.[Csrp2bp fusion transcripts and Ovol2, which is located 37 kb from Csrp2bp and 66 kb from the breakpoint of the chromosomal inversion. While it is possible that the identified genetic rearrangement leads to PPCD1 in the mouse through upregulation of Ovol2 expression, as is the case in humans, the authors provide compelling evidence for the disruption of Csrp2bp as being causative for PPCD1 in the mouse.[CSRP2BP in any of the 8 probands analyzed for CNV. Although identification of other affected pedigrees of sufficient size to perform whole exome sequencing and CNV analysis to identify additional genetic loci should be performed, we also plan to perform and encourage other investigators to perform sequencing of presumed OVOL2 and ZEB1 regulatory sequences to identify other potentially pathogenic sequence variants.Our failure to identify a presumed pathogenic D1 locus., 29 The he mouse., 29 HoweZEB1, as it does in corneal epithelial cells. OVOL2 maintains the epithelial cell state by repressing mesenchymal genes such as ZEB1.[PAX6, OVOL2 induces the transcriptional profile of corneal epithelial cells in fibroblasts, suggesting that OVOL2 may serve as a master regulator of the epithelial cell state.[OVOL2 results in enhanced promoter activity in corneal endothelial cells compared to the wild-type sequence. As it has been previously reported that OVOL2 represses ZEB1 expression, our promoter activity results are consistent with the hypothesis that PPCD-associated OVOL2 promoter mutations lead to PPCD by causing OVOL2 overexpression and subsequent ZEB1 insufficiency.[In silico analyses have predicted PPCD-associated OVOL2 promoter mutations cause a gain or loss of putative transcription factor binding sites, potentially leading to OVOL2 dysregulation.[OVOL2 and ZEB1 mutations on the expression of these transcription factors, the endothelial transcriptome and corneal endothelial morphology and function are ongoing, and are anticipated to clarify whether both PPCD1 and PPCD3 share a common molecular pathway.Additional studies will also need to be performed to elucidate the role of OVOL2 in the corneal endothelium, and to determine whether OVOL2 acts to repress the expression of  as ZEB1. When cotll state. In contrficiency., 27 In sgulation., 25 FurtS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file."}
+{"text": "CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP2 Fox genes perform tissue specific functions during development and mutations can cause cancer and other diseases  transcription factors comprise 19 highly evolutionary conserved, structurally related families, FoxA to FoxS. The defining feature of these genes is the Fox domain which binds to regulatory regions of target genes. Many FoxP subfamily members in vertebrates, FoxP1 to FoxP4 , encoding one of the reelin receptors, and Contactin-associated protein-like 2 (CNTNAP2) gene which codes for a neurexin called CASPR2  were bred in our colony at the Freie Universit\u00e4t Berlin. All procedures were performed according to the guidelines of the governmental law (TierSchG), under permits granted by the local Berlin authorities governing research involving animals. All birds were sacrificed with an Isoflurane overdose.Male zebra finches (Co-Immunoprecipitation (Co-IP) was done using Dynabeads\u00ae Protein G  following the manufacturer\u2019s protocol with a few changes as follows. We first incubated the antibody-Dynabeads mixture for 15 min at room temperature  lysis medium for 15 min on ice. Extracts were centrifuged for 10 min at 1500 g and supernatant was stored at \u221280\u00b0C until used. Cell extracts and co-IP\u2019ed proteins were separated by SDS PAGE (8%\u201310%), transferred to a polyvinylidene fluoride membrane , and blocked with Roti-Immunoblock for 2 h or overnight at 4\u00b0C. The membranes were then incubated with the desired antibody  25 \u03bcl of OptiMEM (GIBCO) containing 30 ng of pGL4.13 (Luciferase gene driven by the SV40 promoter which is known to be regulated by FoxP subfamily members) and 30 ng of pGL4.75  and 250 ng total vector over-expressing the different FoxPs for each well; and (b) 1 \u03bcl of Lipofectamine\u2122 2000 (Invitrogen) in 25 \u03bcl OptiMEM that was premixed for 5 min at RT. After combining (a) and (b) the mix was incubated for 20 min at RT, then it was added to the cells. After 4\u20136 h of incubation we changed the medium to 75 \u03bcl of antibiotic containing medium and incubated for further 48 h at 37\u00b0C in a CO2 incubator.For SV40 we seeded ~30000 HeLa cells in 200 \u03bcl DMEM medium (GIBCO) containing Penicillin-Streptomycin  per well of a 96-well white flat bottom plate . Transfection was performed using Lipofectamine\u2122 2000 (Invitrogen) following manufacturer\u2019s protocol. Briefly, after seeding plates were incubated for 24 h at 37\u00b0C at 5% COVLDLR and CNTNAP2 we used HeK293 cells, and luciferase assays were done as described previously  following manufacturer\u2019s protocol in an Elisa plate reader . Mean background from untransfected wells was subtracted from all other wells. We present Luciferase results as mean Relative Light Units (Luciferase RLU/Renilla RLU) calculated from the normalized values of 4\u20137 independent assays.The pGL4-VLDLR promoter was cloned as described .Proteins for electrophoretic mobility shift assays (EMSAs) were purified using the Protino Ni-NTA Agarose  according to manufacturer\u2019s protocol. Protein was quantified using BCA1 (Sigma). EMSA assays were carried out as published previously, using the Oligo described for VLDLR , and therefore we used FoxP2 and FoxP4 polyclonal antibodies to pull down protein complexes, and all three antibodies for Western blot detection. As a negative control we used IgG antibodies of the same species in which the specific FoxP antibodies were raised. After IP with FoxP2 or FoxP4 antibodies, we detected co-IP\u2019ed FoxP1 Figures . After ISince the majority of FoxP expressing cells in Area X and the surrounding striatum express FoxP1/2/4 . The SV40 promoter has a putative core consensus sequence for binding (TATTTRT) human and murine Foxp1/2/4 , in the same range as reported for mice and human FOXP1/2/4. Also, like its mammalian homologs, zebra finch FoxP4 repressed transcription stronger than FoxP1 or FoxP2 did, and FoxP2 was a weak repressor of the SV40 promoter . There were no significant differences between cells expressing FoxP1 or FoxP4 alone or in any of the combinations tested. We did observe a stronger transcriptional repression in cells expressing FoxP2 in combination with another FoxP subfamily member than when FoxP2 was expressed alone . However, we cannot rule out the formation and action of homodimers of FoxPs in those cells. Taken together, FoxP target genes in cells that express only FoxP2 may be less strongly regulated than those that are co-expressed with FoxP1 and/or FoxP4.We assessed whether the homotypic and heterotypic interaction of zebra finch FoxP proteins lead to differential regulatory activity. To do so we used the SV40 promoter driving Luciferase (VLDLR for a number of reasons: (a) it is a direct target of FoxP2 in humans, mice, and zebra finches . However, FoxP2 and FoxP4 were less effective and not significantly different from each other in their ability to activate the VLDLR promoter (p = 0.999). Combinations of FoxP1/2/4 did activate the VLDLR promoter to a similar degree . Comparing the activation of FoxP2 alone to the FoxP1/2 and FoxP1/2/4 combination (p = 0.0078 and p = 0.012 respectively) we found a significant difference on the VLDLR promoter but not with the FoxP2/4 combination (p = 0.88). There were no differences between the individual activation by FoxP4 and the combinations with FoxP1 (p = 0.10) or FoxP2 (p = 0.98), but there was a significant difference to the FoxP1/2/4 combination (p = 0.034). Taken together FoxP1/2/4 are not only repressors, but are able to bind to the VLDLR promoter and activate it differently depending on the co-expression with other FoxPs.To test whether binding of FoxP1/2/4 to the CNTNAP2 because: (a) CNTNAP2 is associated with speech disorders, as well as ASD, dyslexia and intellectual disability .To further test the regulatory ability of zebra finch FoxPs on a target gene relevant in Area X, we chose CNTNAP2 promoter contained a sequence gap located ~0.75 kb upstream of the start codon of the first exon of CNTNAP2 . FoxP1 was able to bind to the CNTNAP2 oligo and resulted in two shifted bands, consistent with the possibility that it binds in two of the three FoxP2 binding sites . Moreover, in combination, the different FoxPs also failed to regulate expression driven by the CNTNAP2 promoter, . Interestingly, combinations of FoxP4 with either FoxP1 or FoxP2 did not result in repression or activation, in contrast to FoxP1 or FoxP2 alone, suggesting that hetero-dimerization might affect the regulation of FoxPs. Taken together we show that FoxP1 and FoxP2 can bind to and regulate the CNTNAP2 promoter, with opposing activities. CNTNAP2 is an example of how FoxPs may tune the regulation of different targets genes via homo- and hetero-dimerization.To investigate whether binding of FoxP1 and FoxP2 to the r Figure . When teo Figure  FoxP4 faSV40, VLDLR and CNTNAP2 and regulate their transcriptional activity. Zebra finch FoxP1/2/4 repressed the SV40 promoter activity, as reported for mouse and human , which is in the opposite direction of the present findings with HeK293 cells. This highlights the plasticity with which FoxP proteins can regulate target genes in different cellular contexts, depending on different binding of co-factors that change the regulation of the same gene  two ways to associate or interact: the leucine zipper motif and domain swapping, e.g., the exchange of identical structural elements involving the Forkhead domain (Stroud et al., VLDLR and CNTNAP2. All zebra finch FoxP proteins studied bound to the VLDLR oligonucleotide, which was previously shown to bind to FoxP2 (Adam et al., VLDLR oligonucleotide contains a partial FOXP core sequence, ATTT (Stroud et al., ATTTAT (Wang et al., RTTTAY (Pierrou et al., CNTNAP2 oligonucleotide contains three putative FoxP2 binding sites. One TATTTAT (Enard et al., Finally, our data are the first to address neural targets regulated by FoxP1 and FoxP4. We tested the binding of two known targets of FoxP2 in zebra finches, VLDLR and CNTNAP2. Importantly, different FoxP combinations resulted in specific, differential transcriptional regulation. Together, our data demonstrate how versatile and variable FoxP regulation can be in the neural context.In summary, we show that zebra finch FoxP proteins can interact with each other in all combinations in the songbird brain. We show that all neurally expressed FoxPs have the capacity to bind to and regulate the target genes EM and CS: experiment design, analysis of data and wrote the manuscript. EM did all experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Pseudomonas putida P13 and Pantoea agglomerans P5 are well recognized for application as phosphate solubilizing bioinoculants and are used as solid carrier based. Liquid bioinoculants are preferred for economizing production process and longer shelf-life.Phosphorus is one of the low bioavailable macroelements. Use of microorganisms in biofertilizers could release phosphorus from insoluble compounds. 3 dissolved in phosphate buffer, respectively. Formulation 4 was nutrient broth containing 4% glycerol and formulation 5 was diluted nutrient broth containing 4% glycerol. Survival (cfu) and phosphate solubilization index (SI) were evaluated after 3 months.Five low cost liquid formulations were examined. Formulations 1, 2 and 3, were phosphate buffer, 0.2% and 0.5% KNO3 concentration decreased preserving ability. While using KNO3 at 0.2% was accompanied with reaching maximum SI level. Overall, less nutritious formulations (1 and 5) provided maximum preserving ability without bioactivity loss. In the case of strain P13, maximum survival obtained in formulations 2 and 3, whereas SI level decreased. Preserving ability in formulations 1, 4 and 5 was similar but less nutritious formulations (1 and 5), improved bioactivity.Considering strain P5, increase in KNOPseudomonas putida and Pantoea agglomerans.The results introduced two formulations of 1 and 5 as economically efficient liquid bioinoculants for  Nitrogen, phosphorus and potassium, are among the macronutrients which are required in large quantities for plants . Due to Pseudomonas putida strain P13 and Pantoea agglomerans strain P5 are well known as effective phosphate solubilizing bacteria of related bioinoculants in Iran , Tehran branch, Department of Applied Microbiology. Recovery of the two phosphate solubilizing bacteria (PSB) was made by sub-culturing few drops of stored bacterial stocks at \u221270\u00b0C onto nutrient agar medium. Plates were incubated at 30\u00b0C until growth was observed.2PO4 15.44 \u03bcM, NaCl 1.55 mM, Na2HPO4 27.09 \u03bcM . Cultures were then centrifuged at 5000 rpm for 15 minutes at 30\u00b0C and cell mass was collected and washed twice with equal volume of normal saline solution, subsequently. The cell mass was added to liquid formulations under sterile conditions .8 cfu/ml for strain P5 and 1 \u00d7 107 cfu/ml for strain P13 by measuring the 0.5 optical density at 540 nm wavelength OD(540nm). These values are within the range of bacterial cell number allowed for a biofertilizer according to the plant protection organization of Iran. The samples were perfectly sealed and stored at 25\u00b0C in dark conditions.The number of cells in each formulation was adjusted to 2 \u00d7 10The assessment of viable populations of the two strains was made at monthly interval for three months. Serial four-fold dilutions in normal saline solution were made from each sample. An aliquot of 100 \u03bcl from each dilution was dropped on nutrient agar medium as the spread plate technique. The number of colony forming units (cfu) was determined after 24 hours of incubation .(540nm) value of 0.5 was attained. 25 \u03bcl of each bioinoculant (at the OD(540nm) value of 0.5) was spotted onto plates of Sperber\u2032s agar medium to evaluate hydrolysable phosphorus from tri-calcium phosphate qualitatively by forming the clear zone in the opaque medium (SI) was calculated as follows  value of 0.5 were calculated and used as control. The same incubation conditions were applied for preserved bacteria and control.Phosphate solubilization indexes of 24 hours cultures of strains P13 and P5 at ODThe assays were calculated as an average of three independent experiments. Data were analyzed using SPSS version 21. One-way analysis of variance (ANOVA) and Tukey test were used to examine values. P value <0.05 was considered significant.The ability of each formulation to preserve each single bacterial strain was evaluated over a three month period. Data related to population dynamics of strains P5 and P13, are presented in 8 cfu/ml, underwent a reduction in number in all formulations after one month of storage. However, there was no significant reduction in the number of bacteria afterwards. Formulations 2 and 3 induced a reduction in cell number higher than other formulations at the end of the storage period. The simple phosphate buffer tended to preserve better the number of cells in comparison to phosphate buffers containing potassium nitrate (5 \u00d7 106 cfu/ml). Furthermore, increasing the concentration of potassium nitrate in the formulation provoked a further reduction of bacterial population .Strain P5 with the initial population of 2\u00d710Estimating the influence of nutritious formulations (formulations 4 and 5) on P5 viability, better maintaining ability by formulation of undiluted nutrient broth (formulation 4) was not longer than two months and overall better performance was provided by less nutritious formulation (formulation 5) after three months of storage.6 cfu/ml.The results of different formulations for preserving strain P5 indicated that both of the formulations 1 and 5 supported the highest maintaining ability and protected population maintenance of 5 \u00d7 107 cfu/ml), but with the limitation of cell proliferation for duration of one month (in formulation 2) and two months (in formulation 3) after storage. Statistically no significant difference was observed among formulations 1, 4 and 5 . Compared to the control, the addition of potassium nitrate to the phosphate buffer of formulations 2 (SI = 3.98) and formulation 3 (SI = 3.86) enhanced SI ability. Formulation 4, with undiluted nutrient broth, resulted into the lowest index of P solubilization (SI = 3.74).Bacteria of strain P5 with formulations 1 and 5 showed a SIcontrol= 3.5), formulations 1 and 5 enhanced the bacterial ability to solubilize phosphate. The highest SI for P13 was obtained with the simple phosphate buffer of formulations 1 (SI = 3.86) and diluted broth of formulation 5 (SI = 3.81). The presence of potassium nitrate in the phosphate buffer formulation reduced SI (contrary to the results observed for strain P5 in which maximum SI was obtained in the presence of 0.2% KNO3). Formulations 2, 3, and 4, induced a decline on phosphatase activity.In the case of strain P13 as compared to the control . In their study, it was evidenced that the majority of pathogenic bacteria were able to survive in either water or phosphate buffer after 30 weeks of preservation at room temperature, allowing to hypothesize that human and plant pathogenic bacteria can survive in water or phosphate buffer for several years.Considering both bacteria as Gram negative and facultative anaerobes, efficiency of each individual formulation was examined for each strain, separately. Maximum population of strain P5 and one of highest SI level and increasing cell number for at least about one month after storage of the strain P13. Formulation 2, contributed to maximum SI of strain P5 and formulation 3 decreased population level of the strain to its minimum. Our findings about the ability to sustain the Pseudomonas population size of the phosphate buffer based formulations are in line with findings of Vandenbergh and coworkers  were sufficient to maintain Pseudomonas for up to 160 days or more at room temperature.Maximum preserving ability during three months of storage for strain P13 was assured by phosphate buffers containing potassium nitrate. The formulations were confined by reducing oworkers , 10 who Pseudomonas (and other microorganisms meanwhile) at room temperature .The maintained population size for both strains was acceptable in formulation 4, but P. fluorescens preservation at room temperature. The reported potent bacterium maintained its antagonistic activity until 150 days without any remarkable reduction in bioactivity . Nutrient broth medium containing 2% glycerol amendment has been defined as an appropriate formulation for activity .The formulation 5 fitted with two main positive features; keeping both strains bioactive along with promotion of strain P5 to highest population density. However, the formulation was not so effective in maintaining the P13 strain population. Considering the direct relationship of plant growth promotion efficacy with the number of inoculated microorganisms, the population decline of strain P13 in formulation 5 could be counteracted with inoculation of more bacterial biomass.Pseudomonas. It is better to add extenders at on seed level or at sowing time  for on seed level use  can be added to water or microbial culture broth of inoculant formulation at on seed level. Sucrose is a common additive which can be used as an extender or an adhesive in liquid formulations of ing time \u201325. Pantevel use . Additioreported .In prior studies, it has been demonstrated that mixed inoculation of the two phosphate solubilizing bacterial strains P5 and P13, can be performed .In conclusion it would be possible to utilize formulations 1 and 5 for production of efficient liquid bioinoculants composed of single strain and both strains tested."}
+{"text": "The data presented in this article are related to the research paper entitled \u201cAnti-inflammatory effect of avenanthramides via NF-\u03baB pathways in C2C12 skeletal muscle cells.\u201d  [1] This article includes experimental procedures used to analyze the mode of binding between and IkB kinase (IKK\u03b2) and avenanthramides which are a group of phenolic alkaloids found in oats. The protein-ligand docking and the computer simulation method of molecular dynamics (MD) for studying the physical interactions of molecules were performed. Specifications TableValue of the data\u2022The molecule docking model first describes the mode of binding between Avns and IKK\u03b2 molecular domains.\u2022Molecular dynamic (MD) simulations were carried out to establish the protein-ligand complex.\u2022In summary, protein-ligand docking and molecular dynamics simulations methods were performed to understand the potential structure and the nature of molecular clusters with fine interactions with IKK\u03b2.1The data presented in this article are supportive to the data presented in 22.1A standard protocol for protein-ligand docking studies was used. In short, all docking studies were performed using the Schrodinger modeling suite package . The crystallographic structure of the targeted IKK\u03b2 (PDB code: 3RZF) with bound inhibitors was used as a starting point to study the potential mode of Avns binding 2.2MD simulations were performed for IKK\u03b2 in complex with the structurally solved inhibitors. Each system was solvated in a cubic box with positive TIP3P water"}
+{"text": "Cyclin-dependent kinase (CDK) 4/6 inhibition has been demonstrated to improve progression-free survival (PFS) in patients with human epidermal growth factor receptor 2 (HER2\u2212), hormone receptor positive (HR+) in advanced breast cancer . PalbociCDK4/6 inhibitors have been approved by the US Food and Drug Administration (FDA) for initial endocrine therapy in postmenopausal women with metastatic or advanced HR+/HER2\u2212 breast cancer in combination with an aromatase inhibitor and for the treatment of endocrine therapy-resistant HR+/HER2\u2212 advanced or metastatic breast cancer in combination with Fulvesterant (a selective estrogen receptor degrader) . DespiteAnnals of Oncology Condorelli et al. [RB1 gene was noted through plasma ctDNA analyses at the point of disease progression. In the first patient a frameshift event involving exon 8 of RB1 was observed that was predicted to result in a non-functioning truncated version of the protein. This event was not observed through NGS analysis of a liver biopsy acquired before CDK4/6 inhibition. In the second patient of the case-series four RB1 alterations were noted at progression on palbociclib that were not detectable before initiation of therapy. The variant with the highest allele frequency in plasma at progression (Chr13(GRCh37): g.48937094G>A) has been previously shown in lung cancer to result in loss of the Rb protein region responsible for the binding of Rb to E2F-transcription factor complexes [Circulating tumour DNA (ctDNA) describes molecules of cell-free DNA circulating in plasma that originate from a patient\u2019s tumour. ctDNA analyses by next-generation sequencing are demonstrating translational utility within clinical contexts ranging from non-invasive screening , trackini et al.  leverageomplexes . The finRB1 alterations potentially being selected at disease progression following intervention with CDK4/6 inhibitors in patients with metastatic breast cancer. These observations build on a previous invivo investigation of CDK4/6 inhibitor resistance using patient-derived tumour xenograft models that suggested Rb1 inactivation as a resistance mechanism to chronic CDK4/6 inhibition [This study is of interest for the following reasons. Firstly, it provides observational evidence of deleterious hibition . SecondlRB1 alterations and ctDNA analyses demonstrating acquired RB1 alterations\u2014patient 1 received everolimus and exemstane before palbociclib and patient 2 received capecitabine and paclitaxel following ribociclib. Therefore, we cannot be certain that the acquisition of Rb1 alterations solely associate with selective pressure induced by CDK4/6 inhibition. Advancing the findings reported in this case-series will require a larger cohort to determine the incidence of Rb1 alterations as resistance mechanisms in patients with metastatic breast cancer on CDK4/6 inhibitors. Furthermore, more frequent ctDNA monitoring is necessary to follow the dynamics by which RB1 alterations emerge and ascertain the association of their emergence with disease progression.There are however caveats to consider regarding this case-series. The number of patients described within the manuscript is small and there is no indication as to the frequency by which Rb1 alterations are detected at progression on CDK4/6 inhibition in this patient population. Additionally, patients 1 and 3 in the case-series were treated with two lines of therapy in between the biopsies showing lack of RB1 and therefore could conceivably underlie an immune predatory selection pressure toward selection of Rb1 altered populations whilst undergoing treatment with CDK4/6 inhibitors. The fact that CDK4/6 inhibition has recently been shown to increase PD-L1 expression in mouse models of breast cancer provides a clear rationale for anti-PD1 treatment as a combination therapy with CDK4/6 inhibition before the emergence of Rb1 loss of function [Given this work, it is notable that CDK4/6 inhibition has recently been associated with increasing tumour cell antigen presentation through a mechanism involving downregulation of Rb1-E2F induced DNA methyltransferase 1 (DNMT1) activity, increased expression of endogenous retroviral elements and type III interferon production . This refunction ."}
+{"text": "CYP2A6 has been identified as the main gene that codifies the enzyme that metabolizes nicotine. Many alleles have been identified after the discovery of CYP2A6, suggesting a wide interethnic variability and a diverse smoking behavior of the allele carrying individuals. The main purpose of this review is to update and highlight the effects of the CYP2A6 gene variability related to tobacco consumption reported from diverse human populations. The review further aims to consider CYP2A6 in future studies as a possible genetic marker for the prevention and treatment of nicotine addiction. Therefore, we analyzed several population studies and their importance at addressing and characterizing a population using specific parameters. Our efforts may contribute to a personalized system for detecting, preventing and treating populations at a higher risk of smoking to avoid diseases related to tobacco consumption.Tobacco consumption has become a major public health issue, which has motivated studies to identify and understand the biological processes involved in the smoking behavior for prevention and smoking cessation treatments.  Tobacco consumption has become an epidemic affecting more than 1,000 million people worldwide and is considered the main cause of avoidable death causing approximately 6 million premature deaths each year . Thus, Among cigarette compounds, nicotine is responsible for causing dependence by stimulating the smoker and allowing the other compounds to access the body, causing chronic harmful effects and tobacco-related diseases.CYP2A6 gene plays an important role. Its protein product (by the same name) is the principal enzyme responsible for nicotine metabolism to cotinine and other sub-products in the human body. However, more than 45 alleles have been discovered suggesting wide interindividual and interethnic variety.Tobacco consumption is caused by environmental, psychosocial and genetic factors . PrevioCYP2A6 related to tobacco consumption reported in diverse human populations. Moreover, we aimed to consider these polymorphisms in future studies as possible genetic markers for the prevention and treatment of nicotine addiction.The main purpose of this review is to update and highlight the effects of the genetic polymorphisms of The CYP2A6 enzyme belongs to the enzyme superfamily known as the cytochrome P450 system (CYP450), also classified in the drug metabolizing enzymes group. These enzymes are found in the endoplasmic reticulum of the cells of some tissues in the body, particularly in the liver. Moreover, they are phase I enzymes responsible for metabolizing more than 80 % of drugs such as xenobiotics and endogen products in the body  that release neurotransmitters in the brain and cause a pleasant sensation in the smoker. The nicotine availability in the body is mediated by biological factors, mainly those related to its metabolism. Smokers tend to consume the same amount of nicotine each day to acquire the desired effects by modulating their smoking behavior to adjust nicotine availability for the purpose of regulating nicotine levels in the body .It has been reported that CYP2A6 is the main enzyme involved in the nicotine oxidation to cotinine. CYP2A6 catalyzes approximately 80 % (55-92 %) of this reaction via C-oxidation in addition to other metabolic pathways for nicotine and its metabolites  and are inactivated and removed from the body  making their genes ideal candidates for altering smoking behavior .CYP2A6 gene has a 6 kb extension length, and it is composed of 9 exons, which encode for a 494 amino-acid product  are also present. The CYP2A subfamily cluster includes the CYP2A6, CYP2A7, and CYP2A13 genes and other pseudogenes . It is al., 2001).Human CYP-Allele Nomenclature Database (HCAND)  was created in 1999 for the identification and characterization of CYP2A6 alleles (and other CYP450 genes). This database consists of a committee for unifying and assigning nomenclature for the already discovered alleles and alleles to be discovered in the future . To date, 42 well-characterized alleles and some haplotypes that are uncharacterized  have beCYP2A6*1A . CYP2A6al., 2000). In vital., 2009; Wang etal., 2009; Yoshidaal., 2009). Howeveal., 2000; Mwenifual., 2000, 2008; Nak; NakCYP2al., 2002; Rao et al., 2002; Schoedeal., 2002; Xu et aal., 2002); Atlantal., 2002; Sorianoal., 2002)) and Fral., 2002)) also hal., 2002)), Northal., 2002)) and Caal., 2002)) had a al., 2002)) and Swal., 2002)) also ral., 2002) and Tatal., 2002). Africaal., 2002; Nakajimal., 2002)), Africal., 2002; Nakajimal., 2002)), Ghanaal., 2002)), Ethioal., 2002)). Amerial., 2002)) have aal., 2002)) and moal., 2002)), Ecuadal., 2002)), Chileal., 2002)) and Hial., 2002)). On thal., 2002)) of thiCYP2A6*5 is found in a higher frequency in Asian populations, East and Southeast Asian populations such as the Vietnamese had the highest frequency ) comparal., 2009; Nurfadhal., 2009; Oscarsoal., 2009; Schoedeal., 2009), Koreanal., 2009; Kwon etal., 2009; Nakajimal., 2009; Yoshidaal., 2009) and Malal., 2009) populatal., 2009, 2006; Nak; NakCYP2al., 2006, 2004; Nak; NakCYP2al., 2006; Schoedeal., 2006)), Spanial., 2006)) and Caal., 2006)). Then,al., 2006), but inal., 2006; Schoedeal., 2006)) while al., 2006; Koontz al., 2006; Nakajimal., 2006; Oscarsoal., 2006; Schoedeal., 2006) did notal., 2006; Schoedeal., 2006)). In Afal., 2006; Schoedeal., 2006) and othal., 2006; Koontz al., 2006; Mwenifual., 2006; Nakajimal., 2006).CYP2A6*13 has been reported in Japanese ) and Koal., 2002)). It waal., 2002; Nakajimal., 2002).CYP2A6*14 has a higher frequency in Caucasian ) than ial., 2005; Nakajimal., 2005)), but wal., 2005; Nakajimal., 2005).CYP2A6*15 has been reported at a minimum frequency in Japanese ) and Koal., 2002)), but nal., 2002; Mwenifual., 2002; Nakajimal., 2002).CYP2A6*16 has been reported in Caucasian (0.3-3.6 %) and African (0-1.7 %) populations , but noal., 2002; Nakajimal., 2002).CYP2A6*17 has a frequency of approximately 7.3-10.5 % in African populations , but noal., 2004; Fukami al., 2004; Nakajimal., 2004) populatCYP2A6*18 has a higher frequency range in Caucasian ) than ial., 2005; Nakajimal., 2005)), but hal., 2005; Nakajimal., 2005).CYP2A6*19 has been reported in a low frequency in Korean ) and Jaal., 2013; Nakajimal., 2013) populatCYP2A6*20 is present in African population at a frequency range between 1-1.7 % . This aal., 2005; Nakajimal., 2005).CYP2A6*21 has a higher frequency in Caucasian (0.5-2.3 %)  than inal., 2006; Nakajimal., 2006)). It haal., 2006) populatCYP2A6*22 has been reported at frequency less than 0.3 %  in Caucal., 2005) populatCYP2A6*23 has a frequency range among 1-2 %  in AfriCYP2A6*24 has a frequency range among 0.7-1.3 % in African populations , but itCYP2A6*25-*28 has only been reported in African populations, while CYP2A6*26-*27 has a frequency range of 0.7-0.9 %  and CYPal., 2009; Mwenifual., 2009).CYP2A6*31, *34 and *38 have not been studied in any population.CYP2A6*35 has been reported at a frequency between 2.5-2.9 %  in Afrial., 2010) populatCYP2A6*36 and *37 has been reported in Taiwanese populations at a frequency of 0.3 %, but not in African, Caucasian, Chinese nor Japanese , *40 (0.2 %), *41 (1.2 %), *42 (0.2 %), *43 (0.2 %), *44 (0.2 %) and *45 (0.6 %) have only been reported in the African population .CYP2A6*1X2, has been found at higher frequency in Asian populations as Asian ), Indiaal., 2006)), Chineal., 2006; Schoedeal., 2006; Xu et aal., 2006), Koreanal., 2006; Nakajimal., 2006) and Malal., 2006); the Hial., 2006) has a hal., 2006); Caucasal., 2006; Fukami al., 2006; Nakajimal., 2006; Rao et al., 2006; Schoedeal., 2006; Xu et aal., 2006), Spanisal., 2006), Swedisal., 2006), Serbiaal., 2006), North al., 2006) and Canal., 2006). In Afral., 2006)) and Etal., 2006; Nakajimal., 2006; Schoedeal., 2006).Most studies address the majority of a country's population as a general population. However, a few studies focus on more specific population classifications as ethnic and regional groups. In the Asian population, it was studied among Tottori, Shimane, Ehime and Fukuoka people of the respective districts of Yonago, Izumo, Matsuyama and Kurume located in Japan . In ChiCYP2A6 gene, which is highly polymorphic. This variability is due to changes in DNA, which have generated different responses to nicotine and are reflected in the individual smoking behaviors along with other factors. It has been reported that this variability has been generated and distributed over a long time in different human populations, showing well defined ethnic patterns. Although association studies between carriers of certain variants and different smoking behaviors have been numerous with plausible results, population studies reporting frequencies of these variants are few. General population studies exhibit most reliable information than association studies with a variable frequency, because the population requirements are usually more specific, creating a population bias. However, it would be advisable to address this type of methodology in higher risk populations of smoking and those where policies to control smoking are less efficient, and where more smoking-related diseases are reported in the population. Additionally, the population must be characterized by more specific requirements, such as including the ancestry informative markers and avoiding \"self-reporting\" as the unique classification criteria. While there is a Human CYP-Allele Nomenclature Database in which the genetic findings of CYP2A6 are unified, it is necessary to supplement it with updated data as per the population distribution. All of this could contribute to a personalized system that could detect, prevent and treat populations at risk of smoking, and in consequence, avoid tobacco consumption related diseases. The enzyme responsible for metabolizing nicotine is mostly encoded by the"}
+{"text": "Finding additional functional targets for combination therapy could improve the outcome for melanoma patients. In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines . Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well. Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines. The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns. Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients. Malignant melanoma is an extremely dangerous disease with high mortality rates due to the aggressive metastatic potential of melanoma cells. Although the development of new therapies for patients with already metastasized melanoma over the last few years resulted in prolonged survival, for a considerable number of patients these new therapies still do not achieve stable remission for more than a few months  . To. To19]. Stained slides were scanned by a Mirax microscope  and the Panoramic Viewer software  was used to take images.Lung metastases were quantified as previously described . BrieflyAlu sequences was performed as previously described [DNA and RNA extraction from murine blood and tumors as well as quantification of human melanoma cells by quantitative real-time polymerase chain reaction (qRT-PCR) for human escribed .Continuous variables are displayed using boxplots.In all models the interaction of cell line (MeWo vs. MV3) and group (Luc vs. L1 kd) was tested using a likelihood-ratio test. In case of a significant interaction the group comparison was presented separately for each cell line and in case of insignificance the interaction was excluded from the model and an overall group comparison was presented.In vitro outcomes  were analyzed using linear models. Outcomes of the xenograft model were survival, tumor weight, metastases, and number of circulating tumor cells . Survival of mice was defined as time until mice were euthanized when the tumor ulcerated or reached an estimated mass exceeding 10% of the respective animal\u2019s weight. As no censoring was existent it was analyzed using a linear model which was moreover adjusted for the type of death (ulcerated vs. not ulcerated). Tumor weight was analyzed in an analogous model. Number of metastases was analyzed and evaluated/quantified using a negative binomial regression which was adjusted for survival time and tumor weight and included furthermore CTCs as interesting variable. CTCs as outcome variable were logarithmized and analyzed using a linear model, which was adjusted for survival time and tumor weight.Parameter estimates are presented together with 95% confidence intervals (95%-CI). A two-tailed p<0.05 was considered to be statistically significant. All analyses were conducted using Stata 14.1 .Total RNA was extracted from the frozen tumor samples using the miRNeasy Mini Kit  as recommended by the manufacturer. RNA concentrations were determined by the Nanodrop 1000 spectrophotometer . RNA quality was checked using the Experion RNA StdSens Analysis Kit and Experion platform . For all samples RQI value  was greater than 8.Microarray expression analysis was performed using GeneChip\u00ae Human Transcriptome Array 2.0  as recommended by the manufacturer. Data processing was carried out using Signal Space Transformation\u2013Robust Multi-Array Analysis (SST-RMA) method in Affymetrix Expression Console (version 1.4.1.46). Dispersion analysis (ANOVA) was applied to the logarithmic expression values using Affymetrix Transcriptome Analysis Console (version 3.0.0.466). For p-values correction we used the Benjamini-Hochberg method . AnalysiThe newly generated sublines were analyzed for L1CAM expression by flow cytometry.Fig 1A).Sublines with more than 85% (MeWo) and 75% (MV3) reduced L1CAM expression were pooled and used for all further experiments (designated MeWo and MV3 L1 kd). MeWo Luc and MV3 Luc cells with unchanged L1CAM expression, but with puromycin resistance were used as controls .In an XTT assay, MeWo L1 kd cells showed a significant increase in proliferation  compared with the MeWo Luc controls with unchanged surface L1CAM. For MV3 L1 kd there was a decrease in proliferation  in comparison to MV3 Luc controls .We found no significant overall influence of L1CAM knockdown on the cells\u2019 ability for Fig 1D).In a colony forming assay in matrigel, a significant increase of colony numbers for MeWo L1 kd cells in comparison to Luc controls was observed  while there was no significant difference in colony numbers between L1 kd cells and Luc controls for MV3 .In laminar flow assays measuring adherence, tethering and rolling on activated endothelial cells (HUVEC), the number of events increased significantly for MeWo L1 kd cells  while it decreased for MV3 L1 kd cells  or no effect at all (invasion) for both human melanoma cell lines used. These in vitro results do not reflect the in vivo results from the following xenograft model (see below).Summarized, knockdown of L1CAM All animals developed subcutaneous tumors at the injection site and were euthanized when the tumor ulcerated or reached an estimated mass exceeding 10% of the respective animal\u2019s weight. The time span between injections of melanoma cells until euthanization will subsequently be referred to as survival time.Fig 2A).L1CAM knockdown did not significantly change the animals\u2019 survival time in MeWo and MV3 .Within the MV3 cell line the size of the L1CAM knockdown tumors was significantly greater  in comparison to the MV3 control tumors. The size of the MeWo L1CAM knockdown tumors was not significantly different from the control tumors . Metastasis  was 7.26 times higher in the MeWo and MV3 Luc group in comparison to the L1CAM knockdown group . Survival time also showed a significant influence on lung metastasis. With each day of survival, metastatic spread increased by 6.83% . Tumor weight and number of CTCs showed no influence on the number of metastases (p = 0.070 and p = 0.436 respectively).Metastatic spread of the human melanoma cells was exemplarily studied in the animals\u2019 lungs. All control (Luc) metastases displayed high (MeWo) or low (MV3) L1CAM expression and even most L1 kd metastases still showed weak (MeWo) or very weak (MV3) L1CAM expression  in the bone marrow show a tendency towards the results for the animals\u2019 lungs: In the MeWo luc group 33.3% of the animals were positve for DMCs, 23.8% were positive in the MeWo L1 kd group. For MV3, 55.6% were positive for DMCs in the Luc group and 22.2% in the L1 kd group.Melanoma cell numbers (determined by RT-qPCR) in the bone marrow of the animals were low: most of the samples showed no values above background, thus only samples with at least 1 melanoma cell per 10Fig 2D).Regarding influence factors on the number of CTCs we found the mass of the primary tumor to be significant: CTC numbers increased by the factor 3.33 per 1 g increase of the primaries . Survival time and L1CAM knockdown showed no significant influence on CTC numbers (p = 0.454 and p = 0.161 respectively)  genome expression analyses starting with the corresponding primary xenograft tumors of the initially L1CAM high cell line MeWo (MV3 microarray data see below).S1 Table). Analysis of this regulation profile using the Pathway Studio software suggests a central role of Tumor Growth Factor \u03b2  as many genes that have been reported to be directly or indirectly regulated by TGF\u03b2 display a respective up- or downregulation in the L1 kd tumors .Considering all genes with a significant  expression fold change of at least 1.51, in the xenograft tumors formed by the originally L1CAM high MeWo cells, the expression of 981 genes was up- and of 1000 (excluding L1CAM itself) downregulated  regulated genes were screened for EMT related genes:The phenotype of the L1CAM knockdown tumors appeared less mesenchymal than the control tumors\u2019; e.g. significant reduction of N-cadherin (CDH2), Tyrosine-protein kinase receptor UFO (AXL), Matrix Metalloproteinase 2 (MMP2), TGFB1 and Secreted Protein Rich in Cysteine (SPARC) expression. An exception from this reduction of EMT related gene expression, however, was an increased expression of the NOTCH ligand JAG1 (Jagged 1) in the L1CAM knockdown tumors. As this gene was the sole exception among the EMT related genes, we chose JAG1 to exemplarily verify the array results. Interestingly, the increase in JAG1 expression was accompanied by a significant increase in NOTCH1 and 2 expressions, albeit only 1.40 and 1.41 fold, respectively, and a significant increase in NOTCH signaling activator Contactin 1 (CNTN1) expression. With CCNC (Cyclin-C), HIF1A (Hypoxia-inducible factor 1-\u03b1) and KAT2B (K acetyltransferase 2B) three other genes involved in NOTCH signaling are downregulated.Fig 5B).On protein level, a tendency towards increased Jagged 1  and NotcTable 1, analysis see Fig 6). Immunohistochemical stainings showed clear Jagged 1  and Notch 1 expression in the subcutaneous MV3 tumors and lung metastases, however, we did not see a tendency towards higher or lower expression in the L1 kd tumors or metastasis compared with the Luc controls which already displayed a higher Jagged 1 expression than the MeWo Luc tumors . This lack of difference for MV3 is in line with the results on RNA level as no significant difference in Jagged 1 and Notch was detected by analysis of the arrays as well.As L1CAM knockdown also resulted in a reduction in metastasis for the initially L1CAM low MV3 cells, an additional whole genome expression analysis of the corresponding MV3 primary xenograft tumors was performed . Genes wFig 6), up-regulation of RFWD2  in the L1 kd tumors should lead to an increase in p53/p21 pathway activity. This could be verified for the subcutaneous tumors of both cell lines using immunohistochemistry; however, there was a difference between the two cell lines: The L1CAM wildtype MeWo Luc tumors showed no p53 and low p21expression but the respective L1CAM knockdown tumors displayed clear p53 and p21 expression. In contrast to the MeWo Luc tumors, a pronounced p53 expression was found for the MV Luc tumors which did not change for in the L1 kd tumors. However, as for the MeWo tumors, p21 expression did increase in the MV3 L1 kd tumors . According to the presumption that tumor cells with increased p53/p21 pathway activity should display a marked reduction in their metastatic potential, the remaining metastasis in the lungs of the L1 kd animals indeed showed no pronounced change in p53/p21 expression compared with their Luc counterparts with unchanged L1CAM expression .Of the genes regulated in the L1 kd tumors of both the initially L1CAM high (MeWo) and L1CAM low (MV3) cell lines .The only up-regulated gene of the 17 \u2013MAP2K6  indicated an increase in p38 phosphorylation in the L1 kd tumors. The subcutaneous tumors for both cell lines showed an extreme intra-tumoral heterogeneity in p38 phosphorylation ranging from areas with extremely high phospho-p38 to areas with no detectably phospho-p38 which made a reasonable analysis by immunohistochemistry impossible. The L1 kd metastases from both cell lines, however, displayed an increase in phospho-p38 compared with their Luc counterparts . An exception to this was the expression of the Notch ligand Jagged1 which showed an increase in expression. Genes with changed expression in the xenograft tumors after L1CAM knockdown for both melanoma cell lines indicate an increase in p53/p21 and also p38 activity which could be verified on protein level using immunohistochemistry. Both, reduced expression of EMT-network genes and increased p53/p21 and P38 activity probably contribute to the reduction of the metastatic potential of the human melanoma cells after L1CAM knockdown.in vitro experiments with the MeWo and MV3 Luc and L1CAM knockdown cells gave no clear indication how L1CAM influenced the metastatic behavior of the corresponding xenograft tumors in vivo  as both cell lines displayed different changes, e.g. in proliferation, after L1CAM knockdown. Melanoma cells are rather heterogenic in many aspects [in vitro changes in behavior differed between the two cell lines. We used an originally L1CAM high (MeWo) and an L1CAM low (MV3) cell line which might have been differently affected by L1CAM knockdown as their original dependence on L1CAM probably also differed. L1CAM knockdown led to a decrease in in vitro proliferation for MV3, but obviously even increased tumor growth in vivo. However, the in vitro proliferation assay is performed with cells in 2D culture with a standard cell culture medium. The conditions in this assay are thus quite different from the growth conditions in the animals and it appears not always to be very helpful for predicting the in vivo behavior of the cells. Considering the conflicting in vitro data we think that it is all the more remarkable that the influence of the L1CAM knockdown on the in vivo metastatic potential did not differ between the cell lines as the xenograft model of this study clearly demonstrated that L1CAM expression promotes melanoma metastasis. The unchanged CTC numbers in the xenograft indicate that the number of cells that left the primary tumor and entered the bloodstream (to become CTC) and were able to leave the vasculature to invade underlying tissue indeed did not differ between Luc and L1 kd cells. However, this number might not be directly correlated with the number of metastases as the L1CAM knockdown probably affects steps of the metastatic cascade following extravasation as the increase in p38 phosphorylation  indicates that the melanoma cells in the L1 kd metastases are more stressed than the Luc controls, but L1CAM expression  could be detected even in the metastases formed by the L1CAM knockdown cells  indicating that L1CAM is necessary for the outgrowth of metastases. Additionally, involvement of L1CAM in spreading on the abluminal surface of capillaries has been demonstrated in a xenograft model of brain metastasis with human breast and lung cancer cells [The performed ent ones) which mier cells .Fig 4). Interestingly, L1CAM is able to directly or indirectly regulate TGFB1 expression and not only vice versa.Although melanocytes reside in the stratum basale of the epidermis they are not epithelial cells themselves but are derived from the neuronal crest. During malignant transformation these non-epthelial cells do not undergo a classic EMT (as observed in carcinoma), however, many molecules involved in EMT do also play important roles in the malignant transformation of melanocytes although these roles are sometimes different from classic EMT . For exaFor L1CAM low melanoma, a partial compensation for the lack of L1CAM could be an overexpression of JAG1 (Jagged 1), modulating Notch signaling . UpregulFig 6), RASAL2 (Ras GTPase-activating protein nGAP) and MALAT2 (long non-coding RNA Metastasis Associated Lung Adenocarcinoma Transcript 2) have been implicated in EMT networking, with the role of RASAL2 still under discussion [) and p38  pathways could indeed be verified by immunhiostchemistry.Of the genes regulated in the L1 kd tumors of both the initially L1CAM high and L1CAM low cell lines  in subcutaneous (s.c.) tumors of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown . All scale bars: 100 \u03bcm. Stainings show ncreased p53 for MeWo L1 kd tumors only but increased p21 for MeWo and MV3 L1 kd tumors.(TIF)Click here for additional data file.S2 FigImmunohistochemical staining for p53 (left panels) or p21 (right panels) expression (red) in lung metastases of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown . All scale bars: 50 \u03bcm. Stainings show no change in p53 and p21 expression for MeWo and MV3 L1 kd cells.(TIF)Click here for additional data file.S3 FigImmunohistochemical staining for phospho-p38 (red) in lung metastases of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown (respective lower panels). All scale bars: 50 \u03bcm.(TIF)Click here for additional data file.S1 TableIncluded are only fold changes at least +/- 1.51; ANOVA and adjusted p-values <0.05.(DOCX)Click here for additional data file.S2 TableIncluded are only fold changes at least +/- 1.51; ANOVA and adjusted p-values <0.05.(DOCX)Click here for additional data file."}
+{"text": "Higher plants are sedentary organisms which inevitably endure a variety of environmental stresses throughout the life cycle. Abiotic stresses can be atmospheric like cold, heat and UV irradiation; or can also be edaphic like salinity, drought, and heavy metal toxicity  was observed in Zea mays during cold acclimation. This resulted in the global deacetylation at the lysine residues on the histone subunits H3 and H4  along with reduced DNA methylation and dimethylation at H3K9 (H3K9me2) in the unsilenced stretches  crucially phosphorylate histone H3-Thr 3 (H3T3ph) in the pericentromeric/knob regions in Arabidopsis to maintain heterochromatin organization and chromosome functions  and Galactinol Synthase 3 in Arabidopsis. H3K27me3 decrease permitted the de-repression of these cold responsive genes and ensured systemic acclimatization to low temperatures  and COR413 in Z. mays exposed to cold / DREB1s, CORs, Responsive to Dehydration genes (RDs), cold induced genes (KINs), and the cold induced TF, ICE1 during cold stress  chromatin to maintain FLC expression  and their target genes  and the 600 kDa polycomb complex . The MSI-like protein, MSI4/FVE negatively regulated Chorispora bungeana exposed to 4\u00b0C chilling and \u22124\u00b0C freezing stress. Rapid alterations in cytosine methylation occurred throughout the periods of chilling and freezing  , VRN2 and Vernalization Insensitive 3 (VIN3). Cold stress induces the expression of VIN3, a homeodomain finger containing gene encoding protein. VIN3 associates with an unidentified complex and triggers repressive H3K27me3 and H3K9me in the FLC chromatin  and VAL2 stably repress FLC during prolonged cold treatment  in the late phases of vernalization. High ColdAIR levels reduce H3K4me3 in FLC and facilitates flowering after vernalization  recruits epigenetic factors to regulate vernalization-related floral gene expression in winter annual accessions of FLC during vernalization is maintained as a cellular memory via the mitotic inheritance of the repressive histone marks in the chromatin  has verified the increase in gene activating H3K9me3 and decrease in gene suppressing H3K27me3 in VRN1 during vernalization  were observed in both the vernalized and non-vernalized seedlings. This depicts that unlike in VRN1, cold treatment does not modify the chromatin of these genes (Achrem et al., VRN2 and FT expression in the germinating caryopses but refrain from regulating VRN1 expression in the shoot apices (Li and Liu, Arabidopsis seedlings exposed to 30\u00b0C, directly after cold treatment exhibited low H3K27me3 at the FLC locus (Bouche et al., Trans-generational repression of Triticum monococcum where VRN1 initiates the regulatory cascade to down-regulate cold acclimation genes like CORs and CBFs (Galiba et al., Epigenetic regulation under cold stress is a manifestation of multiple crosstalks Figure  and planAR and SW suggested the title of the opinion article. AB designed the article, drafted the entire manuscript and arranged the references. AR incorporated all the necessary modifications. All the authors checked and confirmed the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The first sentence of the sixth paragraph in the Discussion section should have cited references 39\u201345, 56 instead of 39\u201345. The correct sentence should read: It can be therefore safely inferred that the copper metal composing the axe was extracted from Southern Tuscan ores and not from Alpine or Balkanic ores, despite the fact that copper deposits in both areas were certainly known and exploited during the Copper Age .The third sentence of the eighth paragraph in the Discussion section should have cited reference 42 instead of references 42\u201356. The correct sentence should read: Virtually all of the objects found south of the Alps  were made of Tuscan or southern Alpine copper , whereas all of the objects found north (Tyrol) [41] and east (Serbia) [42] of the Alps were produced from Balkanic copper, mainly from Serbia and Bulgaria.Fig 5. 2D projections of the available lead isotope data for Neolithic and Copper Age objects from Italy , Tyrol [41], and Serbia [42] (from the late 5thmillennium to the early 3rdmillennium BC). (a) 206Pb/204Pb vs 207Pb/204Pb plot; (b) 206Pb/204Pb vs 208Pb/204Pb plot.The Fig 5 legend should have cited reference 42 instead of 42\u201356. The correct Fig 5 legend is:"}
+{"text": "AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, The online version of this article (doi:10.1007/s00439-017-1772-0) contains supplementary material, which is available to authorized users. AKT3 is the main candidate for microcephaly, ZBTB18 for AnCC and HNRNPU for epilepsy  is associated with a complex neurological phenotype, including moderate to severe intellectual disability (ID), microcephaly, epilepsy and anomalies of the corpus callosum (AnCC). More than 40 patients with 1q43q44 microdeletions of variable sizes identified by chromosome microarray have been reported. Comparison of their clinical phenotypes has established some genotype-phenotype correlations and has identified three genes, preferentially expressed in the brain and located in a genomic region spanning 1.36\u00a0Mb , as the main genes contributing to the 1qter microdeletion phenotype: AKT3, ZBTB18 and HNRNPU have recently been identified by whole exome sequencing in patients with different neurodevelopmental phenotypes. AKT3 encodes a serine/threonine protein kinase involved in the mammalian target of rapamycin (mTOR) signaling pathway. Gain-of-function point mutations or microduplications leading to abnormal AKT3 and mTOR activation, most of which are limited to somatic brain populations, cause a spectrum of disorders characterized by cerebral hemisphere overgrowth such as hemimegalencephaly (HME), megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) and megalencephaly-capillary malformation (MCAP)  encodes a C2H2-type zinc finger transcription factor negatively controlling the expression of genes involved in neuronal development, including cell division of progenitor cells and survival of postmitotic cortical neurons    U, an abundant nucleoplasmic phosphoprotein able to bind pre-mRNA in vivo, possibly involved in pre-mRNA splicing  in humans or mice , Genematcher  . Clinical data were collected using a standardized questionnaire directly from the referring clinicians. Microcephaly was considered for patients with an occipitofrontal circumference (OFC) of at least \u22122.5 standard deviation (SD) below the mean. A radiologist, a neuropediatrician and a geneticist collegially ascertained brain MRI anomalies. AgCC designates the absence of one or all parts of the CC, DysCC is used for complete CC with an abnormal shape or abnormally small CC, ThCC is used for complete CC with insufficient thickness.We then collected clinical and molecular data of patients with We retrieved the probability of loss-of-function (LoF) intolerance (pLI) calculated by the Exome Aggregation Consortium (ExAC) and the haploinsufficiency score (HI) established by Huang et al.  for genehttp://genetics.bwh.harvard.edu/pph2) and SIFT (http://sift.bii.a-star.edu.sg).Missense variants were assessed in silico for possible pathogenicity using Alamut Visual 2.7 , PolyPhen-2 (https://genome-euro.ucsc.edu) to align microdeletions on a schematic representation of the 1q chromosome. Alignments were performed using different colors explained in the figure legends. Minimal critical regions were defined as the smallest deleted region of overlap found in at least 95%  or 85% (AnCC) of patients harboring a given phenotypic trait. Frequencies were compared using the Fisher\u2019s exact test.We used UCSC  were predicted to be probably or possibly intolerant to haploinsufficiency among the 44 genes other than OR genes (Table S1). Among these genes, AKT3, ZBTB18 and HNRNPU are clearly\u00a0those with the highest pLI scores and HI ranks as well as the highest expression in the brain (Table S1). We then decided to focus our study on these three genes that likely contribute to most clinical features of 1q43q44 microdeletion syndrome, as predicted from previous genotype-phenotype correlation studies. Specifically, 12 microdeletions encompassed AKT3, ZBTB18 and HNRNPU; six completely or partially included AKT3 but not ZBTB18 and HNRNPU; nine encompassed AKT3 and ZBTB18 but not HNRNPU, two ZBTB18 and HNRNPU but not AKT3, and 25 deleted HNRNPU but not AKT3 and ZBTB18 .To decipher the contribution of genes located in 1qter region to the corresponding microdeletion syndrome, we collected clinical data from 17 patients with 1q43-q44 microdeletion (Table S2) and compiled them with those of 37 previously reported patients fulfilling our criteria . Altogether, the 54 deletions span a 10\u00a0Mb region comprising 83 genes, 39 of which encode clustered olfactive receptors (OR). Seven genes ; 47/54 patients had available brain imaging: 20 had AnCC, including ten with agenesis , five with dysgenesis  and five with thin corpus callosum . Finally, 36/54 patients had epilepsy .AKT3 and ZBTB18, regardless of the presence of HNRNPU in the deletions . AKT3 was included in 24/26 deletions identified in individuals with microcephaly, whereas only 2/23 patients with microdeletions sparing AKT3 had microcephaly (p\u00a0=\u00a01.46E\u22129). The number of patients with microcephaly who had deletions including ZBTB18 (n\u00a0=\u00a021/22) and sparing ZBTB18 (5/27) was also significantly different (p\u00a0=\u00a04.25E\u22128). Four of the six deletions including coding sequences of AKT3 only and one of the two deletions encompassing ZBTB18 but not AKT3 were associated with microcephaly. The minimal critical region for microcephaly  mapped to a region encompassing the 5\u2032 upstream region and the five first exons of AKT3  ZBTB18 haploinsufficiency may independently lead to microcephaly with a lower penetrance; and (3) co-deletion of AKT3 and ZBTB18, which are neighboring genes spaced from only ~200\u00a0Kb, may have an addictive effect, resulting in constant microcephaly.The alignments of microdeletions found in patients with a known OFC and comparison of their gene content showed that microcephaly was present in all 20 patients with deletions encompassing both AKT3 deletion was not significantly associated with AnCC. More precisely, 11/26 patients with AKT3 deletion versus 16/21 without AKT3 deletion had a normal CC (p\u00a0=\u00a00.06). Accordingly, all six patients with a microdeletion involving only AKT3 had a normal CC. The proportions of patients with AnCC who had deletions encompassing (17/32) or sparing HNRNPU (5/15) were also not significantly different (p\u00a0=\u00a00.23). In contrast, the number of patients with AnCC was significantly higher in cases of deletions containing ZBTB18 (17/22) compared with deletions sparing this gene . However, these proportions reached statistical significance when considering AgCC instead of AnCC (p\u00a0=\u00a00.01). These results suggest that (1) the main driver for AnCC, and more particularly AgCC, in the 1q region is ZBTB18, although with incomplete penetrance, (2) HNRNPU haploinsufficiency can be associated with ThCC, and (3) deletion of both ZBTB18 and HNRNPU, which are 792\u00a0kb distant, has an additive effect, resulting in more penetrant AnCC phenotype and more frequent AgCC.Considering all types of AnCC, the alignment of deletions revealed that, contrary to microcephaly, HNRNPU and only one had a deletion sparing HNRNPU  or spared (n\u00a0=\u00a025/27) and the absence of seizures in patients with deletion restricted to this gene confirmed that AKT3 was not involved in epilepsy. The difference in the number of epileptic patients with or without ZBTB18 deletion was also not significant . These results suggest that the loss of one HNRNPU allele is the primary cause of epilepsy.Deletions identified in epileptic patients showed a shift toward the telomeric extremity of the 1q region . Of the 36 patients with epilepsy, 35 had a deletion including HNRNPU mutations, six had constitutive de novo mutations and one has a mosaic frameshift mutation . Two of them are located in zinc finger domains in which missense mutations tend to cluster  inherited from his father who was reported to have an intelligence within the normal range , (2) 13 with a deletion including HNRNPU but not AKT3 and ZBTB18 walked at a mean age of 32.6\u00a0months , and (3) two had a large deletion encompassing the three genes and walked at a mean age of 6.5\u00a0years (range 5\u20137\u00a0years). Among non-ambulatory patients, one carried a HNRNPU deletion and the three others had a deletion of the three genes. These data suggest that the loss of one HNRNPU allele has a more deleterious effect on walking acquisition than the loss of ATK3/ZBTB18 genes but larger deletions including all three genes have even more severe consequences. Walking abilities in patients with HNRNPU point mutations were similar to those with deletions of the gene. Patients with ZBTB18 mutations globally walked later than those with deletions including AKT3 and ZBTB18 although this should be confirmed on larger patient series.Since formal evaluations of cognitive functioning were unavailable for most other patients, we used the postural and language milestones to evaluate their developmental level. Walking abilities were available for 22 patients with 1q43q44 deletions older than 2\u00a0years. Four of them were unable to walk and 18 walked independently at a mean age of 35\u00a0months (median age 24\u00a0months). Among ambulatory patients, (1) three had microdeletions encompassing n\u00a0=\u00a05) or full (n\u00a0=\u00a01) sentences. None of the four patients with a deletion of all three genes made sentences, whereas 2/2 patients with deletions including AKT3 and ZBTB18 but not HNRNPU, and 4/10 patients with HNRNPU deletions did. Thus, patients with HNRNPU point mutations globally had more severe speech impairments than patients with deletions including HNRNPU, who had more variable language abilities. Yet, language abilities were more preserved in patients with ZBTB18 point mutations and individuals with deletions including ZBTB18 but sparing HNRNPU, since 6/7 could speak sentences.Language abilities were available for 15 patients with 1q43q44 deletions older than 4\u00a0years of our series only. Three patients did not speak any word, six had acquired a few words, six were able to speak short  in individuals with missense mutations leading to increased mTOR signaling that are usually\u2014but not always\u2014limited to mosaic brain tissues  three deletions sparing AKT3 (one encompassing ZBTB18 and HNRNPU and two HNRNPU but not ZBTB18) were also associated with microcephaly, and (2) all patients with deletion comprising AKT3 but extending to and including ZBTB18 and/or HNRNPU had microcephaly. The OFC is known for nine patients with ZBTB18 mutations [from the literature (Table S6) and our series] and three have microcephaly. This observation, combined with the microcephaly phenotype described in Zbtb18\u2212/\u2212 mice, suggests that microcephaly can also be associated with ZBTB18 mutations and deletions with a lower penetrance. In contrast, data from patients with HNRNPU mutations  AKT3 haploinsufficiency is sufficient to cause microcephaly with high but incomplete penetrance, (2) the heterozygous loss of ZBTB18 may cause microcephaly with a lower penetrance, and (3) other regions located more distally (including ZBTB18) may contribute to microcephaly in addition to AKT3 deletion.The preponderant role of on that:  three deZBTB18 to be the main candidate gene for AnCC in the 1q43q44 deletion syndrome  three patients carrying HNRNPU deletions sparing ZBTB18 and two with HNRNPU point mutations had ThCC (one had DysCC), and (3) patients with ZBTB18 point mutations had either partial AgCC, DysCC or ThCC. Thus, ThCC is the main category of AnCC observed when HNRNPU is deleted and is possibly related to insufficient myelination of crossing axons rather than indicating malformation of the CC. We conclude that ZBTB18 haploinsufficiency predisposes to different types of AnCC, particularly partial AgCC, while HNRNPU anomalies are more specifically associated with ThCC. Furthermore, AgCC is significantly more frequent in patients with microdeletions comprising ZBTB18 extending towards the telomeric end of the 1q region, i.e., encompassing both ZBTB18 and HNRNPU, compared to those encompassing ZBTB18 but sparing HNRNPU. This suggests that the loss of genetic determinant(s) in 3\u2032 of the ZBTB18 coding sequence or that the co-deletion of ZBTB18 and HNRNPU has an additive effect resulting in AgCC. Moreover, the AgCC observed in patients with point mutations or small deletions altering mainly ZBTB18 is partial and characterized by a small splenium with the absence of beak  no patient with 1q43q44 deletion sparing HNRNPU and COX20. COX20 encodes a protein contributing to the assembly of mitochondrial cytochrome C oxidase and has been involved in a recessive disease with healthy heterozygous carriers  epilepsy is present in 90% of patients with deletions comprising HNRNPU and absent in 14/15 patients with deletions sparing HNRNPU, and (2) all patients with constitutive HNRNPU mutations have epilepsy. The only epileptic patient with a deletion sparing HNRNPU had a deletion encompassing ZBTB18. Given that 3/11 patients with ZBTB18 mutations, including two of our series, had seizures; epilepsy may also be a minor phenotypic trait in some patients with ZBTB18 haploinsufficiency. The pro-epileptogenic effect of ZBTB18 alteration could be masked by the strong penetrance of HNRNPU-related epilepsy in patients with loss of both genes.Two thirds of patients with 1q43q44 deletions have epilepsy. The minimal critical region for epilepsy included HNRNPU point mutations from both our series and the literature (n\u00a0=\u00a09) was 13.5\u00a0months (median 8.5\u00a0months), versus 12.3\u00a0months (median 12\u00a0months) in individuals with deletions encompassing HNRNPU but sparing ZBTB18 (n\u00a0=\u00a013) and 12.9\u00a0months (median 12\u00a0months) in patients with deletions encompassing both HNRNPU and ZBTB18 (n\u00a0=\u00a07). These observations suggest that the age at seizure onset is independent of the size of the deletion, and that the loss of one HNRNPU allele is the strongest factor determining the age at seizure onset.The mean age at seizure onset in patients with HNRNPU mutations or deletions. Tonic\u2013clonic seizures and atypical absences are the most frequently reported seizure types. Seizures occur with variable frequencies, are frequently triggered by fever at the onset of the disease and are pharmacoresistant in some patients. Diffuse or focal slow-waves or a slow background activity are recurrently reported on EEG recordings together with various epileptiform features.To date, available data did not reveal specific epileptic features in patients with AKT3 gene  and 5/8 of them aged 3\u201337\u00a0years were not able to speak sentences or to associate several words. Thus, the impression of a relatively preserved development in patients with ZBTB18 mutations from our series should probably be attenuated. These differences are apparently unrelated to the nature (missense versus truncating) of the mutation but likely to the small sample sizes. An overview of acquired developmental milestones in these series of patients shows that those with HNRNPU deletions or mutations have a globally more severe postural and speech delay that those with AKT3/ZBTB18 deletions and those with ZBTB18 mutations. Because the most severe group of patients have deletions encompassing HNRNPU, AKT3 and ZBTB18, co-deletions of these three genes could have an additive detrimental effect on neurodevelopment.Patients with AKT3 is the main gene driving microcephaly, ZBTB18 defect is responsible for AnCC and HNRNPU is the main gene accounting for epilepsy. These correlations can be summarized as follows: (1) AKT3 deletion causes microcephaly with incomplete penetrance but ZBTB18 and HNRNPU deletions may also be involved with a weaker effect; (2) epilepsy and the loss of one HNRNPU allele are strongly associated; and (3) AgCC, is dependent on the loss of ZBTB18 allele but is also influenced by the alteration of neighboring genes. Neurodevelopmental impairment in patients with ZBTB18 LoF is more variable and less severe than that with HNRNPU LoF. Additional studies are required to investigate factors controlling this phenotypic variability in more details, including in particular the possibility of modifiers variants located on the trans allele.In conclusion, the complete neurodevelopmental phenotype of the 1q43q44 microdeletion syndrome is the consequence of the deletion of three main genes spanning 1.36\u00a0Mb. Our data confirm that Figure S1. Alignment of all 1q43-q44 microdeletions included in this study. L1, L2, etc (literature) and D1, D2, etc  in the left margin refer to patients\u2019 identities used in the manuscript (JPEG 531 kb)Figure S2. Alignment of 1q43q44 deletions found in patients with microcephaly, anomalies of the corpus callosum and epilepsy. A. Alignment of deletions found in patients with (red bars) and without (blue bars) microcephaly showed a shift of microcephaly-associated deletions towards the centromere, i.e. encompassing AKT3 . B. Alignment of deletions according to the \u201cCC status\u201d  did not easily suggest the involvement of ZBTB18 . C. Deletions found in patients with (red bars) and without (blue bars) epilepsy were shifted towards the telomeric end of the regions, suggesting the involvement of HNRNPU  (JPEG 2965 kb)Figure S3. Alignment of 1q43q44 deletions found in patients with normal and abnormal corpus callosum. These alignments show the different categories of AnCC  in patients with deletions of different sizes. They show that i) deletions comprising ZBTB18 and AKT3 but not HNRNPU (A) are associated with all types of AnCC, ii) deletions comprising HNRNPU but not ZBTB18 and AKT3 (C) are not associated with AgCC, iii) most AgCC are observed in patients with large deletions including ZBTB18 and mostly extending to the telomere (B). (JPEG 767 kb)Figure S4. Orthologous ZBTB18 protein alignments in the regions surrounding the three affected amino acids altered by missense mutation reported in this study  (TIFF 502 kb)Table S1. Probability of haploinsufficiency intolerance (pLI) calculated by the Exome Aggregation Consortium (ExAC) and haploinsufficiency score (HI) for genes of the 1q43q44 region comprised between genomic positions 239,990,618 to 249,208 (PDF 240 kb)Table S2. Genetic and clinical data from the 17 patients with 1q43q44 deletions (PDF 169 kb)Table S3. Molecular and clinical data from 37 patients with 1q43q44 deletions in the literature (XLSX 29 kb)Table S4. Additional clinical data from the six patients with HNRNPU mutations (PDF 21 kb)Table S5. Clinical and molecular data from patients with HNRNPU mutations in the literature (PDF 113 kb)Table S6. Clinical and molecular data from patients with ZBTB18 mutations in the literature (XLSX 14 kb)Below is the link to the electronic supplementary material."}
+{"text": "PDE4B subtype selective inhibitors are known to reduce the dose limiting adverse effect associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. To achieve this goal, ligand based pharmacophore modeling approach is employed. Separate pharmacophore hypotheses for PDE4B and PDE4D inhibitors were generated using HypoGen algorithm and 106 PDE4 inhibitors from literature having thiopyrano  Pyrimidines, 2-arylpyrimidines, and triazines skeleton. Suitable training and test sets were created using the molecules as per the guidelines available for HypoGen program. Training set was used for hypothesis development while test set was used for validation purpose. Fisher validation was also used to test the significance of the developed hypothesis. The validated pharmacophore hypotheses for PDE4B and PDE4D inhibitors were used in sequential virtual screening of zinc database of drug like molecules to identify selective PDE4B inhibitors. The hits were screened for their estimated activity and fit value. The top hit was subjected to docking into the active sites of PDE4B and PDE4D to confirm its selectivity for PDE4B. The hits are proposed to be evaluated further using  Most of the mortality related to these inflammatory disorders occurs in low and low-middle income countries , a predominant family of phosphodiesterase (PDE) enzymes expressed in immune and inflammatory cells, includes three subtypes PDE4A, PDE4B and PDE4D. Inhibition of PDE4 has been shown to suppress a diverse spectrum of inflammatory responses  in-vivo -5. More  in-vivo , 6, 7, a in-vivo -10.in-vitro and in-vivo. Investigation in ferrets also showed significantly less emesis with this compound compared with the non-selective PDE4 inhibitor cilomilast  and C-terminal domain CR3 (i.e Leu in PDE4D vs Gln in PDE4B)  , 17. Then PDE4B) -18.The availability of PDE4B and PDE4D inhibition data for recently reported PDE4 inhibitors allows the development of pharmacophore models of PDE4B and PDE4D inhibitors -21. PharData set50 of the above series were taken from the original papers. Numbers used in original papers were used to denote molecules belonging to triazine series while numbers used in original papers for molecules belonging to 2-arylpyrimidine and thiopyranoPyrimidine series were suffixed with a and b respectively.Selective PDE4B inhibitors belonging to thiopyrano Pyrimidines,2 arylpyrimidines and triazines class reported recently, along-with their PDE4B and PDE4D inhibitory activities, were used for the present study  19-21).-21.19-213D QSAR pharmacophore modelingPharmacophore generationPharmacophore modeling is the most widely used method for identification of essential structural features required for biological activity. In the present study, HypoGen algorithm was applied to build the 3D QSAR pharmacophore models for both PDE4B and PDE4D inhibitors using DS V2.0 software  . 50) between 3.0 nM and 18755 nM were selected as training set, which was used to engender the hypotheses. The training set selected complies with the requirements specified in the literature. To validate the hypothesis, the test set was prepared using the specified requirements. Test set contains 24 molecules having wide range of activity values. Sketch function of DS was used to sketch the two-dimensional (2D) chemical structures of all molecules which were later converted into 3D structures. Maximum of 250 conformations were generated for each molecule using the best conformation model generation method based on CHARMm force field and Poling algorithm , HBD (hydrogen bond donor), and H (hydrophobic), features were selected based on the training set molecules. The number of features allowed in the model were kept in the range 0-5. The \u2018Uncertainty\u2019 values for all the 75 molecules in the training set were set to 2.0, and the default values for other parameters were kept constant. Subsequently, pharmacophore models were computed and the 10 top scoring hypotheses for both PDE4B and PDE4D inhibition were selected for further study. The qualities of the hypotheses were reliant on the fixed cost, null cost, and total cost values .Assessment of pharmacophore quality Quality of the developed pharmacophore was assessed using three different methods. Initially, cross validation was performed by the Fischer\u2019s randomization test. Secondly, the prediction of the activity values of the test set was performed. The correlation between the experimental and predicted activities was used to assess predictive ability of the model. All queries were addressed using the ligand pharmacophore mapping protocol. Virtual Screening50 less than 20 nM were selected and subsequently subjected to screening using the validated pharmacophore model Hypo1D in the same manner as in the previous step. The hit compounds were chosen that showed Hypo1D estimated fit value less than 4. The validated pharmacophore model (Hypo1B and Hypo1D) of PDE4B and PDE4D inhibitors was used as a query in a sequential manner to search the zinc database. Zinc is a comprehensive database of small molecules containing a total of 17,900,742 drug like molecules . In the i.e. 34b, were docked into the active sites of PDE4B (PDB ID: 4NW7) and PDE4D (PDB ID: 1Y2B). First the protein structures were prepared using the automatic protein preparation module of DS V2.0 software using the default parameters. The structures of the identified hit as well as the standard molecule (34b) were prepared using the prepare ligand module of DS V2.0 software. Docking of the prepared ligands into the active site of the prepared structures of PDE4B and PDE4B was carried out using CDOCKER program available in DS V2.0 software  with default parameters  for PDE4B inhibition consist of three HBA which established the highest cost difference (143.378), best correlation coefficient (0.9571), maximum fit value (5.8678) and lowest root mean square (RMS) of 1.86 . The resTop scoring model (Hypo1D) for PDE4D inhibition consists of two HBA and three H with highest cost difference (164.419), best correlation coefficient (0.9563), maximum fit value (8.1515), and lowest root mean square (RMS) of 1.66 . As in t50 values.The experimental and predicted activities by Hypo1B and Hypo1D for 75 training set molecules are shown in dFit value indicates how well the features in the pharmacophore overlap the chemical features in the molecule. Fit value = weight x [max] where SSE = (D/T)2, D = displacement of the feature from the center of the location constraints and T = the radius of the location constraint sphere for the feature (tolerance).eDifference between the predicted and experimental values. \u2018+\u2019 indicates that the predicted IC50 is higher than the experimental IC50; \u2018-\u2019 indicates that the predicted IC50 is lower than the experimental IC50; a value of 0 indicates that the predicted IC50 is equal to the experimental IC50.3 and hydrophobic groups like halogen atoms in the aromatic ring (Ar) to orient properly for interaction with CR3 will show significant selectivity for PDE4B as compared to PDE4D. This is consistent with the findings described previously in the original papers in which these compounds have been reported  for the test set given by Hypo1B was 0.8579  while thVirtual ScreeningZinc, a comprehensive database of small drug like molecules was used for the sequential virtual screening using the pharmacophore models. Screening of zinc database using the validated pharmacophore model (Hypo1B) of PDE4B inhibitors retrieved a set of 6015 hits, mapping to the pharmacophore model Hypo1B. The hits comprised of some compounds structurally similar to that of the existing PDE4B inhibitors, and some novel scaffolds. 50 less than 20 nM were selected and subsequently subjected to screening using the validated pharmacophore model Hypo1D. 5 hit compounds that showed Hypo1 PDE4D estimated fit value less than 4 were identified (The 397 hit compounds showing Hypo1B estimated ICentified . Among tentified . ZINC091The results of docking studies of ZINC09157416 and 34b with PDE4B and PDE4D further confirmed the selectivity of ZINC09157416 for PDE4B over PDE4D .Ligand-based pharmacophore models for a diverse class of PDE4B and PDE4D inhibitors were developed. The best pharmacophore models Hypo1B and Hypo1D were validated using different methods to evaluate their predictive power over the diverse test set compounds. Hydrogen bond acceptors were identified to be mainly responsible for PDE4B inhibition while both hydrogen bond acceptors as well as hydrophobic groups were found to be responsible for PDE4D inhibition. The highly predictive pharmacophore hypotheses were further used in sequential virtual screening for identification of selective PDE4B inhibitors. Zinc drug like database was used in virtual screening. The hits from the virtual screening were filtered based on the estimated activity and fit value. Five molecules with different backbones were identified as final hits. The most selective hit molecule ZINC09157416 exhibited better selectivity for PDE4B than the standard compound 34b in the docking studies. The activity of the hit compound has not been reported in the literature as we explored by PubChem and SciFinder Scholar search tools. Thus, the sequential virtual screening strategy using 3D QSAR pharmacophores for PDE4B and PDE4D inhibitors proved to be an effective strategy to identify novel selective PDE4B inhibitors."}
+{"text": "During the proton irradiation of thorium targets, large amounts of 103Ru are generated through proton induced fission. The development of a two part chemical separation process to isolate 103Ru in high yield and purity from a proton irradiated thorium matrix on an analytical scale is described herein. The first part employed an anion exchange column to remove cationic actinide/lanthanide impurities along with the majority of the transition metal fission products. Secondly, an extraction chromatographic column utilizing diglycolamide functional groups was used to decontaminate 103Ru from the remaining impurities. This method resulted in a final radiochemical yield of 83 \u00b1 5% of 103Ru with a purity of 99.9%. Additionally, measured nuclear reaction cross sections for the formation of 103Ru and 106Ru via the 232Th103,106Ru reactions are reported within.Ruthenium-103 is the parent isotope of  This decay gives rise to the emission of low-energy Auger/Coster-Kronig electrons (2.3 electrons/decay) . The beam profile was assessed following the experiment by the activation of thin stainless steel plates whose dimensions significantly exceeded those used for the thorium foils. The steel plates were exposed to Gafchromic\u00ae film following the end of irradiation in order to map the beam profile, which was determined to have been quantitatively incident on the desired targets in both experiments.Thin thorium foils were irradiated in two separate experiments at the Los Alamos Neutron Science Center (LANSCE) at LANL using incident proton energies of 100 and 200 MeV as described previously . In eachA 10 g thorium metal target was irradiated at the Isotope Production Facility (IPF), Los Alamos National Laboratory . The target was encapsulated in Inconel cladding and placed into the high energy \u201cA\u201d slot  of the IPF target assembly. IPF targetry and 4\u03c0 water cooling were identical to the design as described previously , 19. The225Ac. The target was dissolved in 200 mL 10 M HCl and 0.1 mL of 2M HF with heating (80\u201390\u00b0C) for approximately 2 hours. A 0.1 mL aliquot of the dissolved target was diluted to 5.1 mL with 0.1M HNO3. This solution was then used as a stock solution for radiotracers that represent radionuclides previously identified in the target [103Ru, eight 5 mL fractions of 10 M HNO3 were added to the column and collected (fractions 8\u201315). All fractions were analyzed by HPGe spectroscopy using the characteristic \u03b3-rays as identified in 225Ac purification process as described previously [The irradiated 10 g thorium target was shipped to Oak Ridge National Laboratory (ORNL) for recovery of e target . For theeviously , 14.103Ru eluted in fractions 8\u201315. Contaminants present included 95Zr, 95Nb, 233Pa, 230Pa, 117mSn and 124,125Sb. These fractions were brought to near dryness and reconstituted in 10 mL 10 M HCl. This solution was then passed through a column containing 1 mL DGA  equilibrated with 10 M HCl. The eluent was collected (fraction 16) and the column was washed with an additional 20 mL 10 M HCl (fraction 17). All separation experiments were performed in triplicate.A second column was developed to remove contaminants from the 103Ru, and 106Ru are plotted in Measured excitation functions of 225Ac, Ra, Ba, lanthanides and bulk thorium along with the majority of 95Zr passed through the anion column in the loading fraction and 10 mL 10 M HCl wash (fractions 1\u20133). Ruthenium is most strongly retained on the anion exchange resin using 1 M HCl [95Nb and 123mTe along with approximately 45% of the Pa. The loading and washing of the column resulted in 103Ru losses of 8\u201315%. Thirty milliliters of 10 M HNO3 resulted in the elution of 85 \u00b1 5% of 103Ru with a radiochemical purity of 82%. The main impurities present in this fraction consisted of 117mSn and 125,126Sb with trace amounts of 230,233Pa, 95Nb, and 95Zr. The cationic species such as 3 solutions and several papers discuss fission product behavior in these systems [103Ru from the DGA column was 98 \u00b1 1% resulting in a final 103Ru recovery of 83 \u00b1 5% with a radiochemical purity of > 99.9%. Ruthenium speciation is a complicated subject with respect to its separation in acid based systems and is likely responsible for the high variability in the loss of 103Ru from the anion column (8\u201315%) [103Ru is an ancillary activity with respect to 225Ac recovery. A flow diagram of the whole process is shown in According to a paper published by Pourmand et al. , Nb, Zr, systems , 27\u201332.  (8\u201315%) . Ideally117mSn and 123mTe have the same identifying \u03b3-ray lines without a viable secondary gamma, a spike of 121mTe was added to the sample to help deconvolute the separation of 123mTe and 117mSn. This information led to the determination that 123mTe was present in the 1 M HCl fractions and 117mSn was present in the 10 M HNO3 fractions. This elemental distribution is further corroborated by prior reports that tellurium is eluted from anion columns in 1 M HCl while tin is retained strongly, and that tin elutes with 103Ru in 10 M HNO3 [As both 0 M HNO3 , 35.106Ru. However as 106Ru decays to 106gRh (29.9 s), 103mRh obtained from a generator would be isotopically pure five minutes after elution. The predicted experimental yield calculated from measured cross sections of 103Ru, with anticipated full scale 225Ac production, is (~ 3 Ci (111 GBq)) at end of bombardment. This would significantly increase the current supply of 103Ru for medical research needs.Ruthenium-103 obtained from this method contains the isotopic impurity 103Ru/103mRh generator system. Solvent extraction generators have been designed employing a carbon tetrachloride extraction, however given the toxicity associated with CCl4 this method is not amenable for biomedical applications [103mRh with minimal breakthrough of 103Ru. This would preferably entail the use of mineral acids that can be readily removed from the product such as HCl or HNO3.Future work needs to be performed in order to determine suitable conditions for a ications , 36. A s103Ru that is produced concurrently with 225Ac. This method results in a final 103Ru chemical recovery yield of 83 \u00b1 5% with a radiochemical purity of > 99.9%. The measurement of energy dependent cross sections for the proton induced fission production of 103Ru and 106Ru at proton energies less than 200 MeV on 232Th targets predict thick target yields of ~111 GBq. This process can be implemented with the existing 225Ac recovery flow sheet at minimal impact to the 225Ac process. Additionally, future work to develop a robust 103Ru/103mRh radionuclide generator is essential to the success of 103mRh for auger therapy.A method was obtained for the recovery and purification of"}
+{"text": "COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts.Hereditary microscopic haematuria often segregates with mutations of We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses.COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested.We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome . Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the \u03b11 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans.Missense mutations of  Isolated microscopic haematuria is a significant risk factor for kidney failure in later life  and can COL4A3, COL4A4 or COL4A5 genes that encode type IV collagen, and the clinical features overlap with the carrier state for autosomal recessive or X-linked Alport syndrome , three requiring renal replacement therapy.COL4A3 and COL4A4) were sequenced in the proband by amplification of each exon and Sanger sequencing. This did not reveal any rare or likely pathogenic variants. A genome-wide linkage study was performed using DNA from 12 affected family members. This excluded linkage with loci containing the genes COL4A3, COL4A4, COL4A5, MYH9 and CFHR5 (LOD < \u22122 at each locus), but did demonstrate linkage (LOD = 3) with loci on chromosomes 6 and 13 that include the COL4A1 gene on chromosome 13  and predicted a change in amino acid sequence of a protein  were identified. Of these, 644 were heterozygous and 270 were novel (i.e. not reported in dbSNP). Two rare variants, both heterozygous, occurred within the linked loci. One variant predicted a missense mutation p.A101T in TMEM14C (NM_016462) on chromosome 6. This gene has not previously been associated with kidney disease and the variant is predicted to be benign, with SIFT and Polyphen scores of 0.42 and 0.023, respectively. The other variant was in the COL4A1 gene and encodes a novel frameshift mutation c.4611_4612insG; p.T1537fs (transcript NM_00185). This was considered a good candidate for causing disease since other mutations in this gene are associated with HANAC syndrome, in which haematuria, kidney cysts and renal impairment are features.Genes associated with autosomal dominant thin basement membrane nephropathy  exhibited renal impairment, haematuria, proteinuria and kidney cysts  and individual II-6. No rare or likely pathogenic mutations of these genes were identified in either of these individuals. We conclude that the COL4A1 mutation is responsible for the familial renal disease in this kindred. We went on to examine whole exome sequencing data from four other families in which glomerular basement membrane abnormalities and microscopic haematuria segregated as a dominant trait in the absence of a COL4A3, COL4A4 or COL4A5 mutation but did not find any additional likely pathogenic variants in COL4A1.To determine whether an additional mutation might be segregating in other family members we went on to sequence Renal failure was unpredictable, with creatinine rising above the normal range in some affected members after the age of 40 years. End-stage renal failure ranged from 63 to 70 years in three of nine siblings in the first generation. In those affected, one or more kidney cysts were detectable by ultrasound from the age of 40 years in 12 family members. Hypertension was variable, but could be present from 35 years of age. No family members reported a history of muscle cramps or cerebral aneurysms, and serum creatine kinase was normal in all 22 family members tested. Some family members had evidence of vascular disease  and 3 had an elevated NAG:creatinine ratio .Little or no proteinuria  was found until the eGFR was <50 mL/min/1.73 mA kidney biopsy was performed in one family member at age 47 years to investigate familial nephropathy and reduced eGFR. Light microscopic examination was normal with the exception of minimal global glomerulosclerosis (1 of 25 glomeruli) and <1% tubular atrophy. Immunostains for type IV collagen were not performed, but stains for immunoglobulins and complement were negative. Electron microscopy demonstrated areas of thinning of glomerular basement membranes (down to 93.5 nm) and subtle lamellation of tubular basement membranes  that have a more restricted distribution of expression and are less broadly conserved across species [Type IV collagen is an important constituent of the extracellular matrix and forms a complex meshwork that provides structural integrity to basement membranes . Individ species .COL4A1 are associated with neurological, vascular and renal disorders. Previously described phenotypes cluster into primarily neurological disease, in which porencephaly (the occurrence of fluid filled cavities in the brain) and cerebral vasculopathy or haemorrhage cause significant neurological damage early in life [COL4A1-associated diseases are incompletely understood. However, it is unlikely that haplo-insufficiency is a major contributor in most cases: previously described pathogenic mutations in humans are almost all missense variants and do not include early nonsense mutations or heterozygous whole gene deletions; only a single collagenous-domain frameshift mutation that reduced transcript levels has been reported to date [Col4A1 allele do not display any detectable phenotype [COL4A1, encoding a region of the protein that contains multiple potential integrin-binding sites, and it has been suggested that disruption of normal interaction between type IV collagen and integrins might be responsible for HANAC syndrome [COL4A1-associated diseases.Pathogenic mutations of  in life , and HAN in life . The mec to date . Moreovehenotype . Mutatiosyndrome , 24. ThiCOL4A1 are exceedingly rare, with only one example observed (encoding p.P438fs) in >120 000 alleles tested in the ExAC project and a single pathogenic allele (p.G696fs) reported previously in the medical literature [211), which is essential for the sulfilimine bond cross-linking adjacent trimers to form hexameric type IV collagen [Frameshift variants of terature . The p.Tcollagen . It alsoSupplementary Video S1). Alternatively, since it is known that \u03b11 type IV chains are expressed in podocytes and present in glomeruli of healthy adults [COL4A1 is considered less likely for the reasons stated above. A homozygous truncating mutation within the C-terminal NC1 domain of COL4A4 has previously been reported in association with autosomal recessive Alport syndrome, but the phenotype of the obligate carrier parents was not documented in this report [One possible mechanism whereby loss of this part of the NC1 domain could cause disease includes disruption of sulfilimine bond(s) by inclusion of one or more defective \u03b11 chain missing the C-terminal part of its NC1 domain within an \u03b11.\u03b11.\u03b12 type IV collagen heterotrimer (See related article by Savige. A further genetic cause of thin basement membrane nephropathy."}
+{"text": "NOD2 contributes to the innate immune response and to the homeostasis of the intestinal mucosa. In response to its bacterial ligand, NOD2 interacts with RICK and activates the NF-\u03baB and MAPK pathways, inducing gene transcription and synthesis of proteins required to initiate a balanced immune response. Mutations in NOD2 have been associated with an increased risk of Crohn\u2019s Disease (CD), a disabling inflammatory bowel disease (IBD). Because NOD2 signaling plays a key role in CD, it is important to further characterize the network of protein interacting with NOD2. Using yeast two hybrid (Y2H) screens, we identified new NOD2 interacting proteins (NIP). The primary interaction was confirmed by coimmunoprecipitation and/or bioluminescence resonance energy transfer (BRET) experiments for 11 of these proteins . These proteins are involved in diverse functions, including endosomal sorting complexes required for transport (ESCRT), cytoskeletal architecture and signaling regulation. Additionally, we show that the interaction of 8 NIPs is compromised with the 3 main CD associated NOD2 mutants . Furthermore, to determine whether these NOD2 protein partners could be encoded by IBD susceptibility genes, a transmission disequilibrium test (TDT) was performed on 101 single nucleotide polymorphisms (SNPs) and the main corresponding haplotypes in genes coding for 15 NIPs using a set of 343 IBD families with 556 patients. Overall this work did not increase the number of IBD susceptibility genes but extends the NOD2 protein interaction network and suggests that NOD2 interactome and signaling depend upon the NOD2 mutation profile in CD. Toll Like Receptors (TLR) and Nod-Like Receptors (NLR) are major receptors of the innate immune system . These pNOD2 is the main CD susceptibility gene. Up to 50% of Caucasian CD patients carry mutations in NOD2 . Fi. FiNOD2 assessed  Tables. assessed .Several clones coding for RICK protein were identified in the colon or lung library, validating the Y2H screening procedure, since RICK is an undisputed NIP. All RICK positive clones contained the C-terminus CARD domain of RICK, a region known to be involved in a heterotypic interaction with the N-terminus CARD domains of NOD2 . ImportaThe NIP candidate CHMP4b was found several times only in the colon library . As CHMPAs a result of Y2H screens, 14 preys were selected to further investigate their interaction with NOD2. Several proteins like RICK, CHMP5, TRIM41, SDCCAG3, VIM, and ANKHD1 were chosen because they were identified independently in the colon and the lung cDNA library. The other selected proteins  included mostly NIP candidates isolated a minimum of 3 times.Co-immunoprecipitation experiments were performed to confirm Y2H interactions. The full length (or near full length) cDNAs coding for the 14 selected NIPs including RICK were cloned in a plasmid to express EGFP fused proteins. Each EGFP-tagged NIPs was co-expressed with Myc tagged NOD2 protein in HEK293T cells by transient transfection. The cells were then lysed in a \u201cstringent\u201d buffer containing 1% TX100 and subjected to immunoprecipitation with anti-myc (to immunoprecipitate myc-NOD2) or anti-GFP antibodies (to immunoprecipitate each EGFP-NIP) and immunoprecipitated proteins were analysed by Western blot using anti-myc and anti-GFP antibodies. RICK and NOD2 were used as positive controls . Six putThe Bioluminescent Resonance Energy Transfer (BRET) approach reveals direct interaction between proteins in the cellular context without cell lysis . FollowiIn order to understand the impact of NOD2 activation on the NIP-NOD2 interaction, BRET experiments in live cells were also carried out with the addition of MDP following transfection. Among all the proteins investigated, only the CHMP5/NOD2 BRET profile appeared clearly modified by the addition of MDP. In that case, an overnight incubation with MDP induced a decrease in BRETmax of the NOD2/CHMP5 interaction, indicating either a conformational change or dissociation of the protein complex after MDP activation . This reAn interaction score was calculated as an attempt to classify the NIPs characterized in this study. This score (arbitrary unit) integrates, the number of hits in Y2H screens, the \u201cstrength\u201d of the co-immunoprecipitation and the profile of the BRET saturation curves and MDP response . In summTo evaluate the functional role of each NIP on the NOD2 dependent NF-\u03baB signaling, reporter assays were carried out in HEK293T cells transfected with plasmid encoding luciferase under the control of NF-\u03baB response elements. As reported elsewhere, overexpressing NOD2 in these cells stimulates NF-\u03baB and a MDP incubation further increased NF-\u03baB activation . The celNod2 is constitutively expressed by the THP-1 monocyte cell line. The expression of Nod2 and 10 NIP genes was studied by qRT-PCR arrays upon MDP, LPS or LPS + MDP stimulation . Nod2, VThe interaction between 8 NIPs and the three main CD associated NOD2 mutants were tested by Y2H. By this analysis , we obseNOD2 mutation.Genetic analyses were performed for genes encoding 15 NIPs with a set of 101 Single Nucleotide Polymorphisms (SNP) chosen to cover the vast majority of gene haplotypes with a frequency higher than 0.05, as estimated on the Hapmap website . StudiesDOCK7, GOLGB1, IKBIP, PRR16, and VIM  was tested for each SNP independently and for all possible haplotypes with a combination of 2 or 3 of the genotyped SNPs in the different family groups. Among the 15 genes, we found a weak distortion of transmission for haplotypes in 5 NIPs: M Tables  and 5.DOCK7, a positive TDT was seen for rs17381383 in the group of CD only families . This marker tags a haplotype with a frequency of 0.142 in the Hapmap CEU panel and 0.15 in our cohort  and for another haplotype in the whole group of CD families (T: 24 U: 9 p = 0.0090)  were found in the IBD family cohort most often with haplotypes containing of the intronic SNP rs13161840. Similarly, distortions of transmission were observed in the NOD2 mutated CD families with haplotypes containing the minor frequency allele of rs300970. However, the p values of all these TDT did not remain significant after applying the Bonferoni correction for multitesting.For r cohort . For GOL 0.0090) . A nomin 0.0090) . PRR16 iVIM is defined by one haplotypic block and 8 haplotypes. The 4 genotyped SNPs were available defining only 3 different haplotypic groups in the 8 haplotypic group defining VIM in HapMap CEU panel. A distortion of transmission was observed for an haplotype with a frequency of 0.557 in NOD2 WT CD families . These novel protein partners extend the NOD2 interactome and provide new leads about mechanisms of NOD2 function, regulation, and subcellular localization. The use of two independent methods to confirm Y2H interactions allowed validation of a total of eleven NOD2 interacting candidates. Importantly, some interactions could not be seen by co-immunoprecipitation, but were positive in BRET experiments, suggesting that the intracellular milieu and/or the subcellular localization can be important for some specific interactions to occur.Based on a generic NF-\u03baB reporter assay, combined with the use of siRNA, none of the NIPs isolated here, except RICK, appear to be major regulators of the NOD2 dependent NF-\u03baB pathway at least in HEK293T cells. These data are however consistent with a recent publication reporting the identification by genome wide RNAi screen in HEK293 cells of new positive and negative regulators of the NOD2 dependent NF-\u03baB pathway . Of noteThe expression of several NIPs is upregulated by MDP and LPS in THP-1 cells suggesting that these proteins may play a role as feedback control to amplify or down regulate the NF-\u03baB signaling pathway. Alternatively some NIPs may be involved in other specific NOD2 functions including autophagy induction, Interleukin production (IL-8 or IL1\u03b2) or in the subcellular localization and traffic of NOD2 during signaling.Importantly, our data indicate that the 3 main NOD2 mutants associated with CD, R702W, G908R and 1007fs  interact differentially with 8 NIPs and this could have functional consequences in term of downstream signaling. Future studies will help define whether these alterations (loss or gain) of interactions between NIPs and NOD2 CD associated mutants could contribute to the pathophysiology and severity of CD.In The NIPs identified in this study are possible candidate genes for susceptibility to CD or UC, another IBD condition. We thus analysed the main NIPs in a cohort of more than 300 European IBD families. This cohort may appear limited when considering the very large cohorts used in GWAS to date, however the type of analysis is quite different and good power can be attained with a modest number of inferred meioses from the family data.NOD2 mutations. Unfortunately, this result was not reproduced in the second family sample. Similarly, a weak association has been reported by Stevens et al. between VIM polymorphisms and CD but without firm validation [RICK, a well-known NIP [Among all the tested NIPs, we observed a positive association with some haplotypes of VIM in the CD family group carrying no lidation , especialidation . We thusnown NIP  was likenown NIP .C10orf67 and SDCCAG3 have been proposed as putative genes associated with CD [C10orf67 was found following a genome scan to isolate genes associated with sarcoidosis and CD. The SDCCAG3 locus is in a region where a SNP associated with CD was found in the vicinity of another IBD gene candidate, CARD9. However, none of the SNPs tested here for SDCCAG3 and C10orf67 showed any association. For SDCCAG3 and C10orf67, we also genotyped the two SNPs previously found to be associated rs10870077 and rs1398024 respectively and we were unable to replicate the previous observation . It is nevertheless very striking that the C10orf67 locus was identified in our study on NOD2 partners and in an independent study of CD genetics based on totally different screening procedure [C10orf67 and C10orf115. Surprisingly the cDNAs sequences of our Y2H positives clones were actually chimeric between the C10orf67 and C10orf115 loci. However, the full cDNA that was used in this study for validation experiments spans only sequences of the C10orf67 gene. Further biochemical studies are thus required to determine the exact nature of protein(s) encoded by transcripts arising from this 10p13 region and whether they interact with NOD2 and could possibly contribute to CD susceptibility. In summary, we report here a set of new NOD2 protein partners that connect into the NOD2 protein network  are shown in parallel with the Nomarski field (left). The white scale bar represents 10\u03bcm.(PDF)Click here for additional data file.S1 TableGrowing yeast colonies were tested and scored by 3 phenotypic assays .(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file."}
+{"text": "In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.Human embryonic (hES) stem cells are widely used as an  Pre-clinical animal studies undertaken over a decade involving transplantation of hES derived cardiomyocytes have also shown considerable promise. Studies describing differentiation and outcome of transplantation using hES cells derived cardiac cells are compiled as Supplementary Table\u00a03. In order to address these questions, it is necessary to understand stage-wise gene expression during early cardiac development and hES cells serve as an excellent model to study this differentiation in vitro.The successful isolation of human embryonic stem (hES) cells in the late nineties5 and plays an important role when pluripotent hES cells become committed. Chromatin modification is one of the fundamental epigenetic mechanisms associated with active or repressed state of gene expression in a cell8. Active states are marked by methyl modifications like H3K4me3 and H3K36me3 brought about by Trithorax group (TrxG) of proteins10 while repression is catalysed by addition of H3K27me3 by Polycomb group (PcG) of proteins11 grouped as Polycomb Repressive Complex 1 (PRC1)  and Polycomb Repressive Complex 2 (PRC2) 13.In addition to various signalling cascades and transcription factors, epigenetics is another player involved in major functional genomics decisionsin vitro15. Dynamics of PcG proteins during differentiation of human ES cells into the three lineages was for the first time reported by our group17. Transcripts of both PRC1 and PRC2 were found up regulated during endodermal differentiation while during ectodermal differentiation only PRC1 transcripts were found to be up regulated. On the other hand, both PRC1 and PRC2 were down regulated during mesoderm lineage formation in vitro. A study by Murry\u2019s group determined the evolution of chromatin modifications like H3K4me3 and H3K27me3 along cardiac lineage formation wherein the dynamic acquisition of H3K4me3 and H3K27me3 by various transcription factors during differentiation at the five different time points was identified18. Bruneau and colleagues19 also revealed a unique epigenetic pattern of H3K27ac, H3K4me1, H3K4me3 and H3K4me3 during the gene expression changes of cardiomyocytes differentiation in vitro.Various efforts are now focussed to characterize the ES cells and their differentiated progeny with respect to epigenetic mechanisms. Importance of PcG proteins for differentiation of ES cells and not in their self-renewal was identified when the ES cells deficient for PRC2 proteins  could still be maintained in their undifferentiated state 23. Though much of the research has focused on providing key conclusions regarding genetic and epigenetic regulatory mechanisms, we are still left with key questions like how are only the right genes expressed or repressed in a given cell type? How does the gene activation or repression machinery gain or keeps its access into the cell? In the present study, we aim to understand the gene expression and histone modification pattern during cardiac differentiation from hES cells using directed differentiation protocol.All these emerging evidences of requirement of unique pattern of epigenetic mechanisms during lineage specification indicate that the disturbed or faulty epigenetic program may contribute to various cardiac disorders. Mutations in a number of histone methyltransferases like JMJD2A, H3K36me3 methyltransferase SETD2, H3K4me3 methyltransferase MLL2, H3K27me3 methyltransferase EZH2 are implicated in congenital heart disordersIn-house derived hES cell line KIND1) was subjected to cardiac differentiation using a directed differentiation protocol , cardiovascular progenitors (D12) and beating cardiomyocytes (D20). A list of genes (expected and predicted) was generated showing a fold change value of >=2.0 and a p-value score\u2009<\u20090.05. Comparisons were made amongst three groups including D0 versus D12, D0 versus D20 and D12 versus D20.About 1400 genes were found to be differentially expressed when KIND1 cells (D0) differentiated into cardiovascular progenitors D12)  on days 0, 12 and 20 of cardiac differentiation protocol was performed by gene ontology study. Genes enriched during the differentiation of undifferentiated cells (D0) into cardiac progenitors (D12) were found to be associated with biological processes related to early mesoderm and cardiac derivatives formation like \u2018extracellular region development\u2019, \u2018vasculature development\u2019, \u2018regulation of cell proliferation\u2019, \u2018skeletal system development\u2019, \u2018embryonic morphogenesis\u2019  as they represent the key players, the repressors and activators of gene expression respectively. Various studies till date have reported the importance of PcG proteins and that the abolishment of PcG proteins like EZH2, EED or SUZ12 disrupts the stem cell identity and their developmental potential. ES cells in their undifferentiated state maintain the bivalent marks in order to keep the developmental genes poised for further activation. In consistence with all the reports, the core proteins EZH2 and MLL2 forming the bivalent marks (H3K27me3 and H3K4me3 respectively) were found expressed in undifferentiated hES cells. As the differentiation proceeded to progenitors and cardiomyocytes, expression of EZH2 began to down-regulate. Expression of MLL2 core component ASH2L was expressed in both cardiac progenitors and cardiomyocytes. Another PcG group proteins like RING1, BMI1, PHC1, that represent the Polycomb repressive Complex 1 (PRC1) group, were up-regulated while the PRC2 group like SUZ12 and EED were significantly down-regulated in progenitor and cardiomyocyte stages as compared to their status in the undifferentiated state hES cells. Histone methyltransferase expressed during the differentiation is the SET domain containing 2 or SETD2 that is involved in the activation of gene transcription and is reported to be crucial for the cardiac formation21 Fig.\u00a0. BRG1 an21 Fig.\u00a0.Figure 4miRNAs and lncRNAs represent a valuable tool to understand the role of cellular proteins. As important regulators of gene expression at the epigenetic level, they activate or repress the transcription. Cardiac formation machinery also includes the number of non-coding RNAs as regulators of gene activation and repression. Among the number of miRNAs identified to be differentially regulated, the miRNAs significantly changed were miR21, miR208a, miR423 and miR27b that have been shown to regulate endothelial and myogenic differentiation. Expression was found increased with the differentiation of hES cells in cardiomyocytes. Similarly, the long non coding RNA represents non-coding ability and act as regulators of development and pathophysiology. ANRIL, CDKN2A/2B and MIAT were among the many lncRNAs identified in our study that might be involved in lineage specific gene expression Fig.\u00a0.Differential regulation of specific gene transcripts was studied by qRT-PCR to validate the microarray results. This included specific transcripts for pluripotent genes , ectodermal (MAP2), mesodermal into cardiac lineage , LTB, along with transcripts representing epigenetic machinery . The results were found consistent with the microarray data Fig.\u00a0 and CTNTThe extensive genetic and epigenetic characterization of KIND1 cells during differentiation into cardiac lineage, by microarray along with the qRT-PCR validation depicts the dynamic expression of various transcripts during cardiac differentiation. Though the array and the qRT-PCR validation involved differences in the expression levels, the technique reveals the global view of the genes along with illuminating the differences in their expression within the cells and their differentiated counterparts. Transcription factor NR2F2 was further studied as it showed increased expression during cardiac both by microarray and qRT-PCR.31, transcriptional regulation of OCT4 in mouse embryonal carcinoma cells as well as in hES cells undergoing neuroectoderm development33. NR2F2 is also crucial during mice heart development for angiogenesis and coronary vessel formation35. We thus interrogated the expression of NR2F2 in our differentiation system to evaluate whether it acts as regulator of early cardiac differentiation. The expression levels of NR2F2  were found to be inversely related to that of OCT4  and were comparable to EZH2, a transcriptional repressor as observed both by microarray analysis and qRT-PCR validation  justifying the formation of mature counterparts on D20 compared to D12. The only down-regulated gene LTB between D12 vs D20 (of the 19 genes) was not included in the down-regulated gene list between D0 and D20. All the 18 genes that were up-regulated on D20 compared to D12 were not expressed on D0. Thus, we believe our results show dramatic maturation of cardiac cells on D20 compared to D12 associated with a change in only 19 genes. Of all the genes being altered during ES cells differentiation, transcription factor NR2F2 reported to repress OCT-4 during neural differentiation33, has not yet been reported during cardiac differentiation but its mutation is known to cause atrial septal defects31. NR2F2 showed up regulation as cells differentiate into cardiac progenitors and cardiomyocytes by both microarray and qRT-PCR . A simultaneous down regulation of EZH2 was noted. Further ChIP experiments were undertaken to study whether a cross-talk exists between NR2F2 and EZH2 for repressing OCT-4 during cardiac differentiation. We first observed that the repressive mark H3K27me3 was occupied on NR2F2 promoter on D0 while not in D12 and D20; this explains the increased expression of NR2F2 as differentiation proceeded. The results clearly showed the occupancy of H3K27me3 on OCT4 promoter in progenitor cells on D12 (and not on D0) resulting in repressed OCT4 expression during differentiation. Since this repressive mark is brought about by histone methyltransferase EZH2, we studied its binding to NR2F2 and OCT4 by ChIP and sequential ChIP. QPCR following ChIP displayed the EZH2 binding on both OCT4 and NR2F2 while sequential ChIP further confirmed that NR2F2 was bound to both EZH2 and OCT4. Based on these results, we propose that NR2F2 recruits EZH2 at the OCT4 further leading to its repression during cardiac differentiation from hES cells.Present study provides novel information on the detailed genetic and epigenetic gene expression pattern underlying cardiac differentiation 41; OCT4A (true stemness marker containing unique exon 1), OCT4B (non-pluripotent variant) and OCT4B1 (putative stemness marker). In order to understand the mechanisms of hES cells pluripotency, it thus remains essential to concentrate upon the OCT4A isoform . Present study is focused on OCT-4A positive ES cells since we used primers specific to OCT4A. Moreover, in the ChIP sequencing studies, the occupancy of H3K27me3 mark on the promoter driving the expression of exon 1 that proves the involvement of \u2018pluripotent OCT4 isoform\u2019. Looking at the binding of EZH2 onto the OCT4 promoters, the primer pair specific to exon 1 which amplifies the distinct OCT4A gene further validates our findings, supporting the mechanism of binding of EZH2 and NR2F2 with OCT4A and repressing its expression for leading the hES cells to cardiac differentiation.OCT4 gene is reported to generate multiple transcripts that are translated into multiple isoforms of OCT4in vitro and in vivo44. Epigenetic remodelers found to be expressed were BRG1, MTA family and HDACs known to promote cardiac cell differentiation by regulating the levels of crucial cardiac transcripts like NKX2.5, TBX5 and GATA448. Similarly, the expression of other components of epigenetic machinery noted in our study is well studied in cardiac differentiation context; though their specific role is still unknown.Epigenetics adds another layer of gene expression control by modulating the transcriptional activities in a trimly and gene specific manner. The extensively studied epigenetic feature for stem cells is the simultaneous appearance of H3K27me3, H3K4me3 termed as bivalency that holds the equal potential to be expressed or repressed and that are resolved as per the signals and MLL2 enzymes specific for H3K27me3 and H3K4me marks respectively in our undifferentiated hES cells that were later subjected to cardiac lineage formation. However, on examining the cardiac progenitors and cardiomyocytes, EZH2 had a lowered expression in cardiac progenitors and much lower in cardiomyocytes, holding the evidence in support of reported studies that EZH2 is crucial for cardiac development 50. Additionally, it has been identified as a direct regulator of vascular endothelial cells by activating the NF-\u0138B pathway51. With respect to cardiac development, LTB is identified as a risk factor altered for cardiovascular diseases and thus the TNF antagonist potentially acts as a likely candidate to reduce the CVD risk in rheumatoid arthritis patients55. However, though there exists a dearth in studies reporting molecular mechanisms associated with LTB in cardiac development and pathogenesis, present study reports reduced levels of LTB during differentiation of hES cells into the cardiac lineage.The sole down regulated gene LTB represents a TNF family ligand that majorly acts as the inducer of inflammatory response and is involved in the organization/development of lymphoid tissue57 or the endothelial cells58 at various stages of differentiation and the data was also compared with the transcriptional pattern in fetal and adult heart in vitro. However, majority of these have employed spontaneous embryoid body formation technique to obtain beating clusters that led to achieving the mixed population comprising of all the lineages. Secondly, the purifying techniques like percol gradient resulted in purified cardiac population of as low as 40%56. Third employed way was studying the cardiomyocytes differentiated from ES cells that were co-cultured onto the layer of END2 cells or inactivated mouse fibroblast feeders59. Despite the extensive characterization data and their comparison with the in vivo counterparts is available, the fact that the resulting population being studied contains the non-mesodermal population still remains. Our study on the other hand employs the directed differentiation approach that ensured the cardiac cell fate of majority of the population. Moreover, the protocol also allows studying the step wise formation of cardiomyocytes involving early nascent mesoderm, cardiac mesoderm formation followed by cardiac progenitors and beating cardiomyocytes. The protocol from the very beginning restricts the formation of ectodermal and endodermal population excluding the endodermal genes reported to be essential for mesodermal direction.Number of studies till date have reported the transcriptional profiling of ES derived beating clustersin vivo and in vitro that would provide deeper insights. Despite this, our study for the first time provides the mechanism of OCT4 repression by the combined actions of NR2F2 and EZH2 that would direct the future efforts to undertake more specific experiments towards understanding cardiac development. Analysing various histone modifications and their dynamics along with the lineage specific gene expression patterns could uncover the regulatory genes involved during early human cardiac development in vitro.To conclude, detailed transcriptomic expression of genetic and epigenetic factors in pluripotent hES cells and during their conversion into cardiac progenitors and cardiomyocytes is reported. With a view to identify a role of NR2F2 during this differentiation, we also show the expression pattern of H3K27me3 mark on NR2F2 and OCT4 genes. Limitations of the study include use of unsorted cardiac population which otherwise would bring much clearer picture of cardiac differentiation mechanisms. Secondly, the deficiency of more confirmations like loss of function tests with respect to NR2F2 and OCT4 both 60 were transitioned to feeder-free condition17 and used to differentiate into tripotent cardiac progenitors. Similar protocols (described in details in supplementary section) were used in the present study to obtain undifferentiated hES cells (D0), cardiovascular progenitors (D12) and beating cardiomyocytes (D20) for various studies.In-house derived human embryonic stem cells (KIND1) derived on human feedershttp://david.abcc.ncifcrf.gov/home.jsp). All data analysis and visualization of differentially expressed genes was conducted using R 3.1.2 (www.r-project.org). Genes with fold values between \u22122 and +2 were omitted from the analysis. Three combinations of data were analysed, namely \u2013 Day 0 Vs Day 12, Day 0 Vs Day 20 and Day 12 Vs Day 20. QRT-PCR analysis for pluripotent , cardiac specific markers  including LTB and other epigenetic machinery  validated microarray data. Human fetal and adult cardiac total RNA  were used to compare the maturity of the differentiated cells and GAPDH was used as an internal control. All the primers used in the study are mentioned in Table\u00a0Microarray was performed using two biological replicates of each time point using Illumina, HT-12 v4.0 platform commercially. hES cells on days 0, 12 and 20 were harvested by mechanical dislodging using a cell lifter in TRIzol reagent  for RNA extraction as per manufacturer\u2019s instructions. Genome-wide gene expression analysis was performed commercially using Illumina, HT-12 v4.0 platform . Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using DAVID Bioinformatics Resources  were cross linked with 1% formaldehyde for 10\u2009mins at room temperature (RT). Formaldehyde was quenched with 1.25\u2009M glycine and cells were rinsed with ice cold 1X PBS, collected and centrifuged at 2500\u2009rpm for 5\u2009mins. Supernatant was aspirated leaving behind the pellet of cross linked cells. Cells were lysed with cell lysis buffer containing protease inhibitor (PI) . Lysed cells were subjected to sonication for about 25 cycles using pulse ON for 30\u2009sec and pulse OFF for 50\u2009sec on  to obtain fragments appropriate for performing qPCR (200\u2013500\u2009bp) or DNA sequencing (100\u2013300\u2009bp) analysis. Sonicated samples were then incubated with 10ug of anti H3K27me3 antibody  for ChIP-sequencing or anti EZH2 antibody  for ChIP-qPCR in blocking solution (0.5% BSA in 1X PBS). The beads with antibody - DNA complexes were washed 3 times with each of lysis buffer, IP1 buffer (lysis buffer with 500\u2009mM NaCl), IP2 buffer  and Tris EDTA (TE) buffer . For eluting the bound complexes, the bead \u2013 complex solution was incubated with 200ul elution buffer (TE buffer with 1% SDS) at 65\u2009\u00b0C for 50\u2009mins. The eluted product was treated with RNase A and proteinase K at 60\u2009\u00b0C for 60\u2009mins. Following this the immuno-precipitated DNA was isolated Phenol:Chloroform:Isoamyl alcohol method.Sequential ChIP was performed using standard protocols. Briefly, the cross linked chromatin sample of undifferentiated and differentiated cells was immuno-precipitated using EZH2 antibody. Following this the complex was eluted using elution buffer with higher salt concentration and DTT  in order to remove traces of EZH2 antibody. The resulting chromatin sample was then immuno-precipitated with anti OCT4 antibody  and continued with standard ChIP protocol described above. The genomic DNA isolated after both ChIP and sequential ChIP experiments was quantified by qPCR  calculated as % of total input DNA. The genes included in study are listed in Table\u00a0For ChIP sequencing step, the DNA isolated after the standard ChIP protocol immuno-precipitated with anti H3K27me3 antibody was quantified using Nanodrop  and then Illumina sequencing was done at sequencing facility of Genome Institute of Singapore (GIS). The data analysis was performed by Sandor Life Sciences Pvt Ltd, Hyderabad, India. The reads obtained were mapped with the Human hg19 and aligned using Bowtie . MACS  were employed for peak calling. The resulting peaks were visualized using Integrative Genome Viewer.Microarray data raw files are available on GEO NCBI data base accession number GSE97268. ChIP sequencing raw files are available on SRA NCBI data base accession number SRP115340.Supplementary Information"}
+{"text": "NKG2D is a critical activatory molecule expressed on cytotoxic cells such as NK cells, \u03b3\u03b4 T cells and subsets of \u03b1\u03b2 T cells . LigatioULBP6 is the most polymorphic gene within the ULBP family and in recent work our team contrasted the molecular and functional correlates of the two most common protein variants, ULBP601 and ULBP602 Figure  3]. Str. Str3]. The mechanisms that underlie the association of NKG2DL alleles with clinical disorders such as auto-immunity  or tumorin vivo murine studies have shown that ectopic expression of NKG2D ligands on tumor cells can be sufficient to mediate control of tumor growth[The critical role of NKG2DL in tumour-specific immune responses is highlighted by the fact that tumours frequently evolve a range of mechanisms to evade NKG2D-mediated responses. These include NKG2DL cleavage from the surface, and NKG2D downregulation which is frequently observed on cytotoxic cells in patients with cancer . Interesor growth. In ordeThe central importance of NKG2D interactions within immune homeostasis has been relatively under investigated but an emerging hope is that detailed interrogation of the significance of natural alleleic variation within the ULBPL family members  will ser"}
+{"text": "TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.25 of 51 cases were found to carry MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.In summary, we here show that subgroups of SDCs display genomic amplifications of  TP53 mutations were detected, involving around 50% of cases of de novo and ex pleomorphic adenoma SDCs [TP53 wildtype (WT) tumors which might harbor alternative alterations in the p53 regulatory network.Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands, most commonly involving the parotid gland. It is one of the most aggressive salivary gland malignancies, most frequently associated with the occurrence of early distant metastasis and poor prognosis . In the oma SDCs , 3. HoweMDM2-amplified well-differentiated or dedifferentiated liposarcomas (LS), which are consistently characterized by a high-level genomic amplification (frequently >15-30 copies as clusters) of sequences derived from chromosomal region 12q13-15 comprising the oncogenes MDM2 and CDK4, showed that treatment with the MDM2 antagonist RG7112 activates the p53 pathway and decreases cell proliferation [TP53, but MDM2 alterations have also been described in subsets of TP53 mutated tumors [MDM2 transgenic mice developing mammary gland tumors suggest a crucial role for MDM2 in epithelial tumors of glandular differentiation [For decades, p53 has been a well-known tumor suppressor that is mutated or functionally inactivated in large subsets of human cancers . Physiolferation . Most frd tumors . It has ntiation .CDK4 together with MDM2 [MDM2+/CDK4- LS shows favorable prognostic features compared to MDM2+/CDK4+ LS [CDK4 amplification status therefore provides genomic information on the structural characteristics of the amplicon with MDM2 being located at 12q15 and CDK4 at 12q13.3-12 and it might add further information on an independent oncogenic mechanism apart from p53 dysfunction. Since CDK4 is the key regulator of the G1-S cell-cycle transition and drives cell-cycle progression, CDK4 inhibitors might offer new strategies for a targeted cancer therapy [In LS, the 12q13-15 amplicon usually, but not always, shows a co-amplification of the cell cycle regulator ith MDM2 . It has CDK4+ LS . Knowled therapy , 12.HMGA2, encoding a high-mobility group protein. Chromosomal breaks of the HMGA2 locus have been described in several benign mesenchymal tumors including lipomas and uterine leiomyomas [HMGA2 amplification was shown in several soft tissue malignancies including liposarcomas where it is almost always co-amplified with MDM2 [HMGA2 are well known in subsets of pleomorphic adenomas (PA) [ex PA have been reported to generally retain HMGA2 rearrangements along with further gene alterations in tumor progression making HMGA2 a potential marker for SDCs arising in PA [Another gene in chromosomal region 12q13-15 frequently subject to structural alterations is iomyomas , and HMGith MDM2 , 15. In mas (PA) , 16. CarMDM2 and CDK4 alterations in SDC and to put them in context with HMGA2 alterations known in SDC. We here report on the rare occurrence of MDM2 and CDK4 amplifications in a large collection of these aggressive salivary neoplasms showing a heterogeneous distribution among TP53 wildtype tumors and those carrying a TP53 mutation.Only very little is known about the role of MDM2 or CDK4 in SDC tumorigenesis. The major aim of this study therefore was to systematically evaluate the involvement of TP53 mutational status, MDM2, CDK4 and HMGA2 genomic amplification as well as HMGA2 rearrangement and p53, MDM2 and CDK4 protein expression. The clinicopathological characteristics of these 51 patients are summarized in Table 51 SDC cases were analyzed for TP53 mutations were detected in 25 of these 51 cases  staining result in 13 cases, all of these cases displaying a TP53 missense point mutation except for one case in which the TP53 mutational status could not be evaluated due to minor DNA quality. In 8 cases with various TP53 mutations, an extreme negative (EN) p53 immunohistochemical staining was detected and 5 cases with TP53 mutations showed a non-extreme (NE) staining level for p53. An extreme negative (EN) p53 staining was detected in 8 cases without TP53 mutation and in 5 further cases in which TP53 mutational status could not be evaluated.MDM2 amplification, one case showed an MDM2 grey-zone amplification in FISH analyses. Two cases with MDM2 amplification strongly stained for MDM2, one MDM2 amplified case and the grey-zone amplified case showed low MDM2 immunohistochemical staining levels. Two of the MDM2 amplified cases synchronously carried TP53 mutations. All MDM2 amplifications and the MDM2 grey-zone amplification occurred in de novo SDCs, none was detected in an SDC ex pleomorphic adenoma.Three out of the total 51 cases displayed an CDK4 amplification, one coinciding with an MDM2 amplification and one in the setting of an MDM2 grey-zone amplification. Nine cases showed a CDK4 grey-zone amplification, one of these cases in the setting of an MDM2 amplification. However, the immunohistochemical CDK4 staining was negative in all cases.In two cases, we detected a HMGA2 FISH could be analyzed in 42 out of 51 cases. 5 cases showed rearrangement and/or amplification of HMGA2, among these 3 de novo and 2 ex PA SDCs. Two of the 5 cases displayed HMGA2 rearrangement and amplification, both occurring in association with an MDM2 amplification; the third MDM2 (and CDK4) amplified case displayed no HMGA2 alteration. One of the HMGA2 rearrangements occurred in the setting of a CDK4 grey-zone amplification, the two further HMGA2 rearrangements were detected in cases without MDM2 and/or CDK4 amplification. One CDK4 amplified tumor with grey-zone MDM2 amplification showed only a low HMGA2 copy number increase.TP53 mutated cases showed a tendency to a worse 5-year OS (20.5% with TP53 mutation vs. 53.3% without TP53 mutation) but statistical significance was not reached (p=0.267)  in our collection of SDCs was 39.6% Figure . TP53 mu) Figure . No statTP53 mutations in de novo and ex PA SDCs [TP53, the major aim of this study was to elucidate the role of another key player within the p53 regulatory network, i.e. the ubiquitin ligase MDM2, in SDC. Due to the co-localization of MDM2 with the cell cycle regulator CDK4 in the chromosomal region 12q13-15 and a documented proneness of this region to genomic alterations, these regulators of cell growth and fate are frequently co-amplified in some human malignancies including a subset of soft tissue tumors [CDK4 amplification status might add further information on an independent oncogenic mechanism apart from p53 dysfunction, opening therapeutic options. Involvement of the chromosomal region 12q13-15 in salivary gland tumorigenesis has been documented before with the HMGA2 gene showing rearrangements and genomic amplification in subsets of PA and SDCs ex PA [HMGA2 furthermore represents an almost constant partner of MDM2 in the 12q13-15 amplicon known in other tumors, we complemented our analysis with the analysis of HMGA2 to get further insight into the structure of the amplicon [Recent genomic profiling studies confirmed a high frequency of  PA SDCs , 3, affee tumors . Apart fCs ex PA . Since Hamplicon .MDM2 amplifications in 3 cases, interestingly all classified as de novo SDCs. Two of these MDM2 amplified cases showed a concurrent rearrangement/co-amplification of HMGA2. According to Bahrami et al. [HMGA2 as a marker for SDCs ex PA, it could be hypothesized if these two cases might also have arisen in totally obscured PA, however, there was no evidence for a pre-existing PA, neither by histopathology nor with respect to clinical history. One of the MDM2 amplified SDCs harbored no HMGA2 alteration while 3 SDCs with HMGA2 rearrangement did not show an MDM2 amplification, implying that alterations of these two genes are obviously not strictly connected in SDCs. Thus, MDM2 amplification appears to be a genomic alteration at least partly independent from HMGA2 status and particularly from pathogenesis (de novo vs. ex PA) in SDC. Since only two of the cases harboring an MDM2 amplification revealed a strong immunohistochemical staining for MDM2, immunohistochemical staining alone appears not to be a reliable marker of MDM2 amplification in SDC. Interestingly, two of the MDM2 amplified cases synchronously carried TP53 mutations, while one MDM2 amplification occurred in a TP53-wildtype setting. In another TP53-wildtype tumor a grey-zone MDM2 amplification was detected. A very recent study on 37 SDCs revealed one MDM2 amplified case, also in the setting of a TP53 mutation [TP53 cannot be expected to substantially gain in oncogenic potential due to a concomitant amplification of MDM2 since it usually acts as an oncogenic factor by downregulation of wildtype p53 protein. In contrast, in tumors harboring particular TP53 mutations and retaining their wildtype TP53 allele, MDM2 amplification might contribute to oncogenesis by additionally disrupting the remaining functional p53 levels. Additionally, several reports argue in favor of an oncogenic role for MDM2 independently from the p53 status [MDM2 amplification obviously may serve as an alternative mechanism leading to dysregulation of the p53 network, thereby offering an option for a targeted MDM2-directed therapy [MDM2 amplification might represent an alternative or additional mechanism in deregulating the p53 network besides well-known and pathogenetically relevant genetic alterations of the PI3K/AKT signaling pathway in SDC [We detected i et al. , who pro3 status , 17, 18. therapy . With re therapy , 20, MDMTP53 mutational status: With 48% of TP53 mutated cases and none of the TP53 wildtype cases showing an extreme positive (EP) staining for p53 protein expression, EP p53 staining reliably went along with a missense TP53 mutation. 32% of TP53 mutated cases showed an extreme negative (EN) p53 staining and 20% of TP53 mutated cases a non-extreme (NE) p53 staining. In contrast, cases without TP53 mutation displayed an EN p53 staining result in 44% and a NE staining in 56%. So, in summary, in case of an EN or a NE p53 staining, a reliable prediction of TP53 mutational status is not possible in SDCs whereas an EP p53 expression is strongly suggestive of an underlying TP53 missense mutation.The immunohistochemical staining for p53 partly correlated with the TP53 mutated cases showed a tendency to a worse 5-year OS , pointing to a CDK4-mediated cell-cycle dysregulation in SDC tumorigenesis. CDK4 inhibitors might therefore offer new strategies for targeted therapeutic approaches in SDCs as recently discussed for several entities [CDK4 amplified or grey-zone amplified cases showed a positive immunohistochemical staining for CDK4 (with solid staining result of the positive control), so immunohistochemical CDK4 staining obviously is not reliably suitable for the detection of CDK4 amplified SDC. Anyway, lack of immunohistochemical CDK4 protein detection does not exclude a pathogenic role of the CDK4 oncogene and may be due to tight protein regulation beyond the threshold of staining sensitivity. Contrasting with our immunohistochemical results and potentially pointing to different characteristics of diagnostically employed antibodies, a previously reported study on five SDC cases demonstrated CDK4 protein expression in four of five cases, accompanied by MDM2 protein expression in two cases; however, CDK4 and MDM2 amplification status was not determined in that study [Out of the entities , 12. Intat study .MDM2 and/or CDK4 amplified/grey-zone amplified subgroups, which is probably due to the limited case numbers contained in the smaller subgroups. Larger cohorts of these aggressive tumors should be analyzed for a better understanding of a potential prognostic impact of MDM2 and/or CDK4 alterations.As described above, in a subset of liposarcomas the small subgroup of MDM2+/CDK4- tumors shows favorable prognostic features compared to MDM2+/CDK4+ tumors . In our TP53 mutational status, MDM2 and CDK4 amplification and HMGA2 rearrangement/amplification as well as protein expression of p53, MDM2 and CDK4. We showed that in subgroups of SDCs, MDM2 and/or CDK4 amplification might play a pathogenic role, in part apparently in association with other genetic alterations. Further work is mandatory to clarify the role of MDM2 and CDK4 alterations in the tumorigenesis of SDCs , the potential prognostic relevance of these alterations in SDCs and the feasibility of MDM2- and/or CDK4-directed therapeutic strategies in SDCs.In this study, we investigated for the first time systematically the involvement of MDM2 and CDK4 in the carcinogenesis of SDCs by analyzing the th edition of the UICC TNM classification for carcinomas of the salivary glands. Patients were followed up at the outpatients department of Cologne or Muenster, respectively, at periodic visits in 3 to 6 months. At the time of analysis, 22 patients had died and 11 patients had developed a histologically confirmed relapse. Mean follow-up time was 22.2 months (range 0 to 161). The study was approved by the local Ethics Committees.The investigation was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. The retrospective study included 51 patients with newly diagnosed salivary duct carcinoma. Among these, 30 cases were derived from the University Hospital of Muenster and 21 cases from the University Hospital of Cologne  material of the patients was obtained from the archives of the Departments of Pathology at the University Hospitals of Cologne and Muenster, respectively. All tumors were re-evaluated microscopically and by means of immunohistochemistry by two experienced pathologists with regard to histopathological diagnosis in accordance with WHO 2005 classification of tumors of salivary glands.From FFPE material of all included cases, two core biopsies out of the tumor area were taken to assemble tissue microarrays (TMA).For statistical analysis, the IBM SPSS Statistics 22 software  was applied and Kaplan-Meier survival analysis and Log rank test were performed. The significance level was set at p<0.05.in-situ hybridization (FISH) analyses and immunohistochemical stainings were conducted on slides from TMAs. FISH analyses were performed as described previously [\u00ae SPEC MDM2/CEN 12 Dual Color Probe for assessment of MDM2 amplification and the ZytoLight\u00ae SPEC CDK4/CEN 12 Dual Color Probe for assessment of CDK4 amplification . HMGA2 FISH analysis was performed according to a previously published assay [MDM2 and CDK4 was defined as an MDM2/centromer 12 (CEN12) or CDK4/centromer 12 (CEN12) ratio \u22652.0 or an average number of MDM2 or CDK4 signals per tumor cell nucleus \u22656 or large clusters of MDM2 or CDK4 signals \u226510%, respectively. Grey-zone amplification was defined as an MDM2/centromer 12 (CEN12) or CDK4/centromer 12 (CEN12) ratio \u22651.8 or as microclusters of \u22655 MDM2 or CDK4 signals in \u226515% of tumor cell nuclei, respectively, based on modified scoring algorithms for HER2 and FGFR1 as published before [HMGA2 rearrangement and amplification were evaluated as published before [Fluorescence eviously , 23 usined assay , 24 usind before . For HMGd before .et al. [Immunohistochemical staining was conducted on a Dako Autostainer  following the manufacturer's instructions. Three \u03bcm sections were cut from the TMAs, followed by heat induced antigen retrieval in low (for p53) or high (for MDM2 and CDK4) pH buffer. For visualization LSAB method with AP/RED was used. Following antibodies and concentrations were used: p53 , MDM2  and CDK4 . Sections were counterstained with hematoxylin and tiled with Cytoseal (Thermo Fisher Scientific Inc.). For scoring of the immunohistochemical stainings for p53, MDM2 and CDK4 only nuclear staining was rated. The staining intensity was evaluated semiquantitavely into 0, 1, 2 or 3 by comparing within the different tumor samples. To determine percentage labelling indices, all tumor cells within the cores were analyzed using high-power (400x) magnification. A sum score was calculated out of the staining intensity and the percentage labelling index, and for MDM2 and CDK4 value ranges for staining level were defined as follows: 0: no staining; >0 to 50: low staining level; >50 to 100: intermediate staining level; >100: strong staining. Sum score of p53 staining was interpreted modified according to Boyle et al.  as folloTP53 mutational status as previously described for a smaller subset of samples [TP53 mutational status was carried out as follows:To complete the data on 2 tumor area corresponding to the tumor area of H&E-stained section was scraped off with a scalpel and collected into plastic tubes. Subsequently, the DNA was automatically extracted using the Maxwell DNA FFPE isolation kit on a Maxwell platform . Fluorometric DNA quantification was performed according to the Qubit dsDNA HS assay .Sections were prepared from FFPE material and stained with hematoxylin & eosin (H&E). Six additional sections of 6 \u03bcm thickness were cut, mounted onto glass slides and used for macrodissection. In total, 1 cmTP53 . Target enrichment was processed by means of the GeneRead DNAseq Panel PCR V2 Kit (Qiagen), following the manufacturer's instructions. All purification and size selection steps were performed utilizing Agencourt AMPure XP magnetic beads . End repair, A-addition and ligation to NEXTflex-96 DNA barcodes  was carried out using the GeneRead DNA Library I Core Kit (Qiagen). Amplification of adapter-ligated DNA was conducted using NEXTflex primers (Bioo Scientific) and the HiFi PCR Master Mix . Next generation sequencing was performed applying 12.5 pM library pools (2% PhiX V3 control) and the MiSeq Reagent v2 chemistry .Pre-verified multiplex PCR primer sets  and all variants below 4% allelic frequency were filtered, ending up with variants listed in Supporting Information TP53 mutations is provided in Supporting Information Fastq files were generated by the MiSeq Reporter software (Illumina) and further analyzed by means of the CLC Biomedical Genomics Workbench software . The total batch of identified TP53 primer sets  and various"}
+{"text": "Scientific Reports7:9384; doi:10.1038/s41598-017-09296-w; Article published online 24 August 2017This Article contains an error in the legend of Figure 5.\u201cMultiple sequence alignment of the prepropeptides of AtCLE18 and LjCLE34. CLE domains are highlighted with a red box and the CLEL domain is underlined in blue. Conservation between amino acid residues of the two sequences is represented by grey  and black (100%) shading\u201d.should read:LjCLE5 and MtCLE12. The LjCLE5 translation start site corresponding to that of MtCLE12 results in a truncated prepropeptide (denoted by the asterisk shaded in black). The blue box represents an alternative start codon that results in the CLE domain region of\u00a0LjCLE5 (underlined in red) being translated in frame. Black highlighted nucleic acid bases are 100% conserved\u201d.\u201cSequence alignment of"}
+{"text": "Rint1) influences cellular homeostasis through maintenance of endoplasmic reticulum, Golgi and centrosome integrity and regulation of vesicle transport, autophagy and the G2/M checkpoint. Rint1 has been postulated to function as a tumor suppressor as well as an oncogene, with its role depending perhaps upon the precise cellular and/or experimental context. In humans, heterozygosity for germline missense variants in RINT1 have, in some studies, been associated with increased risk of both breast and Lynch syndrome type cancers. However, it is not known if these germline variants represent loss of function alleles or gain of function alleles. Based upon these findings, as well as our initial consideration of Rint1 as a potential candidate for Mom5, a genetic modifier of intestinal tumorigenesis in ApcMin/+ mice, we sought to explicitly examine the impact of Rint1 on tumorigenesis in ApcMin/+ mice. However, heterozygosity for a knockout of Rint1 had no impact on tumorigenesis in Rint1+/-; ApcMin/+ mice. Likewise, we found no evidence to suggest that the remaining Rint1 allele was lost somatically in intestinal tumors in ApcMin/+ mice. Interestingly, in contrast to what has been observed in Rint1+/- mice on a mixed genetic background, Rint1+/- mice on a pure C57BL/6J background did not show spontaneous tumor development. We also evaluated colorectal cancer data available in the COSMIC and ONCOMINE databases and found that RINT1 overexpression, as well as the presence of somatic missense mutations in RINT1 were associated with colorectal cancer development. In vitro evaluation of two missense variants in RINT1 suggested that such variants do have the potential to impact RINT1 function.The Rad50 Interacting Protein 1 ( Th. ThRint1Rint1 had no discernible impact on tumorigenesis in ApcMin/+ mice. Mean tumor multiplicity in the Rint1+/-; Mom5129/B6;ApcMin/+ mice was 49.6\u00b14, which was essentially identical to the mean of 49.8\u00b17.4 in Rint1+/+; Mom5129/B6;ApcMin/+ mice  and Rint1 expression is not reduced in colon tumors in ApcMin/+ mice , this phenotype was not enhanced in Rint1+/-; ApcMin/+ mice (data not shown).Our analysis indicated that heterozygosity for n/+ mice . Since Rn/+ mice . AlthougRint1 knockout did not support the hypothesis that heterozygosity for the Rint1 knockout allele promotes tumorigenesis in ApcMin/+ mice. These observations were consistent with data extracted from the COSMIC database, which revealed no evidence for loss of heterozygosity or deletion of RINT1 in colorectal cancers, suggesting that somatic loss of RINT1 was rare during colorectal tumorigenesis. Furthermore, we note that just 0.16% (1 of 607) colorectal tumors in the COSMIC database showed loss of RINT1 expression. By contrast, 6.1% (37 of 607) of colorectal tumors in the COSMIC database overexpressed RINT1. Likewise, comparison of colorectal cancers and normal mucosa in the Oncomine database showed that RINT1 transcript levels were significantly greater in many colon adenocarcinoma subtypes (RINT1were detected in 2% (30 of 1493) of cancers of the large intestine, and ~57% (17/30) of these somatic mutations were missense mutations  developed lymphomas [Rint1+/- mice were on a highly mixed 129 x B6 genetic background, which is known to have a high incidence of spontaneous lymphoma development[Rint1+/- mice observed in Lin et al is attributable to genetic background rather than the Rint1 knockout allele. This possibility cannot be excluded because this original description of the tumor phenotype in Rint1+/- mice did not include a parallel analysis of tumorigenesis in Rint1+/+ mice on this same mixed genetic background. Because of the possibility that genetic background might impact the tumor phenotype in Rint1+/- mice, we sought to determine if genetic background might also impact the embryonic lethality associated with homozygosity for the Rint1 knockout allele. However, as was observed on the mixed 129 x B6 genetic background, homozygosity for the Rint1 knockout allele on a pure B6 genetic background also resulted in embryonic lethality.It has been reported previously that mice heterozygous for the velopment, we cannRint1 on tumorigenesis in ApcMin/+ mice. However, this analysis revealed that heterozygosity for Rint1 did not impact intestinal tumor number or size. We also found no evidence for somatic loss of Rint1 in colon tumors in ApcMin/+ mice. Altogether, our studies with Rint1+/- mice did not support the idea that the Rint1 knockout allele functioned as either a modifier allele of tumorigenesis in ApcMin/+ mice or a cancer predisposing allele on the B6 background. Thus, these studies did not provide robust evidence to support the hypothesis that Rint1 functions as a tumor suppressor. Likewise, analysis of data available in the COSMIC and ONCOMINE databases failed to yield strong evidence that loss of RINT1 was associated with intestinal carcinogenesis in humans. Rather, analyses of available colorectal cancer data in these databases suggested that overexpression of RINT1 as well as somatic missense mutations in RINT1 may be associated with colorectal carcinogenesis. It has been reported previously that germline missense variants in RINT1 are associated with increased risk of breast and Lynch Syndrome type cancers [RINT1 mutations, most of which are missense mutations, on the function of RINT1 is not known.The original, primary purpose of this study was to evaluate the impact of heterozygosity for a knockout of  cancers . HoweverRINT1 variants in vitro and examined the impact of these variants on the interaction of RINT1 with some of its binding partners. Compared to wild type, some of the RINT1 variants we generated showed a more robust association with UVRAG, a known RINT1 interacting protein that is involved in retrograde transport and autophagy [in vitro are similar to some of those associated with Lynch Syndrome type cancers in that they are missense variants in the coil-coil domain, which is thought to mediate the interaction of RINT1 with many of its binding partners. Thus, our results raise the possibility that missense mutations in RINT1 may represent dominant, gain of function alleles that may be functionally equivalent, at least in a certain respect, to RINT1 overexpression. Altogether, these data are more consistent with the hypothesis that RINT1 functions as an oncogene rather than a tumor suppressor gene in the context of colorectal cancer.To explore the possible impact of RINT1 missense variants on RINT1 function, we expressed several missense utophagy . The RIN"}
+{"text": "Fog2 re-expression in the Fog2 null embryos is sufficient to extend their life span, identification of GATA4-FOG2 target genes in cardiomyocytes could shed light on the molecular mechanism of GATA4-FOG2 action in these cells. We report here that cardiac expression of slow skeletal troponin T (Tnnt1) strictly depends on the physical interaction between GATA4-FOG2 in the myocardium of both atria and ventricles.Previous work by us and others has shown that the loss of interaction between GATA4 and FOG2 protein partners is embryonic lethal due to heart failure at embryonic day (E) 13.5; however, the role of this important protein duo in various cardiac compartments  remains to be understood. Although a dual role (both as an activator and a repressor) for the GATA4-FOG2 transcriptional complex has been put forward, the specific genes under GATA4-FOG2 control in the developing heart have remained largely elusive. Since the myocardial-restricted"}
+{"text": "TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate  Many organisms lack formalized gene and protein nomenclature conventions. We apply zebrafish nomenclature conventions to stickleback [Gasterosteus aculeatus], spotted gar [Lepisosteus oculatus], and coelacanth [Latimeria chalumnae] and human nomenclature conventions to Chinese softshell turtle (Pelodiscus sinensis) and anole lizard (Anolis carolinensis) genes and proteins.Nomenclature rules for vertebrate genes and proteins follow accepted conventions. This work presents gene and protein nomenclature for specific species according to their respective naming conventions  software version 3.6b  , TNFAIP8L1 , TNFAIP8L2 , and TNFAIP8L3  display 50\u201357% sequence identity , we determined that the zebrafish genome encodes four members of the vertebrate TNFAIP8 gene family, called tnfaip8 (ENSDARG00000086457), tnfaip8l2a (ENSDARG00000075592), tnfaip8l2b (ENSDARG00000046148), and tnfaip8l3 (ENSDARG00000088709). To investigate relationships of members of the zebrafish tnfaip8 gene family to their human orthologs, we compared conserved syntenies of chromosome segments containing the four zebrafish genes to the human genome. We found that the gene called \u2018tnfaip8\u2019 (ENSDARG00000086457) occupies a chromosome segment on Dre22 whose genes are mostly orthologous to the region of Hsa19 that contains TNFAIP8L1, not to the region of Hsa5 that contains TNFAIP8  but not with TNFAIP8 (Hsa5). The region of Hsa19 between 0 and 10Mb displays conserved synteny with both Dre 22 and Dre2 , the locus originally designated as tnfaip8 was changed to tnfaip8l1, which is in agreement with our earlier analysis (ENSDARG00000086457 22:4769086\u20134787454:-1). We carefully examined EST data that had been previously associated with the incorrectly annotated term \u201czebrafish tnfaip8\u201d (located on UniGene [https://www.ncbi.nlm.nih.gov/unigene] at UGID: 2554148) and determined that there was no evidence of a zebrafish tnfaip8 ortholog in the expression data.In our initial investigation using Ensembl Zv9  and tnfaip8l2b. (ENSDARG00000046148). Conserved synteny analysis verified that the sections of the zebrafish genome on chromosomes Dre19 and Dre16 that contain these two genes are both orthologous to the section of Hsa1 that contains TNFAIP8L2, consistent with their gene nomenclature  and that a true ortholog of human TNFAIP8 is absent from the zebrafish genome.These data lead to the conclusion that zebrafish has one ortholog of TNFAIP8 lies at 118.6Mb on chromosome Hsa5 and is flanked by DMXL1 and HSD17B4. All three genes are transcribed in the same direction (DMXL1 (dmxl1[1of2] and dmxl1[2of2]) and an adjacent ortholog of HSD17B4 lie adjacent to one another, 3.2 kB apart at the left tip of Dre8 at location 0.3 Mb. The 3.2 Kb between zebrafish dmxl1 and hsd17b4 contains no sequence recognizable as a TNFAIP8 ortholog (or any other known genetic element)   as foundrections  rather tebrafish , suggestdmxl1, tnfaip8, and hsd17b4 genes in the same order and orientation as in the human genome , hsd17b4, [TNFAIP8 gene, however, resides in the region of Hsa5 between 117Mb and 125Mb, whose TGD paralog is less unclear. To investigate this point further, we looked for paralogs of genes occupying the left tip of Dre8 (note that hsd17b4 is the tenth gene from the left telomere). Results failed to reveal an obvious paralogon for this region, although Dre5 and Dre10 possess some more distantly related homologs  form a monophyletic group with 99.8% support , two zebrafish co-orthologs to mammalian Tnfaip8l2 (tnfaip8l2a and tnfaip8l2b), and one zebrafish ortholog to mammalian Tnfaip8l3 (tnfaip8l3). The phylogenetic tree suggests, although without strong support (59% for the TNFAIP8/TNFAIP8L1 clade and 48.4% for the TNFAIP8L2/TNFAIP8L3 clade), that VGD1 produced a TNFAIP8/8L1 ancestor gene and a TNFAIP8L2/8L3 ancestor gene, and then VGD2 produced the full group of four genes  that is represented in the current human genome  and percomorphs (including stickleback) teleosts. This loss could have resulted after the divergence with the spotted gar but before the VGD because no tnfaip8 orthologs have been identified in other representative teleost species, including stickleback and zebrafish. It is also possible that the tnfaip8-containing region duplicated with the TGD to produce, tnfaip8a and tnfaip8b ohnologs and that two independent events\u2014one associated with an inversion break point and the other associated with the loss of several adjacent genes\u2014led to the loss of both tnfaip8 TGD ohnologs. Although the two-loss scenario seems less likely by strict parsimony, biologically it seems to be more likely. To help answer this question, we attempted to identify tnfaip8 gene family members in the genomes of the Japanese and European eels , which are representatives of the Anguilliformes, a basally diverging lineage that arose immediately after the TGD [The vertebrate L3 genes . Our ana the TGD . We perfTNFAIP8L1 and TNFAIP8L3 genes  and TNFAIP8L3 (tnfaip8l3a and tnfaip8l3b) were present in the common teleost ancestor genome and that one of the co-orthologs for each gene was subsequently lost, leading to the current condition in teleost genomes. The loss of tnfaip8 may reflect a non-essential function and/or functional redundancy among tnfaip8 family members. In contrast, two co-orthologs to the human TNFAIP8L2 gene remain present in teleosts (tnfaip8l2a and tnfaip8l2b)  . Based ulization . It is afunction . Going ftnfaip8 gene from the teleost lineage and described a genome inversion mechanism by which it likely occurred. Our findings highlight the value of the spotted gar as an orthology bridge that can be used to not only identify gene orthologs shared between mammalian and teleost genomes but also to resolve discrepancies related to ohnolog loss. These sorts of studies have broad applicability and will serve to strengthen rationales for the application of teleost models to study problems in human health and disease.Our findings support the importance of characterizing vertebrate gene histories in developing teleost models for human biomedicine , 48. TelS1 Fig(A) Clustal Omega alignment of human TNFAIP8, TNFAIP8L1, TNFAIP8L2, and TNFAIP8L3 protein sequences. (B) Percent amino acid identity based on pairwise comparisons between each of the TNFAIP8 family members.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file."}
+{"text": "Editorial):\u201cNext, Duchemin-Pelletier et al. present data about the role of CK2\u03b2, the regulatory subunit of the ubiquitous protein kinase CK2 [16]. In MCF10A mammary epithelial cells where CK2\u03b2 expression was downregulated, the authors provide evidence that CK2\u03b2 downregulation can promote the acquisition of characteristics commonly associated with the cancer stem cell phenotype. They demonstrate that a CK2\u03b2 level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Since EMT supports the induction stem-cell phenotype, identifying the CK2 substrates will improve the discovery of new specific markers for breast cell stemness.\u201dThe author wishes to make the following correction to the paper . The folThe reference numbering was also corrected.The author would like to apologize for any inconvenience caused. The manuscript will be updated and the original will remain online on the article webpage."}
+{"text": "A novel highly pathogenic avian influenza A(H5N6) virus affecting wild birds and commercial poultry was detected in the Netherlands in December 2017. Phylogenetic analysis demonstrated that the virus is a reassortant of H5N8 clade 2.3.4.4 viruses and not related to the Asian H5N6 viruses that caused human infections. The samples tested positive by real-time PCR on the matrix gene (Cygnus olor) and a tufted duck (Aythya fuligula) were found dead in this area , as described previously  that was detected in February 2017 but likely represents a separate introduction related to wild bird migration in fall 2017. The reassortment events may have occurred on breeding grounds in Siberia, where large numbers of wild birds congregate, and the virus may have spread by long-distance flights of infected migratory birds  can be infected by the novel reassortant HPAI H5N6 viruses detected in the Netherlands, Greece, Japan, and Taiwan. We recommend further studies in mammals (ferrets or mice) to provide experimental data on the virulence for mammals.Additional information about the emergence of HPAI H5N6 virus in the Netherlands in December 2017."}
+{"text": "Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6\u2019s cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6\u2019s effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6\u2019s expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis.We studied whether regulation of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6/\u2212\u2212 embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells.We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation.We found that Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.Our results suggest that Pax6 normally represses  In rodents, about two thirds of excitatory, pyramidal neurons in the cerebral cortex are generated from progenitor cells that divide beneath the apical surface of the embryonic cortical neuroepithelium in a region called the subventricular zone \u20133. TheseEarly in corticogenesis, Pax6 expression levels are graded across the ventricular zone of the developing cortical neuroepithelium. Levels increase with proximity to the boundary between the lateral cortex and the subpallium. Pax6 exerts regional control of cell proliferation, with its greatest effects in anterior and lateral regions where its levels are highest . It is rPax6 identified Cdca7 (Cell division cycle associated 7) as a gene whose expression is increased by loss of Pax6 function  to caudo-medial [low]. As corticogenesis continues, this gradient of Pax6 expression becomes progressively less steep [Cdca7 normally correlates with that of Pax6 and whether the expression of Cdca7 is affected in mutants that are null for Pax6.Pax6 is expressed at high levels in the ventricular zone of the developing cerebral cortex and at lower levels in the ventricular zone of the lateral ganglionic eminence (LGE). At the earliest stages of murine cortical neurogenesis, around embryonic days 12\u201313 (E12-13), ss steep . At all Pax6 and Cdca7 in adjacent sections from wild-type  and Pax6/\u2212\u2212 (n\u00a0=\u00a04) embryos at E12.5 . In contrast, Cdca7 expression levels were lower in the lateral cortex than in the LGE , Pax6 expression declined more gradually than normal across the PSPB, as at E12.5  and a ribbon whose width corresponded to that of the gene expression domains was drawn through the telencephalon from dorsal to ventral as shown in Fig.\u00a0Pax6 gene. Regression analysis gave values for the slopes on the two sides .We used a quantitative approach to confirm the breakdown in the normal expression of Pax6 and Cdca7, with significant interaction effects for both genes . In wild-types, the gradients of Pax6 and Cdca7 expression were opposite at E12.5  or reaching (in the case of Cdca7) zero by E14.5 . In Pax6/\u2212\u2212 pallium, the gradient of Pax6 expression was much smaller than normal at E12.5  and then reversed  but one appeared, with a slope opposite to normal, over the subsequent 2\u00a0days .In the pallium . Pax6 and Cdca7 were expressed in opposing gradients from E12.5-14.5  sequence to generate an HA epitope-tagged version of Cdca7. This was cloned into the expression vector pCAGGS_GFP, which expresses green fluorescent protein (GFP) tagged with a nuclear localization signal (NLS)  Fig.\u00a0B. The CdLS) Fig.\u00a0C. The leLS) Fig.\u00a0D. GFP+ cLS) Fig.\u00a0E. ImmunoLS) Fig.\u00a0F, G. Sinitiation .Pax6/\u2212\u2212 mutants (Fig.\u00a0We estimated the level of Cdca7 expression from the electroporated cells relative to surrounding non-electroporated cells by randomly selecting 30 GFP-expressing cells and 30 intermingled GFP-non-expressing cells within the lateral cortex, outlining each cell and measuring the intensity of the Cdca7 signal from each using Fiji software. We found that the average intensity of the Cdca7 signal was 4.4-fold higher in the GFP+ than in the GFP\u2212 cells. To put this difference into context, we then carried out the same analysis with 30 GFP-non-expressing cells from the ventral telencephalon, where Cdca7 is highly expressed by some cells; in this case we selected cells with high levels of expression, located in the ventral telencephalic subventricular zone (Fig.\u00a0We examined all GFP+ electroporated cells 16\u00a0h after electroporation at either E12.5 or E14.5 in a series of sections stained for: GFP and Tbr2; GFP and Tbr1; GFP and BrdU. We found no evidence that the elevation of Cdca7 levels altered the distribution of affected cells through the cortical depth 16\u00a0h after electroporation Fig.\u00a0.Fig.\u00a06ThThere was a significant reduction by ~20\u201325% in the proportion of GFP+ cells that were Tbr2+ in embryos electroporated with Cdca7 at E12.5 (Fig.\u00a0Analysis of the proportion of cells labelled by BrdU shortly before animals were sacrificed allowed us to assess whether elevated Cdca7 levels in the lateral cortex had a drastic effect on proliferation, for example bringing effected cells out of the cell cycle. We found that 20\u201330% of GFP+ cells took up BrdU in both control and experimental groups following electroporation at E12.5 or E14.5 Fig.\u00a0. At neitOur findings indicate that the main effect of elevated Cdca7 expression in early embryonic lateral cortical progenitors is to reduce the production of cells expressing Tbr2, which is the hallmark of intermediate progenitors.Pax6/\u2212\u2212 forebrain is a failure of the lateral cortex to expand ahead of more medial regions. This regional defect has been linked to a disproportionate underproduction of Tbr2+ cells in the lateral cortex of Pax6/\u2212\u2212 embryos, a loss which equilibrates the numbers of Tbr2+ cells across all cortical regions [Based on new findings reported here, we suggest that one of the actions of Pax6 in the embryonic cerebral cortex is to suppress Cdca7 expression and thereby enhance the production of Tbr2+ intermediate progenitors. Interestingly, our experiments suggested that this suppressive effect is most prominent in the lateral cortex early in corticogenesis, where cortical levels of Pax6 expression are highest . Since T regions . It is pOn the ventral side of the PSPB in wild-type embryos, Pax6 levels are lower and Cdca7 levels are higher, particularly in the subventricular zone of the subpallium. As an extension of the present study, it would be interesting to test with gene knock-out or knock-down experiments whether the particularly high levels of Cdca7 that we observed in the subpallial subventricular zone contribute to the lack of Tbr2 expression in the cells of this region.CDCA7 was first identified as a MYC direct target and CDCA7 protein is often found to be overexpressed in human solid tumours [Cdca7 expression and whether the repressive effects of Pax6 on Cdca7 expression are direct, or an indirect consequence of effects on Notch signalling, or both.Relatively little is known about the mechanisms of action of Cdca7. Human and mouse CDCA7/Cdca7 are nuclear proteins which contain a zinc finger domain in the C-terminus , 15. In  tumours , 26\u201328.  tumours . The evi tumours . In the  tumours , 31. It Cdca7 overexpression early in corticogenesis reduced not only the production of Tbr2+ intermediate progenitors but also the production of Tbr1+ cells. The divisions of Tbr2+ cells generate Tbr1+ neuronal precursors early in corticogenesis. The length of cell cycles in the cerebral cortex is around 10\u00a0h at E12.5, which is a few hours shorter than the 16\u00a0h between electroporation and sacrifice in our experiments , 33. It Pax6 and Cdca7 mRNA cortical expression gradients following mutation of the Pax6 gene indicated that both become highly abnormal from E12.5 to E14.5. By E14.5, the slopes of both gradients had reversed from normal. This result highlights the complexity of the molecular interactions that establish graded expression patterns. It indicates that Pax6 is not the only factor that can affect differential Cdca7 expression levels across the developing cortex. Changes in the expression of Pax6 mRNA in Pax6/\u2212\u2212 mice are likely to result from a loss of direct or indirect autoregulation of the Pax6 locus, which is known to be a prominent element in the control of expression of the Pax6 gene [Finally, our quantitative analysis of ax6 gene .In summary, this study provides evidence of counter-gradients of Pax6 and Cdca7 during embryonic ages E12.5 to E14.5 in mice and this relationship is disrupted when Pax6 is absent. In addition, elevated Cdca7 in the lateral cortex reduces the production of intermediate progenitors and postmitotic neurons, suggesting that repression of Cdca7 by Pax6 is required for the normal production of intermediate progenitors in this region."}
+{"text": "Drosophila melanogaster, three Robo paralogs  each have specialized roles in regulating midline crossing and the formation of longitudinal axon pathways in the embryonic ventral nerve cord. The number of robo genes differs in other insects, and it is unknown whether the roles and/or signaling mechanisms of Drosophila Robos are shared in other insect species. To directly compare the axon guidance activities of Robo receptors in Drosophila and the flour beetle Tribolium castaneum, I have used a CRISPR/Cas9-based approach to replace Drosophila robo3 with Tribolium robo2/3.Axon guidance receptors of the Roundabout (Robo) family regulate a number of axon guidance outcomes in bilaterian animals in addition to their canonical role in Slit-dependent midline repulsion. In the fruit fly robo3 locus in Drosophila embryos, Tribolium Robo2/3 (TcRobo2/3) protein is properly translated and localized to axons, where it reproduces the normal expression pattern of Drosophila Robo3. In embryos expressing TcRobo2/3 in place of robo3, two distinct subsets of longitudinal axons are guided properly to their normal positions in the intermediate neuropile, indicating that TcRobo2/3 can promote Robo3-dependent axon guidance decisions in developing Drosophila neurons.I show that when expressed from the Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. The CRISPR/Cas9-based gene replacement approach described here can be applied to comparative evolutionary developmental studies of other Drosophila genes and their orthologs in other species.These observations suggest that the mechanism by which  Drosophila melanogaster, where the underlying genetics has been best studied, the medial\u2013lateral and dorsal\u2013ventral position of dendrites, axon terminals, and longitudinal axon pathways within each neuromere is specified by a combination of extracellular guidance cues including Slit and semaphorin ligands, and their neuronal receptors of the Roundabout (Robo) and Plexin families, respectively [During development of the nervous system, neural circuits form as axons and dendrites follow directional cues which guide them to specific regions of the central nervous system (CNS), where local contact-dependent cues promote the formation of synaptic connections. In the insect ventral nerve cord, a stereotypical three-dimensional arrangement of axons and dendrites forms within each segmentally repeated neuromere , 2. In tectively \u20135.Drosophila, the secreted Slit ligand and its Robo receptors regulate midline crossing of axons in the developing embryonic CNS. In addition, two of the three Robo family members in Drosophila (Robo2 and Robo3) specify the medial\u2013lateral position of longitudinal axon pathways. The functional specialization of the three Drosophila Robos depends on both their differential expression in distinct neuronal subsets and distinct activities of the receptors themselves [Drosophila robo3 gene, along with all of the intervening introns, can be replaced with a non-native cDNA encoding robo3 without any detectable changes to its expression pattern or function [robo3 with robo1 or robo2 cDNAs further demonstrated that each of these paralogs can fully substitute for robo3 to promote axon pathway formation in intermediate regions of the neuropile in the Drosophila embryonic CNS. In contrast, neither robo1 nor robo3 can rescue robo2-dependent formation of lateral pathways, suggesting that unique structural features of Robo2 specify this role [Drosophila Robo receptors regulate lateral position in response to a gradient of Slit originating at the CNS midline, the precise mechanism(s) underlying this activity have not been fully characterized [In emselves \u20139. Previfunction . Equivalhis role . Althougcterized .robo2 and robo3 have distinct expression patterns and axon guidance roles in Drosophila, these two genes are a product of a recent gene duplication and do not exist as separate genes outside of dipteran insects. Instead, most insect groups have retained an ancestral robo2/3 gene [Tribolium castaneum, this gene (TcRobo2/3) appears to combine the activities of Drosophila robo2 and robo3 and is required for the formation of both intermediate and lateral axon pathways [Drosophila Robo2 and Robo3 throughout its extracellular portion, with the highest degree of sequence conservation in the Ig domains, especially the Slit-binding Ig1 domain . Despite this size difference, the two conserved cytoplasmic (CC) motifs present in Drosophila Robo3 are also present in TcRobo2/3. These two motifs, named CC0 and CC1, are conserved in most Robo family members, including Drosophila Robo1, Robo2, and Robo3, as well as Robo1 and Robo2 in vertebrates [While 2/3 gene , 12. In pathways . The TcRtebrates .Fig.\u00a01Serobo2 and robo3 was required for both intermediate and lateral pathway formation, and that this dual activity is retained by the robo2/3 gene in most insect species (including Tribolium), while the activities of Drosophila robo2 and robo3 have diverged through subfunctionalization after gene duplication [TcRobo2/3 should be able to substitute for both Drosophila robo3 and robo2 to promote intermediate and lateral pathway formation, respectively. Here, I test this prediction by replacing the robo3 gene in Drosophila with the robo2/3 gene from Tribolium via CRISPR/Cas9-mediated gene replacement. I find that when expressed from the robo3 locus, TcRobo2/3 protein reproduces the endogenous Robo3 expression pattern in the Drosophila embryonic CNS, including proper localization to neuronal axons and distribution on longitudinal axon pathways within intermediate and lateral regions of the neuropile. Further, TcRobo2/3 is able to substitute for robo3 to direct two distinct subsets of longitudinal axons to intermediate regions of the neuropile. These results suggest that Drosophila robo3 and Tribolium robo2/3 regulate axon pathway formation through an evolutionarily conserved mechanism and demonstrate the utility of CRISPR/Cas9-mediated gene replacement for comparative studies in evolutionary developmental biology.We have previously hypothesized that the ancestor of lication , 11. A probo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611). The four fragments were derived from pBluescript , the wild-type robo3 genomic locus , and the robo3 cDNA . This initial donor construct contained an untagged robo3 cDNA flanked by BglII sites. To make the HA-tagged robo3TcRobo2/3 donor, the robo3 coding sequence was excised with BglII and a 4xHA sequence flanked by BamHI (upstream) and BglII (downstream) sites was cloned into the BglII site, thus retaining a single BglII cloning site immediately downstream of the 4xHA tag. The TcRobo2/3 coding sequence was amplified by PCR using primers 279 and 280, then digested with BglII, and cloned into the 4\u00d7\u00a0HA-containing donor backbone. The entire donor region including TcRobo2/3 coding sequence and robo3 flanking regions was sequenced prior to injection.The initial robo3 gRNA sequences were cloned into the tandem expression vector pCFD4 [or pCFD4  via PCR Drosophila strains, transgenes, and mutant alleles were used: Canton-S (wild type), robo31 [robo3robo3 [robo3TcRobo2/3 (this study), P{sema2b-TauMyc} [w1118; snaSco/CyO,P{en1}wgen11 (\u201cSco/CyOwg\u201d). All crosses were carried out at 25\u00a0\u00b0C.The following  robo31 , robo3ro3robo3 , robo3T-TauMyc} , w1118robo3 gRNA and robo3TcRobo2/3 homologous donor plasmids were coinjected into nos-Cas9.P embryos [Sco/CyOwg. Founders  were identified by testing two pools of three F1 females per G0 cross by genomic PCR with primers 229 and 280, which produce a 0.8-kb product only when TcRobo2/3 sequences are present. From each identified founder, 5\u201310 F1 males were then crossed individually to Sco/CyOwg virgin females. After three days, the F1 males were removed from the crosses and tested by PCR with primers 229 and 280 to determine whether they carried the modified allele. F2 flies from positive F1 crosses were used to generate balanced stocks, and the modified alleles were fully sequenced by amplifying the entire modified locus (approx. 6\u00a0kb) from genomic DNA using primers 589 and 590, then sequencing the PCR product after cloning via CloneJET PCR cloning kit (Thermo Scientific). Details of G0 survival, fertility, and modified allele transmission rates are provided in Table\u00a0The  embryos  by BestGDrosophila embryo collection, fixation, and antibody staining were carried out as previously described . The folTribolium Robo2/3 can substitute for Drosophila robo3 to promote axon guidance outcomes in the Drosophila embryonic CNS, I used a CRISPR/Cas9-based approach to replace the robo3 gene with TcRobo2/3. Two guide RNAs (gRNAs) targeting exons 2 and 12 were combined with a homologous donor plasmid containing 1-kb flanking regions to induce homology-directed repair, replacing robo3 exons 2-12 with an HA-tagged TcRobo2/3 cDNA  [robo3TcRobo2/3 donor plasmid into Drosophila embryos expressing Cas9 under the control of the germline-specific nanos promoter [TcRobo2/3 sequences. Additional PCR screening and DNA sequencing were used to identify correctly modified robo3TcRobo2/3 loci among the lines recovered from the positive F1 flies. Further details are provided in Methods and Table\u00a0To test whether  (pCFD4)  was injepromoter , and F1 robo3TcRobo2/3 allele, I first compared its expression to that of the endogenous Robo3 protein in various wild-type and modified genetic strains , there are no detectable large-scale defects in the axon scaffold in robo31 mutants, as assayed by an antibody against horseradish peroxidase (anti-HRP), which labels all of the axons in the CNS . In robo3robo3 homozygous embryos, the Robo3 protein expressed from the modified locus precisely reproduces Robo3\u2019s normal expression pattern  axon segments and longitudinal axons in the medial neuropile. No appreciable levels of TcRobo2/3 appear to accumulate within the cell bodies of normally robo3-expressing neurons, or on the cell body plasma membrane, consistent with proper subcellular localization of the Tribolium Robo2/3 protein in this heterologous context. These observations indicate that the TcRobo2/3 protein is properly translated and trafficked within Drosophila neurons, and suggest that axons that would normally select intermediate or lateral pathways under conditions of wild-type Robo3 expression make equivalent pathway choices when TcRobo2/3 is expressed in its place.In the robo3TcRobo2/3 embryos, I used an antibody against the cell adhesion molecule Fasciclin II (FasII) to label a subset of longitudinal axon pathways in these embryos  by which robo3 and TcRobo2/3 regulate axon guidance outcomes in the Drosophila and Tribolium embryonic CNS, respectively, are evolutionarily conserved. In addition, the gene replacement approach described here should be broadly applicable to evo-devo studies of other genes in Drosophila and other insects.In this paper, I have used a CRISPR/Cas9-based gene replacement approach to directly examine the evolutionary conservation of axon guidance activities within the Robo family of axon guidance receptors. I have replaced the Drosophila axons: (1) The subset of longitudinal axons that normally express Robo3 are still localized to the lateral two-thirds of the neuropile when they express TcRobo2/3 instead of Robo3; (2) FasII-positive axon pathways in the intermediate neuropile form in their proper location when robo3 is replaced by TcRobo2/3; and (3) sema2b-positive longitudinal axons also select their correct intermediate position when robo3 is replaced by TcRobo2/3. These observations, together with a previous report that RNAi-mediated knockdown of TcRobo2/3 disrupts intermediate pathway formation in the Tribolium embryonic CNS [Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. CRISPR approaches have recently been used successfully in Tribolium [TcRobo2/3 with Drosophila robo3. In this case, robo3 should be able to replace the function of TcRobo2/3 to promote intermediate pathway formation, but the lateral pathways (which presumably depend on a robo2-like activity of TcRobo2/3) may not form properly.Three lines of evidence suggest that TcRobo2/3 can act equivalently to Robo3 to guide onic CNS , suggestribolium , so it sDrosophila embryonic CNS suggests that any sequences that are necessary for this role in Robo3 are conserved in TcRobo2/3. Notably, the highest degree of sequence conservation between these two proteins occurs within the extracellular Ig1 and Ig3 domains  are conserved in Robo3 and Robo2/3, but there is little conservation in the cytodomain outside of these motifs . A similar approach should be applicable to other genes assuming that, like all three Drosophila robo genes, the introns that would be removed by the cDNA replacement are dispensable for proper expression of the gene to be modified.The gene replacement approach described here has several advantages for both structure\u2013function studies and evo-devo functional comparisons of orthologs from different species. First, replacing multiple coding exons in the endogenous gene with a single cDNA sequence allows for epitope tagging of the modified locus in addition to replacing the entire coding region with a foreign sequence or a modified cDNA carrying engineered deletions, additions, or sequence swaps. For multi-exon genes, this eliminates the need to replace multiple exons individually. Second, the endogenous promoter and translational start site remain unmodified, minimizing effects on gene expression. Third, using two gRNA targets that flank the majority of the gene should allow recovery of a null deletion allele via non-homologous end joining (NHEJ) in addition to the HDR-mediated gene replacement allele. This would be of particular benefit for genes for which a null allele is not currently available. Finally, this approach allows the same gRNA plasmid and donor backbone to be used for multiple gene replacement experiments, minimizing troubleshooting and optimization for each subsequent experiment. We have used the same donor construct backbone to replace Drosophila and used this strategy to demonstrate the evolutionary conservation of axon guidance mechanism(s) between the Drosophila robo3 and Tribolium robo2/3 genes. When expressed from the robo3 locus, TcRobo2/3 protein is properly translated and localized to neuronal axons in the Drosophila embryonic CNS and can guide developing axons to pathways in the intermediate region of the neuropile in an equivalent manner to Drosophila Robo3. The approach described here should be generally applicable to other genes and developmental contexts and will be useful to interrogate the evolutionary conservation or divergence of additional axon guidance mechanisms in insects.Here I have described a strategy for CRISPR/Cas9-based trans-species gene replacement in"}
+{"text": "Mitogen-activated protein kinase 4 (MPK4) interacts with the (Mitogen-activated protein kinase kinase kinase 1) MEKK1/ Mitogenactivatedprotein kinase kinase 1 (MKK1)/ Mitogen-activated protein kinase kinase 2 (MKK2) complex to affect its function in plantdevelopment or against pathogen attacks. The KEGG (Kyoto Encyclopedia of Genes and Genomes) network analysis of Arabidopsisthaliana revealed close interactions between those four genes in the same plant-pathogen interaction pathway, which warrants furtherstudy of these genes due to their evolutionary conservation in different plant species. Through targeting the signature sequence inMPK4 of papaya using orthologs from Arabidopsis, the predicted sequence of MPK4 was studied using a comparative in silicoapproach between different plant species and the MAP cascade complex of MEKK1/MKK1/MKK2. This paper reported that MPK4was highly conserved in papaya with 93% identical across more than 500 bases compared in each species predicted. Slight variationsfound in the MEKK1/MKK1/MKK2 complex nevertheless still illustrated sequence similarities between most of the species.Localization of each gene in the cascade network was also predicted, potentiating future functional verification of these genesinteractions using knock out or/and gene silencing tactics. Defense mechanisms in plants towards pathogens infection lay ina complicated web of multiple genes interactions in the defenseresponse pathways. Mitogen-activated protein kinase (MAPkinase) cascades are critically involved in regulating plantdefense mechanisms including plant innate immune responses. StudiesStudy of the MPK4/MPK4-like gene is common in the modelplant, Arabidopsis. However, there is still lack of information innon-model plant species including papaya. Interestingly inpapaya, MPK4 was found to magnify the defense responsesagainst papaya dieback disease caused by Erwinia mallotivora . Based oThe unique and complex interaction of MPK4 within the MAPkinase cascade also warrants a crucial in silico study of the MPK4gene concomitantly with other genes in the cascade that interactdirectly with it. Activation and regulation of MPK4 in the MAPkinase cascade were noted to require MEKK1 for cell deathdefense response in Arabidopsis  and MKK1In this study, the MPK4 gene from papaya was extracted basedon Arabidopsis MPK4 (AtMPK4) orthologs and the sequence wasanalysed using bioinformatics approach to predict itscharacterized functions and interactions in the MAP kinasecascade in papaya in comparison to other plants. The MEKK1,MKK1 and MKK2 orthologs were also mined from theArabidopsis genes designated as AtMEKK1, AtMKK1 andAtMKK2, respectively. Homologs of MPK4 in soybean showedMPK4 functioned to negatively regulate defense responses butpositively control growth and development . AnotherSequences of AtMPK4, AtMEKK1, AtMKK1 and AtMKK2 wereobtained from the Arabidopsis Information Resource (TAIR)database  and orthThe base content percentage of each sequence was calculatedusing the GC Calculator from BiologicsCorps . The nucTranslated nucleotides sequences that matched with the databasewere assessed using the ScanProsite software  to prediThe evolutionary path of each gene in different plant species wasevaluated using the neighbour-joining phylogenetic treesconstructed using the Mega software version 6  withbooFurther study was carried out using the Pathway Studio softwareweb version  to prediThe papaya MPK4, MEKK1 and MKK1/MKK2 sequences wereblasted into Blast2GO version 3.2  for the Sequences were obtained from the Phytozome database for eachgene of each available plant, respectively, with nine sequences forMPK4, eight for MEKK1, nine for MKK1 and eight for MKK2.There were no available sequences of MEKK1 in grape andMKK2 in Japanese rice. The length of the MPK4 sequence fromeach plant shows a slight difference from each other,approximately 1100 base pairs (bp). The longest sequence ofMPK4 was from sorghum with 1167bp while the shortestsequence was from cucumber with 1113bp. The MEKK1sequences, however, were dissimilar in nucleotides sequencelength ranging from 663bp (Japanese rice) to 1998bp (tomato).While most of the sequences were approximately 2000bp inlength, only Japanese rice (663bp) were shorter by 1000bp. TheMKK1 sequences were relatively similar to each other with 50 to74bp larger than 1 kilo base pair (kb) length. In contrast, theMKK1 sequence from soybean was only 807bp. The longestsequence of MKK1 obtained was from tomato with 1074bp. Thelongest sequence of MKK2 mined was from the Arabidopsisgenome with 1119bp while the shortest sequence was in sorghumwith only 69bp differences. The least variation in sequence lengthbetween the species corresponded to higher chance of conservedregion within the particular gene. In the case of verification of theMPK4 gene in papaya, we concluded that the MPK4 sequence obtained was closely related to the AtMPK4, likewise maybe itsfunction too. Whereas in MEKK1 and MKK1, high variations oflength sequences were acquired in one to two species which wasout of the comparative range (\u00b1100bp).Comparing the base content analysis of each MPK4, MEKK1,MKK1 and MKK2 sequences, the lowest percentage of individualbase was cytosine while the highest was adenine, both in MKK1of soybean . GmMKK1 Diversity of the three types of stop codon in the CDS sequenceswas observed in MEKK1 and MKK1 sequences, while MPK4 andMKK2 sequences revealed only diversity in TGA and TAA. OnlyJapanese rice, corn and sorghum have TAA in the MPK4sequences while most other species consist of TGA. In the MKK2sequence, TAA is commonly found whereas only papaya andsorghum contain TGA. Almost half of the species have TAG inthe MKK1 gene while the remaining has distribution of 33.33%TAA and 22.22% TGA. Stop codons in MEKK1 are distributedwith only one TAG stop codon (sorghum), and respectively threeand four stop codons of TGA and TAA between plant species.The percentage of stop codon variations was diverged betweengenes as represented . From thAnalyses of peptide sequences from the translated genes of eachspecies via domain and motif predictions using the ScanPrositesoftware revealed similar domain of protein kinase. The featuresstudied from the conserved domains and motifs in the MPK4,MEKK1, MKK1 and MKK2 for each plant species.Thirty out of 35 sequences studied shared similar motif signatureregion of the protein kinase ATP-binding and serine/threonine protein kinase active-site signatures. The domain region in all theMPK4 from each species demonstrated the same 286bp length.These MPK4 sequences had the conserved MAP kinase signatureregion except in sorghum, which exhibited a glycine rich regionprofile and lipid cysteine motifs of S-diacylglycerol and Npalmitoyl.The MPK4 of sorghum contained tyrosine proteinkinase specific active site signature at positions 177-189bp. Incontrast, no MAP kinase signature was obtained from the otherthree genes sequences. These genes share the protein kinasedomain with minor distinction of locations and lengths ofbinding domain ranging from 117 to 286bp. Interestingly, MKK1and MKK2 in cotton were found with the presence of TonBdependentreceptor protein signature but lacking the kinase ATPbindingsite.Study of the predicted domains and motifs indicate variance inlength of the protein kinase ATP-binding site in different genes inwhich MPK4 had the longest region of 25bp, MEKK1 with theshortest region of 23bp while MKK1/MKK2 with 24bp. Theprediction analysis also found the absence of the proton acceptoractivesite in the MEKK1 sequence. Amino acids changes werenoted in D482F in the papaya MEKK1 protein sequence, D220S inthe Japanese rice MEKK1 protein sequence and K159R soybeanMKK1 protein sequence. These changes could be due toevolutionary mutation processes.The identity matrix of each genes sequence alignment calculatedusing the t-coffee software discovered the papaya MPK4 genehad the highest identical value (92.25%) with grape MPK4 genewhile the papaya MEKK1 gene had the highest identical value(59.02%) with cucumber MEKK1 gene. The papaya MKK1 andMKK2 also shared high sequence homology with the grape at77.90% and 78.19%, respectively. Although MEKK1 in grape wasundiscovered, other three genes studied in papaya showedclosest homology in term of sequence composition to thesequences in grape with 77% to 92%.The significant differences observed between each MPK4sequences across the plant species are postulated as a response toevolutionary pressure imposed on the gene to maintain its corefunction as a defense gene against certain host-specificpathogens. For each respective variation in the sequencesthroughout the plant species, they were grouped into two majorclusters consisting of dicotyledonous plants (Class:Magnoliopsida) versus monocotyledonous plants (Class:Liliopsida).The evolution of the MPK4 gene between the plant species wasdistinctive in those two clusters. The papaya MPK4 wasclustered under the same clade with Arabidopsis (96%), whiletomato MPK4 (67%) showed a distant lineage from those groupsas illustrated in . Within The phylogenetic analysis of MEKK1 showed high (71 to 100%)bootstrap support across the species . These vFurther phylogeny analysis for MKK1 suggested that thesequence was closely associated between rice\u2019s and sorghum\u2019s. In contTo sum the sequence analysis of corresponding genes, MPK4scored high in conservation and stability across the plant species.It had less variance of stop codons and GC content, containedspecific domain features, high conservation and sequencehomology identities, and close distance of phylogeny. TheMEKK1, however, lack domain features, and has low (below 60%homology) sequence identity. Nevertheless, its high bootstrap ofmore than 70% confidence is likely true across the cluster groupbetween the species analysed. Both the MKK1 and MKK2 lackedcertain features in some species but were conserved in papaya,with higher homology identity index for MKK2 disputed toMKK1, which had a closer distance referred to papaya sequence.Searches using the Pathway Studio software  successfBased on in silico study of MPK4 and MEKK1/MKK1/MKK2complex interaction using the Pathway Studio software, it wasreported that all four related genes were involved in theregulation of defense response. In a study of the geneticmanipulation of MAP kinase cascade in plants proposed that theMPK4, MEKK1, and MKK1/MKK2 complex negatively regulateddefense responses . Based oWithin the MAP kinase cascade, it was highlighted that themultiple levels of signaling pathway starting from the MAPkinase kinase kinase (MAPKKK) to the MAP kinase kinase(MAPKK), and then to the activation of the MAP kinase (MAPK),leading to signal plant defense modulation . This paUpon infection, the MEKK1 received signal from the receptor andtriggered the MKK1/MKK2 complex with consequent activationof MPK4 -42. The We postulated that signals from MEKK1 will lead to theexpression of MKK1 and MKK2 as these two complexes promoteprotein modification interaction to stimulate MPK4 expression.MKK1/MKK2 functions as an important intermediate, whichinteracts between MEKK1 and MPK4. Within Arabidopsis,MKK1/MKK2 was conveyed to have overlapping functions indefense signaling mediated by MEKK1 and MPK4 . It can Inactive MPK4 was described to be phosphorylated by MKK1 atthe threonine site of MPK4 to trigger defense response .PhosphoThe interaction of MPK4 in the MAPK cascade together with theupstream components, MKK1/MKK2 and MEKK1, was found tobe involved in the phosphorylation pathway as a negativeregulator of systemic-acquired resistance (SAR) . While tAnalysis of the MPK4 cascade via the Cytoscape software  revealedThe detailed studied interactions between genes extracted fromthe Cytoscape database whereby two different interaction typeswere performed to study the genes interaction. The two hybridand two hybrid array detection methods related a physicalassociation type of interaction, while the protein kinase assay andin-gel kinase assay showed a phosphorylation reaction type ofinteraction. The overall data shown supports the interaction ofMEKK1-MKK1/MKK2-MPK4 as proposed in Additional analysis performed using the Blast2GO software withMEKK1, MKK1/MKK2 and MPK4 sequences in papaya to studythe potential functions as indicated in the gene ontology datashowed a transferring phosphorus-containing group of enzyme(Enzyme code (EC): 2.7.11) with 20 hits and 0.0e0 e-value. Theattained gene ontologies of each sequence are summarised.CpMEKK1 showed the highest possibility of classes in signaltransduction and cellular protein modification ; ion binding and kinase activity (molecular functionaspect); and intracellular related function (cellular componentsaspect). CpMPK4 also portrayed five possible gene ontologyclasses of functions including cellular protein modificationprocess ; ion binding, signal transduceractivity and kinase activity (molecular function); and intracellularrelated function (cell components aspect). Both of the postulatedCpMKK1/MKK2 from the single sequence showed a group ofeight classes of functions: cell death, signal transduction, cellularprotein modification process, cell cycle and response to stress; ion binding and kinase activity (molecularfunctions); and cytoplasm related function (cellular component).As gene ontology study of MEKK1, MKK1/MKK2 and MPK4 inpapaya emanated mainly in signal transduction, ion binding andkinase activity, interaction between each gene seemed essentialespecially in the MAP kinase cascade studied. Biological processof response to stress discovered in CpMKK1/MKK2 maydemonstrate more direct approach of the gene to play its functionagainst stress. This result might also hypothesise the lesserinteraction as proposed in MKK2 interaction of the Cytoscaperesult . The simConsidering the MEKK1-MKK1/MKK2-MPK4 module wasexpected to negatively regulate the defense response in papaya, agood suggestion put forth by Bigeard et al. (2015) that a mutantof this cascade be created using knock-out gene approach torepresent a constitutive defense responses including aconstitutive expression of the pathogenesis-related (PR) defensegenes, would indeed elucidate the intricacies of the playersinvolved in conferring resistance of plants against potentialpathogens . Here, oThe analysis of the conserved regions and signature motifs ofMPK4 and MEKK1/MKK1/MKK2 complex genes support therole each gene plays in the MAP kinase cascade in promotingdefense response. Overall, MPK4 indicates high conservation andstability in papaya and other plant species investigated. Theprotein kinase domain and motifs of MPK4 justified with highestimates of the conservation, homology and sequencepropositions across the plant species analysed. The findings fromthis in silico study revealed that the conservation of the candidategenes analysed particularly MPK4 within each plant species arefunctionally reliable as compared to the referenced sequencesvalidated through knock out gene study of MPK4 in Arabidopsis.The in silico study of the MPK4 gene in the MAP kinase cascadeand the MEKK1/MKK1/MKK2 complex showed wide variationsin stop codons, GC-content and homology identity but yetconserved in their respective signature domains to assume theirbasic functional role, and thus to make use of these interactionsbehaviour between genes isolated for future references."}
+{"text": "Up to 30% of HIV infected patients who are receiving HAART do not exhibit a marked increase in the CD4+ T cell count. There is still a concern that immune recovery may not be complete once CD4+ T cells have decreased below 200 cells/\u03bcl. The objective is to assess CD4+ cell recovery in HIV+ patients with CD4 count below 200 cells/\u03bcl) at HAART initiation.This was a retrospective cohort study among 110 HIV+ patients with initial CD4 count < 200 cells/\u03bcl. Baseline Age, sex, CD4 count and viral load were extracted from the patient\u2019s database. After12 months of HAART; CD4 count was done using flow cytometry and viremia by COBAS AmpliPrep/COBAS TaqMan HIV-1 test v 2.0 technology.The mean age of the respondents was 35 years; males being 57% and females were 43%. The mean CD4 count before HAART was 110.18 cells/\u03bcl whereas at 12 months of HAART; this was 305.01 cells/\u03bcl. Though some patients did not achieve a CD4 count of more than 200 cells/\u03bcl or a drop in viral load; there was a significant recovery of CD4+ cells  and viremia following HAART . Participants aged 18-30 years were likely to have less than 200 cells/\u03bcl CD4 count (46.4%)  than participants aged above 40 years (16.7%).HAART was associated with viremia suppression but many patients failed to achieve a CD4 count >200 cells/\u03bcl. HAART before severe immunosuppression is a key factor for immune restoration among HIV+ patients. Human immunodeficiency virus (HIV) is a lentivirus; a member of the retrovirus family that causes Acquired Immunodeficiency Syndrome (AIDS) . Human I+ patients biological follow up that aims at assessing the infection progression as well as response to HAART [Work undertaken by the National Institute of statistics of Rwanda (NISR), in the 2010 RDHS, indicated that in Rwanda, HIV prevalence was 3.7% and 2.2% among women and men, respectively. The city of Kigali, Capital of Rwanda had the highest prevalence (7.3%) in the country . Periodito HAART . The besto HAART \u201310. A loto HAART  and studto HAART . HIV+ arto HAART .CD4 count has been reported to be one of the best surrogate markers for monitoring the progression of HIV infection and low CD4 counts are associated with increased risk of developing AIDS or death . The absThis was a retrospective cohort study that started from March 2015 to February 2016. The study aimed at looking at how CD4+ cells recover when HIV+ patients initiate HAART with a severe immunosuppression. Baseline data including demographic (age and sex), CD4 count, and viral load were collected from HIV+ patient\u2019s database . The inclusion criteria were HIV+ patients who had initiated HAART with a CD4 count of less than 200 cells/\u03bcl.The study was conducted at Centre Medico-Social Cor-Unum located in Kigali; the capital city of Rwanda. This health facility is a member of public health facilities in Rwanda that host voluntary counseling and testing (VCT), antiretroviral treatment services and follow up for many HIV+ patients. In addition, the area is near the National Reference Laboratory of Rwanda where viral load was to be measured and this has facilitated easy and cheap sample transportation at 12 months of HAART. Blood samples were collected from 110 study participants who had been on HAART for 12 months. The CD4 count was done using flow cytometry (BD FACSCount\u2122 system) while viral load was determined using COBAS AmpliPrep/COBAS TaqMan HIV-1 test v2.0 technology.Paired and independent t test were used to compare means between baseline CD4 cell; viremia and at 12 months of HAART initiation; as well as between demographics and CD4 cell/viral load respectively. Moreover, chi square test was used to determine the association between demographics and CD4 count level. The significance level was set at P value <0.005. This study was reviewed and approved by Mount Kenya University (MKU04/DPA/16/2015/3007) and Congregations Around Richmond Involved to Assure Shelter (CARITAS)-Rwanda as Centre Medico-Social Cor-Unum operates under CARITAS in partnership with the Rwanda Ministry of Health. All the patients signed the informed consent before participation to the study.As shown in Viral load median was computed at both time points as shown in Before HAART initiation males had a mean rank viral load 59.9 compared to 49.5 among females but this was not significant . Following HAART mean rank of drop in viral load achieved by males was 55.3 and by females 55.8 . Similarly, there was no significant difference between the age and mean rank of viral load before HAART initiation  as well as age and mean rank of viral load after 12 months of HAART initiation  .CD4 Count and viral load levels were analyzed based on whether a patient achieved a CD4 count of \u2265 200 cells/\u03bcl at 12 months of HAART initiation. Despite HAART, 29 (26.4%) of study participants were still having CD4 count below 200 cells/\u03bcl compared to 81 (73.6%) participants who had a CD4 count of more than 200 cells/\u03bcl as CD4 count. As for viral load, 106 (96.4%) patients achieved a net decrease in viremia to 20 RNA copies/ml while 4 (3.6%) had an increase in their viremia .The World Health Organization recommends a limit CD4 count of 350 cells/\u03bcl or clinical stage based decision for HAART initiation; particularly in countries where viral load cannot be easily determined , 17 and A significant recovery of CD4 positive cells and a drop in viremia following 12 months of HAART among study participants were documented and this is in agreement with findings by  and a stSome patients saw a decrease in their viral load after 12 months of treatment. However; this was not associated with a net recovery of CD4+ cells to reach more than 200 cells/\u03bcl; as following the 12 months of HAART considerable percentage (26.4%) of participants were still having CD4 count below 200 cells/\u03bcl compared to 73.6% of participants who achieved more than 200 cells/\u03bcl as CD4 count. This finding highlights the importance of viral load for the biological follow up of HIV+ patients both before and following HAART compared to CD4 count. Several studies have reported a poor prognosis prediction by CD4 count as it sometimes does not correlate with viral load in what is known as discordant response  and slowPoor immunologic response has been attributed to patient factors like age, the regimen as well as viral factors. However; this was not the case in the present study where young patients were likely to have a poor immune recovery compared to old patients . The preWe conclude that immune recovery is slow among HIV+ patients who start HAART with severe immunosuppression. Early HAART intervention is necessary for achieving effective CD4+ T cell responses and optimal immunological function in HIV+ patients.Several researchers have focused their attention on CD4+ cells recovery among HIV+ patients following HAART in general.This study has enriched the knowledge on how CD4+ cells recover among HIV+ patients when they initiate HAART after a severe immunosuppression;The study has again pointed out the importance of early HAART initiation among HIV+ patients particularly in the context of Rwanda where such data were scarce;The present study will also raise more interests among researchers on host cellular and molecular as well as viral factors behind this poor recovery in CD4+ cells."}
+{"text": "Dw1-Dw4). Subsequent research showed that Dw3 encodes an ABCB1 auxin transporter and Dw1 encodes a highly conserved protein involved in the regulation of cell proliferation. In this study, Dw2 was identified by fine-mapping and further confirmed by sequencing the Dw2 alleles in Dwarf Yellow Milo and Double Dwarf Yellow Milo, the progenitor genotypes where the recessive allele of dw2 originated. The Dw2 locus was determined to correspond to Sobic.006G067700, a gene that encodes a protein kinase that is homologous to KIPK, a member of the AGCVIII subgroup of the AGC protein kinase family in Arabidopsis.Sorghum is an important C4 grass crop grown for grain, forage, sugar, and bioenergy production. While tall, late flowering landraces are commonly grown in Africa, short early flowering varieties were selected in US grain sorghum breeding programs to reduce lodging and to facilitate machine harvesting. Four loci have been identified that affect stem length ( Additionally, shorter grain varieties were selected to reduce lodging and to aid mechanical harvesting. In contrast, sorghum genotypes with longer stems and delayed flowering enhance biomass and sugar production3. In sweet sorghum, stem length is associated with higher sugar yield because stems accumulate high levels of sucrose post floral initiation5. In energy sorghum, 83% of the shoot biomass accumulates in the stem6. Therefore, increasing our knowledge of stem growth will aid the improvement of sorghum hybrids for bioenergy production.Sorghum is the fifth most widely grown cereal crop worldwide (faostat.fao.org). Its drought and heat tolerance make this crop especially important in semi-arid regions. Sorghum is a C4 grass with a diverse germplasm that has been selected for many uses including production of grain, forage, sugar, and biomass for bioenergy. In its native Africa, sorghum grows 4\u20135 meters tall and many genotypes are photoperiod sensitive, resulting in delayed flowering in long day environments. Upon introduction to temperate locations, photoperiod insensitive varieties that flower early were selected for production of grain7 identified four loci (Dw1-Dw4) that control internode length by measuring the height of the stem from the ground to the flag leaf. At each Dw locus the dominant allele increased internode length. Recessive alleles of Dw1 and Dw2 were identified in Milo lines, while recessive alleles of Dw3 were identified in Kafir backgrounds, and dominant alleles at Dw4 were only found in broomcorns7. Dw2 was shown to have pleiotropic effects on panicle length, seed weight, and leaf area9. In addition to internode length, Dw3 influences grain yield, tiller number10, and leaf angle11.Plant height is determined primarily by the length and number of stem internodes. The number of internodes produced by a plant is a consequence of growth duration and the rate of internode production. Quinby and KarperDw3 was the first dwarfing gene to be cloned in sorghum12. Dw3 encodes a homolog of the maize Br2 gene and is an ATP-binding cassette type B1 (ABCB1) auxin efflux transporter. This is in contrast to dwarfing or semi-dwarfing genes in other important crops, such as rice and wheat, which have mutations in genes involved in the gibberellin pathway14. Dw1 was mapped to a region on chromosome 9 between 56.8\u201357.1 Mb15. The gene corresponding to Dw1 was recently identified as Sobic.009G229800 by map-based cloning17. This gene regulates internode cell proliferation17 and encodes a putative membrane protein not previously assigned a function16. The recessive dw1 allele in Dwarf Yellow Milo (DYM), first identified by Quinby and Karper7, contains a stop codon in exon 2 that results in protein truncation16. The dw1 allele originating from Dwarf Yellow Milo has been used extensively in grain sorghum breeding programs.Dw2 has also been used extensively in grain sorghum breeding programs to reduce plant height. Dw2 is linked to Ma1, an important flowering time gene that confers photoperiod sensitivity1. Ma1 is located on chromosome 6 at ~40.3\u2009Mb and encodes PRR3718. Dw2 was previously mapped to a location near Ma1 at ~42\u2009Mb in several QTL mapping studies22 and was associated with a SNP marker at 42.7\u2009Mb in a GWAS study23. In another study, Dw2 was suggested to be a histone deacetylase (Sobic.006G067600) based on GWAS analysis21. Recessive alleles of Ma1 and the dwarfing genes were used in the Sorghum Conversion Program to convert tall late flowering landraces from Africa into short, early flowering genotypes that are useful for grain sorghum breeding. The landraces were crossed to BTx406 (dw1dw2dw3dw4) to introduce one or more of the recessive alleles at the Dw loci into landrace backgrounds19. Recent analysis of the sorghum conversion lines has shown that large portions of chromosome 6 have been introgressed from BTx406 into landrace accessions during conversion and that the peak of introgression frequency aligned with Dw224.Dw2 was map-based cloned using two RIL populations: BTx623 (dw1Dw2dw3dw4) x IS3620c (dw1dw2Dw3dw4) and BTx642 (dw1dw2dw3dw4) x Tx7000 (dw1Dw2dw3dw4). Dw2 was identified as a protein kinase whose closest homolog in Arabidopsis is the kinesin-like calmodulin-binding protein (KCBP)-interacting protein kinase (KIPK), a member of the AGCVIII subfamily that also includes PINOID (PID) and PHOTOTROPIN1 and 2 (PHOT1 and 2).In the current study, dw2 allele present in Double Dwarf Yellow Milo (DDYM), the original source of dw2, arose as a mutation in Dwarf Yellow Milo (DYM)25. Comparison of DYM and DDYM stem internode lengths at anthesis showed that the recessive allele of dw2 in DDYM caused a reduction in the length of every elongated internode compared to the corresponding internodes in DYM  and for Dw3 on chromosome 7 (~59.8\u2009Mb) and these loci affected both total stem length and internode length. An additional QTL (Dw03_67.5) at ~67.5\u2009Mb on chromosome 3 affected total stem length  near Dw2 segregating for the length of the sixth internode below the peduncle. However, the peaks for the fifth and sixth internode are broad and the 2-LOD interval for the peak on chromosome 6 for both internodes includes both Dw2 and Dw06_48.6  analysis (Supplementary Table\u00a0Dw3 (chromosome 7 at 59.8\u2009Mb) and another between a QTL on chromosome 1 and a QTL close to Dw3 (10.7\u2009cM from Dw3 at 61.2\u2009Mb)  that decreases function; therefore, all of the genes in the delimited Dw2 locus  and Tx7000 (Dw2)  from DDYM (dw2), the source of the recessive allele of dw2.The gene corresponding to Dw2 alleles in historically important sorghum genotypes was investigated by sequencing Sobic.006G067700 from the genotypes listed in Tables\u00a07 as dw2 contain the indel in Sobic.006G067700 derived from DDYM. For example, 80\u2009M is a maturity standard with the reported genotype dw1dw2Dw3dw41. 80\u2009M and the other maturity standards were selected from a cross of Early White Milo (Dw2) and DDYM (dw2) and contain the DDYM dw2 allele  were also crossed to genotypes containing the DDYM dw2 allele during their construction26. Several genotypes contained polymorphisms in exons that changed the protein sequence encoded by Sobic.006G067700. However, SIFT27 analysis predicted that those polymorphisms would be tolerated and not disrupt function  are members of the AGC1 group. A BLAST search of the Arabidopsis AGCVIII kinase gene family to the sorghum genome identified 21 sorghum homologs  and Sobic.008G096200 (SbKIPK-like) . Genes in other plants with the greatest sequence similarity to Sobic.006G067700 include LOC_Os12g29580 (rice), GRMZM2G412524 (maize), GRMZM2G128319 (maize), and At3G52890 (Arabidopsis) (Phytozome). At3G52890 encodes an ACGVIII kinase called KIPK, a KCBP-interacting protein kinaseke) Fig.\u00a0.Figure 432. A comparison of Dw2 (Sobic.006G067700) with KIPK and other members of the AGC1-kinase group showed that Dw2 contains a conserved GxGxxG sequence in the P-loop of sub-domain I of the N-lobe, an activation segment in the C-lobe that includes the Mg++ binding sequence DFDLS, an insertion domain typical of plant AGC-kinases, and a T-loop and activation domain [SxxSFVGTxYxAPE] that is a site of phosphorylation32 , it aligned best with the N-terminal domain of KIPK and next best to KIPK2 (AGC1-9). Multiple sequence alignment of Dw2, rice and maize homologs of Dw2, and Arabidopsis KIPK showed regions of sequence similarity throughout the N-terminal domain and several deletions relative to Arabidopsis KIPK that explain the difference in overall length of the N-terminal protein sequences (423 versus 545 amino acids)  by analysis of RNAseq profiles that are part of the sorghum RNA Atlas (Phytozome). Dw2 is annotated as having two transcripts that differ in the 5\u2032UTR. The primary transcript (Sobic.006G067700.2) has a UTR with no introns that extends 537\u2009bp before the start codon, while the secondary transcript (Sobic.006G067700.1) has one intron and extends 923\u2009bp. The analysis of Dw2 expression shown in Fig.\u00a0Dw2 was relatively high in developing panicles, peduncles, growing internodes and leaf sheaths, with lower expression in fully expanded internodes, leaf blades and the lower portion of the root system that includes root tips and fully elongated roots  locus segregating in the second population derived from BTx642 x Tx7000, genotypes recessive for dw1dw3dw4. Indeed, the only QTL segregating for total height in this population was a QTL corresponding to Dw2 (~43.2\u2009Mb). The location of the Dw2 QTL mapped in this study corresponds to most previous reports of the location of Dw221. Higgins et\u00a0al.22 also identified QTL for plant height in this region of chromosome 6 with peaks at 44.3\u201344.5\u2009Mb or 42.1\u2009Mb depending on the population and QTL model. The authors suggested that variation in QTL location was due to the linkage between Dw2 and Ma1 since both influence plant height22. In the current study, the influence of Ma1 alleles is minimal because the BTx642 x Tx7000 RIL population is segregating for a weak allele and null allele of Ma1, respectively33, and BTx623 and IS3620c each contain null alleles of Ma119. During the analysis of Dw2 a nearby QTL located at 48.6\u2009Mb on chromosome 6 was identified that modified the length of internode 6 according to single QTL mapping. MQM revealed that this QTL also affected the length of internodes 4\u20137; however, Dw2 had a greater impact on the length of the fourth and fifth internode. This additional QTL could also have confounded the location of Dw2 in the study of Higgins et al.22.Dw2 and Dw3 influence internode length differentially during development. Dw2 had the greatest additive effect on the length of the internode immediately below the peduncle. The additive effects of Dw2 and Dw3 on the length of this internode were similar. The influence of Dw2 gradually decreased in the internodes below the top internode and there was no detectable impact of Dw2 on the length of internodes 7\u20138 below the peduncle in this population. Dw3 had a much greater effect than Dw2 on internodes 2\u20135 below the peduncle with reduced but significant impact on the length of internodes 6\u20138  showed that Dw2 has an effect on the length of all of the ~12 internodes produced by plants grown in the greenhouse during the fall under short day conditions , a gene located near Dw2 on chromosome 6 (40.3\u2009Mb), was also more effective in one of the three populations used in that study18. The reason for differences in recombination efficiency among populations used to map Ma1 and Dw2 is unknown. However, it is possible that sequence divergence or differences in chromatin/DNA methylation in this region of chromosome 6 between parental genotypes affects local recombination frequency. In both studies, the most useful population for fine mapping involved parental lines of the durra race that were crossed to parental lines of the kafir race (BTx642 (durra) xTx7000 (kafir) (Dw2); 100\u2009M (durra) x Blackhull kafir (Ma1)). Larger populations and additional crosses will be required to determine if the association between genetic background and local recombination frequency is significant.Fine mapping narrowed the region encoding Dw2 locus encoded a histone deacetylase that was previously suggested to be a candidate for Dw221. However, the deacetylase did not contain polymorphisms in the coding regions that distinguish the parental genotypes used for fine mapping, or DYM (Dw2) and DDYM (dw2). DDYM was reported to have originated as a shorter mutant in a field of DYM25. Thus, these two yellow milos should be isogenic except at Dw2. All of the other genes in the delimited Dw2 region were sequenced from DYM and DDYM. Only the kinase encoded by Sobic.006G067700 had a polymorphism that distinguished DDYM from DYM in the delimited Dw2 locus. This polymorphism resulted in a frameshift mutation and a premature stop codon in the first exon. This results in a protein of only 190 amino acids instead of 809 amino acids found in DYM. The kinase domain is located between 424\u2013763 amino acids; therefore, the mutant protein found in DDYM would lack kinase activity.One of the ten genes in the delimited 34. Each of these kinases has been shown to regulate auxin efflux transporters, including ABCB1 and PIN1, with PHOT1 and 2 doing so in a blue-light dependent manner35. In Arabidopsis, KIPK has a close homolog, KIPK2  and the closely related kinase, AGC1-836. In sorghum, Dw2 has one closely related homolog, Sobic.008G096200, and these two genes form their own branch on the phylogenetic tree 40. While KIPK did not phosphorylate the N-terminal end of KCBP under experimental conditions, it is possible that it phosphorylates KCBP under other conditions, and it is possible that KCBP transports KIPK within the cell28.In Arabidopsis KIPK was so named due to its interaction with KCBP, a plant-specific kinesin-like calmodulin binding protein that functions in cell division and trichome formation36. Other PERK-genes, such as PERK1, mediate growth inhibition, possibly in response to cell wall signals41. In Arabidopsis, KIPK1 and 2 double mutants did not produce shoot phenotypes although there were differences in root elongation when plants were grown on elevated sucrose36. Different parts of the N-terminal domain of KIPK1 and 2 mediate the direct interactions with KCBP and the various PERKs36. The 423 amino acid N-terminal sequence of Dw2 aligned well with the ~545 amino acid N-terminus of KIPK despite several deletions that account for the difference in overall length of this domain. The sequence similarity of the N-terminal domains of KIPK and Dw2 indicates that Dw2 has likely retained the ability to interact with one or more members of the PERK family. The best BLAST hits to Arabidopsis PERK8 and 10  in sorghum  were expressed in stem internodes (Phytozome). Therefore, it will be of interest to determine if Dw2 interacts with sorghum PERK8 or 10 homologs.Subsequent work has also shown that Arabidopsis KIPK1 and 2 directly interact with members of the proline-rich extensin-like receptor-like kinase (PERK) family, specifically PERK8, 9, 10, and 1338 potential Dw2 interactions with PERKs and KCBP in sorghum could regulate growth and the flow of materials to the cell wall during and after organ elongation. Alternatively, in trichomes KCBP has been found to organize cytoskeleton components37, thus KIPK may be involved cytoskeletal regulation that is associated with cell elongation. This more general coordinating function may explain why Dw2 is expressed in growing zones of leaf blades, leaf sheaths, stems, and panicles. Lack of growth phenotypes in all organs where Dw2 is expressed  could be due to the presence of a second KIPK-like gene in sorghum (Sobic.008G096200). In fact, Sobic.008G096200 is more highly expressed than Dw2 in the roots, and both genes are highly expressed in the panicle, peduncle, and internodes , another important gene in grain sorghum and energy sorghum development18. In addition to its historical significance, a better understanding of Dw2 function may enable the design of improved sorghum crops.Dw2) and Double Dwarf Yellow Milo  were grown to examine the internode length phenotypes caused by the two Dw2 alleles. For each genotype, three plants were individually grown in 3.8-gallon pots (Custom2000) containing Metro Mix 900 (Sun Gro Horticulture) with supplemental fertilizer (Peters 20-20-20) in the greenhouse in short days during the fall. At anthesis, the plants were harvested and the total stem length and length of each internode were measured.The progenitor genotypes Dwarf Yellow Milo . BTx623 is dw1Dw2dw3dw4 and IS3620c is dw1dw2Dw3dw4; therefore, the population segregated for both Dw2 and Dw3. The population (n\u2009=\u2009380) was grown in the greenhouse in the summer of 2013 with natural day lengths. Three plants of each RIL were grown per pot, one pot per line in the same manner as DYM and DDYM. Plants were harvested at grain maturity. For each plant, the total length of the plant (base of the plant to the base of the panicle) and the length of each internode and peduncle were measured. Internodes were numbered from the peduncle. Plants differed for flowering time, with earlier flowering lines producing fewer elongated internodes. As a consequence, the 6th, 7th, and 8th internodes below the peduncle had smaller sample sizes than the other traits measured . Genotyping and genetic map construction (n\u2009=\u2009398) were performed as described in Truong et al.46 except the DG marker sequences were mapped to version 3 of the sorghum reference genome assembly , using BWA47, and INDEL realignment and joint variant calling were performed with the GATK using the naive pipeline of the RIG workflow51. QTL mapping was performed in R/qtl using interval mapping (IM) with 1000 permutations and an \u03b1\u2009=\u20090.0552. Both the genetic map and QTL mapping were performed as an F7 instead of a RIL population due to excess heterozygosity.The BTx623 x IS3620c RIL population was used for mapping 54. Penalties  for all normalized phenotypes were calculated from 25,000 permutations of two-dimensional genome scans using the TIGGS-HPC cluster at Texas A&M; penalties calculated were negligibly different between phenotypes (i.e. same to the tenths place). Significant QTL identified from an initial IM analysis  were used to seed multiple-QTL model selection analysis 54. The best scoring multiple-QTL model from model selection of each phenotype was then merged into a composite multiple-QTL model. The composite multiple-QTL model was generated by merging all overlapping 2-LOD intervals into one QTL and designating the position of the MLOD (maximum LOD) marker as the QTL position55, where loci with an epistatic interaction were merged independently of strictly additive loci.MQM was performed using the same phenotypes, except peduncle length, and genotypes that were used for IM, except the genetic map was thinned to obtain a marker set with at least 1\u2009cM spacing between markers. Also, measurements of the length of each internode, average internode length, and total internode length were normalized using Empirical Quantile Normal Transformation prior to QTL mapping with R/qtldw1dw2dw3dw4 and Tx7000 is dw1Dw2dw3dw4; therefore, the population derived from a cross of these genotypes will segregate for alleles of Dw2. The BTx642 x Tx7000 RIL population (n\u2009=\u200989) was grown in the field in the spring and summer of 2009. It was planted in a Norwood silty clay loam , thermic Typic Udifluvent) in duplicate in a randomized block design at the Texas A&M Research Farm located near Snook, TX on 03/04/2009. The blocks were arrayed in 20 rows 4.6\u2009m long and spaced 76\u2009cm apart with two buffer rows on each end of the block. Each block was offset from the next by approximately 1.5\u2009m. The plants emerged on 08/04/2009 and were thinned to a within-row spacing of 10\u2009cm at 16 days after emergence (DAE). The average daily maximum temperature was 33.3\u2009\u00b0C and the average daily minimum temperature was 21.1\u2009\u00b0C. The population received 24.9\u2009cm of natural rainfall during the growing season with supplemental flood irrigation as needed. The population was harvested on 23/06/2009 (76 DAE), approximately at anthesis for the population. Three plants of each RIL and parental lines from each of two replicates were harvested. For QTL mapping, the average of the two replications was used. Plants were phenotyped for total height, which was measured from the base of the plant to the top of the panicle.BTx642 is 20 using the enzyme NgoMIV to digest genomic DNA. Reads were mapped to the reference genome and variants were processed as described for the BTx623 x IS3620c RIL population. The genetic map was constructed using R/qtl (n\u2009=\u200993) after removing any markers that did not define a recombination breakpoint. QTL mapping was also performed in R/qtl using IM with 1000 permutations and an \u03b1\u2009=\u20090.0552.DNA was extracted from leaf tissue harvested from each RIL and processed using ZR Plant/Seed DNA MiniPrep (Zymo Research). Digital Genotyping (DG) was performed as previously describedDw2. Lines that had recombination breakpoints in or near Dw2 were used to delimit the locus to the extent possible using additional DG genotypes and SNPs identified by Sanger sequencing genes in the region. Primers used for Sanger sequencing are listed in Supplementary Table\u00a0\u00a9 High-Fidelity DNA Polymerase  using the standard conditions. The PCR product was gel purified using QIAquick Gel Extraction Kit (Qiagen) and prepared for capillary sequencing with BigDye\u00a9 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using standard reaction conditions. Sequencing was performed with the ABI 3130xl Genetic Analyzer (Applied Biosystems) and the results were analyzed with Sequencher v4.8 (Gene Codes Corp.).The BTx642 x Tx7000 RIL population was used for fine mapping Dw2 region were grown to confirm stem and internode length phenotypes. Two pots containing two plants from each RIL were grown in two different greenhouses for a total of eight plants per RIL; otherwise the RILs were grown in the same manner as DYM and DDYM. At anthesis, the plants were harvested and the total length of the stem (measured from the base of the plant to the base of the panicle) and the length of each internode were recorded.RILs with recombination breakpoints in the delimited Dw2 was delimited to the extent possible with available genetic resources, the genes in this region were sequenced to search for functional mutations that distinguish DYM (Dw2) from DDYM (dw2). The genes in the Dw2 locus were identified using the sorghum reference genome version 3.1 gene set . The primers for sequencing the genes are listed in Supplementary Table\u00a0Dw2 differentiate the two genotypes25. For Sobic.006G067600 only the exons were sequenced, for all other genes, the entire gene was sequenced. Sobic.006G067700 was further sequenced in the other important breeding lines to examine the distribution and extent of allelic variation in Dw2.Once the region encoding Dw2. Tx7000 and BTx642 seeds were obtained from Dr. W.L. Rooney . IS3620c seed (PI 659986 MAP) was obtained from the USDA-ARS Plant Genetic Resources Conservation Unit . Seeds were soaked in 20% bleach for 20\u2009minutes and washed extensively in distilled water for one hour. Seeds were germinated on water-saturated germination paper in a growth chamber . Genomic DNA was isolated from 8-day old root tissue using a FastPrep DNA Extraction kit and FastPrep24 Instrument , according to the manufacturer\u2019s specifications. DNA template (350\u2009bp average insert size) was prepared using a TruSeq\u00ae DNA PCR-Free LT Kit, according to the manufacturer\u2019s directions. Paired-end sequencing (125\u2009\u00d7\u2009125 bases) was performed on an Illumina HiSeq2500. Sequence reads were mapped to version 3 of the sorghum reference genome assembly , using BWA v0.7.1247. Base quality score recalibration, INDEL realignment, duplicate removal, joint variant calling, and variant quality score recalibration were performed using GATK v3.3 with the RIG workflow51. Whole genome sequence of Tx7000, BTx6424, and IS3620c are available at the Sequence Read Archive (www.ncbi.nlm.nih.gov/sra).Whole genome sequencing was used to identify polymorphisms that distinguish the parents of the two populations used to map 56. The sequences were aligned using the MUSCLE algorithm58. The tree was estimated using maximum likelihood with the substitution model developed by Le & Gascuel59 and the Gamma distribution. To estimate the reliability of the branches, 1000 boostraps were performed. Protein alignments were performed in Jalview v2.060 using the TCoffee algorithm61 with defaults.Each of the AGCVIII proteins in Arabidopsis was aligned with the sorghum genome using BLAST and the best hits were recorded. The resulting sorghum AGCVIII protein family was used to make a phylogenetic tree in MEGA6Supplementary InformationSupplementary Table S4Supplementary Table S5Supplementary Table S7"}
+{"text": "A pepducin is a lipopeptide containing a peptide sequence that is identical to one of the intracellular domains of the G-protein coupled receptor (GPCR) assumed to be the target. Neutrophils express two closely related formyl peptide receptors belonging to the family of GPCRs; FPR1 and FPR2 in human and their respective orthologue Fpr1 and Fpr2 in mouse. By applying the pepducin concept, we have earlier identified FPR2 activating pepducins generated from the third intracellular loop of FPR2. The third intracellular loop of FPR2 differs in two amino acids from that of FPR1, seven from Fpr2 and three from Fpr1. Despite this, we found that pepducins generated from FPR1, FPR2, Fpr1 and Fpr2 all targeted FPR2 in human neutrophils and Fpr2 in mouse, but with different modulating outcomes. Whereas the FPR1/Fpr1 derived pepducins inhibited the FPR2 function in human neutrophils, they activated Fpr2 in mouse. The FPR2 derived pepducin activated FPR2/Fpr2, whereas the pepducin generated from Fpr2 inhibited both FPR2 and Fpr2. In summary, our data demonstrate that pepducins generated from the third intracellular loop of human FPR1/2 and mouse Fpr1/2, all targeted FPR2 in human and Fpr2 in mouse. With respect to the modulating outcomes, pepducin inhibitors identified for FPR2 are in fact activators for Fpr2 in mouse neutrophils. Our data thus questions the validity of pepducin concept regarding their receptor selectivity but supports the notion that FPR2/Fpr2 may recognize a lipopeptide molecular pattern, and highlight the differences in ligand recognition profile between FPR2 and its mouse orthologue Fpr2. The members of the formyl peptide receptor (FPR) family expressed by neutrophil phagocytes belong to the large group of G-protein coupled receptors (GPCRs) and play key roles in proper recruitment and activation of neutrophils at sites of infection/inflammation \u20133. NeutrStaphylococcus aureus bacteria that potently and selectively activate Fpr1 (fMIFL) and Fpr2 (PSM\u03b12), respectively [10 being prominent examples [When looking at the FPRs in different species it is clear that the receptor family has a complex evolutionary history, as illustrated by the fact that the number of genes in the family vary markedly in mouse and man . Human pectively . We alsoexamples . We haveexamples , 14. Thith century, a unique class of lipopeptide ligands (pepducins) was introduced. The proposed concept for interaction suggested that pepducins modulate receptor signaling through a direct interaction between the peptide part of the pepducins and intracellular signaling active parts of the targeted receptors [2R) all have in common that they modulate FPR2 function [When searching for new mechanistic concepts for allosteric modulation of GPCRs in the early 21eceptors \u201317. Pepdeceptors \u201318. The eceptors . Over theceptors \u201322. It sfunction \u201325. The function . Even th16) and FPR2  on mouse neutrophils [16) and Fpr2  on mouse and human neutrophils were also investigated. We show that the FPR2 derived pepducin activated both FPR2 and Fpr2, whereas the corresponding Fpr2 pepducin potently inhibited FPR2/Fpr2. The FPR1- as well as the Fpr1-derived pepducins interacted with FPR2 and Fpr2, but the outcome of this interaction differed as they activated Fpr2 but inhibited FPR2 function. Taken together, we describe pepducins that inhibit FPR2 activity in one species activate the receptor orthologue in another species, and that all the modulating pepducins studied selectively targeted FPR2/Fpr2 irrespectively from which the receptor the peptide sequence was derived. Although our results disagree with the proposed molecular mechanism for how pepducins achieve their receptor specificity, this group of FPR2 selective ligands clearly serve as excellent tools for further studies of FPR2/Fpr2 functions.In this study, we applied the pepducin approach in an attempt to gain more insights into similarities and differences in the ligand recognition profiles of the human neutrophil FPRs and their mouse orthologous. We have determined the effects of two earlier described human FPR pepducins generated from the third intracellular loop of FPR1 . The pepducins were synthesized by Fmoc solid-phase peptide synthesis and the fatty acid were N-terminally linked on the resin as the last step before deprotection of side chains, followed by HPLC purification on a C18 column and further verification by MALTI-TOF Mass Spectrometry. All peptides and pepducins were dissolved in dimethyl sulfoxide to a concentration of 10 mM and stored at -80\u00b0C until use. Further dilutions were made in Krebs-Ringer phosphate buffer that was supplemented with glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) .Percoll was obtained from Amersham Pharmacia . Dextran and Ficoll-Paque were obtained from GE-Healthcare Bio-Science . The peptides were synthesized and purified by high-pressure liquid chromatography (HPLC) by Alta Bioscience . Cyclosporin H (CysH) was kindly provided by Novartis Pharma  and the peptidomimetic activator F2M2 and inhibitor were kindly provided by Henrik Franzyk . Boc-FLFLF (Boc2) and isoluminol were purchased from Sigma Chemical Co. . The phenol-soluble modulin  was obtained in its N-formylated form from EMC . Recombinant murine tumor necrosis factor alpha (TNF-\u03b1) was from R&D Systems Europe Ltd. . Horseradish peroxidase (HRP) was from Boehringer Mannheim . Pepducins and the FPR2 inhibitor PBP\u2212/\u2212 mice generated as described previously [ad libitum, at the Department of Rheumatology and Inflammation Research, University of Gothenburg. Sex matched WT and Fpr2-/- animals (age 8\u201310 weeks) were used to isolated bone marrow derived neutrophils. The studies performed were approved by the Ethical Committee for Animal Experimentation, G\u00f6teborg, Sweden.C57BL/6 wild-type mice were purchased from Charles River Laboratories and Fpr2eviously , were geHuman peripheral blood neutrophils were isolated from buffy coats from healthy blood donors using dextran sedimentation and Ficoll-Paque gradient centrifugation as described . The rem2+ and Mg2+ (KRG-) was forced through the bone by using a 1-mL syringe with a 27-gauge needle. After dispersing cell clumps and removing the debris, the bone marrow granulocytes were isolated according to an earlier described procedure [Mice (8\u201312 weeks of age) were killed by cervical dislocation, the femurs and tibias were removed and freed of soft tissue attachments, and the extreme distal tip of each extremity was cut off. KRG without Carocedure  with som-6) as a function of time (min). The activity before stimulus addition was recorded as basal line and this activity was not changed in vehicle control samples. Since the FPR/Fpr-mediated superoxide production with respect to the onset and decline are very similar, the peak activities were used to compare the responses induced.NADPH-oxidase activity was determined using an isoluminol and HRP-enhanced chemiluminescence (CL) system that allow for the determination of superoxide production , 30. TheNa\u00efve bone marrow cells produce very low levels of superoxide upon stimulation and to increase this production the cells were routinely primed for one hour at RT followed by TNF-\u03b1  priming for a period of 20 min at 37\u00b0C , allowin50/IC50 values were determined from experiments in which the activities were normalized to the response induced by maximal response or induced by agonist in the absence of inhibitor. The statistical analysis was performed on raw data using Student\u2019s t-test when two groups were compared or one-way ANOVA with Dunnett\u2019s multiple comparisons test in comparison to the control response when three groups were compared. * p \u2264 0.05 was considered significant.Data analysis was performed using Graph Pad Prism 7.0. The EC16, Pal-KIHKKGMIKSSRPLRV), with a peptide sequence identical to the entire third intracellular loop of FPR2 (spanning from amino acid K227 to V242), activates human neutrophils  and PSM\u03b12 (Fpr2) derived from S.aureus  rather than Fpr2  mice and the recently described selective ligands for mouse Fprs [16-induced response was significantly lower in neutrophils isolated from Fpr2-/- mice compared to that from WT cells (16. This suggestion gained further support from receptor desensitization experiments using the Fpr2 selective agonist PSM\u03b12 [16 (16 response in WT cells was significantly reduced by the earlier described Fpr2 selective peptidomimetic inhibitor (Lau-(Lys-\u03b2NSpe)6-NH2) but not the Fpr1 antagonist Boc2 . No activation of mouse neutrophils was obtained using mF2Pal16, however, since pepducins may negatively modulate the targeted receptor, we examined the effects also in \"inhibitory mode experiments\". Our data show that mF2Pal16 potently inhibited the response induced by the Fpr2 specific agonist PSM\u03b12 with an IC50 ~ 30 nM  interacts with FPR2 and inhibits this receptor specifically [16 inhibit the response induced by the FPR2 agonist WKYMVM  is obviously not critical for pepducin action as the two pepducins induced very similar activities but with somewhat different potency. Human neutrophils were not activated by mF1Pal16  but not the Fpr1 inhibitor Boc2 .The mF1Pal F1Pal16 . Similar/- cells . Receptotrophils . In additor Boc2 . Very siThe formyl peptide receptors play important roles in host defense and as immune regulator , 2, suggth century as a novel concept for allosteric modulation of GPCR signaling [Pepducins with a peptide sequence identical to one of the intracellular domains or cytoplasmic tail of a GPCR were introduced in the early 21ignaling , 16, 33.ignaling . The sugignaling . Accordiignaling , 25. Sevignaling . Neverthignaling .16) targets this receptor  indicates that there is no fundamental difference in signaling profile between human and mouse derived peptides, as these two pepducins are almost identical (one amino acid difference) and they have the same effects, i.e., they negatively target human FPR2 and positively target the mouse Fpr2. This suggests that their effects can only be explained by the differences between FPR2 and Fpr2 across species. More importantly, they don\u2019t target the receptor FPR1/Fpr1 predicted by the pepducin specificity concept. Instead, our data strongly suggests that some specific motif in FPR2/Fpr2 may recognize a lipopeptide pattern, as suggested by the fact that this receptor seems to be the primary target for pepducins derived also from other, sometimes totally unrelated, GPCRs [In agreement with the pepducin concept regarding origin and receptor specificity, the pepducin derived from the third intracellular loop of FPR2 targets FPR2, and the one derived from Fpr2  in the FPR2 pepducins is of prime importance for FPR2 activation; the K5 residue could be replaced by an R  without any activity major change, but when replaced by Q  the agonistic effect is completely lost [-/- cells strongly suggests that Fpr1 is not targeted by these pepducins. Although according to the pepducin concept an extracellular receptor antagonist, including Boc2, should not affect the pepducin activity, our earlier studies have demonstrated that this rule does not apply to FPR2 activating pepducins [All activating pepducins examined in this study target not only FPR2 in human neutrophils but also the mouse neutrophil Fpr2. The C-terminus in these pepducins contain 6 amino acids that are identical in all of them, and our data from this and earlier published studies show that this part is of prime importance for activity. This is illustrated by the fact that upon a removal of this part from F1Palon Fpr2  as important regulators of the physiological processes leading to resolution of inflammation [Fpr genes in mice and humans and an incomplete understanding of the evolutionary relationship between human FPRs and their animal counterparts, also the fine-tuning of receptor ligands has obvious implications for how the results obtained should be interpreted [16 or mF1Pal16) can act as an agonist for Fpr2, and our data demonstrate new agonistic and antagonistic pepducins that might be useful tools for future translational studies in mouse models. These two FPR1/Fpr1-derived pepducins are almost identical in sequence (one amino acid difference) and exert similar effects, i.e., they both negatively target human FPR2 and positively target the mouse Fpr2. This suggests that there is no pharmacological profile difference between the two pepducins and the difference must be due to differences between the human and mouse receptors. More importantly, our data puts the pepducin specificity concept into question as these two pepducins don\u2019t target FPR1/Fpr1 as expected. Availability of these well-characterized cross-species ligands is important to advance mouse disease models aimed at understanding the role of human FPRs in immune defense and inflammation. Our data reveal clearly that even small structural variations may abolish cross-species ligand recognition and this holds true for both agonists and antagonists. Cross-species receptor ligands for members of the FPR family, possessing a high degree of receptor-selectivity, are required for translation of activation/inhibition experiments performed with different animal disease models. Using mouse models for innate immune research always raises questions about species-specific differences that have to be considered when extrapolating results from the models to human diseases, but also there are reports describing differences in neutrophil biology between species. For example, mouse neutrophils do not produce defensins, an important group of microbial peptides produced by human neutrophils [Genetically modified mouse strains and mouse disease models provide excellent tools to study the function of neutrophil FPRs ammation . Howeverammation . In addierpreted . A largeerpreted . We now trophils .in vivo studies related to the pathophysiologic functions of FPR2. With its complexity in ligand binding and signaling modulation FPR2 provides an excellent model receptor for our understanding of GPCR signaling and modulation in general, and the potent and unique FPR2/Fpr2 modulating pepducins presented in this study could be excellent tools in future mechanistic and functional studies.In conclusion, we have identified both activating and inhibiting pepducins for mouse neutrophils and these pepducins display a receptor preference for Fpr2. Our data also highlight differences in ligand recognition between FPR2 and Fpr2 that have to be taken into account when choosing receptor specific ligands for translational studies across species. A better understanding of Fpr2 pharmacology is needed and will facilitate S1 Fig4 cells) were pre-incubated without (solid line) or with mF2Pal16 (250 nM) for 5 min before stimulation with the pepducin F1Pal16  and the release of superoxide anions was continuously measured. One representative experiment out of three performed with individual buffy coats is shown.WT mouse neutrophils (5 x 10(TIF)Click here for additional data file.S2 Fig5 cells) were pre-incubated with HRP and isoluminol followed by stimulation with the FPR2 specific agonist WKYMVM  or the pepducin mF1Pal16 . Arrow indicates the addition. The release of superoxide anions was continuously measured. One representative experiment out of five independent experiments performed with individual buffy coats is shown.Human neutrophils (10(TIF)Click here for additional data file."}
+{"text": "Gallus gallus). Broiler breeder hens were exposed to two maternal temperatures (21\u00b0C and 32\u00b0C) \u00d7 three maternal dietary zinc treatments  for 8 weeks. Maternal hyperthermia increased the embryonic mortality and induced oxidative damage evidenced by the elevated mRNA expressions of heat shock protein genes. Maternal dietary zinc deficiency damaged the embryonic development associated with the global DNA hypomethylation and histone 3 lysine 9 hyperacetylation in the embryonic liver. Supplementation of zinc in maternal diets effectively eliminated the embryonic mortality induced by maternal hyperthermia and enhanced antioxidant ability with the increased mRNA and protein expressions of metallothionein IV in the embryonic liver. The increased metallothionein IV mRNA expression was due to the reduced DNA methylation and increased histone 3 lysine 9 acetylation of the metallothionein IV promoter regardless of zinc source. These data demonstrate that maternal dietary zinc addition as an epigenetic modifier could protect the offspring embryonic development against maternal heat stress via enhancing the epigenetic-activated antioxidant ability.The role of maternal dietary zinc supplementation in protecting the embryos from maternal hyperthermia-induced negative effects via epigenetic mechanisms was examined using an avian model ( Maternal heat stress impairs the fetal and embryonic development in mammalian and avian species , 2. MateMT mRNA expression  \u00d7 3  factorial arrangement of treatments was adopted. Thus, a total of 6 treatment groups  were included. All eggs collected from the above 6 maternal treatments were incubated for 21 d.This study with all experimental procedures was approved by Animal Welfare Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences. A total of 144 18-wk-old female broiler breeders  were randomly allotted to 1 of 6 treatments with 6 replicates (4 birds per replicate) for each treatment according to the experimental design. All broiler breeder hens were initially adapted from 18 to 29 wk of age using a conventional corn-soybean meal diet. The body Zn stores were deleted for all the birds from 30 to 32 wk of age using a corn starch-isolated soybean protein purified diet without Zn addition. After that, the birds in 3 dietary Zn treatments from the NT group were housed at 21 \u00b1 1\u00b0C, while those in 3 dietary Zn treatments from the HT group were reared at 32 \u00b1 1\u00b0C for the experimental period from 33 to 42 wk of age. All hens were provided free access to tap water and the experimental diets. Semen was collected from male broiler breeders from 27 to 29 wk of age and mixed, and the volume and numbers of semen and sperm motility were determined before insemination. Artificial insemination was performed for twice a week.4\u00b77H2O) and the Zn proteinate with a moderate chelation strength  \u00d7100.The MeDIP analysis for gene promoter was performed as previously described  with sompG sites . The relMT4 by real-time PCR with the same specific primers as described in MeDIP analysis. ChIP with antibodies against AcH3K9, nonspecific IgG as a negative control (NC) and blank control (BC) was used to determine the fidelity of the ChIP protocol (GAPDH promoter region. The fold enrichment of each target sequence was determined using the following formula [fold enrichment=2\u2212(Ct IpAcH3K9\u2013Ct input)] [The preparation of sonicated chromatin from the embryonic liver tissue samples was performed according to previous publication . The cruprotocol . The qPC input)] .P \u2264 0.05 and at 0.05 < P < 0.10 for a trend.Data were analysed by 2-way ANOVA using the general linear model procedure of the SAS 9.2 , and the model included the main effects of maternal environmental temperature, maternal dietary Zn, and their interaction, and treatment comparisons for significant differences were tested by the LSD method. Embryonic mortality data were transformed to arcsine values before statistical analysis. Each replicate served as the experimental unit for all statistical analyses. Significant differences were set at"}
+{"text": "Thus far, these oxygenation and metabolic parameters have been measured independently by different techniques in separate animals, precluding a comprehensive and correlative assessment of retinal oxygenation and metabolism dynamics. The purpose of the current study is to report an innovative optical system for dual oxyphor phosphorescence lifetime imaging to near-simultaneously measure retinal vascular PO2 and tPO2 in rats. The use of a new oxyphor with different spectral characteristics allowed differentiation of phosphorescence signals from the retinal vasculature and tissue. Concurrent measurements of retinal arterial and venous PO2, tPO2 through the retinal depth, inner retinal OEF, and outer retinal QO2 were demonstrated, permitting a correlative assessment of retinal oxygenation and metabolism. Future application of this method can be used to investigate the relations among retinal oxygen content, extraction and metabolism under pathologic conditions and thus advance knowledge of retinal hypoxia pathophysiology.The retina requires adequate oxygenation to maintain cellular metabolism and visual function. Inner retinal oxygen metabolism is directly related to retinal vascular oxygen tension (PO Furthermore, inadequate oxygen availability can lead to hypoxic injury and eventual cell death. Therefore, assessment of retinal oxygenation is essential to improve knowledge of disease pathophysiology and advance treatments that target alleviation of hypoxia-induced pathologies.The retinal tissue requires an adequate supply of oxygen for cellular metabolism and function. Retinal ischemia due to reduced blood flow has been implicated in the development of vision threatening pathologies such as neovascularization and macular edema2) has been performed by spectrophotometry5, photoacoustic ophthalmoscopy6, and visible optical coherence tomography8. Additionally, retinal vascular oxygen tension (PO2) has been reported using phosphorescence lifetime imaging11. Direct depth-resolved measurements of retinal tissue oxygen tension (tPO2) have been provided by the oxygen-sensitive microelectrode technique at single point locations14 and by phosphorescence lifetime imaging at multiple contiguous locations15. Furthermore, information about the metabolic activity of the retinal tissue has become available by calculation of inner retinal oxygen extraction fraction (OEF) based on retinal vascular oxygen content16 and by estimation of outer retinal oxygen consumption (QO2) from retinal tPO2 depth profiles18.Several techniques have become available for quantitative assessment of oxygen content within the retinal vasculature or tissue. Specifically, retinal oximetry for measurement of hemoglobin oxygen saturation  was administered intravenously (20\u2009mg/kg) for retinal vascular PO2 imaging. Before imaging, pupils were dilated with 2.5% phenylephrine and 1% tropicamide, and a glass cover slip with 1% hydroxypropyl methylcellulose was placed on the cornea to minimize corneal refractive power and prevent dehydration. Rats were placed on an animal holder with a closed-loop water heater to maintain body temperature at 37\u2009\u00b0C and were spontaneously breathing during imaging. One eye of each rat was imaged in temporal or nasal regions within three-disk diameters (600 microns) from the edge of the optic nerve head.All experimental procedures were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Care Committee of the University of Illinois at Chicago. The study was performed in 10 Long Evans pigmented rats. Rats were anesthetized with intraperitoneal injections of ketamine (100\u2009mg/kg) and xylazine (5\u2009mg/kg) and additional injections were given as required to maintain anesthesia. One day prior to imaging, oxyphor G2  was constituted at 1.5\u2009\u03bcM and administered as a 5\u2009\u03bcL intravitreal bolus injection for retinal tPO15 for either retinal vascular PO2 or tPO2 measurement was modified for near-simultaneous measurement of both parameters by dual oxyphor phosphorescence lifetime imaging  and three repeated phase-delayed optical section phosphorescence images from the retinal vasculature were acquired at exactly the same retinal location. The total time for image acquisition was less than 60\u2009seconds.2 in major retinal arteries and veins was measured from the phosphorescence lifetime using the Stern-Volmer equation and the R0 oxyphor\u2019s quenching constant and lifetime in a zero-oxygen environment, as previously described20. PO2 in each blood vessel was calculated by averaging three repeated measurements. A mean arterial and venous PO2 (PO2A and PO2V) was determined for each rat by averaging measurements in each vessel type. Inner retinal OEF was calculated from PO2A and PO2V and using the oxyhemoglobin dissociation curve in rat21, as previously described16. Inner retinal OEF is the fraction of oxygen supplied by the retinal circulation that is extracted by the inner retinal tissue for metabolism, or equivalently, the ratio of inner retinal oxygen metabolism to inner retinal oxygen delivery.Vascular PO2 image was generated from the phosphorescence lifetime measured at each pixel using the Stern-Volmer equation and the G2 oxyphor\u2019s quenching constant and lifetime in a zero-oxygen environment22, as previously described15. A mean depth-resolved retinal tPO2 image was generated from three repeated images.Retinal tissue optical section phosphorescence images were first processed to minimize retinal curvature, then smoothed in vertical (y-axis) and axial (z-axis) dimensions using a 2D anisotropic averaging filter (6\u2009\u00d7\u20094 pixels), corresponding to 18 \u03bcm in both axes. A depth-resolved tPO2 image (superior to inferior), 35 contiguous tPO2 depth profiles were generated by vertically averaging tPO2 over 10 pixels (30 microns) and plotting tPO2 as a function of retinal depth, as previously described15. The outer and inner retina were defined as 50% to 100% and 0% to 50% of the retinal depth, respectively. Maximum outer retinal tPO2, minimum outer retinal tPO2, and mean inner retinal tPO2 were calculated from each tPO2 depth profile. Mean inner retinal tPO2 was plotted along the vertical dimension of the image to demonstrate changes in this parameter with respect to retinal arteries and veins. Furthermore, mean values for each parameter were calculated from all profiles along the tPO2 image.Along the vertical dimension of each retinal tPO2 depth profile, outer retinal QO2 was calculated by fitting a three-layer, one-dimensional, steady-state oxygen diffusion model using a non-linear least squares technique, as previously described23. In this model, oxygen diffuses in one dimension from the choroidal circulation across the outer retinal depth, which is divided into three layers14, based on their oxygen consumption properties. QO2 has been shown to be negligible in layers 1 and 323, which correspond to the photoreceptor outer segments and outer nuclear layer, respectively. In contrast, oxygen is consumed in layer 2 by mitochondria of the photoreceptor inner segments, yielding a quadratic relationship between tPO2 and retinal depth. A mean outer retinal QO2 was calculated by averaging measurements obtained from all tPO2 depth profiles along the vertical dimension of the tPO2 image.From each retinal tPOThe data generated are available from the corresponding author on reasonable request.2 and tPO2 image obtained at the same location overlaid on the retinal reflectance image is shown in Fig.\u00a02 image displays measurements in two arteries and one vein, demonstrating higher PO2A than PO2V, as expected. The tPO2 image shows higher tPO2 near the chorioretinal interface compared to inner retina. Mean inner retinal tPO2 plotted along the vertical dimension (superior to inferior) is shown in Fig.\u00a02 near arteries than the vein.A representative example of a retinal vascular PO2 and tPO2 measurements in all rats are presented in Fig.\u00a02A and PO2V were 41\u2009\u00b1\u20095\u2009mmHg and 25\u2009\u00b1\u20094\u2009mmHg, respectively . Inner retinal tPO2, minimum outer retinal PO2, maximum outer retinal PO2 were 23\u2009\u00b1\u20097 mm Hg, 22\u2009\u00b1\u20095 mm Hg and 33\u2009\u00b1\u20098 mm Hg, respectively. There was no significant difference between PO2V and mean inner retinal tPO2 (P\u2009=\u20090.4). Inner retinal OEF was 0.58\u2009\u00b1\u20090.11 and outer retinal QO2 was 0.57\u2009\u00b1\u20090.27 mLO2/100\u2009g*min. There was no significant correlation between inner retinal OEF and mean inner retinal tPO2 . As demonstrated in Fig.\u00a02 and maximum outer retina tPO2 .Compiled retinal vascular PO2 and tPO2 was demonstrated by phosphorescence lifetime imaging of dual oxyphors delivered intravenously and intravitreally. For the first time, concurrent assessment of oxygen content across the retinal depth and oxygen metabolism metrics of the inner and outer retina was demonstrated. Future application of this method can provide knowledge of relations among these parameters under experimental pathologic conditions and thus yield a comprehensive understanding of retinal oxygenation and metabolism dynamics.A novel optical imaging method for near-simultaneous imaging of retinal vascular PO2A and PO2V were in general agreement with those from previous studies25. Depth-resolved retinal tPO2 images displayed maximum tPO2 near the choroid, consistent with normal physiology. Inner retinal tPO2 measurements using the G2 oxyphor were similar to our previously published data with the R2 oxyphor15 and those reported by the oxygen microelectrode technique14. Alterations in inner retinal tPO2 were consistent with the nearby presence of arteries and veins. Inner retinal OEF was 0.58, \ufeffon average, indicating 58% of the oxygen delivered by the retinal circulation was extracted for metabolism by the inner retinal tissue. The mean inner retinal OEF was higher as compared to our previous study16 and maximum outer retinal tPO2 in the current study was slightly lower than previous reports15. Both of these results are consistent with the presence of reduced systemic oxygenation26, which likely resulted from the effect of anesthesia while the rats were under spontaneous breathing conditions27. Furthermore, the variability of tPO2 adjacent to the chorioretinal interface along the tPO2 image may, at least in part, be attributed to presumed anesthesia-induced systemic hypotension. This may have resulted in reduced choroidal blood flow, thus altering tPO2 gradients within the retinal depth. Overall, the results demonstrate the ability of the optical imaging system to measure retinal vascular PO2, tPO2 and inner retinal OEF.Measurements of retinal PO2 obtained in the current study were similar to our previously reported values17, but lower than those obtained by the oxygen microelectrode technique in light-adapted rats18. This difference is likely due to intraretinal phosphorescence scattering which decreases the depth resolution, minimizes the curvature of tPO2 profiles and calculated values of QO2. Nevertheless, outer retinal QO2 was significantly associated with maximum outer retinal tPO2, in agreement with a previous study14, though a small portion of oxygen utilized by the photoreceptors is supplied by the retinal vasculature14. This result suggests a dependence of the photoreceptor metabolic function on the level of oxygen supplied by the choroidal circulation.Measurements of outer retinal QO2 and tPO2 coupled with derivation of inner retina OEF and outer retinal QO2 can advance knowledge of retinal oxygen dynamics. For example, we demonstrated here, for the first time, that there was no significant correlation between inner retinal OEF and mean inner retinal tPO2 under healthy condition. This result suggests that despite physiological variations, tPO2 is well-maintained due to compensatory alterations in OEF and oxygen delivery, and implies the presence of highly effective regulatory mechanisms. Previous studies have shown alterations in OEF under experimental hypoxia in rats16. In future studies, by relating inner retinal tPO2 with OEF under graded hypoxia/ischemia, diabetes or light flicker stimulation, the OEF threshold necessary to sustain tPO2 may be identified. Additionally, data obtained under graded levels of ischemia may be used to establish a relationship between inner retinal tPO2 and PO2V. Furthermore, combined retinal blood flow measurements with the current method can be used to determine the inner retinal oxygen delivery threshold necessary to maintain retinal tPO2 with direct relevance to retinal ischemic diseases. These thresholds can only be accurately determined with concurrent retinal vascular PO2 and tPO2 measurements in the same animal.Simultaneous measurements of retinal vascular PO2 were obtained from literature and may be different within the retinal tissue environment. Phototoxicity may affect data derived from images acquired repeatedly at the same location with R0 oxyphor28, but any effect of phototoxicity on measurements derived using the G2 oxyphor is not known. Although, the laser irradiance was below the threshold for tissue damage, repeatability of measurements obtained at the same location with the use of dual oxyphors will need further evaluation. The feasibility of the method was demonstrated in a limited number of rats and future studies using larger sample sizes are needed to fully establish the utility of the method.One potential limitation of this method is the scattering of phosphorescence light within the retina and vitreous which can degrade image quality. However, compared to the R2 oxyphor, less scattering is expected from the G2 oxyphor due to its longer phosphorescence emission wavelength. The quenching constants used to calculate POIn conclusion, for the first time, near-simultaneous measurement of retinal vascular and tissue oxygen tension by phosphorescence lifetime imaging was demonstrated. Future application of this method under challenged physiologic or pathologic conditions permits correlation of retinal vascular and tissue oxygen content and can potentially elucidate retinal oxygenation dynamics."}
+{"text": "Porcine circovirus type 2 (PCV2) diagnostics in live pigs often involves pooled serum and/or oral fluid samples for group-level determination of viral load by quantitative real-time polymerase chain reaction (qPCR). The purpose of the study was to compare the PCV2 viral load determined by qPCR of paired samples at the pen level of pools of sera (SP) from 4 to 5 pigs and the collective oral fluid (OF) from around 30 pigs corresponding to one rope put in the same pen. Pigs in pens of 2 finishing herds were sampled by cross-sectional (Herd 1) and cross-sectional with follow-up (Herd 2) study designs. In Herd 1, 50 sample pairs consisting of SP from 4 to 5 pigs and OF from around 23 pigs were collected. In Herd 2, 65 sample pairs consisting of 4 (SP) and around 30 (OF) pigs were collected 4 times at 3-week intervals.A higher proportion of PCV2-positive pens (86% vs. 80% and 100% vs. 91%) and higher viral loads (mean difference: 2.10 and 1.83 log(10) PCV2 copies per ml) were found in OF versus SP in both herds. The OF cut-off value corresponding to a positive SP (>3 log(10) PCV2 copies per ml) was estimated to 6.5 and 7.36 log(10) PCV2 copies per ml for Herds 1 and 2, respectively. Significant correlations between SP and OF results were found in Herd 1 (rho\u2009=\u20090.69) and the first sampling in Herd 2 (rho\u2009=\u20090.39), but not for the subsequent consecutive 3 samplings in Herd 2.The proportion and viral loads of PCV2 positive pens were higher in collective OF (including up to 30 pigs) compared to SP (including 4\u20135 pigs) of the same pens. Also, OF seemed to detect the PCV2 infection earlier with OF values just below 6.5 (Herd 1) and 7.36 (Herd 2) log(10) being associated with a negative SP for the same pen. Nevertheless, a statistically significant correlation between SP and OF could not be found for all sampling time points, probably due to a high within-pen variation in individual pig viral load becoming very evident in SP of only four or five pigs. Consequently, the results imply that OF is well suited for detecting presence of PCV2 but less so for determining the specific viral load of pigs in a pen. Porcine circovirus type 2 (PCV2), a circular, single-stranded, non-enveloped DNA virus has been demonstrated to be present in almost all commercial swine herds worldwide , 2. PCV2Vaccination provides an effective tool to control PCV2 infections \u201317 but iHaemophilus parasuis and Streptococcus suis has since been demonstrated [In the last 10\u00a0years, oral fluid sampling has gained growing interest as an even more cost and time-saving method while also considering animal welfare. The sampling procedure consists of hanging a cotton rope in a pen for the pigs to chew on, followed by wringing of the rope to release the oral fluid. The method was initially described in 2008 , 20, andnstrated . Also fonstrated , 24\u201326.r\u2009=\u20090.76) between viral loads in the two sample types [r\u2009=\u20090.78) between viral loads in oral fluid (20\u201330 pigs per pen) and pooled serum (5 pigs) was found to be significant in another study [Some previous studies have compared detection and load of PCV2 by PCR in serum and oral fluid. A fair agreement between individual PCV2-positive serum and oral fluid samples (kappa\u2009=\u20090.24) but a poor agreement between pooled serum and oral fluid collected from pen-housed pigs  has been found . Anotherle types . A similer study .The inconclusive and limited number of studies regarding the association between viral loads in pooled serum and oral fluid at pen level and possible differences between PCR-assays require further elucidation by including additional herds/samples for the comparison. Moreover, since both oral fluid and pooled serum sampling are done with the purpose of diagnosing PCV2 infection in a group of pigs, determination of the oral fluid viral load corresponding to a positive serum pool is relevant for practical validation of oral fluid as a substitute for serum pools. Consequently, the objectives of this study were to: 1) determine the oral fluid viral load cut-off agreeing best with a PCV2-positive serum pool and 2) compare viral loads of PCV2 in serum pools and collective oral fluid from pigs in the same pen.Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae type 2\u2009+\u20096\u2009+\u200912 and PRRSv) and samples were collected between September 2014 and July 2015 as a part of a larger field trial. None of the herds vaccinated against PCV2 prior to initiation of sampling, but in the field trial in Herd 2, half of the finishers were vaccinated during sample collection as a part of a PCV2 vaccine trial. However, only PCV2-qPCR results from the non-vaccinated group were included in the present study and an overview of the serum results have been presented briefly elsewhere [Serum and oral fluid samples were collected in 2 PCV2-infected finishing herds (Herd 1 and 2). Neither of the herds experienced clinical signs attributable to PCV2 infection  . Herd 1 lsewhere .At the time of sampling in Herd 1 (August 2010), no previous studies had estimated the correlation coefficient between serum pools and oral fluid samples and the interest of the study was therefore to determine whether or not a correlation existed. Therefore, the sample size calculation was based on detecting a correlation coefficient equal to or higher than 0.4 at a significance level of 95% and a power of 0.8 corresponding to a sample size of 47 . In HerdThe study design in Herd 1 was cross-sectional with all samples collected from pigs of 3 different age groups in one day. The study design in Herd 2 was cross-sectional with follow-up consisting of totally 4 repeated samplings of the same pigs/pens at 3-week intervals. Serum and oral fluid were collected simultaneously at each sampling.th pig in the pen depending on the number of pigs per pen, assuring that 5 or 4 pigs per pen were selected in Herd 1 and 2, respectively. In Herd 2, the same 4 pigs per pen were bled at the 4 consecutive sampling time points .The study unit was the pen. Herd 1 had a total of 64 pens of which 50 were randomly (with age-stratification) selected for sampling . In HerdBlood samples were collected from the cranial vena cava in plain tubes. In Herd 1, the blood samples were transported directly to the laboratory after sampling and refrigerated overnight. In Herd 2, samples were kept refrigerated overnight and were shipped to the laboratory by mail the following day.At the laboratory, blood samples were centrifuged to separate serum. Equal amounts of serum from each of the 4/5 pigs per pen were pooled prior to PCV2-qPCR analysis, resulting in one serum PCV2 copy number  per pen. Thus, serum viral loads refer to qPCR-results from pooled serum samples.Sampling of oral fluid was performed as previously described . BrieflyThe oral fluids collected in both herds were grey and dirty in appearance, probably reflecting fecal contamination. Feces as well as saliva can contain PCR inhibitors \u201336 and i1. DNA extraction from serum from Herd 1 was performed differentlyDNA extraction from all oral fluid samples and the serum samples from Herd 2 was performed with a commercially available extraction kit1 to avoid false negative test results. Oral fluid viral loads were subsequently corrected according to the extra dilutions. The qPCR-assay had a detection limit of 103 and a quantification range of 3.3\u2009\u00d7\u2009104\u20133.3\u2009\u00d7\u2009109 PCV2 copies per ml [4 versus 103). Samples were considered positive when the viral load was above the detection limit.The serum and oral fluid samples were tested for PCV2-DNA by qPCR essentially as previously described . During s per ml . BecauseAll PCV2 viral loads were analyzed on a log-transformed scale. Comparison of PCV2 viral loads in serum and oral fluid was made separately for Herd 1 and 2, and because the same pens were repeatedly sampled in Herd 2, and hence could not be considered independent, each sampling time point in Herd 2 was also analyzed separately. Viral loads below the assay detection limit were included in the statistical analyses with a value of 0, since the true distribution of these was unknown and excluding the observations would reduce the actual variation and thereby bias the results.Descriptive statistics consisted of frequency distributions, graphical illustrations and summary statistics. Evaluation of agreement between serum and oral fluid PCV2 viral loads was done both on a dichotomous (PCV2-positive/negative) and a quantitative scale. On a dichotomous scale, the oral fluid cut-off value for obtaining the best agreement with a PCV2-positive serum result (above the test detection limit of 3 log(10) PCV2-copies per ml serum) was estimated by drawing a receiver operating characteristic (ROC)-curve for all possible oral fluid cut-off values against a serum value fixed at the assay detection limit. Best agreement was defined as the oral fluid cut-off value where relative sensitivity and relative specificity were maximized simultaneously. The terms \u2018relative sensitivity\u2019 and \u2018relative specificity\u2019 were used, since the serum result could not be considered \u2018gold standard\u2019 in the classical sense but rather a \u2018reference standard\u2019 (as in a study from 2003 ). All saIn total, 310 serum and oral fluid sample pairs were collected. Of these, 50 sample pairs were from Herd 1 with 4\u20135 pigs bled per pen with a mean of 23 pigs per pen (range: 5\u201333) and 260 sample pairs (65 pens sampled 4 times) were from Herd 2 with 4 pigs bled per pen with a mean of 29 pigs per pen (range: 9\u201332). The number of pigs per pen reflects the maximum number of pigs contributing to the oral fluid sample.Classification of sample pairs into PCV2 positive and PCV2 negative based on the test detection limit is shown in Table\u00a0The 2 ROC-curves (one for Herd 1 and one for the first sampling in Herd 2) for evaluation of best agreement between serum and oral fluid concerning a PCV2-positive serum result are displayed in Fig.\u00a0Figure\u00a0Results from the linear regression models showed that oral fluid viral load was not significantly influenced by the number of pigs per pen when the viral load in serum (Herd 1 and 2) and sampling number (Herd 2) were also included in the models.Overall, both herds had a widespread PCV2 infection with moderate viral loads resulting in relatively few serum pools and even fewer oral fluid samples being negative. A relatively small number of serum pools had viral loads above 7 log(10), which fits well with the observation that no clinical signs clearly related to PCV2 infection were present in either of the 2 herds. This, however, also implies that generalizing results to herds with high viral loads and/or clinical signs of PCV2 infection should be done with caution. Only one sample pair had a positive serum PCV2 result (5.59 log(10) PCV2 copies per ml serum) and a negative oral fluid PCV2 result, which may reflect that not all blood-sampled pigs chewed on the rope. The estimated contribution to the oral fluid sample of more than 80% of the pigs in a pen is supported by a study reporting that 94% of pigs in a pen (pen size: 17\u201324 pigs) had chewed on the presented rope after 30\u00a0min .As expected, a slightly higher proportion of oral fluid samples compared to serum were PCV2 positive which probably reflects that the likelihood of a positive result is higher when more animals are tested . This is similar to previous findings concerning PCV2 , 29 and The estimated oral fluid cut-offs for best agreement (the highest possible relative sensitivity and specificity) with a PCV2-positive serum pool were fairly similar for Herd 1 and 2 (first sampling) with 6.5 and 7.36 log(10) PCV2 copies per ml oral fluid, respectively. However, the oral fluid cut-off was determined with a higher accuracy in terms of relative sensitivity and specificity for Herd 1 than for Herd 2, probably due to the lower variation between individual oral fluid results in Herd 1 compared to Herd 2 (first sampling)  PCV2 copies per ml sample was found in both herds, hence, substantially higher than the 1 log(10) reported by others , 30. WheSecondly, higher PCV2 viral loads may be present in oral fluid compared to the viral loads found in serum. Previous comparisons of serum and oral/tonsillar swabs found either no difference concerning prevalence of PCV2 positives , 45 and A significant correlation between serum and oral fluid viral loads was found for Herd 1 and the first sampling in Herd 2, whereas no significant correlations were found for samplings 2, 3 and 4 in Herd 2. For Herd 1, the estimated correlation coefficient of 0.69 was comparable to the 0.76 and 0.78 previously reported , 29. HowFor the first sampling in Herd 2, the correlation coefficient, even though significant, was only estimated to 0.39, which may be due to the high number of negative serum pools. For samplings 2, 3 and 4 in Herd 2, no statistically significant correlations were found between serum pools and oral fluid. A higher proportion of pigs in the pen were blood sampled in Herd 1 (~ 20%) compared to Herd 2 (~14%), which would seem like a plausible explanation, but the results from the linear regression models showed that the number of pigs per pen at sampling did not influence the oral fluid viral load when the serum viral load (Herd 1 and 2) and sampling number (Herd 2) were accounted for. Another explanation could be the higher variation in positive serum viral loads for all samplings in Herd 2 compared to Herd 1  being associated with a negative serum pool for the same pen. Nevertheless, a statistically significant correlation between serum pools and oral fluid samples could not be found for all sampling time points, probably due to a high within-pen variation in individual pig viral load becoming very evident in serum pools including only four or five pigs in a pen of around 30. Hence, from a practitioner\u2019s point of view, oral fluid might be better suited to identify presence or not of the pathogen than to determine the specific viral load."}
+{"text": "In this article, we review the contributions of genetic polymorphisms to major individual variations in drug pharmacotherapy, focusing specifically on the pharmacogenomics of phase-I drug metabolizing enzymes and transporters. Substantial frequency differences in key variants of drug metabolizing enzymes and transporters, as well as their possible functional consequences, have also been discussed across geographic regions. The current effort illustrates the common presence of variability in drug responses among individuals and across all geographic regions. This information will aid health-care professionals in prescribing the most appropriate treatment aimed at achieving the best possible beneficial outcomes while avoiding unwanted effects for a particular patient.The interindividual genetic variations in drug metabolizing enzymes and transporters influence the efficacy and toxicity of numerous drugs. As a fundamental element in  Pharmacogenomics is the understanding of how individuals differ in their response to drug therapy and the mechanisms underlying variable drug response by utilizing genomics, proteomics, transcriptomics, and metabolomics based knowledge. Every individual has a different genetic makeup, which influences the risk of developing diseases as well as responses to drugs and environmental factors Drug response of individual patients is primarily determined by the pharmacokinetic and pharmacodynamic properties of prescribed drugs, which is directly or indirectly affected by polymorphisms in drug metabolizing enzymes and transporters. Different populations have varied allele frequencies in genes of both drug metabolizing enzymes and transporters. For precision medicine, the molecular and clinical information is integrated in order to understand the biological basis of disease and develop medications with better outcomes for patients It is well known that individuals vary significantly in their clinical responses to administered drugs and the outcomes, which can be inherited or acquired, are always patient-specific Different patients can respond differently to the same drug and dose. Sometimes, the effective drug dose for a particular patient may prove lethal to or result in therapeutic failure in others , leading to serious adverse effects or no effects at all. Continuous drug monitoring is recommended when prescribing drugs with known serious side effects and narrow therapeutic indexes to avoid unexpected and undesirable outcomes The recommended daily maximum dose of simvastatin for the management of blood cholesterol levels is 40\u00a0mg. In a cohort study of 156 patients, 95% of them showed reduced levels of low-density lipoprotein (LDL) cholesterol, whereas the remaining 5% exhibited no reduction was observed for the remaining 5% of the patients, even at doses as high as 160\u00a0mg/day of simvastatin Individual-specific response to medication can be attributed to many multifold and complex factors including the unique genetic makeup . These genetic factors, as well as physiological conditions ; environmental influences ; and pathological factors  can work alone or in combination to influence drug responses Disease conditions of individuals used to be diagnosed based on signs and symptoms, which may be indicative of several different diseases or somewhat related to the family history. In the past, clinicians could only attempt to cure or treat disease upon its onset Genetic polymorphisms may influence a drug\u2019s effect by altering its pharmacokinetics, pharmacodynamics, or both , which aMutations in the gene coding regions could cause alterations in gene expression or protein structure, leading to variations in protein quantity and quality. In the case of enzymes, such mutations affect both the protein function and the rate and kinetic constants. Changes in drug-receptor or drug\u2013enzyme interactions due to structural alterations of enzymes or receptors could also result in variations in drug responses Twin studies have provided evidence supporting the contribution of genetic factors to individuals\u2019 varied drug responses. For instance, in the late 1950s, it was found that dizygotic twins exhibited more metabolic variability than did monozygotic twins for isoniazid metabolism CYP2D6. The highly polymorphic CYP2D6 gene is located on the chromosome 22q13.1, consisting of nine exons and eight introns (GenBank accession No. NM 000106.5) CYP2D6 genetic variants have been described  to date, resulting from point mutations, duplication, insertions or deletions of single or multiple nucleotides, and even whole-gene deletion. Individuals carrying different CYP2D6 allelic variants have been classified as poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs) according to the metabolic nature of the drugs and degree of involvement in drug metabolism of these variants Cytochrome P450 (CYP), which represents a large and diverse group of heme-containing enzyme superfamily, is involved in oxidative metabolism of structurally-diverse molecules like drugs, chemical, and fatty acids. The genetic polymorphism in the genes encoding CYP members was firstly reported for CYP2D6 gene significantly affects phenotypic drug responses. Up to a 10-fold difference in the required dose was observed in order to achieve the same plasma concentration in different individuals in vivo CYP2D6 phenotyping. According to the probe substrate metabolic capabilities among the sampled individuals in a population, patients can be categorized into the following four phenotypic groups: poor, intermediate, extensive, and ultra-rapid metabolizers , respectively CYP2D6 allelic variants . These allelic variants lead to inactive CYP2D6 enzymes CYP2D6*3 (3.3% in Sardinians), CYP2D6*4 (23%\u201333% in Polish and Faroese populations), CYP2D6*5 (5.9%\u20136.2% in Spaniards and African Americans), and CYP2D6*6  were also calculated in diverse populations  in the Faroese population CYP2D6*4/*4 genotype than in patients with one or no CYP2D6*4 alleles (20%) CYP2D6, codeine is not recommended as an analgesic because of the minimal enzymatic conversion from codeine to morphine. Conversely, a higher risk of morphine toxicity may occur in patients with the UM phenotype owing to the rapid conversion of codeine to morphine. The situation would be more devastating in UMs who are lactating mothers because the normal codeine dose can translate into fatal morphine concentrations into the breast milk CYP2D6 allelic variants *10, *17, and *41 exhibit normal catalytic activity but are sometimes associated with intermediate to low metabolic activities CYP2D6*10 allele has been found more common than other alleles  and it causes a greatly decreased (but not deficient) enzyme activity The prodrug tamoxifen is a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer patients CYP2C9 gene . Among them, CYP2C9*2 (R144C) and CYP2C9*3 (I359L) are the most common variants associated with highly-reduced CYP2C9 enzymatic activities in comparison with the wild-type allele (CYP2C9*1) CYP2C9 is another important member of the CYP superfamily. The gene coding for CYP2C9 is located on chromosome 10q24.2, and spans more than 55\u00a0kb in length. CYP2C9 constitutes approximately 18% of the total CYP protein in the human liver microsomes CYP2C9*2 variant results in a markedly decreased enzyme activity due to higher km value and lower intrinsic clearance of drugs like S-warfarin CYP2C9*2 allelic variant has been reported with up to 25% allelic frequencies in the Iranian population CYP2C9*1/*2, homozygous CYP2C9*2 or CYP2C9*3 carriers were lower (0.1%\u20131%) in the Chinese and Japanese populations compared with those in Caucasians and Iranians. Caucasians have approximately 1% CYP2C9*2 and 0.4% CYP2C9*3 homozygotes, respectively *1*2 or the *1*3 genotype, while more than 2% have the *2*2, *2*3, and *3*3 genotypes CYP2C9*2 and CYP2C9*3 is greater than that in the other studied populations CYP2C9*1 allelic frequency variant of >90%, whereas allelic CYP2C9*2 variant was not detected in the Korean, Chinese, and Vietnamese populations but occurs 1% in the Japanese. Furthermore, no individuals from the South African and Zimbabwean populations have been reported to carry the CYP2C9*2 allele -5-phenylhydantoin (phenytoin metabolite) than those with the CYP2C9*1/*1 genotype. Multiple studies have also shown that the CYP2C9*3/*3 genotype is associated with reduced metabolisms and altered pharmacokinetic properties of substrates such as phenytoin, warfarin, losartan, and tolbutamide Both CYP2C9 and CYP2C19 are involved in microsomal hydroxylation of phenytoin to its R and S enantiomers CYP2C19, which is located on the chromosome 10q24 encodes another CYP family member. CYP2C19 can metabolize numerous routinely-administered drugs such as anxiolytics (diazepam), proton pump inhibitors (omeprazole), anticonvulsants (S-mephenytoin), and antimalarial biguanides CYP2C19 variants and approximately 2000 SNPs have been identified , with continuous increase in SNP numbers reported. Among them, CYP2C19*2 and CYP2C19*3 are the most common variants that have been studied extensively. Both of them are null variants and patients carrying these variants are therefore categorized as PMs. CYP2C19*2 is the most common allelic variant caused by a single nucleotide alteration in exon 5 (G\u00a0>\u00a0A), resulting in an abnormal splicing site and conferring reduced enzymatic activities of CYP2C19 The polymorphic CYP2C19*2 variant is found at a high allelic frequency (30%) in South Indians, but occurs with the lowest frequency (2.9%) in the Faroeses. In contrast, CYP2C19*3 is found at higher allelic frequencies in the Japanese (approximately 13%) but lower (0) among the Italians, South Africans, Greeks, European-Americans, and other populations  and most of these variants are responsible for varied enzyme activities ranging from modest to highly reduced catalytic efficiencies among the affected individuals CYP3A4 gene (CYP3A4*2 and CYP3A4*3) were observed in Caucasian whereas high frequencies of allelic variant CYP3A4**18 were observed in Chinese people  is associated with impaired ability to accept electrons from NADPH POR*13 (Q153R) variant leads to severely-impaired steroid biosynthesis in Antley\u2013Bixler skeletal malformation syndrome (ABS) CYPOR genes have been described .CYP oxidoreductase (CYPOR) is the catalytic partner and compulsory element to all CYP-mediated metabolisms. The interaction between the CYP and CYPOR is essential for the metabolic activities of CYPs A drug could produce a beneficial or toxic effect in a particular patient. The nature and extent of the resulting effect is largely dependent on the absorption, distribution, and excretion rates of the drug. Drug transporters primarily control the movement of all drugs and their active or inactive metabolites into or out of cells. Therefore, polymorphisms of drug transporter genes can modify the absorption, distribution, and excretion rates, and ultimately safety and efficacy of the administered drugs. The ABC and solute-carrier (SLC) transporters are two superfamilies of transport proteins are ubiquitous membrane-bound transport proteins that are involved in the absorption, distribution, and elimination of drugs ABCA to ABCG (http://nutrigene.4t.com/humanabc.htm). The impact of some important polymorphisms on the drug transport activities of various ABC transporters is summarized in http://www.bioparadigms.org/slc/menu.asp). Among them, members of the organic anion transporter (OAT), organic anion transporting polypeptides (OATP), and organic cation transporter (OCT) subfamilies are of particular significance in drug disposition ABC transporters often transport drugs and other substances against the concentration gradient using ATP as an energy source ABCB1 gene, also known as the multidrug resistance 1 (MDR1), encodes a P-glycoprotein (Pgp), which is involved in the cellular efflux of numerous chemotherapeutic agents, physiological metabolites, and carcinogens ABCB1 is highly polymorphic with allelic variants found in varied frequencies in different populations  in Asians but low (34%\u201342%) in Caucasians. The substrate-dependent effects of Pgp on pharmacokinetic and pharmacodynamics properties remain obscure due to controversial studies on digoxin disposition. For example, for patients with a mutant allele (3435C\u00a0>\u00a0T) that were administered a single oral dose of digoxin, Sakaeda et al. ABCC2 and G671V variant with 28% allelic frequency in Caucasians have been reported ABCC2  that has been extensively studied for its role in drug resistance development in cancer and human immunodeficiency virus type 1 (HIV-1)-infected patients As the important ABC members, both ABCC1 and ABCC2 are involved in the transport and excretion of several chemotherapeutic agents, toxicants, and organic anion molecules ABCG2 gene have been identified Similar to ABCC1, ABCG2 was first discovered in multidrug-resistant cell lines ABC transporters would be essential to enhance the understanding of the genetic impact on pharmacotherapy.The C421A is widely distributed in many ethnicities with frequencies of 27%\u201335% in Asians, 9%\u201314% in Caucasians, and 1%\u20135% in Africans . GefitinOATPC*5 and OATPC*9 allelic variants are associated with a reduced uptake of OATPC substrates such as estrone sulfate and estradiol-17-\u03b2-D-glucuronide OATPC*5 allele OATPs are a large family of membrane-bound influx transporters that are responsible for the cellular uptake of a wide range of endogenous and exogenous substances including bile salts, hormones, and clinically administered drugs such as antibiotics, cardiac glycosides, and anticancer agents SLCO1B1 and is essential for the hepatic uptake of the simvastatin active metabolite, simvastatin acid SLCO1B1 gene with their allelic frequencies and functional consequences in Asian, African and Caucasian have been discussed in SLCO1B1 is associated with reduced OATP1B1 activity, which is responsible for the higher blood concentrations of simvastatin acid, as well as the consequently increased toxicity and reduced efficacy of simvastatin OATP1B1*15 was associated with increased plasma concentrations of pravastatin and 7-ethyl-10-hydroxycamptothecin (irinotecan active metabolite), whereas OATP1B1*17 variant is linked with an increased cholesterol synthesis mediated by pravastatin On the other hand, OATP1B1, OATP2B1, and OATP1B3 are mainly expressed on the hepatocyte sinusoidal membrane, which can facilitate the hepatic drug uptake SLC22A family and in humans, which are present in the basolateral cell membrane of the renal proximal tubule OCT1, OCT2, and OCT3, have been identified in humans OCT2 is highly expressed in the kidneys. OCTs mediate the cellular uptake of a wide range of structurally-different organic cations including clinically-administered drugs such as metformin and procainamide OCT2 270S variant has been associated with low activity while the 270A variant induces high activity of OCT2 OCTs are proteins encoded by the in vivo by CYP1A2, CYP2B6 and CYP2C19 for its anti-platelet activity CYP2C19, CYP1A2, 2B6*6, and CYP3A5*3 were found to be associated with the varied degree of drug\u2013drug interactions for clopidogrel, due to its highly-complex pharmacokinetics and variable drug response as compare to other anti-platelet drugs Effects of one drug are modified by other concomitantly administered drugs due to drug\u2013drug interactions, which may be attributed to the altered pharmacokinetic or pharmacodynamic properties of one drug induced by the coadministered drug. The polymorphisms in drug metabolizing and transporter genes are an important risk factor of drug\u2013drug interactions and varied interindividual drug responses Mutations in the drug transporter genes also contribute to drug\u2013drug interactions and adverse drug reactions. HMGCR inhibitors such as atorvastatin, rosuvastatin, and pravastatin are actively transported by OATP1B1 and ABCG2 The genetic variations of CYPs and transporters have been described in diverse populations. In this review, we review the different allelic variants that are responsible for altered drug activities in diverse geographic regions. Some populations exhibited extremely high frequencies of allele variants that are associated with several significant clinical consequences. Taking advantage of pharmacogenomics, researchers have assessed some specific genetic variants responsible for the particular drug responses of individuals.Whole genome SNP profiling, haplotyping, multigene analysis, and gene expression studies by biochip or microarrays are all in place to study drug responses of individuals, which would aid in drug discovery, development, and individualized treatments. Given the common variability in drug responses among patients, the optimization of dosage regimen at the individual level is not an easy task. Comprehensive appreciation of the contributing factors associated with interindividual and interethnic differences in medication responses is a must for the development of precision medicine, and help health-care professionals in recommending the proper treatment with the best possible beneficial outcomes while preventing unwanted drug effects in the particular patients. The development of clinical practice strategies based on accurate genotype testing will facilitate the enhanced understanding of altered drug responses and drug\u2013drug interactions. Furthermore, the development of more reliable biomarkers based on polymorphisms in genes responsible for the adverse events will hopefully create strategies for administering drugs based on the genotype and phenotype of patients, to minimize unwanted drug reactions.The authors have declared no competing interests."}
+{"text": "Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection. As exemplified by the highly successful, yellow fever virus (YFV) 17D vaccine developed in the early 1930\u2019s and more recently Japanese encephalitis virus (JEV), vaccination is a feasible strategy for preventing and controlling mosquito-borne flavivirus infections4. In other flavivirus infections where neutralizing antibody titers >10 protect6, similar titers in DENV infection are complicated by the existence of four heterotypic serotypes and heterotypic cross neutralization. While the presence of neutralizing antibodies has been long considered a correlate of protection for flaviviruses, recent data from dengue vaccine trials prove that the presence of antibodies that neutralize DENVs in cell culture do not necessarily confer protection7. New assays and reagents are needed to characterize human antibody responses to dengue virus infections and vaccination and to identify requirements for protection beyond mere neutralizing antibodies.The four serotypes of dengue viruses (DENV1-4) are estimated to cause around 100 million cases of dengue fever or dengue hemorrhagic fever each year8. Additionally, people experiencing a secondary DENV infection with a new serotype face a greater risk of progression to severe DHF (Dengue hemorrhagic fever) and DSS (Dengue shock syndrome). Severe disease is a result of immunopathology, likely mediated by aberrant T cells9 and non-neutralizing antibodies induced by the first infection. Moreover, pre-existing antibodies may increase viral load in secondary infections through the process of antibody-dependent enhancement (ADE) of infection of Fc receptor bearing cells10. As such, a successful DENV vaccine should ideally elicit robust anti-DENV protective immunity against all 4 serotypes to prevent subsequent dengue illness, especially severe illness that can result from ADE infection. To date this has been a difficult challenge to overcome, especially in those seronegative at the time of vaccination.A major challenge to DENV vaccine development is the existence of 4 serotypes and the need for vaccines to confer protection against all 4 serotypes. With an approximate 60% amino acid divergence between the E proteins of the 4 serotypes, immunity to one serotype usually does not confer long-lasting cross-protective immunity to the other serotypes11. Structural studies with human monoclonal antibodies (hmAbs) isolated from dengue patients have provided high resolution maps of epitopes on the viral surface. These studies have also led to the development of new tools and reagents to identify correlates and mechanisms of protective immunity following natural infection or vaccination16.It has been known since the early 1980s through passive transfer experiments that antibodies targeting the E glycoprotein can protect from lethal flavivirus challenge14. Our group and others have characterized DENV-specific antibodies in people exposed to natural and experimental infections or live attenuated vaccines18. Both serotype-specific and cross-reactive strongly neutralizing mAbs have been isolated from the memory B cells of donors with a history of primary and secondary DENV infections20. The location of mAb epitopes on the envelope glycoprotein often, but not always, differs between serotypes and commonly the paratope recognizes a complex quaternary epitope that is present only on fully assembled and intact virions. While the structural footprints of several human neutralizing mAbs on the viral envelope have been determined through the use of cryo-electron microscopy, the resolution of these structural studies can only predict the relative contribution of different fractions of the quaternary epitope to monoclonal and/or polyclonal antibody neutralization phenotypes. Recently, DENV3-specific potently neutralizing antibody, 5J717, was identified as using a complex quaternary epitope to neutralize DENV3 in a manner previously hypothesized for West Nile virus (WNV)22. Importantly, the 5J7 monoclonal antibody footprint interacts with distinct residues encoded in 3 adjacent monomers, A, B\u2019 and B  system for a clinical isolate of DENV4 isolated in Sri Lanka in 1992 and belonging to genogroup I26. To circumvent instability and toxicity during bacterial plasmid amplification in E coli, the DENV4 genome was subcloned into 4 distinct fragments nts Fig.\u00a0. Naturalnts Fig.\u00a0. DENV4 r17. Using cryo-electron microscopy, the foot print of hmAb 5J7 was recently mapped to a quaternary epitope that spans 3 adjacent E protein molecules on the viral envelope15  hinge region was superimposed upon the DENV3 E protein crystal structure to approximate the footprint of an antibody epitope binding region. Following amino acid (AA) and nucleotide alignments between DENV3 and DENV4, interserotypic variant AA were identified between DENV3 and DENV4  5J7 is a strongly neutralizing serotype specific antibody isolated from a patient who recovered from a DENV3 infectione15 Fig.\u00a0. We recee15 Fig.\u00a0 into DEN B\u2019 Fig.\u00a0 for hmAbNV4 Fig.\u00a0. Three dNV4 Fig.\u00a0. NucleotNV4 Fig.\u00a0. DENV4/3All three of the rDENV4/3 viruses  were successfully recovered following electroporation and subsequent passage on C6/36 cells. Using sequence analyses and RT-PCR RFLP analyses, all viral stocks were pure and contained only the rDENV of interest  Fig.\u00a0. In factTo eliminate the possibility that the panel of rDENV4/3 viruses had been modified in such a way as to increase their sensitivity to neutralization by DENV immune sera in a non-specific manner, neutralization assays were performed with immune sera collected at late convalescence (>6 months) from subjects exposed to a primary DENV2 (DT100) or DENV1 (310 and 147) infection and secondary DENV infections (DT 000). These immune sera cross-neutralized DENV3, DENV4 and DENV4/3 M14 and M16 in U937\u2009+\u2009DC-DIGN cells to similar degrees and were not significantly different by a 2-way Wilcoxon matched-pairs rank test. Fig.\u00a0 indicati20. Some of these quaternary structure-dependent epitopes are present on the E dimer but some are only present on the intact virion and may span across dimers to bind to neighboring monomers/dimers, as is seen with the 5J7 epitope. It is becoming apparent that knowing the epitope specificity of an antibody response to a vaccine may be essential for understanding the correlates of DENV protective immunity. Large-scale clinical trials of new vaccine candidates have demonstrated that in vitro gold standard neutralization results do not always predict vaccine efficacy, current data indicate that the standard Vero neutralization assay without regard to epitope specificity does not always correlate with protective immunity30. This is not the first time that failure of in vitro characteristics, previously small-plaque and temperature-sensitive phenotypes failed to be predictive of vaccine efficacy33. Measuring epitope specificity of polyclonal sera is a daunting task, although recent advances in elucidating the contributions of individual hmAb to the overall serological repertoire have demonstrated that only a few neutralizing antibodies may dominate a protective response against influenza virus35. While this approach provides rich molecular information on an individual basis, new reagents are needed for population based studies. Single and multiple mutations in the E protein have been effectively used to measure type specific antibodies in human polyclonal sera by loss of neutralization36. Using epitope-transplanted chimeric DENV viruses, such as the set described herein, gain of neutralization can be measured to easily and accurately measure epitope specific antibody binding and neutralization responses in complex polyclonal sera. Such chimeric viruses may be combined with the 4 serotypes of DENV or they may be used in combination with antigen depletion of sera to measure epitope specificity of polyclonal sera.Human mAbs that bind quaternary epitopes on flaviviruses were first described for WNV followed by the 4 DENV serotypes37.Here, we show incremental gain of binding of the 5J7 monoclonal with the increasing size of the transplanted epitope in both binding and neutralization assays. It is also abundantly clear that additional residues in adjoining monomers significantly enhanced 5J7 binding to DENV4/3 M16 virus, clearly demonstrating that residues on A, B\u2019 and B monomers are critically important for 5J7 recognition, maximal neutralization and quaternary epitope function. More importantly, we show that the larger epitope transplants that include residues of 3 different E proteins can be used to measure the amount of antibody that targets this antigenic site in DENV 3 primary immune polyclonal sera. Because individual monoclonal antibodies like 5J7 can only infer the location of the primary neutralizing antigenic site that is recognized by the polyclonal response, epitope transplant provides a means of demonstrating the importance of the A, B\u2019 and B monomers in the global polyclonal neutralization response against DENV3. We do not always capture the entire DENV3 neutralization titer present in the polyclonal sera, suggesting that additional neutralizing sites and/or genetic variability within the epitope must exist in DENV3, supporting the critical need for identifying and charactering more type specific hmAb for each DENV serotype. Importantly, our data argue that our rDENV 4/3 panel is a powerful tool for measuring DENV3 epitope-specific antibodies in polyclonal sera. Indeed, the DENV4/3 M14 and M16 have very recently been successfully used to track development of antibodies to the 5J7 epitope in a large scale DENV3 cohort study with similar results41. To date, none have presented complex quaternary epitopes that span multiple monomers. We have previously demonstrated that residues in the A monomer of the 5J7 epitope can be transplanted into DENV125. The transplant of the DENV3 5J7 epitope into DENV 4 is unique from previous transplants in that this is the first transplanted epitope in any virus in which residues of adjoining monomers and dimers were also modified to capture an entire complex quaternary epitope. We detected a clear gain of epitope specific responses to 5J7 in the DENV4/3 recombinant viruses as the size of the transplanted region was increased to capture residues adjacent in B and B\u2019 E molecules. With the DENV4/3 panel we have enlarged the scope and complexity of epitopes that can be successfully transplanted, effectively reconstructing a functional epitope that spans multiple monomers in the E glycoprotein raft15. In the DENV4/3 M16 recombinant virus, it is interesting that a single nucleotide change emerged in cell culture and that some minor differences were noted in the peak binding levels of broadly cross reactive fusion loop antibodies. DENV4/2 M16 displayed higher peak binding to 1M7 and 1N5 fusion loop antibodies, perhaps reflecting different maturation states and prM levels on virions. Future studies will focus on unraveling the role of this mutation and its phenotypes in epitope exchange viruses.A variety of approaches are being investigated to present complex epitopes in virus-like particles and on scaffolds, both for diagnostic and vaccine applications42. New developments in reverse genetics can be utilized to generate infectious clones for a number of viruses, including flaviviruses50. Importantly, quaternary epitopes and complex conformational neutralizing epitopes exist in many highly evolving and variable viruses, thus the methods described herein should be applicable to broad, based immunogen design for multiple pathogens. Live virus vaccines for yellow fever virus and more recently Dengue viruses provide new avenues for improving vaccine immunogenicity and safety, using reverse genetic approaches. In particular, the DENV tetravalent vaccine usually elicits immune responses against all four serotypes, regardless of manufacturer54. Nonetheless, the scope of the response varies based on vaccine serotype and the pre-immune status of the host. A detailed understanding of the location and functional activity of conformational and quaternary epitopes in vaccine performance offers novel opportunities for developing epitope specific correlates of protective immunity, engineered serotypes for improved vaccine immunogenicity and performance, and simplified bivalent vaccine formulations. (reviewed in55).The advent of modern molecular techniques and genomics has allowed for a new, highly-rational approach to be taken in the development of tools for epitope analysis and of new vaccine candidates13 The rDENV3 clone, based on a Sri Lankan 1989 strain, was initially described in detail23 and the DENV4 clone was based on the Sri Lankan 1992a sequence26 and has only briefly been described previously13.Vero cells (ATCC# CCL-81), C6/36 cells (ATCC CRL-1660) and DC-SIGN-expressing U937 cells (ATCC CRL-1593.2 transfected with a retrovirus coding human DC-SIGN) and their growth conditions have been previously described57.Convalescent DENV immune blood samples were collected from travelers visiting countries where dengue is endemic. Our protocol for collecting dengue immune blood samples was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol 08-0895). Written informed consent was obtained from all subjects before their participation in the study. Studies at UNC and NIH were performed in accordance with the relevant guidelines and regulationsDENV serum samples used in this study were obtained from the existing dengue traveler collection as mentioned above. The samples were not from acute clinical patients and hence were not PCR confirmed. All samples were coded and analyzed anonymously.24, all changes were isolated to the A fragment of the DENV4 clone genome. The 3 increasing sizes of the 5J7 epitope transplant were synthesized  and incorporated into 3 different DENV4 fully assembled DNA genomes, transcribed, and then the genome length RNAs electroporated into Vero-81 cells to generate the rDENV4/3 panel of viruses, all 3 of which were viable. The first attempt at DENV4/3 M16 acquired a tissue culture adaptation, Env K323Q. The DENV4/3 M16 described in this paper was rederived with this additional Env K323Q residue and is otherwise isogenic with DENV4. Recombinant viruses were subjected to full length sequencing to demonstrate the presence of appropriate subsets of mutations as previously described13.To generate the 3 different size rDENV4/3 viruses , increasing numbers of amino acid residue sequences were transplanted into DENV4 from DENV3. As a result of our quadripartite infectious clone designFor viral titrations, viral stocks were diluted 10-fold serially in dilution medium  supplemented with 2% heat-inactivated fetal bovine serum  and 1x antibiotic/antimycotic. Following a 1hr infection, cells and inoculum were overlaid with overlay medium: OptiMem  containing 5% carboxymethylcellulose, 2% HI-FBS (Hyclone defined), and 1x antibiotic/antibiotic . Following a 4\u20136\u2009day incubation (to achieve countable foci of infection), cell monolayers were extensively washed with 1xPBS followed by fixation in a solution of 80% methanol:20% phosphate-buffered saline (PBS) (v:v). Fixed cell monolayers were blocked in a solution of PBS containing 5% non-fat milk, and incubated with a primary antibody cocktail of 4G2 and 2H2 murine mAbs. Following thorough PBS washes, infectious foci were visualized using HRP-goat-anti-mouse secondary antibody , followed by TrueBlue substrate and foci were enumerated and used to calculate infectious titer.DENV3, DENV4, and the rDENV4/3 panel were subjected to multi-step (MOI\u2009=\u20090.1) growth curve analyses on Vero or C6/36 cells. Briefly, Vero or C6/36 cell monolayers in 12-well plates were inoculated with indicated virus at an MOI\u2009=\u20090.1. At 24hr intervals, all medium was removed and an equivalent amount of fresh medium was added. Supernatants were clarified by centrifugation and stored at \u221280\u2009\u00b0C until viral titrations were performed. Virus grown on Vero cells was titrated on Vero cells, and C6/63-derived virus was titrated on C6/36 cells, enumerated, and plotted over time to generate growth curves.13. Briefly, monolayers of Vero-81 cells in 24 well plated were infected with a virus:antibody/serum cocktail that had previously incubated for 1hr at 37\u2009\u00b0C to allow for Ab:virion binding. Following a 1hr incubation at 37\u2009\u00b0C for infection, cells and inoculum were overlaid with overlay medium (see above). Following a 4\u20136\u2009day incubation at 37\u2009\u00b0C, monolayers were fixed, stained, and geometric mean neutralization titers enumerated using wells containing no antibody as a control for infection.Neutralization on Vero-81 cells has previously been described4 cells were added and the infection was allowed to proceed for 2\u2009hours at 37\u2009\u00b0C. Unbound virus was washed with infection media and the volume of media in each well was increased to 200\u2009\u03bcl, and the cells were returned to 37\u2009\u00b0C for a total of 24\u2009hours. After 24\u2009hours, the cells were washed with PBS, fixed in paraformaldehyde, permeabilized with saponin, blocked with normal mouse serum in permeabilization buffer, and stained with AlexaFluor 488-conjugated  2H2 antibody. Unbound antibody was washed off, and cells were resuspended in Hank\u2019s Buffered Salt Solution  supplemented with 2% fetal bovine serum. Assays were performed twice and in duplicate. Samples were read on a Guava easyCyte 5HT flow cytometer (Millipore) as previously described by our group27. All serum neutralization curves are shown in Supplementary Figures\u00a0Human sera or monoclonal antibodies were serially diluted 3-fold and mixed with sufficient virus to cause 15% infection in U937\u2009+\u2009DC-SIGN cells. Dilution media contained reduced 2%) fetal bovine serum. The virus:antibody mixtures were incubated for 45\u2009minutes in a 96-well plate at 37\u2009\u00b0C. Following this incubation, 5\u2009\u00d7\u200910% fetal b13. Briefly, RNA isolated from viral stocks, DNase treated , was reverse transcribed and the resulting cDNA amplified  using primers specific for each serotype. Positive controls from WT DENV RNA were run for each reaction as was a no template control. Presence of amplicons for rDENV4 and no other rDENV indicates absence of any species of DENV1-3.In order to confirm our recombinant virus stocks were pure and did not contain any WT DENV, highly sensitive RT-PCR and RFLP were performed as previously describedFor RFLP, RNA isolated from viral stocks was converted to cDNA and the portion of the E protein gene containing the transplanted epitope was amplified by PCR. The resulting amplicon was gel purified on a 1% agarose gel, and subjected to specific restriction endonuclease (RE) digests designed to cut the WT amplicon, but not the rDENV amplicon (due to ablation of the RE site by epitope transplantation). While the approximately 1000\u2009bp wild-type amplicon was cut by BsiWI into an 800\u2009bp and 200\u2009bp fragments, the rDENV were not digested, indicating the absence of WT DENV4 in the recombinant viral stocks.Geometric mean neutralization titers were determined by 4-way non-parametric curve fitting with upper and lower bounds of 100 and 0. Nonparametric column statistic  were used to calculate p-values. All statistics were calculated using Graphpad Prism .Supplementary Information"}
+{"text": "Bpifa1 have been associated with OM susceptibility, its role in the ME is not well characterized. We investigated the role of BPIFA1 in protection of the ME and the development of OM using murine models. Loss of Bpifa1 did not lead to OM development. However, deletion of Bpifa1 in Evi1Jbo/+ mice, a model of chronic OM, caused significant exacerbation of OM severity, thickening of the ME mucosa and increased collagen deposition, without a significant increase in pro-inflammatory gene expression. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions and its loss in the presence of inflammation, exacerbates epithelial remodelling leading to more severe OM.Otitis Media (OM) is characterized by epithelial abnormalities and defects in innate immunity in the middle ear (ME). Although, BPIFA1, a member of the BPI fold containing family of putative innate defence proteins is abundantly expressed by the ME epithelium and SNPs in  Acute OM is primarily caused by the normally commensal bacteria non-typeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis2 while chronic forms of OM show a genetic component of 45\u201375%3. The large numbers of OM susceptibility genes that have been identified emphasize the importance of innate immunity in protection against OM development7. More than 20 mouse genetic models of chronic OM have been characterised in recent years and these have proven to be a powerful tool in understanding the pathobiology of OM  One such model is the N-ethyl-N-nitrosourea (ENU) mutant mouse, Junbo, that spontaneously develops chronic suppurative Otitis media (CSOM) under specific pathogen free (SPF) conditions, characterised by development of cellular fluid and hypoxia in the ME and inflammatory thickening of the mucoperiosteum. The causative mutation is a SNP in the gene encoding the transcription factor EVI1, also known as MECOM9. EVI1 is an inducible negative regulator of NFkB10 and a repressor of the TGF\u03b2 signalling pathway11. The heterozygous mutant Evi1Jbo/+ mouse has increased responsiveness to microorganisms and inflammatory stimuli10.Otitis Media (OM), or middle ear (ME) inflammation, is the most prevalent paediatric disease and the leading cause of paediatric surgery, antibiotic prescription and conductive hearing impairment13, where it localises to a non-goblet, non-ciliated population of secretory cells14, minor glands and mucous cells of the sub-mucosal glands15. Secreted BPIFA1 is found in nasal lavage, saliva, sputum and bronchoalveolar lavage (BAL)13 and in the apical surface lining (ASL) fluid from air liquid interface cultures of differentiated tracheobronchial epithelial cells17.The ME is an anatomical extension of the upper respiratory tract (URT) and demonstrates similar physiological and immune mechanisms. The epithelium of the URT and its secretions play a key role in protection against environmental insults and infections. BPIFA1, a member of the bacterial permeability-increasing (BPI) fold containing family of putative innate defence proteins, is one of the most abundant secretory proteins in the URT. Its expression is restricted to the nasal passages, oropharynx, trachea and bronchi18 and chronic rhinosinusitis19. Despite the lack of compelling mechanistic evidence to identify the precise biological function of BPIFA1, an increasing number of studies suggest that it plays a multifunctional host defence role in the conductive airways16. In line with the structural similarity to BPI20, BPIFA1 exhibits antimicrobial activity against a variety of respiratory pathogens and deletion of Bpifa1 in mice is associated with increased susceptibility to pulmonary infections25. Other proposed functions of BPIFA1, include a surfactant-like activity, dispersal of bacterial biofilms, and regulation of ASL volume by modulation of epithelial sodium channel (ENaC) function16.Alterations of BPIFA1 levels are associated with multiple URT diseases such as cystic fibrosisBpifa1 expression in vivo using siRNA in chinchillas led to impaired mucociliary clearance23. BPIFA1 protein was present in the effusions of patients with chronic OM27 and BPIFA1 was also identified as one of the top hits in a Genome Wide Association Study of genes associated with OM susceptibility28. We recently showed production of BPIFA1 in the murine ME, in vivo and in vitro29 and it has been reported that Bpifa1\u2212/\u2212 mice greater than 10 months-of-age show increased susceptibility to spontaneous development of chronic OM30. Together this data suggests that BPIFA1 may be a determinant for predisposition to OM.Recently, evidence of BPIFA1 expression in the ME and its involvement in OM pathogenesis has started to emerge. Knocking down Junbo model of chronic OM. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions. This is the first study to report the use of compound Bpifa1 mutants and it underlines the importance of investigating the additive effect of multiple genetic defects in order to better understand the aetiology of multifactorial diseases such as OM.In this study, we have studied the role that BPIFA1 plays in protection of the ME and its association with OM development. Our data shows that although the loss of BPIFA1 did not lead to the development of spontaneous OM in mice up to 6 months-of age, it significantly exacerbates the inflammatory phenotype in the 31. In order to evaluate the onset of BPIFA1 production during the post-natal development of the bulla, immunohistochemistry (IHC) was performed from the day of birth (P0) to P30 at intervals of 5 days. BPIFA1 was detected in the ME throughout this period. As described previously, between P0 to P10, the ME space is occupied by mesenchyme32. BPIFA1 was detected in the non-ciliated epithelium enclosing this mesenchyme mass, in the region where it retracted from the opposing epithelial surface  or 12 weeks of age .Having established that BPIFA1 is present in the ME epithelium during the process of bulla cavitation, we went on to determine whether loss of the protein caused OM. Hearing thresholds, measured using broad-spectrum Auditory Brainstem Response (ABR) (which is indicative of conductive hearing loss), did not differ significantly in age Fig.\u00a0. Histoloeks Fig.\u00a0 or 6 moneks Fig.\u00a0  and Bpifa1/\u2212+ (2/8) bullae contained fluid and 2/12 Bpifa1\u2212/\u2212 ears and 1/8 Bpifa1/\u2212+ ears were NTHi culture positive. WT bullae (n\u2009=\u200916 ears) did not have fluids and all were NTHi culture negative. Although this data was suggestive of an effect of loss of Bpifa1 on infection these differences did not achieve statistical significance. A similar outcome was achieved when we challenged WT, Bpifa1/\u2212+ and Bpifa1\u2212/\u2212 mice with NTHi using baroinoculation34 and sampled bulla fluids at day 3 post-inoculation (data not shown).As the development of OM is often associated with bacterial infection, we investigated whether the loss of BPIFA1 modulated the ability of mice to respond to the intranasally (IN) inoculated otopathogen NTBpifa1 does not lead to spontaneous development of OM and does not lead to an increased ME infection or overt induction of OM following NTHi inoculation.Taken together, the data suggested that deletion of Evi1Jbo/+ mouse. OM initiates spontaneously in Evi1Jbo/+ mice from P21 onwards and is characterised by significant remodelling of the middle ear epithelium9. At P28, IHC staining for BPIFA1 appeared to be reduced in the ME epithelium of Evi1Jbo/+ mice compared to their age matched WT littermate controls  from WT and Evi1Jbo/+ mice using RT-qPCR. This analysis showed that there was a significant down-regulation of Bpifa1 mRNA in Evi1Jbo/+ mMMCs when compared to WTs  compared to Evi1Jbo/+ mice  from 7 to 10 week old Bpifa1\u2212/\u2212Evi1Jbo/+, Evi1Jbo/+, Bpifa1\u2212/\u2212 and WT mice. Although mMMCs of Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ mice had significantly elevated levels of the pro-inflammatory cytokines Cxcl2, Il1\u03b2, Tnf\u03b1 and the profibrotic mediator, Tgf\u03b2, compared to WT mMMCs, there was no significant difference in the expression levels for any of these genes between WT and Bpifa1\u2212/\u2212 mMMCs or between Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ mMMCs  and fibroblasts isolated from the bullae of Bpifa1\u2212/\u2212Evi1Jbo/+, Evi1Jbo/+, Bpifa1\u2212/\u2212 and WT mice. No significant differences in doubling times were seen in either mMECs  by 10\u201318 months of age30. This apparent difference could be due to strain differences (as we have studied mice on C57BL/6\u2009J and C3H/HeH backgrounds) and could be further examined by studying our Bpifa1\u2212/\u2212 model at a comparable age. However, OM is typically studied in young mice as this better represents the timing of the paediatric disease in humans28 and the relevance of any late development of the disease is unclear. As mice over expressing BPIFA1 showed reduced levels of lung fibrosis when exposed to a sterile irritant37, it is possible that extended exposure to respiratory irritants or previous episodes of bacterial infection may be contributory to the low penetrance OM in aged Bpifa1\u2212/\u2212 mice30.However, our data shows that loss of bpifa1 has previously been shown to predispose mice more severe respiratory infections following an experimental challenge and yet we found that IN challenge of Bpifa1\u2212/\u2212 mice with the major otopathogen NTHi, did not result in significant ME infection, although our data did appear to suggest that Bpifa1\u2212/\u2212 mice infection prevalence was marginally higher. NTHi is a significant causative pathogen isolated from a majority of AOM and COME patients38 and the potential role that BPIFA1 plays in defence against this organism requires further studies.The loss of Bpifa1\u2212/\u2212 mice, we investigated the contribution of the protein to OM disease progression by studying compound mutant Bpifa1\u2212/\u2212Evi1Jbo/+ mice. Evi1Jbo/+ mice represent an established model of OM39 and develop hallmarks of the disease soon after middle ear cavitation occurs. Our analysis suggests that BPIFA1 protein staining was reduced in the remodelled epithelium in Evi1Jbo/+ mice compared to WT. It seems possible that this loss of staining may be due to an alteration of cell populations in the mucosa, with a reduction in the number of BPIFA1 producing cells being present. However we could still readily detect the protein in the ME exudates from Evi1Jbo/+ mice. This is consistent with proteomic data from human ME exudates27 and our own data from the apical secretions of differentiated mMECs29.Given the high levels of BPIFA1 seen in the ME, but the absence of spontaneous OM or lack of significantly increased susceptibility to ME infection by NTHi in our Bpifa1\u2212/\u2212Evi1Jbo/+ bullae demonstrated an exacerbation of OM severity compared to Evi1Jbo/+ mice. This suggests that removal of BPIFA1 from the middle ear environment allows for a more significant remodelling response. This further suggests a shifting of the time required to produce the OM phenotype. Indeed analysis of compound mutant mice at day P21 also revealed an exacerbation of OM severity (results not shown). It seems likely that the loss of BPIFA1 from the middle ear mucosa during the process of cavitation directly exacerbates the development of OM. Previous studies of Evi1Jbo/+ bulla exudates demonstrated elevation of inflammatory gene networks and hypoxia signalling genes compared to venous blood as a baseline control39. We have shown up-regulation of the pro-inflammatory mediators Cxcl2, Tnf\u03b1, Il1\u03b2 and Tgf\u03b2 in the ME mucosa of Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ mice. Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ bullae also appeared to demonstrate epithelial cellular alterations characterised by down regulation of the ciliated cell marker, Foxj1, and the secretory cell marker, Muc5ac, and Evi1Jbo/+ mice demonstrated reduced levels of Bpifa1 expression. However, despite the exacerbated OM phenotype in Bpifa1\u2212/\u2212Evi1Jbo/+ mice, there was no significant additive effect of the loss of Bpifa1 on the expression of these inflammatory and epithelial markers. This may be a consequence of comparison based on the already inflamed Evi1Jbo/+ epithelium that has high basal expression levels of these inflammatory genes. At this time we are unable to identify exactly which cell type(s) express these pro-inflammatory markers but our mucosal cell isolation procedure generates a population of structural  cells without a significant inflammatory cell component.The most striking observation from these studies is that Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ mice do not appear to be explained by proliferation rates of the epithelial cells and fibroblasts measured ex-vivo. It seems likely that inflammatory changes such as connective tissue oedema, dilation of lymphatics and blood vessels, infiltration of inflammatory cells and fibrosis are responsible for the mucosal thickening seen in the ME. An interesting feature observed in the inflamed Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ MEs was fibrosis of the sub-epithelial connective tissue, indicated by collagen deposition and accumulation of myofibroblasts. These features are characteristic of fibrotic pulmonary diseases such as asthma and idiopathic pulmonary fibrosis40 and indicative of epithelial remodelling due to an over-active wound repair response following inflammation. In turn this is suggestive of over-activation of the TGF\u03b2 signalling pathway (a target for EVI1)41. The enhanced thickness of the Bpifa1\u2212/\u2212Evi1Jbo/+ mucosa compared to that of Evi1Jbo/+ mice suggests greater fibrosis due to loss of BPIFA1. BPIFA1 has been previously associated with an allergen-induced asthma-like phenotype in mice42 and overexpression of BPIFA1 has been shown to protect against pulmonary fibrosis37. A recent report suggesting a role for BPIFA1 as an epithelial derived smooth muscle relaxing factor43 is supportive of a potential role for this epithelial protein in mediating crosstalk with the underlying connective tissue.The differences in the mucosal thickening of Bpifa1 in the inflamed microenvironment of the Evi1Jbo/+ ears. This reduction in BPIFA1 may represent transcriptional down regulation of Bpifa1 or perhaps more likely is a consequence of phenotypic changes in the epithelial cell populations. Previous work with BPIFA1 has largely focused on evaluation of a role for the protein in bacterial and viral pathogen challenge studies or in the presence of pre-existing chronic allergic and inflammatory pulmonary disease (for review see16). Importantly, we did not observe any phenotypic abnormalities or signs of organ inflammation in the non-challenged Bpifa1\u2212/\u2212 mice. Thus, the presence of an infectious or inflammatory stimulus seems to be a pre-requisite to uncover functional roles of BPIFA1. This may also explain why Bpifa1 deletion alone does not lead to the spontaneous development of OM in Bpifa1\u2212/\u2212 mice. Consistent with this hypothesis we have recently shown that Bpifa1\u2212/\u2212 mice are more susceptible to influenza A infection through a mechanism that allows Bpifa1 deficient cells to be more readily infected by the virus44.An important observation in this study was the downregulation of epithelial 46. Further studies are required to address this point and whether the mechanism(s) are via alteration in surfactant function, epithelial permeability, and/or impaired antimicrobial action.This work provides new insights into the function of BPIFA1 in the ME. From birth onwards BPIFA1 is secreted at high levels into a film that overlies the epithelium lining the ME, but its absence does not predispose to spontaneous OM or increase the susceptibility to bacterial infection. This indicates that, within normal limits, BPIFA1 is a functionally redundant innate immune protein that plays a homeostatic role in mucosal protection in a manner described for multiple respiratory proteinsWe have explored the role of BPIFA1 in mice bearing an OM predisposing Evi1 mutation, and shown an upregulation of ME epithelial cytokine expression and down-regulation of epithelial markers, with dispersal of BPIFA1 from this surface layer into bulla fluid. Deficiency of BPIFA1 on an Evi1 mutant background does not markedly alter epithelial cytokine and cell marker expression probably via perturbation of epithelial cell populations, and consequently exacerbates inflammatory thickening of the submucosal connective tissues.Bpifa1 on an OM prone background results in the earlier initiation of OM and whether, once initiated, the progression of disease is significantly altered. It is also important to study if loss of BPIFA1 exacerbates the OM phenotype in other OM mutant mouse lines. Given that features such as mucosal fibrosis have been noted in chronic OM in Tlr4 and Evi1 mutant mice47, early fibrosis, and mucosal thickening may suggest accelerated disease in the absence of BPIFA1 rather than substantially altered pathogenesis.Together this suggests that BPIFA1 aids homeostasis in the ME by contributing to the epithelial barrier function, and its role is revealed only under significant epithelial insult. Future time course studies are needed to define whether the deletion of Mice were bred and maintained by the Mary Lyon Centre, MRC Harwell and were housed in specific-pathogen free conditions. All animal experimentation was approved by the Animal Welfare and Ethical Review Body at MRC Harwell. The humane care and use of mice in this study was under the authority of the appropriate UK Home Office Project License.Bpifa1\u2212/\u2212 mice on a C57BL/6 background were obtained from Professor Ralph Shohet at the University of Hawaii. The generation of these mice in which exons 2 and 3 of the Bpifa1 gene have been deleted have been described in detail elsewhere44. C57BL/6 Bpifa1\u2212/\u2212 mice were re-derived at MRC Harwell Institute, UK and backcrossed 5 times to C3H/HeH mice to produce a congenic background. Junbo heterozygote (Evi1Jbo/+) mice were maintained on the C3H/HeH background9. Compound Bpifa1\u2212/\u2212Evi1Jbo/+ mutants and their littermate controls were generated by intercrossing Bpifa1\u2212/\u2212 and Evi1Jbo/+ mice and mice were examined at P28 and at 6 months of age. P0-P30 WT C3H/HeH mice, used for IHC of BPIFA1, were obtained from MRC Harwell Institute. For comparative purposes Bpifa1\u2212/\u2212 mice on the original C57BL/6 background and a mixed C3H/HeH-C57BL/6 background were used in some infection studies. Husbandry of SPF mice and microbiological surveillance is described elsewhere9.9.The hearing thresholds of 8 and 12 week-old mice were assessed using ABR measurements as previously describedMice were euthanized by intraperitoneal overdose of sodium pentobarbital . The mouse was decapitated and head skin removed. The tympanic membrane was examined under 10\u00d7\u2009binocular magnification to establish whether there was evidence of opacity which is indicative of macroscopic bulla fluid accumulation.Bpifa1\u2212/\u2212 mice at 6 months of age were analysed for morphological abnormalities.Tissue samples were fixed in 10% formaldehyde (Surgipath Europe) for 48\u2009hours at room temperature and the skulls decalcified in Kristenson D.F.B agent (Pioneer Scientific) for 72\u2009hours before wax embedding. Four \u00b5m H&E stained sections of the bullae and nasal passages were scanned using the NanoZoomer Digital Pathology system and morphometric measurements made with NanoZoomer software (Hamamatsu). Thickness of the ME mucosa was measured in a defined 1\u2009mm area in the promontory region opposite the tympanic membrane. Average mucosal thickness was calculated using 5 measurements taken at a distance of 250 \u03bcm within this region. Both male and female mice were analysed and no sex-related differences were seen in the development of OM. H&E slides for sections of a total of 42 target tissues from 31 or rabbit anti-\u03b1SMA (1:200) (Abcam 5694) antibodies overnight in a humified chamber at 4\u2009\u00b0C then washed twice in PBS, incubated in 0.5% biotinylated polyclonal goat anti-rabbit secondary antibody  for 30\u2009minutes at room temperature, followed by incubation for 30\u2009minutes with the ABC reagent for signal amplification before colour detection using a NovaRed system . Images were captured with an Olympus BX61 light microscope and Olympus colour view digital camera.Four \u00b5m wax sections were used for IHC. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide in methanol . Non-specific binding was blocked by incubating the sections in 100% goat serum for 30\u2009minutes at room temperature. Sections were washed and incubated in rabbit anti-Bpifa1 (1:750)SR), was performed as previously described48.Intranasal bacterial challenge experiments, using genomically sequenced, streptomycin resistant strain NTHi #375 . The pressure was raised to approximately 1.4 pounds per square inch (Psi) by compressed air through an inlet port. Thereafter, the pressure was increased by approximately 0.7 Psi every 15 secs to achieve a maximum pressure of 5.8 Psi, then after 2\u2009minutes was decreased by venting air via an outlet port at 30 secs intervals to return the chamber to atmospheric pressure.The baroinoculation protocol was adapted from previously published studies in mice29. Fibroblasts were isolated using a differential adherence method described in the same paper. Adherent fibroblasts were cultured by adding 5\u2009ml of mMEC basic 10% FBS media to the tissue culture dish29 and incubating at 37\u2009\u00b0C in a 5% CO2 incubator till confluent. Upon confluence, cells were washed with warm HBSS and sub-cultured twice to obtain a pure fibroblast population. For the third passage, 3mls of Trypsin-EDTA solution (Sigma) was added to the dish and incubated as above for a minute to dislodge the cells. Trypsin was neutralised by addition of 3\u2009ml neutralising medium (mMEC basic plus 10% FBS) and cells were centrifuged at 500\u2009\u00d7\u2009g for 5\u2009minutes. Cell pellets were resuspended in 5 mls of fresh mMEC basic 10% FBS and split into five T25 flasks for expansion of the fibroblast cultures. 4\u2009ml of media was added to each flask and cells were cultured at 37\u2009\u00b0C in 5% CO2 till confluence. When calculating population doubling time (PD), the purified fibroblasts were seeded at 5\u2009\u00d7\u2009104 cells/well in mMEC basic 10% FBS media in 6 well tissue culture plates . Similarly, fresh mMECs were seeded at 5\u2009\u00d7\u2009104 cells/well in mMEC- Plus media29 in submerged culture. Media was changed every 48\u2009hours. Fibroblasts were harvested every 24\u2009hours from day1 to day 3 whereas mMECs were harvested every 48\u2009hours by trypsinisation and quantified using a haemocytometer from day 2 to day 10.The isolation of mMECs was performed as previously described10, C1\u2009=\u2009initial cell count, C2\u2009=\u2009final cell count.Population doubling time was calculated using the formula.48, these were then mixed with an equal volume of 2XSDS lysis buffer. Samples were denatured at 95\u2009\u00b0C for 5\u2009minutes and resolved on a 12% polyacrylamide gel, transferred to a PVDF membrane using a semi dry blotting system (Biorad Trans-blot turbo) then probed with rabbit anti-Bpifa1 primary antibody (1:20031), overnight at 4\u2009\u00b0C. The primary antibody was detected using a polyclonal goat anti-rabbit secondary antibody (Dako P0448) conjugated with HRP (1:2000) and bound antibody was visualised using the EZ-Chemiluminescence detection system  and manual development. Images were scanned as a whole with no modification.Bulla fluids were sampled by pipetting as previously describedBpifa1\u2212/\u2212, Evi1Jbo/+ and Bpifa1\u2212/\u2212Evi1Jbo/+ MEs, mouse mucosal epithelial cells (mMMCs), i.e. ME epithelial cells before undergoing the differential fibroblast separation, were used. Total RNA was extracted, cleaned from residual genomic DNA contamination, quantified and reverse transcribed into cDNA as previously described29. RT-qPCR was performed using Applied Biosystems TaqMan gene expression assays for Cxcl2 (Mm00436450_m1), TNF\u03b1 (Mm99999068_m1), TGF\u03b2 (Mm01178820_m1), Bpifa1 (Mm00465064_m1), FoxJ1 (Mm01267279_m1) and Muc5ac (Mm01276718_m1) on a 7500 Fast Real Time PCR system (Applied Biosystems), with 2\u00d7\u2009Taqman Fast Universal Master Mix . 10 ng cDNA was added to each reaction and three technical replicates were performed for each assay in each batch. Target gene expression levels were normalized to three endogenous controls: Atp5b, Cyc1 and Canx (Primerdesign geNormTM Reference Gene Selection Kit) and analysed using ABI 7500 software v2.0.1 using the 2\u2206\u2206Ct\u2212 method. Data is presented as mean Relative quantification (RQ) and error bars represent standard error of the mean.For comparison of gene expression between WT, t-test was used to compare two groups of data. One way or two-way analysis of variance (ANOVA) with Tukey\u2019s post hoc test was used to compare more than two groups, for parametric data. Fisher\u2019s exact test was used for analysing frequency data such as presence or absence of bulla fluids or genotype ratios . Significance threshold was set at p\u2009<\u20090.05. Data are presented as the mean +/\u2212 standard error of the mean (SEM) unless otherwise stated. Significance levels represented on graphs are as follows: ns\u2009=\u2009not significant; *p\u2009\u2264\u20090.05; **p\u2009\u2264\u20090.01; ***p\u2009\u2264\u20090.001; ****p\u2009\u2264\u20090.0001.The normality of experimental data was determined by performing Levene\u2019s test prior to the statistical analysis. GraphPad Prism (version 6.0) was used to perform all statistical tests. 2 tailed students Supplementary Dataset 1"}
+{"text": "PGD2 is released from guinea pig bladder urothelium/suburothelium and can inhibit detrusor contractile responses. We presently wished to investigate PGD2 actions in guinea pig out\u2010flow region and the distribution of DP1/DP2 receptors. The effects of PGD2 on urothelium\u2010intact trigone and proximal urethra contractility were studied in organ bath experiments. Expression of DP1/DP2 receptor proteins was analysed by western blot. Immunohistochemistry was used to identify distribution of DP1/DP2 receptors. PGD2 in a dose\u2010dependent manner inhibited trigone contractions induced by electrical field stimulation (EFS) and inhibited spontaneous contractions of the proximal urethra. PGD2 was equally (trigone) or slightly less potent (urethra) compared with PGE2. Expression of DP1 and DP2 receptors was found in male guinea pig bladder trigone, neck and proximal urethra. In the trigone and proximal urethra, DP1 receptors were found on the membrane of smooth muscle cells and weak immunoreactivty was observed in the urothelium. DP2 receptors were distributed more widespread, weakly and evenly in the urothelium and smooth muscles. Inhibitory effects by PGD2 on motor activity of guinea pig trigone and proximal urethra are consistent with finding DP1 and DP2 receptors located in the urothelium and smooth muscle cells of the trigone and proximal urethra, and PGD2 may therefore be a modulator of the bladder out\u2010flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome.The proximal urethra and urinary bladder trigone play important roles in continence. We have previously shown that  The lower urinary tract (LUT) consists of the urinary bladder and the urethra with the functions of urine storage and periodic urination. Continence and micturition involve a balance between detrusor activity and urethral sphincter closure. Bladder filling and voiding are controlled by a complex pattern of afferent and efferent signalling in parasympathetic, sympathetic and somatic pathways In the LUT, the bladder trigone is a smooth region in the base of urinary bladder within the two ureteral orifices and the internal urethral orifice which has different embryological origins from the rest part of the bladder. The majority of vessels and nerves of the bladder enter and concentrate in the trigone making it very sensitive to expansion 2 is an important lipid mediator that exerts its biological functions via the G protein\u2010coupled receptors prostaglandin D receptor type 1(DP1) and prostaglandin D receptor type 2 (DP2), the later also known as chemoattractant homologous receptor expressed on Th2 cells (CRTH2). We have previously shown that PGD2 and PGE2 were released from guinea pig urinary bladder and that PGD2 inhibited induced bladder detrusor contractions 2 on the trigone and proximal urethra has been studied for decades. Andersson and colleagues showed that PGE1 and PGE2 relaxed pre\u2010contracted human circularly cut urethral rings 2 was shown to relax the pre\u2010contracted trigone and longitudinally cut human and pig urethra i.e. PGE2 relaxed the pre\u2010contraction of circularly cut hamster and longitudinally cut dog urethra 2 contracted the longitudinal urethra strips but relaxed the circular urethra muscle 2 enhanced the tone and increased spontaneous activities 2 is involved in the regulation of trigone and urethra motility remains unknown. In the human, expression of functional DP receptors was found in corpus cavernosum smooth muscle 1 receptors was found in the smooth muscle and urothelium with a dominant localization to smooth muscle membranes, DP2 was also found on the bladder wall 1 and DP2 receptors in the proximal urethra and trigone regions have not been reported.Prostaglandin D2 and PGE2 on male guinea pig trigone and proximal urethra and report on the efficacy of PGD2 and PGE2 in these tissues. We describe the expression and distribution of DP1 and DP2 receptors in trigone and proximal urethra with respect to their distribution in both urothelium and muscle components.In the present study, we examined the effects of PGDen bloc. Seminal vesicles, deferent ducts, coagulating gland ducts and ejaculatory ducts were removed at duct openings. The trigone was dissected by locating the urethra and ureter openings. A trigone strip about 7 \u00d7 2 mm was made from each guinea pig and with the urothelium intact. The IUS ring from the level of bladder neck to above duct openings of the proximal urethra was dissected with intact urothelium was opened and cut into 1\u20132 strips for organ bath experiments. All tissue strips were tied at both ends with thin cotton threads and equilibrated in 5.5 ml organs bath containing Tyrode's solution  and aerated with 5% CO2 in O2 at 37\u00b0C.All experiments were approved by the local animal ethics committee (Dnr N178/11). Male albino guinea pigs weighing 500\u2013750 g were anaesthetized with midazolam 1 mg/kg + sodium pentobarbital 120 mg/kg and exsanguinated. The urinary bladder and proximal urethra were removed \u22126 M was given to the trigone tissues to inhibit the production of endogenous prostaglandins. After 10 min. incubation with diclofenac, the tissues were washed and diclofenac 10\u22126 M was reapplied to trigone strips throughout the experiment.After 30 min. equilibration, one end of the tissue was connected to an isometric transducer and the other end to a hook at the bottom of the bath. Tissues were carefully washed with Tyrode's three times. The initial resting tension of the trigone and urethra strips was adjusted to 5 mN. Proximal urethra strips were left unstimulated to record the spontaneous contractions. When stable tension developed, trigone strips were electrically stimulated by means of two platinum electrodes on the walls of the organ baths . The evoked contractions were recorded with a computerized acquisition system . When stable contractions were recorded, diclofenac 102 and PGE2 were added to the tissues cumulatively in log increments from 10\u22129 to 10\u22126 M. Each dose was applied for 10 min. Control contraction amplitudes were measured before application of PGD2 and PGE2. Contractile response at 10 min. at every dose of PGD2 and PGE2 were measured and compared with control amplitude. Log concentration\u2010response curves were constructed.PGD2 and PGE2 were given to the urethra strips cumulatively in half\u2010log or log increments from 10\u22129 to 10\u22126 M at 8\u201310 min. intervals in the absence of diclofenac since cyclo\u2010oxygenase inhibitors were reported to inhibit spontaneous contractions in the urinary tract 2 and PGE2 and expressed as area per min. Log concentration\u2010response curves were constructed.After regular spontaneous contractions were developed, PGD2 and PGD2 was applied cumulatively in log increments to trigone and urethral tissues without any compound. The final concentration of ethanol in each organ bath was less or equal to 0.1%.The corresponding amount of ethanol used to dissolve PGE1 receptor C\u2010terminal antibody  or rabbit anti\u2010human DP2 (CRTH2) receptor antibody  diluted in PBS\u2010Tween 20 with 5% skim milk. HRP\u2010conjugated goat anti\u2010rabbit secondary antibodies  and Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific) were used to detect protein signal on autoradiographs .Male guinea pigs were anesthetized as above and the abdominal aorta was flushed distally with 30\u201340 ml warm saline to achieve blood\u2010free tissues. The bladder trigone, neck and proximal urethra were dissected and isolated apart. For protein extraction, each mg wet tissue was subjected to 20 \u03bcl of lysis buffer (pH 7.6) containing 20 mM Hepes, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 25 mM KF, 1 mM sodium orthovanadate, 0.5% Triton X\u2010100, 20% glycerol and 1% protease inhibitor cocktail . Tissues were homogenized using an Ultra\u2010Turrax for 2 min. and then homogenized for 4 min. in a Dounce glass homogenizer. Lysates were centrifuged at 13,000 \u00d7 g gravity for 20 min. at +4\u00b0C. Protein content of the supernatant was determined with the Bradford protein assay . 7 \u03bcg of protein was loaded onto 8\u201316% SDS Pierce Protein Gel  and separated by electrophoresis. Proteins were transferred onto PVDF membranes using dry blot/iBLOT according to the manufacturer's instructions . Membranes were blocked for one hour with 5% skim milk dissolved in PBS\u2010T . Membranes were probed overnight with rabbit anti\u2010human DPen bloc and cleaned from connective tissues. The bladder was flushed of urine before fixation which was by immersion in ice\u2010cold 4% paraformaldehyde 0.1 M phosphate buffer fixative solution for 4 hrs at 4\u00b0C. After fixation, tissues were cryoprotected by incubation in 0.1 M phosphate buffer with 30% sucrose solution for 16\u201320 hrs at 4\u00b0C. The trigone and urethra were then dissected from the bladder. The proximal urethra was cut transversely into several ring segments. Small pieces of tissue were covered with Neg\u201050\u2122 (Thermo Scientific) and quickly frozen in liquid nitrogen cooled isopentane and stored at \u221280\u00b0C for later cryostat sectioning. 10 \u03bcm cryostat sections of vertical trigone and transverse urethra were mounted on gelatin coated slides.Male guinea pigs were anaesthetized and perfused as above. The urinary bladder with short ureter remains and proximal urethra were taken 1 receptor antibody  or labelled with the rabbit anti\u2010human DP2 receptor antibody . All the antibodies were diluted in blocking buffer. The sequential control sections were treated with blocking buffer. Cryostat sections labelled with antibodies were incubated overnight at 4\u00b0C. Before secondary antibody treatment, the sections were rinsed three times for 5 min. in PBS. Then, sections were incubated for 1 hr with secondary antibodies at room temperature. Counterstaining of smooth muscle actin, F\u2010actin and nuclei was made by monoclonal anti\u2010\u03b1\u2010smooth muscle actin antibody , phalloidin  and Hoechst33258 , respectively. The secondary antibodies used in this study were: donkey anti\u2010rabbit antibody labelled with Alexa Fluor 568  and donkey anti\u2010rabbit antibody labelled with Alexa Fluor 488 . After 1 hr incubation, sections were rinsed three times in PBS for 5 min. in the dark. The sections were then mounted in S3023 medium with anti\u2010fading agent  and covered with coverslips.Cryostat sections were blocked in blocking buffer with PBS (pH 7.2) containing 0.5% Triton X\u2010100 and 5% normal bovine serum for 20 min. at room temperature. Sections were either labelled with the rabbit anti\u2010human DPAll immunolabelled sections were observed under an Axioplan 2 imaging fluorescence microscope  equipped with FITC (Chroma 41001), TRITC (Chroma 41002a) and DAPI (Chroma #31000) filters. Sections were photographed with a Nikon D7000 digital camera using NKRemote software  for camera control with 12\u2010bit image acquisition followed by subsequent background subtraction and contrast enhancement in ImageJ (NIH open source).2 and PGD2 were generous gifts from Professor Ernst H Oliw . Diclofenac, atropine, tetrodotoxin and D\u2010tubocurarine were from Sigma\u2010Aldrich. All the data are presented as mean \u00b1 S.E.M. PGE2 and PGD2 log dose\u2013response curves were fitted by a 4\u2010parameter sigmoidal curve model using Prism 5  to estimate the IC50 of PGE2 and PGD2.PGEIn vitro, unstimulated urothelium intact guinea pig urinary bladder trigone strips showed irregular, fast and fast\u2010relaxing (3\u20134 sec.) spontaneous contractions with a frequency of 4.71 \u00b1 0.74 contractions per min (n = 7). When the trigone strips were electrically stimulated , monophasic and reproducible post\u2010stimulation contractile responses were observed. These were nerve\u2010mediated as indicated by their sensitivity to tetrodotoxin. Muscle strips from the level of bladder neck had no spontaneous activity and did not respond to nerve stimulation. Strips of proximal urethra taken below the bladder neck and above the duct openings, representing the major region of the IUS, showed regular, slow and long\u2010lasting (30\u201340 sec.) spontaneous contractions , PGE2 or PGD2 was applied cumulatively from 10\u22129 to 10\u22126 M. PGE2 and PGD2 inhibited the EFS\u2010induced contractions in a dose dependent manner. In some experiments, low concentrations of PGE2 (10\u22129 to 10\u22128 M) elicited a weak enhancement of contractile responses, whereas higher concentrations (above 10\u22128 M) always elicited inhibition. Dose\u2013response curves  and an estimated pIC50 value for PGD2 of 7.08 \u00b1 0.27 (n = 6). The corresponding amounts of ethanol used to dissolve PGE2 and PGD2 had no effect on EFS contractions when applied to the trigone strips.Reproducible repeated contractile responses of isolated trigone were induced by EFS as above. After treatment with diclofenac  for PGE2 and 7.09 \u00b1 0.22 (n = 14) for PGD2. Ethanol at the corresponding concentrations as used to dissolve PGE2 and PGD2 did not modify the IUS spontaneous contractions.Urethral strips from below the neck and above the duct openings, which is the IUS region exhibited regular spontaneous contractions that were unaffected by atropine 5 \u00d7 101 and DP2 (CRTH2) receptor proteins in the guinea pig trigone, bladder neck and proximal urethra was examined by Western blot. Tissue extracts, containing the whole layers of both muscle and urothelium from these regions were exposed to DP1 and DP2 antibodies and which were the same antibodies used in the immunohistochemistry. As shown in Figure 1 and DP2 proteins. In Figure 1 panel, two groups of protein bands at, respectively, the predicted molecular weight for DP1 (40 kD) and at around 95 kD were seen. This result fits the data provided by the manufacturer in tests with the antibody on different cell lines. In Figure 2 panel, only one group of DP2 protein bands was observed at around 75 kD. The predicted band location for DP2 is 40 kD, the reason for observing an increased size of the protein bands is likely a reported post\u2010translational modification, e.g. phosphorylation, glycosylation etc. The expression of DP for DP1 0 kD and 1 and DP2 receptors was seen throughout the trigone urothelium and smooth muscle. DP1 receptors were more prominent in the smooth muscle layer while DP2 receptors were more evenly distributed in the urothelium and smooth muscle layers but not as strong and localized as DP1 receptors in the muscle cells. Figure 1 receptor distribution in the urothelium of the trigone. The border between urothelium and suburothelium was heavily stained for DP1 receptor with localization to the cells immediately below the membrane but only faint fluorescence was seen in the urothelium layer, with a predominant localization to urothelium cell membranes. Figure 1 receptor distribution in the smooth muscle bundles. As shown in Figure 1 receptor antibody was found surrounding the red fluorescence for muscle actin, indicating localization of DP1 receptor mainly in the membranes of smooth muscle cells. The blue fluorescence in Figure i.e. not exposed to primary antibody, only secondary antibodies) showed no staining for DP1 or DP2 receptors.Fluorescence immunohistochemistry results from sequential vertical sections of the trigone with transitional urothelium (uro) and part of the smooth muscle (sm) layers are shown in Figure The morphology of the male guinea pig urethra differs depending on the position at which sections are taken. In this study, we focused on the proximal urethra with the IUS. Figure 1 receptor was seen distributed in the proximal urethra urothelium  and anti\u2010\u03b1\u2010smooth muscle actin (red) shows strong yellow fluorescence indicating co\u2010localization of DP1 receptor on the smooth muscle bundles  and striated muscle (st) in male guinea pig proximal urethra is shown in Figure les Fig. C2. The dcle Fig. C1.2 can exert inhibitory influence on smooth muscle contractile responses induce by EFS and on spontaneous contractions. DP1 and DP2 receptors are found expressed in the trigone and proximal urethra. In the bladder trigone, DP1 receptors are markedly located on the suburothelium layer and smooth muscle cells membranes similar as in the proximal urethra. DP2 receptors are found weakly and evenly on the urothelium/suburothelium layers and also in the smooth muscle.The major novel findings in the present study are that, in male guinea pig trigone and proximal urethra PGDin vitro study using human bladder strips found no effect of PGD2 up to 3 \u00d7 10\u22127 M on resting tension and was contractile at higher concentrations 2 was produced in guinea pig urinary bladder in a urothelium\u2010dependent fashion and exerted its inhibitory effects on EFS induced contractions via DP1 receptors localized on the detrusor membrane 2 together with other relaxing mediators such as PGE2 is produced by the urothelium/suburothelium to relax the underlying smooth muscle. When the bladder reaches a threshold volume, an emptying process is triggered where PGD2 together with other mediators might reduce the amplitude of initial detrusor muscle contractions, but also by relaxation of the internal sphincter might facilitate entry of urine into the proximal urethra. Passage of urine into the proximal urethra is a signal in the initiation of micturition A previous We observed distinct regional inherent activity and responses to EFS along the proximal urethra. An earlier study measuring the urethral pressure profile of the male feline urethra using a silicone rubber catheter with pressure transducers showed increased urethra pressure from proximal to distal with several peaks at the region of prostatic urethra, bulbourethra and penile urethra 1 and DP2 receptor distribution is supported by our Western blot data and by results with similar antibody in a study by Zhang et al. who showed that DP1 and DP2 receptors are present in guinea pig oesophageal nodose ganglia by immunostaining and Western blot with similar antibody, and by RT\u2010PCR 1 and DP2 receptors located in the urothelium and smooth muscle and that DP1 receptors were prominent on the membranes of smooth muscle cells, in agreement with our previous study on bladder detrusor 2 might therefore be suggested to directly bind to the receptors located on the smooth muscle, tentatively regulating the contractility by modulating muscle cAMP level since this is a known mechanism in the PGD2 inhibitory action on smooth muscle The validity of our histochemical results on DPin vitro design using male guinea pig trigone and proximal urethra strips. In vivo experiments dealing with the whole LUT with intact neural system will be necessary to determine the exact functional implications of our results. Studies on female urethra should also be carried out in the future.Some limitations of this study include the fact that it was an 2 on the guinea pig trigone and proximal urethra and is consistent with the expression and distribution of DP1 and DP2 receptors in these regions. PGD2 may therefore be a modulator of the bladder out\u2010flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome. The information is of value for our further understanding of the LUT physiology and provides a foundation for future studies on the human out\u2010flow region.The present study suggests an inhibitory influence of PGDNG conceived and designed the study and performed all the organ bath experiments after discussion with LG. NG and PdV performed the Western blots and NG and KS performed the immunohistochemistry experiments which were evaluated together with LG and PW. NG compiled the data and made all the draft figures which were finalized together with LG. NG drafted the first manuscript which was reviewed and revised by all authors.The authors confirm that there are no conflicts of interest."}
+{"text": "Fibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.We used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification  was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20\u00d7 objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n\u00a0=\u00a012). All highly amplified samples showed very strong FGFR2 mRNA expression . Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0\u20133 patients.We successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong RNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.The online version of this article (doi:10.1007/s10120-017-0758-x) contains supplementary material, which is available to authorized users. Gastric cancer is the third most frequent cause of death from cancer worldwide . WorldwiOverexpression and gene amplification of fibroblast growth factor receptors 2 (FGFR2) has been associated with poor outcome in gastric cancer , 13. DifFGFR2 gene copy number . For analysis of both overall survival (OS) and recurrence-free survival (RFS), univariate Cox proportional hazards models and Kaplan\u2013Meier estimates were computed, complemented by a multivariate Cox model analysis adjusting for clinical covariates. The latter models were moreover subjected to Akaike information criterion (AIC)-based backward variable selection to ensure robust and informative statistical modeling.All analyses were done in R v3.2.3  ; FGFR2 gene amplification and patient outcome data. Univariate Cox PH and Kaplan\u2013Meier analysis suggested that patients with tumors that had FGFR2 gene amplification had shorter RFS than patients with nonamplified tumors   Fig.\u00a0a. After  OS Fig.\u00a0b.Fig.\u00a07PFGFR2 gene amplification with different methods are currently ongoing; however, no FGFR2-targeted agent along with a biomarker assay has been approved yet. FGFR2 mRNA expression rather than FGFR2 gene amplification has been discussed as a biomarker to select patients for FGFR2-targeted therapies in gastric cancer [FGFR2 gene amplification and mRNA expression as biomarkers in gastric cancer. The gold standard to detect FGFR2 gene amplification is FISH. However, the method is expensive and requires fluorescence microscopy, limiting its use in clinical practice. Polymerase chain reaction (PCR)-based methods are cheaper but lack spatial resolution; the latter might be problematic considering that gastric cancer has been shown to have a very high rate of intratumor heterogeneity [FGFR2 gene amplification determined with another in situ technique, dual-color in situ hybridization (DISH), which does not require fluorescence microscopy and is more convenient to use compared to FISH. Very recently, Han et al. performed a study exploring FGFR2 mRNA expression by in situ techniques and FGFR2 protein expression by IHC in 362 surgically resected gastric cancer tissues and 135 matched metastatic lymph nodes from Korea. FGFR2 gene amplification was determined by FISH in 188 of the samples [FGFR2 has been proposed as a target for the treatment of gastric cancer . Severalc cancer . We wantogeneity . We therFGFR2 gene amplification in 578 patients. We were able to generate both FGFR2 gene amplification and mRNA expression for 468 patients. We detected a high level of FGFR2 gene amplification (FGFR2:CEN10 >10) in 2% (12/578) of the samples, and high FGFR2 mRNA expression score 4 was detected in 4% (29/718) of samples. Moderate FGFR2 gene amplification  was detected in 8% (47/578). Previous studies have reported FGFR2 gene amplification in 3.6\u20139% of gastric cancer cases [FGFR2 gene amplification and 5.8% FGFR2 mRNA overexpression, which is very similar to the 2% of cases with high FGFR2 gene amplification and 4% mRNA score 4 patients we detected in a large cohort of Japanese gastric cancer patients [Not all the available samples were analyzed by DISH; 1036 samples were analyzed by RNAscope and only 967 of those were also analyzed by DISH. Some samples could not be analyzed because the core in the TMA was missing or the sample did not have sufficient tumor content. The percentage of successful analysis of samples was acceptable, with 69% for RNAscope and 60% for DISH (Supplementary Table\u00a03). For 27% of the tested samples the RNAScope assay failed; 36% of the assayed DISH samples did not give a result. The samples analyzed in this study were fixed in unbuffered formalin and stored for more than 10\u00a0years, explaining the rather high failure rate. Both in situ techniques detect nucleic acids, and factors preserving or degenerating nucleic acids will affect both methods in a similar way. In line with that expectation, 68% of the samples tested either failed both assays or were successfully analyzed with both methods (Supplementary Table\u00a03). There was no major difference in failure rate between the two methods. The success rate is slightly higher for RNAscope. We cannot exclude the possibility that degradation of nucleic acids in some samples biased the analysis. FGFR2 mRNA was successfully determined in 718 patients and er cases , 37\u201343, FGFR2 gene amplification also showed high levels of FGFR2 mRNA expression. All samples with dense clusters of RNAscope signal, visible under a 1\u00d7 objective, showed a high level of FGFR2 gene amplification with a FGFR2:CEN10 ration >10. Surprisingly, these samples also showed a more homogeneous FGFR2 expression within the tumor sample, suggesting that FGFR2 gene amplification might have been an early event in the development of these gastric cancer cases. The scoring algorithm described in the RNAscope kit instructions does not allow separating this potentially biologically distinct subgroup from regular score 4 cases. We therefore suggest adding a score 5 category for samples with dense clusters of RNAscope signal visible under a 1\u00d7 objective. There were only 12 score 5 samples with high FGFR2 gene amplification in our study. Further studies may help to explore if this subgroup is indeed biologically distinct from other gastric cancer patients.We assessed intratumor heterogeneity using TMAs. The small size of TMA cores as compared to whole-tissue sections is limiting this analysis. As recommended by previous reports, we sought to overcome this limitation by obtaining two cores from different areas to evaluate the tissue heterogeneity, with one sample collected from the intramural area and one from the invasive front area. The statistical power given by the large sample size is thought to compensate for the small size of the TMA cores , 45. In FGFR2 gene amplification with histological subtypes [FGFR2 gene amplification in this study Supplementary material 2 (XLSX 12 kb)Supplementary material 3 (XLSX 11 kb)Supplementary material 4 (XLSX 12 kb)Below is the link to the electronic supplementary material."}
+{"text": "Tardive dyskinesia (TD) is a motor side effect that may arise after long-term treatment of antipsychotic drugs. Its etiology is not well understood, but a number of risk factors have been associated with TD. TD occurrence appears to be familial, thus suggesting a genetic component. We previously reported on an association between the SLC18A2 gene that codes for the vesicular monoamine transporter 2 (VMAT2) that packages monoamines including dopamine from the cytoplasm into synaptic vesicles . In the present study, we examined the dopamine transporter gene SLC6A3 by itself and in conjunction with SLC18A2 for possible association with TD.We genotyped and analyzed the variable-number tandem repeat (VNTR) polymorphism in the 3\u2019 untranslated region of the SLC6A3 gene in our European sample of 187 schizophrenia/schizoaffective disorder patients assessed for TD occurrence based on the Abnormal Involuntary Movement Scale (AIMS). We also explored the interaction between the VNTR and the TD-associated SLC18A2 marker rs363224.Our preliminary analysis did not show the SLC6A3 VNTR to be associated with TD occurrence or severity. There also appeared to be no significant interaction between SLC6A3 VNTR and SLC18A2 rs363224 in TD occurrence or severity (p>0.05).Our findings did not support a major role of the dopamine transporter gene in TD risk or severity, but we will examine additional putative functional markers in this gene."}
+{"text": "The diagnostic performance of cardiac magnetic resonance (CMR) imaging in suspected myocarditis is still limited. Recently, CMR T1 and T2 mapping has been suggested to yield excellent diagnostic accuracies in patients with suspected myocarditis as compared to healthy controls. However, the true diagnostic performance of CMR mapping when compared to endomyocardial biopsy (EMB) and the impact of CMR field strength in patients with a variety of pathologies is still unknown and was therefore assessed in this study.Within the final analyses, 129 consecutive patients with suspected acute or chronic myocarditis were included. Patients had to fulfill indications for CMR imaging according to the JACC White consensus paper.All patients underwent biventricular EMB, cardiac catheterization for exclusion of coronary artery disease, and CMR on a 1.5  and 3.0  Tesla tomography.The CMR protocol included standard Lake-Louise (LL) parameters as well as native and post contrast T1 and T2 mapping. Relative Enhancement was assessed with a T1 weighted TSE sequence, edema ratio with a T2 weighted STIR sequence. T1 Mapping was measured with a MOLLI-Sequence pre and post contast  and T2 Mapping with a Multi Echo Spin Echo sequence on the 1.5T scanner and a T2 prepared SSFP sequence on the 3T scanner. Patients were divided into 2 groups according to duration of symptoms: patients with acute symptoms (\u226414 days) and those with chronic symptoms (>14 days).The diagnostic performance of LL criteria, native T1 and extracellular volume fraction (ECV) as well as T2 mapping is summarized in the table. On 1.5 Tesla, in patients with acute symptoms, native T1 yielded best diagnostic performance (81%) followed by ECV (75%) and T2 (72%). In chronic patients, T2 was the only technique achieving an acceptable accuracy (72%). Accuracies of imaging techniques on 3.0 Tesla CMR were slightly lower as compared to 1.5 Tesla. However, T2 mapping on 3.0 Tesla appeared to be unsatisfactory for the diagnosis of myocarditis.In patients with acute symptoms and suspected myocarditis, mapping techniques provides a useful tool for the confirming or rejecting the diagnosis of myocarditis and are superior to LL criteria. In contrast, only T2 mapping results in an acceptable diagnostic accuracy in patients with chronic symptoms. The impact of field strength on diagnostic accuracies requires further exploration."}
+{"text": "Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4C's membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a \u2018relay\u2019 mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.The chromosomal passenger complex (CPC)\u2014composed of Aurora B kinase, Borealin, Survivin and INCENP\u2014surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes  In addition, these filaments appeared to promote the closure of membrane gaps over time . This behaviour is similar to that observed previously for the C. elegans CHMP4B homologue  Methionine-labelled CHMP4C and MKLP1 polypeptides were prepared from corresponding PCR products amplified, using primers harbouring a T7 promoter and then transcribed and translated in vitro using the TnT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [35S] methionine (Perkin Elmer). The binding reaction contained 150 mM NaCl and subsequent washes varied from 150 mM to 1 M NaCl. GST pull down assays were carried out as described previously [DNA fragments coding for CHMP4C and MKLP1 fragments were generated by PCR and cloned into pDEST15 (Thermo), using Gateway technology to express N-terminal GST-tagged polypeptides in eviously ."}
+{"text": "Complete genome sequences of 13 human respiratory syncytial virus strains were determined from samples obtained from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection. We identified amino acid polymorphisms between the NA1 and ON1 genotypes in the P, G, F, and L proteins. Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infection in infants and young children . CurrentWe obtained nasopharyngeal swab samples from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection and isolated HRSV strains after culturing the swab samples in HEp-2 cells . Thirtee7\u2013Among the 13 sequenced isolates, 6 were identified as genotype NA1 and 7 as genotype ON1, both of which belong to HRSV subgroup A. The complete genome sequences of the NA1 and ON1 strains were 15,204 and 15,277 nucleotides long, respectively. As previously reported, the ON1 strains had a 72-nucleotide duplication in the second hypervariable region of the G gene (The reason the ON1 strain has replaced the previously circulating NA1 strain and has become a globally predominant genotype remains unclear . The comKY654506 to KY654518.The complete genome sequences of the 13 HRSV strains in this study have been deposited at GenBank under the accession numbers"}
+{"text": "The data shows results acquired in a large cohort of 5668 ethnic Arabs involved in a common variants association study for coronary artery disease (CAD) and myocardial infarction (MI) using the Affymetrix Axiom Genotyping platform   Specifications tableValue of the data\u2022Genomic distribution of risk variants for CAD/MI in ethnic Arabs.\u2022Comparative analysis of the genomic distribution of the associated loci for CAD/MI between the Arab population and other ethnic groups.\u2022Regional association plots demonstrate loci on 2q33, 8q13, 9p31 for CAD and on 21q22.11 for MI.\u2022Principal component analysis comparison with 11 other MApMAp3 populations shows variations and clustering of ethnic populations.\u2022The Saudi Arab cohort show similarities with the Caucasian populations.1The summary puts together the clinical features of the studied cases versus controls, the genomic distribution of the implicated variants and the principal component analysis of the data, as well as comparison of the Saudi population with other ethnic groups, Fig. 3.The figure shows the loci on loci 2q33, 8q13, 9p31 associated with coronary artery disease and locus 21q22.11.associated with and myocardial infarction.The figure shows the first and second principal component plot for the Saudi Arab samples (4431 samples: 2165 cases and 2266 controls) with eleven other HapMap3 populations .The figure displays the first and second principal component plot of the Saudi Arab samples (4431 samples: 2165 cases and 2266 controls) without Hapmap3 populations.2The discovery study involved 5668 Saudi Arabs who were subjected to genotyping using Affymetrix Axiom Genome-Wide ASI Array (Asian population). Genotyping data were generated using the Axiom GT1 algorithm and an IBS/IBD analysis in PLINK"}
+{"text": "A large number of studies have reported the effectiveness of psychotropic agents targeting 5-HT2A and D2 receptors in these disorders. In addition to the individual functions of these receptors, the interaction between the two neurotransmitter systems has been studied in the living brain. However, little is known about their regional relationship in individual human brains. We investigated regional relationships between 5-HT2A and D2 receptors using positron emission tomography (PET) and a bicluster analysis of the correlation matrix of individual variation in the two receptor densities to identify groups of distinctive regional correlations between the two receptors.Serotonin 2A  was calculated from PET data for 76 cerebral cortical regions. A correlation matrix was calculated between the binding potentials of [18F]altanserin and [11C]FLB 457 for those regions. The regional relationships were investigated using a bicluster analysis of the correlation matrix with an iterative signature algorithm.Seven healthy volunteers underwent PET scans with [2A and D2 receptors, with the former in regions related to sensorimotor integration  and the latter in most cortical regions. The second cluster identified another distinct profile of correlation coefficients between 5-HT2A receptors in the bilateral hippocampi and D2 receptors in most cortical regions.We identified two clusters of regions. The first cluster identified a distinct profile of correlation coefficients between 5-HT2A and D2 receptors in sensorimotor integration and hippocampal function. A bicluster analysis of the correlation matrix of these neuroreceptors may be beneficial in understanding molecular networks in the human brain.The observation of two distinct clusters in the correlation matrix suggests regional interactions between 5-HT Psychotropic agents targeting 5-HT2A and D2 receptors are used in the treatment of these disorders FLB 457. The scan protocol consisted of 35 frames and lasted 90 minutes. The injected dose and specific activity were 235 \u00b1 4.8 MBq and 220 \u00b1 68 GBq/\u03bcmol at the time of injection, respectively. For evaluation of density of serotonin 5-HT2A receptors, a 90-minute dynamic PET scan was performed after an injection of [18F]altanserin. The scan protocol consisted of 33 frames and lasted 90 minutes. The injected dose and specific activity were 191 \u00b1 5.2 MBq and 153 \u00b1 73 GBq/\u03bcmol at the time of injection, respectively. Each PET scan was preceded by a transmission scan for attenuation correction using a 137Cs source. A head holder was used to minimize head movement. The two scans were performed on separate days .Each subject underwent two PET scans to visualize serotonin 5-HT18F]altanserin PET, arterial blood samples were obtained manually 33 times after the injection of the radioligand to obtain an arterial input function. Each blood sample was centrifuged to obtain plasma and blood cell fractions, and the concentrations of radioactivity in whole blood and plasma were measured. The fractions of the parent compound and its radiometabolites in plasma were determined using high-performance liquid chromatography from 6 samples for each subject.For the FLB 457 altanserin and BPND of [11C]FLB 457 for the 76 regions (the two parameters are referred to as BP hereafter). The relationships between the distributions of the 5-HT2A and D2 receptors were investigated using unsupervised biclustering of regions on the correlation matrix with an iterative signature algorithm (ISA) FLB 457 changes slowly with aging (10% decrease per decade) [BP of [18F]altanserin also changes slowly with aging (less than 10% decrease per decade) altanserin for 5-HT2A receptors and [11C]FLB 457 for D2 receptors in cerebral cortical regions.An excel file contains (XLSX)Click here for additional data file.S2 Filer values) between individual binding potential values of [18F]altanserin for 5-HT2A receptors (columns) and [11C]FLB 457 for D2 receptors (rows) in 76 regions.An excel file contains Spearman\u2019s correlation coefficients ((XLSX)Click here for additional data file."}
+{"text": "Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of  VEGF is crucial in normal angiogenesis during embryonic vascular development  by TSP1  given in the Supplementary Material Section. We also included the SBML file associated with the model.Table Binding of PLC\u03b3 to pVEGFR2 and subsequent phosphorylation and dissociation of PLC\u03b3 from the receptor is described by a Michaelis-Menten type reaction as follows:pVEGFR2 denotes all the species containing phosphorylated VEGFR2 which is determined by BioNetGen. Using this approach lowers the number of reactions generated by the rules and prevents combinatorial explosion in the model.Ras activation by S1P is modeled as a Michaelis-Menten type reaction as follows:S1PRas and Km, S1PRas determining the strength of Ras activation by S1P.with parameters kCurrent through the CRAC channels is modeled according to the following equation. This model is a simplified version of the CRAC component of the mathematical model developed by Schmeitz et al.  in T-celThe steady-state current (Equation 4) as a function of ER calcium concentration is described by an empirically determined Hill function . The set of ordinary differential equations (ODEs) describing the reaction network was numerically integrated using SUNDIAL numerical solver suite  algorithm described in Marino et al. . The parTo accurately capture receptor dynamics and signaling to downstream targets, we opted to use a rule-based modeling approach where molecular details of the species and the corresponding rules for the interactions are implemented in a programing environment such as BioNetGen   via the IP3 sensitive receptors and calcium regulation involving plasma membrane and SERCA pumps. CRAC channels are also a prominent hallmark of the calcium cycling module Figure  see met. The com\u22121 vs. 1.24 \u00d7 10\u22123 s\u22121). This reduction in recycling rate is sufficient to explain the rapid decay of total cellular receptor levels in the absence of NRP1. In fact, it is the combined effects of internalization, recycling, and degradation that determine the rapid decay of VEGFR2 cellular level . Samples traces for four different degradation rates are also shown in Figure We simulate the direct effect of TSP1 by assuming that CD47 binding to TSP1 leads to accelerated VEGFR2 degradation. It is assumed that TSP1/CD47/VEGFR2 undergo internalization together and either recycle back to the membrane or degrade with a higher degradation rate . In cells devoid of C47, VEGFR2 signaling is not affected by TSP1. This implies that TSP1/CD47 interaction is necessary for VEGFR2 degradation. For the simulations here, cells are exposed to 2 nM TSP1 for 10 min, followed by 40 min exposure to 50 ng/ml VEGF. This is similar to the experimental protocol in Kaur et al. . Accordi2 (7000 receptors per cell) to 2.2 receptors/\u03bcm2 (3100 receptors per cell). Figure 2 does not affect TSP1 inhibition of VEGFR2 signaling.The effect of VEGFR degradation and CD47 surface levels on ERK1/2 activation is investigated in Figure Overall, the results here demonstrate that the enhanced degradation of VEGFR2 by TSP1/CD47 interaction is a viable mechanism to explain the global shutdown of VEGFR2 signaling. The dependence on TSP1 concentration is also consistent with the experimental data indicating the viability of the enhanced degradation mechanism.We next consider the hypothesis that TSP1 might insert some of its inhibitory effects on VEGFR2 by recruiting phosphatases to the receptor complex. For the simulations here the cells are exposed to 2 nM TSP1 for 10 min, followed by the addition of VEGF for 40 min. It is assumed that the increase in dephosphorylation rate is the sole mechanism. As shown in Figure Next, we consider a different layer of TSP1 effects involving the experimentally suggested alteration of calcium signaling by TSP1  is modeled in a simple manner by lowering the concentration of TSP1 and monitoring the recovery of calcium and pERK1/2 signals. We consider a mixed mechanism involving enhanced VEGFR2 degradation (10-fold) and increased VEGFR2 dephosphorylation (51-fold). These values are consistent with the two-parameter scan in Figures The effect of depleting CD47 as a therapeutic strategy is summarized in Figures The simulations demonstrate that CD47 depletion may be a more effective strategy than targeting TSP1 in restoring VEGF signaling to calcium and ERK1/2. CD47 depletion is particularly effective in restoring ERK1/2 activation downstream of VEGFR2.in silico approach to computationally investigate potential mechanisms of inhibition. The model incorporated detailed molecular mechanisms using a rule-based model for receptor-receptor interaction and a coarse-grained model for signaling to calcium and ERK1/2 from the activated receptors. The inclusion of detailed calcium cycling module in the model allowed us to study the role of TSP1 in inhibiting calcium signaling and the potential crosstalk with ERK1/2 activation. Fluorescene resonance energy transfer based (FRET-based) experiments in endothelial cells had shown the preassociation of VEGFR2 with CD47 in endothelial and T cells . Indeed, only 18% depletion of CD47 was needed to restore ERK1/2 activation signal compared with 72% TSP1 inhibition. The results highlight the important role of efficient and sustained therapeutic agent delivery to affected tissues to ensure above-threshold inhibition of TSP1 and CD47. They further suggest that CD47 depletion should be considered as an initial approach in pro-angiogenic therapeutic intervention. It is plausible that different endothelial cell types may exhibit differential inhibition thresholds to TSP1 and CD47 which would further necessitate the need for sustained delivery of anti-TSP1 and anti-CD47 agents.Simulations here also proposed the possibility of inhibiting TSP1 or CD47 as potential interventions for restoring VEGF-mediated angiogenic response. The model showed that sequestering TSP1 effectively normalized calcium signaling and ERK1/2 activation, albeit requiring at least 80% inhibition. This high threshold for inhibition might preclude the Further complicating anti-TSP1 therapy, platelets contain significant pre-formed TSP1 that are a depot source of protein. Thus, targeting CD47 is an alternative strategy expected to be more efficient at preserving pro-angiogenic signals. Additionally, VEGF can also signal via NO while CD47 can interact with cell membrane SIRP\u03b1  and has equity interest in the same and in Tioma Therapeutics . The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1+/C2++ KIR3DS1+/Bw4-80Ile+) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1\u2212/KIR3DL1+/Bw4-80Ile+ combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis.The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n\u2009=\u2009148) or absence (n\u2009=\u2009149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n\u2009=\u2009120) or recurrent (n\u2009=\u200928). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating  Toxoplasma gondii infection2T. gondiiOcular Toxoplasmosis, the most common form of posterior uveitis, results from The damage to ocular tissues led to the proposal of phenomena that may be related to pathogenic mechanisms of ocular toxoplasmosis, including autoimmune mechanisms15dim cytotoxic cells and a decrease in immunoregulatory NK CD56bright cells have been identified in children congenitally infected with T. gondii who have active eye lesions. Furthermore, subsets of NK cells and CD8+ T cells play a crucial role as biomarkers of cicatricial lesion of the eyein vitro during T. gondii infections, due to an increased production of interferon-gamma (IFN-\u03b3) in patients with congenital ocular toxoplasmosisIncreases in the numbers of circulating proinflammatory monocytes and NK CD56KIR genes are responsible for coding the KIR receptors of NK cells. These genes comprise a family of 15 genes located on chromosome 19q13.4 characterized as inhibitors  or activators , and two pseudogenes (KIR2DP1 and -3DP1). Moreover, based on the content of the genes, KIR genotypes are divided into two groups designated AA and BX (BB and AB) that differ in the number and type of KIR genesThe effector function of NK cells is regulated by a set of receptors named killer immunoglobulin-like receptors (KIR) expressed on the cell surface that recognize human leukocyte antigen (HLA) class I molecules of target cells9KIR genes have been described as risk or protective factors in different types of non-toxoplasmic uveitis and inflammatory ocular diseases. These diseases include Behcet\u2019s uveitis1315KIR genes are also associated with many other infectious diseases1718202122232425T. gondii infectionKIR genes that encode the immune receptors of NK cells and can trigger local inflammation in the eye has not been elucidated in ocular toxoplasmosis yet. The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the resistance or susceptibility to the development of ocular toxoplasmosis.NK cells have great importance in the control of P\u2009<\u20090.0001, t\u2009=\u20097.00), with the subgroup of patients with primary manifestations of ocular toxoplasmosis  and with the subgroup of patients with the recurrent form of the disease . A higher mean age was also observed for the subgroup of patients with primary manifestations than those who had recurrent manifestations .The characteristics of the study population with respect to age, gender, clinical diagnosis and serological profile are shown in KIR2DL2/3 and KIR3DL1/S1 were in Hardy\u2013Weinberg equilibrium (P\u2009>\u20090.05) in this study population. However, KIR3DL1/S1 for the patient group that developed ocular toxoplasmosis was not in Hardy-Weinberg equilibrium (P\u2009=\u20090.03). KIR framework genes, KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1, as expected were present in all samples, which are important internal controls to check the quality of genotyping.The distribution of genotype frequencies of KIR gene frequencies and AA and BX genotype frequencies are shown in P\u2009=\u20090.003, Pc\u2009=\u20090.04) was observed for the KIR3DS1 activating gene. There was also a positive association between KIR3DS1 and recurrent manifestations of disease  when this patient group was compared to patients without ocular toxoplasmosis, although it was lost after applying the Bonferroni correction. On the other hand, the KIR2DS2 activating gene was associated with decreased susceptibility for ocular toxoplasmosis , but the significance was also lost after applying the Bonferroni correction. No significant difference was observed in the AA and BX genotype frequencies between all groups investigated in this study.The distribution of P\u2009>\u20090.05) in all groups studied. The frequencies of the HLA class I ligands of KIR  were analyzed and were similar between groups , patients with primary manifestations  and patients with recurrent manifestations  than patients without ocular toxoplasmosis.Data on the distribution of KIR genes with their HLA class I ligands are listed in KIR2DL3 inhibitory allele in the homozygous state and presence of its ligands whether homozygous or not (KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1) was associated with resistance to ocular toxoplasmosis when patients without ocular toxoplasmosis were compared with those with ocular toxoplasmosis , primary manifestations  and recurrent manifestations  (The ctively) .P\u2009=\u20090.01; Pc\u2009=\u20090.04) and patients with primary manifestations and without ocular toxoplasmosis  when only two pairs of activating ligands were present ( present . Signifi present .+/C2++ KIR3DS1+/Bw4\u201380Ile+) is responsible for increasing the risk of developing ocular toxoplasmosis . The combination is also responsible for its primary  and recurrent clinical forms  compared to patients without ocular toxoplasmosis  for the association mentioned above was investigated. The combination involving KIR2DS1 and KIR3DS1 in the presence of their respective ligands  and recurrent manifestations of the disease  was observed for the KIR3DS1\u2212/KIR3DL1+/Bw4\u201380Ile+ combination (KIR2DL1 and the Bw4-80Ile ligand in the absence of KIR3DS1) when compared to patients without ocular toxoplasmosis. This correlation was also observed when patients with recurrent manifestations were compared to patients with primary manifestations .The correlation between the distribution of activating and inhibitory KIR and their respective HLA ligands was analyzed . A decreT. gondii infection. To the best of our knowledge, this is the first study of KIR genes and HLA ligands in the immunopathology of ocular toxoplasmosis.Possible causes of ocular manifestations of toxoplasmosis have not been fully elucidated, but it is believed that factors related both to the parasite and the host contribute to the development of this disease4T. gondii infection can occur at any time of life, and although most cases of ocular toxoplasmosis occur due to infections acquired after birth, a significant number of patients can acquire the disease congenitally and the resulting scars trend to be persistent30The difference in the mean ages of the patient groups of this study was carefully discussed previouslyKIR genes, after Bonferroni correction only the KIR3DS1 activating gene was associated with increased risk of developing ocular toxoplasmosis with the other associations being lost. The Bonferroni correction decreases the chance of a significant difference by chance alone, making the data more robustKIR3DS1 with ocular toxoplasmosis can be explained by the absence of KIR3DL1, because 3DS1 and 3DL1 segregate as alleles of a single locus. Thus, the presence of KIR3DS1, and consequently the absence of KIR3DL1, can create an increased potential for the activation of NK cells owing to decreases in the ratio of inhibitory/activating receptors. Previous studies have shown involvement of the KIR3DS1/L1 alleles in various types of non-toxoplasmic uveitis and inflammatory eye diseases triggered by autoimmune factors. Levinson et al.KIR3DS1 gene in their haplotype have an increased risk of developing Vogt-Koyanagi-Harada syndrome, while the presence of KIR3DL1 was associated to protection against the development of the disease. A similar result was observed by Moon et al.KIR3DS1 was associated as a risk factor and KIR3DL1 was associated as a protective factor.Regarding the distribution of KIR3DL1/S1 for the patient group that developed the ocular toxoplasmosis were not in Hardy-Weinberg equilibrium. However, some authors claim that this equilibrium should only be investigated in the control group, because it represents the general population34KIR3DS1 might be changing the distribution of these alleles in individuals with ocular toxoplasmosis, resulting in a deviation from the Hardy-Weinberg equilibrium. Considering that numerous precautions were adopted to prevent bias in this study, we can safely say that the 3DS1 gene exerts a real influence on the development of ocular toxoplasmosis, even though KIR3DL1/S1 were not in Hardy-Weinberg equilibrium.In this study, KIR genes in immune response is highly dependent on the HLA molecules expressed on the target cell surface. Therefore, KIR receptors influence susceptibility for or protection against certain illnesses by means of a balance in activation and inhibition signals that regulate the NK cell effector function937The function of KIR genes were analyzed in the presence of their respective ligands (KIR-HLA), the KIR3DS1-Bw4-80Ile pair was associated with the development of ocular toxoplasmosis irrespective of the type of clinical manifestation (primary or recurrent); while KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1 were associated with protection against the development of ocular toxoplasmosis and its clinical manifestations. In accord with our results, other studies found an association involving the KIR3DS1-Bw4-80Ile pair in other human diseases404142KIR2DL3 and KIR2DL2 are considered alleles, as are KIR3DL1 and KIR3DS1. Although the KIR2DL2-C1 interaction is stronger than the KIR2DL3-C1 interaction, the inhibitory signal generated by the absence of KIR2DL2 (KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1) appears to be sufficient to inhibit the effector function of NK cells and protect against elevated inflammation and tissue damage. It has been shown that abnormal expressions of inhibitory receptors of NK cells, including the KIR2DL3 receptor, may be associated with the development of Behcet\u2019s diseaseIn this study, where the +/C2++ KIR3DS1+/Bw4-80Ile+, although the KIR2DS1 and KIR3DS1 genes are not in linkage disequilibrium as observed among patients with ocular toxoplasmosis  and patients without ocular toxoplasmosis . The combination of one pair (inhibitory) in the absence of the other pair (activating) was analyzed. It was possible to observe that the KIR3DS1\u2212/KIR3DL1+/Bw4-80Ile+ combination decreases the risk of developing ocular toxoplasmosis and its recurrent clinical forms.It is important to highlight the results observed for the number of pairs of KIR-HLA ligands and the correlation between the distribution of activating KIR and their respective HLA ligands. A higher frequency of only two pairs of ligands was observed in patients with ocular toxoplasmosis and in patients with primary manifestations compared to patients without ocular toxoplasmosis. The combination responsible for this association was KIR2DS1et al.These results may suggest that the activating function mediated by KIR2DS1 plus KIR3DS1 and their respective HLA ligands is, in fact, an important factor interfering in ocular toxoplasmosis, since such a combination may affect the balance of inhibiting/activating signals. NK cells are activated when there is an increase of activation signals, even if there is a combination of strong or weak inhibitory signalsT. gondii infection. IL-12 stimulates NK cells to produce IFN-\u03b3 and to promote the development of Th-1 cells that produce IFN-\u03b3, a cytokine involved in the activation of macrophages, the main phagocytes in chronic inflammation47T. gondii infection promotes the production of factors such as IFN-\u03b3 that suppress the immune privilege of this organ50NK, CD4 and CD8 T cells, and type 1 cytokines, such as IFN-\u03b3 and IL-2, play a protective role in T. gondii infection, and the mechanisms involved may be associated with both the pathogenesis and protective effects that control tissue damage. It has been shown that increases in the frequency of circulating NK cells and proinflammatory monocytes in children infected by T. gondii, particularly in those with active ocular lesions, are indicative of a strong and persistent proinflammatory response. Moreover, subsets of NK cells and CD8+ T cells act as biomarkers for cicatricial lesions of the eye51The immune response can determine the development of eye injuries resulting from T. gondii and NK cytotoxicity assays to better understanding the role of NK cells and the expression of KIR in the immunopathogenesis of ocular toxoplasmosis.The current study investigated the KIR-HLA ligand as a risk factor in ocular toxoplasmosis and these results may improve to understanding of the immunopathogenic mechanism involving NK cells in ocular manifestations related to toxoplasmosis. However, others studies should be performed such as histological analyses of the ocular tissue affected by KIR3DS1, KIR3DS1-Bw4-80Ile and KIR3DS1+/Bw4-80Ile++ KIR2DS1+/C2+) and inhibitory signals as protective factors .In conclusion, the results of this study show that activating and inhibitory KIR in the presence of their respective HLA ligands may have influence on the development of ocular toxoplasmosis and its clinical form in this population. In particular this is seen with the strong presence of activating signals as risk factors This study was approved by the Research Ethics Committee of the Medicine School in S\u00e3o Jos\u00e9 do Rio Preto (#1980/2009) and all individuals who agreed to participate signed informed consent forms. The experiments were carried out in accordance with the approved relevant guidelines and regulations.A total of 297 unrelated patients from the Retinopathy Outpatient Service of Hospital de Base of the Medicine School in S\u00e3o Jos\u00e9 do Rio Preto (HB-FUNFARME) and Medical Outpatient Clinic (AME) in S\u00e3o Jos\u00e9 do Rio Preto participated in this study.The study subjects have been described previouslyT. gondii and epidemiological data. Besides, although patients self-reported themselves as European descent, mixed African and European descent, and African descent, due to high miscegenation of the Brazilian population they were defined as a population of mixed ethnicityAll individuals who participated in this study were monitored and evaluated in respect to clinical symptoms, serology for In this study, in order to avoid bias in the results, all patients were selected after clinical examination using the same criteria. Additionally, the probability of variations in the allele frequencies due to ethnic background was minimized by matching patients with ocular toxoplasmosis and patients without ocular toxoplasmosis from similar ethnic backgrounds. Furthermore, gender and residence in the same geographical areas were carefully matched during group selection.KIR genes with statistical power of more than 90% and it was chosen according to frequency of KIR genes recorded in Allele*Frequencies database  observed in a population located in the southeast region of Brazil and defined as a population of mixed ethnicity.The number of patients enrolled is sufficient to demonstrate whether there is an association between ocular toxoplasmosis and The inclusion criteria of patients with ocular toxoplasmosis were positive laboratory diagnosis of toxoplasmosis, the presence of ocular scars/lesions due to toxoplasmosis and live in municipalities in the northwest region of the State of Sao Paulo . The inclusion criteria of patients without ocular toxoplasmosis were positive laboratory diagnosis of toxoplasmosis but without ocular scars/lesions due to toxoplasmosis and living in the same geographical region as the patients with ocular toxoplasmosis. All patients were clinically evaluated by two experienced physicians.The exclusion criteria were: patients with other infectious and parasitic diseases, patients with any type of mental disability, patients with blood dyscrasia and using oral anticoagulants and related patients.Anti-T. gondii antibodies were detected by immunosorbent assay (ELISA) according to the manufacturer\u2019s instructions . The microplates were read using Epoch\u2122 equipment using the Gen5\u2122 2.0 software . The samples were tested in duplicate and in cases of indeterminate results, the samples were retested in duplicate. Positive and negative controls were included in all reactions.Blood samples were collected into tubes without anticoagulant to obtain serum. All patients were clinically assessed using an indirect binocular ophthalmoscope . The evaluation of visual acuity followed the logMAR Early Treatment Diabetic Retinopathy Study chart (ETDRS) criteriaT. gondii. This is therefore a presumptive diagnosis.As no invasive test was performed, the ocular toxoplasmosis diagnostic criteria used were the same as in the clinical practice: injury identified by ophthalmoscopy associated with positive serology for \u00ae DNA Blood Mini Kit, QIAGEN, the Netherlands) following the manufacturer\u2019s instructions. All DNA samples were subjected to an evaluation of concentration and purity using the ratio of the absorbance at optical densities (OD) of 260 and 280\u2009nm with Epoch\u2122 equipment . KIR and HLA-A, -B and -C were genotyped according to manufacturer\u2019s instructions by Polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) protocols with Luminex\u00ae technology . This technique uses PCR-amplified DNA with specific biotinylated primers. The amplified product is hybridized by complementary DNA probes conjugated to fluorescently coded microspheres, with detection using R-Phycoerythrin-conjugated Streptavidin (SAPE). The data were interpreted using a computer program .Blood samples were also collected into tubes containing EDTA anticoagulant for DNA extraction. DNA of all patients was extracted using the commercial kit for silica column extraction . HLA-Bw4 molecules were divided into two groups based on whether isoleucine or threonine was present at position 80 (Bw4-80Ile and Bw4-80Thr). KIR3DS1 binds to Bw4-80Ile molecules. HLA-KIR ligand specificities were considered according to Carr http://www.allelefrequencies.net). Individual genotypes were determined to be AA when the genes KIR2DL1, KIR2DL3, KIR2DL4, KIR2DS4, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1 were present. The presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 characterized the BX genotype.Two types of KIR genotypes have been described based on the content of the genes: AA and BX (BB and AB) . Odds ratio (OR) with a 95% confidence interval (95% CI) was also calculated to evaluate the risk association. The mean ages were compared using the t-test. Differences with P-values\u2009<\u20090.05 corrected by the Bonferroni inequality method for multiple comparisons (Pc) were considered statistically significant. A Hardy-Weinberg equilibrium fit was performed by calculating expected genotype frequencies and comparing that with the observed values for KIR2DL2/3, KIR3DL1/S1, and the HLA alleles using ARLEQUIN software, version 3.1. (http://cmpg.unibe.ch/software/arlequin3).KIR, HLA and KIR-HLA frequencies were obtained by direct counting. The comparisons of the frequencies of HLA ligands, KIR genes, KIR AA and BX genotypes and KIR with or without ligands between groups of patients were performed with the Chi-square test with Yates\u2019 correction or, when necessary, Fisher\u2019s exact test using the program Graph Pad Instat .Publisher\u2019s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching. Published 2, August 2016In the originally published article, the primer sequences used for PCR amplification of immunoglobulin heavy chain genes (IGH) which bind in the constant region were reported incorrectly in Table 5. The correct oligonucleotide sequences are now reported in the corrected Table 5.In the 'Library preparation' section of 'Materials and methods', the text \u201ca pooled set of five isotype-specific IGH constant region primers that contain 8 random nts (Table 5)\u201d has been changed to \u201ca pooled set of ten isotype-specific IGH constant region primers that contain 8 or 12 random nts (Table 5)\u201d.The article has been corrected accordingly."}
+{"text": "The following statistics were accidentally reversed: \"The prevalence in rural areas is nearly double that of urban areas for both early marriage (39% versus 22%) and early childbearing (42% versus 21%).\" This sentence should read: \"The prevalence in rural areas is nearly double that of urban areas for both early marriage (42% versus 21%) and early childbearing (39% versus 22%).\"2)The statistic on the prevalence of sexual initiation in the following sentence was incorrect: \"For instance, the median age of sexual debut is 17.7 years, approximately 1 year prior to marriage, and the prevalence of sexual initiation among 15\u201319 year olds is 27%. This sentence should read\": For instance, the median age of sexual debut is 17.7\u00a0years, approximately 1\u00a0year prior to marriage, and the prevalence of sexual initiation among 15\u201319\u00a0year olds is 49%.\u201dAfter publication of the article , it has"}
+{"text": "Interestingly, orthotopic tumours arising from transplantation of 4T1 murine mammary tumour cells exhibit both phosphorylated Stat3 and mCLCA5 expression. However, we demonstrate that expression is highly compartmentalized to distinct subpopulations of cells, and that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. These findings enhance our understanding of the regulation of CLCA channel expression both in vitro and in vivo, and in particular, demonstrate that expression of mCLCA1 and mCLCA2 during involution is profoundly dependent upon Stat3, whereas the relationship between mCLCA5 and Stat3 activity is reciprocal and restricted to different subpopulations of cells.Mammary gland regression at the cessation of lactation (involution) is an exquisitely orchestrated process of cell death and tissue remodelling in which Stat3 signalling has an essential role. The involution microenvironment of the mammary gland is considered to be pro-tumourigenic and a proportion of cases of pregnancy-associated breast cancer are suggested to originate in tandem with involution. However, the apparent paradox that STAT3 is required for cell death in normal mammary gland, but is associated with breast cancer cell survival, has not been resolved. Herein, we investigate Stat3-mediated regulation of expression of members of the calcium-activated chloride channel regulator (CLCA) family of proteins during involution and mammary carcinogenesis. Using the conditionally immortal mammary epithelial cell line KIM-2, together with mice exhibiting mammary epithelial cell-specific deletion of Stat3 during lactation, we demonstrate that expression of mCLCA1 and mCLCA2 is elevated in concert with activation of Stat3. By contrast, murine CLCA5 (mCLCA5), the murine orthologue of human CLCA2, is significantly upregulated at 24, 72 and 96\u2009h of involution in Stat3 knockout mice, suggesting a reciprocal regulation of these proteins by Stat3  Although the mechanism by which they activate other channel proteins to trigger movement of chloride ions across membranes is not clear, there is some evidence that they can be secreted, and thereby potentially activate chloride channels.16 apoptosis, tumourigenesis and mucus cell differentiation.17 Of particular interest, both CLCA2 and CLCA4 have been shown to inhibit proliferation of breast cancer cells when ectopically expressed in vitro,18 whereas normal breast tissue exhibits high levels of CLCA2 in both acini and small ducts.19 Murine CLCA5 (mCLCA5) is highly homologous to human CLCA2 (hCLCA2) and is considered to be its orthologue.20 Widely expressed in the cytoplasmic granules of granular layer keratinocytes in stratified squamous epithelia,21 mCLCA5 is downregulated in some murine mammary tumour cell lines, including 4T1 cells, where re-expression inhibits proliferation.22 Thus, hCLCA2 and its murine orthologue have been proposed as tumour suppressor genes in breast cancer.24 However, other investigators have demonstrated an upregulation of hCLCA2 in triple-negative breast cancer patients with a poor prognosis,25 perhaps suggesting breast cancer subtype specific roles, and exemplifying the need for further characterization of this poorly understood protein family.CLCA proteins have been associated with diverse functions including cell adhesion,mClca1 and mClca2 that share ~96% complementary DNA sequence identity, and encode mCLCA1 and mCLCA2, respectively.26 These proteins are reciprocally expressed during mammary gland development with mCLCA2 being expressed at high levels during lactation and involution, whereas mCLCA1 expression is suppressed during involution.27 This pattern suggests different functional roles for these highly related genes.Other murine genes include Given the pro-tumourigenic potential of the involution mammary microenvironment, the critical role of Stat3 in orchestrating regression and the intriguing regulation of mCLCA proteins, we hypothesized that mammary epithelial expression of Stat3 would influence the expression of mCLCA proteins.28 Differentiation was confirmed by robust upregulation in expression of the milk protein \u03b2-casein , whereas expression of mCLCA1 is markedly upregulated between 12 and 48\u2009h of involution (first phase), and subsequently maintained from 48\u2009h onwards  microarray data that have been previously published,Stat3fl/fl; BLG-Cre; hereafter referred to as Stat3 KO) and compared these with age-matched controls lacking Cre expression (control). Using qRT-PCR, we compared expression of mCLCA1, mCLCA2 and mCLCA5 during lactation and involution, utilizing samples collected at 42\u2009h involution, close to the onset of the irreversible phase (unpublished data).To further interrogate a possible relationship between Stat3 activity and CLCA protein expression, we utilized mice with a mammary epithelial cell-specific conditional deletion of Stat3 , we identified at least two different cell populations at the edge of the tumour . Strikin35 is likely to be critical to the invasive nature of the neoplastic cells at the tumour margin.We therefore suggest that in individual neoplastic cells, the negative regulation of mCLCA5 by pStat3 remains functional. Although both mCLCA5 and pStat3 are expressed predominantly at the invasive edge of the tumour, minimal co-localization of nuclear Stat3 and cytoplasmic mCLCA5 is observed. It seems likely that mCLCA5 expression may be predominantly in the tumour cells, whereas pStat3 nuclear localization is seen in both the tumour cells and immune and stromal compartments at the invasive front. Thus, activity of pStat3, a known breast cancer oncogene,36 and it is tempting to speculatively attribute such as role to mCLCA5 in this context at the invasive edge of the tumour  Stat3. This suggests that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. It is in accordance with the findings of others, and therefore tempting to speculate, that downregulation of mCLCA5 in the presence of cells expressing Stat3 activity reflects an invasive phenotype in these cells and potential epithelial\u2013mesenchymal transition.37 However, in this context it is important to note the compelling data demonstrating that re-expression of mCLCA5 in 4T1 cells inhibits proliferation22 and that loss of hCLCA2 expression in human MCF10A cells augments proliferation of the cells,24 whereas re-expression elicits a reduction in tumour cell growth.19 In the in vivo 4T1 murine model described in our study, cellular behaviour may be influenced by the presence of subpopulations of neoplastic cells in which either Stat3 activity suppresses mCLCA5 expression, or mCLCA5 is expressed in the absence of Stat3 activity. The influence of the immune cell compartment also needs to be considered particularly given that the 4T1 model involves introduction of syngeneic tumour cells into immune-competent mice. It is possible that mCLCA5 secretion is stimulated in host cells, such as stromal components or immune cells, in an attempt to negatively regulate tumour growth. Currently, the role of mCLCA5 at the invasive front of the tumour is unclear. Future work will require development of inducible models of mCLCA5 expression and/or Stat3 activity in vivo in different cell types to understand the relationship between the key oncogene Stat3 and the intriguing factor mCLCA5.However, our data also raise the question of the role of the observed mCLCA5 expression at the invasive front. One interpretation is that mCLCA5 expression at the invasive edge may be beneficial to tumour growth with mCLCA5 acting in a non-cell autonomous manner to augment tumour cell proliferation.Although in normal mammary gland, Stat3 regulates cell death, breast cancer cells frequently become addicted to Stat3 and require Stat3 activity for survival. Our data demonstrate that Stat3 is a negative regulator of mCLCA5 during mammary gland involution, and that in tumourigenesis in an immune-competent murine model, mCLCA5 expression is suppressed in cells with high levels of nuclear Stat3. Stat3 appears to be a cell autonomous negative regulator of this protein.28 Human LIF (a generous gift from Dr Jennifer Nichols) or recombinant mouse OSM  were used at a final concentration of 10 or 20\u2009ng/ml (LIF), or 25\u2009ng/ ml (OSM). For imaging, KIM-2 cells were differentiated in 35\u2009mm diameter glass bottom dishes . Murine 4T1 cells were purchased from ATCC  and were maintained according to ATCC protocols.Maintenance and differentiation of KIM-2 cells and instigation of hormone withdrawal, were as previously described.38 The primer sequences utilized are detailed in Standard protocols were followed.38 Antibodies employed were: anti-phospho-Stat3 , anti-Stat3 , anti-GAPDH  , and anti-alpha-tubulin .Standard protocols were followed.Stat3fl/fl; BLG-Cre mice40 were utilized and involution induced as previously described.9 All animals were treated according to the local ethical committee and the UK Home Office guidelines.\u03bcl containing 1 \u00d7 105 4T1 cells with matrigel  was injected into the right- or left-fourth mammary gland fat pad of ~12-week-old virgin Balb/c mice under anaesthesia and analgesia.41 Tumour development was monitored, mice were killed and tumours were harvested at day 25 post injection. Tumour tissue  was collected for RNA and protein extraction. Tissue for histological sectioning was dissected. Formalin fixation, histological sectioning and staining followed routine protocols.A final volume of 16\u2009KIM-2 cells in glass bottom dishes were fixed for 10\u2009min in ice cold methanol and immunofluorescence staining for E-cadherin  was carried out following a standard protocol.Paraffin embedded tumour specimens were prepared as three micron sections on positively charged slides . Immunohistochemical staining for mCLCA5 , vimentin  and murine pStat3   was carried out using a routine protocol employing an automated immunohistochemistry system .Immunofluoresence staining for E-cadherin , mCLCA5 , vimentin  and murine total Stat3   was carried out following de-paraffinization and antigen retrieval for 20\u2009min at 90\u2009\u00b0C using Dako Envision Flex Target Antigen Retrieval Solution High pH in a PT Link and Pre-Treatment Module for Tissue Specimens (both Dako). For Fc receptor blocking prior to staining for murine total Stat3 , anti-mouse CD16/CD32 clone 93 was utilized . Isotype- and species-matched immunoglobulins were used as a negative control."}
+{"text": "The data presented in this article are related to the research article entitled \u201cCyr61/CCN1 is involved in the pathogenesis of psoriasis vulgaris via promoting IL-8 production by keratinocytes in a JNK/NF-\u03baB pathway\u201d   Specifications TableValue of the data\u2022This data characterizes the distribution of Cyr61 expression in skin lesion of patients with psoriasis vulgaris.\u2022These data could be used for developing improved strategy in the treatment of psoriasis.1in vitro and anti-Cyr61mAb in vivo   on the shaved back and the right ear, representing a daily dose of 3.125\u00a0mg of the active compound. Control mice were treated similarly with vaseline . Mice received intraperitoneal injections of 200\u00a0\u03bcg/day of either anti-Cyr61 mAb 093G9 or control IgG1  2 days after IMQ treatment. After16 days later mice were sacrificed and skin samples were collected and inspected National Natural Science Foundation of China , Excellent Young Doctor Foundation of Shanghai Ninth People\u05f3s Hospital (201608), Education Ministry Research Fund for the Doctoral Program (20130073110003), Science and Technology Commission of Shanghai Municipality (13JC1402300) and Shanghai Cultivate Outstanding Young Teachers in Colleges and Universities Scientific Research Fund (JDY09062).This work was funded by"}
+{"text": "Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats. Many RBC micronuclei result from double strand DNA breaks that give rise to acentromeric chromosomal fragments that fail to incorporate into nuclei during mitosis and consequently remain in the cell following enucleation. Here, RBC micronuclear DNA is sequenced (Mic-Seq) to define the locations of breaks genome-wide and this assay is used to study ongoing genome instability resulting from insufficient DNA replication origin licensing. Using a mouse model, we show that there is increased instability at discrete sites across the genome, which include genes that are recurrently deleted in the T-lymphocytic leukemias that eventually arise in these mice. Mic-Seq may provide an effective means of predicting locations that are susceptible to genetic damage and these predictions may have prognostic value. Licensing of DNA replication origins begins early during the G1-phase of the cell cycle when ORC and CDC6 recruit CDT1-MCM2-7 to the chromatin [reviewed in Chaos3/Chaos3) females succumb to mammary adenocarcinomas when carried on the C3H genetic background on which it was isolated [IRES-CreERT2/ IRES-CreERT2) mice exhibit near to 100% penetrance of T-lymphocytic leukemia (TLL) within 5 months on the 129Sv background on which they were constructed [The cancers that arise in MCM deficient mice can be highly specific to a particular genetic background. For example, >80% of hypomorphic MCM4  with a preponderance of deletions averaging 400\u2013500 kbp in size \u201310. Seveexamined . Tcf3 is in mice . Importaent mice .Mcm2-7, which occurs in many tumors [Tcf3 gene is also the site of elevated ongoing genome instability even prior to tumor initiation in MCM2 deficient mice on the 129Sv genetic background. The increased instability at this site predicts the high rate of genetic lesions affecting the Tcf3 gene and consequent high rate of TLL in these mice. In addition to loss of Tcf3, MCM2 deficient mice exhibit genetic lesions within the 45S ribosomal RNA gene repeats clusters on Chrs 12, 16, 18 and 19 consistent with the observed nucleolar associated DNA damage foci in aging HSCs. The present study demonstrates that the consequences of reduced MCM expression on local genome instability is reflected in micronuclear DNA sequences allowing prediction of specific genetic lesions in the etiology of cancer and during aging.Replication licensing may become limiting due to genetic deletion of y tumors , oncogeny tumors  and duriy tumors . It is ny tumors . In the To map sites of ongoing genomic instability across the genome we take advantage of the fact that in the hematopoietic system of mammals DNA remnants resulting from genetic damage events during differentiation of hematopoietic stem cells (HSCs) to erythrocytes are retained in the cells as micronuclei following enucleation . To isolSequence tag density in the WBC and GRN fractions of wt mice are largely uniform both between and across individual chromosomes. In contrast, in the MN fraction, although the minimal sequence tag density is similar between chromosomes, there is a significant deviation from the average sequence tag density as a function of position across each chromosome. This, in part, reflects the frequency with which different portions of each chromosome are present in micronuclei. Similar patterns are seen in the MN fractions of two different wt mice where the correlation between experimental repeats is 0.988. Several parameters are defined to describe the observed changes quantitatively . First, Functions describing \u03b2 and \u03c1 for wt animals were established using Chr7 and Chr11 since, by inspection, there are few localized increases in sequence tag density on these chromosomes. Both \u03b2 and \u03c1 are non-linear where the best fit for each is a quadratic function  panel a.To identify localized regions of the genome exhibiting elevated instability, \u03b3 values were estimated by determining the rate of the sequence tag coverage change within smoothed and normalized 20 kbp windows genome wide. Although the overall contribution of \u03b2 and \u03c1 to differential representation of centromere distal and, to a lesser extent proximal ends of whole chromosomes is significant over entire chromosomes, over shorter 20 kb intervals these effects are minimal and the slope of the sequence tag density largely reflects the localized effect \u03b3 e.g. , \u03b3 trackPrior studies have shown that agents that inhibit replication fork progression lead to chromosome breakage at specific locations across the genome referred to as common fragile sites. Characteristics of these sites have been defined where breakage typically occurs at large, late replicating, transcriptionally active genes . To deteThe micronuclear fractions from HU treated mice show a more rapid increase in sequence tag density as a function of distance from the centromere relative to wt mice and the value estimated for \u03b2 (again using Chr7 and Chr11) is increased ~5 fold  panel a.Wwox, Inspection of the micronuclear sequence data from HU treated mice reveals sharp increases in sequence tag coverage distal to discrete locations suggesting that specific sites are disproportionately affected by HU treatment  protein 2 deficient mice . MCM2 deComparison of the sequence tag densities derived from micronuclei of wt and MCM2 deficient 129Sv mice shows that the additional breaks resulting from MCM2 deficiency suppress \u03c1 values and modestly increase \u03b2 values relative to wt cells  panel a.http://www.ncbi.nlm.nih.gov/gene/19791). These are typically composed of 30\u201340 repeats of an approximately 45 kb repeating unit at each location . One poin situ hybridization (FISH) plus spectral karyotyping (SKY) to localize a probe for the 45S ribosomal gene  to specific chromosomes. Results from these studies  and mapping breaks in banded metaphase chromosomes. Numerous studies have characterized sites that are frequently affected in human and mouse cells and led to identification of a set of locations termed common fragile sites that are affected under conditions of chemically induced replication stress in a high proportion of individuals. These sites have been extensively characterized and are significantly associated with the presence of large, transcriptionally active, and late replicating genes over 300 kbp in size . The frein vivo. Many micronuclear sequences result from the presence of double strand DNA breaks that lead to failure of acentromeric portions of chromosomes to segregate to the nucleus during mitosis. Further we take advantage of the fact that micronuclei are retained in maturing RBCs following enucleation in mammals. In contrast to defining fragile sites cytogenetically, harvesting micronuclei from RBCs allows recovery of tens of millions chromosomal remnants each of which defines a breakpoint that can be queried at nucleotide level resolution by high throughput sequencing. Although there is a concern that erythroblasts (just before enucleation) may not reflect the DNA repair and checkpoint responses typical of other cells, application of the Mic-Seq method to define fragile sites in mice treated with hydryoxyurea shows that the method identifies 5 of the 8 molecularly characterized fragile sites previously defined in mouse lymphocytes [Wwox and Immp2 genes. Further sites detected in this study support that the majority of the most sensitive HU induced fragile sites occur within subdomains of the transcribed regions of large (>300 kbp), transcriptionally active, genes similar to prior studies and consistent with the possibility of interference between the transcription and replication machinery under conditions of DNA polymerase inhibition [Here we have used the representation of different genomic regions in micronuclear DNA sequences to infer the frequency of chromosome breaks in erythroid cells phocytes  includinhibition \u201316, 20.Similar to HU treatment, reduced replication licensing results in genome instability, increased DSBs, and chromosomal deletions and rearrangements \u20135, 9. ThShort nascent strand analysis has shown that origin usage does not decline uniformly across the genome, but rather specific locations lose function preferentially, in MCM2 deficient MEFs . These lTcf3 is sufficient to drive TLL formation [Even within early replicating regions of the genome chromosomal breaks detected by Mic-Seq show only a modest correlation with locations where SNS analysis demonstrates that origin usage is most affected. Nonetheless, it is important that many of the locations showing recurrent deletions in the TLLs that arise in MCM2 deficient mice  are siteormation  and the The strongest signals observed by Mic-Seq in MCM2 deficient mice are associated with chromosomes carrying nucleoli and implicate a high rate of DSBs within rDNA clusters as the location of much of the DNA damage, and the source of a large proportion of the additional micronuclei, found in MCM2 deficient mice. This observation confirms and extends prior studies showing that, as mice age, MCM levels are suppressed in HSCs and, coincident with the loss of MCM expression, nucleolar associated \u03b3-H2AX foci accumulate . The preThese results demonstrate that experimental reduction of MCM proteins in young asymptomatic mice is sufficient to cause a profile of genome instability that predicts at least a subset of chromosomal locations where genetic damage is found in both cancers and during aging. The observation that, unlike HU, many locations where MCM2 deficiency causes instability can already be detected in young wt mice suggests that even under normal conditions the distribution of licensed DNA replication origins contributes to base line levels of chromosomal instability. Although replication stress is widely recognized as a potent cause of genomic instability , 25, theAnimal husbandry programs and protocol reviews are in compliance with NIH, USDA, and New York State Standards. Mice were maintained in facilities covered under NIH assurance #A-3143-01, certified by New York State for the use of living animals, and the USDA APIHS registration as research facility #21\u2013124. The studies were approved by the Roswell Park Cancer Institute Animal Care and Use Committee under Protocols 817M and 876M.IRES-CreERT2/IRES-CreERT2 (MCM2 deficient) mice were used in studies addressing the effects of MCM2 deficiency. For studies addressing the effects of hydroxyurea (HU), 3 month old wild type 129Sv mice were administered HU continuously in the drinking water at the concentrations indicated in the text. Blood samples were taken by retro-orbital bleed or cardiac puncture.Five to six week old wild type 129Sv and Mcm2 For flow cytometric analysis of micronuclei  blood saCombined SKY/FISH was performed on wt or MCM2 deficient mouse embryonic fibroblast (passage 3) by the Roswell Park Cancer Institute SKY/FISH core facility. The rDNA probe for FISH analysis was prepared using Nick Translation Reagent Kit 07J00-001 (Abbott Molecular Inc.) Green-dUTP 02N32-050 (Abbott Molecular Inc.) to fluorescently label a 7109 bp EcoRI fragment from human genomic ribosomal gene DNA containing a portion of the 18S ribosomal RNA gene, the intergenic spacer, the 5.8S ribosomal RNA gene and a portion of the 28S ribosomal RNA gene.Between 400\u2013500 \u03bcl of whole blood was washed with 10 ml of phosphate buffered saline (PBS) 3 times and re-suspended in 3 ml PBS. The sample was then layered over 2 ml of Lymphocyte Separation Medium  in a 15 ml centrifuge tube and spun for 15 min at 800 RPM. Lymphocytes (WBCs) at the PBS-media interphases were collected and placed in a 15 ml tube and washed 3 times with 10 ml PBS prior to pelleting for DNA isolation. Separation media was removed from the red blood cell (RBC)/granulocyte (GRN) pellet in the original tube and cells were washed 3 times with 10 ml PBS and pelleted. The cell pellet was then resuspended in 4 ml of RLF lyse buffer and incubated at room temperature for 5 minutes prior to spinning at 800 RPM for 5 minutes. The supernatant was collected as the RBC micronuclear fraction (MN). The pellet is the granulocyte fraction and was washed with 10 ml PBS and pelleted for DNA isolation. WBC and GRN pellets were re-suspended in 2 ml 1X lysis buffer with 1 ug/ml proteinase K and the RBC fraction was brought to 1X lysis buffer and 1 \u03bcg/ml proteinase K using 4X lysis buffer. Samples were incubated at 37\u00b0C overnight and extracted with an equal volume of phenol-chloroform-isoamyl alcohol. Nucleic acid was precipitated with isopropanol, washed with 70% ethanol and re-suspended in 100 ul TE buffer. Samples were treated with 2000 units ribonuclease T1 (Invitrogen) and 3 ug ribonuclease A (Invitrogen) for 30 minutes at 37\u00b0C followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation of the DNA.Libraries were prepared for DNA sequencing using Wafergen prep X kits and TruSeq Index tags . Paired end 100 nt sequences were generated using an Illumina HiSeq2500. A summary of numbers of mapped sequence tags generated for each sequencing library is given in Sequence mapping and coverage generation: Sequences were aligned to the reference mouse genome (mm9) using BWA [sing BWA  with defThe Pearson correlation coefficient is 0.975 between MCM2 deficient samples 1 and 2, 0.938 between HU treated samples 1 and 2, and 0.969, 0.978, and 0.988 between wt samples 1 and 2, 2 and 3 and 1 and 3 respectively.  Based on the extremely high linear correlations, data was pooled for samples from the same experimental condition to reach better accuracy and allow the use of smaller window sizes in subsequent analyses. A window based approach is employed to infer the parameters in the model by counting read pairs in overlapping windows. Proper paired reads with mapping scores greater than 20 are assigned to a window if the middle point of the left-end read is within the window. A 40 kb window was applied where adjacent windows are overlapped by 20 kb and for pooled data, a 20 kb window with a 10 kb overlap is used.Window count data are first normalized against the GRN fraction where deviation in sequence coverage is expected to result from GC bias, other sequencing biases and strain specific bias as the reference genome is not 129Sv. The GRN fraction from the HU untreated control was used as it shows similar bias with MN fractions and windows with read counts outside of three standard deviations are excluded. The data are further normalized to remove any effect related to sequencing depth using scaling factors determined by \u03b1 estimates. The number of read pairs for each sample is related to parameter estimates as more reads often have better coverage/higher \u03b1. \u03b1 values are estimated using the minimum median value of 1000 consecutive data points, this estimate ignores \u03b2 and \u03c1 effects and is an over-estimate. Then the median ratio is selected as a base line for data scaling after excluding chromosomes with strong \u03b1 changes, which include chr 6, 12, 15, 16, 18, 19, and X. The normalization will make any biological effects comparable as they are scaled to the same level. For the pooled data, the following factors are applied: MCM2 deficient (1.742), HU treated (1.000) and wt (2.037).\u03b3 plots: To extract \u03b3 values, pooled coverage data was windowed and smoothed monotonically. Monotonic smoothing is used to suppress the effect of local changes in sequence tag coverage due to GC bias or other effects that are unrelated to the cumulative changes in coverage expected for \u03b3. For chromosomes exhibiting significant levels of \u03c1 (Chr1 and Chr2 in wt samples), a monotonically decreasing smoothing line is fitted before the minimum median value point while after that point an increasing smoothing line is fitted. R package scam [age scam  is used 2 + \u03c1(Chr length\u2014x)2 where deviation from this relationship is defined as \u03b3. Finally, the distance effects of \u03b3 values related to telomeres were normalized by multiplying the corresponding distance and the \u03b3 peaks do not show any relationship with genomic locations after normalization.\u03b3 plots were further normalized by removing \u03b2 and \u03c1 effects estimated using chr7 and 11. To estimate \u03b2 and \u03c1, a quadratic function with 2 quadratic terms for beta and rho separately is fitted using non-linear weighted least-squares method provided by nls R function. Chr7 and chr11 are used for the inference as, by inspection, they have smallest \u03b3 effects. To further estimate the accuracy of the estimation, bootstraps of 50% data points without replacement with 1000 repeats were carried out to estimate standard deviation and 95% confidence interval. The effect of \u03b1, \u03b2 and \u03c1 on sequence tag coverage at various positions across a chromosome is then described as \u03b1 + \u03b2xTo establish cutoff values for normalized \u03b3 peaks, the \u03b3 values (with \u03b2 and \u03c1 effect removed) on chr11 and chr7 are used to estimate the null distribution and the histograms are shown in Major satellite (MaSat) and minor satellite (MiSat) sequences  were conhttps://www.ncbi.nlm.nih.gov/sra/?term=SRP091564) under accession number SRP091564.Sequence data from this study have been submitted to the NCBI short reads archive (SRA),  and GRN (panel b) blood fractions of the 6 week old wt 129Sv mouse shown in (TIF)Click here for additional data file.S2 FigWild type 129Sv mice were treated with varying concentrations of HU (n = 3 for each condition) as indicated and assayed for RBC MN frequency at 1 week or 3 weeks of treatment in panel (a) and by CBCs at three week of treatment in panel (b). Panel (c) is a list of genes over 300 kbp in length that contain the largest \u03b3 peak regions in HU treated mice. These include sites 1\u20133 and 5 (indicated in parentheses following the gene name) as marked in (TIF)Click here for additional data file.S3 FigPanels A and B show 20X images of blood smears from 6 week old129Sv wt (panel a) and MCM2 deficient (panel b) mice stained with acridine orange (green). Arrows indicate micronuclei. Panel c shows RBC micronuclear frequency determined by flow cytometry for wt ) and MCM2 deficient  6 week old129Sv mice (error bars indicate s.d.). Panel d shows SKY/FISH analysis to identify chromosomes hybridizing to a 45S rRNA gene probe sequence where signal is seen as green . Chromosomes 6, 12, 15, 16, 18, and 19 are marked by arrows as indicated. Panel (e) shows the number of chromatids exhibiting 45S rRNA gene signal and panel (f) shows the average signal strength per chromatid  in 13 metaphase spreads (52 chromatids) for each chromosome (error bars indicate s.d.). Panel (g) shows the ratio of sequence tags mapping to the major (MaSat) relative to the minor (MiSat) satellites for wt and MCM2 deficient granulocyte (GRN), micronuclear (MN) and white blood cell (WBC) fractions as indicated.(TIF)Click here for additional data file.S4 FigPanels a-f show MEFs from wt (a-c) and MCM2 deficient (d-f) embryos stained for nucleolin , \u03b3H2AX  and counter stained with DAPI (blue). Panels c and f are overlays of panels a/b and d/e respectively and the proportion of nucleoli containing \u03b3H2AX foci in wt n = 122, 345) verses MCM2 deficient  cells are quantified for two experiments in panel g. Panels h and i compare sequence tag density over the 45S rRNA gene using data from total genomic DNA from thymus to estimate the ability to map sequences across the repeat (h) or short nascent strands prepared from wt (blue) or MCM2 deficient (red) MEFs by nascent strand capture and release (i) extracted from data in [2, 345 ve(TIF)Click here for additional data file.S5 FigPanel a shows \u03b2 and \u03c1 values estimated from chr7 using pooled wt, MCM2 deficient and HU treated samples. To further estimate the accuracy of the estimation, bootstraps of 50% data points without replacement with 1000 repeats were carried out to estimate standard deviation and 95% confidence interval as shown in the table. Panel b shows normalized \u03b1 values for individual wt (3), MCM2 deficient (2) and HU treated (2) samples. Panel c shows a plot of 45S ribosomal RNA gene (rDNA) sequence tag coverage (y-axis) against major satellite (MaSat) coverage for individual wt (3), MCM2 deficient (2) and HU treated (2) samples from both GRN fractions (G) and MN fractions (R). The shaded area indicates the 95% confidence interval excluding the MCM2 deficient MN samples.(TIF)Click here for additional data file.S6 FigChr7 and Chr11 were chosen to estimate experimental variance in \u03b3 values since by inspection they exhibit few local \u03b3 peaks. In panel a, \u03b3 value distributions are shown for each chromosome for hydroxyurea treated (HUEX), MCM2 deficient (Mcm2) and wild type (wt) mice as indicated. Panel b shows the standard deviations estimated from each of the distributions shown in panel a.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file."}
+{"text": "The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.We identified 17 genes significantly differentially expressed between mFTC and wFTC. CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.We conclude that the underexpression of The online version of this article (10.1186/s12885-017-3948-3) contains supplementary material, which is available to authorized users. Thyroid cancer is the most frequent type of endocrine cancer with an incidence of 12 cases per 100,000 individuals . PapillaBRAF mutations have been shown to be useful for predicting recurrence and/or disease persistence , 20 FTA, and 19 normal samples of adjacent/contralateral thyroid tissues from patients with DTC from this series. The histology of all DTC was revised by two pathologists (CE and MSS) and the final classification was made according to the WHO criteria \u2009>\u20091 was considered as gain of expression, and fold change [log2 (2-\u0394\u0394CT)]\u2009\u2264\u20091 was established as normal gene expression. For CRABP1 and CILP genes, fold change [log2 (2-\u0394\u0394CT)]\u2009<\u2009\u22121 was considered as loss of expression, and fold change [log2 (2-\u0394\u0394CT)]\u2009\u2265\u2009\u22121 was established as normal gene expression. The diagnostic performance (sensitivity and specificity) of those cut-off values are presented in Table\u00a0The relative quantification of target genes was determined using the comparative CT method 2systems) . This prsystems .C1QL1, LCN2, CRABP1 and CILP genes. Gene expression values for each sample are normalized read counts, estimated by using RSEM software  and LCN2  expression were significantly associated with the extrathyroidal extension of the tumour. In the multivariate model, gain of C1QL1  and LCN2  expression were independent predictive factors for extrathyroidal extension in DTC. Additionally, gain of C1QL1 expression was observed in the larger (> 4\u00a0cm) DTC (OR\u2009=\u20094.60), but the difference was only borderline in terms of statistical significance (P\u2009=\u20090.056). No associations were found between CRABP1 and CILP expression and prognostic factors of thyroid cancer.In the univariate analysis, gain of the The clinical behaviour of thyroid cancer is still difficult to predict and clinical and histopathological prognostic factors remain the key elements for diagnosis and prognosis of the patients . Due to C1QL1, LCN2, CRABP1 and CILP genes were selected for further validations in a larger series of DTC, using FTAs and a pool of normal thyroid tissues for comparison.In total, 17 genes were found to be differentially expressed in mFTC and wFTC. After customized filtering and validation of expression values by qPCR, CRABP1 was differentially expressed between DTC, FTA and normal thyroid tissue. Expression of CRABP1 was significantly lower in FTC, FVPTC and PTC and in all ten thyroid cancer cell lines than in normal thyroid and FTA. In the ROC analysis, AUC for the CRABP1 had the highest value (0.902), in the comparison of DTC versus normal thyroid. Gene expression from thyroid samples in TCGA reinforced CRABP1 as biomarker in DTC.Remarkably, CRABP1 (cellular retinoic acid binding protein1) encodes a specific binding protein for a vitamin A family member and is thought to play an important role in retinoic acid-mediated differentiation and proliferation processes. Loss of CRABP1 expression in thyroid cancer was shown in previous studies ]. Our results indicate that LCN2 expression may also serve as a useful biomarker in DTC, namely for the FNAB diagnosis of thyroid nodules.e stress . Consist cancers . Our datehaviour . LCN2 haC1QL1 was found differentially expressed between DTC and normal thyroid tissue. We observed a clear cut gain of C1QL1 expression in FTC, FVPTC and PTC. The relative low number of FTC of the present series can justify the lack of statistical significance verified in the comparison of FTC with normal thyroid tissue regarding C1QL1 expression. Gain of C1QL1 expression was significantly associated with the presence of extrathyroidal extension in DTC. In PTC, tumours with gain of C1QL1 expression were significantly larger than tumours without C1QL1 overexpression. Since extrathyroidal extension and tumour size are major prognostic factors of thyroid cancer it may be advance that gain of expression of C1QL1 will probably be a good indicator of clinical evolution. Notably, gain of C1QL1 expression was significantly higher in wFTC than in mFTC, reinforcing the assumption that gain of C1QL1 expression is related with thyroid cancer progression. Although we found an association of C1QL1 overexpression and lymphocytic thyroiditis in DTC, further statistical analyses revealed no significant differences of C1QL1 expression between the subtypes of DTC with lymphocytic thyroiditis and without lymphocytic thyroiditis (P\u2009=\u20090.316). Gene expression from thyroid samples in TCGA reinforced C1QL1 as biomarker in DTC.C1QL1 is member of a subfamily of small secreted proteins of unknown function -C1q-like that are expressed almost exclusively in brain, and produced in differential patterns by specific types of neurons  as input. For evaluation of the combined biomarker panel the sum of log2 (2-\u0394\u0394CT) expression values from genes with gain (C1QL1 and LCN2) and loss (CRABP1 and CILP) in DTC were used. AUC, area under the curve; CI, confidence interval. (DOCX 14 kb)Receiver Operating Characteristics (ROC) curve analysis. ROC curves for individual biomarkers were generated using log2  of C1QL1 (a), LCN2 (b), CRABP1 (c) and CILP (d) in differentiated thyroid cancer (DTC) and normal thyroid (NT) tissues available in The Cancer Genome Atlas (TCGA). Each dot represents the gene expression value of each sample. The lines represent the averages. Statistical significance values: *, P\u2009<\u20090.001. (JPEG 518 kb)Gene expression of Additional file 12: Table S6.Clinicopathological and genetic data of the FTC classified by classes based on gene expression. (DOCX 18 kb)Additional file 13: Table S7.Clinicopathological and genetic data of the FVPTC classified by classes based on gene expression. (DOCX 17 kb)Additional file 14: Table S8.Clinicopathological and genetic data of the PTC classified by classes based on gene expression. (DOCX 17 kb)"}
+{"text": "EPB41L3 in 148 anal and perianal biopsies to determine whether high levels of methylation would be associated with anal intraepithelial neoplasia (AIN). The most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade AIN (lgAIN), 82% of the 59 high grade AIN (hgAIN) and 4 of the 5 anal cancers. A methylation score was developed (0.561*HPV16me+0.439*EPB41L3) which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers (p < 0.0001). The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6% , specificity of 50.7%  and area under the curve of 0.82 (95% CI: 0.75\u20130.89) for separating hgAIN and cancer from benign and lgAIN biopsies. We conclude that methylation of HPV16 and EPB41L3 show highly significant association with increasing severity of AIN and cancer and may be useful as biomarkers in anal disease.We studied DNA methylation patterns of human papillomavirus (HPV) and tumor suppressor gene  Human papillomavirus (HPV) infects a majority of people worldwide. Infection can occur at any age and can either be transient  or could be persistent and last for many decades . High riAnal cancer has been growing in incidence in the past few decades, especially in women and also in men who have sex with men (MSM). Furthermore, anal cancer incidence is higher in HIV-positive MSM with approximately 100 cases compared to 25 cases per 100,000 in HIV-negative MSM  and onlyAnal cytology sampling is problematic because anal folds may hide lesions and this has resulted in recommendations for more frequent sampling to compensate for poor sensitivity . Normal,a priori hypothesis that high levels of methylation at genomic positions associated with hgCIN would also be associated with hgAIN. Here, we focus on the methylation of host gene EPB41L3 and the high risk viral types: HPV16, HPV18, HPV31 and HPV33. EPB41L3 (Erythrocyte Membrane Protein Band 4.1 like 3) is a tumor suppressor gene that inhibits cell proliferation, promotes apoptosis and has been found to be highly methylated in many cancers such as lung, cervix, ovarian and breast [DNA methylation testing of HPV and human genes has been validated as an accurate method for detection of CIN2 and CIN3 \u201322. Leved breast \u201328.There were 30 biopsies with <AIN, 43 lgAIN, 59 hgAIN and 5 cancers among the anal samples and 11 biopsies of high-grade perianal lesions (Table About a third 47/148) of samples were infected with multiple HPV types. 33% of anal biopsies with either <AIN, or lgAIN histopathology were not infected by hrHPV types while 53% and 33%, respectively, were HPV16 positive , but no significant differences in the likelihood ratios were found for methylation of HPV18, HPV31 and HPV33 and these latter markers were dropped from further analysis . The bivariable logistic regression using EPB41L3 and HPV16me was highly significant , there was a highly significant trend of increased methylation with disease progression . Figure EPB41L3, HPV16me and the DNAme score.The DNAme levels of p < 0.0001) for EPB41L3, 0.781  for HPV16me and 0.821  for the DNAme score. Figure The ROC curves comparing the methylation levels in the <AIN and lgAIN samples to the hgAIN and cancer cases Figure  had an ASensitivity and specificity of the DNAme score to detect hgAIN and cancers and the proportion of positive samples identified by the DNAme score is shown in Table EPB41L3 against HPV16me with the relationship having a weak Spearman correlation. We also investigated whether the DNAme score would be able to correctly identify the 11 high-grade perianal samples  of HPV16. The AUC of the methylation score for separating <AIN and lgAIN from hgAIN and cancer was 0.82  for DNA methylation, similar to what has been generally observed for cervical cancer. The DNAme score we developed is a multi-biomarker panel composed of three CpG sites within the EPB41L3. These data demonstrate that DNA methylation provides significant diagnostic information for detecting hgAIN in addition to that provided by HPV16 genotyping and suggests that a methylation test may be used to detect essentially all the cancers. The methylation score in HPV16 negative people did not show a significant discriminating effect. However, we cannot be sure of the lack of value of methylation information in the HPV16 negative samples because the analysis was underpowered for this endpoint. These results indicate the need for larger studies and a search for more genes that could provide additional triage information in people infected with hrHPV types other than HPV16.It is notable that the triage information for hgAIN and cancer provided by the methylation score was significantly higher only in HPV16 positive people ; howeverA limitation of the study is that we used FFPE biopsies. Our results need to be replicated in an adequately powered study of exfoliated anal cells because in routine practice hrHPV positive patients would have methylation tests performed on exfoliated cells collected by a swab or similar device. This would allow efficient triage to HRA, thus reducing costs, anxiety and possible over-treatment of low risk people. Our study had incomplete information on HIV status and the small size of the HIV-negative subset likely produced some ascertainment bias. Another limitation of our study is the use of patients (mostly MSM) recruited from two sites specializing in anal HPV-related disease in London. It remains to be seen if our results can be duplicated in other settings.We conclude that high levels of DNA methylation are associated with hgAIN and anal cancer. This finding should be further explored to better understand the biological mechanisms and the value of DNA methylation testing as a molecular triage of hrHPV positive individuals for high-resolution anoscopy screening.A set of anal and perianal biopsy specimens were obtained from 148 patients  of whom 94 were HIV positive, 40 were HIV negative and 14 had not been tested for their HIV status . The forIn patients undergoing HRA, biopsies were obtained from areas of clinical interest that exhibited acetowhite changes (regardless of vascular changes) on 5% acetic acid application. Most of the biopsies showed morphological changes varying from slight to severe which distinguished them from the surrounding non-acetowhite normal epithelium. The main clinical endpoint for all comparisons was the histology result. The biopsies were graded histopathologically using the AIN terminology  which isWe used H&E sections annotated by MS as a guide to dissect areas of interest on the corresponding four unstained 5 \u03bcm sections, as previously described . If moreThe samples were tested using the PapType High Risk HPV Detection and Genotyping kit  according to the manufacturer's instructions. The kit is able to detect 13 high-risk HPV types  a possible high risk type (HPV66) and two low-risk types (HPV6 and HPV11). The PapType test was performed with 10 \u03bcL of DNA in a final reaction volume of 20 \u03bcL with the addition of 2% Tween 20. The PCR reaction amplifies a variable region of the L1 gene of the HPV genome. A fragment of the human cardiac myosin light chain gene (MLC-1) was co-amplified in the same reaction vessel as a quality and quantity control. For simplicity, we categorized the samples into three HPV genotype groups independently of whether they were singly or multiply infected: those infected by (1) HPV16, (2) by any other high-risk types (including HPV66) and (3) those HPV negative or infected only by a low-risk type (lrHPV). We did not combine HPV18 with HPV16 due to obvious differences in viral disease characteristics as noted in earlier studies .EPB41L3 and viral late genomic regions of HPV16  and HPV18 , HPV31  and HPV33  as previously described [EPB41L3 (HPV assays) in a 25 \u03bcl volume with final concentration of reagents of 1\u00d7 for Coral Load and PyroMark mix, 0.2 \u03bcM of PCR primers. PCR cycling conditions were 15 minutes at 94\u00b0C, followed by 45 cycles of 94\u00b0C, 54\u00b0C, 72\u00b0C each for 30 seconds and a final extension at 72\u00b0C for 10 minutes. The PCR products were pyrosequenced using a PyroMark\u2122Q96 ID (Qiagen) instrument as previously described [Bisulfite conversions on 20 \u03bcl of DNA extracts were done using EZ DNA Methylation Kit  following the manufacturer's instructions. DNA methylation (DNAme) was measured by pyrosequencing for human biomarker escribed . All pyrEPB41L3 and HPV positive samples were imputed using a median regression with age as a predictor and DNA methylation as an outcome. All statistical analyses were performed on the imputed data set [EPB41L3, 22 for HPV16L1, 31 for HPV16L2, 5 for HPV18, 11 for HPV31 and none for HPV33. Of the 11 perianal samples, 4 had missing values for HPV16L1, 1 for HPV16L2 and 3 for HPV18. No value was missing for EPB41L3, HPV31 nor HPV33 , a variable called HPV16me was created by taking the geometric mean of DNAme levels of HPV16L1 and HPV16L2.Statistical analyses for the anal and perianal lesions were not combined. Spearman correlations were calculated between the markers. Since methylation values of HPV16L1 and HPV16L2 regions were correlated  with 95% confidence intervals were estimated and reported. Performance of the markers were assessed univariably by the LR \u03c72 test and the area under the curve (AUC) with 95% confidence intervals (CI) using the Delong method [Univariable and bivariable logistic models were fitted for statistically significant genes with the outcome measures 0 = <AIN and lgAIN, and 1 = hgAIN and cancer. The likelihood ratio (LR) \u03c7g method . SensitiEPB41L3 and HPV16me was computed as a linear predictor of the fitted bivariable logistic regression. Confidence intervals for difference in sensitivities and specificities between the DNAme score at different cut-offs and HPV16 genotyping was computed using 2000 stratified bootstrap replicates as recommended by Carpenter and Bithell [EPB41L3, HPV16me and the DNAme score to test for significant changes of methylation between the groups .A combined DNAme score of  Bithell . Cuzick Multiple imputations analyses were performed to test whether the single imputation led to any bias . We usedp-values were two-sided with significance set at \u03b1 < 0.05. Analyses were undertaken using R statistical software version 3.3.1 [All on 3.3.1 ."}
+{"text": "To the editor: We greatly appreciate the editorial by Penttinen and Friede summarising the data regarding recent observations in the United States (US) of decreased effectiveness of the influenza A(H1N1)pdm09 strains  included in live attenuated influenza vaccines (LAIV) [luenza AHN1pdm09 sBased on evidence presently available to us, we believe that reduced replicative fitness of the A/California and A/Bolivia (H1N1)pdm09 LAIV strains is the most probable root cause for the reduced vaccine effectiveness (VE). From 2010/11 through 2013/14, LAIV VE in children aged 2\u201317 years against matched A(H3N2) and B strains has been comparable to that observed with IIV . LaboratIn the context of reduced replicative fitness, vaccine\u2013virus interference may have contributed to the observed reduced VE. However, vaccine\u2013virus interference specific to the quadrivalent formulation appears to be an unlikely root cause of the reduced VE with LAIV. LAIV3 demonstrated reduced VE against A(H1N1)pdm09 in 2010/11 in the US . AdditioBecause rates of vaccine coverage in the US have historically been higher compared with European countries, questions have been raised regarding the role of prior vaccination in the reduced AH1N1)pdm09 effectiveness N1pdm09 e-10. No sIn 2013/14, with LAIV4 containing the A/California strain, a statistically significant correlation was observed between reduced LAIV VE against (H1N1)pdm09 viruses and higher outdoor temperatures during LAIV lot unloading at US distributors . In laboWe have initiated a multifaceted scientific investigation into the causes of the recently observed reduced effectiveness of LAIV, with the goal of identifying a more effective A(H1N1)pdm09 LAIV strain for potential inclusion in the 2017/18 LAIV formulation. All potential hypotheses continue to be evaluated. We welcome the input and support of the multiple stakeholders involved, including national public health agencies, the World Health Organization, and additional external scientific experts, as we work together to ensure that VE of LAIV is improved in future influenza seasons."}
+{"text": "After 3 days as a Category 3 and 4 hurricane in Haiti and Bahamas, Hurricane Matthew moved along the coast of the southeastern United States during October 6\u22128, 2016  . DrowninDespite public health warnings to avoid flood waters, among all 23 hurricane-related drownings, 18 deaths (78%) occurred in motor vehicles . As little as 6 inches of water might result in loss of control of a vehicle, and 2 feet of water can carry most cars away ("}
+{"text": "The BRCA1-associated protein 1 (BAP1) is an example of a critical player in determining the outcome of the interaction of Genes 3 Original studies by Carbone\u2019s group identified the association of germline mutations in BAP1 with early onset of benign melanocytic skin tumours and a later high incidence of malignant mesothelioma together with uveal and cutaneous melanoma.6 The study conducted on two unrelated US families, referred to as \u2018L\u2019 and \u2018W\u2019 for their origins from Louisiana and Wisconsin respectively, implicated the 3p21 chromosomal region with an unexpectedly high incidence of malignant mesothelioma following minimal exposure to asbestos.2 Sequencing of this locus led to the identification of heterozygous mutations in BAP1, uncovering a novel familial autosomal dominant cancer syndrome. Indeed, biallelic somatic mutations in BAP1 are also frequently observed in human cancers.8 Nearly all cancer types have been reported in carriers of BAP1 germline mutations, but the high incidence of tumours associated with environmental stressors, such as mesothelioma (with asbestos), melanoma and skin tumours (with UV), highlights BAP1 as a critical player in the interaction of genes with environment.BAP1 is an ubiquitin C-terminal hydrolase belonging to a larger group of deubiquitylating enzymes.3 Nuclear BAP1 indeed deubiquitylates BRCA1\u2010associated RING domain 1 (BARD1), which in a heterodimer with BRCA1, forms the E3 ubiquitin ligase that regulates the DNA damage response (DDR).10 Loss of BAP1 resembles BRCA1 deficiency, resulting in hypersensitivity to ionising radiation.3 However, an important unexplored aspect of BAP1 is the significant proportion of the protein that can be sequestered in the cytoplasm by multi-monoubiquitylation of its nuclear localisation signal. In this study Bononi et al. uncovered a cytoplasmic function of BAP1, implicating a novel mechanism for the contribution of BAP1 loss to environmental carcinogenesis.Despite the critical relevance of BAP1 for cancer predisposition, the mechanistic insights underlining its function have so far been elusive. Originally identified in a yeast two\u2010hybrid screen as an interactor of the RING finger domain of the tumour suppressor BRCA1, BAP1 was eventually disproved as a BRCA1 deubiquitylating enzyme.+/\u2212 and BAP1+/+ fibroblasts derived from carriers and control members of families \u2018W\u2019 and \u2018L\u2019. BAP1+/\u2212 fibroblasts express 50% BAP1 protein levels as compared to control fibroblasts, and represent an excellent cellular model to experimentally recapitulate BAP1 deficiency in pre-carcinogenic conditions. In the work published in Nature1 the authors showed in subcellular fractionation experiments a significant accumulation of BAP1 in the cytoplasm and more specifically in the endoplasmic reticulum (ER). A reduced level of BAP1 in BAP1+/\u2212 fibroblasts mainly affected the ER fraction of the protein, suggesting a potential dysfunction of the ER in the mutant cells.1 As BAP1+/\u2212 cells appeared hyposensitive to a wide range of pro-apoptotic drugs and inducers, the authors speculated that BAP1 may contribute to control of the pro-apoptotic Ca2+ release from the ER. BAP1+/\u2212 fibroblasts indeed displayed a reduced level of Ca2+ release from the ER, which was restored by re-expression of chimeric BAP1 mutants selectively targeting ER and cytoplasm, but not the nucleus. Thus, Carbone et al. reported an unanticipated cytoplasmic function of BAP1 that could explain its critical role in determining the biological outcome of its interaction with the environmental stressors.1The authors employed an elegant experimental approach, using BAP12+ flux to the cytoplasm. The data clearly indicated that BAP1 physically interacts with the type-3 inositol-1,4,5-trisphosphate-receptor (IP3R3), and BAP1 expression level is directly correlated with IP3R3 in wild-type and mutant fibroblasts. Deeper biochemical analysis indeed demonstrated that BAP1 enzymatically controls IP3R3 ubiquitination and thus positively influences its stability. Loss of BAP1 therefore results in reduced expression of IP3R3 with correspondingly reduced cytoplasmic Ca2+ release, which in turn determines resistance to apoptotic inducers. The authors also observed an increase of cytoplasmic Ca2+ release and apoptosis in crocidolite asbestos-treated primary human mesothelial cells. Remarkably, depletion of BAP1 or IP3R3 promoted crocidolite asbestos/TNF-\u03b1-induced transformation of primary mesothelial cells, and re-expression of IP3R3 in BAP1-depleted cells partially reverted the phenotype.1 However, experiments with subcellular specific chimeric mutants of BAP1 indicated that both nuclear and cytoplasmic BAP1 contributed to the phenotype. This latter data opens the question regarding the specific contribution of the balance between cytoplasmic and nuclear BAP1 to cell death and genomic stability upon interaction with environmental stressors.As a ubiquitin hydrolase, BAP1 exerts an enzymatic activity which directly influences stability and activation of its substrates. Investigating the cytoplasmic function of BAP1, the authors focused on the possible interaction between BAP1 and ER channels that control CaCell Death & Differentiation2 by the same groups reports an intriguing observation in BAP1+/\u2212 individuals, which might shed light on the molecular basis of the transformation potential of these cells.2 By metabolic profiling of fibroblasts and plasma of carriers and control members from \u2018W\u2019 and \u2018L\u2019 families, the authors identified a clear alteration in cellular metabolism adopted by the mutant cells. BAP1+/\u2212 fibroblasts displayed a typical \u2018Warburg effect\u2019, as their cellular metabolism largely relied on aerobic glycolysis, increased lactate production and reduced mitochondrial respiration.2 Metabolic profiling of plasma from carrier individuals also displayed a similar distribution of the metabolites, and their profile was predictive of the genotype with 100% accuracy. The originality of these data relies on the observation of metabolic rewiring typical of transformed cells in a premalignant cellular setting, thus suggesting that that \u2018Warburg effect\u2019 might facilitate cancer transformation rather than be an adaptive process occurring in malignancy. Whether the observed metabolic change is associated with alteration of the Ca2+ flux and ER/mitochondria function of BAP1+/\u2212 cells will require further investigation and will probably highlight additional layers of complexity on the contribution of BAP1 deficiency to carcinogen hypersensitivity.A second study published in +/\u2212 carrying individuals and suggest new relevant questions, related to BAP1-dependent cancer susceptibility, and to more general gene/environment interactions -environmental signals can trigger BAP1 subcellular localisation and consequentially balance its contribution to genomic stability and cell death. Also, it will be important to assess the contribution of BAP1 somatic mutations to the tumorigenic process, and whether their incidence correlates with a selective mutational landscape. In addition, the contribution of premalignant metabolic rewiring to tumour transformation will stimulate a broad interest in the field. This aspect might also have wider implications for the genes\u2212environment interaction, as it might imply that dietary regimes could influence metabolism and cellular transformation in specific predisposed conditions such as BAP1 mutations. Finally, these studies further highlight the importance of the integration of multidisciplinary approaches to dissect the interactions between genetic and environmental factors, to translate this information and stratify patients for prevention and treatment, and to provide novel biomarkers that predict risk of exposure.In conclusion, these studies have clarified novel important molecular aspects associated with the cancer predisposition of BAP1ractions . It is n"}
+{"text": "Prospective and case-control studies which reported adjusted relative risk estimates and 95% confidence intervals of endometrial cancer associated with a hypertension diagnosis were included. Summary relative risks were estimated using a random effects model. Nineteen case-control studies and 6 cohort studies were included. The summary RR was 1.61  for all studies, 1.73  for case-control studies and 1.32  for cohort studies. The association between hypertension and endometrial cancer was weaker, but still significant, among studies with adjustment for smoking, BMI, oral contraceptive use, and parity, compared to studies without such adjustment. This meta-analysis suggest an increased risk of endometrial cancer among patients with hypertension, however, further studies with more comprehensive adjustments for confounders are warranted to clarify the association.A history of hypertension has been associated with increased risk of endometrial cancer in several studies, but the results have not been consistent. We conducted a systematic review and meta-analysis of case-control and cohort studies to clarify the association between hypertension and endometrial cancer risk. PubMed and Embase databases were searched up to 27 Hypertension is a major cause of morbidity and mortality worldwide and is an established risk factor for coronary heart disease and stroke158910Endometrial cancer is the eighth most common type of cancer in women with approximately 320 000 cases recorded in 2012, accounting for about 4.8% of all cancers in women 1420212223242526272829303132333435363739404142432021242526283032333435363738394222232729313942451620212533352021242528293335th February 2016 for eligible studies. We used the following search terms in the PubMed search: (hypertension OR high blood pressure OR blood pressure OR risk factor) AND . We followed standard criteria for reporting meta-analysesWe searched the PubMed and Embase databases up to 27We included published retrospective case-control studies and cohort studies that investigated the association between hypertension and the risk of endometrial cancer. Adjusted estimates of the relative risk  had to be available with the 95% CIs in the publication. A list of excluded studies and exclusion reasons is provided in The following data were extracted from each study: The first author\u2019s last name, publication year, country where the study was conducted, study period, sample size, number of cases/controls, exposure and subgroups of tumor characteristics  or cancer type (type 1 vs. type 2), relative risks and 95% confidence intervals for the association and variables adjusted for in the analysis. Data were extracted by one reviewer (DA) and checked for accuracy by a second reviewer (AS).We calculated summary relative risks of developing endometrial cancer by history of hypertension using the random-effects model by DerSimonian and Laird2 statistics2 is a measure of how much of the heterogeneity that is due to between study variation rather than chance. I2-values of 25%, 50% and 75% indicates low, moderate and high heterogeneity respectively. We conducted main analyses  and stratified by study design  because of the greater potential for recall and selection bias in retrospective case-control studies and to investigate sources of potential heterogeneity. We also conducted subgroup analyses by other study characteristics such as sample size, number of cases, geographic location, and by adjustment for confounding factors. We also conducted a stratified analysis by whether the articles explicitly stated that participants with prevalent hysterectomies at baseline were excluded, and/or whether participants with incident hysterectomies were censored during follow-up in cohort studies, or excluded from the control group in case-control studies.Heterogeneity between studies was evaluated using Q and IPublication bias was assessed using Egger\u2019s testOut of a total 7879 records identified by the search we included 25 studies with 28385 cases and 300598 participants in the meta-analysis of hypertension and endometrial cancer risk, including six cohort studies394041424320212223242526272829303132333435362\u2009=\u200986%), and it was 1.73  for case-control studies and 1.32  for cohort studies  for studies with such exclusions or censoring and 1.81  for studies without such exclusions or censoring.In a further sensitivity analysis we also conducted a subgroup analysis by whether the studies explicitly stated that they excluded participants with prevalent hysterectomies at baseline and/or stated that they censored participants at the time of incident hysterectomy (cohort studies) or excluded participants who had undergone hysterectomy from the control group (case-control studies). The summary RR was 1.51  for all studies and 1.33  for cohort studies. Further including another cohort study2\u2009=\u200985%) for all studies and summary RR\u2009=\u20091.28  for cohort studies. Mean (median) study quality scores were 7.3 (7.0) for all studies combined, 7.3 (7.0) for case-control studies, and 7.3 (7.0) for cohort studies).In a sensitivity analysis we also included a pooled analysis which assessed the association between quintiles of systolic blood pressure and endometrial cancer riskTo our knowledge this is the first meta-analysis of published observational studies of hypertension and the risk of endometrial cancer and our results confirm that hypertension is a strong risk factor for endometrial cancer with a 61% increase in the relative risk, however, the association was weaker in cohort studies (RR\u2009=\u20091.32) than among case-control studies (RR\u2009=\u20091.73). These findings are consistent with a large cohort study of 290 000 women in Austria, Norway and Sweden which found an increased endometrial cancer risk with increasing levels of diastolic blood pressure and in particular, systolic blood pressureThe present meta-analysis has some limitations. As hypertension is a condition that is strongly related to lifestyle factors and some medical conditions including diet, BMI, physical activity, and diabetes we cannot entirely exclude the possibility that the observed association between hypertension and endometrial cancer risk at least partly could be due to confounding. We found that the association was weaker, but still statistically significant, among studies that adjusted for smoking, BMI, oral contraceptive use, hormone replacement use, parity and age at menopause (RR\u2009=\u20091.14\u20131.34 for studies with such adjustment vs. 1.74\u20132.10 for studies without such adjustment). However, because there was still a significant association in subgroups that adjusted for these factors it could indicate that there is an adverse effect of hypertension on endometrial cancer risk, but that it may be slightly weaker than what was suggested from the overall summary estimates. Because the original studies did not stratify for BMI or diabetes it was not possible for us to investigate whether the association was limited to specific weight classes or if it was modified by diabetes status.We also found that the positive association between hypertension and endometrial cancer persisted when the studies were stratified by whether participants with prevalent hysterectomies at baseline were excluded and/or whether participants with incident hysterectomies were censored, or whether prevalent hysterectomies were excluded from the control group. Hypertension may also be related to hysterectomies5657Case-control studies are more likely to be affected by certain biases, such as recall bias and selection bias. Because we included both case-control and cohort studies there is a possibility that recall or selection bias might have affected the results in the case-control studies and the overall summary estimate. Although the association appeared to be stronger in case-control studies than among cohort studies, there was still a significant association among cohort studies, which suggest that recall bias or selection biases does not entirely explain the observed association. In addition, there was some indication of publication bias with Egger\u2019s test, but this appeared to be restricted to the analyses of case-control studies and all studies combined, and was not observed among the cohort studies.The biological mechanism(s) that may explain an adverse effect of hypertension on endometrial cancer risk are unclear at present. It has been suggested that long-term hypertension may lead to cellular senescence and inhibition of apoptosisStrengths of the present meta-analysis include the comprehensive search strategy, the detailed subgroup and sensitivity analyses, and the large sample size providing a more robust estimate of the association between hypertension and endometrial cancer risk. To date relatively few studies have investigated the association between hypertensive disorders of pregnancy and endometrial cancer risk with one study suggesting an increased risk with hypertensive disorders overall66In conclusion, the results from this systematic review and meta-analysis suggest that women with hypertension may have a 61% increase in the relative risk of developing endometrial cancer. Any further studies should clarify potential effect modification by age, BMI and diabetes status, and the causality of the observed association, as well as the potential underlying mechanism(s).How to cite this article: Aune, D. et al. Hypertension and the risk of endometrial cancer: a systematic review and meta-analysis of case-control and cohort studies. Sci. Rep.7, 44808; doi: 10.1038/srep44808 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Multiple environmental stresses affect growth and development of plants. Plants try to adapt under these unfavorable condition through various evolutionary mechanisms like physiological and biochemical alterations connecting various network of regulatory processes. Transcription factors (TFs) like APETALA2/ETHYLENE RESPONSE FACTORS (AP2/ERFs) are an integral component of these signaling cascades because they regulate expression of a wide variety of down stream target genes related to stress response and development through different mechanism. This downstream regulation of transcript does not always positively or beneficially affect the plant but also they display some developmental defects like senescence and reduced growth under normal condition or sensitivity to stress condition. Therefore, tight auto/cross regulation of these TFs at transcriptional, translational and domain level is crucial to understand. The present manuscript discuss the multiple regulation and advantage of plasticity and specificity of these family of TFs to a wide or single downstream target(s) respectively. We have also discussed the concern which comes with the unwanted associated traits, which could only be averted by further study and exploration of these AP2/ERFs. Plants are mostly affected by multiple stresses like salinity, toxicity, pathogen attack, and wounding. To survive and reproduce in adverse conditions, plants develop various adaptive traits governed through a complicated system. The interconnected and diverse networks operated during downstream signaling cascades are regulated by TFs which bind to the cis elements present in their promoters , Trichodesmium erythraeum (cyanobacterium), Enterobacteria phage RB49 (virus), and Bacteriophage Felix 01 (Virus) are reported to have an AP2 domain , DREB (subgroup A1\u2013A6), ERF (subgroup B1-B6), and others  from Poppy which bind to both DRE as well as GCC box elements thus provide abiotic and biotic stress tolerance in tobacco , ICEr2 and CM2-Conserved DNA Motif 2  to which a bHLH PIF7 (Phytochrome Interacting Factor) interacts and suppresses expression of DREB1C. PIF7 interacts with TOC1 (Timing of CAB expression 1) and PhyB (phytochrome B) to transcriptionally repress the expression  and LHY (Late Elongated Hypocotyl) that bind to the EE (AAAATATCT) and CCA1 (AATCT) motifs of DREB1C/CBF2 promoters and positively regulate their expression . ORCA3 has a regulatory role in primary and secondary metabolism in Catharanthus roseus, therefore it needs to be properly regulated at the transcriptional level. Its promoter carries a 74-bp long autonomous Jasmonate Responsive Element (JRE) having a quantitative sequence responsible for enhanced expression and a qualitative sequence that acts as an on/off switch in response to MeJA. CrMYC2 binds to T/G-box (CACGTG\u2013G box with one mismatch) present in the qualitative sequence of JRE and mediate expression of ORCA3 that in turn regulate early jasmonate-responsive genes  alters its binding affinity to DRE/CRT cis-element  which are then recognized and degraded by N recognins ATE1/2 (Arginyl tRNA Protein Transferases) & PRT6 (Proteolysis 6) as shown in Figure Ubiquitination is another post-translational mechanism through which AP2/ERFs are regulated. It may affect proteins in number of ways, either it process them for proteasomal-mediated degradation, or change cellular location, or alter their activity or enhance/prevent interaction of protein. Adding to the list of tight regulation of DREBs is the ubiquitination by DRIP1/2 (DREB2a-Interacting Protein1) under non-stressed condition. These are RING E3 ligases that interact with DREB2A in the nucleus and leads to its ubiquitination in the cytosol  while some are involved in development (OsERF3 and WOX11 interact with cytokinin-responsive gene RR2 to regulate crown root development in rice)  and AREB2/ABF4 proteins to regulate ABA response  adjacent to DBD that makes DREB2A unstable under normal condition. Deletion of NRD converts DREB2A into its constitutively active form whose overexpression displays reduced growth but improved drought and salt tolerance , GmERF6 (enhanced drought tolerance), and NtERF3 (increased resistance to TMV) positively regulates stress response while AtERF7  negatively regulates stress response  in the alternative respiratory pathway and is involved in seed germination and seedling growth  gene that encodes an isoform of the cytochrome c oxidase zinc binding subunit involved in respiratory chain complex  and Sub1C from rice are induced by ethylene but acts antagonistically to each other in response to flooding  and provide enhanced cold and salt stress tolerance  involved in BR biosynthesis. DWF1/4 leads to accumulation of DELLA protein such as SLR1 (Slender Rice 1) and SLRL1 (SLR1 like) which inhibits GA responsiveness  BR (brassinosteroid) also affect ERF mediated regulation. Hypoxia responsive HRE1 and HRE2 show ethylene response, but unlike RAP2.2 they lead to inhibition of ethylene signaling and provide GA response  gene was found to play an important role in regulating determinacy of spikelet meristem and floral organ identity. MFS1 is associated with unidentified function clade in the family of AP2/ERF which certainly regulates the expression pattern of Long Sterile Lemma (LSL) and the Indeterminate Spikelet1 (IDS1) like genes Supernumerary Bract and OsIDS1 , 10 subgroups, and two soloists  transgenic wheat showed reduced electrolyte leakage and H2O2 content, while increased soluble sugar and proline content , Chit1 (Chitinase 1), RAB18 (ras-related protein 18), LEA3 (Late embryogenesis abundant 3), TIP2 (Delta tonoplast intrinsic protein 2), POX2 (Peroxidase 2), and GST6 (Glutathione S-Transferase 6) while could not bind with DRE cis-element  and displayed better tolerance to bacterial pathogen Ralstonia solanacearum. Constitutive expression of SlRAV2 in tomato also led to enhanced tolerance to R. solanacearum bacterial wilt and increased expression of SlERF5 and PR5 genes  and the zinc finger oxidative stress 2 are predicted targets of this ERF  1long-chain acyl-CoA synthetase1, PAS2 (Pasticcino2), KCR1 (\u03b2-ketoacyl CoA reductase1), ECR , and WSD1 . Wax layer in turn protects plants from high irradiance, desiccation and UV radiation  genes  and post-translational level . Therefore, these aspects need to be exploited at a large level so that specific targets in the complex mechanism could be identified, which could be modulated for development of improved varieties.cis-elements present in the promoter of that gene. We believe that this specificity to identify a single or multiple cis-elements is regulated not only by the rare anomalies in the DBD but also by the motifs flanking it. These bindings are also influenced by protein-protein interactions through different domains. So these domains and motifs, which lead to specificity or plasticity in the interaction of AP2/ERFs with their downstream targets to achieve multiple responses, should be thoroughly studied.AP2/ERFs activate or repress expression of a particular gene by interacting specifically to the Also with recent findings, it is evident that retrograde signaling affects several AP2/ERF-mediated responses. Signaling from mitochondria or chloroplast to the nucleus and its feedback is not properly understood. Therefore, new insight is required in the field of retrograde regulation of AP2/ERFs.AP2/ERFs from different plant species.Recent molecular tools like CRISPR  and CRISPR-associated 9 (Cas9) genes could be applied to different non-model plants where functional mutants are not available. CRISPR/cas9 genome editing tool would have an edge over conventional tedious silencing approach (through pART vectors) or VIGS (through TRV vectors) in the coming era. Therefore, this technique could be exploited for functional characterization of UP and GJ has compiled and written the review. VT and RS has edited and finalized the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "MgF2 conversion layer was first produced by immersing Mg alloy substrate in hydrofluoric acid solution. The outer PCL coating was then prepared using dip coating technique. Morphology, elements profile, phase structure, roughness, mechanical properties, invitro corrosion, and biocompatibility of duplex MgF2/PCL coating were then characterized and compared to those of fluoride coated and uncoated Mg samples. The invivo degradation behavior and biocompatibility of duplex MgF2/PCL coating with respect to ZK60 Mg alloy were also studied using rabbit model for 2 weeks. SEM and TEM analysis showed that the duplex coating was uniform and comprised of porous PCL film (~3.3 \u03bcm) as upper layer with compact MgF2 (~2.2 \u03bcm) as inner layer. No significant change in microhardness was found on duplex coating compared with uncoated ZK60 Mg alloy. The duplex coating showed improved invitro corrosion resistance than single layered MgF2 or uncoated alloy samples. The duplex coating also resulted in better cell viability, cell adhesion, and cell proliferation compared to fluoride coated or uncoated alloy. Preliminary invivo studies indicated that duplex MgF2/PCL coating reduced the degradation rate of ZK60 Mg alloy and exhibited good biocompatibility. These results suggested that duplex MgF2/PCL coating on magnesium alloy might have great potential for orthopedic applications.The present work addresses the performance of polycaprolactone (PCL) coating on fluoride treated (MgF Such closely matched mechanical properties can minimize stress shielding effects caused by difference in elastic modulus between foreign implant and host hard tissue -2,5-diphenyltetrazolium bromide  solution (100 \u03bcl/well) was then added and incubated at 37\u00b0C for 4 hr. At the end, 100 \u03bcl of dimethyl sulfoxide  solution was added to each well. The sample plate was incubated for 1 hr and the absorbance value or the optical density (OD) of the resulting purple colored solution was measured by using an ELISA plate reader  at wavelength 595 nm.MC3T3-E1 preosteoblast cells  were employed in the 5 cells/well were seeded direct onto sterilized MgF2 and MgF2/PCL samples in 24 well plates. These samples were incubated at 37\u00b0C with 5% CO2 atmosphere for 1, 3, and 5 days. After a stipulated time, samples were washed thrice with PBS solution and proceed to fixing with paraformaldehyde  for 15 min at room temperature. The cells were later permeabilized with 0.5% Triton X-100  for 10 min. After subsequent blocking with 2.5% bovine serum albumin  solution for 60 min, cell membrane was immunostained with fluorescein isothiocyanate conjugated phalloidin  solution overnight at 4\u00b0C. Samples were later stained with 10 \u03bcg/ml Hoechst 33342  for 5 min for staining cell nuclei. The stained samples were finally washed with PBS solution thrice, mounted onto slides, and visualized under confocal fluorescent microscope  along with the accompanied FV10i-ASW2.0 viewer software.For cell proliferation studies, 2x105) seeded samples were incubated for 4 hr in humidified atmosphere with 5% CO2 at 37\u00b0C. Following the same procedure steps until BSA blocking as mentioned above for proliferation, the cells were further immunostained using vinculin antibody  and placed overnight at 4\u00b0C, followed by washing thrice with PBS. Afterwards, samples were incubated with Alexa Flour 594  conjugated secondary antibody for 1 hr at room temperature. At the end, cells were stained with FITC conjugated phalloidin and Hoechst 33342 at the specified interval, mounted and analyzed by confocal microscope.For cell adhesion studies, cell . Fur on the right knee of the rabbit was shaved, the exposed skin was thoroughly cleaned with 70% ethanol and disinfected with povidone iodine solution. A full thickness incision measuring 3\u20134 cm in length was made along the lateral aspect of the knee to access the underlying joint. The distal femoral head was accessed by creating another incision on the overlaying soft tissues. The exposed distal femoral head was then drilled with a trephine drill with a 6 mm diameter at a height of 5 mm with constant irrigation with sterile saline solution. The implant  was then carefully placed within the bone defect. Control defect was left without any implant material. Three rabbits were used for each sample. All incisions were then closed with 0\u20135 Vicryl suture and disinfected with povidone iodine solution. Tramadol and Baytril were administered based on recommended doses to manage pain and as prophylaxis respectively. After 2 weeks, the animals were sacrificed via overdose of ethyl ether anesthetic. Tissue samples containing the implants were extracted and fixed using 10% formaldehyde solution. Extracted samples were then scanned with Skyscan 1076 microCT scanner . Data scans were reconstructed and analyzed to determine the progression of implant biodegradation and tissue development. Using CTAnalyzer software , the implanted sample was selected using a series of region of interest to create a volumetric selection. The data set was then segmented through two ranges of gray intensities to separate the implant and the surrounding bone tissue. Volume of the remaining sample was calculated using the same program. Three-dimensional reconstruction was created using the same corresponding data set using CTVox software .To determine the effect of the duplex MgF2/PCL were then dehydrated using subsequent series of ethanol then acetone immersion for 1 day each. After dehydration, the samples were infiltrated and embedded in methyl methacrylate (MMA) resin using benzoyl peroxide as a hardener. MMA embedded blocks were transversally cut using a diamond abrasive cutter  and were polished to 10\u201345 \u03bcm thickness. The slides were washed with distilled water and finally stained with Villanueva Osteochrome Bone stain. Micrographs of the stained tissue sections taken using an optical microscope  equipped with a DP72 digital camera.The fixed tissues ZK60 Mg alloy, duplex MgF2, and duplex MgF2/PCL coatings are shown in 2/PCL coating was found to be uniform and crack-free  and zinc (Zn) elements were present in ZK60 Mg alloy with minute impurities of carbon (C) and oxygen (O) (2 film on the substrate. The oxygen content might be attributed to the formation of Mg(OH)2. Minute traces of carbon are also seen in the film  . Quantitthe film . Small cthe film . The pre surface .2/PCL coating was investigated by TEM micrograph along with selected area electron diffraction (SAD) patterns and mapping profile. Results are shown in 2, and Mg substrate. The thickness of compact MgF2 film was approximately 2.2 \u03bcm and that of outer PCL coating was around 3.3 \u03bcm . The amorphous nature of PCL coating contributed to the diffused ring observed in SAD pattern  peak with tetragonal structure was primarily found along with Mg. However, the intensity of MgF2 peak was quite low compared to that of Mg, suggesting that the fluoride coating layer was thin as depth of CuK\u03b1 line for Mg is around 0.8 \u03bcm at grazing angle of 1\u00b0C [2 by displacement reaction and insoluble MgF2 can form a barrier coating on Mg [2 in MgF2/PCL coatings  coating was significantly increased to 66.3 nm. Minute pores generated on the surface due to acid immersion might be ascribed to the above increment. The surface morphology was described by the precipitation of intermetallic particles (MgZn) on the surface and at grain boundaries  studied that PCL film exhibited poor bonding strength with substrate which might be due to its molecular structure. PCL possess lower ratio of oxygen by its weight, which leads to weak electrostatic interaction with Mg substrate [2 coating. The relatively increased adhesion of MgF2/PCL coatings over PCL coatings was more likely due to the mechanical interlocking as PCL solution was infused into MgF2 film sealing the micro pores and thus forms a stronger bonding layer with MgF2 film. In addition, the higher interfacial adhesion strength of MgF2 film with the PCL might be due to the formation of hydrogen bonding between -OH group present in fluoride treated surface and = O group of PCL [2 film showed the existence of O along with F elements as evident from EDX  degrades to acids [2 could be possibly due to more rigorous destruction of MgF2 coating than that of duplex MgF2/PCL coating. Hence, Mg substrate with MgF2 coating was more degraded compared to that with MgF2/PCL coating at later stage. The reduction in long-term protective ability of MgF2 coating might be due to the existence of micro-cracks in the coating evident in SEM micrographs shown in 2/PCL coating could effectively prevent the penetration of immersion fluids  protection on the ZK60 Mg alloy which retards the corrosion rate. ZK60 Mg alloy exhibited the highest weight loss rate among other samples. An increasing trend in rate of weight loss was noticed for all the samples with increase in exposure time. The % weight loss of coated MgF2 and MgF2/PCL samples were about 13.6% and 4.5%, respectively compared to ZK60 Mg alloy (21.2%) after 14 days of immersion. The continuous increment in weight loss of coated samples with prolonged exposure time indicated the delamination of the coating layers on Mg substrate. It signified that both MgF2 and MgF2/PCL coatings were not providing adequate protection for longer immersion time. In addition, it was found that the corrosion rate was more accelerated in the initial 3 days of exposure compared to prolonged immersion time (14 days) for ZK60 Mg alloy  2 into soluble MgCl2 (H2) (Eq ). Hence,gCl2 (Eq ). Hence,EM image . Also, t2, and MgF2/PCL coating after immersion in PBS solution for 14 days along with EDX are shown in 2, showed network-like cracks with small precipitates evenly distributed over the entire surface of the alloy. The thicker corroded layer might be ascribed to the presence of micro-cracks in the coating caused by the shrinking of surface corrosion product during drying. However, a few white precipitates were observed on duplex MgF2/PCL coated surface. EDX analysis of corroded surface compositions for different samples showed the presence of O, C, Na, K, Cl, and P elements. The existence of elements suggested the formation of MgCl2, Mg(OH)2 and other Na and K rich compounds in the corrosion product. Therefore, contents of these elements on the surface after immersion can be used to estimate the degree of corrosion occurred in each sample. It was observed that ZK60 Mg alloy had much more corrosion products on its surface than MgF2 or MgF2/PCL coated sample. Duplex coated surface showed significantly lower concentrations of P and O with the absence of K or Na compared to ZK60 Mg alloy. The lower concentration of Mg and higher concentration of C in MgF2/PCL coated sample indicated that PCL coating was still present after 14 days of immersion. However, in case of MgF2, traces of Na and P with relatively higher concentration of Mg depicted the destruction of fluoride coating on Mg substrate (2/PCL coated sample after 14 days of immersion. The cracks in MgF2 coating can provide a suitable path for penetration of PBS solution into the coating (2 is more stable than MgO and Mg(OH)2 and efficient in resisting corrosion attack [2/PCL coating systems, outer PCL layer retard the corrosion attack while the inner MgF2 layer prevent the ZK60 Mg alloy substrate from aggressive medium when it crosses from the outer PCL layer. These results suggested that duplex MgF2/PCL coated sample exhibited higher corrosion resistance compared to MgF2 coating or ZK60 Mg alloy.SEM images of surfaces of ZK60 Mg alloy, MgFubstrate . SEM ima coating . Hence,  coating . All corn attack . Hence, 2 and MgF2/PCL coatings after 4 hr of incubation is shown in 2/PCL coatings exhibited higher cell attachment and more spreading than MgF2 coating.The degree of proliferation and adhesion of host cells on the substrate specifies its biocompatibility . Cell ad2 and MgF2/PCL coatings using indirect cell assay with increasing extract concentrations after 1 day of incubation are shown in 2 and 95% to 87% for MgF2/PCL coatings. MgF2/PCL coatings showed higher cell viability than MgF2 coating at all extract concentrations.Results of cell viability studies of MgF2 and MgF2/PCL coating were examined by confocal imaging  shows gradual reduction of the inflammatory phase after 2 weeks , ZK60 Mg alloy (12.34\u00b11.99 mm3) and duplex MgF2/PCL coating (13.56\u00b10.47 mm3) within 2 weeks  and MgF2 film (~2.2 \u03bcm) onto ZK60 Mg alloy. Duplex MgF2/PCL coatings showed better adhesion strength than PCL coatings. No significant change was found in the microhardness of duplex MgF2/PCL coating when compared to ZK60 Mg alloy. Hydrogen evaluation, pH change, and degradation tests showed that the duplex protective layer of MgF2/PCL exhibited an enhancement in corrosion resistance compared to single layered MgF2 or ZK60 Mg alloy. The barrier properties of uniform PCL film along with compact MgF2 film contributed to lowest corrosion rate. Such duplex MgF2/PCL coating also supported cell viability, cell adhesion and proliferation better than MgF2 coated and uncoated alloy sample. Invivo studies indicated that duplex MgF2/PCL coating reduced the degradation rate of ZK60 Mg alloy and exhibited good biocompatibility for 2 weeks of implantation. These results indicated that outer PCL coating might be a promising approach after fluoride treatment. However, long-term invivo studies are required to further validate the application of duplex coated implant for orthopaedic applications.Duplex MgF"}
+{"text": "Although numerous studies explore the effects of emotion on decision-making, the existing research has mainly focused on the influence of intrapersonal emotions, leaving the influence of one person\u2019s emotions on another\u2019s decisions underestimated. To specify how interpersonal emotions shape decision-making and delineate the underlying neural dynamics involved, the present study examined brain responses to utilitarian feedback combined with angry or happy faces in competitive and cooperative contexts. Behavioral results showed that participants responded slower following losses than wins when competitors express happiness but responded faster following losses than wins when cooperators express anger. Importantly, angry faces in competitive context reversed the differentiation pattern of feedback-related negativity (FRN) between losses and wins and diminished the difference between losses and wins on both P300 and theta power, but only diminished the difference on FRN between losses and wins in cooperative context. However, when partner displays happiness, losses versus wins elicited larger FRN and theta power in competitive context but smaller P300 in both contexts. These results suggest that interpersonal emotions shape decisions during both automatic motivational salience valuation (FRN) and conscious cognitive appraisal (P300) stages of processing, in which different emotional expressions exert interpersonal influence through different routes. How would you react to a competitor or a cooperator who displayed happy expressions when you lost money in a poker game? Would you trust a salesperson who smiles at you, or give money to a grumpy beggar muttering angry phrases? While such decisions are routinely made, little is known about how emotional expressions shape interpersonal decisions. Although contemporary scholars show increasing interest in identifying the effects of emotion on decision making, current efforts mainly focus on how individuals\u2019 judgments and decisions are influenced by their own mood state 134So far, some studies have investigated the interpersonal effect of emotional expressions. For instance, it has been shown that people are more likely to cooperate with smiling partners7111217Despite the fact that an interpersonal effect of emotional expressions has repeatedly been demonstrated, the mechanisms underlying this are not fully understood. One line of study implicates emotion contagion in the effect. Emotion contagion occurs when people automatically mimic each other\u2019s facial expressions and bodily postures. Interoceptive feedback from these reactions then produces corresponding emotions19212345While previous research depicts the interpersonal effect of emotional expression in decision making and external moderators thereof26272829The quick detection of the motivational or valence significance of a feedback is associated with feedback-related negativity (FRN), a negative component peaking at fronto-central electrodes roughly 250\u2013300\u2009ms after presentation of relevant feedback information262632333435363827282930354041424329354327293541424748495048495051475348Although some studies have examined the neural dynamics underlying emotional feedback processing32732For these reasons, the current study aims to specify the neural dynamics of interpersonal effects of emotional expressions in decision making, after taking account of the interaction between utiliarian and emotional cues. We carried out an experiment with a modified version of Gehring and Willoughby\u2019s gambling taskWe focused on the FRN, feedback-related P300, as well as theta synchronization to seek converging evidences, because of their strong link with feedback processing2635595961625963646166596869596061715932747576404142We hope to replicate the interpersonal effect of emotional expressions345305t(36)\u2009=\u2009\u22124.86, P\u2009<\u20090.001] of the shortened Chinese version of the Costa and McCrae NEO-FFIForty right-handed university students  ages 18\u201327 years were recruited to participate in this experiment. The mean age of the sample was 20.5 years old (SD\u2009=\u20092.14). They were randomly assigned to either competitive or cooperative games, with 20 participants  in each. All participants reported normal auditory and normal or corrected-to-normal visual acuity, and were free of neurological or psychiatric problems. They were emotionally stable, as evidenced by the significant below-threshold (0) scoring in neuroticism assessment , (see The participants selected more high-risk options after losses (0.55\u2009\u00b1\u20090.02) than after wins (0.43\u2009\u00b1\u20090.03), [39], see .outcome valence\u2009\u00d7\u2009context  for all these components. Critically, no significant main effect of emotion  and interactions involving emotion  were observed, suggesting that emotional expressions did not differ regardless of utilitarian cues and contexts.The analysis of VPP, N170, EPN and SPW amplitudes showed a significant main effect of This study examined behavioral and brain responses to utilitarian feedback combined with emotional expressions in both competitive and cooperative contexts to explore the neural dynamics underlying how emotional expressions shape interpersonal decisions. In line with previous studies 2628More importantly, angry faces in the competitive context reversed the differentiation pattern of FRN, and diminished the difference between losses and wins on both feedback P300 and theta band power, partly consistent with our prediction. FRN323334353647484950272935404142Even though the new interpretation of FRN can account for the current results, the bigger FRN for wins relative to losses is still a little bit elusive. We speculate the comparison between the ERPs associated with two emotional expressions following wins and losses separately may help to explain this. It has been proposed that the negative activity in the 200\u2013300\u2009ms time window, referred to as the FRN or N2 component, has consistently been observed to reflect valence difference697074757659When a competitor displayed happiness, losses were associated with larger FRN and theta power, but smaller P300. In this regard, happy expression in competitive context seems to have no profound influence on the valence of the utilitarian feedback. This might result from that the happy expressions following losses were taken as negative feedback  suggested that both happy and angry expressions share same level of attentional allocation and emotional arousal in the current experimental context, in line with the previous studies reporting no significant difference in early components between angry and happy expressions5959Despite the contributions of this study, some limitations should be noted. For the sake of brevity, we did not examine the potential effects of different magnitudes of monetary rewards. Though findings have not been completely consistent, some research suggests that magnitude of monetary reward may influence modulation of emotion expression on utilitarian feedback. For instance, magnitude of reward has been reported to affect FRNThe present study examined both behavioral and brain responses to utilitarian feedback combined with emotional expressions in both competitive and cooperative contexts to specify the neural dynamics underlying the interpersonal effects of emotional expressions. As predicted, interpersonal emotional expressions affect one\u2019s response latency, FRN, P300, and theta power change associated with outcome evaluation, and such modulation varies depending on context. These findings indicate that emotional expressions shape interpersonal decisions during both automatic motivational salience valuation and the conscious cognitive appraisal stages of processing, in which happy expressions mainly elicit inferential processing while angry expressions elicit affective reactions in exerting interpersonal influence.How to cite this article: Chen, X. et al. The neural dynamics underlying the interpersonal effects of emotional expression on decision making. Sci. Rep.7, 46651; doi: 10.1038/srep46651 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43\u2009kb was identified in nine families , and another one of 45\u2009kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39\u2009kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of  Understanding the genetics of rare developmental macular conditions is key for unravelling the mechanism of development of this structure that is found only in higher primates within mammals1. NCMD shows fully penetrant inheritance and is considered a non-progressive disorder with a wide range of phenotypic manifestations, usually affecting both eyes symmetrically3. Phenotypic presentation varies from mild cases with drusen-like deposits covering the macular region but with little or no visual impairment, to severe cases with marked central chorioretinal atrophy and poor vision. Although generally non-progressive, complications associated with choroidal neovascularization can contribute to visual deterioration.North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disorder in which there is abnormal development of the macula, a crucial structure of the central retina responsible for central vision and colour perception10. A similar phenotype has been assigned to a second locus at 5p15-p13 11. Interestingly, several studies reported evidence for ancestral haplotypes at the MCDR1 locus13. Early sequencing studies of the two disease intervals failed to identify exonic disease-causing variants14. More recently, three novel single nucleotide variants (SNVs) were identified in 11 families at the MCDR1 locus, within a DNase1 hypersensitivity site (DHS), in the non-coding interval between PRDM13 and the neighbouring overlapping genes CCNC/TSTD315. Two tandem duplications including the full coding region of PRDM13, with some additional upstream and downstream sequence included, were also identified16. One MCDR3-linked family of Danish origin3 was found to carry a 900\u2009kb tandem duplication15 that includes the entire coding sequence of IRX1. However, duplications of IRX1 have been observed in several normal individuals from the Database of Genome Variants17 and the significance of this reported variant is uncertain. Thus, the causative mechanism at the 5p15-p13 NCMD locus remains unclear.The molecular genetics of NCMD has been extensively investigated with the disorder being mapped to chromosome 6q16  in multiple families of different ethnic origins since the early 1990sIn this report we present a combination of genomic investigations in a cohort of 18 NCMD families. The aim of this study was to identify any causative molecular changes and mechanism of disease in these families.14, family 2 was originally reported to be linked to the MCDR3 locus11, and families 12 and 13 were linked to MCDR19, with family 13 recently found to carry the SNV V2 upstream of PRDM1315. All families  showed autosomal dominant inheritance and had at least one individual with Grade 3 disease. DNA samples from a total of 56 affected and 33 unaffected family members were available for genetic analysis.Eighteen families with phenotypes consistent with a diagnosis of NCMD were included in the study Table\u00a0. Four faFigure\u00a018 to search for shared identical-by-descent (IBD) chromosomal segments among affected individuals within each family. This analysis was performed in those families for which Illumina single-nucleotide polymorphism (SNP) array data were available for more than one affected family member (families 1\u20135 and 12\u201313). The 6q16 MCDR1 locus was excluded in four families, including the two previously MCDR3-linked families 114 and 211 and unreported families 3 and 4  at the 6q16 locus, and not at the 5p15-p13 locus  methodPRDM1315. In addition to the previously reported V2 family 1315, two more NCMD families were found to harbour the variant V2 (family 11 and the previously described MCDR1-linked family 127).All families, except families 1\u20134 for which linkage at the 6q16 locus had been excluded via haplotype sharing analysis, were tested with Sanger Sequencing for the three previously reported SNVs (V1-V3) upstream of IRX1 and upstream of ADAMTS16  data (CNV browser)20.To investigate the MCDR3 locus for the presence of structural variants (SVs), an aCGH experiment using 10,000 probes spanning the region at chr5:11882\u201310140073 (GRCh37/hg19) was performed in three affected individuals from families 1\u20133 which did not show linkage at the 6q16 locus 1\u20133 Fig.\u00a0. PrecisePolymerase chain reaction (PCR) primers were designed to amplify the novel sequence across the breakpoint between duplicated copies Table\u00a0, Fig.\u00a03 The remaining 8 unmapped families were tested with the established PCR assay for both duplications, and 3 of them (families 8\u201310) were also found to carry the 43\u2009kb duplication Table\u00a0. Thus, a15. Therefore, haplotype sharing analysis was performed using available Illumina SNP array data from 14 affected individuals in 3 families carrying the 6q16 V2 variant (families 11\u201313) and 14 affected individuals in 6 families carrying the 5p15 43\u2009kb duplication (families 2\u20137). Using a cut-off of 2.0\u2009cM and 2.5\u2009cM respectively, the results confirmed that all the genotyped 6q16 individuals collectively shared a RCHH of approximately 2.5\u2009Mb from GRCh37/hg19 coordinate chr6:98962591 (rs150396) to chr6:101468591 (rs1321204) at the MCDR1 locus, and all the genotyped 5p15 individuals collectively shared a RCHH of approximately 0.9\u2009Mb from GRCh37/hg19 coordinate chr5:4327455 (rs155354) to chr5:5210050 (rs1560063) at the MCDR3 locus 26. Different sites were identified to be open/active within the 39\u2009kb shared region at four out of five time points (~10\u201315 weeks of gestation) available during retinal development 16 was suggested. However, no causal relationship between the non-coding variants and PRDM13 expression has been identified.Eye development, like other organogenesis processes, requires the precise spatio-temporal and quantitative expression of genes, orchestrated by a complex network of regulatory mechanisms influencing critical transcription factors and other developmental genes. The lack of readily accessible animal or PRDM13 at the MCDR1 locus and the most likely candidate gene IRX1 at the MCDR3 locus exists warrants further investigation. iPSC technology and CRISPR manipulation in eye cups from normal and affected individuals may help elucidate the molecular mechanism33 and the potential molecular links between the two genes. Importantly, the involvement of ancestral variation at both the 6q16 and 5p15 loci  and family 139 . When possible, retinal imaging was undertaken using colour fundus photography, fundus autofluorescence and OCT imaging. Blood/saliva samples were collected for DNA extraction, genotyping and sequence analyses. The study protocol was approved by the local ethics committees  and conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants, or their parents, before inclusion in the study.All families were ascertained at Moorfields Eye Hospital, London, United Kingdom, expect for family 1Genomic DNA was extracted from whole blood/saliva and genotyped using the Illumina HumanOmniExpress-24 v1.0 beadchip . Genotypes were determined using the Genotyping Module in the Illumina GenomeStudio v2011.1 software.18 was used for the analysis of those affected individuals that were genotyped with the Illumina array. The HH is a type of haplotype described by the homozygous SNPs only  and, therefore, can be uniquely determined on each chromosome. Since affected family members who inherited the same mutation from a common ancestor share a chromosomal segment IBD around the disease gene, they should not have discordant homozygous calls in the IBD region and thus they should share the same HH. The HH approach predicts IBD regions through the identification of RCHHs defined as those regions with a shared HH among affected individuals and a genetic length longer than a certain cut-off value .In order to search for chromosomal segments sharing the same haplotype across affected individuals (within the same family or across different families), the non-parametric HH method2 ratio of the Cy3/Cy5 intensities of a region encompassing at least four probes was\u2009>\u20090.3 or\u2009<\u2009\u22120.6, respectively (software default settings).aCGH was performed at Oxford Gene Technology (OGT)  using a custom design consisting of 10,000 probes spanning the MCDR3 locus at GRCh37/hg19 chr5:11882\u201310140073 , designed with Agilent e-Array software , in three individuals from families 1\u20133  and WGS data from 650 individuals with inherited retinal disease19.WGS was performed using the Illumina HiSeq X10 platform , generating minimum coverage of 30X. Reads were aligned to the hg19 human reference sequence (build GRCh37) with novoalign (version 3.02.08). The aligned reads were sorted by base pair position and duplicates were marked using novosort. Discordant reads were marked with samblaster (version 0.1.20) and sent to a separate file for manual inspection of breakpoints using the IGV (version 2.3.61). SVs were manually investigated using the IGV by identifying peaks of discordant reads which were interpreted as breakpoints. The identified duplicated regions were also screened for the presence of common copy number variants using data from the CNV browserSegregation analysis of the duplication events identified by WGS was performed using primers Table\u00a0 designed26 was interrogated for fetal retina datasets of interest. Bed files from DNA-seq datasets  were downloaded and investigated at the shared duplicated region with R Studio. A second microarray expression dataset on human fetal retina (19\u201320 gestation week) was queried for the genes of interest24 using the platform GENEVESTIGATOR23.The Encyclopedia of DNA Elements (ENCODE)The datasets generated during the current study are not publicly available due to limitations imposed by the scope of participant consent, but are available from the corresponding authors on reasonable request.Supplementary Information"}
+{"text": "To study use of feature tracking technique in evaluation of regional and global diastolic strain rate and to define the best predictor of diastolic dysfunction by studying its correlation with echo grading of diastolic dysfunction.Echocardiographic examination is currently the gold standard for the non invasive assessment of diastolic dysfunction. Although strain is not load independent, studies have shown that systolic strain rates can assess myocardial contractility and are relatively load independent. It remains to be established if diastolic strain parameters correlate with the degree of diastolic dysfunction and if so which parameter correlates best with echo derived grade of diastology.We performed a retrospective, IRB approved, review of 46 patients who underwent cardiac MRI at our facility for evaluation of cardiac amyloidosis. All patients had an echocardiographic assessment of diastolic function . Conventional short axis, vertical long axis and 4-chamber cine SSFP (Single shot Free Precession) images from the cardiac MRI scans were used to generate 2D  and 3D  diastolic strain rate maps using myocardial feature tracking software. The diastolic strain rates were compared with echocardiographic grades of diastology.Patients had an average age of 61.5 years; males 61%, 17 patients had grade 0, 16 had grade 1 and 13 had grade 2/3 diastolic dysfunction. Base level parameters had the strongest correlations with diastolic dysfunction. The strongest correlations were found for base radial 2D  and longitudinal 3D diastolic  diastolic strain rates. Across all levels on average, radial 2D diastolic strain rate had the strongest correlation with diastolic dysfunction (0.46) followed by circumferential 3D (-0.36) and longitudinal 3D (-0.36) diastolic strain rates.Also, the base diastolic parameters separated best according to echo grade of diastolic dysfunction: The base longitudinal 3D differed the most between grade 0 vs. all grades of diastolic dysfunction . The radial 2D diastolic strain rate differed the most between grades 1 vs. Grades 2/3 .MRI feature tracking is a useful tool for detection of diastolic dysfunction. Our study demonstrated that global radial 2D strain rates and global longitudinal and circumferential 3D diastolic strain rates correlate best with the degree of diastolic dysfunction. For segmental analysis, measuring basal 2D and longitudinal 3D diastolic strain rates will provide the best measure of diastology."}
+{"text": "EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida.  Genotyping of Epha2tm1Jrui/tm1Jrui and Epha4rb-2J/rb-2J mice was carried out according to the protocol provided by The Jackson Laboratory (stock # 006028 and stock # 003129 respectively) . Pregnant females were euthanized by cervical dislocation and an incision was made at the abdominal area. The uterine horns were incised and immediately transferred into cold Dulbecco's Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). The embryos were dissected out of the decidua and washed briefly with phosphate buffered saline (PBS) (Sigma) before overnight fixation in 4% paraformaldehyde (PFA) (Sigma). Subsequently, the embryos were washed and agitated in PBS for 10 min at 4\u00b0C. All steps from this point were agitated to ensure thorough washing. Then the embryos were dehydrated by ascending ethanol washes a concentration of 30, 50, and 70% for 20 min on each wash at 4\u00b0C. The embryos were kept in 70% ethanol at 4\u00b0C for downstream experiments. The embryos were analyzed in detail and documented under a high-resolution stereomicroscope (Leica MZ16).in situ hybridization, was performed using digoxygenin-labeled cRNA probes  incapable of having a close neural tube that adheres and fuses in the region whereby these genes are expressed  of the E11.5 mouse embryo in Figure Epha2tm1Jrui/+Epha4rb-2J/+) reveals bilateral elevated neural folds, which remain unfused in the dorsal midline at the point of closure of the neural tube highlighted by boxed region in the figure  embryo  that successfully completes closure 1  in more than 50% of the population of double heterozygotes (Epha2tm1Jrui/+Epha4rb-2J/+). The embryos have lipomyelomeningocele : 79% (Epha2tm1Jrui/+Epha4rb-2J/+): 2% (Epha2tm1Jrui/+Epha4rb-2J/rb-2J): 5% (Epha2tm1Jrui/tm1JruiEpha4rb-2J/+): unknown (Epha2tm1Jrui/tm1JruiEpha4rb-2J/rb-2J) (Table Epha2tm1Jrui/tm1JruiEpha4rb-2J/rb-2J) embryos were resorbed by E8.5. The parental double heterozygotes were considered robust without any gross abnormalities. Double heterozygous pups with abnormalities could not survive because the pups die within 2 h after birth  Table  apart frJ) Table . In cont Figures  due to mEphA2 and EphA4 gene expression was studied in the early E11.5 double heterozygotes  mutant embryos to observe for difference in expression pattern. Figure EphA2  among our crosses. However, we note that the numbers of animals given birth to in any of the crosses are much smaller in number than if the embryos were harvested during embryogenesis. There were between 10 and 12 embryos in each litter but if allowed to birth, the numbers dwindled to between 5 and 6 pups per litter. This gives rise to the possibility that if the embryos are unable to survive being a double heterozygote mutant, it gets resorbed; hence failure to complete embryogenesis successfully. Again, this system is similar to what has been observed in the EphB2; EphB3 double knockout (EphB2\u2212/\u2212EphB3\u2212/\u2212) that suffer embryonic lethality  have close spina bifida, which would translate clinically as spina bifida occulta. The implication of this finding is tremendous; that this is the first spina bifida occulta mouse model arising from failure of primary neurulation. Therefore, our mouse model can potentially explain the embryogenesis of lipomyelomeningocele as well as it is at odds with the current dogma of occulta-type spina bifida arising from failure of secondary neurulation. A previous study had reported Trpm6h (Walder et al., Trpm6h to understand the structure of the neural tube regulated by the Trpm6h protein and whether this occurs during primary neurulation (Walder et al., Our double heterozygotes (EphA2 and EphA4 the phenotype representation would be more severe. A further 7% have a more severe neural tube defect when either both the EphA2 allele or both EphA4 allele is completely deleted simultaneously with a single allele of either EphA2 or EphA4 i.e., Epha2tm1Jrui/+Epha4rb-2J/rb-2J or Epha2tm1Jrui/tm1JruiEpha4rb-2J/+ (Figure EphA2 and EphA4 during neurulation (Figure EphA2 and EphA4 are not expressed at the closure 1 site during neurulation but are expressed in the rhombomeres and the posterior neuropore. This phenomenon also further illustrates the specificity of the perturbation of neural tube development in this model and supports the haploinsufficiency theory. Gene dosage determines severity of the phenotype. The defect is selective enough not to phenocopy craniorachischisis, yet its caudal and anterior neuropores remain remarkably open in the areas where EphA2 and EphA4 would be expressed in the wildtype. It is striking that Closure 1 is achieved (Shum and Copp, EphA2 and EphA4 such as EphA1 and EphA5 which have a far broader expression domain than EphA2 and EphA4 (Abdul-Aziz et al., EphA2 and EphA4 in neural tube closure.These mutant embryos showed gene dosage pattern whereby with every loss of an allele of + Figure . Closure+ Figure . This isn Figure . EphA2 aEpha2tm1Jrui/+Epha4rb-2J/+ shows a defective phenotype encompassing spina bifida occulta, close exencephaly, gastrochisis, caudal dysgenesis and cyclopia. It is interesting to note that in the double heterozygotes, the expression of the \u201cdot\u201d which is visible at the point of adhesion and fusion is absent (Figures Epha2tm1Jrui/+Epha4rb-2J/+ are most likely attributed to the Epha4rb-2J/rb-2J mutant isoform that generates a protein size of 104 kDa (Mohd-Zin et al., Epha2tm1Jrui/tm1Jrui is a complete targeted knockout of the EphA2 protein (Ruiz and Robertson, Epha4rb-2J/rb-2J is not (Mohd-Zin et al., EphA4 gene were reviewed in Mohd-Zin et al. (Epha2tm1Jrui/+Epha4rb-2J/+. The background strain is a mix of C57BL/6J (Cook et al., More than 65% of  Figures  in refer Figures . The muln et al. . The fusn et al. . TherefoEphA2 in the notochord could explain a potential role between Sonic hedgehog and the EphA receptor family seeing that the double heterozygotes in our study display cyclopia (Cooper et al., EphA2 is expressed in the region surrounding the gut, which could explain the potential role of the EphAs in gastrochisis.Skin covering of exencephaly; poses the question whether malformations of the brain such as callosal dysgenesis with interhemispheric cyst is implicated in neural tube closure as they occur during the period of neural tube closure or early during post-closure of the central nervous system (Barkovich et al., Our report provides the genotypic and phenotypic embryonic evidence of the occulta-type NTDs arising from failure of primary neurulation.NA, AA-A, and NA-A: Conceived and designed the experiments; NA, SM-Z, and NA-A: Performed the experiments; All authors: Analyzed the data; AA-A and NA-A: Contributed reagents, materials, analysis tools; NA and NA-A: Wrote the manuscript. All authors read and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as  The process of progenitors undergoing cell fate decisions to determine specific cellular identity and tissue patterning is fundamental in the field of developmental biology. Neural development is an excellent model system to study cell fate processes to understand how neural restricted progenitors differentiate into diverse types of neurons through complex interactions of transcriptional activators and repressors. Multiple regulatory design principles involving combinations of transcription factors have emerged through studies from vertebrate and invertebrate model systems that have significant repercussions in determining and maintaining cell identity . During The dorsal spinal cord functions as an essential center for integration of somatosensory information . ApproprPRDM13 is a transcriptional repressor present in progenitor domains of the dorsal but not the ventral neural tube from hindbrain regions to the spinal cord  Figure . It is aPrdm13 mutant mouse strains. In contrast to the previously reported Prdm13 mutant deleting exons 2 and 3  of PRDM13 \u0394ZF)  . This ZFdm13\u0394ZF) . The phend PTF1A . PRDM13-nd PTF1A  do not dnd PTF1A . A thirdm13\u2206115) . Surpris protein . Westerndm13\u2206115 . This muPrdm13 was identified as an important specification gene in the dorsal neural tube required for the balance of inhibitory  interneurons and excitatory  interneurons in the chick neural tube  and an increase in dI3/5 (TLX1/3+) in both the GFP/GFPPrdm13 and \u0394ZF/\u0394ZFPrdm13 neural tubes was seen at this stage. These phenotypes are identical to the Ptf1a null mouse  (for review see ). Prdm13ral tube . Overexping PAX2 . Supportulations . A complll mouse   in the Prdm13 mutants that is not seen with the loss of Ptf1a , a marker of excitatory neurons, is detected in the lateral region of the domain lacking Gad1  in dorsal neural tube cells in the presence and absence of PRDM13. RNA-seq was performed on GFPal tubes . 837 gengulation .Tlx3 , and used only those sites identified in at least two experiments. We identified 220 PRDM13 binding sites largely located at distal sites relative to the transcription start sites of genes. These targets included a known target of PRDM13, Tlx3 . The gen of Tlx3 . This reenhancer .De novo motif analysis failed to identify a novel binding motif unique to PRDM13, but PRDM13 bound sites were enriched for motifs for known neural TFs such as E-box (bHLH) (40%), Rfx (17%), Pdx (HD) (25%) and Sox (30%) motifs . Indeed,GFPPrdm13 neural tubes  whereas only six are decreased   .Prdm13 mutants. For example, PRDM13 binds the Tlx3 enhancer and restricts its expression at E10.5/E11.5 (eTlx3). Consistently, Tlx3 is elevated 17-fold, while Pax2 is reduced 6.3-fold in the absence of Prdm13  ( control . To dete control . Becausecription  these dacription  where th (Ptf1a) .Ptf1a transcription is regulated through a 2.3 kb autoregulatory enhancer (e2.3Ptf1a)    . PTF1A b.3Ptf1a) . One posr region .Ptf1a autoregulatory enhancer using the chick electroporation assay and a GFP reporter under the control of the 2.3 kb Ptf1a autoregulatory enhancer (e2.3kb-Ptf1a::GFP) , and HEB (Tcf12) (Ptf1a transcription by interrupting PTF1A auto-regulation through binding with PTF1A to the Ptf1a autoregulatory enhancer (We next tested the ability of PRDM13 to regulate transcription through the 1a::GFP) . As was activity . In contmpletely . Finallympletely . Becausempletely   . We propenhancer .Prdm13 and Ptf1a loss of function phenotypes in the second wave of neurogenesis in the dorsal neural tube ). The latter finding is striking because these ventral specification factors are restricted to the ventral neural tube. Three factors with the highest fold increase in the Prdm13 mutant are Olig1, Olig2 and Prdm12 , as well as a number of neural related factors misregulated upon loss of PRDM13 . Notablyd Prdm12 . We usedOlig1 and Olig2 show striking ectopic dorsal expression within GFP/GFPPrdm13 neural tubes  in the ventral spinal cord at these early stages of neurogenesis, and it is required for specification of motor neurons  Figure . Upon loal tubes . This ab mutants . Co-labe mutants . Notably mutants , factorsOlig1 and Olig2, we identified three genomic regions surrounding the Olig genes bound by PRDM13 from ChIP-seq  and is required for generation of V1 interneurons  survived to adulthood and exhibited a retinal phenotype  for function  activity dependent on the PR domain of the protein . Those PDecades of work have gone into defining progenitor domains and resulting neuronal populations in the developing spinal cord mainly by combinatorial TF expression of HD and bHLH factors . RepressPrdm13 mutants they are aberrantly present dorsally, altering the identity of these neurons. Taken together, PRDM13 is a transcriptional repressor essential in the dorsal spinal cord development to modulate the transcriptional activity of neural bHLH factors to generate precise neuronal identities.Here we provide evidence for an additional variation of these models whereby the transcriptional repressor is broadly expressed and it limits inappropriate gene expression driven by progenitor-specific activators, the bHLH factors. PRDM13 is identified as a key transcriptional repressor of a whole battery of genes, not just those in domains adjacent to the domain giving rise to the dorsal inhibitory neurons, but also factors normally restricted to the ventral neural tube and required for neuronal identity in that region  Figure . PRDM13 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Jane E. Johnson (jane.johnson@utsouthwestern.edu).GFPPrdm13 mutant and the fusionGFPPrdm13 mouse model were developed using zinc-finger nuclease technology (Prdm13 gene (CACCAGCGTGAACGCTGActgctGCATCCCGGCTGGCT) were purchased from Sigma-Aldrich  and delivered by pronuclear injection into fertilized mouse eggs along with a donor plasmid. For GFPPrdm13 mutant mice, the donor plasmid encoded GFP followed by a stop codon, and contained two homology regions each 750 bp in length to allow for homologous recombination of the insert into the Prdm13 locus (fusionGFPPrdm13 mouse that was used for ChIP-seq. Homozygous fusionGFPPrdm13 mice live to adulthood and show no changes in PAX2 or TLX1/3 expression at E10.5 (data not shown). Mice were genotyped by PCR with 5\u2019-GCTGCTCCTGGTTCTGTCA-3\u2019, 5\u2019-CCTTTTCTCTGCTGCTCGTC-3\u2019 and 5\u2019-GCTGGAGTACAACTACAACAGCCA-3\u2019 .The chnology . mRNA en13 locus . Out of \u0394115Prdm13 mutant line is a 115 bp deletion generated within the first exon of Prdm13 upon injection of the ZFN mRNA . Homozygous \u0394115/ \u0394115Prdm13 mice live to adulthood, and show no changes in PAX2 or TLX1/3 expression at E10.5. PCR genotyping used primers 5\u2019-GCTGCTCCTGGTTCTGTCA-3\u2019 and 5\u2019-CCTTTTCTCTGCTGCTCGTC-3\u2019  (The  198 bp) .\u0394ZFPrdm13 mutant mouse line was generated by pronuclear injection of CAS9 mRNA and two sgRNA targeting the 3\u2019 end of Prdm13 . The CREPtf1a mouse line replaces the coding sequence for Ptf1a with that for Cre recombinase  and cryosectioned at 20 \u00b5m (E10.5-E11.5) and 30 \u00b5m (E12.5-E16.5) focusing on sections from the upper limb level.20/1X loading dye and injected into the lumen of the closed neural tube at stages HH13-15 along with either a pMiWIII-Myc epitope tagged vector serving as an electroporation control or a TF containing expression vector. The injected embryos were then electroporated with 5 pulses of 25 mV each for 50 msec with intervals of 100 msec. Embryos were harvested 48 hr later at stages HH22-23, fixed with 4% paraformaldehyde for 45 min, and processed for cryosectioning and immunofluorescence.For in ovo chick electroporation assays, fertilized White Leghorn eggs were obtained from the Texas A and M Poultry Department  and incubated for 48 hr at 37\u2070C. The supercoiled reporter plasmids were diluted to 1.5 mg/ml in HImmunofluorescence was performed using the following antibodies: guinea pig anti-PRDM13 , mouse Prdm13 and Ptf1a (Prdm12 (Gad1 and Vglut2 (Slc17a6) . The chick Prdm12 probe was generated starting from a pBSK+\u00a0cPrdm12 cDNA (EST BU233582). Probes for Ccbe1 and Mfng were generated by PCR from mouse neural tube cDNA. Imaging was performed with a Hamamatsu Nanozoomer 2.0HT digital slide scanner.In situ hybridization was performed as previously described . Probes nd Ptf1a , mouse P  , mouse Od Phox2b , mouse Ob , e2Prdm12 , e1Olig , e2Olig , e3Olig . E1Olig-EboxMut, e2Olig-PTF1Mut and e2Olig-allEboxMut were subcloned using gBlocks with E-box mutations . KpnI/SpeI restriction sites were introduced on each end for cloning adjacent to the \u03b2-globin minimal promoter in the reporter cassette. The Ptf1a 2.3 kb enhancer reporters are as described   with pMiWIII  expressi in pCIG , and toto acids) . The HEKGFPPrdm13 embryos either heterozygous or homozygous for GFP were individually placed into cold DMEM/F12 and dissociated in 0.25% trypsin for 15 min at 37 degrees C. Trypsin activity was quenched with 2% fetal bovine serum, and GFP positive cells were purified from the resulting single cell suspension by fluorescence activated cell sorting (FACS). Total RNA from FACS isolated cells was extracted and purified with Mini RNA Isolation Kit . Genotypes were determined and RNA from 9 homozygous and 7 heterozygous embryos were combined, mRNA was purified, reverse transcribed and amplified for sequencing with Illumina\u2019s mRNA-Seq kit  . Gene exfusionGFPPrdm13 embryos were used for GFP ChIP. ChIP was performed as previously described (PTF1A and ASCL1 ChIP-Seq were previously published  (GSE5584escribed . Notablyescribed . LibrariSequence reads for each sample were mapped to the mm10 genome assembly by using Bowtie2 (v.2.2.6) . Mapped enh::GFP expression was measured by ImageJ on the electroporated side of the neural tube with the background levels measured from the non-electroporated side subtracted. Significant differences between control and experimental samples in each case were calculated using a two-tailed two-sample unequal variance (homoscedastic) Student\u2019s t-test in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ns\u00a0=\u00a0not significant. Error bars indicate SEM. The enrichment of PRDM13 ChIP targets in the list of genes that showed significant expression change in mutant samples relative to controls was calculated using the hypergeometric test.In sections from mouse embryos with cells labeled by immunofluorescence  and 3, qRNA-Seq and ChIP-Seq data generated here are available on the GEO database (GSE90938). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Diogo S Castro (Reviewer #1); John LR Rubenstein (Reviewer #2).Thank you for submitting your article \"Repression by PRDM13 is critical for generating precision in neuronal identity\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submissionThe described study utilizes newly generated null mutations in Prdm13 to investigate the function of this transcriptional regulator in the developing dorsal spinal cord. Resulting data supports previous findings that Prdm13 suppresses the fate of excitatory neurons in inhibitory lineages. By a combination of developmental and genomic assays, the authors demonstrate that the recruitment of PRDM13 to bHLH bound enhancers inhibits the expression of ventral transcription factors in the dorsal neural tube. While consistent with current models of neural tube patterning this study provides significant mechanistic insight into the Prdm13-mediated gene regulatory network that regulates neurogenesis in the dorsal spinal cord.While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.2) In 3) Although the authors conclude that \"these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators\" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading. While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.We thank the reviewers for their time and suggestions for improving this study. As you will see below, in performing additional analysis in response to the reviewer\u2019s comments we have significantly strengthened our findings and thus, support for our conclusions.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.Prdm13 mutant neural tubes. Instead we offer a different set of experiments that we believe serves a similar purpose and provides a high confident set of PRDM13 target genes. We now identify PRDM13 target genes based on 3 PRDM13 ChIP-seq data sets that were generated with 3 independent strategies. These include: 1) ChIP-seq data from the original submission that is from E11.5 neural tube tissue using a lab produced antibody generated against the terminal ZnF domain of mouse PRDM13. 2) ChIP-seq data from E11.5 neural tube tissue using an independently produced antibody generated against full-length mouse PRDM13. Here, telencephalon, a PRDM13 negative tissue, was used to control for non-specific binding of the antibody. 3) We generated a mutant mouse strain with a GFP fused in-frame N-terminal to the PR domain of PRDM13. Mice homozygous for this GFP-PRDM13 fusion are indistinguishable from wildtype. ChIP-seq data from E11.5 neural tube tissue from these animals using an antibody directed against GFP was generated.We agree it is important to strengthen the data identifying PRDM13 bound sites in the genome and provide some validation for these sites. While we would have liked to provide data from the suggested experiment, we have been unable to obtain efficient enough ChIP enrichment from the limited source material provided by Ptf1a, Olig2, and Prdm12 were all confirmed as PRDM13 targets providing the validation requested by the Reviewers. We show the genome browser tracks for all three ChIP-seq experiments in supplementary data associated with This exercise had a substantial positive impact on the support for the conclusions reported. We restricted our analysis to PRDM13 bound sites that were called as peaks in at least 2 of the 3 ChIP-seq datasets. This limited the number of PRDM13 target genes identified. The genes highlighted in the study including Prdm13 mutants relative to controls were identified as PRDM13 targets , whereas only 6 of 356 genes that decreased in the Prdm13 mutants were identified as targets . These findings support the role of PRDM13 as a repressor  In Olig1/2. We did this for e1Olig and e2Olig rather than e3Olig for reasons described below, and because e3Olig was not responsive to activation by PTF1A or ASCL1. These experiments demonstrate that the E-boxes are required for e1Olig and e2Olig enhancer activity. In the process of performing these experiments we corrected an error in the data reported in the original manuscript. The e1Olig sequence originally tested was not the correct sequence (consistent with its lack of activity in the reporter assay). In the current revised manuscript, we corrected this error. None of the 3 Olig enhancers direct reporter expression restricted in the endogenous Olig domains. Nevertheless, e1Olig and e2Olig require the Ebox motifs within the enhancers for their activity  Although the authors conclude that \"these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators\" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading.Tlx3 previously in Chang et al., 2013 and this is described and referenced where relevant. In the current study, we provide additional support for those conclusions showing the increase in Tlx3 in the Prdm13 mutant mice. In the revised manuscript, we now point out that although this current study does not show an example of a validated ASCL1/PRDM13 target, this was shown in Chang et al., 2013 for regulation of TLX3 at the site highlighted in We disagree that this statement in the abstract is misleading. In this manuscript, we explicitly provide evidence for PRDM13 repressing multiple bHLH activators including PTF1A, NEUROG1, and NEUROG2. We don\u2019t explicitly show a validated ASCL1/PRDM13 target here, but we have demonstrated this for"}
+{"text": "Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2\u2212 breast tumor cell samples tested, 7 of 34 patients  had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity  . One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2 Breast cancer classification is largely based upon a patient's expression levels of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2).ERBB2) amplification and/or HER2 protein overexpression is detected in approximately 15%\u201320% of breast cancers and associated with more aggressive disease progression, metastasis, and a poorer prognosis  was used, where the values associated with the EGF and NRG1b stimulus and inhibition were combined to arrive at a total amount of HER2 signaling for a particular cell sample.\u2212 sample as having abnormal signaling, a test cut-off value of 250 was established that was 90% of the upper range of the healthy responses and coincidental with the median test value for the sample set of HER2+ cell lines previously tested [\u2212 patients have abnormal pathway signaling, assuming that 15% of the population would be abnormal, a sample size of 33 was required.To classify a HER2y tested . To conf+ cell lines generate different HER2 signaling levels using the CELx HSP test, it was first assumed that normal breast cells have a median CELx HSP test value of 100 with a standard deviation of 100. To detect a 150 absolute difference  in test value between the two types of cells, with a two-sided \u03b1 at 0.05 and 90% power, the required number of patients in the normal population and HER2+ population arms is 10 each. Since only nine HER2+ positive cell lines were available, the sample size of the normal specimens was increased to 16 to offset the effect of the smaller HER2+ sample size.To confirm cells from normal patients and those from the HER2P < 0.05 was considered statistically significant.Pearson correlation analysis was performed with GraphPad Prism 6 to evaluate the relationships among the variables of interest. All dose-response curves were obtained using nonlinear regression curve fitting with GraphPad Prism 6. Box-and-whisker plots were constructed to analyze the different groups' CELx HSP test results."}
+{"text": "Members of the calcium-activated chloride channel (CLCA) gene family have been suggested to possess a variety of functions including cell adhesion and tumor suppression. Expression of CLCA family members has mostly been analyzed in non-neural tissues. Here we describe the expression of mouse and human CLCA genes in the nervous system.We show that from the six mouse CLCA family members only Clca1, Clca2 and Clca4 mRNAs are expressed in the adult brain, predominantly in olfactory ensheathing cells. During mouse nervous system development Clca1/2 is more widely expressed, particularly in cranial nerves, the diencephalon and in the cerebral cortex. While human CLCA2 and CLCA4 genes are widely expressed in brain, and at particularly high levels in the optic nerve, human CLCA3, the closest homologue of mouse Clca1, Clca2 and Clca4, is not expressed in the brain. Furthermore, we characterize the expression pattern of mouse Clca1/2 genes during embryonic development by in situ hybridization.The data published in this article indicate that within the nervous system mouse Clca1/2 genes are highly expressed in the cells ensheathing cranial nerves. Human CLCA2 and CLCA4 mRNAs are expressed at high level in optic nerve. High level expression of CLCA family members in mouse and human glial cells ensheathing nerves suggests a specific role for CLCA proteins in the development and homeostasis of these cells. Calcium activated chloride currents have been characterized in a number of cell types including smooth muscle, skeletal muscle and epithelium. Physiologically, it has been shown that activation of calcium-activated chloride current plays a prominent role in among others the maintenance of smooth muscle tone, epithelial secretion and vertebrate olfactory transduction. The precise molecular identities of the currents are still hotly debated. Proteins belonging to CLC, CLCA, bestrophin and tweety gene families have been proposed to function as calcium activated chloride channels [reviewed in ].The CLCA gene family includes 4 genes in humans, 5 genes in rat and 6 genes in mouse. The nomenclature of the CLCA genes in different organisms is somewhat confusing since the numbering of different genes does not reflect the actual homologies between the genes in different organisms but rather the time of characterization [reviewed in ].Although a lot of evidence shows the involvement of CLCA proteins in mediating chloride conductance, it is still unclear whether CLCA proteins are channels themselves. It has been shown that there are differences in endogenous chloride current characteristics in normal versus CLCA over-expressed cells . Also, aIn addition to their functions as chloride channels or channel modulators, some CLCA family members function as cell adhesion molecules ,8 and tuExpression analysis by RT-PCR of mouse Clca family members, has revealed that mClca1 is expressed at high levels in spleen and bone marrow and mClca2 in mammary gland. Moderate or low expression levels of both genes were found in most tissues with only mClca1 expressed in brain tissue . ExpressTo date, expression analysis has shown that only mClca1 is expressed in the mouse brain. However, in case of mClca3 and 4, brain tissue was not included in the expression analysis. In this study we describe the spatio-temporal expression of mClca1, 2 and 4 genes in the nervous system. We show that these genes are expressed in the olfactory ensheathing cells. In addition we also describe the expression pattern of human CLCA2 and 4 genes in the nervous system. Finally, we describe the expression pattern of mClca1/2 during embryonic development by in situ hybridization.Total RNA from NMRI mouse tissues and total RNA from postmortem adult human brain regions was extracted using RNAWiz (Ambion) according to the manufacturers instructions. Total RNA from human non-neural tissues was obtained from Clontech. First-strand cDNAs were synthesized with Superscript III (Invitrogen) reverse transcriptase using 5 \u03bcg RNA as recommended by the manufacturer.PCR reactions were performed in a volume of 25 \u03bcl, using 1/50 of the first-strand cDNA reaction. Annealing temperature for different sets of primers ranged from 55\u201360\u00b0C. The number of cycles used varied from 25\u201335 for different primer sets. Number of cycles for different primer sets was determined empirically and we always analysed the PCR product in the exponential phase of amplification. PCR with primers specific for housekeeping gene HPRT and GAPDH were used as a control to determine the variation of the amount of cDNA in different PCR reactions.Real-time quantitative (Q) RT-PCR analysis of CLCA mRNA levels in adult mouse and human brain regions and during mouse brain development was performed in triplicates using qPCR Core Kit for SYBR Green I (Eurogentec) with Lightcycler 2.0 (Roche) according to manufacturers instructions. Data was normalized with housekeeping gene HPRT and analyzed with Lightcycler 4.05 software (Roche). Data was not normalized with HPRT in case of mClca1, mClca2 and mClca4 PCRs using equal amounts of cDNAs from different mouse brain developmental stages, since the level of HPRT mRNA is increased during development .Primers used in the experiments are the following:hclca1 sense ACGAACAAGGACACCAGCAAAhclca1 antisense AAGAGATCAGGTATGGGAGCAThclca2 sense TGCATGTCAATCACTCTCCCAhclca2 antisense GAGTTCCTATCCATTGCTCGThclca3 sense GAAGGAGCTCAAACAGACGAChclca3 antisense ACTTTCTACTGAACCAGGCTChclca4 sense GCCACAGTTCATGAGGATAAGhclca4 antisense CACAGACAATACCAGCGTAGmclca1sense CACCAGGATCACTGGCACCAATmclca1 antisense GCATCGATAAGGCTGTTTAGGTCmclca2 sense CGCCAGCATCACAGGCAAGAAGmclca2 antisense GCGTCGATAAGGCTGCTTACATGmclca4 sense TTCAGCAGGACAGCATCTGGmclca4 antisense TGCCACTTGTGCGATGTTGgapdh sense TTCCTACCCCCAATGTGTCCGTCgapdh antisense ACCCTGTTGCTGTAGCCGTATTCAhprt sense GATGATGAACCAGGTTATGAChprt antisense GTCCTTTTCACCAGCAAGCTTGhclca2real sense AGCACCTGGAGAAGACTTTGAhclca2real asense CTTGCTGAGGATTTCGCTTTGAhclca4real sense AGACCTTGATGCCACAGTTCAThclca4real asense TGGTGACAGATCAGTAGTATTTAmclca1 realS CACTGATAACTTGCGTATCTACmclca1 realAS CACAGTTGTGAACCACATTGGmclca2 realS TCACTGATAACTTGCGTATCTATmclca2 realAS ACACTCGTGGACCACCTTCTmclca4 realS AATGACAGCTCCTACCTAGCmclca4 realAS GGCTCCACTGTGTTTGACCT35S]UTP  for labeling. The hybridization specificity was confirmed using [\u03b1-35S]-labeled sense riboprobes synthesized from the same templates. All sense probes resulted in the hybridization signal equivalent to the background. This shows that cRNA labeling of different CLCAs was specific. Primers used to generate probes were the following:DNA fragments for riboprobe generation were subcloned into pCRII-TOPO vector (Invitrogen), sense and antisense cRNA probes were synthesized with the MAXIScript In Vitro Transcription Kit (Ambion) T7 or SP6 RNA polymerase, using [\u03b1-CLCA12 sense ATAGTATCTCTGCACTGGTGCLCA12 antisense GAATGGATATCTAATTTCCATAGCLCA4 sense CCTCCTGGTCTGGGTACCAACLCA4 antisense ATAGACGCAAATAGGAAATTTACSerial saggital and coronal sections (14 \u03bcm) from fresh-frozen NMRI mouse brain were analyzed by in situ hybridization analysis following the previously described protocol . EmulsioPrevious analyses have shown that out of six mouse Clca genes only mClca 1 is expressed in the brain and is expressed at relatively low levels compared to other tissues where the gene is expressed [Quantitative real-time PCR analysis of mClca1, 2 and 4 expression during mouse brain development showed that expression of mClca1 was increasing during postnatal brain development and reached maximum levels in the adult mouse brain. mClca2 expression did not change during mouse brain development and mClca4 expression was low during embryonic development, highest around birth of the animal and the level of respective mRNA was gradually decreasing during postnatal development Fig .In order to analyze the spatial distribution of mClca1, 2 and 4 mRNA expression in the adult brain we performed real-time PCR anaysis using cDNAs from various regions of mouse brain. Strikingly, all the mClca genes expressed in the nervous system, were highly enriched in olfactory bulb Fig . ExpressTo further analyze the cellular distribution of mClca1,2 and 4 expression in brain, we performed in-situ hybridization on adult brain sections. Since mClca1 and 2 genes share 95% identity, we were unable to design probes that distinguish between these genes. Therefore, we consider the expression pattern of mClca1 and 2 together (marked mClca1/2). It should be noted however, that mClca2 was expressed at very low levels in the adult mouse brain Fig  and therWe performed in situ hybridization analysis in order to characterize mClca1/2 expression during mouse development. Our analysis showed that at embryonic day 13 (E13) mClca1/2 is expressed at high levels in the developing urethra, midgut, aorta and heart Fig . High maIn situ hybridization analysis on E17 mouse embryos showed that high levels of mClca1/2 mRNA expression were retained in the developing urethra Fig . ExpressSince three mouse Clca gene family members were expressed in brain, we were interested if any of the four human CLCA genes are expressed in the nervous system. As the numbering of CLCA family members in human and rodents is different, we performed bioinformatic analysis to reveal which rodent Clca genes have closest homology to which human family members. Schematic depiction of human, mouse and rat CLCA loci is shown in Fig Our bioinformatic analysis showed that human CLCA3 is the closest homologue of mouse Clca1, 2 and 4 genes Fig . It sharhCLCA2 and 4 were differentially expressed in the adult brain as revealed by quantitative RT-PCR analysis Fig . The higIn this study we show novel expression sites for mouse Clca1, Clca2 and Clca4 genes. All six mouse Clca genes are located in chromosome 3 and are clustered in the same locus. Our RT-PCR and in situ hybridization analyses reveal that in the murine nervous system, mClca1, 2 and 4 genes are preferentially expressed in the olfactory ensheathing cells. In contrast to our findings, it has been previously shown that mClca2 is not expressed in the mouse brain . Our anamClca 1, 2 and 4 genes share highest similarity with each other within the gene family. At amino acid level mClca1 and 2 share 95% identity and mClca4 shares 81% identity with mClca2 and 80% identity with mClca1. Moreover, these three genes lie next to each other and form a 3' gene cluster in the Clca gene locus. Given their similar expression pattern in the nervous system, it could be argued that either their olfactory ensheathing cell specific expression is driven by a common regulatory element or each of these mouse genes has retained an olfactory ensheathing cell specific promoter element following gene duplication. Other reports have shown that the rat Clca genes most homologous to mouse Clca1,2 and 4, i.e. rbClca and rbClca2 are expressed in rat brain ,21. TheiOur analysis of mClca1/2 expression in the nervous system at early postnatal development revealed that it is expressed also in the layer II-III of the cerebral cortex. Interestingly the expression was seen only in the frontal part of the developing cortex. At E17 mClca1/2 expression was more widespread in the brain, with prominent expression in diencephalon. During embryonic development mClca1/2 expression was seen in the developing nerves of the peripheral nervous system. We could detect expression at E13 in the developing olfactory nerve and spinal nerves and at E17 in the optic nerve, trigeminal nerve and olfactory nerve. It is possible that mClca1/2 expression marks the glial cells ensheathing peripheral nerves.In this study we have also analyzed the expression of human CLCA genes in the nervous system. Human CLCA locus contains 4 genes. The most 3' of the genes is hCLCA3, which is also most closely related to mClca1, 2 and 4. RT-PCR analysis showed that unlike its mouse homologues, hCLCA3 is not expressed in the nervous system. In contrast, our results show that two other members of the family, hCLCA 2 and 4 are expressed in various parts of human brain. It has previously been shown, using RNA dot-blot analysis, that hCLCA4 is expressed rather uniformly in the brain with striking absence in the cerebellum . HoweverIn this study we have shown that mClca1, mClca2 and mClca4 are expressed in the olfactory ensheathing cells of the adult mouse CNS. During mouse development mClca1/2 widely expressed in the CNS but at particularly high levels in cranial nerves. In addition, we found that mClca1/2 is expressed in layer II of the developing cerebral cortex at P9. Our analysis also reveals that hCLCA2 and hCLCA4 are expressed in the CNS of adult humans.TT and DM contributed to the design of the experiments and to the preparation of the manuscript. MP performed the experiments and contributed to the design of the experiments and to the preparation of the manuscript."}
+{"text": "It is now recognised that epithelial-stromal interactions are important in a wide range of disease processes including neoplasia and inflammation. Metalloproteinases are central to matrix degradation and remodelling, which are key events in tumour invasion and metastasis and may also be involved in tissue changes occurring in chronic inflammation. Immunohistochemistry was performed on sections from 50 patients with pancreatic cancer (n = 27), ampullary cancer (n = 12), low bile duct cancer (n = 3), neuroendocrine tumours (n = 3) and chronic pancreatitis (n = 5), using antibodies raised against collagenase (MMP2), stromelysin (MMP3) and tissue inhibitor of metalloproteinase (TIMP1) and developed using the avidin-biotin complex method. Abundance of MMP2, MMP3 and TIMP1 was greater in pancreatic and ampullary cancer than any other pathology and immunoreactivity in the malignant epithelial cells in pancreatic and ampullary cancer was greater than in the stromal tissues . There were strong correlations between the immunoreactivity of the two antibodies for MMP2 (P < 0.0001), between MMP2 and TIMP1 (P < 0.0001) and between MMP3 and TIMP1 (P < 0.0001). The immunoreactivity for TIMP1 in pancreatic and ampullary cancers with lymph node metastases was significantly less compared with those cases without lymph node metastases (P < 0.02) and there was an association between increased immunoreactivity for MMP2 and the degree of tumour differentiation (P < 0.01). The results implicate MMP2, MMP3 and TIMP1 in the invasive phenotype of pancreatic and ampullary cancer."}
+{"text": "Four subtypes have been detected, and pathogenicity, zoonotic potential, or stability may vary between subtypes. Non\u2013travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans. Hepatitis E virus (HEV) is an RNA virus that causes liver inflammation in humans, predominantly in developing countries. In the 1990s, serologic studies among blood donors in industrialized countries showed that anti-HEV seropositivity also occurred among a small percentage (1.1%\u20131.4%) of persons without a travel history to a hepatitis E\u2013endemic region  that were caught in 2 regions in the Netherlands, in the northeast (Groningen) and in the southeast (Limburg)  throughout 2005  were collected from the Meuse River monthly by using a conventional filter adsorption\u2013elution method and concentrated by ultrafiltration by using a cellulose-acetate filter  under high pressure (3 bar)  was performed on 10-fold serially diluted RNA samples with primers HEVORF2con-s1 and HEVORF2con-a1, which were specific for the conserved open reading frame (ORF) 2 region, as described  and corresponded with analysis of a 242-nt fragment of ORF1 and a 148-nt fragment of ORF2. Human HEV sequences were obtained from human serum samples used for diagnosis of acute viral hepatitis (HEV RT-PCR products positive by hybridization (ORF2) or of the correct size (ORF1) were subjected to electrophoresis on a 2% agarose gel, excised, purified by using a Qiaquick Gel Extraction Kit , and sequenced by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit . HEV RT-PCR products for which no sequences were obtained by direct sequencing were cloned into a pCRII-TOPO vector ; Ten-fold serially diluted RNA samples extracted from animal samples and river water were analyzed for HEV ORF2 sequences by RT-PCR. The highest prevalence of HEV ORF2 RNA in 51 (53%) of 97 pooled fecal samples was detected in pig farms housing pigs 5\u201327 weeks of age . A preva3 to 106 PDU/g. In 2 (17%) of 12 river water samples analyzed, HEV RNA was detected at concentrations of 2 PDU/L to 100 PDU/L.HEV RNA concentrations in positive fecal samples varied from 10www.ncbi.nlm.nih.gov) by phylogenetic analysis. This comparison showed that all belonged to HEV genotype 3.Thirty-six newly generated nucleotide sequences of 148 nt of HEV ORF2 were obtained from pooled fecal samples from pig farms that were previously shown to be positive for HEV ORF1 sequences . These sSequences grouped within 4 previously proposed genotype 3 clusters  , of whicTwo sequences in cluster 3f obtained from different pig farms were 100% identical within the 148-nt fragment of ORF2. A total of 242 nt of ORF1 were sequenced and showed that the 2 sequences were 98.8% identical within this region of the HEV genome. This result indicated that the strains were similar but not identical.Dutch environmental and animal HEV sequences obtained during 2004\u20132006 were compared with HEV ORF2 sequences from a study on Dutch hepatitis E patients without a travel history who received a diagnosis during 2004\u20132006  (19). NuHuman sequences grouped within the same 4 HEV genotype 3 clusters, with most of the sequences clustering within Dutch cluster 3c. A relatively high percentage of the sequences in cluster 3c is of human origin (43%). In this cluster, 1 human sequence was 100% identical with a porcine HEV sequence. For confirmation of homology, ORF1 RT-PCRs were performed, but only the pig sample yielded an ORF1 sequence. No direct geographic evidence could be established for an association between the patient and the pig farm; the patient who contracted the HEV infection in 2005 lived in the northern region of the Netherlands, but the pig farm was located in the eastern region. Comparison of postal codes of other Dutch patients without a recent travel history with postal codes of environmental sample locations did not show any geographic clustering.In cluster 3f, only 1 (5%) of 21 sequences identified during 2004\u20132006 was of human origin. Comparison with HEV sequences derived from human serum samples showed 2 close homologies, 1 between sequences from a hepatitis E patient in 2005 and a pig (96.6%) and 1 between sequences from a patient in 2006 and a surface water sample in 2005 (97.3%).Evidence was obtained for the presence of HEV in food, water, and animals in the Netherlands. Although 4 genotypes of HEV are known, 3 of which have been detected in pigs, each HEV ORF2 fragment sequenced in our study was identified as genotype 3. The highest prevalence (53%) was found on pig farms housing pigs 5\u201327 weeks of age, which is consistent with studies reporting that most HEV infections in pigs occurred between 2 and 4 months of age   virus. To confirm this hypothesis, samples should be tested for infectious virus by in vivo infection experiments  showed large differences (43% vs. 5%). This finding suggests that HEV 3c strains may be more pathogenic to humans, more stable in the environment, or are shed in higher numbers. Strains of feline calicivirus with different pathogenicities obtained from distinct outbreaks did not show conserved changes in virus genomes. Nevertheless, strains with higher pathogenicity infected tissue culture cells more efficiently and showed earlier cytopathic effects   suggest that the route of exposure and the virus subtype will play a role in HEV infection and disease in humans."}
+{"text": "In a series of 46 glioblastomas, 16 anaplastic astrocytomas and eight astrocytomas, all tumours retaining one or both alleles of CDKN2A (48 tumours) and CDKN2B (49 tumours) were subjected to sequence analysis (entire coding region and splice acceptor and donor sites). One glioblastoma with hemizygous deletion of CDKN2A showed a missense mutation in exon 2 (codon 83) that would result in the substitution of tyrosine for histidine in the protein. None of the tumours retaining alleles of CDKN2B showed mutations of this gene. Glioblastomas with retention of both alleles of CDKN2A (14 tumours) and CDKN2B (16 tumours) expressed transcripts for these genes. In contrast, 7/13 glioblastomas with hemizygous deletions of CDKN2A and 8/11 glioblastomas with hemizygous deletions of CDKN2B showed no or weak expression. Anaplastic astrocytomas and astrocytomas showed a considerable variation in the expression of both genes, regardless of whether they retained one or two copies of the genes. The methylation status of the 5' CpG island of the CDKN2A gene was studied in all 15 tumours retaining only one allele of CDKN2A as well as in the six tumours showing no significant expression of transcript despite their retaining both CDKN2A alleles. Three tumours  were found to have partially methylated the 5' CpG island of CDKN2A. It appears that in human astrocytic gliomas point mutations of the CDKN2A and CDKN2B genes are uncommon and hypermethylation of the 5' CpG region of CDKN2A does not appear to be a major mechanism for inhibiting transcription of this gene."}
+{"text": "LKB1 and SMAD4 genes have been shown recently to cause a number of PJS and JPS cases respectively, but there remains considerable uncharacterized genetic heterogeneity in these syndromes, particularly JPS. The mouse homologue of CDX2 has been shown to give rise to a phenotype which includes hamartomatous-like polyps in the colon and is therefore a good candidate for JPS and PJS cases which are not accounted for by the SMAD4 and LKB1 genes. By analogy with SMAD4CDX2 is also a candidate for somatic mutation in sporadic colorectal cancer. We have screened 37 JPS families/cases without known SMAD4 mutations, 10 Peutz-Jeghers cases without known LKB1 mutations and 49 sporadic colorectal cancers for mutations in CDX2. Although polymorphic variants and rare variants of unlikely significance were detected, no pathogenic CDX2 mutations were found in any case of JPS or PJS, or in any of the sporadic cancers. \u00a9 2001 Cancer Research Campaign www.bjcancer.comPeutz\u2013Jeghers syndrome (PJS) and juvenile polyposis (JPS) are both characterized by the presence of hamartomatous polyps and increased risk of malignancy in the gastrointestinal tract. Mutations of the"}
+{"text": "We followed 87 cirrhotic patients with esophageal varices and without previous hemorrhage for a mean period of 24 mo to prospectively evaluate the occurance of variceal bleeding within (early) or after (late) 6 mo from entry and the contribution of portal Doppler ultrasound parameters to the prediction of early and late hemorrhage. Clinical, biochemical, endoscopic and portal Doppler ultrasound parameters were recorded at entry. Variceal bleeding occurred in 22 patients (25.3%). Nine (40.9%) bled within the first 6 mo. Cox regression analysis identified variceal size, cherry-red spots, serum bilirubin and congestion index of the portal vein  as the only independent predictors of first variceal hemorrhage. Discriminant analysis was used to find the prognostic index cut off points to identify patients who bled within 6 mo (prognostic group 1) or after 6 mo (prognostic group 2) or remained free of bleeding (prognostic group 3). The cumulative proportion of patients correctly classified was 73% in prognostic group 1, 47% in prognostic group 2 and more than 80% in prognostic group 3. The addition of Doppler ultrasound flowmetry to clinical, biochemical and endoscopic parameter only improved the classification of patients with early bleeding."}
+{"text": "The aim of the study was to determine the relationship between StO2 and mixed venous oxygen saturation (SvO2) in patients with severe left heart failure with or without additional severe sepsis or septic shock.Low cardiac output states such as left heart failure are characterized by preserved oxygen extraction ratio, which is in contrast to severe sepsis. Near infrared spectroscopy (NIRS) allows noninvasive estimation of skeletal muscle tissue oxygenation (StOn = 24) and B (n = 41) included patients without and with additional severe sepsis/septic shock, respectively. Thenar muscle StO2 was measured using NIRS in the patients and in 15 healthy volunteers.Sixty-five patients with severe left heart failure due to primary heart disease were divided into two groups: groups A . StO2 was higher in group B than in healthy volunteers (P = 0.02). In group A StO2 correlated with SvO2 , although StO2 overestimated SvO2 . In group A changes in StO2 correlated with changes in SvO2 . In group B important differences between these variables were observed. Plasma lactate concentrations correlated negatively with StO2 values only in group A .StO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock. However, in patients with severe left heart failure without additional severe sepsis or septic shock, StO2 values could be used to provide rapid, noninvasive estimation of SvO2; furthermore, the trend in StO2 may be considered a surrogate for the trend in SvO2. Skeletal muscle StOTrial Registration: NCT00384644 In 80% of patients pathogenic bacteria were isolated.Included in the study were 65 patients  with primary heart disease . In 24 patients (group A) severe left heart failure or cardiogenic shock but no additional severe sepsis/septic shock was the reason for ICU admission. In 25 patients severe sepsis and in 16 patients septic shock was diagnosed . StO2 was higher in group B patients than in healthy volunteers (P = 0.02). In group A StO2 correlated with SvO2 , but no correlation was observed between StO2 and SvO2 in group B  was detected.In group A StO2-StO2 changes were recorded. Changes in StO2 correlated with changes in SvO2 ; the equation for the regression line was as follows  = 0.84 \u00d7 \u0394StO2 (%) - 0.67. In eight patients from group B 38 pairs of SvO2-StO2 changes were recorded. In group B changes in StO2 did not correlate with changes in SvO2 .In 10 patients from group A 42 pairs of SvO2 values in group A ; there was no correlation between lactate and StO2 in group B.Plasma lactate concentrations correlated negatively with StO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock. However, in patients with severe left heart failure without additional severe sepsis or septic shock, the StO2 value could be used as a fast and noninvasive estimate of SvO2; also, the trend in StO2 may be considered a surrogate for the trend in SvO2.The main result of the study is that skeletal muscle StO2 and slow deceleration in StO2 during stagnant ischaemia in septic patients [per se, because an increase in tissue oxygen tension is normally observed [2 levels seen in our patients with additional severe sepsis or septic shock support this hypothesis. Mitochondrial dysfunction has been implicated by Ince and Sinaasappel [We previously detected high StOpatients . Our datpatients . Studiespatients . Howeverpatients . Reducedobserved . The higaasappel . This miaasappel .2/low SvO2, seen in severe sepsis and septic shock, suggest blood flow redistribution. Thenar muscle StO2 probably correlates with central venous oxygen saturation (ScvO2), which is measured in a mixture of blood from head and both arms. In healthy resting individuals ScvO2 is slightly lower than SvO2 [2 is greater than ScvO2.The high StOhan SvO2 . Blood ihan SvO2 . As a re2 and SvO2 in both haemodynamically stable and shocked patients. In stable patients ScvO2 was similar to SvO2. In patients with failing heart ScvO2 was slightly higher than SvO2 and in shock patients the difference between SvO2 and ScvO2 was even more pronounced . Lee and coworkers [2 measurements could substitute for SvO2 demonstrated problematic large confidence limits [This relationship changes in the presence of cardiovascular instability. Scheinman and coworkers  performeoworkers  describee limits  and poore limits .2 is greater than SvO2 [2 (or probably StO2) as a treatment goal, the relative oxygen consumption of the superior vena cava system may remain stable at a time when oxidative metabolism of vital organs, such as the splanchnic region, may reach a level at which flow-limited oxygen consumption occurs, together with marked decrease in oxygen saturation. In this situation StO2 provides a falsely favourable impression of adequate body perfusion, because of the inability to detect organ ischemia in the lower part of the body.Most authors attribute this pattern to changes in the distribution of cardiac output that occur in the presence of haemodynamic instability. In shock states, blood flow to the splanchnic and renal circulations falls, whereas flow to the heart and brain is maintained . This rehan SvO2 -25. Cons2 of 75% or less ; they were all in septic shock  with low cardiac index (< 2.0 l/min per m2). These patients were probably in an early under-resuscitated phase of septic shock. Low numbers of septic patients with low StO2 values did not allow us to study the agreement between StO2 and SvO2 in such patients; however, there was a wide range in StO2 values with SvO2 below 65%. Additional research is necessary to study muscle skeletal StO2 in under-resuscitated septic patients.In the present study three patients with septic shock had skeletal muscle StO2  and systemic vascular resistance . No correlation was found between systemic oxygen transport variables and skeletal muscle partial oxygen pressure in septic patients. These measurements were taken in the most common cardiovascular state in sepsis; this is in contrast to hypodynamic shock, which is only present in the very final stages of sepsis or in patients without adequate volume replacement [Our data are supported by previous work conducted by Boekstegers and coworkers , who mealacement . In a sulacement .2 and venous oxygen saturation  was observed; the venous effluent was obtained from a deep forearm vein that drained the exercising muscle. StO2 was minimally affected by skin blood flow. Changes in limb perfusion affect StO2; skeletal muscle StO2 decreases during norepinephrine and increases during nitroprusside infusion.In a human validation study  a signif2  correlated with systemic DO2 in a pig model [2 is already used in trauma patients, in whom it identifies the severity of shock [2 can track systemic DO2 during and after resuscitation of trauma patients [In shock with preserved or even increased oxygen extraction, such as haemorrhagic shock, StOig model . Changesig model . Continuof shock . Basal spatients .2 overestimated SvO2 (bias -2.5%) in the present study. This may be due to the NIRS method, which does not discriminate between compartments. It provides a global assessment of oxygenation in all vascular compartments  in sample volume of underlying tissue. This is major limitation of the present study. The noninvasive measurement of only venous oxygen saturation is complicated by the fact that isolation of the contribution of venous compartment to the noninvasive optical signal is not straightforward. New methods like near-infrared spiroximetry, which measures venous oxygen saturation in tissue from the near-infrared spectrum of the amplitude of respiration-induced absorption oscillations, may lead to the design of a noninvasive optical instrument that can provide simultaneous and real-time measurements of local arterial, tissue and venous oxygen saturation [StOturation .2 regional monitoring to guide tissue oxygenation, in addition to the early goal-directed therapy proposed by Rivers and coworkers [In low flow states, in which controversies regarding monitoring persist , it appeoworkers .2 provides a noninvasive estimate of and tracks with StO2. It should be emphasized that in patients with severe heart failure and additional severe sepsis or septic shock, skeletal muscle StO2 provides a falsely favourable impression of body perfusion.In patients with severe left heart failure without additional severe sepsis or septic shock, SvO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock.\u2022 Skeletal muscle StO2 values could be used to provide rapid, noninvasive estimation of SvO2; furthermore, the trend in StO2 may be considered a surrogate for the trend in SvO2.\u2022 StO2 = oxygen distribution; ICU = intensive care unit; NIRS = near infrared spectroscopy; ScvO2 = central venous oxygen saturation; SOFA = Sepsis-related Organ Failure Assessment; StO2 = tissue oxygenation; SvO2 = mixed venous oxygen saturation.DOThe authors declare that they have no competing interest.MP was responsible for conception and design of the study; for acquisition of data, and its analysis and interpretation; and for drafting the manuscript. HM was responsible for conception and design of the study; for acquisition of data, and its analysis and interpretation; and for drafting the manuscript."}
+{"text": "The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested."}
+{"text": "In order to assess the role of endoscopic retrograde cholangiography in evaluating the patients with post-operative biliary leak and of endoscopic nasobiliary drainage in its management, 36 patients with biliary leak seen over a period of 9 years were studied. Thirty-two had biliary leak following cholecystectomy, 3 following repair of liver trauma and 1 following choledochoduodenostomy. Patients presented at an interval of 4 days to 210 days  following laparotomy. Hyperbilirubinemia was noticed in only 13 patients (36.1%), while abdominal ultrasonogram showed ascites or biloma in 24 (66.7%). Endoscopic retrograde cholangiography showed the leak to involve the common bile duct in 55.6%, cystic duct in 33.3% and intrahepatic biliary radicles in 8.3%. Associated lesions included bile duct obstruction due to stricture or accidental ligature in 20%, bile duct stone in 20% and liver abscess in 2.8%.        Endoscopic nasobiliary drainage using a 7 Fr pig-tail catheter was attempted in 14 patients and could be established in 12 of them. Bile duct leak sealed in all but one of these 12 patients after an interval of 3 days to 40 days . A single patient with large defect and a proximal bile duct stricture did not respond and required surgery. Common bile duct stones were removed by endoscopic sphincterotomy in 3 out of 4 patients. One patient with large stone required surgical choledocholithotomy. In conclusion, endoscopic retrograde cholangiography was safe and useful in confirming the presence of leak as well as its site, size and associated abnormalities. Endoscopic nasobiliary drainage proved an effective therapy in post-operative biliary leak and could avoid re-exploration in 71.4% patients."}
+{"text": "The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation.Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation . Deletion of 25 nts from the 3\u2032terminus of the 5\u2032untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2\u20135\u2032UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3\u2032end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA.We have probed the putative secondary structure of the IRF2 5\u2032UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factors to mediate internal initiation of translation.It appears that PTB binding to multiple sites within IRF2 5\u2032UTR leads to a conformational change in the RNA that facilitate binding of other  IRF1 function as an activator of IFN and IFN-inducible genes, whereas IRF2 act as repressor of IRF1 action trans-acting factors. Ribosome binding analysis also indicated that supplementation of purified PTB enhanced the formation of translation initiation complex. This led us to hypothesize that PTB interaction with the IRF2 IRES might trigger a change in the conformation that helps in the recruitment of other hitherto unknown trans acting factors and ribosomes, thus facilitating translation initiation.We have partially mapped the secondary structure of the IRF2 5\u2032UTR using various enzymatic and chemical modification probes. The data validates the predicted MFOLD structure of the 5\u2032UTR RNA. To map the putative contact points of the protein on the IRF2 5\u2032 UTR we have performed primer extension inhibition assay. PTB was found to have multiple contact points on the IRF2 RNA, but mostly at the 3\u2032end of the 5\u2032UTR. Since many contact points were found on the 3\u2032 end of the RNA, a deletion mutant was generated, where 25 nt from 3\u2032end was deleted. UV cross-linking and competition experiments showed a reduced binding of the purified PTB on the mutant RNA as compared to the wild type RNA. The above data was supported by Circular Dichroism (CD) spectra analysis of the RNA, which also suggested a change in the conformation of IRF2 5\u2032 UTR in presence of PTB binding. Proteins from whole cell extracts showed that full length IRF2 IRES interacted predominantly with a 57 kDa (PTB) protein, compared to the mutant IRF2. Further PTB silencing experiments showed reduced PTB binding along with significant reduction in binding of other cellular Earlier we have shown that the putative secondary structure of IRF2 5\u2032UTR RNA (predicted by using the MFOLD program) IRF2 monocistronic RNA was generated from the corresponding DNA construct . The RNAin vitro transcribed IRF2 UTR RNA was incubated with RNase T1 followed by extension using AMV-RT (Promega). The products were analyzed on 8% polyacrylamide-8M urea denaturing gel. For precise mapping of the contact points, a DNA sequencing reaction with the same end labeled primer was run alongside . Since IRF2 5\u2032UTR folds into a fairly stable secondary structure, only few specific RT pauses were observed at G8, G44, G59, G69, G83 and G131 , the pauses corresponding to nuclease (TI and VI) cleavage sites and the toe prints corresponding to PTB binding sites were mapped to MFOLD predicted structure for clarity . The res32P labeled wild type IRF2 5\u2032UTR RNA was incubated with 0.87 nM of purified PTB protein in absence and presence of increasing concentrations of the competitor RNAs. The toe-printing data showed that the contact points of PTB are predominantly at the 3\u2032 end of the IRF2 5\u2032UTR RNA. To investigate this further, 25 nucleotides were deleted at the 3\u2032end from the IRF2 5\u2032 UTR to generate mIRF2 . To compSince 3\u2032 deletion mutant showed reduced PTB binding, we hypothesized that this might also affect the IRF2-IRES activity. To investigate it, the deletion mutant was cloned in the bicistronic plasmid construct . Both wiTo further verify the possible conformational change in the RNA upon protein binding CD spectroscopic studies were performed. CD spectra of IRF2 5\u2032UTR RNA was analyzed in absence and presence of purified recombinant PTB protein. CD spectra were obtained in 240 to 320 nm range at 20\u00b0C in 0.5 ml RNA binding buffer. The results showed distinct change in the wavelengths at which the peak of ellipticity value is reached for RNA incubated with PTB (260 nm) compared to RNA alone (256 nm). Also, at this particular wavelength there was significant increase in the ellipticity value suggesting a change in the conformation of the IRF2 5\u2032UTR RNA in presence of PTB . Interes32P labeled Wt and mIRF2 UTR RNAs were incubated with the extracted total proteins, UV-crosslinked and analysed on SDS-PAGE. The results indicated that wtIRF2 5\u2032UTR interacted strongly with 57 kDa protein (PTB), whereas with mIRF2 5\u2032UTR the intensity of the band corresponding to 57 kDa protein was relatively less. Interestingly, the mIRF2 showed binding with additional proteins, some of those could be the cleavage products of PTB (p25), which might negatively influence IRES activity trans acting factors to enhance translation, PTB depleted HeLa S10 was used in UV cross linking study. PTB was partially silenced in HeLa cells by using increasing concentration of siPTB. 36 hours post transfection S10 extracts were prepared. A significant decrease of PTB protein level was observed at 60 nm & 80 nm concentration of siPTB. UV-crosslinking experiment was performed using wt IRF2 RNA probes and S10 extracts from control or siPTB treated cells. Interactions of several cellular proteins were found to be significantly reduced  has been implicated as an important ITAF for a number of viral as well as cellular IRESs. It is believed that PTB has a chaperone like activity that can bring about a change in the conformation of the IRES structure, which might facilitate the interaction of the ribosomes with the RNA leading to efficiently initiate translation.The mechanism of IRES mediated translation in cellular RNA is still not completely understood. Various IRES In this paper, we have investigated the interaction of PTB with the IRF2-IRES RNA and studied its importance on its translation initiation. Multiple contact points were mapped on the IRF2 IRES RNA. A significant number of those contact points were mapped to the 3\u2032 end of the IRES element. In fact, Apaf-1 IRES is also known to have two potential PTB binding sites at the 3\u2032 end region However recent report suggests that the IRES activity might not completely depend on the overall structure but on different sequence modules that play important role in the ITAF recruitment trans acting factors near the vicinity of the translation start site that would facilitate the recruitment of the ribosomes to the RNA and thus facilitating translation initiation. To further consolidate the observation, S10 extract isolated from HeLa cells, partially silenced for PTB expression was used for binding using Wt IRF2 probe. Several cellular tran-acting factors were unable to bind Wt IRF2 in reduced presence of PTB. Interestingly, we found that supplementing the in vitro translation mix with purified PTB marginally enhanced the formation of the 48S ribosomal initiation complex.Nuclease mapping and chemical modifications partly revealed the secondary structure of the IRF2 5\u2032UTR, which largely supports the MFOLD prediction. DMS modification interference assay indicated perturbation of the RNA structure in presence of PTB. The presence of PTB masked the RNA, rendering it inaccessible to DMS modifications at certain places. Also, a few new DMS modification sites were found under such condition, suggesting changes in local structure due to protein binding. CD spectroscopic analysis reinforces the idea of change in RNA conformation upon protein binding. In fact when CD experiment was performed with the mutant IRF2 IRES RNA and the PTB protein, change in molar ellipticity was found to be less than that obtained with the wild type RNA. PTB has been shown to have chaperone like activity, thus it would be interesting to study how its interaction with the IRF2 5\u2032 UTR RNA and consequent change in the RNA conformation helps in ribosome assembly during internal initiation. Also it is possible that such a change in the conformation of the RNA might recruit certain other trans-acting factors that could facilitate IRES mode of translation initiation in the IRF2 mRNA. However, at this stage it is not clear how PTB or other IRES trans-acting factors (ITAFs) interaction helps in switching on the IRES activity of the cellular IRES elements. It could be a complex interplay of PTB and several other ITAFs. Future experiments would be directed to understand how the ITAFs binding directs the ribosome to land preferentially to the IRES site and translate by internal initiation under stress conditions.Thus we hypothesize that PTB serves as an ITAF which recruits other possible No, an ethics statement is not required for this work.The bicistronic constructs containing respective 5\u2032UTR sequences of full length IRF2 (NCBI accession number NM_002199) and 3\u2032 deletion IRF2  were cloned downstream of the landscape of structure derived form the inactive part of \u0394EMCV IRES sequence between Renilla luciferase (RLuc) and Firefly luciferase (FLuc) genes, in HindIII and EcoRI sites. The construct pR\u0394EnullF HeLa S3 cells were maintained in DMEM (Invitrogen) with 10% fetal bovine serum . Cells were transfected with various bicistronic plasmids and pSV40\u03b2-gal using Tfx 20 reagent (Promega) and luciferase assay was performed using Dual luciferase assay reagent (Promega). Transfection of siRNA was performed in HeLa S3 cells growing in monolayer using Lipofectamine-2000 transfection reagent and optiMEM-I prepared without addition of antibiotic (Invitrogen). Cells were seeded onto 100 mm dishes in duplicates one day prior to transfection in similar manner. Transfections were performed with 40 nm, 60 nm and 80 nm of siPTB (Dharmacon) or 80 nm of nonspecific si (Dharmacon) diluted with optiMEM-I to a final volume of 100 \u00b5l followed by incubation at room temperature for 20 minutes. Subsequently, 4.9 ml of optiMEM-I was added to the transfection mixture, which was then layered onto cells. 4 hours later the medium was replaced with 9 ml of DMEM (with antibiotic) and 10% FBS. 36 hours post transfection the cells were washed with PBS, and were trypsinized followed by HeLa S10 extract preparation. S10 extract was prepared from HeLa cells as described before (22).32P labeled RNA probes corresponding to the 5\u2032UTRs of IRF2 and deletion mutant IRF2 were made from their respective plasmid DNAs after linearizing with EcoRI and transcribed with T7 RNA polymerase and 10 \u00b5Ci/\u00b5l of alpha 32P UTP (NEN) as per manufacturer's guidelines.The IRF2 5\u2032UTR and mIRF2 RNA probes were made from IRF2Fluc and mIRF2Luc DNA plasmids respectively linearized with Eco RI and was transcribed by T7 RNA polymerase. Similarly, the E.coli (BL21 DE3) cells transformed with the expression vector. His-tagged protein was purified using Ni2+-nitrilotriacetic acid-agarose (Qiagen) under non-denaturing conditions and eluted with 250 mM imidazole.The expression of recombinant PTB (NCBI accession number NM_031990) from PET28a-PTB (a generous gift from Dr. J.G. Patton) was induced by 0.6 mM IPTG in in vitro transcribed IRF2 bicistronic RNAs were denatured at 65\u00b0C for 10 minutes and then cooled slowly to room temperature. The RNAs were then digested with 0.5 unit of RNase T1 (SIGMA) for 15 minutes at 37\u00b0C. After incubation the reaction mixture was treated with proteinase K followed by phenol:chloroform extraction and alcohol precipitation. For RNase V1 (Ambion) digestion, the RNA was incubated with 0.01 units of RNase V1 for 10 minutes at 30\u00b0C. After the incubation the reaction mixture was extracted with phenol: chloroform and alcohol precipitated. T1 digestions were carried out in the buffer containing 10 mM Tris.Cl (pH 7.5), 10 mM MgCl2, and 100 mM KCl. The DMS modification was carried out in the buffer containing 50 mM sodium cacodylate (pH 7.5), 5 mM MgCl2, 100 mM KCl at 30\u00b0C for 10 minutes using 1 \u00b5l of DMS diluted 1\u22368 in absolute ethanol. The modified RNAs were precipitated with ethanol. The digested and the modified RNAs were reverse transcribed using AMV RT (Promega) with an end labeled primer annealing to 1\u201320 at the 5\u2032 end of the firefly luciferase gene. The cDNAs were precipitated with 0.3 M sodium acetate and absolute ethanol and then resolved in a 8M urea-8% Acrylamide gel electrophoresis along with a reference sequencing reaction  using the same end-labeled primer. Similarly, purified PTB bound RNA in buffer was modified with 1 \u00b5l of DMS diluted 1\u22368 in absolute ethanol at 30\u00b0C for 10 minutes The modified RNAs were precipitated with ethanol the modified RNAs were reverse transcribed using AMV RT (Promega) with an end labeled primer annealing to 1\u201320 at the 5\u2032 end of the firefly luciferase gene and was analyzed as mentioned before.10 \u00b5g of in vitro transcribed RNA corresponding to the IRF2 bicistronic RNA (derived from the construct pRIRF2F) and binding reaction was performed in a final volume of 20 \u00b5l at 30\u00b0C for 20 minutes. Reaction where PTB was not added serve as a negative control. To the reactions, [32P]-end labeled primer complimentary to 20 nucleotides of the 5\u2032 end of the Firefly luciferase was added and allowed to extend using 3 units of AMV-Reverse transcriptase (Promega) at 30\u00b0C for one hour. The cDNAs were alcohol precipitated, resuspended and compared with the dideoxynucleotide sequence ladders (obtained using same end-labeled primer) by electrophoresis on a 6% polyacrylamide/8M urea denaturing gel.Increasing concentration of purified his-tagged PTB protein (500 and 800 ng) was incubated with 5 pmoles of 2, 3.8% glycerol, 2 mM DTT, 0.1 mM EDTA). CD spectra were obtained in 240 to 320 nm range at 20\u00b0C with IRF2 5\u2032UTR (400 ng) and 800 ng of PTB or bovine serum albumin (BSA).Measurements of CD spectra were performed with a Jasco J-715 spectropolarimeter. Spectra were obtained in 0.5 ml RNA-binding buffer . Briefly, [\u03b1-Protein concentration of the S10 extract prepared from HeLa cells were assayed by Bradford (Bio-Rad). Equal amounts of cell extracts were loaded on SDS-10% polyacrylamide gel. The resolved proteins were electro-transferred to nitrocellulose membranes. Samples were then analysed by mouse monoclonal anti-PTB antibody , followed by secondary antibody . Rabbit polyclonal anti-actin (Sigma) was used as control for equal loading of total cell extracts. Antibody complexes were detected using the enhanced chemiluminescence (ECL) detection kit (Amersham Antibodies).32P UTP and the 25 \u00b5l reactions containing 17.5 \u00b5l RRL (Promega), 1.5 \u00b5l of amino acid mix (Promega) and PTB was supplemented as indicated in the figure legends. Labeled RNA 100,000 cpm was used per reaction. The reactions were incubated at 30\u00b0C for 20 min. and the volume was made upto 150 \u00b5l with the sucrose density gradient buffer . The reactions were overlaid on 5\u201330% linear sucrose density gradients and centrifuged for 3 hrs at 30,000 rpm using SW41 rotor in Beckman ultracentrifuge. 200 \u00b5l fractions were collected from top of the radioactive tube. Counts of each fraction were measured and %cpm was plotted against the fraction number.IRF2 UTR RNA was labeled with We gratefully acknowledge members of our laboratory for their help and suggestions."}
+{"text": "Human endocrine tumors often express the somatostatin receptors SSTR 1\u20135 with different intensity. It has been widely investigated their distribution in pituitary adenomas, brain tumors, adrenal tumors and neuroendocrine tumors in gastrointestinal tract (NET). Some of studies also concern the expression of SSTRs in thyroid tumors but they are mainly limited to parafollicular C cells \u2013 derived medullary thyroid carcinomas (MTC). Results of SSTR 1\u20135 detection in other thyroid pathologies like follicular adenomas and papillary cancers are still scarce and often controversial, depending of investigation method used. The aim of this study was to report the presence of all the 5 subtypes of SSTR (including 2A and 2B SSTR isoforms) in some surgically treated human thyroid tumors by means of immunohistochemistry and real-time PCR method and to correlate the results obtained with both techniques. SSTR 1 protein was expressed in 88.8% of investigated cases, SSTR 2A and 2B both in 44.4%, SSTR 3 in 55.5%, SSTR 4 in 11.2% and SSTR 5 in 33.3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. We found positive confirmation of both methods in 88.8% for SSTR 1, 2A, 3 subtypes, in 22.2% for SSTR 4 and in 100% for SSTR 5. It suggests that somatostatin multiligand analogs or selective SSTR 1 agonists may be used in thyroid tumors treatment. Somatostatin (SST) as well as its synthetic analogs are engaged in regulation of hormones secretion in different ways and this action depends on specific receptors expression which are present on the target cells. Five subtypes of the SST receptor have been identified so far, i.e. SSTR 1\u20135. SSTR 2 is present in two splicing variants (2A and 2B)  and are ex vivo in vitro method in detecting cellular distribution of SST receptors .SSTR 1 which was distributed both in the cytoplasm and the membranes or only in the cytoplasm was shown in 8  specimens  and in the remaining 4 cases  distribution of this SSTR subtype was fluctuated from weak to pale. In 4 specimens  strong to moderate staining of SSTR 2B was observed with both membranous distribution and in the area of the cell cytoplasm. In the other 4 cases the staining intensity was weak or pale. Staining for SSTR 3 was characterized by cytoplasmic distribution with various intensity. In 5  out of 9 examined specimens SSTR 3 was found with strong or moderate intensity in the cell cytoplasm and the remaining 3 cases  showed weak to pale intensity of staining  of investigated cases with exception of 1  specimen showing cytoplasmic distribution of this receptor subtype fluctuating from pale to strong (patient nr 2) . Another 5  cases demonstrated pale to moderate staining. In 1  specimen the intensity of staining was from pale to strong (patient nr 2).Immunoreactivity of investigated thyroid tissues was estimate in pathologically altered cells of thyroid cancers and benign lesions of the thyroid gland. The area of normal surrounding tissue was immunonegative in all investigated cases. Multiple SSTR subtypes were found to coexist in each case of investigated tissue Table . Most ofens Fig. . SSTR 2AWe consolidated result for endogenous control gene of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as negative  and according to this all remaining mRNAs were calculated relative to the amount of HPRT and given in arbitrary units. The diagram presented below show the mean results of 3 experiments for each primer Fig. . SSTR 1 We demonstrated in our investigation that somatostatin subtype receptors are widely distributed in human thyroid diseases with different intensity and cellular localization. It is consists with earlier reports of England et al. who findWe proved that somatostatin receptor subtypes are frequently expressed in pathologically altered thyrocytes, in contrast to normal thyroid follicular epithelium. SSTR 1 protein was expressed in 77,7% of investigated cases, SSTR 2A and 2B both in 44,4%, SSTR 3 in 55,5%, SSTR 4 in 11,2% and SSTR 5 in 33,3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. A good correlation with SSTR 1 receptor gene expression was observed in 77,7% of investigated cases. Immunohistochemical estimation of SSTR 2A and 3 was agree with RT-PCR method in 77,7%, only 22,2% of results of SSTR 4 mRNA estimation were correlated with immunohistochemical staining and SSTR 5 mRNA was good correlated with IHC staining in 100% tissues. It suggests that somatostatin multiligand analogs or selective SSTR 1 agonists may represent a further useful approach for the thyroid tumors treatment. The expression of somatostatin receptor subtypes in thyroid tumors needs father studies concerning greater and more differentiated group of patients.mem: membranous localization; cytopl: cytoplasmatic localization; strong staining (+++), moderate staining (++), weak staining (+) and pale staining (+/-); SST: somatostatin; SSTR: somatostatin receptor subtype; RT-PCR: real time polimerase chain reaction; RT-PCR: reverse \u2013 transcriptase polimerase chain reaction; ISH: in situ hybridisation; IHC: immunohistochemistry; NET: neuroendocrine tumors in gastrointestinal tract; MTC: medullary thyroid carcinoma; MNG: multinodular goiter; mRNA: messenger ribonucleic acid; HPRT: hypoxanthine-guanine phosphoribosyltransferase control gene.The authors declare that they have no competing interests.HP carried out the immunohistochemistry studies and drafted the manuscript. TS obtained thyroid tissues during surgery. RK established histopathological diagnosis. EB carried out the molecular biology studies. MP supervised the experiences, helped to draft and revised the manuscript and provided general support. All authors read and approved the final manuscript."}
+{"text": "Rats were prepared surgically so that peripheral intestinal lymph could be collected from them while a syngeneic tumour (the HSN sarcoma) was growing in each major Peyer's patch of the small intestine. Dendritic lymph cells were isolated from the lymph and injected i.p. into naive, syngeneic rats. Each of the 16 recipients received just under 10(6) such cells and was challenged 10 days later with a subcutaneous dose of 10(4) viable HSN cells. Six weeks after this challenge only 7 of the recipients had a tumour and these were small (mean weight 1.8 g), while 17 controls (which had each been treated with 10(6) thoracic duct lymphocytes from the same donors, and given the same challenge) all had large tumours (mean weight 8.8 g). The remaining 9 test rats were still free of tumours when they were killed and autopsied 4 months after challenge. Dendritic lymph cells from normal rats were 'sensitised' by incubating them overnight on a monolayer of HSN cells. They were then transferred to 5 naive recipients which received the usual challenge. Six weeks later they all had tumours (mean weight 1.3 g) but these were much smaller than those in the 5 controls (mean weight 9.3 g)."}
+{"text": "Based on the important role of CD44 splice variants in colorectal cancer progression and metastasis, we evaluated the use of CD44v6 expression to detect and assess the metastatic potential of colorectal tumour cells circulating in peripheral blood. A nested amplification was designed that allowed to detect 10\u2013100 colon cancer cells. This assay was applied to blood samples from healthy donors. Strong signals were detected in all cases, indicating that it cannot be used to detect colorectal carcinoma cells in whole blood. We then included an enrichment step based on the use of an anti-epithelial cells monoclonal antibody (BerEP4) coupled to magnetic beads. The CD44v6 reverse transcription polymerase chain reaction (RT PCR) assay was performed on cDNA synthesized from blood samples treated with these beads. We analysed 18 samples from 12 patients with a gastrointestinal disease, and 36 samples from ten patients with a colorectal cancer. None of the patients used as negative controls were found to contain epithelial cells in their blood as determined by cytokeratin 19 RT-PCR. By contrast, CD44 transcripts containing exon v6 were detected in nine out of the 18 samples tested (50%). For the colorectal cancer patients, six out of the seven samples (85.7%) that were cytokeratin 19-positive were CD44v6-negative, whereas ten samples out of the 29 not containing epithelial cells were CD44v6-positive (34.5%). This is probably due to the persistence of CD8+ leucocytes in the enriched preparations, as determined by PCR analysis of the CD8 \u03b1-chain. We conclude that detection of CD44v6 transcripts using a sensitive nested RT-PCR assay has no potential value to detect and characterize colorectal cancer micrometastases from blood, even following an initial enrichment step. \u00a9 2000 Cancer Research Campaign"}
+{"text": "MLH1, MSH2 and MSH6 mutation carriers.Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis.MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2.MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.Although the cumulative lifetime risk of LS related cancer is similar,  MLH1MLH1[MSH24MLH1[MSHMSH6[PMS2.MSH2 mutation families compared to MLH1 mutation families [MSH6 mutation families probably have a milder clinical phenotype with a later onset of both CRC and EC and clustering of endometrial carcinoma [PMS2 mutation families are largely unknown. One study reported that PMS2 mutation families have a milder phenotype compared to MLH1 and MSH2 families [The colorectal cancer risk in LS is dependent on sex and the MMR gene involved. The reported lifetime risk for colorectal cancer in the literature varies from 28-100% in males and 25-83% in females ,10-18. Tfamilies ,19. MSH6arcinoma . The risfamilies .MLH1 and MSH2 mutations, while studies evaluating MSH6 mutation families are sparse. The most efficient way to calculate the lifetime risks of CRC and EC in Lynch syndrome would be to calculate these risks based on a cohort of proven mutation carriers. Therefore, the aim of the present study was to calculate the cumulative lifetime risks for CRC and EC in Lynch syndrome using a cohort of proven MLH1, MSH2 and MSH6 mutation carriers.Unfortunately, the precise lifetime risk for CRC and endometrial carcinoma may be biased because the families selected in previous studies were mainly selected on basis of clustering of CRC or fulfilment of clinical criteria (Amsterdam II criteria). Furthermore, it was not always clear whether the affected subjects were proven mutation carriers. In addition, most studies have only evaluated lifetime risks for During the period 1994-2007, an MMR gene mutation was detected in 67 families who were counselled at the Department of Clinical Genetics of the Erasmus MC University Medical Center, because of a clinical suspicion for Lynch syndrome. Clinical data of family members including sex, age, mutation status, age at diagnosis of both LS-associated and other cancers were collected. LS-associated cancer included colorectal, endometrial, stomach, ovaries, upper uroepithelial tract, biliary tract, skin and brain cancer. Also, the site of the tumour, age at death and cause of death were collected. With consent of the patients or (in case the patient was deceased) of a close relative the cancer diagnosis was confirmed by pathology and/or medical reports. All pathology and medical reports were reviewed by the first author (DR) in order to confirm the diagnosis. If a subject reported the occurrence of cancer in the family and no pathology or medical report was available, the cancer was excluded from analysis. In addition, data regarding colonoscopic surveillance of affected and unaffected family members were collected.Only subjects with a proven MMR gene mutation were included in this study.http://www.hgvs.org. A variant was considered a mutation when leading to a predicted truncated protein or based on previously published data. Silent or missense variants which were previously unreported or of unclear status were labelled unclassified variants (UV) and not considered as an MMR gene mutation.Mutation analysis was performed by denaturing gradient gel electrophoresis, sequencing and multiplex ligation-dependent probe amplification (MRC-Holland kits P003 and P008). Mutation nomenclature was used according to international guidelines MLH1, MSH2 and MSH6 mutation carriers. Penetrance for age was calculated using the Kaplan Meier survival analysis method and included the 67 index cases. In case of multiple or recurrent colorectal carcinoma or endometrial adenocarcinoma, only the first diagnosis of either cancer was included in the analysis. The observation time for the different cancers was from birth until the date of first cancer diagnosis, death, date of hysterectomy  or the end of the study (31 December 2007). A p value below .05 was considered statistically significant.Data were submitted for statistical testing using the Statistical Package for the Social Sciences , version 12.0.1. Data are given as median and range or as mean with standard deviation when appropriate. The chi square test, Student's t test and log rank test were used to compare differences between MLH1 mutation, 20 (30%) with an MSH2 mutation and 21 (31%) with an MSH6 mutation. Of the 67 families, 46 (69%) met the Amsterdam II criteria. Mutation analysis was performed in 725 subjects (296 men and 429 women) and a mutation was identified in 246 subjects   mutation carriers developed another LS-associated cancer during their life  were detected with an One of the 69 mutation carriers had previously been diagnosed with EC and developed CRC while being under colonoscopic surveillance. The other 68 mutation carriers were included in a colonoscopic surveillance program after being diagnosed with colorectal cancer. These 68 subjects were treated surgically  for colorectal cancer and colonoscopic surveillance of the remaining colon was performed. Of the remaining 125 mutation carriers none developed colorectal cancer and in 23 (18%) adenomatous polyps had been detected and removed. The person-years of follow up was 1414 years and the mean follow up time of the subjects under colonoscopic surveillance was 7 \u00b1 4 years.MLH1, 77% for MSH2 and 75% for MSH6 mutation carriers . This was due to the fact that before the age of 70 years the risk of developing any Lynch syndrome associated cancer in MSH6 carriers was lower compared to MLH1 or MSH2 mutation carriers  compared to MLH1 and MSH2 mutation carriers. However, the highest increase in risk in male MLH1 and MSH2 mutation carriers was observed between the ages of 40 to 50 years, while the risk in male MSH6 mutation carriers mostly increased between the ages of 50 to 60 years. Although the age related risks were not significant different between the three different MMR genes, there was a trend in male MLH1 and MSH2 mutation carriers to develop CRC at an earlier age than male MSH6 mutation carriers. The cumulative risks for CRC in females were lower compared to males, 57% for MLH1, 52% for MSH2 and 30% for MSH6 mutation carriers  compared to MLH1 and MSH2 mutation carriers.In Figure MSH6 mutation carriers (61%), while the cumulative risks for MLH1 and MSH2 mutation carriers were 25% and 49% respectively. However, the log rank test showed no significant difference in age related cumulative risk (p = 0.58) between MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.For endometrial carcinoma, the highest cumulative risk was observed in the MSH6 mutation carriers  compared to MSH2 mutation carriers , but not significantly higher compared to MLH1 mutation carriers   with MLH1  and MSH2 . The median age of EC onset was significant higher in MSH6 mutation carriers  compared to MLH1 mutation carriers  and MSH2 mutation carriers . There were no significant differences in the age of onset of other LS-associated cancers between MLH1 , MSH2  and MSH6  mutation carriers .The median age of CRC onset in males was significantly higher in In this study, we evaluated 246 individuals from 67 families with a proven mismatch repair gene mutation to determine the cumulative lifetime risk of developing cancer. Previous studies have shown different lifetime risks for developing CRC in Lynch patients.MLH1 and MSH2 mutations. Thirty one percent of the families included in our study carried an MSH6 mutation. This frequency is higher than previously reported [MLH1, MSH2 and MSH6 families are sparse. A study evaluating the risk in 20 MSH6 families showed that colorectal cancer was less frequent and developed 10 years later in MSH6 compared to MLH1 and MSH2. In addition a significant higher lifetime risk of endometrial carcinoma of 71% in MSH6 mutation carriers with a later age of onset (54 years vs. 48 and 49 years for MLH1 and MSH2) was reported [MSH6 mutation families with 156 MLH1 and MSH2 mutation families confirmed the lower risk and later age of onset of CRC in MSH6 families [One of the first studies evaluating the lifetime risk reported a lifetime risk for CRC at age 75 years of 92% in males and 83% in females . Most lareported ,21-23. Sreported . A Germafamilies . These rfamilies .MLH1, MSH2 and MSH6 mutation carriers is similar, each mutated gene has a distinguishable cancer risk profile. In MSH6 mutation carriers the risk at age 70 years for developing CRC was the lowest in both male (54%) and female (30%) when compared to carriers of MLH1 and MSH2 mutations.Our study indicates that, however the cumulative risks of cancer at age 70 years in MSH6 and MSH2 mutation carriers also a significant difference in the age of CRC onset  was found and there was a trend in higher age of CRC onset between male MSH6 and MLH1 mutation carriers. For female mutation carriers, no significant differences were found in the mean age of onset of CRC. This can be explained by the fact that female MLH1 and MSH2 mutation carriers still developed CRC at an older age. The lower risk of CRC onset in female MSH6 mutation carriers under the age of 50 years raises the question whether colonoscopic surveillance guidelines in these subjects can be changed. Current guidelines advise to start with biennial colonoscopy surveillance from the age of 20-25 years [MSH6 mutation carrier with CRC was 34 years. Our data and the data from previous studies support that colonoscopic surveillance can be started at an age of 30 years in female MSH6 mutation carriers [Between male carriers .MLH1 mutation carriers. In MSH2 and MSH6 mutation carriers extracolonic cancers appear to contribute more to the similar cumulative lifetime risk of cancer in MLH1, MSH2 and MSH6 mutation carriers. A higher risk of extracolonic-LS-associated cancer was previously reported in MSH2 mutation carriers compared to MLH1 mutation carriers [MSH6 carriers have the highest endometrial cancer risk followed by MSH2 and MLH1 mutation carriers. Also, this risk increases sharply after the age of 50 years. In view of the disputable effect of endometrial carcinoma surveillance [MSH6 carriers aged 45 years or above prophylactic hysterectomy may be suggested in order to decrease the risk for developing endometrial carcinoma [MSH2 and MLH1 female mutation carriers this option may be more questionable. In MSH2 mutation carriers the risk of other extracolonic and extraendometrial cancers may reduce faith in and benefit of risk reducing surgery. In MLH1 mutation carriers the risk of endometrial cancer may not outweigh the disadvantages of surgery. In case of surgery for another cause, additional hysterectomy should be considered also in MLH1 en MSH2 mutation carriers.However our numbers are too small to draw definite conclusions, CRC seems to be the predominant cancer in carriers ,19. Unfocarriers ,26 our seillance ,28, in farcinoma . In MSH2MLH1, MSH2 and MSH6 mutation carriers.A strength of the present study was that the age related risks where calculated using proven mutation carriers. However, the age related risks might be somewhat lower since not all the unaffected individuals from proven mutation families opted for genetic testing and thus the total number of unaffected mutation carriers in the mutation families may be underestimated. In addition, individuals with a higher risk for mutation carriership, i.e. with an affected first degree relative, more often opt for genetic testing . This maMLH1, MSH2 and MSH6 mutation carriers are similar, each mutated gene has a distinguishable cancer risk profile. It underlines that female MSH6 mutation carriers have a distinct clinical phenotype with a lower CRC risk and a higher risk for developing endometrial carcinoma. Starting with biennial colonoscopic surveillance at an age of 30 years instead of an age of 20-25 years in female MSH6 mutation carriers is more suitable. Moreover, in female MSH6 mutation carriers prophylactic hysterectomy may be considered from an age of 45 years.In conclusion, the present study indicates that, although the cumulative risks at age 70 years of LS related cancer in MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. Starting with biennial colonoscopic surveillance at an age of 30 years in female MSH6 mutation carriers is more suitable and prophylactic hysterectomy may be considered from an age of 45 years.The present study indicates that each mutated MMR gene has a distinguishable cancer risk profile. Female CRC: colorectal cancer; EC: endometrial cancer; LS: Lynch syndrome; MMR: mismatch repair; UV: unclassified variant.The authors declare that they have no competing interests.DR participated in the data collection, performed the statistical analyses and helped to draft the manuscript. AW conceived of the study and participated in the data collection. ML helped to draft the manuscript. DD participated in the data collection. CT participated in the data collection. ES participated in the design of the study and assisted in the statistical analysis. EK helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "P <0\u00b7001) higher than in a healthy control group, but after local therapy the concentration was significantly lower than before treatment (P <0\u00b701). The concentration of conjugated 3OHA was nearly constant in the three groups. It was concluded that other factors than a genetic determined abnormality might be operating in bladder cancer patients which could lead to an abnormal concentration of 3OHA in their urine.The basal concentration of the tryptophan metabolite 3-hydroxyanthranilic acid (3OHA), which has carcinogenic properties, was measured with an enzymatic method of determination which allowed separate measurement of free and conjugated 3OHA. The concentration of free 3OHA in untreated bladder cancer patients was significantly ("}
+{"text": "We screened the 185delAG and 5382insC (BRCA1) and the 6174delT (BRCA2) mutation in 298 Spanish women with breast cancer. Two women (one with Sephardic ancestors) presented the 185delAG mutation and the same haplotype reported in Ashkenazim with this mutation. This suggests a common origin of the 185delAG in both Sephardic and Ashkenazi populations. \u00a9 1999 Cancer Research Campaign"}
+{"text": "Circulating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC.Using both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay.We concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol. Detection of circulating tumor cells (CTC) in blood and disseminated tumor cells (DTC) in the bone marrow and/or lymph nodes, which are thought to be responsible for metastases, may allow a better prediction of the individual prognosis of patients with colorectal cancer -3. RecenIn this study, we aimed to compare two different specific antibodies against the epitope in the EGF-like domain I of EpCAM for immunomagnetic enrichment and subsequent detection of CTC in CRC patients. We used commercially available immunomagnetic beads coated with mAb BerEP4  and magnBoth whole blood and MNC fractions of five healthy donors were tested regarding the specificity of cell extraction and enrichment protocols with immunomagnetic beads coated with BerEP4 and KS1/4. No CK20 signal was observed in all examined blood samples of healthy donors, demonstrating the specificity of the used assays.4 HT29 cells could be detected in 5 ml whole blood using the BerEP4 mAb whereas 103 HT29 cells could be detected in the same volume using the KS1/4 mAb.In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. In serial dilution assays, a minimum number of 103 HT29 cells spiked in 5 ml blood (200 cells/ml) could be detected after isolation of the MNC fraction using the mAb BerEP4 Dynabeads, whereas 102 HT29 cells spiked in 5 ml blood (20 cells/ml) were detected using the mAb KS1/4 coated beads.Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment of blood samples spiked with HT29 cells resulted in a higher sensitivity of the CK20 RT-PCR assay. 10Blood spiking experiments were repeated several times to confirm the above mentioned results.The observed higher sensitivity of tumor cell detection after isolation of the MNC fraction prior to immunomagnetic CTC enrichment prompted us to generally use Ficoll gradient centrifugation before further immunomagnetic enrichment and detection of CTC in the blood of CRC patients.Median age of included CRC patients was 63 years (range 27 - 90) with 16 females and 23 males. Eighteen of 39 (46%) patients presented with metastatic disease to the liver and were classified as UICC stage IV; 7 patients were UICC stage III, and 7 patients stage UICC II and I. Clinical data of the patients are shown in Table Using two different antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment, CTC were detected in 11 of 39 28%) patients with CRC. Among these, immunomagnetic enrichment with mAb BerEP4 beads accounted for 6 CK20 positive patients. Immunomagnetic enrichment using mAb KS1/4 beads showed 5 CK20 positive patients. Interestingly, there were no blood samples being CK 20 positive for both used antibodies (Table 8% patienCK20 PCR transcripts were detected in 3 of 18 (17%) blood samples from UICC stage IV patients after immunomagnetic enrichment with mAb KS1/4 coated beads. When mAb BerEP4 beads were used, we also observed positive CK20 products in 3 of 18 (17%) UICC stage IV patients. Among the 7 patients with UICC stage III, 3 patients showed CK20 positive samples after enrichment of CTC with BerEP4 beads only. Positive CK20 signals were observed in 2 of 7 patients with UICC stage I after enrichment with KS1/4 coated beads only  after MNC gradient enrichment.To our knowledge, this is the first study reporting the use of mAb KS1/4 coated beads to isolate CTC from CRC patients. The sensitivity of the KS1/4 coated bead system was evaluated by HT29 tumor cell dilution experiments and reproducibly allowed the detection of about 10Our study also confirmed the study from Guo et al. which showed better results for MNC population extraction prior to immunomagnetic isolation of tumor cells than the immunomagnetic isolation of circulating tumor cells from whole blood . Guo et BerEP4 and KS1/4 are high affinity and specific mAb frequently used to detect EpCAM positive cells. Interestingly, we noted that CK20 RT-PCR products were commonly not found in both samples when analyzing blood of the same patient after enrichment with either mAb BerEP4 or mAb KS/4. Other authors have already described heterogeneity in reactivity of EpCAM specific antibodies. Balzar et al. suggested that different conformational states of the cell surface EpCAM protein might hide some epitopes leading to subpopulations of EpCAM and thus heterogeneous affinity .Successful CTC enrichment depends on the level of EpCAM expression in the target cell . FurtherThe dissimilar capture of CTC by BerEP4 and KS1/4 mAbs in our analysis might be explained by various expression of the EpCAM molecule among the examined patients. Nevertheless, our KS1/4 system was able to retrieve 10 fold more CTC compared to the BerEP4 system. The use of different EpCAM clones might enhance the detection rate of CTC. Additional investigations to assess the variations of EpCAM molecule expression among different patients will be of critical importance to identify a panel of suitable mAbs which could be used for efficient and reliable CTC isolation.Our results also demonstrate that Ficoll-gradient isolation is a decisive step prior to the immunomagnetic enrichment of CTC from peripheral blood.Our study for the first time shows that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further analysis regarding the clinical importance of heterogenous expression of the EpCAM molecule in CTC of CRC patients is urgently needed.\u00ae, Sarstedt, Germany). To avoid epithelial cell contamination from skin puncture, the first 5 ml of peripheral blood were discarded. After collection, blood samples were immediately processed for further experiments.For blood spiking experiments and for testing the specificity of the various extraction and enrichment protocols peripheral blood samples were drawn from the antecubital vein of 5 healthy donors and collected in EDTA-coated tubes (S-Monovetteth edition) [Five ml blood samples were obtained after induction of general anesthesia (and before start of the operation) through a central venous line from 38 CRC patients (UICC stage I-IV) undergoing surgical therapy at the Department of Surgery, University of Heidelberg, Germany. Patients with histopathologically confirmed CRC were staged according to the classification of the UICC (6edition) . The stu6, 105, 104, 103, 102, 10 and 0 HT29 cells per 5 ml whole blood.For cell spiking experiments and the examination of different cell extraction methods, EpCAM and CK20 positive cells from the human colon cancer cell line HT29 were serially diluted in 5 ml blood samples taken from five different healthy donors. Dilutions performed were: 102 atmosphere. Collection of cells was performed with help of Trypsin-EDTA  and centrifugation at room temperature for 3 minutes at 300 g. Cells were then counted with a hemacytometer and viability was confirmed by Trypan blue stain.The human colon cancer cell line HT29 expressing EpCAM and Cytokeratin 20 (CK20) was purchased from American Type Cell Culture (Austria Branch). Cells were cultured in RPMI-1640 Medium with L-glutamine and HEPES  supplemented with 100 U/ml penicillin, 100 \u03bcg/ml streptomycin , and 10% fetal bovine serum  in plastic flasks at 37\u00b0C in a 5%CO\u00ae, Linaris, Germany density 1.077) and covered with 10 ml phosphate buffered saline solution . The samples were spun in a centrifuge at 4\u00b0C for 30 minutes at 300 g without brake. Concentrated MNCs were harvested from the interface with the help of a disposable pipette. The isolated cells were washed once in PBS, spun in a centrifuge for 10 minutes at 300 g and resuspended in 1 ml PBS. The MNCs were counted with a hemacytometer and then resuspended at 107 cells/ml in PBS.The mononuclear cell (MNC) population was extracted according to the following protocol: 5 ml peripheral blood samples were carefully layered over a 15 ml Ficoll gradient (FicoLite-HThe unspiked and spiked (with HT29 cells) blood samples (5 ml blood for each experiment) of healthy volunteers were processed using two different cell extraction protocols followed by immunomagnetic enrichment using the device of Dynal MPC-L :7 beads per ml blood) using either Dynabeads Epithelial Enrich  coated with mAb BerEP4 or alternatively, Pan Mouse IgG beads  coated (1 \u03bcg antibody/107 beads) with the mAb KS1/4 . The samples were then placed in a roller at 4\u00b0C for rosetting to occur and after 30 minutes the tubes were placed in a magnetic device for 3 minutes; the blood supernatant was carefully removed and, while the tubes were still on the magnet, rosettes were washed three times with cold PBS.1. Whole blood samples underwent directly an immunomagnetic enrichment  was performed prior to further immunomagnetic enrichment. Immunomagnetic enrichment using the two different EpCAM antibodies was performed as described above.CK20 transcripts were detected after immunomagnetic enrichment of tumor cells either derived from blood samples spiked with HT29 cells or from blood samples drawn from CRC patients.For the detection of tumor cells a CK 20 nested RT-PCR was performed as previously shown ,13. In bFor the first PCR cDNA was subjected to amplification of CK20 with primers 1.for (ATGGATTTCAGTCGCAGA) and 558.rev (ATGTAGGGTTAGGTCATCAAAG) in 35 amplification rounds performed at 93\u00b0C for 51 seconds, 60\u00b0C for 63 seconds, and 72\u00b0C for 42 seconds, with a final extension step at 72\u00b0C for 10 minutes. The nested PCR was performed with 8 \u03bcl PCR product of the first PCR with primer 139.for (TCCAACTCCAGACACACGGTGAACTATG) and 429.rev (CAGGACACACCGAGCATTTT GCAG) under amplification conditions as following: 93\u00b0C for 51 seconds and 72\u00b0C for 81 seconds in 35 amplification rounds. PCR products were analyzed by electrophoresis on 2% agarose gels. RNA quality and performance of reverse transcription of the analyzed samples was confirmed by RT-PCR amplification of GAPDH transcripts.DA, LG, and AC performed the experiments and contributed to clinical data collection and manuscript preparation. NR, MWB, and JW contributed to the conduction of the study and were involved in critical review and revision of the manuscript. MK designed the study and was responsible for manuscript preparation and revision. All authors read and approved the final manuscript"}
+{"text": "To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5\u00b0C. Free chlorine concentrations typically used in drinking water treatment are sufficient to inactivate the virus by >3 orders of magnitude. Growing concerns about the public health threat posed by highly pathogenic avian influenza (HPAI) subtype H5N1 has prompted interest in evaluating environmental control measures for this virus. The World Health Organization has noted that more information is needed on the effectiveness of inactivation of subtype H5N1 in water  virus were used in this study /mL (oC under 5% CO2 for 96 h and examined by light microscopy for cytopathic effect (CPE). Culture plates were stained with 1% (w/v) crystal violet in 10% (v/v) neutral-buffered formalin for further examination. Failure to produce CPE indicated that the virus was not capable of infecting the cells. The neutralized buffer control without virus did not cause CPE. All experiments were conducted in duplicate under Biosafety Level 3 agricultural conditions.The infectivity of the samples was quantified by using microtiter endpoint titration  were used to determine the rate of inactivation for the 2 pH levels. Ct values are commonly used to make disinfection recommendations for water treatment and provide a means for comparing biocidal activity for various microorganisms  required to achieve 1, 2, and 3 orders of magnitude inactivation for both strains at the 2 pH levels. Covariance analysis of the decay coefficients indicated no significant difference in the inactivation of the 2 virus strains at pH 8.0 (p = 0.10). Rapid inactivation at pH 7.0 did not allow for statistical evaluation.The results of the chlorination experiments  represenCt values of 6 and 8 mg-min/L to achieve enteric virus inactivation of 3 and 4 orders of magnitude, respectively  in the water environment. The information on chlorine disinfection presented here should be helpful for developing risk management procedures regarding the role of water in the transmission of the virus to humans and poultry.The results of this study confirm that avian influenza (H5N1) is readily inactivated by chlorination. Although the viral inoculum exerted a considerable initial chlorine demand, the maintenance of a free chlorine residual (0.52\u20131.08 mg/L) was sufficient to inactivate the virus by >3 orders of magnitude within an exposure time of 1 minute. Chlorine demand would also be anticipated when the virus is associated with fecal material. These findings indicate that the ability to compensate for an initial chlorine demand followed by exposure to a relatively low level of free chlorine for a short time is sufficient to inactivate the virus by chlorination. For drinking water disinfection at conditions similar to those used in this study, the US Environmental Protection Agency specifies free chlorine"}
+{"text": "The purpose of this study was to evaluate the efficacy of HDR brachytherapy for primary or recurrent vaginal cancer.Between the years 2000 to 2006, 18 patients with primary or recurrent vaginal cancer were treated with brachytherapy (HDRB). Six patients had primary vaginal cancer (stage II to IVA) while 12 were treated for isolated vaginal recurrence . Five patients had previous pelvic radiation therapy. All except one patient received external beam radiation therapy to a median dose of 45 Gy (range 31.2\u201355.8 Gy). The HDRB was intracavitary using a vaginal cylinder in 5 patients and interstitial using a modified Syed-Nesblett template in 13 patients. The dose of interstitial brachytherapy was 18.75 Gy in 5 fractions delivered twice daily. The median follow-up was 18 months (range 6\u201366 months).Complete response (CR) was achieved in all but one patient (94%). Of these 17 patients achieving a CR, 1 had local recurrence and 3 had systemic recurrence at a median time of 6 months (range 6\u201322 months). The 2-year actuarial local control and cause-specific survival for the entire group were 88% and 82.5%, respectively. In subset analysis, the crude local control was 100% for primary vaginal cancer, 100% for the group with recurrence without any prior radiation and 67% for group with recurrence and prior radiation therapy. Two patients had late grade 3 or higher morbidity . Both these patients had prior radiation therapy.Our small series suggests that HDRB is efficacious for primary or recurrent vaginal cancer. Patients treated with primary disease and those with recurrent disease without prior irradiation have the greatest benefit from HDRB in this setting. The salvage rate for patients with prior radiation therapy is lower with a higher risk of significant complications. Additional patients and follow-up are ongoing to determine the long-term efficacy of this approach. Primary or recurrent vaginal carcinoma is an uncommon tumor . The iniBrachytherapy has been shown to be an important component of treatment in these patients. Treatment selection can be adapted to account for stage and location of the tumor. It can be done with either intracavitary or interstitial approach. The majority of published studies with interstitial brachytherapy have reported data using low-dose-rate brachytherapy (LDRB) -9. ThereThe purpose of this study was to evaluate the efficacy and toxicities of HDRB for primary or recurrent vaginal cancer.Between January 2000 and December 2006, 18 patients with primary or recurrent vaginal cancer were treated with HDRB. The median age was 69 yrs (range 43\u201388 yrs). Six patients had primary vaginal cancer . Twelve patients were treated for isolated vaginal recurrence . The stage and grade (G) of endometrial cancer were IB G1-1 patient, IB G2-2 patients, IC G3-2 patient, IIA G 3 \u2013 1 patient and IIB G2 \u2013 1 patient. The median time to recurrence for all patients was 12 months (range 3 months \u2013 18 years). Six of these patients had prior pelvic radiation therapy. The type of previous radiation was EBRT alone 1 patient, EBRT plus brachytherapy 3 patients and brachytherapy alone 2 patients. The median time since previous radiation to recurrence was 4 years (6 months \u2013 18 years). The recurrence was marginal (within 2 cm of previous field) in 2 patients and within the previous field in 4 patients. The sites of disease were proximal vagina 9 patients, distal vagina 6 patients and diffuse disease in 3 patients.All except one patient received a combination EBRT and HDRB. The median dose of EBRT was 45 Gy (range 31.2\u201355.8 Gy) at 1.8 to 2 Gy per fraction. The technique of EBRT was 3D conformal in 9 patients and IMRT in 8 patients. Five out of six patents with vaginal cancer also had concurrent weekly cisplatinum at 40 mg/m2 along with EBRT.The HDRB was intracavitary brachytherapy for superficially invasive tumors (less than 5 mm of invasion), while interstitial brachytherapy was used for more deeply invasive tumors greater than 5 mm. Five patients had intracavitary brachytherapy utilizing Delclos Vaginal Applicators system. Two of these patients had shielded vaginal applicators to protect the uninvolved vaginal mucosa. Orthogonal X-ray films were obtained for planning and verification of applicator placement. The median dose for intracavitary brachytherapy was 20 Gy 12 \u2013 20 Gy) in 3\u20135 fractions prescribed at 0.5 cm from the surface of the applicator. One of these patients had intracavitary brachytherapy only because of previous radiation therapy with EBRT plus HDR brachytherapy. Interstitial brachytherapy using a modified Syed-Nesblett template was performed in 13 patients. We have previously described this technique [2 \u2013 20 GyThe biologically equivalent dose (BED) in terms of equivalent doses given at 2 Gy per day (EQ2) was calculated using the LQ equation . The \u03b1/\u03b2The median follow-up was 18 months (range 6\u201366 months) for the entire group. Complete response (CR) was achieved in all but one patient (94%). This one patient with previous radiation therapy had recurrent papillary serous endometrial cancer and had partial regression of the tumor. Of the 17 patients achieving a CR, 1 had local recurrence and 3 had systemic recurrence at a median time of 6 months (range 6\u201322 months). The one local recurrence was in the patient treated with previous adjuvant radiation therapy for endometrioid adenocarcinoma. Two patients have died of disease at 12 and 18 months, respectively. Both patients had recurrent endometrial cancer with papillary serous and carcinosarcoma histology, respectively. Persistence of local disease was noted in one of these patients. The 2-year actuarial local control, cause-specific and overall survival for the entire group was 88%, 82.5% and 78%, respectively. In subset analysis, the crude local control was 6/6 (100%) for primary vaginal cancer, 6/6 (100%) for the group with recurrence without any prior radiation and 4/6 (67%) for group with recurrence and any prior radiation therapy.No grade 3 or worse early toxicity was observed. Two patients had late grade 3 or 4 morbidity. One patient had rectovaginal fistula 2 years after radiation therapy and other patient had chronic vaginal ulcer with significant narrowing and shortening of vagina . Both these patients had proximal vaginal disease and had prior radiation therapy and their EQ 2 for total doses were 142.98 Gy and 154 Gy, respectively. The cumulative doses were highest in these two patients. No other significant grade 3 or higher morbidity was noted.Radiotherapy is the main therapeutic modality in the management of primary or recurrent vaginal cancer. Brachytherapy remains integral part of definitive radiation therapy for these patients -9. The pet al. on 22 patients with recurrent endometrial cancer reported local tumor control rate and the 5-year disease-specific survival rate of 100% and 96%, respectively [et al. on 20 patients, 10-year local control rate and disease-free survival rates were 74% and 46%, respectively [In a review of the literature regarding salvage treatment options for vaginal recurrence, only two studies have examined the efficacy of HDRB for treatment of vaginal recurrences ,15. The ectively . Similarectively .Neblett template implantation procedure delivering 5 fractions twice a day for a total HDRB dose of 18.75 Gy over 48 \u2013 56 hours. Because of lack of published data, the American Brachytherapy Society (ABS) did not make any recommendation on HDRB interstitial brachytherapy dose and fractionation schedule for vaginal recurrences and preferred LDRB as the technique for interstitial brachytherapy [Interstitial brachytherapy has been traditionally performed using LDRB techniques in this setting. Our technique involved one Syed-ytherapy . Our treet al. first reported on 19 patients treated with the combination of intracavitary and interstitial brachytherapy [n = 26/86), whereas patients with locally advanced disease (stages II-IV) received HDR brachytherapy combined with external beam therapy (n = 55/86). The prescribed dose per fraction varied from 5 Gy to 8 Gy, with a mean dose of 7 Gy. In this large series only 8 patients had interstitial brachytherapy. The 5-year recurrence-free survival were 100%, 77%, 50%, 23%, and 0% for stage 0, I, II, III and IV respectively. Chronic grade 1\u20134 side effects were observed in \u2264 2%  and 1%\u20136% (vagina). Our series only had 6 patients of primary vaginal cancer and all had interstitial brachytherapy with 2-year local control of 100%. The toxicity profile was favorable with no grade 3 or higher toxicities.Similarly, there are only few published studies on HDRB for primary vaginal cancer. Kusher, ytherapy . The medytherapy . Early set al. noted an increasing risk of local recurrences in patients who received <55 Gy when compared with those receiving >55 Gy (53% vs. 17%) [et al. reported local failures in 25%, 33% and 62% of patients for the administered dose of >75 Gy, 60\u201375 Gy and <60 Gy respectively [In LDRB literature for vaginal cancer, an inverse relationship between the total dose and rates of local recurrences have been reported by several authors ,18. Chylvs. 17%) . Similarectively . With a Notwithstanding, our small series with preliminary results shows that HDRB is efficacious for primary or recurrent vaginal cancer. The fractionation schedule used was well tolerated with a low incidence of acute or later toxicities. Additional patients and follow-up are ongoing to determine the long-term efficacy of this approach. The limitation of our retrospective study is small size with limited follow up and heterogeneous patient population evaluated (inclusion of both primary and recurrent disease). The incidence of primary or recurrent vaginal cancer is low so that it is difficult for a single institution to have a large series. That was the rationale to combine heterogeneous disease together to see the efficacy and toxicities of this approach. As HDR equipment is widely available, there are more institution doing HDR interstitial brachytherapy. We may need to consider multi-institutional pooled analysis similar to the LDR experience  to see tSB \u2013 Took part in design and implementation of study, drafted manuscript, performed statistical analysis. DEH \u2013 Lead in drafting manuscript. RM \u2013 Helped in data collection. RPE \u2013 Participated in study design and patient selection. JLK \u2013 Participated in study design and patient selection. PS \u2013 Conceived the study, participated with design and coordinated/helped with patient selection. All authors read and approved the final manuscript."}
+{"text": "Sir,AKT1 in about 2% of endometrial cancer patients (PTEN and PIK3CA (PIK3CA (PTEN (The activating E17K mutations recently discovered in the pleckstrin homology domain of AKT2 (D399N) and the regulatory domain of AKT3 (E438D)) were previously reported in a sequencing screen of 123 genes in 41 primary endometrial cancers  in two additional samples. We validated these mutations as somatic by mass spectrometric genotyping of the tumour and matched normal DNA, after an independent PCR amplification. We also found a novel candidate mutation in the pleckstrin homology domain of AKT2 (D32H), which we could not validate because of insufficient DNA. All these mutations occurred in cancers of the endometrioid subtype that had no signs of metastasis either at primary treatment or during follow-up. Taken together, we find that 5 out of 41 endometrial cancers have mutations in AKT family members for a 12% rate.We found an additional four mutations in AKT family members . Two of PTEN, one of which also had amplification of and a mutation in PIK3CA (AKT1 E17K mutation is not associated with either PTEN or PIK3CA genomic alteration. It is therefore possible that these AKT family mutations have different functional effects from mutations of PTEN and PIK3CA. Given the importance of the PI3 kinase pathway in endometrial cancer oncogenesis (Confirmation that these novel mutations activate the PI3 kinase pathway awaits their functional characterisation. Notably, all the AKT family member mutations found in our data occur at residues conserved across multiple species see . Howevern PIK3CA ; the AKTogenesis , the fun"}
+{"text": "Dmrt1 suggested that SDGs evolve from downstream genes by gene duplication. Orthologous sequences of the major genes of the mammalian sex determination pathway have been reported in the rainbow trout but the map position for the majority of these genes has not been assigned.Rainbow trout have an XX/XY genetic mechanism of sex determination where males are the heterogametic sex. The homology of the sex-determining gene (SDG) in medaka to Amh, Dax1, Dmrt1 and Sox6) were tested for linkage to the Y chromosome of rainbow trout. We exclude the role of all these loci as candidates for the primary SDG in this species. Sox6i and Sox6ii, duplicated copies of Sox6, mapped to homeologous linkage groups 10 and 18 respectively. Genotyping fishes of the OSU \u00d7 Arlee mapping family for Sox6i and Sox6ii alleles indicated that Sox6i locus might be deleted in the Arlee lineage.Five loci of four candidate genes (Sox6 loci supports previously suggested homeology between linkage groups 10 and 18. Enrichment of the rainbow trout genomic map with known gene markers allows map comparisons with other salmonids. Mapping of candidate sex-determining loci is important for analyses of potential autosomal modifiers of sex-determination in rainbow trout.Additional candidate genes should be tested for their linkage to the Y chromosome. Mapping data of duplicated  Dmy, is a homologue of a downstream gene of the pathway in mammalian species  and a 649 bp promoter sequence of Dmrt1 [Genbank: FJ617281] identified a number of transcription factor binding sites or cis regulatory elements  and fugu (Takifugu rubripes). PCR amplification of the fourth intron of Sox6 from OSU and HC produced two products of differing size in each of the clonal lines. Following cloning and sequencing of all four products, OSU products were determined to be 984 and 1067 bp in size, while the sizes of the recovered products in HC were 984 and 1097 bp  indicated that Sox6 is duplicated in the rainbow trout.Boundaries of eight putative introns were identified in Sox6 allele in Arlee. Sequence comparison revealed a polymorphism between OSU and Arlee parental lines that resulted in the loss of an AvaII restriction site in Sox6ii of OSU .PCR amplification of the same intron from Arlee produced only one product 984 bp in size. Cloning and sequencing of the product did not show any sequence variants supporting the presence of a second SU Table . PCR-RFLSox6i to linkage group 18 (acrocentric chromosome 26) and Sox6ii to linkage group 10 (long arm of chromosome 6) supporting their homeology  OSU \u2013 Sox6i/OSU \u2013 Sox6ii (Genotype 1 in figure Sox6i/A \u2013 Sox6ii (Genotype 2) (3) OSU \u2013 Sox6i/A \u2013 Sox6ii (Genotype 3) (4) A \u2013 Sox6i/OSU \u2013 Sox6ii. This last genotype was not observed in the O \u00d7 A family (figure If we assume that Arlee (A) has two identical Genotype  (3) OSU Sox6 loci failed to amplify Sox6i allele in Arlee because of a mismatch in the primer sequence, or because of deletion of Sox6i in the Arlee lineage, Sox6i allele in Arlee can be genotyped as a null allele. Four genotype classes are expected in the O \u00d7 A progeny if Sox6i is genotyped as a null allele: (1) OSU \u2013 Sox6i/OSU \u2013 Sox6ii (Genotype 1 in figure null/A \u2013 Sox6ii (Genotype 2) (3) OSU \u2013 Sox6i/A \u2013 Sox6ii (Genotype 3) (4) A -null/OSU \u2013 Sox6ii (Genotype 4). Genotype analysis of the O \u00d7 A mapping family showed that 23 out of 73 doubled haploids were of genotype 1, 20 doubled haploids were of genotype 2, another 20 doubled haploids were of genotype 3 and only 9 were of genotype 4. Chi-square analysis did not show significant deviation of the observed proportions for the different genotype classes from the theoretical proportions expected under this model .If we assume that primer combination used to amplify different null/OSU \u2013 Sox6ii) genotype can also be explained by failure of the AvaII restriction assays. Genotyping was however confirmed by digestion with NlaIII which also produces a distinctive restriction profile for each of the four genotype classes of the mapping family and thus ruling out this possibility (data not shown).The  and three WT1 loci [We tested five loci for their linkage to the Y chromosome of rainbow trout in this study. In addition, three WT1 loci  have alsWT1 loci .Sox gene (Sry-related HMG box) family have been shown to be involved in the sex differentiation pathway or expressed in embryonic testes or ovaries [Dmrt1, orthologous sequences of Dmrt2 and Dmrt4 have been isolated from rainbow trout [Pax2a as a gene that is expressed early in gonadal development and displays a testis-specific expression profile. Our study identified potential binding sites of Pax2 in the promoter sequences of Dmrt1 and Dax1 suggesting that members of this gene family should be explored in future analyses. Although our analysis focused on candidate genes that already have sequences deposited in public databases, paralogous loci might still be isolated and would also be strong candidates for sex-determination based on the model presented for evolution of sex-determining systems [A number of candidate genes can still be tested for their linkage to the sex locus of rainbow trout. In mammalian species, ten out of the 30 genes discovered so far in the  ovaries ,25. Rain ovaries ,26. In aow trout . Studyinow trout  identifiIt is largely accepted that the sex-determining system in rainbow trout is genetic with male heterogamety. Nonetheless, Quillet et al  reportedSox6 locus. Inheritance pattern of Sox6 loci in O \u00d7 A mapping family indicated possible deletion of Sox6i locus in the Arlee lineage. Our mapping data supports previously suggested homeology between linkage groups 10 and 18 in rainbow trout, and adds four known genes to its genomic map. Enrichment of the rainbow trout genomic map with type I markers allows the identification of conserved syntenic blocks between rainbow trout and other salmonid species, which would eventually allow for understanding the molecular organization of genomes following genome duplication. Although we did not find the primary sex-determining gene in this study, our data is of importance for analyses of potential autosomal modifiers of sex-determination in rainbow trout.Using linkage analysis, we excluded the role of five candidate loci as the primary sex-determining gene in rainbow trout. We also identified a duplication of the Two mapping families and their respective linkage maps have been used for this study. Mapping families are doubled haploid fish generated from F1 hybrids between two clonal lines using androgenesis. Clonal lines were produced by androgenesis and gynogenesis ,30. EachSox6, Dmrt1 and Dax1.This family was produced from the F1 hybrid of the Oregon State University  and Hot Creek  clonal lines. The family was previously described in Zimmerman et al  where itSox6 and Amh. Synteny between the O \u00d7 H and O \u00d7 A linkage maps has been established [This family was produced by androgenesis from a cross between the OSU (XX) and Arlee  clonal lines, previously used to generate a dense linkage map and is described in Young et al  and Nichablished .Sox6, Dmrt1 and Amh through comparisons of their cDNA sequences from Genbank  or TIGR gene index  with genomic sequences of Takifugu rubripes and Danio rerio. Primer sequences are listed in Table In order to amplify genomic DNA fragments of non-coding regions of candidate genes, primers were designed to anneal within exons flanking tentative introns. Boundaries between exons and introns were predicted for 2, 10 pmoles of each primer, 0.25 mM dNTPs  and 1.5 U of Taq DNA Polymerase .All PCR reactions were performed using a Thermolyne Amplitron II thermocycler. PCR reaction conditions began with denaturation at 94\u00b0C for 2 minutes, followed by 30 cycles of 94\u00b0C for 45 seconds, 60\u00b0C for 45 seconds and 72\u00b0C for 45 seconds. The reaction ended with a final extension step of 72\u00b0C for 2 minutes. All reactions were 20 \u03bcl in volume containing: 50 ng DNA template, 1\u00d7 PCR buffer , 2.5 mM MgClFollowing the amplification of non-coding regions of candidate genes, PCR amplification products were resolved in a 2% agarose gel stained with ethidium bromide. Desired DNA fragments were gel-purified using Gene Clean , cloned into the pGEM-T Easy plasmid system  and then sequenced at the Washington State University Laboratory for Biotechnology and Bioanalysis. Target gene sequence verification was performed using the BLASTN and BLASTX algorithms of the NCBI website. Sequence polymorphisms between parental alleles were identified by aligning them using Sequencher 3.11 software . Because cloning can introduce artifact sequence differences, polymorphisms were confirmed by direct sequencing of genomic DNA amplification products using a nested primer (nested primer sequences are not shown).Dax1 and Dmrt1 from a \u03bb Zap II  genomic library created from an O \u00d7 H F1 hybrid. Following sequencing of the Dax1 and Dmrt1 lambda library clones by PCR walking, the structure of the Dax1 gene was deduced based on sequence alignment with the relevant sequences of Dicentrarchus labrax and Oreochromis niloticus. Intron and promoter sequences of Dmrt1 were identified based on alignment of the recovered genomic clone with rainbow trout Dmrt1 published cDNA sequence.A PCR-based approach  was used to isolate lambda clones of Sox6, Dmrt1 and Amh have sequence substitutions between alleles of the different parental lines that alter recognition sequences of restriction enzymes (RE). Utilizing these differences, doubled haploid fish of the mapping family were genotyped for the parental allele containing the polymorphic restriction site by PCR amplification, followed by RE digestion. Products of the RE-digest were size-fractionated in 1.5% agarose gels. Appropriate restriction enzyme digestions were done in 20 \u03bcl volume reactions according to the manufacturer's directions . Gene-specific primers utilized in RFLP-genotyping are listed in Table Non-coding sequences of the candidate genes Dax1. The polymorphism did not change any published recognition sequence of a RE. A Taqman assay  was used to score doubled haploids of the O \u00d7 H mapping family for having either OSU or HC alleles. Primers and probe sequences used in the Taqman assay are listed in Table A single nucleotide polymorphism (SNP) was detected between OSU and HC in the deduced promoter region of Sox6 fourth intron was found to have a different size among the different Sox6 alleles . Genotyping data of candidate genes was incorporated into the pre-constructed O \u00d7 H and O \u00d7 A linkage maps using Map Maker 3.0/EXP . Marker Dax1 and 0.6 kb region upstream of Dmrt1 coding regions were sequenced from the isolated lambda clones. The promoter sequences were analyzed for the presence of putative regulatory elements for transcription factors known to be involved in sex differentiation using MatInspector software .A 2.2 Kb region upstream of MA drafted the manuscript, assisted in designing the study, recovered genomic sequences and genotyped doubled haploid fish of reference families. JB assisted in genotyping analyses and provided technical assistance. RD conducted linkage analysis on the reference families. GT conceived the study, participated in its design and coordinated the roles of the authors. All authors read and approved the final manuscript."}
+{"text": "We identified avian influenza virus A (H5N1) infection in a child in Bangladesh in 2008 by routine influenza surveillance. The virus was of the same clade and phylogenetic subgroup as that circulating among poultry during the period. This case illustrates the value of routine surveillance for detection of novel influenza virus. Human infection with highly pathogenic avian influenza (HPAI) virus A (H5N1) has been associated with severe disease  and often with death  and the International Centre for Diarrhoeal Disease Research, Bangladesh  established nationwide hospital-based surveillance to identify clusters of persons with life-threatening influenza virus infections . His lungs sounded clear. The patient again visited the clinic on February 5 and February 10 and was afebrile on both occasions with a mildly elevated respiratory rate (38 breaths/min) and clear lungs. The mother reported that the child had had loose stools (possibly associated with amoxicillin) on February 10, but she denied that he had diarrhea. The child was never hospitalized; he completed a 14-day course of amoxicillin for suspected enteric fever and recovered fully. His blood culture was negative for any organism, and his final diagnosis was upper respiratory tract infection. On February 13, a convalescent-phase serum sample was collected as part of routine surveillance.Culture of the child\u2019s nasopharyngeal wash sample showed cytopathic effects consistent with influenza virus infection; an aliquot reacted to influenza virus A antiserum but not to antiserum to subtypes H1 or H3. An aliquot of the viral culture material was shipped frozen on April 22, 2008, to CDC, where the isolate was identified as HPAI (H5N1) virus by real-time reverse transcription\u2013PCR. The clinical specimen was recultured on embryonated chicken eggs. The complete genome was sequenced (GenBank accession nos. FJ573465\u2013FJ573472). The virus was identified as an H5N1 clade 2.2 strain on the basis of the hemagglutinin sequence . After iWe report HPAI (H5N1) in a child in Bangladesh; the infection was confirmed by virus isolation from an upper respiratory specimen and by serologic testing. The source of the child\u2019s subtype H5N1 infection is uncertain. One potential exposure was the healthy-appearing chicken that was brought inside the home. The child did not have direct contact with the chicken, although indirect contact was suggested because his mother handled him after slaughtering the chicken. The owner of the poultry shop where the chicken was purchased reported that 5%\u201310% of chickens had died each day during January 2008 (In Bangladesh and other countries with influenza (H5N1) outbreaks among poultry, surveillance for human subtype H5N1 cases is focused on hospital-based case finding for febrile patients with severe acute respiratory illness. This child was not suspected of having subtype H5N1 infection and had had no known poultry contact; his illness would not have met standard criteria for subtype H5N1 testing (The 1 in 5 sampling frame, a major limitation of this study, raises the possibility that undetected mild cases of subtype H5N1 infection have occurred in children in this population. Other limitations include the elapsed time between illness onset and investigation and the identification of only 1 case.The public health value of identifying the cause of severe acute respiratory illness clusters with pandemic potential is clear. This case highlights the value of routine outpatient surveillance for detecting both seasonal and novel influenza A viruses, particularly in settings in which subtype H5N1 strains circulate among poultry. Because exposure of subtype H5N1 to humans increases its opportunities for genetic mutation or reassortment, or both, with human influenza A viruses ("}
+{"text": "We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis.Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p  87, 432\u2013440. doi:10.1038/sj.bjc.6600490www.bjcancer.comCancer Research UK\u00a9 2002  Gallbladder carcinoma (GBC) is a relatively uncommon neoplasm which demonstrates considerable geographic and gender variation in incidence . For unkIt is now well recognised that tumourigenesis is a multistep process resulting from the accumulation of sequential genetic alterations . In addiOur recent genome-wide allelotyping analysis on GBC indicated that allelic losses at multiple sites of the genome are frequent in this neoplasm . Six (25%) were well differentiated, eleven (46%) were moderately differentiated, and seven (29%) were poorly differentiated tubulo-papillary adenocarcinomas. The majority of the tumours were advanced GBCs  with invasion of the gallbladder serosa; the remaining  were early GBCs, with invasion of the submucosa  or muscularis propia  of the gallbladder.Forty-five histologically discrete foci of non-invasive gallbladder epithelia were identified adjacent to 20 GBCs, each consisting of at least 1000\u2009cells. These included 17 histologically normal epithelia and 28 high-grade dysplasias , 8p (n=14), 9q (n=29) and 22q (n=12) regions showing high frequencies of LOH in the genome-wide allelotyping analysis on GBC  spanning a total of 12 chromosomal regions frequently deleted in invasive GBC , 8p n=1, 9q  to examine LOH at 12 chromosomal regions located on the four chromosomal arms examined. To determine whether the losses were progressive in the sequential development of GBC, we determined the frequency of loss of individual microsatellite markers (n=17) and chromosomal regions (n=12) using the Fractional Allele Loss (FAL) index and the Fractional Regional Loss (FRL) index, respectively. The FAL index is defined as the total number of microsatellite markers with LOH in an epithelial sample divided by the total informative markers in the corresponding normal DNA. The FRL index is defined as the total number of chromosomal regions with LOH divided by the total number of informative regions in the corresponding normal DNA.The data were analysed using a series of P<0.05 were regarded as statistically significant.Statistical analysis was performed using the nonparametric Wilcoxon and Fisher Exact tests. The cumulative binomial test was used to examine the likelihood that the occurrence of a particular event  occurs at a particular probability when observed in repeated trials. When the results are compared with a chance occurrence or nonoccurrence, the particular probability of comparison is 0.5. Probability values of Overall frequencies of allelic loss at any 3p , 8p , 9q , and 22q  sites were very high in GBCs. Most of the sites of allelic loss at all chromosomal arms examined were localised, and the extent of the partial allele losses was used to identify 13 discrete minimal regions of non-overlapping allele losses . The patterns of LOH and frequencies of allelic loss at different critical regions identified are shown in Table 2Although most of the losses at all chromosomal arms examined were localised, the data demonstrate that in chromosomes 3p, 8p and 9q the most frequently observed pattern was loss of two or more regions. Thus, allelic loss of a single region in each of those chromosomes by itself was a relatively infrequent event. Two or more regions were lost in 84% at 3p, 73% at 8p, 63% at 9q, and 52% at 22q. Cluster analysis to examine if allelic loss at one chromosomal region was linked to changes at another region  and dysplastic  foci examined . The FALThe pattern of allelic loss was not random, and losses at one or more 3p (54%) and 8p (50%) regions were the most frequently detected abnormalities in histologically normal epithelium . While 9Figure 6n=11) and dysplastic (n=19) epithelial specimens that were informative for at least one marker in each chromosomal arm examined. From these specimens only a single dysplastic focus showed no LOH at any chromosomal arm and was excluded from this analysis. Three patterns of allelic loss were discerned in the 30 histologically normal, dysplastic and neoplastic foci: (a) Early pattern, with only 8p loss or 8p accompanied with 3p. (b) Intermediate pattern, with only 9q loss or 9q accompanied with 3p and/or 8p. (c) Advanced pattern, with 22q loss accompanied with 8p+3p or 8p+9q or 8p+3p+9q. Of interest, normal gallbladder epithelium had only early (55%) and intermediate (45%) patterns, while tumours had advanced (71%) and intermediate (29%) patterns. Dysplastic gallbladder epithelia demonstrated the entire spectrum of patterns, including the advanced (56%), intermediate (30%) and early (14%).To determine the sequential molecular changes involved in the development of GBC, we analysed the pattern of allele losses detected in the GBC tumours and their accompanying normal and dysplastic epithelia. We considered only 14 tumours and 30 accompanying normal (P=5.3\u00d710\u221222).Previous studies in gallbladder carcinoma and other neoplasms demonstrated that at any one locus, loss of parental alleles was not random, and that there was a strong tendency for the identical allele to be lost in all non-neoplastic and neoplastic foci examined . We refeP=9.1\u00d710\u221226).Because in the GBC there is a close morphological relationship between invasive carcinoma and its dysplastic lesions  we then We detected a relatively high frequency of MSI at one or more 3p, 8p, 9q and 22q chromosomal loci in one of 13 (8%) of histologically normal foci, in four of 54 (7%) dysplastic foci and in six of 12 (50%) invasive carcinomas examined (data not shown).Our recent genome-wide allelotyping analysis indicated that allelic losses at multiple sites of the genome are frequent in GBC, and indicated for the first time that in addition to 3p and 8p, LOH on 9q and 22q may also play a role in the pathogenesis of this neoplasm , 8p (100%), 9q (88%), and 22q (92%) sites were very high in our tumours. Overall, thirteen distinct sites of frequent allele loss in GBC at chromosome 3p, 8p, 9q and 22q (summarised in RAR\u03b2) gene (FHIT) gene, spans the FRA3B fragile site at 3p14.2 (ROB01 (DUTT1), has been cloned residing in the U2020 3p12 deletion region at marker D3S1274  in our GBC cases. Recently, a new candidate TSG located in this region, the human RAS effector homologue (termed RASSF1A) gene, has been shown to have tumour suppressing function and undergoes epigenetic inactivation in several cancers  with frequent allelic loss in this tumour. Several candidate TSGs have been detected in those 3p regions with frequent allele losses in GBC. One candidate in the 3p22-24 region is the retinoic acid receptor-beta ( D3S1274 . One of (PRLTS) candidate TSG is centromeric to our 8p21 minimal region  gene, a candidate TSG at this region  occurring early during the sequential pathogenesis of GBC. The present findings of frequent chromosome 3p, 8p, 9q and 22q allele losses in non-malignant gallbladder epithelia confirm and greatly extend the findings that molecular changes commence early  during the sequential pathogenesis of GBC. Our major findings regarding the molecular pathogenesis of GBC are: (1) molecular changes preceded the onset of histologically recognisable changes and 88% of the normal histologically normal foci have allele loss at one or more chromosomal regions examined. (2) There was a progressive increase of the overall LOH frequency expressed by the FAL and FRL indices, with increasing severity of histopathological changes. The development of epithelial cancers requires multiple mutations , are present in many cancers, including GBC (In summary, allele losses of chromosome 3p, 8p, 9q and 22q regions are frequent in GBC. Our data identifies 13 distinct regions of loss on those chromosomal arms, many of which harbour one or more candidate TSGs, which may play a role in GBC pathogenesis. These regions have previously been reported to be frequently lost in several human cancers, suggesting that may harbour TSGs whose inactivation may be critical to the process of tumourigenesis. In addition, our findings in the non-malignant gallbladder epithelium indicate that multiple, non-random and sequential allele specific abnormalities commence early in the multistage pathogenesis of GBC. These findings should be useful for the identification of the TSGs involved in the pathogenesis of GBC, with the potential for defining molecular markers for early detection as well as for the development of gene therapy strategies of this highly malignant neoplasm."}
+{"text": "Gad1, Gabrb3 or Viaat genes leads to the development of non-neural developmental defects such as cleft palate. Studies of the Gabrb3 and Gad1 mutant mice have suggested that GABA function could be required either in the central nervous system or in the palate itself for normal palatogenesis.Previous studies have shown that disruption of GABA signaling in mice via mutations in the Gad1 or Viaat function is required in the fetal CNS for normal palate development. We used oral explant cultures to demonstrate that the Gad1 and Viaat mutant palates were able to undergo palatogenesis in culture, suggesting that there is no defect in the palate tissue itself in these mice. In a second series of experiments we found that the GABAA receptor agonist muscimol could rescue the cleft palate phenotype in Gad1 and Viaat mutant embryos. This suggested that normal multimeric GABAA receptors in the CNS were necessary for normal palatogenesis. In addition, we showed that CNS-specific inactivation of Gad1 was sufficient to disrupt palate development.To further examine the role of GABA signaling in palatogenesis we used three independent experimental approaches to test whether Gad1 and Viaat in the central nervous system for normal development of the palate. We suggest that the alterations in GABA signaling lead to non-neural defects such as cleft palate as a secondary effect due to alterations in or elimination of fetal movements.Our results are consistent with a role for  Gad1, Gabrb3 or Viaat (Slc32a1) has been an enigma Gad1), GABA vesicular transport (Viaat) and the postsynaptic response to GABA (Gabrb3). The most prominent phenotype noted by us and other investigators has been the surprising presence of a cleft palate in neonatal mice homozygous for null mutations in Gad1, Viaat and Gabrb3GAD1 or GABRB3 have been reported Gad1, Viaat and Gabrb3 mice might lead to new insights into the origin of human oral clefts, a common congenital disorder.The mechanism leading to non-neural developmental defects in mice homozygous for null alleles of the genes Gad1 and Gabrb3 mutant mice. These studies have drawn different conclusions depending on the gene examined and the experimental approach taken. In the case of Gabrb3, two studies support a role in non-neural cell types for normal palatogenesis. In one case, transgenic expression of Gabrb3 from a neuron-specific enolase promoter failed to complement the cleft plate phenotype of a Gabrb3 null mutation, suggesting that non-neural expression of Gabrb3 was required for normal palate formation Gabrb3 gene, again suggesting that Gabrb3 function was required in the palate or other craniofacial structures for normal palatogenesis Gad1, previous studies indicated that the cleft palate phenotype of Gad1-/- pups was due to a lack of fetal oral movements as well as inhibition of palate shelf elevation due to the abnormal position of the tongue between the shelves Gad1 phenotype was consistent with a requirement for GABA signaling in the CNS for normal fetal movement that in turn allows normal palate development. However, several papers have reported GABA, Gabrb3 or Gad1 expression in developing craniofacial structures such as the palate, oral epithelium, tooth placodes or condensations, tooth buds and palate medial edge epithelium Gad1 mRNA or protein as well as GABA in these structures is consistent with a functional role for GABA signaling in the palate. In addition, Gad1 gene expression has been detected in several epithelial placodes as well as in non-neural tissues that are developmental signaling centers, suggesting a role for GABA in developmental processes outside of the CNS Viaat, cleft palate and body wall phenotypes were noted in a previous analysis of a Viaat knockout mouse Viaat mutants.Several studies have been performed to determine whether GABA signaling is required in the CNS or in peripheral tissues for normal palate formation in Gad1 and Viaat mutant mice are due to loss of gene function within the CNS during development. We performed a pharmacological rescue experiment to show that the GABAA agonist muscimol can suppress the cleft palate phenotype in Gad1 and Viaat mutant embryos. The ability of muscimol to rescue the cleft palate phenotype of Gad1 and Viaat mutants suggests that multimeric GABAA receptors are downstream of Gad1 and Viaat in palate formation, a pathway that is most consistent with GABA signaling in the CNS. In addition, we used a serum free explant culture system to show that oral explants derived from laczGad1 -/- and lacZViaat -/- embryos were competent to undergo normal palate shelf elevation and fusion when removed from their normal oral context. This experiment provided additional evidence that Gad1 and Viaat function are not directly required for palate formation. A third experimental approach was to inactivate Gad1 specifically in neural precursor cells throughout the CNS. We took advantage of a Gad1 conditional allele to demonstrate that an early neural precursor specific knockout of Gad1 was sufficient to cause a cleft palate phenotype in mutant embryos. Our work strongly supports the idea that the cleft palate phenotype in Gad1 and Viaat knockout mice is due to a loss of GABA signaling in the CNS rather than in craniofacial tissues. We interpret our results in the context of other studies that have documented a role for fetal movement in the development of tissues and structures outside of the CNS. We suggest that the phenotypes seen in the Viaat and Gad1 knockout mice belong to a family of fetal defects in non-neural tissues that are caused by a loss of normal fetal movements due to defects in CNS or muscle function during development.In this study we used three independent experimental approaches to show that the non-neural defects in lacZViaat knockin/knockout allele was generated by gene targeting in ES cells. Genomic clones containing the mouse Viaat locus were isolated from a 129/SvEv lambda phage library. A 5.2-kb fragment immediately upstream of the translation start site was used as a 5\u2032 arm and a 2.8-kb fragment starting downstream of the start codon was used as a 3\u2032 arm. A \u03b2-galactosidase reporter/neomycin resistance cassette was placed into an NcoI site in the Viaat first exon. This NcoI site includes the start codon of the Viaat gene Viaat knockout mice used in this study had been crossed for 4\u201310 generations to the C57Bl/6J background. The lacZGad1 knockin-knockout mice have been described previously Gad1 gene was obtained from Dr. Richard Palmiter. This strain has been described previously All work with mice conformed to the stipulations of the University of Georgia Institutional Animal Care and Use Committee. The University of Georgia animal welfare assurance number is A3437-01 which expires on 11/30/2011. The For paraffin sections, embryos were fixed in Bouin's solution at 4\u00b0C from 3 hours to overnight depending on the embryonic stage and washed in phosphate buffer (pH 7.3) overnight. Then the embryos were then dehydrated in a graded series of ethanol, equilibrated with xylene, embedded in paraffin and sectioned at a thickness of 10 \u00b5m. Sections were stained with Mayer's hematoxylin (Sigma) and eosin solution after dewaxing with xylene, and mounted on slides.Viaat embryos. The embryos were fixed in 0.4% paraformaldehyde (PFA), 100 mM sodium phosphate pH 7.3, 2 mM MgCl2, 5 mM EGTA for 30\u201360 minutes and then rinsed in 100 mM sodium phosphate pH 7.3, 2 mM, MgCl2, 0.01% sodium deoxycholate, 0.02% igepal for at least 3 hours and stained in 100 mM sodium phosphate pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% igepal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal at 37\u00b0C. After staining, embryos were washed in PBS and postfixed in 4% PFA/PBS for 1 hour at room temperature.Whole mount lacZ histochemistry was performed on E9.5-E14.5 day old heterozygous in vitro mouse embryo culture technique 2/5% CO2. The culture medium was replaced after 24 hr. After culture for 1 or 2 days, explants were processed for paraffin embedding and H&E staining as described above.For the explant cultures we developed a new serum free culture system that combined a serum-free Gad1 or Viaat heterozygotes. To increase the likelihood that litters were within comparable ranges of gestational age matings were set up at 9pm and the mice were separated at 9am the next day. Muscimol injections were done within the same time window for each set of pregnant dams. This was done to improve the reproducibility of the results between different litters. In the Gad1-/- rescue experiment, muscimol was injected into the abdominal cavity of pregnant dams every 24 hours from E13.5 to E15.5. For the Viaat-/- rescue experiment, muscimol was injected every 12 hours from E13.5 to E16.0. We used 4 mg/kg of muscimol for each injection. This is a dosage that results in obvious sedation of the mice To generate homozygous offspring for the rescue experiments, we intercrossed RT-PCR analysis of mRNA expression was performed as previously described Viaat (578 bp)5\u2032-GTCGAGGGAGACATTCATTATCAG-3\u2032, 5\u2032-GTACACAGCAGACTGAACTTGGAC-3\u2032Gad1 (302 bp)5\u2032-CCTTCGCCTGCAACCTCCTCGAAC-3\u2032, 5\u2032-GCGCAGTTTGCTCCTCCCCGTTCTT-3\u2032GAPDH (310 bp)5\u2032-GTCTACATGTTCCAGTATGACTCCACTCAC-3\u2032, 5\u2032-CAATCTTGAGTGAGTTGTCATATTTCTCGT-3\u2032Gabrb3 (356 bp)5\u2032-GAAATGAATGAGGTTGCAGGCAGC-3\u2032, 5\u2032-CAGGCAGGGTAATATTTCACTCAG-3\u2032Ucp-1 (593 bp)5\u2032-TAGGTATAAAGGTGTCCTAGGGA-3\u2032, 5\u2032-CGCTTGGGTACTGTCCTGG-3\u2032Viaat and Gad1 for genetic studies and to facilitate the detection of Viaat or Gad1 expressing cells by \u03b2-galactosidase histochemistry. The lacZViaat and lacZGad1 mice add to the existing mouse resources that express lacZ or fluorescent proteins in GABAergic and/or glycinergic neurons .To define the spectrum of mutant phenotypes in the  breathe . All Vial region . HistololacZViaat -/- newborn phenotype to that of lacZGad1-/- newborns. Nearly all (98%) of the newborn lacZViaat homozygotes exhibited umbilical hernias  umbilical hernia phenotype that had not been previously reported. Histological sections of lacZViaat homozygotes revealed that this defect was indeed an umbilical hernia, not an ompahalocele as previously reported for the Viaat homozygous mice Gad1 and Viaat functions are not necessary for body wall formation per se but are instead required for the retraction of the umbilical hernia during development. In both genotypes, palate shelf elevation fails to occur at E14.5 and all Gad1 and Viaat mutants were born with cleft secondary palates . Ucp1 is a specific marker of brown fat lacZViaat homozygotes leads to the displacement of these fat deposits to this abnormal location.We also examined the unusual \u201cbumps\u201d on the dorsal cervical region of the -/- mice . These \u201cGabrb3 receptor subunit and Gad1 mRNAs are expressed in the developing palate and teeth as well as in a variety of other non-neuronal cell types Viaat function is required in the palate or craniofacial structures it should be expressed in these tissues. To determine whether Viaat mRNA is expressed in the developing palate, we examined the expression of Gabrb3, Gad1 and Viaat in mRNA from dissected palatal shelves from E13.5 and E14.5 day old mice. Using RT-PCR we easily detected Gad1 and Gabrb3 transcripts but could not detect Viaat mRNA in this tissue  that expresses Cre under the control of the Nestin regulatory sequences Gad1 used in our experiments has been described previously To perform a genetic test of the requirement for Gad1 we crossed lacZ/+Gad1; NesCre mice to flox/Gad1floxGad1 mice. To confirm that Gad1 had been inactivated specifically in the CNS of the flox/GadlacZGad ; NesCre/+ offspring from this cross we used RT-PCR to measure Gad1 mRNA levels in RNA isolated from dissected palatal shelves and brain from E14.5 embryos. The RT-PCR analysis showed that the amount of Gad1 mRNA in the palatal shelves of the CNS specific Gad1 knockout embryos  was equivalent to controls, while Gad1 mRNA levels in the CNS were greatly reduced as compared to the control embryos . Inactivation of Gabrb3 in mice led to the development of cleft palate in a proportion of the homozygous offspring Gabrb3 was driven by the neuron specific enolase (NSE) regulatory sequences in a genetic background that lacked Gabrb3Gabrb3 function is required in the palate or other non-neural tissues for normal palate development Gabrb3 temporal and spatial expression pattern in the transgenic embryos was provided. This is important since NSE transgenes can be subject to position effects resulting in very large differences in the spatial expression pattern of expression within the CNS between different transgenic lines Gabrb3 was being expressed early enough in the NSE-Gabrb3 transgenic lines tested in this previous study.In contrast, some previously published studies support a developmental function for GABA signaling outside of the CNS. Most of these results came from genetic studies of the gene encoding the \u03b23 subunit of the GABAGabrb3 gene Gabrb3 gene was inactivated specifically in neurons by crossing to a synapsin-Cre transgenic line Gabrb3 specifically in neurons resulted in normal palate development in the mice. Although the synapsin-Cre (SynCre) line is well characterized and has been used to generate neuron specific gene inactivation, the timing of expression may not be ideal for assessing Gabrb3 function in palate formation. The SynCre transgene is specifically expressed in postmitotic neurons and is not widely expressed in the CNS until E13.5 days floxGabrb3 mice the Cre may inactivate the conditional Gabrb3 allele too late in development to interfere with palate elevation and fusion which occur during E13.5-E14.5. This is a particular concern since the authors were relying on the inactivation of two floxGabrb3 alleles in the offspring. Unfortunately, the authors did not monitor Gabrb3 transcript levels in the CNS and palate of the SynCre floxGabrb3/floxGabrb3 mice or the extent of Cre mediated recombination of the Gabrb3flox allele in these experiments A second set of observations supporting a developmental function for GABA in non-neural tissues comes from studies of a neuron-specific knockout of the Fetal akinesia during human or rodent development has been shown to cause defective development of several non-neural tissues and structures in the fetus. For example, it has been shown that normal lung development requires spontaneous fetal breathing movements in humans and mice Gad1 and Viaat knockouts can be understood as additional examples of non-neural phenotypes caused by changes in fetal CNS function that in turn lead to changes in fetal muscle tone or fetal movements. Previous work has shown that fetal movements during palate formation are impaired in the Gad1 mutant mice Gad1 and Viaat homozygous mice are part of a larger spectrum of developmental defects and disorders that are caused by impaired or absent fetal movement. Our work suggests that disruptions of GABA signaling during development could interfere with any one of several developmental processes that depend on fetal movement.Within this context, the craniofacial and body wall phenotypes found in Gad1 or Gabrb3 alleles and human oral clefts Gad1 and Gabrb3 with oral clefts is due to the fundamental requirement for normal GABA neurotransmission for late fetal or neonatal viability. This would eliminate individuals who are homozygous for null or strong loss of function alleles of Gabrb3 or Gad1 from the populations sampled for these studies.In this context is intriguing that there are correlations between the timing of initial fetal movements in mice or humans and the initiation of palatogenesis. In mice, palate elevation and fusion occur at the same time as the first fetal movements are detected Gad1 and Viaat mutant mice are due to a requirement for GABA signaling in the CNS during mouse fetal development. The non-neural defects in these mice are most likely due to defects in fetal movement caused by CNS dysfunction and appear to be part of a spectrum of defects caused by abnormalities in fetal movements. Our results help to clarify the mechanism leading to cleft palate in the Gad1 and Viaat knockout mice and suggest that defects in fetal movement caused by alterations in fetal neuronal GABA signaling may lead to similar defects in humans.In conclusion, our work provides multiple lines of evidence that the non-neural developmental defects in"}
+{"text": "Nicotinamide and carbogen breathing are both effective radiosensitisers in experimental tumour models and are even more effective in combination. This study was to investigate the feasibility of using the agents in combination in patients and to measure their effect on tumour oxygenation. Twelve patients with advanced malignant disease were treated with 4-6 g of oral nicotinamide (NCT) in tablet formulation. Ten of these 12 patients breathed carbogen  for up to 20 min at presumed peak plasma NCT concentration (Cpeak) and had tumour oxygen partial pressure (pO2) measured using the Eppendorf pO2) histograph. The mean Cpeak values were 82, 115 and 150 micrograms ml-1 for NCT doses of 4, 5 and 6 g respectively and were dose dependent. The time of Cpeak was independent of dose with an overall mean of 2.4 h (range 0.7-4 h). NCT toxicity occurred in 9 out of 12 patients and was mild in all but one; carbogen was well tolerated in all patients. Following NCT only two patients had significant rises (P < 0.05) in tumour median pO2. During carbogen breathing, eight out of ten patients had early highly significant rises in pO2 (P < 0.0001), of which six continued to rise or remained in plateau until completion of gas breathing. Six patients had hypoxic pretreatment values less than 5 mmHg, which were completely abolished in three and reduced in two during carbogen breathing. In conclusion, the combination of NCT and carbogen breathing was generally well tolerated and gave rise to substantial rises in tumour pO2 which were maintained throughout gas breathing. These results should encourage further study of this potentially useful combination of agents as radiosensitisers in the clinic."}
+{"text": "In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate.Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin.Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway. TIR1 complex, polyubiquitinylated and degraded via the 26S proteasome abp1 mutant in Arabidopsis ABP1 (At4g02980) exhibit a fairly constant expression in almost all tissues throughout vegetative plant development suggesting that its role is not restricted to embryo development  or via cellular immunization to inactivate ABP1 protein through its in vivo interaction with the recombinant antibody scFv12. The latter recognizes a conformational epitope of ABP1 overlapping the auxin binding site (SS12S and SS12K lines) thus impairing the capacity of the protein to bind and respond to auxin in vivo  and SCARECROW (SCR) are required for QC identity and are also involved in root radial patterning during embryogenesis d plants . Expressd plants . Interes2 mutant . The expPLETHORA family, encoding AP2-domain transcription factors, are also essential for root stem cell maintenance and are related to auxin action. PLTs were recently revealed to control distinct aspects of root development in a dose-dependent manner PLTs was unchanged in roots inactivated for ABP1 but the overall area of expression was consistently reduced compared to controls , thus revealing an insensitivity to auxin of the cells towards the response of division. This data confirms that ABP1 exerts a strict control on cell division and it suggests that ABP1 is required for the capacity of meristematic cells to divide in response to auxin.\u22126M IAA indicating that cell elongation was not reduced . In the same conditions, Col0 plants exhibited 50% inhibition of growth corresponding to the inhibition of cell elongation. For IAA concentrations higher than 10\u22126M, a significant inhibition of root growth was observed despite the initial reduced length of ABP1 inactivated roots. This effect resulted from a reduced elongation of the cells as observed for the control at lower concentrations. Roots inactivated for ABP1 were less sensitive than control to primary root elongation inhibition caused by auxin, indicating that ABP1 is also required for this auxin response. Interestingly, it is not a complete insensitivity to auxin as observed for division but a shift in sensitivity suggesting that ABP1 is likely to contribute to this auxin response together with other regulators.Second, we examined the inhibitory effect of exogenous auxin on root elongation. As shown in Aux/IAA mRNA accumulation in response to auxin in short term ethanol induced control and SS12K lines. Auxin efficiently induced the expression of most Aux/IAA genes in root samples with minor changes in comparison to controls as illustrated for IAA3 (out of 14 genes tested) with the notable exception of IAA5, IAA6 and IAA19 . Roots of rRBr and rRBr,SS12K plants were fixed on acetic acid and methanol. Periodic acid-Schiff's reagent was used to reveal starch granules and tissues were stained with Propidium Iodide before observation. The pictures were shaped and assembled using Photoshop (Adobe) without treatment. Quantitative measurements were realised with ImageJ.The \u03b2-glucuronisade (GUS) assays were performed as described Immunolocalization was performed on 4 day old seedlings. Immunolocalization in roots was performed as described 13C6]-IAA  internal standard for 2 min at a frequency of 30 Hz, using a Retsch MM 301 vibration mill  and a 3 mm tungsten carbide bead. The samples were then incubated for 15 min at +4\u00b0C under continuous shaking. The pH was adjusted to 2.7 with 1 M HCl, and the samples were purified by solid phase extraction on a 500 mg Isolute C8 (EC) column , conditioned with 2 ml methanol and 2 ml 1% acetic acid. After sample application, the column was first washed with 2 ml 10% methanol in 1% acetic acid and then eluted with 2 ml 70% methanol in 1% acetic acid. The dried samples were dissolved in 0.2 ml 2-propanol and 1 ml dichloromethane and 5 \u00b5l 2 M trimethylsilyl-diazomethane in hexane (Aldrich) was added to methylate the samples. After methylation, the samples were trimethylsilylated and IAA was quantified by gas chromatography-selected reaction monitoring-mass spectrometry as described in Root tips were collected from 4 dpg seedlings exposed to ethanol for the indicated time and frozen immediately in liquid nitrogen. The frozen samples were homogenized in 0.5 ml 50 mM sodium-phosphate buffer pH 7.0 containing 0.02% diethyldithiocarbamic acid (Sigma) and 500 pg [RNA was extracted from roots of 4dpg seedlings treated with 5 \u00b5M NAA for 30 min to 6 hours using an Qiagen RNeasy kit and digested with RNAse free DNAse on the column following the manufacturer's instructions . First-strand cDNAs were synthesized from 5 \u00b5g of total RNA using Superscript II reverse transcriptase according to the manufacturer's instructions. Quantitative RT-PCR analyses were performed using SYBR Green QPCR master mix (Roche) with specific primers as reported Figure S1http://www.weigelworld.org/resources/microarray/AtGenExpress/AtGE_dev_samples.pdf/view).Expression pattern of ABP1 in Arabidopsis Expression data were extracted from AtGenExpress developmental series (1.06 MB PDF)Click here for additional data file."}
+{"text": "In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes . We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO3\u2212 transporters and NO3\u2212 transport in grass crop species.A large proportion of the nitrate (NO An important first step towards improving the NO3\u2212 uptake capacity and the NUE of crop plants would be characterisation of the transporters responsible for NO3\u2212 transport. Either the expression of the relevant genes or else the function of the proteins encoded by the genes could then be manipulated through traditional plant breeding or genetic engineering in order to improve NO3\u2212 uptake characteristics. With this goal in mind, the aim of this research was to identify the NO3\u2212 transporters in grass species.Nitrogen use efficiency (NUE) in plants is determined by the efficiency with which the plant acquires and uses nitrogen. Nitrate  family genes 3\u2212. The NRT1 family comprises predominantly low-affinity NO3\u2212 transporters, with the exception of AtNRT1.1 which appears to mediate dual-affinity NO3\u2212 transport 3\u2212 sensor AtNRT1.2 is constitutive and located predominantly in the root epidermis indicating that the encoded transporter may also be involved in NO3\u2212 uptake from the soil AtNRT1.3 in roots is repressed by exposure to NO3\u2212 and is induced by NO3\u2212 deprivation; its functional role, however, remains less clear AtNRT1.4 is expressed primarily in the leaf petiole and appears to be involved in NO3\u2212 storage 3\u2212 efflux and to have a role in the loading of NO3\u2212 into the xylem for transport to the shoot 3\u2212 from maternal tissue to developing embryos 3\u2212 from older to younger leaves through facilitating phloem loading 3\u2212 from the xylem parenchyma in the roots and shoots, thus working synergistically with AtNRT1.5 to control long-distance NO3\u2212 transport.The transport of NONRT2 family are high-affinity NO3\u2212 transporters comprising NO3\u2212 inducible and constitutively expressed members AtNRT2.1 and AtNRT2.2, which are located next to each other on chromosome 1 and appear to encode proteins with similar function 3\u2212 influx and is expressed in the root cortex and epidermis AtNRT2.1 and AtNRT2.2, however, are inducible by provision of NO3\u2212 to NO3\u2212 starved plants AtNRT2.3 other than its expression may increase and decrease in cycles over the life cycle in the roots and shoots (mostly shoot) AtNRT2.4 is expressed predominantly in the root, and expression appears to decrease following exposure of plants to NO3\u2212AtNRT2.5 is expressed in the root and shoot (mostly root) and is repressed by the provision of NO3\u2212AtNRT2.6 remains relatively unchanged in roots and shoots (mostly root) following exposure of plants to NO3\u22123\u2212 in seeds The NRT3 genes in Arabidopsis play a role in NO3\u2212 transport through regulating the activity of NRT2 genes, but are not themselves transporters NRT3 genes appear to be closely related, but NRT3.1 (NAR2.1) appears to play the more significant role in high-affinity NO3\u2212 uptake AtNRT3.2 gene is larger than originally published http://www.arabidopsis.org/).The NRT1, NRT2 and NRT3 gene families in the four fully sequenced grass genomes of rice Brachypodium3\u2212 transporters in grass species. The evolution from a common ancestor of the four species studied is such that they provide a good indication of the diversity of genomes within the grass species; maize and sorghum are the most closely related, having diverged an estimated 12 million years ago NRT genes is presented.Here, bioinformatic analyses are presented of the A commonly accepted, extensively used and well documented method for the determination of genes that share a common evolutionary ancestor  across two genomes is the reciprocal best hit (RBH) NRT1 genes from the PTR genes (the protein products of which transport peptides) are unknown, the analysis here was limited to determining putative grass orthologues of eight functionally characterised NRT1 genes  genes and the complexity of ancestral events, rendered clear orthologous relationships between dicots and monocots, unattainable.As the genetic sequence(s) or protein motif(s) that separate the T1 genes . ResultsNRT1 genes was less than 10\u2212139, with AtNRT1.1 \u2013 AtNRT1.5 and AtNRT1.8 showing greater sequence similarity to their grass counterparts  than AtNRT1.6 and AtNRT1.7 (\u224850% sequence identity across \u226580% of the query sequence). Furthermore, RBH analyses returned near identical hits to the grass genomes for AtNRT1.5 and AtNRT1.8 as well as for AtNRT1.6 and AtNRT1.7. Graphical representation of the dicot \u2013 monocot RBH analysis can be seen in NRT1 family is illustrated in RBH BLASTp E values in both forward and reverse direction for all eight NRT1 genes and their identified grass homologues . As the genes fall into three subclades, it is likely that gene duplication events gave rise to the three groups after the dicot-monocot split. For AtNRT1.2, AtNRT1.3 and AtNRT1.4 we see a much clearer picture, all cluster with a single clade of grass genes containing at least one member from the four grass species (rice and maize have an extra representative in the 1.3 and 1.4 clade respectively). Notably, poplar has no AtNRT1.4-like gene. AtNRT1.5 and AtNRT1.8 sit together with two and one poplar orthologues respectively and branch off from the main tree with two separate but complete clades of grass genes. Due to a slightly higher dicot \u2013 monocot RBH score for 1.5 over 1.8, the grass members of these two clades were named 1.5A and 1.5B. Upon close scrutiny of this branch, we found that the grass 1.5B clade had a nearer phylogenetic neighbour (a PTR gene \u2013 At5g19640) even though BLAST result scores were higher for 1.5 and 1.8. Since both clades sit equidistant from AtNRT1.5 (and neither more closely related to AtNRT1.8) an unambiguous assignment of orthology is not possible. Similarly, AtNRT1.6 and AtNRT1.7 sit together on the tree but unlike 1.5 and 1.8, 1.6 and 1.7 share one poplar orthologue and branch of the main tree with a single clade of grass genes missing both a maize and sorghum representative. Interestingly, the sorghum genome was found to contain a degraded pseudogene version that is related to NRT1.6 and NRT1.7, but this transcript is likely to be non-existent as no ESTs exist in any database.The phylogenetic tree of the eight Arabidopsis mologues  depicts Brachypodium - were initially included in the potential orthologue list after RBH analysis but rejected after refinement and validation. These sequences were not included as they were clearly seen to be nearest neighbours to three Arabidopsis PTR genes . Results of the best hits for AtNRT2.5 were marginally lower . Graphical representation of the dicot \u2013 monocot RBH analysis can be seen in NRT2 family is illustrated in The seven members of the 1 family . RBH anaNRT2 family painted a particularly interesting picture, exclusive of NRT2.5, the grass NRT2 genes sit entirely separate on the phylogenetic tree from the Arabidopsis NRT2 genes  cluster with the Arabidopsis sequences suggesting that the NRT2 genes developed primarily following the divergence of the monocots and dicots. Thus identification of a clear grass orthologues was only achieved for AtNRT2.5. A close investigation of the genomic localisation of these genes revealed clustering of the genes somewhat reminiscent of genes involved in disease resistance. AtNRT2.1 and AtNRT2.2 are neighbouring genes in opposing orientation, AtNRT2.3 and AtNRT2.4 are tandem repeats and AtNRT2.6  was located on a completely separate chromosome to the others. Correspondingly, of the twelve related sequences in grass , while Brachypodium has two sets of closely located NRT2 genes which are immediately adjacent to each other. Interestingly, the pair of NRT2 genes found in each of the grass species is generally separated by a non-NRT2 gene. These non-NRT2 genes do not share any sequence similarity with one another, nor to any other functionally characterised proteins in the databases (BLASTx search against NCBI). The pairs of NRT2 genes in the grass species may have similar function to AtNRT2.1 and AtNRT2.2, although confirmation of this would require proper functional analysis. Analysis of the gene structure of the NRT2 genes showed that the dicot NRT2 genes all contain both exons and introns, while none of the grass NRT2 genes contain introns  (AtNRT3.2), the proteins translated from the other two splice forms  are 311aa in length, the first 281 aa being identical with that of the longest splice form. Furthermore, there is evidence (NCBI Acc# DQ492237) for another splice product from this locus, which gives rise to a protein of 209 aa overlapping with the C-terminal half of the longest splice form. This protein sequence (referred to here as NRT3.2CT) was described and compared to NRT3.1 by Okamoto et al Genome analysis revealed the 4G24730)  and S5. NRT3.1 but all three of these translate into the same protein (http://arabidopsis.org/servlets/TairObject?type=gene&name=AT5G50200.3).When the protein sequence of At4G24730.1 was used in BLAST searches it became apparent that in the other species in this study the N- and C terminal halves of At4G24730.1 are coded for by two distinct genes. Consequently, it was decided that the protein sequences of NRT3.2SF2/3 and NRT3.2CT would be used separately for orthology searches. Notably, three splice forms have been predicted for Brachypodium  compared with NRT3.2 (\u224868%)  and sorghum (Sb07g024380.1), but not in rice. E values, sequence identity and alignment length for these hits were found to be just below those of the top hits . Although considered noteworthy, the maize and sorghum sequences were not used in any subsequent analysis. Similarly, AtNRT3.2 and AtNRT3.2SF2/3 produced the same RBH results, identifying a single NRT3 orthologue in maize, sorghum and rice and a tandem repeat in hypodium . BLAST ENRT3 loci in Arabidopsis lyrata and in a further four dicot species each with a fully sequenced genome. As can be seen from Arabidopsis species is the organisation such that a di-cistronic mRNA can give rise to a fusion product through alternative splicing. In Ricinus communis, Vitis vinifera and Poplar trichocarpa the NRT3.2SP2/3 gene and the NRT3.2CT gene are encoded on the opposite strand of the double helix, and no additional separate NRT3.1-like gene could be found. The exception is Medicago trunculata which appears to lack any NRT3.2CT (or NRT3.1) orthologues in the genome at all. This organisation, with the genes being encoded on opposite strands in direct genomic vicinity, is true also for Carica papaya, Cucumis sativus, and Manihot esculenta (data not shown). These results clearly indicate that the genomic organisation found in Arabidopsis may be the exception rather than the rule for dicots.An attempt was made to explain our finding that At4G24730.1 appeared to be a fusion of what seemed to be two distinct genes in other species. This was done by comparing the genomic organisation of the NRT genes. Several studies have considered the NRT genes in grasses, but have resulted in some confusion, not least in the nomenclature ascribed to the various NRT genes identified. Much of this confusion was due, presumably, to the unavailability of fully sequenced grass genomes leading, for example, to the cloning of orthologues using degenerate primers. For instance, Lin et al NRT gene in rice which was referred to as OsNRT1.1 by Tsay et al NRT1(PTR) family, but it is not a likely orthologue for AtNRT1.1. From the present analysis, and that of Tsay et al AtNRT1.1 than Os03g13274 as originally suggested by Lin et al AtNRT1.1; however this gene does not share as high a similarity with AtNRT1.1 as do the four genes identified in our analysis .The purpose of this study was to determine the cereal orthologues of the characterised Arabidopsis NRT family structure between Arabidopsis and the grasses indicate that Arabidopsis may not be the best model for interpreting NO3\u2212 transport in the grasses. The grasses have 3\u20134 closely related co-orthologues to AtNRT1.1. Recent work indicates that AtNRT1.1 (CHL1) may function as a nitrogen sensor Oryza sativa Build #80 (http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=4530) shows that OsNRT1.1A and OsNRT1.1B are expressed throughout the rice plant, however OsNRT1.1A is predominantly expressed in the root and is expressed more highly than OsNRT1.1B. This indicates that at least two genes potentially fill the same functional role of AtNRT1.1 in grasses. Conversely, the grass genomes lack certain NRT genes that have been characterised in Arabidopsis. Our analysis reveals that the Brachypodium and rice genomes contain only one protein similar to AtNRT1.7 and AtNRT1.6, or no orthologues in the case of maize and sorghum. AtNRT1.6 appears to be involved in transport of NO3\u2212 from the maternal tissue to the developing embryo 3\u2212 from the older leaves Oryza sativa Build #80 shows OsNRT1.7 is expressed in flower, seed and panicle, perhaps indicating that NRT1.7 in the grasses fills a similar functional role to AtNRT1.6. Whether the proposed long distance NO3\u2212 transport functions of the AtNRT1.5 OsNRT1.5A is expressed predominantly in root and panicle, but also in stem, leaf and flower. OsNRT1.5B was mostly expressed in panicle, but also flower and stem.The important differences which exist in NRT2 gene family. The presence of closely located NRT2 genes is reminiscent of the AtNRT2.1/AtNRT2.2 cluster in Arabidopsis and functional analysis of these genes may show similar function between the NRT2 gene clusters. However, with extra duplication in Brachypodium, for example, it will be interesting to identify the functional role played by each of the repeats. Since the NRT2 genes in the grasses lack an intron, it would appear that development of the NRT2 family occurred following the split between the dicots and monocots. It is possible that when they diverged from primitive dicots, the early monocots lost the NRT2 intron(s) whilst the dicots retained the intron(s). However, determination of the development of the NRT2 gene family in plants requires significant further analysis including the genomes of species all along the evolutionary tree, especially other monocots and will need to wait until more genomes are fully sequenced. The NRT2 family is part of the Major Facilitator Superfamily (MFS); of interest, therefore, would be an investigation of the other gene families in this superfamily to establish whether they show the same dichotomy in exon/intron structure. The grass genomes do not contain an AtNRT2.7 orthologue. Since the function of this gene is to load NO3\u2212 into the seeds of Arabidopsis 3\u2212 into seeds and embryos (similarly to the lack of AtNRT1.6 described above). Further analysis is required to determine whether the grass NRT2 genes have a similar function to that of the Arabidopsis NRT2 genes, or whether their evolutionary divergence also results in a divergence in function. Should there be a divergence in function the isolation of any common protein motifs or sequence differences that separate the dicot and grass NRT2s may provide important structure/function information of value in guiding biotechnological approaches to improving NO3\u2212 transport in plants. Although not included in our full analysis (since the genome has not been fully sequenced) the four barley (Hordeum vulgare) NRT2s Brachypodium NRT2 genes (BdNRT2.1/BdNRT2.2 and BdNRT2.3/BdNRT2.4)  ; consequAtNRT3.2SF2/3 gene with the AtNRT3.1CT gene in Arabidopsis compared with the way this protein interacts with the high affinity NRT2 transporters in grasses remains unknown. It will be interesting to determine whether the AtNRT3.2SF2/3 orthologues in the grasses are involved in NO3\u2212 transport. In barley (Hordeum vulgare), three NRT3 genes  have been identified AtNRT3.1 or AtNRT3.2CT genes . We have attempted to take this issue into account by naming all potential co-orthologues A, B, C, etc. However, in the case of the NRT2 gene family where dicot and grass genes do not cluster together in the phylogenetic tree, this approach becomes problematic. Therefore, we named family members NRT2.1, NRT2.2, etc, despite the fact that a grass NRT2.1 may not share a functional role with AtNRT2.1.To assist future research we have developed a nomenclature for grass RT genes . WithoutNRT gene families in Arabidopsis and the grasses has revealed some striking differences in gene family structure. Important questions about the evolution of NRT transporters in plants and, significantly, about the suitability of Arabidopsis as a model for NO3\u2212 transport in the grasses have also been posed. With the current exponential increase in the availability of molecular genetic resources for cereal crop plants it appears likely that the relevance of Arabidopsis research will decline. This analysis provides a framework for future studies of NO3\u2212 transporters and transport in the grasses, and potentially will guide strategies for improvement of NUE in cereal species through genetic manipulation of the NRT genes.In conclusion, the present analysis of the AtNRT family members . The complete database of predicted amino acid sequences from Arabidopsis thaliana and Populus trichocarpa as well as from four monocot species; Zea mays, Oryza sativa, Brachypodium distachyon and Sorghum bicolor were downloaded from public databases (DNA and amino acid sequences of 17  members . were reatabases .Brachypodium and sorghum). This was achieved by using BLASTp, standalone version 2.2.21 Identification of homologues was based primarily on sequence similarity between the 17 Arabidopsis NRTs and the predicted amino acid sequences of the four above mentioned monocots  http://tcoffee.vital-it.ch/). Trees were built using various programs of the Phylogenetic Interference Package (PHYLIP) 3.63 . One thousand bootstrap datasets were generated with SEQBOOT to estimate the confidence limits of nodes. Protein distance matrices were calculated with PROTDIST using the PMB model Geneious v4.8 . Information on genome organisation was obtained from the Phytozome (www.phytozome.org) and TAIR.The RBH obtained sequences for NRT1, were aligned by MAFFT version 6.240 using the L-INS-I method with associated default parameters at  poplar, (B) Brachypodium, (C) maize, (D) sorghum and (E) rice. BLASTp hits of equal e\u2010value in forward and reverse directions are depicted as forward facing and reverse facing arrows respectively. The colour code of all arrows represents the order of hits, as returned by the BLAST program, first best hit (red), second best hit (blue), third best hit (orange), fourth best hit (green), fifth best hit (black). Truncated arrows indicate a minor drop in the e\u2010value score between hits. Also coded are the gene accession numbers; an asterisk (*) indicates the presence of alternative splice forms all scoring identical BLASTp results, an upward facing arrow head (\u2227) indicates a tandem repeat with identical BLAST score, red accessions indicate neighbouring genes in opposing orientation with identical BLAST scores and blue accessions indicate genes in close proximity to each other with identical BLAST scores.(TIF)Click here for additional data file.Figure S2Phylogenetic relationship of potential grass PTR transporters orthologues to AtNRT1.6 and AtNRT1.7. Unrooted Neighbour\u2010joining tree of NRT1 transporters in Arabidopsis (black), poplar (blue) and 4 grass species: rice (orange), sorghum (green), maize (red) and Brachypodium (purple). Highlighted in the box are the three closest PTR homologues to AtNRT1.6 and AtNRT1.7  as well as orthologous grass PTR transporters . This figure provides rationale for exclusion of grass transporters (brown) as orthologues to AtNRT1.6 and AtNRT1.7. Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.2 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Figure S3Conservation of closely localisedNRT2genes. Gene identifiers and chromosome number are provided for NRT2 genes (red) for (A) Arabidopsis, (B) poplar, (C) rice, (D) Brachypodium, (E) maize and (F) sorghum. Sorghum has a third closely localised NRT2 gene (blue). Also depicted are non\u2010NRT2 genes (black) between NRT2 genes. Illustrations are not to scale.(TIF)Click here for additional data file.Figure S4Depiction of AtNRT3 genes. Schematic of the AtNRT3.2 locus (AT4G24730) from TAIR 9 GBrowse (http://gbrowse.arabidopsis.org/). Represented are chromosome, BAC, locus, gene models from TAIR 8 and TAIR 9 and cDNA details.(TIF)Click here for additional data file.Figure S5Alignment of AtNRT3 proteins. Proteins included are AtNRT3.1 and AtNRT3.2CT described previously Okamoto et al (TIF)Click here for additional data file.Figure S6Phylogenetic relationship of the NRT2 family including barley family members. Unrooted Neighbour\u2010joining tree of NRT2 transporters in Arabidopsis (black), poplar (blue) and 5 grass species: rice (orange), sorghum (green), maize (red), Brachypodium (purple) and barley (brown). The four barley members include HvNRT2.1 (HVU34198), HvNRT2.2 (HVU34290), HvNRT2.3 (AF091115) and HvNRT2.4 (AF091116). Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.08 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Figure S7Phylogenetic relationship of the NRT3.1 or NRT3.2CT family including barley family members. Unrooted Neighbour\u2010joining tree of NRT3.1 or 3.2CT family in Arabidopsis (black), poplar (blue) and 5 grass species: rice (orange), sorghum (green), maize (red), Brachypodium (purple) and barley (brown). The three barley members include HvNRT3.1A (HvNAR2.1 \u2010 AY253448), HvNRT3.1B (HvNAR2.2 \u2010 AY253449) and HvNRT3.1C (HvNAR2.3 \u2010 AY253450). Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.09 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Table S1Identifiers, annotations and family similarities for the ArabidopsisNRT genes described in this study. Provided are the TAIR annotation and identifier, UniprotKB identifier and RefSeq identifier for each NRT gene. Amino acid length of each protein is provided as well as the amino acid identity (%) between the NRTx.1 protein and the other family members.(XLS)Click here for additional data file.Table S2Comparison of theNRT2gene intron and exon numbers for dicots and grasses. Gene identifiers and exon and intron numbers are provided for the dicots Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, Ricinus communis and Vitis vinifera; and for the grasses Oryza sativa, Zea mays, Sorghum bicolor and Brachypodium distachyon.(XLS)Click here for additional data file.Table S3Web addresses for the genome databases searched during this study including Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Populus trichocarpa, Sorghum bicolor and Zea mays.(XLS)Click here for additional data file.Table S4List of candidate homologues excluded from analysis after manual inspection of multiple sequence alignments and their corresponding trees.(XLS)Click here for additional data file."}
+{"text": "P and PtdInsP2, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as \u2018endosomal PIs\u2019. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3P and PtdInsP2 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdInsP2 5-phosphatase SAC3/FIG4 are implicated in Charcot\u2013Marie\u2013Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3P5-kinase PIP5K3/PIKfyve have been found in patients affected with Fran\u00e7ois\u2013Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3 P and PtdInsP2 are involved in membrane trafficking P2. PtdIns3P was first discovered through the characterization of PI kinases in fibroblasts and by radioactive labeling of astrocytoma cells, while studies in osmotically stressed yeast strains and in resting mouse cells led to the detection of in vivo PtdInsP2 pools P2 metabolism unbalance on the phenotypic manifestations remains poorly understood, but one may hypothesize that the diseases share some common pathomechanisms involving abnormal trafficking. Phenotypes seen in patients may thus highlight new roles of PIs and implicated regulators, and conversely, the known roles of PIs and regulators may help to understand the pathophysiology of the related diseases.In eukaryotic cells, spatiotemporal regulation of cellular organization and cell\u2013cell communication require tightly regulated second messengers and membrane microdomains. Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking through recruitment of protein effectors to their site of action . Phosphand PtdIns,5P2 are P2 pools \u20137. TheirP by the class III PI 3-kinase . PtdIns3P is subsequently phosphorylated by the type III PI 5-kinase  into PtdInsP2, the reverse reaction being catalyzed by the phosphatase SAC3 (the ortholog of S. cerevisiae FIG4). PtdInsP2 can be dephosphorylated by the 3-phosphatase myotubularins (MTMs), leading to the production of PtdIns5P. Myotubularins also dephosphorylate PtdIns3P into PtdIns P2 through the action of class II PI 3-kinase, 4-phosphatase and 5-phosphatase acting on PtdInsP3 or class I PI 3-kinase acting on PtdIns5P, respectively  and can recruit proteins containing FYVE, PX or PH motifs (P (S. cerevisiae) produces PtdIns3P and binds Rab5  against PIK3C3   . A rare ne study ,28. ThisPIP5K3 gene have been found in cases of autosomal dominant Fran\u00e7ois\u2013Neetens corneal fleck dystrophy , which is characterized by abnormal swollen keratocytes with enlarged vesicles of unknown origin in the cornea  retrograde transport and PIP5K3 interacts with p40, a RAB9 effector implicated in retrograde traffic from the late endosome P2 levels increase upon hyperosmotic shock and lead to vacuolar fragmentation P2 effectors  are a heterogeneous group of genetic peripheral neuropathies affecting motor and sensory nerves and are characterized by progressive distal muscle weakness and atrophy. Nerve conduction velocity (NCV) tests are used to differentiate between the demyelinating forms of CMT  and the axonal form of CMT . Autosomal recessive forms of demyelinating CMT are collectively designated CMT4 . Nerve bSAC3 gene have been identified in four unrelated patients with autosomal recessive CMT neuropathy named CMT4J (OMIM 611228) (Compound heterozygous mutations of the human  611228) . These pSAC3 mutations has been allowed by the observation of phenotype similarities between CMT patients and the \u2018pale tremor mouse\u2019. This mouse contains an insertion of a transposon into an intron of the SAC3 gene resulting in a loss of the protein and presents neuronal degeneration in the central nervous system, peripheral neuropathy and diluted pigmentation. Cultured fibroblasts from pale tremor mice are filled with enlarged late endosomes/lysosomes and show a reduced level of PtdInsP2 compared with wild type in apparent discrepancy with the PtdInsP2 5-phosphatase function of SAC3 P2 levels P2 was found mutated in autosomal recessive demyelinating neuropathy CMT type 4B  . MTMR2 insP2\u201360. The proteins ,61.Mtmr2 knockout mouse models develop a mild CMT4B1-like neuropathy and azoospermia. Myelination of peripheral nerves is abnormal in the knockout mice, with myelin outfoldings and recurrent loops originating at paranodal regions , which has similar pathological features to CMT4B1, with additional glaucoma in some patients P2P2 and subsequent membrane trafficking defects.f CMT4B2 ,84. The nsP2. FurthernsP2. In the MTM1) gene have been identified in patients affected with X-linked centronuclear myopathy, also called myotubular myopathy  (MTM1 mutations have been reported in more than 300 unrelated families (Numerous mutations of the myotubularin ( 310400) . XLCNM ifamilies ,87. Theyfamilies . Almost families ,88.Mtm1 knockout mice recapitulate the histopathological signs of XLCNM and show a progressive myopathy starting a few weeks after birth, while muscle histology appears normal at birth  lead to reduced fluid-phase uptake into coelomocytes P2P2. A role nsP2,94. ThusMTM1 and MTMR2. Even if ubiquitous, MTM1 is more expressed in skeletal muscles  with similar enzymatic activity and which are implicated in diseases affecting two different tissues, skeletal muscles for MTM1 in XLCNM and Schwann cells in peripheral nerves for MTMR2 in CMT4B1, as emphasized by corresponding tissue-specific ablation experiments in both knockout mice. A first explanation could consist in differential regulation of expression between  muscles . MTM1 an muscles ,93. Conv muscles ,95. Sugg muscles ,93. SuchP and PtdInsP2 and their regulators all share a common function in membrane dynamics, with a possible implication in membrane fission and retrieval. Altered levels of PtdInsP2 and PtdIns3P, following a loss of the MTMR2/MTMR13 complex in patients affected with CMT4B1/B2, may cause a defective transport of membranes between a storage compartment and myelin sheaths. Membrane fission is essential for membrane retrieval, and fission defects may also be the underlying cause of CMT dominant intermediate type B (CMTDIB) because of missense mutations in dynamin 2 (DNM2), a large GTPase involved in the tubulation of membranes and in the release of newly formed vesicles  that targets membrane proteins for lysosomal degradation through a monoubiquitination mechanism  have recently been identified in patients with an autosomal recessive form of CNM P2 may also have roles independent of membrane traffic and may be found elsewhere than on endosomes. Questions remain about the mechanisms responsible for PtdIns3P- and PtdInsP2-dependent membrane remodeling. Tissue-specific interactors of these endosomal PIs are probably involved in the tubulation and scission of membranes and in the transport of the resulting vesicles to correct locations in cells. Such interactors are interesting candidates for centronuclear myopathies and CMT neuropathies for which several responsible genes still remain to be identified.A significant amount of work has recently highlighted a role for the endosomal PIs in membrane retrieval and their implication in human diseases. This leads to the emerging concept that the associated diseases may be caused by altered membrane remodeling and retrieval at plasma membrane and membrane storage compartments. Additionally, PtdIns3"}
+{"text": "Patients with high-grade astrocytomas have a poor prognosis. However, considerable variation exists within this group of patients with respect to post-operative survival. In order to determine whether genetic alterations might be of help in subdividing this group, we used allele loss on chromosomes 10 and 17p and epidermal growth factor receptor (EGFR) gene amplification in the tumours as genetic parameters and determined their prognostic value. A series of 47 malignant (grade III and grade IV) tumours were genetically characterised, and four types of tumours were found. Type 1 tumours had loss of heterozygosity on chromosome arm 17p (LOH 17p) as the sole genetic alteration. Patients with this type of tumour were relatively young (mean age 39 years) and had a median survival period of 17 months. Type 2 tumours displayed only allele loss on chromosome 10 (LOH 10), type 3 tumours had LOH 10 + LOH 17p and type 4 tumours contained LOH 10 + EGFR gene amplification. Patients with types 2, 3 and 4 tumours were older  and had a shorter survival  than type 1 patients. Multivariate analysis indicated that the genetic subdivision was a significant prognostic variable. In this study, age proved to be of minor importance with regard to survival. Our study revealed a predominance of frontally located tumours in patients with type 1 tumours, i.e. with LOH 17p only."}
+{"text": "We have screened 33 ovarian tumours of various grades and stages for the loss of heterozygosity (LOH) of markers on chromosome 9. LOH was detected in 26 cases (79%). Eleven tumours (33%) showed LOH of all informative markers. The remaining 15 cases had partial deletions. Of these, six (18%) had losses on 9p only, three (9%) had LOH confined to 9q and six (18%) had losses on both chromosome arms, four of which had a retention of hetereozygosity in between. There was no association between tumour grade stage or histopathology and any losses. High-density deletion mapping was carried out in 12 selected cases that had partial deletions of 9p and/or 9q. The deleted region on 9p included the cyclin-dependent kinase inhibitor 2 (CDKN2) locus and one tumour was found to have a homozygous deletion of CDKN2. LOH on 9q extended over a larger region. We found evidence for two regions of deletion on 9q, one at 9q34 and the other encompassing the nevoid basal cell carcinoma (Gorlin) syndrome locus on proximal 9q."}
+{"text": "Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5\u2032 flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5\u2032 upstream region of transcribed var and during the poised state , \u2018bookmarking\u2019 this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5\u2032 flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5\u2032 flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.In the human malaria parasite  Plasmodium falciparum during the proliferation phase in red blood cells of its human host depends on the successive expression of variant molecules on the surface of the infected erythrocytes. This variation is mediated by the differential expression of a polymorphic parasite protein, P. falciparum erythrocyte membrane protein 1, which is encoded by \u223c60 var genes  and upsD types (var1csa) , non-transcribed or poised state (schizont stage) and silent states of the same var gene in its natural chromosomal context  to be active or stably repressed  along a region of 10 kb comprising exon 1 (8 kb) and 2 kb upstream sequences  and 5\u2032-rapid amplification cDNA end (5\u2032-RACE). For this purpose a first strand cDNA was synthesized from total RNA obtained from chondroitin sulphate A (CSA) and CD36 parasite population using a gene-specific primer (D reverse primer) (var2.4as followed by nested PCR using the gene-specific primer var2.3as and the first PCR as template generated a product around 200 bp (var2CSA gene in parasites in which the gene is either repressed or active.To map the histone modification patterns at the equences . Examina primer) . Then a d 200 bp . The PCRd 200 bp . Our resquantitative Chromatin Immunoprecipitation (qChIP) analyses on var2csa loci in CSA parasites in which the var2csa is being transcribed (\u2018ON\u2019 state termed here var2csaON) or transiently repressed (\u2018POISED\u2019 state termed here var2csaPOISED) and in CD36 parasites in which the var2csa is stably repressed (\u2018OFF\u2019 state termed here var2csaOFF)  are associated with high levels of methyl marks in H3K9  (We investigated the role of H3K9 trimethylation in the control of antigenic variation. To this end we carried out 2csaOFF) . All expng stage . On the od stage . This is in H3K9   called PfGam here. The analysed position for silent PfGam was not associated with significant levels of H3K9 trimethyl marks.An asexual blood stage gene, such as his mark . We alsovar gene transcriptional control. ChIP analyses with antibodies against trimethylated H3K4 show that there is a prominent localized enrichment of trimethyl H3K4 at the 5\u2032 flanking region of the var2csaON  that is highly transcribed in asexual blood stage parasites  and compared it to parasite chromatin preparations where the var2csa is stably repressed (var2csaOFF). These results show that levels of tri- and in particular dimethylation of H3K4 are higher for var2csaPOISED compared with var2csaOFF  need to be recruited to this site in order to co-ordinate expression of var genes.A previous study demonstrated that P. falciparum. We have initiated experiments to elucidate their role in var gene silencing.Specific histone methyl mark binding chromodomain proteins, such as HP1, may translate this information into stably repressed chromatin . An active var promoter state was artificially mimicked by selecting blasticidin-resistant parasites. As the analysis of the 5\u2032 upstream region is compromised by the sequence homology with numerous var genes, the authors analysed a single region in the BSD gene or silent var genes. Based on their results, they concluded that H3K9me3, but not H3K9me2 or monomethylation, correlates with repression of var genes. However, our data clearly show that most changes in the histone methyl and acetyl marks occur at the 5\u2032 flanking region of var genes, indicating that investigating coding regions gives only a limited insight into critical DNA elements involved in epigenetic regulation of antigenic variation. With regard to H3K9me3, the study of Chookajorn et al. supports our finding, because this mark spreads from the 5\u2032 flanking region into the coding region of var genes.A recent study analysed histone marks associated with silent var gene state is maintained during many cell generations. We hypothesize that cellular memory may be acquired by inheriting a var locus enriched with H3K4me at the 5\u2032 flanking region. Active transcription restarts in the same \u2018marked\u2019var gene, possibly by raising histone trimethyl marks again to levels needed for transcription. Dynamic changes of H3 lysine 4 methylation marks in the 5\u2032 flanking region have been observed in higher eukaryotes and yeast and correlated with transcriptional activation or permissive state of genes (The question arises how an active trypanosomes the mutually exclusive transcription of a vsg gene is mediated by a particular subnuclear compartment called the \u2018Expression Site Body\u2019 (Our findings may be relevant for a number of other protozoan pathogens that use antigenic variation and in the mammalian neuronal cells that express a single member of the large odorant receptor gene family . For exate Body\u2019 . In mousP. falciparum generates reiterated var gene expression patterns through multiple blood stage cycles. Our data show that H3K4 lysine and H3K9 lysine methylation/acetylation marks at the flanking region of var genes are involved in setting up distinct var gene states and this in a strictly mutually exclusive manner. Future research will focus on the molecular machinery that can read these marks at particular DNA regions of the 5\u2032 upstream region and translate this information into co-ordinated gene expression pattern of var genes.In conclusion, our findings have important implications for the processes that promote immune escape and pathogenesis in human malaria parasite. The presented data overcome a conceptual gap in our understanding of antigenic variation, namely, how P. falciparum blood stage parasites from FCR3 strain and panning assays for selection of FCR3 parasites that transcribed var genes associated with CD36 and CSA binding were performed according to Parasite histones were purified by modification of a previously described procedure . Precipivar2csa at ring stage and between poised and silent var2csa at schizont stage.The chromatin immunoprecipitation assay was performed as described previously . We did"}
+{"text": "A Cox multivariate model for survival duration identified age , P = 0.004), grade  and any loss of chromosome 10  as the only independent prognostic variables. Specifically, LOH for chromosome 10 was able to identify a subgroup of patients with grade 3 tumours who had a significantly shorter survival time. We conclude that LOH for chromosome 10 is an independent, adverse prognostic variable in high-grade glioma. \u00a9 1999 Cancer Research CampaignLoss of heterozygosity (LOH) for chromosome 10 is the most frequent genetic abnormality observed in high-grade gliomas. We have used fluorescent microsatellite markers to examine a series of 83 patients, 34 with anaplastic astrocytoma (grade 3) and 49 with glioblastoma multiforme (grade 4), for LOH of chromosome 10. Genotype analysis revealed LOH for all informative chromosome 10 markers in 12 (35%) of patients with grade 3 and 29 (59%) grade 4 tumours respectively, while partial LOH was found in a further eight (24%) grade 3 and ten (20%) grade 4 tumours. Partial LOH, was confined to the long arm (10q) in six and the short arm (10p) in three cases, while alleles from both arms were lost in four cases. Five tumours (one grade 3 and four grade 4) showed heterogeneity with respect to loss at different loci. There was a correlation between any chromosome 10 loss and poorer performance status at presentation (\u03c7"}
+{"text": "Growth factor receptor bound (Grb) proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF) stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK). Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported.Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106) on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP) Ang1.Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor. Growth factor Receptor Bound (Grb) adaptor proteins are a group of structurally similar proteins beginning to emerge as key players in a number of cellular functions including cell metabolism, cell survival and cell migration.Signal transduction pathways encompass a series of highly coordinated events involving numerous proteins, varying in both structure and function. Adaptor proteins play a pivotal role in such molecular networks by allowing formation of protein complexes via interactions involving their non-catalytic binding domains. The Between PH and SH2) domain. To date the BPS domain has been shown to play a role in certain Grb/receptor interactions including those involving the activated Insulin receptor (IR) and Insulin-like Growth Factor Receptor ATP (Amersham Biosciences), 20 \u03bcM ATP (Amersham Biosciences) for 30 min at 30\u00b0C [\u0394PH phosphorylation over the amount of immunoprecipitated Tie2 in each of the samples. All experiments were performed twice or more times with similar results.Kinase assays were performed as follows: Cell lysates were immunoprecipitated with anti-Tie2 antibodies for 2 h at 4\u00b0C. Immunoprecipitates were washed 3x in PLC lysis buffer (without sodium fluoride or sodium pyrophosphate)  and 2x i at 30\u00b0C . Kinase The authors declare that they have no competing interests.CS performed all the experiments and wrote the manuscript. DD revised the manuscript and provided final approval for publication. All authors have read and approved the final manuscript."}
+{"text": "Mutations of the tumour suppressor p53 gene are found in a number of spontaneous canine cancers and may contribute to increased cytogenetic alterations and tumour formation. Using reverse transcription and DNA amplification, we isolated p53 cDNA from normal and tumour tissue of ten canine mammary cancer patients. DNA sequencing identified p53 mutations in three of the ten patients. These included tumour-associated p53 gene mutations within exons 2 and 5 and a germ line deletion of exons 3 to 7. These results support a role for p53 inactivation in canine mammary tumour formation and breed predisposition to cancer. Such information could prove invaluable in the successful outbreeding of inherited predisposition to cancer in the dog. A putative polymorphism was also identified at codon 69 in exon 4 and we discuss the possibility that similar polymorphisms may be associated with human breast cancer. \u00a9 1999 Cancer Research Campaign"}
+{"text": "SMN1 exon 7 are reported to account for 94% of affected individuals. Published literature places the carrier frequency for SMN1 mutations between 1 in 25 and 1 in 50 in the general population. Although SMA is considered to be a pan-ethnic disease, carrier frequencies for many ethnicities, including most ethnic groups in North America, are unknown.Spinal muscular atrophy (SMA) is the most common inherited lethal disease of children. Various genetic deletions involving the bi-allelic loss of SMN1 mutation carrier frequencies in African American, Ashkenazi Jewish, Asian, Caucasian, and Hispanic populations, more than 1000 specimens in each ethnic group were tested using a clinically validated, quantitative real-time polymerase chain reaction (PCR) assay that measures exon 7 copy number.To provide an accurate assessment of SMN1 was identified in the African American group (27% compared to 3.3\u20138.1%). This latter finding has clinical implications for providing accurate adjusted genetic risk assessments to the African American population.The observed one-copy genotype frequency was 1 in 37 (2.7%) in Caucasian, 1 in 46 (2.2%) in Ashkenazi Jew, 1 in 56 (1.8%) in Asian, 1 in 91 (1.1%) in African American, and 1 in 125 (0.8%) in Hispanic specimens. Additionally, an unusually high frequency of alleles with multiple copies of Differences in the frequency of SMA carriers were significant among several ethnic groups. This study provides an accurate assessment of allele frequencies and estimates of adjusted genetic risk that were previously unavailable to clinicians and patients considering testing. SMN1 mutations were obtained for five North American ethnic groups by evaluating more than 5000 specimens.Precise allele frequencies for SMN1 mutation frequencies were found between several populations.Significant differences in SMN1 copies. This is considerably higher than other populations and results in a SMA carrier adjusted risk estimate that is five times greater than that of the Caucasian population.Approximately 27% of alleles in the African American specimens had two or more This study provides accurate estimates of allele frequency and adjusted genetic risk for five major ethnic groups in North America.With an incidence of 1 in 6000 to 1 in 10\u2009000 live births, spinal muscular atrophy (SMA) is the most common lethal genetic disease of children.SMN1) gene.SMN1 gene resides in a duplicated region of chromosome 5q13 telomeric to the near identical homologue SMN2. More than 90% of the functional SMN protein is contributed by SMN1. Comparably SMN2 produces only a small fraction of SMN due to a point mutation in exon 7 that disrupts an exon splice enhancer site, preventing normal post-transcription processing.SMN1 and SMN2 genes varies. Published information suggests that 85\u201395% of the population has one copy of the SMN1 gene per allele; however, individuals with more than one copy per allele have been identified.SMN1 exon 7. Of the remaining 6% of SMA cases, most are attributed to a variety of rare molecular lesions distributed throughout the gene, including point mutations, small insertions, and deletions. A minority of cases may involve genes other than SMN1.SMN2 copy number and disease severity, such that affected individuals with three or more copies of SMN2 typically have milder forms of the disease.SMN1 exon 7 copy number by quantitative polymerase chain reaction (PCR).et al reported a frequency for one-copy carriers at approximately 1 in 40 individuals in the general population.SMN1 copies paired with a zero-copy allele (hereafter referred to as \u201c2+0\u201d genotype). By exon 7 quantification these individuals are indistinguishable from wild-type (or \u201c1+1\u201d genotype). It has been estimated that approximately 1 in 600 apparent two-copy specimens may be \u201c2+0\u201d carriers.16SMA is caused by mutations in the Survival Motor Neuron 1  recommend universal carrier screening for SMA.20SMN1 copy number. These ethnicities are relevant since they comprise >95% of the North American population at large and the North American patients electing to participate in genetic testing (2006 US Census estimates).To address this need for accurate allele frequency data, more than 1000 samples from five major ethnic groups, African American, Ashkenazi Jewish, Asian, Caucasian and Hispanic, were tested for SMN1 exon 7 copy number employed a clinically validated, real-time, quantitative PCR assay specific for the single nucleotide change in exon 7 (cd 840 c>t). All reported results could be assigned to validated, non-overlapping genotype groups of 1, 2 or 3 SMN1 copies (the three copies group includes samples with three or more SMN1 gene copies).Samples were collected from residual material following routine clinical testing of individuals presumed to have no family history of SMA. All specimens were made completely anonymous before testing in accordance with approved institutional protocols. Ethnic assignment relied upon patient reported data that were not collected as part of complete family histories. However, these ethnic assignments, which reflect clinical practice, are therefore highly representative of the anticipated clinical experience for SMA carrier screening. The assessment of SMN1 genotypes were observed among several ethnicities , suggesting a much higher frequency of alleles with two or more SMN1 copies than the other four ethnic groups. These unexpected results in this group were confirmed by retesting 10% of the samples using multiplexed ligation dependant probe amplification  as an alternate technique. All MLPA results were concordant with the original real-time PCR data.Significant differences in the frequencies of nicities . The higSMN1 in the African American group is 3.4 to 8.4 times more prevalent when compared to the other ethnic groups. The preponderance of the two-copy allele in the African American group also suggests a much higher frequency of individuals with the SMA carrier \u201c2+0\u201d genotype compared to other ancestries.Using the observed genotype data and assuming Hardy\u2013Weinberg equilibrium, maximum likelihood estimation was employed to determine frequencies for alleles and allele pairings within all sample groups  and 3. TSMN1 copies. These calculations account for rare mutations undetectable by the method described here, and model the \u201c2+0\u201d carrier genotype. Adjusted carrier risk was calculated for each ethnic group based on its unique allele frequencies .Adjusted carrier risk assessment estimates have been previously published based on aggregate results of several studies.quencies . The resSMN1 copy number in the African American population is unknown. Previously published data have shown that individuals with higher copy numbers of SMN1 tend to have fewer copies of SMN2.SMN2 may have converted to SMN1; however, the inverse may also be true. Alleles with multiple copies of SMN1 may be the ancestral form of the duplication, with conversion to SMN2 a more recent mutation event. An assessment of SMN2 copy number is currently underway to understand the SMN1/SMN2 correlation in this sample set.The genetic basis for the unusually high SMN2 copy number, as well as other genes such as plastin 3.Prediction of disease phenotype in SMA is complicated by the modifying effects of SMN1 mutations and for African American patients who bear increased frequencies of two-copy alleles. These data will facilitate the accurate interpretation of clinical testing results and provide additional information for genetic counselling.The recent ACMG guideline for SMA carrier screening"}
+{"text": "Immunodeprived mice survived a high, otherwise lethal dose of cyclophosphamide (Cy) provided they had been \"primed\" with a low dose (50 mg kg-1) of the drug 4 days earlier. These combinations were then tested on 2 oat cell xenograft lines (which are known to reproduce the chemotherapeutic responses of the parent tumours) grown in immunodeprived mice. In the treatment of the first oat cell xenograft, 200 mg kg-1 Cy produced a growth delay of 34 days in the unprimed group and 45 days in the primed group. At a dose of 300 mg kg-1 a growth delay could not be assessed in the control group as 16/17 of these unprimed mice bearing this xenograft died. However, 14/22 tumours went into complete remission in this group before death occurred. In contrast only 3/16 deaths occurred in the group of mice that were primed before receiving the same challenge dose. In these animals 19/26 tumours went into complete remission and were still completely absent when the experiment was terminated at 60 days. Using the second oat cell xenograft, 300 mg kg-1 Cy produced a growth delay of 27 days. However, at this dose level all the animals were dead by day 46. In mice which had been primed with 50 mg kg-1 Cy 4 days before the administration of 300 mg kg-1 a growth delay of 32 days was achieved and 2/9 animals were alive at day 60. This study shows that priming allows larger doses of Cy to be given to immunodeprived mice bearing human tumour xenografts than would normally be tolerated and that the priming does not alter the anti-tumour efficacy of the large challenge dose as measured by tumour growth delay or complete remission rate. As the tumours were human in origin it raises the question whether high dose cyclophosphamide therapy and priming have a role to play in the treatment of patients with oat cell carcinoma."}
+{"text": "The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked \u03b12\u20136 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential. Avian influenza viruses naturally circulate in wild aquatic birds as a diverse population having 16 HA and 9 NA antigenic subtypes In Pakistan the first H9N2 outbreak in poultry was reported in 1998 To analyse the genetic nature of H9N2 viruses in the enzootic region of Pakistan, we sequenced the complete coding regions of all eight segments of twelve H9N2 viruses isolated between May 2005 and March 2008 from poultry flocks in the Punjab and the North West Frontier Province (NWFP) of Pakistan. The results showed that these H9N2 viruses had changed considerably compared with previous H9N2 viruses. The eight gene segments of these viruses no longer clustered in a single established Eurasian H9N2 virus sublineage but there was evidence for complex reassortment of genes from viruses belonging to H9N2 (G1-lineage) HPAI H5N1 (Z-genotype), and HPAI H7N3 viruses. Therefore, these viruses represent novel genotypes of H9N2 viruses, the potential consequences of which should not be overlooked.The twelve viruses under study (UDL viruses) were isolated at the University Diagnostic Laboratory (UDL) Lahore, from farm outbreaks in the Punjab and the NWFP of Pakistan . VirusesIntravenous pathogenicity testing was carried out using standard methods 5\u2032AGCAAAAGCAGG-3\u2032 with the Verso\u2122 cDNA Kit (Thermo Scientific). PCR amplification was performed using Pfu Ultra II fusion HS DNA polymerase (Stratagene) and specific primers for the eight gene segments. For HA, both specific and universal primers GATC Biotech, Constance, Germany). Five virus samples that contained the NS gene homologous to H5N1 viruses and the two samples (Ck/Pak/UDL-01/06 and Ck/Pak/UDL-03/08) that contained the NS gene homologous to H7N3 were re-examined in greater detail. The HA genes of these seven virus samples were subjected to in-depth sequence analysis to exclude the possibility that the samples were from mixed infections with H9N2 viruses and H5N1 or H7N3 viruses. Sequence analyses of the HA genes were performed by sequencing a minimum of 60 clones per sample that was calculated to detect with 95% probability any variant sequences at a level of 5% within the virus sample.Viral RNA was extracted from allantoic fluid using the High Pure RNA Extraction Kit (Roche Diagnostics) and cDNA was prepared using an influenza universal oligonucleotide primer, http://www.ebi.ac.uk/Tools/fasta33/nucleotide.html) were used to retrieve the top fifty homologous sequences to the sequenced gene from public sequence databases. Multiple nucleotide and amino acid sequence alignments for all eight gene segments were performed using ClustalX (version 1.83). Unrooted phylogenetic trees were generated from nucleotide sequences based on the complete open reading frame of all eight gene segments using minimum evolution analysis with maximum composite likelihood and the Tamura-Nei model Sequence data were analysed using the Staden package (pregap4 v1.5 and gap4 v1.0). Blast homology searches ; seven amino acids  in NA. Similarly, M and NP genes also retained conserved G1-lineage defining residues (A157) in M1, one (L10) in M2, and four amino acids  in NP.The pattern of nucleotide similarity indicates that the 12 H9N2 viruses retained 4 genes  similar to the H9N2 G1-lineage, 3 genes  from a lineage of viruses from the Indian sub-continent and the Middle-East, and NS genes from one of two H7N3 lineages or from H5N1 genotype Z viruses that were previously present in Pakistan and neighbouring countries . A set oIn order to identify molecular markers that may correlate with the pathogenic properties of these H9N2 UDL isolates analysis was performed to compare deduced amino acid sequences of the envelope glycoproteins and the internal proteins of the Pakistan virus isolates with representative strains of established H9N2 virus Eurasian lineages.Changes in the HA glycoprotein are central to the switch from LPAI to HPAI, but additional amino acid substitutions in the HA molecule have been associated with inter-species adaptation 183, A190, L 226, I227 and G228 in the receptor binding cleft. Of these, three represent substitutions  when compared with the reference strains of the H9 lineage  in human cells in culture The receptor binding site motif of HA is critical for cellular receptor specificity and determining virus host range  lineage . GlutamiThe major molecular determinants that are known to influence the functional activities of the neuraminidase (NA) glycoprotein are the enzyme active site, the stalk length, the sialic acid binding site also referred to as the hemadsorbing site (HB), and potential glycosylation sites. The neuraminidase sequences of all 12 isolates were compared with N2 NA sequences of H9N2 viruses prevalent in Asia and the Middle East during the last fifteen years. For each virus amino acids in the enzyme active site were conserved and showed no evidence of substitutions associated with resistance to the sialidase inhibitors oseltamivir and zanamivir. The data revealed that, of the 12 viruses analysed, 2 isolates (Ck/Pak/UDL-02/05 and Ck/Pak/UDL-03/05) contained a unique 5 amino acid residue deletion (aa 46\u201350) in the stalk region, the others having no deletion in the NA stalk, as was also observed in H9N2 viruses from the Middle East Analysis of the HB site, which is located on the surface of the NA molecule away from the neuraminidase enzyme active site at positions 366\u2013373, Asn-400, Trp-403 and Lys-432 236I substitution. Again, the significance of glycosylation changes at sites 44 and 234 is unknown; however, addition or loss of potential glycosylation may contribute towards increased virulence The comparison of conserved potential glycosylation sites in NA glycoprotein showed that the majority of H9N2 viruses belonging to established Eurasian lineages contain seven conserved potential glycosylation sites at positions 61, 69, 86, 146, 200, 234 and 402 (N2 numbering). The viruses sequenced in this study contained an additional glycosylation site at Asn 44 due to the substitution of Pro45Ser. This additional glycosylation site had also been reported in a number of other H9N2 viruses including Ck/Ind/2048/03, Ck/HK/G9/97 A comparison of the amino acid sequences of the H9N2 neuraminidases shows that certain amino acid substitutions have become fixed as the viruses have evolved but many are unique substitutions represented in individual viruses (Supplementary 80TIASV84) residues show increased virulence in both mouse and poultry infections The NS gene showed a high level of sequence diversity among the twelve UDL isolates analysed. Of particular note were five isolates  that contained an NS gene closely related to the highly pathogenic H5N1 viruses belonging to clade 2.2 of genotype Z with over 99% nucleotide identity . The NS1RNA segment 8 of one virus (UDL-01/06) was very closely related to that of H7N3 viruses from Pakistan isolated over an eleven year period. This single virus isolate shared a 13 amino acid C-terminal truncation; similar truncations have been reported previously in H7 and H9 subtype viruses isolated from poultry The other 6 viruses  formed a distinct sub-clade and showed no truncation or deletion. They contained a PL motif \u201cKSEI\u201d. This KSEI sequence as a PL motif is uncommon. The large scale sequence analysis of avian influenza viruses reported by Obenauer et al. 44A substitution in PB2 and all isolates had aspartic acid at position 372 in NP which has been found in avian influenza viruses isolated from humans A number of residues in the polymerase proteins  and in the nucleoprotein (NP) are known to play a key role in the host range of AI viruses to increase virulence or replication in the mammalian host. Some may be considered as hall-marks of avian or mammalian viruses, whilst others can influence replication efficiency in mammalian or avian hosts. Two analyses of molecular changes associated with the transmission of avian-origin H5N1 and H9N2 viruses to humans Additional residues in the ribonucleoproteins implicated in enhanced replication in mammalian cells have been defined  and thes66S in PB1-F2 is associated with increased virulence in a mouse model RNA segment 2 encodes a second polypeptide in addition to PB1, termed PB1-F2 It is notable that the three polymerase polypeptides have reassorted as a group and the maintenance of the avian specificity at residue 627 of PB2 is striking. The reason for the maintenance of the three polymerase polypeptides may be chance or it is quite likely that the three PB1, PB2 and PA polypeptides act most efficiently as a set and so there may be pressure to maintain a gene constellation for optimal replication proficiency. This may be analogous to the situation observed in human influenza A viruses in which H1N1 and H3N2 co-circulate but with only very limited gene reassortment 15I is common within H9N2 lineages. The UDL viruses showed heterogeneity at residue 37 with 5 viruses encoding valine and 7 viruses alanine but the substitution T37A/V clusters exclusively with the recent UDL H9N2 viruses from Pakistan. The significance of any of these changes to the potential of the currently evolving H9N2 viruses in Pakistan to increase their host range is not known.Several amino acids in the virus matrix, M1, protein are linked with increased replication in mammals or increased pathogenicity in small animal models . At resiNone of the UDL viruses contained substitutions at amino acid positions 26, 27, 30, 31 or 34 in the M2 proteins suggesting that these viruses have no resistance to amantadine The emergence of these novel genotypes of H9N2 viruses and the sustained prevalence of these viruses in poultry warrant further surveillance of H9N2 viruses by complete genomic analysis. These viruses are endemic in poultry in many countries in Asia and the surrounding regions and the generation of new genotypes within H9N2 viruses by reassortment should not be ignored since it allows for an increased ability of the virus to adapt rapidly to different challenges. The observation shown here that further gene reassortment has occurred subsequent to the emergence of viruses in the Middle East highlights the potential for viruses to evolve rapidly. It is widely recognised that genetic reassortment in avian influenza viruses is common in wild bird and poultry isolates Figure S1Amino acid changes in the surface glycoproteins of H9N2 viruses isolated from poultry in Pakistan between 2005 and 2008. The substitutions in the HA (A) and NA (B) are shown in coloured boxes and arrows represent the common changes at a particular node of the tree and the unique changes within individual isolates are represented by black boxes/arrows. Amino acid numbers are based on the H9 amino acid sequence starting at the initiator methionine residue.(2.41 MB TIF)Click here for additional data file.Table S1H9N2 viruses isolated from chicken flocks in Pakistan.(0.06 MB DOC)Click here for additional data file.Table S2Analysis of host range and pathogenicity determinants in the PB2, PB1, PA, NP, M1 and M2 proteins in H9N2 viruses isolated from poultry in Pakistan.(0.38 MB DOC)Click here for additional data file."}
+{"text": "We analysed chromosome 3p for loss of heterozygosity (LOH) in 48 primary oral squamous cell carcinomas (SCCs) using 15 markers and constructed a deletion map for this chromosome arm. LOH at one or more loci was found in 34/48 (71%) of tumours. The data support the existence of at least three distinct regions of deletion at 3p24-26, 3p21.3-22.1 and 3p12.1-14.2. A significant correlation was observed between the number of regions showing allele loss at 3p and tumour stage, consistent with the progressive accumulation of genetic errors during the development of oral SCC. There were also significant associations between LOH at 3p and disease-free and overall survival of patients with early stage disease. This study is the first to demonstrate the prognostic significance of LOH at 3p for oral cancer and may help to identify patients who should receive more aggressive treatment."}
+{"text": "The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Ig\u03b1/Ig\u03b2 heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation."}
+{"text": "Peromyscus maniculatus) and congeneric species are the most common North American mammals. They represent an emerging system for the genetic analyses of the physiological and behavioral bases of habitat adaptation. Phylogenetic evidence suggests a much more ancient divergence of Peromyscus from laboratory mice (Mus) and rats (Rattus) than that separating latter two. Nevertheless, early karyotypic analyses of the three groups suggest Peromyscus to be exhibit greater similarities with Rattus than with Mus.Deer mice .These data suggest that: 1. the  Peromyscus constitutes the most abundant and speciose group of North American mammals. Though superficially similar in appearance to rats and mice, deer mice represent a more distantly related lineage. Mouse and rat are thought to have diverged from each other ~10\u201312 million years ago (mya) while they last shared a common ancestor with the deer mouse (P. maniculatus) lineage ~25 mya [P. maniculatus species complex is a series of semi-interfertile populations spanning nearly every habitat on the continent and is consequently an emerging tool for the study of natural mammalian genetic variation. Facilitating such research is the existence of captive stocks derived from individual populations. Utilizing two of these stocks, we have developed a comparative genomic map for the deer mouse to further research of this genus and to provide insight into the genome rearrangements seen in rats, mice, and other mammals.The cricetid genus Comparative genomic analyses can reveal substantial amounts of information about the biology and evolution of species and are one of the keys to deciphering the roles that genomic structure and organization play in areas such as development, gene expression, and speciation. These analyses, however, are limited to portions of the genomes that have been mapped in all the species being compared and may be compromised by uncertainty of gene orthology and order between any two species.Although whole-genome sequences are available for many species, proper genome annotation is difficult and typically requires additional resources (e.g. meiotic linkage and radiation hybrid cell maps) . As a reRattus norvegicus) and mouse (Mus musculus), which both belong to the rodent family Muridae. Rodentia is the largest mammalian order, containing > 2000 of the ~4600 recognized species and the murids constitute the majority of these [Two of the most complete mammalian genomic maps are associated with the most used biomedical models, the rat  and P. polionotus stock derived from Ocala Nat'l forest in Florida (PO). While neither population is completely inbred, both originated from a limited number of founders and have been maintained as closed colonies. Thus, identifying fixed differences between the two  was typically not difficult.We employed a standard backcross design for these studies utilizing P. maniculatus was conducted using both the Rattus and Mus maps as references [Rattus chromosomes on our backcross panels. Table Mus and Rattus genomes that are informative for this comparative analysis.Map construction for ferences ,16 and uferences . In all,Our backcross panels facilitated linkage of markers at distances ranging from 1.2 cM to 35.8 cM. There are a few instances, however, where marker co-segregation occurs. These may be due to recombination \"cold-spots\", segmental inversions between the BW and PO strains, or simply the interval distance may be below our mapping panel resolution.Peromyscus genome using loci from Mus Chr 11 [Rattus Chrs 10 and 14 and the resulting chromosomal breakpoint is shared with the Rattus genome relative to the Mus genome  indicating rat genome homology. However, all three markers co-segregated. At a distance of only 1.9 Mb in the Rattus genome, these markers are likely to be closer in the deer mouse than our panel is able to resolve.To explore this possibility, we expanded the existing linkage groups using loci whose orthologs lie on Rattus Chr 14 homology group using two loci from Mus Chr 5 that have orthologs on Rattus Chr 14, Afp and Hip2. Similar to both Rattus and Mus genomes, Afp and Hip2 are linked in the deer mouse at a distance of 33.6 cM (LOD = 4.4)   of Rattus Chr 14 in the deer mouse with only a few markers. Our results again show a greater similarity between Peromyscus genome organization and the Rattus genome than either to the Mus genome.Using a similar strategy, we also expanded the Rattus genome similarity that we found for these two deer mouse linkage groups warranted examination of additional informative regions where synteny breakpoints occur between the Mus and Rattus genomes. While not comprehensive for every chromosome, this strategy accelerated our examination of chromosome evolution for the deer mouse genome and helped determine the best reference genome for future deer mouse genome mapping.The high degree of Rattus Chr 1, focusing our efforts within the regions surrounding the breakpoints between Mus Chrs 7, 17, 10, and 19 . This linkage conservation is again consistent with the Rattus genome but not with Mus.We next chose markers that spanned 9 Figure . Within Mus Chr 17 and Mus Chr 7 homology segments of Rattus Chr 1 and found no linkage in Peromyscus between any Mus Chr 17 markers and the Mus Chr 7 marker Usp29, which is only 17.8 Mb away on Rattus Chr 1. We made no further efforts to extend the Mus Chr 7 linkage group, as Usp29 is only 5.93 Mb from the centromeric end of Mus Chr 7 and additional markers in this region were unlikely to yield a different result. We also tested for linkage between the Mus Chr 17 marker Mas1 and the Mus Chr 7 markers Usp29 and Myod1 on a PO \u00d7 PO/BW backcross panel. This was to ensure that our negative linkage results were not a result of aberrant interspecific chromosomal recombination in the BW \u00d7 BW/PO panel. Again, Mas1 did not link to either locus. The lack of linkage between Mus Chr 17 and Mus Chr 7 homology segments in the deer mouse genome constituted the first example of a common breakpoint between the deer mouse and Mus genomes when compared to Rattus.We next tested for linkage between the Mus Chr 7 section of Rattus Chr 1 using five markers: Usp29, Q8C5Y2, Ube3a, Rab6ip1, and H19. Althoughthe RIKEN cDNA marker Q8C5Y2 has not been accurately mapped in either Rattus or Mus, BLAST results indicated intervals between Q8C5Y2 and Usp29 at 26.4 Mb for the Rattus genome and 37.3 Mb in the Mus genome. In support of our placement, the Mus Chr 7/Rattus Chr 1 marker Ube3a co-segregated with Q8C5Y2 in the deer mouse. Our data for these five Mus Chr 7 markers showed high conservation of linkage and gene order with both Rattus and Mus genomes  Rattus and the deer mouse genome by markers spanning the breakpoint between the Mus Chr 7 and Chr 19 regions of Rattus Chr 1. The Mus Chr 7 marker H19 is linked to the Mus Chr 19 marker Prdx5 at a distance of 6.5 cM (LOD = 12.2). Prdx5 is also linked to a second Mus Chr 19 marker Prpf19 at a distance 4.9 cM (LOD = 13.4 cM).We also found genome homology between Rattus Chr 1 loci show that the two deer mouse linkage groups span two Mus genome breakpoints but only one Rattus genome breakpoint, which shows a continued bias towards similarity with the Rattus genome. Our results also imply that the Mus genome has been more rearranged in this region.Overall, our data for Rattus chromosomes. Rattus Chrs 4 and 6 were priority candidates because of the simple breakpoint arrangements and well-conserved gene orders between Rattus and Mus.To broaden the scope of the deer mouse map and help reduce bias resulting from any localized phenomenon such as segmental inversions, we acquired data for markers from multiple Rattus Chr 4 is represented by the entirety of Mus Chr 6 and approximately 30 Mb of the centromeric end of Mus Chr 5. To test for a conserved organization in the deer mouse genome, we typed two markers from each side of the breakpoint on our backcross panel  and the two Mus Chr 6 markers, Ccdc132 and 1700016G05Rik, are linked to each other at a distance of 7.6 cM (LOD = 14.8). Spanning the breakpoint, we found that Sri and Ccdc132 are linked to each other at a distance of 13.3 cM (LOD = 10.3). Overall, our results span about 30% of Rattus Chr 4 and show conservation of the Rattus gene order. However, the linkage between Sri and Srpk2 was shorter than expected and may be due to a recombination cold-spot, an interspecific inversion, or a deletion.Rattus Chr 6 loci consisted of ten markers: Sos1, Ppp1cb, Preb, Dnmt3a, Allc, 493504H06Rik, Pygl, Clec14a, Pcnx, and 1700001K19Rik. These markers span Mus chromosomes 17, 5, and 12 and our results yielded two separate linkage groups . Within the Mus Chr 5 segment, Ppp1cb is linked to Preb at a distance of 6.1 cM (LOD = 16.8). A second Mus breakpoint is spanned by the linkage between Preb and the Mus Chr 12 marker Dnmt3a at a distance of 5.2 cM (LOD = 16.4). Also from Mus Chr 12, Allc represents the terminal marker and links to Dnmt3a at an interval of 16.5 cM (LOD = 7.6).Our mapping of Mus Chr 12 form a second linkage group in Peromyscus. Although synteny with both Rattus Chr 6 and Mus Chr 12 is conserved, we discovered an inversion with respect to both Mus and Rattus involving markers Clec14a and Pygl  while Clecl4a is linked to the more telomeric marker Pcnx at a distance of 18.2 cM (LOD = 6.3). We also found that Clec14a and Pygl have smaller distance interval at 1.3 cM (LOD = 21.2) than would have been expected from the physical intervals in Mus (11.9 Mb) and Rattus (13.4 Mb). Forming the end of the linkage group, Pcnx was linked to 1700001K19Rik at a distance of 25.3 cM (LOD = 3.9).The five remaining markers from Mus genome breakpoints by the deer mouse linkage groups again indicates a more Rattus-like genome organization. However, the breakpoint between the two Peromyscus linkage groups flanked by the two Mus Chr 12 markers Allc and 493504H06Rik represents a unique rearrangement that differs from both the Rattus and Mus genomes. ZOO-FISH results from Mlynarski et al.  also concur that Rattus Chr 6 is indeed represented by two separate chromosomes in the deer mouse.The spanning of two Rattus genome similarity, we also selected markers to span Rattus synteny breakpoints in relation to the Mus genome. This involved markers that span approximately 89% of Mus Chr 1 but are located separately in Rattus on Chrs 5, 9, and 13  but without segregation to Oprk1 and Tram1, despite a distance 35.3 Mb in the Rattus genome between Ube2j1 and Oprk1.For the Mus Chr 1, we detected linkage between the Rattus Chr 13 markers Acbd3 and Glul at a distance of 21.2 cM (LOD = 9.6) and between Glul and Bcl2 at 35.8 cM (LOD = 3.8) . However, Col3a1 is surprisingly not linked to Col9a1, which represents a deviation from the Rattus genome. To confirm these results, we also tested Mut, a marker that is closely linked to Col9a1 on Rattus Chr 9 , thus identifying a linkage similarity between the Rattus and deer mouse genomes. The breakpoints present in the deer mouse and Mus maps for this region are offset and may represent breakpoint area re-usage and a rearrangement hotspot [Col9a1 and Col3a1 on Rattus Chr 9 are needed to refine the breakpoint location. We discovered additional Rattus Chr 9 similarity using the marker Lama, which represents a second and separate region of Mus Chr 17 than that of Mut .Amongst the 9 Figure . Mut is t Figure . We founPeromyscus genome homology to Mus, we performed an analysis using six loci from Mus Chr 8 that form two linkage groups in the Rattus genome  and Mtus1 is linked to Spata4 at a distance of 16.9 cM (LOD = 9.3). Spata4 is also linked to the terminal marker 9130404D08Rik at a distance of 7.0 cM (LOD = 16.4). The two markers from Rattus Chr 19, Elmod2 and Pmfbp1, are linked 10.3 cM (LOD = 12.5).As with the Rattus Chr 17 and Mus Chr 2, Sec61a2 links to the Rattus Chr 19/Mus Chr 8 marker Pmfbp1 at a distance of 30.1 cM (LOD = 3.7). This linkage indicates a clear deviation from both the Rattus and Mus genomes by the deer mouse genome and highlights the benefit of performing cytogenetic analyses in tandem with meiotic linkage mapping.Based on suggestions from ZOO-FISH results , we also examined an additional chromosomal segment for linkage. Representing Mus genome similarity within the deer mouse using loci from Mus Chrs 17, 5, and 13  were tested for linkage in all possible arrangements despite having already been assigned to other deer mouse linkage groups. No linkage was found other than that which already existed amongst the Rattus Chr 1 markers Tcp10b, Pacrg, and Mas1 . The strong organizational similarity of the deer mouse genome with the Rattus genome rather than the more morphologically similar Mus musculus suggests that a significant amount of rearrangement occurred in the Mus genome after the divergence of the cricetid and murid lineages. Concomitantly, our results suggest that the genomic organizations of Rattus and Peromyscus are more representative of the ancestral muroid genome than the Mus genome, which is in agreement with previous literature that indicated a higher rate of genome rearrangement for Mus [Mus genome is extraordinary with approximately 200 homology blocks. However, the Mus genome is not unique in having higher relative rearrangement rates, as the canine and gibbon genomes have approximately twice the average number of homology blocks [There are three instances, however, where the deer mouse map differs from both the y blocks .Mus and Rattus shared a common ancestor significantly more recently than either have with Peromyscus. Our data does not propose to change this phylogeny but rather merely highlights that DNA sequence variation and chromosome rearrangement are independent processes and greater understanding of both processes can provide different insights into the evolution of the structure and function of the eukaryotic genome.Our results also show that genome rearrangement can act independently from other forms of genome evolution, such as sequence mutation. Although rodent sequence mutation rates are higher compared to other mammals, such measurements of genome evolution show P. maniculatus bairdii; BW) and the old field mouse  and were obtained from the Peromyscus Genetic Stock Center at the University of South Carolina [We chose interspecific backcross analysis in order to maximize genetic polymorphism and for the ease of linkage analysis . The twoCarolina . We set Carolina .plt BW \u00d7 PO) F1 animals with 12 unrelated plt BW animals to generate 116 backcross progeny. Backcrosses to BW can be performed using both female and male F1 hybrid animals, as both matings will give viable offspring. However, all but one of the matings used for this panel were F1 \u00d7 BW (\u2640 \u00d7 \u2642).We created two separate backcross panels, BX116 and BX2, for the linkage analysis. For the BX116 panel, we bred twelve hybrid . Genomic DNA for the BX2 panel animals was extracted using a phenol/chloroform/isoamyl alcohol extraction method to increase yields.Il23a, Mas1, H19, Sos1, and Grb10 were developed or obtained from published Peromyscus data [Trp53 primers were developed from P. maniculatus sequence and were kindly provided by Michael McLachlan. We developed all other primers from P. maniculatus EST sequences . Deer mouse EST clones were used for marker design because of greater PCR amplification success (>80% versus ~60% for the published sets).We obtained primer sequences for some Type I markers from published sets of orthologous gene markers. These are termed UMPS, CATS, and TOASTs -33. Primcus data -36. Trp5Mus musculus genomic sequences using cross-species megaBLAST (NCBI). Some critical markers however, spanned larger introns and resulted in amplicons slightly larger than the ideal size parameters.We designed all the markers to be ~400 bp\u20131500 bp and to span an intron to increase polymorphism detection. This was done by aligning deer mouse DNA sequences to P. maniculatus and P. polionotus DNA using a MJ Research PTC-200 DNA Engine gradient thermal cycler and are defined as follows: 1) Standard: 95\u00b0C for 14.5 min followed by 35 cycles of , 72\u00b0C for 10 min, 4\u00b0C hold. 2) Touchdown65 (TD65): 95\u00b0C for 14.5 min followed by 20 cycles of  followed by 15 cycles of , 72\u00b0C for 10 min, 4\u00b0C hold. If no product was obtained using Touchdown65, the starting annealing temperature was changed to 60\u00b0C or 55\u00b0C, with the final annealing temperature remaining 10\u00b0C lower than the starting temperature.PCR cycling conditions for all Type I markers were optimized for 2), 200 \u03bcM each dNTP, 0.4 \u03bcM forward primer, 0.4 \u03bcM reverse primer, and 1 unit Qiagen HotStar Taq polymerase. Some difficult templates required the use of Qiagen Q-solution at either 1\u00d7 or 0.5\u00d7 strength. Four markers, Sparc, Xbp1, Grb10, and Ugp2 required 2.0 mM MgCl2.PCR was performed using 20 ng of genomic DNA in a 10 \u03bcl reaction containing 1 \u03bcl 10\u00d7 Qiagen HotStar buffer  and incubated at 37\u00b0C for 15 minutes followed by heat-inactivation at 80\u00b0C for 15 minutes. For sequencing reactions, 2.0\u03bcL of purified PCR product was direct sequenced with BigDye (v3.1) (ABI) on an ABI 3130 \u00d7 l according to the manufacturer's protocol.Mus musculus genome. Any predicted simple size polymorphisms between BW and PO were tested by gel electrophoresis using amplification products from BW, PO, and BW/PO mix  DNAs. Markers not showing size polymorphisms were further analyzed for species-specific RFLPs by sequence comparison using Sequencher software (Gene Codes Corporation), the TCAG program available as part of the Biology Workbench software utilities provided at the San Diego Supercomputer Center [Sequence identities were verified by cross-species megaBLAST or BLASTN search to the r Center , or withr Center . CandidaGbl to Hba and Hba to Canx, of which both only used 39 animals due to the small usable data set for Hba. For the BX2 panel, no fewer than 60 animals were typed between any two markers with the exception of Mut and Col9a1, for which only 40 animals could be genotyped in common.We tested the backcross panel parental mice with each marker prior to use on the backcross panel. We typed then typed the markers on all possible backcross animals whose parents exhibited the expected genotype. On the BX116 panel, no fewer than 73 animals were used for data to establish linkage between any two markers with exception of We performed backcross linkage analysis using Map Manager QTX software . A minimBLAST Basic Local Alignment Search ToolPeromyscus maniculatus bairdiiBW CATS Comparative Anchor Tag SequencescDNA complementary DNAChr chromosomecM centimorgan20 distilled deionized waterddHdNTP dinucleotide triphosphateEDTA ethylenediaminetetraacetic acid-disodium saltEST expressed sequence tagLOD logarithm of the odds (to the base 10)Mb megabasemya million years agoNEB New England BiolabsPCR polymerase chain reactionplt platinum coat-color mutationPeromyscus polionotus subgriseusPO RFLP restriction fragment length polymorphismSAP shrimp alkaline phosphataseSNP single nucleotide polymorphismSSLP simple sequence length polymorphismTBE Tris-Borate EDTATE Tris-EDTA TLE Tris-low EDTA TOAST traced orthologous amplified sequence tagsUMPS universal mammalian primer sequenceZOO-FISH cross-species fluorescence in-situ hybridizationCR was the lead researcher, conceived the study, participated in its design and coordination, developed markers and assays, participated in the design of the backcross panel, performed linkage analysis, performed molecular genetic experiments, performed sequence alignments, and drafted the manuscript. AL developed markers and assays, performed molecular genetic experiments, and performed sequence alignments. JG developed markers and assays, participated in the design of the backcross panels, performed molecular genetic experiments, performed sequence alignments, and edited the manuscript. PV developed markers and assays, performed linkage analyses, participated in the design of the study, and contributed to and edited the manuscript. RO participated in the design and coordination of the study and helped to draft the manuscript. MD was the principle investigator on the project, participated in its design and coordination, performed sequence alignments, participated in the design of the backcross panels, and contributed in drafting the manuscript and figures. All authors read and approved the final manuscript."}
+{"text": "Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge \u201ctype specific\u201d epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII. Dengue viruses are mosquito-borne flaviviruses and the agents of dengue fever and dengue hemorrhagic fever. It has been widely assumed that antibodies that neutralize dengue bind to regions on the viral envelope (E) protein that are conserved within each serotype. However, few studies have explored how natural variation influences dengue-antibody interactions. Mouse antibodies that strongly neutralize dengue bind to a region on domain III of E protein. This region has been the focus of much recent work because it might be the target of protective human antibodies as well. We compared a large number of E protein sequences and discovered that the region on E protein domain III targeted by neutralizing antibodies was highly variable between strains of dengue serotype 3. Using a panel of antibodies, we experimentally demonstrate that natural strain variation in dengue serotype 3 has a strong influence on antibody binding and neutralization. Our results challenge the dogma that neutralizing antibody binding regions are conserved within each serotype. The results of this study are relevant to the current global effort to develop and evaluate dengue vaccines. Dengue viruses (DENVs) are mosquito-borne flaviviruses and the agents of dengue fever and dengue hemorrhagic fever (DHF). According to the World Health Organization, over 2.5 billion people are at risk of contracting dengue, 100 million people develop symptomatic infections and up to 50,000 die from DHF each year. The DENV complex consists of 4 serotypes (DENV1-DENV4). DENVs have antibody epitopes that are unique to each serotype and epitopes that are cross reactive between serotypes. People who have recovered from primary DENV infections develop long term, protective immune responses against the homologous serotype only. In fact, individuals exposed to a second infection with a different serotype face a greater risk of developing DHF indicating that pre-existing immunity can exacerbate disease under some conditions As previously infected individuals do not appear to be re-infected with the same serotype, it is widely assumed that neutralizing antibody epitopes are conserved among strains belonging to the same serotype Antibodies, in particular, have emerged as key effector molecules responsible for protective and pathogenic immune responses to DENV In the present study, we have examined the phylogenetic relatedness of the E protein sequences from a large number of viruses representing the different genotypes of DENV3. Many surface exposed amino acids were variable between established genotypes of DENV3. Especially noteworthy was the observation that the EDIII lateral ridge, which is a known site targeted by neutralizing MAbs in related flaviviruses, was variable between DENV3 genotypes. We experimentally demonstrate that naturally occurring amino acid differences in DENV3 EDIII lead to differential binding and neutralization by MAbs.Aedes albopictus C6/36 cells were maintained at 28C in MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml) and streptomycin (100 \u00b5g/ml) in the presence of 5% CO2. Human leukemic monocyte lymphoma cell line U937 expressing DC-SIGN (U937 DC-SIGN) were maintained at 37C in RPMI (Gibco) supplemented with 10% FBS, 50 mM beta mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 \u00b5g/ml) in the presence of 5% CO2. All media were also supplemented with 0.1 mM non-essential amino acids (Gibco) and 2 mM glutamine (Gibco).2. Supernatants were harvested, clarified by centrifugation and, supplemented with 15% FBS and stored in aliquots at \u221280C. Viral titers were determined by plaque assay on Vero-81 cells as previously described 5 PFU/ml were used in experiments. The reference virus strains used in the study were strains West Pacific 74 (DENV 1), S16803 (DENV2), CH53489 (DENV3) and TVP-360 (DENV4) routinely used in the DENV neutralization test. These viruses were obtained from Robert Putnak  and they have been passaged >10 times in different mammalian  and insect  cell lines. For studies on different genotypes of DENV3, we also used UNC3043 , UNC 3009 , and UNC3066 (strain 1342 from Puerto Rico 1977). These viruses were obtained from Dr. Duane Gubler and Claire Wong at CDC, Fort Collins, CO. These viruses had been passaged 3 times in Aedes albopictus C6/36 cells prior to being used in these studies.Working virus stocks were obtained by inoculating C6/36 mosquito cells in MEM (Gibco) supplemented with 2% FBS (Gibco), penicillin (100 U/ml) (Gibco) and streptomycin (100 \u00b5g/ml) (Gibco) 0.1 mM non-essential amino acids (Gibco) and 2 mM glutamine (Gibco) and growing the virus for eight days at 28C under 5% COMAbs 8A1 (IgG1) and 14A4 (IgG1) against DENV3 were provided by Robert Putnak . MAb1H9 (IgM) was provided by John Aaskov  g for 5 h) to pellet the virus. The virus pellet was allowed to dissolve overnight in PBS before layering on a 10%\u201340% iodixanol gradient and being centrifuged at 163,700\u00d7g for 120 min. The virus-containing fractions were harvested. PBS was added to the virus to dilute the iodixanol. The diluted solution was centrifuged  to pellet the virus and remove the iodixanol. The virus pellet was resuspended in PBS and virus protein content was estimated by spectrophotometry. The virus was stored at \u221280\u00b0C.Vero-81 cells were inoculated with UNC 3043 (DENV3 -genotype I), CH53489 (DENV3 - genotype II), UNC 3009 (DENV3 - genotype III), or UNC3066 (DENV3 -genotype IV) at an MOI of 0.1. The virus-containing media was harvested 5\u20137 days after infection and centrifuged to pellet cell debris. The clarified media was laid on top of a 20% sucrose (wt/vol) cushion and centrifuged  and purified using amylose resin affinity chromatography (NEB) according to the manufacturer's instructions.Recombinant EDIII constructs were created using cDNA from the following virus strains to represent each serotype of DENV and genotypes of DENV3: West Pacific 74 (DENV 1), S16803 (DENV2), UNC 3043 (DENV3 -genotype I), CH53489 (DENV3 - genotype II), UNC 3009 (DENV3 - genotype III), UNC3066 (DENV3 -genotype IV), and TVP-360 (DENV4). Envelope gene fragments encoding EDIII from DENV1, DENV3, and DENV 4 (AA295\u2013398) and DENV2 (AA297\u2013399) were amplified using Vent polymerase . Reverse primers used in the study were designed to introduce either Hind III (for DENV2\u20134) or PstI (for DENV1) restriction site at the 3\u2032 ends of the PCR products. PCR products were digested with HindIII or PstI and cloned into pMAL c2X vector (NEB) to generate recombinant EDIII that is fused to maltose binding protein (MBP-EDIII) at the N terminus according to the manufacturer's instructions. MBP-EDIII were expressed in www.stratagene.com) and PCR was conducted according to manufacturer's instruction. Single stranded pMal c2X plasmids  encoding MBP-EDIII fusion proteins with amino acid substitutions were cloned into DH5\u03b1 cells for expression and purification of mutant rEDIIIs. Substitution of amino acids in all mutant constructs was confirmed by sequencing. Expression and purification of mutant rEDIII proteins were essentially same as mentioned in the earlier section.Selected amino acids residues on rEDIII were mutated by site directed mutagenesis using Quickchange multi kit . When selecting sites to mutate, we gave precedence to positions on loops and (not beta sheets) because we did not want to disrupt the overall folding of EDIII. Thus, of the 20 positions we mutated, 16  are located on loops that form the lateral ridge neutralizing epitope recognized by type specific neutralizing MAbs ELISA plates were coated by adding 200 ng/well of purified EDIII-MBP protein or 75 ng/well of purified DENV antigen in Carbonate buffer (pH 9.0) and incubating the plates overnight at 4C. Rabbit anti MBP sera (New England Biolabs) was used to quantify binding of MBP-EDIII to plates. The flavivirus cross reactive MAb 4G2 was used to quantify binding of virus to plates. Two hundred nanograms of EDIII-MBP saturated binding to the ELISA plate and we did not observe appreciable differences in binding between different EDIII proteins created for this study. Similarly, 75 ng saturated virus binding to the plate and we did not observe appreciable differences in binding between different viruses used in the current study.The plates were washed with Tris buffered saline with 0.2% Tween 20 (TBST wash buffer) and blocked with 3% normal goat serum (NGS) in Tris buffered saline with 0.05% Tween 20 (TBST blocking buffer) for 1 hour at 37C. Serially diluted MAbs in TBST blocking buffer were then added to each well and incubated for one hour at 37C. After washing 3 times with TBST wash buffer, the plates were incubated for one hour at 37C with alkaline phosphatase conjugated goat anti-mouse antibody (Sigma). Plates were washed 3 times with TBST wash buffer and developed by adding p-nitrophenyl phosphate substrate (Sigma). Optical density (OD) was measured at 405 nm using a spectrophotometer.et. al., was used with modifications to determine 50% neutralization values for each antibody 7 genome equivalent copies) from each DENV3 genotype was used to infect cells in experiments comparing the neutralization activity of MAbs among the DENV genotypes. Different concentrations of MAbs were mixed with each virus strain in a 96 well tissue culture plate and incubated for one hour at 37C in the presence of 5% CO2. U937DC-SIGN cells (5\u00d7104 cells in 100 \u00b5l) were introduced to each well and incubated for an additional 2 hours at 37C to allow virus to bind to cells. The cells were then washed with media and 200 \u00b5l of fresh media was added to each well and incubated for 24\u201372 hr at 37C with 5% CO2. After washing 2 times with PBS, the cells were fixed and permeabilized using CytoFix/Cytoperm kit (BD bioscience). The cells were then stained with Alexa 488 conjugated anti dengue MAb 2H2 and the percentage of infected cells was measured in a flow cytometer. EC50 values were calculated using GraphPad Prism version 4.00 for Windows  and non linear regression analysis.The flow cytometry based neutralization protocol as described by Kraus, http://www.homepage.mac.com/barryghall/Software.html) and the PAM series matrix was determined to be the most appropriate, with default gap opening and extension values. The alignment was used to identify 32 variable/informative sites that were defined as columns of heterogeneity in the alignment where the same amino acid change occurred in at least three independent sequences. To display the 32 informative sites  that bind to E protein have been mapped to six regions  [10],,[18To directly address if natural amino acid variation in DENV3 EDIII results in altered antibody binding and neutralization, we assembled and mapped a panel of DENV3 EDIII reactive mouse MAbs. All the antibodies selected for this study  bound to EDIII of DENV3 . MAbs 8ATo map the binding sites of the MAbs, we expressed and purified 28 EDIII recombinant proteins with defined mutations. The positions to mutate were selected based on antibody mapping studies done with other flaviviruses MAb 8A1 is a strongly neutralizing, type specific DENV3, EDIII reactive IgG antibody. With this antibody, we observed a greater than 80% loss of binding when amino acids at 301, 302, 304, 326\u2013328, 330, 361, 380, 382 and 386 were mutated on EDIII . As in tMAb 14A4 is a neutralizing EDIII reactive IgG antibody that cross-reacts with DENV3 and DENV1 . This DEThe DENV complex cross reactive MAbs  bound to all the mutant proteins indicating these antibodies likely bind to a cross reactive epitope outside the lateral ridge region . In FiguOne concern with the above mapping studies was that some mutations might disrupt the overall folding of EDIII and non-specifically reduce antibody binding. To address this concern, we performed binding studies with a well characterized DENV subcomplex specific MAb 1A1D2, which binds to and neutralizes DENV1, 2 and 3 but not 4 Several amino acid positions  on EDIII implicated in binding to MAbs 8A1 and 1H9  were alsTo verify that amino acid differences at the EDIII lateral ridge were responsible for MAb binding differences, further studies were conducted with MAb 8A1 and recombinant EDIII proteins. We systematically changed amino acids in the EDIII genotype IV construct to genotype II and defined the minimum number of changes required to restore the 8A1 epitope. In Experiments were conducted to compare the ability of EDIII MAbs to neutralize different genotypes of DENV3. MAbs 8A1 and 1H9 failed to neutralize DENV3 genotype IV , which wA long-held paradigm in flavivirus research has been that DENVs display little if any within intra-serotypic antigenic variation and this has been the basis for the development of current multivalent vaccines and immunotherapeutics Our results show that EDIII lateral ridge antibodies 8A1 and 1H9 bound to DENV3 genotypes I, II and III but not genotype IV indicating that naturally occurring mutations in EDIII can lead to a total loss of MAb binding. Even though MAbs 8A1 and 1H9 bound to DENV3 genotypes I, II and III with similar apparent affinity, we observed striking differences in the ability of the MAbs to neutralize these viruses. The neutralization titers were almost 10 fold different between viruses for 8A1 and 60 fold different for 1H9. Our results indicate that apparent affinity of antibody to virus immobilized on ELISA wells is not always predictive of the neutralization titer. There are amino acid differences on the EDIII lateral ridge of genotype I, II and III viruses  and thesOne potential problem with our studies is the possibility that some of the recombinant EDIII proteins used in the current study might be grossly misfolded and the binding differences might not be due to direct interactions between antibody and the altered amino acid. NMR and antibody binding assays have established that wild type EDIII expressed as an MBP fusion protein is properly folded Several groups have focused on developing DENV vaccines based on recombinant EDIII Recently we reported that people exposed to natural DENV infections have low levels of EDIII reactive antibody several years after recovery from infection Figure S1Phylogenetic tree of 175 DENV3 E protein sequences used to identify informative sites. A phylogenetic tree was generated using Bayesian inference to analyze the evolutionary relationship of 175 unique DENV3 envelope protein amino acid sequences that were available from Gen Bank at the time this study was initiated. The four known genotypes of DENV3 are indicated. The numeric values at the nodes represent Bayesian posterior probabilities and the distance scale bar represents 0.01 changes per site.(0.15 MB PDF)Click here for additional data file.Table S1Binding of mouse MAbs 1A1-D2 to mutant DENV3 EDIII proteins(0.01 MB PDF)Click here for additional data file."}
+{"text": "From September 1995 to July 1996 50 patients were treated for carpal tunnel syndromeas outpatients by endoscopic release in the rooms of an orthopaedic surgeon .The average age was 51.3 years (27\u201361 years). The average length ofsymptoms was 43 months, the postoperative time off work averaged 27 days. Six monthspostoperatively wasting of the thenar persisted in 2 out of 16 patients, a positive Tinel'ssign in 1 out of 46 patients and delayed median nerve conduction in 2 out of 48presenting these symptoms preoperatively. At 6 months the average handgrip strengthhad recovered to 109% of the preoperative value. One out of 49 patients still presentedparesthesia and 1 out of 50 nocturnal dysesthesia. There were minor complications in 7patients (14%), only one patient requires further treatment. We conclude that endoscopiccarpal tunnel release done on outpatients in a private surgery can be reliable, safeand cost efficient."}
+{"text": "Using mathematical modeling of population age-incidence data, we estimate that 1 (95% confidence limits 0 and 3) of these families is expected to meet the Amsterdam criteria I for HNPCC due to chance clustering of colorectal cancer. \u00a9 2001 Cancer Research Campaign"}
+{"text": "The inverse relationship between GLUT4 and RBP4 expression is known to play a role in the pathogenesis of type 2 diabetes. Elevated levels of RBP4 were shown to cause insulin resistance in muscles and liver. Identification of STRA6 as a cell surface receptor for RBP4 provides further link in this axis and hence we analyzed SNPs in these three genes for association with type 2 diabetes in a South Indian population.STRA6 with type 2 diabetes . None of the SNPs in RBP4 and GLUT4 showed any association with type 2 diabetes. Haplotype analysis revealed that two common haplotypes H1  and H2  in STRA6, H6  in RBP4 and H4  in GLUT4 were associated with type 2 diabetes.Selected SNPs in the three genes were analyzed in a total of 2002 individuals belonging to Dravidian ethnicity, South India, by Tetra Primer ARMS PCR or RFLP PCR. Allele frequencies and genotype distribution were calculated in cases and controls and were analyzed for association by Chi-squared test and Logistic regression. Haplotype analysis was carried out for each gene by including all the markers in a single block. We observed a significant association of three SNPs, rs974456, rs736118, and rs4886578 in STRA6, gene coding the cell surface receptor for RBP4, were significantly associated with type 2 diabetes and further genetic and functional studies are required to understand and ascertain its role in the manifestation of type 2 diabetes.SNPs in  Type 2 diabetes is primarily characterized by insulin resistance, relative insulin deficiency and fasting hyperglycemia GLUT4 and RBP4 but with varied results GLUT4 with type 2 diabetes is lacking. SNPs in both RBP4 and GLUT4 have not been analyzed in Indian population for their association with type 2 diabetes. With respect to GLUT4, a study in Welsh population identified a silent polymorphism (Asn130) in exon-4 present in diabetic subjects. They also identified a Val383Ile mutation present in 3 out of 160 type 2 diabetes subjects but in none of the control subjects. But further analysis in British population failed to confirm any association of these mutations with type 2 diabetes GLUT4 include RFLP studies with KpnI enzyme in Italian, Caucasians, Chinese, and Asian Indians, American black and Japanese population GLUT4. Recent studies in other populations have identified common variants and haplotypes in RBP4 to be associated with type 2 diabetes RBP4 to be associated with altered serum lipid levels . Interestingly, none of these studies looked at polymorphisms in STRA6; the only identified high affinity receptor for RBP4.Genetic studies have been carried out with respect to single nucleotide polymorphisms (SNPs) in GLUT4-RBP4-STRA6 may play an important role in the pathogenesis of type 2 diabetes, wherein the adipocyte specific down regulation of GLUT4 leads to an increased expression of RBP4 and this increase in RBP4 expression leads to insulin resistance via STRA6. In this study, SNPs in GLUT4, RBP4 and STRA6 were analyzed for association with type 2 diabetes in a South Indian population. We report for the first time that genetic variants in STRA6 are significantly associated with type 2 diabetes.The triad of The study was conducted by using DNA isolated from blood samples of 2002 unrelated individuals (1002 cases with type 2 diabetes and 1000 normoglycemic control subjects) belonging to Dravidian ethnicity from Kerala, South India. Diabetes related clinical and biochemical parameters were collected for all the case samples whereas fasting blood glucose, BMI and blood pressure were documented for control samples. Clinical and biochemical features of the study population are summarized in STRA6) were in Hardy-Weinberg proportions and there was no deviation from Hardy-Weinberg equilibrium. rs351224 was subsequently omitted from further analysis. dbSNP ID, Minor allele frequency (MAF) and HWE P value for all the selected SNPs are summarized in STRA6, rs736118 and rs4886578 spanned one LD block (D'\u200a=\u200a0.95), rs974456 was not in LD with the two SNPs and rs351224 was omitted from the analysis due to deviation from HWE. Kovacs et al have previously defined the LD pattern for RBP4 for Caucasian population GLUT4, the 4 SNPs spanned two different LD blocks with SNPs rs2654185 and rs5412 in one LD block and SNPs rs5418 and rs5435 in the second LD block.Test for Hardy-Weinberg equilibrium suggested that the genotypes for all the SNPs except rs351224 . Significant P values were also obtained in the additive model analysis after adjustment for age, sex and BMI. The associations remain statistically significant even after correction for multiple testing by Bonferroni correction . As revealed by ODD's Ratio (OR), all three SNPs in STRA6 tend to be associated with a decreased susceptibility for type 2 diabetes. None of the selected SNPs in GLUT4 or RBP4 showed any significant association with type 2 diabetes. A silent polymorphism in GLUT4, rs5435, showed a slight trend towards association (P\u200a=\u200a0.05). Results of association analysis are summarized in All the three SNPs in STRA6, rs351224 was not included in the haplotype analysis because of significant deviation from Hardy-Weinberg proportions  and H2  were significantly associated with type 2 diabetes (P\u200a=\u200a0.001 and P\u200a=\u200a0.002 respectively) even after correction for multiple testing (P\u200a=\u200a0.005 and P\u200a=\u200a0.01)  in the order rs3758538, rs3758539, rs36014035 and rs34571439 is significantly associated (P\u200a=\u200a0.006) with type 2 diabetes (P\u200a=\u200a0.036). When we omitted rs3758538 from the haplotype analysis due to low minor allele frequency, we found that the haplotype (121) was significantly associated with type 2 diabetes with a P\u200a=\u200a0.002 (data not shown). We also analyzed the haplotypes in GLUT4 for association with type 2 diabetes. 87.7% of the total haplotypes were constituted by four common haplotypes with a frequency >5% in case samples. Case-control analysis revealed that the haplotype H4  defined by SNPs rs2654185, rs5412, rs5418 and rs5435 is marginally associated with type 2 diabetes after correction for multiple testing (P/P*\u200a=\u200a0.01/0.04). Results of haplotype analysis with SNPs in GLUT4 are summarized in In case of portions . Therefo\u200a=\u200a0.01) . The hapdiabetes . This asRBP4 with serum lipid levels STRA6 also did not reveal any significant association with type 2 diabetes related phenotypes (data not shown). We also did not find any evidence of multiplicative gene-gene interaction in modulating BMI, fasting blood glucose, HBA1C and age of onset of diabetes though a higher proportion of individuals with diabetes had higher number of risk alleles (STRA6 (rs736118), RBP4 (rs3758539) and GLUT4 (rs5435).Recent studies in other populations have reported a significant association of SNPs in  alleles . PercentGLUT4, RBP4 and STRA6 for association with type 2 diabetes. We provide evidence that SNPs in STRA6 are significantly associated with type 2 diabetes. We also identified two common haplotypes in STRA6 associated with type 2 diabetes. Furthermore, common haplotypes in GLUT4 and RBP4 were also found to be associated with type 2 diabetes. After a case-control analysis in 1002 type 2 diabetes samples and 1000 control samples, we found that the three SNPs in STRA6, rs736118 (P\u200a=\u200a0.003), rs4886578 (P\u200a=\u200a0.001) and rs974456 (P\u200a=\u200a0.001) were significantly associated with type 2 diabetes. rs736118 is a G>A polymorphism leading to a Met to Ile change in the C-terminal end of STRA6. Though Met and Ile are hydrophobic amino acids, evidence from literature suggests that such changes can alter the trafficking and cell surface expression of proteins. For example, a V302M mutation in the cytoplasmic G loop of Kir6.2 (KCNJ2) led to a loss of coassembly with the wild type protein thereby affecting protein trafficking to the cell surface In this study, we used a case-control approach for analyzing SNPs in three candidate genes viz. TCF7L2, the most strongly associated and replicated type 2 diabetes susceptibility loci, is an integral part of Wnt-1 signaling rs4886578 (G>A), is an exon-intron boundary polymorphism whereas rs974456 (C>T) is present in intron 7. It has been shown that SNPs present in the non-coding region can modulate gene expression and SNPs present in exon-intron boundary can alter splice forms RBP4 and GLUT4 for association with type 2 diabetes and related parameters. SNPs in RBP4 have been previously reported to be associated with type 2 diabetes in Mongolian population RBP4 associated with type 2 diabetes. Another study which analyzed SNPs in the RBP4 for association with type 2 diabetes identified a common haplotype associated with type 2 diabetes but failed to observe any single SNP association RBP4 but our haplotype analysis revealed that a common haplotype  is significantly associated with type 2 diabetes (P\u200a=\u200a0.004). Kovacs et al. identified significant association of the common haplotype AGGTGC  with type 2 diabetes RBP4 have reported association with serum lipid parameters but in our study we failed to observe any significant association of the promoter polymorphisms with serum lipid levels or with other diabetes related parameters RBP4 and STRA6 with diabetic retinopathy in a case control analysis using 155 samples with diabetic retinopathy and 150 samples with type 2 diabetes for more than 12 years with no symptoms of diabetic retinopathy.We also analyzed SNPs in GLUT4 is a strong candidate gene for diabetes related studies, we did not find any evidence of individual SNPs in GLUT4 associated with type 2 diabetes in our population. Haplotype analysis with selected SNPs revealed a common haplotype 2121 defined by the order rs2654185, rs5412, rs5418 and rs5435 marginally associated with type 2 diabetes. We also found that the SNP rs5435 showed an increasing trend towards association with considerable difference in minor allele frequency in cases and controls. Probably this SNP in GLUT4 may increase the susceptibility to type 2 diabetes in combination with other SNPs. It should be noted that the sample size used in this analysis is suitable for stronger effects as found in STRA6 but smaller effects like those observed in GLUT4 (rs5435) may be missed and analysis of a larger sample size may confirm such effects. Our gene-gene interaction data reveals that when the sentinel SNPs in STRA6, RBP4 and GLUT4 are combined together they increase the prevalence of type 2 diabetes with increase in the number of risk alleles  as novel type 2 diabetes susceptibility genes not observed in Japanese and Western population Recent Genome Wide Association Studies (GWAS) in different populations have identified various type 2 susceptibility genes GWAS data for type 2 diabetes in South Indian population is still not available. India has the highest number of cases with type 2 diabetes and is referred as the diabetic capital of the world with 58.4 million people with type 2 diabetes. Indian population differs from other population in certain unique biochemical and clinical parameters like increased insulin resistance, higher adiposity, lower adiponectin levels and higher levels of sensitive C-reactive protein GLUT4, RBP4 and STRA6 for association with type 2 diabetes. We provide evidence supporting a novel genetic role for the STRA6 loci in type 2 diabetes. A significant association was observed between two common haplotypes in STRA6 and type 2 diabetes. Additionally haplotype analysis with SNPs in RBP4 and GLUT4 also revealed significant association of a common haplotype in both genes with type 2 diabetes.In conclusion, we analyzed SNPs in three candidate genes namely, Written informed consent forms were obtained from all the participating subjects. The study was conducted following the guidelines of Indian Council of Medical Research and approved by the ethics committee at AIMS.The study was conducted using samples from 2002 unrelated individuals from Kerala, South India as a part of an ongoing project, Amrita Diabetes Awareness and Welfare Study. Case samples were collected from Endocrinology department, Amrita Institute of Medical Sciences (AIMS), Kochi, Kerala. All samples were collected following the guidelines of American Diabetes Association with fasting blood glucose >120 mg/dl and/or 2-hour plasma glucose >200 mg/dl. Case samples were collected after detailed investigation of medical records for symptoms, use of oral hypoglycemic agent and/or insulin, measurement of fasting blood glucose and other related anthropometric parameters. Subjects diagnosed with type 2 diabetes after the age of 60 or subjects who were started on insulin therapy within one year of diagnosis were excluded from the study. Subjects with type 1 diabetes, family history of type 1 diabetes, maturity onset diabetes of the young, monogenic forms of diabetes, or drug induced diabetes were also excluded from the study. Samples from patients outside Kerala and patients migrating from other parts of the country were not included. Case reports of diabetic nephropathy, diabetic retinopathy and fatty liver disease as diagnosed by the physician were also documented during collection.Age, sex and ethnicity matched normoglycemic control subjects were recruited in the study by public advertisement and increased awareness by offering screening for diabetic risk factors at specially organized medical camps. All the participants of these medical camps were subjected to a health questionnaire for detailed investigation of their disease status, family history, socio-economic status, food habits, smoking and alcohol status, BMI and blood pressure (BP). The inclusion criteria for the healthy controls were: a) above 40 years of age, b) no family history of diabetes, c) not taking any oral hypoglycemic agent or insulin, d) blood glucose levels <110 mg/dl. Clinical characterization of the study subjects is summarized in the STRA6, RBP4 and GLUT4 were selected from dbSNP, NCBI, either based on their position in the gene viz. 5\u2032 UTR, exon, exon-intron boundary, 3\u2032UTR and minor allele frequency  reported in dbSNP or based on previous association studies. All the novel SNPs were selected based on their position to influence the expression or function of the protein. rs5435 in GLUT4 has been previously studied in European population. In addition, we selected three more SNPs in GLUT4 based on position and minor allele frequency as reported in dbSNP, NCBI. For RBP4, we selected SNPs based on previous studies in European population whereas for STRA6, SNPs were selected based on their reported population diversity data and position in the gene. There is only one reported non- synonymous SNP, rs736118, in STRA6 with population diversity data and a MAF >0.05 in Asian population so only this SNP from the coding region of STRA6 was included in the study. Other SNPs selected from STRA6 include rs4886578 present in the intron-exon boundary, rs974456 and rs351224 present in the intron. All SNPs were genotyped by tetraprimer ARMS PCR and RFLP PCR. Genotyping success rate was generally >96% except for rs5418 in GLUT4 where the success rate was >87%. A summary of selected SNPs is given in STRA6), rs974456 (STRA6) and rs5418 (GLUT4) were genotyped by Tetraprimer ARMS PCR. Genotyping plates which showed amplification in negative control (without template DNA) were repeated. All ambiguous genotypes were also repeated in duplicates till clear genotypes were available. Approximately 10% (n\u200a=\u200a194) samples were re-genotyped for cross validation and the genotype success rate was >99.5%. rs736118, rs974456 and rs5418 were genotyped by RFLP-PCR using the restriction enzymes Nco1, Xho1 and BamH1 respectively. In case of rs736118 (Nco1) and rs974456 (Xho1) the presence of minor allele resulted in the lack of restriction enzyme site and hence there was no change in the PCR amplicon after restriction digestion. So to ensure the genotyping accuracy all samples homozygous for the minor allele were selectively re-genotyped. Randomly selected 10% samples were genotyped for these SNPs also and the genotyping accuracy was found to be >99.5%. All DNA amplicons and fragments were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining in gel documentation system, Biorad, USA. PCR primer designs and reaction conditions are available on request.Genomic DNA was isolated from peripheral blood leukocytes by salting out procedure http://faculty.vassar.edu/lowry/VassarStats.html) and multivariate regression analysis after adjustment for age, sex and BMI. Correction for multiple testing was done by Bonferroni's inequality method wherever applicable and was defined as P value (single tests) \u00d7 number of tests. Bonferroni correction was not done for multiple traits due to lack of any significant association. Multiallelic analysis with sentinel SNPs in STRA6 (rs736118), RBP4 (rs3758539) and GLUT4 (rs5435) was done by ANOVA after categorizing the participants into groups according to the number of risk alleles they carried. All association analysis was performed using the software SNP and Variation Suite Ver-7 from Golden Helix, Inc.Hardy Weinberg equilibrium (HWE) test was done for each SNP by Pearson's Goodness of fit Chi square test before further analysis. Association analysis of each SNP with type 2 diabetes was done by Chi-square test, for difference in allele frequency between cases and controls. Association of SNPs with type 2 diabetes was also confirmed by logistic regression analysis after adjustment for age, sex and BMI. An additive model was used for logistic regression where homozygotes for the major allele (1_1), heterozygotes (1_2) and homozygotes for the minor allele (2_2) were coded to a continuous numeric variable for the genotype as 0, 1 and 2 respectively. Linkage disequilibrium was estimated for combined data of cases and controls using Haploview version 4.1. Haplotype analysis was done by using the software SNP and Variation Suite Ver-7 from Golden Helix, Inc. Haplotype analysis was done by using all the markers in a gene as a single block. Quantitative traits were analyzed for association with type 2 diabetes by ANCOVA (available at"}
+{"text": "Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB4 as well as LTC4 secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB4 levels in synovial fluid were significantly increased in patients with Lyme arthritis  when compared to the control subjects with NIA  (p < 0.05). No statistical difference of urinary LTE4 levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB4 production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB4 in synovial fluid of patients with Lyme arthritis underline the involvement of LTB4 in the pathogenesis of this disease.The purpose of this study was to evaluate the potential role of LTB"}
+{"text": "We used the nationwide Swedish Family-Cancer Database with 2060 childhood brain tumours diagnosed in the period 1958\u20131996 to analyse the risk of this tumour by parental cancers and in siblings of childhood brain tumour probands. Groups of patients were compared by calculating standardized incidence ratios (SIRs) for brain tumours in offspring. 1.3% of brain tumour patients had a parent with nervous system cancer; SIRs were 2.4 and 1.88 for diagnostic ages < 5 and < 15 years, respectively. The data showed distinct patterns of familial risks for childhood brain tumours, the SIR was 10.26 for brain astrocytoma given a parent with meningioma. Parental colon cancer was associated with offspring ependymoma (SIR 3.70), and parental salivary gland cancers with offspring medulloblastoma . SIR for sibling nervous system cancer from childhood brain tumour probands was 3.55 up to age 61. \u00a9 2000 Cancer Research Campaign"}
+{"text": "In 96 ovarian cancer patients, the present study investigates the clinical significance of pretreatment concentrations of soluble CD44 standard (CD44s) and its isoforms v5 and v6 determined in the serum and the ascitic fluid by means of recently developed enzyme-linked immunosorbent assays (ELISAs). Furthermore, CD44 serum concentrations in the ovarian cancer patients were compared with circulating CD44 levels in 50 healthy age-matched female blood donors. Whereas CD44s was found to be higher and CD44v5 to be lower in ovarian cancer patients than healthy control subjects, no statistical difference between the two cohorts was revealed for CD44 isoform v6. In the ascitic fluid samples, variant isoform v5 and v6 were demonstrated at lower concentrations than serum. Multivariate analysis of overall survival demonstrated that a high pretreatment serum level of soluble CD44 isoform v5 is independently associated with favourable clinical outcome in ovarian cancer. When circulating CD44 isoforms were compared with a panel of serum parameters known to be involved in the immunological network, an inverse correlation between serum CD44v5 levels and indicators of cellular immune system activation, such as soluble interleukin 2 receptor, immunostimulatory protein 90K and neopterin, became apparent."}
+{"text": "Sir,Vulval intraepithelial neoplasia (VIN) is resulted from persistent human papillomavirus (HPV) infection, especially infection of HPV type 16 (HPV16) . SeveralBritish Journal of Cancer that Imiquimod topical application followed by HPV therapeutic vaccine led to the complete regression of VIN in 63% of patients recruited in a Phase II clinical trial at 52 weeks after the treatment. There was significantly increased local infiltration of CD8 and CD4 T cells in responders; whereas non-responders showed an increased density of T regulatory cells in the lesions (\u03b1 and IFN\u03b3, but also promotes the secretion of IL10, an inhibitory cytokine that damps immune responses (\u03b3, and these cells are most likely responsible for the ineffectiveness of Imiquimod therapy. The IL10 secreting T regulatory cells can be amplified by the subsequent vaccination (Activation of TLRs not only induces inflammatory cytokines, such as TNFesponses . TopicalIL10 polymorphism influences IL12 secretion by dendritic cells in response to the TLRs stimulation ("}
+{"text": "Previous molecular cytogenetic studies by comparative genomic hybridization (CGH) on primary tumours of human malignant mesothelioma have revealed that loss of genetic material at chromosome 14q is one of the most frequently occurring aberrations. Here we further verify the frequency and pattern of deletions at 14q in mesothelioma. A high-resolution deletion mapping analysis of 23 microsatellite markers was performed on 18 primary mesothelioma tumours. Eight of these had previously been analysed by CGH. Loss of heterozygosity or allelic imbalance with at least one marker was detected in ten of 18 tumours (56%). Partial deletions of varying lengths were more common than loss of all informative markers, which occurred in only one tumour. The highest number of tumours with deletions at a specific marker was detected at 14q11.1\u2013q12 with markers D14S283 (five tumours), D14S972 (seven tumours) and D14S64 (five tumours) and at 14q23\u2013q24 with markers D14S258 (five tumours), D14S77 (five tumours) and D14S284 (six tumours). We conclude from these data that genomic deletions at 14q are more common than previously reported in mesothelioma. Furthermore, confirmation of previous CGH results was obtained in all tumours but one. This tumour showed deletions by allelotyping, but did not show any DNA copy number change at 14q by CGH. Although the number of tumours allelotyped was small and the deletion pattern was complex, 14q11.1\u2013q12 and 14q23\u2013q24 were found to be the most involved regions in deletions. These regions provide a good basis for further molecular analyses and may highlight chromosomal locations of tumour suppressor genes that could be important in the tumorigenesis of malignant mesothelioma. \u00a9 1999 Cancer Research Campaign"}
+{"text": "Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection.Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1\u2013139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1\u20132 month \u201311 months after exposure).Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre \u226580) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1\u20132 months and 173 at 10\u201312 months and thus were higher than the titers from asymptomatic individuals . Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10\u201311 months after exposure in asymptomatically infected individuals tested negative.Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies. Since 1997, the highly pathogenic avian influenza A (H5N1) virus has spread among poultry and possibly also in wild birds in Asia, Middle-East, Europe and Africa and caused over 470 cases of reported human diseases with more than 280 deaths Human sera were collected at the Hospital for Tropical Disease (HTD) Ho Chi Minh City, Vietnam, from patients with severe H5N1 virus infection confirmed by RT-PCR 5 RLU (\u201crelative\u201d luminescent unit) of H5pp (quantity defined after optimization 2 incubator). Subsequently, 100 \u00b5L of fresh complete medium was added to the virus-antibody mix and 140 \u00b5L of the virus-antibody mix was transferred back to the cells after the old cell medium was discarded. After 48h incubation at 37\u00b0C (5% CO2 incubator), 100 \u00b5L of Steady-Glo (Promega) luciferase substrate was added directly. Luminescence was read 15 minutes after addition with either Micro-beta (Perkin Elmer) or Glomax (Promega) plate readers. The neutralization titer was defined as the reciprocal of the dilution that matches the positivity criteria  after fitting with the Hill model H5 haemagglutinin pseudotyped lentiviral particles (H5pp) were produced, titrated and used as described previously One hundred tissue culture infectious dose 50 (TCID50) of A/Vietnam/CL26/2004 (for the tests done on Vietnamese sera), A/Cambodia/Q040547/2006 (for the tests performed on Cambodian sera collected in 2006) and A/Cambodia/R0405050/2007 (for Cambodian sera obtained in 2007) were incubated with serial two-fold dilutions (starting from 1\u223610) of each serum for one hour at room temperature prior to addition of the virus-antibody mixture onto MDCK cells. The viruses were chosen for their close genetic and antigenic relatedness to the strains responsible for the infection in patients For the purpose of this analysis we pooled serological data obtained by standard MN test from patients with RT-PCR confirmed H5N1 disease in Vietnam as well as asymptomatic infections detected by sero-epidemiological investigations in Cambodia. We used the estimated time interval after known exposure as a surrogate of the symptoms onset for asymptomatic cases. Available measures of antibody titers in each week interval were reported as geometric mean titers (GMTs). Week intervals in which there were no observations were treated as missing values. For H5N1 patients' data, we used fractional polynomial regression as a flexible parametric method for the prediction of the relationship between immune response and time interval. We then plotted the curve along with the confidence interval of the GMTs. Log10 transformed GMTs were calculated for the purpose of plotting the kinetics of the immune responses. For asymptomatic individuals' data, a linear model was fitted to the Cambodia data to assess the significance of the change in titers over weeks. An F statistic at p value <0.05 was considered statistically significant. All statistical analyses, graph construction, and curve-fitting as well as 95% CI were computed using STATA 9.0 . The Two-sample Wilcoxon rank-sum (Mann-Whitney) test was used to compare small numbers of samples. We assessed correlation between the two assays generating the Spearman's correlation coefficient. We then plotted relative differences in values using the Bland and Altman method A total of 42 individuals including 11 (26%) Vietnamese H5N1-infected patients with severe clinical disease and 31 (72%) seropositive Cambodians with asymptomatic infection or mild disease were analyzed. Of note, 6 of the 31 cases presented with an MN titer \u22651\u2236160 and an HI titer varying from 1\u223640 to 1\u223680. Of the 11 Vietnamese patients, 6 died. The Vietnamese H5N1-infected patients with severe disease were older than the Cambodian seropositive asymptomatic individuals . There was no difference in gender proportions between the two groups .The average time of follow-up of 11 Vietnamese patients was 34.2 weeks after symptoms onset . Of these 11, seven (64%) were bled twice or more for anti-H5N1 antibody testing at different points in time   and 3. OIn individuals who were asymptomatic or had mild disease, sera collected 10\u201311 months after exposure showed a 4- to 32- fold reduction in H5N1 neutralizing antibody titers. At this later time point, 10 of 24 individuals (42%) had H5N1 neutralizing antibody titers lower than 1\u223680 (ranging from 1\u223620 to 1\u223640). These ten individuals also had the lowest initial H5N1 neutralizing antibody titers (1\u223680) . Of the Antibody kinetics measured by H5pp were similar to that from MN assay  & 2. TheThe fractional polynomial regression model for symptomatic individuals predicted a rise of neutralizing antibody titers \u22651\u223680 at week 2 and a peak at week 5 or 6 for the 2 assays . InteresThis study analysed the kinetics of the anti-H5N1 virus neutralizing antibody response in naturally infected patients with symptomatic and asymptomatic or mild infection. Overall, the titers of antibodies measured by both MN and H5pp assays were generally higher in hospitalized patients with severe H5N1 disease than in subclinically infected individuals. Serological evidence of infection (titer \u22651\u223680) was still detectable by both assays in severe H5N1 cases for periods up to the limit of follow-up at 2 years, contrasting with a shorter longevity of detectable antibodies in subclinical cases of whom a substantial proportion declined to titers below 1\u223680 within 10 months after presumed exposure. Higher initial titers during the acute phase, rather than differences in decay rate, could explain persistence of significant titers over a longer period of time among sick patients. Indeed, the slopes of the decay of titers were not significantly different between the two groups of cases when using linear regression modelling (data not shown). These findings suggest that conducting serosurveys could still be relevant within several months of the exposure, and that the level of antibody titers in a human population combined with information about the timing of poultry outbreaks and about H5N1 morbidity and mortality among humans could serve as a tool to accurately measure and monitor the extent of transmission as well as the risk factors for transmission and disease in a given area or premise (i.e. markets).Among patients hospitalized in Vietnam for clinically apparent H5N1 infection, we observed that no neutralizing antibodies were detected during the first week after the onset of the disease while an antibody titer \u22651\u223680 was detectable in 70% by day 14 and in 80% of patients by day 21. It cannot be excluded that the patient that did not test positive by day 17 would never seroconvert, as previously described in other patients infected with H5N1- or other influenza viruses The regression model used in this study predicts that the antibody titer in clinically ill patients who develop a specific immune response should be higher than 1\u223680 by day 14 after onset of symptoms and peaks 2 to 10 weeks after symptom onset. The curve suggests that during the peak, the average expected titer should be between 1\u2236320 and 1\u2236640. Interestingly, even 2 years after H5N1 disease, serology should still be positive in most of these severe human cases. To our knowledge, the kinetic data were only documented in 8 severely ill Thai patients naturally infected by H5N1 virus For the patients with subclinical H5N1 infection, we observe a titer approximately 3 times lower at the time of recruitment by comparison with patients who developed severe symptoms. The average titer (1\u2236150) at the time of blood draw (1\u20132 months after exposure) is just two fold dilution higher than the cut-off value, hence the importance of using neutralization-based methods, which seems to be more sensitive than HI test, and choosing the most appropriate virus for these tests The interpretation of our findings is subject to several limitations. The MN tests on the asymptomatic (Cambodian sera) and symptomatic (Vietnamese) sera were done using identical protocols but in two laboratories using different clade 1 H5N1 viruses isolated in Cambodia and Vietnam, respectively. These limitations and the inter-laboratory variations have been highlighted in a recent study There is little data available on the natural history and kinetics of the antibody response to influenza H5N1 infection over time, crucial information required to inform the design of seroepidemiological studies. We have demonstrated a good correlation in the profiles of antibody response of the H5pp and MN titres hence confirming the validity of the H5pp test as a screening test in seroepidemiological studies of H5N1 infection. Our data provide important novel insights into these dynamics of the serological responses in patients with the full spectrum of clinical disease from severe through mild to asymptomatic H5N1 infection. Whilst community based seroepidemiology testing after one year may pick up people with clinically apparent infection it may fail to show the true extent of the community exposure to H5N1 as the antibody response apparently wans faster in those individuals who were mildly symptomatic or asymptomatic. Hence delayed community seroprevalence studies for H5N1 may underestimate the true burden of human infection."}
+{"text": "Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals.The inhibitory or negative Smads, Smad6 and Smad7, block TGF\u03b2 superfamily signals of both the BMP and TGF\u03b2 classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one  Smad6 and Smad7), antagonize and block TGF\u03b2 superfamily signaling Bone morphogenetic proteins (BMP) are members of the transforming growth factor-\u03b2 (TGF\u03b2) ligand superfamily. BMPs have diverse essential roles during limb development which include establishment of the apical ridge and zone of polarizing activity, as well as in the regulation of cell death, chrondrogenesis, myogenesis, digit identity and fracture repair Smad6 expression in the developing chick limb have been reported Smad6 mRNA expression was also found to be upregulated in interdigital regions following Bmp5 protein application Smad7 expression or the dependence of negative Smad expression on BMP signaling have not been described. Thus while some information is available about Smad6 in the developing limb, how the negative Smad gene expression patterns and their expression levels are regulated, and how they might contribute to the dynamic control of BMP or TGF\u03b2/Activin signaling during limb development remains to be determined.In previous studies limited descriptions of Smad6 and Smad7 from HH st12\u201315 whole chicken embryo and HH st20\u201324 chicken limb bud cDNA libraries. Multiple cDNAs encoding three distinct open reading frames were identified. Comparison with known Smad protein sequences indicates that one encodes a Smad6 homolog (Genbank Accession FJ417094), while the other two encode Smad7 homologs , and maps to a contig that is not associated with a particular chromosome within the sequenced chicken genome (Contig: NW_001472907.1). The Smad7a cDNA contains a partial open reading frame that is lacking only the four amino terminal amino acids as determined by comparison with other Smad7 proteins. This sequence maps to the predicted genomic Smad7 locus on the Z chromosome (XM_427238). Thus there are two chicken Smad7 loci: the cDNA we call Smad7a is equivalent to a genomic \u2018Smad7\u2019 locus on the Z chromosome. The sequence we call Smad7b is equivalent to a previously reported \u2018Smad7\u2019 cDNA, which is not encoded by the genomic Smad7 locus.Most vertebrate species have only one cSmad7a sequences with public databases identified two distinct genomic sequences with similarities to cSmad7a. Nucleotides 541\u2013616 of cSmad7a are identical to sequences in an unassigned 1363 nucleotide genomic contig (Contig: NW_001482773.1), while cSmad7a nucleotides 615\u20131235 and genomic DNA sequences at the \u2018Smad7\u2019 locus have only one mismatch. These data suggest the cDNA spans at least two genomic fragments. Analysis of the conceptual protein sequences is consistent with this idea: The cSmad7a ORF encodes a protein of at least 384 amino acids, while the predicted genomic cSmad7 protein sequence is 222 amino acids long. cSmad7a aa207\u2013384 (the translation of nt615\u20131235) and genomic cSmad7 aa45\u2013222 are identical, consistent with the nucleotide alignments. Protein BLAST analysis of aa1\u201344 of genomic cSmad7 does not identify any protein other than genomic cSmad7, while similar analysis of cSmad7a aa1\u2013206 or the unassigned genomic contig (NW_001482773.1) identifies numerous Smad7 homologs. These data imply that the first 45 aa predicted by the conceptual translation of genomic cSmad7 are incorrect, and that the unassigned contig belongs in the genomic sequence in this region.BLAST analysis of cSmad7a extend 5\u2032 only to cSmad7a nucleotide 541, there is likely at least one additional sequence missing from the genomic sequence. The cSmad7a and cSmad7b cDNAs show multiple differences scattered throughout their sequences, and are most closely conserved with sequences at different chromosomal locations. Thus cSmad7a and cSmad7b are encoded by different genes, and are not splice or allelic variants. To the best of our knowledge, the chicken is the first example of an amniote species with two Smad7 genes.As cSmad7a is similar to cSmad7b through their most amino terminal amino acids, and genomic regions of identity to cSmad6 expression by whole mount in situ hybridization from HH stages 18\u201334, focusing on the developing limbs. At HH st18 expression is observed in the fore and hindlimb buds in small anterior and posterior mesenchymal domains . These data are consistent with, and extend, previously described cSmad6 expression patterns cSmad7a and cSmad7b expression by whole mount in situ hybridization from HH stages 17\u201334, again focusing primarily on expression in the developing limb bud. cSmad7a is expressed at HH st19 in the lateral mesoderm extending from a small mesenchymal domain in the posterior forelimb to the anterior hindlimb . We could not detect significant expression of cSmad7b in the developing limbs until HH st29/30 when expression was observed in interdigital mesenchyme and between the radius and ulna .We examined al ridge . From HHal ridge . From HHal ridge . Expressand ulna . cSmad7b HH st32 . cSmad7bcSmad6 and cSmad7a. We focused on cSmad7a rather than cSmad7b, as cSmad7a is expressed in the limb from HH st 19 through at least HH st32. Taken together the Bmp2, Bmp4 and Bmp7 expression domains bear a remarkable similarity to those of cSmad6 and cSmad7a  or applied recombinant BMP protein directly to limb tissue cSmad6 and cSmad7a expression was assessed up to 72 hours later. Using a virus-specific probe to monitor the extent of infection, we observed extensive but incomplete staining by 24 hours post-infection, and that by 48 hours post-infection had spread throughout the limb mesenchyme (data not shown). In the majority of cases both cSmad6 and cSmad7a were upregulated in their normal expression domains after 24 hr and were also ectopically induced in limb territories such as the central and proximal mesenchyme  or the BMP signaling antagonist noggin DNBMPR Ib virus infection down regulates, but does not completely abolish both cSmad6 and cSmad7a expression  st12\u201315 chicken embryo Smad6 and Smad7 genes Chicken Gallus gallus Genome Build 2.1. Sequences with the following Genbank accession numbers were used to generate the phylogenetic tree in Smad6 and Smad7a and Smad7b gene assignments were made based on similarities of the predicted protein sequences with published family members and sequences submitted to Genbank. Accession number for cSmad6: FJ417094; cSmad7a: FJ417093; cSmad7b: FJ417092.Overlapping clones for each gene were sequenced using standard dye termination chemistry. DNA and protein sequences were compared to the non-redundant GenBank databases and published Smad sequences using both NCBI Blast cSmad6, Clone 6, 1.7 kb; cSmad7a, Clone 10, 2.7 kb; cSmad7b Clone 33, 1.0 kb.Fertile White Leghorn chicken eggs  were incubated at 38\u00b0C in a humidified, forced air incubator and embryos collected at appropriate developmental stages. Experimental manipulations were performed on HH st15 to HH st21 right limb buds; the left limb bud served as a control. Embryos were harvested through 72 hr postmanipulation and were fixed overnight in 4% paraformaldehyde. Embryos were processed for non-radioactive whole-mount or section in situ hybridization (20 \u00b5m cryosections) and photographed as described noggin, RCAS BP(A)CABMPR Ia, RCAS BP(A)DNBMPR Ib and RCAS BP(A)CABMPR Ib viruses were described previously and caused phenotypic defects consistent with previously published reports 8 infectious units/ml The RCAS BP(A)<1?twb=.35w?>For limb infections, virus was injected into HH st17\u201321 wing buds. Injections were targeted such that either the entire limb or specific subregions were infected with virus. Targeting was monitored by nonradioactive whole mount in situ hybridization using either a pan-retroviral or an insert-specific in situ probe Heparin acrylic beads were soaked in 0.01 mg/ml recombinant human BMP2 or BMP7 protein for 1\u20132 hours. A tungsten needle was used to cut a small slit in the posterior proximal mesenchyme of HH st17\u201320 limbs, and a bead implanted into the slit. Embryos were incubated for approximately 15 hours, fixed in 4% paraformaldehyde and processed for in situ hybridization analysis."}
+{"text": "ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.Activin receptor 2  was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2\u2032-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and  Restoration of activin signaling and growth suppression occur in response to ACVR2 complementation in ACVR2-mutant colon cancer cells ACVR2 mutations in MSI-H colon cancers in conjunction with loss of ACVR2 protein expression TGFBR2 loss found in MSS colon cancers ACVR2 and distinct ACVR2 promoter methylation, but not genetic mutation. In colon cancer cell lines, mechanisms for ACVR2 loss also segregate according to microsatellite status, with MSI-H cell lines showing ACVR2 polyadenine tract mutation and MSS colon cancer cells demonstrating promoter hypermethylation. Thus we show that disruption of activin signaling occurs in MSI and MSS colon cancers by distinct mechanisms, revealing activin signaling as an important target in the two most common genomic subtypes of colon cancer.In this study, we explored activin signaling pathway disruption and possible mechanisms in primary MSS colon cancer specimens and colon cancer cells. We found that loss of ACVR2 expression occurs in a subset of MSS tumors, which is often associated with retained pSMAD2, the next downstream effector of both TGF\u03b2 and activin signaling. Unlike that of TGFBR2, ACVR2 loss in MSS tumors occurs through a combination of LOH at ACVR2 polyadenine tracts Our previous data suggested at least partial loss of ACVR2 protein expression in a subset of primary MSS colon cancer specimens despite wild type , All specimens with loss of at least one activin receptor pathway component revealed expression of both total SMAD2 and TGFBR2 , and 3/7 (43%) of MSS tumors with loss of ACVR2 protein expression also showed LOH , a common genomic mechanism of tumor suppressor inactivation in MSS colon cancers, we assayed for LOH at the owed LOH Figure 2ACVR2 (akin to corresponding mutations within TGFBR2 in MSS tumors)8 tract in ACVR2 lies in the kinase domain of this receptor, and frameshift mutations as well as other mutations in the kinase domain abolish the phosphorylating capacity of ACVR2. However, we found no tumor specific mutations corresponding with loss of ACVR2 protein expression. These data suggest that other inactivating mechanisms play a role in the loss of ACVR2 expression.We then sequenced the coding microsatellite as well as 3 separate hot spots in the kinase domain of ACVR2 promoter was hypermethylated in colon cancer tissue as compared to normal and if so, whether a specific methylation pattern of ACVR2 correlated with ACVR2 protein loss in primary MSS colon cancer specimens. We initially divided the ACVR2 promoter into three regions, region 1 (+142 to \u2212603), region 2 (\u2212607 to \u2212958), and region 3 (\u2212958 to \u22121484). Our bisulfite sequencing results showed that there was no methylation in region 1 and minimal methylation in region 2, but the 46 CpG dinucleotides between to \u2212958 to \u22121484 nucleotides relative to the transcription start site (region 3)   within region 3 of the in vitro, we assessed mechanisms of ACVR2 loss in colon cancer cells lines based on microsatellite instability. We tested 3 MSI-H and 3 MSS colon cancer cell lines for: 1) mutation in the coding polyadenine tract of exon 10 of ACVR2, 2) ACVR2 promoter hypermethylation, 3) quantitative RT-PCR of ACVR2 mRNA, and 4) ACVR2 protein expression by immunoprecipitation. As previously reported ACVR2 (A8 to A7) in the MSI-H colon cancer cell line HCT116 causes loss of ACVR2 protein . HT29 cells revealed a distinct ACVR2 promoter hypermethylation pattern  phenotype correlated with hylation , suggestTaken together, these data suggest that the clinico-pathologic effects of ACVR2 protein loss may be similar in both MSI and MSS colorectal cancers despite differing underlying mechanisms of loss, implicating ACVR2 loss as an important step in colon carcinogenesis.ACVR2 in one of its polyadenine tracts ACVR2 expression is lost via a combination of LOH and epigenetic silencing of the ACVR2 promoter. These findings underscore the importance of abrogated activin signaling in colon tumorigenesis, as its disruption occurs in both MSI and MSS subtypes of colon cancer by differing distinct mechanisms.Disruption of activin signaling is common in MSI-H colon cancer cell lines through mutation of ACVR2 and TGFBR2 mutations in primary MSI colon tumors In MSI-H colon cancers, both TGF\u03b2 and activin are abrogated due to frameshift mutations in the type II receptor ACVR2 mutations may occur early in tumorigenesis and are associated with increased local growth ACVR2 mutations in MSI colon cancer may be similar to that of TGFBR2 in which the frameshift mutations occur in high grade dysplasia at the interface to malignancy It appears that in MSI colon cancers We show that in MSS colon cancers at least three members of the activin signaling cascade, ACVR2, ACVR1, and pSMAD2 are disrupted. A significant subset of colon tumors displayed a decrease in phosphorylated SMAD with intact ACVR2 and TGFBR2, indicating a separate primary event downstream of the primary receptors. One case of loss of ACVR1, ACVR2 and pSMAD2 was identified, which was TGFBR2 staining positive . This coACVR2 locus in 6% of MSS colon tumors, increasing to 50% in tumors with loss of ACVR2 protein expression. This overall rate is slightly lower than the frequency of ACVR2 LOH reported previously in a cohort with unknown ACVR2 status We detected LOH at the TGFBR2 kinase domain inactivation Our mutational analysis focused on hotspots akin to those implicated in inactivation of the ACVR2 promoter hypermethylation as a mechanism, and contrasts with the mechanisms of ACVR2 loss in the MSI colon cancer cells ACVR2 expression is between nucleotides \u22121297 to \u2212958. Among genes that are targets for epigenetic silencing, hMLH1 is the best studied for methylation, and it has been proposed that the promoter region adjacent to the transcription start site (TSS) is critical for transcriptional silencing of this gene hMLH1 promoter region data is often used as a paradigm, and it is believed that for most genes, analysis of a similar promoter region is critical to correlate DNA methylation findings with loss of gene expression. In this study, we have analyzed >1500 bp proximal to the TSS, and found good correlation between ACVR2 promoter methylation (in regions \u2212958 to \u22121297) and loss of protein expression by IHC. Our data suggests that instead of mere proximity to the TSS, differential access to methylation co-activators or repressors in a promoter determines gene silencing, and for ACVR2 such a region may occur upwards of 900 bp from the TSS.This is the first report describing ACVR2 loss in MSS colon cancers and ACVR2 was associated with the CIN phenotype, while ACVR2 hypermethylation correlated with the CIMP phenotype. While these categorizations allow attribution of loss of ACVR2 expression to promoter hypermethylation and/or chromosomal instability as a mechanism in MSS cancers, alternative mechanisms such as histone modification and/or microRNAs may be at play, particularly in the LOH negative/methylation negative cancers. Our data however, show a significant correlation between loss of ACVR2 expression and LOH/epigenetic silencing. Thus we provide evidence for the existence of either chromosomal instability or epigenetic modification of ACVR2 in colon cancer and identify a cell model for epigenetic silencing of ACVR2. The full clinical impact of this data will require further confirmation in future studies with larger patient samples.We are mindful that the total number of MSS patients with ACVR2 loss that were investigated in this study is not large, underscoring that this is a relatively infrequent event ACVR2 loss include LOH at ACVR2 and ACVR2 promoter hypermethylation between nucleotides \u22121297 and \u2212958, which are associated with CIN and CIMP phenotypes, respectively. Loss of ACVR2 is associated with increased tumor size. Therefore, activin signaling can be inactivated by distinctive mechanisms in MSI and MSS colon cancers, suggesting the importance of this pathway in controlling colonocyte growth.In conclusion, loss of ACVR2, ACVR1 and pSMAD2 expression occurs in a subset of MSS tumors, and the evidence supports that this results in abrogation of the normal growth suppressive activity of activin signaling. Decreased pSMAD2 is commonly associated with wild type ACVR2 and ACVR1. Mechanisms for This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of North Carolina hospitals. All patients provided written informed consent for the collection of samples as part of the under IRB approval as part of the North Carolina Colorectal Cancer Study (NCCCS) see below. Analysis as part of this study was completely de-identified and no identifying information was available to the PI, making this study exempt from a separate consent.Sporadic colon tumors were prospectively collected under IRB approval as part of the North Carolina Colorectal Cancer Study (NCCCS), a population-based, case-control study comprising 503 patients The MSI colon cancer cells lines HCT116, SW48, DLD1, HCA7, HCT15, LoVo, LS174T, SNU175, SNU407, SW48, and RKO, as well as the MSS colon cancer cell lines CaCo2, HT29, SNU81, SNU503, SW480, and T84 were maintained in Iscove's Modified Dulbecco's Medium  and FET were maintained in F12/Dulbeco's Modified Eagles Medium at conditions previously described DNA from the formalin-fixed, paraffin-embedded material was extracted following microdissection using the Takara DEXPAT kit . Genomic DNA from cells lines was obtained using QIAamp DNA mini kit . RNA from cell lines was obtained using the TRIzol reagent .Rabbit anti-TyrGly mACVR2 (482\u2013494) , with its target epitope in the C-terminus region of ACVR2 (beyond the resulting truncation from frameshift in exon 10) was used for immunohistochemistry as previously described Immunohistochemistry was performed as described previously ACVR2 and ACVR1 loci was performed as previously described ACVR2ACVR1 (D2S2686) (see Table S1).Assessment of LOH at the Table S1) was used to determine LOH at chromosome 5q, 17q and 18q. PCR amplifications were performed on genomic DNA templates from both tumor and corresponding normal tissues. The amplified fluorescent PCR products were electrophoresed on an ABI PRISM 3100 Avant Genetic analyzer and analyzed by GeneMapper fragment analysis software (Applied Biosystems). When comparing the signal intensities of individual markers in tumor DNA with that of the corresponding normal DNA, a reduction of at least 40% in the signal intensity was considered indicative of LOH.Analysis for CIN was performed as previously described DNA from matched colon cancer tissues and corresponding normal mucosa from CRC patients as well as DNA from colon cancer cell lines underwent bisulfite modification as described previously ACVR2 gene based upon mapping analysis using CpG island search program (http://www.uscnorris.com/cpgislands/cpg.cgi). Accordingly, we designed multiple primer sets for MSP and bisulfite sequencing that spanned the entire ACVR2 promoter region, including part of exon 1, to determine methylation density across all CpG dinucleotides with methylation specific primers (see Table S1). Bisulfite sequencing was initially performed on colon cancer cell lines as it allowed amplification of larger amplicons, and helped determine critical regions of promoter methylation that were subsequently PCR amplified in the clinical samples.For methylation analysis, we first identified the tentative CpG islands in the promoter region of the ACVR2TGFBR28 microsatellites of ACVR2 in exon 10 Table S1) and followed by sequencing as previously described ACVR2 were amplified from genomic DNA extracted from select ACVR2 expressing and non-expressing colon cancer cell lines using specific primers (see Table S1) and subjected to sequencing using the DNA Sequencing Shared Resource, UCSD Cancer Center. Any new mutations were to be deposited to GenBank.Three hotspots in conserved regions of the kinase domain of ACVR2 in colon cancer cells, we performed a quantitative polymerase-chain reaction. Briefly, all 17 colon cancer cell lines were grown to 60% confluence. RNA was extracted using a Trizol-based protocol. The concentration of RNA dissolved in DEPC-treated water was assessed using a Beckman-Coulter DU640B spectrophotometer . We performed RT-PCR using an oligo dT for incubation at 37\u00b0C to generate cDNA as previously described Table S1) Templates from each cell line were prepared in triplicate per target gene as 10 \u00b5L reactions . Templates were plated on fast optical 96-well plate  and spun for 2 minutes at 2000 rpm. Results were observed and analyzed on the StepOnePlus Real-Time PCR System . GAPDH mRNA amplification was performed in parallel  to obtain a normalized ACVR2-GAPDH after the relative expression of each gene was calculated using standard curves.To detect the amplification of To detect presence of ACVR2 in the colon cancer cell lines, we performed immunoprecipitation with ACVR2 followed by Western blotting as previously described Analysis was performed with the help of the UCSD Moores Cancer Center Biostatistics Core (K.M.) applying Fisher's exact test and Student's t-test with a p value of <0.05 indicating statistical significance. Further, Clopper-Pearson exact confidence interval was used to determine the 95% confidence interval.Table S1Specific primers used in LOH analysis, ACVR2 genotyping as well as ACVR2 promoter bisulfite sequencing. F denotes forward primer, R denotes reverse primer; U denotes unmethylated and M methylated.(0.09 MB DOC)Click here for additional data file."}
+{"text": "The etiology of Kawasaki Disease (KD) is enigmatic, although an infectious cause is suspected. Polymorphisms in CC chemokine receptor 5 (CCR5) and/or its potent ligand CCL3L1 influence KD susceptibility in US, European and Korean populations. However, the influence of these variations on KD susceptibility, coronary artery lesions (CAL) and response to intravenous immunoglobulin (IVIG) in Japanese children, who have the highest incidence of KD, is unknown.CCL3L1 gene-containing duplication and CCR2-CCR5 haplotypes in 133 Japanese KD cases [33 with CAL and 25 with resistance to IVIG] and 312 Japanese controls without a history of KD. We observed that the deviation from the population average of four CCL3L1 copies  was associated with an increased risk of KD and IVIG resistance (adjusted odds ratio (OR) \u200a=\u200a2.25, p\u200a=\u200a0.004 and OR\u200a=\u200a6.26, p\u200a=\u200a0.089, respectively). Heterozygosity for the CCR5 HHF*2 haplotype was associated with a reduced risk of both IVIG resistance  and CAL development .We used unconditional logistic regression analyses to determine the associations of the copy number of the CCL3L1-CCR5 axis may play an important role in KD pathogenesis. In addition to clinical and laboratory parameters, genetic markers may also predict risk of CAL and resistance to IVIG.The  Kawasaki disease (KD) is an acute, self-limiting systemic vasculitis of infants and children According to a current paradigm, KD is thought to be triggered by an infectious agent that elicits an inflammatory response directed at cardiovascular tissues in genetically susceptible hosts CCR5CCL3L1-gene containing segmental duplication CCR5 and CCL3L1 affect susceptibility to KD in parent-child trios from the United States There is evidence to suggest that recruitment of inflammatory cells in KD may be mediated through CC chemokine receptor 5 (CCR5) CCR5 genotypes and CCL3L1 copy number in different populations CCR5 haplotypes and CCL3L1 copy number influence KD susceptibility and two disease-related outcomes: development of CAL and IVIG resistance.However, there is significant variation in the prevalence of KD as well as the frequency of This study was approved by the institutional review boards of Yamaguchi and Kurume University Hospitals in Japan and the University of California San Diego and the University of Texas Health Science Center in San Antonio in the U.S. and written informed consent was given by the parents of all KD subjects and controls.We conducted an unmatched case-control study of 133 cases of KD and 312 controls collected between January 2002 and April 2005. The KD patients were recruited from three sites: the Department of Pediatrics, Yamaguchi University Hospital; Oita Children's Hospital; and Kurume University Hospitals, Japan. All patients met the Japanese criteria for the diagnosis of KD CCR5 polymorphisms are described elsewhere CCR5 were categorized into haplotypes as described previously and were designated as CCR5 human haplogroups A (HHA), HHB, HHC, HHD, HHE, HHF*1, HHF*2, HHG*1, and HHG*2 CCR5 haplotypes that bear the CCR5-\u039432 or CCR2-64I polymorphisms are designated as the CCR5 HHG*2 and HHF*2 haplotypes, respectively CCL3L1 gene-containing segmental duplication was estimated as described previously CCL3L1 copy number captures three separate CCL3L genes  as described previously The methods for genotyping CCR5 haplotypes was estimated using the PowerMarker software CCR5 haplotypes and CCL3L1 copy number with KD-related outcomes. The median number of CCL3L1 copies in the study population was 4 and for this reason the study subjects were trichotomized into those possessing <4, 4 and >4 CCL3L1 copies. In these regression analyses, we included CCR5 haplotypes  and CCL3L1 copy number (less than 4 and greater than 4) in the same regression model. To determine whether CCL3L1 gene copy number modified the KD-influencing effects of CCR5 haplotypes, we used the Mantel-Haenszel test of homogeneity. We used Stata 10.0  for the statistical analysis.Allele frequency and Hardy-Weinberg equilibrium for all the Among the cases there were 55 (41.35%) females and 78 (58.65%) males whereas in the control group there were 190 (60.90%) females and 122 (39.10%) males. KD patients with available echocardiographic data were categorized into 2 groups according to the presence of CAL. There were 33 (27.5%) and 87 (72.5%) patients with and without CAL, respectively. Mean age of disease onset was 43.5 months (range 2\u2013270 months). Of the 95 cases who were treated with IVIG within the first 10 days of onset of fever, 25 (26.32%) were resistant to treatment.CCR5 haplotype was CCR5 HHC, followed by HHF*2 and HHE (CCR5-\u039432 mutation is very rare. The CCR5 locus was in Hardy-Weinberg equilibrium (Exact P\u200a=\u200a0.9808 in controls and 0.5624 in cases). The median CCL3L1 copy number in both cases and controls was four  and >4 CCL3L1 copies  was associated with an increased risk of developing KD  and >4 CCL3L1 copies  remained associated with a significantly higher risk of developing KD. Thus, departure from the population average of 4 CCL3L1 copies  was associated with a significantly increased risk of KD before  and after adjustment for gender .To determine whether CCR5 haplotypes had a significant association with the risk of KD. In previous studies, we found that the copy number of CCL3L1 modified the SLE-, Kawasaki disease-, and HIV-1-disease-influencing effects of CCR5 haplotypes . Evaluation of the association for the outcome of CAL revealed that possession of the CCR2-64I-bearing CCR5 HHF*2 haplotype was associated with a reduced risk of developing CAL which trended towards statistical significance . However, we did not observe a significant association between CCL3L1 copy number and the risk of developing CAL.In our cohort, of the 25 subjects who were resistant to IVIG, 18 (72%) developed CAL. By contrast, of the 68 who responded to IVIG treatment, only 5 (7.3%) developed CAL. This association between IVIG resistance and CAL development was highly significant . We also found that possession of <4 CCL3L1 copies was significantly associated with an increased risk of IVIG resistance . Although possession of >4 CCL3L1 copies was also associated with an increased risk of IVIG resistance   while possession of CCR5 HHF*2 haplotype was associated with a salutary IVIG response . Departure from the population average of 4 copies  was associated with a higher risk of IVIG resistance .We next determined whether ll model , possess=\u200a0.146) , possessCCR5 HHF*2 haplotype was associated with a reduced risk of IVIG resistance as well as development of CAL, we next examined whether these associations were due to homozygosity and/or heterozygosity of the HHF*2 haplotype. We observed that heterozygosity but not homozygosity for HHF*2 was associated with a reduced risk of both CAL  and IVIG resistance .Because we observed that the CCL3L1 associates with susceptibility to KD and IVIG response whereas the CCR2-64I-containing CCR5-HHF*2 haplotype is associated with a reduced risk of both CAL development and IVIG resistance. Our finding that deviation from the average CCL3L1 copy number  found in the Japanese population is associated with increased risk of KD is noteworthy because we have previously found that deviation from median copy number of CCL3L1 is also associated with an increased risk of systemic lupus erythematosus (SLE) FCGR3B was associated with increased susceptibility to SLE and primary Sjogren's syndrome Our results suggest that in Japanese children, copy number variation of the segmental duplication bearing CCL3L1-containing segmental duplication in our study population was associated with increased KD susceptibility as well as an increased risk of IVIG failure is unknown. As noted, CCL3L1 is the most potent CCR5 ligand and CCR5 ligands are associated with pro-inflammatory effects CCL3L1-containing segmental duplication that is higher than the population average is associated with increased leukocyte chemoattraction CCL3L1 transcript (data not shown). In this light, it is conceivable that subjects bearing higher CCL3L1-containing segmental duplications may express higher levels of chemokines following antigenic stimulus that in turn may increase the risk of developing KD and possibly, IVIG resistance. In addition to causing an immunologic blockade of Fc receptor and inducing further antibody production, IVIG therapy is also known to play an important role in down-regulation of the cytokine/chemokine levels CCL3L1 gene copy numbers the currently used dose of IVIG may be insufficient to induce the desired degree of down-regulation of chemokines leading to IVIG resistance. The latter along with increased CCL3L1 associated inflammation may provide a partial explanation as to why we observed a trend for possession of a high CCL3L1 copy number and reduced clinical responsiveness to IVIG.The precise mechanistic basis by which deviation from the average copy number of the CCL3L1 copy number is associated with reduced CCL3-CCL3L1 chemokine expression levels CCL3L1-containing segmental duplication, respectively, may explain why all subjects do not respond to a single dose of IVIG and require additional treatments. This hypothesis is supported by the fact that greater than half of IVIG-resistant patients who receive an additional dose of IVIG become afebrile CCL3L1 gene copy associates with KD and IVIG non-responsiveness are currently lacking and require validation.On the other hand, a low CCR5 polymorphisms in KD susceptibility has been investigated previously. Significant attention has focused on the widely recognized 32-bp deletion (\u039432) mutation present in the coding region of CCR5 that is found mainly in populations of European ancestry. We reported previously that there was an inverse relationship between the global distribution of \u039432 allele and the incidence of KD CCR5-\u039432 allele across generations CCR5-\u039432-bearing HHG*2 haploype were modified by CCL3L1 copy number CCR5-\u039432 allele was lower in cases (6.5%) compared to controls (10.7%).The role of CCR5-\u039432-bearing HHG*2 haplotype is rarely found in Asian populations. The results of two prior studies in subjects with KD of European ancestry CCR5 locus also associate with susceptibility to KD. However, in the present study of Japanese subjects, we did not find an association between CCR5 haplotypes and KD susceptibility. By contrast, we did find an association of CCR5 haplotypes with KD outcomes and IVIG-resistance.The CCR5-HHG*2 haplotype was associated with not only reduced KD susceptibility, but also a lower risk of CAL CCR5 HHF*2 haplotype which bears the CCR2-64I polyrmorphism is associated with a reduced the risk of IVIG-resistance and CAL formation. Whether this association suggests an involvement of CCR2, a receptor critically involved in monocyte trafficking and activation, in KD pathogenesis and therapy responses is unclear because the CCR2-64I polymorphism is in linkage disequilibrium with promoter polymorphisms in CCR5CCR5 HHF*2 haplotype with KD-related outcomes may relate either directly or indirectly to inflammation.Early coronary lesions demonstrate marked infiltration of neutrophils ITPKC gene was associated with response to IVIG in US KD children. The results of the present study extend the notion that host genetic factors may influence IVIG resistance. IVIG has been shown to be effective across a range of autoimmune, inflammatory and infectious conditions, as well as for post-infectious complications Many demographic and laboratory factors such as patient age, white blood cell count, and plasma levels of aspartate amino transferase and C-reactive protein have been identified as risk factors for IVIG resistance CCL3L1 and CCR5 in KD CCR5-\u039432 polymorphism which has been intensively scrutinized in persons of European descent, the CCR2-64I polymorphism has also been associated with variable susceptibility to multiple diseases as well. Interestingly, while the CCR5-\u039432 polymorphism is rare among persons of Asian descent, the CCR2-64I-containing HHF*2 haplotype is very common, and has been associated with salutary effects (reduced risk) among persons of Japanese descent for several diseases with immunologic underpinnings including multiple sclerosis CCR2-64I-containing HHF*2 haplotype was associated with beneficial effects for both of these outcomes. Another limitation of our study is that we had to consider CAL outcomes as a dichotomous variable as the clinical centers in Japan did not uniformly use Z scores to characterize the dimension of the lumen of the coronary arteries Although a limitation of our study is the small sample size, our results are concordant with previous data suggesting a role for variations in Notwithstanding these limitations, our findings underscore that genetic determinants influence not only inter-individual differences in KD susceptibility but also inter-subject variation in cardiac complications and treatment response. In conjunction with previous studies that have focused on the relationship between variations in CCR5 and its ligands with KD"}
+{"text": "This is followed by logarithmic growth from 4 to 8 days with a doubling time of 18 hours, always similar in different animals. Eventually after 8 days and after about 100 million cells an asymptote is reached which is due to a fistula-like communication between blood-stream and peritoneal cavity. This terminal phase (9-10 days) is rapidly followed by death of the mouse.Reliable tumour cell counts can only be achieved between 4 and 8 days."}
+{"text": "It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4.To determine which genome segment of three group A equine rotavirus strains  with P[12] specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P[12] strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] specificity did not exhibit this phenomenon when the PAGE running conditions were varied.The concentration of acrylamide in a PAGE gel affected VP4 gene coding assignment of equine rotavirus strains bearing P[12] specificity. Diarrheal disease is one of the principal causes of morbidity and mortality among young children in the developing world. Infectious diarrhea of neonatal animals is also one of the most common and economically devastating conditions encountered in the animal agriculture industry. Among an array of infectious agents including bacteria, viruses and parasites, group A rotaviruses are the single most important etiologic agents of diarrhea in infants and young children worldwide and in addition, they are the most commonly identified viral cause of diarrhea in neonatal food animals -4. In 19Reoviridae family, consists of eleven segments of double-stranded RNA numbered 1-11 according to their order of migration in polyacrylamide gels, segment 1 being the slowest and segment 11 the fastest  specificity, however, we did not find any predicted structures that were different between the equine VP4 sequences and the others (data not shown). In addition, we examined the VP4 sequences of selected rotavirus strains to look for potential changes in the equine VP4 sequence that might induce some sort of \"pairing\" of the ends of the molecule, however, we did not find any good candidate sequences.The relative position of the VP4 gene of three equine P[12] strains  varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] did not exhibit this phenomenon when the PAGE running conditions were varied. Caution needs to be exercised when PAGE analyses are used for VP4 gene coding assignment of rotaviruses.Table The standard phenol-chloroform method or TRIzol method was employed to extract rotavirus genomic dsRNA as previously reported ,29. AnalThe authors declare that they have no competing interests.All authors read and approved the final manuscript.LML, XW and ENO carried out the PAGE analyses. YH participated in the design of the study and drafted the manuscript."}
+{"text": "The proliferation index after phytohaemagglutinin (PHA) stimulation of isolated peripheral blood mononuclear cells was also investigated. Sixteen patients undergoing hypothermic  and normothermic  cardiopulmormry bypass (CPB) were enrolled in this study. As a control, we evaluated four patients undergoing thoracovascular operations without CPB. Blood samples were collected before CPB but after anaesthesia, every 30 min during CPB, at the end of CPB and 10 min after protamine administration. Both C3a and TGF-\u03b21 increased significantly during CPB and after protamine administration in the hypothermic as well as the normothermic group. In the latter case the increase of C3a and TGF-\u03b21, although more prominent, was not significantl higher than in the former group. Conversely, the proliferation, index of peripheral mononuclear cells had already decreased 30 min after CPB was started and remained depressed throughout the CPB time. These results suggest a possible role of C3a and TGF-\u03b21 in the immunological changes occurring during extracorporeal circulation.The aim of this study was to evaluate plasma levels of two mediators with immunosuppressive properties, complement fraction C3a (C3a) and transforming growth factor-\u03b2"}
+{"text": "Pax6-/- ES cells died rapidly after neuronal differentiation in vitro.Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- \u2194 Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- \u2194 Pax6+/+ chimeras made by embryo aggregation.Here we report the derivation of new lines of We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development. Pax6 cause failure of eye morphogenesis and severe abnormalities of brain development  that were Pax6+/- and homozygous for a reiterated \u03b2-globin repeat transgene  and disrupted to give individual clusters of between 1-5 cells. Cell clusters were then transferred to a fresh gelatinised well containing N2B27 media containing LIF (1000 U/ml) with the addition of BMP4 (10 ng/ml). Primary colonies of ES cells were visible after approximately 5 days in culture. ES cell lines were passaged at least twice in feeder-free conditions in BHK-21 Glasgow MEM  with 15% fetal bovine serum (FBS) and LIF (1000 U/ml). Cell lines were genotyped as described previously [ES cells that were either wild type  supplemented with N2 and B27  for 6 or 8 days  before fixation and processing.The neural differentiation protocol followed a previous description . BrieflyPax6+/- animals were isolated. Each cortex was kept separate, its cells were dissociated into separate wells and were allowed to grow for 7 days in DMEM:F12 1:1 culture media supplemented with 100 \u03bcg/ml transferrin, 25 \u03bcg/ml insulin, 20 nm progesterone, 30 nm sodium selenite, 60 \u03bcg/ml putrescine, 20 ng/ml EGF and bFGF . The genotypes of individual embryos were discovered by PCR analysis as described previously [The cortices of E11.5 embryos generated from timed matings of Antibodies used in this study were against \u03b2-III-tubulin , microtubule associated protein 2 , UK, Pax6 , glial fibrillary acid protein  and BrdU . Visualisation was achieved using Alexafluor-488 or Alexafluor-568  conjugated secondary antibodies and cell nuclei were counterstained using TOPRO3 . For counting, 3-8 areas were selected randomly per well. Apoptotic cells were identified by their dense chromatin condensation visible with TOPRO3 staining. Chromatin condensation was easily recognized, is a defining feature of apoptosis  and has For flow cytometry, cells were stained with propidium iodide to allow discrimination of single cells and analysis of DNA content. Staining reactions were carried out in duplicate. Cells were analyzed on a Beckman-Coulter XL flow cytometer with Expo32 software. 8,000-20,000 cells were analyzed per sample.Pax6-/- ES cells were dissociated using Tryple Express\u2122 and plated in GMEM media containing LIF and FBS. Twenty-four hours later they were co-transfected using Fugene  with pCMV-Script plasmid containing the full length Pax6 cDNA [Sub-confluent ax6 cDNA  and plasPax6+/+ ES cells (wtMM1-9) and three lines of Pax6-/- ES cells (SeyD1-3) containing the Tg transgene [Pax6-/- ES cell lines displayed no abnormalities of growth during routine passage; two (SeyD1 and SeyD2) were analysed in detail.We established 9 lines of ransgene . Karyotyransgene . Pax6-/-Pax6-/- line SeyD1 was assessed by injection into wild type blastocysts to create chimeras. We produced ten Pax6-/- \u2194 Pax6+/+ chimeras aged between embryonic day (E) 10.5 and postnatal day (P) 5 and ten control Pax6+/+ \u2194 Pax6+/+ chimeras aged between E10.5 and E14.5. In controls, Tg+ cells derived from wtMM4 were distributed throughout eye and brain tissues, which exhibited no abnormal phenotypes. In Pax6-/- \u2194 Pax6+/+ chimeras, Tg+Pax6-/- cells derived from SeyD1 showed abnormal distributions in eye and brain tissues, similar to those reported previously for Pax6-/- cells in Pax6-/- \u2194 Pax6+/+ chimeras produced by aggregation of Pax6+/+ and Pax6-/- morulae [Pax6-/- cells derived from SeyD1 formed clusters in the subventricular zone of the embryonic cortex  from previously-derived genitors . In agreu et al.  of theirPax6-/- lines at Day 8 .Proportions of apoptotic cells on Day 6 and Day 8 after LIF withdrawal were low with no significant differences between the wild-type and the Pax6-/- ES cell lines SeyD1 and SeyD2 lines showed a significantly lower proportion of cells positive for \u03b2-III-tubulin and MAP2 (around 20-30%) in Day 14 cultures  population of cells by flow cytometry as described previously [Pax6+/+, 12.6% \u00b1 2.10 s.e.m.; Pax6-/-, 7.8% \u00b1 2.85, n = 3 cultures each). These data indicate that loss of Pax6 in primary neural progenitors does not increase cell death.To test whether primary neural progenitors derived from eviously . No signPax6-/- ES cells resembles that of previously derived Pax6-/- ES cells [Pax6-/- progenitors taken from mutant embryos [Pax6-/- mutant lines produced EBs containing proliferating progenitors in large proportions that were similar to those in EBs of wild type ES cells and of previously derived Pax6-/- ES cells. This agrees with a previous conclusion that the generation of early neurogenic progenitors from ES cells is not perturbed in the absence of Pax6 [Pax6-/- ES-derived cells in the dorsal telencephalon of chimeras. Thirdly, previous studies have shown that primary Pax6-/- progenitors from mutant cortex generate reduced proportions of neurons in culture [Pax6-/- ES cells in vitro. Together these data suggest that our novel Pax6-/- ES cell lines are mimicking the in vivo and in vitro characteristics of neuronal progenitors derived from primary tissue of the Pax6-/- embryo.In many respects, the in vitro development of our new ES cells  and that embryos . Firstly of Pax6 . Secondl of Pax6  showed t culture ; we founPax6-/- ES cell lines developed quite differently to Pax6-/- ES cell lines reported previously. Our Pax6-/- ES cell-derived neurons showed no signs of early failure of process development and the extensive cell death previously reported; on the contrary, they were continuing to develop ever more elaborate processes at an age when virtually all of the previously described cells had died [In one respect, however, our new had died .Pax6-/- ES cells, possible explanations for this difference in viability must centre of the nature of the ES cells themselves. One possibility is variation in genetic background. Our ES cells were derived from Pax6 mutant mice inbred on a 129Sv(Ola) background whereas those used by Nikoletopoulou et al. [Pax6-/- phenotype [Since the culture protocol we used is similar to that used to differentiate previously derived u et al.  were notPax6-/- cells derived from their mutant ES cells in mouse chimeras. They did, however, describe the introduction of Pax6-/- ES cell derived progenitors into chick telencephalon: some of the neurons derived from these cells survived in the chick telencephalon, suggesting that even in these cells the absence of Pax6 does not necessarily cause extensive cell death given an appropriate supporting environment. Therefore induction of Pax6 genotype-dependent neuronal cell death might depend on both the genetic background of the mutant cells and the environment in which they are placed. It seems quite conceivable that loss of Pax6 from ES cells might cause the rapid death of all derived cells with some genetic backgrounds in vitro but not in vivo, whereas loss of Pax6 from ES cells with other genetic backgrounds might generate cells that are viable both in vitro and in vivo, as is the case in our novel Pax6 mutant cell lines.Nikoletopoulou et al.  did not Pax6-/- ES cell derived neurons in culture reflects an increased incidence of cell death in the cerebral cortex of Pax6-/- mice, other studies did not report an increase in apoptotic cells in the Pax6-/- cortex in vivo or among Pax6-/- primary cells in culture [Pax6-/- neurons have been shown to survive for extended periods, even into postnatal life following transplantation into wild type embryos [Pax6 [Pax6-/- ES-derived neurons in chimeras also survived into postnatal life. Overall, most published studies point to the importance of Pax6 in the early regulation of neurogenesis [Although Nikoletopoulou et al.  suggeste culture ,9,30. Da embryos  and follos [Pax6 . In the ogenesis ,9,30-33,Pax6-/- ES cells that develop in ways predicted from previous work on Pax6-/- progenitors and neurons in vivo and in vitro. Neurons derived from our new lines show enhanced viability compared to neurons derived from previously studied Pax6-/- ES cell lines. Our new lines offer a good model for the study of Pax6 gene function in vivo and in vitro.In conclusion, we have successfully derived JQ and MM carried out the ES cell derivation and JQ, MM and TN performed the differentiation analysis. JQ, JM and DP conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "There are relatively few articles addressing long-term follow-up in women with breast cancer at very young ages.BRCA1, BRCA2 and TP53, with 115 of 288 consecutive cases being tested. Kaplan\u2013Meier curves were generated to assess overall survival, contralateral breast cancer and other second primaries.We have updated and extended our population-based analysis of breast cancer diagnosed at the age \u2a7d30 years in North-west England to include an extra 15 patients with mutation testing in P=0.05). Contralateral breast cancer rates were at a steady rate of 0.6 per 1000, although the rates in mutation carriers were \u223c2 per 1000. Altogether, 16 BRCA1, 9 BRCA2 and 6 TP53 mutations have now been found among the 115 cases on whom DNA analysis has been performed. BRCAPRO accurately predicted the number of carriers for BRCA1 and BRCA2 and was sensitive and specific at the 10 and 20% threshold, respectively. However, BRCAPRO did not seem to give any weight to DCIS, which accounted for two BRCA1 carriers and three TP53 carriers and overpredicted mutations at the high end of the spectrum, with only 6 of 11 (54%) with a >90% probability having identifiable BRCA1/2 mutations.Survival analysis of all 288 patients showed poor overall survival, although this improved from a 15-year survival of only 46% in those diagnosed between 1980 and 1989 to 58% in those diagnosed between 1990 and 1997 (Rates of new primaries are predicted to some extent by mutation status. BRCAPRO is useful at determining those patients aged \u2a7d30 years to be tested. BRCA1, BRCA2 and TP53 account for a proportion of early-onset and familial breast cancer. These mutations confer a lifetime breast cancer risk of 43\u201385% (BRCA1/2 and TP53 mutations in families with breast and/or ovarian cancer or Li\u2013Fraumeni (LFS) or LFS-like (LFL) syndrome  and data are lacking for TP53.Mutations in BRCA1/2 and TP53 mutations in early-onset breast cancer in earlier papers. These papers showed a predominance of BRCA1/2 mutations in familial aggregations of breast cancer, with penetrance estimates of breast cancer as high as 80\u201390% by 70 years of age. This paper presents a detailed analysis of survival, contralateral breast cancer and other tumour incidence in index cases aged \u2a7d30 years, both with and without BRCA1/2 or TP53 mutations. It also presents results of including an extended series of BRCA1/2 and TP53 index cases diagnosed with breast cancer at \u2a7d30 years of age.The 288 original patients were ascertained from a population-based series of consecutive breast cancers from the North Western Cancer Intelligence Service (NWCIS) diagnosed with breast cancer at or under 30 years of age between 1 January 1980 and 31 December 1997. Diagnoses were confirmed using hospital records and pathology reports. Those patients who were proven to have a histological diagnosis other than breast carcinoma were excluded. Patients were approached through their consultant for permission to be interviewed and, after informed consent, to provide blood for DNA testing. A detailed three-generation pedigree was obtained along with copies of hospital notes, where available. Family histories of malignancy were confirmed through the cancer registry, hospital notes or death certificates.Patients were classified as familial, LFS, LFL or non-familial groups on the basis of FH at initial diagnosis. Familial patients were defined as those with a FH of breast cancer <65 years of age or ovarian cancer at any age in first- or second-degree relatives at the time of the index cases\u2019 primary breast cancer diagnosis. Both LFS and LFL were defined as in BRCA1/2 or TP53 were included for some analyses of contralateral and other tumour risks to enrich the inherited subtypes. Of these patients, 24 were diagnosed in the northwest after 31 December 1997.A further series of 84 patients diagnosed with breast cancer at or under 30 years of age who had tested positive for mutations in in situ (DCIS) included and excluded. A comparison between cases diagnosed before and after 1990 was also undertaken. Kaplan\u2013Meier curves were derived for the occurrence of contralateral breast cancer after the initial diagnosis and for other cancer diagnoses following initial breast cancer diagnosis with the inclusion of mutation carriers from the additional data set. Kaplan\u2013Meier curves for contralateral breast cancer from diagnosis were derived using the enriched data set with a confirmed FH or mutation: comparisons were made between no FH (sporadic): BRCA1; BRCA2; TP53; and FH but no mutation.Kaplan\u2013Meier (KM) survival curves from diagnosis of breast cancer were derived from the original series alone, with ductal carcinoma BRCA1, BRCA2 and TP53 as described previously  or, in the absence of FISH, if the immunohistochemical score was 3+ .Pathology was taken from the original pathology report and NWCIS entry. All available tumours from patients who had donated blood samples for DNA analysis were reviewed by a single pathologist (FK). Oestrogen receptor (ER) , PR (PGR312 Novocastra) and HER2 (c-erbB2 CB11 Novocastra) immunohistochemistry was performed in cases in which tissue was available. Both ER and PR were scored using the Allred (Quick) score based on the assessment of both proportion and intensity of staining. Her-2 was scored according to standard protocol  from the University of Texas Southwestern Medical Center . Additional information about CancerGene is available at http://www.utsouthwestern.edu. The Manchester scoring system was used for comparison  were derived for survival and contralateral tumour incidence.Assessment of KM curves was undertaken by A total of 276 women were registered on the NWCIS with confirmed early-onset primary breast carcinoma diagnosed between 1 January 1980 and 31 December 1997 in the strict regional boundaries. At original ascertainment between 1993 and December 1997, 116 (42%) women were dead and 160 (58%) were alive. As of December 2008, 144 (52%) women were dead and 132 (48%) were alive. The age of diagnosis and grade of tumour did not significantly differ between the living and dead cases. The mean age at diagnosis was 28 years and 3 months (range 18 years 5 months to 30 years 11 months). Consultant permission to approach the patient was refused for 26 living patients (4 subsequently came forward unprompted). Of the remaining 135 cases, 102 consented to participate, 32 refused and 1 could not be traced. Blood samples were available from a further five deceased patients, and were tested after family consent. Further blood samples were obtained from an additional 8 of 12 women affected at the appropriate age and study period on the NWCIS, but outside the strict regional boundaries. As such, genetic status could be established for 115 women from the NWCIS who developed breast cancer at \u2a7d30 years of age (15 more than our previous report).In all, 46 patients (42%) had a significant FH, which was consistent with LFS or LFL in 6 cases. A further five women had a FH of breast cancer, which was not consistent with the original study (breast cancer in third-degree relatives or >65 years of age). The remaining 64 cases had no known FH of breast or ovarian cancer at the time of diagnosis and were classified as non-familial.BRCA1, BRCA2 and TP53 were identified in 31 women: 26 of 53 (49%) familial and 5 of 62 (8%) non-familial cases  diagnosed with breast cancer at \u2a7d30 years of age. BRCA2 mutations were detected in nine women (8%). Pathogenic TP53 mutations were found in 5 patients (5%) including 3 of 6 (50%) of the LFS/LFL subgroup. The updated analysis demonstrated an additional TP53 mutation: 659A>G in a family fulfilling LFS criteria, four further BRCA1 mutations  in four familial breast cancers and 1 BRCA2 mutation (1058C>A) out of the 15 additional samples obtained since our last report (BRCA1 mutation among sporadic cases increase above 10% (2 of 16). However, the overall detection rate in all triple-negative grade 3 cases including those with a FH was 10 out of 27 (37%).Pathogenic BRCA1 mutations diagnosed at \u2a7d30 years of age after 31 December 1979 and 19 with BRCA2 mutations with the same criteria were identified from our clinic mutation database. In addition, six TP53 carriers diagnosed at \u2a7d30 years of age were found. In all, 13 of the total 84 mutation carriers  have had risk-reducing contralateral mastectomy (RRM) representing 20% of the total. We are not aware of any RRMs being performed in the remaining patients.An additional 30 patients with in situ as their initial diagnosis. Of these cases, two had comedo DCIS and were shown to have a TP53 mutation. They subsequently died from a primary glioma and retroperitoneal sarcoma, respectively. Two patients died from subsequent ipsilateral invasive breast cancer and one from non-cancer-related event. Survival analysis from diagnosis in the whole data set is presented in P=0.05).Of the 288 cases in the population-based series, 18 had carcinoma TP53 carrier, 4 in BRCA1 carriers and 1 in BRCA2. In the enriched mutation carrier data set, there is a 2% annual risk of contralateral breast cancer up until 15 years, after which the number of patients is too small for any stable estimate , no ovarian cancers have occurred. Of 59 women with intact ovaries, 25 are now >40 years of age.The incidence of non-breast primary cancer was 4.2% at 25 years . SurprisP=0.05). In isolated sporadic unilateral cases, the only patient exceeding the 10% threshold (25.8%) with BRCAPRO was a Jewish woman who did not have a mutation. In 11 families with a predicted BRCA1/2 mutation status >90% (10.47 predicted), only 6 BRCA1/2 mutations were found (1 in BRCA2). Two of the TP53 carriers had a predicted BRCA1/2 score of >90% . The prediction of DCIS cases is also presented in BRCA1 carriers with DCIS reached the 10% combined threshold, it is clear that this was because of the very strong FH. Indeed, there seems to be no weighting for DCIS, as sporadic cases had a 0% rating for BRCA1 and even those with a FH were no more likely in BRCAPRO to be a BRCA1 carrier than an unaffected sibling.Performance of the BRCAPRO model and myriad tables can be seen in The myriad model did not perform well with poor sensitivity, especially at the 20% level. The Manchester score was partly developed using initial data from this series before the extended follow-up and addition of 15 women. Using the updated pathology-adjusted Manchester score .BRCA1/2 and TP53 mutations in this cohort , demonstrating the importance of accurately documenting a FH when estimating the likelihood of a mutation carrier. Few mutations were found in those women without a FH. These data would therefore not support the testing of BRCA1/2 in patients below the age of 31 years without a FH of breast cancer. In particular, even in sporadic cases with grade 3 cancers, the detection rate was only 5%. Among grade 3 triple-negative sporadic cases, the rate of mutation detection was only 12.5% (2 of 16). BRCA1 mutation by pathological grade and ER status at various ages in women not selected by FH. These data were also from an unselected series. Women aged between 20 and 29 years with grade 3 ER-negative breast cancers had a 35% chance of a BRCA1 mutation, with a similarly high risk for women aged 30\u201334 years at 26.5%. Only after the age of 34 years did the risks fall below 10%  was highlighted in 1990 . This moTP53 mutation. One patient with a renal carcinoma and a de novo TP53 mutation went on to develop a sarcoma behind the remaining kidney that was subjected to regular screening radiation from intravenous urograms. An oesophageal cancer also occurred in a BRCA2 carrier. However, surprisingly, no ovarian cancers occurred in BRCA1/2 carriers, even though 21 women have lived beyond 40 years of age without an oophorectomy. Population studies have indicated that the subsequent ovarian cancer risk is higher if an individual is diagnosed with breast cancer at a young age, particularly if there is a FH of ovarian cancer (BRCA1/2 mutation rather than another mechanism increasing ovarian cancer risk. The data from our study do not suggest a higher ovarian cancer risk in women with known mutations with early-onset breast cancer, compared with mutation carriers with later-onset disease.The diagnosis of a second primary tumour should prompt the clinician to consider a BRCA1/2 mutations even in early-onset breast cancer cases without a FH are low. An adopted sporadic case would need to be diagnosed at the age of \u2a7d24 years to breach the threshold using BRCAPRO. Nevertheless, BRCAPRO does seem to overestimate the BRCA1/2 probabilities in bilateral cases. It is also interesting to note the handling of DCIS. Although both BRCA1 carriers with DCIS were identified using the 10% combined threshold, they were as likely to be a carrier as an unaffected sibling. It seems that no weight is given to DCIS to increase either the BRCA1 or BRCA2 probability of either the individual or the family in BRCAPRO. Ductal carcinoma in situ at the age of \u2a7d30 years is clearly an important diagnosis, as 45% of the 11 cases had a pathogenic mutation, with DCIS being a particular marker for a TP53 mutation. The myriad tables (TP53 carriers had scores above 94%, yet their FHs were consistent with LFS. We have estimated that a combined score of 40+ using the Manchester score suggests a very high probability of a BRCA1/2 mutation (This study assessed the performance of mutation prediction models in this population-based data set. We chose to assess the models at last follow-up to determine how the models dealt with new contralateral disease. Direct comparisons with the Manchester scoring system are inappropriate, as this system was partly developed from the original data set at the time of diagnosis. Another model, BOADICEA, also used this data set in development (The strengths of this study are that it is population based and has obtained DNA samples on the great majority of living cases. Overall death rates and second primary rates are secure because of the cancer intelligence service.This study does have some limitations. The pathology reports on patients diagnosed before 1990 had little information with regard to grade and do not have information regarding hormone receptor status. It was not possible to determine reliable survival data on the basis of tumour grade as a result of this deficiency. However, in the group that submitted a blood sample, a detailed pathological review was possible in most cases.BRCA1, BRCA2 and TP53 mutations in women aged \u2a7d30 years with a FH. Among sporadic patients, mutations are generally in those with grade 3 triple-negative tumours. Rates of further primary tumours other than contralateral breast cancer are not high, except in TP53 carriers, and very young-onset BRCA1/2 carriers do not seem to be at enhanced risk of ovarian cancer compared with other BRCA1/2 carriers. Contralateral breast cancer rates seem stable at \u223c0.6% annually in all women and at 2\u20133% in mutation carriers.This analysis has shown an improvement in survival for women with very early-onset breast cancer in more recent years. There are high rates of"}
+{"text": "We investigated K-ras mutations in cell samples microdissected by laser capture microscopy at multiple sites from lung tissue sections representing tumour tissue and matched histologically normal tissue obtained from 48 lung cancer patients. K-ras mutations were detected in cell samples from 10 of 38 (26.3%) lung adenocarcinomas and in none of the histologically normal or tumour cell samples taken from 10 lung squamous cell carcinomas. Of the K-ras mutation-positive adenocarcinomas, in 4 cases a mutation was found in only the tumour tissue, in 1 case a mutation was found only in the histologically normal tissue, and in 5 cases mutations were found in both the tumour tissue and histologically normal tissue. Among these 5 cases, 2 had identical mutations in both the tumour tissue and histologically normal tissue, 2 had 1 mutation in the tumour tissue and 2 mutations in the histologically normal tissue, 1 of which was identical to the mutation found in the tumour, and 1 case had 2 codon 12 mutations in tumour tissue and 2 mutations, in codons 9 and 11, in histologically normal tissue. These results showed that K-ras mutations are frequent in histologically normal cells taken from outside lung adenocarcinomas and suggest that some of these mutations may represent early events which could pave the way of lung carcinogenesis. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comMutations in the K-"}
+{"text": "P<0.01). Female sex, increased age, elevated alanine transaminase and urea and early grade 1 PPE were significant independent prognostic factors for the development of CTC grade 2 or worse stomatitis (P<0.01). Early CTC grade 1 diarrhoea predicted CTC grade 2 or worse diarrhoea (P<0.01). Older, female patients with good performance status and impaired liver and renal function who develop early grade 1 PPE alone or in combination with diarrhoea are at highest risk of subsequently developing grade 2 or worse PPE or stomatitis during treatment with PVI 5FU. Reduction of infused 5FU dose should be considered for these patients. Such an approach could both reduce severe toxicity owing to chemotherapy and minimise treatment delays, and should be evaluated prospectively.This study used a prospectively managed clinical database in order to identify 1470 patients with gastrointestinal cancers receiving protracted venous infusion (PVI) fluorouracil (5FU). It aimed to determine the time course of toxicity due to PVI 5FU and to analyse factors predicting toxicity. The initial development of stomatitis occurred more rapidly than diarrhoea or palmar plantar erythema (PPE). The percentage of patients with National Cancer Institute Common Toxicity Criteria (CTC) grade 2 or worse PPE peaked at 9% between weeks 8 and 17, whereas this peak occurred earlier for stomatitis and diarrhoea. The development of CTC grade 1 toxicity in the first 28 days after commencement of chemotherapy was classified as early grade 1 toxicity. Multivariate Cox regression analysis showed that female sex, better performance status, elevated bilirubin, early grade 1 PPE and early grade 1 diarrhoea were independent prognostic factors for the development of CTC grade 2 or worse PPE ( Fluorouracil (5FU)-based chemotherapy is used for the adjuvant and palliative treatment of a variety of different tumour types, particularly for gastrointestinal cancers. A recent meta-analysis demonstrated that the efficacy of 5FU was dependent on its schedule of administration. Infused schedules of 5FU resulted in superior response rates with a small but statistically significant prolongation of survival in advanced colorectal cancer compared with bolus schedules . In addiThe development of chemotherapy-induced toxicity has an adverse impact on quality of life and in the most severe cases may cause death during treatment. The management of toxicities owing to chemotherapy are generally made on an empirical basis only after the development of dose-limiting side effects and generally involve interruption of chemotherapy and dose reduction in order to prevent future episodes. This study aimed to analyse the principal dose-limiting toxicities of infused 5FU therapy observed in a large cohort of patients treated at a single centre. It aimed to identify clinical, laboratory and temporal factors, which could be combined in order to identify those patients who were at greatest risk of toxicity.The Gastrointestinal Unit (GI unit) at the Royal Marsden Hospital (RMH) has maintained a prospective database of all patients receiving chemotherapy. This database contains details of baseline demographic data, haematological and biochemical parameters, chemotherapy administration, tumour response to chemotherapy and survival and toxicity data graded according to the National Cancer Institute Common Toxicity Criteria version 1  7\u2009mg/m2 q 6 weekly. MMC is associated with a mild and predominantly haematological toxicity profile; consequently in patients receiving MMC with PVI 5FU, the toxicities of diarrhoea, PPE and stomatitis were considered to most likely result from 5FU  5FU 300\u2009mg\u2009mTreatment with PVI 5FU with or without MMC was continued up to 24 weeks, unless patients developed progressive disease or excessive toxicity.\u22122 dose reduction, grade 3 toxicities to 100\u2009mg\u2009m\u22122 dose reduction and grade 4 toxicities to 150 mg\u2009m\u22122 dose reduction. Therefore, for this analysis dose-limiting toxicities were defined as grade \u2a7e2 toxicities as this severity of toxicity would usually result in a dose reduction.Nonhaematological toxicities developing during PVI 5FU therapy were managed uniformly as follows. Grade 1 stomatitis and diarrhoea were treated with oral sucralfate and codeine phosphate, respectively, and grade 1 PPE was treated with oral pyridoxine. No dose reduction or delay of 5FU was made for grade 1 toxicities. Grade 2 or worse nonhaematological toxicities led to cessation of 5FU treatment until resolution of toxicity followed by a dose reduction of 5FU except for PPE developing for the first time after 10 weeks of therapy, where treatment was initially recommended at the original starting dose. Otherwise, grade 2 toxicities led to a 50\u2009mg\u2009mP-values of less than 0.01 were considered significant.Time to the development of toxicity was calculated using the inverted methods of Between January 1994 and January 2001, 1470 patients with gastrointestinal cancer received treatment using infused 5FU at the Royal Marsden Hospital (RMH). The baseline demographics of these patients are shown in Figure 1A-3AThe time course of grade 2 or worse toxicities was studied in further detail, as this severity usually required modification of chemotherapy dose. The proportion of patients with grade 2 or worse diarrhoea, PPE and stomatitis was therefore determined during each week of chemotherapy. These results depict both initial as well as subsequent episodes of grade 2 or worse toxicities for all patients who were continuing to receive chemotherapy. The denominator at each time point was the number of patients receiving chemotherapy at that time. The percentage of patients with grade 2 or worse diarrhoea peaked at about 4.5% between weeks 5 and 8. Approximately 30% of patients with grade 2 or worse diarrhoea also had another grade 2 or worse toxicity . The perP<0.001) and stomatitis . This result suggested that the time at which grade 1 toxicity initially developed could be used to identify patients who were at higher risk of subsequently developing a more severe toxicity.While the development of more severe toxicities led to a dose delay or dose reduction, the development of individual grade 1 toxicities alone did not mandate any dose modification. The risk of developing a worse grade toxicity after the development of an initial grade 1 toxicity was analysed in more detail, in order to assess whether the time of onset of grade 1 toxicity had any prognostic significance for the subsequent development of more severe toxicity. Univariate Cox regression analysis demonstrated that each extra day after commencing chemotherapy before the development of a grade 1 toxicity resulted in a lower risk of subsequently developing a grade 2 or worse toxicity in the case of PPE (hazard ratio (HR) 0.99, 95% confidence interval (CI) 0.985\u20130.994; The median time until the first development of any grade 1 toxicity was 28 days. Patients with early grade 1 toxicities were therefore defined as those patients who developed grade 1 but not grade 2 or worse diarrhoea, PPE or stomatitis before completing 28 days of chemotherapy. This time point also predates the times at which the largest proportions of patients have grade 2 or worse toxicities see .P<0.001). A total of 19.9% of patients with early grade 1 diarrhoea subsequently developed grade 2 or worse diarrhoea compared with 11.9% of patients without early grade 1 diarrhoea (P=0.003). Totally, 19.5% of patients with early grade 1 stomatitis subsequently developed grade 2 or worse stomatitis compared with 11.4% of patients without early grade 1 stomatitis (P=0.003).In all, 55.1% of patients with early grade 1 PPE subsequently developed grade 2 or worse PPE compared with 27.3% of patients without early grade 1 PPE  and renal function (urea\u2a7e4.8\u2009mmol/l\u22121) who develop early grade 1 PPE alone or in combination with diarrhoea could be considered for early dose reduction, at the time of development of early toxicity, as this group of patients is at the highest risk of grade 2 or worse PPE and stomatitis.The multivariate Cox regression analysis allows identification of subgroups of patients at higher risk of toxicity with PVI 5FU. Based on the multivariate Cox regression analysis, older, female patients with good performance status, impaired liver function . Severe 5FU toxicity may occur in patients with partial or complete DPD deficiency and these individuals show marked impairment of 5FU clearance (On this basis, measurement of DPD levels has been suggested as a possible method to identify patients at increased risk of 5FU toxicity. However, there are limitations with such an approach. Although the majority of 5FU is metabolised by the liver, assessment of DPD levels is typically based on the levels of the enzyme expressed in peripheral blood mononuclear cells because of their easier accessibility. Unfortunately, assessment of peripheral blood DPD levels correlates only weakly with 5FU clearance and is a poor predictor of 5FU toxicity (An alternative approach to predict and ultimately prevent severe 5FU toxicity is pharmacokinetic monitoring. Patients with DPD deficiency may demonstrate a markedly altered pharmacokinetic profile with significant reduction in the plasma levels of 5FU metabolites. The impaired clearance of 5FU is thought to contribute to its toxicity in these patients. Pharmacokinetic evaluation provides a more direct information about whole-body 5FU catabolism, and small studies suggest that with infused 5FU, higher plasma levels of 5FU correlate with increased toxicity (This study provides a strong empirical basis for the use of information about the temporal development of toxicity in combination with baseline clinical factors in order to assess the risk of toxicity. While it appears logical that patients who develop early grade 1 toxicity would be at higher risk of developing a subsequent more severe toxicity, the extent of the risk and the possible implications for patient management have never previously been evaluated. Thus, the data suggest that older female patients with good performance status and impaired liver function who develop early grade 1 PPE or the combination of early grade 1 PPE and diarrhoea would be at a higher risk of grade 2 or worse stomatitis and PPE. Such patients could receive a reduction in the dose of 5FU at the time of development of early grade 1 toxicity, which could prevent the development of more severe toxicity altogether. This approach, if successful, would be likely to achieve significant benefit in improvements in quality of life as well as minimise treatment interruption due to toxicity.However, in order to evaluate rigorously such an approach, it will be important to validate the models generated in this study prospectively. It will also be important to study the use of early dose reduction for selected patients at high risk of toxicity prospectively. This should involve assessment of response, progression-free survival, overall survival and quality of life in addition to toxicity outcomes in order to determine whether efficacy and palliative benefit is affected using such an approach.An increased understanding of factors associated with toxicity due to chemotherapy may potentially allow early identification of patients at risk of severe toxicity. This study suggests that evaluation of the temporal pattern of toxicity can assist in this process. Similar approaches using data derived from large patient cohorts treated with other chemotherapy agents may allow identification of prognostic factors, including temporal factors associated with toxicity due to other drugs, which could prove useful for clinical management."}
+{"text": "From December 2003 through January 2004, the Phnom Tamao Wildlife Rescue Centre, Cambodia, was affected by the highly pathogenic influenza virus (H5N1). Birds from 26 species died. Influenza virus subtype H5N1 was detected in 6 of 7 species tested. Cats from 5 of 7 species were probably infected; none died. On January 24, 2004, the first confirmed outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Cambodia was reported to the Office International des Epizooties . The center is divided into 3 main sections that cover 37 ha. Birds were kept in sections S1\u20131, S1\u20132, and S2, and the cats were in all sections . The infSpilornis cheela), was reported on December 15, 2003, in S2 (Nycticebus sp.), did not become ill. None of the 27 animal keepers, who were 20\u201350 years of age, were reported to have gotten sick.The first case, in a crested serpent eagle . The cats were sick for 5\u20137 days and exhibited anorexia and lethargy but no respiratory illness.>96.5%) to HPAIV (H5N1) strain H5 sequences from Vietnam and Thailand in 2004 (data not shown). All belonged to the H5 clade 1 (>97.12% identity) and to 2004 Vietnamese and Thai N1 sequences (>96%) (data not shown). The HA and NA sequences of the isolates were deposited in GenBank .Laboratory investigations of the organs from 8 birds sampled in December 2003 were performed . For thoRetrospective investigation of the villages surrounding the PTWRC and Phnom Penh showed that chickens from 2 flocks in which deaths had been reported in mid-December had been provided to the PTWRC, either for the restaurants or for the captive animal feeding. Furthermore, at the time of the outbreak, many wild crows were found dead in the forest surrounding the PTWRC.The 4 cat serum samples, each from a different species, were positive for HPAIV (H5N1) with serum neutralization test . None ofThe sources of introduction of HPAIV (H5N1) within the PTWRC were probably multiple: virus-infected chicken bought to feed the carnivorous species, infected live chickens brought to restaurants near S2 , and contact between infected wild and captive birds. The introduction through infected chickens is supported by the absence of an outbreak at the PTWRC after the feeding of chickens to carnivorous species was discontinued; however, deaths in domestic poultry continued in the area. In addition, almost all carnivorous bird species in S2 died  as did most species usually fed chicken meat in captivity . Diet was also the origin of the outbreak among tigers and leopards in Thailand (Corvus macrorynchos) among the 5 species of Passeriformes in the aviaries showed clinical signs and later was confirmed by RT-PCR to be positive for HPAIV (H5N1). This outbreak confirms that Falconiformes and Strigiformes are sensitive to HPAIV (H5N1) infection and disease (\u2013) and shows that numerous species of these orders can be affected by HPAIV (H5N1) (Anas poecilorhyncha), did not show any clinical signs. Heterogeneity in the susceptibility of wild ducks to HPAIV (H5N1), including asymptomatic infection, has been demonstrated  infection at PTWRC ranged from severe illness and death to complete absence of clinical signs, as described (V (H5N1) . PsittacCatopuma temminckii) and the clouded leopard (Neofelis nebulosa) broadens the host range of the virus among mammals.The serologic evidence of influenza virus (H5N1) infection in 4 species of wild cats is in agreement with previous infection in Thailand  infection. It also confirms the broadening range of susceptible species that may be specific to this clade 1 virus."}
+{"text": "KIP2, IGF2 and H19 genes was studied by quantitative RT-PCR in 24 paired samples of urothelial carcinomas and morphologically normal mucosa obtained by cystectomy, and in bladder carcinoma cell lines. The most frequent alteration in tumour tissue was decreased expression of KIP2 identified in 9/24 (37%) specimens. Decreased IGF2 and H19 mRNA levels were found in five (21%) and three (13%) tumours, respectively. One tumour each overexpressed IGF2 and H19. Loss of H19 expression was only found associated with loss of KIP2 expression, whereas decreased expression of IGF2 mRNA occurred independently. Almost all bladder carcinoma cell lines showed significant changes in the expression of at least one gene with diminished expression of KIP2 mRNA as the most frequent alteration. IGF2 mRNA levels were diminished in several lines, but increased in others. The KIP2 gene could be an important target of genetic and epigenetic alterations in bladder cancer affecting the maternal chromosome 11p15.5. However, reminiscent of the situation in Wilms\u2019 tumours, expression of the IGF2 gene on the paternal chromosome can also be disturbed in bladder cancers. \u00a9 2000 Cancer Research CampaignTo identify targets of genetic and epigenetic alterations on chromosome 11p15.5 in human bladder cancer, expression of the imprinted"}
+{"text": "Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3\u2212/\u2212 mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3\u2212/\u2212 and LtapLp/+ mutants, Dvl3+/\u2212;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.Dishevelled (Dvl) proteins are important signaling components of both the canonical \u03b2-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian  Dishevelled (Dvl) family members and while the roles of Dvl1 and Dvl2 have been described previously, the functions of Dvl3 have remained elusive. Here, we show that the lack of Dvl3 in mice affects the formation of the heart, neural tube, and inner ear. We further show that the defects in these tissues are much more severe when the mice are deficient in more than one Dvl family member, indicating redundant functions for these genes. Congenital heart disease affects approximately 75 in every 1,000 live human births, and approximately 30% of these diseases are due to disruptions in the outflow tract, the region affected in mice lacking Dvl genes. Neural tube defects, similar to those observed in the Dvl mutants, are also common in humans. The animal models described here provide useful tools to elucidate the genetic mechanisms that underlie these abnormalities and may provide novel ways of treating these disorders in the future.Multi-gene families, comprising a set of very similar genes with shared nucleotide sequences, are common in mammals. Individual family members may be expressed in different places and perform separate functions. Alternatively, the genes may have redundant functions, but distinct dosage requirements. Mammals share three  Normal mammalian development is the result of complex signaling networks that regulate and coordinate cell behavior. Wnt signaling controls a broad spectrum of cell fate decisions during embryogenesis and is critical for cell to cell communication in mammalian development. Through the activation of specific target genes, the canonical Wnt pathway tightly regulates cell proliferation, differentiation, adhesion and survival, and controls embryonic patterning Dishevelled (Dvl) proteins, of which three have been identified in humans and mice Islet1 (Isl1), migrate from the pharyngeal mesoderm to contribute to the myocardium of the outflow tract Isl1 expression In the heart, normal development of the cardiac outflow tract requires the addition of cells from the secondary heart field (SHF) dorsal to the primary heart tube Looptail (LtapLp) mice, which carry a missense mutation in Van Gogh-like 2  LtapLp/LtapLp homozygous mutants display the outflow tract abnormality DORV Wnt11Wnt5aWnt5a has been proposed to function in the outflow tract by stimulating intracellular increases in Ca2+ to regulate cells of the CNC.Studies of Vangl2/LtapFz3 and Fz6Dvl1 and Dvl2PCP signaling is also required for the normal development of the auditory sensory organ, the organ of Corti Vangl2/LtapDvl1 and Dvl2Celsr (a homolog of flamingo) Fz3 and Fz6The neural plate undergoes narrowing and lengthening attributable to CE movements during mammalian neurulation. When PCP signaling is disrupted, cells of the neuroepithelium fail to intercalate, preventing the neural tube from fusing at the midline Dvls in mammalian development, we have generated mouse knockouts for each of the Dvl genes. Interestingly distinct phenotypes were revealed, suggesting separate functions for the Dvl proteins. Dvl1 knockout mice are viable and fertile, but display social interaction abnormalities and defects in sensorimotor gating Dvl2 knockout mice Dvl genes is also suggested from their overlapping expression patterns, as well as their high degree of conservation. In support of this, we have previously shown that Dvl1\u2212/\u2212;Dvl2\u2212/\u2212 mutants display craniorachischisis, a completely open neural tube, and abnormalities in the organ of Corti, both novel phenotypes not observed in the single Dvl knockouts To elucidate the role of specific Dvl3 and determine its importance in conotruncal, cochlear and neural tube development. Given the overlapping expression patterns of the Dvls, as well as their high degree of conservation, we further addressed the possibility of functional redundancy and demonstrate that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. We attribute several of the developmental functions of Dvl3 to its role in PCP signaling, enhancing our knowledge of this essential pathway in mammalian development and further defining the specific role of individual Dvl genes.Here we describe the phenotype of mice deficient in Dvl3 knock out mice were successfully generated , as expected from Mendelian ratios. In a mixed genetic background, 121 pups were analyzed, but only 4 out of the expected 30 Dvl3\u2212/\u2212 mice (13.3%) survived. In contrast, at E18.5 Dvl3+/+, Dvl3+/\u2212 and Dvl3\u2212/\u2212 genotypes were recovered in normal Mendelian ratios from heterozygous crosses. These data indicate that 100% of Dvl3 homozygotes in a 129S6 inbred background and approximately 87% in a mixed genetic background die shortly after birth. No gross abnormalities were observed in the few adult Dvl3\u2212/\u2212 mice that did survive in a mixed background.enerated . In ordening age . In a 12Dvl3\u2212/\u2212 newborn pups had difficulty breathing and were often cyanotic. Examination of hearts from Dvl3\u2212/\u2212 mutants at P0 , 31% (37/118) of pups genotyped at weaning age were Dvl3\u2212/\u2212 rescued with the transgene, whereas only 1.6% (2/118) Dvl3\u2212/\u2212 mutants survived without the transgene. E18.5 hearts collected from Dvl3\u2212/\u2212 embryos with the Dvl3 transgene appeared normal and displayed no conotruncal abnormalities . Expression of both Dvl3 and Dvl1 was observed in the developing neural tube at E9.5 , defective neurulation was apparent when we crossed Dvl3 mutants with LtapLp/+ mice. Several Dvl3+/\u2212;LtapLp/+ embryos displayed normal neural tube development  and Dvl3\u2212/\u2212;LtapLp/+ mutants . However, craniorachischisis appears to be a more severe phenotype than exencephaly, indicating a more severe phenotype in Dvl3\u2212/\u2212;LtapLp/+ mutants compared to Dvl3+/\u2212;LtapLp/+ mutants.Homozygous elopment , whereaselopment  and exeng normal  and otheg normal . The freDvl3\u2212/\u2212 and LtapLp;LtapLp mutants display cardiac defects but the single heterozygotes do not, we looked for defects in the hearts of the Dvl3+/\u2212;LtapLp/+ double heterozygotes , display misorientation of stereociliary bundles in sensory hair cells due to disrupted planar cell polarity and the cochlear ducts are also shortened and wider due to defects in CE cell movements Dvl3 mutant embryos, with and without an extra LtapLp mutation in Vangl2. No cochlear defects were observed in either Dvl3+/\u2212 (data not shown) or LtapLp/+ single heterozygotes in the basal and middle regions . 7/11 Dvl3\u2212/\u2212 mutants showed xiphoid bifurication, but this was also observed in several wild type controls.We have previously reported both vertebral and rib malformations in Dvl1 and Dvl2 was evident from our studies of Dvl1\u2212/\u2212;Dvl2\u2212/\u2212 double mutants, which displayed novel phenotypes not observed in the single Dvl knockouts Dvl3 and the other two Dvls we examined the phenotypes of Dvl1;Dvl3 and Dvl2;Dvl3 double mutants. Both Dvl1+/\u2212;Dvl3+/\u2212 and Dvl1\u2212/\u2212;Dvl3+/\u2212 mutants survived to adulthood and were fertile. Normal development was also observed in Dvl1\u2212/\u2212;Dvl3\u2212/\u2212 embryos until E12.5  axis, show an abnormal head shape and a truncated snout, as well as a shortened and kinked tail  was able to rescue the lethal Dvl3\u2212/\u2212 phenotype. Importantly, western blot analysis revealed that none of the Dvl transgenes were overexpressed compared to protein levels from wild type alleles  cannot survive. Surprisingly, we found that addition of the Dvl1TG rescued the Dvl3\u2212/\u2212 phenotype such that Dvl3\u2212/\u2212;Dvl1TG mutants (now Dvl3\u2212/\u2212 with 3 copies of Dvl1 and 2 copies of Dvl2) survived. To test whether this was a complete rescue, we crossed these viable Dvl3\u2212/\u2212;Dvl1TG mutants to Dvl3+/\u2212 mice and genotyped the progeny at weaning  survived, comparable to 9 expected from normal Mendelian ratios, indicating full rescue. However, no Dvl2+/\u2212;Dvl3\u2212/\u2212;Dvl2TG mutants were recovered, even though 9 were expected from normal Mendelian ratios if the Dvl2TG could still rescue. Thus, correction of Dvl2 dosage from three copies to two copies of Dvl2 in Dvl2+/\u2212;Dvl3\u2212/\u2212;Dvl2TG mutants  eliminated the ability of the Dvl2TG to rescue the Dvl3\u2212/\u2212 mutant phenotype, supporting the conclusion that ectopic expression or over-expression of the transgene was not responsible for the rescue of the Dvl3\u2212/\u2212 phenotype.To confirm that the BAC transgenes were actually behaving as wild type alleles and that ectopic or inappropriate expression levels were not responsible for rescuing the mutant phenotypes, we genetically reduced the enotyped . Twelve Dvl1\u2212/\u2212;Dvl2\u2212/\u2212Dvl3\u2212/\u2212 mutants , as did those from the Dvl2+/\u2212 (Dvl3+/\u2212 (not shown) single heterozygote samples. However, several of the sensory hair cells in Dvl2+/\u2212;Dvl3+/\u2212 double heterozygotes had rotated stereociliary bundles  results in incomplete neurulation, suggesting a dosage sensitive redundant role for all three Dvls in this process. Interestingly, Dvl1\u2212/\u2212;Dvl3\u2212/\u2212 mutants did not display neural tube defects, suggesting that the three Dvls are not functionally equivalent. Neural tube defects appear in single Dvl mutants when crossed with LtapLp mice, , indicating genetic interaction with the Dvls and the PCP component, Vangl2 and therefore that the Dvls signal through the PCP pathway to promote neural tube closure.We have shown here that Dvl1 and Dvl3 colocalize in the developing neural tube with similar expression patterns to that of Dvl2 as we described previously Dvl3;LtapLp mutants. Craniorachischisis has been observed in many mutants with disrupted PCP signaling, whereas exencephaly is often associated with defective ciliogenesis  Wnt3a allele mutant, vestigial tail, which shows loss of caudal vertebrae causing a shortening of the tail Wnt5a are also shortened along the A\u2013P axis with a phenotype similar to Dvl2+/\u2212;Dvl3\u2212/\u2212 mutants, and share outgrowth defects in the developing face and lack of tail Dvl mutants using the TOPflash reporter of TCF activity. Interestingly we found that global Wnt signaling was largely unaffected in even the most severely affected mutants, suggesting that only a low level of Dvl is sufficient for functional canonical Wnt signals. However, our data also suggests subtle abnormalities in Wnt signaling. Precise determination of these abnormalities will require careful sectioning of specific tissues and examination of various cell types throughout development.We have previously shown that both The three highly homologous Dvl proteins shared in mammals have very similar broad expression patterns in development. This study completes the initial characterization of the specific, individual roles of each of these proteins and also establishes functional redundancy and overlap in a number of developmental processes. Dvl3 is required for the development of the cardiac outflow tract and signals in the PCP pathway to regulate CE in the developing neural tube and cochlea, as well as cell polarity in the organ of Corti. Dvl1 and Dvl2 are redundant with each other LtapLp mutants were originally acquired from Jackson Laboratory, Wnt1-Cre mice were a kind gift of Dr. Andrew McMahon, Harvard and Isl1-Cre mice were a generous gift from Sylvia Evans, UCSD. We generated the Dvl3\u2212/\u2212 and Dvl3-EYFP mouse mutants as described in Dvl3 mutants was performed with the following primers, Dvl3 forward 5\u2032-TCCGATGAGGATGATTCCACC-3\u2032, Dvl3 reverse 5\u2032-TGAGGCACTGCTCTGTTCTGT -3\u2032, Dvl3 knockout 5\u2032-TTGGCCCACAATGGAGATGCCC-3\u2032, NLpgk neo forward 5\u2032-AGGCTTACCCGCTTCCATTGCTCA-3\u2032. The PCR primers used to distinguish between the transgene and wild type Dvl3 allele were Dvl3lnt2 forward 5\u2032-GGACGCAGGAGATCTTTGAA-3\u2032 and Dvl3Int2 reverse 5\u2032-CATAGCTGGGGTTGAAGCTC-3\u2032 which amplify a band of 155 bp for the wild type allele and 189 bp for the transgene, due to the presence of the LoxP site. Genotyping for the following mouse mutants have previously been described; Dvl1Dvl2Dvl2-EYFP transgene Wnt1-CreIsl1-CreRosa-26-lacZ CreBAT-galAll animal care and experiments were performed under protocols approved by the NHGRI/NIH and UCSD Animal Care and Use Committees. After fixing in 3% aldehyde solution  in 0.1 M phosphate buffer pH 7.5, E18.5 hearts were stored in 100% ethanol following dehydration through a graded ethanol series. Hearts were critically point dried, mounted and coated with 300 Angstrom gold-palladium. A Cambridge Instrument Stereoscan 360 scanning electron microscope  was used to view the prepared samples.P0 hearts were dissected into 10% buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 8 \u00b5m thickness and stained with hematoxylin and eosin using standard methods.Dvl3\u2212/\u2212 hearts, Dvl3+/\u2212 mice carrying Wnt1-CreIsl1-CreDvl3+/\u2212 mice carrying the Rosa-26-lacZ Cre reporter gene 2, 0.02% NP-40, 0.01% sodium deoxycholate at 37\u00b0C overnight. After re-fixing in 4% paraformaldehyde, the slides were counterstained with Nuclear Fast Red (Vector Lab), according to the manufacturer's protocol. Whole embryos from the above crosses were also collected at E10.5 and stained for \u03b2-galactosidase activity using the same procedures. All images were captured using a Spot 2 camera mounted on a Leica DMR light microscope.To label the CNC and SHF cell populations in Dvl mutant mice were crossed with mice carrying the BAT-gal reporter gene To visualize canonical Wnt signaling, Dvl3-EYFP embryos or cochleae were fixed in 4% paraformaldehyde for 30 minutes or overnight at 4\u00b0C. To examine the stereocilia in the sensory hair cells, organs of Corti were stained with fluorescein- rhodamine-conjugated phalloidin (Molecular Probes). Both native YFP signal and fluorescent staining was observed using an Olympus FV 1000 or a Zeiss LSM 510 confocal scanning microscope.To visualize Dvl3 expression, To measure the severity of the patterning defect, the whole cochlea duct was scanned and the cells numbered for length reference. The number of cells in the defective regions (where there were no longer three outer hair cell rows) was then calculated as a percentage of total cells.Figure S1Dvl3 deficient mice. A Dvl3 genomic clone was isolated from a 129 genomic DNA library in FIX II (Stratagene) as previously described Dvl3 genomic fragments were subcloned into pBluescript KS II (Stratagene) to enable efficient generation of a Dvl3 knock-out construct (A). A 3.6 kb BamH-Bgl2 fragment from within intron 1 to the middle of exon 2 and a 2.5 kb Dra1-Not1 (from vector MCS) fragment from within intron 7 to beyond 13 were cloned into pPNT 2 litters contained wild type and heterozygous offspring. \u2212/\u2212Dvl3 embryos were collected from crosses between heterozygotes and western blot analysis using E13.5 whole embryo lysates confirmed the absence of Dvl3 protein (C). PCR primers (listed in Targeted disruption and generation of (0.5 MB TIF)Click here for additional data file.Figure S2Dvl3 transgene. An EYFP-tagged Dvl3 transgene was generated using homologous recombination of BACs (A), as described previously Dvl3 genomic region, including flanking sequences, were identified using overlapping PCR primers covering the entire region from a BAC library (Genome Systems). BAC modifications were performed as previously described Dvl3 and LoxP sites were introduced within intron 2 and the 3\u2032 UTR flanking region. Western blot analysis of transgenic mouse E13.5 embryo lysates was used to confirm expression of the transgene, which was larger than the wild type Dvl3 protein due to the additional EYFP (3B). PCR primers .Generation of EYFP-tagged (3.8 MB TIF)Click here for additional data file."}
+{"text": "Id3, a transcriptional inactivator, was selected for further investigation. Id3 mRNA was expressed at reduced levels in 6 out of 9 ovarian cancer cell lines compared to the HOSE cells while at the protein level, all 7 ovarian cancer cell lines examined expressed the Id3 protein at greatly reduced levels. Expression of Id3 mRNA was also examined in primary ovarian tumours and was found in only 12/38 (32%) cases. A search was conducted for mutations of Id3 in primary ovarian cancers using single stranded conformation polymorphism (SSCP) analysis. Only one nucleotide substitution, present also in the corresponding constitutional DNA, was found in 94 ovarian tumours. Furthermore no association was found between LOH at 1p36 and lack of expression of Id3. These data suggest that Id3 is not the target of LOH at 1p36. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comThe molecular events that drive the initiation and progression of ovarian adenocarcinoma are not well defined. We have investigated changes in gene expression in ovarian cancer cell lines compared to an immortalized human ovarian surface epithelial cell line (HOSE) using a cDNA array. We identified 17 genes that were under-expressed and 10 genes that were over-expressed in the cell lines compared to the HOSE cells. One of the genes under-expressed in the ovarian cancer cell lines,"}
+{"text": "To evaluate the tolerance of the cirrhotic liver to extended warm ischaemia, 47 patients with cirrhosis who underwent liver,resection over a 4-year period were studied retrospectively. Three groups of patients were identified. In group 1 (14 patients) liver resection was performed under conditions of portal triad occlusion ranging from 50 to 75 (mean 57.1) min. Group 2 (12 patients) was treated with portal occlusion for a period ranging from 30 to 42 (mean 33.1) min. Group 3 comprised 21 patients who underwent  hepatectomy using conventional techniques. Mean blood loss was significantly reduced by portal triad occlusion  compared with the conventional techniques (1652 ml in group 3) . Hospital death occurred in three of the 21 patients in group 3 but in no patient who underwent portal triad occlusion. The morbidity rate was lower in the two occlusion groups (four of 26 patients) than in group 3 (six of 21). Bilirubin metabolism was substantially better after surgery in the occlusion groups . Although the serum levels of transaminases were significantly raised until day 3 in patients undergoing long term occlusion, the cirrhotic liver withstood the ischaemia-reperfusion insult, as assessed by changes in hepatic microcirculation, lipid peroxidation and the morphology of hepatic sinusoids. It is concluded that prolonged ischaemia during liver resection can be sustained in patients with cirrhosis and without high-risk factors."}
+{"text": "Breast cancer is the most common cancer and cause of deaths in women around the world. Oncogene amplification usually occurs late in tumor progression and correlates well with aggressiveness of tumor. In fact the function of the S100A4 protein and its role in metastasis is unclear at present. The purpose of the study was to determine the expression of S100A4 protein in the invasion status and metastatic potential of breast cancer by using tissue microarray and to determine its role in breast cancer based on the expression of S100A4 gene product.S100A4 protein expression was examined by immunohistochemistry (IHC) using commercially available tissue microarray containing malignant and normal breast tissue cores from 216 patients.S100A4 was absent in normal breast tissues while positive in 45.1% of infiltrating ductal carcinoma (IDC) node negative and 48.8% of infiltrating lobular carcinoma node negative. In paired samples, S100A4 protein was expressed in 13.5% of IDC node positive cases and 35.1% of matched lymph node metastasis.S100A4 protein expression appears widely expressed in early and advanced breast cancer stages compared with normal breast. Our study suggests S100A4 may play a role in breast cancer progression and may prove to be an independent marker of breast cancer which appears to be down regulated in more advanced stages of breast cancer. Breast cancer is still the most common public health problem in women worldwide . Cancer The metastasis process is not a random process but consist of a complex series of linked and interrelated steps involving multiple host-tumor interaction . Many prBreast cancer is considered to be a systemic disease this would mean that the most breast carcinoma metastasize before diagnosis of the primary lesion . TherefoThe S100 gene family located on chromosome 1q21, comprises more than 20 members whose protein sequences encompass at least one EF-hand Ca++ binding motif ,31. S100Studies to determine the mechanistic basis for S100A4 function have shown a potential role for S100A4 in several different facts of tumor progression including motility, invasion, and apoptosis ,41, and , and 41,Formalin-fixed paraffin-embedded tissue microarrays from 188 lymph node negative breast cancer patients, 50 breast cancer patients with lymph node metastasis  and 8 normal breast tissue cores were analyzed by immunohistochemistry for the expression S100A4 protein. Included in this study were patients with infiltrating ductal carcinoma, infiltrating lobular carcinoma, normal breast tissue and lymph node metastasis. The final number was 122 tissue cores of node negative infiltrating ductal carcinoma, 41 node negative infiltrating lobular carcinoma, seven normal breast tissue, 40 node positive  and 46 tissue cores of lymph node metastasis. We did not evaluate infiltrating lobular carcinoma node positive because of the low sample size (2 cases). Forty six tissue cores were excluded from statistics because very little cancer cells or no breast cancer tissue were seen. The total number after exclusion was 254 tissue cores (37 matched tissues) of 216 patients.Rabbit Anti-Human polyclonal primary antibody against S100A4 protein  was used on deparaffinized tissue microarray slides . A secondary detection system (DAKO Envision) enhanced with conjugated polymer was used to bind with the primary antibody. DAB chromogen was used for permanent color development and detection under microscope.The percentage of carcinoma cells with cytoplasmic/membranous/nuclear staining was recorded on each specimen at 200\u00d7 magnification, using light microscope. The expression of S100A4 was scored in all tumors as: positive \u2265 5% and negative < 5% stained cells. Also the intensity of staining was categorized into three groups: weak, moderate and strong. This was ascertained by a single qualified pathologist.A tissue section of breast cancer was used as positive control for S100A4. Rabbit IgG isotype  was used instead of primary antibody in the immunohistochemical technique on a tissue section each of breast cancer and normal breast as negative control  and heated on microwave. Slides were allowed to cool at room temperature and rinsed with Tris Buffered saline (TBS) mixed with tween 20. Slides were covered with peroxidase blocking solution (DAKO), followed by rinsing with TBS buffer mixed with tween 20. Then 200 \u03bcl of primary antibody (dilution 1:200) was added on the tissue microarray slides, followed by rinsing with TBS mixed with tween 20. Two drops of DAKO Envision/HRP, Rabbit/Mouse (secondary antibody) were added on the slides, followed by rinsing with TBS mixed with tween 20. After that DAB substrate (DAKO) was added on section slides followed by rinsing, immersion into hematoxylin and 4 different concentrations of ethanol. After that slides were immersed in two changes of xylene and cover slipped. All incubation steps after heat induced epitope retrieval were carried at room temperature.The S100A4 protein was expressed in the cell cytoplasm without evidence of nuclear staining.There were a total of 122 cases of infiltrating ductal carcinoma node negative and 41 cases of infiltrating lobular carcinoma node negative. Infiltrating ductal carcinoma node positive consisted of 38 cases. Thirty seven cases had paired primary infiltrating ductal carcinoma tissue with its matched lymph node core. There were also 9 cases of unrelated lymph node cores containing metastatic deposits.A positive expression of S100A4 was observed in 45.1% 55/122) cases of infiltrating ductal carcinoma node negative  cases in the paired samples (primary breast carcinoma and matched lymph nodes) showed presence of S100A4 protein in the primary site, while 13/37 (35.1%) cases showed S100A4 in lymph node  paired cases (primary breast cancer with matched lymph node) showed expression of S100A4 in the lymph node metastasis with absent expression in the primary site. Four (23.5%) cases showed S100A4 expression in the primary breast carcinoma with absent expression in its matched lymph node metastasis. One case showed S100A4 expression in both sites.Cancer is a disease or disorder characterized by uncontrolled growth (division) of the cells -55 and tIn the present study, S100A4 protein expression was examined by immunohistochemistry in infiltrating ductal, infiltrating lobular carcinoma and lymph node metastasis and their relation to tumor promotion and progression. Positive expression of S100A4 was observed in 45.1% of the infiltrating ductal carcinoma node negative cases, while in infiltrating lobular carcinoma with node negative, the expression of S100A4 protein was observed in 48.8%. This shows that S100A4 has a similar expression level in both infiltrating ductal carcinoma and infiltrating lobular carcinoma with node negative. S100A4 staining was not observed in normal breast tissues. S100A4 was expressed in a higher percentage in breast cancer tissue compared to normal tissue which showed direct correlation of S100A4 protein in infiltrating breast cancer node negative. This suggests that S100A4 is over expressed in infiltrating breast carcinoma compared to normal breast. Positive expression of S100A4 protein was observed only in 13.5 % of cases of infiltrating ductal carcinoma node positive while positive expression of S100A4 protein was observed in 35.1% of matched lymph node metastasis. These results showed there was a decrease in expression of S100A4 in infiltrating ductal carcinoma node positive (13.5%) compared with IDC node negative (45.1% positive staining), but interestingly there is an increase of expression of S100A4 protein at metastatic lymph node site. This suggests S100A4 may play a role in advanced breast cancer especially with lymph node metastasis. It was also interesting to note that the expression was seen in one site i.e. either primary tumor or its metastatic lymph node only.The majority  of paired samples showed that when there was a positive expression of S100A4 protein in metastatic lymph node, there was associated negative expression in the primary tumor  of the same patient. One case showed positive expression in both primary tumor and its metastatic lymph node at the same time. This was comparable with another study which showed that S100A4 over expression directly correlated with tumor progression . This stIn conclusion S100A4 protein expression appears to be expressed widely in early and advanced stages of breast cancer compared with normal breast. This study indicates a complex role of S100A4 in breast cancer of different types and stages. The difference in the expression of S100A4 protein suggests it may be useful as an independent marker of breast cancer which appears to be down regulated in more advanced stages of breast cancer. However a larger study with more ILC and metastatic cases may clarify the role and function of S100A4 in breast cancer progression.IHC: Immunohistochemistry; IDC: Infiltrating ductal carcinoma; ILC: Infiltrating lobular carcinoma.NII carried out Immunohistochemical part of the study and lab work, participated in drafting the manuscript. GK carried out the pathological part of the study, participated in drafting the manuscript. HH performed the statistical analysis. MSH initiated the project, participated in drafting the manuscript. All authors read and approved this manuscript."}
+{"text": "Approximately nine out of ten screen-detected prostate cancers were localized  and well or moderately differentiated  grade I and 50% grade II), which suggests a higher proportion of curable cancers compared with cases detected by other means. \u00a9 1999 Cancer Research CampaignApproximately 20 000 men 55\u201367 years of age from two areas in Finland were identified from the Population Registry and randomized either to the screening arm (1/3) or the control arm (2/3) of a prostate cancer screening trial. In the first round, the participation rate in the screening arm was 69%. Of the 5053 screened participants, 428 (8.5%) had a serum prostate-specific antigen (PSA) concentration of 4.0 ng/ml or higher, and diagnostic examinations were performed on 399 of them. A total of 106 cancers were detected among them corresponding to a positive predictive value of 27%, which is comparable with mammography screening for breast cancer. The prostate cancer detection rate based on a serum PSA concentration of 4.0 ng ml"}
+{"text": "Lymph node and peripheral blood lymphocytes were studied simultaneously for surface markers of T and B cells in 22 patients with lymphoproliferative diseases and 8 patients with non-neoplastic lymphadenopathy. This resulted in the classification of the malignancy from involved lymph nodes into 4 groups. Six patients had B cell lymphomata with normal or strong immunofluorescent staining for surface membrane immunoglobulin; 8 patients had B cell chronic lymphocytic leukaemia with pale staining for surface membrane immunoglobulin; 5 patients had T cell lymphomata and 3 patients were not definitely classifiable. In 6 out of 8 patients with B cell CLL, histopathology of lymph nodes showed infiltration with well differentiated lymphocytes and in all T cell lymphomata, the infiltrating cells were poorly differentiated. By the use of these markers, malignant lymphocytes were identified in the circulation in only 3 out of 6 patients with B cell lymphoma, in all patients with B cell CLL but in none of those with T cell lymphoma or unclassifiable lymphoma. Therefore a more conclusive characterization of the malignant lymphocyte in lymphoproliferative diseases must include an examination of involved lymph nodes."}
+{"text": "It was found:C3H/He and CBA/T6T6 mice which share the H2(1) The tumour grows at the same rate with the same median survival time in matched groups of non-immunized mice from both strains after i.p. injection of tumour cells.7 BP8, but this dose, and more intensive treatment with this drug, fails to cure C3H mice.(2) Cyclophosphamide (Cyclo) at 10 mg/kg will cure CBA mice which have received i.p. injections of 10125IUdR-labelled tumour cells and counting 125I loss by whole-mouse counting shows that the cytotoxic effect of Cyclo against BP8 is similar in the 2 mouse strains.(3) Injecting 7 BP8 followed by 10 mg/kg Cyclo.(4) Cyclo itself does not cure CBA mice, for viable tumour cells are recoverable from the peritoneal cavity 10 days after CBA mice have received 10(5) CBA mice cured of BP8 ascites by Cyclo treatment will reject further i.p. inocula of BP8.(6) The strength of immunity induced by irradiated BP8 cells was directly related to the length of exposure to this antigen. An important aspect of Cyclo treatment is that it prolongs the period during which immunity may develop.(7) Immunization of CBA mice with heavily irradiated BP8, with or without Cyclo, failed to show that Cyclo depressed the capacity of CBA mice to develop cytotoxic immunity. There was some indication that animals immunized with irradiated cells plus drug did better than those with irradiated cells alone.(8) A single injection of irradiated BP8 cells into CBA mice induced weak cytotoxic immunity, as assessed by destruction of a subsequent challenge with BP8, but these mice died from tumour more rapidly than non-immunized controls. It is suggested from these data that immunological enhancement may not always be due to blocking of cytotoxic immunity."}
+{"text": "Interleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines.IL11 levels in uterine lavage were measured by ELISA. IL11, IL11 receptor(R) \u03b1, phosphorylated (p) STAT3 and SOCS3 were examined by immunohistochemistry in endometrial carcinomas and in control endometrium from postmenopausal women and normal cycling women. The effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance in endometrial cancer cell lines and non-cancer endometrial epithelial cells was determined by Western blot.IL11 was present in uterine flushings and was significantly higher in women with Grade 1 carcinomas compared to postmenopausal women (p < 0.05). IL11 immunostaining was significantly elevated in the endometrial tumour epithelial cells from Grade 1 and 3 compared to endometrial epithelium from postmenopausal and cycling women. IL11R\u03b1 immunostaining intensity was increased in cancer epithelium in the Grades 1 and 2 tumours compared to epithelium from postmenopausal women. Both IL11 and IL11R\u03b1 localized to vascular endothelial and smooth muscle cells while IL11 also localized to subsets of leucocytes in the cancer tissues. pSTAT3 was found in both the tumour epithelial and stromal compartments but was maximal in the tumour epithelial cells, while SOCS3 was predominantly found in the tumour epithelial cells. pSTAT3 staining intensity was significantly higher in Grade 1 and 2 tumour epithelial cells compared to epithelial cells from cycling and postmenopausal women. SOCS3 staining intensity did not differ between between each tumour and postmenopausal endometrial epithelium but SOCS3 in cycling endometrium was significantly higher compared to postmonopausal and Tumour Grades 2 and 3. IL11 increased pSTAT3/STAT3 in all tumour cell lines, while SOCS3 abundance was increased only in one tumour cell line.The present study suggests that IL11 in uterine washings may be useful as a diagnostic marker for early stage endometrial cancer. It indicates that IL11, along with its specific receptor, IL11R\u03b1, and downstream signalling molecules, STAT3 and SOCS3, are likely to play a role in the progression of endometrial carcinoma. The precise role of IL11 in endometrial cancer remains to be elucidated. Endometrial cancer is the most common gynaecological malignancy . Since iInterleukin (IL) 11 belongs to the IL6 family of cytokines and signals via a heterodimeric complex of IL11 receptor (R) \u03b1 and gp130. The cellular responses of IL11 are induced by the activation of downstream Janus kinases (JAK) that phosphorylate the latent cytoplasmic transcription factors, signal transducer and activator of transcription (STAT) . PhosphoNumerous studies have suggested that IL11 has roles in human gastric, prostate and bone cancer -17. In aIn the current study, we determined the levels of IL11 in uterine lavage in women with endometrial cancer and postmenopausal controls. We compared IL11, IL11R\u03b1, pSTAT3 and SOCS3 protein in human endometrial carcinomas of varying histologic grades with endometrium from postmenopausal and cycling women. We determined the effect of IL11 on its downstream signaling molecules in endometrial cancer and non-cancer endometrial epithelial cells.Endometrial cancer tissue biopsies N = 16) were collected from postmenopausal women undergoing total abdominal hysterectomy for endometrial carcinoma at the Monash Medical Centre Melbourne, Australia. The Human Ethics Committee approved the research project and informed consent was obtained from each patient participating in this study. Details of individual patients are provided in Table 6 were coFor immunohistochemistry studies: There were 16 cancer patients, with an age range of 34-88 years (mean age = 64.4 years with standard deviation = 14.1)  and postmenopausal controls .Uterine lavages (uterine washings) were collected from postmenopausal women (N = 4) and women with endometrial cancer above except for women with Grade 1 carcinoma where washings were collected from 4 women instead of 5 women  as previously described . Uterine2O2 in methanol for 10 min. Non-specific staining was blocked using a blocking solution of 10% normal horse serum  and 2% normal human serum, diluted in 1\u00d7 Tris-buffered saline (TBS) for 30 min. Primary antibodies were diluted to 4 \u03bcg/ml in blocking solution and applied for 18 h at 4\u00b0C. A non-immune isotype IgG negative control  diluted to a matching concentration as the primary antibody, was also included for each tissue. Antibody localisation was detected by sequential application of biotinylated horse anti-mouse IgG  diluted 1:200 in blocking solution for 30 min and an avidin-biotin complex conjugated to HRP . The substrate used was diaminobenzidine (DAB)  forming an insoluble brown precipitate. Sections were then counterstained in Harris hematoxylin . Sections from normal endometrium were used as positive controls and included in each immunostaining run to provide quality control.Immunohistochemistry for IL11 and IL11R\u03b1 was performed as described previously  using a Immunohistochemistry for pSTAT3 and SOCS3 was conducted using polyclonal rabbit anti-mouse  and monoclonal rabbit anti-human (Clone C204)  antibodies respectively as previously shown , at fina2O2 in methanol for 10 min. Non-specific staining was blocked using blocking solutions consisting of 10% normal swine serum (in-house) and 2% normal human serum for pSTAT3 and 10% normal goat serum (Vector Laboratories) and 2% normal human serum for SOCS3, each diluted in 1\u00d7TBS for 30 min. Primary antibodies were diluted in the appropriate blocking solution and applied for 18 h at 4\u00b0C. A non-immune isotype IgG negative control (R&D Systems) diluted to a matching concentration as the primary antibody, was also included for each tissue. Antibody localisation was detected by sequential application of biotinylated swine anti rabbit IgG  or biotinylated goat anti-rabbit IgG (Vector Laboratories) diluted 1:200 in blocking solution correspondingly for 30 min and an avidin-biotin complex conjugated to HRP . The substrate used was diaminobenzidine (DAB) (Zymed), which forms an insoluble brown precipitate. Sections were then counterstained in Harris hematoxylin (Sigma Diagnostics). Sections from normal pre-menopausal endometrium were used as positive controls and included in each immunostaining run to provide quality control.Formalin fixed sections were deparaffinized in histosol and rehydrated in a graded series of ethanol. Endogenous activity was blocked by incubation in 3% HThe endometrial carcinoma cells ECC-1, HEC-1A and Ishikawa cells were cultured in DMEM/F12 (1:1), McCoy's 5A and DMEM  respectively supplemented with 10% fetal calf serum , 1% L-glutamine (Sigma-Aldrich Pty. Ltd) and 1% antibiotic-antimycotic . The non-cancer human endometrial epithelial (HES) cell line  was obtaThe endometrial cancer cell lines ECC-1, HEC-1A and Ishikawa and or HES cells were treated with diluents control, IL11  for 15 minutes or 4 hours. Phosphorylated STAT3 and total STAT3 abundance (15 min) and SOCS3 protein abundance  were analysed by Western blot as previously described  and brieAll in vitro cell culture experiments were performed in two independent experiments in duplicate.Positive staining was scored semiquantitatively by two independent observers, blind to the identity of the tissue, with an intensity score assigned as 0 (negative) to 3 . All statistical analyses were performed using GraphPad Prism. Data was analysed by the non-parametric Kruskal-Wallis test followed by the Dunn's post-hoc test. Differences were considered significant at P < 0.05.IL11 was detectable in 3 of 4 postmenopausal controls and in all the flushings from the Grade 1-3 tumours  and the endometrial epithelial cell line HES, secreted very low levels of IL11 under serum free conditions. The cells were subsequently cultured in serum free conditions to examine the effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance.Overall, all the human endometrial cancer cell lines  cells were treated with IL-11 for 4 hours and SOCS3 abundance examined at 0 (before treatment) and 4 hrs as previously described . SOCS3 pThis study was the first to show that IL11 protein was increased in uterine fluid and endometrial tumour epithelial cells in women with Grade 1 endometrial carcinoma compared to postmenopausal women. It further demonstrated that IL11's main endometrial signalling molecules, pSTAT3 and SOCS3, were produced by endometrial cancer cells. IL11 was shown to signal via pSTAT3 and SOCS3 in human endometrial cancer cell lines.Endometrial glandular epithelial products are primarily secreted apically into the uterine lumen therefore we investigated the levels of IL11 in uterine flushings. In agreement with our study, a previous study has suggested that factors present in uterine washings may confirm the presence of endometrial cancer . IL11 lePrevious studies have shown that in cycling endometrium, IL11 and IL11R\u03b1 predominantly localise to human endometrial glandular epithelium and decidualized human endometrial stromal cells ,28. EndoIn agreement with our study, IL11 localised predominantly to cancer epithelial cells in a recent report . IL11 mRIL11R\u03b1 protein was upregulated in endometrial epithelial tumour cells compared to endometrial epithelium from postmenopausal women. Strong staining for both IL11 and IL11R\u03b1 was identified in tumour vascular endothelial and smooth muscle cells as recently reported .IL11 localised to leukocytes only in the advanced Grade 3 tumours and not in control postmenopausal endometrium. Numerous studies report that tumour associated macrophages promote angiogenesis and correlate with poor prognosis . In endoIn agreement with the present study, IL11 is significantly upregulated in several non-endometrial cancers. IL11 and IL11R\u03b1 transcript levels are linked to breast cancer prognosis - breast tumours with a poor prognostic index show a high level of IL11 . SimilarTumour development and progression depends on cell adherence to extracellular matrix, proliferation, migration and invasion of tumour cells followed by their metastasis into other tissues and on escaping immune detection and destruction. Our previous studies show that IL11 increases the adhesion of human endometrial epithelial cells to various extracellular matrix molecules and to human trophoblast, at least in part by regulating adhesion molecule mRNA expression and protein production . EndometIt remains to be determined whether IL11 similarly regulates tumour cell adhesion, migration and invasion in endometrial cancers. Angiogenesis is also a key determinant of tumour formation  and hencIL11R\u03b1 protein is a proposed candidate target for both human osteosarcoma and also bone metastasis . FurtherOverall, SOCS3 imunostaining intensity was low in epithelium from postmenopausal women and all tissues from the cancer patients. There was higher SOCS3 staining in endometrial glandular epithelium from proliferative phase endometrium compared to all other groups. This suggests that SOCS3 has different functions in cycling endometrium compared to endometrium from postmenopausal women and endometrial cancer.IL11 increases pSTAT3 and SOCS3 protein in differentiating human endometrial stromal cells . STAT3 wSeveral studies have shown that SOCS proteins including SOCS3 are expressed in tumours including head and neck cancer ,40, gastIn the present study SOCS3 staining intensity was absent or very minimal in tumour epithelial cells in the Grade 3 cancer specimens perhaps similarly indicating a reduced sensitivity to SOCS3 in endometrial cancers although this remains to be determined. In normal breast epithelial cells SOCS3 is induced, while in several breast cancer cell lines SOCS3 is weakly activated. In breast tumour cells, it has been postulated that the IFN\u03b3 induced anti-proliferative effects are reduced due to a lower sensitivity to SOCS3 induction . Our in Our study suggests that IL11 in uterine washings may be useful as an early marker of endometrial cancer. It is also the first study to demonstrate that IL11 protein is upregulated in Grade 1 endometrial cancers compared to postmenopausal endometrial epithelium and suggests that IL11 signalling is active in endometrial cancer cells. The present study suggests that IL11, along with its specific receptor and downstream signalling molecules pSTAT3 and SOCS3, are likely to play a complex role in the progression of endometrial carcinoma. Functional studies are required to elucidate the role of IL11 in tumourigenesis and determine its potential as a prognostic marker and therapeutic target for endometrial cancer. Large scale studies are required to determine whether IL11 in uterine washings may be useful as a diagnostic marker for endometrial cancer.The authors declare that they have no competing interests.JY performed immunohistochemistry, quantitative ELISA, western blots, data analysis and assisted in drafting the manuscript. LAS contributed to tissue grading and collection of biopsies. TJ co-ordinated patient recruitment, tissue cancer grading and collected the biopsies. PKN provided technical assistance with the immunohistochemistry. ED conceived of the study, designed and co-ordinated the study, participated in data analysis and interpretation and drafted the manuscript. All authors read and approved the manuscript."}
+{"text": "Flavone acetic acid (FAA), 8.6 gm-2 has been administered by 6h intravenous infusion to 19 patients with advanced colorectal carcinoma and 15 patients with advanced malignant melanoma. The drug associated toxicity was generally mild and as predicted from the phase I study. No responses were seen in either disease."}
+{"text": "Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation.We show that  Adult mammalian muscle has the potential to regenerate by activation of undifferentiated myogenic precursor cells (satellite cells), which are normally quiescent and situated between the basal membrane and the myofibers It was previously suggested that dedifferentiated muscle cells became multipotent and could contribute to development of not only new muscle but also cartilage and bones msx-1 is induced in the blastema msx1 in mouse C2C12 myotubes induces cleavage of myotubes into proliferating, mononucleated cells Factors controlling dedifferentiation in newt limb are not well understood; however, expression of the muscle segment homeobox gene Barx2 in regulation of myofiber plasticity. During early embryonic stages of mouse development, Barx2 is widely expressed in proliferating and differentiating cartilage and muscle tissue Barx2 expression was found to be restricted to the joint region Barx2 cooperates with other muscle-expressed transcription factors to regulate cytoskeletal remodeling events of early myoblast differentiation In this study we investigated the role of the homeodomain transcription factor Barx2 over-expression, while more mature myotubes appear to have lost this ability. These results also suggest that Barx2, like Msx1, can regulate muscle plasticity and that homeobox factors could be a part of a general mechanism that controls the susceptibility of cells to reprogramming.Here we show that ectopic expression of Barx2 in C2C12 myotubes and MyoD-induced C3H10T1/2 myotubes induced apparent dedifferentiation indicated by myotube cleavage and concomitant down-regulation of muscle differentiation markers. We also extended these studies to differentiated primary mouse muscle cell cultures; we observed two types of myotubes in primary cultures: thin slowly contracting myotubes with nuclei aligned along the middle of the fiber (immature myotubes), and thicker, faster contracting myotubes often with small or large nuclei clusters (mature myotubes). Microinjection of Barx2 cDNA into the immature myotubes induced cleavage and formation of mononucleated cells that were able to proliferate, whereas injection of Barx2 cDNA into mature myotubes induced myotube contraction but not cleavage. Thus our data indicate that only immature myotubes can dedifferentiate in response to in vivo and is upregulated early during differentiation of C2C12 and C3H10T1/2 cells and primary myoblasts in culture. However, we do not observe Barx2 in the nuclei of mature myofibres  into immature (stage 1) or mature (stage 2) myotubes . To visuBarx2 can promote dedifferentiation of single immature myotubes.In contrast to the results in immature myotubes, injections of Barx2-plasmid into mature myotubes did not induce cleavage ; howeverBarx2 could induce re-entry of myotube-derived cells into the cell cycle, we examined whether Barx2 might affect cell cycle genes directly. Quantitative RT-PCR analysis of C2C12 cells stably transfected with a Barx2-expression plasmid showed that the cyclin D1 gene was upregulated approximately 9-fold relative to control-transfected cells  and accelerates differentiation SMA expression is known to be upregulated at an early stage of myoblast differentiation, possibly to facilitate migration and remodeling, and is subsequently downregulated and replaced by skeletal muscle actin SMA gene and can directly bind to the SMA promoter together with MyoD and increase transcriptional activation of the promoter by MyoD in undifferentiated myoblasts The data presented here combined with our previous work SMA promoter by Barx2 and MyoD was examined by co-transfection of the SMA promoter-luciferase construct with Barx2, MyoD, or empty pcDNA3 expression plasmids in C2C12 cells  family of bHLH factors act as transcriptional repressors and have roles in maintaining progenitor cells in an undifferentiated state and regulating cell fate decisions during embryogenesis. For example, lack of certain Barx2 early in myoblast differentiation as previously investigated The homeobox protein Barx2 is expressed during development of skeletal and smooth muscle Msx1Msx1Msx1 is involved in epimorphic digit tip regeneration in both humans and mice Barx2 and Msx1 share similar pathways and even target genes in the regulation of dedifferentiation. However, an important difference between the functions of Barx2 and Msx1 is that Barx2 is also able to promote the early stages of myoblast differentiation in cooperation with MyoDMsx1; on the contrary expression of Msx1 in undifferentiated C2C12 myoblasts downregulates MyoD expression and inhibits differentiation into myotubes These finding were similar to the previously described functions of another homeodomain transcription factor - Msx1 a number of homeobox factors are capable of suppressing myogenic differentiation. These include Mohawk which is a transcriptional repressor that blocks the myogenic conversion of C3H10T1/2 cells Pax3 and Pax7 paired homeodomain transcription factors have also been shown to suppress myogenic differentiation and both the paired domain and homeodomain of Pax3 are required for this anti-myogenic effect Msx1, Mohawk, and Pax3/7 homeobox containing transcription factors appear to be general repressors of myoblast differentiation. In contrast our previous and current studies indicate that Barx2 positively regulates the early steps of myoblast differentiation; however, its subsequent down-regulation may be necessary for maintenance of the differentiated state of myofibers.It is interesting that in addition to Barx2 in C2C12 cells could be an important factor in promoting cell cycle re-entry by myonuclei derived from dedifferentiated myofibres. Re-entry into the cell cycle has been also induced in terminally differentiated cultured cardiomyocytes by expression of G1 cell cycle factors Barx2 regulates a transcriptional program that allows cellular remodeling to be coordinated with cell cycle re-entry.The upregulation of cyclin D1 expression after overexpression of Barx2 at early and late stages of muscle differentiation are unclear, they are very likely to involve differential recruitment of activator or repressor complexes by Barx2 Barx2 function at different stages of myogenic differentiation.Although mechanisms underlying differential gene regulation by Xenopus embryos, Hes6 increased the size of the myotome due to increased proliferation; it also decreased markers of terminal muscle differentiation Enforced expression of Hes6 has been found to inhibit differentiation, leading to a higher proportion of thin, immature myotubes relative to thickened, mature myotubes. Hes6 expression also reduced the proportion of cells undergoing cell cycle withdrawal and allowing more cells to re-enter the cell cycle after differentiation Hes6 expression in C2C12 myotubes relative to myoblasts Barx2 and Hes6, previous work showed that the repression domain of Barx2 binds directly to the transducin-like enhancer of split (TLE) corepressor family Hes6 and other HES family repressors Our corroborating finding of increased Oct, Nanog) have been shown to be important in induction of pluripotent stem cells (iPS cells). Recent work comparing zebrafish regeneration blastema with iPS cells showed that, although blastemal cells are not pluripotent, some of the key iPS reprogramming factors including the Pou5 homeobox protein are also important for regeneration, presumably due to their role in generating a multipotent cell state. This suggests some common mechanisms may be involved in induced cellular reprogramming and in the natural dedifferentiation process observed in the blastema Barx2 to the list of homeobox regulators of adult cellular plasticity. In future work it would be of considerable interest to assess the role of Barx2 in blastemal regeneration of muscle, perhaps in the zebrafish context.Homeodomain transcription factors regulate gene expression in response to a large variety of extracellular stimuli, and act as molecular switches for controlling cell differentiation, proliferation, and apoptosis. Particular homeobox genes (Mouse C3H10T1/2 (clone 8) and C2C12 (ATCC) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37C in a humidified 5% CO2 atmosphere without antibiotics.Primary myoblast cultures were prepared as described previously The Zeiss LSM 710 laser scanning confocal microscope (LSCM) was used to obtain images. IMARIS software was used for image analysis.7 C2C12 cells were transfected with either 10 \u00b5g of Barx2/pcDNA3 expression vector or pcDNA3 control plasmid using Lipofectamine2000 reagent (Invitrogen). The Barx2 expression plasmid contains an in-frame NH2-terminal Myc tag and was described previously 1\u00d710Total protein was prepared from C2C12 and C3H10T1/2 cells using RIPA lysis buffer and sonicated. Equal aliquots of protein were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies to MyoD , skeletal fast myosin , myogenin  and a custom-made Barx2 anti-peptide polyclonal antibody (Covance). \u03b2-actin, antibody was used as a reference. HRP conjugated secondary antibodies and a chemiluminescent detection system was used to visualize proteins. All experiments were performed in duplicates.Co-immunoprecipitation of Barx2 with Hes6 was performed as essentially as described in  Briefly, COS1 or C2C12 cells were co-transfected with the Barx2/pcDNA3 and Hes6/pCMVSport6 (Open Biosystems) expression plasmids using Lipofectamine-2000 (Invitrogen). Cell lysates were prepared 48 hours after transfection, pre-cleared with Protein A-Sepharose and then incubated overnight at 4\u00b0C with 5 \u00b5g of anti-Barx2 rabbit antibody or preimmune serum. Complexes were precipitated with Protein A-Sepharose, washed four times and resolved by SDS-PAGE and immunoblotted with anti-Hes6 polyclonal goat antibody (Santa Cruz Biotechnologies) antibody.ctcctgaaccacctgctagaatcc, reverse: ctaaggatgtagacaccaaatccggc, and GAPDH primers: forward: gtgaaggtcggtgtgaacggatttggccg; reverse: ccatggtggtgaagacaccagtagactcc. The Hes6 primer set amplified a 252 base-pair DNA segment of the Hes6 cDNA. The PCR products were resolved by agarose electrophoresis, bands were quantified by densitometry, and the ratio of Hes6/GAPDH PCR products was calculated. Three experiments were performed, and the results were normalized to the values from undifferentiated myoblast cultures. Statistical (t-test) analysis was performed using Microsoft Exel.RNA was isolated from undifferentiated C2C12 cells and C2C12 young and mature myotubes using the RNeasy Plus Mini Kit (Qiagen). The RNA was quantified using a Beckman DU 640 Spectrophotometer. 1 \u00b5g of each RNA sample was used to synthesize cDNA using a First-Strand cDNA Synthesis kit and SuperScript III/RNaseOUT Enzyme mix and 50 ng/ul random hexamer primers. The RT PCR was performed using Perkin Elmer 9600 PCR machine. Each sample was amplified with Hes6 primers: forward: gtatttgtctaccccagacaggtt, R: tcatcctgcgattctgatacc; mouse Cyclin D1 F: tctttccagagtcatcaagtgtg, R: gactccagaagggcttcaatc. Mouse GAPDH (NM_008084) (QIAGEN SaBiosciences) and mouse ribosomal protein S26 (RPS26) F: aggtgcagaaggctgagg, R: ggttctcccgagtgatgaag, were used as controls.RNA was prepared from primary myoblast or C2C12 cell cultures using Trizol reagent (Gibco). Cells were lysed directly in Trizol and RNA was prepared according to manufacturer protocol (Invitrogen). RNA was treated with DNase using the DNA-free kit (Ambion) and reverse transcribed using random primers and MMluV reverse transcriptase (New England Biolabs). Quantitative RT-PCR reactions were performed on an ABI 7300 machine using Superarray Biosciences RT2 SYBR green reagent and the following primers. Mouse Barx2 F: 4 1.25, NaHCO3 26, MgCl2 1, Glucose 25, CaCl2 2.Injections into myofibers were performed using traditional patch-clamp electrophysiology equipment In experiments to assess the proliferation of cells derived from microinjected myotubes, the cultures were maintained for an additional 4\u20135 days and then incubated with 5-bromo-2-deoxyuridine (BrdU)  for one hour. Cultures were then fixed and BrdU detection was performed using an Alexa Fluor\u00ae 594-conjugated secondary antibody according to the manufacturers protocol. Proliferating BrdU-labeled cells were detected as fluorescein-positive cells by confocal microscopy."}
+{"text": "To identify and understand physiological processes and behavior that depends on the InsP3 signaling pathway at a systemic level, we are studying Drosophila mutants for the InsP3R (itpr) gene. Here, we show that growth defects precede larval lethality and both are a consequence of the inability to feed normally. Moreover, restoring InsP3R function in insulin producing cells (IPCs) in the larval brain rescues the feeding deficit, growth and lethality in the itpr mutants to a significant extent. We have previously demonstrated a critical requirement for InsP3R activity in neuronal cells, specifically in aminergic interneurons, for larval viability. Processes from the IPCs and aminergic domain are closely apposed in the third instar larval brain with no visible cellular overlap. Ubiquitous depletion of itpr by dsRNA results in feeding deficits leading to larval lethality similar to the itpr mutant phenotype. However, when itpr is depleted specifically in IPCs or aminergic neurons, the larvae are viable. These data support a model where InsP3R activity in non-overlapping neuronal domains independently rescues larval itpr phenotypes by non-cell autonomous mechanisms.The Inositol 1,4,5- trisphosphate receptor (InsP It is known that InsP3R is widely expressed and its role in various cellular processes has been identified using in vitro studies 3R function in the context of whole organism physiology is not well understood.Calcium is a versatile signaling molecule that has been found to regulate a multitude of processes, from fertilization to cell death. The regulation of such diverse processes depends on the intricate regulation of calcium levels by an extensive toolkit that consists of calcium channels and pumps on the plasma membrane and the membrane of intracellular stores that help in assembling signaling systems with very different temporal and spatial dynamics Drosophila melanogaster, a model system amenable to genetic and physiological manipulations, has therefore been utilized to understand both systemic and cellular requirements for the InsP3R 3R activity could underlie a physiological output. By this process, we have previously demonstrated that InsP3R expression in the neuronal domain and specifically the aminergic interneurons (with the DdcGAL4) rescues larval viability itpr mutant phenotypes can be significantly rescued by restoring InsP3R activity in insulin producing cells (IPCs) with use of the Dilp2GAL4itpr mutants arise as a consequence of disrupted feeding behavior. An independent requirement of InsP3R activity in the prothoracic gland cells that synthesize and secrete the insect molting hormone ecdysone also exists. The Dilp2GAL4 and DdcGAL4 expression domains do not exhibit any obvious overlap suggesting that the Dilp2GAL4 rescue is mediated by a non-cell autonomous mechanism.In this study, we show that larval Drosophila itpr gene exhibit larval and adult phenotypes based on the strength of the heteroallelic combination. Stronger mutant combinations are larval lethal while adult viable combinations exhibit defective wing posture with reduced flight ability and altered flight physiology sv35/ug3itpr has been well characterized; a majority of these larvae die as second instars with a slightly extended lethality profile as compared with itpr null organisms ug3itpr is a hypomorph in which the single point mutation lies in the N-terminal ligand binding domain while sv35itpr is a null allele with a stop codon in the modulatory domain sv35/ug3itpr larvae are smaller in size as compared to wild-type controls  in the background of sv35/ug3itpr. A significant rescue of larval size was observed  or due to a feeding defect in these mutants or a combination of both. The feeding ability of itpr mutants was determined quantitatively by measuring ingestion of colored food  analysis  (which stimulates ecdysone synthesis in the prothoraic gland of the ring gland) producing neurons are ablated  animals . While 5 animals . Delays ng pupae  and pupailp2GAL4 . Pupal ailp2GAL4 . The timecdysone . Ecdysonitpr mutant phenotypes by restoring itpr function in the IPCs and aminergic neurons is that an overlap exists between the two domains. In order to determine this, a membrane bound GFP (UASmCD8GFP) was expressed with Dilp2GAL4 and larval brains of these animals were stained with an anti-Ddc antibody DdcGAL4 expresses in both serotonergic and dopaminergic neurons, itpr mutant phenotypes are not rescued by expression of +UASitpr in the dopaminergic domain  lines for the itpr gene and measured their effect on larval viability by ubiquitous expression with an Actin5cGAL4. Amongst the dsitpr lines tested, one line referred to as 1063UASdsitpr, does not survive beyond the larval stages on expression with the Actin5cGAL4 line  or with either Dilp2GAL4 or DdcGAL4 had no significant effect on larval viability or size (data not shown) as judged by the number and size of pupae formed  mRNA are present in the larval and adult nervous system Drosophila serotonin receptors 5-HT1BDro (d5-HT1B) and 5-HT2Dro have been observed in larval and adult brains 3-coupled 5-HT2CR is a key mediator of the serotonergic suppression of feeding and agonists of this receptor were found to significantly improve glucose tolerance and reduce plasma insulin in murine models of obesity and type 2 diabetes + channels that respond directly to glucose levels and signal insulin release are not present on Drosophila IPCs 3 pathway could be one such mechanism The absence of any cellular overlap between aminergic and DILP producing neurons suggests that these domains regulate feeding and growth through secreted serotonin and DILPs and thus communicate with each other or influence a common subset of downstream cells by binding of serotonin and DILP to their cognate receptors. High levels of sv35/ug3itpr is a heteroallelic combination of single point mutants in the itpr gene that were generated in an EMS (ethyl methanesulfonate) screen. Detailed molecular information on these alleles has been published itpr cDNA (+UASitpr) itpr RNAi experiments were done with the UASdsitpr (1063R-2) line from the National Institute of Genetics Fly Stock Center, Japan. The Dilp2GAL4 strain was from Dr. E. Rulifson DdcGAL4P0163GAL4Actin5cGAL4 (4414), C155GAL4Elav and UASdicer(III) (24651) were obtained from the Bloomington Stock Centre. The other fly strains used were generated by standard genetic methods using individual mutant and transgenic fly lines described above.To obtain molting profiles, staging experiments were performed with minor modifications as described previously g for 5 minutes and the supernatant was transferred to a fresh tube. The supernatant was mixed with PBS and the Abs520 read.Yeast paste containing red dye  was placed centrally on 90mm petri dishes plated with 2% agar in Phosphate Buffered Saline (PBS). Larvae of the appropriate age and genotype were placed on red yeast paste and allowed to feed for 4 hrs (at 60 hrs AEL) or 2 hrs (at 108 hrs AEL). After feeding, each group of larvae were washed in distilled water, dried on blotting paper and placed in 1.5 ml tubes and immediately frozen in liquid nitrogen. Larvae were then homogenized in PBS, centrifuged at 14 nd instar larvae of the indicated genotypes were selected at 56\u201364 h AEL and snap frozen in liquid nitrogen. Total RNA was extracted with Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Approximately 1 \u00b5g of purified total RNA was used for reverse transcription reactions. cDNA was generated using gene specific primers and MMLV reverse transcriptase (Invitrogen) at 42\u00b0C for 1 hour. Polymerase chain reactions (PCRs) were performed using cDNA as template in a 25 \u00b5l reaction. rp49 gene primers were used for internal normalization of every batch of RNA. The same sense and antisense primers were used for RT-PCR and Realtime PCR. Quantitative realtime PCRs were performed on the Rotor-Gene 3000  operated with Rotor Gene software version 6.0.34 using SYBR\u00ae Green JumpStart\u2122 Taq ReadyMix (Sigma). Experiments were performed with rp49 and the gene of interest, using serial dilutions  of the cDNA preparation. The experiment was repeated three times with independently isolated RNA samples. Cycling parameters were 95\u00b0C for 10 min, 45 cycles of 95\u00b0C for 20 s and 53\u00b0C (for rp49) and 55\u00b0C (for 4E-BP and Lipase-3) for 30 s, 72\u00b0C for 30 s, then 1 cycle of 72\u00b0C for 5 min and hold at 50\u00b0C for 1 min. The fluorescent signal produced from the amplicon was acquired at the end of the polymerization step at 72\u00b0C. A melt curve was also performed after the assay to check for specificity of the reaction. Amplification primers were as follows:2rp49, 5\u2032ATGACCATCCGCCCAGCATAC; 3\u2032TTACCTCGTTCTTCTTGAGAC; 4E-BP, 5\u2032CATGCAGCAACTGCCAAATC;3\u2032CCGAGAGAACAAACAAGGTGG ; Lipase-3, 5\u2032TGAGTACGGCAGCTACTTCCCT; 3\u2032TCAACTTGCGGACATCGCTDrosophila (Canton S) was determined by the comparative \u25b5\u25b5Ct method \u2212\u25b5\u25b5Ct where \u25b5\u25b5Ct\u200a=\u200a(Ct(target gene)\u2212Ct(rp49))mutant \u2212(Ct(target gene)\u2212Ct(rp49))Wild type.The fold change in the mutant's target gene cDNA relative to wild-type rp49 were as follows: 5\u2032CGGATCGATATGCTAAGCTGT; 3\u2032ATGCCTAGCTTGTTCGCG.Amplification primers used for the experiment in Drosophila larval brains expressing a membrane bound GFP (UASmCD8GFP) with the Dilp2GAL4 or DdcGAL4 that were fixed in 4% paraformaldehyde for 30 minutes. The following primary antibodies were used - rat anti-Ddc , rabbit anti-GFP antibody  and monoclonal anti-5-HT antibody . The following fluorescent secondary antibodies were used at a dilution of 1\u2236400 - anti-rabbit Alexa Fluor 488 and anti-rat Alexa Fluor 633  and anti-mouse Rhodamine Red X (Jackson Laboratories). Confocal analysis was performed on a Zeiss LSM 510 Meta microscope  or an Olympus Confocal FV1000 microscope using 20X 0.9 N.A. or 63X 1.4 N.A. objectives. Confocal data were acquired as image stacks of separate channels and combined and visualized as three- dimensional projections using the LSM5 version 3.2/SP2 software or FV10-ASW 1.3 viewer.Immunohistochemistry was performed on Salivary glands derived from 60 hr AEL larvae were dissected in PBS, fixed in 4% paraformaldehyde, and stained with DAPI to visualize nuclei. Images were acquired at different focal planes and the total number of nuclei per salivary gland was counted.rd instar larvae of the indicated genotype were run on a 5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane by standard western blotting protocols. The affinity purified anti-DInsP3R rabbit polyclonal antibody (IB-9075) raised against KLH-conjugated peptide CEQRKQKQRLGLLNTTANSLLPFQ derived from the DInsP3R sequence Protein extracts from 3t-tests was performed using Origin software  in all experiments.Computation of means, SEM, and Student"}
+{"text": "A series of plasma globulin studies was carried out on 108 patients who were operated on for splenictrauma during the last 3 years. The reasons for splenectomy or spleen salvage were; gunshot woundsin 22 patients (20.3%); stab injuries in 10 patients (9.2%) and blunt abdominal trauma in 76 patients(70.3%). Plasma gamma globulin determinations were made on the 8th postoperative day and at 3months. In the splenectomy group; plasma gamma globulin determinations demonstrated a significantreduction in serum IgM levels (p < 0.001) but no significant changes in IgA and IgG levels (p > 0.05).No changes were detected in IgA, IgG and IgM levels in the spleen salvage group (p > 0.05).        We believe that the preservation of the traumatized spleen should be the prime aim of surgeons."}
+{"text": "Changes in cardiac output may occur during insufflation for laparoscopic procedures. However, there are limited data regarding its potential effects on cerebral oxygenation.2), end tidal CO2, heart rate, blood pressure and oxygen saturation by pulse oximetry were recorded every 5 minutes prior to insufflation, during insufflation and after desufflation. Minute ventilation was increased to maintain normocapnia and the depth of anesthesia was adjusted or fluids/phenylephrine administered to maintain the blood pressure within 20% of the baseline.Cerebral oxygenation . Eighty-two (8.2%) of the values were less than 80% of the baseline value, while 25 values (2.5%) were less than 75% of the baseline value. Twelve patients had at least one ScO2 value that was less than 80% of the baseline and 6 had at least one ScO2 value that was less than 75% of the baseline. Four patients of the cohort had ScO2 values less than 80% of the baseline for more than 50% of the laparoscopic procedure.The cohort for the study included 70 adults for laparoscopic herniorrhaphy, gastric bypass or cholecystectomy. A total of 1004 ScOAlthough relatively uncommon, significant changes in cerebral oxygenation do occur in some patients during insufflation for laparoscopic surgery. Although CO2 is generally accepted as the optimal gas for insufflation, increases in PaCO2 and resultant alterations in cerebral blood flow may occur.[Laparoscopic surgery has become a popular surgical tool due to its less invasive nature, thereby providing a more rapid recovery and improved cosmetic outcome when compared to traditional open surgery. However, a requirement of the procedure is the insufflation of the peritoneal cavity with an inert gas, most commonly carbon dioxide (COay occur.\u20133 Additiay occur.\u20136 These 2), end tidal CO2 (ETCO2), heart rate (HR), blood pressure (BP) and oxygen saturation by pulse oximetry (SaO2) were recorded every five minutes prior to insufflation, during insufflation and after desufflation. Peritoneal insufflation pressures were maintained at 8\u201312 mmHg. The surgical procedures were performed with the patient in the supine or at a 20\u201330\u00b0 head-up position. During insufflation, minute ventilation was increased as needed, to maintain normocarbia. The blood pressure was maintained within 20% of baseline values by either increasing the concentration of the inhalational agent for increases in BP or the administration of a fluid bolus and/or phenylephrine for decreases in BP. The ScO2 values obtained prior to insufflation were averaged for each patient and used as that patient's baseline rSO2 for subsequent evaluations. The values obtained during insufflation were then compared to the baseline values and categorized as an increase in ScO2 from the baseline or as an absolute decrease in rSO2 of 0\u20139, 10\u201319, 20\u201329 or \u2264 30 or greater, from baseline. The number of values representing an ScO2 decrease to less than 75% and less than 80% of the baseline, were also determined. Patients who had any ScO2 value less than 80% of baseline, were compared to those without cerebral desaturation using a non-paired t-test and a contingency table, with a Fisher's exact test. All data are presented as the mean \u00b1 SD.This study was approved by the Institutional Review Board of the University of Missouri and informed consent was obtained from the patients. Patients scheduled for laparoscopic procedures of the abdomen requiring peritoneal insufflation were considered eligible for inclusion. The choice of anesthetic agents was not standardized and was at the discretion of the attending anesthesiologist. Cerebral oxygen saturation was continuously monitored using near infrared spectroscopy . After the induction of anesthesia, a single NIRS sensor was placed on the right side of the patient's forehead, with the caudad border approximately 1 cm above the eyebrow, with the medial edge at the midline according to the manufacturer's guidelines. Cerebral oxygen saturation (ScOThe cohort for the study included 70 adult patients ranging in age from 27 to 80 years (50.3 \u00b1 13.9 years) and in weight from 40 to 186 kgs (104.9 \u00b1 31.6 kgs). Twelve patients were ASA II and 58 were ASA III. The surgical procedures included laparoscopic herniorrhaphy or cholecystectomy in 34 patients and laparoscopic gastric bypass in 36 patients. The anesthetic technique included premedication with midazolam (1\u20132 mg), followed by intravenous induction with thiopental or propofol. Maintenance anesthesia consisted of 1\u20131.5 MAC  of a potent inhalational anesthetic agent, fentanyl (3\u20135 \u03bcg/kg) and neuromuscular blockade with intermittent doses of either cis-atracurium or rocuronium. The duration of insufflation and laparoscopy varied from 25 to 175 minutes (71.7 \u00b1 33.3 minutes).2 varied from 47 to 95 (78 \u00b1 11). A total of 1004 ScO2 values were obtained during laparoscopy. The ScO2 increased from the baseline during 246 of the 1004 data points and decreased from the baseline in 758 of the1004 data points. For the 758 of the 1004 ScO2 values that decreased from baseline during insufflation, the ScO2 decrease was 0\u20139 less than the baseline in 480 values , 10\u201319 less than the baseline in 252 values (24.9%) and 20\u201329 less than the baseline in 26 values (2.6%) [2 value was greater than 30 less than the baseline value. Eighty-two (8.2%) of the values were less than 80% of the baseline value, while 25 values (2.5%) were less than 75% of the baseline value. Twelve of the 70 patients (17%) had at least one ScO2 value that was less than 80% of the baseline value and 6 (0.09%) had at least one ScO2 value that was less than 75% of the baseline value. Four of the 70 patients of the cohort had ScO2 values less than 80% of the baseline, for more than 50% of the laparoscopic procedure. The demographics of patients with an ScO2 value less than 80% of the baseline during insufflation compared to patients without cerebral oxygen desaturation, are outlined in P =0.1). However, these patients weighed more , had longer durations of insufflation  and were more likely to be undergoing a laparoscopic gastric bypass than a laparoscopic herniorrhaphy or cholecystectomy.The baseline ScOs (2.6%) . No ScO23).[The potential use of NIRS, otherwise known as cerebral oximetry to monitor cerebral oxygenatio was first suggested by Jobsis in 1977.[3). Unlike s3).\u201311 Unlik3).\u201314et al[et al studied the response of the INVOS 3100A to arterial hypoxemia, induced by breathing a hypoxic mixture in 22 healthy adults and demonstrated that the cerebral oximeter reading correlated well with the calculated cerebral saturation.[The ability of NIRS to detect sudden changes in cerebral oxygenation when cerebral blood flow is altered by extreme maneuvers, has been demonstrated by Levy et al NIRS haset al16 Additiet al18 Pollaret al. compared changes in cerebral oxygenation using NIRS monitoring with neurologic damage assessed with biochemical markers, including neuron specific enolase (NSE) and S-100, a protein released during neuronal damage.[2 showed a significant correlation with the postoperative increase in NSE (r2 = 0.57) and S-100 (r2 = 0.52).Clinical and biochemical studies have validated the correlation of NIRS monitoring with eventual neurologic outcome. Although the majority of the literature using NIRS has compared periods of cerebral desaturation with outcome assessed by clinical assessment of neurologic status, Plachky l damage. The cohoet al have demonstrated that monitoring and acting on decreased ScO2 values may actually decrease the incidence of postoperative neurocognitive dysfunction in elderly patients undergoing major abdominal surgery under general anesthesia.[2 value was monitored and maintained at \u2265 75% of preinduction values or a monitor only group, in which cerebral oximetry was monitored, but not visible to the anesthesiologist. Cerebral oxygen desaturation (\u2264 75% of baseline) was observed in 11 patients of the treatment group (20%) and 15 patients in the control group (23%). The treatment protocol to treat cerebral oxygen desaturation, included ensuring adequate ventilation, checking head positioning, increasing the delivered oxygen concentration, allowing the arterial CO2 to increase and increasing the BP with fluids or vasoconstricting medications. Postoperative neurocognitive decline was evaluated using a mini-mental status examination (MMSE). Control patients with cerebral oxygen desaturation had a lower MMSE on postoperative day 7, longer PACU stays (median time of 47 minutes) and longer hospital stays (median of 24 days), when compared with patients in the treatment group whose cerebral oxygen desaturation was reversed by interventions. The latter group had a median PACU discharge time of 25 minutes (P =0.01) and a median hospital stay of 10 days (P =0.007).More recently, Casati esthesia. The 122 et al,[2 decrease to less than 75% of baseline correlates with neurocognitive changes assessed using the MMSE, 6 or approximately 1% of our patients would be at risk. Four of our patients had ScO2 values less than 80% of their baseline during more than 50% of the insufflation time. Patients who experienced an ScO2 value less than 80% of the baseline, weighed more and had a longer duration of peritoneal insufflation. As the majority of the patients were ASA III, aside from body weight, we cannot comment as to whether other co-morbid features increased the likelihood of cerebral oxygen desaturation.In the current study, we noted that the majority of patients experienced a decrease in cerebral oxygenation during insufflation. Although the modest decreases are unlikely to be clinically significant, if we use the criteria of Casati et al, who sugget al evaluated changes in cerebral oxygenation during laparoscopy using another type of cerebral oxygenation monitor , in a study that included 12 adult patients (ASA I or II) undergoing laparoscopic cholecystectomy with a pneumoperitoneum of 10\u201312 mmHg.[2 increased from a baseline of 33.9 \u00b1 1.3 to a maximum of 52.8 \u00b1 3.3 mmHg during laparoscopy. Both HbO2  and HbR (reduced hemoglobin) increased from the baseline following insufflation, which they related to changes in cerebral blood volume induced by the hypercapnia. No change in the oxygenation status of cytochrome aa3 was noted. The maximum increase of HbO2 (7.3 \u00b1 2.8 \u03bcmol/L) occurred 90 minutes after the start of laparoscopy.Previous studies have evaluated changes in cerebral oxygenation using NIRS during insufflation for laparoscopy. Kitajima \u201312 mmHg. Minute v2 from the baseline with insufflation (\u22121.4 \u00b1 1.4 from baseline), with a more significant effect, 30 minutes after insufflation and assumption of a head-up positioning for the surgery (\u22125.1 \u00b1 1.9 from baseline). No change in cytochrome aa3 was noted during the study.A follow-up study by the same group of investigators used the same technology to evaluate changes in cerebral oxygenation and cerebral blood volume during laparoscopic cholecystectomy in 12 adult ASA I and II patients, in whom minute ventilation was increased to maintain normocapnia. They notet al evaluated changes in cerebral oxygenation and cerebral blood volume in 15 ASA I-III children during laparoscopic fundoplication, with an insufflation pressure of 5 to 8 mmHg.[2 increased from 30.0 \u00b1 2.8 to 38.3 \u00b1 5.1 mmHg and PaCO2 increased from 32.0 \u00b1 4.7 to 40.4 \u00b1 5.9 mmHg. Cerebral oxygenation increased by 15.7 \u00b1 8.8% and cerebral blood volume increased by 4.6 \u00b1 8.8%.Using the INVOS cerebral oximeter, de Waal o 8 mmHg. During i2 values that we noted, it is possible that smaller studies including only 10\u201315 patients may miss such changes. Additionally, we collected ScO2 values at 5 minute intervals with 1000 values more, as compared with the other studies where cerebral oxygenation was assessed at 30 minutes intervals. We did not average our values, whereby a low value might be lost when averaged with other values, rather we compared each ScO2 value to the baseline, to assess its relative change. Our cohort mostly included ASA III patients, as opposed to the ASA I-II patients of Kitajima et al. Minute ventilation was increased to maintain normocapnia. As demonstrated by the studies of Kitajima et al,[2 values, although the greatest difference they noted was when assuming the head-up position for the surgical procedure.The data in our current study are somewhat different that what has been reported in the 3 previous studies that have been mentioned.\u201323 We woma et al,22 this met al and therefore we cannot comment as to whether treatment of decreases in ScO2 values would have an impact on the outcome. Recommendations for the treatment of decreases in ScO2 values include checking ventilator function and head positioning, increasing the FiO2, increasing the PaCO2 for values less than 35 mmHg, increasing mean arterial pressure with fluid or vasoconstrictors, followed by decreasing the cerebral metabolic rate of oxygen by the administration of propofol or a barbiturate if the above measures fail.The current study did not separate randomized patients into treatment and non-treatment groups as was done by Casati 2, which have been shown to correlate with neurocognitive changes in other studies. The limitations of the current study are, that we did not evaluate our patients postoperatively to see if changes in ScO2 correlated with postoperative neurocognitive changes and therefore we cannot determine if monitoring and treating changes in ScO2 can prevent postoperative neurocognitive changes, as has been previously demonstrated by the study of Casati et al.[2 values, an analysis to determine additional \u201cat risk\u201d features other than body weight and duration of insufflation, such as co-morbid features or intraoperative conditions , was not feasible. However, given that the deleterious physiologic effects of increased intraabdominal pressure during insufflation on stroke volume, cardiac output and other hemodynamic variables have been well documented,[In summary, we noted that although most patients tolerated insufflation without significant effects on cerebral oxygenation, a subset of patients developed significant decreases in ScOti et al. As all tcumented, it is li"}
+{"text": "Sir,Improved self-management following participation in a clinical trial with better glycemic control has been recently reported in the literature.2 ParticiRetrospective analysis of glycemic control in type 2 diabetic patients who participated in clinical trials was done by comparing the fasting glucose and HbA1C values at screening and randomization visits. We selected the patients who participated in a randomized controlled clinical trial on new therapy for type 2 diabetes mellitus. A total of three such studies were selected. All these studies were approved by the Institutional Ethics Committee. Eligible patients for the evaluation were those who undertook both the screening as well as randomization visits. Those who had undergone only screening were excluded. The interval between the two visits varied between studies depending upon the study design. Antidiabetic medication remained unchanged between the two visits. Fasting glucose, HbA1C, and body weight were measured at both the visits. All these trials included adult, males or females.2) who were inadequately controlled with insulin alone or in combination with oral hypoglycemic agents. Patients were screened at visit 1 and those found eligible continued with stable dose of insulin and oral hypoglycemic agents along with placebo medication for 2 months. At the end of 2 months patients were randomized to receive active study medication. Trial 2 included type 2 diabetic patients with inadequate glycemic control. Patients were drug na\u00efve at the time of inclusion. Patients were screened at visit 1 and received placebo for 1 month after which the eligible patients were randomized. Trial 3 included patients with history of type 2 diabetes mellitus and body mass index between 23 and 45 kg/m2. These were inadequately controlled (HbA1C above 8.0%) and received a combination therapy including metformin and another oral hypoglycemic agent. After screening, all eligible patients were put on maximum tolerated dose of metformin along with pioglitazone for 3 months after which the eligible patients were randomized to receive the study medication.Trial 1 included obese patients with features of metabolic syndrome (body mass index above 25 kg/mP < 0.05 was considered significant. The main outcome was short-term glycemic control measured by glycated hemoglobin (%) and fasting glucose level.Fasting glucose and HbA1C were measured at screening as well as randomization visit in all the three studies. Glucometer was dispensed to all patients for self-monitoring of blood glucose. Patients were given adequate education and were taught to use the glucometer. They were also advised to maintain a diary for daily record of glucometer readings. Data from all three studies were combined as well as analyzed individually. The Student \u2018t\u2019-test was used for statistical analysis and a A total of 61 patients  were studied . Patients in trial 3 had maximum duration of diabetes (17.65\u00b18.02 years) and trial 2 patients had shortest. The median interval between screening (visit 1) and randomization (visit 2) was 60 days in trial 1, 30 days in trial 2, and 90 days in trial 3.P=0.074). Overall analysis has shown a decrease in HbA1C between the two visits. Baseline fasting blood glucose was also highest in trial 1 patients and was lowest in trial 2 patients. Fasting blood glucose increased from visit 1 to visit 2 in trial 1 patients and decreased in trial 2 and 3 patients. The drop in fasting glucose level in trial 3 patients was statistically significant (P=0.01). Overall analysis also showed a decrease in fasting glucose level between the two visits [Baseline HbA1C was highest in trial 1 patients. It increased further in trial 1 patients from baseline to randomization visit, whereas it decreased in trial 2 and 3 patients. Drop in HbA1C was much more in trial 3 patients though statistically insignificant (o visits . Body weet al.[th percentile of HbA1C. Improved glycemic control following participation in a clinical trial can be best understood as a result of enhanced awareness in patients, increased care given by health professionals, frequent monitoring and motivation of patients for better adherence to therapy and lifestyle modification.[The present analysis has shown that study participation itself causes sufficient changes in the glycemic control. Combined analysis of all the three studies has shown that there was sufficient reduction in blood glucose and HbA1c level in patients who participated in clinical trials. Similar observations have also been made before.3 In the fication.5 It is o"}
+{"text": "IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2). However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including  NLRP2 and NLRP7 genes) in the mother. Homozygous inactivating mutations in NLRP7 in women have been associated previously with abnormal imprinting and recurrent hydatidiform moles. We identified a homozygous frameshift mutation in NLRP2 in the mother of the two children with BWS implicating NLRP2 in the establishment and/or maintenance of genomic imprinting/methylation.A small set of genes (imprinted genes) are expressed in a \u201cparent-of-origin\u201d manner, a phenomenon known as genomic imprinting. Research in human disorders associated with aberrant genomic imprinting provided insights into the molecular mechanisms of genomic imprinting and the role of imprinted genes in normal growth and development. Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with developmental abnormalities and a predisposition to embryonic tumours. BWS results from alterations in expression or function of imprinted genes in the imprinted gene cluster at chromosome 11p15. Although BWS may be caused by a variety of molecular mechanisms, to date, all the genetic and epigenetic defects associated with BWS have been limited to 11p15.5. We report a family with two children affected with BWS and an epigenetic defect at 11p15.5 in which the primary genetic defect mapped outside the imprinted gene cluster. Using autozygosity mapping, we found an extended homozygous region on chromosome 19q13.4 (containing  IGF2, KCNQ1OT1 (LIT1) genes and the maternally expressed H19 and CDKN1C (P57KIP2) genes. KCNQ1OT1 and H19 transcripts are not translated but the IGF2 gene product is an important prenatal growth factor and the CDKN1C protein is a candidate tumour suppressor that negatively regulates the cell cycle H19 that has an \u201cinsulator function\u201d regulated by the zinc finger transcription factor, CTCF. The insulator is methylation sensitive, such that when CTCF binds to the unmethylated maternal allele, the IGF2 promoters do not have access to (are insulated from) enhancers downstream of H19. Methylation on the paternal allele prevents CTCF from binding, thus permitting interaction between the IGF2 promoters and the enhancers H19 DMR and in these cases IGF2 shows loss of imprinting (LOI) and biallelic expression KCNQ1 gene and is known as KvDMR1. The unmethylated paternal allele permits transcription of the antisense transcript KCNQ1OT1  and silencing of genes including KCNQ1 and CDKN1C. Maternal methylation at the KvDMR1 is thought to prevent transcription of the KCNQ1OT1 gene and enable expression of CDKN1C. Loss of methylation (LOM) at the KvDMR1 is seen in up to 50% of sporadic BWS and is associated with biallelic expression (loss of imprinting) of KCNQ1OT1 and silencing of maternal CDKN1C expression trans mechanism.Genomic imprinting is an epigenetic modification that causes genes to be expressed according to their parent of origin. Although less than 100 imprinted genes have been identified in human and mice, many imprinted genes appear to have a critical role in prenatal growth and development This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the South Birmingham Research Ethics Committee  reference number CA/5175. All patients provided written informed consent for the collection of samples and subsequent analysis.NLRP2 mutations in this family, a further 11 BWS families, each with a single case of BWS (mean age 10.8 years) with KvDMR1 loss of methylation were analysed for NLRP2 mutations. This cohort included 10 patients who also had loss of methylation at other imprinted loci. Ethnically matched laboratory control samples were analysed to evaluate the significance of novel sequence variants.A consanguineous family of Pakistani origin with two affected children with BWS due to loss of methylation at KvDMR1 were investigated in the first instance. Following the identification of Genomic DNA was extracted from peripheral lymphocytes by standard techniques.In preliminary examinations chromosomal abnormalities were excluded and the methylation status of IC1 and IC2 of imprinting region 11p15.5 were determined.BstU1 yielding different sized fragments which is separated using ABI377 or 3730 (PEG1) and the Angelman/Prader-Willi locus at 15q13 (SNRPN) as described previously Methylation analysis of KvDMR1 was performed as described previously, with PCR amplification of bisulphite modified DNA and digestion with restriction enzyme JMJD2D, ZFP57, NLRP7 and NLRP2 was carried out by direct sequencing. The genomic DNA sequence of these genes was taken from Ensembl (http://www.ensembl.org/index.html) and primer pairs for the translated exons were designed using primer3 software (http://fokker.wi.mit.edu/primer3/input.htm). Amplification was performed according to standard protocols with Bio Mix Red provided by Bioline. PCR products were directly sequenced by the Big Dye Terminator Cycle Sequencing System with the use of an ABI PRISM 3730 DNA Analyzer (Applied Biosystem). DNA sequences were analyzed using Chromas software.For linkage studies a genome wide linkage scan was undertaken using the Affymetrix 250k SNP microarray. Mutation analysis of trans imprinting defect.A family with complex consanguinity  was asceNLRP2 and NLRP7 at 19q13.4. NLRP7 is a homologue of the mouse NLRP2 gene (NLRP7 is not present in the mouse) and the human NLRP2 gene. Sequencing of NLRP2 in the mother identified a homozygous frameshift mutation . Genetic linkage studies were undertaken by genotyping the two children and both parents on an Affymetrix 250k SNP array platform. Five regions of homozygosity (>2Mbases) were shared by the two children but these did not contain a gene known to be implicated in the establishment or maintenance of genomic imprinting. However inspection of the maternal genotypes revealed an \u223c8 Mbase homozygous region containing trans imprinting defect extended beyond KvDMR1, we analysed methylation levels at the TND (6q24), SNRPN (15q13) and PEG1 (7q32) DMRs. Both affected siblings  had normal methylation levels at the TND and SNRPN DMRs but Child 2 demonstrated partial loss of methylation at the PEG1 DMR . The major cause of TNDM is aberrant expression of imprinted genes at chromosome 6q24 and about 20% of cases have LOM at the TND differentially methylated region (DMR). Patients with homozygous ZFP57 mutations have LOM at the TND DMR, but also at other imprinted loci including KvDMR1 ZFP57 mutations in our family. Germline NLRP7 mutations are associated with familial recurrent biparental complete hydatidiform mole (FHM) in which there is epigenetic abnormalities at DMRs in multiple imprinting regions NLRP7 mutations is inherited in an autosomal recessive manner, in contrast to ZFP57 mutation, homozygotes have normal genomic methylation but in female homozygotes there is a failure to establish methylation imprints in their germ cells leading to hydatidiform moles and reproductive wastage . The methylation defects in FHM are specific for imprinted loci, and DNA methylation at non-imprinted genes and genes subject to X-inactivation is unaffected NLRP2 and NLRP7 genes are highly homologous and the two proteins consist of 1,062 and 1,009 amino acids respectively and have identical structure and about 64% amino acid identity. Only one of the two affected children with BWS was homozygous for a NLRP2 mutation and, by analogy with FHM caused by NLRP7 mutations, the BWS phenotype most likely results from homozygosity in the mother, such that familial BWS associated with NLRP2 mutations is inherited in a similar manner to NLRP7 associated FHM and not in a conventional autosomal recessive manner.We identified a homozygous frameshift mutation in the mother of two children with BWS caused by epimutations at IC2 (KvDMR1). Most cases of BWS due to loss of methylation of KvDMR1 are sporadic, but a handful of familial cases have been described with maternally inherited germline IC2 deletions ZFP57\u2013TNDM are associated with imprinting aberrations at multiple loci, and we identified partial loss of methylation at the PEG1 DMR in one of the affected children. Nevertheless it seems that NLRP2 mutations have a less severe effect on imprinting than NLRP7 or ZFP57 inactivation. A subset of children with BWS and an IC2 epimutation display hypomethylation at multiple imprinting centres (DMRs) (in our series these children are more likely to have been conceived by assisted reproductive technologies) NLRP2 mutation in the sporadic cases we studied. It appears that the establishment (or maintenance) of methylation at KvDMR1 is particularly sensitive to genetic and/or environmental insults. We note that one of the affected children demonstrated a partial loss of methylation at PEG1 DMR1 both by bisulphite sequencing and MS-PCR suggesting that NLRP2 mutations may be associated with an incomplete failure of imprinting establishment and/or a partial failure of maintenance methylation at this DMR. Interestingly, investigation of a mouse knockout of ZFP57 has suggested a role in both the establishment of germline methylation imprints and in the postfertilisation maintenance of methylation imprints Both FHM and NLRP2 and NLRP7 encode members of the NLRP  family of CATERPILLER proteins. NLRP family of cytoplasmic proteins comprises 14 members of similar structure that are principally encoded by two gene clusters on chromosome 11p15  and 19q13.4 . Most of the family members are well conserved from C. elegans, D. melanogaster, rat, and mouse to human but there is no rodent homologue for NLRP7 and the gene is found in only a few genomes . Some NLRP proteins are components of the inflammasome that is implicated in the sensing of, and inflammatory reaction to, extracellular pathogens and intracellular noxious compounds NLRP3 and NLRP12 are associated with familial cold autoinflammatory syndrome NLRP2 truncating mutation did not show any evidence of an immune or autoinflammatory disorder. Nevertheless most NLRP family proteins are widely expressed and not restricted to the immune system. In addition, many are expressed in human oocytes and embryos at an early stage of development. Thus Zhang et al. have reported that NLRP4, 5, 8, 9, 11, 12, 13, and 14 were highly expressed in oocytes and then gradually decreased in embryos with a very low level in day 5 embryos, whilst NLRP2 and NLRP7 progressively decreased from oocytes to day 3 embryos then showed a sharp increase on day 5 NLRP2 and NLRP7 having a similar role in early development/imprinting establishment. Although it has been suggested that FHM might result from an immune-related defect in oogenesis or early embryo development (with methylation changes being a secondary phenomenon) the specific association of the methylation defects with imprinted DMRs suggests a more direct role in the establishment or maintenance of imprinting marks. Such a view is supported by the identification of germline NLRP2 mutations in BWS and should prompt further investigation of the role of NLRP2 and NLRP7 in genomic imprinting. The apparent involvement of NLRP proteins in genome methylation and the sensing and inflammatory response to extracellular pathogens and intracellular noxious compounds is intriguing given the suggestion that cytosine methylation may have evolved as a host response to transposons NLRP7 mutations have recurrent molar pregnancies only, but at least three families have been reported in which affected women had liveborn offspring  it might be predicted that clinical heterogeneity/incomplete penetrance would be a feature of maternal NLRP2 inactivation. Although maternal NLRP2 mutations appear to be a rare cause of familial BWS, the identification of these cases is important, as the inheritance pattern differs from the autosomal dominant inheritance (with parent of origin effects) associated with other inherited forms of BWS. The inheritance of NLRP2-associated BWS has similarities to other autosomal recessive disorders in which homozygous mothers are well, but there is a high risk to their offspring ."}
+{"text": "The Infectious Diseases Society of America published in 2000 practical guidelines for the management of cryptococcosis. However, treatment strategies have not been fully validated in the various clinical settings due to exclusion criteria during therapeutic trials. We assessed here the optimal therapeutic strategies for severe cryptococcosis using the observational prospective CryptoA/D study after analyzing routine clinical care of cryptococcosis in university or tertiary care hospitals.Cryptococcus neoformans. Control of sterilization was warranted 2 weeks (Wk2) and 3 months (Mo3) after antifungal therapy onset. 208 HIV-positive or -negative adult patients were analyzed. Treatment failure  at Wk2 and Mo3 was the main outcome measured. Combination of amphotericin B+flucytosine (AMB+5FC) was the best regimen for induction therapy in patients with meningoencephalitis and in all patients with high fungal burden and abnormal neurology. In those patients, treatment failure at Wk2 was 26% in the AMB+5FC group vs. 56% with any other treatments (p<0.001). In patients treated with AMB+5FC, factors independently associated with Wk2 mycological failure were high serum antigen titer  and abnormal brain imaging  at baseline. Haematological malignancy , abnormal neurology at baseline  and prescription of 5FC for less than 14 days  were independently associated with treatment failure at Mo3.Patients were enrolled if at least one culture grew positive with Our results support the conclusion that induction therapy with AMB+5FC for at least 14 days should be prescribed rather than any other induction treatments in all patients with high fungal burden at baseline regardless of their HIV serostatus and of the presence of proven meningoencephalitis. Major information on the best therapeutic strategies for cryptococcal meningoencephalitis derives from therapeutic trials involving HIV-positive Using data from a prospective observational cohort of HIV-positive and -negative patients treated in France for cryptococcosis with or without CNS involvement, we previously showed that lack of 5FC was an independent factor of mycological failure at Wk2 whatever the HIV status and the clinical presentation at baseline The CryptoA/D study enrolled HIV-positive or -negative adults experiencing a first episode of culture-proven cryptococcosis and treated in French university hospitals or tertiary care centers C. neoformans from at least one body site. Cases were classified as meningoencephalitis  or as extrameningeal cryptococcosis. A threshold of \u22651\u2236512 for serum or CSF antigen titer was selected since it is linked to prognosis A case was defined by isolation of Mycological outcome was only evaluated in patients for whom at least one body site was sampled at the time of workup. Mycological failure meant that at least one of the cultured samples contained viable yeasts. Mycological failure or death up to 4 days after Wk2 workup was recorded as treatment failure at Wk2. Mycological failure or later death was recorded as treatment failure at Mo3.We first described and compared the major characteristics of the patients and the Wk2 outcome according to the induction therapy strategies. We then assessed how the current IDSA guidelines Clinical presentation and outcome were compared according to therapeutic regimens with the \u03c72 test for qualitative parameters (or the Fisher's exact test when necessary) or the Kruskal-Wallis's or the Mann-Whitney's test for quantitative parameters. Multinomial logistic regression Induction therapy was assessable for 208 patients including a majority of HIV-positive male patients with meningoencephalitis . At the Induction therapies included AMB  mostly associated with 5FC  or FCZ  mostly alone  . AMB wasDespite more severe infections based on our criteria, mycological failure at Wk2 was significantly less frequent among patients treated with AMB+5FC than any other regimen (20/86 (23%) vs. 47/100 (47%), p<0.001) . This waThe majority of patients (n\u200a=\u200a174) were treated with AMB or FCZ alone, or AMB+5FC. Patients treated with AMB did not significantly differ from those treated with the combination therapy except for the lower percentage of patients with high CSF antigen titer in the AMB-treated group . By contIn HIV-infected patients with meningoencephalitis who received as induction therapy either AMB or FCZ alone or the combination AMB+5FC (n\u200a=\u200a120), factors which significantly discriminated between combination and FCZ alone in the univariate analysis were the presence of fungemia, high serum or CSF antigen titer, and hospitalization in university hospitals with a trend towards modification of prescription in older patients, while only high CSF antigen titer was identified as a factor associated with the initiation of combination therapy vs. AMB alone . In the Among the 86 patients given AMB+5FC and assessable at Wk2, the proportion of men, and high serum or CSF antigen titer was significantly higher than with other treatments. There was also a trend towards more disseminated infections and infection by serotype A among those who experienced mycological failure at Wk2 than among those who were cured. An optimal duration and dosage of each drug in the combination had no impact. Factors independently associated with mycological failure at Wk2 were high serum antigen titer  and abnormal brain imaging  at baseline .(1)FCZ(2)), AMB followed by FCZ (AMB(1)FCZ(2)), AMB+5FC followed by FCZ (AMB+5FC(1)FCZ(2)) and \u201cmiscellaneous\u201d corresponding to other induction therapies and/or multiple changes preventing further description.Of 189 patients surviving the Wk2 workup by more than 4 days, 168 were eligible for analysis of Mo3 outcome . The ind(1)FCZ(2) group had significantly the more severe infections, and those in the FCZ(1)FCZ(2) group the less severe ones ((1)FCZ(2) group (20%) than in the AMB(1)FCZ(2) group (52%) despite no significant difference in the cumulative dose and duration of AMB. Of note, direct examination of the Wk2 CSF samples was still positive in almost 3/4 and 1/2 of the cases in AMB+5FC(1)FCZ(2) and AMB(1)FCZ(2) treated patients, respectively. There was a trend toward less treatment failures recorded at Mo3 in the AMB+5FC(1)FCZ(2) group than in the others.Considering the first 3 treatment groups, patients in the AMB+5FCere ones . MycologIn the univariate analysis, the proportion of abnormal neurology at baseline, haematological malignancies and 5FC prescription lasting less than 14 days was significantly higher in patients recorded as treatment failures at Mo3 than in the others . There wCryptococcal meningoencephalitis is still a major health problem in developed countries with the burden of the AIDS epidemics reaching a prevalence of up to 18% in the most severely immunocompromised patients Evaluation of cryptococcosis severity represents a major factor for the management of the patients. Many parameters are predictive of treatment failure including underlying malignancy, cranial hypertension, dissemination, and high antigen titer Initial antifungal treatment strategy is a critical determinant of outcome. Major studies supporting the prescription of 5FC in combination with AMB for the induction treatment of cryptococcal meningoencephalitis in HIV-positive We indeed showed that outcome at Wk2 was statistically better with AMB+5FC compared to any other regimen including AMB alone despite more severe infections in the former group. Furthermore, the proportion of Wk2 mycological failure did not increase in the most severe cases treated with AMB+5FC in contrast to what occurred in all other treatment groups except FCZ+5FC. The drastic difference in outcome between the two groups is probably due to the fact that more severe cases than those usually enrolled in the therapeutic trials were considered here with 40% of abnormal neurology compared to less than 20% in previous studies In the setting of a therapeutical trial on cryptococcal meningoencephalitis in HIV-positive patients, AMB compared favourably with FCZ, the latter being associated with more frequent early deaths and delayed CSF sterilization When considering the other strategies, our results demonstrate that FCZ alone was associated with 42% mycological failure at Wk2 despite the fact that 40% of the patients in that group did not have meningoencephalitis and that only 15% had high antigen titers. Keeping in mind the small number of patients in each of the FCZ+5FC, AMB+FCZ or AMB+FCZ+5FC groups, none of the combination did better than AMB+5FC. Of note, Bicanic and colleagues recently underlined in South Africa the high relapse rates after treatment with FCZ alone at conventional dosages Another issue in the optimization of treatment strategies for cryptococcosis is the potential utility of protracting induction regimen. Indeed, current recommendations are based on results obtained in a therapeutic trial where switch from AMB\u00b15FC to azoles was mandatory at Wk2 whatever the culture results at that time Thus, this observational study with its limitation compared to a randomized therapeutic trial but the assets of the \u201creal life\u201d confirms that the AMB+5FC combination is the optimal antifungal strategy for the induction treatment of severe cryptococcosis Table S1(0.02 MB XLS)Click here for additional data file.Table S2(0.02 MB XLS)Click here for additional data file.Appendix S1(0.03 MB DOC)Click here for additional data file."}
+{"text": "This study investigated expression of the SART3 antigen in breast cancer to explore an appropriate molecule for use in specific immunotherapy of breast cancer patients. The SART3 antigen was detected in all of the breast cancer cell lines tested, 30 of 40 (75%) breast cancer tissue samples, and 0 of 3 non-tumourous breast tissue samples. SART3 derived peptides at positions 109\u2013118 and 315\u2013323 induced HLA-A24 restricted CTLs that reacted to breast cancer cells from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients. Therefore, the SART3 antigen and its peptides could be an appropriate molecule for use in specific immunotherapy of the majority of HLA-A24-positive breast cancer patients. \u00a9 2001 Cancer Research Campaign"}
+{"text": "The average number of nucleolar organiser regions per cell has previously been shown to correlate well with histological grading techniques for a variety of neoplasms in man, and may thus be of value as an aid to post-surgical prognosis. In this study 50 spontaneously arising, subcutaneous canine mast cell tumours were graded and the histological grade compared with the mean AgNOR count. For well differentiated neoplasms the mean count was 1.4 per cell compared with 6.3 for poorly differentiated neoplasms, while tumours of intermediate differentiation had a mean count of 3.2 per cell. Subsequent follow up studies revealed that the AgNOR count was an accurate prognostic indicator, 73% of dogs with a high mean count (greater than 4.9) being destroyed from tumour related disease compared with 33% with an intermediate count (1.7-4.8). No dog with a count of less than 1.7 has been destroyed because of tumour recurrence to date and the AgNOR count has proved to be a better and more objective prognostic indicator than either histological tumour grade or mitotic index. Since most dogs which develop recurrent mast cell tumours do so within 6 months of initial surgery, an assessment of the predictive value of AgNORs can be obtained more quickly in canine tumours than for comparable human neoplasms."}
+{"text": "Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours."}
+{"text": "Reserpine was found to enhance the cytotoxicity of ACNU on ACNU-resistant C6 glioma (C6/ACNU) cells in vitro. When reserpine was added along with ACNU to the C6/ACNU cells in vitro. When reserpine was added along with ACNU to the C6/ACNU culture in vitro at a concentration of 10 microM, the IC50 of ACNU for C6/ACNU cells decreased to the level of that for C6 cells and ACNU resistance was completely overcome in vitro. Furthermore, intracellular uptake of ACNU increased in both sensitive (C6) and resistant (C6/ACNU) glioma cells when 20 microM reserpine was added to the culture medium. Reserpine (20 microM) enhanced the cellular level of ACNU in C6 cells 1.5-fold and enhanced the level of ACNU in C6/ACNU cells 4-fold. The amount of ACNU incorporated into C6/ACNU cells reached the same level as that incorporated into C6 cells. The enhanced cytotoxicity of ACNU in vitro could be explained by the effective intracellular accumulation of ACNU resulting from the increase of intracellular uptake of ACNU in C6/ACNU cells by reserpine."}
+{"text": "SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.The etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.The 274C/T  Type 1 diabetes mellitus (T1DM) is a multifactorial autoimmune disease in which T-lymphocytes infiltrate the islets of the pancreas and destroy the insulin-producing beta cell populations Mycobacterium avium subsp. paratuberculosis (MAP) in the development of T1DM as an environmental trigger Accumulating line of evidence points to role for SLC11A1 gene (previously known as NRAMP1) is a functional and a positional candidate gene that associates with T1DM as well as susceptibility to mycobacterial infections et al. SLC11A1 gene silencing using RNAi approach in mice reduced the frequency of T1DM and protected against experimental autoimmune encephalomyelitis, advocating thereby for a role for SLC11A1 in autoimmunity. Moreover, it was recently demonstrated that association of variants of the gene encoding SLC11A1 with T1DM may reflect its function in processing and presentation of islet cell self-antigens to dendritic cells (DCs) Regarding genetic susceptibility, the Thus, non-MHC genes could affect the MHC-restricted T-cell response through altered antigen processing and presentation.SLC11A1 locus have been associated with susceptibility to infectious agents and to autoimmune disorders SLC11A1 gene seems to be of particular interest, since it has been shown to affect the levels of gene expression In vitro studies of this polymorphism suggested direct contribution of particular alleles either to autoimmune  or to infectious  disease susceptibility SLC11A1 located within the coding region, the introns, and the 3\u2032-UTR have been shown to influence susceptibility to autoimmune disorders and T1DM To date, a number of polymorphisms at the SLC11A1 polymorphisms in relation to the presence of MAP infection, with T1DM in patients from Sardinia.Our study aimed at examining the association of the SLC11A1 polymorphisms and the presence of MAP specific IS900 signature using total DNA extracted from peripheral blood mononuclear cells collected at the Institute of Diabetology, medical clinic of Sassari University, Italy. Informed written consents from patients including other necessary clearances were obtained before blood samples were drawn. Institutional review board of the University of Sassari approved the study.A total of 131 participants comprising of 59 T1DM patients  and 76 healthy controls  were tesBriefly, 5 ml blood from patients was centrifuged and plasma supernatant used in ELISA. Remaining plasma samples were aliquoted and stored frozen at \u221220\u00b0C for short-term storage (<6 months) and \u221280\u00b0C for long term storage (>6 months).To extract MAP DNA, samples were processed by using the lysor instrument - 500 \u00b5l of glass beads (Sigma) were added, followed by standard phenol-chloroform extraction and DNA precipitation with absolute ethanol and 10M ammonium acetate; the pellet was washed with 70% ethanol and resuspended in 70 \u00b5l of TE buffer.SCL11A1 gene amplification was performed as previously published Human genomic DNA extraction for SCL11A1 polymorphisms, 100 ng of template (genomic) DNA, obtained from blood cells, was amplified. The primer sequences and PCR cycling parameters that we used were previously reported MAP DNA detection was performed by PCR as previously reported NRAMP1Nine polymorphisms  were genotyped across Statistical analysis was performed by using the Chi square test with Yate's correction. Inference was aided by GraphPad InStat MAP DNA was amplified from the blood of 33 out of 59 T1DM patients (55.9%) whereas MAP was detected only in 18 out of 79 healthy controls (22.7%) indicating a statistically highly significant difference .SLC11A1 allele frequencies for 274 C/T polymorphism differed significantly between T1DM patients and control group, this polymorphism was significantly associated with diabetes type 1, in particular allele 1 and 2 generated a P value of <0.0005  is associated with tuberculosis susceptibility in Chinese children SCLA11 polymorphisms were analyzed also by stratification by sex and no differences were observed (data not shown). It may be possible that polymorphisms in different loci of the SCL11A1 gene may confer susceptibility to different intracellular pathogens although genetically very close. Further studies are necessary in order to elucidate the role of SCL11A1 polymorphisms and how it may influence MAP infection in humans.In this study we report the novel association of T1DM with the 274 C/T polymorphism within the"}
+{"text": "MYOC) and forkhead box protein C1 (FOXC1) genes for sequence variations in primary congenital glaucoma (PCG).To screen the myocilin (MYOC variations and 54 cases (negative or heterozygous for cytochrome P4501B1 mutations) for FOXC1 mutations by polymerase chain reaction (PCR) and DNA sequencing.Seventy five PCG patients were screened for MYOC and two sequence variations (GGC375ins and GGC447ins) in FOXC1. No pathogenic variations were identified in MYOC and FOXC1 in our patients.Five single nucleotide polymorphisms  were identified in MYOC and FOXC1 mutations are not involved in pathogenesis of primary congenital glaucoma in our patients. Thus, it is important to screen other loci for involvement in congenital glaucoma in cases which are negative or heterozygous for CYP1B1 mutations to have a better insight in to disease pathogenesis. The north India ,20, thesMYOC and FOXC1 in the pathogenesis of primary congenital glaucoma. Thus, it is important to screen other loci for involvement in congenital glaucoma in cases which are either negative or heterozygous for CYP1B1 mutations to have a better insight in disease pathogenesis.This is the first study from north India showing non-involvement of"}
+{"text": "The 249I and 280M alleles were             associated with PAH . In conclusion,             the increased frequencies of 249I and 280M CX3CR1 alleles in a subgroup of             patients with SSc-associated PAH suggest a role for the fractalkine system in             the pathogenesis of this             condition. Further, the 249I allele might be associated with susceptibility to SSc.Fractalkine (FKN) and its receptor CX3CR1 are critical mediators in the             vascular and tissue damage of several chronic diseases, including systemic             sclerosis (SSc) and pulmonary arterial hypertension (PAH). Interestingly, the V249I             and T280M genetic polymorphisms influence CX3CR1 expression and function. We             investigated whether these polymorphisms are associated with PAH secondary to             SSc. CX3CR1 genotypes were analyzed by PCR and sequencing in 76 patients with             limited SSc and 204 healthy controls. PAH was defined by colorDoppler echocardiography.             Homozygosity for 249II as well as the combined presence of 249II and 280MM were             significantly more frequent in patients with SSc compared to controls (17 vs 6%,"}
+{"text": "BRCA1 and BRCA2 are tumour suppressor genes the alleles of which have to be inactivated before tumour development occurs. Hereditary breast cancers linked to germ-line mutations of BRCA1 and BRCA2 genes almost invariably show allelic imbalance (AI) at the respective loci. BRCA1 and BRCA2 are believed to take part in a common pathway in maintenance of genomic integrity in cells. We carried out AI and fluorescence in situ hybridization (FISH) analyses of BRCA2 in breast tumours from germ-line BRCA1 mutation carriers and vice versa. For comparison, 14 sporadic breast tumours were also studied. 8 of the 11 (73%) informative BRCA1 mutation tumours showed AI at the BRCA2 locus. 53% of these tumours showed a copy number loss of the BRCA2 gene by FISH. 5 of the 6 (83%) informative BRCA2 mutation tumours showed AI at the BRCA1 locus. Half of the tumours (4/8) showed a physical deletion of the BRCA1 gene by FISH. Combined allelic loss of both BRCA1 and BRCA2 gene was seen in 12 of the 17 (71%) informative hereditary tumours, whereas copy number losses of both BRCA genes was seen in only 4/14 (29%) sporadic control tumours studied by FISH. In conclusion, the high prevalence of AI at BRCA1 in BRCA2 mutation tumours and vice versa suggests that somatic events occurring at the other breast cancer susceptibility gene locus may be selected in the cancer development. The mechanism resulting in AI at these loci seems more complex than a physical deletion. \u2002\u2002http://www.bjcancer.com \u00a9 2001 Cancer Research CampaignBreast cancer susceptibility genes"}
+{"text": "Percutaneous endoscopic lumbar discectomy is a relatively new technique. Very few studies have reported the clinical outcome of percutaneous endoscopic discectomy in terms of quality of life and return to work.55 patients with percutaneous endoscopic lumbar discectomy done from 2002 to 2006 had their clinical outcomes reviewed in terms of the North American Spine Score (NASS), Medical Outcomes Study Short Form-36 scores (SF-36) and Pain Visual Analogue Scale (VAS) and return to work.The mean age was 35.6 years, the mean operative time was 55.8 minutes and the mean length of follow-up was 3.4 years. The mean hospital stay for endoscopic discectomy was 17.3 hours. There was significant reduction in the severity of back pain and lower limb symptoms  at 6 months and 2 years. There was significant improvement in all aspects of the Quality of Life  scores except for general health at 6 months and 2 years postoperation. The recurrence rate was 5% (3 patients). 5% (3 patients) subsequently underwent lumbar fusion for persistent back pain. All patients returned to their previous occupation after surgery at a mean time of 24.3 days.Percutaneous endoscopic lumbar discectomy is associated with improvement in back pain and lower limb symptoms postoperation which translates to improvement in quality of life. It has the advantage that it can be performed on a day case basis with short length of hospitalization and early return to work thus improving quality of life earlier. The surgical treatment of lumbar disc herniation constitutes a large part of orthopedic practice and it has evolved considerably in terms of surgical technique and instrumentation.Percutaneous endoscopic discectomy is a relatively new technique for removing lumbar disc herniation. It involves using an endoscope to visualize the disc removal. The discectomy is performed through a posterolateral approach using specially developed instruments. The advantage of percutaneous endoscopic discectomy is that the disc is approached posterolaterally through the triangle of Kambin ,2 withouAlthough many studies -8 have sThe purpose of this study is to determine the outcome of percutaneous endoscopic discectomy in terms of the North American Spine Score (NASS) , MedicalFrom 2002 to 2006, 55 patients with percutaneous endoscopic discectomy performed for herniated intervertebral disc at our instituition had data collected prospectively. All the operations were performed by two surgeons.Inclusion criteria were patients who had radicular symptoms due to discogenic lumbar nerve root compression and failed conservative therapy. The diagnosis of lumbar disc herniation was made on MRI and/or CT scans. Patients with calcified discs shown on CT scans were excluded. Patients who met the inclusion criteria were counseled that percutaneous endoscopic lumbar discectomy was a relatively new technique and offered the alternative of open discectomy as well. 55 patients agreed to have percutaneous endoscopic discectomy.Data on patient demographics, operative time, length of hospitalisation, postoperative complications and how soon they returned to work were obtained. In our institution, all patients who underwent spinal surgery had routine preoperative assessment and 6 month and 2 year postoperative assessments done; the patients were assessed based on the North American Spine Score (NASS \u2013 Disease specific questionnaire) , MedicalStatistical analysis was performed with the use of SPSS version 10.0. Categorical data were compared with the use of chi-square test. Non-parametric statistics were used for the analysis of continuous variables when data were not normally distributed. Significance was defined as p < 0.05.Preoperatively, all patients received one gram of cefazolin intravenously as antibiotic prophylaxis and if the patient is allergic to cefazolin, one gram of intravenous vancomycin was given instead. The patients were placed prone on a radiolucent operative table on a Wilson frame.36 of the 55 patients (66%) were done under local anesthesia. The skin, subcutaneous tissue, fascia and muscle layers were infiltrated with 1 per cent lidocaine. For relaxation and comfort of the patient, sedation with intravenous midazolam or Fentanyl was administered by the anesthetist. 19 patients (34%) were uneasy about having the operation performed under local anaesthesia and so the operation was done under general anaesthesia.Using the C-arm oriented in the postero-anterior imaging position, the midline longitudinal line is marked on the skin surface using a narrow metal rod. The metal rod is then placed transversely across the center of the target disc. A horizontal line is drawn, bisecting the disc under evaluation. The anatomic disc center is located where the transverse line crosses the longitudinal midline. The C-arm is rotated to the lateral projection. The metal rod is held along the side of the patient in the parasagittal orientation at the level of the index disc. While the metal rod is held in this position, the length from the center of that disc to the plane of the posterior skin is recorded. This length is used for the lateral distance of the skin entry point from the posterior midline and this is usually about 12 to 14 cm from the midline. [A guidewire is then inserted through the needle into the disc and the needle removed. A small stab incision is made at the entry site of the guidewire and a tapered cannulated obturator is slid over the guide wire and introduced gently into the foramen and into the disc. A beveled working cannula is then introduced over the obturator which is then withdrawn. An endoscope (Yeung Endoscopic Spine System \u2013 Y.E.S.S. endoscope)  is then The mean age of the patients was 35.6 years (range 15 \u2013 68 years). There were 23 (41.8%) female: 32 (58.2%) male. The mean operative time was 55.8 min (30\u2013100 min). The mean length of hospitalization was 17.3 hours (range 6 to 24 hours). The mean follow-up period was 3.4 years (range 2.0 \u2013 6.5 years). All patients who were working preoperatively returned to work. The mean time to return to work was 24.3 days (10 \u2013 60 days). All returned to their previous occupation (Table 39 (70.9%) patients had L4L5 discectomy done, 12 (21.8%) had L5S1, 2 (3.6%) had L3L4 and 2 (3.6%) had two levels L4L5 and L5S1 done. There were 44 (80%) disc protrusions, 10 (18.2%) extrusions and 1 (1.8%) sequestrated disc.Figure 3 patients (5%) subsequently underwent lumbar fusion for increasing back pain despite good relief of radicular symptoms after endoscopic discectomy. One of these patients was a 25 year old male who presented initially with left L4 radicular symptoms. MRI showed L4L5 and L5S1 degenerate discs with a left L4L5 prolapsed intervetebral disc. A left L4L5 endoscopic discectomy was initially performed for him but on followup, he complained of increasing back pain and had L4L5, L5S1 transforaminal lumbar interbody fusion done 3 months after endoscopic discectomy. Another was a 45 year old male who had right L5 radicular symptoms and back pain preoperatively. MRI showed a right L5S1 posterolateral disc prolapse. He underwent right L5S1 endoscopic discectomy but also had increasing back pain on followup. He eventually had L5S1 posterior lumbar interbody fusion done 7 months post-edoscopic discectomy. For both of these patients, their initial radicular symptoms resolved after endoscopic discectomy. The third patient was a 36 year old female with left L4 radicular pain. MRI showed a L4L5 prolapsed disc. Post- endoscopic discectomy, her radicular sumptoms resolved. However 3 years postoperation, she complained of back pain and left L4 radicular pain again. Postoperation MRI showed diffuse L45 disc bulge and central and lateral recess stenosis. She subsequently underwent transforaminal lumbar interbody fusion.th post-operative day, he complained of severe back pain associated with mild fever. Blood tests showed raised total white count, ESR and CRP. An MRI with contrast showed mainly granulation tissue. He underwent endoscopic washout of the disc space. Tissue cultures from the disc space grew Staphylococcus aureus. His symptoms resolved after the washout. He was treated with intravenous Augmentin for 2 weeks followed by a further 4 weeks of oral Augmentin. He had occasional back pain at 2 years follow-up. There were no complications associated with any of the subsequent surgeries performed after endoscopic discectomy.1 patient developed discitis 4 days post-endoscopic discectomy. This is a 37 year old male who underwent left L45 percutaneous endoscopic discectomy. He was discharged well 1 day post-operation. However, on the 4Based on the SF-36 questionnaire , of which 2 had open discectomy. 1 patient was found to have a sequestrated disc post-endoscopic discectomy and had open microdiscectomy subsequently. Recurrence rate of lumbar disc herniation after open discectomy has been reported as 5\u201311% and most have been treated with a repeated discectomy through the same approach as the initial surgery.,19 Howev3 patients subsequently underwent lumbar intervertebral body fusion for increasing low back pain despite resolution of their initial radicular symptoms. Thus while percutaneous endoscopic discectomy is effective in relieving leg symptoms, it is less effective in treating back pain and this has to be communicated to the patients.In this study, the complications for endoscopic discectomy include 1 case of discitis and 1 case of sequestrated disc, giving a complication rate of 3.6%. It has been reported that the overall complication rate for this kind of surgical procedure averages 2.6%.,22 The cPercutaneous endoscopic lumbar discectomy is a safe and efficacious technique to relieve symptoms of herniated discs and this improvement in back pain and leg symptoms translates to improvement in quality of life. It has the advantage that it can be performed on a day case basis with shorter length of hospitalization and early return to work thus improving quality of life earlier. This is important because patients become candidates for lumbar disc herniation surgery to obtain immediate pain relief and to improve their quality of life.The authors declare that they have no competing interests.CWBP performed case collection, data analysis, literature review and wrote article. WY performed case and data collection. SBT supervised and helped in manuscript preparation. All authors read and approved the final manuscript."}
+{"text": "The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass. Homeobox genes are characterized by the possession of a particular DNA sequence, the homeobox, which encodes a recognizable although very variable protein domain, the homeodomain ,2. Most The homeobox genes have been variously subdivided into superclasses, classes, subclasses or groups, although there has been much inconsistency in the use of these terms. The most commonly recognized groupings are the ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS and ZF groups (or variants of these names), although these are not always given equal rank in classification schemes ,2,4-8. TThe initial analyses of the draft human genome sequence published in 2001 included estimates of the number of human homeobox genes. Venter et al  found 16Using exhaustive database screening, followed by manual examination of sequences, we identified 300 homeobox loci in the human genome. Distinguishing which of these loci are functional genes and which are non-functional pseudogenes was difficult in some cases. Most loci classified as pseudogenes in this study are integrated reverse-transcribed transcripts, readily recognized by their dispersed genomic location, complete lack of intron sequences, and (in some cases) 3' homopolymeric run of adenine residues. A small minority are duplicated copies of genes, recognized by physical linkage to their functional counterparts and the same (or similar) exon-intron arrangement. In general, retrotransposed gene copies are non-functional (and therefore pseudogenes) from the moment of integration because they lack 5' promoter regions necessary for transcription. However, such sequences can occasionally acquire new promoters and become functional as 'retrogenes'. Duplicated gene copies often possess 5'promoter regions (as they are often encompassed by the duplication event); most degenerate to pseudogenes due to redundancy in a process known as non-functionalization, however some can be preserved as functional genes through sub- or neo-functionalization. Thus, in both instances, reliable indicators of non-functionality were sought in order to assign pseudogene status, notably frameshift mutations, premature stop codons and non-synonymous substitutions at otherwise conserved sites in the original coding region.PAX2, PAX5 and PAX8) and retrotransposed pseudogenes that correspond to only part of the original transcript, whether or not it includes the homeobox region or indeed any of the original coding region. Consequently, 13 retrotransposed pseudogenes that lack homeobox sequences are included . We do not include PAX1, PAX9 and CERS1; these are functional genes without homeobox motifs, albeit closely related to true homeobox genes (the other PAX and CERS genes).We currently estimate that the 300 human homeobox loci comprise 235 functional genes and 65 pseudogenes Table . These fDUX4 (DUX1 to DUX5), which have become part of 3.3 kb repetitive DNA elements present in multiple copies in the genome  is the second of two human members of the Nk3 gene family. This gene was previously known as BAPX1; we rename it NKX3-2 to show that it is a member of the Nk3 gene family.\u2218 NKX6-3 [Entrez Gene ID: 157848] is the third of three human members of the Nk6 gene family. We designate this previously unnamed gene NKX6-3 on the basis of clear orthology to mouse Nkx6-3, inferred from sequence identity and synteny.\u2218 VENTX [Entrez Gene ID: 27287] is the only functional member of the Ventx gene family in the human genome. This gene was previously known as VENTX2. We remove the numerical suffix from this gene symbol because we discovered that the sequence formerly known as VENTX1 is actually a retrotransposed pseudogene derived from this gene. Accordingly, we also replace the VENTX1 symbol with VENTXP7 (see below).\u2218 NANOG gene have been described previously . A short cDNA sequence [EMBL: X74862] related to the Msx gene family was reported previously . The pseudogene shares 91% sequence identity with MSX2 mRNA, lacks intronic sequence, and has remnants of a 3' poly(A) tail. It is intriguing, but probably coincidental, that the MSX2P1 pseudogene has integrated at 17q23.2, close to several ANTP-class genes .\u2218 eviously ; the foruse Msx3 . It is nNANOGP1 [Entrez Gene ID: 404635]. We follow Booth and Holland . We follow Booth and Holland , VENTXP2 [Entrez Gene ID: 347975], VENTXP3 [Entrez Gene ID: 349814] and VENTXP4 [Entrez Gene ID: 152101]. These four VENTX retrotransposed pseudogenes have been reported previously, and were originally known as VENTX2P1 to VENTX2P4. The correction of the VENTX2 gene symbol to simply VENTX (see above) means that each of the pseudogene names should also change; we rename them VENTXP1 to VENTXP4. VENTXP1 is transcribed but due to mutations it can no longer encode a homeodomain protein; it can however encode an antigenic peptide (NA88A) responsible for T-cell stimulation in response to melanoma [\u2218 VENTXP5 [Entrez Gene ID: 442384]. We designate this previously unnamed sequence VENTXP5 because it is clearly a retrotransposed pseudogene of VENTX. The genomic sequence of VENTXP5 can now be accessed via the Reference Sequence collection [RefSeq: NG_005091]. The pseudogene shares 83% identity with VENTX mRNA , lacks intronic sequence, and has remnants of a 3' poly(A) tail.\u2218 VENTXP6 [Entrez Gene ID: 552879]. We designate this previously unannotated sequence VENTXP6 because it is clearly a retrotransposed pseudogene of VENTX. Its lack of annotation may reflect the fact that it is located within an intron of an unrelated and well characterized gene, STAU2. The genomic sequence of VENTXP6 can now be accessed via the Reference Sequence collection [RefSeq: NG_005090]. The pseudogene shares 87% identity with VENTX mRNA  and lacks intronic sequence.\u2218 VENTXP7 [Entrez Gene ID: 391518]. A short cDNA sequence [EMBL: X74864] was reported previously and named HPX42 . The pseudogene shares 86% identity with VENTX mRNA , lacks intronic sequence, and has remnants of a 3' poly(A) tail.\u2218 ed HPX42 . This waAY151139], has been annotated as a homeobox gene and is located just 20 kb from NANOG. However, no homeodomain was detected when the deduced protein was analyzed for conserved domains. Also, secondary structure prediction did not predict the expected organisation of alpha helices. Alignment with the NANOG homeodomain reveals identity of the KQ and WF motifs, either side of the same intron position (44/45), but few other shared residues. It is possible, but unproven, that the locus arose by tandem duplication of part, or all, of the NANOG homeobox gene. This gene has generated two retrotransposed pseudogenes: one at 2q11.2 and another at 12q24.33.One other gene could conceivably be included in the ANTP class, but is excluded from our survey. This gene  is the first of three human members of the Alx gene family. This gene was previously known as CART1; we rename it ALX1 because it is related to ALX3 and ALX4; all three genes were formed by duplication from a single ancestral invertebrate gene [\u2218 ate gene .DRGX [Entrez Gene ID: 117065] is the only member of the newly defined Drgx gene family in the human genome. This gene was previously known as PRRXL1 and DRG11, and there is a clear mouse ortholog (Prrxl1). The symbol PRRXL1 is misleading because it infers membership of the Prrx gene family, containing PRRX1 and PRRX2 in the human genome. Several lines of evidence suggest it belongs to a different gene family. First, this gene (at 10q11.23) is not located in the same paralogon as PRRX1 (1q24.3) and PRRX2 (9q34.11) so they are not three paralogs generated during genome duplication in early vertebrate evolution. Second, it has a completely different exon-intron structure from the Prrx-family genes, and it does not contain a Prrx domain or an OAR domain  incorporates the root of the former symbol DRG11, referring to expression of the rodent ortholog in dorsal root ganglia neurons  is a human member of the Dux (double homeobox) gene family. As previously discussed, most members of this gene family are intronless and are probably derived by retrotransposition of an mRNA transcript from a functional intron-containing Dux gene (or duplication of such an integrant). Booth and Holland  is the second of two human members of the Gsc gene family. This gene was previously known as GSCL; we rename it GSC2 to remove the inadvertent implication that it is not a true gene, and also to reflect the clear orthology to chick Gsc2 as inferred by phylogenetic analysis and synteny.\u2218 HOPX [Entrez Gene ID: 84525] is the only member of the newly defined Hopx gene family in the human genome. The mouse version of the gene was first identified first and named Hop (homeodomain only protein) because the encoded protein is just 73 amino acids long, with 61 of these making up the homeodomain  is the only member of the newly defined Leutx gene family in the human genome. We designate this previously unnamed gene LEUTX (leucine twenty homeobox) to reflect the presence of a leucine residue at the otherwise highly conserved homeodomain position 20; other PRD-class homeodomains have a phenylalanine at this position  whilst avoiding inadvertent equivalence to specific genes within the cluster.\u2218 RHOXF2B [Entrez Gene ID: 727940] is the third human member of the Rhox gene family. This locus was referred to in previous studies as PEPP2b  is the only member of the Sebox gene family in the human genome. The human gene is the ortholog of mouse Sebox based on their locations in syntenic chromosomal regions (17q11.2 and 11B5 respectively) and presence of the same intron positions. However, sequence identity is lower than normal for orthologous genes in mouse and human (78% amino acid identity over the homeodomain) and there is evidence that the human gene has undergone divergence. Most surprisingly, the human sequence has two unusual substitutions in the homeodomain  is the only member of the Uncx gene family in the human genome. This gene was previously known as UNCX4.1; we remove the numerals to give UNCX as these do not denote a series within a gene family.\u2218 VSX2 [Entrez Gene ID: 338917] is the second of two human members of the Vsx gene family. This gene was previously known as CHX10; we rename it VSX2 to better reflect its paralogous relationship to VSX1. VSX2 has been used as an alias for this gene in other vertebrate species and the gene symbol CHX10 has the disadvantage of implicitly suggesting presence of at least nine paralogs in human (CHX1 to CHX9), which do not exist.\u2218 DUXA gene . We designate this previously undescribed sequence OTX2P1 because it is clearly a retrotransposed pseudogene of OTX2. The genomic DNA sequence of OTX2P1 shares significant homology with OTX2 transcript variant 2 [RefSeq: NM_172337]. There is an Alu element (AluSx subfamily) insertion, a Made1 (Mariner derived element 1) insertion, and a 1182-nucleotide deletion in OTX2P1 compared to OTX2. The OTX2P1 sequence lacks introns, ends with a poly(A) tail, and harbors critical sequence alterations (including a three-nucleotide insertion introducing a stop codon into the deduced homeodomain).\u2218 DUX1 [EMBL: AJ001481], DUX2 [GenBank: AF068744], DUX3 [GenBank: AF133130] and DUX5 [GenBank: AF133131]. These sequences have been cloned in previous studies . This sequence has been extensively studied as some of its multiple copies exist within the 3.3 kb repetitive elements of the D4Z4 locus at 4q35 . This gene was previously known as CXorf43. The gene encodes a highly divergent atypical (68-amino-acid) homeodomain but not a POU-specific domain, and thus it is debatable whether it should be placed within the POU class. Phylogenetic analyses of homeodomains place it basally in a clade with the POU class  [GeneID: 5461], POU5F1P4 [GeneID: 100009666], POU5F1P5 [GeneID: 100009667], POU5F1P6 [GeneID: 100009668], POU5F1P7 [GeneID: 100009669] and POU5F1P8 [GeneID: 100009670]. Prior to this study, a single retrotransposed pseudogene of the POU5F1 gene had been annotated and designated POU5F1P1 [Entrez Gene ID: 5462]. Another POU5F1-related sequence of unknown status had been annotated and designated POUF5F1L [GeneID: 5461]. We replace the gene symbol POUF5F1L with POU5F1P3 as this sequence is a retrotransposed pseudogene of POUF51. Our analyses of the human genome sequence identified a further six pseudogenes of POU5F1, which we name sequentially POU5F1P2, POU5F1P4 through to POU5F1P8. Each clearly aligns to the mRNA sequence of POU5F1 but with sequence alterations, indicating origin by retrotransposition. POU5F1P2 and POU5F1P6 have frameshift mutations in the homeobox. POU5F1P5 and POU5F1P6 have stop codons in the homeobox. POU5F1P7 and POU5F1P8 are partial integrants of POU5F1 mRNA excluding the homeobox \u2013 POU5F1P7 covers part of the 3' untranslated region and POU5F1P8 a short region around the start codon.\u2218 Hnf1) encodes proteins with a POU-like domain N-terminal to a highly atypical homeodomain. The POU-like domain, as its name indicates, is weakly similar in sequence to the POU-specific domain . This sequence was previously known as IRXA1; we rename it IRX1P1 because it is clearly a retrotransposed pseudogene of IRX1 and not a functional gene. The IRX1P1 sequence aligns to the mRNA of IRX1 but has a frameshift mutation and two stop codons in the homeobox.\u2218 IRX4P1 [Entrez Gene ID: 100009671]. We designate this previously unannotated sequence IRX4P1 because it is clearly a retrotransposed pseudogene of IRX4. The IRX4P1 sequence is a partial integrant derived from a region of the IRX4 mRNA around the stop codon; it lacks the homeobox.\u2218 PBX2P1 [Entrez Gene ID: 5088]. This sequence was previously known as PBXP1; we rename it PBX2P1 because it is clearly a retrotransposed pseudogene of PBX2. The former name of PBXP1 did not indicate its transcript of origin. The PBX2P1 sequence aligns to the mRNA of PBX2 but has a frameshift mutation in the coding region.\u2218 TGIF1P1 [Entrez Gene ID: 126052]. We designate this previously unannotated sequence TGIF1P1 because it is clearly a retrotransposed pseudogene of TGIF1. The locus has many sequence alterations when compared to TGIF1 mRNA, including a 48 nucleotide insertion within the homeobox.\u2218 TGIF2P1 [GeneID: 126826], TGIF2P2 [GeneID: 100009674], TGIF2P3 [GeneID: 100009672] and TGIF2P4 [GeneID: 100009673]. These four sequences were unannotated prior to this study. We designate them TGIF2P1 to TGIF2P4 because they are clearly pseudogenes of TGIF2. Each aligns to the mRNA sequence of TGIF2 but with sequence alterations, indicating origin by retrotransposition. TGIF2P1 has many sequence alterations, including a frameshift mutation in the homeobox. TGIF2P2 and TGIF2P3 are very similar neighboring loci that must have originated by tandem duplication of a retrotransposed TGIF2 mRNA; neither includes the homeobox. TGIF2P4 is a short partial integrant derived from part of the 3' untranslated region of TGIF2 mRNA.\u2218 Drosophila gene cut) generally encodes proteins with one or more CUT domains N-terminal to a typical homeodomain. The CUT domain is a DNA-binding domain of approximately 75 amino acids [The CUT class (named after the no acids ). A fourno acids  have proCUX1 and CUX2). We have also identified a total of three CUT-class pseudogenes in the human genome .We have identified a total of seven CUT-class homeobox genes in the human genome Tables  and 4. ACUX1 [Entrez Gene ID: 1523] and CUX2 [Entrez Gene ID: 23316]. These genes were previously known as CUTL1 and CUTL2 respectively. We rename them CUX1 and CUX2 in accordance with homeobox gene nomenclature convention.\u2218 CUX2P1 and CUX2P2. These sequences were unannotated prior to this study. We designate them CUX2P1 and CUX2P2 because they are clearly retrotransposed pseudogenes of CUX2. Both are short partial integrants derived from CUX2 mRNA, excluding the homeobox \u2013 CUX2P1 covers part of the coding region at the 5' end and CUX2P2 part of the 3' untranslated region.\u2218 SATB1P1 [Entrez Gene ID: 100033410]. We designate this previously unannotated sequence SATB1P1 because it is clearly a retrotransposed pseudogene of SATB1. SATB1P1 is a short partial integrant derived from part of the 3' untranslated region of SATB1 mRNA; it does not encompass the homeobox.\u2218 Drosophila gene pros) encodes proteins with a PROS domain C-terminal to an atypical homeodomain. The PROS domain is a DNA-binding domain of approximately 100 amino acids . This gene was previously known as ATBF1; we rename it ZFHX3 to reflect its close relationship to ZFHX2 and ZFHX4; indeed ZFHX3 was a synonym for this gene.\u2218 LAG1. There are six CERS-class genes in the human genome (CERS1 to CERS6) and five of these (CERS2 to CERS6) encode proteins with a homeodomain sequence [CERS2 to CERS6 in our comprehensive compilation of human homeobox genes, as CERS1 lacks a homeobox motif.The highly unusual CERS (ceramide synthase) class, also known as the LASS (longevity assurance) class, comprises a single gene family that is highly conserved amongst eukaryotes and includes the yeast gene and original member sequence ,82. Thessequence ,82. It iDUX1 to DUX5 sequences are also not shown, as these have undergone secondary amplification and are also most likely non-functional (see above).The chromosomal locations of genes can give clues to evolutionary ancestry, including patterns of gene duplication, and the possible existence of gene clusters. In Figure SHOX (short stature homeobox) in the PAR1 pseudoautosomal region at the tip of the short arms of X and Y being the best known. Haploinsufficiency of SHOX is implicated in the short stature phenotype of Turner syndrome patients who lack one copy of the X chromosome [TGIF2LX, has a distinct homolog on the Y chromosome, called TGIF2LY. These genes map to the largest homology block shared by the unique regions of the X and Y chromosomes, spanning 3.5 Mb. It has been proposed that the ancestor of these two genes arose by retrotransposition of TGIF2 mRNA [The first observation is that there are homeobox genes on every human chromosome. Even the two sex chromosomes harbor homeobox genes, with romosome . There aIF2 mRNA .PKNOX1) and 22 (with GSC2 and ISX). Examination of the remaining autosomes reveals that homeobox genes are quite dispersed with some interesting regional accumulations. The best known examples of close linkage between homeobox genes are the four Hox clusters on human chromosomes 2, 7, 12 and 17, comprising 9, 11, 9 and 10 genes respectively; each of these is shown as just a single line on each ideogram for simplicity  on chromosome 13, and four sets of NKL-subclass genes on 2p/8p (split), 4p, 5q and 10q, hypothesized to be derived from an ancestral array by duplication [There are other concentrations of ANTP-class genes away from the Hox clusters. These are the ParaHox cluster  was subjected to a series of tBLASTn searches ,86 usingDrosophila homeodomains were first combined with all human homeodomains in maximum likelihood and neighbor-joining analyses to enable divergent Drosophila genes to be identified and removed. These include genes lost from human, as well genes known to have undergone unusually rapid evolution in Drosophila. For the Hox3 family the rapidly evolving Drosophila genes bcd, zen and zen2 were then replaced by an ortholog from centipede (Sm Hox3b), and for the Nk4 family the rapid evolving Drosophila gene tin was replaced by an ortholog from annelid (Pd NK4). In addition, six genes from other protostome or cnidarian genomes were added to represent gene families known to be missing from Drosophila . Only 100 bootstrap resamplings were performed on this dataset because of its large size (354 homeodomains). Trees were displayed using TreeExplorer [Phylogenetic analyses were performed with homeodomain sequences, after each had been edited to an alignment of 60 amino acids  method. Bootstrap values supporting gene family designations are shown. Homeodomain sequences derived from pseudogenes are excluded. This ML tree should be compared with the neighbor-joining (NJ) tree shown in Additional file Drosophila melanogaster homeodomains, plus selected additional homeodomains from other protostomes or cnidarians when the gene family is divergent or absent in Drosophila. Divergent Drosophila genes that do not group with human genes were identified by construction of a preliminary, non-bootstrapped ML and NJ trees, and subsequently removed from the dataset. These include genes lost from human, as well genes known to have undergone unusually rapid evolution in Drosophila. For the Hox3 family the rapidly evolving Drosophila genes bcd, zen and zen2 were replaced with Sm Hox3b, and for the Nk4 family the rapid evolving Drosophila gene tin was replaced with Pd NK4. In addition, six genes from other protostome or cnidarian genomes were added to represent gene families known to be missing from Drosophila . Species abbreviations: Am, Apis mellifera (honeybee); Dm, Drosophila melanogaster (fruitfly); Hv, Hydra vulgaris (hydrozoan); Nv, Nematostella vectensis (starlet sea anemone); Pd, Platynereis dumerilii (annelid worm); Ps, Phascolion strombus (sipunculan worm); Sm, Strigamia maritima (centipede). ML performed more poorly than NJ in recovering several well known gene families, notably Hox4, Hox5, Nk4 and Alx. In contrast, ML did recover PROP1 and CG32532 as a true gene family; NJ did not. The invertebrate gene does not always lie as a strict outgroup to all human genes in a family; this effect is expected when using a short alignment. Instead, distinct grouping of invertebrate and human genes is taken as evidence of ancestry from a single gene. A few ambiguous cases were encountered, notably divergence of Drosophila H2.0 in the proposed Hlx gene family, and resolution within the Pax4/6 gene family, which is recovered as two families in NJ but one in ML. As explained in the text, several human gene families contain 'orphan' genes without invertebrate orthologs; these are Barx, Nanog, Noto, Vax, Ventx, Argfx, Dprx, Dux, Esx, Hesx, Hopx, Isx, Leutx, Mix, Nobox, Rhox, Sebox, Tprx, Hdx, Pou5, Hmbox, Satb, Adnp and Zhx/Homez. Zeb and Mkx would be placed in this category based on our ML and NJ trees, although other data suggest that Drosophila zfh1 and CG11617 respectively may be the protostome orthologs [Drosophila ortholog simply lacks the homeobox [rthologs ,94. Tshzhomeobox ,96. PhylClick here for fileNeighbor-joining phylogenetic tree of all human plus selected protostome and cnidarian homeodomains for identification of gene families. Arbitrarily rooted phylogenetic tree of all human plus selected protostome and cnidarian homeodomains constructed using the neighbor-joining (NJ) method. Bootstrap values supporting gene family designations are shown. Homeodomain sequences derived from pseudogenes are excluded. Comparison of NJ and ML trees, and description of the dataset used, is given in the legend to Additional file HOPX.Click here for fileNeighbor-joining phylogenetic tree of human ANTP-class homeodomains, for comparison to maximum likelihood tree. Arbitrarily rooted phylogenetic tree of human ANTP-class homeodomains constructed using the neighbor-joining method. Bootstrap values supporting internal nodes with over 70% are shown. Homeodomain sequences derived from pseudogenes are excluded. The proposed division between the HOXL and NKL subclasses is indicated. The position of EN1 and EN2 is unstable; this tree places them close to the base of the HOXL/NKL divergence, whereas maximum likelihood analysis of the same dataset places them firmly in the NKL subclass  was derived from a compilation of 247 human homeodomain sequences. The three horizontal lines indicate the positions of the three alpha-helices. The numbering scheme refers to amino acid position in the canonical 60-amino-acid homeodomain; insertions relative to this sequence are shown when present. Black shaded resides are invariant between all human homeodomains within each class (or family in the case of the ZF homeodomains). Sequence accession numbers are shown. For each gene family designation, maximum likelihood and neighbor-joining bootstrap support values are indicated (see Additional files Click here for filePhylogenetic input file. All human and invertebrate homeodomains used in phylogenetic analyses are shown, after alignment and removal of insertions to give a uniform 60-amino-acid alignment.Click here for file"}
+{"text": "With a median follow-up of 4 years and 9 months a significant reduction in odds of death  was observed for those with adjuvant treatment (65% at 5 year) compared to the observation group (55%). Improved relative survival was present in stage III (56% vs 44%), and in stage II patients (78% vs 70%). In rectal cancer a non-significant difference in disease-free or overall survival was observed. Distant metastases developed in 76%, while local recurrence alone occurred in 14%. An early start of adjuvant treatment (< 4 weeks) did not affect results. Compliance to 5FU plus levamisole was 69%. Severe toxicity did not occur. In conclusion, one year 5FU plus levamisole was of benefit in stage II and III colonic cancer; in rectal cancer a significant positive effect could not be demonstrated. \u00a9 2001 Cancer Research Campaign\u2002\u2002http://www.bjcancer.comBased on the first favourable results of adjuvant therapy of 5FU plus levamisole in Dukes C colonic cancer in 1990, we conducted a prospective trial. 1029 patients were randomised to receive one year 5FU plus levamisole or no further treatment following curative surgery for stage II or III colon ("}
+{"text": "BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through \u2018temporal windows\u2019 in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through \u2018exit gates\u2019 after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression.We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction. Oct4 and Sox2 (inner cell mass), Fgf5 (epiblast) and Brachyury, Mixl1 and Gsc (primitive streak) Pdx1 (foregut endoderm), Nkx2-5 (cardiac mesoderm) and \u03b2H1 globin (yolk sac erythroid cells) The in vitro differentiation of embryonic stem cells (ESCs) represents an accessible system for analyzing parameters influencing the early stages of lineage specification and commitment. During differentiation, ESCs pass through a series of developmental milestones that mirror those traversed by cells within the embryo Brachyury, Mixl1 and GoosecoidNot only is there a correspondence between the developmental pathways followed by cells in vitro and in vivo, but there is a similar concordance between the factors that induce and pattern ESCs and the embryo during differentiation. For example, induction of the primitive streak, the structural harbinger of mesendoderm formation in the embryo, requires the activity of a number of secreted growth factors . Finally, we observed that the differentiating cells passed through \u2018exit gates\u2019 after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression. Overall, our study suggests that growth factor regulatory loops similar to those present in early embryos also exist within EBs. The timing of growth factor activity is critical for the initiation of mesendoderm formation from ESCs and paracrine signalling contributes to mesendoderm development.In this study we determined the periods within which BMP4, Wnt3a and Activin A induced mesendoderm in differentiating mouse ESCs. These factors acted during discrete \u2018temporal windows\u2019 to induce expression of a GFP reporter gene targeted to the locus of the primitive streak gene, Mixl1GFP/wMixl1, a gene whose expression is restricted to the mesendodermal precursors of the primitive streak We utilised a genetically modified mESC line, Mixl1GFP/w ESCs were differentiated in a chemically defined medium (CDM) + cells were observed on this day, and expression rapidly waned thereafter. This was consistent with observations that GFP maturation and fluorescence lagged behind the peak of Mixl1 mRNA expression that was maximal at d4 of differentiation Mixl1 locus (denoted Mixl1GFP) when present in the cultures from d1\u20132  and d2\u20133 (44.2\u00b19.6%) (+ cells was observed when BMP4 was added from d1.5\u20132.5 (55.8\u00b14.6%). A lower frequency of GFP+ cells was seen in d5 cultures stimulated between d2.5 and d3.5 (21.2\u00b17.4%)  . Experim.2\u00b19.6%)  indicate.2\u00b17.4%) . FinallyMixl1 induction window for each growth factor. These experiments confirmed that BMP4 and Wnt3a were most effective at inducing GFP+ mesendoderm precursors when present in the cultures between d1.5 and d3 . On the basis of these preliminary studies, further differentiation experiments were performed in which the growth factors were included prior to, during and after the hypothesized optimal 5 and d3 . In cont5 and d3 . The incMixl1GFP/w reporter ESC line, (clone Mix 114) Mixl1GFP/w clones were derived from the same parental ESC line, we cannot rule out the possibility that different strains of ESCs might vary in their propensity to differentiate. However, in our experience, ESC lines generally respond to growth factors in a similar manner, with the main differences being in the concentration of factors required for the development of specific cell types .Similar outcomes in experiments performed with an independently targeted Mixl1 locus, and that the variable fluorescence intensity observed indicated that not all Mixl1GFP expressing cells were identical. Interestingly, we have previously observed that this heterogeneity for Mixl1GFP expression is more evident within individual EBs than between EBs Even in our relatively well defined, short-term differentiation experiments, we observed that, at best, 60\u201370% of cells expressed GFP from the Mixl1GFP/w cells. In the first instance, we examined the effects of adding inhibitors for Wnt and TGF-beta pathways on the ability of each ligand to induce MixlGFP expressing cells. ESCs were treated with BMP4 or Wnt3a from d1 or with Activin A from d2, because preliminary experiments showed that addition of this factor prior to d2 inhibited ESC mesendoderm differentiation (data not shown). Inhibitors of BMP4- (noggin), Activin receptor- (SB 431542) or canonical Wnt- (Dkk-1) signalling were added to the cultures at the same time as the growth factors. Cultures were analysed at differentiation d5 for expression of GFP by flow cytometry.We sought to determine whether all three growth factor signalling pathways were required for GFP induction in differentiating + cells observed in d5 cultures which had been treated with Activin A  fell outside the window of optimal responsiveness of the EBs to direct BMP4 or Wnt3a induction of Mixl1GFP+ cells.These results implied that endogenously produced BMP, Wnt and Activin-like molecules might play an important role during mesendoderm induction during ESC differentiation in vitro by complementing the actions of exogenously applied growth factors. To directly assay the paracrine mesendoderm inducing ability of these endogenous factors, we performed mixing experiments in which wild type eric EBs . These c+ cells were observed in the chimeric EBs at d5  was assessed. In the absence of inhibitors, wild type \u2018stimulator\u2019 ESCs treated with BMP4 or Wnt3a between d1\u2013d3 effectively induced GFP expression in \u2018responder\u2019 /w cells . Inclusirequired . In addiP/w ESCs . This arP/w ESCs .Mixl1GFP/w cells closed soon after d3, consistent with the observation that addition of these growth factors after this time did not recruit many new cells into the mesendoderm differentiation program. However, this scenario did not exclude an ongoing requirement for active signaling past d3 for maximal GFP induction and/or maintenance in cells that had already committed to mesendoderm formation, a possibility raised by the effects of signaling pathway inhibitors on paracrine mesendoderm signals shown in Results of studies presented thus far suggested that the window during which BMP4 and Wnt3a efficiently induced GFP expression in Mixl1GFP/w cells were differentiated until d3 in the presence of BMP4 or Wnt3a (both added at d1) or Activin A (added d2). At d3, the factors were removed and cells differentiated for a further two days in the presence or absence of inhibitors affecting each pathway  whilst Dkk-1 and SB 431542 reduced the proportion of GFP expressing cells by \u223c70% (from 67.8\u00b12.0% to 22.3\u00b16.9%) and \u223c80% (from 67.8\u00b12.0% to 11.7\u00b12.3%) respectively (+ cells was seen following addition of Dkk-1 and SB 431542 (\u223c75% and \u223c90% respectively), whilst a lesser reduction in the proportion of GFP+ cells was observed in response to treatment of cells at d3 with noggin (\u223c35%)  . Interesells see . This inMixl1 RNA had begun to wane in cells stimulated by BMP4 or Wnt3a  than that seen with either ligand alone (2.0\u00b10.7% for Wnt3a and 29.6\u00b16.3% for Activin A) and higher than that predicted by addition of contributions representing the individual factors  comprise mixtures of many different adult tissue types, whilst their malignant counterparts (teratocarcinomas) also include persistent undifferentiated stem cell components, termed embryonal carcinoma cells (ECCs) The link between normal embryos and teratocarcinomas had been made when Solter Mixl1, a homeobox gene that marks the primitive streak of the mammalian embryo, was linked to a fluorescent reporter Mixl1 expression.We have built on these earlier observations though our investigations of the induction of mesendoderm precursors by exogenously acting growth factors in differentiating mouse ESCs. Whilst early studies proved that ECCs (and later ESCs) could differentiate to form derivatives of the germ layers, the signals initiating differentiation were provided by serum and a specific dissection of the control mechanisms was not possible. We have used a suspension, embryoid body differentiation system, in which a serum free defined medium enabled us to objectively assess the influence of specific growth factors. Our studies were also aided by the use of a genetically modified ES cell line in which the induction of FGF5+Oct4+ ESC phenotype to a Rex1-Oct4+ epiblast-like state Mixl1GFP/w cells (data not shown), were consistent with the hypothesized anti-differentiative role for nodal  during the earliest stages of differentiation Following the removal of the anti-differentiative signal, LIF, cells gained responsiveness to BMP4 and Wnt3a as mesendoderm inducing signals between d1.5 and d3, at a time corresponding to their upregulation of epiblast associated genes, such as Brachyury and Mixl1 at d3, they only weakly up-regulated the anterior mesendoderm/early endoderm markers Goosecoid, Foxa2, and Sox17. Conversely, Activin A induced higher levels of these genes at d5 of differentiation , by Wnt3a and Activin A. This synergy is also consistent with results reported by Hansson and colleagues who noted the late requirement for Wnt signalling in Activin A induction of Sox17+ definitive endoderm Our data demonstrating that BMP4 propagates mesendoderm-inducing signals in differentiating EBs via the induction of endogenous TGF and Wnt growth factors are also consistent with the autoregulatory induction loops proposed to initiate and maintain gastrulation in mouse embryos + cells observed in cultures where growth factors were removed at d3 compared with cultures that were also treated with inhibitors, suggested that mesendoderm formation remained dependent upon a growth factor for a short period even after it was removed from the culture. Experiments in which we evaluated the effects of adding inhibitors to growth factor induced cultures after d3 confirmed that the requirement for BMP4 was lost earlier than dependence upon Wnt or nodal signalling. This was evidenced by the higher percentages of d5 Mixl1GFP+ cells in noggin treated cultures compared to cultures in which Wnt or Activin signalling was inhibited  Mixl1GFP/w (clone Mix147 and Mix114) 4 cells/ml in non-adherent 6 cm bacteriological dishes  or by spin EB formation with 5\u00d7102 cells seeded per well in non-adherent round bottomed 96 well plates (Nunc) in a chemically defined medium (CDM) Mixl1GFP/w cells. Growth factors were removed from the cultures by pelleting EBs by centrifugation (480\u00d7g), aspirating the media, washing once with PBS, and resuspending the EBs in fresh CDM. The EBs were then transferred to a non-adherent bacteriological dish and returned to a humidified 37\u00b0C incubator (8% CO2 in air). GFP expression was analysed by flow cytometry at day 5 of differentiation unless otherwise stated.The Mixl1w/w and Mixl1GFP/w cells were differentiated as EBs in parallel cultures for 3d. Mixl1w/w EBs were differentiated in the absence of growth factors (control cells) or in the presence of BMP4 (10 ng/ml), Wnt3a (100 ng/ml) or Activin A (100 ng/ml) (stimulator cells). BMP4 and Wnt3a were included from the onset of differentiation whilst Activin A was added at d2. For the first 3d, Mixl1GFP/w cells (responder cells) were differentiated in the absence of added growth factors. After 3d, EBs from both lines were harvested, washed in PBS, and trypsinised to form a single cell suspension. The disaggregated Mixl1w/w and Mixl1GFP/w cells were combined at a ratio of 1\u22361 in CDM. Two thousand cells were placed into each well of low adherent round bottomed 96 well trays (Nunc) and chimeric spin EBs were formed by aggregation of the cells following centrifugation RNA was extracted using an RNeasy Mini Kit (Qiagen) and cDNA synthesized using SuperScript III (Invitrogen Corporation) according to the manufacturer's instructions. PCR gene expression analysis was performed as described previously Figure S1BMP4, Wnt3a and Activin A display a similar pattern of mesendoderm inducing activity in differentiating Mixl1GFP/w clone Mix 114. (A) Flow cytometry analysis of d5 Mixl1GFP/w clone 114 ESCs differentiated in cultures supplemented with 10ng/ml BMP4, 100ng/ml Wnt3a or 100ng/ml Activin A for the given time intervals, indicating the proportion of GFP+ cells. The growth factor treatment for each experiment is indicated and the corresponding no growth factor (-GF) control flow cytometry profiles are shown to the left of each series. (B) Representative brightfield and epifluorescence images of differentiating EBs. The growth factors and period of addition are indicated to the left of each row and day of differentiation when the image was taken in the top right hand corner of each panel. .(12.87 MB TIF)Click here for additional data file.Figure S2Kinetics of mesendoderm gene expression in EBs differentiated in BMP4, Wnt3a and Activin A. Real time PCR analysis for the indicated genes at (A) d3 and (B) d5 in ESCs differentiated in the absence of growth factors (-GF) or presence of BMP4, Wnt3a or Activin A for the indicated periods .(2.19 MB TIF)Click here for additional data file.Table S1Sequences of primers used for PCR analysis shown in (0.04 MB DOC)Click here for additional data file.Table S2Probe sets for real-time PCR analysis shown in (0.03 MB DOC)Click here for additional data file."}
+{"text": "MTUS1) has been recently identified as a candidate tumor suppressor gene which resides in a genomic region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types. It has been suggested that multiple gene promoters and alternative splicing lead to the expression of 5 known MTUS1 transcript variants.Mitochondrial tumor suppressor gene 1 (MTUS1 transcript variants.Here, we characterized the 5' untranslated regions of the different transcript variants. We also cloned and functionally tested the alternatively utilized gene promoters that contribute to the production of different MTUS1 gene are driven by multiple gene promoters.Our results confirmed the early hypothesis that the transcript variants of  MTUS1 gene is located in a region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types, including oral cancer [MTUS1 variants comes from the study on Xenopus Icis gene, a homolog of MTUS1 variants 1 and 2, which regulates microtubule growth and spindle formation prior to anaphase [MTUS1 gene and functionally cloned the alternatively utilized gene promoters that control the production of these MTUS1 transcript variants. This will enhance our understanding on the regulation of this candidate tumor suppressor gene.The region 8p2 that shl cancer . It reprl cancer . Evidencanaphase . Here, wMTUS1 transcript variants, 5'-RACE assays were carried out using human brain reference mRNA (Ambion Inc) and a FirstChoice RLM-RACE kit from Ambion, with primers specific for various transcript variants .To characterize the 5' untranslated regions (5'-UTR) of the . The predicted transcription elements of the putative promoters were listed in Additional file MTUS1 transcript variants, the following 4 fragments were PCR amplified using specific primers  a 773 bp fragment (P1') located at the 5' flanking region of exon 1; 3) a 529 bp fragment (P2) located at 5' flanking region of exon 5; and 4) a 733 bp fragment (P3) located at 5' flanking region of exon 8. The PCR products were then cloned into the KpnI/XhoI sites of pGL4.10 vector. After verification by DNA sequencing, the constructs were transiently transfected into cells using lipofectamine 2000 (Invitrogen). The pGL4.74 vector (Promega) was co-transfected as internal control for normalization of the transfection efficiency. After 48 hours, transfected cells were harvested with ice-cold phosphate-buffered saline, and dual luciferase assay were performed according to the manufacturer's protocol (Promega) using a Lumat LB 9507 Luminometer (Berthold Technologies).The gene promoter prediction was carried out using MatInspector Professional MTUS1 transcription variants that are controlled by 3 different gene promoters, 5' RACE assays were performed as described in Materials and Methods section. The 5'RACE assay designed for the long forms of MTUS1 transcription variants  leads to the cloning of the 3 alternatively utilized exons of 91 bp, 169 bp, and 410 bp, respectively . RT-PCR assays confirm the existence of these non-coding exons (data not shown). The 5'RACE assays designed for transcription variant 4 and 5 confirm the existence of these variants and also refined the 5' untranslated region for variant 5  may be regulated by both P1 and P1' promoters. It is possible that these 2 gene promoters may serve a cooperated control circuit in vivo to regulate the expression of this gene. This interesting phenomenon has been observed in a number of other genes, including gene for bradykinin B1 receptor [Based on sequence conservation and cap-analysis gene expression (CAGE) database, 3 gene promoters were predicted: P1, located at the 5'-flanking region of the gene; P2, located in intron region 5'-flanking exon 5; and P3, located in intron region 5'-flanking exon 8. These gene promoters were supported by sequence conservation  and CAGE database . The genomic locations of these putative promoters support the existence of multiple transcript variants of e Figure . Interesreceptor , and proreceptor . Future MTUS1 gene and demonstrated the presence of multiple gene promoters. Characterization of this gene will now facilitate studies on the physiological and pathophysiological function and transcriptional regulation of the MTUS1.In summary, our results refined the genomic structure of the The authors declare that they have no competing interests.HY and XZ conceived the idea for the project. JY and XL performed the laboratory analyses. JY, HY and XZ drafted the manuscript. All authors read and approved the final manuscript.Table S1. Gene-specific Primers used for the 5'-RACE assay.Click here for fileTable S2. Predicted transcription elements in MTUS1 promoters.Click here for fileTable S3. Primers used to clone promoter regions of the MTUS1 gene.Click here for file"}
+{"text": "AAVS1 . Integration at AAVS1 requires the AAV2 replication (Rep) proteins and a DNA sequence within AAVS1 containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site . The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced trs, but the sequences differ from those in AAVS1. These ITR sequences are required for replication and packaging.Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated AAVS1 RRS and trs can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of AAVS1 DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at AAVS1, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to AAVS1 sequences.In this study we demonstrate that the AAVS1 sequences to substitute for the AAV2 RRS and trs provides indirect evidence that the stable secondary structure encompassing the trs is part of the AAV2 packaging signal.The ability of these  AAVS1 dCTP and oligolabeling beads . Hybrisol  was used for the hybridization. A 1 Kb DNA ladder  and pSub201(-) were used as DNA markers. Several of the bands in the 1 Kb DNA ladder contain DNA that is in pSub201(-) and hybridize to the probe. The pSub201(-) marker was made by combining PvuII and XbaI digests of pSub201(-). The digestions were stopped with 5 mM EDTA before combining.Total DNA from HEK293 cells transfected in parallel with those used for the preparation of virus supernatants was isolated using a DNeasy tissue kit  24 and 48 hours post transfection. Some samples were pre-treated with Dpn I to degrade input plasmid. Two micrograms of DNA from each sample was resolved on a 1% agarose gel and transferred to positively charged nylon membrane  for Southern blotting analysis. Briefly, the DNA was first fragmented by soaking the gel in several volumes of 0.25 M HCl for 10 minutes. The gel was washed for several minutes with water and DNA was denatured by soaking the gel in 1.5 M NaCl, 0.5 N NaOH for 30 minutes. The gel was placed front down on a solid support covered by a piece of Whatman 3 mm paper long enough to drape into a reservoir of 10\u00d7 SSC . The positively charged nylon membrane was placed on the back of the gel below a stack of paper towels. After a 16 hour transfer the membrane was washed several times with 5\u00d7 SSC and dried in bright light. The membrane was probed with random primer labeled pSub201(-). The pSub201(-) probe was made using [\u03b1-2 flask of ~25% confluent HeLa cells was infected for 2 hours in medium without fetal bovine serum (FBS). After 2 hours the medium was replaced with medium containing 10% FBS. Cells were harvested 48 h after infection, and genomic DNA was isolated using a DNeasy tissue kit . Several combinations of AAV2 and AAVS1 primer pairs were used to detect integration by nested PCR. For the nested PCR assay 50 ng of genomic DNA and 100 ng of each primer in a 50-\u03bcl reaction volume were used in the first round of PCR amplification. After an initial incubation for 4 min at 94\u00b0C, the reaction mixture was subjected to 28 cycles of PCR amplification for 1 min at 94\u00b0C, 1 min of annealing at 63\u00b0C, and 3 min at 72\u00b0C, using FastStart DNA polymerase (Roche). One percent of the amplification product was diluted into a new reaction mixture containing the second pair of primers. The PCR parameters were the same as those for the first amplification. The following primer sets were used. In each set the first primer listed was used in the first amplification and the second primer was used in the second amplification. AAV2 rep 5'-CAC CCA GTT CAC AAA GCT GTC AGA AAT G-3' and 5'-TCG CTG GGG ACC TTA ATC ACA ATC TC-3', AAV2 cap 5'-CAG GAC AGA GAT GTG TAC CTT CAG GG-3' and 5'-TGG ACA CTA ATG GCG TGT ATT CAG AGC-3', AAV2 ITR 5'-GCC TCA GTG AGC GAG CGA G-3' and 5'-GCA GAG AGG GAG TGG CCA-3', AAVS1 5'-AGG CAG ATA GAC CAG ACT GAG CTA TGG-3' and 5'-CAG GGA AGG AGA CAA AGT CCA GGA-3'. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Cloning and sequencing of PCR-amplified junctions were performed as described previously [A 25 cmeviously .R.A.O. is a co-inventor on several patents involving AAV vectors. To the extent that this work will increase the value of those patents, he has a competing interest.VJM was the primary contributor to project conception, overall experimental design, plasmid construction, virus production, cell infection, integration assays, data analysis and writing of manuscript. He also performed all experiments.RAO was overall project coordinator, and contributed to experimental design, data analysis and writing of the manuscript.All authors read and approved the final manuscript."}
+{"text": "MEN1 locus on chromosome 11q13 and somatic mutation of the MEN1 gene have been frequently associated with the development of MEN 1-type sporadic endocrine lesions. To further investigate the role of the MEN1 gene in sporadic endocrine tumorigenesis, we analysed DNA from 14 primary parathyroid lesions, 8 anterior pituitary tumours and 3 pancreatic tumours for the presence of somatic MEN1 gene mutations and LOH of seven microsatellite markers flanking the MEN1 locus. In addition, we similarly analysed 8 secondary parathyroid lesions which arose in patients with chronic renal failure. None of the patients studied had a family history of MEN 1. Three primary parathyroid lesions and one pancreatic tumour (glucagonoma) were found to have lost one allele at the MEN1 locus. Somatic mutations were identified by SSCP and sequence analysis in one of these parathyroid lesions (P320L) and in the glucagonoma (E179V). These results support previous findings that inactivation of the MEN1 tumour suppressor gene contributes to the development of sporadic MEN 1-type endocrine lesions but is not associated with the development of parathyroid hyperplasia seen in some renal failure patients. \u00a9 2000 Cancer Research CampaignEndocrine tumours of the pancreas, anterior pituitary or parathyroids arise either sporadically in the general population, or as a part of inherited syndromes such as multiple endocrine neoplasia type 1 (MEN 1). The mechanisms responsible for the development of sporadic endocrine lesions are not well understood, although loss of heterozygosity (LOH) of the"}
+{"text": "The hypoxic toxicity and binding of misonidazole (MISO) requires metabolic reduction. The influence of glucose on the toxicity and binding of MISO was studied because glucose is a major substrate for the supply of NADPH through the hexose monophosphate pathway (HMP). Hypoxic EMT6/Ro cells (10(6) cells ml-1) were incubated with varying concentrations of glucose (0.015 mM to 5 mM). The initial rate of glucose transport was found to increase linearly with the extracellular glucose concentration up to 5 mM (0.038 nmol glucose 10(-6) cells sec-1). About 1.5 percent of the total glucose consumed went through the HMP for hypoxic cells in 5 mM glucose. The rate of HMP progressively decreased as the glucose concentration was lowered. When exposed to 5 mM MISO, the HMP was stimulated. This stimulation declined from 3.2 times in 5 mM glucose to barely detectable below 1 mM glucose. Both the hypoxic toxicity and binding of 5 mM MISO to the acid-insoluble fraction were decreased as the concentration of glucose was lowered. Below 0.5 mM glucose, no significant toxicity due to MISO was observed. There was an initial burden of 2.5 nmol MISO 10(-6) cells bound with little toxicity. After this initial burden, the terminal slope was 1.8 mol MISO bound 10(-6) cells (63 percent decrease in the surviving fraction). These results indicate that glucose concentrations lower than 5 mM can decrease the HMP rate and the toxicity and binding of MISO to hypoxic cells, and imply that calibration curves with normal and low glucose concentrations should be used to estimate the possible hypoxic fraction when MISO is used as a hypoxic probe in vivo."}
+{"text": "HNF1A gene since rare, penetrant mutations in these exons (which are only transcribed in selected HNF1A isoforms) are associated with a later age of diagnosis of Maturity onset diabetes of the young (MODY) than mutations in exons 1\u20137. The age of diagnosis in the subgroup of HNF1A-MODY individuals with exon 8\u201310 mutations overlaps with that of early multifactorial T2D, and we set out to test the hypothesis that these exons might also harbour low-frequency coding variants of intermediate penetrance that contribute to risk of multifactorial T2D.There is considerable interest in the hypothesis that low frequency, intermediate penetrance variants contribute to the proportion of Type 2 Diabetes (T2D) susceptibility not attributable to the common variants uncovered through genome-wide association approaches. Genes previously implicated in monogenic and multifactorial forms of diabetes are obvious candidates in this respect. In this study, we focussed on exons 8\u201310 of the HNF1A exons 8\u201310 in 591 European T2D subjects enriched for genetic aetiology on the basis of an early age of diagnosis (\u226445 years) and/or family history of T2D (\u22651 affected sibling). PCR products were sequenced and compared to the published HNF1A sequence. We identified several variants (rs735396 [IVS9\u221224T>C], rs1169304 [IVS8+29T>C], c.1768+44C>T [IVS9+44C>T] and rs61953349  but no novel non-synonymous coding variants were detected.We performed targeted capillary resequencing of HNF1A are unlikely to contribute to T2D-susceptibility in European samples. Nevertheless, the rationale for seeking low-frequency causal variants in genes known to contain rare, penetrant mutations remains strong and should motivate efforts to screen other genes in a similar fashion.We conclude that low frequency, nonsynonymous coding variants in the terminal exons of  HNF1A encodes the transcription factor hepatocyte nuclear factor 1 alpha and is the gene most commonly implicated in the pathogenesis of symptomatic Maturity-onset diabetes of the young (MODY) HNF1A encodes 3 different isoforms, termed A (encoded by exons 1\u201310), B (encoded by exons 1\u20137) and C (encoded by exons 1\u20136) which arise by alternative splicing and polyadenylation Despite the great success of the genome wide association (GWA) approach in identifying common genetic variants which predispose to T2D, the familial aggregation seen in this condition is far from fully explained HNF1A for example, there is evidence that a private common variant  is a major contributor to T2D pathogenesis in the Oji-Cree population (in whom the MAF is 8.7%) HNF1A variants across the allele frequency spectrum play a role in T2D-susceptibility derives from evidence that common variants in this gene are associated with multifactorial T2D Genes already known to play a role in the pathogenesis of monogenic or multifactorial diabetes are logical candidates in which to initiate the search for low frequency causal variants in T2D. In the case of HNF1A were particularly auspicious candidates in terms of harbouring low frequency, medium-penetrance variants involved in multifactorial diabetes. We set out to test this hypothesis by performing deep resequencing of these exons in a large sample of cases likely to be enriched for such variants, namely those with a strong family history of T2D and/or an early age at diagnosis.In the light of this evidence, we reasoned that the terminal isoform-specific exons of All subjects gave written informed consent and all protocols were approved by the local ethics committees. The Warren 2 Sibpair collection was approved by multiple local ethics committees in the UK including St Mary's Local Ethics Committee (EC3231). The Young Diabetes in Oxford Cohort was approved by the Oxfordshire Research Ethics Committee A (04/Q1604/97).To maximize the likelihood of identifying medium penetrance genetic variants influencing T2D-risk, a total of 591 individuals of British ancestry, ascertained for early-adult onset , and/or family history of T2D  were included .Sample 1 (the \u201cYoung Diabetes in Oxford Cohort\u201d) includes subjects diagnosed with T2D between 18\u201345 years of age, recruited from Oxfordshire GP practices. We excluded those with type 1 diabetes (T1D) by requiring that all subjects had no permanent requirement for insulin therapy within 12 months of diagnosis and no evidence of islet autoimmunity (glutamic acid decarboxylase (GAD) antibody levels <14 WHO units/ml) Sample 2 consists of probands from the Warren 2 sibpair collection (W2SP): these have been described previously To establish the background allele frequency of variants identified in this study in a UK control population, we utilised a subset of the British Birth Cohort of 1958  HNF1A (including intron-exon boundaries) was performed on the ABI3700 platform  using standard protocols (primer sequences are available on request). Sequences were compared to the reference sequence (NM_000545.3) using the unidirectional analysis mode of Mutation surveyor V3.2 . This software package has been shown to have a sensitivity of >99% in the unidirectional analysis modeTargeted capillary resequencing of exons 8\u201310 of The background allele frequency of variants identified in this study was established in a UK control population using custom TaqMan assays  in resequencing the terminal 3 exons of ividuals . Howeverividuals . The onlividuals .HNF1A were noted to have been diagnosed as late as 38 years, and the median age of diagnosis was \u223c27 years In a recent study of MODY families showing classical Mendelian segregation, individuals with mutations in exons 8\u201310 of In our study population, 60 (10%) individuals were diagnosed at 38.5 years or younger and the median age of diagnosis was 55 . Almost all cases had close relatives with T2D, a feature which is likely to reflect enrichment for medium penetrance variants.HNF1A and can be confident that there are no such variants with case allele frequencies exceeding 1.0%. Of course, we cannot exclude the possibility that additional low-frequency susceptibility variants will be found in other regions of this gene, nor in other genes known to be causal for monogenic forms of diabetes. Recent advances in high throughput \u201cnext-generation\u201d resequencing technologies Despite these measures, we failed to detect any novel coding variants in the terminal exons of Table S1Assay by design sequences for genotyping of variants detected in exons 8\u201310 of HNF1A(0.03 MB DOC)Click here for additional data file."}
+{"text": "Most teleost species, especially freshwater groups such as the Esocidae which are the closest relatives of salmonids, have a karyotype comprising 25 pairs of acrocentric chromosomes and 48\u201352 chromosome arms. After the common ancestor of salmonids underwent a whole genome duplication, its karyotype would have 100 chromosome arms, and this is reflected in the modal range of 96\u2013104 seen in extant salmonids . The Atlantic salmon is an exception among the salmonids as it has 72\u201374 chromosome arms and its karyotype includes 12 pairs of large acrocentric chromosomes, which appear to be the result of tandem fusions. The purpose of this study was to integrate the Atlantic salmon's linkage map and karyotype and to compare the chromosome map with that of rainbow trout.in situ hybridization with BAC probes containing genetic markers mapped to each linkage group. The genetic linkage groups were larger for metacentric chromosomes compared to acrocentric chromosomes of similar size. Comparison of the Atlantic salmon chromosome map with that of rainbow trout provides strong evidence for conservation of large syntenic blocks in these species, corresponding to entire chromosome arms in the rainbow trout.The Atlantic salmon genetic linkage groups were assigned to specific chromosomes in the European subspecies using fluorescence It had been suggested that some of the large acrocentric chromosomes in Atlantic salmon are the result of tandem fusions, and that the small blocks of repetitive DNA in the middle of the arms represent the sites of chromosome fusions. The finding that the chromosomal regions on either side of the blocks of repetitive DNA within the larger acrocentric chromosomes correspond to different rainbow trout chromosome arms provides support for this hypothesis. The common ancestor of salmonid fishes, which include Atlantic salmon and rainbow trout, underwent a whole genome duplication sometime within the early Tertiary to the late Cretaceous periods . The eviThe karyotype of Atlantic salmon is the exception among the salmonids as it has an NF of 72\u201374 and includes 12 large acrocentric chromosome pairs, which appear to be the result of tandem fusions . The sma). Cytogenetic mapping utilizing fluorescence in situ hybridization (FISH) with BAC probes containing microsatellite loci that are linked to the male phenotype in Atlantic salmon identified the sex chromosome pair as chromosome 2 [Genetic linkage maps have been constructed for European Atlantic salmon using microsatellites , AFLPs  and SNPsmosome 2 . This almosome 2 .Hybridization experiments were carried out with BAC clones containing genetic markers mapped to each linkage group. It should be noted that there are 29 linkage groups in the Atlantic salmon genetic map. The nomenclature for the linkage groups is historical, and linkage groups 26, 27, 29 and 30 no longer exist . With the exception of two very small chromosome pairs, multiple BAC clones were assigned to each chromosome pair. Dual hybridizations were done to confirm assignment of probes from several pairs of duplicate genes . Figure The BAC clones used to assign the genetic linkage groups to chromosomes are given in Additional File Table Table Oncorhynchus species. When the rainbow trout chromosomes are arranged according to size, the smallest metacentric chromosome pair (LG 5) falls between the largest and second largest acrocentrics, a result which supports the inversion hypothesis for this chromosome. In addition, rainbow trout LG 5 corresponds to a single chromosome arm in Atlantic salmon and in the ancestral vertebrates [Oncorhynchus species. When the location of genetic loci mapped in rainbow trout are indicated on the Atlantic salmon merged female genetic map of the Br5 and Br6 SALMAP families  currently have unknown homologies to complete arm segments [A total of 207,869 BAC-end sequences with an average length of 666 bp were screened for microsatellites using a Perl script created in the Davidson Lab, and 20,606 microsatellites were identified. As of December 2008, 697 of these microsatellite markers have been placed on the Atlantic salmon genetic map using the SALMAP mapping families Br5 and Br6 ,19 and t from the flanking regions surrounding microsatellite markers identified as anchor loci on the Atlantic salmon genetic map. The resulting sequences were subsequently screened for suspected and known repetitive elements in salmonids using a salmonid-specific repeat masker . An additional probe (5'-GTTGCCAAATTCCGAGATCTTGGCGACGAAGCCACATGAT-3') was used to hybridize to the C. briggsae control anchor spots on the CHORI-214 BAC filters. Oligonucleotide probes were radioactively labeled at the 5' end with 32P\u03b3-ATP and T4 polynucleotide kinase (Invitrogen) in a 10 \u03bcL volume reaction at 37\u00b0C for 30 minutes. The labeling reaction included 1 \u03bcL of 10 \u03bcM probe, 10 U of T4 polynucleotide kinase, 2 \u03bcL of 5\u00d7 forward reaction buffer, 2 \u03bcL of 32P\u03b3-ATP (3000 Ci/mmol) and 4 \u03bcL of water. Each of the CHORI-214 high density BAC filters was pre-hybridized for 3 hours in 50 or 100 ml of pre-hybridization solution  at 65\u00b0C. To each pre-hybridized filter 1.6 \u03bcL of radioactively labeled oligonucleotide probe and 1.6 \u03bcL of radioactively labeled C. briggsae overgo reference probe were added and hybridization was carried out at 65\u00b0C overnight (~18 hours). Three 1 hour washes consisting of 1\u00d7 SSC and 0.1% SDS were performed at 50\u00b0C to remove unhybridized probe. Filters then were wrapped in Saran\u2122 wrap and exposed on storage phosphor screens for 18 hours. Hybridization signals were detected using a Typhoon 9410 Phosphor Imager (Amersham Biosciences).Oligonucleotide probes (35\u201340 mer), which also acted as forward primers for PCR, and 20 mer reverse complement PCR primers were designed using primer3 software . These BAC clones were picked from the CHORI-214 Atlantic salmon BAC library and grown in 5 ml of LB medium containing 20 \u03bcg/mL chloramphenicol for 14\u201316 hours at 37\u00b0C with shaking at 250\u2013300 rpm. Glycerol stocks were made by placing 700 \u03bcL of each culture into 1.5 mL Eppendorf\u2122 tubes containing 300 \u03bcL of 50% glycerol. BAC DNA templates for PCR were prepared from selected clones by diluting the glycerol stock 1/40 in dH2O. Colony PCR of the selected clones positive by hybridization for a particular microsatellite's flanking region probe was performed in a T3 or T3000 Thermalcycler (Biometra) using appropriate primers and annealing temperature in 25 \u03bcL using the following procedure: initial denaturation step of 95\u00b0C for 5 min; 35 cycles with a denaturation step of 95\u00b0C for 45 sec, 45 sec at specific annealing temperature, extension at 72\u00b0C for 45 sec followed by a final extension at 72\u00b0C for 10 minutes. The PCR contained 0.05 U Taq DNA Polymerase (Qiagen), 12.5 pmoles of each specific primer, 2.5 \u03bcL 10\u00d7 PCR buffer containing 1.5 mM MgCl2, 50 \u03bcM dNTP and 5 \u03bcL of BAC DNA template. PCR products were separated on a 1.5% agarose gel containing 1\u00d7 TBE and 0.5 \u03bcg/ml ethidium bromide. The DNA fragments were visualized using a UV trans-illuminator (Ultra-Violet Products). Only those BAC clones that yielded clean single amplification products of the expected size were used for FISH analysis.BAC clones, positive by hybridization for a specific microsatellite or identified as containing a BAC-end microsatellite, were identified in the Atlantic salmon physical map Chromosome preparations were obtained from blood of the European strain of Atlantic salmon (2N = 58) by methods described previously . BrieflyWe examined several genetic maps for rainbow trout ,22-24 foKPL and RGD constructed the linkage map and integrated the markers with the BAC-based physical map. KAK, MRM, ABV and RBP carried out the FISH and chromosomal analyses. RGD and RBP correlated the Atlantic salmon and rainbow trout linkage maps and homologous chromosome arms. WSD, RBP, RGD and BFP conceived of the study. WSD, RBP and RGD drafted and prepared the final manuscript. All authors read and approved the final manuscript.Table Assignment of Atlantic salmon genetic linkage groups to chromosomes. This table shows the relationships of the genetic markers from each of the linkage groups, the BACs that contain the specific markers and the chromosome arm that each of the BACs was assigned to using FISH analysis.Click here for fileFigure Atlantic salmon consensus female genetic map from Br5 and Br6 families. This figure shows the female genetic map that was constructed based on the SALMAP Atlantic salmon mapping families, Br5 and Br6.Click here for fileFigure Atlantic salmon male genetic map from the Br5 family. This figure shows the male genetic map that was constructed based on the SALMAP Atlantic salmon Br5 mapping family.Click here for fileFigure Atlantic salmon male genetic map from the Br6 family. This figure shows the male genetic map that was constructed based on the SALMAP Atlantic salmon Br6 mapping family.Click here for fileTable Location of shared markers on Atlantic salmon and rainbow trout chromosome arms. This table shows the locations of the genetic markers that are shared in the Atlantic salmon and rainbow trout linkage maps and indicates their chromosomal positions in these species.Click here for file"}
+{"text": "Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCNDI) was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification. \u00a9 1999 Cancer Research CampaignThe expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 ("}
+{"text": "We analyzed national Haemophilus influenzae (Hi) surveillance data from 1994 and 1995 to describe the epidemiology of Hi invasive disease among persons of all ages. Serotype data were available for 376 (56%) of 669 reported Hi cases among children aged 4 years or younger; 184 (49%) were H. influenzae type b (Hib). Among children aged 4 or younger, incidence  of all Hi invasive disease was 1.8 in 1994 and 1.6 (p < 0.05) in 1995. Children aged 5 months or younger had the highest average annual incidence rate of Hib invasive disease ; children aged 6 to 11 months had the next highest rate (p < 0.05). Of 181 children with Hib invasive disease whose age in months was known, 85 (47%) were too young (aged 5 months or younger) to have completed a primary series with an Hib-containing vaccine. Of the 83 children with known vaccination status who were eligible to receive a primary series (aged 6 months or older), 52 (63%) were undervaccinated, and the remaining 31 (37%) had completed a primary series in which vaccine failed. Among persons aged 5 years or older with Hi invasive disease, the lowest average annual incidence was among those 20 to 39 years of age , and the highest was among those aged 80 years or older . Among persons aged 5 years or older, serotype data were available for 1,372 (71%) of the 1,940 Hi invasive disease cases; 159 (28%) of the 568 Hi cases with known serotype were due to Hib."}
+{"text": "The aim ofthis study is the analysis ofthe results in 62 patients over 70 years ofage with acute cholecystitis treated in our Department from 1970 to 1990. The clinical picture in 47 patients was mild and in 15 severe. In 14 cases  the acute cholecystitis subsided with antibiotics (Group A). In 48more cases  following 1-3 days conservative treatment, operation was undertaken.Besides acute cholystitis there was gangrene of gallbladder in 10, choledocholithiasis in 7 andcholoperitoneum without perforation in 7 cases. Cholecystostomy in 25, cholecystectomy in 15 andcholecystectomy with exploration ofthe bill duct in 8 cases was performed (Group B). There was one deathin group A and 3 deaths .in group B. The hospital stay was 20 days. In conclusion the clinical findings inacute cholecystitis in the aged are usually mild. In the case of failure of medical treatment, after 2-3 daysemergency surgery should be performed."}
+{"text": "The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma.Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein.Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells. HSP90 and HSP70 both play a part in the cellular response to stress. However recently it has become apparent that they also co-operate to chaperone a variety of proteins (clients) in multiple cellular pathways in cells which are not stressed. They often play a vital role in the cellular functioning of the client The HSP90 co-chaperones are a group of diverse proteins which help HSP90/70 in their action. This group includes other heat shock proteins, TPR containing proteins, cyclophilins and others. The TPR domain is a 34 amino acid protein motif thought to be involved in protein:protein interactions. TPR motif containing proteins are widely distributed across multiple classes of proteins involved in a variety of cellular functions We have previously reported the identification of a protein from Drosophila (Dpit47) which shows properties consistent with it being a TPR co-chaperone The TTC4 gene was originally identified through its localisation in a genomic region linked to breast cancer In this paper we show that TTC4 is the human orthologue of the Drosophila Dpit47 protein. The two proteins are highly related in sequence, share a common cellular location, and show analogous interactions with both heat shock and the replication initiation protein CDC6. We show that loss of interaction with CDC6 occurs when some of the point mutations identified in melanoma samples are introduced into the TTC4 gene. This suggests a possible route through which TTC4 could affect the development of malignant melanoma. We also present evidence which suggests that TTC4 has a general role in the progression of cancers in addition to melanoma, since the levels of the protein are raised in a variety of tumour cell lines.Identification of the TTC4 gene was carried out using the Blastp programme at the NCBI. The full length gene in pCR2.1 (Invitrogen) vector was a kind gift of J. Cowell (Roswell Park Cancer Institute). All subsequent cloning steps were carried out by amplifying the TTC4 insert using a high fidelity polymerase (Expand\u2013Roche) to introduce appropriate restriction enzyme sites. The gene sequence and cloning sites were subsequently checked by DNA sequencing (Lark Technologies).http://www.es.embnet.org/Doc/phylodendron/clustal-form.html)The phylogenetic tree was assembled by entering the sequences of the relevant genes into the EMBNET clustal W /phylodendron programmes on line (Human full length cDNA clones for HSP90 protein 1 beta (HSPCB) and HSP70 protein 8 (HSPA8) were obtained form Origene Technologies, Inc. HuCDC6 was obtained from A. Dutta (Dept of Pathology Boston).TTC4 was cloned in frame in the EcoR1 and Xho sites of the pJG4.5 vector.Hu CDC6 was cloned in frame in the BamH1 and Not1 sites of the pEG202 vector.HuHSP90 was cloned in frame in the Not1 and Xho1 sites of the pEG202 vector.HuHSP70 was cloned in frame in the BamH1 and Xho1 sites of the pEG202 vector.The sequences of all clones were verified by sequencing (Lark Technologies).http://proteome.wayne.edu/THPL.html)Yeast manipulations were carried out using standard two hybrid protocols . Quantitative analyses were performed using liquid beta-galactosidase assays as described (Full length TTC4 was subcloned in the pRSETa vector (Invitrogen). The 6Xhis tagged protein was purified under denaturing conditions and the resultant proteins sent to NeoMPS (Strasbourg) for antibody manufacture. Two different rabbit antisera were obtained. The specificity of the antibody was checked by affinity purification of the antibody against 6XhisTTC4 blotted onto Nitrocellulose membrane and competition with GST TTC4. For this GST TTC4 was bound to glutathione sepharose. Undiluted anti TTC4 serum was incubated for 1 hour at room temperature and then briefly spun down to pellet the beads associated with the specific antibodies. The depleted serum was used at a 1/1000 dilution in western blot. For all experiments both antibodies gave the same result.Anti HSP90 (ab13495), anti HSP70 (ab6535) and anti HuCDC6 (ab3220) were from Abcam. Alexa fluor 488 anti mouse and Alexa fluor 594 anti rabbit and Toto3 iodide were from Molecular Probes. Anti alpha-tubulin was from Sigma (clone DM 1A).Extracts for immunoprecipitation were prepared from sub confluent Hela cells. After treatment with trypsin, cells were washed twice with PBS. They were lysed in one volume of PBS, 1% triton X100 and Complete EDTA free protease inhibitors (Roche). After incubation for 10 minutes on ice the lysate was cleared by centrifugation for 5 minutes in a microfuge at 10 000 g at 4\u00b0C.Coupling of antibodies to Protein A sepharose beads and immunoprecipitation was carried out as described in 10 \u00b5g of GST-TTC4 (wild type or mutant) bound to glutathione beads was incubated with 6 \u00b5g of HSP90 in 10 mM Tris pH 7.5, 150 mM NaCl, 0.1% triton X100 and protease inhibitors . After 1 hour at 4\u00b0C the pellets were washed in the same buffer, resuspended in SDS PAGE loading buffer and analysed on a 12% PAGE SDS.2. Normal human melanocytes , and grown in RPMI 1640 medium with antibiotics, glutamine, 10% foetal calf serum and 10% COCells were trypsinised and then deposited on poly lysine treated coverslips. Fixation was carried out using 4 % paraformaldehyde diluted in cytoskeleton buffer . The cells were then treated with permeabilisation buffer . The coverslips were incubated with primary antibodies , either for 2 h at room temperature or 4\u00b0C overnight. They were washed with permeabilisation buffer and then incubated with secondary antibodies as described above. The secondary antibodies used were Alexa fluor 488 anti mouse and Alexa fluor 594 anti rabbit. The DNA was counterstained with Toto3 iodide. Prior to microscopy the coverslips were mounted in mounting medium Vectashield (Vector).This was carried out basically as described Cell pellets were washed twice in PBS and then resuspended in SDS-PAGE loading buffer at a concentration of 50,000 cells/\u00b5l. The equivalent of 150,000 cells was loaded in each lane. Cell debris was removed by centrifugation before separation by SDS \u2013PAGE.Point mutations in TTC4 (pJG4.5) were introduced using the Quickchange site directed mutagenesis kit (Stratagene) as described in the manufacturers instructions.The Drosophila Pit47 protein was used to query the NCBI databases in order to find related proteins from other species. The closest homologue from any species was identified as the human TTC4 protein (29% identity). The degree of homology between the two proteins is shown in Our previous studies had shown that the Drosophila Pit47 protein interacts with HSP90 and HSP70. These interactions are very strong and easily detected using two-hybrid analysis. We therefore used similar methodology to determine whether TTC4 showed analogous interactions. The TTC4, human HSP90 and human HSP70 genes were cloned into two hybrid bait and/or reporter plasmids as described in the To confirm these interactions we performed immunoprecipitation experiments using anti-TTC4 antibodies. Full length TTC4 protein was overexpressed and used to raise two rabbit antibodies see . Both anBoth HSP90 and HSP70 were detected in the anti-TTC4 pellet. Staining of an SDS-PAGE of the immune pellet with colloidal coomassie blue showed that HSP90 was present in near stoichiometric amounts and HSP70 was present at a level of approximately 20% (data not shown). This is similar to what we previously observed for the Drosophila TTC4 homologue A comparison of the TPR sequences of HSP90-binding TPR co-chaperones has suggested that 2 basic residues (Lys-152 and Arg-156 in TTC4) are highly conserved and are involved in the interaction with HSP90 If TTC4 is a true orthologue of Pit47 we would expect that it would show interactions with proteins involved in DNA replication. The Drosophila protein was isolated through a two-hybrid interaction with the large subunit of the DNA polymerase alpha, however when we tried the analogous experiment with TTC4 and the human DNA polymerase alpha we failed to detect an interaction  and 5. WThis interaction was also confirmed by co-immunoprecipitation  of CDC6 Dpit47 is a nucleoplasmic protein at all stages of the cell cycle. We therefore analysed the subcellular location of TTC4 to determine if it was comparable. The antibodies were used to check the subcellular localisation of the TTC4 protein using indirect immunofluoresence in the melanocyte lines Nohm 1 and Hermes 3a /b. In boTo determine whether TTC4 was tightly bound to chromatin, Hela cells were fractionated into cytoplasmic, nucleoplasmic and pellet fractions (the latter consisting mostly of chromatin and nuclear matrix) as described in Analysis of the expression of the Drosophila protein at various stages in the life cycle of the fly suggested that the protein was more abundant at those stages where proliferation was taking place. A comparison of the levels of the TTC4 protein in first trimester placental tissue (contains many dividing cells) vs heart (non dividing)  showed tA comparison was also made of the level of the TTC4 protein in normal (MRC5 fibroblasts) versus tumour derived cell lines. Previous studies have reported a number of abnormalities in the TTC4 gene in melanoma tissues isolated from patients In this paper we have presented a number of lines of evidence which suggest that TTC4 is the human orthologue of the Drosophila pit47 protein: 1) The protein sequences of the 2 proteins show high levels of similarity, and this homology is not confined to the region of the TPR motifs; 2) Both proteins are contained in the nucleus and are most likely found mainly in the nucleoplasmic compartment. In each case several putative bipartite like NLS motifs Data presented in this paper also suggests that TTC4 is involved in the development or progression of cancer. A role for TTC4 in melanoma had already been suggested, since mutations seen in the gene in patient-derived samples and cell lines, seemed to correlate with increased invasiveness). Our data suggests that in addition to structural changes in the protein, melanoma lines contain more TTC4 protein than melanocytes. Furthermore increases in TTC4 protein levels are also observed in a number of other tumour derived cell lines compared to normal cells and tissues. It is therefore possible to suggest that an increased level of TTC4 contributes to the development of a variety of different tumours. An increased level of Dpit47 has also been reported in brain tumours in flies Although the precise role for TTC4 in tumour development is not clear, it may be partly related to a loss of protein interactions. Two of the naturally occurring TTC4 point mutations cause the protein to lose its interaction with hCDC6. Only one of these amino acids is fully conserved in the Drosophila protein but we have observed (GC and SC unpublished results) that mutation of this amino acid in Pit47 destroys its interaction with the Drosophila CDC6. We cannot say that the loss of the interaction with CDC6 is the precise cause of the defect, since the region into which the mutations map is likely to be involved in interactions with a number of proteins. (For instance the same region of pit47 is needed for interaction with DNA polymerase (David Loebel and SC unpublished observation)). In addition interaction of TTC4 with other cellular proteins has also been reported (e.g. TFIIIh"}
+{"text": "Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA\u2013DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments  using newly designed specific PCR primer sets. Fluorescence  A limiting step in understanding any microbial ecosystem resides in our ability to inventory the microorganisms inhabiting the ecosystem, and to assess their metabolic potential, the interactions between them and their biotope. A partial answer to this challenge was (i) culture-independent studies based on the development of molecular microbial diversity analyses using the 16S rDNA gene as a phylogenetic marker , and  experiments helped to provide information about morphology and localization of the WWE3 bacteria within a microbial anaerobic sludge sample. Furthermore, the absence of the H17 helix in the WWE3 16S rDNA secondary structures is unprecedented and seems to be a characteristic of the bacterial candidate division WWE3 and its closest relatives.In the present study, we report that in the course of analysing a metagenomic fosmid library constructed from an anaerobic digester, we detected an unusual group of 16S rDNA bacterial gene sequences. These sequences, which presented many mismatches with the 16S rDNA universal primers, cannot be obtained by the classical polymerase chain reaction (PCR)-based amplification methods. Specific 16S rDNA PCR primers were developed for this group of sequences, named WWE3. Phylogenetic analyses show that this group constitutes a new bacterial candidate division. Fluorescence In order to analyse the microbial diversity and the metabolic potential of a mesophilic anaerobic digester, a large fosmid library was constructed using DNA extracted from the sludge digester of the WWTP of Evry, France. A part of the library (27 648 fosmid clones) was screened by hybridization with 16S rDNA gene targeted-hybridization probes. The 16S rDNA genes of 570 positive clones were directly sequenced using internal primers. While for 541 of these positive clones, the 16S rDNA gene sequences were obtained and affiliated to known bacterial or archaeal phyla, we were unable to obtain a 16S rDNA sequence for 29 clones. Analysis of HindIII fingerprints of these clones showed that their profiles were very similar. Southern blot hybridization using 16S rDNA-targeting probes revealed that 27 out of the 29 clones showed a common 1.6 kb HindIII positive fragment while the remaining two clones possess a positive 1.65 kb fragment. Shotgun sequencing of one of these 29 fosmid clones (DIGA11YD11) revealed that it does contain a complete 16S rDNA gene sequence which affiliates (88% identity) with a single sequence (AY953190) in public databases. The 16S rDNA sequences of the remaining 28 fosmids were determined by direct sequencing with specific primers derived from the DIGA11YD11 16S rDNA . All theExperimental procedures) were tested using the four DIGA11YD11-specific primer sets 1\u20134  16S rDNA libraries were constructed from DNA extracted from seven anaerobic digesters, using the DIGA11YD11-specific primers or a combination of specific and universal primers, (ii) sequence analysis of these 16S rDNA confirmed their inclusion in the WWE3 phylogenetic group and allowed us to design degenerate primers with a broader specificity. The presence of WWE3 bacteria was further investigated and confirmed in other anaerobic or anoxic environments such as swine lagoon slurries (6/10 samples) and freshwater biofilms (6/6 samples). The presence of WWE3 bacteria was tested by PCR on DNA sampled from three digesters over a 6-year period (2000\u20132006). During this period, WWE3 bacteria were detected in 16 out of 23 Evry sludge samples, three out of six from Corbeil and in all three sludge samples from Creil, showing that the WWE3 population size is subject to large variations in these anaerobic digesters.A total of 21 16S rDNA libraries were constructed from 12 DNA samples extracted from eight anaerobic digesters , one swiAn additional 61 bp insertion (type II) was found in the OTU-4 sequences. The two insertions (I and II) are located within the same region but exhibit completely different sequences. The overall intradivergence between WWE3 16S rDNA gene sequences reached 20% and is in the same order of magnitude as that for other uncultured bacterial candidate divisions . The retained sequences were then imported into the ARB database (http://www.arb-home.de) for phylogenetic analyses. An automatic alignment was performed which was manually checked and corrected.The 16S rDNA sequences obtained were edited and assembled with Phrap . Twenty-five 16S rDNA sequences representative of WWE3-defined OTUs, as well as representatives of OP11, WS6, OD1 and TM7 candidate divisions were used for tree construction. A modified version of the \u2018Lane mask\u2019 was used to choose homologous positions for tree construction . Phylogetest 3.0  to identtest 3.0  nucleotiradA annotated gene from DIGA11YD11 fosmid was aligned along with bacterial (sequences subset extracted from family HBG000623) and archaeal and eukaryotic (family HBG049531) radA homologues obtained from HOGENOM (http://pbil.univ-lyon1.fr). Phylogenetic analysis were performed using PhyML , six samples from Corbeil and three from Creil. The other digester samples are described in in situ hybridization experiments were performed as previously described  using the https://www.genoscope.cns.fr/agc/mage/wwwE3scope/.Gene prediction was conducted using the AMIGene software (Sequences determined in this study were deposited in the EMBL database under Accession No. CU367853 to CU367881 for sequences obtained from fosmid clones and CU392752 to CU392838 for sequences obtained from 16S rDNA clone libraries. DIGA11YD11 complete fosmid sequence was submitted under Accession No. CU367853."}
+{"text": "DGAT1) gene by using data sets including sires with accumulated phenotypic observations of daughters . However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits.Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR \u2013 K232A haplotypes indicating parent-of-origin effects on milk production traits.Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits.Non-additive effects like those identified at the bovine  DGAT1) gene is presumably the putative causal mutation for this QTL [DGAT1 K232A alleles have been confirmed in several breeds and populations [DGAT1 K232A locus representing the causal mutation for the QTL was achieved by functional in-vitro studies showing a higher efficiency in triglyceride synthesis of the allele DGAT1 232K compared to the allele DGAT1 232A [DGAT1 locus affecting milk production traits in cattle [DGAT1 promoter containing a SP1 transcription factor binding site motif (CCCGCC) was targeted as functional background for these additional alleles due to the functional relevance of juxtaposed SP1 binding sites for the regulation of gene expression in eukaryotes [DGAT1 promoter VNTR alleles consisting of a variable number of putative SP1 transcription factor binding sites showed an association with daughter yield deviations for milk production traits in a data set comprising bulls with homozygous genotype DGAT1 232A/232A [DGAT1 promoter VNTR alleles is underlined by in-vitro studies providing evidence for SP1 binding to the CCCGCC motif in the DGAT1 promoter and for induction of gene expression by the DGAT1 promoter VNTR alleles [Several mapping studies revealed a QTL for milk production traits in the centromeric part of cattle chromosome 14 (BTA14) [this QTL ,3. The pulations . FurtherAT1 232A . Howevern cattle -10. A VNkaryotes ,12. The 32A/232A . This re alleles .per se powerful granddaughter designs [DGAT1 polymorphisms in a data set comprising direct trait measurement in cows, however, demonstrate that the DGAT1 K232A and the promoter VNTR alleles display a dominant-recessive pattern of effects on milk production traits. This pattern of effects was previously unnoticed in data sets using breeding values or daughter yield deviations. Additionally, we demonstrate differences in the effects of maternally and paternally inherited DGAT1 promoter VNTR \u2013 K232A haplotypes on milk production traits, which indicate a parent-of-origin effect at the DGAT1 locus in cattle.There are several confirmed examples for non-additive QTL effects or QTL acting dependent of parental origin ,15 in a  designs , it is i designs . Our stuDGAT1 K232A and promoter VNTR alleles as well as of the DGAT1 promoter VNTR \u2013 K232A haplotypes based on the maternally inherited alleles/haplotypes are listed in Table DGAT1 232A and the promoter VNTR allele 3, respectively. All possible allele combinations were detected in the maternally inherited DGAT1 haplotypes with the maternally inherited promoter VNTR \u2013 K232A haplotype 3 \u2013 232K showing the highest frequency. Strong linkage disequilibrium was observed between both DGAT1 loci, because DGAT1 232K was almost exclusively detected in linkage phase with the promoter VNTR allele 3. Due to their extremely low frequency, the DGAT1 haplotypes 1 \u2013 232K, 2 \u2013 232K, 4 \u2013 232K, and 5 \u2013 232K could not be included in the analysis of haplotype effects.Frequencies of the DGAT1 promoter VNTR allele 5 compared to all other promoter VNTR alleles are listed in Table 232A/232A homozygous. The DGAT1 promoter VNTR allele 5 showed a significantly superior effect on milk fat content and milk protein content, while milk yield was decreased compared to all other promoter VNTR alleles. The differences for effects on milk fat yield and on milk protein yield were not significant.To enable a direct comparison of this study with results of a previous study in German Holstein sires , the genDGAT1 K232A genotypes solitary showed highly significant effects on all milk production traits in a reduced model . Similar results were observed with the full model including the DGAT1 K232A genotypes as well as the promoter VNTR genotypes. The only exception was for milk protein yield, where no significant effect for the DGAT1 K232A locus was obtained in the full model. In contrast to the lack of association between DGAT1 K232A genotypes and milk protein yield, the DGAT1 promoter VNTR genotype had significant effects on all milk production traits including milk protein yield in the full model.The DGAT1 K232A genotype effects revealed that for all milk production traits the difference between 232A/232K heterozygotes and the 232A/232A homozygotes was larger than the difference between the 232A/232K heterozygotes and the 232K/232K homozygotes. This was most prominent for milk fat percentage in both models. Similarly, for milk yield and milk protein yield the difference between the DGAT1 promoter VNTR 4/4 homozygotes and the */4 heterozygotes  was larger than the difference between the */4 heterozygotes and the */* homozygotes. The calculated dominance values showed significant dominance effects on milk yield, milk fat yield and milk protein yield , milk fat content (DGAT1 232K) and milk protein content  (Table 4 had a lower milk yield and as a consequence a lower milk fat yield and a lower protein yield compared to the mean of the homozygotes with zero (*/*) or two copies (4/4) of the allele. Heterozygotes for the VNTR allele 5 had a higher milk protein content and heterozygotes for the DGAT1 232K showed a significantly higher milk fat content and a tendency towards a higher milk protein content and to a reduced milk yield.The analysis of the contrasts between 3 \u2013 232K haplotype resulted in significantly higher milk yield and milk protein yield and lower milk fat content when inherited paternally. In contrast to that, the 1 \u2013 232A haplotype resulted in significantly lower milk yield and higher milk fat content when passed by the sire to the offspring. For all other VNTR \u2013 K232A haplotypes, there is no influence of the parent-of-origin detectable at a significant level, but in the case of the 3 \u2013 232A haplotype, there were tendencies to a lower milk fat content and lower milk protein content for the paternally inherited haplotype. In a further analysis, we tested if those differences between paternally and maternally inherited DGAT1 haplotypes could also be detected when investigating the DGAT1 K232A alleles only. As indicated in Table DGAT1 K232A alleles obtained results similar to those for the DGAT1 promoter VNTR \u2013 K232A haplotypes.The simultaneous test for a parent-of-origin effect over all haplotypes revealed a significant influence for the traits milk yield, milk fat content and a detectable, but not significant influence on milk protein yield Table . More spDGAT1 promoter VNTR genotypes showed significant effects on all milk production traits in a model also including the DGAT1 K232A genotypes. Our results provide an independent confirmation of previous findings [DGAT1 K232A mutation does not account for the entire QTL variation on milk production traits in the centromeric part of BTA14. These results are in contrast to Grisart et al. [DGAT1 K232A genotype did erase the effect of any other polymorphism at the DGAT1 locus. However, Grisart et al. did not consider the DGAT1 promoter VNTR. In our study, the K232A polymorphism did not significantly affect milk protein yield in a model that also included the promoter VNTR genotype. Compared to previous results obtained in an independent data set [DGAT1 promoter VNTR showed an identical pattern and were very consistent in magnitude with allele 5 being superior for milk fat and milk protein content. In an independent cattle breed, Sanders et al. [DGAT1 K232A mutation and the DGAT1 promoter VNTR for milk fat yield and content also supporting effects of those loci on milk production traits. Recently, F\u00fcrbass et al. [DGAT1 promoter VNTR motif. This observation and the confirmed trait association of DGAT1 promoter VNTR alleles presented in this study provide further evidence that the alleles at the DGAT1 promoter VNTR indeed may exert a direct effect on milk production traits. However, in spite of those data, it cannot be formally excluded that another, yet undetected gene outside the DGAT1 locus may be the molecular background for the additional QTL alleles in this genomic region.In our study, the findings ,9 demonst et al. , who dess et al.  detecteds et al.  proved SDGAT1 locus focused on additive gene substitution effects for the QTL affecting milk production traits on BTA14  as well as in models combining the DGAT1 K232A and promoter VNTR genotype effects. Analogous to the non-additive effects of the alleles at the DGAT1 K232A locus, we obtained indication on dominance effects for the alleles 4 and 5 at the DGAT1 promoter VNTR locus. Interestingly, for the 232K allele and the promoter VNTR allele 5, which exhibit a similar pattern of effects on milk production traits, the pattern of dominance was also similar predominantly affecting milk fat and milk protein content. In contrast, recessive effects for the DGAT1 promoter VNTR allele 4 were identified primarily on yield traits. Our data represent the first report on dominance effects on milk production traits for alleles at the DGAT1 locus.Previous publications on the effect of the 14 (e.g. ) as wellDGAT1 effects. Because Fisher and Spelman [et al. [DGAT1 232K/232K included in their study prevented a powerful analysis of DGAT1 K232A effects with respect to dominance. The dominant physiological effects of the DGAT1 232K variant on milk fat synthesis may be caused by the high efficiency of the 232K DGAT1 protein for triacylglycerol synthesis [max of the 232K DGAT1 molecules might result in the supply of substrates becoming the limiting factor for milk fat synthesis, thus preventing a further substantial difference in milk fat synthesis between individuals carrying one or two copies of the DGAT1 232K allele.Some studies ,6,10,18  Spelman ,6. Howevynthesis . The higDGAT1 allele effects have an impact on allele substitutions effects evaluated by regression analysis on the number of DGAT1 K232A or promoter VNTR alleles. They can alter during selection processes in dependency of the magnitude of dominance and allele frequencies. However, allele effects obtained by regression in two independent populations (this study and [Dominant tudy and ) showed DGAT1 alleles demonstrates that although indirect phenotypes like breeding values or daughter yield deviations may have an increased statistical power to detect QTL, the analyses of additional designs with direct phenotypes may be necessary for a precise estimation of the allele effects at causal mutations.The discovery of dominant/recessive effects of DGAT1 promoter VNTR \u2013 K232A haplotypes regarding effects on milk yield, milk fat content and milk protein yield. The results for the differences between paternally and maternally inherited haplotypes are in line with results looking at alleles at the DGAT1 K232A locus only. These differences of effects between paternally and maternally inherited DGAT1 alleles/haplotypes have to be evaluated carefully to exclude artificial effects, e.g. due to the applied model including fixed effects of the sires, but not of the dams. This might result in a reduced variance of the paternally inherited alleles/haplotypes, because within the model the paternal DGAT1 haplotype effect might already be included in the sire effect. However, for the DGAT1 3 \u2013 232K haplotype the associated effect on milk yield was larger when paternally inherited than when maternally inherited, while for 1 \u2013 232A the contrast was opposite. This result indicates that it is quite unlikely that a systematic bias due to reduced variance of paternally inherited alleles is the only source for the observed differences. We interpret these differences of haplotype contrasts for paternally and maternally inherited DGAT1 K232A \u2013 promoter VNTR haplotypes as parent-of-origin effects. If the DGAT1 locus indeed were a target for parent-of-origin effects, differences between paternally and maternally inherited alleles/haplotypes are expected for the DGAT1 promoter VNTR \u2013 K232 haplotype as well as for the DGAT1 K232A mutation alone as we found in our study.Our study revealed a difference between the paternally and maternally inherited DGAT1 gene is not located in a chromosomal region homologous to known imprinted genome areas in mice or human [2 resource population [DGAT1 studies.It has to be noted that the or human . On the or human ,21. Howepulation , which wDGAT1 locus in cattle and also provides further support for a functional role of the DGAT1 promoter VNTR additional to the DGAT1 K232A alleles on milk synthesis. The precise mechanisms behind the dominance and parent-of-origin effects require further targeted functional studies to be fully understood. Additionally, the DGAT1 allele effects deviating from the commonly applied hypothesis of additive QTL effects highlight the need towards efficient ways to include non-additive effects into QTL mapping models [DGAT1 locus, which seems to be responsible for a substantial proportion of the genetic variance of milk production traits [DGAT1 loci in cattle will be a useful model to study the biological background of dominance and parent-of-origin effects in mammals.Our study indicates dominance and parent-of-origin effects at the g models . The DGADGAT1 K232A and promoter VNTR polymorphisms. Nine of the sires were proven sires with high genetic merit for milk production traits with their daughters born after the sires finished their progeny test, whereas ten sires were young sires with their daughters originating from the progeny test itself. The cows themselves were unselected, except for their paternal ancestor.The data set of the study included a total of 1035 German Holstein cows sampled on ordinary dairy farms located in nine different regions of Germany. The cows descended from 19 different sires . The sires were pre-selected to represent a large variety of genotypes at the DGAT1 gene the non-conservative polymorphism K232A in exon 8 giving rise to a lysine => alanine amino acid substitution as well as the non-coding VNTR in the promoter were included. The DGAT1 K232A mutation was genotyped according to [DGAT1UP 5'-GCACCATCCTCTTCCTCAAG-3'; DGAT1DN 5'-GGAAGCGCTTTCGGATG-3') amplified a 411 bp fragment that was digested by the restriction enzyme CfrI . The resulting fragments  were separated on a 2% agarose gel. A PCR fragment containing the VNTR polymorphism at position 1465 in the promoter of the bovine DGAT1 gene (Genbank no. AJ318490) was amplified by the following primers: DGAT1proUP 5'-TCAGGATCCAGAGGTACCAG-3', DGAT1proDN 5'-GGGGTCCAAGGTTGATACAG-3'. The PCR reaction was carried out in a 10 \u03bcl volume under the following conditions: 50 ng genomic DNA, 5 pmol of DGAT1proUP, 10 pmol of DGAT1proDN, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.2 U Taq Polymerase . A touchdown protocol was performed starting at 70\u00b0C with 2\u00b0C steps to the final annealing temperature of 60\u00b0C. One microliter of the reaction volume was run on an automated fragment analysis system  under denaturing conditions. Allele nomenclature for the DGAT1 promoter VNTR was identical to allele classification in [Within the DGAT1 promoter VNTR \u2013 K232A haplotypes of sires were derived from the genotypes of the respective group of daughters. Consecutively, the paternally inherited haplotype of each daughter was determined by assuming no recombination between DGAT1 promoter VNTR and K232A polymorphism due to the close genomic co-localization of both loci . The maternally inherited haplotype of a cow was deduced from subtracting the paternally inherited haplotype from the daughters' genotype. For daughters sharing their sires' genotype at the DGAT1 K232A as well as the promoter VNTR locus, it was not possible to assign paternally or maternally inherited haplotypes, respectively.DGAT1 alleles. Records were only included, if the cow had a minimum of eight milk recordings within the first lactation. Yield deviations for milk fat percentage and milk protein percentage were calculated according to [For the 1035 cows included in the analysis, first lactation yield deviations (YD)  for milkDGAT1 K232A and promoter VNTR genotypes to ensure a high variety of alleles and haplotypes in the analysis. Instead, the maternally inherited alleles and haplotypes of the cows were used to receive an overview of the DGAT1 allele distributions in the population.The allele and haplotype frequencies for the German Holstein population cannot be estimated from the genotypes of the cows, because the sires of the cows included in the study had been selected with respect to DGAT1 promoter VNTR allele 5 displayed the most pronounced effect on milk production traits compared to the other VNTR alleles, particularly on milk fat percentage [DGAT1 promoter VNTR effects estimated in that study with the effects within the German Holstein cow data set of the present study, the allele substitution effect of DGAT1 promoter VNTR allele 5 compared to all other alleles was calculated using a regression analysis with general linear model procedures  applying the following model:In a previous study, daughter yield deviations (DYDs) of German Holstein bulls were analyzed, and the rcentage . To enabyijk is the YD of daughter j within sire i carrying the DGAT1 K232A genotype k, \u03bc is the overall mean, si is the fixed effect of sire i, K232Ak is the fixed effect of the DGAT1 K232A genotype k; zij is the number of alleles 5 at DGAT1 promoter VNTR  of daughter j within sire i, b is the regression coefficient representing the allele substitution effect of VNTR allele 5 compared to all other alleles, and eijk is the random residual effect. The respective analysis was performed including all individuals as well as considering a subset restricted only to the homozygous DGAT1 232A/232A cows.where DGAT1 232A/232A individuals only to evaluate DGAT1 promoter VNTR effects [DGAT1 K232A and promoter VNTR effects jointly by including all DGAT1 K232A genotypes. General linear model procedures  were used to estimate the effects of both DGAT1 locus genotypes on milk yield, milk fat yield, milk protein yield, milk fat percentage, and milk protein percentage with the following model and a type III sum of squares test:While the previous study design consisted of  effects , the preijkl is the YD of daughter j within sire i carrying the DGAT1 genotypes k at K232A and l at the promoter VNTR, respectively, \u03bc is the overall mean, si is the fixed effect of sire i, K232Ak is the fixed effect of the DGAT1 K232A genotype k, VNTRl is the fixed effect of the DGAT1 promoter VNTR genotype l, and eijkl is the random residual effect. Only DGAT1 promoter VNTR genotypes represented by more than ten daughters in the data set were included in the analysis. For comparison to the outcomes of previous studies, a submodel of eq. 2 without the fixed effect of the DGAT1 promoter VNTR genotype was also used.where y232K allele and for the DGAT1 VNTR alleles 4 and 5 defining three genotype classes with zero, one or two copies of the respective allele in each case.For both, the model and the submodel, the magnitude of the dominance was calculated by contrasting the estimated coefficients for heterozygotes to the average of the coefficient of homozygous genotypes. This was done for the DGAT1 promoter VNTR \u2013 K232A haplotypes on milk production traits were estimated separately applying the following model:Finally, the effects of the maternally and paternally inherited ijkl is the YD of daughter j of sire i with paternally inherited DGAT1 promoter VNTR \u2013 K232A haplotype k and maternally inherited DGAT1 promoter VNTR \u2013 K232A haplotype l, \u03bc is the overall mean, si is the fixed effect of sire i, DGAT1_patk * DGAT1_matl is the combined fixed effect of the paternally inherited DGAT1 promoter VNTR \u2013 K232A haplotype k and the maternally inherited DGAT1 promoter VNTR \u2013 K232A haplotype l, and eijkl is the random residual effect. To test for a parent-of-origin effect, a set of linear hypotheses of the estimated effect coefficients were formulated that compare the estimates of the group that received a specific haplotype from the father to the estimates for the group that received it from the mother . Each linear hypothesis was tested separately (for each haplotype) and finally the whole set of hypotheses was tested simultaneously. This last test is interpreted as an overall test for the parent-of-origin effect. In this analysis, only observations with haplotypes occurring in both parent sexes were included.where yThe author(s) declares that there are no competing interests.CK conceived, designed and coordinated the study, carried out the molecular genetic studies, performed a part of the statistical analyses and drafted the manuscript. RW participated in the molecular analysis and helped drafting the manuscript. CE performed the statistical analyses and GT participated in the statistical analysis and design of the study and helped drafting the manuscript. All authors read and approved the final manuscript.DGAT1 genotype effects on milk production traits. Least square mean values (S.E.) for milk production traits for DGAT1 genotypes in a German Holstein cow data set from a combined model with DGAT1 K232A and VNTR genotype effects and from a reduced model including DGAT1 K232A genotype effects only.Click here for file"}
+{"text": "The finding of adenomatous polyps of the gallbladder is a rare occurrence and an unusual clinicalproblem.\t\tAmong 2,145 patients who underwent cholecystectomy for gallbladder disease only 9 (0.4 per cent)presented with adenomatous polyps. There were 6 women and 3 men, aged 17 to 70 years. Preoperativeultrasonographic diagnosis was made in only 1 of 7 patients with gallstones, in contrast polypoid lesionswithin a gallbladder without stones were easily confirmed by both ultrasonography and oral cholecystographyin the remaining 2 patients. All polyps were 1.0 cm or less in size and without histologic evidenceof malignant change. The clinical significance of this rare condition is discussed, with particularreference to a possible role in development of gallbladder carcinoma. Surgical treatment should beadvocated regardless of clinical manifestation when the polyp exceeds 1.0 cm in diameter or rapidgrowth of the lesion is seen on ultrasonographic follow-up examinations."}
+{"text": "The effect of preheating EMT6 cells in vitro on their response to cytotoxic agents of either 43 degrees C or 37 degrees C has been investigated. Preheating for 3 h at 40 degrees C produced measurable protection  to subsequent treatment for 1 h at 43 degrees C. This preheat treatment was further found to reduce cell killing by BLM and BCNU (drug tolerance) present during 1 h at 43 degrees C. In contrast, no such heat-induced drug tolerance was seen with ADR. An additional effect with ADR was the apparent elimination of heat-induced thermal tolerance at toxic drug doses. However, preheating under these conditions had no effect on the subsequent cytotoxicity of any of these drugs at 37 degrees C. Also, preheating for 1 h at 43 degrees C was found to sensitize cells to BLM and BCNU toxicity at 37 degrees C but to protect against ADR toxicity. The results are discussed in relation to known mechanisms of cell killing by heat and of thermal tolerance."}
+{"text": "Biological response modifiers such as interleukin 2 (IL2) may be most effective in the setting of minimal residual disease. In a phase I-II clinical trial, IL2 was administered to 10 patients in remission of acute myeloid leukaemia and three with multiple myeloma 1-4 weeks after treatment with ablative chemotherapy or chemotherapy and autologous bone marrow transplantation. The aim was to assess the capacity of these patients to tolerate IL2 after intensive therapy and to determine whether regenerating lymphocytes were capable of responding to IL2 with the generation of anti-leukaemic effector cells. Toxicity was severe in two patients treated with escalating doses of IL2 and 19 subsequent infusions administered to 11 patients on a fixed dose schedule for periods of 3-5 days were well tolerated. Major toxicity was confined to hypotension (two courses) which responded rapidly to treatment cessation. No patients required intensive care unit support. IL2 infusions produced no significant adverse effects on marrow regeneration; while there were transient falls in platelet counts there were no episodes of clinical bleeding and neutrophil counts increased from a mean of 1.1 pre-infusion to 2.5 x 10(9)l-1 during the infusion (P = 0.004). A significant biochemical abnormality was hypokalaemia which responded rapidly to correction. Cells with activity against leukaemic progenitor cells appeared in peripheral blood within 48 h of beginning treatment. We conclude that IL2 may be used in minimal residual haematological malignancy, and by producing anti-neoplastic effector cells has the potential, as yet unproven, to prolong disease-free survival of patients entering remission."}
+{"text": "Meiosis is a specialized form of cell division involving one round of chromosome replication followed by two rounds of segregation, thereby producing daughter cells with half the genomic equivalent of the progenitor. In most organisms, double-strand breaks (DSBs) are introduced into the genome following premeiotic S-phase. These breaks are repaired almost exclusively from the homologous chromosome via repair pathways that yield either a crossover or non-crossover recombination product It should be no surprise then that most eukaryotes possess a sophisticated mechanism to control meiotic recombination. Consider the situation in an individual mouse meiocyte. More than 200 DSBs are made; however, only a subset of these precursors are repaired as crossovers, while the rest are repaired as non-crossovers  [4]. ThuAlmost a century after the first observation of crossover control PLoS Genetics identify a role for the Pachytene Checkpoint gene, PCH2, in crossover control in Saccharomyces cerevisiaePCH2, which encodes a putative AAA+-ATPase, was initially identified in yeast as a checkpoint factor due to suppression of a zip1\u0394 arrest in a pch2\u0394 mutant. This and other observations led to the hypothesis that Pch2 helps monitor chromosome synapsis during meiotic prophase PCH2 is widely conserved in organisms that construct a synaptonemal complex and exhibit crossover interference, but is absent from organisms such as Schizosaccharomyces pombe that do not exhibit these features pch2 mutants at the HIS4LEU2 recombination hotspot Two articles in this issue of PLoS Genetics, the Alani and B\u00f6rner groups pch2\u0394 strains. Importantly, these analyses demonstrated that the crossovers formed in pch2\u0394 mutants show reduced interference.In studies published in this issue of pch2\u0394 strains carrying various hypomorphic alleles of the topoisomerase-like protein, Spo11. These hypomorphic alleles decrease the number of DSBs pch2 mutation has little or no effect on spore viability on its own, introducing a spo11 mutation that reduces DSB activity by \u223c20% significantly reduced viability despite approximately wild-type crossover frequencies spo11 hypomorphs that reduce DSB activity up to 80%. Although this is an indirect method of measuring crossover homeostasis, these findings provide compelling evidence that Pch2 has a role in multiple aspects of crossover control during yeast meiosis.Recent studies have indicated that decreased crossover interference is associated with a concomitant decrease in crossover homeostasis pch2\u0394 mutants, Hop1/Red1 and Zip1 exhibit a more uniform axial localization pattern than is observed in wild type. Joshi et al. now demonstrate that chromosome domains that are enriched for Hop1 and Red1 tend to colocalize with future sites of crossover formation, leading to the hypothesis that Pch2 functions to stabilize alternating domains enriched for either Hop1/Red1 or Zip1. Such domains are proposed to be modules that mediate crossover designation and interference. Interestingly, when PCH2 is deleted, not only is axial organization of Hop1/Red1 and Zip1 compromised, but appearance of both crossover and non-crossover products is delayed to similar extents pch2 mutant phenotype are consequences of the same molecular defect, nor is it yet clear precisely how Pch2 protein functions in wild-type cells. Nonetheless, the current findings provide new support for the idea that higher order chromosome structure plays a key role in crossover control So what role could Pch2 play in this process? Pch2 is required for differential organization of chromosome structural proteins Hop1 and Red1 relative to the synaptonemal complex central element protein Zip1"}
+{"text": "BRCA1 and BRCA2 to breast cancer incidence in outbred populations have been based on studies that are either small or have selected for cases diagnosed at an early age. Only one of these has reported an estimate of the breast cancer risk associated with a mutation in these genes, and there is no published ovarian cancer risk estimate derived from a population-based case series. We screened a population-based series of breast cancer cases diagnosed before the age of 55 for mutations in BRCA1 and BRCA2. Pedigree information from the mutation carriers was used to estimate penetrance and the proportion of familial risk of breast cancer due to BRCA1 and BRCA2. We identified eight (0.7%)BRCA1 and 16 (1.3%)BRCA2 mutation carriers in 1220 breast cancer cases . Mutation prevalence was substantially higher in cases diagnosed before 35 years-of-age and with increasing number of relatives affected with breast or ovarian cancer. However, most mutation carriers were diagnosed in the older age groups, and a minority reported a first-degree relative with breast cancer. Breast cancer penetrance by age 80 was estimated to be 48% (95% CI 7\u201382%) for BRCA1 mutation carriers and 74% (7\u201394%) for BRCA2 mutation carriers. Ovarian cancer penetrance for BRCA1 and BRCA2 combined was 22% (6\u201365%) by age 80. 17% of the familial risk of breast cancer was attributable to BRCA1 and BRCA2. At birth, the estimated prevalence of BRCA1 mutation carriers was 0.07% or 0.09% depending on the penetrance function used for the calculation. For BRCA2 the birth prevalence estimates were 0.14% and 0.22%. Mutations in the genes BRCA1 and BRCA2 are rare in the population and account for a small fraction of all breast cancer in the UK. They account for less than one fifth of the familial risk of breast cancer. Eligibility criteria for BRCA1 and BRCA2 mutation testing based on family history and age of onset will identify only a small proportion of mutation carriers. \u00a9 2000 CancerResearch CampaignEstimates of the contribution of"}
+{"text": "Two patients with relapsed Wilms' tumour and renal failure requiring dialysis were given carboplatin and etoposide by pharmacokinetically guided dosing. The target area under the drug plasma concentration vs time curve (AUC) was 6 mg ml-1 min for carboplatin and 18 and 21 mg ml-1 min for etoposide. On course 1 measured AUCs of carboplatin and etoposide were 6 and 20 mg ml-1 min for patient 1 and 6 and 21 mg ml-1 min for patient 2 respectively. Peritoneal dialysis did not remove carboplatin or etoposide from the plasma, however carboplatin but not etoposide was cleared by haemodialysis. Therapy with carboplatin and etoposide is possible in children and adults with renal failure who require dialysis, but in this situation pharmacokinetic monitoring is essential."}
+{"text": "Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-\u03b2), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which  Bmp4 in mice affects the formation of not only the sensory regions but also their associated canals. These results demonstrate for the first time that a single gene, Bmp4, is required for the formation of the entire sensory apparatus for detecting angular head movements.Disruption of the sense of balance is highly debilitating, causing vertigo and nausea. Maintenance of proper balance requires sensory inputs from many body parts, including the inner ears and the eyes. Within the inner ear, the vestibular apparatus plays a key role in the sense of balance and is responsible for detecting head orientation and movements. The portion of the vestibular apparatus that detects angular head movements consists of three fluid-filled, semicircular canals oriented at right angles to each other. At one end of each canal is an enlargement that houses the sensory tissue, crista ampullaris, consisting of sensory hair cells and supporting cells. Bone morphogenetic protein 4 (Bmp4), a secreted signaling molecule, is expressed in these sensory regions during development. However, the lack of  The ability to detect angular head movements in vertebrates lies within the vestibular apparatus of the inner ear All the sensory patches within the vertebrate inner ear including the presumptive cristae are thought to arise from a common prosensory  region at the otic placode and otocyst stages . This prDlx and Hmx, have also been implicated Wnt, Dlx, or Hmx gene functions all result in an early disorganization or absence of Bmp4 expression within the presumptive cristae Multiple factors are thought to regulate the formation of the vestibular apparatus Bmp4 in the presumptive cristae is conserved among several vertebrate species including the zebrafish, frog, chicken, and mouse . All animal procedures were approved and conducted according to the NIH Animal Use and Care Committee guidelines.The sequence . Bmp4Tm1at birth . TherefoNoggin cDNA Noggin is driven by the immediate early Cytomegalovirus promoter (Clontech). Chicken Smad6 cDNA in the pCab-IRES-GFP vector Smad6 is driven under the chicken \u03b2-actin promoter and the immediate early enhancer of Cytomegalovirus pSmad6, pNoggin and their respective control vectors at a concentration of 4 to 6 mg/ml were injected into the lumen of chicken otocysts at E3.5. Plasmids were electroporated into the anterior region of the otocyst using a positive and negative electrode flanking the anterior and posterior poles of the otocyst, respectively. Two 50 milli-second pulses at 10 volts were applied using a CUY21 electroporator.Chicken embryos were staged according to Hamburger and Hamilton Paint-fill analyses and in situ hybridizations were performed as described http://mathforum.org/library/drmath/view/57605.html). A total of 70 to 190 cells were counted per treatment.Anti-hair cell specific antigen (HCA) antibodies (gift of Guy Richardson) were used at 1:5000 dilution, and staining was performed as previously described Bmp4 null embryos, we used three different mouse lines. The first, cre/+Foxg1, was made by inserting cre into the endogenous Foxg1 gene which is expressed in tissues such as the embryonic otocysts, eyes, and foregut cre allele has been demonstrated by crossing cre/+Foxg1 mice with the Rosa26R reporter line Tm1Bmp4 is a null allele of Bmp4loxPBmp4 conditional allele was generated as described  embryos that are severely delayed in development, (2) embryos with eye malformations that are either normal or slightly smaller in their body size, and (3) embryos that are morphologically indistinguishable from loxP/+Bmp4 littermates  using an RNA probe (B4-del) generated against exons 3 and 4 of Bmp4. Half of the embryos analyzed at 9.5 dpc displayed abnormal Bmp4 expression patterns (n\u200a=\u200a4/8). By 10.5 dpc, a higher percentage of cre/+Foxg1; loxP/Tm1Bmp4 embryos show no or reduced Bmp4 expression in tissues such as the eyes and otocysts where Foxg1 is normally transcribed .We evaluated the tissue specificity of Bmp4 is transcribed in an anterior streak of tissue and a posterior focus . By this age, Bmp4 is also expressed in the non-sensory region of the growing cochlear duct Bmp4 expression in the cochlear duct appears normal in all of the cre/+Foxg1; loxP/Tm1Bmp4 specimens examined (data not shown).In a normal otocyst, or focus . The antor focus , arrow aanterior , arrow. cre/+Foxg1; loxP/Tm1Bmp4 inner ears at 13.5 dpc was examined by paint filling the membranous labyrinth. Consistent with the variable Bmp4 expression patterns, the paint-filled cre/+Foxg1; loxP/Tm1Bmp4 specimens also show a range of inner ear phenotypes (loxP/+Bmp4 (n\u200a=\u200a3/14) embryos, or display only a lateral canal truncation . Therefore, this milder phenotype observed in cre/+Foxg1; loxP/Tm1Bmp4 embryos is probably due to insufficiency of Bmp4 caused by the presence of both of the Tm1 and the un-recombined floxed Bmp4 allele rather than an incomplete penetrance of the cre activity. Cochlear ducts of cre/+Foxg1; loxP/Tm1Bmp4 embryos show some variability in length . Taken together, inner ear-specific deletion of Bmp4 using two independent cre lines indicates that Bmp4 is required for the formation of the three cristae and semicircular canals, and possibly the utricle and saccule.We also conditionally deleted uncation ; n\u200a=\u200a5. uncation ; n\u200a=\u200a5. Tm1/loxPBmp4 embryos generated by breeding TgPax2cre; +/Tm1Bmp4 with loxP/loxPBmp4 mice also display lateral canal truncation as well (n\u200a=\u200a4/7), suggesting that a combination of Tm1 and loxP alleles can generate hypomorphs depending on the genetic background. Notably, our Tm1/+Bmp4 mice in Black Swiss background do not circle but a small percentage of Bmp4 heterozygous mice in C57BL/6 background do Some of the cre/+Foxg1; loxP/Tm1Bmp4 are already apparent at 11.5 dpc , other crista markers are also present.Analysis of paint-filled ears of younger embryos indicates that the vestibular defects in 11.5 dpc . To bettBmp4 expression in the inner ear also resulted in the absence of semicircular canal formation. The overall size of the vertical canal pouch is usually smaller than normal, particularly in the posterior region  encoding Smad6 or Noggin translationally coupled to GFP were electroporated into the developing anterior crista region in ovo at E3.5, a time when these crista-associated genes are co-expressed in the presumptive crista. Smad6 is an intracellular inhibitor that competes with Smad4 for binding to phosphorylated Smad1/Smad5/Smad8 proteins, thus preventing their subsequent translocation to the nucleus and activation of Bmp target genes Smad6 expression has been used successfully to address the roles of Bmps in neural induction and placode formation Bmp4-positive anterior crista region , Fgf10 (n\u200a=\u200a0/6), Lmo4 (n\u200a=\u200a0/9) and Ser1 (n\u200a=\u200a0/5) are not affected , but quite consistently seen in response to pNoggin . Since Noggin is a secreted molecule, down-regulation of Gata3 and Msx1 in the mesenchyme near the site of electroporation is also observed  or pNoggin (non-cell autonomous) treatments Bmp4 will require further investigation  syndrome DrosophilaGata3 may have a more global effect on cell fate specification in vertebrate crista beyond formation of the cruciatum (see below). Furthermore, the conserved Gata3 expression in the mesenchyme surrounding the presumptive cristae between chicken and mouse Gata3 expression in the mesenchyme by pNoggin, but not by pSmad6, may contribute to the more severe phenotype caused by pNoggin.Drosophila embryogenesis, pannier (homolog of Gata1) is induced by dpp  in the dorsal embryo dpp becomes dependent on pannierGata3 expression appears to begin before that of Bmp4. However, the relatively normal Bmp4 expression in Gata3 null inner ears does not support Gata3 functioning upstream of Bmp4Gata3 in the cristae is dependent on Bmp4.In other systems, Bmp and Gata pathways are thought to interact. For example, during Gata3, considered to be a non-sensory gene, is expressed in the prospective lateral crista of the mouse . Therefore, both Fgfs and Bmp4 could be involved in mediating canal formation.We have proposed that the sensory cristae may induce the formation of their associated semicircular canals Bmp2 in canal formation, Dlx5 is also a key player in canal formation, and its activity is directly or indirectly regulated by Bmp4 as well. Even though the canal phenotypes in Dlx5-/- mutants are milder than those in Bmp4 conditional mutants, the phenotypes in Dlx5-/-; Dlx6-/- double mutants appear to be more severe Bmp4, and in turn, their activities are maintained by Bmp4. It is also interesting that the expression of Dlx5 in the canal pouch is more susceptible than Hmx3 to the lack of Bmp4. Even though both Dlx and Hmx pathways are required for canal formation, regulation of these two pathways appears distinct. This notion is also supported by studies of Gbx2-/- and Wnt1-/-; Wnt3a-/- mutant embryos, in which Dlx5 expression is down-regulated but Hmx3 expression is relatively normal In addition to the postulated role of Dlx, Hmx, Tbx1, Eya1 and Six1 regulate its activity, directly or indirectly Click here for additional data file.Table S1Summary of phenotypes.(0.04 MB DOC)Click here for additional data file."}
+{"text": "The complex is selectively antimitochondrial and inhibits the growth of a number ofyeast strains in non-fermentable media at concentrations as low as 2.5 \u03bc\u039c and induces themitochondrial mutation petite The effect is largely reversed by the presence of aspirin. The complexis shown to be stable in the cell culture media and in the presence of glutathione, but readily reactswith disulfides of oxidized glutathione and serum albumin. Surprisingly, neither [Au(eppe)2]Cl nor[Au(eppe)2]Cl  (dppe = Ph2PCH2CH2PPh2) showed any mitochondrial selectivity in the samescreening protocol.The silver(I) complex [Ag(eppe)"}
+{"text": "Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5\u2032-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell\u2013cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development. Lineage segregation during development and stem cell differentiation depends on transcriptional regulation and is accompanied by cell morphological changes. Much is known about how transcriptional programs regulate cell differentiation and how general cell morphological regulators control cell motility, polarity, and adhesion during development. By contrast, little is known about how lineage specific transcriptional regulation is coupled to correspondingly unique cell morphological changes during the building of tissues and organs. Understanding this coupling at the cellular level is important because it would help to define the steps that stem and progenitor cells take to differentiate.Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation mammalian blastocysts , which cESCs can be induced to differentiate into different cell lineages in vitro and they have been used extensively to study transcriptional changes during differentiation . Here, wAll ESCs were cultured in the absence of feeder cells  on gelatin-coated dishes. To image ESC differentiation in a LiveCell\u2122 Chamber , time-lapse recording at multiple positions per well were acquired on an inverted microscope  controlled by IPLab4.0 every 5 minutes for 2 days. Closed-loop feedback from a Z Encoder Probe  was used to control focus drifts. For additional detailed materials and methods, please refer to the Supporting Information.Oct3/4, Sox2, or Nanog , which has also been referred to as Cdc42EP1 (Cdc42 effector protein 1), encodes a protein that binds to RhoGTPases. Since RhoGTPases bind to various effector proteins to regulate cell morphogenesis, Borg5 is likely a morphological regulator that could influence TE specific cell morphogenesis and transcription during differentiation.To identify genes that are upregulated early during TE differentiation, we carried out microarray analyses 12 hours after Tc addition and found that 13 genes were upregulated by at least twofold , 17. AmoBorg5 and found that Borg5 mRNA is strongly upregulated after reduction of Oct3/4 in both ZHBTc4 . Thus, Oct3/4 is likely to indirectly suppress Borg5 expression in ESCs.Using quantitative real-time polymerase chain reaction (qRT-PCR), we characterized the expression of h ZHBTc4  and E14 h ZHBTc4 , but noth ZHBTc4 . Therefo in ESCs , 19 and Borg5 protein is expressed at low levels in ESCs, but becomes upregulated shortly after Oct3/4 reduction in both ZHBTc4  and E14 Borg5 cDNA lacking the 5\u2032-untranslated region  or Borg5 cDNA carrying silent mutations (Borg5-ins) that render it insensitive to RNAi (We reasoned that as a RhoGTPase binding protein  Borg5 mi to RNAi  rescued Since Borg5 contains the Cdc42/Rac interactive binding (CRIB) motif, we next asked whether it functions downstream of Cdc42 to regulate cell motility during TE differentiation. We first confirmed that Borg5 interacts with the guanosine triphosphate-bound form of Cdc42 (Cdc42Q61L)  20\u201322]..20\u201322]. Borg5 is upregulated during TE differentiation as early as Cdx2, we asked whether it could influence Cdx2 expression. We created ZHBTc4 ESC lines that stably express either S4 short-hairpin RNA (shRNA) or control shRNA (Supporting Information). Both cell lines expressed Oct3/4 and were responsive to Tc, but ZHBTc4-S4 cells failed to upregulate Borg5 as expected  plays a role in TE differentiation upstream of Cdx2 , 23. UsiTE cells are polarized epithelia that occupy the outside position in the blastocyst to enclose the ICM. We designed a cell-sorting assay to test whether Borg5 could be involved in this process. E14 ESCs were mixed with either ZHBTc4 ESCs expressing control (ZHBTc4-Con) or S4 shRNA (ZHBTc4-S4) and Tc was added to initiate TE differentiation from the ZHBTc4 ESCs (Supporting Information). ZHBTc4-Con and ZHBTc4-S4 cells also express GFP, which serves as a marker for identifying these cells. After 42\u201348 hours of Tc addition, cells were fixed and stained with antibodies to GFP and Oct3/4.In the absence of Tc, ZHBTc4-Con or ZHBTc4-S4 ESCs  intermingled with the E14 ESCs in colonies . HoweverThe above study led us to test whether Borg5 regulates blastocyst formation during preimplantation development. Immunofluorescence staining using affinity purified Borg5 antibodies generated in chicken revealed that Borg5 is localized along the cell\u2013cell borders and in the cytoplasm in the post compaction embryos . PreabsoBorg5 and Cdx2 mRNAs were upregulated during eight-cell to morula stage  differentiate toward different lineages, it is possible to uncover cell morphology regulators that influence transcriptional regulation and vice versa. By analyzing early ESCs differentiation live, we have shown that unique cell morphogenesis indeed accompanies specific lineage differentiation from ESCs. By focusing on the differentiation of the trophectoderm lineage from ESCs, we have identified Borg5 as a morphological regulator that undergoes a similar strong up-regulation to Cdx2, a key transcription factor for trophectoderm differentiation. Our studies strongly suggest that Borg5 and Cdx2 reciprocally regulate each other to couple epithelial morphogenesis to transcriptional changes during development. Further study how Borg5 and Cdx2 regulate each other should help to decipher the steps of cell differentiation and tissue morphogenesis."}
+{"text": "Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters. Many yeast plasma membrane transporters are removed from the cell surface in response to excess substrate, nutrients or stress. It is well established that their endocytosis and subsequent vacuolar degradation are controlled by ubiquitination, and in every case tested, ubiquitination requires the HECT domain E3 ligase Rsp5 How a single ubiquitin ligase recognizes a wide variety of substrates is now beginning to be understood. Rsp5 contains three WW domains, which recognize PY motifs with the typical sequence PPXY or LPXY. Several adaptor proteins containing such motifs have been shown to facilitate the ubiquitination of particular proteins or sets of proteins  and to the four transporters mentioned above. Several other permeases appeared unaffected in arrestin mutant strains, possibly because of functional redundancy of the arrestins. We therefore sought to extend the findings to other proteins, to test the generality of the model and see whether a single mechanism can account for the behaviour of all regulated transporters.Here we identify adaptors required for regulated degradation of the inositol permease Itr1, hexose transporter Hxt6, uracil permease Fur4, and tryptophan transporter Tat2. We show that Bul1 and Bul2 have functions related to those of the arrestins and may share structural features with them. We also show that the Rsp5 adaptor Bsd2 and the RING domain ligase Pib1 provide an additional redundant function required for several transporters to negotiate the multivesicular body pathway. The results reveal common themes but also considerable variation in the requirements for individual proteins to be endocytosed to the vacuole.Our previous studies suggested considerable functional redundancy amongst the yeast arrestins. Our strategy therefore was to eliminate all of them, test the effect on a given transporter, then add back plasmids encoding single arrestins to see which were best at restoring endocytosis. This strategy required all of the relevant arrestins to be identified.Yeast contains eight proteins that have clear arrestin sequence signatures and show readily detectable homology to each other, namely Art2/Ecm21, Art8/Csr2, Art4/Rod1, Art7/Rog3, Art6/Aly1, Art3/Aly2, Art5/Ygr068c and Art9/Rim8 We identified an additional protein, Ylr392c, which also shows a match to the pfam arrestin_N domain  yet is d9-arrestin mutant, lacking all the PY-containing arrestins, which was healthier. To allow studies of the endocytosis of the metal transporter Smf1 this strain also lacked Bsd2, an adaptor that in normal medium causes newly synthesized Smf1 to be diverted from the Golgi to the vacuole. We refer to this strain as the 9-arrestin mutant.We constructed a yeast strain in which all 10 arrestins were deleted. Although viable, this strain grew more slowly than wild-type. This was at least partly because of the loss of Rim8, which is required for robust growth under a variety of conditions. Because Rim8 seems to have a distinct function, for subsequent endocytic studies we primarily used a The inositol transporter Itr1 is endocytosed in response to exogenous inositol rsp5 mutant, and also in the 9-arrestin mutant strain  GFP in response to either uracil or cycloheximide was reduced in the BUL1 was deleted in addition to the arrestins  machinery bsd2 cells (our unpublished observations), suggesting that these lysines may be sites of Pib1 and/or Ear1/Ssh4 dependent ubiquitination.It seems most likely that the additional ubiquitination occurs mainly in endosomes, as Bsd2 and Ear1/Ssh4 are membrane proteins that are themselves substrates for Rsp5 and are targeted to the vacuole apparently without reaching the plasma membrane One obvious question is how these disparate proteins recognize their substrates. Ear1 and Ssh4 contain SPRY domains, which may mediate protein\u2013protein interactions, but they appear to act on every protein tested, from the vacuolar enzyme Phm5, with a single transmembrane domain and a tiny cytoplasmic domain, to large multispanning transporters such as Fur4 and Smf1 erg6 mutant, defective in ergosterol synthesis, the stable compartments do not form These considerations suggest a view of transporter regulation in which specificity is provided by arrestin interactions which promote initial internalization, and this is followed by relatively non-specific further ubiquitination in endosomes. This places great importance on the initial endocytic step, and it is worth considering what else might influence this. Notably, it has been shown that Can1, Tat2 and Fur4 are localized within small highly stable ergosterol-rich compartments in the plasma membrane whose formation depends on specific proteins, and that this slows their endocytosis by sequestering them from the sites at which this happens It seems that there are considerable differences in the way that the presence of individual transporters at the plasma membrane is controlled, though arrestins provide a common theme and may in some cases mediate specific regulatory mechanisms. Further exploration will no doubt reveal additional features, and may assign roles to the four arrestins whose functions are currently unknown. The arrestin-deficient strains we have developed should provide useful tools for future study.MATa his3-\u03941 leu2-\u03940 met15-\u03940 ura3-\u03940) or BY4742 (MAT\u03b1his3-\u03941 leu2-\u03940 lys2-\u03940 ura3-\u03940) and are listed in Schizosaccharomyces pombe HIS5 gene flanked by loxP sites, then using transient expression of the cre protein from a plasmid to remove the HIS5 marker.All yeast strains were derivatives of BY4741 . Bsd2 was expressed from either its own promoter (with Fur4 and Hxt6) or the TPI promoter (with Tat2 and Itr1). Strains were grown in appropriate selective media to maintain the plasmids. Growth rates in Plasmids were based on the YCplac111 600 less than 0.3). For Smf1, synthetic medium with glucose and appropriate supplements was used, and cadmium treatment was performed as described previously m myo-inositol. For Hxt6, cells were grown in YEP medium (1% yeast extract and 2% peptone) with 2% raffinose to an OD600 of approximately 1, then endocytosis induced either by adding 100 \u00b5g/mL cycloheximide, or by transferring to medium containing 0.17% yeast nitrogen base without ammonium sulphate or amino acids but with 5% glucose m uracil. For Tat2, the same protocol was used as for Fur4, except that cells were grown on synthetic medium lacking tryptophan and leucine, and endocytosis was induced by adding 1.5 mg/mL tryptophan.For fluorescence experiments cells were usually grown to exponential phase (ODImmunoblotting was performed on total cell extracts prepared using the alkaline lysis method Fluorescence imaging was performed on live cells in growth medium using a Zeiss LSM510 confocal microscope as previously described"}
+{"text": "HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.Human epidermal growth factor receptor 2  and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method  and the ASCO/CAP scoring method with the FISH equivocal cases  and without the FISH equivocal cases .Sequential hybridization and signal detection for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.Automated BDISH applications for  HER2) oncogene, located on the long arm of chromosome 17 (17q12-q21), is over-expressed or amplified in approximately 20% of breast carcinoma cases [HER2 status in breast cancer is used as a prognostic factor, a predictive factor, and a therapy selection factor [\u00ae; Genentech), which is an FDA approved drug for use as monotherapy or combined chemotherapy for treatment of breast cancer patients with amplified HER2 status. Trastuzumab adjuvant treatment for early HER2 positive breast cancer is effective for improving patient survival and cost-effectiveness analyses of such treatment have shown acceptable ratios [HER2 gene over-expression associated with anthracycline treatment [The human epidermal growth factor receptor 2  analyses for detecting HER2 gene amplification and semi-quantitative HER2 immunohistochemistry (IHC) analyses for detecting over-expressed HER2 protein are performed to determine the HER2 status of breast cancer patients. The optimal scoring method for determination of HER2 gene status is the use of chromosome 17 centromere (CEN 17) enumeration for calculating the HER2/CEN 17 ratio [HER2 gene amplification have better prognosis compared to patients with HER2 gene amplification [HER2 and CEN 17 targets is recommended especially for borderline IHC cases [HER2 FISH assays beyond the requirement for a specialized fluorescence microscope and the difficulty of preserving FISH signal during a long term storage. For example, HER2 FISH testing has exhibited a higher assay failure rate in the hands of some investigators when compared to HER2 IHC testing (5% vs. 0.08%), the FISH assay procedure time is longer than the IHC assay (36 hours vs. 4 hours), and the FISH interpretation time is longer than IHC interpretation time (7 minutes vs. 45 seconds) [HER2 proficiency testing study showed that there was 20% (4 out of 20 samples) discordance with HER2 FISH testing among 5 experienced laboratories [Quantitative 17 ratio . One stu17 ratio . Also, cfication . Dual coHC cases . Howeverseconds) . Furtherseconds) . An interatories . On the ratories .HER2 gene status. The chromogenic in situ hybridization (CISH) assay using a DAB chromogen and H2O2 substrate system for horseradish peroxidase (HRP)-based signal detection has been evaluated and the value of this assay for assessing HER2 status has been demonstrated [HER2 gene signal. However, with the current CISH method, the assessment of the HER2/CEN 17 ratio is conducted by enumerating HER2 and CEN 17 separately using two different tissue sections.There are alternatives to FISH for determining nstrated ,17-22. Cin situ hybridization (ISH) methods use autometallography and enzyme metallography: 1) Nanogold\u00ae with gold enhancement in situ hybridization (GOLDFISH) [in situ hybridization (SISH) [HER2 gene signal. On the other hand, the SISH method produces discrete metallic silver black signals. Horseradish peroxidase (HRP) of the detection system reacts with silver acetate, hydroquinone, and H2O2 and deposits metallic silver particles at the reaction site. The reaction product can be seen as discrete black dots under a brightfield microscope. Advantages of SISH include the high sensitivity for detection of single gene copies, the high resolution for quantifying DNA targets, and the high contrast with tissue counterstaining for visual separation of the signal and tissue morphology [Other brightfield microscopy OLDFISH) ,24 and 2n (SISH) -27. The HER2 SISH assay was evaluated for assessing the inter-observer interpretative reproducibility of the HER2 gene status of 99 clinical cases when compared against the reference standard FISH results [HER2 FISH and single color HER2 SISH was 96.0%  and the discrepancies were mainly observed among tumors with the heterogeneity of tumor cell populations [HER2 and CEN 17 on the same tissue section like two-color FISH assays. Dual ISH staining for HER2 gene and CEN 17 would be beneficial for analyzing chromosome 17 aneusomy and for delineation of cases displaying genotypic intratumoral heterogeneity.Recently, an automated  results . Overallulations . AdvantaHER2 status reproducibly is the use of automation for conducting the test in the same manner among different laboratories located in different parts of the world. Thus, as a step toward standardizing HER2 testing, our objective was to develop an automated brightfield double in situ hybridization (BDISH) assay for simultaneous detection of HER2 and CEN 17 DNA targets on formalin-fixed, paraffin-embedded breast cancer tissue samples. Using this method, HER2 status testing can be conducted in a simplified manner for more accurately identifying the patients who are eligible for trastuzumab therapy and potentially leading to the improvement of breast cancer patient care in the future.One prerequisite for testing HER2 status and BT-474 is a breast ductal carcinoma with amplified HER2 status (50\u201360 copies of HER2) and chromosome 17 polysomy [\u00ae Plus glass slides .MCF7 and BT-474 xenograft tumors were utilized for optimizing the BDISH assay. MCF7 is a breast adenocarcinoma cell line with non-amplified polysomy . ParaffiHER2 status by FISH using the PathVysion\u00ae HER-2 DNA Probe Kit  at the Cleveland Clinic Foundation. However, it should be noted that non-consecutive tissue sections were used for FISH and BDISH analyses.Ninety-four (94) breast cancer cases were used from the Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA under IRB approved protocol. Tissue samples were routinely processed for paraffin-embedding after fixing with an alcoholic formalin fixative. All breast cancer cases had been previously tested for \u00ae XT automated slide processing system  was used for the optimization and performance evaluation of the BDISH assay for HER2 and CEN 17 DNA targets. A protocol was established so that the entire assay procedure consisting of baking, deparaffinization, pretreatment, hybridization, stringency wash, signal detection, and counterstaining was completed as a one-step fully automated assay. Paraffin tissue sections on glass slides were baked at 65\u00b0C for 20 minutes prior to the deparaffinization step with EZ Prep\u2122 (Ventana) at 75\u00b0C for 16 minutes. Deparaffinized tissue sections were pretreated with a combination of heat treatment with Reaction Buffer  and ISH Protease 2 or ISH Protease 3 (Ventana) to unmask DNA targets. Pretreatment conditions were chosen for optimal signal to noise ratio and tissue morphology preservation for the xenograft control slides as well as clinical case tissue slides.The BenchMarkHER2 and CEN 17 signal detection were conducted for a complete BDISH assay , a dinitrophenyl (DNP)-labeled, nick-translated repeat deleted DNA probe was applied to the glass slide for co-denaturing the probe and target at 95\u00b0C. Then, the hybridization step was conducted at 52\u00b0C for 2 hours. After 3 stringency wash steps were performed at 72\u00b0C with 2\u00d7 SCC (Ventana), tissue sections were incubated with monoclonal rabbit anti-DNP antibody (Ventana) for 20 minutes and then with HRP-conjugated anti-rabbit antibody for 16 minutes at 37\u00b0C. The metallic silver deposit for HER2 ISH signal was developed using silver acetate, hydroquinone, and H2O2 reaction in the presence of HRP using the ultraView\u2122 SISH Detection Kit (Ventana). For CEN 17 detection, the INFORM Chromosome 17 Probe (Ventana), a DNP-labeled oligoprobe, was applied to the tissue sections, denatured at 95\u00b0C and hybridized at 44\u00b0C for 2 hours. Then, after 3 stringency wash steps at 59\u00b0C with 2\u00d7 SSC, tissues were incubated with rabbit monoclonal anti-DNP antibody for 20 minutes and then with an alkaline phosphatase (AP)-conjugated anti-rabbit antibody for 12 minutes at 37\u00b0C. Finally, the signal for CEN 17 was visualized with a fast red and naphthol phosphate reaction using ultraView Red ISH Detection Kit. Diaminobenzidine (DAB) chromogen and H2O2 substrate reagents from the ultraView Universal DAB Detection Kit (Ventana), 5-bromo-4 chloro-3-indolyl phosphate (BCIP) substrate and nitro blue tetrazolium (NBT) oxidant reagents from the ISH iVIEW\u2122 Blue Detection Kit (Ventana), and a ready-to-use tetramethyl benzidine (TMB) solution  were also evaluated for CEN 17 signal detection of the BDISH application. Because both HER2 and CEN 17 probes were labeled with the same DNP hapten, CEN 17 signal detection was completed without DNP-labeled CEN 17 probe after HER2 signal detection to ensure that the anti-DNP antibody of CEN 17 detection didn't recognize the DNP hapten of HER2 probe.Sequential ISH procedures for HER2 and/or CEN 17 targets were counterstained with Hematoxylin II (Ventana) for 4 or 8 minutes and Bluing Reagent (Ventana) for 4 minutes. Counterstained slides were first rinsed with distilled water containing DAWN\u00ae  for removing LCS from slides and then rinsed with distilled water until soap was removed completely from the slide. Slides were blotted very gently with paper towels and completely dried at 45\u00b0C or 65\u00b0C in the oven for at least 15 minutes. One drop of Cytoseal\u2122 60 (Richard-Allen Scientific) was applied onto a dried slide and a glass coverslip was carefully placed onto the slide. Excess mounting media was removed from the slides by gently pressing the slides against paper towels. Different coverslipping methods were also evaluated for preserving the fast red staining during the assay development. BDISH results were observed with a Nikon ECLIPSE 90i microscope  equipped with Nikon digital camera DXM1200F (Nikon) without oil immersion objective lenses, up to 60\u00d7. However, for presentation purposes, mainly comparing to FISH images taken with a 100\u00d7 objective lens, brightfield photographs contained in this report were obtained using a 100\u00d7 oil immersion objective lens.Single or double stained tissue sections for HER2 and CEN 17 targets of xenograft tumor controls as previously described [PathVysion HER-2 DNA Probe Kit was used for the FISH test for escribed . PhotogrHER2 amplification with the PathVysion assay (Negative: HER2/CEN 17 < 2.0 and Positive: HER2/CEN 17 \u2265 2.0) and using the ASCO/CAP guideline criteria  with or without the equivocal cases. Scoring BDISH slides was conducted by 4 observers , who were experienced with scoring HER2 FISH slides. Scoring occurred at different sites and at different occasions using different microscopes. Each individual observer evaluated the set of slides at their own pace and judgement. No scores were provided by the observers when the staining quality was deemed not adequate. There was no communication among observers regarding their scoring experience of the BDISH slides. Concordance data of FISH scores vs. consensus BDISH scores among 4 observers and FISH scores vs. individual BDISH scores by 4 observers were determined using SAS 9.1  in calculating frequency tables and Kappa statistics. The consensus among observers was defined as the agreement of three or more observers on a given observation. Scoring of BDISH assays was also analyzed for the sensitivity and specificity against FISH scores with the historical scoring method and the ASCO/CAP scoring method without the equivocal cases. Discordant cases were investigated by a non-observer (HN) for possible causes using BDISH slides.Performance of the BDISH assay was compared to FISH results as the reference standard using the historical criteria for HER2 single ISH, CEN 17 single ISH, and HER2 and CEN 17 BDISH results with formalin-fixed, paraffin-embedded xenograft tumor sections are presented in Figure HER2 signal were recognized as black discrete dots in the nuclei with MCF7 xenograft tumor  even though the same hapten was used for the sequential hybridization method. For image comparison of BDISH and FISH for HER2 gene and CEN 17, FISH images with MCF7 tumor and BT-474 tumor are presented in Figure HER2 genes are seen as red-orange dots and CEN 17 targets are seen as green dots.Images of r Figure  while amh Figure . Single r Figure  and BT-4r Figure . After ss Figure  and ampls Figure . Because\u00ae film coverslipper method  after air-drying slides (data not shown). The most common method of coverslipping tissue sections stained with fast red is the use of an aqueous mounting medium. However, this method did not produce crisp fast red staining for quantitative analyses of BDISH signals (data not shown). For the second color for the BDISH application, DAB, BCIP/NBT, and TMB detection systems that produce brown, blue, and green to blue final product, respectively, were evaluated. However, they did not provide sufficient contrast against the HER2 ISH black signal (data not shown).One successful way to preserve the fast red staining was the use of a toluene-based Cytoseal 60 mounting medium placed onto completely dried tissue sections prior to coverslipping with glass coverslips. We also confirmed that the red signal was successfully preserved with the Tissue-TekHER2 gene and CEN 17 with formalin-fixed, paraffin-embedded xenograft tumor sections, we applied the assay to 94 breast carcinoma cases and scoring BDISH slides was conducted by 4 observers . The consensus among observers was defined as the agreement of three or more observers on a given observation. With the historical scoring method (Negative: HER2/CEN 17 < 2 and Positive: HER2/CEN 17 \u2265 2.0)  (Table After optimizing the BDISH assay for 2) Table , the conHER2/CEN 17 ratios could be readily conducted. Non-amplified HER2 gene cases showed 0\u20134 copies of HER2 genes and 0\u20134 copies of CEN 17 depending on cell cycle stage and how each cell was cut within a tissue section  variegated different genotype tumor cell populations in the same area of tissue section  discordant cases of the BDISH slides presented at least some degree of the genotypic heterogeneity of tumor cell populations. In general, there were two types of the tumor cell heterogeneity with n Figure  and 2) sn Figure . Breast HER2 status testing is important for identifying breast cancer patients who may benefit from receiving trastuzumab therapy. Currently, in the United States, HER2 IHC methods are most commonly used for primary screening for HER2 status, and borderline cases are subjected to dual FISH for HER2 and CEN 17 to determine the HER2/CEN 17 ratio. Because the discordance rate between local and central/reference HER2 status testing with IHC and FISH is significantly high [HER2 status in breast cancer [HER2/neu testing guideline [HER2 status testing include: 1) automating the entire process for slide staining [HER2 testing process within experienced laboratories and pathologists that perform large numbers of HER2 tests [Accurate tly high ,31-33, ttly high . The Ameuideline . Two potstaining  and slidstaining -39 and 2R2 tests .HER2 status testing is to automate the assay procedure for HER2 IHC and HER2 FISH assays so that human errors can be diminished. HER2 IHC assays can be performed using an automated slide staining system, but HER2 FISH assays remain technically challenging and time consuming manual molecular diagnostic assays in most laboratories. An evaluator of FISH slides must have access to specialized fluorescence microscopy in a dark room. Because of unstable FISH staining characteristics, the signals of FISH slides can be bleached easily, even while reviewing and enumerating signals. Furthermore, digital images of the FISH slide need to be captured with a sensitive camera system for each patient case for the HER2 gene status record. Therefore, it is desirable to automate a tissue-based HER2 gene status test that can be observed with a regular brightfield microscope and that produces stained slides that can be archived.One way to improve the accuracy of HER2 and CEN 17 targets within the same nuclei of tissue sections with a manual dual brightfield ISH application [HER2 gene staining. However, based on published images [HER2 gene and CEN 17 presented in the current study are: 1) the automation of the ISH application; 2) the visualization of both HER2 gene and CEN 17 targets in the nuclei of the same cell; 3) the generation of discrete HER2 gene signals; 4) the ability to reproducibly detect endogenous HER2 and CEN 17 signals in the stromal tissues and lymphocytes as a reliable internal assay control; 5) the ability to visualise signal with brightfield microscopy with non-oil immersion lenses; and 6) the capability to permanently archive the slides.While the concept of multi-color brightfield ISH applications was published in 1990's ,41, it wlication . This dud images ,42, TMB HER2 and CEN 17 probes are co-hybridized for dual color HER2 FISH. However, for the BDISH assay, because the stringency conditions for the nick-translated HER2 probe and the CEN 17 oligoprobe were different, it was necessary to conduct sequential ISH staining steps for HER2 gene and CEN 17 targets. For CEN 17 ISH, we have optimized a new detection system with an alkaline phosphatase-conjugated antibody and fast red chromogen and naphthol phosphate substrate reaction. The fast red-based detection was selected to obtain a good contrast of CEN 17 ISH signal against the discrete black dots of HER2 SISH signal. DAB, BCIP/NBT, and TMB detection systems did not provide sufficient contrast against HER2 ISH black signal (data not shown). Because fast red precipitate is soluble in organic solvents, in general, aqueous mounting medium is used for coverslipping. However, the standard coverslipping method with aqueous mounting medium on wet tissue sections did not produce tissue sections with high resolution and therefore detailed tissue structure could not be observed (data not shown). A successful method to preserve fast red staining for CEN 17 and high resolution tissue morphology was, after completely dry the slides, to apply a toluene-based tissue mounting medium  for coverslipping with cover glass or to use a film coverslipper (Tissue-Tek\u00ae film coverslipper). Incomplete drying resulted in faint red background staining particularly around the fast red precipitate sites with this method. Interestingly, the use of aqueous mounting medium onto the dried tissue slides produced yellowish background staining on tissue sections and this method did not produce satisfactory results (data not shown).HER2 gene or CEN 17 and BDISH for both targets was evaluated with xenograft tumors. HER2 and CEN 17 copy numbers have been documented previously using the FISH assay [HER2 and chromosome 17 polysomy (3 copies of chromosome 17 per nucleus) while one of chromosome 17 with HER2 deletion (2 HER2 copies per nucleus) [HER2 amplification with 50\u201360 copies of HER2 genes and 4\u20136 copies of CEN 17 per nucleus [HER2 genes are located not only on chromosome 17, but also are translocated on other chromosomes [HER2 and CEN 17 copy numbers produced with the single target ISH and BDISH methods matched with previously reported results. As both probes are labeled with the same DNP hapten, our first concern was to determine if detecting specific signal for each probe was feasible. We confirmed that the fast red chromogen detection reagents did not produce red signal when the fast red ISH was performed without the CEN 17 probe after detection of HER2 by SISH (data not shown). Thus, the SISH detection and the fast red detection can be combined to perform a sequential double ISH assay with 2 probes labeled with the same hapten. Because the sequential BDISH application uses 2 specific stringency conditions based on the length and sequences of 2 probes, it is not necessary to design 2 probes that require the same stringency for co-hybridization, like double color FISH assays.The specificity of single ISH for SH assay . MCF-7 cnucleus) . BT-474  nucleus . Amplifiomosomes . HER2 anHER2 FISH scores and HER2 and CEN 17 BDISH scores by 4 observers were calculated for assessing the performance of the BDISH assay. There were 9 discordant cases  based on BDISH score disagreement with FISH scores, even by one observer. We have found that the number of equivocal cases influences the concordance rate with the ASCO/CAP scoring method. There were 4 equivocal cases based on FISH scores and all cases showed the BDISH score disagreement by at least 2 observers. A similar observation was reported with an international HER2 testing proficiency study [HER2/CEN 17 ratios between 1.7 and 2.3 that are close to the 'equivocal' defined by ASCO/CAP HER2 scoring method (1.8 \u2013 2.2). They also stated \"equivocal cases are difficult to interpret, even highly experienced and validated laboratories\" [HER2 status assessment of breast carcinoma cases, heterogeneity of HER2 gene status was observed in 40 of 742 cases (5%) [HER2 testing results [HER2 status. Further clinical evaluations of HER2 and CEN 17 BDISH application with patient treatment outcome data are required for more accurate HER2 status assessment of breast cancer patients to be obtained.Concordance rates between a set of gold standard dual color cy study . In theiatories\" . In one ses (5%) . It has  results . With cuHER2 gene and CEN 17 targets in formalin-fixed, paraffin-embedded tissue sections that is highly concordant to the FISH and is reproducibly interpreted among observers. Assessment of HER2 gene status can be conducted without the use of a specialized fluorescence microscope and the time required for completing HER2 gene status assessment can be shortened significantly. Furthermore, this application has the potential to be used for other gene targets, any combination of a gene and its chromosome centromere, and tissue section-based gene assessment tests including gene translocation studies. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphology observation.We have successfully developed an automated BDISH application for HN, BHW, ML, AEM, FG, MF, EW, TM are employed by Ventana Medical Systems, Inc. RRT and MD received grant support and honorarium for speaking from Ventana Medical Systems, Inc.LW, ML, JP, and RRT were responsible for identifying and prequalification of the clinical cases used in this study. HN and TMG were responsible for the BDISH assay development and feasibility studies, staining the clinical samples, and preparing the manuscript draft and image data. BHW and ML were responsible for the final assay development. AEM conducted all statistical analyses for the performance of BDISH assay. FG was the study coordinator and contributed intellectual content of the study. MF designed the probes. EW, MK, MD, RRT, and TMG critiqued the assay performance with their molecular histology expertise during the assay development. FPL, MK, MD, and RRT were the observers for scoring the clinical samples. All authors contributed intellectual inputs to the study. All authors read and approved the final manuscript."}
+{"text": "Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the  Arabidopsis thaliana, this transition is genetically controlled by several pathways, including the autonomous pathway, the photoperiod pathway and the vernalization pathway, which form a regulatory network The transition from a vegetative to a reproductive phase  is a major developmental switch in the plant life cycle that must be properly timed to ensure maximal reproductive success. In Arabidopsis is FLC, a MADS box transcription factor that quantitatively inhibits the floral transition FLC expression is delicately controlled by various activators and repressors. The autonomous pathway, which includes FVEFCAFLOWERING LOCUS D (FLD) FLC expression to promote flowering, whereas FRIGIDA (FRI) activates FLC expression to delay flowering FLC expression in response to a prolonged cold exposure  to accelerate flowering in ArabidopsisFLC, in the Arabidopsis genome there are five close FLC relatives including FLOWERING LOCUS M (FLM), MADS AFFECTING FLOWERING 2 (MAF2), MADS AFFECTING FLOWERING 3 (MAF3), MADS AFFECTING FLOWERING 4 (MAF4) and MADS AFFECTING FLOWERING 5 (MAF5); these FLC relatives also appear to repress the floral transition A key component in this regulatory network in FLC expression. Activation of FLC expression in the presence of FRI is associated with the H3K4 trimethylation and also requires deposition of the histone variant H2A.Z in FLC chromatin FLC expression partly through generating repressive histone modifications in FLC chromatin. FLD is involved in the H3K4 demethylation and deacetylation of FLC chromatin FCA functions closely with FLD and is involved in H3K4 demethylation in FLC chromatin FVE is partly involved in the histone deacetylation of FLC chromatin FLC chromatin by Type I and Type II arginine methyltransferases is also associated with FLC repression FLC repression FLC chromatin such as increased trimethylation of histone H3 at lysine 9 and H3K27, and H4R3 dimethylation Chromatin modification plays an important role in the regulation of FLC inhibits the floral transition partly by reducing expression of a key flowering-time integrator, FTFT was first identified as a component of the photoperiod pathway, which promotes flowering in response to increased day length FT expression is activated by CONSTANS (CO), another component in the photoperiod pathway FT is expressed in the vasculature FT locus and represses its expression, and thus antagonizes the activation by CO FT acts as a flowering-time integrator that integrates signals from the photoperiod pathway and the FLC-mediated flowering pathways to promote the Arabidopsis flowering. Recent studies indicate that chromatin modification may play a role in the regulation of FT expression. It has been shown that LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) directly interacts with FT chromatin and represses FT expression Arabidopsis has revealed that this repressive mark is associated with FT chromatin FT chromatin and its role in FT regulation remain elusive.Drosophila. PRC2 is composed of four core proteins including Enhancer of zeste (E(z); an H3K27 methyltransferase), Extra sex comb (Esc), Suppressor of zeste 12 (Su(z)12) and p55, and deposits trimethyl H3K27 to silence the expression of homeotic genes in Drosophila , directly mediates the repression of AGAMOUS (AG) via H3K27 trimethylation and thus controls floral organogenesis CLF plays multiple roles in plant development, and also directly represses the expression of SHOOTMERISTEMLESS (STM) and a flowering gene, AGAMOUS LIKE 19 (AGL19), during vegetative development FLC repression VRN2 is required for FLC repression by vernalization treatment FLC expression in response to vernalization treatment EMF2, a relative of VRN2 and Su(z)12, also plays an important role in sporophyte development, and maintains vegetative development by repressing the floral induction EMF2-mediated floral repression are unclear Recent studies have also shown that CLF, an Arabidopsis PRC2-like complex subunits CLF, EMF2 and FIE repress the expression of FLC and FLC relatives including MAF4 and MAF5, and that CLF directly binds to and mediates the deposition of H3K27me3 in FLC, MAF4 and MAF5 chromatin. Furthermore, we show that during vegetative development CLF and FIE strongly repress FT expression, and that CLF also directly interacts with and mediates the deposition of H3K27me3 in FT chromatin. Theses results imply that PRC2-like complexes containing CLF, EMF2 and FIE deposit repressive H3K27me3 in and directly repress the expression of these flowering genes, and thus control the flowering program in Arabidopsis.Here we report that FLC repression FLC repression in Arabidopsis plants grown in normal conditions . In addition, the expression of FLC relatives such as FLM, MAF4 and MAF5, like FLC expression, is also regulated by chromatin modification FLC relatives. First, we addressed the role of CLF in the regulation of FLC and FLC relatives. Transcript levels of these genes were examined in seedlings of the clf-81 mutant carrying a lesion in the SET domain of CLF FLC, MAF4 and MAF5 were de-repressed in clf, whereas transcripts of FLM, MAF2 and MAF3 in clf remained at levels similar to wild-type Col , we first examined whether CLF directly interacts with the FLC, MAF4, and MAF5 loci. Specifically, genomic DNA was immunoprecipitated using an antibody recognizing GFP from seedlings of a 35S:GFP:CLF clf transgenic line in which GFP:CLF fully functions and its distribution mimics that of the endogenous CLF FLC-P2) around the transcription start site (TSS) and 5\u2032 part of Intron I of FLC (FLC-I) were greatly enriched, whereas a 5\u2032 promoter region 1.8 kb upstream from the TSS in FLC was not enriched , were not enriched , an H3K27 methyltransferase in the Esc-E(z) PRC2 complex AG and STMclf and wild-type Col seedlings, including H3K27 dimethylation, H3K27 trimethylation and H3K4 trimethylation. Levels of trimethyl H3K27 were strongly reduced in clf relative to Col  in Col and loss of CLF activities significantly reduced the levels of trimethyl H3K27, consistent with the derepression of FLC in clf  in which FLC expression is repressed FLC chromatin in these accessions in the absence of vernalization treatment FLC chromatin can simultaneously carry these two modifications as it is formally possible that these modifications could occur in two subpopulations of FLC chromatin and not in the same physical region of FLC. To examine whether FLC chromatin concomitantly carries both H3K4me3 and H3K27me3, we performed a sequential ChIP in which FLC chromatin from seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Both the region around TSS (FLC-P2) and 5\u2032 part of Intron I of FLC (FLC-I) in part of the FLC chromatin concomitantly harbor H3K4me3 and H3K27me3 (FT-E) and the middle of FT (FT-I) in part of the FT chromatin simultaneously harbor H3K4me3 and H3K27me3 (Ta3ACTIN 2 (ACT2) carrying abundant H3K4me3 (data not shown) but lacking of H3K27me3  and the middle of genomic FT (FT-I) were increased upon loss of CLF activities  complexes containing EMF2 and FIE directly interact with and deposit into the FLC, MAF4, MAF5 and FT chromatin repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that CLF-PRC2 complexes play a significant role in control of the Arabidopsis flowering.Our studies reveal that the FLC repression in Arabidopsis plants grown in normal conditions FLC repression because CLF directly binds to FLC chromatin and loss of CLF function leads to a reduction in H3K27me3 and FLC derepression. SWN, a CLF relative, may also play a role in FLC repression because low levels of trimethyl H3K27 in FLC chromatin have still been detected in clf seedlings , Esc, Su(z)12 and p55, and these components are evolutionarily conserved in animals and plants  The pression . TogetheFCA, FLD and FVE, and these genes do not form a linear pathway FLC expression  located at the bottom of Chromosome 5 CLF represses only MAF4 and MAF5, but not MAF2 or MAF3 in this gene cluster. The CLF-dependent H3K27me3 occurs in MAF4 and MAF5, but is absent from MAF3 and At5g65090 (the gene immediately downstream MAF5), suggesting that the H3K27 trimethylation in MAF4 and MAF5 is not the result of spreading from the neighboring genes. Furthermore, CLF specifically binds to MAF4 and MAF5, but not to MAF3 or At5g65090. This suggests that CLF is specifically recruited to the MAF4 and MAF5 loci, indicating that there are cis-regulatory DNA elements in these two genes that may function similarly to Polycomb-group response elements in DrosophilaFT expression during vegetative development, suggesting that a PRC2-like complex containing CLF, EMF2 and FIE represses FT expression. To date, all known PRC2 complexes in animals and plants contain four core components including p55 or a p55 homolog  has been shown to give rise to extremely early flowering independent of the photoperiods FT to be expressed at low levels for preventing precocious flowering and for the regulation of FT by the photoperiods. PRC2 subunits, likely functioning in the context of a CLF-PRC2 complex, maintain FT expression at basal lower levels in vegetative development, which may serve to provide some room for the elevated FT expression in response to photoperiods and thus enable the photoperiodic control of flowering time in plants.The PRC2-mediated transcriptional gene repressing mechanisms are conserved in animals and plants  as the endogenous control. Primers used are specified in Real-time quantitative PCR was performed on an ABI Prism 7900HT sequence detection system using SYBR Green PCR master mix (Applied Biosystems) as described previously Histone protein extraction and Western analysis were performed as described previously FLC, MAF4, MAF5 and FT were performed using SYBR Green PCR master mix (Applied Biosystems). Primers used to amplify FLC-P1, FLC-P2, ACTIN 2, TUB2 and TUB8 were described previously The ChIP experiments were performed as described previously The sequential ChIP experiments were performed as previously described Figure S1FVE represses MAF4 and MAF5 expression. Total RNAs were extracted from Col, fve and fca seedlings grown in long days. MAF4 and MAF5 were de-repressed in fve, but not in fca.(4.35 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file."}
+{"text": "We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids  are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently. \u00a9 2001 Cancer Research Campaign"}
+{"text": "The EORTC Lung Cancer Cooperative Group undertook a phase II study of paclitaxel in 25 chemotherapy-naive patients with malignant pleural mesothelioma. Paclitaxel was given intravenously at a dose of 200 mg m-2 as a 3 h infusion every 3 weeks, after standard premedication with corticosteroids and antihistamines. This regimen was well tolerated, with < 4% of cycles resulting in severe toxicity. No major objective responses were observed and ten patients had stable disease. Median survival time was 39 weeks and the 1 year survival rate was 30%. In conclusion, paclitaxel at the dose and schedule investigated in this trial had no major activity in the treatment of malignant pleural mesothelioma."}
+{"text": "Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (<25%) and 17 arms had moderate allelic loss frequencies (25\u201350%). Five chromosomal arms were deleted in more than half of the breast cancer samples . Three of these frequently lost chromosomal arms had not been identified as such by comparative genome hybridisation, illustrating the higher sensitivity of microsatellite analysis for the detection of allelic losses. As we present allelic loss data of individual samples, our allelotype should not only aid the identification of new breast cancer genes but also provides a baseline for myriad studies involving these breast cancer cell lines. Genomic DNA was extracted using the Qiagen DNeasy kit. Microsatellite analyses indicated that all samples were unique and monoclonal. Of the 28 breast cancer samples, 22 (79%) were shown to be heterozygous for X-chromosome microsatellites , 9p21 (p16), 10q23 (PTEN), 13q12 (BRCA2), 16p13 (AXIN1), 16q22 (E-cadherin), 17p13 (p53), 17q21 (BRCA1), 18q21 (SMAD4), 19p13 (STK11), and 22q11 (hSNF5). Since constitutional non-neoplastic tissues were not available for any of the breast cancer samples, allelic losses were presumed based on statistical arguments. Multiple microsatellite markers were therefore designed within a 5-centiMorgan locus, thus reducing the odds of a deletion breakpoint between markers within a locus. Analysis of genomic DNA from 25 randomly selected non-neoplastic control samples revealed heterozygosity ratios for all microsatellite markers. Heterozygosity ratios generally were about 0.1 lower than those reported in the NCBI database, but higher ratios were also observed. The number of microsatellites that was analysed for each locus was such that the probability for a heterozygous sample to have a single allele size for each of the microsatellites within the locus was less than 5%, based on the heterozygosity ratios of the used markers. Allelic loss of a chromosomal locus was presumed when a breast cancer sample had a homozygous allele pattern for all microsatellites within the locus (with P<0.05 for each locus). In total, 146 microsatellites were analysed, resulting in an average of 3.6 markers per locus and an average P-value of 0.01. A homozygous allele pattern was observed for the heterozygous control samples at eight of 986 (1%) analysed loci, thus validating the statistical approach and implying an overall error rate for the complete allelotype of about 1%.We analysed 24 commercially available breast cancer cell lines and four breast cancer xenografts for allelic losses at all 41 non-acrocentric chromosomal arms, by PCR amplification of microsatellites. A single locus was analysed for each chromosomal arm, encompassing a known tumour suppressor gene at 5q21  and three (11%) of the 28 breast cancer samples, respectively, with one tumour being double mutant (Wasielewski and Schutte, manuscript in preparation, and (PTEN, BRCA2 and RB1, and BRCA1. Systematic mutational analyses of these tumour suppressor genes in this cohort of commercially available breast cancer samples should reveal whether these known genes indeed were the targets of the frequent allelic losses, or that other new tumour suppressor genes are likely to be located at these chromosomal sites.The wide range from 7% allelic loss at 1q to 82% at 17p that we observed in our breast cancer allelotype indicated a nonrandom deletion of genetic material, suggestive of the location of tumour suppressor genes at the sites of frequent allelic loss. Chromosomal arm 17p contains the"}
+{"text": "Hepatic artery ligation is used for the palliation of patients with malignant liver tumours. Collaterals aredeveloped rapidly and could to some extent explain why the growth is affected for only a short period.With intermittent dearterialization, collaterals seem to be avoided and possibly a more extended effectshould be expected. The most efficient period of dearterialization to avoid collaterals was studied in thisexperiment. Five groups of rats were treated with daily repeated transient dearterializations for 0 (n= 3), 60 (n = 6), 120 (n = 6), 180 (n = 6) and 240 minutes (n = 6) respectively for 5 days and comparedto another group (n = 3) that was permanently dearterialized. After treatment, celiac angiograms wereobtained. All hepatic arteries were reliably occluded and patent after 5 days of daily blockades in all buttwo rats. There were no collaterals demonstrable on the angiograms in the first four groups after 5 daysof intermittent obstruction of the arterial blood flow to the liver. After 240 minutes of dearterializationas well as after collaterals developed and were clearly demonstrated on the angiograms after six days.Liver enzymes were normal even after 4 hours of dearterialization. Repeated occlusions of the hepaticartery was reliably achieved with the implantable minioccluder. Repeated, transient dearterializationsfor 1, 2 or 3 hours could be performed without development of collaterals and without damage to theliver."}
+{"text": "Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals  was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.Plasma from 128 individuals (cohort I \u2013 exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.While these data confirm the detection rate of  Colorectal cancer (CRC) is the second most frequent cancer in Europe and the US affecting 412,900 and 150,000 individuals in 2006, respectively A novel approach of molecular testing for CRC is the analysis of DNA in stool. In a study published by Imperiale et al. p16INK4, MGMT, GSTP1, CDH1, APC and TIMP3 among many others ALX4 and SEPT9 DNA in peripheral blood of colorectal cancer patients compared to controls SEPT9 methylated DNA, CRCs were predicted with a sensitivity of 72% and a specificity of 93% SEPT9 methylation in peripheral blood SEPT9 for the detection of early precancerous lesions we decided to further evaluate this marker in patients with polyps. Therefore, we designed this study, including an exploratory and a validation study, in order to further assess the performance of SEPT9 methylated DNA as a marker in the detection of colorectal precancerous lesions. In the validation study, we included also methylated ALX4 DNA to test if the predictive value for polyps increases if an additional marker is used, this has to be further validated, however. Since early non-invasive detection of polyps could be followed by endoscopic removal of precancerous lesions, this strategy might help to prevent more invasive colorectal cancers.Evidence is increasing that, apart from genetic alterations, epigenetic changes are of similar importance in the pathogenesis of CRC. Particularly tumor suppressor genes can be silenced by the methylation of CpG islands, which are found in the 5\u2032 region of approximately half of all human genes The entire study population was assembled by two seperate cohorts. For cohort I (exploratory cohort), plasma was collected from 128 individuals with different clinical characteristics: healthy controls (n\u200a=\u200a12), patients with symptomatic non-malignant bowel diseases with or without positive FOBT (n\u200a=\u200a17), patients with a history of polypectomy or family history for colorectal neoplasia (n\u200a=\u200a6), patients with chronic inflammatory diseases of the gastrointestinal tract (n\u200a=\u200a20), 45 patients with colorectal precancerous lesions and 28 patients with CRC .For cohort II , plasma samples were collected from 76 individuals prior to any intervention: 49 patients  with colorectal precancerous lesions , from 22 healthy controls  without colorectal lesions and 5 patients  with CRCs  and the MagNAPure LC Total Nucleic Acids Large Volume Extraction Kit (Roche Diagnostics #03264793001) as previously reported 5\u2032-GTAGTAGTTAGTTTAGTATTTATTTT-3\u2032, the reverse primer was 5\u2032-CCCACCAACCATCATAT-3\u2032. Probe sequences were GTTCGAAATGATTTTATTTAGTTGC-FL and LC-Red640-CGTTGATCGCGG GGTTC-PH. The blocker sequence was 5\u2032-CATCATATCAAACCCCACAA TCAACACACAAC-3\u2032. A C3 spacer was introduced at the 3\u2032 end of this sequence.SEPT9 methylation was determined using the HeavyMethyl technique 5\u2032-CGTCGCAACGCGTACG-3\u2032, sALX4r, 5\u2032-CGCGGTTTCGATTTTAATGC-3\u2032. Probe sequences were 5\u2032-ACTCCGACTTAACCCGACGATCG-3\u2032 and 5\u2032-ACGAAATTCCTA ACGCAACCGCT-3\u2032Genomic DNA was analysed by the MethyLight technique after bisulfite conversion as previously reported p-value of <0.05 was considered statistically significant The results of the MethyLight and HeavyMethyl assays were interpreted in a purely qualitative way as previously reported First, we determined the presence of a single DNA methylation marker in plasma from patients with colorectal precancerous lesions, CRC, in healthy controls without any colorectal pathology, patients with chronic inflammation, or other gastrointestinal diseases and patients with a high risk for CRC due to family history or positive FOBT . All metSEPT9 methylated DNA in 1 of 3 measurements was detected in 3 out of 12 healthy controls (25%), 28 out of 45 patients with polyps (62%), 25 out of 28 (89%) patients with colorectal cancers, 18 out of 20 (90%) patients with chronic inflammation and 6 of 6 high risk patients as well as 17 out of 17 patients with symptomatic diseases. Using the cut-off of at least 2 or 3 positive measurements only 2 of 12 healthy controls (17%), 9 of 17 symptomatic patients (53%), 4 of 6 risk patients (67%), 10 of 20 patients with inflammation (50%), 21 of 45 patients with colorectal precancerous lesions (47%) and 22 of 28 CRC patients (79%) were classified as positive  healthy controls. In contrast, SEPT9 methylation was significantly more frequent in patients with polyps (15 of 49 patients (31%); p\u200a=\u200a0.01). Using the cut-off of at least 2 or 3 positive measurements with regard to the presence of SEPT9 methylated DNA in plasma, again only 1 of 22 (5%) healthy controls was classified as positive, whereas patients with polyps exhibited methylated SEPT9 DNA in at least 2 measurements in 6 of 49 (12%) cases. Cancer patients were positive in 2 of 5 cases using either classification  healthy controls and 27 out of 94 polyp patients (29%) as well as 24 out of 33 (73%) CRC patients were classified as positive. The difference in prevalence of SEPT9 DNA between polyp and control samples was statistically significant , 38 of the 49 polyp patients (78%) and in 4 of 5 (80%) cancer patients. Using the more stringent criteria (high specificity) the number of healthy controls with positive DNA detection decreased to 4 of 22 individuals (18%), whereas for patients with polyps 22 of 49 patients (45%) were considered positive (p\u200a=\u200a0.02). Methylated DNA was observed in 2 of 5 cancer patients (In order to improve the detection rate of patients .SEPT9 + ALX4 and classifying a sample as \u201cpositive\u201d if any of the single markers is \u201cpositive\u201d (at least 2 of the 3 measurements), we observed 4 positive cases among the 22 healthy controls (18%), 25 positive cases among the 49 patients with polyps (51%) and 3 positive cases among the 5 cancer patients , whereas 18 of 49 patients with polyps (37%) and, again, 3 of 5 cancer patients were found to be positive. The difference between normals and polyps was highly statistically significant (p\u200a=\u200a0.0013) .Next we turned to the analysis of marker performance with respect to the different types of polyps analysed in our study. Polyps were subclassified with respect to the clinically relevant size of <10 mm and \u226510 mm and histomorphological criteria . Patients with tubular or tubulo-villous adenomas irrespective of size were positive with regard to the presence of the marker panel in blood in 19 of 36 (53%) patients. The frequency of detectable methylated DNA increased in the subclass of patients with adenomas \u226510 mm . In the subgroup of polyps \u226510 mm and the presence of high grade intraepithelial neoplasia methylated DNA of the two markers was even observed in the plasma in 5 of 7 cases 71%; . OverallThe main problem of CRC screening today is low compliance in existing screening programmes. Despite the fact that CRC has all features which qualify this disease for mass screening, including high incidence and prevalence, high mortality in advanced disease, potential for cure in early stages, on average less than 30% of the eligible population in the US and Europe actually undergo these preventive procedures Based on the presence of certain genetic, epigenetic and related changes in the proteome of CRCs it has been tempting to assess the diagnostic accuracy of the presence of these changes in blood and stool for the purpose of CRC screening SEPT9 and ALX4 gene methylation as potential markers for CRCs ALX4 methylation in plasma was 83.3% and 70%, respectively SEPT9 methylation in plasma revealed a sensitivity of 70% and specificity of 90% for the detection of patients with CRC Another non-invasive approach is the detection of epigenetic changes in blood and/or stool of patients with CRC  and S4. ALX4 and SEPT9 methylation in CRC patients ALX4 and SEPT9 methylated DNA in plasma from patients with polyps versus healthy controls. In addition, the combined analysis of the two markers proved to be highly significant in the detection of colorectal polyps with a sensitivity and specificity reaching 71% and 95%, respectively, for the detection of advanced precancerous colorectal lesions. Thus, the performance of this marker panel was further enhanced after the detailed analysis of the histomorphological nature of the lesions that were observed in the patients with polyps. Accordingly, the combination of the two markers proved to be highly sensitive in the detection of advanced adenomas, which are defined clinically and pathologically as lesions that are \u226510 mm in size and exhibit features of potential malignant transformation .Similar to our previous reports on Recently, other groups have also analysed the presence of methylation markers in body fluids from patients with CRCs and adenomas, with a special emphasis on molecular stool analysis. However, the sensitivity of single methylation markers does not exceed 57% and in blood almost none of these markers were found so far  and S4. SEPT9 was increased among high risk patients (67%) and patients with chronic inflammation (50%). Thus, our exploratory study indicates that SEPT9 may be able to identify potentially curable early cancers or even precancerous colorectal lesions in asymptomatic individuals. However, patients with concomitant inflammatory diseases or other increased cancer risk should be directed to colonoscopy according to current clinical routine without non-invasive first line screening We also analysed the methylation status by including other symptomatic patients groups with non-malignant intestinal diseases. Methylation of In conclusion, our study presents the first evidence for a novel approach for the detection of advanced precancerous colorectal lesions using methylation markers in plasma. Advanced precancerous colorectal lesions are associated with an increased frequency of methylated DNA in the plasma. Based on this study, the diagnosis of colorectal precancerous lesions may be possible with a non-invasive, sensitive and reliable plasma-based test using a panel of methylation markers. If confirmed in a larger patient population, a diagnostic test based on this panel could improve early detection of cancerous lesions by allowing preselection of patients with precancerous colorectal lesions that could be subsequently be removed by colonoscopy, thereby offering the potential to prevent colorectal cancer.Figure S1Performance comparison of HM/Methylight assays on tissue samples. 198 Colorectal cancer tissues and 22 normal colon mucosa samples were analyzed by quantitative real-time PCR. Performance for the HM/Methylight is demonstrated by ROC plot analysis.(3.88 MB TIF)Click here for additional data file.Figure S2Performance comparison of MSP/Methylight assays on tissue samples. 198 Colorectal cancer tissues and 22 normal colon mucosa samples were analyzed by quantitative real-time PCR. Performance for the MSP/Methylight is demonstrated by ROC plot analysis.(3.83 MB TIF)Click here for additional data file.Table S1Classification table: Marker panel for detection of advanced polyps.(0.03 MB DOC)Click here for additional data file.Table S2Summary of statistical results.(0.04 MB DOC)Click here for additional data file.Table S3Performance of methylation markers in stool and blood for detection of colorectal adenoma.(0.04 MB DOC)Click here for additional data file.Table S4Results of multipanel assays with methylation markers for diagnosis of colorectal cancer in stool.(0.03 MB DOC)Click here for additional data file."}
+{"text": "Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.At the imprinted  Rasgrf1 locus in mice. It is required for methylation of nearby DNA sequences and can also influence the levels of local histone methylation. The methylation programmer has different effects on paternally and maternally derived chromosomes, directing DNA methylation on the paternal allele and histone H3 lysine 27 trimethylation on the maternal allele. These two methylation marks are not only mutually exclusive; they are also mutually antagonizing, whereby one blocks the placement of the other. Manipulations that cause aberrant changes in the levels of one of these marks had the opposite effect on the other mark. These observations identify novel mechanisms that specify epigenetic states in vivo and provide a framework for understanding how pathological epigenetic changes can arise, including those emerging at tumor suppressors during carcinogenesis.Methylation of DNA and histones exert profound and inherited effects on gene expression. These occur without changes to the underlying DNA sequence and are considered epigenetic effects. Disrupting epigenetic states can cause developmental abnormalities and cancer. Very little is known about how locations in the mammalian genome are chosen to receive these chemical modifications, or how their placement is regulated. We have identified a DNA sequence that acts as a methylation programmer at the  Within this region, there is a differentially methylated domain (DMD) that is subject to acquisition of epigenetic modifications, typically DNA methylation and histone modifications. These modifications are placed in a parent-of-origin specific manner and impose an epigenetic state that dictates allele-specific gene expression at imprinted loci In mammals, imprinted loci are expressed from only one allele. Accompanying and controlling monoallelic expression are allele-specific epigenetic modifications influenced by an Rasgrf1 locus. The ICR, located 30 kbp upstream of the transcriptional start site, is a binary switch consisting of a repeated element and the DMD. The repeated element functions as a methylation programmer, that is necessary for the establishment and maintenance of DNA methylation at the DMD on the paternal allele and sufficient for establishing gametic imprints in both germlines . However, in mice bearing a deletion of the repeats that constitute the Rasgrf1 DNA methylation programmer, only the DNA methylation at the originally defined DMD was lost. The DNA methylation on the additional 1.6 kbp was unaffected, indicating that the range of action of the Rasgrf1 DNA methylation programmer in the tandem repeats is limited to the 400 bp proximal DMD in the male germline  using mouse embryonic fibroblasts (MEFs) and antibodies specific to H3K9me2, H3K9me3, H3K27me2, and H3K27me3. Our initial tests were controls to verify that the antibodies detected histone modifications with proper specificity . For theWe then extended our H3K27me3 and H3K9me3 analysis to six segments . We were able to distinguish the transgenic ICR from the endogenous copy because the transgenic repeats were flanked with loxP sites and had the same structure as the WT-flox allele shown in Rasgrf1, then we predicted our MEFs with a methylated transgene should exclude H3K27me3, whereas our MEFs with an unmethylated transgene should allow its placement. This is also precisely what we observed  after amplification and will be digested, whereas unmethylated templates that underwent bisulfite conversion of either CG in the recognition site to TG will resist digestion. There should be an equal amount of digested and undigested PCR product when the maternal allele is completely unmethylated and the paternal allele is completely methylated. This is what we saw in embryoid bodies and blastocysts that were heterozygous respectively for the Suz12 and Yy1 mutations. This indicated that our COBRA assays accurately reported the presence of both methylated and unmethylated templates expected in these Suz12 and Yy1 expressing materials; however, it is not clear why Suz12 heterozygous ES cells did not show this pattern. When we performed COBRA analysis on SUZ12-deficient embryoid bodies (EB) that had differentiated for six (P6) or nine (P9) days in vitro , suggesting that at Rasgrf1, other CTCF binding partners and functions might be more important, such as YY1.We do not know how DNA and H3K27 methylation antagonize each other's placement; however, the literature highlights several molecular and developmental events, as well as protein factors that may be involved. Among these is the transcriptional state that is known to influence which of two mutually exclusive histone modifications is placed by the competing activities of polycomb (PcG) and trithorax (Trx) group proteins  Rasgrf1 , most liDM1 locus in humans, a repetitive element is associated with heterochromatin accumulation Rasgrf1 DMD and repeat sequences DM1 repeat also is a CTCF-binding insulator. CTCF appears to restrict the boundary of heterochromatinization at DM1, but it is not known if CTCF has a similar effect at Rasgrf1. Sequences with appreciable similarity to the Rasgrf1 tandem repeats are not abundant in the mouse genome. However, the Rasgrf1 repeat unit has striking similarity to the B repeat sequences on Xist . Previous reports describe the Suz12 mutation and preparation of homozygous ES cells and embryoid bodies Yy1 mutation and preparation of trophoblast outgrowths Mice used for DNA methylation analysis across the 12 kbp interval were F1 progeny of PWK and 129S4SvJae parents. Polymorphisms in these strains facilitated the assignment of a given clone from bisulfite PCR to one of the two parental alleles. Mice used to prepare MEFs for ChIP analysis across the 12 kbp interval were from strain 129S4SvJae backcrossed to C57BL/6 and included wild type animals, animals carrying a repeat deletion http://www.millipore.com for certificates of analysis for each catalog item and lot number). The DNA recovered after ChIP was used for Q-PCR with input chromatin and mock immunoprecipitations without antibody serving as controls. Q-PCR was performed in triplicate with SYBR green detection using primers listed in MEFs from 13.5 day old F1 embryos of C57BL/6 and 129S4Jae parents were used for ChIP analysis as described in BstUI digestions. In this assay, cytosine methylation enables digestion, whereas absence of methylation prevents it. Assays for DNA methylation using HhaI digested DNAs were described Treatment of genomic DNA with bisulfite was performed as previously described Figure S1CpG dinucleotides and methylated DNA centered at the DMD. A,B,C. The distribution of A, T, C and G over the 220 kb cluster show that there is a predominant accumulation of cytosines over the DMD and repeat region. The DMD repeat region has large amount of C and G together. There are two CpG islands in the region: one, which is the DMD and the other, which is in the promoter region of Rasgrf1 (bottom panel). D. Southern blot analysis of the two CpG islands using methylation sensitive restriction enzymes and tail DNA show that there is monoallelic methylation at the DMD (left panel) but no methylation of the promoter region CpG island (right panel). P (PstNI), N , E (EcoRI), Br . Bands diagnostic for the methylated (+) and unmethylated (\u2212) states are indicated.(0.55 MB TIF)Click here for additional data file.Figure S2Specificity controls for antibodies used in ChIP. Representative gel analysis of ChIP results indicating the specificity of the antibodies for the histone modification analysis in this study. Antibodies specific to H3K9me2, H3K9me3, H3K27me2, and H3K27me3 show enrichment for H3K9me3 and H3K27me3 at the DMD. Positive control PCRs for H3K9me2 and H3K27me2 Charlie , H3K9me3(0.12 MB TIF)Click here for additional data file.Figure S3Rasgrf1 in wild type MEFs show that paternal DNA methylation (green) is largely even over the region, while maternal DNA methylation (red) is absent over the DMD but present upstream and downstream. Strikingly, H3K9me3 and H3K27me3 are perfectly confined to the DMD. (B) Modifications in the paternal allele in +/Rep\u0394 mice. DNA methylation (black) is lost from the DMD and downstream, allowing encroachment of H3K27me3 into these regions (red).Mutual exclusion of H3K27 and DNA methylation. H3K27 and DNA methylation data from (0.15 MB TIF)Click here for additional data file.Figure S4Rasgrf1 ICR. (A) Xist sequences, including the A, B, C, D and E repeats (17 kb) and Rasgrf1 sequences including the DMD and repeats (5 kb) were aligned in a dot plot matrix. (B) Detail of the dot plot matrix in A that includes the Xist B element and the Rasgrf1 repeats.Dot plot of Xist and the (0.24 MB TIF)Click here for additional data file.Table S1Primers used for PCR amplification.(0.06 MB DOC)Click here for additional data file.Table S2Clones sequenced for analysis in (0.08 MB DOC)Click here for additional data file.Table S3http://www.broad.mit.edu/seq_platform/chip/ and experimentally verified CTCF site data were downloaded from http://insulatordb.utmem.edu/browse.php. After filtering the H3K27me3 ChIP data for sites with a read score of two or higher, the data sets were added as custom tracks on the UCSC Genome Browser and intersected in the intervals spanning 17,553 known genes and 53 imprinted gene regions. The intervals examined included the 100 kb 5\u2032 of each gene (+100), sequences between the 5\u2032 and 3\u2032 ends of the genes (G), 100 kb 3\u2032 of the genes (\u2212100), and the entire stretch from 100 kb 5\u2032 to 100 kb 3\u2032 of each gene region (+100 to 100). The number of times H3K27me3 colocalized with CTCF in the indicated intervals is reported. The frequency of colocalization per kb was calculated for each interval examined, and the values for each of the known gene intervals were used to calculate an expected value for the corresponding imprinted gene intervals, given the total number of kbp in each of the imprinted gene intervals examined. The number of observed and expected colocalizations in the imprinted intervals was then used in Chi-square analysis.Enhanced colocalization of CTCF and H3K27me3 at imprinted loci. Whole genome H3K27me3 ChIP data for imprinted and known genes in MEF cells were downloaded from (0.06 MB DOC)Click here for additional data file.Text S1Supporting methods and references.(0.04 MB DOC)Click here for additional data file."}
+{"text": "FHIT (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour suppressor gene. To define the role of the FHIT gene in the development of ovarian cancer, we have examined 33 ovarian carcinomas, 2 borderline tumours and 10 benign adenomas for the presence of FHIT gene alterations. FHIT transcripts were analysed by RT-PCR and sequencing. Aberrant FHIT transcripts were observed in 5/33 carcinomas (15%) and in 1 of 2 borderline tumours. Loss of normal FHIT transcript was observed in 5/33 carcinomas (15%) but not in 2 borderline tumours or 10 benign adenomas. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of FHIT were detected in 5/24 (21%) and 5/25 (20%) informative ovarian carcinomas, respectively. Expression of Fhit protein was analysed by immunohistochemistry in 44 carcinomas, 19 borderline tumours and 16 benign adenomas. Loss or significantly reduced expression of Fhit protein was observed in 6/44 (14%) ovarian carcinomas but not in any of 19 borderline tumours or 16 benign adenomas. The impaired Fhit protein expression was significantly correlated with the loss of normal FHIT transcription. Most notably, loss of normal FHIT transcript and impaired expression of Fhit protein occurred only in serous adenocarcinomas of grade 2 and 3 . The present data suggest that inactivation of the FHIT gene by loss of expression is one of the important molecular events associated with the genesis of ovarian carcinoma, especially of high-grade serous carcinoma. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comThe"}
+{"text": "PAX2 is a transcription factor with an important role in embryogenic development. However, PAX2 expression was frequently identified in neoplasia responsible for the growth and survival of cancer cells. Due to alternative splicing of exon 6, exon 10 and exon 12 four isoforms of PAX2 are described so far.The expression of an intron 9 containing PAX2 splice variant was analyzed in neoplastic B cell and solid tumor cell lines as well as in primary tumor samples by quantitative RT-PCR. PAX2 proteins were detected by Western Blot in a subset of cell lines.All 14 lymphoma cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence. This splice variant could also be detected in 35 solid tumor cell lines, in leukemia and lymphoma as well as in colon carcinoma and melanoma patient samples and in blood samples of healthy donors. Expression of this new splice variant on protein level was verified by Western Blot analysis.We discovered a previously undescribed intron 9 and exon 10 containing splice variant of PAX2 in B-cell neoplasia and in solid tumors on mRNA and protein level. The PAX gene family was first described in Drosophila and later found to be conserved across species . PAX genAlthough PAX2 is primarily expressed during embryonic development and expression is normally repressed upon terminal differentiation, PAX gene expression was frequently identified in tumor cell lines, including lymphoma, breast, ovarian, lung, and colon cancer, as well as in primary tumor tissue samples  and was Apoptosis was induced in cell lines following RNA interference to silence PAX2 expression, suggesting that endogenous PAX2 gene expression is required for the growth and survival of cancer cells ,10,7. ThFour isoforms of PAX2 are described so far. They are products of alternative splicing of exon 6, exon 10 and exon 12: Exon 6 is present in the PAX2a transcript and absent in the PAX2b transcript . InsertiHere we characterized a previously undescribed intron 9 and exon10 containing splice variant of PAX2 in neoplastic B cell lines and solid tumor cell lines as well as in tumor tissue.14 lymphoma cell lines  and 35 solid tumor cell lines  All human cell lines were purchased from DSMZ  and CLS . Cells were maintained in RPMI 1640 containing 10\u201320% FCS, 2% penicillin/streptomycin and 2% glutamine .Fifteen primary low grade lymphoma, 9 myeloma, 11 acute lymphoblastic leukemia (ALL) samples and 7 AML samples were taken from patients that underwent routine diagnostics like venipuncture or bone marrow aspiration. Primary tumor single cell suspensions were prepared by ficoll hypaque separation. The lymphoma and leukemia samples contained more than 80% of tumor cells; therefore no further separation was done. For multiple myeloma, CD138 positive cells were isolated using Mini MACS technology . Tumor cells were resuspended in guanidium thiocyanate (GTC) buffer and stored at -80\u00b0C. 12 melanoma  and 12 colon carcinoma tissue samples were obtained from patients that underwent surgery for their tumors. Tissue samples were collected and dissected under stringent sterile conditions to prevent RNA contamination and immediately frozen in liquid nitrogen. There were no specific inclusion criteria with exception for the leukemia samples. Only PAX2 mRNA expressing AML and ALL samples were included. All patients had given informed consent for the analysis. Approval by the appropriate ethics committee has been obtained  and analyses have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Blood samples of healthy volunteers served as negative controls.Total RNA was isolated by RNeasy Mini Kit including RNase-Free DNase Set . Reverse transcription and quantitative Real Time RT-PCR  was done as described elsewhere . Primer 2 concentrations are listed in table PCR conditions and target-specific final MgClAnalysis of RT-PCR expression data was done with the LightCycler software (version 3). Sample concentrations were calculated using the plasmid standard curve resulting in marker concentrations. All samples were analysed in duplicate. The average value of both duplicates was used as a quantitative value. To correct for differences of cDNA amount on a per-sample basis, results were provided as ratio to housekeeping gene porphobilinogen deaminase (PBGD) expression. Statistical significance was tested using SPSS 15.0 software. For comparison of PAX2 intron 9 specific mRNA expression levels significance was estimated by the 2-sided Mann-Whitney U test for comparison of two different groups.Western blots were performed on equal amounts of protein obtained by lysis of cells using MPer Protein Extraction Reagent . The protein concentration was measured by BCA method using BCA Protein Reagent . 50 \u03bcg protein extract was loaded onto a 10% SDS-PAGE . Following electrophoreses, proteins were transferred to nitrocellulose membranes, and then blocked with 1%BSA in PBST  overnight at 4\u00b0C. Blots were then probed with rabbit anti-PAX2 primary antibody  at 1:1000 dilution. After washing with PBST the membranes were incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) at 1:5000 dilution . Signal detection was visualized using ECL chemiluminescence reagent . As a control, blots were probed with mouse anti-\u03b2-actin primary antibody  and HRP-conjugated anti-rabbit secondary antibody.RT-PCR analysis of the PAX2 transcript in 14 lymphoma cell lines using a forward primer lying in exon 9 and a reverse primer lying in exon 10 (primer set PAX2_1) showed different PCR products on gel electrophoresis figure :All lymphoma cell lines showed a band of 339 bp of varying intensity. A band of the expected size of 185 bp was detected only in the cell lines KM-H2, EHEB, L540 and to a lesser extent in the cell line DG75. Sequencing analysis of the 339 bp PCR products revealed that this product results from the insertion of the entire intron 9 sequence. Thus, these cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence figure  with a sTo analyze, whether the new splice variant is also expressed in solid tumors, a panel of solid tumor cell lines was tested by RT-PCR with the same primer set spanning the intron 9 (PAX2_1). Analysis of the product size by gel electrophoresis showed, that 7 of the 8 melanoma cell lines, 7 of the 9 colon carcinoma cell lines and 1 of the 7 renal carcinoma cell lines expressed the intron 9 and exon 10 containing splice variant. The other cell lines showed only a band of 185 bp. Additionally, 5 of 5 breast carcinoma cell lines, 3 of 3 lung carcinoma cell lines and 4 of 4 thyroid carcinoma cell lines expressed this splice variant.Next PAX2 positive leukemia patient samples were analyzed: In all 11 ALL samples and all 7 AML samples the new splice variant could be detected on gel electrophoresis. Subsequently, samples from patients with low grade lymphoma and multiple myeloma were analyzed. All 15 low grade lymphoma patient samples and 7 of the 9 multiple myeloma patient samples expressed the intron 9 containing splice variant. The remaining 2 multiple myeloma samples were negative for PAX2 mRNA . However, 22 of 24 blood samples from healthy donors surprisingly were also positive for the intron 9 and exon 10 containing splice variant.-4 (range 1.42 \u00d7 10-4 - 7.61 \u00d7 10-2). Additionally, in all solid tumor cell lines intron 9 specific mRNA was detected. The median expression was 1.49 \u00d7 10-3 (7.96 \u00d7 10-5 - 1.04). Moreover, the expression of the intron 9 positive PAX2 isoform was analyzed in 12 melanoma  and 12 colon carcinoma patient samples as well as in 9 AML and 5 ALL patients. All leukemia samples, 11 of the 12 colon carcinoma and 7 of the 12 melanoma samples were positive for expression of intron 9 specific mRNA. The median expression level in solid tumor samples was 1.17 \u00d7 10-1 (range 1.43 \u00d7 10-2 - 5.44) and in leukemia samples 3.07 \u00d7 10-4 (range 1.22 \u00d7 10-5 - 1.3 \u00d7 10-2)  figure . The exp-2 (range 6.33 \u00d7 10-4 - 1.63 \u00d7 10-1)  figure . The medTo verify the expression of this intron 9 positive splice variant on protein level, PAX2 protein expression was examined by Western Blot analysis in whole cell extracts of 3 colon carcinoma cell lines  and 4 lymphoma cell lines . Protein bands corresponding to known PAX2 isoforms  could be found in all cell lines. Additionally, a band of approximately 37 kDa figure  was idenHowever, in blood samples of healthy volunteers bands corresponding to the known splice variants of PAX2 and to a lesser extent to the new splice variant could be also detected. Expression of PAX2 in lymphoid cells was also observed by others .Therefore, regarding PAX2 targeted therapies like vaccination strategies caution is needed.We found a previously undescribed intron 9 and exon 10 splice variant of PAX2 on mRNA and protein level in B cell neoplasia and solid tumors as well as in peripheral blood of healthy patients. This splice variant has a distinct and a shorter C-terminus than the known exon 10 containing splice variant PAX2c due to the deletion of the last 89 amino acid residues. Alternative processing represents an important mechanism for the generation of various protein isoforms with different functions from one genetic locus . The funFinancial Disclosure: Ulrich Keilholz is holding a patent for the use of PAX2 for cancer immunotherapy. All other authors have declared there are no financial conflicts of interest in regards to this work.Grant Support: EU Integrated Project Cancer Immunology and Immunotherapy, project: WP 02.03 Transcription factors PAX2 and PAX8 as new tumor antigens.AB has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and wrote the manuscript; AR: has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. SS have been involved in acquisition of data and revising the manuscript critically for important intellectual content; ET has made substantial contributions to conception and design and was involved in revising the manuscript critically for important intellectual content, UK: has made substantial contributions to conception and design, as well as analysis and interpretation of data and wrote the manuscript."}
+{"text": "Percutaneous access to the biliary tract was chosen when an endoscopic approach was not possible . Visualization of stones was achieved radiologically in 32 patients and by ultrasound in 24. The procedure was successful in 47 of 56 treated patients (83.9%). Clearance of the biliary tract was obtained in 25 cases (53%), whereas in 22 cases (47%) complete clearing of biliary tract was obtained only after endoscopic extraction of fragments (17 cases) or percutaneous (5 cases). The median number of shock waves in each session was 1725 (range 300\u20133166), which were applied during one (n=30), two (n=22) or three sessions (n=4). The only complications were 1case of symptomatic hyperamylasemia and 3 cases of macrohematuria. In conclusion, extracorporeal lithotripsy combined with endoscopic and/or percutaneous treatment is a real alternative to surgery for difficult stones."}
+{"text": "Between 2003 and 2005, highly pathogenic avian influenza A (H5N1) viruses caused large scale outbreaks in poultry in the Ho Chi Minh City area in Vietnam. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005.Single sera from 500 poultry workers and poultry cullers exposed to infected birds were tested for antibodies to avian influenza H5N1, using microneutralization assays and hemagglutination inhibition assay with horse blood. All sera tested negative using microneutralization tests. Three samples showed a 1\u223680 titer in the hemagglutination inhibition assay.This study provides additional support for the low transmissibility of clade 1 H5N1 to humans, but limited transmission to highly exposed persons cannot be excluded given the presence of low antibody titers in some individuals. Since their re-emergence in late 2003, highly pathogenic avian influenza A (H5N1) viruses have spread across the globe, reaching endemic levels amongst poultry in several countries. The continuing occurrence of sporadic human H5N1 infections has ignited worldwide concern about an imminent influenza pandemic with potentially devastating consequences, especially if a pandemic H5N1 virus would keep its current virulence in humans. This threat persists despite the emergence of pandemic H1N1 in early 2009, either through direct adaption of H5N1 to efficient human transmission, or through reassortment with the novel H1N1 virus in swine or in humans.Based on reported cases, the mortality of human H5N1 infections still exceeds 60% with most patients dying of rapidly progressive respiratory failure. The occurrence of mildly symptomatic and asymptomatic human H5N1 infections has been suggested by seroepidemiological studies after the 1997 H5N1 outbreak in Hong Kong Vietnam has been one of the countries hit hardest by influenza H5N1 with 111 human infections reported since 2003. Beginning in early 2004, culling programs were initiated in Vietnam to contain spread of H5N1 across poultry farms. These programs identified infected poultry and provided in culling of all poultry on farms where infected poultry was found. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005 when large scale poultry outbreaks were occurring in and around the city.The sub-department of Animal Health HCMC (AH) performed active surveillance in poultry farms (any size) in Ho Chi Minh City (HCMC), to identify and subsequently cull ducks and chickens with H5N1 infection, between October 2004 and June 2005. During this period, clade 1 H5N1 viruses were circulating in southern Vietnam Poultry farmers were identified by AH as having worked on a farm where poultry tested H5N1 positive during the 6 months prior to April 2005. Adults (>15 years old) living or working in affected sites at least 1 week prior to identification of H5N1 infected poultry, were included in the study. All cullers who were actively involved in culling during the period December 2004\u2013June 2005, were identified by AH. All poultry workers on positive sites and all cullers were visited by staff from the Institute of Preventive Medicine from Ho Chi Minh City in June and July 2005. Witnessed oral informed consent was obtained from all participants. The study and the consent procedure were approved by the Hospital for Tropical Diseases Ethical and Scientific Committee and the University of Oxford Tropical Research Ethical Committee.A questionnaire was made in English and translated into Vietnamese. Participants were asked about demographic characteristics, exposure to poultry, the use of protective gear during exposure, and the occurrence of clinical symptoms during the period of study.Serum samples from poultry were collected on site and directly transferred to the laboratory of AH for analysis. Samples were tested within 48 hours of collection.A single 5 ml blood sample was obtained from all participants in June or July 2005, transported to the laboratory of the Oxford University Clinical Research Unit (OUCRU) in HCMC, and, after separation of serum, stored in aliquots at \u221220\u00b0C. Aliquots of serum were sent to Hong Kong (MP and WL) on dry ice for analysis. As the time between start of poultry culling and serum sample collection was more than 3 months for all participants, only single serum samples were collected and tested.www.oie.int/eng/normes/mmanual/a_00037.htm) using chicken red blood cells and H5N1 antigen derived from strain A\\Ck\\Scot\\59 . Samples from dead poultry were subjected to real-time RT-PCR for detection of the H5 gene Serum samples from poultry were analyzed by HI as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization of Animal Health  (clade 1) and A/Vietnam/30850/05 (clade 2.3.4) with suppression of viral cytopathic effect as the end point Human sera which tested positive at any dilution in at least one of the two tests, were retested by HI using horse red blood cells A total of 65 positive sites were identified. Of these, one site had dying chicken which were confirmed by RT-PCR to be infected with H5N1. Two additional sites had dying chicken which could not be tested for the presence of H5N1 as they had been destroyed. All other sites had ducks with positive HI tests. The number of poultry on a given site varied from 2 to 1600. The number of samples tested per site varied between 1 and 31 and the number of HI positive samples per site varied between 1 and 30.A total of 183 poultry workers were included in the study. 89 (50%) were male (sex was not recorded for 5 poultry workers) and the median age was 36 years . The majority of infected sites contained ducks . Almost 317 cullers were involved in culling during the period December 2004\u2013June 2005. 195 (61.7%) were male (sex was not recorded for 1 culler) and the median age was 42 years (range 22\u201358 years). The median time between the last exposure and serum sample collection was difficult to assess as most cullers were involved in culling of infected poultry during extended periods of time . The preWe studied the prevalence of antibodies against H5N1 among 500 poultry workers and cullers in the HCMC area in 2005. All poultry workers and cullers had a history of exposure to poultry with evidence of symptomatic infection (dying chicken with positive RT-PCR) and/or asymptomatic infection (HI positive ducks) with H5N1. None of the poultry workers or cullers had antibody titres \u226580 in microneutralization tests, which is the recommended threshold for serological evidence of H5N1 virus infection, indicating that transmission of H5N1 was low. These results are in agreement with studies performed amongst poultry workers and persons exposed to infected poultry in the household in Nigeria, Thailand and Cambodia Interestingly, three cullers showed positive antibody responses in one of the neutralization assays, in two cases below-, and in a third case at the lower threshold of recommended cut-off values. These neutralizing antibody responses were confirmed in a different laboratory using a clinical isolate representative of the circulating virus at the time of the study. According to current guidelines, a second serological test, in this case HI using horse blood, was performed on all samples showing a titer in one of the neutralization assays. HI antibodies were observed in these three sera, albeit at titers one dilution below recommended cut-off values for confirmatory testing The median time between the last exposure and serum sample collection for cullers was difficult to assess as most cullers were involved in culling of infected poultry during extended periods of time . This prApproximately 3% of pre-vaccine sera of US participants in an H5N1 vaccine trial had H5N1 neutralizing antibody in the microneutralization test and in HI assay using horse red blood cells The exact exposures of poultry farmers and cullers are difficult to assess. Dying chicken in an area and time where H5N1 is highly prevalent, is highly indicative of infection in chicken, and therefore of exposure to those involved in farming and culling on farms where chicken were dying. However, the exposure is less clear for individuals who were exposed to infected ducks only. Whilst in the early stages of the current H5N1 outbreak infected ducks were reported dying, the virus adapted rapidly to become asymptomatic in ducks In conclusion, whilst our study provides additional support for the low transmissibility of highly pathogenic clade 1 H5N1 viruses to humans, limited transmission to highly exposed persons cannot be fully excluded given the presence of low antibody titers in some individuals."}
+{"text": "Analysis of time-lapse cinematographic film permitted the construction of pedigrees from 88 well oxygenated cells of a mouse osteosarcoma (MOS). These cells have been chronically treated with various concentrations of the hypoxic cell sensitizer misonidazole (MIS) over periods of up to 96 h. At concentrations of 0.5 and 7 mM there is a 2--3 h increase in cell-cycle time. Concentrations of 2 mM show an intermitotic time delay of 7.6--10.3 h. At 4 mM cells divided only once. With increasing drug concentration there was an increase in the number of abnormal mitoses. These results were compared with cloning efficiency (PE) experiments. PE at 0.5 mM is 80%, at 1 mM 40 and at 2 mM is reduced to 4%. Cells treated with 2mM MIS over a period of 28.6 h resume their normal cycle when the drug is washed from the culture. This may indicate that DNA is not a major target for MIS. It is concluded that this hypoxic cell sensitizer is also toxic for MOS cells in well oxygenated conditions."}
+{"text": "Cyclin D1 is a cell cycle regulator essential for G1 phase progression and is frequently overexpressed in several human tumour types as a consequence of gene amplification or chromosomal rearrangements. We analysed the expression of cyclin D1 in 75 patients with transitional cell carcinoma (TCC) to investigate the possible relationship between its expression and clinical outcome as well as histopathological findings using the immunohistochemical method. We observed strong staining  for cyclin D1 in 19 cases (25.3%) and weak staining  in 19 cases (25.3%). Overexpression of cyclin D1 was not associated with tumour invasion. No significant association was found between overexpression of cyclin D1 and tumour grade (P > 0.05). We assessed the differences of disease-free interval in superficial tumours and actuarial survival probability in invasive tumours according to the status of cyclin D1 expression. Tumours with (++) staining for cyclin D1 recurred much more rapidly than (-) and/or (+) staining tumours . However, overexpression of cyclin D1 was not associated with a shortened overall survival of patients with invasive tumours (P < 0.1). These results suggest that genetic alteration of cyclin D1 appears to be an early event in the tumorigenesis of bladder TCC and is associated with early recurrence in superficial tumours."}
+{"text": "Changes which lead to excessive cyclin production or to loss of cell cycle inhibition by proteins such as p16/MTS1 may release breast tumour cells from the constraints of cell division. In order to establish the frequency of MTS1/p16 gene alteration and its relation with genetic damage to the p53 and cyclin D1 genes, we have studied these gene abnormalities in 164 human primary breast cancers and in six breast cancer cell lines. Two breast cancer cell lines and one primary tumour showed a homozygous deletion of exon 2 of the MTS1 gene. Using single-strand conformation polymorphism and subsequent sequencing analysis, one tumour showed an alteration at codon 67 . Another tumour showed a mutation at codon 98 (without amino acid change) with an additional polymorphism at codon 140. This polymorphism was also found in 13 other tumour samples, but has no effect on (disease-free) survival. From these data we conclude that the occurrence of CDKN2 (p16/MTS1) mutation in primary breast cancer is a rare event and is not likely to be involved in human breast tumour carcinogenesis and progression."}
+{"text": "Fifteen patients (34.9%) achieved partial remission  for a median duration of 6 months; the median survival of these patients has not yet been reached. A further 15 had stable disease for a median of 4 months and a median survival of 7 months. The 10 patients (23.2%) who experienced disease progression had a median survival of 4 months. Three patients are not evaluable. The 1-year actuarial survival rate is 31.1%. The treatment was well tolerated: only 35% of the patients had grade 3 or 4 granulocytopenia on day 14, none experienced episodes of neutropenic fever, and there was no evidence of severe haematological toxicity upon recycling. Only 9% of the patients suffered from gastrointestinal toxicity (grade 3); increased but reversible transaminase levels were observed in 11.6%. In conclusion, the results of this phase II study show that the combination of gemcitabine and vinorelbine is active and well tolerated in NSCLC, and thus encourage its use in elderly or unfit patients. \u00a9 2000 Cancer Research CampaignCisplatin-based combinations are efficacious in increasing the overall survival of patients with non-small cell lung cancer (NSCLC), but their toxicity makes them unsuitable for elderly and unfit patients. The primary objective of this non-randomized phase II study was to evaluate the feasibility and activity of the gemcitabine plus vinorelbine combination in previously untreated elderly and/or unfit patients with measurable stage III or IV NSCLC. Forty-three patients aged \u2265 65 years or with contraindications against cisplatin treatment  received intravenous (i.v.) gemcitabine 1000 mg m"}
+{"text": "H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells. IGF2) gene plays a role in the pathogenesis of the overgrowth disorder Beckwith-Wiedemann syndrome , the growth-restriction condition Silver-Russell syndrome , as well as various human cancers including Wilms tumour, rhabdomyosarcoma, hepatoblastoma, colorectal and breast carcinomas Aberrant imprinting of the Insulin-like growth factor 2  H19 DMR) is located 5\u2032 of the H19 promoter is methylated on the paternal chromosome and is known as the imprinting control region IC1 because it functions as an insulator mediated by the CCCTC binding factor (CTCF) Igf2 allele and in down-regulation of H19Igf2 exists such that deletion or mutation within the IC1 also results in methylation changes in DMRs within the Igf2 gene Igf2 DMRs in a mutually exclusive and parent of origin specific manner influenced by CTCF binding and DNA methylation IGF2 DMRs in mouse and human. Specifically, IGF2 DMR0, located 5\u2032 to the main IGF2 promoters is methylated on the maternal allele only in placentas in the mouse IGF2 allele was inferred using cell lines derived from a BWS patient with paternal uniparental disomy at 11p15.5 loci (UPD) IGF2 DMR0 has been carried out in many cancer studies following the demonstration that hypomethylation of this DMR is associated with loss of imprinting in Wilms tumour IGF2 DMR0 plays a role in reactivation of the silent maternal allele Mouse models have been used to demonstrate that the imprinted expression of the closely linked IGF2 and biallelic silencing of H19. Another group of BWS patients has normal IC1 methylation but abnormal methylation at IC2, a maternally methylated imprinting control region within the KCNQ1 and KCNQ1OT1 gene cluster which seems to operate independent of IGF2 and H19IGF2 and biallelic activation of H19The molecular basis of BWS is heterogeneous with 5% of patients exhibiting gain of methylation at IC1 with biallelic activation of IGF2 DMR0 region in BWS and SRS patients and it is not known to what extent the proposed silencing function of this DMR contributes to the aetiology of BWS and SRS. We therefore examined parent of origin methylation at the DMR0 in various subtypes of BWS and SRS patients and also in a cohort of Wilms tumour samples. Our results indicate that the active paternal IGF2 allele is methylated at the DMR0 in cis with H19 methylation in normal individuals and that in SRS and BWS the DMR0 and IC1 have similar methylation abnormalities. In contrast, in Wilms tumour samples DMR0 methylation is negatively correlated with IC1 methylation. These results imply IGF2 imprinting defects in congenital growth disorders and Wilms tumours arise through different epigenetic mechanisms.To date there have been no reports on the methylation status of the trans or that methylation at the DMR0 is on the paternal allele.We examined the methylation status of DMR0 in peripheral blood leukocyte DNA derived from BWS patients with IC1 hypermethylation (n\u200a=\u200a 7), IC2 hypomethylation (n\u200a=\u200a37) or normal methylation at both IC1 and IC2 (n\u200a=\u200a27) and SRS patients with IC1 hypomethylation (n\u200a=\u200a 2), using bisulphite and pyrosequencing analysis. The region analysed is shown in IGF2 DMR0 in some of our BWS and SRS patients and 6 control individuals in order to confirm our findings. The patients with maternal duplication had reduced methylation levels (44%), while all the paternal UPD and the paternal duplication patients had hypermethylation , cis with methylation at the IC1  or the paternal allele fails to become methylated and is silenced (SRS). These allele-specific methylation changes have the effect of an imprint switch so that both alleles look like either a paternal allele (BWS) or a maternal allele (SRS). Pyrosequencing results were verified by bisulphite sequencing  and two SRS patients with maternal 11p15.5 duplications. We also determined directly the parental origin of methylation at the  the IC1 . These rquencing .We analysed DMR0 methylation in two Wilms tumour patients that had arisen in individuals affected by BWS and a series of non-syndromic Wilms tumour patients. One of the BWS patients had constitutional hypermethylation at IC1, the other had paternal UPD. The non-syndromic Wilms tumour patients comprised of individuals that had tumour-specific IC1 hypermethylation (n\u200a=\u200a10), 11p15.5 LOH with loss of the maternal allele (n\u200a=\u200a13) or normal methylation (n\u200a=\u200a10) at the IC1. Remarkably, all tumours with hypermethylation at IC1 and most of those with LOH, including those arisen in individuals affected by BWS, showed reduced methylation at DMR0 , while those with normal IC1 methylation had normal DMR0 methylation  . Indeed,IGF2 gene in Wilms tumour samples with and without loss of imprinting MspI/ HpaII restriction analysis , while others have reported exceptions in ovarian and bladder cancer During tumorigenesis global hypomethylation occurs in repeat rich regions, LINE and SINE elements, while CpG islands associated with promoters are subject to hypermethylation IGF2-H19 locus and associated with imprinting loss in BWS, SRS and Wilms Tumour. In the growth disorders, germline allele-specific methylation changes have the effect of an imprint switch so that both alleles look like either a paternal allele (BWS) or a maternal allele (SRS) while in cancer both parental marks are lost and reprogrammed. Our findings demonstrate that loss of IGF2 imprinting is a complex phenomenon that occurs with different mechanisms in human disease, probably reflecting different molecular causes and responses.In summary, we show three different reprogramming events occurring at the http://www.geneclinics.org). The study also involved 40 Wilms Tumour patients enrolled by Paediatric Oncology Units affiliated to Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). All tumours were histologically diagnosed.85 BWS and 3 SRS cases were recruited from different Italian Pediatric Departments and were clinically diagnosed according to the criteria described in the literature  which included six CpGs, three of which were previously reported to be hypomethylated in colorectal patients with LOI at IGF25\u2032TGAGGATGGGTTTTTGTTTGGTAT3\u2032 and biotinylated reverse primer 5\u2032TCCTCAATCCACCCAAAATAATAT3\u2032. 10 uL of the biotinylated PCR product was used for each sequencing assay using the following sequencing primers 5\u2032GGGGTGGAGGGTGTA 3\u2032 and 5\u2032-AAAAGTTATTGGATATATAGT 3\u2032. Pyrosequencing was carried on PSQ HS 96 System and PyroMark MD System using Pyro Gold Reagent kits . Allele-specific pyrosequencing was carried out by replacing the above biotinylated primer with allele-specific biotinylated primers, 5\u2032CCCAAAATAATATCTATAAAAAAAAAATTCAC3\u2032, or 5\u2032CCCAAAATAATATCTATAAAAAAAAAATTCAT3\u2032 which recognized the G or A allele of the rs3741210 polymorphism on the reverse strand after bisulphite conversion.We designed a standard pyrosequencing assay for Methylation was quantified using Pyro Q-CpG Software  that calculates the ratio of converted C's (T's) to unconverted C's at each CpG and expresses this as a percentage methylation. Average methylation across the DMR0 for all 6 CpGs were calculated. Median and IQR methylation for different categories of patients were analysed using Prism Graphpad software.We verified allele-specific methylation by conventional bisulphite sequencing of cloned PCR products in all our informative cases.Figure S1Bisulphite sequencing analyses of IGF2 DMR0 Methylation in normal, congenital growth disorders and Wilms Tumour. IGF2 DMR0 Methylation in normal, congenital growth disorders and Wilms Tumour. Methylation of 6 CpGs was determined by bisulphite genomic sequencing on DNA extracted from peripheral blood leukocytes  or tumour tissue (Wilms Tumour) of individuals informative for the rs3741210 polymorphism. Filled circles represent methylated CpGs and open circles unmethylated CpGs. The maternal (MAT) and paternal (PAT) alleles of the IGF2 DMR0 region are indicated.(1.33 MB TIF)Click here for additional data file."}
+{"text": "Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomerase repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity plays a role in carcinogenesis and immortalization. In this work, the status of telomerase activity during the development of human thyroid cancer was determined using telomeric repeat amplification protocol (TRAP) in 14 nodular hyperplasia, 14 adenomas, 23 papillary carcinomas and 11 follicular carcinomas. Positive telomerase activity was detected in 2 of 14 nodular hyperplasias (14%), 4 of 14 adenomas (29%), 12 of 23 papillary carcinomas (52%) and 10 of 11 follicular carcinomas (91%). The cancers that are negative for telomerase activity are mostly in early stage (stage I or II). These results suggest that telomerase reactivation plays a role during the development of thyroid cancer."}
+{"text": "Experimental evidence for this mechanism in urinary bladder, ureter, gut, lung, uterus, tooth-pulp and tongue is reviewed. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by agents that interfere with mechanosensory transduction in the organs considered, including P2X A schem3 homomeric and P2X2/3 heteromeric receptors on subepithelial sensory nerves initiating impulses in sensory pathways to pain centres in the central nervous system (CNS) )3 and/or P2X2/3 receptors on subepithelial sensory nerve terminals to relay nociceptive messages via sensory ganglia and spinal cord to pain centres in the CNS.Compelling evidence has been presented for the role of purinergic mechanosensory transduction where ATP, released from epithelial cells lining the bladder, ureter and gut during distension, acts on P2X3 and P2X2/3 receptors are being explored to treat visceral pain and the possibilities for development of agents that inhibit ATP transport from epithelial cells or enhance ATP breakdown after its release are discussed.Antagonists to P2XThe author declares that he has no competing interests."}
+{"text": "The parental conflict hypothesis predicts that the mother inhibits embryo growth counteracting growth enhancement by the father. In plants the DNA methyltransferase MET1 is a central regulator of parentally imprinted genes that affect seed growth. However the relation between the role of MET1 in imprinting and its control of seed size has remained unclear. Here we combine cytological, genetic and statistical analyses to study the effect of MET1 on seed growth. We show that the loss of MET1 during male gametogenesis causes a reduction of seed size, presumably linked to silencing of the paternal allele of growth enhancers in the endosperm, which nurtures the embryo. However, we find no evidence for a similar role of MET1 during female gametogenesis. Rather, the reduction of MET1 dosage in the maternal somatic tissues causes seed size increase. MET1 inhibits seed growth by restricting cell division and elongation in the maternal integuments that surround the seed. Our data demonstrate new controls of seed growth linked to the mode of reproduction typical of flowering plants. We conclude that the regulation of embryo growth by MET1 results from a combination of predominant maternal controls, and that DNA methylation maintained by MET1 does not orchestrate a parental conflict. In flowering plants, meiosis is followed by the production of haploid structures, the male pollen and the female embryo sac, each containing two gametes. After double-fertilization, the female gametes, the egg cell and central cell, respectively give rise to the embryo and its nurturing annex, the endosperm. The embryo and the endosperm develop within the maternally derived seed integuments. Seed size is controlled primarily by interactions between the endosperm and integuments MET1, using a dominant antisense construct, MET1a/sMET1a/s causes an increase of seed size whereas paternal inheritance has an opposite effect. MET1 is a key player in the control of parental genomic imprinting, which restricts gene expression from one of the two parental alleles Arabidopsis, it was proposed that MET1 controls the expression of two pools of imprinted genes: maternally expressed inhibitors and paternally expressed enhancers of endosperm growth Arabidopsis two imprinted genes dependent on MET1 have been identified FWA and FERTILIZATION INDEPENDENT SEED 2 (FIS2) in the male gametes FIS2 and FWA are expressed in the female central cell FIS2 and FWA are expressed in the endosperm from their maternal allele, while MET1 maintains silencing on the paternal allele FIS2 and FWA as imprinted genes.The parental contributions to seed size were identified in crosses involving diploid and tetraploid plants. Tetraploid mothers produced smaller seeds when crossed to diploid fathers, however tetraploid fathers crossed to diploid mothers produced larger seeds MET1a/s were mediated by removal of silencing of the paternal allele of endosperm growth inhibitors, thus causing seed size increase and vice versa MET1a/s has a dominant effect, which does not allow distinguishing whether seed size variations in wild type (wt)\u00d7MET1a/s crosses originated from the loss of MET1 in the previous parental generation (sporophyte) or in the haploid generation producing the gametes (gametophyte). In addition, MET1a/s lines accumulate epimutations met1-6met1 during male gametogenesis reduces seed size. This result was also in agreement with the demonstration of a gametophytic effect of met1-3 on the silencing of the paternal alleles of the imprinted genes FIS2 and FWAmet1-6 on seed size remained unclear met1-6 loss of function on the diploid parental sporophytic generation was not tested explicitly. To address these concerns, we restricted our analysis to homozygous and heterozygous mutants derived from a self-fertilized heterozygous met1-3/+ mother and compared the effects on seed development of met1-3 loss of function during male gametogenesis, female gametogenesis and the parental diploid generation.It was expected that the contrasting effects of met1-3 causes a loss of DNA methylation in first generation homozygous plants met1 function is caused by a T-DNA insert linked to a gene conferring resistance to the herbicide BASTA. To confirm specific parental contributions of met1-3 to seed size, we analyzed digital images of seeds from crosses that varied MET1 genotype and parent of transmission   , Table 1met1-3, seeds from wt\u00d7met1-3/+ crosses were visually sorted according to their size relative to a wild type control, and BASTA resistance associated to met1-3 was tested. Two populations of seeds were distinguished. All smallest seeds were resistant to BASTA (n\u200a=\u200a323) while all largest seeds were sensitive to BASTA (n\u200a=\u200a336). The 1\u22361 proportion supported the predicted association of the paternal effect of met1-3 to gametogenesis (p\u200a=\u200a0.6126 \u03c72). As we did not analyze the entire population we may have missed a complex genetic component regulating seed size. To ensure that abnormally small seeds or seed lethality were not missing from our bulked seed population, we analyzed all seeds from single crosses between wild-type mothers and pollen from met1-3/+ plants  demonstrating that paternal inheritance of met1-3 causes seed size reduction as a result of the loss of MET1 activity during male gametogenesis. The loss of MET1 during male gametogenesis may allow paternal expression of imprinted growth inhibitors and cause a decrease of endosperm and seed size. Loss-of-function paternal effects are uncommon and until now have only been linked to defects in fertilization in DrosophilaC.elegansArabidopsismet1-3 causes a paternal effect associated with defects after fertilization and thus representing a distinct class of paternal effect mutations.To confirm the link between the small seeds and paternal inheritance of + plants , Table 2MET1a/s pollen caused a decrease of seed size, a symmetrical increase of seed size was observed in seeds from the reciprocal crosses MET1a/s\u00d7wt. met1-3 from met1-3/+ mothers would increase size in 50% of the seeds. Crosses between ovules from met1-3/+ plants and wild-type pollen did exhibit increased seed size relative to wild type controls . This is contrary to the expected consequence of a maternal gametophytic effect of met1-3/+, which should produce a greater proportion BASTA resistance among the largest seeds in a population derived from met1-3/+\u00d7wt crosses. To confirm this finding we compared BASTA resistance and seed size in an entire population of seeds from met1-3/+\u00d7wt crosses from a single plant. We observed that larger seeds did not always inherit the met1-3 allele and the means of size measurements did not differ between seed genotypes  was supplied by J. Finnegan met1-3 (Col) was supplied by J. Paszkowsky and contains a TDNA insert conferring resistance to BASTA met1-3 line was maintained as heterozygous by repeated backcrosses to wild-type plants in order to avoid accumulation of epigenetic defects. Once allowed to self, the resulting segregating homozygous plants were used for emasculation for crosses to wild-type plants and for observation of autonomous development.The wild-type control lines C24 and Col were supplied by the ABRC stock center. The line Plants were grown at 22 C and 60% hygrometry in short days (16 h night) for three weeks followed by long days (8 h night) in Conviron Growth chambers.Developing seeds were cleared with Hoyer's medium and observed with DIC optics with a Leica microscope (DM600). Images were recorded with a Snapshot camera and processed with Metamorph for morphometric measurements. For confocal microscopy, material was prepared and observed as described previously met1 we performed a series of four experiments. We produced crosses between wild type and met1-3/+ plants grown in the same conditions and obtained two populations of 900 seeds with inheritance of met1 from the mother or from the father. We visually separated seeds according to size categories in each population and tested BASTA resistance in a subset representing the largest or smallest seeds. In a second series of crosses we produced crosses between wild type emasculated plants and wild-type or met1-3/+ plants or met1-3/met1-3 plants grown together. The seeds obtained were imaged and seed size was measured as detailed below and the data are reported in In order to evaluate the relationship between seed size and parental inheritance of 2 test (\u03c72). Finally, we used the Kalmagorov-Smirnoff Normality test (K-S) to determine whether seed size phenotypes fit a normal distribution based on comparison to a generated ideal normal distribution of similar mean and standard deviation. Calculations were performed using StatView 5.0.1 , except for \u03c72, which was calculated on Excel\u00d7(Microsoft). p-values provided in the text are followed by the abbreviation of the test used.To determine seed area and height, digital images of seeds on a white background were thresholded in Adobe Photoshop CS2  and at 6 DAP  in wild-type seeds , in seeds resulting from crosses between wild-type ovules and pollen from met1-3/+ plants . Scale bars represent 20 \u00b5m  and 50 \u00b5m . Cytological observations were performed to establish the origin of the reduction of seed size caused by paternal inheritance of met1. The final seed size depends both on the extent of cell proliferation in the embryo and on the degree of endosperm growth during the early phase of seed development 1 to 4 Days After Pollination (DAP). Until the late heart stage we did not observe any reduction of cell proliferation in the embryo of seeds, which inherit met1 paternally or maternally. Patterns of embryo development did not show obvious modifications and met1/+ embryos were viable. In contrast seeds which inherited met1 paternally showed a reduction of endosperm size as early as the beginning of the embryo globular stage. Early endosperm development is characterized by a series of nuclei divisions, not followed by cell divisions leading to a syncytium. The frequency and number of syncytial divisions were not altered by paternal inheritance of met1 and both smaller met1/+ seeds and larger wild-type seeds contained approximately 100 nuclei as expected at 3 DAP. The difference in size between the two populations of seeds increased during development leading to two easily distinguishable classes.(0.49 MB DOC)Click here for additional data file.Figure S2Parental effect of met1-3/+ ovules crossed to wild-type pollen on seed size during seed development, correlated with BASTA resistance (R) or sensitivity (S).(1.73 MB PDF)Click here for additional data file.Figure S3Autonomous development of fruit and seed in MET1a/s. (a) Increase of fruit elongation in MET1a/s plants in absence of fertilization 7 Days After Emasculation (DAE). (b) Autonomous development of ovules in met1a/s silique in absence of fertilization (7 DAE). (c) Cleared autonomous seed from MET1a/s plant 7 DAE show remnants of the central nucleus and the egg cell. (d) Wild-type seed (7 DAE). Bars represent 0.8 mm (a), 0.5 mm (b), and 40 \u00b5m .(0.94 MB PDF)Click here for additional data file."}
+{"text": "Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte hypertrophy. However, experimental perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. Thus, it has not been possible to conclude whether the effects on chondrocyte differentiation are cell autonomous or whether they are mediated by Dlx expression in adjacent tissues, notably the perichondrium. To address this question we first engineered transgenic mice in which Dlx5 expression was specifically restricted to immature and differentiating chondrocytes and not the perichondrium. Col2a1-Dlx5 transgenic embryos and neonates displayed accelerated chondrocyte hypertrophy and mineralization throughout the endochondral skeleton. Furthermore, this transgene specifically rescued defects of chondrocyte differentiation characteristic of the Dlx5/6 null phenotype. Based on these results, we conclude that the role of Dlx5 in the hypertrophic pathway is cell autonomous. We further conclude that Dlx5 and Dlx6 are functionally equivalent in the endochondral skeleton, in that the requirement for Dlx5 and Dlx6 function during chondrocyte hypertrophy can be satisfied with Dlx5 alone.The axial and appendicular skeleton of vertebrates develops by endochondral ossification, in which skeletogenic tissue is initially cartilaginous and the differentiation of chondrocytes via the hypertrophic pathway precedes the differentiation of osteoblasts and the deposition of a definitive bone matrix. Results from both loss-of-function and misexpression studies have implicated the related homeobox genes  The adult vertebrate skeleton appears to be a rather uniform bony tissue. This apparent uniformity belies its embryonic origins, as a mosaic structure originating from diverse progenitors that arise in different germ layers, and its development via two distinct ontological processes: intramembranous and endochondral ossification Dlx homeobox genes encode nuclear transcription factors Dlx5 and Dlx6 are expressed in all anlagen of the endochondral skeleton. Their expression has been noted in precartilaginous limb bud mesenchyme Sox9Dlx5 and Dlx6 are expressed in the post-mitotic prehypertrophic and hypertrophic zones but not in immature chondroblasts in the resting or proliferating zones Dlx5 and Dlx6 are also expressed in the perichondrium/periosteum in the long bones as well as ribs and vertebrae \u2212/\u2212Dlx5 and doubly deficient \u2212/\u2212Dlx5/6 mice have revealed requirements for Dlx5 and Dlx6 during chondrogenesis Dlx5 or Dlx6 alone in chicken limb bud micromass cultures stimulated chondrogenesis Dlx5 in vivo reduced proliferation of epiphysial chondrocytes and resulted in precocious chondrocyte hypertrophy Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte differentiation. However, to date, perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. This general concern is particularly germane when seeking to elucidate a cell-autonomous function for Dlx5 in chondrocyte hypertrophy given endogenous Dlx5 expression in both the differentiating chondrocytes and in the perichondrium, the site of synthesis of secreted factors that regulate this process in the chondrogenic core. Here, we first describe a transgenic line of mice in which exogenous Dlx5 expression is targeted to immature chondrocytes using regulatory elements from the Col2a1 gene. Tissue-specific misexpression of Dlx5 accelerated chondrocyte hypertrophy and promoted precocious ossification in the endochondral skeleton of these transgenic mice. Visualization of transgene expression in the absence of endogenous Dlx5 expression  indicated that transgene expression was limited to the chondrogenic core and not the perichondrium. The subsequent rescue of endochondral ossification defects in \u2212/\u2212; Col2a1-Dlx5Dlx5/6 mice therefore establishes a cell-autonomous function for Dlx5 during chondrocyte hypertrophy and, furthermore, demonstrates functional equivalence of Dlx5 and Dlx6 in the endochondral skeleton.Dlx5 and its cis-linked paralogue Dlx6 function as positive regulators of both chondrogenesis and chondrocyte hypertrophy in the endochondral skeleton Dlx5 during chondrocyte hypertrophy in vivo, we generated transgenic mice in which a Dlx5 cDNA was expressed under control of the promoter and intron 1 enhancer of the Col2a1 gene  had a variable number of copies of the transgene, as measured by semi-quantitative PCR from genomic DNA  or P1 after staining cartilage and mineralizing bone with alcian blue and alizarin red respectively. All three dead founders, or viable neonates bearing the t19Col2a1-Dlx5 allele, examined this way had a common phenotype of hypermineralization of the endochondral skeleton  at two developmental stages and is shown, along with an explanation of the scoring system, in t1/+Col2a1-Dlx5 founder, this hypermineralization was so excessive that the supraoccipital and exoccipital bones were completely fused, as were the first two cervical vertebrae and there was hypermineralization in the chondrocranium surrounding the interparietal bone  showed precocious ossification between these centers in the form of narrow bridges of mineralized tissue that joined the ossified centrum and neural arch. This precocious ossification was further advanced in t19/t19Col2a1-Dlx5 homozygotes (n\u200a=\u200a9) such that the majority of centra were completely fused to the adjacent neural arches , lateral , and ventral . Embryonic vertebrae have discrete ossification centres in the centrum and the neural arches, which normally fuse post-natally. The vertebrae of P1 l arches . The oveenotypes , resultienotypes .Col2a1-Dlx5 neonates were mildly affected. t19/+Col2a1-Dlx5 limbs were indistinguishable from wild type in both overall size and in the extent of mineralization. The limbs of homozygous t19/t19Col2a1-Dlx5 neonates averaged slightly smaller than wild type littermates  in which the long bones were shorter and thicker than non-transgenic controls, particularly in the hindlimb, and the relative extent of mineralization was more advanced in the femur compared to wild type controls . Thereafter, the initiation of mineralization in the centrum at a specific axial level in wild type embryos was somewhat variable such that, by E15.5, the number of vertebral centra in which mineralization had commenced was quite disparate among littermates of the same genotype, varying from zero to twelve . However, no significant difference in the extent of ossification in the long bones of the limbs was apparent up to P0 . Thus, expression of Dlx5 in somitic cartilage did not appear to affect the initial timing of mineralization, but rather contributed to a dose-dependent acceleration of ossification once initiated.We next asked whether the timing of onset of mineralization was affected in o twelve . While t however . We also however . By E17.l arches . In contly fused . We alsot19/+Col2a1-Dlx5 transgenic neonates confirmed that mineralization had occurred throughout the vertebrae, whereas the ossification centres of the centrum and neural arches of wild type pups were separated by blocks of cartilaginous tissue that contained both radially flattened and hypertrophic chondrocytes  and hypertrophic (Col10a1) markers was expanded in the limbs and vertebrae of t19/+Col2a1-Dlx5 embryos compared with wild type littermates  with heterozygous +/\u2212Dlx5/6 mice (C57Bl/6 x DBA background) to specifically reconstitute Dlx5 function in the cartilage of otherwise Dlx5/6 null embryos. \u2212/\u2212Dlx5/6; t19/+Col2a1-Dlx5 neonates showed a rudiment of a mineralized element of varying size, located dorsal to the exoccipital bones, which was interpreted to be supraoccipital in identity  and was rescued to wild type levels in hemizygous Col2a1-Dlx5 embryos while complete rescue of the supraoccipital bones required homozygosity of the transgene in a Dlx5/6 null background. Nonetheless, it is quite likely that some functions of the Dlx5-Dlx6 locus will depend on diverged protein functions; some genes behave differently following loss of Dlx6 versus Dlx5 in the mandibular arch, for example Dlx5, Runx2 (encoding two isoforms that differ at their amino-termini) is a multifunctional regulator of both chondrocyte and osteoblast differentiation Dlx5, Runx2 is expressed in cartilaginous condensations and later, during long bone growth, both genes are expressed in prehypertrophic and hypertrophic chondrocytes. In addition, Dlx5 and Runx2 are expressed in the perichondrium flanking the prehypertrophic and hypertrophic zones and, in both cases, perichondrial expression extends over the proliferating zone . In common with Runx2 then, Dlx5 accelerates chondrocyte differentiation but, in contrast, while Runx2 is capable of initiating chondrocyte hypertrophy de novo, Dlx5 is unable to induce ectopic chondrocyte hypertrophy in tissues where it would not normally occur, nor advance the initial timing of mineralization. Thus, it is likely that Dlx5 requires the synthesis of other rate-limiting critical cofactors for its hypertrophic function. This is an interesting situation given that Dlx5 appears to be a direct upstream regulator of Runx2 during osteoblast differentiation Runx2 in chondroblasts. Indeed, the signals and upstream transcriptional regulators of Runx2 in chondrocytes are not yet known.Like ing zone . As thisDlx5 expression is not without precedent; non-uniform effects of Col2a1-driven transgenes in the endochondral skeleton have been a hallmark of other studies too. For example, the supraoccipital bone and vertebral skeleton was more sensitive to the effects of a dominant negative PTHrP receptor than the limbs, which only exhibited an effect at the highest number of transgene copies and then only in discrete elements Dlx5 and other regulators of cartilage development no doubt reflects differences in the molecular milieu of anatomically distinct chondrocytes and further underscores the mosaic nature of the skeleton.The differential sensitivity of the appendicular and axial skeleton to a given level of ectopic Prior institutional approval was received for the animal work described in this study from the University of Guelph Animal Care Committee, in accordance with Canadian Council on Animal Care guidelines.Dlx5 open reading frame was amplified as a 5\u2032 Flag-tagged HindIII-NcoI fragment, and shuttled into a modified pSlax13bpA) sequence from p3000i3020Col2\u03b11Dlx5 as an NcoI-XbaI fragment, and the FlagDlx5-bpA cassette was cloned back into p3000i3020Col2\u03b11 as a HindIII-SalI fragment, replacing the \u03b2geo-bpA sequences. The construct was digested with NotI and SalI and the linear transgene cassette was microinjected into the pronuclei of fertilized CD-1 oocytes. Genotyping of transgene bearing mice was done with forward primer 5\u2032-AACAGTTCCCCGAAAGAGGT-3\u2032 and reverse primer 5\u2032-GAGCGCTTTGCCATAAGAAG-3\u2032, which amplified a 1040 bp fragment. The t19Col2a1-Dlx5 allele was maintained in a hemizygous state on a CD-1 background; hemizygous animals were occasionally bred to generate homozygous embryos or neonates. Offspring from t19/+Col2a1-Dlx5 x +/\u2212Dlx5/6 crosses were backcrossed to Dlx5/6 heterozygotes to generate transgene-positive, Dlx5/6 null mice. A 438 bp genomic Ihh fragment was amplified with primers: 5-CACTTGTGGTGGAGGATGTG-3\u2032 and 5\u2032-TACCACACGCTTGTCAGCTC-3\u2032. Dlx5/6 heterozygotes were genotyped with the lacZ primer pair: 5\u2032-GCGTTACCCAACTTAATCG-3\u2032 and 5\u2032-TGTGAGCGAGTAACAACC-3\u2032FlagDlx5 and \u03b2-actin mRNA was determined on total RNA prepared from E16.5 embryos using the Trizol Reagent (Invitrogen). 1 \u00b5g of RNA was reverse transcribed  and PCR amplified  with standard protocols. Primer sequences used for RT-PCR were: FlagDlx5 (For 5\u2032-GACTACAAGGACGACGATGAC-3\u2032 and Rev 5\u2032-GAGCGCTTTGCCATAAGAAG-3\u2032) and \u03b2 -actin .The murine 2O/glycerol to 100% glycerol.Alcian Blue and Alizarin Red staining was used to visualize cartilaginous and mineralised skeletal tissues respectively of embryos and neonates. Briefly, eviscerated and skinned (>E16.5) bodies were fixed in 95% ethanol over several days. Embryos were stained in 80% ethanol, 20% acetic acid, 0.01% Alcian blue 8GX for 2\u20134 days, cleared overnight in 1% KOH, stained 1\u20132 days in 0.001% Alizarin Red S in 1% KOH, then rinsed in 1% KOH, and a graded series of HTissues were fixed overnight in 4% paraformaldehyde then dehydrated and embedded in paraffin using standard techniques. 7 \u00b5m sections were stained with hematoxylin and eosin using a standard protocol.in situ hybridisation was done as described previously Dlx5, a 0.87 kb BamHI \u2013 HindIII fragment corresponding to the full length open reading frame Col2a1, a 0.4 kb cDNA corresponding to nt 1\u2013402 of Genbank X57982; Ihh, a 1.8 kb partial cDNA EcoRI fragment Col10a1, a 0.86 kb ApaI - SalI fragment containing nt 1351\u20132215 of Genbank X65121.Whole mount and cryosection Images were taken using a MicroPublisher colour digital camera on a Leica MZ12.5 Stereomicroscope with Qcapture software (QImaging) or on a Leica DMRA2 upright microscope with Openlab software (Improvision) and processed using Adobe Photoshop."}
+{"text": "Forty fixed tissue sections from patients with small cell lung carcinoma (SCCL) have been stained with a panel of 10 monoclonal antibodies using a peroxidase anti-peroxidase method and the incidence of staining has been compared to patient characteristics at presentation and to survival. An inverse association between HMFG2 staining and survival was found with median survival in HMFG2 negative patients 13 months compared to 8 months for HMFG2 positive patients. No such association was found with the other antibodies and no association was found between staining and disease extent or primary versus secondary deposits with this panel of antibodies. Epidermal growth factor receptor was detected in 3/38 presentation biopsies and in these 3 patients mean survival was only 5 months. Further prospective study of HMFG2 as a prognostic indicator in SCCL is suggested."}
+{"text": "Rotavirus G10P[11] strains have long been associated with asymptomatic neonatal infections in some parts of India. We have previously reported G10P[11] strains associated with both asymptomatic infections and severe gastrointestinal disease in neonates from Vellore in southern India, with >90% partial nucleotide and amino acid identity to the VP4, VP6, VP7 and NSP4 genes of the exclusively asymptomatic G10P[11] strain I321.In this study, the whole genome of a G10P[11] isolate (N155) from a neonate with severe gastrointestinal disease was characterized to determine whether there were significant differences in its genetic makeup in comparison to G10P[11] strain I321 and to establish the origin of the G10P[11] strains in Vellore.PCR amplification and complete genome sequencing was carried out for all 11 gene segments of symptomatic G10P[11] rotavirus isolate N155. Nucleotide and amino acid sequence similarity with I321, other human and bovine strains for each gene segment were determined. The origin of each gene was determined based on the degree of identity to bovine or human rotavirus strains.N155 was found to be a reassortant between human and bovine rotaviruses. With the exception of NSP2, gene sequences of strain N155 showed >90% identity to published sequences of I321. Gene segments encoding NSP1, 2 and 3 were of human rotavirus origin for both strains; however, phylogenetic analysis of NSP2 sequences indicated that the human parental strain that led to the origin of these bovine-human reassortant strains was different. There were no significant differences between NSP2 sequences of strains from symptomatic and asymptomatic neonates in the same setting.The study shows that the difference in clinical presentations in neonates may not be due to the limited variability in the genome sequence of G10P[11] strains and that G10P[11] strains in different parts of India could have evolved through reassortment of different parental strains. An abdominal radiograph revealed dilated bowel loops and NEC. The baby was given intravenous fluids and kept nil oral for three days after which time feeds were initiated and well tolerated. The sample was screened for other enteropathogens, including Norovirus genogroups I and II, enteric adenovirus, 3.3http://frodo.wi.mit.edu/) and BioEdit softwares (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) based on corresponding genes sequences available in GenBank. Primer pairs were designed to amplify short fragments of each gene ranging from 387 to 1355\u00a0bp, with each fragment overlapping the next one by about 100\u00a0bp. RT-PCR was carried out with an initial denaturation at 95\u00a0\u00b0C for 5\u00a0min and 35 cycles of amplification using 5U of Taq polymerase (Invitrogen). High-fidelity Taq polymerase (Invitrogen) was used for the amplification of three genes  whose amplicon sizes were >1000\u00a0bp. The final sequence was determined by assembling consensus genome sequences from all fragments for the gene. The primer sequences used for sequencing the beginning and end of each gene in this study are given in RNA extraction and random priming reverse transcription for cDNA synthesis was carried out as described previously. Primers for the amplification of individual genes were designed using primer3 (3.44-TTCCTTATGCAGCTGATCACTCTGTGTCA-C6-NH2-3\u2032) to the 3\u2032 ends of both strands of the viral dsRNA using T4 RNA Ligase . One step RT-PCR using Superscript III RT-PCR kit  was carried out with a primer complementary to the modified amino-linked oligonucleotide (5\u2032-TGACACAGAGTGATCAGC-3\u2032) and a gene specific internal primer.The 5\u2032 and 3\u2032 sequences of the 11 gene segments were determined by using the single-primer amplification method.3.5PCR amplicons were purified using PCR cleanup filter plates  and sequenced with ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit . Sequences were resolved in the ABI PRISM 310 Genetic Analyzer . Deduced amino acid (aa) sequences were derived using the Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics (SIB). The nucleotide (nt) and deduced amino acid sequences of all genes were compared with sequences available in the NCBI  GenBank database using BLAST . Multiple alignments and phylogenetic analysis of nt and deduced aa sequences were performed using Clustal W and neighbor-joining algorithms of the BioEdit (version 7.0.5.3) or Bionumerics  software packages. Dendrograms were confirmed by neighbor-joining and maximum parsimony methods. Genetic lineages were confirmed by bootstrap values of >95% using 1000 pseudoreplicate runs.3.6EU200793 to EU200803.The nucleotide sequences of the 11 gene segments of N155 have been deposited in GenBank under the accession numbers 44.1A total of 18,242 nucleotides encoding 5781 amino acids of the eleven segments of the G10P[11] strain N155 were sequenced. The complete coding sequences (cds) were determined for all genes except VP6 and NSP3, in which the first two and three amino acids, respectively, of the open reading frames could not be ascertained.N155 sequences were compared with published sequences of corresponding genes, with origin determined based on the degree of identity to bovine or human rotavirus strains. The closest sequence match and similarity with I321, human and bovine strains for each gene of N155 is shown in 4.2The VP1, VP2 and VP3 genes of N155 were of bovine origin, with 93%, 94% and 92% identity, respectively, with bovine strains NCDV or UK. The VP6 sequence of strain N155 clustered with SGI rotavirus strains and was more closely related to I321 (97% identity at the nucleotide (nt) level) and VP6 SGI sequences derived from bovine strains than SGI sequences of common human rotavirus strains . On tran4.3The gene encoding NSP1 of N155 was 97% identical to the corresponding sequence of I321, and likely of human origin . Human rThe NSP2 encoding gene of N155 was closely related to sequences from human rotaviruses showing >95% nt and 98% aa identity with recent human isolates from Vellore and Bangladesh of common genotypes (G1P[8] and G9P[8]), and >92% with human prototype strain Wa. Only 80.9% nt identity was seen with I321 and 82.8\u201389.1% with bovine strains . PhylogeIn order to assess the role of this difference in virulence, partial nucleotide sequence analysis of the region encoding aa 15\u2013291 was carried out from 5 additional G10P[11] strains (2 symptomatic and 3 from asymptomatic infections). No significant differences were seen among NSP2 sequences of these isolates . TerminaThe NSP3 gene of N155 was similar to I321 (>95% identity at the nt level) and was highly related to human rotaviruses . Two disThe NSP4 sequence of N155 showed 95% identity with I321 and other G10P[11] strains isolated in Vellore , and werGene segment 11 of strain N155 showed 95% identity at the nt level to bovine strain VMRI .5Sequence analysis of the complete genome of strain N155 isolated from a neonate with symptomatic rotavirus infection confirms that G10P[11] strains found in humans in Vellore are bovine\u2013human reassortant strains. Eight out of 11 gene segments are likely to have originated from bovine strains. With the exception of NSP2, a high degree of identity was seen between N155 and I321 in six of seven genes for which sequence data was available for both strains. Sequence data on I321 for genes encoding VP1, VP2, VP3 and NSP5 were not available for comparison although these have been reported to be of bovine origin through whole genome hybridization studies.This study showed that the NSP2 gene of both I321 and N155 originated from human rotavirus strains, but the strains belonged to two distinct genetic lineages with <80% identity between them. These findings would suggest that independent reassortment events between human and bovine strains led to the emergence of G10P[11] strains in Vellore and Bangalore. Both strains have three genes of human rotavirus origin, but the gene encoding NSP2 clearly originated from different human parental strains. These differences however do not explain the differences in virulence, as shown through more extensive analysis of NSP2 sequences from additional G10P[11] strains from symptomatic and asymptomatic infections.Early studies on I321 had revealed strong cross hybridization of NSP1, NSP2 and NSP3 genes with human rotavirus strain Wa.Based on the whole genome characterization of G10P[11], the 3 genes of human rotavirus origin\u2014NSP1, NSP2 and NSP3 are probably essential for efficient replication of a bovine strain in a human host. The role of these genes in host restriction has previously been discussed in other studies but the data are inconclusive.Complete genome sequences are available from few strains throughout the worldNone."}
+{"text": "BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in  BRCA2 has been linked to the development of prostate cancer, although the precise role that BRCA2 dysfunction plays in the development of prostate cancer is unclear. To address this, we have generated an animal model in which the mouse Brca2 gene is specifically deleted in the adult prostate. These mice develop precancerous prostate lesions, which progress in severity and incidence with the loss-of-function of an additional tumour suppressor, Trp53. Importantly, blocking male steroidal hormone production by castration leads to partial regression of the prostate lesions, however cells continue to proliferate after androgen withdrawal. This suggests human BRCA2 mutant prostate tumours, like the majority of prostate cancers, will respond to hormone therapy, but will relapse, as frequently occurs in this disease. In summary, our model suggests that BRCA2 acts as a tumour suppressor in the prostate and provides a pre-invasive model to test novel therapeutics.In Western countries, prostate cancer is the most common male cancer and the second biggest cause of cancer-related deaths in men. Men with a familial history of either breast or ovarian cancer have an elevated predisposition to prostate cancer, suggesting there is a genetic element to this disease. Indeed, the inheritance of a mutated form of the breast cancer susceptibility gene  Prostate cancer is the most common cancer in men in developed countries, with a rising incidence of the disease. However, the etiology of this malignancy is still unclear. Prostate cancer progresses through a pathologically defined series of steps involving increasing grades of PIN, invasive adenocarcinoma and metastatic cancer BRCA2 predispose to both breast and ovarian cancer making it a good candidate gene for prostate cancer etiology. There is an increased risk of prostate cancer in individuals carrying a mutation in BRCA2, particularly early-onset disease BRCA2 mutation that rose to 7.33 in men under 65 years of age BRCA2 carriers account for between 0.8\u20132% of prostate cancer cases, compared with the prevalence of 0.1% BRCA2 mutations in the general population BRCA2 mutation carriers have been associated with aggressive prostate cancer Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility BRCA2 mutations frequently demonstrating loss-of-heterozygosity with loss of the wild-type allele. BRCA2 plays an important role in the repair of DNA double-strand breaks (DSB) through homologous recombination (HR) BRCA2-deficient cells form chromosomal aberrations spontaneously in culture and are more sensitive to certain DNA damaging agents BRCA2 is thought to principally lead to tumour progression by the failure to repair DNA by HR, leading to genomic instability.BRCA2 is thought to act as a tumour suppressor, with tumours arising from in vivo tumour suppressor role for Brca2 in the mammary gland and have demonstrated a synergistic tumour suppressor activity with Trp53. However, Brca2 heterozygous animals do not show a predisposition to tumour formation and Brca2 null mice result in embryonic lethality Cre-LoxP system has been used to conditionally delete Brca2 in a tissue-specific manner. Deletion of Brca2 from the mouse mammary epithelium either fails to produce mammary-gland tumours or results in mammary-gland tumour formation with long latency (1.4\u20131.6 years) Brca2 mutant mice that were Trp53 heterozygous Brca2 and Trp53 developed mammary tumours with high penetrance at 6 months Mouse models have shown a direct Brca2 in prostate cancer we have used a prostate\u2013specific Cre line and a conditional Brca2 allele to delete Brca2 in adult mouse prostate epithelia. We show that loss of Brca2 in the prostate results in focal hyperplasia and low-grade (LG) PIN. Mice with conditional deletion of Brca2 and Trp53 have a high incidence of high-grade (HG) PIN, which contain cells with elevated DNA damage. PIN lesions in Brca2;Trp53 homozygous mutant prostates persist and continue to proliferate after androgen depletion. This work confirms the role of Brca2 as a tumour suppressor in the prostate and provides a model to test potential therapeutics in Brca2-deficient prostate neoplasia.To understand the role of Brca2 in the prostate we deleted Brca2 from the adult mouse prostate epithelia. To achieve this we mated mice carrying a Brca2 allele that has exon 11 flanked by loxP sites (F/FBrca2) to transgenic mice carrying Cre recombinase under the control of a prostate-specific composite rat probasin promoter, PBCre4Cre line has been used successfully to delete tumour suppressor genes and activate oncogenes to drive prostate neoplasia and tumour progression Brca2 conditional allele results in the loss of a Rad51-interacting domain, and consequently, homozygous germline deletion leads to embryonic lethality To investigate the role of F/FBrca2), Brca2 heterozygous  and Brca2 mutant  animals were generated and analysed for tumour progression at 6 months, 10\u201314 months and 15\u201320 months of age. None of the F/+;PBCre4Brca2 prostates had any observable morphological differences compared to control prostates at any time point analysed   . LG PIN \u200a0.0001) . These fTP53 is frequently mutated in BRCA2 cancers and studies in the mouse have shown a genetic interaction between Brca2 and Trp53Brca2 and Trp53 cooperate in the prostate we deleted both of these genes in the prostate epithelia using the PBCre4 transgene. Cohorts of male control , Brca2 homozygous and Trp53 heterozygous  and Brca2 and Trp53 double homozygous  animals were generated and analysed for tumour progression.The tumour suppressor Brca2 mutants, deletion of Brca2 and Trp53 resulted in the formation of HG PIN lesions. At 10\u201314 months, F/F;Trp53F/+;PBCre4Brca2 animals had focal LG PIN and hyperplasia  . Hyperplasia and LG PIN lesions were similar to those found in F/F;PBCre4Brca2 mutants. Frequently multiple ducts of each lobe had HG PIN, which were present in proximal and distal regions of the prostate and consisted of many atypical cells filling the lumen. Atypical cells were unorganised with poor orientation, severe nuclear pleomorphism and abnormal nuclear to cytoplasm ratios  . In thesm ratios . Mitoticm ratios . In somem ratios . These ad tissue .Brca2 and Brca2;Trp53 mutant prostates. An early response to DNA damage is the phosphorylation of histone H2AX (\u03b3H2AX) Brca2 mutant and Brca2;Trp53 mutant prostates contained cells that were positive for \u03b3H2AX, which were not present in control prostates  . Notably, there was a 20 fold increase in apoptotic cells in areas of HG PIN in Brca2;Trp53 homozygous mutant prostates (2% TUNEL positive cells vs 0.1% in control) (Deletion of control) . A 4 folcontrol) . An antiF/F;PBCre4Brca2 mutant prostates (2.2% Ki-67 positive cells vs 0.4% in control) , and dramatically increased by 30 fold in F/F;Trp53F/F;PBCre4Brca2 HG PIN lesions (12% Ki-67 positive cells vs 0.4% in control) (Ki-67 is a marker of proliferating cells and a prognostic indicator in prostate cancer control) . Levels control) .F/F;PBCre4Brca2 and F/F;Trp53F/+;PBCre4Brca2 mutant prostates had increased AR expression in the cytoplasm and nucleus of epithelial cells that correlated with regions of LG PIN  expressed in the luminal epithelium usually increase in the nucleus during human prostate carcinoma progression f LG PIN . The leval cells .Brca2;Trp53 homozygous mutant HG PIN lesions frequently contained large groups of p63-expressing cells, a marker of basal cells  cells are observed in human prostates al cells . Insteadal cells . Brca2;Tal cells . These ral cells  [36].Brca2 and Trp53 to androgen ablation, we surgically castrated animals at 16 months when HG PIN lesions have already formed and analysed them 4 days post-castration. Castration of control animals resulted in normal prostate regression, with a reduction in lumen size   . HoweverF/F;Trp53F/F;PBCre4Brca2 animals resulted in AR expression in a diffuse pattern throughout most prostate luminal and stromal cells   .Brca2;Trp53 mutants that persisted after castration, compared to control castrated animals (5.5% Ki-67 positive cells vs 0.3% in castrated control)  .BRCA2 mutations have implicated this gene in prostate cancer etiology, but its function in this malignancy is unclear. We have undertaken a genetic analysis of Brca2 function in the adult mouse prostate to define its role in prostate cancer and to create an in vivo model of Brca2-dependent prostate disease progression. Our study has demonstrated that loss of Brca2 in the mouse prostate epithelium results in hyperplasia and LG PIN. These lesions have an increase in the number of cells with DNA damage and apoptotic cells, which could be the result of the impairment of DNA repair pathways. This demonstrates not only that Brca2 can play a role in the initiation of prostate neoplasia but also that other factors are required for prostate tumour progression.Studies on human carriers of deleterious Brca2 and Trp53 in mouse prostate epithelia resulted in a shorter latency and increased frequency of prostate neoplasia compared to deletion of Brca2 alone  mice and F/FTrp53 (targeting exons 2\u201310) mice ARR2PBCre transgenic mice, PBCre4Histological phenotype of samples was assessed on haematoxylin and eosin stained sections. Serial sections were then stained for immunohistochemical analysis. Histological assessment was based on published guidelines and assisted by a pathologist Ki-67 or TUNEL staining was performed by immunohistochemistry on sections and cells stained with nuclear brown DAB chromogen were counted as positive. Cells from at least 4 high power fields were counted per animal, which totalled more than 900 cells per animal. Five animals of each genotype were analysed. Randomly selected fields were counted for control analysis and sections corresponding to histologically identified areas of hyperplasia and PIN were counted for mutant animals. All values are significant with p<0.05 using Student t-test unless otherwise stated.Antibody stains were done on paraffin sections as previously described Figure S1Brca2 and Trp53 are deleted in mutant adult prostates. PCR analysis of Cre, Brca2, and Trp53 on dissected anterior prostate (AP), lateral prostate (LP), dorsal prostate (DP), and ventral prostate (VP) tissue from a F/F;Trp53F/FBrca2 (control) F/F;Trp53F/F;PBCre4and Brca2 (mutant) animal. Wild type (WT) and water (H20) are controls.(0.07 MB DOC)Click here for additional data file."}
+{"text": "Kip1p27 gene has been identified as inductor of cell cycle arrest at the G1 checkpoint to prevent entry of somatic cells into the S phase of the cell cycle when substantial DNA damage has occurred. It has been suggested that decreased expression of the p27Kip1 protein may contribute to the development of human malignancies due to loss of critical antiproliferative mechanisms. In the present study, 95 specimens (T1\u2013T4) from 95 randomly selected patients undergoing radical prostatectomy at the Urological Department of Hannover University (82 patients) as well as in the Josef Hospital Regensburg (13 patients) between 1981 and 1992 for whom tissue blocks for immunohistochemical investigation were available, were investigated for different biological and clinical characteristics as possible predictors for recurrence-free and long-term survival: age, depth of tumour infiltration, histological grade, lymph node status, as well as decreased expression of the p27Kip1 protein. After a median follow-up up of 56 months (24\u2013151 months), seven of 21 (33%) patients (Group 1) with loss of p27Kip1 protein expression or a relative amount of <10% of positively stained tumour cells developed recurrent disease in contrast to 17 of 74 (23%) patients (Group 2) with retained p27Kip1 protein expression (\u226510% of positively stained tumour cells). The median recurrence-free survival was 14 months (5\u201340 months) for patients from Group 1 and 31 months (7\u2013133 months) for Group 2 patients (P = 0.02). In multivariate analysis, loss of p27Kip1 protein expression was identified as the only independent prognostic parameter for recurrence-free survival. In contrast, neither the univariate nor the multivariate analysis showed a correlation between loss of p27Kip1 protein expression and the long-term survival of the patients. Prospective studies are urgently needed to confirm the independent prognostic value of decreased p27Kip1 protein expression together with overexpression of the p53 tumour suppressor protein in patients with localized prostate cancer. The availability of more refined prognostically important biological variables in addition to established prognostic factors like tumour stage or Gleason score might help decision making in patients at high risk for the development of local recurrence or systemic tumour progression. \u00a9 1999 Cancer Research CampaignThe"}
+{"text": "BRCA2 in sporadic breast and ovarian carcinomas are exceedingly rare. This led to the suggestion that large genomic rearrangements could be involved. We performed Southern blots in genomic DNA from 130 primary breast cancers and 83 cancer cell lines  and found no genomic rearrangements. These results suggest that a gene other than BRCA2 is the target of the frequent 13q12.3 allelic deletions in human cancers. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comMutations of"}
+{"text": "To the Editor: We read the article by Pelat et al. (The weekly queries from January 2004 through February 2009 were downloaded from Google Insights for Search (gripe (Spanish for influenza) showed a maximum correlation (\u03c1 = 0.70) 2 weeks before the declared ILI (DILI). When excluding the terms for aviar (avian) and vacuna (vaccine), the correlation peak (\u03c1 = 0.81) was likewise observable 2 weeks before the DILI. The maximum correlation observed for symptom queries was for tos (Spanish for cough) 2 weeks before the DILI (\u03c1 = 0.74); for conditions associated with influenza the correlation was for neumon\u00eda  2 weeks after the DILI (\u03c1 = 0.84). The queries for varicela (Spanish for chickenpox) showed a maximum correlation (\u03c1 = 0.96) 1 week after the declared illness, as observed by Pelat et al (The queries for In conclusion, our study points out the utility of Internet queries for the surveillance of ILI and chickenpox in Spain. In the case of ILI, this information can be used as an early warning tool used complementarily to standard surveillance systems. More detailed studies are necessary regarding the usefulness and limitations of this tool in Spain, as well as in other contexts."}
+{"text": "BRCA1 and BRCA2 has promoted the investigation of various polymorphisms in the p53 gene as possible risk modifiers in BRCA1/2 mutation carriers. Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, and it has been recently reported that a p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at the onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population. In this study, we have evaluated this association in a series of 2932 BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2.The close functional relationship between p53 and the breast cancer susceptibility genes  Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, although not all studies have yielded consistent results (p53 and the breast cancer susceptibility genes BRCA1 and BRCA2 (BRCA1/2 mutation carriers (p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population (BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) . In addition, individuals homozygous for Ins16bp were directly sequenced. The concordance rate was 100% in both instances; accordingly, the GCHBOC mutation carriers were included in all subsequent analyses. Call rates ranged between 92 and 100% across studies.Genotypes for the two polymorphisms \u2013 Ins16bp and Arg72Pro \u2013 were determined for each sample using previously described methodology (BRCA1 and BRCA2 mutation carriers separately (Haplotypes were imputed using the R-package \u2018hapassoc\u2019 . Associaparately  and all BRCA1 and BRCA2 mutation carriers. No evidence of association was found for any of the genotypes or haplotypes analysed with either breast or ovarian cancer risk, including the No Ins-72Pro haplotype, previously reported to be associated with an increased risk to develop a first primary tumour before 35 years of age in BRCA2 mutation carriers (Genotype distributions and frequencies for the Ins16bp and Arg72Pro polymorphisms are shown in To confirm that this negative result was not due to the different analytic approach performed in this study, we carried out a logistic regression analysis, as was done in the original study (p53 with an increased cancer risk in BRCA2 mutation carriers (In summary, the previously reported association of the No Ins-72Pro haplotype in"}
+{"text": "E2f4 null mice. Thus, the regulatory mechanisms maintaining quiescence are robust even to complete loss of conserved transcription factor binding events.Maintaining quiescent cells in G0 phase is achieved in part through the multiprotein subunit complex known as DREAM, and in human cell lines the transcription factor E2F4 directs this complex to its cell cycle targets. We found that E2F4 binds a highly overlapping set of human genes among three diverse primary tissues and an asynchronous cell line, which suggests that tissue-specific binding partners and chromatin structure have minimal influence on E2F4 targeting. To investigate the conservation of these transcription factor binding events, we identified the mouse genes bound by E2f4 in seven primary mouse tissues and a cell line. E2f4 bound a set of mouse genes that was common among mouse tissues, but largely distinct from the genes bound in human. The evolutionarily conserved set of E2F4 bound genes is highly enriched for functionally relevant regulatory interactions important for maintaining cellular quiescence. In contrast, we found minimal mRNA expression perturbations in this core set of E2f4 bound genes in the liver, kidney, and testes of  Quiescence of cellular proliferation is crucial for mammalian tissue homeostasis, and aberrant activation of cell cycle programs can lead to cancer Removal of key E2F components of the multisubunit complexes that control the cell cycle can cause aberrant activation of cellular proliferation in specific tissues during development and in adulthood reviewed in . For insE2f4 null mice to determine the transcriptional importance of E2f4 binding in primary mouse tissues.Despite wide fluctuations and evolutionary turnover of transcription factor binding events between mouse and human We identified the proximal promoter regions that E2F4 occupies in three primary human tissues  directly isolated from donor organs, and an asynchronous human cell line (HepG2), using chromatin immunoprecipitation and promoter microarrays representing 13,000 regions in the human genome On our array platform, we found that E2F4 binds approximately 500 to 700 human genes, depending on the tissue. Among all three quiescent primary human tissues and the proliferating HepG2 carcinoma line, we observed overlap greater than 70% and as high as 84% . This ovA substantial majority of genes bound by E2F4 are bound in most tissues; we identified a core set of approximately 450 genes common to all human tissues in our study . These i\u22124, a core set of approximately 450 genes are bound in common by E2f4 in all mouse tissues  may vary between mouse and human; a variation that may have functional implications for E2f4's role in mouse Second, a feature of the approximately fifty genes where E2F4 binding is conserved is their remarkable enrichment in cell cycle, proliferation, and DNA repair functions. This result is consistent with the hypothesis that when a transcription factor binding event is conserved, this conservation is a good indictor of a functional regulatory connection E2f4 null tissues is nevertheless surprising, despite the largely normal physiology of these mice in adulthood. It has been known since the report of viable E2f4 null mice that the function of most tissues, even those directly impacted during development by absence of E2f4, recovers. Our findings reveal that this recovery extends to the level of gene expression; it appears that the absence of E2f4 can have profound, yet remarkably transient, implications for tissue-specific transcriptional programs. This recovery almost certainly depends on the overlapping roles that other members of the E2f family can play. For instance, it has been previously shown that E2F5 can largely compensate for the absence of E2F4 in vivo We expected that removal of E2f4, as a key member of the DREAM complex would have profound implications for the correct gene expression of the genes bound by E2f4 in vivo. Instead, however, we found that developmental recovery and survival to adulthood corresponds with almost completely normal gene expression in multiple tissues, relative to their phenotypically normal heterozygous littermates. Given the substantial developmental defects described above, the subtle and limited changes in the gene expression profiles of Our results also provide direct support to the hypothesis that transcriptional binding is often neutral in nature We have used comparative genomics approaches to explore the evolutionarily conserved regulatory pathways that E2F4, a key member of the DREAM complex, uses to maintain control of gene expression programs. We find that that most genes bound by E2F4 are common to numerous primary tissues within a species. This suggests that the regulatory architecture controlling quiescence may be similar among tissues that have remarkably different capacities for re-entry into the cell cycle. Understanding this architecture will require further studies to explore how E2f4 acts within specific cell types during development. The striking and specific conservation of E2F4 binding between mouse and human at cell cycle and proliferation genes suggests that only the targets crucial for the function of the DREAM complex are under selective pressure, yet our discovery that complete removal of E2f4 has at best modest effects on gene expression programs dramatically underscores the well-known redundancy within cell cycle regulation. Our study demonstrates the power of using the conservation of transcription factor binding at orthologous mouse-human genes as a tool to identify regulatory connections that appear to be under evolutionary pressure.All chemicals were purchased from Sigma-Aldrich, and used as received unless otherwise noted. E2F4 antisera were obtained from Santa Cruz Biotechnology  and used as described in prior studies Mice used in chromatin immunoprecipitation experiments were F1 males from a B6/C57 male cross with an A/J female (Jackson Laboratories). Islets suitable for ChIP studies were isolated by standard techniques and hand picking at Joslin Diabetes Center on mixed gender mice of the same genetic background (B6/C57xA/J). Other tissues were harvested using standard techniques, soaked or perfused with 1% formaldehyde, and homogenized in neutralization buffer followed by ChIP. For expression studies, E2f4 \u2212/\u2212 homozygous and E2f4 +/\u2212 heterozygous knock-out mice were derived in a B6/C57 and 129S2/SvPas cross background Primary human hepatocytes were obtained from the Liver Tissue Procurement and Distribution Program (NIDDK contract number N01DK92310) at the University of Pittsburgh. Human pancreatic islets and pancreatic acinar tissues were the kind gifts of Gordon Weir, Abdulkadir Omer (Joslin Diabetes Center) and Nicolas Benshoff (University Minnesota) .Mouse tissues were harvested from two E2f4 \u2212/\u2212 and two E2f4 +/\u2212 heterozygous littermates limmaaffyaffyPLM package The The procedure for chromatin immunoprecipitation has been reported previously limma package. Quality of the arrays was assessed using array images of the background and foreground intensities and also of the red/green (Cy5/Cy3) ratios to check for spatial artifacts. MA-plots (log-fold change against average log-intensity for each gene) \u22124). The set of commonly bound genes was defined using the less stringent value of 10\u22123, to capture binding events falling just under the more stringent threshold in one or two tissues. To find orthologous mouse and human genes on the promoter arrays, gene symbols and accession numbers were linked to the Ensembl IDs for each species to match the two gene lists. The data in \u22124. The comparison with Litovchick ChIP data was done by comparing their published binding results to the cut-offs described in The statistical analysis was performed using the We determined the enrichment of functional categories among the bound gene sets using the GOstat tool Given the sequences of the promoters on the ChIP array, a Perl script was used to automate BLAST searches of alignment across the genome and screen the BLAST output and BioPerl was used to extract the genomic coordinates of the promoters. To interrogate for the presence of the E2F4 motif, we used the one-kilobase E2F4 bound regions plus an additional 300 bases upstream and downstream to account for the resolution of the ChIP technique limma package within R was used in the genomic microarray data analysis. Quality assessment included boxplots and images of the background and foreground intensities for both red (Cy5) and green (Cy3) channels, as well as MA-plots. All the arrays used in the analysis exhibited good quality. Expression red/green log-ratios were median-normalized, after background subtraction, within each array. E2F4 binding events were empirically assigned as minimum contiguous regions comprising, at least, one probe for which the red/green ratio was greater than 5, or two adjacent probes with ratios greater than 2.5 and a combined (summed) ratio greater than 6.5, or three adjacent probes with ratios greater than 2 and a combined (summed) ratio greater than 9. A Perl script was used to compute the genomic base pair distance between each binding event and the closest transcription start site. Transcriptomic annotation relied on tables downloaded from the UCSC Genome Browser server. The smoothed histograms in density function (default parameters) to the distance distributions and plotting the respective outcomes.Human E2F4 ChIPs in primary human liver were hybridized in duplicate to human whole chromosome 21 microarrays; mouse E2f4 ChIPs in primary mouse liver were hybridized in duplicate to mouse whole chromosome 16 microarrays . The Figure S1(0.05 MB PDF)Click here for additional data file.Figure S2(0.06 MB PDF)Click here for additional data file.Figure S3(0.09 MB XLS)Click here for additional data file.Figure S4(0.17 MB PDF)Click here for additional data file.Figure S5(0.13 MB PDF)Click here for additional data file.Figure S6(0.08 MB PDF)Click here for additional data file.Figure S7(0.14 MB XLS)Click here for additional data file.Figure S8(0.08 MB XLS)Click here for additional data file."}
+{"text": "The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice."}
+{"text": "Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin.We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains.Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin  In vitro reconstituted or purified 30 nm fibers are flexible and organized into imperfect helical structures Gene expression is regulated at the transcriptional level by a variety of trans-acting factors that bind to specific promoter elements to elicit transcription initiation in response to intra- and extra-cellular signals. Only a fraction of the genome is competent for factor binding and the way DNA is wrapped into chromatin regulates its accessibility thus participating actively in the regulation of gene expression This structural definition partially overlaps the biochemical and functional description of chromatin, which is separated into EC and HC on the basis of nuclease accessibility, histone modifications and transcriptional activity in vivo at the ultrastructural level.Sedimentation studies on recombinant nucleosomal arrays were performed to explore the link between chromatin condensation and histone modifications and showed that acetylation of H4K16 inhibits the formation of 30 nm fibers The aim of this study was to correlate the packing of chromatin, the transcriptional activity and the distribution of histone tail modifications in sections of cell nuclei. Here murine rod photoreceptors were investigated by electron tomography and immunolabelling to study these correlations. Mouse rod cells have an extremely dense HC domain located at the centre of the nucleus and a small EC territory placed at its periphery, close to the nuclear envelope 3. Chromatin appeared arranged in concentric layers; the center of the nucleus being composed exclusively of HC whereas a 500\u2013600 nm wide rim adjacent to the nuclear membrane contained a mixture of HC and less dense EC. A fraction of HC was found in close contact with the nuclear membrane thus forming a thin rim that was connected through HC bridges to the central HC. In contrast to other cell types, the EC compartment was markedly reduced in size and was confined to the nuclear periphery close to the nuclear membrane. Stereological measurements performed on 20 different nuclei showed that the HC compartment represents 71% of the surface when the nucleus is sectioned through its centre which, assuming spherical symmetry corresponds to about 17 \u00b5m3. The thin layer of HC in close contact to the nuclear membrane (within 150 nm from the nuclear membrane) was estimated to 3 \u00b5m3 or 15% of the entire HC compartment. The EC compartment contacts the nuclear membrane and projects into the central HC through multiple invaginations thus forming cavities with an extended HC/EC interface. The staining of the EC compartment showed substantial variations indicating that variable degrees of chromatin condensation coexist in this area.Retinas of freshly sacrificed mice were dissected according to an optimized protocol that preserves the electrophysiological activity of the tissue and were instantly cryo-immobilized by high pressure freezing to prevent chemical fixation and changes in physicochemical conditions that could reorganize the cellular ultrastructure. The retinas were then freeze-substituted, epon embedded and sectioned at room temperature. Tolu\u00efdin blue stained sections displayed the multilayered structure of the retina in which the nucleus, inner segment and outer segment of the photoreceptor were recognized   thus validating the estimated chromatin compaction.Finally, area 4 corresponds to a characteristic HC domain where the boundaries of individual particles cannot be distinguished. A total of 61% of the volume has an intensity above the threshold assigned for the nucleosomes which, assuming a similar staining than for the EC territories, yields a concentration of about 540 10\u22121. This observation indicates that the periphery of the HC territory is formed by closely packed 30 nm chromatin fibres whereas such fibres are not detected in the most central domain.Despite its electron dense nature the HC compartment showed at least two distinct textures indicative of different nucleosomal arrangements. The periphery of the HC territory had generally a granular appearance whereas the most central domain showed a highly homogeneous and smooth texture (2 (p/\u00b5m2) in HC and of 42 p/\u00b5m2 in EC yielding a HC/EC labelling ratio of 2. This ratio is significantly lower than the HC/EC nucleosome density ratio of at least 5 found by tomography suggesting that the accessibility of this epitope is affected by the nucleosomal packing. To quantify the radial variation of the labelling density within the nucleus, the HC domain was contoured and the gold particles were counted in concentric rings of decreasing size, each separated by 150 nm . Ultra-thin cryo sections of the retina were therefore produced to gain equal access to all regions of the nucleus and to optimally preserve the antigenicity of the epitopes y 150 nm Figure 8The distribution of three histone trimethylation marks specific for transcriptionally silenced chromatin were investigated by using antibodies recognizing histone H3 modified on lys 9 (H3K9me3), on lys 27 (H3K27me3) or histone H4 modified on lys 20 (H4K20me3).2 in the most external HC ring (from 0 to 150 nm from the EC/HC interface) and increased regularly to reach 255 p/\u00b5m2 in the center of the nucleus  and 2.8 times higher than in the periphery (140 p/\u00b5m2)  and8. A fiand2 whereas it rises to 87 p/\u00b5m2 in the periphery.The H3K27me3 marks are also enriched in the HC region but in contrast to the previously analyzed trimethylations, show a decreased labelling from the periphery to the center of the HC territory and8. A ce2) but, unexpectedly, is found highly enriched in the condensed HC closest to the HC/EC interface (97 p/\u00b5m2) and in the EC compartment (41 p/\u00b5m2) (and2). An antibody directed against all acetylated forms of H4 gave the same labelling profile  and8. The The distribution of acetylated histone H3 was investigated by using an antibody specific for acetylated K9 and K14. Despite a slightly lower labelling density, the distribution of H3K9K14ac is similar to that of H4K8ac Figure 8 and Unlike the H3K9, H3K27 or H4K20 methylations described above, the methylation of lysine 4 of histone H3 is a post translational modification exclusively associated with actively transcribed genes and particularly with the early transcribed region In order to position RNA polymerase II (RNA Pol II) molecules an antibody targetting the repeated heptapeptide of the C-terminal domain (CTD) of the largest RNA Pol II subunit was used. This antibody recognizes all forms of RNA Pol II since it is directed against the non phosphorylated CTD. The RNA Pol II were found almost exclusively (79%) within the EC compartment where the labelling was 14.5 times higher than in the HC domain. Interestingly the labelling was not distributed randomly within the EC cavities but appeared to be stronger close to the EC/HC interface  in the EC compartment. The histogram representing the distance of the active polymerases to the EC/HC interface shows that 66% of the active enzyme peaks at 20+/\u221220 nm from the interface. Moreover no active RNA Pol II labeling could be detected in the most central parts of the EC cavities. These results show that the transcribing enzyme is located even closer to the interface than the total pool of RNA Pol II.Murine rod photoreceptors are terminally differentiated cells and contain a highly condensed HC domain placed at the center of the nuclei. The small size of the EC compartment, the peculiar chromatin organization and the availability of genome wide gene expression data turn these cells into unique models to study the correlations between chromatin compaction, transcriptional activity and histone modifications. At first sight, rod photoreceptor nuclei show two distinct nuclear domains that reflect the early structural definition of EC and HC based on regions differentially stained with basophilic dyes in vitro for dispersed chromatin, 30 nm fibers are more elusive in the cellular context except for isolated starfish sperm or chicken erythrocyte nuclei placed in hypotonic conditions \u22121 proposed to be generated by tightly packed chromatin fibers A widely accepted model for chromatin compaction involves successive folding events during which linear nucleosome arrays wrap into 30 nm wide helical fibers forming either a solenoid or a zig-zag assembly. Mostly observed \u22121 in the Fourier transform of nuclear images. The highly compact nature of the rod nucleus may have favoured the detection of the fibers and special care was taken to perturb as little as possible the nuclear organisation during specimen preparation. In this respect, the nuclei were not isolated from cells, the physiological ionic strength was preserved during dissection and the freshly dissected tissue was shown to be functional just prior to high pressure freezing. Interestingly 30nm fibers are only detected in the periphery of the HC whereas the central part appears uniform and does not show any periodic signal at 1/30nm\u22121. Our results thus favour a model in which rod HC is organized as closely packed 30 nm fibers which, consistent with the liquid crystal model, appear melted in the most central HC. However, even in these central parts of the nuclei, our immunolabelling data reveal 30 nm wide fibers, suggesting that the diffusion of nucleosomes around the positions they would adopt within a regular 30 nm fiber is limited. The transition between the homogeneous and the granular HC is unrelated to histone acetylation but correlates with a specific trimethylation pattern that could contribute either directly or through the recruitment of specific factors to interfiber interactions.Our studies revealed for the first time a signal at 1/30nmThe rod HC is organized into three concentric layers, each characterized by a specific combination of histone modifications and this arrangement reflects the functional layout of the nuclei. The central HC core has a radius of 0.8 \u00b5m corresponding to 42% of the nuclear radius and contains histones highly enriched in H4K20me3 and H3K9me3 marks, weakly trimethylated on H3K27 and deacetylated. The size of this domain correlates with Fluorescence In Situ Hydridization (FISH) experiments showing that telomeric, centromeric and subcentromeric satellite DNA are found between 0 and 45% of the nuclear radius A peripheral domain surrounds this central core and extends to about 0.2 \u00b5m to the EC/HC interface which corresponds to 42% and roughly 60\u201380% of the nuclear radius since the interface is folded. In this domain the histone tails are not acetylated and show reduced H4K20me3 and H3K9me3 and higher H3K27me3 levels than the central core. The H3K27me3 mark was associated in several systems to facultative HC defined here as genomic regions that have the opportunity to adopt an open or a compact conformation within defined temporal or spatial constraints Finally a 0.2 \u00b5m thick rim of heterochromatin at the EC/HC interface displays the same histone trimethylation pattern than the peripheral HC domain but is surprisingly highly acetylated. FISH experiments have located the gene rich regions of the genome, irrespectively of their transcriptional status, between 70 and 100% of the nuclear radius in vitro transcription experiments indicate that acetylation facilitates chromatin unfolding and transcription in vivo observations confirm that H3 and H4 acetylation on its own is not sufficient to elicit chromatin decondensation at the HC/EC interface. In vitro the extended 11 nm chromatin fibers showing the typical beads-on-a-string appearance were only observed in the absence of the histone H1, which is believed to stabilize the 30 nm fiber by acting on the path of DNA at the exit of the nucleosome Transcription, as probed by the presence of RNA Pol II, is strictly restricted to the EC compartment, which is characterized by the presence of decondensed chromatin, acetylated and monomethylated histone tails, and the absence of H3K9me3, H3K27me3 and H4K20me3 marks. Hyperacetylation of H3 and H4 tails is a hallmark of actively transcribed genes in higher eukaryotes 5/\u00b5m3) and the volume of the EC compartment (about 9 \u00b5m3). This fraction may correspond to genes that can rapidly switch between an activated and a repressed state.Electron tomograms revealed large variations in nucleosomal packing in the EC compartment ranging from extended 11 nm fibers to more densely packed chromatin regions. These variations suggest that all the euchromatin is not in the same transcriptional state and that significant transcriptional regulation may still occurs in the EC compartment through modulation of the chromatin structure. The fraction of the genome exposed in the EC territory can be roughly estimated to be 20 Mbp or less than 1% of the mouse genome from the average nucleosome density . The experiments involving animals were conducted under the supervision of a staff member holding an animal experimentation authorization from the Ministry of Agriculture and Fisheries: having followed the obligatory national training programs for animal handling, including procedures of euthanasia. The institution is guided by the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Their eyes were rapidly enucleated and placed in Artificial CerebroSpinal Fluid (ACSF) buffer  for the dissection. The retina were gently separated from the pigment epithelium and the sclera after removal of the lens, iris and vitreous humor. The dissection was completed in less than 5 min Thirteen to twenty-one weeks old C57/BL6 mice were sacrificed in accordance with the National Institutes of Health For High Pressure Freezing (HPF), retinas were punched  to obtain an oriented disk 1.5 mm in diameter that fits into the specimen carrier. After infiltration in a drop of cryoprotectant containing 10% dextran (Sigma # D1662) and 10% BSA (Sigma # A4503) v/v in ACSF buffer (5 sec), each disk was placed onto a 200 \u00b5m thick flat gold-plated specimen carrier in order to be vitrified in the HPF machine .4 and were then warmed up slowly (1\u00b0C/h) to \u221260\u00b0C in an Automated Freeze Substitution device . After 8\u201312 h the temperature was raised to \u221230\u00b0C (1\u00b0C/h) and the samples were kept at this temperature for 8\u201312 h before being rinsed several times in acetone. The samples were then infiltrated with gradually increasing concentration of Epon in acetone  for 2\u20133 h while raising the temperature. Addition of pure Epon was performed at room temperature. After polymerization of the resin at 60\u00b0C, 70\u2013100 nm thin sections were produced using an ultramicrotome , collected on formvar-carbon coated hexagonal 50 mesh copper grids and post-stained for 5 min with 2% aqueous uranyl acetate, rinsed and incubated for 2 min with lead citrate. The quality of the vitrification was assessed by the absence of recognizable ice crystal ghosts.After HPF, the samples were freeze substitute To determine the proportion of the nuclear volume occupied by the different chromatin compartments we used stereological methods http://www.cgl.ucsf.edu/chimera) after having low pass filtered the tomograms to 1/10 nm\u22121. In order to position the electron dense particles the low pass filtered tomograms were searched for peaks of high intensity separated by more than 12 nm. For modeling, a 12 nm sphere was placed at each peak coordinate.Ten nanometer colloidal gold particles were applied on one side of the grid to be used as fiducial markers to align the tilt series. The specimen was inserted in a transmission electron microscope equipped with a field emission gun and operating at 200 kV . Digital images were recorded at a magnification of 29.000\u00d7 on a Pelletier cooled 2k CCD camera leading to a pixel spacing of 3.6 \u00c5 . Each tilt series was recorded in bright-field mode over a tilt range of \u221265\u00b0 to +65\u00b0 with 1\u00b0 increments giving a total of 131 images automatically collected with the Xplore 3D software (FEI Company). The recorded images were aligned on a common origin by means of cross correlation techniques and the 3-D reconstruction was performed using the Inspect3D tomography reconstruction package (FEI Company) according to the weighted-back projection methods or the Simultaneous Iterative Reconstruction Technique (SIRT). Surface rendering was performed by gray level thresholding using the Chimera software , H4K8ac , pan acetylated H4 (Millipore # 06-866) and H3K4me1 (Abcam # ab8895). Inactive marks of transcription were detected with rabbit polyclonal antibodies directed against H4K20me3 (Abcam # ab9053), H3K27me3 (Millipore # 07-449) and H3K9me3 (diagenode # pAb-056-050). Total Histone H3 was detected with a rabbit monoclonal antibody directed against the C-terminus of histone H3 (Millipore # 05-928). RNA polymerase II was detected with an IgG1-type mouse monoclonal antibody developed at IGBMC (1PB7C2). This immunopurified antibody reacts with the conserved heptapeptide repeat of the largest subunit of eukaryotic RNA polymerase II. The active form of RNA polymerase II was detected with an IgM-type mouse monoclonal antibody directed against the phosphoserine 2 in the heptapeptide repeat of Rpb1 (Covance # MMS-129R). The primary antibodies were revealed with goat anti-rabbit ultra-small gold-conjugates . For the detection at the light microscope level goat anti-rabbit IgG coupled to AlexaFluor 488 were used (Invitrogen # A11008).Sample preparation: Briefly, after dissection, retina were cut into small blocks of about 1mm3, fixed in 2.5% formaldehyde, 0.1% glutaraldehyde in 0.1 M Phosphate Buffer Saline pH 7.4 (PBS) for 1h. Free aldehydes were blocked with several washes in PBS supplemented with 50 mM glycine. The blocks were infiltrated with 2.3 M sucrose in PBS in an Eppendorf tube on a rotating wheel for at least 2 h at 4\u00b0C. The blocks were then mounted on the holders of the cryoultamicrotome and rapidly frozen in liquid nitrogen. Cryosectioning: Ultra-thin cryo-sections (100 nm) were cut using a cryo-ultramicrotome  at \u2212130\u00b0C using diamond knives  and recovered with a drop of 2.3 M sucrose on formvar-carbon coated 50 mesh nickel grids or on coated light microscope slides for immunofluorescence. After thawing, immunolabelling was performed using the automatic Immuno Gold Labelling apparatus . Immunolabelling: After a blocking step of two times 10 mn in glycine  and another 10 mn in 1% BSA/PBS (blocking buffer), the sections were incubated 20 min with the primary antibodiy. After 4 times 2 min washes on 10\u00d7 diluted blocking buffer bound antibodies were revealed using goat anti-rabbit conjugated to ultra-small colloidal gold (US) diluted 1\u223625 PBS with 0.1% acetylated BSA (BSA-c\u2122) . After several washes in PBS/0,1%BSA followed by PBS, the antigen-antibody complexes were stabilized on 1% glutaraldehyde in PBS for 5 min After several washes in PBS followed by water colloidal gold silver enhancement was performed using R-Gent\u2122 (Aurion) for 30 min resulting in an average particle size of 10 nm. Thereafter, sections were carefully washed 5 times (5 min each) with water, stained and embedded in a solution containing 4 parts of 2% methyl cellulose (Sigma-Aldrich # M-6385) and one part of 2% uranyl acetate. Sections were observed using a Philips CM 120 transmission electron microscope at an accelerating voltage of 100 kV.Retina samples were prepared, sectioned and immunolabelled according to Tokuyasu For immunofluorescence the primary antibody was detected with a goat anti-rabbit IgG coupled to AlexaFluor 488 diluted in blocking buffer. Sections were counterstained with DAPI   and embedded in Aqua-Poly/Mount (Polysciences # 18606-20) medium. The results were examined using a DM400B Leica epifluorescence microscope connected to a Cool Snap camera (Roper).2 was quantified for 15\u201320 rod nuclei for each immunolabelling experiment. The regions of interest were delimited and their surfaces were measured using the ImageJ software (http://rsbweb.nih.gov/ij/). Gold particles were counted within each region of interest after high pass filtering of the images and by using an automated peak search algorithm .The number of particles per \u00b5m"}
+{"text": "The effect of localized hypothermia on microcirculatory and metabolic parameters in s.c. DS sarcomas on the hind foot dorsum of Sprague-Dawley rats was investigated. Tumours were cooled by superfusion of the tumour surface with cooled saline solution to 25 degrees C or 15 degrees C. Control tumours remained at 35 degrees C. These temperatures were maintained for 30 min. In tumour oxygenation measurements, hypothermia at 25 degrees C and 15 degrees C caused progressive decreases in the size of the fraction of pO2 measurements between 0 and 2.5 mmHg together with a reduction in pO2 variability. No significant changes in median or mean pO2 or in the fraction of pO2 measurements between 0 and 5 mmHg, and 0 and 10 mmHg were observed. Using laser Doppler flowmetry, red blood cell flux was found to decrease significantly upon 25 degrees C or 15 degrees C hypothermia treatment to 67% and 37% of starting values respectively, whereas no significant changes were seen in control tumours over the whole observation period. Viscosity was measured in blood and plasma samples over a range of temperatures and was found to increase with decreasing temperature. Assessment of tumour glucose levels showed an increased concentration of glucose following 15 degrees C hypothermia, an observation consistent with a 'slowing down' of glycolysis. No changes in lactate or adenylate phosphate levels were observed. As a way of improving tumour oxygenation, localized hypothermia may therefore be a useful means of radiosensitization."}
+{"text": "KI and WU Polyomaviruses in Children, France To the Editor: Two new members of the Polyomaviridae family, provisionally named Karolinska Institutet virus (KIPyV) and Washington University virus (WUPyV), have been recently discovered ; influenza virus types A and B; parainfluenza virus types 1, 2, and 3; and adenoviruses (AdVs) by direct immunofluorescence assay. The aspirates were also tested for human metapneumovirus (HMPV) by an enzyme immunoassay  and for the human bocavirus (HBoV) by PCR  children. The viruses found were RSVs in 175 (32.6%), HBoVs in 54 (10.0%), HMPVs in 50 (9.3%), rhinoviruses/enteroviruses in 11 (2%), influenza A viruses in 8 (1.5%), human AdVs in 6 (1.1%), and parainfluenza type 3 viruses in 4 (0.7%) samples. Aspirates were not tested for coronaviruses; detection of rhinoviruses/enteroviruses was likely low because cell culture is less sensitive than molecular assays.A total of 13 (2.4%) samples were positive for WUPyV; of these 4 (30.8%) were co-infected with another virus. The 13 children with samples positive for WUPyV had a median age of 11.2 (2\u201348) months and the male/female sex ratio was 2.2. KIPyV DNA was detected in samples from 3 (0.6%) boys ; 1 of those samples was co-infected with RSV and HMPV.Sequences of WUPyV and KIPyV isolates varied little from each other and from other GenBank sequences, which suggests that these polyomaviruses are genetically conserved viruses. Clinical characteristics of children infected with WUPyV and KIPyV are retrospectively recorded . All chiOur data are in agreement with the 2 original reports that show that the new KI and WU polyomaviruses may be detected in respiratory secretions from patients with respiratory diseases (KIPyV and WUPyV were mainly detected in samples from children with lower respiratory tract disease, such as bronchiolitis or atypical pneumonia, and in samples from children with exacerbated asthma. These preliminary data on the likely role of these viruses as respiratory pathogens need to be interpreted with caution. Aspirates were obtained only from those with observed symptoms; no asymptomatic controls were tested. Detection of WUPyV and KIPyV in respiratory samples may simply reflect a respiratory transmission route as previously suggested for BK virus and JC virus (As with other polyomaviruses, WUPyV and KIPyV could establish persistent and latent infections with likely asymptomatic reactivations ("}
+{"text": "Between 1974 and 1984 69 adults with acute lymphoblastic leukaemia (ALL) were treated with two different protocols. Fifty-four (78%) of the patients entered complete remission (CR); 27 of these then received a consolidation protocol consisting of daunorubicin, cytosine arabinoside and 6-thioguanine, followed by two courses of intravenous methotrexate 500 mg m-2 with folinic acid rescue. All patients received intrathecal methotrexate and cranial irradiation (24 Gy) followed by maintenance therapy with 6-mercaptopurine and methotrexate for at least 2 years. The median survival for all patients was 23 months from the time of presentation with an actuarial 5-year survival of 21%. The actuarial chance of surviving 5 years in CR for patients receiving the consolidation protocol was 38% compared to 19% for patients receiving no consolidation (P = NS). Only patient age and white cell count at presentation were found to influence the chance of achieving CR and the chance of overall survival. The presence or absence of c-ALL antigen did not influence prognosis. Patients younger than 35 years with low white cell counts at presentation (less than 10 X 10(9)1(-1] had a particularly good prognosis but no patient with T-ALL and no patient older than 50 years old at diagnosis survived more than 18 months."}
+{"text": "Mutations in these genes have been linked to hereditary non-polyposis colon cancer; they also occur in a variety of sporadic cancers. Western blot analysis and immunohistochemistry demonstrated that hMLH1 and hPMS2 are widely expressed nuclear proteins with a distribution pattern very similar to that previously described for hMSH2. These observations showing similar localization of hMLH1 and hPMS2 with hMSH2 are consistent with the biochemical function of these proteins in DNA mismatch repair."}
+{"text": "The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1) and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD) has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239) antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1) that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned. Mitotic entry is the paradigmatic cell cycle transition and example of Cdk regulation. Yet, our understanding of this transition remains superficial. A long-term goal of research in this area is to design drugs that treat cancer by either blocking mitotic entry or driving cells into mitosis in the face of lethal DNA damage. Cyclin B/Cdk1 Cdc2/Cdc28) complexes direct many of the events of mitosis. These events must be launched in swift, coordinated fashion but only after DNA synthesis is completed and DNA damage is repaired. To effect such control, cyclin B/Cdk1 activity is regulated through dynamic post-translational modifications. Wee1 is a universal Cdk1 inhibitor that phosphorylates a tyrosine residue (Y15) in the ATP binding site, thereby blocking Cdk1 activity /Cdk2 complexes--help pave the way for mitosis by reducing cyclin B/Cdk1 Y15 phosphorylation. Microinjection of cyclin A was observed to drive cultured human cells into mitosis and injection of cyclin A/Cdk2 inhibitors could block mitotic entry . InductiWe asked whether cyclin A/Cdk2 complexes might antagonize Cdk1 Y15 phosphorylation by directly binding and inactivating Wee1. Wee1 was present in cyclin A and Cdk2 immunoprecipitates from U2-OS cells and associated efficiently with exogenously expressed Cdk2 . In contCyclin A/Cdk2 complexes are known to preferentially recognize some substrates via short sequence motifs termed 'Cy' or RxL' motifs -25. Humain vivo data, studies in human somatic cell extracts showed that recombinant cyclin A can direct the phosphorylation and inactivation of Wee1 more efficiently than cyclin B, and combined addition of cyclins A and B induced nuclear envelope breakdown more efficiently than addition of either cyclin alone [We mutated each of the RxL sequences in Wee1, singly and in combination, and examined the impact on cyclin A/Cdk2 binding and T239 phosphorylation. Mutations of the RxL sequences diminished stable association of Wee1 with cyclin A/Cdk2 complexes in vivo and in vitro . Mutatioin alone .The RxL1 mutant appeared to be modestly more potent than the T239A mutant in mediating G2 phase arrest under the conditions examined, suggesting that loss of the RxL1 site might impact more than phosphorylation of T239. We therefore examined other properties of Wee1. Wee1 has been described as being localized to the nucleus during interphase and the cytoplasm during mitosis. Whether this redistribution is a cause or effect of nuclear envelope breakdown has remained unclear. Early studies demonstrated that nuclear Wee1 could potently protect cells from premature mitosis, even in the presence of activated cyclin B/Cdk1 complexes in the cytoplasm . These sThe simplest explanation for the restricted nuclear localization of the RxL1 mutant was that this site directs cyclin A/Cdk2-mediated phosphorylation of another residue(s) that drives Wee1 cytoplasmic redistribution. This scenario may yet prove to be true, but examination of the Wee1 primary structure suggested another potential explanation. The RxL1 sequence overlaps with residues that match the loose consensus Crm1-dependent nuclear export signal (NES) -30. SubsOne can draw a number of conclusions from this recent work. First, redistribution of human somatic Wee1 to the cytoplasm is an active, temporally regulated event, rather than a passive byproduct of nuclear envelope breakdown. Based on conservation of NES consensus sequences among vertebrate somatic Wee1 proteins, nuclear export might also be conserved, suggesting the presence of selection pressure to maintain it. Given the lack of conservation of the NES in embryonic proteins, regulated export of Wee1 may exert an additional constraint on mitotic entry unique to somatic cells. In this light, it was somewhat surprising that expression of the NES mutant did not impose increased G2 arrest under the conditions tested . It seemMight cyclin A/Cdk1 complexes also bind RxL1 and inactivate Wee1? Cdk2 appears generally to be the preferred binding partner for cyclin A when Cdk1 and Cdk2 are expressed at their normal levels ,18,32. HThe conclusion that cyclin A/Cdk2 complexes drive mitotic entry raises an apparent conundrum. These complexes also drive DNA synthesis ,20,36, aThere are a number of issues that merit further study. The bifunctional nature of the NES/RxL1 site raises the question of whether physical occupancy of the site by cyclin A/Cdk2 complexes competes with that of Crm1. The stable association of Wee1 with both binding partners  suggestsThe authors declare that they have no competing interests."}
+{"text": "Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of \u03b12,3-linked and \u03b12,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SA\u03b12,6Gal and Maackia amurensis agglutinin (MAA) for SA\u03b12,3Gal in the respiratory tract of normal adults and children.We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the \u03b12,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SA\u03b12,3 binding viruses, such as influenza or human parainfluenza are to be made. There are two main epithelial cell types in the bronchus \u2013 ciliated cells and goblet cells that secrete mucus. Within the submucosa there are also submucous glands present. The goblet cells have a glycoprotein which is acidic due to the presence of sialic sulphate groups and this secretion may vary with various diseases . SA\u03b12,6GThe affinity of the attachment of the HA to cell surface receptors is believed to be an important determinant in tissue tropism of the virus and constitutes part of the species barrier that keeps avian influenza viruses from readily infecting humans. Pigs contain a respiratory epithelium that has been reported to contain both \"avian-virus\" binding SA\u03b12,3Gal and \"human-virus\" binding SA\u03b12,6Gal linkages supposedly explaining why they can be infected with both human and avian influenza viruses . Therefoin situ. The SA\u03b12,6Gal and SA\u03b12,3Gal expression on histological specimens in situ can be done using fluoresceinated lectins and by histochemistry. Since the early 1990's many histology laboratories have used unmasking or retrieval techniques to enhance immunohistochemical detection of antigens.Some of the studies on the types of SA expressed on cell surfaces have been done on sialic acids extracted from cell membrane homogenates. However, for the understanding of influenza pathogenesis and pandemic emergence, it is important to have methods that can define the profiles of SA\u03b12,6Gal and SA\u03b12,3Gal in histological tissue sections In this study we address three issues. The first was to investigate if antigen unmasking or retrieval would affect lectin-ligand expression in histological tissues. The second issue was to compare the findings obtained by using lectin fluorescence with cytochemistry for lectin-ligand analysis in the same tissue samples. Finally, we wanted to use optimized methods to re-evaluate the distribution of the SA\u03b12,6Gal and SA\u03b12,3Gal in human respiratory tissues and then correlate this with the reported presence or absence of influenza infection in different parts of the respiratory tract.Biopsy samples were collected from the archived files of the Histopathology Department of Queen Mary Hospital, Pok Fu Lam, Hong Kong. Five surgically removed lungs from children with congenital cystic adenomatoid malformation (CCAM), seven non-neoplastic bronchial biopsies from patients investigated for possible malignancy, eight normal nasopharyngeal biopsies from patients with suspected nasopharyngeal carcinoma and eight lung biopsy samples from 20\u201340 week abortuses were also used. Seven biopsy tissues of CCAM, representing paediatric lung tissues of ages 1 month to 7 years were also retrieved from the files of the Department of Histopathology, Adelaide Women's and Children's Hospital, Adelaide, South Australia. All tissues had been fixed in 10% neutral buffered formalin, processed into paraffin and stored at room temperature. Tissue blocks from the lungs of five patients who had died of acute bacterial pneumonia were used as a non-influenza comparison. Intestinal tissue from four ducks kindly provided by the Agriculture, Fisheries and Conservation Department, Government of HKSAR, were used as a positive control for SA\u03b12,3Gal binding. The research was approved by the Ethics Committee of the University of Hong Kong/Hospital Authority Western Cluster.We used lectin histochemistry and fluorescence which is the standard method of detection of the SA linkages . Lectin For the initial trial of optimization for oligosaccharide ligand retrieval methods one lung block from a case of CCAM was used. The tissues were sectioned at 5 \u03bcm and deparaffinized. Control sections had no retrieval. Two different methods were used for retrieval: microwave and enzyme digestion. For microwaving, an Energy Beam Sciences microwave processor was used together with 2 types of buffer. 0.1 M EDTA and 10 mM citrate buffer was used and the sections were microwaved for 10,15, 20 and 25 minutes at 95\u00b0C. Two enzyme digestion methods were used: trypsin and pronase, and for both these methods sections were incubated for 15 mins at 37\u00b0C.Single fluorescent studies were performed as follows. The sections were microwaved in 95\u00b0C citrate buffer pH 6.0 for 15 minutes, washed with 0.05 M Tris Buffer Saline (TBS) pH 7.6 and then incubated with either 1/100 FITC conjugated SNA (EY Laboratories), or 1/100 FITC conjugated MAA (EY Laboratories) for 1 hour at room temperature in the dark. Double fluorescent studies were performed according to the method of Mason et al. . Briefly2O2 in TBS for 12 min and after washing with TBS 3 times, 5 minutes each were then incubated with 1/50 HRP conjugated SNA (EY Laboratories) and 1/50 HRP conjugated MAA (EY Laboratories) at room temperature for 1 hour respectively. After 3 further washes in TBS the sections were developed with AEC substrate kit (Vector Laboratories) at RT for 30 minutes followed by counterstaining with Mayer's haematoxylin and mounting with DAKO aqueous mount .Lectin horseradish peroxidase (HRP) detection: the sections were microwaved in 10 mM citrate buffer pH 6.0 for 15 min, blocked with 3% H2O2 in TBS for 12 min and with avidin/biotin blocking kit (Vector). They were then incubated with 1/100 biotinylated MAA1 (or 1/100 biotinylated MAA2) (Vector) for either 1 hour at RT or 4\u00b0C overnight, blocked with 1% bovine serum albumin for 10 mins at RT, and then incubated with strep-ABC complex  diluted 1/100 for 30 mins. at room temperature. Development was performed using the AEC substrate kit (Vector) at room temperature for 10 minutes. The nuclei were counterstained with Mayer's hematoxylin and then the sections were dried and mounted with DAKO aqueous mount (Dako Cytomation). Duck intestine sections were used as controls with and without pre-treatment with SA\u03b12,3 specific neuraminidase from Glyko to ensure that sialic acids were specifically targeted. Stain intensity was measured semi-quantitatively using duck MAA as a control. Similar stain intensity to the duck intestine was graded as strong (++) and a weaker pattern as +.Lectin biotin detection: The sections were microwaved in 10 mM citrate buffer pH 6.0 at 95\u00b0C for 15 min then blocked with 3% HTo determine lectin binding profiles we used data from the Consortium for Functional Glycomics (CFG) web site  using glIn the absence of unmasking techniques and using the lectins SNA and MAA from EY Labs, there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin binding to the surface epithelial cells and mucus containing cells for both SNA and MAA Figure  and thisThe 8 samples of fetal tissue at 20 weeks gestation showed strong binding (++) of MAA to the bronchial epithelium and to the developing pneumocytes with absent SNA detected. With advancing development the SNA binding increased and there was a switch in the MAA1 and MAA2 binding between the alveoli and bronchi Table . It was When dual labelling was performed on the bronchial tissues of adults and of children using SNA and MAA, we identified a heterogeneous distribution of SNA and MAA binding in the epithelium with no clear distinction between ciliated and non-ciliated cells resulting in a mixed and occasionally dual expression of both SA\u03b12,3Gal as well as SA\u03b12,6Gal in the ciliated cells, goblet cells and basal cells Fig . We alsoBecause we found that there was a different binding pattern in the upper and lower respiratory tract for MAA1 and MAA2 using these lectins from Vector labs we then investigated whether the MAA from 2 other suppliers \u2013 EY Laboratories and Roche would identify one or both of the MAA isoforms. We therefore used serial sections from adult lung tissue and stained them using the Vector MAA1 and MAA2, digoxigenin labelled MAA (Roche) and biotinylated MAA from EY Laboratories. While EY Laboratories state that their MAA is a combination of 2 isoforms, this information is not evident from Roche. As expected using MAA1 Fig  the alveAnalysis of the glycan array binding data of MAA1, SNA and H5N1 submitted to the CFG showed that MAL did detect SA\u03b12,3Gal\u03b21,4GlcNac(glycan numbers 235\u2013238) with detection of the sulphated forms (216 and 227) [see Additional file N-acetylneuraminic  acids \u2013 a series of 9-carbon sugars. Influenza virus infection of humans involves binding of the virus haemagglutinin (HA) to these sialyoligosaccharides on the surface of cells of the respiratory tract. In addition, the virus neuraminidase (NA) cleaves the sialic acid on the host cell and is important in releasing newly formed virus from the cell after virus replication is completed so these virions can spread out in search of other cells to infect. Since respiratory mucus is also rich in SA, this provides a potential barrier to the spread of newly formed virions. By cleaving these SA, the influenza virus NA facilitates the virus spread through this mucus layer [Cells of the respiratory tract express a number of glycan containing conjugates on the cell surface, many of which terminate with us layer . Thus thThe crystal structures of HA shows that the terminal sialic acids bind in a groove at the top of the HA molecule . PreviouUsing retrieval methods and selection of lectin conjugate we have demonstrated that the lectin binding to the SA\u03b12,3Gal receptor for avian influenza viruses is more widely expressed in the respiratory tract of humans than previously documented . In partThe difference between our studies and previous ones on sialic acid expression in respiratory epithelial cells can be partially explained by the methods used for lectin analysis as well as the type of lectin conjugate used. Antigen retrieval or unmasking did not become an established procedure in many laboratories until the mid 1990's and the earlier publications used paraffin embedded tissues without retrieval ,8. LaterTwo isoforms of MAA have been recognized for many years and their binding profiles have been characterized. Since MAA1 bound to non-SA\u03b12,3 glycans it has not been used as extensively by some researchers as MAA2. For instance, Shinya et al have recently demonstrated SA\u03b12,3 Gal expression only in the lung but not the bronchus or upper respiratory tract . Since tBecause of this varied distribution of MAA1 and MAA2 throughout the respiratory tract, we hypothesised that this may shed new light on the distribution and binding of H5N1 viruses to the upper and lower respiratory tract. Using this information we therefore used ex-vivo cultures of the upper and lower respiratory tract and infected them with different H5N1, H1N1 and H3N2 viruses and found that contrary to previous suppositions, H5N1 viruses were able to replicate in the upper respiratory tract \u2013 a region which lacked MAA2 binding but had abundant MAA1 binding, thus indicating that the virus is perhaps binding to SA\u03b12,3Gal\u03b21,3/4GlcNac motifs or even to non-sialyated receptors .in vitro are only a partial representation of the true nature of sialic acid expression in the adult respiratory tract [in vitro model with cultured tracheobronchial epithelium showed more similarity to the receptor profile seen in the respiratory tract of children. Therefore it is possible that the human tracheobronchial epithelial culture model is more representative of the respiratory tract of children rather than that of adults, and represents a developmentally earlier model of the human respiratory tract.Our results also indicated that the recent findings of Matrosovich and colleagues who found SA\u03b12,3Gal in ciliated cells and SA\u03b12,6Gal in the goblet cells of tracheobronchial cells cultured In summary, we found that there was an overall increased detection of SA\u03b12,3Gal in the respiratory tract when microwave unmasking is used and when a combination of different isoforms of MAA was used. We therefore advocate the routine use of this method for future investigations on the distribution of receptors for influenza viruses in the respiratory tract. We also urge attention to the exact isoform of MAA present in lectins supplied by different manufacturers . These rThe author(s) declare that they have no competing interests.Dr J Nicholls and Professor J S M Peiris designed the experiments and were responsible for primary analysis of tissues. Dr A J Bourne provided input to the CCAM cases from Australia. Dr H Chen and Y Guan assisted in the interpretation of results. All authors have read and approved the final manuscript.Maackia amurensis 1 (MAA1), Sambucus nigra agglutinin (SNA) and H5N1 binding affinity in glycan array Description: Summary of glycan binding profiles of Maackia amurensis 1 (MAA1), Sambucus nigra agglutinin (SNA) and H5N1 influenza (A/Vietnam/1203/04) (Viet04) in the glycan array. Significant binding sugars are shown in column 2 with their glycan numbers according to Printed Array 2.1 shown in column 1. Strong affinity is **, weak affinity is * and no significant affinity is blank. Many SA\u03b12,3Gal oligosaccharides (underlined) bind to both MAA1 as well as H5N1 (A/Vietnam/1203/4).Click here for file"}
+{"text": "TNF-\u03b1 induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.We have studied the effect of human recombinant tumour necrosis factor-\u03b1 (TNF-\u03b1) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-\u03b1 (10 and 100 ng ml"}
+{"text": "BCL10 gene in 81 primary prostate carcinomas, 20 squamous cell cancers of the head and neck, 15 small-cell lung cancer cell lines, 24 renal carcinoma cell lines and 13 sarcoma cell lines. We failed to find evidence of somatically acquired mutations of the BCL10 gene suggesting that BCL10 does not play a major role in the development of these malignancies. \u00a9 1999 Cancer Research CampaignWe have used single-strand conformation polymorphism (SSCP) analysis to screen for mutations in the"}
+{"text": "Purpose and Method. Hyper-fractionated radiotherapy for treatment            of soft tissue sarcomas is designed to deliver a higher total dose of radiation without an            increase in late normal tissue damage. In a previous study at the Royal Marsden Hospital,           a total dose of 75 Gy using twice daily 1.25 Gy fractions resulted in a higher incidence of late           damage than conventional radiotherapy using 2 Gy daily fractions treating to a total of 60 Gy.           The current trial therefore used a lower dose per fraction of 1.2 Gy and lower total dose of 72           Gy, with 60 fractions given over a period of 6 weeks.           Subjects. A total of 37 patients  with a median           age of 56 years (range 19\u201388 years) were treated.           Results. Of eight patients treated pre-operatively, six showed a            partial response and in two the tumour was static. The maximum acute toxicities were grade            1 in eight, grade 2 in 14 and grade 3 in 15 patients. Late toxicities of the skin were            graded 1 in 10 and grade 2 in nine patients. Five patients complained of pain in the irradiated            bone and soft tissues, which was of moderate severity (grade 2). Stiffness was graded 2 in            three patients and severe (grade 3) in one.Three patients had moderate and one patient had            severe lymphoedema following treatment. The 5-year recurrence-free survival probability of           patients treated radically was 76%. Following excision of local recurrences the study group           had a disease-free survival probability of 86% at 5 years.           Discussion. The regime is well tolerated with comparable local control           and late complication rates to standard daily fractionated therapy.The potential benefit of this           regime needs to be defined in a prospective randomized trial."}
+{"text": "The cyclin kinase inhibitor WAF1/CIP1, also termed CDKN1, mediates p53-induced cell cycle arrest in response to DNA damage. This property makes it an attractive tumour-suppressor candidate for a p53-associated tumour-suppressor gene. In order to investigate the role of WAF1/CIP1 in the pathogenesis of primary human brain tumours we performed single-stranded conformation polymorphism (SSCP) analysis and direct sequencing of exon 2 of the gene in a representative series of 158 brain tumours and corresponding blood samples. In addition, all tumours were examined for mutations in exons 5-8 of the p53 gene. Analysis of WAF1/CIP1 revealed multiple polymorphisms, the most abundant being AGC-->AGA (Ser-->Arg) at codon 31 with an allele frequency of 8.5%. Less common polymorphisms included GTG-->GGG  at codon 25, GCC-->ACC (Ala-->Thr) at codon 64, CGC-->CTC (Arg-->Leu) at codon 32, GGC-->AGC (Gly-->Ser) at codon 14 and GCG-->GTG  at codon 39 each with an allele frequency of 0.3%. These polymorphisms were all located in a conserved region of exon 2. Two of the polymorphisms were also seen in a group of 157 healthy controls indicating that WAF1/CIP1 polymorphisms do not predispose to cancer. None of the tumours included in our series showed a somatic mutation in WAF1/CIP1. All samples were also analysed for loss of heterozygosity on the short arm of chromosome 6 in the region of the WAF1/CIP1 locus. Allelic loss was observed in only one patient with a glioblastoma. Mutations in the p53 gene were found in 22 of 158 tumours. No association was found between any polymorphism of the WAF1/CIP1 gene, p53 mutations and histopathological tumour type. Our data indicate that WAF1/CIP1 mutations are probably not involved in the formation of primary human brain tumours."}
+{"text": "To assess the efficacy of ultrasound (US)-guided percutaneous acetic acid  injection for small hepatocellular carcinomas (HCCs) for long-term prognosis, percutaneous acetic acid injection using 15% to 50% acetic acid was performed in 91 patients with one to four HCCs smaller than 3 cm during the past 6.5 years. During the series of treatment sessions for each patient, the same concentration of acetic acid was used. All tumors could be treated successfully with percutaneous acetic acid injection despite the differences in acetic acid concentration used. The number of treatment sessions to treat similar size of tumor was less when the higher concentration of acetic acid was used. No serious complications occurred as a direct sequela to percutaneous acetic acid injection. None of the tumor treated regrew. The 1-, 2-, 3-, 4-, and 5-year survival rates for 91 patients were 95%, 87%, 80%, 63%, and 49%, respectively. The 1-, 2-, 3-, 4-, and 5-year cancer-free survival rates of these patients were 83%, 54%, 50%, 37%, and 29%, respectively. Both liver function and size of tumor affected both survival rate and cancer-free survival rate significantly, but the number of tumors did not. The concentration of acetic acid did not affect the survival rate. Percutaneous acetic acid using 15% to 50% acetic acid will be effective therapy for small HCCs for long-term prognosis."}
+{"text": "Helicobacter pyloriinfection and gastric cancer was observed in a short study out of these patients. The peculiargeography and some special dietary habits with a possible familial predisposition may havea bearing on the high risk of gastric cancer in the valley.The present study is a comprehensive retrospective analysis of 1341 gastric neoplasms out of10 733 individuals subjected to upper gastrointestinal endoscopy at the main teaching cumreferral hospital in the Kashmir Valley. Of these 78% were males and 22% females, majoritybeing in the age group of 41\u201360 years with 60% of the patients being smokers. On endoscopy,the commonest site of cancer was the body of stomach 40.7%, followed by the antrum 35.5%and the cardiac region 23.8%. Endoscopic features revealed nodular masses 39%, polypoidmasses 21%, malignant ulcers 11%, infiltrative masses 12%, rounded tumor masses 9%,linitus plastica 5% and early gastric carcinoma 3%. Histology revealed adenocarcinoma91%, , leiomyosarcoma 7%,and reticulum cell sarcoma 2%. No significant association between"}
+{"text": "The incidence of B cell leukaemia in 186 consecutive untreated patients with histologically defined B cell neoplasms is described. The lymphomas were classified by the Kiel convention. B cell leukaemia in the context of this paper refers to the situation where a neoplastic clone of B cells in the blood greatly outnumbers normal blood B cells. It is defined as an absolute blood B cell count greater than 0.75 X 10(9)1(-1) where either greater than 90% B cells express kappa immunoglobulin light chains or greater than 80% express lambda light chains. This was found in several patients where the total blood lymphocyte count was within normal limits. All patients with diffuse lymphocytic lymphoma with the histological appearances of B cell chronic lymphocytic leukaemia (ML-BCLL) were found to have B cell leukaemia. However, more than half these patients had blood B cell counts less than 10 X 10(9)1(-1). B cell leukaemia was also a feature in approximately 33% of patients with follicle centre cell tumours and 33% of those with lymphoplasmacytoid tumours. B cell leukaemia was not detected in 34/35 patients with myelomatosis. The 35th patient had plasma cell leukaemia. Only 3/22 patients with high grade lymphoma had B cell leukaemia. In the three principal tumour types associated with B cell leukaemia mu + delta was the most common immunoglobulin heavy chain phenotype. Spontaneous mouse red cell rosette formation also characterised leukaemic B cells in these three groups but high proportions of mouse rosetting cells were seen only in association with ML-BCLL. None of 4 cases of prolymphocytic leukaemia showed mouse red cell rosetting. HLA-DR alpha chain was found on the leukaemic cells of all patients except one with ML-BCLL. B cell lymphopenia was a frequent finding in all histological groups in those patients who did not have B cell leukaemia."}
+{"text": "Patients with cardiac disease, chronic pulmonary disease, poor tissue perfusion, or metabolic abnormalities were excluded.Retrospective study of patients admitted to an intermediate care unit (InCU) at a tertiary care center over a 20-month period with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia. Patients with venous pCO2-vpCO2 paired values were available from 62 patients. The mean \u00b1 SD for EtCO2 and vpCO2 was 35.7 \u00b1 10.1 mmHg and 39.4 \u00b1 10.9 mmHg respectively. EtCO2 and vpCO2 values were highly correlated . The correlations for asthma, bronchiolitis and pneumonia were 0.74 (p < 0.0001), 0.83 (p = 0.0002) and 0.98 (p < 0.0001) respectively. The mean bias \u00b1 SD between EtCO2 and vpCO2 was -3.68 \u00b1 4.70 mmHg. The 95% level of agreement ranged from -12.88 to +5.53 mmHg. EtCO2 was found to be more accurate when vpCO2 was 35 mmHg or lower.Eighty EtCO2 is correlated highly with vpCO2 in non-intubated pediatric patients with moderate to severe respiratory distress across respiratory illnesses. Although the level of agreement between the two methods precludes the overall replacement of blood gas evaluation, EtCO2 monitoring remains a useful, continuous, non-invasive measure in the management of non-intubated children with moderate to severe respiratory distress.EtCO This has had far reaching implications in patient care. End-tidal COsettings.,2 In thece 2000. -5 In botce 2000. -8 EtCO2 sedation -13 and isedation -172 monitoring has proven to be efficacious in diverse clinical areas, its utility in non-intubated patients with pulmonary disease remains undefined. In patients with significant pulmonary disease, it is generally believed that EtCO2 values will not accurately reflect blood gas pCO2 because of ventilation-perfusion mismatch, increased dead space, and/or increased shunt fraction. [Though EtCOraction. -21 In faraction. ,22-25 Ho2 monitoring is less accurate in patients with pulmonary disease, there is a paucity of data assessing its utility as a corollary to blood gas pCO2 in non-intubated pediatric patients with moderate to severe respiratory distress. In this study, we investigated the association of EtCO2 to vpCO2 in hospitalized non-intubated children with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia. We also examined the level of agreement between vpCO2 and EtCO2 to determine if EtCO2 could replace blood gas evaluation in the management of non-intubated pediatric patients with respiratory distress secondary to a pulmonary process.Because of the general assumption that EtCO2 monitoring is standard of care for all patients admitted to the InCU with respiratory distress. The study was approved by the institutional review board of CHB.We performed a retrospective chart review of pediatric patients admitted with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia to the intermediate care unit (InCU) at Children's Hospital Boston (CHB) between July, 2003-February, 2005. The InCU is designed for patients who are moderately to critically ill, who need close monitoring and increased nursing needs, but who do not need invasive monitoring, acute ventilatory support, or vasopressor therapy. Continuous nasal cannula EtCO2 measurement within 10 minutes of each other were eligible for inclusion. Moderate to severe respiratory distress was defined as tachypnea and oxygen saturation < 94% on room air with retractions and decreased aeration on physical examination. Patients with chronic pulmonary disease , cardiac disease, poor tissue perfusion (defined as capillary refill greater than 2 seconds), or underlying metabolic abnormalities were also excluded. Patients undergoing acute respiratory failure defined as immediate subsequent transfer from the InCU to the intensive care unit for invasive respiratory support were also excluded.All patients admitted to the InCU with moderate to severe respiratory distress secondary to above diagnoses and who had a blood gas evaluation and an EtCO2 requirement at time of EtCO2 reading. The patients' pulmonary status including severity of retractions and level of aeration as recorded by InCU nurses on InCU data tracking flowsheet were also included. Retractions and aeration were measured on a scale ranging from 0  to 3 . EtCO2 values were measured using Microstream\u00ae Capnoline\u2122 H nasal cannula  attached to Invivo MDE Escort Prism\u00ae monitors .Data collected on each patient included patient demographics, vitals measurements, and O2 and vpCO2 values was analyzed using the Pearson product-moment correlation coefficient (r) and simple linear regression. Multivariate linear regression was used to assess for variation in bias between EtCO2 and vpCO2 according to clinical and patient characteristics. Bland-Altman analysis was performed to determine the level of agreement between EtCO2 and vpCO2 values.[2 and vpCO2 to the line of unity between the two measurements to further define the relationship between EtCO2 and vpCO2. Statistical analysis was performed using SAS Version 9.1 .The association between EtCO2 values.,27 We al2-vpCO2 values from 62 patients. 40 of the 80 paired measurements were simultaneous. The mean \u00b1 SD time difference between measurements was 0.67 \u00b1 8.19 minutes. The bias between EtCO2-vpCO2 values was not significantly affected by the time difference between measurements (p = 0.6110).There were 80 paired EtCOAge ranged from 5.5 months-20 years with a median age of 5.7 years. Asthma was the admitting diagnosis for 41 patients, bronchiolitis for 9 patients and pneumonia for 12 patients. Other characteristics of our study sample are included in Table SD for EtCO2 and vpCO2 was 35.7 \u00b1 10.1 mmHg and 39.4 \u00b1 10.9 mmHg respectively. EtCO2 and vpCO2 were highly correlated . As shown in Figure The mean \u00b1 SD between EtCO2 and vpCO2 values from the Bland-Altman analysis , respiratory rate (p = .9305), oxygen requirement (p = 0.4222), or clinical assessment measurements such as aeration (p = 0.3876) or retractions (p = 0.4381).The mean bias \u00b1 2 values in which accuracy would be maximized in relation to vpCO2. Figure 2 and vpCO2, which deviated significantly from the line of unity  = 35.5, p < 0.001). The regression line and the line of unity were within 3 mmHg of each other for vpCO2 values under 35 mmHg but parted increasingly in the higher range, differing by 5 mmHg above vpCO2 of 47 mmHg. This suggests that maximum EtCO2 accuracy is achieved when vpCO2 is 35 mmHg or below.Further analysis was conducted in order to identify the range of EtCO2 monitoring in the assessment and management of non-intubated pediatric patients with moderate to severe respiratory distress has remained largely undefined. Historically, studies have found EtCO2 monitoring to have limited accuracy in both intubated and non-intubated patients with pulmonary disease.[2 monitoring is mostly useful in following the trend in ventilatory status and not as a specific correlate to blood gas pCO2.[2 can serve as a direct corollary to blood gas pCO2 in patients without pulmonary disease[2 remains an accurate tool to assess blood gas pCO2.The utility of EtCO disease.,22-25 Begas pCO2.,28 Howevy disease, it rais2 to be highly correlated with venous pCO2 and that this relationship was significant across the range of common respiratory illnesses that cause moderate to severe respiratory distress in children. Only one prior study has evaluated the accuracy of non-invasive capnography in non-intubated pediatric patients with respiratory distress.[2 value to capillary pCO2 in pediatrics patients presenting to the emergency department with respiratory emergencies. Their study also found a high correlation between EtCO2 and blood gas pCO2. .In this study, we found EtCOdistress. Abramo e2 was still highly correlated to blood gas pCO2 and EtCO2 monitoring proved useful in the clinical management of the patients beyond just 'following the trend.'Important differences between this prior study and our current study exist. Specifically, there were differences in the severity and underlying causes of respiratory distress in the study populations. 73% of study participants in the prior study were discharged home from the emergency department and the study included patients with signs of upper airway obstruction that may not have had any direct pulmonary involvement. In our study, patients were admitted with moderate to severe respiratory distress secondary to significant lower tract disease. All patients were determined by the admitting physician not to be safe for floor management and therefore admitted to the InCU. Even in this setting, EtCO2 can replace blood gas pCO2 in the management of non-intubated pediatric patients with moderate to severe respiratory distress. This decision should be based on the level of agreement between the two methods.[2 and venous pCO2, correlation is not a measure of agreement but instead is a measure of association. Perfect agreement exists between two methods only when pairs of measurements lie along their line of unity with a slope of 1 and an intercept of 0. Conversely, perfect correlation between two methods exists when pairs of measurements approximate any straight line.[One of the main objectives of our study was to determine if EtCO methods. Though wght line. For examSD between EtCO2 and vpCO2 in our study was -3.68 \u00b1 4.70 mmHg. This bias did not vary across the averaged values of EtCO2 and vpCO2. Abramo et al[2 and EtCO2 of 3.2 \u00b1 2.4 mmHg. Physiologically, there exists a difference between ideally measured EtCO2 concentration and blood gas pCO2, whether arterial or venous. Assuming a difference between arterial and mixed-venous pCO2 of 2\u20135 mmHg[2 and venous pCO2. Therefore, the observed mean difference of -3.68 closely approximates the true physiologic difference.The Bland-Altman analysis allows for an overall assessment of agreement between two methods. From the Bland-Altman analysis, the bias \u00b1 amo et al found a  2\u20135 mmHg one woul2 and vpCO2 ranged from -12.88 to +5.53 mmHg. This range of EtCO2 values is clinically too imprecise for EtCO2 to replace vpCO2 in the clinical management of patients with respiratory distress. However, knowing the range of EtCO2 values can inform clinical decisions in regards to the management of these patients.The 95% limits of agreement between EtCO2 to accurately reflect the true value of vpCO2, we found on further analysis that the regression line of EtCO2 in Figure 2 and vpCO2 measurements when vpCO2 was below 35 mmHg. At higher vpCO2 values, EtCO2 was not as accurate. Therefore, for values of vpCO2 of 35 mmHg or less, EtCO2 may have an acceptable level of accuracy to serve as indirect measurement of vpCO2 and thereby be a therapeutic guide.In an effort to further define the ability of EtCO2 in assessing the accuracy of EtCO2 to reflect blood gas pCO2 content. Venous pCO2 levels reflect many factors beyond just the ventilatory status of the patient including global oxygen consumption, O2 delivery, tissue perfusion, level of metabolic acidosis, and hypoxia.[2 is not regarded as the gold standard to which EtCO2 should be compared.Our study has several limitations. First, no prior study has used venous pCO hypoxia. Therefor2 in this study was determined mainly by our patient population. Arterial blood gas evaluations in non-intubated pediatric patients are only performed when absolutely necessary secondary to their invasive nature, risk of complication, and high level of pain associated with the procedure.[2 and arterial pCO2 have found a high level of correlation and agreement between the two measures, especially in patients who have good tissue perfusion. [2 values would most accurately reflect the ventilatory status of the patient. Furthermore, by using venous samples for blood gas pCO2 measurement we feel that our study more accurately reflects current practice patterns in the management of pediatrics patients with respiratory distress who are not receiving sedation for invasive ventilatory supportThe use of venous pCOrocedure.,34 In thrfusion. -37 The p2 and blood gas pCO2 were recorded as simultaneous, a number of paired values were not done instantaneously. We limited the time difference between the two measurements to 10 minutes for inclusion in our study. Under ideal circumstances, blood gas evaluations and EtCO2 measurements should be done simultaneously because of the minute to minute fluctuations in CO2 levels. A final limitation of the study was its retrospective nature which may have introduced bias.Another limitation of our study was the timing of measurements. Though most evaluations of EtCO2 and EtCO2. We have also further defined the role of EtCO2 in the management of non-intubated pediatric patients with moderate to severe respiratory distress.Despite these several limitations, we still demonstrated a high correlation and moderate amount of agreement between both vpCO2 as a correlate to vpCO2 in the clinical assessment of pediatric patients with moderate to severe respiratory distress. Though EtCO2 monitoring does not replace blood gas assessment it can serve as an important adjunct in the clinical management of pediatric patients even with significant pulmonary disease. Further study should continue to define the utility of EtCO2 as a measure of blood gas pCO2 in pediatrics patients with moderate to severe respiratory distress.This study demonstrates the utility of EtCO2: End-tidal Carbon Dioxide; vpCO2: Venous Blood Gas Carbon Dioxide; InCU: Intermediate Care Unit; PICU: Pediatric Intensive Care Unit; ED: Emergency Department; CHB: Children's Hospital Boston; SD: Standard Deviation; SE: Standard Error; OR: odds ratio; CI: confidence interval.EtCOThe authors declare that they have no competing interests.JM was the principal investigator, largely responsible for data collection, data analysis, manuscript preparation and revision. JA served the role of research assistant, playing a significant role in data analysis and figure preparation. MA was the primary mentor, specifically involved in study design, data analysis, manuscript preparation and revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Renal cell carcinomas (RCC) are among the most common and most lethal genitourinary malignancies. GOLPH2  has recently been proposed as a biomarker for hepatocellular and prostate cancer. In this study we analysed the expression patterns and the prognostic and diagnostic value of GOLPH2 in RCC.GOLPH2 protein expression was analysed by immunohistochemistry in 104 clinically well characterized RCC cases in comparison with matched normal kidney tissue and in association with clinico-pathological parameters. Statistical analyses including Kaplan Meier analyses were performed with SPSS version 15.0.GOLPH2 was highly expressed in normal renal tubules and in almost half of RCC with a statistically significant predominance in the papillary and chromophobe histological subtypes. No other associations with clinico-pathological parameters were detectable. The Kaplan-Meier curves showed a weak trend for unfavourable prognosis of tumours with high GOLPH2 expression, but failed significance.GOLPH2 protein is expressed in normal renal tissue  and is down-regulated in the majority of clear cell RCC. In papillary and chromophobe RCC GOLPH2 expression is consistently present. In contrast to its diagnostic value in hepatocellular and prostatic carcinomas, a prognostic or diagnostic value of GOLPH2 in RCC appears to be unlikely. Renal cell cancer (RCC) is one of the most common genitourinary malignancies and causes of cancer associated death in the United States of America in 2008 . AlthougGOLM1 gene on chromosome 9q21.33, first described by Kladney et al. in liver tissue of a patient with giant-cell hepatitis [GOLPH2 is a golgi phosphoprotein  of yet unknown function. The 73 kDa Golgi apparatus associated protein is coded by the Until now only few studies on GOLPH2 exist. In liver diseases GOLPH2 has been described as a potential serum marker of hepatocellular carcinoma . RecentlIn this study, we carefully analysed the GOLPH2 protein expression in a well characterized renal cell cancer cohort with matched normal tissue. Central aim was to evaluate the potential diagnostic and prognostic value of GOLPH2. We found GOLPH2 differentially expressed between normal and malignant renal tissue and between the different RCC subtypes, but a prognostic value could not be detected.th September 2004.One-hundred-four patients  diagnosed for renal cancer at the Institute of Pathology, Charit\u00e9 \u2013 Universit\u00e4tsmedizin Berlin between 2003 and 2005 were enclosed in this study. The study has been approved by the Charit\u00e9 University Ethics Committee under the title \"Retrospective Untersuchungen von Gewebeproben mittels immunhistochemischer F\u00e4rbung und molekularbiologischer Methoden\"  on 20Patient age ranged between 28 and 92 years with a median of 62. Histological diagnosis was established according to the guidelines of the World Health Organization. Cases were selected according to tissue availability and were not stratified for any known preoperative or pathological prognostic factor. 83 (79.8%) patients had a clear cell RCC (ccRCC), 16 (15.4) a papillary RCC and 5 (4.8%) a chromophobe RCC. Twenty-one patients had systemic disease (M1) at the time of diagnosis. Clinical follow-up data, as annually assessed survival time was available for all patients. The median follow-up time of all cases was 30 months, ranging from one to 47 months. 21 of the patients died from renal cancer. The pT status was as follows: pT1 \u2013 53 (51.0%), pT2 \u2013 3 (2.9%), pT3 \u2013 45 (43.3) and pT4 \u2013 3 (2.9%). Ten patients (9.6%) had pathologically confirmed nodal metastases . 50 (48.1%) patients had no nodal metastases (pN0). For 44 (42.3%) patients no lymph nodes were histologically examined (pNx). Tumour grades were G1 \u2013 11 (10.6%), G2 \u2013 74 (71.2%), G3 \u2013 15 (14.4%) and G4 \u2013 4 (3.8%) respectively.A tissue-micro-array (TMA) was constructed to represent 108 cases, as previously described . The tisThe TMA blocks were freshly cut (3 \u03bcm) and mounted on superfrost slides (Menzel Gl\u00e4ser). Immunohistochemistry was conducted with the Ventana Benchmark automated staining system  using Ventana reagents for the entire procedure. To detect GOLPH2, a commercially available antibody . To achieve a greater uniformity of the evaluation, the first step was to construct a panel with four illustrative examples pictures, of which a hardcopy lay next to the microscope. We used a 10% threshold to determine positivity, irrespective of the intensity grade. Only tumours without any GOLPH2 immunoreactivity or with staining of less than 10% of the tumour cells were considered negative. To delineate between low and high levels of GOLPH2 expression, tumours with moderate to strong GOLPH2 expression (2&3) and tumours with none to weak (0&1) staining intensity were lumped.2-tests were applied to assess the statistical significance of the associations between GOLPH2 expression and clinico-pathological parameters. Univariate survival analysis was carried out according to Kaplan-Meier, differences in survival curves were assessed with the Log rank test. P values < 0.05 were considered significant.Statistical analysis was performed using SPSS, version 15.0. Fisher's exact test, \u03c7GOLPH2 immunostaining was easy to evaluate with the typical cytoplasmic and in most cases perinuclear accentuated staining pattern. In normal renal tissue we detected GOLPH2 with its characteristic fine granular staining pattern more often and with a stronger staining intensity in the distal tubules than in proximal tubules Figure . For 97 Staining intensities of GOLPH2 in RCC were: negative \u2013 29 (27.9%), 1+ \u2013 30 (28.8%), 2+ \u2013 34 (31.5%) and 3+ \u2013 11 (10.6%). GOLPH2 positivity was significantly higher in papillary and chromophobe RCC if compared to clear cell RCC , GOLPH2 had higher serum levels if compared to healthy individuals and has been proposed as a novel serum marker of HCC ,15. HoweEven though in a previous study from our study group (manuscript in preparation) we were able to confirm the GOLPH2 expression in HCC, we also detected GOLPH2 in various other malignancies on a multi-tissue-micro-array which argues against a liver-specific relevance of this biomarker.In summary, this first systematic analysis of GOLPH2 in renal cell cancer describes that GOLPH2 expression is not restricted to liver or prostate cancer but is also found in a higher proportion of RCC, although no direct diagnostic or prognostic value of GOLPH2 as a tissue biomarker could be confirmed.In this tissue-micro-array-based immunohistochemistry study a differential expression of GOLPH2 in normal and malignant renal tissue was demonstrated. While distal tubular epithelia were mainly strongly positive for this marker, other parts of the nephron and the majority of the clear cell RCC were negative. The diagnostic use of the rather constant positivity of papillary and chromophobe RCC is limited since one third of the clear cell carcinomas were also positive for GOLPH2. No other associations with conventional clinico-pathological characteristics were found and no prognostic value of GOLPH2 was demonstrated which limits the diagnostic value of GOLPH2 in RCC.The authors declare that they have no competing interests.FRF coordinated the study, performed immunohistological and statistical analyses and wrote the paper. MOR contributed to statistical analyses and revised the paper. MD and KJ provided samples and clinico-pathological data and supported statistical analyses. HM provided logistical support for the study and revised the paper. GK conceived and coordinated the study, performed immunohistological and statistical analyses and revised the paper.The pre-publication history for this paper can be accessed here:"}
+{"text": "A mouse of monoclonal cell line (L1) was produced by fusing the mouse myeloma P3X63/Ag8 with CD2F1 spleen cells immunized with a highly immunogenic subline of L1210 leukaemia (L1210/DTIC). A very few positive clones (1%) were isolated and one of these was chosen for detailed study. The monoclonal antibody L1 is an IgM immunoglobulin strongly reacting in a complement-dependent cytotoxicity assay against L1210/Cr leukaemia and its more or less immunogenic sublines. The specificity of the L1 antibody against L1210 leukaemia was studied by extensive screening with normal adult and foetal tissues, lymphoid tissues from several independent strains and a panel of the most common experimental tumours, to all of which it was unreactive. Attempts at immunotherapy were carried out in DBA/2 mice challenged with L1210 leukaemia and treated with L1 (ascites) and complement. Although the in vitro cytotoxic titre of ascites fluid from mice bearing hybridoma was very high (10(-7)), no therapeutic effect was obtained in vivo."}
+{"text": "P = 0.004 and 0.019 respectively). These results suggest that loss of these regions is associated with recurrence of TCC. Further investigation is required to identify genes in these regions, which might be responsible for driving recurrence in TCC of the urinary bladder. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comApproximately 2/3 of patients diagnosed with superficial transitional cell carcinoma of the urinary bladder (TCC) will recur within 2 years. Loss of chromosome 9 and loss of heterozygosity (LOH) at 9q34 in index TCCs identify a subset of patients at high risk of recurrence. This study explores genetic alterations on chromosomes 4, 8, 11 and 17 as predictors of recurrence. A total of 109 carcinomas were investigated at 26 loci. DNA was extracted from microdissected archival normal/tumour tissue and was analysed for loss of heterozygosity (LOH). Fluorescent PCR was performed and genotyping carried out on a Perkin Elmer ABI377 sequencer. LOH of D11S490 or D17S928 was significantly more frequent in index carcinomas of patients who experienced recurrence compared to those with no recurrence ("}
+{"text": "RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal, adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3 protein in 151 paraffin-embedded colorectal tissues  was determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among 120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to tumour differentiation . Also, levels of RARRES3 protein were found to be higher in the normal tissues adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to the adjacent normal tissues . The prognostic implication of RARRES3 protein expression was studied in 107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that altered RARRES3 expression occurs late in colorectal carcinogenesis. RARRES3), also named as TIG3  and mutations of DNA mismatch repair genes coincided with the transition to malignancy  and consisted of 100-\u03bcg RARRES3 peptide mixed with the incomplete Freund's adjuvant. Titres of the antiserum were determined using an enzyme immunoassay. The specificity of the antiserum was determined by Western blotting of cytosol extracts prepared from cells expressing the RARRES3-fusion protein  were obtained from 68 male and 52 female patients with a mean age of 64.2 years. The distribution of tumours according to the level of differentiation and Dukes' stages are listed in Table 1 tumours . Primary tumours .\u03bcg of RARRES3 peptide at 4\u00b0C overnight. Samples were spun at 100\u2009000\u2009g at 4\u00b0C for 10\u2009min before adding the absorbed antisera to the tissue section. Sections were also stained with p21WAF1 and Ki-67 monoclonal antibodies obtained from DAKO Co.Tissue sections were air-dried, deparaffinised, and then boiled twice for 2\u2009min in 10% DAKO ChemMate\u2122 solution  containing 0.05% NP-40 (Nonidet P-40). The DAKO LSAB\u00ae2 Peroxidase kit was used to stain the expression of RARRES3 protein in tissue sections. Tissues were incubated with RARRES3 antiserum or preimmune serum at a dilution of 1 to 800 at room temperature for 1\u2009h. The sections were lightly counterstained with Mayer's haematoxylin. To verify antibody specificity, RARRES3 antisera were preincubated with 8\u201330\u2009Patterns of staining, cellular RARRES3 localisation, staining intensity and percentage of RARRES3 expressed cells were recorded. The evaluation of staining patterns was performed using the immunoreactive score (IRS) proposed by The nonparametric Kruskal\u2013Wallis tests were applied to compare IRS of RARRES3 associated with tumours at various levels of differentiation as well as Dukes' stages. Further, Dunn's procedure was applied to compare the IRS between groups. Logistic regression analyses were used to assess the association and trend between tumour differentiation and chance of positive RARRES3 staining and chance of adjacent normal tissues having RARRES3 expression higher than that of tumours, while controlling for potential confounding factors, that is, subject's gender and age. Survival rates were calculated using the Kaplan\u2013Meier method. Significance was calculated by the log-rank test. To further validate the effect of RARRES3 staining on survival, a multivariate Cox proportional hazard method was used to adjust for age and stage.\u03bcg of RARRES3 peptide resulted in the suppression of staining . The better the tumour tissue differentiation, the higher the IRS was rated. A significant linear trend was found in tumour differentiation and RARRES3 expression (P<0.0001). No significant association was found between RARRES3 IRS and Dukes' stages.Levels of RARRES3 protein varied among 120 tissues of colorectal adenocarcinoma . When RAP for trend <0.0001).We also compared levels of RARRES3 protein between adjacent normal and tumour tissues within the same tissue slide among 120 carcinoma tissues (Table 2P=0.883). Similarly, multivariate analysis showed no difference in survival between patients with negative and weak (P=0.929) or between negative and strong (P=0.292) RARRES3 staining in tumours.Altogether, 107 patients with colorectal adenocarcinoma were examined for prognosis related to RARRES3 expression. A total of 23 tumours were stained negative for RARRES3 protein, 25 tumours had weak RARRES3 expression and 59 tumours had strong RARRES3 expression. Kaplan\u2013Meier survival curves for the different groups are presented in Figure 3RARRES3 is a retinoid-inducible tumour suppressor. This study shows that RARRES3 protein is expressed in mucosal tissues of normal colon and rectum, which is correlated to epithelial differentiation. The premalignant adenoma tissues expressed the RARRES3 protein at high levels. Within tumour tissues, the expression of RARRES3 protein is positively correlated to tumour differentiation. Compared to the adjacent normal mucosal tissues, tumours with moderately and poorly differentiated adenocarcinoma had significantly reduced RARRES3 expression. However, survival analysis showed that RARRES3 protein was not a prognostic marker for patients with colorectal adenocarcinoma.WAF1 . In this study, RARRES3 protein was expressed at high levels in tissues of normal colorectal mucosa and the precancerous lesion adenoma. The decrease in RARRES3 expression was only observed in carcinoma tissues, which are unclassified by the Dukes' system, and also observed in tissues at Dukes' stages A and B. Therefore, the decrease or loss of RARRES3 expression may occur in the early stages of colorectal carcinoma. To our knowledge, deletion at chromosome 11q12, where RARRES3 is localised, has not been reported in tissues of colorectal carcinoma. Also, downregulation instead of mutation of the RARRES3 gene was found during progression of B-cell lymphocytic leukaemia (Genetic alterations have been studied extensively during steps of colorectal carcinogenesis. Mutations in the adenomatous polyposis coli and KRAS oncogenes arose in precancerous lesions. Aberrations in TP53, DCC and SMADs coincide with transition to malignancy (In conclusion, this and previous studies have demonstrated the close association of RARRES3 expression in differentiated epidermis and colorectal epithelium. Progressive loss of RARRES3 expression in tissues of moderately and poorly differentiated colorectal adenocarcinoma supports the tumour-suppressive role of RARRES3 in moderately and poorly differentiated colorectal cancer. However, RARRES3 alone was not a prognostic marker for colorectal adenocarcinoma. RARRES3 protein is shown to suppress the downstream signal pathway of Ras. The significance of the loss of RARRES3 expression along with mutations in Ras will be investigated in the future."}
+{"text": "P = 0.009). There was a parallel increase with severity of lesions for the trend in proportions of cases demonstrating p16 inactivation or cyclin D1 overexpression . For Ki67, a marker of cell proliferation, this trend was not significant (P = 0.08). Human papillomavirus infection was only found in 4 cases among the 48 samples tested (8.3%). In conclusion, progression of disease is accompanied by a parallel and continuous increase in telomerase activity and alterations in cell cycle regulators , as proposed by in vitro models. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comAlteration of the p16/pRb pathway may cooperate with telomerase activation during cellular immortalization and tumour progression. We studied p16 expression status by immunohistochemistry and telomerase activity using the TRAP assay in 21 premalignant lesions of the head and neck epithelium as well as 27 squamous-cell carcinomas. We also examined expression of other components of the pathway (cyclin D1 and pRb) as well as presence of human papillomavirus genomes which can target these molecules. 4 of 9 mild dysplastic lesions (44%), 8 of 12 moderate/severe dysplastic lesions (67%), and 25 of 27 squamous-cell carcinomas (92%) demonstrated high telomerase activity ("}
+{"text": "Intracardiac injection, in hooded Lister rats, of syngeneic MC28 sarcoma cells never induced tumour growth in normal bowel. Tumour growth occurred at the site of a colonic anastomosis if surgery preceded tumour injection but not if it followed tumour injection, even by as little as 1 h. Maximum enhancement of tumour growth occurred when the healing process had progressed between 2 and 8 days, with a peak at 5 to 7 days. The enhancing effect was largely over by the time the healing had progressed 14 days. The syngeneic OES5 breast carcinoma also grew at colonic anastomoses when surgery preceded tumour injection by 5 days, but not in normal colon. The MC28 sarcoma also grew at ileal anastomoses but not in the normal ileum after intracardiac injection. By injecting radiolabelled sarcoma cells, an estimate of the probability of a single bloodborne tumour cell lodging at a colonic anastomosis and leading to a tumour deposit was calculated to be of the order of 1:43 whereas the probability of the cell lodging in normal colon and causing a deposit is less than 1:4 x 10(4)."}
+{"text": "A germ line single nucleotide polymorphism (SNP) in the first intron of the gene encoding MDM2 at position 309, an important modulator of p53, has been described. BRCA1/2 mutation have been associated with increased rates of breast cancers with mutated P53. It was shown that the presence of MDM2 309 SNP correlated with younger cancer onset age in individuals with a p53 mutations. The differential effects of this SNP were also linked to estrogen receptor activation. Here we report on our study of 453 Ashkenazi breast cancer patients of whom 180 were positive for the known Ashkenazi BRCA1/2 mutations\u00ae statistical package , and JMP\u00ae software .DNA from breast cancer patients was obtained for analysis of one of the three common BRCA1/2 mutations and MDM2 SNP309. Data regarding cancer onset and death ages was obtained from our database and Statistical analysis was performed using the SPSSThe percentage of MDM2 SNP309 in control and BRCA 1/2 population which is similar to that reported for other Jewish Ashkenazi populations at 52.2% for the heterozygotes and 25.0% for MDM2SNP309G/G and 22.8% for MDM2SNP309T/T.There was not a statistical significant difference in median age of disease onset in the different MDM2 SNP309 subgroups of the BRCA1/2 carriers. When we further divided the group into under and above 51 years old ( presumed menopause age) in the BRCA1 positive subset we found that there were less patients of the MDM2SNP309 G/G versus the MDM2SNP309 T/T in the over 51 patient group (p = 0.049). This result has been obtained in a relatively small subgroup and is of borderline statistical significance. Interestingly, in the BRCA1/2 mutation carriers, we found a survival advantage for patients harboring the SNP309 G/G genotype (p = 0.0086) but not for the 272 patients not harbouring this mutations.MDM2SNP309G/G main effect on BRCA1/2 positive mutation carriers is linked to its effect on patients survival. Further research is needed in order to understand the reason for this difference. MDM2 regulates p53 by targeting its destruction through the ubiquitin pathway and also by directly blocking p53 transcriptional activity. A singlTwo recent articles have studied this subject again with somewhat conflicting results. Yarden et al , studiedSeveral studies have found negative correlations between MDM2 tumor expression and prognosis in various cancers including breast, ovarian and brain -9. Two sEstrogen seems also to influence the magnitude of the MDM2 SNP309 G/G effects on MDM2 expression. Estrogen receptor which has a DNA binding site close to the Sp1 site which is directly influences by MDM2 SNP309 G/G. Bond, Levine and colleagues have described a significant correlation between MDM2 SNP309 G/G and the prevalence of diffuse large cell lymphoma and soft tissue sarcoma, in women under the age of 51  . This fiWe have obtained DNA samples from a large group of breast cancer patients  for analysis of BRCA1/2 mutations and modifier genes. Here we describe the interactions we found out in this group between MDM2 SNP309 G/G, BRCA1/2 mutations and clinical findings including cancer onset age, survival, and the presence of other cancers.DNA was from breast cancer patients obtained for analysis of their BRCA status and modifier genes , and JMP\u00ae software . Assessment of the correlations between genetic carrier status and MDM2 was carried out using the Fisher Exact test or Chi-Squared test. A p value < 0.05 was considered significant. Similar methodology was used to assess the correlation between the frequency of other cancers and MDM2 SNP309 statusFollow up was calculated from the time of diagnosis to date of last follow-up. The rate of death was estimated using Kaplan-Meier methods. Statistical analysis was performed using the SPSSWe evaluated DNA samples from 180 Ashkenazi breast cancer BRCA1/2 positive . There was a distinct survival advantage in the BRCA1/2 mutation carrier group for the patients homozygous for G/G at the -309 position, compared to the patients harboring the SNP309 G/T or T/T Fig . Cox mulIn the current study we analyzed the relationship between MDM2 309 SNP status and cancer in a large group of Ashkenazi patients including a large group of BRCA1/2 mutation carriers Our results revealed high percentage of MDM2 309 SNP in this population of around a quarter of all women. This high prevalence of MDM2 309 SNP G/G in Ashkenazi women has been demonstrated by others before us. Yarden and colleagues recently presented data supporting the notion that the relative enrichment in MDM2 309 SNP G/G in Ashkenazi is a result of a selection process favoring this single nucleotide polymorphism. CurrentWe did not detect significant difference in cancer onset age in BRCA1/2 carriers harboring the different MDM2 SNP309 Fig  and 1d. The effect of the MDM2 SNP 309 G/G on cancer risk has been recently reviewed in a meta- analysis by Wilkeming and colleagues. These aWhen combining the BRCA1 and BRCA 2 mutation carriers we noted significantly better prognosis for the MDM2 SNP309 G/G carriers than for the other MDM2 SNP309 carriers (p = 0.0086). This are supposedly the tumors with higher levels of MDM2 expression. Cancers in BRCA1 and BRCA2 mutation carriers have substantial difference between them. HoweverSince MDM2 is an oncogene it seems counterintuitive that patients in which higher levels of MDM2 are expected will have longer survival. A possible explanation for such a counterintuitive result is based on our understanding of the role of p53 in carcinogensis. The presence MDM2 SNP309 G/G may lead to the overexpression of MDM2 and therefore cause downregulation of p53 in an effective manner and render mutational inactivation of p53 unnecessary. Thus tumors with MDM2 SNP309 G/G might still harbor a potentially active p53. Indeed, when Allazzouzi and colleagues studied p53 in colon cancer of SNP309 G/G carriers), they found a higher tumor prevalence of non dominant p53 mutations. When p5Therefore a possible scheme to explain a role for MDM2 SNP309 G/G in BRCA tumors could be that overexpression of MDM2 in BRCA1/2 tumors results in down-regulation of p53 and allows the development of BRCA1/2 tumors similar to the effect induced by a mutation in p53. Cells overexpressing MDM2 still contain p53 that in certain conditions. in which MDM2 is inactivated might be functional. As described MDM2 is inactivated by DNA damaging agents which includes chemotherapies. This hypothesis concurs with the hypothesis suggested by Mathoulin-Portie in their study of anthracyclin treated breast cancer women . This has been proposed as one of the main mechanisms of oncogenic transformation induced by this protein. MoreoveRecently two articles assessing the relationship between patients' survival and the MDM2 SNP309 were published. However, it is important to note that the patients in those studies suffered from a different disease than those in the current study and were treated with different therapies than those commonly used for treatments of breast cancer.Our data demonstrate that patients with BRCA1/2 breast cancers harboring any of the three MDM2 SNP309 had similar cancer onset ages. Only by studying the over 51 years old group of patients  we could detect a difference between the different MDM2 SNP309 genotypes \u2013 only in the BRCA1 mutation carriers. The relative percentage of the MDM2SNP309G/G carriers was reduced in this group of patients compared to the younger than 51 years old (p = 0.049). While this results concurs with those of Yarden and colleagues they were obtained from a small number of patients and thus need reaffirmation in larger studies.MDM2 SNP309G/G BRCA1/2 carriers had significantly longer survival compared to the combination of the other MDM2 SNP309 subgroups (p = 0.0086). This somewhat surprising result may be a result of differential sensitivity to adjuvant therapy in this subgroup. Further studies on MDM2 SNP309 may provide important insights as to factors effecting tumorigenesis and drug sensitivity in BRCA1/2 mutation carriers.The authors declare that they have no competing interests.Initiation of research HN, TP. Build up of database TP, TH, LK Collection of samples, DNA and statistical analysis \u2013 TH, SM. Discussion and Analysis of results HN, EP, TP, NS, LK.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/60/prepub"}
+{"text": "Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53\u2236MdmX and Mdm2\u2236MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation. A Molecular Interaction Map (MIM) akin to a circuit diagram of an electric device can give a comprehensive view of cellular processes and help understand complex protein functions in cells. To this end, we generated a MIM focused on the p53-Mdm2-MdmX network proteins and performed computer simulations to help understand how Mdm2 and MdmX may regulate p53. Proper regulation of p53 is important for cell survival: elevated levels of p53 can lead to cell death, and decreased levels of p53 can lead to cancer. Mdm2 and MdmX are structurally homologous proteins that regulate p53. Mdm2 negatively regulates p53 by degradation, but MdmX regulation of p53 is not well understood. Recently, Mdm2 and MdmX have been recognized as potential cancer therapeutic targets. In an effort to better understand how MdmX can alter the p53 activity under various conditions, we used mathematical models based on the MIM network to generate hypotheses that can be tested by experiments. Our simulations suggest that MdmX may increase the dependency of p53 on Mdm2 or dampen p53 oscillations during DNA damage response. The transcription factor p53 is a tumor suppressor that causes cell cycle arrest or apoptosis in response to stress signals Mdm2 is a ubiquitin ligase that negatively regulates p53 by promoting ubiqutin-dependent p53 degradation In our model, MdmX is a key component. MdmX interacts with p53 or Mdm2 to form transcriptionally inactive p53\u2236MdmX or enzymatically inactive Mdm2\u2236MdmX. In order to model the interactions of MdmX with Mdm2 and p53, we selected a system of elementary processes that focused on the non-enzymatic interactions between MdmX and other molecules. Since the values of many kinetic constants for our model are unknown, we selected initial kinetic parameter sets to explore potential interesting behaviors. Then, we searched for regions of parameter space that showed biologically interesting behaviors and reproduced previously observed dynamic behaviors, including DNA damage induced oscillations To lay the foundation of a model, we first assembled the known molecular interactions among p53, Mdm2, and MdmX in the form of a heuristic MIM using the previously described notation http://discover.nci.nih.gov/mim)Four pathways of p53 regulation, functioning simultaneously, are highlighted in P53 activates  the transcription of target genes including Mdm2 and p21 (A1). Phosphorylation  and oligomerization  of p53 enhance the transactivation function (A3).p53 ubiquitination (A10) is mediated by the ring domain of p53-bound Mdm2 , and the ubiquitinated p53 undergoes degradation . Oligomerized p53 is more efficiently ubiquitinated and degraded (A13) than monomeric p53 because oligomerization of p53 enhances p53-Mdm2 binding. Ubiquitinated p53 can be deubiquitinated by Hausp (A12). Mdm2 binds to the transactivation domain (TAD) of p53 (A11), and MdmX competitively binds to the same site of p53 (A18). Binding between p53 and MdmX is stimulated by MdmX phosphorylation, mediated by Casein Kinase 1-alpha  . It is not known whether CK1-alpha is regulated by DNA damage. DNA damage-induced phosphorylations of p53 (A15) and Mdm2 (A17) coherently inhibit  the formation of p53\u2236Mdm2 heterodimer, thereby allowing the active forms of p53 to accumulate. P53 Ser20 phosphorylation attenuates the binding between p53 and MdmX (A21).Mdm2 is auto-ubiquitinated by the Mdm2 ring domain , and the ubiquitinated Mdm2 undergoes degradation . Mdm2 may have to form a homodimer to autoubiquitinate (A26), but it is not clear whether the autoubiquitination is in cis or trans. The Mdm2 autoubiqutination is enhanced (A27) by an interaction between an orphan receptor, TR3, and p53 (A28). Ubiquitinated Mdm2 can be deubiquitinated by Hausp (A29), and the deubiquitination is inhibited (A30) by DNA damage induced phosphorylation of Mdm2 at an unknown site (A31).The ring domains of MdmX and Mdm2 can bind each other to form an oligomer (A36), and MdmX is ubiquitinated by the ring domain of bound Mdm2 (A34). Ubiquitinated MdmX undergoes degradation . Ubiquitinated MdmX can be deubiquitinated by Hausp (A39). DNA damage induced phosphorylation controls the level of MdmX coherently via several pathways. DNA damage-induced MdmX phosphorylation (A38) enhances (A37) the binding between Mdm2 and MdmX (A36) which can result in the increased ubiquitination of MdmX (A35). MdmX phosphorylation (A38) inhibits (A40) the deubiquitination (A39) of MdmX by Hausp, which in turn increases ubiquitinated MdmX showing coherent regulation by DNA damage-induced kinases.Model simulations, based on the explicit MIM presented in In further simulations, an increased basal production rate constant (k6) of Mdm2 resulted in a decrease in p53 activity : negativWe hypothesized that the ability of MdmX to induce a sharp dependence of p53 activity on Mdm2 might be accounted for by the formation of MdmX\u2236p53 and Mdm2\u2236MdmX heterodimers. We tested this possibility in pre-equilibrated systems by examining the effects of heterodimer production rate constants  on the p53 responses to DNA damage. Transient increases of p53 activity   were obsPrevious studies indicated that the cellular response to DNA damage involves oscillatory behaviors at the molecular level The results showed that, in an oscillating system, MdmX (k15\u200a=\u200a10) tended to dampen or reduce the amplitude of oscillations  comparedNext we examined the effect of p53 level on the oscillatory behavior. p53 level was varied by changing the p53 basal production rate, k1. The results are shown in a bifurcation diagram . The oscThus, as expected, steady state of p53 activity (species 14) increases as the basal production rate constant (k1) of p53 increases , it begiAfter specific behaviors of interest were identified from the initial simulations, a wider range of kinetic parameter space was surveyed with the full model  using a In this search, we found regions of parameter space that exhibit oscillations and Nutlin responses that are consistent with experimental observations, and used the resulting parameter sets to simulate the effect of MdmX. These surveys were performed in two steps see . In the Nutlin is a drug that blocks p53 and Mdm2 interactions, and it was shown that Nutlin induces basal transcription activity of p53 without phosphorylation Sensitivity analyses were performed for two selected sets of kinetic parameters that were used for further simulations. The normalized local sensitivity of mRNA  during the first 180 min post DNA damage was considered as p53 activity and measured as output while basal production rate of Mdm2 (k6) was varied. Examples of the transient dynamic behavior observed when the production rates of Mdm2 and MdmX were varied are shown in  of MdmX ; for somover 50% . Such Mdover 50%  at some over 50% . As showover 50% .Late DNA damage response  with a full model  was examIn order to investigate the role of MdmX in the p53-dependent response to DNA damage, we prepared a heuristic MIM of the pathways leading from DNA damage to p53 activity , from whThe known pathways of p53 regulation are highlighted in four panels in Enhancement by p53 multimerization of the binding between p53 and Mdm2 [A13].Phosphorylation by Casein Kinase 1 alpha and its effect in binding between p53 and MdmX .Autoubiquitination of Mdm2  became a major reservoir during the pre-equilibration with low Mdm2 or when Mdm2\u2236MdmX (species 9) became a major reservoir during the pre-equilibration with high Mdm2 . Figure Unlike some previously reported switch-like behaviors arising from steady states Previous studies showed sustained oscillations of p53 during the long-term response to DNA damage Kinetic parameter space was explored in two stages. (1) During the first stage, two oscillatory regions, OSC1 and OSC2, were identified. The characteristic difference between the OSC1 and OSC2 clusters was the phosphorylation rate constant k3 in . Narrow The two groups of parameter sets derived from OSC1 or OSC2 underlie MdmX-sensitive and MdmX-insensitive models. It is likely that physiological condition will be explained better by one of those kinetic parameter sets. Experiments to test whether the over-expressed MdmX can in fact dampen DNA damage induced p53 oscillations will allow us to discriminate between the two models.Our theoretical study showed, with little prior knowledge, potentially interesting roles of MdmX in p53 regulation. Many of the kinetic constants used in the study were not biologically constrained, and some of the assumptions made in the models need to be biologically validated. The lack of prior knowledge demands rigorous experimental validation of the simulated results; however, it also allows the unbiased investigation of possible functions that are not intuitively obvious. The MdmX roles predicted in this study are not simply linear increases or decreases of p53 activity dependent upon the level of MdmX; the study suggests that manipulation of MdmX to alter p53 activity requires careful investigation of the dynamics of three proteins: MdmX, Mdm2, and p53. An experimental system with inducible MdmX and Mdm2 and a reporter protein to monitor p53 activity should allow us to test whether the switch-like behavior (increased dependency on Mdm2 by MdmX) or dampening of oscillations are observed. To observe the switch-like behavior, transient p53 activity has to be monitored, and it may require monitoring the p53 activity relative early  after DNA damage. To observe dampening of oscillations by MdmX, a long period of observation (>12 hr) will be necessary since the known peak-to-peak interval is approximately 6 hours. As shown in one example plot in the result section with the full model, the degree of level change in the switch-like effects by the increased MdmX was approximately 30%\u223c50% increase (low Mdm2) or decrease (high Mdm2) of p53 activity, and that level of change may require high sensitivity to measure p53 activity in experiments.We used highly simplified p53 models in this study because we can interpret the simulation results more easily. However, p53 is involved in many other regulations and feedback loops, and it is not obvious whether the conclusions will still hold in a larger p53 system. Although experimental verification will partially address the question, we could also proceed theoretically by introducing additional components successively to the current model and by surveying parameter space with an updated fitness function. The extension of the current model might include Wip1, which is known to trigger recurrent initiation of ATM activity in some cells One goal of this study was to understand how non-enzymatic interaction of MdmX with p53 and Mdm2 can regulate p53 during the response to DNA damage. The mechanism of p53 regulation by MdmX is poorly understood, at least in part because the role of Mdm2\u2236MdmX heterodimer in p53 ubiquitination and degradation is not clearly understood. It was previously speculated that p53\u2236MdmX heterodimer may serve as a reservoir to maintain a pool of p53 First, a heuristic molecular interaction map (MIM)  was consThe kinetic constants used for initial simulations were generated based on collected kinetic constants from previously published models As input, rate constants of phosphorylation (k3\u200a=\u200ak8\u200a=\u200ak17) and basal production rate of MdmX k15 in  and Mdm2We considered that DNA response may display bi-phasic responses: early and late responses. During initial simulations with the simple model , early rTo set realistic initial condition for early DNA damage response, cells without DNA damage were simulated by pre-equilibrating the model in the absence of DNA damage induced kinase activities (k3\u200a=\u200ak8\u200a=\u200ak17\u200a=\u200a0). For late DNA damage response, simulations were performed without pre-equilibration. DNA damage was simulated by setting all the rate constant of phosphorylation as a same positive value (k3\u200a=\u200ak8\u200a=\u200ak17>0) unless otherwise indicated. The rate constant of phosphorylation was kept constant during the simulation of DNA damage response because quite rapid saturation of ATM activity followed by a constant level of ATM activity up to 24 hr were observed in a previous study The bifurcation diagram shown in To search parameter space, we used a previously described algorithm The searches were done in a two step procedure. First, a subnetwork including a subset of interactions and kinetic constants was selected, based on their previously predicted roles in oscillatory behaviors The normalized local sensitivity of mRNA (species 15 in jLS) of the normalized local sensitivity was obtained by trapezoidal numerical integration The local sensitivity calculation was performed using the Simbiology toolbox in Matlab. The time integral  was measured because the full model include basal p53 activity (transcription activity by p53 monomer and promoter complex). Early DNA damage response was quantified by the maximum p53 activity (species 15) achieved during 0\u223c180 min after DNA damage; this period corresponds to the first rise in p53 activity after DNA damage The network was pre-equilibrated without Nutlin (k11>0) and without DNA damage (k3\u200a=\u200ak8\u200a=\u200ak17\u200a=\u200a0). For the simulation of Nutlin treatment, we simulated a network with k11\u200a=\u200a0, because Nutlin is known to affect Mdm2-p53 interaction only Some of example matlab scripts used for simulations were provided as supplemental information , S6, S7.Figure S1An informal diagram of (1.11 MB EPS)Click here for additional data file.Figure S2Regenerated example plots in (0.08 MB TIF)Click here for additional data file.Figure S3Effects of p53\u2236MdmX and Mdm2\u2236MdmX heterodimer reservoirs on p53 oscillatory behavior. The model is based on (0.64 MB TIF)Click here for additional data file.Figure S4MdmX-induced reduction of the amplitude of oscillating p53. This simulation included the p53-Mdm2 feedback loop (k33>0) with a time delay and without the TR3-mediated positive feedback loop (k14\u200a=\u200a0), and the model is based on (0.28 MB TIF)Click here for additional data file.Figure S5Comparison of experimental and simulated Nutlin responses. Simulations were performed based on the model in (1.35 MB EPS)Click here for additional data file.Figure S6Simulations were performed based on the model in (0.14 MB TIF)Click here for additional data file.Figure S7ijLS) of mRNA Click here for additional data file.Figure S8Region of switch-like behaviors as a function of association rate constants of p53\u2236MdmX and Mdm2\u2236MdmX. Simulations were performed based on the model in (0.26 MB TIF)Click here for additional data file.Figure S9Prediction of late response to DNA damage. Simulations were performed based on the model in (0.81 MB EPS)Click here for additional data file.Figure S10Distribution of each kinetic parameter in cluster OSC1 (A) and OSC2 (B) in (0.18 MB TIF)Click here for additional data file.Table S1Annotations of Mdm2-p53-MdmX MIM.(0.13 MB DOC)Click here for additional data file.Table S2Initial condition.(0.04 MB DOC)Click here for additional data file.Table S3Rank ordered time integral (LSj) of the normalized local sensitivity of the kinetic parameter sets from (A) (0.11 MB DOC)Click here for additional data file.Table S4Ordinary differential equations for (0.03 MB DOC)Click here for additional data file.Table S5Kinetic constants used in previous mathematical models.(0.04 MB DOC)Click here for additional data file.Table S6Assumptions used to generate an initial kinetic parameter set (column 3 in (0.03 MB DOC)Click here for additional data file.Text S1Matlab script: Parameter definition to be used to generate (1 KB TXT)Click here for additional data file.Text S2Matlab script: Function definition for a simple model.(2 KB TXT)Click here for additional data file.Text S3Matlab script: Simulate and plot (0.01 MB TXT)Click here for additional data file.Text S4Matlab script: Simulate and plot (0.01 MB TXT)Click here for additional data file.Text S5Matlab script: Function definition for the full model.(3 KB TXT)Click here for additional data file.Text S6Matlab script: Function required to generate (2 KB TXT)Click here for additional data file.Text S7Matlab script: Simulate and plot (3 KB TXT)Click here for additional data file.Text S8SBML format model. Define the full model  in SBML (0.04 MB SBML)Click here for additional data file."}
+{"text": "Three out of four patients with primary (light chain) amyloid nephrotic syndrome treated with vincristine, doxorubicin and dexamethasone (VAD) induction obtained a partial response and are alive in continuing remission at 4.1, 6.5 and 9.3 years. These preliminary results are of considerable interest and suggest that prospective evaluation of this regimen is warranted in patients with this condition."}
+{"text": "Mouse fibrosarcoma tumours were dissociated and divided into subpopulations of viable cells by centrifugation in linear density gradients of Renografin. Two of these subpopulations, designated Band 2 and Band 4, differed in their clonogenic ability in lung colony assay. The less dense Band 2 cells were significantly more clonogenic than the Band 4 cells (2.9 percent vs 1.4 percent respectively). Each band was further separated on the basis of cell size by centrifugal elutriation. Each size class of cells comprising Band 2 showed higher clonogenic ability than the corresponding size class in Band 4. Thus cell size differences were not responsible for the clonogenic differences between these bands. To determine whether cell-cycle distribution of the tumour cells was responsible for differences in cloning efficiency, flow microfluorometric and premature chromosome condensation methods were utilized. The unseparated and Band 4 populations showed a higher percentage of cells in S and G2 than did the Band 2 populations, but many of the S and G2 tumour cells showed extensive chromosome damage. From this study we conclude that the increased clonogenic ability of the lighter tumour cells is not due to differences in cell size or cell-cycle parameters."}
+{"text": "Pancreas divisum has been postulated as a cause of acute pancreatitis and a chronic pain syndrome in a small subgroup of patients and can be treated with endoscopic dorsal pancreatic duct stent placementand minor papilla sphincterotomy. Twenty patients  were treated endoscopically. Dorsal duct stentswere placed in 19 patients with subsequent needle knife sphincterotomy of the minor papilla overthe stent. Clinical response was judged by comparison of symptoms . The symptom score improved from 9.3 to 5.1 in the pancreatitis groupand from 9.3 to 5.7 in the pain group. A good clinical response was observed in 3 of 7 patients in thepancreatitis group and in 6 of 11 in the pain group at a mean follow-up of 22 months. Complicationsof sphincterotomy were limited to pancreatitis in 6 patients (29%), 5 mild and 1 moderate accordingto published criteria. No patient required more than 4 days hospitalization. Two of 39 stents migratedinto the pancreas, and another stent fractured and remained lodged in the pancreas. Eight of 9 patientsevaluated demonstrated new morphologic duct changes on follow-up pancreatograms.Endoscopic stenting and sphincterotomy of the minor papilla are feasible and may be effective insome patients with pancreas divisum but carries a significant complication rate. The subjective improvementin patients with chronic pain warrants further controlled study."}
+{"text": "Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters  and two genes encoding sensors . The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations . Transcript levels were measured from cells grown in five steady state oxygen levels , and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen.The gene family of hexose transporters in HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied.The expression pattern of the When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions. Saccharomyces cerevisiae contains the sugar transporter genes HXT1 to HXT17, GAL2 and the glucose sensor genes SNF3 and RGT2 -1 min-1 (vvm). For cultures which received less than 20.9% O2 in the gas stream, air was replaced with the equivalent volume of N2, so that total gas flow was maintained constant for all experiments. Cultures which were fed 2.8 or 20.9% O2 were subject to oscillations. To prevent these, approximately 5% of the total cell concentration in the bioreactor was added to the culture as cells in mid to late exponential phase at the time when continuous medium feed was started -1) and 20.9% (0.60 Cmol biomass [Cmol glucose]-1) O2 e]-1) O2  resulted2 approximately 25 h after O2 was provided. Fresh, exponentially growing cells were not added to prevent oscillations since the transcript levels in the added cells may have affected the overall results disproportionately.Oscillations occurred in cultures which were maintained in steady state anaerobic conditions and then provided with 20.9% Oet al. -1) so that the results for each probe in any analysis could be correlated to this internal standard, eliminating experimental variation in different hybridizations and samples. Probes were divided into 2 probe pools with 8 and 9 probes per pool. The identity of the probes was determined by the migration behaviour and the quantity by the peak area. Total polyA RNA was quantified from the cell lysates, after eluting the polyA RNA in dimethyl pyrocarbonate (DMPC) treated H2O, using the RiboGreen RNA quantification kit . Individual mRNA expression levels are given as the standardised  amount per total polyA RNA.Transcriptional analysis was performed with the TRanscript analysis with aid of Affinity Capture (TRAC) assay described by Rautio et al. . Total met al. [HXT1-5, HXT8-10, GAL2, RGT2 and SNF3 were designed to hybridise to the 3' end of the coding regions. The coding regions of HXT13 and HXT17 as well as HXT9 and HXT11 have sequence similarity of 97%. Only the 5' ends of the coding region of these genes differ enough to enable the design of specific probes. HXT6 and HXT7 are 99% identical within the coding region, but differences can be found in the 3' flank of the genes. The flanking region of HXT7 is AT rich which leads to a melting temperature of the probe (50\u00b0C) that is lower than that of the other probes (62\u201371\u00b0C). HXT15 and HXT16 are identical within their coding regions and within 1 kb in both directions from the coding region, except one nucleotide. Therefore only one probe was designed which detected both of these genes.The probes used in the TRAC analyses are listed in Table et al. . The proHXT3 promoter forward1 5'ACCGGTATATCAAATGGCGGTGTA 3', HXT3 promoter reverse 5'TCAGGCATGTTCATTACCTGAGAG 3', HXT6 promoter forward1 5'TGGCATCAAATTTGGGAA 3', HXT6 promoter reverse 5'GAGAGATGCTCCACAGGA 3', HXT13 promoter forward1 5'TGCTGCAATTTGCTATTT 3', HXT13 promoter reverse 5'CATCTCCATCGCTATCAA 3', HXT15 promoter forward1 5'CCATTTTTTCAGAATCCT 3', HXT15 promoter reverse 5'CTGTTTAGATTATCTGCA 3'. Three parallel PCR-reactions for each of the HXT promoters sequenced were carried out using Dynazyme Ext or Phusion high fidelity polymerases , and purified PCR-fragments were sequenced using the same oligonucleotides that were used in the PCR-reactions. In addition, the following oligonucleotides were used in the sequencing: HXT3 promoter forward2 5'GGAACATTCTAGCTCGTT 3', HXT6 promoter forward2 5'TACTTGGAAATTAATGTA 3', HXT13 promoter forward2 5'ATCATTTGTCGTGTTCCT 3' and HXT15 promoter forward2 5'CCAAATATCTTATACGTT 3'.The following oligonucleotides were used in PCR of promoter fragments from the genomic DNA of CEN.PK113-1A: Statistical analysis of the data was carried out using SPSS software . Analysis of variance (ANOVA) and Dunnett's T3 multiple comparison test were used for the comparative analysis of the data. All graphs were prepared using the R environment ,62.ER, LR and MP conceived the study. ER carried out the transcriptional, promoter and statistical analyses, participated in the fermentations and drafted the manuscript. AT and MGW carried out the fermentations and MGW revised the manuscript. LR supervised the work and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. Furthermore, naturally occurring insensitivity to IFN-\u03b1 may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not been identified so far. To explore the molecular basis of IFN-\u03b1 responsiveness, we analysed the expression pattern of about 7000 genes in IFN-\u03b1 sensitive and resistant cell lines and we compared the transcription profiles of cells cultured in the presence or absence of the cytokine using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction upon IFN-\u03b1 treatment by standard 3H-thymidine incorporation and flow-cytometry. The study of 4 sensitive and 2 resistant cell lines allowed the identification of 4 genes  preferentially transcribed in sensitive cells and 2 (SHB and PKC-\u03b6) preferentially expressed in resistant cells. IFN-\u03b1 stimulation resulted in the expression of a panel of 19 known inducible genes in sensitive but not in resistant cells. Moreover a group of 30 novel IFN-\u03b1 inducible genes was identified. These data may provide a useful basis to develop diagnostic tools to select potential IFN-\u03b1 responders eligible for treatment, while avoiding unnecessary toxicity to non-responders. Furthermore, by extending the knowledge of the polymorphic effects of IFN-\u03b1 on gene expression, they offer novel clues to the study of its pleiotropic toxicity. \u00a9 2001 Cancer Research Campaign"}
+{"text": "Fgf2 gene is expressed in the brain neuroepithelium during embryonic development and in astroglial cells throughout life. Previous knockout studies suggested that FGF2 plays a role in the proliferation of neural progenitors in the embryonic cerebral cortex. These studies exclusively used knockout alleles lacking the Fgf2 exon 1. However, the description of putative alternative exons located downstream from the canonical exon 1 raised the possibility that alternatively spliced transcripts may compensate for the lack of the canonical exon 1 in the Fgf2 -/- mice.The Fgf2 transcripts and contains essential heparin and receptor binding interfaces. The expression of Fgf2 exon 3 was prevented by inserting a transcriptional STOP cassette in the Fgf2 genomic locus. These mice demonstrate a phenotype in the adult neocortex characterized by decreased density and number of cortical excitatory neurons and astrocytes, which is virtually identical to that of the Fgf2 -/- mice lacking exon 1. In addition, we also show that the Fgf2 exon 3 knockout mice have decreased proliferation of precursors in the adult cerebral cortex, which had not been previously investigated in the other mutant lines.We generated and characterized a new line of Fgf2 knockout mice lacking the expression of exon 3, which is conserved in all Fgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis during embryonic development as well as in adulthood. Thus, FGF2 may be required for the maintenance of the pool of adult cortical progenitor cells.The results demonstrate that the phenotype of two completely different  Rather, the increase in total cell number can be attributed to an increase in the number of progenitor cells [FGF2, one of the most potent among the fibroblast growth factor ligands, is expressed in the neuroepithelium of the central nervous system ventricular zone (VZ) from early embryonic development, along with the transmembrane FGF receptors FGFR1-FGFR3. FGF2 promotes growth in the cerebral cortex by increasing the number of cortical progenitor cells in the VZ [The expression of FGF2 is steadily down-regulated during embryonic development -5-.Fgf2 null (Fgf2 -/-) mice demonstrated its unique role in regulating neurogenesis. Specifically, there is a 40% decrease in the number of neurons and astrocytes in the cerebral cortex of Fgf2 -/- mice [Fgf2 -/- mice [The analysis of -/- mice , as well-/- mice . This su-/- mice . Pyramid-/- mice . The dat-/- mice . In addi-/- mice .Fgf2 is encoded by three exons, exon 1, 2, and 3. In the original knockout studies, Fgf2 null mice were generated by deleting exon 1 and immediately upstream sequences in the Fgf2 gene locus [Fgf2 mRNA variants have been described that lack the canonical exon 1 and utilize alternative exons  that are located downstream from exon 1 and can be spliced to the second exon [Fgf2 -/- mice. To answer this question, we generated a new recombinant mouse model that lacks exon 3, an exon that is conserved among the Fgf2 transcript variants.ne locus -11-. Howond exon . These sFgf2 knockout mice was generated by inserting a stop codon in front of this exon. This stop sequence was flanked by Cre recognition sites, and as such it is amenable to being excised by Cre recombination, reconstituting the wild type gene. An important question to be addressed is whether these mice would show the same or more profound deficits in CNS development, as compared with the original exon 1 Fgf2 knockout line.Because exon 3 is over 5 kb long, the current strain of Fgf2 genomic sequence, respectively , the entire coding sequence for the \u03b2geo gene  followed by a polyadenylation site and flanked by loxP sites. This cassette was inserted by homologous recombination into an intronic KpnI site, located approximately 1 kb upstream from the third exon of the Fgf2 gene   flanked ely Fig. . The SA\u03b2ene Fig. . After eFgf2 alleles, the following primer pairs were used: upstream primer: 5'-TTGGTACCCTGGAATATTTTAGCCC-3'; downstream primer: 5'-AATAAGTAACCCAGAATATACTGG-3'. These are complementary to sequences in the intron 2 \u2013 3 at the cassette insertion site   with an upstream primer in exon 2 and a downstream primer in exon 3  in 0.1 M Phosphate Buffered Solution (PBS), pH 7.4, followed by post-fixation overnight in 4% PFA and cryprotection in 20% sucrose. Thirty minutes prior to perfusion, the mice were injected with 100 mg/kg 2-bromodeoxururidine (BrdU). Brains were sectioned with a cryostat at 50\u03bcm thickness. For each antibody staining, a series of free-floating sagittal sections (1 out every 10) were blocked for one hour in a 0.1 M PBS/10% normal goat serum solution, pH 7.4, containing 0.2% Triton-X-100 and 0.1% Tween 20. Immunohistochemistry was performed with the following antibodies: neuronal-specific nuclear protein (NeuN) , glial fibrillary acidic protein (GFAP) , T-box brain 1 protein (TBR-1)  , parvalbVolume and total cell numbers were calculated as described  using thgeo gene, which was followed by a polyadenylation site  was inserted upstream of exon 3 in the Fgf2 gene by homologous recombination. In the modified gene locus, \u03b2geo mRNA is expressed instead of exon 3, after which there is premature polyadenylation of the gene product. The truncated product is thought to be without function, since many of the receptor binding interfaces and most of the heparin binding sites are in exon 3 [Fgf2 gene can be reconstituted via Cre recombination, which should excise the SA\u03b2geolox2DTA sequence.The SA\u03b2geolox2DTA DNA cassette, containing a splice acceptor, preceding the entire coding sequence for the \u03b2n exon 3 . With thFgf2 exon 3-deficient mice were born at the normal Mendelian frequency and displayed no alteration of body size, fertility, or survival. To assess Fgf2 exon 3 expression, total RNA was extracted from the brain of 5-month-old Fgf2 exon 3 KO, wild type and heterozygous mice and reverse-transcribed into cDNA. RT-PCR analyses were carried out using an upstream primer corresponding to sequences in exon 2 and a downstream primer corresponding to sequences in exon 3 of the wild type Fgf2 gene. A specific 139-bp amplicon was obtained from Fgf2 wild type and heterozygous mice, but not from their KO littermates  homozygous for the wild type or the Fgf2 exon 3 KO allele. Cortical neurons and astroglial cells, identified by NeuN and GFAP immunostaining, respectively . To assess the number of actively proliferating cells, BrdU was administered 30 min before perfusion and the incorporation of the nucleotide in S-phase cells, visualized by immunostaining, was quantified by stereology in the whole cerebral cortex. Proliferating cells were decreased by 29% in the Fgf2 exon 3 KO mice as compared to their wild type littermates  is likely non-specific because were it to have resulted from read-through of the truncating stop codon, it would also appear at the 21 kD and 22 kD positions since all three isoforms are translated from the same transcripts, and it does not (0% of the intensity of the wildtype 21 kD and 22 kD bands).We produced mice with premature termination of the conserved Fgf2 exon 3 causes significant deficits in several major cortical cell populations. Overall, cortical NeuN+ neuron number was decreased by 23% and that of GFAP+ astrocytes by 34%; one subpopulation of cortical GABAergic interneurons, the PV+ interneurons, was not significantly affected, although we cannot exclude that a larger sample may reveal a deficit in this population, as well. This replicates the results obtained in our previous study of Fgf2 null mice [Fgf2 gene locus. In that study, NeuN+ cells were decreased by 24% and glial cells by 38%. Thus, the degree of deficit in these two cell populations in the two Fgf2 KO models is strikingly similar. The decrease in GFAP+ cells in the adult cortex is also in agreement with the results previously reported by another group [Fgf2 KO mice reported a minimal decrease or no significant change in total cortical volume. Given the decrease in neuron number, it is conceivable that some compensatory mechanism maintains normal cortical volume; an expansion of dendritic or synaptic neuropil is a possibility.This study shows that the lack of ull mice , which wer group . Also, iFgf2 KO mice that are slightly different from those described above [Fgf2 KO mice lacking exon 1. Possible reasons for this discrepancy include the genetic background (C57BL/6J/129/Sv versus Black Swiss/129/Sv in our mice) and the quantitative methods used for estimating cell numbers. We examined both total number and density of cells by stereology using the optical fractionator and unbiased sampling methods, whereas Dono et al. assessed cell density, presumably by counting cells in selected sections.Another group reported findings on the cortical phenotype of ed above . Dono etThe decrease in excitatory neurons expressing TBR-1 is less than what was previously found in the exon 1 KO mice using immunostaining for the neurotransmitter glutamate. However, TBR-1 + excitatory neurons represent only a subset of pyramidal cells, those predominantly found in layer 6 in adult mice. The data suggest that FGF2 may be relatively more important for the genesis of pyramidal cells in other cortical layers.Fgf2 KO study [In our prior exon1 KO study  the mutaKO study . This diFgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis in embryos as well as in adulthood. This is shown by the similar phenotype in the two lines: a decrease in total cortical neuron number, due to a deficit in pyramidal cells as well as in astrocytes, as shown by NeuN, TBR-1 and GFAP immunostaining. Pyramidal neurons and astrocytes are progeny of radial glial cells, which are located in the VZ [Fgf2 exon 1 KO mice [Fgf2 KO mice are proliferating stem/progenitor cells in the adult subventricular zone [The implications of these results are manifold. First, the results demonstrate that the phenotype of two completely different n the VZ . This is KO mice . Anotherlar zone , which alar zone . It can The current results also show that FGF2 is required for normal cell proliferation in the mature cortex. In previous studies on exon 1 KO mice, proliferating cells were assessed only in embryos . The curFgf2 gene by inserting a transcriptional STOP cassette by homologous recombination in the Fgf2 genomic locus. Previous research has shown that there are three putative splice variants of exon 1: 1a, 1b, and 1c [Fgf2 KO models [Fgf2 KO mice. However, whether these FGF2 variants are actually transcribed into mRNA and protein is unclear. The generation of an exon 3 KO in the current study sought to verify the role of FGF2 in development because exon 3 is conserved in the all known FGF2 variants. The results clearly show that the phenotype of the Fgf2 exon 3 KO mice show no greater severity, and that the decrease in cell populations is very similar to that found in the exon 1 studies. Overall, the role of FGF2 in regulating cortical development is confirmed by removing the conserved exon 3. The relatively mild phenotype in this Fgf2 mutant may be explained by compensatory or redundant relationship with other FGF family genes. Furthermore, this line will allow for future studies in which the intact Fgf2 gene can be reconstituted by Cre recombination at selective stages in development or adulthood.This study illustrates a novel way of preventing the expression of a specific exon of the , and 1c . Exon 1aO models -11-. AltFgf2 exon 3 KO mice by molecular, immunohistochemical and stereological analyses and drafted the manuscript. YO participated in the molecular analyses for the characterization of the phenotype of Fgf2 exon 3 KO mice. DS generated the targeting vector for the Fgf2 exon 3 KO mice and carried out the screening of the targeted ES cells. TD participated in study design, helped with data analysis and helped to write the manuscript. LPS generated the knockout mice in the University of Cincinnati Gene-Targeted Mouse Service. HL carried out the Western blot assays. FMV conceived the study, participated in its design, helped with data analyses and interpretation and helped to write the manuscript.KC carried out the characterization of the brain phenotype of All authors read and approved the final manuscript."}
+{"text": "DNA extracted from familial and sporadic colorectal neoplasms was compared with constitutional DNA using a range of hypervariable locus specific probes to assess the extent of allele loss during conversion to malignancy. Chromosome 5 allele loss was observed in 23% of carcinoma samples, as previously found by others. However, we have been able to show for the first time loss of the D5S43 locus on chromosome 5 in adenomas from three patients, two of whom had the precancerous condition adenomatous polyposis coli (APC). These results suggest significant genetic changes involving chromosome 5 are occurring in benign adenomas. Probes for chromosome 1 (loci D1S7 and D1S8) and for chromosome 7 (loci D7S21 and D7S22) revealed no notable alterations in the adenoma samples. Complete loss of alleles for loci on chromosome 7 was not observed in carcinomas but reduced intensity of one parental allele was found in three specimens one of which was known to have multiple copies of this chromosome. Results using probes for chromosome 1 suggest that deletion of the D1S7 or D1S8 loci is not a common event in colorectal carcinogenesis. Loss of chromosome 5 alleles in adenomas from APC patients provides evidence in support of Knudson's hypothesis."}
+{"text": "We have used the polymerase chain reaction to detect DNA sequences related to human papillomavirus type 16, by simultaneous priming with oligonucleotides from the E6 and L1/L2 open reading frames of the HPV16 genome. The HPV16-related sequence is present at low levels in normal oral tissue, in addition to biopsies and cell cultures from patients with benign and malignant disease. Ultimate analysis of the amplified sequences from the E6(120bp) and L1/L2(173bp) regions of HPV16 was achieved by gel electrophoresis and comparative nucleotide sequencing. The oral carcinoma biopsies and tissue cultures contained DNA sequences which were identical to the E6 region of HPV16, but only rarely contained sequences closely related to the L1/L2 region. The PCR technology should permit the detection, identification and cloning of latent viruses from extremely small tissue biopsies."}
+{"text": "N-acetyltransferases (NAT). This study examined the association of (NAT) 1 and 2 genotypes with the risk of smoking-related bladder cancer. A total of 74 pathologically confirmed bladder cancer patients and 184 controls were serially recruited from the National Taiwan University Hospital. History of cigarette smoking and other risk factors for bladder cancer was obtained through standardized questionnaire interview. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 and NAT2 by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Allele frequency distributions of NAT1 and NAT2 were similar between cases and controls. There was a significant dose\u2013response relationship between the risk of bladder cancer and the quantity and duration of cigarette smoking. The biological gradients were significant among subjects carrying NAT1*10 allele or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele. The results are consistent with the hypothesis that NAT1 and NAT2 might modulate the susceptibility to bladder cancer associated with cigarette smoking. \u00a9 1999 Cancer Research CampaignAromatic amines from cigarette smoking or occupational exposure, recognized risk factors for bladder cancer, are metabolized by"}
+{"text": "Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells.Human NhNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB.We have silenced hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast.hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins. Although the two cotranslational events, cleavage of the initiator methionine and N\u03b1-acetylation, are common and highly conserved from bacteria to higher eukaryotes, their function it is not well understood acetyl-CoA  for 2 hours at 37\u00b0C. The samples were centrifuged and the supernatant was incubated with SP-Sepharose (50% in 0.5 M acetic acid) for 10 minutes on a rotor before washing the SP-Sepharose three times with acetic acid 0.5 M and once with methanol. The radioactivity incorporated in the peptides was determined by scintillation counting.The NAT assay was performed basically as described previously ,16 but uHepG2 and Hep3B cells were seeded in 15 mm cover glasses and infected with siRNA expressing adenovirus pAdsiRNA2 or pAdsiRNA350 for 24, 48 and 72 hours, BrdU labeled for 1 hour and processed using the 5-bromo-2'-deoxyuridine Labeling and Detection Kit I  following the manufacturer's instructions.HepG2 and Hep3B cells were harvested after 72 hours of infection with the adenovirus pAdsiRNA2 or pAdsiRNA350 and processed using the CycleTEST PLUS DNA reagent kit (Becton Dickinson) following the manufacturer's instructions. Flow cytometric analysis of the different samples was performed using BD FACScalibur flow cytometer and DNA content was analyzed using the MODFIT software.HepG2 and Hep3B cells were incubated in 15 mm cover glasses and infected with adenovirus pAdsiRNA2 or pAdsiRNA350 for 48 hours prior adding the proteasome inhibitor MG132 (5 \u03bcM)  for an additional 8 hours. Cells were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4). TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer's instructions. Cell apoptosis percentage was obtained as indicated in cell proliferation assay.hNAA20 shRNAs. As it is observed in Figure hNAA20 siRNA (pAdsiRNA2) is expressed. Previous data indicate that there is an inhibition of cell cycle progression when hNatB is inhibited. Therefore we measured cellular proliferation after hNAA20 knockdown and observed a clear inhibition in both cell lines . These differences are not restricted to hNAA20 inhibition in distinct cell lines because hNAA25 repression causes also cell growth arrest, but instead of upregulating p21(WAF/CIP1) expression, as hNAA20 knockdown, there is a clear p21(WAF/CIP1) reduction [The p53 tumour suppressor is a tightly regulated protein that acts by stopping cell-cycle progression or promoting apoptosis when cells encounter stress stimuli such as oncogene activation or DNA damage ,20. For eficient , induceseduction . But we hNAA20 knockdown in HepG2 and Hep3B is not associated with apoptosis induction (data not shown ) but it sensitizes the cells to the proapoptotic treatment with MG132 as it is presented in Figure hNAA20 siRNA, siRNA2. This effect correlates in HepG2 with a reduction of BCL2  [We have observed also that Hep3B cell line is more sensitive to MG132 treatment as Hep3B cells that express the unrelated siRNA present some sensitivity to MG132. This is Hep3B specific because a prolonged exposure of the cells to this proapoptotic stimulus induces an apoptotic cell death that it is not observed in Hela and HepG2 cells (data not shown). These findings are also reminiscent of the increased sensitivity to environmental stress when yeast strains were deleted of yNatB enzymatic complex (naa25-\u0394) ,6.in vitro acetyltransferase assay using as substrate a peptide corresponding to human tropomyosin-1 amino terminus, that has been demonstrated to be a hNatB substrate [NAT enzymatic complexes are composed by a catalytic subunit and one or several accessory subunits. In the case of hNatA there are two possible catalytic subunits  and seveubstrate . We obsein vitro acetyltransferase activity we purified enzymatic complexes with the hNaa20p antibody from Hela cells as this has resulted the most efficient isolation method ; MT1A: Metallothionein-1A; RS28: 40S ribosomal protein S28; TNNC1: Troponin C, slow skeletal and cardiac muscles (TN-C); NAT2: N-acetyltransferase 2 (arylamine N-acetyltransferase); DCUP: Uroporphyrinogen decarboxylase; KAD1: Adenylate kinase isoenzyme 1; B3AT: Band 3 anion transport protein; CRBA1: Beta-crystallin A3; PTN1: Tyrosine-protein phosphatase non-receptor type 1; PPLA: Cardiac phospholamban.The authors declare that they have no competing interests.AA, CG, ML, EL, JP and RA designed research. AA carried out all the experiments concerning cell manipulation and analysis. CG and ML performed the in vitro acetylation assays. All authors designed research and analyzed data. RA coordinated the research and wrote the paper."}
+{"text": "The purpose of this study was to determine the prevalence of BRCA1 mutations in Chinese breast cancer patients in Singapore. BRCA1 analysis was conducted in consecutive patients with breast cancer before the age of 40 years (76 women), or whose relatives had breast or ovarian cancer (16 women). Ten patients had both early onset breast cancer and affected relatives. Genomic DNA from peripheral mononuclear blood cells was studied by using the protein transcription\u2013translation assay (exon 11) and single-strand conformational polymorphism, with subsequent DNA sequencing. All six disease-causing mutations occurred in women under 40 years (8.6%) with three occurring in patients under 35 years . Mis-sense mutations of unknown significance were found in three patients. Two of the ten women with affected relatives under 40 years had BRCA1 mutations. The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer is similar to that observed in Caucasian women. Most Chinese patients with affected relatives were not carriers of BRCA1 mutations. \u00a9 2000 Cancer Research Campaign"}
+{"text": "To understand genetic events which play a role in the development and/or progression of human endometrial cancer, we studied allelotypes on all autosomal chromosomes, as well as chromosome X, with 42 microsatellite markers and 56 endometrial cancers. The most frequent loss of heterozygosity (LOH) was observed on the long arm of chromosome 10 , which was commonly detected in grade 1 cancer. We constructed a detailed deletion map and defined two commonly deleted regions in 10q25-q26; one was the 8 cM region between D10S209 and D10S216, the other was the 12 cM region between D10S217 and D10S590. Replication errors at two or more loci were observed in 24 of 56 tumours (43%), suggesting that disruption of the DNA mismatch repair system also plays an important role in the course of endometrial carcinogenesis."}
+{"text": "Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease.Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased.To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals. Down syndrome (DS) is a result of trisomy or partial trisomy of human chromosome 21 (HSA21) and has an incidence of 1 in approximately 1000 live births in utero precluding analysis of the postnatal consequences of trisomy We utilised a mouse model to investigate how trisomy 21 might affect gene regulatory networks and adult neurogenesis. Mouse chromosome 16 (MMU16) has significant homology with HSA21 A number of features analogous to DS have been demonfstrated in the partially trisomic mouse models and the extent and severity of the phenotypes observed mostly correlate with the size of the trisomic segment. Both Ts65Dn and Ts1Cje mice display craniofacial dysmorphology that directly parallels human DS and defective spatial learning and memory Proliferation defects have been demonstrated in the hippocampal dentate gyrus and neocortical germinal matrix of DS, Ts16 and Ts65Dn foetuses, postnatal Ts65Dn mice and in the hippocampal dentate gyrus and cerebellum of adult Ts65Dn mice Neurogenesis occurs prenatally and postnatally but also throughout adulthood, the latter in predominantly two regions of the brain, the subgranular zone of the dentate gyrus and the SVZ lining the lateral ventricles. Neural stem cells (NSCs) residing in neurogenic regions have the ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes in vivo and ex vivo experiments we show that Ts1Cje adult neurogenesis is reduced as a result of abnormal progenitor production and defects in cell lineage commitment during differentiation rather than defects in NSC development or homeostasis. Moreover, we identified specific regulators of cell cycle progression that are dysregulated in neural precursors isolated from the adult Ts1Cje SVZ. We hypothesise that these defects in adult neurogenesis confound pre-existing developmental defects and are likely to progressively contribute to problems in memory formation and possibly brain functions throughout adult life. We also expect that the defects will impair the ability of the DS brain to respond to neuronal loss and degeneration with repair.Based on a number of In order to identify genes dysregulated in adult Ts1Cje neurogenesis we performed a whole-genome expression analysis using GeneChip\u00ae Mouse Genome 430 2.0 Arrays  on 3 pairs of female Ts1Cje and disomic littermate control primary neural stem and progenitor cells after expansion for 7 days as neurosphere cultures. This data is freely accessible on the Gene Expression Omnibus website under the series accession number GSE17760.2 fold-changes (M) for trisomic versus disomic mice versus the average log2 expression (A). Each dot represents a single probe. Probes that were not expressed naturally did not show any change, i.e., the log2 fold-changes were around 0, but probes that were expressed at worthwhile levels  consistently showed fold changes of around 1.5  may be differentially expressed, although the fold changes were small so that few genes could be individually identified as differentially expressed at conventional significance levels. In order to uncover changes in groups of functionally interconnected genes we utilised two online analysis tools the Database for Innovation, Visualization and Integrated Discovery , Mcm7, Pbk, Phb, Prim1 and Suv39h1) by RT-qPCR in neurosphere cultures generated from 3 pairs of female Ts1Cje and disomic littermate controls independently of the original microarray analysis . Phb was shown to be slightly downregulated in the Ts1Cje neurospheres by RT-qPCR analysis but upregulated in the microarray analysis.Focusing on the IPA network with the largest number of differentially expressed probes  we analyanalysis . SimilarTo examine whether these small abnormalities in gene expression affect the Ts1Cje brain we examined the neurogenic lineage systematically from neural stem cells (NSCs) through neural progenitors, via neuroblasts, to neuronal differentiation and neurite outgrowth. In addition, we also examined neuroblast migration.Gene expression abnormalities in proliferation associated genes imply that there are defects in the proliferation of trisomic NSCs and/or progenitors. We used a double labelling assay with the thymidine analogues 5-chloro-2\u2032-deoxyuridine (CldU) and 5-iodo-2\u2032-deoxyuridine (IdU) to investigate whether there is a reduction in the numbers of NSCs or a defect in the proliferation of NSCs in the adult Ts1Cje subventricular zone (SVZ). Thymidine analogues are incorporated into DNA during the DNA synthesis phase of the cell cycle and then diluted exponentially following cell division when half the analogue is distributed to the daughter cell. The thymidine analogue is undetectable by immunohistochemistry after 3 cell divisions.NSCs are relatively quiescent with a relatively long cell cycle length of \u223c15 days and an S phase of \u223c8 h P\u200a=\u200a0.053; N\u200a=\u200a4 pairs of male Ts1Cje and disomic controls; P\u200a=\u200a0.97; N\u200a=\u200a4 pairs of male Ts1Cje and disomic controls; data not shown). We therefore detected no difference in the number of NSCs in the adult Ts1Cje brain compared to their sex matched disomic littermate controls.IdU positive cells were counted in the subventricular neurogenic zone at 5 equally spaced rostrocaudal levels. We found no significant difference in the number of IdU long-term label retaining cells  and move as chains of migrating neuroblasts from the SVZ to the olfactory bulbs where they become periglomerular and granule cell interneurons The PSA-NCAM staining revealed that the Ts1Cje neuroblasts were reduced in number, but directional pattern of migration appeared normal and there was no obvious defects such as blockages or altered orientation. In the following experiment we examined their rate of migration using a medium-term thymidine analogue labelling assay.P\u200a=\u200a0.056; N\u200a=\u200a3 male and 1 female pairs of Ts1Cje and disomic littermates).Neuroblasts take between 2 and 6 days to migrate from the SVZ to the olfactory bulbs via the RMS Finally, we examined the ultimate product of SVZ neurogenesis, the differentiating neuron.in vitro. Following 7 days of primary culture, neurospheres were dissociated and plated onto poly-ornithine and laminin coated coverslips. Cell types were identified by immunofluorescence using antibodies against type III \u03b2-tubulin, glial fibrillary astrocyte protein (GFAP) and O4 to mark neurons, astrocytes and oligodendrocytes respectively. The number of neurons generated from Ts1Cje neurospheres was reduced with an average of 56% compared to sex matched disomic littermate controls  or the average length (P\u200a=\u200a0.55) of primary neurite  or tertiary (P\u200a=\u200a0.96) neurites . In this study we also observed dysregulated expression of genes involved in proliferation, particularly cell cycle control. We have shown a tendency for delayed neuroblast migration through the adult Ts1Cje SVZ. Importantly, we have uncovered severe defects in Ts1Cje neural differentiation, in particular a reduction in the production of neurons and a tendency for reduced side branching. Given that our work demonstrates neurogenesis defects exist during adulthood suggests that there is potential to slow aspects of the progression of cognitive decline and neurodegeneration in DS.We have used a combination of 2-phase of the cell cycle and Brca2 deficiency has been shown to impair cytokinesis and extend cell division 2/M phase Analysis of our microarray expression data from adult SVZ neurosphere cultures using both the Database for Innovation, Visualisation and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) identified dysregulation of genes involved in proliferation in Ts1Cje samples. The majority of proliferation associated genes showing disrupted expression were subtly downregulated and not from the Ts1Cje trisomic region, indicating that trisomy results in a large number of downstream gene expression abnormalities that combined would affect proliferation in adult neural progenitors. The largest IPA network of differentially expressed genes included a number of genes whose down regulation could delay cell cycle progression. The following discussion is focused around the genes from this network for which expression was most affected by trisomy. The helicase minichromosome maintenance proteins (MCMs) and Prim1  are involved in DNA replication and their reduced expression is likely to result in a delay or elongation of the S phase of the cell cycle 2 and M phases. Indeed, elongation of the G1 but particularly the G2 phase has been observed in trisomic neural precursors Ccnb1 and Skp2, two key regulators of the G2/M and G1/S transitions are decreased in neonatal (PN2) Ts65Dn cerebellar granule cell precursors 2/M phase may be a common theme in trisomic cell division. Notably, we found Ccnb1 to be downregulated in adult neurospheres. Furthermore, all probes located to the Skp loci on our microarray that were expressed in adult neurospheres were reduced in the Ts1Cje compared to disomic controls, with fold changes ranging from 0.64 to 0.87.Based on the changes in gene expression that we observed we would expect a delay in cell cycle progression in the S or Get al., (2009) used cells after at least four passages in culture, whilst we subjected cells to only one passage in culture before conducting the microarrays. We note that we have shown changes in gene expression in trisomic neural precursors even in different pathways to those observed by others despite some similarities in neural differentiation defects Rest regulated genes in the trisomic neural progenitor cells. This could be due to the differences in the source of cells investigated. We used primary neurospheres, whilst Bahn et al., (2002) analysed cells after more than 10 weeks of culture and Canzonetta et al., (2008) analysed cell lines following at least 3 passages. In this context it is important to note that striking differences have been observed between primary neuronal cells and cells cultured for extended periods such that primary cells are expected to reflect the situation in vivo more accurately A recent gene expression study on embryonic neural progenitors cultured as neurospheres from the E14.5 Ts1Cje neocortex also observed dysregulation of genes involved in proliferation, although changes in expression of individual genes differed from our study in vivo using a double thymidine analogue labelling assay. However, we observed a reduction in stem cell/progenitor derived primary neurospheres from the Ts1Cje SVZ and neuroblasts migrating through the RMS. A partial block in the S, G2 or M phases of the cell cycle could explain a low production of neural progenitors and neuroblasts. In concordance with previous findings and a reduction in the numbers of neural progenitors and neuroblasts the anterior-posterior length of the Ts1Cje olfactory bulbs were shorter than the disomic controls To our knowledge, no previous study has differentiated between the effects of trisomy 21 in humans, or 16 in mice on NSCs versus neural progenitor cells. We did not detect a difference between the number of adult Ts1Cje and disomic control NSCs 2 phase, which was also shown to be prolonged compared to the disomic In support of our findings others have reported similar defects in the production of neural progenitors, albeit the majority of studies have been focused on different areas of the brain, at different stages of development. A number of studies of Ts16, Ts65Dn and DS embryonic brain development have identified cell cycle and neurogenesis abnormalities in the neocortex and hippocampus Our neurosphere data imply that there is a major reduction in the capacity of Ts1Cje neural progenitors to differentiate into neurons by more than 50%. The number of neurons produced by the SVZ has not been assessed previously in models of DS. Defects in neural differentiation have been reported for other areas of trisomic brains and recently a greater number of GFAP-positive cells were observed prior to differentiation in neural progenitors isolated from the E14.5 Ts1Cje neocortex Dyrk1A), which is located on the Ts1Cje trisomic region. Indeed, the 3 Dyrk1A probes on our microarray were overexpressed at expected trisomic levels. Dyrk1A has been shown to have a highly dynamic expression pattern throughout mouse brain development suggestive of a key role in regulating the multiple events that occur during neuronal development 2-phases of the neuronal progenitor cell cycle combined with the altered levels of cell cycle regulators found in mice overexpressing Dyrk1A and the phosphorylation of cell cycle regulators by DYRK1A led to the suggestion that it is a regulator of neural progenitor proliferation KIP1, which has been shown to be involved in neuronal differentiation Dyrk1A have been generated and were shown to display hippocampal-dependent spatial learning and memory defects, developmental delay and motor deficits Dyrk1A in the striatum of a transgenic mouse model of Dyrk1A resulted in the restoration of motor coordination, attenuation of hyperactivity and improvement of sensorimotor gating We have demonstrated that the expression of a number of genes involved in control of the cell cycle are disrupted and that there are a number of abnormalities in adult Ts1Cje adult neurogenesis, however the question remains, which trisomic gene or genes are responsible? It is tempting to speculate that the abnormalities we have observed are due to the overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A gene  Neurogenic cells isolated from the adult Ts1Cje brain exhibited disrupted expression of genes encoding proteins involved in cell cycle progression. 2) Ts1Cje brains had normal numbers of long-term retaining NSCs. 3) In contrast, the numbers of self-renewing neural progenitor colonies, were reduced. 4) There was a reduction in the number of neuroblasts migrating through the Ts1Cje RMS compared to disomic controls. 5) Our data suggests that the speed of adult Ts1Cje neuroblasts migration was reduced. 6) The number of neurons produced by adult Ts1Cje neurogenic progenitors showed a striking reduction to less than one half whilst the numbers of astrocytes was increased. 7) Ts1Cje neurons showed a tendency for less elaborate neurites.All breeding and experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee .App  as described previously Sex matched disomic littermates were used as controls for all experiments. All mice were aged to 3 months (84 days). Ts1Cje and disomic mice were generated by mating Ts1Cje males  with C57BL/6 female mice for over 10 generations. Offspring were genotyped using multiplex PCR primers for neomycin and RNA was extracted from 3 Ts1Cje and 3 disomic female littermate control neurospheres (generated from adult subventricular zone (SVZ) cells) cultured for 7 days after isolation using a Qiagen RNeasy micro kit  according to the manufacturer's instructions with the DNase I digestion step. The quality and quantity of RNA was assessed using a 2100 Bioanalyzer . Each RNA sample was processed using the Two-Cycle Target Labelling Assay and hybridised onto an Affymetrix GeneChip\u00ae Mouse Genome 430 2.0 Arrays according to the manufacturer's instructions . Fluorescent signals were detected using a GeneChip\u00ae Scanner 3000 .t-statistics P-values were adjusted for multiple testing www.r-project.org) P-values was completed to estimate the true percentage of statistically significantly expressed genes http://www.ingenuity.com) were used to uncover changes in functionally interconnected genes from a list of the most differentially expressed genes . Primer and probe sequences are detailed in the 2O , on a LightCycler\u00ae 480 System . Conditions for the RT-qPCR were a pre-denaturing step of 95\u00b0C for 10 min, 45 cycles of; 95\u00b0C for 10 sec, 60\u00b0C for 30 sec, 72\u00b0C for 30 sec and finishing with a cooling step at 40\u00b0C for 1 min.RT-qPCR was used to validate the expression of 11 selected genes from the microarray analysis. RNA was extracted independently from the microarray samples from 3 female Ts1Cje and 3 disomic litermate control 7-day-old neurosphere culture pellets using a Qiagen RNeasy micro kit  according to the manufacturer's instructions with the DNase digestion step. RNA quality and quantity was assessed using a 2100 Bioanalyzer . First strand cDNA was synthesised using random hexamers and the SuperScript\u2122III Reverse Transcriptase Kit  according to the manufacturer's instructions. Primers were designed and probes selected using ProbeFinder Version 2.34  for each signal was calculated based on the Second Derivative Maximum method. A set of serially diluted cDNAs synthesised from 3 \u00b5g of adult mouse brain RNA was used to construct a 3-data point standard curve for every PCR assay in each run. A PCR efficiency of between 90\u2013110% and an R-squared value >0.98 were used to define successful assays. Relative quantification of target gene expression in Ts1Cje and disomic samples was carried out using the comparative Ct method. We performed intra-sample data normalization against 3 endogenous control reference genes 2+/Mg2+ free) with 200 U/ml penicillin, 200 \u00b5g/ml streptomycin for 30 min at 4\u00b0C and transferred into mouse tonicity phosphate-buffered saline (MTPBS) for 5 min at 37\u00b0C. The pancreatin/trypsin was inhibited briefly by 2% foetal bovine serum in MTPBS, and the tissue was mechanically dissociated. Cells were collected by centrifugation and cultured in neural stem cell (NSC) proliferation medium for 7 days: DMEM/F12, 5 mM HEPES, 13.4 mM NaHCO3, 0.6% D-glucose, 100 U/ml penicillin, 100 \u00b5g/ml streptomycin, 25 \u00b5g/ml insulin, 60 \u00b5M putrescine dihydrocloride, 100 \u00b5g/ml apotransferrin, 30 nM sodium selenite, 20 nM progesterone, 10 ng/ml bovine recombinant fibroblast growth factor 2 (FGF2), 4 \u00b5g/ml porcine heparin sodium salt, 20 ng/ml murine epidermal growth factor (EGF) and 2 mg/ml bovine serum albumin fraction V. Under these conditions SVZ cells proliferate and form neurospheres. Neurospheres were collected by centrifugation and dissociated in 0.25 mg/ml trypsin, 10 \u00b5g/ml DNase I, 10 mM HEPES and 0.2 mg/ml EDTA in Ca2+/Mg2+ free HBSS at 37\u00b0C for 3 min. The trypsin was inhibited briefly by 140 \u00b5g/ml soybean trypsin inhibitor, in HEM with 10 \u00b5g/ml DNase I. 50,000 cells were plated onto poly-ornithine/laminin-coated 13 mm glass coverslips in differentiation medium  for 8 days.Cells from the SVZ were isolated and cultured as described previously For assessment of neuroblast migration along the rostral migratory stream (RMS) 4 pairs of Ts1Cje and disomic littermate controls  were injected with 5-bromo-2\u2032deoxyuridine  twice daily 12 h apart for 4 days and left without treatment for 2 days. The migration front of BrdU labelled neuroblasts was identified on serial paraffin sections stained for BrdU. Adjacent sections were stained with cresyl violet to reveal histological morphology. To determine the distance migrated by the neuroblasts, the distance between the most rostral extent of the frontal cortex, as a reference point and the migration front was calculated based on the number of intervening sections and section thickness.To determine the number of NSCs and progenitors and to assess their proliferation 4 pairs of male Ts1Cje and disomic littermate controls were injected with 5-iodo-2\u2032-deoxyuridine  twice daily 12 h apart for 7 days, left without treatment for 7 days and then injected with 5-chloro-2\u2032-deoxyuridine  twice daily 12 h apart for 7 days and finally left without treatment for 7 days. The number of CldU positive, IdU positive and double positive cells were counted at five equally spaced rostrocaudal levels approximately 250 \u00b5m apart ranging from the most rostral section containing fimbria of the hippocampi to the section containing the fusion of the rhinal fissure. Stained sections were analysed microscopically and photographs were taken with a digital camera .To ensure that assays involving cell counts were accurate, the nuclei of cells throughout the SVZ from digital images at a magnification of 40\u00d7 of 4 pairs of sex matched adult Ts1Cje and disomic littermates were compared. The longest diameter of 630 randomly selected nuclei from each genotype were measured. We found no significant difference in the mean length of the longest nuclei diameter between Ts1Cje and disomic controls (data not shown) and therefore numbers of labelled SVZ cell profiles could be compared For thymidine analogue incorporation studies, brains were perfusion-fixed in 4% PFA, postfixed in 4% PFA for 48 h, embedded in paraffin and coronally sectioned at 5 \u00b5m in preparation for staining.Immunohistochemistry was performed as described previously In Situ Apoptosis Detection Kit S7110 according to the manufacturer's instructions . The number of TUNEL positive cells was counted at 5 evenly spaced rostrocaudal intervals on sections adjacent to those assessed for thymidine analogue incorporation.Four pairs of sex matched  Ts1Cje and disomic littermate controls were assessed for the number of apoptotic cells in the SVZ using the ApopTag\u00ae Fluorescein 2O2 in methanol, and bound antibody was visualised using horseradish peroxidase-coupled streptavidin, diaminobenzidine and H2O2. Intervening wash steps were TBS with or without 0.5% Triton X-100. Photographs of stained tissue were taken using a low magnification stereomicroscope  and digital camera , PSA-NCAM-positive chains were traced and quantified using the NIH Image analysis software, ImageJ.Whole-mount immunohistochemistry was performed as described previously Stata\u00ae10  software and R language and environment were used to perform multifactorial ANOVAs and other statistical analyses (for further details see Text S1Supporting Text.(0.13 MB DOC)Click here for additional data file.Table S1DAVID data.(0.21 MB XLS)Click here for additional data file.Table S2Affymetrix and RT-qPCR data.(0.03 MB XLS)Click here for additional data file."}
+{"text": "Mutations in the BRCA1/BRCA2 genes account for varying proportions of breast cancer families studied, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations in 17 families from Wales with two or more cases of breast cancer under age 50 and/or ovarian cancer. Eight out of 17 (47%) families had demonstrable mutations. Six out of 17 (35%) carried BRCA1 mutations and 2 out of 17 (12%) carried BRCA2 mutations. Two recurrent mutations in BRCA1 were identified, which appear to represent founder mutations in this population. These data support the existence of additional breast and ovarian cancer susceptibility genes."}
+{"text": "A recent report suggests that the KLF6 gene encoding the Kr\u00fcppel-like factor 6 protein is a frequently mutated, putative tumour suppressor gene in prostate cancer. The aims of the present study were to confirm these initial findings by determining the frequency of exon2 KLF6 mutations in a cohort of European prostate cancer patients, and to investigate whether there was evidence for mutational inactivation of both the KLF6 and TP53 tumour suppressor loci in some tumours. We examined 32 primary prostate tumours and three prostate tumour cell lines for mutations by PCR amplification and direct dideoxy sequencing (KLF6), and by oligonucleotide microarray (p53GeneChip\u2122) analysis and dideoxy sequencing (TP53). Whereas TP53 mutations typical of prostate cancer were found at a frequency consistent with the literature, no KLF6 mutations were found in any of the tumour samples nor in the three prostate cancer cell lines. Amplification was performed in an MJB thermal cycler programmed for 40 cycles , and included an initial denaturation step at 95\u00b0C for 2\u2009min, and a final elongation step of 5\u2009min at 72\u00b0C. Polymerase chain reaction products were purified with Microcon 100 filters from Millipore  and used as template in dideoxy cycle sequencing reactions with fluorescent dye-labelled dideoxynucleotides and DNA polymerase from Applied Biosystems International . Four cycle sequencing reactions were performed, using primers KLF6Ex2FC, KLF6Ex2RC, KLF6Ex2R and KLF6Ex2FA  and used according to recommendations from the manufacturer. Chips were scanned with a Hewlett-Packard GeneArray scanner and signal intensities were evaluated with Affymetrix GeneChip software. The probe array methodology scans all coding exons and splice sites of the human p53 gene for point mutations with high efficiency and accuracy  suggest that KLF6 miscoding mutations in tumours of the prostate are uncommon. While the present report was under initial review, a new study on KLF6 alterations in prostate tumours appeared in the Additional studies are called for in order to clarify whether KLF6 is indeed a common mutational target in prostate carcinogenesis."}
+{"text": "A number of randomized studies have been carried out in the UK and USA to determine the optimal radiotherapy dose schedule for advanced non-small-cell lung cancer (NSCLC). We have examined eight radiotherapy regimens from data taken from four randomized phase III studies carried out in the UK (1264 patients): 10 Gy single fraction; 17 Gy in two fractions over 8 days; 30 Gy in ten fractions over 14 days; 22.5 Gy in five fractions in 5 days; 27 Gy in six fractions over 11 days; 30 Gy in six fractions over 11 days; 36 Gy in 12 fractions over 16 days; and 39 Gy in 13 fractions over 17 days. We compared the clinical results in palliation, toxicity and survival with four regimens taken from one randomized study from the USA (365 patients): 40 Gy in 20 fractions over 4 weeks; 40 Gy 'split course' in ten fractions in 4 weeks; 50 Gy in 25 fractions over 5 weeks; and 60 Gy in 30 fractions over 6 weeks. Using the linear-quadratic (LQ) radiobiological model, we have calculated the radiobiological equivalent dose (BED) for acute-reacting tissues (BED10), late-reacting tissues (BED1.7) and tumour (BED25), and related the predicted response to the observed response in each tissue. There was a good correlation between the predicted response and the reported response in the case of late-reacting tissue toxicity and tumour response. The model confirmed that, in good performance status patients, a higher value for BED25 correlated with a higher degree of local control and survival and that radiotherapy regimens with a higher value for BED1.7 were associated with five cases of cord myelopathy, if the spinal cord was not shielded. In poor performance status patients the model suggested that the optimal regimen was a single fraction of 10 Gy because this resulted in an equivalent degree of symptom control as other regimens, needed only one hospital visit and was less likely to result in cord damage, thus, allowing for the possibility of retreatment at a later date."}
+{"text": "Breast cancer is the most frequent cancer in women and consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics. In this study, we aimed to identify DNA methylation based biomarkers to distinguish patients with locally advanced breast cancer who may benefit from neoadjuvant doxorubicin treatment.ABCB1, ATM, BRCA1, CDH3, CDKN2A, CXCR4, ESR1, FBXW7, FOXC1, GSTP1, IGF2, HMLH1, PPP2R2B, and PTEN) in 75 well-described pre-treatment samples from locally advanced breast cancer and correlated the results to the available clinical and molecular parameters. Six normal breast tissues were used as controls and 163 unselected breast cancer cases were used to validate associations with histopathological and clinical parameters.We investigated quantitatively the methylation patterns in the promoter regions of 14 genes (FOXC1 promoter. Absence of methylation at the ABCB1 promoter correlated with progressive disease during doxorubicin treatment. Most importantly, the DNA methylation status at the promoters of GSTP1, FOXC1 and ABCB1 correlated with survival, whereby the combination of methylated genes improved the subdivision with respect to the survival of the patients. In multivariate analysis GSTP1 and FOXC1 methylation status proved to be independent prognostic markers associated with survival.Aberrant methylation was detected in 9 out of the 14 genes including the discovery of methylation at the ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness. FOXC1 proved to be general prognostic factor, while ABCB1 and GSTP1 might be predictive factors for the response to and efficacy of doxorubicin treatment. Pharmacoepigenetic effects such as the reported associations in this study provide molecular explanations for differential responses to chemotherapy and it might prove valuable to take the methylation status of selected genes into account for patient management and treatment decisions.Quantitative DNA methylation profiling is a powerful tool to identify molecular changes associated with specific phenotypes. Methylation at the  Breast cancer, the most frequent cancer in women, consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics . ConventWhile the contribution of genetic factors to breast carcinogenesis has long been recognized, it has become evident that epigenetic changes leading to transcriptional silencing of tumour suppressor genes are an at least equally contributing mechanism. In tumours a global loss of DNA methylation (hypomethylation) of the genome is observed at early stages of breast carcinogenesis which proceeds with increasing malignancy . The oveThe number of genes that has been identified to be aberrantly methylated in breast cancer is rapidly growing. Thus, high-throughput DNA methylation mapping technologies have the potential to identify distinct methylation signatures correlating with specific clinical stages and subtypes, but also to reveal the large heterogeneity of DNA methylation patterns within a tumour subgroup -9. ConsiGSTP1 promoter in breast cancers and to affect patient survival [ABCB1 [ATM [BRCA1 [CDH3 [CDKN2A [ESR1 [GSTP1 [IGF2 [HMLH1 [PPP2R2B [PTEN [CXCR4 [FOXC1 [FBXW7 [Recently, we reported the haplotype structure to influence the level of DNA methylation of the survival . Here wel [ABCB1 , ATM [12M [BRCA1 , CDH3 [1A1 [CDH3 , CDKN2A  [CDKN2A , ESR1 [12A [ESR1 , GSTP1 [1 [GSTP1 , IGF2 [1P1 [IGF2 , HMLH1 [2 [HMLH1 , PPP2R2B[PPP2R2B , PTEN [12B [PTEN ) or otheN [CXCR4 ), 2. gen4 [FOXC1 ) and 3. 1 [FBXW7 ). In totABCB1 (40 CpGs), ATM (56 CpGs), BRCA1 (46 CpGs), CDH3 (35 CpGs), CDKN2A (30 CpGs), CXCR4 (19 CpGs), ESR1 (50 CpGs) FBXW7 (31 CpGs), FOXC1 (14 CpGs), GSTP1 (21 CpGs), IGF2 (16 CpGs), MLH1 (24 CpGs), PPP2R2B (51 CpGs), and PTEN (39 CpGs). The six normal samples were unmethylated for all analyzed regions except for the highly methylated upstream region of BRCA1, the differentially methylated region of the imprinted IGF2 and the promoter region of ESR1 , FOXC1 (50%), PPP2R2B (56% and 65%), HMLH1 (14%), PTEN (22% and 76%) and GSTP1 (65% and 83%). All samples were unmethylated for the transcription start site of BRCA1, ATM, CDH3, CXCR4 and FBXW7. 10% of the samples exhibited a significant hypomethylation in the far upstream region of the BRCA1 CpG island. Some methylation was found around the transcription start site for ESR1 but also within the normal breast samples. None of the genes displayed an age-dependent variation of DNA methylation at the analyzed loci.We analyzed promoter methylation at 432 CpGs in 14 genes giving rise to 37.000 epigenotypes Figure . The ana1 Figure . Three aABCB1, FOXC1, GSTP1, PPP2R2B, PTEN promoters identifying thus strongly correlated methylation events on different chromosomes  showed abnormal methylation at one locus, 8 (11%) at two loci, 4 (5%) at three, 14 (19%) at four and five loci, respectively, 16 (21%) at six, 9 (12%) at seven, 5 (7%) at eight loci and two tumours (3%) displayed aberrant methylation at nine loci. On average, five loci were thus aberrantly methylated in any sample. Methylation events at the different loci were not randomly distributed and independent from each other Figure . As expes Figure . MethylaABCB1, FOXC1, PPP2R2B, and GSTP1 in both the basal-like and normal-like tumours, while IGF2, MLH1 and PTEN were hypomethylated in the basal-like tumours but not in the normal-like tumours. When analyzing the correlation between the expression level and the DNA methylation status of individual genes, genes with methylated promoters were almost exclusively not expressed, while unmethylated genes could be expressed as well as not be expressed weakening the correlation. The only significant correlation was obtained for GSTP1 . Because of their association with survival (see below) we analyzed the expression levels of GSTP1, ABCB1 and FOXC1 by qRT-PCR . Consequently a significant negative correlation between expression as measured by TaqMan and methylation was found for GSTP1 , while expression and methylation for FOXC1 and ABCB1 were not significantly correlated . Highly expressing genes were unmethylated for the respective promoter region of GSTP1 and FOXC1 and methylated promoters correlated with silenced expression. The weak correlation between expression and DNA methylation for FOXC1 was due to the fact that the gene was already silenced in most tumours independent of its methylation status. Four samples were methylated for ABCB1 but displayed high expression. This might be due to alternative usage of an upstream promoter [The observed methylation patterns were compared to the tumour subclasses as defined by microarray expression profiling . Basal-lpromoter  that is PPP2R2B in the pre-treatment sample was significantly associated with tumour grade (p = 0.019), whereby high-grade tumours were more frequently unmethylated than grade 1 and 2 tumours in the discovery cohort. The same was observed in the validation cohort of unselected breast cancers (p = 0.008). No association between methylation and tumour size was found. Estrogen receptor status positivity was associated with the presence and increased extent of methylation at the PPP2R2B promoter in both the discovery (p = 0.004) and the validation cohort (p = 0.006). Samples unmethylated for ABCB1 and those with increased levels of methylation in the differentially methylated region 2 of IGF2 had more often overexpression of the ERBB2 oncogene , previously analyzed by immunohistochemistry [Methylation was analyzed in the discovery and validation cohorts both as a categorical variable, i.e. the presence/absence of methylation at the respective promoter in association with the tumour characteristics, and as a quantitative variable investigating potential associations between the extent or the distribution of DNA methylation values and the analyzed clinical and molecular parameters  as well as in the validation cohort . Tumours unmethylated for the middle region of the ABCB1 CpG island were associated with mutations in the loop domains L2/L3 (p = 0.022), a region that has previously been shown to be associated with lack of response to doxorubicin based treatment. PPP2R2B did not show any differential degree of methylation in function of the type of TP53 mutation.We compared the observed DNA methylation profiles with the ABCB1, BRCA1, CDKN2A, FOXC1, GSTP1, IGF2, PPP2R2B and PTEN) within the discovery cohort were tested for association with survival by a logrank test. Breast cancer specific survival was significantly improved in patients with methylated promoters for ABCB1, GSTP1 and FOXC1 . Consistently, absence of methylation  in the patient cohort treated with doxorubicin. In the validation cohort treated with different regimens, a significant difference in survival between methylated and unmethylated samples was confirmed for FOXC1 (p = 0.024) with patients unmethylated for the promoter region having again worse survival. Methylation of GSTP1 did not condition improved survival in the validation cohort of patients (p = 0.331). Similarly, only a trend for improved survival was observed for the methylation status of ABCB1 (p = 0.070). The findings for GSTP1 and ABCB1 might indicate a treatment specific effect on survivalThe eight genes displaying variable DNA methylation patterns in a significant number of tumours (TP53 mutation status (p = 0.001), grade (p = 0.001) and the estrogen receptor status (p = 0.002) could slightly better differentiate two survival groups in the analyzed sample collection when compared to the methylation status of the single genes (ABCB1 (p = 0.004), GSTP1 (p = 0.004) and FOXC1 (p = 0.021)), while separation based on the progesterone receptor status and amplification of ERBB2 or TOP2A did not reach statistical significance. However, combination of two of the discovered DNA methylation markers further improved the distinction between doxorubicin treated patients having two, one or none of the genes methylated. No statistical difference on survival in function of the gene was found when comparing patients that had one of the two genes methylated and these were therefore combined for analysis. The best two-gene methylation pair comprised GSTP1 and FOXC1 (p = 8\u00b710-5), followed by GSTP1 and ABCB1 (p = 0.001) and ABCB1 and FOXC1 (p = 0.01). Patients with all three genes methylated (n = 20) had an improved survival compared to patients with all three genes unmethylated . However, the separation lost its statistical significance when patients with mixed methylation patterns for all three genes were added to the analysis. We investigated if expression could be used as an alternative molecular measure to DNA methylation and divided samples in high versus low expression based on the mean expression values. The expression of GSTP1 was significantly associated with survival with patients with low levels of expression having as expected an improved survival (p = 0.048). FOXC1 (p = 0.247) and ABCB1 (p = 0.181) were not significant but again showed improved survival for low expressing patients. When combining DNA methylation and expression, patients with an unmethylated GSTP1 promoter and expressed gene had poorer survival compared to patients with a methylated promoter that did not express GSTP1 (p = 0.047). The same correlation was observed for FOXC1 (p = 0.045) and ABCB1 (p = 0.022).Survival analysis in the doxorubicin treated cohort based on the logrank test indicated that To identify significant parameters contributing to the observed differences in survival, Cox regression analysis was performed. The hazard ratio for each of the contributing factors was estimated separately  or modelled together .ABCB1, FOXC1 and GSTP1 as significant predictors of overall survival. Estrogen receptor status as well as TP53 status and grade were also significant predictors of survival in univariate analysis  and FOXC1  showed a higher risk of dying from breast cancer compared with patients methylated for the same genes  while increasing the HR for FOXC1 . The HR for the other parameters remained unchanged.Univariate analysis identified the methylation status of FOXC1 and GSTP1, stage, grade and estrogen receptor status  remained significant together with high grade (p = 0.002) and ER status (p = 0.001) (data not shown).In order to identify the model with the minimum number of covariates that fitted best the experimental data, we used the Akaike information criterion. The best model with a reduced number of covariates explaining survival included the methylation status of In the presented study we analyzed the methylation patterns in the promoter regions of fourteen genes in 75 pre-treatment samples from locally advanced breast cancers, six normal tissues and six widely used cell lines. Aberrant methylation events were detected in eleven out of the fourteen genes investigated. Discussion of the negative results can be found in the Additional File CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A) [LINE1/ALU) as a surrogate for genome-wide methylation levels, basal-like breast tumours are characterized by an overall loss of methylation . The observed hypomethylation in the far upstream region of BRCA1  . However . Howevestricted .ESR1), we studied its promoter methylation in normal samples as well as in a subset of the tumours. It has previously been shown that its degree of DNA methylation did not correlate well with hormone receptor status [Since the tumour sub-classification based on gene expression is driven to a significant extent by expression of the estrogen receptor (r status . Our datr status .PPP2R2B on 5q31-q32 encodes the regulatory subunit of the protein phosphates 2A complex (PP2A) and has been proposed as a tumour suppressor gene candidate due to its negative control of cell growth and the high frequency of LOH in breast cancers [TP53 mutation status is reported here for the first time and might provide an alternative molecular mechanism for gene inactivation, as also the LOH has previously been associated with TP53 mutations [Another studied gene, cancers . An assoutations . The preutations  was alsoABCB1 CpG island to be associated with ERBB2 amplification, TP53 mutation status, and response to doxorubicin treatment and overall survival in a doxorubicin-exposed cohort of primary breast cancers. Although the number of patients with progressive disease in the current study is limited and requires confirmation in other patient cohorts treated with anthracycline based treatment, there is good evidence that methylation of ABCB1 plays an important role in the response to doxorubicin. Lack of methylation in the central part of the ABCB1 CpG island was found to be associated with the TP53 mutation status and in particular with mutations in the L2/L3 DNA binding domain which have previously been associated with lack of response to treatment in the same patient cohort [TP53 and ABCB1 [ABCB1 has been shown to reduce intracellular doxorubicin concentration in cell cultures [in vivo studies has been conflicting [ABCB1 expression the mice leads to the development of doxorubicin resistance that might be reversed by ABCB1 inhibitors such as tariquidar [Our study is the first to show DNA methylation of the t cohort . This find ABCB1 . How muccultures  and re-ecultures . Althougflicting , a recenriquidar .TOP2A) gene significantly improves the outcome of anthracycline based adjuvant chemotherapy [TOP2A and ERBB2 are co-amplified in our dataset  warranting further investigation to explore the interaction between ABCB1 methylation status and TOP2A/ERBB2 amplification and how the combined effect of these proteins contributes to the drug resistance observed in anthracycline treatment regimens.A possible link between ERBB2 and ABCB1 expression has been observed in a multidrug resistant MCF-7 cell line . The ampotherapy ,38. InteIGF2 exerts its action on cellular growth through the insulin-like growth factor 1 receptor which interferes with anti-ERBB2 treatment through Akt signalling [IGF2 have been associated with overexpression of IGF2 [IGF2 is specifically observed in ERBB2 positive breast cancers providing a new potential mechanistic link between IGF1R expression and ERBB2 status via IGF2 methylation status.gnalling . In muri of IGF2 , which iGSTP1 and FOXC1 methylation status as independent prognostic markers for breast cancer survival in a uniform patient cohort receiving neoadjuvant doxorubicin monotherapy prior to surgery and five years of tamoxifen for all ER positive patients according to a clinical study protocol [FOXC1 methylation status might be a general prognostic marker as it is able to separate patients in good and poor survival groups in the doxorubicin treated as well as in validation cohort while GSTP1 and ABCB1 methylation status might be a predictive marker for doxorubicin monotherapy as the methylation status of these genes were not able to separate patients into survival groups in the validation cohort [GSTP1 methylation decreased when the operation status was taken into account indicating that those tumours that increased further or at least did not regress during neoadjuvant treatment were more often unmethylated for GSTP1 while FOXC1 hazard ratio increased as would be expected for a treatment independent effect. ABCB1 methylation status proved to be a marker for survival in the discovery cohort although it was not independent of other known markers in a multivariate model. The association with survival was less significant when the expression status instead of the DNA methylation status was analysed due to a strong correlation between DNA methylation and expression for GSTP1 only.We identify here the protocol . FOXC1 mn cohort . This isFOXC1, a member of the forkhead protein family, many members of which are involved in the development and progression of cancer [FOXC1 have recently been reported in a candidate re-sequencing approach of breast tumours [FOXC1 was found to be specifically hypomethylated and highly expressed in CD44+ compared to CD24+ stem cell progenitors, but no data correlating survival to the methylation patterns was presented [FOXC2 gene has been found to promote tumour metastasis and invasiveness [We confirm here a very recent report on the presence and extent of DNA methylation in the promoter of f cancer . Mutatio tumours  and FOXCresented . The oveglutathione-S-transferase 1 (GSTP1) is a well established biomarker for hormone dependent cancers. Low activity of GSTP1 resulting from promoter hypermethylation may increase the effective therapeutic dose of the pharmacological agent due to lower conjugation and inactivation of the drug leading to increased survival. In support of this hypothesis it has been shown that GSTP1 expression correlates with doxorubicin resistance in breast cancer cell lines [CpG hypermethylation of the promoter region of the ll lines . The obsll lines ,47.ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness.Methylation at the FOXC1 methylation status might be a widely applicable prognostic factor for breast cancer patients while the methylation status of ABCB1 and GSTP1 might be a predictive factor for doxorubicin and perhaps anthracycline treatment in general. However, further studies are necessary to confirm the predictive value of these markers requiring additional patient cohorts treated with a doxorubicin/anthracycline based monotherapy. Valuable time for treatment might be gained and the serious side-effects of a doxorubicin based regimen might be avoided taking the methylation status for treatment decisions into account. As the analyzed cohort consists of locally advanced primary tumours, it will be interesting to investigate the DNA methylation profiles also in T1/T2 and early stage breast cancer samples. Despite similar RNA expression profiles [The profiles , some biprofiles . Additio2) scheduled for 16 weeks. Patients with an operable tumour (n = 60) after neoadjuvant treatment had surgery followed by radiotherapy immediately after termination of the neoadjuvant chemotherapy. Eight patients had to be given radiotherapy prior to surgery due to local tumour extension, and seven patients were not eligible for surgery and were treated on an individual basis. Women with estrogen and/or progesterone positive tumours were all treated with tamoxifen (30 mg daily for 5 years). The main clinical characteristics of the analyzed 75 samples are given in Table Locally advanced breast cancer patients, admitted to the Haukeland University Hospital in Bergen (Norway) between 1991 and 1998 were part of a prospective study evaluating predictive factors for response to doxorubicin (n = 94). Tumour DNA was available in sufficient quantity to perform methylation analyses from 75 of the patients. Tissue was obtained by an incisional biopsy prior to therapy and was immediately snap-frozen (liquid nitrogen in the theater) as previously reported . DNA wasTP53 mutation status (n = 162) were available and used for validation. Follow-up/survival data was available for 87 of the patients. Tumour DNA extraction, bisulphite treatment and pyrosequencing analyses was performed using the same procedures as for the discovery dataset.163 random, unselected breast cancer samples were used for the validation of the observed associations with clinicopathological factors. Clinical and molecular parameters such as histological grade (n = 162), Estrogen receptor status (n = 158) and DNA samples from normal breast tissue were included as control samples for methylation analysis. Normal breast tissue (n = 6) was obtained from women who underwent a biopsy of the mammary gland because of mammographic abnormalities, but for which histology confirmed the presence of only normal tissue.The sample set was completed by immortalized human mammary epithelial cells (HMEC) and five widely used breast cancer cell lines .TP53 were analyzed in both the discovery and the validation cohorts by temporal temperature gradient electrophoresis (TTGE) followed by Sanger sequencing as previously described with primers covering regions (exons and introns) from exon 2-11 [Mutations in xon 2-11 ,49. 50 oxon 2-11 , and a s2, 100 \u03bcM dNTPs and 2.0 U HotStar Taq polymerase  in a 25 \u03bcl volume. The PCR program consisted of a denaturing step of 15 min at 95\u00b0C followed by 50 cycles of 30 s at 95\u00b0C, 30 s at the respective annealing temperature . TaqMan assays were all purchased from Applied Biosystems: Hs 00943351_g1 (GSTP1), Hs00184500_m1 (ABCB1) and Hs00559473_s1 (FOXC1). Human Breast Total RNA  was used to generate standard curves. PMM1 (Hs00963626_m1) was used as endogenous control and the relative gene expression levels were determined using the standard curve method and normalized to PMM1.50 of the tumours have previously been analyzed for gene expression using genome wide cDNA microarrays . For qua2 tests. Samples were scored as methylated when the methylation degree exceeded the average methylation degree of the normal samples by two times the standard deviation of the normal samples and had at least a methylation degree of 5% (detection limit of the technology). Odds ratio and 95% confidence intervals were calculated. Differences in the distribution of methylation were assessed by the non-parametric Mann-Whitney or the Kruskal-Wallis test. Correlation between the methylation status of the different genes was calculated by the non-parametric Kendall's tau test. Pearson's coefficients were used to study the correlation between methylation and expression levels. All calculations were performed using Statistical Package for Science version 15.0. The Cox proportional hazards model was used to evaluate the effect sizes (given as hazard ratios), 95% Confidence intervals (CI), regression coefficients and statistical significance of known clinicopathological features as well as the methylation status of selected genes. All covariates were treated as categorical variables. To investigate the relationship between multiple explanatory factors and survival, we used the Akaike information criterion (AIC) [TP53 mutation status as well as methylation of ABCB1, FOXC1 and GSTP1 respectively, were considered as covariates to the model. With L being the likelihood function of the model and k indicating the number of parameters of the model, the Akaike information criterion (AIC) is calculated by: AIC = -2log L+2k.Differences in the presence of methylation were determined by a two-sided Fisher's test and \u03c7on (AIC) . AIC evaThe authors declare that they have no competing interests.ED and JAR performed laboratory experiments and data analyses and participated in writing of the manuscript. HS was involved in the statistical analyses. PEL, SG and TA were responsible for the doxorubicin treated patient cohort and clinical database management. ALBD and VNK were responsible for the validation cohorts and IB for the control samples. IGG, ALBD, VNK and JT initiated and designed the study. JT wrote the manuscript and VNK, PEL and ALBD participated in writing the manuscript. All authors have read and approved the final manuscript.GSTP1, FOXC1 and ABCB1Correlation between DNA methylation and RNA expression for . Scatter plots showing the correlation between DNA methylation and RNA expression as measured by TaqMan for GSTP1, FOXC1 and ABCB1.Click here for fileAssociations of the presence/absence and degree of methylation and the clinical and molecular parameters. Associations of the presence/absence and degree of methylation and the clinical and molecular parameters of the samples by Fisher's exact test  or \u03c72 analysis  with odds ratio (OR) and 95% Confidence interval (CI) and their respective p-value in the validation cohort. Statistical significance of the differences in the distribution of the degree of methylation is assessed by the non-parametric Mann-Whitney and Kruskal-Wallis test. Samples are called methylated if the methylation degree exceeded 5% and the average methylation degree of the healthy tissue samples plus at least two times the standard deviation of the healthy tissues.Click here for fileBest models to fit the observed survival data. A) Best models with a varying number of covariates to fit the observed survival data. B) Multivariate analysis of the best model with the minimum number of covariates. The presented model is the only one where all covariates are significant in multivariate analysis.Click here for fileSupplementary discussion. Discussion of negative results.Click here for filePCR and pyrosequencing primers. Sequences of primers used for amplification and pyrosequencing reactions, Genbank accession numbers and nucleotides (Nt) corresponding to the amplified fragments as well as the annealing temperatures for the respective PCR amplifications. CpGs are numbered in the order of appearance from the 5' end of an amplification product. Y = pyrimidine.Click here for file"}
+{"text": "Hydralazine can substantially decrease blood flow and increase hypoxia in transplanted tumours. Previous indirect studies have suggested that hydralazine does not induce such effects in spontaneous tumours. We have now directly investigated the ability of hydralazine to increase hypoxia in both transplanted and spontaneous murine tumours by measuring tumour oxygen partial pressure (pO2) distributions using an Eppendorf oxygen electrode. Spontaneous tumours arose at different sites in CDF1 mice, while transplanted tumours were produced by implanting a C3H mouse mammary carcinoma on the backs of the same mouse strain. Measurements of pO2 were made in anaesthetised mice immediately before and 45 min after an intravenous injection of 5 mg kg-1 hydralazine. In the transplanted tumours hydralazine significantly decreased tumour oxygenation, such that the percentage of pO2 values < or = 5 mmHg increased from 45% to 87%, and median pO2 decreased from 5 to 3 mmHg. Similar significant changes were induced by hydralazine in the spontaneous tumours, the percentage of pO2 values < or = 5 mmHg increasing from 60% to 94% while the median pO2 values decreased from 8 to 2 mmHg. These results clearly show that there is no difference in the response of transplanted and spontaneous mouse tumours to hydralazine."}
+{"text": "We analysed 26 metastases from 25 patients with sporadic cutaneous malignant melanoma for alterations in the CDKN2 gene by a combined polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP)/nucleotide sequencing approach. Eleven alterations  were concordantly and independently detected by both SSCP and nucleotide sequence analysis. Two of the exon 2 changes and the five changes in the non-coding exon 3 region are likely to represent natural polymorphism. Four (15%) of 26 metastases thus had CDKN2 mutations and belonged to 3 (12%) of 25 patients. Semi-quantitative PCR furthermore revealed no sign of homozygous deletions of the CDKN2 exon 2 region. The results support an involvement of the CDKN2 product in the development of a subgroup of sporadic melanomas and encourage the search for alterations in additional genes of the 9p21 region."}
+{"text": "Epigenetic modifications associated with imprinting regulation can be compared to those associated with the more canonical developmental regulation, important for processes such as differentiation and tissue specificity. Here we test the hypothesis that these two mechanisms are associated with different histone modification enrichment patterns.The field of epigenetics is developing rapidly, however we are only beginning to comprehend the complexity of its influence on gene regulation. Using genomic imprinting as a model we examine epigenetic profiles associated with different forms of gene regulation. Imprinting refers to the expression of a gene from only one of the chromosome homologues in a parental-origin-specific manner. This is dependent on heritable germline epigenetic control at a Using high-throughput data extraction with subsequent analysis, we have found that particular histone modifications are more likely to be associated with either imprinting repression or developmental repression of imprinted genes. H3K9me3 and H4K20me3 are together enriched at imprinted genes with differentially methylated promoters and do not show a correlation with developmental regulation. H3K27me3 and H3K4me3, however, are more often associated with developmental regulation. We find that imprinted genes are subject to developmental regulation through bivalency with H3K4me3 and H3K27me3 enrichment on the same allele. Furthermore, a specific tri-mark signature comprising H3K4me3, H3K9me3 and H4K20me3 has been identified at all imprinting control regions.A large amount of data is produced from whole-genome expression and epigenetic profiling studies of cellular material. We have shown that such publicly available data can be mined and analysed in order to generate novel findings for categories of genes or regulatory elements. Comparing two types of gene regulation, imprinting and developmental, our results suggest that different histone modifications associate with these distinct processes. This form of analysis is therefore a useful tool to elucidate the complex epigenetic code associated with genome function and to determine the underlying features conferring epigenetic states. Epigenetic mechanisms play an important role in the control of gene expression. Modification to the packaging of DNA is believed to allow a more open or closed structure and influences association of the transcriptional machinery with the genetic material. The most characterised examples of epigenetic mechanisms to date in mammalian cells include DNA methylation of cytosine and post-translational modifications to the core histone proteins of the nucleosome (reviewed in ), thoughThe transcriptome of a cell is tightly regulated by epigenetic mechanisms to allow correct gene expression patterns at appropriate time points. The dynamic changes in gene expression required during the proliferation, differentiation and commitment of specific cell types are associated with specific epigenetic alterations. In order to simplify the description of this type of regulation we shall hereafter refer to this as developmental regulation.An additional mechanism of gene regulation is that of genomic imprinting, an epigenetic process affecting less than 1% of genes in the mammalian genome. An imprinted gene refers to a gene in which expression occurs solely or predominantly from only one of the parental chromosome homologues (reviewed in ). EitherBoth DNA methylation and post-translational histone modifications have been found to be enriched to a greater degree on one chromosome compared to its homologue at a number of imprinted loci in the mouse and human . DiffMany studies of specific loci have described differential enrichment of particular histone modifications between the two parental chromosomes at some ICRs and imprinted gene promoters or transcription start sites (TSSs). These marks include histone H3 acetylation, H4 acetylation, H3 dimethylation at lysine 4 (H3K4me2) and H3K4me3, which are found preferentially enriched on the unmethylated chromosome or normally active allele in comparison with its homologous counterpart, and H3K27me2, H3K27me3, H3K9me2 and H3K9me3 preferentially enriched on the methylated chromosome or inactive allele -42. H4K2Key to the work presented here, the active alleles of imprinted genes are also developmentally regulated. Imprinted genes that are developmentally expressed have only the 'normally active' allele expressed, whereas developmentally repressed imprinted genes have the 'normally active' allele repressed resulting in two repressed alleles. Very few studies have assessed the nature of epigenetic marks at developmentally repressed imprinted genes. We have used imprinted genes as a model system to compare epigenetic marks associated with the control of imprinting to those associated with developmental regulation to test the hypothesis that distinct epigenetic modifications are employed for these two mechanisms.An increasing number of groups are describing the epigenetic characteristics of various cell types across the whole genome in both mouse and human and these studies produce a large amount of publicly accessible data  and in more differentiated cell types . Secondly, 35% of imprinted genes are enriched with both H3K4me3 and H3K27me3 compared to 16% of all genes in the mouse genome . We discuss the H3K4me3/H3K9me3 profile below; whether the H3K4me3/H3K27me3 profile reflects developmental or imprinting regulation is addressed subsequently.Figure et al. [not to act as an ICR (Gnas Exon1A) does not exhibit this specific tri-mark profile. These findings imply that the presence of H3K4me3, H3K9me3 and H4K20me3 is a true epigenetic signature of ICRs.As H3K9me3 enrichment has previously been linked to H4K20me3 , we alsoet al.  using a n = 26) have a significantly different epigenetic profile for these four marks to imprinted genes without a promoter DMR (n = 22) in mouse ESCs .In order to assess the histone modification enrichment profiles at both germline and somatic DMRs we have compared imprinted genes with and without promoter DMRs for enrichment of H3K9me3, H4K20me3, H3K4me3 and H3K27me3 in mouse ESCs using the same source data as above , we find that all are germline DMRs rather than somatic DMRs indicating that the combined presence of H3K9me3 and H4K20me3 only occurs in the presence of H3K4me3 specifically at germline DMRs, defining the identified tri-mark profile.As seen from Figure et al. [Cd81, Osbpl5 and Tssc4 are biallelically expressed in ESCs [The 'normally active' allele of an imprinted gene can be developmentally regulated; we have therefore sorted mouse imprinted genes by expression status in ESCs using microarray data from Mikkelsen et al. . This mi in ESCs ). By intnot associated with developmental regulation, but rather with imprinting. Additional file If a particular modification is enriched to an equal degree at developmentally expressed and repressed imprinted genes, it is likely that this modification is without promoter DMRs that were assessed, none are enriched for H3K9me3 or H4K20me3 . Together, our results strongly suggest that H3K9me3 and H4K20me3 are not associated with developmental repression of imprinted genes in mouse ESCs, but rather with imprinting control.Histone modification enrichment at developmentally expressed or repressed imprinted genes can be assessed alongside the promoter DMR status. Of all developmentally repressed genes The results depicted in Additional file P < 0.01 for [P < 0.05 for [Our results in Figure 0.01 for  and 30% 0.05 for ; data no0.05 for . This maet al. [P < 0.0005). When comparing imprinted genes to all genes in the mouse genome enriched with both H3K4me3 and H3K27me3 a significant difference is observed for both NPCs and MEFs . A reduction in combined H3K4me3 and H3K27me3, but not complete removal, is observed between ESCs and the more differentiated cell types, suggestive of resolution of bivalency [41% in ESCs compared to 7% in NPCs  and 22% in MEFs   and in combination (H3K4me3 and H3K27me3) with regard to developmental expression status Figure . This imOf the few genes that possess both marks in NPCs and MEFs, we observe a somewhat higher number of developmentally repressed imprinted genes with coenrichment than expressed genes  across all cell types is significantly greater at developmentally expressed than repressed imprinted genes . For H3K27me3, enrichment is not observed at all imprinted genes that do not possess a promoter DMR, but rather at only 55% of these genes in ESCs . Furthermore, when assessing only imprinted genes that do not have a promoter DMR and are developmentally expressed in ESCs , H3K27me3 is enriched only 20% of the time ; H3K27me3 enrichment is found at 30% of developmentally expressed imprinted genes that do possess a promoter DMR (n = 10). This implies that H3K27me3 does not commonly repress the inactive allele in the absence of differential promoter methylation in mouse ESCs and that enrichment is not generally dependent on promoter DMR status. No developmentally expressed genes are enriched with H3K27me3 in NPCs; in MEFs an equal level of enrichment is observed at imprinted genes with promoter DMRs  and without promoter DMRs  of those that are developmentally expressed.The presence of H3K4me3 and H3K27me3 together at 25% and 20% of developmentally The comparative characterisation of histone modification profiles, DMR status and expression profiles for all confirmed imprinted genes provides a valuable resource for those interested in the role of these epigenetic marks in different classes of gene expression and repression and in the regulation of genomic imprinting in particular. Additionally, patterns of histone modification enrichment common to imprinted genes have been identified and highlight differences in the epigenetic profiles of these particular genes compared to other genes. In our analyses, we have been able to infer imprinted rather than developmental repression through the identification of modifications always associated with a germline DMR regardless of the developmental expression status of the gene. Histone modification enrichment profiles found to associate with the developmental expression state are acknowledged to be involved in the developmental regulation of imprinted genes.The repressive marks H3K9me3 and H4K20me3 are unlikely to play a prominent role in developmental repression of imprinted genes. This is apparent through the finding that H3K9me3 and H4K20me3 are not enriched at developmentally repressed genes any more often than at expressed genes in mouse ESCs . The presence of H3K27me3 at up to a quarter of developmentally expressed imprinted genes in both mouse ESCs and MEFs Figure  implies Some inactive imprinted alleles are associated with DNA methylation and others are not. As we have identified H3K27me3 to be involved in imprinting regulation at some genes, we investigated whether this histone mark might play a role in repressing the inactive allele in the absence of DNA methylation, as suggested by others . SeveralIgf2r/Airn imprinted locus. In mouse fibroblasts where Slc22a2 and Slc22a3 are developmentally  repressed, widespread H3K27me3 was enriched on both parental chromosomes [et al. [Grb10. They also established that H3K27me3 marks both chromosomes in neurons where this transcript is developmentally repressed. At the brain-type promoter of Grb10, Sanz et al. [The histone mark H3K27me3 is more often found at developmentally repressed than at expressed imprinted genes in mouse ESCs . Studieet al. [Our analyses show that all ICRs display enrichment of H3K9me3 and H4K20me3 in combination with H3K4me3, constituting an epigenetic signature. Previous reports have identified enrichment of H3K27me3 on the methylated chromosome of several ICRs ,25,35,41et al.  to identet al. ,35,36,38et al. ), which The analyses performed here provide insight into the association of histone modifications with different forms of genomic regulation. In all the cell types assessed, H3K27me3 is frequently found associated with developmental repression of imprinted genes, whereas H3K9me3 and H4K20me3 correlate with repression of the inactive allele of imprinted genes possessing promoter DMRs, rather than with spatiotemporally controlled developmental repression in ESCs. H3K4me3 is found associated with developmental regulation. We further propose that bivalent domains act to developmentally repress imprinted genes, as they do for other genes in the genome. In addition, we have identified an H3K4me3, H3K9me3 and H4K20me3 tri-mark signature at all ICRs without exception in mouse ESCs. Figure It is demonstrated here that high-throughput studies can be mined and analysed for smaller functional categories to generate novel insights into different forms of epigenetic regulation. Through genome-wide analysis, we have shown that alleles inactivated by genomic imprinting can be distinguished epigenetically from developmentally repressed alleles. These findings are consistent with published allele-specific data on individual imprinted loci  status: this worksheet details the promoter DMR status of imprinted genes used for analysis of histone modification profiles at genes with and without promoter DMRs, including references; Chromatin and expression states: this worksheet provides data extracted from six high-throughput studies for all available imprinted genes. Notably, not all imprinted genes were present in the source data files, most likely due to the high proportion of imprinted genes currently holding a predicted status in gene reference databases. If a gene was not present in one source data file the output is given as 'Gene not present'. Genes imprinted only in mice, or where imprinting status is not confirmed in humans, or where no orthologous human gene exists (*), are excluded from further human analyses. The same applies for human-specific imprinted genes (\u2020), which are excluded from all mouse analyses. Isoform-dependent imprinted genes (\u2021) are not included in any further analyses. hESC, human embryonic stem cells; mESC, mouse embryonic stem cells; REH, human pro-B cells; mNPC, mouse neural progenitor cells; MEF, mouse embryonic fibroblasts; TSS, transcription start site; HCP, high CpG promoter; ICP, intermediate CpG promoter; LCP, low CpG promoter; MPSS, massively parallel signature sequencing.Click here for fileHistone modification enrichment at developmentally expressed and repressed imprinted genes in embryonic stem cells (ESCs). Enrichment of H3K4me3, H3K27me3, H3K9me3 and H4K20me3 at transcription start sites of developmentally expressed and repressed imprinted genes were assessed in mouse ESCs using high-throughput enrichment and expression source data from Mikkelsen et al. [P = 0.525).n et al. . The preClick here for fileAllele-specific histone modification enrichment at imprinted genes. In order to compare the results of our genome-wide analyses with allele-specific data, we have characterised previously published histone modification enrichment profiles involved in imprinting and developmental repression at imprinted genes. This data supports our findings and provides independent validation of our results.Click here for file"}
+{"text": "G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions . Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60\u201367%).The effect of expression in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.The results suggest that the expression of either gene has led to inhibition of tumour growth  We have performed a deletion survey of 3p on more than 400 lung, renal, breast, cervical and ovarian carcinomas using a defined set of markers, combining conventional LOH (loss of heterozygosity) with quantitative real-time PCR (qPCR), comparative genomic and NotI microarrays hybridisations in vivo and in vitro. When the transfectants were passaged as tumours in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumours tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumours studied. In similar experiments with wt P53, the exogenous P53 gene was maintained and expressed in all 6 tumours tested, but in a mutated form. On the basis of these experiments we have developed the gene inactivation test (GIT) for a functional definition of TSG. It is based on the comparison of cell growth in vitro and tumour growth in vivo when the gene is/is not expressed. The main idea of the test is that a gene inhibiting growth of tumour cells should be inactivated in growing tumours by genetic or epigenetic mechanisms.The definition of a TSG is based on the demonstration of its regular inactivation by mutation or epigenetic silencing in tumour samples. It is also important to obtain supportive evidence from functional studies. We have previously found  non-random losses of human 3p21-p22 fragments from mouse-human microcell hybrids following progressive growth in SCID mice FUS1, SEMA3B, G21/NPRL2, RASSF1A, RASSF1C, RBSP3 (genes from AP20 and LUCA regions) and other TSGs inhibit tumour cell growth both in vitro and in vivoUsing GIT and growth analysis under cultural conditions we have shown that TCEA1, MLH1, RHOA, 3PK, PL6, 101F6, BLU, TGFBR2) did not show any effect in the tested cell lines However other genes from 3p21.3 (e.g. HYAL1 and HYAL2).Here we describe the functional analysis of two additional genes from the 3p21.3 LUCA region, hyaluronidases -1 and -2  is located in the LUCA region. It contains 6 exons producing a 2.6 kb mRNA (coding for 436 aa protein). It is well expressed in all analysed normal human tissues including lung and kidney. It was not expressed in 18 out of 20 lung cancer cell lines The HYAL2  contains 4 exons producing a 2 kb mRNA (that encode 473 aa). It is well expressed in all analysed human tissues including lung, kidney and many lung cancer cell lines. The protein is attached to the membrane by the glycosylphosphatidyl-inositol-anchor (GPI-anchor) HYAL2 protein was identified as a receptor for the sheep lung cancer retrovirus, JSRV, and a sequestration mechanism inactivating HYAL2 protein was demonstrated. The env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequesters the HYAL2 protein. The absence of HYAL2  leads to ligand-independent activation of the RON receptor tyrosine kinase and its downstream AKT and MAPK signaling pathways The These two genes contribute to intracellular and extracellular catabolism of hyaluronic acid (HA) in a CD44-dependent manner HYAL1 and HYAL2 can have a great importance not only for better understanding of HA catabolism and human carcinogenesis but could provide therapeutic targets for cancer treatment. In this study we have found that the expression of either gene suppressed tumour growth in vivo but not in vitro. These findings are consistent with earlier somatic hybrid studies by Henry Harris, George Klein and Francis Wiener, showing that somatic hybridisation of normal and malignant cells can suppress tumorigenicity in vivo but not cell growth in vitroin vivo suppressive effects have not been clarified, but at least part of them may have acted at the level of tumour-host interactions. The finding of a similar phenomenon with the two genes involved in the present study is a step towards the analysis of this important phenomenon.Thus analysis of HYAL1 and HYAL2. These genes are located in the minimally deleted region of LUCA , which were used in our growth suppression experiments for testing RASSF1A and G21/NPRL2HYAL1 and HYAL2 are well expressed in normal kidney and lung (see for example http://www.genecards.org/index.shtml) we have chosen KRC/Y and U2020 for our study.Expression of HYAL1 and HYAL2 were cloned into the episomal tetracycline-regulated pETE-Hyg vector HYAL1 and HYAL2 showed almost no inhibition of colony formation. When HYAL1 and HYAL2 were expressed the cloning efficiency was 77\u2013100%, compared to the empty vector. As a positive control we used FUS1 gene, a strong TSG cloned in pETE FUS1 was less than 5% and for U2020 cells it was less than 25% compared to controls.HYAL1 and HYAL2 transgenes. The pETE-HYAL1 and pETE-HYAL2 vectors carrying wild type alleles of the tested genes were transfected into KRC/Y expressing tTA and clonal cell lines were selected. Expression of the transgenes was tested by Northern hybridization. Two of the best tetracycline regulated clones for each gene were then used in further experiments (see for example HYAL1 clones 1 and 4 were selected (HYAL1-KRC/Ycl.1 and HYAL1-KRC/Ycl.4) and for HYAL2 clones 13 and 14 (HYAL2-KRC/Ycl.13 and HYAL2-KRC/Ycl.14). Growth curves for HYAL1-KRC/Ycl.4 and HYAL2-KRC/Ycl.13 are shown in We performed also growth curve inhibition experiments using KRC/Y cells stably transformed with HYAL1 (HYAL1-U2020cl.4 and HYAL1-U2020cl.5) and HYAL2 (HYAL2-U2020cl.15 and HYAL2-U2020cl.18). All eight clones expressing either HYAL1 or HYAL2 in KRC/Y and U2020 cells were tested with CyQUANT NF Cell Proliferation Assay based on measurement of cellular DNA content via fluorescent dye binding  as described by Li et al., 1999 et al., 2002 in vivo. Clonal cell lines expressing transgenes were inoculated into SCID mice and the expression of the transgene was controlled by tetracycline administered ad libitum in the drinking water. In this setting TSGs suppressed tumour formation in SCID mice, unless they were eliminated or mutated. All grown tumours were analysed for the presence and expression of the transgene.We investigated HYAL1 and HYAL2 cell clones were inoculated into 22 SCID mice . For each SCID mouse, 5\u00d7106 cells were inoculated (one inoculation per mouse). In control mice, KRC/Y cells transfected with empty pETE vector were used (twelve SCID mice were injected). Half of the SCID mice were then given drinking water containing 1mg/ml tetracycline. Results of these experiments are shown in HYAL1 or HYAL2. All grown 12 tumours (T1\u2013T12) were explanted and tested for the presence of pETE-HYAL1 and pETE-HYAL2 constructs by PCR. Transgenes were detected only in one tumour (HYAL2) and in eleven tumours it was deleted. In tumour T10 where the HYAL2 gene was present by PCR, there was no detectable expression by Northern (data not shown).KRC/Y derived HYAL1 and HYAL2 induce growth inhibition of tumours in SCID mice we tested expression of these genes in lung and renal tumours using qPCR (see As our data suggested that both qPCR see .HYAL1 was 7.8 (from 2.5 up to 77) and for HYAL2 8.8 (2.5\u201353) times . In seven cases expression of HYAL1 fell down more than 10-fold. For HYAL2 eight such cases were found. In six of fifteen lung biopsies we found more than 10-fold decrease of both HYAL1 and HYAL2 expression. In RCC samples expression of HYAL1 declined in 10 of 15 cases  and average decrease was 6.5 times (from 2 to 46). For HYAL2 reduced expression was detected in 9 of 15 samples  and average decrease was 4.9 times (2\u201311.4). Only in three RCC samples expression of HYAL1 decreased more than 10-fold and in one case we detected similar strong decline of HYAL2 expression. No statistically significant difference in cases with different tumour stages was observed both in SCC and RCC cases.Fifteen SCC and fifteen RCC tumours were studied. We assumed that expression fell down if it was at least 2-fold lower than in matched control samples. Expression of both genes was drastically decreased in all lung samples  did not show any effect in the tested cell lines. Six genes  had strong growth inhibitory activity, both in vitro and in vivo.In this work we describe functional characterization of NPRL2/G21 demonstrated growth inhibition in vitro in small cell lung cancer cell line U2020 and renal carcinoma cell line KRC/Y cells almost 90%. RASSF1A gene showed the same results. In SCID mice both genes either did not permit tumour formation or in growing tumours they were inactivated (for NPRL2 it was 10 mice and for RASSF1A it was 8 mice).For example, HYAL1 nor HYAL2 showed significant growth inhibition in colony formation and growth curve assays in vitro  even when the genes were repressed (water with tetracycline). However we have already shown that gene expression leakage in vivo is stronger than in vivo. Moreover it is known that tetracycline is a weaker inhibitor of expression compared to doxycycline in tTA system In contrast, neither itro see \u20133. CyQUAitro see  also demin vivo, most likely by influencing some interaction between the tumour cells and the host, but without directly affecting tumour cell growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Since the products of both genes have a potential to influence intercellular interactions, their impairment may inhibit microenvironmental controls that normally protect the host from tumour growth.Thus, results clearly showed that the expression of either gene has led to the inhibition of tumour growth HYAL1 and HYAL2 are the major mammalian hyaluronidases in somatic tissues. They may act in concert to degrade high molecular weight hyaluronan  to the tetrasaccharide level HYAL2 hydrolyses high molecular weight hyaluronic acid  to produce an intermediate-sized product which is further hydrolysed by HYAL1 to give small oligosaccharides. Hyaluronan is claimed to be involved in tumour invasion and metastatic spread. The levels of HA surrounding tumour cells are often correlated with tumour aggressiveness and poor outcome Overproduction of HA enhances anchorage-independent tumour cell growth HYAL1 and HYAL2 in these studies, no frequent inactivating mutations were identified so far in these genes. Nevertheless it was reported that HYAL1 was inactivated in six of seven head and neck squamous cell carcinoma lines by illegitimate splicing HYAL2 promoter was methylated in more than 50% of non- small cell lung cancer (NSCLC) cases with decreased expression of the HYAL2 .In contrast to the tumour suppressor function of HYAL1 and HYAL2 was investigated in the study of Ji et al.HYAL1 and HYAL2 did not significantly inhibit tumour cells growth neither in vitro nor in vivo. However in this work it was demonstrated that HYAL2 inhibited experimental lung metastases in nu/nu mice. It is difficult to compare this work with our study. They used non-small cell lung cancer cell lines and recombinant adenoviral vectors. Completely different methods were used to measure suppressor activity. Despite these differences main results are similar to our work: HYAL1 and HYAL2 do not have strong growth inhibiting activity in vitro and HYAL2 displayed some tumour suppressor activity in vivo.Suppressor activity of HYAL1 and HYAL2 in SCID mice, we suggested that in primary renal and lung cancers expression of these genes might also be distorted. mRNA quantification was done for these genes in fifteen lung (SCC) and fifteen kidney (RCC) tumours using qPCR. In the majority of the tumour samples mRNA level of both genes was significantly decreased (HYAL1 and HYAL2 mRNA level was 0.67 (P\u200a=\u200a0.005) in lung cancer samples . However in RCC samples it was only 0.4 . In any case, the results indicated that these genes may play important role in the development of lung and renal malignancies.Since we demonstrated tumour suppressor function of ecreased , sometimin vivo, without any inhibition of cell growth in vitro?What genes could be responsible for the suppression of tumour growth in vivo; products required for normal cellular responses to microenvironmental controls; or genes whose products inhibit angiogenesis There are at least three conceivable categories: genes encoding products required for responding to differentiation inducing signals Hyaluronidases are known to play an important role in tumour/host interactions. They may be examples of genes encoding interactive molecules that participate in microenvironmental growth control and carcinogenesis.There may be many other, as yet unidentified suppressor genes with similarly asymmetric inhibitory properties. The large numbers of LOHs that occur in the major human tumours and are not accounted for by known tumour suppressor genes, as well as the existence of \u201ctumour suppressor gene clusters\u201d, as documented on human chromosome 3p Paired tumour/normal samples were obtained from Blokhin Cancer Research Center, Russian Academy of Medical Sciences. Altogether fifteen specimens of lung squamous cell carcinoma (SCC) and fifteen clear cell renal carcinoma (RCC) cases and adjacent morphologically normal tissues  were obtained from patients after surgical resection of primary tumours and and stored in liquid nitrogen.. Top and bottom sections (3\u20135 \u00b5m thick) cut from frozen tumour tissues were examined histologically and only samples containing 70% or more tumour cells were used in the study. The samples were collected in accordance to the guidelines issued by the Ethic Committee of Blokhin Cancer Research Center, Russian Academy of Medical Sciences (Moscow). All patients gave written informed consent that is available upon request. The study was done in accordance with the principles outlined in the Declaration of Helsinki. All tumor specimens were characterized according to the International System of Clinico-Morphological Classification of Tumors (TNM).SCLC  cell line U2020 was described earlier HYAL1, HYAL2 and FUS1 genes was described previously FUS1mut containing Val33Met amino acid substitution was isolated from KRC/Y cell line transformed with wild type FUS1 (unpublished data). These genes were re-cloned into the pETE vector Molecular cloning of the human All molecular, cell biology and microbiology procedures were performed as described previously Construction of U2020 and KRC/Y cell lines producing tetracycline trans-activator tTA was described in HYAL1, HYAL2, FUS1 and FUS1mut genes were purified using R.E.A.L. Prep kit . Transfections were performed using Lipofectamine PLUS Reagent  according to the manufacturer's protocol.Plasmid DNAs containing After transfection, cells were selected with 200U/ml Hygromycin and 200ng/ml doxycycline for four weeks.6 cells were collected and total RNA was isolated with Trizol\u00ae reagent .PCR positive clones from each recombinant were grown in Iscove's cell culture medium supplemented with 10% heat-inactivated FBS, 100 \u00b5g/ml penicillin, 100 \u00b5g/ml streptomycin, 200 U/ml hygromycin and 200ng/ml doxycycline. Each clone was split into two parallel flasks, in one of them cells were grown without doxycycline. After one week, 10HYAL1 and HYAL2 probes were purified using electrophoresis and the Jetquick Gel Purification kit . The probes were labeled with \u03b1-P32 dCTP by random labeling.Northern blotting and hybridization were performed as described before in vivo and in vitro were done as described previously All growth inhibition experiments Briefly, U2020 or KRC/Y cells were transfected with plasmid DNA using Lipofectamine/Plus reagent . Transfected cells were stripped and plated on 100 mm cell culture plates. After selection with 400 \u00b5g/ml Hygromycin for 2 weeks, Giemsa-stained colonies were photographed and counted.6 cells were injected into 4-week old female SCID mice. Each mouse received only 1 injection. Twelve control mice were injected with empty pETE vector (six mice were drinking water with DOX and six without). Twenty two mice were used for the experiments  according to the manufacturer's protocol. Briefly, cells were plated at density of 100\u2013500 cells per well in a 96-well plate . Number of cells in wells was counted every 24 hours: growth medium was removed, 50 \u00b5l of green-fluorescent CyQUANT GR dye (which exhibits strong fluorescence enhancement when bound to cellular nucleic acid) was added to the well and incubated for 30 min at 37\u00b0C. The fluorescence intensity of each sample was measured using a fluorescence microplate reader with excitation at 485nm and emission detection at 530nm. Plotted data points represent averages of triplicate samples, the plotted line is a linear regression fit of all data points. The assay is designed to produce a linear analytical response from at least 100\u201320,000 cells per well in most cell lines.GAPDH was assessed using qPCR . Ratio of mRNA level of target genes in all analysed tumours was measured relative to the reference \u201cnormal\u201d samples .RNA isolated from primary tumour samples was reverse transcribed using the GeneAmp\u00ae RNA PCR Kit (Applied Biosystems) according to the manufacturer's protocol. The relative mRNA level of target genes and The primers and probes for transcripts were as follows:HYAL1:5\u2032- TTTCTGCCCCTGGATGAGC-3\u2032;Forward primer, 5\u2032-CTCACCCAGAGCACCACTCC-3\u2032;Reverse primer, 5\u2032-FAM-CCCAGGCTGTGCTCCAGCTCA-[RTQ1]-3\u2032;Probe, (amplicon size 80 bp);HYAL2:5\u2032- CACCACAAGCACGGAGACCT-3\u2032;Forward primer, 5\u2032-CAGGCACTAGGCGGAAACTG-3\u2032;Reverse primer, 5\u2032-FAM-CCTTCCTGCATCTCAGCACCAACAG-[RTQ1]-3\u2032;Probe (amplicon size 197 bp);GAPDH:5\u2032- CGGAGTCAACGGATTTGGTC\u2013 3\u2032;Forward primer, 5\u2032- TGGGTGGAATCATATTGGAACAT\u2013 3\u2032;Reverse primer, 5\u2032-FAM-CCCTTCATTGACCTCAACTACATGGTTTACAT\u2013[RTQ1]-3\u2032;Probe, (amplicon size 141 bp);The optimal primer and probe concentrations for the target and control genes were as follows:HYAL1 primers, 300 nM, probe, 300 nM (for lung cancer cDNA samples); primers, 200 nM, probe, 200 nM (for RCC cDNA samples);HYAL2 primers, 300 nM, probe, 300 nM (for lung cancer cDNA samples); primers, 200 nM, probe, 200 nM (for RCC cDNA samples);GAPDH primers, 300 nM; probe, 150 nM.The thermocycler conditions were 10 min at 95\u00b0C, then 50 two-step cycles 15sec at 95\u00b0C and 60sec at 60\u00b0 C. qPCR amplification was carried out in triplicate in 25-\u00b5l reaction volume using TaqMan Universal Master Mix (Applied Biosystems) and 10ng template cDNA. The sequences of the amplicons were verified by sequencing in 3730 DNA Analyzer automated sequencer (Applied Biosystems).T method was used as described previously The comparative CHYAL1 and HYAL2 and reference gene for the same SCC and RCC patients. The evaluation of statistical significance of mRNA level was tested for all studied cases. Nonparametric Spearmen's rank test was used to calculate the coefficient of correlation between the level of mRNA decrease for HYAL1 and HYAL2 genes. P-values <0.05 were considered statistically significant. All statistical procedures were performed using BioStat software Nonparametric Wilcoxon test was used to compare mRNA expression differences of"}
+{"text": "To date, BRCA1 and BRCA2 mutations in breast and/or ovarian patients have not been characterized in the Turkish population. We investigated the presence of BRCA mutations in 53 individuals with a personal and family history of breast and/or ovarian cancer, and 52 individuals with a personal history of breast cancer diagnosed below age 50 without additional family history. We have identified 11 mutations (nine BRCA1 and two BRCA2) using combined techniques involving protein truncation test, direct sequencing and heteroduplex analysis. We found eight out of 53 patients (15.1%) with a family history to carry BRCA gene mutations (seven BRCA1 and one BRCA2). Of these, four were found in 43 families presenting only breast cancer histories, and four were found in families presenting ovarian cancer with or without breast cancer. We also demonstrated two BRCA1 and one BRCA2 mutations in three out of 52 (5.8%) early-onset breast cancer cases without additional family history. Three of nine BRCA1 and both BRCA2 mutations detected in this study were not reported previously. These mutations may be specific to the Turkish population. The BRCA1 5382insC mutation, specific to Ashkenazi and Russian populations, was found twice in our study group, representing a possible founder mutation in the Turkish population. \u00a9 2000 Cancer Research Campaign"}
+{"text": "Hepatoblastoma is a malignant paediatric liver tumour. In order to approach the genetic background of this malignancy we have screened a panel of eighteen cases from Europe and Japan for chromosomal imbalances using comparative genomic hybridization (CGH). The most frequent losses included chromosomal regions 13q21\u2013q22 (28%) and 9p22-pter (22%), while the most frequent gains occurred on 2q23\u2013q24 (33%), 20q (28%) and 1q24\u2013q25 (28%). A significant difference in CGH alterations between the tumours from patients of Caucasian and Japanese was revealed where loss of 13q was found only in the Japanese samples. In conclusion, the findings indicate several candidate regions for suppressor genes and oncogenes potentially involved in the hepatoblastomas of different ethnic origin. \u00a9 2000 Cancer Research Campaign"}
+{"text": "In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3\u20136 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 \u00b1 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears. \u00a9 2000 Cancer Research CampaignThe efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the \u03b2-globin gene appeared to be superior for the GTC-based assays. Using competitive \u03b2-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 \u00d7 10"}
+{"text": "Familial predisposition to basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) of the skin are apparent in the autosomal dominant syndromes naevoid basal cell carcinoma syndrome (NBCCS) and multiple self-healing squamous epitheliomata (MSSE) respectively. The gene responsible for NBCCS has been proposed to be a tumour-suppressor gene and is mapped to the same 2 Mb interval on 9q22.3 as the MSSE gene ESS1. In an attempt to further map the NBCCS gene, we have examined loss of heterozygosity (LOH) in 16 sporadic BCCs and two familial BCCs using microsatellite markers located within the candidate gene region. The overall frequency of LOH observed was 67% in the BCCs and partial or interstitial deletions were found in eight tumours, with the highest LOH frequency at markers D9S280, D9S287 and D9S180. To determine if the same genomic region also shows frequent LOH in tumours with a squamous phenotype, we have examined 11 SCCs, four actinic keratoses and 13 cases of Bowen's disease for LOH at 9q22.3. An overall LOH frequency of 50% was observed at D9S180, and occurred in all types of squamous tumours. In contrast, a much lower LOH frequency of only 6% was found at the D9S287 locus. Our observation of different patterns of LOH at 9q22.3 in sporadic BCCs and SCCs implies that more than one tumour-suppressor gene might be located in this genomic region."}
+{"text": "The purpose of the present study was to explore the therapeutic potential of serial administration of shedding-inducing endotoxin in a mouse tumour bladder model. The studies were conducted with two variants derived from the MBT-2 tumour namely, T5 and T50, the latter being far more aggressive than the former. It was found that T5 tumours responded to intravesical lipopolysaccharides (LPS) instillation by a considerable reduction in their pace of growth (P < 0.0001) when treatment was initiated 3 days after tumour implantation, but not when started after 7 days. The T50 variant did not respond to LPS when treated 3 days after implantation, but a considerable reduction in rate of growth occurred when treatment was started after 1-2 days. Shedding induced by intravesically instilled LPS was found to retard considerably the progression rate of experimental bladder tumour."}
+{"text": "Loss of multiple chromosomal regions is another genetic alteration frequently found in lung tumours. We have examined the association between p53 mutations, loss of heterozygosity (LOH) at frequently deleted loci in lung cancer, and tobacco exposure in 165 tumours from non-small cell lung cancer (NSCLC) patients. A highly significant association between p53 mutations and deletions on 3p, 5q, 9p, 11p and 17p was found. There was also a significant correlation between deletions at these loci. 86% of the tumours with concordant deletion in the 4 most involved loci  had p53 mutations as compared to only 8% of the tumours without deletions at the corresponding loci (P< 0.0001). Data were also examined in relation to smoking status of the patients and histology of the tumours. The frequency of deletions was significantly higher among smokers as compared to non-smokers. This difference was significant for the 3p21.3 (hMLH1 locus), 3p14.2 (FHIT locus), 5q11\u201313 (hMSH3 locus) and 9p21 (D9S157 locus). Tumours with deletions at the hMLH1 locus had higher levels of hydrophobic DNA adducts. Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and p53 mutations were significant and independent parameters for predicting LOH status at several loci. In the pathogenesis of NSCLC exposure to tobacco carcinogens in addition to clonal selection may be the driving force in these alterations. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comCarcinogenesis results from an accumulation of several genetic alterations. Mutations in the"}
+{"text": "Weekly intramuscular injections of 2.4million U Bicillin for 3 weeks was initiated as recommended by the Centers for Disease Control.Repeat sonogram 1 week after starting treatment revealed increased ascites and a new pericardialeffusion. Due to the worsening fetal condition, therapy was altered and the patient was admitted forIV penicillin. She received a continuous infusion of 18 million U penicillin G daily for 10 days. Serialsonograms showed improvement offetal ascites and pericardial effusion with 10 days of IV therapy,and complete resolution of hydrops was noted within 3 weeks. The fetus was born at term with nostigmata of congenital syphilis on newborn exam, and laboratory tests suggested adequate treatmentin utero.A 21 year old woman (G"}
+{"text": "BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions  were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations  were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations. \u00a9 2001 Cancer Research Campaignhttp://www.bjcancer.comMutations of the"}
+{"text": "Reconstitution of depleted thymicstroma with thymocytes showed evidence of defects in both developmentalcompartments.We have examined fetal thymic development in the trisomy 16 (Ts16) mouse, which isconsidered to be a model for human trisomy 21, or Down Syndrome. The Ts16 thymuscontains 10 to 20% of the number of lymphocytes found in a normal thymus at acomparable stage. Expression of thymocyte differentiation markers  is severely affected in Ts16 fetuses aged 14\u201318 gestational days.When thymuses from 14-day Ts16 mice were cultured"}
+{"text": "BRCA1 or BRCA2. In 104 families with three or more affected individuals (average 3.8) seeking counselling at family cancer clinics, mutation analysis was performed in the open reading frame of BRCA1 and BRCA2 by the protein truncation test and mutation-specific assays. In 31 of the 104 families tested, mutations were detected (30%). The majority of these mutations (25) occurred in BRCA1. Mutations were detected in 15 out of 25 families (60%) with both breast and ovarian cancer and in 16 out of 79 families (20%) with exclusively cases of breast cancer. Thus, an ovarian cancer case strongly predicted finding a mutation (P < 0.001). Within the group of small breast-cancer-only families, a bilateral breast cancer case or a unilateral breast cancer case diagnosed before age 40 independently predicted finding a BRCA1 or BRCA2 mutation . Therefore, even small breast/ovarian cancer families with at least one case of ovarian cancer, bilateral breast cancer, or a case of breast cancer diagnosed before age 40, should be referred for mutation screening. \u00a9 1999 Cancer Research CampaignFor families with a small number of cases of breast and/or ovarian cancer, limited data are available to predict the likelihood of genetic predisposition due to mutations in"}
+{"text": "TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM.In this study levels of prostaglandin E"}
+{"text": "These data provide a better understanding as the first step in the mechanism of action of the activin in the epithelial ovarian carcinoma. \u00a9 2000 Cancer Research CampaignIn this study, we have investigated the expression of inhibin subunits and activin receptors (ActRs) in normal and malignant ovarian cells. Each product of the inhibin subunits  and activin receptors (ActRs) amplified by reverse transcription polymerase chain reaction were detected as a single band in human granulosa cells, surface epithelial cells (OSE), and the ovarian cancer cell lines OVCAR 3 and SKOV 3. Western blot analysis was performed using polyclonal antibodies against ActR IIa or IIb peptides based on 13 COOH-terminal amino acids; cultured human granulosa cells were used as a positive control. Using ActR IIa antibody, one major band corresponding to approximately 80 kDa and one minor band corresponding to 105 kDa were observed in the samples. One single band at approximately 60 kDa was detected in OVCAR 3 and a 50 kDa band was detected with ActR IIb antibody in cultured granulosa cell, OSE and SKOV 3. Although no detectable change was induced in Smad 4 mRNA in OVCAR 3, Smad 2 mRNA levels were increased during 48 h treatment with activin A (50 ng ml"}
+{"text": "CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations  and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant  of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations. http://www.bjcancer.com \u00a9 2001 Cancer Research Campaignhttp://www.bjcancer.comPhysical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the"}
+{"text": "Fifty-seven patients with advanced prostate cancer resistant to first-line hormonal therapy were treated with estramustine and additionally randomized for treatment with clodronate or placebo. Clodronate treatment was started with 5 days intravenous administration (300 mg day[-1]) and followed by oral treatment (1.6 g day[-1]) for 12 months. Skeletal pain relief was only about 10% better in the clodronate than in the placebo group. The results do not support the superiority of combined intravenous and oral treatment with clodronate compared with oral administration only."}
+{"text": "Estimates were made of the rates at which cancer cells were released directly into the renal vein in patients undergoing radical nephrectomy for primary renal cancer. Cancer cells were counted in blood samples taken from the renal vein using a density gradient centrifugation procedure, and identified using immunocytochemical techniques, on the basis of their cytoskeletal intermediate filament proteins. Cancer cells were released as single cells and multicell emboli in 8/10 patients, in numbers varying widely between 14-7509 emboli ml-1 of blood. Despite a calculated median input into the metastatic process of 3.7 x 10(7) cancer cells per day for at least 180 days, only 3/10 patients had extraperitoneal metastases prior to surgery and only 1 of the remaining disease-free patients subsequently developed distant metastases over a maximum 35 month period. These results are discussed in terms of primary tumour kinetics and metastatic inefficiency."}
+{"text": "Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described.A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells.Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway.We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery. While most animal viruses enter into cells by receptor mediated endocytosis in clathrin coated pits ,2, evideHepG2 and Vero cells were maintained as previously described -13 Dengu# E6361).Confluent HepG2 cells were pretreated at 37\u00b0C with either 20 \u03bcM cytochalasin D or 30 \u03bcg/ml nystatin for 2 hr, or with 15 \u03bcg/ml chlorpromazine or 3 mM amiloride or 50 \u03bcM LY294002 or 0.2 \u03bcM wortmannin for 1.5 hr with 80% DMSO for 20 hours as a positive control. Cells were then trysinized and subsequently washed twice with cold-PBS. Cells were then washed with binding buffer  and resuspended in binding buffer. Annexin V-FITC  was added to the cell suspensions and samples were incubated in the dark for 15 min before analysis by flow cytometry  treatment . The inf2 for 2 days. Transfection efficiencies routinely exceeded 70% efficiency as determined by counting of multiple fields. Transfected cells were subsequently infected with dengue virus serotype 1, 2, 3 or 4 at an MOI of 20 for 1.5 hr followed by acid glycine (pH3) treatment to inactivate un-internalized viruses [2. A further set of cells were serum starved for 30 min and incubated with 5 \u03bcg transferrin conjugated with Alexa 594  at 37\u00b0C for 30 min follow by acid glycine treatment.Plasmid constructs of dominant negative (DIII and EH29) and control (D3\u03942) Eps15 were kindly provided by A. Benmerah . Transfections of HepG2 cells were undertaken using lipofectamine2000 . Briefly, 3 \u03bcg of the appropriate plasmid DNA was complexed with 4 \u03bcl of lipofectamine2000 for 30 min at room temperature and then added to HepG2 cells pre-grown to 70\u201380% confluency on glass coverslips. The cell/complex mix was incubated at 37\u00b0C, 10% CO viruses  and incuBoth dengue infected and transferrin treated transfected cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Transferrin control cells were directly mounted with Vectashield  while dengue virus infected cells were further permeabilized with 0.3% TritonX-100 and methanol. Nonspecific binding was blocked by incubation with 5% BSA for 1 hr at room temperature. Cells were incubated with an anti-dengue E protein monoclonal antibody, HB-114  at 4\u00b0C oNM_004859) and the green fluorescent protein  were determined using the on-line tool from Ambion, Austin, TX  and the selected sequences were subjected to siRNA template design to generate DNA oligonucleotide sequences for use with the Silencer\u2122 siRNA Construction kit (Ambion). Six templates for siRNA generation were selected:Target sites on clathrin heavy chain  or U50974 (siGFP). All sequences were searched against the NCBI's database to confirm specificity. Sense and antisense DNA templates were chemically synthesized  and following the kit instructions based on 4 HepG2 cells was added and cell: complex mixtures incubated under standard conditions. Mock transfections (lipofectamine only) were performed in parallel. All transfections were undertaken in a final volume of 600 \u03bcl with siRNA at a final concentration at 50 nM. Transfections were harvested at 1 to 4 days post-transfection.HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum without antibiotics. Reverse transfections according to the manufacturers protocols were performed with Lipofectamine\u2122RNAiMAX  by mixing the respective siRNA and 1.2 \u03bcl of Lipofectamine\u2122RNAiMAX and adding to a single well of a 24 well plate. After 20 minutes of incubation at room temperature, a suspension of 5 \u00d7 1017 primer was used to synthesize first strand cDNA using ImpromII\u2122 reverse transcriptase . The cDNA was then amplified in a multiplex reaction with 2 specific primer pairs for CHC  and GAPDH  as an internal control. Expected products were 343 bp (GAPDH) and 222 bp (CHC) Cycle conditions were 94\u00b0C for 3 minutes followed by 20 cycles of 94\u00b0C for 30 seconds, 58\u00b0C for 45 seconds and 72\u00b0C for 45 seconds followed by a final extension of 72\u00b0C for 7 minutes. PCR products were analyzed on 1.8% agarose gels containing ethidium bromide.Transfected cells from a single well of a 24-well plate were homogenized in 0.5 ml Trizol reagent  and RNA purified as recommended by the manufacturer. For the RT-PCR analysis, an oligo(dT)4) were grown on coverslips in single wells of a 24 well plate and transfected as above with either siRNAs as stated or mock transfected. At day 3 post transfection cells were infected with dengue virus serotype 2 MOI 20 for 2 hours followed by an acid glycine wash and subsequently incubated for 15 hours under standard conditions. Dengue virus E protein was detected as described above except that cells were also stained with DAPI. Parallel non-infected samples were incubated with transferrin as described above and were additionally stained with DAPI. Where biochemical inhibition was used in conjunction with siRNA silencing, samples were treated with 0.2 \u03bcM wortmannin for 30 minutes immediately preceding dengue virus infection.HepG2 cells . Working concentrations (the highest concentration without apparent cytotoxicity) were established as: cytochalasin D at 20 \u03bcM, amiloride at 3 mM, LY294002 at 50 \u03bcM, wortmannin at 0.2 \u03bcM, nystatin 20 \u03bcM at 30 \u03bcM and chlorpromazine at 15 \u03bcM (HepG2). The lack of cytotoxicity at these concentrations was confirmed by Annexin V staining and flow cytometry  significantly excluded the entry of transferrin Figure . HoweverNM_004859) were generated using in vitro transcription together with 1 siRNA targeted to the green fluorescent protein  for use as a control. To confirm all siRNAs were double-stranded, an aliquot of each siRNA was treated with RNaseIII which digests double-stranded RNA or RNaseA which digests single-stranded RNA. All siRNA constructs were confirmed to be off the appropriate size and to consist of dsRNA (data not shown).Given the significant entry of the dengue virus in the presence of over-expressing dominant negative mutants of Eps15, it is possible that either entry was occurring via multiple pathways, or the Eps15 mutants were not completely inhibiting clathrin mediated entry. To further explore this, RNA interference was used to down regulate the expression of clathrin heavy chain, an integral part of the clathrin vesicle . Five diTo optimize the silencing of the expression of the clathrin heavy chain, the 5 different siCHCs were transfected into HepG2 cells in parallel with transfections of siGFP and lipofectamine alone (mock). On days 1 to 4 days post-transfection, cells were harvested and RNA extracted. Multiplex RT-PCR was undertaken to detect messages from GAPDH and clathrin heavy chain (CHC) simultaneously and results analyzed by agarose gel electrophoresis. Experiments were undertaken independently in triplicate.Results showed a constant signal for GAPDH and the clathrin heavy chain (CHC) for mock and siGFP transfection Figure . MessageOptimal silencing of clathrin heavy chain expression was noted at day 3 post transfection with siRNA constructs siCHC3 and siCHC5. These two siRNAs were again transfected into HepG2 cells as above in parallel with transfections of siGFP and mock (transfection agent only) and on day 3 post transfection cells were infected with dengue virus serotype 2 and an MOI of 20 and cells allowed to grow for 15 hours (the time for one replication cycle of dengue serotype 2 minus two hours) under optimal conditions. At 15 hours cells were either analyzed by microscopy or by our adaptation of the standard plaque assay [Consistent with our results with transfections of dominant negative constructs of Eps15, a significant reduction of transferrin entry was seen with siCHC transfections, but not with mock or siGFP Figure . Dengue Results of dengue virus entry as seen by our adaptation of the standard plaque assay  were conTo determine whether the approximately 20% virus entry seen in cells in which clathrin mediated endocytosis has been inhibited is a result of background, or the result of dengue virus entry by macropinocytosis, we sought to simultaneously inhibit both pathways, clathrin mediated endocytosis through siRNA mediated RNA inhibition and macropinocytosis through biochemical inhibition using wortmannin.Cells were therefore transfected with siCHC5 to silence clathrin heavy chain or mock transfected and on day 3 post-transfection were either treated or not treated with wortmannin for 1 hour before being either incubated with transferrin or infected with dengue serotype 2 at an MOI of 20. Following acid glycine treatment of dengue infected cells, cells were incubated for 15 hours before being either examined by microscopy or the number of infected cells determined by our adaptation of the standard plaque assay [Results Figure  show thaResults from our adaptation of the plaque assay Figure , Panel BDespite flaviviral infections representing a significant world wide public health threat, little advance has been made in dissecting out the mechanism by which flaviviruses enter into either mammalian or insect cells. Studies on Japanese encephalitis virus and West Nile virus with either Vero (African Green monkey cells) or C6/36 cells have suggested that these two viruses enter by clathrin coated pit mediated endocytosis -10. WithRecently Chu et al.,  providedInterestingly Chu and colleagues also investigated the entry of West Nile Virus into the aedes albopictus cell line C6/36 using the same dominant negative mutant of Eps15  and simiMore recently Krishnan and colleagues have investigated the entry of the dengue virus into HeLa cells . This stOur data suggests that the remaining 20% virus entry observed is not the results of incomplete ablation of clathrin mediated endocytosis but represents virus entry by a viable, independent pathway, macropinocytosis. Entry of the dengue viruses  by multiple pathways as shown here raises some interesting questions, particularly with respect to the initial flavivirus: host cell interaction and may require a significant re-evaluation of our understanding of flavivirus entry into host cells.Consistently, inhibition of clathrin mediated endocytosis using dominant negative mutants of Eps 15 results in a reduction of dengue virus entry of approximately 80% as shown by this study and others -9. Our dThe authors declare that they have no competing interests.LS undertook the biochemical inhibition studies and Eps15 transfections and analyzed and interpreted the data. TS undertook the siRNA mediated silencing studies and analyzed and interpreted the data. DRS was responsible for design and implementation of the study as well as drafting the manuscript. All authors read and approved the final version."}
+{"text": "A series of 23 children with primitive neuroectodermal tumours (PNET) were analysed with comparative genomic hybridization (CGH). Multiple chromosomal imbalances have been detected in 20 patients. The most frequently involved chromosome was chromosome 17, with a gain of 17q (11 cases) and loss of 17p (eight cases). Further recurrent copy number changes were detected. Extra copies of chromosome 7 were present in nine patients and gains of 1q were detected in six patients. A moderate genomic amplification was detected in one patient, involving two sites on 3p and the whole 12p. Losses were more frequent, and especially involved the chromosomes 11 (nine cases), 10q (eight cases), 8 (six cases), X (six patients) and 3 (five cases), and part of chromosome 9 (five cases). These recurrent chromosomal changes may highlight locations of novel genes with an important role in the development and/or progression of PNET. \u00a9 1999 Cancer Research Campaign"}
+{"text": "Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys47) was essential for Tsa1 activity, while Tsa1C170S lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or CCStsa1 lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The CCS rad51tsa1\u2013 or tsa1 rad51\u2013induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast  S. cerevisiae, we performed a comparative study and found that one, Tsa1, played a key role in preventing DNA damage and assuring genome stability. Tsa1 also cooperated with other peroxiredoxins in antioxidant defense. These functions of Tsa1 required the presence of a cysteine at the catalytic site of this enzyme. Additional studies revealed that Tsa1 activity, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect cells against toxic levels of DNA damage that occur during aerobic growth.Aerobically growing cells are continuously challenged by potent oxidants produced during normal cellular metabolism. These oxidants, including hydrogen peroxide and organic peroxides, are important components mediating various cell functions. However, they can also cause cell damage when present at toxic levels. Aerobic organisms possess extensive antioxidant systems to regulate oxidant levels. Among these, peroxiredoxins have received considerable attention in recent years as an expanding protein family involved in the enzymatic degradation of hydrogen peroxide and organic peroxides. To better understand the physiological role of the five peroxiredoxins in budding yeast  Saccharomyces cerevisiae and six in human cells 2O2 or alkyl hydroperoxides Escherichia coli AhpC, which uses another protein, AhpF, for the regeneration of the reduced Prx 2O2, some Prxs suffer an oxidation of the peroxidatic Cys to sulfinic acid, which temporarily inactivates these enzymes. This hyperoxidation of 2-Cys Prx enzymes is reversible in cells by sulfiredoxin, an ATP-dependent reductases for Cys-sulfinic acid in Prxs Peroxiredoxins (Prxs), previously termed the thioredoxin peroxidases, have received considerable attention in recent years as a new and expanding family of thiol-specific antioxidant proteins S. cerevisiae Tsa1 was the first characterized Prx, followed by Aph1 (cTpxIII) S. cerevisiae Prxs have different sub-cellular localizations: Tsa1, Tsa2 and Ahp1 are localized in the cytoplasm; Prx1 is localized in mitochondria; and Dot5 is localized in the nucleus 2O2 rather than alkyl hydroperoxide, whereas Ahp1 and Dot5 showed the reverse selectivity The five PRX genes are also quite different from each other. There are discrepancies in the reported relative order of mRNA abundance; however, most studies show that TSA1 mRNA is the most abundant PRX mRNA at different growth stages, followed by AHP15 molecules per log phase cell, higher than Ahp1 which is present at approximately 1.6\u00d7104 molecules per log phase cell and much higher than Tsa2, Prx1 and Dot5 which are present at about 1.8\u20134.8\u00d7103 molecules per log phase cell PRX genes are usually upregulated to various degrees in response to treatment with various oxidants and TSA2 is the most strikingly upregulated of the PRX genes TSA1 gene The transcriptional levels of the PRX genes only play a minor role in the suppression of genome instability, but their deletion increases the spontaneous mutation frequency of a tsa1 mutant rad51 or rad52, or the Rad6-mediated post-replication repair pathway including rad6, cause tsa1 cells to be very sick or to die rapidly under aerobic conditions but not anaerobic conditions, emphasizing the importance of Tsa1 for genome protection tsa1 mutants exhibit reduced clearance of reactive oxygen species (ROS) induced by exogenous treatment with H2O2S. cerevisiae cells begin to use fatty acids as energy sources S. cerevisiae Prxs are only known to have stress-protective functions in contrast to the unique Schizosaccharomyces pombe Prx, Tpx1, which is also involved in H2O2 signaling TSA1, TSA2, AHP1, PRX1 and DOT5 are all deleted are still viable S. pombe mutant lacking Tpx1 S. cerevisiae Prx plays a specific role, defined by its cellular location, substrate specificity and expression pattern. On the other hand, different Prxs might cooperate with each other in antioxidant defense.The physiological role of each Prx has been explored. Notably, Tsa1 has been shown to be a significant contributor to genome stability; its absence leads to a broad spectrum of spontaneous mutations and genome rearrangements that are thought to result from increased oxidative damage to the DNA S. cerevisiae Prxs, we performed a comparative study of Prxs for their respective role in maintaining the genome stability and in sustaining aerobic viability of cells deficient in recombinational repair. Tsa1 played a key role in this regard while Tsa1 and Dot5 cooperated to suppress DNA damage that leads to gross chromosomal rearrangements (GCRs). Tsa2 shared functional similarity with Tsa1 and was capable of largely complementing the absence of Tsa1 when expressed under the control of Tsa1 promoter. Peroxidase active site was essential for Tsa1 to function in suppressing genome instability and cell death. Finally we addressed the fate of mutant cells with compromised Tsa1 activity and deficient recombinational repair.To gain more insight into the physiological role of the TSA1 resulted in elevated mutation rates as determined by the Canr assay that detects all gene inactivating mutations that can occur in the 1.8-kb CAN1 gene, which are most frequently base substitution and frameshift mutations, the hom3-10 assay that detects reversion of a +1 insertion in the HOM3 gene, the lys2-Bgl assay that detects reversion of a +4 insertion in the LYS2 gene, and the GCR assay that measures gross chromosomal rearrangements, indicating the importance of Tsa1 in preventing a broad spectrum of types of genomic instability. Deletion of TSA2, AHP1, PRX1 or DOT5 did not cause a mutator phenotype that could be detected with these mutator assays, confirming the unique role of Tsa1 in maintaining genome stability , whereas the GCR rate of the double mutant appeared to be similar compared to the tsa1 mutant. However, the Canr, hom3-10 and lys2-Bgl rates of the tsa1 tsa2 double mutant were not much higher than the sum of the rates of each single mutant (95% confidence limits). Simultaneous elimination of TSA1 and AHP1, or TSA1 and PRX1 did not appear to change the mutation rates in the four assays compared to tsa1 single mutant. The Canr, hom3-10 and lys2-Bgl mutation rates of the tsa1 dot5 double mutant appeared to be only slightly higher compared to tsa1 single mutant. In contrast, the GCR rate of the tsa1 dot5 double mutant showed a significant increase relative to the tsa1 and dot5 single mutants  was replaced by the TSA2 coding sequence was constructed as illustrated in TSA2 mRNA was 5% of that of TSA1 mRNA in log phase wild-type cells but increased to 70% of the wild-type TSA1 mRNA levels in the tsa1::TSA2 strain  epitope tag, enabling the immunodetection of these proteins with the same anti-HA antibody. The protein level of Tsa1 was much higher than that of Tsa2 in wild-type cells while Tsa2 produced in the tsa1::TSA2 strain was comparable to the physiologic level of Tsa1 , but were higher than the mutation rates of the wild-type strain  were sporulated and dissected, and the spore dissection plates were incubated under aerobic conditions for four days. As previously shown, a tsa1 mutation in combination with a rad51 mutation resulted in lethality  and resolving Cys (Cys170). Both Cys47 and Cys170 were shown to be necessary for the maintenance of the dimeric structure of oxidized Tsa1, and Cys47, but not Cys170, is essential for antioxidant activity measured in vitroC47Stsa1, C170Stsa1 and CCStsa1 that express mutant forms of Tsa1 in which Cys47, Cys170 or both were substituted with serines  but were higher than that of the wild-type strain. We then analyzed the meiosis products of the heterozygous diploid cells containing a tsa1 allele of interest and a rad51 mutation. Tetrad dissection and genotyping of viable spore colonies revealed that a rad51 mutation was lethal in combination with the C47Stsa1 or CCStsa1 alleles  obtained under anaerobic conditions were inoculated into liquid cultures and grown under the same anaerobic conditions for 3 to 4 days. Cultures were diluted into fresh medium to a density of 2\u00d7106 cells/ml under aerobic conditions and samples were taken for flow cytometry (FACS) and microscopic analysis at various time points after shifting to aeration. At time 0, the CCS rad51tsa1 cells exhibited cell cycle profiles and cell morphology that were similar to that of wild-type cells .The pe cells  and to tpe cells . At thisragments . HoweverCCS rad51tsa1 cells displayed a cell morphology and FACS profile suggestive of a protracted G2/M checkpoint arrest , late apoptosis eventually leading to secondary necrosis (annexin V+/PI+) and primary necrosis (PI+) 2O2 for 200 min as previously described CCStsa1 single mutant and rad51 single mutant cells maintained low level annexin V staining at 8 hr and 24 hr after shifting to aerobic conditions . Taken together, these results suggest that the CCS rad51tsa1 and tsa1 rad51 double mutant cells undergo a DNA damage induced apoptosis-like cell death.The nditions . In contme point . CultureCCS rad51tsa1 cells. Deletion of YCA1 or STE20 makes cells more resistant to H2O2 induced apoptosis CCStsa1/TSA1 rad51/RAD51 yca1/YCA1 and CCStsa1/TSA1 rad51/RAD51 ste20/STE20 were sporulated and dissected. Tetrads were analyzed and the genotypes were determined or inferred from the mutation segregation patterns. The colony size of CCS rad51 yca1tsa1 and CCS rad51 ste20tsa1 triple mutants was similar to that of CCS rad51tsa1 double mutants (data not shown). We then tested whether the accumulation of annexin V+ cells in aerobic cultures of CCS rad51tsa1 mutants was Yca1- or Ste20- dependent. Anaerobic culture condition restores cellular viability of these double and triple mutants, allowing us to obtain sufficient number of cells in liquid cultures for analysis. As shown in CCS rad51 yca1tsa1 and CCS rad51 ste20tsa1 cells exhibited annexin V and PI staining features that were similar to those of CCS rad51tsa1 cells at different time points after shifting to aeration. Therefore, neither deletion of YCA1 nor deletion of STE20 reduced the apoptotic like phenotype of CCS rad51tsa1 cells. Thus, the Yca1 and Ste20 mediated apoptosis programs do not by themselves contribute to the lethal effects caused by loss of both Tsa1 and Rad51 function. In addition, a cell-permeant pan caspase inhibitor Z-VAD-FMK failed to improve growth and survival of CCS rad51tsa1 cells (data not shown).We next investigated whether the two independent cell death processes mediated by the proapoptotic caspase Yca1 and the Ste20 kinase, respectively, play a role in the death of 2O2 scavengers, redox signal transducers and molecular chaperones is of significant interest S. cerevisiae results in a complex protein network that may reflect the particular physiological roles of each isoform. In this study, our comparative analysis of the five Prxs demonstrated that Tsa1 was distinguished from other Prxs in that by itself it played a key role in suppressing the accumulation of mutations and genome rearrangements, and maintaining cellular survival and growth in the absence of recombinational repair. The predominant role of Tsa1 in this regard may mask the potential phenotype of other prx mutants. Indeed, analysis of double mutants combining tsa1 and mutation of one of the other four PRXs revealed distinct roles of Tsa2 and Dot5 in suppressing the accumulation of mutations and in preventing killing by exogenous oxidative stress.Prxs have generated considerable of interest during recent years, and understanding their distinct roles as Htsa1 mutation, a tsa2 mutation did not cause sensitivity to H2O2, a mutator phenotype or synthetic lethality when combined with a rad51 mutation. However, the tsa1 tsa2 double mutant exhibited much higher H2O2 sensitivity than tsa1 single mutant. The mutator phenotype of tsa1 tsa2 double mutant was more pronounced than that of tsa1 single mutant due to the increase in base substitution and frameshift mutations as revealed by different mutator assays performed in this study. Furthermore, overexpression of Tsa2 under the control of TSA1 promoter largely restored the genetic defects caused by a tsa1 mutation. These observations indicate that Tsa1 and Tsa2 share functional similarity in removing low endogenous concentrations of H2O2 and in dealing with more aggressive exogenous oxidant challenge. This view is consistent with the fact that Tsa2 is unique in sharing striking homology with Tsa1 among the five Prxs in S. cerevisiae, that Tsa1 and Tsa2 exhibit comparable antioxidant activity in vitro and that both have heat shock protective chaperone activity TSA2 is much lower than that of TSA1, transcription of TSA2 shows greater induction after treating cells with different oxidants, including H2O22O2 sensitivity of the tsa1 single mutant  or by the thioredoxin system, whereas Tsa1C170S is active in the presence of DTT and inactive in the presence of the thioredoxin system C170S was only partially functional in vivo, as shown by the partial complementation of a tsa1 strain expressing this protein. The basis for this activity is unclear as it is not clear what provides the needed redox system in vivo. Consistent with these observations, the only 2-Cys Prx of S. pombe, Tpx1, which is essential for cell viability, is able to support aerobic growth when the resolving Cys is eliminated in the Tpx1C169S mutant protein in vivo evidence suggesting that Tsa1 may be the primary scavenger of endogenous H2O2 in S. cerevisiae. Several aspects of observations are consistent with this view. First, Tsa1's affinity for H2O2 with a Km of \u223c12 \u00b5M 2O22O2 concentrations are high. Third, and more important, Tsa1 is functionally unique with regard to maintenance of genome stability and to interaction with other aspects of DNA metabolism, as high rates of accumulating Canr mutations and chromosomal rearrangements were found only in tsa1 mutants and synthetic lethal interactions with rad51 mutations were only seen with a tsa1 mutation The peroxidase active site is essential for Tsa1 to function in suppressing genome instability and cell death. Tsa1S. cerevisiae prx mutants did not have growth defects and a prx null mutant in which TSA1, TSA2, AHP1, PRX1 and DOT5 were all deleted was still viable S. pombe mutant lacking Tpx1 S. cerevisiae cells do not generate enough H2O2 or other reactive species to produce lethal levels of damage, another cellular antioxidant activity compensates for the loss of the Prxs, or other pathways help limit the damage and prevent cell death. Recombinational repair is such a pathway critical for cell survival and growth of tsa1 mutants, including deletion mutants and mutants with a compromised peroxidase active site (C47Stsa1 and CCStsa1). These results confirm the view that aerobic organisms generate enough H2O2 to damage their own DNA. Scavenging enzymes and appropriate DNA repair are required for growth under aerobic conditions and Tsa1 is the dominant peroxidase activity that keeps the steady-state concentration of H2O2 at non-toxic levels. Consistent with this, E. coli fur, ubiH or ubiE mutations most likely lead to an increased level of ROS and cause cell death in the absence of RecA-dependent recombinational repair fur recA synthetic lethality has been ascribed to elevated chromosomal fragmentation. We investigated the cell death processes of CCS rad51tsa1 and tsa1 rad51 under aerobic conditions. Several hours after shifting from anaerobic culture to aeration, CCS rad51tsa1 cell cultures were characterized by the accumulation of cells with irregular cell size/shape, abnormal nuclear staining, and a broader S/G2/M DNA content peak. At longer time after shifting to aeration, dying cells were characterized by a significant increase in the levels of typical early markers of S. cerevisiae apoptosis-like cell death such as phosphatidylserine externalization. This apoptosis-like phenotype did not depend on the caspase Yca1 and Ste20 kinase, two gene products each thought to control a different apoptosis-like cell death process S. cerevisiae and demonstrates enzymatic peptidase activity analogous to mammalian caspase activity S. cerevisiae2O2 treatment S. cerevisiae that triggers the death of CCS rad51tsa1 mutants. Similarly, it is known that endogenous DNA damage, replication failure and perturbation of transcription induces an apoptosis-like death in S. cerevisiaeIndividual The results reported here and in previous studies can be interpreted as follows. Endogenous oxidative stress due to the absence of appropriate and sufficient Tsa1 activity induces a variety of types of DNA damage, including mutagenic, replication blocking and potential lethal DNA lesions. These lesions are repaired by diversity of mechanisms including base excision repair, nucleotide excision repair, post-replication repair, recombination and potentially other mechanisms. Unrepaired mutagenic DNA lesions cause mutations and GCRs but do not significantly affect cell survival. In contrast, in the absence of appropriate repair, a growing number of potential lethal DNA lesions persist or are converted to lethal double-strand break (DSB), activating the Rad9-dependent G2/M checkpoint. This checkpoint activation facilitates repair of at least some of the DNA damage by back-up repair mechanisms allowing checkpoint proficient cells to proliferate to a greater extent than checkpoint deficient cells. Finally, the presence of irreparable or excessive DSBs appears to initiate a DNA damage-induced apoptotic program, although how such damage signals the apoptotic program is not well understood.S. cerevisiae strains were grown in standard media including yeast extract peptone dextrose medium (YPD) or synthetic complete medium (SC) lacking appropriate amino acids as indicated. Cycloheximide resistant strains (CyhR) containing mutations in the CYH2 gene were selected on YPD agar plates containing 10 \u00b5g/ml cycloheximide. Canavanine resistant mutants (Canr) caused by inactivation of the CAN1 gene were selected on SC\u2013arginine dropout plates containing 60 mg/l canavanine. Hom3+ revertants were selected for on SC\u2013threonine dropout plates and Lys2+ revertants were selected for on SC\u2013lysine dropout plates. Canavanine- and 5-fluoroorotic acid (5FOA)- resistant mutants (Canr-5FOAr) resulted from the loss of the region including CAN1 and URA3 on chromosome V were selected on SC\u2013arginine and \u2013uracil dropout plates containing 60 mg/l canavanine, 1 g/l 5FOA and 50 mg/l uracil 2O2 was assayed on solid media by spotting 10-fold serial dilutions of mid-logarithmic phase cells onto SC plates containing H2O2 at indicated concentrations.MATa, ura3-52, leu2\u03941, trp1\u039463, his3\u0394200, lys2\u0394Bgl, hom3-10, ade2\u03941, ade8, hxt13::URA3tsa1::TSA2 mutant in which the entire TSA1 gene was replaced by TSA2 coding sequence under control of the native TSA1 promoter is illustrated schematically in + CyhS) in which the entire TSA1 gene was replaced by the TRP1-CYH2 cassette as previously described TSA2 flanked by sequences homologous to the upstream and downstream sequence of TSA1, were used to transform MEHY1539 (Trp+ CyhS) to replace TRP1-CYH2 at the TSA1 locus yielding the strain MEHY1710 (Trp\u2212 CyhR) in which the TSA2 is under the control of the native TSA1 promoter. Correct integration of TSA2 at TSA1 locus was confirmed by genomic PCR and DNA sequencing.The strains used for this study were all isogenic to the S288c based parental strain RDKY3615 TSA1  were created as follows. The TSA1 gene with 5\u2032 and 3\u2032 flanking DNA sequence was amplified from genomic DNA using primers containing, respectively, the restriction sites XhoI and HindIII. The resulting PCR product was digested with XhoI and HindIII and cloned in XhoI and HindIII sites of the plasmid pRS416 TCTCCAACCGAAATCATTGC-3\u20325\u2032-GCCTTCACTTTCGTC and AGACGAAAGTGAAGGC-3\u20325\u2032-GCAATGATTTCGGTTGGAG (sequences for Cys47 to Ser substitution are underlined), and mutagenic primers TCTAACTGGACTCCAGGTGC-3\u20325\u2032-GGTACTGTCTTGCCA and AGATGGCAAGACAGTACC-3\u20325\u2032-GCACCTGGAGTCCAGTT (sequences for Cys170 to Ser substitution are underlined). The mutated TSA1 gene was sequenced to verify the presence of the desired substitutions and the absence of other mutations. PCR-generated fragments containing mutant alleles of TSA1 flanked by sequences homologous to the upstream and downstream sequence of TSA1, were used to transform MEHY1539 (Trp+ CyhS) to replace TRP1-CYH2 at the TSA1 locus yielding the strains MEHY1702 (C47Stsa1), MEHY1593 (C170Stsa1) and MEHY1590 (CCStsa1), expressing a given mutant allele of TSA1 under the control of the native TSA1 promoter  according to an established procedure TSA1, TSA2, and ADH1 . Serial dilutions of cDNA from each strain were amplified using the appropriate primers and iQ SYBR Green Supermix (Bio-Rad). A single melt curve peak was observed for each sample used in data analysis, thus confirming the purity and specificity of all amplified products. The relative ratio of TSA1 or TSA2 expression to ADH1 was calculated using the relative quantity (\u0394\u0394CT) analysis formula in the Bio-Rad iQ5 Gene Expression Optical System Software.Wild-type and For Western blots, strains were grown to mid-logarithmic phase in YPD medium and whole cell extracts isolated by trichloroacetic acid extraction r mutations, Hom3+ revertants, Lys2+ revertants, and GCRs in cell populations was determined by fluctuation analysis as described previously 8 cells/ml, harvested, washed and resuspended in sterile water. Sufficient quantities of cells were plated on appropriate selection media to identify Canr mutations, Hom3+ revertants, Lys2+ revertants and GCR events, and appropriate dilutions were plated on YPD for total cell counts. Colonies were counted after four days of growth at 30\u00b0C. The number of mutant cells per culture was calculated and the median value from at least 15 independent cultures for each strain was used to determine the mutation rate as described http://www.math.unb.ca/~knight/utility/MedInt95.htm.The rate of accumulation of CanYPD medium with or without agar was supplemented with Tween 80 and ergosterol to a final concentration of 1.32 ml/l and 6.75 mg/l, respectively. Anaerobic liquid cultures or anaerobic growth of dissected spores in plates were placed in an airtight jar containing a disposable hydrogen- and carbon dioxide-generating envelope (BBL GasPak Plus) and grown anaerobically at 30\u00b0C for 3\u20135 days to yield enough cells for analysis. Anaerobic conditions were monitored with the redox indicator Anaerotest (Merck) placed inside of the airtight jar.6 cells/ml and samples were taken at various time points after shifting to aeration. Cells were collected, resuspended in 70% (vol/vol) ethanol for fixation. The cells were washed in 50 mM Tris buffer (pH 8.0), incubated 24 hr with 5 mg/ml DNase-free RNase, centrifuged and labeled with 50 \u00b5g/ml PI, sonicated and submitted to FACS measurements of DNA content with a FACScalibur cytometer (Beckton-Dickinson). Same fixed cells were also stained with DAPI Cells of the desired genotype were grown in liquid culture under same anaerobic condition for 3 to 4 days. Cultures were diluted in fresh medium to a density of 2\u00d7106 intact yeast living cells were treated by 5% glusulase (Perkin Elmer) and 5 U/ml lyticase (Bio-Rad) at 28\u00b0C for 2 hr in a buffer consisting of 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH 7.4. The labeled cells were then analyzed by FACS using an argon-ion laser (excitation wavelength 488 nm and emission of 530 nm) and the data were processed using BD Cell Quest Pro software (version 5.2).Annexin V/PI co-labeling was performed using the Vybrant Apoptosis Assay kit #3 (Invitrogen) according to the manufacturer's instructions, excepted that about 1\u00d710"}
+{"text": "The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events  and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages.In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies. Genomes of extant species are shaped by extensive gene loss and duplication events. In the radiation of vertebrate species, two whole genome duplication events at the base of the vertebrate lineage (approximately 500 million years (Mya) ago) -3 and a slc1 gene family of neutral and excitatory amino acid transporters (EAATs), whose presence on glia cells or neurons is indispensable for precise and sustained synaptic activity [In the context of lineage specific gene loss and duplication events, we studied members of the vertebrate activity -22. EAATactivity . Moreoveactivity . Based oIn mammals, five EAAT genes EAAT 1-5) together with two neutral amino acid transporters form the 'solute carriers 1' (SLC1) gene family , an increased number of SLC1 genes are expected in their genomes. We indeed identified up to 13 SLC1s in fish genomes. While some of these additional SLC1 genes clearly are the consequence of the teleost specific R3, others are evidently members of two additional subfamilies , that were lost in the lineage leading to human and mouse. Interestingly members of these two subfamilies can also be found in prototherian, sauropsidan, and amphibian genomes, suggesting that these genes are specifically retained in these lineages.slc1 genes in the zebrafish genome. While in the human and mouse 7 different members of the slc1 gene family have been described, we identified and annotated 13 slc1 family genes in the zebrafish genome. Cloning from whole embryo cDNA indicated that all of the 13 annotated slc1 sequences are indeed transcribed. Sequencing of the amplified cDNA fragments revealed no significant deviation from our predicted sequences, except for some sites of single nucleotide polymorphisms.As a basis to study the evolutionary history of a given gene family, we analyzed the abundance of slc1 gene family in zebrafish consists of nearly twice as many genes as present in genomes of human and mouse suggests that at least some of the zebrafish slc1 orthologs have originated from the teleost specific whole-genome duplication. Physical and virtual mapping indicated that none of the sequences within a subgroup of slc1 are located within the same chromosomal cluster . Even teleost genomes only distantly related to zebrafish displayed an almost identical number and phylogeny of slc1 family members as those identified in the zebrafish genome , we decided to extend our analysis to encompass the whole vertebrate tree Fig. . TherefoXenopus tropicalis, as a representative of the amphibians on the other hand has retained members of both subfamilies. Interestingly, additional gene losses occurred in a variety of lineages. For instance, chicken  has lost SLC1A5 and the green anole lizard (Anolis carolinensis) has lost SLC1A6  display various gene losses and retentions Fig. . Monotre1A6 Fig. .Phylogenetic reconstruction including now SLC1 sequences from teleosts, amphibia, sauropsida and mammalia clearly indicates that the newly assigned SLC1A8 and SLC1A9 each form a separate clade Fig. .Callorhinchus milii) and the sea lamprey (Petromyzon marinus). The elephant shark, belonging to the cartilaginous fish, represents an independent evolutionary branch of the vertebrates, whose genomes are thought to have undergone the same number of genome duplications as the mammalian branch [slc1 cDNA sequences. Nevertheless, our comparison revealed only exons for one ortholog per subfamily, with the notable exception of the SLC1A2/A9 subgroup, where we detected a retained SLC1A9 ortholog. Interestingly we also identified two paralogs of the SLC1A2/A9 subgroup in the lamprey, a jawless vertebrate at the base of the vertebrate tree .slc1a3 and slc1a2 subfamily genes.As an example to compare the spatial and temporal expression pattern of duplicated genes, we investigated the expression patterns of the zebrafish in situ hybridization experiments show that slc1a3a transcripts can be found in glia cells of the larval zebrafish brain at 3 days post fertilization  genes in zebrafish, we found as expected evidence for many duplicated genes. However, some zebrafish slc1 genes turned out to be not the result of the teleost specific R3. Further analyses ruled out the possibility that these SLC1 genes originated from the teleost specific genome duplication. One possible explanation for these additional genes is lineage and species specific gene loss. This has long been recognized in unicellular organisms [Modern genomes are shaped by frequent duplication and deletion events. Most dramatic are whole genome duplications that were prominently stated by Susumo Ohno as the most important driving force in the evolution of metazoans . This idrganisms ,28. Howerganisms ,29-31, wAmbystoma tigrinum retina reports the cloning of a SLC1A8/EAAT6, as well as a SLC1A9/EAAT7 gene  [In order to reconstruct the phylogenetic history of this gene family we studied a number of vertebrate species from all major vertebrate lineages. We found representatives of both new subfamilies in all analyzed teleost species, showing that the existence of these genes is common to teleost species and is not restricted to zebrafish. Analysis of amphibian genomes reveal the existence of one member of each SLC1A8/EAAT6 and SLC1A9/EAAT7 subfamily, arguing that the tetrapod ancestral species still contained the full complement of SLC1 subfamilies. In this respect it is interesting to note that a study of glutamate transporters in the tiger salamander ctively) ,33, whicAnolis carolinensis) have retained SLC1A9 but lost SLC1A8. The situation is reversed in the archosauromorpha (represented by the bird species zebrafinch and chicken), where SLC1A9 was lost but SLC1A8 was retained. This suggests that the genome of the last common ancestor of the sauropsida still contained both subfamilies, followed by independent gene loss in the two branches of the sauropsida. Following this logic the genome of the amniote ancestors should have contained members of both subfamilies.A clue to when the first subfamily was lost is provided by the sauropsida. Here the Lepidsauromorpha (represented by the green anole lizard Ornithorhynchus anatinus) have retained the member of SLC1A8, while all other remaining mammals have lost members of both subfamilies. In the most parsimonious scenario the last common mammalian ancestor has already lost SLC1A9 but has retained SLC1A8. Subsequently SLC1A8 was lost at the base of the therian lineage, whereas a member of this subfamily was retained in the branched off protheria species. Hence the SLC1A8 and SLC1A9 subfamilies have been lost independently in a number of lineages. While for instance SLC1A8 has been lost at least twice independently in the archosauromorpha and therian lineage, SLC1A9 has been lost independently in the therian and lepidosauromorpha lineage. In order to infer the ancestral situation we assessed the SLC1/EAAT repository in species that are at the base of the gnathosomes.In mammals only the protherian species  and the sea lamprey (Petromyzon marinus), is available. While the former represents the cartilaginous fish, an independent branch of the vertebrates that has split before the division of the tetrapod and the teleost lineage, the later belongs to a clade of skulled chordate animals lacking jaws. Since both lineages branched off before R3, cartilaginous fish as well as lampreys are expected to have undergone the same number of genome duplications as the mammalian branch. Consequently the gene repertoire between lamprey, sharks, and mammals should be comparable [Genome information for two species of interest, the elephant shark , the most likely position of the inactivated SLC1A9 is on the long arm at position 19q13.3. For this human chromosome extensive rearrangements within the short arm and many intrachromosomal breaks in the long arm have been reported, even since the time of primate rodent divergence . TherefoThe phylogenetic analysis of a gene family spanning multiple taxa can yield important information about their evolutionary history. In the case of the solute carrier family 1, consistent with the duplication-complementation-degeneration model, most of the 7 human or mouse orthologs have one or two teleost orthologs. However, for two teleost SLC1 subfamilies we found no corresponding therian counterpart. This indicates that therians must have lost these two subfamilies, now named SLC1A8/EAAT6 and SLC1A9/EAAT7.These two subfamilies are not teleost specific, but can be found throughout different vertebrates. An analysis of the major vertebrate lineages revealed an intriguing pattern of lineage specific gene losses, shaping the phylogenetic history of SLC1 genes. In all non-therian vertebrate species we found at least one member of the SLC1A8/9 subfamilies. Amphibians have neither lost SLC1A8 nor SLC1A9, lepidosauria  on the one hand have lost the SLC1A8 ortholog, while birds on the other hand have lost SLC1A9. Interestingly egg laying mammals (platypus) are the only mammals that have also retained a SLC1A8 ortholog.By studying one gene family across a number of vertebrate taxa, inferences can be made about the evolutionary history of these genes. Such an analysis is needed to guide the interpretation of functional data obtained for those genes and provides a fascinating scenario to study the evolution of vertebrate gene families.2, 0.33 mM MgSO4) at 28\u00b0C. Stages refer to the development in days post fertilization (dpf).Wild-type fish of the T\u00fcbingen (T\u00fc) inbred strain were bred and crossed as previously described . Embryosslc1 genes were annotated manually. Sequences from the species listed in Additional File http://www.ncbi.nlm.nih.gov; Ensembl, http://www.ensembl.org/index.html; version 50/51, 2008) and WGS http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_SPEC=TraceArchive&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch databases. Human and mouse sequences were used as initial query. Genomic regions coding for SLC1 family genes were identified by using the tblastx alignment algorithm . Gaps islc1 genes  were translated into proteins using the EditSeq software  and obtained protein sequences were used to generate a combined sequence file in FASTA format  configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions  were removed with Gblocks  using t, according to the manufacturer's instructions. Oligo dT primers were used to reverse-transcribe the total RNA with reverse transcriptase . Polymerase chain reaction (PCR) was performed using sequence-specific oligonucleotide primers. Amplified fragments were ligated into the pCR-II vector  and at least three independently amplified cDNA fragments per gene were sequenced to confirm our previously annotated zebrafish sequences. Accession numbers for the zebrafish SLC1/EAAT genes are: SLC1A1/EAAT3 (HM138690), SLC1A2a/EAAT2a (HM138691), SLC1A2b/EAAT2b (HM138692), SLC1A3a/EAAT1a (HM138693), SLC1A3b/EAAT1b (HM138694), SLC1A4 (HM138695), SLC1A5 (HM138696), SLC1A6/EAAT4 (HM138697), SLC1A7a/EAAT5a (HM138698), SLC1A7b/EAAT5b (HM138699), SLC1A8a/EAAT6a (HM138700), SLC1A8b/EAAT6b (HM138701), SLC1A9/EAAT7 (HM138702).slc1 genes except slc1a6/eaat4 were localized in the zebrafish genome by radiation hybrid mapping [All zebrafish  mapping  using thslc1 locus of human or zebrafish were used as initial queries for a tblastx [For microsynteny analyses, genes flanking the  tblastx  search aIn vitro transcription of DIG-labeled probes was performed using the Roche RNA Labeling Kit . RNA probes were hydrolyzed to obtain fragments of about 300 bp length. Whole-mount in situ hybridization on 3 dpf zebrafish larvae was performed as follows. PTU treated larvae were anesthetized on ice and immediately fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 40 min at ambient temperature (RT). The in situ hybridization was performed according to the Zebrafish Book protocol [in situ hybridization machine .protocol  using anslc1 genes, determined the intron/exon pattern of the slc1 genes and performed the synteny analysis. AL cloned the slc1 genes used in this study, wrote the first draft of the manuscript and contributed to expression analysis, CMM performed the expression analysis and HBS contributed to cloning and expression analysis. SCFN and MG wrote the manuscript in its present form, and conceived the project and designed experiments. Al authors have read and approved of the final manuscript.MG contributed to cloning, performed the database searches and the phylogenetic analysis, annotated the slc1 genes in the zebrafish genomeGenomic localizations of . Chromosomal localizations were identified both physically, using radiation hybrid mapping, and in silico. All zebrafish slc1 genes localize to different chromosomal regions. For details on radiation hybrid mapping, see Materials and Methods. *The localization of slc1a6/eaat4 could not unambiguously be determined by radiation hybrid mapping and has been mapped in silico only.Click here for filePhylogenetic analysis of SLC1 genes using a Bayesian algorithm. Bayesian phylogeny of members of the zebrafish (dr), mouse (mm) and human (hs) SLC1 family. The phylogenetic tree was build using 370 representative amino acids determined by the program Gblocks after sequence alignment using MUSCLE. The tree was reconstructed using the bayesian inference method implemented in the MrBayes program (v3.1.2). The number of substitution types was fixed to 6. The Poisson model was used for amino acid substitution, while rates variation across sites was fixed to \"invgamma\". Four Markov Chain Monte Carlo (MCMC) chains were run for 10000 generations, sampling every 10 generations, with the first 250 sampled trees discarded as \"burn-in\". Finally, a 50% majority rule consensus tree was constructed. Note that in comparison to the maximum likelihood calculated tree, roots in the Bayesian tree are slightly different. While in the Bayesian build tree it appears that there is a common ancestor of SLC1A1, SLC1A2, SLC1A3 and SLC1A6, the tree deriving from maximum likelihood phylogeny suggests that there is a common ancestor between SLC1A3, SLC1A6 and SLC1A7/8  and yellow (low coverage) belong to species that have been used to generate the phylogenetic tree covering the major linages. Coverage's shown in red belong to species whose sequences have been annotated but not included in the phylogenetic tree.Click here for fileLinks to transcript and genome information of the annotated sequences. The identity of the predicted and the manually annotated sequences is indicated by different colours. Identical predicted and annotated sequences are shown in green, whereas sequences with different predicted open reading frames are highlighted in red. Sequences covering the whole open reading frame are indicated as \"Full\" sequences and sequences lacking one or more exons are marked as partial sequences. Sequences for which neither a genomic nor a transcript link have been found are given by the letters n.d. (not detected).Click here for fileIntron/Exon sizes of SLC1 genes. The different SLC1 gene families are colour coded. Exons serving as identifiers for the corresponding gene family are highlighted in the identical colour as the gene family. Exons containing the start and the stop codon are highlighted in red. Intron sequences containing multiple place holders (Ns) are indicated.Click here for fileThe SLC1A9 subfamily is also present in cartilaginous fish. Database analysis on the elephant shark  and the sea lamprey (Petromyzon marinus) genome identified sequences that represent parts of the SLC1A9 gene. For the exon 234 , two corresponding slc1a2 exons could be found, one of them representing a retained slc1a9 gene. The phylogenetic tree was build using the maximum likelihood method on the entire nucleotide sequence of corresponding 234 exons. Bootstrap values above 50% (0.5) are shown. Elephant shark sequences are highlighted in dark red, lamprey sequences are shown in claret red and teleost genes are depicted in light red. Note that the FSGD generated two retained SLC1A2 genes and most likely also two SLC1A9 genes of which in modern teleosts only one is still present.Click here for fileProtein fastas of species used in the phylogenetic analysis covering the main vertebrate linages.Click here for fileNucleotide fastas of the sequences used in the exon 234 analysis.Click here for fileOligonucleotide Primers Used for Genomic Mapping.Click here for file"}
+{"text": "Infection without disease may occur under natural conditions after contact with infected birds. Avian influenza A virus subtype H5N1 was transmitted to domestic cats by close contact with infected birds. Virus-specific nucleic acids were detected in pharyngeal swabs from 3 of 40 randomly sampled cats from a group of 194 animals (day 8 after contact with an infected swan). All cats were transferred to a quarantine station and monitored for clinical signs, virus shedding, and antibody production until day 50. Despite unfamiliar handling, social distress and the presence of other viral and nonviral pathogens that caused illness and poor health and compromised the immune systems, none of the cats developed clinical signs of influenza. There was no evidence of horizontal transmission to other cats because only 2 cats developed antibodies against H5N1 virus. Avian influenza has attracted worldwide attention because highly pathogenic avian influenza virus subtype H5N1 can cause fatal infections in humans . PCR and egg culture identified avian influenza virus (H5N1) in the swan and in 13 of 38 other culled birds  (day 4) housed with the swan at the same time. Only the swan developed clinical signs of disease. On day 4, the poultry area was disinfected after all 38 birds were removed.In the same shelter were 194 cats; most had access to an outdoor enclosure near the poultry area and were separated from the birds by a wire-mesh fence. On several occasions, 1 or 2 unidentified cats were observed climbing the fence and entering the poultry area. Ingestion of birds by cats was not observed. Austrian authorities ordered random sampling of the cat population at the shelter because of spatial proximity of poultry and cats and the possible exposure of cats to infective debris of the birds. The bird area was left unoccupied while the cats were under observation. The purpose of this study was to monitor health status and possible transmission within a large cat population with proven natural exposure to H5N1 influenza virus.Pharyngeal swabs of 40 cats were sampled  were still available for further observations. Three cats had died and 24 other cats had been placed in private households. Before discharge from the shelter and within 1 week thereafter, all of these cats were examined and no abnormal health status was observed.2. The larger group contained 139 cats (including cats 1 and 2); the smaller group contained 28 cats. Cat 3 was not available for further examination because it was healthy before leaving the shelter and, to our knowledge, did not die. The smaller group was always separated from the larger group and was kept indoors at the animal shelter in Graz. In the quarantine area, the 167 cats were housed in 2 closed rooms, without any activity restriction, and had free access to food and water. Routine physical examination, including auscultation of the chest, was done on days 22, 29, and 50 for all cats at the quarantine station. In case of an obvious health problem, clinical signs were monitored by daily physical examination and serologic testing. The litter pans of the cats and floors of the quarantine areas were cleaned every day and disinfected every other day.A total of 167 cats were transported in small groups in \u224850 containers for 12 h from the animal shelter to a quarantine area and housed in 2 separate groups from day 22 until day 50. Average floor space for each cat was \u22481.4mOn days 22 and 29, pharyngeal and rectal swabs were obtained and transported in phosphate-buffered saline containing antimicrobial drugs .agtggcatgaccgccct-3\u2032, reverse primer: 5\u2032-cgttagcgcaggttgagca-3\u2032), and 5 pmol/L of probe (5\u2032-FAM-cactgtgatgtgttcgaagtttgagca-TAMRA-3\u2032). The cycler scheme consisted of 2 pre-PCR steps of 50\u00b0C for 15 min and 95\u00b0C for 2 min, followed by 45 cycles of 95\u00b0C for 15 s and 60\u00b0C for 30 s. Cycle threshold values were calculated by using PCR 7300 software (Applied Biosystems). FHV-1 nucleic acid was detected by PCR as described by Reubel et al.  according to the method of Spackman et al. (Plasma samples were tested for feline leukemia virus (FeLV) antigen and antibodies against influenza virus A H5N1, feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Antibodies to influenza virus were detected with a hemgglutination inhibition test according to the procedures of the World Organisation for Animal Health (Two cats that seroconverted for H5N1 virus (cats 1 and 4) were humanely killed on day 50. Necropsy was performed on these 2 cats and on 12 other cats that had died during the observation period; organ homogenates  were tested for influenza virus\u2013specific nucleic acids for each cat.H5N1 virus\u2013positive cats (1 and 2) and H5N1 virus antibody\u2013positive cats (1 and 4) did not show any signs of influenza virus\u2013associated illness after the swan had been placed in the animal shelter (days 1\u201350). Upper respiratory symptoms  were evident in 30 cats, bronchopneumonia in 40 cats, diarrhea in 7 cats, mucosal lesions in 37 cats, and traumatic wounds and lesions in 10 cats. However, for each cat with clinical symptoms that might have been associated with influenza infection, another specific etiologic reason for illness could be documented. Pathomorphologic examination showed no lesions associated with respiratory infection in cats 1 and 4 or in any other cat that had died before day 50. Influenza A virus\u2013specific nucleic acids were not detected in any organ sample tested by PCR. Likewise, all pharyngeal and rectal swabs obtained at the quarantine station were negative for influenza A virus by PCR. Antibodies against influenza virus A H5N1 were detected in 2 cats  with titers 256 on day 50 in both cats.Cats 1, 2, and 4 had negative test results for FeLV and FIV, but all 3 cats had high antibody titers against FCoV. FCV was detected in the swab from cat 2, and a double infection with FCV and FHV-1 was detected in cat 4. Clinical, bacteriologic, and virologic tests identified infection with FeLV in 15 cats, FIV in 12 cats, and antibodies against FCoV in all but 1 cat. A total of 44 swabs showed positive results for FCV-specific nucleic acids, 4 for FHV-1; 13 samples showed a double infection with FCV and FHV-1. Some pathologic bacterial infections of the respiratory and digestive system were confirmed by swab cultures.All veterinarians and staff members at the animal shelter and at the quarantine area were clinically monitored for any influenzalike symptoms. Because results of this monitoring were unremarkable and virus excretion by the cats was not detected, serologic tests were not conducted for these persons.This is the first description of an asymptomatic infection with highly pathogenic H5N1 influenza virus in domestic cats. Although infection was detected in a group of cats by positive PCR results for pharyngeal swabs in 3 cats and seroconversion in 2 cats, there was no evidence for influenza-associated disease. This finding contrasts with reports documenting cats with rapidly and developing and fatal disease caused by influenza A virus subtype H5N1 , oral mucosal lesions, and diarrhea were observed in cats in both groups in the animal shelter and in the quarantine station. A long (12 hours) and uncomfortable transport to the quarantine area, social distress caused by high population density, repeated restraint for examinations and sample collection, and multiple infectious agents may have caused such a high level of illness. Twelve cats died or were humanely killed while in a moribund state between days 22 and 50. All showed signs of disease other than infection with influenza virus A and died of feline infectious peritonitis, cardiomyopathy, enteritis, or nephropathy; none tested positive for H5N1 virus.During the observation period from days 22 to 50, excretion of virus was not detected in the pharynx or feces. Positive results were observed only on day 8 for 3 of the randomly sampled swabs. Therefore, viral shedding is assumed to have lasted <2 weeks in cats 1 and 2. In 1 study, no information was reported on the duration of virus shedding because only severe illness with a lethal outcome was reported or the cats were killed 7 days after experimental infection  or whether the cat did not produce sufficient amount of antibodies. Little information is available on immune responses after infection with influenza virus H5N1 in cats.H5N1 virus can cross species barriers (Until recently, the avian flu situations in Asia and Europe appeared to differ. In Asia, large numbers of poultry have been infected and culled. Human and feline cases are mainly associated with close contact with infected poultry or ingestion of contaminated meat that was not sufficiently cooked. In Europe, mainly wild aquatic birds were infected, and only a few turkey farms were affected by H5N1 infection. Because direct contact with poultry is more limited in Europe than in Asian countries and the main source of food for cats in Europe is either commercial cat food or wild rodents and small birds, virus uptake during hunting and ingestion of poultry and aquatic birds is unlikely. Large aquatic birds are normally not a major source of food for cats, although infected birds may have caused the deaths of 3 cats found on the island of Ruegen, Germany (We have shown that under natural conditions infection of cats with influenza virus H5N1 may occur after contact with infected birds or their excrement without inducing clinical disease. However, horizontal transmission between cats was not observed, although infected cats had been introduced into a large cat population that had other viral and bacterial infections and lived under stressful conditions. Avian flu infection in cats is rarely documented and there is no evidence to date that cats are responsible for transmitting the virus to humans. Although this study does not rule out H5N1 infection leading to disease and possible transmission to other mammals and birds by domestic cats under natural conditions, without ingestion of infected birds, cats do not represent a major risk in the epidemiology of H5N1 influenza. The risk posed by cats could change because the virus can rapidly undergo genetic mutation and reassortment, and efforts should be made to minimize contact of domestic cats with infected birds. To have better insights into whether cats represent a potential risk in the epidemiology of H5N1 influenza, more detailed knowledge is needed about the role of viral load, virus uptake, and immune mechanisms of the host on the outcome of infection with H5N1 influenza virus."}
+{"text": "Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1\u0394 cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1\u0394 cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in  Peroxiredoxins are a family of antioxidant enzymes highly conserved from yeast to human. Loss of peroxiredoxin in mice can lead to severe anemia and malignant tumors, but the underlying cause is not understood. One way to derive new knowledge of peroxiredoxins is through genetic analysis in yeast. We have shown that loss of peroxiredoxins in yeast is associated with an increase in mutation rates. Here, we demonstrate that this elevation of mutation rates in yeast cells lacking a peroxiredoxin is due to increased production of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Our findings suggest a new model in which compromised antioxidant defense causes accumulation of damaged DNA and activation of the DNA damage checkpoint. For yeast cells to survive DNA damage, dNTP production is increased to facilitate DNA replication, but at the price of high mutation rates. This new model could lead to a better understanding of human diseases including cancer. Peroxiredoxins belong to a family of thiol-specific peroxidases widely and abundantly expressed in most living organisms TSA1 was a strong suppressor of gross chromosomal rearrangements and spontaneous mutations TSA1 in the maintenance of genome stability, many genetic interaction partners of TSA1 identified through synthetic genetic array analysis were components of DNA repair machinery or DNA checkpoints TSA1 was found to interact genetically with REV1/REV3 and OGG1, which are critically involved in translesion synthesis (TLS) and the repair of oxidative DNA damage, respectively Loss-of-function studies in mice implicated an essential role of peroxiredoxins in antioxidant defense and tumor suppression Intracellular dNTP levels are one important determinant of cellular response to DNA damage tsa1\u0394 cells, additional events subsequent to the disruption of TSA1 might also be influential in the induction of genome instability. In this study we asked whether perturbation of dNTP pools might contribute to the mutator phenotype observed in tsa1\u0394 cells. We then investigated the cause of dNTP pool expansion. Our findings suggested that constitutive activation of the DNA damage checkpoint and consequent overproduction of dNTPs are the root cause of genome instability in tsa1\u0394 cells.We previously demonstrated that yeast Tsa1p is a house-keeping peroxiredoxin which sufficiently suppressed the mutator phenotype TSA1 was found to be a strong suppressor of mutations and gross chromosomal rearrangements tsa1\u0394 cells TSA1 with the DNA damage checkpoint and particularly the machinery of dNTP synthesis, in order to understand the role of TSA1 in the maintenance of genome stability.Yeast peroxiredoxin tsa1\u0394 cells to various DNA damaging agents. tsa1\u0394 cells were sensitive to hydroxyurea (HU), 4-nitroquinoline 1-oxide (4NQO) and ultraviolet (UV) irradiation  inhibitor Sml1p DUN1, SML1 or CRT1 in tsa1\u0394 cells exerted a significant impact on their sensitivity to HU, 4NQO and UV irradiation. Loss of DUN1 further sensitized tsa1\u0394 cells to H2O2, HU, 4NQO and UV  and 5FC-resistant (5FCR) assays  cells  and frameshifts (13.3%). Large deletions were very rare in tsa1\u0394 cells (3.3%) when compared with WT (13.3%) cells. In addition, all of the mutations found in tsa1\u0394 sml1\u0394 cells with high dNTP levels .Consistent with the activation of Rad53p and Rad9p, the levels of Rad53p target Sml1-3HAp were diminished in RNR genes is transcriptionally activated in response to DNA damage RNR1/2/3/4 transcripts in the presence and absence of HU. In this analysis we included an additional control termed HUG1, a target of Mec1p induced highly by DNA damage RNR transcripts were induced to higher levels in tsa1\u0394 cells than in WT and sod1\u0394 cells. The induction of RNR1 and RNR3 was greatest in both untreated and HU-treated tsa1\u0394 cells. The level of HUG1 transcript was also elevated in tsa1\u0394 cells and this was more pronounced in the presence of HU  rate of spontaneous 5FCR mutations over tsa1\u0394 RAD53 cells  as described Rates of spontaneous forward mutations to confer CANTotal RNA was extracted by phenol/freeze RNA preparation method as described Western blot analysis was performed essentially as described"}
+{"text": "Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia.Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times  or a permanent MCAO . Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed.TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study. Focal cerebral ischemia results in typical pathophysiological events that evolve in time and space. These processes are not limited to the lesion itself but differentially affect perilesional and even remote areas , that is known to mainly determine the degree of injury Nowadays non-invasive techniques like MRI - that are routinely applied in humans - can be also used to define ischemic lesions in rodents over time Hereafter differently impaired regions were separated into (1) the infarct core [necrotic damage], (2) perilesional areas [non-necrotic areas affected by a decreased blood flow mainly in territories of the MCA and branches of the ICA] and (3) remote areas [non-ischemic but indirectly affected regions].Male Wistar rats weighing 270\u2013300 g were used. All animal procedures were approved by the local government  and conformed to international guidelines on the ethical use of animals.2O/O2 (70%\u223630%). A commercial 4-0 monofilament nylon suture coated with silicone rubber on the tip  was introduced through an incision of the right common carotid artery into the ICA to occlude the origin of the right MCA for 30 min (n\u200a=\u200a32), 120 min (n\u200a=\u200a32) or permanently (n\u200a=\u200a11). During surgery body temperature was maintained at a physiological level. Cerebral blood flow was measured by laser Doppler flowmetry (LDF)  in cortical areas supplied by the MCA  from before the onset of MCAO until 15 min after reperfusion. Sham operated animals (n\u200a=\u200a12) underwent the same procedure but without occluding the MCA.Transient MCAO was induced according to Nagasawa and Kogure Brains were removed at various times after reperfusion . Using a rat brain slicer  3 mm coronal sections were dissected, cutting at distances of 2, 5, 8, and 11 mm from frontal pole. Section between 5 and 8 mm  for further use.Slices from the anterior and posterior sections (previously stained in TTC) where mounted onto slides and stained with 0.2% thionine.2O2 before the antibodies  were applied in TBS containing 3% normal donkey serum and 0.2% Triton X-100. Sections were incubated with the antibodies at 4\u00b0C overnight and further processed by the Vectastain Elite ABC Kit  using either a donkey anti-mouse or a donkey anti-goat biotinylated secondary antibody . Finally, immunoreactivity was developed in 3,3\u2032-diaminobenzidine tetrahydrochloride . For co-labeling of HSP72 and HSP27 the primary antibodies were simultaneously applied and detected by fluorescence-labeled secondary antibodies . Slices were analysed via confocal microscopy .Free floating sections were treated with 0.24% HAll animals with a dropdown of LDF signal to \u226440% were included in this study. The LDF signals were 27,7%\u00b18,9% of control values (before insertion of the filament) following occlusion of the MCA and recovered to 80,7%\u00b113,1 during reperfusion. There was a very low mortality rate following 30 min MCAO [9% overall including all reperfusion times (3 out of 32 rats)]. Following 120 min MCAO the mortality rates increased over time . Because of the still higher mortality rate in the 120 min MCAO group within 30 days this reperfusion time was omitted. Until 1 d following permanent MCAO 27% (3 out of 11) of operated rats died.All MCAO rats displayed an infarct whereby the infarct size varied. Following mild MCAO (30 min) the infarct always included the striatum and frequently the neocortex. After a severe MCAO (120 min) the necrotic area extended over the striatum to large portions of the adjacent neocortex, amygdala, and hypothalamus. Permanent MCAO produced the largest and most consistent infarcts with an infarct core always including striatum, neocortex, amygdala, and hypothalamus. The formation of edema was strongest under these conditions.Following 30 min MCAO TTC staining did reliably delineate the infarct  at 1 d after reperfusion . No cleaIntact cells  could beThe extent of neuronal injury could be identified by an absence as well as an attenuation of MAP2 immunoreactivity. After 30 min MCAO a diminished MAP2 expression could be detected as early as 2 h after reperfusion in striatum and neocortex. From 1 d of reperfusion on lack of MAP2 (necrotic areas) could be observed mainly in the striatum and occasionally in parts of the neocortex , 3. FollIn agreement with many other studies HSP27 was detectable at 1 d of permanent MCAO and from 1 d of reperfusion on after mild as well as severe ischemia . RegionsSlices from sham operated animals were adequately stained. We and others found HSP27 constitutively expressed in few astrocytes (mainly in fibre tracts) The TTC method rests on the functioning of mitochondrial enzymes By using immunohistochemical markers not only the infarct core but also perilesional and remote affected regions could be discriminated . BesidesMAP2 not only identifies the infarct core but also reveals ischemic damage in perilesional zones (defined by attenuated MAP2 staining) very sensitively. After mild MCAO in general a relatively broad area of partial MAP2 degradation was revealed. Following severe as well as permanent ischemia the infarct core was extended whereas perilesional regions were narrow. Since MAP2 is involved in maintaining the structural integrity of the neuronal cytoskeleton Transient post-ischemic HSP72 expression might start as early as 3 h after brain injury persisting for about 7 d Our results confirmed a post-ischemic upregulation of HSP27 after focal ischemia mainly in reactive astrocytes For the bilateral hippocampal induction of HSP27 one might discuss several causes. Though a bilateral transient impairment of cerebral blood flow has to be considered we think that secondary responses to ischemia are more relevant  Additionally to the HSP27 expressing astrocytes HSP27 positive neurons were detected. Double immunofluorescence stainings of HSP27 and HSP72 revealed a co-staining in the majority of labeled neurons. The induction of two heat shock proteins strengthens the possibility of these perilesional affected neurons to survive. All in all HSP27 is a useful marker to sensitively detect perilesional and far remote affected regions over a large period of time.Though a standardized protocol for infarct induction was used individual deviations occur inter alia due to varying cerebrovascular anatomy. The variability of infarcts as well as the involvement of perilesional and remote affected regions was most pronounced after mild MCAO . With pr"}
+{"text": "BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82\u201394%), poor for BRCAX with an LCS (40\u201350%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71\u2013100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between  BRCA1 and BRCA2 increase risk of breast cancer and contribute to a proportion of breast cancer families. However, more than half of the reported sequence alterations in BRCA1 and BRCA2 are currently of unknown clinical significance. We analysed gene expression in lymphoblastoid cell lines derived from blood of patients with sequence alterations in BRCA1 and BRCA2 and compared these to lymphoblastoid cells from familial breast cancer patients without such alterations. We then classified these lymphoblastoid cells based on their gene profiles. We found that BRCA1 and BRCA2 samples were more similar to each other than to familial breast cancer patients without BRCA1/2 mutations, and that the type of sequence change in BRCA1 and BRCA2 (missense or truncating) influenced gene expression. We included in the study ten familial breast cancer samples, which carried sequence changes in BRCA1 or BRCA2, that are believed to be of little clinical significance. Interestingly these samples were distinct from other familial breast cancer cases without any sequence alteration in BRCA1 or BRCA2, indicating that further work needs to be performed to determine the possible association of these \u201clow clinical significance\u201d sequence changes with a low to moderate risk of cancer.Inherited mutations in the genes  BRCA1 and BRCA2 account for a considerable proportion of these familial breast cancer cases, with the average cumulative risk in BRCA1 and BRCA2 mutation carriers by age 70 years estimated at 65% and 45%, respectively http://research.nhgri.nih.gov/bic/) currently has more than 1400 and 1800 unique sequence variants listed in the BRCA1 and BRCA2 genes, respectively. These include frameshift, nonsense, missense, splice site alterations and polymorphisms. Greater than a third of the BRCA1 and greater than half of the BRCA2 unique variants are \u201cunclassified variants\u201d without compelling evidence of pathogenicity or functional significance. The majority of unclassified variants recorded in the BIC database are predicted missense changes (more than 400 BRCA1 and 800 BRCA2). However other variants which may be categorised as unclassified variants are in-frame deletions or duplications, variants that may disrupt splicing, or variants in the 3\u2032UTR that may affect RNA stability (www.kconfab.org). BRCA1/2 unclassified variants represent a problem in the clinical setting as it is not known which variants are associated with the high risk of disease reported for classical truncating mutations.Approximately 7% of breast cancer cases occur in women with a strong family history of the disease Several functional assays may be used to determine the significance of unclassified variants, including transcription activation and complementation assays BRCA1 mutation carriers BRCA1 or BRCA2 genes, compared to healthy women undergoing reduction mammoplastic surgery with no family or personal history of any cancer or sporadic breast-cancer-affected controls ATM mutations, some of which are known to be associated with an increased risk in breast cancer BRCA1 or BRCA2 mutation status and to assist in assessing the clinical significance of BRCA1 and BRCA2 unclassified variants.Gene expression profiling has increased our understanding of the molecular events in breast tumor development, has been used to predict prognosis, and has characterised breast tumors into subtypes BRCA1 or BRCA2, to familial breast cancer cases with no known BRCA1/2 mutations (BRCAX). We also considered the possibility that BRCAX individuals with a BRCA1 or BRCA2 sequence variant classified to be neutral/low clinical significance (LCS) using multifactorial likelihood analysis may differ in gene expression profile from BRCAX individuals without such sequence variants. In addition, since truncating alterations comprise the majority of known pathogenic mutations but most BRCA1 and BRCA2 unclassified variants are predicted missense alterations, we compared profiles from individuals with known missense or truncating mutations to determine if mutation effect will affect the mutation-associated expression profile for each gene. We derived gene lists to predict mutation status defined by gene and mutation effect, and then tested the efficacy of these gene lists to predict the gene mutation status of LCLs. We provide evidence that gene lists differ according to gene and mutation effect, and according to the presence of sequence variants of low clinical significance. We also demonstrate that the use of appropriately-derived gene lists improves the prediction of pathogenicity of known mutations.In this study we compared LCL gene expression signatures of breast cancer cases carrying pathogenic mutations in BRCA1 or BRCA2 pathogenic mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). BRCAX breast cancer families are likely to result from mutations in several other genes, and thus represent a heterogeneous group. Moreover, included in the BRCAX group were a subset of 10 BRCAX individuals who carried a BRCA1/2 variant previously classified to be of low clinical significance using multifactorial likelihood approaches BRCAX LCLs containing a BRCA1 or BRCA2 variant of low clinical significance clustered away from the majority of remaining BRCAX samples . For this reason, BRCAX samples were stratified according to the presence of an LCS variant for further analyses.The ultimate aim of this experiment was to establish if gene expression profiles could distinguish between  samples . A t-tesBRCA1 and BRCA2 truncating mutations and rare sequence variants of low clinical significance, but differs from BRCA1 and BRCA2 missense mutations and BRCAX non-BRCA1/2 familial cases.Gene expression is similar for carriers of BRCA1 and BRCA2 samples were more similar to each other than BRCAX samples without an LCS variant. This result suggests that germline effects of heterozygous mutations in BRCA1 and BRCA2 cannot easily be separated using the experimental conditions used in this study. Although BRCAX samples tended to cluster distinctly from BRCA1/2 samples, nine of ten BRCAX individuals who carried a BRCA1/2 variant previously classified to be of low clinical significance fell within the major BRCA1 or BRCA2 mutation cluster. In contrast, six of the nine pathogenic missense mutations of BRCA1 or BRCA2 fell into a BRCA1/2 outlier group, which clustered closer to the BRCAX samples.Unsupervised hierarchical clustering  of LCL eBRCA1/2 pathogenic carriers and BRCAX individuals, we used a Gaussian Process Classifier (GPC). GPC analysis was used previously in an analysis of microarray profiles from irradiated short-term fibroblasts of BRCA1/2 mutation carriers BRCA1 truncating mutation carriers to BRCA2 truncating mutation carriers, and to BRCAX samples without an LCS variant. Samples with BRCA1 or BRCA2 pathogenic missense mutations or classified as BRCAX with an LCS variant were then included to determine their affect on the prediction accuracy. A summary of the prediction accuracy is shown in BRCAX with an LCS, and samples with BRCA1 or BRCA2 missense mutations. This prediction accuracy is above the expected performance, as a random prediction with three classes comprised of a similar sample number would be 33% accuracy. When BRCA1 and BRCA2 samples were compared to only BRCAX samples with an LCS variant, the prediction dropped to 43.46%, and the addition of the BRCAX samples without an LCS variant improved the accuracy. In all comparisons the inclusion of the pathogenic non-truncating mutations of BRCA1 and BRCA2 lowered the prediction accuracy.To determine the accuracy of using gene expression data from LCLs to predict BRCA1 or BRCA2 are generally identified after full sequencing of both genes. Therefore the most common clinical question is whether a variant in or BRCA2BRCA1  is pathogenic or not. We thus performed pair wise analyses to determine if BRCAX samples could be distinguished from those with pathogenic mutations in BRCA1 or BRCA2. Based on observations from hierarchical clustering analyses and the GPC analysis, we also considered the possibility that the effect of pathogenic BRCA1/2 mutations (truncating or missense) affected LCL gene expression. T-tests were performed using the 20,874 detected probes to elucidate gene differences between i) BRCA1 or BRCA2 truncating mutations vs BRCAX without an LCS variant; ii) BRCA1 or BRCA2 missense mutations vs BRCAX without an LCS variant. The number of genes which passed these restrictions and the overlap between them is outlined in BRCAX with an LCS variant  appears to affect the genes that are differentially expressed in the carriers after IR  vs BRCAX (noLCS)] and [BRCA1 (truncating) vs BRCAX (noLCS)] was 16 transcripts, with no overlap between the top 200 genes from [BRCA2 (missense) vs BRCAX (noLCS)] and [BRCA2 (truncating) vs BRCAX (noLCS)]. A total of 715 different genes were represented in the four lists of top 200 gene-lists from comparison of BRCAX (no LCS) to the different BRCA1/2 groups above. The results are summarised in BRCA2 truncating pathogenic carriers were consistently predicted with higher accuracy compared to BRCA1 truncating pathogenic carriers. The accuracy of prediction was improved when the gene list used for prediction was appropriate to the mutation effect (truncating or missense) being tested. When the missense-associated gene list was used, pathogenic truncating mutations were predicted with 35% and 68% accuracy for BRCA1 and BRCA2, respectively. Predictions increased to 71% and 84% for BRCA1 and BRCA2, respectively, using the truncating-associated genes. Similarly, the pathogenic missense mutation carriers were predicted with 83% and 100% accuracy when the missense-associated gene list is used, but this accuracy was lower or remained the same when the truncating-specific gene list was used (83% and 0%). Prediction of BRCAX samples that did not carry an LCS variant was high in all comparisons (82\u201394%). In contrast, prediction of BRCAX samples that did carry an LCS variant was poor (40\u201350%).SVM is a widely accepted classification approach for assessing differences in mRNA expression, and was used to compare after IR , we asseto BRCAX . The gentriction . The topBRCA1 (or BRCA2) and BRCAX samples. The significance of the predictions for the BRCA1 pathogenic missense mutations is summarised in When using SVM, the significance of the predictions can also be represented by the distance the prediction is from the plane, where predictions called with greater confidence are further from the plane that separates the BRCA1 or BRCA2 compared to BRCAX samples without an LCS variant was performed to determine the potential functional relevance of the differentially expressed genes. All BRCA1 and BRCA2 pathogenic mutations resulted in gene expression changes relating to cell cycle, cancer and cellular growth and development, while BRCA1 and BRCA2 missense mutations shared some additional similarities (cell death and cell development pathways). There were also alterations in several pathways that were unique to BRCA1 truncating mutations, BRCA2 truncating mutations, BRCA1 missense mutations, or BRCA2 missense mutations .It is difficult to counsel patients with a strong family history of breast cancer who are found to carry an unclassified variant in BRCA1 or BRCA2 genes from sporadic breast-cancer-affected controls BRCA1 or BRCA2 mutation status, with the ultimate aim of predicting the significance of unclassified variants of BRCA1 or BRCA2. We chose LCLs as a minimally invasive source of germline material that can be maintained as long term cultures, and because previous studies have shown that LCL array profiling is robust to sourcing of LCLs established in different laboratories BRCA1 and BRCA2 carriers to those of non-BRCA1/2 BRCAX familial breast cancer patients, an appropriate reference group for the proposed evaluation of unclassified variants identified in familial breast cancer patients. A relatively early time-point of 30 minutes post-irradiation was chosen to capture gene expression initiation, and minimize possible downstream compensation effects. It has previously been shown that 10Gy IR treatment of normal LCLs has an effect on the transcriptional response, with greatest change in mRNA levels for most genes within one hour post-treatment Expression profiles of short-term fibroblasts have previously been reported to separate carriers of a heterozygous mutation in the BRCAX cases carried BRCA1 or BRCA2 sequence variants that had been previously classified using multifactorial likelihood modelling methods to be neutral or of low clinical significance-that is, these rare variants are extremely unlikely to be a high-risk mutation in either of these genes, but the modelling methods used cannot assess whether they are truly neutral or associated with a much lower risk of disease. We found that the BRCAX samples with such LCS variants were separated from the majority of BRCAX samples without such LCS variants using unsupervised hierarchical clustering. This result indicates that LCS samples differ in expression profile as a result of their BRCA1 or BRCA2 sequence variant, and was substantiated by the class prediction methods: GPC prediction of the BRCAX samples decreased in accuracy when BRCAX samples with an LCS were included. In addition, SVM to detect BRCA1 or BRCA2 mutation-related gene lists yielded differences in the significant genes for comparisons to BRCAX samples without an LCS variant, compared to BRCAX samples with an LCS variant. Accordingly, prediction of BRCAX subgroup status based on the more robust gene list derived from comparisons to BRCAX individuals without an LCS variant was generally poorer for BRCAX samples with an LCS (40\u201350%) compared to those without an LCS (82%\u201394%). These rather provocative results indicate that the possible effect of all rare variants should be considered in development of assays to assess which variants have features of high-risk mutations. Moreover, the similarity in expression profile of these variants to other BRCA1/2 pathogenic mutations suggests that at least some of these LCS variants may confer small-moderate risks of breast cancer, presumably acting in concert with alterations in other genes in the BRCA1/2 pathway to lead to breast cancer. Given the rarity of these variants, alternative statistical approaches will be required to assess the risk of cancer associated with them.A number of BRCA1 mutations and those with pathogenic BRCA2 mutations. Ionising radiation has previously been show to separate fibroblast cells which carry BRCA1 or BRCA2 mutations from sporadic cases with 100% accuracy BRCA1 and BRCA2 cases to familial BRCAX cases as an appropriate reference group for familial breast cases likely to be identified as carriers of BRCA1/2 mutations or unclassified variants, we used LCLs instead of fibroblasts, we selected a lower IR exposure (10Gy vs 15Gy), and we chose a relatively early time point of 30 mins after exposure to IR in order to gain a better understanding of the functional differences in response to IR between the BRCA1, BRCA2 and BRCAX cell lines. Some or all of these factors may explain the difference in the ability of this study to distinguish BRCA1 from BRCA2, both of which are involved in DNA damage repair. However, differences in post-irradiation response between BRCAX individuals and BRCA1/2 mutation carriers are supported by alternative analysis we have conducted of the subset of genes reported to be involved in post-irradiation response, comparing mutation-negative normal female controls to BRCAX individuals without an LCS variant, or to BRCA1 or BRCA2 truncating mutation carriers. Our results indicate substantial differences in radiation response between normal controls and the patient groups, and also considerable differences between the BRCAX group and BRCA1 and BRCA2 carriers The assay conditions used in this study could not distinguish between samples with pathogenic BRCA1 or BRCA2. In the clinical setting, individuals generally present with full sequencing of both genes, and presence of a variant in one gene or the other. We thus assessed the ability to distinguish BRCA1 or BRCA2, separately, from BRCAX individuals. Importantly, since most unclassified variants are predicted to cause amino acid substitutions, we also assessed the relevance of mutation effect for expression profiles. We found that the genes which significantly differed between BRCA1 or BRCA2 and BRCAX LCLs were dependent on mutation effect. Accordingly, the SVM prediction for each mutation effect was best if the appropriate gene list was used, in terms of both accuracy of prediction (BRCA1 or BRCA2 vs BRCAX) and confidence in the classification as determined by the distance of the prediction from the SVM plane. Thus we strongly urge that mutation effect is taken into account if this type of assay is to be developed for use in predicting the clinical significance of BRCA1/2 variants. The current challenge is that few missense variants have been classified with respect to their clinical significance, with the only 23 individual missense variants termed clinically important by BIC, 17 in BRCA1 and six in BRCA2. Moreover, these are restricted in terms of the domains/regions in which they occur, residing in the BRCA1 start site (n\u200a=\u200a2), ring finger (n\u200a=\u200a4) or transactivation domains (n\u200a=\u200a11), and the BRCA2 CDK2 phosphorylation site (n\u200a=\u200a3) or at one codon  in a region of unknown function. It will thus be difficult to accrue a panel of known pathogenic missense variants for use in such predictive assays, and will require a concerted collaborative effort. Assuming sufficient pathogenic variants are identified, the successful execution of such a study may eventually distinguish missense-associated gene expression patterns that are generic to missense mutations, and/or those that are specific to the domain location of missense mutations. In addition, a possibly greater challenge will be identifying assay conditions  that can also identify gene expression differences between patients with rare variants of low clinical significance in BRCA1 and/or BRCA2 and those with truly high-risk pathogenic mutations (truncating or missense) in these genes. Our study, using conditions that were not optimal for separating BRCA1 and BRCA2 mutations nevertheless identified gene expression differences between BRCA1/2 pathogenic mutations and LCS variants, suggesting that larger sample sizes and further experimentation may identify a more robust gene list to separate pathogenic mutations, variants of low clinical significance, and individuals with no sequence alterations in BRCA1/2.The ultimate aim of this experiment was to identify array profiles that would be useful for the classification of unclassified sequence variants of BRCA1 and BRCA2 mutation carrier groups is consistent with the known functions of BRCA1 and BRCA2Pathway analysis confirming altered expression of cancer, cell proliferation and cell cycle pathways in BRCA1 and BRCA2 variants considered to be of low clinical significance have array profiles distinct from other non-BRCA1/2 familial cases, but resembling profiles of pathogenic BRCA1/2 cases, indicating that further work will be required to evaluate their possible association with a low-moderate risk of cancer. We have also shown that it will be important to consider mutation effect when developing array-based assays for predicting the clinical significance of BRCA1 or BRCA2 unclassified variants. Lastly, our findings demonstrate the ability of array profiling of immortalized lines derived from lymphoblastoid cells to detect germline mutations in genes that result in breast and ovarian cancer, and thus have relevance to the investigation of other genetic diseases irrespective of the organs or tissues they affect.In conclusion, we have provided evidence that carriers of BRCA1 or BRCA2 mutation have been identified, and families in which no predisposing mutation has been identified (BRCAX). The recruitment criteria for BRCAX families are: 1) at least one member of the family at high-risk according to the National Breast Cancer Centre Category III guidelines (http://www.nbcc.org.au), and four or more cases of breast or ovarian cancer (on one side of the family), and two or more living affecteds with breast or ovarian cancer, and four or more living first or second degree unaffected female relatives of affected cases, over the age of 18 ; 2) two or three cases of breast or ovarian cancer (on one side of the family) in same or adjacent generations, if at least one of these cases is \u2018high risk\u2019 , and two or more living affected cases with breast or ovarian cancer, and four or more living first or second degree unaffected female relatives of affected cases, over the age of 18.LCLs were derived from breast cancer-affected women recruited into the Kathleen Cuningham Foundation for Research into Breast Cancer (kConFab), a consortium which ascertains multiple-case breast cancer families BRCA1 and BRCA2 pathogenic mutations and variants of low clinical significance (LCS) are described on http://www.kconfab.org/Progress/Classification.shtml. Briefly, LCS variants include BRCA1 or BRCA2 variants described in trans with a deleterious mutation in the same gene in an individual and occur at a frequency of less than 1% in unaffected controls, or considered neutral/low clinical significance as measured using multifactorial likelihood approaches Classifications for A cohort of 72 LCLs were used in this study. The full listing of mutation details for LCLs is shown in BRCA1, 17 of which are predicted to lead to a truncated protein, and six of which were missense mutations ;23 LCLs from women carrying a pathogenic mutation in BRCA2, 19 of which are predicted to lead to a truncated protein, and three of which were missense mutations ;22 LCLs from women carrying a pathogenic mutation in BRCA1 or BRCA2 (BRCAX) after complete sequencing and multiplex ligation-dependent probe amplification gene dosage assay (MLPA) large deletion testing of BRCA1 and BRCA2. Ten samples, carried either BRCA1 or BRCA2 sequence germline variants considered from multifactorial likelihood classification to be LCS ; BRCA2 353 A>G Y42C, 2834 C>T S869L, 3031 G>A D935N (3 samples), 8795 A>C E2856A) BRCA1 or BRCA2 sequence variants other than common polymorphisms.27 LCLs from women from breast cancer families that have tested negative for pathogenic mutations in t tests were performed to determine the significance of gene expression changes. Expression differences were validated for 8/10 genes tested.LCLs were grown in RPMI 1640 media with 15% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine. The cell number was normalised and fresh medium was added to cells 24hr prior to irradiation with 10Gy, using a calibrated Cs137 c-source delivering 1 Gy/1.5 min. Total RNA was harvested 30min later using an RNeasy kit . The Illumina Totalprep RNA amplification kit  was used to amplify and biotinylate 450ng of total RNA. Biotinylated RNA was hybridised overnight at 55\u00b0C to Illumina Human-6 version 1 BeadChips containing >46,000 probes . The microarrays were washed, stained with streptavidin-Cy3, and then scanned with an Illumina BeadArray Scanner. Duplicate arrays were performed for eight cell lines across the different groups for quality control purposes, with duplicates performed on different days. All duplicate arrays showed highest correlation with each other (correlation >0.98). Duplicate samples were not included in analysis. Comparative real-time PCR was performed for ten genes on 6\u20138 samples, using GAPDH to normalise all data, and the comparative cycle threshold method for analysis. Paired student BRCA1/BRCA2 mutation status of irradiated fibroblasts Raw data was imported into Illumina Beadstudio and then exported into Genespring v7.3  for further analysis. Data was normalised  and filtered using an Illumina detection score of >0.99 in at least one sample, which yielded 20,874 probes that were used in all further analyses. The majority of these probes used in the analysis were designed by Illumina to assay the curated portion of the NIH Ref sequence database-16,923 were present in the Ref sequence database, comprising 65% of all Ref sequence-listed probes on the array. Transcripts which had a >2-fold change versus the mean were visualised using unsupervised Hierarchical Clustering  and 2. Twww.ingenuity.com) was used for biological interpretation of gene lists. Analysis of the transcripts found to be up- and down-regulated in irradiated LCLs as identified for the different mutation categories identified those biochemical networks most likely to be affected by a BRCA1 and BRCA2 truncating and missense mutation, relative to BRCAX. Those pathways with multiple hits or a significance score \u226520 were then compared.Ingenuity Pathway Analysis Click here for additional data file.Table S1Detailed Mutation Status of LCLs.(0.03 MB XLS)Click here for additional data file.Table S2BRCA1 Missense vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file.Table S3BRCA1 Truncating vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file.Table S4BRCA2 Missense vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.06 MB XLS)Click here for additional data file.Table S5BRCA2 Truncating vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file."}
+{"text": "We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing's sarcoma (ES). 5C11 specifically reacted with ESs but not with other small round cell tumours in childhood, i.e. neuroblastomas, primitive neuroectodermal tumours (PNETs), rhabdomyosarcomas and malignant lymphomas. 5C11 did not react with any other tumours in children except for hepatoblastomas. No reactivity has been identified in normal tissues with the exception of fetal hepatocytes. Immunoelectron microscopically, 5C11 reactive antigen was located on cell membrane of ES cells. Biochemically, 5C11 immunoprecipitated a cell surface protein having molecular weight of 81,000 Da. 5C11 is the first antibody which can clearly distinguish ES from neurogenic tumours, especially from PNETs which were recently reported to have common features to ESs regarding chromosal abnormality and proto-oncogene expression but show evident differentiation into neurogenic direction. The results strongly indicate the usefulness of 5C11 not only for diagnostic purpose when no specific marker is available but also for studying the histogenesis of ES. In addition, no reactivity in normal tissue implies its potential application as a therapeutic reagent when the management of ES patients is still a great problem in clinical field."}
+{"text": "To establish whether loss of heterozygosity (LOH) for chromosome 16q in Wilms' tumours confers an adverse prognosis, DNA from 40 Wilms' tumour/normal pairs were analysed using highly polymorphic microsatellite markers along the length of 16q. Fifteen per cent of tumours showed LOH for 16q. Although the common region of allele loss spanned the 16q24-qter region, a second distinct region of LOH was identified in 16q21. Five out of six tumours showing LOH were either (1) high stage or (2) low stage with unfavourable histology. In addition, there was a higher mortality rate in patients showing LOH for 16q than those that did not. These data strongly support the suggestion that LOH for 16q is associated with an adverse prognosis."}
+{"text": "ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.Mutations in the  Saccharomyces cerevisiae, are extremely useful for studying the basic principles of genome stability and maintenance.Genomic instability is a hallmark of cancer cells. Most human cancer cells show signs of genome instability, ranging from elevated mutation rates, to gross chromosomal rearrangements including deletions and translocations. Several surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. The current view is that most spontaneous chromosomal rearrangements arise during DNA replication. The activity of the DNA polymerases may be impaired by the presence of secondary structures, bound proteins or DNA lesions; this may lead to stalling or even collapse of replication forks. In response, cellular mechanisms are activated that arrest cell cycle progression, induce DNA repair, and restore replication ELG1 gene was identified in yeast as a mutant that causes enhanced levels of genomic instability ELG1 in yeast leads to increased recombination levels elg1 mutants also exhibit elongated telomeres RAD24 and CTF18. Elg1, Rad24 and Ctf18 form three alternative RFC-like (RLC) protein complexes, by interacting with the four small subunits of RFC S. pombe and hRad17 humans) S. cerevisiae, Rad9/Rad1/Hus1 in S. pombe and humans] The elg1 mutants have synthetic growth defects when combined with genes that are involved in homologous recombination, DNA repair, replication fork restart, checkpoint response and sister chromatid cohesion ctf4: double mutant spores elg1 ctf4 germinate, but fail to undergo cell divisions ctf4 and \u0394elg1 mutant cells show partially overlapping phenotypes. Both mutants have high levels of recombination, chromosome loss, as well as synthetic growth defects with rad52, mec1Genetic screens in yeast have shown that CTF4 was found, together with CTF18, in two independent screens for mutants that affect chromosome transmission fidelity ctf4 and ctf18 exhibit elevated levels of recombination ctf screen for increased chromosome loss ctf7 mutants, allowing them to grow at a higher temperature before arresting A clear linkage exists between replication progression and sister chromatid cohesion. First, Ctf7/Eco1, encoded by another gene that was found in the ELG1 has tight genetic interactions with CTF4 and CTF18, it is not known how it affects genomic stability. Here we performed a genetic screen in order to find high copy number suppressors of the SL interaction between elg1 and ctf4. We found a cohesin subunit and a cohesin loader, pointing to the fact that the synergistic genetic interaction between elg1 and ctf4 is due to enhanced defects in sister chromatid cohesion. Consistent with this possibility we also show that elg1 mutants exhibit defects in cohesion. Our results show that both the Elg1 and the Ctf18 clamp loaders affect sister chromatin cohesion and establish a link between Elg1 activity and cohesin loading.Although ELG1 and CTF4 are synthetic lethal (SL), namely, despite the fact that each single-mutant grows well, the double mutant is inviable ELG1 centromeric plasmid enabling the strain to stay alive. This plasmid has two marker genes: LEU2 and ADE3. In the appropriate genetic background, the presence of the ADE3 gene causes accumulation of a red pigment. Thus, elg1 ctf4 strains carrying the LEU2/ADE3/ELG1 plasmid grow well, and form uniformly red colonies, as they are unable to lose the ELG1-containing plasmid. These cells were then transformed with a yeast genomic library cloned in a high copy number plasmid (containing the URA3 marker). Cells that received a plasmid with a gene that, when over-expressed, can suppress the SL phenotype of elg1 ctf4 are now able to lose the ELG1-ADE3-LEU2 plasmid, and therefore show white/red sectored colonies  protein family, and two non-SMC subunits, Scc1, which is a member of the kleisin family, and Scc3. Smc1, Smc3 and Scc1 form a ring large enough to contain two dsDNA molecules SCC2, encodes a protein that forms, together with Scc4, a complex that helps loading cohesin on the DNA After transformation with the high copy number library, we screened about 30,000 colonies; 62 of them showed some degree of sectoring and were further analyzed. After re-introduction of the plasmid to na\u00efve yeast strains, only 29 clones gave a positive result. Twenty six carried the SCC1 are related to its function as a structural component of cohesin. This raises the question of whether Scc1 is the only subunit of the complex able to suppress the synthetic lethal phenotype of elg1 ctf4 strains. The fact that CTF4 was isolated numerous times but ELG1 was not, suggest that the screen may not have been saturated enough. Alternatively, only Scc1 and Scc2 may be the limiting factors that determine the amount of cohesin complexes loaded onto the DNA. Scc1 may be the limiting component in cohesin formation, and Scc2 availability may be limiting the amount of Scc1 loaded. According to this possibility ctf4 elg1 strain is inviable due to a low level of the cohesin ring, for example, at the centromeres.All the known phenotypes of mutants in SCC1, SCC2, SMC1 or SMC3 from LEU2-marked plasmids in a ctf4 elg1 strain carrying a ELG1/URA3 plasmid. If overexpression of any of these genes can suppress the SL phenotype, the ELG1/URA3 plasmid can be lost, allowing the cells to grow on 5-FOA medium (which selects for Ura- cells). SCC1and SCC2, (and of course ELG1), but not SMC1 or SMC3, can rescue the SL phenotype of the ctf4 elg1 double mutant strain. Thus, in agreement with the second hypothesis expressed above, Scc1 and Scc2 are the only limiting components able to suppress the lethality when overexpressed. Scc1 seems to be a slightly better suppressor than Scc2  were detected in these experiments (data not shown).To investigate whether the suppression by Scc1 was due to bypass of the elg1 and mutants defective in each of the components of cohesin. elg1/ELG1 SCC1/scc1-73 and elg1/ELG1 SMC1/smc1-259 double heterozygous diploids. The double mutants elg1 scc1 and elg1 smc1 were very sick being either completely unable to form colonies or forming tiny, sick colonies that failed to develop.In order to determine the genetic relationship between Elg1 and the Cohesin complex, we attempted to create double mutants between elg1 and mutations in other components of the cohesin complex such as Scc3 and Smc3, and additional genes that interact with cohesin, such as the loaders Scc2 and Scc4 elg1 exhibited synthetic fitness phenotypes already at the permissive temperature when combined with any member of the cohesin complex or with the Scc2-Scc4 loaders, but not with mutations in the separin Esp1, in the securin Pds1 or in Pds5. These synthetic genetic interactions show that Elg1 activity is important in the early stages of cohesin establishment and not in the maintenance or the cleavage of cohesin, strengthening the possibility that both Elg1 and Ctf4 play an important role in the coordination between replication fork progression and cohesin loading.We used the above method to examine the genetic relationship between TetO array is integrated approximately 40 kb from the centromere of chromosome V. These strains constitutively express a GFP-tagged TetR protein, which binds the arrays and can be visualized as a single GFP \u201cdot\u201d. In G2-arrested cells, lack of chromatid cohesion is seen as a \u201cdouble dot\u201d elg1 mutants showed this phenotype in 18.5% of the cells. Mutations in CTF18 exhibited a stronger phenotype (33%). Interestingly, double mutants elg1 ctf18 exhibit the same phenotype as the ctf18 single, indicating epistatic interactions. In contrast, mutations in the gene encoding the third RFC-like protein, Rad24, had only a modest effect, if at all, in sister chromatid cohesion, and no aggravating phenotype was observed when the rad24 mutation was combined with the elg1 or ctf18 mutations, or with the double mutant  interaction ctf4 exhibits defects in sister chromatin cohesion elg1 mutant such as its sensitivity to MMS . Moreover, we show that both proteins localize to chromatin, and that Elg1 is required in order to direct Ctf18 to the chromatin fraction  the elg1 ctf18 double mutant showed a higher sensitivity compared to the single mutants, suggesting that these proteins work in different pathways in the presence of DNA damage elg1 and ctf18 mutants of S. pombeelg1 mutants exhibit elongated telomeres, whereas ctf18 mutants have short telomeres ELG1 or CTF18 have the same effect in suppressing the hst3 hst4 mutant that contains hyperacetylated histone H3 at position K56 CTF4, consistent again with a model in which Elg1, Ctf18 and Ctf4 carry out related functions.In previous studies, different interactions between ELG1 can partially suppress the temperature sensitivity and the cohesion defects of an ctf7/eco1-1 mutant, whereas Elg1 overexpression exacerbates its conditional growth defect. Ctf7 is a pivotal sister chromatid cohesion factor that was found to be important for cohesion establishment ELG1 and ctf7 strengthen our characterization of Elg1 as a cohesion factor. We further extend these conclusions by analyzing the interactions between elg1 and all known cohesion factors. In addition, we show that Elg1 plays a possible role in recruiting the Ctf18 RLC to the chromatin, thus providing a mechanistic explanation for the epistatic genetic interactions between elg1 and ctf18 in sister chromatid cohesion. Consistent with our results  and pSBA419 (LEU2-ELG1-ADE3) have been described URA3-marked and LEU2-marked plasmids pSCC1, pSCC2, pSMC1, pSMC3 and pELG1 were created by cloning the respective genes in YEplac181 (LEU2) or YEplac195 (URA3).A list of all strains used in this paper is presented as elg1 ctf4 ura3 ade2 ade3 leu2/pSBA419) was transformed with a high copy number URA3 library as described Strain MK1001 (elg1 and ctf4 mutants carrying various plasmids was measured as described The rate of recombination of wild type, Logarithmically growing cultures were arrested in G1 with \u03b1 factor, at the beginning of S-phase with hydroxyurea or in G2 with nocodazole and incubated for one hour before being cropped by centrifugation This experiment was carried out as described"}
+{"text": "Salmonella Enteritidis phage type 6 (PT6) increased dramatically in the United Kingdom during 1997. The sharp rise suggests that PT6 contamination has spread rapidly throughout a basic food commodity; however, the source and food vehicle remain unknown. We present evidence from three outbreaks suggesting a possible link between PT6 and eggs. Poor documentation of the egg supply network continues to pose problems for public health investigators. Thorough investigation of all future PT6 outbreaks and case-control studies of sporadic infections are needed to confirm the etiology of PT6 infection."}
+{"text": "The I1307K allele was equally distributed among women with sporadic  and inherited  breast and/or ovarian cancer irrespective of their being diagnosed before or after 42 years of age and among asymptomatic  and cancer manifesting BRCA1/2 carriers . Taken together, the prevalence of the I1307K allele was significantly higher in BRCA1/2 carriers compared to non-BRCA1/2 carriers . The high prevalence of the I1307K allele among BRCA1/2 carriers is not associated with increased cancer risk but seems to be genetically connected because of Jewish ancestry. \u00a9 2000 Cancer Research CampaignThe frequency of the APC I1307K mutation and its association with disease pattern was examined in 996 Ashkenazi women consisting of individuals with either sporadic ("}
+{"text": "BRCA1 and BRCA2 are the two most important genes associated with familial breast and ovarian cancer susceptibility. In addition, PALB2 has recently been identified as a breast cancer susceptibility gene in several populations. Here we have evaluated whether large genomic rearrangement in these genes could explain some of Finnish breast and/or ovarian cancer families.BRCA1, BRCA2 and PALB2. The families have been comprehensively screened for germline mutation in these genes by conventional methods of mutation analysis and were found negative.Altogether 61 index patients of Northern Finnish breast and/or ovarian cancer families were analyzed by Multiplex ligation-dependent probe amplification (MLPA) method in order to identify exon deletions and duplications in BRCA1, deleting the most part of the gene (exon 1A-13) in one family with family history of ovarian cancer. No large genomic rearrangements were identified in either BRCA2 or PALB2.We identified one large deletion in BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements at least in BRCA1. On the contrary, the genomic rearrangements in PALB2 seem not to contribute to the hereditary breast cancer susceptibility.In Finland, women eligible for  BRCA1 and BRCA2 are the two major susceptibility genes, accounting for varying fraction of familial breast and ovarian cancer cases in different populations. In Finland, mutations in these genes explain approximately 20% of breast and ovarian cancer families [BRCA1 and BRCA2 are point mutations and small insertions/deletions, but increasing number of large genomic rearrangements in both genes have been identified in different populations [BRCA1 and BRCA2 mutations due to genomic rearrangements is not expected to vary markedly in different populations, although there might be accumulation of certain mutations due to a founder effect.Breast cancer is the most frequently occurring malignancy in women. ulations -6. Rearrulations ,6. The pBRCA1 or BRCA2 [BRCA1, BRCA2, ATM and RAD50 mutations [th century, and it was not until the 17th century that the vast inland regions were gradually inhabited by a relatively small number of individuals, resulting in several regionally occurring founder mutations [BRCA1 and BRCA2 might still be at least partly responsible for the hereditary predisposition to breast and ovarian cancer in Finland.Previous studies performed in the Finnish population have not observed large genomic rearrangements in or BRCA2 -9. Howevor BRCA2 , the stuor BRCA2 . In Finlutations -12, whicPALB2 was recently identified as a breast cancer susceptibility gene [PALB2 encodes a protein that binds to BRCA2 and this interaction is crucial for certain BRCA2 DNA damage response and tumor suppression functions [PALB2 are expected to be deleterious, and all result in protein truncations. The risk estimates for PALB2 mutations have ranged from two- to fourfold, although some PALB2 mutations have been suggested to have higher penetrance [PALB2 could also explain some of the Finnish breast and/or ovarian cancer families.ity gene ,14 and mity gene -17. PALBunctions . The brenetrance -15. HereBRCA1, BRCA2 and PALB2 genes. MLPA has been proven to be very useful in detecting copy number changes in genomic sequences [In the current study we have used Multiplex ligation-dependent probe amplification (MLPA) in order to identify exon deletions and duplications in the equences . The famequences  and wereBRCA1, BRCA2 and PALB2 and were found negative [Altogether 61 index patients of Northern Finnish breast and/or ovarian cancer families were selected for the study. These families have been screened for germline mutations in negative . 41 of tBRCA1 (primary screening kit P002B and confirmation kit P087), BRCA2 (P045B) and PALB2 (P057) kits  were used according to the manufacturer's instructions. After PCR amplification with IRD800 labeled primers the samples were analyzed with Li-Cor IR2 4200-S DNA Analysis system  and Gene Profiler 4.05 analysis program . For BRCA1 analysis the used deletion control had deletions in exons 1A-2, for BRCA2 analysis the control sample showed constant 50% reduction in band intensity resulting from a SNP locating three base pairs from the ligation site, and for PALB2 analysis the used control had heterozygous deletions in exons 1\u20133 and 5\u201310 in addition to homozygous deletion of exon 4.The SALSA MLPA kits for The information regarding the integrated density of each band received from GeneProfiler was analyzed by MLPA spreadsheets  in Excel Software according to the instructions. Dosage quotients 0.35\u20130.65 were considered deleted and dosage quotients 1.35\u20131.65 duplicated, and samples with quality value (standard deviation of the control ligation products) exceeding 0.1 were rejected. For the DNA sample positive for a genomic rearrangement, analysis was repeated using an independent sample in an independent assay.BRCA1, BRCA2 and PALB2 genes by MLPA. In BRCA2 and PALB2 no deletions or duplications were observed. We did, however, observe one large deletion in BRCA1 (exon 1A-13)  of the identified Northern Finnish BRCA1 positive families. Even though the deletion allele was observed only in one out 61 currently analyzed families, our results suggests that women eligible for BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements, at least in BRCA1. Additional work is still needed in order to determine the prevalence of BRCA2 rearrangements in Finland, as the current study was based only on relatively small number of families.To date, only three different utations , accountBRCA1, our results support the previous conclusions that the genomic rearrangements in BRCA1 and BRCA2 are not a major cause for increased breast cancer susceptibility in Finland, and that the previously reported Finnish founder mutations represent the majority of BRCA1 and BRCA2 positive families [PALB2 gene were identified in the studied index cases of 61 families. This suggests that genomic rearrangements in PALB2 are very rare, which has also been indicated by a previous study [PALB2 appears to be the previously reported founder truncation mutation [Despite the identification of one large genomic deletion in families -9,27. Nous study . At leasmutation .BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements at least in BRCA1. In contrast, the genomic rearrangements in PALB2 seem not to contribute to the hereditary breast cancer susceptibility in Finland.In Finland, women eligible for The authors declare that they have no competing interests.KP carried out the MLPA and data analysis, and drafted the manuscript. HE, JN and SS helped to draft the manuscript. RW participated in study design and in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The Ad5CMV-\u03b1VEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comIncreased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-\u03b1VEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-\u03b1VEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-\u03b1VEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF"}
+{"text": "The amount of NO3 in warm seasons is twofold that of cold seasons , and there was a significant difference between cold and warm seasons (Table 1). Annual wet deposition of ammonia (NH3) was 10 kg/ha/year in the Chitgar Parkland[1].An investigation of air pollution in the Tehran metropolitan area between 1992\u20132000 indicated that there are significant amounts of nitrate ion (NO"}
+{"text": "Also in Table 1, a row of data for men was omitted and should have indicated 36 men (45.6%) with low trust and 43 (54.4%) with high trust. This article has been corrected.1In the Original Investigation titled \u201cCharacteristics of Health Care Organizations Associated With Clinician Trust: Results From the Healthy Work Place Study,\u201d"}
+{"text": "The as-prepared CsPbBr3 PNCs and Cs4PbBr6 PNCs exhibit different optical properties that depend on their morphology, size, and structure. The photoluminescence (PL) emission and quantum yield (QY) of the CsPbBr3 PNCs can be tuned by changing the ultrasound power, radiation time, and the height of the vibrating spear. The optimized CsPbBr3 PNCs show a good stability and high PL QY of up to 85%. In addition, the phase transformation between CsPbBr3 PNCs and Cs4PbBr6 PNCs can be obtained through varying the amount of oleylamine (OAm) and water. The mechanism of this transformation between the CsPbBr3 PNCs and Cs4PbBr6 PNCs and their morphology change are studied, involving ions equilibrium, anisotropic growth kinetics, and CsBr-stripping process.We demonstrate an ultrasonication-assisted synthesis without polar solvent of CsPbBr A weak 4PbBr6 PNCs, typically a green emission arises either from defects or from impurities or from a combination of both [4PbBr6 PNCs did not demonstrate PL emission over the whole visible spectrum due to their wide bandgap (Eg(Cs4PbBr6) = 3.94 eV), while the observed weak PL emission results from a small portion of CsPbBr3 impurities in the Cs4PbBr6 PNCs  emerge, which have been confirmed to result from the formation of Cs4PbBr6 PNCs [3 PNCs. Based on this process it can be concluded that the excess amount of OAm triggers the transformation between CsPbBr3 PNCs and Cs4PbBr6 PNCs.Furthermore, the effect of the amount of OAm on the phase transformation was investigated. As shown in Br6 PNCs . When ad3 PNCs to Cs4PbBr6 PNCs was further confirmed by using TEM. When the amount of OAm is between 0.5 and 1.5 mL, the morphology of PNCs gradually becomes irregular and some hexagonal shapes emerge , suggesting the formation of CsPbBr3 PNCs. Moreover, the PL QY of as-prepared CsPbBr3 PNCs was calculated to be ca. 70%. Interestingly, the CsPbBr3 PNCs show a high stability in ambient environment, as shown in 3 PNCs to Cs4PbBr6 PNCs was achieved  solubility of hexane in water [3 PNCs have a higher stability than Cs4PbBr6 PNCs against water.The addition of led to the decomposition of CsBr3 PNCs  and the Br3 PNCs . This isin water . The abo3 PNCs [3 PNCs could be easily tuned. More importantly, with lower the immersion heights of the vibrating spear higher PL QY of CsPbBr3 PNCs were achieved. The as-prepared CsPbBr3 PNCs show a high PL QY of up to 85% and a considerable photostability and chemical stability. The Cs4PbBr6 PNCs are obtained via direct ultrasonication of precursors or after adding OAm in the pre-synthesized CsPbBr3 PNCs solution. The phase transformation of orthorhombic CsPbBr3 NCs to rhombohedral Cs4PbBr6 NCs is promoted by the capacity of organic ligands to dissolve PbBr2, and by the formation of lead oleate and the dissociation of CsPbBr3 PNCs. Morphology changes are mainly ascribed to the anisotropic growth of the crystals. In addition, a reverse transformation from Cs4PbBr6 PNCs to CsPbBr3 PNCs can be achieved by adding water to pre-synthesized Cs4PbBr6 PNCs. The developed ultrasonication assistance results in the successful control over the phase transformation of PNCs, which can find widespread application in photoelectronic devices. We anticipate that this work can be extended to prepare other halide perovskites.In summary, we demonstrate the effect of small changes in the environment of capping ligands and water on the crystal structure and stoichiometry of PNCs. This study expanded our recent work of synthesizing differently shaped CsPbBr3 PNCs . Similar2CO3, 99%), lead bromide , liquid paraffin (90%), oleic acid , oleylamine , and anhydrous toluene (99.8%) were purchased from Shanghai Aladdin Biochemical Technology Co. The chemicals used in the present work were of analytical grade and used without further purifications.Cesium carbonate (CsCsPbBr3 PNCs: The PNCs were prepared via modifying the procedures reported by Tong and Rao and co-workers [2CO3 (0.15 mmol) and PbBr2 (0.30 mmol) powders were added to a mixture of 10 mL liquid paraffin (LP), 0.50 mL OA and 0.50 mL OAm. Then the reaction medium was processed by tip-sonication at a power of 120 W for 40 min. During the sonication, the colorless reaction medium gradually transformed into a yellow and then an orange-yellow solution, which suggests the formation of PNCs and demonstrates strong fluorescence emission under 365 nm UV light excitation. After completion of the reaction, unreacted precursors and excess ligands were removed by centrifugation at a speed of 3000 rpm for 10 min and then the precipitate was redispersed in 5.0 mL of toluene. Then, the colloidal solution was centrifuged at a speed of 12000 rpm for 5 min and the sediment was redispersed in toluene for further characterization.-workers \u201330. In aCs4PbBr6 PNCs: Cs2CO3 (0.15 mmol) and PbBr2 (0.30 mmol) powders were added to a mixture of 10 mL liquid paraffin, 0.50 mL OA and 3.0 mL OAm, while keeping other synthesis conditions as the same as that of CsPbBr3 PNCs.4PbBr6 PNCs solution and shaken slightly, which is a modification of the work reported by Wu and co-workers [50\u2013250 \u03bcL of water was added to 5.0 mL of the pre-synthesized Cs-workers .4PbBr6 PNCs was characterized by using a field-emission scanning electron microscope . The UV\u2013vis absorption spectra of the samples were measured using a UV\u2013vis spectrometer  over the wavelength range from 300 to 700 nm, at 1 nm intervals. The PL spectra of the PNCs were recorded using a fluorescence spectrophotometer  using a Xe lamp as an excitation source.The crystal surface morphology of the PNCs was characterized by transmission electron microscopy  with an accelerating voltage of 100 kV. High-resolution TEM (HRTEM) was carried out on a JEOL JEM-2100F instrument operating at 200 kV. The crystal phases of the products were measured using an X-ray diffractometer  with a Cu K\u03b1 radiation source (\u03bb = 0.15418 nm) at a counting rate of 2\u00b0 per minute in the scanning angle (2\u03b8) range from 5\u00b0 to 50\u00b0. The surface morphology of CsFile 1Additional PL spectra, SEM image, and UV\u2013vis absorption spectra.File 24PbBr6 to CsPbBr3 PNCs after addition of water.Video showing the transformation from Cs"}
+{"text": "World Arthritis DayA report in this issue found that adults with arthritis had higher prevalences of symptoms of anxiety (22.5%) and depression (12.1%) compared with adults without arthritis ("}
+{"text": "SLC2A3) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study.The gene encoding glucose transporter 3 (GLUT3, SLC2A3/GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls.Cells from genotyped healthy controls were analyzed for SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups.Heterozygous deletion of SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.Despite a robust  \u2022SLC2A3/GLUT3 correlates to SLC2A3 gene copy number in a dose dependent manner.T cell and macrophage expression of \u2022SLC2A3Glycolysis rates are reduced in individuals harboring a deletion of the GLUT3 gene \u2022SLC2A3 is not associated with protection from rheumatoid arthritisDeletion of \u2022SLC2A3 is not associated with risk for multiple sclerosisDeletion of \u2022SLC2A3 deletion.GLUT3 is not a viable therapeutic target for RA as previously proposed based on a protective association of  We also did not observe evidence of an association with multiple sclerosis, despite initial encouraging results in multiplex families with autoimmunity.Prompted by a previous study of protection from RA, we explored the biological effects of SLC2A3 deletion. Foremost, we note that our current data reveal that the number of PennCNV false calls can be significant (tableS4). By their nature, random sampling of rare events can lead to false differences more often than common events [There are a number of possible explanations of why this study did not replicate the previously reported association of protection from RA with the n events . We analSLC2A3 CNV with RA [SLC2A3 CNV.Our result is consistent with two previous genome-wide CNV association studies that also found no association of the  with RA ,16, howeSLC2A3 CNVs in the human population at fairly constant frequencies between ethnically diverse groups . K.R.S is supported by the NIH CDA K01AR071502 (USA). PKG is supported by NIH grant UH2AR067694 (USA).This work was supported by the"}
+{"text": "Nature Communications 10.1038/ncomms6404, published online 20 November 2014Correction to: This Article contains an error in Equation 2 in that the denominator is inverted. The correct form of Equation 2 is:"}
+{"text": "Saccharomyces cerevisiae as well as contributing to the integration of the nuclear export of mature mRNA with preceding steps in the nuclear phase of the gene expression pathway. Nab2 is constructed from an N\u2010terminal PWI\u2010fold domain, followed by QQQP and RGG motifs and then seven CCCH Zn fingers. The nuclear pore\u2010associated proteins Gfd1 and Mlp1 bind to opposite sides of the Nab2 N\u2010terminal domain and function in the nuclear export of mRNA, whereas the Zn fingers, especially fingers 5\u20137, bind to A\u2010rich regions of mature transcripts and function to regulate poly(A) tail length as well as mRNA compaction prior to nuclear export. Nab2 Zn fingers 5\u20137 have a defined spatial arrangement, with fingers 5 and 7 arranged on one side of the cluster and finger 6 on the other side. This spatial arrangement facilitates the dimerization of Nab2 when bound to adenine\u2010rich RNAs and regulates both the termination of 3\u2032 polyadenylation and transcript compaction. Nab2 also functions to coordinate steps in the nuclear phase of the gene expression pathway, such as splicing and polyadenylation, with the generation of mature mRNA and its nuclear export. Nab2 orthologues in higher Eukaryotes have similar domain structures and play roles associated with the regulation of splicing and polyadenylation. Importantly, mutations in the gene encoding the human Nab2 orthologue ZC3H14 and cause intellectual disability.The poly(A) RNA binding Zn finger ribonucleoprotein Nab2 functions to control the length of 3\u2032 poly(A) tails in  Saccharomyces cerevisiae Nab2 (ScNab2) accompanies mature transcripts as they move through nuclear pores to the cytoplasm, after which ScNab2 is thought to be removed from the mRNA by the DEAD\u2010box RNA helicase, Dbp5ScNab2 is then recycled back to the nucleus through nuclear pores using the transport factor, karyopherin \u03b22 .ScNab2 shows genetic interactions with the splicing machineryIn eukaryotes, the separation by the nuclear envelope of transcription from translation enables mRNAs to be modified by capping, splicing, and polyadenylation. These processing steps are mediated by a large number of different proteins that interact with transcripts as they pass through the nuclear phase of the gene expression pathway before they are finally exported to the cytoplasm through nuclear pores.Drosophila orthologue, DmNab2 are also required for proper poly(A) tail length control,ZC3H14 gene have been linked to a nonsyndromic form of autosomal recessive intellectual disability,DmNab2 mutant flies exhibit impaired short\u2010term memory and defects in neuronal patterning in the learning and memory center (mushroom body) of the fly brain.DmNab2 mutant flies rescues function, indicating that ZC3H14 is a functional orthologue of DmNab2.In higher eukaryotes, the human Nab2 orthologue, ZC3H14, and ScNab2 in S. cerevisiae. ScNab2 contains an N\u2010terminal domain that has a Proline\u2010Tryptophan\u2010Isoleucine (PWI)\u2010like fold,ScNab2 Zn fingers (ZnFs) are arranged into three groups: ZnF12; ZnF34; and ZnF567. The Nab2 orthologues in other species\u2014S. pombe Nab2 (SpNab2); C. thermophilum Nab2 (CtNab2); D. melanogaster Nab2 (DmNab2); C. elegans SUT\u20102 (CeSUT\u20102); H. sapiens ZC3H14 (HsZC3H14)\u2014have a similar overall domain architecture  RNA using CtNab2 and ScNab2.ScNab2 Zn fingers 5, 6, and 7 (ZnF567) are critical for function, and studies demonstrate that a nab2 mutant lacking ZnF567 is not functional in budding yeast.ScNab2.Binding to polyadenosine RNA, a primary function of the Nab2 protein family, is mediated by the Zn finger motifs.ScNab2 N\u2010terminal domain (Nab2\u2010N)nab2 mutant in which the N\u2010terminal domain has been deleted (nab2\u2010\u0394N) exhibits severely impaired growth and nuclear accumulation of poly(A) RNA.nab2\u2010\u0394N cells, indicating that the N\u2010terminal domain might contribute to the control of poly(A) tail length.ScNab2 N\u2010terminal domain interacts physically with both Mlp1, a component of the nuclear basket that is located on the nuclear face of nuclear pores,ScNab2 Phe73, which is located on a hydrophobic surface patch on the Nab2 N\u2010terminal domain [Fig. ScNab2 F73A and F73D variants show impaired binding to Mlp1 in yeast lysates and in vitro.The crystal and solution structures of the ScNab2 N\u2010terminal domain also interacts with Gfd1,dbp5(rat8\u20102) RNA helicase mutantgle1\u20108 RNA export factor mutant.ScNab2 N\u2010terminal domain and identified a key contribution made by ScNab2 Tyr34, which is located on the opposite side of the N\u2010terminal domain to ScNab2 Phe73, which is important for Mlp1 bindingnab2\u2010Y34A dbp5(rat8\u20102) double mutant shows a synthetic slow growth phenotype.in vivo.The ScNab2\u2010N interacts with the nuclear pore associated proteins, Mlp1 and Gfd1, suggests that Nab2\u2010N PWI domain could also function as a protein\u2013protein interaction module in higher eukaryotes, although no partners have currently been identified.Although the N\u2010terminal domain is conserved in Nab2 orthologues [Fig. ScNab2 ZnF567CtNab2ScNab2 ZnF1 and ZnF2 interact with one another as do ScNab2 ZnF3 and ZnF4 to form defined structural units.The structures of several Nab2 Zn finger clusters have been established using both X\u2010ray crystallography and NMR.ScNab2 ZnF567, NMR chemical shift perturbations associated with binding either AMP or A3 identified a series of basic and aromatic residues associated with RNA bindingScNab2 ZnF567 residues are strongly conserved between Nab2 orthologues [Fig. ScNab2 variants in which these ZnF residues were substituted showed reduced affinity for A9 RNA.nab2 ZnF567 mutants containing substitutions of these basic and aromatic residues had growth rates similar to wild\u2010type cells, the nab2 ZnF567 mutants generated longer poly(A) tails in vivo and also showed genetic interactions with both Dbp5 and Yra1, consistent with their also influencing the generation of mature mRNPs.ScNab2, structural coherence between ZnF567 was lost in the ScNab2 RNA\u2010binding mutant, nab2\u2010C437S, in which a Ser was substituted for the first Zn\u2010coordinated Cys in ZnF6 [Fig. nab2\u2010C437S yeast mutant exhibited cold\u2010sensitive growth and hyperadenylation of bulk poly(A) tails.nab2\u2010C437S mutant with the dbp5(rat8\u20102) RNA helicase mutant suppressed the growth defect of the dbp5(rat8\u20102) mutant.nab2 ZnF mutants in combination with the dbp5(rat8\u20102) mutant indicated that dbp5(rat8\u20102) suppression by nab2 ZnF mutants was more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to the affinity of the mutant Nab2 for poly(A) RNA.ScNab2 has an unanticipated function associated with generating export\u2010competent mRNPs, and that changes within ZnF567 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.In ScNab2 ZnF567CtNab2 ZnF34511G and A8 RNA, respectively, indicated the basis for selective binding of Nab2 Zn fingers to adenine and identified the importance of H\u2010bonds formed by adenine N6  RNA in vitro and generated longer bulk poly(A) tails in vivo.The crystal structures of ScNab2 Zn fingers 567 and CtNab2 Zn fingers 345 complexed with poly(A) RNA, the spatial arrangement of the fingers precludes them from all binding to the same RNA chain, so that the first and third Zn fingers of the module were bound to one RNA chain, whereas the middle Zn finger was bound to a second RNA chain. Moreover, the crystal structure of ScNab2 ZnF567 complexed with A11GIn vitro binding studiesScNab2 ZnF567 and either A12 or A11G, indicating that it was not a crystallization artifact and also that the 3\u2032 terminal G was not necessary for its formation. Unusually, the dimerization of the ScNab2 protein chains was mediated almost entirely by each RNA chain binding to both protein chains and not by specific interactions between residues on the protein chains themselves, precluding the engineering of ScNab2 variants in which the Nab2\u2010Nab2 interaction was impaired. However, ScNab2 dimerization was impaired in Nab2 variants in which RNA binding was disturbed, with substitution of Phe450 in ZnF6, that is important for the interaction with the two adenines that bind ZnF6  tail length in vivo.ScNab2 F450A variant exhibited some of the strongest genetic interactions with Yra1 and Dbp5.In the structures of both ScNab2 ZnF567\u2010A11G heterotetramer, not all the adenines interact directly with the Nab2 protein chains, suggesting that Nab2 could also form analogous dimers with RNA sequences that only contain adenines in key positions [Fig. ScNab2 binds along the coding region of transcripts as well as to the poly(A) tail,ScNab2 ZnF567 in mediating the compaction of GAL1 transcripts in vitro [Fig. ScNab2 ZnF567, which were able to form dimers, resulted in much more compact complexes than those formed by the ScNab2 F450A variant, in which dimerization was impaired.In the S. cerevisiae, the 3\u2032\u2010end processing machinery comprises cleavage factors IA and 1B and the cleavage and polyadenylation factor (CPF), a complex including the riboendonuclease, Ysh1/Brr5, the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1.S. cerevisiae, the CPF component, Fip1, binds Pap1 directly and tethers it to CPF to stimulate/regulate Pap1 activity.S. cerevisiae poly(A) tails have a length of ~60\u201380 nucleotides  RNA as it loops between Nab2 chains. Consequently, it is not clear whether generating a single Nab2 dimer is sufficient to terminate polyadenylation or whether instead it is necessary to form two dimers. Further work will be required to address this question.Polyadenylation is the final processing step in the nuclear phase of the gene expression pathway. In ScNab2 and SpNab2 can protect poly(A) RNA from degradation by the RNA exosome in vitro.In addition to terminating polyadenylation by dissociating Pap1 from the CPF, Nab2 may also contribute to regulating poly(A) tail length through interactions with the 3\u2032\u20105\u2032 riboexonuclease exosome complex,ScNab2 Zn finger variants has indicated that the ScNab2 ZnF567 interaction with polyadenosine RNA plays a central role in the regulation of poly(A) tail length and mRNA compaction in S. cerevisiae. In this context, the observation that the loss of DmNab2 in Drosophila and ZC3H14 in mice and humans results in longer poly(A) tailsDmNab2 ZnF123 and ZC3H14 ZnF123, which are most similar to ScNab2 ZnF567 [Fig. ScNab2 to contribute to the control of poly(A) tail length. In the future, it will be informative to assess the functional consequences of specific DmNab2/ZC3H14 Zn finger variants, such as DmNab2 C879S and HsZC3H14 C622S in ZnF2 that are equivalent to ScNab2 C437S in ZnF6 [Fig. Overall, extensive analyses of S. cerevisiae, Nab2 also shows genetic interactions with components of the mRNA nuclear export machinery, such as Yra1, Sub2, and Mex67.ScNab2 may also interact directly with Mex67, one possible mechanism for signaling the completion of polyadenylation could be mediated through Pcf11 and the THO complex as a result of the dissociation of poly(A) polymerase Pap1 from the CPF complex.In addition to its function in controlling poly(A) tail length in S. cerevisiae, direct evidence for such a dimer containing full\u2010length Nab2 or the way in which the protein chains are arranged in the Nab2 dimer has not yet been obtained either in vitro or in vivo. It is also unclear whether one or two Nab2 dimers are needed to terminate polyadenylation. Although CPF components, such as Pcf11, appear to participate in signaling the termination of polyadenylation to the mRNA export machinery (TREX/Yra1/Sub2/Mex67:Mtr2) that generates an export\u2010competent mRNP, precise details of the signaling pathway and how polyadenylation termination is transmitted to Pcf11 remain to be established. Similarly, although there is clearly crosstalk between Nab2/ZC3H14 and the splicing machinery, most notably involving genetic interactions between ScNab2 and the splicosome component, Mud2,Although Nab2 dimerization following binding to the growing poly(A) tail could provide a mechanism by which Nab2 terminates polyadenylation in S. cerevisiae. Nab2 orthologues also contribute to these functions in higher Eukaryotes, however, the greater complexity of these systems has made establishing precise molecular mechanisms and signaling pathways more difficult. The spatial arrangement of Nab2 Zn fingers facilitates dimerization when bound to adenine\u2010rich RNAs that is important for the termination of 3\u2032 polyadenylation and transcript compaction, albeit the precise structure of the Nab2 dimers generated in vivo remains to be established. Overall, the wealth of information that has been generated about the structure of Nab2 and its interactions with other components of the nuclear gene expression machinery has laid the foundation for beginning to define the precise ways in which these pathways are coordinated and also provides insight into the contribution made by Nab2 orthologues to these processes in higher Eukaryotes.In summary, a combination of functional and structural studies has provided a wealth of insight into the way in which Nab2 regulates mRNA poly(A) tail length, contributes to mRNA compaction and the integration of nuclear steps in the gene expression pathway with nuclear export in"}
+{"text": "A small amount of Al doping into the HfO2 film can stabilize the tetragonal phase of the HfO2, which helps to achieve a higher dielectric constant (k) and lower leakage current density, as well as a higher breakdown voltage than HfO2 film on its own. Moreover, assimilation of Al2O3 into HfO2 can reduce the hysteresis width and frequency dispersion. These are indications of border trap reduction, which was also verified by the border trap extraction mechanism. X-ray photoelectron spectroscopy (XPS) analysis also verified the HfAlO microstructural properties for various Al compositions. In addition, higher amounts of Al2O3 in HfAlO resulted in better interface and dielectric behavior through trap minimization, although the equivalent-oxide-thickness (EOT) values show the opposite trend.This study presents the characteristics of HfAlO films for a series of Al incorporation ratios into a HfO Inte2 film-based MOSCAPs along with Al2O3 and HfO2 ones. The C\u2013V hysteresis was measured by performing the sweep from inversion to accumulation and (without any delay) again sweeping back to inversion at a frequency of 1 MHz to exclude the response of interface traps. It is obvious from the figure that Al incorporation reduces the hysteresis of HfO2 due to the charges trapped at the oxide/semiconductor interface, as well as inside the dielectric. This reduction can be explained as follows, Al2O3 exhibits micromolecular properties, while TMA has high reactivity, which allows the pinholes of HfO2 to be filled with Al ions and promotes formation of a more condensed film during ALD deposition  and sample H [3:3] showed higher breakdown voltages as they have higher thicknesses and greater Al content based on deposition cycle design.2O3 and HfO2 stacks. Lower Al incorporation increased the dielectric constant of HfO2 film though phase transition, while relative permittivity decreased with an increasing Al2O3 amount. Furthermore, leakage current decreased with breakdown voltage augmentation by Al2O3 assimilation since it provides better trap minimization and a bandgap increase for the pure HfO2 film. Both the border trap and interface traps were better minimized with an increased amount of Al2O3 in the HfAlO alloy, and a similar trend was observed in hysteresis and frequency dispersion analyses. In almost all cases, the HfAlO alloy showed behavior within the range set by single Al2O3 and HfO2 films. The behavior of the HfAlO alloy with respect to the Al: Hf ratio was verified, along with that of the single Al"}
+{"text": "Pakistan is among leading countries of world in prevalence of chronic hepatitis C Daclatasvir plus sofosbuvir is recommended for treatment of CHC. The purpose of study was to determine the sustained virological response in patients with chronic viral hepatitis C genotype 3a irrespective of previous treatment experience or presence of liver cirrhosis.Open label observational study was conducted at ABSTH Gujrat from January 2017 to April 2018 using non-probability purposive sampling. Patients chronically infected with hepatitis C virus having genotype 3a irrespective of presence of cirrhosis or previous treatment experience were included. Treatment naive patients without cirrhosis were given 12 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg. Patients with either compensated cirrhosis or treatment experienced were given 24 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg with weight based ribavirin. Data analysis was done using SPSS 20.024 was available for 101(80.8%) patients out of which 48 were having cirrhosis and 53 were without cirrhosis. SVR24 was achieved by 96 patients (95%). Virological response was better in treatment naive patients and without cirrhosis compared to treatment experienced and those with cirrhosis.Total 125 patients were included in study out of which 42 (33.6%) were male and 83 (66.4%) were female. Early virological response and end treatment response was achieved by 124 (99.2%) patients. Twenty four patients were lost to further follow-up and SVRDaclatasvir plus sofosbuvir is an effective combination in patients with chronic hepatitis C genotype 3a infection. Chronic hepatitis C affects 71 million people globally according to WHO estimates.5Decreased prices of DAAs and advent of generics has boosted up the efforts in treatment of chronic hepatitis C in Pakistan. Pakistan along with Egypt had half of people starting DAAs for treatment of chronic hepatitis C in the world during 2016Twelve week regimen of once daily daclatasvir plus sofosbuvir has showen good results in patients with HCV genotype 3 with sustained virological response of 91%.9Combination of daclatasvir and sofosbuvir is recommended for genotype 3 patients according to AASLD 2017 guidelines having a strong evidence. Recommended duration is 12 weeks in patients without cirrhosis and 24 weeks in patients with cirrhosis when weight based ribavirin is added to the regimen irrespective of previous peglated interferon plus ribavirin treatment.11There are no studies previously published in Pakistan regarding efficacy of daclatasvir plus sofosbuvir in patients with hepatitis C genotype 3a up to best of our knowledge. As this is an effective regimen its efficacy should be evaluated in a population having high incidence of infection. Thus the purpose of study was to determine the sustained virological response in patients with chronic viral hepatitis C genotype 3a irrespective of previous treatment experience or presence of liver cirrhosis.This open label observational study was conducted at Aziz Bhatti Shaheed Teaching Hospital Gujrat from January 2017 to April 2018. Patients were included in study after informed consent and approval of ethical committee of hospital. Patients chronically infected with hepatitis C virus having genotype 3a irrespective of presence of cirrhosis or previous treatment experience with interferon plus ribavirin were included. Patients were given this regimen due to free availability of DAAs in government setups and those who could not afford the first line regimen i.e combination of Velpatasvir plus Sofosbuvir.Presence of chronic hepatitis C was confirmed with baseline quantitative PCR testing and a value of > 15 ng/ml was considered positive. Genotype testing was done for every patient by University of Gujrat Laboratory free of cost and those with genotype 3a were selected using non-probability purposive sampling. Patients having liver cirrhosis were confirmed by abdominal ultrasound done by consultant radiologist and presence of coarse echotexture of liver was considered as liver cirrhosis. Although Shear Wave Elastography and Fibroscan are recommended but non-availability of these modalities led us to rely on ultrasound for presence of cirrhosis. Severity of liver disease was assessed using Child Pugh Score and patients with score 5-6 were defined as Child Class A, 7-9 as Child Class B and 10-15 as Child Class C. Patients with Child class A and B were included while those having Child class C were excluded from study.Treatment experienced patients were further divided in two groups. Patients who did not respond to 24 weeks interferon plus ribavirin were classified as non-responders while those who had a positive PCR after achieving ETR with 24 weeks interferon plus ribavirin treatment were classified as relapsers.24) 24 weeks after completion of treatment.Treatment naive patients without cirrhosis were given 12 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg. Patients with either compensated cirrhosis (Child Class A & B) or treatment experienced were given 24 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg with weight based ribavirin. PCR was done to assess the treatment response at four weeks of treatment , at end of 12 or 24 weeks treatment (End Treatment Response or ETR). Primary end point of study was to determine sustained virological response  were male and 83 (66.4%) were female. Mean age was 47.06 + 10.8 years. 102 (81.6%) patients were treatment naive, 7 (5.6%) treatment non-responders and 16 (12.8%) were relapsers. Fifty eight (46.4%) patients had cirrhosis including 43 treatment naive, 4 non-responders and 11 relapsers patients. Fifty patients with cirrhosis had Child Class A while 8 patients had Child Class B.24 was available for 101(80.8%) patients out of which 48 were having cirrhosis and 53 were without cirrhosis. SVR24 was achieved by 96(95%) patients out of 101. Patients who did not achieve SVR24 included both treatment naive and experienced patients and patients with and without cirrhosis. SVR24 in patients with cirrhosis were 91.66% while in those without cirrhosis were 98.11%. 97.65% treatment naive patients achieved SVR24 however only 90.9% relapsers and 60% non-responders achieved SVR24. Results are depicted in Early virological response (EVR) and end treatment response (ETR) was achieved by 123(98.4%) and 124 (99.2%) patients respectively. Taking in account treatment history; treatment naive, relapsers and non-responders achieved EVR of 99%, 100% and 85.7% and ETR was achieved by 99%, 100% and 100% respectively. One fifth of patients (24) were lost to further follow-up who were alive and did not report back despite multiple reminders. SVR24 rates were more in patients without cirrhosis than as compared to cirrhosis and treatment naive patients as compared to treatment experienced patients. However no statistical significance of cirrhosis and treatment history were found.This study shows that combination of daily daclatasvir plus daily sofosbuvir with or without weight based ribavirin is highly effective in Pakistani population with HCV genotype 3a and independent of presence of cirrhosis or previous treatment with interferon plus weight based ribavirin. SVRResults of this study are comparable to ALLY 3 phase III study by Nelson et al.Patients with compensated cirrhosis has good treatment response in this study. 98.3% patients with compensated cirrhosis achieved EVR and ETR. ALLY 3+ study also determined the role of daclatasvir and sofosbuvir in patients with compensated cirrhosis and resulting in 83.3% patients achieving treatment response at week 4 and 100% at end of treatment.24 rates as compared to treatment naive and without cirrhosis. Alonso et al. also found a high SVR rates (94%) among patients with HCV genotype 3 infection treated with sofosbuvir plus daclatasvir24 = 95%).Welzel et al. conducted a study about efficacy of daclatasvir plus sofosbuvir with or without ribavirin in HCV patients. HCV RNA was undetectable in 73% patients at week 4, 97% at week 12 and >99% at week 24. However they included all genotypes of HCV. In genotype 3, 92% patients achieved SVR which was minimally less in treatment experienced patients having decompensated cirrhosis.Mehta et al. conducted a study in HCV genotype 3 patients in India using combination of sofosbuvir plus daclatasvir and 97.3% patients achieved SVR showing it to be highly effective regimen in genotype 3Study by Ferrieria et al. showed lower SVR rates (84.7%) in patients with genotype 3 taking sofosbuvir plus daclatasvir regimen which do not augment results of this study. However they found out no significant association of presence of cirrhosis or treatment experience with achieving SVR which are consistent with results of this study.15This is perhaps first study in Pakistan determining the role sofosbuvir plus daclatasvir in treatment of chronic hepatitis C genotype 3a infection. Although SVR at 12 week is considered the end point in many studies, SVR at 24 weeks was focused in this study. However 19.2% patients lost to follow up which shows a need for a free of cost screening, treatment and follow up program for treatment of hepatitis C at national level owing to high prevalence of chronic hepatitis C Pakistan.Study showed good outcomes in treatment of hepatitis C genotype 3a which is prevalent genotype in Pakistan.Daclatasvir plus sofosbuvir is a highly effective combination in patients with chronic hepatitis C genotype 3a infection in Pakistani population and independent of treatment history and presence of cirrhosis.ZB: Conceived the study, collected data and did final review and approval of manuscript.SMAS: Planned the study, did statistical analysis and wrote manuscript."}
+{"text": "Does an association exist between the sponsorship and conduct of phase 3 randomized clinical trials for cancer drugs despite negative or absent phase 2 trials for the drug, and does an association exist for overall patient survival and such phase 3 trials?This analysis of 67 studies found that both industry and academia conducted negative phase 3 trials of cancer drugs. No association was found between trial sponsorship and lack of a positive phase 2 trial; there was no association with patient overall survival, although the survival hazard ratio was greater than 1 for 37% of such trials.Phase 3 trials conducted without supporting phase 2 trial evidence risks loss of resources owing to trial failure and may be associated with decreased patient survival. Only 3.4% of cancer drugs evaluated in phase 1 trials are approved by the US Food and Drug Administration, with most failing in phase 3 trials.To investigate whether an association exists between the sponsorship and conduct of a negative phase 3 randomized clinical trial (RCT) investigating a cancer drug that lacked supporting phase 2 trial evidence for that drug, and to evaluate the association with overall survival among patients randomized to the experimental arm of such phase 3 trials.Lancet, Lancet Oncology, JAMA, JAMA Oncology, and Journal of Clinical Oncology published between January 2016 and June 2018 were searched.Articles in the Phase 3 RCTs of cancer drugs that failed to improve the primary end point were selected and any prior phase 2 trial of the same drug that supported the phase 3 trial was selected without any date or journal restrictions.Percentages of negative phase 3 RCTs of cancer drugs that lacked any phase 2 evidence, had a negative phase 2 trial, or had a positive phase 2 study were extracted. Associations were assessed using the Fisher exact test. Pooled hazard ratios and 95% CIs for the overall survival of patients enrolled in these negative phase 3 RCTs were estimated using a random-effects model.Negative phase 3 RCTs with a lack of a phase 2 trial or the presence of a negative phase 2 trial and overall survival of enrolled patients in the phase 3 RCTs.In this meta-epidemiological study, 67 negative phase 3 RCTs on cancer drugs, which included 64\u2009600 patients, met the criteria of being sponsored by industry or academic groups, of which 42 RCTs (63%) were industry sponsored and the remaining 25 RCTs (37%) were academic. A phase 2 trial was not available for 28 of these trials (42%). Of 29 trials (43%) with a phase 2 trial available, 8 trials (28%) failed to meet their primary end points and 5 of those were industry sponsored. There was no association with overall survival for patients participating in these negative phase 3 RCTs . When the pooled analysis was limited to the 27 RCTs with a hazard ratio above 1.00, the overall pooled hazard ratio for overall survival was 1.11 . No association between having a negative or undefined phase 2 trial and trial sponsorship was found using the Fisher exact test.More than 40% of the negative phase 3 RCTs in oncology published in these 5 journals were conducted without a supporting phase 2 trial and were sponsored by both academia and industry. Running such trials not only may risk loss of resources owing to a failed trial but also may be associated with decreased patient survival. Further research and regulations in this area appear warranted. This study assesses whether an association exists between the sponsorship and conduct of a phase 3 randomized clinical trial investigating a cancer drug and the lack of supporting phase 2 trial evidence for that drug. Phase 3 RCTs usually involve more patients and resources than earlier phases and are costly undertakings. According to a 2014 report, the cost of a cancer drug trial was $22.1 million, $11.2 million, and $4.5 million, for a phase 3, phase 2, and phase 1 trial, respectively.2 Thus, a drug failing the phase 3 trial  is an undesirable outcome, even from an economic point of view.Phase 3 randomized clinical trials (RCTs) are the final barrier in establishing the efficacy of cancer drugs. One study estimates that only 3.4% of cancer drugs being evaluated in phase 1 trials are approved by the US Food and Drug Administration, with most (35.5%) failing in the phase 3 stage when all indications are considered.3 Second, a trial involves significant investment of time and logistics from health care professionals and patients. Third, a failed phase 3 trial also means unmet patient expectations. Thus, although negative RCTs can provide us with important knowledge for future research and patient care, precautions can be taken to minimize the frequency of negative RCTs, especially if they can be avoided. The US Food and Drug Administration resource \u201cInformation for Consumers (Drugs)\u201d states that \u201cif the phase 2 trials indicate that the drug may be effective\u2014and the risks are considered acceptable, given the observed efficacy and the severity of the disease\u2014the drug moves to phase 3.\u201d4 Another document on the clinical review of investigational new drug applications also states that a phase 2 trial review is conducted on completion before initiation of phase 3 trials, during which questions such as whether sufficient data are available to plan a phase 3 program are asked.5 However, in a previous study, our group discovered that less than 20% of the negative phase 3 RCTs on cancer had a supportive phase 2 trial.6 One hypothesis for such a practice is that it is financially lucrative for the industry to continue to test compounds that have low probability of success in a phase 3 trial because even a chance finding of a positive outcome will offset all associated costs.7 However, associations of sponsors of such negative phase 3 RCTs with phase 2 trial outcomes or the association of such negative phase 3 RCTs on patient survival has not been studied systematically to our knowledge.However, the larger concern is the effect on human resources. First, patient resources for trial enrollment are scarce given that only 3% of patients with cancer in the United States participate in trials and that nearly 60% of phase 3 trials fail to achieve minimum patient enrollment.In the present study, using a larger data set of negative phase 3 RCTs, we investigated whether an association existed between the sponsorship of negative phase 3 RCTs and the lack of supportive prior phase 2 trial evidence. We also studied what proportion of the industry-sponsored negative phase 3 RCTs had a history of the experimental drug being acquired or licensed from a small pharmaceutical company . We hypothesized that such an arrangement might motivate the sponsor to pursue a phase 3 RCT to recoup the costs of the investment. Finally, we investigated whether there was an association with overall survival (OS) among patients who were enrolled in these negative phase 3 RCTs.PRISMA) reporting guidelines for meta-epidemiological studies.8 We searched the top oncology journals based on their impact factor  and their parent journals (Lancet and JAMA) between January 2016 and June 2018 for any negative phase 3 RCTs of cancer drugs. We excluded the RCTs that did not involve a cancer drug in any arm, such as RCTs of radiotherapies or surgical procedures in both the arms. We extracted the hazard ratio (HR) and 95% CI for OS for each of the included trials, where available. We also categorized the sponsors of these negative phase 3 RCTs into academic (cooperative groups) or industry based on the posted ClinicalTrials.gov registration information. We then searched the literature and meeting abstracts for any prior phase 2 trial of the same drug that supported the phase 3 trial. We categorized these findings into yes, no, and not applicable (NA). Not applicable was applied to drugs that had already been approved for later lines of therapy or in an advanced setting but the phase 3 RCT tested the drug in an earlier or curative setting. For those trials categorized as yes for any prior phase 2 trial, we categorized the outcomes of the phase 2 trials into positive, negative, or inconclusive based on whether the primary end point of the phase 2 trial was met, unmet, or unstated, respectively. Finally, for industry-sponsored trials, to find out if the drug was purchased or licensed from a small pharmaceutical company , we used search terms in Google  and then reviewed the news article or press release for details, if any, regarding a transaction or business arrangement or deal with another pharmaceutical company or entity. All study and data extractions as well as categorizations were made by 2 of us (A.A. and G.W.) and rechecked by the third author (B.G.). Any discrepancies were resolved by consensus among the 3 authors.This study was conducted based on modified Preferred Reporting Items for Systematic Reviews and Meta-analyses , and a 2-sided 75 which included 64\u2009600 patients, that met our criteria for inclusion, of which 42 RCTs (63%) were industry sponsored and the remaining 25 RCTs (37%) were academic  of these negative phase 3 RCTs, it was available for 29 (43%) of them, and it was not applicable for 10 (15%) of them. Excluding the 10 nonapplicable trials, the availability of a phase 2 trial was not associated with negative phase 3 trial sponsorship . Of the 29 trials in which a phase 2 trial was available, 8 trials  failed to meet the primary end point, whereas the phase 2 trial was inconclusive in 5 cases (17%). The phase 2 trial met its primary end point and was considered positive in 16 trials . There was no association between having a negative or undefined phase 2 trial and phase 3 trial sponsorship .We found 67 negative phase 3 RCTs,9 The other was the RCT of lapatinib in bladder cancer, which was also tested in a biomarker-selected population (ERBB1/2 [formerly HER1/2] positive) in phase 3, despite a negative phase 2 trial in an unselected population.34 Of the 5 industry-sponsored trials that were conducted despite a negative phase 2 trial, 4 trials were associated with purchase or licensure of the molecule from a small pharmaceutical company.Of the 8 RCTs that were conducted despite a negative phase 2 trial, 5 were industry sponsored. Two of the 3 academic phase 3 RCTs were conducted despite a negative phase 2 trial because the phase 2 trial showed beneficial effects in a subgroup of biomarker-positive patients. One was the Alliance CALGB 30801 trial of celecoxib in non\u2013small cell lung cancer with cyclooxygenase-2 overexpression that was conducted despite a negative phase 2 trial because cyclooxygenase-2 overexpression was supposed to be a predictive biomarker.36 the overall pooled HR for overall survival was 1.11   with no subgroup differences by trial sponsorship or by phase 2 trial results. However, the HR was greater than 1.00 in 27 RCTs (46%). When the pooled analysis was limited to these 27 RCTs,06-1.16) .Of the negative phase 3 RCTs of cancer drugs, 42% did not have phase 2 trial evidence and only 28% had a positive phase 2 trial. Nearly 14% of negative phase 3 RCTs were conducted despite a negative phase 2 trial. The availability of a phase 2 trial or whether or not the phase 2 trial was positive was not associated with trial sponsorship, but of the 5 industry-sponsored negative phase 3 RCTs, 4 were associated with drug purchase or licensure from a small pharmaceutical company. Patients who were enrolled in the negative phase 3 RCTs were not associated with poorer OS although when limited to 46% of negative phase 3 RCTs reporting an HR greater than 1.00, the pooled HR showed a significant increase in mortality among the participants randomized to the experimental arm of the trials.There are several hypotheses as to why a cancer drug may be tested in a phase 3 RCT despite having no or even negative phase 2 trial evidence. Among industry-sponsored trials, we found that acquisition of the investigational drug from small pharmaceutical companies could be one factor. Financial milestones among the small pharmaceutical companies to move a compound from phase 2 to phase 3 trials can influence decisions. However, in the case of academia-sponsored trials, a belief in the biomarker-based subgroup analysis of phase 2 trials seemed to be a motivation for conducting a larger phase 3 trial in the biomarker-enriched subgroup population.76Negative phase 3 RCTs are not futile or meaningless; they are important to advance our understanding of disease and drugs. Nevertheless, because of the financial, human, logistic, and time resources needed to conduct a phase 3 RCT and the patients\u2019 expectation of therapeutic benefit, phase 3 RCTs should be conducted only when there is some prior evidence to suggest a potential therapeutic benefit. If a cancer drug has failed to meet its primary end point in a phase 2 trial, there is neither therapeutic equipoise nor rationale to test it in a phase 3 RCT, and we question if it is against the ethical norms of participating in a phase 3 RCT. Institutional review boards and ethical committees should take a proactive step in discouraging the conduct of such phase 3 RCTs of cancer drugs that have already failed in phase 2 trials. Indeed, in a recent editorial, the US Food and Drug Administration questions the practice of conducting phase 3 RCTs of cancer drugs known to have poor activity in phase 2 trials.We included only those negative phase 3 RCTs published in journals with high impact factors; thus, our findings should be considered conservative. Indeed, our results reflected the best-case scenarios of the negative phase 3 RCTs, and the results would likely be worse for the negative phase 3 RCTs published elsewhere or not published at all. However, our search for phase 2 trials was conducted comprehensively without any journal or time restrictions. Another limitation of our study was the relative difficulty in accessing information on purchase or licensure of a drug from small pharmaceutical companies. Lack of a control group of positive phase 3 RCTs was another limitation; however, our objectives were to assess the association of negative phase 3 RCTs with sponsorship as well as to assess the association of negative phase 3 RCTs with patient survival, and therefore for our purpose, the cohort of negative trials alone sufficed. Furthermore, even though the trials were categorized into groups based on sponsorship, most trials will involve both industry and academia collaboration. We also acknowledge that in some instances of extraordinary responses in phase 1, it may be more efficient to take a drug directly to a phase 3 RCT; however, the activity and criteria for success (taking to a phase 3 RCT) must be prospectively defined in such early-phase trials.In this study of trials published in 2016 through 2018, approximately 40% of negative phase 3 RCTs in oncology were conducted without supporting phase 2 trials, and such phase 3 trials were sponsored by both academia and industry. On the basis of our results, proactive steps from regulators and ethical committees should be contemplated to encourage greater consideration before allowing conduct of phase 3 RCTs despite negative results from phase 2 trials in the interest of protecting patients and trial resources."}
+{"text": "BRCA1/2 mutations to familial breast cancer in Bahrain has not been explored. The objective of this study was to investigate the spectrum of BRCA1/2 genetic variants and estimate their frequencies in familial breast cancer. We also aim to test the efficiency of the next\u2010generation sequencing (NGS) as a powerful tool for detecting genetic variation within BRCA1/2 genes.Breast cancer is the most common malignancy in women worldwide. About 5%\u201310% are due to hereditary predisposition. The contribution of BRCA1/2 variants. All targeted coding exons and exon\u2013intron boundaries of BRCA1/2 genes were amplified with 167 pairs of primers by NGS.Twenty\u2010five unrelated female patients diagnosed with familial breast cancer were screened for BRCA1/2 variants in two patients, one in BRCA1 gene (c.4850C>A) and other in BRCA2 gene (c.67+2T>C). In addition to the deleterious variants, we identified 24 distinct missense variants of uncertain significance, 10 of them are seen to confer minor but cumulatively significant risk of breast cancer.We have identified two deleterious BRCA1/2 variants may contribute to the pathogenesis of familial breast cancer in Bahrain. It also shows that NGS is useful tool for screening BRCA1/2 genetic variants of probands and unaffected relatives.Our data suggest that  These tumor suppressor genes were reported and identified as breast cancer susceptibility genes for the first time in 1990 and 1995 and are located on chromosome 17q21 and 13q12\u201013, respectively  database  and Kuwait (46.6)  and to test the efficiency of the NGS in the diagnosis and screening programs of breast cancer patients in the region. The overall objective was to better understand the genetic risk factors associated with the disease which could eventually lead to an earlier detection with better prognosis and survival rates.In Bahrain, the breast cancer screening by mammography was introduced in 2005 and essentially subjected to high\u2010risk suspected cases for women aged 40 and above. The screening program since introduced has contributed to the diagnosis of 12.7% of the cases .Genomic DNA was isolated from 200\u00a0\u00b5l peripheral blood anti\u2010coagulated with EDTA on the MagNa Pure LC instrument using MagNa Pure LC DNA Isolation Kit , according to the manufacturer's instructions. The concentration of DNA was determined by using the Nano Drop2.4BRCA1/2 genes were amplified with 167 pairs of primers in three primer pair pools. After the targeted amplification and construction of a library through Ion AmpliSeq\u2122 Library Kit 2.0, the nucleotide sequences of the targeted regions are analyzed by Life Technologies Ion PGM platform on an Ion 316 Chip. Sequence variants are identified by Torrent Suite 4.2 . The clinical significance of each sequence variant is suggested on the basis of reference to ClinVar and BIC .The reference sequences used were NM_007294.3 for 33.1BRCA1/2 genetic variants, we screened BRCA1/2 variants in the 25 recruited patients with personal and family history of breast cancer. All women were diagnosed with unilateral breast cancer. The median age at diagnosis of breast cancer was 47\u00a0years (range 33\u201363). DNA was extracted from patient's peripheral blood samples. We amplified targeted coding regions and exon\u2013intron boundaries of BRCA1/2 genes using multiplex PCR. A total of 167 primer pairs were used in three primer pools covering 16.25\u00a0KB of target genomic sequence. Targeted genomic sequencing was performed using the Ion Torrent PGM System from Life Technologies generating short sequence reads of approximately 200\u00a0bp. We have identified two deleterious BRCA1/2 variants in two patients, one in BRCA1 gene (c.4850C>A) and other in BRCA2 gene (c.67+2T>C). In addition to the deleterious variants, we have identified 24 missense variants of uncertain significance (VUS).To analyse the prevalence of breast cancer with 3.2BRCA1 variant c.4850C>A was diagnosed with right breast cancer at age 33 and had a positive family history of breast cancer . There was no family history of ovarian cancer. Patient presented with right breast lump noticed 2\u00a0days prior to presentation that radiological investigations did not reveal any breast lesions or distant metastasis; however, fine needle aspiration reported malignancy.The carrier of The tumor was classified as a stage 2 loco\u2010regional malignancy, and the histopathology results confirmed the diagnosis of an invasive ductal carcinoma grade 3, measuring 23\u00a0mm in maximum dimension. Two palpable mobile axillary nodes were observed during examination. The patient underwent wide local excision of right breast tumor and right axillary clearance.3.3This is a Bahraini woman, known case of hypertension and hyperlipidemia with history of abdominal hysterectomy and bilateral oophorectomy for endometrial cancer 9\u00a0years prior to her presentation. The patient has a very strong family history of breast cancer including her mother and maternal aunt (diagnosed before age 50). She has presented with right axillary swelling and right arm pain. At investigation, a deep right breast lump was observed occupying the upper outer quadrant. Two palpable metastatic axillary nodes were observed during examination. The tumor was classified as a stage 2 loco\u2010regional malignancy, and the histopathology results confirmed the diagnosis of an invasive papillary carcinoma (grade 2), measuring 4\u00a0cm in maximum dimension. The patient underwent right mastectomy and axillary clearance.3.4BRCA1/2 genes of the 25 patients revealed two deleterious variants in two unrelated patients with total frequency of 8% (2/25), one within BRCA1\u00a0and other within BRCA2   which uses sequence homology to predict whether an amino acid substitution will affect protein function and hence, potentially alter phenotype to the missense substitutions associated with the disease. SIFT predicted 42% of the detected 24 variants to be damaging. The SIFT algorithm and software have been described previously  is identical to the figures previously reported for the Arab population, a decade earlier than western countries  or missense alterations at critical residues in functional domains, we defined one BRCA1 c.4850C>A and one BRCA2 c.67+2T>C as deleterious variants in two patients. Interestingly, both patients showed a very strong family history of breast cancer suggestive of genetic predisposition to breast cancer. The two deleterious variants were observed in two unrelated patients out of the 25 subjects studied providing an overall prevalence rate of 8%. Some studies conducted in other Arab countries such as Tunis and Saudi Arabia have reported frequencies of 16%\u201325% . BRCA1 gene contains 22 exons spanning about 110\u00a0kb of DNA and encoding a 1863 amino acid protein with an N\u2010terminal RING finger domain and two BRCT\u2014 domains. The identified BRCA1 nonsense variant c.4850C>A was predicted to be causative because it creates a premature stop codon at amino acid 1617 leading to premature truncated protein. To the best of our knowledge, the BRCA1 variant c.4850C>A was not reported previously elsewhere except in a recent study conducted in Qatar where two patients were found to be positive carriers for the c.4850C>A variant  including distinct polymorphisms and unclassified sequence variants.Additionally, we have identified 24 sequence variants (11 BRCA1 SNPs (rs1799950 (4%), rs16942 (64%), rs4986850 (20%), rs2227945 (16%), and rs1799966 (64%)) and 5 BRCA2 SNPs (rs766173 (12%), rs144848 (60%), rs4987117 (4%), rs4987047 (8%), and rs11571833 (4%)) are found to confer minor but cumulatively significant risk of breast cancer  and other in BRCA2 gene (c.67+2T>C). The identified BRCA1 variant c.4850C>A has been reported in a Qatari study and in this report only suggesting that it might be novel to the gulf region. We also showed that NGS is a useful tool to explore mutations in BRCA1 and BRCA2 genes and can be used to screen mutations in probands and unaffected relatives from the same family.In conclusion, this is the first report on the breast cancer predisposition factors in the population of Bahrain and our data suggest that The authors declare that there is no conflict of interest regarding the publication of this article.FA conceived the study, participated in its design and coordination, carried out the molecular genetics studies, analyzed the data, and drafted the manuscript. MK arranged for the study funding, participated in the study coordination, and revised the manuscript. ST supervised the sequencing experiment and revised the manuscript. LA looked after patients' recruitment, gathering the clinical data, and revised the manuscript. All the authors read and approved the final manuscript."}
+{"text": "We previously reported the identification of monocarboxylate transporter 4 (MCT4) and glypican-3 (GPC3) as prognostic factors for hepatocellular carcinoma (HCC), which are now considered significant poor prognostic factors for the disease. This study aimed to clarify the detailed interaction of these two factors in HCC to improve our understanding of aggressive HCC phenotypes. A total of 225 Japanese patients with HCC from our previous study were subjected to immunohistochemical analyses.The number of MCT4-positive (MCT4+) HCC cases was 47 (21%), and most MCT4+ HCC showed high GPC3 expression . In 44 MCT4+/GPC3+ HCC cases, intratumoral heterogeneity of GPC3 or MCT4 expression was further evaluated. We observed reciprocal (inverse), synergistic, mixed reciprocal and synergistic, or irrelevant interaction of MCT4 and GPC3 expression in 29 (66%), 5 (11%), 1 (2%), and 9 cases (21%), respectively. The cases exhibiting reciprocal expression of both markers tended to have cirrhosis without a history of neoadjuvant therapy. In summary, although MCT4+ HCC cases are mostly GPC3+, intratumoral expression patterns of MCT4 and GPC3 are frequently reciprocal each other, suggesting that dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC cases. Liver cancer is the leading cause of cancer death worldwide and is the second leading cause of cancer death in men . As hepaWe previously reported that MCT4+ HCC cases were mostly GPC3 positive [The eligible cases included 225 Japanese patients  with HCC who had underwent partial hepatectomy in the University of Miyazaki Hospital from February 1999 to October 2012. The patients included in this study were the same as in our previous report , as wereSerial sections, which were prepared from formalin-fixed, paraffin-embedded HCC blocks of 225 cases, were used for hematoxylin and eosin staining and MCT4 and GPC3 immunohistochemistry  as the pAs reported in our previous studies , 4, we dp\u2009<\u20090.05. Data were analyzed by StatView 5.0 .Fisher\u2019s exact test or the Chi-square test was used for assessment of the relationship between variables. Statistical significance was assumed if MCT4+ HCC and GPC3+ HCC were immunohistochemically identified in 21% (47 cases) and 84% (190 cases) of the 225 cases, respectively. The mean positive area of MCT4 and GPC3 was 20%  and 72% , respectively. MCT4+/GPC3+, MCT4+/GPC3-negative (GPC3\u2212), MCT4-negative (MCT4\u2212)/GPC3+, and MCT4\u2212/GPC3\u2212 HCCs were observed in 44, 3, 146, and 32 cases, respectively , we statistically analyzed the correlation between clinicopathological variables of the reciprocal HCC cases (29 cases) and those of the non-reciprocal ones   of MCT4+ HCC cases in our cohort showed GPC3 positivity, and nearly 80% of MCT4+ HCC cases exhibited reciprocal or synergistic expression pattern between MCT4 and GPC3. Thus, the expression of MCT4 in HCC cells might be influenced by GPC3 expression and vice versa. Of note, 68% of MCT4+/GPC3+ HCC cases demonstrated reciprocal interaction of both markers. These findings may provide a novel therapeutic approach for MCT4+ HCC; dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC.In this study, we used the custom-made anti-GPC3 antibody GC33, which is a mouse monoclonal antibody that recognizes human GPC3. Humanized GC33 (codrituzumab) may serve as a treatment option for HCC because it has a significant antitumor activity to HCC cells in vivo via antibody-dependent cellular cytotoxicity , 20. We GPC3 is silenced partly by promoter hypermethylation in some cancers [GPC3 transcription in HCC may be suppressed by transcription factor zinc fingers and homeoboxes 2 (ZHX2), a well-known repressor of the GPC3 gene [The mechanism of reciprocal interaction of MCT4 and GPC3 in HCCs remains unknown. In the tumor areas showing reciprocal interaction of MCT4 and GPC3, MCT4 was likely induced by the hypoxic tumor microenvironment because MCT4+ HCC cells were observed primarily in the central portions of tumor nests distant from the tumor vessels. In fact, we previously showed that MCT4+ HCC cells were present near necrotic portions, and those tumor cells tended to be positive for the hypoxia marker carbonic anhydrase IX [ cancers , 22, and cancers . AlternaPC3 gene , 25, in Although the reciprocal pattern was predominant, 11% of the cases showed a synergistic expression pattern of MCT4 and GPC3. The mechanism underlying the synergistic interaction of MCT4 and GPC3 in HCC also remains unclear. In the areas of tumors showing synergistic interaction of MCT4 and GPC3, concomitant cell surface immunoreactivities of MCT4 and GPC3 were observed as reported previously , suggestBased on statistical analysis, the reciprocal interaction of MCT4 and GPC3 tended to be observed in non-treated HCCs derived from cirrhosis. Thus, severe cell damage induced by adjuvant therapy in HCC may disturb the reciprocal relationship and may result in a synergistic or irrelevant interaction of MCT4 and GPC3; however, this hypothesis remains highly speculative.In conclusion, we immunohistochemically explored the intratumoral relationship between MCT4 and GPC3 expression in HCC. Although the mechanism underlying the interaction of these molecules in HCC is currently unknown, the observed phenomena may have implications in the development of therapeutic strategies targeting MCT4 and GPC3 in HCC.Expression of MCT4 and GPC3 in HCC cells was immunohistochemically evaluated using formalin-fixed, paraffin-embedded HCC tissue blocks from 225 cases. In each case, one tumor block was randomly selected from the maximal section of the tumor. The expression status of MCT4 and GPC3 was not evaluated in whole tumor sections.Prognostic differences between patients with \u201creciprocal\u201d and \u201cnon-reciprocal\u201d HCC could not be statistically evaluated owing to the small sample size.With respect to the expression regulation mechanism of MCT4 and GPC3 in HCC cells, we could not sufficiently explain the mechanism with this morphological study.Additional file 1. Clinicopathological data of patients with HCC.Additional file 2. Intratumoral expression patterns of MCT4 and GPC3 in 44 cases of MCT4+ GPC3+ HCC."}
+{"text": "Figure 3C on page 7 of this paper erroneously presented two identical western-blot panels (GRP78 and HSPA8).GRP78 and HSPA8 western-blot panels as originally intended.The corrected The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "The pristine and modified Li4Ti5O12 electrodes are characterized at an extended voltage range of 3\u20130.01 V. The reversible capacity reaches a high level of 286 mAh g\u22121, which is a little less than its theoretical capacity (293 mAh g\u22121). Electrodes modified by ZnO thin films with various thickness show elevated rate capability and improved cycle performance.Oxide is widely used in modifying cathode and anode materials for lithium-ion batteries. In this work, a facile method of radio magnetron sputtering is introduced to deposit a thin film on Li The calculated data agree with the measured data very well when the inserted equivalent circuit (as shown in figure 5b) is employed to fit the data. Both the measured plots consist of two compressed semicircles and an inconspicuous oblique tail. The high-frequency semicircle is related to the resistance of SEI film formed on the electrode at a low voltage of 0.7 V. The intermediate frequency semicircle is attributed to the charge transfer resistance between the interface of active materials and electrolyte. The oblique line is associated with the Li-ion diffusion within the active materials. The fitted values of Rs of the pristine and ZnO-coated Li4Ti5O12 composite electrodes are less than 5 \u03a9. The fitted value of the SEI film resistance (Rf) of ZnO-coated Li4Ti5O12 (5 min) composite electrodes is 101 \u03a9, which is much smaller than 364 \u03a9 for the pristine electrode. The fitted values of charge transfer resistances (Rct) are 224 and 1019 \u03a9 for pristine and ZnO-coated Li4Ti5O12 (5 min) composite electrodes, respectively. The reduced SEI film resistance and charge transfer resistance ensured elevated rate performance and stable cycle performance.The measured and calculated Nyquist plots are shown in figure 54.4Ti5O12 composite electrodes were successfully coated with ZnO thin films via a radio frequency magnetron sputtering method. The thickness of ZnO films was tailored by altering depositing time. A mixture layer of Zn-Li alloy and Li2O produced by ZnO coating film during the discharge process was suggested to improve the interface between electrode and electrolyte. As a result, the rate performance and cycle stability were greatly improved by ZnO coating layer.In summary, Li"}
+{"text": "In this study, we aimed to investigate the impact of the new recommendations on HER2 FISH interpretation in invasive breast cancers with immunohistochemically (IHC) equivocal results. 1810 breast cancer cases with IHC equivocal results were enrolled in this study between January 2012 and May 2019. Concomitant IHC was performed on the same tissue blocks detected by FISH testing. According to the 2018 guidelines, all the cases in ISH group 2 were categorized as HER2 negative; three of four cases in ISH group 3 were considered as HER2 positive, while the one scored IHC 1+ was reclassified as HER2 negative; Fifty-three previously ISH equivocal cases were redistributed into ten HER2-positive cases and forty-three HER2-negative cases. In conclusion, the utility of 2018 ASCO/CAP guidelines resulted in a slight decrease in HER2 positive rate, due to the reclassification of cases in ISH group 2 and group 4. The implementation of the new guidelines can reduce reflex FISH test and make the diagnosis of HER2 gene status more definitive.The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recently issued updated guidelines on human epidermal growth factor receptor 2 (HER2) testing by fluorescence  Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) is observed in about 15\u201320% of invasive breast cancer patients2. HER2 positivity is closely related to the poor prognosis of patients5. Clinical trials have demonstrated that breast cancer patients with HER2 overexpression can benefit from the targeted therapy, showing the progression-free survival and the overall survival improvement8. Therefore, accurate assessment of HER2 status is a prerequisite for identifying the subset of breast cancer patients who may benefit from the anti-HER2 targeted therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the two most frequently performed technologies to determine HER2 status.Breast cancer is the most common carcinoma and the second leading cause of cancer-related death among Chinese women9? The diagnostic approach incorporates concomitant IHC review into ISH interpretation in ISH groups 2 to 4 to reach the most definitive HER2 status classification.The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) have periodically issued and updated the HER2 testing guidelines in breast cancers, with first version released in 2007, first revised in 2013 and focused updated in 2018. The new guidelines have addressed five clinical questions, including the following: (1) What is the most appropriate definition for IHC 2+ ? (2) Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy? (3) Should invasive cancers with a HER2/chromosome enumeration probe (CEP17) ratio of \u22652.0 but an average HER2 copy number of <4.0 signals per cell be considered ISH positive (designated as ISH group 2)? (4) Should invasive cancers with an average HER2 copy number of \u22656.0 signals per cell but a HER2/CEP17 ratio of <2.0 be considered ISH positive (designated as ISH group 3)? (5) What is the appropriate diagnostic work \u2013up for invasive cancers with an average HER2 copy number of \u22654.0 but <6.0 signals per cell and a HER2/CEP17 ratio of <2.0 and initially deemed to have an equivocal HER2 ISH test result (designated as ISH group 4)In this study, we retrospectively reviewed HER2 status in 1810 breast cancer patients with equivocal IHC results to assess the impact of these revised guidelines.HER2 FISH results were collected from 1810 invasive breast cancer cases between January 2012 and May 2019. All cases were equivocal for HER2 IHC. Specimens included core needle biopsies, surgical excisions, and biopsy samples from metastatic sites. This research was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. The Committee waived the written informed consents for the data were analyzed anonymously. All experiments were performed in accordance with relevant guidelines and regulations.Automated IHC for HER2  was performed on 4-um-thick tissue sections using an automated slide stainer, the Ventana Benchmark XT . The results were interpreted according to the 2018 guidelines.4-um-thick tissue sections were deparaffinized, rehydrated, and immersed in distilled water for 40\u2009min at above 90\u2009\u00b0C. The slides were incubated for 18\u2009min in protease solution at 37\u2009\u00b0C. After dehydration with alcohol, a total of 10 uL HER2/CEP17 mixture probe  was added to the slides. The slides were then transferred to a hybridization oven . The procedure was as follows: denature at 83\u2009\u00b0C for 5\u2009min, and hybridization overnight at 42\u2009\u00b0C. The second day, the slides were washed in preheated post-hybridization buffer, air dried and then counterstained with 15\u2009\u03bcl DAPI. HER2 FISH signals were interpreted by one technologist (BW) and one pathologist (KS). Thirty nuclei from two non-overlapping areas were counted. When there was a conflict between the scores, another pathologist (XLW) would review the slide and reach the final result. The new guidelines were applied for the interpretation of FISH testing results.10, 318 cases with a HER2/CEP17 ratio \u22652.0 and \u22654.0 HER2 signals per cell (ISH group 1) were diagnosed as HER2 positive. 1406 cases with a HER2/CEP17 ratio <2.0 and <4.0 HER2 signals per cell (ISH group 5) were negative for HER2. There were 29, 4 and 53 cases in ISH group 2, 3 and 4 respectively. The positive rate was 19.40% (351/1810). Clinicopathological characteristics of the patients in each ISH group were summarized in Table\u00a0A total of 1810 cases were enrolled in our study. Most of the cases (n\u2009=\u20091478) were sampled from surgical excisions. Specimens from core needle biopsies (n\u2009=\u2009139) and metastatic sites n\u2009=\u200972) were much less common (Table\u00a0 were mucTwenty-nine 1.60%) cases fell into ISH group 2. Among them, twenty-five cases were sampled from surgical excisions, three from core needle biopsies, and one from lymph node metastasis (Table\u00a0.60% caseThere were only four (0.22%) cases in ISH group 3, which were composed of two specimens from surgical excisions, one from lymph node metastasis, and one from core needle biopsy Table\u00a0. Two casFifty-three 2.92%) of the 1810 cases fell into ISH group 4. Most of the cases n\u2009=\u200935) were sampled from surgical excisions, followed by 14 from core needle biopsies and 4 from metastatic sites  of the cases had a different final interpretation by 2018 guidelines compared with 2013 guidelines, including 30 previously considered HER2-positive cases being categorized as negative, 53 previously ISH equivocal cases being redistributed into 10 positive cases and 43 negative cases. In total, 331 of the 1810 cases were positive for HER2, with a slight decrease in positive rate from 19.4% to 18.3% 20. Another research showed that of 35 patients with this ISH pattern tested by IHC, only 3 cases were IHC 2+ and none of them were IHC 3+14. In parallel with these findings, our data also found that there was no case scored IHC 3+ in this group. So far as we know, only one case with HER2 IHC positive was reported in ISH group 221.The HER2 status in ISH group 2 is uncommon. Twenty-nine cases were included in this group, accounting for 1.60% of the present cohort , which was consistent with other reports22. A ratio of <2.0 can be attributed to the increase in both HER2 and control centromere signals. All the four cases in the present study had gain of chromosome 17 (CEP17\u2009\u2265\u20093.0). A remarkable variability of IHC score for cases in this group was observed across different laboratories21. The positive rate of IHC ranged from 8.3% to 75%. Cases with IHC 0/1+ could also be identified. Similarly, our data showed that two cases were scored as IHC 2+, while the other two were interpreted as IHC 3+ and 1+. Owing to the limited number of cases enrolled in clinical trials, a definitive conclusion on whether the patients with this ISH pattern can benefit from HER2-targeted therapy cannot be reached. Considering the heterogeneity of HER2 IHC results, the 2018 guidelines recommend that cases with concurrent IHC score of 2+/3+ be categorized as HER2 positive.There were only four cases in ISH group 3, with a lower incidence than other reports25. Some laboratories relied on alternative chromosome 17 genes as surrogate to deal with this issue, leading to a change in HER2 status from equivocal to positive in about 50% of patients27. Due to the presence of false-positive HER2 results and the absence of evidence in the therapy efficacy, this approach has not been recommended in the 2018 guidelines. In our study, after concomitant IHC review, 29 cases were scored as IHC 0/1+, 15 were IHC 2+ and 9 were IHC 3+. Previous studies proved that HER2 genetic heterogeneity in breast cancers was most frequent in cases with IHC 2+ and equivocal HER2 amplification29. Therefore, another tissue block was selected for further FISH testing in the 15 cases with IHC 2+. Only one case was turned out to be HER2 positive with a HER2/CEP17 ratio of 2.19 and an average of 8.03 HER2 signals per cell. Finally, ten cases were converted to HER2 positive and forty-three cases were diagnosed as HER2 negative. The suggestion that repeat testing on the other tissue samples from the same patients in the new guidelines is appropriate in this setting.Fifty-three cases were assigned as ISH group 4. Before the introduction of the 2018 guidelines, the patients with equivocal ISH results posed a challenge to oncologists of whether to recommend HER2-targeted therapy. Moreover, implementation of the 2013 guidelines resulted in the detection of more equivocal casesIn conclusion, the utility of 2018 guidelines resulted in a mild decrease in HER2 positive rate, due to the reclassification of cases in ISH group 2 and group 4. The implementation of the new guidelines can reduce reflex FISH test and make the diagnosis of HER2 gene status more definitive."}
+{"text": "Can population-level genomic screening identify those at risk for disease?BRCA1 and BRCA2 variants were found in a higher proportion of patients than was previously reported.In this cross-sectional study of an unselected population cohort of 50\u2009726 adults who underwent exome sequencing, pathogenic and likely pathogenic BRCA1/2 variant carriers may not be sufficient as a screening tool; population genomic screening for hereditary breast and ovarian cancer may better identify patients at high risk and provide an intervention opportunity to reduce mortality and morbidity.Current methods to identify  BRCA1 and BRCA2 (BRCA1/2) genes allows for cancer prevention and early diagnosis in high-risk individuals.Detection of disease-associated variants in the BRCA1/2 variants in an unselected research cohort, and to characterize the features associated with P/LP variants.To identify pathogenic and likely pathogenic (P/LP) BRCA1/2.This is a cross-sectional study of adult volunteers (n\u2009=\u200950\u2009726) who underwent exome sequencing at a single health care system  from January 1, 2014, to March 1, 2016. Participants are part of the DiscovEHR cohort and were identified through the Geisinger MyCode Community Health Initiative. They consented to a research protocol that included sequencing and return of actionable test results. Clinical data from electronic health records and clinical visits were correlated with variants. Comparisons were made between those with (cases) and those without (controls) P/LP variants in BRCA1/2 variants in cohort, proportion of variant carriers not previously ascertained through clinical testing, and personal and family history of relevant cancers among BRCA1/2 variant carriers and noncarriers.Prevalence of P/LP BRCA1/2 variants and 267 (0.5%) were BRCA1/2 carriers. Of the 267 cases (148 [55.4%] were women and 119 [44.6%] were men with a mean [range] age of 58.9 [23-90] years), 183 (68.5%) received clinically confirmed results in their electronic health record. Among the 267 participants with P/LP BRCA1/2 variants, 219 (82.0%) had no prior clinical testing, 95 (35.6%) had BRCA1 variants, and 172 (64.4%) had BRCA2 variants. Syndromic cancer diagnoses were present in 11 (47.8%) of the 23 deceased BRCA1/2 carriers and in 56 (20.9%) of all 267 BRCA1/2 carriers. Among women, 31 (20.9%) of 148 variant carriers had a personal history of breast cancer, compared with 1554 (5.2%) of 29 880 noncarriers . Ovarian cancer history was present in 15 (10.1%) of 148 variant carriers and in 195 (0.6%) of 29 880 variant noncarriers . Among 89 BRCA1/2 carriers without prior testing but with comprehensive personal and family history data, 44 (49.4%) did not meet published guidelines for clinical testing.Of the 50 726 health system patients who underwent exome sequencing, 50 459 (99.5%) had no expected pathogenic BRCA1/2 variants. These findings suggest that genomic screening may identify BRCA1/2-associated cancer risk that might otherwise remain undetected within health care systems and may provide opportunities to reduce morbidity and mortality in patients.This study found that compared with previous clinical care, exome sequencing\u2013based screening identified 5 times as many individuals with P/LP  BRCA1/2 variants among US adults enrolled in the MyCode Community Health Initiative.This cross-sectional study investigates the ascertainment of pathogenic and likely pathogenic  BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) genes were first associated with familial breast cancer risk in the mid-1990s.2 Associated risks for ovarian cancer, prostate cancer, pancreatic cancer, and melanoma have since been documented; these clinical associations are grouped as hereditary breast and ovarian cancer (HBOC) syndrome.3Pathogenic variants in the BRCA1 and BRCA2  gene became available in 1995.4 Risk-assessment strategies, based on personal and family history cancer thresholds, have been built, validated, and incorporated into clinical testing guidelines. These guidelines are routinely used to identify individuals with increased pretest probability of pathogenic BRCA1/2 variants.6 Current established guidelines have been issued by the National Comprehensive Cancer Network (NCCN),7 the US Preventive Services Task Force,8 and the American College of Medical Genetics and Genomics together with the National Society of Genetic Counselors.9Clinical testing for pathogenic variants in the BRCA1/2 variants may be associated with the failure to apply testing guideline criteria and the failure of criteria-based strategies to identify all true positives.11 Such underascertainment has been documented even in women with existing cancer diagnoses13 and has prompted calls for DNA sequence\u2013based population screening.14Clinical underascertainment of individuals with pathogenic BRCA1/2 variants identified by exome sequencing. Sequencing-based screening findings were ascertained independent of participants\u2019 age, sex, or cancer history.15 The cases and controls are part of the ongoing DiscovEHR cohort and were identified through the Geisinger MyCode Community Health Initiative. This large exome-sequenced research cohort has a median of 14 years of linked electronic health records (EHRs), and the patients consented to a research protocol that included sequencing and return of actionable test results.16 The genomic findings were confirmed by clinical laboratory testing and entered into the EHR.In this article, we report the pathogenic variants prevalence, HBOC syndrome\u2013associated cancer history, and comparison of exome sequencing\u2013based screening with indication-based ascertainment in a cross-sectional study of predominantly white (European ancestry) adults who underwent exome sequencing at a single US health care system  from January 1, 2014, to March 1, 2016. These individuals had \u201cexpected pathogenic\u201d  STROBE) reporting guideline.This cross-sectional study received approval from Geisinger Institutional Review Board. Written informed consent was obtained from participants. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  variant in either the BRCA1 or BRCA2 gene. We referred to these cases as BRCA1/2 carriers to be consistent with published literature. Controls are defined as the participants who underwent sequencing in the biobank  and screened negative for P/LP variants in both the BRCA1 and BRCA2 genes. We further subdivided the case population on the basis of (1) prior identification of a variant, (2) personal or family history that would have met established clinical criteria for referral or testing, and (3) gene involvement.BRCA1/2 carriers whose genetic risk was associated with personal history of relevant cancer at any age). The relevant cancers included breast, ovarian, prostate, pancreatic, and melanoma.For the purposes of this article, an HBOC syndrome diagnosis was assigned in cases in which both genotype and phenotype were present. Therefore, HBOC syndrome diagnosis refers to cases in which a relevant cancer occurred  classification in ClinVar with a *2 or *3 status, indicating strong evidence for pathogenicity18; (2) predicted loss of function; or (3) both , this application is not sufficient. Therefore, we employed a previously described diagnostic framework for incidental or secondary genomic findings. The framework\u2019s 5 diagnostic groups include 3 groups with relevant cancer diagnoses  and in which the conventional diagnosis of HBOC is made as well as 2 groups in which relevant clinical findings are absent in the participants  encouraged to complete a targeted 3-generation family history (either in person or online). Each pedigree was independently analyzed by at least 2 licensed genetic counselors , who assessed threshold criteria for referral and testing according to the guidelines of the NCCN,7 US Preventive Services Task Force,8 and American College of Medical Genetics and Genomics\u2013National Society of Genetic Counselors.9 Discordance between the reviewers was resolved by joint review and consensus. A total of 122 relevant 3-generation pedigrees were analyzed, of which 89 (72.9%) were from participants who had not undergone prior BRCA1/2 clinical testing  carriers  between  testing .Twenty participants died before their result became available, and 3 died after the return of result . Medical record review was carried out by 3 clinicians  independently and then jointly to determine the cancer diagnosis and cause of death  and 95% CIs. Two-sided P\u2009<\u2009.05 was considered statistically significant. All analyses were performed using SAS, version 9.4 (SAS Institute Inc).Data were summarized using means and ranges for continuous variables as well as frequencies and percentages for categorical variables. Comparisons between groups were performed using Wilcoxon rank sum test, Pearson \u03c7BRCA1/2 variants and 267 (0.5%) were BRCA1/2 carriers. Of the 267 cases (148 [55.4%] were women and 119 [44.6%] were men with a mean [range] age of 58.9 [23-90] years), 183 (68.5%) received clinically confirmed results in their EHR. Compared with the health system\u2019s overall population, the biobank cohort was older , had a greater percentage of women , had a higher representation of white patients , and was enriched for relevant cancers .Of the 50\u2009726 health system patients who underwent whole exome sequencing, 50 459 (99.5%) had no expected pathogenic BRCA1/2 variants was 1:190, and 1:180 when controlling for relatedness  had BRCA2 variants. There were 118 P/LP variants in BRCA1/2 in the 267 carriers  of the existing clinical significance designations in ClinVar; the single difference was 1 listed in ClinVar as variant of unknown significance that was designated as LP on the basis of updated information.The prevalence of participants with a Sanger sequencing\u2013confirmed P/LP BRCA1 and 66 BRCA2 variants, 107 (90.67%) were putative loss-of-function variants  and only 5 (4.2%) were novel predicted loss-of-function variants. Eleven of 118 variants (9.3%) were missense, and no novel missense variants were classified as P/LP, in accordance with the criteria set forth by the American College of Medical Genetics and Genomics for variants interpretation.17Of the 52 BRCA1/2 carriers and ruled out 1 candidate carrier as a false-positive. The number of BRCA1/2 carriers with any given variant ranged from 1 to 24 (eTable 1 in the BRCA2 pathogenic variants (ClinVar variation ID: 38082 and 9320)22 accounted for 42 BRCA1/2 carriers, and 1 of 3 Ashkenazi Jewish founder mutations was found in 19 BRCA1/2 carriers.3Sanger sequencing confirmed 267 bioinformatically identified BRCA1/2 carriers were already aware of their result through prior clinical BRCA1/2 testing; these were patients who were diagnosed on the basis of traditional detection methods such as personal and family history. Newly diagnosed BRCA1/2 carriers  were more likely than controls (without BRCA1/2 variants) to have EHR evidence of personal or family history of HBOC syndrome\u2013associated cancer diagnoses (28.0% vs 53.4%) , indicating significantly increased cancer risk associated with the BRCA1/2 carriers ascertained in this fashion.Forty-eight (17.9%) of the 267 s 53.4%) . New cars 53.4%) B-D. In 2BRCA1/2 carriers and in 56 (20.9%) of all 267 BRCA1/2 carriers. Among women, 31 (20.9%) of 148 variant carriers had a personal history of breast cancer, compared with 1554 (5.2%) of 29 880 noncarriers . Ovarian cancer history was present in 15 (10.1%) of 148 variant carriers and in 195 (0.6%) of 29 880 variant noncarriers .Syndromic cancer diagnoses were present in 11 (47.8%) of the 23 deceased BRCA1/2 carriers, results were returned to 183 eligible adults (68.5%) and their health care practitioner through a secure EHR portal; this was followed up by a letter and telephone call to the participants. Clinical care based on NCCN guidelines7 was offered, and 3 examples of early cancer detection were described in a report.21 A case of a woman in her 40s with subclinical disease detected on screening was identified after that report  of 78 BRCA2 carriers met the NCCN criteria. In nearly every category, women were more likely than men to meet criteria thresholds  of all cases .The participants\u2019 final diagnostic category was based on all relevant clinical information reviewed, including the EHR and participant self-report when available . The HBOBRCA1/2-associated cancer in 9 people (39.1%), unrelated to BRCA1/2 in 11 (47.8%), and indeterminate in 3 (13.0%). In contrast to the deceased cases, 45 (18.4%) of 244 living patients have a personal history of a relevant cancer (eTables 3 and 4 in the Twenty-three (8.6%) of 267 participants were deceased by June 1, 2017, and the median age at death was 64.7 years  met current NCCN criteria for testing but had not received testing; conversely, 44 individuals (49.4%) did not meet NCCN testing criteria but had expected pathogenic variants. Of importance, those patients who did not meet the established thresholds for BRCA1/2 testing were not spared from relevant cancers, as evidenced by the diagnosis of early asymptomatic cancer in such individuals after the initiation of cancer risk management.21Findings demonstrate that routine exome sequencing\u2013based screening may be a potential factor in long-term care improvement. This study found that only 17.9% of BRCA1/2 testing strategies to individuals with high pretest probabilities and the failure of such criteria-based strategies to be sufficiently sensitive to identify all true positives. The practical difficulties of incorporating sufficient family history risk analysis in routine health care settings are well described.24 The observed gap in relevant cancer history between all living BRCA1/2 carriers and deceased BRCA1/2 carriers signals an opportunity to prospectively identify and manage individuals with elevated risk to reduce the morbidity and mortality associated with their defined cancer risk. Early success in this area is demonstrated by the identification of 4 individuals with early-stage cancer during the screening and management recommended by the exome sequencing\u2013based screening results process.Our evidence demonstrates that this clinical underascertainment may be associated with both the failure to apply criteria-based referral for 26 Underascertainment of these conditions, in which presymptomatic case identification is advantageous,25 should prompt prioritization of further research into genomic sequencing\u2013based population screening.27Our findings show considerable clinical underascertainment of individuals with HBOC syndrome. Underascertainment has also been observed in familial hypercholesterolemia and Lynch syndrome, 2 conditions that  are designated as tier 1  by the Centers for Disease Control and Prevention Office of Public Health Genomics and are included in the Healthy People 2020 recommendations.BRCA1/2 expected pathogenic variants of 1:190, adjusted to 1:180 when controlling for relatedness up to the third degree.25 Consistent with other studies, HBOC syndrome\u2013associated cancer rates were higher in women than in men and in BRCA1 carriers than in BRCA2 carriers.28 Unsurprisingly, those with prior clinical BRCA1/2 testing also had higher cancer rates than those without prior testing. For the subset of 23 deceased BRCA1/2 carriers, only 4 had prior clinical testing and the percentage with syndromic cancer diagnoses was 47.8%, which is similar to published observations of clinically ascertained cases.3Overall, ascertainment through sequencing-based screening in the biobank cohort showed an empirical prevalence for BRCA1 to BRCA2 case ratio of 1.0:1.8 is consistent with ratios in other relevant work.31 Previous research3 shows decreased penetrance for BRCA2 carriers, which may be a contributing factor in the underascertainment of these cases compared with BRCA1 cases; in our study, among those with prior testing and cancer diagnosis, more than 90% had BRCA1-associated cancer risk.The observed BRCA1/2 carriers who did not have prior clinical BRCA1/2 testing, the return of genomic result was the start of a diagnostic process aimed at deciding if the risk variants were associated with current or past disease.20 For those with a personal history of BRCA1/2-associated cancer, a diagnosis of HBOC syndrome was now clear and the recommended management was changed to align with NCCN guidelines and to offer cascade testing for at-risk family members. Those without BRCA1/2-associated cancer at results disclosure did not have a diagnosis of HBOC syndrome, and long-term risk-reduction measures were recommended. For individuals with risk variants but without disease, the variants result was listed in their EHR problem list as a test result and not as an HBOC syndrome diagnosis.32 This distinction is important because continued surveillance is recommended, but syndrome diagnosis may not ever be achieved.32Among the 82.1% of 32 Consistent with the vision of learning health care systems,35 this disclosure and follow-up approach is being informed and improved by ongoing feedback from biobank participants, health care practitioners, and researchers. In addition, the disclosure can be used as a template for other settings where the genome-first care model is implemented.Substantial infrastructure and health care practitioner collaboration were necessary to support the disclosure and follow-up care of the 183 participants whose biobank consent form allowed for clinical reporting.BRCA1/2-positive individuals,7 but patients are informed that important knowledge gaps exist. The capture and aggregation of health outcomes data from this and other cohorts is essential to developing evidence that guides precision management strategies for individuals identified through whole exome sequencing\u2013based screening.36 These health outcomes data, coupled with analysis of the cost of care , will ultimately determine the value of exome sequencing\u2013based screening and inform the implementation of screening strategies for the BRCA1/2 variants.Finally, optimal clinical management of patients identified through sequencing-based screening who do not otherwise meet the criteria for clinical testing is likely to evolve as more longitudinal outcomes data become available from this cohort and others. Given a near-term gap in evidence-based management, patients at Geisinger receive management based on previously developed protocols for BRCA1/2 variants as the control population, which mitigated this bias.In general, the participants in the biobank were older than the health system\u2019s patient population; more important, this study skewed toward those who received regular health care, increasing their opportunity to be enrolled in the biobank research. The higher incidence of breast cancer in the biobank population was likely a reflection of frequency of care and age; however, in our risk analysis, we used the biobank patients without evidence of 3; (2) absence of attempts to assign significance to missense variants beyond that established in ClinVar, and (3) estimated underrepresentation of cases with early-onset disease in the study cohort as a result of HBOC syndrome\u2013associated mortality, given the age difference between the BRCA1/2 cohort and the general health system population.37 The first 2 issues will be addressed in future work. We recognize that, because this study population is reflective of the local population and thus is overwhelmingly white,16 it may not be representative of other cohorts, particularly those from different racial/ethnic backgrounds. As with many areas of genomic medicine implementation, inclusion of greater numbers of individuals from underrepresented race/ethnicity is essential to a complete understanding of BRCA1/2 risk.38 Data from other cohorts will be needed to clarify whether this BRCA1 to BRCA2 ratio finding is generalizable for population screening.The study still likely underestimated prevalence in this health care system\u2013based cohort because of the (1) limitations to the current variant identification strategy BRC1/2 variants in the general population may be substantially higher than was previously estimated, and reliance on personal and family history may be an inadequate measure to ascertain risk for BRCA1/2 variants.The prevalence of"}
+{"text": "Following the publication of  concernsFig 3D \u03b2-actin panel appears similar to Fig 5C \u03b2-actin panelThe authors have explained that an error was made during figure preparation. The authors accidentally used the duplicated micrograph image for the Fulvestrant-treated DU145 cells at 0h to represent the ETOH-treated cells at 0h. The authors have provided a replacement image for The lysate samples of the control and Fulvestrant-treated cells used in Western blot analysis were the same for generation of data presented in both Figs Please see the corrected S1 Dataset(ZIP)Click here for additional data file."}
+{"text": "IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3\u03b2, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3\u03b2 demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic  or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4\u20137%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function  Single-suture, non-syndromic craniosynostosis occurs in approximately 1/2500 live births with a familial recurrence rate of 4\u20137% [FGFR3 [TWIST1 [EFNB1 [TCF12 [A limited number of single gene mutations are known to cause single-suture craniosynostosis (SSC) , but th1 [TCF12 . These gIGF1R that were overrepresented in an SSC case population [COL1A2, ALP, and RUNX2, and matrix mineralization [Several genes have emerged as potential polygenic candidates through our RNA microarray and expression analysis, genomic sequencing, phosphorylation studies, and biomechanical assays. We first performed targeted candidate gene sequencing on 186 patients with SSC and identified rare sequence variants in pulation . Structupulation , 10, whilization \u201314. TakeIGF1R variants [Concurrently, our lab used correlation analysis of osteoblast transcriptomes from 199 human cases (including the 186 from the targeted sequencing project) and 50 controls to detect distinct patterns of gene expression that were associated with craniosynostosis , 16. Twovariants , demonstvariants . This acvariants , osteoblvariants , and chovariants .IGF1 expression. These experiments revealed increased contractility and decreased migration in cells with elevated levels of IGF1, supporting our previous results suggesting that enhanced differentiation is correlated with this subset of SSC cases [IGF1 expression, and gain-of-function mutations in IGF1R made the IGF1/IGF1R pathway an obvious choice for further examination.Subsequent biomechanical assays measuring contractility and migration were performed on primary SSC osteoblasts with and without elevated SC cases . The ideRUNX2 in two cases with familial metopic SSC and hypodontia [TWIST1 cause Saethre-Chotzen syndrome, a hereditary form of craniosynostosis [Using the same patient cohort, we performed copy number variant analysis and identified a duplication of podontia . This obpodontia . In addipodontia , 27, RUNpodontia , 29. Mutnostosis , 31, hownostosis .Twist1/Igf1 and Twist1/Gsk3\u03b2 compound heterozygotes to test hypotheses generated from our previous human expression data. These mouse models demonstrate an increased frequency and severity of craniosynostosis and support the hypothesis that combined dysregulation of the TWIST1 and IGF1 pathways could contribute to an increase risk in humans.In this manuscript we present data from mouse cross-breeding experiments designed to explore possible polygenic inheritance of craniosynostosis using This study was reviewed and approved by the Institutional Review Board of Seattle Children\u2019s Hospital . Written informed consent from parents or guardians of children with SSC was obtained and a consent waiver was obtained for the use of anonymous control samples. See reference [Post-surgical calvarial bone samples which would have otherwise been discarded were collected from 211 cases and 50 controls between the years of 2002 and 2006. Computed Tomography confirmed the diagnosis of isolated sagittal, metopic, or unilateral coronal synostosis in each case. All cases were screened for the presence of major medical conditions such as syndromic craniosynostosis, cardiac defects, seizure disorders, cerebral palsy, health conditions requiring surgical intervention, presence of three or more minor extra-cranial malformations; or presence of any other major malformations. No individuals with these conditions were enrolled. Control samples were obtained from patients undergoing a craniotomy for reasons other than craniosynostosis  or autopsies. Control samples were not obtained from individuals with disorders that affect bone such as skeletal dysplasias. Tissue samples were collected in Waymouth\u2019s Media (WM)  and sent to the lab for processing.2, and 99% humidity. Upon confluence, cells were washed with PBS, trypsinized with 0.05% Trypsin-EDTA , and passaged into T75 flasks. Confluent cells were cryogenically stored in freezing media containing 90% FBS and 10% dimethyl sulfoxide in a liquid nitrogen freezer. As previously descried, alkaline phosphatase activity was used to assure osteoblast lineage and all samples demonstrated high ALP activity [Calvarial tissue was rinsed in WM and the surrounding soft tissue was removed. Using a sterile scalpel blade, the tissue was cut into 1-2mm pieces and 2 pieces per well were cultured in 12-well plates at 37\u00b0C, 5% COactivity .2. At 75% confluence, cells were trypsinized, washed in cold 1X PBS, and RNA was isolated using the Roche High Pure miRNA Isolation Kit  in accordance with the manufacturer\u2019s instructions. Initial thawing of the cells for this process was done in batches, and batch effect was considered during expression analysis. RNA integrity was assessed using the Agilent 2100 Bioanalyzer and only samples with a RIN score of >8.0 were used for further analysis.Cell lines were cultured to sub-confluence in T25 flasks and passaged to a density of 175,000 cells per 25cmcDNA microarray assays were performed on 261 calvarial tissue samples (211 cases and 50 controls) using Affymetrix Human Gene 1.0 ST array technology. See full methods in reference .a priori based on their role in bone development and osteoblast differentiation. Transcript expression levels were assessed visually by applying conditional formatting to expression level values for each gene and analyzed by paired t-test.All microarray data are MIAME compliant and the raw dataset has been deposited in the MIAME compliant Gene Expression Omnibus (GEO) database under accession number GSE27976 . For theIgf1(+/tg)/Igf1(tg/tg) (transgenic overexpression of Igf1), Gsk3\u03b2(+/-) (hemizygous knockout of Gsk3\u03b2), and Twist1(+/-) (loss-of-function point mutation in Twist1 \u201ctwist-box\u201d domain) from Jackson Laboratories, Bar Harbor, ME were bred to congenicity on a C57BL/6J background with a minimum of 9 backcross generations (\u226599.5% identity). See official Jackson Laboratory nomenclature (Gsk3\u03b2(+/-) and Igf1(+/tg), and by Sanger sequencing for Twist1(+/-). Primers are listed in Twist1 and Gsk3\u03b2 mice are known to be embryonic lethal and thus were maintained as heterozygotes [Gsk3\u03b2(+/-) mice have been shown to have significantly lower Gsk3\u03b2 expression than their wildtype counterparts [Igf1 mice were maintained as both homozygotes and heterozygotes. Mouse IGF1 ELISA Kit  was used to validate overexpression of IGF1 in the serum of IGF1 transgenic animals . mice. Litters of interest were sacrificed at P28 by CO2 euthanasia and prepared for microCT scanning.All mouse work was approved by Seattle Children\u2019s Institutional Animal Care and Use Committee and complies with NIH Guidelines for the Care and Use of Laboratory Animals. Commercially available mouse lines nclature . Compounnclature . The genozygotes , 35. Gskterparts  Igf1 micu, et al ). RandomA Skyscan 1076 micro Computed Tomography (microCT) scanner was used to render high-quality 3-dimensional CT images of skulls from P28 mice with our genotypes of interest. Scans were performed at a maximum of 35um slice thickness and with the following parameters; 55Kv, 179uA, 0.5mm aluminum filter, 360 ms exposure, rotation step of 0.7\u00b0, 180\u00b0 scan, and 3 frame averaging. CT scans were reconstructed using NRecon software  and rendered in Drishti Volume Exploration and Presentation Tool   and Twist1(+/-) to Twist1(+/-)/Igf1(+/tg) and an n of 18 for the comparison of Gsk3\u03b2(+/-) and Twist1(+/-) to Twist1(+/-)/Gsk3\u03b2(+/-).Statistical analysis was performed using a chi-squared test for comparison of proportions. Percent of expected affected for each compound heterozygous genotype was calculated by the sum of observed affected in each component genotype. Proportions were then compared using an n of 17 for the comparison of RUNX2, a transcription factor critical for osteogenesis, was correlated with IGF1 expression and was significantly different between High IGF1 cases and unaffected controls  as well as affected controls .Our initial data review identified a striking association between IGF1 expression and many of the 399 candidate genes chosen a priori due to their role in osteogenesis and bone development. Comparison of the expression levels of our candidate genes in the 23 cases with the highest levels of IGF1 expression (High IGF1 Cases) and both the 50 unaffected controls  and the Gsk3\u03b2(+/-), Twist1(+/-), Igf1(+/tg), Igf1(tg/tg), and with the following compound- heterozygous variant genotypes; Gsk3\u03b2(+/-)/Twist1(+/-), Twist1(+/-)/Igf1(+/tg), and Gsk3\u03b2(+/-)/Igf1(+/tg) (Igf1(+/tg), Igf1(tg/tg), or wildtype mice. Fifteen percent of Gsk3\u03b2(+/-) mice had suture fusion, one of which had increased biparietal diameter suggestive of hydrocephalus. One Gsk3\u03b2(+/-) mouse without suture fusion also had increased biparietal diameter. Based on the suture morphology we suspect that this mouse had bilateral coronal suture fusion that was disrupted due to pressure from brain growth and concomitant hydrocephalus. This animal was classified as not having craniosynostosis (Twist1(+/-) mice had suture fusion.We analyzed 3D-rendered CT images of congenic, postnatal day 28 (P28) C57BL/6J mice with the following single-variant genotypes; f1(+/tg) , S1 Fig.Twist1(+/-) and 15% in Gsk3\u03b2(+/-) to 94% in Twist1(+/-)/Gsk3\u03b2(+/-), a significance of p = 0.0038 (accounting for the additive effect of these disease-causing genotypes). Of note, inclusion of the mouse with suspected craniosynostosis in our analysis did not change the interpretation of our results (p = 0.0121). There was no significant increase in the severity of craniosynostosis when comparing these groups. The incidence of fusion was not significantly increased in Twist1(+/-)/Igf1(+/tg) as compared to Twist1(+/-) mice, but the severity  increased from 6% of Twist1(+/-) to 29% of Twist1(+/-)/Igf1(+/tg) (p = 0.0766) /Igf1(+/tg) genotype than predicted by Hardy-Weinberg equilibrium (~10% as compared to the expected 25%), suggesting the possibility of prenatal lethality of this genotype. While none of the Gsk3\u03b2(+/-)/Igf1(+/tg) mice that survived to P28 showed suture fusion, the reduced number of offspring with this genotype precludes our ability to define the true craniosynostosis rate.We observed a disproportionally lower number of mice with the Igf1 and Gsk3\u03b2 and disinhibition of Runx2, that result in a significantly increased rate or severity of craniosynostosis when compared to mice with each genotype in isolation.While there have been great advances in our understanding of the molecular genetic causes of syndromic craniosynostosis, the etiology of most cases of the more common single suture synostoses remains unknown. Its low recurrence rate suggests multifactorial, polygenic, or oligogenic inheritance. In this study, we describe compound heterozygous mutant mice with genetically altered expression of IGF1 and RUNX2 expression in a subgroup of osteoblasts derived from clinical cases of SSC. The IGF1/IGF1R pathway is critical for normal bone growth and density [Igf1(+/tg),Twist1(+/-), and Gsk3\u03b2(+/-) converge to reduce inhibition and increase the transcriptional activity of Runx2. The mouse models selected for our work mimicked activation of the Akt pathway and selective disinhibition of RUNX2 to determine the additive effects of pathway dysregulation  mouse is known to harbor a point mutation in the twist-box resulting in selective disinhibition of RUNX2 leading to coronal synostosis of variable penetrance and expressivity. This was validated in our study demonstrating that 35% of Twist1(+/-) mice developed coronal synostosis.The Igf1(+/tg) or Igf1(tg/tg) transgenic mice developed craniosynostosis, 23% of the Gsk3\u03b2(+/-) mice displayed craniosynostosis and/or presumed hydrocephalus, a previously undescribed phenotype with incomplete penetrance. With this unexpected result, we reviewed recent sequence data from our cohort and identified a missense mutation (c.T185C:p.I62T) in a single case of sagittal synostosis. This variant has not been identified in more than 250,000 alleles sequenced [Twist1(+/-)/Gsk3\u03b2(+/-) compound heterozygous mice. While is is known that C57B/6J mice naturally present with an \u201cinterfrontal bone\u201d [While none of the equenced , 46, resal bone\u201d  and thusTwist1(+/-)/Gsk3\u03b2(+/-) had a significantly higher frequency of craniosynostosis than predicted by the additive effects of each mutation alone (increasing from 35% in Twist1(+/-) and 15% in Gsk3\u03b2(+/-) to 94% in Twist1(+/-)/Gsk3\u03b2(+/-) compound heterozygotes). Furthermore, there was a near significant increase in the rate of bilateral coronal synostosis in Twist1(+/-)/Igf1(+/tg) compound heterozygotes when compared to the rate in the parental strains.Compound heterozygous These data suggest that a functional interaction between the TWIST1/RUNX2 and IGF1 pathways is integral to normal calvarial development. We have provided data supporting that combined dysregulation of these pathways result in the first example of polygenic inheritance of craniosynostosis in an animal model. While our data suggest that dysregulation of the TWIST1/RUNX2 and IGF1/Akt pathways may predispose humans to craniosynostosis, further translational research is necessary before the development of clinical utility.S1 Fignot having craniosynostosis. (**) Mouse died at P23 with potential hydrocephalus based on increased bi-parietal diameter. Postnatal age at the time of sacrifice is noted if other than P28.The phenotype of 91 mice was classified in this study. Each skull image is designated by genotype and pattern of suture fusion (U) Unilateral craniosynostosis, (Ub) Unilateral craniosynostosis with \u201cbridging\u201d phenotype, (B) Bilateral craniosynostosis, or (Bb) Bilateral craniosynostosis with \u201cbridging\u201d phenotype. (*) Irregular and wide suture margins and wide biparietal diameter of this mouse suggests the possibility bilateral coronal suture fusion that was disrupted due to concomitant hydrocephalus. This animal was classified as (PDF)Click here for additional data file.S1 TableIGF1 expression and 50 unaffected controls. (\u00a5) indicates significant difference in expression levels between 23 cases with the highest levels of IGF1 expression and 188 affected control cases (e.g. cases with craniosynostosis with low IGF1 expression). (\u2191) indicates a the gene is positively correlated with IGF1 and (\u2193) indicates that a gene is inversely correlated with IGF1 for the group(s) it is significant in.Alpha list of 399 genes of interest chosen a priori for their role in osteoblast development and bone biology. (\u00a7) indicates significant difference in expression levels between 23 cases with the highest levels of (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableIgf1 and Gsk3\u03b2 sourced from Jackson Laboratories genotyping protocols (jax.org). Primers spanning Twist1 Ser192Pro designed in-house.Primer sequences for (PDF)Click here for additional data file."}
+{"text": "Conserv Physiol 3(1): cov025; doi: 10.1093/conphys/cov025The original version of this manuscript was published with an incorrect figure in the place of figure 2. The manuscript has now been corrected."}
+{"text": "The caption of Fig. Table S1 in the Additional file of this original publication contained some errors. The updated Table S1 is published in this correction article.Additional file 1:Updated Table S1, the full supplementary materials can be downloaded from the original publication. (XLSX 10 kb)"}
+{"text": "Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley. Previous studies have showed that all three MAP kinase genes, MGV1, FgHOG1, and GPMK1, are involved in regulating hyphal growth, sexual reproduction, plant infection, and stress responses in this pathogen. To determine the relationship between the Mgv1 and FgHog1 pathways, in this study, we generated and characterized the mgv1 Fghog1 double mutant. Deletion of FgHOG1 partially rescued the defects of the mgv1 mutant in vegetative growth and cell wall integrity but had no effects on its defects in plant infection and DON production. The mgv1 Fghog1 mutant grew faster and was more tolerant to cell wall stressors than the mgv1 mutant. Swollen compartments and cell burst were observed frequently in the mgv1 mutant but rarely in the mgv1 Fghog1 mutant when treated with fungicide fludioxonil or cell wall stressor Congo red. Conversely, the deletion of MGV1 also alleviated the hyperosmotic sensitivity of the Fghog1 mutant in vegetative growth. TGY assays indicated increased phosphorylation of FgHog1 in the mgv1 mutant, and TEY assays further revealed elevated activation of Gpmk1 in the mgv1 Fghog1 double mutant, particularly under cell wall stress conditions. Overall, our data showed that deletion of FgHOG1 partially suppressed the defects of the mgv1 mutant, possibly by affecting genes related to cell wall integrity and osmoregulation via the over-activation of Gpmk1 MAP kinase and avoiding intracellular turgor elevation. Fusarium head blight, a devastating disease of wheat and barley, is caused by the filamentous ascomycete Fusarium graminearum. In addition to severe yield losses, infested grains are often contaminated with mycotoxins, including deoxynivalenol (DON) and zearalenone  kinase cascades that are involved in regulating various developmental and infection processes in fungal pathogens  gene characterized in F. graminearum, is orthologous to the cell wall integrity (CWI) MAPK SLT2 in the budding yeast and MPS1 in Magnaporthe oryzae  and FgHOG1, also have been functionally characterized in F. graminearum  pathway and M. oryzae Osm1  significantly reduced the phosphorylation level of FgHog1  was generated and transformed into the mgv1 mutant. Transformants resistant to both hygromycin B and geneticin were isolated and screened by PCR for the deletion of FgHOG1  cultures were treated with a mixture of cell wall lytic enzymes for 30 min. In the mgv1 mutant, abundant protoplasts were produced, while germlings from the wild type strain PH-1 released few protoplasts , a compound interfering with the fungal cell wall  of the mgv1 mutant  is a commonly used stain to visualize the fungal cell wall. When germlings harvested from 12 h YEPD cultures were stained with 50 \u03bcg/ml CFW, fluorescence signals were evenly distributed along the cell wall and septa in the wild type and 1 mutant . Howeverartments . Weaker 1 mutant . These dmgv1 mutant was lower than 1%, while that of the mgv1 Fghog1 double mutant was increased to 40%  of CFW to treat the conidia, we found that all conidia of both the mgv1 and mgv1 Fghog1 mutants were unable to germinate and had empty conidium compartments  or became empty  in the wild type . In the me empty . The pre mutants . After ierminate . These rr turgor , the incmgv1Fghog1 mutant is more tolerant to hyperosmotic stress than the Fghog1 mutant in vegetative growth but not in conidial germination:The  Because FgHog1 MAPK is involved in regulating the response to hyperosmotic stress, we then assayed the effects of 0.2 M NaCl on the growth of the mgv1 and mgv1 Fghog1 mutants. As shown in Fghog1 and mgv1 Fghog1 mutants were significantly inhibited in vegetative growth on a CM plate with 0.2 M NaCl. Interestingly, the mgv1 Fghog1 double mutant formed a bigger colony than the Fghog1 mutant, which was contrary to the situation on a regular CM plate  and Fghog1 (94.6%)  . However1 mutant . These dFghog1 mutant . In yeast, a number of genes important for cell wall synthesis and assembly are affected by deletion of SLT2  also has higher resistance to fludioxonil than the mpk1 mutant  significantly inhibits the activation of FgHog1  in F. oxysporum   or CM   cultures and conidial germination in YEPD  were assayed as described  . Growth  pH 6.5) . Conidiaescribed . For promgv1 and Fghog1 single mutants were generated in previous studies  and incubated for 6 or 12 h at 25\u00b0C. In order to check the chitin distribution, germlings harvested from 12 h YEPD cultures were stained with 50 \u03bcg/ml CFW for 5 min. Germlings harvested from 18 h YEPD cultures grown at 25\u00b0C were shifted to 32\u00b0C and incubated for another 6 h to assay the effect of elevated temperature. For CWI assays, germlings harvested from 12 h YEPD cultures were digested with the cell wall lytic solution  at 30\u00b0C for 30 min before examination for protoplasts  to estimate the disease index. For each strain, a DON production assay in TBI cultures was performed as described (For plant infection assays, conidia harvested from 5-day-old CMC cultures by filtration through Miracloth were resuspended to a concentration of 1.0 \u00d7 10escribed . There wescribed , with a TM software. Quantitative changes in the phosphorylation levels of Gpmk1, Mgv1, and FgHog1 were analyzed with the Image LabTM software. Each experiment was repeated four times, independently.Total proteins were isolated from hyphae harvested from 18 h YEPD cultures as described . For assGW, CJ, and J-RX conceived and designed the experiments. JR, CL, and CG performed the experiments. GW and CJ analyzed the data. GW wrote the manuscript. J-RX and CJ improved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Centroscyllium fabricii. The data was acquired by metabarcoding using 16S rDNA. Centroscyllium fabricii, a deep sea shark found at depths below 275\u202fm was sampled during Sagar Sampada cruise no 305 in the Indian Ocean and metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit. V3 region of 16S rDNA region was amplified and the amplicons were sequenced on Illumina MiSeq system using 151\u202fbp \u00d7 2 paired end reads. The data of this metagenome is available in the BioSample Submission Portal as Bio-Project PRJNA431407and Sequence Read Archive (SRA) accession number SRR6507004.This data article describes the bacterial diversity of the deep sea shark,  Specifications tableValue of the data\u2022This is the first data report on bacterial diversity of deep sea fish gut from the Oxygen Minimum zone of Indian Ocean.\u2022Centroscyllium fabricii gut can be regarded as a first step towards recognition of its bioactive potential.Generation of an inventory of bacterial diversity of \u2022The significant proportion of hitherto unstudied bacterial phyla (25.30%) identified in the dataset indicates immense scope for Blue Biotechnology.\u2022The data provided here can also be used to understand host-microbiome interactions and to shed light on the factors that influence the establishment of gut microbiota.1Centroscyllium fabricii are deep water schooling sharks found at depths ranging from 180\u20132250\u202fm With 33,700 species described to date Actinobacteria (27.84%), Proteobacteria (18.99%) and Acidobacteria (10.89%). 25.30% of Operational Taxonomic Units (OTUs) did not have any significant hits against the taxonomic database and was categorized as unknown. 107 genera were identified in the dataset, but the abundance of each was found to be less than 1%. Acenitobacter (0.46%) was predominant at the genus level of taxonomic resolution. A vast majority of OTUs (92.4%) remained unclassified at this level. The top 10 genera including unknown are depicted in DNA metabarcoding centered on the 16S rDNA sequence is a high-throughput approach used to catalog taxonomic diversity of environmental samples. The data presented here was obtained by Illumina MiSeq sequencing of V3 region of 16SrDNA. A total of 23 bacterial phyla were identified in the dataset, as shown in 22.1Centroscyllium fabricii was sampled onboard the Fishery Oceanographic Research Vessel (FORV) SagarSampada cruise #305 in the Indian Ocean by using HSDT  net (8\u00b0 11.4\u201d N and 75\u00b0 54.9\u201d E). The fish was subjected to molecular identification by DNA isolation and PCR amplification of 5\u2019 region of cytochrome c oxidase subunit I (cox1) gene from mitochondrial DNA using universal fish primers F2 and R2 KT905423.1).The deep sea shark, 2.2Metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit  and the hypervariable V3 region of 16S rRNA gene was amplified using primers 341F 5\u2019CCTACGGGAGGCAGCAG 3\u2019 and 518R 5\u2019ATTACCGCGGCTGCTGG 3\u2019. The amplicons were sequenced on Illumina MiSeq system using 151\u2009bp \u00d7 2 paired end reads. The raw sequence files were analyzed for base quality, base composition and GC content. V3 region was extracted from paired end reads by trimming of spacer and conserved region and by building a consensus V3 region from the trimmed paired end reads. High quality V3 sequences were extracted by passing the reads through filters for spacer region, conserved region, read quality and mismatch. Subsequent analyses were performed using Quantitative Insights Into Microbial Ecology (QIIME) pipeline."}
+{"text": "ARID1A (BAF250A), SMARCA2 (BRM), SMARCA4 (BRG1), and SMARCB1 (INI1). SMARCA1 (SNF2L) is another member of the chromatin remodelers, and has not yet been studied in neoplasia. As SMARCA1 is located on chromosome X, could be potentially inactivated by a single hit. We aimed to evaluate GAs and protein expression of SMARCA1 in soft tissue tumors.Vital cellular processes such as proliferation and differentiation are regulated by chromatin remodeling complexes. A variety of neoplasms have been discovered to have genomic alterations (GAs) and loss of immunohistochemical (IHC) expression of chromatin remodelers SMARCA1 GAs. Our institutional archives were queried to collect 26 cases of soft tissue tumors including 10 undifferentiated sarcomas, 5 leiomyosarcomas, 6 liposarcomas, and 5 malignant peripheral sheath tumors (MPNST). IHC for SMARCA1 with an SNF 2C4 monoclonal antibody was performed on whole tissue sections.The publically available cBioPortal.32e34 platform was queried to analyze data on soft tissue tumors from The Cancer Genome Atlas project (TCGA) related to SMARCA1 GAs were present in 8/261 soft tissue sarcomas (3%) in the TCGA dataset. Leiomyosarcomas had most common SMARCA1 GAs in 6/99 cases. SMARCA1 deletions existed in 1/56 dedifferentiated liposarcomas and 1/48 undifferentiated sarcomas. No SMARCA1 GAs occurred in other sarcoma subtypes. SMARCA1 IHC was studied in the sarcoma subtypes with potential SMARCA1 alterations in our institutional cases. SMARCA1 nuclear expression was lost in 3/10 cases (30%) of undifferentiated sarcoma, and 2/5 cases of MPNST (40%). SMARCA1 expression was intact in all cases of leiomyosarcoma and liposarcoma.SMARCA1 in the differentiation process and molecular mechanisms of SMARCA1 inactivation.This is the first study to demonstrate loss of expression of SMARCA1 in soft tissue sarcomas subtypes, including undifferentiated sarcoma. Our study highlights merit for further investigation on the role of  ARID1A (BAF250A), SMARCA2 (BRM), SMARCA4 (BRG1), and SMARCB1 (INI1) [SMARCA1 (SNF2L) is another member of the chromatin remodelers, and has not yet been studied in soft tissue tumors [SMARCA1 gene on chromosome X makes it susceptible to gene inactivation by the single hit mechanism [Chromatin remodeling complexes modify the nucleosomal architecture, which provides compact structure and regulates DNA accessibility ,2. VitalID1A BAF20A, SMARCID1A BAF20A, SMARCechanism . We aimeSMARCA1 in soft tissue tumors to analyze the available data. For performing IHC studies, our institutional records were queried for cases of soft tissue sarcoma diagnosed between 2008 and 2018. Twenty six cases were selected from our institutional archives including 10 undifferentiated sarcomas, 5 leiomyosarcomas, 6 liposarcomas, and 5 malignant peripheral sheath tumors (MPNST). IHC staining was performed on paraffin embedded whole tissue sections on Ventana Discovery with the rat monoclonal antibody SMARCA1  in 1:100 dilution. Normal testis tissue was used as a positive control. SMARCA1 expression was evaluated in tumor cell nuclei. When adjacent normal tissue was present for evaluation, SMARCA1 expression was evaluated in nuclei of adjacent nerve, smooth muscle, adipose tissue, endothelium and stromal cells. SMARCA1 expression on IHC was considered intact when expressed in >5% of nuclei of tissue of interest, and lost when not expressed in >5% of nuclei. Results were tabulated in an IRB approved database.In order to study genomic alterations we queried the publically available The Cancer Genome Atlas project (TCGA) platform cBioPortal.32e34 for SMARCA1 genomic alterations were found in eight (3%) of these cases. Leiomyosarcomas were the most common to harbor SMARCA1 genomic alterations, being found in 6 of 99 cases (6%). Of these deletions were found in 3 cases, missense mutation in 1 case, and amplification in 1 case. SMARCA1 deletions were present in only one case of dedifferentiated liposarcoma among 56 cases of liposarcomas (1.7%) and in only one of 48 (2%) undifferentiated sarcomas. None of the other sarcoma subtypes had SMARCA1 genomic alterations.The dataset from TCGA contained 261 soft tissue sarcomas. SMARCA1 nuclear expression was lost in 3 of 10 cases (30%) of undifferentiated sarcomas. There was no difference in morphology or nuclear features of tumor cells between the positive and negative cases of undifferentiated sarcoma. Representative images are shown in Expression of SMARCA1 was noted in 56% of adjacent normal smooth muscle, endothelium, and stromal cells. In adjacent normal adipose tissue, SMARCA1 was expressed patchy in only 1 case (5%). In these tissues, the expression was patchy and nuclear. SMARCA1 was not expressed in any of the adjacent normal peripheral nerves 0% (n=8). In the 3 cases of undifferentiated sarcoma with loss of SMARCA1, the adjacent normal tissue also did not express SMARCA1. Of the two cases of liposarcoma and MPNST with SMARCA1 loss, one case each had expression in adjacent normal tissue.SNF2L or SMARCA1, and SNF2H or SMARCA5 [Chromatin remodeling complexes maintain stability and compactness of the DNA through the nucleosomes and regulate accessibility of the DNA to transcription factors ,2. The c SMARCA5 ,5,7. Chr SMARCA5 ,8.SMARCA2 and SMARCA4 deficiency was noted in 10% of nonsmall cell lung cancer cases, 80% of SMARCA2/SMARCA4 deficient tumors was also TTF-1 negative, and recently SMARCA4 loss was found to be a predictor of response to platinum based chemotherapy [ARID1A mutations [SMARCB1  has been found altered in malignant extrarenal rhabdoid tumor (mutations), epithelioid sarcoma (deletions), epithelioid malignant peripheral nerve sheath tumor (loss of expression), myoepithelial carcinoma (loss of expression), and extraskeletal myxoid chondrosarcoma (loss of expression) [utations . The losutations . Among sression) . The mecression) .KRAS, BRAF [Synthetic lethality is co-occurrence of two genetic events that can be used for cell death, and exploited for cancer therapy . In contAS, BRAF .SMARCA1 (SNF2L), and none on SMARCA1 in soft tissue sarcoma [SMARCA1 is expressed in a broad range of normal tissues and has been reported to modulate the Wnt/\u00df catenin pathway [SMARCA1 silenced by aberrant methylation in gastric cancer cells [Our search of literature revealed there are occasional rare studies on  sarcoma ,5. SMARC pathway . A recenSMARCA1 genomic alterations may have different effect on protein expression. Loss of SMARCA1 IHC expression in MPNST with no genomic alterations in this subtype suggests that other mechanisms may drive SMARCA1 down regulation on the transcriptional or posttranslational level. Further investigation into loss of expression of SMARCA1 with more cases is required to study the association between SMARCA1 genomic alterations and protein expression, and the utility of SMARCA1 loss of expression for diagnostic and prognostic purposes. More studies on regulation and function of SMARCA1 are required to further understand its role in neoplasia and possibly elucidate interaction with other molecules and chromatin remodeling complexes for better therapy.We found genomic alterations in soft tissue sarcomas in TCGA dataset, namely leiomyosarcomas, liposarcomas and undifferentiated sarcomas. Data on methylation was not available in the dataset. The finding of SMARCA1 IHC loss in undifferentiated sarcoma is in keeping with TCGA molecular alterations. The fact that we did not find SMARCA1 IHC loss of expression in leiomyosarcomas and liposarcomas may be due to the different tumor cohorts and smaller number of cases in our study. In addition, SMARCA1 needs to be further explored in the differentiation process and neoplasia. The molecular mechanisms of SMARCA1 inactivation merit further investigation in order to understand and utilize its role in diagnostic, prognostic and therapeutic purposes.Our study is the first to demonstrate loss of expression of SMARCA1 in subtypes of soft tissue sarcomas, including undifferentiated sarcoma and malignant peripheral nerve sheath tumors. The role of"}
+{"text": "Preclinical data have shown that ERBB2 activating mutations are responsive to HER2 tyrosine kinase inhibitors. The aim of this study is to characterize the landscape of ERBB2 mutations in solid tumors and the potential efficacy of ERBB2 targeting.We analyzed the next-generation sequencing results from 17,878 patients with solid tumors and reported the outcome of 4 patients with advanced ERBB2-mutated tumors treated with a combination of trastuzumab and lapatinib.n\u2009=\u2009282) were known as activating mutations. ERBB2 mutation was not mutually exclusive of ERBB2 amplification which occurred in up to 10% of cases. PI3KCA activating mutations were associated with ERBB2 mutations in 12.4% of cases mainly in breast and lung cancer. Four patients  were treated with a combination of trastuzumab and lapatinib. All of them experienced tumor shrinkage resulting in stable disease in three cases and partial response in one case. One patient developed secondary resistance. Sequencing of the progressing metastasis allowed the identification of the ERBB2 L869R mutation previously associated with resistance to lapatinib in vitro.ERBB2 mutations occurred in 510 patients (2.85%). The tumor types with the highest incidence of ERBB2 mutations were the following: bladder (16.6%), small bowel (8.6%), ampullar (6.5%), skin non-melanoma (6.1%), and cervical cancer (5.5%). 49.4%  contains supplementary material, which is available to authorized users. To the EditorERBB2 mutations in a large panel of tumors analyzed by next-generation sequencing (NGS) and reported the clinical impact of ERBB2 targeting in this setting as well as potential mechanisms of secondary resistance.We investigated the incidence of We analyzed the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE)  and the ERBB2 mutations involving all the domains of the receptor  of the mutations identified were described as oncogenic according to COSMIC and were more frequently detected in the bladder (9.4%), small bowel (7.1%), ampullar (6.5%), cervical cancer (4.1%), and nerve sheath tumor (2.9%), respectively , CDK12 amplification , and PI3KCA mutation .The three most frequent alterations co-occurring with ERBB2 amplification and mutation, 16 (32%) were breast ductal carcinoma, 8 (16%) lung adenocarcinoma, and 6 (12%) bladder urothelial carcinoma.Among the 39 tumors bearing both ERBB2 mutation-bearing tumor were treated with dual ERBB2 blockade with trastuzumab + lapatinib and experienced clinical benefit  may be dependent on the type of ERBB2 alteration and type of tumor. For instance, no clinical activity was observed in bladder and colorectal cancers . Of notePI3KCA mutations represent one of the most frequent co-alterations identified in ERBB2-mutated tumors. The PI3K/Akt/mTOR pathway has been shown to play a potential important role in resistance to ERBB2 targeting therapy in ERBB2-overexpressing breast cancer. Moreover, preclinical studies indicated that inhibitors of this pathway can act synergistically with trastuzumab in resistant models [ERBB2-targeted therapy in ERBB2-mutated tumors.Interestingly, t models . This fiERBB2/EGFR inhibitor such as neratinib in the clinical setting [Unfortunately, targeted therapies suffer from a major limitation, that is, the duration of any observed clinical benefit is invariably limited in length, owing to the relatively rapid acquisition of drug resistance. We report here for the first time a case of a ERBB2 activation loop mutation in a patient with non-amplified ERBB2 mutant colorectal cancer with acquired resistance to trastuzumab combined with lapatinib. The L869R mutation is located within the activation loop of the kinase domain and associated with gain of function activity. This mutation, which was identified in the secondary progressing brain lesion was not present in the primary tumor, was shown to confer resistance to lapatinib in vitro but to be sensitive to second-generation ERBB2 in several tumor types and the promising preliminary activity of dual ERBB2 targeting reported here deserved further clinical investigation aiming to demonstrate that ERBB2-mutational driven therapy can improve patient care irrespective of histology.The significant mutation rate of Additional file 1:Supplementary Methods and Results. (DOCX 622 kb)"}
+{"text": "This study demonstrates that the tetraspanins Tspan5 and Tspan15 which directly associate with the metalloprotease ADAM10 have an opposite impact on its endocytosis and half-life. ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 , which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other\u2019s expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins. Many cell and developmental processes are regulated by a proteolytic cleavage of membrane-anchored proteins in their extracellular region, a process referred to as ectodomain shedding. Several proteases have been shown to be involved in this process, including several members of the ADAM  family of membrane-anchored metalloproteases . ADAM10 The activity of ADAM10 is regulated by both intrinsic properties and extrinsic factors. ADAM metalloproteases are synthesized as zymogens that remain catalytically inactive until the prodomain is released after cleavage by pro-protein convertases during transport to the cell surface . The recCaenorhabditis elegans and the three Drosophila TspanC8 tetraspanins regulate ADAM10 subcellular localization in vivo  undergoes faster internalization than ADAM10Tspan15 after reaching the plasma membrane, an anti-ADAM10 mAb added to the medium at 37\u00b0C should accumulate more rapidly inside the cells in Tspan5-positive/Tspan15-negative cells. We incubated U2OS cells at 37\u00b0C with the DyLight 650\u2013labelled ADAM10 mAbs for different times. After detachment, the cells were incubated with a PE-labelled secondary reagent, and the fluorescence was measured by flow-cytometry. DyLight 650 fluorescence measures the total pool of ADAM10 having passed through the plasma membrane (either still present or endocytosed) and the PE fluorescence the surface ADAM10 fraction. As shown in Antibodies to the ectodomain of proteins that traffic through the plasma membrane can bind to their target, even if the passage at the plasma membrane is transient  and are To validate the previous findings in a different cell model, Tspan5 and Tspan15 were also expressed in HeLa cells. Both tetraspanins induced a similar twofold increase in ADAM10 surface expression levels . Again, So far, the data show that Tspan15 expression is associated with both a higher stability and a diminished endocytosis of ADAM10. We reasoned that if the higher stability of the protein is a consequence of the stabilization at the cell membrane, the surface pool of ADAM10 should be more stable in the presence of Tspan15. To address this question, surface proteins were labelled with biotin, and the cells were incubated at 37\u00b0C for 15 h before lysis, or lysed directly to determine the initial level of surface protein. ADAM10 and CD81 were then immunoprecipitated, and the level of biotin-labelled protein was determined using streptavidin blotting . As a coThe preceding data showing that Tspan5 and Tspan15 differentially regulate ADAM10 trafficking and turn-over were obtained using transfected cells. To analyze the impact of endogenous Tspan5 and Tspan15, we generated Tspan5 and Tspan15 KO cells using the CRISPR/Cas9 gene editing technology.In a first model, ablation of Tspan5 in U2OS cells did not modify ADAM10 surface levels, but reduced its internalization rate by 50%, further suggesting that Tspan5 stimulates ADAM10 endocytosis . In PC3 The anti-Tspan5 mAbs TS5-2 immunoprecipitated only a small fraction of mature ADAM10 in parental cells. In contrast, most mature Tspan5 is associated with ADAM10, as shown by the comparison of the ADAM10 and TS5-2 IPs and by the lack of immunoprecipitation of the upper forms with TS5-1r .There was more Tspan5 in the Tspan5 IP from Tspan15 KO cells , consistTetraspanins are palmitoylated at several conserved juxtamembrane intracellular cysteines. Tspan15, in contrast to Tspan5, lacks such typical tetraspanin intracellular cysteines . BecauseTo test whether Tspan5 and Tspan15 are palmitoylated proteins, the cells were incubated with azido palmitic acid and palmitoylated proteins were labelled with biotin using click chemistry. As expected, Tspan5 efficiently incorporated palmitic acid and the Tspan5plm mutant, in which all four cysteines that are putative palmitoylation sites were changed to alanines, did not , indicatBoth the Tspan5plm mutant and the Tspan15 CCLC mutant were expressed at the cell surface similarly to the WT proteins (data not shown). However, these mutations did not affect the influence of Tspan5 and Tspan15 on ADAM10 surface expression levels in U2OS cells or Notch activity .To get further insights into the mechanisms whereby Tspan5 and Tspan15 differentially regulate ADAM10 endocytosis and Notch signaling, we generated a series of Tspan5/Tspan15 chimeras  and exprA major difference between Tspan5 and Tspan15 is the presence in Tspan15 of a cytoplasmic intracellular C-terminal domain unusually long for a tetraspanin . Replacing the N- and C-cytoplasmic domains of Tspan5 by that of Tspan15 (T5NC15) yielded a chimera that strongly inhibited ADAM10 endocytosis in HeLa cells . ChimeraWhen expressed in U2OS cells, all chimeras in which the cytoplasmic domains of Tspan5 and Tspan15 were exchanged stimulated an increase in ADAM10 surface expression level (about twofold) that was lower than that induced by Tspan15 (Tspan5 overexpression did not result in an increase in ADAM10 surface levels) . At leasWe also analyzed the effect of these chimeras on Notch signaling . The repThe above data show that Tspan5 and Tspan15 have a positive and a negative impact, respectively, on ADAM10 endocytosis. Consistent with this finding, the Tspan5 mAb was efficiently internalized after 1 h at 37\u00b0C in HeLa/Tspan5 cells, and the internalized fraction colocalized with ADAM10, suggesting again that both proteins were internalized together , top. HoTo validate the hypothesis that ADAM10 stabilizes Tspan15 at the cell surface, we generated ADAM10 KO PC3 cells. Although there was no change in Tspan15 mRNA levels (data not shown), there was a 60% decrease in Tspan15 surface expression levels  in theseThis finding may explain why transfection of Tspan5 in PC3 cells led to a \u223c40% decrease of Tspan15 surface expression . The oveThe trafficking and function of the metalloprotease ADAM10 is regulated by the members of the TspanC8 subgroup of tetraspanins. Whereas all TspanC8 members allow ADAM10 to exit from the ER, Tspan10 and Tspan17 were shown to target ADAM10 to late endosomes, and the remaining four members target ADAM10 to the plasma membrane . Here, wAlthough both Tspan5 and Tspan15 target ADAM10 to the plasma membrane, Tspan15 is associated with higher ADAM10 surface expression levels: indeed, transfection of Tspan15 in a Tspan5/Tspan14\u2013positive cell line (U2OS) induced a large increase in ADAM10 surface expression levels, whereas transfection of Tspan5 in a Tspan5/Tspan15\u2013positive cell line (PC3) decreased the ADAM10 surface level. In addition, we observed that the surface staining of PC3 cells with a Tspan15 mAb was nearly ten times higher than that observed with a Tspan5 mAb, suggesting a much higher expression level of Tspan15 at the protein level. Accordingly, Tspan5 knockout had little effect on ADAM10 expression, whereas Tspan15 knockout reduced by 75% the surface expression of ADAM10 suggesting that most surface ADAM10 molecules are associated with Tspan15 at the steady state level. In both the U2OS and PC3 models, we have shown that the higher ADAM10 surface levels observed in the presence of Tspan15 reflects a higher total expression level, which is explained at least in part by a longer ADAM10 half-life.Contrasting with U2OS and PC3 cells, Tspan5 transfection in HeLa cells yields a twofold increase in the ADAM10 surface level. This is most likely because in this cell line, ADAM10 is retained to a large extent in the ER because of a limiting amount of TspanC8 . Thus, tTspan15 is at least in part the consequence of a longer residence time at the cell surface and that the analysis of ADAM10 cell surface expression at the steady state does not reflect the overall amount of ADAM10 having passed through the plasma membrane. Indeed, we have shown in several cellular models that ADAM10Tspan5 undergoes faster endocytosis than ADAM10Tspan15, and that after endocytosis, ADAM10 is targeted to CD63-positive compartments which include late endosomes and lysosomes. Notably, incubation of the cells with an anti-ADAM10 mAb showed a time-dependent accumulation of the mAbs inside the cells, which was inhibited by the expression of Tspan15 (either in U2OS-transfected cells or WT PC3 cells). After 20-h incubation with the anti-ADAM10 mAb at 37\u00b0C, staining of Tspan15 KO PC3 cells is comparable with that of parental cells or Tspan5 KO cells, indicating that although the ADAM10 level is strongly reduced in Tspan15 KO PC3 cells, the amount of ADAM10 trafficking through the plasma membrane is similar in all cell types. Thus, the fact that ADAM10 is mainly associated with Tspan15 at a steady state in PC3 cells is not due to a lower Tspan5 level but mainly if not exclusively to the shorter residence time of ADAM10Tspan5 at the plasma membrane.Our data indicate that the longer half-life of ADAM10Tspan5 and ADAM10Tspan15. Because Tspan14 is the major TspanC8 tetraspanin expressed by HeLa and U2OS cells (besides Tspan5 in this latter case), it is possible that ADAM10Tspan14 is endocytosed at a rate intermediate between that of ADAM10Tspan5 and ADAM10Tspan15. Further work will be required to test this hypothesis when mAbs to Tspan14 are available.Tspan15 was shown to stabilize ADAM10 and to inhibit its endocytosis in all cell lines tested and using all assays performed. This, together with the finding that its deletion is associated with a decrease of the mature form of ADAM10 in the brain , suggestThe endocytosis assays were performed using an antibody uptake assay, raising the question of the influence of the mAbs on ADAM10 endocytosis. The finding that incubation at 37\u00b0C with the anti-ADAM10 mAbs led to a decrease in ADAM10 at the surface of cells lacking Tspan15, that is, in U2OS and U2OS/Tspan5 cells and in tspan15 KO PC3 cells , indicates that the mAb accelerates to some extent the rate of ADAM10 endocytosis. Nevertheless, several lines of evidence indicate that the internalization of the mAb mirrors the endocytosis of ADAM10 under normal conditions. First, the decrease at the cell surface of U2OS cells is for the most part observed when comparing the labelling after cell detachment with that observed after 1 h at 37\u00b0C, suggesting that cell detachment may accelerate endocytosis of antibody-bound ADAM10 in these cells. This could explain why we observe after 1-h incubation \u223c20% endocytosis by confocal microscopy and >50% using the flow cytometry protocol. Second, at time points later than 1 h, both in U2OS cells and Tspan15 KO PC3 cells, the mAbs continue to undergo effective internalization, accumulating into the cells at a much higher level than the initial surface level, whereas the surface level decreases proportionally much less.The finding that overexpression of Tspan5 in U2OS cells does not induce an increase in the ADAM10 surface level contrasts with our previous study using GFP-tagged Tspan5 and Tspan15, which induce a three and five times increase in ADAM10 surface levels, respectively . This isWe have started to study how Tspan5 and Tspan15 differentially affect ADAM10 surface levels by analyzing the activity of Tspan5/15 chimeras. Our data indicate a role for the cytosolic domains of Tspan5 and Tspan15 but also indicate that other domains contribute to their effect on ADAM10 endocytosis. This complex situation contrasts with the recent finding that replacing the C-terminus of Tspan15 by that of Tspan33 was sufficient to target Tspan15 and ADAM10 to apical junctions .It has been suggested that the intracellular domain of ADAM10 interacts with the clathrin adaptor AP2 through a non-canonical AP2-binding motif that was shown to regulate to some extent the internalization of an IL2R\u03b1/ADAM10 chimera . BecauseTspan15 at the plasma membrane could be due to a different recycling rate. In this regard, it was shown that Rab14 and its Guanine nucleotide exchange factor FAM116A play a role in recycling membrane proteins, and that depletion of these molecules in A549 cells, which strongly express Tspan15 (data not shown), induced the accumulation of ADAM10 in an intracellular transferrin-positive compartment  yielded chimeras which had an intermediate effect on ADAM10 surface levels in U2OS cells and were not inhibitors of Notch signaling. Based on these data and the previous demonstration that Tspan5 and Tspan15 differentially regulate ADAM10 membrane compartmentalization , we suggTspan15 at the plasma membrane is a consequence of the interaction with yet unknown proteins that stabilize this complex at lateral contacts, where it could process certain cadherins and perhaps other substrates. Concerning Tspan5, the short membrane residence time of ADAM10Tspan5 may prevent ADAM10 from processing discrete substrates until appropriate, for example, after it is stabilized upon proper signaling. This may also allow ADAM10 levels to be increased in a transient manner in response to a particular signal. This may be especially true in the nervous system where Tspan5 is abundantly expressed . Labelling of antibodies with DyLight 650 or 550 N-Hydroxysuccinimide Esters was performed according to the manufacturer\u2019s instructions (Thermo Fisher Scientific). All fluorochrome-labelled anti-Ig antibodies were from Thermo Fisher Scientific. The plasmids encoding Tspan5 and Tspan15 fused to GFP were previously described (GICLAQN-Tspan5 for T15C5 and Tspan5-ALLQIFGVLLTLL-Tspan15 for T5C15. The sequences of the swap sites at the N-terminus were Tspan15-FSYLWLKYFIFGF-Tspan5 for T5N15 and T5NC15 and Tspan5-PEVSCCIKFSLII-Tspan15 for T15N5 and T15NC5 (the conserved amino acid where the swap was made is shown in boldface and is underlined).The mAb directed to human ADAM10 (11G2), CD9 (TS9), CD81 (TS81), CD63 (TS63), and Tspan5  were generated in our laboratory . The mAbescribed . After Pescribed . The seq6 cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. cDNA synthesis was performed from 1 \u03bcg of total RNA using 200u of SuperScript III Reverse Transcriptase (Invitrogen) primed with random hexamer (Promega), in a 20-\u03bcl reaction volume. Quantitative real-time PCRs (qPCRs) were then carried out on duplicates in a final volume of 25 \u03bcl containing 2\u00d7 GoTaq qPCR Master Mix (SYBR Green) from Promega, 0.4 \u03bcM each forward and reverse primers and 1 \u03bcl cDNA. Quantification was performed with the Mx3005P QPCR Systems and MxPro software (Agilent). The values were normalized to rpl38 expression according to the \u0394Ct method. The oligonucleotides used for amplification have been previously described   were culhttps://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). The corresponding guide DNA sequences were cloned into the lentiCRISPRv2 plasmid  according to the instructions of the Zhang laboratory (https://www.addgene.org/52961/)  and the/52961/) . The plaCells were detached with Trypsin\u2013EDTA, washed in complete DMEM, and incubated for 1 h at 4\u00b0C with 10 \u03bcg/ml primary antibody or hybridoma supernatant. After three washings, the cells were incubated for 30 min at 4\u00b0C with an Alexa647-conjugated goat anti-mouse antibody. The cells were analyzed using an Accuri C6 flow-cytometer (Becton Dickinson).2. The cells were transfected 24 h later with the Notch luciferase reporter and Renilla plasmids using FuGene HD (Promega). 24 h later, the cells were co-cultured with OP9 or OP9-DLL1 at 35,000 cells/cm2. The activities of firefly and Renilla luciferases were determined using a dual luciferase reporter assay (Promega) according to the manufacturer\u2019s instructions.This analysis was performed as previously described : U2OS ce2 and MgCl2 was added to the buffer for maximal tetraspanin/tetraspanin interaction . Western blotting on lysates was performed using appropriate primary and Alexa Fluor 680-labelled secondary antibodies. Western blotting on immunoprecipitations was performed using biotin-labelled antibodies and fluorescent streptavidin except for the anti-Tspan15 and the CD81 mAbs. All acquisitions were performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).Immunoprecipitations were performed as previously described with minor modifications . For immeraction . After 34, 1, 5 \u03bcl reaction buffer additive 1, and after 3 min 3 \u03bcl click-it additive 2. After 1 h at room temperature, 20 \u03bcl 3\u00d7 Laemmli buffer was added to the reaction. The samples were analyzed by Western blot using Alexa680-labelled streptavidin.All reagents used for the analysis of Tspan5 and Tspan15 palmitoylation by click chemistry were obtained from Life technologies. U2OS cells expressing GFP-tagged Tspan5, Tspan15, or the mutants were incubated overnight with 50 \u03bcM click-it palmitic acid (15-azidopentadecanoic acid) in serum-free medium. After three washes, the cells were lysed in PBS containing 1% Triton X-100, and the proteins were immunoprecipitated using GFP-trap beads (ChromoTek). After three washing, the beads were resuspended in 15 \u03bcl of a solution containing 50 mM Tris HCl, pH 8 and 0.5% SDS. Biotinylation of the proteins labelled with the modified palmitic acid was performed by subsequent addition of 15 \u03bcl 40 \u03bcM biotin alkyne in click-it reaction buffer, 1, 5 \u03bcl CuSO4Cl in PBS. For surface labelling, the cells were then incubated directly for 1 h with 10 \u03bcg/ml of antibodies in PBS supplemented with 0.1% BSA at room temperature. For intracellular labelling, the cells were incubated with antibodies in the same buffer supplemented with 0.1% saponin. The binding of primary antibodies was revealed using appropriate secondary reagents. The cells were mounted in Prolong Gold (Thermo Fisher Scientific) supplemented with DAPI and examined with a Leica SP5 confocal microscope .The cells grown in complete medium were fixed for 15 min with 4% paraformaldehyde at room temperature, washed in PBS and then incubated for 15 min in 50 mM NHhttp://icy.bioimageanalysis.org). The purpose of this protocol is to use the surface staining (\u201csurface image\u201d) to generate a mask allowing removing the surface labelling from the total staining . An inherent difficulty of this approach is that the results may vary considerably according to the threshold applied to the images. By definition, the internalized pool is not labelled by the secondary reagent or minimally in rare cases of partial permeabilization upon fixation. The surface and internalized pool can, therefore, be distinguished by the ratio of the two fluorescence intensities, independently of any threshold applied. Thus, one of the first steps in our protocol is to divide the \u201ctotal image\u201d by the \u201csurface image.\u201d A threshold image is then generated, which allows removing from the total image the fraction of the signal that overlaps with the surface labelling, yielding a new image with only the intracellular labelling . The intracellular signal is quantified using the spot detector plugin. The total and surface images are quantified after applying a threshold determined using the K-mean method. In HeLa cells, the relative signals of the internalized fraction to the surface fraction were so different in the various transfectants that using the K-mean method on each cell type was not reliable. We, therefore, applied on all HeLa transfectants a threshold calculated on parental cells using the K-mean method. The percentage of endocytosed mAbs was obtained by dividing the value of the internal signal by that of the total signal. Three to eight fields were acquired for each experiment and averaged. At least three independent experiments were performed for each cell type. All images of internalization assays are maximum-intensity projections of the z-stacks.The cells were incubated for 1 h at 37\u00b0C with the anti-ADAM10 mAb 11G2 coupled to DyLight 650. After fixation or cooling to 4\u00b0C, the surface pool is then labelled with an anti-mouse polyclonal antibody coupled to Alexa Fluor 568 or Alexa Fluor 488. Images were acquired every 0.25 \u03bcm throughout the height of the cells using a Leica SP5 confocal microscope . Quantification was performed using a protocol developed in Icy imaging software   in the presence of saponin to reveal the internalized mAb. Quantification using Icy was performed as above, except that an endocytosis index was obtained by dividing the value of the internal image by that of the surface image.i) as follows: FL4i = [FL4n-(FL2n \u00d7 FL40/FL20)], where FL20 or FL2n and FL40 or FL4n are the PE and DyLight 650 mean fluorescence intensity of nontreated cells (labelled after detachment) and cells treated for n hours. The ratio FL4i/FL40 was then calculated to express the data relatively to the amount present at the cell surface at the steady state level.The cells were incubated at 37\u00b0C with 10 \u03bcg/ml DyLight 650\u2013labelled ADAM10 mAb for different times. After detachment using accutase, the cells were incubated at 4\u00b0C with a PE-labelled secondary reagent, and the fluorescence was measured by flow-cytometry. In each experiment, a fraction of cells was not incubated with the ADAM10 mAb at 37\u00b0C, but at 4\u00b0C after detachment, and subsequently with the PE-labelled secondary reagent. This allows determining the ratio of PE (in FL2 channel) to DyLight 650 (in FL4 channel) fluorescence when the ADAM10 mAb is only at the cell surface, and thus an estimation of the surface fraction of ADAM10 from the DyLight 650 fluorescence in the cells incubated with the mAb at 37\u00b0C. The mean fluorescence intensity in the FL4 channel corresponds to the sum of the fluorescence of the mAb present at the cell surface and internalized. We estimated the fluorescence intensity corresponding to the internalized mAb (FL4Statistical analysis was performed with GraphPad Prism on independent experiments as indicated in the legend to the figures. All graphs show the mean \u00b1 SEM."}
+{"text": "Statins are an important class of medications in reducing the risk of cardiovascular events as well as overall mortality. However, a well-known adverse effect of statins is skeletal muscle toxicity, which may lead to abrupt discontinuation of the statin. In turn, patients may miss out on the benefits of statin therapy. An important factor to consider is a patient's solute carrier organic anion transporter 1B1 (SLCO1B1) gene T521C polymorphism status. Herein, an overview of the pharmacogenetics of SLCO1B1 is provided as well as recommendations for use in practice. Statins are, by and far, the number one prescribed class of medication worldwide . Their mThe most common side effect associated with statins is skeletal muscle toxicity that includes pain without evidence of muscle degradation , pain with evidence of muscle degradation (myopathy), and severe muscle damage with acute kidney injury (rhabdomyolysis) . ControlRegarding this association between statins and skeletal muscle toxicity, an important pharmacogenetic marker to consider is the SLCO1B1 gene. The solute carrier organic anion transporter family member 1B1 (SLCO1B1) protein facilitates the hepatic uptake of statins . The mosThe Clinical Pharmacogenetics Implementation Consortium (CPIC) has published dosing recommendations regarding simvastatin and the activity of the SLCO1B1 enzyme (Table CPIC also published information regarding the percent increase in statin exposure in individuals with the SLCO1B1 c.521 CC genotype [Pharmacogenetic testing for the SLCO1B1 gene can be helpful to clinicians who may have a suspicion that a patient\u2019s statin therapy is causing skeletal muscle toxicity. Also, utilizing SLCO1B1 genotyping can help in deciding which statin may be best for providers initiating statin therapy. Adjustments to the dose of a statin or choosing an alternative can be the difference for a patient especially in those cases where prematurely stopping a statin places the patient\u00a0at risk of losing the cardiovascular benefits of this therapy. Consideration of following an algorithm where SLCO1B1 gene testing is incorporated in practice can help patients initiating a statin or considering an alternative statin . Provide"}
+{"text": "Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of  Plasmodium spp. parasites. In 2016 there was an estimated 216 million cases reported, resulting in 445,000 deaths, mostly of children under 5 [Plasmodium parasites invade erythrocytes and remodel them to obtain supplementary nutrition from the blood plasma and to evade the immune system. Symptomatic malaria disease is caused by intracellular blood stage parasites which are enveloped in a parasitophorous vacuole membrane (PVM) within the erythrocyte. Residing on the PVM is an essential protein translocon called PTEX (Plasmodium translocon for exported proteins) [Almost half the world\u2019s population is at risk of malaria, the disease caused by infection with roteins) , 4.Plasmodium genus. The first is HSP101, a AAA+ heat shock protein chaperone which is predicted to form a hexameric structure to unfold proteins for export [Plasmodium berghei since its gene can be deleted, however its loss reduces export efficiency and virulence [P. berghei resulted in reduced sequestration and virulence [P. falciparum resulted in reduced binding to the endothelial receptor CD36 [PTEX consists of five core components, HSP101, PTEX150, EXP2, TRX2 and PTEX88 [irulence , 6, 7. Pirulence , 10, andThe final core PTEX protein is EXP2 which lacks predicted transmembrane spanning domains typical of membrane pores, yet it is the most membrane associated protein of the PTEX complex , 12. VerToxoplamsa gondii [In addition to a full size PTEX complex of >1200 kDa, we have shown EXP2 forms homo-oligomers of approximately 600 and 700 kDa in size by blue native polyacrylamide gel electrophoresis raising the question of what the smaller forms could be doing [a gondii , 16, 17.a gondii .Export of effector proteins can be inhibited by disrupting the PTEX complex by inducibly knocking down expression of HSP101, PTEX150 and EXP2 , 14 or bP. falciparum we have inducibly knocked down EXP2 expression. We show that EXP2 knockdown is detrimental for parasite growth and the extent of growth arrest is correlated with the degree of knockdown. Upon restoration of EXP2 expression parasite growth can partially recover dependent on the length and strength of knockdown. Upon EXP2 knockdown, growth arrest was probably due to the inhibition of export of essential effector proteins since the export of a protein marker was also curtailed.To explore the role of EXP2 in the asexual blood stages of P. falciparum genomic DNA, the 3\u2019 end of the EXP2 coding region was amplified with forward primer EXP2_1F: AGGAGATCTGGTCACGTATGTGGTGGGTA and reverse primer EXP2_2R A\u2019: CTTATACTGCAGCTTCTTTATTTTCATCTTTTTTTTCATTTTTAAATAAATCTCCACTGGCA. After digestion with BglII and PstI the exp2 DNA fragment was ligated into pPTEX150_HAglmS after the ptex150 flank was removed by digestion with the same enzymes to produce pEXP2-HAglmS.From 100 ng of 3D7 P. falciparum was cultured in human RBCs  at 4% haematocrit (HCT) in AlbuMaxII media  at 37\u00b0C as described previously [P. falciparum strain 3D7 was subsequently cultured [exp2 locus. Once western blot validation with anti-HA IgG indicated the exp2 locus had been appended with the HAglmS construct, clonal parasite lines were isolated by limiting dilution. Successful integration was validated by performing diagnostic PCRs with genomic DNA isolated from each clonal parasite line using the primers: A TGCAGAAACAACTTTGCCACA; A\u2019 TGGCATCTTCTTCTTCAACGGT; B\u2019 TCCTTACGGCTGTGATCTGC.eviously . 100 \u03bcg cultured . TransfeTo knockdown protein expression in EXP2-HAglmS parasites 1M glucosamine (Sigma) was added at the desired concentration to 5 mL of 1% synchronous ring stage parasites in 4% hematocrit. Parasites were grown for 1 cell cycle (~48 h) and then harvested by centrifugation. The cell pellet was treated with 0.09% saponin in PBS with Complete Protease Cocktail Inhibitors (Roche) to remove the haemoglobin. Cell proteins were solubilised in reducing SDS sample buffer and fractionated on 4\u201312% acrylamide Bis-Tris gels (Invitrogen) and transferred via iBlot to nitrocellulose membranes (Invitrogen). Membranes were blocked in 1% casein and probed with mouse EXP2 monoclonal, rabbit anti-ERC IgG and chicken anti-HA IgY (Abcam) as per [For parasite growth and recovery assays 5 mL ring stage parasites at 4% hematocrit and 3% parasitemia were treated with the desired GlcN concentration for 1 day. Thin blood smears were then made and 3 x 100 \u03bcL aliquots of culture removed for LDH assays. The remaining culture was treated with 0.09% saponin with Complete Protease Cocktail Inhibitors (Roche) to remove the haemoglobin. Parasites to be sampled 2 and 3 days after GlcN treatment were set up as above but diluted 1/5 in 4% hematocrit and those to be sampled after 5 days were diluted 1/25. At days 2 and 3 post GlcN treatment samples were removed for analysis as above. Parasites that were to be grown for 5 days were fed on days 2 and 3 with fresh media and washed to remove GlcN or fresh GlcN was added back. After completion of the time course lactate dehydrogenase (LDH) assays that correlate with parasitaemia were performed as described previously  and absoIFAs were performed as described previously . BrieflyP. falciparum in in vitro culture [exp2 null parasites, an alternative approach was adopted where an inducible knockdown system based on the glmS ribozyme was employed [exp2 was substituted with a heterologous 3' UTR containing glmS (exp2 coding sequence for antibody-based detection of the protein. In this knockdown system the glmS ribozyme self cleaves the mRNA upon addition of glucosamine (GlcN) to the parasite culture, leading to removal of the poly-A tail, destabilization of the mRNA and subsequent reduction in protein production. The EXP2-HAglmS construct was introduced into the exp2 locus of the wildtype 3D7 parasite line by homologous recombination , before expression of EXP2 peaks at approximately 20\u201330 hpi , 26. ParIt was expected that a reduction in EXP2 levels would have major effects on the growth and protein export of the parasites on the basis that reducing the function of the other two main PTEX components, PTEX150 and HSP101, had a significant effect , 4. To iNext, parasite proliferation using lactate dehydrogenase (LDH) activity as a marker of biomass was measured and indicated that GlcN-induced knockdown of EXP2 proportionally reduced proliferation after 3 days (cycle 1) . Growth To further explore the relationship between EXP2 levels and parasite growth, 0.5 and 2 mM GlcN that had been added to EXP2-HAglmS and 3D7 rings in cycle 0 was washed out in cycle 1 at rings (2 day GlcN treatment) or trophozoites (3 day GlcN treatment). The parasites were then further grown until cycle 2 (day 5) and compared with untreated parasites (0 mM GlcN) and with parasites continuously treated with GlcN for 5 days. Parasite samples from days 1, 2, 3 and 5 were retained for analysis and LDH assays indicated that in parasites treated with 0.5 mM GlcN for 5 days significant growth inhibition was only observed for EXP2-HAglmS. For parasites in which 0.5 mM GlcN was removed after 2 and 3 days, growth almost fully recovered by day 5 . For 2 mThe EXP2-HAglmS parasites were also monitored by microscopy throughout the knockdown and recovery period. In 0.5 mM GlcN the parasites appeared to lag behind the untreated parasites so that by day 5 the trophozoite stage parasites were smaller than the untreated trophozoites and younger rings stages were also present . By day Western blot analysis was next performed on EXP2-HAglmS to determine how protein levels responded during knockdown and recovery. A second 5 day knockdown and recovery assay was performed and the LDH growth results were similar as before . WesternPfEMP1 export [In light of EXP2's putative PTEX pore function we expected that the growth arrest observed upon reduction of EXP2 levels would be due to the parasite's inability to export critical proteins into the erythrocyte. To determine if protein export in the EXP2 knockdown parasites was curtailed we performed microscopy on skeleton binding protein 1 (SBP1), an early exported protein, in ring stage parasites before growth arrested. Once exported, SBP1 accumulates at Maurer's clefts which are parasite-formed vesicular structures that play a role in 1 export , 29. ImmThe exposure times required to produce satisfactory images of the untreated 3D7 parasites were used for all subsequent imaging and likewise all brightness and contrast settings were identically adjusted for the SBP1 and EXP2 fluorescence channels. Imaging of EXP2-HAglmS parasites indicated that EXP2 levels were markedly reduced following GlcN treatment . FurtherAlthough we have previously reported GlcN mediated export trapping in PTEX150-HAglmS parasites we repeated this here for comparison with EXP2-HAglmS . GlcN trglmS ribozyme onto EXP2 and reduced its expression by up to 84% following treatment with 1 mM GlcN. This appeared to arrest parasite growth in late rings one cycle after adding GlcN to degrade the protein\u2019s mRNA. The EXP2-HA knockdown parasites export SBP1 less efficiently than parasites with normal levels of EXP2 suggesting growth arrest could be due to the failure to export functionally crucial proteins other than SBP1, which itself is not essential [To investigate the role of EXP2 in the PTEX protein export complex we attempted to knockdown EXP2 and examine subsequent phenotypes. Our knockdown strategy appended a HA tag and ssential , 29. Durssential . Both thssential , 14.glmS mediated knockdown of EXP2-HA was responsible for growth reduction is provided by the fact that the HAglmS tag only appeared to append to EXP2 and no other genes since no other HA bands apart from EXP2 were present in western blots. The degree of knockdown of EXP2-HA protein was critically dependent on the concentration of GlcN added to the parasite culture and GlcN had no substantial effect on 3D7 parasites except at high concentrations (\u2265 2 mM) for 3 or more days. Furthermore, when GlcN was removed EXP2 expression and parasite growth were partially restored. Specifically, when 0.5 mM GlcN was washed out after 2 and 3 days treatment, EXP2 protein levels and parasite proliferation almost fully recovered by day 5. At a higher dose of 2 mM GlcN for 2 days, only partial recovery occurred by day 5. GlcN mediated knockdown of EXP2 also greatly reduced export of SBP1 consistent with EXP2 being part of the PTEX complex [That the  complex , 29. Con complex .Our initial data suggested that growth of EXP2-HAglmS parasites was arrested by 0.5 mM GlcN treatment after 2\u20133 days, however closer investigation revealed that growth was more likely just slowed given that the parasites recovered well once GlcN was removed and EXP2 expression resumed. These parasites had about a half to a third the normal amount of EXP2-HA after 2\u20133 days treatment and could probably still export enough proteins and obtain sufficient nutrients to survive and hence recovered following GlcN removal. Whether slowed growth also resulted in fewer merozoites being formed was not examined, but nutrient restriction via knockdown of the new permeability pathway protein RhopH2 has been noted to reduce merozoite numbers per schizont . This coParasites seem to be only able to tolerate the strong knockdown of EXP2 caused by 2 mM GlcN treatment for a few days since after this, growth recovery was poor and dead parasites began to be observed after 3 days of 2 mM GlcN treatment. After 2 days of 2 mM GlcN treatment EXP2 levels had fallen to 20% of normal levels and this enabled many parasites to survive since they partially recovered by day 5 if the GlcN was washed out after 2 days. After 3 days 2 mM GlcN treatment when levels of EXP2 had fallen to ~10%, recovery was poor probably because most parasites had died by then. The ability of ring stage arrested parasites to partially recover growth as long as 2 days after arrest has been previously detected for another PTEX protein HSP101, and is consistent with our EXP2 observations here [P. berghei also reduced export of several different PEXEL proteins and in P. falciparum knockdown of HSP101 function reduced the export of several PEXEL and PNEP proteins [PfEMP1 display and the trafficking of other exported proteins, the precise functional role for most exported proteins is not understood, with a few exceptions [P. falciparum genome with piggyBac transposons has helped determine which genes might be unmutable and essential and are therefore a priority for future study [Both our study and Garten et al (2018)  showed tproteins , 4. A knproteins , 31\u201333. ceptions . A recenre study . AlthougP. falciparum blood stages probably because it is required for the efficient export of functionally important proteins into the erythrocyte compartment. Although this finding has been recently published [In conclusion, we have shown that EXP2 is crucial for survival of ublished  our studublished , still lS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file."}
+{"text": "Following publication of the original article , we haveIn this correction article, all members of the SRLF Trial group are now correctly shown in the collaborator list and all members are listed in Additional file The published apologizes for any inconvenience caused by this error.Additional file 1: Table S1. List of contributors; Table S2. Patients\u2019 characteristics according to the presence of hypoxemia; Table S3. ARDS exclusion criteria among patients with bilateral infiltrates; Table S4. ICU mortality according to ventilator support and hypoxemia class; Table S5. Univariate analysis of variables associated with mortality in hypoxemic patients; Table S6. aHRs of PaO2/FiO2 banding in different sensitivity analyses; Figure S1. Evolution of oxygen support after the day of study."}
+{"text": "After the Expression of Concern was posted on this article , 2, the This resolves the concern about data unavailability that motivated the editorial decision to post an Expression of Concern . The corS1 FileReport of statistical analysis.(PDF)Click here for additional data file.S2 FilePRISMA 5.0 reports.(RAR)Click here for additional data file.S3 FileGlobal data.(XLSX)Click here for additional data file."}
+{"text": "Transcription factors GATA3 and STAT4 mediate the cytokine-induced development of na\u00efve T cells into Th1 or Th2 types. In the present study, genetic analyses of GATA3 SNP rs3824662 and STAT4 SNP rs10181656 were performed to investigate the association of allelic and genotypic variations with the risk of T2D in the Bangladeshi population. A total of 297 unrelated Bangladeshi patients with type 2 diabetes and 247 healthy individuals were included in the study. The allelic and genotypic frequencies of rs10181656 located in the STAT4 gene were not found to be associated with risk of type 2 diabetes. The GATA3 rs3824662 T allele and mutant TT genotype had a significant association with the risk of T2D . Thus, the present study postulates that the genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population.Type 2 diabetes mellitus is a multifactorial metabolic disorder caused by environmental factors and has a strong association with hereditary issues. These hereditary issues result in an imbalance in CD4 Diabetes is a multifaceted metabolic disorder caused by impaired glucose metabolism characterized by hyperglycemia and is mainly classified as type 1 mellitus and type 2 diabetes mellitus. Both environmental and hereditary components play pivotal roles in the onset of diabetes  Impaired+T cells and decreased levels of na\u00efve CD4+T cells, which in turn play an important role in the pathogenesis of type 2 diabetes. GATA 3 binding protein (GATA3) and Signal Transducer and Activator of Transcription-4 (STAT4) transcription factors mediate the cytokine-induced development of na\u00efve T cells into Th1 or Th2 type. GATA3 consists of six exons and encodes a transcription factor with two transactivation domains and two zinc finger domains , while the over-dominant model had no effect. Even when either one allele or both alleles were mutated, the study participants were at risk of type 2 diabetes, which is reflected in the dominant model (comparing GG vs GT+TT)  or recessive model (comparing GG+TT vs TT)   independently had a significant association with the risk of disease  . When st662 GT in= 0.011] . 3DSNP c= 0.011] . Thus, dAlthough diabetes mellitus has been broadly classified as type 1 and type 2, increasing evidence has shed light on the fact that these two diseases overlap in many aspects. For example, classical immunological parameters for type 1 diabetes, such as anti-islet cell antibodies, elevated circulating cytokines and chemokines, are also present in many patients with type 2 diabetes ,47. In aThe STAT4 gene polymorphism is associated with increased risk for the development of early onset type 1 diabetes  but not Our findings demonstrate that genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population. The mechanism behind these roles needs to be investigated further. Moreover, studies with a large number of study participants from different ethnic populations are warranted to confirm the findings of this study.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 DataThis excel file contains the raw data for this study.(XLS)Click here for additional data file."}
+{"text": "The connections were directed by pendant phenyl groups to be between proximal ligand ends on the same faces of the grid. The 623link was separated from the topoisomeric granny knot by recycling size\u2010exclusion chromatography. The identity of each topoisomer was determined by tandem mass spectrometry and the structure of the 623link confirmed by X\u2010ray crystallography, which revealed two 82\u2010membered macrocycles, each in figure\u2010of\u2010eight conformations, linked through both pairs of loops.A molecular  Also formed is a 31#31Links  are mechanically connected closed loops (macrocycles).623link621link) but with a different crossing pattern. The topology can be depicted as two figure\u2010of\u2010eight shapes linked through both pairs of loops  or a granny knot  thiazolothiazole scaffold previously used to assemble a Solomon link  direct the pendant alkene chains above or below the grid plane, the biphenyl linkage significantly restricting the space accessible by the chain ends. The chain length was chosen to avoid over\u2010reaching, permitting only the required strand connections. Ethyl groups were used to increase the solubility of the benzimidazole units. These are smaller than the isopentyl groups used in the Solomon link synthesisTo achieve the required connectivity of the ligand strands, we designed bis\u2010tridentate ligand k Figure\u2005.6m The m414](BF4)8 was synthesized in analogous fashion, using ligand 1 and a small excess of iron(II) tetrafluoroborate . The 1H\u2005NMR spectrum of [Fe414](BF4)8 showed significant downfield shifts of the pyridine protons compared to the parent ligand (1), and a significant upfield shift of the benzimidazole proton Hs , which should face the pyridine ring of the perpendicular ligand strand in [Fe414](BF4)8 and therefore is indicative of the anticipated grid structure.To confirm that the modifications of the ligand would not hamper grid formation, we prepared a model ligand with methyl groups in place of the alkene chains . Complexation of the ligand with an equimolar amount of iron(II) or zinc(II) tetrafluoroborate proceeded smoothly in a mixture of dichloromethane and acetonitrile at room temperature to quantitatively afford the corresponding 2\u00d72 interwoven grids, which were characterised by NMR spectroscopy and X\u2010ray crystallography . The alkene\u2010bearing 2\u00d72 interwoven grid [Fe414](BF4)8 was subjected to ring\u2010closing olefin metathesis (RCM) as a dilute solution (1\u2005mm) in dichloromethane/nitromethane (3:1), employing the Hoveyda\u2013Grubbs second generation catalyst1H\u2005NMR indicated complete consumption of the starting material, and electrospray ionization mass spectrometry (ESI\u2010MS) showed multiply charged peaks corresponding to the successive loss of tetrafluoroborate counteranions from the fully\u2010closed grid . The crude product was demetallated  to afford a mixture of the metal\u2010free knot and link and other reaction byproducts, which were separated by preparative recycling size\u2010exclusion chromatography (GPC) .The 2\u00d72 interwoven grid (BF4)8 or [Fe43](BF4)8 respectively. The 1H\u2005NMR spectrum of each complex was significantly sharper than that of the mixture originally obtained from the RCM reaction .The individual topoisomers were each remetallated by treatment with excess iron(II) tetrafluoroborate in a chloroform/acetonitrile mixture under heating 8 obtained by remetallation of a mixture of the organic topoisomers that had been separated from other species by GPC . However, slow diffusion of toluene into a solution of [Fe42/3](BF4)8 in acetone yielded single crystals of, to our surprise, the demetallated623link. The coordination of thiazolothiazole ligands to FeII is rather weak2 causes it to crystallize from solution as it reversibly forms at very low concentration. The X\u2010ray crystal structure of 2 42/3] should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file."}
+{"text": "Dear Editor,DNA methylation at the 5-position of cytosine (5mC) is a crucial epigenetic mark in regulating biological processes including gene silencing, gene imprinting, and X chromosome inactivation  in MDA-MB-231 cells, which is even higher than that between H3K36me3 and 5mC . Importantly, further genomic distribution analyses revealed that the similarity between 5mC and H3K36me3 is restricted to genic regions as reported previously , which leads to massive overexpression of NSD2 and genome-wide gain of H3K36me2 . As revealed by other studies . We also found that the upregulated genes in KMS11 showed higher upstream DNA 5mC which tended to be lost in KMS11TKO, compared to the downregulated genes . While the 5mC levels of the CpG islands at the transcription start sites (TSSs) of the up- and down- regulated genes were generally low and similar, although statistically significant . This observation raises an intriguing possibility of the upstream hyper 5mC in KMS11 being functionally involved in the downstream gene transcription, which certainly needs future investigation.To further explore the functional importance on gene expression by the H3K36me2 and DNA 5mC axis, we carried out RNA-seq analyses in KMS11 and KMS11TKO, indicating that the global hypermethylation of 5mC is required for cancerous growth driven by NSD2 overexpression in KMS11.As NSD2 overexpression was demonstrated as the driver mutation for multiple myeloma bearing T translocation Below is the link to the electronic supplementary material."}
+{"text": "The highest concentrations originate from urban and agricultural areas, are driven by diurnal winds, and peak in the early morning. These enhanced atmospheric levels can be detected across a ~100km wide nearshore area and represent a significant addition to total oceanic CO2 uptake. A global estimate puts the added sea-air flux of CO2 from these greatly enhanced atmospheric CO2 levels at 25 million tonnes, roughly 1% of the ocean\u2019s annual CO2 uptake. The increased uptake over the 100 km coastal swath is of order 20%, indicating a potentially large impact on ocean acidification in productive coastal waters.Greatly enhanced atmospheric carbon dioxide (CO Agricultural practices can also impact local atmospheric CO2 on a diurnal cycle with large nighttime increases due to respiration and daytime decreases associated with photosynthesis [2 might impact marine air via atmospheric circulation and therefore increase the flux of CO2 into nearshore waters enhancing ocean acidification. Here we present novel observations from nearshore moorings and an autonomous sea surface vehicle that show the magnitude of urban CO2 pollution and allow us to calculate the contribution to sea-air fluxes from this previously unquantified source.The increase in atmospheric carbon dioxide  timeseries from multiple autonomous ocean based sensor platforms (moorings and surface vehicles) to provide a detailed assessment of the impacts of these high frequency variations in atmospheric CO2 concentration on sea-air CO2 fluxes in the nearshore environment.As indicated above, traditional estimates of sea-air CO2 instruments which were developed to measure CO2 concentrations in the air and water [2 instrument and CTD. Measurements were averaged hourly in the final analysis. Wave glider measurements were taken within 5km of the Monterey Bay Time Series (MBTS) Line, a transect extending from Moss Landing out 50km along the Monterey canyon  in the Monterey Bay region. Of these, three were moorings  Reference Network [2 = 0.71), as well as CarbonTracker [2 = 0.56), showed good agreement. We used the calculated baseline pCO2 values since these sources only run until December 2016 while our timeseries extends through early 2018.In this paper \u201catmospheric COnTracker  modeled 2 flux was estimated using the equation:2 in seawater [pCO2 is the difference between pCO2 water and pCO2 air. Gas transfer velocity is parameterized as a function of wind speed squared, while solubility is a function of water temperature and salinity. The common convention is used whereby a negative flux indicates CO2 transfer into the ocean (a sink); while a positive flux indicates release of CO2 into the atmosphere (a source).The net sea-air CO velocity, S is thseawater  and \u0394pCO2 sources, anomalies were calculated for each wave glider data point. At ten minute intervals the wind speed and direction were then used to calculate the source position of the wind; this process was repeated additively over the six hours prior to any given air CO2 measurement to construct a probable path for the measured air parcel. This method ignores local variation in wind direction and speed, mixing, and in particular excludes the effects of topography on offshore winds. In order to reduce these effects, all pixels within a five degree cone around the calculated path were assigned the value of the calculated anomaly, and added to the developing composite image containing all previous tracks. When complete the image was then divided by the number of tracks intersecting each pixel, producing an average map of probable atmospheric CO2 anomaly sources and their strengths. This method should not be seen as providing exact locations or intensities of anomaly sources but it is straightforward and provides a general picture of the origin of strong atmospheric CO2 anomalies.In order to construct a high resolution map of enhanced atmospheric CO2 anomalies to sea-air fluxes, two flux calculations were made. The first used the well-mixed oceanic air baseline CO2 concentrations calculated as described above. This dataset represents atmospheric CO2 concentrations used in studies of sea-air flux that rely on modeled or well-mixed atmospheric CO2 values. This timeseries of fluxes was subtracted from a second timeseries calculated using the observed pCO2 air concentrations. The difference between these two timeseries gives a measurement of flux due to our calculated atmospheric anomalies in air pCO2. This method preserves the convention of negative values indicating increased transport of CO2 into the ocean.To estimate the contributions of the atmospheric CO2 are detected on all platforms during periods of offshore winds. A time series of atmospheric CO2 from the OA1 mooring over 2014 and 2015 illustrates the extent of these anomalies  [2 at both these sites relative to well-mixed marine air.Significant positive anomalies in atmospheric COnomalies . In Fig t Creek)  and clea2 anomalies detected along the MBTS line originate from the Salinas valley and Hecker Pass  and the Salinas Valley . OA1 is also impacted by CO2 anomalies during periods of easterly winds from these topographic features. However, its largest anomalies are registered when winds blow from ~150 degrees. OA1 is situated only a few hundred meters north of the city of Monterey and the monthly averages at this buoy were much better correlated with the urban CO2 measurements from Walnut Creek (r2 = 0.78), than with CarbonTracker modeled atmospheric CO2 (r2 = 0.47)  . This suley area .2 anomalies with distance from shore. The wave glider provides the perfect platform to further explore this relationship. Frequent wave glider measurements extend 50km out to the end of the MBTS Line, and average anomalies of 6-10ppm are detected at the end of this line during periods of offshore winds. This shows that during offshore wind events the plume of high CO2 air extends at least 50km from shore, although its CO2 content is reduced on average from the 15 ppm anomalies seen nearshore Click here for additional data file."}
+{"text": "The effects of exogenous application of Zn2+ and of chelation of endogenous Zn2+ were examined on long-term potentiation (LTP) of stimulus-evoked synaptic transmission at Schaffer collateral (SCH) synapses in field CA1 of mouse hippocampal slices using whole-cell patch clamp and field recordings. Low micromolar concentrations of exogenous Zn2+ enhanced the induction of LTP, and this effect required activation of NMDA receptors containing NR2B subunits. Zn2+ elicited a selective increase in NMDA/NR2B fEPSPs, and removal of endogenous Zn2+ with high-affinity Zn2+ chelators robustly reduced the magnitude of stimulus-evoked LTP. Taken together, our data show that Zn2+ at physiological concentrations enhances activation of NMDA receptors containing NR2B subunits, and that this effect enhances the magnitude of LTP.The role of zinc (Zn Under normal physiological conditions, Zn2+ is released from presynaptic vesicles by low frequency synaptic transmission and interacts with multiple receptors that are involved in the induction of LTP, including NMDAR pyrimidin-4-amine . Slices were then transferred to microcentrifuge tubes and flash frozen with liquid nitrogen and stored in a -80\u00b0C freezer.Western blotting was performed on mouse hemi-sectioned brain slices (300 \u03bcm thick), which included not only the hippocampus but overlying cortex as well. Slices were treated in pairs as control , control + ZnFrozen tissue samples were boiled for 5 minutes and homogenized with high speed vortexing in lysis buffer . The samples were centrifuged afterwards for 5 minutes and the protein concentration was determined using a protein assay kit (Bio-Rad). 15 \u03bcg of protein was separated on 4\u201315% SDS-PAGE gradient precast gel (Bio-Rad). A separate gel was run for each animal. For each gel, equal aliquots were run in duplicate for each slice in adjacent lanes. This pattern was duplicated on the same gel so that all samples from the same animal were transferred to the PVDF membrane at the same time under the same conditions. After electroblotting, the membrane was cut in half and one half was developed with anti NMDAR2B antibody to visualize the total amount of receptor present in the sample and the other half was developed with anti PY (TYR1472) NMDAR2B antibody. Membranes were blocked with casein then incubated with primary antibodies at room temperature overnight followed by 1hr washing in TBST  at RT. Primary antibodies were NMDAR/NR2B and phospho-NR2B Y1472 primary antibodies  and anti-\u03b2-actin to normalize band intensities. Secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch) were visualized using chemiluminescence ECL-Plus reagent .The blots were densitometrically analyzed using Image-J. Quantitative comparisons were performed by normalizing each blot to a control lane and the relative value of the PY signal was divided by the total NMDAR2B signal. Thus a ratio of one is the value of control standard- the PY/NMDA for control is set to 1.P<0.05. Data points averaged for statistics are marked with a dashed bracket over the graphs. All data points analyzed were a 5 minute average per slice from the time points indicated by the brackets and are taken from within the last ten minutes of recording. Means \u00b1 SEM were calculated for each group and then compared to other experimental groups using 1-way ANOVA and Dunnett\u2019s multiple comparison test. The number of slices used for analysis included comparison of slices from within the same mice and between slices of different mice.Electrophysiological data were analyzed initially with Clampfit (v9) . Analyzed data were further processed and presented with Origin 6.1  and CorelDraw 10.0  programs. Statistical analysis were performed with SPSS (v11). Statistical data are presented as mean \u00b1 SEM unless indicated otherwise. Significance level was preset to 2+ in regulating synaptic plasticity, we commenced by bath applying exogenous Zn2+ to mouse hippocampal slices at a concentration of 1\u03bcM to evaluate stimulus-evoked LTP at SCH synapses in field CA1. To test the potential role of Zn2 (1\u03bcM) did not significantly alter fEPSP slope during the 15 minutes of baseline recording prior to high frequency stimulation (TBS). However 1\u03bcM ZnCl2 significantly increased the magnitude of LTP after TBS. Bath application of ZnCl2 at concentrations of 0.01\u03bcM and 10\u03bcM, did not significantly enhance the magnitude of LTP after TBS stimulation , it remained to be determined whether endogenous Zn2+ released by SCH terminals can influence LTP in a similar manner. The next set of experiments were designed to determine the effects of chelating endogenous Zn2+ on LTP, using the membrane impermeable Zn2+ chelator CaEDTA  was unable to enhance LTP in the presence of Ro25-6981, confirming a requirement of NR2B containing NMDA receptor activation for the enhancement of SCH-CA1 LTP by Zn2+. Thus, Zn2+ enhancement of LTP may be mediated by the up-regulation of NR2B-containing NMDARs to contribute to induction of LTP in adult rat hippocampus.The next step in our experiments was to see if NR2B-containing NMDARs subunits were required to enhance TBS induced LTP at CA1 synapses. Ro25-6981 did not significantly alter the magnitude of LTP when bath applied alone, which suggests that NR2B-containing NMDAR do not contribute substantially to control LTP elicited by TBS stimulation. This is consistent with literature suggesting that hippocampal NR2B subunits play a more predominate role in the induction of LTP in younger mice, particularly during development . However2+ crossing the membrane through other channels and activating intracellular signaling molecules [2+ mediated NMDA fEPSP. It appears that the timing of PP2 pretreatment was important; if ZnCl2 and PP2 were co-applied with no initial pre-treatment of PP2, NMDA fEPSPs were still enhanced, while pre-treatment with PP2 blocked the actions of Zn2+. This could be explained by more rapid transmembrane penetration of the charged Zn2+ compared to the larger PP2. Nevertheless, our data do suggest that Zn2+ acts via NR2B SFK phosphorylation to enhance NMDA fEPSP activity.Zinc modulation of NMDA receptors could be the result of Znolecules , 45. A lolecules ,13. In t2, supporting a requirement for SFK activation in modulation of LTP by Zn2+.During induction of stimulus-evoked LTP, there are crucial phosphorylation cascades that are activated, resulting in phosphorylation at multiple sites  on many proteins, and multiple levels of direct and indirect modulation of AMPAR and NMDAR transmission. In our experiments, PP2 did not alter TBS-induced LTP at SCH-CA1 synapses, suggesting that SFK activity is not necessary for the induction and maintenance of this form of LTP. However, PP2 did specifically block the increase in LTP elicited by 1\u03bcM ZnCl2+ enhancement of LTP, we examined the role of phosphorylation at the Y1472. Y1472 has been implicated as a key phosphorylation site of recombinant NR2B-containing NMDAR [2+ could alter the phosphorylation of the NR2B receptor subunit at baseline, without any stimulation to the tissue. Thus allowing us to examine whether or not the NR2B subunit was \u201cprimed\u201d by phosphorylation at Y1472 before application of high frequency stimulus. Western blot analysis revealed that 1\u03bcM ZnCl2 did not increase tyrosine phosphorylation of the NR2B subunit at Y1472 prior to TBS. The addition of the inhibitor PP2 had no influence on the basal state phosphorylation at the Y1472 site. Our findings suggest that exposure to Zn2+ without stimulation does not result in an increase in phosphorylation at the site Y1472. However, this does not rule out a role for Y1472 phosphorylation in the enhancement of LTP, which could occur with the combination of Zn2+ and high-frequency synaptic activation. There could be other sites of phosphorylation that do enhance at baseline or after stimulation which should be explored in this context.To further elucidate mechanisms by which PP2 inhibited Znng NMDAR . Phosphong NMDAR , 46,47. 2+ enhancement of LTP via activation of the SFK pathway is Ca2+-dependent, or enhanced due to a direct activation of the signaling pathway that bypasses Ca2+. Although NR2A containing NMDARs have been shown to predominantly contribute to LTP in adult mice, NR2B-containing NMDARs in Field CA1 do contribute to calcium transients, suggesting a potential regulatory role in LTP formation that could be up-regulated by Zn2+ [2+ may act to release RACK1 from NR2B thus increasing NMDAR activation.The question remains as to whether Zn by Zn2+ . Althoug by Zn2+ . A possi2+ enhancement of LTP are triheteromeric NMDA receptors. Trihetermoric NMDARs contain two NR1 subunits and both NR2A and NR2B subunits in combination, which, as some studies suggest, may comprise more than 50% of the NMDARs in the synapse in the hippocampus [2+ binding as well as to possible differences in pharmacological interaction could provide an alternative site of interaction and modulation. Further examination of the kinetics and functions of triheteromeric NMDA receptors at CA1 synapses in the presence of Zn2+ is warranted [2+ may cause a change in the interaction between NR2A and NR2B that favors one subunit over the other in the NMDA receptor complex, changing the mix of NMDARs in the synapse.Another possible contributor to Znarranted . As a co2+ as a modulator of LTP, not related to a generalized properties of heavy metals in the brain [2+ and exogenously applied Zn2+, bath application of exogenous ZnCl2 has the potential to interact with an extrasynaptic receptor population with a higher percentage of NR2B-containing NMDAR that is activated by glutamate spillover during high-frequency stimulation. In conclusion, the data presented here suggests that zinc influences activity-dependent synaptic plasticity within a relatively narrow range, the balance of which may be critical for regulation of LTP of synaptic strength underlying learning and memory formation. Dysregulation of zinc homeostasis that results in altered concentrations of Zn2+ release may be a key target for therapeutic intervention in a multitude of brain pathologies.The data presented here support the conclusion that low micromolar zinc enhances TBS-induced LTP of SCH-CA1 synapses through phosphorylation of NR2B subunits of NMDA receptors. Our findings suggests a specific endogenous role for Znhe brain . Given tS1 Fig2 . Neither 0.01\u03bcM nor 10\u03bcM ZnCl2 significantly altered LTP magnitude compared to controls .The time course of LTP in untreated control slices (circles) compared to slices treated with 10\u03bcM  or 0.01\u03bcM ZnCl(TIFF)Click here for additional data file.S2 Fig2+-free ACSF plus 25\u03bcM DNQX. ZnCl2 was bath applied after 15 minutes of stable baseline. 100nM ZnCl2  and 10\u03bcM ZnCl2  transiently enhanced NMDAR fEPSPs compared to untreated control slices , an effect that had reversed by one hour of application. (Each point mean \u00b1 SEM of n recordings). (B) Waveform of NMDA fEPSPs in presence of magnesium at 0\u2019 without zinc and after 60\u2019 zinc exposure demonstrating no effect in the presence of magnesium.(A)Time course of NMDAR fEPSPs (amplitude) enhanced and isolated by application of Mg(TIFF)Click here for additional data file.S3 Fig2  and NMDA evoked currents for control . Baseline was recorded for 10 minutes before application of ZnCl2. Bath application of ZnCl2 increased the area compared to baseline. .This is the time course of NMDA evoked currents for 1\u03bcM ZnCl(TIFF)Click here for additional data file.S4 Fig2 . (B) Histogram demonstrating how ifenprodil inhibited NMDA fEPSPS in the presence of 1\u03bcM ZnCl2. (C) Time course of NMDA fEPSP amplitudes in control slices  treated with NVP AAM077 alone, versus slices treated with NVP AAM077 + 1\u03bcM ZnCl2 .(A) Time course of NMDA fEPSP amplitudes in control slices  in the presence of 10\u03bcM ifenprodil alone, versus slices treated with ifenprodil + 1\u03bcM ZnCl(TIFF)Click here for additional data file.S5 FigP<0.05; Student\u2019s t-test). (B) Summary of the normalized slope at 50 minutes after TBS stimulation. Mean \u00b1 SEM of fEPSP slopes 50 minutes post-TBS in slices treated with 1\u03bcM ZnCl2 + PP2 (n = 4) versus slices treated with 1\u03bcM ZnCl2 + PP3 (n = 3). The two groups were significantly different, with PP2, but not PP3, completely blocking the Zn2+ enhancement of LTP .(A) The time course of LTP in PP2 treated slices (circles) compared to slices treated with PP3 (squares). PP3 did not inhibit LTP magnitude compared to PP2 ((TIFF)Click here for additional data file.S6 FigA representative western blot. All samples in this figure are from the same animal. The lanes above each other are duplicate aliquots of the same slice. Upper panels are developed with anti NMDAR2B antibody and the lower panels developed with anti PY NMDAR2B antibody. Quantitation of the ECL developed image was performed using Image J software. Quantitative comparisons were performed by normalizing each blot to a control lane and the relative value of the PY signal was divided by the total NMDAR2B signal. Thus a ratio of one is the value of control standard- the PY/NMDA for control is set to 1. The quantization from this study is reported in (TIFF)Click here for additional data file."}
+{"text": "We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS.TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27 The more common form of the disease is the embryonal subtype (ERMS), characterized by loss of heterozygosity at the 11p15 locus, a region which harbors insulin-like growth factor 2. Alveolar RMS (ARMS) is the more aggressive form of RMS that is characterized by t or t translocations. The translocations result in chimeric transcripts that fuse the 5\u2032 portion of the paired box proteins 3 or 7 (PAX3 or PAX7), including an intact DNA-binding domain, to the transactivation domain of a forkhead transcription factor (FKHR), creating novel PAX3-FKHR ) or PAX7-FKHR ) fusion proteins3. RMS is diagnosed by observation of unique skeletal muscle cell morphology phenotypes and the presence of myogenic markers such as myogenic regulatory factors (MRFs)4, yet these factors appear to be inactive in RMS5.Rhabdomyosarcoma (RMS) is the most common soft tissue pediatric sarcoma, which is thought to largely arise from the skeletal muscle lineage6. The T-box motif binds to the core sequence GGTGTGA known as the T-element7. Distinct from most members of the T-box family, TBX2 is known as a potent transcriptional repressor that functions in both embryonic development, and if deregulated, tumorigenesis8. The oncogenic potential of TBX2 was first identified by its ability to bypass cellular senescence in a Bmi\u2212/\u2212 mouse embryo fibroblast model9. TBX2 has been shown to promote cell proliferation and block cellular senescence by repressing CDKN1A (p21), CDKN2A (p14/19ARF)10, and PTEN11.The T-box family of transcription factors are highly conserved and related throughout all metazoan lineages. They share a common DNA-binding domain known as the T-box motif and participate in diverse types of organogenesis and developmental regulation12. TBX2 and TBX3 are highly related and thought to have arisen from a gene duplication event13. In the case of deregulation, TBX3 also acquires the potential to become an oncogene in melanoma cells14. TBX3 has been found to be overexpressed in melanoma15 and several sarcomas16. TBX3 has been shown to promote tumor metastasis and the migration of breast cancer cells17. In MCF-12A breast epithelial cells and B16 mouse melanoma cells, TBX3 has been shown to directly repress TBX2 under the control of TGF-\u03b2118. Our lab has shown that TBX3 is expressed throughout skeletal muscle differentiation, whereas the expression of TBX3 in RMS cell lines is almost completely abrogated11.Another member of the T-box family, TBX3, is also characterized as a transcriptional repressor and developmental regulator19. PRC1 and PRC2 work synergistically and indispensably together for transcriptional repression of target gene by ubiquitylation of histone H2A lysine 119 residue and methylation of histone 3 lysine 27 (H3K27) residues, respectively20. The catalytic subunit of PRC2 is enhancer of zeste 2 (Ezh2)21.The polycomb repressive complexes 1 and 2 (PRC1/2) are critical in the precise and accurate regulation of development in many physiological systems including skeletal muscle22. In RMS, pharmacological inhibition of EZH2 in ERMS resulted in reduced proliferation and pharmacological inhibition or depletion of EZH2-induced myogenic differentiation in these cells23. EZH2 depletion in ARMS has also been shown to inhibit proliferation and initiate apoptosis24. JARID2, a founding member of Jumonji family of proteins, is known to be a substoichiometric component of PRC2 that appears to function in targeting PRC2 activity25. JARID2 is highly expressed in ARMS and contributes to the inhibition of differentiation in ARMS cells26.Many studies have revealed that EZH2 is overexpressed in a large number of cancersEGR1 is correlated with the induction of differentiation and repressed by PRC2 in ARMS. Discovery of this novel PRC2-TBX3-TBX2 genetic axis has important implications for understanding the mechanisms that drive proliferation and differentiation in RMS and skeletal muscle.In this work, we show that TBX3 represses TBX2 under the control of the PRC2 complex in RMS and skeletal muscle. RMS cells and proliferating skeletal muscle cells contain high levels of EZH2 that represses TBX3 and allows TBX2 expression. Depletion of EZH2 upregulates TBX3 which then down regulates TBX2. We also show that the early growth response gene 27. In skeletal muscle, TBX2 is expressed in proliferating myoblasts, but sharply downregulated upon differentiation while TBX3 is expressed throughout myogenesis and highly expressed during differentiation27. To understand the potential role of TBX3 in RMS, we transiently transfected RMS cell lines representing both ERMS (RD and RD2) and ARMS (RH30 and RH28) with an expression plasmid for TBX311. As anticipated, we observed that TBX3 was upregulated  database using GDAC Firehose. Two cohorts of the top one-third TBX3 and TBX2 expressing samples were combined and duplicates samples were removed. A correlation analysis was performed and a scatter plot was generated for TBX3 and TBX2 expression \u2009=\u2009\u22120.5173, p\u2009<\u20090.0001, N\u2009=\u2009113), which was also evident from two distinct clusters on the heatmap with opposite expression (N\u2009=\u2009263)  Fig. .TBX3 mRNA expression  assays were performed using antibodies against TBX3 and primers against the ter Fig. . Intriguter Fig. . Given t27. TBX3 has been implicated in oncogenesis in breast cancer and melanoma17. To determine if TBX3 inhibited oncogenesis by repressing TBX2 or instead had a TBX2 independent role in promoting oncogenesis in RMS cells, we assayed for the growth properties of RH30 cells expressing exogenous TBX3. We found that proliferation was strongly inhibited by TBX3 and closely correlated with the degree of TBX3 expression in each clonal isolate  by immunofluorescence. RH30 cells expressing exogenous TBX3 did not show the presence of MyHC positive cells  and MYOGENIN (MYOG) in cells under normal growth conditions and under differentiation conditions. We found no upregulation of these markers under either condition  in RH30 cells and stable cell lines expressing shEZH2 were selected. Two clones were further characterized and we noted that in both cell lines, EZH2 was modestly depleted at the level of mRNA  on the Tbx3 promoter and we found that H3K27me3 was enriched on this promoter  and western blot , identified a group of developmental regulator genes that are repressed by PRC2 to maintain pluripotency and thus poised for activation during ES cell differentiation and TBX3 was included in this group of developmental regulators34. These results are consistent with what we observe in this study.The repression of 18. However, unlike what has been observed in those previous studies which showed that TBX3 has oncogenic potential, we found no evidence that TBX3 promotes oncogenesis in RMS. Instead, we found that TBX3 acts as a tumor suppressor in RMS, largely due to its repression of TBX2.TBX3 had previously been shown to represses TBX2 expression by directly associating with the TBX2 promoter in a breast cancer cell line, MCF-12A35. JARID2, a noncatalytic, substoichiometric component of the PRC2 complex, has been shown to be upregulated in RMS, where it promotes cell viability and inhibits differentiation26. The catalytic subunit of EZH2 has been shown to be highly expressed in ERMS and siRNA-mediated depletion or pharmacological inhibition of EZH2 inhibits proliferation and induce differentiation of ERMS23. EZH2 was also shown to be required for viability of ARMS by repressing FBX03224. Treatment of RH30 or RD cells with siEZH2 leads to robust transient depletion of EZH2 after 24\u2009h and induced apoptosis but did not induce MYOGENIN or MYOD1. Transient inhibition of EZH2 has been shown to induce MYOGENIN in skeletal muscle cells38. Our lab has recently shown that transient depletion of EZH2 or JARID2 does result in transient upregulation of MYOGENIN and MYOD1 as others have seen, but sustained depletion of either factor leads to a block in Wnt signaling, which inhibits MYOD1 and blocks differentiation32. RMS cells are deregulated in Wnt signaling39, so a similar mechanism is not anticipated in RMS. Here, we found that RH30 cells with a modest sustained depletion of EZH2 generated by stable selection of a shRNA construct against EZH2 led to a decrease in proliferation and an induction of differentiation, including the upregulation of MYOD1 and MYOG.Upregulation of the PRC2 complex has been shown be correlated with many cancer typesTBX3, which allows the expression of the potent oncogene TBX2. This genetic axis also acts in skeletal muscle and provides a molecular explanation of previous results from our lab. We have shown that TBX2 is expressed in proliferating myoblasts and is sharply downregulated upon differentiation, while TBX3 is expressed throughout myogenesis and upregulated during differentiation11. Here, we establish that the down regulation of EZH2 upon skeletal muscle differentiation leads to the upregulation of TBX3 and the subsequent downregulation of TBX2.We show here that EZH2 represses 28. We found that EGR1 is a target of EZH2 repression. EGR1 has also been shown to be a target of the PAX-FOXO1 fusion that characterizes ARMS40. Thus, EGR1 is both repressed by EZH2 and destabilized by PAX3-FOXO1 in ARMS. It remains to be determined if ERG1 is a target of PRC2 in ERMS cells, but it may be that the lack of PAX3-FOXO1 in these cells is sufficient to allow EGR1 expression. Consistent with this hypothesis, we observed that the rescue of EGR1 expression by EZH2 by inhibition or depletion was relatively modest when compared to the level of EGR1 present in ERMS cells.Our results also showed that TBX3 is not sufficient to induce myogenic differentiation in ARMS, while depletion of EZH2 is sufficient to promote differentiation. These results were correlated with the expression of EGR1, which we have shown acts as a tumor suppressor in RMS that can induce differentiation in ARMS upon ectopic expressionThe PRC2-TBX3-TBX2 axis discovered in this work has significant implications for both RMS and skeletal muscle. The PRC2 complex serves as a central mediator between proliferation and differentiation and understanding the functions of its many downstream targets offers new insight into how skeletal muscle grows and repairs. TBX2 serves as a potent oncogene in RMS and many other cancers when overexpressed and thus, understanding the deregulation of TBX2 through the PRC2-TBX3-TBX2 axis offers insight in new approaches to inhibit tumor growth of RMS and additional cancers. This work also provides a mechanistic understanding of the effect of PRC2 in RMS and offers additional support to the therapeutic value of PRC2 inhibition in RMS.RD and RH30 cells (ATCC) were grown in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS)  according to standard protocols. RD2 and RH28 were obtained from Denis Guttridge, Medical University of South Carolina, and grown as described above. All cell lines were authenticated by Bio-Synthesis  using STR analysis on September 14, 2011. Cell lines are routinely screened for mycoplasma infection using the LookOut Mycoplasma quantitative polymerase chain reaction (qPCR) Detection Kit . All cell lines in this study were found to be mycoplasma negative prior to the study and following the study. C2C12 myoblasts  were grown in DMEM supplemented with 10% FBS. To induce differentiation, cells were grown to 90% confluence and the media switched to DMEM supplemented with 2% horse serum . Cells were grown in differentiation medium for the number of days indicated in each experiment.2O at 10\u2009mg/ml and used at 10\u2009\u03bcg/ml in RH30 cells and 2\u2009\u03bcg/ml in RD cells. Puromyocin  was dissolved in ddH2O at 10\u2009mg/ml and used at 2\u2009\u03bcg/ml in all cell lines. Drugs were diluted in DMEM for use at the indicated concentrations.GSK126  was dissolved in DMSO at a concentration of 28.5\u2009mM and 0.1% DMSO was used as the vehicle control. Blasticidin  was dissolved in ddH11. pEF was used as the vector control and contains the gene for blasticidin resistance. shRNA against TBX2 and scr control were previously described27. shRNA constructs are in the pLKO.1 vector, which contains the gene for puromycin resistance.TBX3 was cloned into the pEF TOPO  vector as previously describedCells were transfected with the indicated plasmids using calcium phosphate according to standard protocols or Turbofect transfection reagent (Fisher Scientific) according to manufacturer\u2019s protocol. Transient transfections were harvested at 48\u2009h post transfection or as indicated. Stable cell lines were made by transfecting cells with linearized plasmids and selecting for drug resistant colonies. Glass rings were used to isolate individual clones. Cells were recovered by trypsinization and transferred to single wells for propagation.41 with the following modifications: A total of 1\u2009\u00d7\u2009107 cells were used for each immunoprecipitation and protein A agarose beads  were used to immunoprecipitate the antibody\u2013antigen complexes. Primers are described in Supplemental Table ChIP assays were performed and quantified as described previouslyCells were grown on cover slips, fixed with paraformaldehyde, blocked with 10% goat serum, 1.0 % NP-40 in phosphate buffered saline (PBS) for 1\u2009h and washed with PBS. Primary antibodies against MyHC were incubated overnight at 4\u2009\u00b0C, washed with PBS, and detected by Alexa Fluor-488 goat anti-mouse antibody (Life Technologies). Cell nuclei were stained by incubating with 1\u2009\u03bcM DAPI (Life Technologies) for 5\u2009min.4 cells per well were seeded in six-well plates and on the indicated days, cells were counted under a light microscope using a hemocytometer. Cell viability was determined by trypan blue staining. Cell counting was performed in duplicate for four blinded biological replicates.A total of 2\u2009\u00d7\u200910HPRT and/or 18s rRNA.Trizol (Life Technologies) was used for RNA extraction from cells. Two micrograms of total RNA was treated with DNase  and reverse transcribed with MultiScribe MuLV reverse transcriptase (Life Technologies). Forty nanograms cDNA was used for qPCR amplification (Life Technologies) with SYBR green PCR master mix (Life Technologies). Negative controls included no RT samples where no reverse transcriptase was added for each RNA sample. All quantitative RT-PCR (qRT-PCR) was performed in triplicate and three independent RNA samples were assayed for each time point. qRT-PCR data were calculated using the comparative Ct method (Life Technologies). Standard deviations from the mean of the [\u0394] Ct values were calculated from three independent RNA samples. Primers used are listed in Supplemental Table 2 incubator at 37\u2009\u00b0C for the indicated hours. Percentage migration rates were calculated using following formula:0\u201d is the area of the scratch immediately after the scratch was made, and \u201cAt\u201d is the area of the scratch after indicated time. Scratch area was measured using ImageJ software (NIH). All scratch assays shown are representative of four independent experiments.Cell mobility was assayed by scraping a straight line with a 20\u2009\u00b5l pipet tip on a monolayer of cells grown to 100% confluency. Cell debris was removed by washing. To obtain the same field during the image acquisition, marks were created near the scratch line. The plate was placed in a CO5) were plated in triplicate. One milliliter of culture medium was added to the top of each plate every 5 days and cells were grown at 37\u2009\u00b0C in a CO2 incubator for 30 days. The plates were stained with 1\u2009ml of 0.05% Crystal Violet (Fisher Scientific) for >1\u2009h and colonies were counted using a dissecting microscope. All assays shown are representative of four independent experiments.Soft agar assays were carried out in 60\u2009mm culture dishes in which 2\u2009ml of 0.7% Noble agar (Fisher Scientific) in 1\u00d7 DMEM with 10% FBS was overlaid with 2\u2009ml of 0.35% agar in 1\u00d7 DMEM with 10% FBS containing the cells. Cells of each clone  and clear lysates obtained by centrifugation. Protein concentration was determined by the Bradford\u2019s assay  and 50\u2009\u00b5g of protein was loaded for each well of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Resolved proteins were then transferred onto a PVDF membrane using a tank blotter (Bio-Rad). Membranes were blocked with 5% milk in 1\u00d7 Tris-buffered saline plus Tween 20 (TBST) and followed by incubation with primary antibody overnight at 4\u2009\u00b0C. After washing membranes with 1\u00d7 TBST, membranes were incubated with the corresponding secondary antibody, washed with 1\u00d7 TBST and incubated with chemiluminescent substrate according to the manufacture\u2019s protocol  and visualized by autoradiography and/or an iBright FL1000 Imager . Blots were quantitated using ImageJ software (NIH) or iBright Analysis software. Four biological replicates were performed for each western blot assay.Tbx3 gene were chosen using an online CRISPR Design Tool (http://tools.genome-engineering.org). Guide containing DNA oligonucleotides was designed and cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 as described42. pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang . Following selection of individual puromycin resistant clones, genomic DNA was isolated using Trizol  and PCR amplified targeted regions were sequenced.Guide sequences for the www.cbioportal.org), mRNA expression data for TBX3 and TBX2 in sarcoma patients  were visualized on a heatmap and clustered. The mRNA expression Z-score (RNAseq V2 RSEM) was set to default (\u00b12). For further analysis, mRNA expression (RNA Seq V2 RSEM) data of TBX3 and TBX2 for sarcoma patients were downloaded from TCGA (https://cancergenome.nih.gov/) database using GDAC Firehose. To better understand the correlation between the expression of TBX3 and TBX2 in these patients, two cohorts of the top one-third TBX3 and TBX2 expressing samples were combined and duplicates samples were removed (N\u2009=\u2009113). A correlation analysis was performed, a Spearman correlation coefficient (rs) was calculated and a scatter plot was generated for TBX3 and TBX2 expression using GraphPad Prism 8.0.Using cBioPortal (t-tests. Spearman correlation coefficient (rs) was calculated for TBX3 and TBX2 mRNA expression in TCGA samples using GraphPad Prism 8.0. Statistical significance are denoted by asterisks as indicated in figures with \u201c***\u201d, \u201c**\u201d, and \u201c*\u201d for p-value\u2009\u2264\u20090.001, 0.01, and 0.05, respectively.Data are presented as means\u2009\u00b1\u2009standard errors. Statistical comparisons were performed using unpaired two-tailed Student\u2019s Supplemental Table 1Supplemental Table 2Supplemental File"}
+{"text": "This article contains quality of life data experienced by individuals before and after implantation of a press-fit or screw-type osseointegrated fixation when fitted with conventional socket-suspended and bone-anchored limb prosthesis, respectively. This specifically-designed survey was developed and administered by Queensland Artificial Limb Services (QALS), an Australian State government organization. It was an integrated part of QALS\u2032 continuous quality improvement procedure for assessing the provision of bone-anchored prosthesis. A total of 12 out of the 65 consumers completed to the survey, giving a return rate of 18%. This benchmark information can contribute to inform the design of (A) other patients' experience surveys including those built-in governmental continuous quality improvement procedure as well as (B) clinical trials looking at the overall effects of surgical implantation of ossoeintegrated fixation on patients' quality of life. Online repository contains the files:  This cohort represented circa 16% and 7% of existing population estimated at 400 in Australia and 950 worldwide, respectively 6.1.2This specifically-designed survey data on the quality of life was administered by Queensland Artificial Limb Services (QALS), an Australian State government organization, as an integrated part of its continuous quality improvement procedure for assessing the provision of bone-anchored prosthesis \u20227 (28%) questions about \u201cOsseointegration Surgery Details\u201d ,\u20225 (20%) questions about \u201cPre-Osseointegration Surgery\u201d ,\u202213 (52%) questions about \u201cPost-Surgery Osseointegration\u201d .First, participants were required to indicate their name, address, date of birth and email. Then, participants answered 25 questions organized around the three following sections:The 65 eligible consumers were asked to participate in this study over the phone by a QALS' agent. Consumers could choose if they preferred receiving the survey by email or by post with pre-paid return envelope.6.1.3\u2022\u25cb4 (16%) questions focusing on efficacy, particularly the level of function ,\u25cb1 (4%) question focusing on experience, particularly the level of satisfaction ,\u25cb3 (12%) questions focusing on knowledge of the osseointegration treatment , particularly the motivation for considering the procedure and the sources of information considered where promotional information included TV and social media.7 (28%) questions providing baseline outcomes that related to the quality of life of QALS\u2032 consumers fitted with socket-suspended prosthesis before implantation of osseointegrated fixation including:\u2022\u25cb2 (8%) questions focusing on surgery , particularly the time of the surgery and the level of satisfaction with the procedure,\u25cb2 (8%) questions focusing on safety or harms , particularly the occurrence of infection 17 (68%) questions assessing the quality of life of QALS\u2032 consumers fitted with bone-anchored prosthesis after implantation of osseointegrated fixation including:\u2022\u25cb4 (16%) questions focusing on efficacy, particularly the level of function ,\u25cb1 (4%) question focusing on experience, particularly the level of satisfaction ,\u25cb3 (12%) questions focusing on knowledge of the osseointegration treatment , particularly the motivation for considering the procedure and the sources of information considered where promotional information included TV and social media.7 (28%) questions providing baseline outcomes that related to the quality of life of QALS\u2032 consumers fitted with socket-suspended prosthesis before implantation of osseointegrated fixation including:\u2022\u25cb2 (8%) questions focusing on surgery , particularly the time of the surgery and the level of satisfaction with the procedure,\u25cb2 (8%) questions focusing on safety or harms , particularly the occurrence of infection \u25cb6 (24%) questions focusing on efficacy or benefits , particularly the level of function \u25cb6 (24%) questions focusing on overall experience , particularly the limitations and level of satisfaction.17 (68%) questions assessing the quality of life of QALS\u2032 consumers fitted with bone-anchored prosthesis after implantation of osseointegrated fixation including:\u20221 (4%) general comments provided by consumers focusing on limitation of their observation time as well recommendations, benefits and shortcomings of the treatment .Analysis of the survey data consisted in extracting information for:Answers to the 10 (40%) dichotomous questions  were expressed in percentage of individual responses .Answers to the 3 (12%) Likert-type scale questions were expressed as percentage of participants in each of the 10 levels between 0 for \u201cnot satisfied\u201d and 10 for \u201cvery satisfied\u201d .\u25cb6 (24%) questions focusing on overall experience , particularly the limitations and level of satisfaction.\u20221 (4%) general comments provided by consumers focusing on limitation of their observation time as well recommendations, benefits and shortcomings of the treatment .Answers to the 12 (48%) open-ended questions were coded accordingly to the recurrence of themes in the replies .Answers to the 10 (40%) dichotomous questions  were expressed in percentage of individual responses .Answers to the 3 (12%) Likert-type scale questions were expressed as percentage of participants in each of the 10 levels between 0 for \u201cnot satisfied\u201d and 10 for \u201cvery satisfied\u201d .Answers to the 12 (48%) open-ended questions were coded accordingly to the recurrence of themes in the replies .7Only aggregated data was presented in this study. Exploration of more detailed analysis revealed that proportionate and disproportionate stratification sampling were unattainable given the diversity of case-mix and the small number of respondents."}
+{"text": "Rotavirus A (RVA) strains bearing the DS-1-like constellation of the non-G, non-P genes (hereafter referred to as the genotype 2 backbone) requires better understanding of their evolutionary relationship. However, within a genotype, there is lack of a consensus lineage designation framework and a set of common sequences that can serve as references. Phylogenetic analyses were carried out on over 8,500 RVA genotype 2 genes systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. In line with previous designations, using pairwise comparison of cogent nucleotide sequences and stringent bootstrap support, reference lineages were defined. This study proposes a lineage framework and provides a dataset ranging from 34 to 145 sequences for each genotype 2 gene for orderly lineage designation of global genotype 2 genes of RVAs detected in human and animals. The framework identified five to 31 lineages depending on the gene. The least number of lineages (five to seven) were observed in genotypes A2 (NSP1), T2 (NSP3) and H2 (NSP5) which are limited to human RVA whereas the most number of lineages (31) was observed in genotype E2 (NSP4). Sharing of the same lineage constellations of the genotype 2 backbone genes between recently-emerging, unusual G1P[8], G3P[8], G8P[8] and G9P[4] reassortants and many contemporary G2P[4] strains provided strong support to the hypothesis that unusual genotype 2 strains originated primarily from reassortment events in the recent past involving contemporary G2P[4] strains as one parent and ordinary genotype 1 strains or animal RVA strains as the other. The lineage framework with selected reference sequences will help researchers to identify the lineage to which a given genotype 2 strain belongs, and trace the evolutionary history of common and unusual genotype 2 strains in circulation.Recent increase in the detection of unusual G1P[8], G3P[8], G8P[8], and G9P[4]  Rotaviruses A (RVA) within the genus Rotavirus of the Reoviridae family, are a major cause of severe acute gastroenteritis in children and the young of various mammals and birds. RVAs possess a triple-layered capsid that contains a genome of 11 segments of double-stranded RNA encoding six structural viral proteins  and five to six non-structural proteins (NSP1-NSP5/NSP6) ,,40], andThe genotype 2 genes were found in RVA of human as well as a diverse range of animal host species origin. Examination of over 8,500 RVA genotype 2 genes retrieved from the NCBI Viral Genomes Resource  revealedOut of 724\u20131,326 sequences per gene of both human and animal rotaviruses , retrievThe maximum likelihood phylogenetic trees representing the framework for the genotype 2 backbone genes are shown in Figs A few exceptions need to be noted in which bootstrap support was lower than 70%, yet lineages previously identified by Doan et al.  were maiAs to the number of lineages, it was noted that lineages were more diverse in genes whose genotype was shared between human and animal RVA strains  among 27 emergent reassortant strains . There were four constellations in G1P reassortSecondly, no G2P strains Thirdly, unlike other emergent reassortant strains, there was no corresponding G2P strain wExamining clinical and surveillance specimens of RVA infecting humans at the level of the whole genotype constellation has provided a grand view of Wa-like, genotype 1 RVA originating from porcine RVA and DS-1-like, genotype 2 RVA originating from bovine RVA in the evolutionary perspective   8] and GS1 FigSequences were retrieved using the virus variation resource based on the search criteria described in the results. Frequency of occurrence of each host species within each genotype 2 gene was tallied and plotted against the genome segments. The legend on the right side indicates the Latin names of the host species together with the corresponding colour in the histogram.(PPTX)Click here for additional data file.S1 File(ZIP)Click here for additional data file."}
+{"text": "Clear cell sarcoma of the kidney (CCSK) is a rare malignant pediatric renal neoplasm with a heterogeneous histological appearance which often results in misdiagnosis. There are no specific immunohistochemical markers which can help in differentiating CCSK from other pediatric renal neoplasms. Recently Cyclin D1 has been investigated as a possible marker in this regard. In this study, we aim to determine the usefulness of Cyclin D1 in differentiating between CCSK and other pediatric renal neoplasms and to compare our results with those of recently published studies.A total of 48 cases of CCSK, Wilms tumor (WT), renal rhabdoid tumor, mesoblastic nephroma, renal Ewing sarcoma and neuroblastoma were included in the study. All cases were stained with cyclin D1. Extent of Cyclin D1 staining was graded according to percentage of positive tumor cells as diffuse (>\u200970%), focal (5 to 70%), and negative (<\u20095%). Intensity of Cyclin D1 staining was graded as strong or 3+, moderate or 2+ and weak or 1\u2009+\u2009.Most or all cases of CCSK, neuroblastoma and renal Ewing sarcoma demonstrated diffuse and strong positivity for Cyclin D1. Most cases of Wilms tumor  also demonstrated diffuse and often strong positivity for Cyclin D1. In most cases of WT, blastemal component was negative.Cyclin D1 is a sensitive but not specific immunohistochemical marker for CCSK and many other pediatric renal malignant neoplasms as well as for neuroblastoma. Hence, careful examination of histological features is important in reaching an accurate diagnosis in CCSKs. However, Cyclin D1 is very helpful in distinguishing between blastema-rich WT and CCSK. Clear cell sarcoma of kidney (CCSK) is an uncommon mesenchymal renal tumor of uncertain histogenesis which comprises approximately 3 to 4% of malignant pediatric renal neoplasms. It occurs in young children and mean age at diagnosis is approximately 36\u2009months. It is more common in males , is centered in the renal medulla and is almost always unifocal. It is usually circumscribed but unencapsulated with a tan, soft cut surface and most tumors are quite large in size. On histological examination, classic CCSK is characterized by nests or cords of epithelioid cells with round to oval nuclei. Myxoid pools and thin and regularly branching fibrovascular septa separate the cords of tumor cells [YWHAE-FAM22- NUTM2B/E gene fusion which is also seen in high grade endometrial stromal sarcoma. Recently, CCSK has also been shown to consistently demonstrate BCOR gene abnormalities including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion which distinguish it from other pediatric renal tumors. Metastases can occur as late as ten years after initial diagnosis. Owing to the essential role of doxorubicin in the therapy of CCSK, it is imperative that pathologists identify it accurately. Failure to do so can prevent a child from getting optimal chemotherapy [However, CCSK is very heterogeneous and diverse in histological appearance and a number of variant histological patterns may be seen. It has a propensity to metastasize to bone, and \u201cbone metastasizing renal tumor of childhood\u201d is one of its synonyms . In the otherapy \u201318.BCOR gene abnormalities are not available especially in developing countries such as ours. Recently, a number of studies have suggested that immunohistochemical stain Cyclin D1 is useful in distinguishing CCSK from some pediatric renal neoplasms [Owing to its marked histological heterogeneity, CCSK can be mimicked by a number of malignant pediatric renal and extrarenal neoplasms including blastema-rich Wilms Tumor (WT), mesoblastic nephroma, neuroblastoma etc. It is often difficult to diagnose CCSK from these tumors on morphology alone , 5. Untieoplasms , 5, 19. eoplasms , 15.The aim of the present study was to determine the usefulness of Cyclin D1 in distinguishing CCSK from other pediatric renal neoplasms and to see whether our findings match those in other recently published studies.The surgical pathology files of the Section of Histopathology, Department of Pathology and Laboratory Medicine, Aga Khan University Hospital and cases were submitted from French Medical Institute for Mothers and Children were searched for cases of CCSK, WT (nephroblastoma), renal rhabdoid tumor (RT), congenital mesoblastic nephroma, Ewing sarcoma of kidney as well as retroperitoneal neuroblastoma reported over a ten year period i.e. January 1, 2008 and December 31, 2017. A total of 48 cases were included in the study. These included 19 cases of CCSK, 9 cases of WT and 4 cases each of renal RT, Mesoblastic nephroma and Ewing sarcoma. In addition, 8 cases of neuroblastoma were also included. Although neuroblastomas are not renal tumors but are retroperitoneal like the former and occur in the same age group as pediatric renal tumors. Due to these features they need to be distinguished from pediatric renal tumor. Hence, they were included in the study.All cases were reviewed by the two principal authors (NU and ZA). A number of immunohistochemical markers including Vimentin , Antismooth muscle actin , S100 protein , CD99 , WT1  protein, clone 6F-H2\u20137, ready to use, DAKO, Glostrup, Denmark), Neurofilament , CD56 , Synaptophysin , Desmin , Cytokeratin AE1/AE3 , Epithelial membrane antigen  were performed in different cases at the time of initial reporting and were reviewed by the authors. Cyclin D1  was retrospectively performed in all 48 cases. The staining for Cyclin D1 was performed on DAKO automated immunostainer. The antibody was optimized using Ventana DAB detection kit  and standard quality control procedures were performed. Cyclin D1 staining was performed following heat induced antigen retrieval using ER2 antigen retrieval buffer. Cases of mantle cell lymphoma were used as positive control. Negative controls were also used. For the purpose of study, extent of Cyclin D1 staining was graded according to percentage of positive tumor cells as diffuse (>\u200970%), focal (>\u20095 to 70%) or negative (<\u20095%); intensity of staining was graded as strong or 3+ (nuclear intensity similar to that of mantle cell lymphoma control), moderate or 2+ (definite nuclear staining weaker than 3+ but easily identifiable at \u00d7\u200940 magnification) and weak or 1+ (nuclear staining identifiable only at high power magnification). This scale for extent and grading was based on that used in two studies published in 2015 [A total of 48 cases were included in the study. These included 19 cases of CCSK, 9 cases of WT and 4 cases each of renal RT, Mesoblastic nephroma and Ewing sarcoma. In addition, 8 cases of neuroblastoma were also included..Patients with CCSK ranged in ages from 1 to 29\u2009years with means and median age of 5.5 and 3\u2009years respectively. Of the 19 patients, 13 (68.4%) were males while 6 (31.6%) were females. Tumor was located in left and right kidney in 8 cases each. In 3 cases, laterality was not known. Tumor size ranged from 8\u2009cm to 14.5\u2009cm with mean tumor size of 11\u2009cm in greatest dimensionAll 19 cases of CCSK demonstrated reactivity for Cyclin D1. In 13 cases (68.4%), intensity of staining was 3+, in 4 cases (21%), intensity was 2+, while in 2 cases (10.5%), it was 1+. Extent of staining ranged from 5 to 90% with average extent of staining being 54.5%  were females, while 3 (33.3%) were males. Ages ranged from 2.5 to 7\u2009years. Mean and median ages were 4.5 and 4\u2009years respectively. In 6 patients (66.7%), tumor was located in the left kidney while in 3 (33.3%), tumor was located in the right kidney. Tumor size was available in 8 out 9 cases and ranged from 7.5 to 17.4\u2009cm in largest dimension with mean tumor size of 11.5\u2009cm.Out of 9 cases of renal WT, 2 did not show any epithelial component and tumor was composed entirely of blastema and stromal components (biphasic). In these 2 cases, Cyclin D1 was negative in both components in 1 case, and showed weak positivity in blastemal component in the other case. The remaining 7 cases showed strong (3+) positivity in the epithelial component (tubules), while blastemal component was negative in all cases. Extent of staining ranged from 5 to 70%. Average extent of staining was 37.5%  were males while 1 (25%) was female. Tumor was located in right kidney in 3 cases (75%) and left kidney in 1 case (25%). Tumor size ranged from 7 to 9\u2009cm with mean size of 8\u2009cm in largest dimension.Out of 4 cases of congenital mesoblastic nephroma, 3 were cellular type and 1 was of mixed type and all demonstrated reactivity for Cyclin D1. Intensity of staining was 3+ in all 4 cases. Extent of staining ranged from 10 to 50% and average extent of Cyclin D1 staining was 30% Fig. C, D. AntPatients with renal RT ranged from 1\u2009year to 14\u2009years in age with mean and median age of 4 and 1.5\u2009years respectively. Of the 4 patients, 3 (75%) were males and 1 (25%) was female. Tumor was located in left kidney in 2 cases (66.7%) and in right kidney in 1 case (33.3%). Tumor laterality was not known in 3 cases. Tumor size ranged from 7\u2009cm to 9\u2009cm in maximum dimension. Mean tumor size was 7.5\u2009cm.Out of 4 cases of renal RT, 3 (75%) demonstrated reactivity for Cyclin D1, intensity of staining was 2+ in all 3 cases while extent of staining ranged from 10 to 30%. Average extent of staining was 20%. Vimentin was positive in all 4 cases. EMA was performed in 3 cases and was positive in all 3. All 4 cases showed loss of INI 1.The ages of 4 patients with renal Ewing sarcoma ranged from 2 to 36\u2009years with mean and median age of 20 and 35\u2009years respectively. Of the 4 patients, 2(50%) were males and 2 (50%) were females. Of the 4 cases, 3 were located in the right kidney (75%) while 1 (25%) was located in the left kidney (25%). Tumor size ranged from 2.5 to 17\u2009cm in largest dimension. Mean tumor size was 11.7\u2009cm.Out of the 4 cases of renal Ewing sarcoma, 3 (75%) demonstrated staining for Cyclin D1. Of these 3 cases, intensity of staining was 3+ in 2 cases (66.7%) and 2+ in 1 case (33.3%). Extent of staining ranged from 10 to 30%. Average extent of Cyclin D1 staining was 16.7%  were females while 3 (37.5%) were males.Out of 8 cases of neuroblastoma in our series, 8(88.9%) demonstrated intense 3+ reactivity for Cyclin D1. Extent of Cyclin D1 staining ranged from 5 to 70%. Average extent of staining was 37.5%  in over 68% of cases and moderately intense (2+) in 21%. Thus over 89% cases showed moderate to marked intensity. Average extent of staining was almost 55%. These findings were indicative of diffuse and strong nuclear positivity for Cyclin D1 in the large majority of CCSK and were similar to the findings in other recent studies by Aw et al. (3) and Mirkovic et al. [All 4 cases of congenital mesoblastic nephroma (CMN) in our study showed markedly intense 3+) focal to diffuse staining for Cyclin D1. All 11 cases of CMN in Mirkovic et al\u2019s study showed focal (4 cases) to diffuse (17 cases) moderate to severely intense Cyclin D1 staining . This sh+ focal tSimilarly, 7 out of 9 cases of WT in our study demonstrated intense 3+ staining for Cyclin D1 in their epithelial component with average extent of staining of almost 40%. All 4 cases of NB in Mirkovic et al.\u2019s study also demonstrated diffuse moderate to strong nuclear staining for Cyclin D1 . Thus, COf the 4 cases of renal Ewing sarcoma in our series, 3 75%) demonstrated focal to diffuse staining for Cyclin D1 which was very intense in 2 cases. All 5 cases of renal Ewing sarcoma in the study by Mirkovic et al. % demonst.Of the 4 cases of renal RT in our study, 3 75%) showed moderately intense staining for Cyclin D1 which ranged in extent from 10 to 50% with average extent of staining of 30%. This finding is in contrast to the results in the study by Mirkovic et al. % showed . All 4 cAll 8 cases of neuroblastoma in our study demonstrated intense 3+ staining for Cyclin D1 which ranged in extent from as low as 5% to as high as 90%. In fact, 6 out of 8 cases (75%) showed diffuse 3+ positivity which ranged in extent between 70 and 90%. This finding is similar to the findings by Mirkovic et al. [As discussed by both Mirkovic et al.  and Aw eCyclin D1 is a sensitive but not specific immunohistochemical marker for CCSK and many other pediatric renal malignant neoplasms as well as for neuroblastoma. Hence, careful examination of histological features is important in reaching an accurate diagnosis in CCSKs. Cyclin D1 however is very helpful in distinguishing between CCSK and blastema-rich WT which is arguably the most difficult morphologic distinction in the differential diagnosis of CCSK. Our results were similar to two other recently published studies , 5. Howe"}
+{"text": "The use of postoperative radiation therapy after breast-conserving surgery was longstanding standard practice. The treatment protocol used a standard fractionation of 50 Gy in 25 fractions plus a boost. Recently, the hypofractionation approach has gained support based on Canadian and English studies that claimed\u00a0equal tumor control and similar toxicity to the standard protocol.We conducted a review of the literature of hypofractionation studies and compared the reported toxicity with the general literature. We placed special emphasis on breast fibrosis after hypofractionation versus standard fractionation. We found a striking difference in the breast toxicity reported by the hypofractionation literature regarding breast fibrosis as compared to standard fractionation. Breast fibrosis should be explored further via additional studies and discussed with potential breast-conserving surgery patients. Recently, the American Society of Radiation Oncology released a task force guideline recommending hypofractionated radiotherapy for all women of any age whether they had received chemotherapy or not . Their eAccording to Hall, \u201claboratory data proved that fewer and large dose fractions result in more severe late reactions, even though the early reactions are matched by an appropriate adjustment in total dose\u201d . Hall alWe calculated the biological effective dose (BED) for both protocols using \u03b1/\u03b2 of 3 Gy for late tissues and \u03b1/\u03b2 of 10 Gy for early tissues.In regards to the standard fractionation (50 Gy in 2-Gy fractions), the BED for late tissues was 83.3 Gy and 60 Gy for early tissues where:BED = nd (1 + n/\u00a0(\u03b1/\u03b2) )Comparatively, for hypofractionation (41.6 Gy in 3.2 Gy per fraction), the BED for late tissues was 86 Gy and 55 Gy for early tissues.Table The UK Standardization Breast Radiotherapy START Trial A and B found that breast appearance and breast hardness were the most common changes among the two groups but the results were similar (42.6% for the 50 Gy in two fractions and 44.6% for the 41.6 Gy in 3.2 Gy per fraction or 39 Gy in 3 Gy per fraction) [START Trial A showed no significant difference between the breast hardness caused by 50 Gy in 2-Gy fractions and 40 Gy in 15 fractions. The trial reported 38.2% of the breasts were \u201cHarder\u201d with hypofractionation versus 42.3% for standard fractionation.Leong et al. found no added swelling to the arm using hypofractionation. The hypofractionation daily dose was 2.25 Gy to 2.5 Gy to a total of 45 Gy to 40 Gy, respectively. The conventional fractionation ranged between 1.8 Gy and 2 Gy to a total dose of 45 Gy to 50.4 Gy, respectively. Of 1,759 patients, 708 were evaluated (40%). The two groups were not equal, 406 patients received hypofractionation, and 302 received conventional fractionation [Whelan et al. reported no difference in toxicity between 50.0 Gy in 25 fractions over a period of 35 days and a dose of 42.5 Gy in 16 fractions. Grade two subcutaneous toxicity had tripled in 10 years as compared to the\u00a030% increase for standard fractionation [Yarnold et al. found that the probability of moderate/marked breast induration after 50 Gy in 2-Gy fractions was between 39 Gy in 13 fractions and 42.9 Gy in 13 fractions with fewer side effects for the 3.3-Gy fraction size .Reddy et al. compared hypofractionation versus standard fractionation for 287 women treated from 2011 to 2017 (42.56 Gy in 16 fractions plus 10 Gy to 12.5 Gy in four to five fractions boost or 50 Gy in 25 fractions plus 10 Gy to 14 Gy in five to seven fractions). The cosmetic assessment was dependent on photographs and the patient\u2019s own assessment. There was no report of breast fibrosis. They reported similar or improved cosmetic outcomes with hypofractionation as compared to standard fractionation .These studies showed a high rate of breast fibrosis with hypofractionation and a similarly high rate of breast fibrosis with standard fractionation.Haviland reported more side effects with a fractionation schedule of 13 sessions at 3.3 Gy versus 50 sessions at 2 Gy or 39 sessions at 3 Gy. There was a statistically significantly increased rate of physician-assessed shoulder stiffness in the 42.9-Gy schedule compared to the 50-Gy treatment in the START pilot . There was no such effect reported for the hypofractionated schedules in START-A and START-B . Table 2Xiaofeng et al. found that 50% to 60% of the patients had developed mild to moderate late breast fibrosis one year after hypofractionation radiation therapy (40 Gy plus simultaneous cavity boost to 48 Gy in 15 fractions) . Yu et aDe La Rochefordiere et al. found that using a dose of more than 2 Gy per fraction is associated with worse cosmesis . Clark eThere is some discrepancy between hypofractionation studies and many other literature reports about the incidence of breast fibrosis reported for standard fractionation. Breast fibrosis can be a potential side effect of hypofractionated radiotherapy that needs to be further studied."}
+{"text": "After publication of this article , the autSoon before the Correction of Parental TW02, 500 and TW02/CD24-, 500Parental TW02, 1000 and TW02/CD24-, 1000Parental TW04, 100 and TW04/CD24-, 100TW04/CD24+, 100 and TW04/CD24-, 500The authors provided an updated version of this figure in which the TW02 CD24-, TW04 parental, and TW04 CD24- panels are replaced with different images. The authors also provided images of mice from the original experiment see , but somWhen the authors contacted the journal to correct Please see the corrected Figs PLOS ONE Editors have concerns about the strength of evidence supporting the in vivo claims made in the article and, therefore, issue this Expression of Concern.We regret that the journal did not correct Figs S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(XLSX)Click here for additional data file."}
+{"text": "Tai Ji Quan vs Multimodal and Stretching Exercise Interventions for Reducing Injurious Falls in Older Adults at High Risk of Falling: Follow-up Analysis of a Randomized Clinical Trial,\u201d1 published February 15, 2019, a data error occurred in the Intervention Adherence subsection of the Results section. The third sentence should have read, \u201cA total of 158 participants in the TJQMBB group (70.5%), 160 in the multimodal exercise group (71.7%), and 159 in the stretching exercise control group (71.3%) attended 37 or more sessions .\u201d This article has been corrected.1In the Original Investigation titled \u201cEffectiveness of"}
+{"text": "Proton pump inhibitors (PPIs) are a mainstay treatment for gastroesophageal reflux disease (GERD) and are mainly metabolized by CYP2C19 in the liver. However, several polymorphisms of CYP2C19 exist that affect the metabolism of PPIs. Due to the large variability of PPI pharmacokinetics among the polymorphisms, this has implications in the management of patients with refractory GERD who may be potentially undertreated. Herein, we discuss the role of CYP2C19 and its relation to PPI therapy, particularly in those with GERD. Heartburn is a symptom commonly experienced by many,\u00a0but it may develop into gastroesophageal reflux disease (GERD) when heartburn occurs twice weekly or more. GERD is a chronic condition where the reflux of gastric contents into the esophagus causes symptoms (e.g. heartburn) or complications. GERD affects approximately 10\u201320% of those in Western countries and is becoming progressively prevalent in developing countries [CYP2C19*17 allele is an ultra-rapid metabolizer (UM) of CYP2C19 substrates\u00a0and may be prone to inadequate treatment of GERD due to the PPI being metabolized much faster than usual. The Dutch Pharmacogenetics Working Group (DPWG) developed specific dosing recommendations for PPIs for UMs with insufficient responses: increase by 50\u2013100% with esomeprazole, increase by 200% with lansoprazole, increase by 100\u2013200% with omeprazole, and increase by 400% with pantoprazole [CYP2C19*2 or *3 loss-of-function alleles have shown to have higher cure rates of GERD due to decreased clearance of the PPI. The DPWG has no recommendations for dose adjustments of PPIs in the PM population.Polymorphisms in CYP2C19 may increase or decrease metabolism of PPIs, which ultimately affect the treatment of patients with GERD who may have these polymorphisms. A patient who homozygously expresses the oprazole . RabepraCYP2C19 allele and anti-reflux surgery (ARS) in those with medically refractory GERD [CYP2C19*1/*17 and *17/*17 genotypes were a significant predictor of ARS compared to the non-ARS controls [A recent study investigating CYP2C19 extensive metabolizer (EM) and UM phenotypes in children found an association with the presence of the ory GERD . Pediatrcontrols .With pharmacogenetic testing, determining a patient's CYP2C19 phenotype would aid in finding potential sources of treatment failure of GERD earlier on, leading to fewer complications and increased quality of life . Althoug"}
+{"text": "Xenopus neural plate, PCP is marked by the enrichment of the conserved proteins Prickle3 and Vangl2 at anterior cell boundaries. Here we show that the apical determinant Par3 is also planar polarized in the neuroepithelium, suggesting a role for Par3 in PCP. Consistent with this hypothesis, interference with Par3 activity inhibited asymmetric distribution of PCP junctional complexes and caused neural tube defects. Importantly, Par3 physically associated with Prickle3 and promoted its apical localization, whereas overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. We also adapted proximity biotinylation assay for use in Xenopus embryos and show that Par3 functions by enhancing the formation of the anterior apical PCP complex. These findings describe a mechanistic link between the apical localization of PCP components and morphogenetic movements underlying neurulation.Vertebrate neural tube formation depends on the coordinated orientation of cells in the tissue known as planar cell polarity (PCP). In the  Drosophila genetic studies. In Drosophila epithelial tissues, PCP is manifested by the distribution of the Frizzled/Dishevelled and Prickle/Van Gogh membrane complexes to opposite domains inside each cell (Planar cell polarity (PCP) is a common phenomenon that refers to the orientation of cells in the plane of the tissue. PCP requires the functions of several conserved core proteins, Prickle, Van Gogh/Strabismus, Dishevelled, Frizzled and Flamingo/Stan, originally identified in ach cell . In addiach cell . Disruptach cell . The exiach cell . Howeverapical cell corners  and recruits it to the apical cell membrane. Demonstrating the importance of this interaction, a specific Pk3-binding domain of Par3 interfered with the polarization of neuroepithelial cells. To further study PCP mechanisms, we developed an efficient in vivo proximity biotinylation approach using Pk3 fused to a bacterial biotin ligase. Using this assay, we demonstrate a novel role of Par3 in promoting the interaction of Pk3 and Vangl2 in neuroepithelial cells. These findings link the subcellular localization of two core PCP components to morphogenetic events underlying vertebrate neural tube closure.To address this issue, we examined the localization and function of Par3 in the Xenopus neural plate, using characterized anti-Par3 antibodies  with different sequences and confirmed their efficacy. . UnilatePrickle, Espinas and Testin) domain  domain , consist) domain . These iXenopus embryos using conventional pull-downs (data not shown). We then decided to examine this interaction using proximity biotinylation, a highly sensitive approach  that disrupts Par3 localization and function in Xenopus embryos and mammalian cultured cells , 5\u2019-GCTTCAGCTAGTGACACATGCAT-3\u2019; control MO2 (CO MO2), 5\u2019- AGCGTTTCAGGCCGATCTCTCAGTC-3\u2019. Vangl2 MO 5\u2019-GAGTACCGGCTTTTGTGGCGATCCA-3\u2019 . For depletion studies, the following MOs were purchased from Gene Tools : Par3MOATCCA-3\u2019 .Xenopus laevis PRID:NXR_0.0095 and cultured in 0.1x Marc\u2019s Modified Ringer\u2019s solution (MMR)  and injected with 5\u201310 nl of a solution containing RNAs and/or MOs. For mosaic expression of PCP complexes, embryos were injected into two dorsal blastomeres of 16\u201332 cell embryos. Amounts of injected mRNAs per embryo have been optimized in preliminary dose-response experiments (data not shown) and are indicated in figure legends.In vitro fertilized eggs were obtained from on (MMR)  as descron (MMR) . Stagingon (MMR) . For micFor phenotype analysis, frequencies of neural tube defects were calculated as means\u00a0\u00b1\u00a0s. d.\u00a0In unilaterally injected embryos, neural tube was scored as defective when the distance between the neural fold and the midline (white line) at the injected side was at least 1.5 times of that at the uninjected side. Body axis extension was estimated by measuring the length of stage 26 embryos. Blastopore defects were scored at stage 12, the defects were considered mild if the blastopore diameter was more than twice of that of uninjected embryos and severe when the blastopore groove was not visible.HEK293T cells (ATCC) were maintained in Dulbecco\u2019s modified Eagles medium (Corning) with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Sigma). This cell line was tested and found negative for mycoplasma contamination. Cells growing at 50\u201370% confluence were transiently transfected using linear polyethylenimine  as described . Each 60\u03b6. Secondary antibodies were against mouse or rabbit IgG conjugated to Alexa Fluor 488, Alexa Fluor 555  or Cy3 . Cryosections and explants were mounted for observation with the Vectashield mounting medium (Vector). Standard specificity controls were performed to confirm lack of cross-reactivity and no staining without primary antibodies. Images that are representative of at least 10 different fields were captured using a Zeiss AxioImager microscope with the Apotome attachment . The data shown are from two to five independent experiments with 5\u201315 embryos per group. Quantification for Par3 and ZO1 distribution in the neuroepithelium has been carried out using ImageJ as described  for 40 min, washed in PBS and dissected. Cryosectioning and immunostaining were performed as described . Embryosescribed . Signifi3VO4, 10 mM NaF), containing cOmplete Mini EDTA-free protease inhibitor cocktail (Roche). After centrifugation at 16,000 g, the supernatant was incubated with anti-FLAG agarose beads (Sigma) at 4\u00b0C for 2 hr or with anti-Myc antibodies (9E10) for 2 hr and Protein A Sepharose  at 4\u00b0C for 2 hr. Myc-trap beads (Chromotek) were used to pull-down Par3-containing protein complexes. The beads were washed three times in lysis buffer, and subjected to SDS-PAGE and immunoblotting using standard protocols , or HA-Vangl2 (50 pg). Embryos were collected at stages 13\u201314 and protein biotinylation was assessed in embryo lysates and pulldowns with mouse anti-HA (12A5) and anti-Myc (9E10) hybridoma supernatants, rabbit anti-Par3  antibodies. Proteins were detected by immunoblotting with goat anti-biotin-HRP antibodies , rabbit anti-Par3 and anti-HA , and mouse anti-Myc, anti-FLAG (M2 Sigma) and anti-GFP  antibodies. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Yukiko M Yamashita as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor.Thank you for submitting your article \"Par3 interacts with Prickle3 to generate apical planar cell polarity complexes in the vertebrate neural plate\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Xenopus neural plate. Morpholino-based Par3 knockdown revealed defects in polarization of planar cell polarity proteins Vangl2 and Pk3. The authors used co-IP and proximity biotinylation assays to show binding of Par3 with Pk3 and identify the critical domains in Par3 for the binding, and reveal an important role of Par3 in localization of Pk3 to the apical membrane domain. They show that the overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. The authors finally demonstrate that Par3 is necessary to promote Pk3-Vangl2 interaction.The authors of this manuscript reveal the role of Par3 apical-basal polarity protein in regulation of planar cell polarity. They found planar polarization of Par3 in the This is an exciting study that reveals novel role for the vertebrate Par3 protein and discovers a novel physical and functional connection between apical-basal and planar cell polarity pathways. The experiments are well done and the interpretations are appropriate. Conclusions are well supported by the evidence. The reviewers raised the following major concerns to be addressed prior to publication.Essential revisions:1) The authors assume that Par3 is upstream of Pk3/Vangl2. What if Par3 and Pk3/Vangl2 reinforce each other localization and function? Vangl2 is not showing proper planar polarization without Par3 . Is Par3 showing planar polarization without Vangl2?2) The authors assume that expression of Par3[274-544] generated phenotypes because it disrupts interaction between endogenous Par3 and Pk3. This disruption of Par3 and Pk3 interaction should be confirmed directly using conventional co-IP approach in cell line in culture.3) How Par3 promotes Pk3-Vangl2 interaction? What may be the molecular mechanism responsible? Is Par3 promoting interaction between Pk3 and Vangl2 in cell line overexpression assays?4) Why GFP-Pk3 is not localizing to the apical domain at all without Par3 overexpression ? The endogenous Par3 should be at the apical membrane domain and it should direct at least some GFP-Pk3 to the apical membrane domain.Xenopus ectoderm? At higher doses, does Par3 MO interfere with apicobasal polarity?5) The authors show that Par3 depletion does not affect apicobasal polarity. Is this due to low doses of the MO used, or is it because of a distinct function of Par3 in 6) In the presence of Par3 MO, Vangl2 is no longer seen at the apical surface. Is the protein now localized to the basolateral membrane or is it internalized into membrane vesicles?7) The effect of Par3 MO on Vangl2 distribution seems to differ from that of Par3 domains or deletion mutants on Pk3 distribution. To conclude that Par3 interacts with Pk3/Vangl2 complex, the experiments should be performed under similar conditions. For example, what happens to Pk3 with Par3 MO, or what is the distribution pattern of Vangl2 when Par3[272-544] construct is used?8) Does Par3 compete with Vangl2 for Pk3, or they form a ternary complex?9) The effect of Par3-N and Par3[272-544] on distribution of wild type Par3 should be shown, so that the mislocalization of Pk3 and the localization of endogenous Par3 can be compared and correlated. Essential revisions:1) The authors assume that Par3 is upstream of Pk3/Vangl2. What if Par3 and Pk3/Vangl2 reinforce each other localization and function? Vangl2 is not showing proper planar polarization without Par3 . Is Par3 showing planar polarization without Vangl2?We agree that Par3 and Vangl2 may potentially exhibit feedback regulation during the establishment of neural plate PCP. We performed a complementary experiment suggested by reviewers and analyzed Par3 localization in neural plates of Vangl2-depleted embryos. Indeed, Par3 planar polarity has been compromised in neuroepithelial cells depleted of Vangl2, indicating that planar polarization of Par3 requires core PCP proteins .2) The authors assume that expression of Par3[274-544] generated phenotypes because it disrupts interaction between endogenous Par3 and Pk3. This disruption of Par3 and Pk3 interaction should be confirmed directly using conventional co-IP approach in cell line in culture.Xenopus ectoderm. The interaction was measured in a proximity biotinylation assay, as the degree of biotinylation of Par3 in the presence of Biotin Ligase-tagged Prickle3. Par3[272-544] had a negative effect on this protein interaction ,supporting the view that it competes with Par3 for Prickle3 binding. Moreover, Par3[272-544] was itself biotinylated, confirming its association with Prickle3. This new experiment is described in the first paragraph of the subsection \u201cFunctional significance of the Par3-Pk3 interaction for PCP\u201d.We found that, in HEK293T cells, the levels of Pk3 and Pk3\u2206PET were upregulated by Par3[272-544], an effect that we did not observe in vivo. We, therefore, examined how Par3[272-544] influences the Prickle3-Par3 association directly in 3) How Par3 promotes Pk3-Vangl2 interaction? What may be the molecular mechanism responsible? Is Par3 promoting interaction between Pk3 and Vangl2 in cell line overexpression assays?We have carried out several experiments addressing the mechanism of Par3 effects on PCP. We did not see the competition of Par3 with Vangl2 for Pk3 binding, on the contrary, we show that Vangl2 binds to Par3 in the presence of Prickle3 . Vangl2 was not detected in pulldowns with Par3 mutant lacking Pk3 binding domains, Par3\u2206\u2206, indicating that Pk3 is necessary for the Par3-Vangl2 association . The simplest interpretation of this result is the formation of the ternary complex between Par3, Prickle3 and Vangl2. Since the promoting effect of Par3 on the total amount of the Pk3-Vangl2 complex was not apparent , we propose that Par3 stimulates the interaction of Prickle3 and Vangl3 within the ternary complex.We describe this experiment in the first paragraph of the subsection \u201cThe association of Par3 with the Pk3/Vangl2 complex and the identification of Pk3-interacting domains\u201d and in the fourth paragraph of the Discussion.4) Why GFP-Pk3 is not localizing to the apical domain at all without Par3 overexpression ? The endogenous Par3 should be at the apical membrane domain and it should direct at least some GFP-Pk3 to the apical membrane domain.We suspect that endogenous Par3 is fully engaged in preexisting binding complexes, including those containing different Prickle family members. We suspect that it may be simply unavailable for the recruitment of exogenous Prickle3.5) The authors show that Par3 depletion does not affect apicobasal polarity. Is this due to low doses of the MO used, or is it because of a distinct function of Par3 in Xenopus ectoderm? At higher doses, does Par3 MO interfere with apicobasal polarity?We have examined several apical and basal polarity markers at different doses of the MO and at additional developmental stages. These new experiments confirm our initial conclusions. Although the knockdown of Par3 is efficient and a strong effect on Vangl2 localization has been observed , we saw no obvious changes in ZO1, aPKC or \u03b2-catenin .Drosophila follicular epithelial cells in Bazooka/Par3 mutants  and in mouse retinal endothelial cells with conditionally knocked out par3 . Together, these findings suggest that the requirement for Par3 in apicobasal polarity may depend on the cell type and developmental context.Similarly, no defects in apicobasal polarity were reported in 6) In the presence of Par3 MO, Vangl2 is no longer seen at the apical surface. Is the protein now localized to the basolateral membrane or is it internalized into membrane vesicles?Cryosections revealed that Vangl2 is enriched basolaterally rather than apically after Par3 depletion .This was described in the subsection \u201cPar3 plays an essential role in neural plate PCP\u201d.7) The effect of Par3 MO on Vangl2 distribution seems to differ from that of Par3 domains or deletion mutants on Pk3 distribution. To conclude that Par3 interacts with Pk3/Vangl2 complex, the experiments should be performed under similar conditions. For example, what happens to Pk3 with Par3 MO, or what is the distribution pattern of Vangl2 when Par3[272-544] construct is used?We found that the staining with our Vangl2 antibodies produces a strong background in cells expressing Par3[272-544] construct. However, a new experiment with Par3 MO demonstrated that Pk3/Vangl2 patches are not anteriorly polarized in Par3-depleted cells . This result is added to the second paragraph of the subsection \u201cPar3 is required for the formation of the apical PCP complex in the neural plate\u201d.8) Does Par3 compete with Vangl2 for Pk3, or they form a ternary complex?Please see the response to point 3.9) The effect of Par3-N and Par3[272-544] on distribution of wild type Par3 should be shown, so that the mislocalization of Pk3 and the localization of endogenous Par3 can be compared and correlated.Prompted by the reviewers, we examined the localization of myc-Par3 in cells expressing Par3N and Par3[272-544]. Par3[272-544] did not affect apical localization of Par3  as described in the first paragraph of the subsection \u201cFunctional significance of the Par3-Pk3 interaction for PCP\u201d, consistent with our assumption that the primary effect of this construct is on Pk3 binding. Par3N caused a pronounced decrease in apical Par3, as shown previously in cultured cells . This result is shown in Figure 7\u2014figure supplement 1 and mentioned in the first paragraph of the subsection \u201cPar3 is required for the formation of the apical PCP complex in the neural plate\u201d."}
+{"text": "The 13-valent pneumococcal conjugate vaccine (PCV13) is the only licensed PCV with serotype 3 polysaccharide in its formulation. Postlicensure PCV13 effectiveness studies against serotype 3 invasive pneumococcal disease (IPD) in children have shown inconsistent results.\u00a0 We performed a systematic review and meta-analysis of observational studies to assess PCV13 vaccine effectiveness (VE) for serotype 3 IPD in children. We systematically searched PubMed, Embase, and the Cochrane library for studies published before 14 August 2017. We identified 4 published studies and 2 conference posters that provided PCV13 VE estimates stratified by serotype. The pooled PCV13 VE against serotype 3 IPD from the random-effects meta-analysis was 63.5% . A\u00a0sensitivity analysis including conference posters gave a pooled VE estimate of 72.4% .\u00a0The pooled data from case-control studies with similar methodologies and high quality support direct PCV13 protection against serotype 3 IPD in children. Meta-analysis of published case-control studies of 13-valent pneumococcal conjugate vaccine gave a pooled vaccine effectiveness of 63.5% against serotype 3 invasive pneumococcal disease (IPD). The pooled data from case-control studies with similar methodologies and high quality support direct protection against serotype 3 IPD in children. Streptococcus pneumoniae remains a significant cause of infection associated with high mortality and morbidity in children and adults ..12].I2 statistic to estimate the percentage of between-study heterogeneity [Using Stata version 14.0 software , we calculated a combined VE estimate using the Stata command Metan to fit a random-effects model . Inverseogeneity , 22.The initial search identified 3016 publications as potentially relevant . After sThe 4 published studies were either matched case-control (studies 1 and 2)\u00a0, 13 or iIn the matched case-control study in the United States (study 1)\u00a0, VE was In the indirect cohort studies, cases of nonvaccine-type IPD were used as controls  and VE eTwo of the published studies provided information on the vaccination status of the serotype 3 cases. In study 3 from Germany , 11 seroStreptococcus pneumoniae Invasive Disease network (SpIDnet), which is funded by the European Centre for Disease Prevention and Control (ECDC) to perform active population-based surveillance of IPD in children in the European Union [The authors of the current manuscript identified 2 scientific posters  , 13\u201315. I2\u00a0=\u00a015.7%, P\u00a0=\u00a0.313; I2 of 0%  with low heterogeneity , there was no significant difference in the serotype 3 geometric mean concentrations postbooster between schedules . In any Of the 6 studies identified, 3 used the indirect cohort method , 15, 24,Our analysis has limitations. First, statistical tests that are used to exclude the hypothesis of a heterogeneity of effect estimates in a meta-analysis (the null hypothesis being the absence of heterogeneity) are not sensitive when the number of studies under review is limited . Second,The incidence of disease at the population level is the product of both direct and indirect effects, and available surveillance data have shown a low incidence of serotype 3 IPD in children after PCV13 introduction. For example, in the United States, the incidence of serotype 3 IPD among children aged <5\u00a0years decreased in the years immediately following PCV13 introduction, dropping from 1.1 cases per 100\u2009000 in 2010 to 0.25 in 2013 . The incOther findings are important to consider when interpreting data on PCV13 impact against serotype 3.\u00a0First, PCV13 vaccination leads to a reduction in carriage acquisition and thus indirect protection against vaccine-type disease. PCV13 does not appear to prevent serotype 3 carriage acquisition to the same extent as other vaccine serotypes; however, studies of serotype 3 carriage in children are difficult to interpret due to relatively low carriage prevalence , 36. NonA coherent hypothesis for PCV13 impact on serotype 3 should take into account all of these data: substantial direct protection; evidence of overall reductions in population-based incidence in the early, but not later, years following introduction; and lower protection afforded by PCV13 against serotype 3 relative to other vaccine serotypes. A\u00a0recent study has also suggested that there has been a genetic shift from a relatively low to a relatively high antibiotic-resistant serotype 3 clade that was not driven by PCV13 use . One posAdditional research is needed. First, it is unknown whether the direct protection against serotype 3 is of shorter duration than against other serotypes. Some prelicensure clinical trials showed that the immune response for serotype 3 following the booster dose was not increased above the levels seen after the infant vaccination series, suggesting potential hyporesponsiveness . DespiteThe data we present here support protection against serotype 3 IPD from direct vaccination of children with PCV13. This is a first step to understand the full impact of PCV13 in protecting against serotype 3 and the global host, environmental, and pathogen factors that determine serotype 3 epidemiology. This information will be critical for understanding the best public health use of the current vaccine and for developing better vaccines. For example, if PCV13 provides direct protection against serotype 3, but incomplete or no protection against carriage acquisition, it would imply a need for broader reliance on directly immunizing at-risk populations and less reliance on indirect protection from infant immunization programs.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciy920_suppl_Supplementary_Table_1Click here for additional data file."}
+{"text": "In addition, the neurons secrete several peptide hormones that act on \u03b2-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet \u03b2-cells through actions on M ATP ion channels that are inactivated by the increased ATP/ADP ratio, and the resulting decrease in hyperpolarizing K+ current depolarizes the cell membrane i). This second messenger acts at several key points in the glucose transduction pathway that can influence islet oscillations, as well as stimulating insulin exocytosis [2+]i that promptly follows cholinergic stimulation in a glucose-rich environment [2+]i spike can potentially reset islet oscillators, and thereby play a key role in the synchronization of islet oscillations. In this study, we use mathematical modeling to demonstrate how this resetting can be achieved, even in a model where factors in addition to Ca2+ are involved in the production of islet oscillations.Acetylcholine and CCh bind to Mocytosis . Isolateironment ,41,44,45in vivo (~5 min) i for each islet using calibration constants i trace from a single group was background subtracted using a linear fit to the data and smoothed by inserting two data points between each pair of points using linear interpolation. A spectrogram of the background-subtracted data was then produced using a custom LabVIEW program  [2+]i data helps to quickly, yet quantitatively, determine the extent of synchronicity among islet groups as well as how the average islet [Ca2+]i oscillation period changes in response to CCh. All spectrograms shown throughout the Results are on the same intensity scale.For data analysis, the average islet i traces. The frequency with the highest magnitude was recorded before and after CCh pulses were applied and converted to an oscillation period (min). Calculated averages are reported with 1 SD in parentheses, unless otherwise noted.To supplement this analysis, the major oscillation period of each islet is plotted before and after the initiation of pulsing. To identify the major oscillation period, fast Fourier transforms were performed on the individual islet ) is then described by the following differential equation:IPamp3  = 0.35 \u03bcM when the 10 sec pulse was on, and 0 otherwise. The parameter 3 concentration changes. The code for the model is available for free from www.math.fsu.edu/~bertram/software/islet.The IOM was modified to simulate the effects of activation of muscarinic acetylcholine receptors by CCh. Activated receptors result in the production of IPm the ER . We inclThe microfluidic platform employed for this study facilitated precise and automated delivery of glucose and the various CCh profiles to groups of 3\u20134 islets. Multiple experiments were performed to investigate the effects of different CCh profiles. Although the layout of the microfluidic channels has been described previously, the use of the piezoelectric transducer in conjunction with the flow rate sensors was not. We found that this active flow rate control was much better at rapidly generating pulses and maintaining stable flow rates than passive methods. The reagents took 48 s (SD 1) to travel the 75 mm long channel from the inputs to the islet chamber. This dispersive flow resulted in attenuation of the 10 \u03bcM CCh pulses by 43.5% (SD 0.4). Thus, the concentration felt by the islets was ~5.7 \u03bcM. This value is lower than the those used in other reports ,26 altho2+ measurement from the 3 different experimental groups  tested. Any observed synchronization in the average Ca2+ trace was attributed to the small islet number within a single group.Control experiments were performed by continuously perfusing 3 groups of islets \u2013S5 Figs 2+ oscillations of individual islets (gray traces) from one of the three groups as well as the mean of the four islets (black trace). Before CCh pulsing commenced, the Ca2+ oscillations were out of phase as shown by the minimal rhythmicity in the mean trace. The inset of 2+ oscillations were dominated by the effects of the cholinergic pulses; all four of the islets shifted their oscillation periods to 2.0 min indicating 1:1 entrainment by the periodic CCh pulses  with a train of twenty CCh pulses with R = 2 min, i.e., 2 min between each pulse. ctrogram  of the a2+ oscillations. Two of these islets had long natural oscillation periods of 6.8 and 10.2 min and the stimulus train made them oscillate faster, though the entrained period (~4 min) was still about twice that of the stimulus train. In contrast, for the remaining two islets, the entrained period was longer than that of their natural periods (~2.9 min). 2+]i trace in the representative experiment is shown in 2+]i peaks measured from this group. Nevertheless, it is clear from the analysis of the average [Ca2+]i trace and the change in oscillation periods of the individual islets that the islets were entrained 1:1. It is notable that the 5 min periodicity of the CCh pulse train, so effective at entraining the islets, is similar to that of blood insulin oscillations in healthy animals i when using a single pulse of ACh i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 1(PDF)Click here for additional data file.S4 Fignd negative control experiment with 4 islets perfused with 11 mM glucose is shown. Gray lines indicate the individual islets and the black line indicates the mean [Ca2+]i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 2(PDF)Click here for additional data file.S5 Figrd negative control experiment with 4 islets perfused with 11 mM glucose is shown. Gray lines indicate the individual islets and the black line indicates the mean [Ca2+]i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 3(PDF)Click here for additional data file.S6 Fig2+]i traces (gray lines) of four islets and the average (black line) are shown when CCh pulses with rest durations of R = 2 min were applied. The timing of the CCh pulses is shown by the \u201cx\u201d at the top of the figure. The inset shows the 4 islets had natural periods ranging from 4\u20135 min prior to pulsing (open circles). During pulsing, oscillation periods transitioned to 2 min (filled circle) indicating 1:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The spectrogram of the average [Ca2+]i from the 4 islets in (A) is shown. A prominent oscillation period band emerges at 2 min for the 38 min long CCh pulsing duration indicating synchronization among this islet group. The spectrogram did not show robust oscillation period bands outside the pulsing duration. Any >10 min oscillation period band is an artifact from the STFT data analysis.(A) The [Ca(PDF)Click here for additional data file.S7 Fig2+]i traces (gray lines) of four islets and the average (black line) are shown when CCh pulses with rest durations of R = 2 min were applied. The timing of the CCh pulses is shown by the \u201cx\u201d at the top of the figure. The inset shows the 4 islets had natural periods ranging from ~3\u201310 min prior to pulsing (open circles). During pulsing, oscillation periods transitioned to ~4 min (filled circle) indicating 2:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The spectrogram of the average [Ca2+]i from the 4 islets in (A) is shown. An oscillation period band emerges at ~4\u20135 min after 20 minutes of CCh pulsing indicating some islet synchronization. The >10 min oscillation period band is an artifact from the STFT data analysis.(A) The [Ca(PDF)Click here for additional data file.S8 Fig2+]i from all 4 islets are shown in grey and the average is shown in black. The timing of the CCh pulses is shown by the \u201cx\u201d. The inset shows the natural period of these 4 islets ranged from 6.8\u201310.2 min (open circle), but all converged to 5 min during pulsing (filled circle) indicating 1:1 entrainment. (B) The synchronized response of the group is evident by the emergence of an albeit weaker oscillation period band at 5 min within the 55 min time span of pulsing in the spectrogram.(A) The individual [Ca(PDF)Click here for additional data file.S9 Fig2+]i from all 4 islets are shown in grey and the average is shown in black. The timing of the CCh pulses is shown by the \u201cx\u201d. The inset shows the natural period of these 4 islets ranged from 4.2\u20137.0 min (open circle), but all converged to 5 min during pulsing (filled circle) indicating 1:1 entrainment. (B) The synchronized response of the group is evident by the emergence of an oscillation period band at 5 min within the 55 min time span of pulsing in the spectrogram.(A) The individual [Ca(PDF)Click here for additional data file.S10 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 90 min are shown with an \u201cx\u201d noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods between 4.1 and 6.8 min (open circles) and transitioned to 5.0 min (1:2). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The 5 min period band in the spectrogram of the mean Ca2+ trace depicts the synchronicity among these islets during the 50 min pulsing duration. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S11 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 90 min are shown with an \u201cx\u201d noting the timing of each CCh pulse. The inset shows that 3/4 islets had natural oscillation periods between 5.1 and 6.8 min (open circles) and transitioned to 5.0 (1:2). The remaining islet had a >15 min natural oscillation period and transitioned into 1:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The prominent band at a period of 5 min in the spectrogram of the mean Ca2+ trace shows the high degree of synchronization among these islets during the 50 min pulsing duration. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S12 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 80 min are shown with an \u201cx\u201d noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods between 3.4 and 6.8 min (open circles) and transitioned to 5.0 (1:2) or 2.9 min oscillations (1:3) during pulsing (filled circles). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) As can be seen from the green band in the spectrogram of the average Ca2+ trace during the 50 min pulsing time span, these islets synchronized, albeit to a lesser degree than other islet groups in this experiment. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S13 Fig2+]i traces of the four islets present (grey lines) and their four islet average (black line) over 90 min are shown with an \u201cx\u201d noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods of 6.8 or 10.2 min (open circles) and transitioned to 10.1 min oscillations (1:1) during pulsing (filled circles). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The green 10 min oscillation period band in the spectrogram shows the synchronization of the doublet-like oscillations in among this islet group. The weaker ~5 min bands correspond to the intra doublet gaps in the average Ca2+ trace.(A) The i (black line). Each of the CCh pulses is shown by \u201cx\u201d. Although randomly spaced, the repeated CCh pulses produced a synchronized population of 4 islets as can be seen from the emergence of a green band in the spectrogram (B) of the 4 islet mean trace. The degree of synchronization rapidly diminished after the final pulse at 77 min.(A) The Ca(PDF)Click here for additional data file.S15 Fig2+ traces (grey lines) from a unique, representative experiment with a set of 4 islets are overlaid with the average [Ca2+]i (black line). Each of the CCh pulses is shown by \u201cx\u201d. Although randomly spaced, the repeated CCh pulses produced a synchronized population of 4 islets as can be seen from the emergence of a green band in the spectrogram (B) of the 4 islet mean trace.(A) The Ca(PDF)Click here for additional data file."}
+{"text": "Rht) are known for yield gains due to lodging resistance and partitioning of assimilates into ear. The available and commercially exploited sources of dwarfism in Indian spring wheat are Rht1 and Rht2 genes inspite of availability of over 20 dwarfing genes. Rht8 a Gibberellic acid sensitive dwarfing gene is another reduced height gene commercially exploited in some Mediterranean countries. Two F2 populations segregating for Rht1 and Rht8 genes with each comprising 398 and 379 plants were developed by crossing European winter wheat cultivars Beauchamp and Capitole with Indian spring wheat cultivar PBW 621. Different genotypic combinations for Rht1 and Rht8 genes were selected from these populations through linked molecular markers and selected F3:4 lines were evaluated for various agronomic traits in a replicated trial. Reduction in plant height with Rht8 and Rht1 averaged 2.86% and 13.3% respectively as compared to the group of lines lacking dwarfing gene. Reduction was spread along all the internodes of wheat culm and reduction was lower as progress towards the lower internode. Grain number per spike and highest yield was observed in lines carrying only Rht1 gene. Reduction in plant biomass was observed with either of the dwarfing gene. Longest coleoptile length and seedling shoot length averaged 4.4 \u00b1 0.09 cm and 19.5 \u00b1 0.48, respectively was observed in lines lacking any of the dwarfing gene. Negligible reduction of 6.75% and 2.84% in coleoptile length and seedling shoot length, respectively was observed in lines carrying only Rht8 gene whereas F3:4 lines with Rht1 gene showed 21.64% and 23.35% reduction in coleoptile length and seedling shoot length, respectively. Additive effect of genes was observed as double dwarfs showed 43.31% and 43.34% reduction in coleoptile length and seedling shoot length.Optimizing wheat height to maximize yield has been an important aspect which is evident from a successful example of green revolution. Dwarfing genes ( Triticum aestivum 2n = 6x = 42) has been dramatically reduced. For example, the average heights of wheat varieties have dropped from 150 cm to 90 cm in UK [Rht1 and Rht2 dwarfing genes were transferred from Japan to USA, USA to CIMMYT, Mexico and from Mexico to all over the wheat cultivated area through Japanese variety Norin-10. Rht8 is mainly commercialized in Europe and transferred through Japanese variety Akakomugi from Japan to Italy and then from Italy to other parts of Europe [Rht1-B1b and Rht1-D1b dwarfing alleles of Rht1 and Rht2 not only reduce the plant stature, but also reduce the coleoptile length which is the deciding factor for early emergence under water deficit or stress conditions and hence, effect the crop stand. It has also been well documented that dwarfing genes Rht-B1b and Rht-D1b are associated with Type I susceptibility to Fusarium head blight in wheat and low anther extrusion [Rht genes have concluded that Rht8 gene reduces the plant height but it has negligible effect on coleoptile length [Rht8 gene and GA insensitive Rht1 and Rht2 genes have been conducted in Australian cultivars [Rht8 gene in combination with Rht1 gene in elite spring wheat background under Indian environmental conditions.To meet the escalating demand of fast growing human population, there is need of continuous increase in wheat grain production. Optimizing plant stature to enhance productivity under ever changing and unpredictable climate is one of the major strategy of plant breeders. Reduced height in wheat is often associated with increase in yield, probably, owing to reduced lodging and enhanced partitioning of assimilates to the grain . The concm in UK  and fromf Europe . Rht1-B1e length . The stuultivars  and in sRht8c) and genotype PBW 621 (having gene Rht1) in main season 2012\u201313. Beauchamp and Capitole are winter wheat varieties released in France in 1978 and 1964, respectively. These lines were selected from a diverse European winter wheat germplasm set of 376 cultivars that had been procured by Punjab Agricultural University, Ludhiana, Punjab, India from National Institute of Agricultural Botany, Cambridge, UK in 2010. PBW 621 is a spring wheat cultivar released in Indian in 2011 and widely grown in NWPZ of India. The F1 plants of these crosses were self-pollinated to generate F2 population. In wheat growing season 2014\u201315, 398 F2 plants from cross Beauchamp x PBW 621 and 379 F2 plants from Capitole x PBW 621 were space planted and plant height data was recorded. Further without any selection all of the F2 plants were allowed to self-pollinate to generate F3 progenies. The presence and absence of dwarfing allele(s) of both the Rht1 and Rht8 loci were determined by using linked molecular markers [Rht1 locus which leads to evolution of Rht-B1b allele  [3 progenies of Beauchamp x PBW 621 and Capitole x PBW 621 cross, respectively were identified as being double dwarfs (Rht1 + Rht8) or single dwarf (either Rht1 or Rht8) or tall . Two plants from each of these progenies were harvested separately and multiplied at Keylong, Himachal Pradesh  and 45 of these F3:4 lines with similar growth period were selected for agronomic evaluation in main season 2016\u201317 at experimental area, Punjab Agricultural University, Ludhiana.Two crosses (Beauchamp x PBW 621 and Capitole x PBW 621) were generated between cultivars Beauchamp and Capitole  procedure [Rht8 and gene specific dominant markers BF, WR1 and MR1 for Rht1 were used. The in-vitro amplification (PCR) and PCR product were resolved as per the protocol described [Rht1 gene [3:4 lines were also confirmed with KASP markers and genotypic composition of these groups are: both the dwarfing genes absent (r1r1r8r8), Rht1 absent and Rht8 present in homozygous form (r1r1R8R8), Rht1 in heterozygous form and Rht8 absent (R1r1r8r8), Rht1 in heterozygous and Rht8 present in homozygous condition (R1r1R8R8), Rht1 in homozygous form and Rht8 absent (R1R1r8r8) and both Rht1 and Rht8 in homozygous form (R1R1R8R8).Young leaves of parental lines and Frocedure . The SSRescribed ,13. Duriht1 gene . KASP is3:4 line with no physical damage and of uniform size were placed in the middle of moist germination paper. The seeds were first sterilized with the fungicide to avoid any infection and placed in wet paper with germ end down and then paper was rolled as a \u2018cigar\u2019. The cigars were then placed vertically into a container with 5\u20137cm of water at the bottom and then the container was placed in an incubator at 20\u00b0C for 10 days under dark conditions. After 10 days, cigars were unrolled and average coleoptile length of seedlings was recorded from the base of the seed to the coleoptile tip. Total shoot length of seedlings was also measured.For seedling traits, 12 seeds of each of the F2 individuals were space planted and plant height was recorded. During main season 2015\u201316, F3 progenies were planted as plant to progeny row. Selected F3 plants were harvested individually and planted at offseason 2016 for multiplication to conduct a replicated trial in coming main season. The experiment for certain agronomic traits was conducted for selected F3:4 lines in main growing season 2016\u201317. These lines along with parents were planted as 7 x 7 square lattice design with three replications. Three plants from internal rows of each plot were randomly selected for observations to eliminate the border effect. Final plant height and individual internodal length was measured in centimeter at plant maturity. Ear length was also recorded. The internode below the spike was designated as peduncle length and successive to peduncle length was defined as first internode, second internode and so on. Days to flowering, total tillers per meter row, spikelet per spike, grain per spike, grain yield per plot, 1000-grain weight, plant biomass and harvest index were also recorded.In 2014\u201315, F3:4 line was calculated with the Analysis of variance of square lattice design using SAS ver. 9.3. The relative effect of different gene combinations was compared as a percent change with respect to tall group.Adjusted mean of each of the FRht1, Rht2 and Rht8 confirmed that PBW 621 carries dwarfing allele of Rht1 and both winter wheat parents Beauchamp and Capitole carry dwarfing allele of Rht8 gene. Genotyping with WMS 261 marker showed a 192 bp band in Beauchamp and Capitole and approximately 165 bp band in PBW 621. Considering Norin-10, the source of dwarfism in Indian cultivar which carries both Rht1 and Rht2 gene [Rht1 and Rht2. Genotypes C-306 and C-518, the tall traditional varieties, widely grown before green revolution were used as a control. None of the parental genotype showed the presence of dwarfing allele of Rht2 gene with DF-WR2 primer combination. With BF-MR1 primer combination for Rht-B1a allele , all of the lines showed amplification but C-varieties and European winter wheat lines showed very high intensity PCR product whereas PBW 621 gave very faint band. For Rht-B1b allele  BF-MR1 primer combination was used and faint band was obtained for European winter wheat lines and C-varieties, whereas comparatively intense amplified product was obtained for PBW 621. From these results, it has been concluded that Rht1 gene is responsible for semi-dwarfing nature of spring wheat line used (PBW 621).PCR assay of parental genotypes for ht2 gene , parents3 progenies which seemed homozygous for plant height at early stages were chosen for molecular marker analysis for Rht1 and Rht8 genes. Leaves from all the plants of a progeny were bulked for DNA extraction so that a particular sample represent F2 genotype. For Beauchamp x PBW 621 cross, 85 F3 progenies were selected for molecular marker analysis. Out of 85, 19 progenies were observed to be positive for Rht8 gene, 49 were heterozygous and 16 progenies were negative for Rht8 gene with WMS 261 marker. Similarly, for cross Capitole x PBW 621, 75 progenies were selected for molecular marker analysis based on the phenotype of the F3 progeny. Among these 75 progenies, 15 were come to be positive, 41 were heterozygous and 15 were observed to be negative for Rht8 gene as revealed by marker WMS 261.F3:4 lines with similar growth period were selected for agronomic and seedling evaluation. Because of the uncertainty about Rht1 gene in the segregants, these 45 lines were genotyped with the KASP markers reported [Rht1 revealed that out of 45, 12 segregants were heterozygous for Rht1. So, based on the genotyping of selected F3:4 lines instead of four homozygous groups, these lines were categorized into six different genotypic classes i.e. both the dwarfing genes absent (r1r1r8r8), Rht1 absent and Rht8 present in homozygous form (r1r1R8R8), Rht1 in heterozygous form and Rht8 absent (R1r1r8r8), Rht1 in heterozygous and Rht8 present in homozygous condition (R1r1R8R8), Rht1 in homozygous form and Rht8 absent (R1R1r8r8) and both Rht1 and Rht8 in homozygous form (R1R1R8R8).Based on the molecular marker data and phenotype data, 45 Freported . KASP baColeoptile length (CL) of European wheat was marginally higher as compared to spring wheat cultivar PBW 621 whereas significant difference for seedling shoot length (SL) was observed between the European winter wheat lines and PBW 621. 3.6 cm and 3.5 cm of CL was recorded for Beauchamp and Capitole, respectively whereas PBW 621 was having 2.9 cm of CL. Total seedling shoot length for PBW 621 was 14.9 cm whereas for Beauchamp and Capitole it was 21.1 cm and 22.6 cm, respectively  than the tall category. Rht1 caused approximately 13.3% reduction in plant height when present in homozygous form which indicated that extent of height reduction was stronger with Rht1 as compare to Rht8. However, average plant height of lines with Rht1 in heterozygous form was 8.7 cm taller than the lines which carry Rht1 in homozygous form, suggesting the partial dominance nature of Rht1 gene  yet no significant variation between plant height of these lines were observed. Parental genotypes i.e. Beauchamp, Capitole and PBW 621 used in the study showed comparable plant height averaging 92 cm, 103 cm and 100 cm respectively. Whereas two populations developed from these parents were highly segregating for plant height. Wild type genotypic class (having no dwarfing gene) was taller among all with average plant height of 119.7 cm while both the dwarfing genes caused reduction in plant height with varying extent. Mean plant height of the lines with ht1 gene .Rht1 gene as compare to lines which carry Rht8 gene. Maximum reduction was observed in double dwarf group carry both dwarfing genes. Difference in the internodal length reduction was observed in the group carrying Rht1 in homozygous form and Rht1 in heterozygous form. The schematic representation of internode elongation pattern for different genotypic classes is shown in Individual internodal length was measured to know whether reduction was spread along all the internodes of wheat culm or due to a particular internode. No difference was observed in number of internodes among different genic groups. The reduction effect on wheat culm was spread along all the internodes, however, significant difference in internodal length was observed for peduncle length and first internode. As progress towards to the successive internodes lesser reduction was observed. Decrease in internodal length was larger in the lines which carry Rht8 gene alone and wild type group lacking dwarfing gene. Grain number per spike was recorded maximum in the lines which carries Rht1 gene either in heterozygous or homozygous form. No significant effect of different dwarfing genes was observed on thousand grain weight. However, slightly lower 1000-grain weight was recorded in the lines carry Rht8 gene as compare to tall lines. Group of lines which carry only Rht1 had comparable 1000-grain weight as that of tall group.Reduction in spike length was observed in lines with either single or double dwarfing gene, when the lines were classified into four homozygous groups. The effect on spikelet number per spike varied for different group. There was no significant difference for grain number per spike observed between the group with R1R1r8r8 category which carried dwarf allele of Rht1 gene and tall allele of Rht8 gene. It has been observed that Rht1 caused average yield increase of 10.77% when present in heterozygous form and 18.17% when present in homozygous form whereas Rht8 caused yield reduction of 18.27%. Decreased in plant biomass was observed in lines that carried either of the dwarfing gene or both dwarfing genes. These decrease in plant biomass and highest plot yield resulted in increased harvest index in the lines carry only Rht1 gene (Highest grain yield per plot was observed in ht1 gene .Rht8 gene in different studies [Rht1 or Rht2) have been reported [Rht8 gene [Rht8 [Rht8 as well as of Rht1 (13.3%) on plant height was observed. Our results also showed partial dominance behavior of Rht1 gene as the group with Rht1 in heterozygous form was little taller as compare to the group with Rht1 in homozygous. Additive effect of these genes was also observed, double dwarf group were smallest among all with 24.2% reduction in plant height.Effect of dwarfing genes on plant height and other traits were reported as highly varied with different genetic backgrounds and environmental conditions. 7\u201318% reduction in plant height was reported with  studies , 20, 21 reported ,18,19. 1ht8 gene  and 14% Rht8 gene and 25.4% reduction by Rht1 gene was reported previously [Rht1 and Rht8 gene together shortened CL by 28.4% whereas in the present investigation 42.94% reduction in CL has been observed. Less effect of Rht8 on coleoptile length and on early growth stages of wheat has been reported in many studies [Coleoptile is the pointed protective sheath that encases the emerging shoot as it grows from the seed to the soil surface and is one of the key trait for breeding wheat for drought tolerance. Emergence ability of seedling is highly influenced by coleoptile length. For a seed to emerge successfully, seed should not be planted deeper than its coleoptile length. Reduction in coleoptile length due to dwarfing genes as observed in present study, has also been reported in several other studies. For example, 6.2% reduction in coleoptile length in the wheat cultivar containing eviously  which ar studies , 9, 23.R1R1r8r8 category is consistent with the \u2018tall dwarf\u2019 model proposed [Rht alleles with other genes which increase both plant height and yield. Report of yield advantage with gibberellin insensitive Rht dwarfing genes over tall controls are also available [Rht1 or Rht2 and decrease by Rht8 have also been reported [Rht1 and Rht2, respectively and 21.0% and 5.3% yield reduction by Rht8 in two different genetic background.The observation of highest yield of proposed . They havailable . Yield ireported . HoweverRht1 gene out yielded as compared to the lines having Rht8 gene. However, for seedling traits lines with Rht8 were better performing than lines with Rht1 gene. Coleoptile length is one of the important trait for deep sowing under drought condition to capture moisture from deeper soil. For conservation agriculture, emergence under deep sowing is needed and cultivars with longer coleoptile can emerge quickly and can have better plant vigor. Hence, understanding of such pleiotropic interactions of dwarfing genes with different genetic background will facilitate effective deployment of dwarfing genes in modern varieties. The results of present study showed that lines which carry both Rht1 and Rht8 gene together has showed very high reduction in CL and SL. Such cultivars will not be suitable under water limiting conditions. Hence, considering the coleoptile length as an indicator for better emergence under drought conditions, it is suggested that in order to breed spring wheat cultivars for drought tolerance or water use efficiency, there is need to replace the residents dwarfing genes of Indian spring wheat cultivars which mainly consists of either Rht1 or Rht2. Wheat breeders in the NWPZ of India must adopt Rht8 as an environmentally adaptable alternative semi-dwarfing gene. The Rht8 gene can also be successfully combined with some Ppd and Vrn genes to offer height reduction and adaptability in the niche micro environments of region so as the duration of the crop can be extended to translate into yield enhancement.The present study revealed pleiotropic effect of dwarfing genes along with effect on plant height. Under favorable conditions, lines with Rht1 and Rht8 developed by European winter wheats and spring wheat cultivars allowed us to gain the understanding of potential of alternate dwarfing gene in Indian subcontinent. Lines with higher coleoptile length and seedling shoot length which is a deciding factor for crop establishment were identified in the study. This gave the candidate plant material to evaluate and select the material for conservation agriculture which is one of the new emerging breeding objective in light of climate change.Segregating population of S1 Table(XLSX)Click here for additional data file."}
+{"text": "MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2. Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2. Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in  WWTR1 is the gene) and YAP (YAP1 is the gene) are developmentally important transcriptional coactivators benzamide (MS-275) in the same above three lines. Treatment with 1 \u03bcM MS-275 for 24 hours resulted in at least a 1.5 fold increase in expression of MST1 and MST2 in the three cell lines  ) was calculated by Spearman's correlation coefficient.Standard deviation for the quantitative western blots was calculated from fold change expression derived from different western blot experiments. For quantitative RT-PCR, standard deviation was calculated from fold change values from each triplicate. Correlation between fraction methylation of CpG islands and expression of the Hippo kinases (RSEM [log"}
+{"text": "We describe CD4 count recovery among HIV positive individuals who initiated antiretroviral therapy (ART) with and without severe immune suppression using complete laboratory data from South Africa\u2019s national HIV treatment programme between 2010 and 2014 and discuss implications for CD4 count monitoring.Retrospective analysis of routinely collected laboratory data from South Africa\u2019s National Health Laboratory Service (NHLS). A probabilistic record linkage algorithm was used to create a cohort of HIV positive individuals who initiated ART between 2010 and 2014 based on timing of CD4 count and viral load measurements. A CD4 count < 50 copies/\u03bcl at ART initiation was considered severe immunosuppression. A multivariable piecewise mixed-effects linear regression model adjusting for age, gender, year of starting ART, viral suppression in follow up and province was used to predict CD4 counts during follow up.1,070,900 individuals had evidence of starting ART during 2010\u20132014 and met the criteria for inclusion in the cohort -46.6% starting ART with CD4 < 200 cells/\u03bcl and 10.1% with CD4 < 50 cells/ \u03bcl. For individuals with CD4 counts < 200 cells/\u03bcl, predicted CD4 counts > 200 cells/\u03bcl, >350 cells/\u03bcl and >500 cells/\u03bcl corresponded with mean follow up durations of 1.5 years (standard deviation [s.d] 1.1), 1.9years (s.d 1.2) and 2.1 years (s.d 1.3 years). For those with CD4 counts < 50 cells/\u03bcl, predicted CD4 count above these threshold corresponded with mean follow up durations of 2.5 years (s.d 0.9 years), 4.4 years (s.d 0.4 years) and 5.0 years (s.d 0.1years) for recovery to the same thresholds. CD4 count recovery varied mostly with duration on ART, CD4 count at the start of ART and gender.For individuals starting with ART with severe immunosuppression, CD4 recovery to 200cells/\u03bcl did not occur or took longer than 12 month for significant proportions. CD4 monitoring and interventions recommended for advanced HIV disease should continue until full recovery. Antiretroviral therapy (ART) reduces mortality and morbidity among HIV positive individuals as well as the onward transmission of HIV. \u20133 UntreastJanuary 2010 and 31st December 2014 and followed up until March 2015. We discuss the implications of the findings on the continued use of CD4 count measurements to monitor the effectiveness of ART.Because the CD4 count is a good predictor of morbidity and mortality before and after ART start, HIV treatment guidelines in the past recommended CD4 count measurements for determining eligibility for HIV treatment and for monitoring its effectiveness. Poor or suboptimal CD4 recovery despite ART and viral suppression has been associated with increased risk of mortality and morbidity from tuberculosis and other AIDS related morbidities.\u201315 SinceSome of the methods of this paper have been described elsewhere . BrieflyRoutine specimen data from the laboratory management information system are archived in the Corporate Data Warehouse (CDW) of the NHLS and are available for analysis. However, the lack of a unique identifier for individuals enrolled into the CCMT programme and the inability to uniquely identify multiple tests over time and treatment sites has limited the use of these data in the past. As part of a broader evaluation of adherence and retention in HIV care within the South African National ART programme, the National Department of Health commissioned analyses of laboratory datasets using big data methods in 2015. Initial analyses of these data describing i) proportions of individuals who had CD4 recovery to given thresholds, ii) the time to recovery to these threshold and iii) speed of CD4 recovery in the first 12 months were conducted in 2015\u20132016 and have been published elsewhere.  The resuThe procedures for record linkage and creating a synthetic cohort of HIV positive individuals has also been described in detail elsewhere.  For thisThe date of ART start was defined as the date of the eligible baseline CD4 count described above. This date was taken to be the date of a CD4 count done three to 12 months before the first viral load date. Where multiple CD4 count test dates and results met this criterion, the earliest date was used. The CD4 count at ART start was defined as the result of this eligible baseline CD4 count while follow up CD4 counts were results from the CD4 count tests done at any point after the ART start date. The duration of follow up was defined as the time interval between the date of ART initiation and the date of the latest CD4 count and was considered to be the same as the duration on ART assuming that individuals remained on ART for the entire duration of follow up. A CD4 count of < 50 cells/\u03bcl at the start of ART was considered severe immunosuppression at ART start. Viral suppression was defined as having a viral load result of <400 copies/ml at any point during follow up while age referred to the individual\u2019s age recorded at the eligible baseline CD4 count or determined from the recorded date of birth. Gender and year of ART initiation was taken to be the gender and year recorded at the eligible baseline CD4 count testing. Province was defined as the province where eligible baseline CD4 count testing was done regardless of whether or not the individual subsequently moved to other provinces. The main outcome of the analysis was the mean CD4 counts at given intervals post ART initiation. For individuals who had multiple CD4 counts done in the same interval of follow up, all measurements were used in determining the mean CD4 count in that interval.Once the data were linked to unique individuals using the linkage algorithm, it was exported to Stata 14.2  for analysis. The characteristics of eligible individuals at cohort entry were described using medians and interquartile ranges for continuous variables and as frequency and proportions for categorical data. A multivariable piecewise mixed-effects linear regression model\u2014adjusting for age, gender, eligible baseline CD4 count, any virological suppression during follow up and province with splines at 3, 6, 12, 24, 36, 48 and 60 months post ART initiation\u2014was used to predict the mean square root of CD4 counts during and at specified time intervals. The model was fit on the square root scale for the CD4 count outcome in order to normalise the distribution of the CD4 counts. The model allowed for the following interactions: i) age and gender, ii) eligible baseline CD4 count and duration on ART (as splines), iii) calendar year and duration on ART, iv) gender and duration on ART and v) province of entry into care and duration on ART. Predicted mean CD4 counts at different time intervals post ART start and for different combinations of age, gender and baseline CD4 count were obtained by squaring the predicted square roots of the CD4 counts. Likelihood ratio tests comparing i) the piecewise regression model with splines at 3, 6, 12, 24, 36, 48 and 60 months post ART initiation with no covariates (representing observed data) to the full model and ii)the same piecewise regression model but with no interaction terms to the full model were used to determine goodness of fit.This study was approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) and the Boston University School of Medicine IRB (H-31968.) Permissions were also obtained from the NHLS Research Office and the National Department of Health. Since this was a secondary analysis of routine laboratory data, no written informed consent was obtained from patients.From 3,977,761 unique individuals who had at least one viral load test and one CD4 count test record in the CDW database between January 2010 and March 2015, 1 070 900 (26.9%) individuals met the criteria for inclusion in the cohort. Compared to individuals included in the cohort, individuals excluded from the cohort were more likely to be male (33.3% vs. 30.1%), older (median 46 years [IQR 38\u201355 years] vs. 44 years [IQR 37\u201352 years]), had a higher median nadir (lowest ever) CD4 count (200 cells/\u03bcl vs. 167 cells/\u03bcl) and were less likely to be from KwaZulu-Natal Province (22% vs. 31.9%). Across all provinces the median CD4 counts at the start of ART were lower among males compared to females, decreased with increasing age from age category 25\u201334 years and increased with calendar years from 2010. Overall the observed CD4 counts increased rapidly from baseline to a mean of 319 cells/\u03bcl ) around 6 months post ART initiation. Beyond that, there were smaller but steady increases in the observed means to 358 cells/\u03bcl (s.d 36 cells/\u03bcl) at around 12 months, 409cells/\u03bcl (s.d 37 cells/\u03bcl) at 24 months, 434 cells/\u03bcl (s.d 40cells/\u03bcl) at 36 months, and 544 cells (s.d 44 cells/\u03bcl) by around 48 months post ART initiation. At all the time intervals, the observed CD4 count levels were lower among males compared to females see .Overall the predicted mean CD4 recovery was rapid during the first eight months on treatment before slowing down in the subsequent periods. The mean predicted CD4 counts during the first three months was 217 cells/\u03bcl . The mean predicted CD4 counts increased to 321 cells/\u03bcl  around 12 months; 396 cells/\u03bcl (95% CI 395\u2013398 cells/\u03bcl) around 18 months; 413 cells/ \u03bcl (95% CI 411\u2013415 cells/\u03bcl) around 24 months; 432 cells/\u03bcl (95% CI 430\u2013435 cells/\u03bcl) around 30 months; 441 cells/\u03bcl (95% CI 437\u2013444 cells/\u03bcl) around 36 months; 455 cells/ \u03bcl (95% CI 452\u2013459 cells/\u03bcl) around 42 months; 458 cells/\u03bcl (95% CI 453\u2013463 cells/\u03bcl) around 48 months and 473 cells/\u03bcl (95% CI 466\u2013480 cells/\u03bcl) around 54 months. For individuals with CD4 counts < 200 cells/\u03bcl; predicted CD4 counts >200 cells, 350 cells/\u03bcl and 500 cells/\u03bcl corresponded with a mean ART durations of 1.5 years (s.d 1.1), 1.9 years (s.d 1.2) and 2.1 years (s.d 1.3 years) respectively. At all durations on ART, the predicted recovery was lower among males compared to females  and amonFor individuals who started ART with CD4 counts < 50 cells/ \u03bcl\u2014severe immunosuppression, the mean predicted CD4 count at 12 months was 177 cells/\u03bcl (95% CI 175\u2013179 cells/\u03bcl) overall. This mean was higher among females compared to males [192 cells/\u03bcl (95% CI 190\u2013193 cells/\u03bcl) vs 160 cells/\u03bcl (95% CI 158\u2013161 cells/\u03bcl)]. For both males and females, the mean predicted CD4 counts at 12 months were <200 cells, consistent with the observation that 71% of this population had not recovered to 200 cells/\u03bcl by 12 months of follow up with 39% not achieving CD4 recovery to 200cells/\u03bcl at all during follow up. Predicted CD4 counts >200 cells/\u03bcl, 350 cells/\u03bcl and 500 cells/\u03bcl corresponded with ART durations of 2.5 years (S.D 0.9 years), 4.4 years (S.D 0.4 years) and 5.0 years (S.D 0.1years) respectively. Because CD4 counts at the start of ART decreased with increasing age, the predicted CD4 count recovery among the oldest group of individuals was also determined at the given time points and for given baseline CD4 counts. Among the 324,667 individuals who were aged \u226550 years at the start of ART, the predicted CD4 counts at 12, 24, 36, 48 and 54 months increased with CD4 counts at the start of ART and were higher among females compared to males. For males with CD4 counts <50 cells/\u03bcl, the predicted mean of the CD4 count peaked at 48 months at 254 cells/\u03bcl while for females it continued to increase to 325 cells/\u03bcl by 54 months. For those with CD4 counts \u2265500 cells/\u03bcl, the predicted CD4 count peaked at 54 months for males and 60 months among females at 612 cells/\u03bcl and 705 cells/\u03bcl respectively see . CompareTo compare the extent of CD4 count recovery across the nine provinces in the country, the predicted CD4 counts at 12, 36 and \u226554 months were determined for males and females aged 15\u201349 years with CD4 counts 50\u2013199 cells/\u03bcl and \u2265200 cells/\u03bcl at ART start. Among males, the mean predicted CD4 counts at 12, 36 and \u226554 months for individuals with CD4 counts of 50\u2013199 cells/\u03bcl at ART start and for those with CD4 counts \u2265 200 cells/\u03bcl were highest in KwaZulu Natal, Free State and Gauteng provinces and lowest in the Eastern Cape, Northern Cape and North West provinces see . SimilarAs median CD4 counts at the start of ART increased with calendar year,  an analyWe used a multivariable piecewise mixed-effects linear regression model adjusting for age, gender, eligible baseline CD4 count, virological suppression during follow up and province to predict the extent and magnitude of CD4 count recovery among individuals aged >15 years old initiating ART. Our analyses found that across all ages, provinces and baseline CD4 counts, CD4 recovery was lower and slower among males compared to females even after adjusting for CD4 count at ART start as well as duration on ART. The analysis also found that among individuals with CD4 counts <50 cells/\u03bcl at the start of ART, the predicted mean CD4 count after 12 months post ART initiation remained below 200 cells/\u03bcl and that it took a mean of 2.5 years for recovery to 200 cells/\u03bcl in this group. Our analyses also consistently found greater CD4 count recovery in KwaZulu Natal, Gauteng and Free State provinces.Although this is the first ever national level analysis of CD4 count recovery data, this analysis confirms what smaller cohorts of up to 90 000 individuals from South Africa and other sub Saharan African settings have demonstrated. These studies demonstrated that i) the CD4 count at the start of ART was the strongest predictor of CD4 count recovery,  ii) CD4 Our study had some important strengths. Firstly this is a national level data set with representation of all provinces in the country. Second, there were long durations of follow up\u2014up to five years for some eligible individuals. This meant the trajectories of CD4 count recovery could be described beyond one to two years. Our study also had a number of limitations: Firstly, this analysis used laboratory data linked to unique individuals through the use of a probabilistic matching algorithm to create a cohort and determine CD4 recovery outcomes. The accuracy of the cohort data depended on the performance of the algorithm used to link CD4 count and viral load data to unique individuals, and on the completeness of data in the database. The algorithm used to construct the cohort had a sensitivity of 89% and a positive predictive value of 85%, which means it matched correctly for the majority of individuals in the cohort.  It is thDespite these limitations, our findings have important implications for policy and practice. CD4 count measurement at entry or re-entry into care should be continued in order to identify individuals with advanced disease and to allow clinicians to define the appropriate package of care. However, ART initiation should proceed regardless of CD4, removing the risk of CD4 count measurement becoming a cause of pre-treatment loss-to-follow-up. The cost-effectiveness of different CD4 screening and monitoring strategies needed to be considered in order to ensure that CD4 guided HIV care is provided in the era of test and treat. Recent data from the South African care and treatment programme shows that as much as 10% of HIV positives who start ART do so with CD4 counts less than 100 cells/\u03bcl despite increase in the median CD4 counts at ART start.  For thosS1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file."}
+{"text": "Data from calculating the vertical electronic excitation of 20 excited states for each dye and from calculating excited state oxidation potential (ESOP) and Frontier HOMO/LUMO isosurfaces are also presented. This data is related to the article \u201cMolecular and excited state properties of isomeric scarlet disperse dyes\u201d  [1].X-ray crystallography and DFT calculations were used to characterize the molecular nature and excited state properties of isomeric photostable azo dyes for textile fibers undergoing extensive sunlight exposure. Structural data in CIF files arising from X-ray analysis are reported and the complete files are deposited with the Cambridge Crystallographic Data Centre as CCDC 1548989 ( The data are Supplementary material for the study describing the \u201cMolecular and excited state properties of isomeric scarlet disperse dyes\u201d The data arise from X-ray crystallographic analysis and computational methods in the characterization of isomeric monoazo dyes Sc2 and Sc3 is shown in Sc2 and 0.0001 for Sc3. Other key crystallographic data for the two dyes are summarized in \u03b8max values, and bond lengths. The latter values are especially helpful in establishing the tautomeric form (azo vs. hydrazone) of the dyes analyzed   .Fig. 1ThSc2 shows intermolecular H-bond distances between the NH2 and CN groups (2.418\u2009\u00c5). Also seen are short contacts corresponding to intermolecular hydrogen bonds for Sc3, namely the NO2 and NH2 groups (2.188\u2009\u00c5), and the NH2 and CN groups (2.512\u2009\u00c5).Data for intermolecular H-bonding interactions between layers of molecules positioned parallel to each other are given in Sc2 and Sc3. From these data the excited state oxidation potential (ESOP) for each dye can be extracted.Calculation of vertical electronic excitation energies for 20 excited states along with the oscillator strength (f) and molecular orbitals involved for each dye led to the raw data shown in 2Single crystal X-ray diffraction analysis was conducted using a Bruker\u2013Nonius X8 Apex2 diffractometer. The frame integration was performed with the program SAINT. The resulting raw data was scaled and absorption corrected using a multi-scan averaging of symmetry equivalent data using SIRPOW 2Cl2 solutions of Sc2 and Sc3 at room temperature gave thin plate-like single crystals that were suitable for X-ray crystallographic analysis. The equilibrium molecular geometries (EMGs) of Sc1, Sc2 and Sc3 were calculated in the neutral forms using density functional theory (DFT) employing the generalized gradient approximation (GGA) at the hybrid exchange-correlation energy functional 3-Parameter (Exchange), Lee et al. (B3LYP) Sc2 and Sc3 were superimposed on the corresponding calculated molecular geometries and the RMS was calculated in each case. The isosurfaces of the HOMO and LUMO were extracted for each dye from the corresponding checkpoint files. In addition, TD-DFT calculations were performed on the EMGs and the geometry of the excited state structure was calculated using single point energy calculations for each dye. Vertical electronic excitation energies for 20 excited states were calculated for each dye and the excited state oxidation potential (ESOP) for each dye was extracted.Slow evaporation of CH"}
+{"text": "In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes  Airn silences genes in the Igf2r imprinted cluster, focusing primarily on silencing of the distant imprinted gene Slc22a3. We found that Airn expression blocks chromosome interactions between the Slc22a3 promoter and the Airn gene locus. By making a large genomic deletion including the Airn gene we showed that these interactions are not essential enhancer/promoter interactions, but may help to guide the Airn RNA to target genes to recruit epigenetic silencing. Our study adds to the understanding of how lncRNAs may act to silence distant genes.Long non-coding (lnc) RNAs are numerous in the mammalian genome and many have been implicated in gene regulation. However, the vast majority are uncharacterised and of uncertain function making known functional lncRNAs valuable models for understanding their mechanism of action. One mode of lncRNA action is to recruit epigenetic silencing to target distant genes on the same chromosome. A well-characterized group of lncRNAs that act in this way to silence genes are imprinted lncRNAs. In this study we examined how the imprinted lncRNA  Long non-coding (lnc) RNAs are a diverse and numerous group of non-protein-coding RNA species longer than 200 nucleotides, some of which have been shown to be involved in gene regulation ,2. A groIgf2r cluster expands from 120kb in most embryonic and adult tissues to almost 10Mb in placenta, while the Kcnq1 cluster expands from 250kb in embryonic tissues to 690kb in VYS . By. ByAirn RSDel region is correct, deletion of these enhancers on the maternal allele where Airn is not expressed should prevent expression of Slc22a3 on this chromosome. Slc22a3 silencing should also be maintained on the paternal allele when these enhancers are deleted  to distant imprinted genes in extra-embryonic tissues [Igf2r cluster in extra-embryonic tissues. Therefore, we performed H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) on VYS endoderm from FVB x CAST reciprocal crosses to determine the allele-specific distribution of this mark in the Igf2r cluster and throughout the genome. Using the Allelome.PRO pipeline to analyze the data [Igf2r cluster, despite imprinted expression in VYS endoderm being limited to a 450kb region from Slc22a3 to Airn  is not the same as an interaction between the Airn genomic locus and the Slc22a3 promoter on the maternal allele (detectable by 3C).Previously it has been shown in placenta using a technique derived from RNA FISH called RNA TRAP (Tagging and Recovery of Associated Proteins) that promoter . SurprisSlc22a3 and Slc22a3 in the Igf2r cluster are covered by a broad enrichment of the PRC2 mark H3K27me3, as are imprinted genes in the Kcnq1 cluster and in other imprinted clusters. Imprinted lncRNAs including Airn, Kcnq1ot1 and Meg3 have been reported to directly interact with PRC2 and EHMT2 [Airn-PRC2 interaction was reported in embryonic stem (ES) cells where Airn is very lowly expressed and no genes in the Igf2r cluster show imprinted expression [Kcnq1 cluster that show extra-embryonic specific imprinted expression [Dlk1 by Meg3 requires PRC2 [Igf2r imprinted silencing by Airn does not require PRC2 or EHMT2 [Slc22a3 and other members of the Igf2r cluster has not been tested, loss of EHMT2 has been shown to lead to loss of imprinted silencing of Slc22a3 [Here we show in VYS endoderm that the repressed alleles of nd EHMT2 ,20,21, apression ,20. PRC1pression ,38, and res PRC2 . Igf2r ior EHMT2 , but whiAirn may silence distant imprinted genes like Slc22a2, Slc22a3 and Pde10a by recruiting and targeted PRC1, PRC2 and EHMT2 to these genes to deposit repressive chromatin modification and cause silencing. This indicates that Airn silences imprinted genes in the Igf2r cluster by two different mechanisms: Airn transcription silences Igf2r by transcription interference that does not require repressive chromatin modifying complexes [Airn RNA product recruits repressive chromatin modifying complexes and targets them to distant, non-overlapped genes like Slc22a2, Slc22a3 and Pde10a to cause silencing. Importantly, the mechanism for targeting silencing remains unknown.Although it is technically difficult to exclude a role for transcription of the lncRNA versus the RNA product, together these results indicate that imprinted lncRNAs like omplexes , and theAirn gene body and the Slc22a3 promoter are enriched on the maternal allele because Airn expression represses these interactions on the paternal allele. We showed that these interactions are not required to upregulate Slc22a3 expression on the maternal allele, indicating that they are not essential promoter-enhancer interactions, but they may serve to place the Airn locus and Slc22a3 promoter in close proximity in the nuclear space. Chromosome interactions can exist in the ground state or be formed during development [Airn locus and the promoter of Slc22a3 , are present on both alleles  Protocol on the Protection and Welfare of Animals. The Animal Research is covered by Federal Austrian legislation  and the overriding EU and international legislation and codes of conduct. No experimental procedures were performed on the animals so no extra permissions were required.Thp) mouse (EM:09898) contains an minimum 5.56Mb deletion on chromosome 17 that includes the Igf2r imprinted cluster allowing parental allele-specific analysis [AirnT) mouse (EM:09895) has a polyadenylation cassette inserted into the Airn gene, 3kb downstream from its start site causing it to be truncated and non-functional [R2\u0394) mouse (EM:09897) has a deletion that includes the Airn promoter and the imprint control element (ICE) of the Igf2r imprinted cluster [Thp, AirnT and R2\u0394 mice have been cryopreserved by the EMMA mouse repository (EMMA ID indicated). The Sod2-flox mice contain loxP sites flanking the exon 3 of Sod2 and were made in a 129 ES cell line [FVB/NJ (FVB) mice were obtained from Charles River. CAST/EiJ (CAST) mice were obtained from the Jackson Laboratory. The FVB.AK-Del(17)T<hp>  was isolated from E9.5 and E12.5 embryos under a dissection microscope. The whole VYS was used for the chromosome conformation capture (3C experiments), while for RNA isolation and for chromatin immunoprecipitation (ChIP) the VYS endoderm was mechanically separated away from the rest of the VYS after 1\u20132 hours of DispaseII digestion at 4\u00b0C, as previously described .Igf2r imprinted locus we used reciprocal crosses of Thp and FVB mice [Airn on interactions we used Thp x AirnT cross, where AirnT mice have a truncated and non-functional Airn [Igf2r imprint control element (ICE). Around 28 VYS were then pooled per genotype and thawed on ice and then incubated with 8ml lysis buffer for 15 minutes on ice (1 tab Complete Protease Inhibitor (Roche) per 25ml lysis buffer ). The samples were then dounced in a 15ml glass dounce (Wheaton) with a loose pestle about 30 times, and then 30 times with a tight pestle, before centrifuging for 5 minutes at 2000g at 4\u00b0C. The supernatant was then removed leaving a nuclear pellet, which was then resuspended in 1 x EcoRI buffer (Fermentas). Samples were then subject to EcoRI digestion and ligation as previously described [Chromosome Conformation Capture (3C) was performed following established protocols with minor modifications ,49. To aFVB mice . To detenal Airn . We collnal Airn . Brieflyescribed . The forSlc22a3 promoter) and a \u201cprey\u201d primer was designed near the EcoRI site for fragments in the target region (e.g. Airn gene body). All 3C interactions detected in the Igf2r imprinted cluster (Thp deletion region on chromosome 17) were normalized by dividing by the mean of 2 interactions with the H19/Igf2 ICE, an independent locus on chromosome 7. To correct for technical and biological variation between experiments the highest interaction level was then set to 1. The primer/probe combinations used are given in We detected 3C interactions by Taqman quantitative PCR following a previously published protocol with minor modifications , and by Tissue from VYS endoderm or placenta was collected and homogenized in TRI reagent, and total RNA isolated according to the manufacturers protocol (Sigma-Aldrich).RSDel x FVB reciprocal crosses was DNase treated using the DNA-free kit (ThermoFisher Scientific), and then converted to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was then conducted using either a Taqman or SYBR Green system using the standard curve method to analysis the data, and normalization to a house keeping gene (cyclophillin A). The RT-qPCR results in Total RNA from E9.5 VYS endoderm and E12.5 placenta collected from RSDel x CAST reciprocal crosses using the TruSeq RNA Sample Prep Kit v2 (Illumina) modified as previously described [RSDel x CAST, maternal deletion cross) and 3x WT and 3x Del . Native ChIP was performed using an H3K27me3 antibody  on E12.5 VYS endoderm from FVB x CAST reciprocal crosses  as previously described [Strand-specific polyA enriched RNA-seq libraries were generated from E12.5 placenta and VYS endoderm from escribed . For eacescribed . ChIP-seescribed .RSDel mouse was backcrossed to FVB, but the region around the Igf2r cluster is likely to have a 129 background as both the R2\u0394 and Sod2\u0394 alleles from which the RSDel mouse is derived were made in 129 ES cells [Allele-specific expression and histone modification enrichment was detected from RNA-seq and ChIP-seq data using the Allelome.PRO program . The SNPES cells ,30. TherWe used the following Allelome.PRO parameters for our analysis:RNA-seq: minread 2 , RefSeq annotation.H3K27me3 ChIP-seq enrichment: FDR 1%, allelic ratio cutoff 0.7, minread 1, 20Kb sliding windows.Note: minread = minimum number of reads that must cover a SNP for it to be included in the analysis.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S4 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S5 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file.S6 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file."}
+{"text": "Sir,in vitro and evaluating the efficacy of type I interferon pathway blockade for therapeutic purposes in adults with dermatomyositis  inhibitor. Finally, four adults with refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I IFN levels and IFN-inducible genes scores (We read with interest the recent article by Ladislau and colleagues reporting on the pathogenicity of type I interferon (IFN) et al. now suggests that JAK inhibition may also be of relevance to the treatment of non-genetic IFN-mediated diseases such as adult dermatomyositis (Juvenile dermatomyositis (JDM) is a multi-systemic autoimmune disease characterized predominantly by progressive proximal muscle weakness and pathognomonic skin rashes . Even thThe following standardized measures were used to assess JDM disease activity and response to treatment in this case (2 weekly). Over the next 6 months he developed extensive ulcerative skin disease (modified skin DAS range 5/5) at which point intravenous cyclophosphamide  treatment was initiated with some improvement in CMAS 44/52 and modified skin DAS 3/5. Persistence of cutaneous disease over time and development of calcinosis over his elbow joints, right ear lobe required sequential treatment with azathioprine, mycophenolate mofetil, infliximab, adalimumab, rituximab, tacrolimus and ciclosporin, intravenous immunoglobulin (IVIG) over the next 7 years. He remained symptomatic and steroid dependent (2 mg/kg/day of prednisolone). Genetic testing was performed using version 2 (VIP2) of our recently developed targeted gene panel for Vasculitis and AutoInflammation Panel (VIP), which contains 166 genes, to exclude known monogenic interferonopathies that may mimic JDM, such as the proteasome associated autoinflammatory diseases  and characteristic heliotrope rash, Gottron\u2019s papules over the small and large joints of his hands (skin DAS of 4/5). He had raised creatine phosphokinase  and lactate dehydrogenase ; modestly elevated erythrocyte sedimentation rate  and normal C-reactive protein . His disease was complicated by pharyngeal involvement; but there was no evidence of interstitial lung disease, gastrointestinal or cardiac involvement. Myositis specific autoantibody detection showed positive anti-TIF1\u03b3 and anti-Ro-52 antibodies; antinuclear antibodies were positive with a titre 1:640, negative for dsDNA antibodies, complement function studies were normal and C1q, C3 and C4 levels were all within normal limits. He was initially treated with corticosteroids (30 mg/kg of intravenous methylprednisolone over 3 days followed by 2\u20133 mg/kg/day with intention to wean over 4\u20135 months) and subcutaneous methotrexate (15 mg/mTreatment with baricitinib (a selective JAK1/JAK2 inhibitor) was initiated at age 11.5 years because of ongoing severe skin disease activity and progressive calcinosis (expanded access protocol NCT01724580). He underwent dose escalation until he reached optimal tolerated treatment doses adjusted to his weight and renal function (6 mg twice a day). Clinical symptoms improved upon treatment with baricitinib at this dose with CMAS improving from 46/52 at time of start of therapy to 50/52 at 6 months of treatment; MMT-8 improving from 59/80 at baseline to 70/80 at 6 months; modified skin DAS 5/5 at baseline to 1/5 at 6 months; physician\u2019s VAS from 4.3/10 to 1.5/10 . CPK remAt 12 months of treatment he stopped taking all his medication against medical advice and this led to a significant flare of his symptoms within 6 weeks with significant deterioration of his skin rash (modified skin DAS 5/5), worsening myalgia and arthralgia, CMAS decrease to 46/52 and MMT-8 to 59/80, physician\u2019s VAS 6/10 , CPK eleBiomarkers of IFN signalling, and the type 1 IFN induced gene expression that were modestly elevated at time of start of treatment (note patient was receiving high doses of corticosteroids at that time point) significantly decreased during treatment with baricitinib at 6 months and 18 months with upregulation at 12 months when all treatment had been stopped due to lack of compliance C\u2013E. We mIn line with the findings from The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary MaterialClick here for additional data file."}
+{"text": "The authors wish to make the following corrections to this paper :Abstract: The sentence \u201cThe parameter rm2 penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test.\u201d should read as: The parameter rm2 penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for a small difference between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test.Section 2.3.2.4: The sentence \u201cWe have used a parameter Rp2 [32] in the present paper, which penalizes the model R2 for the difference between squared mean correlation coefficient (Rr2) of randomized models and squared correlation coefficient (R2) of the non-randomized model.\u201d should read as:p2 [32] in the present paper, which penalizes the model R2 for a small difference between squared mean correlation coefficient (Rr2) of randomized models and squared correlation coefficient (R2) of the non-randomized model.We have used a parameter RConclusions: The sentence \u201cThe parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test and thus confirms whether a model has been obtained by chance or not.\u201d should read as: The parameter Rp2 penalizes model R2 for a small difference between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test and thus confirms whether a model has been obtained by chance or not."}
+{"text": "A non-hydrolytic condensation route allows for precise control over the size distribution of HfO2 nanocrystals and enables the stabilization of the tetragonal phase of HfO2. 2 since it is predicted to outperform the thermodynamically stable lower-symmetry monoclinic phase for almost every application where HfO2 has found use by dint of its higher dielectric constant, bandgap, and hardness. However, the monoclinic phase is much more thermodynamically stable and the tetragonal phase of HfO2 is generally accessible only at temperatures above 1720 \u00b0C. Classical models comparing the competing influences of bulk free energy and specific surface energy predict that the tetragonal phase of HfO2 ought to be stable at ultra-small dimensions below 4 nm; however, these size regimes have been difficult to access in the absence of synthetic methods that yield well-defined and monodisperse nanocrystals with precise control over size. In this work, we have developed a modified non-hydrolytic condensation method to precisely control the size of HfO2 nanocrystals with low concentrations of dopants by suppressing the kinetics of particle growth by cross-condensation with less-reactive precursors. This synthetic method enables us to stabilize tetragonal HfO2 while evaluating ideas for critical size at which surface energy considerations surpass the bulk free energy stabilization. The phase assignment has been verified by atomic resolution high angle annular dark field images acquired for individual nanocrystals.There has been intense interest in stabilizing the tetragonal phase of HfO Precise control of the size of HfO2 nanocrystals enables unequivocal determination of the critical size required to stabilize the tetragonal phase and provides access to well-defined crystals of this technologically important metastable structure that is otherwise stable only above ca. 1720 \u00b0C.16A particularly powerful aspect of nanoscience that enables much new functionality derives from the greatly altered phase equilibria obtained upon confining materials to nanoscale dimensions.2 has been widely accessible for several decades, even without incorporation of dopant atoms, since the critical size at which the surface free energy terms overcome bulk free energy considerations is relatively large, variously estimated to be ca. 15\u201330 nm.2 is much more challenging since: (a) the bulk free energy stabilization of the monoclinic phase over the tetragonal phase is almost 40% greater for HfO2 as compared to ZrO2; and (b) the volume expansion accompanying the tetragonal to monoclinic phase transition is much smaller for HfO2 (ca. 2.7%) as compared to ZrO2 (4.0%).2 vary widely from about 2 to 10 nm but it is clear that this value is substantially smaller than the critical size for ZrO2.2 report the stabilization of at least some fraction of tetragonal HfO2; for instance, signatures of the tetragonal phase have been identified in particles obtained by the thermal decomposition of pure Hf(OH)4,2 prepared by methods such as atomic layer deposition in a low-oxygen environment,Tetragonal ZrO2 is not just an academic curiosity. Indeed, the tetragonal (space group P42/nmc) phase of HfO2 is anticipated to outperform the thermodynamically stable lower-symmetry monoclinic phase (space group P21/c) for almost every application where HfO2 has found use. Perhaps the most important application of HfO2 is in gate dielectric stacks as a high-\u03ba dielectric because of its resistance to silicidation and silicate formation and its much greater dielectric constant as compared to SiO2.2, which is far surpassed by the value of almost 70 predicted for tetragonal HfO2.ca. 6.11 eV) as compared to the monoclinic phase (5.78 eV).27The stabilization of tetragonal HfO2 nanocrystals.2 nanocrystals, again crystallized in the monoclinic phase.2 nanocrystals as per:In comparison to aqueous methods, non-hydrolytic methods, often based on formation of oxo-bridges by elimination of small molecules, provide considerable control of the kinetics of condensation.2 and solid-solution HfxZrx1\u2013O2 nanocrystals and established considerable control over the relative Hf\u2009:\u2009Zr concentrations.2 and ZrO2 nanocrystals as well as the relative Hf\u2009:\u2009Zr ratios of HfxZrx1\u2013O2 nanocrystals.4 and MX4 species, substantial ligand exchange takes place to stabilize haloalkoxides such as M(OR)3Cl, M(OR)2Cl2, and M(OR)Cl3.Brus and co-workers extended this method to prepare HfOWhen two different metal precursors are reacted, the composition of the product depends on the relative condensation rates and reactivities. For metals exhibiting comparable reactivity, such as Hf and Zr, the heterocondensation reaction yields solid-solution nanocrystals and can be written as follows:tBu)4 or La(OiPr)3, greatly modifies the concentration of the active monomer depicted in 2 nanocrystals with only minimal incorporation of La and Ce. This unprecedented control of nanocrystal size allows us to explore ultra-small dimensions and determine the critical size for stabilizing the tetragonal phase of HfO2.However, if the rates of homo- and hetero-condensation are vastly different, the products of the faster reaction are obtained preferentially. In this work, we find that adding a much less reactive precursor, either Ce(Oiv) chloride, cerium(iii) chloride, lanthanum(iii) chloride, lanthanum(iii) iso-propoxide, and tri-n-octylphosphine oxide (TOPO) were purchased from Strem and used without any further purification. Hafnium(iv) tert-butoxide was purchased from Alfa Aesar and used as received. Cerium(iv) tert-butoxide was purchased from Gelest and used without further purification. To synthesize the metal oxide nanocrystals, a metal halide and metal alkoxide are mixed in equimolar amounts with ca. 10 g of TOPO in a three-neck round bottom flask under an Ar ambient within a glovebox. The total concentration of metal precursors is maintained at 4 mmol in all of the reactions. In all of the reactions, 2 mmol of HfCl4 is used as the chloride precursor, whereas the alkoxide precursor is x mmol of La(OiPr)3 or Ce(OtBu)4 and 4 \u2013 x mmol of Hf(OtBu)4. CeO2 nanocrystals were obtained by the reaction of equimolar amounts of CeCl3 and Ce(OtBu)4 as reported in our previous work.Hafnium, were used for characterization of the samples by powder X-ray diffraction. The crystallinity and size distribution of the nanocrystals was evaluated by high-resolution transmission electron microscopy (HRTEM) using JEOL-2010 and FEI Tecnai G2 F20 ST electron microscopes at an operating voltage of 200 kV. Samples for HRTEM analysis were dispersed in hexanes, drop-cast onto 400-mesh carbon-coated copper grids, and allowed to dry in air. The stoichiometry of the nanocrystals was determined by X-ray fluorescence (XRF) measurements, conducted by Oneida Research Services, Inc. in Whitesboro, NY, by inductively coupled plasma-mass spectrometry (ICP-MS) using a Perkin Elmer DRCII instrument, and by energy dispersive X-ray spectroscopy (EDX) on a JEOL JSM-7500F field emission scanning electron microscope (FE-SEM) operated at an accelerating voltage of 20 kV. Samples for ICP-MS analysis were prepared by acid digestion in an aqueous solution of 67% metals grade HNO3. Further characterization was performed using atomic resolution high-angle annular dark-field (HAADF) imaging. A probe-corrected JEOL JEM-ARM200CF instrument equipped with a cold field-emission gun operated at 200 kV was used with a convergence angle of 22 mrad. The HAADF inner detector angle was 90 mrad.A Rigaku Ultima IV diffractometer with a graphite monochromator and a Bruker-AXS D8 Advanced Bragg\u2013Brentano X-ray powder diffractometer, both using Cu K\u03b1 radiation (4 with varying proportions of Hf(OtBu)4 and Ce(OtBu)4. x1\u2013CexO2 nanocrystals and provides good agreement with the hafnium and cerium concentrations derived by elemental analysis 4 and HfCl4 are clearly monoclinic (P21/c) as delineated by the appearance of sharp (200) and (220) reflections (Joint Committee on Powder Diffraction Standards (JCPDS) 78-0050) indexed in 2 nanocrystals crystallize in a cubic fluorite structure (Fm3[combining macron]m) with reflections that can be indexed to JCPDS# 34-0394 4 to Hf(OtBu)4 is increased, the reflections are broadened indicating a pronounced diminution in size even though elemental analysis data presented in 4 and 2 mmol of Ce(OtBu)4 preponderantly yields the tetragonal phase of HfO2 characterized by a pronounced (101) reflection centered at 2\u03b8 = 30.3\u00b0 4, indicates the appearance of a reflection at 2\u03b8 = 31\u00b0 that can be attributed to the (101) reflection of the tetragonal phase and suggests that this set of precursors yields a mixture of tetragonal and monoclinic phases. A reflection at 2\u03b8 = ca. 41\u00b0 in  = 30.3\u00b0 . The simtBu)4 in the reaction mixture, the morphology of the obtained nanostructures evolves from elongated nanorods to quasi-spherical particles. The nanorods preferentially grow along the [100] direction of monoclinic HfO2.x1\u2013CexO2 nanorods are monotonically diminished with reduced concentration of Hf(OtBu)4 with respect to Ce(OtBu)4. The average length is decreased from 12.9 \u00b1 1.8 nm for monoclinic nanocrystals grown using only Hf(OtBu)4 to 3.1 \u00b1 0.4 nm for tetragonal nanocrystals grown using Ce(OtBu)4 as the only alkoxide precursor. The widths of the Hfx1\u2013CexO2 nanocrystals are not substantially altered and remain around 2.5 \u00b1 0.4 nm. The lattice-resolved HRTEM image in 2  imaging has been performed using an aberration corrected microscope. To verify the phase assignment of Hf4 with varying proportions of Hf(OtBu)4 and La(OiPr)3. x1\u2013LaxO2 nanocrystals and the determined hafnium and lanthanum concentrations are in good agreement with the elemental analysis results 3 as listed in 3 and La(OiPr)3 yields LaOCl nanocrystals crystallized in the matlockite PbFCl-type phase.tBu)4, with increasing concentration of La(OiPr)3 in the reaction mixture, the (100) and (200) reflections indexed to the monoclinic phase of pure HfO2 are broadened and diminished in intensity suggesting a pronounced diminution in size. Again, although the powder XRD patterns are substantially altered, the elemental analysis results listed in 2. For the sample prepared by the reaction of HfCl4 and La(OiPr)3, the (100) monoclinic reflection is no longer observed and the (101) tetragonal reflection becomes the most prominent feature in the powder XRD pattern suggesting stabilization of the tetragonal phase of HfO2 with modest La doping.Analogous to the above discussion for Ce(OiPr)3, the Hfx1\u2013LaxO2 nanocrystals again evolve from elongated nanorods to quasi-spherical nanocrystals. The width of the Hfx1\u2013LaxO2 nanocrystals is relatively unchanged at ca. 2.5 \u00b1 0.4 nm for the entire set of samples. However, the length of the nanorods is diminished from 12.9 \u00b1 1.8 nm for monoclinic nanocrystals grown using only Hf(OtBu)4 to 3.3 \u00b1 0.4 nm for Hfx1\u2013LaxO2 tetragonal nanocrystals grown using La(OiPr)3 as the only alkoxide precursor.HRTEM images and size distribution histograms of this set of nanocrystals are shown in x1\u2013LaxO2 nanocrystals as being tetragonal, atomic-resolution HAADF STEM images and fast Fourier transforms (FFT) of the HAADF images were acquired and are depicted in To confirm the phase assignment of HftBu)4 and La(OiPr)3 leads to a substantial and monotonic diminution of nanocrystal size even though elemental analysis results indicate that relatively low concentrations of Ce and La are actually incorporated within the lattice. At the smallest dimensions, the tetragonal phase of HfO2 is stabilized over the monoclinic phase. These results lead to two fundamental questions regarding the mechanistic basis for the size control achieved by addition of La and Ce precursors and the origin of the altered phase stability.The preceding discussion illustrates that the addition of Ce(OtBu)4 and La(OiPr)3, it is important to consider the overall equation depicted in eqn (1), which results in formation of an oxo-bridge with the elimination of an alkyl halide, as well as the catalytic scheme proposed by Vioux shown in To understand the origin of the size control achieved by the addition of Ce(O4 and Zr(OR)4 and ZrCl4 and Hf(OR)4 yield exactly the same compositions of solid-solution HfxZrx1\u2013O2 nanocrystals suggesting that ligand scrambling precedes condensation;xZrx1\u2013O2 and LaxCex1\u2013O2, respectively, albeit with compositions that reflect the relative reactivities of the two metal precursors as modified by the alkoxide ligands.2 with very low dopant concentrations is obtained as the exclusive product upon the reaction of HfCl4 with mixtures of Hf(OtBu)4 and Ce(OtBu)4/La(OiPr)3. The relatively low incorporation of Ce and La within the doped HfO2 lattice, very much lower than the precursor concentrations, corroborates the idea of the lower reactivity of these precursors. tBu)4 and La(OiPr)3 allows for substantial tunability of the size of the obtained doped HfO2 nanocrystals. In 3OR species (derived from ligand exchange as per eqn (3)) mediates conversion of the alkoxo-bridge to an oxo-bridge with elimination of an alkyl halide. Indeed, Vioux has shown that the presence of MX3OR species is imperative for condensation and that MX2(OR)2 and MX(OR)3 species are much less reactive.3OR species formed by ligand exchange serves as the catalyst. The retention of equimolar amounts of halide and alkoxide precursors ensures that the amount of catalyst is kept essentially the same for all of the reactions listed in 4 and Ce(OtBu)4/La(OiPr)3 are much less reactive as compared to those prepared from HfCl4 and Hf(OtBu)4, the addition of cerium and lanthanum precursors essentially reduces the concentration of the active monomer. In other words, the reaction rate is diminished as a result of the lower reactivity of mixed metal Lewis acid\u2013base adducts towards formation of oxo-bridges. As further corroboration for this idea, 2 nanocrystals that are 12.9 \u00b1 1.8 nm. In contrast, the homocondensation of cerium precursors yields CeO2 nanocrystals that are 1.5 \u00b1 0.5 nm indicating much slower kinetics of growth 4 when ramped at a rate of 15 \u00b0C to 500 \u00b0C during thermal analysis is monoclinic HfO2, the thermodynamically stable phase, along with an oxygen-deficient orthorhombic HfO2 phase. The inclusion of TOPO still yields monoclinic HfO2, albeit with a smaller size. These results illustrate that direct reaction of the precursors cannot yield the metastable, kinetically trapped phase. The incorporation of the La and Ce alkoxides is imperative to slow the kinetics of crystal growth. ca. 3.6\u20133.8 nm is the tetragonal phase stabilized. Since 3+ cations (110 pm for seven-coordinated and 116 pm for eight-coordinated sites) as compared to Ce3+ (107 pm for seven-coordinated and 114.3 pm for eight-coordinated) or Ce4+ (97 pm for eight-coordinated),x1\u2013LaxO2 nanocrystals remain monoclinic even upon incorporation of 3.54 at% of La).As a next point, we discuss the origins of the stabilization of the tetragonal phase of HfO2 lattice will result in creation of half an oxygen vacancy to maintain electrostatic neutrality. The Ce4+/Ce3+ redox couple is readily accessible but since we start with tetravalent cerium precursors, the vacancy concentration generated upon cerium-incorporation is likely to be lower than upon lanthanum incorporation. Again, if oxygen vacancies were to provide the driving force for stabilization of the tetragonal phase, one would expect that lanthanum-incorporation should more readily bring about stabilization of the tetragonal phase as compared to cerium incorporation. Again, x1\u2013LaxO2 nanocrystals with 3.54 at% La are monoclinic, whereas Hfx1\u2013CexO2 nanocrystals with only 2.36 at% Ce are tetragonal. The preceding discussion and \u20132).2 nanocrystals becomes such that the surface energy term exceeds the bulk energy component of the free energy term (the difference between the two phases is 196 meV for pure HfO2),The second scenario pertains to the potential role of oxygen vacancies. The substitutional incorporation of trivalent lanthanum cations in the HfOd can be expressed as48G is the volume free energy change across the phase transformation, V is the strain energy, \u03b3 is the specific surface energy, and \u03c1 represents the density. Hunter et al. used this expression to predict a critical size for stabilization of tetragonal HfO2 of d = 3.6 nm.2 nanocrystals prepared by aqueous methods is the almost 20\u201330% diminution of surface energy as a result of surface hydroxylation, which in this case further dilutes the influence of the surface energy contribution.202, which is in remarkably good agreement with predictions from this thermodynamic model. The origin of the stability of the tetragonal phase at these dimensions can thus be attributed to the substantially lower surface energies of this phase, which renders this phase energetically stable under conditions of constrained equilibrium.In 1972, Bailey and co-workers developed an expression for calculating the critical size for stabilizing a metastable state based on a classical thermodynamic treatment of the phase transformation and the competing bulk and surface energy terms. In this formulation, the critical size, 2 has been far more challenging as compared to ZrO2 given the smaller volume expansion accompanying the tetragonal \u2192 monoclinic transformation and the relatively greater stabilization enjoyed by the monoclinic phase. Classical thermodynamic models predict that the tetragonal phase should be stable at dimensions smaller than ca. 3.6 nm but the validity of these predictions have been thus far impossible to determine in the absence of synthetic approaches that can access such ultra-small dimensions with precise control of particle size. In this work, we have developed a non-hydrolytic condensation route wherein the concentration of the active monomer is precisely modulated by replacing Hf(OtBu)4 with less reactive Ce(OtBu)4 or La(OiPr)3 precursors. The latter alkoxides exhibit much slower kinetics of condensation and thus are incorporated within the doped HfO2 lattice only to small extents but play a significant role in modifying the growth kinetics by suppressing the concentration of the active monomer. This approach enables precise control of size in ultra-small dimensions and allows for systematic evaluation of the size-dependence of phase stabilities in this system. The much desired metastable tetragonal phase is stabilized at dimensions less than 3.6\u20133.8 nm, which is in good accord with predictions of thermodynamic models that take into account the competing influences of bulk free energy and specific surface energy.Stabilization of the tetragonal phase of HfOThe authors declare no competing financial interest.Supplementary informationClick here for additional data file."}
+{"text": "In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O Different stem cell models are under investigation for cell therapy purposes, all having their advantages and concerns. Among the different models, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or adult stem cells, mesenchymal stem cells (MSCs), show great promise in the field of regenerative medicine . Indeed,A unique population of MSCs referred as SCAPs , resides in the apical papilla of human immature wisdom teeth. SCAPs appear to be the source of odontoblasts that are responsible for the formation of root dentin ,6. Compa2 secrete FGF2 (Fibroblast Growth Factor 2) and display improved survival rates when injected into experimental animals ) is autophagy. Indeed, autophagy plays a key role in stem cell homeostasis as it acts as a robust quality control mechanism [One well-known process regulated by low oxygen concentration  at low [O2] on these properties. This study established the advantage and limitations of SCAP isolation and expansion at low [O2].Human immature wisdom teeth are commonly extracted in teenagers for orthodontic reasons. In this study, we compared the properties of SCAPs (isolated from three teenagers) that were isolated and expanded in parallel under continuous low or high  as that used for their isolation. SCAPs were isolated from three different individuals using these conditions and named thereafter: UBx-SCAP-N1, N2 and N3 (21% O2) or UBx-SCAP-H1, H2 and H3 (3% O2). These settings are summarized in 2] cell chamber was an Invivo2 300 RUSKINN in which all experiments at low O2 concentration were performed. When stipulated, early passages were up to P7 and late passages were from P11 up to P22. The detailed experimental procedure for the preparation of SCAPs for EXP III is provided below.Experiment III (EXP III): SCAPs from two teeth from the same patient were isolated and expanded at either 3% or 21% O2, the medium (40 mL) was conditioned for 1 h under 3% O2. Teeth were then processed in a sterile hood at 21% or 3% O2 as previously described , (EXP II), grew faster: doubling population times were 50 h at 21% O2 and 31 h at 3% O2 and cumulative population doubling were higher at 3% versus 21% O2  or only the expansion process (at 21% or 3% O2) which was important to improve proliferative efficacy, we undertook EXP III with SCAPs isolated from the same individuals, isolated and grown in parallel under 3% and 21% O2 , was plotted for each patient, at early and late passages , which were used as positive controls  and also that of other markers described as \u201cstemness/proliferative-linked\u201d markers like CD49f, stage-specific human embryonic antigen-4 (SSEA4), CD24 and PDGFR\u03b2 ,45,46,47nditions A. Howevereported , while to 21% O2 A. Expres of MSCs  data not of MSCs ,24. Undecontrols B [49]. 2 behave as classical MSCs, we undertook in vitro differentiation experiments towards mesodermal lineages. Cells from the three individuals at early or late passages were induced to differentiate as described in the Materials and Methods, then stained with an OsteoImage\u2122 kit , with red oil  or Alcian blue (for staining of glycosaminoglycans produced by chondrocytes). As shown in 2] was detected, indicating that cells kept their stemness properties in both conditions in vitro. At later passages, we observed a loss in osteogenic and adipogenic differentiation potential at 3% O2, while chondrogenesis differentiation remained, indicating that low [O2] might preserve this unipotent mesodermal feature of SCAPs.To determine whether SCAPs grown under 21% or 3% O2 and 3% O2 without chloroquine but highly increased after the addition of chloroquine. These results demonstrated that the autophagic flux was well activated in both O2 conditions, at early  at low [O2]. This study, performed with SCAPs from three individuals allowed a large spectrum of analyses regarding their properties such as proliferation, clonogenicity, cell cycle, multipotency and autophagic flux both at early and late passages  in which we observed high proliferation and no apoptosis of cells  after at least one month at 3% O2.Previous analyses performed with stem cells and in particular with BM-MSCs show many advantages for cells grown under low [O2] ,52,53,542] 2 ,55. Howe. Howe2 [2] led to an increased proliferation capability of SCAPs and to a preserved expression of the classical MSC markers with a slight decrease of CD105, as already reported [2 and expanded at either 21% or 3% O2 (as in our EXP II) also demonstrated an advantage of cell growth under low [O2] compared with ambient air [2] in comparison with ambient air. In contrast, PDGFR\u03b2, which is enriched in human placental MSCs with elevated colony forming unit efficiency [2]. In addition, CD24 expression, which may refer to the number of committed progenitors cells in the papilla tissue, was not differentially expressed in our cell growth conditions [2]-dependent markers. CD49f/6 integrin, whose deletion in mouse models leads to a disabling skin disease (epidermolysis bullosa), is part of the laminin receptor in epithelial cells which plays a critical structural role in the hemidesmosome [2], we performed the test at 21% O2 to determine whether cells maintained their clonogenicity advantage. We observed that clonogenicity of SCAPs was variable among the three tested individuals and that the switch from 3% to 21% O2 was deleterious at late passage for SCAP clonogenicity. A direct comparison of clonogenicity efficiency of SCAPs grown at 3% or 21% O2 should help to assess whether low [O2] maintains better the self-renewal capacity of SCAPs. SSEA4, which is highly expressed in human ESCs and iPSCs, is a stemness marker enriched in cells maintaining their stemness properties  at early passages (up to P7) but was restricted to chondrogenesis at late passages under 3% O2, in agreement with previous reports showing that osteogenesis was reduced under low [O2]  ,62. The AP cells . In addiAP cells . In addi2]. As expected, we found that the activation of autophagy was associated with high LC3 puncta and with the induction of BNIP3 (a key mitophagy player) in SCAPs switched from 21% to 3% O2 . This observation questions the potential involvement of BNIP3 in mitophagy since LC3 and BNIP3 usually colocalize when an active mitophagy process is engaged. Further investigation is needed to determine the significance of the heterogeneous expression of autophagy/mitophagy-involved markers in SCAP physiology. Nevertheless, our work suggests that autophagy is not an acute stress response in SCAPs and its impact in stemness and proliferation of cells should be further analyzed.Autophagy is known as a stress-induced mechanism that allows cells to recycle macromolecules and organelles to cope with stress conditions and thereby promote cell survival . Hypoxiato 3% O2 . Interesd injury ,67. In Sd injury ,69. Ther2 did not reach their exhaustion phase, up to 24 passages, in contrast with SCAPs isolated and grown at 21% O2 that we could not keep growing after 20 passages. However, at low [O2] and late passages, SCAPs lost some of their stemness properties in classical differentiation medium, that was established for experiments performed at 21% O2. Future work with a larger sampling of cells will aim at deciphering the role of autophagy in increased proliferation of SCAPs grown at 3% O2. A key future challenge will be also to determine how pre-conditioned SCAPs at 3% O2 survive after transplantation in experimental animal models and to understand better the mechanism of their adaptation to [O2] in vivo.In this study, we have produced SCAP banks from EXP I, II and III and characterized many features of the cells which are now available for further fundamental and applied research. SCAPs directly isolated at 3% O"}
+{"text": "The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10\u00a0days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets. Previous studies have shown that bobwhite quail support influenza virus replication with receptors in their respiratory tract.Gallus domesticus) aged 6\u00a0weeks were also used. The number of birds and the sampling times differed between species due to the space limitations in our BSL3 facilities and not handling two different species on the same day. For each virus, five donor quail and five donor chickens were infected by the natural route with 106 EID50 in 0.5 mL total volume phosphate\u2010buffered saline  as others have done.50) was calculated by the method of Reed and Muench.The three specific viruses used in this study were A/environment/Bangladesh/10306/2011 H9N2) , A/chicken/Bangladesh/10450/2011 (H9N2) (GenBank KC757840), and A/quail/Bangladesh/19462/2013 (H9N2) (GenBank KJ643700). The viruses were propagated and titrated in the allantoic cavities of 10\u2010day\u2010old embryonated chicken eggs at 35\u00b0C for 48\u00a0hours as described previously.N2 (isola50 5.4\u20106.4 log10/mL) in the upper respiratory tract of both chickens and quail (Table\u00a010/mL). In donor chickens, peak shedding of virus occurred at 3 dpi and the titers fell to background levels by Day 7. Similar virus titers were measured for each of the three viruses at 3, 5, and 7 dpi (data not shown). Shedding in quail donors peaked at 4 dpi, and shedding was sustained beyond 8 dpi in three of five donor birds for each virus. At least one donor quail in each group shed virus orally for 10\u00a0days.Each of the H9N2 influenza viruses from chickens, quail, and the environment replicated to a high titer  virus showed no overt disease signs, whereas birds infected with the two isolates from quail  showed varying levels of disease signs (Table\u00a02This study shows that the New World quail (Bobwhite species) is more susceptible to infection with Bangladeshi H9N2 influenza virus than chicken. Each of the H9N2 viruses replicated to a high titer in the respiratory tract of both chickens and quail, with low\u2010level shedding in the feces. The viruses all transmitted by direct contact and airborne transmission in chickens and by airborne transmission in quail, but the efficiency of airborne transmission to chickens varied. The differences in morbidity and mortality caused by the three H9N2 viruses in quail may be associated with sequence differences.In this study, quail were more severely affected by the H9N2 viruses containing five gene segments from HPAI H7N3 than were chickens. H9N2 viruses from Pakistan containing four gene segments  from HPAI H7N3\u2010affected quail more severely than chickens and caused sporadic deaths .Although fewer airborne\u2010contact chickens (3 per group) were used than in New World quail (5 per group), differences in airborne transmissibility are apparent and emphasize the risk factors of New World quail present in live bird markets: Variable transmission occurred in chickens among viruses tested, but high transmission occurred in New World quail. Quail (Old World) have been recognized as efficient spreaders of H9N2 influenza viruses and have been banned from live poultry markets in Hong Kong since 2002."}
+{"text": "BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 \u220617_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 \u220611, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting. BRCA1 and BRCA2 variants can confer an increased risk of breast and ovarian cancers, and many of these variants are known to disrupt mRNA splicing, as seen in BRCA1 and BRCA2 mRNA splicing patterns have been explored in depth using a number of advanced sequencing technologies to highlight significant diversity, both in the number and level of the alternative events expressed naturally by these genes [Germline se genes . This dise genes ,5.BRCA1 and BRCA2 mRNA expression levels may be influenced by such fluctuations. Expression based assays (such as reverse transcriptase-PCR and RNA-seq) measure average mRNA expression in a population of cells. However, the individual cells of the population are likely to be producing transcripts at very different levels, potentially providing an additional measure of mRNA expression patterns associated with spliceogenic variants. Advanced in situ hybridisation (ISH) technologies provide the means to observe actual levels of mRNA transcripts in single cells, in addition to detail on spatial expression within a cell. Measuring gene splicing at the single cell level may provide a more comprehensive method of evaluating the biological and clinical significance of alternative isoforms. In this study, we utilised RNAscope [BRCA1 and BRCA2 spliceogenic variants on mRNA transcript expression in single cells.In particular, individual cells are known to express genes in a stochastic manner, occurring in bursts, irrespective of the transcription factors present ,7, suggeRNAscope , a uniquBRCA1 and BRCA2 mRNA splicing events in single cells, from individuals with or without germline spliceogenic variants. Results showed that the proportion of cells expressing BRCA1 transcripts ranged from 4% (2/50) in the control to 44% (22/50) in the BRCA1c.671-2 A>G carrier, as seen in BRCA2 transcripts ranged from 24% (12/50) in the control to 58% (29/50) in the BRCA2c.7988 A>T carrier. Results from the negative and positive control probes from the RNAscope Multiplex Fluorescent Detection Kit are shown in the BRCA1 mRNA transcript expression ranged from 0 to 17 (mean = 2.1) BRCA1 molecules per cell in the BRCA1c.671-2 A>G carrier, and 0 to 10 (mean = 0.3) in the control. The BRCA2 mRNA transcript expression ranged from 0 to 29 (mean = 3.2) molecules per cell in the BRCA2c.7988 A>T carrier, and 0 to 5 (mean = 0.6) in the control as shown in BRCA1 and BRCA2 mRNA molecules per cell compared to non-treated LCLs , as seen in In this study, RNAscope allowed the detection of specific  to 58% 2/50 in thBRCA1 mRNA expression in LCLs carrying the spliceogenic variants BRCA1c.671-2 A>G and BRCA2c.7988 A>T have reported increased expression of the \u220611 and \u220617_18 splicing events, respectively. However, these studies were unable to elucidate BRCA1 and BRCA2 expression patterns at the single cell level. Using RNAscope, we show that the BRCA1c.671-2 A>G variant carrier LCL showed no significant difference in the total number of detected BRCA1 \u220611 mRNAs compared to the control LCL across the cells assayed in each sample, demonstrated by p = 0.48 in BRCA1 \u220611 splicing event was shown to be significantly greater in the variant carrier compared to the control  and to identify cell-specific overexpression in different tissue types. mRNA isoforms detected in samples at very low levels might actually be highly abundant in a small number of cells, but this observation can only be detected at the single-cell level, as seen in BRCA1 and BRCA2 transcripts, and an increase in the number of cells containing transcripts, in the treated samples compared to the untreated samples. Higher expression in the treated samples facilitated the detection of the targeted alternative splicing event, suggesting that NMD inhibitor treatment is advantageous when analysing BRCA1 or BRCA2 transcript expression with RNAscope.Nonsense-mediated decay inhibitors are often used in splicing analysis to prevent the degradation of mRNA , thus imn = 50), which might have led to an under-representation of some isoforms, such as those that were lowly expressed. Increasing the total number of cells counted in each sample may provide a more accurate survey of isoform expression patterns. However, counting a greater number of cells would be relatively laborious using manual assessment. This challenge could be alleviated by using image analysis software, such as the Aperio RNA ISH Algorithm (Leica Biosystems), or Cell Profiler (http://cellprofiler.org/). It is important to note that there is paucity in published data relating to the efficacy of these image analysis software tools. It is unclear why some cells contained a greater number of C1 (targeting the deletion of interest) probe signals compared to the reference C2  probe signals as seen in BRCA1 and BRCA2 is 4.5 and 4.8 h [BRCA1 (\u22063) and BRCA2 (\u220612) [Our study is proof of concept, demonstrating the utility of RNA in situ hybridisation to characterise the effects of spliceogenic variants at the single cell level. Our study had several potential limitations, including the use of a single control and the number of cells analysed , specific mRNA transcripts are now able to be detected using shorter probes [BRCA1 \u220611 using BaseScopeTM probes spanning the exon 10_12 junction will not identify the BRCA1 \u22069_11 splice event, even though both isoforms contain a deletion of the large exon 11 sequence. Thus, assays targeting exon 11 may be utilised to examine potentially \u2018leaky\u2019 variants, which give rise to multiple alternative isoforms.The unique probe design RNAscope uses to obtain high specificity also limits the number of isoforms that can be detected with this technique. Three probe pairs are required to return a detectable signal, with each pair taking up 36\u201350 nucleotides of the target sequence, while 10\u201320 pairs are recommended to gain optimal detection . This lir probes . HoweverBRCA1 and BRCA2 mRNA expression patterns at the single cell level in LCLs. This technology could potentially play an important role in assessing variable expression levels of BRCA1 and BRCA2 isoforms by establishing whether mRNA isoforms detected in samples at very low levels may actually be highly abundant, but in a small number of cells. Such findings may be associated with an increased risk of disease, yet might go undetected using cell population-based methods because the number of cells expressing the aberrant change is likely to be insufficient to indicate an association with disease. Spliceogenic variants previously dismissed as low-risk might need to be re-evaluated at the single cell level to determine if aberrant mRNA isoforms are being expressed at high levels but in a low number of cells. Furthermore, single cell expression analysis provides an important means for investigating the poorly explored link between many human diseases and gene expression variability (variance) [To our knowledge, this is the first study to reveal ariance) ,16. SuchBRCA1c.671-2 A>G and BRCA2c.7988 A>T) and two from healthy controls, were obtained from the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer . The variant carriers were selected for analysis as they contained variants known to disrupt normal splicing patterns [2 atmosphere. mRNA was extracted from LCLs cultured with and without the nonsense mediated decay (NMD) inhibitor cycloheximide (100 \u00b5g/mL) for 4 h.Four lymphoblastoid cell lines (LCLs), two derived from rare variant carriers (patterns . Cell liBRCA1 \u220611 upregulated in BRCA1c.671-2 A>G and BRCA2 \u220617_18 upregulated in BRCA2c.7988 A>T variant carriers [green and C2red) were designed for BRCA1 and BRCA2. C1green probes were designed to target the deleted mRNA region (BRCA1 exon 11 or BRCA2 exons 17_18). C2red probes were designed to target a large transcript region, with the pretext that at least part of that region is represented in all expressed transcripts as seen in green and C2red probe signals are detected, whereas the expression of each of the targeted alternative splicing events of interest was calculated by subtracting the number C1 signals from the number of C2 signals in each cell, as seen in UBC (positive control) tissue and bacterial gene dapB (negative control) were included for each assay.Labelled probes were designed to detect splicing alterations known to be upregulated in the variant carriers, namely, carriers ,9. Probe\u00ae Fluorescent Multiplex kit  according to the manufacturer\u2019s instructions specified in RNAscope\u00ae Fluorescent Assay for PBMC and Non-Adherent Cells (Part 1), and the RNAscope\u00ae Fluorescent Multiplex Kit User Manual Part 2 [RNA in situ hybridisation was performed once for each sample using the RNAscopel Part 2  . The EC plan-Neofluar 40\u00d7/1.3 oil Dic M27 objective was used with DAPI, TRITC and FITC filters. Fluorescent signals were counted independently for each wavelength in 50 non-overlapping cells. Accurate counting was conducted by scanning through different focal planes in the molecule .BRCA1 C1green (exon 11) and C2red (exons 1\u201310) fluorescent signals in a cell, we determine that all detected transcripts contain exon 11 as seen in red to C1green signals indicates an exon-skipping event (\u220611), as shown in The number of transcripts containing the targeted deletion event in each cell was determined through differences in the number of fluorescently labelled probes detected for each colour channel. For example, where there is an equal number of"}
+{"text": "Cancer is associated with various genetic and environmental risk factors. Beside the mutations or aberrant expression of protein-coding\u00a0genes, the genetic deregulation of non-coding\u00a0RNAs has also an important role during\u00a0tumor progression and metastasis. Long\u00a0non-coding\u00a0RNAs (lncRNAs) are\u00a0a class of ncRNAs larger than 200 nucleotides that may function\u00a0as tumor-suppressor or oncogene.There is a raising trend of cancer incidence among Iranian population during the last decades. Therefore, it is required to prepare a general population specific panel of genetic markers for the early detection of cancer in this population. The tissue-specific expression characteristics and high stability in body fluids highlight the lncRNAs as efficient diagnostic and prognostic noninvasive biomarkers in\u00a0cancer. In present review we summarized all of the lncRNAs which have been reported until now in different tumors among Iranian patients.This review paves the way of introducing a population based noninvasive diagnostic panel of lncRNAs for the early detection of tumor cells among Iranian population. Cancer is one of the main global health challenges and is the leading and second causes of deaths among developed and developing nations, respectively . Cancer In present review we have summarized all of the lncRNAs with significant roles during tumor progression which have been reported until now among Iranian cancer patients to pave the way of introducing a non-invasive population based diagnostic panel of lncRNAs Table . We cateSOX2 overlapping transcript (SOX2OT) is an lncRNA located on 3q26.33. The SOX2 is located within the third intron of SOX2OT gene which is transcribed in the same orientation. There are 13 splice variants for SOX2OT . SOX2OT LincRNA-Regulator of Reprogramming (lincRNA-RoR) regulates the reprogramming process in pluripotent stem cells. Moreover, it is associated with iPSC derivation and ESC pluripotency . AberranLnc-POU3F3 is located on the reverse strand of chromosome 2q12.1 and POU3F3 upstream which is a member of the class III POU family of transcription factors . POU3F3 Prostate cancer associated transcript 1 (PCAT1) acts as an oncogene through inhibition of BRCA2 and stimulation of MYC. It promotes tumor cell invasion through targeting RBM5 and KLF6 in pancreatic and ovarian cancer , 55. It Prcat17.3 and Prcat38 are located upstream of TMPRSS2 gene, which are used to distinguish malignant and nonmalignant prostate tissues. Prcat17.3 and Prcat38 are positively correlated with TMPRSS2 in prostate cancer. Cat2184.4 is located upstream of PMEPA1 which acts as a tumor suppressor in prostate tumorigenesis. It has been shown that there were significant up regulations of Prcat17.3 and Prcat38 and significant down regulation of Cat2184.4 in prostate cancer (PCa) tissues compared with benign prostate hyperplasia (BPH) among a group of Iranian subjects. Moreover, the results showed a significant up regulation of Prcat17.3 level in urine samples of PCa patients compared with BPH patients. According to the ROC curve analysis, it has been demonstrated that the Prcat17.3 urine assay has a better sensitivity in distinguishing PCa from BPH compared with urine PCA3 tests. Therefore, Prcat17.3 and Prcat38 can be suggested alone or in combination with PCA3 as diagnostic markers of PCa among Iranians .HOTAIR is an oncogenic factor transcribed from the antisense strand of HOXC locus Which is involved in epigenetic regulation through interaction with Polycomb Repressive Complex 2 (PRC2) . HOTAIR ANRIL is an lnc-RNA which binds with CBX7 and SUZ12 within the\u00a0PRC1\u00a0and PRC2\u00a0to regulate the\u00a0transcriptional\u00a0repression , 63. It Prostate cancer associated non-coding RNA 1 (PRNCR1) is located in 8q24.21 and highly expressed in aggressive PCa. It has a critical role in PCa progression through regulation of androgen receptor (AR) . BesidesLong intergenic non-protein coding RNA 355 (LINC00355) is associated with various cellular processes such as apoptosis, proliferation, and migration , 73. It SOX2OT\u00a0is an lncRNA that is de-regulated in tumor tissues and is down regulated during the cell differentiation , 37. MorThe findings demonstrated that the relative expression of HOTAIR long non-coding RNA was significantly up regulated in GC tissues compared with normal margins in a sample of Iranian subjects. There was also association between HOTAIR expression level with TNM staging and lymph node metastasis. Moreover, they showed a significant direct association between the levels of HOTAIR and SUZ12 expressions . AnotherTUG1 plays a critical role in epigenetic regulation through interaction with PRC2 or PRC1 . It induGrowth arrest specific 5 (GAS5) is an ncRNA that inhibits the glucocorticoid receptor via binding with its DNA binding domain. It regulates gastric cell proliferation, apoptosis, and migration through CDK6, YBX1, and P53 \u2013102. It ANRASSF1 is an antisense lncRNA of RASSF1 involved in epigenetic regulation through binding with PRC2 that is required for recruiting PRC2 to the RASSF1A promoter region. This process results in accumulation of the H3K27me3 and decreases the RASSF1A protein. It has been observed that there were significant increased levels of ANRIL and ANRASSF1 in GC tumors compared with the normal margins among a subpopulation of Iranian patients . ANRIL wProtein tyrosine phosphatase receptor type G antisense (PTPRG-AS1) is an antisense lncRNA of PTPRG which has important roles in cell growth, differentiation, and neoplastic transformation. PTPRG-AS1 is correlated with ER+\u2009and ER\u2009\u2212\u2009 subtypes, tumor grade, and clinical results . It funcIt has been reported that there were a significant association between HOTAIR polymorphisms and risk of breast cancer in a sample of southeast Iranian population in which rs920778 polymorphism significantly increased breast cancer risk while the rs12826786 and rs1899663 polymorphisms significantly decreased breast cancer risk. Moreover, rs920778 and rs12826786 were significantly correlated with ER status . SimilarH19 is a paternally imprinted gene associated with the human Beckwith-Wiedemann syndrome that is located on chromosome 11p15.5. H19 acts as an oncogene in bladder cancer, breast cancer, and hepatocellular carcinoma. It is associated with E2F1 transcription factor to induce breast cancer cell cycle progression . H19 is Steroid receptor RNA activator (SRA) regulates gene expression by steroid hormones. SRA is involved in regulation of physiological processes such as NR signaling, steroidogenesis, and mesenchymal fate. SRA functions as a molecular scaffold to regulate the transcription by various co-regulators and chromatin-modifying factors in both activating and repressive complexes . GAS5 isPRNCR1 is a critical AR regulator. It has been reported that there were significant increased levels of PRNCR1 expressions in breast tumor tissues compared with normal margins among a sub population of Iranian subjects. Moreover, there were significant association between PRNCR1 overexpression and clinical-pathological features such as tumor size and lymph node involvement . SimilarThe NAV2-AS2 is a lncRNA located in 11p15.1 and the minus strand of NAV2 sequence . NAV2 isLong non-coding upstream of MYCN (lncUSMycN) is transcribed from the 14-kbp upstream of the MYCN transcription start site. It has been reported that the lncUSMycN up regulated N-Myc mRNA expression through non O protein. It has also important roles in cell proliferation and tumorigenesis in neuroblastoma. There was significant up regulation of lncUSMycN among a sample of Iranian breast cancer patients compared with healthy controls. Moreover, levels of lncUSMycN expression was significantly associated with the early stages of breast cancer .LncRNA ZEB1 antisense 1 (ZEB1-AS1) is transcribed from the ZEB1 promoter sequence and it regulates the ZEB1expression levels. ZEB1-AS1 binds with MLL1 to induce H3K4me3 in ZEB1 promoter sequence. It promotes colorectal tumor cell proliferation through p15 inhibition . It has LOC100287225 is an lncRNAs located in the long arm of the chromosome 18, upstream region of deleted in colorectal carcinoma (DCC). DCC is a trans-membrane cell adhesion protein belonging to the immunoglobulin family which is associated with axon attraction and apoptosis induction. It has been demonstrated that there were significant LOC100287225 down regulations in tumor tissues compared with adjacent tumor-free tissue among a sample of Iranian CRC patients , 143.VIM-AS1 RNA is located in 10p13 that is transcribed from a shared bidirectional promoter with Vimentin mRNA . It has Lnc-LAMC2-1:1 is located in 1q25.3 and overlaps with LAMC1. It has been reported the genetic variation in lnc-LAMC2-1:1 was associated with CRC by affecting miRNA binding . The lncOIP5-AS1 functions as a sponge for miR-448 to regulates BCL-2 in lung adenocarcinoma cells . OIP5-ASThere are various diagnostic methods for the early detection of cancer. However, majority of routine methods are invasive. The deregulated pattern of lncRNAs in tumor tissues is mirrored in body fluids which make these factors as a non-invasive candidate with better tolerance for the cancer patients compared with biopsy. Therefore, this review summarized all of the lncRNAs with significant roles during tumor progression which have been reported until now among Iranian cancer patients. This review paves the way of introducing a population based non-invasive panel of lncRNAs for the early detection of cancers among Iranian population."}
+{"text": "The World Health Organization (WHO) prequalification of Two hundred and fifty (250) patients were recruited from the HIV outpatient clinic at ITM. Accuracy and precision of CD4 T cell counting on fresh EDTA anticoagulated venous blood samples were assessed in the laboratory on a Muse Auto CD4/CD4% system. Extensive precision analyses were performed both on fresh blood and on normal and low stabilized whole blood controls. Accuracy ((bias) was assessed by comparing results from Muse CD4/CD4% to the reference . Clinical misclassification was measured at 500, 350, 200 and 100 cells/\u03bcL thresholds.Intra-assay precision was < 5%, and inter-assay was < 9%. CD4 T cell counts measured on Muse Auto CD4/CD4% System and on the reference instrument resulted in regression slopes of 0.97 for absolute counts and 1.03 for CD4 T cell percentages and a correlation coefficient of 0.99 for both. The average absolute bias as compared to the reference was negligible (4 cells/\u03bcL or 0.5%). The absolute average bias on CD4 T cell percentages was < 1%. Clinical misclassification at different CD4 T cell thresholds was small resulting in sensitivities and specificities equal or >90% at all thresholds except at 100 cells/\u03bcL (sensitivity = 87%). All samples could be analyzed as there was no repetitive rejection errors recorded.The Muse Auto CD4/CD4% System performed very well on fresh venous blood samples and met all WHO acceptance criteria for analytical performance of CD4 technologies. In 2017, about 36.7 million people were living with HIV of which 20.7 56.4%) were receiving antiretroviral treatment (ART) % were re.in vitro diagnostics assessment includes an independent analytical performance evaluation of CD4 counting instruments, in particular of those meant for use in resource-limited settings [in vitro diagnostic (IVD) flow cytometer installed in a well-equipped accredited clinical laboratory with excellent performance in external quality assessment (EQA) schemes and operated by highly-skilled laboratory personnel [Ideally, laboratory evaluations of new CD4 technologies are conducted independently from the manufacturer. The WHO prequalification of settings . During ersonnel .Most currently used IVD flow cytometers are sophisticated large, heavy and very expensive instruments which are operated by highly skilled and dedicated personnel. They are usually used in large hospitals, are semi- or fully automated and have a high sample throughput. Fortunately; several less complicated and more affordable instruments, dedicated to CD4 T cell counting, have been developed the past two decades . As a reThe gap between the large IVD instruments and the small POC devices is currently filled by a number of simplified, small flow cytometers which still require a laboratory and trained personnel but which are much easier to set up in small laboratories near the point-of-care. An example of such \u201cintermediate\u201d flow cytometer dedicated to CD4 counting, prequalified by WHO, which has been around for more than 20 years is the FACSCount ,15. In 23-EDTA vacutainer tubes for routine CD4 T cell counting. The same blood sample was used for CD4 T cell counting on Muse Auto CD4/CD4% System. The WHO study protocol requires collection of at least 50 blood samples from patients with <200 CD4 T cells/\u03bcL, 100 blood samples from patients with 200\u2013500 CD4 T cells and 50 blood samples from patients with >500 CD4 T cells [Adult patients presenting for routine CD4 T cell enumeration at the HIV outpatient clinic of the Institute of Tropical Medicine (ITM) Antwerp, Belgium (in April-June 2017) provided a venous blood sample in 3 ml K T cells . The stuThe laboratory evaluation was conducted with two Muse Auto CD4/CD4% Systems. The Muse CD4/CD4% System is a small capillary flow cytometer with dedicated software for CD4 T cell counting of both percentages and absolute CD4 T cell counts. The instruments needs to be installed on a steady table and requires stable external power supply. It has a large color touch screen user interface to enter patient information and reagent batch numbers. The Muse Auto CD4/CD4% reagents are a cocktail of fluorochrome-conjugated antibodies to identify all lymphocytes and CD4+ T lymphocytes. The test uses a lysing solution to lyse red blood cells prior to CD4 T cell counting. The test system comes with a Muse system check which are bead controls for system check and proficiency testing of operator (correct volume pipetting). The test requires mixing 10\u03bcL of fresh blood (<48h) with 10\u03bcL of monoclonal antibodies followed by an incubation of 15 minutes. Subsequently, 380 \u03bcL of lysing solution are added  and after another incubation of 15 minutes the tube can be analyzed on the Muse instrument. The instrument reports both CD4 T cell percentages and absolute CD4 T cell counts (cells/\u03bcL).\u03bcL. The CD4 category was determined by the first CD4 result of a venous blood sample measured on FACSCalibur using Trucount tubes.The coefficient of variation (CV), also known as relative standard deviation (RSD), was used as a standardized measure of dispersion (precision) and was calculated as standard deviation (SD) divided by the mean of 10 repeat observation. The assessment of intra-laboratory variation of test results of Muse Auto CD4/CD4% system included instrument precision, intra-assay variation, inter-instrument variation, and inter-assay variation, using venous blood specimens. Blood samples were stratified in categories around clinically relevant CD4 thresholds: 200 (150\u2013250), 350 (300\u2013400) and 500 (450\u2013550) CD4 cells/Instrument precision (run-to-run) consisted of re-reading a single stained venous blood sample ten times on the same Muse Auto CD4/CD4% System. For intra-assay precision (tube-to-tube variability), staining and reading was repeated 10 times on the same blood sample and analyzed on the same Muse Auto CD4/CD4% System. For inter-instrument precision (instrument-to-instrument variability), each blood sample stained ten times and each tube was analyzed on each of the two Muse Auto CD4/CD4% Systems. The average of the obtained results was calculated for each instrument. The inter-instrument variability (CV inter-instrument) was calculated using the average result and SD of the 2 different instruments for each patient sample.The inter-assay precision (day-to-day variability) was measured on venous blood samples analyzed within 6 hours after blood collection and reanalyzed within 48 hours after blood collection.The CD4 reference method was performed in an ISO15189:2012 certified laboratory which participates in external quality assessment programs (QASI and UK-NEQAS). The service maintenance of the CD4 reference instrument  is assured twice a year by a service engineer from BD Biosciences. The CD4 reference instrument is calibrated daily, and checked by running Multicheck Normal and Low process controls (BD Biosciences) . Four different batches of Normal controls and Low controls were used during the three-month duration of the study in Antwerp.The Muse Auto CD4/CD4% System is supplied with control beads which have to be run daily as system and proficiency check prior to analyzing patients samples. Multicheck Normal and Low controls were run daily on the Muse Auto CD4/CD4% Systems to assess reproducibility of test results during the entire study period. For each control blood sample, the reported results were compared to the sample validation range provided by the manufacturer.Venous blood samples were alternatively analyzed on one of two different Muse Auto CD4/CD4% Systems each day. Absolute CD4 T cell counts and CD4 T cell percentages measured on the Muse Auto CD4/CD4% System were compared to the corresponding CD4 counting results from the FACSCalibur . CD4 counts on FACSCalibur were measured according to the manufacturer's recommendations using Multiset reagents  and BD Trucount tubes . Acquisition on FACSCalibur was automated but the individual sample analyses were done manually using CellQuest software in accordance with the accredited procedure used for routine CD4 counting in the laboratory.The agreement between Muse Auto CD4/CD4% System results and those obtained on the reference method was assessed by Passing-Bablok regression analysis , SpearmaThe bias of Muse Auto CD4/CD4% system test results at different clinically relevant CD4 thresholds  and at 1000 cells/\u03bcL was calculated using Passing-Bablok regression analysis with bootstrapping of the confidence intervals of the bias at different CD4 thresholds . EstimatClinical misclassification was calculated for CD4 counts at clinically relevant CD4 thresholds of 200, 350, 500 cells/\u03bcL, using the reference method results as the \u201ctrue\u201d value for sensitivity and specificity. Inter-rater agreement was calculated with the Kappa coefficient .Statistical analyses were done with MedCalc version 18.2.1 .Invalid results or sample rejection was defined as the inability of Muse Auto CD4/CD4% System to provide a CD4 results from a patient blood sample. If the analysis was aborted/rejected, a second sample was prepared and read. Upon a second rejection, the test result was recorded as sample rejection.In total, two hundred and fifty patients were included in this study. Clinical data was available for 236 patients, of whom 234 were HIV infected.The median age (minimum\u2013maximum) of the participants was 46 years (15\u201385) and 78% were males. Most patients (86%) were receiving antiretroviral treatment. The median (inter quartile range) CD4 T-cell count was 410 (252\u2013582) cells/\u03bcL; 52 had CD4 < 200 cells/\u03bcL (median 129 (65\u2013180) cells/\u03bcL), 113 had between 200\u2013500 CD4 T cells/\u03bcL (median 386 (312\u2013437) cells/\u03bcL), 85 had > 500 CD4 T cells/\u03bcL (median 753 (581\u2013948) cells/\u03bcL). None of the patients presented with malaria; 4 patients had clinical features suggestive of pulmonary tuberculosis. From 14 patients clinical data was not available.The mean instrument precision for absolute CD4 T cell counts and CD4 T cell percentages in venous blood varied between 2.5 and 5.5% depending on the CD4 category. The intra-assay CV varied from 2.8 to 4.6%. The inter-assay CV varied from 3.5 to 8.1%. The inter-instrument CV varied from 2.5 to 4.4%. The individual CV for the different CD4 categories and for each type of precision assay are presented in Four different Multicheck batches (lots) of Normal and Low controls were used over the entire laboratory evaluation including 36 consecutive measurements of a Normal and Low CD4 count control over the entire study duration of 3 months. The Low controls were stabilized blood samples with CD4 counts between 100\u2013200 CD4 T cell counts/\u03bcL. The Normal controls were stabilized blood samples with CD4 T cell counts between 600\u20131000 CD4 T cells/\u03bcL. The average CV for absolute CD4 T cell counts over the entire study duration was <5% for Normal CD4 count controls and <8% for Low CD4 T cell count controls. The overall CV for CD4 percentages was <2% for Normal controls and <6% for Low controls.Agreement between Muse Auto CD4/CD4% System and the reference method is shown in Fitted line equation, the number of analysed samples (n) and Spearman rank correlation are depicted in the legends.Absolute bias on absolute CD4 T cells counts (top left), absolute bias on CD4 T cells percentages (top right). Relative bias on absolute CD4 T cells counts (bottom left), relative bias on CD4 T cells percentages (bottom right).The absolute and relative bias was calculated for absolute CD4 T cell counts and for CD4 T cell percentages .As the relative bias can be large at low CD4 T cell counts, without being meaningfull, the relative bias was recacluated for patients with > 100 CD4 T cell counts resulting in a smaller, and thus better limits of agreement . As the Misclassification analysis is shown in During the study, the rejection rate was 0/250 (0%). All blood samples could be analyzed on Muse Auto CD4/CD4% System and all results were included in the study.Technicians found the Muse Auto CD4/CD4% instrument easy to use after a short 4-hour training.The keypad on the screen used to enter details on the sample, user, batch numbers etc was considered a bit small and technicians with larger fingers  had trouble pressing the correct keys. The small blood volume (10\u03bcL) and reagent volume (10\u03bcL) required to perform the test was a concern at the start of the study but, as supported by the results, that did not have any impact on the precision and accuracy of the measurements. The system check at start-up which includes an operator proficiency check is considered an asset as it will assure accurate pipetting. The instrument has no sample loader and samples are analyzed on the instrument manually. The manufacturer recommends an extra instrument cleaning step after 20 measurements to prevent needle clogging. Since preparation of 20 samples takes about 45min to 1 hour and the analysis of 20 samples on the instrument take another hour, 80 samples can be analyzed on an 8h working day per technician and per instrument. In theory, a first result can be generated after 1 hour.The Muse CD4/CD4% instruments are much smaller than conventional large IVD flow cytometers, easier to move around (mobility) and simple to operate (much less training required).The system reported warnings but the frequency was low (<5%). Most of the warnings were related to a concern about correct automatic gating and required visual inspection and adjustment of the gate. Setting the instrument takes about 15 min (complete system clean + system check) before blood samples can be analyzed. Technicians consistently implemented the wash procedure which can optionally be skipped although it is not recommended to do so. At the end of a day, the flow cell needs to be cleaned by a thorough cleaning procedure (designated \u201cExtreme Clean Procedure\u201d) which takes about 15 min. The technicians did not experience any clogging of the needle during the study. The capillary needle of the flow cell in the instrument is very delicate and replacement should be done very carefully. Technicians never had to replace the flow cell during the study.The laboratory evaluation of the Muse Auto CD4/CD4% system was conducted in accordance with the WHO prequalification evaluation protocol to verify whether this system met the WHO acceptance criteria for CD4 technologies . Those aAccuracy (bias) was assessed by comparing results of the Muse Auto CD4/CD4% to the results obtained by an established reference method/instrument. This is the easiest way to assess accuracy of a new CD4 technology as there are no international reference blood samples to assess the bias Alternatively, the bias can be assessed by comparing the results a CD4 technology to the consensus value of an external quality assessment program (EQA). The consensus value is defined as the average CD4 result of all laboratories participating laboratories in the EQA program and the difference between the individual result and the consensus value is a good estimated of the bias. During the present study, the average difference (bias) between Muse Auto CD4/CD4% and the FACSCalibur results were negligible (0.5% or 4 cells), and well within the maximum allowable relative bias of 10%. This small bias is confirmed by the regression analysis with a slope close to 1 (0.97), and a correlation of 0.99. There was a very slight tendency towards larger underestimation at higher CD4 T cell counts as the relative bias increased from -0.7% to -2.1% between 200 and 1000 CD4 T cell counts .In case the Muse Auto CD4/CD4% would be used for taking decisions with regard to treatment initiation, the present study found that sensitivity at 100 CD4 T cells/\u03bcL was 86% and \u226590% at the higher thresholds with a specificity of \u226595% indicating that the number of misclassified patients would be relatively low .The instrument uses small volumes of blood and reagent (10\u03bcL each). This could be considered as a concern as pipetting of such small volumes may introduce larger errors if pipets are not properly calibrated and technicians not properly trained. However, this was not found to be an issue during this study as the precision studies produced very good results. The obligatory pipetting proficiency test (pass/fail) prior to running blood samples is certainly helping to assure good pipetting practice.Until today, two field studies of the Muse Auto CD4/CD4% have been published, both by the same group ,19. One The observation that the Muse Auto CD4/CD4% is small but precise and accurate instrument is encouraging and instruments like the Muse could be used to monitor HIV disease and to screen patients for the risk of opportunistic infections. Even when CD4 T cell counting will lose importance in monitoring ART, instruments like Muse Auto CD4/CD4% are still considered to be as an essential disease-specific IVD tool for health care facilities with clinical laboratories [The limitation of the present study is that the analytical performance was conducted in a Western accredited laboratory with well-trained technicians. This study did not assess information on robustness and temperature and humidity fluctuations common to rural areas. Although our study validated the analytical performance characteristics of the instrument, more field studies are required to assess the performance of the Muse Auto CD4/CD4% in real-life conditions typical of rural settings.In conclusion, the Muse Auto CD4/CD4% system showed a very good analytical performance on fresh venous blood samples. The instrument was considered easy to use, with minimal training and infrastructure required, making it suitable for decentralization of CD4 T cell counting in rural resource-limited settings with a small laboratory, trained technicians and stable power supply.S1 Database(XLSX)Click here for additional data file."}
+{"text": "Following publication of this work , the folFig 1A (ii) Stat3 panel appears to similar to the Input Src panel when horizontally flipped, and to the IP: Stat3/Src blot panel in Fig 1C (i) when flipped verticallyFig 2A (i) Input: EGFR panel appears similar to Fig 1A (i) Input: EGFR panelFig 3A the Stat3 blots for input samples appear similar in (i) and (ii)Fig 3C the EGFR and Src blots shown for input samples appear to be similar in (i) and (ii)Fig 3B EGFR lanes 1\u20133 appear similar to lanes 7\u20139Fig 6B (i) Src panel appears similar to Fig 3A (i) Src panelFig 6B (ii), in the \u03b2-actin panel there appears to be a vertical discontinuity between lanes 3 and 4, and lane 4 appears to have a different background as compared to other lanes in the panelThe authors have noted that all figure panels represent independent experiments. However, they have explained that in Fig 3A, the same Stat3 input panel was presented in panels (i) and (ii) because the same input control data was relevant to both Stat3 immunoprecipitation experiments. The immunoprecipitation experiments shown in panels (i) and (ii) used the same amount of the same samples. In Fig 6B (ii), authors commented that differences in background could be attributed to lower levels of protein in lane 4 compared to lane 3.While the authors stand by the validity of the experimental data and the conclusions reached in the study, owing to the time passed since the experiments were completed they were unable to provide any of the original images or raw underlying data in the article. Hence, we have been unable to resolve the above concerns.PLOS ONE Editors retract this article.In light of the image concerns and the unavailability of primary data, the"}
+{"text": "Human embryos are usually cultured to blastocyst stage by Day 5 or 6 after insemination. However, some embryos grow slowly and reach blastocyst stage at Day 7. Acceptable live birth rates have been reported after transfer of Day 7 blastocysts resulted from fresh oocyte in vitro fertilization\u00a0(IVF). It is unknown whether an extended embryo culture to Day 7 is necessary for cryopreserved oocyte IVF to obtain more transferrable blastocysts.In this study, 455 oocytes from 57\u2009cycles were warmed, inseminated, and the resulting embryos were cultured by Day 7 to examine blastocyst development after extended culture. Fifty one blastocysts from 16\u2009cycles were biopsied to examine embryo aneuploidies.It was found that 35.1% of the cycles had Day 7 blastocysts, and 3.5% of the cycles had only Day 7 blastocysts. Day 7 blastocysts accounted for 15.6% of total blastocysts. The proportion of top quality of blastocysts was lower at Day 7 than at Day 5 or 6. However, no differences were observed on aneuploid blastocyst rates among Days 5, 6 and 7. Similar clinical pregnancy, ongoing pregnancy and embryo implantation rates were obtained after Day 7 blastocyst transfer as compared with Day 5 or 6 blastocyst transfer.These results indicate that embryos from oocyte warming cycles should be cultured to Day 7 if they do not reach to blastocyst stage by Day 6 so that number of usable blastocysts can be increased. Oocyte cryopreservation has become one of the most important human assisted reproductive technologies, which provides approaches of fertility preservation for women who want to delay childbearing , and forThe procedures for in vitro fertilization (IVF) and embryo culture did not have any difference between cryopreserved/warmed oocytes and fresh oocytes. However, recently, it has been found by time-lapsing analysis and morphokinetic that cryopreserved/warmed oocytes had about 1\u2009h of delayed fertilization and embryo development . DelayedFor cryopreserved oocytes from oocyte bank, oocyte number is very limited, usually 6\u20138 per cycle, thus the number of embryos developed to blastocyst stage is much fewer than that from most fresh oocyte IVF cycles. Therefore, it may be necessary to extend embryo culture to Day 7 so that slow growing embryos can reach to blastocyst stage. In our clinic, we started to Day 7 culture for all oocyte warming cycles from 2019, and our preliminary data showed that implementation of Day 7 culture for oocyte warming IVF cycles increased overall blastocyst number.In this study, 455 cryopreserved donor oocytes were warmed for 57 recipient cycles. Warmed oocytes were inseminated by intracytoplasmic sperm injection and all embryos were extended to Day 7 culture if they did not reach blastocysts by Day 6. As shown in Table\u00a0Day 7 blastocysts had poorer quality as compared with Day 5 or Day 6 blastocysts , 11, 12.Although previous studies found that more Day 7 blastocysts were aneuploid as compared with Day 5 or Day 6 blastocysts, suggesting that slow growing embryos may be resulted from oocyte aneuploidy , 11, 12.In the present study, there were 35 fresh blastocyst transfers and 16 frozen embryo transfer (FET). There were no statistical differences between fresh blastocyst transfer and FET in terms of clinical pregnancy rates (60.0 vs 56.3%), ongoing pregnancy rates (54.3 vs 50.0%) and embryo implantation rates (53.9 vs 58.8%).Acceptable live birth rates have been obtained after Day 7 blastocyst transfers \u201310. HoweFor fresh oocyte donation, recipients usually receive many oocytes and high quality of oocytes usually produces enough blastocysts at Days 5 and 6, thus very few laboratory may culture embryos from donor oocytes to Day 7. This may be the reason that no data is published on the transfer of Day 7 blastocysts resulted from donor oocytes. However, most IVF clinics now switch fresh oocyte to frozen oocyte IVF when donated oocytes are used , and 6 oIn conclusion, our preliminary data with limited case numbers indicate that Day 7 culture is necessary for cryopreserved/warmed oocyte IVF cycles, especially for those cycles with limited number of oocytes being warmed. The proportions of cycles with Day 7 blastocysts and proportions of blastocysts obtained at Day 7 appear to be higher than those observed with fresh oocyte IVF. In addition, our limited data indicate that aneuploid rate with frozen donor oocytes did not differ between Day 7 blastocysts and Days 5/6 blastocysts although quality of Day 7 blastocysts is not as good as quality of Day 5/6 blastocysts. Transfer of Day 7 blastocysts resulted in successful embryo implantation and clinical pregnancy, and both embryo implantation rate and clinical pregnant rate were not different from those with Day 5 or Day 6 blastocyst transfer. Putting together, these data suggest that the implementation of Day 7 culture for those embryos that have not developed to blastocysts by Day 6 is necessary in oocyte warming cycles."}
+{"text": "Dear Editor,De novo lipogenesis and gluconeogenesis in the liver contribute, at least in part, to the dynamic homeostasis of lipid and glucose levels  and CREB regulated transcription coactivators (CRTCs)  was also elevated in Crtc2\u2212/\u2212 mice as judged by real-time quantitative PCR (qPCR)  . Together, these results indicate that CRTC2 inhibits SREBP1c activity during fasting.To investigate the effect of CRTC2 on SREBP1c processing during fasting, we fasted mice for 12 h and evaluated SREBP1c processing. We found that higher levels of mature SREBP1c accumulate in ons Fig.\u00a0. TherefoInsig2a expression and thus attenuated SREBP1c processing in wild-type mice during fasting  enhanced Insig2a is a predominant isoform of Insig2 in the liver  and constitutively active (CRTC2/AA)  treatment, while Crtc2 deficiency by knockdown or knockout attenuated Insig2a-luc activity  studies to determine whether Insig2a is a direct target of CRTC2/CREB. Supporting this notion, glucagon stimulation dramatically increased both CRTC2 and CREB occupancy on the Insig2a promoter . This highlights the important role of the CRTC2/CREB complex in SREBP1c maturation during fasting. Meanwhile, our results fill the gap in our understanding of how glucagon or fasting signaling modulates SREBP1c processing.It is well known that fasting or glucagon attenuates SREBP1c processing .All institutional and national guidelines for the care and use of laboratory animals were followed. Yuanyuan Zhang, Yi Liu, Liqun Chen, Yiguo Wang and Jinbo Han declare that they have no conflict of interest.Yuanyuan Zhang and Jinbo Han designed the study and analyzed the data. Yuanyuan Zhang, Yi Liu and Liqun Chen performed the experiments. Jinbo Han wrote the paper. All authors reviewed and commented on the manuscript.Supplementary material 1 (PDF 807 kb)Below is the link to the electronic supplementary material."}
+{"text": "On page 4 of the original publication , the cor570 was measured using microplate reader at 200 \u03bcl per well.Quantitative measurement was performed by dissolving CV stained pellicle in 4 ml of 85% ethanol and the ODTable"}
+{"text": "Jumping translocations of 1q refer to the break-off of chromosome 1q as a donor fusing to two or more recipient chromosomes. We detected jumping translocations of 1q in three patients with initial diagnosis of myelodysplastic syndrome (MDS) and later progression to acute myeloid leukemia (AML). Review of literature found jumping translocations of 1q in 30 reported cases of MDS and AML. The cytogenetic findings from these 33 cases showed that seven cases had a stemline clone and 26 cases had de novo jumping translocations of 1q in which 5% of cell lineages had additional structural rearrangements. In 75% of cases, the 1q donor jumped to the short arm of recipient acrocentric chromosomes. Approximately 82% of the fusions occurred in the telomeric regions of short and long arms and 18% occurred in the pericentric or interstitial regions of recipient chromosomes. Hypomethylation of the donor 1q pericentromeric region and shortened telomeres in recipient chromosomes were associated with the formation of jumping translocations. Jumping translocations of 1q as an indication of chromosomal instability pose high risk for progression of MDS to AML and a poor prognosis. Further understanding of underlying genomic defects and their clinical significance will improve overall treatment and patient care. Jumping translocations are a rare type of cytogenetic abnormality initially detected in a case of acute monocytic leukemia and later found in various types of leukemias \u20133. JumpiA retrospective review of cases with MDS and AML from the CytoAccess database in Yale Cytogenetics Laboratory found three cases with jumping translocations of 1q . ChromosThis male patient was first evaluated at age 59 with an indication of leukopenia and mild anemia; cytogenetic analysis found a normal result. Two years later, this patient was diagnosed with MDS and chromosomal analysis on a bone marrow aspirate showed an abnormal clone with jumping translocations of 1q. The karyotype was as follows: 46,XY,der(15)t[7]/46,XY,der(21)t[3]/46,XY,dup(1)(q21q42)[2]/46,XY,der(14)t[2]/46,XY[6]. FISH analysis using dual color probes for the PBX1 gene at 1q23.3 and TCF3 gene at 19p13.3 detected three copies of 1q in 32% of bone marrow cells. This patient started chemotherapy and then received a bone marrow transplant in two months. Six months later, the patient progressed to AML and died at age 62. Final chromosomal analysis revealed a karyotype of 46,XY,der,del(20)(q11.2q13.3)[15]. FISH tests confirmed three copies of 1q and a deletion at 20q in 83.5%-96.5% of bone marrow cells.This patient, a 63-year old male, was referred with MDS by an indication of MDS with excess blasts. Chromosomal analysis showed a karyotype of 46,XY,der(15)t[4]/46,XY,der(22)t[2]/46,XY[7]. FISH analysis using dual color probes for the CDKN2C gene at 1p32.3 and CKS1B gene at 1q21.3 confirmed three copies of 1q in 5.5% of bone marrow cells. Over the next six months, the percentage of abnormal cells increased despite two rounds of chemotherapy and the patient progressed to AML. A transit clone with the der(15)t and a deletion at 11q was noted. The last chromosome analysis revealed a karyotype of 46,XY,der(15)t[13]/46,XY,der(22)t[2]. FISH detected three copies of 1q in 94% of bone marrow cells. This patient died eight months after the initial finding of the jumping translocations of 1q.This patient was a male at age 73 when diagnosed with MDS. Chromosomal analysis showed a karyotype of 46,XY,der(15)t[13]/46,XY[7]. FISH confirmed three copies of 1q in 82.5% of bone marrow cells. Two years later, the patient progressed to AML and died soon after the last chromosome analysis which revealed a karyotype of 46,XY,der(15)t[9]/46,XY,der(22)t[4]/46,X,der(Y)t,t[3]. FISH detected three copies of 1q in 94% of bone marrow cells.The cytogenetic findings of successive studies from these three patients all showed persistent jumping translocations of 1q with an increased percentage in bone marrow cells. The chromosome patterns of jumping translocations of 1q are shown in The results from these 33 cases revealed the chromosomal fusion patterns between the 1q donor chromosome and different recipient chromosomes. However, all studies done by routine chromosome analysis lacked the molecular characterization of breakpoints involving the chromosome fusion, underlying genomic imbalances, and tumor gene mutations. A recent study using genomic array testing and targeted gene panel next-generation sequencing detected the 100\u2013107 Mb duplication of 1q with breakpoints in 1q21.1-q21.3 region and recurrent mutations in the TET2 and SF3B1 genes , 13.Approximately one-third of MDS/AML cases with jumping translocations of 1q either emerged from a stemline abnormal clone or evolved additional clonal abnormalities and two-thirds were detected as de novo chromosomal abnormalities. Shortened telomeres and many variant telomeric repeats were detected in the fusion points from jumping translocations of 1q in a case of MDS progressed to AML; this result suggested that the extended proliferation of cancer cells might be related to the loss of telomeric function . Copy nuThe progression of MDS to AML and poor prognosis were noted in the present three cases with jumping translocations of 1q. This result was consistent with previous findings in which jumping translocations of 1q were associated with a high risk of progression to AML and poor prognosis and thus warranted aggressive therapy , 10. CurIn summary, jumping translocations of 1q have been shown as an indication of chromosomal instability likely induced by epigenetic alterations of hypomethylation and shortened telomeres. The jumping translocations of 1q showed unique rearrangement patterns with multiple cell lineages which all have a gain of donor chromosome segment and lack other complex structural or numerical rearrangements. It is not clear whether these epigenetic alterations occur independently as a two-step event or concurrently by driving genetic defects. Further molecular analyses to understand the genetic defects causing jumping translocations of 1q and related disease-causing mechanism can facilitate targeted therapy for this type of MDS and AML."}
+{"text": "ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association,  SYNGR2 gene that influences the ability of the PCV2 virus to replicate, which can affect pig growth and immune system following infection. This research will aid in the development of genetic tests with the ability to predict PCV2 susceptibility. Early identification of pigs that are susceptible to PCV2 infection will result in an improvement in the general health and welfare of pigs.The cost of managing Porcine Circovirus 2 (PCV2) associated diseases in the US alone costs the swine industry more than $250 million a year. This virus is found in all swine populations in the US, but only a few pigs get sick and show signs of disease. Previous anecdotal field data showed differences between pig breeds in both incidence and severity of PCV2-associated diseases, supporting the role of host genetics in disease susceptibility. This research, including over 1,000 experimentally infected pigs with PCV2, is the largest study ever conducted to understand the role of host genetics in PCV2 related illnesses. We found that a pig\u2019s own genetics can impact the ability of PCV2 to multiply and cause disease. Specifically, we found a mutation in the  Circoviridae family and the smallest virus known to infect mammalian cells. Despite its small size, this single-stranded circular DNA virus has been identified as the causative source of a set of systemic disorders known as Porcine Circovirus Associated Diseases (PCVAD), which includes Post-Weaning Multi-systemic Wasting Syndrome (PMWS). PMWS is characterized by severe weight loss, respiratory and enteritic conditions that can lead to mortality [Porcine Circovirus 2 (PCV2) is a member of the ortality . Other sortality . Currentortality .in vitro siRNA and gene editing validation models to elucidate the role of host genetics in pathogenesis by identifying genes and genetic variants that could influence PCV2 susceptibility.Anecdotal field data and initial experimental evidence , 5 descrSubstantial variation in the timing and magnitude of immune response, and in the efficiency of PCV2 replication, was reported in our previous studies of experimental infection with a PCV2b strain , 8. The SLAII) at 24\u201325 Mb while the other was located near the proximal end of SSC12, at 3\u20134 Mb. The SNPs associated with the largest genetic variance, ALGA0039682 and ALGA0110477, in each of these two windows explained 65.1% and 99.7%, respectively of the genetic variance explained by their respective windows.The initial GWAS was performed using a BayesB-based approach where individual SNPs and successive 1 Mb windows of the genome were evaluated for association with phenotypic variation . BayesiaALGA0110477) associated with the largest effect on viral load explained 9.3% of the genetic variance and 6.2% of the phenotypic variance for PCV2 viral load . Interestingly, SNPs in the genomic region encompassing these markers did not show strong evidence of association with viral load despite LD with ALGA0110477. A more nuanced analysis fitting haplotypes across the region rather than individual SNPs using BayesIM [ALGA0110477, providing support for the initial discovery based only on ALGA0110477. The unplaced scaffold containing the SSC12 marker, ALGA0110477, did not contain any annotated candidate genes that might underlie the observed effects, and the available sequence surrounding the marker only extended for 84 bp. Using inverse PCR (iPCR), the proximal sequence was extended to 1,252 bp. This extended sequence was used to interrogate contigs from an early version of a long read-based genome assembly of a pig (accession NPJO00000000), [ALGA0110477 used for identification of candidates genes described below. The recent release of a long read-based improved reference assembly, Sscrofa 11.1 (GenBank accession GCA_000003025), supported more accurate ordering and placement of markers, including ALGA0110477 .The SNP  are located at the proximal end of SLAII with DRA being the closest gene (24.8 Mb) from the SLAII complex. Combined, these two SNPs explained 3.8% of the genetic variance for viral load. While the role of SLAII in antigen recognition and immune response in a variety of infectious diseases is well established, highly polymorphic genes and extended LD are the main factors limiting the discovery of functional variants. The SNP ALGA0039710, was still associated with the largest effect in viral load in an analysis of a subset of samples (n = 268) with extreme phenotypes that included novel SNPs located in genes in the QTL such as DRA, C2, CFB, NELFE, SKIV2L [One of the genomic regions associated with viral load was located on SSC7, in the vicinity of , SKIV2L .Sscrofa 11.1, we used the un-annotated long-read scaffold to identify the genes surrounding the SSC12 marker ALGA0110477. Ab initio gene prediction [-64 and a pBLAST score > 200 were identified. Five of these genes were found to be expressed in RNA-seq data of peripheral blood from pigs subjected to PCV2. These genes are involved in immune response and cytokine signaling (SOCS3), inhibition of apoptosis and promotion of cell proliferation (BIRC5), membrane trafficking and transport (SYNGR2) and transmembrane ion channels (TMC6 and TMC8). The number of isoforms observed across these genes varied from one (SOCS3) to more than 10 (TMC6).Previous to the recent release of the ediction  and pBLAALGA0110477 homozygotes exhibiting extreme viral load following PCV2 challenge uncovered missense (n = 4), synonymous (n = 11), and UTR (n = 10) SNPs and an UTR indel across the 5 candidate genes located in the QTL region. In addition, 1\u20132 kb sequencing upstream of the Transcription Start Site (TSS) for BIRC5, SOCS3 and SYNGR2 uncovered 32 SNPs and 4 short indels. These novel polymorphisms and 580 SNPs from the Porcine SNP60 BeadArray were mapped to the 19 Mb scaffold using BLAT. The highest LD between ALGA0110477 and the polymorphisms mapped on the scaffold was with a SNP from the Porcine SNP60 BeadArray  located 24.5 kb away followed by a group of 3 SNPs from SYNGR2 (r2 = 0.42\u20130.48) including the missense polymorphism SYNGR2 p.Arg63Cys located 123.7 kb away. Using an additive linear mixed model and a subset of pigs with extreme high and low viral loads (n = 268) genotyped for all polymorphisms mapped to the scaffold (n = 629), we found that the SYNGR2 p.Arg63Cys SNP and a 1bp indel located 343 bp upstream of BIRC5 TSS were associated with the largest effects on PCV2 viral load (P < 0.0001). The phenotypic variance explained by each of these novel polymorphisms was substantially larger (21\u201323% +/- 6.1\u20136.4%) compared to the original QTL SNP ALGA0110477 (12.6 +/- 4.8%). As expected, these polymorphisms were associated with large effects on all weekly viremia measures (P < 0.0001), and on PCV2-specific antibodies, starting from 14 dpi for IgM and 21 dpi for IgG (p < 0.0001). The effects on growth during the challenge were most evident after 14 dpi as well as during the entire challenge period .RNA-seq analysis of alternate SYNGR2 p.Arg63Cys SNP  is located in the first loop of synaptogyrin-2 (SYNGR2) in a region conserved across mammals [Arg residue is prevalent in other species  sometimes being replaced by His , Lys  or Gln  while the Cys residue appears to be specific to swine  being lower compared to the heterozygote  and alternate homozygote . The favorable homozygous genotype was also associated with lower weekly viremia  was detected between SYNGR2 p.Arg63Cys and the SNPs associated with the largest effects from the QTL detected on SSC7 (ALGA0039682 and ALGA0039710). The effect of this SNP was confirmed in an independent validation data set consisting of 71 pigs infected with the same PCV2b strain and representing all three SYNGR2 p.Arg63Cys genotypes. This SNP had an effect on viremia starting from 14 dpi to 42 dpi (P < 0.05). The viremia of the homozygous genotype for SYNGR2 p.63Cys allele was lower than the alternate homozygote  and the heterozygote .The SYNGR2 p.63Cys allele (Q = 1.2%) compared to Engle et al. (2014) dataset (Q = 18.3%), which is less favorable for detecting associations in additive statistical models, and 2) lower ability of the ALGA0110477 to capture the low viremic effects of SYNGR2 p.63Cys. While in the Engle et al. (2014) dataset the presence of the ALGA0110477 C variant had a probability of 65% to be located on the same haplotype with SYNGR2 p.63Cys, in McKnite et al. (2014) this variant is found in similar proportions in haplotypes that carry different SYNGR2 alleles .Our inability to uncover the QTL located on SSC12 in our previous report  was baseBIRC5 (BIRC5 g.-343delA) was found to be in high LD (r2 = 0.83) with SYNGR2 p.63Cys allele and as expected was associated with low viral load (P < 0.0001). The deletion was predicted to affect a potential motif for NR5A2, a DNA-binding zinc finger transcription factor. However, no significant difference in expression was observed between BIRC5 genotypes across time points following in vivo PCV2 challenge (P < 0.17). At 14 dpi the homozygotes for the insertion exhibited an elevated nominal expression compared to the other genotypes, but the difference was not significant (P = 0.061).The 1bp deletion located 343 bp upstream of the TSS of SYNGR2 p.Arg63Cys and BIRC5 g.-343delA (r2 = 0.83) hampered the ability to distinguish their individual effects in the in vivo challenge dataset. In contrast, the LD between SYNGR2 p.Arg63Cys and other SYNGR2 SNPs was limited (r2 < 0.26), as well as the LD between BIRC5 g.-343delA and other BIRC5 polymorphisms (r2 < 0.16). A very defined LD block exists from ALGA0110477 to SYNGR2 that includes 16 DNA polymorphisms. Within this block, there were 9 haplotypes with individual frequencies greater than 1% that accounted for 85% of the haplotypes present. A single haplotype (Hap 1) carried the SYNGR2 p.63Cys allele. The frequency of this haplotype in our resource population was 0.28. The remaining eight haplotypes carried the SYNGR2 p.63Arg allele. A haplotype substitution effect demonstrates that Hap 1 was associated with the lowest viral load  and indigenous Chinese breeds , as well as wild pigs such as common Warthog (Phacochoerus africanus), Java (Sus verrucosus) and Visayan (Sus cebifrons) warty pigs. Phylogenetic analysis based on the polymorphisms from this haplotype block separated the breeds into paternal  and indigenous Asian breeds while the maternal breeds (Large White and Landrace) were located in between these two groups  was the closest to Pietrain and Duroc. The other haplotypes present in our resource population were similar to those identified in Berkshire (Hap 4), Hampshire (Hap 5), Meishan (Hap 2), or Jinhua (Hap 9). This finding suggests that SYNGR2 p.63Cys is the predominant allele in Duroc and Pietrain, breeds in which more emphasis is placed on growth related traits compared to breeds such as Large White and Yorkshire. Our analysis of SYNGR2 p.Arg63Cys in pure breeds showed that SYNGR2 p.63Cys has a frequency of 0.25 in Yorkshire, 0.53 in Landrace and 0.78 in Duroc.The haplotype that carried the SYNGR2 p.Arg63Cys substitution is in a conserved domain involved in vesicle formation [SYNGR2 affecting replication of a tick-borne human RNA virus [SYNGR2 may play a role in the internalization and early release of PCV2 from endosomes influencing its replication. The Porcine kidney 15 cell line (PK15) has an epithelial origin and is a well-established model system for PCV2 innate immunity and cellular pathogenesis [SYNGR2 p.63Arg and the insertion of BIRC5 g.-343delA variants associated with high-viremia. Expression of SYNGR2 did not differ across time points following PCV2 infection of PK15 cells, corroborating in vivo findings. In order to validate a role of SYNGR2 in PCV2 replication, we transfected PK15 with siRNA targeting the mRNA of SYNGR2. We evaluated two siRNA (siRNA-01 and siRNA-03) at two different concentrations (10 nM and 20 nM) and found that siRNA-01 was the most efficient to knock-down mRNA level of SYNGR2 compared to the cells subjected to a scramble siRNA control. A substantial reduction (>75%) in SYNGR2 mRNA level was observed starting 24 hours after transfection  when compared to scramble siRNA and non-transfected control cells, indicating a role of SYNGR2 in viral replication . This deletion is predicted to cause a shift in the reading frame and an altered protein (195 residues) beginning at amino acid residue 42 compared to the wildtype SYNGR2 sequence (224 residues). The deleted fragment included the conserved motif located in the first loop while the shift in the reading frame affected the C-terminus of SYNGR2. A significant reduction in viral titer starting at 24 hpi in cells (PK15 edited clones were generated by CRISPR-Cas9 Ribonucleoprotein (RNP) complex approach with a pair of guide RNAs (31_AC/40_AC) targeting the second exon of morphism . Sequencin cells  and 48 hin cells  was obseSYNGR2 was observed in E1 cells compared to wildtype PK15 cells with a significant difference observed at 24 hpi , a novel tick-borne bunyavirus in humans [SYNGR2 mRNA had been upregulated more than 200-fold at 36 hpi with SFTSV. In vitro silencing of SYNGR2 resulted in a decrease in viral replication and a reduction in the number and size of the inclusion bodies, further substantiating the role of SYNGR2 in facilitating SFTSV infection [Substantial variation in efficiency of viral replication and specific immune response was reported in our previous studies of experimental infections with PCV2 , 8. Hostocytosis , 18. Then humans . SYNGR2 nfection .SYNGR2 in PK15 cells was associated with a significant reduction in PCV2 titer, indicating a role of SYNGR2 in promoting viral replication. SYNGR2 p.Arg63Cys, the only missense polymorphism identified in SYNGR2, is characterized by a predicted change in charge and hydrophobicity of the first loop that connects two essential transmembrane domains, and is located in a region conserved across mammals. In rats, the first intraluminal loop and the C-terminus of SYNGR2 were found to be crucial for successful incorporation of the protein into vesicular membranes and vesicle formation [SYNGR2 p.63Arg allele  in Duroc than in Large White or Landrace . This selection pressure and the presence of mild PCV2 strains could have resulted in a rapid increase in the frequency of the SYNGR2 p.63Cys allele in both Pietrain and Duroc.Predominance of the SYNGR2 mRNA levels following in vitro or in vivo infection with PCV2. This may reflect important distinctions between SFTSV and PCV2. For instance, SFTSV is an RNA virus with the capacity to replicate within intracytoplasmic inclusion bodies, or viral factories. As Sun et al. (2016) demonstrated, SYNGR2 is a component of these vesicles and necessary for their formation [SYNGR2 p.Arg63Cys on overall protein folding or conformation of the first loop. A shift in the position of the second transmembrane helix as a result of the substitution in the loop region was predicted by HMMTOP software [In our research we did not observe an increase in ormation . As SFTSormation   partial deletion of a key domain by gene editing, provided evidence of the involvement of SYNGR2 in PCV2 infection. Since the SYNGR2 p.Arg63Cys polymorphism is the only missense mutation within the entire gene and located in this key domain, it is a plausible QTN (Quantitative Trait Nucleotide) candidate for PCV2 susceptibility. However, future studies of the mechanistic role of SYNGR2 and specifically of SYNGR2 p.Arg63Cys substitution will be required to provide additional experimental evidence of their role in PCV2 replication and pathogenesis.In this study, decreased viral titer by 1) exogenous reduction of The experimental design and procedures used during this research project were approved by the Institutional Animal Care and Use Committee of the University of Nebraska -Lincoln.Erysipelothrix rhusiopathiae, Clostridium perfringens, Leptospirosis and Colibacillosis and tested negative for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Prior to experimental infection, the pigs tested negative for presence of PCV2 in peripheral blood by real time quantitative PCR (qPCR) and had a sample/positive ratio (S/P) lower than 0.4 for IgM and 0.3 for passive IgG, the PCV2-specific antibodies [Experimental PCV2b challenge was conducted in nine batches that varied in size from 81 to 141 pigs, with a total of 974 pigs. The genetic makeup of this resource population consisted of crossbred pigs produced by 14 genetic lines generated by seven genetic programs. The dams of the experimental pigs had been vaccinated for PCV2 at 3 weeks of age with a single dose of Ingelvac CircoFLEX vaccine (Boehringer Ingelheim). The suppliers of the pigs also had vaccination programs for Porcine parvovirus, SYNGR2 p.Arg63Cys genotypes infected with the same PCV2b strain at 5 weeks of age was generated using the same experimental conditions. A group of 40 pigs (SYNGR2 p.63Arg/63Cys and 63Arg/63Arg) vaccinated for PCV2 at 3 weeks of age were used as controls. The vaccinated pigs were housed in the same room with the experimentally infected pigs, but in different pens.A validation dataset consisting of a group of 71 pigs representing all three 4.0 50% tissue culture infection dose (TCID50) intranasally and intramuscularly.The PCV2b strain (UNL2014001) used for the experimental infection was obtained from a pig with symptoms characteristic to Post-weaning Multisystemic Wasting Syndrome (PMWS), which is the most common PCVAD syndrome. The strain was sequenced (accession KP016747.1) using dye terminators and the sequence was compared to PCV2 strains available in GenBank . The strPCV2 specific antibodies, IgM and IgG, were profiled weekly from serum using ELISA (Ingenasa) as described in McKnite et al. (2014). Estimates of the number of PCV2b copies, or viremia, was performed using viral genomic DNA isolated by QIAamp DNA Minikit (Qiagen) and quantified by qPCR using TaqMan Master Mix and ABI 7900 Real Time PCR System (Thermo Scientific). The viral load for each pig during the entire challenge was represented as area under the curve (AUC) based on an algorithm that takes into account viral levels observed at each time point following infection  fitting a smooth curve over the 28 days and summing the areas in 0.01 time increments [Sscrofa 11.1 porcine reference genome assembly and used in GWAS via GenSel software package [The DNA was isolated from ear and tail tissue clips using DNeasy or Puregene blood and tissue kits (Qiagen). The experimental animals were genotyped using either the first or second generation of the Porcine SNP60 BeadArray (Illumina) that contain 62,183 and 61,565 SNPs, respectively. Only the common SNPs present in both BeadArray versions  were mapped on  package . DNA sam package .SOCS3, BIRC5 and SYNGR2 and their 2\u20134 kb region upstream of the transcription start sites (TSS) was performed using dye terminators and ABI PRISM 3100 Genetic Analyzer (Thermo Scientific) on high and low viremic samples. Discovery and validation of the polymorphisms detected by RNA-seq was based on alignment of DNA sequences using Sequencher software (Gene Codes). Potential impact of the polymorphisms located in the proximal promoter on important regulatory motifs was evaluated using FIMO (version 4.11.3) [Targeted DNA sequencing of candidate genes in the SSC12 QTL region including  4.11.3)  and the SOCS3, BIRC5, SYNGR2, THA1, TMC6 and TMC8 was performed by multiplex assays using Sequenom MassARRAY platform and Sequenom iPLEX chemistry based on the manufacturer protocols .Genotyping of polymorphisms located in the transcribed regions and proximal promoters of \u03c0 equal to 0.99 that assumed a prior probability of 1% of the SNPs having a non-zero effect. The Markov chain included 40,000 samples with the first 1,000 being removed as burn-in. Markov chain was set to use every 40th sample to estimate posterior distribution for the genetic variance explained by each 1 Mb window of the reference genome. This distribution was used to estimate the probabilities of each 1Mb window having a variance greater than 0 or greater than the average variance explained by each 1Mb window as described in McKnite et al. (2014).The proportion of phenotypic variance explained by host genetics for PCV2-viremia, PCV2-specific antibodies (IgM and IgG) and average daily gain (ADGi) during experimental infection was estimated based on Porcine SNP60 BeadArray genotypes using a BayesB model  and GenSBayes Interval Mapping (BayesIM) was implemented to derive haplotype effects across the genome on PCVAD-related traits as described in Kachman (2015) and Wilson-Wells and Kachman (2016) . BrieflyALGA0110477 to SYNGR2 that includes 16 DNA polymorphisms, was estimated as haplotype substitution effects. Contrasts between haplotypes were estimated using a linear mixed model as described above including one variable for each haplotype with values 0, 1, and 2 corresponding to the animal having 0, 1, or 2 copies of the haplotype in question. The haplotype substitution effects were presented as deviations from the mean of the haplotypes.Associations between the single marker genotypes and phenotypic variation were tested using a linear mixed model fitted by JMP 10.0 (SAS Inst. Inc.) that included marker genotype and batch as fixed effects, litter and pen as random effects while age at infection and IgG were used as covariates. Additive and dominance effects were estimated for each of the targeted DNA polymorphisms. A similar model was used to estimate the interaction between SNPs. The potential effect on viral load of the haplotypes in the defined LD block from ALGA0110477 sequence, a SNP previously unmapped on the Sscrofa 10.2 reference genome. A genomic scaffold (19 Mb) of the proximal end of SSC12 was constructed based on Pacific Biosciences sequencing reads [ALGA0110477 sequence and all SSC12 mapped and unmapped SNPs were determined on the genomic scaffold using BLAT. Annotation of the QTL region on the SSC12 scaffold was based on RNA-seq alignments and BLAST but also using ab initio approaches such as GenScan [Inverse PCR (iPCR), using four  and six cutter  restriction enzymes (New England Biolabs), T4 DNA ligase (New England Biolabs) and nested PCR using AmpliTaq Gold 360 DNA polymerase (Thermo Scientific), was employed to expand the genomic DNA sequence surrounding the short  GenScan , 27 in cTT = 6) and low (NCC = 5) viremic genotypes for ALGA0110477 at 0, 7 and 14 dpi were subjected to RNA sequencing. RNA was extracted from peripheral blood collected in Tempus tubes using the Tempus Spin RNA Isolation Reagent Kit (Thermo Scientific). RNA samples were sequenced using Ion Proton technology as described in the manufacturer protocol (Thermo Fisher Scientific Inc.). The adaptor-free sequencing reads were trimmed and filtered using Trim galore (version 0.4) [Sscrofa 11.1. The number of reads mapped to each gene in the annotated QTL region was obtained using HTSeq (version 0.6.1p1) [In order to profile transcriptome changes and sequence variation related to PCV2 infection, peripheral blood samples collected from the validation group of pigs that exhibited high (Nion 0.4)  with lowion 0.4) . The rea0.6.1p1) .SOCS3, BIRC5 and SYNGR2 across time points following PCV2 infection was quantified using TaqMan Master Mix and CFX384 Real Time PCR (BioRad). The qPCR assays were designed using IDT Realtime PCR Tool software (www.idt.com) and sequences generated based on RNA-seq alignments. RNA was extracted from peripheral blood samples collected in Tempus tubes from a subset of pigs representing all genotypes from the validation data set that displayed extreme viral load (high vs low) (n = 40) from 0 to 21 dpi using the Tempus Spin RNA Isolation Reagent Kit (Thermo Scientific). Complementary DNA (cDNA) was obtained using a mix of random hexamers and poly dT primers using First strand cDNA Synthesis Kit . Expression of ribosomal protein L32 (Rpl32) gene was used for normalization. Mean normalized expression (MNE) values were calculated based on cycle crossing thresholds (CT) obtained for the technical triplicates taking qPCR efficiencies into account [ALGA0110477, SYNGR2 p.Arg63Cys or BIRC5 g.-343delA genotypes and time point following infection were log10 transformed and compared by t-test.Expression of the candidate genes  account . MNE val2) with 5.0x105 cells per well and infected with UNL2014001 PCV2b strain (TCID50 = 104) when 80\u2013100% confluent at MOI = 0.00025. One hour following infection, cells were washed and fresh media was added (DMEM high glucose and 2% FBS). The cells were incubated at 37 \u00b0C with 5% CO2 for up to 5 days. Control cells were maintained the same way and mock-inoculated with plain DMEM high glucose media. Supernatants and cells were collected at specific time points and frozen at \u221280 \u00b0C. Viral DNA was extracted from supernatants using QIAamp DNA Mini kit (Qiagen). RNA, viral and host DNA was extracted from PK15 cells using AllPrep DNA/RNA Mini kit (Qiagen). TaqMan Master Mix and CFX384 Real Time PCR Detection System were used for quantification of PCV2 and expression profiling of BIRC5 and SYNGR2 from PK15 cells. Dideoxy sequencing of the cDNA and genomic DNA was used to profile the sequences and to genotype BIRC5 and SYNGR2 variants in PK15 cells.The porcine kidney cell line (PK15) was grown in DMEM high glucose media supplemented with 10% FBS and 1% Penicillin-Streptomycin . Cells were cultured in 12-well plates (4 cm2) with 2.5x105 cells per well when ~80% confluent with two siRNA oligos  targeting SYNGR2 mRNA and the AllStars Negative Control siRNA  at 10nM and 20nM concentrations. Transfection was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen) following the manufacturer\u2019s protocol. Cell samples were collected 24, 48, 72, and 96 hours post transfection in PBS and centrifuged at 16,000xg for 1 minute to pellet the cells. RNA was extracted using RNAeasy Mini kit (Qiagen). Real Time PCR was used to profile SYNGR2 expression. siRNA oligo 01 and the AllStars Negative Control siRNA (20nM) were used for subsequent transfections prior to infection. The siRNA transfected cells were inoculated 24 hr after transfection following the same infection protocol described above. Statistical differences in viral titer between cell lines across time points were tested using a linear model fitted by JMP 10.0 (SAS Inst. Inc.) that included batch and cell line as fixed effects. Pairwise comparisons between least-squares means of the viral titers were based on the Tukey test.PK15 cells were transfected 24 hours after plating in 12-well plates (4 cmSYNGR2 were designed and ordered (IDT), three located upstream (5\u2019) and three located downstream (3\u2019) of the SYNGR2 p.Arg63Cys polymorphism. Each guide RNA was hybridized with fluorescently labeled Alt-R CRISPR-Cas9 tracrRNA ATTO 550 (IDT) and Alt-R S.p. Hifi Cas9 Nuclease V3 (IDT) following the manufacturer\u2019s protocol to form Ribonucleoprotein (RNP) complexes. These RNP complexes were reverse transfected into PK15 cells using Lipofectamine RNAiMAX transfection reagent (Invitrogren) at a final concentration of 10nM. After 48 hours post transfection, genomic DNA was extracted using QIAamp Blood DNA Mini Kit (Qiagen) and amplified via PCR using LongAmp Taq DNA polymerase (NEB) with primers located in the introns flanking the second exon of SYNGR2 . The amplicons were subjected to T7 endonuclease I (NEB) digestion following the manufacturer\u2019s protocol and visualized by agarose gel electrophoresis to assess cutting efficiency of each individual guide RNA. The ability of guide RNA pairs (upstream/downstream) to generate partial deletions of the second exon was assessed following the same RNP transfection protocol with a final RNP concentration of 20nM (10nM/guide RNA). After 48 hours post transfection, genomic DNA was extracted, amplified, and visualized via agarose gel electrophoresis.Six potential guide RNAs targeting the second exon of SYNGR2 mRNA sequence  to amplify full-length transcripts, which were sequenced using Dideoxy sequencing. A single clone (E1) homozygous for a 106bp deletion was plated and infected with PCV2b inoculate as previously described. Wildtype PK15 cells were concurrently infected and served as a control.A single guide RNA pair (sg31_AC/sg40_AC) was selected to generate PK15 edited clones. After 24 hours post transfection, the cells were collected and sorted using Fluorescence Activated Cell Sorting (FACS) into 96 well plates to generate single cell clones. Genomic DNA from each single cell clone was extracted using QuickExtract DNA extraction solution (Lucigen) following the manufacturer\u2019s protocol and genotyped by PCR amplification and gel electrophoresis. RNA was extracted from selected clones using All Prep DNA/RNA Mini kit (Qiagen) and PCR was performed with primers located in the 5\u2019 and 3\u2019 UTRs of the SYNGR2 led to nonsense-mediated mRNA decay, expression of SYNGR2 was profiled by qPCR at 0, 24 and 48 hrs in edited and wildtype PK15 cells.To determine if induced changes in the mRNA sequence of S1 FigSscrofa 11.1 assembly. The y-axis represents the contribution of each SNP to the genetic variance. Alternate colors represent autosomes, from SSCs 1 to 18, chromosome X and Y followed by a set of SNPs without a genomic location.Each dot represents the proportion of genetic variance explained by an individual SNP. The x-axis represents the position of each SNP in the swine genome using (TIF)Click here for additional data file.S2 FigSscrofa 10.2 assembly including ALGA0110477 were excluded from the analysis. Each dot represents the model frequency associated with 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using Sscrofa 10.2 assembly. The Y-axis represents the model frequency of the association between a QTL and PCV2b viral load.Alternate colors represent autosomes, from SSC1 to 18. Unmapped SNPs in the previous (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and PCV2 viremia. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S5 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and IgM following PCV2 infection. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S6 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and IgG following PCV2 infection. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIFF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.S11 Fig(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "Single nucleus sequencing reveals evidence of inter-nucleus recombination in arbuscular mycorrhizal fungi. Published 5, December 2018Recently a reader contacted us about potential inconsistencies between the data in Supplementary File 6 and Figure 2. Figure 2 is directly built from data in Supplementary File 6. After checking the data and the scripts used to generate Figure 2, we found that an earlier version of Figure 2 was mistakenly uploaded.The earlier version Figure 2 was based on an analysis of the isolate SL1 that allowed the maximum number of Blast hits for regions surrounding SNPs to be of 1, instead of 2 as written in the method section of our paper. The results of this parameter change produced minor differences that do not alter the overall patterns of nuclear diversity originally observed. In all cases, the figure shows that isolates A4 and A5 harbour a structured nuclear genome organization, while in the isolate SL1 nuclear genetic diversity appears to be more diverse.As a result, Figures 2 (SL1 portion), Supplementary File 10 (SL1 portion), Supplementary File 11 (SL1 portion), Supplemental File 5 (SL1 portion) and Supplementary File 4 (SL1 portion) have been corrected.Corrected Figure 2:Original Figure 2:Corrected Supplementary file 10:Original Supplementary file 10:Corrected Supplementary file 11:Original Supplementary file 11:Supplementary file 4 and Supplementary file 5 have also been corrected.In Supplementary 4 we also identified a correction of number of SNPs in A4 after stringent filtering, from 2363 to 2362. The increase in SNP counts for SL1 with the updated parameter after stringent filter changes the reduction of SNP from 94.20% to 93.81%. The corresponding passage in the article has now been corrected:Original text: \u201cThis conservative approach has likely removed some true positives from our analysis - for example this filtering method resulted in an average 94.2%\u201dThe corrected text now reads as follows:\u201cThis conservative approach has likely removed some true positives from our analysis - for example this filtering method resulted in an average 93.8%\u201dOur conclusions are unaffected by these changes, and we apologize for this oversight.The article has been corrected accordingly."}
+{"text": "Atherosclerosis is a chronic inflammatory disorder of large- and medium-sized arteries and remains one of the most common causes of age-related death worldwide. It is well established that effector T cell responses promote both atherosclerotic plaque growth and instability . In contin vitro [FOXP3 exon 7, whereas, a reduced portion of FOXP3\u03942 is associated with an unstable plaque phenotype [FOXP3 appears to represent a layer of regulation that can reveal functional differences of Treg cell populations in different patient categories.Treg cells are a potential mediator of future intervention strategies for atherosclerosis. These cells depend on the lineage-defining transcription factor FOXP3 for their development and function. Human FOXP3 isoforms are generated through the exclusion of exon 2 and/or exon 7. In contrast, mice do not produce any isoforms of FOXP3. Full-length FOXP3 (FOXP3fl) and FOXP3 lacking exon 2 (FOXP3\u03942) isoforms confer a suppressive ability to Treg cells in vitro \u20136, whereFOXP3 exon 7 [The two dominant FOXP3 isoforms, FOXP3fl and FOXP3\u03942, are coexpressed in Treg cells and normally makes up 95% of the total amount of FOXP3 protein. The expression pattern does however vary. For instance, we have found that IL-1\u03b2 could promote splicing of 3 exon 7 . Another3 exon 7 . However3 exon 7 . As we hin vivo function of FOXP3 isoforms in humans.What will the future bring to FOXP3 isoforms and their involvement in chronic inflammatory disorders? First, we hope that the studies involving FOXP3 isoforms will be expanded upon as it remains essential to validate the current findings in other patient cohorts. This should go hand in hand with developing better tools for identification of specific FOXP3 isoforms. Another interesting line of research will be to better define the molecular mechanisms of action of distinct FOXP3 isoforms including their interactomes and their differential ability to control gene expression. Finally, we have together with collaborators, identified patients with atypical forms of the immunodysregulation polyendocrinopathy enteropathy X-linked\u00a0syndrome that lack expression of specific FOXP3 isoforms. We believe that these patients will give us a better understanding of the"}
+{"text": "Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion.Malaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion.A systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of fhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods.The search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the ppfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.Based on the review, there is evidence of the presence of  Plasmodium falciparum is the most prevalent malaria species in the WHO African region, accounting for 99.7% of estimated malaria cases in 2017 [The World Health Organization (WHO) estimated that there were 219 million cases of malaria and 435,000 malaria deaths and nearly half of the world\u2019s population was at risk of malaria infection in 2017 [P. falciparum, over 90% of RDTs used for the diagnosis of malaria in sub-Saharan Africa are HRP2-based [Plasmodium falciparum specific RDTs specifically recognize HRP2 antigen that encodes for the pfhrp2 gene and whose antibodies cross-react with histidine-rich protein 3 (pfhrp3) antibodies due to high degree of similarity in amino acid sequence [pfhrp2 and pfhrp3 genes. Plasmodium falciparum parasites lacking the pfhrp2/3 gene do not express HRP2 protein antigen threatening the usefulness of HRP2 RDTs in malaria diagnosis [P. falciparum parasites with pfhrp2 and pfhrp3 gene deletions were reported in the Amazon basin in 2010 by Gamboa et al. [pfhrp2/3 gene deletions outside the Amazon region in Africa and India [P. falciparum with missing pfhrp2/3 genes pose a public health threat as a large number of malaria infected patients will go undetected by the HRP2 RDTs and, therefore, remain untreated leading to increased risk of malaria morbidity and mortality, and continued malaria transmission [Efforts to reduce the burden of malaria in Africa have mostly included the use of long-lasting insecticide-treated nets (LLINs), indoor residual spraying (IRS) with insecticides, intermittent preventive therapy (IPT), diagnosis and treatment. Case management which involves testing and treatment with artemisinin-based combination therapy (ACT) is a major intervention for malaria control , 2. The P2-based , 2. Plassequence . Howeveriagnosis , 6. The a et al. . Howeversmission , 6.pfhrp2-deleted parasites meets or exceeds the lower 90% confidence interval for 5% prevalence, or a plan for change over a longer time frame if deletions are present but\u2009<\u20095% [pfhrp2 and pfhrp3 gene deletions [pfhrp2/3 gene deletion, countries, such as Eritrea have introduced non-HRP2 alternative RDTs that are able to detect gene-deleted parasites [P. falciparum parasite confirmation in Africa and the limited options available of WHO approved non-HRP malaria RDTs [pfhrp2 RDTs are based on quality data generated from well conducted studies using recommended methods to avoid unnecessary costly switch of RDTs [pfhrp2/3 gene deletion studies in Africa have varied. There have been variations in; (1) the size of the studies, (2) source of participants used , (3) clinical classifications of the participants including symptomatic versus asymptomatic individuals, and (4) investigation of pfhrp2 deletion alone versus pfhrp2 and pfhrp3 double deletions and flanking genes and (5) the laboratory methods.The WHO recommends a policy switch to more effective alternative non-HRP2 RDTs, when the prevalence of but\u2009<\u20095% . In Afrieletions \u201318. Due arasites . Howeverarasites . The thrria RDTs , 7. It i of RDTs . Howeverpfhrp2 and pfhrp3 gene deletion [pfhrp2 gene deletion and the methods and approaches being used for its estimation, confirmation and reporting in Africa.Due to this variability in study designs, methodologies and reporting, the WHO Global Malaria Programme published a standard protocol on the recommended approaches and methods required for investigation, confirmation and reporting of deletion . This repfhrp2 gene deletion in P. falciparum parasites in Africa since 2010 when the first deleted parasites were identified in clinical samples in the Amazon region, (2) assess the methodologies and approaches being used for pfhrp2/3 gene deletion estimation, confirmation and reporting in Africa.The review aimed to (1) assess the status of pfhrp2/3 gene deletion in Africa between January 2010 and June 2019. Literature search was done using PubMed and MEDLINE google Scholar for all articles published in English about pfhrp2/3 gene deletion in Africa. The following were used as search words; \u2018Malaria\u2019, \u2018Plasmodium falciparum\u2019, \u2018pfhrp2\u2019, \u2018pfhrp3\u2019 \u2018Gene deletion\u2019, \u2018Malaria Rapid diagnostic tests\u2019, \u2018Africa\u2019. All searches were restricted to paper titles and abstracts.A systematic search of literature was conducted electronically for published studies on pfhrp2/3 gene deletion in clinical samples in Peru until June 2019 [pfhrp2 deletion, (3) conducted in Africa, and (4) published during the selected review period. In order to expand on the scope, the papers referenced or cited in the selected papers were also reviewed for additional evidence. The WHO recommended protocol for investigation, confirmation and reporting of pfhrp2/3 gene deletion  fulfilled the criteria for inclusion and were retained for review (Fig.\u00a0The review considered published articles on P. falciparum pfhrp2 gene deletion based on the WHO recommended methods for confirmation and reporting of pfhrp2 and pfhrp3 gene deletions is shown in Table\u00a0pfhrp2/3 gene deletion across Africa is shown in Fig.\u00a0The summary of findings from the reviewed articles on pfhrp2/3 gene deletions in Africa were assessed and reviewed where studies were conducted and reported between 2010 and June 2019. There was wide variation in methods and approaches used across studies (Table\u00a0pfhrp2 gene deletions (Table\u00a0pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion alone with wide variation in laboratory methods.The status, methods and approaches that have been used for confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa where P. falciparum is the predominant parasite and where huge volumes of HRP2 based RDTs are used for malaria diagnosis [pfhrp2/3 gene deletion across malaria endemic countries in Africa range from the highest 62% in Eritrea to the lowest 1.45% and 0.4% in Mozambique and Angola, respectively [pfhrp2/3gene deletion in these studies could be due to selection pressure caused by exclusive use of HRP2 RDTs over time as suggested in previous studies [pfhrp2/3 negative parasites that remained undetected and continued to increase [pfhrp2/3-negative parasite populations leading to their increase in frequency [pfhrp2/3-negative parasites once they emerge [pfhrp2/3 deleted parasites reported by studies in Western Kenya and Southern Mozambique may be due to the absence of these ideal conditions for pfhrp2 selective pressure, such as are high transmission settings and use of malaria microscopy as the major diagnostic tool [pfhrp2/3 gene deletions above the 5% recommended WHO cut-off, Eritrea has introduced non-HRP2 RDTs to detect pfhrp2 and pfhrp3 deleted parasites [A total of 14 research articles satisfied our criteria for inclusion in the review Table\u00a0. These aiagnosis \u201318. Baseectively , 12, 20.ectively . Gene deectively . The obs studies . In Eritincrease . This serequency . Low maly emerge . The exttic tool . Due to arasites .pfhrp2 and pfhrp3 gene deletion across all studies. The major differences and limitations in methods and approaches across the studies are highlighted below.There was wide variation in the approaches and methods used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion [pfhrp2 and pfhrp3 deletion studies [pfhrp2/3 gene deletion studies is because parasite density is generally higher in the former compared to the low-density infections in the latter and hence provide better quality samples for confirmation of gene deletions by molecular tests [pfhrp2/3 gene deletion investigation including blood donors in Mali [pfhrp2 gene deletions as 6.4%, 4.8% and 1.45%, respectively [pfhrp2 gene deletions up to 62% and 23%, respectively [pfhrp2/3 gene deletion studies. The Mali study however gives contrary findings and reports a significant association between asymptomatic population and pfhrp2/3 gene deletion [pfhrp2/3 gene deletion studies across malaria endemic countries in Africa as the use of appropriate study participants is fundamental for the gene deletion study outcomes.All the reviewed studies but one used a cross-sectional design as recommended by the WHO protocol for confirmation and reporting deletion \u201318. The  studies . The reaar tests , 7. Howe in Mali . The DRCectively , 16, 21.ectively . The WHOdeletion . These vpfhrp2/3 deletion recommends the recruitment of a minimum of 370 or 318 symptomatic individuals with suspected P. falciparum infection to estimate a prevalence of 3.2% and 8.0%, respectively per sampling region or province [pfhrp2 gene deletion of\u2009>\u200980% at one of the study hospitals [P. falciparum DNA samples [P. falciparum population [pfhrp2/3 gene deletion across countries.The WHO protocol for investigation of ospitals . The Zam samples . Based opulation , 24. Nonpfhrp2 and pfhrp3 double deletion versus pfhrp2 single deletion alone: The WHO protocol for pfhrp2/3 studies recommends estimation and reporting of both pfhrp2 and pfhrp3 in P. falciparum gene deletion studies [P. falciparum-based RDTs are designed to specifically recognize HRP2 antigen, however pfhrp2 and pfhrp3 are homologous genes whose antigens may cross-react due to high degree of similarity in their amino acid sequence [pfhrp2 and pfhrp3 gene deletions warranting investigation of both genes. Evidence from the Eritrean study showed that every sample that was pfhrp2 deleted was also pfhrp3 deleted suggesting a possible association [pfhrp2 and pfhrp3 double deletion while others reported pfhrp2 deletion alone. Gene deletions studies in Ghana, Mali and Rwanda investigated and reported single pfhrp2 deletion alone [pfhrp2 and pfhrp3 double deletions [pfhrp2 gene deletion alone is a possible underestimation as some of the samples may test positive with HRP2 RDT even when parasites are pfhrp2 deleted due to cross-reactivity with HRP3 antibodies. These observations and variations in methods call for harmonization and standardization of investigative and reporting approaches for pfhrp2/3 gene deletions.Reporting of  studies , 7. Thissequence , 25, 26.ociation . Howeveron alone , 9, 17, eletions \u201316, 18. pfhrp2/3 gene deletion in P. falciparum parasites have been previously published [pfhrp2/3 deletion should be negative by HRP2 based RDT and positive with expert microscopy or Pf-pLDH RDT [P. falciparum mono-infection and exclude other non-P. falciparum species. Samples that are PCR confirmed as P. falciparum are amplified in the exon1 and exon 2 regions of the pfhrp2/3 gene to detect the presence or absence of the gene [pfhrp2 or pfhrp3 in the exon region are considered pfhrp2 and pfhrp3 deleted after ascertaining the quality of parasite DNA by amplification of MSP1 and MSP1 single copy genes [pfhrp2/3 gene deletion study as opposed to the use of dried blood spots as preferred samples [pfhrp2/3 gene deletion outcome is poorly understood. Four of the reviewed articles that reported pfhrp2 deletion in four countries missed an essential procedural requirement of demonstrating the quality of parasite DNA by PCR amplification of MSP1 and MSP2 single copy genes of P. falciparum [P. falciparum MSP1 and MSP1 single copy genes as an essential confirmation of P. falciparum DNA quality in suspected pfhrp2/3 deleted samples is a major methodological flaw that creates uncertainty on the validity and correctness of the reported deletion estimates [pfhrp2/3 in the subtelomeric region is optional and not essential requirement for confirmation and reporting of P. falciparum parasite gene deletion [pfhrp2 and pfhrp3 gene regions and can extend in the neighbouring flanking genes [The recommended laboratory-based testing methods required for confirmation of suspected ublished , 25\u201335. pLDH RDT , 33, 35.py genes , 6, 7. H samples . The efflciparum , 15, 17.lciparum . Detailelciparum . Failureng genes . Under tpfhrp2/3 surveys that aims to achieve representativeness of a country\u2019s malaria parasites population across all epidemiological settings [pfhrp2 and pfhrp3 gene deletion in parasites collected across the various states and provinces [P. falciparum parasites population [The WHO recommends the design of settings . The improvinces , 31. Howpulation , 16\u201318. P. falciparum infections as measured by microscopy, others used PCR confirmed or number of RDT-/microscopy\u2009+\u2009discordant samples that is a smaller denominator [pfhrp2/3 gene deletion estimates with possible overestimation or underestimation. The WHO standard protocol recommends the use of total P. falciparum infections measured by microscopy the suitable denominator to avoid overestimation of gene deletion estimates [P. falciparum species and false positives that are misclassified by poor quality microscopy. This potentially inflates the denominator leading to under estimation of deletions [Across all the reviewed studies, there were differences in the denominators used in the final computation of gene deletion estimates. While others used total ominator \u201318. The stimates . Howevereletions , 12, 13.pfhrp2/3 gene deletion across studies in malaria endemic countries in Africa. The direct implication of the use of non-Standardised and non-harmonized methods for confirmation and reporting of parasite gene deletion is the risk of unnecessary recommendations for a costly switch from HRP2 based RDTs to non-P. falciparum RDTs. Non-HRP2 RDTs are more expensive, less sensitive with poor field thermal stability [P. falciparum is predominant and where large volumes of HRP2 based RDTs are used for malaria diagnosis [P. falciparum RDTs in the context of gene deletions.Our review found a wide variation in methodologies and approaches for investigation of tability . Unnecesiagnosis , 2. HoweP. falciparum, the search identified only 13 published articles on pfhrp2/3 gene deletion in Africa. This observation could explain the limited data available on the occurrence and status of pfhrp2/3 gene deletion in malaria endemic countries in Africa. The WHO recommends initiation of surveys and surveillance systems to allow early detection and containment of pfhrp2/3 gene-deleted parasites in countries at risk of this threat [pfhrp2/3 gene deletion, the WHO recommends the need for periodic monitoring to assess if levels are increasing or exceeded the 5% prevalence cut-off required for change of diagnostic policies [Despite the high burden and dominance of s threat . Specifis threat . Howevers threat , 37. Evepfhrp2 and pfhrp3 gene-deleted parasites in all malaria epidemiological setting including low and high transmission zones [pfhrp2/3 gene-deleted parasites to spread and cause disease [pfhrp2/3 deleted parasites are drug sensitive compared to gene harbouring parasites and whether current treatment is effective for pfhrp2/3 deleted parasites.Previous studies have demonstrated the possible occurrence and survival of on zones , 15, 31. disease , 9, 11. pfhrp2/3 gene-deleted parasites in P. falciparum infected samples [pfhrp2/3 could still occur due to low quality parasite DNA particularly in low parasitaemia samples. Missed deletions could occur if an infection with a deleted parasite occurs subsequent to an infection with a wild type parasite, since circulating HRP2 can persist for up to a month [Studies have shown the failure of HRP2 based and the ability of non-HRP2 RDTs to detect  samples , 11, 12. samples , 38, 39. samples . However a month . In highpfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. The available evidence on the occurrence of pfhrp2/3 deletion comes from a limited number of countries leaving it largely unknown and unreported in many malaria endemic countries in Africa. Countries that are considering plans to confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.Based on the review, there is evidence of the presence of"}
+{"text": "HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1\u20133 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley.The cytokinin dehydrogenase gene  Triticum monococcum L.), emmer wheat (T. dicoccum (Schrank) Sch\u00fcbl.) and barley (Hordeum vulgare L.)\u2013occurs during the 8th millennium bc  250 matrix); periods indicate positions occupied by amino acids with weakly similar properties (<0.5 and >0 in the Gonnet PAM 250 matrix). The barley sequence is the HvCKX2.1 consensus sequence as determined in this study . The other sequences are taken from Genbank: maize, (TIFF)Click here for additional data file.S3 FigThe ellipses indicate the regions within which 95% of the data points for each haplotype are expected to fall. Black dots and ellipses, haplotype 1; cyan, haplotype 2; orange, haplotype 3.(TIFF)Click here for additional data file.S4 FigtSNE was run with perplexity = 30, iterations = 2000 and theta = 0.5. Black dots and ellipses, haplotype 1; cyan, haplotype 2; orange, haplotype 3.(TIFF)Click here for additional data file."}
+{"text": "Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5\u2019-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography. Metabolism and gene expression are stringently regulated physiological process that are concurrently modulated by a wide array of enzymes and regulatory protein. The concerted control mechanism exerted by the milieu of governing factors lead to dynamic regulation of the cellular machinery and contribute to incessant cell proliferation and tumorigenesis . Metabol\u22121 , 150 mM NaCl, 3 mM EDTA, and 0.05% NP-40 surfactant. The association kinetics for PKM2 was monitored for 400 s, followed by dissociation kinetics for 300 s. Different concentrations of Histone H3 (100 nM- 1000 nM) diluted in running buffer were injected across the sensor surface. SPR signals and resultant sensogram for PKM2-Histone H3 interactions were analyzed. Resultant sensogram was analyzed by subtracting change in the signal from activated/blocked control panel from that obtained as a consequence of binding of histone H3 to PKM2. At last the surface was regenerated with 75 mM NaOH. All the data were recorded at room temperature . Autolab SPR Kinetic Evaluation software was used for data analysis.2, 5% sucrose, 0.1mg/ml of BSA, and 1 mM DTT.500 nM of PKM2 was incubated with 100 nM of FAM-labeled nucleosome on ice overnight. Buffer composition was as 20mM HEPES buffer, pH 7.6, 50mM KCl, 5mM MgClVarying concentration of ExoIII enzyme was added to the 10ul of reaction mixture. After incubation at 23\u2009\u00b0C reaction was terminated with 2X stop buffer containing 20mM EDTA and 2% SDS and addition of ~20\u03bcg of glycogen. DNA was extracted using phenol\u2013chloroform\u2013isoamyl alcohol (25:24:1). The samples were subjected to denaturing electrophoresis in a 7% polyacrylamide\u20147 M urea gel in 1X TBE buffer. The gel was scanned by using Typhoon Phosphorimager FLA9000 .Nucleosome sliding experiment was performed using an electrophoretic mobility shift assay (EMSA)-based method as described previously \u201347 with GCG\u2303C has been inserted at dyad position 73 after each 208 bp and is usually obscured in nucleosome. Remodeling results in exposed restriction site and subsequent digestion of the DNA. To summarize 5nM of the pJ201 (34 \u00d7 601) chromatinized plasmid was incubated with 50 nM of Chd7 remodeler and 2.5mM of ATP with or without PKM2 at 23\u00b0C for 120 mins. Buffer used was 20mM HEPES, pH 7.6, 50mM KCl, 5 mM MgCl2, 1 mM EDTA, 1mM DTT, 0.1 mg/ml of BSA. Post incubation restriction digestion was performed using 1 U of HhaI enzyme for a time period of 30 min. The reaction was allowed for the defined time and stopped using Glycogen stop buffer . Samples were loaded on to 1.4% agarose gels. Electrophoresis was carried at 120V for 2hrs and gel was stained with ethidium bromide [To study the effect of PKM2 on nucleosome accessibility at chromatin level restriction digestion of pre-assembled nucleosome array of plasmid was carried out using HhaI enzyme. HhaI digestion site  bromide .kd value of very stable interactions is difficult and at the same time might be misleading under mild or physiological conditions. The complex decay in such cases is extremely slow and kd value should be only a qualitative measure and not quantitative. We can conclude from the aforementioned binding parameters that immobilized PKM2 interacts with Histone H3 and there is a gradual increase in the response unit with increase in Histone H3 concentration. A kinetic equilibrium is obtained represented by plateau in the association phase of the plot. Affinity Constant (KD) value for the binding kinetics is 6.39E-06M (Affinity error 3.20E-07) and calculated from the equilibrium equation. The obtained value is mean of the experiment repeated in triplicate. However the rate of complex decay is remarkably low as we do not observe any significant dissociation of the PKM2-Histone H3 complex suggesting that some other factor might be needed to break the association between the two biomolecules. This dissociation phase kinetics is interesting and needs to be further investigated. Fluorescence spectroscopy experiment using FITC labelled PKM2 and Histone H3 further validate the interaction between the two proteins. Interestingly we observe a decrease in the fluorescence intensity of labelled PKM2 when titrated with increasing concentration of Histone H3. With subsequent increase in the Histone H3 concentration in the PKM2 microenvironment the fluorescence is gradually quenched and a steady depreciation in the FITC fluorescence intensity is recorded which finally becomes stabilized and no further change in the intensity corresponding to higher concentrations of Histone H3 is observed 2 tetramer and two units H2A\u2013H2B dimers onto the Widom 601 nucleosome positioning sequence DNA is critical for the nucleosome assembly. Microscale reconstitution by employing Salt gradient dialysis with 1:1.5 DNA-to-octamer ratio as previously determined and subsequent titrations by varying the molar ratio PKM2 envince that nucleosome reconstitution can be possibly altered by PKM2 interaction. As can be inferred from native PAGE electrophoresis at a 1:2.4 PKM2-to-octamer ratio PKM2 interacts with the nucleosome and forms a complex of higher molecular weight , Lane 2 observed . The DisExonuclease III possess the phosphomonoesterase activity and catalyzes the 3' exonucleolytic digestion of the duplex DNA. The activity is hindered by the DNA-histone interaction and at regulated low levels of ExoIII, digestion of the nucleosome monomers yield a compact core of 145 bp which defines the boundary of the nucleosome. On extensive ExoIII digestion the nucleosome core is further chopped to yield discrete bands that are integer multiples of 10 bases signifying sequential disruption of the DNA\u2013histone contacts at every turn of the helix with the advance of ExoIII . We usedAs evident from the restriction digestion assay PKM2 interacts with nucleosome and reduces the accessibility to the complex. We were interested in exploring the possible outcomes of this finding and how it might contribute to altered gene expression. Chd7 is a member of the SNF2-protein superfamily and is reported to play a role in DDR (DNA Damage Repair). To cite as per the reports any aberrant expression or somatic mutations of Chd7 often in many cases may lead to cancer. Chd7 exhibits an intrinsic ATP-dependent nucleosome remodeling activity by sliding the histone octamer from one end of the DNA fragment to the centered position . This suSince DNA exist as chromatin in the nucleus and DNA-directed processes have majorly evolved with the chromatin  we furthTo dissect the impact of PKM2 on nucleosome positioning in a chromatin array and investigate if it could hinder Chd7 driven remodeling we used 34-mer nucleosome array for our study. The pJ201 (34 \u00d7 601) construct has restriction site for HhaI at the dyad position. The 34-mer reconstituted chromatin when digested by HhaI do not show any digested fingerprint , lane1 sHere, we report that PKM2 potentially acts as a modulator of chromatin remodeling activity by directly interacting with chromatin and thus contribute to oncogenesis. We highlight a rather unaddressed role of PKM2 that may be pivotal in exacerbating genomic integrity and trigger cell proliferation. The metabolic functions of PKM2 as key catalytic enzyme of glycolytic cycle and regulator of the energy flux in a cell is well established. However, the non-metabolic functions of PKM2 and its various aspects need to be discerned. The oligomeric switch as a consequence of oncogenic signal mediated post-translational modification subsequently facilitate nuclear translocation. Using fluorescence labelled NCP (nucleosome core particle) and 34-mer chromatin array we have demonstrated direct interaction of PKM2 with the nucleosome complex. We examined the possible interaction of PKM2 through nucleosome reconstitution. Gel mobility shift assay result of the reconstituted complex validate the potential binding of PKM2 to nucleosome. It can be inferred that PKM2 may have a role to play in nucleosome assembly and thereby dictate a likely control over gene expression. However the implications of this interaction is immense and needs to be further explored. Intuitively the nature of this interaction was further verified by establishing an evident association of PKM2 with Histone H3. Employing native PAGE gel electrophoresis we confirmed that PKM2 binds with Histone H3 and there is a possible interaction with Histone H4 (as seen in the native PAGE) which was although not as promising as Histone H3. The possibility of interaction with Histone H4 is a subject of study and further interpretation.Our finding was consistent as confirmed by SPR and fluorescence spectrometry experiments. This study was further extended to the validation of 34-mer chromatin array emphasizing the veracity of this interaction to the chromatin level. The results further prove the possibility of wider involvement of PKM2 in the chromatin structural organization and the downstream implication it may exert on gene regulation.Nucleosome restrict the accessibility of transcription factors and impede transcription and other chromatin transactions. This can be attributed to the 14 contact points between DNA and the histone octamer. However, protein complexes like chromatin remodelers can dynamically reposition and slide nucleosome to alter gene expression suggesting multi-layered regulation To dissect the impact of PKM2 on nucleosome accessibility we performed Exonuclease III digestion of PKM2-Nucleosome complex. Constrained digestion of nucleosome at low concentration of enzyme yield a nucleosome core with 145 bp wrapped around it which is further cleaved with the increase in enzyme units. However, in case of PKM2 bound nucleosome it is remarkable to observe from the lack of cleaved bands on the denaturing gel that digestion cannot proceed and the enzyme activity is significantly compromised. Results suggest that PKM2 probably impose a barrier to the remodeling enzymes, insulate nucleosome and obstruct access.As previously noted, in presence of nucleosome, transcription machinery face a roadblock and the highly coordinated process of gene regulation is impaired. Several chromatin remodelers relieve this transcriptional blockade by facilitating nucleosome sliding. CHD family of remodelers harness the energy generated from ATP hydrolysis to alter the chromatin structure. The interplay between the repair machinery and the remodeler is crucial to gain an access to DNA double-strand breaks (DSB) and prevent genomic instability . Chd7 isNucleosome mapping reactions with HhaI using 34-mer pre-assembled nucleosome array imply that PKM2 impair remodeling in chromatin without altering the structure. In contrast to chromatin digestion in the presence of the \u0394Chd7 which was on account of the exposed HhaI site at the dyad position we fail to observe any digestion in presence of PKM2. This was evidently on account of inhibition of nucleosome sliding.To summarize, we have shown that PKM2 can directly interact with the nucleosome through Histone H3 and obscure the chromatin from undergoing epigenetic modifications catalyzed by remodeling complexes. The basis of this prediction was evident from the suspended remodeling activity of Chd7 in the presence of PKM2 which can possibly lead to dysfunctional DDR machinery and genomic instability . As per In a cancer cell metabolic overhaul often commensurate with dysfunctional metabolic enzymes that synergistically impact the epigenetic blueprint and promote altered gene expression. In this research article we report a so far unaddressed role of PKM2 as a modulator of chromatin remodeling activity. Our study show that PKM2 can directly interact with nucleosome through Histone H3 and inhibit Chd7 mediated remodeling. At present non-metabolic functions of PKM2 is an intensely researched domain and its role in cancer progression and tumorigenesis is widely being acknowledged. Besides acting as a key nuclear kinase it is known to regulate the expression of several oncogenes. Here, we attempt to answer how PKM2 can directly alter the expression of genes by hindering nucleosome positioning and inhibiting remodeling. Exonuclease III digestion of Pyruvate Kinase M2- NCP complex suggest that PKM2 obscure nucleosome and create a roadblock for the Chd7 remodeler and thereby impede nucleosome sliding. Chd7 is implicated in a number of developmental disorder and is reported to play a role in cancer as well. As an important factor for DNA Damage Repair machinery any impairment in its functions can have detrimental implications. It can be inferred that PKM2 by negatively modulating Chd7 catalyzed chromatin remodeling may probably exacerbate genomic instability and in turn contribute to cancer. Through this study we aim to highlight the impact of PKM2 on chromatin architecture and its impact on the epigenetic landscape of the cell.S1 Fig(A) Interaction of Chd7 with PKM2 with increasing molar concentration of PKM2. (B) Interaction of PKM2 with Chd7 with increasing molar concentration of Chd7.(PDF)Click here for additional data file."}
+{"text": "GST genes have been reported to be associated with increased susceptibility to cancer development and anticancer drug resistance. In this study, we investigated the association between genetic variants in GSTM1 and GSTP1 and patients\u2019 clinicopathological parameters. The prognostic values of such associations were evaluated among bladder cancer patients.The glutathione S-transferases (GSTs) are a superfamily of phase II detoxifying enzymes that inactivates a wide variety of potential carcinogens through glutathione conjugation. Polymorphic changes in the GSTM1 and GSTP1 in bladder cancer patients was assessed using polymerase chain reaction followed by DNA sequencing. Overall survival was estimated using the Kaplan-Meier method and multiple logistic regression and correlation analysis were performed.Genotyping of GSTM1 null genotype was significantly associated with poor overall survival compared with the wild-type GSTM1 genotype. There was a trend towards better overall survival in patients with wild-type GSTP1 allele (AA) compared with GSTP1 (AG/GG) genotype. Interestingly, Kaplan-meier survival curve for GSTM1 null patients adjusted for sub-cohort with amplified HER2 gene showed poor survival compared with the GSTM1 null/ non-amplified HER2 gene. Also the same population when adjusted with HER2 protein expression, data showed poor survival for patients harboring GSTM1 null/high HER2 protein expression compared with low protein expression.The GSTM1 null genotype on bladder cancer patients\u2019 outcome. Further investigations are required to delineate the underlying mechanisms of combined GSTM\u2212/\u2212 and HER2 status in bladder cancer.This study focuses on the impact of  Bladder cancer is the 9th most common cancer and a leading cause of cancer-related death worldwide. It has been estimated that around 550,000 new bladder cancer cases and 199,922 deaths occurred in the year 2018 worldwide and these numbers are expected to double in the upcoming years . TherefoIt is well documented that occupational exposure to chemical carcinogens including aromatic amines and polycyclic aromatic hydrocarbons is associated with the risk of bladder cancer development , 4. KellProtecting against carcinogen-induced and chemotherapy-induced oxidative stress involves a series of event characterized by the activation of phase-II cellular detoxifying enzymes; Glutathione S-transferases (GSTs) or N-acetyltransferases (NATs) . GSTs enGSTM1 gene is located on chromosome 1p13.3 and the most common polymorphic variant of GSTM1 gene is the homozygous deletion (GSTM1 null genotype) characterized by abolished enzyme activity [GSTM1 and the risk of cancer, but the association remains controversial among different populations. Previous epidemiological studies showed an association between the homozygous deletion of GSTM1 and increased risk of lung, colorectal and head and neck cancers [GSTM1 null and the risk of several types of cancers [activity . Many st cancers \u201317. Howe cancers \u201321.GSTP1 polymorphism at codon 105 is an A to G substitution resulting in an amino acid switch from isoleucine to valine  and lowering the catalytic activity of GSTP1enzyme [GSTP1 Val105 genotype was shown to be associated with a variety of tumors, such as ovarian, breast, colon, lymphoma, and pancreas [GSTP1 variants modulate the risk of urinary bladder cancer has also been investigated [GSTP1 gene polymorphisms and the risk of bladder cancer: while a number of studies identified an obvious association between GSTP1 polymorphisms Ile105Val and bladder carcinoma risk [GSTP1 Ile105Val polymorphism and bladder cancer [GSTP1 is encoded by a single gene located on chromosome 11 . The comP1enzyme . The decpancreas . The hypstigated , 25. Howoma risk \u201328, other cancer , 30.ERBB2 amplification and signalling [HER2 may be involved in cancer susceptibility and clinical management of cancer patients. In the present study, we aim to investigate the prognostic value of GSTM1 and GSTP1 genetic polymorphisms in patients with bladder cancer and evaluate their association with patients\u2019 clinicopathological parameters. We also attempted to evaluate the clinical significance of HER2 status in cases confirmed to have GSTM1/ GSTP1 variants with bladder cancer prognosis.HER2 is a trans-membrane glycoprotein receptor tyrosine kinase of the epidermal growth factor receptor family EGFR/ErbB. It plays an important role in the development and progression of many tumor types including breast, gastric and bladder cancers . Recent gnalling . HER2 isgnalling , 33, 34.Formalin-fixed paraffin-embedded (FFPE) tissue samples were obtained from histologically confirmed bladder cancer patients who underwent bladder resection between 2005 and 2012 at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia. The study group consists of 93 patients; only specimens containing more than 80% cellular composition were used in the analysis. All patients have not been subjected to any chemotherapy or radiotherapy prior to sample collection. Clinical and pathological data including age, gender, tumor grade, tumor stage, lymph node, vascular invasion, metastasis, and survival were gathered from patients\u2019 medical records and summarized in Table Genomic DNA was extracted from FFPE tissue samples using QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer\u2019s instructions. Purified DNA was eluted in 50\u2009\u03bcl elution buffer and stored at \u2212\u200980\u2009\u00b0C until use. Purity and concentration of eluted DNA was analyzed using a spectrophotometer system .GSTM1 (present/null) and GSTP1 Ile105Val polymorphisms was performed as described previously [GSTM1 and GSTP1 oligonucleotide primers were purchased from MWG-Biotech  to amplify the GSTM1 fragments, , GSTP1  PCR was performed on a Thermal Cycler 480 apparatus . Thermo cycler parameters included: an initial denaturation at 94\u2009\u00b0C/ 15\u2009min; followed by 35\u2009cycles of denaturation at 94\u2009\u00b0C/ 1\u2009min, annealing at 57\u2009\u00b0C /1\u2009min, and extension at 74\u2009\u00b0C/ 1\u2009min; and a final extension at 72\u2009\u00b0C/10\u2009min. Confirmation of PCR products were examined by 2% agarose gel electrophoresis and visualized using a Syngene UV transilluminator.Genotyping for the detection of eviously . GenotypGSTP1 PCR products, sequencing kit  was used according to the manufacturer\u2019s instructions using Genetic analyzer 3500 . The resulting sequence data was analyzed using Applied Biosystems sequence analysis software (v 5.4). GSTP1 genotypes were determined as wild type Ile/Ile (AA), heterozygous type Ile/Val (AG) or homozygous variant type Val/Val (GG) as shown in Fig. GSTM1, the PCR products were separated on a 2% agarose gel and determined as null/ present genotypes.To sequence the amplified HER2 immunostaining was undertaken earlier . The expGST polymorphisms with aggressiveness of bladder cancer. Odds Ratios (OR) and their 95% Confidence Intervals (95% CI) were used to calculate the results. The wild type of all genotypes was used as the reference group. Interactions between GSTM1 and GSTP1 polymorphisms and aggressiveness bladder cancer phenotypes were analyzed using Spearman correlation analysis. In all tests, the values p\u2009\u2264\u20090.05 were considered as statistically significant.Statistical data analysis was performed using SPSS . Appropriate, Chi-square test and Fisher\u2019s exact test were used to establish any significant differences in polymorphism incidences between bladder cancer cases. Multivariate Cox regression model were used to evaluate the prognostic significance of GSTs genes, HER2 and other clinicopathological factors. Cumulative survival probabilities were estimated using the Kaplan-Meier method, with log-rank comparison test. Multiple logistic regression analysis was performed to assess the association between In the current study, 93 patients with urinary bladder carcinoma were genotyped for two polymorphisms in two important genes of the glutathione-s-transferase family involved in xenobiotic metabolism. The distribution of the clinicopathological characteristics of the bladder cancer patients is presented in Table GSTM1 and GSTP1 to the susceptibility of bladder cancer . Our data revealed that a total of 44 bladder cancer patients out of 93 (47.31%) had a GSTM1-deleted genotype (\u2212/\u2212). GSTM1 specific bands showing on agarose gel electrophoresis was seen in 45 out of 93 patients (48.38%). No further investigations were carried out to discriminate between heterozygous deletion (+/\u2212) and wild-type (+/+) GSTM1 variants hence both heterozygous deletion and wild-type variants are considered GSTM1 present , whereas the lower percentage was with GSTM1 null and the GSTP1 wild allele 14 (15.05%) shown in Fig. GSTs different groups.A higher frequency within our cohort was found between those carrying GSTM1 null genotype was associated with poor overall survival in comparison to GSTM1 present genotype, log rank p\u2009=\u20090.038 . GSTP1 AG carriers had the worst overall survival compared to GSTP1 AA or GG genotypes carriers . When merging GSTM1 survival and GSTP1 polymorphisms .Kaplan-Meier curve showed that HER2) gene amplification, after breast and gastric cancers, and also demonstrates frequent overexpression of HER2 protein [GSTM1 and GSTP1 polymorphisms in respect to HER2 status of the same cohort. HER2 protein expression and gene amplification data [GST genotypes and HER2 status, bright field double in situ hybridization (BDISH) and immunohistochemistry (IHC) data were used to analyze HER2 gene amplification and protein expression within the GSTP1/ GSTM1 analyzed cohort. Our data indicated no association between HER2 protein level and both GSTP1 (p\u2009=\u20090.07) and GSTM1 (p\u2009=\u20090.75) polymorphic status . Such a relationship was not established for amplified HER2 gene and GSTM1 null/present variants  showed a significant impact on patients\u2019 overall survival. Figure GSTM1 null and amplified HER2 gene , though this was not the case with non-amplified HER2 patients  compared to GSTM1 null/ low HER2 protein expression counterpart  are members of a large gene family of cytosolic phase II xenobiotic metabolizing enzymes involved in catalyzing and detoxifying a variety of carcinogens including reactive electrophilic compounds [GST of carcinogen-detoxifying gene family as well as in NAT2 confer increased risk of bladder cancer [increased expression of GST family members, especially GSTP1 and GSTM1, was reported in several human solid tumors and is believed to confer resistance to various platinum-base chemotherapy drugs and metformin through regulation of many genes and molecular pathways [Globally, bladder cancer is a leading cause of mortality , 38. It ompounds . Membersompounds , 40. It r cancer . Moreovepathways , 42. MecGSTP1 and GSTM1 variants in a cohort of 93 bladder cancer patient from Saudi Arabia. We also evaluated the association between GSTP1 and GSTM1 gene polymorphisms with a set of clinical and pathological parameters as well as the prognostic value of both genes polymorphisms in bladder cancer patients.In our investigation we examined the frequency of GSTM1 and GSTP1 gene variants was represented in Table GSTM1 present and null is equally distributed in our cohort 48.38 and 47.31% respectively. This data is in agreement with previous report on the frequency of the GSTM1 null genotype in the Caucasian population [GSTM1 null genotype was 59.1% in patients with muscle invasive bladder cancer (MIBC) [GSTM1 null genotype varies significantly among populations from different ethnic groups [GSTP1 gene polymorphism when we considered patients holding at least one copy of the dominant allele, data indicated that the frequency of AA and AG genotypes were found to be significantly high in our study group with a combined ratio of 77.4% for both genotypes compared to the GG genotype (6.45%). The reported frequency of GSTP1 AA/AG genotypes is around 67% of the Iranian patients [GSTP1 AA/AG variants was observed in in the Caucasian population with bladder cancer [The frequency and distribution of pulation . In an ir (MIBC) . Nonethec groups . As for patients  and Indipatients . HoweverGSTP1 and GSTM1 genes and patients\u2019 outcome. Our results indicated a significant association between the null GSTM1 genotype and poor overall survival among bladder cancer patients. The association between GSTs and poor survival was previously highlighted in many cancer types including bladder cancer [GSTP1 genotypes, our data show trend for better survival for patients with the wild allele homozygote AA in comparison to heterozygote AG and variant allele homozygote GG genotypes or to GG/AG combined though data are not significant. When GSTP1 GG/AG and GSTM1 null genotypes were present together, poor overall survival increased in comparison to GSTP1 alone.We next sought to evaluate the association between polymorphism of the r cancer \u201350. As fGSTs leads to reduced detoxification potential which may result in increased DNA adduct levels in the target tissues and eventual mutations in the driver genes leading carcinogenesis. Therefore, the association of GSTP1/ GSTM1 variants with highly malignant disease and poor prognosis in cancer patients was suggested [The accumulating data suggested that genetic polymorphism of uggested .GSTM1 were at high risk of developing bladder cancer [GSTM1 null and other cancers such as breast [GSTM1 null distribution in bladder cancer patients than in healthy individuals [GSTM1 in our cohort did not show any significant difference in comparison to the wild-type allele which may indicate that the null genotype is not the only factor in determining the increased risk and aggressiveness of bladder cancer but is certainly one of many combined genetic factors that contribute to the pathogenesis of the disease. To-Figueras et al. suggested a relation between GSTM1 null genotype and p53 mutation in increasing the risk of lung cancer susceptibility among smokers [GSTP1 Ile105Val polymorphism were characterized by frequent mutations in the tumor suppressor gene p53 and high-grade/ high stage tumors in bladder cancer [p53 mutation (data not published) which may suggest the involvement of p53 mutation in association with GSTP1 in the risk of bladder cancer development and drug resistance. This suggestion is valid knowing that GSTP1 gene contains a functional canonical p53 binding motif and the capacity of p53 to transcriptionally activate the human GSTP1 gene [Previous studies on patients from different ethnic origins revealed that individuals with the null r cancer , 51\u201354. s breast , lung [5s breast  and colos breast . Anwar e smokers . In an er cancer . In an iTP1 gene .GSTP1/GSTM1 variants and Human Epidermal growth factor Receptor 2 (HER2) gene/ protein status in bladder cancer patients. Our data indicated that patients with high HER2 protein expression/ gene amplification and null GSTM1 genotype had significant poor survival compared to patients with low HER2 expression and null GSTM1 genotype, suggesting that combining HER2 status with GSTM1 genotype may have a prognostic value for bladder cancer patients. The exact mechanism of the influence of GSTM1 and HER2 on bladder cancer is yet to be elucidated. Together, our data showed that GSTM1 gene deletion either alone or in combination with HER2 may serve as markers for bladder cancer prognosis.In the same context and for the first time we investigated the relationship between different GSTP1 Ile105Val genotype, GSTM1 genotype alone or in combination with HER2 status and patients\u2019 clinicopathological features. This is consistent with previous published reports [GSTP1 Val/Val genotype and GSTM1/GSTT1 double null genotypes.We observed no association between the  reports , 58, and reports  who founGSTM1 null genotype is significantly associated with poor overall survival in urinary bladder cancer patients. Furthermore, combined GSTM1 deletion and amplified HER2 gene might be considered as the worse prognostic genotype combination in bladder cancer. To the best of our knowledge, this is the first study to investigate the association between GSTs genes polymorphisms and HER2 status in Saudi bladder cancer patients. One of the limitations of the current investigation is scarcity of the sample size and clinical data used for correlation analysis. Therefore, further analyses using larger sample size are needed to investigate the functional significance of combined GSTM1 deletion and HER2 on bladder cancer prognosis. Furthermore, larger epidemiological studies are needed to assess the relationship between these genes and response to therapies (chemotherapy and anti-HER2 therapy) which may support their use as potential predictive biomarkers for bladder cancer treatment.The present study revealed that Additional file 1: Figure S1. Histograms showed the frequency of expression patterns of HER2 protein receptors in 93 of bladder cancer by IHC.Additional file 2: Figure S2. Kaplan-Meier survival curves demonstrating the overall survival of GSTP1 adjusted with HER2 status. (A)GSTP1 genotypes with HER2 gene amplification. (B)GSTP1 genotypes with HER2 gene Non-amplification. (C)GSTP1 genotypes with HER2 Protein expression. (D)GSTP1 genotypes with No HER2 Protein expression."}
+{"text": "Salmonella enterica serotype I 4,[5],12:i:- has been increasingly isolated from swine. However, its pathogenic potential is not well characterized. Analysis of swine cases confirmed a strong positive association between isolation of I 4,[5],12:i:- and lesions of enteric salmonellosis and suggested a similar pathogenic potential as that for Salmonella Typhimurium. Salmonella enterica serotype I 4,[5],12:i:- has emerged as a major public health threat in Europe  is a National Animal Health Laboratory Network accredited laboratory that receives >75,000 case submissions annually, of which most are derived from swine production systems located throughout the United States. Histopathologic analysis is performed by veterinary diagnostic pathologists on \u224810,000 cases from swine per year. The ISU-VDL laboratory information management system provided the initial dataset for this analysis.Salmonella I 4,[5],12:i:- from samples submitted from pigs was associated with microscopic lesions consistent with enteric salmonellosis, we compared cases from which Salmonella I 4,[5],12:i:- was isolated with cases from which neither Salmonella I 4,[5],12:i:- nor Salmonella Typhimurium were isolated; these samples were collected during July 2016\u2013December 2017. We also reviewed cases from which Salmonella Typhimurium or 1 of 3 Salmonella serogroup B serovars  were isolated to determine the potential comparative pathogenicity of Salmonella I 4,[5],12:i:-. All of these cases met the following qualifying criteria: animals were 3\u201313 weeks of age, a Salmonella serovar as outlined above was isolated by direct culture performed on enteric tissues, and histopathologic analysis was performed on the large intestine. To determine the association between the presence of Salmonella I 4,[5],12:i:- and lesions consistent with enteric salmonellosis, we also reviewed 40 additional cases that met the previously stated inclusion criteria but from which neither Salmonella I 4,[5],12:i:- nor Salmonella Typhimurium were isolated; we randomly selected these cases from an Excel  data file by using the RAND function.To determine whether isolation of Salmonella were routinely processed and reported (https://www.sas.com) to perform all analyses. We used the Pearson \u03c72 test and odds ratios to determine the association between isolation of Salmonella I 4,[5],12:i:- and pathologic diagnosis of enteric salmonellosis. We considered a p value <0.05 significant.Enteric samples submitted for isolation of reported . MicroscSalmonella I 4,[5],12:i:- from 138 porcine cases that met all of the qualifying criteria. We isolated Salmonella Typhimurium from 18 cases, and other potentially lesser pathogenic Salmonella serogroup B serovars, including Salmonella Derby, Agona, and Heidelberg, from 35 cases.We isolated Salmonella I 4,[5],12:i:-   of 40 cases  . We conf<0.0001)  for 100 isolated  Table 1.40 cases  Table 2.Salmonella was isolated from 6 of the 40 cases, 3 of which had lesions consistent with enteric salmonellosis. We confirmed compatible histologic lesions of salmonellosis in 17 (94%) of 18 cases in which Salmonella Typhimurium was isolated and 11 (31%) of 35 cases in which another serogroup B Salmonella was isolated. Other agents of enteric disease that were concurrently detected in some cases included rotaviruses, coronaviruses, coccidians, and hemolytic Escherichia coli. However, none of these agents caused lesions consistent with those used to define salmonellosis in this report  was isolated than for cases from which Salmonella Typhimurium (94%) was isolated. Based on these data, we believe that Salmonella I 4,[5],12:i:- is a likely cause of enteric salmonellosis that has a similar or perhaps slightly lower pathogenic potential in swine than Salmonella Typhimurium. Pathogenicity studies are needed to further characterize the pathogenic potential and fitness of Salmonella I 4,[5],12:i:- compared with that of Salmonella Typhimurium in swine.Although increased isolation of Salmonella I 4,[5],12:i:- has evolutionary advantages that have resulted in its predominance as one of the most common Salmonella serotypes responsible for swine enteric salmonellosis. Accordingly, it is essential to determine the putative attributes that facilitate its rapid spread and ecologic success. Specifically, antimicrobial drug resistance genes and genes that encode resistance to heavy metal micronutrients should be evaluated, given their current and common use in US swine production.We suspect that Salmonella enterica I 4,[5],12:i:- associated with lesions typical of swine enteric salmonellosis.Additional information on"}
+{"text": "Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or \u03b2-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and \u03b2-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and \u03b2-catenin, however unlike Wnt8, in a GSK3\u03b2 independent manner. These findings suggest that Wnt8 and Dkk2 converge on \u03b2-catenin using distinct transduction pathways both independently required to activate Wnt/\u03b2-catenin signaling and induce neural crest cells.Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/\u03b2-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/\u03b2-catenin signaling. Dkk2 depletion in  The neural crest is a population of cells unique to the vertebrate embryo. They are induced at the neural plate border during gastrulation, and around the time of neural tube closure, leave the neuroepithelium to produce a diverse array of cell types, contributing to multiple tissues, including the heart, the peripheral nervous system, and the craniofacial skeleton.Neural crest cells are generated through a sequence of events orchestrated by the modulation of several signaling pathways and the activation of a complex network of transcription factors . A largeXenopus embryos and promote head formation (Xenopus embryos (The Dickkopf (Dkk) family of secreted glycoproteins acts primarily as negative modulators of Wnt signaling, this is especially true for Dkk1 and Dkk4 . Dkks inormation . Dkk1 in embryos . Similar embryos . During  embryos . The rol embryos , howeverHere we show that Dkk2 knockdown prevents neural crest formation in vivo and in neuralized animal cap explants injected with Wnt8. Furthermore, Dkk2 gain-of-function increases the neural crest progenitor pool, reminiscent of Wnt8, Lrp6 and \u03b2-catenin gain-of-function phenotypes. We demonstrate that Dkk2 mediates its neural crest-inducing activity by activation Wnt/\u03b2-catenin signaling in a GSK3\u03b2 independent manner. We propose that during neural crest formation, Lrp6 mediates two independent signaling events triggered by Wnt8 and Dkk2 converging on \u03b2-catenin to specify the neural crest.dkk2 mRNA . A Dkk2MO was designed to specifically interfere with translation of kk2 mRNA . The spees of MO . Unilate embryos . Concomited side . To confjunction , resultijunction . The phexpansion . snai2 ehenotype . Later iype  was significantly repressed in Dkk2-depleted (Dkk2SMO-injected) animal cap explants, while co-injection of a control MO (CoMO) had no effect on snai2 induction  by BMP inhibition or mesoderm (bra) by fibroblast growth factor 8b (FGF8b) in these explants , consistent with a potential role in neural crest induction. This expression increases over time to reach a maximum around stage 20 and then progressively declines  results in axis duplication, while Wnt activation post-MBT expands neural crest progenitors . For thefficient . This redkk2 mRNA (500 pg) or plasmid DNA (25 pg) in one cell at the 2 cell stage. Dkk2 overexpression in both cases resulted in a lateral expansion of snai2 expression domain in the vast majority of the embryos . This phenotype is very reminiscent of Wnt8, Lrp6 and \u03b2-catenin gain-of-function phenotypes , Dr. Tatiana Piotrowski  and Dr. Sergei Sokol , respectively. A flagged version of Xenopus Dkk2 was generated by adding a Flag tag (DYKDDDDK) at the C-terminal using the following primers F: GAATTCGCCACCGAGATGAACATGGTTTTGCTGGGGA; R: ctcgagTTACTTGTCGTCGTCGTCCTTGTAGTCTATTTTCTGGCAAATG. The PCR product was cloned into pCS2+ (pCS2+\u00a0Dkk2 Flag). Control (CoMO), Dkk2 , Lrp6 , 200 pg (wnt8) or 50 pg  was injected per embryo. MOs, mRNAs and plasmid DNA were injected in one blastomere at the 2 cell stage (NF stage 2) and embryos were analyzed by in situ hybridization at the neurula stage (NF stage 14\u201317). To identify the injected side, 500 pg of \u03b2-galactosidase mRNA was coinjected as a lineage tracer. Only embryos with co-localized expression of the lineage tracer with the cell type marker were considered for analysis. Control and injected embryos were treated in the dark with 10 \u03bcM of GSK3 inhibitor  at the gastrula stage (NF stage 11/11.5), and collected at NF stage 14\u201317. For the axis duplication assay, embryos were injected in the equatorial region in both ventral blastomeres at 4 cell stage (NF stage 3) and analyzed at NF stage 32. For animal cap explant experiments, both blastomeres at the 2 cell stage (NF stage 2) were injected in the animal pole region. Explants were dissected at the blastula stage (NF stage 9) and cultured for 8 hr in NAM 0.5X. In rescue experiments, the injections of mRNAs/plasmid DNA and MOs were performed sequentially. For whole embryo injections and animal cap explant assays each experiment was performed on at least three independent batches of embryos.8 25 pg; , noggin n 10 pg; , dkk2  to visualize the lineage tracer  on the injected side and processed for in situ hybridization. Antisense digoxygenin-labeled probes  were synthesized using template cDNA encoding snai1 , snai2 , containing HaltTM Protease Inhibitor Cocktail . After two consecutive centrifugations to eliminate lipids, the lysate was concentrated on an Amicon Ultra Centrifugal Filter , 5 \u03bcl of the concentrated lysate was resolved on a 10% NuPAGE Bis-Tris gel and transferred onto a PVDF membrane using the iBlot system (Invitrogen). Blots were subsequently incubated overnight with one of the following primary antibodies: monoclonal anti-Flag M2 antibody  and anti\u00a0\u03b1-tubulin antibody . The blots were then washed and incubated with anti-mouse IgG coupled to horseradish peroxidase . Peroxidase activity was detected with the Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and imaged on a ChemiDoc MP Biorad gel documentation system . Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) according to the manufacturer recommendations.Embryos were injected at the 4 cell stage with 10 ng of Power SYBR Green PCR Master Mix  on a QuantStudio 3 Real-Time PCR System  with the following primer sets: bra , sox2 , snai2 , luciferase  and odc . The PCR conditions were as follows: denaturation 95\u00b0C (15 s), annealing and extension at 60\u00b0C (1 min) for 40 cycles.Total RNAs were extracted from embryos or animal cap explants using the RNeasy micro RNA isolation kit . The RNA samples were digested with RNase-free DNase I before RT-PCR. The amount of RNA isolated was quantified by measuring the optical density using a Nanodrop spectrophotometer . Approximately 250 ng of total RNAs from animal caps was reverse transcribed using the Superscript VILO cDNA Synthesis Kit  and 2 \u03bcl of 1:1000 dilution of the synthesized cDNA was amplified using  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior and Reviewing Editor.Thank you for submitting your article \"Dkk2 promotes neural crest specification by activating Wnt/\u03b2-catenin signaling in a GSK3\u03b2 independent manner\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is an interesting paper that addresses the role of Dkk2 in neural crest formation, showing that it is required for the expression of both Snai2 and Sox10. Loss of Dkk2 can be rescued by \u03b2-catenin and Lrp6, as well as Wnt8. Over-expression of Dkk2 leads to an expansion of Snai2. While a GSK3\u03b2 inhibitor expands Snai2 expression anteriorly, the prospective neural crest territory remains sensitive to Dkk2 MO, suggesting it functions in a GSK3\u03b2 independent manner.Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or \u03b2-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/\u03b2-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by \u03b2-catenin and Lrp6 injections in Dkk2-depleted embryos?5) In the absence of lineage tracing, the loss of 6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/\u03b2-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on \u03b2-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of \u03b2-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate \u03b2-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?4) Better quantification would be appropriate.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio\u2026?6) This statement in the Discussion is debatable:\"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/\u03b2-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/\u03b2-catenin regulated developmental process.\"Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested? Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.dkk2 expression data as a main figure of the manuscript . Dkk2 is broadly expressed during embryogenesis and is not distinctly enriched in the neural crest territory. It is likely that the regionalized expression of other components of the pathway, such as Fzd receptors and Wnt ligands, which are expressed at higher levels, provides the spatiotemporal cues to achieve a localized response. The expression pattern of dkk2 is presented and discussed in the Results section of the manuscript .Although the Xenbase RNA-seq data indicates that Wnt8 and Lrp6 are expressed at higher levels than Dkk2 it is difficult to correlate this information to the amount of protein present at the time of neural crest induction. As suggested we have moved the 2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or \u03b2-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?Regarding the mRNA expression levels of Dkk2, Wnt8 and Lrp6 see response to point #1. As suggested we have performed Western blot analyses to evaluate the accumulation overtime of a Flag-tagged version of HuDkk2 (see 3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/\u03b2-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.snai2, dkk2 overexpression did not significantly upregulate other known direct targets of Wnt/\u03b2-catenin signaling such as pax3, znf703 or kremen2. This result suggests that dkk2 alone is a poor activator of Wnt/\u03b2-catenin signaling, consistent with the view that it is acting in concert with a Wnt ligand to activate the pathway. The expression of the mesoderm marker myoD was unaffected in dkk2-depleted embryos ruling out any possible secondary effects .We have performed the suggested experiment and found that with the exception of 4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.pax3, sox8, snai1, twist1 and sox9 . We found that early neural crest markers  such as pax3, sox8 and snai1 were only mildly affected \u2013 their expression levels were unchanged but their expression domain appeared to be shifted laterally \u2013 while other markers  such as twist1, and sox9 to a lesser extent, were downregulated, consistent with the phenotype observed for sox10 and snai2. This result suggests that dkk2 may not participate in neural plate border specification but rather in the neural crest progenitors formation and/or maintenance.We have performed additional experiments to increase the repertoire of neural crest genes analyzed, which now include 5) In the absence of lineage tracing, the loss of snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by \u03b2-catenin and Lrp6 injections in Dkk2-depleted embryos?sox2 expression domain .We have analyzed the expression of keratin, a gene expressed in the non-neural ectoderm, and show that its overall expression is reduced/shifted laterally consistent with the expansion of We also show that neural crest-derived craniofacial cartilages in addition to pigment cells are affected upon Dkk2 depletion , pointing to a loss of neural crest lineages in these embryos.6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?snai2 and sox10 expression, associated with an expansion of sox2 expression domain. This phenotype was observed in both whole embryos and animal cap explants. We also used a control MO, which did not affect the expression of these genes. Importantly, this phenotype was efficiently rescued by expression of Lrp6 and \u03b2-catenin, two components of Wnt/\u03b2-catenin signaling pathway. We believe that this combination of approaches makes a compelling case for a specific requirement of dkk2 in neural crest formation. It is difficult to estimate the efficacy of a translation blocking MO without a good antibody, and we have not been able to identify one that could detect endogenous dkk2 in Xenopus. As suggested, we have performed qRT-PCR to evaluate the proportion of spliced versus unspliced transcripts generated upon injection of increasing doses of MO. These data are presented in Figure 1\u2014figure supplement 1.In this study, we used two MOs interfering with Dkk2 mRNA translation and splicing, respectively. Both gave a similar phenotype resulting in the inhibition of Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/\u03b2-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.Both studies are now referenced in the text .2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.We agree with the reviewer, and this is something we are planning to explore in order to elucidate the precise mechanism of Dkk2-mediated signaling during neural crest specification, however we feel that this is beyond the scope of the current study.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on \u03b2-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of \u03b2-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate \u03b2-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?Our model predicts that signaling by Dkk2 and Wnt8 are both independently required to induce neural crest cells, this is specifically supported by the BIO experiment. Further work will address the mechanisms underlying this dual requirement during neural crest formation.4) Better quantification would be appropriate.For all injection experiments we provide a rigorous quantification of the phenotypes, which are summarized in the form of graphs for all figures, supplemented with numerical data provided as \u201csource data\u201d files.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio?We have included a more representative image for Dkk1+BIO .6) This statement in the Discussion is debatable:\"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/\u03b2-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/\u03b2-catenin regulated developmental process.\"We have revised this statement .7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.sox2 expression does not behave in a way that is compatible with the model of pluripotency retention in the neural crest lineage \u2013 a model that is still up for debate. At the neurula stage, we consider sox2 as a marker for neural progenitors and not as much an indicator of pluripotency.We agree with the reviewer, 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested?dkk2 and wnt8 are both independently necessary to induce neural crest we did not expect that overexpressing dkk2 would be sufficient to restore neural crest formation in embryos injected with GSK3. We have performed the suggested experiment and found that Dkk2 expression was indeed unable to rescue neural crest formation in embryo overexpressing GSK3 (see Because our model predicts that signaling by GSK3 see ."}
+{"text": "COPD) is a multifactorial and heterogeneous disease that creates public health challenges worldwide. The underlying molecular mechanisms of COPD are not entirely clear. In this study, we aimed to identify the critical genes and potential molecular mechanisms of COPD by bioinformatic analysis. The gene expression profiles of lung tissues of COPD cases and healthy control subjects were obtained from the Gene Expression Omnibus. Differentially expressed genes were analyzed by integration with annotations from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, followed by construction of a protein\u2010protein interaction network and weighted gene coexpression analysis. We identified 139 differentially expressed genes associated with the progression of COPD, among which 14 Hub genes were identified and found to be enriched in certain categories, including immune and inflammatory response, response to lipopolysaccharide and receptor for advanced glycation end products binding; in addition, these Hub genes are involved in multiple signaling pathways, particularly hematopoietic cell lineage and cytokine\u2010cytokine receptor interaction. The 14 Hub genes were positively or negatively associated with COPD by wgcna analysis. The genes CX3CR1,PTGS2,FPR1,FPR2, S100A12,EGR1,CD163, S100A8 and S100A9 were identified to mediate inflammation and injury of the lung, and play critical roles in the pathogenesis of COPD. These findings improve our understanding of the underlying molecular mechanisms of COPD.Chronic obstructive pulmonary disease ( ARG1arginase 1BPbiological processCCcellular componentCOPDchronic obstructive pulmonary diseaseCScigarette smokeCX3CR1C\u2010X3\u2010C motif chemokine receptor 1DALYdisability\u2010adjusted life yearDEGdifferentially expressed geneEGR1early growth response 1FCfold changeFGGfibrinogen gamma chainFPR1formyl peptide receptor 1FPR2formyl peptide receptor 2GOGene OntologyGSgene significanceKEGGKyoto Encyclopedia of Genes and GenomesMFmolecular functionMMmodule membershipORM1orosomucoid 1PPBPproplatelet basic proteinPPIprotein\u2010protein interactionPTGS2prostaglandin\u2010endoperoxide synthase 2S100A12S100 calcium binding protein A12S100A8S100 calcium binding protein A8S100A9S100 calcium binding protein A9VCAM1vascular cell adhesion molecule 1wgcnaweighted gene coexpression network analysisYLLsyears of life lostChronic obstructive pulmonary disease (COPD), characterized by long\u2010term poorly reversible airway limitation and persistent respiratory symptoms, is a common and preventable disease As a large\u2010scale and efficient technique for acquiring genomic data, microarray\u2010based gene expression profiles have been widely used to seek new insights for biomarkers in many human diseases GSE27597 and GSE106986). DEGs were identified by r software  and subsequently analyzed using bioinformatic methods including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments and construction of protein\u2010protein interaction (PPI) and weighted gene coexpression analysis (wgcna) networks. We screened the DEGs for potential association with the development and progression of COPD. Our work may further the understanding of the potential molecular mechanisms of COPD.We selected two Gene Expression Omnibus microarray datasets on COPD . GSE27597 comprises the expression profile from 64 lung tissue samples from COPD cases and 16 samples from healthy donors GSE106986 includes molecular profiling of 19 lung tissue samples, containing 14 samples from COPD cases and 5 from smokers GSE27597 and GSE106986 were conducted on the Affymetrix Human Exon 1.0 ST GeneChip  and Agilent\u2010026652 Whole Human Genome Microarray 4x44K v2 , respectively.#AuthorQuery RepTwo gene expression datasets, r software and annotation packages. The two datasets were merged into one array dataset and then batch normalized using r packages (sva and limma 3.40.6). The DEGs between COPD cases and control subjects were identified using the limma package in r 3.60. A P\u2010value <0.05 after being adjusted by false discovery rate and |log2FC\u00a0>\u00a01, where FC represents fold change, were applied together as the cutoff for DEGs screening.The probe set IDs of two datasets were converted into gene symbols using the david online tool (https://david.ncifcrf.gov/) http://kobas.cbi.pku.edu.cn/) GO enrichment of the DEGs on the biological process (BP), molecular function (MF) and cellular component (CC) categories was performed using a https://string-db.org/) is frequently used for identifying the protein interactions cytoscape is an open source bioinformatic software platform used for integrating gene expression profiles and visualizing molecular interaction networks. cytoscape plugin cytoHubba provides multiple topological analysis methods on Hub genes, regulatory networks and drug targets for experimental biologists cytoscape version 3.6.1  with cytoHubba plugin according to the rank of connection degree (number) for each gene, which is represented by the different degrees of color (from red to yellow): the role of the gene is greater in the PPI network with the darker color of the gene STRING database (wgcna may be used for screening modules (clusters) of highly correlated genes and for calculating module membership (MM) measures, in which the module eigengene or an intramodular Hub gene is used to summarize such modules, and eigengene network methodology is used for relating modules to one another and to external sample traits wgcna package was used to identify coexpression modules for the merged and standardized array datasets (GSE27597 and GSE106986). In brief, first, a weighted adjacency matrix containing pairwise connection strengths was constructed based on the selected soft threshold power (\u03b2\u00a0=\u00a011) on the matrix of pairwise correlation coefficients. Then, the connectivity measure per gene was calculated by summing the connection strengths with other genes; modules were defined as branches of a hierarchical clustering tree by using a dissimilarity measure, and each module was assigned a color. Subsequently, module preservation r function was used to assess the module structure preservation. Finally, the module eigengene was used for summarizing the gene expression profiles of each module, and each module eigengene was regressed on case trait (COPD) and smoking status by using the linear model in the limma r package.GSE27597 and GSE106986 by the sva and limma packages, 139 DEGs were identified using the limma package  , proplatelet basic protein (PPBP), prostaglandin\u2010endoperoxide synthase 2 (PTGS2), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), vascular cell adhesion molecule 1 (VCAM1), S100 calcium binding protein A12 (S100A12), arginase 1 (ARG1), early growth response 1 (EGR1), CD163, fibrinogen gamma chain (FGG), orosomucoid 1 (ORM1), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9).The 139 DEGs were applied for construction of PPI networks using STRING. After removing the discrete and partially connected nodes, the PPI network data of DEGs were imported into the cytoHubba of wgcna network was first constructed using lung tissue expression data from cohorts GSE27597 and GSE106986, independent of COPD status and smoking status (ever/current smoking versus nonsmoking). A total of 2942 DEGs were selected (a corrected P <\u00a00.05) and subsequently used to identify modules of coexpressed genes using a hierarchical clustering procedure. The corresponding branches of the resulting dynamic clustering tree and module are shown as colored bands underneath the cluster tree. We then merged the highly similar dynamic clustering modules into the merged dynamic modules (cut height\u00a0=\u00a00.25)  versus each MM. We found that the 14 Hub genes were also either positively or negatively associated with COPD , Red  and Grey  genes most significantly correlated with GS for COPD were also the important MM elements , an important mediator of inflammation, was shown to be involved in inflammation response and associated with COPD pathogenesis PTGS2 may protect smokers against the development of COPD FPR1 and FPR2 were reported to be involved in recruitment and activation of inflammatory cells induced by CS, and play important roles in lung inflammation, injury and the pathogenesis of COPD S100A8, S100A9, and S100A12 might induce neutrophil and monocyte chemotaxis, adhesion to fibrinogen and diapedesis, and neutrophil migration to inflammatory sites S100A8, S100A9 and S100A12 may be regulated by RAGE, which was shown to contribute to CS\u2010induced airway inflammation in COPD S100A8, S100A9 and S100A12 is important in the development of COPD.EGR1 is an autophagy regulator gene that plays important roles in cellular homeostasis, airway remodeling and control of inflammatory immune response; it is also a significant risk factor for susceptibility to COPD EGR1 may be induced by CS and involved in proinflammatory mechanisms that are likely associated with the development of COPD \u2212/\u2212 mice were observed to resist CS\u2010induced autophagy, apoptosis and emphysema EGR1 in CS\u2010induced inflammatory immune response and COPD, and effective inhibition of EGR1 may attenuate airway remodeling and inflammation associated with the pathology of COPD.CD163, a carefully regulated component of the innate immune response to infection and a macrophage receptor for bacteria, was shown to play important roles in functional pulmonary defense elements and the inflammatory immune response of the respiratory system CD163 on lung alveolar macrophages may be implicated in the pathogenesis of COPD and poor lung function ARG1 was shown to contribute to asthma pathogenesis by inhibiting nitric oxide production, modulating fibrosis, regulating arginine metabolism and inhibiting T cell proliferation, and it involves the initiation of adaptive T helper 2 cell\u2010mediated allergic lung inflammation by regulating group 2 innate lymphoid cells ARG1 ablation in the lung may enhance peripheral lung function but have little effect on airway inflammation ARG1 in COPD needs to be studied in the future.ORM1 appears to function in regulating the activity of the immune system during the acute\u2010phase reaction and has been identified as an acute exacerbation of COPD\u2010specific immunomodulatory mediator PPBP serves as a potent neutrophil chemoattractant and activator, and its elevated expression in the bronchial mucosa might be involved in the pathogenesis of COPD VCAM1 was shown to express in endothelial cells of atopic asthma cases, but not COPD cases FGG was found to be involved in blast lung injury resistance via promoting tissue\u2010protective adenosine signaling CX3CR1, PTGS2, FPR1, FPR2, S100A12, EGR1, CD163, S100A8 and S100A9, potentially mediated inflammation and injury of the lung, and play critical roles in the pathogenesis of COPD. The roles of five Hub genes, including PPBP, ARG1, FGG, VCAM1 and ORM1, identified to be associated with COPD in this study need to be confirmed in the future. These findings could improve our understanding of the underlying molecular mechanisms of COPD and provide us with insights for drug target discovery for the disease.In conclusion, we have identified 139 candidate DEGs associated with the progression of COPD. The results from bioinformatic analysis are in agreement with those from previous cell and animal models and human studies. Our results showed that nine Hub genes, The authors declare no conflict of interest.XK, FY and QW conceived and designed the project. XH and YL acquired the data. XG and ZZ analyzed and interpreted the data. XH and YL wrote the paper."}
+{"text": "MRE11 germline single nucleotide polymorphism (SNP), rs1805363 (G\u200a>\u200aA), to be associated with poor outcome following radiotherapy (RT) and increased expression of MRE11 isoform 2 in a limited panel of bladder cancer cell lines and tumours.DNA double strand breaks are the cytotoxic lesions produced by ionising radiation. Critical for the repair of these lesions is the DNA damage response protein MRE11 which, in a complex with RAD50 and NBS1, mediates DNA damage signalling and double-strand break repair. We previously found the presence of an MRE11 isoform 2 would be more radio resistant than cells expressing MRE11 isoform 1.To look for further evidence in support of the SNP/isoform association in a larger panel of germline and tumour samples donated by patients diagnosed with invasive bladder cancer, and to test the hypothesis that bladder cancer cells expressing G\u200a>\u200aA SNP. Loss of heterozygosity was determined by genotyping tumour DNA in 17GA germline patients. The Cancer Genome Atlas was mined to correlate presence of the GA germline genotype with MRE11 isoform expression. We used colony formation assays and \u03b3H2AX foci kinetics after ionising radiation in RT112 MRE11 knockdown cells expressing ectopic MRE11 isoform 1 or 2.Germline DNA from 189 patients with invasive bladder cancer  was genotyped for the rs1805363 MRE11 isoform 2 expression was found to be significantly higher (p\u200a=\u200a0.007) in patients\u2019s samples containing the A minor allele compared to those with only the G major allele (n\u200a=\u200a23). In the TCGA database we found 16% (66 out of 406) of bladder tumours heterozygous for the SNP and only two homozygous, and a significant relative increase of isoform 2 usage (p\u200a=\u200a0.017). We identified no significant difference in radio sensitivity between bladder cancer cells expressing either MRE11 isoform.Of the 189 germline DNA samples, 22 contained both the A minor allele and G major allele with the remaining wild type containing only the G major allele. LOH was identified in seven of 17 available tumour samples. Tumour MRE11 isoform 2 was not found to be associated with increased cellular sensitivity to radiation. We conclude that the previously reported association between the germline rs1805363 SNP and poor survival in MIBC patients following RT is unlikely to be related to the DNA damage response function of MRE11 isoform 2.In this study the  Organ-confined muscle-invasive bladder cancer (MIBC) is routinely treated using either radical cystectomy or bladder-sparing approaches including chemoradiotherapy (CRT) or radiotherapy alone, with or without neoadjuvant chemotherapy. Cystectomy and radiotherapy have been shown to result in similar cause-specific survival rates  but no rMRE11 results in two transcript variants encoding MRE11 isoform 1 and MRE11 isoform 2, respectively. The two MRE11 isoforms are similar, with the only difference in the translated protein being the absence of a short 27 amino-acid-long exon, exon 16, in MRE11 isoform 2. At the transcript level MRE11 isoform 2 has a longer 5\u2019 UTR than the MRE11 isoform 1.DNA double strand breaks (DSB) are the cytotoxic lesions caused by ionising radiation and are repaired via two major pathways: non-homologous end joining and homologous recombination . MRE11 iMRE11 (rs1805363) which was associated with worse cancer-specific survival following radiotherapy  was diluted 1:2000 in PBST and applied for 1 hr in room temperature. Cells were washed with PBS as before and mounted by using proLong diamond antifade mountant with DAPI . Cells were visualised for \u03b3-H2AX foci kinetics, P values were calculated using Kruskal Wallis test with Dunn\u2019s multiple comparisons in GraphPad Prism. Counting of \u03b3-H2AX foci was determined using Image J software. For clonogenic assays, surviving fraction was calculated on the basis of the number of colonies on non-irradiated plates. Data are presented as the log of the surviving fraction with error bars representing the SEM.Clonogenic assays, western blots, and immunofluorescence data are representative of three independent experiments. For G\u200a>\u200aA SNP. Of these patients, 141 were diagnosed with muscularis propria invasion (stage T2+) and 48 with lamina propria invasion (T1). Of the 189 samples, 22 were identified as containing both A and G alleles, with the remainder identified as only containing the major G (WT) allele. Loss of heterozygosity (LOH) within heterozygous tumours was investigated by genotyping extracted tumour DNA, which was available for 17 of the germline genotyped casesidentified as positive for both A and G alleles. LOH was identified in 7 of the 17 samples .We obtained 189 samples of germline DNA from the University of Birmingham\u2019s Bladder Cancer Prognosis Programme which were genotyped for the rs1805363 MRE11 isoform 2 expression was found to be significantly higher (p\u200a=\u200a0.007) in patients positive for the rs1805363 A minor allele  compared to patients positive for only the G major allele (mean proportion of MRE11 isoform 2 in the G only germline genotyped patient group: 2.3%). Isoform 2 was also found to be generally increased in non-wild-type samples in TCGA , as well as in direct comparison to isoform 1 (p\u200a=\u200a0.017) , the area of tissue being too small for sufficient material to be collected (5/12) or the tissue being identified as highly inflamed (1/12). Of the remaining 24 blocks, 10 were from patients\u2019 germline positive for both A and G alleles and 14 from patients positive for only the G (WT) allele. RNA extracted from patient tumours was then used to quantify the relative expression of isoform 1 and 2 within the samples and grouped according to germline genotype . One sam017) see . Althoug017) see A. Furthe017) see B.MRE11 isoform 2 could influence cellular radiosensitivity and DNA damage response. We used an RT112 bladder cancer cell line knocked down for endogenous MRE11 and re-expressing an MRE11 construct containing either the full length MRE11 coding sequence (MRE11 isoform 1) or the MRE11 coding sequence lacking MRE11 exon 16 (MRE11 isoform 2). As this question referred to the function of the translated protein we did not include the MRE11 isoform 1 or 2 5\u2019UTR sequences in these constructs.We then investigated whether the increased presence of MRE11 isoform 1 or isoform 2 in these cells led to a greater than 2.5-fold increase in full-length MRE11 protein level , which corresponds to approximately 30% of the endogenous full-length MRE11 level in the parental cell line  cells  higher levels in the MRE11 knockdown cells compared to the parental cell line that persisted for 2, 6 and 24 hrs after irradiation. Consistent with the level of expression achieved compared to the parental cells, MRE11-transfected cells showed a partial reduction in \u03b3-H2AX foci at 2\u200ahr after 2 Gy irradiation, at an intermediate level between the parental and MRE11 knockdown cells. The reduction in \u03b3-H2AX after rescue with either the MRE11 full length or \u0394exon16 constructs was 1.3-fold at 30 minutes after 2 Gy IR, similar to the MRE11 protein levels achieved relative to the parental cells. There was no significant difference in \u03b3-H2AX foci at any of the time points between the MRE11 full length and the \u0394exon16 cells (\u03b3-H2AX foci level (No IR) for all four cell lines were re-achieved at the 6 hr time point. This suggests that the absence of exon 16 has minimal effects on cell survival and repair of DNA damage after\u00a0IR.A colony survival assay was performed to determine the radiosensitivity of the MRE11 RT112 cell lines. The MRE11 knockdown cell line displayed a significant increase in radiosensitivity compared to the parental RT112 cell line at doses of 2 to 10 Gy. This was rescued by the re-expression of either the MRE11 full length (isoform 1)  or the MRE11 exon16-negative construct (isoform 2) , while MRE11 knockdown cells had 62.5-fold and 52.6-fold reduction on survival compared to parental cells at 8 and 10 Gy. However, there was no significant difference in radiosensitivity between cells expressing the full-length version MRE11 and exon16-deleted (6) cells B. To det16 cells A and B. MRE11 rs1805363 SNP in patients with invasive bladder cancer, we genotyped the rs1805363 status of germline DNA and in this study provided further evidence for a correlation between the presence of the MRE11 germline rs1805363 A minor allele and an increase in the tumour expression of MRE11 isoform 2 over isoform 1. However, it would be useful to establish if the tumour genotype is a more accurate indicator of the expression of MRE11 isoform 2 in invasive bladder tumours.In line with our previous report on the MRE11 isoforms from RNA-seq data. In addition, it would be of interest to study other isoforms, as for example ENST00000540013.5 coding for a much shorter protein without exon 16, which seems to be supressed in the non-wild-type cases  than reported earlier  or cells expressing MRE11 lacking exon 16 (MRE11 isoform 2). We therefore conclude that the presence or absence of MRE11 exon 16 may not influence cell survival or DSB repair capacity after IR, contrary to our initial hypothesis, although we appreciate that our conclusions are limited by the use of only one cell line system.Given that the presence of the rs1805363 A minor allele in the germline DNA of patients with invasive bladder cancer was associated with worse survival after radiotherapy , we hypoMRE11 isoform 2 we were interested in directly comparing the protein function of our MRE11 constructs. Therefore, we selected cells for our assays that stably re-expressed our constructs. Unfortunately, this strategy did not allow us to address whether there was a difference in the protein stability or turnover between the two isoforms. Ideally, the protein levels of MRE11 isoform 1 and isoform 2 would have been assessed in patient samples where we had quantified the relative expression of mRNA. However, unfortunately with no available antibody distinguishing between the two isoforms and with only a 27aa difference between the two protein variants, it was not possible to reliably distinguish the isoforms using techniques such as western blotting or immunohistochemistry. We did have an antibody raised to exon 16 but this unfortunately proved unsuitable for western blotting, and so we could not verify its specificity for use in determining the isoforms\u2019 presence in the samples. Interestingly, the phosphorylation of MRE11 threonine 597, which resides within exon16, has been found to promote MRE11 degradation in PTEN deficient cells [In our cellular analysis of nt cells , which dMRE11 isoform 1 compared to 2 is that we only re-expressed either MRE11 isoform 1 or 2 in our KD cells. However, in the human tissue we analysed MRE11 isoform 1 was present in conjunction with isoform 2. Therefore this method may be an oversimplification of the underlying biology and expressing different ratios of the two isoforms may be a more representative model in which to study their function. Indeed, in cells, MRE11 exists as a homodimer with dimerisation being critical for the correct assembly of the MRN complex and the binding and aligning of DNA ends [A limitation in the system we used to investigate the efficiency of DSB repair by DNA ends . It is pDNA ends .MRE11 isoform 2 levels to radiotherapy.Aside from its role in DNA damage repair, MRE11 also functions in the repair of stalled and collapsed replication forks . While wThe authors report no funding.The authors have no conflict of interest to report.AW performed experiments, interpreted the data and drafted and revised the manuscriptJN performed experiments, interpreted the data and drafted and revised the manuscriptLB provided histopathological expertise for tumour identification and interpretation, and revised the manuscriptRA performed bioinformatics analysis, interpreted the data and drafted and revised the manuscriptN H-Z performed bioinformatics analysis and interpreted the dataMZ contributed patient samplesKC contributed patient samplesNJ contributed patient samplesRB contributed patient samples, interpreted the data and revised the manuscriptAK conceived the study, supervised the work and revised the manuscript.All authors approved the final version of the manuscript and agreed to be accountable for the accuracy and integrity of the work.Click here for additional data file."}
+{"text": "The authors would like to provide clarifications and correct errors in Figs PLOS ONE study, as both studies used the same repository of cells and treatments, and therefore some data was re-used. A revised The wildtype flow cytometry DNA panels in PLOS ONE study [The NE study  was carrNE study ; howeverNE study  are novechl1 mutant cells. The incorrect panel is replaced in the revised In scc2-4 cells was incorrectly used in the NZ 1hr panel for wildtype cells. The incorrect panel is replaced in the revised In chl1 mutant cells. The incorrect panel is replaced in the revised In The authors confirm that the above errors occurred in figure assembly and do not affect the analyses or findings of the study. The authors apologize for the errors.The legends for https://doi.org/10.6084/m9.figshare.11412783.v2.The underlying data for the article is provided on Figshare: S1 Figchl1 mutant cells (N = 100 cells for each strain). B) 1N peak quantification for both wildtype and chl1 mutant cells arrested in nocodazole for 3 hours at 23\u00b0C. C) Flow cytometer data reveals DNA contents in wildtype and chl1 mutant cells after nocodazole arrest at 23\u00b0C for 2.5 hours and 3 hours. D) Flow cytometer data reveals DNA content in wildtype and chl1 mutant cells analyzed in A) Morphology quantification for both nocodazole-arrested wildtype and (TIF)Click here for additional data file."}
+{"text": "Motion sensing superpixels (MOSES) is a systematic computational framework to quantify and discover cellular motion phenotypes. Published 26, February 2019During final figure preparation the similar velocity kymographs for 0% serum EPC2:CP-A and EPC2:OE33 in Figure 3A were switched. This mistake was noticed by the authors when rereading the published version. Also in Figure 3A, the red and green font colours used for the cell line names were accidentally switched; these colours should match those in Figure 1 as the same example videos were used for both figures. Figure 3A has been corrected accordingly. These corrections do not affect any of the scientific findings or conclusions of the original paper.The corrected Figure 3 is shown here:The original Figure 3 is shown here for reference:"}
+{"text": "End-tidal carbon dioxide (EtCO2) monitoring is a noninvasive tool used to estimate Paco2 values.Alterations in the partial pressure of carbon dioxide, arterial  higher than EtCO2. Overall, 187 of all Paco2-EtCO2 pairs (42.0%) agreed. There was larger variation in the Paco2-EtCO2 difference during the first 8 hours compared with 9 to 24 hours after admission to the pediatric intensive care unit. Development of pediatric acute respiratory distress syndrome within 24 hours of admission was associated with a lower likelihood of Paco2-EtCO2 agreement  compared with no development of pediatric acute respiratory distress syndrome. A diagnosis of pediatric acute respiratory distress syndrome 1 to 7 days after admission was associated with a larger first-day Paco2-EtCO2 difference compared with those who never developed pediatric acute respiratory distress syndrome .The analysis included 137 patients  and 445 paired Paco2-EtCO2 agreement was low, especially among patients with pediatric acute respiratory distress syndrome. Low Paco2-EtCO2 agreement early in hospitalization may be associated with future development of pediatric acute respiratory distress syndrome. Data on EtCO2 should not be substituted for data on Paco2 during the first 24 hours.In this study of pediatric traumatic brain injury, Pa co2) and end-tidal carbon dioxide (EtCO2) and associated factors in children with traumatic brain injury (TBI).This secondary analysis of a cohort study examines agreement between partial pressure of carbon dioxide, arterial (Pa However, arterial sampling can be challenging in children, and complications are not uncommon.4 Capnography allows for noninvasive, continuous measurement of end-tidal carbon dioxide (EtCO2) with lower cost and fewer complications compared with arterial cannulation, and it is widely used in clinical pediatric practice as a substitute for arterial cannulation.5 End-tidal CO2 reasonably estimates Paco2 in healthy adults.6 A mean difference of 0 to 5 mm Hg between Paco2 and EtCO2 is generally accepted as good agreement, and good agreement has been demonstrated in patients during general anesthesia who are undergoing prolonged neurosurgical procedures, in clinically stable patients in the pediatric intensive care unit (PICU), and in adult patients with TBI without hypotension, metabolic acidosis, or severe lung injury.10 However, the utility of EtCO2 as a substitute for Paco2 in critically injured children with TBI has not been examined, to our knowledge.Globally, traumatic brain injury (TBI) is a leading cause of pediatric morbidity and mortality.co2 less than 30 mm Hg in the initial 48 hours after admission.11 A recent study showed that adhering to this recommendation improved discharge outcomes.12 The Brain Trauma Foundation does not, however, include a recommendation either for or against the use of EtCO2 measurements because of the lack of data comparing Paco2 with EtCO2 to prevent unwanted hypocarbia and hypercarbia. To evaluate the validity of using EtCO2 as an indicator of Paco2 in children and adolescents hospitalized with TBI, we performed a secondary analysis of a prospective cohort study to describe the agreement between Paco2 and EtCO2 early after TBI and determine the factors associated with Paco2-EtCO2 agreement.Recently, the Brain Trauma Foundation published the third edition of medical management guidelines for severe pediatric TBI, recommending avoidance of prophylactic hyperventilation and Pa12 Waiver of consent was granted by the University of Washington Institutional Review Board for the parent project because this research was a secondary data analysis that was deemed to involve no more than minimal risk to participants and waiver would not adversely affect the rights and welfare of the participants. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.After approval by the University of Washington Human Subjects Division, from December 17, 2018, to January 10, 2019, we conducted a secondary analysis of data from the recently published Pediatric Guideline Adherence and Outcomes program study.Harborview Medical Center is a 450-bed mixed level I adult and pediatric trauma hospital that serves the 5-state  Pacific Northwest region. The 18-bed PICU admits approximately 120 pediatric trauma patients with TBI annually.2 values recorded within 30 minutes of a Paco2 measurement during the first 24 hours after admission and (2) had 1 or more Paco2-EtCO2 pairs with a systolic blood pressure (SBP) measured at any point within the 60 minutes prior to the Paco2 value  had 1 or more EtCOco2, EtCO2, SBP, oxygenation index, oxygenation saturation index, and Glasgow Coma Scale score were abstracted from the electronic health record. Race and ethnicity were self-reported.Data on demographic characteristics, mechanisms of injury, and injury severity measures were obtained from the Harborview Trauma Registry. Ascertainment of diagnosis of TBI via computed tomography, surgical procedures performed, Paco2 measured via arterial blood gas was entered into the electronic health record by laboratory personnel. End-tidal CO2 was measured via Medtronic capnography and manually entered by PICU nurses or respiratory therapists into the electronic health record at a minimum of every hour. Data on SBP associated with EtCO2 and Paco2 levels were recorded with a digital sphygmomanometer or by invasive arterial monitoring and were automatically entered from SpaceLabs monitoring system at least every hour, or more frequently as dictated by clinical care. All paired data points were collected while the patients were intubated and mechanically ventilated.Clinical data collected during the first 24 hours of clinical care after admission to the PICU were examined. Pa13 Patients with at least 3 qualifying values of the oxygenation index or the oxygenation saturation index during a minimum of 6 hours were determined to meet oxygenation criteria for PARDS. Medical record abstraction was performed for all patients meeting oxygenation criteria to evaluate for the presence of a new parenchymal infiltrate detected on a chest radiograph (based on review by the attending radiologist and secondary review by a pediatric intensivist [E.Y.K.]) and for the origin of edema (based on documentation by the attending physician).Patients were determined to have pediatric acute respiratory distress syndrome (PARDS) if they met Pediatric Acute Lung Injury Consensus Conference PARDS criteria within 7 days of their injury.2 value recorded within 30 minutes was used to create 1 Paco2-EtCO2 data pair. The nearest SBP value within 60 minutes prior to the Paco2 value being measured was then added to create a Paco2-EtCO2-SBP trio. Values measured during apnea trials for brain death were excluded because the patients were no longer receiving routine TBI care.For every arterial blood gas measurement obtained during routine medical care, the nearest EtCO2 measured by capnography is a mixture of relatively CO2-rich alveolar gas diffused from capillaries with CO2-poor gas in ventilated lung regions with poor perfusion . Dead space includes the components of the respiratory system that are ventilated but do not participate in gas exchange and alveoli that receive ventilation but are not perfused because of physiologic or pathologic factors, as well as the airways and breathing apparatus. The Paco2 value is typically 2 to 5 mm Hg higher than the EtCO2 value under physiologically stable conditions.14 Therefore, Paco2-EtCO2 pairs were categorized as either in agreement, if the Paco2 value was between 0 and 5 mm Hg greater than its paired EtCO2 value, or not in agreement.End-tidal COco2 measurement and when EtCO2 was recorded.Potential explanatory variables were determined a priori based on literature review and research team discussion, and they included severity of chest injury, severity of nonhead-nonchest injury, presence of PARDS (within 24 hours or 1-7 days after admission), hypotension, patient age, time since admission, and the time difference between PaThe presence of severe chest injury or nonhead-nonchest injury with a region-specific Abbreviated Injury Score greater than 2 was coded as a binary variable. Hypotension was defined as SBP less than 70 mm Hg\u2009+2\u2009\u00d7\u2009(patient age in years) coded as a binary variable. The PARDS status for each patient was categorized as follows: did not develop within the first week of PICU admission, developed within 24 hours of PICU admission, or developed after 24 hours but within 1 week of PICU admission. Time after admission was dichotomized into 0 to 8 hours and 9 to 24 hours, and age was categorized into younger than 1, 1 to 4, 5 to 9, 10 to 14, and 15 to 17 years.co2 and EtCO2 was assessed for the overall study population and stratified by PARDS status at 24 hours using Bland-Altman plots with limits of agreement adjusted for repeated measures.15 We also assessed the proportion of Paco2-EtCO2 pairs with Paco2 values 0 to 5 mm Hg greater than their paired EtCO2 value.We used descriptive statistics to examine clinical injury characteristics. Agreement between Paco2-EtCO2 pairs in agreement with pairs that did not agree. Stratified Bland-Altman plots were created to visually compare trends in agreement across levels of age and whether patients developed PARDS within the first 24 hours and within 1 to 7 days of PICU admission. We assessed constant and proportional differences between Paco2 and EtCO2 within each Paco2-EtCO2 pair, and we estimated Pearson correlation coefficients using Passing and Bablok regression and Deming regression.17The characteristics that may have affected the likelihood of agreement between measures were initially assessed descriptively, comparing Paco2 and EtCO2, we used univariate logistic regression models clustered by patient; to minimize confounding, we used multivariable logistic regression models clustered by patient adjusted for age, PARDS within the first 24 hours of PICU admission, severe TBI, severe chest injury, severe nonhead-nonchest injury, and shock. Anatomic dead space in the pediatric population could have considerable variation and may affect Paco2-EtCO2 agreement. Although we were not able to measure dead space individually, we chose to adjust for age and cluster by patient to address this issue. To account for the nonindependence of data points within patients, we used a fixed-effects model to assess the association between hypotension (the only time-varying a priori selected explanatory variable) and Paco2-EtCO2 agreement within individual patients.To determine the characteristics associated with agreement between Paco2 and EtCO2 difference graphically to assess the association between time after PICU admission and Paco2-EtCO2 agreement. To explore how the median first-day Paco2-EtCO2 difference after PICU admission differed by the timing of PARDS diagnosis, we also constructed side-by-side boxplots of patients who had no PARDS, received a diagnosis within 24 hours of PICU admission, or received a diagnosis 1 to 7 days after PICU admission. Two-sample t tests were used to determine if there were statistically significant differences in the mean Paco2-EtCO2 difference between these groups. The 2 analyses were repeated among patients who survived the first 24 hours of PICU admission to examine the association of early death with our findings.We described the temporal trends of Paco2-EtCO2 differences with subsequent PARDS among patients who had not developed PARDS within the first 24 hours, we performed a receiver operating characteristic curve analysis and examined the sensitivity and specificity of various cutoff values. All statistical analyses were performed using R, version 3.5.1 , and Stata, version 14.2 package (StataCorp),19 and analyses were approved by the study epidemiologist (B.M.).To explore the association of Paco2-EtCO2 pairs were excluded owing to a lack of data within the predefined measurement time window  .Of the 199 potentially eligible patients, 137 (103 boys and 34 girls) met the inclusion criteria; 62 patients and 6 Paa points . The medco2-EtCO2 pairs (15.1%) were recorded for patients who experienced PARDS within 24 hours of PICU admission (co2-EtCO2 pairs (20.7%) were recorded for patients who developed PARDS between 1 and 7 days after PICU admission. Compared with data pairs in agreement, pairs not in agreement were more commonly from patients with PARDS diagnosed within 24 hours of admission (56 of 258 [21.7%] vs 11 of 187 [5.9%]).A total of 67 of the 445 Padmission , and 92 co2-EtCO2 pairs, Paco2 was on average 2.7 mm Hg  higher than its paired EtCO2 value . A total of 43 patients (31.4%) had at least 1 paired data point with disagreement. Among the 378 Paco2-EtCO2 pairs obtained from patients who did not experience PARDS within the first 24 hours of admission, Paco2 was on average 1.4 mm Hg  higher than EtCO2 . Among the 67 Paco2-EtCO2 pairs obtained from patients who developed PARDS within first 24 hours of admission, Paco2 was on average 9.9 mm Hg  higher than its paired EtCO2 value .Among all 445 PaO2 value A. Paco2 an EtCO2 B. Paco2 O2 value C. Paco2 co2 and EtCO2 are most similar are shown by Passing and Bablok regression ; there was no meaningful change in association after multivariable model adjustment.co2-EtCO2 difference during the first 24 hours of admission among those who developed PARDS within 24 hours was 9.41 mm Hg greater than those who never developed PARDS . The mean Paco2-EtCO2 difference during the first 24 hours of admission among patients who developed PARDS 1 to 7 days after admission was 4.02 mm Hg greater than those who never developed PARDS  ; restricting the sample to 24-hour survivors resulted in no meaningful change in findings. In the receiver operating characteristic curve analysis, the Paco2-EtCO2 difference among those who did not develop PARDS within 24 hours was associated with subsequent PARDS development between 1 and 7 days after admission . In addition, a Paco2-EtCO2 difference of 3 mm Hg or greater had a sensitivity and specificity of 0.72 for the onset of PARDS between 1 and 7 days after PICU admission, given that PARDS had not developed within the first 24 hours .The mean Pa1 mm Hg) . The meaco2-EtCO2 pairs were in agreement, and correlation was only moderate; (2) the presence of PARDS in the first 24 hours of PICU admission was associated with a lower likelihood of Paco2-EtCO2 agreement; and (3) even if PARDS was not diagnosed in the first 24 hours, the median Paco2-EtCO2 differences were higher among a subset of patients who developed PARDS later in the first week after injury than among those who never developed PARDS. Together, these findings suggest that, despite the appeal of EtCO2 monitoring, EtCO2 data should not be substituted for Paco2 data early after pediatric TBI.The main findings of this study of hospitalized pediatric patients with TBI are that (1) overall, fewer than half of all Pa2 as a Paco2 surrogate. First, we used Bland-Altman analysis, which revealed less than 50% agreement of EtCO2 with Paco2. Second, we used Passing and Bablok regression, which demonstrated that Paco2 and EtCO2 had positive constant and proportional differences between measurements. Finally, we used the Pearson correlation coefficient and demonstrated that Paco2 and EtCO2 were only moderately correlated. Collectively, these indicators demonstrated a moderate level of agreement and correlation between Paco2 and EtCO2. To our knowledge, this study is the first to use multiple measures of accuracy to address Paco2-EtCO2 agreement, and the results suggest a need to further define the role of capnography and the need to incorporate Paco2 measurements early after pediatric TBI.We used 3 complementary statistical approaches to understand the accuracy of EtCO10 reported that among adults with TBI, 77.3% of Paco2 and EtCO2 values were within 5 mm Hg of each other. In contrast, our pediatric study demonstrated lower agreement and did not identify hypotension or severe chest injury as contributing factors to Paco2-EtCO2 agreement. Differences in the definition of Paco2-EtCO2 agreement and in the study design  may contribute to these differences in results. However, to reflect clinical practice, we defined agreement using a narrower range (Paco2-EtCO2 <5 mm Hg) and paired the data within clinically reasonable intervals, which likely decreased measured levels of agreement. Although the number of infants and young children in our study is too small to make definitive conclusions, the relatively larger amount of dead space in infants and children might further increase the Paco2-EtCO2 difference and lower Paco2-EtCO2 agreement.Lee et alco2 values that are higher than EtCO2 values obtained simultaneously.14 Although there is generally no physiologic basis for why Paco2 would be lower than EtCO2, a negative Paco2-EtCO2 difference was observed among many of our Paco2-EtCO2 pairs. It is possible that this negative difference resulted from the accumulation of exhaled CO2 in ventilation circuitry prior to EtCO2 measurement or poor equipment calibration, but most likely it was due to our method of aligning Paco2-EtCO2 pairs during a period of up to 30 minutes, thus allowing the potential for intervening changes in patients\u2019 clinical conditions to result in recorded peaks in EtCO2 values being paired with recorded dips in Paco2 values.Anatomic and physiologic dead space results in Pa22 It is thus reasonable to expect that many critically injured children with TBI, especially those with PARDS or other pulmonary pathologic characteristics, may have an elevated alveolar dead space fraction and thus lower Paco2-EtCO2 agreement. This study identified PARDS as an independent factor associated with a lower likelihood of Paco2-EtCO2 agreement, which provides new information on the utility of capnography among children with TBI with concurrent lung disease. These data also demonstrated that, for patients who had not received a diagnosis of PARDS during the first 24 hours of PICU admission, Paco2-EtCO2 differences were still greater among those who developed PARDS after the first 24 hours of admission compared with those who never developed PARDS, suggesting that patients may have had a ventilation-perfusion mismatch and worsening dead space fraction before meeting the full criteria for PARDS. Future work is needed to further evaluate Paco2-EtCO2 agreement as a potential early indicator of PARDS development in pediatric TBI.We specifically examined PARDS because children hospitalized with TBI are at risk for PARDS owing to a variety of factors, including intracranial hypertension, severe systemic inflammation, direct traumatic lung injury, and secondary lung injury from aspiration or pneumonia.2 measurements in TBI, this study provides new evidence to fill this knowledge and practice gap. Our data show that, despite the relatively constant mean Paco2-EtCO2 difference and despite the moderate agreement correlation during the first 24 hours of PICU admission, there was even larger variability in the Paco2-EtCO2 difference in the first 8 hours of PICU admission compared with the subsequent 16 hours. Therefore, in addition to the overall observation that Paco2-EtCO2 agreement was low in the first 24 hours after TBI and in the presence of PARDS with large and variable alveolar dead space, EtCO2 may be even more unreliable as a surrogate for Paco2 during the first 8 hours after TBI. Therefore, serial Paco2 measurements during the first 24 hours of admission should still be considered the optimal modality to monitor CO2 levels, especially when ongoing resuscitation may be needed and optimizing cerebral perfusion is critical. Given the focus on prevention of prophylactic hypocarbia in severe pediatric TBI, these findings suggest a need for the Brain Trauma Foundation guidelines to address arterial measurements for Paco2.Because neither the Trauma Quality Improvement Program nor the Brain Trauma Foundation guidelines address the utility of EtCOco2-EtCO2 agreement and hypotension with temporal vicinity, inclusion of PARDS diagnosis as an explanatory variable based on laboratory and clinical information, and a comprehensive description of age subgroups. These methodical strengths enabled us to examine in greater detail the association of Paco2-EtCO2 agreement with hypotension and PARDS. In addition, compared with using severe chest trauma to investigate the association of respiratory disorders with Paco2-EtCO2 agreement, the use of the PARDS diagnosis provided more specific information regarding oxygenation dysfunction and resultant ventilation-perfusion mismatch. To our knowledge, this is the first analysis to effectively obtain timely data on blood pressure and PARDS diagnosis and to use them to examine Paco2-EtCO2 agreement.This study has several strengths, such as a relatively large sample size, a priori analytic framework, use of age-specific definitions of hypotension, examination of the association between Pa2 data points were recorded within 10 minutes of Paco2 measurement, some paired data points had a time gap of up to 30 minutes, which may have reduced the precision of our findings. We did not examine data beyond 24 hours. Despite using fixed-effects multivariable regression models and an a priori analytic framework, we cannot exclude residual confounding that could not be captured by measurable variables.We acknowledge the study limitations. Our findings might not be fully generalizable because the data are from a single level I trauma center and the excluded patients who did not have paired data points differed in several clinical characteristics. Although more than 40% of EtCOco2-EtCO2 agreement is below 50%, especially in the presence of PARDS. Agreement is worst in the first 8 hours after PICU admission, and poor Paco2-EtCO2 agreement early in hospitalization may be associated with future development of PARDS. End-tidal CO2 data should not be a substitute for Paco2 data during the first 24 hours.In children hospitalized with TBI, Pa"}
+{"text": "Herein a highly active non-precious transition metal catalyst for the homogeneous hydrogenation of carbon dioxide to formate is presented. Detailed ligand optimisation enabled the development of a nickel-based catalytic system with exceptional productivity. ii) salts in combination with tailored multidentate ligands enabled the effective transformation of carbon dioxide with an exceptional TON of up to 4.65 \u00d7 106. This unprecedented productivity based on the novel nickel catalyst not only outmatches that of existing systems containing first row transition metals, but also established catalysts based on precious transition metals.Herein a highly active non-precious transition metal catalyst system for homogeneous hydrogenation of carbon dioxide to formate is presented. The application of selected nickel( The first system showed feasibility of CO2 hydrogenation in water with a homogeneous nickel catalyst.2 hydrogenation, again relying on the strong Verkade's base. A bimetallic nickel\u2013gallium catalyst was able to perform the reaction at room temperature with TONs of up to 3150 2 and DBU as the base was performed (In the first set of COerformed .4)2 a low TON of 36 could be obtained methyl)amine) a higher TON of 184 was obtained (3 (tris(2-(diphenylphosphino)ethyl)phosphine) a TON of only 41 could be achieved ethyl)amine) showed a high productivity with a TON of 846 (3Cy (NP3Cy = tris(2-(dicyclohexylphosphino)ethyl)amine) a decreased turnover number of 384 was obtained 2.Consequently, further investigations focused on the application of the versatile NPnformate/nDBU) of approximately 0.33 2, NP3 ligand and DBU as the basic requirement for the exceptionally high activity  to a 1\u2009:\u20091 mixture of Ni2+/NP3 resulted in a highly decreased TON and a low AAR . This productivity denotes an overall yield of roughly 6.30 g (31.62 mmol) of isolable [DBUH] formate and further shows the stability of the nickel catalyst system in this study.3 system 3) or zinc triflate (Zn(OTf)2) did not enhance the activity of the established system (3) inhibits catalysis and nearly no formate could be observed (4) a clear enhancement in the catalytic activity was seen achieving a TON of up to 5440 and an AAR of 1.09 (4) has a clearly inhibiting effect resulting in only a poor yield of formate in a TON of 400 2. Analysis after the catalytic experiment revealed a reduction of the initially formed NP3Ni(ii) complex, leading to the formation of a major species with a chemical shift of 21 ppm. Confirmation of the defined species NP3Ni(0) could be obtained with a further control experiment. Consequently, a mixture of NP3/Ni2+, DBU and molecular hydrogen (p = 3 bar) resulted in a 31P-NMR spectrum comparable to that of a post-reaction mixture  salts enabled the effective transformation of CO2 in the presence of molecular hydrogen in acetonitrile solution. Under optimized conditions, using 0.002 \u03bcmol nickel catalyst, an exceptional TON of approximately 4.65 \u00d7 106 was achieved. This unprecedented productivity based on the novel nickel catalyst not only outmatches that of existing systems containing first row transition metals, but also established catalysts based on precious transition metals. Further development and detailed mechanistic investigations on the catalyst system are ongoing in our laboratories.In conclusion, a non-precious transition metal catalyst system could be developed for homogeneous hydrogenation of CO4)2\u00b76H2O and NP3: under an argon atmosphere, a Schlenk tube was charged with a freshly prepared stock solution (c3Ni/NP = 2.5 \u03bcmol mL\u20131) of Ni(BF4)2\u00b76H2O and NP3. In the case of highly diluted reactions the stock solution was further diluted (see the ESI2 (30 bar) until saturation, followed by hydrogen to a total pressure of 90 bar at room temperature. The reaction mixture was stirred and heated to the respective reaction temperature in an aluminium heating cone. After the reaction period, the autoclave was cooled to room temperature and then carefully vented. Mesitylene was added as the internal standard and the resulting solution was analysed by 1H-NMR-spectroscopy. The TONs were found to be reproducible within \u0394TON = \u00b110% in at least two independent runs in selected experiments.The general procedure for homogeneous hydrogenation of carbon dioxide to formate using Ni(BFThere are no conflicts to declare.Supplementary informationClick here for additional data file."}
+{"text": "TM6SF2 and MBOAT7 genes as risk factors for alcoholic liver cirrhosis and non-alcoholic fatty liver disease. In this study, we tried to evaluate the association between TM6SF2 variant rs58542926 and MBOAT7 variant rs641738 and the risk of hepatic fibrosis or liver cirrhosis of different etiology. In parallel, we also aimed to evaluate whether these two SNPs modify the effects of the PNPLA3 rs738409 risk variant for the development of hepatic fibrosis and liver cirrhosis. The study was conducted at the Department of Gastroenterology, Lithuanian University of Health Sciences Hospital, and included 334 patients with liver cirrhosis, 128 patients with liver fibrosis, and 550 controls. SNPs were genotyped by quantitative PCR, using TaqMan allelic discrimination assays. Overall, TM6SF2 rs58542926 as well as MBOAT7rs641738 were not linked to hepatic fibrosis, alcohol or hepatitis C virus induced liver cirrhosis in an Eastern European population. These genetic variations also did not mediate the effect of PNPLA3 rs738409 SNP for liver developing liver fibrosis or liver cirrhosis.Previous large-scale genetic studies identified single nucleotide polymorphisms (SNPs) of the  Liver cirrhosis is one of the dominant causes of global disease burden and is associated with life threatening complications . In 2016PNPLA3) gene SNP is linked with liver injury [TM6SF2) and membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) gene polymorphisms have been identified as risk factors for alcoholic liver cirrhosis [TM6SF2 variant rs58542926 has also been associated with an increased risk of developing advanced hepatic fibrosis and cirrhosis in patients with non-alcoholic fatty liver disease (NAFLD) [MBOAT7 polymorphism rs641738 was associated with the severity of liver fibrosis in individuals with chronic hepatitis C virus infection [MBOAT7 rs641738 SNP was also reported as an independent predictor of severe liver steatosis in patients with chronic hepatitis C [Genome wide association studies (GWAS) are now widely used to identify associations between single nucleotide polymorphisms (SNPs) and the risk of different diseases employing next-generation sequencing based techniques. Over the last decade a significant number of GWAS have been performed in the field of hepatology , revealir injury ,12. More (NAFLD) . Furthernfection  and NAFLnfection . In a reatitis C .TM6SF2 variant rs58542926 and MBOAT7 variant rs641738 and the risk of hepatic fibrosis or liver cirrhosis of different etiology in an Eastern European patient cohort. Additionally, we also aimed to evaluate whether these two SNPs modify the effects of the PNPLA3 rs738409 risk variant on the development of hepatic fibrosis and liver cirrhosis.In this study we analyzed the association between p < 0.001) than patients with liver fibrosis and controls. Men were predominant in the liver fibrosis group (61.7%. p < 0.05). The major cause of liver cirrhosis was alcohol, the second most common cause was chronic HCV infection. In the liver fibrosis group, the most common cause of liver injury was HCV infection. To eliminate the potential bias of differences in age and sex distribution among the groups, these parameters were included as covariates in logistic regression analysis. The frequencies of alleles and genotypes were in Hardy\u2013Weinberg equilibrium for all SNPs analyzed .TMS6SF2 rs58542926 and MBOAT7 rs641738 polymorphisms in patients with different etiology of liver cirrhosis, hepatic fibrosis and the control groups are presented in TM6SF2 rs58542926 allele frequencies in controls, patients with hepatic fibrosis, liver cirrhosis, alcohol-induced and HCV-induced cirrhosis were: T allele 7.5%, 5.5%, 8.4%, 8.1% and 7.5%; C allele 92.6%, 94.5%, 91.6%, 91.8% and 92.5%, respectively. None of the TM6SF2 rs58542926 alleles or genotypes were linked with the risk of developing liver fibrosis or cirrhosis. MBOAT7 rs641738 SNP alleles and genotypes showed similar distributions across all four groups of the study. In controls, the frequencies of T and C alleles were 43.3% and 56.7%, in the liver fibrosis group 41.4% and 58.6%, in the cirrhosis group 44.6% and 55.4%, in alcohol-induced cirrhosis 42.1% and 57.9%, in HCV-induced cirrhosis 45.8% and 54.2%, and in patients with cirrhosis due to other causes 51.2% and 48.8%, respectively. No significant differences between MBOAT7 rs641738 alleles and genotypes among the different study groups were observed.Allele and genotype distributions for the PNPLA3 rs738409 and liver fibrosis/cirrhosis [PNPLA3 rs738409 and MBOAT7 or TM6SF2 genetic variants. This analysis showed no significant differences between combined MBOAT7 and TM6SF2 SNPs and PNPLA3 risk genotypes.In this part of the study we used genotyping data from our previous study on irrhosis . Table 4TM6SF2 rs58542926 and MBOAT7 rs641738 genes polymorphisms and the risk of hepatic fibrosis and liver cirrhosis. Overall, TM6SF2 and MBOAT7 SNPs were not linked with hepatic fibrosis and liver cirrhosis of different etiology within our study. Additionally, we ran combined analysis to assess whether TM6SF2 and MBOAT7 SNPs mediate the risk of liver fibrosis or liver cirrhosis in the presence of certain PNPLA3 genotypes. The effects of PNPLA3 have been well established in previous research, but we wanted to see if the risk of liver fibrosis is changed by a certain combination of PNPLA3 and TM6SF2 or MBOAT7 genotypes. To the best of our knowledge, this is the first study of an Eastern European population that assessed the impact of TM6SF2 rs58542926 and MBOAT7 rs641738 on developing liver injury.In the current study, we aimed to evaluate the associations between TM6SF2 gene is important for the secretion of very-low density lipoprotein from hepatocytes. Its reduced function leads to increased liver triglyceride and lipid droplet content which contributes to NAFLD [TM6SF2 gene have been linked with a number of liver conditions; however, reported results are varying in between. TM6SF2 rs58542926 was identified as a modifier of hepatic fibrogenesis [TM6SF2 rs58542926 is linked with development of severe liver steatosis in patients with chronic hepatitis C (CHC), although no significant association between TM6SF2 and severe liver necro-inflammation or fibrosis was found [TM6SF2 rs58542926 not only had an impact on liver steatosis, but was also relevant in the development of severe fibrosis in individuals with CHC [rs58542926 has a modest effect on NAFLD [TM6SF2 genotype did not impact histological features with biopsy-proven NAFLD in Japanese patients [TM6SF2 on fibrosis in NAFLD, CHC and CHB patients, Eslam et al. used a large cohort of Caucasians and found that rs58542926 has more influence on the serum metabolic profile and predisposed hepatic steatosis, rather than acting directly on liver inflammation and fibrosis [TM6SF2 variant and hepatic fibrosis [TM6SF2 rs58542926 between fibrosis and cirrhosis groups was different, but the significance level was not reached and this genetic variant alone or combined with PNPLA3 risk genotype was not linked with liver fibrosis or cirrhosis.to NAFLD ,18,19. Gogenesis  and was ogenesis ,21. Coppas found . Furtherwith CHC . Meanwhion NAFLD . Moreovepatients . To conffibrosis . AnotherMBOAT7 rs641738 SNP with the entire spectrum of NAFLD in individuals of European descent was first reported by Mancina et al. [MBOAT7 genetic variant was not associated with hepatic steatosis, but significantly linked to hepatic fibrosis by Krawczyk M. and colleagues [MBOAT7 rs641738 was not linked with risk of developing NAFLD [rs641738 and liver steatosis, but an association with hepatic inflammation and fibrosis in CHC was detected [MBOAT7 locus rs641738 was not linked with liver fibrosis or cirrhosis. When combining different genotypes of this SNP with a well know PNPLA3 rs738409 variant no significant differences were observed.The association of a et al. . The samlleagues . In an Ang NAFLD . Yet anodetected . NeverthPNPLA3 SNP sub-analysis needs further re-evaluation in larger well-defined cohorts in order to evaluate observed synergistic effects of different genetic variations.There are certain limitations associated with the design of our study that need to be acknowledged. The major limitation of our study is related to etiological heterogeneity of liver cirrhosis cohort and not a large number of patients within the subgroup analysis. Most of our patients in the liver fibrosis group had HCV induced fibrosis. Moreover, relatively small sample sizes within combined TM6SF2 rs58542926 and MBOAT7 rs641738 genotyping study included 1012 individuals . For combined analyses of carriers of PNPLA3 rs738409 risk genotype, we used data from our previous study with the same cohort [The e cohort  and inclTM6SF2 rs58542926 and MBOAT7 rs641738 SNPs were genotyped by real-time PCR (RT-PCR), using TaqMan\u00ae allelic discrimination assays with a 7500TM Fast real-time PCR system .Genomic DNA from samples was isolated from whole blood mononuclear cells using a salting-out method and stored at \u221220 \u00b0C until analysis, as described previously . TM6SF2 TM6SF2 and MBOAT7 alleles and genotypes frequencies between cases and controls were compared by Pearson\u2019s goodness-of-fit \u03c72 and Fisher exact tests. Associations between control and cases groups with SNP alleles and genotypes were calculated using logistic regression analysis with adjustment for age and sex. 2 tests. For additional analysis, we divided the cohort in two groups with respect to PNPLA3 rs738409 genotypes. p-value was adjusted for multiple comparisons and the value of <0.025 (0.05/2) was considered to be statistically significant. Statistical analysis of the genotyping data was performed using PLINK software version 1.07 [ion 1.07 .TM6SF2 rs58542926 as well as MBOAT7 rs641738 were not linked to hepatic fibrosis, alcohol or hepatitis C virus induced liver cirrhosis in an Eastern European population. These genetic variations also did not mediate the effect of PNPLA3 rs738409 SNP on the development of liver fibrosis or liver cirrhosis.In conclusion,"}
+{"text": "The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases , a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG) Myotonic dystrophy type 2  is an autosomal dominant multisystemic neuromuscular disorder that occurs in adults , 2. Althn repeat located in the first intron of the CNBP  gene in the 3q21.3 chromosomal region (DM2 is caused by the expansion of an unstable (CCTG)l region . Like inl region . It disrCNBP repeat tract is generally described as (TG)n(TCTG)n(CCTG)n. Normal alleles as a rule have <25 copies of the decisive CCTG repeat, whereas expanded alleles have as many as 75\u201311,000 copies w(CCTG)x(NCTG)y(CCTG)z. In contrast, unstable expanded alleles obviously have a pure CCTG stretch without the NCTG interruption making them more susceptible to strand slippage and unequal crossing over processes in cell division tability , 6, 7. Cdivision .CNBP repeat motif has been conducted for the German population. In the present report, an estimate of the DM2 prevalence in Germany is given and a statistical analysis of the normal allele length frequency and distribution was performed. Alleles of the most frequent repeat lengths (n = 31) as well as of the longest normal alleles (n = 3) were sequenced to determine the detailed motif composition and its variance within the German population. Furthermore, we present two small families, one with a transgenerational contraction into the premutational range and another with a transgenerational expansion of a premutation allele to a full expansion.The composition of the complex repeat motif and allele frequency of normal and expanded alleles has already been studied in the Slovak and American population , 7. So fCNBP repeat sequence, using a fluorescently marked 6-F-primer (DM2-Cl3N58-F) 5\u2032-GGC CTT ATA ACC ATG CAA ATG-3\u2032, binding 47 bp upstream of the TG repeat sequence. The reverse primer (DM2-Cl3N58-R) 5\u2032 GCC TAG GGG ACA AAG TGA GA 3\u2032 binds directly after the last single TCTG tetraplet following the repetitive sequence  on an Applied Biosystems 3130 Genetic Analyzer. Sequencing conditions were the following: Step 1: 96\u00b0C (2 min), step 2: 96\u00b0C (20 s), step 3: 56\u00b0C (20 s), step 4: 60\u00b0C (3 min), and step 5: 60\u00b0C (5 min). Steps 2\u20134 were repeated in 26 rounds. Data were analyzed with the software Chromas lite (Technelysium). All sequenced patients had given informed consent for research purposes in the DM2 field, as required by the declaration of Helsinki 2013.There are no well-documented prevalence data for DM2 except of the reports from Finland  and RomeFor this study, fragment analysis data were collected from 739 probands of presumed German origin in order to define the polymorphic spectrum of DM2 repeat tract lengths in the German population. Figure ~55 allele to a full expansion (family 1) and the contraction of a full expansion to a (CCTG)~30 allele The composition of the complex repeat motive of normal length alleles in the German population was analyzed by Sanger sequencing of DNAs from 34 DM2 patients with repeat expansion determined by routine diagnostic fragment analysis. Thirty one of the 34 DM2 positive patients were specifically selected because they possessed one of the three most frequent repeat lengths . The remaining three DNAs were derived from those three DM2 patients with a full expansion that revealed the three longest healthy range alleles found in our patient cohort . Table CNBP repeat the CCTG tract was interrupted by three tetraplets (NCTG)3: GCTG CCTG TCTG. The last CCTG repeat unit always comprised 7 CCTGs whereas the first CCTG unit before the interrupting (NCTG)3 was found to vary between 5 and 6 CCTG tetraplets. Of the 31 sequenced alleles 26 revealed (CCTG)5(NCTG)3(CCTG)7 (84%) while only 5 alleles (16%) had 6 leading CCTG tetraplets [(CCTG)6(NCTG)3(CCTG)7]. In contrast to the highly stable CCTG tract, the (TG)v and (TCTG)w motifs at the beginning of the CNBP repeat stretch vary widely within the range of v = 17\u201325 (TG)v and w = 7\u201310 (TCTG)w. The most frequent normal allele was identified to contain 9 TCTG tetraplets [(TG)v(TCTG)9(CCTG)5(NCTG)3(CCTG)7], with a frequency of 42% of the 31 analyzed alleles and a range of (TG)v of v = 19\u201323.In case of the 31 patients with the high abundance alleles , in all of the analyzed healthy range alleles of the 22 (TCTG)w(CCTG)8(NCTG)5(CCTG)6. Specifically, in both alleles the interruption comprised five tetraplets: GCTG CCTG TCTG CCTG TCTG. The allele of 168 bp was found to be an exception with 23 uninterrupted CCTG tetraplets  was similar to each other: (TG)21(TCTG)9(CCTG)5(NCTG)3(CCTG)7 (138bp) while the other for (TG)21 (TCTG)10(CCTG)5(NCTG)3(CCTG)7 (142bp). Another patient with homozygous allele lengths but heterozygous motif composition revealed a rare allele (TG)21 (TCTG)7(CCTG)6(NCTG)3(CCTG)9 where the second CCTG motif had 9 instead of the constant number of 7 CCTGs (data not shown).Interestingly, only 2 of the 11 DM2-negative probands that appeared to be homozygous in the fragment analysis were indeed homozygous while the other 9 probands had repeat tracts with an identical length but a different motif composition where particularly TG and TCTG repeats varied widely. One of these true homozygous patients was homozygous for the allele (TG)The estimation of DM2 prevalence in Germany of about 9 in 100,000 people is equivalent to findings in the Italian population . For theCNBP repeat tract seem to be essentially similar among European populations. Comparing the German population's combined tract lengths [(TG)v(TCTG)w(CCTG)x(NCTG)y(CCTG)z] with those of the Slovak population underscores this hypothesis. The distribution of most healthy range alleles was found to be almost identical to the statistical results of the Slovak population . Radvanszky et al. identified 138 bp as the most common tract length, which was found to be the most frequent in the German population, too. The second-most frequent allele length of 134 bp among the German population was in third place in the Slovak population, whereas the third-most frequent tract length of 142 bp in the German population was at second place among the Slovak allele lengths . Notablyv(TCTG)w stretch. Accordingly, sequencing analysis of probands with only one apparently homozygous allele in the fragment analysis showed that fragment length alone does not predict homozygosity per se due to structural differences of the complex repeat. Indeed, a monomorphic structure was rarely observed (<20%). This is in accordance with other studies where heterozygosity was found to be roughly 90% in different European populations (y(CCTG)z part in contrast was fully stable in all 31 probands. The most common (CCTG)5(NCTG)3(CCTG)7 allele accounted for 26 of the 31 alleles (84%) while only 5 (16%) belonged to the (CCTG)6(NCTG)3(CCTG)7 allele. However, other rare alleles are present in the German population as observed in a \u201chomozygous\u201d but actually heterozygous DM2 negative proband, where a (CCTG)6(NCTG)3(CCTG)9 allele with z = 9 instead of z = 7 was present. Our results correlate well with the observations in the Slovak population where 75.3% of probands contained the (CCTG)5(NCTG)3(CCTG)7 and 17.4% the (CCTG)6(NCTG)3(CCTG)7 allele (7) while only few other alleles could be detected. The interrupting motif (NCTG)y was always found to consist of the three consecutive tetraplets (CGTG)(CCTG)(TCTG) for the three most frequent alleles which has also been previously reported by Radvanszky et al. ulations , 6, 7. Sbserved <0%. This 5 consisted of an identical GCTG CCTG TCTG CCTG TCTG sequence in both alleles.Notably, the structural composition of 2 of the 3 longest analyzed normal alleles (152 and 156 bp) varied only in the amount of TCTG tetraplets. The CCTG interruption (NCTG)1(CCTG)1(TCTG)1 or five (GCTG)1(CCTG)1(TCTG)1(CCTG)1(TCTG)1 tetraplets, respectively. Thus, the high polymorphic structure of the CNBP repeat is also recognizable from its interrupting tetraplets. However, uninterrupted alleles have previously been identified as being distributed over the whole size spectrum of 190 normal sized repeats (118\u2013184 bp) and constituted 2.6% in the randomly selected Slovak individuals  shows incomplete penetrance which might depend on different parameters not known to date but could encompass specific changes in the repeat structure to patients' physical conditions or even environmental impacts. Collecting data from more patients with \u201csmall\u201d DM2 expansions and known repeat tract structure as well as a follow up over time is necessary to estimate penetrance and should be a goal of future research to provide better genetic counseling for DM2 patients.Our observations in the German population lead to the assumption that alleles with a combined All subjects gave written informed consent in accordance with the Declaration of Helsinki.AM, TS, and WK data collection, data analysis, and data interpretation. AM, TS, WK, and SR drafting and critically revising the manuscript. BH-K, E-MH, and KS genetic testing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PM and handling Editor declared their shared affiliation."}
+{"text": "Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine  EXOC7 permits isoform switching during epithelial to mesenchymal transition (EMT) in breast cancer cells, such that the mesenchymal EXOC7 isoform promotes a migratory phenotype through its ability to recruit remodelers of the actin cytoskeleton [The exocyst complex is composed of eight distinct components EXOC1-8) and tethers secretory vesicles to the plasma membrane prior to SNARE-mediated fusion and exocytosis. Isoform variants exist for several of the EXOCs and these are derived from alternative splicing of the canonical EXOC transcripts or paralogous gene expression. We have previously reported on the EXOC3 paralog EXOC3L2 and established that it is required for directional migration of endothelial cells and can associate with other EXOCs [-8 and teExoc3 and is also known as tumor necrosis factor \u03b1-inducible protein 2 (TNF\u03b1IP2) and EXOC3L3. M-Sec plays a key role in the formation of tunneling nanotubes (TNTs)[M-Sec is another paralog of es (TNTs), 5. A cres (TNTs). Importaes (TNTs), which ies (TNTs). TogetheExoc3l2 and using a structural homology model based on M-Sec we compare the properties of the canonical isoform of EXOC3L2 with this alternative isoform. The existence of this splice variant of Exoc3l2 expands the potential for EXOC3L2 functional diversity, as an integrated component of the exocyst or as an independent agent, and will need to be accounted for in future studies of EXOC3L2.Here we report an alternative splice variant of murine C57BL6 adult mice were acquired from Charles River Laboratories and housed at the National Veterinary Institute, Uppsala, Sweden. The animal experiments described in this study were conducted under the ethical permit C222/11, which was granted and approved by the Uppsala Animal Experiments Ethics Board , Uppsala District Court, Uppsala, Sweden.RNA was isolated from mouse tissues using the E.Z.N.A. total RNA isolation kit  according to the manufacturers protocol for animal tissues homogenized by needle and syringe. The purity and concentration of the RNA isolates were analyzed using a NanoDrop 2000 . A cDNA library of the RNA isolates was synthesized using the iScript cDNA synthesis kit  according to the manufacturers instructions.Exoc3l2 splice junction were manually designed. Primers were obtained using the Invitrogen Custom DNA Oligos service (Thermo Fisher Scientific). The following primers were used in the study: positive strand primers: 4:5, GTCACAGACGTGAAGGCTCA; P9, GAAGCTCTGGATGGCATCGT; P10:11, GTTTCGGCGGCTGGAGTC; Psj1, GGAGAGAATGGCTTATTGGCTTG,P and negative strand primers: 11, CTCGGAGTGTCCTCCAACTG; Psj2, CAAGCCAATAAGCCATTCTCTC.PPrimers were designed and confirmed to target the templates of interest using the Primer-BLAST tool hosted at the National Center for Biotechnology Information (NCBI) website. Primers that spanned the novel \u00ae DNA polymerase with Buffer II and MgCl2 (Thermo Fisher Scientific). Taq polymerase and Buffer II were added as per the manufacturers instructions; additionally, one PCR volume contained 0.5 mM MgCl2, 5% dimethyl sulfoxide (DMSO), 0.1 \u03bcM dNTPs, 0.5 \u03bcM forward primer, 0.5 \u03bcM reverse primer, and 1.5 \u03bcl of sample DNA (1 \u03bcg/\u03bcl). The PCR was performed on a MiniOpticon PCR System (BioRad Laboratories) or a 2720 Thermal Cycler  with the following protocol: one cycle of 95\u00b0C for 3 min; 39 cycles of 95\u00b0C for 30 sec; 60\u00b0C for 30 sec; 72\u00b0C for 1 min; and one cycle of 72\u00b0C for 7 min. PCR products and the GeneRuler 100 bp or 1 kb DNA ladder (Thermo Fisher Scientific) were diluted in DNA Gel Loading Dye  and separated by electrophoresis on 2% agarose gels, prepared in 40 mM Tris, 20 mM acetate and 1 mM EDTA (TAE buffer) with 0.01% GelRed fluorescent DNA stain . Gels were scanned using a Gel Do EZ imaging system (BioRad Laboratories) or an Odyssey Fc Imaging System  and images were collected using the ImageLab Software (BioRad Laboratories) or Image Studio\u2122 Software (LI-COR Biosciences).PCR was carried out with AmpliTaq Goldnucleobytes.com) and Adobe Illustrator software.cDNA from bands of interest was extracted from the agarose gel using a QIAquick Gel Extraction Kit  according to the manufacturers protocol for spin-columns. Extracted cDNA concentrations were determined using a NanoDrop 2000 . Sanger sequencing was conducted by the Uppsala Genome Center, Science For Life Laboratory, Uppsala, Sweden. Sequence data was viewed and presented using 4Peaks , according to the manufacturers instructions and performed on an iCycler iQ real-time PCR detection system (BioRad). Melt curves were analyzed using iCycler iQ associated software.Exoc3l2 Sanger sequencing results was performed using the online version of the MAFFT multiple sequence alignment program [Multiple sequence alignment for the  program , and the program . Alignme program , 12. The program . The graExoc3l2 gene is annotated at the Ensemble database as ENSMUSG00000011263 and at the NCBI database as XM_006540475.1. Two transcript variants of Exoc3l2 are currently proposed, the longer of the two would encode a 789 amino acid protein (Uniprot D3YUP5) and consists of twelve exons, while the short transcript is derived from the last four exons of the long transcript and would encode a 242 amino acid protein  or common to both long and short transcripts (P9 and P11). PCR of mouse liver cDNA with primers P9 and P11 produced an amplicon that migrated on agarose gel close to the predicted Exoc3l2 product length of 308 bp, while primers P4:5 and P11 produced an amplicon that migrated close to the predicted Exoc3l2 product length of 818 bp  and reverse (Psj2) primer that targeted the alternative splice junction between exon 6 and 11  [Exoc3l2 is important when designing primers and probes for studying transcript expression, and also when attempting to target specific epitopes of the translated protein with antibody based techniques.A polymorphism (rs597668) near the ase (AD) , and whiExoc3l2, and confirmed its expression in a number of major organs. Based on the recently published structure of M-Sec we built a structural homology model of EXOC3L2 and observed that the C-terminal region lost from this alternatively spliced isoform is similar to that lost from a proposed splice variant of M-Sec (XM_006515792.3). This region contains key residues implicated in M-Sec\u2019s ability to recruit RalA and the exocyst and in doing so facilitate TNT formation. Residues with similar properties are found at comparable locations within the region lost from this alternatively spliced isoform of EXOC3L2. Identification of this splice variant of murine Exoc3l2 expands the family of exocyst-like proteins and future studies are warranted to determine the functional relevance of this alternatively spliced isoform of EXOC3L2.Using a strategy of reverse transcription PCR, exon junction-specific primers and Sanger sequencing we have identified a novel splice variant of murine S1 FigEqual concentrations of mouse heart (adult) and brain cDNA were amplified using primers P4:5+11 and P10:11+11 for Exoc3l2 and the \u03b2-actin house keeping gene ACTB. The similar strength of ACTB bands in heart and brain samples indicates that equal concentrations of cDNA (determined prior to PCR by nanodrop) were used from both tissues; therefore, the relatively weaker 818 bp bands for Exoc3l2 in brain samples suggests lower levels of Exoc3l2 transcription in this tissue. Bands at 250 bp representing the splice variant Exoc3l2a were detected in 2/3 mouse heart samples, but in none of the three brain samples.(EPS)Click here for additional data file."}
+{"text": "For this article, the survey data on the effect of perceived excessive workload on faculty members\u2019 commitment was presented. This data was gathered from an academic environment using the full time faculty members of Covenant University. The descriptive research design method was employed. The initial sample size used for the analysis was 228 faculty members but only 189 copies of questionnaire were returned. Statistical package for social sciences (SPSS) was used for the coding of the data. The validity and reliability of the research instrument were carried out using Cronbach Alpha. Descriptive analysis was used for the presentation of the data. This data is made publicly available to assist further study in the area of workload and employees commitment. Specifications TableValue of the data\u2022The data provided in this article shows the basic criteria for measuring excessive workload in organizations and could be adopted for further research work.\u2022This dataset indicates that there is a link between excessive workload and commitment of employees, thus, its availability will foster the need to emphasize on the subject matter.\u2022Improved research work on this survey data would enhance faculty members\u2019 commitment by increased morale, motivation and reduced absenteeism.\u2022Additional investigation of the survey data by the universal professional research bodies will increase the understanding of the effect of work overload on faculty commitment across countries.\u2022Data shared in this data article will open up doors for new research collaborations.1sine qua non for a university\u05f3s survival Number 1 shows the response to the statement \u201cEffective work life balance initiative helps to reduce excessive workload\u201d. 5 (2.6%) respondents were neutral about the statement. 85 (45.0%) respondents agreed that effective work life balance initiative helps to reduce excessive workload while 99 (52.4%) of the respondents strongly agreed that effective work life balance initiative helps to reduce excessive workload.Number 2 presents the responses to the research question are the work life balance initiatives in your organization suitable? Only 1 respondent strongly disagreed with the question representing 0.5%, 3 respondents disagreed representing 1.6%, 14 (7.4%) respondents were neutral about the question. 125 (66.1%) respondents agreed while 46 (24.3%) respondents strongly agreed to the question.Number 3 presents the summary of the data on the responses to the research statement \u201cI often feel that the work life balance initiatives are not effective.\u201d18 (9.5%) respondents strongly disagreed with the statement, 28 (14.8%) disagreed with the statement, 22 (11.6%) respondents were neutral on the statement, 64 (33.9%) respondents agreed with the statement and 57 (30.2%) respondents strongly agreed to the statement.Number 4 provides responses to the statement \u201cI sometimes stay back to finish my work for the day\u201d. 5(2.6%) of the respondents strongly disagreed, 4 (2.1%) of the respondents disagreed while 8 (4.2%) respondents were neutral. 94 (49.7%) of the respondents agreed and 78 (41.3%) strongly agreed that they often stayed back to finish the work for the day.Number 5 shows responses to the statement on I always make sure I go early to work or other related activities. 10 (5.3%) respondents strongly disagreed with the statement, 1 (0.5%) respondent disagreed, 9 (4.8%) respondents were neutral on the statement, 139 (73.5%) respondents agreed and 30 (15.9%) respondents strongly agreed.Number 6 Presents responses to whether the respondents were interested in everything that goes on in the organization. 9 (4.8%) of the respondents strongly disagreed, 18 (9.5%) of the respondents disagreed while 19 (10.1%) of the respondents were neutral as regards the notion. 76 (40.2%) agreed and 67 (35.4%) strongly agreed that they were interested in everything that goes on in the organization.The evolving competition in the university system has placed pressure on employees\u2019 commitment particularly in a recessed economy where organisations are reducing the overhead cost by reducing the workforce and increasing the workload of the surviving employees. Active engagement of faculty members in research, teaching and community impact are 2Data were collected from the sample of two hundred and twenty eight (228) faculty members of Covenant University with the aid of structured questionnaire designed by the researcher based on the similar studies. Stratified and simple random sampling techniques were used. Copies of questionnaire were administered directly to all categories of faculty members ranging from Assistant Lecture to Professor. What informed the choice of the selected Covenant University was because of the fact that Covenant University has been best private university in Nigeria for over four years consecutively. Faculty members were also purposefully selected because of the university drive for quality research outputs and excellent teaching delivery toward achieving their vision of becoming one of the best universities in the world. Two hundred and twenty eight (228) copies of questionnaire were administered; one hundred and eighty nine (189) copies of the questionnaire were retrieved from the field giving the response rate of (82.9%). Meanwhile, the researchers also sought for the permission of the management of the selected institution before the questionnaire were administered to them. In addition, every participant was adequately informed about the objective of the study. In addition, all respondents were given opportunity to stay anonymous and their responses were treated with upmost confidentiality."}
+{"text": "This increase in FIO2, however, often leads to errors in the calculation of oxygen consumption (2 concentrations. A possible solution to this problem could be the use of the \u2018Eschenbacher transformation\u2019 (ET) as an alternative correction factor. This study examines the concentration of FIO2 at which the HT and the ET are valid, providing plausible data of oxygen consumption corresponding to the wattage achieved during cycle ergometry. Ten healthy volunteers underwent spiroergometric testing under standard conditions (FIO2 = 20.9%), as well as at FIO2 = 40% and 80%. When compared with the predicted values of 2 of 40%. At FIO2 concentrations of 80%, however, the 2 allows a reliable evaluation of stress tests in patients requiring high doses of supplemental oxygen.Spiroergometric measurements of persons who require oxygen insufflation due to illness can be performed under conditions of increased inspiratory oxygen concentration (FIO With this method, patients inhale oxygen-enriched air from a gas reservoir. The performance of the patient can thus be determined by means of spiroergometry, without causing unnecessary physical and psychological stress due to breathing difficulties [2 lead to errors in the calculation of the oxygen uptake ?\" .2 leads to an overestimation of the measured 2 of 40% or greater, the error rate in the calculation of 2 = 100%, no valid results can be obtained. Similar findings on the inaccuracy of this measurement method are also provided in studies done by Wilmore et al. (1973) [2 \u2265 40%, 2 concentrations, with an ever-increasing 2 values. The authors also showed a parallel continuous decrease in RER, eventually reaching zero at high FIO2 concentrations. Walsh and Banister (1995) [2 concentrations. They found an increased 2 of 40%. However, higher FIO2 concentrations were not tested in this study. Yet further investigations confirm the effects of increased FIO2 on 2 concentrations [There is consensus in the literature that spiroergometry performed under conditions of increased FIO\" 2012) . At an F. 1973) . A validr et al.  and Stan3 . A valthe V\u02d9O2 . This he . At an 2, so the RER (presented as a ratio of 2) takes place, John Scott Haldane (1860\u20131936) defined a correction factor for the exact calculation of the inspiratory volume (VI). VI is elementary for the calculation of E) [2 = 20.9%), plausible 2 levels lead to different, nonlinear results: (1) The measurement of 2 of 40%. (2) At an FIO2 between 40\u201360%, O2 concentration measurements must be repeatedly calibrated. (3) At an FIO2 between 60\u201380%, inaccurate values are to be expected. (4) At an FIO2 greater than 80%, implausible data are provided. (5) At an FIO2 of 100%, calculation of the 2 levels greater than 40%.Ulrich et al.  explain n uptake . On the ume (VE) , 16. Forr (1986)  showed t Haldane 60\u20131936 d2, Hermann Eschenbacher developed the so-called \"Eschenbacher Transformation\" (ET) in 1987 [2 levels.To achieve a greater level of accuracy regardless of the selected FIO in 1987 , it seem2. The following hypotheses will be discussed:Following the description of the two computational transformations in terms of their basic theories and implementations , the pre2 = 20.9%) and FIO2 = 40%.Both transformations yield 2 > 40%), the HT produces invalid results in the calculation of At higher concentrations . Subsequent measurements were performed under conditions of increased oxygen concentration (FIO2 = 40% and FIO2 = 80%), each preceded by a 30-minute rest period. All stress tests were carried out with a stepwise increasing workload protocol on a cycle ergometer  and evaluated using the HT. After a two-minute reference phase at a load of 20 watts, the applied step protocol provides a ten-minute test phase with five two-minute stages at increasing loads (50-80-110-140-170 watts). The examination terminated with a two-minute recovery phase (20 watts). The so obtained raw data were also used to calculate the To test these hypotheses, 10 healthy, middle-aged men underwent spiroergometric examination with three different FIOrgometry  were car2 = 0; (2) it takes into account that for RER fraction  VI is different to VE; (3) the transformation must calculate the values corresponding to the load even with increased FIO2; and (4) it must be possible to calculate 2 = 100%. A transformation that fulfills these conditions is a further development of the Haldane Transformation, at least for measurements under elevated FIO2. So far there are no verified and validated studies with this transformation.The new approach of the ET is based on the complete spectrum of all oxygen concentrations. For this, the ET has to meet four basic conditions: (1) the formula is not based on the assumption that VNThe prediction values for ceptable , 14. Was2 on the ventilatory threshold was made to investigate further findings.From the obtained data, a first view at a potential influence of higher FIOSpirometry and diffusing capacity studies were performed with the MasterScreen Body/Diffusion and software JLab 5.72, the spiroergometric examinations with the Vyntus CPX and software SentrySuite 2.17, all provided by CareFusion Germany 234 GmbH . Masks and equipment were supplied by HansRudolph, Inc. . Statistical evaluation of the data sets  was done using the statistics software R . Images were created using Microsoft Excel . Differences were considered significant at p \u2264 0.05. To correct for multiple comparisons, p-values were adjusted with the Benjamini-Hochberg procedure. Using the Bland-Altman limit analysis , the calAll subjects participated voluntarily and received a small compensation. The participants provide their written informed consent to participate in this study. The Declaration of Helsinki was adequately addressed, and the study was approved by the ethics committee of the Hamburg Medical Association (register number PV5327).2 differs from classical spiroergometry only in that the subject breathes a gas mixture with a higher proportion of oxygen compared to indoor climate, from a reservoir equipped with a special valve system. The spiroergometry unit includes ECG leads, blood pressure and pulse oximetry, as well as a respiratory mask. The mask is directly connected to the Digital Volume Transducer and the collection hose for gas measurement. A two-way valve for inhalation and exhalation is connected and is coupled via an adapter to the gas bag into which the subject breathes. The O2 filling hose is joined directly to the O2 source and the gas bag is filled with the predetermined FIO2 concentration through a valve  were included in the study. The medical examination, spirometry, diffusing capacity measurements and the three spiroergometric measurements were carried out from September to December 2016, always over the course of a day. The ambient conditions could be kept almost constant despite the different measuring times . All participants fulfilled the inclusion criteria. 2 under standard conditions was 20.78 \u00b1 0.06%. A mean of 39.85 \u00b1 0.18% and 78.57 \u00b1 0.56% FIO2 were obtained for the measurements with increased FIO2 . 2 concentrations.The mean FIO2-level. At FIO2 of 20.78% a similar behavior of both examined transformations is shown. At FIO2 = 39.85% and especially at 78.57%, the HT has a much higher fluctuation range than the ET. The ET values show significantly less deviation, but are generally a little lower than the HT values. With increasing FIO2-concentration, the ET shows more accurate results , while the HT shows a constant large dispersion and deviation.2 = 20.78%), there were only slight differences in the results calculated with the HT and ET. Both transformations provide plausible results for Under normal ambient air conditions  did not occur until 140 watts (compared to 125 watts at FIO2 = 20.78%). In tests with an FIO2 = 78.57%, the first ventilatory threshold (VT1) was reached 136 \u00b1 63 seconds later than that achieved under normal conditions, at a workload of 158 watts. The second ventilatory threshold (VT2) also occurred later  in the protocol; in the tests with an FIO2 of 39.85%, the VT2 was not observed in seven subjects. The VT2 at an FIO2 = 78.57% was not achieved by any of the participants within the test protocol (2 = 20.78%), the time of VT2 could be determined in six of the ten subjects.Regardless of the transformation method, a shift of the ventilatory threshold in the transition from aerobic to anaerobic cellular respiration was observed with increasing workload in all ten subjects at higher FIOprotocol . Convers2 conditions, pointing to an improved load adaptation , ventilation (VE) and maximum oxygen uptake  and with an FIO2 of 39.85%, we could show that both transformations yield plausible 2 = 20.78%, this phenomenon is observed for both FIO2 concentrations. Despite this, both transformation equations can be used to generate plausible data for the FIO2 concentrations of 20.78% and 39.85%.On the basis of the 10 measurements taken under normal conditions (FIO2 > 40%)\u2014can only partially be confirmed and requires further exploration. Investigation into the first hypothesis shows that the use of the HT for 2 values \u2264 40%; oxygen insufflation at higher concentrations result in data inconsistent with predicted values. Indeed, our test results show significant deviation from predicted 2 > 80%. Determination of exhaled carbon dioxide (2 = 80%). The exact concentration of FIO2 for which the HT can still validly be employed cannot be definitively deduced from this study. The results obtained show a broad scattering of 2 = 40%.The second hypothesis\u2014that the HT does not produce valid 2 concentrations of 40% and 80%, the 2 \u2013can thus be confirmed. Furthermore, the scattering of results does not differ significantly for the varying oxygen concentrations. It can therefore be concluded that the ET is a suitable alternative to the HT for measurements at elevated oxygen concentrations.When the ET is applied at increased FIO2. Based on the power calculation as suggested by Kuzma (1998) [This is a pilot study with a small number of subjects (n = 10). In future studies this field should be further investigated with patient groups and other forms of increased FIOa (1998) , the sama (1998) . Another2 (here 80%). The general application of this method cannot therefore be recommended. The use of the alternative ET at higher FIO2 concentrations, however, produces valid results for 2 of 40% and 80%. In our opinion, the results of the study presented here allow us to extend the range of applications of spiroergometry. Through the implementation of the ET, spiroergometry at elevated FIO2 could provide significant added value for the stress testing of patients with oxygen insufflation.As with the classic spiroergometry the spiroergometry under increased oxygen concentration has to generate valid data. However, results of the HT used so far for the calculation of 2, the subjects also showed a shift of the ventilatory thresholds into higher load ranges. The next step, therefore, would be to confirm the findings obtained here in healthy subjects with those of patients suffering from lung disease. The applicability of \"spiroergometry under increased oxygen concentration\" could be extended and thus also be of benefit to seriously ill patients.In measurements with increased FIOS1 Appendix(PDF)Click here for additional data file.S1 FigThis picture is 2018 Vyaire Medical, Inc.; Used with permission (RD_0816-0130).(TIF)Click here for additional data file."}
+{"text": "We report a preterm female infant with intrauterine growth retardation, dysmorphic facies, missing rib, small hands and feet, and hemihypertrophy. The results of whole genome SNP microarray analysis showed approximately 77 Kb interstitial deletion of the short arm of chromosome 11 (11p15.4). We report novel clinical findings of this rare genetic condition. The deletion in this region in relation to hemihypertrophy (overgrowth syndrome) is novel. We report a novel case of 11p15.4 deletion and a review of the literature.Microdeletion of the short arm of chromosome 11 is a rare chromosomal anomaly. The \u201cpure\u201d deletion of 11p15 region with no other chromosomal imbalance is extremely uncommon. Beckwith-Wiedemann syndrome (BWS)/overgrowth syndrome is known to be associated with genetic and/or epigenetic alterations that modify imprinted gene expression on chromosome 11p15.5  11p15.4 x 1. The delThe clinical features of Beckwith-Wiedemann syndrome (BWS) include hemihypertrophy and/or macroglossia. Hypoglycemia is reported in 30-50% of the babies with BWS .  may play a role in the development of BWS. The ZNF214 and ZNF215 genes are located in one of the three regions on chromosome 11p15, called the Beckwith-Wiedemann syndrome chromosome region-2 (BWSCR2). The H19 (103280), ICR1 (616186), and KCNQ1OT1 (604115) are located at 11p15.5; and the CDKN1C (60856) is located at 11p54 [The genetics of BWS is complex and involves multiple genes on chromosome 11p15 \u20136. Threeat 11p54 , 8. Paternally imprinted gene would deactivate the activity of CDKN1C gene .The cyclin-dependent kinase inhibitor 1C (CDKN1C) gene encodes for making a protein that helps regulate growth. This protein acts as a tumor suppressor. It is also involved in controlling fetal growth and stops the developing fetus from becoming too large. The activity of the CDKN1C gene depends on which parent it was inherited from. the regulation of several genes that are normally controlled by IC2, including CDKN1C. Reduction in the activity this gene, which normally controls cell growth and division, leads to overgrowth and the other features of BWS. Rarely, BWS has been caused by the deletion of a small amount of DNA from the maternally inherited copy of the IC2 region.BWS is a condition that causes overgrowth and other signs and symptoms that affect various parts of the body. More than half of all BWS cases result from alterations in methylation of the imprinting center 2 region (IC2). The maternally inherited copy of the IC2 region shows a decrease in the methylation, and this abnormality adversely affectsIntrauterine growth restriction has been reported to be associated with both paternal and maternal 11p15 abnormalities , 10. At Deletion of the 11p15.4 region is associated with autosomal dominant hereditary persistence thalassemia  and autosomal recessive beta-thalassemia (OMIM: 141900) . It remains unclear what exactly is the relation between this deletion and the BWS/hemihypertrophy associated genes.We report on a preterm infant who had intrauterine growth retardation and developed hemihypertrophy during the postnatal stay. The microdeletion of 11p15.4 that was found in our case is likely to be paternally derived, since the patient's half-sister had an inherited anemia (family history). The additional features  in our patient have not been previously reported with deletion of the 11p15.4 region. Microdeletions of 11p15 are rare. There have been limited reports of patients with interstitial deletions involving band 11p15.4. To our knowledge none have reported clinical features in a neonate with the same deletion as in our case. Here, we provide additional human genetic evidence that the 11p15.4 deletion contains regulatory elements that play a mechanistic role in the hemihypertrophy BWS phenotype and intrauterine growth retardation (IUGR). microdeletion 11p15.4. These findings may help identify the gene implicated in BWS and IUGR.In summary, we report a novel case of a dysmorphic preterm neonate with"}
+{"text": "MEIS1, BTBD9 and MAP2K5/SKOR1 are the only known genes located within these loci and their association with RLS was subsequently confirmed in a number of follow up GWAS. Following this finding, our group reported the MEIS1 risk haplotype to be associated with its decreased expression at the mRNA and protein levels. Here we report the effect of the risk variants of the three other genes strongly associated with RLS. While these variants had no effect on the mRNA levels of the genes harboring them, we find that the homeobox transcription factor MEIS1 positively regulates the expression of the transcription co-repressor SKOR1. This regulation appears mediated through the binding of MEIS1 at two specific sites located in the SKOR1 promoter region and is modified by an RLS associated SNP in the promoter region of the gene. Our findings directly link MEIS1 and SKOR1, two significantly associated genes with RLS and also prioritize SKOR1 over MAP2K5 in the RLS associated intergenic region of MAP2K5/SKOR1 found by GWAS.Restless Legs syndrome (RLS) is a common sleep disorder for which the genetic contribution remains poorly explained. In 2007, the first large scale genome wide association study (GWAS) identified three genomic regions associated with RLS.  RLS is a circadian sensorimotor dysregulation disorder that can lead to severe sleep disturbances and impaired quality of life3. Epidemiological studies have established that RLS is a common neurological disorder with a prevalence of up to 15% in Central Europe and North America12.Restless Legs syndrome (RLS), also known as Willis-Ekbom disease (WED) or Wittmaack-Ekbom syndrome, is a common sleep-related sensorimotor disorder characterized by an urge to move the legs to relieve uncomfortable sensations. These sensations occur, or worsen, during rest, such as before falling asleep or during the night13. One of these loci was in an intronic region of the homeobox gene MEIS1. A second locus was in the BTBD9 gene, which encodes a BTB (POZ) domain. The third locus was between the MAP2K5 gene, which encodes a mitogen-activated protein kinase and the SKOR1 gene, which encodes a transcription factor 13. During the same year, an independent GWAS studying cases and controls from Iceland also identified an association between BTBD9 and periodic limb movements in sleep (PLMS), a motor feature strongly associated with RLS14. A third independent GWAS conducted with cases from the United States also replicated the association of MEIS1 and BTBD9 with RLS15. Two years later our group further confirmed the association between MEIS1 and RLS as we established that the intronic risk haplotype (rs12469063/rs2300478: GG/GG) led to decreased expression of the gene at both the mRNA and protein levels in both brain tissues and lymphoblastoid cell lines (LCL) derived from patients16.In 2007, a genome-wide association study (GWAS) conducted using German and French-Canadian RLS cases and control individuals identified variants in three genomic regions located on chromosomes 2p, 6p and 15q17. However, there can be negative side effects to the administration of dopaminergic drugs, like augmentation of overall RLS symptoms18. Iron deficiency anemia, end stage renal disease and multiple pregnancies increase the risk of RLS. The fact that iron supplements relieve symptoms in many of these cases strongly implicated iron in disease pathogenesis19. Studies suggest a dysfunction in the transfer of iron between the serum and the central nervous system, possibly involving the blood brain barrier22.The first line of treatment for RLS are dopamine agonists like levodopa, suggesting that reduced dopaminergic activity is involved in the pathogenesisMEIS1, BTBD9, MAP2K5 and SKOR1). The positive interaction between MEIS1 and SKOR1 was further explored using specific protein-DNA binding studies and promoter reporter assays.To extend on these previous reports and explore the pathogenic molecular mechanisms we looked for interactions between the different RLS-associated genes. First, we investigated the effects of the risk variants on the mRNA expression levels of the genes harboring them and the other RLS associated genes  to test for variations in the expression levels of these genes in RLS patients with these risk variants, similar to our previous report16.Given our previous report highlighting the decreased levels of BTBD9  that is located in intron 5 of BD9 Fig.\u00a0. When mRMAP2K5 and SKOR1. No effect was observed to be driven by this risk allele  on the mRNA expression of either MAP2K5  located in the intergenic region between 2K5 Fig.\u00a0 or SKOR1OR1 Fig.\u00a0; unfortuSKOR1 expression, which varied according to the MEIS1 risk haplotype. Using LCL we observed a significantly decreased expression (P\u2009=\u20090.0202) of SKOR1 in RLS cases who were homozygous carriers of the MEIS1 risk haplotype (rs12469063/rs2300478: GG/GG) by comparison to individuals who were homozygous for the MEIS1 non-risk haplotype (AA/TT). The same SKOR1 expression measures were made using thalamus samples obtained from individuals carrying the same MEIS1 haplotypes and these confirmed a similar decrease of expression(P\u2009=\u20090.0174). Analysis of the pons samples from the same individuals did not show a significant decrease of expression (P\u2009=\u20090.1519)  were found to be reliable as all were prone to produce various patterns of non-specific signals.After establishing the effect of each risk variant on the expression level of the genes harboring them, we looked for links between each of the risk variants and the other susceptibility genes. No significant effect on expression was observed between these genes except for the case of 19) Fig.\u00a0. The samMEIS1 risk haplotype (GG/GG) to be associated with decreased levels of both its mRNA and protein16. Hence, we decided to test if silencing MEIS1 would result in a reduction of SKOR1 mRNA. Using an siRNA  specifically targeting MEIS1 we reduced the expression of the gene (~70% mRNA decrease) in HeLa cells and found a coincident 64% decrease of SKOR1 mRNA level (P\u2009<\u20090.0001)  Fig.\u00a0. This obMEIS1 is a homeobox gene that encodes a DNA binding transcription factor, we hypothesized that it might regulate the expression of SKOR1 by binding to its promoter region. A literature search revealed that MEIS1 and members of another family of homeobox genes, PBXs  form in vivo heterodimeric DNA binding complexes with each other and their DNA binding is more intense when together than when either Meis1 or Pbx1 are alone26. We searched for the presence of MEIS1/PBX1 consensus binding sites27 , a second one at ~1.76\u2009kb upstream (TGACAGagTGAg) and a third one at ~2.6\u2009kb downstream of the ATG, in intron 2 of the canonical isoform of SKOR1 (GGACAGtaTGAT). Our search was focused on the hexameric consensus binding site of MEIS1. Moreover a search for the octameric consensus binding site of MEIS1 (TGATTG/TAT), as reported by Penkov et al., did not reveal any such sites in upstream regulatory region and the fist intron of SKOR1 gene28. It is also noteworthy that an examination of Meis1 ChIP-Seq (Chromatin immunoprecipitation followed by high throughput sequencing) data made in the mouse (GEO database #GSM2188919), shows two Meis1 binding sites upstream Skor1 gene (mm8 genome assembly) which suggest they are likely real elements involved in the activation of Skor1 . The purified proteins were used in electrophoretic mobility shift assay (EMSA) experiments testing the three different human MEIS1/PBX1 binding sequences (~8.7\u2009kb and ~1.76\u2009kb sites upstream to the ATG and the ~2.6\u2009kb site downstream to the ATG)   murine forms of the proteins (Meis1 or Pbx1) we prepared purified proteins using TG) Fig.\u00a0. The ~2.TG) Fig.\u00a0 and therTG) Fig.\u00a0. Overallype Fig.\u00a0, this in31. The average age at enrollment of RLS patients was 52.5\u2009\u00b1\u200914.9 years, with 38.5% men. Patients were diagnosed based on the international RLS study group (IRLSSG) criteria32. The average age at enrollment of the control population was 53.0\u2009\u00b1\u200916.1 years, with 38.2% men. There is no significant difference in sex and age between the cases and controls. The frequency of the risk allele (C) of rs4776976 is 0.848 in RLS cases and 0.776 in controls, respectively; and the genotype distribution fits with the Hardy-Weinberg equilibrium. The p value for the association test of the C risk allele is 0.0001267. The RLS risk allele (C) of rs4776976 is the most common allele in the European population (0.8) while the non-risk T allele of rs4776976 is the less frequent (0.2); as observed on gnom Aggregation Database33.An association study including unrelated and consecutively recruited RLS patients in Montreal, Canada (n\u2009=\u2009401) and controls (n\u2009=\u2009588) of European ancestry, recruited as previously describedSKOR1 expression and so our mRNA expression data was revisited using the two possible genotypes (C and T)  and so it was divided into two separate fragments (P1 contained the ~8.7\u2009kb binding site and nearby rs4776976 SNP (C or T) and P2 the ~1.76\u2009kb   Fig.\u00a0.MEIS1 siRNA, a significant decrease (\u223c40%) of luciferase expression occurred when driven by the P1 fragment with the rs4776976 C risk allele (P\u2009<\u20090.001), by comparison the P1 fragment with the T rs4776976 allele does not affect the luciferase expression. This suggests that the presence of C allele that represents the common RLS risk allele results in binding of MEIS1/PBX1 to the P1 segment of SKOR1 promoter region. When the luciferase expression was driven by the P2 fragment a significant decrease (P\u2009<\u20090.01) also occurred, thus confirming this potential MEIS1/PBX1 binding site was recognized by MEIS1 in our assay. This experiment demonstrates that MEIS1 physically acts as a direct activator of SKOR1 and shows that the rs4776976 SNP plays an important role for the binding of MEIS1 on SKOR1 promoter, where the presence of C allele results in SKOR1 gene being regulated by MEIS1.In the presence of 34. We previously showed that the RLS associated risk haplotype within the MEIS1 non-coding regions is associated with a decreased expression of this gene in the RLS patient LCL and thalamus samples16. Here we show that one of the consequences of this reduced expression is reduced expression of SKOR1. No other risk variants in the other GWAS RLS susceptibility loci show any significant correlation with the expression levels of the genes harboring them. Considering that MEIS1, a homeobox gene, is a transcription factor with precise temporal and spatial gene expression during development35, this link could be a result of transcriptional regulatory function of MEIS1 on SKOR1, which we confirmed using EMSA and a luciferase reporter assay.It is important to understand if genes identified using GWA studies interact with each other, how they exert their biological effects and which are the variants driving the associationSKOR1 ATG start site that is a regulatory SNP (rSNP), as it affects the MEIS1 regulation of SKOR1. We found that when the risk allele is present, reduced MEIS1 expression leads to reduced SKOR1 expression, whereas with the non-risk allele there are no changes in the expression of SKOR1 . These data suggest that a reduced level SKOR1 expression might be relevant to the development of a subset of RLS cases, and therefore it will be important to understand what are the downstream effects of reduced SKOR1 expression. Finally, these data also suggest that the variant driving the association with RLS in the SKOR1 locus is rs4776976.We also report a new SNP in the \u223c8.7\u2009kb upstream region of the SKOR1 strongly suggest that for the GWAS locus MAP2K5/SKOR1, the gene associated with RLS is SKOR1 and not MAP2K5. SKOR1 or SKI family transcriptional corepressor 1 is highly expressed in the central nervous system of human and mouse. In human, it appears to become more restricted to Purkinje cells of the cerebellum during adulthood36. The murine Skor1 interacts with a homeodomain transcription factor (Lbx1) to cooperatively repress transcription selectively in dorsal horn interneurons of the developing spinal cord37. It also appears to be a transcriptional corepressor which among others can regulate cell fate determination in murine dorsal spinal cord37. The necessity of Lbx1/Skor1 for generation of GABAergic phenotypes in dorsal horn interneurons of spinal cord potentially implicate them in the modulation of pain and sensory input processing of RLS40.Our findings directly linking MEIS1 and MEIS1 genomic region as well the MEIS1 protein as a transcription factor with the SKOR1 promoter sequences opens an avenue for future studies on the regulatory function of MEIS1 in the RLS pathogenesis mechanism as well as emphasizing the importance of the other candidate gene, SKOR1. Keeping in mind SKOR1\u2019s expression pattern, as well as its proposed function as a developmental co-repressor, we can proceed to the subsequent research into RLS molecular mechanisms with a greater focus on the regulatory function of MEIS1 and SKOR1, two highly significantly associated genes with RLS.In a post GWAS era, one of the initial strategies to progress from the tag SNPs to the mechanisms underlying the disorders is to use expression studies on human patient cells harboring risk variants. In this study, establishing a link between the risk haplotype within the 22.Brain tissue specimens  were obtained from autopsy brain tissues of 31 individuals with an RLS diagnosis from the Harvard Brain Tissue Resource Center. Final diagnosis was made by an RLS expert based on available questionnaires and medical records, but blinded to the genotype information; these samples were previously used in our earlier RLS studies2 in RPMI 1640 medium (Invitrogen) supplemented with 15% (v/v) heat-inactivated fetal calf serum (Sigma-Aldrich), 0.29\u2009mg/ml of l-glutamine, 100 U/ml of penicillin and 100\u2009\u00b5g/ml of streptomycin (Invitrogen). Healthy cells were harvested at an approximate density of 1\u2009\u00d7\u2009106 cells/ml.Selected LCL from RLS patients previously established by transformation with EB virus using standard protocols were grown at 37\u2009\u00b0C and 5% CO\u00ae Lipid Tissue kit (Qiagen) or from 5 million lymphoblastoid cells using the RNeasy\u00ae kit (Qiagen). Single-stranded cDNA synthesis was performed from 1\u2009\u00b5g of total RNA using a mix of oligo-dT and random primers and the Quantitect\u00ae Reverse Transcription kit (Qiagen) or the SuperScript\u00ae VILO\u2122 cDNA Synthesis Kit (Invitrogen). Quantitative RT-PCR was performed using the TaqMan method (Applied Biosystems) with probes and primers designed by Applied Biosystems recognizing BTBD9 (Hs00537653_m1) and MAP2K5 (Hs00177134_m1) and a custom probe designed at the junction of two exons of SKOR1 (AJ1RULU). PCR conditions were as follows: 50\u2009\u00b0C for 2\u2009min, 95\u2009\u00b0C for 10\u2009min, followed by 40 cycles at 95\u2009\u00b0C for 15\u2009sec (denaturation) and 60\u2009\u00b0C for 1\u2009min . Fluorescent signals were captured using the ABI PRISM\u00ae 7900HT Sequence Detection System (Applied Biosystems). The level of expression was determined by converting the threshold cycle (Ct) values using the 2\u2212\u2206\u2206Ct method. Expression levels were normalized using the human 18\u2009S ribosomal RNA (rRNA) gene with commercial primer-probe mix (Applied Biosystems) or the human polR2A control (Hs00172187_m1). All experiments were run in triplicate. Independent cDNA synthesis was carried out twice. ANOVA test and post-hoc Tukey\u2019s test was used for statistics of all tissues with three genotypes. t-test was used for statistics of BTBD9 expression in thalamus and pons with only two genotypes.Total RNA was extracted from 0.2\u2009g of frozen brain tissue using the RNeasyMEIS1 was realised by transfection of 100pmol of the MEIS1 siRNA  for 48\u2009hours in HeLa cells using the jetPRIME\u00ae (Polyplus) transfection reagent. The negative control cells were transfected with low GC duplex control siRNA. Total RNA was then extracted following a classical protocol using TRIzol\u00ae (Invitrogen) and SKOR1 expression measured as described above. This experiment was run in triplicate and t-test was used for statistics.The artificial 70% decreased expression of SKOR1 promoter has been tested at the three potential sites with an electrophoretic mobility shift assay. Using vectors (PCS2) designed to express either wild-type or mutated  MEIS1 or Pbx1 (provided by Dr. Featherstone), purified proteins were prepared using in vitro transcription/translation (IVTT) . On the other hand, DNA probes were prepared by annealing of the complementary following oligonucleotides  in SSC buffer, marked with dATP 32\u2009P using the Large (Klenow) Fragment DNA polymerase (Invitrogen) and purified using Microspin\u2122 G50 columns . The resulting proteins and DNA probes were incubated for 20\u2009minutes at room temperature with Poly DiDc 0,1 U/\u03bcL (Novagen) and BindBuffer  for DNA-protein interaction and the mix was then loaded on a 30% acrylamide:bisacrylamide gel which has been pre-run at 100\u2009V for 30\u2009minutes. Electrophoresis is performed at 125 Volts for 45\u2009minutes. The gel is then dried under vacuum at 80\u2009\u00b0C for 45\u2009minutes and autoradiography is performed with a time of exposure of 48\u2009hours.The binding of MEIS1 and Pbx1 on SKOR1 promoter into PGL3 vectors expressing the firefly luciferase gene. The ~20\u2009kb of SKOR1 promoter region is too long for its cloning as one piece so we cut this region into two segments; each one containing one of the potential MEIS1/Pbx1 binding site. P1 is the most upstream segment from the ATG and contains the ~8.7\u2009kb MEIS1/Pbx1 binding site. This fragment of 5,287\u2009bp was amplified with Phusion DNA polymerase (New England Biolabs) from BAC clone RP11-207J8 and using the following primers: fwd_5\u2032-gag ctc tta cgc gtg cta gcc cgg gct cga gag ggt gcc tgt ggt gtg gga cgg tag g-3\u2032, rev_5\u2032-ctg act aat tga gat gca gat cgc aga tct taa att gtc ttg acc cct tgc tgg ttt tt-3\u2032. This P1 fragment was produced with 2 alternatives  and using QuikChange\u00ae Site-Directed Mutagenesis (Agilent). The resultant PCR amplicons were then cloned into the XhoI site of pGL3-promoter vector (Promega) containing a SV40 promoter upstream of the firefly luciferase gene with the sequence and ligation independent cloning (slic) method41. P2 is the closest segment to the ATG start codon and contains the ~1.76\u2009kb MEIS1/Pbx1 binding site. This fragment of 1,535\u2009bp was amplified from same BAC clone using the following primers: fwd_5\u2032-gct ctt acg cgt gct agc ccg ggg tcg acg cca aaa aga ggg aag aac c-3\u2032, rev_5\u2032-cta att gag atg cag atc gca gat ctc gag acc agg tcc cac ttg act tg-3\u2032. The cloning of P2 fragment was identical to P1. Resultant clones were screened for the presence of the insert and the sequence of positive clones was verified using Sanger sequencing (Genome Quebec Innovation Centre).To perform luciferase assays, we inserted genomic fragments from the 2 and Student\u2019s t-tests were used to determine differences in sex and age, respectively. The association test between the marker rs4776976 and RLS was performed using the PLINK program42.Genotyping of rs4776976 (C/T) was done using TaqMan assays C__11771023_10, following the manufacturer\u2019s instructions. The genotypes were called using the Applied Biosystems 7900 Fast Real-Time Polymerase Chain Reaction (PCR) System and Safety Data Sheet (SDS) software (v. 2.2.2). The goodness-of-fit test with one degree of freedom (df) was applied to check for the genotype distribution deviation from the Hardy\u2013Weinberg equilibrium. The \u03c7MEIS1 or negative control) using the attractene transfection reagent (Qiagen). After 48\u2009hours, cells were rinsed with PBS and lysed with 100 ul of passive lysis buffer from the Dual-Luciferase\u00ae reporter assay (Promega). Firefly and Renilla luciferase activity was then read using the same kit and a Synergy 4 microplate reader (Biotek). ANOVA test was used for statistics. The luciferase expression was measured as a ratio between firefly and Renilla expression.At day 1, 100,000 HeLa cells per well were plated in a 24-well plate. At day 2, transfection was performed with 100\u2009ng or 200\u2009ng of one of the following vector expressing the firefly luciferase , 100\u2009ng of the vector containing the Renilla luciferase and 25 pmol of siRNA .All statistical analyses of expression studies were performed using GraphPad Prism 5.0 software (GraphPad Software Inc.) by one-way ANOVA followed by post-hoc Tukey\u2019s test for significant results. Student\u2019s t test was only used for Fig.\u00a0Lymphoblastoid cell lines (LCL) were prepared from individuals who signed an informed consent form before entering the study; the biobanking of LCL has been reviewed and approved by the institutional review board of McGill University . RLS brain tissues were provided by the Harvard Brain Tissue Resource Center, which is supported in part by a Public Health Service Grant (R24MH068855), with permission from the RLS Brain Bank Tissue Review Committee through the RLS Foundation. Control brain tissues were provided by the Douglas-Bell Canada Brain Bank . In the case control association study of rs4776976 with RLS, all participants signed informed consent at enrollment, and the study protocols were approved by the institutional ethics review boards. This study was approved by Comit\u00e9 d\u2019\u00e9thique de la recherche du Centre hospitalier de l\u2032Universit\u00e9 de Montr\u00e9al and McGill University ethics, all methods were performed in accordance with the relevant guidelines and regulations of McGill University (REB NEU-14-051).Supplemental Data"}
+{"text": "Carassius auratus in the Dongting water system  can perform normal meiosis. In artificial autotriploid Carassius auratus , female individuals undergo normal meiosis and produce mature gametes, while male individuals cannot. To better understand the effects of triploidization on meiosis in fish, we study the structure, methylation level, and expression level of meiosis-related genes  in diploid Carassius auratus , Carassius auratus red var., 3nCC and 3nRR. The results show that, compared with their diploid ancestors (2nCC and RCC), Dmc1 and Ph1 genes are hypomethylated in all 3nCC and female 3nRR, while are hypermethylated in male 3nRR. Correspondingly, Dmc1 and Ph1 genes are highly expressed in all 3nCC and female 3nRR, while are lowly expressed in male 3nRR. These results indicate that high expression of meiosis-related genes can contribute to restoration of bivalent pairing during meiosis in autotriploid Carassius auratus. This study provides new insights into the effect of DNA methylation on the fertility in triploid fish.Triploid is usually considered to be unable to perform normal meiosis due to the abnormal behavior of the three sets of chromosomes. But autotriploid  Polyploidization, the addition of a complete set of chromosomes to the genome, represents one of the most dramatic mutations known to occur Mallet . InterspDmc1 (DNA meiotic recombinase 1) and Ph1 (polyhomeotic-like protein 1) as important meiosis-related genes control the behavior of chromosomes during meiosis. The expression product of Dmc1 is a recombinant enzyme used in meiosis and plays a vital role during synapsis  (\u2640) \u00d7 Megalobrama amblycephala  (\u2642)  was found in the Carassius auratus  were randomly selected from the Dongting Lake water system. 3nCC came from the self-crossing offspring of the male and female triploid Carassius auratus that were randomly selected from the Dongting Lake water system. Red crucian carp  and 3nRR were provided by the Engineering Center of Polyploid Fish Breeding of the National Education Ministry located at Hunan Normal University. All materials were obtained during breeding season (April) (2017\u20132018) and were cultured in open pools (0.067\u00a0ha) with suitable pH (7.0\u20138.5), water temperature (22\u201324\u00b0), dissolved oxygen content (5.0\u20138.0\u00a0mg/L), and adequate forage. Fish treatments were carried out according to the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in China and approved by the Animal Care Committee of Hunan Normal University (Permit number: 4237). All samples were anesthetized with 100\u00a0mg/L MS-222  before blood collection. Peripheral blood cells were extracted surgically.Diploid Chromosome preparations were performed using the peripheral blood cell cultures of all samples. The chromosomes were prepared in accordance with Qin et al. . SpeciesTo detect the DNA content of 3nCC blood and sperm, a flow cytometer (Partec GmbH) was employed. Detailed steps were performed according to Xiao et al. .Ten RCC, 10 2nCC, and 10 3nCC at age 9\u00a0months were randomly selected for histological observation of gonad structure, while 10 3nRR were randomly selected for observation of 21\u00a0months. Detailed steps were performed according to Qin et al. .Extract RNA according to the instruction of Total RNA Kit I (Omega). The first-strand cDNA was synthesized using a PrimeScript\u2122 RT reagent Kit with gDNA Eraser . The obtained cDNA was stored at \u2212\u200920\u00a0\u00b0C.Ph1 and Dmc1 were designed based on the nucleotide sequences found in zebrafish and other Cyprinidae fish  was used to study the expression of hmittgen . Detailehmittgen .Ph1 and Dmc1 genes were found using the Ensembl (http://asia.ensembl.org/index.html) website. Methylation primers were designed by predicting the promoter region CpG islands by the MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) website . The Dmc1 gene coding regions of RCC, 3nRR, 2nCC, and 3nCC are all 1029\u00a0bp in length, encoding 342 amino acids. The sequences had been deposited in GenBank . Sequence analysis using Clustal W (2.1) and DNAMAN revealed the existence of high homologies between 3nRR and 3nCC at the protein level, with amino acid identities of 92.3% for Ph1, 100% for Dmc1  compared with 2nCC and RCC, but were lowly expressed in 3nRR male individuals (p\u2009<\u20090.05).Comparison of expression levels of rol Fig.\u00a0. The resPh1 and Dmc1 gene promoter regions of RCC, 3nRR, 2nCC, and 3nCC was shown in Table Ph1 and Dmc1 in male individuals of 3nRR were 0.983 and 0.883 (Table Ph1 and Dmc1 genes (Fig.\u00a0The methylation status of the 83 Table , respectnes Fig.\u00a0.Table 4MTriploids are traditionally considered sterile (Ramsey and Schemske Ph1 and Dmc1 are all important meiosis-related genes. The expression products of the former are necessary for meiotic synapse, and the latter is inducing specific binding of centromere. DMC1 deficiency leads to the abnormal formation of synaptic complex and the disordered separation of homologous chromosomes, and the deletion or mutation of Dmc1 in human and mice can lead to spermatogenesis disorder and premature ovarian failure (Kagawa and Kurumizaka Ph1 gene can promote homologous chromosome pairing, and it is also necessary for homologous chromosome pairing (Riley and Chapman Ph1 and Dmc1 gene coding regions of 3nCC and 3nRR were 99% and 100% respectively compared with their diploid ancestors. It is concluded that the Dmc1 and Ph1 genes of 3nCC and 3nRR have no significant difference in nucleic acid sequence and can encode amino acids normally. These results suggest that autotriploid has no significant effect on the structure of Ph1 and Dmc1 genes.Ph1 and Dmc1 genes are hypomethylated in all 3nCC individuals and female 3nRR individuals, while hypermethylated in male 3nRR individuals. The results of quantitative real-time PCR showed that Ph1 and Dmc1 genes were highly expressed in all 3nCC individuals and female 3nRR individuals, but a low expression in male 3nRR individuals. During the breeding season, 3nCC can extrude normal gametes and 3nRR can extrude eggs of different sizes without extruding semen. In meiosis, polyploids adopt a special mechanism of diploid meiosis to avoid the problem of gametes\u2019 imbalance caused by complex chromosome combinations in metaphase I (Maestra et al. Ph1 and Dmc1 genes, homologous chromosome pairing is promoted, resulting in the production of eggs of different sizes. Due to the high expression of the Ph1 and Dmc1 genes, the 3nCC has diploidized to some extent. The level of gene expression is regulated by the degree of methylation of the gene promoter region. In the data presented here, we cannot explain why the Ph1 and Dmc1 genes are hypermethylated in male 3nRR.Genome merging and doubling brings about plenty of gene redundancy (Soltis et al."}
+{"text": "Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kD heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of hsp70 using RNAi did not affect longevity.It has been known for over 20 years that  Drosophila 70 kD inducible heat shock protein (HSP70) is synthesized in salivary glands, Malpighian tubules, brain, wing imaginal discs and many tissues after a heat shock  have been previously obtained , and standardized to a single fly equivalent.The temporal profiles of GAL4 expression were obtained by quantification of \u03b2-galactosidase activity with a CPRG assay  staining  supplemented with protease inhibitors . Extracts were mixed with 50\u00b5l 2\u00d7 SDS loading buffer , incubated for 5 min at 100\u00b0 and centrifuged for 5 min at 10,000 g. 20 \u00b5g samples were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose membrane. HSP70-myc was detected with the mouse 9E10 monoclonal anti-myc antibody  at 1:5000 or the mouse 5A5 monoclonal anti-human HSP70 antibody  at 1:1000. \u03b1-Tubulin was used as loading control and detected with the mouse DM1A monoclonal anti-chicken \u03b1-Tubulin  at 1:5000. Primary antibodies were incubated at 4\u00b0 overnight. After washes, the nitrocellulose membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  at 1:5000 at 4\u00b0 overnight. The ECL advance Western blotting detection kit  was used for signal detection. Results were visualized and imaged with ChemiGenius bio-imaging system . ImageJ  was used for the measurement of optic density of specific bands. HSP70-myc level was normalized to \u03b1-Tubulin for quantification. Two-way ANOVA with Bonferroni post-test was performed with GraphPad Prism 5 to compare HSP70-myc expression levels.Control animals were obtained by crossing w males with homozygous UAS-hsp70 females (UAS control) and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS-hsp70 females with homozygous GAL4 driver males. For each cross, 20-30 males and 50 virgin females were mated for two days. At least 200 0-4 h eggs were collected from each cross and transferred with forceps onto petri plates filled with media . The plates were then incubated at 25\u00b0. Hatching rates were assessed 26-30h after egg transfer by scoring the number of empty eggshells. Pupation and adult emergence rates were scored 5-6 days and 10-12 days after egg transfer respectively.Control animals were obtained by crossing w males with homozygous UAS-hsp70 females (UAS control) and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS-hsp70 females with homozygous GAL4 driver males. 0-2 days old males and females were collected from control and experimental crosses and aged at 25\u00b0 in separate vials with media  until tested. Paraquat oxidative stress tests were performed as previously described  and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS females with homozygous GAL4 driver males. 0-2 days old males and females were sorted and collected from control and experimental crosses under nitrogen anesthesia for less than three minutes. The longevity of each genotype is obtained from four vials of males and four vials of females  kept at 25\u00b0. Animals were transferred to vials with fresh media and the mortality recorded at least twice a week. Four to eight replicates were measured. Each replicate used independent cultures and parents. Kaplan-Meier (Log-rank) analysis with GraphPad Prism 5 was used to determine the significance of experimental animals that displayed a mean longevity higher or lower than both kinds of control animals.hsp70 by the UAS-hsp70-RNAi transgenes. Supplemental Table S1 contains the data and statistical analysis of the survival during development. Supplemental Table S2 contains the data and statistical analysis of the stress resistance experiments. Supplemental Table S3 contains the data and statistical analysis of the longevity experiments with the UAS-hsp70 transgene. Supplemental Table S4 contains the data and statistical analysis of the longevity experiments with the UAS-hsp70-RNAi transgene. The reagent table provides the information and source of transgenic strains, DNA plasmids and antibodies used in this study. Supplemental material available at figshare: https://doi.org/10.25387/g3.9916223.Strains and plasmids are available upon request. All data necessary for confirming the conclusions of the article are present within the article, figures and supplemental files. Supplemental Figure S1 contains western-blot data obtained with an HSP70 antibody. Supplemental Figure S2 shows the outcome of the survival data to thermal, oxidative and metabolic stresses of 3 days and 10 days old females overexpressing HSP70. Supplemental Figure S3 shows the outcome of the longevity data of females overexpressing HSP70. Supplemental Figure S4 reports the cloning steps performed to obtain the pCX9 plasmid. Supplemental Figure S5 contains western-blot data that show the down-regulation of E. coli \u03b2-galactosidase  (P < 0.0001) but modest effect on the expression level.Transgenic UAS-hsp70 fly strains that carry the Hsp70Ab cDNA under the control of a UAS promoter have already been obtained and described (tibodies  and S1.  of GAL4 . Therefo of GAL4 . Three a of GAL4 . At ten  0.0001) . AlthougP < 0.0001) but not with the D42 driver (P = 0.5751). With both drivers, the location of the UAS transgene has a significant effect on the expression level and the range of expression obtained is wider than with the da-GAL4 driver. Between the genotypes with the lowest and highest expression level, there is a \u223c40% (10 days) to \u223c130% (3 days) difference with da-GAL4, a \u223c135% (3 days) to \u223c165% (10 days) with DJ694 and a \u223c410% (3 days) to \u223c560% with D42 (10 days).In agreement with the GAL4 expression profile, HSP70-myc levels increased significantly between three and ten days with the DJ694 driver . High temperature stress was tested by exposure at 36\u00b0. Oxidative stress was assessed by feeding the free radical generator paraquat. Desiccation stress was also examined with a dry starvation assay. At least three and up to 10 replicates were processed per stress, sex, age, UAS-hsp70 insertion and GAL4 driver. The results from each replicate are compiled in the The longevity of flies with both UAS-hsp70 and GAL transgenes was measured and compared to control flies lacking the GAL4 or UAS transgene (Table S3). Consistent results are only seen across all UAS-hsp70 insertions in males with the ubiquitous driver . All insLife span extensions were consistently obtained across replicates with only two (#3.2 and #10.1) out of the seven insertions tested. Both insertions driven by DJ694 or D42 extend the longevity of males in all replicates except for #3.2 that revealed an extension in 5 out of 6 replicates with D42. The extensions associated with #3.2 appear to be also consistent in the females but with a lower magnitude. The extensions associated with #10.1 is seen in females with DJ694 but not with D42. The increased longevity with these insertions is not correlated with the level of hsp70 expression. With D42, #3.2 and #10.1 express the highest and lowest levels respectively. With DJ694, #3.2 and #10.1 express high levels that are similar to the #9.1 insertion that does not display any change in all replicates. Since the extension of longevity is not observed with all the insertions tested and is not correlated with HSP70 levels, the longevity phenotypes observed with the #3.2 and #10.1 are an artifact of the transgene insertion site.hsp70 cDNA section was excised from the pCX1 plasmid . Seven out of the nine RNAi insertions completely prevented the detection of the transgenic HSP70-myc whereas it remains detectable with R5.2 and unaffected with R5.12. Next the effectiveness of the R3.12 insertion to reduce endogenous hsp70 expression was examined with the three GAL4 drivers . As one would expect from the anatomical location of GAL4 expression, almost complete or strong reduction of heat-induced HSP70 is observed with daGAL4 and DJ694 respectively whereas D42 expression is too localized to affect whole animal extracts. Finally, the effects of the DJ694-driven R3.12 and R5.14 insertions on endogenous HSP70 expression were compared (The compared . Both inThe longevity with both UAS-hsp70-RNAi and GAL transgenes was measured and compared to control flies lacking the GAL4 or UAS transgene (Table S4). Two out of the three RNAi insertions tested with all drivers did not show any effects in both sexes . These rHsp70 mRNA (Hsp70 are present (Hsp70 mutants have demonstrated that HSP70 is required for basal and induced thermotolerance of larvae and adults (Hsp70 with any of the three GAL4 drivers did not alter the response of adult flies to thermal, oxidative or metabolic stresses. Although it has long been known that increasing Hsp70 expression is sufficient to increase the thermotolerance of cultured cells (in vivo in the whole organism (Hsp70 expression is sufficient to affect thermotolerance of embryos (Hsp70 overexpression on the resistance to oxidative and wet starvation stresses has been investigated in the presence of additional Hsp70 copies and did not have any consequences with or without heat shock (Hsp70 expression alone is insufficient to affect the resistance of the organism to thermal, oxidative or metabolic stress.The UAS/GAL4 system allowed extensive and precise control of Hsp70 expression within a constant environment. The manipulation of HSP70 level during the pre-adult stages did not cause any visible morphological defect, lethality or developmental delays. Under normal conditions, p70 mRNA . Develop present  but the at shock . The UASHsp70 expression with RNAi transgenes did not affect longevity. Although the western-blot analysis confirmed the effectiveness of the transgenes, it remains possible that there is residual expression. The null Hsp70 mutant does completely prevent expression and has been recently reported to exhibit a modest reduction of the life span (Hsp70 is required for longevity and that only minimal amount is needed accounting for the lack of correlation between longevity and Hsp70 RNA levels in wild-type strains (Hsp70 levels above wild-type levels without any environmental modifications that affect the expression of additional genes. Ubiquitous GAL4-mediated Hsp70 overexpression reduced male longevity while ubiquitous overexpression by heat treatment of males carrying extra Hsp70 copies had the opposite effect (Hsp70 has detrimental effects on longevity that are mitigated or abolished by the simultaneous expression of one or several gene products induced by the heat treatment. After extended periods of recovery from heat shock HSP70 intracellular granules are formed to control HSP70 activity by sequestration (Hsp70 with the da-GAL4 driver during the pupal stage may not be detrimental enough to cause lethality and instead caused the emergence of sick animals. It is unlikely that the detrimental effect is due to overexpression in the nervous system or muscles since it is not observed with the D42 or DJ694 drivers. It will be worth investigating additional GAL4 drivers with different tissue-specificity in order to identify the anatomical location responsible for the detrimental effect in males.The reduction of Hsp70 in the nervous system, muscles in both sex or ubiquitously in females had neither beneficial nor detrimental effects on life span indicating that HSP70 is insufficient to extend longevity. It is important to note that identical results were obtained in muscles with the MHC-GAL4 driver and independent UAS-hsp70 strains (Hsp70 copies would suggest that HSP70 extends longevity in combination with one or several gene products induced by the heat treatment. The identification of HSP70 partners could easily be investigated by measuring the longevity of animals carrying a UAS-hsp70 transgene as well as a UAS transgene for a gene to be tested.The overexpression of  strains . However"}
+{"text": "Cdk6, which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6, other Pax6-regulated cell cycle genes are likely to be primary mediators of Pax6\u2019s actions on cortical progenitor cell cycles. Following acute cortex-specific deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6\u2019s actions. We then used flow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase.Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene  Cerebral corticogenesis requires precise spatio-temporal control of the rates of progenitor cell division. Previous research on the developing mouse embryo established that the transcription factor Pax6 is a key regulator of this process . Pax6 isCdks drive progression through the eukaryotic cell cycle by forming complexes with cyclins . In mostHere we addressed several questions. Are the effects of Pax6 on the cell cycle likely to be mediated primarily through actions on Cdk6, or does Pax6 control multiple cell cycle regulators in parallel? Do the effects of Pax6 vary between cell cycle phases? Are levels of Cdk6 normally constant throughout the cell cycle of cortical progenitors? Does Pax6 modulate Cdk6 levels equally or differentially across the phases?Pax6-/- mutants  to test for changes in the expression of a set of cell cycle regulators following acute cortical deletion of Pax6. The set we tested contained all cell cycle regulators that were shown in previous work to have altered cortical expression levels in constitutive  mutants . Here wePax6Sey allele . Dissociated cell suspensions diluted to 1.8 \u00d7 106 ml-1 were fixed and permeabilised in 100% ethanol and stored at -20\u00b0C.All mice were bred in-house. The University of Edinburgh Animal Welfare and Ethics Board and a license from the Home Office UK issued under the UK Animals (Scientific Procedures) Act 1986 regulated all procedures. For constitutive inactivation of Pax6, we used the 6- here; . For conP allele  and a grP allele  with BAC1CreERT2 . Dams wecDNA was synthesized from RNA with a Superscript reverse transcriptase reaction  and qRT-PCR was done with a Quantitect SYBR Green PCR kit  and a DNA Engine Opticon Continuous Fluorescence Detector . Primer pairs are listed in Table Aliquots of permeabilised and fixed cells suspended in fluorescence activated cell sorting (FACS) buffer were reacted with Hoechst 33342 . Primary antibodies used were: anti-Pax6 ; anti-Tuj1 ; anti-PH3 ; anti-Ki67 ; anti-Cdk6 . We used secondary-only and fluorescence-minus-one controls to set thresholds for primary antibody-specific staining. Cells were analyzed using an LSRII flow cytometer and FlowJo V10 software . In all of our experiments, we used the same gate for forward scatter for both wild-type and mutant progenitor cells. Since forward scatter values in flow cytometry are directly linked to cell size, this indicates that the wild-type and mutant cells were in the same size range.-1 IdU and then 1.5 h later with the same dose of BrdU. They were sacrificed after 30 min and histological sections were cut through the cortex. Primary antibodies used were anti-BrdU/IddU (which recognizes both BrdU and IddU)  and anti-BrdU . Nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes). Analysis was as described in  and littermate control Pax6fl/+;Emx1CreER embryos at E9.5. All embryos contained a floxed-stop GFP reporter allele  in gene expression at E12.5 and this was sustained at E13.5  of progenitors in Pax6+/+ and Pax6-/- E12.5 and E14.5 cortex using double labeling with iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) . At E14.5, Tc was \u223c15% shorter in Pax6-/- cortex than in Pax6+/+ cortex   . At E12.Pax6+/+ and Pax6-/- embryos . This was only the case in G1 at E14.5 (p < 0.01). These findings on rostral versus caudal differences are in excellent agreement with previous work showing a rostral [high] to caudal [low] gradient of Pax6 expression at E12.5 and E13.5 that flattens by E14.5  directly affect the lengths of these phases.Our new results support a central role for Cdk6 in Pax6\u2019s regulation of cortical progenitor cell cycles. Figure  Figures . OscillaThis proposal has similarity to the mechanism proposed to regulate an engineered minimal control network in fission yeast . This covice versa. An additional complexity would be explaining the dynamics of this scenario. Changes in Pax6 protein levels would need a certain time to influence transcription of the Cdk6 gene and the mRNA would then need to be translated to alter Cdk6 protein levels. Although we do not exclude the possibility that fluctuations in Pax6 levels might contribute to the temporal pattern of change of Cdk6 levels, the simplest suggestion is that Pax6 reduces Cdk6 levels overall and that the oscillations in Cdk6 levels during the cell cycle are driven by other mechanisms. Our previous work showed that Pax6 overexpression significantly reduced Cdk6 levels . G1 length is increased in neurogenic progenitors compared with proliferative progenitors . This isDM, MM, and Y-TH performed the experiments. DP and JM helped to design the experiments, obtained the funding, and supervised the work. DP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Density functional theory was applied to simulate the structure of crystalline KAlF4 while a quantum chemistry ab initio simulation was performed to explore the structure of molten KAlF4. Two crystal polymorphs demonstrated to be present in solid KAlF4. At the temperature below 673 K, it belongs to the tetragonal crystal system within the P4/mbm space group, while the high temperature phase is attributed to the monoclinic crystal system within the P21/m space group. Both polymorph KAlF4 phases are characterized by a layered structure consisting of K+ and [AlF6]3\u2212 octahedra, each of the [AlF6]3\u2212 octahedra equivalently shares four corners with other four [AlF6]3\u2212 octahedra along the layer. The layered structure became unstable at higher temperatures and crashed when the temperature exceeded the melting point. It demonstrated that the molten KAlF4 consisted of predominant [AlF4]\u2212 and a small amount of [AlF6]3\u2212. The Raman spectrum of molten KAlF4 simulated by using a quantum chemistry ab initio method agreed well with the experimental Raman spectrum.In situ high temperature X-ray diffraction and Raman spectroscopy were used to investigate the temperature dependent micro-structure of KAlF Akdeniz concluded that the Raman peaks observed at 621 cm\u22121 for NaAlF4 are related to the Al-F bond of isolated [AlF4]\u2212 clusters with a bond length of 1.69 \u00c5 18]) vers4, a simulation based on a quantum chemistry ab initio method was also carried out. A series of aluminum fluoride model clusters with typical characteristic structures were proposed and optimized for geometric configuration before the simulation. The restricted Hartree-Fock (RHF) 3\u2212 octahedra, each [AlF6]3\u2212 octahedron equivalently shares four corners with other four [AlF6]3\u2212 octahedra within the layer. The [AlF6]3\u2212 octahedron consists of a six-coordinated aluminum atom which is linked to six fluorine ions including four bridging fluorine ions and two non-bridging fluorine ions. 4 crystal, drawn by the VESTA software 3\u2212 octahedra, where each [AlF6]3\u2212 octahedron shares four corners with other [AlF6]3\u2212 octahedra within the layer. The potassium ions between the [AlF6]3\u2212 octahedron layers were used to keep the charge balanced. Comparing with the room temperature phase, the structure of the high temperature phase is more distorted due to the temperature impact, resulting in a more complicated XRD spectrum, as evidenced in 4 was calculated  from room temperature to 823 K and at a heating rate of 5 K/min afterwards. The high temperature spectra were recorded 5 min after the sample reached the target temperature. 6]3\u2212n clusters (by ab initio method) which refers to our previous work 3\u2212 octahedra with the high temperature phase. As clearly shown in 4 structure as it evolves from the room to high temperature structure. The room temperature and high temperature phase are drawn in a simple diagram. In 4 turns from the tetragonal to monoclinic structure approximately at 723 K, the space group changes from P4/mbm to P21/m. The cell length becomes much longer, and one of the cell angles increases from 90.000 to 108.478\u00b0. As a consequence, the distribution of the bond length and bond angle of KAlF4 becomes more complicated.Two new Raman bands at 319 and 481 cmAs the sample temperature increased further, the sample was fully melted at 893 K and is characterized by the molten structure as evidenced in 4]\u2212 is the main species in molten KAlF4. 4]\u2212n model clusters presented in molten KAlF4. After being optimized for geometry configuration, these model clusters were used for a quantum chemistry ab initio method to calculate the Raman spectra. The characteristic vibrational wavenumber of the Al-F symmetric stretching vibrational modes of the [AlF4]\u2212n clusters were then estimated accordingly.According to the literatures ,30, [AlF4]\u2212 tetrahedra in the neighbor of an isolated [AlF4]\u2212 tetrahedron in the melt according to the crystal KAlF4 structure in KAlF4. In 4]\u2212n cluster. As the number of molecules in the clusters increased, the characteristic wavenumber of the Al-F symmetric stretching vibrational mode of [AlF4]\u2212n appeared to increase. The symmetric stretching vibrational wavenumber of the model cluster with seven molecules of [AlF4]\u2212 was therefore estimated to be 625.6 cm\u22121, which was in good agreement with the values observed in the experiment and reported in the literature \u2212 tetrahedron, appeared at 863 K. Simultaneously, the intensity of the Raman band of 628 cm\u22121 increased while the intensity of the Raman band of 536 cm\u22121 decreased. The six-coordinated Al atom began to turn into four-coordinated. At 893 K, KAlF4 had been completely melted, revealing the symmetric stretching vibrations of AlIV-Fnb of the [AlF4]\u2212 tetrahedron and little symmetric stretching vibrations of AlVI-Fnb of the [AlF6]3\u2212 octahedron were in the melt.As shown in 4 was investigated by in situ high temperature X-ray diffraction and Raman spectroscopy. The major vibrational modes of temperature dependent Raman spectra of KAlF4 were assigned based on the calculated spectra by both density functional theory and quantum chemistry ab initio methods.The structure evolution of KAlF4 was found to present in two different polymorph phases at room and high temperature. Both polymorph KAlF4 phases are characterized by a layered structure consisting of K+ and [AlF6]3\u2212 octahedra, where each [AlF6]3\u2212 octahedron equivalently shares four corners with other four [AlF6]3\u2212 octahedra along the layer. While the room temperature phase is in the tetragonal cell (space group P4/mbm), the high temperature phase undergoes a monoclinic distortion (space group P21/m) together with an ordering of the K atoms.Below the melting point, solid KAlF4]\u2212 tetrahedra when the temperature exceeds the melting point. It was hence indicated that in molten KAlF4, there is a principal amount of [AlF4]\u2212 and a small amount of [AlF6]3\u2212. This was further confirmed by quantum chemistry ab initio simulations.The layer structure decomposed with increasing temperature and finally turned to [AlF"}
+{"text": "ELF5) and ETS\u2010homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis\u2010regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site\u2010specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC\u2010seq in these lines revealed novel peaks of open chromatin at the 5\u2019 end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C\u2010seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP\u2010seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA\u2010mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.E74\u2010like factor 5 ( Of note, mutations in CFTR do not correlate with lung disease severity, the major cause of morbidity in CF.Many monogenic disorders have significant phenotypic variability that may be attributed to genetic elements outside the causative locus. These sites (modifier loci) are often identified by genome\u2010wide\u2010association studies (GWAS).CFTR. The high frequency of the disorder facilitated a replicated GWAS, which identified single nucleotide polymorphisms (SNPs) in an intergenic region of chromosome 11p13 that significantly associated with CF lung disease severity.EHF) and E74\u2010like factor 5 (ELF5) on the 5\u2019 side and Apaf\u20101\u2010interacting protein (APIP) on the 3\u2019 side.CF impacts about 70,000 people worldwide EHF is abundantly expressed in differentiated epithelial tissues, particularly in the prostate, pancreas, salivary gland and airway.CFTR regulation, as it binds to an airway selective enhancer element of CFTR and represses its expression.EHF is a strong candidate CF modifier gene and also a potential CF therapeutic target since elevated CFTR substrate may enhance the efficacy of correctors and potentiators.EHF expression, since ChIP\u2010seq with an antibody specific for ELF5 showed its occupancy at the EHF promoter and first intron in the breast cancer cell line T47D.In contrast, the role of ELF5 has been studied in breast cancer, where it drives lung metastasis by recruiting myeloid\u2010derived suppressor cells.cis\u2010regulatory elements (CREs) at chr11p13 that may control the expression of these two epithelial selective ETS transcription factors. Using CRISPR/Cas9 technology, three key CREs were deleted from the region in airway epithelial cells, and subsequent changes in gene expression and chromatin architecture were examined. We also took an unbiased screening approach to identify additional CREs at chr11p13 by activating multiple DNase I hypersensitive sites (DHS) across the region using CRISPRa,EHF by investigating open chromatin and 3D architecture in cell types with different abundance of ELF5. These genomic context studies revealed a critical role for ELF5 in coordinating the complex regulatory environment at chr11p13. Our results are relevant to the involvement of both ELF5 and EHF in lung disease, since both genes are expressed in human bronchial epithelium.Although to date, the highest p\u2010value SNPs identified in the replicated GWAS do not correlate with the transcription of genes at 11p13, this may reflect a paucity of data on gene expression at the single\u2010cell level. However, revealing the regulatory interactions of the EHF and ELF5 TFs is critical to understanding their role in lung epithelial biology. Here we investigate the 22.1A549,2.2Luciferase promoter:enhancer constructs were generated as described previously.2.3EHF intron 6, 11.2521 and 11.2516 were identified using Benchling Two pairs of gRNAs flanking 2.4RNA was extracted from cells using TRIzol (In Vitrogen) according to manufacturer's instructions. Reverse transcription used TaqMan Reverse Transcription Reagents (Life Technologies) by the standard protocol. qPCR assays were performed using SYBR Green reagents (Life Technologies) and primers listed in Table 2.5Chromatin immunoprecipitation (ChIP)\u2010seq was performed as previously described 2.64C\u2010seq was performed as previously described 2.7ELF5 and EHF promoters were designed within 1\u00a0kb 5\u2019 to the transcriptional start sites (TSS). Two to four sgRNAs were designed to target DHS cores at chr11p13 using Benchling Single guide RNAs (sgRNAs) targeting 2.8Omni\u2010ATAC\u2010seq was performed on 50,000 Calu3, LNCaP and T47D cells as described previously2.9ELF5 depletion in T47D cells was performed using Silencer select siRNA  as previously described.2.10t tests. All statistical analysis was performed in Prism software (GraphPad).Error bars denote standard deviation (SD) or standard error of the mean (SEM) as noted in figure legends, which also define the statistical analysis used. This was either two\u2010way ANOVA plus multiple comparisons tests or Student's unpaired 33.1EHF locus coincides with an extended track of the H3K27ac active histone mark, indicative of a stretch enhancer in airway epithelial cells.EHF intron 6 lies at the 3\u2019 end of this feature and coincides with a region of high occupancy by multiple transcription factors (ENCODE data EHF intron 6 (right) are shown in Figure ELF5, EHF and APIP genes was assayed by RT\u2010qPCR  expression.The R Figure B. RemovaEHF are recruited to the intron 6 element or that loss of this site alters chromatin architecture at the locus, thus facilitating occupancy of activating factors at the EHF promoter or its enhancers. To investigate the latter hypothesis, we first assayed CTCF occupancy at specific sites across the chr11p13 region by ChIP\u2010qPCR. CCCTC\u2010binding factor (CTCF) is an architectural protein involved in higher order chromatin organization. It is recruited to invariant sites in the genome  boundaries) in addition to variant structural elements that may be cell\u2010type specific.EHF intron 6 element binds CTCF with high affinity in several airway cell types,EHF intron 6 element was associated with a substantial (~3\u2010fold) decrease in CTCF occupancy at the adjacent site DHS11.2516 , most notably close to 11.2516 and within the first intron of EHF. With a viewpoint located 20kb 5\u2019 to the EHF promoter (11.2516), interactions with the ELF5 and EHF promoters were notably reduced and those with HB11.1485 element were slightly diminished (black arrows). These results are consistent with our earlier data from WT cells showing that DHS11.2516 and the EHF intron 6 element interact directly.EHF promoter viewpoint shows a modest loss of interactions across the whole region, most prominently between HB11.1485 and the ELF5 promoter (black dotted line). In summary, these data suggest that looping of the CRE at DHS11.2516 to the EHF and ELF5 promoters is somewhat dependent on the EHF intron 6 element and moreover that this element contributes to the maintenance of locus architecture.To examine the impact of loss of the s Figure D. A mini3.2ELF5 and EHF promoters (though not APIP), by luciferase assays in 16HBE14o\u2010 bronchial epithelial cells.ELF5, EHF and APIP expressions were measured using RT\u2010qPCR .The 11.2521 CRE was shown previously to be a strong enhancer of both the ELF5 and EHF promoters in transient assays,ELF5 upon deletion of this CRE suggests that features of the genomic architecture of the region may have a dominant role in regulating gene expression. To address this hypothesis, we measured CTCF occupancy at known binding sites across the region using ChIP\u2010qPCR. The 11.2521 CRE itself binds CTCF with low affinity in 16HBE14o\u2010 cells though not in other airway cell types.Since the 11.2521 CRE is an enhancer of the ELF5 promoter, loss of the 11.2521 CRE increased its interactions with DHS11.2512/11.2513 and an uncharacterized element immediately 3\u2019 to EHF (black arrows). However, the deletion had no impact on interactions with the HB11.1485 viewpoint, while associations with the EHF promoter were modestly reduced (black dotted line). These data suggest that the 11.2521 CRE may inhibit ELF5 promoter interactions with previously uncharacterized enhancer elements, and that its functions depend upon specific chromatin architecture at chr11p13.Next, to investigate the impact of removal of the 11.2521 CRE on chromatin architecture across chr11p13, we performed 4C\u2010seq on the deletion clones and compared them to WT A549 c9 cells , by luciferase assays in 16HBE14o\u2010 cells.ELF5, EHF and APIP expressions were measured using RT\u2010qPCR .The 11.2516 CRE was also shown previously to be a strong enhancer of both the R Figure B. RemovaEHF intron 6 (~1.4\u2010fold increase), though these alterations did not reach statistical significance . These data are consistent with our previous results showing direct interaction of 11.2516 with these sites.ELF5 promoter, EHF promoter and EHF intron 6 (black arrows). Loss of the 11.2516 CRE also coincided with a modest reduction of interactions between the EHF promoter and the rest of the region, with the exception of an unidentified element located between HB11.1485 and 11.2516 (black arrow), which showed an enhanced association. These data suggest that 11.2516 has an important role in the maintenance of chromatin architecture at the chr11p13 region.Next, to examine the impact of removal of the 11.2516 CRE on chromatin architecture, we performed 4C\u2010seq on the deletion clones and compared them to WT A549 c9 cells Figure D. Again,3.4ELF5 transcript abundance is very low ELF5 to EHF transcripts is highest in T47D cells, and the reverse is seen in the LNCaP line \u2010seq ELF5).None of the airway cell lines used in this work and our earlier analysis of the chr11p13 region w Figure . HoweverELF5 and EHF loci in the different cell lines  between the ELF5 promoter viewpoint, the novel peak of open chromatin at 11.2513 and a site in EHF intron 1, also associated with an ATAC\u2010seq peak. Interactions with the 3\u2019 TAD boundary and an element 3\u2019 of 11.2527 were also seen.Since expression of EHF promoter viewpoint, the major specific difference seen in Calu3, LNCaP and T47D cells was a great reduction in interactions with the HB11.1485 5\u2019 sub\u2010TAD boundary when the ELF5 gene is highly expressed (T47D) (black arrows). A minor reduction of interactions with the EHF intron 6 CRE was also observed in LNCaP and T47D cells (black arrows). In addition, in Calu3 cells only, interactions were evident with the airway enhancers (11.2516 and 11.2521), EHF intron 6 and the 11.2525/11.2526 sites closest to the highest p\u2010value SNP (rs10742326) from the GWASELF5.Using the 3.5ELF5 this locus may have an important role in the functional genomics of the chr11p13 region. Our earlier in vitro studies suggested that the 11.2516 and 11.2521 CREs enhanced activity of both the ELF5 and EHF promoters p65\u00a0subunit [p65], and Replication and transcriptional activator [Rta] (SP\u2010dCas9\u2010VPR) to target sites. A minimum of two guide RNAs were designed to target the ELF5 and EHF promoters and multiple airway DHS across chr11p13 in 16HBE14o\u2010 and A549 cells . Expression of ELF5 in 16HBE14o\u2010 cells was also increased significantly by CRISPRa at the EHF intron 6 (~3.5\u2010fold), 11.2521 (~7.5\u2010fold), 11.2522 (~3\u2010fold), 11.2523 (~5\u2010fold), 11.2529 (~4.5\u2010fold) and 11.2530 (~4\u2010fold) DHS . The modest response to CRISPRa at chr11p13 suggests that either additional sequences, which are not associated with regions of open chromatin in airway cells, are key in transcriptional activation of ELF5 and EHF, or that a more complex regulatory mechanism exists for the genes in this region. The latter explanation would be consistent with our 4C\u2010seq observations suggesting close interactions between the ELF5 and EHF genes and their CREs.The data shown in the previous sections suggest that in cells that express s Figure . Targetis Figure . APIP ex3.6ELF5 and EHF loci and their CREs might underlie the complex regulatory environment at chr11p13. We first performed siRNA\u2010mediated depletion of ELF5 in T47D cells, which express ELF5 and EHF, and assayed expression of both genes by RT\u2010qPCR. Primers located in exons 5 and 6 for ELF5 and exons 3 and 4 for EHF  analysis of two replica experiments is shown in Figure EHF locus, most notably in introns 1 and 6 and close to its 3\u2019 end (black arrows). Each of the peaks of ELF5 binding also coincides with a peak of open chromatin in T47D cells, mapped by ATAC\u2010seq , suggesting that ELF5 may have a major impact across 11p13, both through controlling other genes and by autoregulation. These results are highly relevant to primary bronchial and tracheal epithelial cells, which also express both ELF5 and EHF, albeit with much lower ELF5 abundance than in T47D cells.A previous study4ELF5 and EHF within the region and defined important aspects of the chromatin architecture relevant to the regulation of genes at chr11p13.EHF locus.Intense interest in the chr11p13 region arose through its association with lung disease severity in CF; however, the genetic mechanisms underlying the functions of this region are not well understood. Our data reveal a complex regulatory network at chr11p13 that coordinates the expression of the ETS transcription factors ELF5 and EHF. We previously identified several putative airway epithelial cell\u2010selective enhancer elements of EHF intron 6 and the two airway epithelial cell\u2010selective enhancers at 11.2516 and 11.2521 using CRISPR/Cas9 protocols had unexpected effects on gene expression, and were associated with modest changes in CTCF occupancy and local higher order chromatin structure. Firstly, removal of the two enhancers that were shown to activate the ELF5 and EHF gene promoters in transient luciferase assaysELF5 or EHF expression. Removal of either element enhanced ELF5 transcript abundance and had no impact on EHF levels. Though these data do not support a simple enhancer role for the 11.2521 and 11.2516 CREs, these elements are both marked with active histones (H3K27Ac) and furthermore lack repressive marks in several airway cell types, supporting their role as enhancers in the genomic context.ELF5 and EHF gene expression levels in their endogenous location, perhaps providing functional redundancy. These factors may include features of the higher order chromatin structure and organization. This interpretation would be consistent with the luciferase assays showing enhancer activity, since within the reporter gene constructs potential CREs are sited next to a gene promoter without consideration for architectural barriers such as CTCF sites. It is also possible that eRNA transcription arising at these CREs may repress nearby gene expression.EHF intron 6  and an altered chromatin architecture associated with expression of ELF5. Two sites are of particular interest: 11.2513, is a peak of open chromatin in primary HTE cellsEHF locus, which is not evident in primary HTE cells, but is clearly seen in both ELF5\u2010expressing cell types studied here. Upon removal of the 11.2521 enhancer from airway cells, the ELF5 promoter gains interactions with this element , which are essential for cell motilitySPDEF),MUC5AC) gene,EHF locus and represses its expression, it is likely that this ETS factor also plays an important role in lung biology. Modulation of ELF5 expression would be predicted to impact many of the EHF\u2010mediated processes discussed above. Although EHF remains the primary candidate for influencing lung phenotype differences observed in CF patients, ELF5 likely also plays an important role through its regulation of EHF expression. These observations are not only relevant to CF but also to other airway diseases associated with inflammation, defective wound response and lung tissue remodelling, including asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis.Returning to the role of chr11p13 as a modifier of lung disease severity, we do not address here the mechanisms underlying the association of high p\u2010value SNP close to the chr11.2525 site. However, our observations on cell\u2010specific recruitment of CREs across the modifier region and most importantly on the direct regulation of The authors confirm that there are no conflicts of interest.HS, JSE, KML and AH conceived and designed the project. HS, JSE, KML, SY, JLK, MN, CC, EMM and S\u2010HL designed and performed experiments. HS and AH wrote the manuscript.\u2009Click here for additional data file.\u2009Click here for additional data file."}
+{"text": "All proliferating cells need to match metabolism, growth and cell cycle progression with nutrient availability to guarantee cell viability in spite of a changing environment. In yeast, a signaling pathway centered on the effector kinase Snf1 is required to adapt to nutrient limitation and to utilize alternative carbon sources, such as sucrose and ethanol. Snf1 shares evolutionary conserved functions with the AMP-activated Kinase (AMPK) in higher eukaryotes which, activated by energy depletion, stimulates catabolic processes and, at the same time, inhibits anabolism. Although the yeast Snf1 is best known for its role in responding to a number of stress factors, in addition to glucose limitation, new unconventional roles of Snf1 have recently emerged, even in glucose repressing and unstressed conditions. Here, we review and integrate available data on conventional and non-conventional functions of Snf1 to better understand the complexity of cellular physiology which controls energy homeostasis. Cell growth and proliferation require a high amount of energy for biosynthetic pathways. Cells take energy from nutrient intake and both unicellular and multicellular eukaryotes have evolved systems that allow dynamic sensing of energy sources, mainly sugars. The class of Snf1/AMPK (Sucrose non-fermenting/AMP-activated protein kinase) plays a key role as a guardian of cellular energy Saccharomyces cerevisiae and its conventional roles in the regulation of metabolism, stress response and aging. In addition, we also focus on recent advances showing emerging functions of Snf1 on the modulation of key processes such as endocytosis and cellular trafficking as well as cell cycle, proliferation and metabolism.Here we discuss the mechanisms of action of Snf1, a member of the Snf1/AMPK family in Protein kinase Snf1 in yeast is a heterotrimeric complex made by the catalytic \u03b1 subunit Snf1, a regulatory \u03b2 subunit  and the \u03b3 subunit Snf4 SNF1 gene) was identified in a screening of mutants unable to grow in presence of sucrose as carbon source 5The catalytic \u03b1 subunit  are present. They share partially redundant functions, since only the triple mutant sip1\u0394sip2\u0394gal83\u0394 strain showsgrowth defects when glycerol or ethanol are added as carbon sources 711In SNF1, the gene encoding the \u03b3 subunit, SNF4, was identified by isolation of a sucrose non fermenting mutant 10SNF4 deletion causes a decreased kinase activity of Snf1, whereas deletion of the AIS domain of Snf1 fully complement the phenotype of a snf4\u0394 strain 19snf4\u0394 strain Similarly to sak1\u0394tos3\u0394elm1\u0394 strain shows the same phenotype of a snf1\u0394 strain, such as growth defects in presence of limiting glucose or alternative carbon sources like glycerol or ethanol Snf1 complex is activated through phosphorylation of the Thr210 of the \u03b1 subunit by one of the three constitutively active upstream kinases Sak1, Tos3 and Elm1 22Although Snf1 phosphorylation is a key step for its activation, a non-phosphorylatable Snf1 mutant (Snf1-T210A) retains a low catalytic activity, originating intermediate phenotypes 24262728On the other side, in response to high glucose concentrations Snf1 is inactivated through dephosphorylation of Thr210 by the Gcl7 protein phosphatase , which is targeted to Snf1 by the adaptor subunit Reg1 303233Active Snf1 phosphorylates serine and threonine residues contained in the consensus pattern \u03a6-x-R-x-x-S/T-x-x-x-\u03a6, where \u03a6 is a hydrophobic residue Differently from its mammalian homolog AMPK, yeast protein kinase Snf1 is not allosterically activated by AMP 37The structure of the kinase domain of Snf1 showed that it is a dimer, which represents an inactive form of the kinase, since Thr210 is inaccessible for phosphorylation by the activating kinases (i) phosphorylation of Ser214, inside the activation loop, downregulates Snf1 function (ii) SUMOylation of the catalytic \u03b1 subunit Snf1 inhibits its activity, possibly by attenuating its levels in the cell and/or favoring the inactive conformation of the kinase (iii) the SAGA acetyl transferase complex deubiquitylates Snf1 affecting the stability of the complex and its kinase activity  (iv) the ubiquitin-associated motif (UBA) of the \u03b1 subunit Snf1 indirectly regulates SNF1 gene expression and Snf1 interaction with the \u03b3 subunit Snf4 Interestingly, some recent evidence indicates additional mechanisms that regulate Snf1 activity: http://www.yeastgenome.org, 92 of which are also Snf1 substrates (identified by high throughput or low throughput assays). In Figure 2 we clustered them on the base of their function. Apart from the most known interactors involved in Snf1 complex regulation, transcription, histone modification, signaling and metabolism, there are many proteins which regulate translation, ribosome function, intracellular transport/trafficking and cell cycle. In addition, some of them are also proteins of the ubiquitin/proteasome machinery, chaperones and Fe/S cluster proteins . Remarkably, only a few of them have been extensively investigated for the physiological relevance of Snf1-dependent phosphorylations, suggesting that many functions of Snf1 are still to be discovered.A total of 216 proteins physically interacting with Snf1 are annotated in SGD , maltose (the MAL regulon) and galactose (GAL4) HXT2, HXT4) TPS1, essential for the metabolism of trehalose Mig1 is the most important glucose-regulated transcriptional repressor 4852CAT8 gene is activated by Snf1 through inhibition of Mig1 Besides Mig1, Snf1 regulates the activity of other transcription factors. Cat8 and Sip4, which bind Carbon Source Responsive Elements (CSRE), regulate the expression of gluconeogenic genes 4961ADY2 gene. In fact, Snf1 stimulates the binding of Gcn5 and the acetylation of histone H3 at ADY2 promoter, promoting the transcription of this gene Snf1 has been reported to phosphorylate Ser10 of histone H3 and to promote the acetylation on Lys14 of histone H3 by Gcn5, a component of the SAGA complex INO1 expression 65Besides its role in regulating the transcription of several genes involved in metabolism, Snf1 directly regulates, through phosphorylation, important metabolic enzymes. In fact, together with the class of transcription factors and regulators, proteins linked to metabolism are the most abundant among Snf1 interactors . Probably the most impactful function exerted by Snf1 as a direct regulator of metabolism is the regulation of the acetyl-CoA carboxylase Acc1 Grr1-dependent degradation of Pfk27 Snf1 was also shown to phosphorylate Pfk27, the second isoform of 6-phosphofructo-2-kinase Moreover, Snf1 phosphorylates Gpd2, the glycerol-3-phosphate dehydrogenase required for anaerobic growth, thus inhibiting glycerol synthesis during the diauxic shift. In fact, it was reported that Snf1 phosphorylates Gpd2 on Ser72 priming Gpd2 for subsequent phosphorylation on Ser75, probably by Yck1 In yeast, the main pathway activated by glucose is the PKA pathway, involved in metabolism, growth and proliferation 71727176Notably, recent data nicely complement observations of a cross-talk between Snf1 and PKA. Indeed, the adenylate cyclase Cyr1 and Snf1 interact in a nutrient-independent manner The Target Of Rapamycin Complex I (TORC1) is a highly conserved nutrient-responsive regulator of cell growth and metabolism in all eukaryotes 80818381858780Kog1 is phosphorylated by Snf1 under glucose deprivation, as AMPK does on the ortholog Raptor in vitro kinase assay and epistasis analysis, Sch9 has been shown to be also a target of Snf1, indeed total Sch9 phosphorylation is reduced in snf1\u0394 mutant In the presence of nutrients, TORC1 phosphorylates and activates Sch9, the ortholog of S6K in yeast which, together with other substrates, drives ribosome biosynthesis 9192Interestingly, Orlova and coworkers showed that rapamycin treatment results in a significant increase of Thr210 phosphorylation on Snf1 snf1\u0394 cells, the translocation of GFP-Atg8 to the vacuole is reduced by 50% compared to the wild type TORC1 activity is involved in the regulation of autophagy, a cellular recycling system that degrades proteins and organelles by delivery to the vacuole in response to nutrient deprivation 98ENA1, responsible for Na+ ions detoxification Besides nutritional deprivation, Snf1 is also involved in the response to other cellular stresses. Snf1 activity protects against toxicity caused by cadmium 106SNF1 gene and Snf1 activation cause increased and decreased resistance to ER stress, respectively snf1\u0394 cells are more sensitive to tunicamycin, a known inducer of ER stress In addition, protein kinase Snf1 regulates the Unfolded Protein Response (UPR), the evolutionary conserved pathway activated when improperly folded proteins accumulate and induce endoplasmic reticulum (ER) stress 109111Thus, even though these data indicate an interesting role for Snf1 in the regulation of ER stress response, more work is needed to understand the underlying molecular mechanism.Interesting results from Simpson-Lavy and collaborators recently show cross-talk between Snf1 and protein kinases involved in DNA damage (Mec1/ATR and Tel1/ATM) Given the role of AMPK kinase in the regulation of energy homeostasis, it is not surprising the existence of a strong relationship between aging and the AMPK pathway 115116117118The first evidence of the involvement of Snf1/AMPK in the aging process in yeast occurred several years ago when Asharafhi and co-workers discovered that Snf1 activity increased in replicative aging even in the presence of abundant glucose in the environment SNF1 gene have a very short CLS 125On the contrary, in a yeast model of Chronological Life Span , Snf1 activity is critical for the extension of CLS in caloric restriction condition and cells deleted for Taken together these data indicate that Snf1 activity promotes longevity in CLS while accelerates RLS, showing distinct and opposite mechanisms in the regulation of aging, as also reported for other proteins in yeast 129Recent data suggest that Snf1 function is not limited to conditions of carbon limitation. One of such functions is the regulation of proteins involved in endocytosis and cellular trafficking, indeed several interactors and substrates of Snf1 are in this cluster . Snf1 interacts with and phosphorylates the \u03b1-arrestin Rod1 27131Snf1 also regulates Arf3 135snf1\u0394 and snf1as mutant, whose activity can be chemically inhibited) show a slow growth phenotype and an increased fraction of cells in G1 phase, in synthetic medium supplemented with 2% glucose 2525Several reports indicate that Snf1 could be active in the presence of high glucose 54133137The relevant role of the catalytic activity of Snf1 in regulating proliferation in glucose-repressed condition is further supported by the fact that Snf1 loss reduces the expression of G1-specific genes 25SNF1-T210A mutant shows a slow growth phenotype and a delayed G1/S transition. 25Snf1-T210 is weakly phosphorylated in 2% glucose, confirming that it is partially active in that growth condition 21139Snf1 exerts its function in mitosis too and active Snf1 is localized at the division site from the time of bud emergence to cytokinesis snf1\u0394 cells, further supporting the emerging roles of Snf1 in non-limiting nutrient condition too It is amazing that cells lacking Snf1 and growing in synthetic media containing 2% glucose, show an extensive transcriptional reprogramming, being the most upregulated genes mainly involved in transmembrane transport and metabolic processes such as aminoacid biosynthesis, iron homeostasis and redox metabolism Many studies have reported that AMPK activity is altered in several diseases, such as inflammation, diabetes and cancer 145147145Saccharomyces cerevisiae is a powerful model for studying fundamental aspects of eukaryotic cell biology and to validate the increasing downstream targets of the class of Snf1/AMPK protein kinases which control the complexity of cellular physiology.Therefore, the expansion of the repertoire of AMPK substrates, as well as more in-depth studies of the molecular mechanisms by which AMPK is activated, will help to better understand the roles of this kinase in the regulation of human pathologies. In this context, the yeast unicellular organism"}
+{"text": "It is a clear liquid at room temperature, allowing it to be mistaken for water if unlabelled. Ingestion of H2O2 can have serious sequelae even in low volumes and concentrations.We present a case study of H2O2 poisoning and discuss the pertinent radiographical findings.Hydrogen peroxide (H It is a clear liquid at room temperature, allowing it to be mistaken for water if unlabelled. Ingestion of H2O2 can have serious sequelae even in low volumes and concentrations. We present a case study of H2O2 poisoning and discuss the pertinent radiographical findings.Hydrogen peroxide  and mildly hypertensive (155/100 mmHg) but not hypoxic (SpO2 97% on room air). A clinical examination revealed dysphonia with mild erythema and oedema of the oropharynx and uvula; the abdominal and respiratory examinations were otherwise unremarkable. Blood and biochemical investigations were also normal.A healthy 21-year-old male unintentionally ingested approximately one mouthful of 3% HA CT scan was performed to exclude gastrointestinal perforation, which demonstrated pneumatosis and mucosal thickening throughout the stomach and proximal duodenum, as well as extensive portal venous gas \u20133.\u22121 was commenced prior to transfer to a tertiary intensive care unit.The patient was intubated owing to concerns for developing airway involvement and gastrointestinal perforation, and conservative treatment with intravenous piperacillin/tazobactam 4.5 g four times daily and intravenous pantoprazole infusion at 8 mg hourAn upper endoscopy performed 3 days after the ingestion was normal, with no evidence of mucosal injury. A repeat CT scan at this time revealed interval partial resolution of the bowel wall thickening and complete resolution of the pneumatosis and portal venous gas . The pat2O2 is an oxidizing agent that is available in concentrations ranging from 3% to 90%.1 It is found in numerous household products, including disinfectants, hair dyes, bleaches and stain removers. It is a clear, colourless liquid at room temperature,2 allowing it to be mistaken for water if unlabelled, as in this case. Recently, H2O2 has also been sold for consumption in small volumes following promotion of its purported natural health benefits,3 despite multiple documented fatalities from its ingestion2 and no evidence to demonstrate health benefits of any kind. Upon contact with the enzyme catalase in the gastric mucosa, H2O2 undergoes rapid decomposition into oxygen and water (2H2O2 \u2192 2H2O + O2 + heat).4 If the amount of oxygen liberated exceeds the maximum solubility of blood, bubbles migrate through the epithelial interstices and gas embolism may occur, manifesting as pneumatosis3 or gas within the portal venous system,5 brain6 and coronary arteries.7 Toxicity is also caused by direct caustic injury to the gastric mucosa, resulting in gastritis and potential rupture, as well as cytotoxicity from lipid peroxidation.1 This particular case is unusual because gas embolism usually only occurs with ingestion of the stronger 35% H2O2 solution, with only a few other published case reports of portal venous gas following ingestion of the 3% solution.H2O2, a common household item, can have serious sequelae even in low volumes and concentrations.Ingestion of HRadiographical findings may include gastritis, pneumatosis, perforation and portal venous gas."}
+{"text": "Myo10 in pigmentation, with a phenotype unlike those of Myo5a or Myo7a. Adult mice homozygous for a disrupted Myo10 allele on a C57BL/6N background displayed a high degree of penetrance for white patches on their abdomen and dorsal surface. Forepaw syndactyly and hind paw syndactyly were also observed in these mice. Tail epidermal wholemounts showed a complete lack of melanocytes in the hair follicles and interfollicular epidermis. Myo10 has previously been implicated in human pigmentation. Our current study reveals involvement of Myo10 in murine skin pigmentation.Myosins are molecular motors that are well known for their role in cell movement and contractile functions. Although extensively studied in muscle physiology, little is known about the function of myosins in mammalian skin. As part of the Sanger Institute Mouse Genetics Project, we have identified a role for  ATPadenosine triphosphateHair\u2010GELHair Gene Expression LibraryMyo10Myosin 10Myo5aMyosin 5aMyo7aMyosin 7aTrp1tyrosinase\u2010related protein 1UVultravioletWTwild type1Skin pigmentation is a highly variable and conserved trait among living organisms. It plays a critical role in social communication, camouflage, mimicry and protection against the harmful effect of UV radiation.Myo5a), Myosin 7a (Myo7a) and Myosin 10 (Myo10) are unconventional myosins and are known to have functions in melanocytes and neurons.Myo5a and Myo7a are known in skin pigmentation, whereas the in vivo involvement of Myo10, while previously studied in primary human cell culturesMyosins are among the motor proteins involved in melanosome cargo transportation. Functionally, myosins are a large superfamily of ATP\u2010dependent motor proteins that translocate actin filaments. Other functions include cell adhesion, motility, endocytosis, exocytosis and cytokinesis.2Does Myosin 10 play a role in murine skin pigmentation?3Myo10tm2/tm2 C57BL/6N mice from the Sanger Institute Mouse Genetics Project,Myo10 , Myo10tm2/tm2 mice typically displayed abnormal dorsoventral coat patterning with white belly spots and extensive depigmented areas in the tail. Depigmentation was limited to the tail tip in control mice . Unlike Xenopus Myo10 mutants, Myo10tm2/tm2 mice did not show any cranial or skeletal abnormalities.To assess the efficiency of knockout of the s Figure A. SimilaMyo10tm2/tm2 mice prompted us to investigate whether the skin had any melanocyte abnormalities. Confocal imaging of tail epidermal wholemounts revealed a complete lack of pigmented melanocytes in the hair follicles and interfollicular epidermis  showed two distinct clusters of interactions for Myo5a and Myo10  database.Myo10 is predominantly expressed in melanocytes in embryonic and neonatal mice, it is also expressed in other skin compartments such as epidermis and dermal fibroblasts  Myo10 expression in WT and Myo10tm2/tm2 tail. Myo10 probe spans exons 28\u201029. RQ value is relative to endogenous control gene B2m. (B) Haematoxylin and eosin staining of the tail and dorsal skin of Myo10tm2/tm2 and WT mice. (C) Myo10tm2/tm2 tail skin sections immunolabelled with the antibody to the melanocyte stem cell marker c\u2010Kit show the absence of melanocytes in hair follicles when compared to WT (arrows). Asterisks indicate nonspecific staining. (D) STRING interaction network of Myo10 and Myo5a shows two distinct classes of interacting proteins. (E) Gene expression results obtained from Hair\u2010GEL database show high expression of Myo10 in melanocytes at E14.5 and widespread expression in all skin subpopulations at P5. FPKM, Fragments Per Kilobase of transcript per Million mapped reads; Scale bars 100\u00a0\u03bcm.Click here for additional data file.Data S1\u2002Materials and MethodsClick here for additional data file."}
+{"text": "Reactive oxygen and nitrogen species have cell signaling properties and are involved in a multitude of processes beyond redox homeostasis. The peroxiredoxin (Prdx) proteins are highly sensitive intracellular peroxidases that can coordinate cell signaling via direct reactive species scavenging or by acting as a redox sensor that enables control of binding partner activity. Oxidation of the peroxidatic cysteine residue of Prdx proteins are the classical post-translational modification that has been recognized to modulate downstream signaling cascades, but increasing evidence supports that dynamic changes to phosphorylation of Prdx proteins is also an important determinant in redox signaling. Phosphorylation of Prdx proteins affects three-dimensional structure and function to coordinate cell proliferation, wound healing, cell fate and lipid signaling. The advent of large proteomic datasets has shown that there are many opportunities to understand further how phosphorylation of Prdx proteins fit into intracellular signaling cascades in normal or malignant cells and that more research is necessary. This review summarizes the Prdx family of proteins and details how post-translational modification by kinases and phosphatases controls intracellular signaling. The relative reactivity of these species spans several log orders of magnitude to affect the half-life and distance traveled within the intracellular environment. The hydroxyl radical is the most reactive of these species with a half-life on the order of 10\u22129 s, while H2O2 is much less reactive in comparison with a half-life of 10\u22123 s [In biological systems, redox reactions can regulate intracellular levels of reactive oxygen (ROS) and nitrogen species (RNS) to control a multitude of intracellular processes. These reactive species include both radical and non-radical forms of ROS and RNS such as superoxide anion  [2O2 load as well as the extracellular secretion of H2O2, which can reach 2 \u00b5M in stimulated neutrophils [There are diverse sources of ROS that emerge from several organelles within the cell. The intracellular concentration of Hto 10 nM . Mitochopiration , but thepiration  and the  in vivo . Complex in vivo ,8 and II in vivo  produce by MnSOD . The stevely low  based onM\u22121\u00b7s\u22121)  and mitoM\u22121\u00b7s\u22121) . Other oM\u22121\u00b7s\u22121) , the endM\u22121\u00b7s\u22121) ,15 and lM\u22121\u00b7s\u22121) . The plaM\u22121\u00b7s\u22121) , prostagM\u22121\u00b7s\u22121) , lipoxygM\u22121\u00b7s\u22121)  and xantM\u22121\u00b7s\u22121)  and tranM\u22121\u00b7s\u22121) , respecttrophils . Reactive species have a complex relationship in cancer. An overabundance of reactive species can cause cancer initiation and drive progression and accumulation of additional DNA mutations to support further growth . Ant2O2 is a particularly important reactive molecule with second messenger function. Spatiotemporal features of the molecule enable it to possess reactivity, yet move through biological macromolecular microenvironments intracellularly within different organelle compartments and intercellularly to neighboring cells. Organ and cellular systems coordinate the balance of the H2O2 through enzymatic-catalyzed reductant proteins in conjunction with the pro-oxidant sources mentioned above to form an interconnected network that maintains homeostasis or drives oxidative signaling. The enzymatic metabolism of H2O2 is primarily catalyzed through the action of catalase, glutathione peroxidases (GPx) and peroxiredoxins (Prdx). While all three enzymatically metabolize H2O2, important biochemical and biological differences exist. Insight into the basal function of the three enzymes in vivo can be drawn from the phenotypic effects observed in gene knockout studies. Deletion of catalase displays no overt pathological changes under basal conditions in mice, while GPx1 knockout mice were found to be smaller sized [Her sized  and harber sized ,29,30. Ter sized . Deletioer sized ,33. Prdxer sized .2O2 via an iron heme porphyrin complex [2O2 is enzymatically scavenged through oxidation followed by reduction of the active site heme iron. The active site heme is initially oxidized to an oxyferryl species, which is subsequently reduced by a second H2O2 molecule to regenerate enzymatic function. Sequestration of catalase to a single organelle enables control of peroxisomal H2O2 levels but also requires H2O2 derived from other intra- and extracellular sources to diffuse to the peroxisome in order for catalase-dependent catalysis to occur. Catalase exhibits a high H2O2 turnover rate [m close to 100 mM in human erythrocytes [2O2 concentrations are low. Contrasting catalase, GPx family members are found within several organelles. There are eight family members in the glutathione peroxidases (GPx) family (GPx1-8). GPx1-4 and 6 utilize a selenocysteine active site and glutathione (GSH) as a co-factor to reduce H2O2 [. GPx1 and 4 are present in most tissues, with GPx1 expression found in the cytoplasm and mitochondria, while the phospholipid hydroperoxide reducing GPx4 is found in the plasma membrane and cytoplasm [5 M\u22121 s\u22121 [Catalase is localized within peroxisomes and catalyzes the decomposition of H complex . A two-sver rate , but seqhrocytes , yield auce H2O2 ,38. GPx1 M\u22121 s\u22121 . These p2O2 to water. The family is subdivided into groups classified as 2-Cys (Prdx1-4), atypical 2-Cys (Prdx5) and 1-Cys (Prdx6) isoforms based on their structure and mechanism of action [p) is conserved among all family members at roughly 50 amino acids from the N-terminus. The Cysp is highly reactive to H2O2 due to its surrounding amino acids, which yield rate constants on the order of 106 to 108 M\u22121 s\u22121 [p under conditions of heightened H2O2 concentrations, resulting in a cysteine sulfinic acid, which can be further oxidized to a Cys sulfonic acid to cause irreversible inactivation of the enzyme [p [2O2 induced sulfinylation reactions that are characteristically observed in typical 2-Cys Prdxs [The Prdx family has six members (Prdx1-6) that are present in many cellular compartments. Prdx1, 2 and 6 are located in the cytoplasm, and nucleus; Prdx3 is localized to the mitochondria; Prdx4 is found in the endoplasmic reticulum; and Prdx5 is located in the peroxisomes, cytoplasm and mitochondria . The Prdf action . The act M\u22121 s\u22121 . Typicale enzyme ,44. Typinzyme [p ,44,45. Tys Prdxs ,45,46.p to the mechanistically important resolving Cys located on the opposing Prdx homodimerization partner near the C-terminus. Prdx1 and 2 homodimers can associate non-covalently to form larger decameric complexes that are ordered as a pentamer of dimers to form a doughnut-like structure [p is oxidized by H2O2 to a sulfenic acid moiety, which then forms a disulfide bond with the resolving Cys on the homodimerization partner [p can become overwhelmed in the presence of high levels of H2O2 and become overoxidized to form Cys sulfinic or further sulfonic moieties that lack peroxidase activity. The rate constant of the sulfenic acid form of Prdx2 with H2O2 to form the sulfinic Prdx is on the order of 104 M\u22121 s\u22121 [p can be adducted with GSH (rate constant 500 M\u22121 s\u22121) under physiological concentrations to protect from overoxidation and recycle with Grx1 [Homodimerization of Prdx proteins in a an N-terminus head to C-terminus tail fashion enables 2-Cys family members to align the Cystructure ,46. In 2 partner   enable protein tyrosine kinases (PTKs) to phosphorylate downstream targets without PTP-dependent removal of the phosphate moiety. PTPs are more readily targeted by H2O2 due to the enhanced reactivity of the low pKa active site cysteine residue, which causes increased susceptibility to oxidation compared to PTKs. Cell signaling cascades, therefore, utilize local H2O2 to coordinate signal transduction pathways to promote growth [2O2 within different intracellular compartments, cells require not only the production of H2O2 but the inactivation of peroxidases to drive phenotypic responses. As Prdx proteins are highly sensitive and enzymatically responsive to low levels of H2O2, the cell has developed pathways to inhibit peroxidase activity within the cell during cell proliferation. Suppression of Prdx1 activity by phosphorylation has been shown to play a role during the cell cycle in two different intracellular compartments, namely at the plasma membrane during growth factor signaling and within the nucleus during the transition to mitosis.Outside of oxidation of the Cysch as pH , ionic sch as pH  and tempch as pH . Expressch as pH ), but toe growth ,68. To d2O2 through the action of Nox [2O2 production by NOX1 is not sufficient alone to promote and sustain signaling [2O2, thereby allowing target proteins such as the Src family kinases to become oxidized [2O2 near lipid rafts is necessary to both simultaneously activate Src family kinases and inactivate PTPs. H2O2-mediated inhibition of PTPs is therefore crucial for growth factor signaling as activation of PTKs alone is not adequate to promote sufficient levels of protein tyrosine phosphorylation [A classic counter-example to the redox relay cell signaling coordination by Prdx described above is provided by phosphorylation of Tyr194 on Prdx1 by Src . Local in of Nox  at the pn of Nox ,72. Of iignaling ,69. Rathp was found to be protected from overoxidation in phosphorylated protein during co-treatment of cells with growth factor and H2O2. In vitro studies of the phosphorylated protein found the dimeric form of the Prdx1 was present without decamers. In vivo wound healing experiments exhibited a peak in Tyr194 phosphorylation after 1 day, which remained for 1 week.Phosphorylation on Tyr194 in response to treatment of cells with EGF or PDGF displayed isoform selectivity for Prdx1 in comparison to Prdx2 . Studies2O2 accumulation through inactivation of Prdx1 by phosphorylation is also important in the nucleus during mitosis [2O2 [2O2 enables active Cdk1-cyclin B to transition cells to late mitosis where Prdx1 can be dephosphorylated to promote deactivation of Cdk1 via dephosphorylation by the now activated Cdc14B. The importance of the centrosomal H2O2 during the transition to mitosis was evaluated by expressing catalase fused to a centrosomal targeting sequence, which inhibited entry into mitosis. Local H mitosis . During sis [2O2 . Inactiv2O2 in vitro and ex vivo. In the absence of TOPK via siRNA decrease, cells were more sensitive to UVB-induced apoptosis. Melanoma cells expressing wild-type but not Ser32Ala Prdx protein was more resistant to UVB irradiation. Whether Prdx2 can also be phosphorylated by TOPK is unknown but may not be seen in experiments with RPMI7951 cells, which only express high levels of Prdx1. While the studies above indicate post-translational modifications that serve to reduce peroxidase activity, not all Prdx phosphorylation is repressive to enzymatic activity. The T-cell-originated protein kinase (TOPK) can phosphorylate Prdx1 on Ser32 to enhance peroxidase activity . Investi2 (PLA2) activity, which is unique to the Prdx family. As Prdx6 phosphorylation increases PLA2 activity, phosphorylation of the protein may mirror protein overexpression seen in bladder [2 activity [2 (aiPLA2) functions to degrade surfactant phospholipids and aide DPPC  synthesis through the deacylation-reacylation pathway, by providing the required substrate, lyso-PC. Phosphorylation of Prdx6 provides an important functional role as a PLA2; phosphorylated Prdx6 maintains activity at both acidic and cytosolic pH [2 activity through the MAPK pathway [2 activity compared to Thr177Ala protein. Phosphorylation of Prdx6 has been found to attenuate phospholipase A bladder , liver [ bladder  and lung bladder ,79,80 caactivity . Acidic solic pH . Treatme pathway . ERK1, E\u2212/\u2212Prdx6 PMVEC displayed dramatically decreased plasma membrane translocation of the NOX2 components Rac1 or p47phox following Ang II stimulation. Upon treatment with the known MAPK inhibitor, U0126, Prdx6 phosphorylation, PLA2 activity and ROS were significantly decreased. This result suggested that MAPK activation mediates phosphorylation of Prdx6 resulting in its translocation to the cellular membrane, whereby, PLA2 activity can function to promote NOX2 activation [2 activity when compared to wild-type protein [Phosphorylation of Prdx6 on Thr177 was found to be critical for activation of NOX2 in pulmonary microvascular endothelial cells (PMVEC) by angiotensin II (Ang II) or PMA . Phosphotivation . Whethertivation ,83. Chhu2O2 in the cell, phosphorylation of Prdx proteins has been tied to cell death. Interestingly, Prdx1 phosphorylation at Thr90, which aids in driving the cell cycle through mitosis in the nucleus as described above, can also be targeted by tumor suppressor proteins. The difference between activation of survival or death pathways based on phosphorylation of the same protein at the same amino acid highlights the importance of location and timing of Prdx phosphorylation and the possible involvement of additional phosphorylation of Prdx1 at Thr183 to control activity. The following sections will detail the mechanistic studies that implicate Prdx phosphorylation with death. While some examples below highlight neurodegenerative pathways as examples of inactivation by phosphorylation, further cancer-focused work needs to be undertaken to extend these mechanisms as both Cdk5 and LRRK2 activity have been implicated in cancer. Cdk5 activity has been noted to be involved in proliferation, migration, invasion, metastasis, the epithelial to mesenchymal transition, the DNA damage response and angiogenesis in many forms of human cancer . The LReference ,86. 2O2 cause Prdx1 to bind Mst1, which was found to promote Mst1 activation and enhance apoptosis [2O2. In contrast, phosphorylation of the Thr90 residue of Prdx1 by Mst1 and possibly Mst2 has been described to inactivate peroxidase activity [\u2212/\u2212Prdx1 mouse embryonic fibroblasts (MEFs) showed heightened levels of the DNA damage biomarker phosphorylated Ser139 H2AX following treatment with H2O2. This finding suggests that the inactivation of Prdx1 by Mst1 could potentially cause a positive feedback loop whereby excess H2O2 further activates Mst1. That inactivation of Prdx1 by phosphorylation can promote Mst1 activation, while knockdown of Prdx1 can inhibit Mst1 activity, suggests that a more complex pattern of regulation is present that may involve both oxidation and phosphorylation.Mammalian sterile 20\u2013like kinase-1 (Mst1) and Mst2 have been shown to suppress tumor formation in the liver and intestines in vivo ,88,89,90poptosis . Loss ofactivity . Full-leactivity . Whetheractivity . Express+ treated cells in vitro and MPTP treated mice in vivo. Furthermore, Cdk5 activation was shown to mediate dopaminergic (DAergic) neurodegeneration by downregulation of Prdx2 peroxidase activity which ultimately leads to significant elevations of intracellular ROS and DAergic neuron death [Prdx2 phosphorylation has been primarily investigated in pathological brain diseases and injury. Mitochondrial dysfunction and excessive oxidative stress are believed to be critical facilitators of the pathogenesis of Parkinson\u2019s disease (PD). Neurons have oxidative metabolism systems in place, such as Prdxs, to manage and prevent the accumulation of deleterious levels of oxidative stress but can be overwhelmed when ROS scavengers are decreased or inactivated. In the context of PD, Prdx2 associates with a Cdk5 kinase complex via p35 or the proteolytically cleaved products of p35, p10 and the hyper-activated p25 . Cdk5/p3on death . This obon death . Anotheron death . Expresson death , p35 seeon death . Cdk5-de\u2212/\u2212p35 neurons. Glutamate-induced neuronal cell death was rescued by viral expression of wild-type or Thr89Ala Prdx2 but not Thr89Glu Prdx2. Ischemia-dependent neuronal cell death generated by 4-vessel occlusion in hippocampal CA1 neurons could also be rescued by expression of wild-type Prdx2 but not Thr89Glu in vivo [The Cdk5-Prdx2 pathway has also been shown to cause neuronal death in ischemia models . Glutama in vivo . PhosphoDrosophila model that expressed activated kinase mutant LRRK2, where overexpression of Prdx3 rescued flies from decreased lifespan, depletion of dopaminergic neurons and dysfunctional mitochondria in flight muscles of flies [Drosophila with the Prdx mimetic ebselen, which diminishes H2O2 and peroxynitrite. Prdx3 is expressed in mitochondria and has been shown to be important for maintaining mitochondria mass and membrane potential in Myc-transformed fibroblasts and breast cancer cells . The debof flies . The impde novo expression of unmodified protein during signaling events. While information regarding the dephosphorylation of Prdx family members is relatively scarce, some information is available. The protein phosphatase PP2A has been identified to dephosphorylate Prdx1 when phosphorylated at Thr90, which could be blocked with okadaic acid [\u2212/\u2212Pin1 MEFs. The association between Pin1 and Prdx1 was enhanced in the presence of 50 \u03bcM H2O2 and could be inhibited by pharmacologically blocking Cdk1 activity or mutation of Thr90 Prdx1 to Ala. Further experiments found that Pin1 could also bind Prdx2, 3 and 4 but not in cell lysates containing Thr-Pro motif mutants Thr89Ala Prdx2, Thr146Ala Prdx3 and Thr162Ala Prdx4. Reports detailing the importance of interactions between kinases and Prdx family members suggest dephosphorylation may represent an equally significant process. The absence of a Prdx phosphate removal mechanism would require phosphorylated Prdx protein to be degraded or sequestered for future coordination of various signaling pathways. Protein recycling through proteasomal degradation may occur, but this would represent an energy-intensive process requiring aic acid . Dephosp\u2212/\u2212Ptp1b MEFs exhibited significantly higher levels of Prdx4 phosphorylation in the presence or absence of G-Csf stimulation when compared to Ptp1b-proficient MEFs. It is unclear which Tyr was phosphorylated in \u2212/\u2212Ptp1b MEFs or the functional consequences, but the authors suggested that the Tyr phosphorylation may decrease Prdx4 peroxidase activity. Overall, the existence of phosphatases that dephosphorylate Prdx family members is an area that requires further exploration. Dephosphorylation of Prdx4 has also been demonstrated by Ptp1b . GranuloThe current state of knowledge of phosphorylation as a means to modulate Prdx function has been relatively understudied. While biochemical details exist for modification at Ser32, Thr90, Thr183 and Tyr194 and attempts have been made to evaluate Thr18, the consequences of phosphorylation of Prdx1 at multiple sites are unclear. Phosphorylation of Prdx1 Thr90 and Thr183 would be expected to perform similar functions as both sites are targeted by Mst1, decrease peroxidase activity and promote the formation of decameric complexes, but not all combinations are as straightforward. There are questions regarding what occurs when contrasting sites are phosphorylated that cause distinct changes to the oligomeric tertiary structure or peroxidase activity. Phosphorylation of Ser32 has been shown to enhance peroxidase activity, but whether this can exert a dominant effect to rescue the decreased peroxidase functionality of Prdx phosphorylated at Thr90, Thr183 or Tyr194 is unknown. The general structure of a phosphorylated Thr90 or Thr183 Prdx1 decamer may also collapse following Tyr194 phosphorylation, which has been shown to lead to homodimers of Prdx1 primarily. Multi-site phosphorylation may be precluded from occurring in some cases due to changes in the three-dimensional protein structure that prevent sequential kinase binding from occurring, but many other proteins have been shown to have multi-layered signal transduction cascades of regulation. The existence of positive and negative phosphorylation feedback loops to control post-translationally modified proteins temporally has been well documented in transcription factors, kinases and other enzymes. In our current era where large resource experimental datasets have become more readily available due to technical advancements, large proteomic datasets can be mined to determine if protein modifications are present under a myriad of conditions. Published datasets have emerged that support existence of novel phosphorylated forms of Prdx family members. As modifications of plants have been well described in a previous review by Liebthal et al. , we willPhosphorylation of other members of the Prdx family has been less well described. There is evidence that the cdc2-cyclin B complex is capable of phosphorylating Prdx1, 2, 3 and 4 in vitro but cell compartmentalization concerns were suggested by the authors to preclude Prdx3 and 4 from the likelihood of interacting with Cdk proteins . While C2 activity but peroxidase specific phosphorylation sites have not yet been detailed. Six phosphorylation sites of Prdx6 have been described in large proteomic datasets (+/+bi-1) and \u2212/\u2212bi-1 mice found that Tyr phosphorylation was increased in \u2212/\u2212bi-1 liver, brain, heart, lung and kidney tissues [In comparison to Prdx1 and 2, the number of phosphorylation sites present in large datasets is less abundant for the rest of the protein family. Delineation of the effects of phosphorylation of Prdx3 for Thr146 in LRRK2 kinase mutant cells and flies has been described and suggests phosphorylation is a mechanism utilized to alter peroxidase activity, but other sites have yet to be studied ,100. Thrdatasets . Tyr89 i tissues . The ide2O2, ROOH and ONOO\u2212 concentration within multiple cellular organelles. Research efforts have therefore primarily focused on how oxidation of the peroxidatic Cys perturbs cell signaling. While there are many important unrealized details and properties of Prdx oxidation that remain, the effects of other forms of post-translational modifications to Prdx proteins represent another exciting area of exploration. Dynamic changes to oxidation, phosphorylation and other forms of post-translation modifications can change three-dimensional structure and function of Prdx proteins to coordinate the various processes outlined above. In addition, control of Prdx through phosphorylation further integrates redox and signaling cascades. Inactivating phosphorylations of Prdx proteins likely prevent Prdx redox relay reactions as the peroxidatic Cys loses peroxidase activity. Prdx phosphorylation cascades would thus enable attenuation of Prdx-dependent oxidative equivalent transfer to partner proteins and inhibit redox signaling. The role of peroxidase activating phosphorylation sites such as Ser32 of Prdx1 on redox relays is also unknown but may perhaps oppose peroxidase inactivating phosphorylation sites.The highly sensitive peroxidatic cysteine thiol of Prdx proteins makes them well suited to sense and control cell signaling due to minor changes in HPost-translational modifications of Prdx family members have been identified to play a significant role in several signaling cascades, but there remains to be a complete understanding of the function of several putative sites in many family members. The introduction of phosphomimetic and non-phosphorylatable mutants via genomic editing through systems such as CRISPR/Cas9 would be useful to determine the functional consequences of Prdx phosphorylation in normal non-transformed cells as well as cancer cells. Candidate phosphorylation sites to be studied further could be identified from proteomic screens in cancer cells such as those indicated in bold in"}
+{"text": "RNA binding proteins (RBPs) are strongly linked to the pathophysiology of motor neuron diseases. Recent studies show that RBPs, such as TIA1, also contribute to the pathophysiology of tauopathy. RBPs co-localize with tau pathology, and reduction of TIA1 protects against tau-mediated neurodegeneration. The mechanism through which TIA1 reduction protects against tauopathy, and whether TIA1 modulates the propagation of tau, are unknown. Previous studies indicate that the protective effect of TIA1 depletion correlates with both the reduction of oligomeric tau and the reduction of pathological TIA1 positive tau inclusions. In the current report, we used a novel tau propagation approach to test whether TIA1 is required for producing toxic tau oligomers and whether TIA1 reduction would provide protection against the spread of these oligomers. The approach used young PS19 P301S tau mice at an age at which neurodegeneration would normally not yet occur and seeding oligomeric or fibrillar tau by injection into hippocampal CA1 region. We find that propagation of exogenous tau oligomers (but not fibrils) across the brain drives neurodegeneration in this model. We demonstrate that TIA1 reduction essentially brackets the pathophysiology of tau, being required for the production of tau oligomers, as well as regulating the response of neurons to propagated toxic tau oligomers. These results indicate that RNA binding proteins modulate the pathophysiology of tau at multiple levels and provide insights into possible therapeutic approaches to reduce the spread of neurodegeneration in tauopathy.The online version of this article (10.1007/s00401-018-1937-5) contains supplementary material, which is available to authorized users. RNA binding proteins (RBPs) are strongly implicated in neurodegeneration. Mutations in RBPs are associated with motor neuron diseases and form a strong component of the pathology of these diseases. Recent studies demonstrate that RBPs, stress granules (SGs) and the translational stress response are also key pathways mediating the pathophysiology of tauopathy , 9, 43. The current study uses tau seeding and propagation models to investigate the mechanisms through which TIA1 impacts on tauopathy. In tauopathies, misfolding or aggregated tau forms oligomers and fibrils . EmerginThe current study investigates the activities of tau species in propagating tau pathology and neurodegeneration and demonstrates a key role for TIA1 and SGs in neurodegeneration mediated by extracellular, propagated tau oligomers. The critical role of TIA1 enabled further investigation into the pathophysiological mechanisms through which TIA1 contributes to tauopathy. This study begins by comparing the effects in vivo and in vitro of different tau species extracted from P301S tau mouse brains and demonstrates that tau oligomer and fibrillar fractions both propagate tau pathology in vitro and in vivo, but exhibit striking differences in the neurodegenerative outcomes. The oligomeric tau fraction induced tau pathology in neuronal soma that co-localized with SG markers and elicited profound neurodegeneration. In contrast, over the 3-month period of this study, the fibrillar tau fractions induced tau inclusions predominantly in synapses and dendrites, but showed no association with SG markers, nor did they elicit neurodegeneration. Next, we further use this model for mechanistic studies. We show that TIA1 reduction elicited neuroprotection through both cell autonomous and cell non-autonomous mechanisms. Mice with reduced TIA1 produce less toxic tau oligomers and also exhibit reduced vulnerability to toxic tau oligomers generated from elderly P301S MAPT mice. These results demonstrate that oligomeric tau propagates toxic tau pathology through a mechanism mediated by TIA1 and pathological SGs, suggesting a broad role for SGs in the mechanisms of tau-mediated neurodegeneration.Tia1\u2212/\u2212 mice (B6.129S2(C)-Tia1tm1Andp/J) were generated in and obtained from Anderson lab in Harvard University, Dana Farber Cancer Institute . . 7]. OurTo further confirm the existence of oligomeric and fibrillar forms of tau in S1p or P3 injected mice brain, tau oligomer marker TOMA2 and fibrillar tau marker thioflavin S were used to detect tau inclusions in CA3 and LEnt, respectively. The data indicate that the S1p fraction primarily induced oligomeric tau aggregation, while the P3 fraction primarily induced fibrillar tau aggregation . We also examined a cohort of injected PS19 mice that were killed at 15\u00a0days after injection. In both cohorts, the fractions containing tau aggregates exhibited only modest diffusion into CA3 and no diffusion into distant LEnt region . The limited diffusion of the tagged tau suggests that the tau aggregation present in CA3 and LEnt over both short- and long-term models resulted predominantly from the templating of human tau synthesized in the recipient neurons.The mechanism of propagation and toxicity in neurons is largely unknown. The localization of propagated tau oligomers to neuronal soma raised the possibility that they might interact with the TIA1/SG pathway, which also localizes to the neuronal soma. To test for co-localization of tau oligomers with TIA1 positive SGs, brain tissue sections from mice treated with the S1p or P3 fractions were probed with anti-TIA1 antibody and the oligomeric tau-specific antibody TOMA2  tau than fibrillar tau (P3 fraction). Neurodegeneration associated with propagation of the oligomeric or fibrillar tau was assessed by immunohistochemical labeling with neuronal marker NeuN in CA3 and LEnt regions Fig.\u00a0a. The reWe hypothesized that the neurodegeneration induced by the propagated oligomeric tau might elicit a stress reaction in the affected neurons and that this stress response might be associated with cytoplasmic translocation of TIA1 and SG formation. This is important because recent studies indicate that tau pathology associates with TIA1 positive SG responses in neurons , 43, 44.Tia1/++ and PS19::Tia1/\u2212+ mice and harvested at 6\u00a0months. The TOMA2 staining data showed that TIA1 reduction greatly reduced the basal level of TOMA2 oligomeric tau in PS19::TIA+/\u2212 mice, as well as blocking the propagation of oligomeric tau in S1p-injected mice  via AAV9-mediated transduction in hippocampal neurons over-expressing human WT or P301L tau. The efficiency of TIA1 knock down was greater than 50% of TIA1 as detected in cell lysate by immunoblot . We documented propagation of S1p-induced tauopathy (CP13 staining) in these cultures . We proceeded to test whether the S1p fraction from PS19::Tia1/\u2212+ mice was less toxic than the equivalent fraction from PS19::Tia1/++ mice. We exposed DIV14 cultures of hippocampal neurons to samples of S1p fraction isolated from PS19::Tia1/++, PS19::Tia1/\u2212+ and WT C57BL/6 mice. Medium was collected after 24\u00a0h. Analysis of LDH levels from the medium of the neurons demonstrated a 30% decrease in LDH from cultures exposed to PS19::Tia1/\u2212+ S1p compared to PS19::Tia1/++ , TIA1 and SGs. Indeed, binding of TIA1 to tau oligomers generated by templating of extracellularly propagated tau could promote their stabilization and accumulation . This suTia1/\u2212+ mice were highly resistant to neurodegeneration mediated by the S1p oligomeric fractions from PS19 mice, and S1p oligomeric fractions from PS19::Tia1/\u2212+ mice exhibited reduced toxicity in cultures of hippocampal neuron over-expressing tau. Although the relative contributions to neurodegeneration from cell synthesized tau and intercellular propagated tau are not well understood for PS19 P301S tau mice aging normally, the current study used templated tau fractions to greatly accelerate production of tau pathology, which means that most of the tau pathology studied resulted from tau propagation. The neuroprotection provided by TIA1 reduction demonstrates its requirement in neurodegeneration mediated by propagated, templated tau oligomers. Thus, the current study demonstrates for the first time that TIA1 regulates neurodegeneration mediated by both intrinsic and extrinsic tau, with TIA1 regulating toxicity by determining the amount of toxic tau oligomers and the response of neurons to the toxic tau oligomers. The discovery that these neurodegenerative pathways share a unified mechanism mediated by tau oligomers, TIA1 and SG biology, suggests that therapeutic approaches targeting this pathological mechanism will be able to inhibit both the cell synthesized (intrinsic) and intercellular propagated tau pathways in tauopathy, leading to increased therapeutic efficacy. We suggest that inhibiting the interaction of TIA1 with tau might be one such approach.The direct role of TIA1 in the mechanism of toxicity mediated by propagated tau was proven by showing that TIA1 reduction decreased degeneration associated with propagated tau. PS19::Supplementary material 1 (DOCX 11731 kb)Below is the link to the electronic supplementary material."}
+{"text": "Concerns have been raised about several results reported in this article :The second and last FACS plots in Fig 2C (U251/Empty Vector and A172/Notch2) appear similar, although the listed percentages are different.In Fig 3, U251 NF-\u03baB panel lanes 1, 2 appear similar to A172 PCNA panel lanes 2, 1, respectively, when flipped vertically and horizontally. Additionally, the U251 MMP2 panel appears similar to the A172 PI3K panel, the U251 Cyclin D1 panel appears similar to the A172 Bcl-2 panel, and there are similarities between the U251 Caspase 3 panel and the A172 Notch2 panel.In Fig 4, several mice appear to be included in multiple panels as representing different experimental results. The authors reported that errors were made in generating Fig 4 such that images from control experiments were used as representing Scramble Notch1 siRNA (Scr siRNA) and Empty Vector results, and images from the Scramble Notch1 siRNA (Scr siRNA) group were used as representing Notch2 results.The tumor sizes reported in Fig 4 exceed sizes commonly accepted for mouse tumor studies. In response to journal queries, the authors did not provide scientific or ethical justification for the tumor sizes, and they clarified that no specific criteria  were used to determine when animals were euthanized. They explained that they typically terminated the experiment after 3\u20134 weeks, when quality of life was \u201cseriously affected\u201d in the group bearing the largest tumors.In Fig 6, there is overlap between the lower half of the Notch1 siRNA and the upper half of the Notch2 plasmid images. The authors commented that this resulted from a figure preparation error and that the images are both from the Notch2 siRNA experiment.In the published Fig 6, there are areas where the TUNEL staining pattern (green) appears similar although the DAPI staining (blue) differs, when comparing the Control, Scramble Notch1 siRNA (Scr siRNA), and Empty Vector panels; and when comparing the Notch1 siRNA and Notch2 plasmid panels. There are also areas in the Control and Scr siRNA panels where DAPI staining patterns appear similar, although regions adjacent to these areas differ.The authors offered updated versions of Figs 4 and 6 in which the panels in question were replaced to address image duplications, but they noted that the original data supporting results reported in this article are no longer available.PLOS ONE Editors retract this article in light of the above issues which call into question the validity and reliability of the reported results and raise concerns about animal welfare considerations in the study design.The AZ agreed with the retraction. The other authors did not respond or could not be reached."}
+{"text": "They arrived to the laboratory 10 hrs and 34 hrs after the final football session and blood samples were collected at fasted (0 min) and 45, 90, 240 and 360 min post a high fat load meal. There were non significant increase for postprandial TG AUC  and iAUC  between 10 and 34 hrs after the 1FOOT. For the 3FOOT, there was a non significant decrease in postprandial TG AUC  and iAUC  from 10 to 34 hrs, respectively. Performing three consecutive days of football exercise may offer no greater protective effect for postprandial TG before a period of reduced activity, compared to a single session.Elevated postprandial triglyceride (TG) is associated with increased risk of cardiovascular disease. The time window for the last bout beneficial effect on postprandial lipaemia after football play is unknown. The aim of the present study was to examine whether playing affects postprandial TG during 1.5 day of reduced activity. Eighteen males were randomly allocated to perform either 1 (1FOOT; n = 9; age = 33.0 \u00b1 5.0 yrs; body mass index = 24.2 \u00b1 3.6 kg/m Sedentary behavior is an important issue in public health . ProlongSeveral different models of sedentary behaviour exist, including training cessation for athletes, increasing sitting time, reducing daily ambulatory activity and bed rest. All of these likely induce negative physiological effects that may provide information relating to metabolic dysfunction. The negative effect of low activity levels on postprandial TG have been reported in some studies ,8,9,10. Several studies have shown endurance, resistance and high intensity exercise to independently lower postprandial TG \u201316. Howe2) or 3 football  sessions across a 5-day study period. Participants arrived to the laboratory 10 hrs (day 4) and 34 hrs (day 5) after the final football session and blood samples were collected at fasted (0 min) and 45, 120, 240 and 360 min post a high fat load meal. The study was approved by the Qatar Anti Doping Laboratory ethics committee (number F2015000112), and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants prior to commencing.Eighteen males were randomly allocated to perform either 1  cardiovascular, pulmonary or metabolic disease, (2) absolute contraindications to exercise testing as established by the American College of Sports Medicine, (3) dietary restrictions regarding the meals provided. In order to control for confounding variables, participants were instructed to (1) fasting was standardized for 10 and 34 hrs before the fat meal, (2) abstain from caffeine, tobacco, and vitamin supplements for 24 hrs before blood sampling, and (3) awake between 06:00 and 07:00 hrs prior to laboratory testing. Participants were asked to refrain from any other physical activity, besides that of the exercise intervention, for the 36 hrs before the start of pre intervention measures.th rib landmarks (waist) and the girth of the buttocks at the level of the posterior protuberance. Body fat was estimated using the InBody Bioelectrical Impedance Analysis Systems .Body mass and stature were recorded using a combined scale and stadiometer  with body mass index calculated. Waist and hip circumferences were measured at visual narrowing of the waist between the iliocristale and 10All participants performed the Yo Yo intermittent endurance test level 1 (YYIETL1), in an air-conditioned sports hall on artificial turf to establish maximal heart rate (HR) and cardiorespiratory fitness. The YYIETL1 test consists of 2 x 20 m shuttle runs performed at increasing running speeds, interspersed with 5 s of active recovery, during which the participants jogged around a cone placed 2.5 m behind the start/finishing line. The speeds were controlled by audio and the test was terminated when the participant was no longer able to maintain the required speed for 2 consecutive runs. The total distance (metres) covered represented the test result.On day 1 participants arrived to the laboratory at approximately 0800 hrs after an overnight fast (10 hrs) . FollowiParticipants were instructed to wear an Actigraph activity monitor  across the 5-day experimental period to monitor step counts, this included the three days of the intervention  as well as the two days of trials (10 and 34 hrs). The activity monitor was placed on the hip as this position has shown to be the most reliable for estimating energy expenditure . ParticiAll sessions for both groups consisted of 60 min (2 x 30 min) small sided game (5 vs 5) performed on an artificial outdoor court (36.5 x 27.5 m). All matches were played at the same time of day (20:00 hrs) and each player in turn played as goalkeeper for the matches. The activity profiles during the football match were recorded using the same Actigraph activity monitor and session rating of perceived exertion (sRPE) was recorded within 5 min of exercise completion using the CR-10 Borg scale and player load calculated (sRPE x minutes) . PerceivFollowing the last football session and the evening prior to the first visit to the laboratory, all participants consumed a standardised meal  providing 960 kcal; 36 g fat, 120 g carbohydrates and 42 g protein. Participants were instructed to consume the sandwich, and nothing else, after the last football session. Participants consumed the same meal (Subway sandwich) the evening of trial one (day 1) to ensure the dietary intake was replicated prior to the second trial (day 2) experiment.g and at room temperature) and was collected and stored at -80 oC for further analysis. Analyses of all samples from the same participant were done in a single batch. The spectrophotometric analyses were performed on an automated clinical chemistry system  to determine the level of TG, insulin, glucose and non-esterified fatty acids (Abbott-Cell Dyn 3700).Blood samples were collected into 9-ml potassium-EDTA Monovettes  or serum separate tubes (SST)-gel/clot activator. Serum was obtained by centrifugation . Incremental area under the curve was calculated using the trapezoid rule and calculated by subtracting, from the postprandial area, the mean baseline value extrapolated over 6 hrs; thus they reflect changes occurring after the meal. Whole-body insulin sensitivity was assessed with the homeostasis model assessment (HOMA) of insulin resistance index from serum fasting data as: insulin (\u03bcU/ml) \u00d7 glucose (mmol/l)/22.5)  and withData were tested for normality with the Kolmogorov-Smirnov test, with all data shown to be normally distributed. A 2x2x5 (group x hours after the match x blood sampling time points) analysis of variance (ANOVA) was used for all comparisons between the two trials. When significant overall differences between the trials was found, a Bonferonni post hoc test was applied. Students\u2019 unpaired t-test was used to compare baseline characteristics between 1FOOT and 3FOOT groups. Statistical significance was set at p< 0.05 and 95% confidence intervals (CI) and Cohen\u2019s d effect sizes (ES) were calculated. The criteria to interpret the magnitude of the ES were: > 0.2: small, > 0.6: moderate, > 1.2: large and >2.0: very large) . All staEstimated energy expenditure was greater for day two football session  than day one  and day three  in the 3FOOT group. No other difference was found between the 3FOOT consecutive days of football session for any of the metrics relating to time in different zones for energy expenditure. In the 3FOOT condition we found no difference for sRPE , although a large effect size was calculated for day one vs day three; ES = 1.17. There were no differences for PRS  between the 3 days for the 3FOOT group. We found no differences for sRPE between 1FOOT (282 \u00b1 56) and 3FOOT (313 \u00b1 16) (ES = 0.75) for the only (1FOOT) and day 3 of football matches (3FOOT). There were no differences between groups for the number of steps for the three days prior to the first laboratory visit and there was no difference for the number of steps between 1FOOT (4897 \u00b1 1504 steps/day) and 3FOOT (4753 \u00b1 2071 steps/day) during day 4 of the experiment (10 hrs), inferring that participants performed a similar activity level prior to day 5 of the experiment (34 hrs).There was a non-significant increase in postprandial TG AUC  and iAUC   between Insulin iAUC increased from 34 to 10 hrs  in the 3The aim of the present study was to examine whether different frequency of playing football (1 session vs 3 sessions) affects postprandial TG during 1.5 day of reduced activity. We found no difference for postprandial TG incremental area under the curve from 10 to 34 hrs following either one single or three consecutive 60 min sessions of playing football. Our findings have important clinical implications, notably that there does not appear to be an additive effect from three consecutive days of playing football in the evening 10 and 34 hrs post the last bout of exercise, compared to solely one football session.In comparison to our study, Zhang et al.,  reportedThe fact we showed no difference between the 10 and 34 hrs may be explained by several reasons. While we did not perform pre intervention measure of postprandial TG, several studies, including previous work from ourselves, have shown the lowering effects of exercise from control to post training intervention. Energy expenditure and lipoprotein lipase (LpL) are suggested to be important factors for postprandial TG reduction, however, evidence has shown a dissociation of energy expenditure from the magnitude of postprandial TG attenuation . While t2max every 30 min, improved postprandial TG area under the curve [The light ambulatory contractions of habitual daily activity performed during the 10 and 34 hrs time period may have been enough to maintain lower levels of postprandial TG, since LpL has shown to be sensitive to small changes in ambulatory daily activity, albeit this was reported in rats . While the curve . Thus, sIt is important to consider the participant characteristics, as well as the synergistic interaction between previous training experience and acute exercise, may be a factor for the results in our study. Though our participants were not highly trained individuals, they had previous experience playing football. Research has shown that trained individuals may have a low chylomicron-TG half-life which may partly be due to a reduction in the fasting TG pool size and partly to a direct effect of chronic exercise on the TG removal system . It is aThe 3FOOT group showed a substantial increase ~108%) in insulin incremental area under curve from 10 to 34 hrs, compared to the 1FOOT group (~ 16%). This change in the 3FOOT is probably a manifestation of the 40% lower concentrations at 45 min on day 4 (10 hrs). The negative effect of low levels of activity on insulin response have been shown after only a few days 3. Pafili et al 8% in ins. The altThe different changes in insulin from day 4 to 5 seen between 1FOOT and 3FOOT may be a combination of a decreased concentration on day 4 , but also a large increase on day 5 (after the period of inactivity). We speculate a greater muscle damage in 3FOOT which may have been detected 34-hour post. Paschalis et al.,  demonstrOur results may also be explained by the composition of the high fat meal consumed. Chong et al.,  demonstrIn conclusion there was no difference for postprandial TG incremental area under the curve from 10 to 34 hrs following either one single, or three consecutive days of playing 60 minutes of football. Performing three consecutive days of football exercise may offer no greater protective effect for postprandial TG before a period of reduced activity, compared to a single session. Two-hour insulin response increased from 10 to 34 hrs, suggesting impaired sensitivity."}
+{"text": "TNFAIP3 gene encodes A20, an inhibitor of nuclear factor-\u03baB pathway, and is a susceptibility gene for autoimmune diseases. We investigated deleterious variants in the coding regions of TNFAIP3 gene of Japanese AIH patients or those with cirrhosis. The deleterious variants in the coding regions of TNFAIP3 gene were analyzed by the cycle sequencing method and the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis were compared with those of Japanese controls. The deleterious alleles in TNFAIP3 were not associated with AIH. A significant association was shown for the deleterious alleles in TNFAIP3  4.28, 95% confidence interval (CI) 1.53\u201311.95) with AIH with cirrhosis at presentation. The serum IgM levels in AIH patients with deleterious alleles in TNFAIP3 were tended to be lower than those without . The frequency of deleterious alleles in TNFAIP3 was higher in the AIH subset without the DRB1 risk alleles than that with . The deleterious alleles in TNFAIP3were associated with AIH with cirrhosis.Autoimmune hepatitis (AIH) is an autoimmune liver disease and cirrhosis is sometimes complicated with AIH at diagnosis, influencing its prognosis.  Liver cirrhosis is sometimes complicated with AIH at diagnosis and influences its prognosis. AIH patients with cirrhosis had a worse survival2. The genetic and environmental factors are involved in the pathogenesis of AIH. A genome-wide association study (GWAS) on European population revealed that human leukocyte antigen (HLA) is the strongest genetic risk factor for AIH3. HLA-DRB1*03:01 and DRB1*04:01 were associated with AIH in European populations4. DRB1*04:01 and DRB1*04:05 were associated with AIH in Japanese populations8. DRB1*08:02 and DRB1*08:03 were also predisposing for the disease, when these alleles were possessed by individuals with DRB1*04:058. The previous GWAS also suggested associations of single nucleotide variants in other genes outside of HLA3. Several candidate-gene approach studies reported weak genetic associations of single nucleotide variants with Japanese AIH in other genes than HLA; STAT49, PTPN2210, ICOS11, TNIP112, and SH2B313.Autoimmune hepatitis (AIH) is an autoimmune liver disease with chronic progressionTNFAIP3 (tumor necrosis factor-\u03b1 induced protein 3) gene encodes A20, an inhibitor of nuclear factor-\u03baB (NF-\u03baB) activation, and is a susceptibility gene for autoimmune diseases including systemic lupus erythematosus15 or rheumatoid arthritis17. A20 is a negative regulator of the NLRP3 inflammasome and myeloid cell specific deletion of A20 caused spontaneous arthritis in mice18. Analogically, inflammasome was activated in monocytes of non-transplanted AIH children and liver-transplanted children with de novo autoimmune hepatitis, but increased expression of A20 was observed in monocytes of liver-transplanted children without de novo autoimmune hepatitis19. Thus, A20 plays some important roles against autoimmune diseases. Recent studies reported that loss of function (nonsense or frameshift variants) or deleterious missense variants (variants that changed amino acid residues on positions highly conserved across orthologs) in TNFAIP3 dominantly caused an autoinflammatory disease with Beh\u00e7et\u2019s disease-like symptoms, the haploinsufficiency of A20 syndrome (HA20)22. However, the symptoms of HA20 also included those of autoimmune diseases, arthritis, nephritis, vasculitis, or hepatitis23. Thus, we investigated the variants in the coding regions of TNFAIP3 gene of Japanese AIH patients by the cycle sequencing method and tried to compare the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis with those of Japanese controls.TNFAIP3 gene were amplified by 9 primer sets to detect variants by the direct sequencing method. Seven single nucleotide variants were found in the coding regions of TNFAIP in 360 AIH patients. No variant was detected in the splice sites of TNFAIP3 gene and no insertion or deletion was found. Two (rs200595071 and rs769014911) of these variants were synonymous and the other five were non-synonymous. Of these five non-synonymous variants, three variants  were predicted to be deleterious  by all five protein prediction algorithms, and two [c.380T\u2009>\u2009G  and c.2140C\u2009>\u2009T ] were predicted to be neutral  by all. Thirteen alleles of these three deleterious variants  4.28, 95% confidence interval (CI) 1.53\u201311.95, Table\u00a0Deleterious allele frequencies in the AIH patients and the Japanese controls are shown in Table\u00a0TNFAIP3 were analyzed .The demographic features of AIH patients with or without deleterious alleles in ed Table\u00a0. The serTNFAIP3 and DRB1 was also investigated .The gene-gene interaction between TNFAIP3 were predisposing for AIH with cirrhosis in a Japanese population. TNFAIP3 encodes A20, an inhibitor of the NF-\u03baB signaling pathway, and is a susceptibility gene for autoimmune diseases and HA2023. A20 is a negative regulator of the NLRP3 inflammasome and plays some important roles against autoimmune diseases19. TNIP1 is a predisposing gene in AIH12, and encodes an adaptor protein binding to A20. These data suggested the common signaling pathways in the pathogenesis of AIH, other autoimmune diseases, and HA20.The present study revealed that deleterious variants in 2. Cirrhosis at presentation was also a risk factor of development of hepatocellular carcinoma24. However, an age at onset was older in AIH patients with cirrhosis at presentation2, but that of AIH patients with deleterious variants in TNFAIP3 was not older  were detected in Japanese AIH patients, but none of them were reported to cause HA20. One of the deleterious variants in TNFAIP3 (c.305A\u2009>\u2009G) was appeared to be related to the poor clinical outcome of rheumatoid arthritis16, suggesting that deleterious variants in TNFAIP3 are modulating symptoms of autoimmune diseases.The progression pattern of AIH patients with cirrhosis at presentation would be smoldering and latent and the prognosis of AIH with cirrhosis was worseis Table\u00a0. Thus, dTNFAIP3 was also analyzed between patients with or without the DRB1 risk alleles. The frequency of deleterious variants in TNFAIP3 was higher in the AIH patients without the DRB1 risk alleles than those with. These data suggested that deleterious variants might not predispose AIH in individuals with the strongest genetic risk factor, the DRB1 risk alleles. It was also possible that deleterious variants would be readily found in the individuals without the DRB1 risk alleles. Serum IgM levels in AIH with deleterious variants in TNFAIP3 were tended to be lower than those without , linkage disequilibrium was not strong between these two loci , suggesting that deleterious variants in TNFAIP3 and the DRB1 risk alleles would be independently associated with AIH. However, the independence could not be confirmed by logistic regression analysis, because the genotypes of the Japanese controls were not available.The association of deleterious variants in ut Table\u00a0. Since ATNFAIP3 with Japanese AIH with cirrhosis. Since the sample size of this study was limited and the frequencies of deleterious variants were low, the association was modest. The associations should be confirmed in future large scale studies.To the best of our knowledge, this is the first report of the association of deleterious variants in 24. All the AIH patients were native Japanese living in Japan and satisfied the criteria of International Autoimmune Hepatitis Group for diagnosis of AIH25. The allele frequencies of single nucleotide variants in TNFAIP3 gene in Japanese population were referred to 3.5KJPN panel from the genome cohort study of Tohoku Medical Megabank Organization (https://ijgvd.megabank.tohoku.ac.jp/)28. The distribution pattern of the age and gender of the controls was described elsewhere . This study was reviewed and approved by NHO Central IRB and University of Tsukuba Research Ethics Committee. Written informed consent was obtained from each individual. This study was conducted in accordance with the principles expressed in the Declaration of Helsinki.A total of 360 AIH patients were recruited from National Hospital Organization (NHO) hospitals. The clinical information of the enrolled patients was obtained and 38 had cirrhosis at presentationTNFAIP3 gene was performed by the direct sequencing method of PCR products amplified with previously reported primers16 for exon 2 to exon 9 and BigDye Terminator v3.1 Cycle Sequencing Kit  using Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific Inc.). Deleterious alleles were defined by missense variants annotated as deleterious by all five protein prediction algorithms of PolyPhen-2 HumDiv , PolyPhen-2 HumVar , SIFT , Mutation Taster , and Likelihood Ratio Test Predictions 34. DRB1 genotyping results of the AIH patients were reported previously8. DRB1*04:01, *04:05, *08:02, or *08:03 were designated as the DRB1 risk alleles for AIH8.Genotyping of 35. The clinical phenotypes of AIH patients with deleterious alleles were compared with those without by Fisher\u2019s exact test using 2\u2009\u00d7\u20092 contingency tables or Mann-Whitney\u2019s U Test. The distribution of deleterious allele frequencies was compared between AIH patients with or without the DRB1 risk alleles by Fisher\u2019s exact test using 2\u2009\u00d7\u20092 contingency tables under the allele model. Correction for multiple testing was performed by calculating false discovery rate Q-value36.The distribution of deleterious allele frequencies in AIH patients or AIH patients with cirrhosis was compared with those in Japanese controls by Fisher\u2019s exact test using 2\u2009\u00d7\u20092 contingency tables under the allele modelSupplementary Figure S1"}
+{"text": "The opposite syndrome (microduplication) has not yet been characterized. Our main objective is the recognition of a new clinical entity - 12q14 microduplication syndrome. - as well as confirming the role of HMGA2 gene in growth regulation.Array Comparative Genomic Hybridization (CGH), Karyotype, Fluorescence in situ Hybridization, Quantitative-PCR analysis and Whole exome sequencing (WES) were performed in a girl presenting overgrowth and obesity. Array CGH identified a 1.5\u2009Mb 12q14.3 microduplication involving HMGA2, GRIP1, IRAK3, MSRB3 and TMBIM4 genes. Karyotype and FISH showed that duplication was a de novo insertion of 12q14.3 region on chromosome 9p resulting in an interstitial microduplication. Q-PCR confirmed the duplication only in the proband. WES revealed no pathogenic variants.Phenotypic comparison with patients with 12q14 microdeletion syndrome showed a reciprocal presentation, suggesting a phenotypically recognizable 12q14 microduplication syndrome as well as confirming the role of HMGA2 gene in growth regulation.\u00a0It is also indicative that other genes, such as IRAK3 and MSRB3 might have of role in weight gain and obesity. HMGA2, GRIP1 and in some cases LEMD3, IRAK3 and TMBIM4 genes [HMGA2 gene gains of function were acquired rearrangements related with potentially malignant conditions. The majority were HMGA2 fusions with partner genes and subsequently formation of chimeric transcripts with gain of HMGA2 gene function and tumor formation [Copy number variations on chromosome 12q14 have been mainly reported as a microdeletion syndrome OMIM #166700) first described by Menten et al. in 2007 and classified as an autosomal dominant bone dysplasia. The patients showed failure to thrive in infancy evolving to proportionate short stature, mild intellectual disability and osteopoikilosis [6700 firsormation .HMGA2 and IRAK3 genes [HMGA2 gene as a common feature in the 12q14 microdeletion syndrome [HMGA2, GRIP1, IRAK3, MSRB3 and TMBIM4 genes and the phenotype associated with the duplication apparently mirrors the microdeletion syndrome phenotype.Previous reports on microdeletions within 12q14 region ranged from 1.83\u2009Mb to 10.12\u2009Mb with a 378Kb smallest region of overlap containing the syndrome . There aOur patient was born to non-consanguineous 37\u2009year-old Human Immunodeficiency Virus (HIV) positive mother and 22\u2009year-old healthy father. She has an\u00a018 year-old healthy sister on her mother side. Anti-retroviral medication was carried out throughout both pregnancy and delivery according to national guidelines, and this treatment was not complicated by any adverse effects. The girl was born by caesarean section at 38 and 2/7\u2009weeks, and Apgar scores were 9 and 10 at 1st and 5th minutes respectively. Birth weight was 4015\u2009g (P95), length 50.5\u2009cm (P75\u201390) and OFC 36\u2009cm (P95). She was admitted to the neonatal intensive care unit soon after birth for moderate respiratory distress. She had mild hypotonia and feeding difficulties. Head ultrasound was normal. Supraventricular extrasystoles conditioning tachyarrhythmia were noted on electrocardiogram, and she received propranolol treatment transiently. Echocardiogram was normal. No other anomalies were noted. Upon follow up after discharge she was noted to have increased growth parameters with marked obesity, weighing 12.7Kg at six months (+\u20096.7SD). At 8\u2009months she was referred for genetic evaluation. She had developed mild dysmorphic features, with slight bilateral ptosis, scarce eyelashes, anteverted nostrils and macroglossia. She had inverted nipples and increased inter nipple distance. Both hands and feet seemed edematous and she had tapering fingers. She had normal skin. Follow up studies at 12 and 18\u2009months showed cholesterol, apolipoprotein A1 and B, and endocrine studies  were normal. Serological markers for HIV were negative. She had no signs of urogenital abnormalities, visceromegaly or lipomatosis on abdominal ultrasounds. Radiologic studies did not show any signs of skeletal dysplasia. At 18\u2009months her weight was 20.2Kg (+\u20095.1SD), height was 91\u2009cm (+\u20093.6SD), occipitofrontal circumference (OFC) 50\u2009cm (+\u20092.8SD) and body mass index (BMI) 24.4 (+\u20096.7SD)  according to manufacturer recommendations Results were analyzed using Cytogenomic software 2.9.2.4 and Human Genome 19 (GRCh37) assembly.HMGA2 gene. Applied Biosystems CopyCaller v2.0 software was used to calculate the copy number probe and the confidence of the statistical method.Copy number quantification was carried out by quantitative real time PCR (qPCR) on a StepOnePlus\u2122 Real-Time PCR System  using the taqman Hs01969532_cn probe (Applied Biosystems) locate within exon 6 of Conventional G Banding with Trypsin and Leishman (GTL banding) and Fluorescence in situ hybridization (FISH) studies were performed on the proband and progenitors peripheral blood lymphocytes using standard protocols and according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016 .green color) and 12 (aqua color) (Abbot\u00ae) and also a SureFISH probe specific for 12q14.3 HMGA2 gene region (red color)  were used.Centromeric FISH probes for chromosomes 9 (Whole exome sequencing (WES) was performed as a trio on proband and progenitors using Agilent SureSelect Human All Exon V6 and Illumina Hiseq2000 plataform. GRCh37 was used as Human Genome Reference.Standard deviation scores were calculated according to the World Health Organization (WHO) Growth Reference Data Z-scores .HMGA2, IRAK3, GRIP1, MSRB3, TMBIM4, HELB, RPSAP52 and LLPH . The duplication region contains 8 genes, including HMGA2 gene revealed one signal in each q14.3 arm of both chromosomes 12 and a third signal on chromosome 9p arm  encodes a protein that belongs to the non-histone chromosomal high mobility group (HMG) protein family. These proteins function as architectural factors and contain structural DNA-binding domains that may act as transcriptional regulating factors. The HMGA2 gene consists of five exons spanning a genomic region of 160\u2009kb. Exons 1\u20133 encode A-T hook domains that bind to the minor grove of AT- rich DNA. In humans, haploinsufficiency of theHMGA2 gene is capable of causing a poor growth phenotype with failure to thrive and short stature, as seen in the 12q14 microdeletion. The association between growth retardation and short stature and the loss of the HMGA2 gene has been established [HMGA2\u00a0presenting exclusively short stature and none of the other 12q14 microdeletion syndrome features. HMGA2 deletion was also present in this patient\u2019s mother and maternal grandmother, and also in a maternal aunt, all presenting short stature as well. The patient and his relatives therefore did not fulfill the criteria described for a 12q14 microdeletion syndrome, suggesting instead a strong association between the HMGA2 gene deletion and short stature [The ablished , 12. BuyHMGA2 [Ligon et al. by other side described a patient with overgrowth and advanced bone age who had a de novo pericentric inversion of chromosome 12, with breakpoints at p11.22 and q14.3, apparently truncating HMGA2 . The pathmga2 inactivation are born with reduced body size and extremely decreased fat levels [HMGA2 is an early event in the pathway to tumor formation. Normally, the expression of HMGA2 is restricted to undifferentiated mesenchymal cells. Chromosomal rearrangements frequently result in the activation of the normally silent HMGA2 allele in terminally differentiated cells. It has been suggested that such activation can lead to mesenchymal tumor formation [HMGA2 3\u2032-UTR contains target sites for the let-7 miRNA and some rearrangements may lead to increased levels of HMGA2 protein due to the loss of mi-RNA-mediated repression [In vivo data shows that mice with homozygous t levels  whereas t levels . It is gormation , 16. It ormation . Additiopression .HMGA2 is expressed either solely from the translocated allele associated with a gain of function or from the nondisrupted allele when the disrupted allele does not acquire a regulatory domain [HMGA2 has been identified as the target gene of in mesenchymal tumors, namely in lipomas, the exact mechanism by which HMGA2 overexpresssion drives benign tumorigenesis in a target cell is still widely unknown [Ashar et al. suggest that in lipomas y domain , 16. Alt unknown .HMGA2 seems to act in a narrow window early in the adipogenesis where it is involved in expanding the population of preadipocytes and keepping the cells in an undifferentiated state. The authors concluded that in an in vitro model both wild type and truncated HMGA2\u00a0resulting in an overexpression of the gene abrogated growth inhibition and adipogenesis [HMGA2 gene develop undifferentiated lipomas and abundant adipose tissue [HMGA2 gene rather a rearrangement (truncated HMGA2 gene) could also involve different regulatory mechanisms. Publications of new constitutional cases with similar phenotypes will be necessary to confirm this possible new entity.According to Heriksen et al. ogenesis , which se tissue . This coHGMA2 with a dosing effect could explain the overgrowth of our patient.Taking into account the clinical data, all previous reports and the phenotype associated with the microdeletion, the duplication of The absence of lipomas or other tumors thus far in our patient remains to be elucidated. Nonetheless, given the patient\u2019s young age these features can still arise later in life, and follow up medical screening is mandatory.HMGA2 gene and FISH demonstrated that the third copy was inserted on chromosome 9p. The presence of an insertion on p arm of this chromosome could also interfere with gene overexpression or subexpression. Literature revision on genes located chromosome 9p and association for overgrowth or obesity showed no results except a paper suggesting a susceptibility loci on 9p22 for adiposity phenotypes [In our patient array CGH revealed the presence of three copies of the enotypes . AlthougGRIP1 (OMIM#604597)gene has been implicated in the regulation of glutaminergic and perhaps also GABAergic signaling, both of which are involved in many neuronal mechanisms, and it is known that bialelic mutations of this gene cause Fraser syndrome [GRIP1 is also necessary for dendritic development [GRIP1 variants have been described in patients with neuronal and behavioral autistic phenotypes [GRIP1 gene only one did not present developmental delay [GRIP1 is involved in our patient duplication and apart from neonatal hypotonia, development and language have occurred within the normal timing frame.syndrome . Additioelopment . Furtherenotypes . Of the IP1 OMIM#04597geneIRAK3 and increased obesity [IRAK3 is downregulated in obesity, but also that its expression is increased following weight loss [IRAK3 duplication are not known. Dosing of blood adiponectin was not performed in our patient. As our patient presents severe obesity, implication of IRAK3 gene cannot be excluded.IRAK3 (OMIM#604459) encodes a member of the interleukin-1 receptor-associated kinase protein family. Members of this family are essential components of the Toll/IL-R immune signal transduction pathways and mutations in this gene have been associated with a susceptibility to asthma . It also obesity . Hulsmanght loss . ClinicaMSRB3 (OMIM#613719) is predominantly located to the endoplastic-reticulum and is thought to play a role in cell growth. Bialelic mutations of this gene cause autosomal recessive deafness 74 [MSRB3 protects mammalian and Drosophila cells against endoplastic-reticulum stress and has been shown to increase the longevity of Drosophila [MSRB3 deficiency induces cell growth inhibition in mouse embryonic fibroblasts. Haploinsufficiency of MSRB3 could therefore contribute to the growth restriction seen in all patients with 12q14 microdeletion. Correspondingly, duplication of MSRB3 with a simultaneous gain of function could cause overgrowth, either by inducing growth itself or by not regulating it.fness 74 . Overexposophila . These aTMBIM4 is a candidate gene for susceptibility to type 2 diabetes and obesity [TMBIM4 duplication can put our patient at a higher risk of type 2 diabetes. obesity . As metaLLPH is an intrinsically disordered protein predicted to function in nucleolus, possibly as a molecular hub for protein-protein interaction and regulating neuronal morphogenesis and synaptic transmission [HELB is a DNA Helicase  and it has been hypothesized that HELB is recruited to sites of DNA damage and has a role in recovery from replication stress [LLPH and HELB genes have not been published.smission . HELB isn stress . NeverthHMGA2 gene in growth regulation and suggest that other genes, such as IRAK3 and MSRB3 might have a role in weight gain and obesity.Patients with 12q14 microdeletion syndrome present low birth weight and failure to thrive, proportionate short stature and developmental delay, and in some cases osteopoikilosis. Our patient, presenting overgrowth, obesity and tall stature with advanced bone age, is the first described case with 12q14 duplication. Phenotypical comparison of both entities shows that they mirror each other. We propose that this phenotype could represent a new microduplication 12q14 syndrome. WES studies showing no other known pathogenic variants support the causative effect of the duplication. In conclusion, our results strongly support the role of"}
+{"text": "Following the publication of this article , the folFigure 3A panel IV and Figure 3B panel IV appear to contain areas of similarity;Figure 3A panels I, V, and VI appear to contain areas of similarity;Figure 3B, panels I and II contain areas of similarity;Figure 3B, parts of panels V and VI contain areas of similarity;Journal of Food and Chemical Toxicology [Figure 6 of a previously published article in the xicology  by four xicology  appear txicology  appears xicology ; Figure xicology  appears xicology ;Repeated regions of similarity in flow cytometry plots within Fig 6 IV, VI, and II;Figure 2A ERK1/2  bands in lanes 5 and 6 appear similar when adjusted for brightness/contrast;Figure 2A p38  panel lanes 2 and 6 appear similar when adjusted for brightness/contrast;Figure 2A ERK1/(P) panel contains vertical discontinuities across all lanes when adjusted for brightness/contrast;Figure 5 p53 contains background irregularities when adjusted for brightness/contrast such that lane 2 appears to have a background different to other lanes.The authors have been unable to provide any primary data underlying the figures. The authors commented that the regions of similarity in histopathological images arose due to similarity in lesions studied. For the similarities noted in flow cytometry plots, the authors commented that this could have arisen due to similarity in equipment and protocols.PLOS ONE Editors retract the article.In the absence of the data underlying the figures and in light of the above concerns, the The authors did not comment on the retraction decision."}
+{"text": "The role of many lncRNAs in cancer remains elusive including that for a Prostate Cancer Associated Transcript 92 (PCAT92). PCAT92 shares the locus on chromosome 13 with ABCC4 gene, known to be implicated in prostate cancer. It has been shown that PCAT92 and ABCC4 are up-regulated in prostate cancer samples from multiple transcriptome datasets. Among the prostate cancer cell-lines LNCaP showed maximum overexpression of PCAT92 compared to control cell-line RWPE-1. We have shown that knockdown of PCAT92 in LNCaP cells reduces cell viability and proliferation and down-regulates ABCC4 transcript/protein expression. The shared region between PCAT92 and ABCC4 has a binding site for an oncogenic transcription factor (ZIC2) which is also upregulated in the majority of datasets studied here. ZIC2 binding to the predicted ABCC4 promoter has been confirmed using pull-down assay. Interestingly, under PCAT92 knockdown condition, there is a reduction in the ZIC2 binding to ABCC4 promoter indicating the potential involvement of PCAT92 in the recruitment of ZIC2. We have identified distinct regions on PCAT92 with potential to bind to ZIC2 non-DNA binding Zinc-finger domain and potential for triplex formation near ABCC4 promoter region, which have been experimentally validated. Together, these observations and localization in the nucleus suggests that PCAT92 may play a role in prostate cancer by increasing the local concentration of ZIC2 by forming RNA-DNA triplex near ABCC4 promoter thus helping in recruitment of ZIC2 for ABCC4 regulation. Long non coding RNAs (lncRNAs) are bringing a new level of understanding of cancer biology. Using high-throughput sequencing, a large number of cancer-associated lncRNAs are being discovered and involvement of many of them in cancer is slowly emerging. The mechanism of MALAT1, one of the earliest lncRNAs associated with cancer, is still emerging. Using mouse knockout models, it was shown that a set of metastasis associated genes were deregulated, strongly indicating its basis as a regulator of gene expression \u20134. OvereMore recently, using high-throughput transcriptome sequencing, it has been shown that almost a quarter of abundant transcripts in prostate cancer are lncRNAs with many that are still unannotated . This isOne of more established mechanism of action of lncRNAs is by regulation of genes within the same locus. For example, PCA3, located in intron 6 of the PRUNE2 gene, is co-expressed along with PRUNE2 in the nucleus to form a double-stranded RNA . The lncWe have previously shown that PCAT92 is located closely to ABCC4 gene on chromosome 13 . ExpressZinc-finger transcriptional regulators ZIC1 through ZIC5 have been associated with increase in transcriptional activity of many genes . ZIC2 isHere our work demonstrates how PCAT92 transcript is regulating the expression of ABCC4 by recruiting ZIC2 at the ABCC4 promoter region and forming triple helix near the promoter site to stabilize the binding of ZIC2 at ABCC4 promoter region.PCAT92 and ABCC4 are only 4305 bases apart on chromosome 13 in a divergent configuration sharing the 5\u2019 regulatory region Figure . PromoteIt was reported that PCAT92, ABCC4 and ZIC2 are overexpressed in multiple prostate cancer datasets in the public repository (SRP002628 and ERP000550) . The oveFigure Binding affinity of ZIC2 at the ABCC4 promoter region in cell line is studied using ChIP-RTPCR with anti-ZIC2 antibody. Primers flanking the predicted ZIC2 binding site on ABCC4 promoter region was made. As shown in Figure in vitro. The transcribed RNA was then titrated onto the purified zinc finger domains of ZIC2 using Nano Isothermal Titration Calorimetry (NanoITC). This validated the interaction between the predicted PCAT92 RNA strand and the zinc finger domains of ZIC2. Antisense strand to the predicted PCAT92 RNA region was also cloned and transcribed to be used as negative control. As shown in Figure in-vivo validation of interaction between PCAT92 and ZIC2, we performed RNA Immunoprecipitation (RIP). The cells were fixed and RNA and protein were crosslinked. Using anti-ZIC2 antibody RNA-protein complexes were pulled down. RNA-Protein complex were broken by reverse crosslinking and the resulting RNA was subjected to cDNA synthesis. HEIH, one of the well-established non-coding RNA  ATP using T4 Polynucleotide Kinase . Zinc finger domains of ZIC2 protein were PCR amplified and cloned in pET28a expression vector. The clone was sequenced verified and the induced using 0.5mM IPTG, at 16\u00b0C overnight. Induced protein was purified using His Tag affinity chromatography and eluted with the buffer containing 200 mM imidazole. 2\u03bcg of ZIC2-HIS protein and 100pmol of radiolabeled target dsDNA, was mixed in the binding buffer. The reaction solutions were incubated for 30 minutes at 37\u00b0C and subject to electrophoresis on 6% polyacrylamide gels prepared in 1\u00d7TBE (Tris-borate-EDTA) buffer for 16 to 18 hours at 100V at 4\u00b0C. Imaging was done using phospho imager .To find all putative regions within PCAT92 transcript with triplex forming potential against the 4kb shared 5\u2019UTR region on the chromosome, Triplexator computational pipeline was used  with all*DD) triple helical structure comprising U*AT and C*GC (Sequence 1) base triplets is generated conforming to a 12-fold helix [A parallel RNA.DNA.DNA  experiments were performed at 25\u00b0C using Nano ITC . The protein sample was prepared as explained in Electrophoretic Mobility Shift Assay study. The predicted region of PCAT92 was cloned in pBS-SK plasmid followed by synthesis of sense and antisense RNA from the clone using T3 RNA polymerase  and T7 RNA polymerase . 50\u03bcl of each sense RNA (60\u03bcM) and antisense RNA (60\u03bcM) was titrated into 300\u03bcl protein solution (5\u03bcM) with a fixed stirring speed of 300 rpm at 25 \u00b0C. Both RNA and Protein were dialysed in the same buffer . Similarly to under the interaction between the PCAT92 RNA and ABCC4 DNA we performed ITC for the predicted PCAT92 and ABCC4 region. The oligos corresponding to both were obtained. 50\u03bcl of each PCAT92 RNA (10\u03bcM) and random RNA (10\u03bcM) was titrated into 300\u03bcl ABCC4 DNA solution (5\u03bcM) with a fixed stirring speed of 300 rpm at 25 \u00b0C. Both RNA and DNA were dialysed in the same buffer . For both the titration experiment, 5\u03bcL of RNA was dropped for 10 injections. To achieve complete equilibration, the spacing time between each injection was set to 300 seconds. All measurements were repeated at least twice. Using Nano ITC NanoAnalyze software, the raw data was integrated, corrected for nonspecific heats and analysed according to independent model."}
+{"text": "AZFc microdeletion in their blood leukocytes. However, if AZF genes were involved in impaired spermatogenesis, a higher frequency of chromosomal microdeletions was expected. In this study the frequency of AZFc microdeletion was compared with TTY2 gene family, i.e., TTY2A2A and TTY2A12A in blood leukocytes of NOA patients and normal fertile control. In the present study 30 normal fertile individuals with mean age of 35.0 \u00b1 6.0 and 30 NOA patients with mean age of 34.0 \u00b1 7.0 were screened for microdeletion of TTY2L2A and TTY2L12A at Yq11 and Yp11 respectively and sequence-tagged site (STS) markers for AZFc gene using multiplex PCR technique. At the first step karyotyping was done for all subjects using standard G-banding technique to identify patients with normal karyotype as well as non-affected normal controls for molecular analysis.About 10\u201315% of non-obstructive azoospermia (NOA) patients show AZFc microdeletion in normal and NAO patients whereas one TTY2L2A microdeletion in normal control (3.3%) and 4 in NOA (13.3%) was observed (p < 0.05). However our data indicated that 6 of 30 NOA patients (20%) showed TTY2L12A microdeletion whereas there was no observed microdeletion in normal control (p < 0.01).Results showed no AZF genes in infertile men. Therefore, screening these genes along with AZF genes might be valuable for infertile patients. The reason why these genes are deleted from Y chromosome is not known but might be associated with genomic instability induced by environmental physico-chemical genotoxic agents.Results indicate that the studied genes might be involved in impaired spermatogenesis more effective than the routinely screened  Therefore the aim of this study was to evaluate the frequency of microdeletion in TTY2 genes versus AZFc in peripheral blood leukocytes of NOA patients.As known, sperm DNA damage is clearly associated with male infertility and abnormal spermatogenesis 2.2.1.This experimental study was performed on whole blood samples obtained from 30 non-obstructive azoospermia patients and 30 normal men candidate for Assisted Reproductive Technology (ART) referred to Fertility and Infertility Center of Shariati Hospital . Normal and patients' demographic data is presented in 2.2.Karyotype analysis was performed to exclude those individuals with chromosomal abnormalities especially those involved in male infertility such as Klinefelter's syndrome from molecular analysis. Whole blood obtained from normal individuals and non-obstructive azoospermia patients in heparinized tubes was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin (100 U/mL)/streptomycin (100 \u00b5g/mL), and 0.1 mL phytoheamaglutinin . Cells were exposed to thymidine 17 hours prior to colcemid treatment at final concentration of 0.4 \u00b5g/mL one hour before harvesting. Cells were harvested at 72 h after culture initiation. Harvesting, slide preparation and G-banding were performed according to routine standard procedure. At least 20 metaphases were analyzed for each subject. The resulting karyotypes were described according to the norms established by the International system for Human Cytogenetic Nomenclature 2.3.AZFc and TTTY2 microdeletion analysis. Peripheral blood samples were collected in tubes containing EDTA from cytogenetically normal infertile patients. All tests were performed on genomic DNA that was extracted from peripheral blood leukocytes using a commercially available DNA-isolation kit for mammalian blood . For each participant, AZFc-specific STSs that spanned the sub regions (sY254 and sY255) as well as STS for SRY  as internal controls were used to amplify specific regions of the Y chromosome using multiplex PCR TTY2L12A and TTY2L2A regions with SRY  as internal control. The primers used in this study have been previously used by Yapijakis et al. AZFc and TTY2L2A and TTY2L12A gel electrophoresis images in normal and azoospermia patients respectively.The patients and normal individuals with 46,XY, normal karyotypes were selected for 2.4.U-test as well as one way analysis of variance (ANOVA) was used to compare differences between the study groups. Sigma Plot (2004) for Windows was used to draw figures, p < 0.05 was considered as statistically significant.Statistical analysis was done using SPSS software . The Mann-Whitney 3.p < 0.001) clearly indicates the status of fertility in both groups. Both normal individuals and non-obstructive azoospermia patients did not show any microdeletions in their AZFc reigon . In non-obstructive azoospermia group six patients showed deletion in TTY2L12A (20%), whereas four patients had deletions in TTY2L2A (13.3%). In addition, none of the patients showed deletions in both studied TTY2 genes simultaneously. Results obtained for TTYA2L2A and TTY2L12A microdeletion were significantly different compared to control fertile group (p < 0.05 and p < 0.01 respectively). There was no correlation between microdeletions and age of the patients.Results are summarized in c reigon  and 4. N4.AZF region is mainly done with the use of PCR on blood leukocytes In the diagnostic work-up of infertile men, screening of Y chromosome microdeletion usually in AZF genes, AZFc is the most commonly deleted interval in men with azoospermia or severe oligozoospermia AZFc deletion frequency in azoospermia patients is reported to be 10\u201315%. Although, recent report indicates that a high frequency of microdeletions in AZFc region especially DAZ gene might occur de novo during spermatogenesis when sperm nuclei are studied AZF) region of Y chromosome especially in the DAZ genes was shown previously to occur in lymphocytes of men exposed to low dose and background level of ionizing radiation in vitroAmong TTY2L2A and TTY2L12A genes, expect one individual who exhibited microdeletion for TTY2L2A. The reason for this observation is not understood, but might be a de novo chromosomal alteration due to environmental exposure. Similar observation was previously reported for DAZ deletion in normal population exposed to high natural background radiation TTY2-associated deletions observed in leukocytes of infertile azoospermia patients might suggests that TTY2-like transcripts may play a significant role in the complex process of spermatogenesis. As shown in TTY2L2A and TTY2L12A respectively which is very high frequency compared to the overall frequency reported for AZFc microdeletion and more importantly in those patients with no deleted AZFc. These results might clearly indicate the involvement of the studied genes in spermatogenesis failure and male infertility. Perhaps their implication in male infertility might be more important than AZF genes which are routinely screened. A similar frequency of microdeletions of TTY2L2A and TTY2L12A genes is reported in oligoazoospermia and azoospermia patients form Greek patients TTY2L2A and TTY2L12A genes in azoospermia patients with no AZFc microdeletion and higher frequency of microdeletions in azoospermia patients with unkown AZF situation TTY2L2A and TTY2L12A genes are related to AZF deletion, then only in a lower percentage of patients' deletion in TTY2 related genes could be detected. Moreover the deletion in TTY2L12A gene has nothing to do with AZF genes because it is located on the short arm of Y chromosome. Another possibility of lower frequency reported by Shaveisi-Zadeh et al. TTY2 gene family is in its early stage. There are only limited reports associating these genes with male infertility. Although the possible causes of different frequency of microdeletions reported for these genes has already been discussed but for implementing testing these genes in paraclinic along with other Y microdeletions, there is a need for more researches and more data from different populations with different ethnicity and geographic distributions. So that we could be able to make a meta-analysis from different reports to provide a frequency range of microdeletions for TTY2 gene family for clinical use. As known for routinely screened Y microdeletions among infertile or subfertile men, there is a very big variation in the frequency of occurrence from 1\u201355% TTY2 gene family leading us to observe these microdeletions with different frequencies.As shown in TTY2 genes is in line with our previous observation for DAZ microdeletion and indicate that there are hot spots in these genes that make them vulnerable to damage and eventually deletion from entire DNA in Y chromosome Our observations regarding to AZFc microdeletion might clearly indicate involvement of TTY2 genes in spermatogenesis failure and male infertility.In conclusion a high frequency of microdeletions observed in our study on non-obstructive azoospermia patients without"}
+{"text": "Uncovering how new members of multigene families acquire new functions is an important topic in evolutionary and developmental genetics. CORL proteins  are a family of CNS specific proteins related to mammalian Sno/Ski oncogenes. Drosophila CORL (dCORL) participates in TGF-\u03b2 and insulin signaling during development and in adult homeostasis but roles for the two mouse CORL proteins (mCORL) are essentially unknown. A series of studies were conducted to test the hypothesis based on previous results that mCORL1 is more similar to dCORL than mCORL2. Neither an updated alignment nor ectopic expression in adult wings were able to distinguish mCORL1 or mCORL2 from dCORL. Transgene experiments employing a dCORL endogenous function in mushroom body neurons showed that mCORL1 is distinct from mCORL2 and dCORL. mCORL1 and mCORL2 are also distinct in biochemical assays of Smad-binding and BMP signaling. Taken together, the data suggests testable new hypotheses for mCORL2 function in mammalian TGF-\u03b2 and insulin signaling based on known roles for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species. The dominant model for the growth of multigene families and the acquisition of novel functions by new genes is based on gene duplication with one copy of the pair maintaining the original function while the other copy accumulates advantageous mutations leading to a novel function. We recently explored the consequences of this \u2018duplication then neofunctionalization\u2019 model for new genes in mammals and flies. For each, we employed the conserved Dpp/BMP signaling pathway (subfamily of the TGF-\u03b2 family) that regulates embryonic dorsal-ventral axis formation in flies and vertebrates as our assay. Neofunctionalization for two new genes contrasted with the conservation of a third new gene.lolal is not present in mammals. It encodes a BTB domain protein that functions in a chromatin-remodeling complex maintaining Dpp transcription during dorsal-ventral axis formation  in the larval brain. In Drosophila mushroom body neurons dSmad2 signaling upstream of EcR-B1 transcriptional activation is facilitated by the Smad binding dCORL protein (mCORL1 (mSKOR1) was identified as a Sno/Ski family member that functions as a transcriptional co-repressor in cell culture. In embryos mCORL1 is expressed only in dorsal interneurons of the cerebellum  and both rescue Dpp mutants during dorsal-ventral patterning. Underlying the rescue, BMP2 is 82% and BMP4 is 81% similar to Dpp in the ligand region , mCORL1 (AK049035) and mCORL2 (NM_001109743) generated with Clustal Omega at escribed .Wing experiment stocks were: MS1096.Gal4 (Df(4)dCORL / ciD (Df(4)dCORL/Dci, 2) UASt.mCORL1 on III; Df(4)dCORL/Dci or 3) UASt.mCORL2 on III; Df(4)dCORL/Dci for rescue and overexpression experiments. Fly crosses for rescue and overexpression experiments were maintained at 25\u00b0 prior to heat shock. Larvae were heat shocked 40-64 hr after egg lay for 1 hr @ 37\u00b0. After one hour of recuperation at 18\u00b0 they were returned to 25\u00b0. Wandering 3rd instar larvae were picked 6 days after egg lay and checked for GFP before dissection (Df(4)dCORL status . This allowed the interpretation of the lobe with a clone in the MB as either mutant rescue or wild type repression of EcR-B1. At least seven brains were examined per genotype . The number of brains with clones in the MB for each genotype is reported in the figure legend for each experiment.Flip out clone stocks were: yw hs.FLP; AY.Gal4 UASt.GFP; yw) were picked, sorted and their brains dissected as in Post-heat shock larvae that have stopped wandering but not yet begun pupariation . A schematic of the structure of CORL proteins and an assessment of the similarity of dCORL, mCORL1 and mCORL2 in the Smad-binding Sno homology domain based on a new alignment is in The premise that similar sequences lead to similar functions is the reason investigators use BLAST to search for proteins similar to their favorite protein with the hope that functional data exists for a match, pointing to a specific hypothesis they can test in their system. A tree of the CORL/Sno/Dac family that share the Smad-binding Sno homology domain was valuable in suggesting we look in mushroom body neurons for dCORL function as this was the only established location for Activin subfamily signaling at the time , between the two mCORL proteins and dCORL in the Sno homology domain is greater than initially reported due to updated methods underlying the new alignment. In this region mCORL1 and mCORL2 (94% similar with 14 differences over 195 residues) are almost as similar as human BMP2 and BMP4 (96%) in the ligand region. The conservation of these two with dCORL, 89% for mCORL1 (27 differences) and 90% for mCORL2 (24 differences) is very high. This is greater than the conservation of Dpp with BMP2 (82%) or BMP4 (81%), proteins that can reciprocally substitute for each other across species dCORL mutant brains was due to the loss of dCORL, we rescued EcR-B1 expression with heat shock driven flip-out clones of UASt.dCORL (Df(4)dCORL mutants expressing UASt.GFP or UASt.GFP with either UASt.dCORL or UASt.mCORL1 or UASt.mCORL2 .Previously, to show that the loss of EcR-B1 mushroom body (MB) expression in Df(4)dCORL brains are easily visible in the presumptive EcR-B1 domain of the MB body. This location is characterized by proximity to Tailless expressing MB neuroblasts that are unaffected in Df(4)dCORL brains (Df(4)dCORL brains fully rescues EcR-B1 dCORL; Pursuing another line of experimentation employed in our prior analysis of dCORL, we examined flip-out clones expressing dCORL, mCORL1 and mCORL2 in the MB of wild type siblings of The data are shown at high magnification in dCORL mutants. Genetic evidence from both assays of an endogenous function of dCORL (regulation of EcR-B1 expression) support the alternative hypothesis that mCORL1 has a divergent function with mCORL2 and dCORL more similar.In this assay, MB clones of mCORL1 did not repress EcR-B1 expression ; Table 1dCORL mutants  consistent with loss of EcR-B1 expression in The binding data shows that mCORL2 does not bind Smad3  even in Consistent with the binding assay, parallel studies of TGF-\u03b2 and BMP signaling showed that mCORL1, mCORL2 and c-Ski can repress TGF-\u03b2 signaling  but onlyTaken together the sequence, genetic and biochemical data support the alternative hypothesis that dCORL and mCORL2 are the closest functionally with mCORL1 divergent. The data does not address which mCORL came first, as either of the two duplicates could diverge while the other maintained the ancestral function.dCORL mutants, 2) repression of EcR-B1 when overexpressed in wild type, 3) mCORL2 failure to bind the mCORL1 partner mSmad3 and 4) mCORL1 inability to antagonize BMP signaling in luciferase assays. We recognize that the data are not black and white with similarities between mCORL1 and mCORL2 in the wing assays and modest rescue of EcR-B1 by mCORL1 in dCORL mutants. However, the preponderance of evidence indicates that mCORL2 and dCORL share the ancestral function and that mCORL1 has a distinct function.None of the data supported the initial hypothesis that mCORL1 is more similar to dCORL than mCORL2. Instead most experimental data were consistent with the alternative hypothesis that mCORL1 has a distinct function from mCORL2 and dCORL: 1) rescue of EcR-B1 in From a larger perspective the data informs our understanding of multigene family evolution, specifically the accumulation of advantageous mutations. The six differences between the mCORL genes in the Sno homology domain are quantitatively similar to the eight differences between human Smad2 and Smad3 in the DNA-binding domain (Regarding functions for mCORL2 based on its biochemical properties and the reported roles of dCORL (In summary, our data that mCORL2 and dCORL share functions while mCORL1 has diverged reinforces the current working hypothesis that neofunctionalization of a new gene can be achieved with a handful of advantageous amino acid changes in a highly conserved functional domain. We also encourage our colleagues to experimentally test roles for mCORL2 in development and adult homeostasis that reflect functions for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species."}
+{"text": "CCL3\u2212/\u2212 mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3 One mechanism that has been identified as a driver of BMME dysfunction is the inflammatory chemokine CCL3.The bone marrow microenvironment (BMME) is comprised of a complex network of cellular and molecular components that regulate hematopoiesis and hematopoietic stem cells (HSCs). The BMME contributes to the initiation and progression of hematologic malignancies such as chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and myeloproliferative neoplasms (MPN)5. In hematologic malignancies CCL3 is a well-known mechanism of BMME disruption in multiple myeloma, both activating osteoclastic bone resorption, and inhibiting bone forming osteoblastic cells6. We have previously identified CCL3 as an abundant leukemia-derived factor in a murine model of blast crisis chronic myelogenous leukemia (bcCML) that also displayed a profound loss of mature osteoblastic cells in the BMME7. CCL3 has also been implicated as a factor leading to microenvironmental dysfunction in a variety of malignancies including myelodysplastic syndrome (MDS), metastatic disease of renal cell carcinoma, and B-Cell lymphomas11.CCL3 is a chemotactic factor, signaling through the chemokine receptors CCR1 and CCR5 to recruit lymphocytes to sites of infection, and CD8+ T-cells to active lymph nodes12. In addition, when a mutant allele of the gene Ptpn11 that is associated with juvenile myelomonocytic leukemia (JMML) is homozygously expressed in murine osteoprogenitors mice develop a myeloproliferative neoplasm reminiscent of JMML. The murine disease correlates with high levels of CCL3 expression and is reversed with the administration of CCL3 receptor antagonists13. In human AML approximately 75% of patients exhibit elevated expression of CCL37, and overexpression of CCL3 in Nup98/HoxD13 expressing pre-leukemic cells leads to transformation to AML14. These studies confirm an essential role for CCL3 in the emergence and maintenance of LSCs in these murine models of myelogenous leukemias. Therefore, CCL3 is an appealing therapeutic target in hematologic malignancies.In a murine model of CML, malignant cells that do not express CCL3 cannot maintain a leukemia stem cell (LSC) population and consequently do not give rise to leukemic disease15. Further, the CCL3 receptor CCR5 is a coreceptor for the cellular entry of human immunodeficiency virus (HIV)16, and the FDA has approved the drug maraviroc, a small molecule CCR5 antagonist, for the treatment of HIV infection15. Since leukemia initiating cells share a number of characteristics with normal stem cells, such as increased self-renewal and a more quiescent state, targeting of LIC may also impact HSCs. In fact, the role of CCL3 in support of hematopoiesis is controversial. CCL3 was previously demonstrated to inhibit hematopoietic progenitor proliferation in colony forming assays18, to enhance myelopoiesis in vitro19, and to be dispensable for HSC maintenance in vivo20. To determine the necessity of CCL3 for maintenance of normal hematopoiesis we performed a comprehensive in vivo analysis of homeostatic hematopoiesis and the BMME in CCL3 knockout (CCL3\u2212/\u2212) mice21.The potential for translation of CCL3 targeting is further supported by the prior development of small molecule inhibitors of the CCL3 receptors CCR1 and CCR5. Multiple CCR1 inhibitors have been developed for the treatment of a variety of diseases such as rheumatoid arthritis and multiple sclerosis, and, having proceeded through phase 1 and 2 clinical trials, have been found safe for use in humans22. However, while previous reports have shown no overt hematopoietic abnormalities in CCL3\u2212/\u2212 mice a detailed analysis of HSC function in the absence of CCL3 signaling has not been performed. To perform an unbiased multidimensional analysis of the bone marrow we generated CyTOF data to phenotype bone marrow cells based on 31 antigens , and positive for Sca1 and cKit , short-term repopulating HSCs (ST-HSC), multipotent progenitor (MPP) 2, and MPP3, while the lymphoid biased MPP4 population was not increased 7. Under homeostatic conditions CCL3\u2212/\u2212 mice show no difference from controls in the number of Lineage-CD45-CD31-CD51+ Sca1- osteoblastic cells as measured by flow cytometric analysis  of primary AML samples that were analyzed7. Therefore there is potential for translation of CCL3 pathway inhibition to the clinic. Additional support for the feasibility of CCL3 inhibition as a therapy for hematologic malignancies is provided by the availability of inhibitors for CCL3 receptors. CCL3 signals through the chemokine receptors CCR1 and CCR55, and several small molecule antagonists have been developed for both of these receptors for a variety of diseases and with varying degrees of success27. CCR1 antagonism has been seen as a potential strategy for diseases such as rheumatoid arthritis and multiple sclerosis; however, clinical trials for the antagonists developed have generally failed in phase 2 after being found safe, but not efficacious for treatment27. Antagonists of CCR5 have also been developed for the treatment of HIV infection, as CCR5 is a co-receptor for cellular entry of HIV. One such antagonist, Maraviroc, was developed by Pfizer and is currently FDA approved for the treatment of HIV infection28. Notably, though CCL3 is emerging as a potential therapeutic target, its role in homeostatic regulation of HSCs has not been carefully studied.The chemokine CCL3 is relevant to human disease as a therapeutic target and previous findings in murine models, including our own, have demonstrated a role in hematologic malignancies\u2212/\u2212 mice. This is consistent with previous reports of CCL3 acting as an inhibitor of stem cell proliferation18. A careful analysis of the heterogeneous HSPC compartment in the bone marrow of CCL3\u2212/\u2212 and control mice revealed that in addition to HSCs, myeloid biased MPP2 and MPP3 populations were significantly increased, while lymphoid biased MPP4 populations were unchanged. Further, the levels of monocytes and granulocytes were lower in CCL3\u2212/\u2212 mice compared to WT. This indicates that CCL3 is involved in the differentiation process of these cell types, leading to a decrease of mature cells in the periphery and an increase in their progenitors in the bone marrow. Together these data suggest that CCL3 regulates the self-renewal capacity of the most immature hematopoietic cells, as well as the differentiation of progenitors of the myeloid lineage.To establish the role that CCL3 plays in regulation of homeostatic hematopoiesis and the BMME we characterized a global CCL3 knockout murine model. Results from flow cytometric analysis of HSPC populations demonstrate that lack of CCL3 signaling results in an expansion of the HSC pool in the bone marrow of CCL3\u2212/\u2212 HSPCs repopulate peripheral blood B cells, T cells, and myeloid cells with a reduced contribution compared to WT controls. These results suggest that increased proliferation and accumulation of HSPCs in the bone marrow is caused by a defect in myeloid differentiation and a relative loss of functional HSPCs in CCL3\u2212/\u2212 mice.Increased numbers of phenotypic HSC populations in the bone marrow do not necessarily correlate with increased functional repopulating ability. For this reason we analyzed the ability of sorted HSPCs to repopulate the hematopoietic system of myeloablated recipients. Following competitive transplantation, CCL37, loss of CCL3 expression has no effect on the osteoblastic populations, including MSCs. However, in female mice there is a loss of osteoclastic cells in the bone marrow, suggesting that the change in osteoclastic activity is the cause of altered trabecular architecture.The differences that were observed can be caused either by direct signaling to HSPCs, or indirect signaling through alterations in the BMME. Micro-CT analysis of trabecular and cortical bone in the femur revealed no change in overall bone volume, though some minor differences in microarchitecture were observed in female mice, suggesting that CCL3 may regulate trabecular bone remodeling in a sexually dimorphic manner. Though it has previously been shown that CCL3 overexpression in malignant conditions leads to loss of mature osteoblastic cells, and over-activation of osteoclasts leading ultimately to a loss of boneOverall, our data demonstrate that CCL3 plays an active role in the homeostatic maintenance of myeloid differentiation. Since CCL3 has previously been shown to be critical for the maintenance and emergence of leukemia initiating cells in multiple murine models, it will be important to evaluate any impact of CCL3 inhibition on the normal hematopoietic system as well as the malignancy being treated. However, these results, coupled with the availability of CCR1 and CCR5 antagonists suggest that the CCL3 signaling pathway may be an attractive therapeutic target in myelogenous leukemias.Ccl3tm1Unc/J (CCL3\u2212/\u2212) mice fully backcrossed onto a C57BL/6J were purchased from Jackson Laboratory. B6.SJL-PtprcaPep3b/BoyJ expressing the CD45.1 allotype were purchased from the mouse breeding core facility at the University of Rochester School of Medicine and Dentistry.Mice were maintained within the Vivarium facility at the University of Rochester School of Medicine and Dentistry in accordance with protocols approved by the University\u2019s Institutional Animal Care and Use Committee, the University Committee on Animal Resources. Wild type C57BL/6J, and B6.129P2-5 molecular weight dextran to precipitate RBCs and the WBC containing supernatant was collected.Bone marrow cells were collected by flushing the long bones of the hind limbs of mice using a 25-gauge needle and FACS buffer . Peripheral blood cells were collected by submandibular bleeds into EDTA coated tubes. CBC counts were performed on a HESKA Hematrue. Peripheral blood white blood cells (WBCs) were collected by incubating blood in a 2% solution of 5\u2009\u00d7\u2009107 cells were collected as described above and suspended in 100\u2009\u03bcL PBS supplemented with 2% heat inactivated fetal calf serum (FACS buffer) and stained with the appropriate antibodies , resuspended in PBS with 50\u2009\u03bcM 127IdU cell identification reagent (DVS-Fluidigm cat# 201127) and incubated for 30\u2009minutes at 37\u2009\u00b0C. Cells were washed twice with MaxPar staining buffer, resuspended in 100\u2009\u03bcl MaxPar staining buffer containing the appropriate extracellular antibodies .For CyTOF analysis 10\u2009\u00d7\u200910\u2212/\u2212 or C57bl/6 (CD45.2+) control mice were mixed with 5\u2009\u00d7\u2009105 competitor whole bone marrow cells from B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice in a total of 0.1\u2009mL of FACS buffer. Recipient 6\u20138 week old B6.SJL-Ptprca Pep3b/BoyJ mice received a split dose of radiation of 5\u2009Gy each separated by 24\u2009hours. The second dose of radiation occurred 1\u20132\u2009hours prior to injection of transplanted cells via tail vein.One hundred sorted LSK, Flt3- bone marrow cells from CCL3Dissected hindlimbs were scanned on a Viva CT40  using a 55-kVp, 145-\u03bcA current and a 300-ms integration time at a resolution of 12.5\u2009\u03bcm. Analysis of trabecular bone was performed on a 1.25\u2009mm region 50\u2009\u03bcm below the growth plate of the femur and tibia. Cortical analysis was performed 4375\u2009mm from the growth plate of the femur and tibia and encompassed a region of 375\u2009\u03bcm.7. Immunohistochemical staining for F4/8029 and Osteocalcin30 was performed as previously described.Dissected hindlimbs were fixed, decalcified, processed and embedded in paraffin as previously describedSupplementary information"}
+{"text": "After this article  was publcyr1-, sxa2-) in cyr1-, sxa2-, cpc2-) in In the 8h, 16h, and 32h panels for JY546 panels shown in In the JY1578 appears to have different contrast levels between the cells and surrounding background.In sxa2-, pmp1-) in sxa2-,oe-cpc2+).The image for strain JY1716 in sxa2-,oe-cpc2+).The image for strain JY710, 16h panel is provided in The authors are unable to clarify the concerns raised about background irregularities in cyr1-, sxa2-, pmp1-) cells in sxa2-, pmp1-) panel, and in sxa2-, pyp2-) and JY1717 panels. In the updated sxa2-, pmp1-) panel is included in The authors apologize for the PLOS ONE Editors issue this Expression of Concern.Original data are no longer available for the other figure panels discussed above and other results reported in the article. Due to the nature and extent of image issues and lack of underlying image data for the figures in question, the PLOS ONE Editors apologize for our delay in resolving this matter and thank the authors for replying promptly to journal queries.Some of the above concerns were raised in 2014. The S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."}
+{"text": "Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 ( However, unlike animal cell cycles, in plants two CDKs, A and B regulate the transition from G2 to mitosis3. CDKA is closely related to the ancestral cdc2 of fission yeast and is able to complement temperature sensitive cdc2- mutants. In tobacco BY2 cells, CDKA transcript4 and protein5 levels are constant throughout the cell cycle, whereas activity peaks at S/G25. The CDKB family  is unique with the highly conserved PSTAIRE domain of cdc2 (and CDKA) altered to PPTLARE/PPTLRE3. Also, Arabidopsis CDKB genes are unable to complement cdc2-/cdc286. CDKB transcripts5, protein and activity all peak at G2/M4.The eukaryotic cell cycle is a conserved phosphorylation cascade in which key substrates require phosphorylation or dephosphorylation prior to the next step in the cell cycle. At the G1/S and G2/M transitions, major phosphoregulation occurs, catalysed by cyclin-dependent protein kinases (CDKs) that are largely conserved in unrelated species8. When Wee1 is over-expressed in fission yeast (Schizosaccharomyces pombe) cells arrest in G2 resulting in highly elongated cells7. Conversely, the phosphatase, Cdc25, dephosphorylates the same tyrosine residue activating the CDK9. The role of CDC25 in the plant cell cycle is less clear. A truncated version of the yeast CDC25 gene containing only the catalytic domain is present in the Arabidopsis genome10 and can induce a short cell length when expressed in fission yeast11. However, its role in the plant cell cycle seems to be limited to the DNA damage replication checkpoint12 as the plants grow and develop normally. Thus perturbation of Arath;CDC25 expression in Arabidopsis resulted in hypersensitivity to hydroxyurea while over-expression resulted in tolerance compared to wild type. Moreover, although Arath;CDC25 has phosphatase activity10 it also has arsenate reductase activity13 suggesting that in plants CDC25 may have additional roles outside of the cell cycle.In fission yeast, Wee1 and Mik1 kinases phosphorylate Tyr15 of the CDK to inactivate it and prevent entry into mitosisSpcdc25) was expressed in plant cells to study the effects of CDK de-phosphorylation14. Expression of the fission yeast CDC25 gene in both tobacco15 and Arabidopsis16, resulted in phenotypes that are consistent with its action in dephosphorylating and activating CDK. Expression of Spcdc25 in tobacco BY2 cells resulted in a reduced mitotic cell size and a reduction in the length of the G2 phase17. Moreover, in these cells, cytokinin levels were greatly reduced and the cells were insensitive to the cytokinin biosynthetic inhibitor, lovastatin indicating a link between CDK de-phosphorylation and cytokinin signalling. In addition, Spcdc25 expression in tobacco cell suspension cultures altered carbohydrate status resulting in an increase of starch and soluble sugars and a higher sucrose:hexose ratio. These changes are inducible in WT by cytokinin treatment, thus, Spcdc25 expression in tobacco had a cytokinin-like effect18. In whole plants, this cytokinin-independent phenotype was supported by an ability of Spcdc25 expressing stem explants to produce shoots in the absence of exogenous cytokinin19. Consistent results were obtained in Arabidopsis plants expressing Spcdc2516, which showed a reduction in primary root length and increased production of lateral roots. Another effect of Spcdc25 expression in tobacco was precocious flowering with a dramatic reduction in both the time to flowering, and the number of leaves and nodes formed prior to flowering20. Moreover, study of flowering of tobacco nodal stem segments in vitro revealed that the typical acropetal flowering gradient in WT plants did not occur in the Spcdc25 transgenic plants21. However when Spcdc25 was expressed in Arabidopsis, flowering time was not affected (Rogers and Francis lab. unpublished data).Given the uncertainties around plant CDC25, fission yeast CDC25  resulted in increased overall WEE1 protein, reduction in CDKA histone H1 kinase activity and an increase in phosphorylated CDKA25. This was accompanied by an increase in cell size and a delay in the G2/M transition in synchronised cells. However, surprisingly, when Arath;WEE1 was expressed in tobacco BY2 cells, there was a shortening of G223. This was reversed by co-expression of the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1), which interacted with WEE1, presumably removing it through the 26\u2009S proteasome.Cultured hypocotyls of Arath;WEE1 expression in tobacco cells are mirrored by effects on the development of whole plants, and is consistent with a perturbation of the native tobacco WEE1, creating a dominant-negative-like effect.Data are presented here showing that the anomalous effects of Arath;WEE1 expression in tobacco  flowered significantly earlier, after about 100 days  were detected compared with the empty vector line (EV) or with WT  and peaked earlier (4\u20135\u2009h) compared with the minus DEX control in which expression of Arath;WEE1 was not induced , the WEE1 protein level decreased . Hence, changes in WEE1 protein through the cell cycle followed the altered timing of mitosis in the induced cells.To establish the mechanism of the cell cycle changes, effects on the timing of changes in WEE1 protein during the cell cycle were investigated. In synchronised BY2 cell lines transformed with an inducible sed Figs\u00a0; S6. WheArath;WEE1 by addition of DEX, WEE1 kinase activity was maximal in early S phase and decreased by 31% in late G2 reaching a minimum during mitosis, consistent with the observed decrease in WEE1 protein level. In induced cultures, the WEE1 kinase activity was again maximal in early S phase and decreased by 29% in G2 and by a further 28% when the mitotic index peaked. Thus, WEE1 kinase activity also followed WEE1 protein levels and the altered timing of the mitotic peak.A WEE1 kinase inhibition assay was used to investigate whether the WEE1 protein levels correlated with changes in the timing of WEE1 kinase activity. WEE1 activity was measured as the inhibitory action of immunoprecipitated WEE1 protein on CDK activity, using histone H1 as substrate Fig.\u00a0. SamplinArath;WEE1 line 1 with and without DEX induction. CDKA activity was relatively constant regardless of the addition of DEX  and Nicta;CDKB;1 (referred to here as CDKB) in the inducible DEX Figs\u00a0; S8. How/G2 Figs\u00a0; S8. ThuNicta;WEE1 in exponential phase BY2 cell cultures carrying the Arath;WEE1 inducible construct following DEX induction, was compared with Nicta;WEE1 expression in exponential phase WT BY2 cell cultures  when Arath;WEE1 expression was induced by DEX in BY2 cells carrying the inducible construct compared to exponential phase WT BY2 cell cultures  and number of leaves produced before flowering (a 2.8 fold reduction) was almost identical. However in contrast to tobacco plants expressing Spcdc25, expression of Arath;WEE1 in tobacco plants did not result in additional flowering from lateral branches. Based on grafting experiments20 it was hypothesised that the anticipation of flowering in the Spcdc25 expressing plants may be result from an earlier competence of the shoot apical meristem to respond to the floral stimulus15. A similar mechanism may be operating in the tobacco plants expressing Arath;WEE1. It is also possible that Arath;WEE1 tobacco plants have similar perturbations in cytokinin signalling and carbohydrate status that were noted in Spcdc25 expressing tobacco plants19, although this would require further verification.The flowering phenotype seen in the Arath;WEE1 contrasts with the effect of Spcdc25 in increasing lateral root production noted by26. However, it is consistent with later reports of a restriction in root growth elicited by Spcdc25 expression in tobacco and attributed to a replacement of cytokinin effects in the roots15. Shorter primary roots were also found when Arath;WEE1 was over-expressed in Arabidopsis24 and is consistent with a negative effect of increased WEE1 on root meristematic cell division.The reduction in primary root length and lateral root production in tobacco plants expressing Spcdc25 and Arath;WEE1. However it contrasts with the phenotype seen in Arabidopsis plants over-expressing Arath;WEE1 where cultured hypocotyls from the Arath;WEE1 over-expressors produced fewer shoots than WT24. In fact the phenotype of the tobacco plants expressing Arath;WEE1 in this respect is more similar to the Arath;WEE1 knockout mutant lines, which produced more shoots from cultured hypocotyls than WT24.The spontaneous formation of shoots in the absence of added cytokinins was also seen both in tobacco expressing Spcdc25 and Arath;WEE1 in tobacco. This is surprising given the opposing functions of the enzymes encoded. The difference between the expression of Arath;WEE1 in tobacco and Arabidopsis confirms that Arath;WEE1 does indeed induce the expected phenotype when expressed in its native environment. However the effects of its expression in tobacco are more consistent with a dominant negative effect, somehow repressing the action of the native Nicta;WEE1.Thus at a plant and organ level there are strong similarities between the effects of expressing Arath;WEE1 also had a positive effect on cell division, very similar to that seen with the expression of Spcdc2517. This effect was independent of the insertion location or the construct since multiple tobacco BY2 lines of both constitutively expressed and inducible Arath;WEE1 created multiple times in the lab all had the same phenotype. In most of the transgenic lines, the reduction in mitotic cell area in Arath;WEE1 expressing tobacco BY2 cells was not quite as severe as that seen when Spcdc25 was expressed, and indeed the Arath;WEE1 expression did not induce the formation of double files of cells as was seen in the Spcdc25 expressing cell lines17. However in one line, c-WEE1 line 10 where mitotic cell area was as low as seen in Spcdc25 expressing lines, double files of cells were also visible. This indicates a threshold effect for the production of double cell files. As previously suggested15 the double cell files are reminiscent of the initial divisions in the pericycle that lead to the production of lateral root primordium. It is possible that the increase in lateral roots seen in some Spcdc25 expressing tobacco plants may be related to the severity of the effect on meristematic cell size. The reduction in root mass and in lateral roots in Spcdc25 and Arath;WEE1 expressing plants may therefore be consistent with a less severe cellular phenotype when the transgene is expressed constitutively as was the case here and in Bell et al.14 as opposed to an inducible vector26.At a cellular level expression of Arath;WEE1 or Spcdc25 with both showing a dramatic reduction in the length of the G2 phase and a lengthening of G1\u2009+\u2009M phase17. In Spcdc25-expressing cells the anticipated mitotic peak was matched by an earlier increase in CDKB activity. Consistent with previous reports5, CDKB activity was also high at G2/M in uninduced cells. However, it peaked much earlier, in S phase in the cultures expressing Arath;WEE1. The anticipation of the mitotic peak when Arath;WEE1 expression was induced, was also accompanied by a premature fall in WEE1 protein and kinase activity, consistent with the changes in WEE1 seen in WT cells23. Thus at a cellular level the induced Arath;WEE1 expressing cell cultures are consistent with an early induction of mitosis after a short G2 resulting in a smaller mitotic cell size.Effects on cell cycle progression again were strikingly similar between BY2 cells expressing Arath;WEE1 in tobacco BY2 cells resulted in the opposite phenotype to that found with Solly;WEE1 expression in BY2 cells25 and indeed over-expression of Nicta;WEE1 in the tobacco BY2 cells essentially had no effect. The results here also contrast with the effects on cell size seen when Arath;WEE1 was over-expressed in Arabidopsis plants24 where root meristematic cells were larger than in WT.Expression of Arath;WEE1 in the BY2 cells was causing an overall reduction of WEE1 protein perhaps due to a reduction of the native Nicta;WEE1 transcript. However overall WEE1 protein was higher, and neither Nicta;WEE1 or overall WEE1 transcript  changed dramatically on induction of Arath;WEE1 expression in exponentially growing BY2 cells. This indicates that the phenotypic effect is not due to a cell cycle-independent activation of the RNAi degradation pathway, which can be activated even with sense expression of transgenes27. A sense silencing mechanism is also less plausible given that in all three vector systems  used to express Arath;WEE1 in BY2 cells the orientation of the constructs is such that read through of antisense transcript from the selectable marker construct is not possible. This was shown to be a key factor in sense-mediated silencing27.One hypothesis to explain these unexpected results was that the expression of Nicta;WEE1 may form the underlying mechanism for the activation of a premature mitosis with the resulting phenotypic effects seen at a cellular, organ and whole plant level. In both WT and uninduced BY2 cells, Nicta;WEE1 transcripts are most abundant during S phase. This is consistent with the slightly later accumulation of WEE1 protein during S\u2009+\u2009G2 phase. However, when Arath;WEE1 is expressed, the peak of Nicta;WEE1 transcripts in S phase seems to be replaced by a later expression peaking in M/G1.\u00a0Arath;WEE1 expression in these induced cultures is expressed more evenly through the cell cycle with a slight peak in S phase. This pattern is broadly consistent with reports on the expression of the 35S promoter during the cell cycle which show either a peak in S phase28 or constant expression throughout all phases29. One possible mechanism is that Arath;WEE1 transcript production and translation into protein during S\u2009+\u2009G2 results in a feedback to Nicta;WEE1 transcription, delaying the accumulation of native WEE1 transcripts. This could be mediated through the large number of transcription factors that are thought to regulate WEE1 expression that include AtTCP1530, SOG131 and many others. An alternative mechanism may act at the protein level. The accumulation of Arath;WEE1 protein in S/G2 may activate the proteasome machinery prematurely due to differences in its sequence  and P36SS (5\u2032GCACACTAGTCGACTCAACCTCGAATCCTAT-3\u2032) and cloned into the BIN HYG TX vector32 under an attenuated form of the 35S promoter (as described in33) for constitutive expression, or into the inducible vector pTA700234 using Xho I/Spe I. Individual clones were sequenced and a clone for each construct in which the amino acid sequence was intact was chosen for further work. For expression in whole tobacco plants, Arath;WEE1 was cloned into pkanII-SPYCE(M)35 as described in Lentz Gr\u00f8nlund et al.36. Nicta;WEE1 was cloned into the pTA7002 vector as described in Cook et al.23.For expression of Nicotiana tabacum) BY2 cells was achieved using a modified version of the method described by37 with the addition of 20\u2009\u00b5M acetosyringon (Sigma-Aldrich) during co-cultivation of the Agrobacterium (LBA4404) with the BY2 cells. Transformants were selected on solidified BY2 medium (0.8% agar) supplemented with 250\u2009\u00b5g/ml Timentin and 80\u2009\u00b5g/ml hygromycin. Calli were cultured in 50\u2009ml BY2 medium, 250\u2009\u00b5g/ml Timentin and 80\u2009\u00b5g/ml hygromycin until stationary phase (1\u20133 weeks). Cultures were subjected to at least four rounds of sub culturing before being used in synchrony experiments.Stable transformation of tobacco  to a final concentration of between 1\u2009\u00b5M and 100\u2009\u00b5M. Induction of Nicotiana\u00a0 tabacum var Samsun plants grown in soil were surface sterilised in 5% hypochlorite solution containing 100 ul/l Triton X-100 for 5\u2009min with gentle agitation. Leaves were rinsed three times in sterile distilled water and cut into 1\u2009cm2 squares using a razor blade. Leaf squares were co-cultivated for 20\u201330\u2009min in 100\u2009ml of Rhizobium radiobacter (Agrobacterium tumefaciens) LBA4404 cell suspension (containing the WEE1 construct) at OD600 of 0.5 in 1\u2009\u00d7\u2009MS medium in 140\u2009mm diameter Petri dishes. Leaf squares were then transferred to shooting medium . Following 48\u2009h at 22\u2009\u00b0C in the light, Leaf squares were then transferred to shooting medium including 50 ug/ml hygromycin and 200 ug/ml carbenicillin and incubation was continued for 4\u20136 weeks with a weekly subculture until calli and shoots were visible. Shoots were then excised and further cultured in rooting medium  to induce rooting. Plantlets were transferred to soil and grown to maturity. Expression of the transgene was analysed by PCR using primers AtWEE1fw (AGCTTGTCAGCTTTGCCT) and AtWEE1rv (TCAACCTCGAATCCTATCA). Two lines expressing the transgene (lines #2 and #8) were selected for further experiments.Young leaves from \u22122), and a relative humidity 50\u201375% as described in38. The leaves were numbered from the base (1 oldest) and when the first flower bud emerged, the length of leaves without the petiole was measured and leaves above 10\u2009cm in length were counted. The age of the plants is given as days of growth after sowing.Wild type and transgenic tobacco plants were grown from seed in a growth chamber at 22/18\u2009\u00b0C day/night thermoperiod with 16 hrs illumination  as described in38. The length of the main root was measured and lateral roots counted semi-automatically with Smart Root software. For visualisation of root primordia the clearing method was used. The roots were fixed in acetone overnight and then fixed in phosphate buffer and mounted in 65% aqueous glycerol. They were observed with an Olympus BX51 microscope equipped with anvApogee U4000 digital camera.Sterilized tobacco seeds were sown on a square Petri dish containing MS medium  containing 3% sucrose, 2\u2009cm apart. After 21 days of cultivation at 25\u2009\u00b0C with 16\u2009h illumination with PFD (photon flux density) approximately 100 \u03bcmol m38. After 21 days of cultivation, the number of shoots and protruded shoot primordia were counted.Tobacco stem segments, 1\u2009cm long, were placed onto MS medium  containing 3% sucrose, or SIM (shoot inducing medium) consisting of MS medium with 3% sucrose, 0.1\u2009mg/l NAA , and 2\u2009mg/l BAP (benzylaminopurine) as described in39. The mitotic index was measured at hourly intervals after removal of aphidicolin by scoring \u2265300 Hoechst-stained cells per slide in random transects using fluorescence microscopy . Mitotic cell area was measured for approximately 300 cells per experiment.BY2 cells were subcultured every 7 d and division was synchronized as previously described33. Total RNA was extracted from BY2 cells using TRI reagent  and residual genomic DNA was removed by DNase treatment . RNA (5\u2009\u00b5g) was reacted with Superscript II reverse transcriptase . To study expression of Arath;WEE1, primers were designed which do not amplify the endogenous tobacco Wee1 gene : Arath;WEE1fw, and Arath;WEE1R: GTGCATCTCCTTCTTCTACT. Thermocycle conditions were: 35 cycles of 95\u2009\u00b0C (1\u2009min), 55\u2009\u00b0C (1\u2009min), 72\u2009\u00b0C (1\u2009min). Two sets of specific primers for Nicta;WEE1:  were used to analyse the expression of the endogenous tobacco WEE1 gene, Nicta;WEE1 (Tm\u2009=\u200960\u2009\u00b0C and 55\u2009\u00b0C respectively). The first set was used for the quantification of Nicta;WEE1 expression in synchronised cells while the second set were used to quantify expression in exponential phase cultures. For detection of Nicta;WEE1 transgene expression only, a primer was designed to bind to the vector sequence: (35STRS 5\u2032-ACGCTGAAGCTAGTCGACTC) and used in conjunction with NtWEE15R 5\u2032-TTATCCCCATCGGCAGCATCAG. Histone H4 primers (H4F: 5\u2032-GGCACAGGAAGGTTCTGAGGG ATAACA and H4R: 5\u2032-TAACCGCCGAAACCGTAGAGAGTCC) were used to verify cell cycle stage, and primers to 18S rRNA: PUV2 5/-TTCCATGCTTAATGTATTCAGA and PUV4, 5/-ATGGTGGTGACGGGTGAC were used as a control17 (Tm\u2009=\u200960\u2009\u00b0C). Thermocycler conditions were as above.RT-PCR was performed as described in40 .For all semi quantitative RT-PCR experiments, cycle number was reduced and optimised rigorously as described previously40 Fig.\u00a0 so that 42. The WEE1 antibody and Western blotting were described in35. The antibody was used at a dilution of 1:1000 followed by \u03b1-rabbit IgG at 1:2500 . ECL reagents  were used to visualise the proteins.Proteins were extracted from Arabidopsis or tobacco leaves essentially as described inet al.42. Immunoprecipitations were carried out using antisera raised to Nicta;CDKA;1 and Nicta;CDKB1 as described in Sorrell et al.4. H1 protein kinase assays were as previously described42 using 2\u2009\u00b5l of antiserum. Incorporation was assayed by quantitation of autoradiographs using the GeneGenius .For histone kinase assays proteins were extracted from 5\u2009ml of synchronised cultures and assayed essentially as described in Cockcroft Supplementary Figures"}
+{"text": "There are errors in the placement of references 17 and 18. The sixth sentence of the fifth paragraph in the Introduction section should have no citations. The seventh sentence should have cited references 17 and 18, in addition to references 19 and 20.The correct sentences should read: The same drawback is also observed with smaller affinity tags. This problem has been solved through the use of proteolytic enzymes whose cleavage sites enable the formation of native A\u03b2  [17], [18], [19], [20]."}
+{"text": "Alzheimer\u2019s disease (AD) is the most common form of dementia characterized by the deposition of extracellular amyloid-\u03b2 (A\u03b2)-containing plaques, the formation of intraneuronal neurofibrillary tangles as well as neuroinflammatory changes. As the key player in the brain innate immune system, microglia has now taken a center stage in AD research. A large number of AD risk loci identified by genome-wide association studies are located in or near the genes highly expressed in microglia. Among them, the triggering receptor expressed on myeloid cells 2 (TREM2) has drawn much attention. A rare variant in TREM2 increases AD risk with an odds ratio comparable to the strongest genetic risk factor apolipoprotein \u03b54 allele. In the past 6 years, extensive studies have dissected the mechanisms by which TREM2 and its variants modulate microglial functions impacting amyloid and tau pathologies in both animal models and human studies. In addition to the full-length TREM2, research on the soluble form of TREM2 (sTREM2) has facilitated the translation of preclinical findings on TREM2. In this review, we summarize our current understanding of the biology and pathobiology of sTREM2 including its origin, its emergence as a disease biomarker, and its potential neuroprotective functions. These aspects are important for understanding the involvement of sTREM2 in AD pathogenesis and may provide novel insights into applying sTREM2 for AD diagnosis and therapy. Alzheimer\u2019s disease (AD) is the most common cause of dementia in the elderly, typically presenting with a slow but progressive behavioral and cognitive impairment. Key histopathological hallmarks of AD include the deposition of extracellular neurotoxic plaques primarily composed of A\u03b2, and intracellular neurofibrillary tangles resulting from the aggregation of hyperphosphorylated tau  since the rare R47H variant of TREM2 increases AD risk almost three-fold  has facilitated the translation of preclinical findings on TREM2. sTREM2 was produced from either the proteolytic cleavage of the membrane-anchored TREM2 receptor or the alternative splicing of TREM2 lacking the transmembrane domain . However, the predicted molecular weight of this transcript is ~27 kDa, which is larger than the 20 kDa deglycosylated sTREM2 protein detected in the CSF. Therefore, alternative splicing may not be the major source of sTREM2 protein.The existence of sTREM2 at the protein level was first reported in 2008 by immunoblotting of the precipitates from either human CSF or cultured dendritic cells supernatant fluid  gene cluster as a key modulator of sTREM2 concentration in the CSF. Variants in the MS4A gene region have been previously linked to the risk of developing LOAD  is associated with reduced CSF sTREM2 levels, increased AD risk and accelerated age at onset. The study further provided functional evidence that CSF sTREM2 levels can be modified via modulating MS4A4A expression or targeting MS4A4A with a specific antibody. However, the study only focused on the common variants with a minor allele frequency larger than 0.02. Therefore, additional studies in a larger sample size will be necessary to fully identify the genetic modifiers of CSF sTREM2 levels. Moreover, future studies are necessary to uncover the molecular mechanisms by which the MS4A gene cluster modulates sTREM2 level. The MS4A transmembrane proteins are thought to play a role in intracellular protein trafficking  and other CNS inflammatory diseases  are less potent in both suppressing apoptosis and triggering inflammatory responses in microglia. Thus, our results may constitute previously unknown molecular mechanisms linking TREM2 variants with increased risk of AD.Initially, sTREM2 was postulated to block the function of full-length TREM2 by competing with the ligand binding, in a way similar to the soluble version of another TREM family member, TREM1. Nevertheless, accumulating evidence suggests that sTREM2 possesses important biological and pathobiological functions rather than acting as a decoy receptor opposing full-length TREM2 signaling. One study presented the first evidence that sTREM2 efficiently prevents macrophage apoptosis when cultured under low concentrations of colony stimulating factor 1 (Wu et al., 181 levels in the CSF predicted slower rate of clinical progression. These findings suggest that higher CSF sTREM2 levels are associated with attenuated cognitive and clinical decline in AD.Since the CSF levels of sTREM2 are intimately associated with AD progression, it has been proposed that sTREM2 might modulate AD pathology. Accumulating evidence from the association analysis between CSF sTREM2 levels and regional brain volumes in AD cohorts suggests that sTREM2 functions are likely neuroprotective. One study reported that higher CSF sTREM2 levels were correlated with increased gray matter volume in MCI patients, when controlled for age, sex, and p-tau-associated brain atrophy (Gispert et al., Trem2 deficient 5xFAD mice, while no such findings were observed in mice expressing the R47H variant (Song et al., It was reported that sTREM2 bound to plaques and neurons when the common variant of human TREM2 was expressed on a bacterial artificial chromosome in The majority of AD therapies currently under investigation target A\u03b2 accumulation by modulating the activity of key secretases involved in APP processing. Alternatively, antibodies were developed to prevent A\u03b2 aggregation and/or to promote A\u03b2 plaque clearance (Cao et al., X-FC and LZ reviewed the literature and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Rf4) and hence cannot be utilized directly in the hybrid rice breeding. To convert the partial restorer to complete restorer, a cross was made between Swarna and a stable restorer KMR3R possessing Rf3 and Rf4 genes and developed BC1F5 and BC2F4 populations by marker-assisted back cross breeding (MABB). The SSR marker DRRM-RF3-10 linked to Rf3 gene located on chromosome 1, clearly distinguished restorers from partial restorers. All the improved lines of Swarna possessing Rf3 and Rf4 genes showed complete fertility restoration in test crosses with higher grain yield heterosis. Few rice hybrids developed by using converted restorers were evaluated in multi location testing under the All India Co-ordinated Rice Improvement Project (AICRIP). The results indicated that new rice hybrids expressed higher heterosis with matching grain quality attributes like Swarna. This study provides significantly novel and relevant restorers to enhance and economize future hybrid rice breeding programs.The major constraints in hybrid rice breeding are availability of limited number of parental lines with specific desirable traits and lower frequency of restorers among elite breeding lines. The popular, high-yielding mega-rice variety Swarna, has been identified to be a partial restorer (as it has only one of major fertility restorer genes,  O. sativa L.) is the most important staple food crop for more than half of the world\u2019s population and it is cultivated in an area of 44.5 million hectare in India with the production of 106.5 million tonnes during the year 20161. After the advent of high yielding semi-dwarf rice varieties, hybrid rice technology has been touted as a major strategy for enhancing the genetic yield potential of rice. The success of hybrid rice technology has been very well demonstrated in China, which produces 146.5 million tonnes of rice from 30.32 million hectares1. This significant increase in production in China is mainly due to cultivation of hybrid rice (with >50% area and production under rice hybrids). Several technical challenges, market and policy constraints has limited the development and diffusion of hybrid rice outside China2. In India, hybrid rice is cultivated in an area of ~3 million hectares3, which is about 6.7% of total area of rice cultivation. Hybrid rice accounts for less than 10% of the area under rice cultivation in Bangladesh, Indonesia, and the Philippines and just 10% in Vietnam.Rice (5. The most widely used CMS system in rice is based on wild abortive (WA) cytoplasm derived from Oryza sativa f. Spontanea7. The WA-CMS system is highly stable with complete pollen sterility8. Hybrid rice seed production using CGMS/three line system involves a CMS (A) line, a maintainer (B) line and a restorer (R) line carrying the fertility restorer genes.Hybrid rice technology aims to increase the yield potential of rice by exploiting the phenomenon of hybrid vigour or heterosis. Cytoplasmic male sterility coupled with fertility restoration controlled by nuclear genes is a very useful tool in exploiting heterosis in self pollinated crops. In rice, three CMS systems viz. Wild Abortive (WA), Boro II (BT) and Honglian (HL) are deployed for commercial hybrid rice seed production7. Rice fertility restoration is governed by two independent dominant genes and one of the genes appeared to be stronger in action9 than the other. Bharaj et al.10 reported that fertility restoration is governed by two dominant genes, the restorers with dominant alleles of the two genes in homozygous or heterozygous condition will be fully fertile and the genotypes having dominant alleles of one of the two genes in homozygous or heterozygous condition but homozygous recessive alleles of the other gene will behave partially sterile or partially fertile and vice-versa.The restorers with strong restoration ability have two major genes along with modifier genes and a restorer with semi-restoring ability have either one of the two major genesRf3 and Rf4 and have been mapped on chromosome 1 and 10 using Zhenshan 97A near-isogenic lines (NILs) mapping population12. The chromosomal location of Rf genes has been resolved using RFLP (Restriction Fragment Length Polymorphism) markers and it showed that effect of the locus on chromosome 10 is larger than chromosome 113. Mapping of two Rf genes of WA-CMS system on the long and short arm of chromosome 10 using SSLP (Simple Sequence Length Polymorphism) markers was done by Jing et al.14. The SSR primer RM258 located on chromosome 10 was found linked to the restorer gene at a distance of 9.5 cM15. Fertility restorer gene linked to RM6100 was mapped at a distance of 6\u20137\u2009cM on chromosome 10 in the restorer lines viz., PRR 78\u2009R, IR 40750 and MTU 999216. The candidate gene based marker, DRRM-RF3-10 associated with Rf3 locus showed maximum selection accuracy in identifying restores in comparison with other reported markers viz., RM10305, RM10318, DRRM-RF3-5 and DRRM-RF3-617.The major loci restoring the fertility has designated as 1 for their pollen and spikelet fertility. According to the male fertility of F1 plants, the test lines can be classified as maintainers, restorers, partial restorers and partial maintainers9. Molecular markers linked to the fertility restoration trait are useful for evaluation of large number of germplasm/breeding lines to identify restorers in rice without progeny testing18. Based on molecular screening with markers linked to Rf4 and Rf3 genes19 reports that lines possessing both the Rf genes showed higher fertility than the genotypes containing Rf3 or Rf4 individually. In hybrid rice breeding, elite lines and released varieties from varietal improvement programme are being tested for their fertility restoration ability by crossing with the CMS lines to identify restorers and maintainers. The restorer frequency among indica lines is only 40%20. The majority of the popular, mega varieties released in India like Swarna, Samba Mahsuri, and MTU1010 were found to be partial or incomplete restorers and hence cannot be utilized as such to produce experimental hybrids.The process of screening for the trait of fertility restoration involves test crossing with a set of CMS lines and evaluation of hybrids/F21, occupying more than 30% of total rice area in eastern India22. Swarna possesses good grain and cooking quality with desirable grain type. However, as mentioned earlier, it cannot be utilized in hybrid breeding as it is a partial restorer. Therefore in the present investigation, an attempt was made to convert the partial restorer Swarna to a complete restorer by transferring major fertility restorer gene(s) through marker-assisted back cross breeding (MABB), and demonstrated the complete fertility restoration ability of the improved lines and developed new rice hybrids with attributes like Swarna.The major issues in hybrid rice breeding are lack of parental lines with desirable specific traits and lower frequency of restorers and maintainers among elite breeding lines. One of the major constraints, which limit the spread of hybrid rice area in shallow low lands and coastal areas, is non availability of long duration hybrids, which can mature in 145\u2013150 days or more. The popular mega rice variety, Swarna has been widely adopted by farmers for its high yielding ability and adaption under low input conditions, shallow low lands. It is cultivated in an approximate area of 4.5\u2009M ha1 hybrids were produced by crossing Swarna and KMR3R with four CMS lines viz., APMS 6A, IR79156A, IR68897A and IR58025A. The F1 hybrids were evaluated for their pollen and spikelet fertility during both kharif (wet) and rabi (dry) seasons  and seven plants were selected based on genome recovery. The chromosome 4, 7, 8 and 11 were shown complete recurrent parent type polymorphism, whereas the remaining allele ranged from 20% (Chromosome 5) to 55% (Chromosome 3). In BC2F1, out of 22 plants, three plants were selected based on foreground and background selection, which had shown complete genome recovery on chromosome 2, 3, 5 and 6 with 91% of recovery of Swarna genome.A set of 728 hyper-variable SSR markers were utilized for surveying polymorphism between Swarna and KMR3R across the 12 linkage groups of rice. Of these, 89 were polymorphic between the parents (12.22% polymorphism). In this study, chromosome 8 and 6 recorded the highest and lowest polymorphism percentage of 19.60% and 5.88%, respectively  along with better agro morphological traits (i.e. similar to Swarna) was backcrossed with Swarna to produce BC2F1 seeds. Simultaneously, the selected BC1F1 plant with maximum recurrent parent genome was selfed to produce BC1F2 seeds. By foreground selection in BC2F1 and BC1F2 generation plants carrying Rf3 gene in homozygous and heterozygous conditions were identified  possessing superior phenotypes were subjected to background selection analysis. BC2F1 and BC1F2 plants with maximum recurrent parent genome (RPG) were selfed to produce BC2F2 and BC1F3 seeds. The BC2F2 and BC1F3 plants were raised and subjected to foreground selection for the presence of Rf3 gene. Marker-assisted screening resulted in identification of 35 plants to carry Rf3 in homozygous condition in BC2F2 and 40 plants in BC1F3 generation. The identified homozygous plants were selfed to raise BC2F3 and BC1F4 generations. Phenotypically superior plants were identified based on morphological traits at BC2F3 and BC1F4 generations and were selfed to produce the subsequent generation seeds. BC2F4 and BC1F5 lines were evaluated in three rows and plants were subjected to stringent phenotypic selection and agro morphological evaluation.One superior BCied Fig.\u00a0. A total2F4 and BC1F5 generations derived from the cross Swarna x KMR3R, were evaluated in alpha lattice design along with parents and checks in 6 sq. m plot during kharif \u00a02015 at Indian Institute of Rice Research, Rajendranagar, Hyderabad. The observations were recorded on five plants in each replication and mean data is presented in  and 23.9\u2009cm (KMR3R). The panicle length of converted restorer lines were significantly longer (>7\u2009cm) than Swarna. With respect to mean grain yield per plant of selected backcross derived lines, ranged from 19.0 to 25.0\u2009gm, which is significantly higher than the recurrent parent, Swarna (15.28\u2009gm). The test weight of Swarna (1000 grain weight) was 15.9\u2009gm with short bold grains, whereas KMR3R possessed 26.5\u2009gm of test weight with long bold grain type. The converted restorer lines test weight ranged from 15.1 to 21.9\u2009gm with the grain type of short slender to short bold. Days to 50% flowering (DFF) of selected backcross derived lines ranged from 91 to 121 days  and possessing plant height of more than 100\u2009cm were crossed with above mentioned CMS lines to produce F1 hybrids. F1 hybrids were evaluated for pollen and spikelet fertility percentage and grain yield heterosis along with different duration checks. Based on days to 50% flowering three hybrid groups viz., early (<100 days), medium (101\u2013110 days) and late (>110 days) were constituted for evaluation.  in different zones in India. The hybrid entries with a yield superiority of more than 5% and 10% over the best hybrid and varietal checks either on over all mean basis or on zonal mean basis are promoted from IHRT to next stages of testing in Advance Varietal Trial 1 (AVT 1) and AVT 223.Based on the performance of converted improved restorer lines in the research station trial, two high yielding hybrids namely IIRRH-111 and IIRRH-112 were nominated for multi-location evaluation in AICRIP (All India Co-ordinated Rice improvement project)-IHRT trial during kharif 2016 multi-location testing. With respect to quality data collected from the AICRIP progress report (2016), this hybrid possessed short bold grains as that of Swarna and preferred intermediate amylose content (26.22%) with 60% high head rice recovery. In AICRIP testing rice hybrids were evaluated for not only yield and also for their grain quality characters. The grain quality characters of hybrids developed by crossing CMS lines with improved restorers are presented in Table\u00a0The rice hybrid IIRRH-112 showed a superior performance over the checks  and registered yield superiority over checks during 24. In India, as a result of concerted efforts by both public and private sectors, a total 102 hybrids have been released for commercial cultivation in the country. In spite of having great potential to enhance rice production and productivity, area expansion under hybrid rice has not increased significantly in the last few years due to major constraints like non availability of long duration hybrids suitable for shallow lowlands and coastal areas, poor grain and cooking quality, marginal heterosis, higher seed cost, non-involvement of public sectors in hybrid rice seed production25. The popular, high yielding, mega rice variety Swarna, with good grain and cooking quality, cannot be utilized as such in hybrid breeding due to their partial restoration of fertility of CMS lines (pollen and spikelet fertility is less than 80%)  and Improved Samba Mahsuri, Kavya and MTU 1010  particularly Rf3 gene located on chromosome 1. Backcross breeding is the most commonly used method for incorporating any essential genes into a rice cultivar. Backcross breeding along with marker-assisted foreground and background selection contributes immensely to accelerate recurrent parent genome (RPG) recovery27.An earlier study28 considered to be an efficient strategy for transferring targeted specific genes to elite lines. MABB is a most effective breeding strategy in rice for improving simple and complex traits. Some of the popular rice varieties released for cultivation in India through MAS/MABB for biotic and abiotic stress tolerance are Pusa Basmati 1 with bacterial blight (BB) resistance genes xa13 and Xa21 involving single back cross generation followed by selfing and pedigree based selection29 and Improved Samba Mahsuri with three BLB resistance genes viz., xa5, xa13 and Xa21 involving four back crossing30. Swarna Sub1 with Sub1 QTL for the submergence tolerance31 and IR64 Drt 1 with two yield QTLs under drought situations namely qDTY2.2 and qDTY4.1 (IR64 NILs) for drought tolerance32 are the best examples of abiotic stress tolerance varieties derived through MAS.Marker-assisted backcross breeding which includes MAS for the target gene(s) known as foreground selection and MAS for the recovery of recurrent parent genome known as background selectionRf3 gene. We could recover Swarna genome in the backcross derived lines of converted restorers of BC1F5 and BC2F4 generations by 80% and 92.3%, respectively. The agro morphological evaluation of converted restorers (RP5934-66 to RP 5934-100) in the background of Swarna exhibited desirable traits for an ideal restorer lines viz., improved plant height, longer panicles, more number of productive tillers with heavy pollen load along with traits specific for Swarna, like brown glume or golden husk colour with stay green type etc  in order to develop improved parental lines in the background of an elite Indian mega- variety of rice and thereby confirming the major and minor role of Rf4 and Rf3. The grain quality characters of the converted (i.e. improved) restorer lines in the genetic background of Swarna were similar to that of the original parent. In a panel test for grain quality traits conducted at ICAR-IIRR, Hyderabad, it was observed that, the Rf3 gene introgressed lines were indistinguishable from Swarna in terms of color of paddy, grain size and shape. Further, replicated field trials that were carried out at twelve different locations across India under the all India coordinated rice improvement project (AICRIP), showed that yield levels of the improved lines were significantly higher than Swarna and the check lines indicating that there is no apparent yield penalty associated with presence of the Rf3 gene3. Considering these points, improved version of restorers developed in the genetic background of Swarna would be of great use in hybrid rice breeding for developing late duration hybrids. Large scale seed production of potential hybrids involving long duration improved restorers with different WA-CMS lines are under pipeline for multi location testing. Some of the improved lines were also demonstrated their potentiality as higher heterotic hybrids in the early and medium duration category , whereas Swarna is a very popular rice variety and occupying major area under cultivation.Introgression of fertility restorer genes without recovery of other characters of recurrent parent would have been a futile exercise, if the developed lines do not meet expected grain and cooking quality with higher yields. We feel that this was made possible because of the extensive phenotypic selection and also because of the use of molecular markers for background selection. The selected BCles Fig.\u00a0. This waRf genes through marker-assisted backcross breeding. Thus, in the present study, we have successfully developed restorers with characteristic features of popular mega Indian rice variety Swarna for utilization in hybrid rice breeding, specially for development of late maturity hybrids.To the best of our knowledge, this is the first attempt to convert partial restorer to complete restorer by transferring Rf gene(s) was KMR3R (Jaya/IR29723-143) a restorer parent of popular rice hybrid KRH-2. KMR3R has been identified to carry both fertility restorer genes Rf4 and Rf3 genes18. To determine the fertility restoration status of recurrent and donor parent, crosses were made with four CMS lines namely APMS 6A, IR 79156A, IR 68897A and IR 58025A to produce F1 hybrids during rabi 2011. These F1 hybrids were evaluated for their pollen and spikelet fertility and grain yield heterosis during two rice crop seasons viz., kharif 2012 (i.e. wet season 2012) and rabi 2013 (i.e. dry season 2013) to confirm their fertility restoration status.The experimental materials consist of Swarna (MTU 7029) a popular mega rice variety, which occupies larger area under cultivation in India, derived from the cross Vasistha x Mahsuri and is a partial restorer and it was utilized as the recurrent parent for improvement of its fertility restoration ability. The donor parent for the fertility restoration trait/2-KI) solution43, on a glass slide and three randomly selected microscopic fields were counted. Stained, well filled and round pollen grains were counted as fertile, while unstained, shrivelled and empty pollen grains were considered as sterile. Pollen fertility was calculated and expressed in percentage as given belowPollen fertility was measured using anthers collected from the spikelets at one or two days before anthesis. The anthers from each spikelet were smeared in a drop of 1% Iodine-potassium iodide , partially fertile (51\u201380%), partially sterile (21\u201350%) and completely sterile (0\u201320%)The panicles that emerged from the primary tiller were bagged before anthesis to avoid out crossing and the number of filled grains and chaffs in the panicle were counted at the time of maturity. The ratio of filled grains to the total number of spikelets was expressed as spikelet fertility as given below:Plants were classified into four classes based on spikelet fertility percentage, namely, fertile (more than 85% spikelet fertility), partially fertile (50\u201385%), partially sterile (21\u201350%) and completely sterile (0\u201320%)44. PCR analysis was carried out using 30\u201350\u2009ng DNA as template, 5 pmoles of each primer, 0.05\u2009mM dNTPs, 10X PCR buffer (TaKaRa Taq\u2122 DNA Polymerase) and 1 U Taq DNA polymerase in a total volume of 10\u2009\u00b5l. Template DNA was initially denatured at 94\u2009\u00b0C for 5\u2009min followed by 35 cycles of PCR amplification with the following parameters: a 30\u2009s denaturation at 94\u2009\u00b0C, a 30\u2009s annealing at 55\u2009\u00b0C and 1\u2009min of primer extension at 72\u2009\u00b0C. A final extension was done at 72\u2009\u00b0C for 7\u2009min. The amplified products were electrophoretically resolved on a 3% agarose gel containing 0.5\u2009\u00b5g/ml of ethidium bromide in 0.5X TBE buffer and visualized under UV and results were documented. The recurrent parent Swarna, donor parent KMR 3\u2009R along with few popular varieties  were screened with 20 reported SSR markers linked to Rf4 & Rf3 genes to identify polymorphic marker between restorer and partial restorers for foreground selection . The backcross breeding procedure followed is presented in the Fig.\u00a01 plants of Swarna x KMR3R were screened using DRRM-RF3-10 identified polymorphic SSR markers between donor and recurrent parent. The true F1s were then backcrossed with Swarna to generate BC1F1s, which were confirmed for the presence of fertility restorer gene(s) Rf3 and Rf4 with the help of DRRM-RF3-10 and PPR3 markers (i.e. foreground selection). The plants which were positive for the restorer genes Rf4 and Rf3 were subjected to background selection with a set of 89 identified polymorphic SSR markers to identify a single BC1F1 plant, possessing maximum recovery of the recurrent parent genome (RPG). This plant was selfed to generate BC1F2s and also backcrossed with Swarna to generate BC2F1s. The MABB process involving foreground and background selection as explained above was repeated among the BC2F1 plants and the best BC2F1 plant was selfed to generate BC2F2 seeds. The BC1F2 and BC2F2 were analyzed with markers specific for Rf3 and Rf4 to identity homozygous plants. Homozygous BC1F2 and BC2F2 plants were then advanced through pedigree method of selection for further phenotypic evaluation based on duration of flowering (days), plant height (cm), number of tillers, panicle length (cm) and single plant yield (g). Phenotypically superior plants were advanced for further evaluation.A cross was made between Swarna x KMR3R to improve the fertility restoration trait of the recurrent parent Swarna, during 1F5 and BC2F4 generations along with the parents were transplanted in the main field with the spacing of 20\u2009\u00d7\u200920\u2009cm and fertilizer dosage of 120:80:60 (N:P:K) kg/ha during kharif 2014. The experimental plots were arranged in alpha lattice design in four blocks with two replications. Standard agronomic practices were followed while raising the rice crop. Data was recorded on randomly selected five plants in each replication for the agronomic traits, viz. flowering duration, plant height (cm), number of productive tillers, panicle length (cm), grain yield per plant, and 1000-grain weight. The plants were visually observed for the following traits viz., heavy pollen load during anthesis, strong culm, non-lodging type and Swarna\u2019s specific traits of stay greenness and golden hull colour for selecting superior phenotypic plants as restorers. The data was tabulated and statistically analyzed using standard Microsoft office excel and SPSS package.Thirty days old seedlings at BC1F5 and BC2F4 generations possessing Rf4 and Rf3 genes were crossed with two CMS lines viz., APMS 6A and CRMS\u00a032A to produce F1 hybrids during kharif 2014 (i.e. wet season 2014). Thirty days old seedlings of experimental hybrids along with checks were transplanted to the field with the spacing of 20\u2009\u00d7\u200920\u2009cm in two replications. Data on days to flowering, plant height, productive tillers, panicle length, pollen and spikelet fertility percentage, grain yield and grain yield heterosis were estimated. Seeds of Swarna and selected experimental hybrids were stored for three months after harvesting and grain quality tests were carried out by standard grain quality evaluation protocols (as explained in30) with the rice grains having 12 to 14% moisture content. Superior rice hybrids were identified to produce hybrid seed in larger plots during rabi 2016 (i.e. dry season 2016) and nominated to multi-location evaluation of AICRIP trials during kharif 2016 (i.e. wet season 2016).The phenotypically superior lines from BC"}
+{"text": "SLC26A4 gene cause hearing loss with enlargement of the vestibular aqueduct (EVA). Some patients also have a thyroid iodination defect that can lead to multinodular goiter as part of Pendred syndrome. A haplotype of variants upstream of SLC26A4, called CEVA, acts as a pathogenic recessive allele in trans to mutations affecting the coding regions or splice sites of SLC26A4. Our first hypothesis is that CEVA, acting as a pathogenic recessive allele, is correlated with a less severe phenotype than mutations affecting the coding regions and splice sites of SLC26A4. Our second hypothesis is that CEVA acts as a modifier of the phenotype in patients with EVA caused by mutations affecting the coding regions or splice sites of both alleles of SLC26A4 or EVA caused by other factors.Recessive mutations of coding regions and splice sites of the . To test our first hypothesis, we compared the thyroid and auditory phenotypes of subjects with mutations affecting coding regions of both alleles of SLC26A4 with those of subjects carrying CEVA in trans to mutations affecting the coding regions. To test our second hypothesis, we compared the phenotypes associated with the presence versus absence of CEVA among subjects with no coding region mutations, as well as among subjects with mutations affecting coding regions of both alleles.This was a prospective cohort study of 114 individuals and 202 ears with EVAtrans to a mutation of SLC26A4 have a normal thyroid phenotype and less severe hearing loss in comparison to individuals with mutations affecting coding regions of both alleles of SLC26A4. In subjects with no mutant alleles of SLC26A4, hearing loss was more severe in subjects who carry the CEVA haplotype in comparison to non-carriers. There was no correlation of CEVA with the phenotype of subjects with mutations affecting coding regions of both alleles.Subjects carrying CEVA in SLC26A4. CEVA may act as a genetic modifier in patients with EVA caused by other factors.CEVA, acting as a likely pathogenic recessive allele, is associated with a less severe phenotype than alleles with a mutation affecting the coding regions or splice sites of  SLC26A4 gene on chromosome 7q (OMIM 274600) [SLC26A4 can cause bilateral EVA and a thyroid iodination defect that can lead to multinodular goiter as part of Pendred syndrome (PS) (OMIM 274600) [SLC26A4 can also be associated with nonsyndromic EVA (NSEVA), also referred to as nonsyndromic recessive hearing loss DFNB4 (OMIM 600791) [Enlarged vestibular aqueduct (EVA) is the most common temporal bone malformation detected in ears with sensorineural hearing loss . Some in 274600) . The iod 600791) .SLC26A4 with the presence or absence of the PS thyroid phenotype. Variants originally reported to be specifically associated with NSEVA/DFNB4 [Until recently, there was no known correlation of specific mutations or variants of VA/DFNB4 . HoweverVA/DFNB4 , 9.SLC26A4 with the thyroid phenotype [SLC26A4 (M2) [SLC26A4 [We have reported correlations of the number of mutant alleles of henotype , 11, bi\u2212henotype , 13, andhenotype . Pendred[SLC26A4 , 13.SLC26A4-linked haplotype, called Caucasian EVA (CEVA), comprised of 12 variants (10 single-nucleotide substitutions and two single-nucleotide deletions) upstream of SLC26A4 [SLC26A4. Some of the variants are intergenic and some are found within introns of the other genes which include BCAP29, DUS4L, COG5, HBP1, and PRKAR2B. None of these five genes are known to be associated with EVA or any phenotype consistent with EVA. We showed that a chromosome 7 with CEVA and no mutations of coding regions or splice sites of SLC26A4 acts as a mutant allele with incomplete penetrance in trans to the allele with a mutation affecting the coding regions or splice sites in M1 patients [We recently reported an  SLC26A4 . The fre SLC26A4 . CEVA wa SLC26A4 . The 12 patients . The prepatients .SLC26A4 allele with the CEVA haplotype is correlated with a less severe phenotype than mutations affecting the coding regions or splice sites of SLC26A4. The aim of our current study was to test this hypothesis by comparison of the phenotypes of M2 subjects without CEVA with those of M1 subjects carrying CEVA in trans to a mutation affecting the coding regions or splice sites. A second aim of our study was to test the hypothesis that CEVA acts as a genetic modifier of the phenotype caused by mutations affecting coding regions or splice sites of both alleles of SLC26A4 (M2) or by factors other than SLC26A4 mutations (M0).Based on these findings, we hypothesized that the SLC26A4 genotypes and CEVA haplotypes have been previously reported [SLC26A4 (M0).This study was approved by the Combined Neurosciences Institutional Review Board (IRB) of the National Institutes of Health . Written informed consent was obtained from all adult subjects and parents of minor subjects. Race and ethnicity were classified according to our IRB reporting designations. All Caucasians with EVA on at least one side were included in this study. We originally defined a vestibular aqueduct as enlarged if the diameter\u2009>\u20091.5\u2009mm , but subreported , 18, 19.reported . Two prereported  , Pendred syndrome (PS), or indeterminate (I). Ultrasonography was used to evaluate thyroid size and texture , 11. SubSeverity of hearing loss was classified using a four-frequency (0.5/1/2/4-kHz) pure-tone air-conduction threshold average (PTA) calculated from the most recent complete audiogram for each ear with EVA , 13. WheSLC26A4 genotype-haplotype combination. The following comparisons were made: (1) M2 vs. M1/CEVA (2) M2 vs. M2/CEVA (3) M0 vs. M0/CEVA. The Mann-Whitney test was used to compare pure-tone threshold average (PTA) for ears with different genotype-haplotype combinations. These analyses were performed using GraphPad Prism 7 for Mac OS X . Stata  was used to perform multivariate linear regression analysis to investigate associations of hearing loss in M2 R/R and M1 C/R EVA patients as a function of genotype status (M1 or M2), CEVA (reference or CEVA) haplotype status, age, sex, and EVA laterality. We performed the same analysis in M0 EVA patients but without genotype status as a variable since all patients were M0.Fisher\u2019s exact test was used to investigate associations of sex, EVA laterality , and thyroid phenotype with SLC26A4, we compared the phenotypes of M2 subjects without CEVA to those of M1 subjects with CEVA in trans to an allele with the reference haplotype and a mutation affecting the coding regions or splice sites but no CEVA. Among subjects with a determinate thyroid phenotype (Pendred syndrome or nonsyndromic), 10 of 11 M2 subjects without the CEVA haplotype had a Pendred syndrome thyroid phenotype and six of six M1 subjects with CEVA in trans had a nonsyndromic phenotype . Twenty-five (93%) of 27 M2 subjects without CEVA and nine (82%) of 11 M1 subjects with CEVA in trans had bilateral EVA. This difference was not significant (p\u2009=\u20090.564). The median pure-tone average  in the M2 without CEVA group was significantly different  from the median pure-tone average  in the M1 with CEVA group . These results indicate that, as a recessive Mendelian allele in trans to an allele with a mutation of the coding regions or splice sites of SLC26A4, an allele with CEVA but no coding region or splice site mutations is associated with a normal thyroid phenotype and less severe hearing loss in comparison to alleles with a mutation of the coding regions or splice sites and the reference haplotype.To test the hypothesis that CEVA, acting as a recessive mutant allele, is correlated with a less severe phenotype than mutations affecting the coding regions or splice sites of SLC26A4 mutations, we compared the phenotypes of M0 subjects without CEVA with the phenotypes of M0 subjects heterozygous or homozygous for the CEVA haplotype . The median pure-tone average  in the M0 without CEVA group was significantly different  than the median pure-tone average  in the M0 with homozygous CEVA group  than M0 subjects heterozygous for CEVA  . These results indicate that CEVA modifies the severity of hearing loss in M0 EVA ears.To test the hypothesis that CEVA acts as a modifier of the phenotype of patients with EVA caused by factors other than subjects ). Among SLC26A4 alleles with the CEVA haplotype within the cohort of M2 subjects (p\u2009=\u20090.3346). These results do not support the hypothesis that CEVA modifies the severity of hearing loss caused by mutations affecting the coding regions or splice sites of both alleles of SLC26A4.We did not observe any significant differences between hearing loss severity and number of SLC26A4, termed CEVA, that acts as a pathogenic recessive allele in trans to an allele with a mutation affecting the coding regions or splice sites of SLC26A4 in M1 EVA patients [We recently reported a haplotype of 12 variants upstream of patients . When Ampatients , resultipatients .SLC26A4, individuals carrying an SLC26A4 allele with the CEVA haplotype in trans to a mutation of SLC26A4 have a normal thyroid phenotype (i.e. nonsyndromic EVA or DFNB4) and tend to have less severe hearing loss. Although our previous comparisons of phenotypes of M2 subjects with those of M1 subjects revealed that the M1 phenotypes were less severe than the M2 phenotypes, some of the M1 subjects had the reference haplotype in trans to the mutant allele, and some of the M2 subjects had CEVA in cis with one or both mutant alleles. Our current result and conclusion are novel because we performed a comparison that was not confounded by inclusion of M1/reference or M2/CEVA subjects.In contrast to individuals with mutations affecting coding regions or splice sites of both alleles of SLC26A4. Alternatively, our observation may result from ascertainment bias or other unknown factors among the M0 subjects. In contrast, we did not observe any correlations between hearing loss severity and number of SLC26A4 alleles with the CEVA haplotype within the cohort of M2 subjects. However, the power of these analyses was limited by the number of M2 subjects with CEVA (four). We did not perform this analysis in M1 subjects since CEVA is etiologic in those subjects and cannot also be considered a modifier. Furthermore, we cannot be certain of the etiology of EVA in M1 subjects without CEVA. We could therefore not test the hypothesis that CEVA acts as a genetic modifier in M1 subjects.Our analysis of M0 subjects suggests that CEVA, acting as a genetic modifier, increases the severity of hearing loss associated with EVA caused by factors other than mutant alleles of SLC26A4 that cause hearing loss and nonsyndromic EVA or modify the severity of hearing loss in ears with EVA. However, the co-segregation of EVA with SLC26A4-linked markers in M1 families, and the near-zero probability of EVA in the siblings of M0 EVA probands, indicate that such variants would be extremely rare Mendelian causes of nonsyndromic EVA [It is possible there are pathogenic variants in regions or genes unlinked to omic EVA . Our resSLC26A4 exons and splice sites for individuals with hearing loss and EVA because the detection of CEVA provides a definitive diagnosis for individuals with a mutation affecting the trans allele of SLC26A4 [SLC26A4 genotype-haplotype result are unlikely to develop the thyroid abnormality and increased severity of hearing loss associated with Pendred syndrome and mutations affecting the splice sites or coding regions of both alleles of SLC26A4. Finally, this study indicates that CEVA may act as a genetic modifier to increase the severity of hearing loss in ears with EVA caused by factors other than mutations affecting the coding regions or splice sites of SLC26A4.We previously recommended inclusion of testing for CEVA along with analysis of  SLC26A4 . Our curSLC26A4, is associated with less severe auditory and thyroid phenotypes than alleles with a mutation affecting the coding regions or splice sites of SLC26A4. CEVA may act as a genetic modifier in patients with EVA caused by other factors.CEVA, acting as a likely pathogenic recessive allele of"}
+{"text": "Mecp2 promoter methylation in adolescent and adult brain tissue of offspring exposed to prenatal immune activation on gestation day 9 and offspring of saline exposed mice. PCR based methylation assays using Sequenom EpiTYPER was used to quantify DNA methylation at promoter CpG methylation of Long Interspersed Elements-1 (LINE1 or L1) and Mecp2. The dataset\u00a0also includes global DNA methylation and Mecp2 promoter methylation profile at 6 and 12 weeks following early dietary intervention with omega-3 (n-3) PUFA.Prenatal exposure to infection and inflammation increases the risk of neurodevelopmental disorders such as schizophrenia and autism. The etiology could be partly through transgenerational and modifiable DNA methylation changes in the adult offspring's brain. This data descriptor presents a dataset of global DNA methylation (using LINE1 assay) and  Lists are sorted on ID column with summary information . Other columns are body weight in grams at 12 week or 6 week, MIA group or saline control group, assigned diet (n-3 or n-6), sex, mean LINE1 promoter methylation, mean Mecp2 promoter methylation. Missing data marked as \u2018na\u2019 are either not measured or samples with <70% data.This manuscript describes methylation datasets from offspring exposed to prenatal infection and subsequent inflammation in the striatum and prefrontal cortex  and matched controls . Half of the animals in each group received dietary intervention with n-3 poly unsaturated fatty acids (PUFA) from weaning. 2C57BL/6\u00a0N mice were bred and mated in the Laboratory Animal Unit (LAU), The University of Hong Kong. The animals were maintained under ad libitum food and water, kept in 12:12 h normal light-dark cycle (lights off at 19:00) and temperature and humidity-controlled  animal vivarium. Pregnant females were not disturbed, except for weekly cage cleaning. All experiments were performed in accordance with relevant institutional and national guidelines and regulations approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) at The University of Hong Kong and every effort was made to minimize the number of animals used and their suffering.PolyI:C administered C57BL6N MIA mouse model was generated as described elsewhere 2.1The pups were weaned, weighed, and littermates of the same sex were caged three to four per cage on PND 21. Both saline control group and polyI:C group were split into two halves with diet enriched with n-3 was matched to a standard rodent American Institute of Nutrition 93 (AIN93) diet or diet enriched with n-6. Diets differ only in the ratio of n-3/n-6 fatty acids used and provides 16% energy from fat diets supplied by Harlan Laboratories, Madison, and water. Please see study design Approximately 2\u00a0gm n-3/day was administered through diet as an early dietary intervention. The calorific value and total fat content of both n-3 and n-6 diet was balanced. A detailed list of nutrient contents of the rodent AIN93 and modified diet is shown in 2.2At 6-week and 12-week of age, the mice were sacrificed by cervical dislocation and brains removed quickly and transferred to chilled PBS solution. Striatum and prefrontal cortex were collected in 1.5ml tubes using microdissection on a cold platform referring to the Allen Mouse Brain Atlas 2.3EZ DNA methylation kit from Zymo Research, CA, USA was used to treat five hundred nanograms of genomic DNA with sodium bisulfite in duplicate following the manufacturer's standard protocol. The kit exploits the three-step chemical modification that converts unmethylated cytosine to uracil and the methylated cytosine will be protected from sodium bisulfite 2.4Mecp2 promoter were amplified using previously reported primers in LINE1 elements and 2.5Mecp2 is provided in the dataset as values from 0 to 1 (0 represents not methylated and 1 represent fully methylated).MALDI-TOF MS readings were interpreted by EpiTYPER software and generates quantitative information about individual CpGs in each analyzed amplicon. Blank, fully methylated and fully unmethylated controls were confirmed for their corresponding epigram and methylation levels. CpGs with missing data (>20%) and samples with less than 70% data recorded across the CPGs were deemed unfit for subsequent analysis. All flagged data from EpiTYPER such as low mass, high mass  were discarded. Mean CpG methylation for LINE1 and"}
+{"text": "Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema  is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010\u20132015 to identify and serotype  Streptococcus pneumoniae (pneumococcus) is the leading cause of bacterial pneumonia in children and is the most common pathogen isolated in pleural effusions and empyemas .We asked 61 hospital laboratories and pediatric departments located throughout Portugal to report all cases of possible PCPP (in patients <18 years of age) for which pleural fluid was available for analysis and to submit these samples for characterization. We included in our study only samples recovered during January 2010\u2013December 2015, but we performed no audit that would ensure reporting compliance. Because our network includes all secondary and tertiary care hospitals in which PCPP is likely to be treated and a pleural fluid sample obtained, we assume our catchment population is the entire population of Portugal <18 years of age. During 2010\u20132015, this population steadily decreased, from 1,929,331 in 2010 to 1,802,196 in 2015  . For this study, we classified serotypes as vaccine serotypes  and nonvaccine serotypes.We identified all bacteria as S. pneumoniae by using conventional PCR to target the lytA and wzg genes (2010\u20132011) and later (2012 and later) by using real-time PCR (rPCR). We serotyped positive samples by conventional PCR, rPCR, or a combination of both. We confirmed negative results by conventional PCR by using rPCR on samples stored at \u221280\u00b0C.When identification of disease etiology by conventional microbiologic methods failed, we sent pleural fluid to the central laboratory, where total DNA was extracted from 200 \u03bcL of the patient sample by using the High Pure PCR Template Preparation Kit  or DNeasy Blood and Tissue Kit  according to the manufacturers\u2019 instructions. We used a conventional PCR amplification of 2 human genes (encoding human \u03b2-actin and RNaseP) as control for the quality of the purified DNA. We initially tested the presence of S. pneumoniae genes (lytA and wzg) (We used a multiplex PCR for the amplification of the genes encoding human \u03b2-actin and RNaseP (lytA and wzg genes) (2 (50 nmol/L), and water, for a final volume of 25 \u03bcL. Cycling conditions were as follows: 95\u00b0C for 10 min, followed by 50 cycles at 95\u00b0C for 15 s and 60\u00b0C for 1 min. We performed detection of lytA and wzg in singleplex PCR. We defined negative results as those with cycle threshold (Ct) >40 and positive results as those with Ct<35  by using the Platinum quantitative PCR SuperMix-UDG . PCR reactions contained 5 \u03bcL of DNA, 5 pmol/L of each primer .S. pneumoniae in the pleural fluid by molecular methods, but we confirmed the remaining 92 cases (68%) to be PCPP by molecular methods. The total numbers of requests for molecular testing and samples positive for S. pneumoniae were approximately constant over the years  PCPP cases occurred in nonvaccinated children (median age 2.6 [IQR 1.4\u20138.5] years), and the vaccination status was unknown for 28 (26%) patients. The remaining 47 (43%) PCPP cases occurred in children who received >1 vaccine dose (median age 3.2 [IQR 3\u20135] years). All vaccinated children received PCV7, PCV13, or both; PCV10 had not been administered to any child in the study.Among the 109 PCPP case-patients (17 identified by culture and 92 by molecular methods), 56 were male and 52 female; the sex of 1 patient was not available. Patient age ranged from 4 months to 17 years (median age 4 [IQR 2.3\u20136] years); 27 cases were in children >0.198 by Fisher exact test). Whenever possible, conventional PCR serotyping enabled the identification of serotypes 7F, 6B, and 9V, whereas rPCR detected only the groups 9V/9A, 7F/7A, and 6 without further discrimination. On the 17 culture-positive samples, only 5 serotypes were found: 1 and 3 (n = 7 each) and 14, 10A, and 19A (n = 1 each). Apart from serotype 10A, which was detected exclusively in 1 isolate, all other serotypes were also detected by PCR from culture-negative samples. Most of the PCPP case-patients for whom a serotype was unambiguously identified were infected with the addPCV13 serotypes (n = 47 [43%]), followed by the addPCV10 serotypes (n = 25 [23%]), whereas PCV7 serotypes were responsible for a small fraction of PCPP cases (n = 7 [6%]). Among the remaining cases, 6 were caused by nonvaccine serotypes, and in another 6 cases, we were unable to classify capsular types as vaccine or nonvaccine type. These cases included samples for which rPCR tested positive for serogroup 6 (n = 1), 7F/7A (n = 4), or 9V/9A (n = 1).Except for 18 (16%) samples, we were able identify the serotypes of the pneumococci responsible for illness . OverallAmong the 47 vaccinated children, 35 were age-appropriately vaccinated . Of thesS. pneumoniae among children age-appropriately vaccinated with PCV13 (n = 22/27) and among the other case-patients for whom vaccination status was known (n = 59/71) (p = 1 by Fisher exact test).We compared the number of addPCV10 and addPCV13 PCPP cases  among children age-appropriately vaccinated with PCV13 and among the other case-patients for whom vaccination status was known and serotype could be unambiguously determined. Serotype 3 was overrepresented among PCPP cases in children age-appropriately vaccinated with PCV13 (p<0.001 by Fisher exact test). We detected a similar number of positive samples for S. pneumoniae in pleural fluid samples was greatly improved by the use of molecular methods, as previously reported , which would increase the likelihood that any PCPP cases in this group would be caused by serotype 3.>3 years of age (n = 12/17), but the distribution was similar to that of serotype 3 case-patients not vaccinated with PCV13 (p = 0.353 by Fisher exact test), so it does not seem likely that this was attributable to faster waning of the immune response to this serotype. In fact, cases of serotype 3 PCPP occurred in younger children than did cases caused by serotype 1, a serotype also included in PCV13 and for which only 1 vaccine failure was detected. Because the synthesis of the serotype 3 capsular polysaccharide proceeds through a synthase mechanism, the polysaccharide is not covalently linked to the peptidoglycan and can be released during growth, thereby potentially reducing opsonophagocytosis (The effectiveness of PCV13 against serotype 3 has been questioned in several studies. In a large surveillance study performed in the United States, no reduction in IPD caused by serotype 3 was observed despite reductions in IPD incidence and evident decreases in IPD caused by PCV13 serotypes 19A and 7F (The proportion of children asymptomatically colonized with serotype 3 increased in the period preceding PCV13 introduction in Portugal (In agreement with our observations, other reports document serotype 3 vaccine failures. A study in Greece found 5 cases of complicated pneumonia caused by serotype 3 pneumococci among vaccinated children, although most vaccine failures occurred in children who received a single dose of PCV13, which could offer only limited protection (S. pneumoniae in pleural fluid samples and emphasizes the potential role of molecular techniques when evaluating disease incidence. Another limitation of our study is lack of detailed information about the immune status or other underlying conditions in age-appropriately vaccinated children with PCPP by vaccine serotype. However, given the high prevalence of serotype 3 in this group, it is unlikely that all cases could be explained by host characteristics, indicating that specific properties of serotype 3 must be responsible for this behavior.Although our study was prospective and involved both pediatric and microbiology departments, it was not designed to estimate the incidence of PCPP because it did not identify cases in which there were clinical or radiographic criteria for complicated pneumonia and for which pneumococci were identified in either blood or respiratory samples. Although this certainly resulted in an underestimation of PCPP, the use of conventional PCR and rPCR techniques greatly enhanced the ascertainment of PCPP cases by improving the detection of In summary, we describe data that are compatible with a lower individual effectiveness of PCV13 against PCPP, a presentation of IPD for which PCV13 is specifically recommended. The public health consequences of such possible lower protection might be mitigated by a reduction in circulating serotype 3 by vaccination with very high coverage, such as those achieved through inclusion in NIPs. Carriage studies and continued surveillance are necessary to determine the group effect of the introduction of PCV13 in the NIP to clarify the effectiveness of PCV13 in the prevention of infections caused by serotype 3."}
+{"text": "We hypothesized that MRI, when used in an optimal This prospective case-control study was approved by the institutional review board. 3D MRI acquisitions including saturation-recovery T1 mapping and DIXON imaging was performed at 4.0 T on 9 human LA samples collected from patients who underwent cardiac surgery. Histological quantification of fibrosis and fat was obtained. MRI T1 maps were clustered based on a Gaussian Mixture Model allowing quantification of total, interstitial and fatty fibrosis components. Fat maps were computed from DIXON images and fat fractions were calculated. MRI measurements were performed on the same location as the histological analysis (plane) and on the entire sample volume (3D).High correlations and levels of agreement were observed between MRI and histology for total (r = 0.93), interstitial (r = 0.93) and fatty fibrosis (r = 0.98) and fat (r = 0.96). Native T1 correlated with the amount of fibrosis from MRI and histology. The 3D MRI total, interstitial and fatty fibrosis ranges were between 6% and 23%, 4% and 17.3%; and 1.4% and 19.7% respectively.ex vivo MRI was able to quantify different LA myocardial components with high agreement in 2D with histology and moreover to provide 3D quantification of such components whereas in vivo application remains a challenge.High Field  Most. Most2].o by MRI . Subsequin vivo quantitative myocardial tissue characterization non-invasively 8], p[8], pin , p[8], pvasively 10]11].[11].in v.[11].in  effects . Few att effects . However effects 15]..in vivo ex vivo setting  to study the ability of MRI to characterize complex fibrotic and fat components of the LA wall. The DIXON method has been proposed to generate fat maps of the myocardium . The. Thek-spin vivo observations at close field strength 25],[25],in v,[25],in strength 9]10][2[10][2in [2[10][2istrength 27]..in vivo in vivo studies analyzing left ventricular myocardial biopsies in subjects undergoing aortic valve replacement (r = 0.65) 30].[30].in v.[30].in ex vivo. This issue has been raised by Kellman et al. ..versus hIn vivo assessment of myocardial fat has been proposed by spectroscopy or imaging [in vivo when applied to the thin (2 mm) left atrial wall [ex vivo results to patients in the clinical setting and uniquely provide compartmental quantitative tissue characterization of the LA wall in vivo. Such data at the tissue level would fill an important gap in currently available information in vivo as LA biopsies are not performed and could increase our understanding of LA cardiomyopathy and pave the way for novel management strategies. imaging  but the ial wall . Improveex vivo T1 mapping sequence used here in vivo in a moving heart with a sufficient spatial resolution to reliably study the thin left atrial wall. However, our primary goal was here to demonstrate the ability of MRI to characterize the complex LA myocardial compartments and define the determinants of native T1 in \u201cideal\u201d conditions. This technique may be immediately useful in analyzing myocardial biopsies to reduce sampling errors or in animal validation studies in the whole heart before technical improvements make them ready for translation to in vivo human studies. Further studies should be performed to overcome the challenging technical issues which will allow translation to a moving heart.This study has several limitations. First, there is a relatively small sample size. This reflects the high difficulty to obtain tissue samples in patients. Second, it is not currently possible to apply the ex vivo on human LA myocardium provided highly accurate quantification of myocardial components including fibrosis, fat and fatty fibrosis, as revealed by highly significant correlations with histology. Increased native T1 was strongly related to increased interstitial fibrosis and the extent of adipose tissue tended to decrease T1 values. Furthermore, MRI was able to propose a 3D distribution of these myocardial tissue components over the sample.T1 mapping combined with DIXON MRI sequences acquired S1 Fig(DOCX)Click here for additional data file."}
+{"text": "GSTM1 and GSTT1 gene deletions in causing predisposition to adult ALL.Biotransformation of xenobiotics are critical for their metabolism and removal from the body which is carried out by xenobiotic metabolizing enzymes. Individuals carrying variants of genes that encode these enzymes have an altered ability to metabolize xenobiotics which may lead to an increased risk of acute lymphoblastic leukemia. The current study aimed to investigate the impact of GSTM1 and GSTT1 deletions. The genotype frequency obtained for patients was compared to controls using odds ratio and chi-square.The current case-control study involved 62 adult ALL patients and 62 age and gender matched healthy controls. Whole blood samples processed with standard phenol chloroform protocol for DNA isolation were genotyped using multiplex PCR approach for simultaneous identification of GSTM1 and GSTT1 in a group of adult ALL patients from Pakistan were 47% and 11% respectively. Deletion of GSTM1 and GSTT1 did not show statistically significant association with adult ALL (p=0.86 and p=0.35 respectively). The combined GSTM1/GSTT1 deletion was observed in 2% patients and was not significantly associated with ALL in adults (p=0.85).The null genotype frequency of GSTM1 and GSTT1genes does not influence ALL susceptibility among adult patients. Cancer susceptibility associated with GST polymorphism varies with ethnic and geographic differences. Therefore, further investigation on different populations is needed to understand the role of these genetic variations in modifying adult ALL risk.The results reveal that homozygous null polymorphism of  Acute lymphoblastic leukemia (ALL) is a blood malignancy distinguished by an excessive buildup of lymphoid progenitor cells in blood and bone marrow. ALL constitutes one fourth of all cancer cases occurring in childhood making it the most common pediatric cancer.5The development of ALL includes both genetic and environmental factors with DNA damage in hematopoietic precursor cells being acrucial step.GSTM1 and GSTT1, exhibit null polymorphism indicating homozygous deletion of the genes.GST deletion has also been studied in the development of hematologic malignancies.GSTM1 and GSTT1among adult ALL patients in comparison to controls was studiedand their association with the occurrence of adult ALL was examined.The glutathione S-transferases (GSTs) constitute an enzyme super family responsible for detoxification of carcinogens. Detoxification represents the second phase of carcinogen metabolism in human body, followed by bioactivation of procarcinogens (phase I). GSTs detoxify reactive intermediates produced during the first phase of metabolism by conjugating soluble glutathione with them.The current case control study was assessed and ethically approved by the Institutional Ethical Committee of the Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi. The study comprised 62 adult ALL cases and an equal number of controls. Patients were recruited from department of oncology, Jinnah Postgraduate Medical Centre (JPMC) Karachi, Pakistan after clinical diagnosis. Questionnaire was filled to collect personal information of patients while medical records were checked to extract their clinical history. Patients with history of other cancers were excluded. Healthy individuals from the population qualified as controls and were matched to cases on age and gender. All participants were provided with an information sheet explaining study objective along with potential risks and benefits associated with their participation prior to their enrolment in this study. A written informed consent declaring their voluntary participation was obtained from them.Five ml of whole blood was withdrawn from patients as well as controls in sterile acid citrate dextrose (ACD) vacutainer for genotype analysis. Collected blood samples were stored at -20\u00b0C till further use. They were treated with standard phenol chloroform procedure for genomic DNA isolation.GSTM1 and GSTT1genes.GSTM1, 5\u2032 TTCCTTACTGGTCCTCACATCTC 3\u2032 and 5\u2032 TCACCGGATCATGGCCAGCA 3\u2032 for GSTT1which resulted in 219bp and 459bp fragments respectively. \u03b2-globin gene was indicative of successful PCR. Primers used for its amplification were 5\u2032 CAACTTCATCCACGTTCACC 3\u2032 and 5\u2032 GAAGAGCCAAGGACAGGTAC 3\u2032 that gave a 268bpamplified product. Multiplex PCR was carried out with 200ng extracted DNA in total reaction volume of 20 \u00b5l. The other PCR componentsincluded 1x (NH4)2SO4 buffer, 1.5mM MgCl2, 200 \u03bcM dNTPs mix, 1.5 units of Taq DNA polymerase, 0.6 \u03bcM of sense and antisense primer for GSTM1, 0.3 \u03bcM of each primer for GSTT1 and 0.4 \u03bcM of each primer for \u03b2 globin gene.PCR cycle conditions included an initial melting step at 94\u00b0C for four minutes, 35 PCR cycles, each including 30 s denaturation at 94\u00b0C, 35 s annealing at 58\u00b0C and 30 s elongation at 72\u00b0C, followed by a seven minutes long final elongation step at 72\u00b0C. Eight microliters ofthe PCRproducts were loaded on 2.5% agarose gel and electrophoresis was carried outfor 40 min at 100 V. The ethidium bromide stained gel containing amplified fragments wasvisualized under gel documentation system.Multiplex PCR assay was used to screen out homozygous deletions in GSTM1 and GSTT1for the disease. Chi-square test was performed to test association of homozygous deletion in GSTM1 and GSTT1 genes with development of adult ALL using VassarStats, a freely available online tool, under the null hypothesis of \u201cno association\u201d.16Odds ratio (OR) with 95% confidence interval (CI) was calculated to measure the risk conferred by null allele of The present study involved 62 adult ALL patients ranging from 15 to 55 years of age. The mean age (\u00b1SD) of patients at diagnosis was24.7 \u00b1 1.1 years. Patients under 25 years of age constituted 53% of the total number of cases while 89% of patients were less than 35 years. Patients included both genders but male cases predominated female cases with a ratio of 2.4:1.GSTM1 and GSTT1 null polymorphism resulted in banding pattern shown in GSTM1deletion resulted in the absence of 219bp amplified product while absence of 459bp fragment meant homozygous deletion ofGSTT1gene. Amplification of \u03b2-globin gene resulting in 268bp fragment confirmed the success of PCR when both GST genes were deleted.Multiplex PCR assay for co-identification of GSTT1 null genotype (MlT0); lane 2, GSTM1 null and GSTT1 null genotype (M0T0); lane 3, 8, 9 and 10, GSTM1 null genotype (M0T1); lane 4, 5, 6, 7, 11, 12, GSTM1 and GSTT1 wild type genotype (M1T1)Lane M: 50 bp DNA ladder; lane 1, GSTM1 and GSTT1genes among patients and controls and the odds of their association with ALL are shown in GSTM1 and GSTT1 deletion among study participants (n=124) was46% and 8.7% respectively. GSTM1 was deleted in 47% adult ALL patients (n=62) and in 45% controls (n=62). The null genotype of GSTT1 was 11% for cases and 6% for healthy individuals. The Pearson chi-square test revealed neither GSTM1 deletion nor GSTT1 null genotype was significantly associated with adult ALL . GSTM1 and GSTT1 were simultaneously deleted in 1.6% cases as compared to 3.2% controls .Frequency distribution of homozygous null polymorphism in controls . This diGSTM1 and GSTT1 resulting from homozygous deletion of the respective genes lead to the lack of active enzymes. This loss of enzyme activity affects metabolism of several carcinogens and may therefore influence an individual\u2019s risk of cancer development.GST genetic variants are also good candidates for association studies in leukemia as they have a potential to alter metabolism of leukemogens and to cause lack of protection against ROS leading to cellular DNA damage.17Glutathione S-transferases take part in detoxification of different carcinogens, environmental toxins and chemotherapeutic drugs. The null genotypes of GSTM1 and GSTT1deletions as predisposing factor of adult ALL revealed that the genotype frequencies of both genes were not significantly higher in ALL patients as compared to healthy controls (47% vs. 45% for GSTM1 and 11% vs. 6% for GSTT1). The use of healthy controls over hospital based controls was intentionally preferred to reduce possible bias in results due to the risk conferred by deletion polymorphism of GSTM1 and GSTT1to non-cancer diseases.The current study evaluating the impact of GST allelesis not uniform across human population and ethnic as well as geographic variations have been observed. In Asians, the frequency of GSTM1deletionhas been reported to vary between 42% and 54% (n=1511) while 35% to 52% for GSTT1 null polymorphism (n=575).GSTM1 and GSTT1 deletion to be 45% and 21% in Iranian (n=229) while 33% and 18% in North Indian (n=198) population respectively.20Distribution of Indian n=8 populatGSTM1 null polymorphism  or GSTT1 gene deletion  with ALL among adults. Most genetic association studies examining role of GSTM1 and GSTT1 deletion as a modulator of ALL risk have focused children affected with ALL. Only a limited number of studies have evaluated this risk with adult ALL owing to the fact that ALL has a higher occurrence in children as compared to adults. A study evaluating 71 adult ALL patients for GSTM1 and GSTT1 reported significant association of GSTT1 null variant  with adult ALL but no association of GSTM1 null genotype. The participants of this study were Caucasians.GSTM1 null genotype but were contradictory for GSTT1 as they showed negative association of GSTT1 deletion with ALL .GSTM1 or GSTT1 null genotype with ALL. The results of this study are comparable to our findings, however, the patients included in the study were all under 25 years of age.GST null polymorphisms with acute leukemia risk in children while no association with adults.GST null allele distribution and its association with ALL.The current study reports no association of either GSTM1 null, GSTT1 null, and GSTM1/GSTT1 double null genotypes with ALL in adult patients from Pakistan. ALL risk may not only be affected by genetic factors but also with environmental factors. Therefore, studies with a better study design and a larger sample size including environmental exposure data in addition to genetic variants of xenobiotic metabolism are required to provide more insight into the etiology of adult ALL.In conclusion, results from the current study suggest no statistically significant association of"}
+{"text": "Once established in poultry, the virus rapidly spread between turkey and chicken farms in neighboring states. Enhanced biosecurity is required to prevent the introduction and dissemination of HPAIV across the poultry industry.Eurasia highly pathogenic avian influenza virus (HPAIV) H5 clade 2.3.4.4 emerged in North America at the end of 2014 and caused outbreaks affecting >50 million poultry in the United States before eradication in June 2015. We investigated the underlying ecologic and epidemiologic processes associated with this viral spread by performing a comparative genomic study using 268 full-length genome sequences and data from outbreak investigations. Reassortant HPAIV H5N2 circulated in wild birds along the Pacific flyway before several spillover events transmitting the virus to poultry farms. Our analysis suggests that  Highly pathogenic avian influenza virus (HPAIV) A(H5N1) emerged in 1996 in Guangdong, China (A/goose/Guangdong/1/1996 [Gs/GD]), and has since caused outbreaks in poultry, infections in wild birds, and often fatal clinical cases in humans in >80 countries . ThiWe sequenced and analyzed the full-length genome sequences of 249 H5N2 and 19 H5N8 HPAIVs collected during the 2014\u20132015 outbreaks in the United States. Viruses came from 32 wild birds, 7 raptors, 14 backyard poultry farms, and 196 commercial poultry sites. We used a molecular epidemiologic approach involving genome sequences and outbreak information to determine the evolution and spread patterns of these viruses.The National Animal Health Laboratory Network conducts PCR testing for avian influenza in the United States, and per title 9, Code of Federal Regulations, nonnegative samples are forwarded to the National Veterinary Services Laboratories  for confirmation testing and genome sequencing. In December 2014, interagency wild bird surveillance started including testing for the HPAIV Gs/GD lineage in wild birds in the Pacific flyway, and by summer 2015, surveillance was further expanded to include testing for this virus in all flyways (http://www.dnastar.com/t-nextgen-seqman-ngen.aspx). We deposited nucleotide sequences in GenBank . We synthesized complementary DNA and amplified all 8 segments by reverse transcription PCR using SuperScript III  ( GenBank  Table 5.http://platform.gisaid.org) and sequenced the 249 viruses from the United States. In all, 32 viruses were from wild waterfowl in 10 states ; 7 viruses were from captive and wild raptors in 5 states ; and 210 viruses were from backyard and commercial poultry farms in 13 states . Data from Minnesota  and Iowa , where most commercial poultry were affected, predominated in this sample.Of the 265 H5N2 sequences analyzed, we retrieved the 16 viruses from Canada from the GISAID EpiFlu database (https://www.tableau.com/).To demonstrate the phylogenetic organization of the HPAIV outbreak, we built a maximum-likelihood phylogenetic tree of the concatenated genome  with RAxML version 8.0.0 (http://www.fluxus-engineering.com/sharenet_rn.htm) with epsilon set to 0   of HPAIV hemagglutinin (HA) sequences from Asia, Europe, and North America, we tested whether the HPAIV H5 outbreak in North America resulted from a single virus introduction or multiple virus introductions into the bird population of North America. We then developed a more refined model to estimate viral diffusion and transmission between wild and domestic populations and assess the most likely route of spread. We incorporated HPAIV HA sequences from the US and Canada 2014\u20132015 outbreaks  to investigate the relatedness of isolates Figure 2.Both group 1 and 2 viruses were found in wild birds and gallinaceous poultry  Figure 3Our phylogenetic analysis suggests that the outbreak in the Midwest was initiated through multiple independent HPAIV H5N2 introductions with group 1 viruses of wild bird origin , 3. To fBayesian simulation results showed that turkey farms and chicken farms were equally supported as the source of infection of domestic flocks after establishment of the virus in Midwest US farms . AlthougWe identified 14 common substitutions across the entire genome of HPAIV H5N2 when comparing with US index virus A/northern pintail/WA/40964/2014 (H5N2). These substitutions include 10 nonsynonymous substitutions in group 1 and 2 viruses  and 4 nonsynonymous substitutions in group 2 viruses   Table 3.Phylogenetic reconstruction of source\u2013sink dynamics supports multiple independent introductions of HPAIV H5N8 into the United States during late 2014 from wild birds. Despite the frequent detection of HPAIV H5 in wild birds of the Pacific flyway up to February 2015 , and epidemiologic links could not be found between Pacific Coast and Midwest farms for any virus subgroup. The widespread detection of the HPAIV H5 lineage in healthy wild birds  to infect chickens; directly inoculated and in-contact exposed survivors did not seroconvert (50 of H5N2 group 2 viruses for poultry (103.2\u20133.6 EID50) was lower than that of group 1 viruses for poultry (105.1 EID50) and index wild birds (105.7 EID50).The epidemiology of the HPAIV H5 detections and pathobiologic features suggest that the early H5N8 and H5N2 group 1 viruses detected in the United States were highly adapted to waterfowl and poorly adapted to chickens and turkeys  were identified among H5N2 group 2 viruses from the United States and a virus isolate from a chicken in British Columbia  reemerged and caused outbreaks in wild birds and domestic poultry across Europe, Asia, and Africa  A(H5Nx) clade 2.3.4.4 isolates from 2014\u20132015 outbreak in North America."}
+{"text": "How to cite this article: Aquino MC, Lim D, Chew PTK. Micropulse P3\u2122(MP3) Laser for Glaucoma: An Innovative Therapy. J Curr Glaucoma Pract 2018;12(2):51-52. Laser therapy remains a useful, noninvasive option in the widening array of glaucoma treatment modalities.Traditional ciliary body lasers are generally cyclodestructive and concern about its use stem from the risks of sight threatening sequelae. Transcleral diode laser cyclophotocoagulation (TSCPC)  is a tre1 It is hypothesized that this form of ciliary body treatment lowers intraocular pressure (IOP) by enhancement of existing uveoscleral outflow channels without photocoagulating the target site.A new form of ciliary body laser termed micropulse P3\u2122 (MP3) using 810 nm infrared diode has been developed. It uses a novel probe design (micropulse P3\u2122 glaucoma device)  of a bal2 and randomized3 studies on advanced, refractory glaucomas have provided evidence that MP3 lowers IOP by an average of 45% from baseline with minimal to nil complications without any incidence of hypotony and phthisis bulbi after 18 months of follow-up. With the micropulse laser machine set at 31.3% duty cycle  the desired effect is reached. Reports on MP3 by other investigators adapting the technology demonstrated the same efficacy. Williams, Moster et al.4 treated patients with refractory glaucoma and reported up to 51% IOP reduction at the last follow-up (7.8 months \u00b1 4.5). Maslin and Noecker5 reported their experience after 12 months of follow-up and found an average of 41.6% IOP reduction without any hypotony. Radcliffe et al.6 observed a mean IOP reduction of 29.8% after 3 months. To show the non destructive effects of MP3 on the ciliary body, Shan Lin et al.7 performed ultrasound biomicroscopic imaging before and after treatment. No ultrasonographic evidence of morphological changes, destruction of adjacent structures nor suprachoroidal effusion were seen following the using of MP3. With encouraging results in lowering IOP, proven safety in advanced and medically refractory glaucoma, MP3 use and its clinical applications continue to be widen.Micropulse technology in glaucoma therapy has paved the way for an efficient and safe procedure applicable to all glaucoma types. The pioneering work and experience of Chew et al. both in the pilot89 New treatment protocols are being evaluated to address cases with minimal or no response to initial MP3. Its role as a potential replacement or adjunct to medical therapy is currently being evaluated. MP3 is now being increasingly used in combination with cataract surgery. MP3 as a temporizing therapy to lower IOP prior to glaucoma filtering surgery in medically refractory cases increases the safety and reduces the risks of devastating sight threatening complications (such as hypotonous maculopathy) before definitive glaucoma filtering surgeries especially in eyes with extremely high IOP preoperatively. Like all new technology, the expanding indications of MP3 remain to be explored.MP3 is increasingly used by eye surgeons in different centers worldwide. Modifications to the power, treatment duration, duty cycle or a combination of the three parameters have been described in the literature."}
+{"text": "The regulation of mechanistic target of rapamycin (mTOR) signaling contributes to the metabolic effects of a calorie restriction (CR) diet. We assayed the effect of CR on the activity of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) in the liver of mice at six different times across the day. CR effects on mTORC1 and mTORC2 activities were time-of-day dependent. CR induced mTORC1 activity at one time, reduced at two times and has no effect during other times. CR induced mTORC2 activity at one time of the day and has no effects at other times. Circadian clocks are implemented in the regulation of mTOR signaling in mammals and mechanisms of CR. We assayed the effect of CR on mTOR signaling in the liver of mice deficient for circadian transcriptional regulators BMAL1 and CRYs. The CR induced suppression of mTORC1 activity was observed in both clock mutants, while up regulation of mTORC2 was observed in the liver of CRY deficient but not in the liver of BMAL1 deficient mice. Our finding revealed that CR has different time dependent effect on the activity of mTOR complexes 1 and 2 and suggest that circadian clock protein BMAL1 is involved in the up regulation of mTORC2 upon CR in mammals. Calorie restriction (CR) is a feeding regimen that increases longevity in organisms from yeast to primates . The mecThe activity of mTORC1 and mTORC2 is regulated by nutrients, hormones and growth factors . In mammAd Libitum (AL group) access to the food. CR mice received the food at ZT14 . We and others previously reported that feeding CR mice at ZT14 (or ZT12) did not significantly shift the phases of expression of core circadian clock genes [To assess the effect of CR on mTOR signaling, we compared daily rhythms mTORC1 and mTORC2 activities in the liver of mice that were subject to 30% CR for 2 months (CR group) with the activities in the liver of mice that had ck genes ,19\u201323 in-/-Bmal1 and -/-Cry1,2 mice [-/-Cry1,2 mice but not in in -/-Bmal1 mice. Thus, the circadian clock proteins BMAL1 and CRYs are not required for CR mediated down regulation of mTORC1 activity during rest/fasting period.mTORC1 activity was assayed based on the phosphorylation of ribosomal protein S6 on S235/236 sites (S6 is not a direct target but it is often used as a surrogate marker of mTORC1 activity) ,5 in the-/- mice \u201326 under-/-Bmal1 mice with high level at ZT10-18. In contrast to the effect in wild type mice CR did not induce mTORC2 activity but resulted in significant reduction at ZT10-14. mTORC2 responded differently to CR in the liver -/-Cry1,2 mice: the phosphorylation of AKT-S473 was reduced at ZT6/ZT10 and increased at ZT14/ZT18. Thus, circadian clock protein BMAL1 but not CRYs is important for the CR mediated induction in mTORC2 activity.We assayed mTORC2 activity through phosphorylation of its downstream target AKT on S473 . Results-/-Bmal1 at ZT10 and ZT14. The phosphorylation was increased at ZT18 and decreased at ZT10 in -/-Cry1,2 mice. Thus, we observed a good correlation between AKT and PRAS40 phosphorylation.TORC2-dependent phosphorylation of AKT stimulates the kinase activity, and we decided to check the effect of CR on the phosphorylation of PRAS40 at S246, it is known that this site is phosphorylated by AKT . We assaWe reported here that CR affected daily changes in mTORC1 and mTORC2 activity in the liver. mTOR plays an important role in the control of metabolism and aging. High mTORC1 activity promotes aging and suppression of mTORC1 increases longevity. mTORC2 might also be involved in the control of longevity but, in contrast to mTORC1, it might be a positive regulator of longevity . Changes-/-Bmal1 and at ZT14 in -/-Cry1,2 mice; mTORC2 activity was high at ZT22 in wild type, intermediate in -/-Bmal1 and low in -/-Cry1,2 mice. Together that suggests that the change in feeding pattern is not the only regulator of CR induced effects on mTOR signaling.It is important to mention that feeding is a regulator of mTOR activity and CR can change a feeding pattern, therefore, some of the observed effects can be due to the changes in the feeding. It is also well documented that phases of clock gene expression in the liver are strongly affected by time of the feeding . Feeding-/-Bmal1 mice [-/-Cry1,2 mice consume about 15% less food [-/-Bmal1 and did not changed in -/-Cry1,2 mice. Thus, the difference in the response of mTOR complexes to CR between wild type and mutants cannot be explained through the difference in total food intake: wild type and -/-Bmal1 mice have the same food intake but different responses.Difference in the amount of the food consumption between wild type and mutants might be another contributing factor. There is no significant difference in food consumption between wild type -/- mice , while Cess food . We comp-/-Bmal1 became rhythmic under CR, while -/-Cry1,2 did not. mTORC2 activity was rhythmic under AL in wild type and -/-Bmal1 mice, it lost the rhythms in wild type under CR but it was still rhythmic in -/-Bmal1 mice. The peaks in mTORC1 and mTORC2 activities coincided at ZT14 for AL wild type mice, while mTORC1 peaked at ZT 16 and mTORC2 peaked at ZT 20  in the liver of -/-Cry1,2mice. Interestingly, at wildness, animals do not have continuous access to the food and our data suggest that intact circadian clock is necessary for temporal separation of the peaks in mTORC1 and mTORC2 activities under conditions of limited nutrients supply.mTORC1 and mTORC2 share the same catalytic subunit, the TOR kinase ,5. It waat ZT 20  in CR wi-/-Bmal1 mice, suggesting that BMAL1 has some clock independent functions in CR. Indeed, CR regulated expression and activity of the clock transcriptional factor BMAL1 is necessary for the regulation of CR-controlled signaling pathways and for the full benefits of CR. Interestingly, while -/-Bmal1 mice demonstrate multiple metabolic abnormalities as well as accelerated aging and reduced lifespan [Based on the presented data, we proposed the following hypothesis: the balance between the complexes established under AL feeding was changed by CR and shifted toward mTORC2. Circadian clock and circadian clock proteins contributed to this shift through compartmentalization of mTORC1 and mTORC2 activities in time. The effect of CR was further compromised in lifespan , this phlifespan . BMAL1 ilifespan ,14 and alifespan  activiti-/-Bmal1 and -/-Cry1,2 mice were previously described [Ad libitum (AL) group had unrestricted access to food. Animals on Caloric restriction (CR) diet received their food once per day at ZT14. CR was started at 3 months of age. Animals were on 30% CR for two months before tissue collection. All groups had unrestricted access to water. All tissue collection experiments were performed for 5-month-old wild type, -/-Bmal1 and -/-Cry1,2 mice. All animal studies were conducted in accordance with the regulations of the Committee on Animal Care and Use at Cleveland State University.Wild type and circadian mutant mice were on C57BL/6J background. escribed ,25. AnimoC. For quantitative analysis, liver samples were run individually to estimate variability between biological replicates. For a representative WB, liver lysates from three different mice were pooled together for each time point. Lysates were prepared with lysis buffer with Protease/Phosphatase Inhibitor Cocktail  using a sonicator. Protein concentration was determined using Bradford protein assay kit. Protein loading was checked by Ponceau staining. List of primary antibodies is in Analysis of protein expression was performed on liver tissues collected every four hours around the clock and stored at -80At least 3 animals for every time point, for each feeding type and for each genotype were used for all experiments. Data are shown as average +/- standard deviation. IBM SPSS Statistics 20 and GraphPad software packages were used for analysis. The effect of diet (AL versus CR) and time of day were tested for significance with two-way repeated ANOVA corrected for multiple comparison using Bonferroni. P<0.05 was considered as statistically significant difference.Supplementary Table 1Supplementary Table 2"}
+{"text": "Ortega et al. published a comprehensive review which examines the relationship between nutritional genomics and Type 2 diabetes mellitus (T2DM) from a wide perspective . The autPPAR-\u03b3 gene is localized in the chromosome 3p25.2 region. The most studied, prevalent and also benign Pro12Ala variant was identified in the PPARG2 isoform [GNGT1) gene localized in the region 7q21.3 [PPAR-\u03b3 gene is localized in the region 3p25.2 and the rs number of the Pro12Ala variant is rs1801282 [Peroxisome proliferator activated receptors (PPARs) are a group of nuclear receptor subfamily which modulate energy metabolism and act as ligand activated transcription factors by small lipophilic molecules to regulate gene expression ,3. PPAR- isoform . It is k isoform . In a st isoform . It has  isoform . In addin 7q21.3 . The PPAs1801282 .CLOCK variant sampled in both text and Table 3 is incorrect. The authors cited a PREDIMED study by Corella et al. [TCF7L2 and obesity in T2DM. The correct reference seems to be another PREDIMED study performed by the same group [CLOCK rs4580704, CRY1 rs2287161 variants in T2DM, but other circadian related gene variants were not addressed. In a study investigating the gene\u2013diet interactions of the CLOCK variants in metabolic syndrome patients, it was found that TT carriers of rs1801260 variant had higher insulin sensitivity, lower plasma insulin concentration and lower HOMA-IR after one year of low fat consumption [Ortega et al. also mentioned gene\u2013diet interactions related to variants of circadian related genes in T2DM. It is known that the circadian clock is involved in the control of metabolic functions and regulation of blood glucose concentration . In an ea et al.  investigme group . The autsumption . Each 1%sumption . CLOCK variant. In this study, it was quoted that obesity status should be taken into consideration when classifying patients to obtain more accurate information about an interaction between diet and TCF7L2 rs7903146 variant for T2DM risk [Additionally, there is no information in the text about the PREDIMED study, which is associated with TCF7L2\u2013diet interactions, given as reference incorrectly for the 2DM risk .This review successfully discussed the relationship between nutritional genomics and T2DM in light of current data in detail. Readers could obtain more accurate data with considering the aforementioned issues."}